Matrix-free calcium in isolated chromaffin vesicles

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Abstract
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 A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (.DELTA.pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 .mu.M. 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 NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 .mu.M. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 .mu.M was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl. Calculation of the total Ca2+ present in the secretory vesicles gives an apparent intravesicular Ca2+ concentration of 40 mM, which is a factor of 104 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.