Effect of metabolic inhibitors and ouabain on amphetamine- and potassium-induced release of biogenic amines from isolated brain tissue

In experiments on isolated rat cerebral cortex, it was observed that d-amphetamine released norepinephrine from cerebral cortex into the incubation medium. Amphetamine-induced release of norepinephrine was markedly potentiated in the presence of the metabolic inhibitors sodium cyanide, 2,4-dinitrophenol and iodoacetate. Release of norepinephrine by the metabolic inhibitors in the absence of amphetamine was minimal. Amphetamine-induced release of dopamine from corpus striatum was also potentiated by sodium cyanide. The EC₅₀ for amphetamine-induced release of dopamine was 20μM with amphetamine alone and 0.58 μM in the presence of sodium cyanide. Release of norepinephrine and dopamine by elevated concentrations of potassium chloride was again potentiated by the metabolic inhibitors. Similarly, ouabain (10–25 μM) produced minimal release of norepinephrine but potentiated amphetamine- and potassium-induced release of catecholamines. The metabolic inhibitors markedly reduced the ATP content of the chopped tissue during the incubation while ouabain had no effect. The results suggest that the potentiation of amphetamine- and potassium-induced release of biogenic amines from isolated brain tissue is not simply due to depletion of tissue ATP levels but may be related to sodium or potassium transport. The Na⁺ , K⁺ -ATPase sodium pump is inhibited either: (1) when ATP levels are reduced by metabolic inhibitors, or (2) when Na⁺ , K⁺ -ATPase is inhibited by ouabain. Inhibition of the sodium pump would lead to an increase in the intraneuronal sodium concentration. A model is described in which norepinephrine and sodium are co-transported across the membrane during amphetamine-induced release of the biogenic amine. Elevation of the intraneuronal sodium concentration would provide increased levels of the co-substrate for transport and could account for the potentiation of release produced by metabolic inhibitors and ouabain.     

Role of the equilibrative and concentrative nucleoside transporters in the intestinal absorption of the nucleoside drug, ribavirin, in wild-type and ent1(-/-) mice

Ribavirin is frontline treatment for hepatitis C virus infection. To determine the role of nucleoside transporters in the intestinal absorption of orally administered ribavirin, we perfused the intestines of Ent1(-/-) and wild-type mice, in situ, with [(3)H] ribavirin (20, 200, and 5000 μM) in the presence and absence of sodium. The decrease in luminal ribavirin concentration over 30 min was measured at 5 min intervals. Blood samples were collected approximately every 10 min. Ribavirin plus phosphorylated metabolite concentrations (hereafter referred to as ribavirin) were determined in tissue, blood, and plasma by HPLC fractionation and scintillation counting. There was no significant difference between wild-type and Ent1(-/-) mice in intestinal loss of ribavirin at any ribavirin concentration studied. Perfusions without sodium drastically reduced the intestinal loss of ribavirin in both wild-type and Ent1(-/-) mice. After 20 μM ribavirin perfusions, Ent1(-/-) intestinal tissue contained 8-fold greater ribavirin than wild-type mice (p < 0.01). Ribavirin concentrations in the wild-type intestinal tissue were 70-fold higher after 200 vs 20 μM perfusions (p < 0.001), indicating saturation of intestinal ribavirin efflux and possibly other processes as well. Ribavirin plasma concentrations were significantly higher in wild-type mice (2.7-fold) vs Ent1(-/-) mice at 30 min after the 20 μM perfusion (p < 0.01). These results suggest that, at lower intestinal concentrations of ribavirin, concentrative and equilibrative nucleoside transporters are important in the intestinal absorption of ribavirin. At higher intestinal concentrations, these transporters are saturated and other processes in the intestine (transport and/or metabolism) play an important role in the absorption of ribavirin.     

