Platelets enhance contractility in perfused rat mesenteric arteries: Involvement of endothelin-1

We investigated the effects of platelet supernatant on pressor responses to norepinephrine in isolated perfused rat mesenteric arteries. Perfusion of the arteries with platelet supernatant for 2 h markedly enhanced the pressor responses to norepinephrine (10⁻⁶ and 3×10⁻⁶ M). This enhancement was significantly inhibited by phosphoramidon (10⁻⁴ M), an endothelin converting enzyme inhibitor. Both BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-

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-γ-methylleucyl-

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-1-methoxycarbonyltryptophanyl-

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-norleucine] (10⁻⁶ M), an endothelin ET_B receptor antagonist, and bosentan (Ro47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2´-bipyrimidin-4-yl]-benzenesulfonamide) (10⁻⁵ M), a nonselective endothelin receptor antagonist, also prevented the potentiation of responses to norepinephrine evoked by platelet supernatant, but FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methyl-pentanoyl] amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl) propionic acid) (10⁻⁶ M), an endothelin ET_A receptor antagonist, had little effect. Suppressor doses of endothelin-1 (3×10⁻¹⁰ M) or sarafotoxin S6c (S6c) (3×10⁻¹⁰ M) potentiated significantly the norepinephrine-induced vasoconstriction, in the same preparation. Moreover, supernatant-induced enhancement of pressor responses to norepinephrine was markedly suppressed by TGF-β1 neutralizing antibody. Transforming growth factor-β1 (TGF-β1) (40 pM) also significantly enhanced the pressor responses to norepinephrine (10⁻⁶ M) and this enhancement was significantly inhibited by phosphoramidon. These results suggest that platelet-derived TGF-β1 stimulates the vascular production of endothelin-1 and thereby enhances vasoconstrictor responses to norepinephrine. Platelet-induced enhancement of vasoconstrictor responses to norepinephrine seems to be mainly mediated by endothelin ET_B receptor, in rat mesenteric arteries.     

Trace iron determination in aminoisophthalic acid using differential-pulse cathodic stripping voltammetry at carbon paste electrodes

Application of differential-pulse cathodic stripping voltammetry using a carbon paste electrode (consisting of carbon powder and liquid paraffin) have been investigated for trace determination of iron in 5-aminoisophthalic acid (AIPA). Samples were dissolved in 1 M HCl, pH was adjusted to 4–5 after addition of EDTA. Voltammetric measurements were performed after filtration. No sample decomposition (mineralization) was necessary. The method showed a good linearity between current and concentration from 3×10⁻⁷ to 5×10⁻⁵ mol dm⁻³ of iron, with a detection limit of 3×10⁻⁷ mol dm⁻³ (resp. 1 ppm in solid AIPA). The results agreed well to those obtained by atomic absorption spectrometry (AAS) using electrothermic atomisation. For AAS measurement, however, microwave digestion of samples was necessary.     

Anodic voltammetry of cefotaxime

In this study electrooxidation of cefotaxime was investigated using specially activated glassy carbon (GC), platinum and carbon paste (CP) electrodes in different supporting electrolyte solutions and at different pHs. The data revealed that the shapes of the voltammograms and the numbers of the oxidation steps changed depending on the nature of the electrode. The nature of the supporting electrolyte was also important for the response of the electrode. From an analytical point of view, the activated GC electrode was the most favourable one. In 0.2 M H₃PO₄ with an activated GC electrode the calibration graph gave two lines with different slopes in the concentration ranges of 2×10⁻⁵–1×10⁻⁴ and 2×10⁻⁴–6×10⁻⁴. The results of the recovery test and statistical analysis showed that the voltammetric method could be used for the determination of cefotaxime.     

