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.     

Antihypertensive and vasorelaxant activities of Laelia autumnalis are mainly through calcium channel blockade

The aim of the present study was to evaluate the possible mechanism of the vasorelaxant action of methanol extract from Laelia autumnalis (MELa) in isolated rat aortic rings, and to establish its antihypertensive activity in vivo. MELa (0.15→50 µg/mL) induced relaxation in aortic rings pre-contracted with KCl (80 mM), showing an IC₅₀ value of 34.61 ± 1.41 µg/mL and E_max value of 85.0 ± 4.38% (in endothelium-intact rings) and an IC₅₀ value of 45.11 ± 4.17 µg/mL and E_max value of 80.0 ± 12.1% (in endothelium-denuded rings). Serotonin (5-HT, 1 × 10^− 4 M) provoked sustained contraction, which was markedly inhibited by MELa (0.15→50 µg/mL) in a concentration-dependent and endothelium-independent manner. Pretreatment with MELa (15, 46, 150, 300 and 1500 µg/mL) also inhibited contractile responses to norepinephrine (NE 1 × 10^− 11 M to 1 × 10^− 5.5 M). In endothelium-denuded rings, the vasorelaxant effect of MELa was reduced partially by ODQ (1 µM), but not by tetraethylammonium (5 µM), glibenclamide (10 µM), and 2-aminopyridine (100 µM). The extract also reduced NE-induced transient contraction in Ca²⁺-free solution, and inhibited contraction induced by increasing external calcium in Ca²⁺-free medium plus high KCl (80 mM). The antihypertensive effect of MELa was determined in spontaneously hypertensive rats (SHR). A single oral administration of the extract (100 mg/kg) exhibited a significant decrease in systolic and diastolic blood pressure and heart rate (p < 0.05) in SHR rats. Our results suggest that MELa induces relaxation in rat aortic rings through an endothelium-independent pathway, involving blockade of Ca²⁺ channels and a possible cGMP enhanced concentrations and also causes an antihypertensive effect.     

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.     

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.     

Inhibition of voltage-gated L-type calcium channels by labedipinedilol-A involves protein kinase C in rat cerebrovascular smooth muscle cells

Labedipinedilol-A, a novel calcium channel blocker with α/β-adrenoceptor blockade properties, inhibits L-type calcium channels (LTCCs) in rat cerebrovascular smooth muscle cells (CSMCs). We used conventional whole cell patch-clamp electrophysiology to investigate Ba²⁺ currents (I_Ba) through LTCCs in rat CSMCs enzymatically dissociated from rat cerebral arteries. Labedipinedilol-A (1, 10 µM) reversibly inhibited I_Ba in a voltage-dependent manner without modifying the I_Ba current–voltage relationship. The I_Ba was also abolished by the LTCC blocker nifedipine (1 µM), but enhanced by the LTCC activator Bay K8644 (100 nM). Labedipinedilol-A shifted the steady-state inactivation curve of I_Ba to more negative potentials. Additionally, labedipinedilol-A had greater inhibitory activity on I_Ba holding at − 40 mV than at − 80 mV. This might contribute to labedipinedilol-A's more selective effect on vascular muscles compared to cardiac muscles. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and norepinephrine-enhanced I_Ba were also inhibited by labedipinedilol-A. Pretreatment with the PKC inhibitor chelerythrine (5 µM) attenuated labedipinedilol-A-mediated I_Ba inhibition. However, the Rho kinase inhibitor Y-27632 (30 µM) had little effect on labedipinedilol-A inhibition of I_Ba. Labedipinedilol-A inhibition of voltage-dependent LTCCs may be, at least in part, due to its modulation of the PKC pathway.     

Inactivation of nitric oxide synthases and cellular nitric oxide formation by N-iminoethyl-