Cyanide and migratory birds at gold mines in Nevada,USA

Since the mid-1980s, cyanide in heap leach solutions and mill tailings ponds at gold mines in Nevada has killed a large but incompletely documented number of wildlife (>9,500 individuals, primarily migratory birds). This field investigation documents the availability of cyanide at a variety of typical Nevada gold mines during 1990 and 1991, describes wildlife reactions to cyanide solutions, and discusses procedures for eliminating wildlife loss from cyanide poisoning. Substantial progress has been made to reduce wildlife loss. About half of the mill tailings ponds (some up to 150 ha) in Nevada have been chemically treated to reduce cyanide concentrations (the number needing treatment is uncertain) and many of the smaller heap leach solution ponds and channels are now covered with netting to exclude birds and most mammals. The discovery of a cyanide gradient in mill tailings ponds (concentration usually 2–3 times higher at the inflow point than at reclaim point) provides new insight into wildlife responses (mortality) observed in different portions of the ponds. Finding dead birds on the tops of ore heaps and associated with solution puddling is a new problem, but management procedures for eliminating this source of mortality are available. A safe threshold concentration of cyanide to eliminate wildlife loss could not be determined from the field data and initial laboratory studies. New analytical methods may be required to assess further the wildlife hazard of cyanide in mining solutions.     

The interaction between chronic oral slow-release theophylline and single-dose intravenous erythromycin

Eight volunteers were each given 300 mg of erythromycin lactobionate by i.v. infusion over 15 min in the presence and absence of chronic dosing with slow-release theophylline. Pharmacokinetic profiles were obtained for theophylline in the presence and absence of erythromycin and for erythromycin in the presence and absence of theophylline. A very small, clinically unimportant, but statistically significant increase occurred in mean (+/- S.E.M.) serum theophylline concentration from 4.9 +/- 0.3 mg/l to 5.2 +/- 0.3 mg/l in the presence of erythromycin (P = less than 0.01). The theophylline pharmacokinetic parameters did not change significantly. The only changes in erythromycin pharmacokinetics were an increase in the renal excretion (0-12 h) from 5.5 +/- 4.0 mg to 11.2 +/- 6.0 mg (P less than 0.03) and an increase in renal clearance (0-2 h) from 9.0 +/- 6.0 ml/min to 21.6 +/- 15 ml/min (P less than 0.05) in the presence of theophylline.     

Stability,tissue metabolism,tissue distribution and blood partition of azosemide

Stability of azosemide after incubation in various pH solutions, human plasma, human gastric juice, and rat liver homogenates, metabolism of azosemide after incubation in 9000g supernatant fraction of various rat tissue homogenates in the presence of NADPH, tissue distribution of azosemide and M1 after intravenous (IV) administration of azosemide, 20 mg kg^?1, to rats, and blood partition of azosemide between plasma and blood cells from rabbit blood were studied. Azosemide seemed to be stable for up to 48 h incubation in various pH solutions ranging from two to 13 at an azosemide concentration of 10 μg mL^?1; more than 93.4% of azosemide was recovered and a metabolite of azosemide, M1, was not detected. However, the drug was unstable in pH 1 solution: 75.8% of azosemide was recovered and 2.16 μg mL^?1 of M1 (expressed in terms of azosemide) was formed after 48 h incubation in pH 1 solution at an azosemide concentration of 10 μg mL^?1. Azosemide was stable in both human plasma and rat liver homogenates for up to 24 h incubation at an azosemide concentration of 1 μg mL^?1, and in human gastric juice for up to 4 h incubation at an azosemide concentration of 10 μg mL^?1. However,-all rat tissues stdied had metabolic activity for azosemide in the presence of NADPH, with heart having a considerable metabolic acitivity: approximately 22% of azosemide disappeared and 9.32 μg of M1 was formed per gram of heart (expressed in terms of azosemide) after 30 min incubation of 50 μg of azosemide in 9000g supernatant fraction of heart homogenates. The tissue to plasma ratios of azosemide (T/P) were greater than unity only in the liver (1.26) and kidney (1.74); however, M1 showed high affinity for all tissues studied except the brain and spleen when each tissue was collected at 30 min after IV administration of azosemide to rats. The equilibrium plasma to blood cell concentration ratios of azosemide were independent of azosemide blood concentrations: the values were 2.78–4.25 at azosemide blood concentrations of 1, 10, and 20 μg mL^?1 three rabbits. There was negligible ‘blood storage effect’ of azosemide, especially at low blood concentrations of azosemide, such as 1 and 10 μg mL^?1.     

