AN ACROLEIN-DERIVATIZED cAMP ANTISERUM TO STUDY cAMP SIGNALING AND VISUALIZATION IN THE ENTERIC NERVOUS SYSTEM-IMPLICATIONS FOR GUT INFLAMMATION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree of Doctoral of Philosophy in the Graduate School of The Ohio State University
By
Jorge E Guzman *****
The Ohio State University 2004
Dissertation Committee: Fievos L. Christofi, Ph.D., Adviser Approved by Helen J. Cooke, Ph.D. Richard H. Fertel, Ph.D. Scott, Melvin, M.D. Bob Stephens Ph.D.
______Adviser Department of Physiology
ABSTRACT
Background & Aims: The general aim was to elucidate the role of cAMP signaling in
intact neural circuits of the enteric nervous system in normal or inflamed gut. Forskolin
binding, AC-ir and FIChR/cAMP recordings suggest AC/cAMP signaling occurs in a
heterogeneous population of gut neurons. An acrolein-derivatized cAMP antiserum was
developed and used for the visualization, quantitation, classification and polarity of gut
neurons. We tested hypothesis 1 that cAMP signaling occurs in functional classes of neurons other than AH/Dogiel Type II intrinsic primary afferent neurons (IPANs).
Hypothesis 2 tested if cAMP signaling in the two neural plexuses contributes to cAMP- dependent reflexes. Hypothesis 3 tested if amplification of the R/Gs/AC/cAMP- dependent pathway occurs in intestinal inflammation induced by Trichinella spiralis infection. Experimental Design: Forskolin stimulation was used for visualization of cAMP immunoreactivity, quantitation and functional classification of neurons according to their morphology and polarity. This technique was complimented by physiological, electrophysiological, molecular, immunochemical, ELISA or biochemical approaches in normal or T. spiralis infected jejunum. Results: Overall, 15-20% of myenteric and 60% of submucous neurons generate cAMP. Myenteric cAMP-visualized neurons had polarized projections for descending reflexes. Visualized cells were classified as IPANs with Dogiel II shape, descending myenteric interneurons (Dogiel I/filamentous), ii descending LM motor neurons (Dogiel I/simple), short-projection interneurons (Dogiel
I/lamellar), VIP-ergic secretomotor neurons (filamentous), novel Dogiel I/lamellar or simple submucous neurons. Co-localization occurred with cAMPir in VIP but not NPY – secretomotor-neurons. Notable differences between the plexuses exist in cAMP/cGMP cross-talk, PDE IV activity, A1/A2aR cAMP coupling, polarity, proportions and numbers of each type. Synaptic blockade or neurosecretion studies indicated that cAMP contributes to synaptic communication. Acute inflammation causes AH cell hyper- excitability, elevates AC expression in calbindin-D28 and calretinin-positive neurons and amplification in ganglionic cAMP content in response to various stimulants.
Inflammation, histamine or forskolin also induce phosphorylation of CREB that is blocked by cAMP-dependent PKA inhibition. H1/H2 histamine or COX 2 inhibitors attenuate AH hyper-excitability. Conclusion. The cAMP antiserum provided new insights into cAMP function in the enteric nervous system. Cyclic AMP signaling is involved in specific neural circuits, polysynaptic pathways, neurosecretion, motility reflexes, neuronal hyperexcitability in inflamed gut and neuronal plasticity.
iii
Dedicated to my parents, Hector E. Guzman and Anita E. Guzman
iv
ACKNOWLEDGMENTS
I have had the distinct honor and privilege to work under Dr. Fievos L. Christofi, who has been an extraordinary advisor, mentor and most of all a great friend. I am grateful for his relentless support of my scientific, intellectual and professional growth. I am also grateful to his wife and children for their kindness, understanding, and especially for treating me like a member of their family.
I would also like to express my gratitude and many thanks to my advisory committee members, Dr. Helen Cooke, Dr. Richard Fertel, Dr. Scott Melvin, and Dr. Bob
Stephens.
I am also grateful to Dr. Zhixiong Chen, Dr. Jun-Ge Yu, Dr. Hamdy Awad, Asad
Javed, and Iveta Grants for all their technical advise and aid. Their kindness cannot be overstated.
There are components of this dissertation that would not have been possible without the cooperation and consideration granted me by the following laboratories and their staffs: Dr. Hamdy H. Hassanain and his staff for the molecular expertise, Dr. Helen
Cooke and her staff for their short circuit current and molecular expertise, Dr. Jeff Palmer from Johnson & Johnson for the T. spiralis expertise, Dr. Jack Grider from the University of Virginia for the 3 chamber model, and Dr. Wiemelt from the Wistar Institute for providing antibody for preliminary experiments.
I am grateful to Dr. Fred Sanfilippo for acting as my medical science mentor.
v I would also like to express my appreciation to Dr. Allan Yates for his leadership as director of the Medical Scientists Program.
I am also thankful to NIH for awarding me a four-year fellowship to conduct my studies.
I would also like to thank my parents, Hector E. Guzman, and Anita E. Guzman and my brother Jon Guzman. Their love and constant support has been unwavering and words cannot express the gratitude that I feel. I would also like to express my thanks to
Esther Flores for her support.
vi
VITA
June 03, 1971 ------Born- Bogota, Colombia
1994 ------B.S. General Biology U. of Oregon, Eugene, Oregon
1997-1999 ------Medical School, Years I & II
1999-Present ------Graduate Research Associate
The Ohio State University
PUBLICATIONS
A. Peer-reviewed Manuscripts (2001-2004)
1. F.L. Christofi, H. Zhang, J Xue, Y-Z Wang, J-G Yu, M Kim, J Guzman, HJ Cooke (2001). Differential Gene expression of Adenosine A1, A2a, A2b and A3 receptors in the human enteric nervous system. J. Comp. Neurol. 439:46-64.
2. Uma Sandaram, Hamdy Hassanain, Zacharias Suntres, Jun-Ge Yu, Helen J. Cooke, Jorge Guzman, Fievos L. Christofi (2003). Rabbit Chronic Ileitis lead to up-regulation of adenosine A1/A3 gene products, oxidative stress, and immune modulation. Biochemical Pharmacology. 65: 1529-1538.
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3. H.J. Cooke, J. Wunderlich, J. G Yu, Y-Z Wang, J. Xue, J. Guzman, Najma Javed and F. L. Christofi (2004). Mechanically-Evoked Electrogenic Chloride Secretion in Rat Distal colon is Triggered by Endogenous Nucleotides acting at P2Y Receptors. J. Comp. Neurol. 469:16-36.
4. H.J. Cooke, J. Wunderlich, J. G Yu, Y-Z Wang, J. Xue, J. Guzman, Najma Javed and F. L. Christofi (2004). Mechanical stimulation Releases Nucleotides that Trigger Neural Reflex Chloride Secretion in Guinea-Pig Distal Colon. J. Comp. Neurol. 469:1-15.
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FIELDS OF STUDY
Major Field: Physiology
ix
TABLE OF CONTENTS
Page
Abstract ------ii
Dedication ------iv
Acknowledgments ------v
Vita ------vii
List of Tables ------xvii
List of Figures ------xix
List of Abbreviations ------xxiii
Chapters:
1. INTRODUCTION ------1
1.1 ENTERIC NERVOUS SYSTEM ------1
1.1.1 Structural organization ------1
1.1.2 Basic Enteric Reflex ------3
1.1.3 Morphological Classification of Enteric Neurons ----- 5
1.1.4 Electrophysiological classification of AH and S neurons ------7
1.2 ROLE OF CYCLIC AMP SIGNALING IN THE ENS ------9
1.2.1 Slow synaptic transmission and self-reinforcing networks ------9
x 1.2.2 Role of IPANS and S neurons in gut reflexes ------14
1.3 SUBMUCOSAL NEURONS------16
1.3.1 Chemical coding of submucosal plexus neurons (SMP) ------16
1.3.2 Cyclic GMP ir in SMP neurons ------18
1.3.3 Circuit of the SMP ------18
1.3.4 Interneurons in the SMP ------19
1.3.5 Cyclic AMP signaling in the S MP ------19
1.4 ADENYLYL CYCLASE/ CYCLIC AMP SIGNALING PATHWAY ------20
1.4.1 Components of the cAMP signaling pathway ------20
1.4.2 Adenylate cyclase ------22
1.4.3 Activation by Gsα and Forskolin ------26
1.4.4 Phosphodiesterases ------26
1.4.5 Protein Kinase A, C and CaMKII regulation of AC ------29
1.4.6 Phosphatases ------33
1.4.7 Compartmentalization of cAMP ------33
1.5 ROLE OF CYCLIC AMP IN PATHOPHYSIOLOGY ------35
1.5.1 CNS disease ------35
1.5.2 G-protein couple receptor mutations ------36
1.5.3 Role of Phophodiesterase inhibitors in disease ------37
1.5.4 Role of neural reflexes in pathophysiology of the gut ------37
xi 1.5.5 Is cyclic AMP signaling altered in the ENS following infection with Trichinella Spiralis? ------39
1.6 SPECIFIC AIMS ------40
2. IMMUNOCHEMICAL VISUALIZATION OF CYCLIC AMP IN MYENTERIC CULTURE NEURONS OF GUINEA-PIG SMALL INTESTINE ------43
2.1 INTRODUCTION ------43
2.2 MATERIALS AND METHODS ------46
2.3 RESULTS ------51
2.3.1 Cyclic AMP immunofluoresence ------52
2.3.2 Cyclic GMP immunofluoresence ------52
2.3.3 Shapes of cAMP ir neurons ------55
2.3.4 Chemical coding of cAMP ir neurons ------55
2.3.5 Distribution of cAMP ir ------56
2.3.6 Slow EPSP-mimetic agents ------56
2.4 DISCUSSION ------61
2.4.1 Morphological heterogeneity in cAMP ir neurons ------62
2.4.2 Receptor activation elicits cAMP ir in a subset of myenteric neurons ------63
2.4.3 Cyclic AMP and Cyclic GMP cross-reactivity ------63
2.4.4 Phosphodiesterase isoenzyme ------64
2.4.5 Subcellular distribution of cAMP ir in isolated myenteric neurons ------65
2.4.6 Glial cells exhibited cyclic AMP ir forskolin ------66 xii
3. SYNTHESIS AND TESTING OF THE ACROLEIN DERIVATIZED CYCLIC AMP ANTISERUM ------68
3.1 INTRODUCTION ------68
3.2 MATERIALS AND METHODS ------69
3.2.1 Production of anti-cAMP antibodies according to Dr. Wiemelt and co-workers (1997) ------69
3.3 RESULTS ------73
3.3.1 KLH-cAMP acrolein conjugation ------73
3.3.2 Antibody detection after immunization ------75
3.3.3 Serum purification and affinity for cAMP ------75
3.3.4 Cyclic AMP vs. CMP specificity ------78
3.3.5 Lack of affinity of cAMP antiserum for nucleotide and their products ------81
3.4 DISCUSSION ------84
3.4.1 Coupling of KLH to cAMP and antiserum production ------84
3.4.2 Serum affinity vs. protein G purification of the cAMP antiserum ------85
3.4.3 Specificity of the cAMP antiserum for cAMP vs. cGMP ------85
3.4.4 Cross-reactivity of the cAMP antiserum to various nucleotides and their bi-products ------87
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4. USE OF THE NEW ACROLEIN-DERIVATIZED CYCLIC AMP ANTISERUM IN THE QUANTITATION, MORPHOLOGICAL DIVERSITY AND POLARITY OF CYCLIC AMP VISUALIZED NEURONS IN THE INTACT NEURAL PLEXUSES OF THE GUINEA-PIG SMALL INTESTINE ------91
4.1 INTRODUCTION ------91
4.2 METHODS AND PROCEDURES ------96
4.2.1 Fixation procedure ------98
4.2.2 Immunohistochemistry staining ------100
4.2.3 Data analysis and statistics ------101
4.3 RESULTS ------101
4.3.1 Cyclic AMP visualization in intact myenteric plexus with immunofluorescence ------102
4.3.2 Immunohistochemical visualization of cAMP ir neurons in the intact myenteric plexus ------107
4.3.3 Optimization of cAMP visualization in the enteric nervous system ------115
4.3.4 Myenteric neurons regulated by phosphodiesterase Type IV ------118
4.3.5 Lack of one to one correlation between cAMP ir and depolarization of AH and S neurons ------121
4.3.6 Cyclic AMP ir interstitial cell of Cajal ------121
4.3.7 Cyclic AMP and cyclic GMP ir ------121
4.3.8 Cyclic GMP and cAMP cross-talk ------122
xiv 4.4 DISCUSSION ------126
5. MUCOSAL REFLEX ACTIVATION OF SUBMUCOSAL NEURONS IS MEDIATED BY CYCLIC AMP SIGNALING ----- 137
5.1 INTRODUCTION ------137
5.2 MATERIALS AND METHODS ------142
5.3 RESULTS ------144
5.3.1 Immunofluorescence (IF) ------144
5.3.2 Immunohistochemistry (IHC) ------144
5.3.3 Chemical coding of cAMP ir neurons ------145
5.3.4 Adenosine effects on cAMP ir ------155
5.3.5 Cyclic AMP drives cell-to-cell communication in SMP neurons ------159
5.3.6 Short circuit current (Isc) ------159
5.4 DISCUSSION ------162
6. AMPLIFICATION OF AC/Camp SIGNALING IN MYENTERIC NEURONS DURING TRICHINELLA SPRIALIS INDUCED ACUTE INFLAMMATION ------170
6.1 INTRODUCTION ------170
6.2 METHODS AND PROCEDURES ------174
6.2.1 Immunofluorescent co-labeling studies and Laser Scanning Confocal Imaging ------175
6.2.2 Analysis of cAMP production in isolated myenteric ganglia ------176 xv 6.2.3 Western blot analysis ------176
6.2.4 Electrophysiology ------177
6.2.5 Tissue preparation for biochemical analysis ------179
6.3 RESULTS ------180
6.3.1 Up-regulation in the cAMP response to immune-neuromediators ------181
6.3.2 CREB phosphorylation in neurons expressing CaBPs in inflamed gut ------186
6.4 DISCUSSION ------195
7. OVERALL DISCUSSION AND CONCLUDING REMARKS ---- 206
LITERATURE CITED ------228
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LIST OF TABLES
Tables Page
1. Mammalian adenylyl cyclase isoforms and their tissue distribution ---- 23
2. Mammalian AC isoforms and their regulators ------25
3. Family of mammalian PDE isoforms, their specificity for cAMP over cGMP , their regulation, modulation and pharmacology ------27
4. Most common isoforms of PDE in the brain and gut ------29
5. Drug and concentration used in experiments ------48
6. Fixation reagents and concentrations ------49
7. Characteristics of antibodies used for cAMP ir and chemical coding -- 50
8. Drugs and Stimulation Parameters and Their Target Site of Action To be used determining their actions on [cAMP]ir ------98
9. Fixation reagents and concentrations ------99 10. Immunofluorescent labeling studies and Laser Scanning Confocal Imaging ------99
11. Immunohistochemistry ------100
12. Morphological classification and projections of cAMP-visualized Myenteric Neurons in intact myenteric plexus of the LMMP preparations in guinea-pig small intestine by immunofluorescence ------107
13. Morphological classification of cAMP-visualized Myenteric Neurons in intact myenteric plexus of the LMMP preparations in guinea-pig small intestine by immunohistochemistry of all clearly visible neurons/cm2 ------115
xvii 14. Comparative analysis of cAMP visualized neurons in the ENS using different processing techniques ------117
15. Comparative analysis of the pharmacological characterization of the acrolein-derivatized cAMP antiserum between myenteric and submucous plexuses ------118
16. cAMP antibody failed to stain Biocytin labeled Forskolin responsive myenteric neurons ------121
17. Drugs and Stimulation Parameters and their Target Site of Action To be used for determining their actions on [cAMP]ir ------143
18. Cyclic AMP immunofluorescence and co-labeling in submucous neurons ------144
19. Increaseas in inflammatory markers in the jejunal mucosa and plasma of guinea-pigs infected with T. Spiralis during the peak of acute inflammation ------181
20. Cyclic AMP dependent CREB phosphorylation in myenteric neurons in T. Spiralis infected and age-matched control jejunum -- 186
21. Partial reversal of enhanced excitability in AH/IPANs by receptor blockade of immune mediators ------188
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LIST OF FIGURES
Figure Page
1. Major plexuses of the small intestine ------2
2. Sensory and motor neurons in the MP and SMP ------4
3. Examples of 5 major morphological types of myenteric neurons in the guinea-pig Ileum ------6
4. Chemical coding of SMP neurons ------17
5. Model for activation and deactivation of mammalian adenylate cyclase- 21
6. A model of mammanlian adenylate cyclase (AC) with indicated sites for catalysis and binding of the regulators ------24
7. Examples of signaling pathways for G-protein families that converge to activate CREB and more downstream AP-1 activation ------32
8. Cyclic AMP immunoreactivity (ir) in myenteric culture neurons ------53
9. Quantitation of cAMP immunoreactivity (ir) in culture myenteric neurons ------54
10. Calbindin D-28 ir IPANs display cAMP ir responses in culture of myenteric plexus ------57
11. Morphological diversity of cAMP visualized myenteric neurons ------58
12. Cyclic AMP immunoreactivity (ir) in a multipolar neuron expressingVIP ir ------59
13. The slow EPSP-mimetic agent; substance P (1uM) evoked a cAMP response in a discrete subset of myenteric neurons ------60
14. Timeline for acrolein-derivatized cAMP antiserum ------70
xix 15. Antibody activity against the immunogen acrolein-cAMP-KLH complex immunized in rabbits 46 and 48 ------74
16. Sera vs. affinity purified cAMP antiserum ------76
17. Affinity purified antiserum is more sensitive than protein-G purification of the cAMP antiserum ------77
18. The cAMP antiserum has a higher affinity for cAMP than for cGMP ---- 79
19. The affinity purified cAMP antiserum has higher affinity for cAMP than cGMP ------80
20. A competitive ELISA assay to test cross-reactivity of the cAMP antiserum from rabbit 46 (A) and 48 (C) respectively to various nucleotides and their products ------82
21. The cAMP antiserum is specific only for cAMP ------83
22. Multipolar cAMP ir MP neurons in the guinea-pig small intestine ------104
23. Filamentous and Simple shape cAMP ir MP neurons in the guinea-pig small intestine ------105
24. Multipolar and uniaxonal cAMP ir neurons display polarity ------106
25. Multipolar cAMP ir MPneurons of the guinea-pig small intestine ------109
26. Filamentous cAMP ir MP neurons of the guinea-pig small intestine ----- 110
27. Simple shape cAMP ir MP neurons of the guinea-pig small intestine ---- 111
28. Lamellar shape cAMP ir neurons of the guinea-pig small intestine ------112
29. cAMP signaling activates morphologically identified myenteric neurons that are known to belong to certain functional types of neurons involved in neural reflexes ------113
30. Phosphodiesterase activity in the myenteric plexus of the guinea-pig small intestine ------119
31. Quantitation of cyclic AMP in the intact myenteric plexus (MP) using immunohistochemistry techniques ------120
xx 32. Receptor activation with slow EPSP-mimetic agents cause cAMP ir in interstitial cells of Cajal ------123
33. The cAMP antiserum discriminated between cAMP and cGMP in microdissected SMP neurons of the guinea-pig small intestine ------124
34. Forskolin and ODQ effects on SMP neurons of the guinea pig small intestine ------125
35. cAMP immunofluorescence staining in SMP neurons ------147
36. Multipolar cAMP ir SMP neurons of the guinea-pig small intestine ---- 148
37. Filamentous cAMP ir SMP neurons ------149
38. Simple shape cAMP ir SMPneurons ------150
39. Lamellar and clusters of cAMP ir SMP neurons ------151
40. Heterogeneity of cAMP visualized submucous neurons and quantitative analysis of cAMP responsive neurons ------152
41. Cylclic AMP immunoreactivity (ir) and chemical coding of SMP neurons in the guinea-pig small intestine ------153
42. Quantitative and chemical coding of SMP neurons ------154
43. Blockade of adenosine A1 receptors augments cAMP responses in submucous neurons of the guinea-pig small intestine ------156
44. Quantitative and chemical coding of cAMP ir SMP neurons ------157
45. A subset of submucous neurons that generate cAMP do so via A2a receptor activation ------158
46. Comparmentalization of cAMP ir ------158
47. CylclicAMP signaling in cell to cell communication ------160
48. Effects of forskolin on short circuit current (Isc) and synaptic blockage - 161
49. A hypothetical model to show that interneurons are likely to exist in the SMP and how various synaptic blockers can influence cAMP cell to cell communication in this plexus ------169
xxi
50. Amplification of the cAMP response in myenteric neurons during acute inflammation with T. Spiralis nematode infection of guinea-pig jejunum ------183
51. Up-regulation in the expression of AC ir in calbindin D-28-positive myenteric neurons during acute inflammation induced with T. spiralis nematode infection of guinea-pig jejunum ------184
52. Up-regulated of AC ir in cells expressing CaBPs in myenteric ganglia of T. spiralis nematode infected guinea-pig jejunum ------185
53. Nuclear CREB phosphorylation in normal and T. spiralis infected guinea-pig jejunum ------191
54. Cyclic AMP-dependent CREB phosphorylation in myenteric neurons of normal uninfected and T. spiralis infected jejunum ------192
55. Effects of anti-inflammatory agents on AH cell hyperexcitability and accommodation recorded in LMMP preparations with T. spiralis nematode infection in guinea-pig jejunum ------194
56. A working hypothesis of amplification in the AC/cAMP signaling pathway leading to hyperexcitability in AH intrinsic primary afferent neurons in acute infection with T. spiralis in guinea-pig jejunum ------197
57. Working hypothesis of the functional types of cAMP-dependent neurons according to cAMP visualization studies using an acrolein-derivatized cAMP antiserum ------207
xxii
LIST OF ABBREVIATIONS
AC Adenylyl Cyclase
CalbD28- Calcium binding protein-marker of IPANS (AH neurons)
Ca+2 Calcium Ion cGMP Cyclic guanylyl monophosphate-stimulation of cGMP-dependent protein kinase (PKG).
ChaT Choline Acetyltransferase
CNS Central nervous system
CM Circular muscle
CREB Cyclic AMP Related Element Binding Protein
Cyclic adenosine 3’,5’ monophosphate-Cyclic AMP, cAMP
CGRP Calcitonin-gene related peptide
DiI 1,1’-didodecyl-3,3,3,3’-tetramethyl-indocarbocyanine perchlorate
EC Enterochromaffin cells
ENS Enteric nervous system
EPSP Excitatory postsynaptic potential
Fepsp Fast EPSP
Fsk FORSKOLIN
F.T.S. Fiber tract stimulation
GRP gastrin relating peptide
5-HT rotonin, sensory mediator, slow EPSPP mediator
IBMX Isobutyl-1- methylXanthine
Ir munoreactivity
IPAN trinsic primary afferent neuron
LM longitudinal muscle xxiii LM longitudinal muscle
LMMP crodissected longitudinal muscle-myenteric plexus preparation
MPO Myeloperoxidase
Mg +2 Magnesium
ODQ Guanylate cyclase inhibitor
PACAP Pituitary Adenylyl Cyclase Activating Peptide
PDEI Phosphodiesterase inhibitor
PGE2 Prostaglandin E2
PGP9.5 Protein gene related product-marker for neurons
PI post infection
PKA Protein kinase A
PKC Protein kinase C
PLC Phospholipase C
Ro R0-201724 cAMP dependent phosphodiesterase inhibitor
SEPE Slow excitatory postsynaptic excitation
SOM- omatostatin
SP Substance P
TTX Tetrodotoxin
VIP Vasoactive intestinal peptide
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CHAPTER 1
INTRODUCTION
1.1 ENTERIC NERVOUS SYSTEM
1.1.1 Structural organization
The physiological role of the gastrointestinal (GI) system is to digest food, absorb nutrients (as well as electrolytes and water) and excrete waste materials as feces. In addition, a formidable immune system exists in the GI tract that acts to neutralize foreign antigens and pathogens by inducing emesis or diarrhea[1]. These important physiological functions are under the control of the enteric nervous system (ENS) that functions to coordinate motility (movements of smooth muscle), secretion (of electrolytes and water), mucosal absorption (of amino acids, peptides, monosaccharides, bile acid, vitamins, fatty acids) and vasomotor control of local arterioles[2]. The ENS consists of networks of neurons and glial cells (supporting cells), which are contained within the walls of the tubular digestive tract, pancreas and biliary system. In the tubular digestive tract the ENS consists of two ganglionic plexuses: the submucous (Meissner’s) plexus (SMP) whose main role is to control mucosal function and blood flow, and the myenteric (Aurbach’s) plexus (MP) that contains a lot more neurons than the SMP and primarily controls motility[3] (Fig 1). The focus of our attention, as described later, is on cAMP signaling in
1 Interstitial cells Circular muscle Longitudinal muscle Serosa
submucosa mucosa
Submucosa plexus
myenteric plexus
Figure 1. Major plexuses of the small intestine. The myenteric and submucosal plexuses make up the two major ganglion plexuses in the enteric nervous system. The myenteric plexus is located between the longitudinal and circular muscle layers. Interstitial cells are located on either side of the myenteric plexus in close communication with smooth muscle and function as pacemakers for the gut musculature. The submucosal plexus is located between the circular muscle layer and mucosa. In each plexus, neurons are organized into functional units called ganglia. Ganglia are connected to each other via interganglionic fiber tracts (arrows). Together, the intact neural networks of the two interconnected nerve plexuses provide the basis for all neural reflex activity in the GI tract and coordinate motility, secretion and vasomotor activity. Microdissected myenteric and submucous layers are used in electrophysiological, immunochemical and cAMP visualization studies described later.
2 the intact neural networks of the two nerve plexuses that can be microdissected as
submucosa (SMP) or myenteric plexus with attached smooth muscle (LMMP).
1.1.2 Basic Enteric Reflex
The intestine can function autonomously and as such, they display normal neural
reflex activity even after it is isolated from the CNS and from the host [4-7]. The most
basic reflex of the gut is the peristaltic reflex that serves to mix luminal contents with
secretions, aid in digestion, bring nutrients close to absortive cells and propel luminal
contents and waste material in the aboral direction [8]. All the neural components to
initiate the reflex are contained within the wall of the gut in the ENS [9]. These
components include sensory receptors, sensory cells, intrinsic primary afferent neurons
(IPANs), ascending and descending interneurons, inter-plexus neurons, vasomotor
neurons, ascending and descending secretomotor and muscle motor neurons, as well as
excitatory and inhibitory motor neurons[6, 9-15]. Various luminal nutrients such as
glucose, amino acids or fatty acids activate receptors on enterochromaffin cells (EC) to
release 5-HT and other sensory mediators (i.e. ATP, adenosine) that initiate neural
reflexes leading to a coordinated response involving motility and secretion. 5-HT-
activates 5-HT1P/or 5-HT3 or 5-HT4 receptors on IPANs located in the myenteric or
submucous plexus[16] (Fig 2) that process and integrate sensory information and initiate
neural reflexes.
It is estimated that there are as many neurons that make up the intrinsic nervous
system of the gut (ENS) as there is in the spinal cord (≈ 100-200 million neurons) - therefore it is not surprising that it has taken over a hundred years to categorize all the different types of neurons into meaningful information and functional categories [17, 18].
3 Smooth muscle
4 1
Myenteric Plexus 2
Submucous Plexus 3
5-HT-R 5-HT
EC Mucosa
Lumen
Nutrients (i.e.glucose, amino acids) or mechanical stimulation of the mucosa
Figure 2. Sensory and motor neurons in the MP and SMP. Intrinsic primary afferent neurons
(IPANs) located either in the MP (1) or SMP (2) send an afferent process to the mucosa innervating
5-7 villi (not shown). Luminal contents such as glucose, amino acids, fatty acids,vitamins, pH or bile acid can activate enterochromaffin cells (ECs) located in the mucosa to release serotonin (5-HT) which then activates 5-HT receptors (5HT1P/L R on submucous IPANs or 5HT3 Rs on myenteric
IPANs) (large curved dark arrow) on IPANS, which in turn activates the other neurons in the reflex.
Eventually, secretomotor neurons (3) and smooth muscle motor neurons are activated leading to secretion and motility, respectively. {Vasomotor reflexes are not depicted in the submucous plexus}
4 A combination of various complimentary techniques including electrophysiology [19-
24], pharmacology [25-27], and visualization with lucifer yellow [28], immunohistochemistry/neurochemistry [6, 14, 15], receptor studies [27, 29-31], retrograde/anterograde tracing of neuronal projections [32], and ultrastructural analysis
[11, 33], have so far classified eighteen functional groups of enteric neurons in guinea- pig small intestine and therefore provided considerable insight into how the ENS functions to control motility, secretion and absorption [6, 14, 15]. The ENS in the guinea pig is the most well characterized, but recent studies are confirming many of these functional classes of neurons in other species including humans [34], although significant species differences do exist [35].
1.1.3 Morphological Classification of Enteric Neurons
Enteric neurons were originally classified based on their morphological characteristics and projections to the mucosa into three main groups, namely Dogiel Type
I, Dogiel Type II and Dogiel Type III by Dr. Dogiel at the turn of the century using tracing techniques. Almost a century later, this original classification was revised to include 5 categories of neurons based on their morphologies following microinjection with neurobiotin through an intracellular recording electrode [9]. The various morphologies of enteric neurons are shown in (Fig 3). Conventional intracellular recordings are very tedious, not all neurons can be reliably recorded from and often, sampling error occurs that can lead erroneous conclusions about the population response.
To date, there are no techniques available for the simultaneous analysis and visualization of the projections and morphologies of large populations of neurons that are activated during mucosal or distension reflexes. However we have developed a new
5 Dogiel Type I I/Dendritic
Dogiel Type II
Dogiel Type I /Simple
Dogiel Type I/Filamentous
Dogiel Type I /Lamellar
Figure 3. Examples of the five major morphological types of myenteric neurons of the guinea-pig ileum. Multipolar cells consist of Dogiel Type II and Dogiel Type II/Dendritic that function as IPANs and possibly as interneurons in the ENS. Uniaxonal cells consist of three groups of neurons: Dogiel
Type I/Filamentous, Dogiel Type I/simple shape and Dogiel Type I/Lamellar neurons. Both filamentous and lamellar neurons have been shown to function as interneurons in the myenteric plexus. Small filamentous and some simple neurons which include excitatory and inhibitory motor neurons project to the longitudinal muscle while others project locally to the circular muscle.
6 technique using an acrolein-derrivatized cAMP antibody for visualization of the shapes
and projections of large numbers of cAMP- responsive neurons [36, 37] in the ENS from
intact gut preparations; the intact gut has intact neural circuits involved in gut neural
reflexes that can be studied. Cyclic AMP immunoreactivity is indicative of a rise in
intracellular free cAMP immunoreactivity that can be used to identify large populations
of neurons that are activated during a reflex. This technique is very useful because it
identifies all responsive SMP/MP neurons that have functional adenylyl cyclase
(AC)[38].
1.1.4 Electrophysiological classification of AH and S neurons.
Intracellular sharp-tip microelectrode recordings have broadly classified
myenteric neurons according to their electrical properties, S (S/type 1) and AH (AH/Type
2) neurons [39]. The S neuron is more excitable than an AH neuron and can fire
repetitively (up-to- 10 to 15 spikes) during intrasomal injection of a depolarizing current
pulse. The Na+ – dependent action potential in S neurons is followed by a very short after-hyperpolarizing potential of <1 s. Transmitter release evokes fast or slow excitatory postsynaptic potentials (fast EPSPs and slow EPSPs, respectively) in S neurons, whereas
AH neurons primarily receive slow EPSPs especially in the MP of the guinea-pig small intestine. Fast EPSPs are mediated by acetylcholine acting at nicotinic receptors or ATP acting at P2X receptors [40] and last up to 50 msec, where as slow EPSPs are characterized by a slowly rising membrane depolarization that is sustained for several seconds to minutes after termination of the fiber tract stimulation [39]. S neurons generally have 1 long axonal process, whereas AH neurons are multipolar and may have
2-6 axonal processes [41] (Fig 3).
7 The AH neuron has a characteristic long lasting after-hyperpolarizing potential
(AHP) and exhibits a broad action potential (AP) that is driven by the opening of voltage-
sensitive Na+ and Ca2+ channels, resulting in two inward currents [20]. The AP is
terminated by decrease in Na+ and Ca2+ condunctances and opening of at least three different K+ conductances (gK+) that drive the early AHP, followed by the late AHP, which is driven by an intermediate conductance K+ channel (IKCa2+) or a Ca2+-dependent gKCa2+ [20, 39, 42-45]. In fact recent studies have proven that in a subset of AH neurons
2+ the conductance of IKCa channels that underlie the slow AHP which is a mechanism by which IPANS gate excitability, are subject to inhibition by PKA-dependent phosphorylation and that PKA plays an integral role in their gating[42]. It is hypothesized that phosphorylation of these channels (IKCa2+) leads to slowly activating depolarizations
(i.e slow EPSPs) in AH neurons which result from closure of these Ca2+-activated K+ channels (causing a decrease in gKca2+) leading to suppression of the AHP– this converts the cell to an excitable mode for several seconds to minutes, [39, 44, 45]. PKA inhibition increased conductance of (IKCa2+)) channels in AH neurons and caused hyperpolarization of some of the AH neurons suggesting phosphorylation by PKA was important in regulating the conductance of these channels. AH neurons also tend to have high resting membrane potential (due to higher resting gK+) associated with lower input
resistance than in S neurons making these neurons more ideal for initiating and regulating
enteric neural reflexes. As describe later, Trichinella spiralis infection leads to
hyperexcitability in AH neurons (IPANs) that we think involves alterations in AC/cAMP
signaling via putative phosphorylation of the calcium dependent-potassium channels.
8 1.2 ROLE OF CYLCIC AMP SIGNALING IN THE ENS
1.2.1 Slow synaptic transmission and self-reinforcing networks
The AH neuron is now understood to represent intrinsic primary afferent neurons
(IPANS), and involved in processing sensory information from the lumen or smooth muscle, which will be discussed later. Dual electrode recordings provided proof that AH neurons communicate with each other via slow synaptic transmission (slow EPSPs) [23,
24, 46]. It has been suggested that large groups of AH neurons form self-reinforcing networks that propagate sensory information up-and-down the gut and around its circumference during mucosal or distension reflexes [9]. Slow synaptic transmission in
AH/IPANS is hypothesized to occure via activation of adenylyl cyclase (AC) and the consequent rise in intracellular free cAMP levels. AH neurons also communicate directly with interneurons, secretomotor, ascending (excitatory) and descending (inhibitory) motor neurons via fast and slow EPSPs by triggering the release of various neurotransmitters (i.e. Vasoactive intestinal peptide, acetylcholine, subtance P) [47].
Indirect electrophysiological suggest that slow synaptic transmission in AH neurons may, in part, be the result of activation of AC and elevation of intraneuronal cAMP. Electrophysiological experiments on myenteric neurons showed that direct exposure of the AC activator forskolin mimicked slow EPSPs in AH neurons [48]. In a few cells tested, exposure to membrane-permeant analogues of cAMP or intracellular injection of cAMP analogues also mimic the slow EPSP response in AH/type 2 neurons
[49, 50]. The phosphodiesterase inhibitors isobutylmethylxanthine (IBMX) and Ro-20-
1724 alone sometimes increase excitability of AH neurons, and can enhance the response to forskolin in AH neurons [49]. Electrophysiological data suggested that forskolin
9 activates AH but not S neurons [48]. However, this could be questioned on the basis of the small number of S neurons that were tested with forskolin – they may not be representative of the entire population that is made up of more than 10 functional classes of S neurons. It is difficult to record or target many of these neurons due to their small size or shape or small numbers in the S cell population. Therefore, electrophysiological data remains equivocal on the types of neurons that have functional AC and cAMP- dependent slow EPSPs in small intestinal myenteric plexus neurons – the former question will be studied in detail. Many GI messengers like histamine [51], Vasoactive Intestinal
Peptide (VIP) [52], Serotonin (5-HT) [53-55], Substance P (SP) [56, 57], and Calcitonin
Gene Related Peptide (CGRP) [58] were also shown to mimic slow EPSPs in AH neurons. It was hypothesized that paracrine mediators (histamine), monoamine transmitters (5-HT) and neuropeptides (CGRP, SP, VIP, CCK) increase excitability in
AH neurons via activation of AC/cAMP signaling.
Direct measurement of cAMP content was done in enzymatically- dissociated myenteric ganglia (EDMG) from guinea-pig small intestine in response to forskolin,
IBMX or neurotransmitters to stimulate cAMP production. Radioimmunoassays were used to quantitate the cAMP content in the ganglia [59-62]. The slow EPSP-mimetics SP,
CGRP, VIP, CCK, and GRP increased ganglionic cAMP in a dose-dependent manner
[63]. Forskolin greatly enhanced the activity of SP and CGRP but had no influence in furthering the cAMP response to VIP, CCK and GRP implying that slow EPSPs to these neurotransmitters are differentially regulated by activation of AC [63]. A criticism of using isolated ganglia for cAMP studies is that enteric glial cells that are abundant in the ganglia, contribute significantly to the total cAMP response produced during stimulation
10 with forskolin [64]. Also, cAMP content is not necessarily representative of intracellular free cAMP levels that are involved in the physiologicall response. In other studies, 5-HT stimulated cAMP production in EDMG in a dose dependent manner consistent with electrophysiology data that 5-HT was involved in cAMP production [60]. The 5-HT antagonist renzepride blocked all 5-HT evoked cAMP production, implying that the 5-HT action at 5HT1P receptors was through AC [60]. Another study showed that the histamine H2 agonist dimaprit also stimulated cAMP production in a dose-dependent manner in the ganglia, and the response was blocked with its antagonist, indicating that
H2 receptor activation leads to cAMP production [61].
In electrophysiological studies, as noted earlier, it was suggested that S neurons
(uniaxonal) were unresponsive to forskolin and therefore do not utilize cAMP as a second messenger [65]. However, a later study showed that the pituitary adenylyl cyclase activating peptide (PACAP) produced slow EPSP-like responses and activated 96% of all
AH cells and 36% of all S-type 1 neurons, suggesting that a significant proportion of S neurons either have cAMP responses or displayed PACAP responses through a separate cAMP-independent mechanism[26].
Adenosine is known to exert many of its effects via cAMP-dependent and independent mechanisms [66]. The discovery that adenosine (AD) inhibited forskolin- induced excitation in AH neurons suggested that possibly adenosine receptors were coupled to AC/cAMP signaling and that cAMP was somehow involved in slow EPSP activation [67]. Further studies showed AD also acted like an inhibitory neurotransmitter by hyperpolarizing AH/type neurons, increasing membrane conductance and suppressing the excitability of certain slow EPSP-mediators like histamine and VIP [68], but not
11 others – for instance, AD enhanced the slow EPSP-like responses of 5-HT, SP and CGRP implying that adenosine’s dual actions were receptor specific [69]. This suggested that two distinct AD receptors were coupled to two AC isoenzymes in the same or different
AH cell.
Later studies provided proof for 2 distinct adenosine receptors in the ganglia. The adenosine A1 agonist (CCPA) inhibits slow EPSPs in all myenteric AH and neurons [25,
70]. A subset of AH neurons in the MP were shown to be depolarized by A2 receptor agonists CGS 21680 and NECA (mixed A1/A2) at lower concentrations but at higher concentrations, they seemed to reverse the response resulting in hyperpolarization of the
AH cells [71]. A separate study showed that the adenosine A2a agonist, CGS 21680, reduced the potassium conductance of guinea pig submucosal plexus neurons by activating PKA which is involved in depolarizing AH neurons [72].
Recent studies in Dr. Christofi’s lab strongly suggest that AC/cAMP signaling is not restricted to the AH/Multipolar cell phenotype, and many functional classes of neurons are likely involved [73, 74] A recent study looked at AC/cAMP signaling by quantitating with BODIPY- Forskolin (FSK) fluorescence binding to membrane bound
AC - it was used to study the distribution of AC within the MP and SMP plexuses [74].
This study deduced that AC is expressed in distinct functional subsets of AH calbindin-
D28-positive and S neurons with calretinin ir in the ENS, but a majority of other unidentified neurons also expressed BODIPY-FSK binding sites. This meant that the majority of AC-neurons represented neurons other than IPANS/AH neurons or cholinergic neurons.
12 Another study showed that AC 1, AC 3 and AC 4 isoforms were present in calbindin-D28-positive IPANS (AH cells) and calretinin positive cholinergic neurons (S cells). Subsets of IPANS variably expressed different isoforms [73]. AC 4 was the most common isoenzyme in calbindin-D28 identified IPANS/AH neurons. The expression of these isoenzymes also varied depending on the region of the gut studied, with the duodenum expressing higher levels of AC 4 compared to other forms. This finding might have functional importance, since different regions of the small intestine serve different roles. The differential expression of AC isoenzymes in AH cells might be an important finding to understanding the functions of specific subsets of AH neurons in enteric neural reflexes. These distinct isoforms of AC may play a crucial role in gating the response of the ENS in response to various slow EPSP-mimetics. This study further supported that one or more classes of S neurons or uniaxonal Dogiel Type I neurons express AC and therefore agrees with earlier experiments with PACAP [26]. It is possible that AC has other functions in the ENS other than modulating cell excitability such as driving glycogenolysis, survival (i.e. apoptosis) as well as driving neuronal plasticity by altereing gene expression (i.e memory) [75]. This could also explain why earlier electrophysiological studies could not find S neurons reactive to forskolin. Alternatively, as pointed out before, the response in S neurons to FSK was missed due to sampling error. An important implication from this study is that different groups of EPSP mimetic agents may activate distinct AC isoenzymes in subsets of IPANS. Different second
2+ messengers (i.e. Gβγ, PKC, Gi, Gs, Ca ) modulate the activity of AC isoforms, so it is possible these regulatotry mechanisms are also involved in modulating IPAN cell excitability.
