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PubMedDate not supplied

Opposing effects on modulation of angiogenesis by protein kinase C and cAMP-mediated pathways.

The role of cAMP-mediated pathway in modulating angiogenesis was investigated. We have shown previously that activators of protein kinase C (PKC) caused a marked increase in angiogenesis, while the specific inhibitor of PKC, Ro 318220 suppressed angiogenesis. Here we show that forskolin, which activates adenylate cyclase and elevates the intracellular levels of cAMP, and the Sp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Sp-cAMPS), caused a dose-dependent suppression of collagenous protein biosynthesis and angiogenesis in the chick chorioallantoic membrane system (CAM). The opposite modulation of angiogenesis by activators of PKC and elevated cAMP levels was further confirmed by the suppression of 4 beta-phorbol-12-myristate-13-acetate (4 beta-PMA)-stimulated angiogenesis by either forskolin or Sp-cAMPS. On the contrary, the Rp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Rp-cAMPS), which antagonises endogenous cAMP biochemical actions, had no effect on angiogenesis alone and did not suppress 4 beta-PMA stimulated angiogenesis. However, Rp-cAMPS antagonised the effect of forskolin and Sp-cAMPS on 4 beta-PMA induced angiogenesis. Similar results were obtained in the human umbilical vein endothelial cell tube formation assay. In this system, the PKC inhibitor, Ro 318220, caused a dose-dependent inhibition of 4 beta-PMA reversed this effect. Also, forskolin and Sp-cAMPS caused an inhibition in tube formation. These results indicate that increased levels of intracellular cAMP have a negative effect in normal angiogenesis and cause a large reduction of the promotion of angiogenesis resulting from PKC activation.

Allantois↗
PubMed1997

Localization of low-KM cAMP phosphodiesterase in rat nephron segments.

To evaluate the roles of cAMP degradation on hormonal actions in the kidney, we examined the segmental distribution and activities of cAMP-phosphodiesterase (PDE), a key enzyme for cAMP hydrolysis, in dissected segments of the rat nephron and characterized the isozyme compositions with subtype-specific inhibitors. Summary of the nephron distribution of PDE activities showed that cAMP-PDE activities were detected in all nephron segments and the distal convoluted tubules (DCTs) had the highest activity. Both in proximal convoluted tubules (PCTs) and medullary collecting ducts (MCDs), more than 80% of the total cAMP-PDE activities were inhibited by a nonspecific PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). In PCTs, rolipram (type-IV PDE inhibitor) was an equally potent inhibitor of IBMX, while 8-methoxymethyl-IBMX (MM-IBMX, type-I PDE inhibitor) and cilostamide (type-III PDE inhibitor) inhibited cAMP-PDE by 35 and 57%, respectively. In MCDs, inhibition by rolipram (40%) was less than that by MM-IBMX (70%) or cilostamide (66%). Rolipram potentiated the parathyroid hormone (PTH)-induced cAMP content in PCTs most effectively. Rolipram and MM-IBMX increased the vasopressin (AVP)-stimulated cAMP content equally in MCDs. Cilostamide had no effect on the cAMP content stimulated by PTH or AVP in PCTs and MCDs. These results indicate that PDE activities were unevenly distributed along the rat nephron and cAMP-PDE was composed of at least three isoforms, namely type I, III and IV in PCTs and MCDs. Among these, type-IV PDE was responsible for hydrolysis of cAMP in PCTs, and type-I and IV PDE were responsible for that in MCDs.

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

Cardiac fibroblasts express the cAMP-adenosine pathway.

The extracellular "cAMP-adenosine pathway" refers to the local production of adenosine mediated by cAMP egress into the extracellular space, conversion of cAMP to AMP by ectophosphodiesterase, and the metabolism of AMP to adenosine by ecto-5'-nucleotidase. The goal of this study was to assess whether the cAMP-adenosine pathway limits cardiac fibroblast growth. Studies were conducted in ventricular cardiac fibroblasts maintained in 3-dimensional cultures. Addition of exogenous cAMP to cardiac fibroblasts increased extracellular levels of AMP, adenosine, and inosine in a concentration-dependent and time-dependent manner. This effect was attenuated by blockade of total phosphodiesterase activity (3-isobutyl-1-methylxanthine), ectophosphodiesterase activity (high concentration of 1, 3-dipropyl-8-p-sulfophenylxanthine), or ecto-5'-nucleotidase (alpha, beta-methylene-adenosine-5'-diphosphate). Treatment with exogenous cAMP inhibited cell growth as assessed by DNA synthesis ((3)H-thymidine incorporation), cell proliferation (cell counts), and protein synthesis ((3)H-leucine incorporation). Antagonism of A(2) (KF17837) or A(1)/A(2) (low concentration of 1, 3-dipropyl-8-p-sulfophenylxanthine), but not A(1) (8-cyclopentyl-1, 3-dipropylxanthine), adenosine receptors blocked the growth-inhibitory effects of exogenous cAMP, but not the growth inhibitory effects of 8-bromo-cAMP (stable cAMP analogue). The growth-inhibitory effects of exogenous cAMP were enhanced by the combined inhibition of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl) adenine] and adenosine kinase (iodotubercidin). In conclusion, the extracellular cAMP-adenosine pathway exists in cardiac fibroblasts and attenuates cell growth. Pharmacological augmentation of this pathway could abate pathological cardiac remodeling in heart disease.

