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Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K (i) of uranyl was 139+/-30 microg uranyl/mg protein of BBMV. Uranyl at 140 microg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V (max) 2.2+/-0.2 and 0.96+/-0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K (m) (1.54+/-0.33 and 1.54+/-0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V(max) is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V (max) was reduced from 247+/-13 pmol/mg protein in control BBMV to 119+/-3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

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In a previous work, we showed that picomolar concentrations of prostaglandin E2 (PGE2) inhibit Na-K-ATPase activity and ouabain binding in a clone of Madin-Darby canine kidney (MDCK) cells. In the present study, we demonstrate that the inhibitory effects of PGE2 on Na-K-ATPase activity, ouabain-sensitive Rb+ uptake, and ouabain binding in MDCK cells were diminished by treatment of the cells with nonsteroidal anti-inflammatory drugs. These results suggested that products of arachidonic acid synthesized through the cyclooxygenase pathway are involved in the inhibitory mechanism of PGE2. Treatment of the cells with arachidonic acid resulted in inhibition of ouabain binding, and the inhibition was eliminated by cyclooxygenase inhibitors. These observations further support the involvement of cyclooxygenase products in the PGE2-induced inhibitory process. Finally, we demonstrated that dopamine inhibits Rb+ influx and ouabain binding in MDCK cells similarly to PGE2. Cyclooxygenase inhibitors suppressed the inhibition of ouabain binding by dopamine, thus also suggesting the involvement of cyclooxygenase products in the inhibitory effect of dopamine.

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To better understand the link between lysosomal cystine accumulation and the renal impairment seen in cystinosis, we have studied the effect of cystine loading in vivo, on renal function of rats, and in brush-border membrane vesicles (BBMV) prepared from the kidney cortex of the treated rats. Intraperitoneal injection of cystine dimethyl ester (CDME) (400 mumol, twice a day, for 5 days) led to an increased urine volume and excretion of glucose, phosphate, and protein. Kinetic analysis of alpha-methylglucoside initial flux in BBMV showed reduction in maximal transport capacity (Vmax, from 10.1 +/- 1.3 to 8.5 +/- 0.7 nmol.min-1.mg protein-1; P < 0.01) with no change in Michaelis constant (Km, 4.80 +/- 0.08 and 4.90 +/- 0.05 mM). The number of phlorizin binding sites declined (from 6.5 +/- 0.7 to 4.1 +/- 0.4 pmol/mg protein; P < 0.01) with no significant change in the affinity for phlorizin (0.64 +/- 0.08 and 0.59 +/- 0.06 microM). In the cortex homogenate, cystine concentration, which was undetectable in controls, increased to 0.97 +/- 0.09 nmol 1/2 cystine/mg protein. Two hours after CDME administration, ATP content declined to approximately 50% of control values. This decline was transient, and ATP content was recovered to control values 5 h after CDME administration. The treatment did not affect ouabain-sensitive adenosinetriphosphatase activity (40.0 +/- 3.9 and 38.6 +/- 4.7 nmol Pi.mg protein-1.min-1) or the number and affinity of ouabain binding sites (Bmax = 1.48 +/- 0.25 and 1.44 +/- 0.18 pmol/mg, and Kd = 0.68 +/- 0.09 and 0.72 +/- 0.12 microM, respectively). (ABSTRACT TRUNCATED AT 250 WORDS)

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In the present study we report on a direct effect of prostaglandin E2 (PGE2) on ouabain binding and Na-K-adenosinetriphosphatase (Na-K-ATPase) activity in a clone of Madin-Darby canine kidney cells, a renal cell line with collecting duct properties. Incubation of the cells with low concentrations (pM) of PGE2 produced a concomitant reduction of approximately 50% in the activity of Na-K-ATPase in the cell homogenate and in ouabain binding to the intact cells (half-maximal inhibition of approximately 0.1 pM). The inhibition was apparent within 10 min of preincubation of the cells with PGE2. Scatchard analysis of the binding demonstrated that the treatment with PGE2 reduced the number of ouabain binding sites without a change in the dissociation constant. PGE1 and PGF2 alpha (10 nM) did not affect ouabain binding or Na-K-ATPase activity. The fast, potent, and specific effect of PGE2 suggests that the diuretic/natriuretic effect of prostaglandins of the E series in the collecting tubule, in addition to the interference with the activity of arginine vasopressin, may result from a direct reduction in the number of the Na-K-ATPase active units, via a prostaglandin receptor.