13 A very recent study in our laboratory used a cAMP-dependent protein kinase A
fluorosensor (FICRhR) to monitor in vivo the intraneuronal cAMP response after forskolin / PACAP stimulation on live myenteric cultured neurons [73, 76]. This was the first direct functional study to prove that slow EPSP mimetics (forskolin and PACAP) cause an increase in intraneuronal free cAMP levels. We were interested in further studying cAMP signaling in the intact neural circuits of the ENS. In particular, it would be useful to be able to visualize and quantitate the large numbers of enteric neurons that may communicate via cAMP signaling. All techniques to this date could not do this! To date, we had to rely on FICRhR/cAMP imaging of single neurons in culture,
ELISA/cAMP content measurements in ganglia with neurons and glia, indirect electrophysiogical studies or immunofluorescent /binding studies on AC without functional analysis. A primary goal of my thesis was therefore to develp and adopt a new technique using an acrolein-derivatized cAMP antibody for visualization and quatitation of the shapes and projections of large numbers of cAMP- responsive neurons [36, 37, 77-
82] in the ENS from intact gut preparations; the intact gut in microdissected myenteric or submucous plexus preparations from guinea-pig small intestine has intact neural networks involved in gut neural reflexes that can be studied (Fig 1). Cyclic AMP ir is indicative of a rise in intracellular free cAMP that can be used to identify large populations of neurons that may be activated during a reflex. This technique would be very powerful because it could potentially identify all responsive neurons that have functional AC [73, 74] and can generate cAMP.
1.2.2 Role of IPANS and S neurons in gut reflexes
14 IPANS in the myenteric plexus were shown to be responsive to distension and tension in the smooth muscle [83, 84]. The nomenclature of intrinsic primary afferent neurons (IPANs) is used to describe the first neuron in the reflex pathway, in an effort to avoid calling these special cells sensory neurons. It was difficult to deduce if these cells were by definition sensory cells, since data indicates that their activation is indirect and once activated it does not result in a sensation (conscious knowledge of the state of the intestine) [9].
Studies combining of DiI retrograde/anterograde labeling techniques and experiments that recorded from myenteric plexus neurons, while at the same time stimulating the mucosa (electrical, chemical or mechanical stimulus) [85, 86] or causing tension changes in the external muscles (circular or longitudinal) [87, 88], have further strengthened the notion that AH neurons act as intrinsic primary afferent neurons
(IPANs) such that they constitute an afferent limb in the gut neural reflex [9]. Therefore, it is now accepted that IPANs serve a sensory role in the gut and that they convey sensory inputs from the lumen or the smooth muscles to the ENS for processing, integration and motor output (Fig 2). IPANs are the only vertebrate primary afferent neurons identified with cell bodies outside the central nervous system and their 3-D projections do make them ideal for detecting mechanical deformation of the mucosa, chemical and radial stretch and muscle tension of the gut [89], but it is not directly known if, once activated, these cells use cAMP as a second messenger to propagate the slow EPSP to other IPANs, interneurons, descending (inhibitory), ascending (excitatory) and secretomotor neurons to turn on the mucosal reflex.
15 Local chemical stimulation of the mucosa by micropuff application, while
simultaneously recording from a myenteric neuron, showed that exposure of the mucosa
to various agents (low/high pH, short chain fatty acids, or 5-HT), elicited slow EPSPs in
AH and fast/slow EPSPs in S neurons [90, 91]. The slow EPSP in AH neurons was not
block by high Mg2+(10mM)/low Ca2+(0.2mM) which meant that these responses were not dependent on synaptic transmission. However, EPSPs in second order neurons (now believed to be S neurons) were entirely inhibited in the presence of such synaptic blockade [91, 92].
1.3 SUBMUCOSAL NEURONS
1.3.1 Chemical coding of submucosal plexus neurons (SMP)
SMP neurons from guinea pig ileum have been grouped into 6 distinct classes based on their morphology, projections, chemical coding, and function [6, 14, 15, 93]
(Fig 4). Based on their chemical coding SMP neurons can be divided into four large groups. VIP ir neurons (Non-cholinergic secretomotor/vasodilator) that project to the mucosa and arterioles make up 45% of all SMP neurons. VIP ir neurons can be further sub-divided into two groups, one that projects to the mucosa and other to the MP (inter- plexus interneurons) [94]. These inter-plexus interneurons are believed to send one-way processes directly to the MP, although their function is still unknown [15]. NPY ir
(cholinergic secretomotor/ (non-vasodilator) neurons that project to the mucosa and mucosal glands make up approximately 29% of the total SMP neurons. Calretinin ir
(cholinergic secretomotor/vasodilator) neurons make up 15% of the population in the
SMP. The last major group consists of IPANs that project to the mucosa and MP and make up 11% of the population. It is assumed, based on functional studies and chemical
16 Longitudinal Muscle Myenteric Plexus Circular Muscle 5 6 1 Submucous 4 Plexus 1 3 2
Mucosa
Figure 4. Chemical coding of SMP neurons. Six classes of neurons have been identified in the submucous plexus based on chemical coding. They are organized as cholinergic (ChAT ir) and non- cholinergic (VIP ir) neurons. VIP ir neurons make up two of the classes in the SMP, those that project strictly to the mucosa (2) and those that project to the myenteric plexus (less than 5% of the VIP ir neurons, 5). One class of ChAT ir neuron project to both the mucosa and myenteric (IPANS, 1) while the other ChAT groups project strictly to the mucosa (3) and are either NPY(3) or calretinin ir (4). One group (< 1%, 6) is still undefined since these neurons are not ir for either VIP or ChAT. Choline acetyltransferase, ChAT; vasoactive intestinal peptide, VIP. Figure adapted from Song, ZM et al 1998,
J of Comparative Neurology 399: 255-268.
17 coding, that the major neurotransmitter in these neurons is tachykinins. The last two groups of neurons in the SMP are excitatory and inhibitory neurons to the muscularis mucosae [6, 15, 94].
1.3.2 Cyclic GMP ir in SMP neurons
Previous studies in the SMP showed that cGMP ir occurred in all VIP ir secretomotor neurons, but never in NPY ir neurons suggesting that cGMP was a key player in the physiology of the enteric nervous system [95]. The majority of neurons that display cGMP ir were filamentous, simple shape, and Dogiel type I with lamellar dendrites but it was shown that none of the Dogiel II neurons (IPANs) display cGMP ir suggesting that this second messenger is not involved in the function of these neurons.
Comparable studies for cAMP visualization using a cAMP antiserum were lacking. My studies sought to identify and classify the types of submucous neurons with functional
AC.
1.3.3 Circuit of the SMP
Fast and/ or slow EPSPs have been recorded in SMP neurons after exogenous application of 5-HT agonists, mechanical and electrical stimulation of the mucosa of myenteric-free preparations [93]. However since it is impossible to record from IPANs projecting directly underneath the mucosa; most of these recordings were done in second order neurons making it difficult to map out the circuitry in the SMP [9]. In an effort to overcome this mechanical limitation and study recruitment patterns of SMP neurons investigators have employed optical recordings such as voltage sensitive dyes [96, 97] or c-fos expression [98]. These techniques identify sub-populations of neurons activated in response to specific stimuli but could not examine the pattern of enervation between
18 identified neurons. Therefore other investigators have used dual electrical recordings from SMP neurons while simultaneously stimulating with 5-HT to activate cell bodies of
SM cholinergic neurons in nearby ganglia [99]. These experiments showed that cholinergic neurons provide both diverging and converging inputs to VIP neurons providing a mechanism to augment activation of VIP-secretomotor neurons. They hypothesize that AH neurons are likely to be good candidates for providing diverging patterns due to their multipolar morphology [99].
1.3.4 Interneurons in the SMP
The reduction of cholinergic inputs after the removal of the myenteric plexus suggested the existence of interneurons in the SMP [28]. Since vagal fibers do not appear to innervate the submucosal plexus it was concluded in these studies that inputs must originate from submucosal interneurons projecting to other SM neurons [100].
Furthermore, hexamethonium blocked various neural elements that mediate reflex vasodilatation of SM arterioles further supporting interneuron involvement in the SMP
[93, 99, 101, 102].
1.3.5 Cyclic AMP signaling in the SMP
In comparison to the myenteric plexus, even less is known about the contribution of cAMP signaling to cell communication in the submucous plexus. A brief report on a few neurons suggested that both AH/Dogiel type II neurons (IPANs) and S/Dogiel type I neurons (S neurons) in the SMP were depolarized by forskolin stimulation [103]. Later it was shown in Dr. Christofi’s lab that using Bodipy-FSK that AC was present in various types of neurons in the SMP [74]. My studies used our new acrolein-derivatized cAMP antiserum to identify and quantitate submucous neurons that display cAMP ir in response
19 to AC activation [79-81]. The chemical coding of cAMP visualized neurons could be determined using dual-labeling with antibodies against various neurotransmitters known to be present in functional subsets of neurons. To assess the physiological relevance of cAMP signaling in intact neural networks of the submucous plexus, short-circuit current
Ussing chamber studies were done to assess the role of cAMP in neurosecretion (i.e. chloride secretions).
1.4 ADENYLYL CYCLASE/CYCLIC AMP SIGNALING PATHWAYS
1.4.1 Components of the cAMP-signaling pathway
The discovery on the physiological role of cyclic AMP in 1966 by Sutherland
[104] prompted the unknown field of signal transduction that has continued to grow almost exponentially. Over the years cAMP has been shown to play a key role in a variety of processes that include: cell metabolism, cell division, growth, differentiation, secretion, neoplastic transformation, assembly/disassembly of microtubules, and now more recently protein and DNA synthesis as well as regulating gene transcriptional events which ultimately influence memory [105-107]. However, its exact role in neurons in the physiological regulations of excitability, transmitter release, spatial-temporal changes in neuronal activity or in determining neuronal plasticity {i.e. involved in learning and memory remains elusive [108-112].
Extracellular ligands (hormones, neurotransmitters, growth factors, etc) bind to their specific receptors located in the cell surface of cells (G-protein couple receptors;
GPCRs) which then activate GTP-binding proteins (G-proteins) that are coupled to the these receptors [106, 113-115] (Fig 5). G-proteins are heterotrimeric in nature since they
20 Off Ligand
β γ β γ AC α AC α Effector GDP GTP Receptor GTP
Ligand On ATP cAMP Ligand
AC β γ AC β γ α α
GTP GTP GDP + Pi
Figure 5. Model for activation and deactivation of mammalian AC. The activation and inactivation of cycle of G proteins (i.e Gα) regulating the activity of adenylyl cyclase. Redrawn, revised, and adapted from Patel, T.B. et al , GENE 269;13-15, 2001. See text for details.
21 are made from three subunits: α, β, and γ. Hundreds of GPCRs have been cloned and
their unique patterns of expression among a limited number of cell types contribute
greatly to the obvious specificity of ligand action. Receptor occupancy causes a
conformational change in the G-protein from an inactive state (GDP bound to the α
subunit) to the active state (GTP bound to the α subunit)[75, 116]. The GDP-GTP
exchange causes disassociation of the complex from the receptor and causes disassembly
by the trimer into a free α subunit and a βγ complex. The free, active GTP-bound α
subunit (Gsα) now influences that activity of various effector proteins including that of adenylyl cyclase (AC) [116, 117] (Fig 5).
Functional analysis suggested that certain ligands activate while others inhibit AC through stimulatory (Gsα) or inhibitory (Giα) G proteins, respectively [118]. However recently it has been suggested that a significant number of GPCRs are coupled to multiple
G proteins therefore involving cross-talk between different second messenger systems
[119]. For example, PACAP receptors are known to be couple to stimulatory G proteins
(Gsα) leading to activation of AC [120, 121]. However the same receptor was also shown
to activate Gq, which in turn stimulates the polyphosphoinositide cycle leading to
activation of PKC [122]. This can lead to either inhibition or stimulation of AC
depending on the profile of the cell.
1.4.2 Adenylyl Cyclase
Since the discovery of the first adenylyl cyclase (AC) gene in 1989 a great deal of
interest has been generated in an effort to understand the function, molecular basis and
diversity of mammalian adenylyl cyclases [113, 123, 124]. So far a total of nine
22 membrane-bound and one soluble mammalian isotypes of adenylyl cyclase numbered
(AC 1-10) have been cloned and identified in various tissues [119, 125, 126] (Table 1).
Tissue Regions 1 2 3 4 5 6 7 8 9 10 Ref Brain Hypocampus + + 0 + 0 + + + + u [118] Cortex + 0 0 0 0 + + 0 0 u [138] Olfactory 0 + + + + + + 0 0 u [118] Basal ganglia 0 0 0 0 + + + 0 0 u [118] Forebrain 0 0 0 0 0 + + 0 + u [118] Retina + 0 + 0 0 0 0 0 0 u [139] Adrenal + 0 0 0 0 0 0 0 0 u [140] Medulla Lung 0 + + + + + + u u u [141] Aorta u u + u u u u u u u [142] Testis u u + u + + + 0 u + (s) [143] Kidney 0 0 0 + + + + 0 + u [144] Cardiac 0 0 + + + + + 0 + u [145] Muscle Liver 0 0 0 + + + + 0 + u [146] Spleen u u u u u + + u u u [147] Skeletal u u u u u u u u + u [146] Muscle Small Ileum + + + * * [73] intestine Jejunum + + + * * of the doudenum + + + * * guinea pig 1-10 Adenylyl cyclase isoforms; 0, none; +, present; u, unknown, *< 5% of the myenterpic plexus (MP) of the guinea pig small intestine. In the guinea pig small intestine AC isoforms listed above were always co-localized in calbindin-D28 ir neurons (IPANs).
Table 1. Mammalian adenylyl cyclase isoforms and their tissue distribution
Except for the soluble AC (sAC), all other isotypes are predicted to contain 2 highly variable segments of transmembrane glycoproteins comprised of six transmembrane helices and 2 highly conserved (96 % homology between all the AC isotypes) large cytoplasmic regions (C1a and C2a) which are believed to function as the catalytic and regulatory portions of the enzyme [127] (Fig 6). Once activated, AC
23 Extracellular
M1
C 1b (CaM,PKA) Plasma (PP2B, Ca2+) (6) Membrane
C1a
Fsk (1) ATP Cytosol (5) Gi,o,zα G C2a (2) sα Gβγ C2b (3) PKC (4)
Figure 6. A model of mammalian adenylyl cyclase (AC) with indicated sites for catalysis and binding of the regulators. (1) Forskolin (Fsk) stabilizes the interaction between catalytic domains C1a and C2a near the binding site for ATP which enhances catalysis of AC. (2) Gsα functions in a similar mechanism as Fsk, but binds at a different site in between C1a and C2a. (3, 4) Both Gβγ and PKC can either activate or inhibit AC depending on the AC isoform by binding to the C2a catalytic domain. (5)
Giα/oα/zα isotypes bind in between C1a and C2a catalytic domains to inhibit some AC isoforms. (6)
Calcium calmodulin (CaM) can activate some AC isoforms by binding to a regulatory site of catalytic
2+ domain C1a, however Ca alone, PKA, and PP2B (P site inhibitors) all inhibit some AC isoforms by acting at C1a site. Redrawn, revised and adapted from Tang, W.J. & Hurley, J.H. Mini Review,
Molecular Pharmacology, 54:231- 240, 1998.
24 converts ATP to cAMP (Fig 5) which in turn activates cAMP-dependent protein kinase
to regulate diverse physiological processes in various types of tissues [117, 128, 129].
Reconstituted systems using purified and recombinantly expressed protein
components support that all the membrane bound AC isotypes are activated by GTP-
bound α subunit (Gαs) (Fig 4) [130-133] . However, AC can also be directly modulated by intracellular calcium, G-protein subunits, such as Gβγ, the inhibitory Gi and Go and/ or indirectly by calcium via protein kinase A (PKA) or calmodulin [119, 134-137] (Table
2). Therefore, the potential for cross talk between cAMP and other second messengers is significant in various tissues and cells incuding neurons with multiple AC isoforms. This can also result in either an increase or a decrease in intracellular free cAMP and/ or no change in cAMP depending on the coupling of the AC isoenzyme and the G-protein. For example it is now known that feedback inhibition of AC by PKA occurs and this may prove to be important in desensitization of the cAMP signaling pathway [116, 117, 119]
Effector/Isoform 1 2 3 4 5 6 7 8 9 10 Ref Forskolin + + + + + + + + 0 0 [148] Gsα + + + + + + + + + 0 [148] Giα - 0 - U- - UUUU [148] Gzα - U UU- U UUUU [148] Goα - 0 UU- - UUUU [148] *Gβγ - + 0 + 0 +/- + U 0 U [148] Ca2+-Cam + 0 + 0 0 0 0 + 0 U [148] Ca2+ - U - U - - U 0 - U [149] P-site analogues ------U [126] PKC + + + - + U + U U U [119] PKA U U UU- - UUUU [119] 1-10, Adenylyl cyclase isoforms; 0, none; U, unknown; -, inhibition; +, excitation
Table 2. Mammalian AC isoforms and their regulators
25 1.4.3 Activation by Gsα and Forskolin
Crystal structure analysis of the catalytic domains of AC after activation by either
forskolin (diterpene forskolin derived from the root of the Indian plant Coleus forskolii)
or the Gsα subunit have shown that both molecules increase catalytic activity by
increasing the affinity and therefore stabilizing dimerization of the cytoplasmic domains
(C1a and C2a) [130, 150-153]. It is unknown if an endogenous molecule like forskolin
actually exist in a physiological sense, although it does appear to mimic ATP in a
structural and evolutionary sense [148, 154]. Functional studies have shown that
forskolin can increase catalytic activity (Vmax) by a factor of 60 which is more than 10 times its activity under physiological conditions by altering the interactions of C1a and
C2a [154]. Forskolin also facilitates cAMP production by directly altering the conformation of the active site for improved catalysis by lowering the transitional state[151, 152] . The above studies were done utilizing reconstituted systems using purified and recombinant expressed protein components (in vitro), and therefore it is
unlikely that in the intact AC the dimerization promoting role of forskolin and Gsα are as
important since in this condition the C1 and C2 are covalently bound [151, 152].
1.4.4 Phosphodiesterases
Phosphodiesterases (PDE) play a critical role in tonically degrading intracellular
cAMP and hence play a fundamental role in shaping the intracellular cAMP signal [155-
159]. Thus far 40 isoforms of PDEs have been cloned, each showing distinct kinetic and
regulatory properties as well as, in some instances, showing targeted expression within a
particular cell type [116, 117, 157, 160]. From these isoforms eleven families of PDEs
exist (Table 3). PDE isoforms are important for regulating cAMP in several ways: (1) 26 PDE Specificity Regulation Modulation Pharmacology Reference Family PDE Inhibitors PDE1-A cGMP>cAMP All Ca2+- PKA Vinpocetine [163] PDE1-B cGMP>cAMP Calmodulin - 8- PDE1-C non-specific dependent - methoxymethyl- IBMX PDE2 Non-specific CGMP Unknown EHNA [164] stimulated PDE3A Non-specific CGMP PKA & Milrinone [165] PDE3B inhibited PDEIK Enoximone Imazodan Cilostamide Quazinone Siguazodan Trequinsin Zardaverine PDE4A All cAMP- Unknown PKA Rolipram [158] PDE4B specific R0-20-1724 PDE4C Etazolate PDE4D Zardeverine PDE5A cGMP specific Unknown PKA Zaprinast [166] Dipyridamole MCBCQ MY-5445 PDE6α cGMP specific GT binds to unknown Zaprinast [166] PDE6β and PDE6γ displaces γ- subunit allows activation of αβ catalytic domain PDE7A cAMP specific Unknown unknown Rolipram [167] insensitive PDE8A cAMP specific Unknown unknown IBMX- [162] PDE8B insensitive Dipyridimole PDE9 cGMP specific Unknown unknown IBMX- [168] insensitive Zaprinast PDE10A1 Non-specific Unknown unknown cAMP [169] PDE10A2 IBMX PDE11A Non-specific Uknown unknown unknown [170] PDE11B
Table 3. Family of Mammalian PDE isoforms, their specificity for cAMP over cGMP, their regulation, modulation and pharmacology
27
they control endogenous levels of cAMP, (2) they control the degree of intracellular
concentration and temporal characteristics of cAMP elevation after activation of AC and
(3) recently they have been shown to play a big role in regulating the compartmentalization of cAMP utilizing A-Kinase anchoring proteins (AKAPs) [158-
161]. Specific families of phosphodiesterases have been identified in certain areas of the
CNS and ENS, however thus far phosphodiesterase families 4, 7 and 8 seem to play the most important role in regulating cAMP levels in the small intestine (Table 4) [162] .
The role of specific PDE’s in neural regulation of cAMP signaling is unknown. My
studies using PDE inhibitors in cAMP visualization studies shed some light on this
question.
28 Family of Phosphodiesterases Brain Regions Other areas of importance Cerebral cortex Cerebellum IV Brainstem *Small intestine Neostriatum Hippocampus Olfactory bulb *Caudate Nucleus *Small intestine Cerebral cortex colon Frontal lobe Hippocampus Med. Oblongata VII *Occipital Lobe *Putamen Substantial Nigra Temporal Lobe Thalamus Nucl Accumbens Spinal Cord Frontal Cortex *Small intestine Posterior Lobe *Colon Entorhinal Cortex Hippocampus Olfactory bulb VIII Striatum Thalamus Midbrain Pons Medulla Spinal Cord * based on tissue northern blots
Table 4. Most common isoforms of PDE in the brain and gut
1.4.5 Protein Kinase A, C and CaMKII regulation of AC
Intracellular function of cAMP in mammalian cells is regulated by cAMP-
dependent protein kinase A (PKA) [171]. Under basal conditions, low levels of
intracellular cAMP, PKA exist as an inactive holoenzyme made of two catalytic subunits
(C) and two regulatory subunits (R) bound together. Thus far four isoforms of regulatory
subunits (RIα RIβ, RIIα RIIβ) and 2 catalytic (Cα, Cβ) subunits, have been cloned in the mammalian PKA, each encoded by a unique gene [112, 172]. The combination of these
29 subunits to make a tetrameric holoenzyme, (inactive PKA), is likely to differ among tissues since it has already been demonstrated that distinct subunit expression patterns occur in the brain [118, 173]. PKA activation occurs when two cAMP molecules binds to each of the regulatory (R) subunits causing conformational change in the R subunits.
This results in the dissociation of the holoenzyme into its constituent subunits, catalytic and regulatory subunits [124, 174, 175]. Now the free and active catalytic (C) subunit can then control a variety of cellular processes such as phophorylating cytoplasmic and nuclear protein substrates, including enzymes and nuclear promoter regions that are involved in memory consolidation [176]. It is unknown how specific phosphorylation occurs via PKA but the recent discovery of compartmentalization of signal pathways may shine some light in this fundamental process [177, 178].
It is widely accepted that once PKA is activated its catalytic subunits function to regulate various forms of synaptic plasticity and thus shaping memory formation especially in long- term memory [176]. This was first shown in gill-withdrawal reflex experiments using sensory cells of Aplysia (A. californica) [76]. These investigators showed that PKA activity remained up to 12 hrs even in the absence of any further stimulation indicating that PKA activity is critical for long-term memory formation.
Recently this was further emphasized when investigators showed that cAMP/PKA cascade mediates molecular machinery for converting short-term memory into long term memory by activating CREB, MAPK [109, 110], and the immediate early gene, c-fos.
CREB phosphorylation by PKA binds to the promoter region of c-fos and activates it resulting in AP-1 transcriptional regulation [108]. The immediate-early gene c-fos is a critical player of the AP-1 transcriptional factor [179, 180] (Fig 7).
30 Many studies have revealed that cross talk between PKA and other kinases is
critical to fine-tuning and encoding incoming information [176, 181-183]. For example
many studies have showed that some of the twelve protein kinase C (PKC) isoenzymes
can regulate the function of certain AC isotypes [184] (Table 2). PKC activation is also
believed to contribute to slow EPSPs in AH and S neurons [185]. Whether PKC/AC
interactions occur is not known. The exact mechanism on how PKC regulates AC
enzymes remains unclear, however, it is hypothesized that its regulation might play a
substantial role in synaptic plasticity and learning [186, 187]. Based on all available data
thus far it does seem that PKC is essential in the transformation of short-term into long-
term memory [188-190].
Little is known about the Ca2+ signal required to modulate directly and or indirectly the activity of a particular AC isotype. For example some studies have shown oscillatory changes in intracellular Ca2+ can activate calmodulin-dependent protein kinase
II (CaMKII), which once activated, can phosphorylate specific AC isoenzymes and can therefore indirectly inactivate or activate their catalytic function [191] (Table 2). It has
been shown that these kinases are appropriately positioned to control synaptic strength by
31 Extracellular Plasma Membrane RAC DAG PKC Ca2+ GαS AC Gβ Gγ Gα1 P PAK RAS GDP GTP Gγ Gβ MEKK1 ATP cAMP GDP RAS GTP P SEK1 RAP 1 B-RAF 3 3 2 JAK/SAPK Cytosol MEK PKA 3 c-FOS P44/42-MAPK 1 3 (ERK) 3 RSK2 c-JUN
CREB P CREB P 4 c-fos promoter
CRE AP-1 c-JUN c-FOS
Nucleus c-JUN c-FOS 5 AP-1
Figure 7. Examples of signaling pathways for G-protein families, that converge to activate CREB and more downstream AP-1. Stimulation of AC by various signaling pathways (i.e Ca2+, CaMKII, PKC or by G-protein couple receptors to AC, not shown) activate PKA which can directly activate cAMP – responsive element binding protein (CREB) (1). PKA can also indirectly activate CREB by phosphorylation of RAP-1 (2) which then activates the Ras pathway (3). Phosphorylated CREB (P-
CREB) then binds to the c-fos promoter region (4) causing c-fos and c-JUN to form a complex which then binds to the AP-1 transcription site (5) to initiate gene transcription.
32 sensing local changes in intracellular calcium and therefore phosphorylate nearby
synaptic channels or ACs [176].
1.4.6 Phosphatases
So far the underlying mechanism of how protein phosphatases (i.e phosphatase 1
or calcineurin) regulate AC is largely unknown. Since many protein kinases including
PKA and PKC are substrates of some phosphatases, these enzymes might regulate the
function of ACs indirectly via inactivating one of the above kinases [118, 192]. Recent
evidence suggests that calcineurin indirectly regulates the activity of AC 9 via Ca2+ but it is not known if calcineurin can dephosphorylate AC 9 directly [116]. However, it is known that phosphatases generally act downstream from PKA to dephosphorylate proteins, thus regulating indirectly the activity of PKA [193]. Some studies have shown that inhibition of phosphatase activity (i.e calcineurin or PP1) in the hippocampus and cortex enhanced learning efficacy and prolonged memory in both young adult and aged miced [194]. The enhanced cognitive ability was associated with many downstream effectors including cAMP-responsive element binding protein (CREB) transcription factor [194, 195]. Recent studies have suggested that mutations or alteration in the function of phosphatases might be involved in Alzheimer’s and other neurological diseases (i.e Parkinson’s) [196, 197] since it is now becoming more evident that the balance between protein phosphorylation and dephosphorylation determines the survival of neurons [198, 199].
1.4.7 Compartmentalization of cAMP
Localized or gradients of second messengers within the cytosol of a cell are determined by several factors such as: localized site of generation, degradation-
33 accumulation, and the diffusion rate for a particular second messenger. For example,
cyclic AMP is produced completely at the plasma membrane, but its degradation can
occur throughout the cytoplasm. It is estimated cAMP diffuses at a rate of ≈
500µm2/second that is much higher than that of Ca2+ with a diffusing rate of ≈ 10-
50µm2/second [200-204]. Basal cAMP levels vary from 5-20 pmol/mg depending on the
type of tissue or cell line, but can increase to as much as 100-1000 pmol/mg after
activation of ACs [36]. Recently the discovery of more than thirty five functional A-
kinase anchor proteins (AKAPs) homologues that are thought to bind to an array of
proteins (i.e PKA, PKC, Epac, CaMKII and PDEs) has further added insight into the
spread, compartmentalization and further regulation of cAMP function [178, 205-209].
AKAPs have been localized to strategic cellular and intracellular locations (i.e. plasma
membrane, cytoskeleton, nucleus, postsynaptic densities, etc) to ensure discrimination of
phosphorylation of substrate/effectors in response to different signals [116, 117, 178]. It
was shown that mutations in one of the RII (RIα RIβ, RIIα RIIβ) subunits of PKA prevented its binding to AKAPs and eliminated visible compartmentalization of cAMP
[210-214]. This indicated that restricting domains in cAMP rise was likely to be important for limiting the activation to specific sub-population of PKA molecules that anchor though AKAPs.
Recently a novel cAMP receptor; Epac (exchange protein directly activated by cAMP) has been discovered that binds to cAMP with the same affinity as the regulatory units of PKA emphasizing a new dimension and complexity in the cAMP signaling pathway [215] since it is hypothesized that by binding to this protein cAMP can directly open other ion chanels. Epac might be selectively expressed in specific cells to further
34 regulate the roles of cAMP. Therefore the function of cAMP within a particular cell
might depend on the relative abundance and distribution of Epac and PKA that further
supports spatial heterogeneity, (i.e. localized increases in cAMP) [216].
1.5 ROLE OF CYCLIC AMP IN PATHOPHYSIOLOGY
1.5.1 CNS disease
VIP and PACAP 27, members of the secretin family, have shown tremendous
therapeutic potential for diseases like Alzheimers, amyotropic lateral sclerosis,
Parkinson’s, AIDS related neuropathy, diabetic neuropathy, autism, stroke, and nerve
injury (i.e spinal cord injury) [217]. PACAP and VIP can both cross the blood brain
barrier and once inside PACAP has been shown to exhibit neurotrophic or
neuroprotective action using in vitro models of glutamate-induced neuronal cell death, cell death induced by a toxic envelope protein of HIV in cultured neurons, or an in vivo model of transient global ischemia [217] by increasing adenylate cyclase activity.
Recently it was shown that PACAP released by neurons can increase the rate of glutamate re-uptake by increasing the glutamate transporter expression in astroglia and therefore reducing the incidence of neuronal cell death [218]. Another study showed that ischemia-induced apoptosis of rat hippocampus neurons could be prevented by intracerebrovascular or intravenous infusion of PACAP. AC stimulation by PACAP inhibited the activation of Jun N-terminal/stress-activated protein kinase (JNK/SAPK) which is activated within 3-6 hours of global ischemia [219] (Fig 7). Daily injections of
VIP agonist, stearyl-Nle17-VIP, to mice deficient in apolipoprotein E, a lipid carrier
molecule linked to the etiology of Alzheimer’s disease caused enhanced cholinergic
35 activity resulting in improved retardation with peptide-treated animals developing cognitive skills as fast as control animals [220]. Interestingly, treatment with PACAP failed to show any significant effects suggesting that these neuropeptides function in very specific manners within the CNS [220]. Neuropetides released by the trigeminal ganglion such as calcitonin gene-related peptide, substance P, and PACAP have revealed clear links between their concentration and primary headaches: such as chronic paroxysmal headaches, and cluster headaches suggesting that adenylate cyclase activity greatly influences the pathology of CNS diseases [221].
1.5.2 G-protein Coupled Receptor mutations
The cAMP/PKA signaling pathway has been implicated in several pathologic conditions. For example, binding of Cholera toxin, a secretory product of the bacterium vibrio cholera, to mucosal epithelial cells is partially responsible for the devastating clinical symptoms (dysentery diarrhea) in patients infected with this bacterium. This exotoxin inhibits the guanosine triphosphatase (GTPase) activity of Gα causing this subunit to remain active it its GTP-bound form (Fig 4) and therefore causing ligand- independent stimulation of AC. The ensuing accumulation of cAMP in the intestinal epithelial cells results in excessive chloride conductance and water into the lumen of the gut thereby leading to large fluid lost (dysentery diarrhea). Pertusis toxin release by
Bordetella pertusis, the causative agent of whooping cough, inhibits the exchange of
GDP for GTP in the Gi subunit resulting in an inactive Gi. This results in disregulation of the AC and overproduction of cAMP with similar clinical outcomes as in Cholera toxin.
McCune-Albright syndrome is caused by a somatic mutation in the gene
(GNAS1) that encodes the Gαs subunit producing a dysfunctional subunit that
36 overstimulates AC [222]. This results in a constitutive (ligand-independent) activation of
AC [223]. In this disease the high levels of cAMP results in overproduction of hormones
and/or cellular proliferation of many tissues therefore explaining the clinical phenotypes
seen in these patients: short stature, subcutaneous ossification, obesity, sexual precocity
and hyperfunction of multiple endocrine glands [222].
1.5.3 Role of Phosphodiesterase inhibitors in disease
Since elevated levels of cAMP is proving to be beneficial for many neurological diseases (i.e Alzheimer’s and Parkinson’s diseases or multiple sclerosis) or inflammatory diseases like chronic obstructive pulmonary disease and more recently in the management of viral infections (i.e AIDS), phosphodiesterase inhibitors have been aggressively researched in an effort to better manage these incurable diseases [224].
Originally the potent anti-inflammatory effects of Ro-20-1724 and rolipram showed great promise as potential therapeutic agents in IBD and they were able to reduce the function of many immune cells [225, 226]. However their therapeutic potential ended quickly since these drugs exhibited drastic emetic and gastric side-effects (i.e emesis, nausea, and increased acid secretion) [224, 227]. However, new generations of selective
phosphodiesterase inhibitors such as Ariflo or Roflumilast with much less side-effects
are now being tested in the clinics for exercise-induced asthma and allergic rhinitis with
excellent efficacy [224, 228]; perhaps they could also prove more useful in treating IBD.
1.5.4 Role of Neural reflexes in Pathophysiology of the gut
The intestinal tract is the largest organ that is open to the external environment.
The GI mucosa represents a huge surface area that faces the harsh external environment,
and it is therefore not surprising that it is engineered with the proper detectors to combat
37 any intruders (toxins, bacteria, viral or any other noxious agent), but at the same time, is programmed to efficiently absorb necessary nutrients. It is perfectly designed to communicate with nearby organs such as the stomach, gallbladder, and pancreas, etc via entero-enteric reflexes, while at the same time reporting back to the CNS information such as the smell of food triggering hunger or the desire to eat. Over 80% of the immune system is contained within the GI system; therefore it is not surprising that when it is exposed to any noxious agent, foreign intruder or there is mis-communication between the CNS and the ENS, the response can be extremely debilitating such as in irritable bowel syndrome, one of the largest idiopathic GI diseases in North America [229]. Some of these patients complain of abdominal pain, explosive watery diarrhea, and weight loss
[230]. Others experience less debilitating symptoms such as swollen abdomen and constant gas production. Infection with the nematode parasite Trichenella spiralis by eating pork that is not fully cooked, leads to a massive diarrhea in the host to expel the parasite [231]. Inflammatory bowel disease in general, Crohn’s disease or ulcerative colitis are associated with diarrhea, abdominal pain, wait loss, and particularly devastating is mal-absorption of nutrients [232]. The role of the ENS in these and other
GI diseases is becoming increasingly recognized. Our lack of understanding of the ENS, enteric neural reflexes, cell-to-cell communication within the ENS and especially sensory signaling mechanisms within the ENS has slowed progress towards better therapies for the various GI disorders. We must first understand how the gut senses/tastes nutrients and initiates reflexes, and how it responds to noxious stimuli or foreign invaders, before we can implement more rational therapies. We must also better understand how sensory and motor signals are processed via second messengers such as cAMP (Ca2+ or others).
38
1.5.5 Is Cyclic AMP sensory signaling altered in the ENS following infection
with Trichenella spiralis?
When exposed to any noxious agent or parasite (i.e. the nematode Trichinella
spiralis), the gut response can be extremely aggressive and debilitating. In the guinea pig,
infection with T. spiralis results in an inflammatory response that is associated with changes in the motility and secretory functions of the small intestine resulting in hyperexcitable gut and diarrhea in an effort for the animal to expel the nematode.
Intracellular recording from myenteric neurons day 3-10 post-infection shows marked alteration in the synaptic behavior of IPANs/AH neurons including: enhanced excitability, more depolarized membrane potentials, low membrane conductance’s, low thresholds for action potential discharge, decreased amplitude of AHPs, enhanced synaptic potentials, increase c-fos ir and increase in cytochrome oxidase activity, a marker for neuronal metabolic activity. These results indicate T. spiralis infection causes
changes in neuronal transcriptional events that could result in reorganization of the
enteric nervous system. The alterations in excitability characteristics of IPANs mimic
effects of forskolin on these neurons and activation of AC/cAMP signaling.
The hyperexcitability observed in AH cells could be attributed to permanent
changes in the expression of adenylate cylcase, the receptors linked to this enzyme, or
any component of the R-G proteins – AC-cAMP signaling cascades modulating cell excitability. Such changes would greatly affect the physiology of the gut. An important goal in my thesis is therefore to test the general hypothesis that up-regulation in
AC/cAMP signaling in the ENS occurs in T. spiralis inflamed intestine.
39
1.6 SPECIFIC AIMS
The physiological or pathophysiologic role of cAMP signaling in the ENS
remains unclear and somewhat controversial.
Specific Aim 1. The first specific aim was to further elucidate the role of cAMP in
the intact neural circuits of the myenteric and sumbucous plexuses in the guinea-pig
small intestine. To do this, an acrolein derivatized cAMP antibody technique was
developed, adapted and used to visualize, quantitate and identify the projections and sub-
cellular distribution of free intracellular cAMP in enteric neurons in both cultured and
intact nerve-gut preparations.
During the early part of my thesis work, it became necessary to develop a new acrolein-derivatized cAMP antiserum in order to complete studies in intact neural tissues.
This was because the original antiserum provided to us by Dr. Wiemelt at the Wistar
Institute (Pennsylvania) ran out. Our new antiserum as it turns out is more sensitive and more selective for cAMP over other nucleotides.
General Hypotheses that were addressed in Chapters 2-7 {Please note later that these hypotheses may refer to more than 1 chapter/topic}.
Hypothesis 1.1 Our newly developed acrolein-derivatized cAMP antiserum is a suitable technique for visualization, quantitation, morphological classification, identification of
40 the polarity or neurons, dual labeling and chemical coding, and spatial analysis of
intracellular free cAMP in the intact ENS.
Hypothesis 1.2. Cyclic AMP signaling occurs in several functional classes of neurons in
the ENS other than IPANs.
Hypothesis 1.3. Differences that exist in cAMP signaling and function between myenteric and submucous plexuses will be revealed by cAMP visualization. In submucous plexus, cAMP signaling lead to chloride secretion, and to motility in the myenteric plexus.
Specific aim 2. To determine if alteration occur in the R/Gs/AC/cAMP-dependent signal transduction pathway in a model of acute intestinal inflammation induced by
Trichinella spiralis nematode infection in guinea-pig jejunum.
General Hypothesis:
Hypothesis 2.1. To test the hypothesis that in Trichinella spiralis inflamed jejunum, amplification in the Rc/GP/AC/cAMP signaling cascade and transcriptional regulation contributes to neuronal plasticity that is not restricted to the AH/Dogiel Type II (IPAN) celll phenotype.
Significance: Outcomes of my experiments described in Chapters 2-6 and summarized in
Chapter 7, provide significant new information on the role of cAMP in cell to cell communication in the ENS under both physiological and pathophysiological
41 circumstances. New insights are revealed on the functional subsets of neurons that can generate cAMP. It distinguished between cAMP-responsive and non-responsive classes of neurons, their projections, polarity and morphological diversity. My studies provided for the first time a complete analysis of all neurons that generate cAMP, using the acrolein derivatized cAMP antiserum. Comparative analysis between myenteric and submucous plexuses revealed clear differences in cAMP signaling mechanisms. My studies established that cAMP signaling plays a fundamental role in motility and secretory reflexes. In acute infections associated with gut inflammation, studies determined the role of cAMP in the hyperexcitable nervous system that is known to lead to diarrhea. These studies provide direct proof that neuronal AC/cAMP signaling is up regulated in the inflamed gut. Overall, the combination of cAMP visualization with other functional/anatomical techniques provides a unique approach to further study enteric neural reflexes.
.
42
CHAPTER 2
IMMUNOCHEMICAL VISUALIZATION OF CYCLIC AMP IN MYENTERIC
CULTURE NEURONS OF GUINEA-PIG SMALL INTESTINE
2.1 INTRODUCTION
Indirect electrophysiological experiments provided evidence to support the hypothesis that slow synaptic transmission (slow EPSP) is the result of activation of adenylyl cyclase (AC) [50, 51] and elevation of intraneuronal cAMP since direct exposure of the AC activator forskolin, intracellular injection of cAMP analogues [49,
50] or exposure to phosphodiesterase inhibitors mimicked slow EPSPs in AH neurons but not in S neurons [48, 49, 51].
Many GI messengers including histamine, VIP, 5-HT, SP, PACAP, CGRP, forskolin or PDEIs were also shown to mimic slow EPSPs in AH neurons and elevate cAMP in myenteric ganglia suggesting that an increase in intraneuronal free cAMP was necessary for slow EPSPs [52, 56, 57, 59, 61, 63, 68, 233, 234]. A criticism of using isolated ganglia for cAMP studies is that enteric glial cells that are abundant in the
43 ganglia, contribute significantly to the total cAMP produced during stimulation with forskolin [64].
Earlier electrophysiological studies suggested that S type 1 neurons (uniaxonal) were unresponsive to forskolin and therefore do not utilize cAMP as a second messenger
[48]. However, this assumption was challenged when it was shown that PACAP activated both AH and S-type 1 neurons, implying that responsiveness to a certain stimulant was dependent on receptor subtypes within a particular neuron [26]. It also suggested that some S neurons either had cAMP responses or display PACAP responses through a separate mechanism.
More recent investigations using two different techniques, BODIPY Forskolin
(FSK) and AC immunocytochemistry to study the distribution of AC ir within the MP and SMP plexuses further showed AC localization in immunochemically identified subsets of AH and S neurons in the ENS and that the majority of neurons expressing
ACir/ BODIPY-FSK binding sites were not neurons with calbindin D28 (AH neurons) or calretinin ir (S neurons) as was expected. This meant that the majority of AC- neurons represented neurons other than IPANs/AH neurons or cholinergic neurons [74]. These studies further supported that S neurons did contain AC and therefore agrees with earlier experiments with PACAP [26].
Another study done in our laboratory used a cAMP-dependent protein kinase A fluorosensor (FICRhR) to study the spatial and temporal changes in intraneuronal cAMP response after forskolin / PACAP stimulation on live myenteric culture neurons [73, 76].
This was the first functional study to show an increase in cAMP after exposure to slow
EPSP mimetic agents, (Forskolin and PACAP).
