a novel phosphorylated lipid counteracts activation of the renal plasma membrane (ca2+ + mg2+)atpase...

Bioscience Reports, Vol. 18, No. 2, 1998

A Novel Phosphorylated Lipid Counteracts Activation of theRenal Plasma Membrane (Ca2+ + Mg2+)ATPase byEndogenous Phosphatidylinositol-4-Phosphate

Adilson Guilherme,1 Cledson Reis-Silva,1 Jorge H. Moraes-Albuquerque,1Mecia M. Oliveira,2 and Adalberto Vieyra1,3

Received March 23, 1988

The plasma membrane (Ca2+ + Mg2+)ATPase is activated by acidic phospholipids in recon-stituted systems. In this report it is shown that reversible phosphorylation of endogenousphosphatidylinositol regulates the renal plasma membrane (Ca2+ + Mg2+)ATPase, and thata novel phosphorylated lipid that can be isolated from the same membrane stronglycounteracts the stimulatory effect of phosphatidylinositol-4-phosphate.

KEY WORDS: Phosphoinositides; plasma membrane; calcium pump.

INTRODUCTION

The basolateral plasma membrane of kidney tubules contains a variety of hormonalreceptors for agonists that elicit both cytosolic Ca2+ fluctuations and changes inphospholipid turnover (Gesek and Schoolwerth, 1990). It is generally accepted thatthe (Ca2+ + Mg2+)ATPase resident in the basolateral membranes of renal proximaltubule cells is responsible for the fine-tuned regulation of the intracellular Ca2+

activity despite the existence of large Ca2+ fluxes across the epithelium (Coelho-Sampaio et al., 1991; Friedman and Gesek, 1995; Vieyra, 1996).

The plasma membrane (Ca2+ + Mg2+)ATPase is modulated by oligomerization,acidic phospholipids, trypsinization and protein kinase-dependent phosphorylation(Enyedi et al., 1987; Kosk-Kosicka and Bzdega, 1990; Carafoli, 1991, 1992; Falch-etto et al., 1992; Penniston and Enyedi, 1994). Most, if not all, of these treatmentsappear to promote a decrease in the interaction of the autoinhibitory calmodulinbinding domain with its receptor regions in the pump molecule (Falchetto et al.,1991). It has also been shown that phosphatidylinositol-4-phosphate PtdIns(4)P acti-vates the sarcoplasmic reticulum (Ca2+ + Mg2+)ATPase by increasing the rate of

1Departmento de Bioquimica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.

2Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro21941-590, Brazil.

3To whom correspondence should be addressed.

790144-8463/98/0400-0079$15.00/0 © 1998 Plenum Publishing Corporation

80 Guilherme, Reis-Silva, Moraes-Albuquerque, Oliveira, and Vieyra

breakdown of the phosphorylated intermediate formed during the catalytic cycle(Lee et al., 1995).

As far as we know, there is no evidence that modifying the turnover of acidicphospholipids in the plasma membrane can promote short-term modifications in theactivity of the Ca2+ pump. In other transport ATPases, synthesis and degradation ofassociated PtdIns(4)P are influenced by Ca2+ and it is postulated that Ca2+-inducedconformational changes could affect the rate of phosphorylation and dephosphoryl-ation of the neighboring membrane lipids (Schafer et al., 1987). However, there isno evidence of functional changes associated with this lipid turnover.

The aim of this work was to investigate the influence of phosphorylation ofendogenous plasma membrane phospholipids in the activity of renal(Ca2+ + Mg2+)ATPase.

