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At least 937 records · Page 52Linked to original sources
PubMed1998

Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: evidence against a cAMP-dependent effect.

In intact guinea pig ventricles, acetylcholine (ACH) has been shown to attenuate the positive inotropic effects of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, by reducing protein phosphorylation without altering cAMP levels. In the present study, we tested the hypothesis that the cAMP-independent inhibitory action of ACH is also evident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PLB) were determined in unlabeled and 32P-labeled guinea pig ventricular cardiomyocytes, respectively. IBMX increased PKA activity ratio and phosphorylation of PLB in a dose-dependent manner. When cardiomyocytes were incubated simultaneously with IBMX (0-1 mM) and ACH (2 microM), ACH attenuated PLB phosphorylation stimulated by low concentration (1O-100 microM) but not by high concentrations (> 200 microM) of IBMX. EC50 value for IBMX-induced phosphorylation of PLB was 32 +/- 6 microM and increased nearly 3-fold after addition of ACH while PKA activity ratio remained unchanged. The rank order of cyclic nucleotide derivatives to phosphorylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibutyryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAMP. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosphorylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of ACH on PLB phosphorylation cannot be explained by a reduction in IBMX-stimulated cAMP levels but may involve the activation of protein phosphatases.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Diminished response of ovarian cAMP to luteinizing hormone in experimental uremia.

In prepuberal female rats with acute bilateral nephrectomy or chronic subtotal nephrectomy, the increase of ovarian cAMP concentration in response to submaximal doses of luteinizing hormone (LH 10 micrograms) and human chorionic gonadotropine (hCG 2.5 IU) was diminished (CO + 2.5 IU hCG 488 +/- 49 pmoles cAMP/mg protein; NX + 2.5 IU hCG 366 +/- 56. P less than 0.05). The cAMP response to follicle stimulating hormone (FSH) was unchanged. The abnormality was found both after administration of LH in vivo and incubation of ovaries with LH in vitro. Similarly, plasma estradiol concentrations in response to submaximal hCG stimulation were diminished. Basal cAMP concentrations and cAMP concentrations after maximal stimulation were unchanged. The defect was observed both in ovaries of untreated prepuberal rats, of pregnant mare serum (PMS)-treated rats (follicular phase) and PMS/hCG-treated rats (luteal phase). Diminished ovarian cAMP response to LH was observed both in parathyroid intact and in parathyroidectomized rats. Administration of 1,25(OH)2D3 in physiological doses (60 ng/kg) to acutely uremic rats restored diminished ovarian cAMP response to submaximal LH stimulation irrespective of parathyroid status. The effect of 1,25(OH)2D3 could not be reproduced by hypercalcemia resulting from intraperitoneal calcium injection. In vivo administration of indomethacin further diminished ovarian cAMP response in uremic animals and had no effect in control animals. Incubation of ovaries with PGE1 and PGE2 increased basal and stimulated cAMP concentrations and abolished the difference between control and uremic animals. The diminished response of ovarian cAMP content to submaximal doses of hCG was not corrected by bromocriptine (1 mg/kg) despite normalization of hyperprolactinemia. The present study shows diminished ovarian cAMP and plasma estradiol response to LH in experimental uremia. It documents a role of 1,25(OH)2D3 and prostaglandins in the genesis of this abnormality.

Alprostadil↗
PubMed1993

ADH resistance of LLC-pk1 cells caused by overexpression of cAMP-phosphodiesterase type-IV.

