Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated.
2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O₂^.−) generation with IC₅₀ values of 0.33±0.05 μg ml⁻¹ and 0.49±0.04 μg ml⁻¹, respectively.
3. Abruquinone A also inhibited O₂ consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O₂^.− in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation.
4. Abruquinone A inhibited both the transient elevation of [Ca²⁺]_i in the absence of [Ca²⁺]_o (IC₅₀ 7.8±0.2 μg ml⁻¹) and the generation of inositol trisphosphate (IP₃) (IC₅₀ 10.6±2.0 μg ml⁻¹) in response to fMLP.
5. Abruquinone A did not affect the enzyme activities of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA).
6. Abruquinone A had no effect on intracellular guanosine 3′ : 5′-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) levels.
7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/CB was inhibited by abruquinone A with IC₅₀ values of 2.2±0.6 μg ml⁻¹ and 2.5±0.3 μg ml⁻¹, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73–78 kDa in activated neutrophils.
8. Abruquinone A inhibited both the O₂^.− generation in PMA-activated neutrophil particulate NADPH oxidase (IC₅₀ 0.6±0.1 μg ml⁻¹) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC₅₀ 1.5±0.2 μg ml⁻¹).
9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.

     

Modelling of the pharmacodynamic interaction between phenytoin and sodium valproate

1. Treatment of epilepsy with a combination of antiepileptic drugs remains the therapeutic choice when monotherapy fails. In this study, we apply pharmacokinetic-pharmacodynamic modelling to characterize the interaction between phenytoin (PHT) and sodium valproate (VPA).
2. Male Wistar rats received a 40 mg kg⁻¹ intravenous dose of PHT over 5 min either alone or in combination with an infusion of VPA resulting in a steady-state concentration of 115.5±4.9 μg ml⁻¹. A control group received only the infusion of VPA. The increase in the threshold for generalized seizure activity (ΔTGS) was used as measure of the anticonvulsant effect.
3. PHT pharmacokinetics was described by a pharmacokinetic model with Michaelis-Menten elimination. The concentration-time course and plasma protein binding of PHT were not altered by VPA. The pharmacokinetic parameters V_max and K_m were, respectively, 294±63 μg min⁻¹ and 7.8±2.4 μg ml⁻¹ in the absence of VPA and 562±40 μg min⁻¹ and 15.6±0.9 μg ml⁻¹ upon administration in combination with VPA.
4. A delay of the onset of the effect relative to plasma concentrations of PHT was observed. The assessment of PHT concentrations at the effect site was based on the effect-compartment model, yielding mean k_e0 values of 0.128 and 0.107 min⁻¹ in the presence and absence of VPA, respectively.
5. A nonlinear relationship between effect-site concentration and the increase in the TGS was observed. The concentration that causes an increase of 50% in the baseline TGS (EC_50%TGS) was used to compare drug potency. A shift of EC_50%TGS from 13.27±3.55 to 4.32±0.52 μg ml⁻¹ was observed upon combination with VPA (P<0.01).
6. It is concluded that there is a synergistic pharmacodynamic interaction between PHT and VPA in vivo.

     

Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT₁-like'' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996.
2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg⁻¹) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine.
3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg⁻¹) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg⁻¹) and mesulergine (300 and 1000 μg kg⁻¹) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg⁻¹), the 5-HT_1B/1D receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg kg⁻¹) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses.
5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT₇ receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht₇ gene product, the 5-HT₇ receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT₇ receptors.

     

RPR 106541, a novel,airways-selective glucocorticoid: effects against antigen-induced CD4+T lymphocyte accumulation and cytokine gene expression in the Brown Norway rat lung

