Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. I. Characterization of Na+/Ca2+ exchange activity.
GH3 rat anterior pituitary cells possess a Na+/Ca2+ exchange transport mechanism which is present in purified plasma membrane vesicles prepared from these cells. Imposition of an outwardly directed Na+ gradient in vesicles results in a marked concentrative uptake of Ca2+ which is abolished by the Ca2+ ionophore A23187. Transport activity depends on a sustained Na+ gradient. Dissipation of the driving force by treatment with Na+ ionophores or by passive gradient collapse abolished transport activity. The exchange reaction is completely reversible since addition of extravesicular Na+ enhances Ca2+ efflux from Ca2+ loaded vesicles. A kinetic analysis of Na+/Ca2+ exchange indicates saturation kinetics for both substrates with apparent values of Km for Na+ and Ca2+ of 17 mM and 5 microM, respectively, and a Vmax of about 8 nmol/min/mg of protein for Ca2+ uptake at 25 degrees C. In addition to Na+/Ca2+ exchange, the transporter functions in a Ca2+/Ca2+ exchange mode with an apparent Km of 20 microM and Vmax of 16 nmol/min/mg of protein for Ca2+ influx. Na+/Ca2+ exchange is not inhibited by protonophores indicating that Ca2+ flux does not occur via coupled Na+/H+, Ca2+/H+ exchange. Transport is inhibited by derivatives of the pyrazine diuretic amiloride. The pH dependency of Ca2+ uptake displays a sigmoidal relationship with stimulation of activity at alkaline pH and inhibition at acid pH. Furthermore, the reaction is electrogenic (i.e. more than 2 Na+ transported per Ca2+) as demonstrated by stimulated uptake of lipophilic cations during exchange and by effects of artificially imposed membrane potentials on the rate of Ca2+ transport. Plasma membrane vesicles prepared from bovine anterior pituitary glands also display Na+/Ca2+ exchange with many of the same characteristics. These results support the notion that Na+/Ca2+ exchange functions in Ca2+ homeostasis in pituitary cells.
D000584:Amiloride / Q000031:analogs & derivatives; D000818:Animals; D001693:Biological Transport, Active; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D007700:Kinetics; D009861:Onium Compounds / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology*; D051381:Rats; D012964:Sodium / Q000378:metabolism*
D002121:Calcium Channel Blockers; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D000001:Calcimycin; D000584:Amiloride; D012964:Sodium; D002118:Calcium; C013289:tetraphenylphosphonium