3-iodothyroacetic acid,a metabolite of thyroid hormone,induces itch and reduces threshold to noxious and to painful heat stimuli in mice

Background and purpose

Itch is associated with increased sensitization to nociceptive stimuli. We investigated whether 3-iodothyroacetic acid (TA1), by releasing histamine, induces itch and increases sensitization to noxious and painful heat stimuli.

Experimental Approach

Itch was evaluated after s.c. administration of TA1 (0.4, 1.32 and 4 μg·kg⁻¹). Mice threshold to noxious (NHT) and to painful heat stimuli were evaluated by the increasing-temperature hot plate (from 45.5 to 49.5°C) or by the hot plate (51.5°C) test, respectively, 15 min after i.p. injection of TA1 (0.4, 1.32 and 4 μg·kg⁻¹). Itch, NHT and pain threshold evaluation were repeated in mice pretreated with pyrilamine. Itch and NHT were also measured in HDC^+/+ and HDC^−/− following injection of saline or TA1 (1.32, 4 and 11 μg·kg⁻¹; s.c. and i.p.). pERK1/2 levels were determined by Western blot in dorsal root ganglia (DRG) isolated from CD1 mice 15 min after they received (i.p.): saline, saline and noxious heat stimulus (46.5°C), TA1 (0.1, 0.4, 1.32, 4 μg·kg⁻¹) or TA1 1.32 μg·kg⁻¹ and noxious heat stimulus.

Key Results

TA1 0.4 and 1.32 μg·kg⁻¹ induced itch and reduced NHT; pyrilamine pretreatment prevented both of these effects. TA1 4 μg·kg⁻¹ (i.p.) reduced pain threshold without inducing itch or modifying NHT. In HDC^−/− mice, TA1 failed to induce itch and to reduce NHT. In DRG, pERK1/2 levels were significantly increased by noxious heat stimuli and by TA1 0.1, 0.4 and 1.32 μg·kg⁻¹; i.p.

Conclusions and Implications

Increased TA1 levels induce itch and an enhanced sensitivity to noxious heat stimuli suggesting that TA1 might represent a potential cause of itch in thyroid diseases.     

Histamine mediates behavioural and metabolic effects of 3-iodothyroacetic acid,an endogenous end product of thyroid hormone metabolism

BACKGROUND AND PURPOSE

3-Iodothyroacetic acid (TA1) is an end product of thyroid hormone metabolism. So far, it is not known if TA1 is present in mouse brain and if it has any pharmacological effects.

EXPERIMENTAL APPROACH

TA1 levels in mouse brain were measured by HPLC coupled to mass spectrometry. After i.c.v. administration of exogenous TA1 (0.4, 1.32 and 4 μg·kg⁻¹) to mice, memory acquisition-retention (passive avoidance paradigm with a light-dark box), pain threshold to thermal stimulus (51.5°C; hot plate test) and plasma glucose (glucorefractometer) were evaluated. Similar assays were performed in mice pretreated with s.c. injections of the histamine H₁ receptor antagonist pyrilamine (10 mg·kg⁻¹) or the H₂ receptor antagonist zolantidine (5 mg·kg⁻¹). TA1 (1.32 and 4 μg·kg⁻¹) was also given i.c.v. to mice lacking histidine decarboxylase (HDC^−/−) and the corresponding WT strain.

KEY RESULTS

TA1 was found in the brain of CD1 but not of HDC mice. Exogenous TA1 induced amnesia (at 0.4 μg·kg⁻¹), stimulation of learning (1.32 and 4 μg·kg⁻¹), hyperalgesia (0.4, 1.32 and 4 μg·kg⁻¹) and hyperglycaemia (1.32 and 4 μg·kg⁻¹). All these effects were modulated by pyrilamine and zolantidine. In HDC^−/− mice, TA1 (1.32 and 4 μg·kg⁻¹) did not increase plasma glucose or induce hyperalgesia.

CONCLUSIONS AND IMPLICATIONS

Behavioural and metabolic effects of TA1 disclosed interactions between the thyroid and histaminergic systems.     

Antithrombotic effect of Z4A5 on coronary thrombosis in a canine model of acute unstable angina

Background and Purpose

The glycoprotein IIb/IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking coronary thrombosis. In this study, the anti-platelet and antithrombotic actions of Z4A5, a new glycoprotein IIb/IIIa receptor inhibitor, were evaluated in a canine model of acute unstable angina.

