PROTEIN-KINASE-C ACTIVATION DURING CA2-INDEPENDENT VASCULAR SMOOTH-MUSCLE CONTRACTION()

The cellular signaling mechanisms that modulate the sustained vascular smooth muscle contractions that occur in vasospasm are not known. We and others have hypothesized that a kinase cascade involving protein k inase C (PKC) modulates sustained vascular smooth muscle contraction. The purpose of this investigation was to develop a model in which the traditional contractile pathways involving myosin light chain phosphor ylation are not activated and determine if the PKC pathway is activate d under these conditions. The phosphorylation of caldesmon, myosin lig ht chain (MLC20), and the specific PKC substrate, MARCKS (myristoylate d, alanine-rich C-kinase substrate) was measured in bovine carotid art erial smooth muscle (BCASM) stimulated with phorbol 12,13-dibutyrate ( PDBu) under Ca2+-containing and Ca2+- free conditions. PDBu stimulatio n led to increases in caldesmon and MARCKS phosphorylation to the same degree in the presence or absence of Ca2+. PDBu stimulation but did n ot lead to increases in MLC20 phosphorylation over basal levels in Ca2 +-free conditions. Immunoblot analysis of BCASM using PKC isoform-spec ific antibodies demonstrated the presence of one ''Ca2+-dependent'' PK C isoform: alpha, and two of the ''Ca2+-independent'' isoforms: epsilo n and zeta. These data suggest that Ca2+-independent isoforms of PKC m ay play a role in the sustained phase of BCASM contractions through a kinase cascade that involves caldesmon and MARCKS phosphorylation but not MLC20 phosphorylation. (C) 1998 Academic Press.