L.R. Leão-Ferreira

Modulation of the (Na+ + K+)ATPase activity by Angiotensin-(1–7) in MDCK cells

In the present paper the effect of Ang-(1-7) on the distal tubule (Na(+)+K+)ATPase activity was evaluated by using MDCK cells as a model. Confluent cell monolayers were incubated with increasing concentrations of Ang-(1-7) for 30 min. Thereafter, the (Na(+)+K+)ATPase activity was evaluated and a dose-dependent (from 10(-12) to 10(-7) M) inhibition was observed. The maximal inhibitory effect (54%) was reached at the concentration of 10(-8) M. The inhibitory effect of Ang-(1-7) was not affected by the AT2 receptor selective antagonist PD123319 (from 10(-10) to 10(-7) M) but was blocked in a dose-dependent manner by the AT1 receptor selective antagonists losartan (10(-10) M), candesartan (10(-17) M), irbesartan (2 x 10(-12) M) and telmisartan (2 x 10(-16) M). The signaling pathway triggered by stimulation of the AT(1) receptor was also investigated. The PI-phospholipase C (PI-PLC) inhibitor U73122 (5 x 10(-8) M) blocked the inhibitory effect elicited by Ang-(1-7). Involvement of the protein kinase C (PKC) was evidenced by the sensitivity of the inhibitory effect of Ang-(1-7) to calphostin C (6.32 x 10(-7) M) and the lack of additive effects when the cells were co-incubated with Ang-(1-7) and 3.2 x 10(-8) M PMA. Altogether, these results demonstrate that Ang-(1-7) inhibits the (Na(+)+K+)ATPase activity of the prototypic distal tubule cell MDCK through the AT1 receptor-mediated stimulation of PI-PLC/PKC signaling pathway.

Synthesis and antiviral activities of new pyrazolo[4,3-c]quinolin-3-ones and their ribonucleoside derivatives.

Several new pyrazolo[4,3‐c]quinolin‐3‐one ribonucleosides (5a–g) and their corresponding heterocycle moieties (3a–g) were synthesized and evaluated against vaccinia virus (VV) and herpes simplex virus type 1 (HSV‐1). The derivatives 3c and 3d showed modest inhibitory activity against vaccinia virus reaching 70% at a concentration of 100 µM. All heterocyclic compounds (3a–f) showed a modest inhibition against HSV‐1, reaching the maximal inhibitory effect around 20–30%. The antiviral effects of most of the pyrazolo[4,3‐c]quinolin‐3‐one ribonucleosides (5a–f) on VV and HSV were not impressive.

PLA2/PGE2 are involved in the inhibitory effect of bradykinin on the angiotensin-(1–7)-stimulated Na+-ATPase activity of the proximal tubule

Recently, we demonstrated that bradykinin (BK) counteracts the stimulatory effect of Ang-(1-7) on the Na(+)-ATPase activity from basolateral membrane of the proximal tubule through B2 receptor. In the present paper, the signaling pathway involved in the inhibitory response of the Na(+)-ATPase activity to BK was investigated. The following results indicate that the phospholipase A2 (PLA2)/COX/prostaglandin E (PGE2) pathway is implicated in this process: (1) The inhibitory effect of BK on Ang-(1-7)-stimulated enzyme is abolished in a dose-dependent manner by quinacrine (10(-9)-10(-6)M), a nonspecific PLA2 inhibitor, and by PACOCF3 (10(-7)M), an inhibitor of a Ca(2+)-independent PLA2. However, AACOCF3 (2 x 10(-4) M), an inhibitor of the cytosolic PLA2, does not modify the inhibitory effect of BK. (2) The inhibitory effect of BK on the Ang-(1-7)-stimulated enzyme is reversed by cyclooxygenase (COX) inhibitors diclofenac (10(-12) M) and indomethacin (10(-12) M). (3) PGE2 (10(-12)-10(-5) M) inhibits the Na(+)-ATPase activity in a dose dependent manner. (4)The inhibitory effects of PGE2 and BK on the Na(+)-ATPase activity are not cumulative. (5) PGE2 (10(-12)-10(-8) M) counteracts the stimulatory effect of Ang-(1-7) on the enzyme activity in a dose-dependent manner.

DNA methylation: A promising target for the twenty-first century

Background: Over the last few years DNA methylation and its involvement in diseases such as cancer has become of great interest for applied research. Since reversal of aberrant DNA methylation may influence the behavior of tumors, the methylation of DNA CpG sites is a potential target for the development of inhibitors for use in cancer treatment. Objective/methods: We briefly review the structural and mechanistic features of DNA methylation, including a structural analysis of the three main human DNA methyltransferases and some (pre)clinical results. Results/conclusion: Despite side effects, data obtained to date still support the vision that DNA-methylation, possibly associated with the use of histone deacetylases (HDACs) and/or artificial transcription factors (ATFs), is a promising target for improving anticancer therapy in the 21st century.

