Definition of the alpha-KTx15 subfamily

Three novel scorpion toxins, Aa1 from Androctonus australis, BmTX3 from Buthus martensi and AmmTX3 from Androctonus mauretanicus were shown able to selectively block A-type K⁺ currents in cerebellum granular cells or cultured striatum neurons from rat brain. In electrophysiology experiments, the transient A-current completely disappeared when 1 μM of the toxins was applied to the external solution whereas the sustained K⁺ current was unaffected.

The three toxins shared high sequence homologies (more than 94%) and constituted a new ‘short-chain’ scorpion toxin subfamily: -KTx₁₅. Monoiododerivative of ¹²⁵I-sBmTX3 specifically bound to rat brain synaptosomes. Under equilibrium binding conditions, maximum binding was 14 fmol/mg of protein and the dissociation constant (K_d) was 0.21 nM. This K_d value was confirmed by kinetic experiments (k_on=6.0×10⁶ M⁻¹ s⁻¹ and k_off=6.0×10⁻⁴ s⁻¹). Competitions with AmmTX3 and Aa1 with ¹²⁵I-sBmTX3 bound to its receptor on rat brain synaptosomes showed that they fully inhibited the ¹²⁵I-sBmTX3 binding (K_i values of 20 and 44 pM, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. A panel of toxins described as specific ligands for different K⁺, Na⁺ and Ca²⁺ channels were not able to displace ¹²⁵I-sBmTX3 from its binding site. Thus, ¹²⁵I-sBmTX3 is a new ligand for a still unidentified target in rat brain. In autoradiography, the distribution of ¹²⁵I-sBmTX3 binding sites in the adult rat brain indicated a high density of ¹²⁵I-sBmTX3 receptors in the striatum, hippocampus, superior colliculus, and cerebellum.     

Structure-activity requirements of bombesin for gastrin-releasing peptide- and neuromedin B-preferring bombesin receptors in rat brain

The pharmacological profile of [¹²⁵I][Tyr⁴]bombesin binding to gastrin-releasing peptide- and neuromedin B-preferring sites has been investigated in rat cerebral cortex and olfactory bulb membranes, respectively. [¹²⁵I][Tyr⁴]bombesin specific binding to cerebral cortex membranes was displaced biphasically by gastrin releasing peptide and [D-Phe⁶]bombesin-(6–13)-ethyl amide. In the presence of 10 nM neuromedin B, displacement curves for bombesin-related peptides were monophasic with gastrin releasing peptide displaying approximately 100-fold higher affinity than neuromedin B. In olfactory bulb membranes, [¹²⁵I][Tyr⁴]bombesin binding was also displaced biphasically by gastrin releasing peptide, [D-Phe⁶]bombesin-(6–13)-ethyl amide and neuromedin B. In the presence of 10 μM [D-Phe⁶]bombesin-(6–13)-ethyl ester, displacement curves were monophasic with neuromedin B possessing approximately 10-fold higher affinity than gastrin-releasing peptide. Under these conditions, successive deletion of N-terminal amino acids from bombesin-(1–14) was well tolerated at both sites, with little loss in affinity up to bombesin-(5–14). A 5- to 10-fold drop in affinity was observed at both sites with bombesin-(6–14), whilst the octapeptide acetyl-bombesin-(7–14) displayed similar affinities to bombesin-(1–14). Bombesin-(8–14), -(9–14) and -(10–14) were essentially inactive (IC₅₀ > 10 μM). C-terminal deletion of Met¹⁴ (bombesin-(1–13)) resulted in 100-fold loss of affinity at the gastrin-releasing peptide site and complete loss of affinity at the neuromedin B site. Fragments smaller than bombesin-(1–13) were virtually inactive at either site. Replacement of consecutive amino acids in the minimal active fragment, acetyl-bombesin-(7–14), with L-alanine revealed the critical importance of Trp⁸ and Leu¹³ for binding to both sites.     

