PD24-10 NICOTINIC RECEPTORS ON NERVE TERMINALS INDUCE ACETYLCHOLINE RELEASE IN CANINE BLADDER

Abstract

INTRODUCTION AND OBJECTIVE: Carbachol, a mixed muscarinic and nicotinic agonist similar to acetylcholine, is often used for in-vitro bladder contraction with the implicit assumption that it causes contraction by only activating bladder smooth muscle muscarinic receptors. We sought to determine whether nicotinic receptors may also be involved in canine detrusor muscle contractions in- vitro. METHODS: Mucosa denuded canine bladder muscle strips from sham operated animals were used from a larger study of nerve transfer for bladder reinnervation. Strips were fixed between force transducers and positioners and suspended in Tyrode's solution bubbled with 95% O2/5% CO2 at 37 C. After stretching to a length of optimal force production, maximal responses to 120 mM KCl were determined then various agents were added for 20 minutes before inducing contraction with the nicotinic agonists epibatidine and nicotine. RESULTS: Epibatidine induced contractions that were approximately 40% of the maximal response to 120 mM KCl whereas nicotine only induced contractions that were 20% of KCl. The muscarinic receptor antagonist atropine (10 uM) completely blocked 10 uM epibatidine or 1 mM nicotine induced contractions but desensitization of purinergic receptors with 10 uM α,β methylene ATP only blocked these contractions by 40%. Blocking sodium channels with 1 uM tetrodotoxin (TTX) had no statistically significant inhibitory effect on epibatidine or nicotine induced contractions. The α7 nAChR-selective agonist AR-R17779 had no effect and the α7 selective antagonist MLA had no effect at α7 selective concentrations on epidbatidine induced contractions. The α4β2 selective agonist TC2559 also had no effect whereas the α3β4 selective agonist NS3861 and the non-selective ganglionic receptor agonist DMPP induced small contractions. Also, the α3β4 selective antagonist SR-16584 (1-3 uM) blocked eipibatidine induced contractions by 50% . At relatively high concentrations, the skeletal muscle neuromuscular junction nicotinic receptor antagonists atracurium besylate (5 uM) and tubocurarine (0.1 uM) blocked epibatidine induced contractions. At concentrations reported to be selective for ganglionic nicotinic receptors, the neuronal nicotinic receptor antagonists hexamethonium (100 uM) and mecamylamine (10uM) blocked epibatidine induced contractions. CONCLUSIONS: Because of atropine blockade but only minor blockade by α,β methylene ATP desensitization, the nicotinic agonists induce bladder contractions indirectly by releasing predominately acetylcholine from intramural nerve terminals. Because TTX was ineffective, these nicotinic receptors do not need to induce action potentials and thus are likely located near the neuromuscular junction. Based on the pharmacological data, the α3β4 receptor subtype is likely involved in contraction of the canine bladder. Source of Funding: NINDS R01NS070267