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S. _ degree ithamcology and ' Toxicology Date M- 0-7539 MS U is an Affirmative Action/Equal Opportunity Institution —._— . 7- ‘¥, 7. 7 _ _ .__ PLACE IN RETURN BOX to remove this checkoutfrom your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE moo chlMpGS—p.“ MODULATION OF DESENSITIZATION OF NICOTINIC ACETYLCHOLINE RECEPTORS (nAChRs) IN ENTERIC NEURONS By Erika Nicole Brown A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology and Toxicology 2000 ABSTRACT MODULATION OF DESENSITIZATION OF NICOTINIC ACETYLCI'IOLINE RECEPTORS (nAChRs) IN ENTERIC NEURONS By Erika Nicole Brown The enteric nervous system (ENS) is the division of the autonomic nervous system (ANS) that controls gut functions. Acetylcholine (ACh), acting at nAChRs and muscarinic acetylcholine receptors (mAChRs), is a neurotransmitter in the ENS. NAChRs and mAChRs are expressed by the same neurons, but if these receptors interact is unclear. The hypothesis of this study is that nAChR desensitization is modulated by intracellular signaling mechanisms. Specifically, mAChR activation accelerates nAChR desensitization in the ENS. Whole-cell patch clamp methods were used to record nAChR mediated inward currents in guinea pig myenteric neurons maintained in primary culture. Drugs were used to activate and/or inhibit intracellular signaling pathways modulating nAChR desensitization. The major findings of this research were that: desensitization rate is influenced by intracellular ATP and GTP; desensitization and recovery are influenced by mAChR activation; ACh and nicotine cross-desensitize suggesting activation of the same nAChR; protein kinase C but not cyclic AMP-dependent pathways modulate nAChR desensitization. These results suggest that simultaneous activation of nAChRs and mAChRs coupled to PKG-linked signaling pathways modulate nAChR desensitization. This may be important in regulating enteric neurotransmission. Copyright by ERIKA NICOLE BROWN 2000 DEDICATION I would like to dedicate this thesis to my parents, George and Carolyn Brown, Sr., and to my brother, George E. Brown, Jr. They have supported me throughout my graduate career, and they continue to do so through emotional, financial, and spiritual support. Thank you Mom, Dad, and George Jr. iv ACKNOWLEDGMENTS To my advisor, James J. Galligan, Ph.D., I would like to give thanks to him for allowing me to perform research studies in his laboratory. I would like to thank him for his continuous support, guidance, and knowledge he was willing to share with me and others in the laboratory and in course lectures. Lastly, I would like to thank Dr. Galligan for times when he was willing to help me out with problems or concerns I had either in my course work , laboratory research, or future career goals. Thank you Dr. Galligan. To the members of my graduate committee, Dr. William Atchison, Dr. Peter Cobbett, Dr. David Kreulen, Dr. Stephanie Watts, and Dr. James Galligan, and to the other faculty members of Pharmacology and Toxicology department, I would like to thank you for your support and guidance throughout my graduate career at Michigan State University. Thank you to all. TABLE OF CONTENTS LIST OF FIGURES ................................................................................................... viii LIST OF ABBREVIATIONS ......................................................................................... x INTRODUCTION .................................................................................................... 1 ENTERIC NERVOUS SYSTEM ...................................................................... 1 Development of the ENS ..................................................................... 1 Structure of the ENS ...................................................................... 1 Neurotransmitters in the ENS ........................................................... 3 Morphology of enteric neurons ......................................................... 3 Functional classes of enteric neurons ................................................. 5 Synaptic transmission of the ENS ........................................................ 6 Diseases and Disorders of the ENS .................................................... 8 Summary of the ENS .................................................................... 1O BACKGROUND OF mAChRs AND nAChRs ................................................. 10 MAChRs and nAChRs ................................................................... 10 Structure of mAChRs ..................................................................... 1 1 Subtypes of mAChRs ..................................................................... 12 Co-expressionofmAChR gene subtypes .......................................... 13 Function and G—protein pathways of mAChR subtypes ..................... 14 MAChRs in the ENS ....................................................................... 15 Summary of mAChRs ..................................................................... 15 Structure of nAChRs ...................................................................... 16 Subtypes of nAChRs ...................................................................... 17 Function of nAChRs ....................................................................... 19 Desensitization stateoanChRs ....................................................... 21 Summary of nAChRs ..................................................................... 25 Functional significance of desensitization ......................................... 25 Overall Significance ....................................................................... 26 HYPOTHESIS ............................................................................................... 27 RESEARCH DESIGN AND METHODS .................................................................. 28 PROTOCOL .................................................................................................. 28 EXPERIMENTS ............................................................................................. 28 MAChR activation modulates nAChR desensitization ....................... 29 Cross-desensitization ........................................................................ 29 Forskolin induced modulation of nAChR desensitization .................. 30 Phorbol 12, 1 3-dibutyrate (PDBu) induced nAChR desensitization. .30 Bethanecol-induced nAChR desensitization .................................. 31 vi GENERAL METHODS .............................................................................. 31 PRIMARY TISSUE CULTURE OF MYENTERIC NEURONS ....................... 32 WHOLE-CELL PATCH CLAMP RECORDINGS ........................................... 33 DRUG APPLICATION ................................................................................. 34 STATISTICAL PROCEDURES ................................................................... 34 RESULTS .................................................................................................................. 35 MUSCARINIC ACTIVATION COUPLED ACh INDUCED nAChR DESENSITIZATION .................................................................................. 35 CROSS-DESENSITIZATION OF nAChRS IN MYENTERIC NEURONS ...... 47 TIME CONTROL OF ACh AND NICOTINE ............................................. 52 PDBu INDUCED MODULATION OF nAChR DESENSITIZATION ............... 53 F ORSKOLIN INDUCED MODULATION OF nAChR DESENSITIZATION...67 BETHANECOL INDUCED NICOTINE-nAChR DESENSITIZATION ............. 85 DISCUSSION ............................................................................................................ 87 INTRACELLULAR SIGNALING ACTIVATION ON nAChR DESENSITIZATION ...................................................................................... 87 NON ~ADENYLATE - CYCLASE SITE OF ACTION ................................... 88 TIME CONTROL-EIOH VEHICLE ON nAChR DESENSITIZATION ............. 89 MAChR EFFECTS ON nAChR ................................................................. 90 CROSS-DESENSITIZATION OF nAChR .................................................... 90 TWO TYPES OF DESENSITIZATION CHARACTERISTICS IN S-TYPE MYENTERIC NEURONS ............................................................................. 91 NAChRs IN MYENTERIC NEURONS ........................................................ 92 CONCLUSIONS ........................................................................................................ 93 BIBLIOGRAPHY ....................................................................................................... 96 vii LIST OF FIGURES Figure 1 . Desensitization rate of the nAChR ........................................................... 37 Figure 2. Traces of recovery rate of the nAChR from desensitization ...................... 39 Figure 3. Recovery rate of the nAChR from desensitization .................................... 41 Figure 4. Nicotine effects on nAChR desensitization and recovery in presence of scopolamine .......................................................................................... 43 Figure 5. Cross-desensitization effects of the nAChR ............................................. 45 Figure 6. Time control effects of ACh-induced nAChR desensitization .................. 48 Figure 7. Time control effects of nicotine-induced nAChR desensitization ............. 50 Figure 8. Traces of PDBu and 4-a-PMA effects on ACh-induced nAChR desensitization ....................................................................................... 54 Figure 9. PDBu effects on ACh-induced nAChR desensitization ............................ 56 Figure 1 O. 4-a-PMA effects on ACh-induced nAChR desensitization ..................... 58 Figure 11. Traces of PDBu and 4-a-PMA effects on nicotine-induced nAChR desensitization ..................................................................................... 60 Figure 12. PDBu effects on nicotine-induced nAChR desensitization ..................... 62 Figure 13. 4-a-PMA effects on nicotine-induced nAChR desensitization ............... 64 Figure 14. Traces of forskolin effects on ACh-induced nAChR desensitization ..... 68 Figure 15. Forskolin effects on ACh-induced nAChR desensitization ..................... 70 Figure 16. DDF effects on ACh-induced nAChR desensitization ............................. 72 Figure 17. Traces of forskolin effects on nicotine-induced nAChR desensitization ..................................................................................... 74 Figure 18. F orskolin effects on nicotine-induced nAChR desensitization ............... 76 viii Figure 19. DDF effects on nicotine-induced nAChR desensitization ....................... 78 Figure 20. Traces of bethanecol effects on nicotine-induced nAChR desensitization ..................................................................................... 81 Figure 21 . Bethanecol effects on nicotine-induced nAChR desensitization ............ 83 Figure 22. Proposed model for nAChR desensitization in the ENS ........................ 