1. The effects of a novel 17-thiosteroid, RPR 106541, were investigated in a rat model of allergic airway inflammation.
2. In sensitized Brown Norway rats, challenge with inhaled antigen (ovalbumin) caused an influx of eosinophils and neutrophils into the lung tissue and airway lumen. In the lung tissue there was also an accumulation of CD4⁺ T lymphocytes and increased expression of mRNA for interleukin-4 (IL-4) and IL-5, but not interferon-γ (IFN-γ). These findings are consistent with an eosinophilia orchestrated by activated Th2-type cells.
3. RPR 106541 (10–300 μg kg⁻¹), administered by intratracheal instillation into the airways 24 h and 1 h before antigen challenge, dose-dependently inhibited cell influx into the airway lumen. RPR 106541 (100 μg kg⁻¹) caused a significant (P<0.01) (98%) inhibition of eosinophil influx and a significant (P<0.01) (100%) inhibition of neutrophil influx. RPR 106541 was approximately 7 times and 4 times more potent than budesonide and fluticasone propionate, respectively.
4. When tested at a single dose (300 μg kg⁻¹), RPR 106541 and fluticasone each caused a significant (P<0.01) (100%) inhibition of CD4⁺ T cell accumulation in lung tissue. Budesonide (300 μg kg⁻¹) had no significant effect. RPR 106541 and fluticasone (300 μg kg⁻¹), but not budesonide (300 μg kg⁻¹), significantly (P<0.05) inhibited the expression within lung tissue of mRNA for IL-4. RPR 106541 (300 μg kg⁻¹) also significantly (P<0.05) inhibited expression of mRNA for IL-5.
5. The high topical potency of RPR 106541 in this model, which mimics important aspects of airway inflammation in human allergic asthmatics, suggests that this glucocorticoid may be useful in the treatment of bronchial asthma.

     

Role of transforming growth factor-β pathway in the mechanism of wound healing by saponin from Ginseng Radix rubra

1. The effects of saponin from Ginseng Radix rubra on extracellular matrix metabolism, the activation and synthesis of TGF-β1, and the modification of TGF-β receptor in fibroblasts were examined to elucidate the contribution of the TGF-β pathway to the mechanism of wound healing by saponin.
2. Fibronectin synthesis was analysed by the immunoprecipitation method. Activation and synthesis of TGF-β1 were measured by ELISA. The expressions of TGF-β receptors in fibroblasts were examined at protein and mRNA levels by the cross-linking method and Northern blot analysis, respectively.
3. The fibronectin synthesis increased 2.3- and 3.9-fold at fibroblasts treated with 1 and 10 μg ml⁻¹ of saponin, respectively, compared with that in non-treated cells. Fibronectin synthesis stimulated with 10 μg ml⁻¹ of saponin was inhibited with 69% by 5 μg ml⁻¹ of an anti-TGF-β1 antibody. mRNA of TGF-β type I receptor increased 4.8- and 4.4-fold at fibroblasts treated with 1 and 10 μg ml⁻¹ of saponin, respectively, and that of TGF-β type II receptor also increased 3.4- and 3.2-fold at fibroblasts treated with 1 and 10 μg ml⁻¹ of saponin, respectively. The significant increases of TGF-β type I and II receptors and of fibronectin synthesis were observed at the same concentrations of saponin. TGF-β1 content increased 1.74- and 1.87-fold at conditioned medium of fibroblasts treated with 100 and 250 μg ml⁻¹ of saponin, respectively, higher concentrations than those which accelerated fibronectin synthesis. Furthermore, the active TGF-β1 content was below 10% of total TGF-β1 at each concentration of saponin.
4. These results indicate that saponin stimulates fibronectin synthesis through the changes of TGF-β receptor expressions in fibroblasts.

     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E₂ (PGE₂) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE₂ EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml⁻¹, interleukin-1β (IL-1β) 1 u ml⁻¹ and lipopolysaccharide (LPS) 10 μg ml⁻¹ induced nitrite formation which could be inhibited by the competitive NOS inhibitor N^G-nitro-L-arginine-methyl-ester (L-NAME). IL-1β alone (1–50 u ml⁻¹) induced PGE₂ formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ to induce both the iNOS and COX-2 pathways, and IL-1β 3 u ml⁻¹ to induce COX-2 without iNOS activity.
3. Cells treated with IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ for 48 h either alone, or with the addition of L-NAME (0 to 10⁻² M), demonstrated inhibition by L-NAME of PGE₂ (3.61±0.55 to 0.51±0.04 pg/10⁴ cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/10⁴ cells; P<0.001) production. Restoration of the PGE₂ response (0.187±0.053 to 15.46±2.59 pg/10⁴ cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10⁻⁶ M, but with the addition of the NOS substrate L-arginine (0 to 10⁻⁵ M).
4. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10⁻⁴ M), demonstrated increased PGE₂ formation (1.23±0.03 to 2.92±0.19 pg/10⁴ cells; P< 0.05). No increase in PGE₂ formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE₂ formation. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10⁻³ M) also demonstrated an increased PGE₂ response (2.56±0.21 to 4.53±0.64 pg/10⁴ cells; P<0.05).
5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

     

