Immunolocalization of anion exchanger AE2, Na+/H+ exchangers NHE1 and NHE4, and vacuolar type H+-ATPase in rat pancreas

Abstract

We have studied the expression and localization of several H+ and HCO3- transporters, whose presence in the rat pancreas is still unclear. The Cl-/HCO3- exchanger AE2, the Na+/H+ exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H+-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at ≅160 kD for AE2, at ≅90 kD and ≅103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl- uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO3- secretion. The molecular identity of the ductal Cl-/HCO3- exchanger remains unclear.