This paper was published as American Journal of Physiology: Cell Physiology, 2008, 295 (6), pp. C1590-C1601. It is available from http://ajpcell.physiology.org/cgi/content/abstract/295/6/C1590. Doi: 10.1152/ajpcell.00365.2008Metadata only entryArterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca2+ concentration ([Ca2+]i) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca2+ signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes , , and ; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKC mirrored the rise in [Ca2+]i, while that of eGFP-PKC or - occurred more slowly. Endothelin-induced translocation of eGFP-PKC was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca2+]i increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKC translocation, while eGFP-PKC translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca2+ and PKC responses to different vasoconstrictors in ASM
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