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cAMP Production (camp + production)
Selected AbstractsRegulation of MC1R signalling by G-protein-coupled receptor kinasesEXPERIMENTAL DERMATOLOGY, Issue 9 2004J. C. García-Borrón The melanocortin 1 receptor (MC1R) is a key regulator of melanocyte proliferation and differentiation and a major determinant of human skin phototype and skin cancer risk. Although the regulation of MC1R gene expression is fairly well understood, little is known about regulatory mechanisms acting at the protein level. In particular, no information is available on homologous desensitization of MC1R signalling. We studied MC1R and Mc1r desensitization and found that: 1) MC1R and Mc1r in melanoma cells undergo homologous desensitization, demonstrated by decreases in cAMP contents upon continuous exposure to agonists, 2) desensitization is not dependent on PKA, PKC, calcium mobilization or MAPKs but is agonist dose dependent, suggesting a role of receptor occupancy, 3) melanoma cells express two members of the GRK family of serine/threonine kinases, GRK2 and GRK6, 4. These kinases are expressed in normal melanocytes, 5) in cotransfection experiments performed with HEK 293T cells, GRK2 strongly impairs agonist-dependent signalling by MC1R or Mc1r, 6) expression of a dominant negative GRK2 mutant in melanoma cells increases their cAMP response to MC1R agonists, 7) cotransfection of HEK 293T cells with GRK6 and MC1R inhibits both basal and agonist-dependent signalling, and 8) cAMP production in agonist-stimulated melanoma cells is strongly impaired by enrichment with GRK6 following stable transfection. Therefore, GRK2 and GRK6 are key regulators of MC1R signalling and may be important determinants of normal and pathological skin pigmentation. [source] Molecular and functional characterization of novel CRFR1 isoforms from the skinFEBS JOURNAL, Issue 13 2004Alexander Pisarchik In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1,, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1, was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1,-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis -elements (CRE, CaRE, SRE, AP1 or NF-,B) demonstrated that only CRFR1, was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4,-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction. [source] Association of a melanocortin 4 receptor (MC4R) polymorphism with performance traits in Lithuanian White pigsJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2006R. Jokubka Summary The melanocortin 4 receptor is expressed in virtually all brain regions of mammals and plays an important role in energy homeostasis. Polymorphisms in this gene may thus be related to growth and obesity. In pigs, a non-synonymous polymorphic site was described (Asp298Asn) and demonstrated to affect cAMP production and to alter adenylyl cyclase signalling. Association studies revealed significant linkage of this mutation with production trait in pigs. In this study, 207 Lithuanian White pigs were genotyped at the MC4R locus and analysed on relationships between genotype and breeding values for several performance traits. The observed allele and genotype frequencies did not deviate significantly from Hardy,Weinberg equilibrium (wildtype allele 0.59; mutant allele 0.41) and are comparable with those described in other Large White populations. The mutant Asn298 allele of the MC4R gene was significantly associated with increased test daily gain, higher lean meat percentage and lower backfat thickness. There was a trend towards an improved feed conversion ratio (p = 0.065) in animals with the mutant allele whereas no significant effect was found on lifetime daily gain. These results indicate that the MC4R polymorphism should be integrated in selection programmes in the Lithuanian White to improve carcass composition. [source] Phosphodiesterase inhibition by naloxone augments the inotropic actions of ,-adrenergic stimulationACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 8 2009W. K. PARK Background: In a shock state, naloxone generates the cardiovascular pressor effect by displacing the endogenous opiate-like peptide ,-endorphin, resulting in restoration of the normal response to catecholamines. In addition to this opioid antagonistic effect, the non-opiate receptor-mediated effect has also been proposed. The aim of this study was to define the mechanism of non-opiate receptor-mediated action of naloxone. Methods: In guinea-pig ventricular tissues, cumulative concentration,response curves for isoproterenol as well as for forskolin and 3-isobutylmethylxanthine (IBMX) were obtained by increasing the concentration stepwise. To assess the effect on the phosphodiesterase (PDE), the effects of naloxone on contractile forces induced by isoproterenol (0.05 ,M) in the presence of IBMX, cilostamide (a PDE III inhibitor), or rolipram (a PDE IV inhibitor) were observed. Naloxone-induced changes in cAMP production by isoproterenol both in the absence and in the presence of IBMX were measured. Naloxone-induced changes in cAMP production by forskolin in the presence of IBMX were also measured. Results: Naloxone (30 ,M) produced a leftward shift of the isoproterenol concentration,response curve (0.01,2 ,M) without changing the maximal response. Forskolin (0.5,10 ,M) produced a concentration-dependent increase in contractile forces. Naloxone increased the maximal inotropic response of forskolin. Naloxone showed no effect on the IBMX concentration,response curve. In the presence of IBMX (200 ,M), naloxone did not alter the contractions evoked by isoproterenol or forskolin. Whereas naloxone increased contractile forces significantly (approximately 25%) more than that of isoproterenol in the presence of rolipram, no alteration of contractile forces in the cilostamide-incubated muscles was observed. Naloxone caused a concentration-related increase of cAMP in the absence of IBMX, but caused no change in its presence. Conclusions: The enhancement of myocardial contractility by naloxone in the presence of stimulation of adenylyl cyclase activity appears to be mediated by inhibition of PDE, specifically PDE III. [source] Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblastsAGING CELL, Issue 6 2006Ji-Heon Rhim Summary Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3,,5,-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs ,/, both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gi, in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC ,/, and AC4/6. [source] Agonist-induced internalization of histamine H2 receptor and activation of extracellular signal-regulated kinases are dynamin-dependentJOURNAL OF NEUROCHEMISTRY, Issue 1 2008A-Jing Xu Abstract Histamine H2 receptor (H2R) is a member of G protein-coupled receptor family. Agonist stimulation of H2R results in several cellular events including activation of adenylate cyclase and phospholipase C, desensitization of the receptor, activation of extracellular signal-regulated kinases ERK1/2, and receptor endocytosis. In this study, we identified a GTPase dynamin as a binding partner of H2R. Dynamin could associate with H2R both in vitro and in vivo. Functional analyses using dominant-negative form of dynamin (K44E-dynamin) revealed that cAMP production and the following H2R desensitization are independent of dynamin. However, the agonist-induced H2R internalization was inhibited by co-expression of K44E-dynamin. Furthermore, activation of extracellular-signal regulated kinases ERK1/2 in response to dimaprit, an H2R agonist, was attenuated by K44E-dynamin. Although H2R with truncation of 51 amino acids at its carboxy-terminus did not internalize after agonist stimulation, it still activated ERK1/2, but the degree of this activation was less than that of the wild-type receptor. Finally, K44E dynamin did not affect ERK1/2 activation induced by internalization-deficient H2R. These results suggest that the agonist-induced H2R internalization and ERK1/2 activation are partially dynamin-dependent. Furthermore, ERK1/2 activation via H2R is likely dependent of the endocytotic process rather than dynamin itself. [source] SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brainJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Li-Qing Jin Abstract Previously a distinct D1 -like dopamine receptor (DAR) that selectively couples to phospholipase C/phosphatidylinositol (PLC/PI) was proposed. However, lack of a selective agonist has limited efforts aimed at characterizing this receptor. We characterized the in vitro and in vivo effects of SKF83959 in regulating PI metabolism. SKF83959 stimulates (EC50, 8 µm) phosphatidylinositol 4,5-biphosphate hydrolysis in membranes of frontal cortex (FC) but not in membranes from PC12 cells expressing classical D1A DARs. Stimulation of FC PI metabolism was attenuated by the D1 antagonist, SCH23390, indicating that SKF83959 activates a D1 -like DAR. The PI-linked DAR is located in hippocampus, cerebellum, striatum and FC. Most significantly, administration of SKF83959 induced accumulations of IP3 in striatum and hippocampus. In contrast to other D1 DAR agonists, SKF83959 did not increase cAMP production in brain or in D1A DAR-expressing PC12 cell membranes. However, SKF83959 inhibited cAMP elevation elicited by the D1A DAR agonist, SKF81297, indicating that the compound is an antagonist of the classical D1A DAR. Lastly, we demonstrated that SKF83959 enhances [35S]guanosine 5,- O -(3-thiotriphosphate) binding to membrane G,q and G,i proteins, suggesting that PI stimulation is mediated by activation of these guanine nucleotide-binding regulatory proteins. Results indicate that SKF83959 is a selective agonist for the PI-linked D1 -like DAR, providing a unique tool for investigating the functions of this brain D1 DAR subtype. [source] Functional expression of corticotropin-releasing hormone (CRH) receptor 1 in cultured rat microgliaJOURNAL OF NEUROCHEMISTRY, Issue 2 2002Wei Wang Abstract Corticotropin-releasing hormone (CRH), known as a key regulator of the hypothalamic,pituitary,adrenal axis response to stress, elicits its biological effects by binding to two membrane receptors (CRH-R1 and CRH-R2). The present studies examined the presence of functional expression of CRH receptors in cultured microglia