Fast-mixing and rapid-filtration techniques were used to analyze the kinetics of potassium-depolarization-dependent (delta K+ = 47.5 mM) influx of 45Ca into synaptosomes, in the time range from 50 msec to 5 sec. The results are consistent with the presence in synaptosomes of a homogeneous population of voltage-sensitive Ca channels. With 1 mM Cao in the medium, the delta K+-dependent Ca influx has a single-exponential time course with the half-life, t1/2 approximately 0.5-0.7 sec. Ca influx, measured between 0.1 and 10 mM Cao, shows half-saturation (KCa) at 1.5 mM Cao and has the limiting value (JCamax) of 5.9 nmol/sec/mg protein, or a current of approximately 0.06 pA/micron2 surface area. The estimated density of functional Ca channels is 0.6-6 micron-2. Voltage- and time-dependent inactivation of Ca channels was measured in synaptosomes predepolarized in 52.5 mM Ko+ with Ca omitted from the medium. Channel inactivation is a single-exponential process with a half-life of t1/2 approximately 2.3 sec. Channel recovery in 5 mM Ko+ media is likewise a single-exponential process with a half-life of t1/2 approximately 4.3 sec. The slower rate of voltage-dependent channel inactivation than of decay of Ca influx suggests that Ca entry into synaptosomes terminates by a mechanism that depends on Ca influx itself. Synaptosomes contain 200 fmol/mg protein, or approximately 6 micron-2 high-affinity (KD = 0.12 nM) 3H-nitrendipine binding sites; however, nitrendipine at concentrations greater than 10(4) X KD is without effect on the phasic influx of Ca measured at 215 msec with either 1.0 or 0.1 mM Cao. This suggests that Ca channels characterized in this study belong to a class of dihydropyridine-insensitive channels.

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We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb2+‐induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb2+ interacts with the adrenal enzyme at multiple sites. Pb2+ activates the enzyme through high‐affinity (KA(Pb) = 2.4 × 10−12M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar‐(Ki = 7.1 × 10−9M) and micromolar‐ (K′i = 2.8 × 10−7M) affinity sites, respectively. Activation of PKC by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Ca2+‐deficient, Pb2+‐containing medium, enhances the Pb2+‐induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb2+ required for half‐maximal activation of the secretory response from 7.5 × 10−10 to 5.7 × 10−11M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb2+‐induced secretion in the absence of TPA. These results indicate that (a) Pb2+ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb2+ results in increased sensitivity of exocytosis to Pb2+ but is not obligatory for Pb2+‐triggered secretion.