Presynaptic Regulation of Carotid Body Type I Cells by Histaminergic and Muscarinic Receptors

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Carrie Marie Thompson Wright State University

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PRESYNAPTIC REGULATION OF CAROTID BODY TYPE I CELLS BY HISTAMINERGIC

AND MUSCARINIC RECEPTORS

A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science

CARRIE MARIE THOMPSON B.S., Wright State University, 2006

2010 Wright State University

WRIGHT STATE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

DATE OF DEFENSE

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Carrie Marie Thompson ENTITLED Presynaptic Regulation of Carotid Body Type I Cells by Histaminergic and Muscarinic Receptors BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF Master of Science.

Christopher Wyatt, Ph.D. Thesis Director

Timothy Cope, Ph.D. Department Chair

Committee on Final Examination

Robert Putnam, Ph.D.

Mark Rich M.D., Ph.D.

Andrew T. Hsu, Ph.D. Dean, School of Graduate Studies

ABSTRACT

Thompson, Carrie Marie. M.S., Department of Neuroscience, Cell Biology, and Physiology, Wright State University, 2010. Presynaptic Regulation of Carotid Body Type I Cells by Histaminergic and Muscarinic Receptors.

Type I cells are one of two main cell types located within the carotid body. These cells respond to hypoxia, hypercapnia, and acidosis by releasing excitatory and inhibitory neurotransmitters. This causes increased firing of the carotid sinus nerve and restores blood gas levels to their physiological values. While previous studies have shown whether individual neurotransmitters are excitatory or inhibitory, this work demonstrates how the interplay between two neurotransmitters may potentially shape the output of the carotid body. Histamine, which has previously been shown to have no effect on intracellular Ca2+ in type I cells, may function to modulate the actions of an excitatory neurotransmitter such as acetylcholine. Using Ca2+-imaging techniques, this work shows that histamine inhibits the acetylcholine muscarinic receptor-induced rise in intracellular Ca2+. Histamine, acting on the Gi-coupled H3 receptor, may exert its actions by inhibiting the activation of adenylate cyclase and therefore reducing levels of cAMP. The observed inhibition of muscarinic Ca2+ signaling seems independent of protein kinase A (PKA), as two PKA inhibitors did not mimic the inhibition. Data suggest that Gi-coupled receptor activation may inhibit muscarinic Ca2+ signaling in type I cells by the inhibition of Exchange Proteins Activated by cAMP (Epac).

Some of this work has been published previously:

Thompson, C. M., et al (2010). Neuroscience Letters 471, 15-19

iii

TABLE OF CONTENTS

Page

I. INTRODUCTION ...... 1

Anatomy ...... 1

History ...... 4

Type I Cell Signal Transduction ...... 5

Membrane Hypothesis ...... 5

Mitochondrial Hypothesis ...... 9

Neurotransmitters ...... 11

Dopamine ...... 11

Serotonin ...... 14

ATP ...... 15

Acetylcholine ...... 17

Histamine ...... 21

Hypothesis ...... 24

Summary ...... 24

II. MATERIALS AND METHODS ...... 25

Dissection and Dissociate of Neonatal Rat Carotid Body Type I Cells ...... 25

Fura-2 AM ...... 27

Calcium Imaging ...... 30

Perfusion of Isolated Type I Cells ...... 31

Statistical Significance ...... 32

Chemicals ...... 32

III. RESULTS ...... 34

Effect of Methylcholine on [Ca2+]i...... 34

Effect of Histamine on [Ca2+]i ...... 37

Effect of the H3 Receptor Agonist on [Ca2+]i...... 37

Effect of the Adenylate Cyclase Inhibitor ...... 37

Effect of Protein Kinase A Inhibitors ...... 40

A Recently Discovered Pathway: Exchange Proteins Activated by cAMP ...... 45

IV. DISCUSSION ...... 55

Future Experiment ...... 58

Conclusion ...... 58

V. REFERENCES ...... 62

LIST OF FIGURES

Figure Page

1. A schematic demonstrating the anatomy of the carotid body ...... 3

2. Heymans’ experiment in which two living canines were attached via parabiosis ...... 7

3. A schematic demonstrating the release of neurotransmitters from carotid body type I cells ...... 13

4. Fluorescence emission of Fura-2 AM at differing wavelengths ...... 29

5. Inhibitory effect of histamine on muscarinic receptor induced calcium signaling in rat carotid body type I cells ...... 36

6. Inhibitory effect of selective histamine H3 agonist on muscarinic receptor induced calcium signaling in carotid body type I cells ...... 39

7. Inhibitory effect of adenylate cyclase inhibitor SQ22536 on muscarinic receptor induced calcium signaling in carotid body type I cells ...... 42

8. PKA inhibition does not significantly attenuate muscarinic receptor induced calcium signaling in rat carotid body type I cells ...... 44

9. PKA inhibition does not significantly attenuate muscarinic receptor induced calcium signaling in rat carotid body type I cells ...... 47

10. Inhibition of Exchange Proteins Activated by cAMP (Epac) significantly inhibited muscarinic receptor induced calcium signaling in rat carotid body type I cells ...... 49

11. Low dose activation of Epac does not potentiate the effects of muscarinic receptor induced calcium signaling in rat carotid body type I cells ...... 52

12. Higher does activation of Epac potentiates the muscarinic receptor induced calcium signaling in rat carotid body type I cells ...... 54

13. A schematic demonstrating how the activation of histaminergic and muscarinic receptors affect intracellular calcium levels ...... 61

vii

LIST OF TABLES

Table Page

1. Experimental Compounds ...... 33

viii

ACKNOWLEDGEMENTS

There are several people who contributed to this project whom all have my deepest appreciation and respect. Dr. Christopher Wyatt has spent the past year guiding me through the entire research process. He has been a wonderful mentor and has provided me with opportunities I never thought I would have when I entered my graduate program. Additionally, the rest of the Wyatt lab, Heidi Jordan and Barbara

Barr, have also been a tremendous support. Furthermore, I would like to thank Dr.

Robert Putnam and Dr. Mark Rich for their guidance, suggestions, and feedback for my research project.

Lastly, I would like to thank my wonderful friends and family for their constant encouragement. Most importantly, I would like to thank my parents, Rhonda and Doug, and my sister, Natalie, who have always had faith in me even when I didn’t have it in myself. They have reminded me of the light at the end of the tunnel even during my most stressful of times.

I: INTRODUCTION

ANATOMY

The carotid bodies (CB) are small organs located at the bifurcation of the common carotid artery in mammals (Fig. 1 A). These organs function as the primary peripheral chemoreceptors to modulate breathing in regards to changes in levels of arterial PO2, PCO2, and [H+]. In response to hypoxia, hypercapnia, or acidosis, the CB releases a series of neurotransmitters that eventually lead to hyperventilation.

The CB has duel innervation. First is the carotid sinus nerve (CSN), a branch of the glossopharyngeal nerve, cranial nerve IX, whose cell bodies lie in the petrosal ganglion. The CSN afferently projects to the nucleus tractus solitarius of the medulla

(Finley and Katz, 1992). The superior cervical ganglion, via the ganglioglomerular nerve, innervates the CB as well. The ganglioglomerular nerve is the efferent component that functions to primarily innervate the blood vessels controlling the blood flow to the CB (Gonzalez et al, 1994). A large area of the CB is covered with blood vessels (McDonald, 1981) with up to a third of its volume being blood vessels (de Castro and Rubio, 1968). The CB is one of the most richly supplied organs in the body with perfusion from branches of the common carotid artery, mainly via the internal carotid body artery (Gonzalez et al, 1994). The chemosensory organ is drained via tributaries of the internal jugular vein (Gonzalez et al, 1994).

The CB parenchyma consists of two major cells types: type I (glomus) cells and type II (sustentacular) cells (Fig. 1 B). The majority of cells, type I cells, are the sensory receptors responsible for detecting levels of arterial PO2, PCO2, and [H+]

1 Figure 1

A schematic demonstrating the anatomy of the carotid body. A. The carotid body shown at the bifurcation of the common carotid artery (CC), lying on the internal carotid artery (IC) across from the external carotid artery (EC). B. Type I cells enveloped by type II cells in the carotid body. Type I cells are innervated by the carotid sinus nerve (CSN).

