PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries

Abstract

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca2+]i) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 × 10-6 M] in the absence or presence of ERK1/2 inhibition (U-0126, 10-5 M). After PDBu ± ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmonScr789, myosin light chain 20 (MLC20), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca2+]i. PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmonSer789 and MLC 20 levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 × 10-7 M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC20 phosphorylation in the thick filament pathway and increases Ca2+ sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmonScr789 was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase. Copyright © 2005 the American Physiological Society.