Signalling to contractile proteins by muscarinic and purinergic pathways in neurally stimulated bladder smooth muscle

Abstract

Urinary bladder smooth muscle contraction is triggered by parasympathetic nerves, which release ATP and acetylcholine (ACh) that bind to purinergic and muscarinic receptors, respectively. Neuronal signalling may thus elicit myosin regulatory light chain (RLC) phosphorylation and contraction through the combined, but distinct contributions of these receptors. Both receptors mediate Ca2+ influx whereas muscarinic receptors may also recruit Ca2+-sensitization mechanisms. Using transgenic mice expressing calmodulin sensor myosin light chain kinase (MLCK) in smooth muscles, the effects of suramin/α,β-methylene ATP (α,β-meATP) (purinergic inhibition) or atropine (muscarinic inhibition) on neurally stimulated elevation of [Ca2+]i, MLCK activation, force and phosphorylation of RLC, myosin light chain phosphatase (MLCP) targeting subunit MYPT1 and MLCP inhibitor protein CPI-17 were examined. Electric field stimulation (EFS) increased [Ca2+]i, MLCK activation and concomitant force in a frequency-dependent manner. The dependence of force on [Ca2+]i and MLCK activation decreased with time suggesting increased Ca2+ sensitization in the late contractile phase. RLC and CPI-17 phosphorylation increased upon stimulation with maximal responses at 20 Hz; both responses were attenuated by atropine, but only RLC phosphorylation was inhibited by suramin/α,β-meATP. Antagonism of purinergic receptors suppressed maximal MLCK activation to a greater extent in the early contractile phase than in the late contractile phase; atropine had the opposite effect. A frequency- and time-dependent increase in MLCK phosphorylation explained the desensitization of MLCK to Ca2+, since MLCK activation declined more rapidly than [Ca2+]i. EFS elicited little or no effect on MYPT1 Thr696 or 850 phosphorylation. Thus, purinergic Ca2+ signals provide the initial activation of MLCK with muscarinic receptors supporting sustained responses. Activation of muscarinic receptors recruits CPI-17, but not MYPT1-mediated Ca2+ sensitization. Furthermore, nerve-released ACh also initiates signalling cascades leading to phosphorylation-dependent desensitization of MLCK.