In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform.
Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.
Cardiovascular Division, Charles A. Dana Research Institute, Harvard-Thorndike Laboratory, Beth Israel Hospital, Boston, Massachusetts.
R A
RA
Khalil
C
C
Lajoie
K G
KG
Morgan
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D004056:Differential Threshold; D005289:Ferrets; D005455:Fluorescent Antibody Technique; D016257:Fura-2; D007425:Intracellular Membranes / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000378:metabolism*
D007527:Isoenzymes; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2