94 ix LIST OF ABBREVIATIONS 4-a-PMA: 4-a-phorbol 12-myristate 13-acetate 5-HT: 5-hydroxytryptamine ACh: acetylcholine AH: after-hyperpolarization ANS: autonomic nervous system ChAT: choline acetyltransferase CNS: central nervous system DDF: 1, 9-dideoxy-forskolin ENS: enteric nervous system fEPSPs: fast excitatory postsynaptic potentials GABA: y-aminobutyric acid GI: gastrointestinal I73: current at seven seconds Ipoakl peak current mAChRs: muscarinic acetylcholine receptors nAChRs: nicotinic acetylcholine receptors NE: norepinephrine NO: nitric oxide NOS: nitric oxide synthase NPY: neuropeptide Y PDBu: phorbol 12, 13-dibutyrate Pl: phosphatidyl inositol PKA: protein kinase A PKC: protein kinase C sEPSPs: slow excitatory postsynaptic potentials SP: substance P TMDs: transmembrane domains VIP: vasoactive intestinal peptide INTRODUCTION ENTERIC NERVOUS SYSTEM (ENS) Development of the ENS The ENS is the division of the autonomic nervous system (ANS) that is localized in the wall of the gastrointestinal (GI) tract and is responsible for controlling GI function. GI functions controlled by the ENS include motility, blood flow, exocrine and endocrine secretions, and regulation of immune and inflammatory processes (Goyal and Hirano, 1996). The ENS differs from the sympathetic and parasympathetic divisions of the ANS in its embryonic origin. The ENS arises from precursor cells from the vagal neural crest. Precursor cells migrate to the gut beginning at the proximal portion of the GI tract and spreading to the distal portion of the GI tract (Goyal and Hirano, 1996). Structure of the ENS The ENS is able to function without connections with the central nervous system (CNS). The ENS is capable of mediating coordinated reflexes such as intestinal peristaltic contractions through the activity of intramural sensory neurons, intemeurons and motor neurons (Goyal and Hirano, 1996; Costa et al, 1986). The ENS contains approximately 100 million neurons and the function of individual neurons and neurochemistry are similar to neurons in the CNS. Communication 1 between the CNS and ENS occurs via efferent neurons of the sympathetic and parasympathetic nervous system, and also via sensory afferent neurons from dorsal root ganglia and vagal afferent fibers. This communication is known as the central autonomic neural network which contributes to the neuronal control of GI functions (Goyal and Hirano, 1996). In the ENS, nerve-cell bodies are grouped together to form ganglia. Ganglia in the ENS are connected via interconnecting nerve strands which contain nerve axonal processes and form two ganglionated plexuses called the myenteric (Auerbach’s) and the submucosal (Meissner’s) plexuses (Goyal and Hirano, 1996; Costa et al, 1986). The myenteric plexus resides between the longitudinal and circular smooth muscle layers and it controls motor function of the GI tract. The myenteric plexus contains intrinsic sensory neurons, intemeurons, and excitatory and inhibitory motor neurons. The submucosal plexus resides in the connective tissue between the circular muscle and mucosal layer where it controls secretomotor function of the mucosal epithelium and local blood flow. The submucosal plexus also contains sensory neurons, intemeurons, and motorneurons (Goyal and Hirano,1996, Costa, Fumess, and Gibbins, 1986). Myenteric and submucosal ganglia contain tightly packed nerve cell bodies, terminal bundles of nerve fibers, and glial cells (Goyal and Hirano, 1996). There are more glial cells in the ENS than neurons and enteric glia are similar to astrocytes found in the CNS (Gershon and Rothman, 1991). Glial cells have laminar processes that are extend over the surface of enteric neurons (Gershon 2 and Rothman, 1991). In response to cytokine stimulation, glial cells are able to express interleukins and MHC class II antigens which suggests a role for glia in regulating inflammation in the GI tract (Goyal and Hirano, 1996). Neurotransmitters in the ENS There are more than 20 neurotransmitters released by myenteric and submucosal neurons. These neurotransmitters include: norepinephrine (NE), 5- hydroxytryptamine (5-HT), y-aminobutyric acid (GABA), ATP, nitric oxide (NO), neuropeptide Y (NPY), opioids, somatostatin, substance P (SP), and vasoactive intestinal peptide (VIP). The major excitatory neurotransmitter is ACT (Costa et al, 1986, Gayal and Hirano, 1996, Kunze and Fumess, 1999). Therefore, cholinergic neurotransmission plays a critical role in controlling normal Gl function. Morphology of enteric neurons The morphological types of enteric neurons found in the ENS are Dogiel Type I, Dogiel Type II, and Dogiel Type III (Hirst et al, 1974; Nishi and North, 1973; Fumess and Costa, 1987; Costa et al, 1986; Brookes et al, 1990; Song et al, 1996). Dogiel Type I neurons have flat cell bodies, numerous short lamellar dendritic processes, and one long axon (Brookes et al, 1990). These neurons are motor neurons and intemeurons. Dogiel Type I neurons are “S” neurons classified using electrophysiological criteria (Hirst et al, 1974; Nishi and North, 1973; Fumess and Costa, 1987; Brookes et al, 1990). 3 Stimulation of interconnecting nerve strands produces fast excitatory postsynaptic potentials (fEPSPs). F EPSPs last less than 20 milliseconds and are associated with increased membrane conductance and reversal potentials near zero millivolts. ACT released from nerve terminals acts at nAChRs on S neurons to mediate fEPSPs (Hirst et al, 1974; Nishi and North, 1973; North et al, 1980; Fumess and Costa, 1987). Dogiel Type II neurons are multipolar neurons that have large oval smooth cell bodies and several long axonal processes (Fumess et al, 1998 and Song et al, 1996). Axonal processes arise from cell bodies to project either to the mucosal layer or project circumferentially to other myenteric ganglia (Fumess et al, 1998). Dogiel Type II neurons are electrophysiologically characterized as AH (after- hyperpolarization) neurons and they are sensory neurons (Hirst et al, 1974; Nishi and North, 1973; Fumess et al, 1998; Kunze and Fumess, 1999). AH neurons receive synaptic input via slow excitatory postsynaptic potentials (sEPSPs). A prominent electrophysiological property of AH neurons is the action potential and hyperploarization that is slow in onset that lasts up to twenty seconds (Nishi and North, 1973; Hirst et al, 1974). The long-lasting after-hyperpolarization results from an increase in calcium dependent potassium (Hirst and Spence, 1973; North, 1973; Morita et al, 1982; Hirst et al, 1985; North and Nishi, 1974). Filamentous type neurons have smooth ovoid cell bodies, single long axons and several filamentous dendrites (Song et al, 1996). These neurons project anally in the myenteric plexus (Song et al, 1996). There function is not known. 4 Functional classes of enteric neurons in the ENS There are three functional types of enteric neurons. Excitatory and inhibitory motor neurons including secretomotor, intemeurons, or intrinsic sensory neurons (Kunze and Fumess, 1999). Excitatory and inhibitory motor neurons have cell bodies that are localized along the wall of the GI tract. Motor neurons in the stomach are activated by the stimulation of the vagus nerve, but vagus nerve stimulation has less effect in the small intestine and colon (Gayal and Hirano, 1996; Kunze and Fumess, 1999). Excitatory motor neurons project to circular muscle and run circumferentially in the direction of muscle cells (Gabella, 1972; Kunze and Fumess, 1999). The primary neurotransmitter is ACT as cholinergic transmission can be largely blocked pharmacologically by muscarinic cholinergic antagonists. With the addition of tachykinin receptor antagonists, neuromuscular transmission is completely blocked (Costa et al 1996; Kunze and Fumess, 1999). Excitatory motor neurons function is to cause contractions. Inhibitory motor neurons project to muscle close and anal to their cell bodies (Fumess et al, 1995; Kunze and Fumess, 1999). The transmitter released from inhibitory motor neurons is not clear but it is likely that NO, ATP, VIP, and pituitary adenyl cyclase-activating peptide participate in inhibitory neuromuscular transmission (Stark and Szurszewski, 1992; Kunze and Fumess, 1999). Inhibitory motor neurons function to cause smooth muscle relaxation. lntemeurons are localized in myenteric and submucosal ganglia, and they have orally directed or anally directed projections (Costa et al, 1996; Kunze and 5 Fumess, 1999). There are four classes of intemeurons consisting of one ascending intemeuron class and three descending intemeuron classes. The one ascending intemeuron class is cholinergic and is immunoreactive for the calcium binding protein, calretinin (Costa et al, 1996; Kunze and Fumess, 1999). Descending intemeuron classes are cholinergic but not purely as they also release other neurotransmitters. It has been found that with the addition of PPADS, a purinergic antagonist can inhibit fast excitatory transmission from some descending intemeurons (Lepard et al, 1997). The three descending intemeuron classes consist of intemeurons immunoreactive for choline acetyltransferase (ChAT)l somatostatin, ChATI NOSI VIP, andChATIS-HT (Costa et al, 1996; Kunze and Fumess, 1999). From further studies, it has been found that ChAT/NOS intemeurons are likely to be involved in inhibitory responses of the GI tract (Young et al, 1995; Yuan et al, 1995; Mann et al, 1997; Kunze and Fumess, 1999). Intrinsic sensory neurons have cell bodies localized in the wall of the intestine and project axons to other nerve cells and to the mucosa (Fumess et al , 1997). They are the first neurons in the intrinsic nerve circuitry of the GI tract to be activated by sensory stimuli such as acid, short-chain neutralized fatty acid, and 5-HT. 5-HT is important because of its intermediate action in reflexes initiated from the mucosa (Kunze et al, 1995; Bertrand et al, 1997; Kirchgessner et al, 1992; Kunze and Fumess, 1999). Synaptic transmission of the ENS AHIDogiel Type II neurons are intrinsic sensory neurons. When their synaptic inputs are activated by chemical stimuli or distention (stretch), they produce sEPSPs. Rarely, fEPSPs are recorded from AH neurons on those that are recorded, and recordings are usually low in amplitude (Iyer et al, 1988; Kunze and Fumess, 1999). Intrinsic sensory neurons give rise to varicose terminals which synapse with other neurons in adjacent. Experiments have also shown that sEPSPs from intrinsic sensory neurons originate from the synaptic input from other intrinsic sensory neurons. These observations suggest that intrinsic sensory neurons are interconnected networks and excitatory and therefore self-reinforcing (Kunze and Fumess,1999). lntemeurons project orally and anally along the intestine. Ascending intemeurons connect with other ascending intemeurons, and they also receive synaptic input from intrinsic sensory neurons. In return, they process this input to provide synaptic output to motor neurons orally along the intestine. 0f the three classes of descending intemeurons, the choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) containing classes have been found to be involved in descending reflexes because they make connections with the inhibitory motor neurons (Yuan et al, 1995; Kunze and Fumess, 1999). Pharmacological studies have shown that the ChAT and 5-HT containing classes are not involved in descending reflexes due to the lack of connections with inhibitory motor neurons, and other studies have shown that the ChAT and somatostatin containing classes receive majority of their synaptic inputs from other chat neurons and few from 7 intrinsic sensory neurons suggesting that they are auxiliary descending intemeurons involved in descending reflexes (Yuan et al, 1994; Kunze and Fumess, 1999). The circular muscle cells are electrically coupled together and receive input from motor neurons. The motor neuron axonal processes branch within the circular muscle (Kunze and Fumess, 1999). Excitatory motor neurons have axons projecting short distances along the wall of the intestine, where majority of the them synapse adjacent to the muscle at 2 mm and few synapse anally at 8 mm, to cause contraction (Brookes et al, 1991; Kunze and Fumess, 1999). Inhibitory motor neurons project about 2-14mm anally along the muscle to cause relaxation. From this, muscle along the intestine undergo contractions and relaxations, and the strength of each depends on the quantity and frequency of neurons activated (Brookes et al, 1991; Kunze and Fumess, 1999). Diseases and Disorders of the ENS There are many disorders and diseases that cause a disruption of GI motility, secretory, and inflammatory responses of the GI tract. Motility disorders include achalasia, gastric stasis and outlet obstruction, acute intestinal ileus and chronic intestinal pseudo-obstruction, megacolon, and other general motility disorders. Achalasia is caused by an inability of the lower esophageal sphincter to relax. This is due to a dysfunction of inhibitory neurons that release VIP and NO (Gayal and Hirano, 1996). Gastric stasis and outlet obstruction result from a congenital 8 disorder called infantile hypertrophic pyloric stenosis in which the neurons innervating the circular muscle do not release NO due to the lack of NOS (Gayal and Hirano, 1 996). Acute intestinal ileus and chronic intestinal pseudo—obstruction result from a loss of motor activity in the intestine. Megacolon results from a congenital disease called Hirschsprung’s disease which is identified by the lack of enteric neurons in the colon (Gayal and Hirano, 1996). Other general disorders are characterized by the involvement of more than one segment of the GI tract. This consists of either of two groups, the hyperganglionosis, cited as neuronal dysplasia or ganglioneuromatosis, or hypoganglionosis, resulting from acquired diseases leading to enteric neuron destruction (Gayal and Hirano, 1996). Secretory dysfunctions can result in diarrhea. Diarrhea can be caused by chemicals and toxins exposed to the lumen of the intestine. Such substances are