3 (Stea and Nurse, 1991, Heymans et al, 1930). When PO2 or pH falls or PCO2 gets too high, the type I cells respond by releasing neurotransmitters that cause an increase in action potential frequency along the CSN. In addition to their connections with nerve endings, type I cells also have gap junction connections with one another (Gonzalez et al, 1994). This allows for cellular communication among type I cells. Type II cells have a more glial-like function. The processes of these cells surround the junctions formed by type I cells and nerve endings (Prabhakar, 2000) and may also serve to phagocytize damaged nerve endings (McDonald, 1981). However, these cells lack any excitable properties. Some studies have shown other functions of the type II cells. For example,

Pardal et al (2007) found that the type II cells serve as a stem cell population for type I cells.

HISTORY

Jacques-BénigneWinslow first described the carotid bodies in the 1600’s

(Kellogg, 1981). Without a prior classification and improving technology, another anatomist, Hubert Luschka, classified the CB as a gland in 1862 (McDonald, 1981).

Much more detailed information became available in the 1920’s due to the work of

Fernando de Castro. The CB was initially considered neurosecretomotor, but de Castro

(1926) discovered that the cell bodies of the CSN lay within the petrosal sensory ganglion thus proving that the CB was sensory. Originally thought to be a gland, de

Castro (1928) concluded that the CB was in fact a sensory organ. He continued his work by hypothesizing that the CB functioned to sense changes in arterial blood composition, such as a decrease in PO2 or pH or an increase in PCO2, and that the type I

4 cells functioned as the chemosensors (de Castro, 1928). It was another researcher,

Corneille Heymans, who proved that these changes in blood composition acted on the chemosensors in the CB to elicit a response. His experimental design consisted of two living canines attached via parabiosis, one canine’s head was connected by arteries of the second canine (Fig. 2). In his experiment the second canine was exposed to hypoxia, and the first canine began to hyperventilate (Heymans et al, 1930). For this work,

Heymans won the Nobel Prize in Physiology or Medicine in 1938 (De Castro, 2009).

TYPE I CELL TRANSDUCTION

Carotid body type I cells respond with depolarization to adverse chemostimuli, such as hypoxia, due to the inhibition of chemosensitive K+ channels (Lopez-Barneo et al, 1988). In the rat there are two different types of chemosensitive K+ channels, the high-conductance Ca2+ -dependent K+ channels and the TASK-like background K+ channels (Peers, 1990, Buckler et al, 2000, Buckler, 1997). The membrane depolarization results in voltage-gated Ca2+ entry causing a rise in intracellular calcium

([Ca2+]i) (Buckler and Vaughan-Jones, 1994) and consequential neurotransmitter release (Gonzalez et al, 1994). Exactly what causes the K+ channels to be inhibited in the first place is debatable and has given rise to two different chemotransduction hypotheses: the membrane hypothesis and the mitochondrial hypothesis.

MEMBRANE HYPOTHESIS

The membrane hypothesis is based on the fact that during hypoxia, plasma membrane K+ channels are inhibited (Lopez-Barneo et al, 1988) resulting in type I

Figure 2

Heymans’ experiment in which two living canines were attached via parabiosis. Canine

A was connected to Canine B through the vessels of their necks and thoracic cavities.

Canine A was subjected to hypoxia, and Canine B responded by hyperventilating.

7 cell depolarization due to voltage-gated Ca2+ channels. The type of K+ channel involved varies among species (Prabhakar, 2000). This introduction will focus only on two classes of K+ channels that are chemosensitive in the rat.

The first class, high-conductance Ca2+ -dependent K+ (BK) channels, are open at resting membrane potential; therefore, they may be able to regulate action potentials of cells (Lahiri et al, 2006). BK channels are thought to play a vital role in regulating type

I cells during hypoxic responses (Peers, 1990). The enzyme hemeoxygenase-2 (HO-2) is the suggested oxygen sensor (Williams et al, 2004). HO-2 causes the production of carbon monoxide (CO), which tonically activates the BK channels (Williams et al, 2004,

Kemp, 2005). During a hypoxic challenge CO levels drop to a level that can no longer sustain the activation of BK channels (Kemp, 2005). However, the fact that HO-2 null mice demonstrate similar CB output in response to hypoxia as control mice shows that

HO-2 may not be the sole oxygen sensor (Ortega-Saenz et al, 2006).

The BK channels have also been studied by pharmacological and patch-clamp methods. Only one BK channel blocker, ChTX, has been shown to depolarize type I cells

(Wyatt and Peers, 1995). Using the patch clamp technique it has been shown that hypoxia affects the BK channels depending on the exact patch-clamp method that is used. With whole-cell and inside-out configuration, BK channels of type I cells are sensitive to hypoxia (Riesco-Fagundo et al, 2001). However, when the outside-out configuration is applied, the same channels are no longer sensitive to hypoxia (Wyatt and Peers, 1995). This indicates that something is lost in the cytoplasm due to the various patch-clamping method that is used, so the membrane alone is probably not responsible for type I cell depolarization.

8 The second category of oxygen-sensing K+ channels that were found to be open at resting membrane potential in type I cells are the Twik-related acid sensing K+

(TASK-like) channels (Lahiri et al, 2006). These channels were first described as voltage-insensitive background K+ channels (Buckler, 1997), and upon further research, these channels were found to be similar to TASK channels and thusly named TASK-like channels (Buckler et al, 2000). Recently, research has shown that the TASK-like channels expressed in type I cells are TASK-1, TASK-3 and a TASK-1/TASK-3 heteromer, which is responsible for the majority of oxygen-sensing background K+ current (Kim et al, 2009). Hypoxia inhibited TASK channels leading to depolarization in

CB type I cells and consequent rise in [Ca2+]i (Buckler et al, 2000, Buckler, 1997).

Finally, mitochondrial inhibitors and uncouplers inhibited TASK channel currents. The fact that these channels are sensitive to mitochondrial metabolism implies that these channels may detect blood O2 levels by a mitochondrial mechanism. Thus, functional mitochondria may be required for hypoxic inhibition of the chemosensitive TASK-like channels in type I cells (Buckler et al, 2006).

MITOCHONDRIAL HYPOTHESIS

The process of oxidative phosphorylation, occurring in the mitochondria, requires oxygen as the final electron acceptor to produce ATP via cellular respiration.

In times of low oxygen, or hypoxia, this process will slow or be halted. The fact that oxygen is central to this process allowed for the development of the mitochondrial hypothesis of type I cell transduction (Lahiri et al, 2006). Mills and Jobsis (1972) proposed that a particular mitochondrial complex in type I cells must have an unusually

9 low affinity for oxygen to allow for oxygen sensing. It was also found that during hypoxia the mitochondrial membrane of oxygen-sensing type I cells depolarizes, but in non-oxygen-sensing chromaffin cells of the adrenal gland and sensory neurons of the dorsal root ganglion, the mitochondrial membrane does not depolarize until anoxia

(Duchen and Biscoe, 1992b, Duchen and Biscoe, 1992a). This information provides further support for the mitochondrial hypothesis and indicates that type I cell mitochondria respond to hypoxia in a specialized manner.

Mitochondrial electron transport chain inhibitors and uncouplers as well as ATP synthase inhibitors have been used pharmacologically to mimic the effects of hypoxia.

This is seen by an increase in [Ca2+]i in type I cells which causes a release of neurotransmitters that act post-synaptically on the CSN. Heymans et al (1931) used cyanide, a known mitochondrial IV complex inhibitor, to increase CB activity.

Oligomycin, an ATP synthase inhibitor, was used to show a link between mitochondrial function and the increased firing activity of the CSN due to hypoxia is via the inhibition of ATP mitochondrial production (Mulligan et al, 1981, Wyatt and Buckler, 2004).

Inhibitors of complex I, complex III, and complex IV caused an increase in [Ca2+]i as well as inhibition of background K+ channels (Wyatt and Buckler, 2004). These mitochondrial inhibitors also prevent the type I cells from further responding to hypoxia suggesting a common mechanism that mediates both processes (Mulligan et al,

1981, Wyatt and Buckler, 2004).

How the mitochondria of type I cells couple to the K+ channels of the plasma membrane is not exactly known. One possible mechanism is that ATP regulates TASK- like K+ channels such that when ATP levels decrease, the K+ channels are inhibited

10 (Varas and Buckler, 2006). Another hypothesis in this matter involves AMP-activated protein kinase (AMPK). Due to the decreased ATP levels, an increase in the AMP/ATP ratio activates AMPK, which in turn inhibits the K+ channels. The inhibition of the K+ channels then depolarizes the cell activating the voltage-dependent calcium channels.

