bluuebunny/crossref_metadata_2025 huggingface dataset:
HS‐599: a novel long acting opioid analgesic does not induce place‐preference in rats
Authors: Lattanzi R , Negri L , Giannini E , Schmidhammer H , Schutz J , Improta G
Published: January 2005

When administered subcutaneously HS‐599, a new didehydroderivative of buprenorphine (18,19‐dehydrobuprenorphine), produced a long‐lasting antinociceptive response in rats. Its potency exceeded twice that of buprenorphine. In the tail‐flick test it acted as a full agonist but in the plantar test only as a partial agonist. Whereas the μ‐opioid antagonists naloxone and naltrexone antagonized HS‐599 antinociception the δ‐opioid antagonist naltrindole and the κ‐opioid antagonist nor‐binaltorphimine did not.

Unlike buprenorphine and morphine, HS‐599 never induced conditioned place‐preference in rats.

In radioligand binding assays, compared with buprenorphine HS‐599 had 3 fold higher μ‐opioid receptor affinity but lower δ‐ and κ‐opioid receptor affinity.

In isolated guinea‐pig ileum preparations, HS‐599 only partially inhibited the electrically‐stimulated contraction, acting as a partial opioid agonist. When tested against the μ‐opioid receptor agonist dermorphin, it behaved as a non‐equilibrium antagonist. Conversely, in mouse vas deferens (rich in δ‐opioid receptors) and rabbit vas deferens preparations (rich in κ‐opioid receptors) HS‐599 acted as a pure equilibrium antagonist, shifting the log‐concentration‐response curves of the δ‐opioid agonist deltorphin I and the κ‐opioid agonist U‐69593 to the right.

In conclusion, HS‐599 is a novel buprenorphine derivative with higher affinity, selectivity and potency than the parent compound, for μ‐opioid receptors. It produces intense and long‐lasting antinociception and does not induce place‐preference in rats.

British Journal of Pharmacology (2001) 134, 441–447; doi:10.1038/sj.bjp.0704280
Comment on a Published Paper
Authors: Dodds Helen M , Rivory Laurent P
Published: January 2005

British Journal of Pharmacology (2001) 134, 448–449; doi:10.1038/sj.bjp.0704282
The new compound, LASSBio 294, increases the contractility of intact and saponin‐skinned cardiac muscle from Wistar rats
Authors: Sudo R T , Zapata‐Sudo G , Barreiro E J
Published: January 2005

A new compound designated as LASSBio 294 (L‐294), 3,4‐methylenedioxybenzoyl‐2‐thienylhydrazone, was synthesized as an alternative therapeutic for cardiac dysfunction.

L‐294 increased in a dose‐dependent manner the spontaneous contractions of isolated hearts from Wistar rats with maximal effect (128.0±0.7% of control) observed at 25 μM.

The positive inotropic effect of L‐294 was also observed in electrically stimulated cardiac tissues from Wistar rats. The maximal increment of twitches, at 200 μM, was 163.1±18.4% for atrial, 153.5±28.5% for papillary and 201.5±18.5% for ventricular muscles.

In saponin skinned ventricular cells: (a) L‐294 present in the period of sarcoplasmic reticulum (SR) loading with Ca2+ shifted the dose and caffeine‐induced contracture curve; (b) L‐294 (100 μM) increased 40% the Ca2+ uptake into SR; (c) L‐294 did not significantly alter the sensitivity of contractile proteins to Ca2+ in SR‐disrupted skinned ventricular cells.

Retrograde perfusion of the isolated heart from Wistar rats with L‐294 (100 μM) did not cause any significant change in rhythm, heart rate (control, 220±14.7 b.p.m.; 246±24.6 b.p.m. for L‐294), PR interval (control, 66.0±2.4 ms; 64.0±2.3 ms for L‐294) or QRS duration (control, 28.8±3.4 ms; 32.0±2.0 ms for L‐294).

These results suggest a novel mechanism for a positive cardioinotropic effect through an interaction with the Ca2+ uptake/release process of the SR. The effect of L‐294 could be explained by a pronounced increased accumulation of Ca2+ into the SR.

British Journal of Pharmacology (2001) 134, 603–613; doi:10.1038/sj.bjp.0704291
Modulation of serotonergic neurotransmission by short‐ and long‐term treatments with sigma ligands
Authors: Bermack J E , Debonnel G
Published: January 2005

Sigma receptors were first described in 1976 as opiate receptors but were later determined to be a distinct class of receptors with two subtypes, sigma1 and sigma2. Although the endogenous ligand is yet to be elucidated, the sigma1 receptor has recently been cloned.

Behavioural models used to test potential antidepressants have shown sigma ligands to produce antidepressant effects but their mechanism of action is unknown.

The goal of the present study was to assess the effects of various sigma1 ligands on the firing activity of serotonin (5‐HT) neurons of the dorsal raphe nucleus (DRN) using extracellular in vivo recordings in anaesthetized rats.

The sigma1 ligands (+)‐pentazocine and 4‐(N‐benzylpiperidin‐4‐yl)‐4‐iodobenzamide (4‐IBP) (2 mg kg−1 day−1) increased markedly 5‐HT firing activity after 2 days of treatment and maintained the same increased firing rate after long‐term (21 days) treatments. Furthermore, the increased firing rate produced by 2 and 21 day treatments with (+)‐pentazocine was prevented by the co‐administration of N,N‐dipropyl‐2‐(4‐methoxy‐3‐(2‐phenylethoxy)phenyl)‐thylamine (NE‐100) (10 mg kg−1 day−1) a selective sigma1 antagonist, confirming the sigma1 receptor's modulation of these effects. In contrast, the sigma1 ligands (+)‐N‐cyclopropylmethyl‐N‐methyl‐1,4‐diphenyl‐1‐1‐ethyl‐but‐3‐en‐1‐ylamine hydrochloride (JO‐1784) and 2‐(4‐morpholinoethyl 1‐phenyl‐cyclohexane‐1‐carboxylate hydrochloride (PRE‐084) had no effect.