ethanol, bile salts, heat-stable toxins of Escherichia coli, and cholera toxin. These substances act by stimulating mucosal receptors on the luminal layer of the intestine to cause intestinal secretomotor reflexes (Gayal and Hirano, 1996). Inflammatory and immunologic responses can cause bowel diseases. Such diseases are caused by a known toxin called Clostidium difficile toxin A (C. difficile toxin A) which result in necro-inflammatory responses that involve intrinsic neural pathways. Activation leads to mast—cell degranulation and release of inflammatory mediators such as prostaglandins, histamine, and 5-HT which stimulate secretomotor reflexes. Pharmacological experiments suggest that C. difficile toxin A acts at extrinsic primary afferent neurons containing SP. This conclusion is 9 based on studies in which these neurons were destroyed with capsaicin resulting in the inhibition of secretory and inflammatory responses to C. difficile toxin A. Another cause of bowel disease could be due to psychological stress suggesting an interaction between the CNS and the ENS (Gayal and Hirano, 1996). Summary of the ENS The ENS plays an important physiological and pathophysiological role of the ANS by being responsible for controlling the function of the GI tract with and/or without connections to CNS. One of the reasons for studying the ENS is because it is a system of its own in which neuronal network patterns, neuronal receptors and its neurotransmitters, and the functions of motility are still being studied and analyzed. A second reason is to compare and contrast the differences and similarities between the CNS and ENS because as mentioned above the ENS is a system of its own and can function without CNS input. From this, scientists can obtain knowledge on how to treat GI diseases. BACKGROUND OF mAChRs AND nAChRs MAChRs and nAChRs The first characterization of mAChRs and nAChRs was the demonstration that mAChRs are selectively activated by muscarine and blocked by atropine (Hulme et al 1990). NAChRs were identified as being selectively activated by nicotine and 10 blocked by curare (Hulme et al 1990). Subsequently, molecular biological studies would show that there are multiple genes whose products make up the mAChRs and nAChRs, therefore, making these receptors more complex than originally defined (Hulme et al, 1990). Even though mAChRs and nAChRs belong to different gene superfamilies, they share ACT as their neurotransmitter for activation (Hulme et al, 1990). This holds true for the ENS because even though the ENS has many neurotransmitters responsible for the regulation of GI function, the major neurotransmitter is ACT (Gayal and Hirano,1996, Galligan, 1993). In enteric ganglia, ACT acts at mACh Rs and nAChRs to excite enteric neurons (Bertrand and Galligan, 1995). Structure of mACh Rs MAChRs belong to the family of G-protein coupled receptors that link to one or more intracellular signaling pathways. There are approximately 20 members of the G-protein coupled superfamily which includes the catecholamine receptors, serotonin receptors, peptide receptors, mas oncogen, rhodopsin family, and several other receptors of unknown specificity (Hulme et al, 1990). Of these members, the mAChRs have the greatest homology with adrenergic receptors (Hulme et al, 1990). MAChRs transverse the membrane seven times creating an extracellular NH,+ terminus and an intracellular CO; terminus and extracellular and intracellular loops connecting seven transmembrane domains (TMD l-Vll). There 11 is overall sequence variation in the NH; terminus, third intracellular loop, and in the CO,‘ terminus, but there are conserved subsections of the third intracellular loop and CO; terminus. Also, there is great homology between TMD Il-Vll. Seven residues of the C01; terminus of third intracellular loop are recognized as a determinant for G-protein coupled specificity. The third intracellular loop has substrate sites that are recognized by kinases and also influences mAChR expression. With other G-protein coupled receptors, the CO; terminus influences the activation of effector systems, but this is yet to be observed with the mAChR C02“ terminus (Hulme et al, 1990). For mAChR ligand binding, the TMD III has aspartate amino acid residues that participate in binding (Hulme et al, 1990). MAChR subtypes have been recognized thus far to be either coupled via a pertussis toxin (PTX) insensitive G-protein that activates Pl hydrolysis (6,) or via a PTX sensitive G-protein that inhibits adenylate cyclase (G,) (Hulme et al, 1990). Subtypes of mAChRs There are five subtypes of the mAChR,1-5. The definition and nomenclature of the five subtypes have been partially characterized by binding, pharmacological, mechanistic, and molecular studies (Hulme et al, 1990). Pharmacologically, the subtypes are named as M1, M2, M3, M4, and M5, and structurally, the subtypes are defined as m1-m5 based upon their amino acid sequences (Hulme et al, 1990). To provide a source for the receptor clones of the mAChR subtypes, cDNA libraries were obtained, and from this, the m1 gene has been cloned from the human, rat, 12 pig, and mouse, the m2 and m3 genes have been cloned from the human , rat, and pig, and the m4 and m5 genes have been cloned from the human and rat (Hulme et al, 1990). From this, questions arose of whether if there are other muscarinic receptor genes present and this brought up the analysis of a DNA hybridization of a partial m1 cDNA which resulted in the production of up to ten hybridizing bands from rat and up to six hybridizing band from human (Hulme et al, 1990). In northern blot studies, the m1 and m3 genes are localized in the brain and exocrine glands, the m2 gene is found in cardiac and smooth muscle, the m4 gene is localized mainly in neural tissue, and the m5 gene is found in the brain (Hulme 1990). Co-expression of mAChR gene subtypes From in situ hybridization studies, m1, m2, m3, and m4 genes are localized in autonomic ganglionic neurons. But by studying the signal transduction pathway of the mAChR subtypes, observations show that the m1 and m3 receptors subtypes couple to the activation of PI hydrolysis, whereas the m2 and m4 receptor subtypes couple to the inhibition of adenylate cyclase (Hulme et al, 1990). These coupling systems are only observed in heterologous expression systems, therefore, questions arise of why this is not observed in native cells. These question include: (1) Do effector systems in vivo couple differently than when expressed in vitro? This question can only be answered in cells expressing native mAChRs in studies using pharmacological agonists and antagonists that are able to act specifically to 13 identify function of the mAChR subtypes. (2) Can a specific effector system be coupled to different receptor subtypes but be regulated by different signals? This would allow cell flexibility to a signal modifying a response. (3) Are different subtypes localized on different cellular domains, that is, on presynaptic nerve terminals or postsynaptically? These factors would determine the different interactions between the subtypes on a cell (Hulme et al, 1990). Function and G-protein pathways of mAChR subtypes In the ENS, mAChRs mediate slow synaptic responses and long lasting changes in neuronal excitability (Morita et al, 1982, Galligan, 1998). MAChR agonists that are able to mimic slow synaptic potentials are bethanecol, carbachol, methylcholine, muscarine, and oxotremorine (Morita et al, 1982, Cassell and McLachlan, 1987, Hulme et al, 1990), and at this time there is no agonists that are able to act at mAChR subtypes specifically. MAChRs can be blocked nonspecifically by atropine, hyoscine, and scopolamine (North 1982, Morita et al, 1982, and Wessler et al, 1987). The M1-mAChR subtype elicits slow excitatory responses and can be found in autonomic ganglia and in the CNS. This subtype is thought to act through a Gq protein that is coupled to Pl hydrolysis. Responses can be blocked by pirenzepine (Hulme et al, 1990). The M2 subtype exhibits slow inhibitory responses and can be found in the CNS and peripherally. It is thought to act through a GI protein and can be blocked by AF-DX116 (Hulme et al, 1990). The M3 subtype elicits slow 14 excitatory responses and can be found in secretory glands and smooth muscle and in the CNS. It is thought to act through a Gq protein that is coupled to Pl hydrolysis and can be blocked by hexahydrosiladifenidol (Hulme et al, 1990). The M4 subtype elicits slow inhibitory responses and can be found in the brain and some smooth muscle. It is thought to act through a Gi protein and can be blocked by himbacine (Hulme et al, 1 990). The ma subtype was detected by RNA hybridization from to tissue collected from the rat brain, therefore, this mAChR subtype is localized in the brain (Hulme et al, 1990). The physiological function and pathway of this receptor subtype is unknown. From this, a hypothesis can be formed that M1 activation is responsible for mediating muscarinic effects in the ENS. MAChRs in the ENS MAChRs in the ENS mediate sEPSPs in myenteric S neurons (North et al, 1982; Galligan, 1998). Although the specific signaling pathways involved in mediating sEPSPs are not known, there is evidence that PKC and adenylate cyclase activators are able to mimic sEPSPs on myenteric ganglia (Bertrand and Galligan, 1995; Palmer et al, 1987; Galligan, 1998). Summary of mAChRs MAChRs are G-protein coupled receptors that mediate slow synaptic responses through either of the four subtypes to cause neuronal excitability and muscle contraction and relaxation responses. These responses are regulated by 15 intracellular signaling pathways which are able to cause modulation of receptors, mAChRs and other receptor subtypes, and also influence the expression of receptors. Pharmacologically, mAChRs can be activated by agonists such as ACT, bethanecol, carbachol, methylcholine, muscarine, and oxotremorine, and they are able to be blocked nonspecifically by atropine, hyoscine, and scopolamine, and specifically by pirenzepine, AF -DX1 16, hexahydrosiladifenidol, and himbacine. In the ENS, mAChRs mediate sEPSPs, but the intracellular signaling involved regulating these responses are not known. Therefore, future studies should focus on how mAChR activation modulates GI function through specific intracellular signaling pathways. Structure of nAChRs NAChRs belong to a superfamily of membrane bound ligand-gated ion channels that mediate cell to cell communication by transforming neurotransmitter signals into an ion flux at the postsynaptic neuron or muscle (Corringer et al, 2000). Other members of this superfamily include receptors for glutamate, GABA, 5-HT, glycine, and ATP. The nAChR structure, based on the number of times it transverses the membrane (four TMDs), closely resembles the GABA,, GABAC, 5-HT,, and glycine receptors (Corringer et al, 2000). NAChRs are one of the largest members and among this group. They contribute to cell to cell communication by being distributed at presynaptic and/or postsynaptic sites (Corringer et al, 2000). NAChRs are found throughout the CNS and PNS, neuromuscular junctions, and adrenal glands. 