bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostanoid receptors involved in the relaxation of human pulmonary vessels
Authors: Walch Laurence , Labat Carlos , Gascard Jean‐Pierre , De Montpreville Vincent , Brink Charles , Norel Xavier
Published: January 2005

To characterize the prostanoid receptors on human pulmonary smooth muscle involved in vasodilatations, isolated arteries and veins were contracted with norepinephrine (10 μM) and vessels were subsequently challenged with different prostanoid‐receptor agonists in the absence or presence of selective antagonists.

Prostaglandin D2 (PGD2) and the selective DP‐receptor agonist, BW245C, induced relaxations in the contracted human pulmonary venous preparations. The pD2 values were: 6.88±0.11 (n=17) and 7.31±0.12 (n=5), respectively. The relaxant responses induced by PGD2 were reduced by the selective DP‐receptor antagonist, BWA868C, and the estimated pA2 value was 7.84±0.16 (n=4). PGD2 and BW245C did not relax contracted human pulmonary arteries.

The selective IP‐receptor agonists, iloprost and cicaprost, both induced relaxations in the contracted human vascular preparations. The pD2 values for iloprost were: 7.84±0.08 (n=6) and 8.25±0.06 (n=4) and for cicaprost: 8.06±0.12 (n=5) and 8.11±0.09 (n=5) in arteries and veins respectively.

Prostaglandin E2 (PGE2) and the EP2/EP3‐receptor agonist, misoprostol, partially relaxed the contracted venous preparations and the pD2 values were: 8.10±0.15 (n=15) and 6.24±0.33 (n=3), respectively. These relaxations suggest the presence of an EP receptor in the human pulmonary veins. The contracted human pulmonary arteries did not relax when challenged with PGE2.

In human pulmonary venous preparations, the PGE2‐induced relaxations were neither modified by treatment with TP/EP4‐receptor antagonist, AH23848B (10 and 30 μM, n=6), nor by the DP/EP1/EP2‐receptor antagonist, AH6809 (3 μM, n=6).

These data suggest that the relaxation induced by prostanoids involved DP‐, IP‐receptors and to a lesser extent an EP‐receptor on human pulmonary venous smooth muscle. In contrast, only the IP‐receptor is involved in the prostanoid induced relaxations on human pulmonary arterial smooth muscle.

British Journal of Pharmacology (1999) 126, 859–866; doi:10.1038/sj.bjp.0702393
Characterization of airway and vascular responses in murine lungs
Authors: Held Heinz‐Dieter , Martin Christian , Uhlig Stefan
Published: January 2005

We characterized the responses of murine airways and pulmonary vessels to a variety of endogenous mediators in the isolated perfused and ventilated mouse lung (IPL) and compared them with those in precision‐cut lung slices.

Airways: The EC50 (μM) for contractions of airways in IPL/slices was methacholine (Mch), 6.1/1.5>serotonin, 0.7/2.0>U46619 (TP‐receptor agonist), 0.1/0.06>endothelin‐1, 0.1/0.05. In the IPL, maximum increase in airway resistance (RL) was 0.6, 0.4, 0.8 and 11 cmH2O s ml−1, respectively. Adenosine (1 mM), bombesin (100 μM), histamine (10 mM), LTC4 (1 μM), PAF (0.25 μM) and substance P (100 μM) had only weak effects (<5% of Mch) on RL.

Vessels: The EC50 (μM) for vasoconstriction in the IPL was LTC4, 0.06>U46619, 0.05<endothelin‐1, 0.02. The maximum increase in pulmonary artery pressure (PAP) was 11, 41 and 48 cmH2O, respectively. At 250 nM, the activity of PAF was comparable to that of LTC4. At 100 μM only, substance P caused a largely variable increase in PAP. Serotonin, adenosine, bombesin, histamine and Mch had no or only very small effects on PAP.

Hyperresponsiveness: In both the IPL and slices, U46619 in subthreshold concentrations (10 nM) reduced the EC50 to 0.6 μM. In the IPL, U46619 raised the maximum airway response to Mch 5 fold and the maximum PAF‐induced vasoconstriction 4 fold.

Conclusion: Murine precision‐cut lung slices maintain important characteristics of the whole organ. The maximum reagibility of murine airways to endogenous mediators is serotonin<Mch <U46619<ET‐1. The reagibility of the murine pulmonary vasculature is serotonin<LTC4∼PAF <U46619<ET‐1. The airway and vessel hyperreactivity induced by U46619 raises the possibility that thromboxane contributes directly to airway hyperresponsiveness in various experimental and clinical settings.

British Journal of Pharmacology (1999) 126, 1191–1199; doi:10.1038/sj.bjp.0702394
Evidence for novel caffeine and Ca2+ binding sites on the lobster skeletal ryanodine receptor
Authors: Zhang Jin Jun , Williams Alan J , Sitsapesan Rebecca
Published: January 2005

The effects of Ca2+, ATP and caffeine on the gating of lobster skeletal muscle ryanodine receptors (RyR) was investigated after reconstitution of the channels into planar phospholipid bilayers and by using [3H]‐ryanodine binding studies.

The single channel studies reveal that the EC50 (60 μM) for activation of the lobster skeletal RyR by Ca2+ as the sole ligand is higher than for any other isoform of RyR studied.

Inactivation of the channel by Ca2+ (EC50=1 mM) occurs at concentrations slightly higher than those required to inactivate mammalian skeletal RyR (RyR1) but lower than those required to inactivate mammalian cardiac RyR (RyR2).