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor.
2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E₂ (PGE₂) with at least a 1,000 fold selectivity for COX-2 (IC₅₀=41±14 nM) over COX-1 (IC₅₀>50 μM). Indomethacin was a potent inhibitor of both COX-1 (IC₅₀=18±3 nM) and COX-2 (IC₅₀=26±6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B₂ (TXB₂) by Ca²⁺ ionophore-challenged human platelets (IC₅₀>50 μM and 4.1±1.7 nM, respectively).
3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a K_i value of 140±68 μM for the initial reversible binding to enzyme and a k₂ value of 0.11±0.06 s⁻¹ for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62±26 μM and 0.06±0.01 s⁻¹, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t_1/2=1–3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site.
4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC₅₀=63±5 μM at 0.1 μM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1.
5. DFU inhibited lipopolysaccharide (LPS)-induced PGE₂ production (COX-2) in a human whole blood assay with a potency (IC₅₀=0.28±0.04 μM) similar to indomethacin (IC₅₀=0.68±0.17 μM). In contrast, DFU was at least 500 times less potent (IC₅₀>97 μM) than indomethacin at inhibiting coagulation-induced TXB₂ production (COX-1) (IC₅₀=0.19±0.02 μM).
6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 μM), DFU inhibited COX-1 with an IC₅₀ value of 13±2 μM as compared to 20±1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac>indomethacin∼naproxen>nimesulide∼ meloxicam∼piroxicam>NS-398∼SC-57666>SC-58125>CGP 28238∼etodolac>L-745,337>DFU.
7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED₅₀ of 1.1 mg kg⁻¹ vs 2.0 mg kg⁻¹ for indomethacin) and hyperalgesia (ED₅₀ of 0.95 mg kg⁻¹ vs 1.5 mg kg⁻¹ for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED₅₀=0.76 mg kg⁻¹ vs 1.1 mg kg⁻¹ for indomethacin).
8. In a sensitive model in which ⁵¹Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg⁻¹, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg⁻¹), meloxicam (3 mg kg⁻¹) or etodolac (10–30 mg kg⁻¹). A 5 day administration of DFU in squirrel monkeys (100 mg kg⁻¹) did not affect chromium leakage in contrast to diclofenac (1 mg kg⁻¹) or naproxen (5 mg kg⁻¹).
9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

     

Characterization of prejunctional 5-HT1 receptors that mediate the inhibition of pressor effects elicited by sympathetic stimulation in the pithed rat

1. A study was made of the effects of 5-carboxamidotryptamine (5-CT) on pressor responses induced in vivo by electrical stimulation of the sympathetic outflow from the spinal cord of pithed rats. All animals had been pretreated with atropine. Sympathetic stimulation (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. Intravenous infusion of 5-CT at doses of 0.01, 0.1 and 1 μg kg⁻¹ min⁻¹ reduced the pressor effects obtained by electrical stimulation. The inhibitory effect of 5-CT was significantly more pronounced at lower frequencies of stimulation. In the present study we characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-CT.
2. The inhibition induced by 0.01 μg kg⁻¹ min⁻¹ of 5-CT on sympathetically-induced pressor responses was partially blocked after i.v. treatment with methiothepin (10  μg kg⁻¹), WAY-100,635 (100 μg kg⁻¹) or GR127935T (250 μg kg⁻¹), but was not affected by cyanopindolol (100 μg kg⁻¹).
3. The selective 5-HT_1A receptor agonist 8-OH-DPAT and the selective 5-HT_1B/1D receptor agonists sumatriptan and L-694,247 inhibited the pressor response, whereas the 5-HT_1B receptor agonists CGS-12066B and CP-93,129 and the 5-HT_2C receptor agonist m-CPP did not modify the pressor symapthetic responses.
4. The selective 5-HT_1A receptor antagonist WAY-100,635 (100 μg kg⁻¹) blocked the inhibition induced by 8-OH-DPAT and the selective 5-HT_1B/1D receptor antagonist GR127935T (250 μg kg⁻¹) abolished the inhibition induced either by L-694,247 or sumatriptan.
5. None of the 5-HT receptor agonists used in our experiments modified the pressor responses induced by exogenous noradrenaline (NA).
6. These results suggest that the presynaptic inhibitory action of 5-CT on the electrically-induced pressor response is mediated by both r-5-HT_1D and 5-HT_1A receptors.