Role of angiotensin II in the response to endothelin-1 of goat cerebral arteries after ischemia–reperfusion

As angiotensin II may underlie the deleterious effects of some vascular diseases, we have examined the role of this peptide on the cerbrovascular endothelin-1 action after ischemia–reperfusion. In anesthetized goats, 1 hour-occlusion followed by 1 hour-reperfusion of the left middle cerebral artery (MCA) was induced, and then segments 3-mm in length from branches of the right MCA (control) and the left MCA (ischemic) were obtained for isometric tension recording. Endothelin-1 (10^− 11–10^− 7 M) produced a contraction that was higher in ischemic than in control arteries, and in control but not in ischemic arteries this contraction was potentiated by angiotensin II (10^− 7 M). Losartan (3 × 10^− 6 M), antagonist of AT₁ receptors, did not affect the response to endothelin-1 in control arteries, but reduced it both in ischemic arteries and angiotensin II-treated control arteries. PD123,319 (3 × 10^− 6 M), antagonist of AT₂ receptors, or the inhibitor of nitric oxide synthesis l-NAME (10^− 4 M) did not alter the arterial effects of endothelin-1. Therefore, angiotensin II may potentiate the constriction to endothelin-1 in normal cerebral arteries by activating AT₁ receptors. The observed cerebrovascular increased response to endothelin-1 after ischemia–reperfusion might be related in part to activation of AT₁ receptors under this condition.     

Mechanism of prostaglandin E2-, F2α- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E₂-, prostaglandin F_2α- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F_2α)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E₂ caused maximum relaxation of endothelin-1 precontracted vessels (EC₅₀: 1.8×10⁻⁸ M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor,

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-NAME (N^G-Nitro-

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-arginine methylester). CGRP-(8–37) (calcitonin gene-related peptide fragment (8–37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK₁ receptor blocker, GR 82334 ([

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-Pro⁹[Spiro-γ-Lactam]Leu¹⁰,Trp¹¹]physalaemin (1–11)), markedly attenuated the response. Both prostaglandin F_2α and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1α(Z),2β,3β,5α]]-(+)-7-[5-([1,1′-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), a thromboxane receptor antagonist (EC₅₀: for prostaglandin F_2α 7.9×10⁻⁹ M, and for latanoprost acid 4.9×10⁻⁹ M).

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-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8–37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E₂ could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

Biopharmaceutical classification of drugs using intrinsic dissolution rate (IDR) and rat intestinal permeability

The solubility and dissolution rate of active ingredients are of major importance in preformulation studies of pharmaceutical dosage forms. In the present study, passively absorbed drugs are classified based on their intrinsic dissolution rate (IDR) and their intestinal permeabilities. IDR was determined by measuring the dissolution of a non-disintegrating disk of drug, and effective intestinal permeability of tested drugs in rat jejunum was determined using single perfusion technique. The obtained intrinsic dissolution rate values were in the range of 0.035–56.8 mg/min/cm² for tested drugs. The minimum and maximum intestinal permeabilities in rat intestine were determined to be 1.6 × 10⁻⁵ and 2 × 10⁻⁴ cm/s, respectively. Four classes of drugs were defined: Category I: P_eff,rat > 5 × 10⁻⁵ (cm/s) or P_eff,human > 4.7 × 10⁻⁵ (cm/s), IDR > 1(mg/min/cm²), Category II: P_eff,rat > 5 × 10⁻⁵ (cm/s) or P_eff,human > 4.7 × 10⁻⁵ (cm/s), IDR < 1(mg/min/cm²), Category III: P_eff,rat < 5 × 10⁻⁵ (cm/s) or P_eff,human < 4.7 × 10⁻⁵ (cm/s), IDR > 1 (mg/min/cm²) and Category IV: P_eff,rat < 5 × 10⁻⁵ (cm/s) or P_eff,human < 4.7 × 10⁻⁵ (cm/s), IDR < 1(mg/min/cm²). According to the results obtained and proposed classification of drugs, it is concluded that drugs could be categorized correctly based on their IDR and intestinal permeability values.     