Lifetime analysis demonstrates that cytosolic Ca2+, as the sole activating ligand, cannot fully open the lobster skeletal RyR (maximum Po approximately 0.2). The mechanism for the increase in open probability (Po) is an increase in both the frequency and the duration of the open events.

ATP is a very effective activator of the lobster RyR and can almost fully open the channel in the presence of activating cytosolic [Ca2+]. In the presence of 700 μM Ca2+, 1 mM ATP increased Po to approximately 0.8.

Caffeine, often used as a tool to identify the presence of RyR channels, is relatively ineffective and cannot increase Po above the level that can be attained with Ca2+ alone.

The results reveal that caffeine increases Po by a different mechanism to that of cytosolic Ca2+ demonstrating that the mechanism for channel activation by caffeine is not ‘sensitization’ to cytosolic Ca2+.

By studying the mechanisms involved in the activation of the lobster RyR we have demonstrated that the channel responds in a unique manner to Ca2+ and to caffeine. The results strongly indicate that these ligand binding sites on the channel are different to those on mammalian isoforms of RyR.

British Journal of Pharmacology (1999) 126, 1066–1074; doi:10.1038/sj.bjp.0702400
Activation of adenylate cyclase by human recombinant sst5 receptors expressed in CHO‐K1 cells and involvement of Gαs proteins
Authors: Carruthers Alan M , Warner Andrea J , Michel Anton D , Feniuk Wasyl , Humphrey Patrick P A
Published: January 2005

The coupling of the human somatostatin sst5 receptor recombinantly expressed in Chinese hamster ovary (CHO‐K1) cells to adenylate cyclase was investigated using receptor selective ligands.

Forskolin (10 μM)‐stimulated adenosine 3′ : 5′‐cyclic monophosphate (cyclic AMP) accumulation was inhibited by somatostatin‐14 and a number of receptor‐selective agonists with a rank order of agonist potency typical of the sst5 receptor. L‐362,855 and BIM‐23056 behaved as full agonists. At higher somatostatin‐14 concentrations there was sub‐maximal inhibition resulting in a bell‐shaped concentration‐effect relationship. Pertussis toxin (PTx; 100 ng ml−1, 18 h) pre‐treatment abolished agonist‐mediated inhibition of cyclic AMP accumulation and markedly enhanced stimulation of cyclic AMP at higher agonist concentrations.

The concentration of prostaglandin E2 (PGE2) in the incubation media was raised 14 fold by 1 μM somatostatin‐14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors.

Pre‐treatment with cholera toxin (ChTx; 20 μg ml−1, 18 h) markedly inhibited sst5 receptor‐mediated increases in cyclic AMP formation in intact cells. Somatostatin‐14‐stimulated cyclic AMP accumulation was also observed in sst5 receptor containing CHO‐K1 membranes and was inhibited by the synthetic peptide Gαsacetyl‐354‐372‐amide (100 μM) by 65.9±3.5%, implicating a Gαs protein involvement in this response.

Activation of Gαs proteins by somatostatin‐14 could be demonstrated with [35S]‐guanosine 5′‐[γ‐thio]triphosphate ([35S]‐GTPγS) binding and subsequent immunoprecipitation of 35S labelled Gαs proteins with anti‐Gαs serum.

These data show that the sst5 receptor is very efficiently coupled in a negative manner to adenylate cyclase. However, at higher agonist concentrations the receptor can also mediate activation of adenylate cyclase by a mechanism apparently involving Gαs protein activation.

British Journal of Pharmacology (1999) 126, 1221–1229; doi:10.1038/sj.bjp.0702401
Pharmacological characterization of the bradykinin B2 receptor: inter‐species variability and dissociation between binding and functional responses
Authors: Paquet J ‐L , Luccarini J ‐M , Fouchet C , Defrêne E , Loillier B , Robert C , Bélichard P , Cremers B , Pruneau D
Published: January 2005

The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non‐peptide receptor antagonists WIN 64338 (phosphonium, [[4‐[[2‐[[bis(cyclohexylamino)methylene]amino]‐3‐(2‐naphtalenyl)1‐oxopropyl]amino]‐phenyl]‐methyl]tributyl, chloride, monohydro‐chloride), and FR173657 (E)‐3‐(6‐acetamido‐3‐pyridyl)‐N‐[‐N‐[2,4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl)oxymethyl]‐phenyl]N‐methylamino carbonyl methyl] acrylamide.

[3H]‐BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO‐K1) expressing the cloned human (hB2‐CHO) or rat (rB2‐CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non‐peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer.

BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2‐CHO, rB2‐CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D‐Arg0‐[ Hyp3, Thi5, D‐Tic7, Oic8]‐BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2‐CHO, rB2‐CHO and INT407 cells, respectively.

In human isolated umbilical vein, Hoe 140, D‐Arg0‐[Hyp3, D‐Phe7, Leu8]‐BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively).

FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively).

In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter‐species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents.

British Journal of Pharmacology (1999) 126, 1083–1090; doi:10.1038/sj.bjp.0702403
Pro‐ and anti‐inflammatory actions of thrombin: a distinct role for proteinase‐activated receptor‐1 (PAR1)
Authors: Vergnolle Nathalie , Hollenberg Morley D , Wallace John L
Published: January 2005

Thrombin has well characterized pro‐inflammatory actions that have recently been suggested to occur via activation of its receptor, proteinase‐activated receptor‐1 (PAR1).

In the present study, we have compared the effects of thrombin to those of two peptides that selectively activate the PAR1 receptor, in a rat hindpaw oedema model. We have also examined whether or not thrombin can exert anti‐inflammatory activity in this model.