Following a 21‐day treatment with (+)‐pentazocine there was a marked reduction in the number of neurons found per track. This decrease was not seen after chronic treatment with 4‐IBP and may represent a depolarization block.

These results suggest a modulation of serotonergic neurotransmission by some sigma receptors and provide a potential mechanism for the ‘antidepressant effects’ reported and provide evidence toward sigma1 ligands as potential antidepressants with a rapid onset of action.

British Journal of Pharmacology (2001) 134, 691–699; doi:10.1038/sj.bjp.0704294
Nitric oxide regulates human eosinophil adhesion mechanisms in vitro by changing integrin expression and activity on the eosinophil cell surface
Authors: Conran N , Ferreira H H A , Lorand‐Metze I , Thomazzi S M , Antunes E , De Nucci G
Published: January 2005

The nitric oxide synthase (NOS) inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME), inhibits both rat and human eosinophil chemotaxis in vitro. Here, the role of nitric oxide (NO) in human eosinophil cell surface integrin expression and function was investigated.

Human peripheral blood eosinophils were treated with L‐NAME (0.01 – 1.0 mM) and their adhesion to human fibronectin and serum observed. Adhesion of cells to fibronectin and serum increased by 24.0±4.6 and 43.8±4.7%, respectively, when eosinophils were treated with 1.0 mM L‐NAME. Increased adhesion by L‐NAME could be abolished when cells were co‐incubated with VLA‐4‐ and Mac‐1‐specific monoclonal antibodies (mAbs).

The NO donor, sodium nitroprusside (2.5 mM), significantly inhibited eosinophil adhesion to fibronectin and serum by 34.3±4.5 and 45.2±5.6%, respectively. This inhibition was accompanied by a 4 fold increase in the levels of intracellular cyclic GMP.

Flow cytometrical analysis demonstrated that L‐NAME induced an increased expression of CD11b (Mac‐1) on the eosinophil cell surface of 36.3±7.4%. L‐NAME had no effect upon CD49d (VLA‐4) expression.

Treatment of human eosinophils, in vitro, with H‐[1,2,4] oxadiazolo quinoxalin‐1‐one (ODQ) (0.1 mM), an inhibitor of soluble guanylate cyclase, also significantly increased eosinophil adhesion to fibronectin and serum by 73.5±17.9 and 91.7±12.9%, respectively. This increase in adhesion could also be inhibited by co‐incubation with the Mac‐1 and VLA‐4‐specific mAbs.

In conclusion, results indicate that NO, via a cyclic GMP‐dependent mechanism, inhibits the adhesion of human eosinophils to the extracellular matrix (ECM). This inhibition is accompanied by a decrease in the expression and function of the eosinophil's adhesion molecules, in particular, the expression of the Mac‐1 integrin and the function of the VLA‐4 integrin.

British Journal of Pharmacology (2001) 134, 632–638; doi:10.1038/sj.bjp.0704295
Despite substantial degradation, 2‐arachidonoylglycerol is a potent full efficacy agonist mediating CB1 receptor‐dependent G‐protein activation in rat cerebellar membranes
Authors: Savinainen Juha R , Järvinen Tomi , Laine Krista , Laitinen Jarmo T
Published: January 2005

Two endocannabinoids, arachidonoyl ethanolamide (AEA) and 2‐arachidonoylglycerol (2‐AG) bind and activate G‐protein‐coupled cannabinoid receptors, but limited data exist on their relative ability to activate G‐proteins.

Here we assess agonist potency and efficacy of various cannabinoids, including 2‐AG, HU‐310 (2‐arachidonoyl glyceryl ether, a third putative endocannabinoid), HU‐313 (another ether analogue of 2‐AG), AEA, R‐methanandamide (an enzymatically stable analogue of AEA), and CP‐55,940 at rat brain CB1 receptors using agonist‐stimulated [35S]‐GTPγS binding to cerebellar membranes and whole brain sections. Degradation of endocannabinoids under experimental conditions was monitored by HPLC.

To enhance efficacy differences, agonist dose‐response curves were generated using increasing GDP concentrations. At 10−6 M GDP, all compounds, except HU‐313, produced full agonists responses ∼2.5 fold over basal. The superior efficacy of 2‐AG over all other compounds became evident by increasing GDP (10−5 and 10−4 M).

In membrane incubations, 2‐AG was degraded by 85% whereas AEA and HU‐310 were stable. Pretreatment of membranes with phenylmethylsulphonyl fluoride inhibited 2‐AG degradation, resulting in 2 fold increase in agonist potency. Such pretreatment had no effect on AEA potency.

Responses in brain sections were otherwise consistent with membrane binding data, but 2‐AG evoked only a weak signal in brain sections, apparently due to more extensive degradation.

These data establish that even under conditions of substantial degradation, 2‐AG is a full efficacy agonist, clearly more potent than AEA, in mediating CB1 receptor‐dependent G‐protein activity in native membranes.

British Journal of Pharmacology (2001) 134, 664–672; doi:10.1038/sj.bjp.0704297
Mechanisms of hydralazine induced vasodilation in rabbit aorta and pulmonary artery
Authors: Ellershaw D C , Gurney A M
Published: January 2005

The directly acting vasodilator hydralazine has been proposed to act at an intracellular site in vascular smooth muscle to inhibit Ca2+ release.

