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PubMed1989

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

The separate or combined effects of insulin and the cAMP antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), were examined on fatty acid-stimulated ketogenesis in hepatocytes from normal fasted rats. Addition of 0.4 mM oleic acid or 0.4 mM octanoic acid resulted in a linear increase in ketone production measured over 60 min. When oleic acid was the substrate, incubation with 1 to 30 microns Rp-cAMPS alone or 0.1 to 10 nM insulin alone caused a variable decrease in the production of ketones which did not exceed an average value of 30% in any one experiment. The simultaneous addition of Rp-cAMPS and insulin resulted in a greater than additive inhibition which reached average values between 47-60% when the theoretical combined inhibitory effect of the insulin alone plus the Rp-cAMPS alone was less than 18%. No significant effects of either insulin or Rp-cAMPS, alone or in combination, were seen when octanoic acid was the substrate. These data imply that Rp-cAMPS can potentiate insulin inhibition of hepatic ketogenesis through inhibition of a cAMP-mediated process.

3-Hydroxybutyric Acid↗

PubMed1991

Inhibitory effects of ethanol on the calcium-dependent potentiation of vasoactive intestinal peptide-stimulated cAMP and cGMP accumulation in rat pinealocytes.

In rat pinealocytes, ethanol has been shown recently to inhibit the alpha 1-adrenergic potentiation of vasoactive intestinal peptide (VIP)-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses, with the cGMP response being more sensitive to the inhibition. Two intracellular events known to be of importance to the potentiation mechanism are activation of protein kinase C and elevation of intracellular calcium [( Ca2+]i). In this study, we examined the effects of ethanol on these two intracellular mechanisms with an activator of protein kinase C, and two agents that elevate [Ca2+]i, depolarizing concentrations of K+ and A23187. Using dispersed pinealocytes, ethanol (up to 175 mM) was found to have no effect on the 4 beta-phorbol 12-myristate 13-acetate (PMA, an activator of protein kinase C) potentiated VIP-stimulated cAMP response. As for the cGMP response, full potentiation requires both activation of protein kinase C and simultaneous elevation of [Ca2+]i. This could be achieved by stimulating the VIP-treated cells with a combination of PMA and 15 mM K+. In the presence of ethanol, the amplification effect of PMA and K+ on the VIP-stimulated cGMP response was inhibited with an IC50 value of 125 mM. In contrast, similar concentrations of ethanol had no effect on the corresponding cAMP response. These findings suggest that the potentiation of cAMP response by protein kinase C is not affected by ethanol. When depolarizing concentrations of K+ were used to potentiate the VIP-stimulated cAMP and cGMP accumulation, ethanol inhibited the K+ potentiation of VIP-stimulated cAMP and cGMP responses with IC50 values of 50 and 30 mM, respectively. The A23187 potentiation of VIP-stimulated cGMP response was also sensitive to the inhibitory effect of ethanol with an IC50 value of 120 mM. In comparison, the corresponding IC50 value for the cAMP response was greater than 175 mM. Based on our findings, we conclude that ethanol likely inhibits a Ca(2+)-dependent event(s) that is critical to the alpha 1-adrenergic-mediated potentiation of VIP-stimulated cAMP and cGMP responses.

Animals↗

PubMed1994

Ethanol enhancement of ligand-stimulated cAMP production by cultured human placental trophoblasts.

