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