METHODS

Incorporation of 32P in membrane lipids was assayed as follows. Purified basola-teral membranes of sheep kidney proximal tubules were obtained as described else-where (Guilherme et al., 1991). Then the membranes (1 mg protein/ml) wereincubated for 30 min at 37°C in 30 mM Mes-Tris buffer (pH 7.0), 1 mM [y-32P]ATP(220uCi/umol), 0.2 mM EGTA, and 1.25% (v/v) Triton X-100 in the absence orpresence of 1.1 mM MgCl2 (final volume 0.5ml). After addition of achloroform: methanol: HCl mixture (200:100:0.75), the lipids were extracted (Kates,1972), resolved by thin layer chromatography (TLC) using oxalate impregnatedplates (Merck, Darmstadt, Germany) and chloroform: methanol: acetone: aceticacid:water (80:26:30:24:16) as solvents (Horowitz and Perlman, 1987), andexposed to X-ray film. The principal membrane lipids and the standards applied inparallel were visualized using iodine vapour. The phosphorylated lipid of greatermobility, which migrates near phosphatidylcholine, was then preparatively separatedusing bidimensional TLC (Yavin and Zutra, 1977) for biological assay (Fig. 4) andchemical characterization (Table I). The appearance of a single peak of PtdIns(4)32Pin the lower radioactive area of the plate was confirmed by anion-exchange highperformance liquid chromatography (HPLC) (Pignataro and Ascoli, 1990) usingpurified standards (Oliveira et al., 1993). After separation of the lipids by TLC, theradioactive regions of the plates were scraped, eluted withchloroform: methanol: water (10:20:8) (Pignataro and Ascoli, 1990), and counted ina liquid scintillation counter to measure the relative amounts of the two labeledlipids. Densitometric analysis of the autoradiograms gave the same results. The totalincorporation of 32P in lipids was measured by counting aliquots of the organicphase of the chloroform: methanol: HC1 extract.

Ceramide 1-32P standard was prepared using a modification of the proceduredescribed by Schneider and Kennedy (1973) and adapted by Preiss et al. (1986).Type III ceramide (0.5 mg) (Sigma Chemical Co., St. Louis, MO, USA) suspendedin 0.3 ml chloroform with 2 mM cardiolipin was dried under N2. The ceramide/cardiolipin mixture was then resuspended in 0.1 ml of 7.5% n-octyl-b-glycopyrano-side and sonicated for 5 min in a bath sonicator. The lipid mixture was then addedto a reaction mixture (0.4 ml) containing 50 mM imidazole-HCl (pH 6.6), 50 mM

Phosphorylated Lipid and Calcium Pump 81

Table I. Chemical Characterization of the Phosphorylated Lipidic Molecule that CounteractsActivation of (Ca2+ + Mg2+)ATPase by PtdIns(4)P

Rfa 0.66Fatty acid content (in comparison with glycerophospholipids)b =50%Methanolysis in mild alkaline conditionsc NoHydrolysis by phospholipase A2d NoIncrease of phosphorylation in a Triton X-100-resistant membrane complex* 420%Hydrolysis by alkaline phosphatasef 100%Hydrolysis by phospholipase Cg NoPeriodate sensitivity of the phosphoryl grouph NoHgCl2 sensitivityi NoAcid butanol hydrolysisj No

aTLC separation shown in Fig. 1a.b Phosphorylated lipids were separated by TLC, dried and methanolyzed in 0.5 M HO (inmethanol) during 18 h at 80°C. The fatty-acid methyl esters were extracted with hexane andanalyzed by gas-liquid chromatography on a 5% phenyl-methyl silicone column (40 to 120°Cat 15°C/min; 120 to 300°C at 6°C/min).

c Phosphorylated lipids were methanolyzed in mild alkaline conditions (Smith and Lester,1974), and then separated by TLC.

dThe non-methanolyzed total lipid extract obtained as described under Methods was incubatedwith phospholipase A2 from Crotalus durissus (Sigma Chemical Co., St. Louis, MO).

The membranes were solubilized with Triton X-100 and centrifuged as previously described(Vieyra et al. 1986). The pellet (Triton X-100-resistant membrane complex) was phosphoryl-ated as described under Methods. Percent increase of phosphorylation was calculated withrespect to the whole mixture.

fThe phosphorylated total lipids were incubated with 2 U/ml of calf intestinal alkaline phos-phatase (Sigma Chemical Co., St. Louis, MO, USA) in 50 mM Tris-HCl (pH 8.0), 100 mMZnCl2, and 1 mM MgCl2 for 3 h at 37°C. After this treatment, the radioactivity of the areacorresponding to the new lipid completely disappeared. 32Pi was detected by paper electroph-oresis and by extraction as a phosphomolybdate complex (Vieyra et al., 1986; Guilherme etal., 1992).

gThe total lipid extract from phosphorylated membranes was incubated in 100 mM Tris-HCl(pH 7.2), 20 mM CaCl2 and 10 U/ml of phospholipase C from Clostridium perfringens (SigmaChemical Co., St. Louis, MO) for 12 h at 37°C.

