Phosphate transport by capillaries of the blood-brain barrier.

Capillaries were isolated from bovine brain cortex and used for phosphate transport studies. The influx of phosphate through capillary membranes was studied by incubation with [32Pi]phosphate followed by a rapid filtration technique. Phosphate uptake by brain capillaries was mediated by a saturable high-affinity system which is independent of the sodium concentration in the incubation medium. The apparent half-saturation constant (Km) and maximal influx (Vmax) were estimated to 160 microM and 0.37 nmol/mg protein/30 s. Transport was inhibited by the phosphate analogues arsenate and phosphonoformic acid with apparent inhibition constants of 5 and 11 mM, respectively. The metabolic inhibitors cyanide and ouabain had no effect on the transport activity. Competition experiments showed that phosphate uptake was inhibited up to 41% by various anions (pyruvate, acetate, citrate, glutamate, and sulfate). In addition, phosphate uptake was significantly decreased by two selective inhibitors of anionic exchangers, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Chloride was not a substrate of the phosphate carrier as the replacement of external chloride, by nitrate, thiocyanate, or gluconate, did not increase phosphate transport. Aminohippuric acid and N'-methylnicotinamide, two specific substrates of anionic and cationic drug exchangers, did not compete with the phosphate carrier of cerebral capillaries. However, trans-stimulation with bicarbonate increased phosphate transport by 28%, and this stimulation was inhibited by 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that the carrier of the cerebral capillaries could exchange phosphate with bicarbonate.

Effects of p-aminohippurate and pyrazinoate on urate excretion in Cebus monkeys.

Changes in the fractional excretion of urate (FEurate) induced by various concentrations of p-aminohippurate (PAH) and pyrazinoate in plasma were evaluated in anesthetized Cebus monkeys using the standard clearance technique. PAH at concentrations between 2 and 40 micrograms/ml produced modest uricosuria (delta FEurate, ca 50%). At higher concentrations of PAH (up to 1200 micrograms/ml) the values of FEurate were not different from controls. Pyrazinoate at concentrations between 1 and 700 micrograms/ml reduced FEurate. The depressant effect of pyrazinoate was not influenced by the presence of PAH. These results are consistent with the idea that the secretory component for urate is distinct from that for PAH in this monkey.

Transport of methotrexate in cortical slices of monkey kidney: effect of organic anions and vincristine.

Thin cortical slices of cynamolgus and rhesus monkey kidney were used to study the renal transport of methotrexate (MTX). In experiments with renal cortical slices, MTX uptake at 25 degrees C was linear over the initial 30 min and was temperature-dependent. The Km was 0.094 mM for MTX uptake at 25 degrees C and Vmax was 0.098 mumol/g of tissue/30 min. In the presence of either 1 mM 2,4-dinitrophenol, p-aminohippurate or acetylsalicylate, MTX uptake was competitively inhibited. 2,4-Dinitrophenol had the greatest and acetylsalicylate had the least inhibitory effect. Folinic acid, folic acid and ouabain produced little or no effect on MTX uptake. MTX efflux from preloaded slices (preincubated with 0.5 mM MTX for 45 min) was a first-order process with T 1/2 of 7.13 +/- 0.86 min. In the presence of vincristine or p-aminohippurate the half-lives for MTX were 15.25 +/- 0.91 and 4.59 +/- 0.47 min, respectively. Thus vincristine, an organic base, was found to augment MTX uptake, due to a reduction in the rate of efflux of MTX from the cortical tissues, whereas p-aminohippurate, an organic acid, was found to decrease MTX intracellular concentration by blocking influx and stimulating efflux. It was concluded that the renal transport of MTX in monkey kidney is mediated predominantly by an organic anion secretory process and that there is probably little or no reabsorptive transport.

Effect of anion exchange inhibitors and para-aminohippurate on the transport of urate in the rat proximal tubule.

The present studies were designed to examine the effect of some anion exchange inhibitors and para-aminohippurate (PAH) on urate transport in the proximal tubule of the rat utilizing microperfusion techniques. The addition of SITS, DIDS, or furosemide to the luminal perfusion solution resulted in a decreased rate of absorption of water and 2-14C-urate. In addition, the presence of PAH in the luminal microperfusion solution resulted in a lower rate of urate absorption. The absorptive flux of urate was significantly higher, however, when PAH was added to the solution microperfusing the capillaries. The capillary to lumen secretory flux of urate was significantly higher when PAH or unlabeled urate was added to the luminal perfusion solution and significantly lower when PAH was added to the capillary perfusion solution. The addition of SITS to either the capillary or luminal microperfusion solution resulted in lower secretory and absorptive fluxes of urate. These studies suggest that both the secretion and reabsorption of urate in the proximal convoluted tubule of the rat is influenced by some anion exchange inhibitors and PAH. The results are considered in conjunction with recent in vitro data suggesting that urate transport is mediated by a process of anion exchange.

