bluuebunny/crossref_metadata_2025 huggingface dataset:
G‐protein activation by putative antagonists at mutant Thr373Lys α2A adrenergic receptors
Authors: Wurch T , Colpaert F C , Pauwels P J
Published: January 2005

Replacement of a threonine by a lysine at position 373 in the C‐terminal portion of the third intracellular loop of the human α2A‐adrenergic receptor (α2A AR) has been reported to generate a constitutively active mutant receptor in analogy with similar mutations in the α1B and β2 AR (Ren et al., 1993). In the present study, the mutant Thr373Lys α2A AR receptor was investigated by measuring the formation of inositol phosphates in either the absence or presence of mouse Gα15 protein in Cos‐7 cells.

Increased affinity, potency and/or efficacy for the agonists [(−)‐adrenaline, UK 14304, clonidine, guanabenz and oxymetazoline] was observed, consistent with a precoupled mutant α2A AR: G‐protein state. The basal inositol phosphates response was similar at the wild‐type (wt) and mutant α2A AR, but was enhanced at the mutant α2A AR upon co‐expression with the mouse Gα15 protein. This enhanced response could not be attenuated in the presence of any of the tested α2 AR antagonists (10 μM), suggesting that inverse agonist activity did not occur at the mutant α2A AR.

Ligands that so far have been identified as antagonists at the wt α2A AR demonstrated either no intrinsic activity (MK 912, WB 4101, RS 15385, RX 811059 and RX 821002) or positive efficacy [Emax, % vs. 1 μM UK 14304: dexefaroxan (27±7), idazoxan (34±9), atipamezole (27±4), BRL 44408 (59±5) and SKF 86466 (54±9)] at the mutant α2A AR, but only in the presence of the mouse Gα15 protein. The ligand potencies corresponded with their respective pKi values at the mutant α2A AR receptor.

The partial agonist effect of SKF 86466 was resistant to pertussis toxin treatment (100 ng ml−1) and not affected by co‐expression of the rat Gαi1 protein. It was virtually absent in the presence of 10 μM RS 15385. SKF 86466 was without intrinsic activity upon co‐expression of the mouse Gαq protein.

Some putative α2 AR antagonists exerted a partial agonist activity that was highly dependent on the presence of specific G‐protein α‐subunits, suggesting that these ligands cause selective G‐protein activation at the mutant α2A AR.

British Journal of Pharmacology (1999) 126, 939–948; doi:10.1038/sj.bjp.0702379
A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission
Authors: Hernández Medardo , Barahona María Victoria , Bustamante Salvador , García‐Sacristán Albino , Orensanz Luis M
Published: January 2005

The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission.

In U46619 (10−7  M)‐contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10−6  M), adenosine and related analogues induced relaxations with the following potency order: 5′‐N‐ethylcarboxamidoadenosine (NECA)=5′‐(N‐cyclopropyl)‐carboxamidoadenosine (CPCA)=2‐chloroadenosine (2‐CA)>adenosine>cyclopentyladenosine (CPA)=N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methylcarboxamide (IB‐MECA)=2‐[p‐(carboxyethyl)‐phenylethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS21680).

Epithelium removal or incubation with indomethacin (3×10−6  M) and L‐NG‐nitroarginine (L‐NOARG, 3×10−5  M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine.

1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 10−8 M) and 4‐(2‐[7‐amino‐2‐(2‐furyl) [1,2,4]‐triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol (ZM 241385, 3×10−8  M and 10−7  M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8‐phenyltheophylline (8‐PT, 10−5  M) and DPCPX (10−6  M), which block A1/A2‐receptors, reduced such relaxations.

In strips treated with guanethidine (10−5  M), atropine (10−7  M), L‐NOARG (3×10−5  M) and indomethacin (3×10−6  M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10−4  M) induced contractions of preparations. 8‐PT (10−5  M) increased both contractions. DPCPX (10−8  M), NECA (10−4  M), CPCA, (10−4  M) and 2‐CA (10−4  M) did not alter the contractions to EFS.

The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids or NO, through activation of A2B‐receptors located in the smooth muscle. This relaxation may modulate the ureteral NANC excitatory neurotransmission through a postsynaptic mechanism.

British Journal of Pharmacology (1999) 126, 969–978; doi:10.1038/sj.bjp.0702386
The inhibition of nicotine‐evoked relaxation of the guinea‐pig isolated basilar artery by some analgesic drugs and progesterone
Authors: Rhodes Keith F , Buckingham Julia C , Kennard Christopher
Published: January 2005

The purpose of this study was to investigate the mechanism of nicotine‐evoked relaxation of the guinea‐pig isolated basilar artery and to study the effects of drugs associated with the aetiology or treatment of migraine on the nicotine response.

The guinea‐pig isolated basilar artery, pre‐contracted with prostaglandin F2α (PGF2α), in the presence of atropine (3 μM) and guanethidine (3 μM), relaxed on addition of nicotine (0.1 mM) in approximately 50% of preparations. The responses to nicotine were of short duration and blocked in preparations pre‐treated for 10 min with capsaicin (1 μM) and are therefore probably a consequence of the stimulation of trigeminal C fibre terminals.

Responses to nicotine were reduced in the presence of 5‐carboxamidotryptamine, 5‐hydroxytryptamine and sumatriptan in that order of potency. This is consistent with a 5‐HT1 receptor mechanism. These agonists evoked small additional contractions in vessels pre‐contracted with PGF2α.

