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2؉ , independently of CaM, inhibited isoproterenol-stimulated AC. Data suggest that agonist augmentation of stimulated cAMP levels is due to activation of AC8 in mouse parotid acini, and strongly support a role for AC5/6 in the inhibition of stimulated cAMP levels.To date, 10 different ACs, 1 each with distinct regulatory properties, have been cloned; their existence suggests that they may be differentially regulated. The enzymes exhibit type specific stimulatory and inhibitory regulation by G-protein ␣ and ␤␥ subunits, Ca 2ϩ , CaM, forskolin, P-site inhibitors, protein kinases A and C (PKC) (2-6), and calcineurin (7) entry plays an important role in promoting AC synthesis. These data, combined with findings that AC8 is expressed in mouse parotid acini (1) and that Ca 2ϩ /CaM stimulates AC and augments the effects of forskolin on cyclase activity in membrane fractions and intact cells (21,22), are consistent with results obtained in HEK 293 cells expressing AC8 (8, 11).Interpretation of the mechanism(s) involved in the cross-talk that occurs between the Ca 2ϩ and cAMP signaling pathways in cells is complex and requires not only identification of AC subtypes expressed, but also tools that provide definitive answers as to regulation of AC synthesis in specific cell types. Thus, the goal of the present study was to determine the involvement of AC8 in agonist-induced augmentation of stimulated cAMP levels in mouse parotid acini by examining the effects of carbachol and the microsomal Ca 2ϩ -ATPase inhibitor, thapsigargin, on isoproterenol-induced cAMP accumulation in acini from AC8-KO mice. Our data show that carbachol and thapsigargin augmented stimulated cAMP accumulation in acini from wild type (WT) mice as previously reported (1), whereas these agents not only prevented augmentation, but inhibited isoproterenol-induced cAMP accumulation in AC8-KO mice. Augmentation of stimulated cAMP accumulation, however, was not affected in acini from AC1-KO mice. Agonist-induced inhibition of stimulated cAMP accumulation was reversed in a nominally Ca 2ϩ -free buffer and in the presence of lanthanum (La 3ϩ ), but not by KN-62, an inhibitor of CaM kinase, or by the CaM antagonist, calmidazolium. Studies with isolated parotid membranes revealed that Ca 2ϩ , independently of CaM, inhibits AC activity in a concentration-dependent manner, consistent with the expression of the Ca 2ϩ -inhibited AC5/6 isoforms in parotid gland. Results demonstrate that capacitative Ca 2ϩ entry is associated with the activation of AC8 in mouse parotid acini and support an involvement of AC5/6 in the inhibition of cAMP synthesis.

Muscarinic receptor interaction leading to augmentation of isoproterenol-stimulated cAMP accumulation in mouse parotid acini involves Ca2+ (28). The effectiveness of capacitative Ca2+entry and intracellular Ca2+release on this response was determined in time course studies by using three independent tools to manipulate the free intracellular Ca2+ concentration: the muscarinic agonist carbachol, thapsigargin, and ionomycin. Time course studies revealed that Ca2+ release from intracellular stores by carbachol produced an early rapid increase (0.25–0.5 min) in stimulated cAMP levels, whereas capacitative Ca2+ entry resulted in a sustained increase in stimulated cAMP levels that was blocked by La3+. Capacitative Ca2+ entry, alone, was involved in thapsigargin and ionomycin augmentation of stimulated cAMP accumulation. The inability of phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine and milrinone, to prevent agonist augmentation of cAMP levels, as well as the finding that the type VIII adenylyl cyclase (ACVIII) is expressed in parotid acini, suggests that capacitative Ca2+ entry augments stimulated cAMP accumulation, at least in part, via activation of this adenylyl cyclase isoenzyme.

In this study of nervous control of exocrine secretion, electrical field stimulation (FS) evoked a marked, tetrodotoxin-sensitive increase in the amylase output from in vitro segments of rat pancreas. Blockade of the large cholinergic component of the response by atropine revealed a smaller noncholinergic nerve-mediated secretion. This noncholinergic secretion was unaffected by phentolamine but abolished by propranolol, as were the secretory responses to norepinephrine and other beta-adrenergic agonists. FS also produced an increase in the efflux of radiolabeled norepinephrine from preloaded tissue that was tetrodotoxin sensitive and calcium dependent. Although FS and the adrenergic secretagogues had no effect on 45Ca2+ metabolism or acinar cell electrical properties of atropine-treated rat pancreas, they both evoked increases in tissue cAMP levels. These increases in cAMP concentration were also blocked by propranolol. The phosphodiesterase inhibitor isobutylmethylxanthine potentiated both the elevation of cAMP levels and the amylase secretion evoked by adrenergic stimulation. Since both specific beta 1- and beta 2-adrenergic agonists elevated cAMP levels and caused amylase secretion, it appears that both beta-receptor subtypes are present in the rat pancreas. Of the selective beta 1- and beta 2-antagonists used, the most pronounced reduction, but not complete blockade, of the FS- and norepinephrine-induced cyclic nucleotide and secretory effects was obtained with the beta 1-antagonist metoprolol. It is concluded that stimulation of adrenergic nerves in the rat pancreas evokes an amylase secretion that is mediated via the activation of mainly beta 1-type adrenergic receptors and the utilization of cAMP as an intracellular second messenger.

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