Effects of pyrazinoate and p-aminohippurate on renal urate excretion by the dog and guinea pig.

In anesthetized dogs, pyrazinoate reduced the fractional excretion of urate when the concentration of the drug in plasma ranged from 3 to 300 micrograms/ml. At higher concentrations, fractional excretion rose progressively. p-Aminohippurate at concentrations in plasma ranging from 1 to 1000 micrograms/ml had no effect on the fractional excretion of urate. In guinea pigs, pyrazinoate exerted only a faint uricosuric action and p-aminohippurate was without effect. Both substances were tested over the range of 1 to 1000 micrograms/ml of plasma. The results in dogs help explain some previous reports of negative experiments with pyrazinoate; the doses used were not optimal for detecting responses. The results in guinea pigs suggested that urate secretion in this animal does not fit either of two previously recognized mechanisms.

Organic anion infusions exacerbate experimental acute renal failure.

UNLABELLED: This investigation proposed to determine whether high organic anion (OA) loads per nephron increase renal susceptibility to acute ischemic and nephrotoxic injury. Anesthetized Sprague-Dawley rats were infused with a control infusate, Na2SO4, or an OA (hippurate, p-aminohippurate, cephalothin; 0.125-1.0 mg/min). After a 40-min control period, acute renal injury was induced by either bilateral renal pedicle cross-clamping (X25 min) or by HgCl2 administration (12 mg/kg i.v.). Glomerular filtration rate (clearance of [125I]iothalamate) was determined every 20 min before and after renal injury. Non-OA-infused rats lost 51 +/- 4% (ischemia) and 40 +/- 4% (HgCl2) of control GFR. OA infusion exacerbated this loss of renal function (ischemia, 89 +/- 2%; HgCl2, 84 +/- 4%). Renal histology demonstrated that OA-treated acute renal failure (ARF) rats had more vacuolar degeneration of proximal tubular cells (HgCl2, ischemia) and greater tubular dilatation (ischemia) than did non-OA-treated ARF rats. These functional and histologic responses to OA infusion were not OA dose dependent. Discontinuation of OA infusion did not cause a subsequent rise in GFR. Na2SO4 infusion had no detrimental effects on ischemic ARF. Control rats subjected to prolonged OA infusion (1 mg/min X 5 h) maintained stable GFR and had normal renal histology. ARF rats infused with low-dose cephalothin had terminal serum cephalothin concentrations within a range commonly seen in humans. CONCLUSION: organic anion infusions can exacerbate early functional and histologic parameters of experimental ARF.

Comparison of the renal excretory mechanisms of cefmenoxime and other cephalosporins: effect of para-aminohippurate on renal clearance and intrarenal distribution of cephalosporins in rabbits.

The renal excretory mechanism of cefmenoxime in rabbits was compared with that of 6 other cephalosporins (cefotaxime, deacetylcefotaxime, cefotiam, cefazolin, cephaloridine, and cefsulodin). The clearance ratios (Cf-Drug/CInulin=CRf) of cefmenoxime (337) and cefazolin (73) were considerably higher than those of the 5 other cephalosporins (0.9-20). When p-amino-hippurate (PAH) was administered concurrently with each of the cephalosporins, the CRf values of the cephalosporins except for cefsulodin were significantly decreased. These findings indicate that cefmenoxime and the 5 other cephalosporins except cefsulodin are actively incorporated in the proximal tubular cells and secreted into the tubular lumen. In the case of cefotiam and cefsulodin, glomerular filtration tended to exceed urinary excretion with highest dose of PAH (40 mg/kg/minute), suggesting the possibility of tubular reabsorption of these drugs. On the other hand, glomerular filtration of cefmenoxime and the 4 other cephalosporins did not exceed urinary excretion. The drug concentration ratio of the cortex to medulla indicated that the tubular cell level of cefmenoxime was lower than, higher than, and similar to those of cephaloridine, cefotaxime, and the remaining cephalosporins, respectively. These results demonstrate that the renal excretory mechanisms of cefmenoxime is similar to that of cefazolin but not to that of the remaining cephalosporins.

Succinate dehydrogenase activity in rat kidney after repeated administrations of p-aminohippurate or sodium chloride.

