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 Ca2+-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) greater than or equal to 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.