44 Compartmentalization of signal transduction has emerged as a key player in
regulating specificity in cAMP-PKA signaling [209]. The recent discovery of
macromolecular complexes, which include phosphodiesterase and phosphatases anchored
to AKAPs strategically located near G-proteins coupled to AC, further suggests that
cAMP signaling is tightly regulated within the soma and processes of a cell [235].
Microinjection of a competing peptide for PKA binding to AKAPs eliminated visible
compartmentalization of cAMP in culture heart cells [202]. Another study showed that
AKAPs target specific PKA isoforms to define subcellular compartments by anchoring to
regulatory (R) subunits of PKA and that disruption of this process inhibited the localized
effects of G-proteins coupled to AC in hippocampal neurons (i.e. modulation of K+ currents) suggesting that the composition of these macromolecular structures was essential for tightly regulating the microanatomy and spatial spread of cAMP within neurons [236].
The aim of this study was to test the suitability of an acrolein derivatized cAMP antiserum for quantitation, visualization and cellular distribution of cAMP in cultured myenteric neurons as had been previously established in non-neuronal cultured cells by our collaborator Dr. Weimelt at the Wistar Institute [36].
The acrolein-derivatized antiserum detects the free intracellular free cAMP inside cells that is trapped by acrolein. This initial study was essential to establish the suitability of this technique for enteric neurons and to assess the specificity and selectivity of the antiserum. In other words, it was necessary to establish suitable conditions for further studies on cAMP signaling in the intact ENS. We also looked for compartmentalization of the cAMPir using thin optical sectioning with the laser confocal imaging system.
45 2.2 MATERIALS AND METHODS
Myenteric ganglia isolation and incubation
The entire small intestine of adult male guinea pigs (250-350grams) is isolated
and placed in cold (8-10 ºC) oxygenating (5% CO2 and 95% O2) Krebs solution containing in mM: NaCl 120; KCl; 5.0; MgCl2, 1.2; NaH2PO4, 1.35; NaHCO3, 14.4;
CaCl2, 2.54; glucose, 12.7. The entire small intestine was then cut into 3-5cm segments.
A glass rod (0.5cm diameter) is then inserted into the lumen of each intestinal segment, followed by cutting gently (avoiding transmural cuts) in a straight line along the mesentery border. LMMP-CM segments were placed in cold Krebs (8-10 ºC) to minimize ischemic damage. Equal amounts of isolated LMMP were placed in 2 separate 50 ml tubes each containing 25 ml of enzyme cocktail (1.4 mg/ml of collagenase type 1X
(Sigma), 1.0 mg/ml of proteinase (Sigma), and 0.15 mg/ml of DNASE (Sigma) dissolved in 50 ml of Krebs) and digested for 30 min in a shaking water bath at 37 ºC [237-241].
The digested LMMP-CM strips were then aliquoted in 15 ml tubes and centrifuged at
3,000 rpm for 10 min followed by discarding the enzyme solution and gentle resuspension of the pellet with filtered (0.45µm pore filters) cold Krebs [242]. The same
procedure is repeated 3 times to completely eliminate further digestion. Using a 20µl micropipeter, networks or single ganglia with visible internodal strands were harvested and placed into cold filtered Medium 199 (Sigma) supplemented with 10% fetal bovine serum, 5mg/ml of glucose, 10,000 units/ml of penicillin, and 25µg/ml of amphotericin
[242].
Culturing of the ganglia
46 Forty to sixty ganglia were placed in poly-coated glass bottom dishes (MatTek
Corporation) or 16-well plates in a volume of 20µl Krebs (Fischer Scientific) [242]. The
total volume is increased to 200µl using supplemented medium 199 that has been
warmed to 37 ºC and then the neurons are cultured in a 95% CO2 incubator (Fischer
Scientific) for 48 hours at which point the total volume is increased to 2ml of the supplemented medium 199 containing 1mg/ml of nerve growth factor (NGF) (Fisher
Scientific) [242]. The medium was changed every 2-3 days to insure sufficient nutrient supply and minimize contamination. Sometimes, the mitotic inhibitor, cytosine arabinoside (10µM) was added on the 5th day and thereafter until the neurons were used in our studies to reduce glial cell and fibroblast growth [64] and permit better visualization of the neurons for LSM imaging with the cAMP antiserum. Neurons were generally cultured for 3-21days prior to exposing cells to various treatments to elevate intracellular free cAMP levels.
Drugs
The adenylyl cyclase activator forskolin (0.1-100µM) and Ro-20-1724 (10µM)
were both dissolved in DMSO (Sigma) with final concentration of DMSO in Krebs never
higher than 0.001% of the total volume. DMSO vehicle controls never gave a response.
Tissues were incubated in the presence of appropriate drugs for 30 min to allow for the
accumulation of cAMP. This incubation time was based on preliminary/pilot experiments
on the time course of generating cAMPir detectable with our cAMP antiserum at various
dilutions.
47
Drug or Solution or cocktail (30 min) Concentration Ranges
Phosphodiesterase Inhibitors Ro-20-1724 (Ro) 0.1-1mM (Sigma) IBMX 0.1-1mM (BioMOL) Adenylyl cyclase activator Forskolin 0.1 - 100µM (Sigma) Forskolin + phosphodiesterase Inhibitor CGMP cocktail Na Nitroprusside + N-acetylcesteine + 100µM, 5mM, 1mM (respectively) IBMX (Sigma) Blockade of neural activity Ro + Forskolin + TTX 1mM, 100µM, 1µM (respectively) Ro + TTX 1mM, 1µM (respectively) (Sigma) Membrane Depolarization Ro + KCl 1mM, 60mM (respectively) (Sigma)
Neurotransmitters Ro + Vasoacitve intestinal peptide (VIP) 1mM, (1µM & 10µM VIP) (Sigma) Ro + substance P (SP) 1mM, (1µM & 10µM SP) (Sigma)
Table 5. Drug and concentration used in experiments
Fixation Procedure
Following incubation with the appropriate drug, cultured myenteric neurons were
immediately fixed in a 5.5% acrolein (v/v) made in sodium acetate-buffered solution for
30 min at 4°C. The cultures were washed in a quenching solution containing 1% glycine
for 30 min at room temperature followed by a reduction step with sodium
cyanoborohydride (1%) for 30 min, followed by three washes in 50mM Tris-HCl solution
(pH 7.5) and 0.4 M NaCl, prior to adding the cAMP antibody.
48
Reagent/Solutions Company Concentration (%)
Acrolein (fixation) Sigma 5.5% (v/v) in 0.1M Na Acetate (pH 4.75)
Glycine Sigma 1% (g/L) in 0.1M Na Acetate (pH 4.75) (quenching) Na+Cyanoborohydride Sigma 1% (g/L) in 0.1M Na Acetate (pH 4.75) (reduction)
Table 6. Fixation reagents and concentrations
Immunofluorescence staining for cAMP using the acrolein-derivatized cAMP antiserum
Nonspecific staining was reduced by blocking for one hour with 10% donkey serum (Jackson) and 2% bovine albumin (Sigma) in 0.5% Triton X-100 (v/v) made in
50 mM Tris-HCl (pH 7.5) and 0.4 M NaCl, for one hour. Blocking serum was suctioned with a transfer pipette followed by blotting gently with absorbent paper to make sure that nearly all the blocking serum was removed. Myenteric culture neurons were then incubated overnight at 4°C with a cAMP rabbit polyclonal antiserum (1/100-1/1000)
made in 50 mM Tris-HCl (pH 7.5) and 0.4 M NaCl, followed by biotinylated goat anti-
rabbit IgG and FITC-avidin each incubated for two hours at room temperature. Double
labeling was done using the neuronal marker PGP 9.5 (1/100-1/200 dil), VIP (1/100 dil),
Calbindin-D28 (1/100 dil), followed by Texas Red (TR) donkey anti-mouse, TR donkey
anti-sheep, TR horse anti-mouse, respectively. Cultures were then imaged using the Carl
Zeiss LSM 410 confocal imaging system in Dr. Christofi’s laboratory. Negative controls
were done for the secondary antibody by omitting the primary antibody and for the
specific antigen by preabsorption of the primary antiserum with its corresponding
immunogenic peptide (when available) or in the case of the cAMP antiserum with cAMP.
49
Cyclic AMP Immunofluorescence
Prim. Host Type Source Dil Second. Ab Source Dil Ab cAMP rb poly Wiemelt 1:50- Biotin-SP-donkey anti- Jackson 1:400 1000 rabbit IgG (1:100) (+ Avidin-FITC) PGP9.5 ms mono Accurate 1:100 TR-horse anti-mouse Vector 1:40- IgG 1:200 VIP sp poly Chemicon 1:100 TR-donkey anti-sheep Jackson 1:40 IgG
Calb ms mono Sigma 1:100 TR-donkey anti-mouse Vector 1:40- IgG 200
Abbreviations: sp, sheep; rb, rabbit; ms, mouse; calb, calbindin D28; TR, Texas Red; Ab, antibody; Dil, dilution.
Table 7. Characteristics of antibodies used for cAMP ir and chemical coding
Labeling was viewed by using the Zeiss LSM 410 laser scanning confocal imaging system (Carl Zeiss, Germany). An argon/krypton laser was used to excite tissues at 488/568 nm. The emitted fluorescence selected at 515-550 (FITC) and 590 (TR) band- pass filters were captured by a photomultiplier tube (PMT) and displayed as a 512x512 pixel RGB image on the monitor of an IBM-compatible custom LSM mainframe computer. The average image of 4-8 scans was collected and saved as a .tiff image for later analysis. The number of neurons displaying cAMPir, as well as pixel intensity of cAMPir was calculated for analysis.
50 Single images were always captured with the pinhole set at 35 corresponding to
an optical slice in the z direction of approximately 2µm in an effort to objectively
compare fluorescence intensities between different drug treatments or different
conditions. During some of the experiments the pinhole was reduced further to permit
thinner sections with an optical thickness of 0.7-1.0µm in an effort to test if
compartmentalization of cAMP ir occurs around the nucleus of the neuron, or in different
regions of the neuron.
Data analysis and Statistics
All data are expressed as means ± standard errors of the means (SEM). Statistical significance was evaluated by paired or unpaired Student’s t-test, ANOVA with
Bonferroni’s multiple comparison posthoc test or Fisher exact test, depending on experimental design. A p value of < 0.05 was considered significant. Additional details of specific analyses are described under appropriate sections.
2.3 RESULTS
Our results indicate that this antiserum is very specific for detecting free
intracellular cAMP, and that some of the cAMP ir neurons do not contain Dogiel Type II
morphological characteristics as was expected from previous electrophysiological data, which collaborates previous findings that neurons other than AH/Dogiel Type II are responsive to forskolin [73, 74]. The rabbit polyclonal antibody (that recognizes acrolein- derivatized cAMP) is useful in the quantitation and visualization of intracellular free cAMP levels in cultured cells [36]. We tested the feasibility and suitability of this
51 antibody in cAMP quantitation, visualization and cellular distribution in cultured
myenteric neurons (n=14 guinea-pigs). A biotinylated goat anti-rabbit IgG and FITC-
avidin were used to visualize the cAMP by laser confocal imaging. In cultured neurons
identified by PGP ir, forskolin increased cAMP ir dose (0.001-100µM) - and time-
dependently, and the forskolin response was greated in the presence of the
phosphodiesterase inhibitors (Figs 8 A-C & 9C). Cyclic AMPir was dependent on
antibody dilution (Fig 9A), type of stimulus (Figs 8 & 9) and region within the cell (Fig
9).
2.3.1 Cyclic AMP immunofluoresence
About 41% of 1,055 PGP9.5 immunoreactive (ir) neurons display cAMP ir
responses (Fig 8D). A Dogiel Type I neuron with lamellar dendrites is depicted in Fig
8B. A Dogiel Type II neuron is shown by an arrow in Fig 8C. The cAMP ir is abolished
by preabsorption of primary antibody by 0.1-1mM cAMP (Fig 9B).
2.3.2 Cyclic GMP Immunofluorescence
Activation of adenylyl cyclase and not guanylate cyclase mediates a rise in
intracellular free cAMP levels in myenteric neurons. Myenteric neurons were treated with
10-100µM Forskolin + (1mM IBMX or 1mM Ro-20-1724), Ro-20-1724 alone or a cyclic
+ GMP cocktail (100µM Na Nitroprusside, 1mM IBMX, 5mM N-acetyl-L-cysteine).
Confocal imaging and data analysis were performed to quantitate immunofluorescence intensity in the cytoplasm of neurons. In cultured myenteric neurons, forskolin (10-100
µM) + Ro-20-1724 (0.1 mM) treatment elevated cAMP immunofluorescence pixel intensity from 15.0 ± 1.2 to 228 ± 3.0 units/10 µm2 area (n=18 animals); this represents a
1500% increase in pixel intensity of cAMP ir (Figure 9C). The cAMP ir response in
52 ABC
cAMP+PGP
D 1200 1000 PGP9.5 800 PGP9.5 + cAMP 600
Neurons 400 200 Number of Myenteric 0 Neuronal Immunoreactivity
Figure 8. Cyclic AMP Immunoreactivity (ir) in myenteric culture neurons. (A-C) Neurons were treated with 0.001-100µM forskolin (FSK), 1mM IBMX or 1mM RO-20-1724 prior to fixation with acrolein.
All neurons were double stained with both anti-cAMP antiserum at 1/100-1/400-fold dilution and PGP
9.5 at 1/100-fold dilution. PGP 9.5 ir neurons are shown in red and overlay images for both cAMP (green) and PGP 9.5 (red) appear yellow (arrows). cAMP visualized neurons appear green due to FITC tagged secondary antibody. (D) Proportion of PGP ir neurons that display cAMP ir after 30 minutes of uniform incubation with FSK and the phosphodiesterase inhibitor.
53 RO+F Preabsorption A B RO+F p< 0.001 250 250 F+IBMX Basal ty ty 200 200 tesi n tesi n
150 xel I 150 xel I 100 100 Average Pi
Average Pi 50 50
0 0 1 10 100 1000 10000
cAMP Antibody Dilution
Basal Ro C IBMX+F D Nucleus RO+F Cytoplasm cGMP Stimulation 300 250 Processes p< 0.001 P> 0.05 p< 0.001 250 ty ty 200 tesi tesi n n 200 150 xel I xel I 150 p< 0.001 100 100 Average Pi Average Pi 50 50
0 0
Figure 9. Quantitation of cAMP immunoreactivity (ir) in culture myenteric neurons. (A) Quantitation of cAMP ir against various dilutions of the primary cAMP antibody. (B) Preabsorption of the cAMP antiserum (1/50) with cAMP (1mM) for 60 minutes at 37°C blocks the Ro+F response. (C) Comparing cAMP ir in myenteric neurons under basal and stimulated conditions. (D) Regional differences in cAMP response after F and phosphodiesterase stimulation for 30 minutes and 37°C. F, forskolin; RO, Ro-20-
1724. 54 neurons activated by forskolin (+ Ro-20-1724 or IBMX) was 290% greater than in
neurons stimulated with a cGMP cocktail (p< 0.01, n=4 animals; Fig. 9C). More importantly, the cGMP cocktail evoked a cAMP ir response in ≤ 5% of neurons that
could respond with an increase in cAMP ir to forskolin stimulation (data not shown).
Therefore, there is negligible or no cross-reactivity with cGMP in cultured myenteric
neurons and the antiserum detects intracellular free cAMP levels. The Bonferroni t-test
showed significant differences between all groups except between the cGMP and Ro-20-
1724 (or IBMX, not shown) treatments (p< 0.001). IBMX gave the same response as Ro-
20-1724 (p>0.05). There was no difference in the response to forskolin in the presence of
either one of these phosphodiesterase inhibitors (Fig. 9C).
2.3.3 Shapes of cAMPir neurons
The shapes of neurons were revealed by cAMPir in response to forskolin or VIP
(Fig 11). Neurons with Filamentous/Dogiel Type I, Simple/Dogiel Type I,
Lamellar/Dogiel Type I, and Multipolar Dogiel Type II morphologies were observed.
Uniaxonal and filamentous Dogiel Type I neurons made up the highest population.
2.3.4 Chemical coding of cAMP ir neurons
As shown earlier, neurons with cAMP responses were confirmed by PGP 9.5 ir.
Forskolin elevated cAMPir in calbindin ir neurons that represent the AH/Dogiel Type
II/IPAN phenotype (Fig 10); preabsorption of antibody with 0.5mM cAMP abolishes the
cAMP ir response (green fluorescence) in Fig 10. Cyclic AMP ir was also co-expressed
in a subset of VIP ir neurons suggesting that the cAMP technique provides a suitable
method for visualization, quantitation and transmitter analysis of intraneuronal free
cAMP in peptidergic neurons (Fig 12).
55 2.3.5 Distribution of cAMP ir
Cyclic AMP ir is not expressed uniformly within the myenteric neurons after myenteric neurons are stimulated with 100µM Forskolin and 1mM RO for 30 minutes to increase intracellular cAMP levels. Confocal Imaging was used to quantitate immunofluorescence intensity in various regions of cAMP positive cells. The nucleus and processes had average intensities of 122 and 128 respectively (Fig. 9D). The average intensity in the cytoplasm was 233 (Fig. 9D). Significant differences between the cytoplasm and the processes or the nucleus were observed (p<0.001).
2.3.6 Slow EPSP-mimetic agents
The slow EPSP-mimetic agents SP and VIP (1-5µM) increased cAMP by 50% of the maximum response to forskolin in <15% of neurons for each agent. Responses to both SP and
VIP were often restricted to cytoplasm (Figs 11D, E, Fig 13) and VIP also elevated cAMP in varicose fibers (Fig 11 D-E). VIP stimulation caused cAMP ir in Dogiel Type II neurons and
Dogiel Type I neurons (Fig 11 D-E), similar to forskolin stimulation (Fig. 11 A-C).
56 Figure 10. Calbindin D-28 ir IPANs display cAMP ir responses in cultures of myenteric plexus. (A)
Blocking the cAMP antiserum with cAMP prior to staining the cultured myenteric neurons. Neurons were stimulated for cAMP elevation with 100µM FSK and 1mM RO-20-1724 for 30 minutes. Cells were incubated overnight with a preabsorption cocktail (anti-cAMP antibody (1/50 dil) + 1mM cAMP) and primary antibody for calbindin D28 (1/100 dil). Immunoreactive neurons for calbindin D28 were visualized with a secondary antibody conjugated to Texas Red. (B) Cyclic AMP responses in myenteric neurons immunoreactive for calbindin D28 ir; they appear yellow (arrows) since they are double labeled with secondary antibodies tagged with FITC (for cAMP detection) and Texas Red (for calbindin D-28 detection).
57 ABC
DEF
G 45 40 Total number of neurons 35 Filamentous ve Cells
si 30 Single axon & smooth cell soma 25 Smooth cell soma 20 Multipolar er of Respon
b 15 10 Num 5 0 Morphological Types of Neurons
Figure 11. Morphological diversity of cAMP visualized myenteric neurons. (A-C) Visualization of the various shapes of forskolin (FSK)- stimulated cAMP responsive neurons. (D-F) Morphological shapes of cAMP ir neurons in response to stimulation with the slow EPSP-mimetic agent vasoactive intestinal peptide (VIP). VIP (5µM) was used to stimulate neurons in the presence of priming doses of FSK (1-
2µM FSK + 1mM RO-20-1724) for 30 minutes. (G) Quantitative analysis of the various shapes of cAMP ir neurons after stimulation with the slow EPSP-mimetic agent VIP. 58 Figure 12. Cyclic AMP ir in a multipolar neuron expressing VIP ir. Cells were stimulated for cAMP elevation with Forskolin + Ro for 30 minutes. Cells were then fixed with acrolein and incubated overnight with anti-cAMP antibody (1/50) and anti-VIP (1/100). (A) Black and white image of the VIP immunoreactive neuron (IR). (B) cAMP response in the same VIP IR neuron shown in A.
59 Figure 13. The slow EPSP-mimetic agent; substance P (1µM) evoked a cAMP response in a discrete subset of myenteric neurons.
60 2.4 DISCUSSION
Previous studies had used an acrolein-derivatized cAMP antiserum to study cAMP ir in primary neurons, astrocytes, oligodendrocytes and Schwann cells [36, 77,
78]. This is the first study to use an acrolein-derivatized cAMP antiserum to visualize, localize and quantitate free intracellular cAMP in mammalian neurons in response to phosphodiesterase inhibitors, forskolin and neuropeptides. Since a subset of the cAMP ir neurons were of Dogiel Type II morphology representing AH neurons (Figs. 10 & 11A-
B), our finding are consistent with previous indirect electrophysiological and radioimmunoassay studies respectively, which suggested that cAMP signaling may mediate slow synaptic transmission in AH neurons [48-50, 59, 61, 62]. However, the majority of the cAMP ir neurons were of Dogiel type I morphology with a single axon belonging to the S/Type I neuronal classification. This latter finding in not consistent with earlier electrophysiological findings in intact LMMP tissues which indicated that only AH/Dogiel II neurons were responsive to the AC activator, forskolin, and hence had cAMP responses (Fig 11C) [48, 242-244]. Similar findings were obtained in cultured neurons but investigators only recorded from 3 S/Dogiel type I neurons, making it impossible to deduce that all classes of S/Type I neurons do not respond to forskolin and hence have no cAMP responses [242] {i.e. > 10 classes of S neurons exist in the myenteric plexus}. In fact, AC or forskolin binding sites are found on calbindin-D28
(AH), calretinin (S/Type I, cholinergic neurons) and other unidentified types of neurons.
The apparent discrepancy between effects of forskolin on neuronal excitability and cAMPir may be due to sampling error in elecrophysiological studies that did not permit recording from forskolin-responsive classes of S/Type 1 neurons [26]. The small number
61 of Dogiel II neurons with cAMP ir compared to AH responsive neurons could be due to a
+ direct effect of forskolin at K Ca2+ channels. Micropuff application of forskolin (0.5-
+ 5mM) may achieve a high enough concentration to act directly on and close K Ca+2 channels to elicit a slow EPSP like response in AH (Dogiel Type II) neurons. The
Bodipy-forskolin and AC ir data suggest that localization of adenylyl cyclase is not exclusive to Dogiel II neurons of the myenteric plexus [73, 74]. The link between a rise in intraneuronal cAMPir and electrical behavior of myenteric neurons remains an open question.
2.4.1 Morphological heterogeneity in cAMP ir neurons.
Our data is the first functional study to provide direct evidence that forskolin stimulates both Dogiel Type II neurons (AH) and Dogiel Type I neurons (S) (Figs 11A-
C). This data directly corraborates previous work which had shown that AC ir occurred in both classes of neurons representing AH and S neurons [73, 74]. Some of the cAMP ir neurons display filamentous morphology (Figs 11C, 11F), which is consistent with a previous study which showed that a subset of filamentous neurons were responsive to receptor activation with PACAP [26], a pituitary adenylyl cyclase activating peptide.
It is suggested, based on our data, that since 41% of all PGP 9.5 ir (a marker that labels > 90% of myenteric neurons) that forskolin stimulation is not restricted to Dogiel
II neurons since these neurons only make up approximately 26% of the neuronal population [15]. It is unlikely that the culturing process caused phenotypic changes in myenteric neurons making it difficult for meaningful interpretation according to cAMP visualization of neurons, since previous electrophysiological, pharmacological and
62 immunochemical studies had shown no such changes when compared to the intact
LMMP preparations [242].
VIP stimulation provides further evidence that multiple types of neurons including Dogiel type I myenteric neurons [68, 73, 74] contain VIP receptors coupled to adenylyl cyclase activity.
2.4.2 Receptor activation elicits cAMP ir in a subset of myenteric neurons
Previous studies had shown that receptor activation with slow EPSP-mimetic agents such as vasoactive intestinal peptides (VIP), substance P (SP) and biogenic amines
5-hydroxytryptamine (5-HT), pituitary adenylyl cyclase-activating peptide (PACAP) all mimicked slow EPSP-like responses to the AC activator forskolin in AH neurons suggesting the involvement of AC/cAMP signaling [26, 68]. This is the first study to directly prove that some of these receptors are coupled to AC activity in myenteric neurons since stimulation with SP and VIP caused a rise in cAMP ir in a subset of cultured myenteric neurons. However, the numbers of neurons displaying cAMP ir were significantly lower and often display less immunoreactivity (pixel intensity; data not shown) when compared to uniform stimulation with forskolin, which is consistent with selective localization of receptors to particular subsets of neurons in the ENS. This is true for neuropeptide transmitters in the ENS including VIP, SP, and PACAP [16].
2.4.3 Cyclic AMP and Cyclic GMP cross-reactivity
The average pixel intensity after stimulating with the cGMP cocktail (maximum stimulation) was similar to stimulation with Ro-20-1724, (Fig. 9C) or IBMX alone (data not shown), suggesting that cross-reactivity of the cAMP antiserum with cGMP was non- significant, but stimulation with either of these phosphodiestarase inhibitors was
63 significant when compared to basal levels (no treatment). Our findings with cAMP or
cGMP-based stimulation protocols are consistent with previous findings in other cells,
and establish that the cAMP antiserum negligibly cross-reacts with cGMP even after
maximum stimulation of guanylate cyclase (GC) activity [36] in cultured neurons.
Various models used for measuring cGMP content have shown that endogenous levels of
intracellular cGMP levels are tightly titrated in almost all tissue and cell types even after
maximum stimulation, therefore never reaching high enough concentration to cross-react
with the highly selective cAMP antiserum [36, 245]. Our findings are consistant with a
previous study by Furness that showed that cGMP ir occurs only in approximately 2% of
all myenteric neurons [95]. In addition preabsorption of the cAMP antiserum with cAMP
blocked most of the cAMP ir in neurons treated with forskolin ± phosphodiesterase
inhibitors (Fig 9B), whereas preabsorption of the antiserum with cGMP failed to inhibit the forskolin response (figure not shown) further supporting the antibody’s specificity.
An important finding is the observation that 41% of neurons can generate cAMPir whereas only 2% can generate cGMP [37]. This implies that cAMP signaling is more
prominent than cGMP signaling in the ENS. Secondly, cAMP and cGMP do not function
in the same classes of neurons.
2.4.4 Phosphodiesterase isoenzyme
There was no statistical differences when culture neurons were stimulated with
forskolin ± Ro-20-1724 (specifically inhibits phosphodiesterase type IV) or IBMX (non-
specific phosphodiesterase) suggesting that either one of these phosphodiesterase
inhibitors (Fig 9C) had equal activity for inhibiting cAMP degradation, which is
consistent with previous electrophysiology and radioimmunoassays studies on myenteric
64 neurons [49, 63, 246]. Once we established no statistical differences between these two
phosphodiesterase inhibitors, we used Ro-17-2074 since it is more specific at inhibiting
phosphodiesterase type IV, which has been shown to be more abundant in neurons [247-
250]. In addition it has been shown in neuronal studies that IBMX is also a receptor
antagonist at purinergic receptors that are coupled to AC, and it is known that
endogenous adenosine modulates AC, cAMP, and neuronal excitability in AH neurons.
The use of IBMX would therefore provide equivocal results that are more difficult to
interpret [50, 251, 252]. Data with Ro-20-1724 implicate PDE IV in the regulation of
basal cAMP levels in myenteric neurons. In fact, PDE IV plays a significant role in
degrading cAMP, because Ro could unmask a large increase in cAMP in cultured
neurons.
2.4.5 Subcellular distribution of cAMPir in isolated myenteric neurons
Similar to previous FICRhR/cAMP fluorescence studies, uniform application of
forskolin caused a rise in cAMP ir in the cytoplasm, around the nucleus and neuronal
processes but with no evidence of spatial or compartmentalized gradients [73]. However,
the average pixel intensity was 47% higher in the cytoplasm than in either the nucleus or
the processes (axons and neurites) suggesting that the majority of cAMP was localized in
the cytoplasm (Fig 9D), a phenomenon also seen in the FICRhR studies [73]. Lack of gradients, which were visible in sensory neurons of Aplysia, is likely caused in part by the long incubation of forskolin (30 min), which would allow the diffusion of cAMP throughout the neuron. Lack of gradients could also be explained by the distribution of
AC throughout the cytoplasm and neurite processes [73, 74]. The recent discovery of strategically located intracellular A-kinase anchoring proteins (AKAPs) that are believed
65 to bind an array of proteins such as phophodiesterases have further added insight into the spread, compartmentalization and degradation of cAMP [205]. It was shown in neonatal cardiac myocytes that compartmentalization could be made to disappear in the presence of a phosphodiesterase inhibitor, which could also explaine the lack of gradients
/compartmentalization in our experiments, since we stimulated for 30 min in the presence of a phosphodiesterase inhibitor [202, 203] . We did run several time-dependent experiments with forskolin in the presence and absence of a phosphodiesterase inhibitor but failed to see gradients (data not shown) – however, this study was done in minute intervals and second interval resolution is required to account for diffusion [76, 253-255].
It is difficult to deduce why gradients were not visible, but it could also be a limitation in the sensitivity of the cAMP antiserum. The presence of perinuclear cAMP ir (Fig 9D) could have resulted from diffusion of cAMP from its original site of production which has been shown to occur in sensory cells of Aplysia [256]. In addition, diffusion of cAMP into cell bodies has important biological implications because increases in perinuclear cAMP are known to affect gene expression in many systems including neurons [76, 256].
2.4.6 Glial cells exhibited cyclic AMP ir to forskolin
A significant number of glial cells did express cAMP ir (data not shown) consistent with previous work which showed that the majority of cAMP content produced in stimulated enzymatically isolated myenteric ganglia was attributed to glial cells and not to myenteric neurons [64]. However ,based on data obtained in the intact myenteric plexus, LMMP preparations (see Chapter 4 later) forskolin stimulation failed to significantly elevate cAMP ir in glial cells suggesting that this phenomenon is only significant in isolated ganglia, but not in the intact system.
66 It is likely that alterations occur in the AC/cAMP signaling cascade in cultured
neurons – this is so because for instance, 1 mM Ro-20-1724 is required to block PDE
activity in cultured neurons and optimize the cAMP ir response or cAMP content
response/RIA [59-62] whereas only 10µM Ro-20-1724 ir necessary in intact LMMP to
protect cAMP inside the AH neurons and maximize the observed increase in the cAMP-
dependent increase in excitability.
Similarily, the difference is glial cell responsiveness between intact LMMP and
cultured myenteric plexus could be due to an up-regulaton of AC/cAMP signaling in
cultures. Further studies are needed. It may be that abnormal (culture) or disease states
lead to AC/cAMP amplification as is described in detail in Chapter 6, showing that
Trichinella spiralis infection causes a permanent functional up-regulation in the
Rc/AC/cAMP signaling in the ENS.
Summary
Overall, this study established the suitability and specificity of the acrolein- derivatized cAMP antiserum for studies on cAMP signaling in enteric neurons and glia in primary cultures. The antiserum is sensitive enough to also detect changes in intraneuronal cAMP levels that can occur in response to receptor activation. Multiple functional cell types use AC/cAMP signaling. The antiserum can reliably distinguish between cAMP and cGMP, and is useful in studies on chemical coding and dual labeling of neurons, and both basal and stimulus evoked alterations in intraneuronal cAMP levels.
67
CHAPTER 3
SYNTHESIS AND TESTING OF THE ACROLEIN DERIVATIZED
CYCLIC AMP ANTISERUM
3.1 INTRODUCTION
It was reported by Wiemelt and co-workers in 1997 [36] that an acrolein- derivatized cAMP antiserum was suitable for visualization and quantitation of intracellular free cAMP in cultured cells. Therefore we tested the selectivity and specificity of his acrolein-derivatized cAMP antiserum for free intracellular cAMP levels in primary cultures of myenteric neurons. Wiemelt’s acrolein-derivatized antiserum was extremely specific for recognizing free intracellular cAMP in cultured myenteric neurons as described in Chapter 2, and his original antibody was extremely useful for early pilot / feasibility studies on cAMP visualization in intact enteric neural circuits of the guinea pig small bowel [37, 79], described later.
However, unfortunately for us, the original cAMP antiserum developed by
Wiemelt et al [36] ran out and it was the only acrolein-derivatized cAMP antiserum available in the world. I tested commercially available antisera (not acrolein-derivatized) for ~ 6 months with no success in labeling enteric neurons for cAMPir. Therefore, we
68 decided to develop a new cAMP antiserum that recognizes acrolein-derivatized cAMP
since our goal was to use this antiserum to quantitate, visualize, classify and identify the
projections of enteric neurons in the intact neural microcircuits of the enteric nervous
system of the guinea-pig small intestine in response to forskolin and slow EPSP mimetic
and other agents.
Therefore my goal was to develop a new acrolein-derivatized cAMP antiserum
for cAMP visualization of intact neural circuits of the gut.
3.2 MATERIALS AND METHODS
3.2.1 Production of anti-cAMP antibodies according to Dr. Wiemelt and co-
workers (1997) with modifications (Fig 14).
The cAMP antisera were raised in rabbits against cAMP (Sigma) conjugated to
keyhole limpet hemocyanin (KLH) (Sigma). In general, 25 mM of cAMP was incubated
with KLH (3.0 mg) in 3.0 ml of 1% acrolein (v/v) in 0.1 M sodium acetate buffer, pH
4.75 (acetate buffer) for 4 hours (h) at room temperature (RT). Glycine (1mg/ml) was added for 30 min to quench the reaction and react with unbound acrolein, followed by a
reduction step with sodium cyanoborohydride (1gm/ml) (Sigma) for 30 min to reduce double-bonded oxygen and nitrogen and therefore finishing the cross-linking reactions.
The solution was then dialyzed overnight against four changes of distilled water at 4°C,
and then frozen for lyophilization.
A volume of 3 ml of 25 mM cAMP w/ 1.0% acrolein was coupled to 1.5, 3 and 6 mg KLH, respectively. Each was then dialyzed (6000-8000 MW cut off) against water.
To determine the cAMP-KLH ratio an ELISA assay (Cayman Chemical) was used after
69 Week 0 KLH-cAMP-acrolein complex 1 ELISA to test KLH:Acrolein:cAMP 2 complex Immunized rabbit 46 and 48 3
5 1st bleed 1st boost 6 7 2nd bleed 2nd boost 8 9 3rd bleed rd 3 boost 10
4th bleed and & non-competitive ELISA, 12 (sera 1:800) & purified antiserum for 4th boost + tested affinity purified & testing on LMMP and cultures lyophilized cAMP antiserum on LMMP 13 14 Sacrifice and final bleed Affinity purified 5ml from 15 each rabbit+ELISA using sera
Tested on LMMP & SMP affinity purified 18 antibody+ELISA to test aff. pur. against Western to test purity of affinity OA-Acrolein & OA-cAMP-Acrolein purified antiserum 19 Second western to confirm that Protein G 20 bound had removed albumin
Protein G bound ELISA 22 Purified ½ of remaining serum (aff. pur.) 23 for both rabbits cAMP vs cGMP competitive 24 ELISA studies 25 cAMP and cGMP competitive and non competitive studies
cAMP vs cGMP competitive studies on LMMP and SMP intact tissue 27
cAMP vs cGMP and related nucleotide 29 competitive ELISA studies cAMP vs ATP competitive studies using intact LMMP and SMP 30 cAMP vs related nucleotide and products 31 non-competitive ELISAs
Figure 14. Timeline for acrolein-derivatized cAMP antiserum 70 the following set up. Based on the Bradford protein assay the initial protein
concentration was 0.1 µg/µl for all three conditions (1.5mg, 3.0mg and 6.0mg of KLH)
and each condition was then titrated on the plate in 5 fold increments therefore starting
with an initial concentration of 0.00016µg/µl. A standard curve for the assay was not
used since we were only interested in the coupling ratio of each condition and not in the
concentration of uncoupled cAMP (See Results, Fig 15).
We followed the exact immunization schedule in rabbits as published before [36].
Briefly, antigens were introduced in Freund's complete, TiterMax (Cytex) since it had been shown previously to produce the best immunogenic response. After the first immunization five boosts were given to reach optimal response by the rabbits.
To produce anti-cAMP antibodies both rabbits were injected subcutaneously with
500 mg of cAMP –KLH conjugate dissolved in 0.5 ml of a 1:1 mixture of TiterMax adjuvant and phosphate-buffered saline (PBS) [36]. The first boost was given with
200 mg conjugate in 0.5 ml TiterMax/PBS at 21 days, and then subsequently every
14 days for a total of four boosts. Blood from both rabbits (46 and 48) was analyzed at every every boost until the rabbits were sacrificed.
Affinity purification steps- Five mls of sera from both rabbits were centrifuged and filtered at 0.2µm to remove all cellular material. Sera was then diluted with 0.05 M
Tris/HCl pH 7.5, and passed over a 5ml column containing bound cAMP bound (5ml) to
4% agarose beads (Pharmacia AKTA). Bound antibodies in the column through acid-
sensitive interactions were eluted with 0.1 M glycine pH 2.5. Samples were then collected in separate 50-ml polypropylene tubes containing 5ml of 1 M Tris/HCl (pH 8.0) to neutralize the pH, then washed with 10mM Tris-HCl (pH 8.8) until the eluate pH was
71 8.8. Antibodies bound to the column by base sensitive interactions were eluted by 45ml washes X 2 with 0.1 M triethanolamine (pH 11.5) over the column, and collecting the eluate in separate 50-ml polypropylene tubes containing 5ml of 0.5M Tris/HCl (pH 7.5) and 0.5M NaCl. All bound Ab fractions were pooled and concentrated using an YM10 membrane with a stirred cell ultra filtration (10,000 MS cut off). Samples were then concentrated to less than 5 mls X 2 with an exchange of ultrapure water and then stored at -20ºC for usage.
Protein G bound purification steps- Collection tubes were prepared by adding
0.1ml of 1M Tris base. 10mls of sera from rabbit 48 was centrifuge to remove particulates followed by pH adjustment to 7.0. The column (Pharmacia Biotech # 17-
0404-01) was washed with 5 bed volumes of 20mM Sodium Phosphate buffer, pH 7.0 to equilibrate the column with binding buffer. The antibody was eluted with 1-3 bed volumes of 0.1M glycine, collecting fractions into tubes containing 1M Tris Base. Pool fractions of containing the antibody was then dialyzed against three changes of PBS, at least 100 times the sample. A Bradford assay was then used to determine the antibody
(IgG) concentration, follow by storage at -20ºC.
The specificity of the cAMP antiserum was tested using standard enzyme-linked immunosorbent assay (ELISA). The wells were coated with ovalbumin, (0.002µg/ul) in
0.05m Tris/HCl, 0.3 M KCl, 20mM EDTA (pH 8.0), 100µl/well, overnight shaking, (60 rpm) in a mini-orbital shaker (MOS) at room temperature (RT). Next day, the contents of the well were discarded, followed by incubation for one hour at RT with acrolein 2% in
Na+acetate buffer (0.1M) pH 4.75, containing 20mM cAMP to bind cAMP to the ovalbumin. Contents in well were again discarded, followed by incubation for 30 min
72 with 1.0% glycine in Na+ acetate buffer (0.1M), pH 4.75 in order to remove any open
sites in acrolein that did not bind to cAMP. Contents were discarded again followed by
incubation for 30 min with 1% Na+cyanoborohydrate made in Na+acetate buffer (0.1M), pH 4.75 in order to finalized the convalent (permanent) bonds between the ovalbumin- cAMP-acrolein complex. This was followed by a wash in PBS-Tween 20 (0.3%) in order to eliminate none-specific staining and then a final wash with ddH2O. The wells were then incubated for one hour with the anti-cAMP Ab at final concentration ranges of
(1/250-1/400) in PBS-Tween 20 (0.2%) at 100 µl/well follow by a wash in PBS-Tween
20 (0.3%) for 30 minutes. The secondary antiserum (goat anti-rabbit-horseradish peroxidase) was added at a concentration of 1/5000 in PBS-Tween (0.3%) at 100µl/well
for 15 min, follow by a wash in PBS-Tween (0.3%). The samples were then incubated for
10 min in tetramethyl sodium benzoate. The reaction was quenched by the addition of
50 µl of 1 M sulfuric acid, and absorbance was read at 650 nm. For competition assays, diluted cAMP antiserum (1/250-1400) was incubated with the appropriate competitor (1-
10mM) for 30 min at 37 °C, and this solution was added to the ELISA plate and tested
[36].
3.3 RESULTS
3.3.1 KLH-cAMP acrolein conjugation
A competitive ELISA assay to indirectly assess the coupling of cAMP to KLH showed that 6.0 mg of KLH improved the ratio of cAMP bound to the hapten protein
(more cAMP bound/ total protein) when compared to 1.5 or 3.0 mg of KLH (Fig. 15).
