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PubMed1990

Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.

A genomic DNA fragment from Saccharomyces cerevisiae which contains the SRA5 (=PDE2) gene, coding for a low Km cAMP-phosphodiesterase, was transfected into Chinese hamster ovary cells. Clones carring the cAMP-phosphodiesterase gene were capable of growth in the presence of cholera toxin, which slows the growth of untransfected cells by elevating their cAMP levels. The cholera toxin-resistant transfected cell lines expressed high levels of cAMP-phosphodiesterase mRNA and cAMP-phosphodiesterase activity. Basal intracellular cAMP levels were not significantly affected by the presence of the yeast cAMP-phosphodiesterase gene, but elevation of cAMP levels in response to cholera toxin or prostaglandin E1 was suppressed. Induction of the cAMP-responsive tyrosine aminotransferase promoter by cholera toxin was also blocked in cell lines carrying the yeast cAMP-phosphodiesterase gene. Cholera toxin-resistant transfected cell lines were sensitive to the growth inhibitory effects of N6,02'-dibutyryladenosine 3',5'-monophosphate, which can be used to bypass the effects of the yeast cAMP-phosphodiesterase.

1-Methyl-3-isobutylxanthine↗
PubMed1975

Two novel stimuli of cyclic adenosine 3',5'-monophosphate (cAMP) in human lymphocytes.

Polystyrene latex particles (PLP) and zymosan particles (ZP), two commonly employed phagocytic stimuli, were noted to bind to purified human peripheral blood lymphocytes. This interaction was not accompained by ingestion but did lead to a marked increase in intracellular cyclic AMP. The cAMP response to PLP was proportional to the particle cell ratio which, in turn, correlated with the number of membrane-associated particles. After the addition of PLP to lymphocytes, the cAMP response occurred within 2 min, peaked between 4 and 15 min, and returned to baseline by 30 to 60 min. The cAMP response to ZP was similar in onset and duration to that seen with PLP but was less marked (2- to 4-fold vs 25- to 50-fold) and more variable in magnitude. This is probably a reflection of the smaller number of cells interacting with ZP. At high PLP to cell ratios almost all of the lymphocytes bound PLP but only 10 to 28% of the mixed lymphocyte population bound ZP. Two lines of evidence established conclusively that the cAMP response was taking place in the lymphocytes themselves rather than in contaminating cells. 1) When lymphocytes were purified additionally by filtration through a nylon wool column (99 to 100% lymphocytes), they were found to undergo a similar cAMP response to PLP. Since the nylon filtration procedure also removes almost all of the B cells, this further indicates that T cells are capable of undergoing the response. 2) Immunofluorescence studies with anti-cAMP antibody revealed an increase in intralymphocytic cAMP which was primarily adjacent to the site of PLP or ZP attachment. The likely explanation of this data is that PLP and ZP perturb the lymphocyte surface leading to regional activation of membrane-bound adenylate cyclase and subsequent cAMP accumulation. Although the physiologic significance of these observations remains to be determined, the results: 1) provide histologic confirmation for the concept of cAMP compartmentablization, 2) clarify conflicting results regarding the localization of cAMP accumulation during the phagocytosis of PLP by mixed leukocyte populations, and 3) suggest that this experimental system may allow an analysis of the mechanism by which perturbations of the lymphocyte surface modulate cAMP.

Binding Sites↗
PubMed1990

Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP.

A truncated regulatory subunit of cAMP-dependent protein kinase I was constructed which contained deletions at both the carboxyl terminus and at the amino terminus. The entire carboxyl-terminal cAMP-binding domain was deleted as well as the first 92 residues up to the hinge region. This monomeric truncated protein still forms a complex with the catalytic subunit, and activation of this complex is mediated by cAMP. The affinity of this mutant holoenzyme for cAMP and its activation by cAMP are nearly identical to holoenzyme formed with a regulatory subunit having only the carboxyl-terminal deletion and very similar to native holoenzyme. The off rate for cAMP from both mutant regulatory subunits, however, is monophasic and very fast relative to the biphasic off rate seen for the native regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are also very similar for the mutant and native holoenzymes. Like the native type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity. The positive cooperativity seen for MgATP binding to the native holoenzyme, however, is abolished in the double deletion mutant. The Hill coefficient for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of magnitude for both mutant forms of the holoenzyme in the presence of MgATP. A similar shift is seen for the native holoenzyme. Further characterization of the MgATP-binding properties of the wild-type holoenzyme indicates that a binary complex containing catalytic subunit and MgATP is required, in particular, for reassociation with the cAMP-bound regulatory subunit. This binary complex is required for rapid dissociation of the bound cAMP and is probably responsible for the observed reduction in cAMP-binding affinity for the type I holoenzyme in the presence of MgATP.

