<<

FMRFamide-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS IN THE CROP- OF THE , LUMBRICUS TERRESTRIS

HUMBOLDT STATE UNIVERSITY

By

Jamey Krauss

A Thesis

Presented to

The Faculty of Humboldt State University

In Partial Fulfillment Of the Requirements for the Degree Master of Arts In Biology

May, 2007

FMRFamide-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS IN THE CROP-GIZZARD OF THE EARTHWORM, LUMBRICUS TERRESTRIS

HUMBOLDT STATE UNIVERSITY

By

Jamey Krauss

Approved by the Master’s Thesis Committee:

Bruce O’Gara, Major Professor Date

Jacob Varkey, Committee Member Date

Casey Lu, Committee Member Date

Joe Szewczak, Committee Member Date

Mike Mesler, Graduate Coordinator Date

Chris A. Hopper, Interim Dean, Date Research, Graduate Studies & International Programs

ABSTRACT

FMRFamide-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS IN THE CROP-GIZZARD OF THE EARTHWORM, LUMBRICUS TERRESTRIS

Jamey Krauss

In this study, I examined the effects of FMRFamide on the isolated crop-gizzard of the earthworm, Lumbricus terrestris. The peptide induced contractions of both the longitudinal and circular muscles of the crop-gizzard at concentrations examined (10-9 to

10-5 M). The responses were quantified by measuring increases in basal tonus, peak tension, integrated area, mean contraction amplitude, and contraction rate. FMRFamide application induced concentration-dependent decreases in basal tonus increase, peak tension, integrated area, and mean contraction amplitude of the longitudinal muscles.

However, FMRFamide application induced a biphasic response in contraction rate where at low concentrations (10-9 to 10-7 M) there was an increase in contraction rate, but at high concentrations (10-6 – 10-5 M) the rate decreased and approached control values.

FMRFamide application induced a complex multiphasic effect in basal tonus increase, peak tension, and integrated area of the circular muscles. At low concentrations (10-9 –

10-8 M) there was a decrease in each FMRFamide-induced response, whereas at higher concentrations (10-7 – 10-6 M) the FMRFamide-induced responses increased before falling at the highest exposure (10-5 M). Additionally, FMRFamide induced a concentration-dependent biphasic effect on mean contraction amplitude, whereas the contraction rate revealed an excitatory trend as FMRFamide concentrations increased.

iii

The main aim of this study was to determine which signal transduction pathways were activated by FMRFamide in the crop-gizzard. It was discovered that the crop-gizzard lacks amiloride-sensitive sodium channels gated by FMRFamide. Second messenger pathway manipulation experiments suggested that the phosphatidylinositol and arachidonic acid pathways are involved in the FMRFamide-induced responses.

FMRFamide-induced responses were reduced by the protein kinase C inhibitors, H-7 (5

×10-5 M) and BIM I (10-5 M), calcium-calmodulin kinase II inhibitor, KN-62 (10-5 M),

-6 phospholipase A2 inhibitor, 4-BPB (10 M), and the phospholipase A2 and phospholipase

C inhibitor, U-73122 (18 × 10-6 M). However, there was no evidence to suggest that the cAMP or NO-induced cGMP second messenger pathways were involved in the

FMRFamide induced responses. FMRFamide-induced responses were unaffected by the protein kinase A inhibitor, H-89 (10-6 M), adenylyl cyclase inhibitor, MDL-12,330A (10-5

M), and guanylyl cyclase inhibitor, ODQ (10-6 M). Additionally, application of the cAMP analog, 8-Br-cAMP (10-5 M), NO donor, SNAP (10-5 M), and cGMP analog, 8-Br- cGMP (10-5 M) produced contractile responses that did not resemble those induced by

FMRFamide. Certain drug treatments alone induced distinct contractile responses of the crop-gizzard, indicating the role of specific transduction mechanisms in mediating crop- gizzard spontaneous activity. Normal crop-gizzard spontaneous activity was altered by the calmodulin inhibitor, W-7 (10-4 M), and the tyrosine kinase inhibitor, genistein (5 ×

10-5 M).

iv

ACKNOWLEDGEMENTS

Several individuals have been instrumental in assisting me throughout my thesis project. First and foremost, I must extend my greatest thanks to Dr. Bruce O’Gara who was an integral part in my research and development as a graduate student. Bruce provided a wealth of knowledge, guidance, and support during my years at Humboldt

State University. My committee members, Dr. Jacob Varkey, Dr. Casey Lu, and Dr. Joe

Szewczak, also contributed valuable insight, expertise, and support during the course of this project. Additionally, I must acknowledge Bruce and Jacob for their contributions to my professional school aspirations. I look forward to staying connected to the O’Gara and Varkey labs as I head to UCSF School of Pharmacy.

Finally, I want to thank my parents for their constant love and support over the years. I feel truly blessed to belong to a family that is as special as ours. Last, but surely not least, I thank Miranda Haggarty for her unwavering commitment to me during my stay at Humboldt State.

v

TABLE OF CONTENTS

ABSTRACT ...... iii

ACKNOWLEDGEMENTS ...... v

TABLE OF CONTENTS...... vi

LIST OF TABLES...... viii

LIST OF FIGURES ...... ix

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 10

Drugs and Saline ...... 10

Isolated Crop-Gizzard Preparation...... 11

FMRFamide Response Determination...... 15

Attempt to Examine FMRFamide-Gated Sodium Channels...... 17

Protocol to Examine the Effects of Pharmacological Manipulation upon Second Messenger Pathways...... 18

Statistics...... 19

RESULTS ...... 20

Quantification of Crop-Gizzard Responses to FMRFamide ...... 20

FMRFamide-Induced Longitudinal Contractions ...... 22

FMRFamide-Induced Circular Contractions...... 26

The Absence of Amiloride Sensitive FMRFamide-Gated Sodium Channels ...... 30

Effects of Manipulating Second Messenger Pathways on FMRFamide-Induced Responses...... 31

Effects of Manipulating the Phosphatidylinositol Second Messenger Pathway ...... 34

vi

Effects of Manipulating the Arachidonic Acid Second Messenger Pathway ...... 39

Effects of Manipulating the cAMP Second Messenger Pathway...... 40

Effects of Manipulating the NO-Induced cGMP Second Messenger Pathway ...... 43

Manipulations of Second Messenger Pathways Alter Spontaneous Activity of Crop-Gizzard ...... 45

DISCUSSION...... 51

Crop-gizzard Responses to FMRFamide...... 51

FMRFamide-Induced Longitudinal Contractions ...... 52

FMRFamide-Induced Circular Contractions...... 53

The Absence of Amiloride Sensitive FMRFamide-gated Sodium Channels...... 54

Effects of Manipulating Second Messenger Pathways on FMRFamide-induced Responses...... 55

Effects of Manipulating the Phosphatidylinositol Second Messenger Pathway ...... 56

Effects of Manipulating the Arachidonic Acid Second Messenger Pathway ...... 59

Effects of Manipulating the cAMP Second Messenger Pathway...... 60

Effects of Manipulating the NO-induced cGMP Second Messenger Pathway ...... 61

Manipulation of Second Messenger Pathways Alters Spontaneous Activity of the Crop-Gizzard ...... 62

LITERATURE CITED ...... 68

vii

LIST OF TABLES

Table Page

1 Effects of phosphatdylinositol and arachidonic acid pathway manipulations on FMRFamide-induced contractions of the crop-gizzard. A Dunnett’s multiple comparison test was utilized to compare the drug (FMRFamide + drug) and recovery treatments (FMRFamide) with the control treatment (FMRFamide). Statistically significant treatments are represented in bold print. The concentrations of FMRFamide (10-7M) was kept constant through all pathway manipulation experiments...... 36

2 Effects of cAMP and nitric oxide-induced cGMP pathway manipulations on FMRFamide-induced contractions of the crop-gizzard. A Dunnett’s multiple comparison test was utilized to compare the drug (FMRFamide + drug) and recovery treatments (FMRFamide) with the control treatment (FMRFamide). Donor and analog experiments required a paired t-test to compare mean value responses between control (FMRFamide) and drug (analog or donor) treatments. Recovery treatments were not applied in donor and analog experiments (gray-shaded boxes). Statistically significant treatments are represented in bold print. The concentrations of FMRFamide (10-7 M) was kept constant through all pathway manipulation experiments...... 41

3 Direct effects of drugs on the phosphatidylinositol, arachidonic acid, and mitogen-activated protein kinase second messenger pathways in the crop-gizzard. A Dunnett’s or Student-Newman-Keuls multiple comparison test was utilized to compare the drug and combined FMRFamide + drug treatments to the control FMRFamide treatment. Statistically significant groups are represented in bold print. Integrated area analysis was limited to only the FMRFamide and FMRFamide + drug treatments and required a paired t-test to compare the mean value responses. The concentration of FMRFamide (10-7 M) was kept constant through all pathway manipulation experiments...... 47

viii

LIST OF FIGURES

Figure Page

1 Chemical structure of FMRFamide (Sigma-Aldrich Inc., Saint Louis, MO). Amino acids phenylalanine (F), methionine (M), arginine (R), and phenylalanine (F) joined by peptide linkages. FMRFamide is the prototypical member of the RFamide family and all related peptides retain the amino acids arginine and amidated phenylalanine on the c-terminus as highlighted above...... 5

2 The isolated Lumbricus terrestris crop-gizzard experimental setup. The crop-gizzard was suspended in a saline-filled organ chamber where a constant circulation was maintained via a saline inflow and a suction outflow. An isometric force transducer, placed above the crop-gizzard, monitored contractions of the organ. The signals from the transducer were displayed and recorded using a computer-based data acquisition system. (A) The crop-gizzard positioned along a longitudinal axis in preparation to record longitudinal muscle contractions. Microsurgical sutures were used to secure the crop-gizzard in the organ chamber, with one affixed to the force transducer and the other affixed through the organ chamber saline inflow intersection. (B) The crop-gizzard positioned along a circular axis in preparation to record circular muscle contractions. A polyester thread was passed through the lumen of the crop-gizzard and subsequently tied to the force transducer. A lone microsurgical suture was attached to the medial lateral juncture of the crop-gizzard and then affixed through the organ chamber saline inflow intersection...... 13

3 Parameters measured to quantify the response of the crop-gizzard to FMRFamide. (A) Peak tension is the greatest tension produced in response to the peptide. The maximal increase in basal tonus is measured as the highest valley between phasic contractions. (B) Integrated area is measured as the area under the contraction curve when FMRFamide was applied until the basal tonus has returned to the baseline value...... 16

4 Contractile response of the crop-gizzard induced by 10-9 M FMRFamide. An approximately 4-min application of FMRFamide caused a series of phasic contractions superimposed upon an increase in basal tonus. Responses of the crop-gizzard to other FMRFamide concentrations were similar in form, although they differ in magnitude...... 21

ix

5 Longitudinal muscle contractile recordings showing the effects of increasing FMRFamide concentrations on a single isolated crop-gizzard. An arrowed line indicates the duration of FMRFamide application. A saline wash immediately followed the peptide application. The molar concentration of the added peptide is stated on the left of each recording...... 23

6 Concentration-response curves of the effects of FMRFamide on basal tonus, peak tension, and integrated area from longitudinal muscles of the crop-gizzard. (A) The effects of FMRFamide on maximal increase in basal tonus. (B) The effects of FMRFamide on peak tension. (C) The effects of FMRFamide on integrated area. Unless otherwise noted, in this and subsequent figures each point represents the mean of ten different crop-gizzard preparations and the vertical bars represent standard errors...... 24

7 Concentration-response curves of the effects of FMRFamide on percent changes in mean contraction amplitude and contraction rate from longitudinal muscles of the crop-gizzard. (A) The effects of FMRFamide on the percent change in mean contraction amplitude. (B) The effects of FMRFamide on the percent change in contraction rate. The dashed line in each plot indicates the control values for each measurement...... 25

8 Circular muscle contractile recordings of the effects of increasing FMRFamide concentrations on a single isolated crop-gizzard. An arrowed line indicates the duration of FMRFamide application. A saline wash immediately followed the peptide application. The molar concentration of the added peptide is stated on the left of each recording...... 27

9 Concentration-response curves of the effects of FMRFamide on basal tonus, peak tension, and integrated area from circular muscles of the crop-gizzard. (A) The effects of FMRFamide on maximal increase in basal tonus. (B) The effects of FMRFamide on peak tension. (C) The effects of FMRFamide on integrated area. Unless otherwise noted, in this and subsequent figures each point represents the mean of eleven different crop-gizzard preparations and the vertical bars represent standard errors...... 28

10 Concentration-response curves of the effects of FMRFamide on percent change in mean contraction amplitude and percent change in contraction rate from circular muscles of the crop-gizzard. (A) The effects of FMRFamide on the percent change in mean contraction amplitude. (B) The effects of FMRFamide on the percent change in contraction rate. The dashed line in each plot indicates the control values for each measurement...... 29

x

11 The effects of 10-4 M amiloride on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments. An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment. Parallel line breaks in this and following figures represent segments of deleted data corresponding to extended saline wash periods. All contractile responses were produced from a crop-gizzard orientated to record longitudinal muscle contractions by the force transducer...... 32

12 Quantification of the effects of 10-4 M amiloride on the 10-7 M FMRFamide- induced response. (A) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no significant effect on maximal increase in basal tonus 2 ( ! r = 1.19; p = 0.552; df = 2 [Friedman ANOVA on ranks]). Basal tonus data were not normally distributed and consequently the data was displayed using a box plot. The line in the center of the box represents the median, the lower and upper limits of the box represent the 25th and 75th percentile respectively, and the whisker bars represent the 10th and 90th percentiles. (B) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no 2 significant effect on peak tension ( ! r = 3.82; p = 0.1482; df = 2 [Friedman ANOVA on ranks]). (C) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no significant effect on integrated area (F = 2.68; p = 0.093; df = 2, 20 [One way repeated-measures ANOVA]). A vertical bar chart was used to display the normally distributed data, with the column bars representing the treatment means and the whisker bars representing respective standard errors...... 33

13 The inhibitory effects of 5 × 10-5 M H-7 on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment...... 35

14 The inhibitory effects of 10-5 M BIM I on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment...... 35

xi

15 The inhibitory effects of 10-5 M KN-62 on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment...... 38

16 The inhibitory effects of 10-6 M 4-BPB on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment...... 38

17 The different effects of 10-5 M 8-Br-cAMP and 10-7 M FMRFamide on a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the two treatments (dashed lines). Treatment applications were varied in order throughout the sample set...... 42

18 The different effects of 10-5 M SNAP and 10-7 M FMRFamide on a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the two treatments (dashed lines). Treatment applications were varied in order throughout the sample set...... 42

19 The direct effect of 10-4 M W-7 on a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed lines). In this and following figures, contractions were continuous between the drug and FMRFamide-drug treatments...... 46

20 The direct effect of 18 x 10-6 M U-7122 on a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed lines)...... 46

21 The direct effect of 5 x 10-5 M genistein on a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed lines)...... 49

xii

INTRODUCTION

Two significant regions of the Lumbricus terrestris gut are the thin-walled crop, wherein food is stored, and the thick cuticle-lined gizzard that extends from the crop and mechanically grinds ingested material (Brusca 1990). Food is passed through the entire alimentary canal via body movements associated with locomotion, as well as muscular activity generated by the gut musculature (Brusca 1990; Edwards and Bohen 1996). The musculature of the crop-gizzard is organized into inner circular and outer longitudinal muscles that produce wave-like contractions to propel the ingesta into the intestine. The crop apparently initiates and drives the muscular activity with frequent and vigorous contractions, whereas the gizzard contracts secondarily with weak and irregular contractions (Wu 1939a; Mill 1978; Krajniak and Khlor 1999). The organization of individual muscle fibers is similar to that seen in the earthworm Eisenia foetida and garden snail Helix aspersa. Specifically, Royuela et al. (1995, 2000) described the muscle ultrastructure of the earthworm and snail intestines as a variant of obliquely striated muscle with a high thick-thin filament volume in no true sarcomeric organization, numerous mitochondria, and a well developed sarcoplasmic reticulum, all contributing to the generation of a slow vigorous force with a high resistance to fatigue.

