cAMP modulation of a Ca2+-dependent K+ conductance in rat submandibular acinar cells

Rat submandibular acinar cells exhibit a tetraethylammonium (TEA)-insensitive, Ca2+-activated K+ conductance, which may play an important role in Ca2+-dependent fluid secretion by this gland (T. Ishikawa, M. Murakami, and Y. Seo. Pfluegers Arch. 428: 516-525, 1994; T. Ishikawa and M. Murakami. Pfluegers Arch. 429: 748-750, 1995). We have now investigated whether this conductance would be modified during the potentiation of Ca2+-activated fluid secretion by an adenosine 3',5'-cyclic monophosphate (cAMP)-mediated mechanism in the rat submandibular gland. In isolated, vascularly perfused rat submandibular gland, we found that the adenylate cyclase activator forskolin (10 and 30 microM) enhanced a transient initial phase of a biphasic fluid secretion induced by the Ca2+ ionophore A-23187 (3 microM). We also unexpectedly found that forskolin reduced a smaller sustained phase of the secretion. The dual effects of forskolin were mimicked by the application of the cocktail of 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and dibutyryl cAMP (DBcAMP) (0.1 and 0.5 mM). Basolateral K+ efflux studies showed that 1) forskolin significantly enhanced a large transient net K+ efflux induced by A-23187 in a dose-dependent manner; 2) the forskolin-induced enhancement was also mimicked by a cocktail of IBMX and DBcAMP; and 3) the A-23187-induced K+ efflux enhanced by these agents was not inhibited by TEA (5 or 10 mM), a concentration known to completely block Ca2+ voltage-dependent large-conductance K+ channels (maxi-K+ channels). With use of microspectrofluorometry with fura 2, we then showed that an increase in cytosolic Ca2+ concentration induced by the nonfluorescent, brominated derivative 4-bromo-A-23187 (3 microM) was not altered by forskolin (30 microM) in freshly isolated rat submandibular acinar cells. With use of whole cell patch-clamp techniques, we demonstrated that a TEA-insensitive K+ conductance induced by A-23187 in cells dialyzed with pipette solutions containing cAMP (0.1 mM) was significantly larger than that in cells treated with control pipette solutions. Taken together, these results indicate that potentiation of the Ca2+-activated fluid secretion by a cAMP-mediated mechanism is accompanied by potentiation of a TEA-insensitive, Ca2+-activated K+ conductance in this gland.