The influence of N -ethyl- and N -benzyl-1,2-diphenyl ethanolamines (compounds E and B, respectively) was examined on the spontaneously contracting rabbit jejunum and the rat uterus together with their influence on the contractions induced by some spasmogens in the guinea-pig ileum and oxytocics and CaCl₂in the pregnant rat uterus. Both E and B inhibited the spontaneous contractions of the rabbit jejunum with ID₅₀values of 0.13 and 0.03 μmol ml⁻¹. Their inhibitory activities were not antagonized by α- or β-adrenoceptor blockers but significantly reversed by CaCl₂(0.015 μmol ml⁻¹). The compounds also antagonized nicotine, ACh-, histamine-, 5-HT- and CaCl₂-induced contractions by 44–100%. Compound E seemed to be several times more potent than B in inhibiting the spontaneous uterine contractions with an ID₅₀of (7 nmol ml⁻¹). Their inhibitory effects were not antagonized by β₂-adrenoceptor or H₂-receptor blocking drugs. Both compounds (40 nmol ml⁻¹) antagonized in a competitive manner CaCl₂-induced contractions in the K⁺-depolarised uterus and PGE₂and oxytocin-induced uterine contractions. The ID₅₀values were in the range of 1.6–10.7 nmol ml⁻¹. The results suggest that E and B compounds may be considered as putative L-Ca²⁺channel blockers with certain selectivities. The E compound seemed to be more selective against uterine L-Ca²⁺channels and the B compound against intestinal smooth muscles. Thus, the compounds may be of potential value in treatment of some colics, the irritant bowel syndrome, dysmenorrhoea and premature deliveries. 1999 Academic Press@p$hr     

STUDIES ON THE SPASMOLYTIC AND UTERINE RELAXANT ACTIONS OF N -ETHYL AND N -BENZYL-1,2-DIPHENYL ETHANOLAMINES: ELUCIDATION OF THE MECHANISMS OF ACTION

The influence of N -ethyl- and N -benzyl-1,2-diphenyl ethanolamines (compounds E and B, respectively) was examined on the spontaneously contracting rabbit jejunum and the rat uterus together with their influence on the contractions induced by some spasmogens in the guinea-pig ileum and oxytocics and CaCl₂in the pregnant rat uterus. Both E and B inhibited the spontaneous contractions of the rabbit jejunum with ID₅₀values of 0.13 and 0.03 μmol ml⁻¹. Their inhibitory activities were not antagonized by α- or β-adrenoceptor blockers but significantly reversed by CaCl₂(0.015 μmol ml⁻¹). The compounds also antagonized nicotine, ACh-, histamine-, 5-HT- and CaCl₂-induced contractions by 44–100%. Compound E seemed to be several times more potent than B in inhibiting the spontaneous uterine contractions with an ID₅₀of (7 nmol ml⁻¹). Their inhibitory effects were not antagonized by β₂-adrenoceptor or H₂-receptor blocking drugs. Both compounds (40 nmol ml⁻¹) antagonized in a competitive manner CaCl₂-induced contractions in the K⁺-depolarised uterus and PGE₂and oxytocin-induced uterine contractions. The ID₅₀values were in the range of 1.6–10.7 nmol ml⁻¹. The results suggest that E and B compounds may be considered as putative L-Ca²⁺channel blockers with certain selectivities. The E compound seemed to be more selective against uterine L-Ca²⁺channels and the B compound against intestinal smooth muscles. Thus, the compounds may be of potential value in treatment of some colics, the irritant bowel syndrome, dysmenorrhoea and premature deliveries. 1999 Academic Press@p$hr     

Leucocyte recruitment induced by type II phospholipases A2 into the rat pleural cavity

Bothropstoxin-I (BthTX-I) and bothropstoxin-II (BthTX-II) are Lys-49 and Asp-49 phospholipases A₂ (PLA₂s), respectively, isolated from Bothrops jararacussu venom. Piratoxin-I (PrTX-I) is a Lys-49 PLA₂ isolated from Bothrops pirajai venom. In this study, the ability of BthTX-I, BthTX-II and PrTX-I to recruit leucocytes into the rat pleural cavity and potential mechanisms underlying this effect were investigated. Intrapleural injection of either BthTX-I or PrTX-I (10–100 μg/cavity each) caused a significant leucocyte infiltration at 12 h after injection. The maximal cell migration was observed with the dose of 30 μg/cavity (14.9±15.5 and 17.6±1.6×10⁶ cells/cavity, respectively). Leucocyte counts consisted mainly of mononuclear cells, but significant amounts of neutrophils and eosinophils were also observed. Intrapleural injection of BthTX-II (10–100 μg/cavity) caused a marked leucocyte infiltration at 6 and 12 h after injection. The maximal response was observed with the dose of 100 μg/cavity (57.3±3.4×10⁶ cells/cavity, 6 h). The leucocyte counts were mainly composed of neutrophils and mononuclear cells. The treatment of either BthTX-I (30 μg/cavity, 12 h) or BthTX-II (30 μg/cavity, 6 h) with the PLA₂ inhibitor p-bromophenacyl bromide (p-BPB) had no effect on the total and differential leucocyte counts induced by these proteins. The same treatment partially reduced the PrTX-I-induced pleural leucocyte infiltration. In rats depleted of the histamine and 5-hydroxytryptamine (5-HT) stores by chronic treatment with compound 48/80, the total leucocyte counts in response to BthTX-I, BthTX-II and PrTX-I was not significantly affected compared to control animals. In addition, BthTX-I, BthTX-II and PrTX-I (100 μg/ml each) significantly degranulated pleural mast cells in vitro leading to the release of [¹⁴C]5-hydroxytryptamine ([¹⁴C]5-HT). p-BPB and heparin (50 IU/ml) significantly reduced the [¹⁴C]5-HT release induced by these PLA₂s. Our results demonstrate that BthTX-I, BthTX-II and PrTX-I recruit leucocyte into the pleural cavity of the rat by mechanisms unrelated to enzymatic activity and pleural mast cell degranulation.     