This study investigated the mechanism of action of hydralazine on rabbit aorta and pulmonary artery by comparing its effects on the tension generated by intact and β‐escin permeabilized vessels and on the cytoplasmic Ca2+ concentration, membrane potential and K+ currents of isolated vascular smooth muscle cells.

Hydralazine relaxed pulmonary artery and aorta with similar potency. It was equally effective at inhibiting phasic and tonic contractions evoked by phenylephrine in intact vessels and contractions evoked by inositol 1,4,5 trisphosphate (IP3) in permeabilized vessels.

Hydralazine inhibited the contraction of permeabilized vessels and the increase in smooth muscle cell Ca2+ concentration evoked by caffeine with similar concentration dependence, but with lower potency than its effect on IP3 contractions.

Hydralazine had no effect on the relationship between Ca2+ concentration and force generation in permeabilized vessels, but it slowed the rate at which maximal force was developed before, but not after, destroying sarcoplasmic reticulum function with the calcium ionophore, ionomycin.

Hydralazine had no effect on membrane potential or the amplitudes of K+ currents recorded from isolated smooth muscle cells over the concentration range causing relaxation of intact vessels.

The results suggest that the main action of hydralazine is to inhibit the IP3‐induced release of Ca2+ from the sarcoplasmic reticulum in vascular smooth muscle cells.

British Journal of Pharmacology (2001) 134, 621–631; doi:10.1038/sj.bjp.0704302
Systemic ETA receptor antagonism with BQ‐123 blocks ET‐1 induced forearm vasoconstriction and decreases peripheral vascular resistance in healthy men
Authors: Spratt James C S , Goddard Jane , Patel Neil , Strachan Fiona E , Rankin Andrew J , Webb David J
Published: January 2005

The effect on systemic haemodynamics of BQ‐123, a selective endothelin A (ETA) receptor antagonist, was investigated in healthy men by giving, on separate occasions, ascending intravenous doses of 100, 300, 1000 and 3000 nmol min−1 BQ‐123, each for 15 min, in a randomized, placebo‐controlled, double‐blind study. The response of forearm blood flow to brachial artery infusion of endothelin‐1 (ET‐1; 5 pmol min−1 for 90 min) was also studied using bilateral forearm plethysmography, after systemic pre‐treatment, on separate occasions, with one of two doses of BQ‐123 (300 and 1000 nmol min−1 for 15 min) or placebo.

Systemic BQ‐123 dose‐dependently decreased systemic vascular resistance (P<0.01 for all doses vs placebo) and mean arterial pressure (P<0.05 for 300 nmol min−1 and P<0.01 for 1000 and 3000 nmol min−1) during the 60 min following infusion. There were concurrent increases in heart rate and cardiac index. BQ‐123, when infused systemically for 15 min, appeared to reach a maximum effect at 1000 nmol min−1.

Intra‐brachial ET‐1 infusion, after pre‐treatment with placebo, caused a slow onset progressive forearm vasoconstriction without systemic effects. This vasoconstriction was attenuated by pre‐treatment with BQ‐123 at 300 nmol min−1 and abolished by BQ‐123 at 1000 nmol min−1 (P<0.01 vs placebo).

These effects occurred at concentrations of BQ‐123 in the plasma (510±64 nmol l−1) that were ETA receptor selective, and were not accompanied by an increase in plasma ET‐1 that would have indicated ETB receptor blockade.

We conclude that ETA‐mediated vascular tone contributes to the maintenance of basal systemic vascular resistance and blood pressure in healthy men.

British Journal of Pharmacology (2001) 134, 648–654; doi:10.1038/sj.bjp.0704304
Nitric oxide (NO) modulation of PAF‐induced cardiopulmonary action: interaction between NO synthase and cyclo‐oxygenase‐2 pathways
Authors: Fabi Fulvia , Calabrese Roberto , Stati Tonino , Del Basso Paola
Published: January 2005

To further investigate into the mechanisms of PAF‐induced cardiopulmonary actions, we examined the effects of the nitric oxide synthase (NOS) inhibitor L‐Nω‐nitro‐L‐arginine (L‐NNA), of the specific cyclooxygenase‐2 (COX‐2) inhibitor NS 398, and of the combined presence of both COX and NOS inhibitors on the PAF responses in the heart lung preparation of guinea‐pig (HLP).

In HLPs perfused with homologous blood, dose‐response curves for the haemodynamic and bronchial effects of PAF (1 – 32 ng) were carried out in the absence or presence of L‐NNA (200 μM). L‐NNA caused an increase in the resting pulmonary arterial pressure (PAP) without affecting the other basal values, and strongly potentiated the bronchoconstriction and pulmonary hypertension elicited by PAF. An enhancement of the PAF‐induced actions on right atrial pressure (RAP) and cardiac output (CO) was also observed. All the effects of L‐NNA were antagonized by L‐arginine (2 mM).

The presence of L‐NNA in the perfusing blood of HLPs failed to affect the pulmonary hypertensive and bronchoconstrictor responses induced by the thromboxane A2 mimetic U46619 (0.05 – 1.6 μg), 5‐hydroxytryptamine (0.1 – 1.6 μg), and histamine (0.1 – 1.6 μg), thus suggesting that these PAF secondary mediators are not responsible for the hyper‐responsiveness to PAF induced by L‐NNA.

Blocking COX‐2 pathway with NS 398 (15 – 30 μM) did not alter the cardiopulmonary resting variables. However, a reduction of the PAF‐mediated pulmonary hypertension, but not of bronchoconstriction, was observed. When L‐NNA was added to the perfusing medium of HLPs pre‐treated with NS 398 or with indomethacin (15 μM), the basal PAP values were enhanced. However, in the combined presence of COX and NOS inhibitors, only a slight increase in the hypertensive responses to the highest doses of PAF was observed, whereas the PAF mediated actions at bronchial and cardiac level were unaffected.

