MECHANISMS OF CA-2+ TRANSPORT IN PLASMA-MEMBRANE VESICLES PREPARED FROM CULTURED PITUITARY-CELLS .1. CHARACTERIZATION OF NA+/CA-2+ EXCHANGE ACTIVITY

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  • 1 January 1984
    • journal article
    • research article
    • Vol. 259  (15) , 9395-9403
Abstract
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+/Ca+ exchange indicates saturation kinetics for both substrates with apparent values of Km for Na+ and Ca2+ of 17 mM and 5 .mu.M, respectively, and a Vmax of about 8 nmol/min per mg of protein for Ca2+ uptake at 25.degree. C. In addition to Na+/Ca2+ exchange, the transporter functions in a Ca2+/Ca2+ exchange mode with an apparent Km of 20 .mu.M and Vmax of 16 nmol/min per 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/Ca2+) as demonstrated by stimulated uptake of lipophilic cations during exchange and by effects of artifically 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. Na+/Ca2+ exchange probably functions in Ca2+ homeostasis in pituitary cells.

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