INTRACELLULAR ACIDIFICATION ASSOCIATED WITH CHANGES IN FREE CYTOSOLIC CALCIUM - EVIDENCE FOR CA2+/H+ EXCHANGE VIA A PLASMA-MEMBRANE CA2+-ATPASE IN VASCULAR SMOOTH-MUSCLE CELLS

The purpose of this study was to define the mechanism whereby agonists that increase free cytosolic calcium (Ca-i(2+)) affect intracellular pH (pH(i)) in smooth muscle, Rat aortic vascular smooth muscle cells grown on coverslips were loaded with BCECF/AM or fura-2/AM for continuous monitoring of pH(i) or Ca-i(2+), respectively, in a HCO3-/CO2-containing medium, Recovery from rapid increases in Ca-i(2+) produced by 1 mu M angiotensin (Ang) II (Delta Ca-i(2+) -229+/-43 nM) or 1 mu M ionomycin (Delta Ca-i(2+) -148+/-19 nM) was accompanied by a fall in pH(i) (Delta pH(i), -0.064+/-0.0085 P < 0.01, and -0.05+/-0.012 pH units, P < 0.01, respectively), Neither the fall in pH(i) nor the rise in Ca-i(2+) elicited by Ang II was prevented by pretreatment with agents which block the action of this agonist on pH(i) via the stimulation of the Cl/HCO3 exchangers (DIDS, 50 mu M) or the Na+/H+ antiporter (EIPA, 50 mu M). In the presence of DIDS and EIPA, Ang II produced a fall in pH(i) (Delta pH(i), -0.050+/-0.014, P < 0.01) and a rise in Ca-i(2+) (Delta Ca2+ 252+/-157 nM, P < 0.01), That the change in pH(i) was secondary to changes in Ca-i(2+) was inferred from the finding that, when the rise in Ca-i(2+) elicited by Ang II was prevented by preincubation with a Ca2+ buffer, BAPTA (60 mu M), the fall in pH(i) was abolished as well (Delta pH(i), 0.0014+/-0.0046). The pH(i) fall produced by Ang II and ionomycin was prevented by cadmium at a very low concentration (20 nM) which is known to inhibit plasma membrane Ca2+-ATPase activity (Delta pH(i) -0.002+/-0.0006 and -0.0016 pH units, respectively), Cadmium also blunted Ca-i(2+) recovery after Ang II and ionomycin, These findings suggest that the fall in pH(i) produced by these agents is due to H+ entry coupled to Ca2+ extrusion via the plasma membrane Ca2+-ATPase, Our results indicate that agonists that increase Ca-i(2+) cause intracellular acidification as a result of Ca2+/H+ exchange across the plasma membrane, This process appears to be mediated by a plasma membrane Ca2+-ATPase which, in the process of extruding Ca2+ from the cell, brings in [H+] and thus acidifies the cell.