16 The nAChR is composed of a pentameric structure that protrudes out extracellularly and intracellularly to form a five fold axis that is perpendicular to the membrane (Corringer et al, 2000). This structure forms a pore that becomes narrower within the transmembrane (Corringer et al, 2000). As a whole structure, the nAChR consists of an NH; and a CO,’ terminus localized synaptically, three TMDs with short extracellular and intracellular loops, a large variable third intracellular loop, and a fourth TMD (Corringer et al, 2000). There are five subunits that make up the nAChR oligomer, and they fall into two main classes. These classes include the a subunit class and the non-a subunit class. The a subunit class consists of nine subtypes, a1-ct9. This class has two adjacent cysteines present which are responsible for agonist binding (Corringer et al, 2000). The non-or class consists of the 8,7,8, and 8 subunits which the [3 subunit has four subtypes, [31-84. This class does not bind agonists (Corringer et al, 2000). Subtypes of nAChRs There are two subtypes of nAChRs, the muscle nAChR and the neuronal nAChR. Four homologous subunits make up the muscle subtype of nAChR, and the subunit composition consists of a,[3,y,8, or 3. These subunits assemble into a heteropentamer with a stoichiometry of (112,8 1 ,y, and 8, or sometimes a (Rangwala et al, 1997; Corringer et al, 2000). The ion permeability ratio of Na‘, K’, Ca2+ is 12120.2 (Adams et al, 1980; Vemino et al, 1 992). Neurons express a varied range 17 of nAChR subtypes which makes characterizing the neuronal subtype difficult (Rangwala et al, 1997). The subunit composition is composed of a and B with the a subunit consisting of eight subtypes, a2-a9, and the [3 subunit consisting of three subtypes, 82-84. The neuronal subtype is expressed in the brain, sensory end organs, and peripheral ganglia, where the a and 8 subunits provide functional and pharmacological diversity (Rangwala et al, 1997). Neuronal nAChRs have best been described in brain tissue by high affinity nicotine binding in which the nAChR is composed of a4 and B2 subunits with a stoichiometry of a42823, and it has also been characterized by the high affinity a-bungarotoxin receptor which is composed of homopentamer of a7 subunits (Rangwala et al, 1 997; Chen and Patrick, 1997). The ion permeability ratio of Na‘, K“, Ca2+ is 1:1 :5 (Nutter and Adams, 1995). Parts of TMD l and II of all the subunits make up the lining of the channel. For both types of nAChRs, the a subunit is organized around the channel so that the two agonist binding sites are at the interfaces between the a subunit and the non-a subunit (Gerzanich et al, 1998). Pharmacologically, muscle nAChRs can be activated by such agonist as ACT (Dionne and Stevens, 1 975) and can be blocked by tubocurarine (Ascher et al, 1978). For the neuronal types, nAChRs can be activated by ACh, choline, cytisine, DMPP, (-) epibatidine, and (-) nicotine (Albuquerque at al, 1997). These receptors can also be blocked nonspecifically by hexamethonium and decamethonium (Ascher et al, 1 979), and specifically , they can be blocked by a-bungarotoxin and methyllycaconitine (OJ-nAChRs), dihydro-b- 18 erythroidine (a482-nAChR composition), and mecamylamine (a384-nAChR composition) (Albuquerque at al, 1997). Function of nAChRs NAChRs mediate fEPSPs in the peripheral nervous system and the neuromuscular junction. These fast excitatory responses cause muscle to contract and neurons to become excitable. NAChRs depolarize neurons via their ion channels to permit the flow of cations across the membrane. NAChRs regulate neurotransmitter release presynaptically, and they cause synaptic currents from synaptic and presynaptic locations on postsynaptic sites (Conroy and Berg 1997). The neuronal nAChRs can become functional with the (1382 combination 0:382 or 0:384 combinations, or with the a7 combination (Gerzanich et al, 1998; Blumenthal et al 1999). Also, the a5 subunit has been found in the a384 or a382 combination but the exact stoichiometry of this combination has not been determined. The a5 subunit has been found not to form functional nAChRs and is organized along the channel so that it does not interface with the sides that are involved in forming agonist binding sites (Gerzanich et al, 1998). One prominent property of nAChRs is that they desensitize rapidly and the rate of desensitization and recovery from desensitization can be altered by receptor modification. Desensitization was first described at the neuromuscular junction (Katz and Thesleff, 1957). Agonist binding to the receptor activated the channel and thus, ion passage through the channel (Katz and Thesleff, 1957; Reitstetter 19 et al, 1999). Prolonged exposure to the agonist can lead to a desensitized, inactivated state where the ion passage through the channel decreases (Reitstetter et al, 1999). Recovery from desensitization occurs when agonist is removed (Reitstetter et al, 1999). In the neuronal nAChRs, desensitization has a role in synaptic plasticity and nicotine dependence in smokers (Reitstetter et al, 1999). Although neurotransmission at nAChRs is an important mechanism in the ENS, there have been few studies of desensitization of these receptors in the ENS or how desensitization is modulated by simultaneous activation of other neurotransmitter receptors expressed by the same neurons. Desensitization of the nAChR is thought to be modulated by phosphorylation when intracellular signaling pathways are activated during neurotransmission. It has been determined by using heterologously expressed nAChR in Xenopus oocytes that the a and 8 subunit composition influences desensitization of nAChRs (Fenster et al, 1997). It was found that nAChRs containing a4 subunits were more sensitive to nicotine activation than nAChRs containing a3 subunits, and also that nAChRs containing 82 subunits reach maximal desensitization at lower concentrations of nicotine compared to nAChRs containing 84 subunits. It was also shown that the a subunit influences the rate of desensitization and the rate of recovery from desensitization (Fenster et al, 1997). In another study using rat striatal synaptosomes, it was found that prolonged treatment with nicotine results in incomplete recovery from desensitization suggesting that high concentrations of nicotine can produce long-lasting inactivation of the nAChR (Rowell and Duggan, 20 1998). Using nAChRs expressed in Xenopus oocytes, it was found that the 0:5 subunit increases desensitization and calcium permeability of 0:382 containing nAChRs (Gerzanich et al, 1998). Studies have also shown, using rat chromaffin cells, thatchanges in intracellular calcium following activation intracellular signaling pathways (Khiroug et al, 1998). For example, nAChR desensitization is accelerated by PKA mediated phosphorylation, and PKA activation keeps the nAChR in a long-term desensitized state (Paradiso and Brehm, 1998). It has also been shown that PKC and PKA mediated phosphorylation of nAChRs increases the rate of desensitization (Nishizaki and Sumaikawa, 1998). Therefore, the properties of desensitization of the nAChR are dependent on subunit composition, changes in intracellular calcium, and activation of PKA and PKC dependent pathways. Desensitization state of nAChRs Subunit composition development seems to play a part in the desensitization state of the nAChR. In muscle, stabilization of the subunit composition on nAChR desensitization plays a part in the kinetics of gating modes during development because in Xenopus oocytes, the embryonic state (088w) tended to interchange between short and long open time gating modes, but in the adult (0:858), nAChRs have a short open time gating mode (Naranjo and Brehm, 1993). The decay after ACT- induced maximal response at the nAChR is described by the sum of two exponential components and the recovery rate from the desensitized state is also 21 the sum of two exponential functions, a fast and slow time constant (Naranjo and Brehm, 1993). This suggests that a fast onset to desensitization can lead to a fast recovery when agonist is removed from nAChR binding sites. A proposed model of nAChR desensitization is based on two ACT molecules binding to the nAChR to cause a high probability of the channel to become open. At this stage, the receptor is considered to be in an unliganded closed state (A2R) (Naranjo and Brehm, 1993). From linear relationship analysis, the closed state undergoes a conformational change to either the open state (A2R*) or the desensitized state (A2D) (Naranjo and Brehm, 1993). From this study, the authors conclude that desensitization rates of specific agonists may depend on the destabilization of a particular gating mode. Also, the gating modes may be due to a limited number of protein conformation sets in which each gating mode is modulated. And they also proposed that modulation may be due to a short-term post-translational modification (Naranjo and Brehm, 1993). In another study, the authors wanted to test what a high agonist concentration would have on intrinsic channel kinetics (Jones and Westbrook, 1996). A high concentration results in receptor saturation and minimizes the unbound state of the nAChR so that the nAChR current produced reflects only the bound to open transition, and the decay phase reflects the bound to desensitization state (Jones and Westbrook, 1996). They concluded that nAChRs, under normal physiological conditions, are more likely to unbind than to desensitize with short pulses of agonist (Jones and Westbrook, 1996). 22 A study on neuronal development has found that there are increases in ACT sensitivity and receptor number, but there are also decreases in the rate of receptor desensitization and apparent affinity for ACT (Margiotta and Gurantz, 1989). Also, the CAMP-dependent mechanism develops to enhance the ACT response. These changes were due to a shift in the small and large conductances and also by a change in the large conductance kinetics (Margiotta and Gurantz, 1989). The authors concluded that neuronal development plays a role in controlling receptor regulation and expression of different nAChR subtypes (Margiotta and Gurantz, 1989) There are studies that suggest that nAChRs are phosphoproteins (Gordon et al, 1977; Teichberg et al.1977; Huganir and Greengard, 1983; Huganir et al, 1984; Miles et al, 1987). Studies using the Torpedo electric organ indicate that CAMP-dependent PKA, PKC and tyrosine-specific protein kinase target phosphorylation sites on nAChRs (Huganir et al, 1984). Rat muscle cell studies have shown that phosphorylation by PKA increases the nAChR desensitization rate, and also that, forskolin and its analogs activate CAMP-dependent pathways to increase nAChR desensitization by phosphorylation of the receptor (Albuquerque et al, 1986; Middleton et al, 1986). In another study, the authors wanted to test nAChR modulation on the single channel level, and they found that forskolin increased agonist-induced desensitization without changing the properties of single channel currents (Middleton et al, 1988). The authors concluded that forskolin acts by promoting phosphorylation of nAChRs by PKA, and also that at low 23 concentration of forskolin, there is no direct affect of nAChR function (Middleton et al, 1988). In neuronal nAChRs, research studies have found that PKC an cAMP- dependent pathways regulate nAChR function by phosporylation. In a study using ACT- induced currents in voltage clamped sympathetic ganglion neurons, the authors tested the effect of PKC mediated phosphorylation on the regulation of neuronal nAChR desensitization (Downing and Role, 1987). This study found that the desensitization rate is enhanced by PKC, and the study concluded that modulation of the nAChR function involves PKC mediated phosphorylation changes (Downing and Role, 1987). In other studies using chick ciliary ganglion neurons, effects of cAMP enhances ACT response of the neurons by a mechanism involving an increase in number of functional nAChRs, which does not require protein synthesis nor an increase in total number of nAChRs (Margiotta et al, 1987; Vijayaraghavan et al, 1 990). Results from these studies show that cAMP mediates the recruitment of functional receptors from intracellular receptors or from nonfunctional receptors on the cell surface and causes a two to three fold increase in ACh response (Margiotta et al, 1987; Vijayaraghavan et al, 1990). These studies concluded that this regulation provides a reversible mechanism of cell to cell interaction to modulate synaptic transmission (Margiotta et al, 1987; Vijayaraghavan et al, 1990). Phosphorylation of neuronal nAChRs differs from muscle type in that neuronal nAChRs are phosphorylated on the 0: subunits rather than the non-ligand 24 binding subunits. Two studies using 0:4 nAChRs purified from rat brains and the other using rat and chick 0:7 nAChRs expresses in E. Coli have shown that these subunits have intracellular domains containing serine residues that are phosphorylated by CAMP-dependent pathways (Nakayama et al, 1993; Moss et al, 1996). These studies conclude that PKA plays a role in protein phosphorylation which mediates cellular regulation of neuronal nAChR function (Nakayama et al, 1993; Moss et al, 1996). Summary of nAChRs NAChRs are ligand-gated ion channels that form a pentamer shape that transverses the membrane forming a central pore that is aligned by TMDs 1 and II. These receptors when activated by agonists cause muscle to contract and neurons to become excitable. These responses are regulated by desensitization following prolonged agonist exposure. The functional basis of activation and desensitization of nAChR can be caused by the subunit combination composition of the nAChR. Because nAChRs make up a diverse group of ligand-gated ion channels, it makes them difficult to study and analyze. Understanding the nAChRs in the CNS via there function and subunit composition is becoming clearer with pharmacological, molecular and desensitization studies, but similar studies of nAChRs in the ENS have not been done. Therefore, in future studies, it would be important to identify how subunit composition and nAChR modulation plays a role in GI tract function. Functional significance of desensitization 25 There are multiple neurotransmitters and neurotransmitter receptors in the ENS. Drugs which either mimic or block the action of these neurotransmitters can alter gastrointestinal function in vivo and in vitro. Most previous studies have examined neurotransmission occurring during activation of only a single receptor. A question that has not been addressed is: Are there functional interactions between different receptors activated simultaneously during enteric neurotransmission? One way receptors can interact functionally is by modulation of desensitization. Activation of nAChRs excites enteric neurons and increases intracellular Caz“. Activation of M, receptors also excites enteric neurons and increases intracellular Ca”. Acceleration of nAChRs desensitization following M, activation may provide a negative feedback inhibition on nAChRs to minimize Ca2+ entry and neuronal excitation during bursts of high synaptic activity. Overall significance Because ACT is the principal excitatory transmitter in the ENS, cholinergic neurotransmission plays a critical role in controlling normal gastrointestinal function. Changes in cholinergic neurotransmission may be associated with gastrointestinal motility disorders such a diabetic gastropareisis, intestinal pseudo-obstruction, and constipation-irritable bowel syndrome. Therefore, it is relevant to study desensitization of nAChRs which is modulated by activation G-protein coupled receptors that activate intracellular signaling mechanisms. 