Angiotensin II stimulates renal proximal tubule Na(+)-ATPase activity through the activation of protein kinase C

Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.     

PI-PLCbeta is involved in the modulation of the proximal tubule Na+-ATPase by angiotensin II

In previous papers we showed that Ang II increases the proximal tubule Na+-ATPase activity through AT1/PKC pathway [L.B. Rangel, C. Caruso-Neves, L.S. Lara, A.G. Lopes, Angiotensin II stimulates renal proximal tubule Na+-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316, L.B.A. Rangel, A.G. Lopes, L.S. Lara, C. Caruso-Neves, Angiotensin II stimulates renal proximal tubule Na+)-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316]. In the present paper, we study the involvement of PI-PLCbeta on the stimulatory effect of angiotensin II (Ang II) on the proximal tubule Na+-ATPase activity. Western blotting assays, using a polyclonal antibody for PI-PLCbeta, show a single band of about 150 KDa, which correspond to PI-PLCbeta isoforms. Ang II induces a rapid decrease in PIP2 levels, a PI-PLCbeta substrate, being the maximal effect observed after 30 s incubation. This effect of Ang II is completely abolished by 5 x 10(-8) M U73122, a specific inhibitor of PI-PLCbeta. In this way, the effect of 10(-8) M Ang II on the proximal tubule basolateral membrane (BLM) Na+-ATPase activity is completely abolished by 5 x 10(-8) M U73122. The increase in diacylglycerol (DAG) concentration, an product of PI-PLCbeta, from 0.1 to 10 nM raises the Na+-ATPase activity from 6.1+/-0.2 to 13.1+/-1.8 nmol Pi mg(-1) min(-1). This effect is similar and non-additive to that observed with Ang II. Furthermore, the stimulatory effect of 10 nM DAG is completely reversed by 10(-8) M calphostin C (Calph C), an inhibitor of PKC. Taken together these data indicate that Ang II stimulates the Na+-ATPase activity of proximal tubule BLM through a PI-PLCbeta/PKC pathway.     

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.     

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.     

Protein kinase C-induced phosphorylation modulates the Na(+)-ATPase activity from proximal tubules

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from renal proximal tubule basolateral membranes (BLM) by protein kinase C (PKC). Two PKC isoforms were identified in BLM, one of 75 kDa and the other of 135 kDa. The former correlates with the PKC isoforms described in the literature but the latter seems to be a novel isoform, not yet identified. Both PKC isoforms of BLM are functional since a protein kinase C activator, TPA, increased the total hydroxylamine-resistant 32P(i) incorporation from [gamma-32P]ATP into the BLM. In parallel, TPA stimulated the Na(+)-ATPase activity from BLM in a dose-dependent manner, the effect being reversed by the PKC inhibitor sphingosine. The stimulatory effect of TPA on Na(+)-ATPase involved an increase in the V(max) (from 13.4+/-0.6 nmol P(i) mg(-1) min(-1) to 25.2+/-1.4 nmol P(i) mg(-1) min(-1), in the presence of TPA, P<0.05) but did not change the apparent affinity for Na(+) (K(0.5)=14.5+/-2.1 mM in control and 10.0+/-2.1 mM in the presence of TPA, P>0.07). PKC involvement was further confirmed by stimulation of the Na(+)-ATPase activity by the catalytic subunit of PKC (PKC-M). Finally, the phosphorylation of an approx. 100 kDa protein in the BLM (the suggested molecular mass of Na(+)-ATPase [1]) was induced by TPA. Taken together, these findings indicate that PKCs resident in BLM stimulate Na(+)-ATPase activity which could represent an important mechanism of regulation of proximal tubule Na(+) reabsorption.     

Ouabain-insensitive Na(+)-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.     

Angiotensin II stimulates renal proximal tubule Na-ATPase activity through the activation of protein kinase C

Recently, our group described an AT₁-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na⁺-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na⁺-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na⁺-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na⁺-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.     

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.     

Angiotensin-(1-7) reverts the stimulatory effect of angiotensin II on the proximal tubule Na(+)-ATPase activity via a A779-sensitive receptor.