Indomethacin (0.3–10 μM), aspirin (10–30 μM), and nitro‐L‐arginine methyl ester (L‐NAME, 0.1 mM) reduced nicotine‐evoked relaxation of the basilar artery, suggesting the involvement of both nitric oxide and cyclo‐oxygenase products in this response.

Progesterone (1 μM) markedly reduced the response to nicotine, a possible reflection of the ion channel blocking activity of high concentrations of this compound.

The guinea‐pig basilar artery is a preparation in which the effects of drugs on responses to stimulation of trigeminal nerve terminals can be studied in vitro and may thus be of interest in assessing the actions of drugs used in treatment of headache.

British Journal of Pharmacology (1999) 126, 1003–1008; doi:10.1038/sj.bjp.0702391
Identification of glucoside and carboxyl‐linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil
Authors: Shipkova Maria , Armstrong Victor William , Wieland Eberhard , Niedmann Paul Dieter , Schütz Ekkehard , Brenner‐Weiß Gerald , Voihsel Martin , Braun Felix , Oellerich Michael
Published: January 2005

Mycophenolic acid (MPA), is primarily metabolized in the liver to 7‐O‐MPA‐β‐glucuronide (MPAG). Using RP‐h.p.l.c. we observed three further MPA metabolites, M‐1, M‐2, M‐3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP‐glucuronic acid, UDP‐glucose or NADPH; (C) hydrolysis of metabolites was investigated using β‐glucosidase, β‐glucuronidase or NaOH; (D) cross‐reactivity of each metabolite was tested in an immunoassay for MPA (EMIT).

Mass spectrometry of M‐1, M‐2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively.

Incubation of microsomes with MPA and UDP‐glucose produced M‐1, with MPA and UDP‐glucuronic acid MPAG and M‐2 were formed, while with MPA and NADPH, M‐3 was observed.

β‐Glucosidase hydrolysed M‐1 completely. β‐Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M‐2 was reduced by approximately 30%. Only M‐2 was labile to alkaline treatment.

M‐2 and MPA but not M‐1 and MPAG cross‐reacted in the EMIT assay.

These results suggest that: (i) M‐1 is the 7‐OH glucose conjugate of MPA; (ii) M‐2 is the acyl glucuronide conjugate of MPA; (iii) M‐3 is derived from the hepatic CYP450 system.

British Journal of Pharmacology (1999) 126, 1075–1082; doi:10.1038/sj.bjp.0702399
The putative melatonin receptor antagonist GR128107 is a partial agonist on Xenopus laevis melanophores
Authors: Teh Muy‐Teck , Sugden David
Published: January 2005

GR128107 (3‐(1‐acetyl‐3‐methyl‐piperidine)‐5‐methoxyindole) has previously been reported to be a competitive melatonin receptor antagonist in blocking melatonin inhibition of [3H]‐dopamine release from rabbit retina, a response mediated by the MT2 receptor subtype.

GR128107, like melatonin, induced a rapid (maximum response in 60–90 min) pigment aggregation in a clonal line of Xenopus laevis melanophores. GR128107 behaved as a partial agonist (pEC50 8.58±0.03, n=3) with an Emax of 0.83 (relative to melatonin, pEC50 10.09±0.03, n=3).

The concentration‐response curve for pigment granule aggregation to both melatonin and GR128107 was displaced in a parallel, rightward manner by melatonin receptor antagonists with very similar potencies; estimated pKB RJ252 (against melatonin 4.60/against GR128107 4.54) <GR135533 (6.40/6.14) < Luzindole (6.45/6.49) < S20929 (6.58/6.65) < 4‐P‐PDOT (6.73/6.85).

Both melatonin‐ and GR128107‐induced pigment granule aggregation was prevented by pre‐treatment of melanophores with pertussis toxin (10–1000 ng ml−1).

Prolonged pre‐treatment of melanophores with melatonin desensitized the pigment aggregation response to GR128107. In desensitized cells, the maximal aggregation produced by GR128107 was only 0.27±0.01 (n=4) and the pEC50 was reduced (vehicle 8.57±0.12; melatonin pre‐treated 7.84±0.09, n=4). The maximal response to melatonin in desensitized melanophores was unchanged but the pEC50 was reduced (vehicle 10.49±0.03; melatonin pre‐treated 9.83±0.04, n=4).

These results demonstrate that GR128107 induces pigment granule aggregation in Xenopus melanophores by activating a cell membrane melatonin receptor coupled via a pertussis toxin‐sensitive G‐protein.

The partial agonist activity of GR128107 in melanophores may be apparent because of the very high density of melatonin receptors in these cells (Bmax 1223 fmol mg protein−1) compared to the low density of sites in rabbit retina (Bmax 3.1 fmol mg protein−1). This suggestion is supported by the finding that GR128107, like melatonin, acted as a full agonist and inhibited forskolin‐stimulation of cyclic AMP accumulation in NIH‐3T3 cells expressing a high density of human mt1 or MT2 receptors.

British Journal of Pharmacology (1999) 126, 1237–1245; doi:10.1038/sj.bjp.0702404
Lung clearance of intratracheally instilled 99mTc‐tobramycin using pulmonary surfactant as vehicle
Authors: Van 't Veen Annemarie , Gommers Diederik , Verbrugge Serge J C , Wollmer Per , Mouton Johan W , Kooij Peter P M , Lachmann Burkhard
Published: January 2005

The use of pulmonary exogenous surfactant as a vehicle for intratracheally administered antibiotics to improve local antimicrobial therapy has been proposed. The present study investigated lung clearance rates in the rat of intratracheally instilled technetium labelled tobramycin with and without the addition of surfactant to the antibiotic solution.

