THE CA2-EXTRUDING ATPASE OF THE HUMAN PLATELET CREATES AND RESPONDS TO CYTOPLASMIC PH CHANGES, CONSISTENT WITH A 2 CA2+()NH+ EXCHANGE MECHANISM/

The Ca2+-extruding ATPase pump of the human platelet was studied in si tu by measuring Ca2+ extrusion from quin2-overloaded platelets (Johans son, J.S., Haynes, D.H. 1988. J. Membrane Biol. 104:147-163). Cytoplas mic pH (pH(cyt)) was measured by BCECF fluorescence in parallel experi ments. The pump was studied by raising the cytoplasmic free Ca2+ to 2. 5 mum and monitoring active Ca2+ extrusion into a Ca2+-free medium. Th e pump was shown to perturb pH(cyt), to not respond to changes in memb rane potential and to respond to imposed changes in pH(cyt) in a manne r consistent with the Ca2+ pump acting as a 2 Ca2+/nH+ exchanger. (i) Raising the external pH (pH(ext)) from 7.40 to 7.60 lowers the V(max) of the pump in basal condition (V(max,1)) from 110 +/- 18 to 73 +/- 12 mum/min (= mumol/liter cell volume/min). (ii) Lowering pH(ext) to 7.1 3 raised V(max,1) to 150 +/- 15 mum/min. (iii) In an N-methYl-D-glucam ine (NMDG+) medium, the pump operation against high [Ca2+]cyt acidifie s the cytoplasm by -0.36 +/- 0.10 pH units, and the pump becomes self- inhibited. (iv) Use of nigericin to drive pH(cyt) down to 6.23 reduces the V(max,1) to 18 +/- 11 mum/min. (v) Alkalinization of the cytoplas m by monensin in the presence of Na+ raises the V(max,1) (basal state with K(m,1) = 80 nm) to 136 +/- 24 mum/min, but also activates the pum p fourfold (V(max,2) = 280 +/- 28 mum/min; K(m,2) = 502 +/- 36 nm). (v i) Transient elevation of pH(cyt) by NH4Cl at high [Ca2+]cyt activates the pump eightfold (V(max,2) greater-than-or-equal-to 671 +/- 350 muM /min). The large activation by alkaline pH(cyt) at high [Ca2+]cyt can be explained by Ca2+-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.