Enhancement of the hypotensive effects of intrathecally injected endocannabinoids by the entourage compound palmitoylethanolamide

The intrathecal (i.t.) injection of 50 and 100 nmol anandamide to urethane anesthetized rats induced a dose-dependent decrease in the mean blood pressure (− 10.6 ± 1.6 mmHg and − 15.0 ± 1.7 mmHg, respectively; n = 6) whereas a lower dose of this endocannabinoid (25 nmol) was devoid of effect. Similar responses were obtained both with the non-metabolizable analog methanandamide and with the endocannabinoid N-arachidonoyldopamine. When the sub-effective dose (25 nmol) of each compound was co-injected with palmitoylethanolamide (100 nmol), significant decreases in the blood pressure were observed (− 12.3 + 1.3 mmHg for anandamide; − 12.1 ± 0.8 mmHg for methanandamide; − 12.1 ± 0.8 mmHg for N-arachidonoyldopamine; n = 4–6). Palmitoylethanolamide also enhanced the hypotensive responses to the 50 nmol-dose of both anandamide and methanandamide. The hypotensive response induced by co-administration of palmitoylethanolamide and 25 nmol anandamide was prevented both by the cannabinoid CB₁ receptor antagonist SR 144716A (20 nmol; i.t.) and by the vanilloid TRPV1 receptor antagonist capsazepine (20 nmol; i.t.) and enhanced by pretreatment with URB602 (3.5 nmol; i.t.), a putative inhibitor of palmitoylethanolamide degradation. These results suggest that in the spinal cord palmitoylethanolamide acts as an entourage compound for the hypotensive effects of i.t. administered endocannabinoids. The facilitative action of palmitoylethanolamide affects the vanilloid TRPV1 as well as the cannabinoid CB₁ receptor-mediated effects of endocannabinoids on the blood pressure control.     

Kinetic analysis of glutamate-induced chloride current in Aplysia neurones: a ‘concentration clamp’ study

L-Glutamate (Glu) applied by the ‘concentration clamp’ technique to isolated neurones of Aplysia induced a chloride current (I_Cl) by activating a single population of the channel. The concentration-response curve for the peak I_Cl gave a dissociation constant of 1.3 × 10⁻⁴ M and a Hill coefficient of 1.8. The current-voltage relationship was linear in the voltage range examined (−60 to +10 mV). The activation phase of the I_Cl followed a single-exponential time course and desensitization was complete with a double-exponential time course. The time constant for activation and desensitization decreased with increasing concentrations of Glu but were voltage-independent. The process of recovery from desensitization was also double-exponential. The single-channel conductance estimated by ensemble noise analysis was 50±4.7 pS (n=4). These results suggest that the Glu receptor-Cl channel complex in Aplsia neurones consists of a single population with two binding sites for the agonist.     

A novel compound N,N-(Z)-N-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition

Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC₅₀ of 0.74 ± 0.15 µM for S6 cells or 1.07 ± 0.23 µM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.     

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.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the α-phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

