Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V-m) and intracellular concentrations of Ca2+, Na+ ([Na+](c)), Cl-, and H+ (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 mu M, 20 s), RSMGA showed strong delayed hyperpolarization (V-h,V- ACh; - 95 +/- 1.8 mV) that was abolished by ouabain. In the HCO3--free condition, the V-h,V- ACh was also blocked by bumetanide, a blocker of Na+-K+-2Cl(-) cotransporter (NKCC). In the presence of HCO3- (24 meq, bubbled with 5% CO2), however, the V-h,V-ACh was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride ( EIPA), a Na+/H+ exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na+](c) was totally blocked by bumetanide in the absence of HCO3-, but only by one-half in the presence of HCO3-. ACh induced a prominent acidification of pH(i) in the presence of HCO3-, and the acidification was further increased by EIPA treatment. Without HCO3-, an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pHi during the application of NH4+ ( 20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO3-. In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na+](c). The intracellular Na+-induced activation of electrogenic Na+/ K+-ATPase causes V-h,V- ACh. The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO3-.