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The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pH1) and HCO3 excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pHi was measured with 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECF-AM) using a microfluorimetric method. Basal pHi 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 /HCO-cotransport were not affected by this hormone. After acute Cl-removal, pH1 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 pH1 recovery after Cl-readmission (P < 0.025), indicating stimulation of Cl-/HCO3 exchange activity. These findings were reproducedwithN',2'-O-Dibutyryladenosine-3'-5'-cyclicmonophosphate (DBcAMP). The Cl-channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 AM) significantly decreased the effects of secretin and DBcAMP on the pH1 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 C1-channel activation should enhance HCO3 entrance through electrogenic Na+/HCO-symport, which in turn stimulates the Cl-/ HCO3 exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile. (J. Clin. Invest. 1993. 92:1314-1325.) Key words: bile duct epithelium. intra-

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