Ethacrynic acid influx and efflux in kidney cortex slices: Dependence on sodium gradient

Ethacrynic acid (EA) accumulation in rat kidney cortex slices was inhibited by reduction of sodium concentration in the incubation medium. Preloading of slices with sodium reduced EA accumulation at medium sodium concentration of 30–140 mM. Ouabain (10^?3 M) inhibited EA accumulation in kidney cortex slices of rabbit, guinea-pig, Psammomys obesus, rat, mouse and Acomys cahirinus in decreasing order. Ouabain inhibited the sodium pump in kidney cortex slices of these species in the same order. Ethacrynic acid efflux was faster from slices of rat kidney medulla than from kidney cortex. the efflux rate from cortical slices increased when sodium concentration in the incubation medium was lowered. Probenecid (10^?3 M) in the medium enhanced the efflux rate of EA from kidney cortex slices. It was concluded that EA fluxes in kidney cortex slices fit the model of a sodiumactivated carrier mechanism and that the sodium gradient across the cell membrane determines the direction of net EA transport.     

Mechanisms of diethyldithiocarbamate-induced loss of cytochrome P-450 from rat liver

A single, intraperitoneal injection of diethyldithiocarbamate (DDTC) to adult, male Sprague-Dawley rats decreased hepatic cytochrome P-450 (P-450) concentrations. This effect was dose-dependent over a range of 250 to 750 mg/kg and most prominent 24-36 hr after dosing. Depletion of hepatic glutathione (GSH) by diethylmaleate (DEM) administration significantly decreased P-450 8 hr after concurrent treatment with DDTC at a dose which given alone had little effect on P-450 concentrations. When hepatic microsomes were incubated with DDTC in the presence of NADPH, P-450 was converted to cytochrome P-420 (P-420). Similar incubations employing [35S]DDTC demonstrated strict NADPH-dependent binding of labeled sulfur to microsomal membranes, suggesting that diminished P-450 concentrations are related to the metabolic activation of DDTC. Addition of reduced GSH to incubation mixtures blocked the binding of 35S to microsomal membranes, as well as conversion of P-450 to P-420. DDTC inhibited NADPH-ADP3+ mediated peroxidation of microsomal lipids in vitro, suggesting that the effect of DDTC on P-450 does not result from stimulation of lipid peroxidation, but may be influenced by the levels of hepatic GSH. DDTC treatment 1 hr after P-450 was pulse labeled by an intravenous injection of [3H]delta-aminolevulinic acid resulted in a 2-fold increase in the rate of loss of radioactivity associated with membrane-bound P-450 heme during the next 20 hr. Within this time interval, hepatic heme oxygenase (HO) activity increased and at 8 hr after dosing was 7-fold greater than control values in the livers, but was unchanged in the kidneys and spleens of DDTC-treated animals. An elevation of hepatic delta-aminolevulinic acid synthetase (delta-ALAS) activity occurred at 8 and 24 hr after DDTC treatment. Since this enzyme is rate limiting in the biosynthesis of heme, its increased activity may represent a compensatory response to offset the DDTC-mediated loss of P-450 heme.     

Renal transport of 2'-deoxytubercidin in mice

Previous results [J. F. Kuttesch, Jr. and J. A. Nelson, Cancer Chemother, Pharmac. 8, 221 (1982)] from this laboratory indicate that mechanisms exist for renal secretion of 2'-deoxyadenosine and possibly for reabsorption of adenosine in humans and in mice. Since significant metabolism of these purine nucleosides occurs even in the presence of adenosine deaminase inhibitors, the renal handling of a compound which is not significantly metabolized by the deaminase or by kinases was studied. Unlike 2'-deoxyadenosine itself, the 2'-deoxyadenosine analog, [4-amino-7-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrrolo-(2,3-d)pyrimidine; 2'-deoxytubercidin], is not significantly metabolized by mammalian tissues. In mice, the renal plasma clearance of 2'-deoxytubercidin exceeded that of inulin by about 3-fold. Also, mouse kidney slices concentratively accumulated 2'-deoxytubercidin by a saturable and metabolically dependent process. The uptake by mouse kidney slices was inhibited by classical substrates for the organic cation secretory system (tetraethylammonium, choline and N1-methylnicotinamide) but was not markedly inhibited by classical substrates for the organic anion secretory system (p-aminohippurate, phenol red and probenecid). Since 2'-deoxytubercidin inhibited the active, concentrative uptake of [14C]tetraethylammonium, but failed to inhibit the uptake of p-[14C]aminohippurate by mouse kidney slices, it is concluded that 2'-deoxytubercidin may be secreted by the organic cation system. Additional studies are required, however, to unequivocally establish the relationships between 2'-deoxytubercidin, 2'-deoxyadenosine and tetraethylammonium renal secretory mechanisms.     