Chronic ethanol (EtOH) use during pregnancy can be associated with fetal injury including the fetal alcohol syndrome (FAS). A contributing factor in this fetal injury may be the effect of EtOH on the placenta. In this study, we have examined the effect of in vitro EtOH treatment on adenosine 3':5'-cyclic monophosphate (cAMP) production by cultured trophoblasts, in response to various ligands. Epinephrine (10(-6) M) rapidly stimulated cAMP with a peak between 2.5 and 5 min, which gradually returned to basal levels over 3-4 hr. EtOH treatment for > 16 hr resulted in an up-regulation of epinephrine-stimulated cAMP production. Inhibition of phosphodiesterase with Rolipram enhanced the effect of EtOH on cAMP production, suggesting that the effect of EtOH treatment was not due to phosphodiesterase inhibition. In cultured trophoblasts, EtOH treatment increased both epinephrine and 16,16'-dimethylprostaglandin E2 (dm-PGE2)-dependent cAMP production at varying ligand concentrations, suggesting an increased capacity to respond. When trophoblasts were treated with forskolin, a stimulator of adenylyl cyclase, cAMP production was enhanced in EtOH-treated cells. This suggests that EtOH treatment enhances adenylyl cyclase activity in these intact, cultured cells. Unlike trophoblasts from term human placenta, JAR choriocarcinoma cells did not respond to epinephrine, adenosine, or dm-PGE2. The choriocarcinoma cells appeared to have lost the ability to respond to these ligands. Although the JAR cell adenylyl cyclase was stimulated by forskolin, EtOH treatment did not alter forskolin-stimulated cAMP production. In summary, EtOH-induced up-regulation of cAMP production appears to be cell specific, being present in normal human trophoblasts but not in undifferentiated choriocarcinoma cells.

16,16-Dimethylprostaglandin E2↗

PubMed1996

Calcium modulates circadian variation in cAMP-stimulated melatonin in chick pineal cells.

In chick pineal cells, melatonin synthesis is regulated by both calcium and cAMP. Calcium-dependent regulation of melatonin is suggested by the monotonic decrease in melatonin observed with decreasing extracellular calcium ion concentrations ([Ca2+]0), the stimulation of melatonin with Bay K8644, and the inhibition of nocturnal melatonin by several calmodulin antagonists. At submicromolar [Ca2+]0, a stimulation of melatonin was observed in the presence of 8-Br cAMP, but not with Bay K8644, suggesting that this amount of stimulation of melatonin by 8-Br cAMP is independent of Ca2+ influx through dihydropyridine-sensitive Ca2+ channels. At micromolar [Ca2+]0, there was a further increase in the stimulation of melatonin by 8-Br cAMP that was not blocked by nifedipine, a dihydropyridine-sensitive Ca2+ channel antagonist. Micromolar [Ca2+]0 is required for the greater stimulation of melatonin by 8-Br cAMP during the night than during the day. Melatonin was stimulated by 8-Br cAMP to higher levels during the night than during the subjective day under normal [Ca2+]0 (1.3 mM). This difference in the amount of melatonin stimulated by 8-Br cAMP during the subjective night versus the subjective day was blocked by lowering [Ca2+]0 to a submicromolar concentration (0.2 microM). Both nifedipine and calmidazolium partially blocked nocturnal increases in melatonin, but were ineffective during the day. These results suggest that Ca2+ plays an important role in the differential ability of cAMP to stimulate melatonin during the night versus the day.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Modulation of IFN-mediated Ly-6E antigen induction by cAMP in a T cell lymphoma: opposite effects on the responses to IFN-gamma and IFN-alpha/beta.

This study was initiated to examine the role of cyclic nucleotides in the regulation of the expression of the Ly-6E cell surface Ag by IFN. As a model system, we used the YAC T cell lymphoma where this Ag is constitutively absent but is highly inducible by both IFN-gamma and IFN-alpha/beta. Treatment with cAMP or cGMP analogs did not cause Ly-6E expression in the absence of IFN. However, treatment with cAMP analogs, but not with cGMP analogs, markedly altered Ly-6E expression triggered by IFN, both at the mRNA and at the cell surface protein levels. Interestingly, these effects depended on whether Ly-6E induction was mediated by IFN-gamma or IFN-alpha/beta. Thus, the response to IFN-gamma was enhanced up to tenfold, whereas the response to IFN-alpha/beta was suppressed by 90-95%. Similar differential modulations of Ly-6E induction were also exerted by forskolin and cholera toxin, which are known to elevate intracellular cAMP concentration through distinct mechanisms. A YAC cell variant (C10) was isolated, where cAMP analogs or cAMP inducers could not modify Ly-6E induction. Although resistant to the biological effect of cAMP, the C10 mutant exhibited normal IFN-mediated Ly-6E responses. Moreover, in this mutant, Ly-6E induction was still affected by the PKC activator PMA, as in wild-type YAC cells. Altogether, our data demonstrate that cAMP (and cGMP) is not directly involved as second messenger in Ly-6E induction mediated by IFNs. However, a rise of cAMP modulates in an opposite fashion the Ly-6E-inducing actions of IFN-gamma and IFN-alpha/beta, which suggests that the two types of IFN utilize separate biochemical pathways to regulate Ly-6E expression.

