Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules

Julliana F. Sant’Anna1 • Vanessa S. Baldez1 • Natalie A. Razuck-Garrão1 • Thiago Lemos 1 • Bruno L. Diaz2 • Marcelo Einicker-Lamas1

Received: 20 April 2020 / Accepted: 15 February 2021 / Published online: 11 March 2021
Ⓒ University of Navarra 2021

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respec- tively. Furthermore, the addition of 10−8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.
Keywords Lysophosphatidic acid . Kidney . Calcium . Bioactive lipids . PKC . Renal physiology

Highlights • Lysophosphatidic acid (LPA) can be a modulator of Ca2+- ATPase.
• PKC-dependent control of intracellular Ca2+ in renal cells.
• LPA included in the bioactive lipids network controlling renal function.
* Bruno L. Diaz [email protected]
* Marcelo Einicker-Lamas [email protected]


For many years, lipids were only referred as structural com- ponents of plasma membranes limited to give support for the different proteins and selective control the passage of certain ions and molecules. However, these concepts were revised, and mem- brane lipids are known to be involved in different cellular func- tions with particular action in cell signaling processes [17].
A milestone in the study of lipid signaling was the discov- ery that certain classes of membrane lipids could be cleaved generating biologically active molecules, in an orchestrated process that allows cells to translate an external signal into a cellular response [8]. The great example for this was the phosphoinositide turnover cycle described by Hokin and Hokin in the 1950s [21], which culminates in the 1980s, with the discovery of phosphatidylinositol-4,5-bisphosphate (PtdIns4,5-P2) hydrolysis by phospholipase C (PLC), leading to the formation of diacylglycerol (DAG) and inositol-1,4,5- triphosphate (InsP3), which are involved in Ca2+ release from intracellular stores and activation of classical protein kinase C (PKC) [32, 42, 44]. In the kidney proximal tubule cells, plas- ma membrane Ca2+-ATPase (PMCA) is responsible for Ca2+ transport from the cytosol to the peritubular space [49, 50], being involved not only in Ca2+ reabsorption but, especially, in the fine adjustment of intracellular Ca2+ levels [13, 47]
However, the capacity of lipids to participate in the regu- lation of cellular processes is not only ascribed to its role as intracellular mediators. A broad range of bioactive lipids had been discovered to function as extracellular mediators, mainly by the activation of different G protein-coupled receptors (GPCR). Among the already known bioactive lipids, lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are probably the most well studied [1, 18]. LPA (mono acyl glycerol-3-phosphate), is the smallest glycerolipid, pre- senting its basic structure as glycerol, linked to a phosphate group and the esterification of a fatty acid moiety at C-1 or C-2 [3]. The enormous interest in research on LPA is due to a variety of biological responses obtained in different cell types, where its functions may be related to the control of many physiological and pathological processes by triggering its dif- ferent receptors [24], which were initially referred as EDG receptors [20] and were already identified within the renal tissue [15, 31, 37]. LPA is present in all tissues and cells, including blood, with plasma concentrations ranging from 0.1 to 1 mM, whereas serum exhibit higher concentrations up to 10 mM [7]. LPA can be generated through the hydrolysis of membrane phospholipids after cell activation by a broad range of agonists in two different ways: (i) by the action of a lysophospholipase D (lyso-PLD) which cleaves lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPS) to generate LPA and
(ii) via phosphatidic acid (PA), either derived from membrane phospholipids or DAG by the action of phospholipase D (PLD) and diacylglycerol kinase (DGK), respectively [4].
Thus, LPA emerges as an important bioactive lipid due to the diversity of its biological activities, such as induction of migra- tion, proliferation, smooth muscle contraction, platelet aggrega- tion, neurogenesis, and cytoskeleton reorganization [12]. In spite of its physiological action, LPA is also involved in the pathogen- esis of some clinical disorders such as atherosclerosis, cancer [9, 22], neuropathies [23], reproductive disorders [53], and fibrosis in some organs like lungs and kidney [40, 43].
Here we show the involvement of LPA signaling in the regulation of intracellular Ca2+ levels through the regulation of the PMCA in a PKC-dependent event that occurs in renal proximal tubule cells.

