Effects of Bafilomycin A1 on Cytosolic pH of sheep Alveolar and Peritoneal Macrophages: Evaluation of the pH-Regulatory Role of Plasma Membrane V-ATPases

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (mϕ) from sheep. Cytosolic pH was measured using 2′7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir <6.8) by approximately 35 %. Recovery of pHi was abolished by co-treatment of mϕ with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal mϕ from sheep. Sheep mϕ also appear to possess a Na⁺/H⁺ exchanger. However, Na⁺/H⁺ exchange either is inactive or can be effectively masked by V-ATPase-mediated H⁺ extrusion at physiological pHi values.