Transport of Ca2+ and Na+ across the chromaffin-granule membrane

Abstract

Bovine chromaffin-granule ghosts accumulate 45Ca2+ in a temperature- and osmotic-shock-sensitive process; the uptake is saturable, with Km 38 microM and Vmax. 28 nmol/min per mg at 37 degrees C. Entry occurs by exchange with Ca2+ bound to the inner surface of the membrane. It is inhibited non-competitively by Na+, La3+ and Ruthenium Red (Ki 10.7 mM, 7 microM and 2 microM respectively), and competitively by Mg2+ (ki 0.9 mM). Uptake was not stimulated by ATP. Na+ induces Ca2+ efflux; Ca2+ can re-enter the ghosts by a process of Ca2+/Na+ exchange. La3+ inhibits Ca2+ efflux during Ca2+-exchange, and Ca2+ efflux induced by Na+, suggesting that Ca2+ uptake and efflux, and Ca2+/Na+ exchange, are catalysed by the same protein. Na+ enters ghosts during CA2+ efflux, but the kinetics of its entry are not exactly similar to the kinetics of Ca2+ efflux. Initially 1-2 Na+ enter per Ca2+ lost, but at equilibrium 3-4 Na+ have replaced each Ca2+. There is no evidence that either Ca2+ uptake or efflux by Ca2+/Na+ exchange is electrogenic, suggesting that the stoichiometry of exchange is Ca2+/2Na+. This exchange reaction may have a role in depleting cytoplasmic Ca2+ after depolarization-induced Ca2+ entry through the adrenal medulla plasma membrane; there is some evidence that there may be an additional entry mechanism for Na+ across the granule membrane.

Uptake of Ca2+ by isolated secretory vesicles from adrenal medulla

“…Similar problems have arisen previously when secretory vesicle ghosts were used to investigate Ca2+ transport across the vesicle membrane [16]. Addition of EGTA to this preparation resulted in an instantaneous leakage of Ca2+ from the vesicles, which was only slightly modified by Na + or K +.…”

“…In this context it is interesting to note that the data concerning the effect of Na + on the inhibition of Ca2+ uptake by intact secretory vesicles (see Results) are compatible with an exchange of two Na+ for one Ca2+ across the vesicular membrane. It has been reported that Ca2+ -Ca2+ exchange across the vesicular membrane is inhibited 'by Mg 2+ and Ruthenium red [16]. Since we did not find an effect of both substances on Ca2+ uptake, Ca2+ -Ca2+ exchange does not contribute significently to the uptake of Ca2+ by intact secretory vesicles which results in a net increase of intravesicular Ca2+ [I].…”

“…Addition of EGTA to this preparation resulted in an instantaneous leakage of Ca2+ from the vesicles, which was only slightly modified by Na + or K +. Despite it has been described that roughly two Na + may enter the ghosts during efflux of one Ca2+ [16]. In this context it is interesting to note that the data concerning the effect of Na + on the inhibition of Ca2+ uptake by intact secretory vesicles (see Results) are compatible with an exchange of two Na+ for one Ca2+ across the vesicular membrane.…”

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

“…The presence of a Ca2+-dependent Ca2+ efflux system was first observed with chromaffin secretory vesicle ghosts (Phillips, 1981).…”

“…The data are in accordance with an exchange system of 1 Ca2+ to 2 Na+ (KriegerBrauer and Gratzl, 1982). Investigations with chromaffin secretory vesicle ghosts provided evidence that in addition to the Na+/Ca2+ exchange, a Ca2+/Ca2+ exchange system exists within its membrane (Phillips, 1981). Since Na+-dependent Ca2+ uptake could also be demonstrated in the membrane of secretory vesicles from neurohypophysis (Saermark et al, 1982), such transport systems seem to be of general importance for the Ca2+ metabolism of secretory cells.…”