of rat. CRH-R1 mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and receptor chemical cross-linking assay in cultured microglia. CRH-R2 mRNA was undectable by RT-PCR. The radioligand binding analysis using [125I]Tyr-rat/human CRH revealed a high affinity binding site (Kd of 1.2 nm and Bmax of 84 fmol/mg of protein). Competition studies using CRH and related peptides indicated kinetic and pharmacological characteristics consistent with the CRH-R1 receptor subtype. Receptor chemical cross-linking assay demonstrated a single band of CRH receptor with a molecular weight of ,77 kDa, which was inhibited in the presence of excess unlabeled rat/human CRH in a dose-dependent manner and inhibited by a CRH receptor,antagonist astressin. Functional coupled cAMP production in cultured microglia was stimulated by exogenous addition of CRH and related peptides in a dose-dependent manner and blocked by astressin. Our findings suggest the functional expression of CRH-R1 receptor in rat microglia, indicating an important mechanism of interaction between immune and neuroendocrine systems in brain physiological and,pathological conditions. [source] Potentiation of PGE2 -mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Se-Young Choi The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all- trans retinoic acid. The incubation with 5 µm all- trans retinoic acid for 4,6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E2 (PGE2)-induced cAMP production was dramatically increased, whereas forskolin- and AlF -induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all- trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE2 -induced cAMP production. In addition, the binding of [3H]PGE2 to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE1 = PGE2 > PGD2 = PGF2, = PGI2) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP2 receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE2 upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP2 mRNA level was about seven times higher in differentiated cells, while the dopamine ,-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP2 receptor results in an increase in the PGE2 -evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation. [source] Genetic Correlations Between Initial Sensitivity to Ethanol and Brain cAMP Signaling in Inbred and Selectively Bred MiceALCOHOLISM, Issue 6 2001Shelli L. Kirstein 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 =,BEC = 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. [source] Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathwayMOLECULAR CARCINOGENESIS, Issue 11 2007Xingya Wang Abstract Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1,4). In this study, we investigated the role of EP receptors in PGE2 -induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM,10 µM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2 -induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2,5,-dideoxyadenosine, at concentrations that inhibited PGE2 -induced cAMP, significantly blocked PGE2 -induced VEGF secretion in PC-3 cells. We conclude that PGE2 -induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways. © 2007 Wiley-Liss, Inc. [source] cAMP microdomains and L-type Ca2+ channel regulation in guinea-pig ventricular myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2007Sunita Warrier Many different receptors can stimulate cAMP synthesis in the heart, but not all elicit the same functional responses. For example, it has been recognized for some time that prostaglandins such as PGE1 increase cAMP production and activate PKA, but they do not elicit responses like those produced by ,-adrenergic receptor (,AR) agonists such as isoproterenol (isoprenaline), even though both stimulate the same signalling pathway. In the present study, we confirm that isoproterenol, but not PGE1, is able to produce cAMP-dependent stimulation of the L-type Ca2+ current in guinea pig ventricular myocytes. This is despite finding evidence that these cells express EP4 prostaglandin receptors, which are known to activate Gs -dependent signalling pathways. Using fluorescence resonance energy transfer-based biosensors that are either freely diffusible or bound to A kinase anchoring proteins, we demonstrate that the difference is due to the ability of isoproterenol to stimulate cAMP production in cytosolic and caveolar compartments of intact cardiac myocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment. Unlike other receptor-mediated responses, compartmentation of PGE1 responses was not due to concurrent activation of a Gi -dependent signalling pathway or phosphodiesterase activity. Instead, compartmentation of the PGE1 response in cardiac myocytes appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the caveolar compartment associated with ,AR regulation of L-type Ca2+ channel function. [source] Indoloquinolizidine,Peptide Hybrids as Multiple Agonists for D1 and D2 Dopamine ReceptorsCHEMMEDCHEM, Issue 9 2009Marc Vendrell Abstract Multiple-specificity ligands are considered promising pharmacological tools that may show higher efficacy in the treatment of diseases for which the modulation of a single