Neural control of the crop-gizzard is essential to gut motility and the overall efficacy of . Both the crop and the gizzard are under extrinsic neural control with innervations arising from the circumpharyngeal connectives, via a subepithelial nerve plexus, and from the ventral nerve cord, via septal nerves (Wu 1939a). In addition,

1 2 intrinsic neural control is demonstrated in isolated crop-gizzard experiments where the isolated organ spontaneously contracts up to ten hours in a saline-filled chamber (Wu

1939a; Vassileva et al. 1982; Krajniak and Khlor 1999). The subepithelial nerve plexus runs the entire length of the alimentary canal and consists of a network of nerves from the stomatogastric system, a peripheral nervous branch originating beneath the cerebral ganglia and extending from the circumpharyngeal connectives (Mill 1978). Wu (1939a) demonstrated that direct electrical stimulation of the L. terrestris stomatogastric system produced increases in tonus and contractile rate, whereas direct electrical stimulation of the ventral nerve cord inhibited the contractile rhythm. This dual innervation reveals the presence of two distinct neural pathways, one excitatory and one inhibitory that control the crop-gizzard. In addition, Millot (1943a) revealed that segmental nerves entering the surrounding body wall of the crop-gizzard form synapses in the peritoneum from which nerves pass to the gut via unique septal pathways and elicit antagonistic responses in the tone of the gut muscles. Specifically, direct electrical stimulation of the anterior segmental nerve produces a distinct fall in tone, whereas the direct electrical stimulation of the medial and/or posterior segmental nerves produces a rise in tone (Millot 1943a).

Wu (1939a) and Millot (1943b) concluded that excitatory and inhibitory activity is produced by cholinergic and adrenergic nerves respectively, eliciting the stimulatory and inhibitory effects upon the tone of the crop-gizzard. Contractile experiments involving intact and isolated crop-gizzard preparations show acetylcholine to induce an excitatory effect, whereas epinephrine induces a dose-dependent inhibitory effect (Wu 1939a;

Millot 1943a, 1943b; Vassileva et al. 1982; Krajniak and Khlor 1999).

3 In addition to acetylcholine and epinephrine, there is evidence that several other neurotransmitters are involved in regulating annelid gut activity. The following neurotransmitters either alter gut contractile activity or have been localized to gut tissue via immunocytochemistry: norepinephrine, GABA, serotonin, dopamine, octopamine, proctolin, annetocin, Eisenia tetradecapeptides (ETP), and Eisenia inhibitory pentapeptides (EIPP) (Anctil et al. 1984, 1990; Telkes et al. 1996; Ukena et al. 1996a;

Reglödi et al. 1997; Krajniak and Khlor 1999; Barna et al. 2001; Oumi et al. 1994; Ukena et al. 1995; Ukena et al. 1996b). In the polychaete, Chaetopterus variopedatus, Anctil et al. (1984, 1990) described the presence of norepinephrine-immunoreactive neural processes associated with the intestine and also demonstrated norepinephrine-induced augmentation of intestinal tone. Telkes et al. (1996) detected GABA-immunoreactive neurons in the cerebral ganglia, stomatogastric ganglia, enteric plexus, and individual cells in the gut epithelium of L. terrestris, while Ukena et al. (1995a) demonstrated a

GABA-induced excitatory response on the E. foetida gut. Serotonin appears to perform a significant role in the peripheral nervous system of oligochaetes since neural processes in the body wall and the enteric nervous system reveal strong serotonin immunoreactivity

(Reglödi et al. 1997). Additionally, Krajniak and Khlor (1999) demonstrated that serotonin induced a concentration-dependent inhibitory response on the L. terrestris crop- gizzard. Immunoreactive evidence revealed the distribution of dopamine, octopamine, and proctolin in the stomatogastric ganglia and enteric plexus of E. foetida (Barna et al.

2001). Also, Barna et al. (2001) demonstrated that dopamine and octopamine induced a concentration-dependent excitatory effect on the E. foetida , whereas proctolin did

4 not induce any significant contractile effect. Oumi et al. (1994) isolated annetocin, an oxytocin-related peptide, from E. foetida, and Ukena et al. (1995a) demonstrated that this neurotransmitter stimulated contractions in the earthworm crop-gizzard. Finaly, Ukena et al. (1995b; 1996) isolated both ETP and EIPP from the gut of E. foetida, and documented their respective excitatory and inhibitory actions on the spontaneous activity of the crop- gizzard.

Investigations of additional neurotransmitters that induce activity on the gut of annelids have been an area of emergent research in recent years. In particular, attention has been directed on the RFamide neuropeptide family, where the actions of FMRFamide have generated great interest. FMRFamide, a neuropeptide composed of four linked amino acids (Figure 1), was first discovered in the cerebral ganglia of the mollusc,

Macrocallista nimbosa (Price 1977). After the initial discovery, FMRFamide and related peptides were isolated from a large number of , including several species from the phylum Annelida. To date seven RFamide neuropeptides have been isolated from Polychaeta and Hirudinea species and include FMRFamide, FTRFamide,

FLRFamide, YLRFamide, YMRFamide, GGKYMRFamide, and GDPFLRFamide

(Krajniak 2005). FMRFamide induces a range of responses from annelid muscles, including excitatory effects on the , intestine, and body wall of the earthworm

E. foetida, and the heart, , and body wall of Hirudo medicinalis (Ukena et al.

1996; Csokyna et al. 2005; Thompson et al. 1992; O’Gara et al. 1999a; Norris et al.

1990). FMRFamide-induced relaxation on annelid muscles occurs in the esophagus of

Nereis virens, the crop-gizzard of E. foetida, and the body wall of Sabellastarte

5

Figure 1. Chemical structure of FMRFamide (Sigma-Aldrich Inc., Saint Louis, MO). Amino acids phenylalanine (F), methionine (M), arginine (R), and phenylalanine (F) joined by peptide linkages. FMRFamide is the prototypical member of the RFamide family and all related peptides retain the amino acids arginine and amidated phenylalanine on the c-terminus as highlighted above.

6 magnifica (Barratte et al. 1990; Ukena et al. 1996; Diaz-Miranda et al. 1992).

Additionally, Krajniak and Khlor (1999) revealed that FMRFamide induces a complex concentration-dependent contractile response on the crop-gizzard of L. terrestris, consisting of a biphasic change in rate and a decrease in contraction amplitude. The distinct effects of FMRFamide on a variety of annelid muscle tissues suggest the existence of different FMRFamide-activated signaling pathways in the different muscle types.

To date no neuropeptides in the RFamide family have been isolated from oligochaetes. However, in a recent unpublished report, FMRFamide sequence similarity

(EMBL: CF416445) was revealed in the humus earthworm Lumbricus rubellus (Jones et al. unpublished). In addition, FMRFamide-like immunoreactivity was shown in the nervous system of oligochaetes (Fujii et al. 1989). In particular, Fujii et al. (1989) described FMRFamide-like immunoreactive neural processes associated with the E. foetida gut. A recent immunocytochemical study of the L. terrestris nervous system revealed the presence of FMRFamide-like immunoreactive cells in the cerebral ganglia, ventral nerve cord, the stomatogastric ganglia and nerves, as well as the wall of the foregut (Reglödi et al. 1997). Reglödi et al. (1997) reported staining of FMRFamide-like immunoreactive fibers between the inner circular and outer longitudinal muscle bands of the foregut. Reglödi et al. (1997) speculated that these fibers have their origin in stomatogastric ganglia or ventral nerve cord; thus illustrating evidence for the possibility of FMRFamide control on the Lumbricus gut. Krajniak and Khlor (1999) proposed that

7 the complex FMRFamide-induced response they observed from the crop-gizzard was attributed to the activation of two different RFamide receptor subtypes, an excitatory one with high affinity for FMRFamide and an inhibitory one with low affinity for the peptide.

Currently, no reports exist attempting to explore the signal transduction processes involved in the FMRFamide-induced contractile responses of the Lumbricus crop- gizzard.

Signal transduction pathways are known to detect, amplify, and integrate diverse external signals to generate physiological responses such as changes in enzyme activity, gene expression, or ion channel activity. In particular, members of the RFamide neuropeptide family appear to activate the phosphatidylinositol second messenger pathway to mediate synaptic transmission, ion channel regulation, or contractile responses. In the crayfish, Procambarus clarkia, Friedrich et al. (1998) revealed that

DRNFLRFamide (DF2) induced a long-lasting synaptic response in abdominal extensor muscle cells through protein kinase C, an enzyme activated by the second messenger diacylglycerol (DAG) via the phosphatidylinositol signal pathway. O’Gara et al. (1999a) demonstrated that FMRFamide-induced contractile activity on the pharynx of the leech,

H. medicinalis, is at least partially mediated via protein kinase C. The additional phosphatidylinositol pathway second messenger, inositol triphosphate (IP3), has been reported to mediate FMRFamide-induced contractile responses in retractor muscles of the snail, H. aspersa, and in the heart of the pond snail, Lymnaea stagnalis (Falconer et al.

1993; Willoughby et al. 1999). Furthermore, DF2 was shown to activate calmodulin

8 dependent protein kinase, an enzyme modulated by the phosphatidylinositol pathway, in contractile responses of the crayfish abdominal extensor muscle (Noronha et al. 1995).

RFamide neuropeptides activate additional signal transduction processes including cyclic adenosine monophosphate (cAMP), nitric oxide-induced cyclic guanosine monophosphate (NO-induced cGMP), arachidonic acid second messenger pathways, independent G protein-coupled receptors, and directly opening an amiloride- sensitive sodium ion channel to elicit responses in tissue. The existence of cAMP second messenger pathways in RFamide-induced cellular responses is seen in the heart of the bivalve, Mercenaria mercenaria, and the body wall of nematode, Ascaris suum, where each tissue exhibits cAMP-dependent contractile responses after the application of FMRFamide and other RFamide neuropeptides (Higgins et al. 1978;

Reinitz et al. 2000). The NO-induced cGMP second messenger pathway appears to mediate the FMRFamide-induced slow activation of a sodium current in neuron R14 of the sea slug, Aplysia californica (Ichinose and McAdoo 1989). Arachidonic acid appears to serve as a second messenger in mediating the FMRFamide-induced slow activation of potassium currents in the neuron L7 of A. californica (Piomelli et al. 1987). Cottrell

(1993) revealed that different RFamides activate an independent G protein-coupled receptor that in turn slowly activates potassium currents in H. aspersa neurons.

Additionally, Cottrel (1997) discovered that FMRFamide directly gates an amiloride- sensitive sodium channel in neurons of H. aspersa, the only report of a peptide directly activating an ion channel. From these studies, it appears FMRFamide and related

RFamide peptides induce a variety of cellular responses via a number of signal

9 transduction processes. However, the role and importance of these signal processes in the FMRFamide-induced activity of the annelid gut are largely unexplored.

In this report, I confirmed the results generated by Krajniak and Khlor (1999) in demonstrating the sensitivity of the L. terrestris crop-gizzard to the neuropeptide

FMRFamide. Moreover, I recorded additional response parameters that more completely describe the response of the crop-gizzard to FMRFamide. To generate a more comprehensive view of the contractile activity induced by the peptide, I examined and compared the effects of FMRFamide applications on the longitudinal and circular muscles of the crop-gizzard. Finally, I investigated a number of signal transduction pathways for their potential involvement in mediating the FMRFamide-induced effects or spontaneous activity of the crop-gizzard.

MATERIALS AND METHODS

Earthworms, Lumbricus terrestris, were obtained from a local bait shop

(Bucksport Sporting Goods, Eureka, CA) and maintained individually in 300-ml paper cups containing Magic Worm Bedding (Magic Products, Inc., Amherst Junction, WI).

The cups were covered with perforated plastic lids and stored in an incubator at 14 °C in constant darkness.

Drugs and Saline

During the dissection and experiment, the crop-gizzard was superfused with a physiological saline (normal earthworm saline) containing 26 mM Na2SO4, 25 mM NaCl,

4 mM KCl, 1 mM MgCl2, 6 mM CaCl2, 2 mM TRIS base, 55 mM sucrose and adjusted to pH 7.40 with HCl (Drewes and Pax 1975). All experiments were conducted at room temperature (21 – 25 °C). Peptides or drugs were purchased from the following suppliers: FMRFamide, amiloride (N-amido-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride), 4-bromophenacyl bromide (4-BPB), W-7 (N-[6-aminohexyl]-5-chloro-1- napthalenesulfonamide hydrochloride), KN-62 (1-[N,O-bis-(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one), MDL-12,330A (cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine hydrochloride), U-73122 (1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17- yl)amino]hexyl]-1H-pyrrole-2,5-dione), 8-Br-cAMP (8-bromoadenosine 3’,5’-cyclic

10 11 monophosphate sodium salt), 8-Br-cGMP (8-bromoguanosine-3’,5’-cyclomonophosphate sodium salt), and SNAP (S-nitroso-N-acetylpenicillamine) (Sigma-Aldrich, Inc., Saint

Louis, MO); H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) and genistein (4’,5,7-trihydroxyisoflavone) (Axxora, LLC, San Diego,

CA); OBAA (3-[4-octadecyl]-benzoylacrylic acid) (Biomol Inc., Plymouth Meeting,

PA); BIM I (2-[1-(3-dimethylaminopropyl)-1H-indol-3-3-yl]-3-(1H-indol-3- yl)maleimide hydrochloride) (EMD Biosciences, Inc., La Jolla, CA); ML-7 (1-[5- iodonaphthalene-1-sulfonyl]-1H-hexahydro-1,4-diazepine hydrochloride) (Seikagaku

Corporation, Tokyo, Japan). FMRFamide was dissolved in NANOpure distilled water

(Barnstead International, Dubuque, IA) and frozen (-20 °C) in small aliquots. 4-BPB, H-

89, W-7, KN-62, ML-7, ODQ, MDL-12,330A, genistein, and SNAP were prepared as concentrated stock solutions (10-1 – 10-2 M) dissolved in DMSO (Sigma-Aldrich, Inc.,

Saint Louis, MO). OBAA and U-73122 were prepared as concentrated stock solutions

(10-2 M; 1.8 × 10-3 M) dissolved in ethanol. All drugs were diluted to their final concentration in normal earthworm saline just prior to the experiment.