Experimental Approach

Z4A5 was given i.v. as a bolus followed by 60 min of continuous infusion at doses of 30 μg·kg⁻¹ + 1 μg·kg⁻¹·min⁻¹, 30 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹ or 300 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹. Its antithrombotic effect was evaluated in a model of coronary thrombosis, the injured, stenosed left circumflex coronary artery, in which platelet-dependent cyclic flow reductions (CFRs) were induced by vascular compression and constriction to simulate clinical acute unstable angina. Platelet aggregation and coagulation parameters were determined in platelet-rich plasma and platelet poor plasma respectively.

Key Results

The Z4A5 infusion induced a dose-dependent reduction in CFR frequency, which returned to baseline levels after the termination of the infusion at low doses. At medium dose that inhibited most part of platelet aggregation, it increased tongue bleeding time marginally with no dramatic changes in haemodynamic and coagulation parameters. Furthermore, the inhibition of ADP-induced platelet aggregation and prolonged bleeding time observed during Z4A5 infusion reverted to baseline levels after the termination of the infusion.

Conclusions and Implications

Z4A5 is an effective antithrombotic agent for coronary artery thrombosis with a rapid-on and rapid-off pharmacological profile, and could be used as an alternative treatment of coronary artery ischaemic syndromes.     

Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine

Background and Purpose

Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system.

Experimental Approach

Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA).

Key Results

Both p.o. (0.008–1.0 μg·kg⁻¹) and intraplantar (0.0006 μg per paw) administration of crotalphine induced antinociception in PGE₂-induced hyperalgesia. Antinociception by p.o. crotalphine (1 μg·kg⁻¹) was blocked by AM630 (50 μg per paw), a CB₂ receptor antagonist, and by antiserum anti-dynorphin A (1 μg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB₂ (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630.

Conclusions and Implications

Crotalphine-induced antinociception involves peripheral CB₂ cannabinoid receptors and local release of dynorphin A, which is dependent on CB₂ receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.     

Effect of low doses of cannabidiolic acid and ondansetron on LiCl-induced conditioned gaping (a model of nausea-induced behaviour) in rats

Background and Purpose

To determine the minimally effective dose of cannabidiolic acid (CBDA) that effectively reduces lithium chloride (LiCl)-induced conditioned gaping reactions (nausea-induced behaviour) in rats and to determine if these low systemic doses of CBDA (5–0.1 μg·kg⁻¹) relative to those of CBD could potentiate the anti-nausea effects of the classic 5-hydroxytryptamine 3 (5-HT₃) receptor antagonist, ondansetron (OND).

Experimental Approach

We investigated the efficacy of low doses of CBDA to suppress acute nausea, assessed by the establishment of conditioned gaping to a LiCl-paired flavour in rats. The potential of threshold and subthreshold doses of CBDA to enhance the reduction of nausea-induced conditioned gaping by OND were then determined.

Key Results

CBDA (at doses as low as 0.5 μg·kg⁻¹) suppressed nausea-induced conditioned gaping to a flavour. A low dose of OND (1.0 μg·kg⁻¹) alone reduced nausea-induced conditioned gaping, but when it was combined with a subthreshold dose of CBDA (0.1 μg·kg⁻¹) there was an enhancement in the suppression of LiCl-induced conditioned gaping.

Conclusions and Implications

CBDA potently reduced conditioned gaping in rats, even at low doses and enhanced the anti-nausea effect of a low dose of OND. These findings suggest that combining low doses of CBDA and OND will more effectively treat acute nausea in chemotherapy patients.     

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.

     

The efficacy of parecoxib on systemic inflammatory response associated with cardiopulmonary bypass during cardiac surgery

Aims

Cardiopulmonary bypass (CPB) during cardiac surgery is well known to be associated with the development of a systemic inflammatory response. The efficacy of parecoxib in attenuating this systemic inflammatory response is still unknown.

Methods

Patients undergoing elective mitral valve replacement with CPB were assessed, enrolled and randomly allocated to receive parecoxib (80 mg) or placebo. Blood samples were collected in EDTA vials for measuring serum cytokine concentrations, troponin T, creatinekinase myocardial‐brain isoenzyme CK‐MB concentrations and white cell counts.

Results

Compared with the control group, IL‐6 and IL‐8‐values in the parecoxib group increased to a lesser extent, peaking at 2 h after the end of CPB (IL‐6 31.8 pg ml⁻¹ ± 4.7 vs. 77.0 pg ml⁻¹ ± 14.1, 95% CI −47.6, −42.8, P < 0.001; IL‐8 53.6 pg ml⁻¹ ± 12.6 vs. 105.7 pg ml⁻¹ ± 10.8, 95% CI −54.8, −49.4, P < 0.001). Peak concentrations of anti‐inflammatory cytokine IL‐10 occurred immediately after termination of CPB and were higher in the parecoxib group (115.7 pg ml⁻¹ ± 10.5 vs. 88.4 pg ml⁻¹ ± 12.3, 95% CI 24.7, 29.9, P < 0.001). Furthermore, the increase in neutrophil counts caused by CPB during cardiac surgery was inhibited by parecoxib. The increases in serum troponin T and CK‐MB concentrations were also significantly attenuated by parecoxib in the early post‐operative days. Peak serum concentrations of CK‐MB in both groups occurred at 24 h post‐CPB (17.4 μg l⁻¹ ± 5.2 vs. 26.9 μg l⁻¹ ± 6.9, 95% CI −10.9, −8.1, P < 0.001). Peak troponin T concentrations occurred at 6 h post‐bypass (2 μg l⁻¹ ± 0.62 vs. 3.5 μg l⁻¹ ± 0.78, 95% CI −1.7, −1.3, P < 0.001).

Conclusion

Intra‐operative parecoxib attenuated the systemic inflammatory response associated with CPB during cardiac surgery and lowered the biochemical markers of myocardial injury.     

Are women more susceptible than men to drug-induced QT prolongation? Concentration-QTc modelling in a phase 1 study with oral rac-sotalol

Aim

To study the differences in QT_c interval on ECG in response to a single oral dose of rac-sotalol in men and women.

Methods

Continuous 12-lead ECGs were recorded in 28 men and 11 women on a separate baseline day and following a single oral dose of 160 mg rac-sotalol on the following day. ECGs were extracted at prespecified time points and upsampled to 1000 Hz and analyzed manually in a central ECG laboratory on the superimposed median beat. Concentration–QTc analyses were performed using a linear mixed effects model.

Results

Rac-sotalol produced a significant reduction in heart rate in men and in women. An individual correction method (QT_cI) most effectively removed the heart rate dependency of the QT_c interval. Mean QT_cI was 10 to 15 ms longer in women at all time points on the baseline day. Rac-sotalol significantly prolonged QT_cI in both genders. The largest mean change in QT_cI (ΔQT_cI) was greater in females (68 ms (95% confidence interval (CI) 59, 76 ms) vs. 27 ms (95% CI 22, 32 ms) in males). Peak rac-sotalol plasma concentration was higher in women than in men (mean C_max 1.8 μg ml⁻¹ (range 1.1–2.8) vs. 1.4 μg ml⁻¹ (range 0.9–1.9), P = 0.0009). The slope of the concentration–ΔQT_cI relationship was steeper in women (30 ms per μg ml⁻¹ vs. 23 ms per μg ml⁻¹ in men; P = 0.0135).

Conclusions

The study provides evidence for a greater intrinsic sensitivity to rac-sotalol in women than in men for drug-induced delay in cardiac repolarization.     

Anti-thrombotic effects and bleeding risk of AJvW-2, a monoclonal antibody against human von Willebrand factor

