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

The effects of secretin on ion transport mechanisms involved in regula tion of intracellular pH (pH(i)) and HCO3- excretion were characterize d in bile duct epithelial (BDE) cells isolated from normal rat liver. pH(i) was measured with xyethyl)-5(6)-carboxy-fluorescein-acetomethyle ster (BCECFAM) using a microfluorimetric method. Basal pH(i) of BDE wa s 7.04+/-0.06 in Hepes and 7.16+/-0.10 in KRB and was unaffected by se cretin (50-200 nM). Recovery rates from an acid load in Hepes or in KR B media (with and without amiloride) were also not altered by secretin , indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not a ffected 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 induce s 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 , 2'-O-Dibutyryladenosine-3'-5'-cyclicmonophosphate (DBcAMP). The Cl- ch annel 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 e stablish that secretin stimulates the activity of the Cl-/HCO3- exchan ger in BDE cells, probably by activating Cl- channels via the intracel lular messenger cAMP. This in turn depolarizes the cell, stimulating e lectrogenic 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. Th ese mechanisms could account for secretin stimulated bicarbonate secre tion in bile.