bluuebunny/crossref_metadata_2025 huggingface dataset:
Strain‐dependency of leukotriene C4 generation from isolated lungs of immunized mice
Authors: Zuany‐Amorim Claudia , Vargaftig B. Boris , Maclouf Jacques , Pretolani Marina
Published: July 2012

The antigen‐induced leukotriene C4 (LTC4)‐like‐material release from isolated perfused lungs of actively sensitized Swiss, Balb/C and CBA/J mice was compared. The intra‐tracheal (i.t.) instillation of 1 and 100 μg ovalbumin to lungs from Swiss mice was followed by a dose‐dependent generation of LTC4‐like material into the effluent, as detected by radio‐immunoassay and h.p.l.c., followed by an enzyme‐immunoassay. In contrast, lungs from sensitized Balb/C and CBA/J mice failed to exhibit LTC4‐like‐material release in amounts above the basal values. No histamine secretion was observed when lungs of the three strains of mice were challenged with ovalbumin.

The i.t. instillation of 1 or 10 μg platelet‐activating factor (PAF) or of 100 μg arachidonic acid to lungs from non‐sensitized mice, induced the release of comparable amounts of LTC4‐like‐material in the effluent, irrespective to the strain. However, N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine (fMLP, 0.1, 10 μg), was more effective in inducing the release of LTC4‐like‐material from lung from Swiss and CBA, than from Balb/C, mice.

The intraplantar injection of 0.01 μg ovalbumin to sensitized Swiss mice induced an intense oedema formation, as measured plethysmographically, while Balb/C mice required a dose of antigen at least 10 fold higher for a similar response. CBA/J mice did not respond to antigen challenge in terms of oedema formation. The intraplantar injection of PAF or fMLP to non‐immunized mice induced an oedema of similar intensity in all the strains considered. Accordingly, the different responses to ovalbumin of the three strains of mice is not accounted for by different paw responsiveness to inflammatory mediators.

Swiss and CBA/J mice exhibited higher titers of circulating IgG antibodies, as measured by passive cutaneous anaphylaxis (PCA), than Balb/C mice. Conversely, lower IgE titers were measured in the serum of sensitized Swiss and CBA/J mice, as compared to Balb/C.

Our results demonstrate a strain‐dependency of antigen‐induced LTC4 release from lungs from sensitized mice. This difference is related to the ability of sensitized animals to develop immediate hypersensitivity responses, such as paw oedema formation, but not to the antibody subclass involved in the immunization. Strain‐dependent factors may influence the intensity of the response to antigen stimulation. It is thus essential to characterize the different components of the immune response when mouse models for studying hypersensitivity reactions are developed.
Separation and characterization of a novel isoenzyme of cyclic nucleotide phosphodiesterase from rat cerebrum
Authors: Mukai Jun , Asai Tomoko , Naka Michiko , Tanaka Toshio
Published: July 2012

Anion‐exchange chromatography on a Mono‐Q column of the supernatant fraction, after ultracentrifugation, from a homogenate of rat cerebrum, prepared under isotonic conditions in the presence of protease inhibitors, yielded a novel isoenzyme of cyclic nucleotide phosphodiesterase (PDE) with properties unlike those of known PDEs. The isoenzyme was insensitive to stimulation by Ca2+/calmodulin and cyclic GMP, and it hydrolyzed both cyclic AMP and cyclic GMP with KM values of 0.109 ± 0.008 μm and 1.78 ± 0.04 μm, respectively. The ratio of Vmax of hydrolysis of cyclic GMP to that of cyclic AMP was 1.90 ± 0.07. Nicardipine (PDE I inhibitor), SK&F 94120 (PDE III inhibitor), rolipram (PDE IV inhibitor) and zaprinast (PDE V inhibitor) had very weak inhibitory effects on the PDE activity of the isoenzyme. These results suggest that the isoenzyme is a novel and previously unreported species of PDE, which we tentatively designate PDE VIII.
AT1 receptor‐mediated stimulation by angiotensin II of rat aortic fibronectin gene expression in vivo
Authors: Kim Shokei , Ohta Kensuke , Hamaguchi Akinori , Omura Takashi , Tominaga Kazunari , Yukimura Tokihito , Miura Katsuyuki , Tanaka Motoharu , Iwao Hiroshi
Published: July 2012