Both thrombin and the two PAR1 activating peptides induced significant oedema in the rat hindpaw following subplantar injection.

The oedema induced by thrombin was abolished by pre‐incubation with hirudin, and was markedly reduced in rats in which mast cells were depleted through treatment with compound 48/80 and in rats pretreated with indomethacin. In contrast, administration of the PAR1 activating peptides produced an oedema response that was not reduced by indomethacin and was only slightly reduced in rats pretreated with compound 48/80.

Co‐administration of thrombin together with a PAR1 activating receptor resulted in a significantly smaller oedema response than that seen with the PAR1 activating peptide alone. This anti‐inflammatory effect of thrombin was abolished by pre‐incubation with hirudin.

These results demonstrate that the pro‐inflammatory effects of thrombin occur through a mast‐cell dependent mechanism that is, at least in part, independent of activation of the PAR1 receptor. Moreover, thrombin is able to exert anti‐inflammatory effects that are also unrelated to the activation of PAR1.

British Journal of Pharmacology (1999) 126, 1262–1268; doi:10.1038/sj.bjp.0702408
Evidence for in vitro expression of B1 receptor in the mouse trachea and urinary bladder
Authors: Trevisani Marcello , Schmidlin Fabien , Tognetto Michele , Nijkamp Frans P , Gies Jean‐Pierre , Frossard Nelly , Amadesi Silvia , Folkerts Gert , Geppetti Pierangelo
Published: January 2005

Motor responses to des‐Arg9‐bradykinin and bradykinin were studied in the isolated mouse trachea (precontracted with carbachol, 10 μM) and the urinary bladder of either Swiss, C57Bl/6J or bradykinin B2 receptor knockout (Bk2r−/−) mice after 1–6 h in vitro. The expression of mRNA for the mouse B1 receptor in tracheal and urinary bladder tissues was also studied by using Northern blot analysis.

In isolated tracheae, des‐Arg9‐bradykinin produced a relaxant response that increased over time: no response was observed after 1 h of incubation, whereas after 6 h the maximum response (1 μM) was 68–84% of the relaxation produced by isoproterenol (1 μM) in the three mouse strains. The relaxant response to bradykinin (1 μM) observed at 1 h (38–51% of isoproterenol) was increased (62–65% of isoproterenol) after 6 h in Swiss and C57Bl/6J mice, but was absent in Bk2r−/− mice. In the presence of cycloheximide, des‐Arg9‐bradykinin did not cause any response at 6 h.

Similar findings were obtained in the urinary bladder: at 1 h des‐Arg9‐bradykinin (1 μM) did not cause any motor effect, whereas at 6 h it caused a contraction that was 28–59% of that produced by carbachol (1 μM) in the three mouse strains. Cycloheximide blocked the response to des‐Arg9‐bradykinin. Bradykinin (1 μM) contracted urinary bladders at 1 h (34–35% of carbachol), as well as at 6 h (66–77% of carbachol) in Swiss and C57Bl/6J strains, but was without effect in Bk2r−/− mice.

Northern blot hybridization with a specific cDNA probe against mouse B1 receptor mRNA using total RNA extracted from tracheae and urinary bladders freshly removed from Swiss and Bk2r−/− mice revealed minimal expression. However, marked hybridization was detected 150 min after in vitro exposure in both tissues.

Evidence is provided that in vitro exposure of mouse trachea and urinary bladder causes a time‐dependent induction of B1 receptors that cause relaxation and contraction, respectively.

British Journal of Pharmacology (1999) 126, 1293–1300; doi:10.1038/sj.bjp.0702410
Increased dopaminergic and 5‐hydroxytryptaminergic activities in male rat brain following long‐term treatment with anabolic androgenic steroids
Authors: Thiblin Ingemar , Finn Anja , Ross Svante B , Stenfors Carina
Published: January 2005

The effects of treating groups of rats with four different anabolic androgenic steroids (AAS) (testosterone, nandrolone, methandrostenolone, and oxymetholone) on 5‐hydroxytryptamine (5‐HT) and dopamine (DA) neurones in different brain regions were examined. The AAS was injected six times with 1 week's interval and the rats were sacrificed 2 days after the final injection. 5‐HT and its metabolite 5‐hydroxyindoleacetic acid (5‐HIAA), DA and its metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured. The effect on DA and 5‐HT synthesis rate was analysed as the accumulation of 3,4‐dihydroxyphenyl‐alanine (DOPA) and 5‐hydroxytryptophan (5‐HTP), respectively, after inhibition of the amino acid decarboxylase with NSD‐1015 (3‐hydroxy‐benzylhydrazine dihydrochloride). Additionally, the monoamine oxidase (MAO) activity was analysed in the hypothalamus.

The DOPAC+HVA/DA ratio was increased in the striatum in all treatment groups. However, the synthesis rate of DA was significantly increased only in the methandrostenolone treated group.

The 5‐HIAA/5‐HT ratio was increased in all treatment groups in the hippocampus, in the frontal cortex in the methandrostenolone‐treated animals and in the hypothalamus in the testosterone‐ and oxymetholone‐treated rats, while the 5‐HT synthesis rate was not affected by the AAS‐treatments.

The MAO‐A activity was increased in the oxymetholone‐treated rats while the other treatment groups were unaffected. The MAO‐B activity was not changed.

The results indicate that relatively high doses of AAS increase dopaminergic and 5‐hydroxytryptaminergic metabolism in male rat brain, probably due to enhanced turnover in these monaminergic systems.

