Ceramide Is a Potent Activator of Plasma Membrane Ca2+-ATPase from Kidney Proximal Tubule Cells with Protein Kinase A as an Intermediate*

The kidney proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca2+-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca2+-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca2+ levels. In this paper we show that nanomolar concentrations of ceramide (Cer50 = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca2+-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca2+-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca2+-ATPase activity and PKA-mediated phosphorylation of the Ca2+ pump molecule. Ceramide also stimulates phosphorylation of renal Ca2+-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca2+ pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca2+-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells.

Important molecular transport processes take place across the epithelium of kidney proximal tubules. The basolateral membranes (BLM) 4 of kidney proximal tubules cells contain different active transporters, or ion pumps, such as the very abundant Na ϩ K ϩ -ATPase, which is considered to be the molecular machinery responsible for Na ϩ reabsorption (1). Other ion pumps are not so numerous, but some, such as the plasma membrane calcium pump, play important roles in the fine regulation of intracellular ion concentrations. Our group has recently shown that Ca 2ϩ -ATPase is exclusively located and active in caveolin-cholesterol-rich membrane microdomains or lipid rafts in the kidney BLM (2). These membranes have also been shown to house different cell signaling systems that are initiated by the activation of either different lipid kinases, with further generation of bioactive molecules (3,4), or protein kinases associated with the BLM (5)(6)(7)(8).
The location of the BLM Ca 2ϩ -ATPase in caveolin-cholesterol-rich domains adds ceramides to the emerging potential regulatory network in these membranes, because those microdomains are also rich in sphingolipids (9). Rafts are thought to be present in the outer leaflet of the cell membrane, where sphingomyelin, the precursor of ceramide (Cer) in a pathway catalyzed by sphingomyelinases, appears to be predominantly located. An important concept is that the assembly of the outer leaflet lipid rafts would alter the inner leaflet, thus enabling the different steps required for signal transduction to be coordinated (for review, see Ref. 10).
Many studies during the past decade have shown that smaller lipid rafts are merged into large membrane domains when sphingomyelin is hydrolyzed and Cer is generated (Refs. 11 and 12; see also Ref. 10 for review). The generation of Cer molecules and their self-association leads to dramatic changes in plasma membranes with further formation of small Cer-rich microdomains, which are able to fuse spontaneously to others resulting in large domains called platforms (12)(13)(14). Therefore, Cer-enriched microdomains seem to play an important role in facilitating and amplifying signaling processes, via different types of cell surface receptors, resulting in clusters of receptors and other cell signaling machinery that facilitate the effective transduction of different signals (10).
The importance of Cer in different cell processes is not only related to its physicochemical properties. It can be also considered a cell signaling molecule with different roles in different subcellular compartments (for review, see Ref. 15).
Classically, Cer generation occurs either by the activation of sphingomyelin or via de novo synthesis (16,17). In view of its structural analogy with diacylglycerol, it has been suggested that Cer fulfills a second messenger function by binding directly to different intracellular targets (18,19). Some of these targets have already been studied, such as different sets of protein kinases and protein phosphatases (20). Although some studies report atypical PKC, principally PKC, as the primary ceramide-activated PKC (20,21), there are some reports of a direct association of Cer with PKC␣ and PKC␦ in kidney cells (22,23).
More than 10 years ago it was shown that Cer could also be phosphorylated by a ceramide kinase, resulting in another bioactive sphingolipid, ceramide-1-phosphate (C1P), which has a broad spectrum of cellular targets (23)(24)(25) including ion transporters (26).
In view of the preferential formation of Cer in the lipid rafts and because the Ca 2ϩ -ATPase is exclusively located in cholesterol-caveolin-rich lipid rafts in the BLM (2), we decided to investigate the effects of Cer and C1P on the kidney proximal tubule BLM Ca 2ϩ -ATPase and on the cell signaling cascade that could be involved.