Animals↗

PubMed1988

cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium.

We have examined the regulation of three early developmentally regulated genes in Dictyostelium. Two of these genes (D2 and M3) are induced by pulses of cAMP and the other (K5) is repressed. Expression of these genes has been examined in a number of developmental mutants that are specifically blocked in various aspects of the signal transduction/cAMP relay system involved in aggregation and control of early development. The mutant strains include Synag mutants, which are blocked in receptor-mediated activation of adenylate cyclase and do not relay cAMP pulses; FrigidA mutants, which are blocked in receptor-mediated activation of both adenylate cyclase and the putative phosphoinositol bisphosphate (PIP2) turnover pathway and appear to be mutations in the gene encoding one of the G alpha protein subunits; and a StreamerF allele, which lacks cGMP-specific cGMP phosphodiesterase. From the analysis of the developmental expression of these genes under a variety of conditions in these mutant strains, we have drawn a number of conclusions concerning the modes of regulation of these genes. Full induction of D2 and M3 genes requires cAMP interaction with the cell surface receptor and an "oscillation" of the receptor between active and adapted forms. Induction of these genes does not require activation of the signal transduction pathway that leads to adenylate cyclase activation and cAMP relay, but does require activation of other receptor-mediated intracellular signal transduction pathways, possibly that involving PIP2 turnover. Likewise, repression of the K5 gene requires pulses of cAMP. Expression of this gene is insensitive to cAMP pulses in FrigidA mutants, suggesting that a signal transduction pathway is necessary for its repression. Results using the StreamerF mutant suggest that the rise in cGMP in response to cAMP/receptor interactions may not be directly related to control of the pulse-induced genes. In addition, we have examined the effect of caffeine, which M. Brenner and S.D. Thomas (1984, Dev. Biol., 101, 136-146) showed preferentially blocks the cAMP relay system by blocking receptor-mediated activation of adenylate cyclase. We show that in many of the mutants and in an axenic wild-type strain, caffeine causes the induction of pulse-induced gene expression to almost wild-type levels or in some cases to higher than wild-type levels. Our data suggest that caffeine works by activating some step in the signal transduction pathway that must lie downstream from both the receptor and at least one of the G proteins and thus has effects other than simply blocking the receptor-mediated cAMP relay system.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMedDate not supplied

Influence of atropine, metiamide and vagotomy on cAMP of resting and stimulated gastric mucosa.

In normal rats the effect of atropine and metiamide was studied on cAMP levels of resting and histamine-, pentagastrin-, carbachol- and insulin-stimulated gastric mucosa. In vagotomized rats gastric mucosal cAMP levels were investigated under basal conditions and after insulin. Atropine and metiamide did not alter cAMP levels of resting gastric mucosa. Truncal vagotomy caused an increase in gastric mucosal cAMP, which was not affected by insulin. All gastric secretagogues caused a significant rise in gastric mucosal cAMP levels, which was not antagonized by atropine. Metiamide effectively blocked the rise in cAMP concentration after histamine and pentagastrin, but not that evoked by cholinergic stimulation. The results are consistent with the view that the in vivo rise of gastric mucosal cAMP after carbachol or insulin is not due to a direct cholinergic action. From the spectrum of inhibitory actions of metiamide it looks as if the increase in rat gastric mucosal cAMP concentration after histamine and pentagastrin administration is mediated by H2-receptor stimulation.

Animals↗

PubMed1991

Diuretics modify [Arg8]vasopressin-stimulated cAMP but not atrial natriuretic peptide-stimulated cGMP formation in renal cells.