The influx of calcium causes the release of neurotransmitters from the type I cells of the

CB (Evans, 2006).

NEUROTRANSMITTERS

This thesis will focus on the interaction of neurotransmitters at the level of the carotid body type I cells. The rise in intracellular calcium ([Ca2+]i) during chemostimuli causes the release of a series of neurotransmitters. The neurotransmitters can either act presynaptically (in a paracrine or an autocrine fashion) or postsynaptically acting on the CSN (Fig. 3). The balance between the release of excitatory and inhibitory neurotransmitters determines the nature of the response of the CSN.

DOPAMINE

Dopamine (DA) is one of the monoamines produced, stored, and released by the

CB during hypoxia. It was once considered to be the main neurotransmitter of the CB since DA is found at its highest levels peripherally in the CB (Gonzalez et al,

1994). To produce DA, type I cells require the enzymes tyrosine hydroxylase (TH).

Type I cells also contain VMAT 1 and VMAT2 for the storage of DA. In addition, the proteins SNAP25 and syntaxin1 were shown in type I cells as they are required for the release of DA (Koerner et al, 2004).

11 Figure 3

A schematic demonstrating the release of neurotransmitters from carotid body type I cells. Neurotransmitters released from type I cells can bind to receptors on the surface of the cell releasing them, on the surface of neighboring type I cells, or on afferent fibers of the carotid sinus nerve.

13 DA acts on D2 receptors found on the surface of type I cells and the CSN (Dinger et al, 1981, Mir et al, 1984). During hypoxic challenges the D2 receptor agonist inhibited the expected rise in [Ca2+]i; however, once the D2 receptor antagonist was applied, the rise in [Ca2+]i was restored (Carroll et al, 2005). This indicates that DA is inhibitory presynaptically in most species of animals (Jiang and Eyzaguirre, 2004). In D2 knockout mice the action potential frequency of the CSN was lowered compared to wild-type mice indicating that DA is probably not essential to CB function, but it plays some role in shaping the CB output (Prieto-Lloret et al, 2007). It should be noted that while DA is considered to be inhibitory in most species, the rabbit is the exception; DA has excitatory effects on the CSN (Iturriaga et al, 2009).

SEROTONIN

The second monoamine localized in the CB is serotonin (5-HT). 5-HT5a receptors are expressed in the CB, PG, and SCG meaning that serotonin may have effects both pre- and post-synaptically (Wang et al, 2000). Additionally, 5-HT2 receptors, Gq- coupled receptors, are located on type I cells (Zhang et al, 2003). Application of serotonin caused the depolarization of type I cells acting on 5-HT2 receptors. Protein kinase C (PKC), which is activated during Gq-coupled receptor stimulation, inhibits background K+ channels accomplishing the depolarization (Zhang et al, 2003).

Zhang discovered several different effects that are accomplished using the 5-

HT2, PKC-mediated pathway in the CB (2003). First, it was discovered that the spontaneous activity seen in type I cell clusters is due to serotonin acting in an autocrine / paracrine mode, and that serotonin is responsible for the presynaptic

14 modulation of type I cell transduction during hypoxia. Moreover, the spontaneous activity seen postsynaptically on afferent petrosal ganglion neurons is due to serotonin acting presynaptically on type I cells causing the release of the excitatory neurotransmitters acetylcholine and ATP (Zhang et al, 2003). Taken together, these experiments demonstrate the role of serotonin in a positive feedback loop that modulates the excitatory effects seen both pre- and post-synaptically in the CB.

ATP

Originally thought to be the main excitatory neurotransmitter released from the

CB, the role of acetylcholine (ACh) has been debated for some time (McQueen, 1977,

Donnelly, 2009, Dasso et al, 1997, Wyatt and Peers, 1993). Some of the debate lies in the fact that when cholinergic receptors are blocked, the petrosal ganglion nerves still depolarize during a hypoxic challenge (Zhong et al, 1997, Nurse and Zhang, 1999, Zhang et al, 2000). Using a co-application of a blocker of P2 purinoceptor, an ATP receptor, and a cholinergic receptor blocker, researchers were able to completely abolish the hypoxic-induced depolarization of the petrosal ganglion nerves (Zhang et al, 2000).

This reflects the co-release of two main excitatory neurotransmitters in CB transduction, ACh and ATP.

During a hypoxic challenge, ATP is released from the CB (Buttigieg and Nurse,

2004). ATP acts on P2X receptors with the specific P2X2 and P2X3 subunits (Zhang et al,

2000, Prasad et al, 2001). The two subunits can form either homomeric or heteromeric

P2X receptors (Rong et al, 2003). Using knockout mice, Rong was able to show that

P2X2-/- and P2X2/P2X3Dbl -/- mice had a decreased ventilatory response to hypoxia

15 compared to their wild-type littermates. Additionally, the two knockouts groups also showed a significant decrease in CSN activity during a hypoxic challenge compared to their wild-type littermates (Rong et al, 2003). Taken with the fact that P2X3-/- mice showed no difference compared to the wild-type animals to a hypoxic challenge, P2X2 subunits are thought to be vital to the CB chemotransduction in response to hypoxia

(Rong et al, 2003).

Immunohistochemical staining and pharmacological techniques have shown that

P2X2 and P2X3 subunits are located in the carotid body but not specifically on type I cells in both rats and mice (Zhang et al, 2000, Prasad et al, 2001, Rong et al, 2003).

Nonetheless, the receptors containing the two subunits are found postsynaptically on the afferent terminals closely associated with type I cell clusters and on petrosal ganglion neurons (Prasad et al, 2001, Rong et al, 2003). The location of purinergic receptors in the CB-CSN preparation explains why the application of ATP did not cause a rise in [Ca2+]i in type I cells (Xu et al, 2003) but did cause the depolarization of the CSN

(Rong et al, 2003).

Moreover, ATP also acts on P2Y receptors, which are located on both type I and type II cells (Xu et al, 2003, Xu et al, 2005). Its action on type I cells, via P2Y1 receptors, is to inhibit the rise in [Ca2+]i induced by hypoxia (Xu et al, 2005). Therefore, ATP is excitatory postsynaptically, while inhibitory presynaptically possibly acting as a negative feedback regulator of type I cells. Interestingly, ATP caused a rise in [Ca2+]i in type II cells, the glial-like cells found in close association with type I cells found in CB, acting on P2Y2 receptors (Xu et al, 2003). Since ATP is released from type I cells during

16 hypoxia, and type I and type II cells are so closely associated, it is hypothesized that the type II cells may act in some type of modulatory role during a hypoxic response.

ACETYLCHOLINE

Acetylcholine (ACh) is yet another neurotransmitter released from the carotid body due to a chemostimulus (Eyzaguirre and Zapata, 1968). As previously stated, it is co-released with ATP acting as one of the main excitatory neurotransmitters of the CB

(Zhang et al, 2000). Before it was discovered that both ATP and ACh are co-released acting as excitatory transmitters, the role of ACh in CB transduction was debated. Some results could not support the excitatory role of ACh in the actions of the CB (McQueen,

1977, Donnelly, 2009). However, a vast amount of research has been able to clearly demonstrate an excitatory role for ACh. Some of the various actions in chemotransduction via ACh include membrane depolarization of type I cells (Wyatt and

Peers, 1993, Nurse and Zhang, 1999), increase in [Ca2+]i in type I cells(Jiang and

Eyzaguirre, 2004, Dasso et al, 1997), and depolarization of the CSN (Nurse and Zhang,

1999, He et al, 2005).

Research has shown that type I cells possess the proteins necessary to produce, store, and degrade ACh. Choline acetyltransferase (ChAT), the rate-limiting enzyme required for ACh synthesis, vesicular ACh transporter (VAChT), the protein responsible for mobilizing ACh into synaptic vesicles, and acetylcholinesterase (AChE), the enzyme that functions to degrade ACh, are all localized in type I cells (Nurse and Zhang, 1999,

Wang et al, 1989). In addition autonomic microganglion cells also supply type I cells with much of its ACh, specifically in the rat CB (Gauda et al, 2004).

17 Acetylcholine activates two different cholinergic receptors: nicotinic and muscarinic ACh receptors. Both types of receptors are found on the surface of type I cells and postsynaptically on the CSN (Wyatt and Peers, 1993, Shirahata et al, 2007,

Dinger et al, 1991, Zhong and Nurse, 1997, Shirahata et al, 2004). Stimulation of both receptors on the surface of type I cells results in an increase in [Ca2+]i (Dasso et al, 1997,

He et al, 2005).

