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

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 A1 receptor-selective antagonist. Inosine (10−7 M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and Gαi-3 protein-coupled A1 receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A1 receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A1 receptor-mediated inhibition of adenylyl cyclase.