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PubMed1998

Carbonyl reduction of timiperone in human liver cytosol.

This in vitro study using human liver enzymes was undertaken in order to compare the mechanism of metabolic reduction of timiperone, a potent butyrophenone neuroleptic, with that of haloperidol. Conversion of timiperone to reduced timiperone in liver cytosol was confirmed. The carbonyl reductase inhibitors (menadione IC50 5-18 microM; ethacrynic acid IC50 26-51 microM) potently inhibited both timiperone reductase and haloperidol reductase activity, while 4-methylpyrazole (alcohol dehydrogenase inhibitor) had no effect and phenobarbital (aldehyde reductase inhibitor) had a weak inhibitory effect. The formation of reduced timiperone was highly correlated with the formation of reduced haloperidol(r = 0.87, n = 6, P < 0.02). Timiperone reductase activity was approximately 40% lower than haloperidol reductase activity (at a substrate concentration of 100 microM, two-tailed t-test, P < 0.05). The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of reduced timiperone formation were much lower than reduced haloperidol formation (K(m) values: 29.7 +/- 15.1 versus 381.3 +/- 1.1 microM, n = 3, P < 0.01; Vmax: 0.81 +/- 0.19 versus 6.00 +/- 1.47 nmol/mg/min; n = 3, P < 0.05). However, the ratio Vmax/K(m) (clearance) for timiperone was 1.3-2.4 times higher than for haloperidol, indicating that metabolic clearance of timiperone by carbonyl reductase may be similar to or slightly higher than for haloperidol.

Alcohol Oxidoreductases↗
PubMed1992

Expression of human mu or alpha class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents.

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.

Antineoplastic Agents↗
PubMed1995

Differences in hepatic drug metabolizing enzymes and their response to lindane in rat, rabbit and monkey.

Untreated rabbit liver microsomes demonstrated the highest content of cytochrome P450 and activity of NADPH cytochrome c reductase compared to rat and monkey. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of microsomes from untreated rabbit demonstrated a greater quantity of 50 KDa polypeptide than in rat and monkey. The activity of glutathione-S-transferase towards 1-chloro-2,4-dinitrobenzene and the band intensity of 26 KDa polypeptide was found to be at maximum in untreated rabbits, while rat liver demonstrated the highest activity of glutathione-S-transferase towards ethacrynic acid. The extent of hepatic microsomal lipid peroxidation was at maximum in untreated rats. The activity of catalase was higher in untreated monkeys compared to untreated rats and rabbits. Lindane at a dose of 10 mg kg-1 body weight for a period of six days increased the hepatic content of cytochrome P450 and the activities of NADPH cytochrome c reductase, aminopyrine N-demethylase, glutathione-S-transferases, haem oxygenase and lipid peroxidation, decreased non-protein thiols and concomitantly intensified the 50 and 26 KDa polypeptides in the microsomes and 100,000 x g supernatants respectively, in the rat but not in the rabbit or monkey. The results demonstrate that lindane is a bifunctional inducer in the rat and non-functional in rabbit and monkey. It also increased the activities of hepatic drug metabolizing enzymes with concomitant production of oxidative stress in the rat, whereas in rabbit and monkey it did not alter the drug metabolizing enzymes nor produced any oxidative stress.

Aminopyrine N-Demethylase↗
PubMed1987

Mechanism of osmotic diuresis studied by infusion of NaHCO3 and mannitol in dogs.

