Plasticity of non-adrenergic non-cholinergic bladder contractions in rats after chronic spinal cord injury

Abstract

The purpose of this study was to examine the pharmacologic plasticity of cholinergic, non-adrenergic non-cholinergic (NANC), and purinergic contractions in neurogenic bladder strips from spinal cord injured (SCI) rats. Bladder strips were harvested from female rats three to four weeks after T9–T10 spinal cord transection. The strips were electrically stimulated using two experimental protocols to compare the contribution of muscarinic and NANC/purinergic contractions in the presence and the absence of carbachol or muscarine. The endpoints of the study were: (1) percent NANC contraction that was unmasked by the muscarinic antagonist 4-DAMP, and (2) P2X purinergic contraction that was evoked by α,β–methylene ATP. NANC contraction accounted for 78.5% of the neurally evoked contraction in SCI bladders. When SCI bladder strips were treated with carbachol (10 µM) prior to 4-DAMP (500 nM), the percent NANC contraction decreased dramatically to only 13.1% of the neurally evoked contraction (p=0.041). This was accompanied by a substantial decrease in α,β–methylene ATP evoked P2X contraction, and desensitization of purinergic receptors (the ratio of subsequent over initial P2X contraction decreased from 97.2% to 42.1%, p=0.0017). Sequential activation of the cholinergic receptors with carbachol (or with muscarine in neurally intact bladders) and unmasking of the NANC response with 4-DAMP switched the neurally evoked bladder contraction from predominantly NANC to predominantly cholinergic. We conclude that activation of muscarinic receptors (with carbachol or muscarine) blocks NANC and purinergic contractions in neurally intact or in SCI rat bladders. The carbachol-induced inhibition of the NANC contraction is expressed more in SCI bladders compared to neurally intact bladders. Along with receptor plasticity, this change in bladder function may involve P2X-independent mechanisms.

Diabetic plasticity of non-adrenergic non-cholinergic and P2X-mediated rat bladder contractions

“…The desensitization of P2X1 purinergic contractions on the diuretic and diabetic detrusor seems to be enhanced by the application of CCh, confirming a specific muscarinic receptor inhibitory interaction taking place in smooth muscle cells [12, 13]. In fact, the IC 50 doses of atropine for M2/M3 muscarinic receptor inhibition are in the nM range [4], thus we predict that 1 μM was enough for blocking all muscarinic receptor isoforms, supporting the idea that muscarinic receptor-activation is a key event in mediating the impairment of NANC and P2X-mediated bladder contractions.…”

“…As performed in our previous studies, two stimulation protocols were achieved on each half bladder [12, 13]. In all cases the intact P2X purinergic contractile response was evaluated with the application of the specific P2X agonist α, β-Methylene-Adenosine Triphosphate (αβMeATP) using a single dose of 50 μM at the beginning of each contractile experiment.…”

“…The pharmacological inhibition of cholinergic receptors using atropine [13] or specific M-type muscarinic antagonists such as 4-Diphenylacetoxy- N -methylpiperidine (4-DAMP), unmasks a remaining NEC known as the non-adrenergic non-cholinergic (NANC) contractile response [2, 12]. In rat detrusor the NANC response is mostly mediated by the activation of P2X-type purinergic receptors by co-released ATP, and can be revealed by pharmacological inhibition of cholinergic transmission [9, 13, 26].…”

“…Moreover, the specific inhibition of bladder M-type muscarinic receptors with 4-DAMP prevents the attenuation of the NANC responses, thus suggesting that activation of muscarinic receptors may desensitize a purinergic contractile component mediated by P2X-type receptors [12, 13, 26]. …”

“…By following a similar approach as in previous studies [12, 13], we calculated the changes in NEC and NANC by measuring strip contractile changes while preventing muscarinic receptor activation with a high dose of atropine. We also determined the degree of desensitization for the purinergic component by comparing P2X purinergic receptor contractions at the beginning and end of the stimulation protocols.…”