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SUMMARYCertain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation fromAngomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affectA. deaneirespiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2consumption. In theA. deaneigenome, sequences encoding most proteins of the respiratory chain are present. The symbiont genome lost part of the electron transport system (ETS), but complex I, a cytochrome d oxidase, and FoF1-ATP synthase remain. In conclusion, this work suggests that the symbiont influences the mitochondrial respiration of the host protozoan.

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.

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg·ATP 2− complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg 2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca 2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis. © 2004 Elsevier Ltd. All rights reserved.

Glial fibrillary acidic protein (GFAP) is the main intermediate filament protein used as a marker for the identification of astrocytes in the central nervous system of vertebrates. Analogous filaments have been observed in the glial cells of many mollusks and annelids but not in crustaceans. The present study was carried out to identify by light microscopy immunohistochemistry, immunoelectronmicroscopy and immunoblotting, GFAP-like positive structures in the visual system of the crab Ucides cordatus as additional information to help detect and classify glial cells in crustaceans. Conventional electron microscopy, light microscopy of semithin sections and fluorescence light microscopy were also employed to characterize cells and tissues morphology. Our results indicated the presence of GFAP-like positive cell processes and cell bodies in the retina and adjoining optic lobe. The labeling pattern on the reactive profiles was continuous and very well defined, differing considerably from what has been previously reported in the central nervous system of some mollusks, where a diffuse spotted fluorescence pattern of labeling was observed. We suggest that this glial filament protein may be conserved in the evolution of the invertebrate nervous systems and that it may be used as a label for some types of glial cells in the crab.

We have observed the effect of copper and zinc on the biology of Euglena gracilis. The cells displayed different sensitivities to these metals, as the apparent LC50 for Cu2+ was 0.22 mM, and for Zn2+ it was 0.88 mM. While Zn2+ was able to increase cell proliferation even at 0.1 mM, the minimal CuCl2 concentration tested (0.02 mM) was sufficient to impair cell division. Higher concentrations of these metals not only inhibited cell division in a concentration-dependent manner, but also interfered with the metabolism of E. gracilis. A higher accumulation of proteins and lipids per cell was observed at the DI50 concentration for metal-treated cells. These results suggest that the test concentration of both metals leads to a failure in completing cell division. Ultrastructural analysis indicated a chloroplast disorganization in copper-treated cells, as well as the presence of electron dense granules with different shapes and sizes inside vacuoles. Microanalysis of these granules indicated an accumulation of copper, thus suggesting a detoxification role played by the vacuoles. These results indicate that E. gracilis is an efficient biological model for the study of metal poisoning in eukaryotic cells. They also indicate that copper and zinc (copper being more poisonous) had an overall toxic effect on E. gracilis and that part of the effect can be ascribed to defects in the structure of chloroplast membranes.

The neural system appears before the vascular system in the phylogenetic tree. During evolution, vascular system generation takes advantage of the pre-existing vascular endothelial growth factor (VEGF) in order to form its networks. Nevertheless, the role of VEGF in neuronal and glial cells is not yet completely understood. In order to support the hypothesis of a neural role for VEGF, we searched for VEGF- and VEGF receptor (VEGFR)-like immunoreactivities (immunohisto/cytochemistry and Western blotting) in the eyestalk of the invertebrate Ucides cordatus (Crustacea, Brachyura, Ucididae). Our results showed that both neurons and glial cells expressed VEGF-immunoreactivity, and that VEGFR was evidenced in neural cells. This is the first report about the VEGF/VEGFR-like immunoreactivities in the nervous tissue of a crustacean, and enables U. cordatus to be included in the repertoire of animal models used for ascertaining the role of VEGF in the nervous system.

The present study aimed to investigate the in vitro effects of isoschaftoside isolated from Syngonium podophyllum on pig kidney Na+,K+-ATPase. The Na+, K+-ATPase activity was determined by colorimetric measurement of inorganic phosphate (Pi), resulting from ATP hydrolysis. Isoschaftoside significantly decreased the renal Na+, K+-ATPase activity at the highest concentration as well as at a lower concentration. Our work suggests that isoschaftoside is a promising compound for the treatment of hypertension.

Trypanosoma cruzi, the causative agent of Chagas' disease in humans, is an intracellular protozoan parasite with the ability to invade a wide variety of mammalian cells by a unique and remarkable process in cell biology that is poorly understood. Here we present evidence suggesting a role for the host phosphatidylinositol (PI) 3-kinases during T. cruzi invasion. The PI 3-kinase inhibitor wortmannin marked inhibited T. cruzi infection when macrophages were pretreated for 20 min at 37 degrees C before inoculation. Infection of macrophages with T. cruzi markedly stimulated the formation of the lipid products of the phosphatidylinositol (PI) 3-kinases, PI 3-phospate, PI 3,4-biphosphate, and PI 3,4,5-triphosphate, but not PI 4-phosphate or PI 4,5-biphosphate. This activation was inhibited by wortmannin. Infection with T. cruzi also stimulated a marked increase in the in vitro lipid kinase activities that are present in the immunoprecipitates of anti-p85 subunit of class I PI 3-kinase and anti-phosphotyrosine. In addition, T. cruzi invasion also activated lipid kinase activity found in immunoprecipitates of class II and class III PI 3-kinases. These data demonstrate that T. cruzi invasion into macrophages strongly activates separated PI 3-kinase isoforms. Furthermore, the inhibition of the class I and class III PI 3-kinase activities abolishes the parasite entry into macrophages. These findings suggest a prominent role for the host PI 3-kinase activities during the T. cruzi infection process.

myo-inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1-10 microM with maximal transport obtained at 8 microM. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, inhibited the Na+-dependent and Na+-independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 degrees C. The addition of glucose (5-50 mM) or mannose (10 mM) did not change the myo-inositol uptake, whereas the addition of 10 mM nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mM) and phoridzin (5 mM), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters - one Na+-dependent and the other Na+-independent.

In the present paper, the presence of a ouabain-insensitive Na+-stimulated, Mg2+-dependent ATPase activity in T. cruzi epimastigotes CL14 clone and Y strain was investigated. The increase in Na+ concentration (from 5 to 170 mᴍ), in the presence of 2 mᴍ ouabain, increases the ATPase activity in a saturable manner along a rectangular hyperbola. The was 18.0 ± 1.0 and 21.1 ± 1.1 nmoles Pi x mg-1 x min-1 and the half-activation value (K50) for Na+ was 34.3 ± 5.8 mᴍ and 37.7 ± 5.3 in CL14 clone and in Y strain, respectively. The Na+-stimulated ATPase activity was inhibited by 5-[aminosulfonyl]-4-chloro-2-[(2-furanylmethyl)-amino] benzoic acid (furosemide) in a dose-dependent manner. The half-inhibition value (I50) was 0.22 ± 0.03 and 0.24 ± 0.07 mᴍ, and the Hill number (n) was 0.99 ± 0.2 and 2.16 ± 0.29 for CL14 clone and Y strain, respectively. These data indicate that both cell types express the ouabain-insensitive Na+-ATPase activity, which might be considered the bio­chemical expression of the second Na+ pump

The presence of (Na++K+)ATPase activity in CL14 clone and NIH NTY strain of Trypano­soma cruzi epimastigotes is demonstrated. A Na+ plus K+ stimulated ATPase activity is found in both strains. The optimal Na+/K+ ratio is 5:1 and 9:1 in CL14 clone and NIH NTY strain, respectively. In both strains, vanadate completely inhibits the ouabain-sensitive ATPase activ­ity indicating that it belongs to the P-type (E 1/E2) family of ion-transporting ATPases. The I50 for vanadate is 0.66 ± 0.04 and 0.04 ± 0.02 μᴍ in CL14 clone and NIH NTY strain, respectively. These data indicate that both strains of T. cruzi epimastigotes express the oua­ bain-and vanadate-sensitive (Na++K+)ATPase activity. On the other hand, the discrepancy between the parameters analyzed for the inhibitors suggests that they express different iso­ forms of this enzyme.

PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB / phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB / phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB / phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae . Biological significance Certain V. cholerae strains are pathogenic to humans, causing cholera, an acute dehydrating diarrhoeal disease endemic in Southern Asia, parts of Africa and Latin America, where it has been responsible for significant mortality and economical damage. Its ability to grow within distinct niches is dependent on gene expression regulation. PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. However, Pho regulon genes also play roles in virulence, motility and biofilm formation, among others. In this paper we report that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways, in Pi abundance. Moreover, we showed, for the first time, that the interrelationship between PhoB–RpoS–(p)ppGpp–poly(P) in V. cholerae , is somewhat diverse from the model of inter-regulation between those systems, described in Escherichia coli . The V. cholerae dependence on PhoB/PhoR for the RpoS mediated stress response and cellular growth under Pi abundance, suggests that this system's roles are broader than previously thought.

BACKGROUND: The aim of this work was to investigate the mechanisms by which chronic malnutrition (CM) affects vas deferens function, leading to compromised reproductive capacity. Previous studies have shown that maternal malnutrition affects the reproductive tracts of adult male offspring. However, little is known about the effects of CM, a widespread life-long condition that persists from conception throughout growth to adult life. METHODOLOGY/PRINCIPAL FINDINGS: Young adult male rats, which were chronically malnourished from weaning, presented decreased total and haploid cells in the vas deferens, hypertrophy of the muscle layer in the epididymal portion of the vas deferens and intense atrophy of the muscular coat in its prostatic portion. At a molecular level, the vas deferens tissue of CM rats exhibited a huge rise in lipid peroxidation and protein carbonylation, evidence of an accentuated increase in local reactive oxygen species levels. The kinetics of plasma membrane Ca(2+)-ATPase activity and its kinase-mediated phosphorylation by PKA and PKC in the vas deferens revealed malnutrition-induced modifications in velocity, Ca(2+) affinity and regulation of Ca(2+) handling proteins. The severely crippled content of the 12-kDa FK506 binding protein, which controls passive Ca(2+) release from the sarco(endo) plasmic reticulum, revealed another target of malnutrition related to intracellular Ca(2+) handling, with a potential effect on forward propulsion of sperm cells. As a possible compensatory response, malnutrition led to enhanced sarco(endo) plasmic reticulum Ca(2+)-ATPase activity, possibly caused by stimulatory PKA-mediated phosphorylation. CONCLUSIONS/SIGNIFICANCE: The functional correlates of these cellular and molecular hallmarks of chronic malnutrition on the vas deferens were an accentuated reduction in fertility and fecundity.

It has been demonstrated that reabsorption of Na⁺ in the thick ascending limb is reduced and the ability to concentrate urine can be compromised in undernourished individuals. Alterations in phospholipid and cholesterol content in renal membranes, leading to Na⁺ loss and the inability to concentrate urine, were investigated in undernourished rats.Sixty-day-old male Wistar rats were utilized to evaluate (1) phospholipid and cholesterol content in the membrane fraction of whole kidneys, (2) cholesterol content and the levels of active Na⁺ transporters, (Na⁺ + K⁺)ATPase and Na⁺-ATPase, in basolateral membranes of kidney proximal tubules, and (3) functional indicators of medullary urine concentration.Body weight in the undernourished group was 73 % lower than in control. Undernourishment did not affect the levels of cholesterol in serum or in renal homogenates. However, membranes of whole kidneys revealed 56 and 66 % reduction in the levels of total phospholipids and cholesterol, respectively. Furthermore, cholesterol and (Na⁺ + K⁺)ATPase activity in proximal tubule membranes were reduced by 55 and 68 %, respectively. Oxidative stress remained unaltered in the kidneys of undernourished rats. In contrast, Na⁺-ATPase activity, an enzyme with all regulatory components in membrane, was increased in the proximal tubules of undernourished rats. Free water clearance and fractional Na⁺ excretion were increased by 86 and 24 %, respectively, and urinary osmolal concentration was 21 % lower in undernourished rats than controls.Life-long undernutrition reduces the levels of total phospholipids and cholesterol in membranes of renal tubular cells. This alteration in membrane integrity could diminish (Na⁺ + K⁺)ATPase activity resulting in reduced Na⁺ reabsorption and urinary concentrating ability.

Renal tubular cells are the main target of ischemic insult associated with acute renal injury. Low oxygen and nutrient supplies result in ATP depletion, leading to cell death and loss of renal function. A possible mechanism by which bone marrow-derived cells support renal tissue regeneration relies on the capacity of mononuclear cells (BMMC), particularly mesenchymal stem cells (MSC), to secrete paracrine factors that mediate support for kidney regeneration.BMMC/MSC and renal cells (LLC-PK(1) from pig and IRPTC from rat) were co-cultured under stressful conditions (ATP depletion and/or serum free starvation), physically separated by a microporous membrane (0.4 μm), was used to determine whether bone marrow-derived cells can interact with renal cells in a paracrine manner.This interaction resulted in stimulation of renal cell proliferation and the arrest of cell death. MSC elicit effective responses in renal cells in terms of stimulating proliferation and protection. Such effects are observed in renal cells co-cultured with rat BMMC/MSC, an indication that paracrine mechanisms are not entirely species-specific.The paracrine action of BMMC/MSC was influenced by a renal cell stimulus released during stress, indicating that cross-talk with injured cells is required for renal regeneration supported by bone marrow-derived cells.

Muscle fibers are formed during embryonic development by the fusion of mononucleated myoblasts. The spatial structure and molecular composition of the sarcolemma are crucial for the myoblast recognition and fusion steps. Cyclodextrins are a group of substances that have the ability to solubilize lipids through the formation of molecular inclusion complexes. Previously, we have shown that methyl-β-cyclodextrin (MbCD) enhances muscle differentiation. Here, we analyzed the effects of α-cyclodextrin (aCD) during myogenesis. Myogenic cultures treated with aCD showed an increase in myoblast fusion and in the expression of myogenin, sarcomeric tropomyosin and desmin. aCD-conditioned media accelerates myogenesis in a similar way as aCD does, and increased levels of IL-4 were found in aCD-conditioned media. aCD-induced effects on myogenesis were inhibited by an anti-IL4 antibody. These results show that α-cyclodextrin induces myogenic differentiation by the release of IL-4.

Nuclear migration is regulated by the LIS1 protein, which is the regulatory subunit of platelet activating factor (PAF) acetyl-hydrolase, an enzyme complex that inactivates the lipid mediator PAF. Among other functions, PAF modulates cell proliferation, but its effects upon mechanisms of the cell cycle are unknown. Here we show that PAF inhibited interkinetic nuclear migration (IKNM) in retinal proliferating progenitors. The lipid did not, however, affect the velocity of nuclear migration in cells that escaped IKNM blockade. The effect depended on the PAF receptor, Erk and p38 pathways and Chk1. PAF induced no cell death, nor a reduction in nucleotide incorporation, which rules out an intra-S checkpoint. Notwithstanding, the expected increase in cyclin B1 content during G2-phase was prevented in the proliferating cells. We conclude that PAF blocks interkinetic nuclear migration in retinal progenitor cells through an unusual arrest of the cell cycle at the transition from S to G2 phases. These data suggest the operation, in the developing retina, of a checkpoint that monitors the transition from S to G2 phases of the cell cycle.

Background. Bioactive lipids are important in tissue injury and regeneration. Ceramide (Cer) is known for its proapoptotic action and sphingosine-1-phosphate (S1P) for inducing proliferation and cell survival; diacylglycerol (DAG) and lysophosphatidic acid (LPA) are involved in various signalling pathways including modulation of ion transport. LPA signalling through its receptor LPA1 is also related to the progression of fibrosis. This study investigated the modulation of lipid signalling pathways induced by administration of bone marrow-derived mononuclear cells (BMMC) in chronic kidney disease. Methods. Unilateral ureteral obstruction (UUO) was followed by intravenous injection of ∼2×10 7 BMMC. Controls were UUO group treated with buffered solution and sham-operated group. Animals were killed 14 days after surgery, and lipid phosphorylation assays and immunoblotting were performed on the kidney homogenates. Results. More DAG was available in the UUO rats (2.4 ± 0.4 and 2.4 ± 0.3 vs 1.0 ± 0.2 pmol 32 PA mg −1 min −1 ,i n UUO and UUO + BMMC vs SHAM). Sphingosine kinase was 150 ± 12% more active in UUO + BMMC than in UUO and SHAM. Cer levels were 76 ± 7% lower in the UUO + BMMC than UUO. LPA receptor type 1 (LPA1) expression was 169 ± 7% higher in the UUO group than in UUO + BMMC and SHAM. BMMC maintain control levels of Ca 2+ -ATPase expression altered by UUO by 40%. Conclusions. BMMC infusion modulated diverse lipid signalling pathways and protein expression, shifted sphingolipid metabolism toward a regenerative pattern and favourably reduced the levels of a receptor involved in the progression of tissue fibrosis. These results strengthen the benefits of BMMC treatment and give insight into its paracrine mechanisms of action.

The basolateral membranes of kidney proximal tubule cells have (Na(+)+K(+))-ATPase and Na(+)-ATPase activities, involved in Na(+) reabsorption. We showed that ceramide (Cer) modulates protein kinase A (PKA) and protein kinase C (PKC), which are involved in regulating ion transporters. Here we show that ceramide, promotes 60% inhibition of Na(+)-ATPase activity (I(50) approximately 100nM). This effect was completely reversed by inhibiting PKA but did not involve the classic PKC signaling pathway. In these membranes we found the Cer-activated atypical PKC zeta (PKCzeta) isoform. When PKCzeta is inhibited, Cer ceases to inhibit the Na(+)-ATPase, allowing the cAMP/PKA signaling pathway to recover its stimulatory effect on the pump. There were no effects on the (Na(+)+K(+))-ATPase. These results reveal Cer as a potent physiological modulator of the Na(+)-ATPase, participating in a regulatory network in kidney cells and counteracting the stimulatory effect of PKA via PKCzeta.

Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-β-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/β-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. β-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac β-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.

This review reports the role of oxidative stress in impairing the function of lung exposed to particulate matter (PM). PM constitutes a heterogeneous mixture of various types of particles, many of which are likely to be involved in oxidative stress induction and respiratory diseases. Probably, the ability of PM to cause oxidative stress underlies the association between increased exposure to PM and exacerbations of lung disease. Mostly because of their large surface area, ultrafine particles have been shown to cause oxidative stress and proinflammatory effects in different in vivo and in vitro studies. Particle components and surface area may act synergistically inducing lung inflammation. In this vein, reactive oxygen species elicited upon PM exposure have been shown to activate a number of redox-responsive signaling pathways and Ca(2+) influx in lung target cells that are involved in the expression of genes that modulate relevant responses to lung inflammation and disease.

The growing number of patients suffering from chronic renal disease is a challenge for the development of innovative therapies. Benefits of cell therapy in acute renal diseases in animal models have been reported but seldom for chronic lesions. We present evidence for the improvement of renal morphology in a model of tubulointerstitial fibrosis. Wistar rats were submitted to unilateral ureteral obstruction (UUO), treated with bone-marrow mononuclear cells (UUO+BMMC) infused via the cava vein, and killed on day 14. Labeled BMMC were seen in renal tissue after 7 days in the group UUO+BMMC. UUO+BMMC also showed a reduction in ED1(+) cells and tubular apoptotic cells together with enhanced tubular proliferation. Myofibroblasts were also reduced after BMMC which is consistent with a decrease in collagen deposition (picro Sirius staining) and RT-PCR data showing lower levels of procollagen-I mRNA. Simultaneously, nestin+ cells increased in the interstitium and decreased in the tubules. Double stained nestin(+)/alpha-SMA(+) cells were present only in the interstitium, and their levels did not change after BMMC infusion. These data indicate a renoprotective effect of BMMC through increased tubular cell regeneration, inhibition of tubular cell apoptosis and partially blocking of the inflammatory and fibrotic events that occur after unilateral ureteral obstruction.

1. Intrauterine malnutrition has been linked to the development of adult cardiovascular and renal diseases, which are related to altered Na + balance. Here we investigated whether maternal malnutrition increases placental oxidative stress with subsequent impact on renal ATP-dependent Na + transporters in the offspring. 2. Maternal malnutrition was induced in rats during pregnancy by using a basic regional diet available in north-eastern Brazil. Placental oxidative stress was evaluated by measuring thiobarbituric acid-reactive substances, which were 35―40% higher in malnourished dams (MalN). Na + pumps were evaluated in control and prenatally malnourished rats (at 25 and 90 days of age). 3. Identical Na + /K + -ATPase activity was found in both groups at 25 days (approximately 150 nmol P i /mg per min). However, although Na + /K + -ATPase increased by 40% with growth in control rats, it remained constant in pups from MalN. 4. In juvenile rats, the activity of the ouabain-insensitive Na + -ATPase was higher in MalN than in controls (70 vs 25 nmol P i /mg per min). Nevertheless, activity did not increase with kidney and body growth: at 90 days, it was 50% lower in MalN than in controls. The maximal stimulation of the Na + -ATPase by angiotensin (Ang) II was 35% lower in MalN than in control rats and was attained only with a much higher concentration of the peptide (10 ―10 mol/L) than in controls (10 ―14 mol/L). 5. Protein kinase C activity, which mediates the effects of AngII on Na + -ATPase was only one-third of normal values in the MalN group. 6. These results indicate that placental oxidative stress may contribute to fetal undernutrition, which leads to later disturbances in Na + pumps from proximal tubule cells.

In this study, the role of phospholipid biosynthetic pathways was investigated in the establishment of the mutualistic relationship between the trypanosomatid protozoan Crithidia deanei and its symbiotic bacterium. Although the endosymbiont displays two unit membranes, it lacks a typical Gram-negative cell wall. As in other intracellular bacteria, phosphatidylcholine is a major component of the symbiont envelope. Here, it was shown that symbiont-bearing C. deanei incorporates more than two-fold (32)Pi into phospholipids as compared with the aposymbiotic strain. The major phospholipid synthesized by both strains was phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol. Cellular fractioning indicated that (32)Pi-phosphatidylcholine is the major phospholipid component of the isolated symbionts, as well as of mitochondria. Although the data indicated that isolated symbionts synthesized phospholipids independently of the trypanosomatid host, a key finding was that the isolated bacteria synthesized mostly phosphatidylethanolamine, rather than phosphatidylcholine. These results indicate that phosphatidylcholine production by the symbiont depends on metabolic exchanges with the host protozoan. Insight about the mechanisms underlying lipid biosynthesis in symbiont-bearing C. deanei might help to understand how the prokaryote/trypanosomatid relation has evolved in the establishment of symbiosis.

Inositol is the precursor for most Trypanosoma cruzi surface molecules, including phosphoinositides, glycosylinositolphospholipids and glycosylphosphatidylinositol anchors. As the parasite is an inositol auxotroph, the inositol transport system might be a potential target for new trypanocide drugs, as some of its properties are different from its mammalian counterpart. Here, we investigated the modulation exerted by effectors of PKA and PKC on this transport system to comply with the parasite physiology. Pre-incubation of the cells with either dibutyryl-cyclic AMP (25 microM) or forskolin (30 microM) decreased the myo-inositol uptake by half, this effect being reversed by KT5720 (PKA inhibitor). Conversely, pre-incubation of the cells with PMA (2.8 microg/ml) or serum (5%) had a approximately 50% stimulation in myo-inositol uptake, being this effect reversed by staurosporine (0.5 microM) or sphingosine (10 microM). These results allow us to conclude that the myo-inositol transport system in T. cruzi epimastigotes is inhibited by PKA and stimulated by PKC effectors.

In invertebrates, a few studies have suggested apoptosis as the mechanism of choice to protect the retina after exposure to ultraviolet (UV) radiation. We demonstrated previously, by electron microscopy, that the retina and lamina ganglionaris (or lamina) cells of the crab Ucides cordatus displayed subcellular signs of apoptosis after exposure to UVB and UVC. Here, we first ascertained, by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, that UV irradiation indeed produced the previously reported results. We next tested, in the visual system of U. cordatus, whether the expression (as analyzed by immunohistochemistry and observed with laser scanning microscopy) and levels (as examined by Western blotting) of catalase, Bax, and p53 were affected by the same dose of UV radiation as that used previously. Our data revealed that the intensity of catalase, Bax, and p53 labeling was stronger in irradiated retina and lamina cells than in non-irradiated retina and lamina. However, no significant difference was observed in the concentrations of these proteins isolated from the whole optic lobe. The results thus suggest that UVB and UVC induce apoptosis in the crustacean retina and lamina by increasing catalase expression and activating the Bax- and p53-mediated apoptosis pathways.

Hydrogenosomes are found in organisms that lack typical mitochondria. Cardiolipin is a phospholipid located exclusively in bacterial membranes and the inner membrane of mitochondria. Here we show, by cell fractionation, thin-layer chromatography, high-pressure liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry that hydrogenosomes of Tritrichomonas foetus , a cattle vaginal parasite, contain cardiolipin, which is strong evidence for its endosymbiotic origin.

Some protozoa of the Trypanosomatidae family have a close relationship with an endosymbiotic bacterium. As the prokaryote envelope has a controversial origin, a sterol 24-methyltransferase inhibitor (20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol; 22,26-azasterol) was used as a tool to investigate lipid biosynthetic pathways in Crithidia deanei, an endosymbiont-bearing trypanosomatid. Apart from antiproliferative effects, this drug induced ultrastructural alterations, consisting of myelin-like figures in the cytoplasm and endosymbiont envelope vesiculation. Concurrently, a dramatic reduction of 24-alkyl sterols was observed after 22,26-azasterol treatment, both in whole cell homogenates, as well as in isolated mitochondria. These effects were associated with changes of phospholipid composition, in particular a reduction of the phosphatidylcholine content and a concomitant increase in phosphatidylethanolamine levels. Lipid analyses of purified endosymbionts indicated a complete absence of sterols, and their phospholipid composition was different from that of mitochondria or whole protozoa, being similar to eubacteria closely associated with eukaryotes.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca(2+) enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca(2+)-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca(2+)-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.

Neurofilaments (NFs) have not been observed in crustaceans using conventional electron microscopy, and intermediate filaments have never been described in crustaceans and other arthropods by immunocytochemistry. Since polypeptides, labeled by the NN18-clone antibody, were revealed on microtubule side-arms of crayfish, we have tested, in this study, whether proteins similar to mammalian NFs are present in the protocerebral tract (PCT) of the crab Ucides cordatus. We used immunohistochemistry for light microscopy with monoclonal antibodies against three different NF subunits, high (NF-H), medium (NF-M), and light (NF-L). Labeling was observed with the NN18-clone, which recognizes NF-M. In order to confirm the results obtained with the immunohistochemical reactions, Western blotting, using the three primary antibodies, was performed and the presence of NF-M was confirmed. The NN18-clone monoclonal antibody recognized a protein of approximately 160 kDa, similar to the mammalian NF-M protein, but NF-L and NF-H were not recognized. Conventional transmission electron microscopy was used to observe the ultrastructural components of the axons and immunoelectron microscopy was used to show the distribution of the NF-M-like polypeptides along cytoskeletal elements of the PCT. Our results agree with previous studies on crustacean NF proteins that have reported negative immunoreactions against NF-H and NF-L subunits and positive immunoreactions against the mammalian NF-M subunit. However, the protein previously referred to as P600 and recognized by the NN18-clone, has a very high molecular weight, thus, being different from mammalian NF-M subunit and from the protein revealed now in our study.

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.

Selective pressures from polluted environments have led to the development of resistance systems in aquatic organisms. Using different techniques, this study examined a cadmium defense mechanism of the freshwater unicellular protozoa Euglena gracilis, and found it to be an efflux pump similar to the multidrug resistance P-glycoprotein. Cd(2+)-treated E. gracilis were able to extrude Rhodamine 123 at 21 degrees C, but not at 4 degrees C. Furthermore, verapamil, a P-glycoprotein modulator, partially blocked the efflux process (at 21 degrees C), and enhanced the Cd(2+) toxic effects on these cells. Western immunoblots of cell lysates, using the anti-P-glycoprotein antibody JSB-1, revealed a 120-KDa protein, which was expressed, in high amounts on Cd(2+)-exposed cells (74% above the control values). Moreover, cells treated with JSB-1 became more sensitive to the harmful effects of cadmium, showing a decreased survival rate. Taken together, these results suggest that a MDR phenotype has evolved in Euglena as one of the mechanisms for cadmium detoxification.

The protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironmental and lymphoid compartments. Acute infection results in severe atrophy of the organ and early release of immature thymocytes into the periphery. To date, the pathophysiological effects of thymic changes promoted by parasite-inducing premature release of thymocytes to the periphery has remained elusive. Herein, we show that sphingosine-1-phosphate (S1P), a potent mediator of T cell chemotaxis, plays a role in the exit of immature double-negative thymocytes in experimental Chagas disease. In thymuses from T. cruzi-infected mice we detected reduced transcription of the S1P kinase 1 and 2 genes related to S1P biosynthesis, together with increased transcription of the SGPL1 sphingosine-1-lyase gene, whose product inactivates S1P. These changes were associated with reduced intrathymic levels of S1P kinase activity. Interestingly, double-negative thymocytes from infected animals expressed high levels of the S1P receptor during infection, and migrated to lower levels of S1P. Moreover, during T. cruzi infection, this thymocyte subset expresses high levels of IL-17 and TNF-α cytokines upon polyclonal stimulation. In vivo treatment with the S1P receptor antagonist FTY720 resulted in recovery the numbers of double-negative thymocytes in infected thymuses to physiological levels. Finally, we showed increased numbers of double-negative T cells in the peripheral blood in severe cardiac forms of human Chagas disease., Author Summary The formation of mature lineage-committed T cells requires the specialized environment of the thymus, a central organ of the immune system supporting the development of self-tolerant T cells. Key events of intrathymic T-cell development include lineage commitment, selection events and thymic emigration. This organ undergoes physiological involution during aging. However, acute thymic atrophy can occur in the presence autoimmune diseases, malignant tumors and infections caused by intracellular pathogens. The present study shows that the protozoan parasite Trypanosoma cruzi changes the thymic microenvironmental and lymphoid compartments, resulting in premature release of very immature CD4−CD8− double-negative thymocytes, TCRneg/low, which bear a pro-inflammatory activation profile. Strikingly, we also found elevated levels of these undifferentiated T lymphocytes in the peripheral blood of patients in severe cardiac forms of chronic Chagas disease. Importantly, we provided evidence that migration of CD4−CD8− T cells from infected mouse thymus is due to sphingosine-1-phosphate receptor-1-dependent chemotaxis. These findings point to an important role for bioactive signaling sphingolipids in the thymic escape of immature thymocytes to the periphery in Chagas disease.

Chagas' disease is a debilitating and often fatal disease caused by the protozoan parasite Trypanosoma cruzi. The great majority of surface molecules in trypanosomes are either inositol-containing phospholipids or glycoproteins that are anchored into the plasma membrane by glycosylphosphatidylinositol anchors. The polyalcohol myo-inositol is the precursor for the biosynthesis of these molecules. In this brief review, recent findings on some aspects of the molecular and cellular fate of inositol in T. cruzi life cycle are discussed and identified some points that could be targets for the development of parasite-specific therapeutic agents.

Inositol has been cited as being essential for the growth of micro-organisms and animals. Its action rests mostly in the formation of a set of inositol-containing lipids, including phosphatidylinositol and its phosphorylated derivatives. To evaluate the functional responses coupled to the phosphoinositide metabolism in Trypanosoma cruzi we used myo-inositol and its six fluorinated analogues. Their uptake into epimastigotes was characterised using tritium-labeled myo-inositol and monodeoxyfluoro-myo-inositols. The analogues were tested for their ability to inhibit [3H]-myo-inositol incorporation into phosphoinositides and the proliferation of epimastigotes and amastigotes. The results showed differences between T. cruzi and mammalian systems in the responses to the fluorinated analogues. We found that the 3-, 5- and 6-fluoro analogues did not enter the cells but had an inhibitory effect on the incorporation of the radioactive inositol into lipids and on the amastigotes' and epimastigotes' replication. The most effective inhibitor, 1-D-6-deoxy-6-myo-inositol, had no effect on mammalian cell division.

Euglena gracilis , a unicellular flagellated alga, can display numerous shape changes. These changes are most probably caused by a pellicle and an internal cytoskeleton. In this paper we studied the distribution of the cytoskeletal proteins actin, α-actinin, tropomyosin and tubulin in dark-adapted Euglena , using immunofluorescence microscopy. We found that F-actin, α-actinin, tropomyosin and tubulin have a distribution that is coincident in the plasma membrane and, in addition, α-actinin and tropomyosin are seen in small patches in the cytoplasm, and tubulin in the flagella. We have also studied the distribution of the endoplasmic reticulum, nucleus, and Golgi apparatus of these cells, using fluorescent probes. Both the endoplasmic reticulum and the Golgi apparatus have a meshwork pattern distributed throughout the cytoplasm, and the nucleus has a chromatin evenly distributed in the nucleoplasm.

BACKGROUND: Several studies have correlated perinatal malnutrition with diseases in adulthood, giving support to the programming hypothesis. In this study, the effects of maternal undernutrition during lactation on renal Na(+)-transporters and on the local angiotensin II (Ang II) signaling cascade in rats were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Female rats received a hypoproteic diet (8% protein) throughout lactation. Control and programmed offspring consumed a diet containing 20% protein after weaning. Programming caused a decrease in the number of nephrons (35%), in the area of the Bowman's capsule (30%) and the capillary tuft (30%), and increased collagen deposition in the cortex and medulla (by 175% and 700%, respectively). In programmed rats the expression of (Na(+)+K(+))ATPase in proximal tubules increased by 40%, but its activity was doubled owing to a threefold increase in affinity for K(+). Programming doubled the ouabain-insensitive Na(+)-ATPase activity with loss of its physiological response to Ang II, increased the expression of AT(1) and decreased the expression of AT(2) receptors), and caused a pronounced inhibition (90%) of protein kinase C activity with decrease in the expression of the α (24%) and ε (13%) isoforms. Activity and expression of cyclic AMP-dependent protein kinase decreased in the same proportion as the AT(2) receptors (30%). In vivo studies at 60 days revealed an increased glomerular filtration rate (GFR) (70%), increased Na(+) excretion (80%) and intense proteinuria (increase of 400% in protein excretion). Programmed rats, which had normal arterial pressure at 60 days, became hypertensive by 150 days. CONCLUSIONS/SIGNIFICANCE: Maternal protein restriction during lactation results in alterations in GFR, renal Na(+) handling and in components of the Ang II-linked regulatory pathway of renal Na(+) reabsorption. At the molecular level, they provide a framework for understanding how metabolic programming of renal mechanisms contributes to the onset of hypertension in adulthood.

Plasma membrane Ca2+-ATPase is involved in the fine-tuned regulation of intracellular Ca2+. In this study, the presence of Ca2+-ATPase in caveolae from kidney basolateral membranes was investigated. With the use of a discontinuous sucrose gradient, we show that Ca2+-ATPase is exclusively located and fully active in caveolin-containing microdomains. Treatment with methyl-β-cyclodextrin – a cholesterol chelator – leads to a spreading of both caveolin and completely inactive Ca2+-ATPase toward high-density fractions. These data support the view that Ca2+ fluxes mediated by Ca2+-ATPase in kidney epithelial cells occur only in caveolae, being strictly dependent on the integrity of these microdomains.

Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca2+ at the μM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP2− complex is the true substrate of the enzyme and that free Mg2+ is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.