The influence of surfactant on 99mTc‐tobramycin lung clearance rates was studied dynamically with a gamma‐camera in anaesthetized spontaneously breathing animals and in mechanically ventilated animals.

The results show that instillation of 99mTc‐tobramycin with use of surfactant as vehicle significantly increases 99mTc‐tobramycin lung clearance compared to instillation of 99mTc‐tobramycin solution alone (P=0.006 between the two spontaneously breathing groups of animals and P=0.02 between the two ventilated groups of animals, ANOVA for repeated time measurements). The half life (t½) of composite clearance curves in spontaneous breathing animals was 147 min for animals receiving 99mTc‐tobramycin versus 61 min for animals receiving 99mTc‐tobramycin with surfactant. In mechanically ventilated animals this was 163 min versus 51 min, respectively.

It is concluded that exogenous surfactant, used as vehicle for intratracheally instilled 99mTc‐tobramycin, increases lung clearance rate of 99mTc‐tobramycin in rats.

British Journal of Pharmacology (1999) 126, 1091–1096; doi:10.1038/sj.bjp.0702405
Effect of protein kinase A inhibitors on the production of Aβ40 and Aβ42 by human cells expressing normal and Alzheimer's disease‐linked mutated βAPP and presenilin 1
Authors: Marambaud P , Ancolio K , Alves da Costa C , Checler F
Published: January 2005

We previously established that the formation of both α‐ and β/γ‐secretase‐derived products generated by human embryonic kidney 293 cells (HEK293) expressing either wild type or mutant βAPP could be stimulated by agonists of the cyclic AMP/protein kinase A pathways. This cyclic AMP‐dependent effect modulates post‐translational events since it is not prevented by actinomycin D or cycloheximide.

We show here that two protein kinase A inhibitors, H89 and PKI, both trigger dose‐dependent inhibition of the basal constitutive production of Aβ40 and Aβ42 by HEK293 cells expressing wild type βAPP751.

H89 also potently inhibits the total Aβ produced by the neocortical neuronal cell line TSM1.

These two inhibitors also drastically reduce the recovery of Aβ40 and Aβ42 produced by HEK293 cells expressing the Swedish (Sw) βAPP and M146V‐presenilin 1 (PS1) mutations responsible for cases of the early‐onset forms of Familial Alzheimer's disease (FAD).

By contrast, H89 and PKI do not significantly affect the recovery of the physiological α‐secretase‐derived fragment APPα.

Our study indicates that protein kinase A inhibitors selectively lower the formation of Aβ40 and Aβ42 in human cells expressing normal and mutant βAPP and PS1 without affecting the physiological α‐secretase pathway in these cells. Selective inhibitors of protein kinase A may be of therapeutic value in both sporadic and Familial Alzheimer's disease, since they may decrease the production of Aβ that is thought to be responsible for the neurodegenerative process.

British Journal of Pharmacology (1999) 126, 1186–1190; doi:10.1038/sj.bjp.0702406
Characterization and tissue location of the neural adenosine receptor in the rat ileum
Authors: Coupar Ian M
Published: January 2005

The aim of the present investigation was to characterize and determine the tissue location of the adenosine receptors present in the rat ileum using a method that detects drug action on the cholinergic nerves innervating the longitudinal and circular muscles.

The non‐selective adenosine agonist, NECA (10 and 100 nM) caused significant concentration‐related reductions in the circular muscle responses to transmural stimulation over the frequency range of 2.5–40 Hz, but did not affect the responses of the longitudinal muscle, nor did it reduce the muscle responses of the guinea‐pig ileum.

The affinity order of antagonists at inhibiting the effect of NECA on the circular muscle was: CPDPX>8‐PT>DMPX with apparent pA2 values of 9.31, 7.54 and 5.63 respectively. CPDPX (10–100 nM) caused parallel displacements of the concentration‐effect curves to CPA with a pKb value of 9.15 and Schild slope of 1.03.

The agonists previously tested in the rat jejunum peristaltic reflex preparation were also shown to inhibit responses of the rat ileum in the following decreasing order of potency: CPA>NECA>2‐CADO>R‐PIA>S‐PIA>>PAA. In addition, CHA and CCPA were also potent agonists. NECA (100 nM) and CPA (32 nM) did not inhibit carbachol (1 μM)‐induced tone of tissues pre‐treated with TTX (1 μM).

In conclusion, the rat ileum contains inhibitory A1 adenosine receptors situated on cholinergic nerve endings innervating the circular muscle.

British Journal of Pharmacology (1999) 126, 1269–1275; doi:10.1038/sj.bjp.0702411
Inverse agonist abolishes desensitization of a constitutively active mutant of thyrotropin‐releasing hormone receptor: role of cellular calcium and protein kinase C
Authors: Grimberg Hagit , Zaltsman Ilona , Lupu‐Meiri Monica , Gershengorn Marvin C , Oron Yoram
Published: January 2005

C335Stop is a constitutively active mutant of the TRH receptor (TRH‐R). To investigate the mechanism of the decreased responsiveness of C335Stop TRH‐R, we studied cellular Ca2+ concentrations ([Ca2+]i) in AtT20 cells stably transfected with C335Stop TRH‐R cDNA, or Ca2+‐activated chloride currents in Xenopus laevis oocytes expressing this mutant receptor after injection of cRNA. The competitive TRH‐R binding antagonist, chlorodiazepoxide (CDE), was used as an inverse agonist to study the contribution of constitutive activity to desensitization.