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Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)

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Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated from epididymal rat adipocytes revealed inhibition of cAMP production at low concentrations of PGE2 (less than 10 mM) and stimulation at higher concentrations. This biphasic effect of PGE2 was obtained when adenylyl cyclase was stimulated with GTP or NaF. In the presence of forskolin only the inhibitory phase by PGE2 was observed. Sulprostone, a PGE2 analogue, did not affect cAMP synthesis in the presence of either GTP or NaF; however, in the presence of forskolin, it inhibited cAMP production similarly to PGE2. Treatment of the membranes with cholera or pertussis toxin did not alter the biphasic effect of PGE2 on cAMP production. These findings raise the possibility that PGE2 acts through several receptor subtypes which are coupled to GTP binding proteins different from the classical Gi or Gs proteins.

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Albumin enhances prostaglandin E2 (PGE2) binding to isolated epididymal adipocyte membrane and also binds PGE2 with low affinity. On the other hand, S-100, ovalbumin and albumin-stearate failed to bind PGE2, as shown by ultrafiltration, and also failed to enhance PGE2 binding to the isolated adipocyte membranes. These results suggested that albumin enhances PGE2 binding possibly by serving as a carrier for the prostaglandin molecules. 3 mM warfarin or 1 mM phenylbutazone inhibited PGE2 binding to albumin by 70% and 95%, respectively, but both drugs failed to affect the enhancement of PGE2 binding to the isolated adipocyte membrane in the presence of albumin. These results exclude the possibility that PGE2 bound to albumin is more accessible to the prostaglandin receptor than free PGE2 in solution. Finally it is shown that fatty acid binding protein (FABP), a cytosolic protein which binds specifically PGE1 but not PGE2, enhances PGE1 and PGE2 binding to isolated adipocyte membranes similarly to albumin. The physiological implications of these findings are discussed.

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Both NaCl and NaF promoted PGE2 binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the 'salt effect' of sodium on PGE2 binding, the effects of Mg2+ and guanyl nucleotides on PGE2 binding in the presence of NaCl or NaF were compared. Mg2+ decreased PGE2 binding; high NaF concentration abolished this inhibition, while increased NaCl concentrations did not affect the Mg2+ inhibition. In the presence of Mg2+ the effects of NaCl and NaF were additive. The enhancement of PGE2 binding by fluoride, unlike sodium, was dependent on the presence of Mg2+. Incubation of the membranes with GDP beta S, Gpp(NH)p, GTP or GTP gamma S increased PGE2 binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE2 binding at low NaF concentrations and inhibition of PGE2 binding at high NaF concentrations. No changes in the stimulatory action of NaCl on PGE2 binding were observed in the simultaneous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE2 binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE2 binding. The implications of these findings are discussed.

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Detailed studies of PGE2 binding to isolated rat adipocyte membranes revealed two different classes of binding sites, namely high affinity-low capacity binding sites and low affinity-high capacity binding sites. Addition of albumin or GTP to the incubation medium enhanced the specific binding of PGE2 by decreasing the dissociation constant of the low affinity-high capacity binding sites. Albumin also increased the affinity of PGE2 binding to native canine renal medullary membranes and enhanced the binding of PGE2 to prostaglandin receptors solubilized from these membranes. Pretreatment of the adipocyte membranes with the alkylating agent NEM completely abolished the enhancement of PGE2 binding by GTP, while the enhancement of PGE2 binding by albumin was only partially inhibited. The enhancement of PGE2 binding by GTP was shown to be dependent on the presence of Mg+2, while the albumin effect was independent of Mg+2. These results suggest that the affinity of the prostaglandin receptors is modulated by more than one mechanism.

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