Adenosine Triphosphate↗
PubMed1990

Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase.

Mouse L929 cells were used to study the mechanism of cAMP induction of alkaline phosphatase (AP) activity. Following treatment with 200 microM 8-chlorophenylthio-cAMP (CPT-cAMP), alkaline phosphatase enzyme activity was observed to increase 80-fold after 24 h. The CPT-cAMP dose response of the alkaline phosphatase enzyme activity correlated well with the CPT-cAMP activation of cAMP-dependent protein kinase in L cells. A cDNA clone for the alkaline phosphatase was isolated and used to demonstrate a 10-fold increase in alkaline phosphatase mRNA levels after a 24-h treatment of L cells with CPT-cAMP. Increased mRNA levels were first detected 4-6 h, after CPT-cAMP treatment, and the level of alkaline phosphatase mRNA decreased rapidly after removal of CPT-cAMP. In vitro nuclear transcription studies showed that a 3-fold increase in alkaline phosphatase gene transcription was detectable 6 h after CPT treatment, and this increase was blocked by cycloheximide. In order to determine if the catalytic (C) subunit of cAMP-dependent protein kinase was able to mediate the induction of AP, L cells were transfected with expression vectors containing the metallothionein promoter and coding for the C alpha isoform of the catalytic subunit of cAMP-dependent protein kinase or for a catalytic subunit in which lysine 72 had been mutated to methionine (C alpha K72M). Zinc treatment of stably transfected cells expressing the wild-type C subunit showed an increase in protein kinase activity and an increase in AP activity. Zinc treatment of cells containing the mutant C subunit expression vector produced an increase in the amount of a protein which was recognized by C subunit antibodies on Western blots, but these cells showed no increase in protein kinase activity or in AP activity. We conclude that the C subunit is sufficient for transcriptional induction of the AP gene and that the phosphotransferase activity of the C subunit is required for this induction.

Alkaline Phosphatase↗
PubMed1990

Alpha 1-adrenoceptor augmentation of beta-stimulated cAMP formation is enhanced by estrogen and reduced by progesterone in rat hypothalamic slices.

These experiments examined the influence of estradiol and progesterone given in vivo on norepinephrine (NE) regulation of cAMP synthesis in hypothalamic and preoptic area slices in vitro. Administration of progesterone to estrogen-primed female rats attenuated NE-induced slice cAMP accumulation. This hormone-dependent reduction in NE-stimulated cAMP synthesis was observed in slices incubated with TTX and in slices prepared from hypophysectomized rats, suggesting that progesterone effects on NE receptor activation of cAMP-generating systems are not secondary to the release of neurotransmitters that inhibit adenylyl cyclase or to changes in pituitary hormone secretion. Progesterone suppression of NE-induced cAMP formation could be prevented by incubating slices in the presence of a phorbol ester. In additional studies, the activity of beta-NE receptors was assessed by measuring isoproterenol (ISO)-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor RO-20-1724, and the activity of alpha 1 receptors was evaluated by measuring phenylephrine (PHE) augmentation of the ISO response. Estradiol reduced the cAMP response to ISO in both hypothalamic and preoptic area slices, and this effect was not reversed by subsequent progesterone treatment. Estradiol also enhanced PHE augmentation of ISO-stimulated cAMP synthesis. Moreover, administration of progesterone subsequent to estradiol eliminated alpha 1-receptor augmentation of the ISO response. An alpha 1 enhancement of the ISO response is observed if the progestin receptor antagonist RU 38486 is administered before progesterone. Progesterone also abolished PHE potentiation of vasoactive intestinal polypeptide-stimulated cAMP accumulation. In contrast, neither phorbol ester nor muscarinic (carbachol) potentiation of the cAMP response to ISO was affected by progesterone. The data suggest that ovarian steroids regulate the coupling of both alpha 1 and beta receptors to the membrane effector systems that generate intracellular cAMP.

