Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling.

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 GDPßS. The inhibitory effect of 10⁻6 M guanine on Na+-ATPase activity was reversed by 10⁻6 M GDPßS, 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.

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.

Adenine-induced inhibition of Na(+)-ATPase activity: Evidence for involvement of the Gi protein-coupled receptor in the cAMP signaling pathway.

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.

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.

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.

Stimulation of the proximal tubule Na+-ATPase activity by adenosine A(2A) receptor.

The aim of this work was to determine the molecular mechanism involved in the stimulation of the pig kidney proximal tubule Na+-ATPase by adenosine (Ado). To study the role of A2 Ado receptors, we added in all experiments 10(-6)M DPCPX, an A1 receptor-selective antagonist, since we have previously shown that Ado inhibits the enzyme activity through this receptor. Ado increased the Na+-ATPase activity with maximal effect observed at 10(-6)M. The presence of both A(2A) and A(2B) receptors were demonstrated by immunoblotting using specific polyclonal antibodies. The stimulatory effect of Ado was completely abolished by 5 x 10(-9)M DMPX, an antagonist of A2 receptor, and 10(-7)M SCH 58261, an A(2A) receptor-selective antagonist. DMPA (10(-7)M), a specific agonist of A(2A) receptor mimicked the stimulatory effect of Ado. Involvement of a Gs protein/adenylate cyclase/PKA pathway was evidenced by: (a) the reversion of Ado-induced effect by GDPbetaS; (b) stimulation of the Na+-ATPase activity in a similar and non-additive manner to Ado by 10(-8)M cholera toxin, 10(-7)M GTPgammaS, 10(-6)M forskolin, 10(-7)M cAMP or 1.25 U catalytic subunit of PKA; (c) the reversion of the stimulatory effect of Ado by 10(-8)M PKA inhibitor peptide; (d) Ado-produced two-fold increase of the PKA activity, which was completely reversed by 10(-6)M DMPX. These are the first evidences showing the modulation of a renal primary active sodium transporter by Ado through A(2A) receptor.

Antiviral activity of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis against human immunodeficiency virus type 1 (HIV-1).

The antiviral effect of the CH(2)Cl(2)/MeOH-soluble fraction from the alga Dictyota menstrualis on HIV-1 replication was evaluated in vitro. The antiretroviral activity was attributed to two diterpenes: (6R)-6-hydroxydichotoma-3,14-diene-1,17-dial, named Da-1, and (6R)-6-acetoxi-dichotoma-3,14-diene-1,17-dial, named AcDa-1. Da-1 or AcDa-1 were added to the culture medium of HIV-1-infected PM-1 cells at different times post-infection or during virus adsorption/penetration. The results indicated that the compounds affected an early step of the virus replicative cycle. Virus binding and entry into the host cells were evaluated in the presence of each diterpene, but no inhibitory effect was observed. To evaluate provirus DNA synthesis/integration into the host genome, the viral protease coding sequence was amplified from total cellular DNA. Proviral DNA was not detected in infected cells incubated with the diterpenes. To investigate the effect of the diterpenes on the reverse transcription of the viral genomic RNA, the recombinant HIV-1 reverse transcriptase (RT) was assayed in vitro in the presence of each diterpene. Da-1 and AcDa-1 inhibited the RNA-dependent DNA-polymerase activity of HIV-1 RT in a dose-dependent manner. Taken together, our results demonstrate that both diterpenes inhibit HIV-1 RT and consequently virus replication.

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.

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.

Inhibition of vaccinia virus replication by adenosine in BSC-40 cells: involvement of A(2) receptor-mediated PKA activation.

In the present study, we show that adenosine (Ado) affects vaccinia virus (VV) replication in BSC-40 cells. In order to identify its effects on VV replicative cycle we analyzed the synthesis of virus macromolecules in cells incubated with 0.5 mM Ado. A 50% reduction in the steady-state level of virus DNA synthesis was observed. Consequently, virus post-replicative gene expression was also affected. A prolonged synthesis of the F11L early virus protein was also observed and it is likely related to a slow decline in the steady-state level of early mRNAs, as suggested by northern blot analysis of the VGF early transcript. The involvement of cAMP-signaling pathway as mediator of Ado response was also evaluated. Ado stimulated a three-fold increase in cAMP levels in BSC-40 cells and cAMP-mimetic agents reduced virus yield in a dose-dependent manner. Co-incubation of infected cells with H-89 reduced the inhibitory effects of 8-Br-cAMP and Ado on VV yields suggesting PKA involvement. A(2) receptor-mediated activation of PKA was indicated by antagonism of Ado response by theophylline and DMPX. Together, these results indicate that virus DNA replication is the main target of Ado. The mechanism involved is not related to reduction of the pyrimidine nucleotide synthesis. Furthermore, Ado-induced PKA activation modulates negatively an unidentified step of the virus replicative cycle.