Electrochemical behavior and determination of rutin on a pyridinium-based ionic liquid modified carbon paste electrode

In this paper the electrochemical behavior of rutin on a pyridinium-typed ionic liquid modified carbon paste electrode (IL-CPE) was investigated and further used for rutin sample determination. The IL-CPE showed strong electrocatalytic effects to the oxidation of rutin. In phosphate buffer solution (PBS, pH 2.5; 0.1 M) a pair of well-defined cyclic voltammetric redox peaks of rutin appeared with the redox peak located at 512 mV (Epa) and 448 mV (Epc) (vs. SCE), respectively. The redox peak current was increased about 27.5 times more than that on traditional carbon paste electrode (CPE). The electrochemical parameters of rutin on the IL-CPE were calculated with the results of the charge transfer coefficient (α), the number of electron transfer (n) and the electrode reaction rate constant (k_s) as 0.53, 1.80 and 2.39 s⁻¹, respectively. The cathodic peak currents increased linearly with the concentration of rutin in the range from 5.0 × 10⁻⁷ to 1.0 × 10⁻⁴ M with the detection limit as 3.58 × 10⁻⁷ M (3σ). The relative standard deviation (RSD) of 10 successive detection of 5.0 × 10⁻⁵ M rutin was 4.2%. The method was successfully applied to the determination of rutin content in tablets samples with good recovery. The modified electrode showed good stability and reproducibility without the influence of the coexisting substances.     

Negative regulation by dexamethasone of fluvastatin-inducible CYP2B expression in primary cultures of rat hepatocytes: role of CYP3A

Fluvastatin (Fluva), a synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, induces CYP2B1/2 in rat liver and primary cultured rat hepatocytes. However, the overall profile of CYP induction, which includes induction of CYP4A, suggests that Fluva is not a typical "phenobarbital (PB)-like" inducer. Several treatments affecting diverse cell signaling pathways have been reported to modify PB-inducible CYP2B expression in primary cultured rat hepatocytes. We examined the effects of selected treatments on the ability of Fluva to induce CYP2B1/2 mRNA. Only dexamethasone (Dex) produced effects on Fluva-inducible CYP2B1/2 mRNA expression that differed from those produced on PB-inducible CYP2B1/2 mRNA expression. Dex concentrations up to 10(-7) M of potentiated PB (10(-4) M)-mediated CYP2B1/2 mRNA induction, while higher Dex concentrations produced a progressive reduction in PB-induced CYP2B1/2 mRNA levels. By contrast, Dex concentrations up to 10(-8) M had no effect on Fluva (3 x 10(-5) M)-induced CYP2B1/2 mRNA levels, while Dex concentrations of 10(-7) M and higher markedly suppressed Fluva-mediated CYP2B1/2 mRNA induction. The concentrations of several glucocorticoids that produced suppression of Fluva-induced CYP2B1/2 mRNA levels were the same concentrations that induced CYP3A mRNA. Treatment with pregnenolone 16 alpha-carbonitrile also produced a concentration-dependent suppression of Fluva-induced CYP2B1/2 mRNA levels. Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA was concentration-dependently reversed when hepatocytes were cotreated with troleandomycin, a selective CYP3A inhibitor. The amounts of Fluva detected in culture medium and cells were reduced significantly when hepatocytes were incubated with Dex. However, Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA expression was not overcome when hepatocytes were incubated with Fluva concentrations greater than 3 x 10(-5) M, suggesting that mechanisms other than CYP3A-catalyzed metabolism may contribute to Dex-mediated suppression of Fluva-induced CYP2B1/2 expression.     

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents

Q.H. Gong, S.J. Wieland, J.E. Fletcher, G.E. Conner and M.S. Jiang. Effect of a phospholipase A₂ with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A₂ with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca²⁺-activated K⁺current; the sensitivity for voltage activation of the K⁺ current was enhanced by increasing free Ca²⁺ in the recording pipette from 10⁻⁸ M to 2 × 10⁻⁶ M. In contrast, peripheral blood lymphocytes possessed voltage-activated K⁺ currents that were inhibited by increasing intracellular Ca²⁺.Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10⁻⁶ M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K⁺ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K⁺ currents in human lymphocytes, suggesting a specific increase in intracellular Ca²⁺ levels in both cell types.The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca²⁺ levels. Exposure of either lymphocytes or epithelial cells to toxin (10⁻⁶ M) resulted in a transient increase in Ca²⁺. However, while the Ca²⁺ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca²⁺-regulated K⁺ channels may reflect an irreversible action of the B.fasciatus phospholipase A₂ on a Ca²⁺-dependent regulatory process.     