26 HYPOTHESIS Desensitization of the nAChR in the ENS is modulated by simultaneous activation of membrane bound G-protein receptors that activate intracellular signaling mechanisms. These intracellular signaling mechanisms alter the functional properties of nAChRs. Specifically, mAChR activation accelerates desensitization of nAChRs in the ENS. 27 RESEARCH DESIGN AND METHODS PROTOCOL Myenteric neurons maintained in primary culture and whole cell patch clamp methods were used to record inward currents caused by nAChR agonists. Neurons were voltage clamped at -60mV (resting membrane potential for myenteric neurons) (Hodgkiss and Lees, 1983; Fumess and Costa, 1987) and agonists were applied by gravity fed flow tubes positioned within 100 um of the neurons. Desensitization caused by ACh and nicotine was studied. The agonist concentration was 1mM based on previous dose-response curves performed in the laboratory and in the literature showing that the maximal response occurs at 1mM concentration for the two agonists (Zhou and Galligan 1998, and Fenster et al 1997). Agonists were applied for 7 seconds to ensure complete desensitization of nAChRs. Peak desensitization was measured as the current remaining at 7 seconds (I,,) as a fraction of peak current (lmk). Desensitization rate was measured using an exponential standard fit and the Levenberg- Marquardt least square minimizing algorithm to determine the time course of decay of the agonist current during continuous application. Recovery rate was measured as the time required for complete recovery of agonist induced current after desensitization. EXPERIMENTS 28 MAChR activation modulates nAChR desensitization To test that desensitization of nAChRs is modulated by mAChR activation, the first study tested that ATP and GTP is needed to mediate the function of nAChRs. In this study, ACh was applied and ATP and GTP were eliminated from pipette solution. Peak desensitization, desensitization rate, and recovery rate were measured. The results were compared to those obtained under control conditions in which the standard pipette solution contains 1mM ATP and 0.25mM GTP. The second study tested the affects of the mAChR antagonist scopolamine (1 (M) on ACh and nicotine-induced desensitization of nAChRs. In this study, scopolamine was added to the extracellular solution and the drug- containing flow tubes. Peak desensitization, rate and recovery rate were measured and the results were compared to obtained with scopolamine free extracellular solution. Cross-Desensitization This study tested the hypothesis that ACh and nicotine act at the same set of nAChRs by attempting to demonstrate that they cross desensitize. In this study, nicotine was applied for 2 seconds to obtain control peak responses. Then ACh was applied for 7 seconds to desensitize the nAChR and then nicotine was re- applied for 2 seconds. The complementary experiment was also done, where ACh was applied for 2 seconds, then nicotine was applied for 7 seconds to desensitize the nAChR and then ACh was re-tested. The amplitude of the test 29 response (after desensitization) was compared to the initial control response for each agonist and this ratio was taken as an index of cross-desensitization. Forskolin induced modulation of nAChR desensitization This tested the effects of forskolin application on myenteric neurons. This study examined the possible role that CAMP-dependent activation has in modulating nAChR desensitization. Scopolamine (18M) was present throughout these studies. In this study, ACh (1mM) application was applied for 7 seconds to myenteric neurons with additions of varied concentrations of forskolin (100nM, 18M, and 108M). The complementary experiment was also done in which nicotine (1mM, 7 seconds) was applied in the presence of varied concentrations of forskolin (10nM, 100nM, 10M, and 108M). To test that forskolin was not acting nonspecifically to alter nAChR function, 1,9-dideoxy-forskolin (DDF), an inactive analog of forskolin, was applied. Concentrations of DDF used were 10nM, 100nM, 10M, and 100M. Phorbol 12, 13-dibutyrate (PDBu) induced nAChR desensitization PDBu was used to examine the possible role PKC activation and PI hydrolysis pathway may have on modulating nAChR desensitization. Scopolamine (1mM) was present throughout these studies. This was tested using ACh (1mM, 7 seconds) application with varied concentrations of PDBu, 10nM, 30nM, 100nM, and 300nM. The complementary experiment was also done in which nicotine (1mM, 7 seconds) was applied to the neurons with the same concentrations of PDBu. To test that PDBu was not acting nonspecifically to alter nAChR function, 30 4-0:-Phorbol 12-myristate 13-acetate (4-0:-PMA), and inactive phorbol ester analog, was applied in the presence of ACh (1mM, 7 seconds) and nicotine (1mM, 7 seconds). The concentrations of 4-0:-PMA used were 10nM, 30nM, 100nM, and 300nM. Bethanecol-induced nAChR desensitization This tested the effects of bethanecol, a muscarinic receptor agonist, application on nAChR desensitization. Bethanecol was used to examine the possible role muscarinic receptor activation may have on modulating the nAChR desensitization. In this study, 1mM-7 second nicotine application was applied to neurons which was followed by an application of nicotine plus bethanecol after a 5 minute recovery period in which the neurons were bathed in Krebs’ solution. The concentrations of bethanecol used were 18M, 108M, 100nM, and 1mM. GENERAL METHODS The specific hypothesis proposed was tested by using whole cell patch clamp methods to make electrophysiological recordings from myenteric neurons maintained in primary culture. This method allows adjustments to the intracellular environment by using a patch-clamp pipette, and it also allows the transmitter ACh and other drugs to be applied to the cell via computer - controlled, solenoid-gated flow tubes. Drugs are used to activate or block receptors and intracellular signaling pathways. Any changes in nAChR desensitization are measured after drug treatment. 31 PRIMARY TISSUE CULTURE OF MYENTERIC NEURONS (Zhou and Galligan 1998) The procedures used in these studies are similar to previously published methods using similar preparations. Newborn (1 to 2 days old) guinea pigs were anesthetized by halothane inhalation, stunned, and exsanguinated by severing major neck blood vessels. The ileum (20cm) were placed in 4°C Krebs bicarbonate buffer of the following composition (mM): NaCl, 117; KCI, 4.7; CaCl,, 2.5; MgClz, 1.2; NaH2P04, 1.2; NaHCOa, 25; glucose, 11. The longitudinal muscle myenteric plexus was stripped free using a moist cotton swab and then cut into 5mm pieces. Dissected tissues were divided into two equal aliquots and each aliquot was transferred to 1mL of sterile-filtered Krebs solution containing 1600 U of trypsin (Sigma Chemical Co., St. Louis, M0) for 30 minutes at 37°C. Following trypsin incubation, tissues were triturated 30 times through a fire- polished Pasteur pipette and centrifuged at medium speed for 10 minutes using a bench-top centrifuge. The supernatant was discarded and the pellet resuspended in 1mL Krebs solutions containing 4000 U crab hepatopancreas collagenase (Calbiochem-Novabiochem, Corp., La Jolla, CA). The low protease activity of this collagenase preparation allows incubation in the presence of Ca2+ and Mg”. The presence of Ca2+ in the incubation medium preserves the viability of neurons to a greater extent than obtained when incubation is done in the Ca2*lMgz” - free media. The suspension was triturated using fire-polished Pasteur pipette and the centrifuged for 10 minutes. The pellet was resuspended 32 in Easgle’s minimum essential medium (MEM) containing 10% fetal bovine serum, gentamycin (10mM/mL), penicillin (1OOUImL) and streptomycin (50mglmL) (all from Sigma). Aliquots (200mL) was plated onto sterile, poly-L- lysine (MW 30-70K, Sigma) - coated 35 mm plastic culture dishes containing 3mL of MEM. After two days of incubation, cytosine arabanoside (10mM) was added to the MEM to limit smooth muscle and fibroblast proliferation. Cultures were maintained at 37°C in a tissue culture incubator containing a 5% CO2 atmosphere. Cultures were maintained up to 2 weeks after plating with medium replacement every 2 days. WHOLE-CELL PATCH CLAMP RECORDINGS These studies focused on the characterization of the agonist-induced desensitization of the nAChR in cultured neurons. The extracellular solution was the standard Krebs solution described above and was maintained at 37°C and superfused through the culture dish at a flow rate of 4 mUmin. Patch electrodes had a tip resistance of 3-7 MQ. Electrode liquid junction potentials and series resistance was compensated. The standard intracellular (pipette) solution for patch clamp recording is as follows (mM): CsCl, 160; MgCl2, 2.9; EGTA, 10; HEPES, 10; ATP, 0.5; GTP, 0.25; pH was adjusted to 7.4 using CsOH. The CaCl2/EGTA ratio yielded a resting level of free [Ca2+] of less than 100 nM. Experiments were performed using an Axopatch 2008 patch clamp amplifier, a Digidata 1200 AID converter and pCLAMP 6.01 programs for acquisition, storage 33 and analysis of data (Axon Instruments, Burlingame, CA). Data was filtered at 2 kHz using a 4 pole Bessel filter (Warner Instruments, New Haven, CT) and were digitized at a rate of 5 kHz and stored on the computer hard drive. Studies were performed on myenteric neurons. DRUG APPLICATION Drugs were applied via flow tubes gated by computer controlled solenoid valves. Four of these flow through tubes were glued together at the tip and each were connected via polyethylene tubing to a reservoir (10 mL) syringe containing a known concentration of drug. The reservoir platform was positioned above the recording chamber so that drugs were gravity fed through the flow tubes. Platform height was adjusted to provide a flow rate of 0.1 mL/min. Patch electrodes were mounted on a micromanipulator and drugs were applied by positioning the flow tube over the neuron. This method permitted rapid adjustments between control and drug-containing solution (equilibrium<100 ms). STATISTICAL PROCEDURES For each of the experiments described in this study, a minimum of 5 myenteric neurons were used for each treatment. Data were expressed as meantS.E.M., Student’s t-test for paired and unpaired data, and one-way ANOVA for data containing three or more treatment groups were used for comparisons. 