Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: Implications for modulation of the membrane-associated protein kinase A

• Published in: Archives of biochemistry and biophysics Vol. 486; no. 1; pp. 44 - 50
• Main Authors: Assaife-Lopes, Natália, Wengert, Mira, Pinheiro, Ana Acacia de Sá, Landgraf, Sharon Schilling, Paes-de-Carvalho, Roberto, Leão-Ferreira, Luiz Roberto, Caruso-Neves, Celso
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2009
• Subjects: Adenosine; Adenosine - metabolism; Adenosine A1 Receptor Antagonists; Adenosine deaminase; Adenosine Deaminase - metabolism; Animals; cAMP; Cell Membrane - metabolism; Cell signaling; Cyclic AMP - metabolism; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic AMP-Dependent Protein Kinases - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; In Vitro Techniques; Inosine; Inosine - metabolism; Inosine - pharmacology; Kidney; Kidney Tubules, Proximal - drug effects; Kidney Tubules, Proximal - metabolism; Protein kinase; Proximal tubule; Purine receptor; Receptor, Adenosine A1 - metabolism; Renal tissue; Signal Transduction - drug effects; Swine; Xanthines - pharmacology; Proximal tubule; Adenosine; Protein kinase; Purine receptor; Inosine; Renal tissue; Adenosine deaminase; cAMP; Kidney; Cell signaling
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2009.03.008

In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [ 35S] GTPγS binding to the BLM and it was inhibited by 10 −6 M DPCPX, an A 1 receptor-selective antagonist. Inosine (10 −7 M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G αi-3 protein-coupled A 1 receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A 1 receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A 1 receptor-mediated inhibition of adenylyl cyclase.