Accumulation of intracellular HCO3- by Na(+)-HCO3- cotransport in interlobular ducts from guinea-pig pancreas.

摘要

1. Short segments of interlobular duct were microdissected from guinea-pig pancreas following enzymatic digestion. After overnight culture, intracellular pH (pH1) and Na+ concentration ([Na+]i) were measured by microfluorometry in duct cells loaded with either the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or the sodium-binding benzofuran isophthalate (SBFI). 2. The transporters responsible for maintaining pHi above equilibrium were investigated by using the NH4Cl pulse technique to acid load the cells. In the absence of HCO3-/CO2, the recovery of pH1 was Na+ dependent, abolished by 0.2 mM amiloride and by 10 microM N-methyl-N-isobutylamiloride and was therefore attributed to Na(+)-H+ exchange. 3. In the presence of HCO3-/CO2, amiloride only partially inhibited the recovery from acid loading. The amiloride-insensitive component was abolished by 0.5 mM H2DIDS and unaffected by depletion of intracellular Cl- and was therefore attributed to Na(+)-HCO3- cotransport. 4. Stimulation with 10 nM secretin did not cause a significant change in pH1 despite a significant increase in HCO3- efflux. However, in the presence of secretin, addition of 0.5 mM H2DIDS caused a decline in pH1 that was three times more rapid than that obtained with 0.2 mM amiloride. 5. In secretin-stimulated ducts, Na+ uptake increased when HCO3-/CO2 was added to the bath and this increase was strongly inhibited by 0.5 mM H2DIDS. 6. We conclude that Na(+)-HCO3- cotransport contributes approximately 75% of the HCO3- taken up by guinea-pig pancreatic duct cells during stimulation with secretin. It is proposed that electrical coupling between HCO3- efflux at the luminal membrane and electrogenic Na(+)-HCO3- cotransport at the basolateral membrane explains why secretin causes little change in pH1.