Nicotinic receptors are ligand-gated ion channels, and their activation opens voltage-gated calcium channels. Binding of a nicotinic agonist to the receptor increases the cell membrane’s permeability to calcium resulting in an increase in [Ca2+]i within milliseconds. When these receptors were activated in a calcium-free medium, no rise in

[Ca2+]i was seen indicating that the source of calcium freed by these receptors is extracellular (Dasso et al, 1997). Activation of nicotinic receptors is responsible for much of the increase in [Ca2+]i (Shirahata et al, 1997). Calcium responses to nicotinic agonists were transient as the calcium levels returned to baseline levels while still in the presence of the agonist indicating nicotinic receptor desensitization (Dasso et al,

1997).

Much of this thesis will focus specifically on the regulation of muscarinic receptor signaling in type I cells. There are five sub-types of muscarinic receptors, the other cholinergic receptor; all of which are G protein-coupled. It is commonly accepted that the odd-numbered muscarinic receptors (M1, M3, and M5) are Gq-coupled, and that the even-numbered muscarinic (M2 and M4) receptors are Gi-coupled (Ishii and Kurachi,

2006). Activation of the odd-numbered receptors will activate phospholipase C (PLC), which causes the cleavage of phosphatidyl 4,5-biphosphate (PIP2) into diacylglycerol

18 (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG enables the activation of protein kinase C (PKC) while IP3 binds to intracellular receptors that cause the release of calcium from internal stores. The released calcium can further activate PKC. PKC has been shown to inhibit BK channels in the membranes of rat type I cells leading to membrane depolarization (Peers and Carpenter, 1998). Additionally, the activation of

G q coupled receptors inhibits TASK channels, which would also lead to cell depolarization (Chen et al, 2006). Furthermore, the activation of muscarinic receptors on rat type I cells has been shown to inhibit TASK channels via a PLC/PKC-mediated mechanism (Ortiz and Varas, 2010). The inhibition of these K+ channels would cause the type I cells to depolarize possibly due to the activation of an odd-numbered, Gq- coupled muscarininc receptor.

The even-numbered receptors are negatively coupled to adenylate cyclase and will prevent the accumulation of cAMP and calcium and inhibit the activation of protein kinase A (PKA). The activation of PKA has been shown to inhibit TASK channels, so by inhibiting PKA activation, the cell membrane may not undergo depolarization and therefore voltage-gated Ca2+ entry would not occur (Xu et al, 2007).

Due to the high level of homology between the five subtypes, the receptor subtypes in the CB and petrosal ganglion of most species have not yet been classified

(Caulfield and Birdsall, 1998). However, in the cat CB, the M1 and M2 receptors have been localized to type I cells by using RT-PCR and immunocytochemistry as well as pharmacologically, but the M2 receptors were not found on petrosal ganglion nerves while the M1 receptors were (Shirahata et al, 2004, Fitzgerald et al, 1997). During the application of a M1 antagonist during a hypoxic challenge, there was a decrease in post-

19 synaptic activity; the opposite was found during the application of a M2 antagonist

(Fitzgerald et al, 1997). These results indicate that activation of the M1 receptor stimulates type I cells, and M2 receptor activation inhibits them, and they further support the idea that odd-numbered receptor activation is excitatory while even- numbered receptor activation is inhibitory. Non-specific muscarinic receptor activation increases [Ca2+]i by causing a calcium influx from extracellular sources just as nicotinic receptor activation does, but additionally, muscarinic receptor activation releases calcium from intracellular stores (Dasso et al, 1997). This is most likely due to the activation of one of the Gq- coupled, odd-numbered muscarinic receptors.

The ratio of nicotinic to muscarinic receptors may also play a role in determining if ACh is excitatory or inhibitory in CB function, and this may be a reason why certain researchers could not support the excitatory role of ACh. For example, Hirano’s research showed that in the rabbit CB, ACh inhibited CSN activity, and in the cat CB, ACh stimulated CB activity (Hirano et al, 1992). It was found that the rabbit CB had a 12:1 ratio of muscarinic to nicotinic receptors. Conversely, in the cat, the ratio of nicotinic to muscarinic receptors in the CB was 2:1 (Hirano et al, 1992). Even though the specific muscarinic receptor subtypes were not identified, it is assumed that it was a Gi-coupled receptor, which would inhibit activity. This data shows another marked difference in

CB actions among species.

Besides being released via reactions to various chemostimuli, ACh is spontaneously released from type I cells (Zhong et al, 1997, Nurse and Zhang, 1999,

Zhang et al, 2000). Resulting from this spontaneity is a spiking activity seen postsynaptically on the CSN (Nurse and Zhang, 1999, Wyatt et al, 2007). The excitatory

20 actions are most likely due to the activation of nicotinic receptors since the use of the nicotinic antagonist mecamylamine inhibited most of the spontaneous activity (Nurse and Zhang, 1999). Furthermore, the spontaneously released ACh will presumably act in an autocrine or paracrine fashion on type I cells since the type I cells have cholinergic receptors.

HISTAMINE

Histamine is yet another transmitter found to be associated with CB function.

Koerner et al (2004) found that the proteins required to produce, store, and release histamine are found in type I cells. Histidine decarboxylase, the rate-limiting enzyme for histamine production, is expressed throughout the CB. The vesicular monoamine transporters, VMAT1 and VMAT2, were also found in type I cells. Finally, SNAP25 and syntaxin1 were shown to be in type I cells (Koerner et al, 2004).

There are four receptor subtypes in which histamine acts upon: H1, H2, H3, and

H4. Several groups have localized the various receptors. All four receptors were found on the surface of type I cells (Koerner et al, 2004, Burlon et al, 2009, Lazarov et al,

2006). Additionally, H1 and H3 receptors were located on petrosal ganglion neurons and in portions of the NTS (Lazarov et al, 2006). The four histamine receptor subtypes are coupled to G-proteins, which make use of second messenger systems.

The H1 receptor is coupled to the G q family of G-protein coupled receptors.

Activation of H1 receptors activates phospholipase C, which in turn, causes the cleavage of phosphotidylinositol 4,5-biphosphate (PIP2) into diacylglycerol (DAG) and inositol-

1,4,5-trisphosphate (IP3) (Hill et al, 1997). DAG causes the activation of protein kinase

21 C (PKC). IP3 binds to intracellular receptors of membrane-bound organelles causing a release of calcium from these intracellular stores. The increase in [Ca2+]i also further stimulates the activation of PKC.

H2 receptors are coupled to adenylate cyclase by way of Gs receptor protein, and when stimulated, cause the accumulation of cAMP (Hill et al, 1997). cAMP then activates protein kinase A (PKA) and calcium channels allowing calcium to enter the cell. Furthermore, a PKA-dependent pathway was shown to inhibit TASK-like channels in type I cells, channels responsible for maintaining resting membrane potential in type

I cells, resulting in cell depolarization and increase in [Ca2+]i (Xu et al, 2007).

Both H3 and H4 receptors are negatively coupled to adenylate cyclase via Gi coupled proteins, and therefore, oppose the actions of H2 receptors. By preventing the activation of adenylate cyase, there is no resulting cAMP accumulation, and therefore, no calcium or PKA accumulation. H3 receptors are thought to be auto-receptors that function to regulate the synthesis and release of histamine and other transmitters

(Arrang et al, 1985, Arrang et al, 1983). Recent research has shown that the G subunit of H3 receptors inhibits voltage-dependent calcium channels, which would further prevent calcium influx (Morrey et al, 2008).

As reported, histamine is released during hypoxia (Koerner et al, 2004); however, the role of histamine during hypoxia has not been exactly pinpointed. In the working heart-brainstem preparation (WHBP) of rats, the CB was perfused with NaCN and H1 and H3 agonists. All three experiments’ effects showed an increase in phrenic nerve activity, which would cause an increase in respiration, indicating an excitatory role for histamine (Lazarov et al, 2006). Recall that both H1 and H3 receptors are also

22 found on the surface of petrosal ganglion neurons and within the NTS (Lazarov et al,

2006), so the excitatory effects of histamine may be due to the activation of histamine receptors in any of these three locations. The injection of histamine may have also caused the afferent artery to constrict, which would induce hypoxia. As a result, histamine may have appeared to be an excitatory transmitter. A third possibility is that histamine may be potentiating the effects of excitatory neurotransmitter, such as acetylcholine, that is released from the carotid body.

