H⁺-ATPase-mediated cytoplasmic pH-responses associated with elevation of cytoplasmic calcium in cultured rabbit nonpigmented ciliary epithelium

Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H⁺-ATPase activity in cultured rabbit nonpigmented ciliary epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 μM), by exposure to a mixture of epinephrine (1 μM) + acetylcholine (10 μM) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 nm bafilomycin A₁, a specific H⁺ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H⁺-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H⁺-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO₂. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H⁺-ATPase-mediated protein export under acid conditions.