Crosstalk between the signaling pathways triggered by angiotensin II and adenosine in the renal proximal tubules : Implications for modulation of Na+-ATPase activity

We have previously demonstrated that adenosine (Ado) reverses the stimulatory effect of angiotensin II (Ang II) on Na(+)-ATPase activity via the A(2A) receptor. In this work, the molecular mechanism involved in Ado-induced shutdown in the signaling pathway triggered by 10(-8)M Ang II was investigated. It was observed that: (1) both 10(-12)M PMA (a PKC activator) and 5x10(-8)M U73122 (an inhibitor of PI-PLCbeta) prevent the reversion effect induced by 10(-6)M Ado (only observed in the presence of 10(-6)M DPCPX (an A(1) receptor antagonist)) on Ang II-stimulated Na(+)-ATPase and PKC activities; (2) Ang II-stimulated PKC activity was reversed by 10(-6)M forskolin (an adenylyl cyclase activator) or 10(-8)M PKA inhibitory peptide and 10(-8)M DMPX (an A(2) receptor-selective antagonist). Considering that PMA prevents the inhibitory effect of Ado on Ang II-stimulated Na(+)-ATPase and PKC activities, it is likely that the PMA-induced effect, i.e. PKC activation, is downstream of the target for Ado-induced reversion of Ang II stimulation of Na(+)-ATPase activity. We investigated the hypothesis that PI-PLCbeta could be the target for Ado-induced PKA activation. Our data demonstrate that Ang II-stimulated PI-PLCbeta activity was reversed by Ado or 10(-7)M cAMP; the reversibility of the Ado-induced effect was prevented by either DMPX or PKA inhibitory peptide. These data demonstrate that Ado-induced PKA activation reduces Ang II-induced stimulation of PI-PLCbeta.

Adenosine reverses the stimulatory effect of angiotensin II on the renal Na+-ATPase activity through the A2 receptor

In the present paper, we report the modulation of the Angiotensin II (Ang II)-stimulated Na+-ATPase activity of the proximal tubule basolateral membrane by adenosine (Ado). Preincubation of isolated basolateral membrane with 10(-8)M Ang II increases the Na+-ATPase activity from 7.5+/-0.3 (control) to 14.6+/-0.9 nmol Pi x mg(-1)x min(-1)nmol Pi x mg(-1) x min(-1) (p<0.05). Incubation of Ang II-stimulated enzyme with 10(-6)M Ado, in the presence of the A1 receptor antagonist DPCPX (10(-6)M), completely reverses the Ang II-induced effect bringing the Na+-ATPase activity to the basal level. The following evidences demonstrate involvement of the A2 receptor/Gs protein/adenylyl cyclase/PKA signaling pathway in the inhibitory effect induced by Ado on the Ang II-stimulated Na+-ATPase activity in the presence of the DPCPX: 1) the inhibitory effect of Ado is abolished by the A2 receptor selective antagonist DMPX (10(-8)M); 2) the effect induced by Ado is blocked by 10(-8)M GDPbetaS and mimicked by 10(-9)M cholera toxin and 10(-8)M GTPgammaS; 3) the stimulatory effect of Ang II is reduced by 10(-6)M forskolin, an activator of adenylyl cyclase, or 10(-6)M cAMP; 4) Ado stimulates PKA activity; 5) the inhibitory effect induced by this nucleoside is reversed by the PKA inhibitor peptide.

Inhibition of renal Na+-ATPase activity by inosine is mediated by A1 receptor-induced inhibition of the cAMP signaling pathway.

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.

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.

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor.

Recently, we demonstrated that angiotensin-(1-7) (Ang-(1-7)) stimulates the Na(+)-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B(2) receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1-7)-stimulated Na(+)-ATPase activity was evaluated. Preincubation of Na(+)-ATPase with 10(-9) M Ang-(1-7) increases enzyme activity from 7.9+/-0.9 to 14.1+/-1.5 nmol Pi mg(-1) min(-1), corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10(-14)-10(-8) M), reaching maximal inhibitory effect at 10(-9) M. Des-Arg(9) bradykinin (DABK), an agonist of B(1) receptor, at the concentrations of 10(-9)-10(-7) M, does not mimic the BK inhibitory effect, and des-Arg(9)-[Leu(8)]-BK (DALBK), a B(1) receptor antagonist, at the concentrations of 10(-10)-10(-7) M, does not prevent the inhibitory effect of BK on Ang-(1-7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B(2) receptor, abolishes the inhibitory effect of BK on the Ang-(1-7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10(-7) M. Taken together, these data indicate that stimulation of B(2) receptors by BK can counteract the stimulatory effect of Ang-(1-7) on the proximal tubule Na(+)-ATPase activity.

Guanine-induced inhibition of renal Na+-ATPase activity: Evidence for the involvement of the Gi protein-coupled receptor

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 10⁻⁶ M. This effect was reversed by DPCPX (8-cyclopentyl-1,3-dipropylxanthine), an antagonist of A₁ receptors, but it was not changed by 10⁻⁸ M DMPX (3,7-dimethyl-1-propargylxanthine) or 10⁻⁸ 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 10⁻⁶ M and this effect was abolished by 10⁻⁶ M GDPβS. The inhibitory effect of 10⁻⁶ M guanine on Na+-ATPase activity was reversed by 10⁻⁶ M GDPβS, 10⁻⁶ M forskolin, 10⁻⁶ M pertussis toxin and 10⁻⁸ 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.