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and H-deoxyglucose-6-phosphate release from human red blood cells

, and . Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and ³H-deoxyglucose-6-phosphate release from human red blood cells. Toxicon 27, 1297–1305, 1989.—At a low concentration of Naja naja kaouthia cardiotoxin (3 μM) Ca²⁺, Sr²⁺ and Ba²⁺ (2 mM), had little to no effect on ³H-deoxyglucose-6-phosphate (³H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (≥ 10 μM), Ca²⁺ (2 mM), but not Sr²⁺ or Ba²⁺, significantly enhanced ³H-dGlu-6-p and hemoglobin release. Mn²⁺ (2 mM) almost completely inhibited ³H-dGlu-6-p release and hemolysis at both the 3 μM and 10 μM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 μM), Ca²⁺ at low concentrations (0.5 mM) enhanced ³H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 μM) induced ³H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. ³H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between ³H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca²⁺-dependent. The Ca²⁺-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A₂ contamination in the toxin fraction. Mn²⁺ is an effective antagonist of cardiotoxin action.     

Selective effects of [D-Ser(O-t-butyl),Leu]enkephalyl-Thr and [D-Ser(O-t-butyl),Leu]enkephalyl-Thr(O-t-butyl), two new enkephalin analogues, on neurotransmitter release and adenylate cyclase in rat brain slices

The selectivity and potency of two new enkephalin-derived δ-opioid receptor agonists, DSTBULET ([D-Ser²(O-t-butyl), Leu⁵]enkephalyl-Thr⁶) and BUBU ([D-Ser²(O-t-butyl),Leu⁵]enkephalyl-Thr⁶(O-t-butyl)) were determined with functional tests in vitro of μ-, δ-, and κ-opioid receptor activation in the rat brain. Both peptides concentration dependently (1 nM-1 μM) inhibited the release of radiolabeled acetylcholine (ACh) from striatal slices (pD₂ 7.6–7.9), an effect exclusively mediated by δ-opioid receptor activation. Fentanyl isothiocyanate (FIT), an irreversible δ-antagonist, completely blocked the inhibitory effects of DSTBULET and BUBU. Up to a concentration of 1 μM, the peptides did not affect striatal [³H]dopamine (DA) release nor cortical [³H]noradrenaline (NA) release, processes which are known to be inhibited by opioids activating κ and μ-receptors, respectively. Furthermore, both DSTBULET and BUBU caused a strong inhibition (pD₂ 8.2–8.3) of D-1 dopamine receptor-stimulated cyclic AMP efflux from striatal slices, an effect known to be mediated by μ- and/or δ-opioid receptor activation. However, the peptides were without effect when D-1 and D-2 dopamine receptors were stimulated simultaneously, a situation in which only μ-agonists are able to inhibit the resulting cAMP efflux. In conclusion, DSTBULET and BUBU appear to display a high selectivity and potency toward functional δ-opioid receptors in the brain.     

NK-1 receptors mediate the tachykinin stimulation of salivary secretion: selective agonists provide further evidence

The relative contribution of NK-1, NK-2 and NK-3 receptors to the sialogogic response to i.v. tachykinins was investigated in urethane-anesthetized rats. [Pro⁹,Met(O₂)¹¹]substance P (SP), a selective NK-1 receptor agonist, was about 10 times more potent than SP itself and its action was unaffected by pretreatment. On the other hand [Nle¹⁰]neurokinin A (NKA)-(4–10) and [MePhe⁷]neurokinin B (NKB), two selective agonist for NK-2 and NK-3 receptors, respectively, were ineffective.     

Time-dependent alteration of epidermal growth factor receptor in rat stomach by ethanol feeding

This study investigated the time-dependent effects of ethanol (EtOH) feeding on epidermal growth factor binding and epidermal growth factor-mediated functions in the stomach. Adult male rats were fed either an isocaloric control or EtOH-containing liquid diet (36% total calories as EtOH) for 2, 4 and 6 weeks. At the end of each feeding period, animals were sacrificed and the stomach was dissected for the sample preparation. EtOH caused a time-dependent alteration (r = 0.89) of the ¹²⁵I-epidermal growth factor binding to the gastric mucosal membrane (% control: week 2, 114%; week 4, 64%^* and week 6, 45%^*, n = 5, ^*P < 0.05). Protein kinase analysis also showed that EtOH caused a time-dependent decrease of epidermal growth factor-stimulated autophosphorylation of epidermal growth factor receptor protein (180 kDa) during three feeding periods. Western blot analysis, using anti-tyrosine phosphorylated epidermal growth factor receptor (active form) antibody, revealed a major immunoreactive protein band (180 kDa) in all samples pre-incubated with 1 μM epidermal growth factor. Consistent with data from kinase analysis, treatment of EtOH decreased the immunoreactivity of the active form of epidermal growth factor receptor (180 kDa) in the stomach. In conclusion, EtOH feeding caused a time-dependent alteration of epidermal growth factor receptor in the stomach, which may be one of the mechanisms underlying the gastric pathology associated with alcohol abuse.     