British Journal of Pharmacology (1999) 126, 1301–1306; doi:10.1038/sj.bjp.0702412
Effects of short chain alkanols on the inducible nitric oxide synthase in a glial cell line
Authors: Syapin Peter J , Rendon Alexia , Huron David R , Militante Julius D
Published: January 2005

Ethanol inhibits inducible nitric oxide synthase (iNOS) expression in C6 glioma cells by an unknown mechanism. Because relatively high concentrations are needed for inhibition in drug‐naïve cells (IC50∼150 mM), suppression due to cytotoxicity is one possible mechanism that has not been ruled out. Therefore, the present study examined the effects of ethanol and other alkanols on C6 glioma cell viability and iNOS activity to better understand the mechanism for inhibition.

iNOS expression was induced in cell culture with lipopolysaccharide and phorbol ester treatment. Nitrite accumulation in culture medium, the in vitro conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline, and immunoblotting were used to quantify iNOS induction and activity. Trypan blue exclusion, extracellular release of lactate dehydrogenase, and quantity of total cell protein were used as measures of viability.

Short chain alkanols, methanol through 1‐heptanol, concentration‐dependently inhibited nitrite accumulation. Longer chain alkanols, 1‐octanol and 1‐decanol, did not except at cytotoxic concentrations. Experiments indicated short chain alkanol inhibition was not due to direct actions on iNOS catalytic activity, but that it transpires during iNOS induction. Immunoblots showed reduced iNOS protein levels.

Correlation analysis ruled out iNOS inhibition as being due to decreased cell number, total cell protein, or cell viability. In contrast, there was significant correlation with physical measures of lipophilicity.

In conclusion, inhibition of iNOS expression by ethanol and other short chain alkanols is not due to cytotoxicity. Instead, the strong correlation with lipophilicity suggests the inhibition derives from an interaction with unknown hydrophobic cellular sites.

British Journal of Pharmacology (1999) 126, 1253–1261; doi:10.1038/sj.bjp.0702417
Pharmacological activities of TEI‐8362, a novel inhibitor of human neutrophil elastase
Authors: Mitsuhashi Hiroaki , Nonaka Takashi , Hamamura Ichiro , Kishimoto Tadashi , Muratani Emiko , Fujii Katsuhiko
Published: January 2005

TEI‐8362, 4‐(N‐(3‐((3‐carboxypropyl)amino)‐8‐methyl‐1‐oxo‐4‐azaisochromen‐6‐yl)carbamoyl)‐4‐((phenylmethoxy)carbonylamino)butanoic acid (C26H28N4O9) is a novel inhibitor of human neutrophil elastase (HNE). We evaluated its pharmacological profile in vitro and in vivo.

TEI‐8362 demonstrated potent inhibition of HNE with a Ki value of 1.38×10−9 M. Its selectivity for HNE among a variety of proteases ranged from 163 fold to 68,000 fold in favour of HNE.

The pulmonary haemorrhage that occurred after i.t. instillation of HNE to hamsters was inhibited by either i.t., i.v., or inhalant administration of TEI‐8362.

Intratracheal administration of lipopolysaccharide induced pulmonary neutrophilia. Twenty‐four hours after lipopolysaccharide administration, the additional treatment with formyl‐methionyl‐leucyl‐phenylalanine resulted in a specific neutrophil‐dependent acute lung injury. In this model, lung injury was significantly attenuated by i.t., i.v., or inhalant administration of TEI‐8362.

These pharmacological actions of TEI‐8362 suggest that this drug has therapeutic value in the treatment of destructive lung diseases due to neutrophils.

British Journal of Pharmacology (1999) 126, 1147–1152; doi:10.1038/sj.bjp.0702425
A nitric oxide‐mediated mechanism regulates lipolysis in human adipose tissue in vivo
Authors: Andersson Kurt , Gaudiot Nicolas , Ribiere Catherine , Elizalde Montserrat , Giudicelli Yves , Arner Peter
Published: January 2005

Possible nitric oxide (NO)‐mediated effects on lipolysis were investigated in vivo in human subcutaneous adipose tissue using microdialysis, as well as in vitro on isolated fat cells of non‐obese, healthy volunteers. NO donors were added to the ingoing dialysate solvents.

Changes in lipolysis and local blood flow were investigated by measuring glycerol levels and ethanol ratios, respectively, in the microdialysates.

It was shown that the NO synthase inhibitor, NG‐monomethyl L‐arginine (L‐NMMA), but not the biologically inactive enantiomer NG‐monomethyl D‐arginine (D‐NMMA), increased glycerol levels in the microdialysates without causing a change of local blood flow. In addition, L‐NMMA increased glycerol levels in the microdialysate when local blood flow was stimulated with hydralazine.

Nitric oxide gas as well as the NO donor, nitroglycerine, reduced glycerol release from isolated adipocytes in vitro.

Expression of inducible nitric oxide synthase (iNOS) in human adipose tissue was shown by Western blot analysis. Biologically active NOS was demonstrated by measuring total enzymatic activity.

In conclusion, the data demonstrate that inhibition of NO release in subcutaneous adipose tissue results in an increased lipolysis in vivo. These effects, which were also observed in vitro, are independent of local blood flow changes. Furthermore, the demonstration of enzymatic NOS activity and the expression of inducible nitric oxide synthase (iNOS) in adipose tissue indicate that locally synthesized NO may play a role in the physiological control of lipolysis in human adipose tissue.