Since the optical density (O.D.) for 6.0mg of KLH is lower for any given concentration
73 A C co th agai n ant concent (2 to assay begi acrol and 4 Figure 15. o e p assayth 0 m n i
m Raw OD at 650 A s c nst p n Raw OD at 650 A erum o r e M cAMP,2 0. 0. 0. 0. 0. 0. 1. . 0. 0. 0. 8 n etitiv ev AcAM i m 0. 0. 0. 0. 0. 0. 0. . n i t 2 3 4 5 6 7 0 1 8 9 n 0 r 1 2 3 6 p - iou 0 h 4 5 7 ( at g c e A e i
of AMP-KLH titiv i and t e cA e as ons ) A s Ant m con 9/ t h m P 1 e say in co i of t MP-KLH ratio e h 3 Enzy bo unogen ELISA assaywasrun d ese experim % m i Dil dy tion h 6 ac p R e acrolei 10/ wellsbou r etitiv
act eactant m com u a ro ,
bbit tion t e 1 c lein h i I
o v e is ELISA e ELISAassayaft m n nt
p i t 46 j l of acrolein-cAMP- y m 5 ugat
e 10/ n s wer a was 0. agai x t n -cAM unoassay . (
nd d 10
1 o B e 5 withacro assesst (acrol ) nst
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P 1µg m used added P-KLH
10/ r t 4 g i t h o /m Ki o / 2 e i r
µl Bleed Dates ou ei t con 5 l o h
o m n-cAM f t t e (C ru o
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coupl ov f th r al om
r p i nn unogen 3 aym r e ELISAwelltoco - m tib KL alb cAMP-KLH r pl i l
e- n t t P 74 ody i ex wer g t h
a h n -KLH ab u H complex ree n C at g
m 9/ h so rabbi . acrol of cAM e assay, 2 in (D) 1 h con rpti 3 ) em e com w
pre ei d o The newcAMP 1. t as b ) 3. 6. s i n 10/ n-cAM t and com 4 R 5 mg (no wit 0 mg i 0 mg p P - Com on a incubated withcom 6 a l t o 1 c e 1 an bbit o t u r s calcul x) at e, ov h o KLH n m ad
lein K d d d p
K K P p
i 48 lex +cA 10/ L to allwells.Differ 48 de
l L -KLH com variou L et m u albu - H H
ti H e w c e . wereproduci d towell 1
0 a ng A rcial The prot 5 ted usi M m ith co serum 10/ i s P-
n MP
cAM n bl bo ov i cen 2 nstead eed dat n p und 5 g t alb ei l wast A e m tratio P D B n co x usedi b h u cAMP. e ant Ne e B n r m ofKLH). cial cAMP g ant e ncent Ad in e i n e w cA s-her r bo st n s adford pr co
ed i t de o
dy n i Ov f Ac 3’5’ cAMP Ac bo KL 3’5’ cAMP rabbi m r t d towell e MP (C) was at n h , unl p di ro ro albu ano e i lex H on at es lein lein A se t ot used
s i k
min rum n- 4 ei e t 6 n h
e of cAMP, it suggests that more sites are available for binding cAMP. Therefore based on
this assay data, we inferred that more cAMP is likely to be bound to 6.0mg KLH and
therefore we chose this concentration of KLH in the acrolein-cAMP-KLH complex for
immunizing both rabbits. These results do not reveal the coupling efficiency of cAMP to
KLH since radioactivity measurements were not done which is very different from
Wiemelt’s protocol.
3.3.2 Antibody detection after immunization
A non-competitive ELISA assays showed that both rabbits were progressively
producing higher levels of antiserum against the immunogenic cAMP-acrolein-KLH
complex after every boost as expected (Fig. 15C). At this stage it appeared that both
rabbits were responding to the boosts since there was a progressive increase in the
activity of antiserum produced. Rabbit 46 showed a marked increase in O.D. only after 2
weeks of immunization (1st bleed) whereas rabbit 48 progressively produced more and more antiserum with every bleed as expected according to Wiemelt’s protocol. Rabbit 48 seemed to be a better respondent to immunization.
3.3.3 Serum purification and affinity for cAMP
Pre-absorption of pure serum (sera not processed yet) with 3.33mM cAMP showed no difference when compared to no blocking indicating that the cAMP antiserum
(IgG) contained in the sera was not specific for recognizing cAMP and that the high OD response was not specific (Fig. 16). Since pre-absorption of the affinity purified cAMP antiserum (IgG) with 3.33mM cAMP abolished its ability to recognize the ovalbumin- cAMP-acrolein complex bound to the wells suggested that purification was absolutely necessary (Fig. 17A) since this purification step provided an antiserum that specifically
75 A 2.5
2.0 B 46 sera 46 sera + cAM P Acrolein 50A 1.5 46 IgG 3’5’ cAMP 46 IgG + cAMP Ovalbumin D. at 6
O. 1.0
Raw 0.5
0.0 1e-7 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 [IgG] & Sera Dilutions (log scale) C 2.5
2.0 D 48 IgG 48 IgG + cAM P
50A Acrolein 1.5 48 sera 3’5’ cAMP 48 sera + cAM P Ovalbumin
D. at 6 1.0 O.
0.5 Raw
0.0
1e-7 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 [IgG] & Sera Dilutions (log scale)
Figure 16. Sera vs. affinity purified cAMP antiserum. A competitive ELISA assay to show that pre-absorption of serum (sera) from either rabbit 46 (A, dark and clear circles) or rabbit 48 (C, dark and clear triangles) with 3.33mM cAMP failed to block its affinity for the acrolein-cAMP-ovalbumin complex bound to the wells. However, pre- absorption of the affinity purified antibody (IgG) from rabbits 46 (A, dark and clear triangles) and 48 (C, dark and clear circles) with 3.33mM cAMP blocked its reaction with acrolein-cAMP-ovalbumin bound to the wells. Pre- absorption was done at room temperature for 30 minutes in a mini-orbital shaker with a concentration of 1/250 for either IgG (AP) or sera. (B,D). Acrolein-cAMP-ovalbumin complex (20mM cAMP, 2% acrolein and 10mg/ml of ovalbumin) was used to coat the wells. (B, D) The complex in the wells is used to mimic the antigen that was used to immunized the rabbits. The ovalbumin is used to block non-specific binding since albumin commonly found in affinity purified antiserums. 76 A 0.24 0.22 B 0.20 46 IgG 48 IgG Acrolein 50A 0.18 46 IgG + cAM P 3’5’ cAMP 0.16 48 IgG + cAM P Ovalbumin
D. at 6 0.14 O.
0.12 0.10 Raw 0.08 0.06 0.04 0.02 1e-7 1e-6 1e-5 1e-4 1e-3 [IgG] (log scale) 2.0 affinity purified IgG/preabsorp. C affinity purified IgG D protein G purified IgG/preabsorp. 1.5 protein G purified IgG Acrolein
50A 3’5’ cAMP Ovalbumin 1.0 D. at 6 O.
0.5 Raw
0.0 1e-5 1e-4 1e-3 [IgG] (log scale)
Figure 17. Affinity purified antiserum is more sensitive than protein G purification of the cAMP antiserum. (A) A competitive ELISA assay to show that pre-absorption of the affinity purified (AP) antibody (IgG) from rabbits 46 (dark circles and dark triangles) and 48 (clear circles and clear triangles) with 3.33mM cAMP blocked most of its reaction with the acrolein-cAMP-ovalbumin complex bound to the wells. Pre-absorption was done at room temperature for 30 minutes in a mini-orbital shaker with a concentration of IgG (AP) or sera at 1/250. (C) A non- competitive ELISA assay to compare the affinity purified IgG (clear circles) and protein G purified antiserum (clear triangles) from rabbit 48 to recognize cAMP in the acrolein-cAMP-ovalbumin complex. (C) However pre-absorption (competitive assay) of affinity purified (dark circles) and protein G purified (dark triangles) cAMP antiserum with 3.3mM cAMP completely block its ability to recognized the acrolein-cAMP-ovalbumin complex bound to wells (B and D). Pre-absorption was done at 1/250 dilution for the cAMP antiserum. The secondary antibody, goat anti-rabbit peroxidase (1/5000), was incubated for 15 minutes in PBS-Tween 0. 3% since the primary antibody was raised in rabbit. (B,D) The acrolein-cAMP-ovalbumin complex (20mM cAMP, 2% acrolein and 10mg/ml of ovalbumin) was used to coat the wells prior to running the competitive ELISA. (B, D) The complex in the wells is used to mimic the antigen that was used to immunized the rabbits. Ovalbumin is used to block non-specific binding.
77 recognized cAMP. No difference was observed between ± preabsorption of affinity purified antiserum (IgG) for either rabbit 46 or 48 because the non-specific response for sera was so high that the y-scale does not allow one to observe the difference. However a smaller y-scale shows that pre-absorption does significally block the ability of the affinity-purified antiserum (IgG) to recognize the ovalbumin-cAMP-acrolein complex bound to the wells (Fig 17A). Additionally rabbit 48 appeared to produce a more selective cAMP antiserum since at the same antibody concentrations it yieled a higer O.D in the absence of competition with cAMP and a larger difference in OD after blocking with cAMP compared to rabbit 46 (Fig 17A). The purity of the cAMP antiserum (IgG) using the affinity purification technique for rabbits 46 and 48 were checked by a western, which showed that this purification technique did not remove the entire albumin (data not shown). Therefore a second and more sensitive purification technique was used (protein
G bound), followed by a second western which confirmed that the protein G bound purification removed most of the albumin (data not shown). However, since the protein G bound antiserum was less sensitive at detecting the ovalbumin-cAMP-acrolein complex in the absence of pre-absorption with cAMP, it suggested that this form of purification had altered the cAMP antiserum (IgG) affinity for cAMP (Fig. 17C).
3.3.4 Cyclic AMP vs. cyclic GMP specificity
Preabsorption of the affinity purified antibody (AP IgG; 1/250 dilution) from rabbits 46 and 48 at various concentrations of either cAMP or cGMP indicated that the cAMP antiserum was more selective for cAMP (Fig. 18, 19C). This selectivity was more evident in the non-competitive ELISA assay when the same concentration of cAMP antiserum recognized primarily the ovalbumin-cAMP-acrolein and not the ovalbumin-
78 A Rabbit 46 B 0.10 Acrolein 0.10 3’5’ cAMP 0.09 Ovalbumin 0.09
50A 0.08 cAMP cGMP 0.08
D. at 6 0.07 O. 0.07
Raw 0.06 0.06 0.05 0.05 0.01 0.1 1 mM of Competing cAMP or cGMP 0.01 0.1 1 C D 0.10 0.10 Rabbit 48 Acrolein 3’5’ cAMP 0.0.0909 Ovalbumin cAM P cGM P
50A 0.0.0808
D. at 6 0.0.0707 O.
Raw 0.0.0606
0.0.0505
0.0.0101 0.0.11 11 mM of Competing cAMP or cGMP
Figure 18. The cAMP antiserum has a higher affinity for cAMP than for cGMP. A competitive ELISA assay to determine the specificity of the affinity purified cAMP antiserum (IgG) (1/250 dilution) from rabbits 46(A) and 48 (C) to distinguish between cAMP (dark circles) and cGMP (clear circles). For both animals the acrolein-cAMP-ovalbumin complex was bound to the wells and pre-absorption was done at room temperature for 30 minutes in a mini-orbital shaker with a concentration of IgG (AP) of 1/250 at various concentration the nucleotides. The secondary antibody, goat anti-rabbit peroxidase (1/5000), was incubated for 15 minute in PBS-Tween 0. 3% since the primary antiserum was raised in rabbits. (B,D)
The acrolein-cAMP-ovalbumin complex (20mM cAMP, 2% acrolein and 10mg/ml of ovalbumin) was used to coat the wells. 79 B1 B2 A 0.35 Acrolein Acrolein 3’5’cAMP cGMP 0.30 Ovalbumin Ovalbumin 50A 0.25 0.20 D. at 6
O. 0.15
0.10 Raw 0.05 0.00 P P P P M AM GM G c cAM ’c 5’c 46 8 ’5 3’ it t 4 3 8 bb bi 46 t 4 ra ab it bi C r bb ab ra r D 0.40 Acrolein 0.35 3’5’ cAMP
50A Ovalbumin 0.30 cGMP cAMP 0.25 D. at 6 O.
0.20
Raw 0.15 0.10 0.05
1e-7 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 Competing concentrations of cAMP or cGMP (log scale)
Figure 19. The affinity purified cAMP antiserum has higher affinity for cAMP than cGMP. (A) A non- competitive ELISA assay to determine the ability of affinity purified cAMP antiserum (IgG) (1/250 dilution) from rabbits 46 and 48 to distinguish between cAMP and cGMP. Either acrolein ± cAMP or cGMP-ovalbumin complex was bound to the wells. (C) A competitive ELISA assay to further titrate the specificity (sensitivity) of the affinity purified cAMP antiserum (IgG) (1/400 dilution) from rabbit 48 to distinguish between cAMP and cGMP at various diluting mM concentrations of either cAMP or cGMP. Only the acrolein-cAMP-ovalbumin complex was bound to all wells for graph C prior to running the assay and all competitive assays were pre-incubated with appropriate dilution of the cAMP antiserum (1/250) for 30 min in a mini orbital shaker and at RT. The secondary antibody, goat anti-rabbit peroxidase (1/5000), was incubated for 15 minute in PBS-Tween 0. 3%. (B1, B2, D) The acrolein- (cAMP or cGMP)-ovalbumin complex (20mM cAMP or cGMP, 2% acrolein and 10mg/ml of ovalbumin) was used to coat the wells.
80 cGMP-acrolein complex both bound to the wells (Fig. 19A). The antiserum from rabbit
48 appears to have higher affinity for cAMP (Figs. 18C & 19C) since in the competitive assay a larger OD difference existed between pre-absorption and none (Figs. 18C&19C).
In the non-competitive assay rabbit 48 resulted in the larger OD suggesting that it recognized more of the cAMP bound to the plates or are more sensitive in detecting cAMP (Fig. 19A).
3.3.5 Lack of affinity of cAMP antiserum for nucleotides and their products
The cAMP antiserum does not recognized various nucleotides and its products since pre-incubation with various nucleotides and their bi-products (1mM) for 30 minutes at 37ºC in a mini-orbital shaker prior to running the ELISA assay failed to compete for cAMP or cGMP (Fig. 20). In the first competitive ELISA assay (Fig. 20) the cAMP antibody from both rabbits recognized ATP; however when the ELISA assay was repeated by binding the nucleotides to the plates to better mimic our conditions in the laboratory in intact tissues or cultured neurons whereby the cAMP generated inside the cells is fixed to intracellular proteins in complex with acrolein. Similarly, acrolein fixation would fix other nucleotides to intracellular proteins as with cAMP. The cAMP antiserum failed to recognize ATP as was expected (Fig. 21), indicating that it is selective for cAMP alone. Later studies in basal/unstimulated tissues, lack of cAMPir responses indicates that no nucleotide including ATP that is in mM concentrations inside cells can react with the cAMP antiserum.
81 A 0.8 B
Acrolein 3’5’ cAMP
0 0.6 Ovalbumin 5 D. at 6 O. 0.4 Raw
0.2
e n P P PP P P P P P P P P P in e a in P P P P PP PP see nee MM TT DPD M M M M os inph h M DD T sinin o n cA AA A A McAM CM G iMn sty yp G MUMM U UTU o s nno :5 cA A :A3 A C:5 Gd ntoo r 5 ’G U U in o 3 2 ’c 3 ’Ac yeo to 5 In 5’ 3 5 Kd o ’ ’ ’ A y 3 2 3 K
CD0.8
0.7
0 Acrolein 5 3’5’ cAMP 0.6 Ovalbumin
D. at 6 0.5 O.
0.4 Raw 0.3
0.2
0.1 n P P P n e a P e P P P P PM P P MP s i P h e in P P P P P nPs e n e e AM T MP A P o y p n h M M PT s iT ino n c MAT AD AD c M CMM GMin Mt si pG UM MUD DU o sn o 5 A A A3 A A : 5 Cd tGo o 5ry ’G U U in U o n 3 : c 2 : ’c 3 A o’c n o 5 In 5’ 3 y5 de ot ’ ’ K3’ y 3 2 A K
Figure 20. A competitive ELISA assay to test cross-reactivity of the cAMP antiserum from rabbits 46
(A) and 48 (C) respectively to various nucleotides and their products. All plates were coated with acrolein-cAMP-ovalbumin. Cyclic AMP antiserum (1/250) was pre-incubated for 30 min in a mini orbital shaker at RT with various nucleotides and their products (1mM). (B,D) The acrolein-cAMP- ovalbumin complex (20mM cAMP, 2% acrolein and 10mg/ml of ovalbumin) was used to coat the wells.
82 A B1 B2 B3 1.2 Acrolein Acrolein Acrolein 3’5’ cAMP cGMP Remaining nucleotides Ovalbumin Ovalbumin Ovalbumin 1.0
0.8 0 5
D. at 6 0.6 O.
Raw
0.4
0.2
0.0 e P P P P P P P ee nin P P P P ee nne P PT P P P MP P iinn ah MP MP DP PT isn o MM TA DD MM MM M MM ss hp GM M D UT ins n AA A AA AA cAA CC GG nono yrpy G’ UU UU U onso 5 c c 3 ’c :5’c die oto 55 inI : 5’ : 3 3 5 A d t t 3 ’ 2 ’ 3’ A yoyo 3 2 KK
Figure 21. The cAMP antiserum is specific only for cAMP. (A) A non-competitive ELISA assay to test the specificity of the cAMP antiserum (1/400) from rabbit 48 to bound nucleotides and its products: acrolein-cAMP-ovalbumin (B1), acrolein-cGMP-ovalbumin (B2), and acrolein- -ovalbumin (B3) complexes. The secondary antibody, goat anti-rabbit peroxidase (1/5000), was incubated for 15 minute in
PBS-Tween 0.3%. 20mM (cAMP, cGMP, and remaining nucleotides, ), 2% acrolein and 10mg/ml of ovalbumin was used to coat the wells with the acrolein-(cAMP, cGMP etc)-ovalbumin complexes (B1-
B3). Note: This is analogous to what happens in gut tissues, LMMP or SMP, and therefore the cAMP antiserum only sees cAMP.
83 3.4 DISCUSSION
3.4.1 Coupling efficiency of KLH to cAMP and antiserum production
We decided to make our own acrolein-derivatized cAMP antiserum since our pilot studies showed for the first time that this antiserum was very helpful for studying the intact enteric circuits [37].
Since we were unable to use radioactivity to measure the coupling efficiency of
KLH to cAMP but we had a starting point based on Wiemelt’s protocol, we made an assumption that by doubling the concentration of KLH and keeping the concentration of cAMP (25mM) the same, would provide more binding sites for cAMP and therefore improve the likelihood of immunizing the rabbits. We tested this theory prior to immunizing the rabbits by running a competitive assay (ELISA) (Fig 15A) and showed that indeed a higher concentration of KLH (6 mg concentration) contained more bound cAMP. Therefore we chose this complex to immunize both rabbits (46 & 48) because it had more of the epitope (cAMP) present since we made another assumption that this would produce a higher immunogenic response. However, unlike Wiemelt’s protocol we did not use radioactivity to measure total protein therefore we could not calculate the concentration of cAMP bound to KLH.
In an effort to provide checkpoints at various stages of the antibody synthesis, non-competitive ELISAs were done in order to assess if the rabbits were producing antiserum against the cAMP-KLH-acrolein complex, 14 days after the first immunization and compared to subsequent bleeds prior to each boost (Fig 15C).
84 3.4.2 Serum vs affinity purified vs. protein G purification of the cAMP
antiserum.
ELISA studies showed that pre-absorption of cAMP with sera from either rabbit
(Figs. 16A&C) failed to specifically recognize cAMP, suggesting that unpurified sera was non-specific and not useful. Pre-absorption of the affinity purified (AP) cAMP antiserum with cAMP blocked most of the immunoreactivity (Fig. 17A), indicating that purification of the serum was necessary for eliminating background and optimizing specificity of the cAMP antiserum. The purified antiserum produced by rabbit 48 appeared to be more sensitive at specifically detecting cAMP compared to rabbit 46 since it had a lower O.D. at the same antibody concentration (Fig. 17A) after cAMP pre- absorption and much higher OD in the absence of cAMP pre-absorption.
The first standard western blot showed that the affinity purification technique employed by original authors of the cAMP antiserum failed to remove impurities
(albumin) and suggested in our hands that the antiserum needed further purification.
However to our surprise further purification (protein G bound) reduced the sensitivity of the cAMP antiserum (Fig. 17C). Although protein G bound methods are advantages for removing albumin, the method tends to dilute the final concentration of the antiserum which was consistent with our results since it reduced the final concentration from
0.129µg/ul for affinity purified to 0.085µg/ul for protein G bound. Protein G bound methods may also reduce affinity of antiserum for cAMP.
3.4.3 Specificity of the cAMP antiserum for cAMP vs CGMP.
Minimal cross reactivity of the cAMP antiserum with cGMP was evident (Fig.
19A); however this is more likely an inheritance of the assay due to the second antbody,
85 since the background was so low (conversation with Jeff Call). To address this
possibility we incubated the anti-cAMP antiserum at serial dilutions of cAMP and cGMP
and found that cAMP out-competed cGMP at almost all concentrations of either cyclic
nucleotide (Fig. 19C). Only at concentrations above 10mM was cGMP able to compete with cAMP (Fig. 19C). However, endogenous cGMP concentrations never reach mM levels even after maximum stimulation [245]. The cAMP antiserum from rabbit 48 appears to be approximately 10X more sensitive towards cAMP than for cGMP in
ELISA, suggesting that this antiserum is even more selective than Wiemelt’s antiserum
(Figs. 18C& 19C). A possible explanation for the difference is that we immunized both rabbits using a more efficient cAMP-KLH-acrolein coupling complex (Fig. 15A).
However since rabbit 46 produced an antiserum that is less sensitive towards cAMP, it cannot be argued as the reason. A more plausible reason is that rabbit 48 was more sensitive towards the immunization, which is the reason why two rabbits were used simultaneously. Since the rabbits were not inbred, they were not genetically identical therefore this would provide some explanation for the difference between both rabbits.
Although immunization techniques are now highly standardized an immunogenic response is highly dependent on the efficiency of inoculation. Any slight variability in the technique of inoculation (i.e. human error) would result in very different immunogenic responds among two animals. In an effort to minimize this possibility the same person immunized both rabbits under the same abjunt conditions, concentration of complex and the same number and site of inoculations for both rabbits. Other factors that contribute to variation in response include sex, age and weight. However both rabbits were the same sex, age and approximate weight so this is less likely to be a factor in the observed
86 differences. Overall, individual variability in immune respons, a better KLH-cAMP-
acrolein complex mixture and avoiding lyophilization of our purified antiserum, as done
by Dr. Wiemelt, are reasons for a more sensitive cAMP antiserum in rabbit 48.
3.4.4 Cross-reactivity of the cAMP antiserum to various nucleotides and its
bi-products.
To further analyze the specificity of the antiserum, 1mM concentrations of
various competitors were incubated with the cAMP antiserum prior to adding it to the
ELISA wells containing bound OA:Acrolein:cAMP (Fig 20). With the exception of cAMP, cGMP and ATP, all other purines and their bi-products failed to compete with cAMP bound to the wells. As shown later in other studies in intact LMMP or SMP tissue, neural staining with the cAMP antiserum detects cAMPir and no other nucleotides, and any minimal cross-reactivity with these other nucleotides is not relevant.
Dr. Wiemelt used 1/1000 of the primary cAMP antiserum. The competition of ATP for cAMP was less obvious in rabbit 48 again consistent with this antiserum being more sensitive at recognizing cAMP. UTP, several of its bi-products (UMP, UDP), inosine and
5’-GMP which were not tested by Dr. Wiemelt also failed to compete with the cAMP antiserum further proving the specificity of the antiserum. In an effort to further dissect the reason why ATP competed with cAMP we designed a noncompetitive assay to mimic our conditions/protocol used in the laboratory using rabbit 48 antiserum since it showed to be more specific towards cAMP (Fig. 21). This non-competitive ELISA assay mimicked the situation in our studies on cAMP visualization in the intact MP and SMP, where free cAMP, cGMP or related nucleotides if present bind to intracellular proteins in complex with acrolein after processing the tissue with the fixation cocktail (Chapter 2,
87 Table 6). In this noncompetitive assay the cAMP antiserum failed to recognize ATP or
cGMP or any other related nucleotide (Fig. 21) which was also observed in tissues under non-stimulatory conditions (Krebs; shown later in Chapter 4) suggesting that endogenous intracellular ATP or related nucleotides present in all cells do not cross-react with the acrolein derivatized cAMP antiserum. This can also be argued for cGMP for some neurons since it too would be present in subsets of enteric neurons however not at the high concentrations tested in this non-competitive assay (Fig. 21) and is further supported by the effects of guanylate cyclase inhibitor ODQ and cGMP cocktail shown in
Chapter 4.
When it became necessary to develop a new cAMP antiserum, we decided that custom development of the antibody by a company was more time and cost effective than developing it ourselves. However, I took an active role in every step of the process:
1) I contacted 4 different companies, including one in Germany to discuss in detail what
I wanted done.
2) After talking to Dr. Wiemelt, who developed the original cAMP antiserum, I
provided Dr. Jeff Call from USU Biotechnology Center with specific instructions and
protocol information on the design, testing, and making the immunogenic complex.
3) I designed all ELISA protocols for testing cAMP antiserum
4) I analyzed, graphed all data.
5) I advised Dr. Jeff Call on the necessity of further purification steps, after conducting
extensive tissue experiments in SMP, LMMP or whole-thickness tissues using
unpurified serum. These studies indicated that further purification steps were
essential.
88 6) I discussed, in detail, with Dr.Call all subsequent purification steps.
7) In this manner, it still took 9 months to establish the suitability of the cAMP
antiserum for visualization of the enteric neurons in intact gut preparations.
Summary
We successfully developed a new acrolein-derivatized AP cAMP antiserum for our studies in cAMP visualization in the intact neural plexuses of the small intestine using micordissected SMP or LMMP tissues. One rabbit (rabbit 48) produced several ml of AP cAMP antiserum that was plenty for all our subsequent studies and for many years to come. The antiserum appears to be even more sensitive than that obtained from the
Wistar Institute and highly specific for cAMPir over other nucleotides. Studies in intact
LMMP and SMP confirm this (see later chapters). So, we were now ready to fully address our specific aims 1 and 2 covering 4 general hypotheses.
89 Preview – List of experimental conditions done to establish affinity, selectivity, specificity, and sensitivity of cAMP antiserum in enteric neurons of intact gut preparations. Most of these will be discussed in detail in later sections:
1) FSK + RO-20-1724 >>> RO-20-1724 = Rolipram >>> Krebs.
2) OA preabsorption eliminates all background IFor IHC
3) AP antiserum IgG testing in tissues
4) Serum testing in cultures and gut tissues
5) Further purification of AP antiserum
6) ELISA testing in rabbits 46 and 48
7) cAMP preaborption vs cGMP in tissues
8) GC inhibition with ODQ to block FSK cAMP response in SMP tissues
9) cGMP cocktail in tissues
10) Cultured myenteric neurons vs intact LMMP, whole-thickness vs SMP
11) Activation of G-protein (Gi/o and Gs) coupled receptors in SMP or LMMP
12) Dilution of antiserum
13) Different secondary antibodies for IF or IHC in intact LMMP or SMP
14) Different methods to tag cAMPir/visualizing it using IHC, IF/FITC, TR, PAP,
HRP, avidin
15) Dose response curves for agonists
16) Polarity/projections of neurons (oral-distal-circumferential)
17) Chemical coding-dual labeling of neurons in intact LMMP, SMP, or whole-
thickness tissue
90
CHAPTER 4
USE OF THE NEW ACROLEIN-DERIVATIZED CYCLIC AMP ANTISERUM
IN THE QUANTITATION, MORPHOLOGICAL DIVERSITY AND POLARITY
OF CYCLIC AMP VISUALIZED NEURONS IN THE INTACT NEURAL
PLEXUSES OF THE GUINEA PIG SMALL INTESTINE
4.1 INTRODUCTION
The mammalian enteric nervous system (ENS) is distributed throughout the gastrointestinal tract and is made up of the myenteric and submucosal plexuses [1, 2].
The ENS is often referred to as the ‘little brain’ of the gut because it can function autonomously, without any input from the CNS to initiate and coordinate peristaltic and luminal / secretory reflexes [29, 30]. More than 20 candidate neurotransmitters and neuromodulators exist in the ENS including neuropeptides, monoamines, classical transmitters, nitric oxide, carbon monoxide and purines [6, 14-16, 95, 257].
Enteric neural reflexes are triggered by release of 5-HT or other sensory mediators (1) from chemo- and mechanosensitive enterochromaffin cells (EC) residing in the epithelium [258, 259]. 5-HT activates intrinsic primary afferent neurons (IPANs) in the enteric nervous system (ENS) to initiate reflexes [9]. In the guinea-pig small intestine, myenteric AH neurons function as IPANs [9, 29, 260]. Synaptic communication between
AH neurons occurs via slow excitatory postsynaptic potentials (slow EPSPs), and they
91 have been suggested to form self-reinforcing networks for feed-forward excitation in the
ENS for initiation of reflexes [84, 87, 260, 261]. In IPANs, Sustained Slow Excitatory
Postsynaptic Potentials (SSEPs) that can last several hours are believed to be involved in long-term memory [260]. Such plasticity in neural activity in AH neurons may involve the prototypical second messenger cAMP, as well as diacylglycerol and protein kinase C.
Adenosine 3' 5'-cyclic monophosphate (cAMP) is suggested to be a key mediator of slow synaptic sensory transmission in AH neurons of the ENS [48-50, 59] . A primary transmitter for the slow EPSP is a tachykinin (SP) [262-264], although there are many other candidates that mimic slow EPSPs that include gut neuropeptides (CGRP, PACAP,
VIP, GRP, SP), immune mediators (histamine, PGE2), monoamines (5-HT) and purines
(ATP, adenosine) [16]. Indirect electrophysiological studies suggest that slow EPSPs in
AH neurons are mediated by both cAMP [48-51, 59] and PKC-dependent pathways [184,
265]. Based on electrophysiological studies, it has been concluded that AC / cAMP signaling occurs exclusively in the AH cell phenotype [16, 39]. However, those original studies were done predominantly in AH neurons, and given the small number of S/Type 1 neurons included in their recordings, it is likely that not all classes of neurons represented by the S/Type 1 electrophysiological behavior were represented in their analysis [41,
266-269] . In fact, other studies with fluorescent binding of Bodipy forskolin [74] or AC immunoreactivity provide evidence for AC expression in cell types other than AH neurons identified by the calbindin immunoreactivity, suggesting that cAMP signaling occurs in other cell types as well [73]. A recent report indicates that slow EPSPs in
S/Type 1 neurons are mediated by activation of PKC [244]. Functional analysis of intraneuronal free cAMP levels in identified myenteric neurons is a first step towards
92 identification of the various classes of neurons involved in cAMP signaling i.e. prove whether subsets of both S/Type 1 and AH/Type II neurons are involved.
It was reported by Wiemelt and co-workers in 1997 [36] that an acrolein- derivatized cAMP antiserum was suitable for visualization and quantitation of intracellular free cAMP in cultured cells. After the original cAMP antiserum developed by previous investigators [36] ran out, we developed a new AP cAMP antiserum that recognizes acrolein-derivatized cAMP [36, 77, 78] and used it for the first time in the intact neural microcircuits of the myenteric plexus of the guinea-pig small intestine to quantitate, visualize, classify and identify the projections of myenteric neurons displaying a cAMP response to forskolin or other treatments. The original antiserum developed by our collaborators [36] was successfully used in pilot / feasibility studies on cAMP visualization in cultured neurons and intact enteric neural circuits of the guinea pig small bowel [37, 79, 80]. As shown in Chapter 2, that cAMP antiserum was suitable in the quantitation, visualization, and identification of morphological diversity, chemical coding, and receptor activation of AC and elevation of cAMPir in cultured myenteric neurons.
To date, little is known about the classes of neurons and their projections that utilize cAMP as a second messenger [231, 270] . At least 18 classes of myenteric neurons can be distinguished by their morphology, chemical coding, projections and / or neurophysiology [6, 14, 15]. Morphological classification alone can discriminate several functional cell phenotypes including IPANs (Dogiel Type II), descending interneurons
(Uniaxonal filamentous), longitudinal muscle motor neurons (uniaxonal simple neurons), other motor neurons, and short-inter-plexus interneurons [15]. No direct proof exists for
93 cAMP signaling in particular classes of neurons in the enteric nervous system (ENS) or in the CNS. Electrophysiological data provide indirect and incomplete characterization of neurons with a functional AC/cAMP signaling pathway [48, 59, 244, 271]. Biochemical analysis of cAMP content in isolated ganglia may represent alterations in intracellular cAMP levels in both neurons and glia [64], and cannot discriminate between different classes of responsive neurons or between free and bound, sequestered, or compartmentalized cAMP. Bodipy forskolin binding studies or AC immunoreactivity studies do not provide functional proof for cAMP signaling in enteric neurons [73, 74], although they do provide proof for multiple types of neurons with AC. Our approach to visualize cAMP immunoreactivity is a functional endpoint that targets a main second messenger for slow synaptic transmission that is of basic importance in sensory neurotransmission in enteric neural reflexes. Study of, and alterations in, the AC / cAMP signal transduction in Dogiel type II neurons, that represent AH / IPANs in this region of the gut and species or other cell phenotypes [231, 272, 273] provides a common target shared by many transmitters, paracrine or immune mediators in the integrated neural circuits of the gut [231]. Responses in Dogiel Type I neurons could potentially provide information on other functional classes of neurons utilizing cAMP.
In comparison to myenteric neurons, even less is known about cAMP signaling in submucosal neurons[74, 103, 244, 271]. However, it is expected that differences in cAMP signaling may occur between the two neural plexuses. Reasons are as follows:
1) The two nerves plexuses subserve different physiological functions.
2) Chemical coding and functional types of neurons differ
3) Electrophysiology differs in responsive types of neurons to the AC activator forskolin
94 4) Slow EPSPs believed to involve cAMP dependent mechanisms. Types of excitatory
mediators causing slow EPSPs differ i.e. myenteric plexus – many candidates
including CGRP, 5HT, SP, VIP, ADP, CCK, GRP etc. vs submucous plexus, CGRP
and ADP
5) BODIPY FSK dual labeling with calbindin, calretinin, etc binding studiew
established different distribution of FSK/AC binding sites in the two neural
plexuses[74].
6) Puncturing of neurons with the sharp electrode during electrophysiology recordings
might cause release of calcium which could directly or indirectly activate AC in
myenteric neurons. There is evidence for mechanically sensitive AC in neurons [274].
This is the first report establishing a key role for the second messenger cAMP in functional subsets of neurons in the ENS with specific morphologies and polarized projections, arguing against the original view that cAMP signaling is restricted exclusively to the AH/IPAN cell phenotype, or that cAMP signaling serves additional roles in the ENS other than gating neuronal excitability in AH and S neurons. However, our data also indicate that cAMP plays a very discrete role in enteric neural circuits.
Overall, the role of cAMP signaling in the ENS is not well understood. The new acrolein-derivatized cAMP antiserum is used to test the specific hypothesis that activation of adenylyl cyclase (AC) and elevation of intraneuronal free cAMP levels is not restricted to the Dogiel Type II (AH) / IPAN cell phenotype in the ENS. Comparative analysis between the two nerve plexuses was used to explore potential differences in cAMP signaling between myenteric and submucous neurons.
95 Studies on cAMP visualization are done in intact LMMP and SMP gut tissues and the following end points are analyzed:
1) Polarity
2) Numbers of myenteric or submucous neurons displaying cAMPir
3) RO responses
4) FSK responses
5) Cross-talk between cAMP and cGMP
6) Morphological identification of cAMPir neurons
7) Types of neurons with cAMPir compared to AC ir or BODIPY FSK binding or
electrophysiology
Therefore the overall aim of this study was to use our newly developed acrolein- derivatized cAMP antiserum to directly visualize cAMP ir neurons in the intact enteric nervous system and to quantitate and classify myenteric neurons based on their projection and shapes in order to gain more insight on the role of cAMP in the circuits of the enteric nervous system.
4.2 METHODS AND PROCEDURES
Preparation of intact myenteric plexus for cAMP visualization. Male albino
Hartley guinea pigs (Harla Sprague Dawley, Indianapolis, IN, USA) weighing 200-400 g were stunned and exsanguinated. Approximately 10 cm of the ileum from the ilea cecal junction was discarded and then segments of small intestine were quickly removed and placed in an ice-cold Krebs solution with 1µM nicardipine that bubbled with a mixture of
96 95% O2 and 5% CO2. Ileum and jejunum segments cut along the mesenteric border and prepared for microdisection of either the SMP or LMMP ± CM.
After sacrificing the guinea pig, the ileal cecal junction was identified, and oral-
distal orientation noted, a segment of small intestine (5-10 cm long) was removed and
immersed in Krebs’ solution (~32ºC). Feces or luminal contents were flushed out with
Krebs’ solution being careful to minimize distension and damage. The clean tissue was
then transfered to a clean petri dish (Sigma). Noting orientation of tissue (i.e. oral or
aboral ends) tissues were cut along either side of the mesentary border with straight-blade
scissors and then pinned flat under visual control with a dissecting microscope keeping
longitudinal and radial stretch to 25%. The preparation was cut in a rectangular shape
with very straight edges under visual magnification of 15-25x in transmitted light. Using
two pairs of blunt-ended jewelers’ forceps, the mucosa was picked up together with the
submucosa close to the anal end and to one of the mesenteric borders. The tissue was
gently peeled back sufficiently to reveal the underlying circular muscle layer. The
circular muscle was gently peeled along the entire length of the specimen, avoiding the
center of the preparation until most of the circular muscle was removed. Some
experiments were done in the presence of the CM and it did not seem to make a
difference in the results (see later). In the end, we end up with longitudinal muscle-
myenteric plexus (LMMP) or LMMP-CM for cAMP studies.
Preparation of intact microdissected submucosa plexus-The longitudinal and
circular muscle layers with the myenteric plexus were carefully removed by blunt
dissection to give sheets of submucosa + mucosa containing intact submucosa plexus
(SMP). The mucosa was then gently peeled away and SMP preparations (as LMMP
97 preparations) were cut into 1cm2 segments and pinned using micro-pins (Fine Science
Tools) in modified 35 X 10mm or 100 X 15mm Falcon dishes (Sigma) containing 0.5cm
Sylgard 184 (Dow Corning Corporation).
The segments were then equilibrated for 45 minutes at 37ºC in Krebs solution containing in mM: NaCl 120; KCl; 5.0; MgCl2, 1.2; NaH2PO4, 1.35; NaHCO3, 14.4;
CaCl2, 2.54; glucose, 12.7 prior to drug treatment.
Condition Target Site/Mechanism Vendor
IBMX (1mM), + N-Acetyl-Cysteine (5mM), + Guanylyl Cyclase (↑) Sigma Na+Nitroprusside (1mM) cocktail Ro 20-1724 (10µM) CAMP –dependent BIOM. phosphodiesterase (↓) Ro + Forskolin (0.01-10µM) Adenylyl cyclase (↑) Sigma Ro ± Forskolin ± 1H-[1,2,4]oxadiazolo[4,3- guanylate cyclase (↓) Sigma a]quinoxalin-1-one (ODQ) (1µM) ↑activates (agonist), ↓ inhibits (antagonist)
Table 8. Drugs and Stimulation Parameters and Their Target Site of Action To be used for determining their actions on [cAMP]ir.
4.2.1 Fixation Procedure
Following incubation with the appropriate drugs, myenteric or submcuous plexus
tissue (LMMP or SMP) were immediately fixed in a 6.0% acrolein (v/v) made in sodium
acetate-buffered solution for 30 min at 4°C. The tissues were then washed in a quenching
solution containing 1% glycine for 30 min at room temperature (RT), followed by a
reduction step with 1% sodium cyanoborohydride for 30 min at RT (Table 9 below). The
tissues were then washed three times in 50mM Tris-HCl solution (pH 7.5), 0.4 M NaCl at
RT and then left overnight at 4ºC in this solution before adding the primary cAMP
antiserum. Next day tissues were further microdissected by removing any remaining
98 circular muscle left behind and then cut into 1cm2 pieces and prepared in appropriate sylgard dishes for adding then blocking with appropriate agents.The cAMP antiserum was added after preabsorption of tissues with 2% bovine albumin (Sigma) and 10% donkey serum (Jackson) to block the nonspecific staining to ovalbumin (OA). It was shown in preliminary experiments that the cAMP antiserum can bind to OA. This step is critical since I found it eliminates all nonspecific background binding.
Reagent/Solutions Company Concentration (%)
Acrolein (fixation) Sigma 6.0% (v/v) in 0.1M Na Acetate (pH 4.75)
Glycine Sigma 1% (g/L) in 0.1M Na Acetate (pH 4.75) (quenching) Na+Cyanoborohydride Sigma 1% (g/L) in 0.1M Na Acetate (pH 4.75) (reduction)
Table 9. Fixation reagents and concentrations
Prim. Host Type Source Dil Second. Ab Source Dil Ab cAMP rb poly Christofi 1:50- Biotin-SP-donkey anti- Jackson 1:400 1000 rabbit IgG (1:100) (+ Avidin-FITC) Abbreviations:sp, sheep; rb, rabbit,
Table 10. Immunofluorescent labeling studies and Laser Scanning Confocal Imaging
Image analysis was done using the LSM 410 Zeiss Confocal Imaging system.
LSM imaging was carried out for cAMP immunoreactivity using a secondary IgG
antibody tagged with fluorescein or Texas Red described in Liu and co-workers [73, 74,
275]. Images were captured as RGB images and saved as .tiff images. LSM images
99 represented an average of 2-4 images; optical sections were 0.7 – 4.7 µm thick, but all comparisons were made at the same optical thickness [73, 74, 275].
4.2.2 Immunohistochemistry Staining
IHC Methods- Nonspecific staining was blocked for one hour at RT with 5-10% normal donkey serum, follow by one hour with 3% hydrogen peroxide to minimize endogenous peroxidase activity. Specificity of the cAMP antiserum was tested by either omitting the primary antibody (i.e cAMP antibody) or by preabsortion with either cGMP
(1mM) or cAMP (1mM) prior to adding the cAMP antiserum to the tissue. FITC, rhodamine or Texas Red was utilized as a fluorescent tag for secondary antibodies.
Peroxidase anti-peroxidase (PAP) Method: After incubation with cAMP antiserum, the tissues were washed and incubated for 24-48 hours at RT with goat anti- rabbit IgG (Jackson) or biotinylated donkey anti-rabbit IgG (Jackson), followed by a 15 min rinse. Tissues were then incubated for 2 hours at RT with rabbit peroxidase anti- peroxidase complex (Jackson). Color reaction was developed using a VIP peroxidase substrate kit (Vector) (Table 11). {Other methods used are described elsewhere}.
Prim. Host Type Source Dil Second. Ab Source Dil Ab cAMP rb poly Christofi 1:50- Biotinylated-dk anti-rb Jackson 1:40 400 IgG/ (PAP/VIP) (1:400) Abbreviations:rb, rabbit,
Table 11. Immunohistochemistry
Mounting tissues- Slides were dried using a hair dryer at medium heat for 10 min, followed by incubation for 10 min at increasing concentrations of alcohol (70%, 80%,
95% and 100% respectively). The final step was incubation in xylene for 10 min. A drop 100 of GEL/MOUNT (Biomeda) is then added to tissue, followed by careful placement of
cover slip (size 1, Fischer Scientific) over the tissue.