Quantitative assessment of the negative catalytic effects of a cationic surfactant myristyl-γ-picolinium chloride on the specific-acid catalyzed epimerization of 15(S)-15-methyl prostaglandin F2α

The specific-acid catalyzed epimerization of 15(S)-15-methyl PGF_2α was found to be significantly inhibited in the presence of a cationic surfactant, myristyl-γ-picolinium chloride, at pH 2.5 and 25°C. The micellar inhibition observed is attributed to electrostatic repulsion between hydronium ion and the cationic polar heads of the micellar phase. As expected, at a given concentration of prostaglandin, the observed rate decreased as the surfactant concentration increased. When the concentration of prostaglandin was in the order of 7 × 10⁻² mg/ml (2 × 10⁻⁷M), the observed epimerization rate in a 1.0% surfactant solution was found to be approximately 120 times slower than that observed in the absence of surfactant. A quantitative analysis showed that the epimerization rate constant in the micellar phase is in the order of 6 × 10⁻⁷ s⁻¹ (cf. 2 × 10⁻⁴ s⁻¹ in the absence of the surfactant).However, the extent of micellar inhibition decreased as the initial concentration of prostaglandin increased in a series of solutions containing a constant amount of the surfactant. In order to quantitatively interpret this result, the apparent partition coefficient (ψm) of the prostaglandin between the micellar phase and the aqueous bulk phase was determined using a partitioning technique. Heterogeneity of the binding sites was detected in the analysis of ψm using the Scatchard equation.The primary binding sites are postulated to occur by a short penetration of prostaglandin molecules in the palisade layer. The secondary binding sites can be provided by either a simple adsorption process of prostaglandin onto the surface of micelles or the formation of mixed micelles. Although the primary binding sites show approximately 75-fold greater affinity towards the prostaglandin than the secondary binding sites, the number of available sites of the latter is approximately 25 times greater than that of the former. It is proposed that the Scatchard equation be used in the quantitative analysis of the effects of the substrate concentration at a constant surfactant concentration upon the observed micellar catalysis, a subject which has been unjustifiably neglected in the past.     

Biphasic effects of mianserin and desipramine on serotonin-evoked current and Cl efflux in Xenopus oocytes

Serotonin (5-HT, 1 μM) elicited two phases of Cl⁻ inward current in Xenopus oocytes injected with rat brain mRNA: a transient current (T-current), which was generated rapidly (within 1 min), and a sustained current (S-current), which persisted for 10 min. Each type of 5-HT-evoked response was time-dependent after mRNA injection. The T-current was generated at 20-30 h and the S-current at 30–40 h. Although mianserin at 0.1 μ M completely inhibited the T-current, 10 μ M mianserin was required to suppress the S-current. 5-HT also caused Cl⁻ efflux from oocytes preloaded with ³⁶Cl⁻, Cl⁻ efflux during 1 min, corresponding to the T-current, was inhibited by 0.1 μ M mianserin. A higher concentration of mianserin (10 μ M) was required to block the efflux for 10 min, corresponding to the S-current, as well as the current response. Desipramine selectively inhibited the T-current and Cl⁻ efflux for 1 min. The mechanisms underlying the different sensitivity to mianserin of oocytes injected with rat brain mRNA are discussed.     

Tyrosine kinase inhibitors suppress prostaglandin F2α-induced phosphoinositide hydrolysis, Ca elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F_2α- and carbachol-induced inositol-1,4,5-trisphosphate (IP₃) production, [Ca²⁺]_i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F_2α and carbachol induced contraction in a concentration-dependent manner with EC₅₀ values of 0.92×10⁻⁹ and 1.75×10⁻⁸ M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F_2α, but not those evoked by carbachol, on IP₃ accumulation, [Ca²⁺]_i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F_2α analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 μM) markedly reduced (by 67%) prostaglandin F_2α-stimulated increase in [Ca²⁺]_i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC₅₀ of 82 μM and increased IP₃ generation in a concentration-dependent manner with an EC₅₀ of 90 μM. The effects of vanadate were abolished by genistein (10 μM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F_2α- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca²⁺]_i evoked by prostaglandin F_2α. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F_2α receptor activation and increases in intracellular Ca²⁺ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.     

