MP07-02: Long-term bladder decentralization in canines changes the location and pharmacology of neuromuscular nicotinic receptors in the intramural ganglion neurons

Nagat Frara*, Dania Giaddui, Alan S. Braverman, Danielle S. Porreca, Ekta Tiwari, Lucas J. Hobson, Courtney L. Testa, Philadelphia, PA, Justin M. Brown, Boston, MA, Michael Mazzei, Ida J. Wagner, Michel A. Pontari, Mary F. Barbe, Michael R. Ruggieri, Sr., Philadelphia, PA

Introduction: It was previously shown that nicotinic receptor activation induces detrusor muscle contractions via facilitating fast synaptic transmissions between preganglionic and postganglionic neurons. In a canine bladder, it has been reported that smooth muscle contractility is preserved after decentralization indicative of functional muscarinic receptors. Here, utilizing ex vivo studies, we were interested in evaluating alterations in the expression of nicotinic receptors mediating bladder function after long-term extensive decentralization in canines.

Methods: This study is an add-on investigation to a long-term project designed to develop novel surgical approaches for restoration of bladder, urethral and anal sphincter control to lower motoneuron lesioned pelvic organs. Four female canines were extensively decentralized by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7 and hypogastric nerves. Controls included sham-operated and unoperated animals. Mucosa-denuded smooth muscle strips were mounted in muscle baths and maximal contractile responses to 120 mM potassium chloride (KCl) were measured. Then, strips were incubated with different antagonists for 20 minutes, and contractions to nicotinic receptor agonist epibatidine (10µM) were determined. Responses are expressed as percentages of KCl-induced contraction.

Results: While the muscarinic receptor antagonist atropine (1µM) completely blocked epibatidine in strips from both control and decentralized bladders, the sodium channel blocker tetrodotoxin (TTX, 1µM) inhibited epibatidine-induced contractions in strips from decentralized, but not control bladders. Neuromuscular nicotinic receptor antagonists atracurium (5µM) or tubocurarine (0.1µM) inhibited epibatidine-induced contractions in strips from control, but not decentralized bladders. The neuronal (a3ß4) selective antagonist SR 16584 (10µM) inhibited epibatidine contractions in both groups.

Conclusions: Atropine blockade of epibatidine contractions indicates that these nicotinic receptors induce release of acetylcholine that acts on muscarinic receptors to induce bladder muscle contraction. TTX does not block epibatidine contractions in strips from control bladders, suggests that nicotinic receptors are likely located on nerve terminals and their antagonist pharmacology suggests that these consist of both a3ß4 neuronal and (a1)2ß1de neuromuscular subtypes. TTX blockade of contractions in decentralized group suggests that long-term extensive decentralization causes nicotinic receptors to relocate from nerve terminals to along the axons of presynaptic neurons distant from neuromuscular junction such that agonist induced depolarization triggers TTX sensitive action potentials that induce acetylcholine release at the neuromuscular junction. The antagonist pharmacology of these axonal nicotinic receptors is consistent with neuronal (a3ß4) nicotinic receptor subtypes. Changes in nicotinic receptor profile after decentralization might suggest a physiological relevance of these receptors as a compensatory mechanism to recover bladder function and a potential target for drug treatment.

Source of Funding: NINDS R01NS070267.