Involvement of nitric oxide in 3-nitropropionic acid-induced depression of spinal reflexes in neonatal rat spinal cord in vitro

The objective of the present investigation is to study the involvement of nitric oxide (NO) in 3-nitropropionic acid (3-NPA)-induced depression of spinal reflexes. Experiments were conducted on preparations of hemisected spinal cord isolated from 4 to 8 day old rats. Stimulation of a dorsal root evoked reflex potentials (monosynaptic, MSR; polysynaptic, PSR) in the corresponding segmental ventral root. Superfusion of 3-NPA (3.4 mM) depressed the spinal reflexes in a time-dependent manner and the reflexes were abolished after 35 min. The time required to produce 50% depression of the reflexes (T-50) was 17.8 ± 5.3 min for MSR and 17.5 ± 2.1 min for PSR. L-NAME (Nω-nitro-l-arginine methyl ester; 100 µM), a nitric oxide synthase inhibitor, antagonized the 3-NPA (3.4 mM)-induced depression of reflexes and increased the T-50 values (34 and 30 min for MSR and PSR, respectively) significantly (P < 0.05). In addition, hemoglobin (Hb, 100 µM), a NO scavenger, blocked the 3-NPA-induced depression of reflexes significantly (P < 0.05). T-50 values in Hb pretreated cords were 57 and 45 min for MSR and PSR, respectively which were greater than the cords pretreated with L-NAME. The nitrite (NO₂⁻) content of the 3-NPA exposed cords was 84 µM/g of tissue which was significantly greater than the control (13 µM/g; P < 0.05). Pretreatment of cords with L-NAME or Hb antagonized the 3-NPA-induced increase in NO₂⁻. The results indicate that NO produced by 3-NPA is involved in the 3-NPA-induced depression of spinal reflexes.     

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.     

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.     

Modulation of opioids via protection of anandamide degradation by fatty acid amide hydrolase

Lack of involvement of the opioid system with the endocannabinoid, arachidonylethanolamide (anandamide) was possibly due to hydrolysis by fatty acid amide hydrolase (FAAH). Cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597) is an inhibitor of FAAH, increases brain anandamide levels and enhances anandamide-induced antinociception in male ICR mice (25–30 g). The combination of URB597 (10 mg/kg, i.p.) and anandamide (40 mg/kg, i.p.) produced maximal antinociception in the mouse tail-flick test [68.7 ± 16.8 percent maximum possible effect (%MPE)], versus either substance alone (27.3 ± 7.9%MPE and 4.6 ± 2.3%MPE, respectively) and is significantly blocked (p < 0.05) by the cannabinoid CB₁ receptor antagonist, SR141716A (rimonabant), the kappa opioid receptor-selective antagonist, nor-Binaltorphimine (10 μg i.t.; 12.7 ± 4.0%MPE) and the mu opioid receptor antagonist, naloxone (1 mg/kg, s.c.; 6.0 ± 3.8%MPE), but not by the delta opioid receptor-selective antagonist, naltrindole (2 mg/kg, s.c.; 29.7 ± 8.2%MPE) or the cannabinoid CB₂ receptor antagonist, SR144528. In addition, nor-BNI (10 μg i.t) administration to FAAH^−/− knockout mice produced a nociceptive response. The URB597/anandamide combination was not active in the CB₁^−/− knockout mice, but retained activity in the MOR^−/− knockout mice. The sub-active combination of (URB597 10 mg/kg, i.p/anandamide 10 mg/kg, i.p.; 15.5 ± 4.3%MPE) shifted the dose response curve of morphine to the left (morphine alone ED₅₀ = 4.6 mg/kg [3.7–5.6] versus morphine/URB597/anandamide (ED₅₀ = 2.5 mg/kg [1.9–3.4]). These data are the first demonstration that anandamide, if protected from degradation, acts via the CB₁ receptor to interact with kappa opioid receptor systems in opioid analgesia.     

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.     

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.     