Acute treatment with CDE resulted in a rapid (within minutes) decrease in [Ca2+]i and an increase in the response amplitude to TRH with no measurable change in receptor density. Conversely, removal of chronically administered CDE caused a rapid increase in [Ca2+]i and a decrease in TRH response amplitude.

CDE abolished heterologous desensitization induced by C335Stop TRH‐R on muscarinic m1‐receptor (m1‐R) co‐expressed in Xenopus oocytes.

Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca2+]i and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH‐Rs.

Chelerythrine, a specific inhibitor of protein kinase C (PKC), reversed the heterologous desensitization of the response to acetylcholine (ACh). The phosphoserine/phosphothreonine phosphatase inhibitor, okadaic acid, abolished the effect of chelerythrine.

Down‐regulation of PKC by chronic exposure to phorbol 12‐myristate 13‐acetate (PMA) or acute inhibition with chelerythrine caused a partial resensitization of the response to TRH.

Western analysis indicated that the α subtype of protein kinase C was down‐regulated in cells expressing C335Stop TRH‐Rs. Following a 5 min exposure to PMA, the residual αPKC translocated to the particular fraction.

We propose that cells expressing the constitutively active mutant TRH‐R rapidly desensitize their response, utilizing a mechanism mediated by an increase in [Ca2+]i and PKC.

British Journal of Pharmacology (1999) 126, 1097–1106; doi:10.1038/sj.bjp.0702415
Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta
Authors: Brailoiu Eugen , Filipeanu Catalin M , Tica Andrei , Toma Catalin P , De Zeeuw Dick , Nelemans S Adriaan
Published: January 2005

We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved.

Compounds were delivered into the cytoplasm of de‐endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10−5 M).

Intracellular administration of Ang II (incorporation range: 0.01–300 nmol mg−1) resulted in a dose‐dependent contraction, insensitive to extracellular administration (10−6 M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non‐selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]‐Ang II (P<0.05).

Intracellular administration of CV11947 or PD 123319 right shifted the dose‐response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg−1 Ang II was incorporated.

Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P<0.05).

Intracellular administration of Ang I and saralasin also induced contraction (P<0.05). Both responses were sensitive to intracellular CV11947 (P<0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril.

Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre‐treatment with saralasin or CV11947 (P<0.05), but not with PD 123319. Extracellular saralasin induced no contraction.

Intracellular Ang II induced contraction was not affected by pre‐treatment with heparin filled liposomes, but completely abolished in Ca2+‐free external medium.

These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+‐influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+‐stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

British Journal of Pharmacology (1999) 126, 1133–1138; doi:10.1038/sj.bjp.0702421
Down‐regulation of microglial cyclo‐oxygenase‐2 and inducible nitric oxide synthase expression by lipocortin 1
Authors: Minghetti Luisa , Nicolini Alessia , Polazzi Elisabetta , Greco Anita , Perretti Mauro , Parente Luca , Levi Giulio
Published: January 2005

Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO).

The anti‐inflammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective effect of LC1 could be explained by an inhibitory effect on microglial activation.

We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclo‐oxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS), respectively.

Dexamethasone (DEX, 1–100 nM) and LC1‐derived N‐terminus peptide (peptide Ac2‐26, 1–100 μg ml−1) dose‐dependently inhibited the production of both PGE2 and NO from LPS‐stimulated microglia. The inhibitory effects of DEX on NO and of the peptide on NO and PGE2 synthesis were partially abrogated by a specific antiserum, raised against the N‐terminus of human LC1. The peptide Ac2‐26 did not affect arachidonic acid release from control and LPS‐stimulated microglial cultures.

Western blot experiments showed that the LPS‐induced expression of COX‐2 and iNOS was effectively down‐regulated by DEX (100 nM) and peptide Ac2‐26 (100 μg ml−1).

In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms.

British Journal of Pharmacology (1999) 126, 1307–1314; doi:10.1038/sj.bjp.0702423
In vitro prostanoid release from spinal cord following peripheral inflammation: effects of substance P, NMDA and capsaicin
Authors: Dirig David M , Yaksh Tony L
Published: January 2005

Spinal prostanoids are implicated in the development of thermal hyperalgesia after peripheral injury, but the specific prostanoid species that are involved are presently unknown. The current study used an in vitro spinal superfusion model to investigate the effect of substance P (SP), N‐methyl‐d‐aspartate (NMDA), and capsaicin on multiple prostanoid release from dorsal spinal cord of naive rats as well as rats that underwent peripheral injury and inflammation (knee joint kaolin/carrageenan).

In naive rat spinal cords, PGE2 and 6‐keto‐PGF1α, but not TxB2, levels were increased after inclusion of SP, NMDA, or capsaicin in the perfusion medium.

Basal PGE2 levels from spinal cords of animals that underwent 5–72 h of peripheral inflammation were elevated relative to age‐matched naive cohorts. The time course of this increase in basal PGE2 levels coincided with peripheral inflammation, as assessed by knee joint circumference. Basal 6‐keto‐PGF1α levels were not elevated after injury.

