Cyclic AMP Levels, Adenylyl Cyclase Activity, and Their Stimulation by Serotonin Quantif ied in Intact Neurons

In molluscan central neurons that express cAMP-gated Na+ current (INa,cAMP), estimates of the cAMP binding affinity of the channels have suggested that effective native intracellular cAMP concentrations should be much higher than characteristic of most cells. Using neurons of the marine opisthobranch snail Pleurobranchaea californica, we applied theory and conventional voltage clamp techniques to use INa,cAMP to report basal levels of endogenous cAMP and adenylyl cyclase, and their stimulation by serotonin. Measurements were calibrated to iontophoretic cAMP injection currents to enable expression of the data in molar terms. In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity. Serotonin stimulation of adenylyl cyclase and [cAMP] was inversely proportional to cells' resting adenylyl cyclase activity. Average cAMP concentration at the membrane rose from 3.6 to 27.6 μM, levels consistent with the expected cAMP dissociation constants of the INa,cAMP channels. These measures confirm the functional character of INa,cAMP in the context of high levels of native cAMP. Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.

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Supramaximal CCK and CCh concentrations abolish VIP potentiation by inhibiting adenylyl cyclase activity

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.5.g1202 ◽

1998 ◽

Vol 275 (5) ◽

pp. G1202-G1208 ◽

Cited By ~ 3

Author(s):

Toshiharu Akiyama ◽

Yoshihide Hirohata ◽

Yoshinori Okabayashi ◽

Issei Imoto ◽

Makoto Otsuki

Keyword(s):

Adenylyl Cyclase ◽

Cyclase Activity ◽

Intracellular Camp ◽

Amylase Secretion ◽

Adenylyl Cyclase Activity ◽

Amylase Release ◽

Pancreatic Acini ◽

Camp Pathway ◽

Kinase C ◽

Intracellular Camp Accumulation

Exocrine pancreatic secretion stimulated by vasoactive intestinal polypeptide (VIP), which acts through the adenylyl cyclase-cAMP pathway, is potentiated by stimulation with other secretagogues such as CCK and carbachol (CCh). However, the potentiating effect is abolished by the same secretagogues at supramaximal concentrations. In the present study, we examined the mechanisms by which supramaximal concentrations of CCK octapeptide (CCK-8) or CCh reduce the VIP-induced potentiation of amylase secretion from isolated rat pancreatic acini. VIP-stimulated amylase secretion was potentiated by submaximal stimulatory concentrations of CCK-8 and CCh but was reduced by the same reagents at higher concentrations. Supramaximal concentrations of CCK-8 or CCh also reduced forskolin-induced potentiation of amylase release but did not reduce that induced by 8-bromo-cAMP. Moreover, supramaximal concentrations of CCK-8 or CCh inhibited VIP-stimulated intracellular cAMP production as well as adenylyl cyclase activity. 12- O-tetradecanoylphorbol 13-acetate (TPA) also reduced the magnitude of the potentiation of amylase release caused by VIP plus CCK-8 or CCh, although TPA itself decreased neither VIP-stimulated adenylyl cyclase activity nor intracellular cAMP accumulation. These results indicate that supramaximal concentrations of CCK-8 and CCh reduce the potentiating effect of VIP and forskolin on amylase secretion by inhibiting the adenylyl cyclase activity. In addition, protein kinase C is suggested to be partly implicated in this inhibitory mechanism. The mechanisms that lead to such inhibition may be interlinked but distinct from each other.

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