h Assayed as described by Smith and Lester (1974) and Randriamampita and Tsien (1993).iAssayed as described by Kates (1972).jTreatment with 6 N HC1:1 -butanol (1:1) was carried out according to Kaller (1961). Afterextraction, the membrane lipids were suspended in 0.3 ml of a 6 N HCl : 1 -butanol mixture(1 : 1) and incubated for 1 h at 100°C. Then the samples were dried, resuspended inchloroform: methanol (1:1) and resolved by TLC. After this treatment, the Rf of the unknownlipid remained unchanged when compared with that from untreated samples. Parallel treat-ment of the ceramide 1-32P standard followed by TLC showed deacylation to sphingosine 1-32P.

NaCl, 12.5mM MgCl2, 1 mM EGTA, 5 mM [y-32P]ATP (=250uCi/umol), and0.8 U/ml of E. coli recombinant DAG-kinase (Calbiochem, San Diego, CA, USA).After 1 h at room temperature the reaction was stopped by addition ofchloroform:methanol:HC1 (100:100:1), and the organic phase separated andwashed twice with methanol: 1 N HC1 (1:1). The contaminant glycerophospholipidswere removed by mild alkaline hydrolysis in 40 mM NaOH as described by Smithand Lester (1974). The ceramide 1-32P standard and the labeled phosphorylated lipid


of greater mobility formed in the incubated membranes were simultaneouslyresolved by TLC using chloroform :methanol: acetone: acetic acid: water(80:26:30:24:16) as solvents.

Except when otherwise indicated (Ca2+ + Mg2+)ATPase was assayed in amedium containing 1.25% (v/v) Triton X-100, 30 mM Mes-Tris (pH 9.0), 5 mM [y-32P]ATP, 10 mM MgCl2, 120 mM KC1, 1 mM ouabain, 10 mM NaN3, 160 mMsucrose, 0.2 mM EGTA and 217 um CaCl2 (12 uM free Ca2+). Temperature of theassays was 37°C and incubation time was 20 min. 32Pi release was determined aspreviously described (Vieyra et al., 1986; Guilherme et al., 1991).

The effect of the novel phosphorylated lipid on the activation by PtdIns(4)P of(Ca2++ Mg2+)ATPase activity, was also tested in reconstitution experiments afterisolation of these lipids, as follows. The membranes (1 mg/ml) were first incubatedin the phosphorylating conditions described above. PtdIns(4)32P and the novel phos-phorylated product were chromatographed, separated and visualized by auto-radiography. The radioactive regions were scraped and eluted withchloroform :methanol: water (10:20:8) (Pignataro and Ascoli, 1990). In each experi-ment the pooled extracts of six plates were dried under N2, resuspended in 1 ml of30 mM Mes-Tris buffer (pH 7.0) and sonicated for 2 min at room temperature in aThornton apparatus (Brazil). Aliquots of these suspensions were mixed with non-preincubated membranes and sonicated for 5 min at room temperature, prior toassay of (Ca2+ + Mg2+)ATPase activity in the reaction medium described above sup-plied with 20 ug/ml phosphatidylcholine. The amount of eluted lipid added (in pmolof incorporated 32P) is indicated on the abscissa of Fig. 4. Control samples run inparallel using phosphatidylcholine or extracts from other areas of the plate withoutlipid had neither an activating nor an inhibitory effect on (Ca2+ + Mg2+)ATPaseactivity.

Several chemical properties of the novel phosphorylated lipid were investigatedusing the methods described in the legend of Table I. The reagents used in all experi-ments were of the best grade available. 32Pi was purchased from the Brazilian Insti-tute of Nuclear and Energetics Research. [y-32P]ATP was synthesized according toMaia et al. (1983). All experiments were repeated at least four times using differentmembrane preparations. The autoradiograms in the figures correspond to typicalexperiments repeated at least three times.

