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 Ca(2+) concentration ([Ca(2+)](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 Ca(2+) signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes alpha, delta, and epsilon; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCalpha mirrored the rise in [Ca(2+)](i), while that of eGFP-PKCdelta or -epsilon occurred more slowly. Endothelin-induced translocation of eGFP-PKCepsilon was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca(2+)](i) increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCalpha translocation, while eGFP-PKCdelta 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 Ca(2+) and PKC responses to different vasoconstrictors in ASM.
Nelson
Carl P
CP
Department of Cell Physiology & Pharmacology, Univ. of Leicester, LE1 9HN, UK. cpn5@le.ac.uk
Willets
Jonathon M
JM
Davies
Noel W
NW
Challiss
R A John
RA
Standen
Nicholas B
NB
D000818:Animals; D001158:Arteries / Q000166:cytology; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D032389:Myocytes, Smooth Muscle / Q000187:drug effects*; D011493:Protein Kinase C / Q000187:drug effects* / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D007527:Isoenzymes; D014662:Vasoconstrictor Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C