Cyanide metabolism in the isolated, perfused, bloodless hindlimbs or liver of the rat

Female CD1 rats weighing 250-300 g were anesthetized with ip pentobarbital, 50 mg/kg, and either the liver or the hindlimbs were surgically isolated and perfused in situ with a Krebs-Henseleit buffer, pH 7.4, at 38 degrees C, containing 40 g/liter dextran and 30 mg/liter papaverine. Perfusion pressure was continuously monitored, and in most experiments, flow was maintained at the physiological rate of 8.5 ml/min. In-line Clark-type electrodes allowed the continuous measurement of oxygen extraction. Potassium cyanide to 0.15 mM was usually added to the perfusate just prior to the start of a run. After a period of equilibration, samples of the perfusate were taken periodically for cyanide (CN) and thiocyanate (SCN) analyses. The results were used to determine CN extraction ratios or clearance and rates of SCN formation. When it was apparent that a steady state had been reached with respect to the above, sodium thiosulfate (TS) was added to the perfusate (to 0.1, 1.0, or 2.0 mM), and periodic samples were again collected after an equilibration period. In the absence of albumin, TS rapidly and significantly increased the rate of conversion of CN to SCN in both the liver and the hindlimbs. The rate of CN clearance in milliliters per minute per kilogram perfused tissue was 20-fold greater in the liver than in the hindlimbs. However, when the results from hindlimbs were extrapolated to the total body skeletal muscle mass, the rate of CN clearance by the total liver mass was only 1.5-fold greater than in total muscle mass. In the absence of TS, total muscle mass cleared CN at a rate that was 2.6-fold greater than the total liver mass, but the rates in both tissues were very much less than in the presence of TS. The extraction ratio for CN in the liver was 0.8 and the clearance was dependent on the flow rate. The extraction ratio for CN in the hindlimbs was 0.2, and the clearance was independent of the flow rate. Thus, CN clearance by the liver probably increases (within limits) with increasing portal blood flow. Evidence was obtained for the existence of a significant CN "sink," particularly in the liver, which presumably represents reversible binding to unknown tissue constituents.     

Dose-response assessment of naphthalene-induced genotoxicity and glutathione detoxication in human TK6 lymphoblasts

The dose-response relationship for the induction of micronuclei (MN) and the impact of glutathione (GSH) detoxication on naphthalene-induced cytotoxicity and genotoxicity were investigated in human TK6 cells. TK6 cells were exposed to 10 concentrations ranging from 0.0625 to 30μM naphthalene in the presence of β-naphthoflavone- and phenobarbital (βNP/PB)-induced rat liver S9 with a nicotinamide adenine dinucleotide phosphate-generating system. Three approaches were used to identify a no-observed-effect level (NOEL) for naphthalene-induced genotoxicity: (1) laboratory criteria of ≥ twofold increase over the concurrent solvent controls (NOEL = 10μM), (2) ANOVA with Bonferroni correction (NOEL = 2.5μM), and (3) the benchmark dose approach (BMCL(10) = 3.35μM). The NOEL and point of departure micronucleus frequency for naphthalene-induced MN are between the tested naphthalene concentrations of 2.5-10.0μM in this experimental system. Supplementation of the exposure system with physiological relevant concentrations of 5mM GSH eliminated naphthalene-induced cytotoxicity and genotoxicity; no increased cytotoxicity or genotoxicity was observed at concentrations of up to 500μM naphthalene in the presence of GSH compared with 2.5-10.0μM in the absence of GSH. Naphthalene bioactivation by βNP/PB-induced rat liver S9 exhibits a nonlinear dose-response for the induction of MN in TK6 cells with a NOEL of 2.5-10μM that in the presence of GSH is shifted upward greater than 50- to 200-fold. These data demonstrate a nonlinear dose-response for naphthalene-induced genotoxicity that is eliminated by GSH, and both observations should be considered when assessing human risk from naphthalene exposures.     

