Maturation and the role of PKC-mediated contractility in ovine cerebral arteries

Abstract

Ca2+-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca2+-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca2+ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.

α1-Adrenergic receptor subtype function in fetal and adult cerebral arteries

“…We also have shown that in ovine CA adrenergic-mediated vasoconstrictor responses differ significantly in fetus and adult (27,28,51,52). Recently, we have shown (8) significant changes in the Ca 2ϩ sensitization pathway (PKC and ERK1/2) with developmental maturation from fetus to adult.…”

Neural programming of mesenteric and renal arteries

“…It is likely that other targets of ␣-adrenergic signaling that control either calcium signaling or calcium sensitivity of the myofilaments are also developmentally regulated. Interestingly, a study of fetal vs. mature ovine middle cerebral arteries also demonstrated increased expression of CPI-17 protein and a proposed switch, based on chemical inhibitors, from Rho kinase to PKC-dependent enhancement of force production (20). Consistent with that study, we did not detect any change in the mRNAs of signaling intermediates, such as RhoA and Rock1 and 2 (data not shown), but a more comprehensive analysis of the developmentally regulated gene program is required.…”

Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle

“…PKC inhibits myosin light chain phosphatase activity by activating PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), resulting in increased levels of myosin light chain phosphorylation (47,62). MAP kinase and its activator MEK have been shown to be activated via PKCmediated phosphorylation in various cell types (2,21,37,43). Furthermore, MAP kinase and PKC have been shown to translocate to the surface membrane upon phenylephrine-induced smooth muscle contraction (61).…”