     

Pharmacokinetic-pharmacodynamic modelling of the anti-lipolytic and anti-ketotic effects of the adenosine A1-receptor agonist N6-(p-sulphophenyl)adenosine in rats

1. The purpose of this study was to develop and validate an integrated pharmacokinetic-pharmacodynamic model for the anti-lipolytic effects of the adenosine A₁-receptor agonist N⁶-(p-sulphophenyl)adenosine (SPA). Tissue selectivity of SPA was investigated by quantification of haemodynamic and anti-lipolytic effects in individual animals.
2. After intravenous infusion of SPA to conscious normotensive Wistar rats, arterial blood samples were drawn for determination of blood SPA concentrations, plasma non-esterified fatty acid (NEFA) and β-hydroxybutyrate levels. Blood pressure and heart rate were monitored continuously.
3. The relationship between the SPA concentrations and the NEFA lowering effect was described by the indirect suppression model. Administration of SPA at different rates and doses (60 μg kg⁻¹ in 5 min and 15 min, and 120 μg kg⁻¹ in 60 min) led to uniform pharmacodynamic parameter estimates. The averaged parameters (mean±s.e., n=19) were E_max: −80±2% (% change from baseline), EC₅₀: 22±2 ng ml⁻¹, and Hill factor: 2.2±0.2.
4. In another group, given 400 μg kg⁻¹ SPA in 15 min, pharmacodynamic parameters for both heart rate and anti-lipolytic effect were derived within the same animal. The reduction in heart rate was directly related to blood concentration on the basis of the sigmoidal E_max model. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The EC₅₀ values (mean±s.e., n=6) were 131±31 ng ml⁻¹ and 20±3 ng ml⁻¹ for heart rate and NEFA lowering effect, respectively.
5. In conclusion, the relationship between blood SPA concentrations and anti-lipolytic effect was adequately described by the indirect suppression model. For SPA a 6 fold difference in potency was observed between the effects on heart rate and NEFAs, indicating some degree of tissue selectivity in vivo.

     

Capsaicin and neurokinin A-induced bronchoconstriction in the anaesthetised guinea-pig: evidence for a direct action of menthol on isolated bronchial smooth muscle

1. For many years menthol has been used in the treatment of respiratory disorders although, a bronchodilator effect of menthol has yet to be described. Using the bronchoconstrictors capsaicin (acting via stimulating the release of neuropeptides from sensory afferents) and neurokinin A (NKA) we have raised airways resistance in the guinea-pig (GP) and studied the effect of menthol on both capsaicin and NKA-induced bronchoconstriction in vivo. In vitro the effect of menthol on acetylcholine (ACh) and KCl precontracted GP bronchi was also studied.
2. GP (n=13) were anaesthetized (urethane 1.5 g kg⁻¹, i.p.) and a bolus injection of capsaicin (7.5 μg ml⁻¹, i.v.) or infusion of NKA (1 μg min⁻¹, i.v.) was given either in the presence of air (0.81 min⁻¹) or air impregnated with menthol vapour (7.5 μg l⁻¹) freely breathed from a tracheal cannula via a T-piece. Airways resistance (R_aw) and ventilation were measured throughout. Bronchi of mean internal diameter (1029+73.6 μm; n=24) were removed from GP (n=16) and mounted in the Cambustion myograph. Bronchial rings were maximally precontracted with 80 mM KC1 or 2 mM ACh. Relaxation due to a cumulative dose of menthol (1–3000 μM) was measured.
3. Menthol produced a significant (P<0.05) 51.3% reversal of the capsaicin-induced increase in R_aw, and also inhibited the significant (P<0.05) reduction in minute ventilation (V_e) associated with the capsaicin-induced increased in R_aw. Menthol also caused a significant (P<0.05) 41% reversal of the NKA-induced increase in R_aw. The NKA-induced decrease in V_e was again significantly (P<0.05) reversed with menthol inhalation. Menthol caused a significant (P<0.001) dose-dependent relaxation of KCl and ACh precontracted bronchi.
4. We have shown that menthol attenuates both capsaicin and NKA-induced bronchoconstriction in vivo and relaxes KCl and ACh preconstricted bronchi in vitro. Menthol inhibition of NKA and capsaicin-induced bronchoconstriction could be, in part, explained by a direct action of menthol on bronchial smooth muscle.