Using in vitro CB and petrosal ganglion preparations in cats, Del Rio demonstrated that exogenously applied histamine perfused and superfused over the preparation in cats caused an increase in CSN firing frequency, but when histamine was applied to isolated petrosal ganglia cell bodies, there was no effect on the CSN. In this same study the superfusion of the H1 receptor antagonist reduced the CSN firing frequency, while the H3 receptor antagonist potentiated the effect of histamine on the firing frequency of the CSN (Del Rio et al, 2008). This supports the idea that histamine is presynaptically excitatory.

While the previously discussed research implicated histamine as an excitatory transmitter, Burlon et al (2009) found that histamine had no effect on isolated CB type I cells in rats. Histamine and H1, H2, and H3 agonists did not raise [Ca2+]i. This data does not support Del Rio’s findings that histamine is an excitatory presynaptic neurotransmitter (Burlon et al, 2009).

The differences in results may be due to differences in species. However, Del

Rio assumed that histamine was excitatory presynaptically because his research found no effect of histamine on the petrosal ganglion cell bodies, but he did not apply

23 histamine to CSN nerve endings within the carotid body. The excitatory effects of histamine are most likely post-synaptic as seen with Lazarov’s experiments.

HYPOTHESIS

It has been shown that activation of muscarinic receptors is excitatory (Dasso et al, 1997); however, the response due to the activation of histamine receptors is not yet fully understood (Burlon et al, 2009, Lazarov et al, 2006). This thesis will test the hypothesis that activation of histamine receptors will modulate the signaling properties of muscarinic receptors. Potentiation of the muscarinic response could partially explain the findings of Lazarov et al (2006) that histamine is excitatory in the carotid body.

SUMMARY

The carotid bodies, located at the bifurcation of the common carotid artery, serve as the primary peripheral chemoreceptors. Of the two different cell types located in the carotid body, type I cells act as the chemosensors, while the type II cells primarily function as glial cells. Type I cells detect changes in arterial blood gas and pH, and when non-homeostatic levels are sensed, depolarize resulting in a release of neurotransmitters. The neurotransmitters can either modulate the release of other neurotransmitters being released from type I cells, or they can act postsynaptically on the carotid sinus nerve. Output from the brainstem causes hyperventilation to restore arterial blood gas and pH levels to homeostasis.

The focus of this thesis is to determine the interplay between muscarinic and histamine receptor activation. This work will test whether or not histamine can

24 modulate the Ca2+ response to muscarinic receptor activation. Finally, this work aims to characterize any observed modulation using pharmacological methods.

II: MATERIALS AND METHODS

All studies described in this paper were performed in accordance with protocols approved by the Wright State University Institutional Laboratory Animal Care and Use

Committee (IACUC). These protocols are in accordance with the National Institute of

Health guide for the care and use of laboratory animals (NIH publications No. 80-23) revised 1996.

DISSECTION AND DISSOCIATION OF NEONATAL RAT CAROTID BODY TYPE I CELLS

On each experimental day, two or three neonatal Sprague Dawley rats (aged 10-

20 days) were placed into an induction chamber supplied with 4.5% isofluorane and oxygen to anesthetize the animal. After the rat became unconscious it was moved from the induction chamber and placed so that a gas mask supplying the mixture of gases was continuously supplied to the animal. To ensure that the animals were completely unconscious, the foot pinch withdrawal reflex was tested. Only after it was ensured that the animal was completely asleep were the following steps taken: The rat’s forelegs were taped down and an incision was made along the sternum exposing the underlying subcutaneous fascia. Then the salivary glands as well as muscle lying lateral to trachea were removed using fine forceps (Moria, Fine Science Tools, USA) to expose the

25 bifurcation of the common carotid artery deep to those structures. Great care was taken to ensure that none of the surrounding blood vessels were damaged.

Using a dissection microscope (Omâna, Japan) under low magnification, the fat and fascia were removed to further expose the bifurcation of the common carotid artery. First the overlying glossopharyngeal nerve was removed followed by the retraction of the occipital carotid artery to expose the carotid body. The carotid body was almost always found anchored in connective tissue to the internal carotid artery.

The organ was then carefully removed with the forceps and placed directly into ice- cold, oxygenated, Dulbecco’s phosphate buffered saline (DPBS) without Ca2+ or Mg2+

(Sigma). Following removal of the carotid body, the rats were euthanized by decapitation while still deeply anesthetized. The bodies were disposed of according to the Lab Animal Research specifications. In the lab, the remaining connective tissue was removed from the four to six carotid bodies using the fine forceps underneath the

Omâna dissecting microscope.

The cleaned carotid bodies were then placed in a digestive enzyme solution

(0.4mg ml-1 collagenase type I, 220u mg-1 (Worthington Biochemical Corporation), 0.2 mg ml-1 trypsin type I, 8550 BAEE u mg-1 (Sigma) in DPBS with low CaCl2 (86 M) and

MgCl2 (350 M) for 20 minutes at 37° C to digest the connective tissue holding the carotid body together. The carotid bodies were teased apart and incubated at 37 °C for

7 minutes. Following the last digestion, the carotid body tissue was removed from the

Petri dish using a fire-polished, silanized (Sigmacote, Sigma) Pasteur pipette and placed into a test tube. The tissue was triturated and centrifuged at 110g for 5 minutes. After, the cells were resuspended in tissue culture medium (Ham’s F12 (Sigma) supplemented

26 with 10% heat inactivated fetal bovine serum (Biowest), centrifuged again for 5 minutes at 110g, resuspended in tissue culture medium, and plated onto 12 mm2 poly- d-lysine (0.01% w/v) coated glass coverslips for imaging. Coverslips were placed in Petri dishes and maintained at 37 °C in a humidified, 5% CO2/air incubator. Cells were allowed to adhere to the coverslips for 2 hours, and all cells were used for experimentation within 8 hours.

FURA-2AM

Fura-2AM is a molecular probe that is used to determine intracellular calcium concentrations through fluorescence. (Fig. 4). The acetoxymethyl ester portion of the probe allows for the molecule to diffuse easily through the plasma membrane of the cell, enabling researchers to avoid using more invasive techniques for loading. Once inside the cell, the AM portion is cleaved off by the cell’s esterases, and the cell- impermeant fluorescent indicator is left behind. Upon binding Ca2+, Fura-2 exhibits an absorption shift that can be observed by scanning the excitation spectrum between 300 and 400 nm, while monitoring the emission at ~510 nm (invitrogen.com). In these experiments the Fura-2 was excited by exposing it to both 340 nm and 380 nm wavelengths of light while monitoring emissions at 510 nm. Calculations made by looking at the ratios of emitted light evoked by 340 nm/380 nm excitation allowed for us to determine the intracellular concentration of calcium at specific instances in time

(see formula below):

27 Figure 4

Fluorescence emission of Fura-2 at differing wavelengths. This graph indicates the difference in fluorescence of Fura-2 due to the excitation wavelength and intracellular free-calcium concentrations. The dye was excited at wavelengths indicated on the X axis. When the intracellular calcium level is zero, the fluorescence intensity at 340 nm is less than that at 380 nm when viewed at 510 nm. As the free calcium level increases, the fluorescence intensity at 340 nm increases as the fluorescence intensity at 380 nm decreases when viewed at 510 nm.

Fluorescence Intensity Fluorescence

Wavelength (nm)

29 [Ca] = Kd (Sf2/Sb2)(Rexp - Rmin/Rmax-Rexp)

Kd = 224 x 10-9 M

Sf2 = intensity 380 nm Ca free Sb2 = intensity 380 nm Ca saturated

Rexp = measured experimental ratio Rmin = 0 Ca ratio Rmax = Ca saturated ratio

Thus the measured experimental ratio is directly proportional to the calcium concentration within the cell and can be calculated by calibrating the system using a 0 mM Ca2+ solution and a saturating Ca2+ solution (typically 5 mM). Additionally, by using two different wavelengths of light to measure concentration, variables, such as dye bleaching and cell thickness, can be cancelled out preventing unwanted artifact. In this thesis all fluorescent values are given as ratio units rather than Ca2+ concentrations.

Previous students in the Wyatt laboratory have not been able to acquire satisfactory

Sb2 readings to allow accurate calibration.