RESULTS AND DISCUSSION

Incubation of purified basolateral membranes of proximal tubule cells with [y-32P]ATP in the presence of nanomolar Ca2+ concentrations (0.2 mM EGTA; freeCa2+< 10-8M) leads to a rapid incorporation of 32P into the membranes, and thisphosphorylation is resistant to hydroxylamine, as previously shown (Guilherme etal., 1992). Thirty-two per cent of the 32P incorporated is found in lipids with anunusual pattern of distribution. Figure la shows how this label is distributed: about65% is found in PtdIns(4)32P (Rf=0.28; identified in Fig. 1b); the rest is incorporatedin a novel phosphorylated lipid of greater mobility (Rf=0.66; Fig. 1a). Phosphoryl-ation of PtdIns(4)P decreases rapidly upon removal of the [y-32P]ATP (Fig. 1c, filledcircles), indicating that it undergoes a rapid turnover in the membrane, whereas the


novel phosphorylated lipid remains unchanged (Fig. 1d; filled circles), showing thatthe phosphatase responsible for its dephosphorylation is not present in the plasmamembrane fraction. Although the present study does not address the enzymaticmechanism in basolateral membranes which is involved in the labeling of the novellipid, it should be mentioned that Mg2+ ions are not required. This is another differ-ence with respect to PtdIns(4)P, which is not labeled in the absence of MgCl2 (seebelow Fig. 5).

The participation of a fluoride-sensitive phosphomonoesterase has been impli-cated in the regulation of PtdIns(4)P in sarcoplasmic reticulum (Schafer et al., 1987).To investigate the possible involvement of membrane-bound phosphatase in theturnover of PtdIns(4)P shown in Fig. 1c, 1 mM sodium fluoride (NaF) was added

Fig. 1. Formation of PtdIns(4)P and of a novel phosphorylated lipidic molecule in basolateralplasma membranes of kidney proximal tubule cells. (a) Autoradiography of the phosphorylatedlipids labeled in the presence of 1.1 mM MgCl2. (b) Anion-exchange HPLC shows a single peak ofPtdIns(4)32P after elution of the lower radioactive area of the plate. Effect of substrate removal inthe level of the previously formed PtdIns(4)32P (c) and the novel phosphorylated lipid (d). Themembranes were incubated in the phosphorylation medium indicated above and after 30 min(arrows; zero time on the abscissae) the samples were supplied with hexokinase (16 U/ml) and20 mM glucose (filled circles) or with glucose alone (control; empty circles). The lipids were separatedby TLC and the radioactive areas of the plates were cut out. Then, the lipids were extracted, chroma-tographed, separated as above, and counted in a liquid scintillation counter.


Fig. 2. Opposing effects of sodium fluoride in the formation of PtdIns(4)32P and of the novel phos-phorylated lipidic molecule, and correlation with plasma membrane (Ca2+ + Mg2+)ATPase activity,(a) Phosphorylation of membrane lipids was assayed as indicated under Methods (with 1.1 mMMgCl2), in the absence or in the presence of 1 mM NaF. Membrane lipids were extracted, dried,weighed and resuspended in chloroform :methanol: water. Aliquots were pipetted onto a filter, driedand counted, and the results were expressed as pmol 32P-phosphorus incorporated per mg of totallipids. (b) Autoradiogram of the phosphorylated lipids shown in Fig. 1a, extracted and separatedfrom membranes incubated in the absence or in the presence of NaF. (c) (Ca2+ + Mg2+) ATPaseactivity. The membranes were preincubated in the phosphorylation medium described underMethods (with 1.1 mM MgCl2), in the absence or in the presence of 1 mM NaF. Then, aliquotswere five-fold diluted in the (Ca2+ + Mg2+)ATPase assay medium and ATP hydrolysis was measuredas described under Methods.

to the phosphorylation assay used in Fig. 1a. In the presence of NaF, there is a2.3-fold increase in total lipid phosphorylation (Fig. 2a). Autoradiography shows acorresponding increase in PtdIns(4)32P, together with a decrease in phosphorylationof the unknown lipid (Fig. 2b). At the same time, (Ca2+ + Mg2+)ATPase activity alsoincreases (Fig. 2c). Activation of the ATPase becomes evident when uM Ca2+ isadded to the assay after 30-min preincubation with [y-32P]ATP and EGTA. Theseresults clearly indicate that formation of PtdIns(4)P in the natural membraneenvironment is associated with activation of the Ca2+ pump. The fact that chemicalrecognition of lipids was not possible in the plate areas from which PtdIns(4)32P andthe novel lipid are removed, suggests that interaction of the Ca2+ pump with bothendogenous lipids occurs with a low stoichiometry of phosphorylatedlipids: ATPase.

