Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle

Abstract

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles

“…Nevertheless, the initial ACh-induced contraction of both smooth muscles correlates with an increase in MLC phosphorylation of ϳ40%, suggesting that myosin phosphorylation in ESO is catalyzed by a kinase different from MLCK. Indeed, the addition of activated MLCK in permeabilized ESO cells elicited a small contraction and a minor increase in MLC phosphorylation (from 11 to 18%) compared with LES (from 6 to 25%) (54). Similarly, ACh-induced MLC phosphorylation and contraction in LES were inhibited by CaM and MLCK inhibitors but not by PKC inhibitors, whereas in ESO, they were inhibited by PKC inhibitors but not by CaM or MLCK inhibitors (54).…”

“…IP 3 triggers Ca 2ϩ release from the sarcoplasmic reticulum, activation of CaM-dependent MLCK, MLC phosphorylation, and contraction (55). In sharp contrast, ACh-induced MLC phosphorylation and contraction in ESO are inhibited by PKC inhibitors but not by CaM or MLCK inhibitors (54). In this case, ACh-induced contraction involves activation of M 2 muscarinic receptors, which are coupled to both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) via G i3 (55).…”

“…LES, however, maintains spontaneous myogenic tone and relaxes in response to stimulation of nonadrenergic, noncholinergic neurons. Previous work has suggested that fundamental differences exist in the signal transduction pathways involved in excitation-contraction coupling in these two smooth muscles (54,55) for the initial phase of contraction (47). In both cases, contractile stimuli such as acetylcholine (ACh) trigger an increase in MLC phosphorylation (54).…”

“…Previous work has suggested that fundamental differences exist in the signal transduction pathways involved in excitation-contraction coupling in these two smooth muscles (54,55) for the initial phase of contraction (47). In both cases, contractile stimuli such as acetylcholine (ACh) trigger an increase in MLC phosphorylation (54). In the case of LES, both phosphorylation and contraction are inhibited by CaM and MLCK inhibitors and are unaffected by PKC inhibitors (3,54).…”

“…In both cases, contractile stimuli such as acetylcholine (ACh) trigger an increase in MLC phosphorylation (54). In the case of LES, both phosphorylation and contraction are inhibited by CaM and MLCK inhibitors and are unaffected by PKC inhibitors (3,54). ACh-induced contraction of LES involves activation of M 3 muscarinic receptors, which are coupled to phosphatidylinositol phospholipase C (PI-PLC) via G q/11 .…”