2-Methoxy-3,8,9-trihydroxy coumestan: a new synthetic inhibitor of Na+,K+-ATPase with an original mechanism of action

The aim of the present work was to analyse the interaction between Na⁺,K⁺-ATPase and one of our recent synthesized coumestans, namely the original molecule 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). Rat brain (mainly α2 and α3 Na⁺,K⁺-ATPase isoforms) and kidney (α1 isoform) fractions enriched with Na⁺,K⁺-ATPase were utilized to compare the inhibition promoted by PCALC36 with that of classical inhibitors like ouabain and vanadate. Analysis of inhibition curves revealed that unlike ouabain, which was about a thousand times more potent to inhibit brain isoforms than kidney isoform, PCALC36 had a similar affinity for brain ( μM) and kidney ( μM) isoforms. The inhibitory effect of PCALC36 was not antagonized by 1-10 mM K⁺, as observed with ouabain. Whereas vanadate was more potent in ionic conditions promoting the E2 conformation of the enzyme, the inhibitory effect of PCALC36 was equal in ionic conditions favouring either the E1 or E2 conformations. Equilibrium binding assays with []ouabain revealed that the addition of 2-10 μM PCALC36 did not change the K_d of ouabain but decreased its maximal binding (B_max) in a concentration-dependent manner (from 76.6 to 44.0 pmol/mg protein). This inhibitory effect of PCALC36 was not reverted after an extensive washing procedure indicating that it forms a very stable complex with Na⁺,K⁺-ATPase. We conclude that PCALC36, a new molecule with a non-steroidal skeleton, inhibits the Na⁺,K⁺-ATPase by a mechanism of action different from the cardiac glycosides and could thus serve as a structural paradigm to develop new inotropic drugs.     

Oxidative metabolism of mescaline in the central nervous system. IV. In vivo metabolism of mescaline and 2,3,4-trimethoxy-beta-phenylethylamine

Metabolic rates of mescaline (3,4,5-trimethoxy-β-phenylethylamine) and of its non-hallucinogenic isomer, 2,3,4-trimethoxy-β-phenylethylamine were studied in whole and in different areas of mouse brain in vivo. The effects of Pargyline and of Probenecid on the concentrations of the amines and their corresponding metabolites, together with the results obtained from intraventricular injections of mescaline suggested the formation of the trimethoxyphenylacetic acids in the brain.The metabolic differences between mescaline and 2,3,4-trimethoxy-β-phenylethylamine are discussed in terms of possible implications of metabolic parameters with psychotomimetic activity.     

Desensitization to locally injected PGF2α as reflected in the vascular permeability and collagen and noncollagenous protein synthesis of carrageenin-induced granulation tissue in rats

Administration of PGF_2α at doses of 50 and 250 μg into pouch fluid (calculated concentration of PGF_2α was about 4 and 14 μg/ml of pouch fluid, respectively) of 7-day-old carrageenin granuloma in rats depressed vascular permeability, as measured by leakage of radioiodinated human serum albumin into the pouch fluid. However, 3 hr after PGF_2α treatment at a dose of 50 μg. the vascular permeability had recovered to control level; 3 hr after a dose of 250 μg PGF_2α, the vascular permeability had not recovered completely. The uptake of [³H]proline into collagen hydroxyproline and noncollagenous protein of carrageenin granuloma tissue was also examined. It was found that the first injection of PGF2a at a dose of 50 or 250 μg depressed the uptake of [³H]proline into both protein fractions, but 3 hr after the first injection of PGF_2α, there was no significant difference in uptake between the control and the PGF_2α-treated group. A study of the metabolism of PGF_2α in a homogenate of granulation tissue and in pouch fluid showed little activity in the former and very little activity in the latter. In the homogenate, about 70 per cent of the originally added [³H]PGF_2α remained unmetabolized after 3 hr of incubation. In the inflammatory fluid, less than 20 per cent of the radioactivity present at 35 min after the injection had dissappeared from the pouch fluid at 3 hr after the [³H]PGF_2α injection; all the remaining radioactivity was found to be unmetabolized [³H]PGF_2α. Consequently, it was suggested that the tissue became desensitized to the injected PGF_2α. To confirm this suggestion, another 50 or 250 μg of PGF_2α was injected 3 hr after the first PGF_2α injection. However, a decrease in vascular permeability and in uptake of [³H]proline into both protein fractions after the second injection of PGF_2α were not observed. It was concluded that, 3 hr after the first PGF_2α treatment, the sensitivity of the carrageenin-induced granulation tissue to PGF_2α had decreased.     