EFFECT OF POLICOSANOL SUCCESSIVE DOSE INCREASES ON PLATELET AGGREGATION IN HEALTHY VOLUNTEERS

Policosanol is a cholesterol-lowering drug with hypocholesterolemic effects demonstrated in experimental models, healthy volunteers and type II hypercholesterolemic patients. In addition, antiplatelet effects of policosanol have been shown in experimental models and healthy volunteers. The effect of successively increasing doses of policosanol on platelet aggregation was investigated in a randomized, placebo-controlled, double-blind study conducted in 37 healthy volunteers. The volunteers were on a placebo-baseline period (two tablets per day) for 7 days and thereafter they received randomly, under double-blind conditions, placebo or policosanol (10mgday⁻¹) for 7 days. After this period dosage was doubled to 20mgday⁻¹for the next 7 days and then again doubled to 40mgday⁻¹, while the control group received placebo tablets all the time. Platelet aggregation as well as coagulation time was measured at baseline and after each dosing step. Results showed that antiplatelet effects of policosanol were successfully enhanced throughout the study, thus suggesting a dose-dependent relationship. No significant effect was reached during the first dosing period, but significant reductions of epinephrine and ADP-induced platelet aggregation were observed after the second one. Finally, a significant inhibition of platelet aggregation induced by all the agonists was observed at the last dosing step. Coagulation time remained unchanged during the trial.     

Effects of Rhodocodon madagascariensis extracts on embryo–larval development of medaka fish, Oryzias latipes

Rhodocodon madagascariensis, also named Urginea mascarenensis, is a malagasy plant belonging to the Hyacinthaceae family. As for the other members of the endemic malagasy genus Rhodocodon, the chemical and toxicological properties of this species have not yet been studied. The present study concerns the analysis of the toxicity of R. madagascariensis to medaka embryo–larval development. The incubation of medaka fish embryos or larvae in a medium containing R. madagascariensis extract resulted in a dose dependent reduction in development of embryos leading to lethality and a drastic reduction in survival rate of exposed larvae. Survival rates were reduced up to 100% with an extract concentration of 4 mg mL⁻¹. The LD₅₀ was estimated to be 1 mg mL⁻¹. Anatomopathological studies did show some neuro-embryonal modifications in the encephalic region. The data presented in this paper thus extends the use of medaka embryos as a valuable model to detect and analyse the effects of plant toxins.     

Application of a Combined “Effect Compartment/Indirect Response Model” to the Central Nervous System Effects of Tiagabine in the Rat

Pharmacological inhibition of GABA uptake transporters provides a mechanism for increasing GABAergic transmission, which may be useful in the treatment of various neurological disorders. The purpose of our investigations was to develop an integrated pharmacokinetic–pharmacodynamic (PK/PD) model for the characterization of the pharmacological effect of tiagabine, R-N-(4,4-di-(3-methylthien-2-yl)but-3-enyl)nipecotic acid, in individual rats in vivo. The tiagabine-induced increase in the amplitude of the EEG 11.5–30 Hz frequency band (), was used as pharmacodynamic endpoint. Chronically instrumented male Wistar rats were randomly allocated to four groups which received an infusion of 3, 10, or 30 mg kg ^–1 ml min ^-1 kg^–1, 1.50.1 L kg^–1 and 200.2 min.A time delay was observed between the occurrence of maximum plasma drug concentrations and maximal response. A physiological PK/PD model has been used to account for this time delay, in which a biophase was postulated to account for tiagabine available to the GABA uptake carriers in the synaptic cleft and the increase in EEG effect was considered an indirect response due to inhibition of GABA uptake carriers. The population values for the pharmacodynamic parameters characterizing the delay in pharmacological response relative to plasma concentrations were k_eo=0.030 min ^–1 and k_out=81 min^–1, respectively. Because of the large difference in these values the PK/PD model was simplified to the effect compartment model. Population estimates were E₀=155 6 V, E_max=100 5 V, EC₅₀=287 7 ng ml^–1, Hill factor=1.8 0.2 and k_eo=0.030 0.002 min ^–1. The results of this analysis show that for tiagabine the combined effect compartment-indirect response model can be simplified to the classical effect compartment model.     

Endothelium-derived nitric oxide partially mediates salbutamol-induced vasodilatations

This study examined the ability of salbutamol (selective β₂-adrenoceptor agonist) to cause endothelium-dependent relaxation in rat aortic rings and depressor response in conscious rats. Salbutamol (0.01–100 μM) concentration dependently relaxed preconstricted aortic rings. The relaxant response was partially attenuated by either mechanical removal of the endothelium or treatment with N^G-nitro-l-arginine methyl ester (L-NAME, 100 μM). In conscious rats, either i.v. infused phenylephrine (5 μg/kg per min) or i.v. bolus injected L-NAME (12.8 mg/kg), but not the vehicle, caused similar sustained increases in mean arterial pressure (MAP). I.v. infused salbutamol (2–128 μg/kg per min, each dose for 5 min) dose dependently decreased MAP in vehicle-treated rats; the depressor responses were potentiated by hypertension induced by phenylephrine. In contrast, the magnitudes of the depressor response to salbutamol in L-NAME-treated rats were less than those in rats pretreated with phenylephrine or the vehicle. I.v. bolus injections of salbutamol (0.25–16 μg/kg) also caused dose-dependent and transient decreases in MAP in vehicle-treated rats. The magnitude but not the duration of the depressor response to salbutamol was less in rats treated with L-NAME, compared to those in rats given phenylephrine or the vehicle. These results suggest that endothelium-derived nitric oxide is partially involved in β₂-adrenoceptor-mediated vasodilatation.     