From this inflammation‐evoked increase in basal PGE2 levels, SP and capsaicin significantly increased spinal PGE2 release in a dose‐dependent fashion. Capsaicin‐evoked increases were blocked dose‐dependently by inclusion of S(+) ibuprofen in the capsaicin‐containing perfusate.

These data suggest a role for spinal PGE2 and NK‐1 receptor activation in the development of hyperalgesia after injury and demonstrate that this relationship is upregulated in response to peripheral tissue injury and inflammation.

British Journal of Pharmacology (1999) 126, 1333–1340; doi:10.1038/sj.bjp.0702427
Cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild‐type and D79N α2A‐adrenoceptor transgenic mice
Authors: Zhu Q‐M , Lesnick J D , Jasper J R , MacLennan S J , Dillon M P , Eglen R M , Blue D R
Published: January 2005

We investigated the cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild‐type and D79N α2A‐adrenoceptor mice. The in vitro pharmacology of these agonists was determined at recombinant (human) α2‐adrenoceptors and at endogenous (dog) α2A‐adrenoceptors.

In wild‐type mice, rilmenidine, moxonidine (100, 300 and 1000 μg kg−1, i.v.) and clonidine (30, 100 and 300 μg kg−1, i.v.) dose‐dependently decreased blood pressure and heart rate.

In D79N α2A‐adrenoceptor mice, responses to rilmenidine and moxonidine did not differ from vehicle control. Clonidine‐induced hypotension was absent, but dose‐dependent hypertension and bradycardia were observed.

In wild‐type mice, responses to moxonidine (1 mg kg−1, i.v.) were antagonized by the non‐selective, non‐imidazoline α2‐adrenoceptor antagonist, RS‐79948‐197 (1 mg kg−1, i.v.).

Affinity estimates (pKi) at human α2A‐, α2B‐ and α2C‐adrenoceptors, respectively, were: rilmenidine (5.80, 5.76 and 5.33), moxonidine (5.37, <5 and <5) and clonidine (7.21, 7.16 and 6.87). In a [35S]‐GTPγS incorporation assay, moxonidine and clonidine were α2A‐adrenoceptor agonists (pEC50/intrinsic activity relative to noradrenaline): moxonidine (5.74/0.85) and clonidine (7.57/0.32).

In dog saphenous vein, concentration‐dependent contractions were observed (pEC50/intrinsic activity relative to noradrenaline): rilmenidine (5.83/0.70), moxonidine (6.48/0.98) and clonidine (7.22/0.83). Agonist‐independent affinities were obtained with RS‐79948‐197.

Thus, expression of α2A‐adrenoceptors is a prerequisite for the cardiovascular effects of moxonidine and rilmenidine in conscious mice. There was no evidence of I1‐imidazoline receptor‐mediated effects. The ability of these compounds to act as α2A‐adrenoceptor agonists in vitro supports this conclusion.

British Journal of Pharmacology (1999) 126, 1522–1530; doi:10.1038/sj.bjp.0702429
Bradykinin‐induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells
Authors: Luo Shue‐Fen , Pan Shiow‐Lin , Wu Wen‐Bin , Wang Chuan‐Chwan , Chiu Chi‐Tso , Tsai Yih‐Jeng , Yang Chuen‐Mao
Published: January 2005

Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs).

BK and Lys‐BK caused an initial transient peak of [Ca2+]i in a concentration‐dependent manner, with half‐maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively.

Kinin B2 antagonists Hoe 140 (10 nM) and [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK (1 μM) had high affinity in antagonizing BK‐induced Ca2+ response with pKB values of 8.90 and 6.99, respectively.

Pretreatment of TECs with pertussis toxin (100 ng ml−1) or cholera toxin (10 μg ml−1) for 24 h did not affect the BK‐induced IP accumulation and [Ca2+]i changes in TECs.

Removal of Ca2+ by the addition of EGTA or application of Ca2+‐channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK‐induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK‐induced responses.

Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+‐free buffer and subsequently induced Ca2+ influx when Ca2+ was re‐added to this buffer. Pretreatment of TECs with TG completely abolished BK‐induced initial transient [Ca2+]i, but had slight effect on BK‐induced Ca2+ influx.

Pretreatment of TECs with SKF96365 and U73122 inhibited the BK‐induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor‐gated Ca2+ ‐channels and phospholipase C in TECs, respectively.

These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C‐mediated IP accumulation and Ca2+ mobilization via a pertussis toxin‐insensitive G protein in canine TECs. These results also suggest that BK‐induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK‐induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.

British Journal of Pharmacology (1999) 126, 1341–1350; doi:10.1038/sj.bjp.0702431
Conformational restraints revealing bioactive β‐bend structures for hα CGRP8–37 at the CGRP2 receptor of the rat prostatic vas deferens
Authors: Wisskirchen F M , Doyle P M , Gough S L , Harris C J , Marshall I
Published: January 2005

The main aim of this study was to identify putative β‐bends and the role of the N‐ and C‐terminus in the CGRP receptor antagonist hα CGRP8–37, which was measured against hα CGRP inhibition of twitch responses in the rat prostatic vas deferens.

With a bend‐biasing residue (proline) at position 16 in hα CGRP8–37 (10−5 M) an inactive compound was produced, while alanine at the same position retained antagonist activity (apparent pKB 5.6±0.1 at 10−5 M). Proline at position 19 within hα CGRP8–37 (10−5 M) was an antagonist (apparent pKB 5.8±0.1).