Oxidative metabolism of mescaline in the central nervous system. 3. Side chain degradation of mescaline and formation of 3,4,5-trimethoxy-benzoic acid in vivo

After intraperitoneal injection of 8-[¹⁴C]mescaline. HCl into mice 0.05 per cent of the injected radioactivity was found within 1 hr in the respiratory gases. This observation prompted us to search for mescaline metabolites with a degraded side chain. 3,4,5-trimethoxy-benzoic acid was identified in brain and liver by derivative formation and mass spectrometry. N-acetyl-mescaline was also identified by mass spectrometry. Some new cationic mescaline metabolites were detected in brain but the available data did not allow us to establish conclusively the structure of these compounds     

Disposition,elimination and metabolism of O-ethylO-4-nitrophenyl phenylphosphonothioate after subchronic dermal application in male cats

The metabolism, distribution, and excretion of the insecticide O-ethylO-4-nitrophenyl phenylphosphonothioate (EPN) were studied in the male cat. Each cat was given a daily dermal dose of 0.5 mg/kg [¹⁴C] EPN for 10 consecutive days. Fifteen days after the last dose, the cats had excreted 62% of the cumulative dose in the urine and 10% in the faces. No ¹⁴CO₂ was detected in the expired air. O-Ethyl phenylphosphonic acid (EPPA) was identified as the major urinary and fecal metabolite. Phenylphosphonic acid (PPA) was the second highest metabolite. Only traces of the intact EPN were recovered in the urine and feces. The disposition studies performed 1, 5, 10 and 15 days after the administration of the last dose showed that EPN was the major compound identified in the brain, spinal cord, sciatic nerve, adipose tissue, plasma and kidney. Most of the radioactivity in the liver was identified as EPPA followed by PPA. The time course of plasma EPN, determined after the 10th daily dose was biphasic. The slower process had a half-life of 17.0 days. After tissue distribution was completed, tissue elimination was adequately represented as a single first-order process.     

Metabolism of anethole. I. Pathways of metabolism in the rat and mouse

The metabolic fate of the naturally occurring food flavouring trans-anethole has been investigated in rats and mice. A single 50-mg/kg dose of trans-[methoxy-14C]anethole was given orally to female Wistar albino rats and by ip injection to male CD-1 mice. The major routes of elimination of 14C were the urine and expired air (as 14CO2). Excretion of 14C in the faeces and as volatile compounds in the expired air was very low (total less than 2% of the dose). Urinary metabolites were separated by solvent extraction, TLC and HPLC and were characterized by MS and GC-MS directly and following methylation or trimethylsilylation, the results being compared where possible with authentic standards. Eleven 14C-containing urinary metabolites were identified in the rat and ten in the mouse. These compounds arose from side-chain oxidation, side-chain cleavage and various conjugations. The major urinary metabolites were two isomers of 1-(4'-methoxyphenyl)propane-1,2-diol, 2-hydroxy-1-methylthio-1-(4'-methoxyphenyl)propane and 4-methoxyhippuric acid, the first three all being excreted as glucuronides. In addition to these 14C-labelled metabolites, 4-hydroxypropenylbenzene, the unlabelled product of oxidative O-demethylation of trans-[14C]anethole, was excreted extensively in urine as the glucuronide.     

Novel serotonin receptors in Fasciola: Characterization by studies on adenylate cyclase activation and [3H]LSD binding

Serotonin (5-HT) receptors coupled to adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in the liver fluke Fasciola hepatica have been characterized by adenylate cyclase activation studies and by direct binding studies using $\text{[}{}^3\text{H}\text{]-d-}\text{lysergic acid}$ diethylamide ([³H]LSD) as a radioligand. Inhibition of 5-HT stimulation of adenylate cyclase by a series of 5-HT antagonists revealed a potency order of LSD = 2-bromo-LSD > methiothepin > metergoline = cyproheptadine > methysergide > spiroperidol. [³H]LSD binding to a cell-free fluke particle preparation was rapid, stereospecific, and proportional to protein concentration. Scatchard analysis indicated multiple binding sites which, when resolved into two components, gave for the high affinity site an apparent dissociation constant of 25 nM and a receptor concentration of 160 fmoles/mg protein. The ability of a series of compounds to compete for [³H]LSD binding sites correlated closely with their ability to inhibit 5-HT stimulation of adenylate cyclase. [³H]LSD binding sites were most concentrated in the anterior region of the fluke which was consistent with the higher levels of 5-HT activated adenylate cyclase found in this region. GTP and 5′-guanylyl imidophosphate, a poorly hydrolyzable GTP analog, decreased the affinity of the agonist 5-HT for the binding sites but had little effect on the affinity of the antagonist 2-bromo-LSD. Calcium at concentrations above 300 μM significantly reduced both [³H]LSD binding and 5-HT activation of adenylate cyclase. The results indicate that [³H]LSD can be used to label the 5-HT receptors coupled to adenylate cyclase activity. The pharmacological specificity and other characteristics of the fluke receptors appear to differ from the properties of reported mammalian 5-HT receptors. As a result, serotonin receptors in the flukes represent sites that may be amenable to selective manipulation by new chemotherapeutic agents useful in the treatment of these parasite infections.     

Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide: A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?7}}\text{M}\text{)}$ but not of acetylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 4 × 10}{}^{\mathrm{?4}}\text{M}\text{)}$. For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K_i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K_d of 4.5 × 10^?7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.     

Maternal-foetal distribution studies in late pregnancy. I. Distribution of [N-methyl-14C]betaine in tissues of beagle dogs and miniature pigs

[N-Me-14C]Betaine was administered iv as a single dose (5 mg/kg) to pregnant beagle dogs and miniature pigs late in gestation. Two hr after administration of the radiolabel, when the compound was in equilibrium, the dams were killed and the foetuses were removed for determination of the radioactivity in maternal and foetal tissues. Eight litters of dogs (56 foetuses) and four litters of pigs (30 foetuses) were examined. The distribution of betaine in both species showed distinct differences between maternal and foetal tissues, indicating definite placental barriers; the placental distribution factor was estimated to be 52.3% in dogs and 97.8% in pigs. The blood/brain distribution factor was 84.6% in maternal dogs, 89% in maternal pigs, 65.7% in foetal dogs and 0% in foetal pigs. In the dog, maternal liver was the largest depot of the administered betaine, followed by foetal liver. Foetal heart, lung and kidney tissues also incorporated radiolabelled betaine. The highest concentrations of betaine in the pig were found in maternal kidney and liver.     

Maternal-foetal distribution studies in late pregnancy. II. Distribution of [1-14C]acrylamide in tissues of beagle dogs and miniature pigs

[carbonyl-14C]Acrylamide was administered iv as a single dose (5 mg/kg) to pregnant beagle dogs and miniature pigs late in gestation. After a 2-hr equilibration period, the animals were killed and foetuses were removed for determination of the amount of radioactivity in maternal and foetal tissues. In total, six dog litters (33 foetuses) and seven pig litters (45 foetuses) were examined. In dogs, acrylamide was distributed readily to both maternal and foetal tissues with a placental distribution factor of 17.7%. The blood/brain distribution factor was insignificant (5.9%) in maternal dogs and 0% in the foetuses. Maternal liver was the largest depot of the administered acrylamide in the dog, followed by the maternal kidney. In pigs, the placental distribution factor was 31%, and the blood/brain distribution factor was insignificant in both maternal and foetal pigs. Liver and kidney of maternal pigs also contained the greatest amount of radioactivity. Although there appears to be some placental protection of the foetuses from the xenobiotic in the maternal circulation, foetal brain would be exposed to the effect of any acrylamide present in the foetal circulation, since the foetuses of both species had blood/brain distribution factors that were either small or zero, reflecting the absence of a blood-brain barrier.     

Studies on the metabolic fate of sucrose esters in rats

The metabolism in rats of sucrose esters of stearic acid and palmitic acid was studied in vivo and in vitro using esters labelled with ¹⁴C at the sucrose or fatty-acid moiety. In excretion studies, the ratio of expired radioactivity to absorbed radioactivity after oral administration of the sucrose esters labelled at the sucrose moiety was similar to that after the administration of [¹⁴C]sucrose. A similar correlation between the ester labelled at the fatty-acid moiety and the free [¹⁴C]fatty acid was also observed. No intact sucrose ester was detected in the urine. Studies in vitro using everted intestinal sacs showed that there was virtually no transport of ¹⁴C-labelled sucrose esters from the mucosal to the serosal solution through the intestinal tissues, and that the enzymes in the intestinal mucosa played a more important role in the hydrolysis of sucrose esters than did those in the digestive fluid. In studies of intestinal absorption through the mesenteric lymphatic system, during the 24 hr after ingestion 1.8% of the administered radioactivity was recovered in the lymph after dosing with [U-¹⁴C]sucrose monostearate whereas 20% was recovered in the lymph after dosing with sucrose [1-¹⁴C]monostearate. This difference in levels of recovery of administered radioactivity indicated that sucrose monostearate was absorbed only after hydrolysis. No intact ester was detected in the lymph or in the portal or femoral blood. The results of all of these experiments show that the sucrose esters are hydrolysed to sucrose and fatty acids prior to intestinal absorption.     