To examine whether mannitol and NaHCO3 are equally potent inhibitors of proximal tubular fluid reabsorption, experiments were performed in 10 anaesthetized volume-expanded dogs during continuous infusion of ethacrynic acid. At plasma pH 7.5, a rise in plasma osmolality of 40 mosmol kg-1 reduced the remaining tubular fluid reabsorption in five dogs by 14 +/- 3% during NaHCO3 infusion and by 28 +/- 1% during mannitol infusion. Bicarbonate reabsorption increased by 25 +/- 5% during NaHCO3 infusion and decreased by 14 +/- 1% during mannitol infusion. At equal rates of bicarbonate reabsorption the inhibitory effects on tubular fluid and NaCl reabsorption were slightly less during mannitol than during NaHCO3 infusion. In five other dogs studied at constant plasma concentration of sodium, changes in bicarbonate reabsorption were avoided by raising plasma pH to 7.7 during NaHCO3 infusion and by reducing plasma pH to 7.4 during mannitol infusion. Tubular fluid reabsorption was reduced 32 +/- 4% by NaHCO3 and 34 +/- 4% by mannitol infusion, indicating equal inhibitory effects. The mechanism may be that the osmotic force for paracellular reabsorption of water and NaCl across the tight junction is equally reduced by equiosmolal increments in the NaHCO3 and mannitol concentration of the proximal tubular fluid.

Animals↗
PubMed2006

Supporting cell proliferation after hair cell injury in mature guinea pig cochlea in vivo.

In cold-blooded animals, lost sensory hair cells can be replaced via a process of regenerative cell proliferation of epithelial supporting cells. In contrast, in mammalian cochlea, receptor (hair) cells are believed to be produced only during embryogenesis; after maturity, sensory or supporting cell proliferation or regeneration are thought to occur neither under normal conditions nor after trauma. Using bromodeoxyuridine (BrdU) as a proliferation marker, we have assessed cell proliferation activity in the mature organ of Corti in the cochlea of young guinea pigs following severe damage to the outer hair cells induced by kanamycin sulfate and ethacrynic acid. Although limited, we have found BrdU-labeled nuclei in the regions of Deiters cells when BrdU is given for 3 days or longer. When BrdU is given for 10 days, at least one labeled nucleus can be observed in the organ of Corti in approximately half of the ears; proliferating cells typically appear as paired daughters, with one nucleus being displaced away from the basement membrane to the position expected of the hair cells. Double-staining with antibodies to cytokeratin, vimentin, and p27 have shown that the BrdU-labeled nuclei are located in cells phenotypically similar to Deiters cells. Most of the uptake of BrdU occurs 3-5 days following ototoxic insult, and the number of BrdU-labeled cells does not decrease until 30 days following insult. These findings indicate that Deiters cells in the mature mammalian cochlea maintain a limited competence to re-enter the cell cycle and proliferate after hair cell injury, and that they can survive at least for 1 month.

Animals↗
PubMed2001

Astrocytes as a predominant cellular site of (99m)Tc-HMPAO retention.

Technetium-99m-d,l-hexamethylpropylene amine oxime ((99m)Tc-HMPAO) retention in the brain monitored by single photon emission computed tomography (SPECT) is currently used as a marker of cerebral blood flow. The purported mechanism by which (99m)Tc-HMPAO accumulates in the brain is through its intracellular conversion from a lipophilic form to more hydrophilic derivatives within the brain parenchyma. The issue of the contribution of different cell types on (99m)Tc-HMPAO retention was investigated in vitro by studying the accumulation of (99m)Tc-HMPAO in primary cultures of mouse cortical astrocytes and neurons. Results show that (99m)Tc-HMPAO retention predominates in astrocytes over neurons by a factor of approximately 2.5 (0.26 +/- 0.05 vs. 0.095 +/- 0.042 fmol/mg protein after 120 minutes, respectively). Diethyl maleate (60 micromol/L), ethacrynic acid (1 mmol/L) and buthionine sulfoximine (1 mmol/L), 3 agents which significantly reduced glutathione levels also decreased (99m)Tc-HMPAO retention in both astrocytes (29%, 3%, and 46% of control, respectively) and neurons (69%, 11% and 63% of control). Decrease did not always correlate with glutathione levels, however, which suggests that other factors could be involved. The possibility that cell energy status determines (99m)Tc-HMPAO retention was also assessed. Agents that activate (glutamate, azide) or inhibit (cytochalasin B) glucose utilization in astrocytes, as measured by the (3)H-2-deoxyglucose method, were without effect on (99m)Tc-HMPAO retention. In conclusion, the data presented indicate that astrocytes may constitute a prominent site of (99m)Tc-HMPAO retention and most likely contribute significantly to the SPECT signal. In addition, the data also suggest that specific alterations in glial cell metabolism could explain flow-independent changes in (99m)Tc-HMPAO retention in the brain as observed by SPECT in some pathologies.

