ETA RECEPTOR-MEDIATED INHIBITION OF INTRACELLULAR PH REGULATION IN CULTURED BOVINE CORNEAL EPITHELIAL-CELLS

The contributions were determined in primary cultures of bovine cornea l epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPas e (i.e. V-type) activity to the regulation of intracellular pH (pH(i)) . Furthermore, we characterized the effects on pH, regulation of expos ure to 1 mu M ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2',7' Bis (carboxyethyl)-5, 6-carboxynuorescein acet oxymethyl ester (BCECF-AM), the control pH(i) was 7.1 in NaCl (nominal ly HCO3-free) Ringers. Inhibition of NHE with 100 mu M dimethylamilori de (DMA) rapidly decreased pH(i) by 0.37 units. Similarly, selective i nhibition of V-type H+-ATPase with 10 mu M bafilomycin A(1) decreased pH(i) by 0.22 units. Following acid loading in NaCl Ringers with a 20 mM NH4Cl prepulse, pH(i) recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 mu M DMA or 20 mu M bafilomycin A(1). Based on decreases in H+ efflux resulting from selective inhibi tion of NHE and V-type H+ pump activity, NHE activity accounts for 76% of the pH(i) recovery following acid loading. Under control condition s, ET-1 (1 mu M) had no effect on pH(i) whereas ET-1 completely suppre ssed pH(i) recovery following acid loading in NaCl or NMGCl Ringers. T his inhibitory effect was largely due to stimulation of ETA because in the presence of BQ-123 (10 mu M), a selective ETA receptor antagonist , pH(i) recovery was completely restored, Suppression of pH(i) recover y also occurred following stimulation of protein kinase C (PKC) with 1 0(-7) M phorbol myristate (PMA) whereas 10-7 M 4 alpha phorbol 12,13 d idecanoate (PDD) had no effect. ET-1 failed to suppress pH(i) recovery after inhibition of PKC with 0.5 mu M calphostin C suggesting that th e inhibition of pH(i) recovery by ET-1 is a consequence of PKC stimula tion. Similarly, inhibition of Ca2+-dependent calmodulin stimulated Ca M II kinase with KN-62 (10 mu M) reversed the suppression of pH(i) rec overy by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 mu M phenylarsine oxide, 10 nM okadaic acid, 10 mu M cyclosporin A(1) or 20 mu M BAPTA, also obviated the su ppression of pH(i) recovery by ET-1. Therefore ETA receptor mediated i nhibition of pH(i) regulation following acid loading could be a conseq uence of either PKC or CaMII kinase stimulation. Each one of these kin ases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V -type H+ pump. This alteration may prevent them from becoming adequate ly stimulated to elicit pH(i) recovery in response to acid loading. (C ) 1998 Academic Press.