Purification scheme for cytochrome P-450 of blue crab callinectes sapidus rathbun

Cytochrome P-450 from the hepatopancreas of male blue crab, Callinectes sapidus Rathbun, was partially purified using n-octylamine Sepharose 4B and hydroxylapatite chromatography methods. Pure P-450 is needed for amino acid analysis, reconstitution, and immunological studies. The initial step in purifying blue crabP-450 was to develop a stabilization buffer (0.1 M potassium phosphate, pH 7.3, 1 mM DTT, 1 mM EDTA, and 20% glycerol including 1% USA. 5 μM trypsin inhibitor, 0.1 mM barbital, and 0.1 mM phenobarbital) to prevent or minimize proteolytic conversion toP-420. The CO-difference spectrum has a maximum at 450 nm, and an absolute oxidized spectrum maximum at 418 nm, characteristic of hemoproteins. The highest specific content obtained was 1.8 nmol/mg. Its SDS-acrylamide electrophoresis pattern had one faint band with an estimated molecular weight of 50 000. Type 2 substrate binding to microsomes had apparent dissociation constants (A'^app) of 6–10 mM (aniline), and 0.3 mM and 29 × 10⁻⁴ mM (n-octylamine). Their A_min were 403 and 410 nm. and their A_max were 430 and 428–430 nm, respectively, TheP-450 eluted from the affinity column using Triton N101 (a nonionic detergent had values of 0.9 and 0.4 mM for aniline and n-octylamine, respectively. The type 1 ligand SKF-525A had a value of 1.4 × 10¹ mM with A_min 416 and A_max 375 nm for microsomes. The binding of these ligands convert crabP-450 to P-420, More work is needed 10 determine if the n-octylamine binding data indicate twoP-450 isoenzymes. If cytochromeP-450 of crabs is inducible the discussed purification scheme wouid be useful in elucidating the role played by cytochromeP-450 in mediating zenobiotic-induced biological effects.     

Novel and selective potentiometric membrane sensor for amiloride determination in pharmaceutical compounds and urine

A new PVC membrane sensor is described as a potentiometric sensor for amiloride. The sensor having amiloride–sodium tetraphenyl phthalate (ion-pair) as an electroactive material and dibutyl phthalate (DBP) as an anion excluder in PVC matrix in the percentage ratio of 4:66:30 (ion-pair: DBP:PVC) (w/w). The membrane sensor exhibits suitable response to amiloride in a concentration range of 1.0 × 10⁻² to 1.0 × 10⁻⁶ mol L⁻¹ with a limit of detection of 9.9 × 10⁻⁷ mol L⁻¹. The slope of the system was −54.3 ± 1.0 mV decade⁻¹ over pH range of 2.0–7.0. Selectivity coefficients for amiloride relative to a numbers of potential interfering substances were investigated. The sensor was highly selective for amiloride over a large number of similar compounds. The sensor showing a fast response time of 6 s and was used over a period of 2 months with a good reproducibility. The sensor was successfully applied to determination of amiloride in pharmaceutical samples with satisfactory results.     

VITAMIN K1 ATTENUATES HYPOXIA-INDUCED RELAXATION OF RAT CAROTID ARTERY

Vascular responses to hypoxia are heterogeneous and involve the release of vasodilators substances such as nitric oxide (NO) and prostacyclin (PGI₂). In vitro studies have shown that Vitamin K₁ modulates the release of arachidonic acid (AA) in vascular cells, and thus inhibits the capacity of blood vessels to synthesise vasodilator AA metabolites. The aim of our work was to investigate the effects of Vitamin K₁ on the hypoxia-induced vasorelaxation. Hypoxia was induced by changing the gas from 95% O₂/5% CO₂ to a mixture containing 95% N₂/5% CO₂. Rat carotid arteries were pre-contracted with phenylephrine (Phe, 10⁻⁸ mol/l) and when the contraction reached a plateau, the bath was bubbled with 95% N₂/5% CO₂ for 15 min. In intact rings, there was a total relaxation after 15 min of exposure to hypoxia. Removal of the endothelium strongly reduced hypoxia-induced relaxation. In intact rings, indomethacin and -NAME reduced the hypoxic relaxation after 5 min of exposure but not after 10 or 15 min. Exposure of endothelium-intact rings to Vitamin K₁ (5×10⁻⁶ and 5×10⁻⁵ mol/l), -NAME+indomethacin as well as the combination of -NAME+indomethacin+Vitamin K₁ reduced the hypoxic relaxation after 5 and 10 min of exposure but not after 15 min. At 5×10⁻⁷ mol/l Vitamin K₁ did not attenuate hypoxia-induced relaxation. It was also found that Vitamin K₁ (5×10⁻⁶ and 5×10⁻⁵ mol/l) inhibited ACh-induced relaxation in normoxic conditions. These results show that the effect of Vitamin K₁ on attenuating hypoxia-induced vasorelaxation is concentration-dependent and probably related to its action on endothelial cells.     