In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist

(S)1- {;2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl };-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR140333) is a new non-peptide antagonist of tachykinin NK₁ receptors. SR140333 potently, selectively and competitively inhibited substance P binding to NK₁ receptors from various animal species, including humans. In vitro, it was a potent antagonist in functional assays for NK₁ receptors such as [Sar⁹,Met(O₂)¹¹]substance P-induced endothelium-dependent relaxation of rabbit pulmonary artery and contraction of guinea-pig ileum. Up to 1 μM, it had no effect in bioassays for NK₂ ([βAla⁸]neurokinin A-induced contraction of endothelium-deprived rabbit pulmonary artery) and NK₃ ([MePhe⁷]neurokinin B-induced contraction of rat portal vein) receptors. The antagonism exerted by SR140333 toward NK₁ receptors was apparently non-competitive, with pD'₂ values (antagonism potency evaluated by the negative logarithm of the molar concentration of antagonist that produces a 50% reduction of the maximal response to the agonist) between 9.65 and 10.16 in the different assays. SR140333 also blocked in vitro [Sar⁹,Met(O₂)¹¹]substance P-induced release of acetylcholine from rat striatum. In vivo, SR140333 exerted highly potent antagonism toward [Sar⁹,Met(O₂¹¹]substance P-induced hypotension in dogs (ED₅₀ = 3 μg/kg i.v., bronchoconstriction in guinea-pig (ED₅₀ = 42 μg/kg i.v.) and plasma extravasation in rats (ED₅₀ = 7 μg/kg i.v.). Finally, it also blocked the activation of rat thalamic neurons after nociceptive stimulation (ED = 0.2 μg/kg i.v.).     

Distribution of tritiated tetrodotoxin administered intraperitoneally to pufferfish

S. , Y. , M. , N. , K. , T. , N. and K. . Distribution of tritiated tetrodotoxin administered intraperitoneally to pufferfish. Toxicon 25, 1283 – 1289, 1987. — Tetrodotoxin was recoil-tritiated by the ³He(n,p)³H reaction and purified by gel filtration. The [³H]tetrodotoxin gave only one spot in both cellulose acetate strip electrophoresis and thin layer chromatography. The specific toxicity of tetrodotoxin did not decrease during the recoil tritiation and the [³H]tetrodotoxin showed a specific radioactivity of 25 × 10⁻⁶ Ci/mmole. In spite of the low specific radioactivity, the [³H]tetrodotoxin was able to be used to investigate the anatomical distribution of tetrodotoxin in pufferfish. When intraperitoneally injected into ‘torafugu’ puffer, [³H]tetrodotoxin accumulated in most tissues, the level being highest in the skin, followed by the liver, intestines and muscle. With time, the [³H]tetrodotoxin radioactivity level in the injected pufferfish decreased in most tissues, except for skin and gallbladder. Based on these results, the metabolism of tetrodotoxin in pufferfish is discussed.     

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

Characterisation of calcitonin gene-related peptide receptors in rat atrium and vas deferens: evidence for a [Cys(Et)(2, 7)]hCGRP-preferring receptor

The present study was performed in order to characterise calcitonin gene-related peptide (CGRP) receptor subtypes in rat left atrium and vas deferens by using [R-(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide (BIBN4096BS), a novel CGRP receptor antagonist. When CGRP was used as an agonist, BIBN4096BS exhibited an almost 10-fold higher affinity for CGRP receptors in rat left atrium compared to those in the vas deferens, indicating that CGRP acts through different CGRP receptor subtypes in these two tissues. In addition, BIBN4096BS was almost 10-fold more potent in antagonizing [Cys(Et)^2,7]hCGRP and human adrenomedullin-induced responses than CGRP-induced responses in rat vas deferens. This might indicate receptor heterogeneity in rat vas deferens. Accordingly, the present work provides first experimental evidence that the rat vas deferens contains two CGRP-like receptor subtypes. Namely, the CGRP₂ receptor and a “novel” receptor that possesses low efficacy for CGRP and that is selectively stimulated by [Cys(Et)^2,7]hCGRP or adrenomedullin and which can be blocked with high affinity by BIBN4096BS.     

