Expression of CPI‐17 and myosin phosphatase correlates with Ca²⁺ sensitivity of protein kinase C‐induced contraction in rabbit smooth muscle

Abstract

Various smooth muscles have unique contractile characteristics, such as the degree of Ca2+ sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)‐induced Ca2+ sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI‐17, and the thin filament regulator h‐calponin. Immunohistochemical and Western blot analyses indicated that CPI‐17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI‐17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI‐17 concentration of the phasic vas deferens. In contrast to CPI‐17 expression patterns, phasic muscles contained more MLCP myosin‐targeting subunit than tonic tissues. Calponin expression was not statistically different. Addition of phorbol ester to α‐toxin‐permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI‐17 and myosin light chain (MLC) at submaximal [Ca2+]i. These responses were several‐fold greater in femoral artery as compared to vas deferens. We conclude that the expression ratio of CPI‐17 to MLCP correlates with the Ca2+ sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI‐17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI‐17 and high MLCP content. This implicates CPI‐17 inhibition of MLCP as an important component in modulating vascular muscle tone.

Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

“…Vascular smooth muscles have an abundance of CPI-17 protein relative to other kinds of smooth muscles (42). Both ROCK and PKC inhibitors inhibited agonist-induced sustained contractions in MYPT1-deficient vascular smooth muscle similar to control smooth muscles, suggesting a contribution of CPI-17 phosphorylation by ROCK in regulating PP1c␦ activity.…”

Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

“…Although Rho kinase is capable of activating CPI-17 in vitro, albeit to a lesser extent when compared with PKC, its predominant effect in vivo appears to be via phosphorylation of the large, 110-130 kDa regulatory myosin phosphatase targeting subunit (MYPT1), and inhibition of MLCP activity [27]. In both vascular and visceral smooth muscle, CPI-17 expression varies widely and is most abundant in tonic smooth muscle, consistent with a role for CPI-17 in sustained contraction [28,29]. CPI-17 expression correlated with the ability of phorbol esters to increase contraction and MLC 20 phosphorylation at submaximal Ca 2+ concentrations ('Ca 2+ sensitization').…”

The contribution of Ca2+ signaling and Ca2+ sensitivity to the regulation of airway smooth muscle contraction is different in rats and mice

“…These results suggest that Rho kinase but not PKC downregulates the activity of rat MLCP under conditions of high [Ca 2ϩ ] i . The minimal involvement of PKC may result from the low expression level of CPI-17, which largely varies depending on the type of SMCs and animal species (15,38). However, it remains to be determined whether the high Ca 2ϩ sensitivity associated with high [Ca 2ϩ ] i was mediated by the activation of RhoA/Rho kinase pathway to inactivate MLCP or whether the activity of rat MLCP is inherently low.…”