Isolated Crop-Gizzard Preparation

Approximately 24 hrs prior to an experiment, were transferred from their home cups into individual Petri dishes containing a 8 x 8 cm paper towel section dampened with distilled water and returned to the incubator until dissection. This procedure allowed the gut to clear of ingesta. Prior to dissection, earthworms were immobilized by placing them in an ice bath for approximately 10 min. Following

12 immobilization, the worms were pinned dorsal side up in a frozen wax-bottomed dissection tray and covered with ice-cold normal earthworm saline. A dorsal midline incision was made from just anterior of the clitellum to the anterior end of the worm. The crop-gizzard was freed from the body wall by severing the connective septa on the lateral and ventral sides. The crop-gizzard was removed from the worm by severing the gut anterior to the crop and posterior to the gizzard. The isolated crop-gizzard was removed from the dissection tray and temporarily placed into a small saline-filled Petri dish. To record contractions of the longitudinal muscles (Figure 2A), separate microsurgical needles, bent into hook shapes and attached to 8-0 monofilament nylon suture material

(Ethicon 2808G, Sommerville, NJ), were inserted through the ends of the crop and the gizzard. The suture that was attached to the gizzard was anchored to the bottom of a small perfusion organ chamber (volume 0.8-ml; constructed from a 3-ml syringe), while the suture attached to the crop was affixed to an overhead isometric force transducer

(FORT-10, WPI, Sarasota, FL). The crop-gizzard was placed under approximately 15 –

20 mN of tension and allowed to relax for approximately 1 hr under saline perfusion.

Most crop- arranged in this orientation immediately produced spontaneous contractile activity. To record contractions of the circular muscles (Figure 2B), a 100% polyester Talon® sewing thread was passed through the lumen of the crop-gizzard and a lone microsurgical bent needle with its adjoined nylon suture was inserted through the medial lateral juncture of crop-gizzard. The nylon suture was anchored to the bottom of a new small perfusion organ chamber (volume 0.8-ml; constructed from a 5-ml syringe),

13

Force A Transducer

Suction Outflow Crop-gizzard

Organ Chamber

Display Saline Inflow

B Force Transducer

Suction Outflow Crop-gizzard

Organ Chamber

Display Saline Inflow

Figure 2. The isolated Lumbricus terrestris crop-gizzard experimental setup. The crop-gizzard was suspended in a saline-filled organ chamber where a constant circulation was maintained via a saline inflow and a suction outflow. An isometric force transducer, placed above the crop-gizzard, monitored contractions of the organ. The signals from the transducer were displayed and recorded using a computer-based data acquisition system. (A) The crop-gizzard positioned along a longitudinal axis in preparation to record longitudinal muscle contractions. Microsurgical sutures were used to secure the crop-gizzard in the organ chamber, with one suture affixed to the force transducer and the other affixed through the organ chamber saline inflow intersection. (B) The crop-gizzard positioned along a circular axis in preparation to record circular muscle contractions. A polyester thread was passed through the lumen of the crop-gizzard and subsequently tied to the force transducer. A lone microsurgical suture was attached to the medial lateral juncture of the crop-gizzard and then affixed through the organ chamber saline inflow intersection.

14 while the polyester thread was tied to the overhead isometric force transducer. The crop- gizzard was placed under approximately 10 – 15 mN of tension and allowed to relax for approximately 1 hr under saline perfusion. All crop-gizzards arranged in this orientation revealed weaker contractile activity compared to the set-up for recording longitudinal muscle contractions. At the conclusion of the 1 hr relaxation period, spontaneous longitudinal contractions were approximately 4 mN in strength whereas spontaneous circular contractions were approximately 1 mN in strength.

The output of the force transducer was fed into a transducer interface (ETH-200,

CB Sciences, Dover, NH), whose subsequent amplified output was fed into a computer- based data acquisition system (WINDAQ 200, Dataq Instruments, Akron, OH). The signal from the force transducer was digitized at 50 samples/sec and recorded to disk.

Data analysis of recorded signals was performed using playback software of the data acquisition system and Advanced CODAS software (Dataq Instruments, Akron, OH).

The organ chamber was continuously perfused with saline through an inlet at the bottom of the chamber at an approximate rate of 1 ml/min; saline was removed from the top of the chamber by suction via a 26-gauge syringe needle. The application of control or experimental salines was controlled by a attached to reservoirs (5-ml and 60-ml) containing each of the salines. Prior to experimental manipulations, the experimental reservoir held an appropriate measured volume of drug-containing saline. The switching of a valve connected to the experimental and control saline reservoirs initiated the application of drug-containing saline. An air bubble introduced into the perfusion line

15 indicated the beginning and end of each treatment. Each application of FMRFamide- containing saline was 4-ml in volume (5 times the volume of the organ chamber) and approximately 4 min in duration and followed by a sufficient saline wash to remove the

FMRFamide-containing saline from the organ chamber and return the crop-gizzard to a predetermined control tension (as FMRFamide-induced effects wore off). Event markers were inserted within the data acquisition file to accurately note the beginning and end of each treatment (i.e., an air bubble entering the organ chamber).

FMRFamide Response Determination

When determining concentration-response relationships, the lowest FMRFamide concentration was applied first (10-9 M) and higher concentrations (10-8 to 10-5 M) were added in sequential order. FMRFamide-induced responses were quantified by measuring the maximal increase in basal tonus (greatest sustained increase in muscle tonus; Figure

3A), peak tension (greatest tension produced in response to the peptide; Figure 3A), integrated area (area under the contraction curve during the response of the peptide;

Figure 3B), contraction rate (average number of contractions during the response period), and mean contraction amplitude (average contraction height during response period) during the observed contraction period. Contraction rate and mean contraction amplitude recordings were adjusted to percent change values compared to control contractions prior to FMRFamide application (control period was approximately 10 min.). FMRFamide- induced longitudinal contractions were recorded from 10 crop-gizzards, while

FMRFamide-induced circular contractions were recorded from 11 crop-gizzards. Data

16

A Peak Tension

Maximum In crease of Basal T onus

B Integrated Ar ea

FMRFamide Figure 3. Parameters measured to quantify the response of the crop-gizzard to FMRFamide. (A) Peak tension is the greatest tension produced in response to the peptide. The maximal increase in basal tonus is measured as the highest valley between phasic contractions. (B) Integrated area is measured as the area under the contraction curve when FMRFamide was applied until the basal tonus has returned to the baseline value.

17

collected from FMRFamide response periods were plotted to generate concentration- response curves where response thresholds and relationships were determined.

Attempt to Examine FMRFamide-Gated Sodium Channels

An amiloride-sensitive sodium channel that is directly gated by FMRFamide occurs in the snail H. aspersa (Cottrel 1987). To examine the possibility of FMRFamide acting upon this sodium channel in the earthworm crop-gizzard the following protocol was used (n = 11). An isolated crop-gizzard was positioned in the organ chamber to record muscle contractions from the longitudinal axis since early experiments revealed

FMRFamide elicited the larger responses from the longitudinal muscles. After the relaxation period, the crop-gizzard was exposed to a control period consisting of a 4-ml volume of 10-7 M FMRFamide and the response was recorded. This FMRFamide concentration produced a clearly observable contractile response (Figure 5). Following

FMRFamide application, the crop-gizzard was washed in normal saline for 35 – 40 min and afterward exposed to 9-ml (approximately 15 min of exposure time) of the sodium channel blocker amiloride (10-4 M). Pretreatment with 10-4 M amiloride was followed by the experimental period consisting of a 4-ml volume of 10-7 M FMRFamide and 10-4 M amiloride. After an additional 35 – 45 min saline wash, a second application of 4-ml of

10-7 M FMRFamide was performed to measure the recovery of the crop-gizzard from the amiloride treatment.

18 Protocol to Examine the Effects of Pharmacological Manipulation upon Second Messenger Pathways

Experiments conducted to examine the possible role of specific second messenger pathways in mediating the FMRFamide-induced responses used the following protocol.

An isolated crop-gizzard was positioned in the organ chamber to record muscle contractions from the longitudinal axis. After the initial relaxation period, the crop- gizzard was exposed to a control application consisting of a 4-ml volume of 10-7 M

FMRFamide and the response was recorded. Following FMRFamide application, the crop-gizzard was continuously washed in normal saline for 35 – 45 min. Drugs that inhibit signal transduction pathways were then applied to the tissue for approximately 30 min prior to the experimental period consisting of a 4-ml volume of 10-7 M FMRFamide and the drug. Following the FMRFamide and drug application, the tissue was washed in normal saline for 35 – 45 min and a second control application was performed consisting of a 4-ml volume of 10-7 M FMRFamide to measure recovery of the crop-gizzard from the drug treatment. When drugs were dissolved in DMSO or ethanol, the solvent concentration (0.01% – 1.0% of final solution) was added to each treatment applied to the crop-gizzard.

The use of second messenger analogs or nitric oxide donors required slight experimental adjustments. Following the relaxation period, the crop gizzard was exposed to a control application consisting of a 4-ml volume of 10-7 M FMRFamide, followed by a 40 – 60 min saline wash, and then the experimental period consisting of a 4-ml volume of the specific drug. To ensure an unbiased response, the procedure was reversed (drug

19 applied first, followed by FMRFamide) for an equal number of crop-gizzards in each experiment. Because SNAP was dissolved in DMSO, during the control period the crop- gizzard was exposed to an identical concentration of the solvent (0.1%).

Statistics

Data collected from the drug investigations were subjected to statistical analysis to determine if drug treatments induced significant effects. Statistics were performed using Sigma Stat 1.0 (SigmaStat Inc., San Rafael, Ca) or NCSS 2004 (Number Cruncher

Statistical Systems, Kaysville, UT). Normality was determined with preliminary descriptive statistics using the Kolmogorov-Smirnov test with Lilliefors’ correction.

When the crop-gizzard was exposed to three or more treatments, normally distributed data were subjected to a repeated measures analysis of variance test (ANOVA) and the values are presented as mean ± standard error. Data that were not normally distributed were subjected to a Friedman repeated measures ANOVA on ranks and the values are presented as medians (25th percentile, 75th percentile). If either ANOVA result was significant (pvalue< 0.05), multiple comparison tests (Dunnett’s or Student Newman-Keuls test) were utilized to identify differences between treatments. When the crop-gizzard was exposed to only two treatments, paired t-tests (pvalue= 0.05) were used to compare each treatment if the data were normally distributed. However, when data were not normally distributed, a Wilcoxon signed rank test was utilized to compare each treatment.

RESULTS

Quantification of Crop-Gizzard Responses to FMRFamide

Contractions of the isolated crop-gizzard and its responses to FMRFamide application were monitored with a force transducer (Figure 2). At the end of a preliminary relaxation period of about 1 hr, the crop-gizzard exhibited a constant basal tonus with spontaneous contractions ranging from approximately 2 contractions per minute for longitudinal muscle recordings to approximately 1 contraction per minute for circular muscle recordings. After the relaxation period, a 4-min application of

FMRFamide to the crop-gizzard produced a contractile response (Figure 4). During the

FMRFamide-induced response, the phasic contractions often exhibited an increase in peak tension and contraction rate. The FMRFamide-induced responses were quantified by measuring the maximal increase in basal tonus, the peak tension, the integrated area under the contraction curve (Figure 3), the percent change in contraction amplitude, and the percent change in contraction rate. Changes in basal tonus and peak tension are proportional to the amount of work produced by the crop-gizzard; and for at least some neurotransmitters each of these measured variables is pharmacologically separable

(O’Gara et al. 1999b). Integrated area allows the entire contraction curve to be analyzed and is influenced by both response duration and contraction rate.

20 21

10-9 M FMRFam ide

5 mN

5 min

FMRFamide

Figure 4. Contractile response of the crop-gizzard induced by 10-9 M FMRFamide. An approximately 4-min application of FMRFamide caused a series of phasic contractions superimposed upon an increase in basal tonus. Responses of the crop-gizzard to other FMRFamide concentrations were similar in form, although they differ in magnitude.

22

FMRFamide-Induced Longitudinal Contractions

Preferential recording of longitudinal muscle contractions was achieved by positioning a force transducer above an organ chamber where a crop-gizzard was orientated along a longitudinal axis (Figure 2A). Contractile recordings depicting each

FMRFamide application (10-9 – 10-5 M) upon a single crop-gizzard are displayed in

Figure 5. Spontaneous or induced-contractions were more frequent during the initial stages of an experiment and as the isolation time increased, the contractile activity lessened. Each FMRFamide-induced response revealed an excitatory effect upon the crop-gizzard along with a concentration-dependent decrease in response. The excitatory effect and concentration dependence of the decrease in response amplitude are shown in

Figure 6, which plots the effects of FMRFamide upon basal tonus, peak tension, and integrated area. The amplitude of each response variable decreased with higher concentrations of FMRFamide up to at least 10-5 M (FMRFamide concentrations higher than 10-5 M were not tested). Positive basal tonus measurements at each concentration reveal the excitatory effect induced by FMRFamide (Figure 6A); FMRFamide did not induce relaxation at any concentration tested. In addition, the shape of each concentration-response curve shows the decreasing amplitude of each of these parameters as peptide concentration increased (Figure 6).

Concentration-response relationships for percent change in mean contraction amplitude and contraction rate are plotted in Figure 7. The application of FMRFamide at

23

5 mN

5 min

-9 10

FMRFam ide

10-8

-7 10

10-6

-5 10

Figure 5. Longitudinal muscle contractile recordings showing the effects of increasing FMRFamide concentrations on a single isolated crop-gizzard. An arrowed line indicates the duration of FMRFamide application. A saline wash immediately followed the peptide application. The molar concentration of the added peptide is stated on the left of each recording.

24

3.0 A

)

N

m 2.5 (

e s a

e 2.0 r c

n I 1.5 s u n

o

T 1.0

l a s a 0.5 B 0.0 -9 -8 -7 -6 -5 10 10 10 10 10 8 B 7

) 6

N

m 5 (

n o i 4 s n e 3 T

k a 2 e

P 1 0 -9 -8 -7 -6 -5 10 10 10 10 10 1200 C

) 1000

s

·

N 800 m

(

a

e r 600 A

d e t 400 a r

g e t

n 200

I

0 -9 -8 -7 -6 -5 10 10 10 10 10

FMRFamide Concentration (M) Figure 6. Concentration-response curves of the effects of FMRFamide on basal tonus, peak tension, and integrated area from longitudinal muscles of the crop-gizzard. (A) The effects of FMRFamide on maximal increase in basal tonus. (B) The effects of FMRFamide on peak tension. (C) The effects of FMRFamide on integrated area. Unless otherwise noted, in this and subsequent figures each point represents the mean of ten different crop-gizzard preparations and the vertical bars represent standard errors.

25

60 A

50

40 %) (

e

d 30

u t n i i l 20 e p g m

n a A 10

h

n C o

i

t 0 t c n e a c r

t -10 r e n o P

C -20

n a e -30 M -40 -9 -8 -7 -6 -5 10 10 10 10 10

B 480

420

360 n %) i

(

e e t g 300 a n a R

h 240 n C o

i t

t n c 180 e a c

r t r e n 120 o P C 60

0

-60

-9 -8 -7 -6 -5 10 10 10 10 10 FMRFamide Concentration (M) Figure 7. Concentration-response curves of the effects of FMRFamide on percent changes in mean contraction amplitude and contraction rate from longitudinal muscles of the crop-gizzard. (A) The effects of FMRFamide on the percent change in mean contraction amplitude. (B) The effects of FMRFamide on the percent change in contraction rate. The dashed line in each plot indicates the control values for each measurement.

26

increasing concentrations revealed an inhibitory effect upon contraction amplitude. The threshold concentration for the inhibition of contraction amplitude was between 10-8 and

10-7 M. However, FMRFamide had a concentration-dependent biphasic effect upon contraction rate. At low concentrations (10-9 – 10-7 M) there was an increase in contraction rate with a peak contraction rate attained at 10-7 M, whereas at high concentrations (10-6 – 10-5 M) the rate decreased and approached control values.