The present study was undertaken to examine whether sulfonamide-derived diuretics affect [Arg8]vasopressin (AVP)-stimulated or atrial natriuretic peptide (ANP)-stimulated cyclic nucleotide formation in cells cultured from rat or dog kidney. In rat renal cells, all four sulfonamide-derived diuretics examined significantly suppressed 10(-9) M AVP-stimulated cAMP formation at concentrations of 10(-4) and 10(-3) M, while basal cAMP formation was unchanged by the diuretics. When cells were stimulated with 10(-7) M AVP, low ceiling diuretics (indapamide and trichlormethiazide) did not suppress cAMP formation, while high ceiling diuretics (furosemide and azosemide) significantly suppressed cAMP formation at concentrations of 10(-4) and 10(-3) M. The suppressive effect of the diuretics on AVP-stimulated cAMP formation in vitro paralleled the reported diuretic potency of the agents in vivo. In dog renal cells, all four diuretics significantly suppressed 10(-9) M AVP-stimulated cAMP formation at concentrations from 10(-6) to 10(-5) M, while these diuretics did not change basal cAMP levels. High ceiling diuretics suppressed 10(-7) M AVP-stimulated cAMP formation, whereas low ceiling diuretics did not. The difference in effective doses between rats and dogs seems to be consistent with the species difference observed in vivo. None of the diuretics affected basal levels of intracellular cGMP or ANP-stimulated cGMP formation in cultured rat renal cells. In addition to the inhibition of the Na/K/Cl co-transporter, it is suggested that most sulfonamide-derived diuretics act, at least in part, by inhibiting the actions of AVP.

Animals↗

PubMed1992

Effect of vasodilators, including nitric oxide, on the release of cGMP and cAMP in the isolated perfused rat kidney.

In isolated Tyrode-perfused rat kidneys, the release of the cyclic nucleotides cAMP and cGMP was measured in response to several vasodilators, including nitric oxide (NO). During vasoconstrictions induced by methoxamine, a basal release of both cyclic nucleotides was detected in the renal effluent (357 +/- 32 fmol/min for cGMP and 3097 +/- 219 fmol/min for cAMP). Injection of acetylcholine (ACh; 11 nmol), sodium nitroprusside (SNP; 0.8 nmol) and atrial natriuretic factor (ANF; 80 pmol) caused a marked release of cGMP. The cGMP release induced by ACh was not altered by indomethacin (3 microM) but was markedly reduced by the NO synthase inhibitor nitro-L-arginine (L-NNA; 200 microM). Authentic NO (0.16-80 nmol) caused dose-dependent vasodilatations that were accompanied by increases in the overflow of cGMP. The vasodilatations caused by forskolin (6 nmol) and prostacyclin (PGI2; 3-52 nmol) were not accompanied by an overflow of cGMP. The vasodilator responses to 5-hydroxytryptamine (5-HT; 0.25-2 mumol), obtained in presence of the 5-HT2 receptor blocker ritanserin (10 nM) and the 5-HT3 blocker ICS 205930 (10 nM), were markedly reduced by L-NNA; however, they were not accompanied by the renal release of cGMP. Both forskolin and PGI2 induced the release of cAMP from perfused rat kidneys; ACh, 5-HT and 5-carboxamidotryptamine (5-CT) also evoked a significant release of cAMP into the renal effluent. The release of cAMP induced by ACh and 5-HT was reduced by indomethacin and L-NNA. Higher doses of NO released cAMP from the perfused rat kidneys. Our data illustrate that both cAMP and cGMP can be released by vasodilator substances into the venous effluent of isolated perfused rat kidneys. The dilator responses to 5-HT were sensitive to the NO synthase inhibitor L-NNA and were accompanied by the release of cAMP and not by the release of cGMP. Our data suggest that the dilator responses may be due to NO released from endothelial cells, which then activates adenylyl cyclase either directly or indirectly.

Acetylcholine↗

PubMed1996

Differentiation of group 2 and group 3 metabotropic glutamate receptor cAMP responses in the rat hippocampus.

The effects of group 2- versus group 3-selective metabotropic glutamate (mGlu) receptor agonists were examined against forskolin (10 microM)-, vasoactive intestinal peptide (VIP; 1 microM)- and 5'-N-ethylcarboxamidoadenosine (NECA; 10 microM)-stimulated cAMP accumulations in adult rat hippocampal slices (in the presence of adenosine deaminase). Group 2 mGlu receptor-selective ((1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD) and (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG I)) and group 3 mGlu receptor-selective (L-2-amino-4-phosphonobutyric acid (L-AP4) and L-serine-O-phosphate) agonists greatly inhibited forskolin-stimulated cAMP formation ( > 80% at maximally effective concentrations). In contrast, stimulation of cAMP by VIP or NECA was inhibited by group 3, but not by group 2, mGlu receptor agonists. In fact, group 2 mGlu receptor agonists greatly potentiated cAMP accumulation evoked by NECA. Both the inhibitory effects of 1S,3R-ACPD on forskolin-stimulated cAMP and the potentiating effects on NECA-stimulated cAMP accumulation were reversed by the competitive group 1/2 mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG). However, (+)-MCPG had no effects on L-AP4 inhibition of cAMP. Thus, the effects of group 2 versus group 3 mGlu receptor agonists on cAMP coupling can be pharmacologically as well as functionally differentiated in the rat hippocampus.

Adenosine↗

PubMed1994

Stimulation of formation of cAMP by 5-hydroxytryptamine in myenteric ganglia isolated from guinea pig small intestine.

Effects of 5-hydroxytryptamine (5-HT) and related agonists and antagonists on formation of cAMP were determined for enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine. Formation of cAMP was stimulated by 5-HT in both dose- and time- dependent manners. The stimulatory action of 5-HT was suppressed by the 5-HT1P antagonist, renzapride, but not by the 5-HT3 antagonist, tropisetron (formerly ICS 205-930). Neither renzapride nor cisapride increased the levels of cAMP. Levels of cAMP were suppressed by concentrations of tropisetron greater than 1.0 microM. Levels of cAMP were unaffected by the 5-HT3 agonist, 2-methyl-5-hydroxytryptamine. A putative 5-HT4 receptor agonist, 5-methoxytryptamine, stimulated formation of cAMP, but to a lesser extent than 5-HT. We conclude that 5-HT acts to stimulate adenylate cyclase and the formation of cAMP in myenteric ganglia. The 5-HT1P receptor is the most likely subtype involved in 5-HT action on cAMP formation.

5-Methoxytryptamine↗

PubMed1989

Specific inhibition of FSH-stimulated cAMP accumulation by delta 9-tetrahydrocannabinol in cultures of rat Sertoli cells.

delta 9-Tetrahydrocannabinol (THC), the major psychoactive component in marihuana, is a reproductive toxicant in both man and animals. THC acts at both the level of the pituitary-hypothalamic axis and the testis, specifically the Leydig cell; an effect on the Sertoli cell has not been shown. Since THC inhibits cAMP accumulation in several cell types, we have examined the effect of THC on Sertoli cell function using altered cAMP accumulation as a marker of toxicity. THC reduced the FSH-induced accumulation of cAMP at concentrations which were neither cytotoxic nor affected cellular ATP levels. This inhibition was evident after 3 hr and did not affect the dose of FSH which gave half-maximal stimulation, suggesting that THC does not compete with FSH for binding to its receptor. The ability of THC to inhibit cAMP accumulation was not affected by incubation in the presence of phosphodiesterase inhibitors, making it unlikely that it acts via stimulation of phosphodiesterase activity. This THC-induced inhibition of Sertoli cell cAMP is specific for FSH; it does not affect the ability of forskolin, cholera toxin, isoproterenol, or prostaglandin E1 to stimulate Sertoli cell cAMP. Furthermore, inhibition occurs in the presence of pertussis toxin, suggesting that this effect of THC is independent of the inhibitory adenylate cyclase pathway. Inhibition of Sertoli cell cAMP also occurs with other cannabinoids which are present in marihuana, but which are not psychoactive. These data indicate that a part of the testicular toxicity of THC may be due to a specific alteration of the hormonal control of Sertoli cell function via an inhibition of FSH-stimulated cAMP accumulation.

Adenosine Triphosphate↗

PubMed1978

Progesterone-induced meiosis in Xenopus laevis oocytes: a role for cAMP at the "maturation-promoting factor" level.

Cholera toxin inhibition of progesterone-induced meiosis of Xenopus laevis oocytes in vitro has been correlated with increased cAMP levels. Inhibition of germinal vesicle breakdown (Gvbd) and cAMP increase occurred after a lag period of 2 hr, when cholera toxin was injected, or 4--5 hr, when applied externally. The ability of the maturation-promoting factor (Mpf) to provoke Gvbd when injected into recipient oocytes was found to be dependent upon whether the oocytes had been exposed to cholera toxin alone or to toxin and progesterone. With the former, cAMP levels were elevated and Mpf activity was abolished, whereas with the latter, the increase in cAMP was less pronounced and Mpf activity was observed. Injection of cAMP or its 8-thio derivatives shortly before the appearance of progesterone-induced Mpf abolished Gvbd. If injected earlier or later, no inhibition was observed. In contrast, cholera toxin inhibited maturation even when added several hours before progesterone, suggesting a sustained accumulation of cAMP. No Gvbd occurred when 8-thio-methyl-cAMP was injected together with Mpf. These data suggest that cAMP is involved in the control of the formation/amplification and/or activity of Mpf-a result which may be of general significance in cell division mechanisms.

Animals↗

PubMedDate not supplied

Inhibition of IFN-gamma induction of class II MHC genes by cAMP and prostaglandins.

Triggering of the cyclic AMP (cAMP) signal transduction pathway inhibits the the interferon gamma (IFN-gamma)-mediated induction of class II major histocompatibility (MHC) genes. We have investigated the mechanism of the inhibition of IFN-gamma induction of the murine A alpha class II MHC gene by cAMP and E series prostaglandins (PGEs). 151 base pairs of the A alpha promoter were sufficient to confer positive regulation by IFN-gamma and negative regulation by cAMP which accurately mirrored the regulation of the endogenous A alpha gene. cAMP also inhibited the IFN-gamma activation of the Fc gamma receptor I (Fc gamma RI) gene promoter, an "early" promoter which is activated immediately after treatment of cells with IFN-gamma. PGEs, which cause an elevation in intracellular cAMP, inhibited the induction of the A alpha promoter, and inhibition was greater in the presence of tumor necrosis factor alpha (TNF alpha). A mutational analysis of the A alpha promoter showed that all four conserved class II promoter elements, the S, X1, X2, and Y boxes, play a role in mediating A alpha promoter activation by IFN-gamma. Mutations in these elements did not diminish the cAMP inhibition of promoter activation by IFN-gamma. Thus, conserved class II promoter sequences which mediate most known examples of positive and negative regulation, including cAMP inhibition of constitutive class II expression, do not mediate cAMP inhibition of IFN-gamma activation of the A alpha promoter. We suggest that this inhibition may be mediated by a novel class II promoter element or by disruption of an early step in the IFN-gamma signal transduction pathway.

Animals↗

PubMed1987

Partial purification and characterization of cAMP-dependent protein kinase from Fasciola hepatica.