EXPERIMENTAL PROCEDURES
Materials-Buffers, bovine serum albumin, Tris-buffered saline, CHAPS, and protease inhibitors were obtained from Sigma. Percoll was from GE Healthcare. Distilled water, deionized using the Milli-Q system of resins (Millipore Corp., Marlborough, MA), was used to prepare all solutions. 32 P i was obtained from IPEN (São Paulo, Brazil). [␥-32 P]ATP was prepared as described by Maia et al. (27). Ceramide (from bovine brain), C1P, histone H8, protein A-agarose from Staphylococcus aureus in saline suspension, the PKA ␣-catalytic subunit inhibitor 5-22 peptide (PKA i ), and the PKC inhibitor calphostin C were purchased from Sigma. The PKA ␣-catalytic subunit (from bovine heart) was purchased from Calbiochem (La Jolla, CA). All of the other reagents were of the highest purity available. The antibody against plasma membrane Ca 2ϩ -ATPase (clone 5f10) was from Affinity Bioreagents (Golden, CO); the antibody (C-16) against isoform 1 of Ca 2ϩ -ATPase (PMCA1) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The phospho-Ser/Thr PKA substrate polyclonal antibody was from Cell Signaling Technology (Beverly, MA). Nitrocellulose membranes (Hybond) and the ECL TM system were from Amersham Biosciences. X-Omat TM diagnostic film was from Eastman Kodak Co. Pig kidneys were obtained from a slaughterhouse under the supervision of licensed veterinarians.
Isolation of Basolateral Membranes-The kidneys were rapidly removed after the animals had been killed and transported in a chilled solution containing 250 mM sucrose, 10 mM Hepes-Tris (pH 7.6), 2 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, and 0.15 mg/ml of soybean trypsin inhibitor. The external portion of the cortex (cortex corticis) was carefully removed, and purified BLM derived from kidney proximal tubules were prepared using the Percoll gradient method (28). Controls for contamination with other membranes were carried out as previously described (5,29). The specific activity of the basolateral membrane marker Na ϩ K ϩ -ATPase (260.8 Ϯ 1.6 nmol/mg per min) was enriched 5-fold over the initial kidney cortex homo-genate. The membranes were stored in 250 mM sucrose in liquid N 2 , which preserved marker and Ca 2ϩ -ATPase activities for at least 5 months.
Protein Determination-A 10-l aliquot (in triplicate) was used for protein assays, which were performed by the Folin phenol method described by Lowry et al. (30) with the addition of 5% SDS in the samples, using bovine serum albumin as standard.
Determination of Plasma Membrane Ca 2ϩ -ATPase Activity-The membranes (0.2 mg/ml) were preincubated at 37°C for 10 min in a medium (1 ml) containing 50 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane buffer (pH 7.4), 5 mM MgCl 2 , 10 mM NaN 3 , 0.5 mM ouabain, 20 M free Ca 2ϩ (0.2 mM EGTA, 238.3 M CaCl 2 ), and the concentrations of Cer or C1P indicated in the figures or figure legends. After 10 min of sonication (240 W, 25 kHz, 24 -25°C; Unique Sonifier Cleaner, Indaiatuba, São Paulo, Brazil), the samples were supplemented with 120 mM KCl and then with 5 mM ATP. Where indicated, 10 nM PKA i (the 5-22 inhibitor peptide of PKA) or 10 nM calphostin C (the inhibitor of PKC) were added to the preincubation medium. The reaction was stopped after 20 min by adding 1.5 ml of activated charcoal in 0.1 N HCl to each tube. After centrifugation in a clinical centrifuge (2,000 rpm), aliquots of the supernatants (0.5 ml) were transferred to new glass tubes to measure the amount of P i released using the colorimetric method of Taussky and Shorr (31). Ca 2ϩ -ATPase activity was calculated as the difference between the activities measured without and with 2 mM EGTA. The free Ca 2ϩ concentration was calculated using a computer program that took into account the different species involved in the equilibrium between EGTA, Ca 2ϩ , the different ATP forms, Mg 2ϩ , H ϩ , and K ϩ (32).
SDS-PAGE and Immunoblotting-Electrophoresis of BLM proteins, followed by immunodetection of Ca 2ϩ -ATPase by the 5f10 or PMCA1 antibodies and of phospho-Ser/Thr residues (in the sequence recognized by PKA), was carried out as described recently (2,33).