FMRFamide-Induced Circular Contractions

Preferential recording of circular muscle contractions was achieved by positioning a force transducer above an organ chamber where the longitudinal axis of the crop- gizzard was positioned perpendicular to the thread transmitting contractile force to the transducer (Figure 2B). Contractile recordings depicting each FMRFamide application

(10-9 – 10-5 M) upon a single crop-gizzard are displayed in Figure 8. FMRFamide application produced concentration-dependent changes in contractions produced by the circular muscles of the crop-gizzard (Figure 9). At low concentrations (10-9 – 10-8 M) there was a decrease in each FMRFamide-induced response, whereas at higher concentrations (10-7 – 10-6 M) the FMRFamide-induced responses increased before falling at the highest exposure (10-5 M). This multiphasic effect of FMRFamide was seen throughout each response parameter and emphasized by similar shaped response curves

(Figure 9). Additionally, positive basal tonus measurements at each concentration reveal the excitatory effect induced by FMRFamide (Figure 9A).

27

2 mN

5 min

-9 10

FMRFamide

10-8

-7 10

10-6

-5 10

Figure 8. Circular muscle contractile recordings of the effects of increasing FMRFamide concentrations on a single isolated crop-gizzard. An arrowed line indicates the duration of FMRFamide application. A saline wash immediately followed the peptide application. The molar concentration of the added peptide is stated on the left of each recording.

1.2 A

) 28 N

m 1.0 (

e s

a

e 0.8 r

c n I

s 0.6 u

n o

T 0.4

l a

s a 0.2 B

0.0 10-9 10-8 10-7 10-6 10-5

3.0 B

2.5

) N

m 2.0

(

n

o i

s 1.5

n e T

k 1.0 a

e P 0.5

0.0 -9 -8 -7 -6 -5 10 10 10 10 10 350 C

)

s 300

·

N 250

m (

a e 200 r

A

d

e 150 t a

r g 100 e t n I 50

0 10-9 10-8 10-7 10-6 10-5

FMRFamide Concentration (M) Figure 9. Concentration-response curves of the effects of FMRFamide on basal tonus, peak tension, and integrated area from circular muscles of the crop-gizzard. (A) The effects of FMRFamide on maximal increase in basal tonus. (B) The effects of FMRFamide on peak tension. (C) The effects of FMRFamide on integrated area. Unless otherwise noted, in this and subsequent figures each point represents the mean of eleven different crop-gizzard preparations and the vertical bars represent standard errors.

29

150 A

125

%) (

e

d 100

u t n i

i l

e p 75 g m n a A

h n 50 C o

i t

t c n e a c r 25 t r e n

o P C 0 n a

e M -25 -50 10-9 10-8 10-7 10-6 10-5

B 450 400

350

n %) i

( 300

e e t g a n 250 a R

h

n C o 200 i t t n c e a c r 150 t r e n P o 100 C 50 0 -50 10-9 10-8 10-7 10-6 10-5 FMRFamide Concentration (M) Figure 10. Concentration-response curves of the effects of FMRFamide on percent change in mean contraction amplitude and percent change in contraction rate from circular muscles of the crop- gizzard. (A) The effects of FMRFamide on the percent change in mean contraction amplitude. (B) The effects of FMRFamide on the percent change in contraction rate. The dashed line in each plot indicates the control values for each measurement.

30 The FMRFamide-induced responses were also quantified via mean contraction amplitude and contraction rate. Contractile recordings revealed a concentration- dependent biphasic effect on mean contraction amplitude, whereas the contraction rate revealed an excitatory trend as FMRFamide concentrations increased (Figures 8, 10).

Concentration-response relationships for percent change in mean contraction amplitude and contraction rate are plotted in Figure 10. At low concentrations (10-9 – 10-8 M) there was a decrease in mean contraction amplitude to the control value, whereas at high concentrations (10-7 – 10-5 M) the mean contraction amplitude increased. However, the application of FMRFamide at increasing concentrations revealed an excitatory effect upon contraction rate with a sharp increase between 10-9 – 10-7 M and subsequently reaching a plateau between 10-7 – 10-5 M.

The Absence of Amiloride Sensitive FMRFamide-Gated Sodium Channels

The main aim of this investigation was to attempt to explain the signal transduction processes that mediate the FMRFamide-induced responses on the crop- gizzard. My early results revealed that FMRFamide elicited the greatest response on the longitudinal muscles of the crop-gizzard. Additionally, an application of 10-7 M

FMRFamide proved to elicit an average characteristic response (versus other tested

FMRFamide concentrations) in the longitudinal preparations. Consequently, 10-7 M

FMRFamide was selected as my control application with the crop-gizzard orientated to record longitudinal muscle contractions in the amiloride sensitive sodium channel and signal transduction experiments.

31 It is known that in the snail H. aspersa, FMRFamide can directly gate a Na+ channel that is blocked by amiloride (Cottrell 1997). Presence of FMRFamide-gated sodium channels on the surface of the crop-gizzard could serve as a potent source for muscle cell depolarization and ultimately be responsible for the observed FMRFamide- induced contractile activity. A decrease in the FMRFamide-induced response due to an amiloride application would suggest the presence of amiloride-sensitive sodium channels.

An application of 10-4 M amiloride did not alter the response induced by an application of

10-7 M FMRFamide (Figure 11). Statistical analysis revealed that no significant differences existed between contractile response measurements (basal tonus, peak tension, or integrated area) between treatments (Figure 12). These results suggest that the crop-gizzard lacks amiloride-sensitive sodium channels gated by FMRFamide.

Effects of Manipulating Second Messenger Pathways on FMRFamide-Induced Responses

A collection of second messenger pathways was examined to determine their possible role in the FMRFamide-induced responses of the crop-gizzard. Given the apparent absence of FMRFamide-gated sodium channels, FMRFamide-induced effects on the crop-gizzard are likely to be mediated by second messenger transduction systems.

Fifteen different drugs were used to manipulate specific steps within a number of second messenger pathways. The ability of specific drugs to alter FMRFamide-induced responses would indicate the role of specific transduction mechanisms in mediating crop- gizzard responses.

32

2 mN

5 min

-7 -7 -7 10 M FMRFamide 10 M FMRFamide 10 M FMRFamide + -4 10 M Amiloride

Figure 11. The effects of 10-4 M amiloride on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments. An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment. Parallel line breaks in this and following figures represent segments of deleted data corresponding to extended saline wash periods. All contractile responses were produced from a crop-gizzard orientated to record longitudinal muscle contractions by the force transducer.

33

A C 2.00 600

)

) N

1.75 s

· m 500

(

N e 1.50 s m a

( 400

e

r 1.25 a c e r n I

1.00 300 A

s d u e n

0.75 t o

a 200 r T

g l 0.50 e a t s 100 n a

0.25 I Basal B 0.00 0 FMRFamide FMRFamide + Amiloride FMRFamide FMRFamide FMRFamide + Amiloride FMRFamide

Tonus

Increase B 12 (mN)

) 10 N m (

8 n o i s 6 n e T

k 4

a e Peak Tension (mN) P 2

0 FMRFamide FMRFamide + Amiloride FMRFamide

Figure 12. Quantification of the effects of 10-4 M amiloride on the 10-7 M FMRFamide-induced response. (A) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no significant effect on maximal increase in basal tonus ( 2 = 1.19; p = 0.552; df = 2 [Friedman ANOVA on ! r ranks]). Basal tonus data were not normally distributed and consequently the data was displayed using a box plot. The line in the center of the box represents the median, the lower and upper limits of the box represent the 25th and 75th percentile respectively, and the whisker bars represent the 10th and 90th percentiles. (B) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no significant effect on peak tension ( 2 = 3.82; p = 0.1482; df = 2 ! r [Friedman ANOVA on ranks]). (C) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no significant effect on integrated area (F = 2.68; p = 0.093; df = 2, 20 [One way repeated- measures ANOVA]). A vertical bar chart was used to display the normally distributed data, with the column bars representing the treatment means and the whisker bars representing respective standard errors.

34 Prior to manipulation experiments, the consistency of FMRFamide-induced responses was determined in a set of eight control experiments. A crop-gizzard preparation was exposed to three 10-7 M FMRFamide applications in intervals identical to the second messenger pathway manipulation protocol. Recordings of basal tonus increase (F = 2.34; p = 0.132; df = 2, 14 [One way repeated-measures ANOVA]), peak tension (F = 2.53; p = 0.115; df = 2, 14 [One way repeated-measures ANOVA], and integrated area (F = 2.34; p = 0.132; df = 2, 14 [One way repeated-measures ANOVA]) proved statistically indistinguishable between the three FMRFamide treatments.

Effects of Manipulating the Phosphatidylinositol Second Messenger Pathway

RFamide-activated signal pathways that utilize protein kinase C (PKC) have been described to affect the duration and magnitude of musculature responses (Noronha et al.

1995; Friedrich et al. 1998; O’Gara et al. 1999a). H-7 is a nonselective protein kinase inhibitor, but it has been reported to exhibit the greatest selectivity and potency toward protein kinase C (Kawamoto and Hidaka 1984; Hidaka et al. 1984). Application of 5 ×

10-5 M H-7 reduced the response induced by the application of 10-7 M FMRFamide

(Figure 13). Statistical analysis revealed significant reductions in contractile response measurements (basal tonus, peak tension, or integrated area) between treatments (Table

1). The effects of H-7 upon FMRFamide-induced responses were not reversible.

However, evidence of a possible crop-gizzard recovery following washout of H-7 is seen

35

2 mN

5 min

10 -7 M FMRFamide 10-7 M FMRFamide 10-7 M FMRFamide + 5 × 10-5 M H-7 Figure 13. The inhibitory effects of 5 × 10-5 M H-7 on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment.

4 mN

5 min

-7 -7 -7 10 M FMRFamide 10 M FMRFamide 10 M FMRFamide + 10-5 M BIM I

Figure 14. The inhibitory effects of 10-5 M BIM I on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment.

Table 1. Effects of phosphatdylinositol and arachidonic acid pathway manipulations on FMRFamide-induced contractions of the crop-gizzard. A Dunnett’s multiple comparison test was utilized to compare the drug (FMRFamide + drug) and recovery treatments (FMRFamide) with the control treatment (FMRFamide). Statistically significant treatments are represented in bold print. The concentrations of FMRFamide (10-7M) was kept constant through all pathway manipulation experiments.

Target Drug Increase in basal tonus (mN) Peak tension (mN) Integrated area (mN·s) (M) Control Drug Recovery Value of Control Drug Recovery Value of Control Drug Recovery Value of [n] statistic statistic statistic

Phosphatidyl inositol pathway PKC H-7 1.19 ± 0.26 0.50 ± 0.34 0.56 ± 0.32 F = 6.59 3.56 ± 0.77 1.90 ± 0.29 1.91 ± 0.28 F = 4.95 400.0 ± 93.2 220.0 ± 60.0 195.5 ± 39.0 F = 3.78 PKA (5×10-5 M) p = 0.01* p = 0.024* p = 0.048* [n = 8]

PKC BIM I 0.75 0.38 0.48 2 3.59 1.50 1.44 2 280.6 160.4 221.3 2 -5 14.0 12.8 10.4 (10 M) (0.71, 1.03) (0.24, 0.51) (0.26, 0.52) ! r = (2.16, 4.55) (0.89, 2.06) (0.77, 2.40) ! r = (210.2, 683.1) (108.0, 248.4) (134.6, 338.3) ! r = [n = 10] p = 0.001* p = 0.002* p = 0.006*

MLCK ML-7 0.97 ± 0.19 0.92 ± 0.19 0.70 ± 0.15 F = 1.87 2.58 2.07 1.23 2 425.6 ± 105.8 387.9 ± 103.9 267.4 ± 103.9 F = 1.76 -5 = 8.17 (10 M) p = 0.183 (1.44, 3.83) (1.43, 4.06) (1.00, 1.67) ! r p = 0.20 [n = 10] p = 0.017*

Ca2+- KN-62 0.85 ± 0.11 0.67 ± 0.15 0.71 ± 0.18 F = 0.919 4.76 ± 1.30 1.84 ± 0.32 2.54 ± 0.64 F = 4.01 517.1 ± 98.0 284.3 ± 68.8 329.8 ± 59.2 F = 7.81 CaMK II (10-5 M) p = 0.425 p = 0.046* p = 0.0067* [n = 7]

Arachidonic acid pathway PLPA2 4-BPB 1.13 ± 0.15 0.66 ± 0.18 0.41 ± 0.09 F = 8.75 3.81 ± 0.72 1.70 ± 0.47 0.81 ± 0.12 F = 19.4 291.2 293.6 133.0 2 -6 7.75 (10 M) p = 0.03* p = 0.0001* (231.8, 473.7) (132.1, 347.7) (70.2, 279.1) ! r = [n = 8] p = 0.02*

PLPA2 OBAA 1.29 0.77 0.48 2 4.21± 0.91 2.33 ± 0.61 1.88 ± 0.50 F = 4.78 504.6 ± 100.0 333.8 ± 82.7 309.5 ± 96.6 F = 3.26 -7 4.08 (10 M) (1.02, 2.24) (0.47, 1.80) (0.32, 0.52) ! r = p = 0.03* p = 0.074 [n = 7] p = 0.13 * Statistically significant (p < 0.05) 36

37 with the slight recovery in the amplitude of the increase in basal tonus measurement

(Figure 13, Table 1).

BIM I, an additional potent and selective PKC inhibitor (Toullec et al. 1991), was applied to the crop-gizzard to determine the importance of the phosphatidylinositol pathway in the FMRFamide-induced response. Application of 10-5 M BIM I reduced the response induced by the application of 10-7 M FMRFamide (Figure 14). Statistical analysis revealed significant reductions in contractile response measurements (basal tonus, peak tension, or integrated area) between treatments (Table 1). The crop-gizzard demonstrated a more pronounced recovery from the BIM I application (versus H-7) as evident with the increase in FMRFamide-induced basal tonus and integrated area following drug washout.

Myosin light chain kinase (MLCK), a calcium-calmodulin-dependent enzyme that is activated via the phosphatidylinositol signal pathway, is known to regulate contractile activity in smooth muscle (Rasmussen et al. 1987). ML-7, a selective inhibitor of myosin light chain kinase, has been reported to reduce smooth muscle phasic contractions (Saitoh et al. 1987; Morano 2003). However, the application of 10-5 M ML-7 failed to produce a significant change in the FMRFamide-induced responses of the crop-gizzard (Table 1).

Despite the significant decrease reported in peak tension for the recovery treatment, a multiple comparison test revealed that the FMRFamide-induced response during the drug treatment was statistically similar to the control FMRFamide treatment.

An additional calcium-calmodulin kinase, termed calcium-calmodulin kinase II

(Ca2+-CaMK II), is known to interact with myosin light chain kinase and the SK ion

38

5 mN

5 min

10-7 M FMRFamide 10-7 M FMRFamide 10-7 M FMRFamide + 10-5 M KN-62

Figure 15. The inhibitory effects of 10-5 M KN-62 on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment.

2 mN

5 min

1 0-7 M FMRFamide 10-7 M FMRFamide 10-7 M FMRFamide + 10-6 M 4-BPB

Figure 16. The inhibitory effects of 10-6 M 4-BPB on the 10-7 M FMRFamide-induced contractile activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific treatment was added to the organ chamber. A saline wash immediately followed each treatment.

39 channel, events that can initiate muscle cell contraction (Ikebe and Reardon 1990; Tansey et al. 1992; Kong et al. 2000). KN-62 is a selective calcium-calmodulin kinase II inhibitor that has been suggested to influence smooth muscle contractions (Tokumitsu et al. 1990). Application of 10-5 M KN-62 reduced the response induced by the application of 10-7 M FMRFamide (Figure 15). Statistical analysis revealed significant reductions in contractile response measurements (peak tension and integrated area) between treatments

(Table 1). However, there was no significant change in the increase in basal tonus response. The effects of KN-62 on peak tension were reversible.