Ca2+ IMAGING

Cells were visualized with a Nikon TE2000U inverted microscope with a CFI super fluor x40 oil immersion objective. Fura-2 was excited with 50 msec exposures to

340 nm and 380 nm light at 0.2 – 0.5 Hz using a Lambda 10 – 3 filter wheel (Sutter) and emitted fluorescence measured at 510 nm using a CoolSNAP HQ2 CCD camera. Light was provided by a Lambda-LS xenon arc lamp (175 watt, Sutter). Because of the extremely high wattage of this lamp, the light was first passed through neutral density filters of 0.7 optical densities (Chroma, USA) before reaching cellular specimens to

30 prevent any photodamage to the cells (Burlon et al, 2009). Neutral density filters are capable of decreasing the intensity of light from all wavelengths equally, therefore lowering the power of light from the source.

Data acquisition and analysis was controlled using Metafluor 7.1.2 imaging software (Molecular Devices). 340/380 ratio images were generated on-line and time courses showing changes in the fluorescence ratio assessed by placing regions of interest over the type I cell images. Only cells that responded to an 80 mM K+ challenge

(KCl replaced NaCl in equimolar amounts, 80 mM KCl was used to evoke maximal Ca2+ influx) with a rapid, robust, and reversible increase in fluorescence ratio were selected for study.

PERFUSION OF ISOLATED TYPE I CELLS

Isolated cells, plated on round coverslips, were placed into the perfusion chamber (0.4 ml) and washed with HEPES buffered saline solution (34 – 36° C) for approximately 5 – 7 minutes. Temperatures were maintained at 34 – 36° C by passing solutions through an in-line heater (SH-27F, Warner Instruments, USA) which was feedback controlled by an automatic temperature controller (TC-344B, Warner

Instruments, USA).

The dissociation procedure used here yields predominantly individual, isolated type I cells. Occasionally clusters of cells were observed, but these were not recorded from as locally released neurotransmitters within the cluster would confuse any results obtained. Regions of interest (ROI) were drawn over isolated cells using Metafluor software, and the calcium signal was taken as the average within the ROI, data were

31 background subtracted (using an ROI placed over a region where there were no cells to obtain background signal). Changes in the calcium signal were measured from the baseline (average of three exposures immediately prior to drug exposure) to the peak response during exposure. Only cells that responded to KCl exposure, recovered to baseline within three minutes, showed no spontaneous Ca2+ spiking and had level baselines that did not rise throughout the recording were used in this study. A complete recovery from the KCl challenge had to occur to ensure that the Ca2+ handling machinery of the type I cells were healthy. Recordings from individual cells represents n = 1. Compounds were only ever applied once to a coverslip of cells to avoid the possibility of any desensitization or sensitization occurring.

Solution changes were accomplished by switching the solution inflow to a chamber containing the solution of choice; all solutions were perfused by gravity.

Solution exchange in the chamber was usually complete within 5 seconds.

STATISTICAL SIGNIFICANCE

Data are presented as means standard error of the mean. Differences between individual means were determined by unpaired Student’s t-test. A value of P < 0.05 was taken to indicate statistical significance.

CHEMICALS

The drugs used in this research are included in Table 1.

Table 1

Compound Target Source Acetyl- -methylcholine chloride muscarinic receptor agonist Sigma Histamine dihydrochloride histamine receptor agonist Sigma (R)-(-)- -methylhistamine H3 receptor agonist Tocris dihydrobromide SQ 22536 adenylate cyclase inhibitor Tocris H89 PKA inhibitor Tocris KT 5720 PKA inhibitor Tocris Brefeldin A Epac inhibitor Tocris 8-pCPT-2’-O-Me-cAMP AM Epac agonist Biolog

Compound Concentration (vehicle) Exposure Acetyl- -methylcholine chloride 100 µM (extracellular soln’) 30 sec Histamine dihydrochloride 300 µM (extracellular soln’) 2 min (R)-(-)- -methylhistamine 30 nM (extracellular soln) 2 min dihydrobromide SQ 22536 100 µM (extracellular soln’) 5 min H89 10 µM (0.1% DMSO) 2 min KT 5720 1 µM (0.1% DMSO) 2 min Brefeldin A 100 µM (1% DMSO) 6 min 8-pCPT-2’-O-Me-cAMP AM 1 – 3 µM (0.1-0.3% DMSO) 6 min

III: RESULTS

A concentration of 80mM K+ was applied to cells to ensure that they were both living and viable. Only cells that responded with a rapid, robust, and reversible rise in

[Ca2+]i were selected for study. Thus cells were selected with intact calcium entry mechanisms and healthy calcium handling properties. To avoid the possibility that type

I cells released neurotransmitters that could act upon their immediate neighbors and confuse interpretation of data, clusters of three or more cells were not used in this study.

EFFECT OF METHYLCHOLINE ON [Ca2+]i

Methylcholine has been shown to increase [Ca2+]i by releasing calcium from intracellular stores and by allowing calcium to enter from outside the cell (Dasso et al,

1997). The muscarinic agonist acetyl- -methylcholine (MC, 100 m, submaximal concentration (Dasso et al, 1997)) evoked maximal calcium signaling responses of 0.40

0.04 ratio units (R.U.) in 63.2% of isolated type I cells examined after a 30 second challenge (n = 38, Fig. 5 A and B). The response was often seen immediately after the drug reached the cell, but sometimes the response was delayed by seconds. The calcium ratio returned to baseline, and occasionally, spiking responses were seen after the removal of MC.

Figure 5

Inhibitory effect of histamine on muscarinic receptor induced calcium signaling in rat carotid body type I cells. A. An example trace of the fluorescence ratio when an isolated type I cell is exposed to acetyl- -methylcholine (MC, 100 m, 30 sec). K indicates exposure to a high potassium solution (80 mM). B. A histogram showing the percentage of type I cells that responded to MC (63.2%) and to MC in the presence of histamine dihydrochloride (H, 300 M, 53.0%). C. An example trace of the fluorescence ratio when a type I cell is exposed to MC (100 M) in the presence of H

(300 M). D. A histogram indicating the average magnitude of the response of type I cells to MC (0.401 R.U.) and to MC in the presence of H (0.122 R.U.). ** indicates statistical significance (p < 0.003).

EFFECT OF HISTAMINE ON [Ca2+]i

The application of histamine did not have an effect on [Ca2+]i in isolated type I cells (Burlon et al, 2009). Therefore, histamine was tested to see if it could modulate the actions of an excitatory stimulus. As shown in Fig. 5, MC caused rises in [Ca2+]i in type I cells and has previously been shown to be excitatory (Dasso et al,

1997, Ortiz and Varas, 2010). The histaminergic agonist histamine dihydrochloride

(300 M, maximal concentration (Burlon et al, 2009)) was applied for 60 seconds prior to a co-application of MC (100 M). The MC-induced calcium response in the presence of histamine was significantly reduced (p < 0.003) to an average of 0.12 0.04 R.U (Fig.

5 C and D). In these same conditions, only 53.0% of the isolated type I cells responded

(n = 17, Fig. 5 B).

EFFECT OF THE H3 RECEPTOR AGONIST ON [Ca2+]i

Since the histamine attenuated the MC response, the next step was to determine through which receptor histamine was acting. Type I cells express Gi proteins coupled to the H3 receptor (Burlon et al, 2009, Lazarov et al, 2006). Using the H3 specific agonist, (R)-(-)- -methylhistamine dihydrobromide at a concentration of 30 nM (60 sec prior to the 100 M 30 sec co-application of MC), the MC response was significantly reduced to 0.06 0.01 R.U (n = 9, p < 0.003, Fig. 6 B, C, and D).

EFFECT OF THE ADENYLATE CYCLASE INHIBITOR

The mechanisms by which H3 receptor activation inhibited MC induced Ca2+ signaling are not known. However, Gi coupled receptors exert their actions via the

37 Figure 6

Inhibitory effect of a selective histamine H3 agonist on muscarinic receptor induced calcium signaling in rat carotid body type I cells. A. An example trace of the fluorescence ratio when an isolated type I cell is exposed to acetyl- -methylcholine (MC,

100 m, 30 sec). K indicates exposure to a high potassium solution (80 mM). B. A histogram marking the percentage of type I cells that responded to MC (63.2%) and to

MC in the presence of (R)-(-)- -methylhistamine dihydrobromide (H3, 30 nM, 55.5%).

C. An example trace of the fluorescence ratio when a type I cell is exposed to MC (100

M) and H3 (30 nM). D. A histogram indicating the average magnitude of the response of type I cells to MC (0.401 R.U.) and to MC in the presence of H3 (0.060 R.U.). ** indicates statistical significance (p < 0.003).