Fibronectin plays an important role in various vascular diseases. A subpressor (200 ng kg−I min−1) or pressor (1000 ng kg−1 min−1) dose of angiotensin II was continuously infused into rats by osmotic minipump for various times, to investigate the effects on aortic fibronectin gene expression. In rats infused with a subpressor dose of angiotensin II in which blood pressure was normal for 3 days, aortic fibronectin mRNA levels started to increase by 1.4 fold at 12 h and reached the maximal levels (increased by 3.1 fold) at 3 days. Treatment with TCV‐116 (3 mg kg−1 day−1), a non‐peptide selective AT1 receptor antagonist, completely inhibited the angiotensin II‐induced increase in aortic fibronectin mRNA, while hydralazine (10 mg kg−1 day−1) did not block this effect. Similar results were also obtained for a pressor dose of angiotensin II. Thus, angiotensin II directly stimulates aortic fibronectin gene expression in vivo, which is mediated by the AT1 receptor but not by blood pressure.
Effects of cyclopiazonic acid on contractility and ecto‐ATPase activity in guinea‐pig urinary bladder and vas deferens
Authors: Ziganshin Airat U. , Hoyle Charles H.V. , Ziganshina Lilia E. , Burnstock Geoffrey
Published: July 2012

Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic ATPase, was tested on guinea‐pig urinary bladder and vas deferens for its ability: (1) to modify contractile responses to electrical field stimulation (EFS), exogenous ATP, α,β‐methylene ATP (α,β‐meATP), carbachol, noradrenaline (NA), histamine, and KCl; (2) to affect ecto‐ATPase activity; (3) to modify the release of ATP evoked by EFS.

In the urinary bladder, CPA (10 μm) potentiated contractile responses to EFS, exogenous ATP (100 μm), α,β‐meATP (1 μm), carbachol (0.5 μm), histamine (30 μm) and KCl (30 μm). In the vas deferens, CPA (10 μm) potentiated responses to EFS, ATP, α,β‐meATP, NA (100 μm) and KCl. CPA at a concentration of 1 μm had no effect on ATP‐induced relaxation of carbachol‐precontracted guinea‐pig taenia coli, and at a concentration of 10 μm it markedly increased spontaneous contractile activity of taenia.

Ecto‐ATPase was estimated to have Vmax and Km values of 0.98 nmol Pi 30 min−1 mg−1 wet tissue and 881 μm ATP in the urinary bladder, and 0.75 nmol Pi 30 min−1 mg−1 wet tissue and 914 μm ATP in the vas deferens, respectively. CPA at a concentration of 10 μm significantly inhibited ecto‐ATPase activity by 18% in the urinary bladder and by 24% in the vas deferens.

In the guinea‐pig vas deferens, CPA significantly potentiated ATP release evoked by EFS from 2.2 ± 0.8 (6) pmol ATP min−1 g−1 wet tissue to 35.2 ± 4.8 (6) pmol ATP min−1 g−1 wet tissue (P < 0.01).

In conclusion, the potentiation of contractile responses of the guinea‐pig urinary bladder and vas deferens by CPA has a non‐specific character. CPA inhibited ecto‐ATPase activity and increased ATP release, but these effects do not appear to contribute to the potentiation of P2x‐purinoceptor‐mediated responses since the contractile actions of all the agonists studied were potentiated to the same extent.
Modulation by endogenous prostanoids of the vasoconstrictor activity of endothelin‐1 in the canine isolated, perfused spleen
Authors: Grassi‐Kassisse Dora M. , Antunes Edson , Withrington Peter G. , de Nucci Gilberto
Published: July 2012

Endothelin‐1 (ET‐1, 0.4–200 pmol) was injected into the arterial circuit of the isolated perfused spleen of the dog in which splenic arterial perfusion pressure and spleen weight were recorded continuously.

Serial collection was made of splenic venous effluent before and after intra‐arterial injection of ET‐1 and assayed by direct radioimmunoassay for prostaglandin E2 (PGE2), 6‐oxo‐PGF1α and thromboxane B2 (TXB2).

ET‐1 caused graded arterial vasoconstriction of prolonged duration with small reductions in spleen weight at higher doses.

ET‐1 cause a dose‐related release of PGE2, 6‐oxo‐PGF1α and TXB2 into the splenic venous effluent. The mean peak increase above the basal levels following 200 pmol of ET‐1 was 800% for PGE2, 233% for 6‐oxo‐PGF1α and 205% for TXB2.