1-Methyl-3-isobutylxanthine↗
PubMed2006

A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes.

Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-AR regulation of I(Ca,L), whereas only PDE4 controlled Glu-R regulation of I(Ca,L) thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.

Animals↗
PubMed1996

Functional and structural assessment of intercellular communication. Increased conduction velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes.

Remodeling of conduction pathways in the hypertrophic response to myocardial injury is a potential mechanism leading to the development of anatomic substrates of lethal arrhythmias. To delineate the responsible mechanisms and to directly relate changes in intercellular coupling at gap junctions with electrophysiological alterations, we studied the effects of cAMP, a mediator of cardiac hypertrophy, on action potential conduction velocity and connexin expression in neonatal rat ventricular myocyte cultures. Conduction velocity was measured with an optical activation mapping technique in cells loaded with the voltage-sensitive dye RH-237. Action potentials were conducted 24% to 29% more rapidly (P < .005) after incubating cultures for 24 hours with the cAMP analogue dibutyryl cAMP (db-cAMP, 1 mmol/L). However, db-cAMP caused no change in the maximum rate of rise of the action potential upstroke, Vmax. Electron and immunofluorescence microscopy revealed a significant increase in the number and size of gap junctions in db-cAMP-treated cells. Immunoblotting showed that the total amounts of the ventricular gap junction proteins connexin43 and connexin45 (Cx43 and Cx45, respectively) increased 2- to 4-fold. Immuno-precipitation of metabolically labeled connexin proteins revealed a dose-dependent increase in the rate of Cx45 protein synthesis in myocytes exposed to db-cAMP ( > 2-fold after a 4-hour exposure) but no change in the Cx43 synthesis rate. Northern blot analysis demonstrated a time-dependent increase in the amount of Cx43 mRNA, with a maximum 3.3-fold increase after 4 hours of exposure to 1 mmol/L db-cAMP; cycloheximide did not block this effect. In contrast, Cx45 mRNA levels were not altered significantly after db-cAMP treatment. Thus, cAMP causes a significant increase in conduction velocity that appears to be attributable largely to enhanced expression of proteins responsible for intercellular communication. Cx43 and Cx45 levels appear to be upregulated by cAMP by disparate molecular mechanisms.

Action Potentials↗
PubMed2003

Regulatory role of cAMP on expression of Cdk4 and p27(Kip1) by inhibiting phosphatidylinositol 3-kinase in corneal endothelial cells.

PURPOSE: Fibroblast growth factor (FGF)-2 is a potent mitogen of corneal endothelial cells (CECs). Results in an earlier study showed that FGF-2 activates phosphatidylinositol (PI) 3-kinase to stimulate the cell cycle machinery. The current study was designed to determine whether adenosine 3',5'-monophosphate (cAMP) antagonizes FGF-2 by inhibiting PI 3-kinase/Akt pathways, thus leading to regulation of cyclin-dependent kinase 4 (Cdk4) and p27(Kip1) (p27) expression. METHODS: Cell proliferation was assayed by counting cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of Cdk4, p27, PI 3-kinase, Akt, and beta-actin was analyzed by immunoblot analysis. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. RESULTS: 8-Bromoadenosine cAMP (8-Br-cAMP), a diffusible cAMP analogue, inhibited the PI 3-kinase/Akt signaling pathways. The 8-Br-cAMP and PI 3-kinase inhibitor (LY294002) produced equivalent stimulation and inhibition, respectively, of p27 and Cdk4 protein levels. They also equally inhibited cell proliferation, nuclear translocation of Cdk4, and phosphorylation of p27. Negative regulation of PI 3-kinase by 8-Br-cAMP was mediated by a direct inhibition of PI 3-kinase activity, which subsequently blocked phosphorylation of Akt at both the Ser473 and Thr308 sites. In addition, 8-Br-cAMP promoted a rapid turnover of Akt protein, and 8-Br-cAMP markedly reduced the half-life of Cdk4 protein. This inhibitory activity of cAMP was not mediated by PKA, but 8-Br-cAMP inhibited membrane localization of the p85 regulatory subunit of PI 3-kinase. CONCLUSIONS: These data support the hypothesis that cAMP inhibits the proliferation of CECs, preventing them from entering the S phase by negatively regulating PI 3-kinase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event.

Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.

Animals↗
PubMed1993

Restoration of the cAMP second messenger pathway enhances cardiac preservation for transplantation in a heterotopic rat model.

Current organ preservation strategies subject graft vasculature to severe hypoxia (PO2 approximately 20 Torr), potentially compromising vascular function and limiting successful transplantation. Previous work has shown that cAMP modulates endothelial cell (EC) antithrombogenicity, barrier function, and leukocyte/EC interactions, and that hypoxia suppresses EC cAMP levels. To explore the possible benefits of cAMP analogs/agonists in organ preservation, we used a rat heterotopic cardiac transplant model; dibutyryl cAMP added to preservation solutions was associated with a time- and dose-dependent increase in the duration of cold storage associated with successful graft function. Preservation was also enhanced by 8-bromo-cAMP, the Sp isomer of adenosine 3',5'monophosphorothioate, and types III (indolidan) and IV (rolipram) phosphodiesterase inhibitors. Neither butyrate alone nor 8-bromoadenosine were effective, and the cAMP-dependent protein kinase antagonist Rp isomer of adenosine 3',5'monophosphorothioate prevented preservation enhancement induced by 8-bromo-cAMP. Grafts stored with dibutyryl cAMP demonstrated a 5.5-fold increase in blood flow and a 3.2-fold decreased neutrophil infiltration after transplantation. To explore the role of cAMP in another cell type critical for vascular homeostasis, vascular smooth muscle cells were subjected to hypoxia, causing a time-dependent decline in cAMP levels. Although adenylate cyclase activity was unchanged, diminished oxygen tensions were associated with enhanced phosphodiesterase activity (59 and 30% increase in soluble types III and IV activity, respectively). These data suggest that hypoxia or graft ischemia disrupt vascular homeostasis, at least in part, by perturbing the cAMP second messenger pathway. Supplementation of this pathway provides a new approach for enhancing cardiac preservation, promoting myocardial function, and maintaining vascular homeostatic properties.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Downregulation of mdr-1 expression by 8-Cl-cAMP in multidrug resistant MCF-7 human breast cancer cells.

8-Cl-cAMP, a site-selective analogue of cAMP, decreased mdr-1 expression in multidrug-resistant human breast cancer cells. A sixfold reduction of mdr-1 mRNA expression by 8-Cl-cAMP began within 8 h of treatment and was associated with a decrease in the synthesis of P-glycoprotein and with an increase in vinblastine accumulation. A reduction in mdr-1 expression after 8-Cl-cAMP treatment was also observed in multidrug-resistant human ovarian cancer cell lines. 8-Cl-cAMP is known to change the ratio between the two regulatory subunits, RI and RII, of protein kinase A (PKA). We observed that RI alpha decreased within 24 h of 8-Cl-cAMP treatment, that RII beta increased after as few as 3 h of treatment, and that PKA catalytic activity remained unchanged during 48 h of 8-Cl-cAMP treatment. The results are consistent with the hypothesis that mdr-1 expression is regulated in part by changes in PKA isoenzyme levels. Although 8-Cl-cAMP has been used to differentiate cells in other model systems, the only differentiating effect that could be detected after 8-Cl-cAMP treatment in the MCF-7TH cells was an increase in cytokeratin expression. Evidence that the reduction of mdr-1 mRNA occurred at the level of gene transcription was obtained by measuring chloramphenicol acetyltransferase (CAT) mRNA in MCF-7TH cells transfected with an mdr-1 promoter-CAT construct prior to 8-Cl-cAMP treatment. Thus, 8-Cl-cAMP is able to downregulate mdr-1 expression and suggests a new approach to reversal of drug resistance in human breast cancer.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Transcriptional regulation of the human GnRH II gene is mediated by a putative cAMP response element.

