A Radioimmunoassay for cyclic AMP (cAMP) Obtained by Acetylation of Both Unlabeled and Labeled (<sup>3</sup>H‐cAMP) Ligand, or of Unlabeled Ligand only

Skomedal, Tor; Grynne, B.; Jb, Osnes; Sjetnan, Anne E.; Øye, Ivar

doi:10.1111/j.1600-0773.1980.tb02443.x

Cited by 50 publications

(11 citation statements)

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“…The reaction was stopped by removing the buffer and adding 5% TCA. cAMP in the neutralized TCA extract was determined by radioimmunoassay as previously described [38]. …”

Section: Methodsmentioning

confidence: 99%

Inhibitory effects of prostaglandin E2on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

et al. 2014

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BackgroundSeveral studies have described an increased cyclooxygenase-2 (COX-2) expression in pancreatic cancer, but the role of COX-2 in tumour development and progression is not clear. The aim of the present study was to examine expression of COX-2 in cancer cells and stromal cells in pancreatic cancer specimens, and to explore the role of PGE2 in pancreatic stellate cell proliferation and collagen synthesis.MethodsImmunohistochemistry and immunofluorescence was performed on slides from whole sections of tissue blocks using antibodies against COX-2 and α-smooth muscle actin (αSMA). Pancreatic stellate cells (PSC) were isolated from surgically resected tumour tissue by the outgrowth method. Cells were used between passages 4 and 8. Collagen synthesis was determined by [3H]-proline incorporation, or by enzyme immunoassay measurement of collagen C-peptide. DNA synthesis was measured by incorporation of [3H]-thymidine in DNA. Cyclic AMP (cAMP) was determined by radioimmunoassay. Collagen 1A1 mRNA was determined by RT-qPCR.ResultsImmunohistochemistry staining showed COX-2 in pancreatic carcinoma cells, but not in stromal cells. All tumours showed positive staining for αSMA in the fibrotic stroma. Cultured PSC expressed COX-2, which could be further induced by interleukin-1β (IL-1β), epidermal growth factor (EGF), thrombin, and PGE2, but not by transforming growth factor-β1 (TGFβ). Indirect coculture with the adenocarcinoma cell line BxPC-3, but not HPAFII or Panc-1, induced COX-2 expression in PSC. Treatment of PSC with PGE2 strongly stimulated cAMP accumulation, mediated by EP2 receptors, and also stimulated phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of PSC with PGE2 or forskolin suppressed both TGFβ-stimulated collagen synthesis and PDGF-stimulated DNA synthesis.ConclusionsThe present results show that COX-2 is mainly produced in carcinoma cells and suggest that the cancer cells are the main source of PGE2 in pancreatic tumours. PGE2 exerts a suppressive effect on proliferation and fibrogenesis in pancreatic stellate cells. These effects of PGE2 are mediated by the cAMP pathway and suggest a role of EP2 receptors.

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“…The reaction was stopped by removing the buffer and adding 5% TCA. cAMP in the neutralized TCA extract was determined by radioimmunoassay as previously described [38]. …”

Section: Methodsmentioning

confidence: 99%

Inhibitory effects of prostaglandin E2on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

et al. 2014

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“…Experiments were conducted in the presence of the β 1 AR blocker CGP20712 (300 nM), the non-selective PDE inhibitor IBMX (0.5 mM) or PDE3 and 4 inhibitors cilostamide (1 µM) and rolipram (10 µM), as indicated. cAMP accumulation was measured by radioimmunoassay as previously described [36]. Protein was measured with the Coomassie Plus Protein Assay (Pierce, Rockford, IL) according to the manufacturer's protocol and cAMP accumulation was normalized to the amount of protein in each sample.…”

Section: Methodsmentioning

confidence: 99%

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

et al. 2014

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Background and purposeDespite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

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“…The columns were washed with 20 ml distilled water and 10 ml 5 mM sodium tetraborate/60 mM ammonium formate, and inositol phosphates were eluted with 10 ml 1 M ammonium formate/0.1 M formic acid. cAMP was determined by radioimmunoassay as previously described [42]. …”

Section: Methodsmentioning

confidence: 99%

Mechanisms involved in PGE2-induced transactivation of the epidermal growth factor receptor in MH1C1 hepatocarcinoma cells

Tveteraas

Müller

Aasrum

et al. 2012

J Exp Clin Cancer Res

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BackgroundIt is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.MethodsThe rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay.ResultsIn the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family.ConclusionThe results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).

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A Radioimmunoassay for cyclic AMP (cAMP) Obtained by Acetylation of Both Unlabeled and Labeled (³H‐cAMP) Ligand, or of Unlabeled Ligand only

Cited by 50 publications

References 9 publications

Inhibitory effects of prostaglandin E2on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

Inhibitory effects of prostaglandin E2on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Mechanisms involved in PGE2-induced transactivation of the epidermal growth factor receptor in MH1C1 hepatocarcinoma cells

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A Radioimmunoassay for cyclic AMP (cAMP) Obtained by Acetylation of Both Unlabeled and Labeled (3H‐cAMP) Ligand, or of Unlabeled Ligand only

Cited by 50 publications

References 9 publications

Inhibitory effects of prostaglandin E2on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

Inhibitory effects of prostaglandin E2on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Mechanisms involved in PGE2-induced transactivation of the epidermal growth factor receptor in MH1C1 hepatocarcinoma cells

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A Radioimmunoassay for cyclic AMP (cAMP) Obtained by Acetylation of Both Unlabeled and Labeled (³H‐cAMP) Ligand, or of Unlabeled Ligand only