Effects of Manipulating the Arachidonic Acid Second Messenger Pathway

Arachidonic acid production has been linked to a variety of cellular events that are involved in muscle cell contraction (Gong et al. 1992; Alcorn et al. 2002; Kiss 2005).

Phospholipase A2 (PLPA2), a necessary enzyme involved in the arachidonic acid pathway is potently inhibited by 4-BPB (Synder et al. 1992). Inhibition of PLPA2 prevents production of arachidonic acid within a cell. Application of 10-6 M 4-BPB reduced the response induced by the application of 10-7 M FMRFamide (Figure 16). Statistical analysis revealed significant reductions in all contractile response measurements (basal tonus, peak tension, and integrated area) between treatments (Table 1). The inhibitory effects of 4-BPB upon FMRFamide-induced responses were not reversible.

Another drug shown to inhibit PLPA2 is OBAA (Kohler 1991). However, OBAA only partially altered the FMRFamide-induced responses in the crop-gizzard.

Application of 10-7 M OBAA produced a significant reduction in the 10-7 M

40 FMRFamide-induced peak tension response, but failed to alter the FMRF-induced increase in basal tonus and integrated area (Table 1).

Effects of Manipulating the cAMP Second Messenger Pathway

A variety of steps in the cAMP signal transduction pathway were manipulated to determine if this signaling system was involved in the FMRFamide responses of the crop- gizzard. H-89 is a selective and potent inhibitor of cAMP-dependent protein kinase A, an enzyme that has been reported to be involved in smooth muscle contractile responses

(Geilen 1992; Makhlouf and Murthy 1997). However, application of 10-6 M H-89 failed to alter the FMRFamide-induced increase in basal tonus, peak tension, and integrated area (Table 2).

MDL-12,330A is an adenylyl cyclase inhibitor and prevents the production of the second messenger cAMP (Lippe and Ardizzone 1991). However, application of 10-5 M

MDL-12,330A did not produce significant changes in the FMRFamide-induced responses

(Table 2).

In a final investigation into the cAMP signal transduction pathway, a membrane permeable cAMP analog, 8-Br-cAMP, was applied to the crop-gizzard and the recorded responses were compared to the prior or subsequent responses induced by FMRFamide.

If FMRFamide-induced responses are dependent upon this system, then both 8-Br-cAMP and FMRFamide application should produce some similarity in their respective induced responses. Application of 10-5 M 8-Br-cAMP failed to produce a distinct contractile response on the crop-gizzard despite a small increase in basal tonus (Figure 17; Table 2).

Table 2. Effects of cAMP and nitric oxide-induced cGMP pathway manipulations on FMRFamide-induced contractions of the crop-gizzard. A Dunnett’s multiple comparison test was utilized to compare the drug (FMRFamide + drug) and recovery treatments (FMRFamide) with the control treatment (FMRFamide). Donor and analog experiments required a paired t-test to compare mean value responses between control (FMRFamide) and drug (analog or donor) treatments. Recovery treatments were not applied in donor and analog experiments (gray-shaded boxes). Statistically significant treatments are represented in bold print. The concentrations of FMRFamide (10-7 M) was kept constant through all pathway manipulation experiments.

Target Drug Increase in basal tonus (mN) Peak tension (mN) Integrated area (mN·s) (M) Control Drug Recovery Value of Control Drug Recovery Value of Control Drug Recovery Value of [n] statistic statistic statistic cAMP pathway PKA H-89 0.90 ± 0.20 0.71 ± 0.16 0.63 ± 0.13 F = 2.13 2.04 1.89 1.04 2 364.1 ± 83.1 318.6 ± 60.3 254.1 ± 58.7 F = 2.79 -6 3.68 (10 M) p = 0.16 (1.61, 4.52) (0.95, 4.52) (0.60, 3.62) ! r = p = 0.10 [n = 8] p = 0.16

adenylyl MDL- 1.27 1.36 1.11 2 3.10 3.11 3.85 2 428.4 692.2 521.5 2 = 3.47 = 5.25 = 4.75 cyclase 12,330A (0.62, 1.94) (0.86, 2.72) (0.84, 1.42) ! r (2.46, 7.81) (2.07, 7.14) (1.87, 7.54) ! r (326.5, 856.5) (382.4, 1321) (316.2, 1089) ! r (10-5 M) p = 0.18 p = 0.072 p = 0.09 [n = 8]

cAMP 8-Br-cAMP 1.05 ± 0.27 0.65 ± 0.17 t = 2.96 2.20 2.33 W = -15.0 501.2 ± 142.7 355.2 ± 94.2 t = 2.41 analog (10-5 M) p = 0.016* (0.96, 6.97) (1.19, 3.11) p = 0.49 p = 0.039* [n = 10]

NO-cGMP pathway guanylyl ODQ 0.89 ± 0.16 0.67 ± 0.11 0.55 ± 0.13 F = 2.72 2.27 1.31 1.53 2 464.4 278.8 196.0 2 -6 3.38 5.25 cyclase (10 M) p = 0.10 (1.29, 4.33) (0.91, 2.39) (0.65, 3.11) ! r = (197.1, 498.0) (169.6, 336.0) (61.5, 334.7) ! r = [n = 8] p = 0.18 p = 0.072

NO donor SNAP 1.12 ± 0.12 0.48 ± 0.13 t = 3.32 5.72 ± 1.77 3.89 ± 1.24 t = 1.47 817.1 ± 279.7 551.4 ± 304.4 t = 2.03 (10-5 M) p = 0.016* p = 0.19 p = 0.039* [n = 7]

cGMP 8-Br-cGMP 0.87 0.75 W = 1.0 4.60 ± 1.30 5.04 ± 1.86 t = -0.245 487.6 ± 128.9 829.3 ± 350.0 t = -0.81 analog (10-5 M) (0.77, 1.55) (0.24, 1.81) p = 1.0 p = 0.82 p = 0.46 [n = 6] *Statistically significant (p < 0.05) 41

42

4 mN

5 min

10-5 M 8-Br-cAMP 10-7 M FMRFamide

Figure 17. The different effects of 10-5 M 8-Br-cAMP and 10-7 M FMRFamide on a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the two treatments (dashed lines). Treatment applications were varied in order throughout the sample set.

2 mN

5 min

10-7 M FMRFamide 10-5 M SNAP

Figure 18. The different effects of 10-5 M SNAP and 10-7 M FMRFamide on a single isolated crop- gizzard. The figure displays the contractile responses of a single crop-gizzard to the two treatments (dashed lines). Treatment applications were varied in order throughout the sample set.

43 There were no observable differences in contractile responses (mean contraction amplitude, peak tension, and contraction rate) between the 8-Br-cAMP treatment and the normal spontaneous activity just prior to drug application. However, statistical revealed significant differences in the increase in basal tonus and integrated area between the

FMRFamide and 8-Br-cAMP applications (Table 2). The peak tension responses were similar between the FMRFamide and 8-Br-cAMP treatment.

Effects of Manipulating the NO-Induced cGMP Second Messenger Pathway

The nitric oxide-induced cGMP signal transduction pathway was explored to determine if the FMRFamide-induced responses were mediated through this system.

This specific pathway is known to mediate relaxation events in smooth muscle (Makhlouf and Murthy 1997). Manipulation of cGMP levels prior, during, or subsequent to

FMRFamide applications would reveal the role of the cGMP in the FMRFamide-induced responses of the crop-gizzard. ODQ is a selective inhibitor of nitric oxide-sensitive guanylyl cyclase, an enzyme necessary to produce cGMP (Garthwaitel et al. 1995).

Application of 10-6 M ODQ failed to produce significant changes in FMRFamide- induced increase in basal tonus, peak tension, or integrated area (Table 2).

If the crop-gizzard possesses a NO- induced cGMP transduction system coupled to the muscle contraction mechanism, then application of the nitric oxide donor SNAP would be expected to alter contractile activity of the organ. If FMRFamide-induced responses are dependent upon this system, then both SNAP and FMRFamide application should produce some similarity in their respective induced responses. Application of 10-5

44 M SNAP failed to produce a distinct contractile response on the crop-gizzard despite a small increase in basal tonus (Figure 18; Table 2). There were no observable differences in contractile responses (mean contraction amplitude, peak tension, and contraction rate) between the SNAP treatment and the normal spontaneous activity just prior to drug application. However, statistical analysis revealed significant differences in contractile response parameters (basal tonus and integrated area) between the FMRFamide and

SNAP applications (Table 2). The peak tension responses were similar between the

FMRFamide and SNAP treatments.

In a final investigation into the nitric oxide-induced cGMP signal transduction pathway, a cGMP analog, 8-Br-cGMP, was applied to the crop-gizzard in attempt to elevate cGMP levels inside the muscle cells. If FMRFamide-induced responses are dependent upon this system, then both 8-Br-cGMP and FMRFamide application should produce some similarity in their respective induced responses. Application of 10-5 M

8-Br-cGMP failed to induce a distinct contractile response upon the crop-gizzard despite a small increase in basal tonus (Table 2). There were no observable differences in contractile responses (mean contraction amplitude, peak tension, and contraction rate) between the 8-Br-cGMP treatment and the normal spontaneous activity just prior to drug application. Additionally, application of 8-Br-cGMP produced statistically similar changes in the increase in basal tonus, peak tension, and integrated area when compared to the FMRFamide application (Table 2).

45 Manipulations of Second Messenger Pathways Alter Spontaneous Activity of Crop-Gizzard

Certain drug applications alone produced distinct responses on the crop-gizzard.

Pretreatment of the crop-gizzard with particular drugs prior to FMRFamide-drug treatments produced marked changes in basal tonus and peak tension. The direct effects of these specific drugs demonstrate that the second messenger pathways targeted by each drug are involved in regulating the spontaneous contractile activity of the crop-gizzard.

Additionally, in some experiments the combined FMRFamide-drug application produced distinct responses on the crop-gizzard that differed from normal spontaneous activity or responses induced by peptide and drug applications alone. However, these discoveries provide little evidence to which transduction pathways are used during FMRFamide- induced responses.

W-7 is a compound that is known to act as a calmodulin antagonist, calcium- calmodulin phosphodiesterase inhibitor (Ca2+-CaMP), and myosin light chain kinase inhibitor (Asano 1989). Application of 10-4 M W-7 demonstrated a marked increase in basal tonus and peak tension when compared to normal crop-gizzard spontaneous activity

(Figure 19). Statistical analysis revealed that the increase in basal tonus in the W-7 treatment was significantly larger than the FMRFamide-induced increase in basal tonus

(Table 3). However, the combined FMRFamide-drug treatment failed to generate a marked increase in basal tonus (Figure 19, Table 3). The peak tension responses in the

FMRFamide and W-7 treatments were statistically indistinguishable, but in the combined

FMRFamide-W-7 treatment the peak tension was significantly reduced. Additionally, the

46

3 mN

5 min

-7 -4 10 M FMRFamide 10 M W-7 10-7 M FMRFamide + 10-4 M W-7

Figure 19. The direct effect of 10-4 M W-7 on a single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed lines). In this and following figures, contractions were continuous between the drug and FMRFamide-drug treatments.

5 mN

5 min

10-7 M FMRFamide 18 x 10-6 M U-73122 10-7 M FMRFamide + 18 x 10-6 M U-73122 Figure 20. The direct effect of 18 x 10-6 M U-7122 on a single isolated crop -gizzard. The figure displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed lines).

Table 3. Direct effects of drugs on the phosphatidylinositol, arachidonic acid, and mitogen-activated protein kinase second messenger pathways in the crop-gizzard. A Dunnett’s or Student-Newman-Keuls multiple comparison test was utilized to compare the drug and combined FMRFamide + drug treatments to the control FMRFamide treatment. Statistically significant groups are represented in bold print. Integrated area analysis was limited to only the FMRFamide and FMRFamide + drug treatments and required a paired t-test to compare the mean value responses. The concentration of FMRFamide (10-7 M) was kept constant through all pathway manipulation experiments.

Target Drug Increase in basal tonus (mN) Peak tension (mN) Integrated area (mN·s) (M) FMRFamide Drug FMRFamide + Value of FMRFamide Drug FMRFamide + Value of FMRFamide FMRFamide + Value of [n] Drug statistic Drug statistic Drug statistic

Phosphatidyl inositol pathway CaM W-7 0.75 1.24 0.26 2 3.12 2.74 1.38 2 362.8 173.1 W = -28.0 2+ -4 10.7 10.2 Ca -CaMP (10 M) (0.37, 0.96) (0.75, 1.80) (0.24, 0.62) ! r = (2.24, 4.00) (2.61, 4.10) (0.72, 2.97) ! r = (218.0, 402.7) (135.0, 308.4) p = 0.301 MLCK [n = 12] p = 0.005* p = 0.006*

Phosphatidyl inositol & Arachidonic acid pathways PLPC U-73122 1.86 ± 0.24 2.09 ± 0.19 0.72 ± 0.13 F = 22.0 7.03 ± 1.34 7.36 ± 1.34 5.31 ± 1.26 F = 1.88 1192.7 ± 201.9 760.7 ± 129.9 t = 1.92 -6 PLPA2 (18 x 10 M) p = 0.0001* p = 0.189 p = 0.097 [n = 8]

MAP kinase pathway tyrosine Genistein 1.35 ± 0.25 2.01 ± 0.42 1.39 ± 0.29 F = 3.57 3.97 10.59 9.10 2 824.7 ± 199.7 1370.0 ± 286.2 t = -1.90 -5 = 7.61 kinase (5 x 10 M) p = 0.045* (2.99, 9.00) (4.97, 16.2) (5.71, 16.1) ! r p = 0.084 [n = 12] p = 0.022* * Statistically significant (p < 0.05) 47

48 integrated area responses in the FMRFamide and FMRFamide-W-7 treatments were statistically indistinguishable.

U-73122 is a potent phospholipase C inhibitor and selective PLPA2 inhibitor

(Yule and Williams 1992; Bleasdale et al. 1990). Application of 18 x 10-6 M U-

73122produced a complex response on the crop-gizzard that demonstrated similarities and differences to the FMRFamide-induced response (Figure 20). The U-73122 application produced a marked increase in basal tonus and peak tension when compared to normal crop-gizzard spontaneous activity. Statistical analysis revealed that the increase in basal tonus between FMRFamide and U-73122 treatments were similar, but in the combined FMRFamide-U-73122 the increase in basal tonus was significantly reduced

(Table 3). Additionally, peak tension and integrated area responses were statistically indistinguishable between all measured treatments.

Genistein is a specific inhibitor of tyrosine kinase, an enzyme involved in the mitogen-activated protein kinase pathway, and has been shown to inhibit the contraction of several smooth muscle types (Akiyam et al. 1987; Wigetunge et al. 1992; Chopra et al.

1997; Palmier et al. 1999). Application of 5 x 10-5 M genistein alone or combined with

10-7 M FMRFamide induced distinct contractile responses upon the crop-gizzard (Figure

21). The genistein application produced a marked increase in basal tonus and peak tension when compared to normal crop-gizzard spontaneous activity. Statistical analysis revealed that the genistein-induced increase in basal tonus and peak tension were significantly larger than the FMRFamide-induced responses (Table 3). Additionally, the combined FMRFamide-genistein treatment demonstrated a similar increase in peak

49

10 mN

5 min

-7 -5 -7 10 M FMRFamide 5 x 10 M Genistein 10 M FMRFamide + 5 x 10-5 M Genistein Figure 21. The direct effect of 5 x 10-5 M genistein on a single isolated crop-gizzard . The figure displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed lines).