     

Vascular actions of octreotide in the portal hypertensive rat

1. We have investigated the actions of the somatostatin analogue octreotide in the portal hypertensive Wistar rat in vivo and in rat small mesenteric artery and aorta in vitro.
2. In small mesenteric artery, octreotide (0.1–0.3 μM) failed to produce any direct contraction, nor did it affect contractions to noradrenaline (NA, 10 μM) or endothelium-dependent relaxations to acetylcholine.
3. In rat aorta, octreotide (0.3 μM) and somatostatin (1 μM) failed to affect contractions to NA (1 μM), or concentration-contractile response curves to NA.
4. In rat vas deferens, octreotide and somatostatin significantly reduced contractile responses to electrical stimulation with pD₂ values (−log IC₅₀) of 8.19±0.10 (n=4) and 8.16±0.26 (n=4), respectively. Hence, the lack of effect of these agents in aorta or mesenteric artery was not due to lack of efficacy or inappropriate choice of concentration.
5. In the anaesthetized portal hypertensive rat, intravenous injection of octreotide (1–100 μg kg⁻¹) did not significantly affect systemic blood pressure, nor did it affect mesenteric vascular conductance as measured by laser doppler flow probes. However, octreotide (100 μg kg⁻¹) significantly reduced vascular conductance to 74.2±7.7% of control (n=6) in porto-systemic shunt vessels as measured by laser doppler flow probes.
6. Phenylephrine (1 μg kg⁻¹) significantly raised blood pressure and significantly decreased vascular conductance in both mesenteric (66.6±3.7% of control) and porto-systemic shunt vessels (58.7±10.0% of control).
7. It was concluded that octreotide has selective effects on porto-systemic shunt vessles in vivo in the portal hypertensive rat.

     

Mediation of 5-HT-induced external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs by the putative 5-HT7 receptor

1. The vasodilator effects of 5-hydroxytryptamine (5-HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho-inhibitory 5-HT_1B/1D receptors and postjunctional 5-HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5-HT with the 5-HT_1B/1D receptor antagonist, GR 127935, unmasks a dose-dependent vasodilator effect of 5-HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5-HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 μg kg⁻¹, i.v.).
2. One-minute intracarotid (i.c.) infusions of 5-HT (0.330 μg min⁻¹), 5-carboxamidotryptamine (5-CT; 0.010.3 μg min⁻¹), 5-methoxytryptamine (1100 μg min⁻¹) and lisuride (31000 μg min⁻¹) resulted in dose-dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5-CT>>5-HT⩾5-methoxytryptamine>lisuride, whereas cisapride (1001000 μg min⁻¹, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85±7 μg kg⁻¹, i.c.), but not cisapride (mean dose of 67±7 μg kg⁻¹, i.c.), specifically abolished the responses induced by 5-HT, 5-CT and 5-methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8-OH-DPAT (33000 μg min⁻¹) produced dose-dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200±33 μg kg⁻¹, i.c.) the agonist-induced vasodilator responses.
3. The external carotid vasodilator responses to 5-HT, 5-CT and 5-methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (±)-pindolol (4 mg kg⁻¹) or ritanserin (100 μg kg⁻¹) plus granisetron (300 μg kg⁻¹), but were dose-dependently blocked by i.v. administration of methiothepin (10 and 30 μg kg⁻¹, given after ritanserin plus granisetron), mesulergine (10 and 30 μg kg⁻¹), metergoline (1 and 3 mg kg⁻¹), methysergide (1 and 3 mg kg⁻¹) or clozapine (0.3 and 1 mg kg⁻¹). Nevertheless, the blockade of the above responses, not significant after treatment with the lower of the two doses of metergoline and mesulergine, was nonspecific after administration of the higher of the two doses of methysergide and clozapine.
4. Based upon the above rank order of agonist potencies and the antagonism produced by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, our results indicate that the 5-HT receptor mediating external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs is operationally similar to the putative 5-HT₇ receptor mediating relaxation of vascular and non-vascular smooth muscles (e.g. rabbit femoral vein, canine coronary artery, rat systemic vasculature and guinea-pig ileum) as well as tachycardia in the cat.

     

Effects of ibuprofen enantiomers and its coenzyme A thioesters on human prostaglandin endoperoxide synthases