Animals↗
PubMed1990

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation.

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate.

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1989

Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined. The incorporation of [32P]Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase. The incorporation of [32P]Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains. The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in [32P]Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis. We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP. The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells. The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases. Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells. The effect was most prominent in membranes from cyr1-2 and ras2 cells. These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.

1-Phosphatidylinositol 4-Kinase↗
PubMed1989

Sequence-selective DNA binding to the regulatory subunit of cAMP-dependent protein kinase.

The fluorescence of Trp-226 in the regulatory subunit of bovine type II cAMP-dependent protein kinase is unaffected by the binding of cAMP, but is quenched by the binding of 2'-dansyl-cAMP (DNS-cAMP). Up to 67% of the fluorescence of Trp-226 can be quenched by resonant energy transfer to the DNS-cAMP bound to the first site, and 96% of the fluorescence can be quenched by saturating both sites with DNS-cAMP. The observed efficiencies of energy transfer gave a distance of 16 A between Trp-226 and the DNS-cAMP bound at the first site and a distance of 12.7 A between Trp-226 and the DNS-cAMP bound at second site. The fluorescence of Trp-226 was suppressed by incubation of RII with the self-complementary octanucleotide TGACGTCA (CRE) due to binding of the oligonucleotide to RII. A detailed study of the binding equilibrium showed that each RII(cAMP)2 molecule binds 1 molecule of CRE with Kd = 80 nM. The corresponding Kd value for cAMP-depleted RII was found to be 25-fold higher. RII was also found to bind randomly selected DNA fragments with an average Kd value much higher than that of CRE. These observations show for the first time that the binding of oligonucleotide to RII is cAMP-enhanced and sequence-selective.

Animals↗
PubMed1988

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells.

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Effect of intravenous glucagon on the urinary excretion of adenosine 3',5'-monophosphate in man and in rats. Evidence for activation of renal adenylate cyclase and formation of nephrogenous cAMP.

To examine the effect of glucagon in vivo on renal formation and excretion of cAMP, clearance studies were performed in patients with hypoparathyroidism and in parathyroidectomized rats. Four patients with idiopathic hypoparathyroidism and 2 patients with pseudohypoparathyroidism were studied during an intravenous glucagon infusion (20 micrograms/kg body weight). In all patients, glucagon induced a significant increase in nephrogenous cAMP and a 2- to 3-fold increase in fractional excretion of phosphate. The average increase in nephrogenous cAMP was from a baseline of 784 +/- 229 to 18,748 +/- 3,842 pmol/100 ml glomerular filtrate (GF) (p less than 0.01) and occurred 30-60 min after the beginning of the glucagon infusion. The effect of intravenous glucagon, given as a bolus, was further examined in parathyroidectomized rats. Glucagon elicited a significant increase in the urinary excretion of the nucleotide. The excreted cAMP was compared with its filtered load for each urine collection period. In the first two collections, 0-15 and 15-30 min, the filtered load of cAMP was higher than its urinary excretion. During the following periods, 30-90 min, the excreted urinary cAMP exceeded by far its filtered load, suggesting a net nephrogenous contribution to the excretion of the nucleotide. Infusion of exogenous cAMP to parathyroidectomized rats induced significant increments in the filtered load and urinary excretion of the nucleotide. Tubular secretion of extrarenal cAMP could not be detected during the cAMP infusion. These results provide evidence supporting in vivo a possible parathyroid-independent formation of nephrogenous cAMP after glucagon administration, in men and in rats. The glucagon-induced increase in nephrogenous cAMP seems to account, at least partly, for some of the renal actions of this hormone.