Intra‐arterial infusion of indomethacin significantly reduced the basal release of all three eicosanoids and significantly elevated the basal splenic vascular resistance. The release of all three eicosanoids in response to ET‐1 and adrenaline (Ad) was significantly reduced by indomethacin and the accompanying increases in the splenic arterial vascular resistance were significantly potentiated at low doses of ET‐1. The splenic arterial vascular responses to Ad were unchanged by indomethacin infusion.

These results indicate that the release of eicosanoids may modulate the splenic vascular responses to ET‐1.
The interaction between salmeterol and β2‐adrenoceptor agonists with higher efficacy on guinea‐pig trachea and human bronchus in vitro
Authors: Källström Britt‐Louise , Sjöberg Jacob , Waldeck Bertil
Published: July 2012

In guinea‐pig tracheal preparations precontracted with 1 μmol 1−1 carbachol, formoterol, procaterol, fenoterol, salmefamol, salbutamol and terbutaline (in that order of potency) caused a concentration‐dependent and almost complete, relaxation. However, under these conditions, the maximum relaxation by salmeterol was approximately 30% of the maximum attainable relaxation.

We have therefore explored the ability of salmeterol to inhibit the relaxant response to β2‐adrenoceptor agonists of different chemical structure and relatively higher efficacy in smooth muscle preparations from guinea‐pig trachea and human bronchus.

With 1 μmol 1−1 salmeterol in the organ bath, the concentration‐effect curves for the other agonists were shifted to the right in a variable way by 1.8‐2.8 log units, fenoterol and salbutamol being the extremes.

When 20 μmol 1−1 sulfonterol, another low efficacy β2‐adrenoceptor agonist, was substituted for salmeterol, the difference in the magnitude of the rightward shift between fenoterol and salbutamol was eliminated.

In the human bronchus, formoterol and terbutaline had a higher apparent efficacy than salmeterol. With 1 μmol 1−1 salmeterol in the organ bath, the concentration‐effect curve for formoterol was shifted 2.7 log units to the right.

Salmeterol inhibits, competitively, relaxant responses to β2‐adrenoceptor agonists with higher efficacy. The degree of inhibition seems to be dependent on the agonist used. This contrasts with results obtained with sulfonterol and suggests that salmeterol interacts with the β2‐adrenoceptor in a complex way.
Antagonistic properties of McNeil‐A‐343 at 5‐HT4 and 5‐HT3 receptors
Authors: Sagrada A. , Schiavi G.B. , Cereda E. , Ladinsky H.
Published: July 2012

This study describes the in vitro interaction of the muscarinic ligand McNeil‐A‐343 with two 5‐hydroxytryptamine (5‐HT) receptor subtypes, the 5‐HT4 and 5‐HT3 receptors, using functional as well as radioligand binding studies.

In the rat oesophageal muscularis mucosae, precontracted with carbachol, McNeil‐A‐343 was a competitive antagonist (pA2 6.2) of the 5‐HT4 receptor which mediates the relaxation induced by 5‐HT. The compound per se relaxed the oesophagus at high concentration only (≥ 10 μm), an effect unchanged by desensitization of the 5‐HT4 receptor with 10 μm 5‐methoxytryptamine. In the same preparation in the absence of tone, McNeil‐A‐343 displaced the carbachol concentration‐response curve to the right, yielding an apparent affinity (pA2) of 4.9 for muscarinic receptors.

In the rat isolated superior cervical ganglion preparation, after blockade of muscarinic and nicotinic receptors, McNeil‐A‐343 caused a concentration‐dependent depolarization that was unaffected by 100 nm ondansetron. The concentration‐fast depolarization curve to 5‐HT, mediated by the 5‐HT3 receptor, was displaced to the right by McNeil‐A‐343, which showed an apparent affinity (pA2) of 4.8 for the 5‐HT3 subtype.

In binding studies, McNeil‐A‐343 recognized a single population of 5‐HT4 receptors in pig caudate nucleus, with a pAi of 5.9. The binding affinity of McNeil‐A‐343 for 5‐HT3 receptors in NG 108‐15 cells was approximately four times lower (pK1 5.3). Binding affinities (pK1) for muscarinic receptor subtypes in rat tissues were 5.3 (Mb cortex), 5.2 (M2, heart) and 4.9 (M3, submandibular glands), respectively.