4.2.3 Data analysis and statistics
All data are expressed as means ± standard errors of the means (SEM). Statistical significance was evaluated by paired or unpaired Student’s t-test, ANOVA with
Bonferroni’s multiple comparison posthoc test or Fisher exact test, depending on experimental design. A p value of < 0.05 was considered significant. Additional details of specific analyses are described under appropriate sections.
4.3 RESULTS
It was reported by Wiemelt and co-workers [36] that an acrolein-derivatized
cAMP antiserum was suitable for visualization and quantitation of intracellular free
cAMP in cultured cells. In this study, the newly developed antiserum was used for the
first time in intact tissues to quantitate, visualize, classify and identify the projections of
enteric neurons displaying a cAMP response to the AC activator forskolin or other
treatments. The use of the antiserum in our studies on enteric neurons was described in
our laboratory in several preliminary reports in abstracts on cultured myenteric neurons
[37] or neurons in the intact neural circuits of the submucous or myenteric plexus
preparations of the guinea pig small intestine [79-82]. LMMP preparations were treated
with 1-10 µM forskolin (± Ro 20-1724) for 1-30 min, fixed with acrolein, (Table 9) and
processed for cAMP immunofluorescence [36]. Tissues were incubated with the cAMP
antiserum (1:25 – 1:400) for 24-48 hrs followed by addition of secondary antibodies and
processed for visualization (Tables 10 and 11).
101 4.3.1 Cyclic AMP visualization in intact myenteric plexus with
immunofluorescence
In intact microdissected LMMP preparations (n=5 animals), forskolin stimulation
(1.0 µM) permitted the detailed visualization and classification of 328 fluorescently tagged myenteric neurons by their cAMP ir (Table 12). Five functional types of neurons could be distinguished by cAMP visualization, their morphology, and projections. This was possible because cell processes can be followed for 6-7 ganglia at the most for a distance of ~ 1mm, then they disappear. An axonal process is defined as a process that is at least 2 times the length of the long axis of the cell somal {According to Furness’ original classification of biocytin-histochemical visualization of neurons. In some cases, visualized neurons have a very short descending process that ends in the adjacent ganglion or two ganglia over, with a specialized terminal portion. At 1µM forskolin, or
10µM forskolin (with Ro 20-1724), no visible glia are seen in the ganglia that display cAMPir.
The shapes of visualized neurons are as follows:
1) Multipolar Dogiel Type II neurons with 2-6 long tapering neurites / or axonal processes (Fig. 22); dendritic Dogiel Type II neurons were more rare (Fig. 22A).
Neurons had circumferential projections, but sometimes 1 process could be seen traveling in the anal direction (Figs. 22B, N; 24L, M). Occasionally 1 process is seen ending abruptly into a swelling as it leaves the myenteric plexus (Fig. 22P) – it likely represents a process that was interrupted as it left the plexus on its way through the CM, the SMP to the epithelial layer. All Dogiel II neurons send one process to the mucosa, the sensory process.
102 2) Filamentous neurons with 3 to 9 neurites of varying lengths (Fig. 23A-J) and a
single long axon projecting in the anal direction observed in ~ 80% of such neurons (Fig
23G-J), but never in the oral direction. In the remaining neurons of this category, a short axon projected in the circumferential direction within the same ganglion or an adjacent ganglion (Fig 24G, H, J).
3) Simple shape neurons with one or two short processes (Fig 23K-O) and a single axon traveling in the anal direction. As shown in Figs 24 C, D, E and F, the varicose axon of
this type of neuron could be seen traveling thru the longitudinal muscle. Often, the long
process could be seen leaving a ganglion in a fiber tract (Figs 24N, O) traveling to
another ganglion (Fig 24B) or traveling in the LM (Fig 24C, D, F). Some overlap exists
in neurons in categories 3 and 4.
(4) Simple neurons with a small cell soma < 20µm diameter in its long axis
(ranging from 12 – 18 µm) without any neurites, and a prominent axon that often became varicose shortly after leaving the cell body and always projected in the anal direction whenever it could be visualiz (Fig 24C).
(5) Rare cAMP visualized neurons, usually with very faint/weak labeling had
lamellar dendrites with no obvious staining on any axonal process. These represented
classical Dogiel Type I neurons with lamellar dendrites. Many other neurons had
immunoreactivity for cAMP but only the cell soma was labeled that invariably had a
smooth / ovoid shape consistent with Dogiel Type II or filamentous neurons. Clusters of
cAMP ir neurons were visible with 2 - 12 neurons / ganglion (Fig 24O-T, n=900 ganglia
imaged individually).
103 A B C D D E F
G H IKJL
M N O P
Figure 22 Mutipolar cAMP ir neurons in the guinea-pig small intestine. AH/Dogiel Type II myenteric neurons visualized by cAMP ir after uniform application of forskolin (1.0 µM) and Ro-20-1724 (10 µM) for 30 min. Left to right is oral to distal orientation of tissues. Arrows indicate axonal processes that could be traced for several ganglia in either circumferential or distal directions and never in the oral direction. Arrow head in panel (P), terminal swellings of Dogiel II processes where process was cut off entering into another layer. Scale bar = 30 µm. 104 AB C D
E FG HIJ
K L M N O
Figure 23. Filamentous and simple shape cAMP ir neurons in the guinea-pig small intestine. Cell body shapes of Dogiel Type I/Filamentous (A-J) and Dogiel Type I/Simple (K-O) myenteric neurons visualized by cAMP ir after uniform application for 30 minutes with forskolin and Ro-20-1724. All neurons had a simple long axonal process (arrow) that traveled in the distal direction (not shown here) or ended in the same or adjacent ganglion. Scale bar = 30 µm. 105 A BCD LM
LM
Anal Oral E FGH LM
LM
I JKL
M N O P
QRS T
30 µm
3
Figure 24.Multipolar & uniaxonal cAMP ir neurons display polarity. Polarity and projection of Dogiel Type I/Filamentous, and Dogiel Type I/simple and Dogiel Type II myenteric neurons visualized by cAMP ir after uniform application of forskolin and Ro-20-1724 for 30 min. (A,B, M,N) Simple neurons with single anal projection; (B) neurons sending an anal process to an adjacent ganglion. (C,D, E,F) Anal process goes thru the LM. (G,H, I, J) Filamentous neurons with anal projections. (L) Dogiel II neurons with an anal process and local process that is abruptly interrupted as it travels to the mucosa. (P-T) Clusters of cAMP visualized neurons with anal, short/ganglionic or circumferential projections. LM , longitudinal muscle; Many of the fibers are varicose in nature. 106
Cell type # of neurons Polarity of projections Anal Oral Circumf. LM Dogiel Type I/Filamentous 82 82 0 0 Dogiel Type I /Simple 158 120 0 0 38 Dogiel Type I /Lamellar dendrites 17 ND ND ND Dogiel Type II 26 8 0 18 Smooth cell soma/no processes 45 ND ND ND ND, not determined because the processes were not visible.
Table 12. Morphological classification and projections of cAMP-visualized Myenteric Neurons in intact myenteric plexus of the LMMP preparations in guinea-pig small intestine by immunofluorescence (n =328 neurons).
4.3.2 Immunohistochemical visualization of cAMP ir neurons in the intact
myenteric plexus.
Immunohistochemical analysis provided the best visualization and detailed morphological identification of the various neurons capable of generating cAMP.
Analysis of 995 cAMPir myenteric neurons from a total of 2,378 neurons in 3 different animals revealed 5 very distinct categories of neurons in the myenteric plexus after uniform application of forskolin and Ro 20-1724 for 30 min. Data is summarized in
Table 13 on page 114.
The 5 classes of neurons identified were:
1. Dogiel Type II neurons with circumferential or descending processes.
2. Dogiel Type II neurons with dendritic processes projecting as above.
3. Dogiel Type I neurons with filamentous processes in the descending or
circumferential direction.
107 Dogiel Type I small or simple descending neurons with some fibers projecting in the LM.
4. * Dogiel Type I neurons with lamellar dendrites and single descending or short
circumferential processes were prominent with IHC-unlike IF labeling. This is the
main cell type that was revealed by IHC that could not easily be detected by IF.
Dogiel Type II and Dogiel Type II dendritic neurons consisted of 19% of the total population and these multipolar neurons projected either circumferential or in the descending direction but never in the oral direction (Fig 25A, B). Dogiel Type I /
Filamentous neurons made up the largest group of cAMP ir at approximately 38% of the population analyzed. These neurons projected in an aboral direction and occasionally some of the neurons projected circumferentially for short distances (Fig 26). Dogiel Type
I/ Simple myenteric neurons made up the second largest group at approximately 28% and nearly all of these neurons projected in an aboral direction (Fig 27). Occassionaly some of the neurons had a varicose fiber that ran through the longitudinal muscle. Dogiel Type
I with lamellar dendrites made up approximately 15% of the population and projected in a similar fashion as that of filamentous neurons. However it was rare to see a lamellar neuron projecting annally (Figure 28). Uniform stimulation with forskolin and Ro-20-
1724 caused cAMP ir in clusters of 2-12 neurons with very specific shapes and polarity suggesting that cAMP mediates a specific reflex within this plexus, (Fig 29). Forskolin stimulation caused cAMP ir Dogiel Type II (i.e IPANs), Dogiel Type I/Filamentous and
Dogiel Type I/ Lamellar neurons while in other ganglia only Dogiel Type I/Filamentous and Dogiel Type I/Simple neurons expressed cAMP ir indicating that cAMP is discretely and selectively localized in enteric ganglia, presumably forming a discrete cAMP
108 A
B
Figure 25. Multipolar cAMP ir neurons of the guinea-pig small intestine. Morphology of cAMP visualized myenteric neurons in intact MP preparations in response to uniform application of forskolin and Ro-20-1724. Shapes of Dogiel Type II (A) and Dogiel Type II Dendritic neurons (B) with circumferential or anal projections. Oral to anal is left to right. Scale = 30 µm. 109 Figure 26. Filamentous cAMPir MP neurons of the guinea-pig small intestine. Shapes of Dogiel Type I / filamentous neurons visualized by cAMP ir. All neurons eventually projected in the anal direction (not shown). Scale bar = 30 µm.
110 Figure 27. Simple shape cAMP ir neurons of the guinea-pig small intestine. Shapes of Dogiel Type I simple neurons visualized by cAMP ir. All neurons eventually projected in the anal direction or had a short circumferential process that disappeared in the same or adjacent ganglion. Scale bar = 30 µm.
111 Figure 28. Lamellar shape cAMP ir MP neurons of the guinea pig small intestine. Shapes of Dogiel Type I neurons with lamellar dendrites visualized by cAMP ir. Scale bar = 30 µm.
112 Fig 29. Cyclic AMP signaling activates morphologically identified myenteric neurons that are known to belong to certain functional types of neurons involved in neural reflexes. (i)
Visualization of cAMP ir myenteric neurons with simple shape morphology (a&b) with darkly stained axons that projected circumferentially (upper 4 arrows) but eventually projected in the anal direction (not shown). In the same ganglia Dogiel Type II (c) and possibly a filamentous (d) neuron also stained for cAMP ir with projection in the anal direction. (ii) An axon (arrows) projecting in the anal direction (thick arrow heads), cell (a) is seen leaving a ganglion, and passing through another ganglion (arrow heads). Often axons were seen passing through 2-7ganglia (not shown). Sometimes an ICC (c) was seen in close contact with a neuron (b). (iii & iv) Shapes of Dogiel Type II (a) and Dogiel Type I/Lamellar neurons (arrows) are seen in these plates. Usually no more than 2-3 Dogiel Type I/Lamellar neurons were seen in a given ganglia and these neurons usually projected short distances and circumferentially. (v) Shapes of Dogiel Type II/Dendritic (c), Dogiel Type I/Simple (a) and
Dogiel Type I/Lamellar (b) are seen in this ganglia with cAMP staining of varicose fibers
(arrows) from a Dogiel Type II (d) and other varicose fibers passing in between two neurons
(a &b). Dogiel Type I/Lamellar neurons often stained lighter than other shapes of neurons and sometimes it was not possible to see their axons (b). Cluster of 4-12 neurons are seen in plates, (iii, vi & vii) suggesting even more complexity in the cAMP-dependent neural circuits of the enteric nervous system. In some clusters some of the neurons did not stained for cAMP ir (arrow), (viii & ix) indicating that only a subset of neurons contained AC.
113 (i) (ii) a
a a c c d c
b (iv) (v) (vi) b
a d (iii)
a c b (viii) (ix) (vii)
Figure 29. (see previous page for legend)
114 dependent neural circuit. Our data indicate that cAMP is primarily generated in neurons with aboral or circumferential projections, since most of the Dogiel Type I/Filamentous and Dogiel Type I/Simple shape neurons and some of the IPANs projected in the anal direction (Fig 29 and Table 12 &13).
Morphology n % Neurons* Projections
Dogiel Type II 96 9.6 ± 0.1 circumf. or descend.
Dogiel Type II Dendritic 95 9.5± 2.3 circumferential or descend.
Dogiel Type I / Filamentous 376 38.3 ± 2.0 descending or short circumf.
Dogiel Type I / small or simple 279 27.9 ±0.34 descending or with varicose shapes with varicose fibers in LM
Dogiel Type I/lamellar dendrites 149 15.0 ± 1.5 descend. or short circumf. processes
*The percent (%) of each cell type was calculated as the average ± SEM from 3 animals
Table 13. Morphological classification of cAMP-visualized Myenteric Neurons in intact myenteric plexus of the LMMP preparations in guinea-pig small intestine by immunohistochemistry-identification of all clearly visible neurons / cm2
in a cm2 stretched/pinned LMMP. A total of 995 myenteric neurons were used in the analysis of
2,378 neurons visualized from 3 animals.
4.3.3 Optimization of cAMP visualization in the enteric nervous system
Different immunohistochemistry/immunofluorescence techniques and secondary antibodies were tested in order to optimize the number of cAMP ir neurons under the same stimulatory conditions and animals, but regardless of the technique utilized cAMP ir was always higher in the SMP than the myenteric plexus suggesting that technical issues were not a reason for the difference in the number of cAMP ir neurons observed between the two plexuses (Table 115 14). Comparing the number of cAMP ir neurons between the MP and SMP under the same maximal stimulatory conditions always showed more cAMP ir in the SMP than the myenteric plexus (Table 15) further supporting that neither technical issues nor stimulatory conditions were
a reason to explain the differences in number of cAMP ir neurons between the two plexuses. A 2
to 3 fold difference exists in the number of cAMP visualized neurons between myenteric and
submucous plexuses.
116 Secondary Antibody Visualization Cell Numbers / cm2 Number of Animals (dilution) Method Biotinylated -dk anti-rb IgG (1:50-1:100) LMMP-CM FITC-avidin 1:40 225±10 n=17 dil. Biotinylated-dk anti-rb IgG FITC-avidin 1:40 (1:80) SMP dil. 1022±110 n=19 Biotinylated -dk anti-rb IgG (1:80) LMMP-CM ABC 1:100 dil 661±58 n=3 Biotinylated-dk anti-rb IgG (1:80) SMP ABC 1:100 dil 1,750±70 n=3 Biotinylated -dk PAP (rb) 1:400 anti-rb IgG (1:20 to 1:80 dil) LMMP-CM dil/VIP substrate 737±140 n=3 HRP-gt anti-rb IgG (1:25 to 1:400 dil) LMMP-CM VIP substrate 570±39 n=6 gt anti-rb IgG PAP 1:400 (1:20) LMMP-CM dil/VIP substrate 413‡ n=1 * in myenteric neurons of LMMP-CM, 1µM forskolin + 10µM Ro-20-1724 was used to stimulate cAMPir; the concentration of acrolein derivatized cAMP antiserum was kept constatn for all conditions at 1:20 dilution (maximum sensitivity for low levels of cAMP to insure max. counts of neurons).
** 0.1µM forskolin + 10µM Ro-20-1724 was used to stimulate cAMPir in SMP.
‡ Background was very high in tissue.
Table 14. Comparative analysis of cAMP visualized neurons in the ENS using different processing techniques.
117
Pharmacoligical Agent Cell Numbers / cm2 p value Number of Animals Krebs (IHC/ABC) LMMP 0 n=6 Krebs (IHC/ABC) SMP 17 ± 9 <0.05 n=3 Krebs (IF) LMMP 0 n=11 Krebs (IF) SMP 0 n=13 Ro-20-1724, 10µM (IHC) LMMP 18 ± 32 (3/7 tissues) n=7 Ro-20-1724, 10µM (IHC) SMP 465 ± 60 (many light stain) <0.05 n=3 Ro-20-1724, 10µM (IF) LMMP 0 n= 12 Ro-20-1724, 10µM (IF) SMP 0 n=22
Ro-20 + 0.1µM Forskolin (IF) 225 ± 10 n= 17 LMMP Ro-20 + 1-10µM Forskolin (IF) SMP 1022 ± 110 <0.001 n=19
Table 15. Comparative analysis of the pharmacological characterization of the acrolein derivatized cAMP antiserum between myenteric and submucous plexuses.
4.3.4 Myenteric neurons regulated by phosphodiesterase Type IV
Phosphodiesterase activity seems to play a larger role in the regulation of basal cAMP levels in Dogiel Type I/Lamellar and Dogiel Type I/simple shape neurons since only these subsets of neurons expressed cAMP ir after incubation with Ro-21-1724 alone
(Fig 30). In addition it was rare to see more than two or three neurons per ganglion suggesting that only a very specific subset of neurons within a ganglion contained a more active phosphodiesterase enzyme. However forkolin (10µM) augmented the Ro-20-1724
(10µM) response by approximately 3,585% suggesting that phosphodiesterase Type IV activity was low in the myenteric plexus (Fig 31).
118 Figure 30. Phosphodiesterase activity in the myenteric plexus of the guinea-pig small intestine. cAMP visualization of myenteric neurons in response to the phosphodiesterase inhibitor Ro-20-1724 (RO) alone. Dogiel Type I/simple shape neurons were the most responsive to RO alone, type (A,B,D,F,I) follow by Dogiel Type I/Lamellar neurons (A and G, arrow). Occasionally a Dogiel Type II neuron with a smooth cell body (C) was seen with very light staining of its axons (arrows). A Dogiel Type II/Dendritic neuron is shown in G with arrow. Dogiel Type I/Filamentous neurons were not clearly detectable. Clusters were never more than 2-4 neurons and their morphology was often unclear (D,E, G-J). Scale bar = 30µm.
119 1000 P < 0.001
900 2 m
c 800 s/ n
o 700
neur 600 r i P 500 M A c 400 pf
er 300 b m
u 200 N P > 0.05 100
-0- 0 Krebs Ro Ro + F
Figure 31. Quantitation of cyclic AMP ir in the intact myenteric plexus (MP) using immunohistochemistry techniques. MP neurons were stimulated with forskolin and Ro-20- 1724 (Table 8) to increase free intracellular cAMP levels. The primary cAMP and secondary antiserum were 1/50 and 1/40 respectively (Table 11). N= 3 animals
120 4.3.5 Lack of one to one correlation between cAMP ir and depolarization of
AH and S neurons.
Forskolin stimulation of four AH/Dogiel Type II and 3 S/Dogiel Type I
(filamentous) neurons caused a slow EPSP-like response but ther was no detectable increase in cAMP ir suggesting that the rise of free intracellular cAMPir is below the sensitivity of the antiserum or that forskolin is acting by some other unknown mechanism
(See summary of electrophysiology data in (Table 16).
Shape FSK IR Depol. AB Processing method Positive Aft/bf cAMP ir
Dogiel Type II + 1.12 36 0 FITC-Don, anti-Rab IgG none Dogiel Type II + 1.38 21 0 Goat-Antirabbit IgG, PAP none Dogiel Type II + 1.72 9 5 HRP-Goat anti-rabbit IgG, VIP none Dogiel Type II + 1.67 36 0 HRP-Goat Anti-rabbit IgG, VIP none Filamentous + 1.42 23 1 FITC-Don, anti-rabbit IgG none Filamentous + 1 6 1 FITC-Don, anti-rabbit IgG none Lamellar + 1.04 8 1 HRP-Goat anti rabbit IgG, VIP none +; deporalized by forskolin, FKS; forskolin, Depol; depolarization, AB; anodal brakes
Table 16. cAMP antibody failed to stain Biocytin labeled Forskolin-responsive myenteric neurons.
4.3.6 Cylcic AMP ir in interstitial cells of Cajal.
Stimulation for 30 min of the myenteric plexus with the slow EPSP mimetic agents VIP (1-5µm) or PACAP (1-5µM, data not shown) cause cAMP ir in interstitial cells of Cajal (ICCs) with various morphological shapes suggesting that cAMP signaling is critical in these non-neuronal cells Fig 32B &C. (n=2 tissues).
4.3.7 Cyclic AMP and cyclic GMP ir
Forskolin (0.1-10µM) with PDE inhibition by Ro-20-1724 (10µM) was the most effective stimulator of intraneuronal cAMP levels. Similar to earlier findings this
121 antiserum displays excellent selectivity and specificity of the cAMP ir response since it is blocked by preabsorption of the cAMP antibody with cAMP (0.5-1mM) but not when pre-incubated with cGMP (1mM; Fig 33A). It is also barely stimulated by a cGMP cocktail (Table 8) which is consistent with previous testing of the cAMP antibody [36,
37] and the cGMP ir response is reduced by > 50% by the guanylate cyclase (GC) inhibitor ODQ (1µM) (Fig 33B) [276, 277] suggesting that the cGMP produced by the cGMP cocktail (Table 8) was due to stimulation of the soluble and not the particulate
form of guanylate cyclase [276-278]. More importantly, cGMP contribution to the cAMPir responses is at best negligible in intact ENS preparations.
4.3.8 Cyclic GMP and cAMP Cross-Talk
Furthermore, ODQ does not inhibit forskolin - cAMP responses – in fact it enhances them by 86% (n=4 animals) (Fig 34C), suggesting cross-talk between cAMP and cGMP in the intact neural circuits of the submucous plexus. ODQ inhibition of soluble GC seems to recruit neurons in additional ganglia that can display cAMPir but does not increase the number of neurons displaying cAMP ir/ganglion suggesting that inhibition of soluble GC artificially blocks endogenous inhibitory cGMP-dependent signaling that serves to gate/inhibit the excitatory AC/cAMP signaling (Figs 34 A&B).
The distribution of cAMP responsive neurons in ganglia, ODQ increases the number of cAMP visualized neurons/ganglia for ganglia with certain number of positive cells (Fig
34A). Thus, GC functions in this role only in a subset of ganglia within the enteric nervous system.
122 A 500nM VIP + 10 M RO
B 5 M VIP + 10 M RO
C 5 M VIP + 10 M RO
Figure 32.. Receptor activation with slow EPSP mimetic agents causes cAMP ir in interstitial cells of Cajal (ICCs). Both VIP (above) and PACAP (data not shown) mimic the forskolin response in ICCs and cause cAMP ir in various different shapes of ICCs. Scale bar = 30µm.
123 A B 2
2 1200 p = 0.0001 400 p = 0.0001 p = 0.031 1000
p = 0.0001 urons/cm 300 ne
eurons/cm 800 r n i r P i 600 200
400 100 200
0 Number of cAM 0 N= 11 17 19 3 2 N= 5 5 s P Number of cAMPP F P eb O + M M r R O P Q K cA cG D R M O M M cG + 1m 1m
Figure 33. The cAMP antiserum discriminated between cAMP and cGMP in microdissected SMP neurons of the guinea-pig small intestine. (A) Immunofluorescent visualization and quantitation of cyclic AMP ir in the intact submucosal plexus (SMP). SMP neurons were stimulated with Ro-20 1724 ± forskolin (Table 8) to increase free intracellular cAMP levels. Preabsorption with 1mM cAMP but not cGMP of the cAMP antiserum blocked the forskolin + Ro-20 1724 response. Tissues were fixed with acrolein and incubated with primary anti- cAMP as outlined in Tables 9 and 10. Laser confocal imaging was used to count the cells. (B) The guanylate cyclase inhibitor ODQ effectively reduced the cGMP cocktail response (also see Table 8).
124 A 300 * 250 RO+Forskolin * 2 RO+Forskolin+ODQ 200 * *
150 *
100 * Number of ganglia/cm
50
0 12345678910111213 Number of Neurons
BCp=0.00835 1200 p = 0.026 2000 1800
h
t 1000 1600 i 1400 800 1200 urons/unit 1000 ne
area 600 r
i 800 P 400 600 Number of Neurons cAM
Number of ganglia w 400 200 200
0 0 N= 3 3 N= 6 5 Ro+F +ODQ Ro+ F + ODQ
Figure 34. Forskolin and ODQ effects on SMP neurons of the guinea-pig small intestine. (A) Comparing the distribution of cAMP ir after forskolin ± ODQ stimulation.*,p<0.05 for Ro+F+ODQ vs Ro+F; n=3 animals. (B) ODQ increases the number of ganglia with one or more neurons displaying cAMP ir but not the number of neurons/ganglion. (C) ODQ increases the total number of neurons displaying cAMPir. 125 4.4 DISCUSSION
This study is the first to use the cAMP visualization technique in the functional
identification of classes of myenteric neurons that utilize the Gs/AC/cAMP signaling
pathway in the intact mammalian ENS. After forskolin stimulation, cAMP may diffuse
readily to all parts of neurons, making it possible to classify the neurons by their
morphology. FlCRhR / cAMP visualization in living myenteric neurons provided
consistent evidence for cAMP elevation in all regions of a neuron [73]. Localized
expression of several AC isoenzymes in cell soma, neurites and axonal processes [73, 74]
is likely to contribute to the complete visualization of neuronal morphology achieved
with cAMP visualization (this study).
Activation of AC and visualization of cAMP ir was useful in elucidating all the
classes of enteric neurons that can generate cAMP. Cyclic AMP visualized neurons were
identified as Dogiel Type II with circumferential or descending projections, Dendritic
Dogiel Type II, descending filamentous neurons or small/simple descending Dogiel Type
I neurons or those with lamellar dendrites. The distribution of fluorescent Bodipy
forskolin binding to AC in live gut LMMP or SMP tissue from guinea pig small intestine
[74] or AC ir [73] in several classes of myenteric neurons is consistent with our findings for functional cAMP visualization.
Cyclic AMP visualized Dogiel Type II neurons in the guinea-pig small intestine are IPANs with AH cell characteristics, although they can also serve as interneurons in the myenteric microcircuits [279, 280]. Dendritic Dogiel II neurons have AH cell characteristics, pronounced slow EPSPs, lack fast EPSPs, account for 10% of Dogiel II neurons {~ 3% of all neurons} and may convey signals in long aboral / descending
126 reflexes [15] or they may be a type of intrinsic sensory neuron. Interestingly, equal
proportions of Dogiel II and Dogiel II dendritic neurons were identified, eventhough the
overwhelming majority of AH/Dogiel II neurons (≥ 90%) are Dogiel IIs, suggesting a
prominent role for cAMP in Dendritic Dogiel II neurons.
Cyclic AMP visualized filamentous neurons were described previously to form
very long anally directed interneuronal chains that connect with both myenteric and
submucous neurons [281]. They receive slow EPSPs as do AH neurons, and were classified in some studies as a type of AH neuron [26, 39]. These neurons display slow
EPSP-like responses to the AC activating peptide PACAP [26]. They may represent
interneurons involved in descending peristaltic reflexes and in mucosal secretory reflexes
[282]. Some filamentous neurons may be mechanosensitive and thus may serve a dual
role as intrinsic primary afferent neurons [87, 88].
Dogiel Type I/Simple (simple) shape neurons have been functionally categorized
as either excitatory or inhibitory longitudinal muscle motor neurons with short
projections and are hypothesized to make up approximately 24% of the total population
of myenteric neurons in the guinea-pig small intestine. In fact, cAMP visualized neurons
could be seen sending a varicose fiber through the longitudinal muscle confirming their
identity as LM motorneurons. These represent primary short projections or
descending/presumably inhibitory longitudinal muscle motor neurons. Cyclic AMPir in
the LM varicose fibers implies a role for cAMP in neuroeffector transmission to LM. To
obtain a more accurate number, double labeling studies will need to be done for calretinin
and cAMP since these calcium binding protein labels approximately 87% of all small
longitudinal motor neurons in the guinea pig small intestine [15]. In addition, NPY
127 +/GABA+ neurons denote small/simple shape inhibitory motorneurons to LM. Retrograde labeling studies with DiI from LM can provide unequivocal proof for their identity as LM motorneurons.
Based on previous functional and immunohistochemistry studies Dogiel Type
I/Lamellar neurons are categorized functionally as descending interneurons and
hypothesized to make up approximately 2% of the entire population within the myenteric
plexus [15]. It has been suggested that the total number of neurons in the myenteric
plexus ranges from 4-8,000 neurons/cm2 in the guinea pig small intestine [18] and since
lamellar neurons are hypothesize to make up 2% of this total population [14, 15] would
result in approximately 180 lamellar neurons/cm2. In functional IHC studies on cAMP ir these subpopulation of neurons consisted of about 149 lamellar neurons/cm2, therefore
approximately 83% of all possible lamellar neurons were cAMP ir after uniform
stimulation with forskolin. However these neurons often did not stain as darkly as other
neurons and sometimes it was difficult to include these neurons in the population since
axons were sometimes not visible, therefore it is possible that these numbers might be
higher. Triple labeling studies are needed for cAMP and neurfilament triple protein
(NFP) and 5-HT ir since colocalization of NFP and 5-HT has been previously shown to
exist exclusively in these neurons [14].
The distribution of enteric neurons with cAMP ir differed significantly from that of neurons with cGMPir described previously in the MP and SMP [95]. In the myenteric plexus, about 2% of neurons express cGMPir compared to 15-20% for cAMP ir (our data) [95]. Therefore, there is little possibility of substantial co-localization of GMPir responses in the 15-20% of myenteric neurons that are capable of generating cAMP;
128 however, this does not exclude the possibility of co-localization of cAMP and cGMP responses.
The acrolein- derivatized cAMP antiserum has a much lower affinity for cGMP and the cGMP cocktail gives weak staining for cGMPir and the proportion of neurons displaying cGMP ir are a small percentage of those that display cAMP responses to forskolin – this is consistent with a previous report using a cGMP antiserum [95]. In the submucous plexus, co-localization of cAMP and cGMP responses in the same types of neurons is much more likely than in the myenteric plexus. Others showed that 60% of cGMPir SMP neurons had simple shapes, 16% were filamentous and 12% had Dogiel
Type I morphology but not Dogiel type II neurons were cGMP ir. In contrast, the acrolein derivatized cAMP antiserum used in our studies revealed that 28% of cAMP visualized neurons had simple or small neuronal morphology and 19.1% of all responsive neurons to forskolin were of Dogiel Type II morpohology. Therefore, direct evidence with the cAMP antiserum indicates that AC/cAMP and not GC/cGMP signaling occurs in a subset of intrinsic primary afferent (Dogiel Type II / AH) neurons. With other types of neurons, a greater proportion of 38% of cAMP-responsive neurons had filamentous morphologies in comparison to 16% for cGMPir. Similar proportions of Dogiel Type I neurons with lamellar dendrites display cAMPi (this study) and cGMPir [95].
New findings also indicate that there is negative cross-talk between cGMP and cAMP in the enteric nervous system. Functional interactions between cGMP and cAMP were tested with the guanylate cylcase ODQ inhibitor. In the myenteric plexus, ODQ had no effect on the AC/cAMP response to forskolin, but it augmented the response to forskolin in submucous neurons (Fig. 34). ODQ is selective for GC because it was able to
129 block the response to the cGMP cocktail in the submucous plexus; in the myenteric
plexus, the cGMP cocktail produces very weak cAMPir or no response. The number of
neurons / ganglion with cAMP ir to forskolin remained the same after ODQ but the
number of ganglia with cAMP ir increased significantly (Fig 34). This indicates that cGMP provides an ongoing inhibitory modulation of cAMP ir that limits the rise and spread of cAMP ir from neuron to neuron in the submucous plexus (VIP ir neurons are always ir for cGMP). Cyclic AMP dependent neural secretion does occur in the intestine via activation of AC/cAMP signaling in submucous neurons [283, 284]. Therefore, one possibility that deserves further consideration is the negative influence of cGMP (and A1 inhibition of AC/cAMP signaling) in the regulation of cAMP dependent excitability of submucous neurons, and cAMP dependent neural-secretion in response to mechano- or – chemosensitive stimulation [285, 286].
Rolipram and Ro-20-1724 elevate cAMP ir in enteric neurons indicating that a
PDE4 regulates the basal levels of cAMP in the neurons. At the concentrations used, the
PDE inhibitors can elevate the excitability in some AH neurons [73], and is consistent
with the hypothesis that the resting excitability of AH neurons is tightly regulated by
PDE4. PDE4 plays a more prominent role in regulating intracellular cAMP levels in
submucous than in myenteric neurons, since > 10 times more neurons display cAMP ir in
submucous neurons than myenteric neurons in response to PDE4 inhibition. Other
enzyme inhibitors were not investigated, and it is possible that different PDEs found in
neural tissues (i.e., PDE7 or PDE8) [287] are involved in the regulation of resting cAMP
levels in the myenteric plexus. Furthermore, if increases in intracellular cAMP are linked
to the excitability of enteric neurons, then it can be predicted that PDE4 should cause a
130 greater increase in excitability of submucous neurons than myenteric neurons, while other
PDEIs would have more effect in myenteric than submucous neurons.
Three times as many neurons/cm2 displayed cAMP in submucous neurons
representing > 60% of population compared to 15-20% of population of myenteric
neurons (with IHC). The more discrete localization of cAMP signaling in the myenteric
plexus argues for very different physiological roles of the second messenger in the two
nerve plexuses. It also suggests that SMP may be a better model to study coupling of the
cAMP signaling to electrical activity / EFS stimulation or single cell recording, and that
we would have better chance in ascertaining whether a 1:1 relationship exists between
forskolin activation of AH or S neurons and cAMP ir. EFS stimulation was able to
elevate cAMP ir in 5% of possible cAMP-responsive myenteric neurons (i.e. calculated
with forskolin stimulation) and a greater proportion of 20% of submucous neurons (data
not shown). This provides direct proof that cAMP is linked to the electrical behavior of
enteric neurons. The remaining responses in cAMP visualized neurons may represent
other functions of cAMP signaling in neuronal plasticity, transcriptional regulation or
even metabolism within the neuron, or more likely the cAMP antiserum is not sensitive
enough to answer this important physiological question – it cannot detect small transient
elevations in physiologic levels of cAMP in neurons.
Our findings indicate that cAMP is an important second messenger in a variety of enteric neurons serving very different functions in enteric neurophysiology. This establishes a more prominent and complex role for cAMP in enteric neuroregulation than could be predicted from intracellular recordings from AH or S neurons [261, 288] In the submucous plexus, activation of both AH and S neurons by forskolin has been reported
131 [103] but in the myenteric plexus, only AH neurons have been reported to express
functional AC linked to their excitability [244]. A significant point of departure from
electrophysiological data on AC/cAMP signaling is the proportion and types of
responsive myenteric neurons.
Indirect electrophysiology data suggested that all Dogiel Type II/AH neurons
(IPANs) were responsive to forskolin stimulation, however in our hands forskolin
stimulation caused an increase cAMP ir in approximately 4-5% of all the possible Dogiel
type II neurons/cm2 in the LMMP. Some possible explanations are offered for the discrepancy between previous electrophysiology work and our cAMP ir data: (1) Dogiel
Type I/Filamentous neurons had previously been miscategorized as AH neurons (that are known to have Dogiel II morphology) when in fact these neurons were of Dogiel Type I morphology. Recorded neurons at that time were not typed morphologically – such neurons were refered to as activated AH neurons, and later studies proved that they had
AH/S like properties [15, 26]. (2) In addition, the likelihood of a sampling error during intracellular recording is very high since recordings can only be done in those neurons that have a stable membrane potential (i.e healthy). This is significantly different from our technique where uniform stimulation with forskolin stimulates all the ganglia in a none-biased manner and neurons do not have to be punctured or damaged in any way. In our hands, not every ganglion responded to forskolin stimulation and yet it is known that every ganglion contains Dogiel Type II - neurons therefore in theory every ganglion should have cAMP responses to forskolin. During intracellular recording there is a lot of navigation from ganglion to ganglion until one finally obtains a stable recording which
132 artificially provides a bias sampling of the ganglia because only those ganglia that
contain healthy neurons would provide a suitable environment for recording.
Previous studies have shown that forkolin can directly activate GABA-gated Cl-
channels in rat brain synaptoneurosomes and on potassium channels in molluscan
neurons [289, 290], therefore suggesting that the effects of forkolin are not solely
mediated by activation of AC. More recent studies have shown that intracellular cAMP
may by pass PKA and stimulate Epac ion channels [291, 292]. Preliminary
electrophysiology data in our laboratory, obtained by Dr. Chen, showed that forskolin
slow EPSP-like responses occurred in biocytin identified neurons and subsequent cAMP
visualization failed to localize a cAMPir response in the biocytin filled Dogiel II neurons
(Table 13D) (Dr Chen Abstract AGA 2004) suggesting that (1) the cAMP antibody is not
sensitive enough to detect intracellular concentrations of cAMP that correlate with the
depolarization of the AH neuron, (2) that forskolin is acting via another unknown
mechanism (i.e directly on potassium channels) or assay/fixation conditions are not
suitable for detecting histochemical identification of the biocytin filled neuron and
immunochemically identification of cAMP ir. The possibility that forskolin puff in AH
+ 2+ neurons can activate K Ca conductance directly can only be tested in the future by using forskolin ± KT 5720 (PKA inhibitor) or 2’5’DDA, (p-site/AC inhibitors) must be done[42, 293-295].
A subset of interstitial cells of Cajal (ICCs) are believed to to function as the pacemakers for slow waves in the gut musculature and disfunction of these cells is now hypothesized to cause a variety of disease such as infantile hypertrophic pyloric stenosis
(a very common disease in children of the pyloric sphincter), Hirschsprung’s disease and
133 intestinal pseudo-abstruction [296, 297]. Therefore understanding how these cells influence smooth muscle of the gut has become very important. Current work on ICCs showed that forskolin stimulation of culture ICCs reduced the frequency of spontaneous pacemaker activity that itactivate a sustained outward current. These effects of forkolin were not mimicked by membrane permeable cAMP analogues or its effect was not blocked by PKA inhibitors suggesting that forskolin might be acting via another mechanism [289, 290, 298]. However in our hands forskolin and the slow EPSP-mimetic agents VIP and PACAP caused cAMPir in ICCs in LMMP-CM preparations (Figure
24B) suggesting that in the intact tissue adenylyl cyclase activity on ICCs might function very different from culture system. Electron microscopy and immunhistochemistry techniques have shown that VIP nerve terminals lay in very close contact with ICCs supporting our data those VIP receptors in ICCs might be modulated by VIP release from the enteric nervous system [296]. In addition since PACAP often cross-reacts with certain VIP receptors subtypes (VIP-2) and this subtype has been recently localized to
ICCs, suggesting that both PACAP and VIP may elevate cAMP ir via the same receptors.
VIP receptors are involved because a VIP antagonist prevents the VIP-cAMP response
[299]. Lastly the shapes of ICCs have been primarily studied using electron microscopic ultrastructure and now more recently using antibodies raised against the tyrosine-kinase receptor c-kit which is now the golden marker for ICCs [296]. However our cAMP ir on
ICCs not only provides a more clear morphology of these unique cells (conversation with an expert in the field Dr. Kenton Sanders) but more importantly a vey defined functional end point.
134 At least 18 classes of myenteric neurons can be distinguished by their chemical coding, projections and / or neurophysiology [15]. Of these 18 classes, cAMP signaling is found in subsets of IPANs, descending interneurons, descending inhibitory neurons and LM motor neurons with polarized projections for circumferential or descending pathways. The striking polarity of cAMP visualized neurons indicates that activation of the Gs/AC/cAMP signaling pathway may be involved in descending reflexes in the gut.
To date, little was known about cAMP signal transduction in normal or disease states of the gut [231, 270, 300] or the classes of neurons and their projections that utilize cAMP as a second messenger. The polarized neural circuits of the myenteric plexus utilizing the Gs/AC/cAMP signaling pathway are organized for aboral peristaltic reflexes.
Other studies in Dr. Christofi’s laboratory, and in collaboration with Dr. Jack
Grider, (University of Virginia), the hypothesis that cAMP signaling is involved in descending reflexes. Experiments targeted ascending and descending reflexes in a 3- chamber model of the intestine. The middle chamber serves as the sensory chamber and oral/distal chambers serve as the motor chambers. Stroking of the mucosa or stretch of muscle leads to ascending contraction (recorded in oral chamber) or descending relaxation (recorded in distal chamber). Application of myr PKI, a selective PKA inhibitor, leads to partial inhibition of both ascending and descending reflexes. However, mrPKI application to the motor chambers only inhibits the descending reflex. These data strongly support and are consistant with our functional cAMP visualization data indicating that cAMP neural circuits are polarized for descending reflexes.
135 In summary, it can be concluded that:
1) Cyclic AMP signaling is not restricted to Dogiel II/AH neurons. In fact, it occurs in 5 functional classes of myenteric neurons.
2) Cyclic AMP-dependent neural circuitry is polarized for descending reflexes.
3) Clear differences exist between myenteric and submucous neural cAMP dependent circuitry – in PDE IV modulation of basal cAMP activity, co-localization of cAMP and cGMP responses, polarity of cAMP visualized neurons, types of cAMP-neurons, numbers of cAMP neurons.
4) Cyclic AMP visualization is a powerful technique for functional studies of neural circuits in the ENS.
136
CHAPTER 5
MUCOSAL REFLEX ACTIVATION OF SUBMUCOSAL NEURONS IS
MEDIATED THROUGH CYCLIC AMP SIGNALING
5.1 INTRODUCTION
All aspects of digestive function are under the regulatory influence of extrinsic
(CNS) and intrinsic (ENS) neurons. Since the initial hypothesis [8] that intestinal propulsion were driven by local neuronal reflexes (i.e ENS) within the intestine much of the subsequent work focused on the myenteric plexus of the intestine [24, 301] since it is the predominant player in driving peristalsis. However, more recent work has clearly demonstrated that autonomous reflexes also exist in the submucosal plexus [28, 302-
305]. Furthermore even though the myenteric plexus strongly influences submucosal functions [305, 306] in vitro mucosal-submucosal preparations devoid of myenteric plexus have shown that reflex-evoked dilation of arterioles and mucosal secretion from enterocytes are confined strictly within submucosal neurons [98, 307-314].
