Modulation of murine gastric antrum smooth muscle STOC activity and excitability by phospholamban

Abstract

We investigated intracellular Ca2+ waves, spontaneous transient outward currents (STOCs), and membrane potentials of gastric antrum smooth muscle cells from wild-type and phospholamban-knockout mice. The NO donor sodium nitroprusside (SNP) increased intracellular Ca2+ wave activity in wild-type antrum smooth muscle cells, but had no effect on the constitutively elevated intracellular Ca2+ wave activity of phospholamban-knockout cells. STOC activity was also constitutively elevated in phospholamban-knockout antrum smooth muscle cells relative to wild-type cells. SNP or 8-bromo-cGMP increased the STOC activity of wild-type antrum smooth muscle cells, but had no effect on STOC activity of phospholamban-knockout cells. Iberiotoxin, but not apamin, inhibited STOC activity in wild-type and phospholamban-knockout antrum smooth muscle cells. In the presence of SNP, STOC activity in wild-type and phospholamban-knockout antrum smooth muscle cells was inhibited by ryanodine, but not 2-APB. The cGMP-dependent protein kinase inhibitor KT5823 reversed the increase in STOC activity evoked by SNP in wild-type antrum smooth muscle cells, but had no effect on STOC activity in phospholamban-knockout cells. The resting membrane potential of phospholamban-knockout antrum smooth muscle cells was hyperpolarized by approximately −6 mV compared to wild-type cells. SNP hyperpolarized the resting membrane potential of wild-type antrum smooth muscle cells to a greater extent than phospholamban-knockout antrum smooth muscles. Despite the hyperpolarized membrane potential, slow wave activity was significantly increased in phospholamban-knockout antrum smooth muscles compared to wild-type smooth muscles. These results suggest that phospholamban is an important component of the mechanisms regulating the electrical properties of gastric antrum smooth muscles.

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The stomach generates myogenic activity in which electrical slow waves generate rings of contraction that spread from the corpus towards the pyloric sphincter (Hirst & Edwards, 2006). Slow waves generated by interstitial cells of Cajal (ICC) conduct into smooth muscle cells and depolarize the membrane, causing a transient contraction due to mechanically productive Ca2+ entry via voltage-gated (L-type) Ca2+ channels (Costa et al. 2005). Removal of Ca2+ from the cytoplasm by the plasma membrane Ca2+-ATPase and the sarcoplasmic reticulum (SR) membrane Ca2+-ATPase (SERCA) repolarizes the membrane and relaxes the smooth muscle cells (Sanders, 2008). Refilling of the SR by SERCA also ensures that sufficient Ca2+ will be available for regulatory SR Ca2+ release in the form of sparks or puffs (Sanders, 2008). These spontaneous release events activate a number of effectors including CaM kinase II and Ca2+-sensitive ion channels, which regulate a variety of smooth muscle contractile responses (Amberg et al. 2002; Kim & Perrino, 2007). At physiological temperatures the spatio-temporal recruitment of Ca2+ puffs or sparks results in an intracellular Ca2+ wave that activates STOCs to hyperpolarize the membrane and reduce muscle excitability (Gordienko et al. 1998; Jaggar et al. 2000; Hennig et al. 2002).

Modulation of SR Ca2+ uptake by SERCA is an important regulator of smooth muscle phasic and tonic behaviour (Ishida & Paul, 2005). SERCA activation elevates the SR Ca2+ load (Inesi et al. 1990), which contributes to relaxation by lowering the Ca2+ concentration of the myoplasm [Ca2+]c (Karaki et al. 1997), and increasing Ca2+ spark frequency and STOC activity (Wellman & Nelson, 2003). Inhibiting SERCA activity with thapsigargin results in diminished Ca2+ clearance from the cytosol, and decreases in the amplitudes of slow waves and the associated Ca2+ transients and phasic contractions of gastric antrum smooth muscles (Ozaki et al. 1992). SERCA activity is endogenously regulated by the SR membrane protein phospholamban (PLB) to vary the rate of Ca2+ influx into the SR via its phosphorylation levels, from maximal SERCA inhibition of 50% by dephospho-PLB to no SERCA inhibition by phospho-PLB (Paul et al. 2002). Cardiac, skeletal, vascular and bladder smooth muscles from phospholamban-knockout (PLB-KO) mice demonstrate clear differences in contractile properties from the corresponding wild-type muscles (Kadambi & Kranias, 1997).

In smooth muscle cells, NO or NO donors increase cGMP levels, resulting in the activation of several downstream signalling cascades which ultimately result in Ca2+ removal from the myoplasm, membrane hyperpolarization and relaxation (Petkov & Boev, 1996; Cohen et al. 1999; Yu et al. 2003). In gastric motility disorders where enteric neuropathies are implicated, such as impaired gastric accommodation, directly targeting smooth muscle cGMP levels with NO donors or phosphodiesterase inhibitors improves symptoms and gastric smooth muscle function (Tack et al. 2002; Sarnelli et al. 2004; van den Elzen & Boeckxstaens, 2006). We previously reported that SNP increased cGMP-dependent PLB phosphorylation, and relaxed murine gastric fundus and antrum smooth muscles (Kim et al. 2006; Kim & Perrino, 2007). Ryanodine, 2-APB, or cyclopiazonic acid inhibited the relaxation of gastric fundus and antrum smooth muscles (Kim et al. 2006). These findings suggest that increased SERCA activity due to PLB phosphorylation leads to increased SR Ca2+ release and higher STOC activity resulting in hyperpolarization and relaxation. Similarly, SNP hyperpolarizes and relaxes cerebral and coronary artery smooth muscles by increasing the frequency of Ca2+ sparks and STOCs via a NO–cGMP pathway (Porter et al. 1998). In support of these findings, we found that the frequency of intracellular Ca2+ waves in gastric antrum smooth muscles from PLB-KO mice are constitutively elevated and are unaffected by caffeine (Kim et al. 2008). In this study, we extended these findings and investigated the function of PLB in mediating the effects of SNP on intracellular Ca2+ waves, STOC activity, and membrane potential of murine gastric antrum smooth muscles using gene-targeted PLB-KO mice. Our results show that PLB is an important determinant of the electrical properties of gastric antrum smooth muscles, and plays a critical role in the hyperpolarization of antrum smooth muscles in response to the NO donor SNP.