McNeil‐A‐343 is an antagonist at 5‐HT4 and 5‐HT3 receptors; the interaction of the compound with these receptor subtypes (notably the 5‐HT4) occurs in a range of concentrations which generally overlaps that relevant to the interaction with muscarinic receptors.
Pharmacological evidence of distinct oci‐adrenoceptor subtypes mediating the contraction of human prostatic urethra and peripheral artery
Authors: Hatano Akihiko , Takahashi Hitoshi , Tamaki Makoto , Komeyama Takeshi , Koizumi Takako , Takeda Masayuki
Published: July 2012

The α1‐adrenoceptor subtypes mediating contractions of the smooth muscle in human prostatic urethra and branches of internal iliac artery were characterized in isometric contraction experiments.

Phenylephrine produced concentration‐dependent contractions in both the urethra and artery. These responses were competitively inhibited by prazosin, WB4101 and 5‐methyl‐urapidil, and the slopes of Schild plots for all these antagonists were close to unity.

The pA2 values for prazosin were not significantly different between the urethra (9.42 ± 0.11; mean ± s.d.) and artery (9.50 ± 0.27), while the pA2 values for WB4101 and 5‐methyl‐urapidil in the human prostatic urethra (8.94 ± 0.19 and 8.42 ± 0.14, respectively) were significantly greater than in the branches of human internal iliac artery (7.94 ± 0.21 and 7.43 ± 0.22, respectively; P < 0.01).

Pretreatment with chlorethylclonidine (CEC) at concentrations ranging from 0.1 μm to 100 μm attenuated the maximum contraction to phenylephrine in a concentration‐dependent manner in both the urethra and artery. However, the urethra was significantly less affected by CEC than the artery. The pD'2 values (negative logarithm of the molar concentration of antagonist which reduced the maximum contraction to one half) in the urethra and artery were 4.35 ± 0.27 and 5.20 ± 0.37, respectively (P < 0.01).

The present results indicate that there are distinct populations of α1‐adrenoceptor subtypes in the human prostatic urethra and branches of the internal iliac artery. The α1‐adrenoceptors responsible for the contraction of the human internal iliac artery branches are predominantly α1B‐subtype, whereas those in the human prostatic urethra are considered to be not α1B, but α1C or possibly α1A or α1A/D‐subtype.
The anticonvulsant and behavioural profile of L‐687,414, a partial agonist acting at the glycine modulatory site on the N‐methyl‐D‐aspartate (NMDA) receptor complex
Authors: Tricklebank M.D. , Bristow L.J. , Hutson P.H. , Leeson P.D. , Rowley M. , Saywell K. , Singh L. , Tattersall F.D. , Thorn L. , Williams B.J.
Published: July 2012

The anticonvulsant and behavioural effects of the glycine/NMDA receptor partial agonist, L‐687,414 (R(+)‐cis‐β‐methyl‐3‐amino‐1‐hydroxypyrrolid‐2‐one) have been investigated in rodents.

L‐687,414 dose‐dependently antagonized seizures induced by N‐methyl‐D, L‐aspartic acid (NMDLA, ED50 = 19.7 mg kg−1), pentylenetetrazol (PTZ, ED50 = 13.0 mg kg−1) and electroshock (ED50 = 26.1 mg kg−1) when given intravenously 15min before test, in male Swiss Webster mice but was most potent against audiogenic seizures induced by a 120 dB bell in DBA/2 mice (ED50 = 5.1 mg kg−1, i.p., 30min before test).

L‐687,414 also induced impairments of performance in a rotarod test in both Swiss Webster and DBA/2 mice and the ratio [rotarod MED:anticonvulsant ED50] varied between 0.9 and 5, depending on the convulsant used.

Similar behaviours to those seen after administration of the non‐competitive NMDA receptor antagonist, MK‐801 (head weaving, body rolling, hyperlocomotion) were seen in the mouse after giving L‐687,414, although the peak effect occurred at a dose (100 mg kg−1) which was 5–20 times the anticonvulsant ED50S, depending on the convulsant used. Unlike MK‐801, however, doses of L‐687,414 that were behaviourally stimulant did not increase dopamine turnover in the nucleus accumbens.

Consistent with the interaction of L‐687,414 with the glycine/NMDA receptor, the anticonvulsant, ataxic and motor stimulant effects of the compound were significantly attenuated by the glycine/NMDA receptor agonist, D‐serine (10–100 μg per mouse, i.c.v.).