Whereas Fig. 1d indicates that the membrane preparation does not contain aphosphatase able to dephosphorylate the lipid of greater mobility, Fig. 2b showsthat phosphorylation of this compound is strongly inhibited in NaF-containingmedium. It may be that the kinase involved in its phosphorylation is inhibited byNaF, as in the case of some kinases activated by growth factors (Erikson and Mailer,1986). Alternatively, the lipid kinase may be more active only in the dephosphoryl-ated form, as are a variety of protein kinases (Dent et al., 1990).

The effect of NaF on membrane lipid phosphorylation at a shorter time (2 min)is seen in Fig. 3. In this shorter interval the level of PtdIns(4)32P (Fig. 3a) is lowerthan at 30 min (25 and 72 pmol/mg, respectively) and there is no effect of NaF on


its level (27 pmol/mg), whereas the formation of the novel phosphorylated lipid (Fig.3b) shows the same strong inhibition found at 30 min (Fig. 2b). Phosphorylation ofthe novel lipid decreases by 80% with respect to the control without NaF (1.1 and5.1, respectively). In this condition, the plasma membrane (Ca2+ + Mg2+)ATPaseactivity is the mirror image of the phosphorylation of this lipid (Fig. 3c) suggestingthat the phosphorylated lipid may act as a natural inhibitor of the Ca2+ pump.

Fig. 3. Correlation between the level ofthe novel phosphorylated lipid and(Ca2+ + Mg2+)ATPase activity, (a and b)The membranes were phosphorylated inthe conditions described under Methods(with 1.1 mM MgCl2), in the absence orpresence of 1 mM NaF, except that incu-bation time was 2 min. Radioactive lipidswere extracted, resolved and counted asdescribed under Methods. (c) (Ca2+ +Mg2+)ATPase activity of the membranespreincubated for 2 min was assayed asdescribed under Methods.


To further explore the effects of both phospholipids on (Ca2+ + Mg2+)ATPaseactivity, they were eluted from the chromatograms and mixed with solubilizedenzyme not preincubated in phosphorylating conditions. Measurement of enzymeactivity after addition of MgATP2– and Ca2+ shows that addition of the elutedPtdIns(4)P stimulates (Ca2++ Mg2+)ATPase activity by more than 60% (Fig. 4,empty bar). The novel phosphorylated molecule activates the enzyme in a saturablemanner when assayed alone (Fig. 4, hatched bars), suggesting that it may act as apartial agonist of the effect of endogenous phosphoinositides. Since it is able tocounteract the stimulation by PtdIns(4)P when both are present (Fig. 4, filled bars),it may be that activation of the (Ca2+ + Mg2+)ATPase is controlled by the reciprocalchanges in the phosphorylation of these lipids in the membrane. The fact that theeffects shown in Fig. 4 are observed in the presence of excess phosphatidylcholine(20 ug/ml) clearly indicates that both modulators interact in a specific lipid-bindingdomain of the renal enzyme. Specific binding domains for negatively charged lipidshave been described in the (Ca2+ + Mg2+)ATPase from red cells (Brodin et al., 1992).

In an attempt to characterize the novel phosphorylated lipid, several chemicalproperties were evaluated, and they are summarized in Table I. It was first isolated,methanolyzed and extracted with hexane to analyze the fatty acid methyl esters.Gas-liquid chromatography performed in parallel with a sample of PtdIns(4)P elutedfrom the same thin-layer chromatogram shows that the novel compound contains= 50% of the amount of the fatty acids present in glycerolipids. This indicates thatthere exists only one acylation site in the lipid molecule.

Fig. 4. The novel phosphorylated lipid counteracts the activation byPtdIns(4)P of (Ca2+ + Mg2+)ATPase. Empty bar shows the stimulation ofthe non-preincubated enzyme by PtdIns(4)P eluted from the chromatogramshown in Fig. la. Hatched bars show the effect of the novel phosphorylatedlipid isolated after bidimensional chromatography. Filled bars show theeffect on (Ca2+ + Mg2+)ATPase activity when both lipids are added togetherat different concentration ratios.


