Presentation Authors: Alan Braverman*, Nagat Frara, Danielle Salvedeo, Mary Barbe, Michael Ruggieri, Sr., Philadelphia, PA
Introduction: 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 female 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 antagonists were added for 20 minutes before inducing contraction with the nicotinic agonists epibatidine and nicotine itself.
Results: Epibatidine induced contractions that were 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 Î¼M epibatidine or 1 mM nicotine induced contractions but desensitization of purinergic receptors with 10 uM Î±,Î² methylene ATP did not. Blocking sodium channels with 1 uM tetrodotoxin (TTX) had no statistically significant inhibitory effect on epibatidine or nicotine induced contractions. Desensitizing nicotinic receptors by exposure to nicotine blocked contractile responses to epibatidine and vice versa. The skeletal muscle neuromuscular junction nicotinic receptor antagonist atracurium besylate (5 uM) blocked both epibatidine and nicotine induced contractions. Epibatidine contractions were also completely blocked by another skeletal muscle neuromuscular junction nicotinic receptor antagonist tubocurarine (1 uM) or the ganglionic nicotinic antagonists hexamethonium (100 uM) or mecamylamine (10uM).
Conclusions: Because of atropine blockade but not blockade by Î±,Î² methylene ATP desensitization, the nicotinic agonists induce bladder contractions indirectly by releasing 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. The nature of these nicotinic receptors appears to be somewhat unusual in that they can be blocked by antagonists thought to be selective for skeletal muscle neuromuscular junction nicotinic receptors (atracurium and tubocurarine) as well as ganglionic nicotinic receptors (hexamethonium and mecamylamine).
Source of Funding: NINDS R01NS070267 to MRR and MFB.