RESUMEN
In this work, the (Na(+)/K(+))-ATPase activity was evaluated during the early stages of the postnatal development of rat retina and showed an almost three-time increase from P0 to P14. Expression of the three catalytic subunit isoforms (α1, α2, and α3) of the (Na(+)/K(+))-ATPase was also evaluated by immunoblot in the same period, but no correlation to the catalytic activity increment was observed. On the other hand, immunolocalization of these three α-catalytic isoforms in the developing retina showed an age-related pattern. Involvement of IGF-I in the stimulation of the (Na(+)/K(+))-ATPase was investigated. Our results demonstrate that the exogenous IGF-I (10 ng/mL) stimulates enzyme activity at the age of P7 only. Incubation of retinas with 10 µM I-OMe-AG 538 (inhibitor of the IGF-I receptor) indicates that the basal (Na(+)/K(+))-ATPase activity is sustained by endogenous IGF-I in P7 animals. These data were corroborated by an age-dependent decrease in the immunodetection of endogenous IGF-I as well as in the phosphorylation level of its cognate receptor in rat retina homogenates. The signaling pathway involved in IGF-I-induced modulation of the (Na(+)/K(+))-ATPase was also investigated. Our data show that the inhibitory effects induced by I-OMe-AG 538 and the PI 3-kinase inhibitor Ly 294002 on the basal (Na(+)/K(+))-ATPase activity were non-cumulative. Furthermore, IGF-I induced phosphorylation of PKB in a Ly 294002-sensitive manner. Together, these data demonstrate that the PI 3-kinase/PKB signaling pathway is involved in the IGF-I-sustained basal (Na(+)/K(+))-ATPase activity during the first 7 days of the postnatal development of rat retina.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Retina/enzimología , Retina/crecimiento & desarrollo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Animales Recién Nacidos , Dominio Catalítico , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinasas , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de SeñalRESUMEN
We have previously demonstrated that adenosine is deaminated to inosine in the isolated basolateral membrane (BLM) of kidney proximal tubules. This work investigates the possible effect of inosine on proximal tubule Na(+)-ATPase activity. Inosine reduced Na(+)-ATPase activity by 70%. This effect of inosine was completely attenuated by 10(-8) M DPCPX, an A(1) receptor-selective antagonist, but it was not affected by either 10(-8) M DMPX or 10(-7) M MRS1523, A(2) and A(3) receptor-selective antagonists, respectively. The inhibitory effect of inosine was blocked by: (1) 10(-6) M GDPbetaS, a trimeric G protein inhibitor; (2) 1microg/ml pertussis toxin, a Gi protein inhibitor; (3) 10(-6) M forskolin, an adenylyl cyclase activator; (4) 10(-9) M cholera toxin, a Gs protein activator; (5) 10(-6)M cAMP. Our results demonstrate that the inhibitory effect of inosine on the sodium pump is mediated by the A(1) receptor/Gi/cAMP pathway.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , AMP Cíclico/metabolismo , Inosina/farmacología , Túbulos Renales Proximales/enzimología , Receptor de Adenosina A1/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Antagonistas del Receptor de Adenosina A1 , Antagonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A3 , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Receptor de Adenosina A3/metabolismo , Receptores de Adenosina A2/metabolismo , Porcinos , Xantinas/farmacologíaRESUMEN
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.
Asunto(s)
Adenosina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inosina/metabolismo , Túbulos Renales Proximales/metabolismo , Antagonistas del Receptor de Adenosina A1 , Adenosina Desaminasa/metabolismo , Animales , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Técnicas In Vitro , Inosina/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Receptor de Adenosina A1/metabolismo , Transducción de Señal/efectos de los fármacos , Porcinos , Xantinas/farmacologíaRESUMEN
It has been recently demonstrated that HIV-1 reverse transcriptase is the target of two diterpenes, (6 R)-6-hydroxydichotoma-3,14-diene-1,17-dial (compound 1) and (6 R)-6-acetoxydichotoma-3,14-diene-1,17-dial (compound 2), that inhibit HIV-1 replication in vitro. In this work, the effects of both diterpenes on the kinetic properties of the recombinant HIV-1 reverse transcriptase (RT) enzyme were evaluated. RNA-dependent DNA-polymerase (RDDP) activity assays demonstrated that both diterpenes behave as non-competitive inhibitors with respect to dTTP and uncompetitive inhibitors with respect to poly(rA).oligo(dT) template primers. The K(i) values obtained for compounds 1 and 2 were 10 and 35 microM, respectively. Neither of these diterpenes affected the DNA-dependent DNA-polymerase (DDDP) activity of the HIV-1 RT. The RDDP activities of AMV-RT and MMLV-RT enzymes were also inhibited by compounds 1 and 2. In contrast to the HIV-1 enzyme, the DDDP activities of AMV-RT and MMLV-RT enzymes were significantly reduced by compound 1. Taken together, our results demonstrate that compound 1 is a more effective inhibitor of the viral reverse transcriptases from HIV-1, AMV and MMLV than compound 2. The kinetic behavior analyses of the HIV-1 RT demonstrate that both diterpenes have similar mechanisms of inhibition of RDDP activity.