This study indicates that (i) the cardiopulmonary actions induced by PAF are specifically modulated by endogenous NO through the NOS pathway, and (ii) COX‐2 isoform is involved in the pulmonary hypertensive, but not bronchoconstrictor, effects of PAF. Furthermore, an interaction between PAF stimulated COX, particularly COX‐2, and NOS pathways appears to take a functional role at both bronchial and cardiovascular level.

British Journal of Pharmacology (2001) 134, 777–788; doi:10.1038/sj.bjp.0704311
Formation of functional α3β4α5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach
Authors: Groot‐Kormelink Paul J , Boorman James P , Sivilotti Lucia G
Published: January 2005

The α5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related β3 subunit, α5 forms functional recombinant receptors if expressed together with a pair of typical α and β subunits, but its effect on the properties of the resulting αβα5 receptor depends on the α and β subunits chosen and on the expression system. We used a reporter mutation approach to test whether α5, like β3, is incorporated as a single copy in human α3β4α5 receptors expressed in oocytes.

As previously reported, the main indication of the presence of α5 in α3β4α5wt was an increase in apparent receptor desensitization (compared with α3β4 receptors). If the α3β4α5 receptor bore a 9′T mutation in the second transmembrane domain of either α3 or β4, α5 incorporation produced a decrease in ACh sensitivity (by 4 fold for α3LTβ4α5 vs α3LTβ4 and by 40 fold for α3β4LTα5 vs α3β4LT). The much greater effect observed in α3β4LTα5 receptors accords with the hypothesis that α5 takes the place of a β subunit in the receptor.

Introducing a 9′T mutation in α5 had no effect on the agonist sensitivity of α3β4α5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations.

Introducing the 9′T mutation in α3 or β4 in the triplet receptor reduced the EC50 for ACh by a similar extent (7 and 9 fold, respectively), suggesting that α3β4α5 receptors contain two copies each of α and β and therefore only one copy of α5.

British Journal of Pharmacology (2001) 134, 789–796; doi:10.1038/sj.bjp.0704313
Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant α7 and α4β2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes
Authors: Coates Kristen M , Flood Pamela
Published: January 2005

Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the α7 subunit or α4 and β2 subunits expressed in Xenopus laevis oocytes.

Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human α7 and α4β2 nAChRs, with IC50 values of 20 and 50 μM respectively. Inhibition of the α7 nAChRs occurred within a clinically relevant concentration range, while inhibition of the α4β2 nAChR was observed only at higher concentrations. The Ketalar formulation inhibited nAChR function more effectively than was expected given its ketamine concentration. The surprising increased inhibitory potency of Ketalar compared with pure ketamine appeared to be due to the activity of BCl, which inhibited both α7 (IC50 value of 122 nM) and α4β2 (IC50 value of 49 nM) nAChRs at concentrations present in the clinical formulation of Ketalar.

Ketamine is a noncompetitive inhibitor at both the α7 and α4β2 nAChR. In contrast, BCl causes a parallel shift in the ACh dose‐response curve at the α7 nAChR suggesting competitive inhibition. Ketamine causes both voltage‐dependent and use‐dependent inhibition, only in the α4β2 nAChR.

Since α7 nAChRs are likely to be inhibited during clinical use of Ketalar, the actions of ketamine and BCl on this receptor subtype may play a role in the profound analgesia, amnesia, immobility and/or autonomic modulation produced by this anaesthetic.

British Journal of Pharmacology (2001) 134, 871–879; doi:10.1038/sj.bjp.0704315
Mechanism of trypsin‐induced endothelium‐dependent vasorelaxation in the porcine coronary artery
Authors: Nakayama Tetsuzo , Hirano Katsuya , Nishimura Junji , Takahashi Shosuke , Kanaide Hideo
Published: January 2005

To investigate the mechanism underlying the trypsin‐induced endothelium‐dependent relaxation, cytosolic Ca2+ concentration ([Ca2+]i) and tension development of smooth muscle were simultaneously monitored in the porcine coronary artery, and [Ca2+]i of in situ endothelial cells were monitored in the porcine aortic valvular strips, using fura‐2 fluorometry.

During the contraction induced by 30 nM U46619, a thromboxane A2 analogue, 100 nM trypsin induced a rapid transient significant decrease in both [Ca2+]i (from 67.9±5.1 to 15.7±4.4%) and tension (from 97.5±9.2 to 16.8±3.5%) of smooth muscle only in the presence of endothelium (100% level was assigned to the level obtained with the 118 mM K+‐induced contraction). [Ca2+]i and the tension thus returned to the levels prior to the application of trypsin by 5 and 10 min, respectively.

The initial phase of this relaxation was partly inhibited by 100 μM Nω‐nitro‐L‐arginine (L‐NOARG), and was completely inhibited by L‐NOARG plus 40 mM K+ or L‐NOARG plus 100 nM charybdotoxin and 100 nM apamin, while the late phase of the relaxation was inhibited by L‐NOARG alone.

Trypsin induced a transient [Ca2+]i elevation in the endothelial cells mainly due to the Ca2+ release from the intracellular stores, at the concentrations (1 – 100 nM) similar to those required to induce relaxation.

In conclusion, trypsin induced an elevation in [Ca2+]i mainly due to Ca2+ release in endothelial cells, and thereby caused endothelium‐dependent relaxation. The early phase of relaxation was due to nitric oxide and hyperpolarizing factors, while the late phase was mainly due to nitric oxide in the porcine coronary artery.