Mechanism of antagonizing cyanide-induced lethality by alpha-ketoglutaric acid

alpha-Ketoglutaric acid (alpha-KG) has been shown to be an effective antagonist for cyanide-induced lethality. The mechanism of this antagonism is hypothesized to result from alpha-KG binding with cyanide. Several investigative approaches were taken to determine the existence of this binding. First, mixtures of various molar ratios of alpha-KG:cyanide were injected into a high pressure liquid chromatograph. The addition of cyanide reduced the peak area of alpha-KG at a molar ratio of greater than 1:5. Second, blood from naive male ICR mice was spiked with alpha-KG and cyanide. Headspace above these blood samples was injected into a gas chromatograph and analyzed for released hydrogen cyanide. alpha-KG reduced the peak area of hydrogen cyanide released into the headspace at molar ratios of greater than 1:2.5. Third, the effect of cyanide on the ultraviolet spectrum of alpha-KG was determined as an indication of binding. In the presence of cyanide the absorption peak at 316 nm for alpha-KG was eliminated. Inhibition of cytochrome oxidase is an accepted target enzyme for cyanide-induced lethality. Fourth, further evidence of alpha-KG's mechanism was determined by the effect of alpha-KG on brain cytochrome oxidase (BRCYTOX) and its ability to antagonize cyanide-induced inhibition of BRCYTOX. BRCYTOX activity was determined in the presence of alpha-KG and was found to be unaffected between 0.01 and 0.06 M of alpha-KG. Greater concentrations of alpha-KG inhibited BRCYTOX activity. The complete inhibition of BRCYTOX activity by 10(-5) M cyanide was prevented with 0.05 and 0.06 M alpha-KG. Fifth, BRCYTOX activity of animals pretreated with saline and then an LD80 dose (8.5 mg/kg) of cyanide was 80% inhibited, while BRCYTOX activity of animals pretreated with 2 g alpha-KG/kg, i.p., and then an LD80 dose (7.75 mg/kg) of cyanide was not different from control values. Thus, these data suggest that alpha-KG does bind with cyanide, and this binding can account for the antagonism of cyanide-induced lethality.     

Dissolving microneedles to obtain rapid local anesthetic effect of lidocaine at skin tissue

Abstract

Dissolving microneedles (DMs) were applied to lidocaine for local anesthesia of the skin. Three DM array chips were prepared where lidocaine was localized at the acral portion of DMs (type 1), loaded in whole DMs (type 2), and lidocaine was loaded both in whole DMs and the chip (type 3). DM chips were 15-mm diameter with 225 DMs, each 500-μm long with a 300-μm diameter base. The lidocaine contents were (type 1) 0.08?±?0.01?mg, (type 2) 0.22?±?0.01?mg and (type 3) 8.52?±?0.49?mg. Lidocaine was released from type 1 and 2 DM array chips within 10?min. Pharmacological activity of DMs were compared to lidocaine cream by the suppression of idiospasm of hair-removed rat skin. Type 1, 2 and 3 DMs showed faster onset time, 5?min, than lidocaine cream. Type 2 and 3 DMs showed stronger anti-idioplasmic activity than type 1 DMs. Pharmacokinetic study showed that tissue lidocaine levels, 62.8?±?3.6 (type 1), 89.1?±?9.9 (type 2) and 131.2?±?10.2(type 3) μg/g wet weight at 5?min after the removal of DM were obtained higher than lidocaine cream, 26.2?±?12.5?μg/g wet weight. Those results suggest the usefulness of type 2 DMs to obtain fast onset time for the local anesthesia in the skin.     