A comparison of the stability of doxorubicin and daunorubicin in solid state

The degradation of doxorubicin and daunorubcin in the solid state was studied using an HPLC method with UV detection (LiChrospher RP-18, 5 μm, 250 mm × 4 mm; mobile phase: acetonitrile-solution A 1:1, v/v (solution A: 2.88 g of laurisulfate sodium and 1.6 ml of phosphoric acid(V) in 1000 ml); flow rate – 1.4 ml min⁻¹; UV detection – 254 nm). The degradation of doxorubicin was a first-order reaction depending on the substrate concentration and daunorubicin degraded according to the kinetic model of autocatalysis. The dependence ln k_i = f(1/T) was described by the equations ln k_DOX = 40.0 ± 15.6 – (19804 ± 5682) (1/T) and ln k_DAU = 35.9 ± 11.3 – (16581 ± 3972) (1/T) at 76.4% RH. The dependence ln k_i = f(RH%) was described by the equations ln k_DOX = (8.80 ± 3.60) × 10⁻² (RH%) – (21.50 ± 2.57) and ln k_DAU = (6.63 ± 1.22) × 10⁻² (RH%) – (13.35 ± 1.68). The thermodynamic parameters (E_a, ΔH^≠a, ΔS^≠a) of the degradation of doxorubicin and daunorubicin were calculated. Although the degradation of doxorubicin was slower at increased temperature (353–373 K) and relative air humidity (50.9–90.0%), the differences between the influence of temperature and relative air humidity on the stability doxorubicin and of daunorubicin were not significant.     

The vasorelaxant effect of caffeic acid phenethyl ester on porcine coronary artery ring segments

Caffeic acid phenethyl ester (CAPE) is a naturally occurring compound isolated from honeybee propolis whose cardiovascular properties remain uncertain. The purpose of this study was to investigate the possible mechanisms of CAPE-induced vasorelaxation in porcine coronary artery rings. It was found that both the quiescent and precontracted coronary artery ring segments were relaxed by CAPE (10^− 7–10^− 4 M). N^ω-nitro-l-arginine (L-NNA), methylene blue and removal of endothelium significantly attenuated CAPE-induced relaxation of both quiescent and precontracted artery rings. This relaxing effect of CAPE on coronary arteries was also significantly reduced by propranolol, and SQ22536, but not by indomethacin. In addition, the dose–response curves of KCl (2.5–100 mM) and CaCl₂ (10^− 5–10^− 2 M) were displaced downwards in the presence of CAPE. These results suggest that the relaxant effect of CAPE on porcine coronary artery rings might involve the action of nitric oxide (NO) and adrenergic β-receptor, together with their second messenger, cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), respectively, but not involve the synthesis of prostaglandin.     

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.     

Effects of propofol on responses of rat isolated renal arteriole to vasoactive agents

Rat renal arterial rings were suspended in organ chambers for isometric tension recording. The effects of propofol on the resting tone, KCl-, norepinephrine (NE)-, serotonin- and thromboxane A₂ analog U46619-induced contractions were observed. The relaxation responses to propofol on KCl-, NE- and U46619-induced contractions were assessed in the absence or presence of cyclooxygenase inhibitor, nitric oxide synthetase inhibitor or specific K⁺ channel inhibitors. Propofol did not significantly affect the resting tone, but inhibited the contractions induced by KCl-, NE-, serotonin- and U46619. Propofol (1–100 µM) concentration-dependently relaxed 60 mM KCl-, 10 µM NE-, and 1 µM U46619-induced contractions with the values of RC₅₀ (concentration to decline the precontraction by 50%) being 18.9 µM, 70.6 µM and 12.7 µM, respectively. Propofol-induced relaxation was attenuated by indomethacin, but not by either N^G nitro-l-arginine methyl ester (L-NAME) or any K⁺ channel specific inhibitors used. The vasorelaxations induced by acetylcholine, sodium nitroprusside and amrinone were not affected by the presence of propofol. The present results indicate that propofol antagonizes provoked contractions of the arteriole and suggest that inhibition of extracellular Ca²⁺ influx and synthesis of vasodilator prostanoid may be involved in propofol-induced relaxation of the arteriole.     

