The purpose of this study was to define the mechanism whereby agonists that increase free cytosolic calcium (Cai2+) affect intracellular pH (pHi) 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 pHi or Cai2+, respectively, in a HCO3-/CO2- containing medium. Recovery from rapid increases in Cai2+ produced by 1 microM angiotensin (Ang) II (delta Cai2+ -229 +/- 43 nM) or 1 microM ionomycin (delta Cai2+ -148 +/- 19 nM) was accompanied by a fall in pHi (delta pHi, -0.064 +/- 0.0085 P < 0.01, and -0.05 +/- 0.012 pH units, P < 0.01, respectively). Neither the fall in pHi nor the rise in Cai2+ elicited by Ang II was prevented by pretreatment with agents which block the action of this agonist on pHi via the stimulation of the Cl/HCo3 exchangers (DIDS, 50 microM) or the Na+/H+ antiporter (EIPA, 50 microM). In the presence of DIDS and EIPA, Ang II produced a fall in pHi (delta pHi, -0.050 +/- 0.014, P < 0.01) and a rise in Cai2+ (delta Ca2+ 252 +/- 157 nM, P < 0.01). That the change in pHi was secondary to changes in Cai2+ was inferred from the finding that, when the rise in Cai2+ elicited by Ang II was prevented by preincubation with a Ca2+ buffer, BAPTA (60 microM), the fall in pHi was abolished as well (delta pHi, 0.0014 +/- 0.0046). The pHi 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 Ca(2+)-ATPase activity (delta pHi -0.002 +/- 0.0006 and -0.0016 pH units, respectively). Cadmium also blunted Cai2+ recovery after Ang II and ionomycin. These findings suggest that the fall in pHi produced by these agents is due to H+ entry coupled to Ca2+ extrusion via the plasma membrane Ca(2+)-ATPase. Our results indicate that agonists that increase Cai2+ cause intracellular acidification as a result of Ca2+/H+ exchange across the plasma membrane. This process appears to be mediated by a plasma membrane Ca(2+)-ATPase which, in the process of extruding Ca2+ from the cell, brings in [H+] and thus acidifies the cell.
J T Daugirdas, J Arrieta, M Ye, G Flores, D C Battle