Nitric oxide formation from glyceryl trinitrate by rabbit aortic strip: detection by rabbit taenia coli concurrent with vasorelaxation.

1. The purpose of the present study was to assay NO formation from GTN biotransformation by the rabbit aortic strip (RAS) at times concurrent with its vasorelaxation. Such an assay is an important test of the prodrug hypothesis where it is postulated that glyceryl trinitrate (GTN) is biotransformed to nitric oxide (NO), the active species that initiates vascular smooth muscle relaxation. To test such a hypothesis, we propose that a sample of smooth muscle, poorly responsive to GTN, yet sensitive to the effects of NO could be used to detect RAS production of NO from GTN. 2. Muscle strips of rabbit taenia coli (RTCS) and RAS in close apposition, were mounted in tissue baths, and muscle relaxation was recorded with isometric force transducers. Tissues were submaximally precontracted with 30-35 mM K+ depolarizing solution and exposed to increasing concentrations of GTN (0.1 nM-10 microM). 3. EC25 for GTN-induced relaxation of RTCS in the presence of RAS was significantly decreased to that for RTCS in the absence of RAS (5.9 +/- 3.0 x 10(-8) M and 5.5 +/- 3.7 x 10(-6) M, respectively). Mean maximal levels of GTN-induced relaxation of similarly precontracted RTCS also differed in the presence and absence of RAS, viz., 80.8 +/- 2.1% and 29.8 +/- 8.3% respectively. 4. RTCS was found to relax upon administration of NO gas bubbled through the incubation medium. Analysis of tissue bath medium revealed that the NO concentration to which RTCS was exposed attained a maximum of 33 nM. Relaxation of RTCS by NO gas was inhibited by 1 microM reduced haemoglobin. 5. For GTN-incubation with intestinal and vascular smooth muscle preparations, NO formation was greater with RAS compared to RTCS. Thus, in the two-issue bioassay, the RAS was the predominant source of NO formation from GTN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of a hepatocyte-erythrocyte coincubation system to studies of cyanide antidotal mechanisms

A coincubation system composed of hepatocytes in primary monolayer culture and erythrocytes suspended in the culture medium was developed and used as a model for investigations of mechanisms of cyanide antidote action at the cellular level. Hepatocyte ATP was used as the cytotoxicity indicator. Treatment of rat hepatocytes in the coincubation system with KCN (1.0 mM) for 10 min at 37 degrees C selectively reduced hepatocyte ATP levels to 33 +/- 15% of control (no KCN added) levels. 4-dimethylaminophenol (DMAP), cobalt(II) chloride, sodium nitrite, sodium thiosulfate, or a combination of the last two antidotes added to the KCN-containing medium significantly reversed ATP depression and the response was concentration dependent. The relative effectiveness, on a molar basis, was estimated to be DMAP greater than CoCl2 much greater than NaNO2 congruent to Na2S2O3. NaNO2 and DMAP induced methemoglobin formation in the absence of cyanide and cyanmethemoglobin formation in its presence; erythrocytes were required in the medium for effectiveness. CoCl2 produced neither cyanmethemoglobin nor thiocyanate in appreciable quantities nor required erythrocytes for antagonism. Na2S2O3 converted cyanide to thiocyanate and reversed ATP depression without erythrocytes in the medium. The addition of erythrocytes increased these rates significantly and to a greater extent than albumin. The overall results are consistent with previously proposed modes of action for these antidotes. However, the enhancement in cyanide metabolism and ATP recovery with Na2S2O3 and erythrocytes in the system was unexpected and raises the possibility that erythrocytes may contribute to cyanide disposition and antagonism in vivo when this antidote is administered.     