British Journal of Pharmacology (1999) 126, 1639–1645; doi:10.1038/sj.bjp.0702430
Prostaglandin endoperoxide‐dependent vasospasm in bovine coronary arteries after nitration of prostacyclin synthase
Authors: Zou Minghui , Jendral Michelle , Ullrich Volker
Published: January 2005

In the present study we used a bioassay to study the effects of peroxynitrite (ONOO−) on angiotensin II (A‐II)‐triggered tension in isolated bovine coronary arteries in order to show the consequences of the previously reported PGI2‐synthase inhibition by ONOO− in this model. The following results were obtained:

1 μmol L−1 ONOO− impaired A‐II‐induced vasorelaxation and caused a second long lasting constriction phase. Indomethacin (10−5M) prevented both effects. U51605, a dual blocker of PGI2‐synthase and thromboxane (TX)A2‐synthase mimicked the effects of ONOO−.

The selective TXA2/prostaglandin endoperoxide (PGH2) receptor antagonist SQ29548 antagonized the second vasoconstriction phase after ONOO−‐treatment. Since a generation of TXA2 and 8‐iso‐prostaglandin F2α could be excluded a direct action of unmetabolized PGH2 on the TXA2/PGH2 receptor was postulated.

ONOO− dose‐dependently inhibited the conversion of 14C‐PGH2 into 6‐keto‐PGF1α in isolated bovine coronary arteries with an IC50‐value of 100 nM.

Immunoprecipitation of 3‐nitrotyrosine‐containing proteins with a monoclonal antibody revealed PGI2‐synthase as the only nitrated protein in bovine coronary arteries treated with 1 μmol  l−1 ONOO−.

Using immunohistochemistry a co‐localization of PGI2‐synthase and nitrotyrosine‐containing proteins was clearly visible in both endothelial and vascular smooth muscle cells. We concluded that ONOO− not only eliminated the vasodilatory, growth‐inhibiting, antithrombotic and antiadhesive effects of PGI2 but also allowed and promoted an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2.

British Journal of Pharmacology (1999) 126, 1283–1292; doi:10.1038/sj.bjp.0702434
Mechanisms of melatonin‐induced vasoconstriction in the rat tail artery: a paradigm of weak vasoconstriction
Authors: Lew Michael J , Flanders Samantha
Published: January 2005

Vasoconstrictor effects of melatonin were examined in isolated rat tail arteries mounted either in an isometric myograph or as cannulated pressurized segments. Melatonin failed by itself to mediate observable responses but preactivation of the arteries with vasopressin (AVP) reliably uncovered vasoconstriction responses to melatonin with maxima about 50% of maximum contraction. Further experiments were conducted with AVP preactivation to 5–10% of the maximum contraction.

Responses to melatonin consisted of steady contractions with superimposed oscillations which were large and irregular in isometric but small in isobaric preparations. Nifedipine (0.3 μM) reduced the responses and abolished the oscillations. Charybdotoxin (30 nM) increased the magnitude of the oscillations with no change in the maximum response.

Forskolin (0.6 μM) pretreatment increased the responses to melatonin compared to control and sodium nitroprusside (1 μM) treated tissues. The AVP concentration required for preactivation was 10 fold higher than control in both the forskolin and nitroprusside treated groups.

In isometrically‐mounted arteries treated with nifedipine, melatonin receptor agonists had the potency order 2‐iodomelatonin>melatonin>S20098>GR196429, and the MT2‐selective antagonist luzindole antagonized the effects of melatonin with a low pKB of 6.1±0.1.

It is concluded that melatonin elicits contraction of the rat tail artery via an mt1 or mt1‐like receptor that couples via inhibition of adenylate cyclase and opening of L‐type calcium channels. Calcium channels and charybdotoxin‐sensitive K+ channels may be recruited into the responses via myogenic activation rather than being coupled directly to the melatonin receptors.

It is proposed that the requirement of preactivation for overt vasoconstrictor responses to melatonin results from the low effector reserve of the melatonin receptors together with the tail artery having threshold inertia. Potentiative interactions between melatonin and other vasoconstrictor stimuli probably also result from the threshold inertia. A simple model is presented and a general framework for consideration of interactions between weak vasoconstrictor agonists and other vasoconstrictor stimuli is discussed.

British Journal of Pharmacology (1999) 126, 1408–1418; doi:10.1038/sj.bjp.0702435
Characterization of receptors for calcitonin gene‐related peptide and adrenomedullin on the guinea‐pig vas deferens
Authors: Poyner David R , Taylor Gillian M , Tomlinson A Elaine , Richardson Alan G , Smith David M
Published: January 2005

The receptors which mediate the effects of calcitonin gene‐related peptide (CGRP), amylin and adrenomedullin on the guinea‐pig vas deferens have been investigated.

All three peptides cause concentration dependant inhibitions of the electrically stimulated twitch response (pD2s for CGRP, amylin and adrenomedullin of 7.90±0.11, 7.70±0.19 and 7.25±0.10 respectively).

CGRP8–37 (1 μM) and AC187 (10 μM) showed little antagonist activity against adrenomedullin.

Adrenomedullin22–52 by itself inhibited the electrically stimulated contractions of the vas deferens and also antagonized the responses to CGRP, amylin and adrenomedullin.

[125I]‐adrenomedullin labelled a single population of binding sites in vas deferens membranes with a pIC50 of 8.91 and a capacity of 643  fmol  mg−1. Its selectivity profile was adrenomedullin> AC187>CGRP=amylin. It was clearly distinct from a site labelled by [125I]‐CGRP (pIC50=8.73, capacity=114  fmol  mg−1, selectivity CGRP>amylin=AC187>adrenomedullin). [125I]‐amylin bound to two sites with a total capacity of 882 fmol mg−1.