1. A murine anti-human vWF monoclonal antibody, AJvW-2, was developed that inhibited the interaction between platelet glycoprotein Ib (GPIb) and von Willebrand factor (vWF) during the ristocetin- (IC₅₀=0.7±0.1 μg ml⁻¹) and botrocetin- (IC₅₀=1.8±0.3 μg ml⁻¹) induced aggregation of human platelets.
2. AJvW-2 inhibited the high shear stress (10.8 N m⁻²) induced aggregation of human platelets dose-dependently with an IC₅₀=2.4±0.3 μg ml⁻¹, but had no effect on low shear stress induced platelet aggregation (1.2 N m⁻²) up to 100 μg ml⁻¹.
3. AJvW-2 also inhibited the high shear stress (5.0 N m⁻²) induced adhesion of human platelets to collagen I with the same efficacy (IC₅₀=2.4±0.3 μg ml⁻¹), but had no effect at low shear conditions (1.5 N m⁻²).
4. AJvW-2 inhibited the botrocetin-induced aggregation of platelets from guinea-pig, rat, rabbit, dog and pig at the same concentration range as human platelets; it likewise also inhibited the high shear stress induced aggregation and adhesion to collagen I of guinea-pig platelets.
5. AJvW-2 prevented arterial thrombus formation in guinea-pigs at a dose of 100 μg kg⁻¹ without prolonging the template bleeding time, whereas the GPIIb/IIIa antagonist lamifiban mediated inhibition of thrombosis at 1000 μg kg⁻¹ was accompanied by a significant prolongation of the bleeding time.
6. These results suggest that AJvW-2 is a potent inhibitor of the GPIb-vWF interaction and a potential novel antithrombotic agent with lower bleeding risk than GPIIb/IIIa antagonists.

     

The role of A3 adenosine receptors in central regulation of arterial blood pressure

1. Pharmacological studies have suggested that A₃ receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed.
2. In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N⁶-2-(4-aminophenyl)-ethyladenosine (APNEA), a non-selective A₃ adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective antagonist of A₁ adenosine receptors.
3. Anaesthetized rats, after DPCPX (12 μg⁻¹ kg i.c.v.), were treated with APNEA (0.4–4 μg kg⁻¹ i.c.v.) resulting in a transitory and dose-dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 μg kg⁻¹. In contrast, pretreatment 48 h before, with 4 μg kg⁻¹ i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 μg kg⁻¹ i.c.v.), after DPCPX, induced a decrease in ABP of −66±5.4 mmHg and after 3 min a decrease in heart rate of −62±6.0 beats min⁻¹. Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR.
4. These results suggest that a population of A₃-receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

     

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.

     

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.

     

Comparative study between peripherally and centrally acting sublethal and lethal doses of Leiurus quinquestriatus scorpion venom in rabbits: The usefulness of the sodium channel blocker lidocaine

Background

Scorpion envenomation is common among desert dwellers, affecting several systems and resulting in multiple organ dysfunction (MOD) or failure (MOF), mainly due to their action on Na⁺ channels. Although scorpion venoms toxins do not pass the blood brain barrier, their CNS effects are prominent, occurring in conjunction with, or as an aftermath of peripheral actions of the venom.

Objective

To determine the ability of venom of the common scorpion Leiurus quinquestriatus (LQQ) to induce MOD or MOF when injected into rabbits in micro quantities centrally (intracerebroventricularly, i.c.v.) or macro amounts peripherally (s.c. or i.v.). Also, to assess if the Na⁺ channel blocker lidocaine can protect rabbits from the resultant manifestations.

Methods

Rabbits were injected with LQQ venom centrally or peripherally, in either sublethal or lethal doses, and MOD or MOF determined by assessing: cardiac output (CO), estimated hepatic blood flow (EHBF), biochemical parameters indicative of cardiac/hepatic/renal and pancreatic functions, blood pressure (BP), survival, lung/body index (LBI, indicative of pulmonary edema), and/or histological changes in hearts, lungs, livers plus kidneys. In pre-treatment experiments, lidocaine was injected 40 min before venom and protective ability examined.

Results

LQQ venom in sublethal doses caused comparable significant reductions (vs control) in CO and EHBF when injected i.c.v. (2 μg kg⁻¹) or s.c. (0.2 mg kg⁻¹). Both routes caused gradual dose-related enhanced levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, creatinine, glucose and amylase, indicating MOD. Also, characteristic venom-induced changes in BP were evident after lethal doses of venom i.v. (0.5 mg kg⁻¹) or i.c.v. (3 μg kg⁻¹). Histological changes in the organs plus LBI were comparable after i.c.v. and i.v. venom injection, with animals ultimately exhibiting MOF. Lidocaine (1 mg kg⁻¹ i.v., then infusion 50 μg kg⁻¹ min⁻¹, 30 min before venom), protected the animals from MOF evoked by lethal doses of the venom (whether injected centrally or peripherally), as evidenced by the amelioration of the venom’s effects on blood pressure, LBI, survival and multiple organ histopathological manifestations.