39 inhibition of adenylate cylase, which prevents the accumulation of cAMP. The drug SQ

22536 (SQ), an adenylate cyclase inhibitor, was therefore co-applied with MC to determine if its effects mimicked those of the H3 specific agonist. SQ (100 M, 4 min) also significantly inhibited the average magnitude of the response to 0.17 0.04

R.U as did the activation of the H3 receptor (p < 0.02, Fig. 7 C and D). In the presence of

SQ, the percentage of cells that responded to MC was reduced to 44.4% (n = 18, Fig. 7

B).

EFFECT OF THE PROTEIN KINASE A INHIBITORS

Inhibition of adenylate cyclase mirrored the effects seen with the activation of the H3 receptor. It was therefore investigated whether inhibiting the activity of protein kinase A (PKA) was responsible for this. Inhibition of adenylate cyclase will attenuate production of cAMP. This in turn will inhibit any constitutive activity of PKA. Inhibition of PKA has been shown to activate K+ leak channels, and therefore, oppose depolarization and calcium entry in type I cells (Xu et al, 2007). Thus activation of Gi- coupled receptors may potentially inhibit Ca2+ signaling in type I cells by an inhibitory action on PKA.

The first PKA inhibitor used was H89 (10 M, 60 sec). The drug did not have any significant effect on the average magnitude of the MC response or in the number of cells that responded to MC (Fig. 8 C). 64.7% of the cells responded to MC with an average response of 0.35 0.07 R.U. (n = 17, Fig. 8 B and D). Importantly, it has previously been shown that application of H89 at this concentration inhibited PKA (Xu et al, 2007).

40 Figure 7

Inhibitory effect of the adenylate cyclase inhibitor SQ22536 on muscarinic receptor induced calcium signaling in rat carotid body type I cells. A. An example trace of the fluorescence ratio when an isolated type I cell is exposed to acetyl- -methylcholine (MC,

100 m, 30 sec). K indicates exposure to a high potassium solution (80 mM). B. A histogram marking the percentage of type I cells that responded to MC (63.2%) and to

MC in the presence of SQ 22536 (SQ, 100 M, 44.4%). C. An example trace of the fluorescence ratio when a type I cell is exposed to MC (100 M) and SQ (100 M). D. A histogram indicating the average magnitude of the response of type I cells to MC (0.401

R.U.) and to MC in the presence of SQ (0.173 R.U.). ** indicates statistical significance (p

< 0.02).

42 Figure 8

PKA inhibition does not significantly attenuate muscarinic receptor induced calcium signaling in rat carotid body type I cells. A. An example trace of the fluorescence ratio when an isolated type I cell is exposed to acetyl- -methylcholine (MC, 100 m, 30 sec).

K indicates exposure to a high potassium solution (80 mM). B. A histogram showing the percentage of type I cells that responded to MC (63.2%) and to MC in the presence of H89 (10 M, 64.7%). C. An example trace of the fluorescence ratio when a type I cell is exposed to MC (100 M) and H89 (10 M). D. A histogram indicating the average magnitude of the response of type I cells to MC (0.401 R.U.) and to MC in the presence of

H89 (0.350 R.U.).

44 To ensure that PKA inhibition actually had no role in attenuating the MC response, a second PKA inhibitor was used, KT 5720 (1 M, 60 sec). Again, the PKA inhibitor was unable to mimic the effects of the activation of the H3 receptor (Fig. 9 C). The KT compound reduced the average magnitude of response to 0.26 0.04 R.U. (n = 27, Fig. 9

D) with 48.10% of type I cells responding (Fig. 9 B), however, these results were not significantly different from control MC responses.

A RECENTLY DISCOVERED PATHWAY: EXCHANGE PROTEINS ACTIVATED BY cAMP

Since the two PKA inhibitors were unable to significantly reduce the excitatory effects of MC, a mechanism independent of PKA was potentially responsible for inhibiting muscarinic receptor mediated calcium signaling. Exchange Proteins

Activated by cAMP (Epac) mediate PKA independent signal transduction properties of cAMP (Bos, 2003). Activation of Epac has been shown to mediate changes in intracellular calcium (Holz et al, 2006).

Therefore, a non-selective Epac inhibitor was used to see if the inhibition of Epac mimicked the effects of H3 receptor activation. The drug, Brefeldin A (BFA, 100 M,

(Rocher et al, 2009)) was applied 5 minutes prior to the application of MC (Fig. 10 C).

BFA significantly reduced the average magnitude of the calcium response to 0.14 0.04

R.U. (p < 0.02, Fig. 10D) with only 50.0% of the type I cells responding (n = 12, Fig. 10

B). It was noted that in 92% of the cells that were tested a rise in baseline calcium was seen with the application of BFA prior to MC exposure (Fig. 10 C). The rise was small but observed repeatedly with an average magnitude of 0.06 R.U.

45 Figure 9

K indicates exposure to a high potassium solution (80 mM). B. A histogram marking the percentage of type I cells that responded to MC (63.2%) and to MC in the presence of KT 5720 (KT, 1 M, 48.1%). C. An example trace of the fluorescence ratio when a type I cell is exposed to MC (100 M) and KT (1 M). D. A histogram indicating the average magnitude of the response of type I cells to MC (0.401 R.U.) and to MC in the presence of KT (0.259 R.U.).

Figure 10

Inhibition of Exchange Proteins Activated by cAMP (Epac) significantly inhibited the muscarinic receptor induced calcium signaling in rat carotid body type I cells. A. An example trace of the fluorescence ratio when an isolated type I cell is exposed to acetyl-

-methylcholine (MC, 100 m, 30 sec). K indicates exposure to a high potassium solution (80 mM). B. A histogram marking the percentage of type I cells that responded to MC (63.2%) and to MC in the presence of brefeldin A (BFA, 100 M,

50.9%). C. An example trace of the fluorescence ratio when a type I cell is exposed to

MC (100 M) and BFA (100 M). D. A histogram indicating the average magnitude of the response of type I cells to MC (0.401 R.U.) and to MC in the presence of BFA (0.141

R.U.). ** indicates statistical significance (p < 0.02).

Since BFA is not a selective Epac inhibitor and may influence Ca2+ signaling by multiple mechanisms (see dicussion, (Holz et al, 2006)), a known Epac activator was used to determine if activation of Epac could modulate MC induced Ca2+ signaling. 8- pCPT-2’-O-Me-cAMP AM is a potent membrane permeable Epac activator (Vliem et al,

2008). It was applied for 5 minutes at a concentration of 1 M prior to the co- application of MC at 100 M (Fig. 11 C). The MC response was 0.46 0.07 R.U. (n = 29,

Fig. 11 D) in 48.3% of isolated type I cells; however, this was not a significant increase in magnitude. 8-pCPT-2’-O-Me-cAMP is a new compound, and there are few publications where it has been applied to isolated cells (Vliem et al, 2008). The effects of 8-pCPT-2’-O-Me-cAMP AM (3 µM) on MC evoked Ca2+ signaling were therefore examined. A five-minute pre-application with the higher dose caused a significant increase in the average magnitude 0.97 ± 0.24 (n = 11, p < 0.004, Fig. 12) with 54.5% of the cells responding to MC.

50 Figure 11

Low dose activation of Epac does not potentiate the effects of muscarinic receptor induced calcium signaling in rat carotid body type I cells. A. An example trace of the fluorescence ratio when an isolated type I cell is exposed to acetyl- -methylcholine (MC,

100 m, 30 sec). K indicates exposure to a high potassium solution (80 mM). B. A histogram marking the percentage of type I cells that responded to MC (63.2%) and to

MC in the presence of 8-pCPT-2’-O-Me-cAMP AM (x-cAMP, 1 M, 48.3%). C. An example trace of the fluorescence ratio when a type I cell is exposed to MC (100 M) and x-cAMP (1 M). D. A histogram indicating the average magnitude of the response of type I cells to MC (0.401 R.U.) and to MC in the presence of x-cAMP (0.463 R.U.).

52 Figure 12

Higher dose activation of Epac potentiates the muscarinic receptor induced calcium signaling in rat carotid body type I cells. A histogram indicating the average magnitude of the fluorescent response of type I cells to acetyl- -methylcholine chloride (MC, 0.401

R.U.) to MC in the presence 8-pCPT-2’-O-Me-cAMP AM (x-cAMP, 1 M, 0.463 R.U.), and to MC in the presence x-cAMP (3 M, 0.968 R.U.) **indicates statistical significance (p <

0.005).