Recently, we demonstrated that the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubules is reversed, in a dose-dependent manner, by Ang-(1-7) [Biochim. Biophys. Acta 1467 (2000) 189]. In the present paper, we characterized the receptor involved in this phenomenon. The preincubation of the Na(+)-ATPase with 10(-8) M Ang II increases the enzyme activity from 7.50+/-0.02 (control) to 12.40+/-1.50 nmol Pi mg(-1) min(-1) (p<0.05). Addition of 10(-9) M Ang-(1-7) completely reverts this effect returning the ATPase activity to the control level. This effect seems to be specific to Ang-(1-7) since Ang III (10(-12)-10(-8) M) does not modify the stimulation of the renal proximal tubule Na(+)-ATPase activity by Ang II. Saralasin abolishes the Ang-(1-7) effect in a dose-dependent manner being the maximal effect obtained at 10(-11) M. The increase in A779 concentration (from 10(-12) to 10(-7) M), a specific Ang-(1-7) antagonist, also abolishes the Ang-(1-7) effect. On the other hand, PD123319 (10(-8)-10(-6) M), an AT(2) antagonist receptor, and losartan (10(-12)-10(-7) M), an AT(1) antagonist receptor, does not modify the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) reverts the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubule through a A779-sensitive receptor.     

Leishmania amazonensis: Heme stimulates (Na + K)ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways

In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na⁺ + K⁺)ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50 nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH₃ and U73122, specific inhibitors of PI-PLC. (Na⁺ + K⁺)ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50 nM. This effect was completely reversed by 10 nM calphostin C, an inhibitor of PKC. Thus, the effect of 50 nM heme on (Na⁺ + K⁺)ATPase activity is completely abolished by ET-18-OCH₃ and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na⁺ + K⁺)ATPase activity through a PI-PLC/PKC signaling pathway.     

Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Biphasic effect of protein kinase C on rat renal cortical Na+, K+-ATPase.

We examined the dependence of rat renal Na+, K+-ATPase activity on protein kinase C (PKC) stimulation. Infusion of either phorbol 12, 13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) into rat abdominal aorta resulted in dose-dependent changes of renal cortical Na+, K+-ATPase activity. Low doses of these esters (3 x 10(-11) mol/kg/min) increased activity of Na+, K+-ATPase whereas high doses (3 x 10(-9) mol/kg/min) decreased it. The changes in Na+, K+-ATPase activity induced by PDBu and PMA were prevented by staurosporine, a PKC inhibitor. 4Alpha phorbol didecanoate (4alpha PDD), phorbol ester which does not activate PKC had no effect on cortical Na+, K+-ATPase. PDBu and PMA did not change Na+, K+-ATPase activity in the renal medulla. The stimulatory effect of PDBu (3 x 10(-11) mol/kg/min) was neither mimicked by amphotericin B, a sodium ionophore nor blocked by amiloride, an inhibitor of Na+/H+-exchanger. The inhibitory effect of 3 x 10(-9) mol/kg/min PDBu was not mimicked by amiloride indicating that the observed effects of PKC stimulation are not secondary to alterations in intracellular sodium concentration. The inhibitory effect of PDBu was prevented by infusion of ethoxyresorufin, an inhibitor of cytochrome P450-dependent arachidonate metabolism. These results suggest that the inhibitory effect of PKC on renal cortical Na+, K+-ATPase is mediated by cytochrome P450-dependent arachidonate metabolites.     

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.     

Role of protein kinase C in the signal pathways that link Na+/K+-ATPase to ERK1/2

We have shown before that Na(+)/K(+)-ATPase acts as a signal transducer, through protein-protein interactions, in addition to being an ion pump. Interaction of ouabain with the enzyme of the intact cells causes activation of Src, transactivation of EGFR, and activation of the Ras/ERK1/2 cascade. To determine the role of protein kinase C (PKC) in this pathway, neonatal rat cardiac myocytes were exposed to ouabain and assayed for translocation/activation of PKC from cytosolic to particulate fractions. Ouabain caused rapid and sustained stimulation of this translocation, evidenced by the assay of Ca(2+)-dependent and Ca(2+)-independent PKC activities and by the immunoblot analysis of the alpha, delta, and epsilon isoforms of PKC. Dose-dependent stimulation of PKC translocation by ouabain (1-100 microm) was accompanied by no more than 50% inhibition of Na(+)/K(+)-ATPase and doubling of [Ca(2+)](i), changes that do not affect myocyte viability and are known to be associated with positive inotropic, but not toxic, effects of ouabain in rat cardiac ventricles. Ouabain-induced activation of ERK1/2 was blocked by PKC inhibitors calphostin C and chelerythrine. An inhibitor of phosphoinositide turnover in myocytes also antagonized ouabain-induced PKC translocation and ERK1/2 activation. These and previous findings indicate that ouabain-induced activation of PKC and Ras, each linked to Na(+)/K(+)-ATPase through Src/EGFR, are both required for the activation of ERK1/2. Ouabain-induced PKC translocation and ERK1/2 activation were dependent on the presence of Ca(2+) in the medium, suggesting that the signal-transducing and ion-pumping functions of Na(+)/K(+)-ATPase cooperate in activation of these protein kinases and the resulting regulation of contractility and growth of the cardiac myocyte.     