50 tension to that seen in genistein application alone. However, the increase in basal tonus and integrated area responses in the FMRFamide and FMRFamide-genistein treatments were statistically indistinguishable.

DISCUSSION

The results of the experiments presented in this thesis confirm the work of

Krajniak and Khlor (1999) and indicate that the motility of the Lumbricus terristris crop- gizzard is, in part, regulated by FMRFamide or other members of the RFamide family.

FMRFamide application induced contractions of the outer longitudinal and inner circular muscles of the crop-gizzard. In addition, pharmacological manipulation of signal transduction pathways demonstrated that the FMRFamide-induced and spontaneous longitudinal contractions of the crop-gizzard appear to be mediated through calmodulin and the phosphatidylinositol, arachidonic acid, and mitogen-activated second messenger pathways.

Crop-gizzard Responses to FMRFamide

The results in this investigation create a more complete understanding of the role of FMRFamide in crop-gizzard motility. In addition to replicating previous findings on the FMRFamide-induced responses on the longitudinal muscles (Krajniak and Khlor

1999), my experiments are the first to document the FMRFamide-induced responses of the circular muscles of an isolated crop-gizzard. Moreover, I measured five distinct contractile parameters from each peptide-induced response to produce the most comprehensive report of FMRFamide-induced activity on the L. terrestris gut to date.

51 52

FMRFamide-Induced Longitudinal Contractions

Previous experiments have effectively reported that FMRFamide induces a dose- dependent reduction in contractile responses of the longitudinal muscles of the earthworm crop-gizzard (Ukena et al. 1995a; Krajniak and Khlor 1999). In the present study, all FMRFamide treatments elicited an excitatory effect on the crop-gizzard

(demonstrated by an increase in basal tonus after each peptide application) with the amplitude of most contractile responses decreasing as FMRFamide concentrations increased. My experiments demonstrated that FMRFamide application induced a concentration-dependent decrease in contraction amplitude and a biphasic effect on contraction rate, results that were also reported by Krajniak and Klor (1999). However, small discrepancies existed between my findings and those by Krajniak and Khlor. The threshold for the decrease in contraction amplitude appeared between 10-8 – 10-7 M

FMRFamide in my experiments versus the reported threshold of 10-9 – 10-8 M

FMRFamide by Krajniak and Khlor. Additionally, my experiments reported an increase in contraction rate from 10-9 – 10-7 M FMRFamide followed by a decrease in contraction rate from 10-7 – 10-5 M FMRFamide, whereas the work by Krajniak and Khlor revealed an increase in contraction rate from 10-9 – 10-6 M FMRFamide and a decrease in contraction rate from 10-6 – 10-5 M FMRFamide. These differences in threshold concentrations between the different studies could be attributed to the number of experiments performed (my investigation utilized 10 crop-gizzard preparations versus 5

53 used by Krajniak and Klor), procedural techniques, or equipment; however, the trends reported in the two studies are qualitatively similar.

The concentration-dependent FMRFamide-induced decrease in contractile responses is further demonstrated in the three new response parameters used in this investigation. The maximal increase in basal tonus, peak tension, and integrated area all demonstrated similar concentration-response relationships as each measurement decreased with increasing FMRFamide concentrations. The addition of these three newly reported parameters adds greater support to the conclusion drawn by Krajniak and Khlor

(1999) that FMRFamide largely induces concentration-dependent reduction in contractile activity in the longitudinal muscles of the L. terrestris crop-gizzard.

FMRFamide-Induced Circular Contractions

Manipulation of the crop-gizzard orientation in relation to the force transducer enabled spontaneous circular muscle contractions to be recorded over an extended period of time. Direct recordings of FMRFamide-induced circular muscle contractions from an isolated L. terrestris crop-gizzard have not been recorded until this investigation. The application of increasing FMRFamide concentrations on this preparation produced a different concentration-response relationship versus the longitudinal preparation. The magnitude of the circular muscle responses was far smaller than the longitudinal muscle responses, suggesting that longitudinal muscle activity dominates the FMRFamide- induced crop-gizzard response. In addition, high FMRFamide concentrations (10-7 – 10-6

M) elicited a concentration-dependent increase in each of the measured parameters,

54 whereas the FMRFamide-induced longitudinal responses decreased with increasing concentrations. However, low FMRFamide concentrations (10-9 – 10-8 M) appeared to parallel the concentration-dependent decrease demonstrated in the longitudinal muscle recordings.

The concentration-dependent biphasic effect on the circular muscles may suggest the presence of multiple FMRFamide receptor subtypes on the crop-gizzard. Specific receptor subtypes may show greater affinity to different FMRFamide concentrations and ultimately induce different contractile responses. FMRFamide and FLRFamide were reported to elicit biphasic responses in F2 neurones of Helix aspersa at high and low concentrations (Chen et al. 1995). Additional RFamide related neuropeptides have been reported to act on multiple receptor subtypes in H. aspersa central neurons and elicit distinct responses (Cottrel and Davies 1987; Chen et al. 1995). Several experiments have isolated and sequenced annelid RFamide related peptides including FLRFamide,

FTRFamide, YMRFamide, YLRFamide, GGKYMRFamide, and GDPFLRFamide

(Krajniak and Price 1990; Barratte et al. 1990; Evans 1991; Salzet 1994). Thus, it appears likely that the L. terrestris possesses several RFamide neuropeptides and that multiple RFamide receptor subtypes may exist that have the potential to bind preferentially to FMRFamide or other members of the RFamide family.

The Absence of Amiloride Sensitive FMRFamide-gated Sodium Channels

One hypothesis was that FMRFamide directly gates an amiloride-sensitive sodium ion channel on the longitudinal muscle cell membrane inducing a fast influx of

55 sodium ions that ultimately triggers cell contraction. This specific sodium channel is the only documented peptide-gated ion channel (Cottrel 1997). In a recent investigation of the leech pharynx, amiloride failed to block the FMRFamide-induced contractile responses (O’Gara et al. 1999a). Similarly, my results demonstrated that amiloride- sensitive sodium channels were absent in the L. terrestris crop-gizzard and with no reports of new peptide-gated ion channels it appears unlikely that ligand-gated ion channels mediate the FMRFamide-induced contractile responses in the earthworm gut. It is worth noting that a low sequence similarity was reported between the amiloride sensitive FMRFamide-gated sodium channel and epithelial sodium channels and degenerins, suggesting that peptides may indeed activate other ion channels in other systems (Lingueglia et al. 1995).

Effects of Manipulating Second Messenger Pathways on FMRFamide-induced Responses

The bulk of this study concentrated on an investigation into the possible role of specific second messenger pathways in mediating the FMRFamide-induced responses of the earthworm crop-gizzard. Fifteen drugs were applied to the crop-gizzard in attempt to manipulate known signal transduction components that might mediate the FMRFamide- induced responses. To my knowledge, this is the first investigation into the FMRFamide- induced activation of second messenger pathways in oligochaetes. The results of this study suggest that the actions of FMRFamide on the crop-gizzard are mediated through the activation of protein kinase C, calcium-calmodulin kinase II, and arachidonic acid.

56 Furthermore the normal crop-gizzard spontaneous activity appears to be regulated by the activation of tyrosine kinase, calmodulin protein, and myosin light chain kinase.

Effects of Manipulating the Phosphatidylinositol Second Messenger Pathway

My investigation revealed that the phosphatidylinositol second messenger pathway appeared to be a likely mediator of the FMRFamide-induced contractile responses in the crop-gizzard. The ligand-activated phosphatidylinositol pathway proceeds via stimulation of phospholipase C by a specific G protein, allowing the activated enzyme to hydrolyze phosphatidylinositol into the second messengers, diacylglycerol (DAG) and inositol triphosphate (IP3). DAG is the major physiological activator of protein kinase C whereas IP3 mobilizes intracellular calcium that in turn can activate calmodulin-dependent kinases. The activation of protein kinase C or calcium- calmodulin kinases can lead to several cellular events including muscle cell contraction.

H-7 and BIM I, two different protein kinase C inhibitors, were applied to the crop-gizzard to determine if protein kinase C is involved in the FMRFamide-induced responses. Both drugs irreversibly inhibited the FMRFamide-induced responses by a similar magnitude suggesting that protein kinase C is a likely mediator in the responses.

Active protein kinase C is known to induce tonic contraction of smooth muscle through myosin light chain phosphorylation and inhibition of myosin light chain phosphatase

(Vorotnikov et al. 2002; Rasmussen el al. 1987; Masuo et al. 1994). In experiments involving H-7 and BIM I, O’Gara et al. (1999a) concluded that the actions of

FMRFamide on the contractile activity of leech pharynx were at least partially mediated

57 via protein kinase C. In a signal transduction investigation involving spontaneously active muscle strips from liver flukes, Graham et al. (2000) reported that a FMRFamide- related neuropeptide stimulated mechanical activity through a protein kinase C-dependent pathway. Thus, it appears likely that FMRFamide induces contractile activity upon the earthworm crop-gizzard via the activation of protein kinase C.

Additionally, I examined the role of calcium-calmodulin kinases in mediating the

FMRFamide-induced contractile responses. It is well known that the activation of a dedicated calcium-calmodulin-dependent kinase, termed myosin light chain kinase, phosphorylates myosin light chain and activates the contractile myosin adenosine triphosphatase (ATPase), events necessary for gastrointestinal smooth muscle contraction in vertebrate systems (Makhlouf and Murthy 1997; Vorotniknov et al. 2002). Smooth muscle myosin light chain kinases are also found in invertebrates and have been reported to be activated by similar calcium-dependent events (Gallagher et al. 1997). However, in my experiments the application of ML-7, a drug designed to inhibit myosin light chain kinase, failed to alter the FMRFamide-induced contractile responses on the crop-gizzard.

Although it seems unlikely that myosin light chain kinase is absent in mediating contractile events in the crop-gizzard, ML-7 proved ineffective in reducing the

FMRFamide-induced contractile response parameters. Perhaps ML-7 failed to effectively select the myosin light chain kinase in the crop-gizzard, suggesting that ML-9 or other novel myosin light chain kinase inhibitors might demonstrate better selection and effectiveness. Saitoh et al. (1987) reported that ML-9 and synthesized ML-9 derivatives inhibited myosin light chain kinase in smooth muscle preparations.

58 KN-62 is a drug designed to inhibit the multifunctional calcium-calmodulin- dependent kinase II, an enzyme known to phosphorylate and regulate multiple cellular targets including myosin light chain kinase in smooth muscle cells (Ikebe and Reardon

1990; Tansey et al. 1992). This study revealed that application of KN-62 significantly decreased the FMRFamide-induced contractile responses of the crop-gizzard, suggesting that this specific calcium-calmodulin kinase is involved in the FMRFamide-induced contractile responses of the crop-gizzard and might serve to activate the myosin light chain kinase. Despite the reported link of calcium-calmodulin-dependent kinase II to myosin light chain kinase, the calcium-calmodulin enzyme has also been shown to affect gastrointestinal cell excitability via the SK channel, a calcium-activated potassium channel (Xia et al. 1998; Kong et al. 2000). The opening of the SK channel limits the possibility of repetitive cell depolarizations, an event that is necessary in regulating action potential frequency. Kong et al. (2000) demonstrated that KN-93, an additional calcium-calmodulin-dependent kinase II inhibitor, decreased the open probability of the

SK channels in colonic myocytes. If SK channels are present in the crop-gizzard, channel inhibition would increase the excitability of muscle cells and possibly induce contractile activity. However, application of KN-62 significantly reduced all

FMRFamide-induced contractile parameters in the crop-gizzard, suggesting that SK channels were not involved in the contractile responses. Thus, it appears that calcium- calmodulin kinase II activation of myosin light chain kinase is involved in the

FMRFamide-induced contractile responses of the crop-gizzard.

59 Effects of Manipulating the Arachidonic Acid Second Messenger Pathway

The ligand-activated arachidonic acid pathway proceeds via stimulation of phospholipase A2 by a specific G protein, allowing the activated enzyme to hydrolyze phospholipids into arachidonic acid and additional lipid products. Although the arachidonic acid second messenger pathway is best known to produce prostaglandin molecules inside cells, recent evidence reports that arachidonic acid can activate several cellular targets including protein kinase C (Khan et al. 1995). In investigating the arachidonic acid second messenger pathway, I found that inhibiting phospholipase A2 with 4-BPB significantly attenuated the FMRFamide-induced responses of the crop- gizzard. Additionally, the crop-gizzard failed to recovery from the 4-BPB dose, supporting the potent irreversibility of the drug (Kits et al. 1997; Kiss 2005). My evidence suggests that arachidonic acid plays a role in mediating the FMRFamide- induced responses, possibly through the stimulation of protein kinase C, cell depolarization, or generation of arachidonic acid metabolites that promote contraction. In a vertebrate smooth muscle preparation, arachidonic acid was reported to increase basal tonus levels and myosin light chain phosphorylation, and inhibit the dephosphorylation of myosin light chain (Gong et al. 1992). Additionally, an arachidonic acid metabolite was shown to activate a G protein-gated potassium channel in cardiac myocytes, an event that contributes to contraction deceleration in the heart (Kurachi et al. 1989). Lipoxygenase metabolites of arachidonic acid were also shown to mediate FMRFamide-induced SK channel activity in Aplysia sensory neurons (Buttner et al. 1989). However, as previously mentioned in this report, SK channels are unlikely to exist on the crop-gizzard. In an

60 experiment involving vertebrate smooth muscle strips and cholecystokinin (CCK), an evolutionarily related peptide to FMRFamide, the contractile effects induced by the peptide were inhibited by 4-BPB, suggesting that arachidonic acid is a mediator in the peptide-induced responses (Alcon et al. 2002). Furthermore, Kiss (2005) recently revealed that the activation of neuronal potassium channels by Mytilus inhibitory peptide, a neuropeptide found in molluscs, was irreversibly inhibited by 4-BPB, demonstrating that arachidonic acid is involved in mediating cellular hyperpolarization. The role of arachidonic acid as an intracellular signal is linked to a variety of cellular responses, and with this report, arachidonic acid appears to be involved in the FMRFamide-induced contractile responses of the crop-gizzard.

Effects of Manipulating the cAMP Second Messenger Pathway

FMRFamide or related RFamide neuropeptides have been shown to induce cAMP-dependent responses in a variety of invertebrate tissues (Higgins et al. 1978; Trim et al. 1998; Reinitz et al. 2000). However, there was little evidence suggesting that the cAMP second messenger pathway was involved in the FMRFamide-induced responses in the crop-gizzard. H-89 and MDL-12,330A, drugs designed to inhibit protein kinase A and adenylyl cyclase respectively, failed to significantly alter any of the FMRFamide- induced responses. A similar conclusion was reported in a cAMP second messenger pathway investigation of the FMRFamide-induced contractile responses on the leech pharynx (O’Gara et al. 1999a). Additionally, application of a membrane-permeable cAMP analog, 8-Br-cAMP, on the crop-gizzard produced responses that were distinctly

61 different in appearance compared to FMRFamide-induced responses. However, similar peak tension responses were observed in the 8-Br-cAMP and FMRFamide applications.

Thus, although elevated levels of cAMP inside crop-gizzard muscle cells induce contractile activity, it is unlikely to be responsible for the FMRFamide-induced activity.