Material and methods

All the reagents used for general solutions, buffers, BSA, and protease inhibitors were from Sigma Chemical Co.; Percoll was from Pharmacia. Water used in all the experiments was distilled and deionized using the Milli-Q system of resins (Millipore Corp.). 32Pi was obtained from the Instituto de Pesquisas Energéticas e Nucleares (IPEN, Brazil). [ -32P]ATP was prepared as described by Maia et al. [19]. Phorbol-12-myristate-13 acetate (PMA), histone H8, PLA2 inhibitors, and the PKC inhibitor calphostin C, were pur- chased from Sigma Chemical Co. All other reagents were of the highest purity available. Nitrocellulose membranes (Hybond) and the ECLTM system were from GE Healthcare. Acrylamide and bis-acrylamide were from Bio-Rad. Pig kid- neys were purchased from a licensed slaughterhouse (Frigorífico Novo Meriti, São João de Meriti, RJ, Brazil), minutes after slaughtering the pigs. The chosen slaughter- house is in accordance to the Brazilian Federal Law No. 5.760 that establishes the requirements for this activity, in- cluding the supervision of the processes by a veterinarian.

Basolateral membrane fractions from kidney proximal tubules
Basolateral membrane (BLM)-enriched fractions were obtain- ed from adult pig kidneys, as previously described [21]. Briefly, kidneys were maintained in a cold solution, contain- ing 0.25 M sucrose, 10 mM Hepes-Tris (pH 7.6), 2 mM EDTA, 1 mM PMSF, and 0.15 mg × mL−1 trypsin inhibitor. Then the cortex outermost portion, which is enriched in prox- imal convoluted tubules, was dissected, and the slices were homogenized and centrifuged to isolate the BLM-enriched fraction [51]. Protein content was evaluated by the phenol- Folin method [30], using bovine serum albumin as standard. Na++K+-ATPase activity was used as a control to ascertain the BLM isolation procedure, being its activity eight times higher in the BLM fractions compared to whole homogenates.

Ca2+-ATPase activity
Inorganic phosphate (Pi) resulted from ATP hydrolysis was measured colorimetrically [45]. Basolateral membrane Ca2+- ATPase activity was calculated by the difference of ATP hy- drolysis measured with and without (2 mM EGTA) 20 μM of free Ca2+. To test the modulatory action of LPA, BLM frac- tions were pre-incubated with increasing concentrations (10, 40, 100, 200, and 400 nM) of exogenous LPA (Avanti Polar Lipids) for 15 min, and the ATPase activity was measured as described [10, 45]. In the last set of experiments, PLA2 activ- ity assays were performed in the absence or presence of dif- ferent specific PLA2 inhibitors.

PKC activity
PKC activity was determined by the difference between the values in the incorporation of 32Pi from [γ32-P]ATP to his- tones, obtained in the absence and presence of the PKC inhib- itor, calphostin C, as previously described [11].

SDS-PAGE and immunodetection
Proximal tubule BLM fractions were submitted to SDS- PAGE according to Laemmli et al [26]. Then, proteins were transferred to a nitrocellulose membrane that was blocked using 5% non-fat milk and incubated with anti-plasma membrane Ca2+-ATPase primary antibody 5F10 (Affinity Bio Reagents) with further detection using ECLTM system and Hyperfilm (Eastman Kodak Co.) as previously described [11].

PMCA regulatory phosphorylation
BLM fractions were used and the phosphorylation reaction was started with the addition of [y-32P]ATP, and after 15 min at 37 °C, the reaction was stopped with the addition of 1% CHAPS as described [10]. The phosphorylation was measured after addition of 10 mM NaF (phosphatase inhibi- tor) for 10 min in the presence of 10 nM and 100 nM LPA (Avanti Polar Lipids) in different calphostin C concentrations depicted in the respective figure.