British Journal of Pharmacology (2001) 134, 815–826; doi:10.1038/sj.bjp.0704318
The effects of heparin on the adhesion of human peripheral blood mononuclear cells to human stimulated umbilical vein endothelial cells
Authors: Smailbegovic Amir , Lever Rebecca , Page Clive P
Published: January 2005

The effects of unfractionated heparin (UH) and a selectively O‐desulphated derivative of heparin (ODSH), lacking anticoagulant activity, on the adhesion of human peripheral blood mononuclear cells (HPBMNC) to human stimulated umbilical vein endothelial cells (HUVECs), were investigated.

For comparison, the effects of poly‐L‐glutamic acid (PGA), a large polyanionic molecule without sulphate groups and two different molecular weight sulphated dextrans (DS 5 k and DS 10 k) were studied.

UH (50 – 1000 u ml−1) significantly (P<0.05) inhibited the adhesion of HPBMNC to HUVECs, stimulated with IL‐1β (100 u ml−1), TNF‐α (1000 u ml−1) or LPS (100 μg ml−1), when the drugs were added together with stimuli to HUVECs and coincubated for 6 h. Such effects on adhesion occurred with limited influence on expression of relevant endothelial adhesion molecules (ICAM‐1 and VCAM‐1).

UH (100 – 1000 u ml−1), when added to prestimulated HUVECs, significantly (P<0.05) increased adhesion of mononuclear cells to endothelium at the higher concentrations tested, without any effect on adhesion molecule expression. In contrast, the opposite effect was observed when human polymorphonuclear leucocyte adhesion was examined, under the same experimental conditions, suggesting that the observed potentiation of HPBMNC adhesion is cell specific.

The effects of UH on HPBMNC adhesion were shared by the non‐anticoagulant ODSH (600 – 6000 μg ml−1) but not by sulphated dextrans or PGA (300 – 6000 μg ml−1).

Heparin affects the adhesion of HPBMNC to stimulated endothelium, in both an inhibitory and potentiating manner, effects which are unrelated to its anticoagulant activity and not solely dependent on molecular charge characteristics.

British Journal of Pharmacology (2001) 134, 827–836; doi:10.1038/sj.bjp.0704321
Divergent effects of corticotropin releasing hormone on endothelial cell nitric oxide synthase are associated with different expression of CRH type 1 and 2 receptors
Authors: Cantarella Giuseppina , Lempereur Laurence , Lombardo Gabriella , Chiarenza Andrea , Pafumi Carlo , Zappalà Giovanna , Bernardini Renato
Published: January 2005

Endothelium is a target for an array of factors involved in inflammation. Endothelial cells express receptors for CRH, a neuropeptide produced during inflammation. We report both the concentration‐dependent inhibitory effect of CRH upon cytokine‐stimulated nitrite release by H5V murine endothelioma cells, and its stimulatory one in HUVEC cells.

Western blot analysis showed that CRH inhibits cytokine‐stimulated iNOS protein in H5V cells, and, instead, potentiated it in HUVEC cells.

H5V cells expressed both CRH receptors (CRH‐R1 and R2) mRNAs, whereas HUVEC cells expressed the CRH‐R2 mRNA solely.

CRH increased medium nitrites and iNOS protein expression in H5V cells pretreated with the selective CRH‐R1 antagonist CP 154,526. However, the selective CRH‐R2 antagonist anti‐Svg‐30 failed to produce similar effects. In fact, anti‐Svg‐30 inhibited CRH‐induced increase of nitrite release and iNOS expression in HUVEC cells.

Our results confirm the activating role of CRH on endothelial cells, although it suggests its possible inhibitory role in the late phase of the inflammatory response. NO‐mediated effects of CRH on endothelial cells could be exploited in therapeutic strategies related to inflammatory and/or degenerative diseases.

British Journal of Pharmacology (2001) 134, 837–844; doi:10.1038/sj.bjp.0704322
Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide
Authors: Bisogno Tiziana , Hanuš Lumír , De Petrocellis Luciano , Tchilibon Susanna , Ponde Datta E , Brandi Ines , Moriello Aniello Schiano , Davis John B , Mechoulam Raphael , Di Marzo Vincenzo
Published: January 2005

(−)‐Cannabidiol (CBD) is a non‐psychotropic component of Cannabis with possible therapeutic use as an anti‐inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA).

CBD and its enantiomer, (+)‐CBD, together with seven analogues, obtained by exchanging the C‐7 methyl group of CBD with a hydroxy‐methyl or a carboxyl function and/or the C‐5′ pentyl group with a di‐methyl‐heptyl (DMH) group, were tested on: (a) VR1‐mediated increase in cytosolic Ca2+ concentrations in cells over‐expressing human VR1; (b) [14C]‐AEA uptake by RBL‐2H3 cells, which is facilitated by a selective membrane transporter; and (c) [14C]‐AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase.

Both CBD and (+)‐CBD, but not the other analogues, stimulated VR1 with EC50=3.2 – 3.5 μM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 – 70% of the effect obtained with ionomycin (4 μM). CBD (10 μM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive.

(+)‐5′‐DMH‐CBD and (+)‐7‐hydroxy‐5′‐DMH‐CBD inhibited [14C]‐AEA uptake (IC50=10.0 and 7.0 μM); the (−)‐enantiomers were slightly less active (IC50=14.0 and 12.5 μM). CBD and (+)‐CBD were also active (IC50=22.0 and 17.0 μM).

CBD (IC50=27.5 μM), (+)‐CBD (IC50=63.5 μM) and (−)‐7‐hydroxy‐CBD (IC50=34 μM), but not the other analogues (IC50>100 μM), weakly inhibited [14C]‐AEA hydrolysis.