Incorporation of a bend‐forcing structure (beta‐turn dipeptide or BTD) at either positions 19,20 or 33,34 in hα CGRP8–37 (10−5 M) antagonized hα CGRP responses (apparent pKB 6.0±0.1 and 6.1±0.1, respectively). Replacement by BTD at both positions 19,20 and 33,34 within hα CGRP8–37 competitively antagonized responses to hα CGRP (pA2 6.2; Schild plot slope 1.0±0.1).

Hα CGRP8–37 analogues (10−5 M), substituted at the N‐terminus by either glycine8, or des‐NH2 valine8 or proline8 were all antagonists against hα CGRP (apparent pKB 6.1±0.1, 6.5±0.1 and 6.1±0.1, respectively), while hα CGRP8–37 (10−5 M) substituted in three places by proline8 and glutamic acid10,14 was inactive.

Replacement of the C‐terminus by alanine amide37 in hα CGRP8–37 (10−5 M) failed to antagonize hα CGRP responses.

Peptidase inhibitors did not alter either the agonist potency of hα CGRP or the antagonist affinities of hα CGRP8–37 BTD19,20 and 33,34 and hα CGRP8–37 Gly8 (against hα CGRP responses).

In conclusion, two β‐bends at positions 18–21 and 32–35 are compatible with high affinity by BTD and is the first approach of modelling the bioactive structure of hα CGRP8–37. Further, the N‐terminus of hα CGRP8–37 is not essential for antagonism, while the C‐terminus interacts directly with CGRP receptor binding sites of the rat vas deferens.

British Journal of Pharmacology (1999) 126, 1163–1170; doi:10.1038/sj.bjp.0702432
4‐trifluoromethyl derivatives of salicylate, triflusal and its main metabolite 2‐hydroxy‐4‐trifluoromethylbenzoic acid, are potent inhibitors of nuclear factor κB activation
Authors: Bayón Yolanda , Alonso Andrés , Crespo Mariano Sánchez
Published: January 2005

The effect of two derivatives of salicylate, 2‐hydroxy‐4‐trifluoromethylbenzoic acid (HTB) and 2‐acetoxy‐4‐trifluoromethylbenzoic acid (triflusal), on the activation of NF‐κB elicited by tumour necrosis factor‐α (TNF‐α) on human umbilical vein endothelial cells (HUVEC) was tested.

The expression of the mRNA of vascular cell adhesion molecule‐1 (VCAM‐1) was studied as an example of a gene the expression of which is regulated by NF‐κB. To extend these findings to other systems, the induction of nitric oxide synthase in rat adherent peritoneal macrophages was studied.

Both HTB and triflusal were more potent than aspirin or salicylate as inhibitors of the nuclear translocation of NF‐κB. The calculation of the IC50 values showed ∼amp;2 mM for HTB, 4 mM for aspirin and >4 mM for salicylate.

Comparison of the potency of these compounds on VCAM‐1 mRNA expression showed complete inhibition by both triflusal and HTB at a concentration of 4 mM whereas aspirin and salicylate produced only 36–43% inhibition at the same concentration.

Inhibition of NF‐κB activation was also observed in rat peritoneal macrophages stimulated via their receptors for the Fc portion of the antibody molecule with IgG/ovalbumin immune complexes. This was accompanied by a dose‐dependent inhibition of nitrite production by the L‐arginine pathway via iNOS. IC50 values for this effect were 1.13±0.12 mM (triflusal), 1.84±0.34 (HTB), 6.08±1.53 mM (aspirin) and 9.16±1.9 mM (salicylate).

These data indicate that the incorporation of a 4‐trifluoromethyl group to the salicylate molecule strongly enhances its inhibitory effect on NF‐κB activation, VCAM‐1 mRNA expression and iNOS induction, irrespective of the presence of the acetyl moiety involved in the inhibition of cyclo‐oxygenase.

British Journal of Pharmacology (1999) 126, 1359–1366; doi:10.1038/sj.bjp.0702441
Effect of α‐trinositol on interstitial fluid pressure, oedema generation and albumin extravasation in experimental frostbite in the rat
Authors: Berg A , Aas P , Gustafsson T , Reed R K
Published: January 2005

The anti‐inflammatory effect of α‐trinositol (D‐myo‐inositol‐1,2,6‐trisphosphate) on oedema formation, microvascular protein leakage and interstitial fluid pressure (Pif) in rat skin after frostbite injury, was investigated. α‐Trinositol (40 mg kg body weight−1) was administered intravenously as a bolus both before and/or in the interval between freezing and thawing of the tissue.

Pif was measured in rat paw skin with micropipettes connected to a servo‐controlled counterpressure system. Oedema formation was estimated by measuring the increase in total tissue water content (wet weight minus dry weight divided by dry weight). Albumin extravasation (i.e., the difference between the plasma equivalent space for 125I‐ and 131I‐human serum albumin (HSA) circulating for different time intervals) was used to estimate the microvascular leakage.

Compared to untreated animals, α‐trinositol given pre‐ and/or post‐freeze reduced total tissue water and albumin extravasation as well as the fall in Pif in injured tissue significantly (P<0.05). α‐Trinositol given only post‐freeze reduced total tissue water and albumin extravasation from 4.46±0.93 and 2.37±1.12 to 2.51±0.29 and 0.36±0.18 ml g dry weight−1, respectively (P<0.05).