When methanolysis of the dry extract of total lipid is performed in mild alkalineconditions, the PtdIns(4)32P disappears along with the unlabeled glycerophospholip-ids detected in the chromatogram in control conditions. In contrast, non-radioactivesphingomyelin and the novel phosphorylated molecule were insensitive to this treat-ment. This observation suggests the existence of an ether or amide linkage betweenthe fatty acid and the 32P-containing moiety of the molecule. The attachment of thefatty acid by an ether linkage, as in the case of plasmalogens, can be ruled out since:(a) the lipid contains half of the expected fatty acids for a plasmalogen; (b) migrationof the compound after mild alkaline methanolysis did not change, whereas a mol-ecule containing a single ether-linked fatty acid after cleavage of the ester linkageshould migrate with a different Rf. Moreover, the novel phosphorylated moleculewas insensitive to phospholipase A2 and HgCl2 (Table I).

Some complex lipids have been reported to be insoluble in Triton X-100, dueto the formation of heavy vesicles that minimize contact of these molecules withwater medium (Brown and Rose, 1992). The autoradiograms of the lipids in theprecipitated fraction obtained after centrifugation of the Triton X-100-treated baso-lateral membranes show a more than 400%-enrichment with the new lipid (Table I),whereas PtdIns(4)32P and the other glycerolipids remain almost completely in thesupernatant. It is of interest to mention that: (a) (Ca2+ + Mg2+)ATPase in the pellethas a lower specific activity than that in the supernatant (data not shown); (b) lipidphosphorylation decreases and (Ca2+ + Mg2+)ATPase activity of the pellet increaseswhen 1 mM NaF is added. These data indicate that the enzyme co-precipitates in acomplex with the phosphorylated lipid, and that a close physical relationshipbetween the ATPase and the novel lipid allows the occurrence of the NaF-sensitiveinhibition of the pump in the insoluble fraction. These observations, taken as awhole, argue for the existence in the lipidic molecule of a hydrophobic fatty acidmoiety together with chemical groups able to form extended hydrogen bonds withwater medium.

The position of the phosphoryl residue in the molecule was investigated bycombined analysis of the effects of phospholipase C, alkaline phosphatase, and per-iodate (Table I). The linkage between the phosphate and the hydrophilic moietyof the molecule is not a phosphodiester bridge since it is resistant to cleavage byphospholipase C. Conversely, 32P-phosphate is completely released to the mediumby treatment with alkaline phosphatase, indicating a phosphomonoester linkage.Since this lipid is completely insensitive towards periodate, it can be concluded thatthere are no vicinal diols, amino alcohols or neighboring thiol groups to be oxidized.

Although the unknown lipid is resistant to periodate oxidation, the possibilitythat it is ceramide 1-P was further investigated in the experiments shown in Fig. 5.Simultaneous thin-layer chromatography of ceramide 1-32P and of the novel lipidshows that they have different Rf values. Moreover, the mobility of the unknownlipid is not changed after treatment with 6 N HC1 and 1-butanol, which promoteshydrolysis of ceramide 1-phosphate to sphingosine 1-P (Table I) (see Dressier andKolesnick, 1990).

In conclusion, the results described in this paper show that phosphorylation ofendogenous inositides of the basolateral membranes of kidney proximal tubule cellsleads to a significant increase in the activity of the (Ca2+ + Mg2+) ATPase together


Fig. 5. Autoradiogram of ceramide 1-32P and ofthe novel phosphorylated lipid. Ceramide 1-32Pwas prepared as described under Methods. Themembranes were phosphorylated in the absenceof MgCl2, a condition in which only theunknown lipid is labeled. Simultaneous TLC ofceramide 1-32P standard and of the lipids isolatedfrom the membranes was carried out as describedunder Methods.

with the formation of PtdIns(4)P. Since this is reversible and is potentiated by NaF(Fig. 2b), it may be that phosphatidylinositol-4-kinase and phosphatase activitiesparticipate in the control of the catalytic cycle of the renal plasma membrane(Ca2+ + Mg2+)ATPase. In addition, this work also shows that a novel phosphorylatedlipid can counteract the stimulatory effect of endogenous PtdIns(4)P.

ACKNOWLEDGMENTS

We thank Dr. Gerry Smith for helpful suggestions and Dr. Martha Sorensonfor critical reading of the manuscript. This work was supported by grants fromCNPq, FINEP, FAPERJ and FUJB/UFRJ (Brazil).

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a novel phosphorylated lipid counteracts activation of the renal plasma membrane (ca2+ + mg2+)atpase...

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