Adenylyl Cyclases↗
PubMed1985

The separate estimation of cAMP intracellularly bound to the regulatory subunits of protein kinase I and II in glucagon-stimulated rat hepatocytes.

A method is described for the separate determination of cAMP intracellularly bound to the regulatory moieties (RI and RII) of protein kinase I and II. The cAMP endogenously bound to RI or RII in hepatocyte extract was adsorbed to protein A-agarose beads coated with antibodies against RI or RII. The endogenously bound cAMP was eluted from the washed beads with dilute acetic acid before being assayed. By all criteria tested, the present method did not perturb the intracellularly established equilibrium between bound and free cAMP. Stabilization of R X cAMP complexes was achieved by including sulfate in the extraction medium and sulfate/glycerol during the subsequent steps. Hepatocytes were isolated from fed male rats and contained about 0.25 pmol of RI and 0.2 pmol of RII per 10(5) cells. An intracellular titration of the cAMP binding sites of RI and RII was achieved by incubating the cells with various concentrations (1 pM to 10 nM) of glucagon. The fractional saturation of RI and RII was always similar, being 20% in nonstimulated cells. 50% saturation occurred when free cAMP was 0.46 pmol/10(5) cells. A Scatchard plot of the data for the endogenous cAMP binding suggested that cAMP interacted with RI and RII in a slightly positively cooperative manner. About 5% of the intracellularly bound cAMP was sedimentable at 10,000 X g. The apparent affinity of these particulate-associated binding sites was similar to that of soluble RI and RII. Under the conditions used no evidence was obtained for cAMP binding to other proteins than RI and RII.

Animals↗
PubMed1985

Parietal cell protein kinases. Selective activation of type I cAMP-dependent protein kinase by histamine.

cAMP-dependent protein kinases have been characterized in parietal cells isolated from rabbit gastric mucosa. Both Type I and Type II cAMP-dependent protein kinase isozymes are present in these cells. Type II isozymes were detected in 900, 14,000, and 100,000 X g particulate fractions as well as 100,000 X g cytosolic fractions; Type I isozymes were found predominately in the cytosolic fraction. When parietal cells were stimulated with histamine, an agent that elevates intracellular cAMP content and initiates parietal cell HCl secretion, cAMP-dependent protein kinase activity was increased in homogenates of these cells as measured by an increase in the cAMP-dependent protein kinase activity ratio. Histamine activation of cAMP-dependent protein kinase was correlated with parietal cell acid secretory responses which were measured indirectly as increased cellular uptake of the weak base, [14C]aminopyrine. These results suggest that cAMP-dependent protein kinase(s) is involved in the control of parietal cell HCl secretion. The parietal cell response to histamine may be compartmentalized because histamine appears to activate only a cytosolic Type I cAMP-dependent protein kinase isozyme, as determined by three different techniques including 1) ion exchange chromatography; 2) Sephadex G-25 to remove cAMP and allow rapid reassociation of the Type II but not the Type I isozyme; and 3) 8-azido-[32P]cAMP photoaffinity labeling. Forskolin, an agent that directly stimulates adenylate cyclases, was found to activate both the Type I and Type II isozymes. Several cAMP-dependent protein kinases were also detected in parietal cell homogenates, including a Ca2+-phospholipid-sensitive or C kinase and two casein kinases which were tentatively identified as casein kinase I and II. At least two additional protein kinases with a preference for serine or lysine-rich histones, respectively, were also detected. The function of these enzymes in parietal cells remains to be shown.

Adenylyl Cyclase Inhibitors↗
PubMed1986

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist.

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.

Animals↗
PubMed1983

Hormonal regulation of a plasma membrane phosphodiesterase in differentiating granulosa cells. Reciprocal actions of follicle-stimulating hormone and a gonadotropin-releasing hormone agonist on cAMP degradation.

