Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na+ were found to take up Ca2+ when incubated in K+ media or in sucrose media containing micromolar concentrations of free Ca2+. Li+- or choline+loaded ghosts did not take up Ca2+. The Ca2+ accumulated by Na+-loaded ghosts could be released by the Ca2+ ionophore A23187, but not by EGTA. Ca2+ uptake was inhibited by external Sr2+, Na +, Li +, or choline +. All the 45Ca2+ accumulated by Na+-dependent Ca2+ uptake could be released by external Na +, indicating that both Ca2+ influx and efflux occur in a Na+-dependent manner. Na + -dependent Ca2+ uptake and release were only slightly inhibited by Mg2+. In the presence of the Na+ ionophore Monensin the Ca2+ uptake by Na +-loaded ghosts was reduced. Ca2+ sequestered by the Na+-dependent mechanism could also be released by external Ca2+ or Sr2+ but not by Mg2+, indicating the presence of a Ca2+/Ca2+ exchange activity in secretory membrane vesicles. This Ca2+/Ca2+ exchange system is inhibited by Mg2+, but not by Sr2+. The Na + -dependent Ca2+ uptake system in the presence of Mg2+ is a saturable process with an apparent Km of 0.28 μM and a Vmax= 14.5 nmol min−1 mg protein−1. Ruthenium red inhibited neither the Na+/Ca2+ nor the Ca2+/Ca2+ exchange, even at high concentrations

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC 12 cells. Permeabilization with α-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC 12 cells

Matrix-free calcium in isolated chromaffin vesicles

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23 187, as determined by atomic emission spectroscopy. In the presence of N H Q , which causes the collapse of the secretory vesicle transmembrane proton gradient (ApH), Ca2+ uptake decreases. Under these conditions A23 187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 pM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4C1 we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 pM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 pM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4C1. Calculation of the total Ca2+ present in the secretory vesicles gives an apparent intravesicular Ca2+ concentration of 40 mM, which is a factor of lo4 higher than the free intravesicular concentration of Ca2+. It can be concluded, therefore, that the concentration gradient of free Ca2+ across the secretory vesicle membrane in the intact chromaffin cells is probably small, which implies that less energy is required to accumulate and maintain Ca2+ within the vesicles than was previously anticipated

3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg2+ and Zn,2+ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca2+ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis