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PubMed2005

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA.

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.

Amino Acid Sequence↗
PubMed2004

Activation of cAMP signaling facilitates the morphological maturation of newborn neurons in adult hippocampus.

Previous studies have demonstrated that activation of the cAMP cascade, including the cAMP response element-binding protein (CREB), increases the proliferation and survival of newborn neurons in adult mouse hippocampus. In the present study, we determined whether the cAMP-CREB cascade also influences the morphological maturation of newborn neurons in the subgranular zone of the hippocampus. Rolipram, a selective inhibitor of the cAMP-specific phosphodiesterase type 4, was administered to activate the cAMP cascade, and neuronal morphology was determined by analysis of Golgi-impregnated neurons in the subgranular zone of hippocampus. Rolipram administration significantly increased the number of branch points and length of dendrites relative to vehicle treatment. Increased branch number and length were accompanied by increased levels of phosphorylated CREB, the active form of this transcription factor, in immature neurons. In contrast, the morphology of Golgi-impregnated neurons was not significantly influenced by rolipram treatment in inducible transgenic mice expressing a dominant-negative mutant of CREB in hippocampus. We also tested the influence of cAMP analogs in primary hippocampal cultures and found that activation of the cAMP pathway increased and inhibition of the cAMP cascade decreased the number of branches and length of processes as observed in vivo. These findings indicate that the cAMP-CREB cascade plays an important role in the differentiation and maturation of newborn neurons in hippocampus.

Animals↗
PubMed2005

Low-threshold exocytosis induced by cAMP-recruited CaV3.2 (alpha1H) channels in rat chromaffin cells.

We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (-50, -40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 microM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the "low-threshold exocytosis" induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the alpha1H (CaV3.2) channel isoform.

Animals↗
PubMed1999

Method of transfection affects the cAMP-mediated induction of the RIIbeta subunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium.

mRNA for the regulatory subunit RIIbeta of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIbeta gene contains no cAMP response element (CRE), and the induction of RIIbeta mRNA is slow and requires on-going protein synthesis. When a construct containing the 5'-flanking region of the RIIbeta gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RIIbeta mRNA, but not the mRNA for the CRE-containing RIalpha gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbetamRNA (but not RIalpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.

Animals↗
PubMed1994

Inhibition of gonadotrophin-induced ovulation in rabbits by perfusion with dibutyryl cAMP via reduction of ovarian prostaglandin production.

The role of cAMP in ovulation, oocyte maturation and prostaglandin production was assessed using a rabbit ovary preparation perfused in vitro. Dibutyryl cAMP (10(-3), 10(-4) or 10(-5) mol l-1) was added to the perfusate of one ovary. The contralateral, control ovary was perfused with medium alone. Thirty minutes after the onset of perfusion, 50 iu hCG was added to the perfusate of all ovaries. Dibutyryl cAMP inhibited hCG-induced ovulation in a dose-related fashion. No difference in ovum maturity or degeneration was found between control ovaries and ovaries treated with dibutyryl cAMP. Ovarian progesterone production was not affected by exposure to dibutyryl cAMP. The concentrations of 6-keto PGF1 alpha (the stable metabolite of prostacyclin) and PGF2 alpha in the perfusate of ovaries treated with dibutyryl cAMP were 49.6% and 32.0% of the control values, respectively, 12 h after hCG administration. Inhibition of 6-keto PGF1 alpha production by dibutyryl cAMP was dose related. Production of PGE2 was unaffected by dibutyryl cAMP. These data raise the possibility that continuous exposure to dibutyryl cAMP may inhibit hCG-induced ovulation in the perfused rabbit ovary via a reduction in PGF2 alpha and prostacyclin production.

6-Ketoprostaglandin F1 alpha↗
PubMed2006

Illness and injury among children attending summer camp in the United States, 2005.

OBJECTIVES: The purpose of this work was to describe illnesses and injuries sustained by campers at summer camps, calculate illness and injury rates, and identify risk factors for camp-related illness and injury. METHODS: A convenience sample of camps selected from the US summer camp population participated in this pilot illness and injury surveillance study. Camps completed weekly reports detailing the number of campers on site and the number of adverse events sustained and provided specific information about each adverse event. RESULTS: A total of 177 camper illnesses and injuries occurred during 122,379 camper-days, with a median rate of 1.15 adverse events per 1000 camper-days. The majority of reported events were illnesses (68.0%), 11.8% of which were communicable and seen in multiple individuals at camp. Of the injury events (32.0%), cut/scratch/scrape were the most common diagnoses (33.3%), followed by fracture (14.6%) and sprain/strain (10.4%); horseback riding and capture the flag were identified as injury-producing activities. CONCLUSIONS: Internet- and telephone-based surveillance systems can be successfully used to conduct illness and injury surveillance among children attending summer camp. Data collected via such systems can be used to calculate illness and injury rates, to describe patterns of illness and injury, and to identify risk factors for camper-related illness and injury. Given the millions of children attending summer camp yearly, a surveillance system such as this can provide the data needed to develop evidence-based prevention interventions to decrease the number of youth whose camp experiences are negatively affected by illness and injury.

Adolescent↗
PubMed2006

cAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation.

In the regulation of steroid biosynthesis, a process mediated by the steroidogenic acute regulatory (StAR) protein, both cAMP-dependent and -independent pathways are involved. While the cAMP-dependent regulatory events represent, by far, the most robust increase in steroid synthesis and are well established, the knowledge regarding cAMP-independent mechanisms is lacking. The present investigation was designed to elucidate the potential involvement of the latter in regulating StAR expression and steroidogenesis in mouse Leydig tumor cells (mLTC-1 cells). Treatment of mLTC-1 cells with a number of factors including insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor, transforming growth factor (TGF)alpha, interleukin-1 (IL-1), and colony-stimulating factor-1, increased the levels of StAR mRNA, StAR protein, and progesterone to varying degrees and utilized signaling pathways that are not associated with elevations in intracellular cAMP levels. Importantly, phosphorylation of StAR in response to these stimuli was undetectable, which is in marked contrast to observations with human chorionic gonadotropin (hCG), indicating factors that do not alter intracellular cAMP, regulate the steroid biosynthesis in a StAR phosphorylation-independent manner. In addition, the roles for factors involved in cross-talk between the protein kinase pathways, PKA and PKC, were demonstrated. Further characterization of signaling by one such cAMP-independent factor, TGFalpha, demonstrated that the mechanism, whereby it increased StAR expression and steroid synthesis, was dependent on de novo protein synthesis and mediated via activation of the EGF receptor. TGFalpha was also able to augment hCG-stimulated cAMP synthesis, StAR protein and StAR phosphorylation, and influence hCG binding and LH receptor mRNA expression. Furthermore, TGFalpha increased phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and cAMP-response element-binding protein (CREB), processes inhibited by the mitogen-activated protein kinase/ERK inhibitor U0126 and by expression of non-phosphorylatable CREB-M1 respectively. Inhibition of ERK activity enhanced TGFalpha-mediated StAR protein expression (but not its phosphorylation) and decreased progesterone synthesis, events correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). Collectively, these findings demonstrate that, in mouse Leydig cells, cAMP-independent signaling events regulate steroidogenesis in a StAR phosphorylation-independent manner.

3-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Studies on the intracellular cAMP metabolism of tonsillar lymphocytes.

cAMP metabolism, including the intracellular cAMP level and adenylcyclase activity, was studied in tonsillar and peripheral lymphocytes. The basal activity of adenylcyclase was significantly higher in tonsillar lymphocytes than in peripheral lymphocytes, although the basal level of intracellular cAMP did not differ. The responsiveness of the intracellular cAMP level and adenylcyclase activity stimulated by 10(-5) M isoproterenol, or 10(-5) M prostaglandin E1, which was thought to stimulate this enzyme through a specific receptor, was significantly lower in tonsillar lymphocytes than in peripheral lymphocytes. Moreover, the responsiveness of adenylcyclase activity to 10(-2) M NaF, which was thought to stimulate this enzyme directly, was also significantly lower in tonsillar lymphocytes. Tonsillar T lymphocytes represented a lower cAMP basal level than tonsillar B lymphocytes, but no difference was observed in the responsiveness of intracellular cAMP to the above agents. Tonsillar lymphocytes derived from patients with recurrent tonsillitis represented significantly lower cAMP responsiveness than those from patients of other chronic tonsillitis. Furthermore, in the tonsillar lymphocytes from patients with a recent attack of acute tonsillitis, a more depressed responsiveness of the intracellular cAMP level to the above agents was recognized. The cAMP basal level was not changed by these pathological alterations of tonsillitis.

Acute Disease↗
PubMed1995

Individual lymphocyte subset composition determines cAMP response to isoproterenol in mononuclear cell preparations from peripheral blood.

The aim of the present study was to examine to what extent isoproterenol-stimulated cAMP production in mononuclear cells, isolated from resting human subjects, was correlated with lymphocyte subset composition. Peripheral blood was collected from 14 healthy male subjects, who rested in the sitting position. Mononuclear cells were prepared by density centrifugation and subset composition was determined by two-colour flow cytometry. Cyclic AMP production was determined by a radioimmunoassay after inhibition of phosphodiesterase and stimulation with isoproterenol. NK-cells (CD3-CD56+) were positively correlated to total cAMP concentrations (basal cAMP plus increase in cAMP). Tcytotoxic cells (CD8+) were positively correlated to the relative increase in cAMP. Thelper cells (CD4+) were negatively correlated to total cAMP concentrations and B-lymphocytes (CD(19+20)+) were negatively correlated to the relative increase in cAMP. The results support the view that NK-cells and Tcytotoxic cells have higher beta 2-adrenoceptor densities and more receptors with high affinity than Thelper and B-cells. In conclusion, our study demonstrates that cAMP levels in blood mononuclear cells obtained from normal subjects show substantial variation. This variation is at least in part due to differences in lymphocyte subset composition in the same subjects. Therefore, it is important to know the lymphocyte subset composition when evaluating cAMP accumulation in mononuclear cell preparations from peripheral blood.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Inhibition of tumor cell proliferation by type IV collagen requires increased levels of cAMP.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).

Amino Acid Sequence↗
PubMed1990

A system to select for mutant LLC-PK1 cells affected in cAMP mediated hormonal response using a photoactivatable analogue of vasopressin.

The photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenyl-amidino)lysine] vasopressin (apa-LVP) could be used to elicit stimulation of cAMP production in LLC-PK renal epithelial cells, detectable up to 24 h after photoactivation by flash photolysis. This is in contrast to cells treated with vasopressin, or apa-LVP without photoactivation, where cAMP synthesis is down regulated within 4 h. The prolonged stimulation of cAMP production induced by photoactivation of apa-LVP was demonstrated to be cytotoxic to LLC-PK1 cells, whereas the vasopressin receptor negative LLC-PK1 mutant M18 was resistant to the cytotoxic effect. A selection strategy was developed for mutants resistant to this long-term stimulation of cAMP production, whereby multiple cycles of treatment with apa-LVP and photoactivation were used. Mutants so selected were then characterized using a novel screening system for detection of the production of urokinase-type plasminogen activator in response to cAMP agonists. One mutant was examined and found to be impaired in hormonal responsiveness, whereby hormone and forskolin stimulated cAMP-mediated responses were markedly reduced. It exhibited resistance to the long-term stimulation of cAMP production elicited by apa-LVP and photoactivation. This implies that apa-LVP can be used to select for novel mutants specifically impaired in cAMP metabolism and in particular down-regulation of cAMP response.

Adenylyl Cyclases↗
PubMed1999

Effects of ethanol on alpha-adrenergic and beta-adrenergic agonist-stimulated beta-endorphin release and cAMP production in hypothalamic cells in primary cultures.

We have previously shown that low concentrations of ethanol rapidly stimulate beta-endorphin (beta-EP) release from hypothalamic neurons in primary cultures and that chronic exposures to these concentrations of ethanol desensitize beta-EP neurons to ethanol challenges. We have also shown that chronic ethanol desensitizes dibutyryl cAMP-, adenosine-, and prostaglandin E1-stimulated beta-EP release and the cAMP content in hypothalamic neurons. In this study, we determined the effects of ethanol (50 mM) on beta-adrenergic agonist (isoproterenol) or alpha-adrenergic agonist (l-phenylephrine)-induced beta-EP release and cellular contents of cAMP to identify whether ethanol causes heterologous desensitization of the adenylate cyclase system in this neuronal cell population. Both isoproterenol and l-phenylephrine increased beta-EP levels in culture media and elevated the cAMP content in cell extracts in a concentration (0.1 and 10 microM)-dependent fashion between 3 to 6 hr. A 50 mM dose of ethanol increased beta-EP and cAMP levels at 3 hr, but it did not elevate beta-EP and cAMP levels after 48 hr of exposure. Acute exposure (3 hr) of these cells to ethanol moderately enhanced the isoproterenol-stimulated and l-phenylephrine-stimulated levels of media beta-EP and intracellular levels of cAMP. However, chronic exposure (48 hr) to ethanol reduced the magnitude of both alpha- and beta-adrenergic receptor agonist-stimulated beta-EP release and cAMP production. These results confirm our previous findings that the ethanol action on beta-EP secretion is mediated by the cAMP system and further suggest that chronic ethanol causes heterologous desensitization of the adenylate cyclase system in the beta-EP neuronal cell population.

Adrenergic alpha-Agonists↗
PubMed1999

NO-Evoked macrophage apoptosis is attenuated by cAMP-induced gene expression.

BACKGROUND: Previous work has suggested that an increase in expression of cyclooxygenase-2, concomitant formation of E-type prostanoids, and in turn intracellular cAMP conveys macrophage resistance against apoptosis. MATERIALS AND METHODS: We analyzed the effects of lipophilic cAMP analogs on nitric oxide (NO)-induced apoptosis in RAW 264.7 macrophages and human primary monocyte-derived macrophages. Parameters comprised DNA fragmentation (diphenylamine assay), annexin V staining of phosphatidylserine, caspase activity (quantitated by the cleavage of a fluorogenic caspase-3-like substrate Ac-DEVD-AMC), and mitochondrial membrane depolarization (DeltaPsi), analyzed using DiOC(6)(3). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c, and expression of the antiapoptotic protein Bcl-X(L). A cAMP response-element decoy approach confirmed cAMP-dependent gene induction. RESULTS: We verified resistance of murine and human macrophages against NO donors such as S-nitrosoglutathione or spermine-NO by pre-exposing cells to lipophilic cAMP analogs or by pretreatment with lipopolysaccaride, interferon-gamma, and N(G)-nitroarginine-methylester for 15 hr. Cellular prestimulation decreased NO-evoked apoptosis, as apoptotic parameters were basically absent. Macrophage protection was not achieved during a short period of preexposure, i.e., 1 hr. To verify gene induction as the underlying protective principle, we treated RAW cells with oligonucleotides containing a cAMP-responsive element in order to scavenge cAMP response element-binding protein prior to its promoter-activating ability. Decoy oligonucleotides, but not an unrelated control oligonucleotide, weakened cAMP-evoked protection and re-established a p53 response following NO addition. CONCLUSION: Gene induction by cAMP protects macrophages against apoptosis that occurs as a result of excessive NO formation. Decreasing programmed cell death of macrophages may perpetuate inflammatory conditions in humans when macrophages become activated in close association with innate immune responses.

Animals↗
PubMed2000

cAMP-dependent plasticity at excitatory cholinergic synapses in Drosophila neurons: alterations in the memory mutant dunce.

It is well known that cAMP signaling plays a role in regulating functional plasticity at central glutamatergic synapses. However, in the Drosophila CNS, where acetylcholine is thought to be a primary excitatory neurotransmitter, cellular changes in neuronal communication mediated by cAMP remain unexplored. In this study we examined the effects of elevated cAMP levels on fast excitatory cholinergic synaptic transmission in cultured embryonic Drosophila neurons. We report that chronic elevation in neuronal cAMP (in dunce neurons or wild-type neurons grown in db-cAMP) results in an increase in the frequency of cholinergic miniature EPSCs (mEPSCs). The absence of alterations in mEPSC amplitude or kinetics suggests that the locus of action is presynaptic. Furthermore, a brief exposure to db-cAMP induces two distinct changes in transmission at established cholinergic synapses in wild-type neurons: a short-term increase in the frequency of spontaneous action potential-dependent synaptic currents and a long-lasting, protein synthesis-dependent increase in the mEPSC frequency. A more persistent increase in cholinergic mEPSC frequency induced by repetitive, spaced db-cAMP exposure in wild-type neurons is absent in neurons from the memory mutant dunce. These data demonstrate that interneuronal excitatory cholinergic synapses in Drosophila, like central excitatory glutamatergic synapses in other species, are sites of cAMP-dependent plasticity. In addition, the alterations in dunce neurons suggest that cAMP-dependent plasticity at cholinergic synapses could mediate changes in neuronal communication that contribute to memory formation.

Animals↗
PubMed2000

Preglomerular microcirculation expresses the cAMP-adenosine pathway.

UNLABELLED: The purpose of this study was to investigate whether the extracellular cAMP-adenosine pathway (i.e., transport of cAMP out of cells followed by extracellular conversion of cAMP to adenosine) exists in preglomerular microvessels (PGMVs). Incubation of PGMVs for 1 h with 30 microM cAMP increased the amount of extracellular adenosine from 163 +/- 18.6 (n = 18) to 9810 +/- 604 (n = 12) pmol/mg of protein (P < 10(-6)). The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM; n = 6) and the ecto-phosphodiesterase inhibitor 1, 3-dipropyl-8-p-sulfophenylxanthine (DPSPX; 1 mM; n = 6) significantly (P < 10(-6) and P < 10(-5), respectively) reduced the cAMP-induced increase in extracellular adenosine. Incubation of PGMVs for 1 h with isoproterenol (beta-adrenoceptor agonist; 1 microM) + IBMX (0.1 mM) increased the amount of extracellular cAMP from 0.800 +/- 0.047 to 22.3 +/- 2.20 pmol/mg of protein (P < 10(-6); n = 41). In PGMVs incubated with isoproterenol (1 microM) + IBMX (0.1 mM) for 1 h, there was a significant (P < 10(-4)) linear (r(2) = 0.6) relationship between intracellular and extracellular cAMP levels. Incubation of PGMVs for 1 h with 1 microM isoproterenol increased the amount of extracellular adenosine from 163 +/- 18.6 (n = 18) to 297 +/- 38.3 (n = 12) pmol/mg of protein (P =.002). Propranolol (beta-adrenoceptor antagonist; 1 microM; n = 7), IBMX (1 mM; n = 14), and DPSPX (1 mM; n = 12) blocked (P =.037, P =.015, and P =.026, respectively) isoproterenol-induced increases in extracellular adenosine. CONCLUSIONS: PGMVs transport endogenous cAMP to the extracellular compartment and metabolize extracellular cAMP to adenosine. This pathway can increase extracellular levels of adenosine during beta-adrenoceptor activation of adenylyl cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Attenuation of macrophage apoptosis by the cAMP-signaling system.

Previous studies revealed that expression and activation of cyclooxygenase-2 (Cox-2) conveyed a protective principle in murine macrophages, thus attenuating pro-apoptotic actions of chemotherapeutic agents or programmed cell death as a result of massive nitric oxide (NO) generation. Expression of Cox-2 was achieved by treatment of cells with lipopolysaccharide/interferon-gamma or nontoxic doses of NO releasing agents. We reasoned E-type prostanoid formation, and in turn an intracellular cAMP increase as the underlying protective mechanism. To prove our hypothesis, we analyzed the effects of lipophilic cAMP-analogs on NO, cisplatin, or etoposide induced apoptosis in RAW 264.7 macrophages. Selected apoptotic parameters comprised DNA fragmentation (diphenylamine assay), annexin V staining of phosphatidylserine, caspase activity (quantitated by the cleavage of a fluorogenic caspase-3-like substrate Ac-DEVD-AMC), and mitochondrial membrane depolarisation (delta psi). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c to the cytosol, and expression of the anti-apoptotic protein Bcl-xL. Prestimulation with lipophilic cAMP-analogs attenuated apoptosis with the notion that cell death parameters were basically absent. To verify gene induction by cAMP in association with protection we established activation of cAMP response element binding protein (CREB) by gel-shift analysis and moreover, treated macrophages with oligonucleotides containing a cAMP-responsive element (CRE) in order to scavenge CREB. Decoy oligonucleotides, but not control oligonucleotides, attenuated cAMP-evoked protection and reestablished pro-apoptotic parameters. We conclude that gene induction by cAMP protects macrophages towards apoptosis that occurs as a result of excessive NO formation or addition of chemotherapeutica. Attenuating programmed cell death by the cAMP-signaling system may be found in association with Cox-2 expression and tumor formation.