Mucosal reflexes initiated by stroking or touch of the mucosa, balloon distention and distortion of the mucosal surface cells and activation of enterochromaffin cells (EC), alterations in luminal pH, nutrients or nitrogen puffs on the mucosa, all lead to neurosecretion via the submucous plexus. In addition, mucosal reflexes lead to activation
137 of peristalsis via activation of myenteric IPANs with their cell bodies in the myenteric plexus and their sensory process projecting to the mucosa [91, 269]. Intracellular recordings from myenteric AH/IPANS proved that mucosal stimulation can cause excitation of these neurons – they lead to alterations in motility [9, 87]. Finally, the activity of neurons in submucous and myenteric plexuses must be coordinated for motility, secretion and vasomotor functions in the gut to occur during normal or pathophysiological circumstances [35].
Indirect electrophysiology and radioimmunoassay studies have suggested that adenosine 3' 5'-cyclic monophosphate (cAMP) is a key mediator for slow synaptic transmission in the myenteric plexus [26, 42, 48, 50, 59, 61, 265, 315, 316]. However, little is known about the role of adenylyl cyclase in the submucosal plexus (SMP). One study, in a few neurons, showed that elevation of cAMP after application of forskolin or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused depolarization of the membrane potential in S and AH neurons [103]. However, a later study by another investigator suggested that only AH neurons were excited by forkolin and IBMX [271], and hence had a functional AC/cAMP signaling pathway.
A more recent study using BODIPY forskolin in Dr. Christofi’s lab, showed that the majority of neurons expressing AC in the SMP were non-cholinergic neurons (i.e VIP ir neurons) [74], which supports previous findings [103], that a subset of S neurons are responsive to forskolin and IBMX stimulation. A recent review states that forskolin elevates cAMP in enteric ganglia and mimics the slow EPSP in AH neurons but has no effect on the excitability in S-type neurons [244]. Therefore, the role of cAMP in the submucous plexus remains unclear and somewhat controversial. Detailed analysis of
138 cyclic AMP visualized submucous neurons and their projections as done in the myenteric plexus (Chapter 4) would be very useful in our efforts to better understand the role of cAMP in the microcircuitry of the submucous plexus.
The chemical coding and functional identity of submucous neurons that can generate cAMP remains unknown. SMP neurons consist of non-cholinergic (45%) and cholinergic ir neurons (55%). Non-cholinergic neurons are almost always VIPir neurons, which have been shown to function as secretomotor neurons as well as interneurons in submucosal reflexes [6, 15, 94]. NPYir neurons makes up the largest group of cholinergic neurons and usually project to the mucosa and mucosal glands to function as secretomotor neurons and possibly as interneurons [317, 318]. Intrinsic Primary afferent neurons (IPANs) and/ or calbinin ir neurons make up approximately 10-20% of the entire
SMP neuronal population and the second largest population of cholinergic neurons [94,
319]. IPANs located in the SMP generally send one afferent process to the mucosa [13] and function as sensory neurons of the gut [9]. These neurons may also function as interneurons within the SMP [93]. Calretinin ir neurons make up approximately 6% of the entire SMP neuronal population and the third largest group of cholinergic neurons and can function as secretomotor and interneurons to regulate tone in local arterioles
[102, 317].
Previous experiments showed that forskolin elicits an increase in short circuit current (Isc) indicative of electrogenic chloride ion transport by stimulating SMP neurons as well as by acting directly on enterocytes [284, 285] in guinea pig ileum. Later experiments showed that forskolin and IBMX enhanced the response of Isc to VIP stimulation and that VIP enhanced the chloride secretory response by stimulating
139 cholinergic SMP neurons but had no effect on non-cholinergic neurons [283]. These
studies provided some proof for cAMP-dependent neurosecretion.
Previous intracellular studies had shown that stimulation with either the adenosine
A2 receptor agonist (A2R), 2-chloroadenosine (CADO) or the A2aR agonist, (2-[p-
(carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS21680),
depolarized SMP neurons and that this effect could be blockd by cyclopentyltheophyline
(CPT) [293]. In the same studied it was shown that adenosine acting via presynaptic A1
receptors inhibited release of acetylcholine [293]. Later studies by the same investigator
showed that theA2R agonist CADO inhibited Ca2+ dependent potassium conductances on submucosal S neurons by activating the cAMP/PKA pathway and that this depolarization of S neurons was inhibited by the very specific PKA antagonist, KT5720 [(8R*,9S*,
11S*)-(-)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,
11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]-trin-den-1-one] [72, 320].
The possibility that other mediators can stimulate cAMP exists, and these could include neural, immune, inflammatory paracrine or autocrine substances. Little more is known about cAMP dependent signaling in submucous neurons or neural circuits, in constrast to myenteric neurons where a variety of neuropeptides, amines, and immune mediators could act through cAMP signaling [60, 61, 231]
Evidence for recruitment patterns of SMP neurons and poly-synaptically coupling.
Fast and/or slow EPSPs have been recorded in SMP neurons after exogenous application of 5-HT agonists, mechanical and electrical stimulation of the mucosa of myenteric-free preparations [93]. However since it is impossible to record from IPANs
140 projecting directly underneath the mucosa, most of these recordings were done in second order neurons making it difficult to map out the sensory-motor circuitry in the SMP. In an effort to overcome this mechanical limitations and study recruitment patterns of SMP neurons investigators have employed optical recordings such as voltage sensitive dyes to
[96, 97] or c-fos expression [98]. These techniques identified sub-populations of neurons activated by specific stimuli but could not examine the pattern of innervation between identified neurons. Therefore other investigators have used complex dual electrical recordings from SMP neurons while simultaneously stimulating with 5-HT to activate cell bodies of SMP cholinergic neurons in nearby ganglia [99]. These difficult experiments showed that cholinergic neurons provide both diverging and converging inputs to VIP neurons providing a mechanism to augment activation of VIP-secretomotor neurons. They hypothesize that AH neurons are likely to be good candidates for providing diverging patterns due to their multipolar morphology [99], but this remains speculative.
The possibility of interneurons in the SMP was first suggested after the removal of the myenteric inputs that reduced the number of cholinergic inputs to SMP neurons
[306]. Since vagal fibers do not appear to innervate the submucosal plexus it was concluded in these studies that inputs must originate from submucosal interneurons projecting to other SMP neurons [100]. Furthermore, since hexamethonium blocked neural elements that mediate reflex vasodilation of SM arterioles further supports that interneurons are involved in the SMP [93, 99, 101, 102].
These studies also serve to stress how little we know about submucous neural circuitry and especially cAMP signaling. A functional analysis with the acrolein-
141 derivatized cAMP antiserum and studies on synaptic transmission may shed more light
on relevant functional connections between submucous neurons in the intact neural
plexus that are not possible with other techniques. Chemical coding of cAMP visualized
neurons was assessed in double immunofluorescence labeling studies. Pharmacological
interventions were used to prove if cAMP contributes to receptor activation or synaptic
transmission in polysynaptic pathways in the intact submucous plexus. The role of cAMP
in neurosecretion was studied in Ussing chambers on short-circuit current/Isc analysis.
Cyclic AMP visualization would reveal the polarity of cAMP-dependent neurons.
5.2 MATERIALS AND METHODS
Male albino Hartley guinea pigs (Harlan Sprague Dawley, Indianapolis, IN, USA)
weighing 200-400 g were stunned and exsanguinated. Approximately 10 cm of the ileum
from the ilea cecal junction was discarded and then segments of small intestine were
quickly removed and placed in an ice-cold Krebs solution with 1µM nicardipine that
bubbled with a mixture of 95% O2 and 5% CO2. Ileum and jejunum segments cut along the mesenteric border. The longitudinal and circular muscle layers with the myenteric plexus were carefully removed by blunt dissection to give sheets of submucosa ± mucosa containing intact submucosa plexus (SMP) as described in Chapter 4. The SMP preparations were cut into 1cm2 segments and pinned using micro-pins (Fine Science
Tools) in modified 35 X 10 mm or 100 X 15 mm Falcon dishes (Sigma) containing
0.5cm Sylgard 184 (Dow Corning Corporation). The segments were then equilibrated for
45 min at 37 ºC in Krebs solution containing in mM: NaCl 120; KCl; 5.0; MgCl2, 1.2;
142 NaH2PO4, 1.35; NaHCO3, 14.4; CaCl2, 2.54; glucose, 12.7 prior to drug treatment (Table
17).
Fixation Procedure- Following incubation with the appropriate drug (Table 17),
submcuous plexus segements were immediately fixed in a 6.0% acrolein (v/v) made in
sodium acetate-buffered solution for 60 min at 4°C. The tissues were then washed in a
quenching solution containing 1% glycine for 30 min at room temperature followed by a
reduction step with 1% sodium cyanoborohydride for 30 min (see Table 9 on Chapter
4). Follow by three washes in 50mM Tris-HCl solution (pH 7.5), containing 0.4 M NaCl,
prior to adding the cAMP antibody (Table 18).
Condition Target Site/Mechanism Vendor
RO-20-1724 (10µM) CAMP –dependent BIOM. phosphodiesterase ↓ RO + forskolin (0.01-10µM) Adenylyl cyclase ↑ Sigma RO + PGE2 (1-10 µM) secretomotor neurons ↑ Sigma RO + SP (1-5µM) Slow EPSP-mimetic ↑ Sigma RO + ATPγS (0.1-10µM) P2 receptor agonist ↑ Sigma RO + ATP (0.1- 50µM) P2 receptor agonist ↑ Sigma RO + CGRP (1-10µM) Slow EPSP-mimetic ↑ Sigma RO + 5HT (1-10µM) Sensory mediator from Sigma enterochromaffin cells ↑ RO + CGS 21680 (0.01-1µM) ±CSC (1-5µM) A2a ↑ / ↓ respectively Sigma (RO+ Forskolin) + High Mg2+ (10mM) / low Blocks synaptic transmission Sigma Ca2+ (0.5mM) (RO+ Forskolin)+ TTX (1 µM) Block axonal transmission Sigma
(Forskolin + R0) + Hexamethonium (100 µM) Nicotinic ganglionic blocker Sigma
Mecamylamine (75µM) Nicotinic ganglionic blocker Sigma CCPA (1µM) A1 receptor agonist ↑ N-phenylanthranilic acid (100µM) Chloride channel ↓ Sigma
↑activates (agonist), ↓ inhibits (antagonist)
TABLE 17. Drugs and Stimulation Parameters and Their Target Site of Action To be used for determining their actions on [cAMP] ir. 143
i. Cyclic AMP Immunofluorescence and colabeling in submucous neurons.
Prim. Host Type Source Dil Second. Ab Source Dil Ab cAMP rb poly Christofi 1:50- Biotin-SP-donkey anti- Jackson 1:400 1000 rabbit IgG (1:100) (+ Avidin-FITC) PGP9.5 ms mono Accurate 1:100 TR-horse anti-mouse IgG Vector 1:40- 1:200 VIP sp poly Chemicon 1:100 TR-donkey anti-sheep Jackson 1:40 IgG NPY sp poly Chemicon 1:50 TR-donkey anti-sheep Jackson 1:40 IgG nNOS sp poly Chemicon 1:50 Cy5-anti-sheep IgG Jackson 1:40 Abbreviations:sp, sheep; rb, rabbit; ms, mouse;TR, Texas Red; Ab, antibody; Dil, dilution.
Table 18. Characteristics of antibodies used in immunolocalization of cAMP.
5.3 RESULTS
5.3.1 Immunofluorescence (IF)
Unlike the myenteric plexus, the submucosal neurons do not display any specific polarity based on cAMP ir after uniform stimulation with forskolin and Ro-20 1724.
They project in the oral, distal, or circumferential directions. Based on immunofluorescence (IF) staining various morphological shapes of neurons are clearly visible such as Dogiel type II, Dogiel Type II/dendritic, Dogiel type I/filamentous, Dogiel type I/simple, Dogiel type I with Lamellar dendrites as well as clusters of neurons (Fig
35).
5.3.2 Immunohistorchemistry (IHC)
144 Figures 35, 36 (A-B), 37-39 (E-F) show examples of the various morphological
shapes of neurons displaying cAMP ir after uniform stimulation with 0.1µM forskolin
and 10 µM Ro-20 1724. In the intact submucous plexus uniform application of Ro-20
1724 (10µM) caused cAMP ir in 465 ± 60 neurons/cm2 when compared to basal levels
(Krebs) (Fig 40A). Forskolin (0.1µM) augmented this response by 273% when averaged
between three animals (Fig 40A). Random analysis of cAMP visualized submucosal
neurons from 3 different animals and similar stimulatory parameters as above, but using
an IHC technique revealed similar morphological groups when compared to IF staining
(Table 13 on page 114). A total of 3, 678 neurons/cm2 from three animals were cAMP ir
after uniform stimulation with forskolin (0.1µM) and Ro-20-1724 (10µM), but only 416
neurons/cm2 were analyzed since their shapes were clearly visible (not in clusters). Out of
the 416 neurons 29% of them were of Dogiel Type II (multipolar) and 71% were of
Dogiel Type I morphology (unipolar) (Figure 40B). Approximately 8% Dogiel Type II neurons contained dendritic processes, while another 21% of the population did not.
Dogiel Type I/Filamentous made up the largest group of uniaxonal neurons at 38%, followed by simple shape at approximately 25%. Uniaxonal neurons with lamellar dendrites made up the smallest group at 9% (Fig 40B).
5.3.3 Chemical coding of cAMP ir neurons
Pulled data from 3 different animals showed that uniform stimulation with forskolin and Ro-20-1724 for 30 minutes caused 48% of the PGP ir neurons to co-label for cAMP ir (Figs 41A-B, 42A). From 60 SMP ganglia randomly imaged and a total of
469 neurons carefully analyzed, approximately 64% of the VIP neurons were co-labeled
145 for cAMP ir (Figs 41 C-D, 42B). However, none of the NPY or nNOS ir neurons were ever co-labeled with cAMP ir (Figs 41 E-H, 42B).
146 Figure 35. cAMP immunofluorescent staining in SMP neurons. Visualization of cAMP immunofluorescence staining revealed Dogiel Type II (A-G), Dogiel Type II/Dendritic (H-I), Dogiel Type I/Filamentous (J-K), Dogiel Type I/Simple (L-M), Dogiel Type I/Lamellar (N-O) shapes and clusters (P-R) of SMP neurons after uniform stimulation with forskolin (0.1µM)and Ro-20- 1724(10µM) for 30 minutes. Scale bar = 30µm. 147 A
B
Figure 36. Multipolar cAMP ir neurons of the guinea-pig small intestine. (A) Dogiel/Type II and (B) Dogiel/Type II Dendritic SMP neurons visualized by cAMPir after uniform stimulation with forskolin (0.1µM) and Ro-20 1724 (10µM) for 30 minutes. Scale bar = 30µm.
148 Figure 37. Filamentous cAMP ir neurons. Dogiel/Type I Filamentous SMP neurons visualized by cAMPir after uniform stimulation with forskolin (0.1µM) and Ro-20 1724 (10µM) for 30 minutes. Scale bar = 30µm. Scale bar = 30µm.
149 Figure 38. Simple shape cAMP ir neurons. Dogiel/Type I Simple SMP neurons visualized by cAMPir after uniform stimulation with forskolin (0.1µM) and Ro-20 1724 (10µM) for 30 minutes. Scale bar = 30µm.
150 E
F
Figure 39. Lamellar and clusters of cAMP ir neurons. Dogiel Type I/ Lamellar SMP neurons (E) and a variety of clusters of SMP neurons (F) visualized by cAMPir after uniform stimulation with forskolin (0.1µM) and Ro-20 172-4 (10µM) for 30 minutes. Scale bar = 30µm.
151 A 2 2000 P < 0.0001 m /c 1800 ons 1600 1400 ed neur iz l 1200 1000 P < 0.0001
MP visua 800 A 600
er of c 400 b
m 200 Nu 0 Krebs RO-20-1724 RO-20-1724 + FSK
B 45
40
35 ized neurons l 30
25
20
15
10
5 Percentage of cAMP visua 0 ) ) II I (I & r l (I s ll I) lla ie c u a ( e g ti to Sm le m o ri en p a D d m im L en la S D Fi
MULTIPOLAR UNIPOLAR Filamentous > Dogiel I/small/simple = Dogiel II >> Dogiel I/Lamellar
Figure 40. Heterogeneity of cAMP visualized submucous neurons and quantitative analysis of cAMP responsive neurons. (A) Quantitation of SMP cAMP ir neurons comparing basal, phosphodiesterases inhibition and forskolin (FSK) stimulation. (B) Morphological categorization and their percentage of cAMP visualized neurons after stimulating intact SMP tissues with the adenylyl cyclase cocktail (Table 12). The cAMP antiserum was 1/50 and the secondary antiserum was 1/80 using the ABC method (1/100); see Table 14, Chapter 4. 152 A B
PGP cAMP CD
cAMP VIP EF
cAMP NPY GH
cAMP nNOS
Figure 41. cAMP immunoreactivity (ir) and chemical coding of SMP neurons. (A -D). Visualization of cAMP ir staining revealed that a subset of forskolin (0.1µM) and Ro -20-1724 (10µM) stimulated submucosal neurons showed co-labeling, (arrows) for cAMP, PGP, and VIP ir and the neurons that did not show co-localization are marked with the arrow heads. (E-H) Cyclic AMP ir was never colocalized in NPY or nNOS ir neurons, (short thick arrows) and visa versa NPY or nNOS ir neurons were never cAMP ir (curve arrows). SMP tissues were then fixed with acrolein and incubated 24-48 hours with anti-cAMP antibody (1/100-1/400) and PGP 9.5 (1/100) as outlined in methods. Confocal imaging was done to visualize neurons. Scale = 30µm. 153 2 A 3000 p<0.001
2500 ir neurons/cm 2000
1500
1000 MP and PGP
500 er of cA b 0
Num PGP RO+FSK N=3 N=17 NPY B 300
250 Neurons VIP
ctive 200
150 64%
100 nNOS
er of Immunorea 50 b 0% 0% -0- -0- 0 Num + + + + + + - Y P Y S S P + IP P P O O I IP V N M N N N /V V / / cA l / n n + l + + l ta + -/ P ta P P ta o P P o M M o T M M T A T M A cA cA c cA c
Figure 42. Quantitatiion and chemical coding of SMP neurons. (A) Quantitation of cyclic AMP ir in the intact submucosal plexus (SMP) in identified NPY, nNOS or VIP immunoreactive neurons. SMP neurons were stimulated with the adenylate cyclase cocktail (Table 12) to increase free intracellular cAMP levels. Tissues were fixed with acrolein and incubated with primary anti-cAMP and PGP 9.5 antibodies for 24-48 hours as describe in methods. Laser confocal imaging was used to count the cells. (B) Chemical coding of cAMP ir neurons after uniform stimulation with forskolin and Ro-20- 1724 (RO) for 30 minutes.
154 5.3.4 Adenosine effects on cAMP ir
Preincubation of SMP tissues with the A1 receptor (A1R) antagonist CPT (1µM)
for 20 minutes prior to stimulating with the phosphodiesterases inhibitor Ro-20 1724
(10µM) augmented the Ro-20 1724 response when compared to Krebs (basal) or Ro-20-
1724 alone (Fig 43A). On average from 2 different animals CPT increased the number of neurons/cm2 by nearly 1,269% when compared to Ro-20-1724 alone (Fig 43B).
Receptor activation with the A2a receptor agonist CGS 21680 (0.001-1µM) in the
presence of Ro-20 1724 (10µM) caused cAMP ir in approximately 65% of the forskolin
responsive neurons suggesting that the majority of SMP neurons expressing functional
adenylyl cyclase were coupled to an A2a receptor (Fig 44A). A2aR activation increased
the intensity and number of cAMP visualized neurons. Pre-incubation for 20 min with the
A2A receptor antagonist CSC (1.0µM) prior to stimulating with CGS 21680 and Ro-20-
1724 reduced approximately 45% of the CGS 21680 cAMP-dependent response in the intact submucous plexus (Fig 44A). Chemical coding of cAMP ir neurons after stimulation with the A2aR agonist CGS 21680 showed that approximately 31% of the
VIP ir neurons were co-localized with cAMP ir indicating that a minor subset of these neurons contained the A2a receptor coupled to adenlylate cyclase (Fig 44B). However, similar to forskolin stimulation, NPY ir neurons were never co-localized with cAMP ir neurons further suggesting that NPY neurons do not utilized cAMP (Fig 44B).
Comparing the distribution of cAMP ir neurons/cm2 between forskolin and receptor
stimulation suggest that a majority 65% of submucous neurons are responsive to the A2a
R agonist with a cAMPir increase (Fig 45).
155 A
B 2 700 p=0.0128
600
500 Neurons/cm
r 400
300 p=0.0224
200
100
Number of cAMP i 0 s O T eb R P r C K + O R
Figure 43. Blockade of adenosine A1 receptors augments cAMP responses in submucous neurons of the guinea-pig small intestine. (A) Visualization of cAMP ir staining in the intact SMP after A1R inhibition with CPT (1µM) and the phosphodiesterase inhibitor Ro-20-1724 (10uM). Scale bar = 30µm. (B) Quantitation of the number of cAMP ir neurons after blockade of the A1R with CPT in the intact SMP plexus.
156 A 1000 p=0.001 2 p=0.002
750 Neurons/cm r i
P 500
250 Number of cAM -0- 0 N= 6 6 5 20 RO+FSK RO+CGS RO+CSC+CGS RO B 12 0 NPY VIP
10 0
80 noreactive Neurons 60
40
20
-0-
Number of Immu 0 cAMP+ NPY+ cAMP+ cAMP+/ VIP+ cAMP+ / NPY+ alone alone VIP+ Total alone
Figure 44. Quantitation and chemical coding of cAMP ir SMP neurons. (A) Comparing the number of cAMP ir neurons between nonspecific adenylyl cyclase activator (forskolin) and receptor activation of SMP neurons with the Adenosine A2A R agonist and antagonist. The A2a effect is sensitive to CSC A2a antagonist. {Graphs include pulled data from both IH and IF techniques.} (B) Chemical coding of the type of SMP neurons that expressed CGS 21680/CSC functional A2A receptors coupled to Gs/AC stimulation of intraneuronal cAMP levels. For details on IH and IF see Tables 11 (Chapter 4) and Table 15.
157 200 * 180 2 RO+ Forskolin 160 * RO+CGS21680 140
120 * * 100
80
Number o f ganglia/cm 60 * * 40 20 * 0 12345678910111213 Number of neurons
Figure 45. A subset of submucous neurons that generate cAMP do so via A2a receptor activation. Comparison of the distribution of cAMP ir neurons/ganglia/cm2 that display A2a receptors with more generalized stimulation with forskolin. This figure only includes pulled data from IF techniques. IF details are describe in Table 15. * P< 0.01, n = 3 animals.
Figure 46. Compartmentalization of cAMP ir. Receptor activation with the A2a agonist CGS 21680 (0.1µM) caused cAMP ir in a Dogiel Type I/ Filamentous neuron localized to the left side of the cells (arrows). Scale bar = 30µm. 158 Short exposures to forskolin without any PDE inhibitors did not reveal any discrete
localizations of cAMP-IR, whereas, activation of A2aRs for a brief period (i.e. 10s, 30
sec, 1min or 2min, 10-6 or 10-8M) causes a localized cAMP response in specific regions of the neuron (Fig 46 above).
5.3.5 Cyclic AMP drives cell-to-cell communication in SMP neurons
Cyclic AMP is involved in cell-to-cell communication since both CCPA (A1 receptor agonist) and hexamethonium (nicotinic blocker) were able to reduce the forskolin response by 57% and 43% respectively (Figs 47A-B). It also indicates that cAMPir reflects electrical activity in enteric neurons since axonal and synaptic blockade with TTX (0.5µM) or high Mg2+ / low Ca2+ Krebs reduced the number of cAMP ir neurons by 50% of the forskolin response (Figs 47 C-D).
5.3.6 Short circuit current (Isc)
Electrical field stimulation (EFS) of submucous neurons in 2 mucosa –
submucosa preparations elevated cAMP ir in 300 ± 48 neurons / cm2. Forskolin and EFS
elicited a similar concomitant increase in short-circuit current (Isc). The increase in Isc
induced by forskolin stimulation was reduced to 66% by TTX (0.2µM), to 40% by
mecamylamine (75µM), to 55% by CCPA (1µM) and to 60-70% with the chloride
channer blocker n-phenylanthranilic acid (100 µM) (Fig 48A).
159 AB p = 0.002, N=3 p = 0.005, N=5
800 800 Bb Interneuron Interneuron Ach 2 Aa 2 AC N AC
A1 X 600 AC AC 600 ons/cm HEX 43%
57% neur P 400 400
200 200 Number of cAMP neurons/cm Number of cAM 0 0 F A F + P + EX O C O H R +C R + 2 CD2 p = 0.022, N=4 p = 0.025, N=4 1000 1000 ons/cm 800 Cc 800 Dd
X neur P 600 600 44% 60% of cAM 400 400 er m Nub Number of cAMP neurons/cm 200 200
0 0 2+ / F a 2+ F X C O+ TT O+ g R + R ow M L h ig H
Figure 47. Effects of forskolin on short circuit current (Isc) and synaptic blockage. Forskolin evoked cyclic AMP-dependent communication between submucosal neurons was reduced by CCPA (A1 receptor agonist), tetrodotoxin (TTX), hexamethonium (nicotinic blocker) and low Ca2+/ High Mg2+. See materials and methods for concentrations of drugs (Table14) and fixation procedures and antibody dilutions (Table 15). CCPA binds to receptors located both in the cell soma and presynapticaly (Aa). Hexamethonium blocks nicotinic receptors located postsynaptically (Bb). The final effect of both TTX (sodium channel blocker), Low Ca2+/High Mg2+ and A1 activation is to block release of neurotransmitter presynaptically as illustrated in Figures Cc and Dd. 160 A Forskolin 10 µM P 11 µA/cm2 t1/2 2.7 min CCPA 1µM P 29 µA/cm2
t1/2 0.3 min
VEH P 31 µA/cm2
t1/2 3.4 min
TTX 0.2µM 2.5 min P 61 µA/cm2 t1/2 1.7 min
VEH P 18 µA/cm2
t1/2 11 min MEC P 29 µA/cm2
t1/2 0.6 min
VEH 2.5 min
BC 40 10.0 *
) *
2 30
) 7.5 * m
* n i c A/ (m
µ 20 x ( * * 5.0 ma Isc T
∆ * 10 2.5
0 CCPA Cl CHANNEL 0.0 CONTROL MEC TTX CONTROL CCPA Meca TTX (1µM) (75µM) (0.2µM) (100 µM) (1µM) (75µM) (0.2µM) Forskolin(1µM) Forskolin(1µM)
Figure 48. Forskolin (10µM) caused an increase in short-circuit current (Isc). (A) The increase in Isc induced by forskolin stimulation was reduced by TTX (0.2µM), mecamylamine (75µM), CCPA (1.0 µM) and n-phenylanthranilic acid (100µM). (B) CCPA, mecamylamine, and TTX reduced the time to reach maximum (Tmax) Isc. * , P<0.05.
161 5.4 DISCUSSION
Earlier electrophysiology work suggested that all AH/Dogiel Type II neurons
(AH neurons) and most S/Dogiel Type I neurons (S neurons) in the SMP were
depolarized by forskolin stimulation [103]. Later it was shown using Bodipy Forskolin
that adenylyl cyclase was present in neurons other than AH confirming these earlier
recordings in the SMP [74]. Our study using an acrolein derivatized cAMP antiserum
further confirms this earlier work and showed that both AH and S neurons display cAMP
ir after inhibition with either Ro-20-1724 (Fig 40A) or rolipram (data not shown); both
selective inhibitors of phosphodiesterase type 4 activity. This was not the case for the
myenteric plexus, which showed little or no response to these inhibitors.
These inhibitors are selective for PDE type IV and therefore, under physiological
conditions, intraneuronal cAMP levels in submucous neurons are strongly regulated by
the PDE isoform. The basal cAMP response in myenteric neurons is not modulated by
PDE IV. It is possible that cAMP signaling in myenteric neurons is modulated by
different PDE isoforms, which were insensitive to Ro-20-1724 or rolipram. Recent
studies have shown that different phosphodiesterase isoforms are strategically located in
specific regions of the brain to mediate and regulate synaptic transmission [159].
Forskolin augmented the Ro-20-1724 response by 285% which is consistent with the
myenteric plexus (Chapter 4, Fig 31), but the number of neurons displaying cAMP ir
was approximately 3 fold higher in the SMP, since the LMMP contains 3X less
neurons/cm2 when compared to the myenteric plexus in the guinea pig ileum (Fig 40A)
[321]. This means that in the submucous plexus, cAMP signaling plays a more prominent
162 role than in myenteric plexus, and such signaling is tightly regulated by PDE IV. Very
different roles are emerging for cAMP in the two plexuses.
Based on cAMP ir visualization, multipolar neurons make up approximately 28% of all SMP while uniaxonal neurons make up about 72% of the remaining response (Fig
40B), which is different from the myenteric plexus, where only 18% of multipolar
(Chapter 4, Table 13) neurons display cAMP ir after forskolin stimulation suggesting that cAMP signaling might be more important in multipolar neurons within the SMP. A recent study showed that Dogiel Type II neurons or IPANs in the SMP are different from
those in the myenteric plexus since only 10% of them labeled for the calcium binding
protein (calbindin), a marker for these neurons suggesting that only a subset of Dogiel
Type II neurons are accounted by this chemical marker in the SMP [322]. Furthermore a
subset of these calbindin ir neurons were also calretinin ir further providing evidence that
IPANs in the SMP are chemically and therefore likety to be functionally very different
from those in the myenteric plexus since this co-localization had never been observed in
the enteric nervous system [322]. Some of the cAMP ir neurons with filamentous
morphology could be a subset of the VIP secretomotor neurons since these neurons have
been described as filamentous in shape in the SMP plexus [317]. The small shape or
lamellar neurons expressing cAMP ir could represent the secretomotor/vasomotor
calretinin ir neurons since these neurons have been described as having similar shapes as
this study [323]. However unlike the myenteric plexus there was no polarity expressed in
the SMP suggesting that cAMP plays a more ubiquitious role within this plexus unlike
the myenteric plexus, where cAMP functions more discretely in descending reflex
pathways.
163 Since 64% of the VIP-ergic neurons can generate cAMP it suggests that cAMP
signaling is very important in the function of these secretomotor neurons (Fig 41). This also implies that VIP/secretomotor neurons can be further subcategorized into cAMP- dependent secretomotor neurons and cAMP-independent secretomotor neurons. Since
100% of all VIP ir neurons are also cGMP ir [95] it can be deduced tht a majority of VIP secretomotor neurons are regulated by the activity of both AC and guanylate cyclase
(GC). Such cross-talk between cAMP and cGMP does occur – cGMP provides an ongoing inhibition of AC/cAMP signaling in submucous neurons. Suppression of GC activity with ODQ eliminates the inhibitory influence and reveals a more robust cAMP response.
The implication of such negative cross-talk between cAMP and cGMP is in the
modulation of neurosecretion in the gut. These second messengers provide a unique
mechanism of neural regulation not shared by myenteric neurons. The functional
relevance of cross-talk remains to be confirmed in Ussing chamber short-circuit
current/Isc studies.
Adenosine A1 and A2aRs are often coupled to inhibition or stimulation of
AC/cAMP signaling in neurons and other cell types respectively [324]. In the ENS, A2aR
activation mimics slow synaptic transmission in both nerve plexuses, suggesting a
possible cAMP dependent mechanism. In fact, the slow EPSP-like response to 2-
chloradenosine in submucous neurons is sensitive to PKA inhibition and therefore may
involve cAMP [320]. In the myenteric plexus, A2aR activation in a subset of AH neurons
leads to enhanced excitability [70, 71]. We show in this study that CGS 21680 causes a
rise in cAMPir in 65% of possible cAMP-responsive submucous neurons (Fig 42A). The
164 response was sensitive to blockade by the A2aR antagonist CSC indicating that A2aR activation leads to the cAMP response in the neurons (Fig 44A). Therefore, as predicted from electrophysiological studies, our cAMP visualization data provide unequivocal proof that A2aR activation of AC leads to a rise in intracellular cAMP that underlies the slow EPSP-like response observed in S/Type I neurons of the submucous plexus. This is a significant finding on purinergic signaling in neurons because it is the first time that unequivocal proof has been obtained linking A2aR/AC/cAMP signaling and elevation of intraneuronal free cAMP in the cytoplasm of an intact neuron. All previous studies in
CNS or other tissues relied on isolated membrane preparations (contaminated by glial component) and cAMP content after providing the ATP substrate to the assay. In our study, we show that intraneuronal cAMP increases in response to A2aR activation in morphologically identified and visualized neurons. This technique should prove useful in direct CNS studies on A2a, A2b, A3, and A1 – AC coupling in neurons.
A subset of VIP secretomotor neurons contain functional A2a receptors (Fig 44A) making up 31% of VIPir neurons. This represents ~ 50% of VIP secretomotor neurons that can generate cAMP (in response to forskolin). Thus, VIP secretomotor neurons can be further subdivided according to those with or without functional A2aRs. Our novel finding is that the activity of subsets of VIP secretomotor neurons is differentially regulated and distinguished by AC/cAMP signaling, cAMP/cGMP negative cross talk and A2aR activation. Previous studies in the CNS have shown that A2A activation causes VIP neurotransmitter release in subset of VIP ir neurons [325], therefore our data suggest that a similar mechanism may exist in the ENS. Comparing the distribution of cAMP visualized neurons/ganglion/cm2 indicates that neurons containing A2aRs are 165 found in subsets of ganglia of variable size (3-10 neurons/ganglion) that can generate
cAMP in response to forskolin. A2aR activation did not lead to cAMP ir in NPY
secretomotor neurons, which is consistent with forskolin stimulation of the SMP where
NPY neurons were never co-localized with cAMP ir (Figs 42B & 44B) further
supporting that A2a receptors are coupled to AC only to specific functional groups of
neurons within the SMP.
Since approximately 60% of all possible SMP neurons expressed cAMP ir to
forskolin stimulation suggest that nearly 39% of all possible submucous neurons
expressed A2a receptors implying that coupling of this receptor to AC is critical in
submucosal neurons. Interestingly short exposures to forskolin without any PDE
inhibitors did not reveal any discrete localizations of cAMP-IR, whereas, activation of
A2aRs for a brief period (i.e. 10s, 30 sec, 1min or 2min, 10-6 or 10-8M) causes a localized cAMP response in specific regions of the neuron (Fig 46). This is consistent with discrete localization of A2aRs on cell somal membranes near the sites of activation of AC leading to localized elevation of cAMP ir in the neurons [326]. The forskolin response was less discrete, because AC isoforms are expressed throughout the cell and general AC activation with forskolin raises cAMP ir everywhere in the cell. Compartmentalization into cAMP microdomains occurs in cardiac myocytes [155, 202] and many other cell types [202]. The role of PDEs has been delineated with the use of fluorescent indicators that permit real-time measurements of cAMP [327-330].
Endogenous adenosine provides an ongoing inhibitory tone on excitability and
synaptic transmission in myenteric neurons [70]. In the submucous plexus, endogenous
adenosine provides an inhibitory tone on both the 5-HT and prostaglandin limbs of the 166 stroking/mechanically – evoked neural reflex and chloride secretory response [285, 286].
A potential site of action of endogenous adenosine is AC/cAMP signaling in enteric neurons. A1Rs are found on AH/IPANs and at presynaptic sites in submucous neurons
[70, 293]. In fact, the A1 antagonist CPT augmented cAMP ir in both myenteric (data not shown) and submucous plexuses (Figs 43A, B) providing unequivocal proof for this notion. However, the effect of CPT was several fold greater in submucous than myenteric neurons, indicating that endogenous adenosine exerts a much greater A1 inhibitory modulation of AC/cAMP signaling in the resting state in the submucous plexus than myenteric plexus. The A1R agonist reduced the facilitation or amplication of cAMP synaptic transmission suggesting that cAMP is involve in driving cell to cell communication. Based on our data and previous studies that showed that presynaptic inhibition with A1 agonist (CCPA) reduces the cholinergic response in S neurons further supports the existence of interneurons in the SMP [72]. The possibility of polysynaptic hardwiring in the SMP has already been hypothesized based on dual electrical recording in the SMP plexus [101].
Results from indirect short circuit studies had indicated that cyclic AMP dependent neural secretion occurred in the intestine via activation of AC/cAMP signaling in submucous neurons and this was mediated by VIP and NPY secretomotor neurons
[283, 284]. Based our experiments above where forkolin activated only VIP-ergic neurons (Fig 44B) but not NPY neurons it is possible that a component of Isc circuit is driven by cAMP. Since several synaptic blockers, the adenosine A1 receptor agonist
(CCPA), nicotinic blocker (Hexamethonium), a Na+ channel and nerve conductance
167 blocker (TTX) and synaptic blockade with low Ca2+/high Mg2+ reduced the number of cAMP ir neurons and the cAMP response to forskolin it is suggestive that cAMP is involved in mediating cell-to-cell communication in the SMP (Fig 47). In a different
experiment CCPA, TTX or the nicotinic blocker (mecamylamine) was able to reduce the
forskolin stimuated Isc response, further strengthening the hypothesis that cAMP is
involved in driving synaptic transmission within the SMP either directly or indirectly.
Our findings in submucous neurons are suggestive of the existence of interneurons in the integrative circuit of the submucous plexus. A hypothetical scheme for these polysynaptic pathways for cAMP dependent signaling is shown in Fig 49.
Therefore, one possibility that deserves further consideration is the negative influence of cGMP (and A1 inhibition of AC/cAMP signaling) in the regulation of cAMP dependent excitability of submucous neurons, and cAMP dependent neural-secretion in response to mechano- or –chemosensitive stimulation [285, 286].
Conclusions
Cyclic AMP visualization in the submucous plexus identified sensory and
secretomotor neurons that generate cAMP. A1 and A2aRs are negatively or positively
coupled to AC/cAMP signaling. In contrast to MP, cAMP-dependent submucous
neurons do not have polarized projections. Our findings suggest a prominent role for
cAMP signaling in polysynaptic pathways that drive neurosecretion.
168 AH 4
AH VIP/S 2 Slow EPSP/AC 1 3 response
Nicotinic receptors A1 receptors High Mg2+/low Ca2+ Sodium channel blocker (TTX)
Figure 49. A hypothetical model to show that interneurons are likely to exist in the SMP and how various synaptic blockers can influence cAMP cell to cell communication in this plexus. Forkolin can directly activate either an IPAN (cell 1) or a secretomotor neuron (cell 3). In the absence of any blockers the activated IPAN (cell 1) can release neurotransmitter to activate an interneuron (cell 2), does not contain AC) and this cell then releases transmitter to further stimulate the secretomotor neuron (cell 3) which enhances cAMP ir. In the presence of the A1 agonist CCPA which acts at the cell body of the IPAN (blue dots) or at presynaptic sites at varicosities (blue dots) to prevent neurotransmitter release and thereby reducing the influence of the interneuron (cell 2) on the secretomotor neuron (3). The same principal is likely to occur with High Mg2+/low Ca2+ since it too prevents neurotransmitter release. The presence of a nicotinic blocker (Hexamethonium) blocks the effects of the IPAN (cell 1) on the S/Type 1 interneuron that receives fast nicotinic synaptic inputs and therefore reducing its cAMP responses on the secretomotor neuron. The Na+ channel blocker (TTX ) blocks all neurotransmitter release from the IPAN, the interneuron and the secretomotor neurons so any cAMP ir observed was due to direct effects of forskolin in the neuron. These blockers prevent facilitation of cAMP-dependent synaptic transmissions in polysynaptic pathways.
169
CHAPTER 6
AMPLIFICATION OF AC/cAMP SIGNALING IN MYENTERIC NEURONS
DURING TRICHINELLA SPIRALIS INDUCED ACUTE INFLAMMATION
6.1 INTRODUCTION
Enteric neural reflexes are triggered by release of 5-HT or other sensory mediators [331] from chemo- and mechanosensitive enterochromaffin cells (EC) residing in the epithelium. 5-HT activates intrinsic primary afferent neurons (IPANs) in the enteric nervous system (ENS) to initiate reflexes resulting in coordination of motility and secretion. In the guinea-pig small intestine, myenteric AH neurons function as IPANS.
Synaptic communication between AH neurons are via slow excitatory postsynaptic potentials (slow EPSPs), and they have been suggested to form self-reinforcing networks for feed-forward excitation in the ENS for initiation of reflexes. AH neurons display
Sustained Excitatory Postsynaptic Potentials (SSEP) evoked with low frequency stimulation (1Hz) that can last several hours. The SSEP is the only known physiologic mechanism for inducing hyperexcitability in the ENS [260].
Enteric parasitism has a direct impact on nerve cell function and neurotransmission pathways in both the ENS and the CNS [231, 273, 300]. Infection with
Trichinella spiralis nematode leads to acute inflammation in the jejunum associated with neuronal plasticity in both neurotransmitter content and function, hyperexcitability in AH
170 neurons, facilitation of nicotinic synaptic transmissions in S neurons, and long-term
changes in gene regulation [231, 273] . The only intracellular recording study done in T.
spiralis infected gut [231, 273] focused primarily on AH neurons and the morphology of
recorded neurons was not identified by dye filling of cells. Therefore, it remains
unresolved whether the excitability of S neurons which represent various classes of
interneurons or motor neurons in addition to AH / IPANs is also enhanced in T. spiralis
inflamed gut.
The hyperexcitability in myenteric AH/ IPANs during acute infection with T.
spiralis [273, 300] is very similar to that observed in sensory AH neurons of Aplysia
[332]. All changes in electrical properties observed in AH neurons in infected gut are mimicked by interventions known to elevate intraneuronal cAMP levels [16]. This suggests that up-regulation in the AC / cAMP signaling pathway during acute inflammation may lead to AH cell hyperexcitability.