Spectrophotometric determination of pefloxacin in pharmaceutical preparations

It has been established that the antibiotic pefloxacin (Abaktal) methane-sulphonate reacts with Fe(III) at pH 1.00–8.00 to form a water-soluble complex with maximum absorbance at 360 nm. The composition of the complex, determined spectrophotometrically by the application of Job's, molar-ratio and Bent—French's methods, was pefloxacin: Fe(III) = 1:1 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The relative stability constant, obtained by the methods of Sommer and Asmus was 10^5.02 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The molar absorptivity of the complex at 360 nm was found to be 4.8 × 10³ l mol⁻¹ cm⁻¹, Beer's law was followed for pefloxacin concentrations of 2.15–85.88 μg ml⁻¹. The lower sensitivity limit of the method was 2.15 μg ml⁻¹. The relative standard deviation (n = 10) was 0.57–1.07%. The method can be applied to the rapid and simple determination of pefloxacin in aqueous solutions and tablets.     

Characterization of polymethoxylated flavones in Fructus aurantii by liquid chromatography with atmospheric pressure chemical ionization combined with tandem mass spectrometry

Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) was operated in positive mode (PI) to characterize polymethoxylated flavonoids (PMFs) through its specific radical cations by collision-induced dissociation (CID). The fragments of [M + H − n × 15]⁺ produced by loss of one or more methyl group from the protonated molecule, as well as [M + H − 29]⁺, [M + H − 31]⁺, [M + H − 33]⁺, [M + H − 43]⁺, [M + H − 46]⁺, and [M + H − 61]⁺ fragment ions were detected, which were diagnostic for the polymethoxylated species, and could be adopted to form the multiple MS (MSⁿ) “fingerprint” of PMFs. Based on this “fingerprint”, 29 PMFs were screened out from extracts of Fructus aurantii, among which two of them were identified as sinensetin and tangeretin. It was proved that the PI was suitable for structural characterization of PMFs by APCI-MSⁿ.     

Gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acidB receptor (GABABR) binding sites are distinctive from one another: molecular evidence

γ-Hydroxybutyric Acid (GHB) is thought to be a weak partial agonist at the γ-aminobutyric acid_B Receptor (GABA_BR), but the precise relationship of the GHB receptor (GHBR) to the GABA_BR remains unclear. In order to test the hypothesis that the GHBR is not identical to the GABA_BR, we conducted two groups of experiments. First, GABA_BR subtype 1 (R1) and/or subtype 2 (R2) were over expressed in HEK 293 cells and membrane binding studies on the transfected cells done using [³H]GHB and [³H] (2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([³H]NCS-382). The latter is a specific antagonist at the GHB binding site. Second, [³H]GHB and [³H]NCS-382 autoradiographic binding studies were done on the brains of mice in which the gene for GABA_BR1a was deleted. Such mice do not have a functioning GABA_BR. There was no detectable specific [³H]GHB or [³H]NCS-382 binding in HEK 293 cells transfected with GABA_BR1, R2, or R1/R2. Binding to [³H]CGP54626A, a high affinity GABA_BR antagonist, was absent in GABA_BR1a^−/− mice. There was no difference in [³H]NCS-382 binding observed in the brains of GABA_BR1a^−/−, GABA_BR1a^+/− or GABA_BR1a^+/+ mice. Specific [³H]GHB binding was observed in the brain of GABA_BR1a^−/− mice but was significantly lower than in wild type mice. These data support the hypothesis that the GHB binding site is separate and distinct from the GABA_BR.     

