Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct

Abstract

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na+-dependent and amiloride- sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl-/HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pH(i) changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na+-independent vacuolar-type H+ pump, and (c) BLM-located, Na+-dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene- 2,2'-disulfonic acid sensitive mechanism, possibly a Na+-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na+-dependent HCO3- transporter. An Na+- independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-. Exposing all segments of the duct to CO2/HCO3- significantly increased the appearance of H+ pumping in the plasma membrane. This effect completely reversed on removal of HCO3- from the medium. Stimulation of the ducts with various agonists in the absence of HCO3- also induced H+ pumping in the plasma membrane. All forms of H+ pump activation were blocked by loading the duct cells with the Ca2+ buffer 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Direct measurement of [Ca2+](i) showed that HCO3- and all agonists reported to affect pancreatic fluid secretion increased [Ca2+](i) in microdissected intralobular ducts and the perfused common ducts. The overall studies provide the first detailed characterization and localization of H+ and HCO3- transporters in the pancreatic duct; report the presence of several new, unsuspected transporters in the LM and BLM; and should allow better understanding of fluid and electrolyte secretion by the pancreatic duct.