Human neuronal medulloblastoma cells (TE-671) were recently demonstrated to express the two forms of GnRH (GnRH-I and GnRH-II). We have used this cell line as a model system to demonstrate regulation of the human GnRH-II gene by cAMP. RT-PCR and Southern hybridization demonstrated that GnRH-II mRNA is strongly up-regulated ( approximately 6-fold) by (Bu)(2)cAMP. The concentration of GnRH-II that was released into the medium of TE-671 cells treated with the cAMP analog was significantly higher than that of the untreated cells. TE-671 cells that were stimulated by (Bu)(2)cAMP demonstrated morphological changes and strong immunoreactive GnRH-II staining in part of the cell population. After screening of the GnRH-II promoter sequence, we identified a putative cAMP response element consensus site. The GnRH-I and GnRH-II promoters were isolated by PCR using human genomic DNA and cloned into the luciferase reporter plasmid. By measuring the basal activity of the promoters that were transfected to TE-671 cells, we found a much stronger basal activity of the GnRH-II promoter compared with that of GnRH-I. Treatment of transfected TE-671 cells with (Bu)(2)cAMP resulted in a strong activation of the GnRH-II promoter compared with a modest activation of the GnRH-I promoter. To determine the functionality of this putative cAMP response element site, we mutated this site. TE-671 cells that were transfected with cAMP response element mutant constructs demonstrated a diminished basal activity of the GnRH-II promoter. Treatment of the transfected cells with the cAMP analog demonstrated a decrease to 0.03% of the activity of the mutated promoter compared with that of the wild type. These results clearly demonstrate the importance of the putative cAMP response element site for the basal activity as well as for induction of the GnRH-II promoter by cAMP.

Base Sequence↗
PubMed1979

Effects of local anesthesia on gingival cAMP levels.

The basal levels of cAMP in the attached gingiva of Rhesus monkeys and the changes in tissue cAMP levels produced by infiltration anesthesia with lidocaine and lidocaine containing 1:100,000 epinephrine were studied. The basal level of cAMP in uninjected monkey gingiva ranged from 12 to 20 picomoles of cAMP per mg of gingival protein. This level was 75 times greater than the cAMP content of monkey blood plasma. Infiltration of the attached gingiva with saline or plain lidocaine for 5 minutes did not produce any significant changes in tissue cAMP levels. Infiltration of the gingiva with lidocaine containing 1:100,000 epinephrine, on the other hand, caused a very marked increase in tissue cAMP levels. Thirty seconds after infiltration with lidocaine with 1:100,000 epinephrine there was a 250% increase in cAMP content of the anesthetized tissue versus the uninjected control tissues. The maximal increase in tissue cAMP levels was observed 5 minutes after infiltration when the cAMP content of the gingiva was 1000 to 1100% above the control level. It is proposed that regulation of tissue cAMP levels by epinephrine or other agents may prove of therapeutic usefulness in regulating inflammation and healing of tissues after surgery or other trauma.

Anesthesia, Dental↗
PubMed1999

Activation of cAMP-dependent protein kinase A in prefrontal cortex impairs working memory performance.

Activation of the adenylyl cyclase-cAMP-protein kinase A (PKA) intracellular signaling cascade is necessary for long-term memory consolidation in brain regions such as the hippocampus. However, the role of the PKA cascade in the working memory functions of the prefrontal cortex (PFC) is unknown. The present study examined the effects of manipulating PKA activity in the PFC using the cAMP stereoisomers Sp-cAMPS and Rp-cAMPS, which activate and inhibit PKA, respectively. Animals received bilateral infusions of Sp-cAMPS and/or Rp-cAMPS into the PFC immediately before testing on the delayed alternation task, a test of spatial working memory that depends on the integrity of the PFC. Low doses of Sp-cAMPS (0.21, 2. 1, or 21 nmol/0.5 microl) produced a marked, dose-dependent impairment in working memory performance. The impairment produced by infusion of Sp-cAMPS (21 nmol/0.5 microl) was fully reversed by co-infusion of Rp-cAMPS (21 nmol/0.5 microl), consistent with actions on PKA. Rp-cAMPS (21 or 42 nmol/0.5 microl) by itself had no effect on performance. These results indicate that activation of the PKA intracellular signaling cascade in the PFC impairs working memory performance. The current findings contrast with studies of long-term memory consolidation, in which inhibition of PKA with agents such as Rp-cAMPS impaired memory consolidation (Bernabeu et al., 1997; Bourtchouladze et al., 1998), whereas enhancement of the PKA pathway improved memory (Bernabeu et al., 1997; Barad et al., 1998). These results demonstrate that discrete cognitive processes subserved by different cortical regions are mediated by distinct intracellular mechanisms.