Similarly, spontaneous contractions of flatworm muscle are increased by 8-Br-cAMP, but contractions induced by another RFamide peptide could not be linked to the cAMP system (Graham et al. 2000). Additionally, Occor et al. (1985) revealed that FMRFamide did not affect cAMP levels in snail sensory neurons and Flamm et al. (1987) reported the same conclusion in lobster pyloric cells. Despite evidence that RFamides work through the cAMP second messenger pathway in some preparations, the FMRFamide-induced response of the earthworm crop-gizzard is unlikely to be mediated through the cAMP second messenger pathway.

Effects of Manipulating the NO-induced cGMP Second Messenger Pathway

FMRFamide did not appear to activate the nitric oxide-induced cGMP second messenger pathway or elevate cGMP levels in the crop-gizzard responses. The application of ODQ, SNAP, and 8-Br-cGMP, drugs designed to inhibit guanylyl cyclase, release nitric oxide, and increase cGMP levels respectively, failed to alter or mimic

FMRFamide-induced responses. Despite the similar contractile responses elicited in the experiment involving FMRFamide and 8-Br-cGMP treatments, mere resemblance of contractile responses is insufficient to infer a causal relationship. However, the two remaining drugs, ODQ and SNAP, produced convincing results that FMRFamide was not

62 activating the nitric oxide-induced cGMP pathway or increasing cGMP levels. Worden et al. (1994) reached a similar conclusion when they demonstrated that a FMRFamide- related neuropeptide enhanced contractility in the lobster dactyl opener muscle, but the peptide failed to elevate cGMP levels inside the muscle cells. Many studies have shown that the nitric oxide-induced cGMP signaling pathway is involved in the relaxation of smooth muscle preparations from a variety of organs (Ahn et al. 1997; Olsson and

Holmgren 1997; Choi and Farley 1998; Elphick 1998); however, my results along with the work of Krajniak and Khor (1999) have not found any evidence of a FMRFamide- induced relaxation on the crop-gizzard. Activation of the nitric oxide-induced cGMP signaling system by neuropeptides remains unexamined in annelids, but the results from this study suggest that this second messenger pathway is absent in the FMRFamide- induced responses on the earthworm crop-gizzard.

Manipulation of Second Messenger Pathways Alters Spontaneous Activity of the Crop-Gizzard

In a few experiments, second messenger pathway manipulations provided evidence that normal crop-gizzard contractile activity is mediated by specific signal pathways. For instance, the drugs W-7 and genistein produced distinct contractile responses when applied to the crop-gizzard prior to FMRFamide application. It was apparent that these drug-induced contractile responses provided evidence that calmodulin and tyrosine kinase are involved in mediating crop-gizzard spontaneous activity.

Additionally, treatment of W-7 and U-73122 with the FMRFamide application produced

63 distinct contractile responses on the crop-gizzard that generated causal conclusions regarding the involvement of calmodulin and the phosphatidylinositol signal pathway in the FMRFamide-induced responses.

U-73122, a potent phospholipase C inhibitor and selective phospholipase A2 inhibitor, produced contractile responses that were similar to the FMRFamide-induced increase in basal tonus and peak tension. From these results, it appears that the phosphatidylinositol pathway and/or arachidonic acid pathway may be involved in the normal spontaneous activity of the crop-gizzard. It has been reported that arachidonic acid and lipoxygenase metabolites can induce smooth muscle relaxation, most notably during vasodilation events (Pfister and Campbell 1992; Zhang et al. 2005). However, recent investigations into vertebrate smooth muscle preparations involving U-73122 and other phospholipase C inhibitors have reached opposing conclusions regarding the role of the phosphatidylinositol pathway in the mediation of spontaneous contractile activity

(Balemba et al. 2005; Tanaka et al. 2003). Perhaps the normal spontaneous activity of the crop-gizzard is partially mediated by the inhibitory products of the arachidonic acid pathway; thus supporting the increased contractile responses generated by the phospholipase A2 inhibitor, U-73122. I have already discussed that the FMRFamide- induced responses appear to be mediated by the phosphatidylinositol pathway based upon the effects of the H-7, BIM I, and KN-62 treatments. When FMRFamide was combined with U-73122, the contractile responses diminished with significant reductions found in the basal tonus levels. U-73122 is known to inhibit invertebrate neuropeptide-induced responses through the inactivation of the phosphatidylinositol pathway (Satake et al.

64 2003). Therefore it seems likely that the decreased contractile responses generated from the combined FMRFamide-U-73122 treatment support the conclusion that FMRFamide activates the phosphatidylinositol pathway in the crop-gizzard responses.

The application of genistein, a tyrosine kinase inhibitor, elicited a significant elevation in basal tonus and peak tension when compared to the FMRFamide-induced responses. Additionally, in the combined FMRFamide-genistein treatment, the induced contractile responses maintained the elevated levels seen in the individual genistein treatment, suggesting the absence of tyrosine kinase in the FMRFamide-activated signal transduction. However, the actions of genistein alone indicate that tyrosine kinase is involved in the regulation of the crop-gizzard spontaneous activity. Protein tyrosine kinases occupy a large class of enzymes found in vertebrates and invertebrates that are categorized into two groups, receptor or cellular tyrosine kinases. Each type of tyrosine kinase has been shown to play a vital role in signaling pathways (mitogen-activated protein kinase pathway) that initiate cellular processes such as proliferation, differentiation, and development (Blenis 1993; Seger and Krebs 1995). In addition, tyrosine kinase has been shown to increase contractile responses in a variety of vertebrate smooth muscle preparations primarily through voltage-gated calcium influx (Chopra et al. 1997; Palmier et al. 1999; Tolloczko et al. 2000). Genistein has been demonstrated to block the tyrosine kinase-activated calcium influx and induce muscle relaxation

(Wigetunge et al. 1992; Chopra et al. 1997; Palmier et al. 1999). Despite the evidence of genistein-induced relaxation in vertebrate smooth muscle, there exists no reported tyrosine kinase signal pathway investigation in invertebrate smooth muscle to date. In

65 my investigation, it appears that genistein is inhibiting a tyrosine kinase signal pathway that regulates crop-gizzard spontaneous activity via a unique signaling process that relaxes basal tonus and reduces contractile strength.

W-7 is a drug known to have wide ranging actions including inhibition of calmodulin, calcium-calmodulin phosphodiesterase, and myosin light chain kinase. W-7 elicited a response that was significantly greater than the FMRFamide-induced response.

The W-7-induced contractile response on the crop-gizzard was similar to contractile recordings from a bovine smooth muscle experiment, where application of W-7 alone induced contractions, possibly through a histamine-releasing mechanism (Asano 1990).

Wu (1939a) demonstrated that histamine induced an elevation of basal tonus on the

Lumbricus crop-gizzard. It has also been reported that W-7 might enhance intracellular calcium motility in cardiac muscle cells through the modulation of the calcium induced- calcium release mechanism during excitation-contraction coupling (Suziki et al. 1999).

My results demonstrate that the normal spontaneous activity of the crop-gizzard appears to be mediated by a calmodulin-dependent event due to the excitatory effects induced by

W-7. However, when the FMRFamide application was combined with W-7, the contractile responses were diminished with significant reductions found in both basal tonus increase and peak tension. Thus, it appears W-7 does inhibit some contractile

FMRFamide-activated signal transduction pathways. W-7 has been shown to block

Substance P-induced contractions in smooth muscle through the inhibition of calmodulin and internal calcium motility, or myosin light chain phosphorylation (Bitar et al. 1990;

Washabau 2002). Additionally, Suenaga et al. (2001) demonstrated that protein kinase C

66 activation induced a contractile response in a smooth muscle preparation, and the response was delayed and weakened by W-7. From my earlier assertion that the

FMRFamide-induced contractile response of the crop-gizzard is mediated by the phosphatidylinositol pathway and together with the FMRFamide-W-7-induced effect, I believe that W-7 is inhibiting portions of the phosphatidyl inositol pathway that are necessary for the FMRFamide-induced response. Thus, experiments involving W-7 exposed a new excitatory response on the L. terrestris crop-gizzard and strengthened the link between FMRFamide and the phosphatidylinositol pathway.

In this thesis, I have presented the most comprehensive examination of

FMRFamide-induced activity on the oligochaete foregut to date. An early success of my project was the validation of the original FMRFamide-induced dose-dependent response curves reported by Krajniak and Khlor (1999). My attempt to reveal a more complete contractile profile of FMRFamide-induced activity on the crop-gizzard was achieved by constructing concentration-response curves for five different parameters on both the longitudinal and circular muscles. The main aim of the investigation was to determine

FMRFamide-activated signal transduction pathways that mediate the contractile responses of the crop-gizzard longitudinal muscles. I found no evidence that

FMRFamide activated an amiloride-sensitve sodium channel, but my results demonstrated that the phosphatidylinositol, arachidonic acid, and mitogen-activated protein kinase second messenger pathways are involved in mediating the peptide-induced responses. Additionally, I found that the calmodulin protein and the phosphatidylinositol second messenger pathway appear to be involved in the regulating normal crop-gizzard

67 spontaneous activity. Thus, this investigation is the first to identify specific signaling pathways that are involved in both the FMRFamide-induced responses and normal spontaneous activity of the L. terrestris crop-gizzard.

The role of FMRFamide or other RFamides in oligochaetes is still largely unknown. However, with this investigation and the past works by Krajniak and Khlor

(1999), Reglodi et al. (1997), Ukena et al. (1995a), and Fugii et al. (1989), FMRFamide appears to adopt a convincing role in regulating crop-gizzard activity. The significance of FMRFamide alongside the established gut neuromodulators acetylcholine and epinephrine is a question that remains unanswered. The identification of specific signaling pathways involved in the FMRFamide-induced response helps increase the understanding of the actual response mechanism and can prove valuable in future cellular, molecular, or pharmacology investigations in the crop-gizzard or other systems.

LITERATURE CITED

Ahn HY, Chang KC, Chung MH, Kim MS, Moreland RS (1997) Cyclic AMP and cyclic GMP relax phorbol ester-induced contractions of rat aorta by different mechanisms. Life Sciences 60: 2333-2340

Akiyama T, Ishida J, Nakagawa S, Ogawara H, Wantanabe S, Itoh N, Shibuya M, Fukami Y(1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. Journal of Biological Chemistry 262: 5592-5595

Alcon S, Morales S, Camello PJ, Pozo MJ (2002) Contribution of different phospholipases and arachidonic acid metabolites in the response of gallbladder smooth muscle to cholecystokinin. Biochemical Pharmacology 64: 1157-67

Anctil M, Laberge M, Martin N (1984) Neuromuscular pharmacology of the anterior intestine of Chaetopterus variopedatus, a filter-feeding polychaete. The Journal of Comparative and Biochemical Physiology C 79: 343-351

Anctil M, De Waele JP, Miron MJ, Pani AK (1990) Monoamines in the nervous system of the tube-worm Chaetopterus variopedatus (Polychaeta): Biochemical detection and serotonin immunoreactivity. The Journal of Cell and Tissue Research 259: 81-92

Asano M (1990) Effects of the calmodulin antagonist W-7 on isometric tension development and myosin light chain phosphorylation in bovine tracheal smooth muscle. The Japan Journal of Pharmacology 52: 471-481

Balemba OB, Salter MJ, Heppner TJ, Bonev AD, Nelson MT, Mawe GM (2005) Spontaneous electrical rhythmicity and the role of the sarcoplasmic reticulum in the excitability of guinea pig gall bladder smooth muscle cells. The American Journal of Physiology: Gastrointestinal and Liver Physiology 290: G655-G664

Barna J, Csoknya M, Lázár Z, Barthó L, Hámori J, Elekes K (2001) Distribution and action of some putative neurotransmitters in the stomatogastric nervous system of the earthworm, Eisenia foetida (Oligochaeta, Annelida). Journal of Neurocytology 30: 313-325

Barratte B, van Minnen J, Masson M, Dhainaut-Courtois N (1990) Localisation par voie immunohistochimque d’un materiel apparente au FMRFamide chez Annelides Polychetes Nereidae (Nereis diversicolor, Nereis virens, Perinereis cultrifera). Comptes Rendus de l’Academie Sciences III 311: 495-500

68 69 Bitnar KN, Hillemeier C, Biancani P (1990) Differential regulation of smooth muscle contraction in rabbit by substance P and bombesin. Life Sciences 47: 2429-2434

Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S (1990) Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. Journal of Pharmacology and Experimental Therapeutics 255: 756-768

Blenis J (1993) Signal transduction via the MAP kinases: proceed and your own RSK. Proceedings of the National Academy of Science of the United States of America 90: 5889-5892

Brezina V (1988) Guanosine 5’-triphosphate analogue activates potassium current modulated by neurotransmitters in Aplysia neurons. Journal of Physiology (London) 407: 15-40

Brusca RC, Brusca GJ (1990) Invertebrates. Sinauer Associates, Inc. Sunderland, MA 406-408

Buttner N, Siegelbaum SA, Volterra A (1989) Direct modulation of Aplysia S-K+ channel by a 12-lipoxygenase metabolite of arachidonic acid. Nature 342: 553- 555

Chen ML, Sharma R, Walker RJ (1995) Structure-activity studies of RFamide analogues on central neurons of Helix aspersa. Regulatory Peptides 58: 99-105

Choi J, Farley JM (1998) Effects of 8-bromo-cyclic GMP on membrane potential of single swine tracheal smooth muscle cells. The Journal of Pharmacology and Experimental Therapeutics 285: 588-594

Chopra LC, Hucks D, Twort CH, Ward JP (1997) Effects of protein tyrosine kinase inhibitors on contractility of isolated bronchioles of the rat. American Journal of Respiratory Cell Molecular Biology 16: 372-378

Cottrell GA, Davies NW (1987) Multiple receptor sites for a molluscan peptide (FMRFamide) and related peptides of Helix. Journal of Physiology (London) 382: 51-68

Cottrell GA (1993) The wide range of actions of the FMRFamide-related peptides and the biological importance of peptidergic messengers In: Pichon Y (ed) Comparative Molecular Neurobiology. Birkhäuser, Basel, pp 279-285

Cottrell GA (1997) The first peptide-gated ion channel. Journal of Experimental Biology 200: 2377-2386

70 Csoknya M, Lengvári I, Benedeczky I, Hámori J (1992) Immunohistochemical and ultrastructural study of the enteric nervous system of earthworm, Lumbricus terrestris. Acta Biologica Hungarica 43: 241–251

Diaz-Miranda L, Escalona de Molta G, Garcia-Arraras JE (1992) Monoamines and neuropeptides as transmitters in the sedentary polychaeta, Sabellastarte magnifica. Actions on the longitudinal muscles of the body wall. Journal of Experimental Zoology 263: 54-67

Drewes FC, Pax RA (1975) The effect of physiological salines on the crop-gizzard of the earthworm, Lumbricus terrestris. Canadian Journal of Zoology 53: 1391-1394

Edwards, CA and Bohlen, PJ (1996) The biology and ecology of earthworms (3rd Edition). Chapman & Hall, London, pp 78-80, 84-86

Elphick MR, Melarange R (1998) Nitric oxide function in an echinoderm. Biological Bulletins 194:260-266

Evans BD, Pohl, J, Kartsonis NA, Calabrese RL (1991) Identification of RFamide neuropeptides in the medicinal leech. Peptides 12: 897-908

Falconer SWP, Carter AN, Downes CP, Cottrell GA (1993) The neuropeptide Phe-Met- Arg-Phe-NH2 (FMRFamide) increases levels of inositol 1,4,5-triphosphate in the tentacle retractor muscle of Helix apsersa. Experimental of Physiology 78: 757- 766

Flamm RE, Fickbohm D, Harris-Warrick RM (1987) cAMP elevation modulates physiological activity of pyloric neurons in the lobster stomatogastric ganglion. Journal of Neurophysiology 58: 1370-1386

