pH-regulatory mechanisms in in vitro perfused rectal gland tubules of Squalus acanthias

Abstract

Isolated in vitro perfused rectal gland tubules (RGT) were preincubated with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and pH-regulatory mechanisms were studied. A reduction of bath Cl- concentration from 269 to 6 mmol/l increased the fluorescence ratio 488/436 [corresponding to cytosolic pH (pHi)] slightly but significantly (n=10). Depolarization by Ba2+ (1 mmol/l) or a bath solution containing 30 mmol/l K+ (n=4-6) increased the fluorescence ratio (pHi). These data suggest that HCO3- uptake and/or H+ extrusion is dependent on Cl- and/or voltage. A reduction of bath Na+ from 278 to 5 mmol/l reduced the ratio significantly (n=3). Addition of trimethylamine (Trima+, 20 mmol/l) alkalinized cytosolic pH (n=7). Similarly, addition of NH4+ (20 mmol/l) led to an initial alkalinization and a strong acidification when NH4+ was removed (n=59). The initial pHi-recovery rates after NH4+ removal were quantified and the responsible H+ extrusion and/or HCO3- import systems were examined. The recovery was almost completely abolished when the extracellular Na+ concentration was reduced to 5 mmol/l. In the presence of normal Na+, recovery was slower in the absence as compared to the presence of HCO3- (n=5). It was inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (0.5 mmol/l, n=11) in the presence of HCO3- and in the absence of HCO3- by the Na+/H+-exchange blocker HOE694 (0.5 mmol/l, n=6). These data suggest that acid extrusion probably occurs by basolateral Na+-2HCO3-/Cl- exchange in the presence of HCO3- and by basolateral Na+/H+ exchange in the absence of HCO3-. Luminal perfusion with a solution containing a low Cl- concentration (6 mmol/l) increased the fluorescence ratio (pHi) (n=5). The ratio (pHi) was further increased and pH recovery further delayed by basolateral addition of Trima+ (20 mmol/l, n=3). These data suggest that the HCO3-/Cl- exchanger is present in the luminal membrane. Luminal HCO3-/Cl- exchange and basolateral Na+-2HCO3-/Cl- exchange may work in tandem to secrete HCO3- and exchange it for luminal Cl-.