Immunoprecipitation of Ca 2ϩ -ATPase-The BLM Ca 2ϩ pump was immunoprecipitated using the following procedure. The BLM of kidney proximal tubules (5 mg/ml) were first incubated for 10 min in the absence or presence of 5, 20, and 50 nM Cer, before the addition of the phosphorylation medium (see below) supplemented with 10 mM NaF. After 20 min at 37°C, the reaction was arrested with 1% CHAPS, and the tubes were incubated at room temperature for 30 min to allow the membranes to be solubilized. In the meantime, protein A-agarose was mixed with the 5f10 antibody (equal volumes of each original stock) and gently stirred for 20 min before the addition of an equal volume of bovine serum albumin (1 mg/ml) in 0.01% CHAPS. This mixture was then added to the solubilized membranes and incubated overnight at 4°C with gentle agitation. The immunoprecipitate and the supernatant were separated by centrifugation at 1,000 ϫ g for 4 min (4°C). The immunoprecipitated Ca 2ϩ -ATPase samples were washed three times with cold Tris-buffered saline, solubilized in Laemmli buffer for SDS-PAGE, electrophoresed in parallel with a supernatant sample, transferred to nitrocellulose membranes, and probed with the 5f10, PMCA1 or phospho-Ser/Thr PKA antibodies.
Determination of Protein Kinase A Activity-PKA activity was determined using histone H8 as a substrate for potential kinases present in BLM. Briefly, the [␥-32 P]phosphoryl group of radiolabeled ATP was transferred to the histone in an incubation medium consisting of 4 mM MgCl 2 , 20 mM Hepes-Tris (pH 7.0), histone H8 (1.5 mg/ml), and BLM preparation (0.5 mg/ml) in a final volume of 0.1 ml. The reaction was initiated by adding 10 M [␥-32 P]ATP (7-10 Ci/mol). After 10 min at 37°C, the reaction was stopped by adding 0.1 ml trichloroacetic acid (40% w/v), and the tubes were immediately placed in an ice bath. The content of each tube was filtered through 0.45-m Millipore filters, which were then washed with 20% trichloroacetic acid and phosphate buffer (2 mM, pH 7.0) to remove the unused [␥-32 P]ATP. The radioactivity incorporated into the histone was determined by liquid scintillation counting (Tri-Carb 2100; Packard, Downers Grove, IL), and the PKA activity was measured as the difference between radioactivity incorporated in the presence and absence of 10 nM M PKA i , the 5-22 PKA inhibitor peptide. A control with 100 nM cAMP confirmed the association of PKA activity with the BLM. To investigate a possible direct, cAMP-independent effect of Cer on PKA, the same assay was carried out using purified PKA ␣-catalytic subunit instead of BLM.
Determination of Protein Kinase C Activity-We used the same method described for the determination of PKA activity, but PKC activity was measured as the difference between tubes incubated in the absence and presence of 10 nM calphostin C (PKC inhibitor). Phorbol 12-myristate 13-acetate (PMA, 1 pM), a phorbol ester, was also used to confirm the presence of functional PKC in the BLM fractions.
Regulatory Phosphorylation of Ca 2ϩ -ATPase-Phosphorylation of Ca 2ϩ -ATPase was also determined as described recently (33) with slight modifications. The membranes were preincubated with the reaction medium employed to measure the Ca 2ϩ -ATPase activity (no ATP) in the presence of Cer (50 nM), PKA i (10 nM), and calphostin C (10 nM) in the combinations shown in the corresponding figure legends. The phosphorylation reaction in the PKA assays was started by adding 5 mM cold ATP. After 20 min the reaction (0.2 ml; 100 g of BLM protein) was stopped by adding 50 l of sample buffer (2.3 g of dithiothreitol, 3 g of SDS, 12 ml of 1.0 M Tris, pH 6.8, 15 ml of 10% glycerol in a 50-ml final volume in water), and 0.1 ml of the solubilized samples were applied to each slot for SDS-PAGE (10% acrylamide) as described above. The gel proteins were transferred to a nitrocellulose membrane, and phospho-Ser/ Thr residues were recognized with the PKA substrate polyclonal antibody as described above. Phosphorylation of Ca 2ϩ -ATPase by PKC was assayed in the same medium employed for the PKA assay, except that [␥-32 P]ATP (0.5-1 Ci/mol) was used as follows. After electrophoresis and transfer, the nitrocellulose membranes were exposed overnight to a phosphor screen and analyzed using a PhosphorImager Storm 860 (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA) to measure the intensity of the 140-kDa [ 32 P]phosphorylated bands recognized by the 5f10 antibody. Phosphorylation of Ca 2ϩ -ATPase by the BLM-resident PKC was quantified by the difference in band intensities in the absence and presence of 10 nM calphostin C.
Data Analysis-The means were compared by one-way analysis of variance, taking into account the treatment of experimental groups. The differences were evaluated using the multiple comparative Bonferroni test. In all cases the n values correspond to the results obtained from different BLM preparations, except for the purified PKA ␣-catalytic subunit assay. Linear regression analysis with errors in both variables was used to study the extent to which stimulation of Ca 2ϩ -ATPase activity in the presence of Cer correlates with Cer-induced PKAmediated phosphorylation of the Ca 2ϩ pump.

Fig. 1 (open circles)
shows that increasing concentrations of Cer in the nanomolar range (pA Ϸ 8.5) stimulate Ca 2ϩ -ATPase activity up to ϳ50% over control. We also tested C1P, the phosphorylated derivative of Cer, which was ineffective in modulating Ca 2ϩ -ATPase activity (filled circles). The observed effect of Cer could be a consequence of structural changes in the membrane similar to those observed upon sphingomyelinase activation and Cer release (12)(13)(14) or to direct activation of protein kinases, which are targets for Cer in intracellular compartments. Because PKA and PKC appear to be involved in the modulation of Ca 2ϩ -ATPase from different sources (33)(34)(35)(36)(37)(38) including renal tissue (5, 7), the following experiments were conducted to investigate whether the stimulatory effect of Cer on active Ca 2ϩ transport in kidney involves PKA or PKC, two of the renal BLM-resident kinases (6,7).
The observation (Fig. 2) that Ca 2ϩ -ATPase is no longer stimulated by Cer when peptide 5-22 (PKA i ), the highly specific inhibitor of PKA, is added simultaneously supports the hypothesis that nanomolar Cer activates this type of protein kinase in kidney membranes and that stimulation of the Ca 2ϩ pump is a consequence of triggering this signaling cascade. The ability of Cer to activate cAMP-dependent protein kinase was therefore tested. Fig. 3 shows that Cer induces a 3-fold increase in PKA, saturating in the same concentration range as the effect on Ca 2ϩ -ATPase activity. This effect mimics that found with cAMP (hatched column in Fig. 3). However, because the effects of Cer and cAMP are additive (black column), it is clear that they both activate PKA but in different ways. The experiments presented in Fig. 4 show that Cer, in the concentration that promotes half-maximal activation of Ca 2ϩ -ATPase, strongly stimulates phosphorylation catalyzed by the purified PKA ␣-catalytic subunit, supporting the view that Cer activates PKA by a mechanism independent of cAMP.
Activation of PKA by Cer is associated with subsequent phosphorylation of Ca 2ϩ -ATPase, as demonstrated in Figs. 5, 6, and 8. Fig. 5 (A and C) shows that addition of 50 nM Cer promotes a PKA i -sensitive increase in the phosphorylation of a 140-kDa band detected by the specific phospho-Ser/Thr PKA antibody. The same 140-kDa band was shown to be Ca 2ϩ -ATPase by the specific antibody 5f10 (Fig. 5B). The fact that 10 nM of the PKA inhibitor completely abolishes stimulation of both Ca 2ϩ -ATPase and phosphorylation of the pump molecule in the presence of 50 nM Cer (compare Fig. 5 with Fig. 2) reinforces the view that activation of Ca 2ϩ pumping activity requires phosphorylation.    To exclude the possibility that the increase in phosphorylation occurs in a different protein with the same molecular mass as the Ca 2ϩ -ATPase, the Ca 2ϩ pump incubated under phosphorylating conditions (Figs. 2 and 5) was immunoprecipitated with the specific antibody 5f10 and then analyzed for incorporation of the ATP ␥-phosphoryl group into Ser or Thr residues in a sequence recognized by PKA (Fig. 6). The experiments shown in this figure confirm that the phosphorylated band at 140 kDa, enhanced by Cer, is indeed phospho-(Ca 2ϩ -ATPase) (Fig. 6, compare A and D with C). Moreover, Fig. 7 shows that the phosphorylated 140-kDa protein (C) immunoprecipitated by the 5f10 antibody (A) was also recognized by the PMCA1 antibody (B).
The correlation between phosphorylation of the Ca 2ϩ pump into Ser/Thr residues, in a sequence recognized by PKA, and Ca 2ϩ -ATPase activity in the activation phase of the curve depicted in Fig. 1 shows a straight line (Fig. 8). The linearity of the correlation and the maintenance of basal levels of both Ca 2ϩ -ATPase activity and phosphorylation status of the pump after simultaneous addition of 20 nM Cer and 10 nM PKA i gives additional support to the view that stimulation of the Ca 2ϩ pump by Cer occurs via activation of PKA (see also Figs. 2, 5, and 7). It should be emphasized that the increase in both phosphorylation of the Ca 2ϩ -ATPase and Ca 2ϩ -ATPase activity reach a plateau in parallel, with saturation attained at ϳ50 nM Cer (compare Fig. 6 with Fig. 1). Fig. 9 shows that incubation of BLM with 200 nM Cer also increases PKC activity (defined by the difference between total and calphostin C-sensitive phosphorylation). This stimulation is equivalent and not additive to that promoted by 1 pM PMA, a classic activator of PKCs (39). Fig. 10 shows that 50 nM Cer increases the calphostin C-sensitive phosphorylation of Ca 2ϩ -ATPase, even though histone phosphorylation by 50 nM was not distinct from the basal level. This point will be discussed below.
Although Cer strongly increases BLM PKC, this process is not associated with the stimulation of Ca 2ϩ -ATPase by Cer, because calphostin C does not prevent its effect on Ca 2ϩ -ATPase activity (Fig. 11). Indeed, activation of PKC in different ways by hormones and autacoids promotes inhibition rather than stimulation of Ca 2ϩ -ATPase in the BLM (Refs. 5 and 7; see also inset to Fig. 11). Calphostin C alone does not promote a significant increase in Ca 2ϩ -ATPase activity (hatched column in Fig. 11), and this observation is consistent with the unmodified basal phosphorylation status of the Ca 2ϩ pump when calphostin C is added alone (hatched column in Fig. 10).

DISCUSSION
Efficient regulation of fluid transport across the tubular epithelium is one of the principal functions of kidneys. Different cell signaling systems act on active transporters located in the BLM (1). Vectorial transport in polarized epithelial renal cells is the result of two coordinated major steps of transport across the luminal and basolateral faces of these cells. Sphingolipids  have been shown to be involved in both differential sorting of membrane components and cell signaling processes, either by organizing regulatory membrane microdomains (rafts and caveolae) or by direct triggering of cellular effectors (15,20,40). Therefore, it is plausible that small changes in membrane sphingolipids such as Cer, which is believed to be a second FIGURE 8. Correlation between PKA-mediated phosphorylation of Ca 2؉ -ATPase and Ca 2؉ -ATPase activity in the presence of ceramide. Abscissa, basolateral membranes were phosphorylated with cold ATP in the presence of the nanomolar Cer concentrations shown in parenthesis. Ca 2ϩ -ATPase was immunoprecipitated, and its phosphorylation was determined with the use of a phospho-Ser/Thr PKA substrate antibody, as described in the legend to Fig. 6 (see also "Experimental Procedures"). Each phosphorylation value was calculated by the ratio between the intensity of the specific phospho-Ser/Thr PKA signal and the corresponding total Ca 2ϩ -ATPase detected with the 5f10 antibody. Ordinate, Ca 2ϩ -ATPase activity was determined as described under "Experimental Procedures" in the presence of the Cer concentrations used in the phosphorylation assays. The symbols indicate Ca 2ϩ -ATPase activity (mean Ϯ S.E. of at least six experiments) at each Cer-induced phosphorylation of the 140-kDa band (mean Ϯ S.E., n ϭ 5). The straight line was obtained by linear regression with errors in both variables (r ϭ 0.98). The arrow and filled circle indicate Ca 2ϩ -ATPase activity and phosphorylation level in the presence of 20 nM Cer plus 10 nM PKA-inhibitor (5-22 peptide).   messenger (18,19), can play a pivotal role in the modulation of renal transporters. The evidence that Ca 2ϩ -ATPase is exclusively located and active in caveolin-cholesterol-rich lipid rafts in the BLM (2) led to the hypothesis that raft lipids may be involved in regulating intracellular Ca 2ϩ concentrations and that the special components of these structures could participate in modulating the machinery responsible for the finely tuned control of cytosolic Ca 2ϩ , the plasma membrane Ca 2ϩ pump. Cer is also implicated in activating or inhibiting several effectors proteins, which may potentially influence Ca 2ϩ signaling (10,23,41).
It has been shown that Cer with short fatty acid chains (C 2and C 8 -Cer) activates both soluble and native erythrocyte Ca 2ϩ -ATPase in a concentration range near and above the critical micellar concentration (42). In this work we explored the possibility that, in addition to a direct effect at high concentrations, probably related to physical changes in the pump molecule, Cer, in the nanomolar range expected for a second messenger, could be capable of interacting with plasma membrane signaling cascades in the close vicinity that are functionally coupled to Ca 2ϩ -ATPase.
In this paper we show that Cer stimulates both PKA and PKC (Figs. 3 and 9) and also modulates Ca 2ϩ -ATPase activity with Cer 50 ϭ 3.5 nM (Fig. 1). The data in Figs. 3 and 4 show that Cer activates PKA by a mechanism that appears to be independent of that promoted by cAMP. cAMP-independent activation of the ␣-catalytic subunit of PKA by Cer (Fig. 4) could suffice to sustain the phosphorylating capacity of the kinase and the concomitant stimulus of plasma membrane Ca 2ϩ -ATPase (Figs. 1 and 2), after the PKA-regulatory subunit dissociates or when cell cAMP levels are low. It is particularly interesting that the mRNA encoding the isoform PMCA1 is found in proximal tubules (43), and this isoform is phosphorylated in the presence of Cer (Fig. 7) in a region that is detected by the specific phospho-Ser/Thr PKA antibody. It has previously been reported that the consensus sequence for PKA phosphorylation is present only in PMCA1 (44). This sequence is located between the calmodulin-binding domain and the C terminus of the pump molecule (45).
The PKA-mediated stimulation of Ca 2ϩ -ATPase by Cer (Figs. 2 and 8) could be especially relevant during renal injury. It has been demonstrated that Cer levels in kidney are elevated because of an increased Ca 2ϩ -stimulated sphingomyelinase activity after cellular Ca 2ϩ homeostasis is disrupted (46,47). In whole cells, activation of active Ca 2ϩ pumping could be hypothesized as a mechanism compensating against sustained and harmful increases in cytosolic Ca 2ϩ . Because of the high affinity effect of Cer on Ca 2ϩ pumping activity (Cer 50 ϭ 3.5 nM; Fig. 1), it seems probable, in these situations, that Cer could really influence the evasion of uncontrolled cytosolic Ca 2ϩ increase, even though there is only a modest increase in the Cer content of kidney cells after sphingomyelinase activation (47). In other cells, Cer production also follows cytosolic Ca 2ϩ elevation (48), supporting the general view that Cer plays an important role in the recovery of normal cellular Ca 2ϩ levels.
