Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

Abstract

Intracellular pH (pHi) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4',5'-dimethylfluorescein. Steady-state pH was 7.05 +/- 0.1 in the nominal absence of bicarbonate, and 7.15 +/- 0.1 in the presence of 28 mM HCO3-/5% CO2. Following an acid load imposed by a NH4Cl prepulse, pHi was regulated in the absence of HCO3- by a Na+-dependent process inhibitable to a large extent by 1 mM amiloride and 0.1 mM dimethylamiloride. In the presence of 28 mM HCO3-/5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1 mM) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO3-. The presence of intracellular Cl- did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl-. Removal of extracellular Na+ led to a fall of pHi, which was greatly attenuated in the absence of HCO3-. This acidification was largely reduced by 1 mM DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO3-. The presence of a Cl-/HCO3- exchanger was supported by demonstrating DIDS-sensitive 36Cl- uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na+/H+ antiport, a Na+-HCO3- symport and a Cl-/HCO3- exchange, the latter two being DIDS sensitive.

Mechanisms of endothelial cell swelling from lactacidosis studied in vitro

“…Swelling in these experiments could be prevented by amiloride analogs, as an indication that a Na ϩ /H ϩ exchanger (NHE) participates in the swelling process. NHEs have been widely studied as membrane mechanisms involved in the regulation of intracellular pH (pH i ) and are present in endothelial cells (4,10). Five NHE isoforms (NHE1-NHE5) represent the major acid-extruding transporters in the absence of bicarbonate (HCO 3 Ϫ ).…”

“…In vivo, however, HCO 3 Ϫ is usually available, and HCO 3 Ϫ -dependent systems contribute to pH i regulation; among these, the Na ϩ -dependent Cl Ϫ /HCO 3 Ϫ exchanger (NCBE) has been found activated in acidosis in bovine aortic endothelium (4). In addition, Na ϩ -HCO 3 Ϫ cotransport is activated in corneal endothelium (1,2,10). Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchange (CBE) is known to maintain pH i after alkalinization.…”

“…-dependent transporters so far have not been discussed with respect to endothelial swelling, although their participation in the regulation of endothelial pH i is known (1,2,4,10); at physiological pH e , HCO 3 Ϫ -dependent transport systems contribute more to pH i homeostasis than NHE. In most cell types, the contribution of NHE to pH i homeostasis increases with decreasing pH i (4,24).…”

“…It remains to be determined whether NCBE or Na ϩ -HCO 3 Ϫ cotransport is responsible for HCO 3 Ϫ -dependent swelling, because both are present in endothelium (1,2,4,10). NCBE, however, is less likely to be involved in the swelling response, because uptake of HCO 3 Ϫ by this transporter and the ensuing buffering of protons generates CO 2 , which can readily leave the cell.…”

The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells.

“…If such mechanisms were involved, then the acidosis produced by Na+ removal should be faster and/or larger in the presence of HCO3-(see e.g. Jentsch, Korbmacher, Janicke, Fischer, Stahl, Helbig, Hollwede, Cragoe, Keller & Wiederholt, 1988). In our experiments, however, the degree of acidification produced by Na+ removal was not markedlv different in the presence and the (nominal) absence of HCO3- (Table 1), suggesting only a minor role for such mechanisms in acid extrusion.…”