Stimulatory effect of zinc on bone formation in tissue culture

The present investigation was undertaken to clarify the in vitro effect of zinc on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old males) and cultured for periods up to 96 hr in Dulbecco's Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-7) to 10(-3) M zinc sulfate. All cultures were incubated at 37 degrees in 5% CO2/95% air. Zinc uptake by bone was increased significantly in cultures with concentrations of zinc greater than 10(-6) M. Bone calcium content was increased significantly by the presence of 10(-4) M zinc. This increase was blocked by the presence of 10(-6) M cycloheximide. Bone alkaline phosphatase activity was elevated in the presence of zinc (10(-6) to 10(-3) M), but the effect was inhibited by 10(-7) M cycloheximide or 10(-8) M actinomycin D. Zinc (10(-4) M) also significantly increased ATPase activity in the bone, whereas it did not alter significantly by pyrophosphatase, acid phosphatase and beta-N-acetylglucosaminidase activities. Furthermore, bone collagen content was raised by 10(-6) to 10(-4) M zinc. This elevation was prevented by 10(-7) cycloheximide or 10(-8) M actinomycin D. Bone DNA content and [3H]thymidine incorporation by the bone were not altered significantly by 10(-4) M zinc. These findings indicate that the zinc had a direct stimulatory effect on bone mineralization in vitro, and that bone protein synthesis was a necessary component of this response. Zinc may stimulate bone formation in tissue culture.     

Endothelial dysfunction after exposure to cobalt chloride enhanced vascular contractility

Brief exposure to cobalt chloride augmented vascular contractility. We hypothesized that endothelial dysfunction plays a role in the augmentation of aortic contractility, after brief exposure to cobalt chloride. Rat aortic ring preparations were mounted in organ baths, exposed to cobalt chloride (0.3-300μmol/L) for 30min, and then subjected to contractile agents or relaxants 1 and 5h after the end of exposure. Presence of cobalt chloride did not affect the contractile response to phenylephrine. Brief exposure to cobalt chloride, however, even at 5h after the end of exposure, not only augmented contractile responses to KCl or phenylephrine but also attenuated the relaxant response to acetylcholine. The mechanical denudation of endothelium or inhibition of endothelial nitric oxide synthase with 100μmol/L N(ω)-nitro-l-arginine methyl ester abolished the augmentation of contractile responses. Pre-treatment with 150units/mL of superoxide dismutase also abrogated the augmented contractile responses. Brief exposure to cobalt chloride did not affect the contractile response to phorbol dibutyrate in the presence or absence of calcium, or the expression of HSP70. In conclusion, endothelial dysfunction plays an important role in the augmentation of aortic contractility, after brief exposure to cobalt chloride.     

Histamine release from the isolated guinea-pig heart by compound 48/80

Histamine release was observed in isolated guinea-pig heart perfused by the constant flow method, after bolus injection of 50-200 micrograms of compound 48/80. Practically all the histamine is released within the first 3 min after 48/80. This effect is inhibited by the addition of sodium cyanide (300 microM) to a glucose-free perfusion fluid and also by the removal of calcium of the medium in presence or absence of EDTA (100 microM). This dependence on metabolism and calcium is highly suggestive of a non-cytotoxic action of 48/80 on the mast cells of guinea-pig heart.     

离子色谱法测定厄贝沙坦中氰化物

目的建立离子色谱法测定厄贝沙坦中氰化物的含量。方法采用IonPac AS7（250 mm×2 mm）离子交换色谱柱,淋洗液采用0.1 mol.L^-1氢氧化钠和0.2 mol.L^-1乙酸钠,流速为0.3 mL.min-1。结果氰根离子在0.008～0.12μg.mL^-1线性关系良好（r2=0.999 7）,检测限为0.002μg.mL^-1。在所建立的色谱条件下,氰根离子与各常见阴离子之间的分离度良好。结论该方法简单快速,专属性强,结果准确,可用作厄贝沙坦中氰化物的含量的检测。     

