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Phosphorylated adenosine derivatives as low-affinity adenosine-receptor agonists. Methodological implications for the adenylate cyclase assay.

ABSTRACT: In cellular systems provided with activatory (Ra-site) receptors for adenosine, such as rat cerebral microvessels and rat liver plasma membranes, the adenosine-receptor antagonist 8-phenyltheophylline (10 microM) significantly decreased adenylate cyclase activity if ATP was the substrate and only if GTP was present. With dATP as substrate, adenylate cyclase activities in both preparations remained unaffected by 8-phenyltheophylline. In rat cerebral-cortical membranes, with inhibitory (Ri-site) receptors for adenosine, 8-phenyltheophylline significantly enhanced adenylate cyclase activity only in the presence of GTP and if ATP was the substrate. In rat cardiac ventricular membranes, which are devoid of any adenylate cyclase-coupled adenosine receptor, the methylxanthine had no GTP-dependent effect, irrespective of the substrate used. All assay systems contained sufficiently high amounts of adenosine deaminase (2.5 units/ml), since no endogenous adenosine, formed from ATP, was found chromatographically. In order to demonstrate a direct influence of phosphorylated adenosine derivatives on adenylate cyclase activity, we investigated AMP in a dATP assay system. AMP was verified chromatographically to remain reasonably stable under the adenylate cyclase assay conditions. In the microvessels, AMP increased enzyme activity in the range 0.03-1.0 mM, an effect competitively antagonized by 8-phenyltheophylline. In the cortical membranes, 0.1 mM-AMP inhibited adenylate cyclase, which was partially reversed by the methylxanthine. The presence of GTP was again necessary for all observations. In the ventricular membranes, AMP had no effect. Since the efficacy of adenosine-receptor agonists and, probably, that of other hormones on adenylate cyclase activity can be more efficiently measured with dATP as the enzyme substrate, this nucleotide seems preferable for adenylate cyclase measurements in systems susceptible to modulation by adenosine.

SUBMITTER: Schutz W

PROVIDER: S-EPMC1153611 | biostudies-other | 1984 May

REPOSITORIES: biostudies-other

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Project description:BackgroundThe incidence and mortality of bladder cancer (BCa) are increasing, while the existing diagnostic methods have limitations. Therefore, for early detection and response prediction, it is crucial to improve the prognosis and treatment strategies. However, with existing diagnostic methods, detecting BCa in the early stage is challenging. Hence, novel biomarkers are urgently needed to improve early diagnosis and treatment efficiency.MethodsThe gene expression profile and gene methylation profile dataset were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs), differentially methylated genes (DMGs), and methylation-regulated differentially expressed genes (MeDEGs) were gradually identified. A cancer genome map was obtained using online gene expression profile interaction analysis, and survival implications were produced using Kaplan-Meier survival analysis. GSEA was employed to predict the marker pathways where DEGs were significantly involved. The study used bisulfite PCR amplification combined with bisulfite amplicon sequencing (BSAS) to screen for methylation analysis of multiple candidate regions of the adenylate cyclase 2 (ADCY2) based on the sequence design of specific gene regions and CpG islands.ResultsIn this study, DEGs and DMGs with significantly up- or down-regulated expression were selected. The intersection method was used to screen the MeDEGs. The interaction network group in STRING was then visualized using Cytoscape, and the PPI network was constructed to identify the key genes. The key genes were then analyzed using functional enrichment. To compare the relationship between key genes and the prognosis of BCa patients, we further investigated ADCY2 and found that ADCY2 can be a potential clinical biomarker in BCa prognosis and immunotherapy response prediction. In human BCa 5637 and MGH1 cells, we developed and verified the effectiveness of ADCY2 primers using BSAS technology. The findings revealed that the expression of ADCY2 is highly regulated by the methylation of the promoter regions.ConclusionThis study revealed that increased expression of ADCY2 was significantly correlated with increased tumor heterogeneity, predicting worse survival and immunotherapy response in BCa patients.

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Phosphorylated adenosine derivatives as low-affinity adenosine-receptor agonists. Methodological implications for the adenylate cyclase assay.

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