The studies of animal models of nephrogenic diabetes insipidus (NDI) suggest that abnormally high activity of cAMP phosphodiesterase (cAMP-PDE), may cause unresponsiveness to the diuretic effect of AVP. We explored whether overexpression of one of the cAMP-PDE type isozymes, PDE-IV, in [8-Arg]-vasopressin (AVP) sensitive renal epithelial LLC-PK1 cells can prevent the hormone-elicited cAMP increase. LLC-PK1 cells were stably transfected with ratPDE3.1 cDNA (which encodes for rolipram-sensitive PDE-IV), inserted in plasmid pCMV5 and then were compared with sham-transfected LLC-PK1 cells and wild LLC-PK1 cells. In the stably transfected clone (LLC-PK1-S #16), the rolipram-sensitive PDE-IV activity was about five times higher than in controls, whereas activities of other types of PDEs were not different. The presence of cognate mRNA for PDE-IV was confirmed by Northern blot. Whereas in the control cells (wild LLC-PK1 cells and sham-transfected LLC-PK1 cells), the incubation with 10(-7) M AVP increased cAMP more than tenfold, the LLC-PK1-S#16 cells with overexpressed cAMP-PDE were resistant to cAMP-increasing effects of AVP and forskolin. However, in the same LLC-PK1-S#16 cells the cGMP increases in response to nitroprusside were not diminished. The AVP-dependent cAMP accumulation in LLC-PK1-S#16 cells with overexpressed PDE-IV was restored by addition of roliprams which decreased cAMP-PDE activity to the levels similar to those in wild LLC-PK1 cells and sham-transfected LLC-PK1-#A1 cells. In contrast, inhibitors of other PDE isozymes (PDE-I or PDE-III) had little or no effect. Our findings show that excessive activity of cAMP-PDE, in this case of isozyme PDE-IV, can cause resistance to AVP which is analogous to that observed in collecting ducts of mice with hereditary nephrogenic diabetes insipidus.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

Constitutive activation of Galphas within forebrain neurons causes deficits in sensorimotor gating because of PKA-dependent decreases in cAMP.

Sensorimotor gating, the ability to automatically filter sensory information, is deficient in a number of psychiatric disorders, yet little is known of the biochemical mechanisms underlying this critical neural process. Previously, we reported that mice expressing a constitutively active isoform of the G-protein subunit Galphas (Galphas(*)) within forebrain neurons exhibit decreased gating, as measured by prepulse inhibition of acoustic startle (PPI). Here, to elucidate the biochemistry regulating sensorimotor gating and to identify novel therapeutic targets, we test the hypothesis that Galphas(*) causes PPI deficits via brain region-specific changes in cyclic AMP (cAMP) signaling. As predicted from its ability to stimulate adenylyl cyclase, we find here that Galphas(*) increases cAMP levels in the striatum. Suprisingly, however, Galphas(*) mice exhibit reduced cAMP levels in the cortex and hippocampus because of increased cAMP phosphodiesterase (cPDE) activity. It is this decrease in cAMP that appears to mediate the effect of Galphas(*) on PPI because Rp-cAMPS decreases PPI in C57BL/6J mice. Furthermore, the antipsychotic haloperidol increases both PPI and cAMP levels specifically in Galphas(*) mice and the cPDE inhibitor rolipram also rescues PPI deficits of Galphas(*) mice. Finally, to block potentially the pathway that leads to cPDE upregulation in Galphas(*) mice, we coexpressed the R(AB) transgene (a dominant-negative regulatory subunit of protein kinase A (PKA)), which fully rescues the reductions in PPI and cAMP caused by Galphas(*). We conclude that expression of Galphas(*) within forebrain neurons causes PPI deficits because of a PKA-dependent decrease in cAMP and suggest that cAMP PDE inhibitors may exhibit antipsychotic-like therapeutic effects.

Acoustic Stimulation↗
PubMed1998

MMSP tumor cells expressing the EWS/ATF1 oncogene do not support cAMP-inducible transcription.

Malignant Melanoma of Soft Parts (MMSP) is associated with the EWS/ATF1 fusion protein that arises due to chromosomal fusion of the Ewings Sarcoma oncogene (EWS) and the cellular transcription factor ATF1. EWS/ATF1 can activate several cAMP-inducible promoters, suggesting that cellular transformation in MMSP might involve constitutive activation of cAMP-inducible promoters. To assess this possibility we have examined the status of the cAMP-signaling pathway in the available MMSP-derived cell lines (DTC1 and Su-ccs-1) and find that both cell lines share several features. First, in contrast to previous effects observed in transient assays, three chromosomal promoters containing ATF binding sites are not constitutively activated by endogenous EWS/ATF1 in MMSP cells. Second, all the components that are known to be required for cAMP-inducible transcription are present. Third, phosphorylation of the cAMP-response-element-binding protein (CREB) can be efficiently induced by cAMP. Fourth, cAMP is unable to activate transcription, as assessed by a GAL4/ATF1 reporter assay and analysis of the c-fos and adenovirus early promoters. Thus, cell lines derived from MMSP have a block to cAMP-signaling that lies downstream of CREB phosphorylation. In light of the cAMP-responsiveness of almost all mammalian cell types, our findings suggest that the inability to respond to cAMP might be an important feature of MMSP cells.

Activating Transcription Factor 1↗
PubMed1998

Etoposide and cisplatin induced apoptosis in activated RAW 264.7 macrophages is attenuated by cAMP-induced gene expression.

Previous observations suggest expression of cyclooxygenase-2 to convey macrophage protection towards apoptotic cell death. We reasoned prostaglandin formation and in turn a cAMP increase as the underlying protective principle. Here we report that exposure of macrophages to lipopolysaccharide/interferon-gamma or lipophilic cAMP analogs such as dibutyryl-cAMP or 8-bromo-cAMP for 15 h attenuated DNA fragmentation and accumulation of the tumor suppressor p53 in response to the chemotherapeutic agents cisplatin and etoposide, compared to cells that received chemotherapeutic agents only. In contrast, a 1 h lasting preexposure period revealed no protection. The demand for a long incubation period with cAMP-derivates implied cAMP-mediated gene activation as the underlying principle. Therefore, we treated cells with oligonucleotides containing a cAMP-response element (CRE) binding site. Using this decoy-approach we scavaged activated cAMP response element binding protein prior to its promoter activating ability. Incubating macrophages with decoy, but not with control oligonucleotides, reduced cAMP evoked protection and simultaneously restored p53 accumulation in response to chemotherapeutic agents. Our studies demonstrate that cAMP-initiated gene activation regulates the sensitivity towards DNA damaging agents via inhibition of a p53 dependent pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Role of the adenosine 3',5'-cyclic monophosphate (cAMP) in enhancing the efficacy of siRNA-mediated gene silencing in neuroblastoma cells.

Gene-silencing activity mediated by siRNA has been demonstrated in mammalian cells; however, the mechanism of its regulation is not well understood. Since downregulation of a number of genes occurs during adenosine 3',5'-cyclic monophosphate (cAMP)-induced differentiation of neuroblastoma (NB) cells, it is possible that cAMP may play a role in regulating siRNA activity during differentiation. To study this, we utilized an NB cell line (NBP2-PN25) that expresses a short-lived green fluorescent protein (d2EGFP) under the CMV promoter. These cells were transfected with a retroviral plasmid that expresses U6 promoter-driven expression of siRNA targeted to d2EGFP and then were treated with cAMP-elevating agents (200 microg/ml RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and 1 microg/ml prostaglandin A1, a stimulator of adenylate cyclase) for 2 or 24 h. The siRNA activity was measured by determining the level of intensity of d2EGFP protein by flow cytometry, and the level of d2EGFP mRNA by real-time PCR. The results showed that cAMP-elevating agents enhanced U6-driven siRNA activity directed towards d2EGFP in NB cells 24 h after treatment. One of the mechanisms of action of cAMP is mediated via phosphatidylinositol 3-kinase (PI3K) inhibition; therefore, we have investigated the effect of a PI3K inhibitor on siRNA activity. This study showed that inhibition of PI3K also enhanced U6-driven siRNA activity towards d2EGFP. cAMP-stimulating agents increased U6 transcript levels, perhaps suggesting that increased siRNA activity may in part be due to an increase in transcriptional activity. When NB cells were transfected with a synthetic siRNA directed to d2EGFP, both cAMP elevation and PI3K inhibition similarly enhanced siRNA activity. Sodium butyrate, which inhibits the growth of NB cells similar to the effect produced by cAMP, did not affect U6-driven siRNA activity towards d2EGFP. Protein kinase C (PKC) activation or inhibition also failed to affect siRNA activity in NB cells. This study also showed that cAMP elevation and PI3K inhibition increases U6-driven siRNA activity directed towards an endogenous gene, p53. Our data suggest a role for the cAMP pathway in affecting the efficacy of siRNA system during differentiation of NB cells.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1996

Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.

3T3 Cells↗
PubMed1996

Mechanism responsible for glucose-lactose diauxie in Escherichia coli: challenge to the cAMP model.

BACKGROUND: The inhibition of beta-galactosidase expression in glucose-lactose diauxie is a typical example of the glucose effect in Escherichia coli. It is generally believed that glucose exerts its effect at least partly by reducing the intracellular cAMP level. However, there is no direct evidence that the inhibitory effect of glucose on the expression of the lac operon is mediated by a reduction of the cAMP level in the glucose-lactose system. RESULTS: To examine the roles of cAMP and the cAMP receptor protein (CRP) in the glucose effect, the intracellular levels of these factors were determined during diauxic growth in a glucose-lactose medium. We found that the levels of cAMP and CRP in a lactose-grown phase were not higher than those in a glucose-grown phase, although the cAMP levels increased transiently during the lag phase. The addition of exogenous cAMP eliminated diauxic growth but did not eliminate glucose repression. Glucose repression and diauxie were observed in cells which lack cAMP but produce a cAMP-independent CRP. In addition, inactivation of the lac repressor by the disruption of the lacI gene or the addition of IPTG, eliminated glucose repression. CONCLUSION: We conclude that the repression of beta-galactosidase expression by glucose is not due to the reduction of the cAMP-CRP level but due to an inducer exclusion mechanism which is mediated by the phosphoenolpyruvate-dependent sugar phosphotransferase system.

Cyclic AMP↗
PubMed2006

cAMP activation of PKA defines an ancient signaling mechanism.

cAMP and the cAMP binding domain (CBD) constitute a ubiquitous regulatory switch that translates an extracellular signal into a biological response. The CBD contains alpha- and beta-subdomains with cAMP binding to a phosphate binding cassette (PBC) in the beta-sandwich. The major receptors for cAMP in mammalian cells are the regulatory subunits (R-subunits) of PKA where cAMP and the catalytic subunit compete for the same CBD. The R-subunits inhibit kinase activity, whereas cAMP releases that inhibition. Here, we use NMR to map at residue resolution the cAMP-dependent interaction network of the CBD-A domain of isoform Ialpha of the R-subunit of PKA. Based on H/D, H/H, and N(z) exchange data, we propose a molecular model for the allosteric regulation of PKA by cAMP. According to our model, cAMP binding causes long-range perturbations that propagate well beyond the immediate surroundings of the PBC and involve two key relay sites located at the C terminus of beta(2) (I163) and N terminus of beta(3) (D170). The I163 site functions as one of the key triggers of global unfolding, whereas the D170 locus acts as an electrostatic switch that mediates the communication between the PBC and the B-helix. Removal of cAMP not only disrupts the cap for the B' helix within the PBC, but also breaks the circuitry of cooperative interactions stemming from the PBC, thereby uncoupling the alpha- and beta-subdomains. The proposed model defines a signaling mechanism, conserved in every genome, where allosteric binding of a small ligand disrupts a large protein-protein interface.

Binding Sites↗
PubMed1982

Genetic analysis of the role of cAMP in mediating effects of interferon.

The effects of interferon (IFN) on Fc receptor-mediated phagocytosis, intracellular cAMP levels, antiviral activity, and growth inhibition were analyzed in a cloned macrophage-like cell line, J774.2, and variants derived from it. Purified IFN increased Fc receptor-mediated phagocytosis in J774.2 cells, and in cAMP-responsive nonphagocytic variants but was without effect in cAMP-unresponsive nonphagocytic variants, in adenylate cyclase-deficient variants, and in cAMP-dependent protein kinase-deficient variants. Under conditions in which IFN augmented phagocytosis, it increased intracellular levels of cAMP. Parental cells were highly sensitive to IFN-mediated growth inhibition. In contrast, cAMP-dependent protein kinase-deficient variants were only 1/100th as sensitive to growth inhibition by IFN. All cell lines tested, both responsive and unresponsive to cAMP, were equally protected by IFN against infection with vesicular stomatitis virus, demonstrating that the antiviral state was independent of cAMP. These results indicate that, in transformed macrophages, stimulation of phagocytosis and inhibition of growth by IFN are mediated through intracellular cAMP, whereas the antiviral state induced by IFN is independent of cAMP.

Adenylyl Cyclases↗
PubMed1986

A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation.

We describe the gene M4-1, whose unique pattern of developmental expression will allow us to study the molecular mechanisms controlling expression in undifferentiated cells in addition to repression in response to cAMP during development and reinduction during dedifferentiation. M4-1 is a Dictyostelium gene expressed in the undifferentiated cell. We have shown that M4-1 continues to be expressed very early during the developmental cycle but is repressed at a later stage of development, at a time coincident with the establishment of oscillations in the cAMP pool. Studies on the expression of the M4-1 gene in shaking culture, under conditions that mimic early development, have established that pulsatile stimulation of cells with cAMP is sufficient to repress M4-1 expression. Consistent with this, cells that are exposed to high levels of cAMP are unable to respond normally to cAMP oscillations and continue to express M4-1 at vegetative levels. These data indicate that low-level oscillations of cAMP are required for the repression of M4-1 expression rather than the continuous high levels of cAMP responsible for the regulation of a different class of Dictyostelium genes. We suggest that cAMP may mediate developmental expression of the Dictyostelium genome by different mechanisms. We also show that cell-cell interaction, a developmental event that occurs subsequent to the cAMP pulse, does not normally influence the regulation of M4-1. Finally, we have shown that when cAMP-pulsed cells are induced to dedifferentiate, M4-1 RNA sequences rapidly reappear in nuclei and cytoplasm, suggesting that regulation of M4-1 expression is primarily mediated at the level of transcription.

Cell Aggregation↗
PubMed1990

Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase.

Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway.

Biological Transport↗
PubMed1997

Compartmentalization of cAMP signaling in mesangial cells by phosphodiesterase isozymes PDE3 and PDE4. Regulation of superoxidation and mitogenesis.

Some major pathobiologic processes in renal mesangial cells, elicited in response to immunoinflammatory stimuli, are modulated via cAMP-protein kinase A (PKA) signaling pathways; namely, generation of reactive oxygen metabolites (ROM) and accelerated proliferation of mesangial cells. We investigated the role of cAMP phosphodiesterase (PDE) isozymes in these regulatory mechanisms. Generation of ROM in cultured rat mesangial cells was inhibited by selective inhibitors of PDE4, rolipram and denbufylline, whereas PDE3 inhibitors, cilostamide and lixazinone, had no effect. Conversely, cilostamide or lixazinone suppressed mitogenic synthesis of DNA in mesangial cells, but 1 microM rolipram or 1 microM denbufylline showed no inhibitory effect. The efficacy of PDE isozyme inhibitors (IC50) to suppress [3H]thymidine incorporation or ROM generation paralleled IC50 values for inhibition of cAMP PDE. Incubation of mesangial cells with either rolipram alone or with cilostamide alone increased significantly in situ activity of PKA in mesangial cells, assessed by (-cAMP/+cAMP) PKA activity ratio, and the stimulatory effects were additive. Results indicate that in mesangial cells a cAMP pool that is metabolized by PDE4 activates PKA and thereby inhibits ROM generation; another cAMP pool that is metabolized by PDE3 activates another PKA (isozyme or pool) which suppresses proliferation of mesangial cells. We propose that in mesangial cells, a cAMP-PKA pathway that regulates mitogenesis is determined by activity of PDE3, whereas another cAMP-PKA pathway is directed by activity of PDE4 and controls ROM generation. Therefore, two PDE isozymes within one cell type compartmentalize distinct cAMP signaling pathways.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I.

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.

Amino Acid Sequence↗
PubMed2000

The localization and activity of cAMP-dependent protein kinase affect cell cycle progression in thyroid cells.

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases (PKAs), typically determined by their specific regulatory subunits. We describe changes in the cAMP signal transduction pathway during cell cycle progression in synchronized rat thyroid cells. Both PKA type II (PKAII) localization and nuclear cAMP signaling are significantly modified during G(0) and G(1)-S transitions. G(1) is characterized by PKA activation and amplified cAMP signal transduction. This is associated with a decrease in the concentration of RI and RII regulatory subunits and enhanced anchoring of PKAII to the Golgi-centrosome region. Just prior to S, the cAMP pathway is depressed. Up-regulation of the pathway by exogenous cAMP in G(1) inhibited the subsequent decay of the Cdk inhibitor p27 and delayed the onset of S phase. Forced translocation of endogenous PKAII to the cytosol down-regulated cAMP signaling, advancing the timing of p27 decay and inducing premature exit from G(1). These data indicate that membrane-bound PKA amplifies the transduction of cAMP signals in G(1) and that the length of G(1) is influenced by cAMP-PKA.