Animals↗
PubMed2000

Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells.

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Two independent pathways mediated by cAMP and protein kinase A enhance spontaneous transmitter release at Drosophila neuromuscular junctions.

cAMP is thought to be involved in learning process and known to enhance transmitter release in various systems. Previously we reported that cAMP enhances spontaneous transmitter release in the absence of extracellular Ca(2+) and that the synaptic vesicle protein neuronal-synaptobrevin (n-syb), is required in this enhancement (n-syb-dependent; Yoshihara et al., 1999). In the present study, we examined the cAMP-induced enhancement of transmitter release in the presence of external Ca(2+). We raised the intracellular concentration of cAMP by application of either forskolin, an activator of adenylyl cyclase, or by 4-chlorophenylthio-(CPT)-cAMP, a membrane-permeable analog of cAMP, in the presence of external Ca(2+), while recording miniature synaptic currents (mSCs) at the neuromuscular junction in n-syb null mutant embryos. The frequency of mSCs increased in response to elevation of cAMP, and this effect of cAMP was completely blocked by Co(2+) (n-syb-independent pathway). In contrast, in wild-type embryos the cAMP-induced mSC frequency increase was partially blocked by Co(2+). In a mutant, DC0, defective in protein kinase A (PKA), nerve-evoked synaptic currents were indistinguishable from the control, but mSCs were less frequent. In this mutant the enhancement by cAMP of both nerve-evoked and spontaneous transmitter release was completely absent, even in the presence of external Ca(2+). Taken together, these results suggest that cAMP enhances spontaneous transmitter release by increasing Ca(2+) influx (n-syb-independent) as well as by modulating the release mechanism without Ca(2+) influx (n-syb-dependent) in wild-type embryos, and these two effects are mediated by PKA encoded by the DC0 gene.

Adenylyl Cyclases↗
PubMed2002

Decrease in cAMP levels modulates adhesion to fibronectin and immunostimulatory ability of human dendritic cells.

The aim of the present study was to analyze the early events elicited by tumor necrosis factor alpha (TNF-alpha) on monocyte-derived dendritic cells (moDC) adhesion to fibronectin (FN) and the involvement of cAMP in the signal transduction mechanism. The intracellular concentration of cAMP and moDC adhesion to FN decreased after TNF-alpha treatment. An inverted dose-dependency for TNF-alpha effect was observed for adhesion and cAMP levels. The presence of a phosphodiesterase (PDE) inhibitor (IBMX) and cAMP analogs (8Br-cAMP, Db-cAMP) reversed the observed TNF-alpha effects. The role of cAMP was analyzed further by examining the cAMP levels in nonadhered and adhered, TNF-alpha-treated moDC. Nonadhered moDC showed lower cAMP levels compared with adhered moDC. Furthermore, nonadhered moDC showed higher IL-12 content and allostimulatory ability compared with adhered moDC. The higher allostimulatory capacity was abolished in the presence of cAMP analogs and a PDE inhibitor. These results suggest that cAMP levels correlate with TNF-alpha-induced changes of moDC adhesion and allostimulatory capacity.

Cell Adhesion↗
PubMed2003

Neurotrophins elevate cAMP to reach a threshold required to overcome inhibition by MAG through extracellular signal-regulated kinase-dependent inhibition of phosphodiesterase.

Inhibitors of regeneration in myelin, such as myelin-associated glycoprotein (MAG), play an important role in preventing regeneration after CNS injury. Elevation of cAMP, either with dibutyryl-cAMP (db-cAMP) or by priming with a variety of neurotrophins, overcomes inhibition by MAG and myelin. However, activation of cAMP is not generally regarded as a signaling pathway for neurotrophins. Here we show that the NGF-like neurotrophins overcome inhibition by MAG by activating tyrosine kinase receptors. We also show that activation of extracellular signal-regulated kinase (Erk) by BDNF is required to overcome inhibition by MAG, and that activated Erk transiently inhibits phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP. Inhibition of PDE4 then allows cAMP to increase and so initiates the pathway to overcome inhibition. Furthermore, we also show that basal levels of Erk activation and basal cAMP levels contribute to the effects of db-cAMP by pushing the combined levels of cAMP above a threshold required to overcome inhibition. Together, these results not only show how NGF-like neurotrophins can elevate cAMP and overcome inhibition but also point to a novel mechanism of cross talk in neurons from the Erk to the cAMP signaling pathways.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

[Changes in cAMP and cGMP levels in neonatal rat histaminergic neurons of tuberomammillary nucleus following 48-hour morphine exposure and effects of sinomenine intervention].

