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Very little is known about the physiological role of nicotinic receptors in canine bladders, although functional nicotinic receptors have been reported in bladders of many species. Utilizing in vitro methods, we evaluated nicotinic receptors mediating bladder function in dogs: control (9 female and 11 male normal controls, 5 sham operated), Decentralized (9 females, decentralized 6–21 mo), and obturator-to-pelvic nerve transfer reinnervated (ObNT-Reinn; 9 females; decentralized 9–13 mo, then reinnervated with 8–12 mo recovery). Muscle strips were collected, mucosa-denuded, and mounted in muscle baths before incubation with neurotransmitter antagonists, and contractions to the nicotinic receptor agonist epibatidine were determined. Strip response to epibatidine, expressed as percent potassium chloride, was similar (∼35% in controls, 30% in Decentralized, and 24% in ObNT-Reinn). Differentially, epibatidine responses in Decentralized and ObNT-Reinn bladder strips were lower than controls after tetrodotoxin (TTX, a sodium channel blocker that inhibits axonal action potentials). Yet, in all groups, epibatidine-induced strip contractions were similarly inhibited by mecamylamine and hexamethonium (ganglionic nicotinic receptor antagonists), SR 16584 (α3β4 neuronal nicotinic receptor antagonist), atracurium and tubocurarine (neuromuscular nicotinic receptor antagonists), and atropine (muscarinic receptor antagonist), indicating that nicotinic receptors (particularly α3β4 subtypes), neuromuscular and muscarinic receptors play roles in bladder contractility. In control bladder strips, since tetrodotoxin did not inhibit epibatidine contractions, nicotinic receptors are likely located on nerve terminals. The tetrodotoxin inhibition of epibatidine-induced contractions in Decentralized and ObNT-Reinn suggests a relocation of nicotinic receptors from nerve terminals to more distant axonal sites, perhaps as a compensatory mechanism to recover bladder function. [ABSTRACT FROM AUTHOR]

To compare gastroesophageal junction of the human with the pig, [M.sub.2] and [M.sub.3] receptor densities and the potencies of [M.sub.2] and [M.sub.3] muscarinic receptor subtype selective antagonists were determined in gastric clasp and sling smooth muscle fibers. Total muscarinic and [M.sub.2] receptors are higher in pig than human clasp and sling fibers. [M.sub.3] receptors are higher in human compared with pig sling fibers but lower in human compared with pig clasp fibers. Clasp fibers have fewer [M.sub.3] receptors than sling fibers in both humans and pigs. Similar to human clasp fibers, pig clasp fibers contract significantly less than pig sling fibers. Analysis of the methoctramine Schild plot suggests that [M.sub.2] receptors are involved in mediating contraction in pig clasp and sling fibers. Darifenacin potency suggests that [M.sub.3] receptors mediate contraction in pig sling fibers and that [M.sub.2] and [M.sub.3] receptors mediate contraction in pig clasp fibers. Taken together, the data suggest that both [M.sub.2] and [M.sub.3] muscarinic receptors mediate the contraction in both pig clasp and sling fibers similar to human clasp and sling fibers. doi: 10.1152/ajpi.00129.2009 gastroesophageal reflux; receptor immunoprecipitation; smooth muscle contractility; animal models

Bladder muscle specimens from seven patients with neurogenic bladder dysfunction were analyzed to determine whether the muscarinic receptor subtype mediating contraction shifts from [M.sub.3] to the [M.sub.2] subtype as found in the denervated, hypertrophied rat bladder. Seven bladder specimens were analyzed from six female and one male patients. Six of the patients had traumatic cervical spinal cord injuries ([C.sub.4]-[C.sub.7]), and the other patient had an [L.sub.1] congenital myelomeningocele. This was compared with results from bladder specimens obtained from eight organ transplant donors. The affinities of three subtype-selective muscarinic receptor antagonists for inhibition of carbachol-induced contractions were determined. The affinity of the [M.sub.3] selective antagonists darifenacin or p-fluoro-hexahydrosiladifenadol (p-F-HHSiD) was determined in six of the seven spinal injury patient specimens. The affinity was consistent with [M.sub.2]-mediated contractions in four of these six specimens, intermediate between [M.sub.2] and [M.sub.3] in one specimen, and within the [M.sub.3] range in one specimen. The other specimen, tested only with the [M.sub.2] selective antagonist methoctramine, showed an [M.sub.3] affinity. In the organ donors, the affinity of p-F-HHSiD was within the [M.sub.2] range for six of seven specimens, whereas the affinity of darifenacin was within the [M.sub.3] range for five of six and intermediate between [M.sub.2] and [M.sub.3] for the other specimen tested. The affinity of methoctramine in both organ donor specimens tested was within the [M.sub.3] range. Whereas normal detrusor contractions are mediated by the [M.sub.3] receptor subtype, in patients with neurogenic bladder dysfunction as well as certain organ transplant donors, contractions can be mediated by the [M.sub.2] muscarinic receptor subtype. spinal cord injury

