pH-regulatory mechanisms in in vitro perfused rectal gland tubules of Squalus acanthias.
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-.
Bleich
M
M
Physiologisches Institut, Albert-Ludwigs-Universität, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Warth
R
R
Thiele
I
I
Greger
R
R
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000008:administration & dosage / Q000378:metabolism; D002712:Chlorides / Q000008:administration & dosage / Q000378:metabolism; D004284:Dogfish; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008744:Methylamines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012491:Salt Gland / Q000378:metabolism*; D012964:Sodium / Q000008:administration & dosage / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000502:physiology; D013450:Sulfones / Q000494:pharmacology
D001639:Bicarbonates; D002712:Chlorides; D006146:Guanidines; D008744:Methylamines; D000644:Quaternary Ammonium Compounds; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; D012964:Sodium; C023336:trimethylamine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid