Hypothesis / aims of study
Underactive bladder (UAB) is the clinical manifestation of detrusor underactivity (DU), which is defined as a contraction of reduced strength and/or duration resulting in prolonged bladder emptying and/or a failure to achieve complete bladder emptying within a normal time span. Available treatments for UAB include, Bethanechol, a non-selective muscarinic agonist, which mimics the action of acetylcholine (ACh) in bladder, but off-target side effects can reduce its effectiveness. A recent pilot study on DU patients demonstrated the clinical potential of an alternative pharmacological approach of facilitated release of endogenous ACh by blocking the negative auto-feedback of muscarinic M4 receptors. We postulate that similar clinical outcomes in UAB can also be achieved through activation of pre-junctional muscarinic M1 receptors, which are known to exert a positive auto-feedback in ACh release. Cevimeline is a muscarinic agonist, FDA approved for dry mouth with reported >8 fold higher selectivity for M1 compared to M3 muscarinic receptors. Because the expression and function of M1 receptors in rat bladder is very well characterized, a comprehensive description of how cevimeline increases the contractile activity of rat bladder should give insight into the potential therapeutic role of M1 agonists in UAB.
Study design, materials and methods
Longitudinal, urothelium intact bladder strips were removed from euthanized Sprague-Dawley rats (10-12 weeks old) and mounted in 37°C organ bath constantly gassed with 95% oxygen-5% carbon dioxide for isometric tension studies. Strips were stretched to 1 g of tension for eliciting spontaneous phasic contractions. Nerve-mediated contractions (tetrodotoxin-sensitive) were generated by electrical field stimulation of strips (EFS: 5 ms pulses, 1-64Hz, 2s train at 20V) in presence or absence of Cevimeline with or without an M1 receptor antagonist Pirenzepine [50nM]. EFS frequency response curve were generated by stimulating at 1, 2, 4,8, 6, 32 and 64 Hz (one stimulation at each frequency) with 1-min intervals between stimulations. EFS evoked contraction amplitude was normalized by the response to 120mM KCl. Effect of Cevimeline on spontaneous phasic contractions was assessed on strips not exposed to EFS.
α,βme-ATP (10 μM), an ATP analogue, was used to desensitize purinergic receptors and leave only ACh-mediated EFS contractions. TTX sensitive EFS contractions evoked at ≥ 16Hz were significantly enhanced (Two -way ANOVA followed Tukey’s multiple comparison; *p<0.01) by M1 selective concentrations [1.6µM] of Cevimeline and blocked by an M1 selective antagonist, Pirenzepine (50nM, Fig. 1d) or by atropine (5μM). Cevimeline acts on M1 and M3 receptors in a concentration dependent fashion to raise the basal tension and augment the spontaneous contractions of bladder strips. Pirenzepine per se reduces the basal tension and spontaneous contractions, but M1 selective concentration of Pirenzepine (50nM) potentiated the effect of Cevimeline on basal tension and spontaneous contractions, whereas non-selective concentrations of Pirenzepine (>50nM) competitively blocked the concentration dependent activation of M1 and M3 receptors by Cevimeline to enhance basal tension and spontaneous contractions .
Interpretation of results
It is known that TTX sensitive contractions elicited by EFS at low frequencies are dominated by ATP release, whereas contractions evoked by ≥8Hz involves M1 dependent ACh release from post-ganglionic cholinergic nerves in rat bladder. Therefore, the increased magnitude of nerve evoked contractions by Cevimeline involves M1 dependent modulation of ACh release. Concentration dependent effect of Cevimeline on spontaneous contractions of urothelium intact strips could involve activation of urothelial M1 receptors augmenting the ATP release at lower concentrations and a direct M3 activation at higher concentrations is presumably facilitated by the Pirenzepine (50nm) mediated displacement of Cevimeline bound to M1 receptors. M1 selective Cevimeline concentrations [0.1-2µM] tested here correspond to the rat plasma concentrations produced after acute intravenous administration of Cevimeline at doses 0.3-5mg/kg, which reportedly increased the frequency of non-voiding contractions in normal young rats. Animal and clinical studies on UAB report a deficient sensation of bladder filling and it is known that spontaneous contractions can enhance this sensation. Therefore, further studies are warranted to determine whether long term oral dosing of Cevimeline targeting M1 receptor improves the bladder sensory mechanisms and bladder emptying in animal models of UAB.