PMCA immunoprecipitation
After phosphorylation assay, the samples were incubated at room temperature for 30 min. Next, anti-plasma membrane Ca2+-ATPase primary antibody 5F10 (Affinity Bio Reagents) was added to each condition and left under mild stirring for 1 h until addition of protein A/G, followed by overnight incubation under constant agitation at 4°C. Immunoprecipitate was obtained by centrifugation 1000×g for 5 min (4 °C) followed by three times wash with Tris-buffered saline and centrifugation for 10 min at 4 °C and 5000×g. Then the precipitate was solubi- lized in sample buffer, heated at 100°C for 10 min, and centrifuged at 5000×g for 2 min. After centrifugation, supernatant aliquots were submitted to 10% SDS-PAGE, and after that, the gel was transferred to a nitrocellulose membrane and then exposed for 48 h to a “phosphor screen” prior to the analysis on PhosphorImager Storm 860 (Molecular Dynamics) to identify the ~140 kDa [32P] phosphorylated band. The Ca2+-ATPase identity was confirmed with further incubation of the nitrocellu- lose membrane with the 5F10 antibody and revealed for western blotting as described above. Bands were quan- tified densitometrically using Scion Image. Further, we excised the 5F10 positive band from the nitrocellulose membrane and put each in vials individually containing 2 mL of liquid scintillation (4g PPO in 1L toluene) to determine the radioactivity in each experimental condi- tion in a liquid scintillation counter (Tricarb, Packard).

PLA2 activity determination
BLM fractions were submitted to dialysis for 24 h at 4°C and constant agitation to split off thiol groups that result in a high background that potentially interfere to the analysis. PLA2 activity was measured using cPLA2 kit assay (Cayman Chemical Company). To determine the specific PLA2 activity, membrane fractions were pre-incubated with some inhibitors for different PLA2 like bromoenol lactone (BEL), cPLA2i, and arachidonyl trifluoromethyl ketone (ATK, AACOCF3).

Statistical analysis
Statistical analysis was carried out using the one-way ANOVA test and Newman-Keuls post-test. Statistical signif- icance was set at p<0.05. Data were analyzed using the GraphPad Prism 5.0 program. Results Basolateral membrane (BLM) from kidney proximal tubule cells harbors a plethora of signaling systems, involving differ- ent kinds of receptors, protein kinases, and protein phospha- tases, strategically co-located with their regulatory targets. Previous reports from our group had already demonstrated the importance of bioactive lipids in this context. In the pres- ent work, we provide new evidence that allow us to include lysophosphatidic acid (LPA) in this regulatory network pres- ent in the BLM. Assuming a potential generation of LPA in the BLM or LPA availability to kidney cells due to its relative abundance in the blood plasma, we decided to investigate whether LPA would be able to modulate the plasma membrane Ca2+- ATPase activity. Figure 1a shows that even at the lowest con- centration tested (10 nM), there was a significant inhibition of the Ca2+-ATPase activity, being 40 nM the concentration that promoted 50% inhibition of the ATPase activity when com- pared to control condition (40 nmol Pi × mg−1 × min−1 vs 20 nmol Pi × mg−1 × min−1, respectively). All the tested con- centrations are on the physiologic threshold found in plasma [2, 29]. It is well known from the literature that the main LPA receptors expressed in whole kidney are LPA1, LPA2, and LPA3 [15, 31]. The next step was to check the role of the putative LPA receptors in the modulation of the BLM Ca2+- ATPase by using Ki16425, a LPA1, and LPA3 nonselective antagonist [36]. Pre-treating BLM fractions for 15 min with two different concentrations (10−7 and 10−8 M) of the LPA receptors antagonist, we show a blockage of the inhibitory action of LPA (10−7 M) on the BLM-Ca2+-ATPase activity already reported, being the effect more pronounced with 10−7 M Ki16425 (Fig. 1b). The addition of Ki16425 to the assay in Fig. 1 LPA modulates plasma membrane Ca2+-ATPase from kidney BLM fractions through its specific GPCRs. a Ca2+-ATPase activity was measured without or with pre-incubation of BLM fractions with increas- ing concentrations of exogenous LPA (0; 40; 100; 200; 400 nM). PMCA activity was measured as described under Methods. Results are expressed as means ± SEM from different experiments done in triplicate (n= 5). the LPA-dependent inhibition of the plasma membrane Ca2+ ATPase. Ca2+ ATPase activity was determined in the absence or in the presence of Ki16425 (10−7 and 10−8 M), an LPA1, and LPA3 nonselective antag- onist, after 15 min of pre-incubation. Results are expressed as means ± SEM from different experiments done in triplicate (n= 4). *Statistically different from control condition, first bar (p < 0.05) the absence of LPA had no effect on Ca2+-ATPase activity. Thus, the result depicted in Fig. 1 confirms the physiological relevance of LPA receptors signaling in the regulation of the BLM-Ca2+-ATPase activity. Our next step was to determine the signaling pathways involved in the LPA-dependent Ca2+-ATPase activity inhibi- tion in the BLM. Different studies provided evidences that link LPA1 and LPA3 receptors to Gq proteins, which are known to trigger PLC/PKC pathway, being this signaling cas- cade already implicated in the Ca2+-ATPase activity inhibition upon treatment of the BLM fraction with different hormones and autacoids [5, 6, 14]. To determine whether PLC activation could be involved on Ca2+-ATPase activity inhibition by Fig. 2 PLC inhibition blocks the inhibitory action of LPA on the Ca2+- ATPase activity. BLM fractions were pre-incubated for 15 min without or with the PLC inhibitor U73122 (10−8M) and then stimulated or not with LPA (10−7 M). The Ca2+-ATPase activity was measured according to Methods. Results are expressed as means ± SEM from different experi- ments done in triplicate (n= 7). *Statistically different from control, first bar (p < 0.05) LPA, we used a specific PLC inhibitor, U73122 (10−8 M). Figure 2 shows that 15-min pre-incubation of BLM with U73122 in the absence of LPA had no effect on Ca2+- ATPase activity, while it fully prevents the inhibitory action of LPA. PLC activation leads to the formation of DAG and IP3 that converges to the activation of classic PKC. To confirm the involvement of PLC/PKC pathway, we decided to use the DAG analog, PMA that is well known to be capable of acti- vating classic and novel PKC present in the BLM [11, 33]. In fact, 10−8 M PMA treatment mimics the inhibitory action of LPA on BLM Ca2+-ATPase activity (Fig. 3a). When PMA and LPA were added together in the assay, we do not observe an additive effect on Ca2+-ATPase activity inhibition, which suggests that LPA treatment really triggers PLC leading to DAG production within the BLM. These results allowed us to postulate that LPA/PLC/PKC pathway would be coupled to the regulation of the BLM Ca2+-ATPase, thus participating on Ca2+ active transport through basolateral membrane on kidney proximal tubule cells. There are several reports showing phosphorylation/dephosphorylation on specific regulatory do- mains of Ca2+-ATPase, which are directly associated to acti- vation or inhibition of the pump. In this context, our next step was to evaluate the role of PKC on Ca2+-ATPase activity inhibition mediated by LPA. Pre-incubation of BLM with a classic PKC inhibitor, calphostin C (10−8 M), leads to a completely impairment of the inhibitory action of LPA on the Ca2+-ATPase activity (Fig. 3b). In addition, we decided to test whether LPA treatment would increase BLM- associated PKC activity. Figure 4a clearly shows that the PKC activity in the presence of LPA (10−7M) is approximate- ly two times higher than that observed in control condition. Albumin, used as LPA adjuvant, had no effect on BLM PKC activity, as expected. The same was true for calphostin C Fig. 3 Evidences for the role of the PLC/PKC signaling axis in the mod- ulatory effect of LPA on the plasma membrane Ca2+-ATPase activity. a PMA mimetize the inhibitory action of LPA on Ca2+-ATPase activity. Ca2+-ATPase activity assay was performed in the presence of LPA or PMA in the concentrations shown in abscissa. Results are expressed as means ± SEM from different experiments done in triplicate (n= 5). LPA effect on BLM Ca2+-ATPase activity is a PKC-dependent event. Ca2+-ATPase activity was determined after 15 min pre-incubation of BLM with the PKC inhibitor, calphostin C (10−8 M), on the presence or absence of LPA (10−7 M). Results are expressed as means ± SEM from different experiments done in triplicate (n= 8). *Statistically different from control, first bar (p < 0.05) incubated alone with control BLM fractions, which was al- ready demonstrated in other work from our group [5, 11, 14, 27, 34]. In order to elucidate the whole sequence of events, from LPA exposure to PKC activation resulting in the Ca2+ ATPase inhibition, we decided to investigate whether PKC directly phosphorylates the pump. Figure 4b demonstrates that pre-incubation of the BLM fractions with LPA (10−7 M) in- creased by ≈30% the phosphorylation of the pump, being this effect completely abolished when the BLM fractions were Fig. 4 LPA triggers a BLM-PKC that phosphorylates the plasma mem- brane Ca2+-ATPase. A PKC activity is stimulated by LPA. PKC activity was measured as described under Methods, after 15 min of pre-incubation with LPA (10−7 M) or BSA 1% (used because it is the LPA adjuvant). LPA increases the PKC activity by near 100%. Results are expressed as means ± SEM from different experiments done in triplicate (n= 5). *Statistically different from control, first bar (p < 0.05). B PMCA phos- phorylation is significantly increased after BLM pre-incubation with LPA (10−7 M). Ca2+-ATPase phosphorylation was measured in the presence of LPA and calphostin as described in Methods and shown in abscissa. (A) The phosphorylation of the PMCA band (140 kDa) was determined after exposure of the nitrocellulose membrane containing the immobilized proteins to a phosphor image screen, as detailed in Methods. (B) Immunodetection of the BLM Ca2+-ATPase using the monoclonal anti- body 5F10. (C) Densitometric intensity of the PMCA phosphorylation, calculated by the ratio between the intensity of the phosphorylated band (A) and the intensity of the band after western blotting (B). Results are expressed as means ± SEM from different experiments done in triplicate (n= 5). *Statistically different from control, first bar (p < 0.05) pre-treated with 10−8 M of calphostin C. The incubation of BLM fractions in control conditions with calphostin C led to no effect on the plasma membrane Ca2+-ATPase activity (data not shown), as referred above. A complementary analysis was performed by excising the 5F10 positive band from the nitro- cellulose membrane to determine the amount of radioactivity in the different experimental conditions. As observed in the phosphor screen image and quantification, the radioactivity was higher in the LPA-treated BLM fractions (1200±85 cpm), when compared to the control ones (703±62 cpm) and to the calphostin-treated BLM fractions (530±55 cpm). The above results are clearly related to the exposure of the BLM to LPA. Being LPA levels considered high within the body fluid compartments, it is easy to imagine that this PMCA regulator system present in BLM would be constitutively ac- tive. On the other hand, we decided to evaluate the possible contribution of a local formation of LPA within the BLM through the action of a BLM-harbor PLA2, being this enzyme responsible for the direct conversion of PA to LPA. Figure 5 shows that the pre-incubation of BLM fractions with BEL (10−5M), a Ca2+-independent PLA2 (iPLA2) selective inhibi- tor or ATK (10-6M), a cytosolic PLA2 (cPLA2), and iPLA2 inhibitor, induces a significant reduction (≈ 50%) on PLA2 activity, whereas pre-incubation with a specific cPLA2 inhib- itor had no effect on PLA2 activity. Thus, we can postulate that the presence of a constitutive BLM-PLA2 would favor, at least locally, a basal LPA amount that would be able to keep the BLM plasma membrane Ca2+-ATPase under regulation. Putting together these results, we showed for the first time the modulatory action of LPA on the BLM plasma membrane Ca2+-ATPase inhibition through the direct activation of a DAG-dependent PKC phosphorylation. Fig. 5 PLA2 activity in kidney BLM fractions. PLA2 activity from kidney BLM fractions was determined as described under Methods. To access the different PLA2, we used different inhibitors: BEL (10−5 M), AKT (10−6 M), and a specific cPLA2 inhibitor. Results are expressed as means ± SEM from different experiments done in triplicate (n= 5).