Animals↗
PubMed2001

Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells.

Multidrug resistance is a major obstacle for the successful use of chemotherapy. The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump. We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine. The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay. We examined the ability of hyperthermia (42 degrees C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products. Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid. CH(R)C5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells. Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity. Hyperthermia (42 degrees C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CH(R)C5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CH(R)C5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time). Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CH(R)C5 and AuxB1 cells. Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1985

Drug effects on the hydraulic conductivity of the isolated rabbit ciliary epithelium.

Three aspects of hydrostatically driven volume flow across the isolated rabbit ciliary epithelium have been examined. (1) Hydraulic conductivity was increased in a dose-dependent manner by amiloride, but not by furosemide, indicating that amiloride may interfere with junctional permeability. The absence of a furosemide effect suggests that regulation of passive fluid movement may be independent of net ionic fluxes of either chloride or bicarbonate. Ethacrynic acid had no effect on hydraulic conductivity at concentrations below 10(-2) M. At 10(-2) M, hydraulic conductivity was decreased, suggesting that fluid pathways may be linked to ionic pathways. (2) Successive in vivo daily treatments with adrenergic agonists result in changes in the intraocular pressure response. Dose-effect curves to in vitro agonists from eyes pre-treated on successive days indicate that isoprenaline caused a lack of response in hydraulic conductivity after 3 d of in vivo treatment, adrenaline caused responses after 2 d which were about half of those found on day 0, while phenylephrine and noradrenaline had no influence on hydraulic conductivity. These data suggest that beta-agonists cause a deactivation or desensitization of some moiety which is coupled with regulation of hydraulic conductivity in a manner similar to that seen for the beta-receptor-coupled adenylate cyclase. (3) While cyclic GMP was without effect, various modifiers of cyclic GMP activity increased hydraulic conductivity. Alteration of cellular cyclic GMP levels appears to exert some regulation over ciliary epithelial permeability.

Amiloride↗
PubMed1982

Sites of action of loop diuretics.

Isolated canine kidneys were perfused at a constant systolic pressure of 140 mm Hg with autologous blood. Infusion of maximally effective amounts of furosemide (F) increased the fractional excretion of Cl (FEcl) from 0.03 to 0.18. Parallel changes in FENa and FEH2O were observed and, at maximal response, urine/plasma osmolality was 0.91. Ethacrynic acid (EA) was then added to the infusion fluid and produced a further increase in FECl to 0.28. Urine/plasma osmolality returned to unity. When the order of administration of the two diuretics was reversed, the response to EA alone was greater than after F (from FECl 0.05 control to 0.30 after EA) and the urine became isosmotic (urine/plasma osmolality = 1.0). After F was added, FECl increased further to 0.39. K secretion induced by F or by EA was demonstrable in all experiments. These data indicate that: 1) EA is more effective than F; 2) both drugs can completely inhibit Cl- transport in the thick ascending limb; 3) EA is more effective than F in blocking NaCl transport at sites distal to the loop of Henle; and 4) the proximal tubule is a site of action of F and possibly of EA. Whether the two drugs act entirely on membranal transport systems or, in part, by modifying the production of release of intrarenal substances and/or by diverting blood from one region to another cannot be ascertained from this work.

Animals↗
PubMed1996

Direct detection of ototoxicant-induced reactive oxygen species generation in cochlear explants.