54 IV: DISCUSSION

These data demonstrate that two neurotransmitters released from the carotid body type I cells by chemostimuli can functionally interact. Both acetylcholine (ACh) and histamine are released by the rat carotid body (Koerner et al, 2004, Eyzaguirre and

Zapata, 1968) with ACh being excitatory presynaptically via action on both muscarinic and nicotinic receptors (Dasso et al, 1997, Wyatt and Peers, 1993), and histamine being without any excitatory presynaptic activity in isolated cells (Burlon et al, 2009). The data presented in this study indicate that histamine, acting via autoinhibitory H3 receptors, can inhibit calcium signaling initiated by the activation of muscarinic ACh receptors.

Thus the hypothesis that histaminergic and muscarinic signaling can interact in the carotid body type I cells is correct. However, the interaction inhibited rather than potentiated the muscarinic Ca2+ signal, and as a result, it is unlikely that this mechanism can explain why histamine appears to be excitatory in the intact carotid body (Lazarov et al, 2006).

Furthermore, these data define the potential mechanism by which histamine inhibits muscarinic receptor-induced Ca2+ signaling in rat carotid body type I cells. The inhibition of adenylate cyclase, such as would be seen during activation of Gi-coupled histamine H3 receptors, inhibits muscarinic receptor-activated Ca2+ signaling in isolated type I cells (Fig. 7). Inhibition of adenylate cyclase leads to a decrease in the generation of cAMP and, therefore, inhibition of PKA (Glass and Krebs, 1980). It has been previously been hypothesized that regulation of PKA could perhaps modulate Ca2+ entry

55 into type I cells by activation or inhibiting TASK channels and hyperpolarizing or depolarizing cells (Xu et al, 2007). However, this was unlikely to mediate the histamine-induced inhibition of muscarinic receptor induced Ca2+ signaling as histamine does not inhibit depolarization-induced Ca2+ entry in type I cells (Burlon et al,

2009). Indeed it is now shown here that PKA inhibitors have no effect on the muscarinic receptor-activated Ca2+ signaling in isolated type I cells (Fig. 8 and 9). These experiments suggest that the inhibitory effect of histamine on rises in [Ca2+]i and the similar effects of muscarinic Ca2+ signaling seen with inhibition of adenylate cyclase by

SQ22536 (Fig. 7) cannot be mediated by PKA.

Evidence now suggests that 100 M methylcholine (as used in this study) induces a rise in [Ca2+]i that is predominantly driven by release of Ca2+ from intracellular stores in carotid body type I cells (Ortiz and Varas, 2010). Inhibition of adenylate cyclase, and hence a reduction in cAMP, must somehow interact with the muscarinic receptor-induced release of Ca2+ causing it to be attenuated. There is evidence that the cAMP-activated Epac family of exchange factors are involved in mobilizing Ca2+ from intracellular stores (Kang et al, 2003). Additionally it has been postulated that Epac may sensitize Ca2+ release channels such as the IP3 receptor to their endogenous ligands (Holz et al, 2006). It was therefore conceivable that if a fall in cAMP inhibited endogenous Epac activity, Ca2+ stores would become less sensitive to IP3 liberated during Gq-coupled muscarinic receptor stimulation. Thus if Epac could be inhibited pharmacologically, the effects of inhibition of adenylate cyclase would be reproduced (i.e., inhibition of muscarinic Ca2+ would occur). Furthermore, if Epac was

56 stimulated pharmacologically, the reverse should be seen, and the Ca2+ signal following muscarinic receptor activation should be enhanced in the presence of an Epac agonist.

To test this hypothesis, the putative Epac inhibitor brefeldin A (BFA) was applied to type I cells prior to a methylcholine challenge. The Ca2+ response to methylcholine was inhibited in the presence of BFA (Fig. 10). However, BFA also caused an additional transient rise in Ca2+ when perfused over the cells. It is known that in addition to inhibiting Epac, BFA causes retraction of the Golgi apparatus in cells

(Dinter and Berger, 1998). Therefore, the small transient Ca2+ response seen upon application of BFA may be a result of BFA releasing Ca2+ from the Golgi apparatus

(Vanoevelen et al, 2005). If this is the case, then BFA may not be inhibiting muscarinic receptor activated Ca2+ signaling by an inhibitory action on Epac. IP3 receptors are present on the Golgi apparatus ((Pinton et al, 1998), and as such, Gq-coupled muscarinic receptors may release Ca2+ from the Golgi. If BFA has disrupted this store and caused it to be depleted of Ca2+, then the muscarinic Ca2+ signals would be reduced.

Importantly, even when [Ca2+]i is monitored carefully, an effect of BFA on cell Ca2+ signaling cannot be attributed solely to its effect on Epac (Vanoevelen et al, 2005).

Due to this uncertainty over the specificity of BFA for Epac, a new membrane permeant selective Epac agonist 8-pCPT-2’-O-Me-cAMP AM was used to examine the

Epac hypothesis (Vliem et al, 2008). This compound should activate Epac, and in theory may sensitize Ca2+ stores to IP3. Consequently, Ca2+ signals evoked by Gq-coupled muscarinic receptors on the type I cells should be enhanced. A five-minute exposure to

8-pCPT-2’-O-Me-cAMP AM (3 M) did augment the Ca2+ responses to methylcholine

57 (Fig. 12). Thus Epac may be involved in regulating intracellular Ca2+ release in type I cells.

FUTURE EXPERIMENTS

Future studies aim to examine the mechanism by which inhibition of Epac inhibits Ca2+ signaling. The first method to do this would be to use caged IP3 to perfuse over the isolated type I cells. Caged IP3 is IP3 that has been modified so that it is inactive and membrane permeable (Rainbow et al, 2009). Once the caged IP3 has crossed the cell membrane, it is exposed to UV light to activate the IP3. Then a Ca2+ signal can be measured. If the Ca2+ signal is enhanced in the presence of an Epac activator, then Epac can presumably sensitize the IP3 receptors to their ligand (IP3).

The effects of the Epac activator on signaling via a different Gq-coupled receptor could also be studied. For example, the 5-HT2 serotonin receptor subtypes are located on the surface of type I cells (Zhang et al, 2003) and their activation is excitatory.

Activation of these receptors will initiate similar signaling pathways to the type I cell muscarinic receptors and the modulatory effects of Epac on this signaling could also be examined. These experiments will not reveal the mechanism by which Epac is working but will reveal if it can modulate the signaling of multiple receptor types rather than muscarinic receptors alone.

CONCLUSION

Carotid body type I cells release a number of neurotransmitters, such as acetylcholine and histamine, in response to adverse arterial blood chemostimuli. This

58 work shows that these two neurotransmitters can interact with one another to affect the [Ca2+]i levels of type I cells (Fig. 13). Activation of muscarinic receptors causes a rise in [Ca2+]i due to the liberation of Ca2+ by way of IP3 from intracellular stores; however, if both muscarinic and histaminergic receptors are stimulated simultaneously, the Ca2+ signal is significantly attenuated. The inhibitory effects of histamine were shown to be functioning via H3 receptor activation. These Gi-coupled receptors work by inhibiting the activation of adenylate cyclase and therefore the accumulation of cAMP. Instead of functioning in a PKA-dependent manner, the inhibition due to H3 receptor activation is most likely due the inhibition of Exchange Proteins Activated by cAMP (Epac). Therefore, Epac may be functioning to sensitize IP3 receptors on the surface of intracellular stores to release calcium. Further work needs to be completed to determine the precise role Epac plays in this mechanism. Critically, experiments need to be performed in cells that are actively responding to a chemostimulus. By studying these active cells rather than quiescent cells we will be able to determine the importance of the signaling pathways defined in this thesis during chemotransduction.

Figure 13

A schematic demonstrating how the activation of histaminergic and muscarinic receptors affect intracellular calcium levels. The activation of the cell membrane muscarininc receptor (M) is Gq-coupled and therefore activates Gq. This leads to the activation of phospholipase C (PLC). The activation of PLC causes the cleavage of phosphatidyl 4,5-biphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to its receptor (IP3R) located on the membrane of intracellular stores, which will cause the release of calcium. The activation of the Gi- coupled H3 receptor will inhibit the activation of adenylate cyclase, the accumulation of cAMP, and the activation of Epac. The inhibition of Epac will prevent the IP3R from being sensitized, and therefore will not allow for an increase in intracellular calcium due to the release from intracellular stores.

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