The activity of a plasma membrane cAMP-phosphodiesterase in cultured ovarian granulosa cells was regulated by follicle-stimulating hormone (FSH) and the gonadotropin-releasing hormone (GnRH) agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). Degradation of cAMP was similar in cultures treated with FSH alone or FSH plus GnRHa when the labeled cyclic nucleotide was added from 24 to 42 h of culture. However, at 48 h and subsequent times of incubation, cAMP phosphodiesterase activity was significantly higher in cells incubated with FSH plus GnRHa. Phosphodiesterase activity was progressively increased by GnRHa concentrations between 10(-13) and 10(-10) M, and was maximally stimulated by 10(-9) M GnRHa. In comparison with control cells, FSH lowered the Vmax of cAMP catabolism by the high (1 microM cAMP substrate) and the low (50 microM) affinity phosphodiesterase, while GnRHa raised enzyme activity toward control levels. These actions of FSH and GnRHa were specific for a plasma membrane phosphodiesterase that was accessible to extracellular cAMP, since extracellular substrate was hydrolyzed, no intracellular uptake of [3H]cAMP was observed, and only a small fraction (10%) of cAMP was catabolized in the incubation medium in the absence of cells. Further, the actions of FSH and GnRHa on the membrane enzyme were the opposite of those observed when total phosphodiesterase activity was measured in cellular sonicates. Hormonal changes in phosphodiesterase activity were not due to leakage of the enzyme from damaged cells since a constant percentage of cAMP hydrolysis in the medium was observed during culture. Analysis of cAMP catabolites in granulosa cells indicated that the phosphodiesterase reaction product, 5'-AMP, was rapidly converted to adenosine by a plasma membrane 5'-nucleotidase, independent of the cellular hormonal status. These results indicate that the opposing actions of FSH and GnRHa upon granulosa cell differentiation include modulation of cAMP degradation at the plasma membrane level.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Gating properties of the cAMP-gated channel in toad olfactory receptor cells.

1. Inside-out membrane patches were excised from different parts of isolated olfactory receptor cells of the toad, and the gating properties of cAMP-gated channels were investigated under low concentrations of divalent cations. 2. At +50 mV, an outward current was observed when 1 mM cAMP was applied to the cytoplasmic side of membrane patches excised from cilia. cGMP had a similar effect. The dose-response relation for the cAMP-induced current could be fitted with the Hill equation with a coefficient of 1.5 for both cAMP and cGMP, and half-maximal concentration (K1/2) values of 19 microM (cAMP) and 16 microM (cGMP). 3. cAMP at low concentration (1 microM) induced step-like currents representing the opening of individual channels with a unitary conductance of about 30 pS. The I-V relation for the unitary events showed a weak outward rectification. 4. The relation between variance and mean current amplitude was well described with a parabola. Even at a saturating ligand concentration (1 mM cAMP) the current fluctuations did not disappear, indicating that fully liganded channels still switched between open and closed states. The maximum open probability was about 80% (+40 to +60 mV). 5. The current fluctuations at 1 mM cAMP were analysed with power spectral analysis. At +50 mV, frequency components lower than 10 Hz were well described with a Lorentzian function with a corner frequency of 1.7 +/- 0.2 Hz (+/- S.D.). In addition, higher flat frequency components were observed. At -50 mV the corner frequency became 0.6 +/- 0.1 Hz. 6. Membrane patches having a single cAMP-gated channel were obtained from the dendro-somatic membrane. These channels were very similar to the ciliary channels in electrophysiological characteristics. 7. The single cAMP-gated channel current showed flickering bursts that were interrupted with gaps. In saturating cAMP condition (1 mM), both burst- and gap-time histograms were fitted with single-exponential functions with time constants of 148 and 141 ms, respectively. 8. The channels were present at a high density of around 1750 microns-2 on the ciliary plasma membrane, as compared to 6 microns-2 on dendro-somatic membrane.

Animals↗
PubMed1997

GABA activity mediating cytosolic Ca2+ rises in developing neurons is modulated by cAMP-dependent signal transduction.

