Role of sarcoplasmic reticulum in control of membrane potential and nitrergic response in opossum lower esophageal sphincter

Abstract

1. We previously demonstrated that a balance of Ca 2+-activated Cl - current (I Cl(Ca)) and K + current activity sets the resting membrane potential of opossum lower esophageal sphincter (LES) circular smooth muscle at ∼-41 mV, which leads to continuous spike-like action potentials and the generation of basal tone. Ionic mechanisms underlying this basal I Cl(Ca) activity and its nitrergic regulation remain unclear. Recent studies suggest that spontaneous Ca 2+ release from sarcoplasmic reticulum (SR) and myosin light chain kinase (MLCK) play important roles. The current study investigated this possibility. Conventional intracellular recordings were performed on circular smooth muscle of opossum LES. Nerve responses were evoked by electrical square wave pulses of 0.5 ms duration at 20 Hz. 2. In the presence of nifedipine (1 μM), substance P (1 μM), atropine (3 μM) and guanethidine (3 μM), intracellular recordings demonstrated a resting membrane potential (MP) of -38.1±0.7 mV (n=25) with spontaneous membrane potential fluctuations (MPfs) of 1-3 mV. Four pulses of nerve stimulation induced slow inhibitory junction potentials (sIJPs) with an amplitude of 6.1±0.3 mV and a half-amplitude duration of 1926±147 ms (n=25). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific guanylyl cyclase inhibitor, abolished sIJPs, but had no effects on MPfs. Caffeine, a ryanodine receptor agonist, hyperpolarized MP and abolished sIJPs and MPfs. Ryanodine (20 μM) inhibited the sIJP and induced biphasic effects on MP, an initial small hyperpolarization followed by a large depolarization. sIJPs and MPfs were also inhibited by cyclopiazonic acid, an SR Ca 2+ ATPase inhibitor. Specific I Cl(Ca) and MLCK inhibitors hyperpolarized the MP and inhibited MPfs and sIJPs. 4. These data suggest that (1) spontaneous release of Ca 2+ from the SR activates I Cl(Ca), which in turn contributes to resting membrane potential; (2) MLCK is involved in activation of I Cl(Ca); (3) inhibition of I Cl(Ca) is likely to underlie sIJPs induced by nitrergic innervation.