Only the (+)‐isomers exhibited high affinity for CB1 and/or CB2 cannabinoid receptors.

These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield synthesis, and the weak affinity for CB1 and CB2 receptors, (−)‐5′‐DMH‐CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent.

British Journal of Pharmacology (2001) 134, 845–852; doi:10.1038/sj.bjp.0704327
Antagonism of AT2 receptors augments Angiotensin II‐induced abdominal aortic aneurysms and atherosclerosis
Authors: Daugherty Alan , Manning Michael W , Cassis Lisa A
Published: January 2005

We have recently demonstrated that chronic infusion of Angiotensin II into apoE−/− mice promotes the development of abdominal aortic aneurysms. To determine the involvement of specific Angiotensin II receptors in this response, we co‐infused Angiotensin II (1000 ng kg−1 min−1 for 28 days) with losartan (30 mg kg−1 day−1) or PD123319 (3 mg kg−1 day−1) to antagonize AT1 and AT2 receptors, respectively.

Infusion of Angiotensin II promoted the development of abdominal aortic aneurysms in 70% of mature female apoE−/− mice. The formation of aortic aneurysms was totally inhibited by co‐infusion of Angiotensin II with losartan (30 mg kg−1 day−1; P=0.003). In contrast, the co‐infusion of Angiotensin II with PD123319 resulted in a marked increase in the incidence and severity of aortic aneurysms.

To determine whether AT2 antagonism also promoted Angiotensin II‐induced atherosclerosis, Angiotensin II was infused into young female apoE−/− mice that had little spontaneous atherosclerosis. In these mice, co‐infusion of PD123319 led to a dramatic increase in the extent of atherosclerosis. This increase was associated with no change in plasma lipid concentrations and only transient and modest increases in blood pressure during co‐infusion with PD123319.

While antagonism of AT1 receptors totally prevented the formation of aneurysms, antagonism of AT2 receptors promoted a large increase in the severity of Angiotensin II‐induced vascular pathology.

British Journal of Pharmacology (2001) 134, 865–870; doi:10.1038/sj.bjp.0704331
Effects of inhibition of PDE4 and TNF‐α on local and remote injuries following ischaemia and reperfusion injury
Authors: Souza Danielle G , Cassali Geovanni D , Poole Steve , Teixeira Mauro M
Published: January 2005

The effects of phosphodiesterase (PDE)4 and TNF‐α inhibition were assessed on the local and remote injuries following intestinal ischaemia and reperfusion (I/R) injury in rats.

The PDE4 inhibitor rolipram dose‐dependently (1 – 10 mg kg−1) suppressed the local (intestine) and remote (lung) increases in vascular permeability and neutrophil recruitment following mild I/R injury. SB207499 (ariflo), a structurally‐distinct PDE4 inhibitor, also suppressed the injuries following mild I/R injury.

In a severe model of I/R injury, treatment with rolipram (10 mg kg−1) partially reversed the local and remote increases in vascular permeability, neutrophil recruitment, intestinal haemorrhage and intestinal LTB4 concentrations. The anti‐TNF‐α anti‐serum was more effective than rolipram at inhibiting local and remote injuries and prevented the lethality associated with severe I/R.

Rolipram and anti‐TNF‐α prevented the increase in the concentrations of TNF‐α in the lung and intestine, but rolipram only partially inhibited the elevation of this cytokine in serum. Rolipram had little effect on the increases of IL‐1ß concentrations in lung and serum, whereas treatment with anti‐TNF‐α markedly increased the concentration of this cytokine. Concentrations of IL‐10 rose significantly in the lung and serum and these increases were blocked by rolipram or anti‐TNF‐α.

The capacity of PDE4 inhibitors to block the recruitment of neutrophils into tissues, the production of LTB4 and of the pro‐inflammatory cytokines TNF‐α, IL‐1ß and IL‐6 appear to underlie their anti‐inflammatory effects in our model of I/R injury. Overall, PDE4 inhibition was less effective than inhibition of TNF‐α for protection against I/R injury.

British Journal of Pharmacology (2001) 134, 985–994; doi:10.1038/sj.bjp.0704336
A novel transcription factor inhibitor, SP100030, inhibits cytokine gene expression, but not airway eosinophilia or hyperresponsiveness in sensitized and allergen‐exposed rat
Authors: Huang Tung‐Jung , Adcock Ian M , Chung K Fan
Published: January 2005

We examined the effect of SP100030, a novel inhibitor of activator protein‐1 (AP‐1) and nuclear factor (NF)‐κB transcription factors, in a rat model of asthma.

Sensitized Brown‐Norway rats were treated with SP100030 (20 mg kg−1 day−1 for 3 days) intraperitoneally prior to allergen challenge. Allergen exposure of sensitized rats induced bronchial hyperresponsiveness (BHR), accumulation of inflammatory cells in bronchoalveolar lavage (BAL) fluid, and also an increase in eosinophils and CD2+, CD4+ and CD8+ T‐cells in the airways together with mRNA expression for IL‐2, IL‐4, IL‐5, IL‐10, and IFN‐γ.

Pre‐treatment with SP100030 inhibited BAL lymphocyte influx (P<0.03), specifically reduced CD8+ T‐cell infiltration in the airway submucosa (P<0.03), and mRNA expression for IL‐2, IL‐5, and IL‐10 (P<0.05). Neutrophil, eosinophil, and CD4+ T‐cells accumulation in the airways and BHR were not affected by SP100030.

Our results indicate that suppression of IL‐2 and IL‐5 mRNA expression may not necessarily lead to suppression of BHR. The expression of IL‐5 mRNA may contribute to the airway accumulation of eosinophils, but does not correlate with the extent of eosinophilia.

