Received for publication June 20, 2008.
Revised November 13, 2008.
Accepted for publication November 13, 2008.

Role of PKC and Calcium Entry in KCl-Induced Vascular Smooth Muscle Calcium Sensitization and Feed Back Control of Cellular Calcium Levels

Abstract

The degree of tonic force (F) maintenance induced in vascular smooth muscle (VSM) upon K⁺-depolarization (KCl) can be greatly reduced by inhibition of rhoA kinase (ROCK). We explored the possibility that a PKC isotype may also play a role in causing KCl-induced Ca²⁺ sensitization. In isometric rings of rabbit artery, the PKC inhibitors, Go 6983, GF-109203X and a cell permeable (myristoylated) pseudosubstrate inhibitor of PKC (PI_PKC), inhibited KCl-induced tonic F. Rather than inhibiting KCl-induced F, a myristoylated pseudosubstrate inhibitor of PKC/ that inhibited phorbol dibutyrate-induced F slightly potentiated KCl-induced tonic F. Whereas the ROCK inhibitor, H-1152, reduced basal phosphorylation of myosin light chain (MLC) phosphatase (MLCP) targeting subunit at threonine 853 (MYPT1-pT853), 3 µM and 10 µM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PI_PKC elevated basal [Ca²⁺]_i (Ca) and potentiated KCl-induced tonic increases in Ca while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca²⁺ entry through voltage-operated Ca²⁺ channels (VOCCs) reduced KCl-induced Ca²⁺ sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKC are constitutively active and function in "resting" muscle to regulate the basal levels of MYPT1-pT853 and Ca, respectively. In this model, KCl-induced increases in Ca activate PKC to feed forward and cause additional MYPT1p-T853 above that induced by constitutive ROCK, permitting Ca²⁺ sensitization and strong F maintenance. Active PKC also feeds back to attenuate the degree of KCl-induced increases in Ca.

Key words: calcium sensitization, calcium signaling, contraction, myosin phosphatase, protein kinase C, rho-kinase