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

Abstract

The current study set out to compare the temporal relationships of Rho activity, Rho kinase (ROCK) activity and tone following cholinergic stimulation in the presence and absence of three different bronchodilators.Bovine trachea challenged with a half-maximally effective concentration of carbachol (CCh) was flash-frozen at different times, then assayed for Rho (rhotekin pull-down assay) and ROCK (Western blot; radiometric assay) activities.Rho was activated within 30 s, followed by ROCK (peak at 2 min); both returned to baseline by 20 min, although tone continued to rise over that period. Increasing the concentration of CCh greatly increased the magnitudes and rates of stimulation of Rho, ROCK and tone. These CChinduced changes were next compared in tissues pre-treated with isoproterenol, salmeterol or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Neither the time course nor the magnitude of Rho-activation were reduced by the b-agonists; SNAP slowed Rho activation but it did not alter the peak magnitude. These observations were mirrored in ROCK activation and contraction. When tissues were pre-constricted with CCh and then challenged with the bronchodilators, however, all three agonists reversed cholinergically stimulated Rho, ROCK and myosin light chain kinase activities as well as tone.In conclusion, bronchodilators can suppress RhoA and Rho kinase activities, although their major effect appears to be on myosin light chain kinase activity.

The effects of the small GTPase RhoA on the muscarinic contraction of airway smooth muscle result from its role in regulating actin polymerization

“…RhoA-mediated ROCK activation and regulation of MLC phosphatase have been demonstrated during the cholinergic activation of airway smooth muscles (24,34,50). We found that stimulation with ACh increased the phosphorylation of the myosin regulatory subunit (MYPT1) of MLC phosphatase at Thr850, and that ACh-induced MYPT1 phosphorylation was completely inhibited by the inactivation of RhoA.…”

“…We conclude that the RhoA-mediated regulation of ACh-induced contractile tension in ASM results from its role in mediating actin polymerization rather than from effects on MLC phosphatase or MLC phosphorylation. cytoskeleton; calcium sensitization; myosin light chain phosphorylation; myosin light chain phosphatase; myosin phosphatase; calyculin A; myosin light chain kinase THE EFFECTS OF THE SMALL GTPase RhoA in regulating the Ca 2ϩ sensitivity of smooth muscle contraction are well documented and have been described for many smooth muscle tissue types, including airway smooth muscle (10,14,15,19,21,26,34,35,45,47,53). The effects of RhoA on the Ca 2ϩ sensitivity of contraction have been ascribed to its role in regulating the catalytic activity of smooth muscle myosin light chain (MLC) phosphatase (23,33,47,53).…”

On Ca2+ sensitivity and the airways: not just any smooth muscle

“…Whilst they show that activation of RhoA, Rho kinase, MYPT1 phosphorylation and constriction were sequential as expected, with RhoA activation reaching a peak after ,1 min but tension still increasing at 5 min [8], after 5 min there was a disassociation of tension from RhoA/Rho kinase activity, as tension continued to increase over the next 10 min whilst RhoA and Rho kinase activity declined by ,.50%. This differs from VSM, where agonist-induced RhoA activation is reported to be sustained and parallels force [9].…”

“…Therefore, the study by LIU et al [8] in the current issue of the European Respiratory Journal is a welcome addition to the literature, although it raises some intriguing questions.…”

“…LIU et al [8] examined the time course of tension development and the RhoA/Rho kinase signalling pathway in bovine trachealis, following sub-maximal cholinergic stimulation. Whilst they show that activation of RhoA, Rho kinase, MYPT1 phosphorylation and constriction were sequential as expected, with RhoA activation reaching a peak after ,1 min but tension still increasing at 5 min [8], after 5 min there was a disassociation of tension from RhoA/Rho kinase activity, as tension continued to increase over the next 10 min whilst RhoA and Rho kinase activity declined by ,.50%.…”