Pif fell from −0.8±0.2 mmHg pre‐freeze to −3.4±1.0 mmHg (P<0.05) at 20 min after tissue injury (circulatory arrest) and was attenuated by treatment with α‐trinositol.

We conclude that α‐trinositol exerts its anti‐oedematous effect by acting on the extracellular matrix, attenuating the lowering of Pif as well as on the microvascular wall, thereby decreasing the protein extravasation.

British Journal of Pharmacology (1999) 126, 1367–1374; doi:10.1038/sj.bjp.0702442
Thermogenic effects of sibutramine and its metabolites
Authors: Connoley Ian P , Liu Yong‐Ling , Frost Ian , Reckless Ian P , Heal David J , Stock Michael J
Published: January 2005

The thermogenic activity of the serotonin and noradrenaline reuptake inhibitor sibutramine (BTS 54524; Reductil) was investigated by measuring oxygen consumption (VO2) in rats treated with sibutramine or its two pharmacologically‐active metabolites.

Sibutramine caused a dose‐dependent rise in VO2, with a dose of 10 mg kg−1 of sibutramine or its metabolites producing increases of up to 30% that were sustained for at least 6 h, and accompanied by significant increases (0.5–1.0°C) in body temperature.

Based on the accumulation in vivo of radiolabelled 2‐deoxy‐[3H]‐glucose, sibutramine had little or no effect on glucose utilization in most tissues, but caused an 18 fold increase in brown adipose tissue (BAT).

Combined high, non‐selective doses (20 mg kg−1) of the β‐adrenoceptor antagonists, atenolol and ICI 118551, inhibited completely the VO2 response to sibutramine, but the response was unaffected by low, β1‐adrenoceptor‐selective (atenolol) or β2‐adrenoceptor‐selective (ICI 118551) doses (1 mg kg−1).

The ganglionic blocking agent, chlorisondamine (15 mg kg−1), inhibited completely the VO2 response to the metabolites of sibutramine, but had no effect on the thermogenic response to the β3‐adrenoceptor‐selective agonist BRL 35135.

Similar thermogenic responses were produced by simultaneous injection of nisoxetine and fluoxetine at doses (30 mg kg−1) that had no effect on VO2 when injected individually.

It is concluded that stimulation of thermogenesis by sibutramine requires central reuptake inhibition of both serotonin and noradrenaline, resulting in increased efferent sympathetic activation of BAT thermogenesis via β3‐adrenoceptor, and that this contributes to the compound's activity as an anti‐obesity agent.

British Journal of Pharmacology (1999) 126, 1487–1495; doi:10.1038/sj.bjp.0702446
Analysis of the behaviour of selected CCKB/gastrin receptor antagonists in radioligand binding assays performed in mouse and rat cerebral cortex
Authors: Harper E A , Griffin E P , Shankley N P , Black J W
Published: January 2005

The previously described complex behaviour of the CCKB/gastrin receptor antagonist, L‐365,260, in radioligand binding assays could be explained by a variable population of two binding sites. We have investigated whether other CCKB/gastrin receptor ligands (PD134,308, PD140,376, YM022 and JB93182) can distinguish between these sites.

In the mouse cortex assay, Hill slopes were not different from unity and the ligand pKI values did not differ when either [125I]‐BH‐CCK‐8S or [3H]‐PD140,376 was used as label as expected for a single site (G2).

In the rat cortex, where previous analysis of replicate (n=48) L‐365,260 data indicated the presence of two CCKB/gastrin sites (G1 and G2), the competition data for PD134,308, PD140,376, YM022 and JB93182 could be explained by a homogeneous population of CCKB/gastrin sites because the Hill slope estimates were not significantly different from unity. However, the estimated affinity values for JB93182 and YM022 were significantly higher and that for PD134,308 was significantly lower than those obtained in the mouse cortex when the same radioligand was used. In view of our previous data obtained with L‐365,260, the rat cortex data were also interpreted using a two‐site model. In this analysis, SR27897 expressed ∼9 fold, PD134,308 ∼13 fold and PD140,376 ∼11 fold selectivity for the G2 site. In contrast, JB93182 expressed ∼23 fold and YM022 ∼4 fold selectivity for the G1 site. If the two‐site interpretation of the data is valid then, because of its reverse selectivity to L‐365,260, JB93182 has been identified as a compound which if radiolabelled could provide a test of this receptor subdivision.

British Journal of Pharmacology (1999) 126, 1496–1503; doi:10.1038/sj.bjp.0702448
Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity
Authors: Naisbitt Dean J , Hough Sally J , Gill Helen J , Pirmohamed Munir , Kitteringham Neil R , Park B Kevin
Published: January 2005

Bioactivation of sulphamethoxazole (SMX) to chemically‐reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro.

Flow cytometry revealed binding of N‐hydroxy (SMX‐NHOH) and nitroso (SMX‐NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX‐NO was reduced by exogenous glutathione (GSH).

SMX‐NHOH and SMX‐NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism.

Fluorescence HPLC showed that SMX‐NHOH and SMX‐NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma.

Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX‐NHOH and SMX‐NO than those inducing loss of membrane viability, with SMX having no effect. Lymphocytes were significantly (P<0.05) more sensitive to the direct cytotoxic effects of SMX‐NO than neutrophils.

