IN-SITU DETERMINATION OF [CA2-PROTEIN KINASE-C ISOFORM(](I) THRESHOLDFOR TRANSLOCATION OF THE ALPHA)

Because of inherent difficulties in maintaining physiological conditio ns 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 whil e, in parallel, the distribution of the Ca2+-dependent alpha-PKC isofo rm 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 ab sence of agonists, increasing [Ca2+](i) caused < 25% of maximal transl ocation. In the presence of phenylephrine, maximum translocation occur red at [Ca2+](i) greater than or equal to 198 nM. Phenylephrine augmen ted translocation of alpha-PKC primarily by increasing the slope of th e [Ca2+](i)-PKC translocation relationship. These results indicate tha t the [Ca2+](i) threshold of alpha-PKC translocation in situ is less t han that reported in most in vitro assays and are consistent with an e ffect of agonist-induced generation of other second messengers that ca use cooperative interactions leading to translocation.