EFFECT OF SECRETIN ON INTRACELLULAR PH REGULATION IN ISOLATED RAT BILE-DUCT EPITHELIAL-CELLS

The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pH(i)) and HCO3- excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pH(i) was measured with 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECFAM) using a microfluorimetric method. Basal pH(i) of BDE was 7.04+/-0.06 in Hepes and 7.16+/-0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not affected by this hormone. After acute Cl- removal, pH(i) rose 0.24+/-0.08 pHU at a maximal rate of 0.125+/-0.06 pHU/min (H+ flux rates = 6.02+/-3.27 mM/min) and recovered after Cl- readmission (0.188+/-0.08 pHU/min; H+ flux rates = 11.82+/-5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl- removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3- influx. Secretin significantly increased both the maximal rate of alkalinization after Cl- removal (P < 0.012) and of pH(i) recovery after Cl- readmission (P < 0.025), indicating stimulation of Cl-/HCO3- exchange activity. These findings were reproduced with N6,2'-O-Dibutyryladenosine-3'-5'-cyclicmonophosphate (DBcAMP). The Cl- channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 muM) significantly decreased the effects of secretin and DBcAMP on the pH(i) changes promoted by acute Cl- removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3- exchanger in BDE cells, probably by activating Cl- channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na+/HCO3- symport. The cell depolarization induced by Cl- channel activation should enhance HCO3- entrance through electrogenic Na+/HCO3- symport, which in turn stimulates the Cl-/HCO3- exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile.