Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K(+) cycling and to active Na(+) extrusion.

BACKGROUND: Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi. METHODS: (32)Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na(+), H(+) and K(+) fluxes were also investigated. The transport capacities of different evolutive forms were compared. RESULTS: Epimastigotes grew significantly more slowly in 2mM than in 50mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na(+). We found that the parasites express TcPho84, a H(+):Pi-symporter, and TcPho89, a Na(+):Pi-symporter. Both Pi influx mechanisms showed Michaelis-Menten kinetics, with a one-order of magnitude higher affinity for the Na(+)-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K(+) ionophore) or SCH28028 (inhibitor of (H(+)+K(+))ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H(+) gradient energizes uphill Pi entry and that K(+) recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na(+)-ATPase, decreased only the Na(+)-dependent Pi uptake, indicating that this Na(+) pump generates the Na(+) gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently. CONCLUSIONS: Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na(+) or H(+)/K(+) fluxes. GENERAL SIGNIFICANCE: This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. METHODS: We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. RESULTS: T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. CONCLUSIONS: Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. GENERAL SIGNIFICANCE: This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.

Calcium inhibition of the ATP in equilibrium with [32P]Pi exchange and of net ATP synthesis catalyzed by bovine submitochondrial particles.

A previous communication (Fagian, M. M., Pereira da Silva, L. and Vercesi, A. E. (1986) Biochim. Biophys. Acta 852, 262-268) indicated that intramitochondrial calcium inhibits oxidative phosphorylation by decreasing the availability of adenine nucleotides to both the ADP/ATP translocase and the F0F1-ATP synthase complex. In this work we analyzed the interactions of calcium-nucleotide and magnesium-nucleotide complexes with the ATP synthase during catalysis of ATP in equilibrium with [32P]Pi exchange and net synthesis of ATP by submitochondrial particles. Concerning the ATP in equilibrium with [32P]Pi exchange reaction, calcium was ineffective as divalent cation when assayed alone. Furthermore, the addition of calcium increased the magnesium concentration required for half-maximal activation of the exchange, without changing Vmax. With respect to net ATP synthesis, the inhibition by calcium was shown to be due to formation of the CaADP- complex, which competes with MgADP- for the active site of the F0F1-ATP synthase. Moreover, ATP hydrolysis was competitively inhibited by CaATP2-, showing that calcium is able to interact with the enzyme in both forward and backward reactions in the same manner. That high calcium concentrations are required for significant inhibition of ATP synthesis indicates that this inhibition is relevant under conditions in which cytosolic calcium concentrations rise to pathological levels. Therefore, this mechanism may be responsible, in part, for the decrease in cellular ATP content that has been observed to occur when calcium accumulates in the cytosol.

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

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

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.

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

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

The endocannabinoid system in renal cells: regulation of Na(+) transport by CB1 receptors through distinct cell signalling pathways.

BACKGROUND AND PURPOSE: The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70-80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na(+) in LLC-PK1 cells. EXPERIMENTAL APPROACH: Changes in Na(+) /K(+) -ATPase activity were assessed after treatment with WIN55,212-2 (WIN), a non-selective lipid agonist, and haemopressin (HP), an inverse peptide agonist at CB1 receptors. Pharmacological tools were used to investigate the signalling pathways involved in the modulation of Na(+) transport. KEY RESULTS: In addition to CB1 and CB2 receptors and TRPV1 channels, the mRNAs encoding for enzymes of the ECS were also expressed in LLC-PK1. WIN (10(-7) M) and HP (10(-6) M) altered Na(+) re-absorption in LLC-PK1 in a dual manner. They both acutely (after 1 min) increased Na(+) /K(+) -ATPase activity in a TRPV1 antagonist-sensitive way. WIN's stimulating effect persisted for 30 min, and this effect was partially blocked by a CB1 antagonist or a PKC inhibitor. In contrast, HP inhibited Na(+) /K(+) -ATPase after 30 min incubation, and this effect was attenuated by a CB1 antagonist or a PKA inhibitor. CONCLUSION AND IMPLICATIONS: The ECS is expressed in LLC-PK1 cells. Both CB1 receptors and TRPV1 channels regulate Na(+) /K(+) -ATPase activity in these cells, and are modulated by lipid and peptide CB1 receptor ligands, which act via different signalling pathways.

Localization and cation dependence of a Ca2+- or Mg2+-ATPase from electrocytes of Electrophorus electricus, L.

Electrocyte membranes of Electrophorus electricus exhibit high ATPase activity, as demonstrated by cytochemical and biochemical techniques. This activity is visualized as electron-dense deposits in electron micrographs, and appears to be localized only at the innervated face of the electrocyte. ATP hydrolysis can be detected cytochemically or biochemically only in the presence of calcium or magnesium. The effects of Ca or Mg on ATPase activity can be described by Michaelis-like functions with similar apparent Km values for Ca and Mg (0.41 mM and 0.23 mM, respectively). Vmax, however, is fivefold higher in the presence of Mg. The effects of the two cations are not additive, and pH dependence of ATP hydrolysis is identical in the presence of Ca or Mg (maximal at pH 8-9). Therefore, it can be concluded that Ca and Mg activate the same enzyme, the differences in Vmax being attributable to influences in kcat.

Catalytic and structural modifications of sarcoplasmic reticulum and plasma membrane (Ca(2+) + Mg2+) ATPases induced by organic solutes that accumulate in living systems.

Organic solutes such as urea, methylamines, polyols and amino acid can accumulate in the cytoplasm of cells to compensate for hyperosmotic conditions in the external medium. Whereas urea is considered to be typical of solutes that destabilize structure and function of proteins, methylamines, polyols and some amino acids appear to have the opposite effect, and can also compensate for the perturbing effects of urea. These effects have been extensively analyzed for a variety of proteins in terms of global changes in enzyme structure and acceleration or inhibition of overall reaction rates. Here the influence of these solutes on sarcoplasmic reticulum and plasma membrane (Ca(2+) + Mg2+) ATPases is reviewed. The focus is on the changes induced by "perturbing" and "stabilizing" solutes at specific steps of the catalytic cycles of these enzymes, which can run forward (leading to ATP hydrolysis) and backward (leading to ATP synthesis). Structural changes promoted by osmolytes are correlated with functional changes, especially those that are related to energy coupling.

A novel phosphorylated lipid counteracts activation of the renal plasma membrane (Ca2+ + Mg2+)ATPase by endogenous phosphatidylinositol-4-phosphate.

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

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+).

The plasma membrane (Ca(2+) + Mg(2+))ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca(2+) ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca(2+) strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca(2+)-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca(2+) is related to opposite long-term hydration effects on the substrate binding domain and the Ca(2+) binding domain.

Patterns of drug consumption in relation with the pathologies of elderly Mexican subjects resident in nursing homes.

PURPOSE: To describe the patterns of drugs consumed by the male and female elderly living in Mexican private and public nursing homes. METHODS: Three hundred and fifty elderly participants from four nursing homes (2 private and 2 public) were selected for the six month study: 108 subjects were excluded; the remaining 242 were between 65 and 100 years old; 123 were females and 119 males. A complete clinical history was taken and clinical files were reviewed. RESULTS: Of the 242 elderly studied, 193 took diverse medications and 28.5% were at risk of some type of drug interaction. The groups of drugs more frequently consumed were vitamins and anti-anemic medications, followed by cardiovascular drugs. Females consumed greater number of drugs. They also consumed more drugs simultaneously. CONCLUSIONS: There is a need to monitor the elderly for their drugs pattern use.

Interactions of the regulatory ligands Mg2+ and MgATP2- with the renal plasma membrane Ca(2+)-ATPase: effects of osmolytes that stabilize or destabilize protein structure.

In this report we analyze the kinetics of activation of the plasma membrane Ca(2+)-ATPase from kidney proximal tubules by the regulatory ligands Mg2+ and MgATP2-, and we examine modifications in the effects of these ligands that are promoted by organic solutes of natural occurrence that stabilize or destabilize protein structure and function. The solutes tested were trimethylamine-N-oxide (TMA-O), sucrose and urea. TMA-O and sucrose were chosen as representative of the different methylamines and polyols, respectively, that accumulate in living organisms. The results lead to the conclusion that free Mg2+ and the MgATP2- complex both activate the rate-determining E2-->E1 transition during the catalytic cycle of the enzyme, by binding to nonidentical and independent regulatory sites. They also indicate that TMA-O, sucrose and urea not only promote global modifications in the enzyme structure, but also modify specific interactions of the ligands Mg2+ and MgATP2- at their regulatory sites.

Surface charges and interfaces: implications for mineral roles in prebiotic chemistry

There exists an extensive literature on the possible roles of minerals in the prebiotic stages of the chemical evolution of life (Bernal 1951, Cairns-Smith 1982, Wachtershauser 1992, Vieyra et al. 1995, Tessis et al. 1999, see Lahav (1994) for a review). Among the original proposals, minerals have been considered in: (a) processes that would discriminate molecular chirality; (b) condensation reactions of biomolecular precursors; (c) prebiotic catalysis; (d) biochemical templates; and (e) autocatalytic metabolism. In this communication it is emphazised the complex properties of both surface reactions and interfaces between minerals and aqueous solutions simulating Archean scenarios. The properties of pyrite surface net charge and of its interface with a solution simulating primitive seawater are discussed and their implications to the autocatalytic model (Wachtershauser 1988a 1992) are presented in order to demonstrate their relevance. The proposed roles of iron-sulfide minerals (mainly pyrite) as physical support for primitive bidimensional metabolism and chiral discriminator (Wachtershauser 1988a, Huber & Wachtershauser 1998) are revised. It is shown that: (a) the net surface charge can be modulated by the pyrite-aqueous solution interface; (b) mononucleotides attachment to pyrite require a cationic bridge; and (c) direct absorption of acetate - a molecule proposed as carbon source in primitive aqueous environments - also modulates the interface properties and would have masked pyrite's bulk structure. These results indicate that physicochemical changes of mineral surfaces - caused by environments simulating Archean aqueous scenarios - should be taken into account in the proposals of mineral prebiotic roles.

Glycerol inhibits or uncouples the plasma membrane (Ca(2+)+Mg2+)ATPase of kidney proximal tubules depending on the Ca2+ concentration.

In this report it is shown that glycerol (0.5-10% v/v) stimulate the C12-E9-solubilized renal (Ca(2+)+Mg2+)ATPase in the presence of low concentrations of free Ca2+ (< 10(-6) M). At 4% (v/v), the polyol decreases the K0.5 for Ca2+ from 1.15 to 0.22 microM at the high-affinity site, and a very-high-affinity Ca2+ component appears. This component has a K0.5 < or = 10(-9) M and its maximal velocity is about one-third that of the fully activated enzyme (at 10-20 microM Ca2+), which is not affected by glycerol (21.1 and 20.2 nmollmg-1.min-1 in the absence and presence of the polyol, respectively). The low-affinity, inhibitory component of the Ca2+ curve (50-1000 microM) is also unaffected by glycerol. With 0.07 microM free Ca2+ and soluble enzyme, the stimulatory effect of glycerol saturates at approximately 10% (v/v). In contrast, with 17 microM free Ca2+, glycerol has little effect up to 10% (v/v), and then progressively inhibits ATPase activity. These data indicate that the effect of the polyol is modulated by the occupancy of the high-affinity Ca2+ sites. In native vesicles, the stimulation promoted by low concentrations of glycerol at low concentrations of Ca2+ is accompanied by inhibition of active Ca2+ transport, indicating that, in these conditions, the polyol uncouples ATPase activity and ATP-driven Ca2+ influx.

Uncoupling by trehalose of Ca2+ transport and ATP hydrolysis by the plasma membrane (Ca2+ + Mg2+) ATPase of kidney tubules.

Trehalose, the disaccharide of glucose, inhibits both initial rate and maximal capacity of ATP-dependent Ca2+ transport in inside-out vesicles of basolateral membrane from kidney proximal tubules. This inhibition (I0.5 = 60 mM) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by trehalose. In the presence of 600 mM trehalose, Ca2+ uptake was almost completely inhibited, but the Ca(2+)-stimulated ATPase activity was unaffected; thus trehalose uncouples the Ca2+ transport from the ATPase activity. The Ca2+ transport inhibition by trehalose is reversible, since the inhibition disappeared when the vesicles were pre-incubated with 600 mM trehalose and then diluted in reaction medium to measure Ca2+ accumulation. Other mono- and disaccharides such as glucose, fructose, galactose, sucrose, maltose and lactose were tested but were not so effective as trehalose. The uncoupling of Ca2+ transport from hydrolysis can be explained by an interaction of trehalose with the phospholipid environment of the enzyme that induces conformational changes in specific domains of the enzyme so as to impair the coupling process.

Trypanosoma cruzi epimastigotes express the ouabain- and vanadate-sensitive (Na(+)+K+)ATPase activity.

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 (E1/E2) family of ion-transporting ATPases. The I50 for vanadate is 0.66 +/- 0.04 and 0.04 +/- 0.02 microM in CL14 clone and NIH NTY strain, respectively. These data indicate that both strains of T. cruzi epimastigotes express the ouabain- 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 isoforms of this enzyme.

Ouabain-insensitive Na(+)-ATPase activity in Trypanosoma cruzi epimastigotes.

In the present paper, the presence of a ouabain-insensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in T. cruzi epimastigotes CL14 clone and Y strain was investigated. The increase in Na+ concentration (from 5 to 170 mM), in the presence of 2 mM ouabain, increases the ATPase activity in a saturable manner along a rectangular hyperbola. The Vmax 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 mM 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 mM, 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.

Polyols that accumulate in renal tissue uncouple the plasma membrane calcium pump and counteract the inhibition by urea and guanidine hydrochloride.

Sorbitol and mannitol, two stereoisomeric osmolytes, inhibit the ATP-dependent Ca2+ transport in inside-out vesicles derived from basolateral membranes from kidney proximal tubules. This inhibition (I0.5 = 400 and 390 mM respectively) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by these osmolytes. In the presence of 1 M sorbitol or mannitol, Ca2+ uptake is inhibited by 70 and 75%, respectively. Since the Ca(2+)-stimulated ATPase activity is unaffected, sorbitol and mannitol uncouple the Ca2+ transport from the ATPase activity. The inhibition of Ca2+ transport by these osmolytes is reversible, since the inhibition disappears when the vesicles are preincubated with 1 M sorbitol or mannitol and then diluted 25-fold in reaction medium to measure Ca2+ accumulation. On the other hand, these osmolytes protect the (Ca2+ + Mg2+) ATPase from the inhibition of Ca2+ transport and ATPase activity by urea and guanidinium. These data suggest that the high concentrations of polyols that renal cells accumulate during antidiuresis, may regulate Ca2+ transport across the plasma membrane. In addition, polyols may protect the (Ca2+ + Mg2+) ATPase from the deleterious structural effects of urea, a compound that also accumulates during antidiuresis.

Single sublethal dose of microcystin-LR is responsible for different alterations in biochemical, histological and physiological renal parameters.

Microcystins (MCYSTs) are very stable cyclic peptidic toxins produced by cyanobacteria. Their effects on hepatic tissue have been studied extensively, and they are considered to be a potent hepatotoxin. However, several effects of MCYST on other organs have also been described, but generally in studies using higher doses of MCYST. In the present work, we investigated the effect of a single sublethal dose of MCYST-LR (55 µg/kg) in Wistar rats and analyzed different aspects that influenced renal physiology, including toxin accumulation, excretion, histological morphology, biochemical responses and oxidative damage in the kidney. After 24 h of exposure to MCYST-LR, it was possible to observe an increased glomerular filtration rate (6.28 ± 1.56 vs 2.16 ± 0.48 µl/min per cm(2)) compared with the control group. Increase of interstitial space and collagen deposition corresponded to a fibrotic response to the increased production of reactive oxygen species. The observed decrease of Na(+) reabsorption was due to inhibition of the activity of both Na(+) pumps in proximal tubules cells. We suggested that this modulation is mediated by the effect of MCYST as a phosphatase protein inhibitor that maintains the sustained kinase-mediated regulatory phosphorylation of the ATPases. The observed alteration of Na(+) active transporters lead to damage of renal function, since are involved in regulation of water and solute reabsorption in proximal tubules. The results of this report reinforce the importance of understanding the molecular effects of a single sublethal dose of MCYST-LR, which, in this study, was responsible for macro-alterations found in the renal parenchyma and renal physiology in rats.