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

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 α, δ, and ε; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCα mirrored the rise in [Ca2+]i, while that of eGFP-PKCδ or -ε occurred more slowly. Endothelin-induced translocation of eGFP-PKCε was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca2+]iincreases using 1,2-bis-( o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCα translocation, while eGFP-PKCδ 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.