Although CGRP has been shown to act at a CGRP2 receptor on the vas deferens with low sensitivity to CGRP8–37, this antagonist displaced [125I]‐CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4°C to 25°C, suggesting the anomalous behaviour of CGRP8–37 is not due to temperature differences between binding and functional assays.

British Journal of Pharmacology (1999) 126, 1276–1282; doi:10.1038/sj.bjp.0702437
A non‐pungent triprenyl phenol of fungal origin, scutigeral, stimulates rat dorsal root ganglion neurons via interaction at vanilloid receptors
Authors: Szallasi A , Bíró T , Szabó T , Modarres S , Petersen M , Klusch A , Blumberg P M , Krause J E , Sterner O
Published: January 2005

A [3H]‐resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl‐scutigeral, ovinal, neogrifolin, and methyl‐neogrifolin), scutigeral (Ki=19 μM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization.

Scutigeral induced a dose‐dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 μM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 μM).

[3H]‐RTX binding isotherms were shifted by scutigeral (10–80 μM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 μM.

Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 μM) upon intraocular instillation.

In accord with being non‐pungent, scutigeral (5 μM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage‐clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 μM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin.

In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non‐pungent vanilloids.

British Journal of Pharmacology (1999) 126, 1351–1358; doi:10.1038/sj.bjp.0702440
Characterization of the electrophysiological and pharmacological effects of 4‐iodo‐2,6‐diisopropylphenol, a propofol analogue devoid of sedative‐anaesthetic properties
Authors: Sanna Enrico , Motzo Costantino , Usala Marcello , Serra Mariangela , Dazzi Laura , Maciocco Elisabetta , Trapani Giuseppe , Latrofa Andrea , Liso Gaetano , Biggio Giovanni
Published: January 2005

Several derivatives and analogues of the general anaesthetic 2,6‐diisopropylphenol (propofol) have been recently synthesised with the aim of exploring the structure‐activity relationships.

In the present study, the effects of one such compound, 4‐iodo‐2,6‐diisopropylphenol (4‐I‐Pro), on γ‐aminobutyric acid type A (GABAA) receptors in vitro were compared with its in vivo effects in rodents. Human GABAA receptors were expressed in Xenopus oocytes, and the actions of 4‐I‐Pro on receptor function were compared with those of propofol by two‐electrode voltage‐clamp recording.

Similar to propofol, 4‐I‐Pro directly activated Cl− currents in the absence of GABA at all combinations of receptor subunits tested. However, the efficacy of 4‐I‐Pro in inducing direct activation of α1β2γ2S receptors was markedly less than that of propofol.

Similarly to propofol, 4‐I‐Pro potentiated in a concentration‐dependent manner GABA‐evoked Cl− currents measured at different GABAA receptor constructs.

As expected, intraperitoneal injection of propofol induced sedation, ataxia, and loss of the righting reflex in rats. In contrast, administration of 4‐I‐Pro failed to produce any of these behavioural effects.

Administration of 4‐I‐Pro to rats reduced in a dose‐dependent manner the incidence of tonic‐clonic seizures induced by pentylenetetrazol and induced an anticonflict effect as measured in the Vogel test.

Microdialysis revealed that, like propofol, administration of 4‐I‐Pro reduced acetylcholine release in the hippocampus of freely moving rats.

These results demonstrate that para‐substitution of the phenol ring of propofol with iodine yields a compound that exhibits anticonvulsant and anticonflict effects, but is devoid of sedative‐hypnotic and anaesthetic properties. Thus, 4‐I‐Pro possesses pharmacological characteristics more similar to anxiolytic and anticonvulsant drugs than to general anaesthetics.

British Journal of Pharmacology (1999) 126, 1444–1454; doi:10.1038/sj.bjp.0702449
Parallel pathways mediate inhibitory effects of vasoactive intestinal polypeptide and nitric oxide in canine fundus
Authors: Bayguinov Orline , Keef Kathleen D , Hagen Brian , Sanders Kenton M
Published: January 2005

The gastric adaptation reflex is activated by the release of non‐adrenergic, non‐cholinergic (NANC) inhibitory transmitters, including nitric oxide (NO) and vasoactive intestinal polypeptide (VIP). The role of NO in this reflex is not disputed, but some investigators suggest that NO synthesis is stimulated by VIP in post‐junctional cells or in nerve terminals. We investigated whether the effects of these transmitters are mediated by independent pathways in the canine gastric fundus.

VIP and NO produced concentration‐dependent relaxation of the canine fundus. Nω‐nitro‐L‐arginine (L‐NNA) reduced relaxation induced by electrical field stimulation (EFS; 0.5–8 Hz), but had no effect on responses to exogenous VIP and sodium nitroprusside (SNP, 10 μM).

Oxyhaemoglobin reduced relaxations produced by EFS and SNP. Oxyhaemoglobin also reduced relaxation responses to low concentrations of VIP (<10 nM), but these effects were non‐specific and mimicked by methaemoglobin which had no effect on nitrergic responses.

A blocker of guanylyl cyclase, 1H‐[1,2,4]oxidiazolo [4,3,‐a]quinoxalin‐1‐one, (ODQ) inhibited responses to EFS, SNP and DETA/NONOate (an NO·donor), but had no effect on responses to VIP. cis‐N‐(2‐phenylcyclopentil)‐azacyclotridec‐1en‐2‐amine monohydrochloride (MDL 12,330A), a blocker of adenylyl cyclase, reduced responses to EFS, VIP and forskolin, but did not affect responses to SNP.

