KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase

Abstract

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.

Effects of ω-hydroxylase product on distal human pulmonary arteries

“…4B). These results confirm that the addition of 20-HETE opposes the processes triggered by KCl, which usually involve membrane depolarization, an activation of voltage-operated Ca 2ϩ channels, Ca 2ϩ entry, a formation of Ca-calmodulin complexes, and an activation of various enzymatic systems, including the myosin light chain (MLC) kinase (MLCK), which phosphorylates the MLCs (16,30).…”

Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells

“…Initially, the Rho-kinase inhibitor Y-27632 was thought to have no effect on the high-K + -induced contraction in smooth muscle tissues (Uehata et al, 1997). However, more recent studies show that the late plateau-phase (but not the initial phase) of high K + -induced contraction in smooth muscle tissues is very sensitive to the Rho-kinase inhibitors, not only to Y-27632 but also to the structurally different HA1077 (Mita et al, 2002;Urban et al, 2003;Janssen et al, 2004). Since Y-27632 does not block an initial Ca 2+ transient induced by high K + , the initial development of contraction is not prevented by the presence of the inhibitor.…”

Regulation of airway smooth muscle RhoA/ROCK activities by cholinergic and bronchodilator stimuli

“…Ca 2+ release stimulates myosin light chain kinase (MLCK) activity directly [6,7], but may also open plasmalemmal Cl -channels [8,9] leading to membrane depolarisation and voltage-dependent Ca 2+ influx. The M 3 receptors are also coupled to G 12,13 , which activates the monomeric G-protein Rho and its downstream effector Rho-kinase (ROCK) [10]. One of the targets of ROCK is myosin light chain phosphatase (MLCP), whose activity is suppressed.…”

“…There has been a great deal of interest recently in the involvement of the Rho/ROCK signalling pathway in excitation-contraction coupling [11][12][13][14][15][16][17] and airway hyperresponsiveness [18][19][20][21][22]. However, the data available are limited in many ways.…”