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VASCULAR BIOLOGY

Visualizing the temporal effects of vasoconstrictors on PKC translocation and Ca²⁺ signaling in single resistance arterial smooth muscle cells

¹Department of Cell Physiology and Pharmacology, and ²Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Henry Wellcome Building, Leicester, United Kingdom

Submitted 14 July 2008 ; accepted in final form 26 September 2008

Arterial 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.

protein kinase C; arterial smooth muscle; fluorescence imaging; intracellular Ca²⁺ concentration; diacylglycerol

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