RESUMO
The prion protein (PrPC) binds copper and affects copper metabolism, albeit among a poorly understood functional landscape. Much of the data on physiological roles of PrPC were obtained in mice of mixed genetic background deficient of the PrPC-coding gene Prnp. This strategy is currently under scrutiny due to the flanking gene problem, in particular related with a polymorphism, typical of both the 129Sv and 129Ola mouse substrains, in the Sirpa gene located in the vicinity of Prnp. Here we report an investigation of biochemical properties of Cu(I)-ATPases as a function of genotype in two strains of PrPC-deficient mice. We found that both the brain and liver of Prnp-null mice of mixed B6;129Sv background had diminished activity, accompanied by increased catalytic phosphorylation of Cu(I)-ATPase, as compared with the respective wild-type animals. However, no such differences were found between Prnp-null and wild-type mice of a B10;129Ola background. Activity of Cu(I)-ATPase was strongly reduced in brain tissue from mice of 129Sv strain, when compared with wild-type either of B6;129Sv, and especially of mice of the B6 strain. No differences between wild-type and Prnp-null brain tissue were noted in the expression of either Atp7a or b genes, and RFLP analysis indicated that the Sirpa129 polymorphism was present in both the B6;129Sv and B10;129Ola Prnp-null mouse colonies used in this study. The results suggest a novel substrain-dependent effect of 129Sv, but not 129Ola, genotype upon the regulation of the Cu(I)-ATPase catalytic cycle in Prnp-null mice, rather than either a Prnp-dependent, or a 129 strain-dependent effect.
Assuntos
Encéfalo/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Proteínas Priônicas/metabolismo , Animais , Hipocampo/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Priônicas/genética , Especificidade da EspécieRESUMO
There is some evidence to show a possible role of guanosine in the modulation of cellular function, in particular, in the neuronal system. However, nothing is known about the role of guanine in renal function. The aim of the present work was to investigate the role of guanine on modulation of Na+-ATPase activity in isolated basolateral membrane (BLM) of the renal cortex. Guanine inhibited the enzyme activity in a dose-dependent manner with maximal effect (56%) obtained at 10â»6 M. This effect was reversed by DPCPX (8-cyclopentyl-1,3-dipropylxanthine), an antagonist of A1 receptors, but it was not changed by 10â»8 M DMPX (3,7-dimethyl-1-propargylxanthine) or 10â»8 M MRS (2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate), antagonists of A2 and A3 receptors, respectively. Furthermore, it was observed that guanine increased [γ-³5S]GTP-specific binding with the maximal effect observed at 10â»6 M and this effect was abolished by 10â»6 M GDPßS. The inhibitory effect of 10â»6 M guanine on Na+-ATPase activity was reversed by 10â»6 M GDPßS, 10â»6 M forskolin, 10â»6 M pertussis toxin and 10â»8 M cholera toxin. These results indicate that guanine binds to a DPCPX-sensitive receptor promoting the activation of Gi protein and leading to a decrease in cAMP level and, consequently, inhibition of BLM Na+-ATPase activity.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Transporte de Cátions/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanina/farmacologia , Córtex Renal/metabolismo , Antagonistas do Receptor A1 de Adenosina/farmacologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Guanina/metabolismo , Técnicas In Vitro , Córtex Renal/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Cinética , Suínos , Xantinas/farmacologiaRESUMO
In the present work, we demonstrate that adenine reduced Na(+)-ATPase activity in isolated basolateral membrane (BLM) of proximal tubule in a dose-dependent manner. Adenine metabolism was ruled out by TLC analysis of the potential [(3)H]adenine derived-metabolites. Specific binding of [(3)H]adenine to isolated BLM was observed in a dose-dependent manner with K(d) and B(max) of 242.6+/-27.6 nM and 2749.9+/-104.9 fmolmg(-1), respectively. Adenine increased the [(35)S]GTPgammaS specific binding and it was completely abolished by 10(-6)M GDPbetaS (G protein inhibitor) but it was not modified by DPCPX, DMPX and MRS1523, selective antagonists for A(1), A(2) and A(3) receptors, respectively. Furthermore, the inhibitory effect of adenine on the Na(+)-ATPase activity was blocked by 10(-6)M GDPbetaS, 1 microg/ml pertussis toxin (Gi protein inhibitor), 10(-6)M foskolin (adenylyl cyclase activator) and 10(-8)M cAMP. These data demonstrate that adenine inhibits the proximal tubule Na(+)-ATPase activity through the Gi protein-coupled receptor.