Expression of NHE1 and NHE4 in rat pancreatic zymogen granule membranes.
We previously characterized a Na+/H+ exchange activity in rat pancreatic zymogen granules [Anderie, I., and Thévenod, F. (1996) J. Membrane Biol, 152, 195-205]. Here we have identified the Na+/H+ exchanger (NHE) isoforms present in zymogen granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited zymogen granule Na+/H+ exchange in a concentration dependent manner, maximally to 53 +/- 5% of controls at 100nM. The remaining Na+/H+ exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC50 approximately 25 microM) or benzamil (EC50 approximately 100 microM). Amiloride inhibited weakly suggesting that "amiloride-resistant" and "amiloride-sensitive" NHE are expressed in zymogen granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in zymogen granule membranes was confirmed by immunoblots of zymogen granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic zymogen granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in zymogen granule membranes of rat exocrine pancreas.
Anderie
I
I
II, Department of Physiology, University of the Saarland, Homburg/Saar, Germany.
Blum
R
R
Haase
W
W
Grinstein
S
S
Thévenod
F
F
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics*; D015870:Gene Expression; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism; D008297:Male; D016253:Microscopy, Immunoelectron; D010179:Pancreas / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004792:Enzyme Precursors; C081518:Slc9a4 protein, rat; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; C013407:benzamil; D000584:Amiloride; C039614:ethylisopropylamiloride