1. Ibuprofen enantiomers and their respective coenzyme A thioesters were tested in human platelets and blood monocytes to determine their selectivity and potency as inhibitors of cyclo-oxygenase activity of prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2.
2. Human blood from volunteers was drawn and allowed to clot at 37°C for 1 h in the presence of increasing concentrations of the test compounds (R-ibuprofen, S-ibuprofen, R-ibuprofenoyl-CoA, S-ibuprofenoyl-CoA, NS-398). Immunoreactive (ir) thromboxane B₂ (TXB₂) concentrations in serum were determined by a specific EIA assay as an index of the cyclo-oxygenase activity of platelet PGHS-1.
3. Heparin-treated blood from the same donors was incubated at 37°C for 24 h with the same concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 μg ml⁻¹). The contribution of PGHS-1 was suppressed by pretreatment of the volunteers with aspirin (500 mg; 48 h before venepuncture). As a measure of LPS induced PGHS-2 activity immunoreactive prostaglandin E₂ (irPGE₂) plasma concentrations were determined by a specific EIA assay.
4. S-ibuprofen inhibited the activity of PGHS-1 (IC₅₀ 2.1 μM) and PGHS-2 (IC₅₀ 1.6 μM) equally. R-ibuprofen inhibited PGHS-1 (IC₅₀ 34.9) less potently than S-ibuprofen and showed no inhibition of PGHS-2 up to 250 μM. By contrast R-ibuprofenoyl-CoA thioester inhibited PGE₂ production from LPS-stimulated monocytes almost two orders of magnitude more potently than the generation of TXB₂ (IC₅₀ 5.6 vs 219 μM).
5. Western blotting of PGHS-2 after LPS induction of blood monocytes showed a concentration-dependent inhibition of PGHS-2 protein expression by ibuprofenoyl-CoA thioesters.
6. These data confirm that S-ibuprofen represents the active entity in the racemate with respect to cyclo-oxygenase activity. More importantly the data suggest a contribution of the R-enantiomer to therapeutic effects not only by chiral inversion to S-ibuprofen but also via inhibition of induction of PGHS-2 mediated by R-ibuprofenoyl-CoA thioester.
7. The data may explain why racemic ibuprofen is ranked as one of the safest non-steroidal anti-inflammatory drugs (NSAIDs) so far determined in epidemiological studies.

     

Gabapentin potentiation of the antiepileptic efficacy of vigabatrin in an in vitro model of epilepsy

1. An enhancement of promoted release of γ-aminobutyric acid (GABA) and a change in GABA-metabolism have been suggested as mechanisms of action of gabapentin. Vigabatrin is supposed to act mainly via inhibition of GABA-transaminase but it also interferes with GABA-release and GABA-uptake. On the basis of these mechanisms of action, a pharmacodynamic interaction of the two antiepileptic drugs could be supposed which might be of relevance in the sense of a rational polypharmacy.
2. To address the aforementioned hypothesis, experiments were carried out on hippocampal slices (n=107) of guinea-pigs (n=70). Epileptiform field potentials (e.f.p.) were induced by omission of magnesium from the bath solution and recorded in the stratum pyramidale of the CA3 region. Gabapentin (30–600 μM; 5.1–102.72 μg ml⁻¹), vigabatrin (50–200 μM, 6.45–25.8 μg ml⁻¹) and the GABA_A-receptor antagonist bicuculline (100 μM) were added to the bath solution for 3 h.
3. Gabapentin, in concentrations up to 600 μM, failed to decrease the repetition rate or duration of e.f.p. (n=19). However, vigabatrin, evoked a dose-dependent reduction of the repetition rate of e.f.p. For a concentration of 100 μM (12.9 μg ml⁻¹) there was a reduction down to 48±5% (mean±s.e.mean) of the initial value within 3 h (n=11). With simultaneous administration of vigabatrin (100 μM) and gabapentin (60 μM) for 3 h (n=15), the repetition rate of e.f.p. decreased down to 8±3%, which is significantly different from the values obtained after administration of 100 μM vigabatrin alone (P<0.0001). Both, the antiepileptic effect of vigabatrin alone and the enhancement by gabapentin were blocked by the GABA_A-receptor antagonist bicuculline (100 μM, n=16).
4. These results demonstrate that gabapentin is able to augment the antiepileptic effects of vigabatrin significantly. It is possible that a change in the GABA-release machinery is induced by vigabatrin which then can be augmented by gabapentin.

     

