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Intracellular cAMP Levels (intracellular + camp_level)
Selected AbstractsShort-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in opposing agonist-induced Ca2+ responses in endothelial cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2008M Campos-Toimil Background and purpose: We previously reported that agonist-induced rises in cytoplasmic Ca2+ concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC) were inhibited after a short-term (2 min) pre-treatment with cAMP-elevating agents. The aim of this work was to study the effects of longer term (8 h) pre-treatment with dibutyryl-cAMP (db-cAMP) or rolipram, a specific inhibitor of phosphodiesterase-4 (PDE4), on [Ca2+]i, cAMP levels and PDE activity and expression in HUVEC. Experimental approach: [Ca2+]i changes were measured in isolated HUVEC by Fura-2 imaging. Intracellular cAMP levels and PDE4 activity were assessed by enzyme-immunoassay and radio-enzymatic assay, respectively. PDE expression was measured by northern and western blot analysis. Key results: Long-term pre-treatment of HUVEC with rolipram or db-cAMP significantly increased ATP-, histamine- and thrombin-induced [Ca2+]i rises. Short-term pre-treatment with rolipram was associated with an increase in cAMP, whereas long-term pre-treatment was associated with a decrease in cAMP. Long-term pre-treatment with rolipram or db-cAMP induced a significant increase in PDE4 activity and the expression of 74 kDa-PDE4A and 73 kDa-PDE4B was specifically enhanced. All these effects were suppressed by cycloheximide. Conclusions and implications: Our data suggest that sustained inhibition of PDE4 by rolipram induced an increase in PDE4 activity, possibly as a compensatory mechanism to accelerate cAMP degradation and that PDE4A and PDE4B were implicated in the regulation of [Ca2+]i. Thus, isozyme-specific PDE4 inhibitors might be useful as therapeutic agents in diseases where [Ca2+]i handling is altered, such as atherosclerosis, hypertension and tolerance to ,-adrenoceptor agonists. [source] Glucose sensing in the intestinal epitheliumFEBS JOURNAL, Issue 16 2003Jane Dyer Dietary sugars regulate expression of the intestinal Na+/glucose cotransporter, SGLT1, in many species. Using sheep intestine as a model, we showed that lumenal monosaccharides, both metabolisable and nonmetabolisable, regulate SGLT1 expression. This regulation occurs not only at the level of transcription, but also at the post-transcriptional level. Introduction of d -glucose and some d -glucose analogues into ruminant sheep intestine resulted in >,50-fold enhancement of SGLT1 expression. We aimed to determine if transport of sugar into the enterocytes is required for SGLT1 induction, and delineate the signal-transduction pathways involved. A membrane impermeable d -glucose analogue, di(glucos-6-yl)poly(ethylene glycol) 600, was synthesized and infused into the intestines of ruminant sheep. SGLT1 expression was determined using transport studies, Northern and Western blotting, and immunohistochemistry. An intestinal cell line, STC-1, was used to investigate the signalling pathways. Intestinal infusion with di(glucos-6-yl)poly(ethylene glycol) 600 led to induction of functional SGLT1, but the compound did not inhibit Na+/glucose transport into intestinal brush-border membrane vesicles. Studies using cells showed that increased medium glucose up-regulated SGLT1 abundance and SGLT1 promoter activity, and increased intracellular cAMP levels. Glucose-induced activation of the SGLT1 promoter was mimicked by the protein kinase A (PKA) agonist, 8Br-cAMP, and was inhibited by H-89, a PKA inhibitor. Pertussis toxin, a G-protein (Gi)-specific inhibitor, enhanced SGLT1 protein abundance to levels observed in response to glucose or 8Br-cAMP. We conclude that lumenal glucose is sensed by a glucose sensor, distinct from SGLT1, residing on the external face of the lumenal membrane. The glucose sensor initiates a signalling pathway, involving a G-protein-coupled receptor linked to a cAMP,PKA pathway resulting in enhancement of SGLT1 expression. [source] B96Bom encodes a Bombyx mori tyramine receptor negatively coupled to adenylate cyclaseINSECT MOLECULAR BIOLOGY, Issue 3 2003H. Ohta Abstract A cDNA encoding a biogenic amine receptor (B96Bom) was isolated from silkworm (Bombyx mori) larvae, and the ligand response of the receptor stably expressed in HEK-293 cells was examined. Tyramine (TA) at 0.1,100 µm reduced forskolin (10 µm)-stimulated intracellular cAMP levels by approximately 40%. The inhibitory effect of TA at 1 µm was abolished by yohimbine and chlorpromazine (each 10 µm). Although octopamine (OA) also reduced the cAMP levels, the potency was at least two orders of magnitude lower than that of TA. Furthermore, unlabelled TA (IC50 = 5.2 nm) inhibited specific [3H]TA