34 RESULTS MUSCARINIC ACTIVATION COUPLED ACh INDUCED nAChR DESENSITIZATION ACh, acting at nAChRs and mAChRs are the predominant mechanisms of excitatory transmission in the ENS. These receptors are co-expressed by many myenteric neurons and when activated may cause an alteration in nAChR function. Therefore, these studies were conducted to characterize some of the properties of nAChRs and to study the effects of muscarinic receptor blockade on nAChR desensitization in myenteric neurons. Whole cell patch-clamp recordings (inward currents were measured) were obtained from myenteric neurons maintained in primary culture, and a maximum concentration of ACh (1mM) was applied via gravity fed flow tubes. In this study, a standard intracellular pipette solution containing ATP (1 mM) and GTP (0.25) was used. The results from this study show that at seven seconds application of ACh, there was a maximum of 81% (ACh I7s I ACh Ipeek = 0.19:0.02, n=26) desensitization. NAChRs recovered completely from desensitization in five minutes (ACh ImaXSmln l ACh ImaXinitial = 0.931004, n=5). When recordings were obtained using ATP/GTP-free pipette solutions, a seven ACh application caused a maximum desensitization of 60% (ACh I7: / ACh Ipeek = 0.41004, n=18, P<0.05) and recovery from desensitization was complete in 5 minutes (ImaXSInIn / ACh ImaXlnitial = 1.111012, n=8, P>0.05). These data suggest that ATP and Ior GTP-dependent mechanisms modulate desensitization of nAChRs 35 in myenteric neurons (Figure 1). Also in this study, the time course of desensitization was determined. From curve fitting analysis, a sum of a two exponential fit was the best model to describe ACh-induced nAChR desensitization at seven seconds application of ACh. The time constants for desensitization were 1.9103 seconds for the slow time constant and 0.6103 seconds for the fast time constant. ACh acts at nAChRs and mAChRs. MAChRs couple to intracellular signaling that use ATP and/or GTP as substrates. Therefore, the possibility that mAChR activation altered nAChR desensitization was tested. Scopolamine (1 uM), a muscarinic antagonist, was added to the extracellular solution. The agonist application protocol described above was used. It was found in the presence of scopolamine that ACh induced a maximum desensitization of 57% (ACh I73 / ACh Ipeak = 0.431004, n=16, Ps00001) compared to control. Recovery from desensitization was complete in 30 seconds. Therefore, this suggests that simultaneous activation of mAChRs can modulate nAChR desensitization (Figure 2 and Figure 3). Lastly, to show that scopolamine did not act nonspecifically to alter nAChR function, nicotine, which does not activate muscarinic receptors, was applied in the presence of scopolamine. It was found that scopolamine did not affect nAChR desensitization caused by nicotine (Nic I7s/ Ipeak = 0341004, n=7; NICscopoIamine I7s / Ipeek = 0.331007, n=6, P209). Recovery was complete in 5 minutes (Figure 4). Also in this study, the time course of desensitization was calculated using an 36 Figure 1. Desensitization rate of the nAChR. (A) Trace of an inward current caused by ACh. Ipeek is the maximum current flowing through the nAChR and Itime is the current flowing through the nAChR where time of application is 1 to 10 seconds. (B) Plot of Itime l Ipeak vs. Time application. ACh with ATP and GTP compared to ACh without ATP and GTP in pipette solution and ACh in the presence of scopolamine with ATP and GTP in pipette solution desensitizes faster with the nAChR approximately 80% desensitized at 7 seconds. (*P value<0.05) 37 Desensitization Rate of the nAChR peak 1.0- 0.8: 1 mM ACh Time = 1-10 seconds —0- ACh (ATP. GTP) —.— ACh (wlo ATP. GTP) _-_ ACh (1,:M scopolamine, ATP. GTP) é ' I 7 6 ' 6 7 16 Time application (seconds) 38 Figure 2. Recovery rate of the nAChR from desensitization. (A) Initial current peak vs. 0.5,1,5 minute recovery period between ACh application in presence of ATP and GTP in the pipette solution (Control); nAChR recovery takes 5 minutes. (B) Initial current peak vs. 0.5, 1 , 5 minute recovery period between ACh application without ATP and GTP present in intracellular solution. (C) Initial current peak vs. 0.5, 1, 5 minute recovery between ACh application in presence of scopolamine. Traces show that nAChR recovery in presence of scopolamine takes approximately 30 seconds. 39 Traces of Recovery Rate of the nAChR from Desensitization A. ACh with ATP, GTP B. ACh without ATP, GTP Initial 0.5 min 1 m In 5 m In Initial 0.5 min 1min 5 min I- I! 'I II I l . . C. ACh with scopolamine, ATP, GTP Initial 0.5 min 1 min 5 min 'flflfir 40 Figure 3. Recovery rate of nAChR from desensitization. Recovery plot of Itime / Iinitial vs. Recovery period where time equals 0.017 to 10 minutes. Data suggest that recovery rate is affected by muscarinic receptor activation. *P value<0.05 compared to ACh with ATP and GTP and 'P value<0.05 compared to ACh with scopolamine, ATP, and GTP. 41 Recovery Rate of nAChR from Desensitization O ACh with ATP, GTP (n=6-27) . ACh without ATP, GTP (n=5-8) I ACh with 1 uM scopolamine, 125, ATP, GTP (n=12-15) ## ‘ I ' l ' I ' I ' l 7% 0.5 1.0 1.5 2.0 2.5 4.5 5.0 5.5 Recovery (minutes) 42 Figure 4. Nicotine effects on nAChR desensitization and recovery in presence of scopolamine. (A) Desensitization rate plot shows that scopolamine does not affect nicotine induced nAChR desensitization. (B) Scopolamine does not affect recovery rate from nicotine induced nAChR desensitization. 43 Nicotine Effects on nAChR Desensitization and Recovery in Presence of Scopolamine —O—1mM Nicotine (n = 8-12) -O—1mM Nicotine with 10M 1.0- Scopolamine (n = 6) ;\ T ' I ' I ' I ' I 2 4 6 a 10 Time application 0 1mM Nicotine (n = 5-8) 0 1mM Nicotine w/1ttM Scopolamine (n = 9-10) 2' ' 4' ' 6 Recovery (minutes) (1).: 44 Figure 5. Cross-desensitization effects of the nAChR. Plot of 1mM agonist application vs. Ipeek. Data suggest that ACh and nicotine act at the same nAChR. *P value<0.05 when compared to initial ACh, #P value<0.05 when compare to nicotine, and +P value<0.05 when compare to ACh and nicotine mixture. 45 Cross-Desensitization Effects of the nAChR ACh & 0 ACh Nicotine f Nicotine ACh ‘ I n=16 I -500- *# n=16 .I i-1000- n=,6 *J.+ . .I. -1500- "=16 * -2000- 46 exponential standard equation for the function and a Levenberg-Marquardt for the search method to obtain the best model to fit nAChR decay during a seven second application of nicotine. From this curve fitting analysis, a sum of a two exponential fit was the best model for nAChR decay at seven seconds application of nicotine. The time constants were 2.2104 seconds for the slow time constant and 0.2101 seconds for the fast time constant. These were not significantly different from ACh time constant parameters (P>0.05). CROSS-DESENSITIZATION OF nAChRs IN MYENTERIC NEURONS ACh and nicotine are the two nAChR agonists that were used in these experiments to induce nAChR responses. From this a question arose as to whether there were more than one population of nAChRs present on myenteric neurons, and if there were more than one, then does co-application of ACh and nicotine act at the same nACHR subunit subtype composition to result in a cross-desensitized (inactivated) nAChR. In this study, ACh was applied for two seconds, to obtain a peak response, which was followed by five minutes of recovery. Then ACh was applied for seven seconds to desensitize the nAChR followed by two seconds of nicotine application. The results indicate that application of ACh and nicotine mixture following ACh and nicotine application reduces Ipeek to -258158 pA (P<0.05) compared to the initial ACh Ipeek of -15651168 pA and to nicotine Ipeak of -1 1501144 pA (P<0.05), and the recovery of the nAChR had a partial Ipeak recovery of -9231116 pA (P<0.05). Therefore, these data suggest that ACh and nicotine act at the same nAChR in 47 Figure 6. Time control effects of ACh-induced nAChR desensitization. (A) Histogram of I7sec / Ipeak vs. AChinitial (1mM) and AChcontrol (1mM). ACI'Iinitial and ACI'Icontrol were not significantly different, and therefore, this indicates that repeated ACh application in presence of scopolamine (1 uM) recovers in five minutes. (B) Histogram of I7sec l Ipeak vs. ACI‘Iinitial (1mM) and ACh (1mM) plus 0.05% EtOH. ACI‘Iinltial and ACh plus 0.05% EtOH were not significantly different and indicated that the 0.05%EtOH vehicle does not alter nAChR desensitization. (C) Histogram of I7sec/ Ipeak vs. AChinitial (1mM) and ACh (1mM) plus 0.005% EtOH. AChinitial and ACh plus 0.005% EtOH were not significantly different and indicated that the 0.005%EtOH vehicle does not alter nAChR desensitization. 48 Time Control Effects of ACh-induced nAChR desensitization 0.6- T . n=4 T g n=4 8 0.4-l E 0.2- 0.0 1mM AChinitial 1mM AChoontro, B c 06‘ 0.6- T n=7 T .16 _T T .2 n=7 —8 0.44 n-10 ":10 —§ 0.4. g 0.2- _,§ 0.21 0 0.0 1mM AChm, 1mM ACh 1mM ACh 1mM ACh 49 Figure 7. Time control effects of nicotine-induced nAChR desensitization. (A) Histogram of I7sec l Ipeak vs. Nicotinetnttai (1mM) and Nicotineeontrol (1mM). NICOtInBinitial and NICOIInBcontrol were not significantly different, and therefore, this indicates that repeated Nicotine application recovers in five minutes. (B) Histogram of I7sec I Ipeak vs. NICOIInBinitial (1mM) and Nicotine (1mM) plus 0.05% EtOH. NICOtInBinitiel and Nicotine plus 0.05% EtOH were not significantly different and indicated that the 0.05%EtOH vehicle does not alter nAChR desensitization. (C) Histogram of I7sec / Ipeak vs. NICOtIneinitiel (1mM) and Nicotine (1mM) plus 0.005% EtOH. NICOIII'IBinitial and Nicotine plus 0.005% EtOH were not significantly different and indicated that the 0.005%EtOH vehicle does not alter nAChR desensitization. 50 Time Control Effects of Nicotine-induced nAChR desensitization 0.6- 0.6- .: 0.4 _a ‘ I _'§ 0.2 n=4 n24 0'0 1mM 1mM Nicotinem, Nicotinemm C 0.6- T T [1:5 "=5 0.4~ ":5 T 1mM 1mM Nicotine Nicotinem, + 0.05% EtOH |7sec I 'peek 1mM 1mM Nicotine Nicotinem, + 0.005% EtOH 51 myenteric neurons (Figure 5). TIME CONTROL OF ACh AND NICOTINE A time control for nAChR inward current recordings was examined using a seven second application of 1 mM ACh (1 uM scopolamine present) and 1mM nicotine, and because 0.05 and 0.005% concentrations of ethanol (EtOH) were used as the vehicle for forskolin, DDF, PDBu, and 4-0:-PMA solubility. EtOH was added to the control drug applications. This experiment was performed to show that desensitization of nAChRs was reproducible and that run-down of ACh or nicotine responses does not occur during the time course of these studies. Therefore, in the presence of ACh-scopolamine, the 0.05% EtOH time control had an initial ACh desensitization response of 55.514% (n=10) and a ACh-0.05% EtOH desensitization response of 61 15% (n=1 0, . P2 0.05). The 0.005% EtOH time control had an initial ACh desensitization response of 40.216% (n=7) and a ACh-0.005% EtOH desensitization response of 4916% (n=7, P20.0.05) (Figure 6). In the presence of nicotine, the 0.05% EtOH time control had an initial nicotine desensitization response of 57.813% (n=5) and a nicotine-0.05% EtOH desensitization response of 54.614% (n=5, P2005). The 0.005% EtOH time control had an initial nicotine desensitization response of 6013% (n=5) and a nicotine- 0.005% EtOH desensitization response of 66.216% (n=5, P2005). These data indicate that recovery from 7second-1mM ACh and nicotine - induced nAChR desensitization is complete in 5 minutes, and that the vehicle does not alter nAChR 52 desensitization (Figure 7). PDBu INDUCED MODULATION OF nAChR DESENSITIZATION Previous studies have shown that mAChR activation couples to Gq protein which activates phosphatidyl inositol (Pl) intracellular signaling pathway (Hulme1990). Therefore, the effects of PDBu, a PKC activator was examined to test for a possible role of PKC activation in modulating nAChR desensitization. ACh (1mM) was applied for seven seconds in the presence of varied concentrations (10nM, 30nM 100nM, and 300nM) of PDBu. The data suggest that PKC activation and the PI hydrolysis pathway are involved in modulating the nAChR because at 10nM, 30nM, 100nM and 300 concentrations of PDBu, there was a significant increase in nAChR desensitization of 45% (I75 I Ipeak = 0.551007, n=5, P5005), 71% (I7: / Ipeek = 02910.05, n=7, P5005), 56% (I7s/ Ipeak = 0.441004, n=5, P5005), and 62% (I75 I Ipeak = 0.381004, n=6, P5005), respectively, compared to the initial ACh desensitization response of 41% (I75 / Ipeak = 0.591007, n=5), 51% (I70l Ipeek = 04910.04, n=7), 35% (I7s/ Ipeak = 0.651007, n=5), and 51% (I73, Ipeek = 0.491002, n=6), respectively (Figure 8 and Figure 9). To test that PDBu was not acting nonspecifically to alter nAChR function, 4- a-Phorbol 12-myristate 13-acetate (4-0:-PMA), an inactive phorbol ester analog, was applied in the presence of ACh (1 mM) applied for seven seconds. The data show that there was no change in nAChR desensitization at any 4-0t-PMA concentration (10nM I7s/ Ipeak = 0.481005, n=5, P208; 30nM I7s/ Ipeek = 0.61006, 53 Figure 8. Traces of PDBu and 4-0:-PMA effects on ACh-induced nAChR desensitization. (A) Traces of an inward current caused by ACI'Iinltiel (1 mM) and ACh (1mM) plus 30 nM PDBu in the presence of 10M scopolamine. ACI‘Ilnitlel and ACh plus 30 nM PDBu were significantly different. (B) Traces of an inward current caused by ACI‘Iinitial (1 mM) and ACh (1 mM) plus 30 nM 4-0:-PMA in the presence of 1mM scopolamine. ACI'Iinitial and ACh plus 30 nM 4-0:-PMA were not significantly different, and therefore, this indicates that PDBu acts specifically to modulate nAChR desensitization. Traces of PDBu and 4-0:-PMA Effects on ACh-induced nAChR Desensitization 1mM ACh & 1mM AChinitial 30nM PDBu ‘l 1 5minutes Krebs + 2minutes 30nM | PDBu _J | I 10M Scopolamine Present ACh 8. 