The type 8 adenylyl cyclase is critical for Ca2+ stimulation of cAMP accumulation in mouse parotid acini

Capacitative Ca2+ entry stimulates cAMP synthesis in mouse parotid acini, s uggesting that one of the Ca2+-sensitive adenylyl cyclases (AC1 or AC8) may play an important role in the regulation of parotid function (Watson, E. L ., Wu, Z., Jacobson, K. L., Storm, D. R., Singh, J. C., and Ott, S. M. (199 8) Am. J. Physiol. 274, C557-C565). To evaluate the role of AC1 and AC8 in Ca2+ stimulation of cAMP synthesis in parotid cells, acini were isolated fr om AC1 mutant (AC1-KO) and AC8 mutant (AC8-KO) mice and analyzed for Ca2+ s timulation of intracellular cAMP levels. Although Ca2+ stimulation of intra cellular cAMP levels in acini from AC1-KO mice was indistinguishable from w ild type mice, acini from AC8-KO mice showed no Ca2+-stimulated cAMP accumu lation. This indicates that AC8, but not AC1, plays a major role in couplin g Ca2+ signals to cAMP synthesis in parotid acini. Interestingly, treatment of acini from AC8-KO mice with agents, i.e. carbachol and thapsigargin tha t increase intracellular Ca2+, lowered cAMP levels. This decrease was depen dent upon Ca2+ influx and independent of phosphodiesterase activation. Immu noblot analysis revealed that AC5/6 and AC3 are expressed in parotid glands . Inhibition of calmodulin (CaM) kinase II with KN-62, or inclusion of the CaM inhibitor, calmidazolium, did not prevent agonist-induced inhibition of stimulated cAMP accumulation. In vitro studies revealed that Ca2+, indepen dently of CaM, inhibited isoproterenol-stimulated AC. Data suggest that ago nist 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 inhibitio n of stimulated cAMP levels.