Inhibitory effect of a new steroidal saponin,OSW-1, on ovarian functions in rats

1. This study was undertaken to determine the effects of OSW-1 (3β, 16β, 17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1→3)-(2-O-acetyl-α-L-arabinopyranoside)) on the pituitary-ovarian system and the functions of aortic smooth muscle.
2. A single s.c. injection of OSW-1 (9 μg kg⁻¹) on the morning of pro-oestrus inhibited the occurrence of the expected next pro-oestrus, whereas administration of OSW-1 at a dose of 4.5 μg kg⁻¹ did not affect the oestrous cycle. OSW-1 treatment on the day of dioestrus-l did not affect the oestrous cycle.
3. At doses of 4.5 and 9 μg kg⁻¹ OSW-1, the serum oestradiol (E₂) levels at the expected next pro-oestrus were significantly lower than in control (pro-oestrus). The serum luteinizing hormone (LH) levels 4 days after 9 μg kg⁻¹ OSW-1 treatment were also markedly lower than those of control. OSW-1 (4.5 μg kg⁻¹) did not affect the levels of inhibin, progesterone and gonadotrophins on the same day.
4. OSW-1 did not inhibit the preovulatory LH surge which occurs on the afternoon of pro-oestrus day.
5. The expression of mRNA coding for the cholesterol side chain cleavage cytochrome P-450 (p450scc), an ovarian steroidal limiting enzyme, was suppressed at 24 and 96 h after OSW-1 treatment.
6. Administration of OSW-1 (9 μg kg⁻¹) tended to reduce the relaxation of isolated thoracic aorta ring preparations induced by acetylcholine, while there was no difference in the relaxation induced by sodium nitroprusside.
7. Our results show that OSW-1 inhibits ovarian E₂ secretion and that the decrease in E₂ secretion may contribute to its effects on the oestrous cycle and the sensitivity of the thoracic aorta to relaxation. The decrease in the levels of ovarian steroids induced by OSW-1 may be due to its direct inhibitory action on the gene expression of the steroidal enzyme and on the proliferation of granulosa cells in the ovary.

     

Characterization of angiotensin II formation in human isolated bladder by selective inhibitors of ACE and human chymase: a functional and biochemical study

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle.
2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO₂Me) and a recently developed human chymase substrate Pro¹¹-,D-Ala¹²)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used.
3. Angiotensin I (AI) (0.3 μM) induced a contractile response amounting to 58±5% (n=12) of the initial K⁺ (124 mM)-induced contractions. This response was reduced to 36±3% (n=8) by the ACE-inhibitor enalaprilat (10 μM), while pretreatment with soybean trypsin inhibitor (STI 200 μg ml⁻¹) or CH 5450 (10 μM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19±5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1±1% (n=6).
4. The substrate (Pro¹¹-,D-Ala¹²)-AI (3 μM) produced contractions which amounted to 57±4% (n=13) of the initial K⁺ (124 mM) contractions. These contractions were not affected by enalaprilat (10 μM). On the other hand, STI (200 μg ml⁻¹) and CH 5450 (10 μM) added separately, depressed the (Pro¹¹-,D-Ala¹²)-AI-induced contractions to 34±5% (n=6) and 24±4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition.
5. Experiments with detrusor membrane fractions incubated with AI (50 μM) were performed. In the presence of enalaprilat (100 μM), carboxypeptidase inhibitor CPI (10 μg ml⁻¹) and aprotinin (15 μM), CH 5450 (10 nM–1 μM) caused a concentration-dependent inhibition of AII formation.
6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

     

Effects of dopamine and selective dopamine agonists upon platelet accumulation in the cerebral and pulmonary vasculature of the rabbit

1. A selection of novel compounds were shown to exhibit dopaminergic activity in vitro.
2. ¹¹¹Indium-labelled platelets were continuously monitored in the cerebral and pulmonary vasculature of anaesthetized rabbits. The effects of dopamine and selective dopamine receptor agonists on ADP and thrombin induced platelet accumulation were recorded.
3. Pretreatment with dopamine (2 mg kg⁻¹ min⁻¹, i.v.) significantly reduced ADP (20 μg kg⁻¹, i.v.) induced platelet accumulation in the pulmonary vasculature whereas lower doses had no effect.
4. Dopamine (100 μg kg⁻¹ min⁻¹ intra-carotid, i.c.) potentiated thrombin (90 u kg⁻¹, i.c.) induced platelet accumulation in the cerebral vasculature whereas higher doses (1–2 mg kg⁻¹ min⁻¹) inhibited accumulation.
5. The selective dopamine receptor agonists tested did not significantly inhibit platelet accumulation induced by ADP or thrombin. Two of these selective agonists, at doses higher than the intended therapeutic doses, induced thrombocytopaenia and an associated increase in platelet accumulation in the lung in response to thrombin.
6. These results extend previous in vitro studies regarding the dual actions of dopamine upon platelets and show for the first time the effects of selective dopamine receptor agonists upon platelet aggregation in vivo.