*Statistically different from control, first bar (p < 0.05) Discussion BLM from kidney proximal tubule cells harbors different ion transporters that are involved in the major processes related to renal function, such as the Na+ active transporters—specially the Na++K+-ATPase—and the plasma membrane Ca2+- ATPase that is responsible for the fine tune regulation of in- tracellular Ca2+ levels. Due to the key events related to these transporters, BLM also possess a great variety of cell signaling pathways for quick modulation and thus adaptation to the physiological alterations faced by the renal tissue. Among the different signaling molecules, our group has been studying the involvement of bioactive lipids in the modulation of these transepithelial ion fluxes [10, 11 16 27, 42, 46]. Here we provided experimental evidences to include LPA in the hall of bioactive lipids involved in the regulation of Ca2+ homeo- stasis in kidney proximal tubule cells, and as a consequence, in all the Ca2+-dependent events that take place within the renal tissue. It is important to highlight that there are very few reports in the literature exploring LPA as a modulator of Ca2+ levels in different tissues, although LPA is a major cir- culatory bioactive lipid in mammals. The different subsets of LPA receptors were already stud- ied in different cell types, and it is clear that depending on the tissue or organism, each subset is functionally coupled to dif- ferent G proteins [39]. In different kidney cell types, LPA receptors have also been detected [37]. Activation or alteration in the normal pattern of expression is especially relevant either in the maintenance of renal functions as well as in cases of kidney disorders like ischemia/reperfusion injury and urethral unilateral obstruction [25, 40, 48]. Most of the LPA actions within the renal tissue are related to LPA1 and LPA3 receptors, being both coupled to Gq proteins in different cell types. Gq/PLC/PKC axis is a very common pathway involved in different renal fea- tures some of them triggered by important hormones and autacoids such as angiotensin II [5, 6, 38]. This led us to explore the possibility that LPA would also act through this signaling pathway, which was completely confirmed as we were able to block the inhibitory effect of LPA on the Ca2+-ATPase activity either by the use of LPA1/LPA3 antagonist (Fig. 1b) or by inhibiting PLC and PKC (Figs. 2 and 3, respectively). Although the same signaling path- way was already described in mouse embryonic stem cells [46], this is the first time that this pathway is associated to kidney epithelial cells and Ca2+ fluxes. On the other hand, there are also some reports in the literature showing that LPA can induce Ca2+ influx from the external milieu leading to an increase in cytosolic Ca2+ levels [35, 52]. Thus, LPA could efficiently sustain an increase in the intracellular Ca2+ levels through the combined effects of Ca2+ influx and PMCA inhibition, which would be essen- tial for different Ca2+-dependent events. Unraveling LPA biosynthesis on BLM helped us to con- sider that PA, which is present in the BLM [27, 34], could be metabolized by an iPLA2. Líbano-Soares and coworkers (2008) had elegantly demonstrated the presence and activity of iPLA2 associated to pig BLM from kidney proximal tu- bules where the generation of prostaglandins induced by bra- dykinin led to Na+-ATPase inhibition [28]. These observa- tions allow us to indicate that PLA2 can provide substrates for at least two branches of different cell signaling cascades involved in ion transporter modulation: (i) providing arachi- donic acid from membrane glycerolipids, PA included, which will be processed by cyclooxygenases and lipoxigenases to generate prostaglandins and other lipid mediators and (ii) yielding LPA that would be capable to activate different sets of LPA receptors triggering different cell signaling cascades into the renal cells. It is important to mention that although it is expected the presence of lyso-phosphatidylcholine (LPC) within the BLM, this is not a substrate for PLA2. Instead, LPC is metabolized by lyso-PLD or autotaxin to further gen- erate LPA [25, 34, 46, 48]. The ability of LPA as a potent stimulatory agent for differ- ent protein kinases had been demonstrated [19, 41]. The LPA- stimulated PKC led to direct phosphorylation and inhibition of the plasma membrane Ca2+ pump, thus showing that different ser/thr residues play key roles in the switch to control the BLM Ca2+-ATPase activity. In a previous study, our group had provided evidence for a stimulatory phosphorylation site in the BLM plasma membrane Ca2+-ATPase, which occurs after incubation of the BLM fractions with ceramide [10]. Conversely to the observed here, this stimulatory phosphory- lation is due to the activation of a cAMP-dependent protein kinase (PKA). Conclusion The results presented above allow us to include LPA in the complex hall of bioactive lipids that can be generated and act directly within the BLM from kidney proximal tubule cells to further control different renal functions by playing a pivotal role in the regulation of intracellular Ca2+ levels. It is plausible to imagine that this and other cell signaling pathways trig- gered by LPA in the BLM would be involved in the modula- tion of other renal transporters, thus reflecting the physiolog- ical relevance of the LPA-dependent events to renal physiology. Acknowledgements The authors would like to thank Mr. Celso Pereira for helpful technical services. Author contribution Statement: The authors declare that all data were generated in-house and that no paper mill was used. JFS: Experimental design, performed experiments, data interpretation, discussion of the results, and written and revision of the manuscript VSB: Experimental design, performed experiments, data interpreta- tion, and discussion of the results NAR-G: Experimental design, performed experiments, data interpre- tation, discussion of the results TL: Experimental design, performed experiments, data interpretation, and discussion of the results BLD: Experimental design, performed experiments, data interpreta- tion, discussion of the results, and written and revision of the manuscript ME-L: Experimental design, performed experiments, data interpreta- tion, discussion of the results, written and revision of the manuscript, and financial support Funding This work was supported by grants from CNPq, CAPES/ PROBITEC, and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). 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