Characterization of rabbit primary proximal tubule kidney cell cultures grown on Millicell-HA membrane filters

With the aim of developing a kidney cell culture system that can be used to assess renal toxicity in vivo, freshly isolated rabbit proximal tubules were plated on Millipore cellulose filters mounted in plastic inserts (Millicell-HA). DNA synthesis peaked on day 6 of culture and cells reached confluency by days 12–14. The integrity of the monolayer was confirmed by exclusion of [¹⁴C]inulin and cell viability demonstrated by linearity of protein synthesis over a 24-hr period. In confluent cultures, the organic anion, [¹⁴C]p-aminohippuric acid (PAH) and cation [¹⁴C]tetraethylammonium bromide (TEA) were shown to be transported from the basolateral to the apical side at a rate 5–6 times greater than that from the apical to basolateral side during the first 60 min of exposure. Probenecid decreased PAH transport by 60% and N-methylnicotinamide and quinine inhibited TEA transport by 40 and 56%, respectively. Uptake of [¹⁴C]-methylglucopyranoside into the cells was three times greater when label was added to the apical side than when label was added to the basolateral side. Apical uptake of glucose was sodium dependent and inhibited by more than 90% with phloridzin. Thus, kidney proximal tubule cells in the filter insert culture system display functional polarity which appears to mimic function in vivo and may be useful for examining mechanisms of nephrotoxicity.     

A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 1: system characterisation

Purpose: To establish an in vitro system for the rapid assessment of the affinities of potential substrates for the di/tri/oligopeptide transport system (DTS). Methods: Monolayers of Caco-2 cells were cultured in plastic wells for 7–9 days and the uptake of Gly-[³H]L-Pro, a specific and relatively stable substrate for the DTS was used as an affinity probe. Gly-[³H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [³H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3 min. The uptake of radiolabel was determined by liquid scintillation counting. Results: High specific-uptake (>85%) of Gly-[³H]L-Pro was obtained with cells grown for 7–9 days. Gly-[³H]L-Pro uptake had a substantial active concentration-dependent component (K_m of 0.39±0.02 mM, V_max of 0.98±0.04 nmol min⁻¹ (mg protein)⁻¹. This process was shown to be specific for the DTS as evidenced by the significant inhibition by compounds reported to be transported by this system and the lack of inhibition by amino acids. The use of low competitor concentrations (1 mM) enabled a range of inhibition values (0–89%) of a series of competitors (amino acids, dipeptides and β-lactam antibiotics) to be estimated, illustrating that structurally similar compounds can be ranked for affinity to the DTS. Conclusion: A screening system, using Caco-2 cells and the dipeptide Gly-[³H]L-Pro as a displaceable probe, was developed to assess a variety of compounds for recognition by the di/tri/oligopeptide transport system. This fully describes the first system that allows structurally related compounds to be ranked on the basis of their affinity for the DTS recognition site.     

Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus

The effects of selective opioid agonists on the evoked release of [³H]dopamine and [³H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embroyonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [³H]dopamine from dopaminergic substantia nigra/ventral tegmental aera neurons induced by 23 mM k⁺ appeared to be inhibited exclusively by activation of κ-opioid receptors, as [³H]dopamine release was inhibited selectively by the κ- agonists U69,593 and dynorphin-(1–13) (EC₅₀ 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the κ-selective antagonist nor-binaltorphine (K_i 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory μ-opioid receptors only, as evoked [³H]noradrenaline release was inhibited selectively by the μ agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (EC₅₀ 45 nM), a response that was antagonized by the preferential μ antagonist naloxone (K_i = 0.7 nM). The δ-opioid receptor agonist [D-Ser²(O-butyl), Leu⁵]enkephaly-Thr⁶ did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 μM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to κ receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of μ receptors. It is therefore concluded that in rat brain neurotransmitter release from dopaminergic and noradrenergic neurons, originating from the substantia nigra/vental tegmental area and the locus coeruleus, is liable to inhibition by homogenous populations of κ- and μ-opioid receptors, respectively, independent of the input of non-opioid neurons from distict nuclei.     

In vitro dermal absorption of pesticides: V. In vivo and in vitro comparison of the herbicide 2,4-dichlorophenoxyacetic acid in rat, guinea pig, pig, human and tissue-cultured skin

