CVIC Lab.CardioVascular-Ion Channel Laboratory

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 μM, 20 s), RSMGA showed strong delayed hyperpolarization (V_h,ACh; -95 ± 1.8 mV) that was abolished by ouabain. In the HCO₃^–-free condition, the V_h,ACh was also blocked by bumetanide, a blocker of Na⁺-K⁺-2Cl^– cotransporter (NKCC). In the presence of HCO₃^– (24 meq, bubbled with 5% CO ₂), however, the V_h,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 HCO ₃^–. ACh induced a prominent acidification of pH _i in the presence of HCO₃^–, and the acidification was further increased by EIPA treatment. Without HCO ₃^–, an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH_i during the application of NH₄⁺ (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO ₃^–. 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,ACh. The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO₃^–.