EVIDENCE FOR INVOLVEMENT OF A ZYMOGEN GRANULE NA+ H+ EXCHANGER IN ENZYME-SECRETION FROM RAT PANCREATIC ACINAR-CELLS/

We have characterized a Na+/H+ exchanger in the membrane of isolated z ymogen granules (ZG) from rat exocrine pancreas and investigated its r ole in secretagogue-induced enzyme secretion. ZG Na+/H+ exchanger acti vity was estimated by measuring Naf or Li+ influx and consequent osmot ic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternativ ely, intra-granule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na +- or Li+-dependent ZG lysis was enhanced by increasing inward to outw ard directed H+ gradients. Na+-dependent ZG lysis was not prevented by an inside-positive K+ diffusion potential generated by valinomycin wh ich argues against parallel operation of separate electrogenic Na+ and H+ permeabilities and for coupled Na+/H+ exchange through an electron eutral carrier. Na+- and Li+-dependent ZG lysis was inhibited by EIPA (EC(50) similar to 25 mu M) and benzamil (EC(50) similar to 100 mu M), but only weakly by amiloride. Similarly, absorbance changes due to re lease of acridine orange from acidic granules into the medium were obt ained with Na+ and Li+ salts only, and were inhibited by EIPA, suggest ing the presence of a Na+/H+ exchanger in the membrane. Na+ dependent lysis of ZG was inhibited by 0.5 mM MgATP and MgATP-gamma-S by about 6 0% and 35%, respectively. Inhibition by MgATP was prevented by incubat ion of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin a ntagonists calmidazolium (0.75 mu M), trifluoperazine (100 mu M) and W -7 (500 mu M), suggesting that the ZG Na+/H+ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na+ dependen ce of secretagogue (CCK-OP)-stimulated amylase secretion was investiga ted in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na+ containing buffer (30 mM NaCl/105 mM KCl buffer ; 6.4 +/- 0.4% of total amylase above basal) compared to buffer withou t Na+ (0 mM NaCl/135 mM KCl buffer; 4.7 +/- 0.4% of total amylase abov e basal, P < 0.03). EIPA (50 mu M) reduced CCK-OP-induced amylase secr etion in Na+ containing buffer from 7.5 +/- 0.6% to 4.1 +/- 0.8% (P < 0.02). In the absence of Na+ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 mu M EIPA. The data suggest that an am iloride insensitive, EIPA inhibitable Na+/H+ exchanger is present in Z G membranes, which is stimulated by calmodulin antagonists and could b e involved in secretagogue-induced enzyme secretion from rat pancreati c acini.