Ceramide also promotes an increase in the basal BLM PKCmediated phosphorylation of soluble histone H8, but at concentrations higher than those capable of stimulating Ca 2ϩ pump phosphorylation (compare Figs. 9 and 10). Previous reports have shown that PKC, resident and/or recruited to lipid rafts, promotes the stimulation of local protein phosphorylation (49 -51). Because renal Ca 2ϩ -ATPase is located in caveolae (2), a lower supply of Cer could suffice to stimulate PKC in these sphingolipid-enriched membrane microdomains (9) and, consequently, PKC-mediated phosphorylation of the neighboring Ca 2ϩ pump.
From the results presented here, the following question emerges: why does phosphorylation of Ca 2ϩ -ATPase after stimulation of PKA by Cer (Figs. 5-7) lead to its activation (Figs. 1 and 2), whereas Cer-induced stimulation of PKC (Fig. 9) and subsequent phosphorylation of Ca 2ϩ -ATPase (Fig. 10) has no influence on the Ca 2ϩ pump (Fig. 11)? It is known that renal Ca 2ϩ -ATPase is inhibited when PKC␣ is triggered by hormones and autacoids (5,7) or by direct activation of PKC by PMA (see inset to Fig. 11). Therefore, the lack of influence of Cer on Ca 2ϩ -ATPase activity, notwithstanding the clear calphostin C-sensitive phosphorylation of the pump (Fig. 10), might signify that Cer promotes pathways with counteracting effects on Ca 2ϩ -ATPase. The inhibition resulting from PKC activation would be counteracted by stimulation of PKA, which up-regulates the Ca 2ϩ pump. Activation rather than inhibition of the Ca 2ϩ pump by protein kinase C has been observed in human platelets and erythrocytes (35,36). The fact that PKCmediated phosphorylation of renal Ca 2ϩ -ATPase promotes inhibition instead of stimulation (Refs. 6 and 9; inset to Fig. 11) may be due to differences in the relative abundance and primary structures of the Ca 2ϩ pump isoforms found in circulating cells and kidney cells (43).
The combined results of Figs. 2, 3, and 5-8 indicate that a signaling cascade starting with Cer is an efficient pathway for activating Ca 2ϩ -ATPase in renal proximal tubule cells, with a resident PKA as an intermediate. In these membranes, activators of PKA such as cholera toxin or forskolin also stimulate the ouabain-insensitive Na ϩ -ATPase (6, 51); another example of the participation of this kinase in the regulation of ion pumping activity. These observations reveal the importance of PKA in the fine control of Ca 2ϩ and Na ϩ fluxes in a nephron segment where more than two-thirds of the glomerular ultrafiltrate is reabsorbed. We recently demonstrated that PKA mediates activation of the Ca 2ϩ -ATPase from electrocytes (33) via a special activating pathway in which calmodulin and calmodulin-dependent kinase II are early components. The data presented in this paper, showing that nanomolar concentrations of Cer increase Ca 2ϩ -ATPase activity by 50%, allow us to propose that Cer can be considered a lipidic calmodulin-like activator of ATP-dependent Ca 2ϩ transport across the plasma membrane.
Finally, two other mechanisms could be coupled to Cer-mediated regulation of the Ca 2ϩ -ATPase. First, the well known influence of Cer in merging small lipid rafts in which Ca 2ϩ -ATPase is exclusively localized in kidney BLM (2) could favor Cer-guided oligomerization of ATPase units followed by their activation (52,53). Second, Cer-stimulated and PKA-mediated phosphorylation of the pump may help calmodulin in its direct activation of Ca 2ϩ -ATPase by reducing the binding of the autoinhibitory domain (54).
To the best of our knowledge, the present work shows for the first time that Cer can modulate Ca 2ϩ -ATPase, an ion pump located in the plasma membrane, by triggering membrane-associated PKA. The above results therefore suggest that Cer participates in the regulatory network of bioactive sphingolipids and glycerolipids resident in the BLM from proximal kidney tubules. In this network, PKA could be seen as an intermediate in a route that links the sphingomyelinase and the adenylyl cyclase pathways, where both Cer and cAMP (Fig. 3) can act synergistically to increase the activity of Ca 2ϩ pumping across the renal plasma membrane.