A pharmacological examination of venom from the Papuan taipan: (Oxyuranus scutellatus canni)

The Papuan taipan (Oxyuranus scutellatus canni) is the third most venomous terrestrial snake in the world, however, little is know about the pharmacology of the venom. In the chick biventer cervicis muscle, venom (10 μg/ml) abolished nerve-mediated twitches (time to 90% inhibition (t₉₀) 44±5 min, n=9). This inhibition was unaffected by prior incubation of the venom with the phospholipase A inhibitor 4-bromophenacyl bromide (4-BPB; 0.72 mM) (t₉₀ 48±7 min, n=8). The mouse phrenic nerve diaphragm preparation displayed greater sensitivity to venom (10 μg/ml) (t₉₀ 25±1 min, n=6). In the chick biventer muscle, venom (10 μg/ml) significantly inhibited responses to acetylcholine (1 mM) and carbachol (20 μM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. Venom (10 μg/ml) did not affect direct muscle stimulation. Venom (3–30 μg/ml) produced dose-dependant contractions of the guinea-pig ileum. Contractile responses were significantly inhibited by indomethacin (1 μM) or prior incubation of the venom with 4-BPB (0.72 mM) indicating involvement of a PLA component. In rat phenylephrine (0.3 μM) precontracted aortae, venom (3–100 μg/ml) produced endothelium-independent relaxation which was unaffected by prior incubation of venom (30 μg/ml) with 4-BPB (0.72 mM). In anaesthetised rats, 10 μg/kg (i.v.) venom produced rapid respiratory and cardiovascular collapse while 5 μg/kg (i.v.) venom produced only a small transient decrease in mean arterial blood pressure. Prior administration of 5 μg/kg (i.v.) venom enabled subsequent administration of 10 and 100 μg/kg (i.v.) venom without respiratory or cardiovascular collapse. Further work is required to identify specific toxins with the above pharmacological activity.     

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.     

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.     

Effect of a metabotropic glutamate receptor 5 antagonist, MPEP, on the nociceptive response induced by intrathecal injection of excitatory aminoacids, substance P, bradykinin or cytokines in mice

Metabotropic glutamate receptors (mGluRs) are expressed abundantly in the spinal cord and have been shown to play important roles in the modulation of nociceptive transmission and plasticity. In this study, the involvement of metabotropic glutamate receptor 5 (mGluR₅) in the nociceptive response induced by intrathecal injection (i.t.) of excitatory aminoacids, substance P (SP), bradykinin (BK) and cytokines in mice was demonstrated. The administration of 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 10–50 nmol/site, i.t.) caused a significant inhibition in the nociceptive response induced by glutamate and trans-ACPD with maximal inhibitory effects of 36 ± 7% and 56 ± 5%, respectively. MPEP completely failed to affect the nociception induced by α-amino-3-hydroxy-5-mehtyl-4-isoxazolepropionic acid (AMPA; 135 pmol/site), kainate (110 pmol/site) and N-methyl-d-aspartate (NMDA; 450 pmol/site). MPEP also reduced the nociceptive response induced by SP (135 ng/site, i.t.), BK (0.1 µg/site), tumor necrosis factor-alpha (TNF-α; 0.1 pg/site) and interleukin-1beta (IL-1β; 1 pg/site) with maximal inhibitions of 29 ± 5%, 37 ± 5%, 83 ± 3% and 88 ± 1%, respectively. Together, these results indicate the involvement of mGluRs, more specifically of subtype-5, in the nociceptive response induced by i.t. injection of excitatory aminoacids, SP, BK and cytokines in mice.     

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.     

An enhanced postnatal autoimmune profile in 24 week-old C57BL/6 mice developmentally exposed to TCDD

Developmental exposure of mice to the environmental contaminant and AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes persistent postnatal suppression of T cell-mediated immune responses. The extent to which prenatal TCDD may induce or exacerbate postnatal autoimmune disease remains unknown. In the present study, time-pregnant high affinity AhR C57BL/6 mice received a single oral administration of 0, 2.5, or 5 µg/kg TCDD on gestation day (gd) 12. Offspring of these mice (n = 5/gender/treatment) were evaluated at 24 weeks-of-age and showed considerable immune dysregulation that was often gender-specific. Decreased thymic weight and percentages of CD4⁺CD8⁺ thymocytes, and increased CD4⁺CD8⁻ thymocytes, were present in the female but not male offspring. Males but not females showed decreased CD4⁻CD8⁺ T cells, and increased Vβ3⁺ and Vβ17a⁺ T cells, in the spleen. Males but not females also showed increased percentages of bone marrow CD24⁻B220⁺ B cell progenitors. Antibody titers to dsDNA, ssDNA and cardiolipin displayed increasing trends in both male and female mice, reaching significance for anti-dsDNA in both genders and for ssDNA in males at 5 µg/kg TCDD. Immunofluorescent staining of IgG and C3 deposition in kidney glomeruli increased in both genders of prenatal TCDD-exposed mice, suggestive of early stages of autoimmune glomerulonephritis. Collectively, these results show that exposure to TCDD during immune system development causes persistent humoral immune dysregulation as well as altered cell-mediated responses, and induces an adult profile of changes suggestive of increased risk for autoimmune disease.     