OBJECTIVE: To explore the effects of long-term morphine exposure on cAMP and cGMP levels in primary cultured tuberomammillary nucleus (TM) neurons of neonatal rats and the effects of sinomenine on morphine-dependent TM cells. METHODS: TM neurons after a 7-day primary culture were further cultured in the medium containing 100 micromol/L morphine for 48 h to prepare the cell model of morphine dependence. Serial doses of histamine or sinomenine were administered 30 min naloxone treatment, the cAMP and cGMP levels of the TM cells were determined by enzyme immunoassay. cAMP and cGMP levels were also determined in normal TM cells treated by histamine or sinomenine. RESULTS: After treatment with 100 micromol/L morphine for 48 h, cAMP and cGMP levels in the TM neurons were increased markedly. Treatment with 100 micromol/L naloxone added in the culture media caused an overshoot of cellular cAMP and a marked declination of cGMP, resulting in significantly increased cAMP/cGMP ratio. Sinomenine at 30 and 100 micromol/L and histamine at 40 micromol/L failed to obviously affect cAMP and cGMP levels in normal TM neurons, but sinomenine at 300 micromol/L and histamine at 80 micromol/L significantly increased the intracellular cAMP level. After pre-treatment with sinomenine at the above 3 doses or histamine at 40 micromol/L, the TM neurons with morphine dependence exhibited significant reduction in intracellular cAMP level but increment in cGMP level after naloxone treatment, with significantly reduced cAMP/cGMP ratio. CONCLUSION: Long-term morphine (100 micromol/L) exposure for 48 h can induce marked changes of cAMP and cGMP levels in the TM neurons. The central histaminergic nervous system may be responsible for the development of morphine dependence and withdrawal. Sinomenine can significantly reduce the cAMP level and enhance cGMP level of morphine-dependent TM neurons precipitated by naloxone, which results in a near-normal ratio of cAMP and cGMP.

Animals↗
PubMed1990

Effects of cAMP and IBMX on the chick retinal pigment epithelium. Membrane potentials and light-evoked responses.

cAMP is known to alter electrical measures of retinal pigment epithelial (RPE) function (standing potential and DC ERG. To locate the origin of these effects, studies were performed on in vitro preparations of chick retina-RPE-choroid and RPE-choroid, which were separately perfused with cAMP or db-cAMP on the retinal (apical) and choroidal (basal) tissues surfaces. Similar studies were performed using the phosphodiesterase inhibitor IBMX. cAMP and db-cAMP produced essentially identical results. cAMP placed in the retinal or choroidal baths hyperpolarized the RPE basal membrane, increased the apparent basal membrane resistance (Rba), and decreased the ERG c-wave. Experiments in RPE-choroid preparations suggested that these effects of cAMP were not secondary to effects on the neural retina. Effects were observed at 1.0 microM and increased with concentration (1.0 microM-500 microM), with choroidal application producing smaller effects than retinal. The c-wave decrease with retinal cAMP was due principally to the increase in Rba. However, the light-evoked subretinal K+ ([K+]0) decrease, measured with K(+)-specific microelectrodes, showed a small reduction that may have contributed to the c-wave change and that explained an observed decrease in slow PIII. Retinal cAMP also led to a small decrease in ERG a-wave amplitude, which in view of the effects on [K+]0, is consistent with a small reduction in the light response of photoreceptors. The cAMP-induced hyperpolarization of the RPE basal membrane may result from a decrease in basal membrane anion conductance. The results of retinal or choroidal perfusion with IBMX (0.1 mM) in the retina-RPE-choroid preparation were similar and were complicated by effects on photoreceptors. IBMX increased the amplitude of the light response of photoreceptors as indicated by an increase in amplitude of the light-evoked [K+]0 decrease and the a-wave of the ERG. IBMX also produced a transient increase in dark-adapted [K+]0 that altered the membrane potentials of the RPE and obscured any direct effects of IBMX on the RPE. However, IBMX perfusion of the RPE-choroid suggested that the effect on the RPE was similar to that of cAMP perfusion. The IBMX-induced increase in the photoresponse also led to increases in other components of the DC ERG, including the c-wave, fast oscillation and light peak.

1-Methyl-3-isobutylxanthine↗