Animals↗
PubMed2000

Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation.

Together with a transient accumulation of intracellular cAMP, thyrotropin (TSH) stimulation of the FRTL-5 thyroid cell induces phosphorylation and activation of a cAMP-specific phosphodiesterase (PDE4D3). Here we have investigated the impact of PDE4D3 activation on hormone responsiveness. Stimulation of FRTL-5 cells with TSH caused an increase in PDE activity within 3 min, with a maximal stimulation reached after 5 min. Preincubation with the protein kinase A (PKA) inhibitor H89 or (R(p))-cAMPS, but not with the inactive isomer H85, blocked this activation. Preincubation with PKA inhibitors also blocked the shift in mobility of the PDE4D3 protein. Under these conditions, H89, but not H85, potentiated the cAMP accumulation induced by TSH. Incubation of FRTL-5 cells with the PKA activator 8-(4-chlorophenylthio)adenosine-cAMP caused an increase in PDE activity and a decrease in the endogenous cAMP, confirming the presence of a PKA-PDE feedback loop. MA-10 Leydig tumor cells stably transfected with either a wild type PDE4D3 or a PDE4D3 with mutations in the PKA phosphorylation sites showed an increase in PDE activity when compared with control cells. Human choriogonadotropin or Bt(2)cAMP treatment induced a stimulation of PDE activity in cells transfected with wild type PDE4D3, whereas the activation was absent in mutant- and control-transfected cells. The increase in cAMP accumulation elicited by human choriogonadotropin was reduced in cells transfected with the wild type PDE4D3, but not in cells transfected with the mutant PDE. Rolipram, a specific inhibitor of PDE4, restored the cAMP accumulation in the PDE4D3-transfected cells. These data provide evidence that a rapid activation of PDE4D3 is one of the mechanisms determining the intensity of the cAMP signal.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Inhibition by extracellular cAMP of phorbol 12-myristate 13-acetate-induced prostaglandin H synthase-2 expression in human pulmonary microvascular endothelial cells. Involvement of an ecto-protein kinase A activity.

Exposure of human pulmonary microvascular endothelial cells (HPMECs) to phorbol 12-myristate 13-acetate (PMA) leads to the increase of prostaglandin H synthase (PGHS)-2 protein levels. Under same conditions and according to its constitutive nature, no significant variation of PGHS-1 protein was noted. The elevation of the intracellular cAMP rate is known to enhance PGHS-2 levels through a protein kinase A pathway in various cells. To determine whether the extracellular cAMP also regulates the inducible expression of PGHS, cultured HPMECs were exposed to cAMP alone or in combination with PMA. The PMA-induced PGHS-2 protein was attenuated by the extracellular cAMP. In addition, PGHS-2 activity evaluated through 6-keto-PGF1alpha generation, which was enhanced by PMA was inhibited by extracellular cAMP. Furthermore, in HPMEC medium, PMA-induced PGHS-2 expression was accompanied by the generation of a transferable activity (TA) able to abolish platelet aggregation. This resulting TA was dependent from PGHS-2 pathway, because NS-398, a selective inhibitor of PGHS-2, suppressed its production. The inhibitory TA released by treated HPMECs was also prevented by extracellular cAMP. The specific protein kinase A (PKA) inhibitor blocked the extracellular cAMP effect on both PMA-induced 6-keto-PGF1alpha synthesis and inhibitory TA generation, suggesting the involvement of PKA signaling at the outer surface of HPMECs. Accordingly, we established, in phosphorylation experiments, the presence of an endothelial ecto-protein kinase activity, able to phosphorylate the synthetic substrate kemptide in a cAMP-dependent mode. Reverse transcription-polymerase chain reaction analysis showed that PMA-induced PGHS-2 mRNA was markedly reduced by extracellular cAMP. Together, these findings provide the first experimental evidence that extracellular cAMP is able to reduce HPMEC PGHS-2 expression in terms of mRNA, protein, and enzyme activity through an ecto-PKA pathway. In addition, they outline the potential role of endothelial PGHS-2 in the limitation of platelet activation during inflammatory processes.

Carcinogens↗