The joint AP‐1 and NF‐κB inhibitor, SP100030, selectively inhibits CD8+ T‐cells, and mRNA expression of both Th1 and Th2 cytokines in vivo, but does not inhibit allergen‐induced airway eosinophilia and BHR.

British Journal of Pharmacology (2001) 134, 1029–1036; doi:10.1038/sj.bjp.0704344
Pharmacological characteristics of solid‐phase von Willebrand factor in human platelets
Authors: Radomski Anna , Stewart Michael W , Jurasz Paul , Radomski Marek W
Published: January 2005

The pharmacological characteristics of solid‐phase von Willebrand factor (svWF), a novel platelet agonist, were studied.

Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase‐contrast microscopy, flow cytometry and zymography.

Incubation of platelets with svWF (0.2 – 1.2 μg ml−1) resulted in their adhesion to the ligand, while co‐incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation.

6B4 inhibitory anti‐glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N‐Acetyl‐Pen‐Arg‐Gly‐Asp‐Cys, an antagonist of GPIIb/IIIa.

Platelet adhesion stimulated with svWF was associated with a concentration‐dependent increase in expression of GPIb, but not of GPIIb/IIIa.

In contrast, collagen (0.5 – 10.0 μg ml−1) caused down‐regulation of GPIb and up‐regulation of GPIIb/IIIa in platelets.

Solid‐phase vWF (1.2 μg ml−1) resulted in the release of MMP‐2 from platelets.

Inhibition of MMP‐2 with phenanthroline (10 μM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF.

In contrast, human recombinant MMP‐2 potentiated both the effects of svWF on adhesion and up‐regulation of GPIb.

Platelet adhesion and aggregation stimulated with svWF were reduced by S‐nitroso‐n‐acetyl‐penicillamine, an NO donor, and prostacyclin.

Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively.

Mechanisms of activation of GPIb by svWF involve the release of MMP‐2, and are regulated by NO and prostacyclin.

British Journal of Pharmacology (2001) 134, 1013–1020; doi:10.1038/sj.bjp.0704345
Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors
Authors: Sinclair C J D , Powell A E , Xiong W , LaRivière C G , Baldwin S A , Cass C E , Young J D , Parkinson F E
Published: January 2005

Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5′ iodotubercidin (ITU) and 5‐amino‐5′‐deoxyadenosine (NH2dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells.

Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1‐C6 cells) or rCNT2 (rCNT2‐C6 cells) nucleoside transporters.

We tested the effects of ITU and NH2dAdo on [3H]‐adenosine uptake and conversion to [3H]‐adenine nucleotides in the three cell types. NH2dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [3H]‐adenosine uptake and [3H]‐adenine nucleotide formation at concentrations 100 nM in rENT1‐C6 cells, while concentrations 3 μM were required for C6 or rCNT2‐C6 cells.

Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1‐C6 cells.

This study demonstrates that the effects of ITU, but not NH2dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors.

British Journal of Pharmacology (2001) 134, 1037–1044; doi:10.1038/sj.bjp.0704349
Role of spinal NMDA receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced by magnesium deficiency in rats
Authors: Begon Sophie , Pickering Gisèle , Eschalier Alain , Mazur André , Rayssiguier Yves , Dubray Claude
Published: January 2005

Magnesium (Mg)‐deficient rats develop a mechanical hyperalgesia which is reversed by a N‐Methyl‐D‐Aspartate (NMDA) receptor antagonist. Given that functioning of this receptor‐channel is modulated by Mg, we wondered whether facilitated activation of NMDA receptors in Mg deficiency state may in turn trigger a cascade of specific intracellular events present in persistent pain. Hence, we tested several antagonists of NMDA and non‐NMDA receptors as well as compounds interfering with the functioning of intracellular second messengers for effects on hyperalgesia in Mg‐deficient rats.

Hyperalgesic Mg‐deficient rats were administered intrathecally (10 μl) or intraperitoneally with different antagonists. After drug injection, pain sensitivity was evaluated by assessing the vocalization threshold in response to a mechanical stimulus (paw pressure test) over 2 h.

Intrathecal administration of MgSO4 (1.6, 3.2, 4.8, 6.6 μmol) as well as NMDA receptor antagonists such as MK‐801 (0.6, 6.0, 60 nmol), AP‐5 (10.2, 40.6, 162.3 nmol) and DCKA (0.97, 9.7, 97 nmol) dose‐dependently reversed the hyperalgesia. Chelerythrine chloride, a protein kinase C (PKC) inhibitor (1, 10.4, 104.2 nmol) and 7‐NI, a specific nitric oxide (NO) synthase inhibitor (37.5, 75, 150 μmol kg−1, i.p.) induced an anti‐hyperalgesic effect in a dose‐dependent manner. SR‐140333 (0.15, 1.5, 15 nmol) and SR‐48968 (0.17, 1.7, 17 nmol), antagonists of neurokinin receptors, produced a significant, but moderate, increase in vocalization threshold.

These results demonstrate that Mg‐deficiency induces a sensitization of nociceptive pathways in the spinal cord which involves NMDA and non‐NMDA receptors. Furthermore, the data is consistent with an active role of PKC, NO and, to a lesser extent substance P in the intracellular mechanisms leading to hyperalgesia.

British Journal of Pharmacology (2001) 134, 1227–1236; doi:10.1038/sj.bjp.0704354
Differential involvement of the sigma1 (σ1) receptor in the anti‐amnesic effect of neuroactive steroids, as demonstrated using an in vivo antisense strategy in the mouse
Authors: Maurice Tangui , Phan Vân‐Ly , Urani Alexandre , Guillemain Isabelle
Published: January 2005

The sigma1 (σ1) receptor cDNA was cloned in several animal species. Molecular tools are now available to identify its endogenous effectors, such as neuroactive steroids, and to establish its precise physiological role. In particular, the σ1 receptor is involved in memory processes, as observed in pharmacological and pathological rodent models of amnesia.

