THE UPTAKE OF CALCIUM BY ISOLATED CHROMAFFIN GRANULES OF THE ADRENAL-MEDULLA

Bovine chromaffin secretory granules were purified by isopycnic Metriz amide gradient centrifugation and their Ca2+ sequestration pathways we re characterized. The rate of Ca2+ sequestration at 37 degrees C was f irst order, with a maximal uptake of 26.9 +/- 0.46 (mean +/- S.D., n = 3) nmol Ca2+/mg protein and a first order rate constant (k) of 0.046 +/- 0.002 mim(-1). At 4 degrees C the rate of uptake was substantially attenuated, with only 2.47 +/- 0.2 (mean +/- S.D, n = 3) nmol Ca2+/mg protein sequestered in 60 min. Ca2+ sequestration was 93% inhibited b y 180 mM NaCl [I-50% of 78.7 +/- 9.3 mM NaCl (mean i S.D., n = 11)] bu t only slightly inhibited by KCl or MgCl2. Ca2+ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after inc ubation with 30 mu M monensin. Ca2+ sequestration was dependent on ext ravesicular Ca2+ with half-maximal sequestration at pCa(2+) 6.81 +/- 0 .028 (mean +/- S.D., n 3). Sequestered Ca2+ could be exchanged with ex ternal Ca-45(2+), the exchange rate was first order (k of 0.042 +/- 0. 004: mean +/- S.D., n = 3) and saturated at 27.7 +/- 1.1 nmol Ca2+/mg (mean +/- S.D., n = 3). The Ca2+/Ca2+ exchange system was totally inhi bited by NaCl or KCl but only slightly by MgCl2. About 75% of sequeste red Ca-45(2+) could be released by incubation with NaCl, but only 8% w as released by incubation with KCl. Half-maximal release of sequestere d Ca-45(2+) required 69.3 +/- 12.2 mM NaCl (mean +/- S.D., n = 3). The Na+-induced release of sequestered Ca-45(2+) was rapid, t(0.5) of 2.8 0 +/- 0.63 min (mean +/- S.D., n = 3) and inhibited at 4 degrees C. Th e concurrent incubation of chromaffin granules with Ca-45(2+) and eith er annexin proteins V or VI resulted in attenuated uptake of Ca-45(2+) . These results suggest that Ca2+ uptake in adrenal chromaffin granule s is regulated by Na+ and Ca2+ gradients and also possibly by annexins V and VI.