Roles of CaM kinase II and phospholamban in SNP-induced relaxation of murine gastric fundus smooth muscles

Abstract

The mechanisms by which nitric oxide (NO) relaxes smooth muscles are unclear. The NO donor sodium nitroprusside (SNP) has been reported to increase the Ca2+ release frequency (Ca2+ sparks) through ryanodine receptors (RyRs) and activate spontaneous transient outward currents (STOCs), resulting in smooth muscle relaxation. Our findings that caffeine relaxes and hyperpolarizes murine gastric fundus smooth muscles and increases phospholamban (PLB) phosphorylation by Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) suggest that PLB phosphorylation by CaM kinase II participates in smooth muscle relaxation by increasing sarcoplasmic reticulum (SR) Ca2+ uptake and the frequencies of SR Ca2+ release events and STOCs. Thus, in the present study, we investigated the roles of CaM kinase II and PLB in SNP-induced relaxation of murine gastric fundus smooth muscles. SNP hyperpolarized and relaxed gastric fundus circular smooth muscles and activated CaM kinase II. SNP-induced CaM kinase II activation was prevented by KN-93. Ryanodine, tetracaine, 2-aminoethoxydiphenylborate, and cyclopiazonic acid inhibited SNP-induced fundus smooth muscle relaxation and CaM kinase II activation. The Ca2+-activated K+ channel blockers iberiotoxin and apamin inhibited SNP-induced hyperpolarization and relaxation. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-alpha]quinoxalin-1-one inhibited SNP-induced relaxation and CaM kinase II activation. The membrane-permeable cGMP analog 8-bromo-cGMP relaxed gastric fundus smooth muscles and activated CaM kinase II. SNP increased phosphorylation of PLB at Ser16 and Thr17. Thr17 phosphorylation of PLB was inhibited by cyclopiazonic acid and KN-93. Ser16 and Thr17 phosphorylation of PLB was sensitive to 1H-[1,2,4]oxadiazolo-[4,3-alpha]quinoxalin-1-one. These results demonstrate a novel pathway linking the NO-soluble guanylyl cyclase-cGMP pathway, SR Ca2+ release, PLB, and CaM kinase II to relaxation in gastric fundus smooth muscles.

Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum

“…In addition, autonomous CaM kinase II activity and PLB-Thr17 phosphorylation was increased, suggesting that CaM kinase II activation is mediated by localized Ca 2ϩ release events from the SR in murine gastric smooth muscles (19). Furthermore, the findings that PLB phosphorylation results in increased SERCA activity and increased SR Ca 2ϩ release suggest that PLB phosphorylation would exert a positive feedback effect on CaM kinase II, by maintaining enhanced SR Ca 2ϩ release (19,20,21). Similarly, PLB gene ablation would also be expected to elevate CaM kinase II activity by maintaining enhanced SR Ca 2ϩ uptake and release (32,43).…”

Relaxation Patterns of Human Gastric Corporal Smooth Muscle by Cyclic Nucleotides Producing Agents

“…Since these stimulators are known to inhibit slow wave and produce hyperpolarization, then suppressing contraction, it inhibits phasic contraction and basal tone in GI tract (Andersson and Nilsson, 1972;Jin et al, 1993;Smith et al, 1993). These findings imply that cyclic nucleotide activators such as FSK and SNP play an inhibitory role for basal tone as well as spontaneous contraction in human stomach (Huizinga et al, 1991;Ozaki et al, 1992;Jin et al, 1993;Smith et al, 1993;Kim et al, 2006;Kim and Perrino, 2007).…”

“…β-Adrenergic nerve and VIP containing neurons in myenteric plexus, which project to the smooth muscle layers, have been reported to exist in the gut, and their stimulation inhibits slow wave and contraction via increasing cAMP and/or cGMP (Andersson and Nilsson, 1972;Jin et al, 1993;Smith et al, 1993). Furthermore, FSK and SNP, cyclic nucleotide (cAMP and cGMP) activators, have also been shown to inhibit electrical and contractile activity of GI smooth muscle (Huizinga et al, 1991;Ozaki et al, 1992;Jin et al, 1993;Smith et al, 1993;Kim et al, 2006;Severi et al, 2006). In the present study, we found the inhibitory effect of ISO in human stomach: ISO inhibited spontaneous contraction and basal tone in a reversible manner (Fig.…”

“…For the cAMP-induced relaxation: 1) decrease of Ca 2＋ influx (Bulbring and Tomita, 1980;Ozaki et al, 1992;Smith et al, 1993;Koh and Sanders, 1996;Zhu et al, 2005), and 2) regulation of sarcoplasmic reticulum (SR) Ca 2＋ uptake and Ca 2＋ extrusion (Casteels and Raeymakers, 1979;Meisheri and van Breemen, 1982;Nishimura and van Breemen, 1989). For the cGMP-mediated relaxation: 1) decrease of Ca 2＋ influx (Ozaki et al, 1992;Quignard et al, 1997;Ruiz-Velasco et al, 1998;Geeson et al, 2002;Zhu et al, 2005), 2) increase of sarcoplasmic reticulum (SR) Ca 2＋ uptake, 3) involvement of protein kinase (Kim et al, 2006;Kim and Perrino, 2007), and 4) increase of calcium activated K ＋ current (IKCa) (Yu et al, 1993;Geeson et al, 2002;Kim et al, 2006). Therefore, the inhibitory effect of cAMP and/or cGMP on smooth muscle motility seems to be associated with regulation of Ca 2＋ homeostasis.…”

Regulation of sarcoplasmic reticulum Ca2+reuptake in porcine airway smooth muscle

“…Studies in cardiac and slow-twitch skeletal muscles have long established that SERCA can be regulated by phospholamban (PLN), such that phosphorylation of PLN (e.g., by cyclic nucleotides) disinhibits SERCA (5,9,37). Although PLN expression and phosphorylation under physiological conditions have been demonstrated in gastrointestinal (27,28) and vascular smooth muscles (6,8,18,25,30,59), data on the role of PLN in ASM are very limited (38).…”