Three cyclic AMP (cAMP)-dependent protein kinases, designated A1, A2, and B, were isolated from the liver fluke Fasciola hepatica using Phenyl-Sepharose and DEAE-cellulose chromatography. These enzymes differed with respect to activation by cAMP and their molecular weights. The half-maximal activation constant for cAMP-dependent protein kinases A1 and B was 20 nM, while that of A2 was about five-fold higher (110 nM). The estimated molecular weights for cAMP-dependent protein kinases A1 and A2 (both 98,000) suggest a dimeric form for these enzymes; whereas, the higher molecular weight for cAMP-dependent protein kinase B (187,000) indicates that this enzyme is a tetramer. The physical and kinetic properties of the catalytic subunit of fluke cAMP-dependent protein kinase were similar to those reported for the mammalian enzyme. The molecular weight of the catalytic subunit was estimated to be 41,000. The pH optimum for the enzyme was 6.0, 6.5, or 7.0 when casein, histone, or protamine were used as substrates. The protein substrate specificity was in the order histone greater than arginine-rich histone greater than casein greater than protamine greater than lysine-rich histone. Free Mg2+ 'stimulated' enzyme activity at low concentrations (0.5 to 5 mM), whereas at higher concentrations (greater than 5 mM) it became inhibitory. Of the divalent cations tested, only Co2+ and Mn2+ could substitute for Mg2+. Kinetic studies indicated that the reaction mechanism of this enzyme is sequential and that MgATP and MgADP are competitive ligands. Reconstitution experiments using the subunits of fluke and bovine heart cAMP-dependent protein kinase showed that there is sufficient structural homology between these enzymes such that the catalytic subunit from one species can combine with the regulatory subunit of the other species to form inactive holoenzyme. Thus, the present results indicate that cAMP-dependent protein kinase from F. hepatica is similar but not identical to the mammalian enzyme.

Animals↗

PubMed1986

Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin.

Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3-5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5-fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals.

Animals↗

PubMed1993

Glucose stimulates cAMP accumulation in the oral bacterium Actinomyces viscosus.

Actinomyces viscosus T14V, a Gram-positive bacterium found in the oral cavity, was found to be insensitive to glucose-mediated catabolite repression. Basal levels of beta-galactosidase (18-26 U) were observed at all phases of growth regardless of the culture conditions. Further, beta-galactosidase could not be induced with lactose, or with a known inducer of the enzyme, isopropyl-beta-D-thiogalactoside, or with dibutyryl cAMP. Glucose, on the other hand, stimulated cAMP accumulation in a concentration-dependent manner. Fructose and sucrose mimicked the effects of glucose on cAMP accumulation, whereas galactose, mannose and maltose had lesser stimulatory effects. Other carbon sources, i.e., lactose, alpha-methylglucoside, ribose, xylose and succinate were without effect. Glucose and alpha-methylglucoside were found to stimulate cAMP accumulation in toluene-permeabilized cells, in the presence of the phosphodiesterase inhibitor, theophylline. Glucose did not stimulate cAMP levels in other Gram-positive bacteria including Streptococcus mutans, S. sanguis and S. salivarius but did cause cAMP accumulation in other strains of A. viscosus. The results suggest that glucose effects on cAMP metabolism are independent of the induction of beta-galactosidase as presently defined for Escherichia coli, and that the effects appear to be selective to the A. viscosus bacteria. The results also suggest that glucose stimulates cAMP accumulation via activation of adenylate cyclase.

Actinomyces viscosus↗

PubMed1992

P0 gene expression in Schwann cells is modulated by an increase of cAMP which is dependent on the presence of axons.

The role of cAMP in the regulation of P0 gene expression was investigated in Schwann cells of normal, regenerated, and permanently transected rat sciatic nerve. Forskolin treatment of endoneurial segments of rat sciatic nerve resulted in increased cAMP and P0 mRNA levels in normal and regenerated nerves but not in permanently transected nerves, where axonal regeneration is prevented. This increase of cAMP and P0 mRNA occurred within 30 and 90 min, respectively. P0 mRNA levels in the endoneurial segment of the permanently transected nerve were not increased with dibutyryl cAMP. The Schwann cells of the permanently transected nerve, however, retained the ability to myelinate 15 embryonic day (E15) dorsal root ganglia (DRG) neuron and neurite networks cultured in vitro. P0 mRNA levels increased within 4 days in transected endoneurium segments cocultured with E15 DRG neurons and neurites and further increased in 21 day myelinating cocultures. Although cAMP was not detectable in 4 day cocultures, it increased to detectable levels in 21 day cultures, suggesting that cAMP is involved in the myelinating process. These results indicate that the presence of the axon is required for the observed increase of cAMP and P0 mRNA levels and suggest that the increase of cAMP occurs within the axon which then presumably activates a different Schwann cell second messenger pathway to induce P0 gene expression.

Animals↗