Conclusion

LQQ venom, whether injected centrally or peripherally caused comparable systemic dose-dependent MOD or MOF, with the latter attenuated by the Na⁺ channel blocker lidocaine, indicating a role for Na⁺ channels.     

Pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rats

Aim:

M₂ES is PEGylated recombinant human endostatin. In this study we investigated the pharmacokinetics, tissue distribution, and excretion of M₂ES in rats.

Methods:

¹²⁵I-radiolabeled M₂ES was administered to rats by intravenous bolus injection at 3 mg/kg. The pharmacokinetics, tissue distribution and excretion of M₂ES were investigated using the trichloroacetic acid (TCA) precipitation method.

Results:

The serum M₂ES concentration-time curve after a single intravenous dose of 3 mg/kg in rats was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: C_max=28.3 μg·equ/mL, t_1/2=71.5 h, AUC_(0–∞)=174.6 μg·equ·h/mL, Cl=17.2 mL·h⁻¹·kg⁻¹, MRT=57.6 h, and V_ss=989.8 mL/kg for the total radioactivity; C_max=30.3 μg·equ/mL, t_1/2=60.1 h, AUC_(0–∞)=146.2 μg·equ·h/mL, Cl=20.6 mL·h⁻¹·kg⁻¹, MRT=47.4 h, and V_ss=974.6 mL/kg for the TCA precipitate radioactivity. M₂ES was rapidly and widely distributed in various tissues and showed substantial deposition in kidney, adrenal gland, lung, spleen, bladder and liver. The radioactivity recovered in the urine and feces by 432 h post-dose was 71.3% and 8.3%, respectively. Only 0.98% of radioactivity was excreted in the bile by 24 h post-dose.

Conclusion:

PEG modification substantially prolongs the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior. M₂ES is extensively distributed in most tissues of rats, including kidney, adrenal gland, lung, spleen, bladder and liver. Urinary excretion was the major elimination route for M₂ES.     

IL-1β reduces tonic contraction of mesenteric lymphatic muscle cells,with the involvement of cycloxygenase-2 and prostaglandin E2

Background and Purpose

The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, ‘lymphangions’. Here we have studied the effects of the inflammatory cytokine IL-1β on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC).

Experimental Approach

We measured IL-1β in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1β or h-TNF-α (5, 10, 20 ng·mL⁻¹), with or without COX inhibitors [TFAP (10⁻⁵ M), diclofenac (0.2 × 10⁻⁵ M)], PGE₂ (10⁻⁵ M)], IL-1-receptor antagonist, Anakinra (5 μg·mL⁻¹), or a selective prostanoid EP₄ receptor antagonist, GW627368X (10⁻⁶ and 10⁻⁷ M).

Key Results

Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1β or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1β concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1β-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1β increased COX-2 protein and PGE₂ production in RMLMC.. PGE₂ induced relaxations in RMLMC, comparable to h-IL-1β. Conversely, COX-2 and EP₄ receptor inhibition reversed relaxation induced by IL-1β.

Conclusions and Implications

The IL-1β-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE₂. In experimental colitis, IL-1β and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1β restored tonic contraction.     

Effects of intracerebroventricular leptin administration on food intake,body weight gain and diencephalic nitric oxide synthase activity in the mouse

1. Intracranial administration of leptin reduces both food intake and body weight gain in the mouse. Inhibitors of nitric oxide (NO) synthase produce similar effects.
2. To investigate the role of the brain L-arginine/NO pathway in mediating this effect of leptin, we have evaluated food intake and body weight gain after daily (5 days) intracerebroventricular (i.c.v.) administration of leptin (0.5–2 μg) alone or in association with L-arginine (10 μg). Moreover, we measured diencephalic nitric oxide synthase (NOS) activity after a single i.c.v. leptin (0.25–2 μg) injection and after consecutive doses of leptin (0.25–2 μg) over 5 days. The time course of the effect of leptin on NOS activity was also evaluated.
3. I.c.v. injected leptin (1 and 2 μg) significantly and dose-dependently reduced food intake and body weight gain with respect to vehicle (food intake: 5.97±0.16 g 24 h⁻¹ and 4.27±0.18 g 24 h⁻¹, respectively, vs 8.05±0.34 g 24 h⁻¹, P<0.001, n=6 for each group; body weight gain: −10.7±0.46% and −15.2±0.65%, respectively, vs 5.14±0.38%, P<0.001, n=6 for each group). This effect was antagonized by L-arginine (food intake: 7.90±0.37 g 24 h; body weight gain: 5.11±0.31%, n=6). Diencephalic NOS activity was significantly reduced by the highest doses of leptin with respect to vehicle (vehicle: 0.90±0.04 nmol citrulline min⁻¹ g⁻¹ tissue; leptin 1 μg: 0.62±0.03 nmol citrulline min⁻¹ g⁻¹ tissue, P<0.001; leptin 2 μg: 0.44±0.03 nmol citrulline min⁻¹ g⁻¹ tissue, P<0.001, n=6 for each group). Similar results were obtained in animals treated with daily consecutive doses of leptin. The inhibitory effect appeared rapidly (within 30 min) and was long lasting (up to 12 h).
4. Our results suggest that the brain L-arginine/NO pathway may be involved in the central effect of leptin on feeding behaviour and body weight gain in mice.