During PAH excretion and 18 h after repeated PAH administrations to rats, renal cortical SDH activity was unchanged in comparison with untreated controls. On the other hand, 18 h after repeated administrations of 0.9% or 1.8% NaCl solution, SDH activity was decreased by about 20% in kidney cortex. In outer medulla SDH activity was decreased 18 h after all pretreatments. The decline of SDH activity was observed whenever an increased urinary Na excretion occurred, except that PAH was repeatedly administered. The probability is discussed that substrate-induced stimulation of the carrier system for weak organic acids is accompanied by an increase of mitochondrial activity in kidney cortex.

Effect of para-aminohippurate on renal transport of oxalate.

Infusion of para-aminohippurate (PAH) at rates below the maximum rate of transport (Tm) of PAH in the rat resulted in a significant decrease in the fractional excretion of oxalate (FEox) from 128.1% to 113.9% (P less than 0.01). Fractional delivery of oxalate (FDox) to the early proximal tubule, however, was unchanged from control values, whereas FDox to the late proximal tubule was significantly decreased from 126.4% to 107.4% (P less than 0.01). Infusion of PAH at rates above Tm of PAH resulted in a decrease in FDox to the early proximal tubule to 105.3% and to the late proximal tubule to 105.5%, and in FEox to 100.5%. These changes were not the result of alterations in urinary sodium or bicarbonate excretion. Microinjection studies indicated that PAH did not affect the tubular absorption of oxalate. These studies suggest that PAH inhibits the tubular secretion of oxalate and that there may be more than one secretory system for oxalate with differing affinities for oxalate and PAH in the early and late proximal tubules.

Phlorizin stimulation of p-aminohippurate uptake in rat kidney cortex slices.

Phlorizin, 0.5 mM, increases the uptake of tritiated p-aminohippuric acid (PAH) in rat kidney cortex slices in vitro. Phlorizin also diminishes the rate of 3H-PAH washout from preloaded slices into PAH-free medium. At higher concentrations, phlorizin (5.0 mM) reduces slice uptake of 3H-PAH following short incubations but increases 3H-PAH accumulation after more prolonged incubations. Section freeze-dry autoradiography demonstrates that phlorizin inhibits secretion of 3H-PAH from cell to lumen in proximal tubules. Consequently, the increased 3H-PAH uptake and delayed washout induced by phlorizin may be attributed to effects at the antiluminal cell membrane. Phlorizin stimulation of PAH uptake occurs despite inhibition of secretion across the luminal membrane. Intracellular accumulation of 3H-phlorizin, demonstrable by autoradiography, provides direct evidence that cellular accumulation affords the glycoside access to both the luminal and antiluminal membrane in proximal tubules. These interactions between phlorizin and PAH suggest shared features of the membrane transport systems for secretion and reabsorption of sugars and organic acids in kidney.

Changes in serum TSH level after intraventricular injection of various neuromediators in rats.

The influence of various neuromediators on pituitary TSH secretion in rats has been investigated. Noradrenaline 50 microgram/rat, dopamine 50 microgram/rat, serotonine-creatinine-sulphate 100 micogram/rat, gamma-aminobutyric acid 100 microgram/rat, pilocarpine 1 mg/rat, histamine 100 microgram/rat were administered into the lateral ventricle of the brain. All agents were dissolved in Parker's fluid. Two control groups of animals were given Parker's fluid and subjected to surgical manipulations, respectively. Plasma TSH level was estimated after 30 min by means of radioimmunoassay. The increase in the TSH level was observed after the injection of serotonine and noradrenaline (4.0 and 3.1 ng/ml, respectively) as compared with control group (0.7 ng/ml).

Inhibition of probenecid uricosuria by pyrazinamide and para-aminohippurate.

Both para-aminohippurate (PAH) and pyrazinamide inhibited the uricosuric response to probenecid administration. The mechanism of this inhibition of probenecid uricosuria was assessed in 18 male subjects. The decrease in uricosuria was assessed in 18 male subjects. The decrease in uricosuric response to probenecid observed after pyrazinamide administration or PAH infusion occurs by different mechanisms. Administration of PAH and probenecid together resulted in reduced excretion of both drugs. PAH was weakly uricosuric and did not appear to inhibit urate secretion. PAH inhibition of probenecid uricosuria is accounted for by inhibition of probenecid secretion. Probenecid excretion was not affected by pyrazinamide administration. Inhibition of probenecid-induced uricosuria by pyrazinamide is most likely due to inhibition of urate secretion. The urate secretory carrier inhibited by pyrazinamide appears to be independent of that responsible for secretion of probenecid and PAH. Probenecid secretion appears to be required for its uricosuric effect.