Calphostin C induces tyrosine dephosphorylation/inactivation of protein kinase Fa/GSK-3α in a pathway independent of tumor promoter phorbol ester-mediated down-regulation of protein kinase C

The signal transduction mechanism of protein kinase FA /GSK-3α by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase Fa /GSK-3α could be tyrosine-dephosphorylated and inactivated to ∼ 10% of control in a concentration-dependent manner by 0.1–10 μM calphostin C (IC₅₀, ∼ 1 μM), as demonstrated by immunoprecipitation of kinase Fa /GSK-3α from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase Fa /GSK-3α immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 μM calphostin C (IC₅₀, ∼ 0.05 μM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase Fa /GSK-3α, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase Fa /GSK-3α is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA /GSK-3α in tumor cells. © 1996 Wiley-Liss, Inc.     

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and G? 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.     

Nitric oxide decreases renal medullary Na+, K+-ATPase activity through cyclic GMP-protein kinase G dependent mechanism.

The aim of this study was to investigate the effect of nitric oxide on renal Na+,K(+)-ATPase and ouabain-sensitive H+,K(+)-ATPase activities. The study was performed in male Wistar rats. The investigated substances were infused under general anaesthesia into abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. NO donor, S-nitroso-N-acetylpenicillamine (SNAP), infused at doses of 10(-7) and 10(-6)mol/kg/min decreased medullary Na+,K(+)-ATPase activity by 29.4% and 45.2%, respectively. Another NO donor, spermine NONOate, administered at the same doses reduced Na+,K(+)-ATPase activity in the renal medulla by 31.7% and 46.5%, respectively. Neither of NO releasers had any effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase. Infusion of NO precursor, L-arginine (100 micromol/kg/min), decreased medullary Na+,K(+)-ATPase activity by 32.2%, whereas inhibitor of nitric oxide synthase, L-NAME (10 nmol/kg/min), increased this activity by 20.7%. The effect of synthetic NO donors was mimicked by 8-bromo-cGMP and blocked by inhibitors of soluble guanylate cyclase, ODQ or methylene blue, as well as by specific inhibitor of protein kinase G, KT5823. In addition, inhibitory effect of either SNAP or 8-bromo-cGMP on medullary Na+,K(+)-ATPase was abolished by 17-octadecynoic acid (17-ODYA), which inhibits cytochrome P450-dependent metabolism of arachidonic acid. These data suggest that NO decreases Na+,K(+)-ATPase activity in the renal medulla through the mechanism involving cGMP, protein kinase G, and cytochrome P450-dependent arachidonate metabolites. In contrast, NO has no effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase.     

Regulation of Na+, K+ -ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.     

Involvement of the Gi/o/cGMP/PKG pathway in the AT2-mediated inhibition of outer cortex proximal tubule Na+-ATPase by Ang-(1-7)

The molecular mechanisms involved in the Ang-(1-7) [angiotensin-(1-7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1-7) has a biphasic effect on the proximal tubule Na+-ATPase activity, with the stimulatory effect mediated by the AT1 receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na+-ATPase by Ang-(1-7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT1 receptor-mediated stimulation. In this condition, Ang-(1-7) at 0.1 nM inhibited the Na+-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT2 receptor, and by 1 muM GDP[beta-S] (guanosine 5'-[beta-thio]diphosphate), an inhibitor of G protein. Ang-(1-7) at 0.1 M induced [35S]GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and 1 mug/ml pertussis toxin, an inhibitor of G(i/o) protein, reversed the Ang-(1-7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1-7) on the Na+-ATPase activity was completely reversed by 0.1 microM LY83583, an inhibitor of guanylate cyclase, and by 2 muM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1-7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 microM LY83583. Taken together, these data indicate that Ang-(1-7) inhibits the proximal tubule Na+-ATPase by interaction with the AT2 receptor that subsequently activates the G(i/o) protein/cGMP/PKG pathway.