The proposal that free-radical generation contributes to the ototoxicities of several chemical agents was studied utilizing electron paramagnetic resonance (EPR) spectrometry to detect directly ototoxicant-induced reactive oxygen species formation in cochlear tissue. Guinea pig cochlear explants in chelexed artificial perilymph (AP: 200 microliters) were exposed to an ototoxicant or AP for 10 min. Ototoxic agents included gentamicin sulfate (4.0 mM), kanamycin monosulfate (4.0 mM), ethacrynic acid (0.5 mM), furosemide (0.3 mM), cisplatin (0.1 mM), trimethyltin chloride (0.1 mM), and quinine HCl (3.0 mM). Following incubation, 20 microliters of AP/ototoxicant mixture was replaced by the filtered spin trap, 5,5-dimethylpyrroline-N-oxide (DMPO). After 10 min, the EPR spectrum of the mixture was obtained. Four line EPR spectra of relative intensities 1:2:2:1, associated with hydroxyl radical (OH)/DMPO adduct formation, were evidenced by reaction mixtures containing cochlear explants exposed to each ototoxicant. Cisplatin, quinine and the loop diuretics produced weak OH-associated EPR signals in the absence of a cochlear explant, which were amplified in its presence. Deferoxamine quenched all OH spectral peaks. Peroxide levels, assayed in parallel experiments, were diminished by each ototoxicant relative to those seen following AP exposure, suggesting possible H2O2 conversion to OH. These data support the proposal that various ototoxic agents are capable of reactive oxygen species generation or promotion in cochlear tissues.

Analgesics, Non-Narcotic↗
PubMed1987

Vitamin E protection against chemical-induced cell injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism.

Vitamin E protection against chemical-induced toxicity to isolated hepatocytes was examined during an imbalance in the thiol redox system. Intracellular reduced glutathione (GSH) was depleted by two chemicals of distinct mechanisms of action: adriamycin, a cancer chemotherapeutic agent that undergoes redox cycling, producing reactive oxygen species that consume GSH, and ethacrynic acid, a direct depleter of GSH. The experimental system used both nonstressed vitamin E-adequate isolated rat hepatocytes and compromised hepatocytes subjected to physiologically induced stress, generated by incubation in calcium-free medium. At doses whereby intracellular GSH was near total depletion, cell injury induced by either chemical was found to follow the depletion of cellular alpha-tocopherol, regardless of the status of the GSH redox system. Changes in protein thiol contents of the cells closely paralleled the changes in alpha-tocopherol contents throughout the incubation period. Supplementation of the calcium-depleted hepatocytes with alpha-tocopheryl succinate (25 microM) markedly elevated their alpha-tocopherol content and prevented the toxicities of both drugs. The prevention of cell injury and the elevation in alpha-tocopherol contents were both associated with a prevention of the loss in cellular protein thiols in the near total absence of intracellular GSH. The mechanism of protection by vitamin E against chemical-induced toxicity to hepatocytes may therefore be an alpha-tocopherol-dependent maintenance of cellular protein thiols.

Animals↗
PubMed1983

The use of diuretics in varying degrees of renal impairment: an overview.

Administration of diuretics during acute renal failure in animals has been demonstrated to be of value with mannitol and/or loop-blocking diuretics, furosemide or ethacrynic acid. There is evidence that if these drugs are given very early in the controlled experimental environment that there will be some beneficial effect in maintaining renal function. However, in man the temporal relationship between the acute onset and the successful response to the administration of the drugs is, at best, coincidental and the use of diuretics in acute renal failure may not produce the same results as seen in the laboratory. One of the best guides to the underlying disease when there is acute decompensation in renal function is the utility of the renal failure index which utilizes urine and plasma sodium and urine and plasma creatinine ratios. Large doses of loop-blocking diuretics can be of benefit in patients with mild to moderate chronic renal insufficiency and fluid retention and/or hypertension. When renal insufficiency is severe in the pre-dialysis setting, furosemide, bumetanide or muzolimine may be of some benefit; however, as renal failure worsens the response of the kidney is sluggish and it is wise to begin to dialyze when glomerular filtration deteriorates below 5 ml per minute.

Acute Kidney Injury↗
PubMed2004

Biochemical characterization and distribution of glutathione S-transferases in leaping mullet (Liza saliens).