In the majority of developing neurons, GABA can exert depolarizing actions, thereby raising neuronal Ca2+. Ca2+ elevations can have broad consequences during development, inducing gene expression, altering neurite outgrowth and growth cone turning, activating enzyme pathways, and influencing neuronal survival. We used fura-2 and fluo-3 Ca2+ digital imaging to assess the effects of inhibiting or activating the cAMP signal transduction pathway on GABA activity mediating Ca2+ rises during the early stages of in vitro hypothalamic neural development. Our experiments stemmed from the finding that stimulation of transmitter receptors shown to either activate or inhibit adenylyl cyclase activity caused a rapid decrease in Ca2+ rises mediated by synaptically released GABA. Both the adenylyl cyclase activator forskolin and the inhibitor SQ-22,536 reduced the Ca2+ rise elicited by the synaptic release of GABA. Bath application of the membrane-permeable cAMP analogs 8-bromo-cAMP (8-Br-cAMP) or 8-(4-chlorophenylthio)-cAMP (0.2-5 mM) produced a rapid, reversible, dose-dependent inhibition of Ca2+ rises triggered by synaptic GABA release. Potentiation of GABAergic activity mediating Ca2+ rises was observed in some neurons at relatively low concentrations of the membrane-permeable cAMP analogs (20-50 microM). In the presence of tetrodotoxin (TTX), postsynaptic Ca2+ rises triggered by the bath application of GABA were only moderately depressed (13%) by 8-Br-cAMP (1 mM), suggesting that the inhibitory effects of 8-Br-cAMP were largely the result of a presynaptic mechanism. The protein kinase A (PKA) inhibitors H89 and Rp-3', 5'-cyclic monophosphothioate triethylamine also caused a large reduction (>70%) in Ca2+ rises triggered by synaptic GABA release. Unlike the short-term depression elicited by activation of the cAMP signal transduction pathway, Ca2+ depression elicited by PKA inhibition persisted for an extended period (>30 min) after PKA inhibitor washout. Postsynaptic depression of GABA-evoked Ca2+ rises triggered by H89 (in the presence of TTX) recovered rapidly, suggesting that the extended depression observed during synaptic GABA release was largely through a presynaptic mechanism. Long-term Ca2+ modulation by cAMP-regulating hypothalamic peptides may be mediated through a parallel mechanism. Together, these results suggest that GABAergic activity mediating Ca2+ rises is dependent on ongoing PKA activity that is maintained within a narrow zone for GABA to elicit a maximal Ca2+ elevation. Thus, neuromodulator-mediated changes in the cAMP-dependent signal transduction pathway (activation or inhibition) could lead to a substantial decrease in GABA-mediated Ca2+ rises during early development.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Plasma cAMP: a hepatorenal link influencing proximal reabsorption and renal hemodynamics?

The existence of a hepatorenal link is suggested by several pathophysiological observations (indirect actions of glucagon on the kidney, hepatorenal syndrome), but the nature of this link remains unidentified. We propose that extracellular circulating cyclic AMP could be this link. Cyclic AMP (cAMP) is the intracellular second messenger of glucagon (G) action in the liver, and this organ is known to release cAMP in the blood in relatively large amounts after G administration. On the other hand, the proximal tubule (mainly the pars recta) is known to take up cAMP through the organic acid transport system. We observed that the glucagon-induced rise in phosphate excretion, which requires supraphysiologic concentration of G, was significantly correlated with the simultaneous rise in plasma cAMP and could be mimiked by i.v. infusion of cAMP alone. Moreover, we showed that a significant hyperfiltration (similar to that induced by supraphysiologic G) can be observed if cAMP (mimicking G-induced hepatic release) is coinfused with a much lower, physiologic, amount of G. Taken together, these observations suggest that: (1) cAMP is a hepatorenal link and that plasma cAMP permanently influences the intensity of reabsorption in the pars recta of the proximal tubule; and (2) that cAMP participates, in conjunction with G, to control GFR. Insulin is known to exert an inhibitory influence on G-induced cAMP release by the liver and will thus weaken the indirect (cAMP-mediated) influence of G on renal function. This "pancreato-hepatorenal cascade" may explain the natriuretic effects of G and antinatriuretic effects of insulin, and probably contributes to disturbances observed in some pathophysiological situations such as the edema of liver cirrhosis or hyperfiltration of diabetes.

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