In order to establish the involvement of σ1 receptors in memory, a 16‐mer oligodeoxynucleotide antisense to the σ1 receptor cDNA (aODN), and its mismatched control (mODN) were prepared and centrally administered into the mouse brain. The anti‐amnesic effects induced by the selective σ1 agonist PRE‐084 and the steroid dehydroepiandrosterone (DHEA) sulphate or pregnenolone sulphate were examined in ODN‐treated animals.

The aODN treatment failed to affect the dissociation constant (Kd) but significantly decreased the number of σ1 sites (Bmax) labelled with [3H]‐(+)‐SKF‐10,047 in the hippocampus and cortex. In these structures, the in vivo binding levels were also diminished, according to the dose and number of injections, as compared with control animals injected with saline or mODN.

Cannulation and injections failed to affect the open‐field behaviour of the animals. However, the anti‐amnesic effects of PRE‐084 and DHEA sulphate against the dizocilpine‐induced impairments were blocked after aODN treatment in the short‐ and long‐term memory tests. The anti‐amnesic effects of pregnenolone sulphate remained unchanged.

These observations bring a molecular basis to the modulatory role of σ1 receptors in memory, and reveal that the anti‐amnesic action of neuroactive steroids may not similarly involve an interaction with σ1 receptors.

British Journal of Pharmacology (2001) 134, 1731–1741; doi:10.1038/sj.bjp.0704355
Macrophage Stimulating Protein (MSP) evokes superoxide anion production by human macrophages of different origin
Authors: Brunelleschi Sandra , Penengo Lorenza , Lavagno Luisa , Santoro Claudio , Colangelo Donato , Viano Ilario , Gaudino Giovanni
Published: January 2005

Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive.

We show here that human recombinant MSP (hrMSP) evokes a dose‐dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte‐derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N‐formylmethionyl‐leucyl‐phenylalanine and similar to phorbol myristate acetate‐evoked one.

To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c‐Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3‐kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP‐evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3‐kinase. We also show that MAP kinase and p38 signalling pathways are involved.

These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.

British Journal of Pharmacology (2001) 134, 1285–1295; doi:10.1038/sj.bjp.0704356
Somatostatin release by glutamate in vivo is primarily regulated by AMPA receptors
Authors: Hathway G J , Humphrey P P A , Kendrick K M
Published: January 2005

We have used in vivo microdialysis in anaesthetized rats to investigate whether levels of striatal somatostatin (SRIF) can be increased in response to application of the ionotropic glutamate receptor agonists AMPA and NMDA.

Application of both AMPA and NMDA (10, 50, 100 and 500 μM) for 20 min periods produced concentration‐dependent increases in the extracellular levels of SRIF. A 500 μM dose of each compound was shown to be the most potent concentration tested, increasing levels of SRIF by 32 fold (NMDA) and 35 fold (AMPA). At lower concentrations (10 μM) NMDA failed to evoke significant amounts of SRIF while AMPA increased levels of the peptide 2.3 fold.

Application of the respective receptor antagonists APV (NMDA receptor) and DNQX (AMPA receptor) abolished the abilities of the agonists to evoke release of SRIF. Interestingly DNQX abolished the ability of NMDA to evoke release of the peptide as well.

The ability of both AMPA and NMDA to evoke increases in the levels of extracellular SRIF further illustrates the reciprocal relationship that exists between SRIF and glutamate in the striatum which impacts particularly on dopaminergic functioning in this region.

British Journal of Pharmacology (2001) 134, 1155–1158; doi:10.1038/sj.bjp.0704362
Effect of P‐glycoprotein modulators on the pharmacokinetics of camptothecin using microdialysis
Authors: Tsai T H , Lee C H , Yeh P H
Published: January 2005

By performing microdialysis, this study investigated the pharmacokinetics of unbound camptothecin in rat blood, brain and bile in the presence of P‐glycoprotein mediated transport modulators (cyclosporin A, berberine, quercetin, naringin and naringenin). Pharmacokinetic parameters of camptothecin were assessed using a non‐compartmental model.

Camptothecin rapidly crosses the blood‐brain barrier (BBB) within 20 min after camptothecin administration. The disposition of camptothecin in rat bile appeared to have a slow elimination phase and a peak concentration after 20 min of camptothecin administration. The area under the concentration versus time curve (AUC) for camptothecin in bile significantly surpassed that in blood, suggesting active transport of hepatobiliary excretion.

In the presence of cyclosporin A camptothecin AUC, in the brain, was significantly elevated but no significant change in the presence of berberine, quercetin, naringin and naringenin.

With treatment by smaller doses of quercetin (0.1 mg kg−1), naringin (10 mg kg−1) and naringenin (10 mg kg−1), they significantly diminished the camptothecin AUC in bile, but was not altered by the treatment of berberine (20 mg kg−1), a higher dose of quercetin (10 mg kg−1), and cyclosporin A treated (20 mg kg−1) and pretreated groups.

The distribution ratio (AUCbile/AUCblood) of camptothecin in bile was decreased in the cyclosporin A, quercetin, naringin and naringenin treated groups. However, the distribution ratio in the brain was increased in the cyclosporin A groups, but was decreased in the groups treated with quercetin, naringin and naringenin. These results revealed that P‐glycoprotein might modulate hepatobiliary excretion and BBB penetration of camptothecin.

British Journal of Pharmacology (2001) 134, 1245–1252; doi:10.1038/sj.bjp.0704363