In vitro dermal absorption tests were conducted with the ¹⁴C-ring-labelled herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 7–8 μg/cm². Skin absorption was determined for 48 hr after exposure using an in vitro flow-through system. Skin absorption was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution and the percentage recovery in the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline, and Hanks' HEPES buffered saline with 4% serum albumin were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 47 ± 4.3% [tissue cultured Testskin; Hanks' receiver (HR)], 40 ± 4.5% (rat; HR), 19 ± 1.8% (human; HR), 14 ± 2.3% (hairless guinea pig; HR), 14 ± 8.8% (pig; Ringer's receiver). The percentage recovery of the radiolabel in soapy water skin washes at 24 hr, methanol washes and skin digests at 48 hr, and of ¹⁴C-labelled volatiles collected in air traps at 48 hr after exposure are reported. Comparative in vivo studies were conducted for 14 days after exposure and demonstrated 32 ± 3.9 and 28 ± 7.8% recovery of ¹⁴C in the urine of rats (dose rate, 3 μg/cm²) and guinea pigs (dose rate, 4 μg/cm²), respectively. Total faecal recovery was 2 ± 0.3 and 9 ± 3.5% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 day after dosing demonstrated a total tissue recovery of ¹⁴C activity of 1 ± 0.1 and 2 ± 0.5% in rats and guinea pigs, respectively. Including the ¹⁴C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 49 ± 10.4 and 40 ± 9.9% in rats and guinea pigs, respectively. Recovery of ¹⁴C activity from soapy water skin washes conducted at 24 hr after exposure was 28 ± 8.1 and 43 ± 9.0% for rats and guinea pigs, respectively. Recovery in skin patches was 18% (guinea pigs) and 26% (rats). In summary, the in vitro/in vivo concordance for the rat dermal absorption data was good but the in vitro data for hairless guinea pigs underestimated the in vivo absorption, and therefore for 2,4-D, rat skin may provide a better model of percutaneous absorption.     

Interaction between theophylline and ouabain in the rabbit heart: Effect on (Na + K)-ATPase

In electrically stimulated rabbit ventricular strips, theophylline (0.3–30 M) antagonized the increased contractility produced by ouabain (0.8 μM). Initial velocity of specific [³H]ouabain binding to homogenates prepared from the muscle strips was used to determine the fraction of binding sites occupied by ouabain. Theophylline decreased the binding of ouabain to (Na⁺ + K⁺)-ATPase. It is concluded that the effect of theophylline on ouabain-induced positive inotropism may be mediated by decreased ouabain binding to (Na⁺ + K⁺)-ATPase.     

Use of palladium(II) chloride as colour-forming reagent in determination of pralidoxime chloride in water and tablets

Pralidoxime chloride (PAM-2Cl) has been determined spectrophotometrically in Britton—Robinson buffer solution at pH = 6.45; the method is based on measurement of the absorbance of the Pd(II)-pralidoxime complex at 327 nm. Studies of the composition of the complex by Job's continuous variation method, the molar ratio method and Bent—French's method yielded a Pd(II):pralidoxime ratio of 1:1. The conditional stability constant (K′) of the complex at the optimum pH of 6.45 and an ionic strength (μ) of 0.3 M was found to be 10^5.2. The molar absorptivity was 1.05 × 10⁴ 1 mol⁻¹ cm⁻¹. Beer's law was obeyed at concentrations up to 60 μM. The detection limit was 0.55 μg ml⁻¹. The relative standard deviation (N = 10) was 0.28–1.03%. The method was accurate and sensitive for the analysis of PAM-2Cl in water and tablets.     

Synthesis, organotropism and hepatocellular uptake of two tritium-labeled epimers of dihydromicrocystin-LR, a cyanobacterial peptide toxin analog

J. A. O. , S. E. , A. M. and J. E. . Synthesis, organotropism and hepatocellular uptake of two tritium-labeled epimers of dihydromicrocystin-LR, a cyanobacterial peptide toxin analog. Toxicon, 28, 1439–1446, 1990.—Two tritium-labeled epimers of dihydromicrocystin-LR, a derivative of the cyanobacterial peptide hepatotoxin microcystin-LR, were synthesized by reduction with sodium boro[³H]hydride and purified with reversed-phase liquid chromatography. The epimers were hepatotoxic in mice; the i.p. ₅₀ was 120–135 μg/kg. They were concentrated in the liver and to some extent in the intestine and the kidney after an i.v. injection. Freshly isolated rat hepatocytes showed a rapid uptake of both epimers. The cellular uptake of the epimers was almost complete within 5 min at concentrations 1 μM (0.5 μM dihydromicrocystin-LR + 0.5 μM microcystin-LR) and 4 μM (0.5 μM + 3.5 μM). The uptake of the earlier eluting epimer was about three times higher than that of the later eluting epimer.