binding to the membranes of B96Bom-transfected HEK-293 cells more potently than did OA (IC50 = 1.4 µm) and dopamine (IC50 = 1.7 µm). Taken together with the result of phylogenetic analysis, these findings indicate that the B96Bom receptor is a B. mori TA receptor, which is negatively coupled to adenylate cyclase. The use of this expression system should facilitate physiological studies of TA receptors as well as structure,activity studies of TA receptor ligands. [source] Cimetidine inhibits epidermal growth factor-induced cell signalingJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 3 2007Tatsuya Fujikawa Abstract Background:, Cimetidine, a histamine-2 (H2) receptor antagonist, has been demonstrated to have anticancer effects on colorectal cancer, melanoma and renal cell carcinoma. In the current study, we clarified that cimetidine inhibits both epidermal growth factor (EGF)-induced cell proliferation and migration in hepatocellular carcinoma (HCC) cell lines. Method:, HCC cell lines (Hep3B, HLF, SK-Hep-1, JHH-2, PLC/PRF/5 and HLE) were used and cell proliferation was assessed by [3H]-thymidine incorporation assay. Cell migration was measured by in vitro cell migration assay. Biological effects of cimetidine were assessed with human EGF receptor (EGFR)-expressing mouse fibroblast cells (NR6-WT). The autophosphorylation of EGFR and the activation of other downstream effectors were analyzed by immunoprecipitation and immunoblotting. The concentration of intracellular cyclic AMP (cAMP) was measured by competitive enzyme immunoassay. Results:, Cimetidine inhibited both EGF-induced cell proliferation and migration in Hep3B, HLF, SK-Hep-1 and JHH-2, while cimetidine did not affect EGF-induced cell proliferation and migration in PLC/PRF/5 and HLE. Cimetidine was revealed to disrupt the EGF-induced autophosphorylation of EGFR and its downstream effectors, mitogen activated protein kinases and phospholipase C-,. To define the molecular basis of this negative regulation, we identified that cimetidine significantly decreased intracellular cAMP levels and that decrement of cAMP inhibited autophosphorylation of EGFR. The cell permeable cAMP analog, CPT-cAMPS reversed the cimetidine-induced inhibition of EGF-induced cell proliferation and cell migration by restoring autophosphorylation of EGFR. Conclusion:, Cimetidine inhibited EGF-induced cell proliferation and migration in HCC cell lines by decreasing the concentration of intracellular cAMP levels. Cimetidine may be a candidate chemopreventive agent for HCC. [source] Transcriptional Regulation of 2,,3,-Cyclic Nucleotide 3,-Phosphodiesterase Gene Expression by Cyclic AMP in C6 CellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2000M. Gravel Abstract: It was recently shown that the two transcripts encoding the isoforms of 2,,3,-cyclic nucleotide 3,-phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up-regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides -126 and -102 was essential for the cAMP-dependent induction of CNP1 expression. Gel retardation analysis showed that two protein-DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the -126/-102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein-2 (AP-2) binding site. It is interesting that mutagenesis of this region resulted in a significant reduction in transcriptional responses to cAMP, implying a possible role for the AP-2 factor in the expression of CNP1. In addition, we have shown that putative binding sites for activator protein-4 and nuclear factor-1 adjacent to the AP-2 site are required for efficient induction of CNP1 expression by cAMP. Taken together, our results show that the cAMP-dependent accumulation of CNP1 mRNA appears to depend on the synergistic interaction of several regulatory elements. [source] GPR119, a novel G protein-coupled receptor target for the treatment of type 2 diabetes and obesityBRITISH JOURNAL OF PHARMACOLOGY, Issue S1 2008H A Overton GPR119 is a G protein-coupled receptor expressed predominantly in the pancreas (,-cells) and gastrointestinal tract (enteroendocrine cells) in humans. De-orphanization of GPR119 has revealed two classes of possible endogenous ligands, viz., phospholipids and fatty acid amides. Of these, oleoylethanolamide (OEA) is one of the most active ligands tested so far. This fatty acid ethanolamide is of particular interest because of its known effects of reducing food intake and body weight gain when administered to rodents. Agonists at the GPR119 receptor cause an increase in intracellular cAMP levels via G,s coupling to adenylate cyclase. In vitro studies have indicated a role for GPR119 in the modulation of insulin release by pancreatic ,-cells and of GLP-1 secretion by gut enteroendocrine cells. The effects of GPR119 agonists in animal models of diabetes and obesity are reviewed, and the potential value