Role of p38 mitogen-activated protein kinase in ozone-induced airway hyperresponsiveness and inflammation

Ozone is a potent oxidant and causes airway hyperresponsiveness and neutrophilia. To determine the role of p38 mitogen-activated protein kinase (MAPK) activation, we studied the effect of a p38α inhibitor SD-282 (Scios Inc, Fremont, CA USA) on ozone-induced airway hyperresponsiveness and neutrophilia. Balb/c mice received SD-282 (30 or 90 mg/kg i.p) or vehicle 1 h before exposure to either ozone (3 ppm, 3 h) or air. Three hours after exposure, lungs were analysed for cytokine levels and bronchoalveolar lavage was performed. Another set of mice were dosed 6 h after exposure and 1 h before assessing airway hyperresponsiveness. SD-282 (90 mg/kg) significantly inhibited ozone-induced airway hyperresponsiveness (− LogPC₁₅₀: SD-282: − 1.73 ± 0.14 vs. vehicle: − 0.99 ± 0.15, P < 0.05). Bronchoalveolar lavage neutrophil numbers were time-dependently increased in vehicle-dosed, ozone-exposed mice, greatest at 20–24 h after exposure. SD-282 (30 and 90 mg/kg) significantly inhibited ozone induced neutrophil numbers at 3 h and 20–24 h after ozone SD-282 significantly inhibited ozone-induced increases in phosphorylated p38 MAPK expression, and in cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and IL-1β but not MIP-1α gene expression. We conclude that p38 MAPK is involved in ozone-induced airway hyperresponsiveness and lung neutrophilia. Inhibition of p38 MAPK with small molecule kinase inhibitors may be a means of reducing ozone-induced inflammation and airway hyperresponsiveness.     

Study of forced degradation behavior of Eletriptan hydrobromide by LC and LC–MS and development of stability-indicating method

The objective of the present study was to report the stability profile of novel antimigrain drug Eletriptan hydrobromide based on the information obtained from forced degradation studies. The drug was subjected to acid (0.1–1 mol L⁻¹ HCl), neutral and base (0.1–1 mol L⁻¹ NaOH) hydrolysis and to oxidative decomposition (3–15% (v/v) H₂O₂). Photolysis and thermo degradation at 75 °C were carried out in methanol solution and in solid state with both Eletriptan hydrobromide bulk drug and the tablet formulation. The products formed under different stress conditions were investigated by LC and LC–MS.The experimental conditions for LC were chosen by employing experimental design and multicriteria decision making methodology. These powerful tools enabled the accomplishment of satisfactory resolution with the shortest possible analysis time. Analytes were separated on a C₁₈ column (XTerra™, 150 mm × 3.9 mm, 5 μm) with the mobile phase composed of methanol–water solution of TEA (pH 6.52, 1%, v/v) (30:70, v/v) pumped at 1 mL min⁻¹ flow rate. The column temperature was set at 50 °C and the detection at 225 nm using DAD detector. The LC method was suitably modified for LC–MS analysis which was further used to characterize the arisen degradation products. The possible degradation pathway was outlined based on the results.The drug appeared to be instable towards every stress condition but oxidation. The stability was not jeopardized even under more exaggerated conditions such as increased temperature of the solutions to 75 °C, increased strength of acid/alkali solutions and prolonged testing period.Validation of the LC-DAD method was carried out in accordance with ICH guideline. The method met all required criteria and was applied when testing the commercially available tablets.