Stimulation of insulin secretion by the imidazoline α2-adrenoceptor antagonist efaroxan is mediated by a novel, stereoselective, binding site

We have studied the stereospecificity of three responses mediated by the α₂-antagonist efaroxan in rat islets of Langerhans. α₂-Adrenergic inhibition of insulin secretion was relieved most effectively by the (+) enantiomer. In contrast, direct stimulation of insulin secretion and antagonism of the inhibitory effects of diazoxide were both preferentially mediated by the (−) enantiomer. Culture of islets in the presence of efaroxan resulted in loss of responsiveness to subsequent re-addition of the drug. On the basis of these results we propose the existence, in islets, of a novel ‘non-adrenergic’ binding site at which efaroxan acts as an agonist.     

AMELIORIATION OF CHEMICALLY-INDUCED HEPATOCYTE INJURY BY CYCLOSPORINE A

Cyclosporine A, beside its current applications, possesses potential hepatoprotective effects. This study was directed to investigate the effect of Cyclosporine A pretreatment on hepatic injury due to carbon tetrachloride (CCl₄) and -galactosamine. Rats were injected by two successive doses of Cyclosporine A (5mgkg⁻¹day⁻¹). Six hours after the second dose, 1mlkg⁻¹of CCl₄was administered i.p. Effects associated with Cyclosporine A pretreatment were examined by using isolated hepatocytes and hepatocytes that were immobilized and continuously perfused. -Galactosamine (5m ) was added directly to the perfusion medium. After isolation, hepatocytes were examined histologically by light and electron microscopy, immobilized and perfused for further metabolic functional activity evaluation. Cyclosporine A pretreatmentin vivoproduced hepatoameliorative effects of various degrees which were statistically significant as manifested by: (1) an increased trypan blue exclusion after CCl₄; (2) an improved ureagenesis after CCl₄; (3) a reduction in the lipid droplets accumulation in the cytoplasm produced by CCl₄administration; (4) well preserved cytoplasmic organelles as mitochondria, endoplasmic reticulum ER, nuclear chromatin structures that were altered by CCl₄; and (5) an increased hepatocytes survival in the agarose gel matrix, reduction of LD leakage and improvement of ureagenesis after -galactosamine addition to the perfusion medium. The beneficial effect of Cyclosporine A pretreatment in modifying hepatotoxicity of chemical insults merits further studies.     

Oxidative stress, protein carbonylation and heat shock proteins in the black tiger shrimp, Penaeus monodon, following exposure to endosulfan and deltamethrin

The impact of commonly used pesticides, endosulfan and deltamethrin, on the molecular stress level in black tiger shrimp Penaeus monodon, was assessed using classical oxidative stress biomarkers, protein carbonylation profiles, and levels of heat shock proteins. Results showed that 4 days exposure to 0.1 μg L⁻¹ deltamethrin significantly (p < 0.05) increased lipid peroxidation (LPO) level in gills (64.3 ± 3.2 compared to 34.2 ± 5.3 nmol MDA equiv. g⁻¹ tissue at day 0). However, no pesticide treatment had significant effect on the activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). Carbonylated protein profiles were determined on gills following 2,4-dinitrophenylhydrazine derivatization and 2D-PAGE along with Western blotting. Immunoblotting with dinitrophenol-specific antibody revealed 17 protein spots carbonylated in response to 4 days exposure to 0.1 μg L⁻¹ deltamethrin while 24 protein spots specifically oxidized at day 0 were no longer detected after deltamethrin treatment. On the other hand, endosulfan exposure at 0.1 and 1 μg L⁻¹ induced up to 2.1-fold increase of HSP90 level in muscle. This approach is providing new insights into the molecular impacts of deltamethrin and endosulfan on an economically important crustacean. While deltamethrin has shown a pro-oxidant effect in gills, endosulfan exposure rather induced proteotoxic effects in muscles. This argues that LPO level, protein carbonylation specificities, and HSP90 levels may be potential discriminating biomarkers to assess the chemical stress level in farm shrimp.