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 μM MTF and 50 μM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2′-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10–1000 μM MTF and 100–500 μM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5–50 ng/ml epidermal growth factor or 5–100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.     

Prejunctional α-adrenoceptors regulate nitrergic neurotransmission in the rabbit urethra

We evaluated the effects of prejunctional α-adrenoceptors on nitric oxide (NO)-mediated urethral relaxation in rabbits using a muscle bath technique and high-performance liquid chromatography coupled with a microdialysis procedure. The amount of NO₂⁻/NO₃⁻ released during electrical field stimulation was measured by an NO₂⁻/NO₃⁻ analyzer based on the Griess method. Pretreatment with phenylephrine (0.01 μM) and yohimbine (0.1–10 μM) significantly reduced the relaxation responses induced by electrical field stimulation. In contrast, pretreatment with clonidine (0.01 μM) and prazosin (0.01–1 μM) enhanced the relaxation responses. Cys-NO-induced relaxations of rabbit urethral smooth muscle were not affected by pretreatment with α-adrenoceptor agonists and antagonists. The amount of NO₂⁻/NO₃⁻ released by electrical field stimulation increased after pretreatment with clonidine (0.01 μM) and prazosin (0.01–1 μM), but decreased after pretreatment with phenylephrine (0.01 μM) and yohimbine (0.1–10 μM). The results suggest that the release of NO from nitrergic nerves in the rabbit urethra is reduced and increased by stimulation of prejunctional α₁- and α₂-adrenoceptors, respectively.     

Ingestion of toxic Microcystis aeruginosa by dairy cattle and the implications for microcystin contamination of milk

Microcystin (MCYST) toxins can be produced by the bloom-forming cyanobacterium Microcystis aeruginosa. They are chemically stable compounds and have both acute and chronic effects on the health of mammals, including cattle and humans. Cattle will drink water containing lethal cell concentrations of M. aeruginosa. When cattle consume sub-lethal doses of microcystins, the fate of those toxins is unknown. We provided drinking water containing 1×10⁵ cells ml⁻¹ M. aeruginosa (strain MASH01-A19) to four lactating Holstein–Friesian dairy cattle for 21 days to determine if MCYST-LR produced by the cyanobacteria, could be detected in milk produced by the cattle. Cattle consumed up to 15 mg MCYST-LR at an ingestion rate of 1.21 μg kg (live weight) d⁻¹. Analysis by HPLC and ELISA indicated that no detectable amounts of microcystin from the cyanobacteria were present in the milk obtained from the treated animals. Based on the level of quantitation of the ELISA analyses, the maximum possible concentration in the milk was less than 2 ng l⁻¹. This is more than three orders of magnitude less that the concentration that could be considered problematic for milk of 0.86 μg l⁻¹ which we calculated using the World Health Organization derived tolerable daily intake for MCYST-LR and the per capita daily consumption of milk in Australia.     

Thromboxane A2-mediated Cl secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A₂ is a novel endogenous secretagogue of Cl⁻ secretion in the distal colon. Here, we examined if the Cl⁻ secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A₂ production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl⁻ secretion in the rat colon. A selective thromboxane A₂ receptor antagonist (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b,e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[α-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl⁻ current in a concentration-dependent manner. The IC₅₀ values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E₂-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl⁻ current. The present study suggests that the PAF-induced Cl⁻ secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A₂.