Substantial expression of the immediate early gene product c-fos occurs in AH
neurons during the acute phase of infection with T. spiralis [231, 273]. In normal uninfected guinea pigs, AH cells do not express c-fos when activated by forskolin, distention or peristalsis, but other cell types do [333]. The role of AC / cAMP up- regulation during acute T. spiralis infection in induction of c-fos or other nuclear events is unknown, but evidence from many other cell types including neurons supports the hypothesis that alterations in the AC / cAMP signaling cascade leads to transcriptional regulation via CREB phosphorylation and expression of c-fos and AP-1 transcription
[327-329].
171 Adenosine 3' 5'-cyclic monophosphate (cAMP) is suggested to be a key mediator
of slow synaptic sensory transmission in AH neurons of the ENS [26, 39, 63, 334]. A primary transmitter for the slow EPSP is a tachykinin (SP) [260], although there are many other candidates that mimic slow EPSPs that include gut neuropeptides (CGRP,
PACAP, VIP, GRP), immune mediators (histamine, PGE2), monoamines (5-HT) and
purines (ATP, adenosine) [16]. Slow EPSPs in AH neurons are likely to be mediated by
both cAMP [26, 39, 63, 334] and PKC-dependent pathways [265, 316].
It has been suggested that, in the myenteric plexus of the guinea pig small intestine, only AH neurons display slow excitatory responses to forskolin and hence cAMP responses [16, 39]. Other studies with fluorescent binding of Bodipy forskolin[74] or AC immunoreactivity suggest that cAMP signaling is not restricted to this cell phenotype[73]. Our recent findings using a new acrolein-derivatized cAMP antiserum to visualize the shapes and projections of cAMP-responsive neurons with acute exposure to forskolin strongly support other cell phenotypes in cAMP signaling (see
Chapters 4 and 5).
Mast cells lie in close proximity to nerves in rodent and human gut [335] and endogenous histamine or other mast cell mediators may be an important contributor to the hyperexcitability in AH neurons observed in T. spiralis infection as well.
The hyperexcitability in enteric neurons after T. spiralis infection may be
attributed in part to ongoing release of inflammatory mediators such as histamine,
prostaglandins, leukotrienes or 5-HT or various neurotransmitters that are known to act
through the AC/cAMP signaling pathway [16, 26, 39, 63, 260, 273, 334]. In addition to a
direct influence and ongoing activation of AC/cAMP signaling, it is possible that they
172 can induce long-term changes in transcriptional regulation and gene expression culminating in the persistent hyperexcitability observed in enteric neurons to enhance sensitivity of the postsynaptic neuronal membrane in AH cells to these mediators.
To date, little is known about cAMP signal transduction pathways in disease states of the gut [231, 270] or the classes of neurons that utilize cAMP as a second messenger. Our approach targets a main second messenger for slow synaptic transmission that is of basic fundamental importance in sensory neurotransmission in enteric neural reflexes. Study of, and alterations in, the AC / cAMP signal transduction in AH / IPANs and other cell phenotypes provides a common target shared by many transmitters, paracrine or immune mediators in the integrated neural circuits of the gut.
The primary aim of this study was to test the specific hypothesis that neuronal plasticity in myenteric neurons of the Trichinella spiralis – inflamed jejunum results from amplification in the adenylyl cyclase / cAMP signaling pathway. Studies were done in jejunum of guinea-pigs infected with T. spiralis muscle-stage larvae, and all analysis was performed at the peak of acute inflammation on day 6-8 post-infection with the nematode. A secondary aim was to determine the contribution of immune/inflammatory mediators like histamine to AH cell hyperexcitability in acute inflammation, since they can potentially excite these neurons via a cAMP-dependent mechanism. Studies involved localization and expression of AC ir in calbindin-D28 (AH) or other cell phenotypes, immune / neural modulation of cAMP production in isolated myenteric ganglia, cAMP- dependent nuclear CREB phosphorylation and electrophysiology. This report establishes a key role for the second messenger cAMP in neuronal plasticity and functional
173 disturbances in the ENS during acute intestinal inflammation with T. spiralis nematode infection.
This study is being revised for resubmission and publication in the
prestigious journal of Gastroenterology.
6.2 METHODS AND PROCEDURES
Animals and induction of inflammation. All procedures involving the use of live
guinea-pigs for these studies were reviewed and approved by the Institutional Animal
Care and Use Committee of The Ohio State University (Columbus, OH). Male Hartley
strain guinea pigs, Cavia procellus (Harland Sprague Dawley, Indianapolis, IN) weighing
300g – 500 g served as host animals and were orally inoculated with 8 – 9 x 103 T. spiralis muscle-stage larvae that had been isolated using an established protocol [273] and were administered by the oropharyngeal route via a feeding tube in a 0.2 ml 0.85% saline vehicle bolus. Other guinea-pigs served as age-matched controls and received 0.2 ml of saline without the worms.
Microdissection and tissue preparation. Whole mounts of the longitudinal muscle layer and adherent myenteric plexus ganglia (LMMP) were prepared from age-matched control guinea-pigs and T. spiralis infected guinea pigs on days 6-8 postinfection (PI) that were sacrificed by a stunning blow to the head and subsequent exsanguinations [273].
Jejunal LMMP preparations were utilized for intracellular electrophysiological experiments and immunochemical co-labeling studies.
174 6.2.1 Immunofluorescent co-labeling studies and Laser Scanning Confocal
Imaging
Primary antibodies: 1) AH/ IPANs were identified using a monoclonal calbindin-D28 antiserum (chicken gut antigen, Sigma, 1:100 dilution). 2) S/Type 1 neurons (belonging to several classes of cholinergic neurons) were identified with a goat polyclonal calretinin antiserum (guinea-pig antigen, Chemicon, 1:100 dilution) [15]. 3) AC expression was identified using a rabbit polyclonal AC antiserum that does not discriminate between various AC isoforms (R-32, rat antigen, Santa Cruz, 1:100 dilution). 4) Nuclear CREB phosphorylation (pCREB) was identified using a rabbit polyclonal pCREB antiserum (rat antigen, Upstate, 1:250 dilution). 5) Glial cells were identified using a monoclonal antibody to s-100 protein (bovine antigen, Biogenex, 1:100 dilution).
Secondary antibodies: Texas Red or fluorescein conjugated secondary IgG antibodies
(anti-mouse IgG, anti-rabbit IgG or anti-goat IgG) were purchased from Vector Lab
(Burlingame, CA) or Jackson ImmunoResearch Laboratories (West Grove, PA) and used at 1:50 to 1:100 dilution.
Details of immunofluorescent labeling techniques and incubations with primary or secondary antibodies are as previously [73, 74]. Briefly, primary antibodies were incubated for 24-48 hours at 4ºC and secondary antibodies were incubated for 3-4 hours at room temperature. Image analysis was done using the LSM 410 Zeiss Confocal
Imaging system. LSM dual imaging was carried out for FITC/Texas Red fluorescence of secondary IgG antibodies tagged with fluorescein or Texas Red are described in Liu and co-workers [73]. Images were captured as single or dual RGB images and saved as .tiff
175 images. LSM images represented an average of 2-4 images; optical sections were 0.7 –
4.7 µm thick, but all comparisons were made at the same optical thickness.
6.2.2 Analysis of cAMP production in isolated myenteric ganglia
Myenteric ganglia from LMMP tissues from the entire jejunum were
enzymatically isolated according to established methodologies [334]. The effect of
inflammation on cAMP content was determined in 200 ganglia / tube suspended in Krebs
buffer (pH 7.4) pre-equilibrated at 37ºC and oxygenated with 95% O2 / 5% CO2 for 45 min. The cAMP response in myenteric ganglia was compared between age-matched controls and T. spiralis infected guinea-pigs under baseline conditions (Krebs buffer) or after 30 min treatment with the cAMP-dependent phosphodiesterase inhibitor (PDEI) Ro-
20-1724, the AC activator forskolin, the mast cell mediator histamine, or the neurotransmitter SP. Cyclic AMP content was determined by ELISA (Cayman Chemical,
Ann Arbor, MI) and responses were normalized to equal amounts of protein / tube to account for variability in the size of ganglia harvested in each tube (i.e. 200 ganglia/tube).
6.2.3 Western blot analysis
LMMP tissues or isolated myenteric ganglia were frozen in liquid nitrogen for western blot analysis. The frozen specimens were homogenized and centrifuged (3,000 g for 5 min at 4ºC), the pellets were resuspended in lysis buffer [275]. The resuspended pellets were incubated for 30 min on ice, sonicated and the insoluble material was removed by centrifugation (10,000 g for 3 min at 4ºC). Protein concentration of each well was determined (Bio-Rad kit). Ten to 50µg of solubilized proteins were separated by
10% denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes according to published techniques. 176 Immunopositive bands were visualized with the ECL kit (Amersham, Piscataway, NJ).
The protein bands visualized on Hyperfilm ECL (Amersham, Piscataway, NJ) were scanned and the intensity of each band was measured by ScanWizard 5 software
(Microtek, Redondo Beach, CA). The primary antibodies were used at a dilution of
1:300-1000 and the secondary antibodies were diluted 1:2000-1:3000 (Jackson
ImmunoResearch Laboratories, West Grove, PA).
6.2.4 Electrophysiology
Electrophysiological and pharmacological methods. Conventional sharp-tip intracellular microelectrode recordings were made from AH neurons in jejunal LMMP preparations from T. spiralis infected guinea-pigs or age-matched controls on days 6-8 post-infection (PI) [273]. The microelectrode (with direct current resistance of 70-90
MΩ) was filled with 3 M KCl solution and coupled to an electronic amplifier (Intra 767,
World Precision Instruments, Sarasota, FL) that was equipped with a bridge circuit for simultaneous injection of electrical currents into the cell while recording the transmembrane electrotonic responses in myenteric neurons. After impalement and stabilization of resting membrane potential, neurons were classified according to their active and passive electrical membrane properties as AH or S neurons [26, 273, 334].
This study and our analysis focused entirely on AH neurons.
Many of the electrical properties of AH cells used to classify them are altered in inflammation. However, certain hallmark properties of AH cells are sufficiently retained making it feasible to identify them during study. The following criteria were used in AH cell classification: 1) Duration and size of the AHP during threshold stimulation that
177 produces a single action potential (inflamed ≥ 1.0 sec, normal ~ 7.0 -12.0 sec). 2)
Summation of the AHP during incrementing levels of depolarizing current pulses (0.1nA-
0.5nA) that evoke multiple action potentials. 3) Distinguishing sag in the hyperpolarizing potential. 4) Ca2+ shoulder / hamp in the action potential during high sweep. 5) TTX-
insensitive component to the action potential. 6) TTX-insensitive component of
spontaneous APs in T. spiralis inflamed gut [332]. The occurrence or absence of fast
EPSPs is not a reliable criterion since AH neurons in inflamed gut also receive prominent
fast EPSPs [273].
The cell resting membrane potential, cell input resistance, spontaneous action
potential (AP) discharge, anodal-break discharge and accommodation at threshold
(number of APs per current pulse) were compared between age-matched controls and T.
spiralis infected gut for various pharmacological interventions. Data was stored and
analyzed using a MacLab 4-s data acquisition and analysis system (AD Instruments,
Milford, MA) integrated on a Macintosh Power PC computer.
The tissue was first equilibrated for 45 min in Krebs buffer bubbled with
95%O2/5%CO2 at 37ºC. Test agents (cyclooxygenase inhibitor, histamine blockers, leukotriene blockers) for pharmacological studies of responses in AH neurons were delivered to the tissue chamber by addition of the substance to the perfusing Krebs solution. To test effects of inhibitors or receptor blockers, the excitability characteristics of AH neurons were assessed before, after a 30 min drug exposure in the superfusion solution and following washout / recovery from the drug.
Krebs solution was of the following composition (in mM): NaCl (120), KCl (5),
CaCl2 (2.5), MgCl2 (1.2); NaH2P04 (1.35), NaHCO3 (14.4) and glucose (11.5).
178 6.2.5 Tissue preparation for biochemical analysis.
About 15 cm of jejunum [273] was removed from each guinea-pig immediately
after being euthanized and rinsed free of luminal contents with an ice-cold Kreb’s
solution. The jejunum was cut open along the mesenteric border and pinned to a Sylgard
dish for removal of the mucosa by scraping; approximately 0.5g for each sample were
frozen at -70°C in liquid nitrogen for later analysis. Mucosa was homogenized with a
Brinkman Polytron in a sufficient volume of ice-cold 50 mM potassium phosphate buffer,
pH 7.4, to produce a 20% homogenate for analysis of biochemical markers.
Analysis and assays on mucosal homogenates and plasma were done as described
previously[336]. The activity of myeloperoxidase (MPO) in sonicated whole mucosa
homogenates was carried out by using an assay kit (R&D Systems, Minneapolis, MN,
USA). Tumor necrosis factor alpha (TNF-α leukotriene B4 (LTB4), thromboxane B2
(TXB2) and prostaglandin E2 (PGE2) were measured using a specific enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA). Determination of lipid peroxidation was carried out by using an assay kit (R&D Systems, Minneapolis, MN,
USA). This procedure allows for the measurement of malondialdehyde (MDA) and 4- hydroxynonenal (4-HNE) concentrations. Results are expressed as means ± SEM
obtained from four separate animals in each group.
Data analysis and Statistics
All data are expressed as means ± standard errors of the means (SEM). Statistical
significance was evaluated by paired or unpaired Student’s t-test or ANOVA with
Bonferroni’s multiple comparison posthoc test or Fisher exact test, depending on
179 experimental design. A p value of < 0.05 was considered significant. Additional details of
specific analyses are described under appropriate sections.
6.3 RESULTS
A total of 41 animals were used for biochemical, immunochemical and cAMP
studies. An additional 39 animals were used for electrophysiological studies.
Markers of neutrophilic infiltration (MPO), mast cells (mast cell tryptase),
inflammation/cycloooxygenase (LTB4, PGE2 and TXB2) or cytokine pathways (TNF-α) were significantly elevated in mucosa and plasma of T. spiralis infected guinea pigs with some exceptions (Table 19). MPO increased by 209%, COX metabolites by 150%
(PGE2) or 262% LTB4. A 7570% increase in mucosal TNFα occurred with nematode
infection but was undetectable in plasma. No change in lipid peroxidation products were
observed in the mucosa. Plasma levels of TXB2 (and LTB4) were also elevated by
1100%. The findings stress the multiplicity of signaling pathways contributing to acute inflammation in the T. spiralis model, and stress the importance of mast cells/mediators and TNFα.
180
Sample Normal Inflamed P values % Increase Mucosa
MPO (ng/mg DNA) 11.0±2.8 34.0±3.5 .0012* 209
Mast cell tryptase 0.02±0.005 0.34±0.04 .0016* 1,600 (abs/mg DNA)
LTB4 (ng/ug DNA) 0.21±0.004 0.76±12.0 .0028* 262
PGE2 (ng/mg DNA) 27.82±3.45 70.46±12.8 .0100* 150
TNF-α (pg/ml) 0.43±0.10 33.39±6.37 .0001* 7,574
Lipid peroxidation 0.058±0.005 0.05±0.004 >.05 none (abs/mg DNA)
Plasma
TXB2 (pg/ml) 15.2±5.06 179.9±27.47 .0034* 1,100
PGE2 and TNFα were not detectable in plasma.
Table 19. Increase in inflammatory markers in the jejunal mucosa and plasma of guinea- pigs infected with T. spiralis during the peak of acute inflammation.
6.3.1 Up-regulation in the cAMP response to immune-neuromediators
Various immune, inflammatory and neural mediators may activate AC to produce their effects in the ENS [231], and our findings indicate that mast cell tryptase, LTB4,
PGE2 and TNFα are elevated in acute inflammation in mucosa or plasma of infected guinea-pigs. Therefore, we tested the hypothesis that the observed up-regulation of AC expression leads to amplification in the response to such mediators in myenteric neurons.
Experiments in isolated myenteric ganglia indicated that clear amplification in the cAMP response occurs under basal / unstimulated conditions or in the presence of Ro-20-1724,
181 the mast cell mediator histamine, the AC neurotransmitter substance P (SP) or the AC
activator forskolin (Figure 50). Various treatments caused a 110 – 190% greater cAMP
response in the T. spiralis infected jejunum (p<0.05) compared to age-matched controls.
Adenylyl cylcase up-regulation in T. spiralis inflamed jejunum
Dual labeling studies revealed significant up-regulation in the expression of AC ir
(Figure 51) in calbindin-D28 ir myenteric neurons (i.e. representing AH / IPANs) in T. spiralis infected jejunum. Differential up-regulation occurred in calbindin-D28 ir neurons, calretinin ir neurons and s-100 ir glia. Up-regulation in glia was negligible
(Figure 52A). In western blot analysis of protein lysates from isolated myenteric ganglia,
T. spiralis infection caused an 830% up-regulation in a 147 kDa immunogenic band representing AC (Figure 52B).
182 p = 0.013 p = 0.039 5 25 50 p = 0.036
4 20 40
3 15 30
2 10 20
1 5 10
0 0 0 Krebs (Control) Ro-20 1724 Histamine (10 µM) (100 µM) les/200 myenteric ganglia) o p < 0.005 p = 0.030 20 40 Normal Inflamed 15 30
10 20
5 10 Cyclic AMP content (pm 0 0 Substance P Forskolin (1 µM) (10 µM)
Figure 50. Amplification of the cAMP response in myenteric neurons during acute inflammation induced with T. spiralis nematode infection of guinea-pig jejunum. The cAMP response in the ganglia is significantly elevated under basal conditions in Krebs buffer, in the presence of the cAMP-dependent phosphodiesterase inhibitor Ro-20-1724, the mast cell mediator histamine, the neuropeptide transmitter substance P or the AC activator forskolin.
183 A. Noninfected B. Noninfected
Neg. Control Calb + AC for AC
C. Inflamed D. Inflamed
Calb + AC Calb + AC
Figure 51. Up-regulation in the expression of AC ir in calbindin-D28 – positive myenteric neurons during acute inflammation induced with T. spiralis nematode infection of guinea-pig jejunum. (A) Overlay LSM image of AC / calbindin-D28 immunoreactivities in a myenteric ganglion from normal, uninfected jejunum with primary antibody for AC omitted. (B) Low level of AC expression in calbindin-D28 neurons in a ganglion from uninfected gut. (C, D) Strong up-regulation in AC expression in calbindin-D28 ir neurons is revealed in inflamed gut. AC ir was visualized by a secondary antibody conjugated to fluorescein; calbindin-D28 ir was visualized with a secondary antibody conjugated to Texas Red. Yellow staining is indicative of strong co-localization and expression of AC ir and calbindin-D28 ir. Scale bar = 30µm.
184 A. 100 B.
80
147 kDa 1 2 3 4 60
40 AC expression 20 % of cell pop. with increase in 0 Calb Calret S-100
Figure 52. Up-regulation of AC ir in cells expressing CaBPs in myenteric ganglia of T. spiralis nematode infected guinea-pig jejunum. (A) AC expression was significantly elevated in 73% of calbindin-D28 (Calb) ir neurons, 43% of calretinin (Calret) ir neurons and ≤ 1% in s-100 – labeled glia. (B) Up-regulation of the expression of AC was confirmed by western blot analysis. Lane 1, pooled ganglia from 4 animals; lanes 2 - 4, expression of AC ir in 3 lysate preparations each from a different infected guinea-pig. Using densitometry, values are: immunogenic band in lane 1 = 0.849, band in lane 2 = 9.66, band in lane 3 = 6.73, band in lane 4 = 7.18 (Average density in ganglia from inflamed animals = 7.857 ± 0.910). This represents a 725% increase in AC expression in inflamed gut in comparison to age-matched controls. 25 µg of protein lysate was used in each lane. For Calb neurons, n=140 ganglia (700 neurons); Calret, n=140 ganglia (560 neurons); s-100, n=200 ganglia. Difference in AC up-regulation between Calret and Calb cell populations is significant at p<0.00001 with Fisher exact test; difference between Calret and s-100 ir populations, p<0.00001.
185 6.3.2 CREB phosphorylation in neurons expressing CaBPs in inflamed gut.
All data on CREB phosphorylation experiments are summarized in Table 17. In order to assess the possibility that cAMP-dependent nuclear CREB phosphorylation occurs in T. spiralis infected gut, we first sought to establish a link between activation of AC and pCREB.
The AC activator forskolin (± Ro-20-1724) elevated nuclear pCREB ir in neurons that are dually labeled for calbindin-D28 ir that are therefore AH / IPANs (Figure 53A-D). In protein lysates of isolated myenteric ganglia, the AC activator forskolin, the cAMP – dependent phosphodiesterase inhibitor Ro-20-1724 alone or the mast-cell mediator histamine caused an increase in nuclear pCREB of a 47 kDa immunogenic band (Figure 54A-D). The potency profile of various treatments for causing CREB phosphorylation is histamine > forskolin = T. spiralis infection > Ro-20-1724 >> baseline levels (Table 17).
Condition n value* pCREB Response*** ______band density**______Fold increase a. Krebs/basal 6 (4) 0.81± 0.26 --- b. 10µM Ro-20-1724 3 (3) 5.19±0.89 6.4 c. Forskolin (10µM) + Ro-20-1724 (10µM) 3 (3) 13.01±3.34 16.1 d. T. spiralis inflammation (6-7 days PI) 2 (2) 10.34±0.36 12.8 e. Histamine (10µM or 100µM) 6 (2) 32.67±4.37 40.3
% inhib by KT 5270 f. 100µM Histamine +10µM Ro-20 3 (1) 31.78±1.00 100µM Histamine+10µM Ro-20+1µM KT 5720 3 (1) 3.90±0.30 -87.8%
g. 10µM Forskolin + 10µM Ro-20 3 (1) 19.60±0.78 -47.7% 10µM Forskolin + 10µM Ro-20 +1µM KT 5720 3 (1) 10.25±0.40 * number of determinations; ** calculated by densitometry, adjusted volume optical density / mm2 ; ***, compared to Krebs/basal CREB phosphorylation; ( ), represents number of animals used in analysis; p<0.001 for a vs b, c, d, e; p<0.01 for b vs c; p>0.05 for c vs d; p<0.001 for e vs d or c.
Table 20. Cyclic AMP dependent CREB phosphorylation in myenteric neurons in T. spiralis infected and age-matched control jejunum.
186 Forskolin stimulation of AC causes a significant increase in the mean number of
neurons / ganglionic field which display strong nuclear pCREB ir in calbindin-D28,
calretinin or other unidentified neurons in normal non-infected jejunum (Figure 54E).
Twice as many calbindin-D28 positive neurons display pCREB than calretinin positive
neurons. The response to forskolin (+ Ro-20-1724) was much greater than that to Ro-20-
1724 alone.
T. spiralis infection and acute inflammation elevates pCREB in isolated
myenteric ganglia of a 47kDa immunogenic band by about 1300% compared to age-
matched control animals (Figure 54B). T. spiralis infection elevates pCREB ir in 85% of calbindin-D28 ir neurons (Figures 53E-H, Figure 54F); this is similar to the number of calbindin-D28 ir neurons that display pCREB ir in response to maximum stimulation with the AC activator forskolin.
The pCREB responses to forskolin or histamine in non-infected tissues were
blocked by a 30 min pre-incubation of isolated ganglia with 1µM KT 5720 (1µM), a protein kinase A inhibitor (Figure 54D, Table 20).
iii. Effects of anti-inflammatory agents on AH cell hyperexcitability
All electrophysiological data with blockers or inhibitors is summarized in Table
21.
A prominent role for mast cell mediator release has been established [231, 273].
This was confirmed in the current study by showing a 1600% elevation in mast cell
tryptase in T. spiralis infected guinea-pigs. If on going release of histamine from mast
cells contributes to AH cell hyperexcitabity then pharmacological blockade of histamine
receptors should attenuate it. Histamine is a slow EPSP mimetic in AH neurons that may
187
Electrophysiol. Control Day 6 PI Day 6 PI Day 6 PI Day 6 PI Parameters (Ap value) +Pyrilamine +WY48252 Meclofenamic +Cimetidine (Cp value) Acid (Bp value) (Dp value)
AH Neurons 22 26 19 10 12
Accomodation 1.91±.33 8.72±.74 2.3±.35 2.7±.41 3.75±.68 (number of APS (0.0001) (.21;.0001) (.09;.0001) (.005;.0002) /Impulse)
Input Resistance 89.0±12.4 224.0±44.8 194.0±15.0 181.0±22.0 204.0±16.0 (MΩ) (0.005) (.0001;.29) (.0002;.28) (.0001;.38)
Resting Potential 62.1±4.8 47.3±2.9 52.7±1.2 51.2±1.8 49.8±1.4 (mV) (0.005) (.04;.07) (.07;.21) (.04;.29)
Spontaneous AP 9% 67% 47% 40% 66% Discharge (0.0001) (.007;.18) (.06;.16) (.001;.62) (% of neurons)
Anodal Break 9% 83% 68% 50% 58% Excitation (<.00001) (.0001;.27) (.019;.08) (.004;.14) (% of neurons)
Acontrol vs. Day 6 PI for each parameter Bcontrol vs. Day 6 PI with Histamine antagonists; Day 6 PI vs. Day 6 PI with Histamine antagonists Ccontrol vs. Day 6 PI WY48252; Day 6 PI vs. Day 6 PI with WY48252 Dcontrol vs. Day 6 PI with meclofenamic acid; Day 6 PI vs. Day 6 PI with meclofenamic acid
Table 21. Partial reversal of enhanced excitability in AH/IPANs by receptor blockade of immune mediators.
188
2+ act by stimulating AC / cAMP signaling leading to closure of Kca channels [16], and the histamine cAMP response in isolated ganglia was shown to be enhanced in T. spiralis
(Figure 50). The effects of H1 and H2 histamine receptor antagonists on AH cell
hyperexcitability in T. spiralis inflamed guinea-pig jejunum are summarized in (Figure
55). Examples of the effects of histamine blockers in AH cells is shown in Figure 55. AH
cell hyperexcitability is characterized by a reduction in accommodation (reflected in an
increase in the number of action potentials / depolarizing current pulse), increase in cell
input resistance, depolarization of the resting membrane potential, increase in the
occurrence of spontaneous action potential discharge, and anodal break excitation.
H1/H2 receptor blockers only partially blocked the enhanced electrical properties of AH neurons. They mainly reversed effect(s) on accommodation (Figure 55). Little or no reversal of other enhanced electrical properties was observed including input resistance, anodal break excitation, resting potential or spontaneous AP discharge
(Figure 55). A 30 min period of washout of the antagonist was sufficient to cause recovery of AH cell hyperexcitability (not shown) indicating an ongoing stimulation of the neurons by endogenous inflammatory mediators. TTX (0.3µM) did not affect resting membrane potential or cell input resistance of AH neurons from guinea pigs infected with
T. spiralis or the reduction in excitability achieved in the presence of each anti- inflammatory agents (not shown). Therefore, AH cell hyperexcitability involving ongoing release of endogenous mediators is due to direct stimulation at postsynaptic membranes of AH neurons, and not due to synaptic transmission or influence from other neurons. Similarly, a COX inhibitor (meclofenamic acid) or an LT antagonist
189 (WY48252) was only effective at preventing effects on accommodation, with little effect on other electrophysiological parameters. Histamine blockers had no effect on the electrical properties of AH neurons in age-matched control animals (p>0.05, not show for simplicity).
190 A BCD
Forskolin Noninfected Calb pCREB PCREB+ Calb pCREB + Calb E F GH
Ts. piralis Ts. piralis Ts. piralis Ts. piralis pCREB Calb
Figure 53. Nuclear CREB phosphorylation in normal and T. spiralis infected guinea-pig jejunum. (A-D) Forskolin stimulates CREB phosphorylation in myenteric ganglia of uninfected guinea pigs. (A) Single channel image of calbindin-D28 ir. (B) Single channel image of pCREB ir in the same ganglion. (C) Overlay image showing strong stimulation of pCREB ir in the ganglion with forskolin (D) Krebs control overlay image showing lack of pCREB ir. (E-H) Myenteric neurons with calbindin-D28 ir show strong nuclear pCREB ir in T. spiralis infected jejunum. Each image is a representative image from each of 4 animals. Yellow denotes high level of pCREB ir in calbindin-D28 neurons. Scale bar = 30µm.
191 Figure 54.Cyclic AMP-dependent CREB phosphorylation in myenteric neurons of normal uninfected and T. spiralis infected jejunum. (A) Western blot analysis shows that 1 µM forskolin (FSK) +10 µM Ro-201724 (Ro 20) causes CREB phosphorylation in isolated myenteric ganglia in normal uninfected jejunum. Densitometric analysis (adj. volume O.D/mm2) revealed that FSK + Ro 20 increased the immunogenic band from a basal level of 0.696 (lane 1) to 8.79 (lane 2) or 10.65 (lane 3) in two sets of ganglia (200 ganglia/tube) from different animals. Each immunogenic band represents pCREB ir in a 25 µg protein lysate. (B) Western blot analysis indicates an increase in the 47 kDa immunogenic band corresponding to pCREB in protein lysates from ganglia of T. spiralis infected jejunum compared to non- infected age-matched controls; densitometry indicates an optical density of ~ 0.5 for basal (lane 1) vs ~ 10.0 for two sets of ganglia from different animals infected with T. spiralis (lanes 3 and 4). (C) Western blot analysis from ganglia in 2 normal animals indicates that histamine alone is able to elevate CREB phosphorylation at 10µM or 100µM histamine concentrations; optical density (OD /mm2 )of the pCREB immunogenic band in lane 1 is 0.46, lane 2 is 6.53 (Ro 20, 10µM), lane 3 is 5.54 (Ro 20, 10µM), lane 4 is 24.34 (10µM Histamine, 10µM Ro 20), lane 5 is 43.27 (100µM Histamine, 10µM Ro 20), lane 6 is 43.26 (10µM histamine), lane 7 is 39.54 (100µM histamine), lane 8 is 31.4 (100µM histamine). (D) The PKA inhibitor KT 5720 (1µM) blocks the CREB phosphorylation to forskolin or histamine stimulation; the OD/mm2 for lane 1 is 31, lane 2 is 4.0, lane 3 is 20.0, lane 4 is 10.1. The same amount of protein lysate was used in each lane (25 µg) of Figures A-D. Each sample contained 200 ganglia for a total of 4000 ganglia harvested, counted individually and used for these experiments illustrated in Figure 5. (E) In LMMP of normal jejunum, FSK (±Ro-20-1724) increases CREB phosphorylation in calbindin-D28 ir neurons, fewer (less than 50%) calretinin ir neurons (calbD28+/pCREB+ vs calretinin+/pCREB+, p<0.0001) and other unidentified neurons. The pCREB response is also much greater with forskolin than with the PDE inhibitor Ro-20-1724 alone; Krebs vs Ro-20-1724 (Ro 20), p<0.0001; Ro-20-1724 vs Ro-20-1724 + FSK, p< 0.0001. Data is presented as mean number of neurons/ ganglionic field randomly selected in each ganglion (that does not represent entire ganglion); n = 35 to 80 ganglia / group and a total of 421 ganglia were imaged for analysis. (F) T. spiralis infection stimulates CREB phosphorylation in 76% of calbindin-D28 ir neurons in inflamed jejunum in comparison to 90% of neurons observed with 10µM FSK stimulation in normal uninfected jejunum (similar results with 1µM FSK not shown). Difference between normal/Krebs control vs FSK stimulation, p < 0.001; basal vs T. spiralis infection, p < 0.001; FSK vs T. spiralis, p < 0.01.
192 47 kDa A. 47 kDa 1 2 3 B. 1234
Basal FSK Basal Ro 20 Inflamed + Ro 20 Non-infected C. D. 1 234 56781234 47 kDa
t Basal Ro 20 Ro 20 Hist is T K T H K S K + t+ F + + Hist is + K 20 H 0 S 2 F o + o + R 0 R 2 20 E. o o F. R R CalbD28 +/pCREB +
ld 6 r e i i Calret +/pCREB + 100 F 5 pCREB + 80 4 60
3 ed pCREB 40 2 20 elevat Mean Number of 1 / w % of CalbD28 Neurons 0 Neurons/Ganglionic -0--0--0- d 0 te + ed c SK 0 m Krebs Ro 20 Ro 20 fe F 2 la n 0 o f 0 ni 2 In 2 + FSK o o R o N R R
Figure 54. (See preceding page for figure legend)
193 A T. spiralis controls B Cimet + Pyril (1µM) C (H1/H2 blockade) 10 2 APs
9 APs Impulse)
/ 8
6 20 mV † 0.5 nA * 200 ms 4 ∆ * *∆ * 2
0 -54 mV t d i Accommodation(# APs
-49 mV c 48252 Control Y I Day 6 PI I + W + Meclof. a + Pyril. Cime
Figure 55. Effects of anti-inflammatory agents on the AH cell hyperexcitability and accommodation recorded in LMMP preparations with T. spiralis nematode infection in guinea-pig jejunum. (A-B) Effect of the histamine H1 and H2 receptor antagonist cocktail (pyrilamine and cimetidine, respectively). (A) A representative electrical response to intrasomal injection of depolarizing current pulses (200 msec, 0.1 Hz) in an untreated AH neuron from infected gut. (B) Treatment with histamine receptor antagonists for 30 min increased accommodation and reduced the number of action potentials from 9 to 2 action potentials. (C) Histogram analysis from pooled data showing that blockade of histamine receptors by the H1/H2 receptor antagonist cocktail, a leukotriene antagonist or a COX inhibitor significantly reduced the number of action potentials per impulse in T. spiralis infected gut (refer to Table 2 for n values and effects of drugs on other electro-physiological properties of AH neurons). Histamine or the leukotriene antagonist or the COX inhibitor had no effects on the excitability of AH neurons in age-matched control animals (not shown for simplicity). *, Significant difference exists between each condition and 6 Days PI, p<0.05. ∆, no significant difference from control. †, significant differences from control at p<0.05; therefore, not fully recovered.
194 6.4 DISCUSSION
This study provides immunochemical and functional evidence of amplification in
the Gs/AC/cAMP signaling pathway in myenteric neurons of the jejunum following
infection with the T. spiralis nematode. Cyclic AMP is a main mediator of slow synaptic
sensory transmission (slow EPSPs) in AH / IPANs of the ENS [26, 39, 63, 334]. The role
of IPANs is to sense or “taste” the changing luminal environment and respond to
nutrients and distention with an increase in their excitability. Communication between
IPANs is via slow EPSPs and hence cAMP amplification and hyperexcitability in IPANs
of the T. spiralis inflamed gut is expected to facilitate feed-forward activation and gut motility reflexes leading to an abnormal motility pattern [300, 337, 338].
Our current model of Gs/AC/cAMP signaling for AH/IPANS in T. spiralis
infected gut is presented in Figure 56. Cyclic AMP production in myenteric ganglia of
T. spiralis infected guinea pigs is augmented in the basal / unstimulated state or in the
presence of the PDE inhibitor R0-20-1724, suggesting that elevation of basal cAMP that
is regulated by PDE IV plays a role in neuronal plasticity in inflamed but and may
contribute to AH cell hyperexcitability (and perhaps other cells). The effect of Ro-20-
1724 indicates that cAMP-dependent PDE plays a significant role inactivating cAMP
inside cells. In previous studies, we showed that the concentration of PDE inhibitor used
(10µM) is sufficient to evoke a slow EPSP-like response in AH neurons [73].
As anticipated, H1/H2 blockers reduced AH cell excitability suggesting that
ongoing release of histamine from mast cells is also involved in their hyperexcitability.
The excitatory effect of histamine on AH cells is believed to occurs via AC/cAMP
signaling – histamine was shown to augment cAMP levels in enteric ganglia from the
195 infected gut [16]. Therefore, it is likely that histamine release contributes to basal
elevation in neuronal cAMP levels, intraneuronal signaling and perhaps enhanced
excitability in T. spiralis inflamed gut. However, the enhanced AH cell excitability also
involves metabolites of the cyclooxygenase pathway that may include leukotrienes and
prostaglandins since a COX inhibitor (meclofenamic acid) or a leukotriene receptor
antagonist (WY) mimicked the effect of H1/H2 blockers on reducing excitability.
Therefore, these mediators may also contribute to amplification of cAMP signals. We observed similar reductions in hyperexcitability by histamine blockers, the COX inhibitor or LT antagonist and it is tempting to speculate that interactions at the AC pathway may occur between histamine and COX metabolites in T. spiralis.
In the current study, we found that AC up-regulation occurs in both calbindin-
D28 and calretinin ir neurons in T. spiralis infected guinea pigs, as well as other neurons
that were not identified by their chemical coding. Calbindin neurons are AH/IPANS in
this region of the gut and species. Calretinin ir neurons may represent cholinergic LM
motor neurons [15]. The differential up-regulation in neurons and glia indicates that acute
inflammation leads to a discrete effect on AC/cAMP signaling in myenteric neurons.
Differences are also notable between IPANS and cholinergic neurons. This likely
represents an adaptation in response to inflammation. It was beyond the scope of this
study to carry out a detailed functional pharmacological analysis of AC up-regulation in
AH and S neurons with forskolin, slow EPSPs mimetics and evoked slow EPSPs in
normal and T. spiralis infected guinea pigs to prove whether hyperexcitability in AH or S
neurons involves an AC/PKA-dependent mechanism.
196 Ro-20 1724 Forskolin _ + Immune PDE modulators Gi Histamine, PGE , LTB AC _ Transmitters 2 4 R2 SP, slow EPSP mimetics ATP cAMP R1 s R G PKA Protein + C Phosphorylation Contributes to pCREB Cell membrane Enhanced Nucleus Long-term change Excitability c-fos in gene expression and slow EPSPs
Figure 56. A working hypothesis of amplification in the AC / cAMP signaling pathway leading to hyperexcitability in AH intrinsic primary afferent neurons in acute inflammation with T. spiralis in guinea-pig jejunum. Neurotransmitters like substance P (SP) or slow EPSP mimetics (i.e. VIP, CGRP, PACAP, GRP, 5-HT) including immune / inflammatory mediators such as histamine or PGE2 and perhaps LTB4, TXB2, TNFα, proteases interact with specific cell surface receptors (R1, R2 represent multiple receptors each) to activate Gs / AC and elevate intraneuronal cAMP levels leading to closure of Ca2+-dependent K+ channels leading to an increase in cell excitability characterized by a slow EPSP-like response. In T. spiralis infected gut, AC up-regulation leads to an exaggerated cAMP response to these mediators leading to sustained AH cell hyperexcitability. During nematode infection, ongoing release of endogenous immune / inflammatory mediators or neurotransmitters contribute to the AH cell hyper- excitability by activation of the AC / cAMP signaling pathway, and by a PKA – dependent pCREB phosphorylation and AP-1 transcriptional regulation. Ongoing elevation of basal cAMP levels leads to activation of PKA and translocation of the catalytic unit (C) of PKA to the nucleus leading to CREB phosphorylation and transcriptional regulation of various genes via c- fos activation, including AC up-regulation (this study) or other enzymes or target proteins; R, regulatory unit.
197 Neural and non-neural plasticity in the expression of specific AC isozymes occurs
under both pathologic and physiologic circumstances including Alzheimer’s disease,
alcoholism, opiate-induced tolerance and dependence and during circadian rhythms
[118]. Our study is the first to provide direct evidence that jejunal inflammation due to T. spiralis infection causes a tremendous AC up-regulation as well. In the ENS, we previously reported that AC I, III and IV isozymes were differentially expressed in AH / calbindin or other neurons and in different regions of the small intestine [73]. The antiserum used to show up-regulation of AC ir in gut neurons does not discriminate between AC isoforms, and therefore, it remains unknown which of these isoforms was up-regulated in the inflamed jejunum. The AC isozymes of the AC superfamily are differentially modulated (stimulated or inhibited) by Gsα, Giα (Gαi1,2,3), Ca2+, Gβγ, protein kinase C-α, nitric oxide, protein kinase A or protein phosphatase 2A [118]. Since these AC isozymes serve a critical role in integrating multiple signals in neurons, and multiple functional types of neurons can generate cAMP in the ENS it is likely that AC up-regulation would have critical and complex consequences to cell signaling in enteric neurons.
Molecular mechanisms underlying alterations in the cAMP signal transduction cascade in T. spiralis inflamed gut were investigated indirectly by examining nuclear
CREB phosphorylation, a key step involved in transcriptional regulation. Translational and transcriptional modifications of the various components of the AC / cAMP pathway are likely involved [273]. Our findings indicate that nearly 80% of calbindin ir /IPANS
display elevation in CREB phosphorylation during T. spiralis infection and the effects are
mimicked by AC stimulation with forskolin in uninfected gut neurons. In T. spiralis
198 infected gut, it is likely that endogenous immune / inflammatory mediators act on cell
surface receptors to activate AC and elevate cAMP levels to enhance excitability, but also cause nuclear CREB phosphorylation via PKA activation. This premise is supported by
findings that the mast cell mediator histamine or forskolin induced pCREB that could be
blocked by the PKA inhibitor KT 5720. Thus, elevation of intraneuronal cAMP levels
activates protein kinase A leading to translocation of the catalytic unit to the nucleus and
CREB phosphorylation [76].
Phosphorylated CREB is known to bind to the promoter region of c-fos to activate
it leading to AP-1 transcriptional regulation. The immediate-early gene c-fos is an
essential component of the AP-1 transcription factor [179]. Substantial c-fos expression
occurs in 50% of intrinsic primary afferent neurons during the acute phase of infection
with T. spiralis in guinea pig small intestine [231, 273]. Therefore, it is likely that c-fos
induction involves AC / cAMP signaling in T. spiralis infected gut. Protein products of
immediate-early genes are involved in cellular stimulus-transcription coupling effectively
acting as “third” messengers regulating transcription of target genes that affect neuronal
phenotypic expression in the ENS in response to extracellular stimuli [180, 339] and in
response to T. spiralis infection [231, 273]. C-fos induction also occurs in rat
lumbrosacral spinal cord neurons after colorectal distension. However, it should be
pointed out that neural plasticity and long-term changes in gene expression in the ENS of
the T. spiralis infected animals is not restricted to cAMP-dependent pathways. Ca2+- dependent signaling pathways also play a crucial role in induction of c-fos gene
expression, CREB phosphorylation and AP-1 transcriptional regulation [180].
199 The fact that acute application of receptor blockers or enzyme inhibitors of immune/inflammatory products could at best partially reverse the electrophysiological changes and hyperexcitability characteristics, suggests that once enteric neurons are exposed to endogenous inflammatory mediators (and in this case we assume they have been continuously exposed for several days in vivo), that any functional changes initiated as a result are difficult to surmount with an acute application of exogenous substances.