Levels of cyclic GMP were enhanced by the NO donor S‐nitroso‐n‐acetylpenicillamine (SNAP) but were unaffected by VIP (1 μM). The increase in cyclic GMP in response to SNAP was blocked by ODQ.

The results suggest that at least two transmitters, possibly NO and VIP, mediate relaxation responses in the canine fundus. NO and VIP mediate responses via cyclic GMP‐ and cyclic AMP‐dependent mechanisms, respectively. No evidence was found for a serial cascade in which VIP is coupled to NO‐dependent responses.

British Journal of Pharmacology (1999) 126, 1543–1552; doi:10.1038/sj.bjp.0702450
μ‐opioid receptor modulation of calcium channel current in periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR‐1
Authors: Connor Mark , Schuller Alwin , Pintar John E , Christie MacDonald J
Published: January 2005

The actions of opioid receptor agonists on the calcium channel currents (IBa) of acutely dissociated periaqueductal grey (PAG) neurons from C57B16/J mice and mutant mice lacking the first exon of the μ‐opioid receptor (MOR‐1) were examined using whole cell patch clamp techniques. These effects were compared with the GABAB‐receptor agonist baclofen.

The endogenous opioid agonist methionine‐enkephalin (met‐enkephalin, pEC50 6.8, maximum inhibition 40%), the putative endogenous μ‐opioid agonist endomorphin‐1 (pEC50 6.2, maximum inhibition 35%) and the μ‐opioid selective agonist DAMGO (Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol enkephalin, pEC50 6.9, maximum inhibition 40%) inhibited IBa in 70% of mouse PAG neurons. The inhibition of IBa by each agonist was completely prevented by the μ‐receptor antagonist CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2). The δ‐opioid receptor agonists DPDPE ([D‐Pen2,5]‐enkephalin, 1 μM) and deltorphin II (1 μM), and the κ‐opioid receptor agonist U‐69593 (1–10 μM), did not affect IBa in any cell tested.

The GABAB agonist baclofen inhibited IBa in all neurons (pEC50 5.9, maximum inhibition 42%).

In neurons from the MOR‐1 deficient mice, the μ‐opioid agonists met‐enkephalin, DAMGO and endomorphin‐1 did not inhibit IBa, whilst baclofen inhibited IBa in a manner indistinguishable from wild type mice.

A maximally effective concentration of endomorphin‐1 (30 μM) partially (19%), but significantly (P<0.005), occluded the inhibition of IBa normally elicited by a maximally effective concentration of met‐enkephalin (10 μM).

This study indicates that μ‐opioid receptors, but not δ‐ or κ‐opioid receptors, modulate somatic calcium channel currents in mouse PAG neurons. The putative endogenous μ‐agonist, endomorphin‐1, was a partial agonist in mouse PAG neurons.

British Journal of Pharmacology (1999) 126, 1553–1558; doi:10.1038/sj.bjp.0702457
Modulation of the gating of ClC‐1 by S‐(–) 2‐(4‐chlorophenoxy) propionic acid
Authors: Aromataris E C , Astill D St J , Rychkov G Y , Bryant S H , Bretag A H , Roberts M L
Published: January 2005

Using whole‐cell patch‐clamping and Sf‐9 cells expressing the rat skeletal muscle chloride channel, rClC‐1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined.

RS‐(±) 2‐(4‐chlorophenoxy)propionic acid (RS‐(±) CPP) and its S‐(−) enantiomer produced pronounced effects on ClC‐1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration‐dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S‐(−) enantiomer respectively. R‐(+) CPP at similar concentrations had no effect on gating. RS‐(±) CPP did not block the passage of Cl− through the pore of rClC‐1.

ClC‐1 is gated by Cl− binding to a site within an access channel and S‐(−) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl−. Comparison of the EC50 for RS‐(±) CPP and S‐(−) CPP indicates that R‐(+) CPP can compete with the S‐(−) enantiomer for the site but that it is without biological activity.

RS‐(±) CPP produced the same effect on rClC‐1 gating when added to the interior of the cell and in the extracellular solution.

S‐(−) CPP modulates the gating of ClC‐1 to decrease the membrane Cl− conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.

British Journal of Pharmacology (1999) 126, 1375–1382; doi:10.1038/sj.bjp.0702459
Modulation of peroxynitrite‐ and hypochlorous acid‐induced inactivation of α1‐antiproteinase by mercaptoethylguanidine
Authors: Whiteman Matthew , Szabó Csaba , Halliwell Barry
Published: January 2005

Peroxynitrite is a cytotoxic species that can be formed, among other mechanisms, by the rapid reaction of superoxide with nitric oxide. Peroxynitrite formation has been implicated in a wide range of neurodegenerative and chronic inflammatory diseases, as has the formation of hypochlorous acid by myeloperoxidase.

There is considerable interest in the development of peroxynitrite scavengers as therapeutic agents. The thiol compound mercaptoethylguanidine has been suggested to fulfil this role since it has recently been shown to be not only a potent inhibitor of inducible nitric oxide synthase but also a scavenger of peroxynitrite. Indeed, it has been shown to be protective in some experimental models of circulatory shock and inflammation at plasma levels in the approximate range 100–300 μM.