     

Receptor subtypes Y1 and Y5 are involved in the renal effects of neuropeptide Y

1. Systemic infusion of neuropeptide Y (NPY) reduces renal blood flow and can concomitantly increase diuresis, natriuresis and calciuresis in anaesthetized rats. The present study was designed to investigate whether the apparently contradictory NPY effects on renal blood flow and urine formation and composition are mediated by distinct NPY receptor subtypes.
2. NPY and its analogues, peptide YY (PYY), [Leu³¹, Pro³⁴]NPY and NPY₁₃₃₆, were infused in incremental doses of 0.3, 1 and 3 μg kg⁻¹ min⁻¹ for 45 min each and the results compared to those obtained in vehicle-infused rats. Renal blood flow was monitored in 15 min intervals, while urine excretion and composition were determined in 15 min collection periods. Plasma renin activity and aldosterone concentrations were measured at the end of the final infusion period.
3. Relative to vehicle NPY, PYY and [Leu³¹, Pro³⁴]NPY dose-dependently reduced renal blood flow and increased diuresis, natriuresis and calciuresis with roughly similar potency; NPY₁₃₃₆ slightly but significantly increased renal blood flow but had no effect on diuresis, natriuresis and calciuresis. None of the peptides significantly affected endogenous creatinine clearance or kaliuresis.
4. Plasma renin activity was significantly reduced by PYY. Quantitatively similar reductions were observed with NPY and [Leu³¹, Pro³⁴]NPY but failed to reach statistical significance with the given number of experiments. NPY₁₃₃₆ did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations.
5. In another series of experiments infusion of PYY₃₃₆ (2 μg kg⁻¹ min⁻¹ for 120 min) did not reduce renal blood flow but significantly enhancd diuresis and natriuresis to a similar extent as the NPY 2 μg kg⁻¹ min⁻¹.
6. In a final series of experiments the Y₁-selective antagonist, BIBP 3226 (1 or 10 μg kg⁻¹ min⁻¹) dose-dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.130 μg kg⁻¹). BIBP 3226 (10 μg kg⁻¹ min⁻¹) also inhibited the effects of a 120 min infusion of NPY (2 μg kg⁻¹ min⁻¹) on renal blood flow but had only minor inhibitory effects on enhancements of diuresis and did not significantly affect enhancements of natriuresis.
7. We conclude that NPY reduces renal blood via a classical Y₁ subtype of NPY receptor. In contrast enhancements of diuresis, natriuresis and calciuresis occur via a distinct subtype which resembles the receptor that mediates NPY-induced enhancement of food intake.

     

Andrographolide suppresses the expression of inducible nitric oxide synthase in macrophage and restores the vasoconstriction in rat aorta treated with lipopolysaccharide

1. We investigated whether andrographolide, a diterpenoid lactone found at Andrographis paniculata, influences the induction of the inducible nitric oxide synthase (iNOS) in RAW264.7 cells activated by bacterial endotoxin (LPS), as well as in the rats with endotoxic shock and in aortic rings treated with LPS.
2. Incubation of RAW264.7 cells with andrographolide (1 to 50 μM) inhibited the LPS (1 μg ml⁻¹)-induced nitrite accumulation in concentration- and time-dependent manners. Maximum inhibition was observed when andrographolide was added together with LPS and decreased progressively as the interval between andrographolide and LPS was increased to 20 h.
3. Western blot analysis demonstrated that iNOS expression was markedly attenuated in the presence of andrographolide for 6–24 h, suggesting that andrographolide inhibited iNOS protein induction.
4. Thoracic aorta incubation with LPS (300 ng ml⁻¹) for 5 h in vitro exhibited a significant decrease in the maximal contractile response to phenylephrine (10⁻⁹–10⁻⁵ M). Andrographolide (30 μM) restored the contractile response to control level.
5. In anaesthetized rats, LPS (10 mg kg⁻¹, i.v.) caused a fall in mean arterial blood pressure (MAP) from 116±4 to 77±5 mmHg. The pressor effect of phenylephrine (10 μg ml⁻¹, i.v.) was also significantly reduced at 30, 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with andrographolide (1 mg kg⁻¹, i.v., 20 min prior to LPS) maintained a significantly higher MAP when compared to LPS-rats given with vehicle. Administration of andrographolide 60 min after LPS caused a increase in MAP and significantly reversed the reduction of the pressor response to phenylephrine.
6. Our results indicated that andrographolide inhibits nitrite synthesis by suppressing expression of iNOS protein in vitro. And, this inhibition of iNOS synthesis may contribute to the beneficial haemodynamic effects of andrographolide in endotoxic shock.