Partitioning of SMX‐NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone.

Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity.

British Journal of Pharmacology (1999) 126, 1393–1407; doi:10.1038/sj.bjp.0702453
Role of Na+/Ca2+ exchange in endothelin‐1‐induced increases in Ca2+ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB‐R7943
Authors: Yang Huang‐Tian , Sakurai Kiyoharu , Sugawara Hiromi , Watanabe Tomoo , Norota Ikuo , Endoh Masao
Published: January 2005

The effects of endothelin‐1 (ET‐1) on intracellular Ca2+ ion level and cell contraction were simultaneously investigated in rabbit ventricular cardiac myocytes loaded with indo‐1/AM. The role of Na+/Ca2+ exchange in ET‐1‐induced positive inotropic effect (PIE) was examined by using KB‐R7943 (2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate), a selective inhibitor of reverse mode Na+/Ca2+ exchange.

ET‐1 at 0.3 pM–1 nM increased cell contraction and Ca2+ transient (CaT) with EC50 values of 2.9 pM and 1.2 pM, respectively, and the increase in amplitude of CaT was much smaller relative to the PIE: ET‐1 at 1 nM increased peak cell shortening by 237%, while it increased peak CaT by 167%. For a given level of PIE, ET‐1‐induced increase in CaT was much smaller than that induced by elevation of [Ca2+]o and by isoprenaline. Therefore, ET‐1 shifted the relationship between peak CaT and cell shortening to the left relative to the relationship for increase in [Ca2+]o, an indication that ET‐1 increased myofibrillar Ca2+ sensitivity.

KB‐R7943 at 0.1 μM and higher inhibited contraction and CaT induced by 0.1 nM ET‐1 and at 0.3 μM it abolished the increase in CaT while inhibiting the PIE by 48.1%. Over concentration range of 0.1–0.3 μM, KB‐R7943 neither inhibited baseline contraction and CaT nor the isoprenaline‐induced response, although at 1 μM and higher it had a significant inhibitory action on these responses.

These results indicate that in rabbit ventricular myocytes both increases in CaT and myofibrillar Ca2+ sensitivity contribute to the ET‐induced PIE, and the activation of reverse mode Na+/Ca2+ exchange may play a crucial role in increase in CaT induced by ET‐1 in rabbit ventricular cardiac myocytes.

British Journal of Pharmacology (1999) 126, 1785–1795; doi:10.1038/sj.bjp.0702454
Substrates of multidrug resistance‐associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel
Authors: Linsdell Paul , Hanrahan John W
Published: January 2005

The effects of physiological substrates of multidrug resistance‐associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel currents were examined using patch clamp recording from CFTR‐transfected mammalian cell lines.

Two MRP substrates, taurolithocholate‐3‐sulphate (TLCS) and β‐estradiol 17‐(β‐D‐glucuronide) (E217βG) caused a voltage‐dependent block of macroscopic CFTR Cl− currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96±10 and 563±103 μM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453±44 and 3760±710 μM (at 0 mV) respectively.

Reducing the extracellular Cl− concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54±1 μM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl− permeation through CFTR by binding within the channel pore.

Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel.

The apparent affinity of block by TLCs is comparable to that of other well‐known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore.

These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane.

British Journal of Pharmacology (1999) 126, 1471–1477; doi:10.1038/sj.bjp.0702458
GABAB receptor isoforms GBR1a and GBR1b, appear to be associated with pre‐ and post‐synaptic elements respectively in rat and human cerebellum
Authors: Billinton Andrew , Upton Neil , Bowery Norman G
Published: January 2005

Metabotropic γ‐aminobutyric acid (GABA) receptors, GABAB, are coupled through G‐proteins to K+ and Ca2+ channels in neuronal membranes. Cloning of the GABAB receptor has not uncovered receptor subtypes, but demonstrated two isoforms, designated GBR1a and GBR1b, which differ in their N terminal regions. In the rodent cerebellum GABAB receptors are localized to a greater extent in the molecular layer, and are reported to exist on granule cell parallel fibre terminals and Purkinje cell (PC) dendrites, which may represent pre‐ and post‐synaptic receptors.

The objective of this study was to localize the mRNA splice variants, GBR1a and GBR1b for GABAB receptors in rat cerebellum, for comparison with the localization in human cerebellum using in situ hybridization.

Receptor autoradiography was performed utilizing [3H]‐CGP62349 to localize GABAB receptors in rat and human cerebellum. Radioactively labelled oligonucleotide probes were used to localize GBR1a and GBR1b, and by dipping slides in photographic emulsion, silver grain images were obtained for quantification at the cellular level.

Binding of 0.5 nM [3H]‐CGP62349 demonstrated significantly higher binding to GABAB receptors in the molecular layer than the granule cell (GC) layer of rat cerebellum (molecular layer binding 200±11% of GC layer; P<0.0001). GBR1a mRNA expression was found to be predominantly in the GC layer (PC layer grains 6±6% of GC layer grains; P<0.05), and GBR1b expression predominantly in PCs (PC layer grains 818±14% of GC layer grains; P<0.0001).

The differential distribution of GBR1a and GBR1b mRNA splice variants for GABAB receptors suggests a possible association of GBR1a and GBR1b with pre‐ and post‐synaptic elements respectively.

British Journal of Pharmacology (1999) 126, 1387–1392; doi:10.1038/sj.bjp.0702460