target is therapeutically inadequate. We prepared a set of novel ligands for D1 and D2 dopamine receptors by combining two indolo[2,3- a]quinolizidine scaffolds with various tripeptide moieties. The binding and functional properties of these molecules were determined by radioligand binding studies in brain striatum membranes and by intracellular cAMP production assays in cells expressing different dopamine receptor subtypes. Some indoloquinolizidine,peptide hybrids, mainly with the trans configuration, showed dual agonist activity at both D1 and D2 dopamine receptors and may therefore be useful for testing the therapeutic potential of multivalent drugs on these targets. [source] SPATIAL AND TEMPORAL ASPECTS OF cAMP SIGNALLING IN CARDIAC MYOCYTESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 11 2008Radu V Iancu SUMMARY 1,1 -Adrenoceptor and M2 muscarinic receptor regulation of cAMP production plays a pivotal role in autonomic regulation of cardiac myocyte function. However, not all responses are easily explained by a uniform increase or decrease in cAMP activity throughout the entire cell. 2Adenovirus expression of fluorescence resonance energy transfer (FRET)-based biosensors can be used to monitor cAMP activity in protein kinase A (PKA) signalling domains, as well as the bulk cytoplasmic domain of intact adult cardiac myocytes. 3Data obtained using FRET-based biosensors expressed in different cellular microdomains have been used to develop a computational model of compartmentalized cAMP signalling. 4A systems biology approach that uses quantitative computational modelling together with experimental data obtained using FRET-based biosensors has been used to provide evidence for the idea that compartmentation of cAMP signalling is necessary to explain the stimulatory responses to ,1 -adrenoceptor activation as well as the complex temporal responses to M2 muscarinic receptor activation. [source] Gonadotrophin receptor blocking antibodies measured by the use of cell lines stably expressing human gonadotrophin receptors are not detectable in women with 46,XX premature ovarian failureCLINICAL ENDOCRINOLOGY, Issue 3 2004Massimo Tonacchera Summary background, Premature ovarian failure (POF) is defined by cessation of ovarian function after puberty and before the age of 40. The syndrome is characterized by amenorrhoea, oestrogen deficiency and elevated levels of gonadotrophins. Autoimmunity has been proposed as a mechanism for some cases of destruction or malfunction of ovarian follicles. POF is often associated with type I and type II polyglandular autoimmune syndromes. It has also been postulated that receptors such as the LH and FSH receptors might become targets for blocking antibodies and such antibodies could be a cause of ovarian failure. patients and methods, Sixty-nine patients with POF isolated or associated with other endocrine autoimmune diseases (autoimmune thyroid diseases, Addison's disease, type 1 diabetes mellitus, multiple sclerosis, myasthenia gravis) were studied. All the patients had secondary amenorrhoea. The patient group had a median age of 33·1 years (range 15,57). Ovarian failure had been diagnosed at a median age of 29 years (range 15,39). The median time since diagnosis was almost 1 year but in six patients gonadal insufficiency had appeared 10,30 years earlier. All had a normal chromosomal karyotype (46, XX). Patients with POF were characterized by duration of amenorrhoea > 1 year, with elevated FSH and LH levels and undetectable or low oestrogen levels. Cell lines stably expressing recombinant human LH (CHO-LHr) and FSH (CHO-FSHr) receptors were prepared and used to search for antibodies able to inhibit LH- or FSH-stimulated cAMP production. Immunoglobulins extracted from sera of patients with POF were incubated with CHO-LHr and CHO-FSHr in the presence of human recombinant CG and FSH, respectively. results and conclusions, None of the immunoglobulin G (IgG) preparations from patients with POF was able to inhibit the activity of the FSH- and CG-stimulated cAMP production. [source] Potentiation of PGE2 -mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Se-Young Choi The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all- trans retinoic acid. The incubation with 5 µm all- trans retinoic acid for 4,6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E2 (PGE2)-induced cAMP production was dramatically increased, whereas forskolin- and AlF -induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all- trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE2 -induced cAMP production. In addition, the binding of [3H]PGE2 to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE1 = PGE2 > PGD2 = PGF2, = PGI2) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP2 receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE2 upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP2 mRNA level was about seven times higher in differentiated cells, while the dopamine ,-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP2 receptor results in an increase in the PGE2 -evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation. [source] | ||||||||||||||||