The effect is analogous to a “run-away freight train”. Since the effects of histamine
H1/H2 blockers mainly affect accommodation in AH neurons, the persistent changes in cell input resistance, a depolarized cell membrane potential, occurrence of anodal breaks and spontaneous action potentials in T. spiralis inflamed gut is likely due to long-term adaptive and / or plasticity changes that have been activated as a result of the amplified
AC/cAMP transduction cascade and its induction of transcriptional activation. This is consistent with the increased expression of c-fos [273], cytochrome oxidase[273] and pCREB ir (this study) in T. spiralis infected gut. All changes in electrical properties observed in AH neurons in gut infected with T. spiralis are mimicked by forskolin, and that includes the effect on accommodation that was shown to be blocked by removing the influence of endogenous mediators.
Together with evidence from other cell types including neurons [327-329, 333], our data supports the hypothesis that alterations in the Gs/AC/cAMP signaling cascade leads to transcriptional regulation via CREB phosphorylation and putative expression of c-fos and AP-1 transcription. Therefore, cAMP-dependent CREB phosphorylation may be one mechanism leading to AC up-regulation in AH / IPANS, S/calretinin LM motor neurons or other unidentified classes of myenteric neurons that likely represent
200 descending inhibitory interneurons, descending interneurons, and inhibitory LM motor
neurons deduced from cAMP visualization studies. It is unclear what the sequence of
events is leading up to amplification in Gs/AC/cAMP signaling pathway and AH cell
hyperexcitability. A model is illustrated in Figure 56.
A primary transmitter for the cAMP- and PKC-co- dependent slow EPSP in AH
sensory neurons is the neuropeptide SP [260], although many other candidates mimic
slow EPSPs ( i.e. CGRP, PACAP, VIP) including paracrine mediators such as histamine
released from mast cells and COX metabolites (i.e. PGE2) [16]. The observed amplification of the histamine (via H1/H2 Rs) or SP (via NK3 Rs) induced elevations in
intraneuronal levels of cAMP indicate that receptor-coupled Gs/AC/cAMP signal in the
neurons – transmitted by neural or immune mediators- are also exaggerated in T. spiralis
infection. Up-regulation of AC and ongoing activation by endogenous modulators can
explain the cAMP amplification response and perhaps the hyper-excitability in the
neurons, although the contribution of other mechanisms such as up-regulation of Gs-
coupled receptors, down-regulation of Gi/o coupled receptors, down stream alterations in
the Gs/AC/cAMP signaling pathway (i.e. PKA, channel activity) or cross-talk between
cAMP and other second messengers remain unknown.
Glial cells with s-100 ir do not contribute to the Gs/AC/cAMP amplification
observed in isolated myenteric ganglia, since they do not display up-regulation in AC ir.
Forskolin- induced cAMP responses are elevated by ~ 100% to 200 % in inflamed gut,
indicating that infection leads to an increase in the catalytic activity of AC and
amplification in intraneuronal cAMP levels. This notion is consistent with the observed
up-regulation in AC expression in calbindin ir (AH neurons) leading to an exaggerated
201 cAMP response in parallel with AH cell hyperexcitability. The impact of up-regulating
AC ir on the excitability of calretinin ir neurons in T. spiralis inflamed gut remains
unresolved.
It remains unknown if other inflammatory mediators found to be elevated in T.
spiralis infected jejunum including LTB4, TNFα, TXB2 or the serene protease tryptase also activate the cAMP pathway or elevate excitability of the AH neurons. In particular, tryptase was elevated by 1600% in inflamed gut. Mast cell tryptase which is generally released during trauma and inflammation activates about 60% of enteric neurons by cleaving PAR-1 and PAR-2 receptors [340]. The effect of tryptase has been linked to elevation of intracellular Ca2+ levels, but cAMP cannot be excluded [340]. Furthermore,
Ca2+ can activate AC I and AC III known to be expressed in AH / IPANs, and therefore cross-talk between Ca2+ and cAMP remains a potential mechanism in AH cell hyperexcitability.
In both acute jejunal inflammation with T. spiralis [272] and chronic inflammation (TNBS – colitis) [341], there appears to be exclusive enhancement of electrical properties of AH neurons. However, certain features of AH cell hyperexcitability clearly differ in the two models of inflammation: Common features in the two models include 1) reduced action potential accommodation observed as an increase in number of APs / impulse, 2) increased frequency of anodal breaks and spontaneous action potentials and 3) a reduction in the AHP amplitude. Features exclusive to the T. spiralis model include 1) a depolarized resting membrane potential, 2) a 200-300% increase in cell input resistance, 3) a 400-500% shortening of the AH response and 4) alterations in AP characteristics. Differences in AH cell
202 hyperexcitability may be attributed to different models of inflammation (i.e. acute versus
chronic), different regions, or inflammation induced effects on different signal
transduction pathways.
It remains unknown whether AC/cAMP signaling is altered in TNBS colitis,
although its involvement is unlikely, since no changes in membrane potential or cell input
resistance occurs in TNBS colitis as it does in T. spiralis. In TNBS-induced colitis,
enhanced excitability in AH neurons and reduced accommodation is suggested to be a
+ + + result of up-regulation of a Cs sensitive non-selective cation current Ih (for K , Na )
[341]. Up-regulation of the Ih is responsible for abbreviating the AHP and making the AH cell more excitable. It is unresolved whether inflammatory / immune mediators also stimulate ongoing activation of Ih in T. spiralis inflamed jejunum. If so, it would argue against Ih up-regulation.
AH neurons in this and the original study by Palmer and co-workers [231, 273] were classified solely according to their electrophysiological behavior, raising the possibility that a subset of recorded neurons may represent another cell phenotype.
Particularly, since many of the electrical properties of AH cells used to classify them are altered in acute inflammation, some recorded AH neurons may represent descending filamentous uniaxonal neurons that are known to have AH/S cell electrophysiological characteristics with a shorter AHP, higher cell input resistance and exhibit less accommodation than AH / Dogiel Type II neurons. Up to 10%-20% of recorded AH neurons (in this study) may have been filamentous neurons[15, 26, 39]. Therefore, it remains unknown whether T. spiralis infection leads to hyperexcitability in both
203 S/uniaxonal neurons and AH / multipolar / IPANS of the myenteric plexus because S
uniaxonal neurons may represent various classes of interneurons or motorneurons [15].
Concluding Remarks
To date, little was known about cAMP signal transduction in disease states of the
gut [231, 270, 300]. Our investigation targeted a main second messenger for slow
synaptic transmission that is of fundamental importance in sensory neurotransmission in
gut reflexes. This study supports the hypothesis that neuronal plasticity and
hyperexcitability in Trichinella spiralis inflamed gut is the result of amplification and immune-modulation of AC/cAMP signaling leading to CREB phosphorylation and transcriptional regulation in primary afferent, cholinergic and other neurons.
The observed amplification in the cAMP signaling cascade in AH / IPANs of T.
spiralis infected guinea-pigs provides a common Gs/AC target for evoking hyperexcitability via excitatory neurotransmitters (SP, PACAP, VIP,CGRP,GRP,5-HT) and paracrine (adenosine), immune (histamine, tryptase, 5-HT, adenosine) or inflammatory mediators (PGE2, LTB4, TXB2, TNFα) in the integrated neural circuits of the gut. Such neural plasticity in the AC/cAMP signaling pathway may contribute significantly to enteric neural dysfunction, since hypersensitivity in IPANs would undoubtedly affect mucosal and distension reflexes and contribute to alterations in motility and secretory reflexes that are known to occur in acute inflammation [231].
Until now, the only other mechanism for neuronal plasticity in the ENS was the SSEP
[260] that may involve PKC, although AC/cAMP and PKA activation were not ruled out.
204 It would be very important to know if neuronal plasticity and up-regulation in the
Gs/AC/cAMP signaling pathway persists during the post-inflammation period, since
motor, secretory and neurotransmitter abnormalities exist up to 60 days post-infection
with T. spiralis, other nematode infections or in other inflammation models [231, 342].
Relevant to this is a recent preposition that acute-infection may lead to post-infectious
irritable bowel syndrome (IBS),[300] supported by the clinical finding that patients with
acute infectious gastroenteritis develop symptoms of IBS[270, 300, 338]. It also remains unknowin whether neuronal plasticity in AC/cAMP signaling occurs in experimental models of inflammatory bowel disease.
205
CHAPTER 7
OVERALL DISCUSSION AND CONCLUDING REMARKS
Electrophysiolgy, forskolin (FSK)- binding studies using fluorescently tagged bodipy-FSK, FICRhR / cAMP imaging, electrophysiological and biochemical studies; have provided important information and the basis of my studies outlined in this dissertation. Despite such studies, the role of cAMP signaling in intact neural circuits of the gut remained unknown. In an effort to address this question we developed an acrolein-derivatized cAMP antiserum to monitor and quantify cAMP-responsive neurons in the enteric nervous system. The antiserum was raised by immunizing rabbits with
KLH-acrolein-cAMP hapten and used for the quantitation, visualization, classification and polarity of neurons displaying a cAMP response.
Our Working hypothesis of the cAMP-dependent neural pathways involved in the enteric nervous system of the guinea-pig small intestine is illustrated in Figure 57. The functional types of cAMP-dependent neurons are deduced by cAMP visualization studies using the new acrolein derivatized cAMP antiserum.
206 Longitudinal Muscle
4 5 1 Myenteric 6 3 Plexus ***2
Circular Muscle 9 cAMP Interplexus Interneuron 12 VIP Submucous 7 cAMP cAMP 15 Plexus 8 cAMP 11 10 NPY VIP/cAMP
13 14 VIP/A2aR/cAMP
Mucosa
Figure 57. Working hypothesis of the functional types of cAMP-dependent neurons according to cAMP visualization studies using the an acrolein-derivatized cAMP antiserum. Types of neurons that display cAMP ir in the enteric nervous system after forskolin stimulation. Myenteric Plexus (1) Dogiel Type II (IPAN, Interneuron, AH), (2) Dogiel Type II /Dendritic (Long descending Interneuron, AH), (3) Dogiel Type I/Filamentous (Long SOM interneurons, S/AH ), (4) Dogiel Type I/Small or simple (short descending LM motor neurons, S), (5) Dogiel Type I/Lamellar Dendrites (CM motor neurons or interneurons, S), (6) unidenditfied neuron with very short axon and simple shape. Submucous plexus: (7) Dogiel Type II (IPAN, interneuron, AH), (8) Dogiel Type II/Dendritic (AH), (9) Dogiel Type I/Simple, (10) Dogiel Type I / Lamellar dendrites, (11) NPY ir secretomotor neurons not displaying cAMP ir (12) VIP ir interplexus neurnons which a subset of this could have been cAMP ir, (13) a subset of VIP ir secretomotor neurons were cAMP ir, (14) A subset of VIPir secretomotor neurons contained A2a receptors coupled to cAMP production and therefore display cAMP ir. (15) Interneurons revealed functional studies on cAMP-dependent synaptic transmission. 207 The shapes of the neurons and their polarity can be studied with the cyclic AMP
antiserum for several reasons:
(1) Acrolein at low pH traps free intracellular cAMP that is generated by AC activation
and forms covalent bonds with intracellular proteins. The antiserum recognizes acrolein-
cAMP-protein complexes inside cells.
(2) PDEI prevents the breakdown of cAMP and permits its accumulation inside the cell.
The ncAMP can then diffuse freely throughout the cell.
(3) All functional components of neurons (cell body, neurites, axons, and varicosities)
can generate cAMP and have functional AC.
(4) A long incubation of 30 min is used to generate maximum amounts of cAMPir in the
neurons in intact tissues and amplify the signals for optimal visualization of the neurons.
A shorter incubation of 1-2 min is sufficient to generate ncAMPir for partial visualization
of neurons.
(5) The antiserum is sensitive and selective for ncAMPir in gut neurons.
The apparent potency of various treatments for elevation of ncAMPir in
myenteric neurons is forskolin + Ro-20-1724 > forskolin > Ro-20-1724 = rolipram >
cGMP cocktail > Krebs/vehicle. The intracellular P-site inhibitor on adenylyl cyclase,
2’5’dideoxyadenosine caused a concentration dependent inhibition of the forskolin
response (IC50 = 180µM). Pre-absorption of the cAMP antiserum with 1mM cAMP
abolished the ncAMPir. Forskolin evokes an elevation in ncAMPir in ~700-800
neurons/cm2 stretched LMMP (25 % stretch) in comparison to 1,750 neurons/cm2 in the
SMP. Adenosine A1 or A2aR activation inhibits or elevates ncAMPir, respectively.
Cyclic AMP visualized neurons were identified as Dogiel Type II/dendritic or smooth
208 cell soma neurons with circumferential or descending projections (19.1%), descending
uniaxonal neurons with filamentous (38.3 %) or lamellar dendrites (15%); sometimes
these neurons had short intraganglionic projections, or small/simple descending Dogiel
Type I neurons (27.9 %) that sometimes projected to the longitudinal muscle. Cyclic
AMP visualized submucous neurons lacked polarity and was very sensitive to Ro-20-
1724 or the A1 antagonist CPT compared to myenteric neurons.
The suitability of the cAMP antiserum in detecting cAMP immunoreactivity in gut neurons was first established in cultured myenteric neurons and later in intact neural plexus preparations from either microdissected myenteric (LMMP) or submucosal (SMP) tissues. Specificity and selectivity of the antiserum for intracellular free neuronal cAMP immunoreactivity (ncAMPir) was established by ELISA competitive and non- competitive assays without tissues, or in tissues by pre-absorption of the antiserum with cyclic nucleotides, stimulation of cAMP levels using the AC activator forskolin, agonists that stimulate receptors linked to AC, a cGMP cocktail or cAMP-dependent phosphodiesterase inhibitors. Assay conditions were optimized for visualization of the maximum number of neurons displaying an ncAMPir response by using various staining protocols, secondary antibodies, IHC or IF detection methods.
In cultured myenteric neurons, the antiserum proved to be sensitive enough to detect changes in ncAMPir levels, which occur in response to receptor activation and showed that multiple functional cell types according to their shapes use AC/cAMP signaling. The antiserum reliably distinguished between cAMP and cGMP, and was useful in studies on chemical coding and dual labeling of neurons, and both basal and stimulus evoked alterations in intraneuronal cAMP levels. A significant proportion of
209 PGP 9.5 neurons and calbindin-D28 neurons representing AH/IPANs displayed cAMP responses to forskolin, consistent with previous electrophysiological data showing that
AH cell excitability is modulated by a rise in ncAMP levels.
Clear differences were observed between cAMP responses in neurons grown in culture or in intact myenteric plexus preparations (LMMP). About twice as many neurons
(41%) displayed cAMP responses to forskolin in culture compared to 15-20% in LMMP at best. The reasons for differences are not clear, although it is likely that culturing of the neurons and enzymatic dissociation of ganglia may alter the behavior of the neurons and their responsiveness to mediators acting through AC. This is suggested from studies showing that near millimolar concentrations of cAMP-dependent phosphodiesterase inhibitors (PDEI) were needed to protect cAMP levels generated in cultures in response to forskolin. In contrast, in tissues, low micromolar concentrations of PDEIs are sufficient to protect ncAMP levels and permit cAMP visualization of the shapes and projections of neurons or increases in the resting excitability of AH neurons. In fact, higher concentrations in intact LMMP tissues are toxic with ncAMPir being compromised. Therefore, permanent alterations in one or more steps in the AC signaling pathway (i.e. such as activity of Type IV PDE targeted by Ro-20-1724) likely occur in cultured neurons. Receptor activation with the slow EPSP-mimetic agents (i.e VIP or SP) also caused cAMP ir in cultured neurons but not in the intact myenteric plexus suggesting an alteration in the coupling of this G-protein/AC coupled receptors. Glial cells (data no shown) also displayed ncAMP ir in response to forskolin, but this was not seen in either the intact myenteric or SMP plexuses suggesting that in the intact tissue these cells apparently do not utilize the AC/cAMP signalling pathway – alternatively, the levels in
210 glial cells surrounding neurons in intact ganglia may generate lower levels of cAMP that
are undetectable by the antiserum. The intriguing possibility exists that cultured neurons
represent a pathophysiologic model whereby there is an amplification of the AC/cAMP
signaling pathway – As discussed later, in another model of acute inflammation induced
by T. spiralis, AC/cAMP amplification does occur involving both ongoing release of
endogenous mediators as well as cAMP-dependent transcriptional regulation.
Our findings in intact myenteric plexus tissues revealed that ncAMPir in
responses to forskolin stimulation are not restricted to Dogiel II (AH) neurons. In fact, it
occurs in 5 functional classes of myenteric neurons including putative descending
interneurons (Dogiel Type I / filamentous neurons), small descending motor neurons to
longitudinal muscle (seen by confocal imaging), short interneurons, putative circular
muscle motor neurons (i.e. those with lamellar dendrites that need to be proven by DiI
retrograde labeling studies) and intrinsic primary afferent neurons (Figure 57). Studies
done using a 3-chamber model (with collaboration/consultation from Dr. Jack Grider)
were done to address the hypothesis that "cyclic AMP signaling is involved in descending
reflexes, given that all cAMP-visualized neurons had either descending or circumferential
projections and none (<0.1%) had ascending projections." A 3-chamber flat sheet model
of intestine was used to determine the physiological role of cAMP in ascending and
descending reflexes, evoked by stretch of the CM or stroking the mucosa in the middle
sensory chamber. Application of the selective PKA inhibitor myr-GRTGRRNAI-NH2
(1µM) to the sensory chamber reduced both ascending contraction and descending relaxation. Its application in the outer motor chambers reduced exclusively the descending reflex. In contrast, myr-GRTGRRNAI-NH2 could only inhibit the descending
211 response regardless of whether it was applied to the motor or sensory chambers. Our data refutes the original hypothesis that cAMP signaling is restricted to the AH / sensory neuron phenotype, and provides direct proof that the majority of neurons with AC/cAMP signaling may represent uniaxonal (S) motor or interneurons with polarized projections for descending reflexes. Finally, data support the hypothesis that ncAMPir is linked to neuronal excitability and cAMP is differentially involved in ascending and descending gut reflexes evoked by mucosal stroking or stretch. Stretch or mucosal stroking reflexes and their respective neural circuits can be distinguished by cAMP signaling.
Other clear differences existed between myenteric and submucous neural cAMP dependent circuitry as evidenced by: PDE IV modulation of basal cAMP activity, co- localization of cAMP and cGMP responses, types of cAMP-neurons and numbers of cAMP ir neurons suggesting that cyclic AMP visualization is a powerful technique for functional studies of neural circuits in the ENS. However, further studies are needed to identify functional subsets of enteric neurons that utilize cAMP signaling in sensory transmission in order to systematically analyze and understand how the ENS regulates normal function. Cyclic AMP visualization in combination with further electrophysiology, chemical coding and or / or DiI retrograde labeling can provide unequivocal identification of the functional neurons displaying cAMP responses that are linked to the electrical behavior of enteric neurons. It still remains to be determined what proportion of cAMP visualization responses are linked to the excitability of AH/Type 2,
S/AH neurons or S/Type 1 neurons. Such analysis requires a combination of electrophysiological recording, cAMP triple labeling with biocytin and neurochemical coding or retrograde labeling analysis.
212 Various studies have shown that specific endogenous phosphodiesterases are
strategically located in various regions of the brain to tightly regulate intracellular cAMP
signaling. There is little information regarding the isoforms of phophodiesterases present
in the ENS, therefore it is possible that PDE type IV is not as abundant in the MP and
therefore would explain the low number of cAMP ir neurons in the MP after treatment
with Ro-20-1724. Therefore, future experiments need to address this possibility by
monitoring protein levels and activity of these various isoforms.
We have demonstrated that cAMP cell-to-cell communication occurs in
submucous neural circuits as evidenced by nerve conduction/neurotransmitter blockers,
blockade of nicotinic ganglionic transmission or adenosine A1R activation, showing that
they all reduced the forskolin-evoked ncAMPir or neurally mediated Isc/secretory responses. These observations further support the involvement of interneurons in the submucous plexus, however further studies are needed to address this possibility. Our findings with the guanylate cyclase inhibitor ODQ support the hypothesis that ongoing activation of guanylate cyclase inhibits or attenuates cAMP dependent spread of neuronal activity in the submucous plexus since ODQ caused a recruitment of more neurons that could generate ncAMPir in ganglia.
Adenosine has been shown to function as a neuromodulator in a variety of neuronal cell types in the CNS. Based on previous work on A1 and A2a receptors [16] together with our current data, we have demonstrated that adenosine also modulates the activity of the enteric nervous system. Similar to previous work which has shown selectivity of adenosine to specific regions of the brain, our ncAMP ir data also shows that adenosine provides dual modulation of ncAMPir via A1 inhibitory and A2a
213 excitatory receptors. This establishes the suitability of the antiserum in monitoring
receptor-mediated alterations in ncAMPir, and provides the first unequivocal direct
evidence that adenosine modulates intracellular cAMP levels in neurons. This raises
important questions regarding its role in neurosecretion, and with respect to the later
discussion, in neuroprotection in the inflamed gut.
Compartmentalization and discrete subcellular localization of ncAMPir was
observed in SMP neurons treated with an A2aR agonist - Previous studies suggested that
this compartmentalization involves A-kinase anchoring proteins (AKAPs) that are very
important in understanding AC/cAMP/PKA signaling. One could use inhibitory peptides
to prevent the interaction between PKA and AKAPs [209]. In fact, studies using AKAP
inhibitory peptides have been shown to abolish channel activity in myocytes. The resting
excitability of gut AH neurons should be disrupted and become abnormal in their
kinetics, shape and/or duration by disruption of PKA anchoring by the inhibitory peptide,
if anchored PKA is important in the tight regulation of cAMP-dependent closure of ion
2+ channels, and in particular the intermediate conductance KCa channels whose closure leads to slow EPSP-like responses.
Earlier electrophysiology work suggested that all AH/Dogiel Type II neurons
(AH neurons) and most S/Dogiel Type I neurons (S neurons) in the SMP were
depolarized by forskolin stimulation [103]. Later it was shown using Bodipy Forskolin
that adenylyl cyclase was present in neurons other than AH confirming these earlier
recordings in the SMP [74]. Our study using an acrolein derivatized cAMP antiserum
further confirms this earlier work and showed that both AH and S neurons display cAMP
ir after inhibition with either Ro-20-1724 (Fig 40A) or rolipram (data not shown); both 214 selective inhibitors of phosphodiesterase type 4 activity. This was not the case for the
myenteric plexus, which showed little or no response to these inhibitors (Fig 30).
These inhibitors are selective for PDE type IV and therefore, under physiological
conditions, intraneuronal cAMP levels in submucous neurons are strongly regulated by
the PDE isoform. The basal cAMP response in myenteric neurons is not modulated by
PDE IV. It is possible that cAMP signaling in myenteric neurons is modulated by
different PDE isoforms which were insensitive to Ro-20-1724 or rolipram. Recent studies
have shown that different phosphodiesterase isoforms are strategically located in specific
regions of the brain to mediate and regulate synaptic transmission [159]. Forskolin
augmented the Ro-20-1724 response by 285% which is consistent with the myenteric
plexus (Fig 31, chapter 4), but the number of neurons displaying cAMP ir was
approximately 3 fold higher in the SMP, since the LMMP contains 3X less neurons/cm2 when compared to the myenteric plexus in the guinea pig ileum (Fig 40A) [321]. This means that in the submucous plexus, cAMP signaling plays a more prominent role than in myenteric plexus, and such signaling is tightly regulated by PDE IV. Very different roles are emerging for cAMP in the two plexuses.
Based on cAMP ir visualization, multipolar neurons make up approximately 28% of all SMP while uniaxonal neurons make up about 72% of the remaining response (Fig
40B), which is different from the myenteric plexus, where only 18% of multipolar
(Chapter 4, Table 13) neurons display cAMP ir after forskolin stimulation suggesting that
cAMP signaling might be more important in multipolar neurons within the SMP. A
recent study showed that Dogiel Type II neurons or IPANs in the SMP are different from
those in the myenteric plexus since only 10% of them labeled for the calcium binding
215 protein (calbindin), a marker for these neurons suggesting that only a subset of Dogiel
Type II neurons are accounted by this chemical marker in the SMP [322]. Furthermore a subset of these calbindin ir neurons were also calretinin ir further providing evidence that
IPANs in the SMP are chemically and therefore likety to be functionally very different from those in the myenteric plexus since this co-localization had never been observed in the enteric nervous system [322]. Some of the cAMP ir neurons with filamentous morphology could be a subset of the VIP secretomotor neurons since these neurons have been described as filamentous in shape in the SMP plexus [317]. The small shape or lamellar neurons expressing cAMP ir could represent the secretomotor/vasomotor calretinin ir neurons since these neurons have been described as having similar shapes as this study [323]. However unlike the myenteric plexus there was no polarity expressed in the SMP suggesting that cAMP plays a more ubiquitious role within this plexus unlike the myenteric plexus, where cAMP functions more discretely in descending reflex pathways.
Since 64% of the VIP-ergic neurons can generate cAMP it suggests that cAMP signaling is very important in the function of these secretomotor neurons (Fig 41). This also implies that VIP/secretomotor neurons can be further subcategorized into cAMP- dependent secretomotor neurons and cAMP-independent secretomotor neurons. Since
100% of all VIP ir neurons are also cGMP ir [95] it can be deduced tht a majority of VIP secretomotor neurons are regulated by the activity of both AC and guanylate cyclase
(GC). Such cross talk between cAMP and cGMP does occur – cGMP provides an ongoing inhibition of AC/cAMP signaling in submucous neurons. Suppression of GC
216 activity with ODQ eliminates the inhibitory influence and reveals a more robust cAMP response.
The implication of such negative cross talk between cAMP and cGMP is in the modulation of neurosecretion in the gut. These second messengers provide a unique mechanism of neural regulation not shared by myenteric neurons. The functional relevance of cross-talk remains to be confirmed in Ussing chamber short-circuit current/Isc studies.
Adenosine A1 and A2aRs are often coupled to inhibition or stimulation of
AC/cAMP signaling in neurons and other cell types respectively [324]. In the ENS, A2aR activation mimics slow synaptic transmission in both nerve plexuses, suggesting a possible cAMP dependent mechanism. In fact, the slow EPSP-like response to 2- chloradenosine in submucous neurons is sensitive to PKA inhibition and therefore may involve cAMP [320]. In the myenteric plexus, A2aR activation in a subset of AH neurons leads to enhanced excitability [70, 71]. We show in this study that CGS 21680 causes a rise in cAMPir in 65% of possible cAMP-responsive submucous neurons (Fig 42A). The response was sensitive to blockade by the A2aR antagonist CSC indicating that A2aR activation leads to the cAMP response in the neurons (Fig 42A & 57). Therefore, as predicted from electrophysiological studies, our cAMP visualization data provide unequivocal proof that A2aR activation of AC leads to a rise in intracellular cAMP that underlies the slow EPSP-like response observed in S/Type I neurons of the submucous plexus. This is a significant finding on purinergic signaling in neurons because it is the first time that unequivocal proof has been obtained linking A2aR/AC/cAMP signaling and elevation of intraneuronal free cAMP in the cytoplasm of an intact neuron. All
217 previous studies in CNS or other tissues relied on isolated membrane preparations
(contaminated by glial component) and cAMP content after providing the ATP substrate
to the assay. In our study, we show that intraneuronal cAMP increases in response to
A2aR activation in morphologically identified and visualized neurons. This technique
should prove useful in direct CNS studies on A2a, A2b, A3, and A1 – AC coupling in
neurons.
A subset of VIP secretomotor neurons contains functional A2a receptors (Figs
44A & 57) making up 31% of VIPir neurons. This represents ~ 50% of VIP
secretomotor neurons that can generate cAMP (in response to forskolin). Thus, VIP
secretomotor neurons can be further subdivided according to those with or without
functional A2aRs. Our novel finding is that the activity of subsets of VIP secretomotor
neurons is differentially regulated and distinguished by AC/cAMP signaling,
cAMP/cGMP negative cross-talk and A2aR activation. Previous studies in the CNS have
shown that A2A activation causes VIP neurotransmitter release in subset of VIP ir
neurons [325], therefore our data suggest that a similar mechanism may exist in the ENS.
Comparing the distribution of cAMP visualized neurons/ganglion/cm2 indicates that neurons containing A2aRs are found in subsets of ganglia of variable size (3-10 neurons/ganglion) that can generate cAMP in response to forskolin. A2aR activation did not lead to cAMP ir in NPY secretomotor neurons, which is consistent with forskolin stimulation of the SMP where NPY neurons were never co-localized with cAMP ir (Figs
42B and 44B) further supporting that A2a receptors are coupled to AC only to specific functional groups of neurons within the SMP.
218 Since approximately 60% of all possible SMP neurons expressed cAMP ir to
forskolin stimulation suggest that nearly 39% of all possible submucous neurons
expressed A2a receptors implying that coupling of this receptor to AC is critical in
submucosal neurons. Interestingly short exposures to forskolin without any PDE
inhibitors did not reveal any discrete localizations of cAMP-IR, whereas, activation of
A2aRs for a brief period (i.e. 10s, 30 sec, 1min or 2min, 10-6 or 10-8M) causes a localized cAMP response in specific regions of the neuron (Fig 46). This is consistent with discrete localization of A2aRs on cell somal membranes near the sites of activation of AC leading to localized elevation of cAMP ir in the neurons [326]. The forskolin response was less discrete, because AC isoforms are expressed throughout the cell and general AC activation with forskolin raises cAMP ir everywhere in the cell. Compartmentalization into cAMP microdomains occurs in cardiac myocytes [155, 202] and many other cell types [202]. The role of PDEs has been delineated with the use of fluorescent indicators that permit real-time measurements of cAMP [327-330].
Indirect short circuit studies had previously hypothesized that cyclic AMP
dependent neural secretion occurred in the intestine via activation of AC/cAMP signaling
in submucous neurons and this was mediated by VIP and NPY secretomotor neurons
[283, 284]. Based our experiments above where forkolin activated only VIP-ergic
neurons (Fig 44B) but not NPY neurons it is possible that a component of Isc circuit is
driven by cAMP. Since several synaptic blockers, the adenosine A1 receptor agonist
(CCPA), nicotinic blocker (Hexamethonium), a Na+ channel and nerve conductance blocker (TTX) and synaptic blockade with low Ca2+/high Mg2+ reduced the number of
219 cAMP ir neurons and the cAMP response to forskolin it is suggestive that cAMP is involved in mediating cell-to-cell communication in the SMP (Fig 47). In a different experiment CCPA, TTX or the nicotinic blocker (mecamylamine) was able to reduce the forskolin stimuated Isc response, further strengthening the hypothesis that cAMP is involved in driving synaptic transmission within the SMP either directly or indirectly.
Three times as many neurons/cm2 display cAMP responses (ncAMPi) in submucous neurons representing >70% of population compared to 15-20% of population of myenteric neurons. The more discrete localization of cAMP signaling in the myenteric plexus argues for very different physiological roles of the second messenger in the two nerve plexuses. It also suggests that SMP may be a better model to study coupling of the cAMP signaling to electrical activity / EFS stimulation or single cell recording, and that we would have better chance in ascertaining whether a 1:1 relationship exists between forskolin activation of AH or S neurons and ncAMPi. EFS stimulation was able to elevate ncAMPi in 5% of possible cAMP-responsive myenteric neurons (i.e. calculated with forskolin stimulation) and a greater proportion of 20% of submucous neurons. This provides direct proof that ncAMPi is linked to the electrical behavior of a subset of enteric neurons. The remaining responses in cAMP visualized neurons may represent other functions of ncAMPi in neuronal plasticity, transcriptional regulation, metabolism, or regulating transport of proteins to their target sites, etc. If we find that neurons with
Dogiel Type I and Dogiel Type II morphologies display a rise in ncAMPi, and blockade of nerve conduction does not abolish such responses, it could be concluded that somal excitability in both AH and S neurons is linked to activation of AC/cAMP signaling. Co- labeling studies will be used to assess the chemical coding of various classes of enteric
220 neurons that can be predicted by their shapes from cAMP visualization studies. Various
inhibitors of synaptic transmission etc. can be used to assess the role of various receptors
in neuronal communication via cAMP signaling.
Acute inflammation with Trichinella spiralis infection causes neural plasticity,
long-term changes in gene regulation and hyperexcitability of AH primary afferent
neurons. AH hyperexcitability is mimicked by treatments that elevate intraneuronal
cAMP levels. The hypothesis was tested that neuronal plasticity in myenteric neurons of
the Trichinella spiralis – inflamed jejunum results from amplification and immune
modulation in the adenylyl cyclase / cAMP signaling pathway. In these studies, guinea-
pigs were orally inoculated with 8,000 T. spiralis muscle-stage larvae or received saline
without the worms. Studies done at the peak of inflammation (days 6-8 post-infection)
involved cAMP analysis in isolated myenteric ganglia, electrophysiology, CREB
phosphorylation (pCREB), AC expression and analysis of inflammatory markers. Data
indicated that infection caused AH cell hyper-excitability and increased myeloperoxidase, tryptase, LTB4, PGE2, TNFα and TXB2 by 150% to 7,500%. An 830% increase in AC expression (147kDa) occurred in 73% of calbindin-D28 and 43% of calretinin- immunoreactive neurons. In inflamed jejunum, Ro-20-1724, forskolin, histamine or substance P treatment caused a 110% to 190% greater cAMP response. T. spiralis,
forskolin, or histamine caused a 600% - 4,000% increase in pCREB (47kDa band) in
calbindin-D28 and calretinin-positive neurons. CREB phosphorylation was blocked by
inhibiting protein kinase A activity. Blockade of histamine receptors or other
inflammatory targets partially blocked AH hyper-excitability. Our findings support the
hypothesis that neuronal plasticity and hyperexcitability in Trichinella spiralis inflamed 221 gut is the result of amplification and immune-modulation of AC/cAMP signaling leading to CREB phosphorylation and transcriptional regulation in primary afferent, cholinergic and other neurons. Such neural plasticity may contribute significantly to enteric neural dysfunction since; neurotransmitters, neuromodulators or immune mediators target the
AC/cAMP pathway.
The Trichinella model has been suggested to provide a useful approach to the study of post-infectuous Irritable Bowel Syndrome. In fact, dysmotility and abnormal responses in transmitter release persist in this model as late as 2 months post-infection.
The possibility was raised in our submitted manuscript to Gastroenterology that persistence of the AC/cAMP amplification during the post-infective period would provide a mechanism for the abnormal changes occurring in post-infective IBS. Studies in Dr. Christofi’s laboratory are aimed at pursuing the role of AC/cAMP amplification and transcriptional regulation leading to neuronal plasticity and hyperexcitability in sensory signaling in post-infective T. spiralis inoculated animals.
Overall, cyclic AMP signaling plays a critical role in ENS in both normal and inflamed gut, where it involves transcription and neural plasticity. It is involved in specific neural circuits, polysynaptic pathways, neurosecretion, and motility reflexes. The cAMP antiserum provided new insights into cAMP function in the ENS.
T. Spiralis is a human parasite that affects human beings especially in third world countries when people eat improperly cooked pork, making this parasite an ideal model to study acute inflammation since it has direct human implications. We have shown for the first time that acute inflammation with this nematode alters the expression of AC and more downstream expression of pCREB which might provide an explanation for the
222 hyperexcitability observed in AH neurons. However our model also showed that cells
other than Dogiel Type II (AH) neurons show an increase in AC expression, notably
calretinin cholinergic neurons, suggesting that infection leads to an augmentation in
AC/cAMP signaling several types of enteric neurons. As noted earlier, a majority of
neurons with cAMP responses were Dogiel Type I neurons – calretinin positive neurons
have Dogiel Type I morphology and display an increase in AC expression after infection
with the nematode.
It was recently hypothesized that acute infection with T. Spiralis, a Rotavirus or
other organisms leads to permanent alterations in the enteric nervous system and post-
infectious IBS. The chronic symptoms of IBS include diarrhea or constipation, a bloated
stomach and painful abdominal episodes. Thus far we have evidence that the AH neuron
is the target neuron that becomes hyperexcitable during acute T. Spiralis inflammation or chronic TNBS-induced colitis. However, it is likely that multiple channels and signaling pathways may be involved in the hyperexcitability of neurons in the two models.
Therefore further work is needed to further understand the various intracellular pathways that ultimately drive excitability of these neurons and to further understand how different stimuli (T. Spiralis vs TNBS) can selectively turn on this specific pathways.
Although our T. Spiralis data suggest that the AC/cAMP signaling pathway is
upregulated since we saw an increase in AC expression, cAMP content, and an increase
in P-CREB during acute infection with the nematode it remains unknown if other
pathways such the PKC, Ca2+ or Ca2+/ Calmodulin, which are known to indirectly
activate AC are also involved. Therefore future experiments will have to address these
questions by blocking PKC blockers or Ca2+ chelators.
223 Infection with the T. Spiralis nematode causes nausea, vomiting, followed by
activation of stereoptype reflexes in the intestine, which involve severe toxic diarrhea,
and power propulsion in an effort by the host to try to expel the nematode. It is unknown
which specific pathways within the enteric nervous system that get activated after acute
infection with T. Spiralis. However our data suggest that AC/cAMP signaling is one of the mechanisms utilized to activate descending reflexes. Therefore, it is hypothesized that descending reflexes would be exaggerated in the T. spiralis infected animals – this remains to be tested in 3-chamber studies. It is also likely that peristalsis (both ascending and descending components of the reflex would also be affected because the sensory AH neurons display hyperexcitability, and they initiate both ascending and descending reflexes.
Hyperexcitability in sensory AH neurons and facilitation of synaptic transmission
that are known to occur in T. spiralis infection can activate signaling pathways
(Ca2+/calmodulin, AC etc….) leading to immediate early gene transcriptional events that
translate to permanent changes in neuronal function.
7. Role of mast cells in AC/cAMP upregulation i.e histamine connection & Dr.
Woods hypothesis about immune neural connections.
Another emerging hypothesis on the cause of IBS involves ongoing stimulation of
mast cells from extrinsic nerve fibers releasing transmitters causing ongoing release of
histamine resulting in tonic activation of enteric neurons (primarily IPANs) which in turn
activate motor neurons which ultimately activates smooth muscle, glands and local blood
vessels resulting in symptoms observed in IBS such as diarrhea, bloated stomach and
severe abdominal pain. Our findings provide support for this hypothesis and show that
224 ongoing release of histamine contributes to the cAMP dependent AH cell hyperexcitability. However, our findings go further to suggest that cAMP-dependent transcriptional regulation upregulates the AC/cAMP pathway and may account for neuroplasticity in the inflamed gut.
What is the relationship of the cAMP ir response to the total electrical activity of the neurons? It has been difficult for the laboratory to show a one-to-one relationship between an electrophysiological response and visualization of the same neuron using ncAMP ir. The discrepancy can be explained by several factors: There are technical issues - The acrolein fixation process causes the biocytin inside the cell to leak out making it difficult to re-identify the same neuron. Attempts were done to overcome this technical difficulty by fixing the tissues with PFA and acrolein in an effort to preserve the biocytin but this compromise the cAMP which would now leak out of the cell. The number of viable AH or S neurons in microdissected/damaged gut tissues used for electrical recording, biases the sampling, since we can only record and report on viable healthy neurons that can be punctured by the microelectrode. In intact tissues used for cAMP visualization, the entire population of neurons is studied at once, and therefore major discrepancies can exist between a neuron that responds to forskolin with an increase in ncAMPir and a slow EPSP like response in recorded AH neurons. Therefore, the smaller proportion of Dogiel II (AH) neurons that respond to forskolin with an ncAMPir response could represent a more accurate assessment. Alternatively, the cAMP antiserum may not be sensitive enough to detect minute physiological changes in ncAMPir that are needed close to their site of action to influence channel activities and alter neuronal excitability. A main difference in studies on ncAMPir in intact tissues
225 compared to intracellular recordings from individual neurons in intact tissues is that
puncturing the neuron to record it could affect AC – a priming effect on AC by
mechanical puncturing of the neuron could increase sensitivity to forskolin.
Other potential targets besides AC/cAMP signaling exist that may contribute to
the hyperexcitability in AH neurons and facilitation of synaptic transmission.
Hyperexcitability in AH neurons results from phophorylation of the calcium-dependent-
potasium channels (Kca) causing closure of these channels leading to membrane
depolarization and increase in neuronal excitability. Direct effects of various
inflammatory mediators on Ih channels involved in the repolarization of the AHP
response in AH neurons could influence the excitability of enteric neurons in some
inflammatory conditions (i.e. shown for TNBS colitis). Inflammation could also alter the
number of excitatory (i.e. neuropeptide) or inhibitory (adenosine) receptors, the G-
proteins (Gi, Go, Gs) or other mechanisms coupled to AC. It has already been shown that
in patients treated with β1-receptor agonists to help with CHF eventually develop resistance to the drugs because of changes in the number of receptors coupled to AC. It is also possible that inflammation causes changes in the regulatory mechanisms of AC by altering the ratio of Gs/Gi proteins coupled to AC, which drastically affect AC activity.
For example decreasing the amount of Gi would eliminate inhibition of AC and therefore cause an increase in cAMP production compared to baseline. Ongoing exposure to inflammatory mediators released during inflammation can also cause changes in protein production which could alter cross-talk between AC and PKC, Gq / PLC or Gi/Gs etc.
There is a lot of evidence to support that MAPKinases are involved in every step of the
226 inflammatory response and it is likely that these pathways are also altered during acute
inflammation with T. spiralis which has been shown to indirectly activate AC activity.
Since the myenteric plexus role is to drive primarily peristalsis in a stereotypical
manner it is not surprising to see polarity in the myenteric and not in the submucous since
its main function is to regulate secretion, absorption and local vessel function. The
importance of this is significant because when the gut is exposed to a noxious agent
(campylobacteriosis) or a parasite (T. spiralis) the gut functions to evacuate the noxious agent in an anal direction. Our data suggest that a possible player in this reflex response is cAMP since the majority of the neurons projected in the anal direction.
Besides affecting neuronal excitability in the neurons, the AC/cAMP signaling
has been shown to be involved in nearly every process within the neuron from
metabolism (providing energy), transcriptional and translational events and targeting
proteins to their specific location.
227
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