1mM ACh 40LPMA ‘F "'l 2 min 30nM 40PMA I l I | 10M Scopolamine Present 55 Figure 9. PDBu effects on ACh-induced nAChR desensitization. Histograms A, B, C, and D show that ACh pretreated with 10 nM, 30 nM, 100 nM, and 300 nM concentrations of PDBu, respectively, significantly increases nAChR desensitization of 45%, 71 %, 56%, and 62%, respectively. This was compared to the initial ACh desensitization response of 41 %, 51 %, 35%, and 51 %, respectively. (*P value<0.05) 56 PDBu Effects on ACh-induced nAChR Desensitization B ‘ a: 0.6- T ‘l’ 0.6- n=5 . n=5 T n=7 E04- EM * : - z 1 n=7 .12 02 £02 00 : , 1mm)... “TM/’01 mm}. mm +10MPEBJ amaze; D l 06 05 . T I "=5 n=6 * $04" * E04: T 'r _ n=6 : ' m6 \ rB02 _,g 02- 0° mm,” ; 0W0: 0° WW’GL. : 1nMPOI T “I'm +ZIDMHEI 57 Figure 10. 4-0:-PMA effects on ACh-induced nAChR desensitization. Histograms A, B, C, and D show that ACh pretreated with 10 nM, 30 nM, 100 nM, and 300 nM 4-0:-PMA, respectively, do not significantly increase nAChR desensitization of 52%, 40%, 46%, and 36%, respectively. This was compared to the initial ACh desensitization response of 50%, 43%, and 41 %, respectively. These data suggest that activation of PKC by PDBu acts specifically to modulate nAChR desensitization. (P value>0.05) 58 4-a-PMA Effects on ACh-induced nAChR Desensitization B .l. 0.6- 0.6- I l I T J n=6 n=6 n=5 30.“ ":5 $0.4. . : . 302- 43 02 0.0 : : 1nMPCI‘t'u 1nMACh 1mM/30‘,“ “TM/301 + 10M 4—0:-PMA 1' 111M4-0t-PMA C D T 051 T T 0.64 T n=5 J n=5 [1% ‘ n=5 EOA- 3' Q4. 1 : 4 1102' ,3 o.2~ 0.0 = 0,0 : 1mm 1rrMACh 1rrM AChInitial 1rrM ACh + 1(DIM 4-0t—PMA + 300nM 4-a-PMA 59 Figure 11. Traces of PDBu and 4-0t-PMA effects on nicotine-induced nAChR desensitization. (A) Traces of an inward current caused by NICOtInBinitial (1 mM) and Nicotine (1 mM) plus 30 nM PDBu. NICOIIneinitial and Nicotine plus 30 nM PDBu were significantly different. (B) Traces of an inward current caused by NICOIInBlnltieI (1 mM) and Nicotine (1 mM) plus 30 nM 4-0:-PMA. Nicotinetntttai and Nicotine plus 30 nM 4- a-PMA were not significantly different, and therefore, this indicates that PDBu acts specifically to modulate nAChR desensitization. 60 Traces of PDBu and 4-0:-PMA Effects on Nicotine-induced nAChR Desensitization , , 1mM Nicotine + 1mM Nicotine PDBu , 2 min 30nM '1 | PDBU I I I 1mM Nicotine + 1mM Nicotine 30 nM 4-0:-PMA '7 "I 2 min 30nM I 4-a-PMA I 61 Figure 12. PDBu effects on nicotine-induced nAChR desensitization. Histograms A, B, C, and D show that nicotine pretreatment with 10 nM, 30 nM, 100 nM, and 300 nM concentrations of PDBu, respectively, significantly increases nAChR desensitization of 64%, 72%, 77%, and 74%, respectively. This was compared to the initial nicotine desensitization response of 53%, 65%, 64%, and 61%, respectively. (*P value<0.05) 62 PDBu Effects on Nicotine-induced nAChR Desensitization n=7 -Iar- B 0'61 0.61 . T n=5 * 0.4" I 044 n=5 02 : ° ‘ 02- .3 0.0 . : . . 0 0 MM Ncotrneh.“ 1rrM Ncotlne - + 10nM PDBu D 0.6] 0.6. 0.4- T (14) n=7 * E T : 02- n=7 g 0.2- .1? 0.0 00 1mM Nicotine; 1rrM Mootine' 1rrMNcctinefi; 1mMN00tine' +301MPIBJ J +1(IhMPDBu 63 1rrM Nadine“; 1rrM Ncotine 1 +3ID'MPDBJ Figure 13. 4-0:-PMA effects on nicotine-induced nAChR desensitization. Histograms A, B, C, and D show that nicotine pretreatment with 10 nM, 30 nM, 100 nM, and 300 nM concentrations of 4-0:-PMA, respectively, do not significantly increase nAChR desensitization of 65%, 69%, 72%, and 71 %, respectively. This was compared to the initial nicotine desensitization response of 60%, 64%, 62%, and 72%, respectively. These data suggest that PKC activation by PDBu act specifically to modulate nAChR desensitization. (P value>0.05) 64 l,,,,/I,,,,, I7...“peek 4—0:-PMA Effects on Nicotine-induced nAChR Desensitization n=11 ‘IITMW 1n'MN00tine: +ZilM4-0c-PMA D 1. T n=5 n=5 T 04- 0:5 Q4. T "‘5 t 02- I 0 2. 2 0.0 t : immune,“ 1rrMNcn'rle +10M4a-PMA 04- l [1:5 I 0.4- n=5 3 ‘ 02' : 02- .13 0.0 f _ : 1rrMme 1rrMN0dire +1CDM4-0t-HVA 65 0 = ' 1rrMNtm're'fl 1nMN01 mil/won,iii 1rrM/sol +1Q'M U]: + 1(XhM II]: D 067 T 0.61 T n=5 , T {0.4- * _§ 0.4- n=5 n=5 . T \ Hi ,2... 4902. 1 06 0.0 : 1mm 1rTMACh 1m A01“ 1mm: +1 M [I]: + 1W [I]: 73 Figure 17. Traces of forskoIin effects on nicotine-induced nAChR desensitization. (A) Traces of an inward current caused by NiCOtinelnitial (1 mM) and Nicotine (1mM) plus 1 uM forskolin. NiCOtineinItial and Nicotine plus 1 uM forskolin were significantly different. (B) Traces of an inward current caused by NiCOtinelnitial (1mM) and Nicotine (1mM) plus 1 uM DDF. NiCOtineinitiaI and Nicotine plus 1 uM DDF were not significantly different but was significantly different at 10 mm DDF. Therefore, this indicates that forskolin acts nonspecifically at the nAChR to modulate nAChR desensitization. 74 Traces of Forskolin Effects on Nicotine-induced nAChR Desensitization A 1mM Nicotine + 1mM Nicotinemm 1““ Forskolin q H 5 minutes Krebs + 2 minutes 1uM Forskolin l l B 1uM Nicotine 1mM Nicotinemm, + 1mM DDF w III 5 minutes Krebs T- 2 minutes 1pM DDFl 75 Figure 18. Forskolin effects on nicotine-induced nAChR desensitization. Histogram A shows that nicotine pretreatment with 10 nM forskolin does not significantly increase desensitization of 67% compared to the initial nicotine desensitization response of 60%. Histograms B, C, and D show that nicotine pretreatment with 100nM, mm and 10nM forskolin significantly increases nAChR desensitization of 78%, 78%, and 79%, respectively, compared to the initial nicotine desensitization response of 69%, 66%, and 69%, respectively. (*P value<0.05) 76 Forskolin Effects on Nicotine-induced nAChR Desensitization A e T 04- . TF5 T 0.4 I i "=5 i ‘ n=9 T \ 0.2- : 02. ”:9 .2 ti — N 1mm 1rrMNoaine' IWWmm: 1rrMNootine: +10MFasIdn +1ooMFaskoIin c o o.4~ 04‘ T T a n=11 * E ‘ n=7 , 3|}— _ I _ \ °2‘ n=11 \ °2‘ n=7 .2 .2 o.c - . g. 1mMNicotineml 1rrMNootine 1rthstocitine“lial 1rrMNodine + 1pM Forskolin +10MForskolin 77 Figure 19. DDF effects on nicotine-induced nAChR desensitization. Histograms A, B, and C show that nicotine pretreatment with 10 nM, 100 nM, and 1 mm DDF do not significantly increase nAChR desensitization of 68%, 68%, and 79%, respectively, compared to initial nicotine desensitization responses of 59%, 69%, and 66%, respectively. Histogram D shows that nicotine pretreatment with 10nM DDF significantly increases nAChR desensitization of 83% compared to initial nicotine desensitization response of 50%. These data indicate that forskolin acts at the nAChR nonspecifically to modulate desensitization. (*P value<0.05) 78 0.4- peat 17$, /| DDF Effects on Nicotine-induced nAChR Desensitization B T ":9 T 04‘ I T "=9 E ‘ n=5 n=5 \ Q2. ._Q M I788c /| . = . : 0.0 : : ”MN‘mmm 1"MN°°""e 1lTMNcm'reflfi 1rrMNooune + 101M [I]: + 101M U]: D 0.6- 1 T fi n=5 a 0.4q T : * "'5 4g 02- ‘[ fi nfi : ‘ 0.0 : - 1nMNa1inem 1rrMch1ine 1rrM Nodinem 1nMNootine +1pMIIF +10MED= 79 P2005), 100nM (I7. I Ipeak = 033:004, n=9, P2005), and 10uM (I7. I Ipeek = 0491:007, n=5, P2005) concentrations of DDF compared to initial ACh desensitization responses (I7s/ Ipeak =O.42:I:0.06, n=5, l7s/ lpeek = 0451006, n=9, and I7: I Ipeak = 049:1:007, n=5, respectively), but at 1uM DDF there was a significant increase in nAChR desensitization of 72% (I73 I lpeak = 0281003, n=8, P5005) compared to initial ACh desensitization response of 53% (I7. I 'peak = 0.471005). Therefore, this suggests that forskolin has the potential to act nonspecifically at the nAChR to modulate nAChR desensitization (Figure 14 and Figure 16). The effects of forskolin on nicotine-induced nAChR desensitization was examined to determine its role in altering the function of nAChRs on myenteric neurons. In this study, nicotine (1mM) was applied for seven seconds to the neurons in the presence of forskolin (10 nM, 100 nM, 1 mm, and 10 mm). 100 nM, 1 mm, and 10 mm concentrations of forskolin, there was a significant increase in nAChR desensitization of 78% (nicotine l7s/ Imak = 022:004, n=9, P5005), 78% (nicotine I7: I lpeak = 0221003, n=11, Ps0.05), and 79% (nicotine I7: I Ipeak = 021:1:003, n=7, Ps0.05), respectively, compared to the initial nicotine desensitization response of 69% (nicotine l7s/ lpeak = 0311:004, n=9), 66% (nicotine I7. I Ipeak = 0341004, n=11), and 69% (nicotine l7s / lpeak = 0.311003, n=7), respectively (Figure 17 and Figure 18). DDF was applied to the neurons to test for a nonspecific effect of forskolin on nicotine-induced nAChR desensitization. In the presence of nicotine (1mM) applied for seven seconds with the addition of 10nM, 100nM, mm and 10uM 80 Figure 20. Traces of Bethanechol effects on nicotine-induced nAChR desensitization. Traces of an inward current caused by Nicotine (1mM) and Nicotine (1mM) plus 100 mm bethanechol. Nicotine and Nicotine plus 100 mm bethanechol were significantly different. This indicates that mAChR activation plays a role in nAChR modulation in myenteric neurons. 81 Traces of Bethanechol Effects on Nicotine-Induced nAChR Desensitization . . 1mM Nicotine 8. 1mM N'COt'm 100pM Bethanecol -, ] 5 minutes Krebs I j 82 Figure 21. Bethanechol effects on nicotine-induced nAChR desensitization. Histograms A and 8 show that nicotine pretreatment with 1 mm and 10 mm bethanechol do not significantly increase nAChR desensitization compared to the initial nicotine response. Histograms C and D show that 100 mm and 1 mm concentrations of bethanechol significantly increases nAChR desensitization. These data suggest that mAChR activation is involved in modulating nAChR desensitization. (*P value<0.05) 83 Bethanechol Effects on Nicotine-induced nAChR Desensitization A B 0.4- T 0.4- 1' n=6 I . "=5 T E ‘ n=6 j ”.45 : 02. \ 02" .1 .t. QC : . : 0.0 . 3 _ 4 1M Nootinem 1rrM Nootine 1rrM NW 1rrMNcmne + 1pM Bethanecol +10M8ettmednl C D T 0.4- 0.4. I n=6 * fl . "=9 * E. ‘ T : T I 0.2. "=6 g” "=9 4,3 _" : . ‘ 0.0 : 4 1rrM nicotine.“ 101M Noohne 1rrM Nadine,“ 1n'MNodine + 100mm“ +1rrMBetImedioI concentrations of DDF were used to compare to the nicotine-induced nAChR desensitization. The data show that there was not a significant effect observed with 10nM (I7s/ lpeak = 0321003, n=9, P2005), 100nM (I7sI lpeak = 032:1:006, n=5, P2005), and 1uM (I'ls/ Imk = 0211004, n=5, P2005) concentrations of DDF compared to initial nicotine desensitization responses(l7sl lpeak = 0411006, n=9, |7sl 'peak = 031:1:002, n=5, and l7s/ lpeak = 034:008, n=5, respectively), but at 10uM DDF, there was a significant increase in nAChR desensitization of 83% (I7s I lpeak = 017:1:005, n=5, P3005) compared to initial nicotine desensitization of 50% (I73 I lpeak = 0501:006, n=5). Therefore, this further suggests, as with ACh and DDF, that forskolin acts at the nAChR nonspecifically to modulate desensitization (Figure 17 and Figure 19). BETHANECHOL INDUCED NICOTINE - nAChR DESENSITIZATION To further test that muscarinic receptor activation may modulate nAChR desensitization, bethanechol was applied to myenteric neurons. In this study, nicotine (1 mM) was applied for seven seconds in addition to various concentration of bethanecol (1uM, 10nM, 100uM, and 1mM). The data suggest that mAChR activation plays a role in nAChR modulation of myenteric neurons because at 100nM and 1mM concentrations, there was a significant increase in nAChR desensitization of 72% (I7s/ lpeak = 0281002, n=6, P3005) and 75% (In I Ipeak = 0251002, n=9, P5005), respectively, compared to the initial nicotine desensitization response of 59% (l7sl lpeak = 0411005, n=6) and 64% (I73 I lpeak = 85 036:1:004, n=9) (Figure 20 and Figure 21). 