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.

Animals↗
PubMed1975

Effect of sodium nitrate loading on electrolyte transport by the renal tubule.

Effects of sodium nitrate were compared with sodium chloride loading on transport of electrolytes by the nephron. Maximal levels of free water clearance/clomerular filtration rate (CH2O/GFR) averaged 8.4% with nitrate loading and 14.4% with saline loading. Since ethacrynic acid and chlorothiazide exert their major natriuretic effect in the distal nephron, the increment in Na ad Cl reabsorbed beyond the proximal tubule. The administration of these agents resulted in an increase in fractional sodium excretion (CNa/GFR) of 21.1%, urinary sodium excretion (UNaV) of 1,126 mueq/min, and urinary chloride excretion (UClV) of 848 mueq/min during nitrate loading compared with an increase in CNa/GFR of 37.6%, UNaV of 2,362 mueq/min, and UClV of 2,397 mueq/min during saline loading. The smaller diuretic-induced increment in Na and Cl excretion in the nitrate studies suggests, as do the hydrated studies, that less Cl and Na are reabsorbed in the distal nephron during nitrate than saline loading. At every level of UNaV, fractional bicarbonate reabsorption was higher, urine pH was lower, and urinary potassium excretion (UKV) was higher in the nitrate studies. Thus, compared with saline loading, sodium nitrate decreases chloride and sodium reabsorption in the distal nephron. The higher hydrogen and potassium secretion in the nitrate studies may be consequent to the decreased ability of the distal nephron to reabsorb chloride.

Animals↗
PubMed1981

The impermeability of the basic cell membrane to thromboxane-B2' prostacyclin and 6-keto-PGF 1 alpha.

Washed rabbit red blood cells (RBCs) were suspended in electrolyte solution containing 3H-labeled prostacyclin (PGI2), thromboxane (TxB2) or 6-keto-PGF 1 alpha and 14C-labeled sucrose or thiourea. following 1 to 30 min incubation with 14C-sucrose, 3H-TxB2 or 3H-6-keto-PGF 1 alpha, the 14C or 3H space of packed RBCs remained essentially constant, yielding mean values (+/- S.E.) for all time periods of 6.1 +/- 0.3, 9.5 +/- 0.5 and 6.5 +/- 0.4%, respectively. After 1 min of incubation at 4 degrees or 23 degrees C at a pH of 7.4 or 8.5 with trace amounts (10(-9)M) of 3H-PGI2 or in the presence of added PGI2 (10(-5)M) or ethacrynic acid (1.6 x 10(-4)M), the apparent PGI2 space of packed RBCs ranged from 16 to 27%, decreasing to about 7% by 30 min. When RBCs were resuspended in fresh 3H-PGI2 every 5 min, their 3H content increased very slowly (apparent PGI2 space less than 40% at 30 min) as compared to thiourea (distribution space greater than 80% within 5 min). Over 90% of this 3H activity was lost from the RBCs in less than 2 min during elution at 4 degrees or 23 degrees C. It is concluded that RBC membranes and thus, presumably, the basic cell membrane in general, is not fundamentally permeable to PGI2, 6-keto-PGF 1 alpha or TxB2. Hence, the effective entry of these cyclooxygenase products into some cells or their passage across tight-junctional capillaries or epithelial membranes must require facilitated or active transport processes as was shown to be the case for E, F and A PGs. This implies that the distribution, pharmacological action and metabolism of these and presumably all related cyclooxygenase products are selective rather than unrestricted.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Central effect of agents which alter sodium transport on water drinking in the rat.