Friedrich RW, Molnar GF, Schiebe M, Mercier J (1998) Protein kinase C is required for long-lasting synaptic enhancement by neuropeptide DRNFLRFamide in crayfish. Journal of Neurophysiology 79: 1127-1131

Fujii K, Ohta N, Sasaki T, Sekizawa Y, Yamada C, Kobayashi H (1989) Immunoreactive FMRFamide in the nervous system of the earthworm, Eisenia foetida. Zoological Science 6: 951–961

Gallagher PJ, Herring PB, Stull JT (1997) Myosin light chain kinases. Muscle Research and Cell Motility 18: 1-16

Garthwaite J, Southam E, Boulton CL, Nielsen EB, Schmidt K, Mayer B (1995) Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Molecular Pharmacology 48: 184-188

71 Geilen CC (1992) A selective inhibitor of cyclic AMP-dependent protein kinase, N-[2- bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H-89), inhibits phosphatidylcholine biosynthesis in HeLa cells. FEBS Letters 309: 381

Gong MC, Fuglsang A, Alessi D, Kobayashi S, Cohen P, Somlyo AV, Somlyo AP (1992) Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. The Journal of Biological Chemistry 267: 21492- 21498

Graham MK, Fairweather I, McGeown JG (2000) Second messengers mediating mechanical responses to the FARP GYIRFamide in the fluke Fasciola hepatica. American Journal of Physiology- Regulative, Integrative, and Comparative Physiology 279: 2089-2094

Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984) Isoqoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036-5041

Higgins WJ, Price DA, Greenberg MJ (1978) FMRFamide increases the adenylate cyclase activity and cyclic AMP level of molluscan heart. European Journal of Pharmacology 48: 425-430

Ichinose M, Macadoo DJ, (1989) The cyclic GMP-induced inward current in neuron R14 of Aplysia californica: similarity to FMRFamide-induced inward current. The Journal of Neurobiology 20: 10-24

Ikebe M, Reardon S (1990) Phosphorylation of smooth myosin light chain kinase by smooth muscle Ca2+/calmodulin-dependent multifunctional protein kinase. The Journal of Biological Chemistry 265: 8975-8978

Kawamoto S, Hidaka H (1984) 1-(5-isoquinolinesulfonyl)-2methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets. Biochemical and Biophysical Research Communications 125: 258-264

Khan WA, Blobe GC, Hannun YA (1995) Arachidonic acid and free fatty acids as second messengers and the role of protein kinase C. Cellular Signalling 7: 171-184

Kiss T (2005) G-protein coupled activation of potassium channels by endogenous neuropeptides in snail neurons. The European Journal of Neuroscience 21: 2177- 85

Kits KS, Lodder JC, Veerman MJ (1997) Phe-Met-Arg-Phe-amide activates a novel voltage-dependent K+ current through a lipoxygenase pathway in molluscan neurons. The Journal of General Physiology 110: 611-628

72 Kohler T, Friedrich G, Nuhn P (1991) Phospholipase A2 inhibition by alkylbenzoylacrylic acids. Inflammation Research 32: 70-72

Kong ID, Koh SD, Bayguinov O, Sanders KM (2000) Small conductance Ca2+-activated K+ channels are regulated by Ca2+-calmodulin-dependent protein kinase II in murine colonic myocytes. The Journal of Physiology 524: 331-337

Krajniak KG, Price DA (1990) Authentic FMRFamide is present in the polychaete Nereis virens. Peptides 11: 75-77

Krajniak KG, Klohr RW (1999) The effects of FMRFamide, serotonin, and acetylcholine on the isolated crop-gizzard of the earthworm, Lumbricus terrestris: Comparative Biochemistry and Physiology A 123: 409-415

Krajniak KG (2005) Annelid endocrine disruptors and a survey of invertebrate FMRFamide-related peptides. Integrative and Comparative Biology 45: 88-96

Kurachi Y, Ito H, Sugimoto T, Shimizu T, Miki I, Ui M (1989) Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel. Nature 337: 555- 557

Laverack MS (1963) The physiology of earthworms. Macmillan Company, New York, pp 18-24

Lingueglia E, Champigny E, Lazdunski M, Barbry P (1995) Cloning of the amiloride- sensitive FMRFamide peptide-gated sodium channel. Nature 378: 730-733

Lippe C, Ardizzone C (1991) Actions of vasopressin and isoprenaline on the ionic transport across the isolated frog skin in the presence and the absence of adenylyl cyclase inhibitors MDL 12330 and SQ22536. Comparative Biochemistry and Physiology 99: 209-211

Makhlouf GM, Murthy KS (1997) Signal transduction in gastrointestinal smooth muscle. Cell Signaling 9: 269-276

Masuo M, Reardon S, Ikebe M, Kitazawa (1994) A novel mechanism for the Ca2+- sensitizing effect of protein kinase c on vascular smooth muscle: inhibition of myosin light chain phosphatase. The Journal of General Physiology 104: 265-286

Mill, PJ (Ed.) (1978) Physiology of annelids. Academic Press, London, pp 115-234, 509- 541

Millot N (1943a) The visceral nervous system of the earthworm: I. Nerves controlling the tone of the alimentary canal. Proceedings of the Royal Society B 131: 271-295

73 Millot N (1943b) The visceral nervous system of the earthworm: II. Evidence of chemical transmission and the action of sympatheticomimetic and parasympatheticomimetic drugs on the tone of the alimentary canal. Proceedings of the Royal Society B 131: 362-373

Morano I (2003) Tuning smooth muscle contraction by molecular motors. Molecular Medicine 81: 481-487

Noronha KF, Mercier AJ (1995) A role for calcium/calmodulin-dependent protein kinase in mediating synaptic modulation by a neuropeptide. Brain Research 673: 70-74

Norris BJ, Calabrese RL (1990) Action of FMRFamide on longitudinal muscle of the leech, Hirudo medicinalis. Comparative Biochemistry and Physiology 167: 211- 224

Occor KA, Byrne JH (1985) Membrane responses and changes in cAMP levels in Aplysia sensory neurons produced by serotonin, tryptamine, FMRFamide and small cardioactive peptide B (SCPB). Neuroscience Letters 55: 113-118

O’Gara BA, Brown PL, Dlugosch D, Kandiel A, Ku JW, Geier JK, Henggeler NC, Abbasi A, Kounalakis N (1999a) Regulation of pharyngeal motility by FMRFamide and related peptides in the medicinal leech, Hirudo medicinalis. Invertebrate Neuroscience 4: 41-53

O’Gara BA, Illuzzi FA, Chung M, Portnoy AD, Fraga K, Frieman VB (1999b) Serotonin induces four pharmacologically separable contractile responses in the pharynx of the leech Hirudo medicinalis. General Pharmacology 32: 669-681

Olsson C, Holmgren S (1997) Nitric oxide in fish gut. Comparative Biochemistry and Physiology A 118: 959-964

Oumi T, Ukena K, Matsushima O, Ikeda T, Fujita T, Minakata H, Nomoto K (1994) Annetocin: An oxytocin-related peptide isolated from the earthworm, Eisenia foetida. Biochemical and Biophysical Research Communications 198: 393-399

Palmier B, Vacher M, Harbon S, Leiber D (1999) A tyrosine kinase signaling pathway, regulated by calcium entry and dissociated from tyrosine phosphorylation of phospholipase Cgamma-1, is involved in inositol phosphate production by activated G protein-coupled receptors in myometrium. Journal of Pharmacology and Experimental Therapeutics 289: 1022-1030

Pfister SL, Campbell WB (1992) Arachidonic acid- and acetylcholine-induced relaxations of rabbit aorta. Hypertension 20: 682-689

74 Piomelli D, Volterra A, Dale N, Siegelbaum SA, Kandel ER, Schwartz JH, Berlardetti F (1987) Lipoxygenase metabolites of arachidonic acid as second messengers for presynaptic inhibition of Aplysia sensory neurons. Nature 328: 38-43

Price DA, Greensberg MJ (1977) Structure of molluscan cardioexcitatory neuropeptide. Science 197: 670-671

Rasmussen H, Takuwa Y, Park S (1987) Protein kinase C in the regulation of smooth muscle contraction. The Federation of American Societies for Experimental Biology 1: 177-185

Reglodi D, Lubics A, Szelier M, Lengvari I (1997) Serotonin immunoreactivity in the peripheral nervous system of oligochaeta. Acta Biologica Hungarica 48: 439-451

Reglodi D, Slezak S, Lubrics A, Szelier M, Elekes K, Lengvari I (1997) Distribution of FMRFamide-like immunoreactivity in the nervous system of Lumbricus terrestris. Cell Tissue Research 288: 575-582

Reinitz CA, Herfel HG, Messinger LA, Stretton AOW (2000) Changes in locomotory behavior and cAMP produced in Ascaris suum by neuropeptides from Ascaris suum and Caenorhabditis elegans. Molecular and Biochemical Parasitology 111: 185-187

Royuela M, Fraile B, García-Anchuelo R, Paniagua R (1995) Ultrastructurally different muscle cell types in Eisenia foetida (Annelida, Oligochaeta). Journal of Morphology 224: 87-96

Royuela M, Fraile B, Arenas MI, Paniagua R (2000) Characterization of several invertebrate muscle cell types: a comparison with vertebrate muscles. Microscopy Research and Technique 48: 107-115

Saitoh M, Ishikawa T, Matsushima S, Naka M, Hidaka H (1987) Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. Journal of Biological Chemistry 262: 7796-7801

Salzet M, Bulet P, Wattez C, Malecha J (1994) FMRFamide-related peptides in the sex segmental ganglia of the Pharngobdellid leech Erpobdella octoculata. Identification and involvement in the control of hydric balance. European Journal of Biochemsitry 221: 269-275

Saitoh M, Ishikawa T, Matsushima S, Naka M, Hidaka H (1987) Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. The Journal of Biological Chemistry 262: 7796-7801

75 Satake H, Kawada T, Nomoto K, Minakata H (2003) Insight into tachykinin-related peptides, their receptors, and invertebrate tachykinins: a review. Zoological Science 20: 533-549

Seger R, Krebs EG (1995) The MAPK signaling cascade. The Federation of American Societies for Experimental Biology 9: 726-735

Suenaga H, Kasuya Y, Kamata K (2001) Effects of calmodulin antagonist (W-7) on phorbol ester (PMA)-induced contractile response in isolated rat aorta. The Journal of Smooth Muscle Research 37: 1-7

Suzuki YJ, Wang W, Morad M (1999) Modulation of Ca2+ channel-gated Ca2+ release by W-7 in cardiac myocytes. Cell Calcium 25: 191-198

Synder DW, Sommers CD, Bobbitt JL, Mihelich ED (1992) Evidence that phospholipase A2 (PLA2) inhibitors can selectively block PLA2 mediated contractions of guinea pig lung pleural strips. Journal of Pharmacology and Experimental Therapeutics 262: 1147-1153

Tanaka Y, Okamoto T, Imai T, Horinouchi T, Tanaka H, Shigenobu K, Koike K (2003) Phospholipase C inhibitors suppress spontaneous mechanical activity of guinea pig urinary bladder smooth muscle. Biological and Pharmaceutical Bulletin 26: 1192-1194

Tansey MG, Word RA, Hidaka HA, Singer HA, Schworer CM, Kamm KE, Stull JT (1992) Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells. Journal of Biological Chemistry 267: 12511-12516

Telkes I, Csoknya M, Buzás P, Gábriel R, Hámori J, Elekes K (1996) GABA- immunoreactive neurons in the central and peripheral nervous system of the earthworm, Lumbricus terrestris (Oligochaeta, Annelida). Cell and Tissue Research 285: 463-475

Thompson KJ, Calabrese RL (1992) FMRFamide effects on properties of heart cells isolated from the leech, Hirudo medicinalis. Journal of Neurophysiology 67: 280- 291

Tokumitsu, H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, Hidaka H (1990) KN- 62, 1-[N,o-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase-II. Journal of Biological Chemistry 265: 4315-4320

76 Tollozcko B, Tao FC, Zacour ME, Martin JG (2000) Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells. Lung Cellular and Molecular Physiology 278: L1138-L1145

Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Loriolle F, Duhamel L, Charon D, Kirilovsky J (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. Journal of Biological Chemistry 256: 15771-15781

Trim N, Brooman JE, Holden-Dye L, Walker RJ (1998) The role of cAMP in the actions of the peptide AF3 in the parasitic nematodes Ascaris suum and Ascaridia galli. Molecular and Biochemical Parasitology 93: 263-271

Ukena K, Oumi T, Matsushima O, Ikeda T, Fujita T, Minakata M, Nomoto K (1995a) Effects of annetocin, an oxytocin-related peptide isolated from the earthworm, Eisenia foetida, and some putative neurotransmitters on gut motility of the earthworm. The Journal of Experimental Zoology 272: 184-193

Ukena K, Oumi T, Matsushima O, Takahashi T, Muneoka Y, Fujita T, Minakata H, Nomoto K (1995b) A novel gut tetradecapeptide isolated from the earthworm, Eisenia foetida. Peptides 16: 995-999

Ukena K, Oumi T, Matsushima O, Takahashi T, Muneoka Y, Fujita T, Minakata H, Nomoto K (1996) Inhibitory pentapeptides isolated from the gut of the earthworm Eisenia foetida. Comparative Biochemistry and Physiology A 114: 245-249

Vassileva PV, Stoyanov IN, Vassileva VI (1982) On the contractile activity of the alimentary canal of the earthworm (Lumbricus terrestris). Comparative Biochemistry and Physiology C 71: 127-129

Vorotnikov AV, Krymsky MA, Shirinsky VP (2002) Signal transduction and protein phosphorylation in smooth muscle contraction. Biochemistry (Moscow) 67: 1309- 1328

Washabau RJ, Holt DE, Brockman DJ (2002) Mediation of acetylcholine and substance P induced contractions by myosin light chain phosphorylation in feline colonic smooth muscle. The American Journal of Veterinary Research 63: 695-702

Wijetunge S, Aalkjaer C, Schachter M, Hughes AD (1992) Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells. Biochemical and Biophysical Research Communications 189: 1620-1623

77 Willoughby D, Yeoman MS,Benjamin PR (1999) Inositol-1,4,5-trisphosphate and inositol-1,3,4,5-tetrakisphosphate are second messenger targets for cardioactive neuropeptides encoded on the FMRFamide gene. Journal of Experimental Biology 202: 2581-2593

Worden MK, Kravitz EA, Goy MF (1995) Peptide F1, an N-terminally extended analog of FMRFamide, enhances contractile activity in multiple target tissues in lobster. The Journal of Experimental Biology 198: 97-108

Wu KS (1939a) On the Physiology and Pharmacology of the earthworm gut. Journal of Experimental Biology 16: 184-197

Wu KS (1939b) The action of drugs, especially acetylcholine, on the annelid body wall (Lumbricus, Arenicola). Journal of Experimental Biology 42: 251-257

Xia XM, Falkler B, Rivard A, Wayman G, Johnson-Pais T, Keen JE, Ishii T, Hirschberg B, Bond CT, Lutsenko S, Maylie J, Adelman JP (1998) Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395: 503-507

Yule DI, Williams JA (1992) U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells. Journal of Biological Chemistry 267:13830-13835

Zhang DX, Gauthier KM, Chawengsub Y, Holmes BB, Campbell WB (2005) Cyclooxygenase- and lipoxygenase-dependent relaxation of arachidonic acid in rabbit small mesenteric arteries. The American Journal of Physiology- Heart and Circulatory Physiology 288:302-309