     

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.

     

Plasminogen-stimulated airway smooth muscle cell proliferation is mediated by urokinase and annexin A2, involving plasmin-activated cell signalling

BACKGROUND AND PURPOSE

The conversion of plasminogen into plasmin by interstitial urokinase plasminogen activator (uPA) is potentially important in asthma pathophysiology. In this study, the effect of uPA-mediated plasminogen activation on airway smooth muscle (ASM) cell proliferation was investigated.

EXPERIMENTAL APPROACH

Human ASM cells were incubated with plasminogen (0.5–50 μg·mL⁻¹) or plasmin (0.5–50 mU·mL⁻¹) in the presence of pharmacological inhibitors, including UK122, an inhibitor of uPA. Proliferation was assessed by increases in cell number or MTT reduction after 48 h incubation with plasmin(ogen), and by earlier increases in [³H]-thymidine incorporation and cyclin D1 expression.

KEY RESULTS

Plasminogen (5 μg·mL⁻¹)-stimulated increases in cell proliferation were attenuated by UK122 (10 μM) or by transfection with uPA gene-specific siRNA. Exogenous plasmin (5 mU·mL⁻¹) also stimulated increases in cell proliferation. Inhibition of plasmin-stimulated ERK1/2 or PI3K/Akt signalling attenuated plasmin-stimulated increases in ASM proliferation. Furthermore, pharmacological inhibition of cell signalling mediated by the EGF receptor, a receptor trans-activated by plasmin, also reduced plasmin(ogen)-stimulated cell proliferation. Knock down of annexin A2, which has dual roles in both plasminogen activation and plasmin-signal transduction, also attenuated ASM cell proliferation following incubation with either plasminogen or plasmin.

CONCLUSIONS AND IMPLICATIONS

Plasminogen stimulates ASM cell proliferation in a manner mediated by uPA and involving multiple signalling pathways downstream of plasmin. Targeting mediators of plasminogen-evoked ASM responses, such as uPA or annexin A2, may be useful in the treatment of asthma.     

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.

     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious,hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3–4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 μg kg⁻¹ h⁻¹, i.v.)
2. The early (1–2 h) hypotension in SD rats (−11±3 mmHg; n=7) was significantly less than that in TG rats (−35±3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108±3 mmHg; 24 h LPS, 112±4 mmHg; TG rats: baseline, 171±2 mmHg; 24 h LPS, 169±3 mmHg). At this stage in the SD rats there was a renal vasodilatation (Δ vascular conductance, 29±10 [kHz mmHg⁻¹]10³) but not in TG rats (Δ vascular conductance 2±3[kHz mmHg⁻¹]10³).
3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 μg kg⁻¹ bolus, 600 μg kg⁻¹ h⁻¹) between 24 and 31 h in SD rats caused a fall in MAP of 16±2 mmHg accompanied by hindquarters vasodilatation (Δ vascular conductance 11±3 (kHz mmHg⁻¹)10³). In TG rats, under the same conditions, the fall in MAP was −60±6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (Δ vascular conductance, 23±5, 32±7, and 14±4 (kHz mmHg⁻¹)10³, respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats.
4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was −17±4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5±3, −4±5, and 12±4 (kHz mmHg⁻¹)10³, respectively.
5. Administration of the angiotensin (AT₁)-receptor antagonist, losartan (10 mg kg⁻¹, i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (−12±3 and −14±4 mmHg, respectively).
6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.