Animals↗
PubMed2001

Genetic correlations between initial sensitivity to Ethanol and brain cAMP signaling in inbred and selectively bred mice.

BACKGROUND: Several lines of evidence have suggested a role for cAMP (adenosine 3',5'-cyclic monophosphate) signaling in the acute and chronic effects of ethanol. This study investigated whether there is a genetic correlation between cAMP synthesis in the brain and the acute effects of ethanol [alcohol sensitivity or acute functional tolerance (AFT)]. METHODS: By using nine inbred strains of mice, we measured initial sensitivity and AFT to ethanol with a test of balance on a dowel. Initial sensitivity was defined by the blood ethanol concentration (BEC0) at the loss of balance on a dowel after an ethanol injection [1.75 g/kg intraperitoneally (ip)]. When mice were able to regain balance on the dowel, BEC1 was determined, and a second ethanol injection was given (2 g/kg ip). Upon final regaining of balance, BEC2 was determined. AFT was defined by the difference between BEC1 and BEC2 (AFT = DeltaBEC = BEC2 - BEC1). Cyclic AMP synthesis was measured in whole-cell preparations in the cerebellum and other brain areas of mice of the nine inbred strains. RESULTS: Significant differences in BEC0 and AFT were seen among the mice of the nine inbred strains. Cerebellar basal and forskolin- and isoproterenol-stimulated cAMP production differed significantly between the strains, and BEC0 was found to correlate significantly with forskolin- and isoproterenol-stimulated cAMP accumulation in the cerebellum (r = 0.70 and 0.94, respectively). When we measured cAMP production in mesencephalic and telencephalic tissue in three strains of mice that differed significantly in isoproterenol-stimulated cAMP accumulation in the cerebellum, significant differences between strains were found only in telencephalic tissue. The relative relationship between the rank order of the three strains for cAMP accumulation in the telencephalon and initial sensitivity to ethanol was identical to that seen with the cerebellum. However, AFT did not correlate with cAMP accumulation in the cerebellum or any other brain area tested. CONCLUSIONS: These results suggest that cAMP-generating systems of the cerebellum and possibly the brain areas contained in telencephalic tissues (e.g., basal ganglia) may have an important relationship to an animal's initial sensitivity to the incoordinating effects of ethanol.

Animals↗
PubMed2000

Modeling the transformation of exogenously supplied cAMP in the perfused rat liver.

A kinetic model describing the behavior of extracellularly supplied cAMP in the perfused rat liver was derived and compared with experimental data. The model was based on the following conditions and assumptions: a) labeled cAMP is being constantly infused (step input); b) permeation of the cell membrane is an essentially irreversible step (k(in) as transfer coefficient); c) the adenine moiety of cAMP incorporates into a nucleotide pool (km1 as transformation coefficient), which cannot permeate the cell membrane; d) the adenine moiety of cAMP can be transferred from the nucleotide pool to a nucleoside + free base pool (km2 as transformation coefficient), which is able to permeate the cell membrane (k(ef) as transfer coefficient). These events were described by a series of differential equations for which an analytical solution was obtained. Total cellular incorporation of label derived from [3H]cAMP was measured in the isolated perfused rat liver. The equations of the model were fitted to these experimental data by means of a least-squares procedure. In the fitting procedure the previously determined k(in) value (0.55 ml min(-1) ml cellular space(-1)) was used. The model is able to describe the experimental data (correlation coefficient = 0.993 +/- 0.008) with km1, km2 and k(ef) values of 17.11, 0.0948 and 1.385 min(-1), respectively. Simulations revealed the following sequence of decreasing intracellular pool sizes: nucleotide pool > nucleoside + free base pool > intracellular cAMP. The intracellular cAMP concentrations correspond to only 3.2% of the extracellular ones. This low proportion explains why it was generally difficult to detect cAMP in the cell space when this compound was added to an isolated cell system. The model and the parameters determined in the present work can be used to predict intracellular cAMP concentrations in the perfused liver for specific extracellular concentrations.

Animals↗