Differences between the neuronal handling of 3H-7- and 3H-7,8-(−)noradrenaline: implications for the release of the labelled neurotransmitter by nerve stimulation

Neighbouring rabbit aortic strips were exposed to a tracer concentration of 3H-7- or 3H-7,8-(-)noradrenaline (bearing 30-35% of its label in position 8) and to 0.5 mumol/l unlabelled (-)noradrenaline for 60 min and then washed in amine-free Krebs solution. Catechol-O-methyl transferase and extraneuronal amine uptake were inhibited throughout. After 114 min of wash-out, the tissue contained less tritium when loaded with 3H-7-(-)noradrenaline than when loaded with 3H-7,8-(-)noradrenaline, and the fractional rate of loss of tritium was greater for the former than for the latter tissues. In the presence of cocaine (to prevent neuronal re-uptake), the same percentage of tissue tritium was released by nerve stimulation (six consecutive periods of stimulation at 1 Hz for 5 min each) in spite of the above difference between tissue tritium levels of the two differently labelled amines. In the absence of cocaine, a higher percentage of tissue tritium was released by nerve stimulation (1 or 3 Hz, 5 min each) for 3H-7- than 3H-7,8-(-)noradrenaline. Unchanged 3H-(-)noradrenaline amounted to 35% of tritium in the stimulation-evoked overflow for 3H-7- and to 50% for 3H-7,8-(-)noradrenaline (frequency of stimulation, 1 Hz). When monoamine oxidase (MAO) was inhibited, no differences were observed between the neuronal handling of 3H-7- and 3H-7,8-(-)noradrenaline, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 5-benzylacyclouridine,a potent inhibitor of uridine phosphorylase,on the metabolism of circulating uridine by the isolated rat liver

5-Benzylacyclouridine (BAU) is a specific inhibitor of uridine phosphorylase, the first enzyme in the catabolism of uridine. It was found that 20 and 100 μM BAU dramatically reduced the rapid clearance of trace amounts of either [¹⁴C]uridine or hyperphysiologic concentrations of non-labeled uridine by the isolated rat liver perfused with an artificial oxygen carrier. In the absence of exogenously added uridine, non-treated livers maintained circulating concentrations of 1–2 μM uridine. In the presence of 20 μM BAU, these concentrations were increased 2- to 3-fold higher than physiologic levels (1.4±0.6 μM) and remained elevated for the duration of the experiment (120–160 min). In the presence of 100μM BAU, uridine concentrations rose continuously at rates of between 80 and ISOnmoles per hr per g of liver, and the clearance of a single radioactive spike of uridine was reduced extensively. The half-life of a uridine spike was extended 2-fold in the presence of 20 μM BAU and 5- to 6-fold in the presence of 100 μM BAU. Exogenously added uridine (15 and 40 μM) was cleared rapidly by nontreated livers, with a half-life of approximately 10 min. However, BAU at a concentration of 20 μM increased the half-life of 15 or 40 μM uridine added to the perfusate by approximately 10-fold. A 100 μM concentration of BAU inhibited the removal of 40 μM circulating uridine, but with 15 μM uridine there was a continuous increase in the circulating concentration similar to that seen in the absence of added uridine. We conclude that extensive inhibition of uridine phosphorylase occurs at 100 μM BAU and partial inhibition at 20 μM BAU. These data indicate independent catabolic and excretory functions of the rat liver with respect to uridine.     

Inactivation of [125I]-angiotensin II binding sites in rat renal cortex epithelial membranes by dithiothreitol

Preincubation of renal epithelial membranes with DTT produced a dose-dependent inhibition of specific [125I]-angiotensin binding, with an IC50 of 1 mM and total loss of binding at 5-10 mM DTT. Inactivation of specific [125I]-angiotensin II binding by DTT was temperature sensitive; the t1/2 at 22 degrees was 6 min compared with 30 min at 4 degrees. A rapid inactivation rate was dependent on the presence of NaCl. In the presence of 120 mM NaCl the t1/2 for inactivation by DTT was 6 min and 33 min in the absence of NaCl. Protection against DTT inactivation was obtained by preincubating membranes with unlabelled angiotensin II greater than angiotensin I greater than renin substrate while the dipeptide, Ileu-His was only effective in protecting the binding site at high concentrations (10 mM). Preincubations with DTT (1 mM) caused a 43% decrease in Bmax from 217.0 +/- 39.5 to 123.7 +/- 30.9 fmol bound/mg protein while the KD was not significantly affected.     

Pharmacokinetics and tissue distribution of ipriflavone, an isoflavone derivative, after intravenous administration to rabbits

Pharmacokinetic parameters of ipriflavone and its main metabolites, M1 and M5, after intravenous administration of spray-dried ipriflavone, SIP (10, 20, and 30 mg/kg as ipriflavone) and tissue distribution of ipriflavone, M1, and M5 after intravenous administration of SIP (20 mg/kg as ipriflavone) were evaluated in rabbits. Saturable metabolism of ipriflavone were observed after intravenous administration; at an ipriflavone dose of 30 mg/kg, the dose-normalized (based on 10 mg/kg) AUC was significantly greater (72.4 and 64.0 versus 103 microg min/mL), Cl was significantly slower (138 and 156 versus 97.6 mL/min/kg), and terminal half-life (94.8 and 129 versus 211 min) and mean residence time (91.3 and 116 versus 186 min) were significantly longer than those at 10 and 20 mg/kg. The AUC of M1 was also significantly greater at ipriflavone dose of 30 mg/kg. The terminal half-life, AUC, and renal clearance of M5 were also significantly different at ipriflavone dose of 30 mg/kg than those at 10 and 20 mg/kg. Ipriflavone was widely distributed in most rabbit tissues studied and the tissue-to-plasma (T/P) ratios of ipriflavone were greater than unity in all tissues (or organs) studied except spleen, indicating that ipriflavone has high affinity to rabbit tissues studied, and this could be supported by considerably high values of the apparent volume of distribution of ipriflavone at steady state (11 400-16 900 mL/kg). M1 and M5 were also detected in most rabbit tissues with considerable amount of M1 (T/P ratio of 9.43) and M5 (T/P ratio of 4.66) in the kidney.     

p38 Mitogen-activated protein kinase regulates Bax translocation in cyanide-induced apoptosis.

Execution of cyanide-induced apoptosis is mediated by release of cytochrome c from mitochondria. To determine how cyanide initiates cytochrome c release, Bax translocation was investigated in primary cultures of cortical neurons. Under nonapoptotic (control) conditions, Bax resided predominantly in the cytoplasm. After 300-microM cyanide treatment for 1 h, Bax translocated to the mitochondria, as shown by immunocytochemical staining and subcellular fractionation; Western blot analysis confirmed "cytosol-to-mitochondria" translocation of Bax. Temporal analysis showed that Bax translocation preceded cytochrome c release from the mitochondria, which was initiated 3 h after cyanide treatment. In double-immunofluorescence labeling for both Bax and cytochrome c, it was observed that cytochrome c was released only in cells showing Bax in mitochondria. The role of p38 mitogen-activated protein (MAP) kinase in Bax translocation was studied. The p38 MAP kinase was activated 30 min after cyanide, and its phosphorylation level of activity began to decrease 3 h later. SB203580, a p38 MAP kinase inhibitor, blocked translocation of Bax to mitochondria, whereas SB202474, a control peptide, had no effect on translocation. Inhibition of p38 MAP kinase by SB203580 blocked all downstream effects of Bax translocation, including cytochrome c release, caspase activation, and internucleosomal DNA fragmentation. These results demonstrated that Bax translocation is critical for cyanide-induced cytochrome c release and that p38 MAP kinase regulates Bax translocation from cytosol to mitochondria.