The central effect of ouabain (OUA), ethacrynic acid (EA) and diphenylhydantoin (DPH) on water drinking was studied in rats. OUA and EA inhibit cellular Na efflux, increasing intracellular (IC) Na content, while DPH inhibits cellular Na influx lowering IC sodium content. The animals were injected into the third ventricle (3V), fourth ventricle (4V), lateral ventricle (LV), or lateral hypothalamus (LH). Ouabain (50 pg) and EA (50 ng) when injected into the 3V or LV significantly decreased the water intake induced by 24 hr water deprivation. No effect was observed after injections in other loci, thus supporting the hypothesis that sensors could be located in the circumventricular organs, in an area without the blood brain barrier. DPH (270 ng) injected into the 3V or LV significantly enhanced water intake in rats deprived of water for 14 hr, but it did not increase water intake in water-repleted rats. DPH also enhanced water intake induced by cellular-dehydration (2 M NaCl IP). These results suggest that changes in the Na content of sensor cells located on or near the walls of the 3V could be responsible for the movement of water between the cells and their medium, in agreement with the osmoreceptor hypothesis for thirst.

Animals↗
PubMed1967

Uphill transport of urea in the dog kidney: effects of certain inhibitors.

To study the renal medullary transport and accumulation of urea in dogs independent of water transport, we obliterated the medullary electrolyte gradient by a sustained ethacrynic acid diuresis. Infusions of urea were also given at various rates to vary urinary urea concentration. In the steady state, the kidneys were removed, and slices were analyzed for water, urea, and electrolytes. In every experiment in 15 dogs over a range of urinary urea concentration from 19 to 230 mmoles per L and urine flow from 0.5 to 9.7 ml per minute per kidney, an intrarenal urea gradient persisted, and urinary urea concentration was always lower than papillary water urea concentration. The magnitude of this uphill urinary-papillary gradient (mean +/- SE = - 21 +/- 2.9 mmoles per L) was not affected by hemorrhagic hypotension or a nonprotein diet. In 12 additional experiments begun similarly, inhibitors were infused into one renal artery. Both iodoacetate, an inhibitor of anaerobic glycolysis, and acetamide, an analogue of urea, markedly and significantly reduced both the intrarenal urea gradient and the uphill urinary-papillary gradient. In contrast, cyanide, an inhibitor of oxidative metabolism, had no observable effect on the urea gradients. The data are best explained by postulating an active transport system for urea in the medullary collecting duct deriving its energy from anaerobic glycolysis.

Amides↗
PubMed1977

Effects of acetazolamide on proximal tubule C1, Na, and HCO3 transport in normal and acidotic dogs during distal blockade.

It has been suggested that the establishment of a tubular fluid to plasma chloride gradient in the late proximal tubule by the reabsorption of bicarbonate (and other anions) in the early proximal tubule is responsible for a significant part of sodium chloride and water reabsorption in the proximal tubule. In the present study the effects of acetazolamide on proximal tubule water and electrolyte excretion were examined in 6 normal dogs and 10 chronic ammonium chloride-loaded dogs during distal blockade produced by ethacrynic acid and chlorothiazide administration. During distal blockade control urine/plasma osmolality and urine/plasma sodium were close to unity in all experiments. Urine/plasma chloride and urine/plasma bicarbonate were 1.21+/-0.02 and 0.75+/-0.07 in normal and 1.24+/-0.01 and 0.04+/-0.01 in acidotic dogs, respectively. After the administration of acetazolamide (20 mg/kg i.v.), there was a significant increase in urine flow, absolute and fractional excretion of sodium, bicarbonate, and chloride in all animals. Associated with these effects, urine/plasma osmolality and urine/plasma sodium remained unchanged but urine/plasma chloride decreased significantly to 1.15+/-0.01 in normal and to 1.19+/-0.01 in acidotic dogs. In acidotic dogs there was a significant correlation between the increase in bicarbonate, sodium, or chloride excretion after acetazolamide and the plasma bicarbonate level (range 6.8-12.5 meq/liter). These data demonstrate a significant effect of acetazolamide on bicarbonate, sodium, and chloride reabsorption in the proximal tubule even in the face of severe acidosis. Moreover, the data suggest that the decrease in chloride reabsorption (and accompanying sodium) after acetazolamide is related to the decrease in bicarbonate reabsorption and the associated decrease in the transtubular chloride gradient.

Acetazolamide↗