Role of PKC, tyrosine kinases, and Rho kinase in α-adrenoreceptor-mediated PASM contraction

Abstract

Our objectives were to identify the relative contributions of intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in the pulmonary artery smooth muscle (PASM) contractile response to the alpha-adrenoreceptor agonist phenylephrine (PE) and to assess the role of PKC, tyrosine kinases (TK), and Rho kinase (ROK) in that response. Our hypothesis was that multiple signaling pathways are involved in the regulation of [Ca2+]i, myofilament Ca2+ sensitization, and vasomotor tone in response to alpha-adrenoreceptor stimulation of PASM. Simultaneous measurement of [Ca2+]i and isometric tension was performed in isolated canine pulmonary arterial strips loaded with fura 2-AM. PE-induced tension development was due to sarcolemmal Ca2+ influx, Ca2+ release from inositol 1,4,5-trisphosphate-dependent sarcoplasmic reticulum Ca2+ stores, and myofilament Ca2+ sensitization. Inhibition of either PKC or TK partially attenuated the sarcolemmal Ca2+ influx component and the myofilament Ca2+ sensitizing effect of PE. Combined inhibition of PKC and TK did not have an additive attenuating effect on PE-induced Ca2+ sensitization. ROK inhibition slightly decreased [Ca2+]i but completely inhibited myofilament Ca2+ sensitization. These results indicate that PKC and TK activation positively regulate sarcolemmal Ca2+ influx in response to alpha-adrenoreceptor stimulation in PASM but have relatively minor effects on myofilament Ca2+ sensitivity. ROK is the predominant pathway mediating PE-induced myofilament Ca2+ sensitization.

Cellular mechanisms of thromboxane A2-mediated contraction in pulmonary veins

“…The effect of combined inhibition of PKC, ROK, and TK was also assessed. The concentrations of inhibitors in the present study are within the range of concentrations used in previous studies (3,4,12). Pulmonary venous rings were pretreated with these inhibitors for 30 min before administration of U-46619.…”

“…Although we did not directly assess the roles of these signaling pathways on Ca 2ϩ influx or Ca 2ϩ release, we did test the hypothesis that the attenuating effect of these signaling pathway inhibitors was due to a reduction in the U-46619-induced increase in myofilament Ca 2ϩ sensitivity. The involvement of PKC (1,3,4,6,9,13,14,25,30,37), ROK (3-5, 10, 13, 25, 26), and TK (3,4,13,23,28) in Ca 2ϩ sensitization has been reported in intact and permeabilized arteries from different species. PKC may modulate Ca 2ϩ sensitivity via downregulation of myosin light chain phosphatase (MLCP) (18) or by phosphorylation of caldesmon and calponin (11,16).…”

“…Recent evidence suggests that activation of ROK by the active GTPase RhoA may be a key mediator of Ca 2ϩ sensitization in response to G protein-coupled receptor activation (3,13,29). ROK phosphorylates the regulatory subunit of MLCP and inhibits its catalytic activity, thus resulting in increased myosin light chain phosphorylation, Ca 2ϩ sensitization, and increased tension (29).…”

“…ROK phosphorylates the regulatory subunit of MLCP and inhibits its catalytic activity, thus resulting in increased myosin light chain phosphorylation, Ca 2ϩ sensitization, and increased tension (29). Y-27632, a selective ROK inhibitor (32), effectively decreases Ca 2ϩ sensitivity induced by various agonists such as carbachol, histamine, phenylephrine, endothelin-1, and U-46619 (3,4,13,17,32,38). In the present study, Y-27632 caused a rightward shift in the [Ca 2ϩ ] i -tension relationship in U-46619-pretreated pulmonary venous strips, which supports the concept that the ROK-associated pathway is involved in U-46619-induced Ca 2ϩ sensitization in canine pulmonary veins.…”

Role of PKCα and PKCι in phenylephrine-induced contraction of rat corpora cavernosa

“…There are several reports in other cell systems that RhoA-and PKCmediated pathways interact. 41,53,54 However, the mechanism of Rho-PKC interaction is still poorly defined.…”