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

Abstract

Biancani. Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles. Am J Physiol Cell Physiol 287: C384 -C394, 2004; 10.1152/ajpcell.00390.2003.-Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high Ca 2ϩ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca 2ϩ levels. The link between activation of the Ca 2ϩ -independent protein kinase C⑀ (PKC⑀) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281: G467-G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKC⑀-dependent, Ca 2ϩ -independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKC⑀, but not antibodies to PKC␤II, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKC⑀ from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKC⑀, MEK, ERK1/2, and ILK. protein kinase C; myosin light chain kinase; phosphatase; integrinlinked kinase SMOOTH MUSCLE CONTRACTION is regulated primarily by the reversible phosphorylation of myosin (1, 27, 58), depending on the balance between myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities, and both enzymes are subject to regulation. Most contractile stimuli elicit an increase in intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) via entry of Ca 2ϩ from the extracellular space and/or release from intracellular stores, whereupon Ca 2ϩ activates calmodulin (CaM)-dependent MLCK. Activated MLCK phosphorylates the 20-kDa myosin light chains (MLC) of myosin II at Ser 19 to activate cross-bridge cycling and the development of force or shortening of the muscle. Relaxation generally follows removal of the stimulus, whereupon [Ca 2ϩ ] i returns to resting levels as Ca 2ϩ is removed from the cytosol. MLCK (32) is i...

Signal-Transduction Pathways that Regulate Smooth Muscle Function I. Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles

“…Exogenously added phosphatase inhibitors or antibodies may reduce phosphatase activity throughout the cell and release all inhibited kinases. Nevertheless the data confirm that inhibition of a phosphatase linked to MLCK and increased MLCK activity do not play a role in contraction of Eso, which may instead be linked to inhibition of phosphatases in the PKC-dependent contractile pathway (41).…”

“…The ratio of PKC activity (membrane/ cytosolic) was significantly increased 1, 2, and 5 min after okadaic acid stimulation. In addition, contraction of Eso circular smooth muscle cells induced by okadaic acid, by microcystin-LR, and by an antibody against the muscle-specific catalytic subunit of phosphatase (PP1) were blocked by PKC⑀ antibodies, by MAPK (ERK1 and ERK2) antibodies, and by the MEK antagonist PD98059, supporting the conclusion that inhibition of Eso smooth muscle phosphatases activates a PKC-MAPK-dependent contractile pathway (41).…”

Differential expression of multidrug resistance protein 5 and phosphodiesterase 5 and regulation of cGMP levels in phasic and tonic smooth muscle

“…The underlying features of phasic muscles (e.g., distal stomach and intestine) are the ability to generate rhythmic contractions and relaxations, whereas tonic muscles (e.g., sphincters and proximal stomach) have sustained tone and relax transiently in response to relaxant transmitters (4,23,30,38). In both phasic and tonic muscle, phosphorylation of MLC 20 is a prerequisite for contraction; the levels of MLC 20 phosphorylation are determined by the opposing activities of MLCK and MLC phosphatase, and both of these enzymes' activities are well regulated in smooth muscle.…”

“…The phasic and tonic behavior of smooth muscles may also be related to differences in content and isoform composition of contractile proteins and intracellular signaling molecules that regulate the activities of MLCK and MLCP (4,6,7,13,15,17,[21][22][23]36). However, differences in the mechanisms involved in the termination of cGMP signaling in phasic and tonic muscle is not known.…”

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

“…In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1␤ or TNF-␣, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.colon; contraction; cytokines; signaling GASTROINTESTINAL smooth muscle exhibits regional variations in expression of structural, contractile, and signaling proteins that can influence its response to a variety of regulatory agents (e.g., acetylcholine, biogenic amines, neuropeptides, pyrimidines/purines; lipids, and cytokines) (3,12,27,37,57). Functional differences are evident between muscle layers (e.g., circular vs. longitudinal muscle of the intestine) or within the same layer (proximal tonic vs. distal rhythmic circular muscle of the stomach) (10,19,31,35,38,58,59).…”