The natriuretic effect of the organic acid para-aminohippurate.

Infusion of para-aminohippurate (PAH) during renal clearance experiments has been shown to increase urinary sodium excretion (UNAV). In addition, it has recently been suggested that accumulation of PAH and/or related compounds may be responsible for the increased UNAV per nephron in chronic renal failure. Therefore, studies were designed to determine the mechanism for the natriuretic effect of PAH. PAH administration at (0.19 mM per minute) into the dog renal artery led to an average increase in UNAV of 56+/-11 muEq per minute. Mannitol given at this rate (0.19 mM per minute) led to an increase in UNAV of 26+/-10 muEq per minute, which was less than the increase in UNAV with PAH. NA2SO4 given at this (0.19 mEq per minute) or a higher rate (0.60 mEq. per minute) was associated with an increase in UNAV of 53+/-18 and 228+/-38 muEq per minute, respectively. In the PAH and SO4 studies the increase in UPAHV or USO4V was not different from the increase in UNAV. These results suggest that the natriuretic effect of PAH is mainly due to its effect as a nonreabsorbable anion and offer no support for an osmotic effect or a direct effect on active sodium transport.

Effect of substrate pretreatment on renal organic ion transport in the adult rat.

The ability of renal cortical slices to accumulate PAH and NMN was not significantly affected by pretreatment of adult rats with large doses of PAH. Pretreatment of adult rats with THAM significantly increased PAH accumulation but had no effect on NMN. Inulin and PAH clearance and filtration fraction were significantly decreased by PAH pretreatment but unaffected by THAM pretreatment. The effects of pretreatment on transport are probably due to non-specific toxicity.

Effect of para-aminohippurate on renal glutamine metabolism in the rat.

After para-aminohippurate (PAH) infusion into rats, urine pH decreased and urine ammonium excretion increased. Because augmented urine flow and decreased urine pH could not explain entirely the enhanced ammonium excretion, an increased ammonia production was postulated as a contributing influence. This was supported by the in vitro findings that PAH could increase slice ammoniagenesis from glutamine. The ability of PAH to stimulate ammoniagenesis in vitro was attributed to enhanced phosphate-dependent glutaminase activity. We conclude that PAH infusions at certain concentrations in vivo can alter ammonium excretion through increased renal ammonia production. The latter may be secondary to enhanced phosphate-dependent glutaminase activity.

Modification of in vivo lacrimal gland secretion by 2,4-dinitrophenol.

Mecholyl-stimulated flow from the excretory duct of the rabbit lacrimal gland in vivo was recorded continuously and samples of Mecholyl-stimulated fluid were collected before, during, and after 30 min of local intra-arterial infusion of 2,4-dinitrophenol (DNP). Compared with the control period, Mecholyl-stimulated flow during the DNP infusion was 60-70% less; [K+] was the same and [Na+] and [Cl-] were higher in the Mecholyl-stimulated fluid. The changes that occurred during the DNP infusion were not produced by infusion of the solvent (NaHCO3) or an acid with similar pK (para-aminohippuric acid) nor were the changes related to any effect of DNP on the general circulation. The magnitude of the changes during the DNP infusion increased as the dose of DNP increased and the inhibition of flow changes in [Na+] and [Cl-] during the DNP infusion were reversible. The results are compatible with the hypothesis that stimulation by Mecholyl involves oxidative metabolsim at the duct level but they do not exclude the possibility that oxidative metabolism may also be involved in formation of the primary fluid at the acinar level.

[Extent and duration of drug-induced stimulation of renal excretion of p-aminohippuric acid]. / Ausmass und Dauer der Stimulation der renalen Ausscheidung von p-Aminohippursäure durch Pharmaka

Repeated pretreatment with p-aminohippuric acid (PAH), probenecide, cyclopenthiazide, and phenobarbital stimulates the renal excretion of PAH. For an interval of at least 6 hrs following i.p. application pretreated rats excrete more PAH excretion than the controls. All the drugs studied were found to stimulate renal PAH excretion within a period of 3 hrs by 50-60% of the control value. The required duration of pretreatment varies with the substance used. With cyclopenthiazide, the excretion of PAH is demonstrable for 2-3 weeks. Phenobarbital has a brief stimulating action. Correlations of a binding of drugs to structures of their tubular cell and the length of the stimulating action are discussed.