One protein inactivated by peroxynitrite is the major inhibitor of serine proteinases in human body fluids, α1‐antiproteinase. At high (250–1000 μM) concentrations, mercaptoethylguanidine was found to be effective in preventing peroxynitrite‐mediated tyrosine nitration and α1‐AP inactivation.

By contrast, lower concentrations of mercaptoethylguanidine (1–60 μM) enhanced the inactivation of α1‐antiproteinase by peroxynitrite.

At all concentrations tested (1–1000 μM), mercaptoethylguanidine decreased the inactivation of α1‐antiproteinase by hypochlorous acid.

We suggest that products of reaction of mercaptoethylguanidine with peroxynitrite or peroxynitrite‐derived products could cause damage to α1‐antiproteinase, and possibly other proteins in vivo, whereas scavenging of hypochlorous acid by mercaptoethylguanidine could contribute to its anti‐inflammatory action in vivo.

British Journal of Pharmacology (1999) 126, 1646–1652; doi:10.1038/sj.bjp.0702465
Effect of potassium channel modulators in mouse forced swimming test
Authors: Galeotti Nicoletta , Ghelardini Carla , Caldari Bernardetta , Bartolini Alessandro
Published: January 2005

The effect of intracerebroventricular (i.c.v.) administration of different potassium channel blockers (tetraethylammonium, apamin, charybdotoxin, gliquidone), potassium channel openers (pinacidil, minoxidil, cromakalim) and aODN to mKv1.1 on immobility time was evaluated in the mouse forced swimming test, an animal model of depression.

Tetraethylammonium (TEA; 5 μg per mouse i.c.v.), apamin (3 ng per mouse i.c.v.), charybdotoxin (1 μg per mouse i.c.v.) and gliquidone (6 μg per mouse i.c.v.) administered 20 min before the test produced anti‐immobility comparable to that induced by the tricyclic antidepressants amitriptyline (15 mg kg−1 s.c.) and imipramine (30 mg kg−1 s.c.).

By contrast pinacidil (10–20 μg per mouse i.c.v.), minoxidil (10–20 μg per mouse i.c.v.) and cromakalim (20–30 μg per mouse i.c.v.) increased immobility time when administered in the same experimental conditions.

Repeated administration of an antisense oligonucleotide (aODN) to the mKv1.1 gene (1 and 3 nmol per single i.c.v. injection) produced a dose‐dependent increase in immobility time of mice 72 h after the last injection. At day 7, the increasing effect produced by aODN disappeared. A degenerate mKv1.1 oligonucleotide (dODN), used as control, did not produce any effect in comparison with saline‐ and vector‐treated mice.

At the highest effective dose, potassium channels modulators and the mKv1.1 aODN did not impair motor coordination, as revealed by the rota rod test, nor did they modify spontaneous motility as revealed by the Animex apparatus.

These results suggest that modulation of potassium channels plays an important role in the regulation of immobility time in the mouse forced swimming test.

British Journal of Pharmacology (1999) 126, 1653–1659; doi:10.1038/sj.bjp.0702467
Functional evidence of α1D‐adrenoceptors in the vasculature of young and adult spontaneously hypertensive rats
Authors: Villalobos‐Molina Rafael , López‐Guerrero J Javier , Ibarra Maximiliano
Published: January 2005

The role of α1D‐adrenoceptors in the vasculature of spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY), of different ages was assessed in pithed rats by the use of the selective α1D‐adrenoceptor antagonist BMY 7378 (8‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]‐ethyl]‐8‐azaspiro [4.5]decane‐7,9‐dione dihydrochloride). BMY 7378 displaced the pressor effect of phenylephrine in young pre‐hypertensive pithed SHR rats, but produced no effect in young WKY rats (dose ratio of 3.4 and 1.6, respectively), while in adult rats BMY 7378 produced a greater shift in the phenylephrine response curve than in younger animals (dose ratio of 3.2 and 6.2 in WKY and SHR, respectively). The presence of α1D‐adrenoceptors in the vasculature of pre‐hypertensive rats, suggests its role in the pathogenesis/maintenance of increased blood pressure.

British Journal of Pharmacology (1999) 126, 1534–1536; doi:10.1038/sj.bjp.0702468
P2Y receptor‐mediated Ca2+signalling in cultured rat aortic smooth muscle cells
Authors: Pediani J D , McGrath J C , Wilson S M
Published: January 2005

ATP, UTP, ADP and ADP‐β‐S elicited Ca2+‐signals in cultured aortic smooth muscle cells although ADP, UDP and ADP‐β‐S gave ∼40% of the maximal response seen with ATP and UTP. Adenosine, AMP or α,β‐methylene‐ATP had no effect. These responses were attributed to P2Y2/4and P2Y1receptors, which we assumed could be selectively activated by UTP and ADP‐β‐S respectively.

The response to UTP was reduced (∼50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP‐β‐S. This suggests P2Y2/4receptors simultaneously couple to pertussis toxin‐sensitive and ‐resistant G proteins whilst P2Y1receptors couple to only the toxin‐resistant proteins.

Repeated stimulation with UTP or ADP‐β‐S caused desensitization which was potentiated by 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) and attenuated by staurosporine.

TPA completely abolished sensitivity to ADP‐β‐S but the response to UTP had a TPA‐resistant component. In pertussis toxin‐treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA‐resistant part of this response seems to be mediated by pertussis toxin‐sensitive G proteins.

Loss of sensitivity to UTP did not occur when pertussis toxin‐treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin‐sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP‐β‐S.

British Journal of Pharmacology(1999)126, 1660–1666; doi:10.1038/sj.bjp.0702470