Major pelvic ganglion electrocautery (MPGE) and spinal cord injury in the rat induce bladder hypertrophy and a change in muscarinic receptor subtypes mediating bladder contraction from predominantly [M.sub.3] to a combination of [M.sub.2] and [M.sub.3]. To determine whether this is a result of bladder hypertrophy or denervation, we studied the following groups: sham-operated controls, urinary diversion (DIV), MPGE together with urinary diversion (DIV-DEN), bilateral MPGE (DEN), bladder outlet obstruction (BOO), and MPG decentralization (MPG-DEC). The degree of bladder denervation was determined by the maximal carbachol response normalized to the response to electric field stimulation. Receptor subtype density was determined by immunoprecipitation. The affinity of subtype-selective muscarinic antagonists for inhibition of carbachol-induced contractions was used to determine the subtype-mediating contraction. DEN, MPG-DEC, and BOO bladders were hypertrophic whereas DIV bladders were atrophic compared with sham operated. Bladder contraction in sham-operated, DIV, and DIV-DEN was mediated by the [M.sub.3] receptor subtype, whereas the [M.sub.2] subtype participated in contraction in the DEN, MPG-DEC, and BOO groups. The hypertrophied bladders had an increase in total and [M.sub.2] receptor density while all experimental groups showed a reduction in [M.sub.3] receptor density. Thus bladder hypertrophy, independent from bladder denervation, causes a shift in the muscarinic receptor subtype mediating bladder contraction from [M.sub.3] toward [M.sub.2]. denervation; outlet obstruction; urinary diversion

[M.sub.3] muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, [M.sub.2] receptors participate in contraction because [M.sub.3]-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the [M.sub.2]-selective antagonist methoctramine in the denervated bladder is consistent with [M.sub.3] receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the [M.sub.2] receptor and one by the [M.sub.3] receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both [M.sub.2] and [M.sub.3] receptors can induce contraction. In the denervated bladder, the [M.sub.2] and the [M.sub.3] receptors interact in a facilitatory manner to mediate contraction. urinary bladder; synergy; denervation; second messengers

Denervation induces an increase in M2 muscarinic receptor density with no change in M3 receptor density in the rat urinary bladder. Concomitant with these changes, the affinity of a series of muscarinic receptor antagonists for inhibition of carbachol-induced smooth muscle contraction switches from being consistent with M3 receptor-mediated contraction in control bladders to M2-mediated or a combination of M2 and M3 receptor-mediated contraction in denervated bladders.

The muscarinic receptor mediated regulation of bladder contractility was investigated in vitro using muscle strips from rat bladder and a variety of antagonists and receptor blockers. Localization of receptor type was undertaken using reverse transcriptase polymerase chain reaction. Results reveal the presence of three muscarinic receptors controlling the contractility of rat bladder, prejunctional M1 which facilitates contraction, prejunctional M2 which inhibits contraction and postjunctional M3 which modulates contraction.

Aims: We sought to determine whether somatic lumbar nerve transfer to the pelvic nerve's anterior vesical branch after sacral decentralization for detrusor muscle reinnervation also leads to aberrant innervation of the bladder outlet. Methods: Twenty‐six female mongrel hound dogs underwent transection of sacral dorsal and ventral spinal roots (ie, sacral decentralization). Immediately afterward, 12 received genitofemoral nerve transfer and 9 received femoral nerve branch transfer. Five were left sacrally decentralized. Controls included 3 sham‐operated and 6 unoperated. Eight months postsurgery, the bladder and urethra were injected with retrograde tracing dyes cystoscopically. After 3 weeks, detrusor and urethral pressures were assayed electrophysiologically immediately before euthanasia and characterization of neural reinnervation. Results: Electrical stimulation of spinal cords or roots did not lead to increased urethral sphincter pressure in nerve transfer animals, compared with decentralized animals, confirming a lack of functional reinnervation of the bladder outlet. In contrast, mean detrusor pressure increased after lumbar cord/root stimulation. In sham/unoperated animals, urethral and bladder dye injections resulted in labeled neurons in sacral level neural structures (dorsal root ganglia [DRG], sympathetic trunk ganglia [STG], and spinal cord ventral horns); labeling absent in decentralized animals. Urethral dye injections did not result in labeling in lumbar or sacral level neural structures in either nerve transfer group while bladder dye injections lead to increased labeled neurons in lumbar level DRG, STG, and ventral horns, compared to sacrally decentralized animals. Conclusion: Pelvic nerve transfer for bladder reinnervation does not impact urethral sphincter innervation. [ABSTRACT FROM AUTHOR]