86 DISCUSSION The data presented suggest that intracellular signaling pathways are involved in the modulation of nAChR desensitization. This conclusion is supported by studies in which mAChRs were blocked and also those in which activation of the cAMP-dependent pathway and the Pl pathway with forskolin and PDBu, changed nAChR desensitization. This was further implicated with direct activation of the muscarinic receptor with bethanechol application increased nAChR desensitization. Therefore, in the ENS, interactions occurring during simultaneous activation of nAChRs and mAChRs and/or other G-protein coupled receptors coupled to intracellular signaling pathways are responsible for modulating nAChR desensitization and also may be important in regulating enteric ganglionic neurotransmission. INTRACELLULAR SIGNALING ACTIVATION ON nAChR DESENSITIZATION Data presented show that nAChR desensitization is altered specifically by PKC activity which significantly increases nAChR desensitization at 10 to 300 nM concentrations of PDBu. NAChR desensitization was also shown to be altered nonspecifically by forskolin. This conclusion is supported by previous published data in muscle and neurons which have shown that agonist induced modulation of nAChR desensitization occurs following phosphorylation via activation of intracellular proteins such as PKA in muscle (Huganir and Greengard, 1983: 87 Huganlr, 1986), PKC in muscle (Safran et al, 1987), tyrosine specific protein kinases in muscle (Huganir, 1984; Hopfleld, 1988), and PKA and PKC in neurons (Khirong et al, 1998; Fenster et al, 1998). To further show that PKC activation is a regulator in modulating nAChR desensitization, bethanecol, a mAChR agonist, significantly increased nAChR desensitization at 100 uM and 1 mM concentrations. This is significant because published data suggest that mAChR activation couples to Pl hydrolysis to lead to PKC activation (Song et al, 1999; Hulme et al, 1990). NON - ADENYLATE - CYCLASE SITE OF ACTION The data show that by mimicking the activation of intracellular signaling pathways with forskolin and PDBu increases nAChR desensitization compared to that induced by ACh and nicotine alone. To show that these responses were specific, inactive analogs of forskolin and PDBu were used. The data show that there was specificity with intracellular activation of PKC and/or Pl hydrolysis pathways by PDBu because 4-oi-PMA did not modulate nAChR desensitization in the ENS. This was not the case for intracellular activation of cAMP dependent pathways by forskolin because of the DDF, an inactive analog of forskolin, caused a significant increase in nAChR desensitization. This has been shown in a previous experiment done by Wagoner and Pallotta in 1988 who they showed that receptor desensitization mediated by forskolin was not due to cAMP-dependent phosphorylation but due to forskolin interaction with the hydrophobic moieties on or near the nAChR. This was further suggested by the effects of the inactive analog, DDF. 88 This nonspecific action of forskolin to alter nAChR activity is possibly due to the lipophilicity of forskolin which gives forskolin the ability to cross the neuronal membrane and interact with nAChRs. Nonspecificity was confirmed by using an inactive analog of forskolin, DDF, which significantly altered the nAChR activity at high concentrations of DDF at 1 (M for ACh induced nAChR desensitization and 10 uM for nicotine induced nAChR desensitization. For future reference, to confirm that cAMP - dependent pathways are not involved in modulating nAChR desensitization in the ENS, studies need to be performed to analyze the levels of cAMP when ACh or nicotine are exposed to the nAChR for seven seconds. TIME CONTROL - EtOH VEHICLE ON nAChR DESENSITIZATION These recordings involved an EtOH-Krebs solution vehicle. 50% EtOH in these experiments were used to make the analogs soluble in solution so that application of the analog was feasible. To show that this vehicle alone has no effect on nAChR desensitization or run-down, a time control was done using nAChR agonists, ACh and nicotine, as well as the protocol that was used for the forskolin, DDF, PDBu, and 4-a-PMA experiment showed that there was an insignificant effect on nAChR desensitization. Therefore, low EtOH concentrations do not affect nAChR desensitization. MAChR EFFECTS ON nAChR In these studies, the mAChR was blocked by scopolamine to show that nAChR desensitization induced by ACh is mediated by mAChR activation. This was shown to be true due to the significant decrease in nAChR desensitization. To show that 89 this was not a nonspecific interaction of scopolamine at the nAChR, nicotine, another nAChR agonist that acts specifically at nAChRs and not mAChRs, was applied to the neurons in addition to scopolamine being present. Scopolamine did not affect nicotine induced nAChR desensitization. Therefore, scopolamine has a specific effect on blocking the mAChR. CROSS-DESENSITIZATION OF nAChR The data presented from the cross-desensitization study with ACh and nicotine application showed that these two agonists when co-applied act at the same subtype of nAChRs localized on myenteric neurons. But what these results do not indicate are the number and subunit subtype compositions of nAChR populations. Preliminary data obtained from this laboratory (not presented) has shown that the subunits involved in the nAChR subtype composition are a3, a5, [32, and [34, but the positions of these subunits on the nAChR of myenteric neurons are not known. During neurotransmission in the ENS, there are many neurotransmitters that are released and act postsynaptically at their receptors to excite enteric neurons. The activation of the receptors cause a cascade of intracellular mechanisms to become activated to mediate the activity of other receptors to modulate the excited state of enteric neurons. This process acts as a homeostatic feedback mechanism to keep the neurons from being over extended in their excited state. DESENSITIZATION CHARACTERISTICS IN S-TYPE MYENTERIC NEURONS From the exponential standard fit analysis of nAChR responses, the decay phase was calculated to have a sum of a two exponential fit for both ACh and nicotine, 9O which were not significant from one another. These results indicate that the decay phase, which represents the desensitized or inactivated state of the nAChR, has two phases, a fast and a slow phase. A fast phase represents a desensitized state that has been exposed to a short time to agonist application, and in this case, the results indicate that the time constant (1) of approximately 600 milliseconds for ACh and approximately 200 milliseconds for nicotine were calculated for the time course between the maximum inward peak current and the first state of nAChR desensitization. The slow phase represents a desensitized state that has been exposed to a longer period of agonist application, and from the results, approximately two seconds for both ACh and nicotine were calculated as the time course between the peak response and the second state of nAChR desensitization. From previous published data, the decay phase for muscle-nAChRs resulted in a sum of a two exponential fit meaning these nAChRs exhibited a fast and slow phase of nAChR desensitization (Naranjo and Brehm, 1993). In ciliary ganglion neurons, the decay phase of nAChRs resulted in a sum of a two exponential fit (Blumenthal et al, 1999), which means that the nAChRs exhibited a fast, slow, and a steady state phase because of an exponential fit from the peak response to the steady state response instead of an exponential fit from the peak response to the plateau response of the nAChR. Also, the seven second application of ACh and nicotine was used to guarantee that the nAChR was in a desensitized state. From this, the nAChR desensitized state was useful in determining the recovery rate of the nAChR at a seven second application. 91 NAChRs IN MYENTERIC NEURONS As mentioned above the subunit subtype compositions of nAChRs in the ENS are not known. Also, the mechanism involved in modulating nAChR desensitization in the ENS is not known. In studies from neuronal nAChRs, results indicate PKC activation mediates phosphorylation on the nAChR to modulate nAChR desensitization (Downing and Role, 1987). The results from the research studies presented in this thesis indicate that PKC activation specifically mediates nAChR modulation, and that also, activation of the mAChR plays a role in mediating nAChR desensitization possibly through a PI hydrolysis dependent pathway. For nAChRs, subunit composition has been determined to have either a a7- homopentamer, a384, a382, a482, and 0:484 compositions. The desensitization rates of these subunit compositions have been observed and are continuously being studied. Also these subunit compositions have been mainly studied in the CNS and other cell preparations, such as Xenopus oocytes , but the subunit composition for nAChRs localized in the ENS has not been determined. From the data that has been published on subunit composition of nAChRs (Fenster et al, 1997), the a3a582 subunit composition of the nAChR is closely mimicked by nAChRs localized in the ENS. Also the desensitization rate of 0:382 subunit composition and the ENS nAChRs both have a two exponential fit. Because nAChR subunit composition in the ENS has not been studied extensively, the subunit composition or the number of subunit compositions of ENS nAChRs can not be determined. This raises the question of whether there are a number of different 92 subunit compositions of nAChRs localized in the ENS. If there are different subunit nAChR compositions in the ENS, then does subunit composition have an affect on how the nAChR desensitizes and does nAChR subunit composition have an affect on how desensitization of nAChRs in the ENS are modulated? CONCLUSIONS In summary, the results from these research studies indicate that the desensitization and recovery rate are influenced by intracellular ATP and GTP and muscarinic activation. During a seven second application of ACh and nicotine, the nAChR response decays exponentially with a fast and slow phase. ACh and nicotine co-application results in cross-desensitization of the nAChR which suggest that activation of the same nAChR. PKC activation results in a specific involvement in modulating the nAChR by ACh and nicotine in myenteric neurons. This is consistent with other published data which indicates that PKC activation plays a role in mediating phosphorylation of the nAChR to alter its function (Figure 22). Lastly, muscarinic activation modulates nAChR desensitization in myenteric neurons. In conclusion, data presented in these studies show that interactions occurring during simultaneous activation of nAChRs, mAChRs, and intracellular signaling pathways modulate nAChR desensitization. Therefore, activation of these receptors and intracellular signaling pathways may be important in regulating enteric ganglionic neurotransmission. 93 Figure 22. Proposed model for nAChR desensitization in the ENS. Based on the time constants for the fast and slow phase of nAChR desensitization, this model proposes that at approximately 600 milliseconds for ACh and 200 milliseconds for nicotine that the nAChR has reached the peak of the first phase of desensitization (AzD). For stability in function, the nAChR, during the first phase of desensitization prefers to stay in the desensitized state which is indicated in the model by the short and long arrow directions. The long arrow represents the direction the nAChR will tend to stabilize in the desensitized state versus the short arrow represents the less likelihood for the nAChR to reverse back to the open state. With continued exposure of the agonist, the nAChR has reached the second phase of desensitization (AzD*) at approximately two seconds for both ACh and nicotine. At the AzD*, the nAChR is indicated as being modulated. The long arrow represents the direction that the nAChR prefers to be in the modulated state, which forms a more stabilized nAChR, and the short arrow indicates the less likelihood for the nAChR to reverse back to the desensitized state. Overall, this model shows that a stabilized nAChR, when exposed to long periods of agonist, prefers to be in the modulated state. Therefore, the modulated state of the nAChR makes the recovery from desensitization more difficult, which leads to an increase time recovery for long periods exposure of agonists to the nAChR. Proposed Model for nAChR Desensitization in the ENS 2 [A] R . : AZR. ., A2R* (unbound) (bound/closed) (open) \ / A20 (desensitized) l. A2D* (modulated) 95 BIBLIOGRAPHY Adams, D. J., Dwyer, T. M., and Hille, B. The permeability of endplate channels to mononvalent and divalent metal cations. Journal of General Physiology 75: 493- 510, 1980. Albuquerque, E. X., Deshpande, S. S., Aracava, Y., Alkondon, M., and Daly, J. W. 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