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 [(35)S] GTPgammaS 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(alphai-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.
Assaife-Lopes
Natália
N
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS Bloco G, 21949 Rio de Janeiro, RJ, Brazil.
Wengert
Mira
M
Pinheiro
Ana Acacia de Sá
AA
Landgraf
Sharon Schilling
SS
Paes-de-Carvalho
Roberto
R
Leão-Ferreira
Luiz Roberto
LR
Caruso-Neves
Celso
C