Catecholamine metabolism in brain slices. Determination of relevant precursor pool and the effects of elevated K+

Catecholamine synthesis from [3H]tyrosine was studied in slices of striatum, cerebellum and substantia nigra of mice. If low concentrations of tyrosine (less than 5.5 microM) were added to the incubation medium, the slices released significant amounts of tyrosine into the medium during the incubation. Kinetic analysis of the same experiments indicated that medium tyrosine and not tissue tyrosine was the appropriate precursor for both dopamine (DA) synthesis and protein synthesis in striatal slice. Concentrations of medium tyrosine of 8.25 microM or greater were sufficient to prevent changes of medium tyrosine during incubation and thus maintained a constant specific activity of precursor. Increasing concentrations of medium K+ increased both the accumulation of [3H]DA and its release from striatal slices. However, accumulation was stimulated at a concentration of K+ (14 mM) that had no significant stimulatory effect on release, suggesting that the stimulatory effects of K+ on synthesis and release are mediated by separate processes. Release of 14CO2 from [1-14C]tyrosine closely paralleled the accumulation of [3H]DA from [3H]tyrosine. Release of preloaded [14C]DA closely paralleled that of [3H]DA synthesized from [3H]tyrosine, suggesting a common functional pool. The principal DA catabolite produced was dihydroxyphenylacetic acid (DOPAC). The appearance of labeled DOPAC in the media was greatly enhanced by K+ stimulation.     

Distribution and metabolism of DDT, carbaryl and methyl parathion in S-and R-strains of Spodoptera littoralis (Boisd)

The distribution and metabolism of DDT, carbaryl and methyl parathion were studied in the haemolymph, foregut, midgut, hindgut and in the remaining tissues combined together; the fat body, cuticle and muscles constitute the major part of such tissues. The latter tissues in both S- and R-strains contained the highest amounts of the 3 insecticides.DDE, 1-naphthol and an unidentified compound were the only metabolites of DDT and carbaryl, respectively, that could be detected in the fat + cuticle tissues of both S- and R-strains. Metabolism of methyl parathion to p-nitrophenol took place in all studied tissues. In all cases metabolism appeared to be faster in R-larvae than in S-larvae.     

The metabolic profile of sodium o-phenylphenate after subchronic oral administration to rats

We examined the metabolites of o-phenylphenol (OPP) in the urine of male and female rats dosed with 2% sodium o-phenylphenate (OPP-Na) in food from the age of 5 wk for 136 days. The urinary metabolites of OPP-Na produced during the 24 hr after OPP-Na feeding accounted for 55% of the dose in male rats and 40% in females. The main metabolites were OPP-glucuronide and 2,5-dihydroxybiphenyl (2,5-DHBP)-glucuronide. OPP metabolites in the free form accounted for only 1% of the total phenolic metabolites excreted. 2,5-DHBP was rapidly converted to the corresponding quinone in aqueous solvents but not in organic solvents. There was a clear sex difference in the proportions of urinary metabolites; the amount of 2,5-DHBP excreted by male rats in 24-hr urine was more than seven times that excreted by females. This result may be related to the finding that bladder tumours occur in male but not female rats fed OPP-Na in the diet (Hiraga & Fujii, Fd cosmet. Toxicol. 1981, 19, 303).     

Assay and purity control of oxytetracycline and doxycycline by thin-layer chromatography — a comparison with liquid chromatography

A thin-layer chromatographic (TLC) method using densitometry is described for the assay and purity control of oxytetracycline and doxycycline. With a mobile phase of dichloromethane—methanol—water (59:35:6, v/v/v) and a silica gel thin-layer, previously sprayed with 10% sodium edetate solution adjusted to pH 9.0, all the potential impurities of oxytetracycline or doxycycline are well separated from the main components and from each other. Results obtained with TLC are compared with those obtained by previously established liquid chromatography (LC) methods using poly(styrene-divinylbenzene) stationary phases. A good correlation was obtained (r > 0.9999). For TLC the relative standard deviation (RSD) for the assay of the main component was <2%, for LC the RSD was <1%.