of such compounds in future therapies for these conditions is discussed. British Journal of Pharmacology (2008) 153, S76,S81; doi:10.1038/sj.bjp.0707529; published online 26 November 2007 [source] Heterologous desensitization of the sphingosine-1-phosphate receptors by purinoceptor activation in renal mesangial cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2004Cuiyan Xin Sphingosine-1-phosphate (S1P) is considered a potent mitogen for mesangial cells and activates the classical mitogen-activated protein kinase (MAPK) cascade via S1P receptors. In this study, we show that S1P signalling is rapidly desensitized upon S1P receptor activation. A complete loss of S1P sensitivity occurs after 10 min of S1P pretreatment and remains for at least 8 h. A similar desensitization is also seen with the S1P mimetic FTY720-phosphate, but not with the nonphosphorylated FTY720, nor with sphingosine or ceramide. Prestimulating the cells with extracellular ATP or UTP, which bind to and activate P2Y receptors on mesangial cells, a similar rapid desensitization of the S1P receptor occurs, suggesting a heterologous desensitization of S1P receptors by P2Y receptor activation. Furthermore, adenosine binding to P1 receptors triggers a similar desensitization. In contrast, two other growth factors, PDGF-BB and TGF,2, have no significant effect on S1P-induced MAPK activation. S1P also triggers increased inositol trisphosphate (IP3) formation, which is completely abolished by S1P pretreatment but only partially by ATP pretreatment, suggesting that IP3 formation and MAPK activation stimulated by S1P involve different receptor subtypes. Increasing intracellular cAMP levels by forskolin pretreatment has a similar effect on desensitization as adenosine. Moreover, a selective A3 adenosine receptor agonist, which couples to phospholipase C and increases IP3 formation, exerted a similar effect. Pretreatment of cells with various protein kinase C (PKC) inhibitors prior to ATP prestimulation and subsequent S1P stimulation leads to a differential reversal of the ATP effect. Whereas the broad-spectrum protein kinase inhibitor staurosporine potently reverses the effect, the PKC- , inhibitor CGP41251, the PKC- , inhibitor rottlerin and calphostin C show only a partial reversal at maximal concentrations. Suramin, which is reported as a selective S1P3 receptor antagonist compared to the other S1P receptor subtypes, has no effect on the S1P-induced MAPK activation, thus excluding the involvement of S1P3 in this response. In summary, these data document a rapid homologous and also heterologous desensitization of S1P signalling in mesangial cells, which is mechanistically triggered by PKC activation and eventually another staurosporine-sensitive protein kinase, as well as by increased cAMP formation. British Journal of Pharmacology (2004) 143, 581,589. doi:10.1038/sj.bjp.0705980 [source] Experimental study of a type 3 phosphodiesterase inhibitor on liver graft functionBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 1 2001T. Ikegami Background: The number of liver transplant recipients is increasing but donor organ shortages have become more severe. The effect of milrinone, a type 3 phosphodiesterase inhibitor (PDEI), on non-heart-beating donor grafts was evaluated using an orthotopic liver transplantation model in rats. Methods: Type 3 PDEI or normal saline (control group) was given intravenously to the donor animals for 60 min continuously (50 µg kg,1 min,1 ) before 60 min of warm ischaemia followed by cold preservation and subsequent transplantation. Survival, serum chemistry, bile output, histopathological findings and tissue cyclic 3,,5,-adenosine monophosphate (cAMP) concentrations were then compared. Results: Five of seven animals in the PDEI group were alive at 7 days, compared with only one of seven rats in the control group (P < 0·01). Serum levels of alanine aminotransferase 2 and 6 h after reperfusion, and hyaluronic acid levels 6 h after reperfusion, were significantly lower in the PDEI group than in the control group. Bile output from the transplanted graft was significantly greater in the PDEI group than in controls 2 h after reperfusion (P < 0·01). The mean necrotic area 6 h after reperfusion was also reduced in the PDEI-treated grafts (P < 0·01). cAMP levels in liver tissue at the end of both warm and cold ischaemia, and 2 and 6 h after reperfusion, were significantly higher in the PDEI group compared with those in the control group. Conclusion: Type 3 PDEI attenuated the graft injury caused by warm and cold ischaemia and subsequent reperfusion injury via an increase in intracellular cAMP levels. This treatment may be a novel pharmacological intervention for safe and efficient usage of liver grafts from non-heart-beating donors. © 2001 British Journal of Surgery Society Ltd [source] | |||||||||||||