Dorso-ventral patterning of the vertebrate limb and dermatoglyphs with special reference to the pad pattern of mice]

The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHi regulation of exposure to 1 microM ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2',7' Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHi was 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 microM dimethylamiloride (DMA) rapidly decreased pHi by 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 microM bafilomycin A1 decreased pHi by 0.22 units. Following acid loading in NaCl Ringers with a 20 mm NH4Cl prepulse, pHi recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 microM DMA or 20 microM bafilomycin A1. Based on decreases in H+ efflux resulting from selective inhibition of NHE and V-type H+ pump activity, NHE activity accounts for 76% of the pHi recovery following acid loading. Under control conditions, ET-1 (1 microM) had no effect on pHi whereas ET-1 completely suppressed pHi recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETA because in the presence of BQ-123 (10 microM), a selective ETA receptor antagonist, pHi recovery was completely restored. Suppression of pHi recovery also occurred following stimulation of protein kinase C (PKC) with 10(-7) m phorbol myristate (PMA) whereas 10(-7) m 4 alpha phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHi recovery after inhibition of PKC with 0.5 microM calphostin C suggesting that the inhibition of pHi recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 microM) reversed the suppression of pHi recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 microM phenylarsine oxide, 10 nm okadaic acid, 10 microM cyclosporin A1 or 20 microM BAPTA, also obviated the suppression of pHi recovery by ET-1. Therefore ETA receptor mediated inhibition of pHi regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases 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 adequately stimulated to elicit pHi recovery in response to acid loading.