The results show that L‐687,414 is a potent, orally active anticonvulsant with a more benign pharmacological profile than antagonists acting at the ion channel of the NMDA receptor complex. The compound is a useful tool with which to probe the functional role of the glycine co‐agonist site in vivo.
Failure of CGS15943A to block the hypotensive action of agonists acting at the adenosine A3 receptor
Authors: Patel M. , Sheehan M.J. , Strong P.
Published: July 2012

Adenosine receptor agonists were evaluated for their activity at the putative adenosine A3 receptor which mediates a ‘xanthine‐resistant’ hypotensive response in the anaesthetized rat. The compounds tested were: the A1/A3 receptor agonist, N‐[2‐(4‐aminophenyl)ethyl]adenosine (APNEA), the nonselective adenosine receptor agonist, 5′‐N‐ethylcarboxamidoadenosine (NECA), the adenosine A1 receptor‐selective agonists, N‐[(lS,trarts)‐2‐hydroxycyclopentyl]adenosine (GR79236) and N6‐cyclopentyl adenosine (CPA), the A2a receptor‐selective agonists, 2‐[[2‐[4‐(2‐carboxyethyl) phenyl] ethyl] amino]‐N‐ethylcarboxamidoadenosine (CGS21680) and 2‐phenylaminoadenosine (CV1808), and the moderately A2b selective agonist, N‐[(2‐methylphenyl)methyl]adenosine (metrifudil).

In confirmation of literature findings, APNEA (1–1000 nmol kg−1) induced hypotension and bradycardia; the hypotension was not blocked by pretreatment with the xanthine antagonist, 8‐P‐sulphophenyltheophylline (8‐sPT; 40 mg kg−1, i.v.), whereas the bradycardia was attenuated. The non‐xanthine antagonist, 9‐fluoro‐2‐(2‐furyl)‐5,6‐dihydro [1,2,4]triazolo{1,5‐c}‐quinazin‐5‐imine (CGS15943A; 3 mg kg−1 i.v.), also attenuated the bradycardia without affecting the hypotension.

The adenosine A1 receptor‐selective agonists, GR79236 and CPA, both produced dose‐dependent falls in blood pressure and heart rate which were antagonized by 8‐sPT (40 mg kg−1) and CGS15943A (3 mg kg−1).

The adenosine A2a receptor‐selective agonists, CGS21680 and CV1808, produced only a hypotensive response which was antagonized by 8‐sPT (40 mg kg−1) and to a much greater extent by CGS15943A (3 mg kg−1), consistent with the response being mediated solely by A2a receptors.

The modestly A2b receptor‐selective agonist, metrifudil, produced a dose‐dependent fall in blood pressure and at higher doses a fall in heart rate. The hypotension induced by metrifudil was not antagonized by either 8‐sPT (40 mg kg−1) or CGS15943A (3 mg kg−1) even though the bradycardia was abolished, suggesting that this agonist activates the putative A3 receptor.

The non‐selective adenosine receptor agonist, NECA, produced a hypotension and bradycardia that was attenuated by 8‐sPT (40 mg kg−1), confirming previous work. The non‐xanthine antagonist, CGS15943A (3 mg kg−1), also attenuated the hypotension and bradycardia. The bradycardia was blocked to a much greater extent, suggesting that NECA may therefore induce hypotension partly by activating the putative A3 receptor.

In conclusion, we have confirmed that the putative A3 receptor mediating hypotension in the anaesthetized rat is not blocked by 8‐sPT, and further shown that it is not blocked by CGS15943A. The A2a agonists CGS21680 and CV1808 showed no discernible activity at the A3 receptor, whereas APNEA, NECA, CPA and metrifudil appear to activate this receptor. The adenosine A1 receptor agonist, GR79236, shows considerable selectivity for the A1 receptor but may activate the A3 receptor at high doses.
Identification of renal natriuretic peptide receptor subpopulations by use of the non‐peptide antagonist, HS‐142‐1
Authors: Rutherford Richard A.D. , Matsuda Yuzuru , Wilkins Martin R. , Polak Julia M. , Wharton John
Published: July 2012

The renal actions of natriuretic peptides are dictated by the distribution of guanylyl cyclase‐linked (NPRA and NPRB) and non‐guanylyl cyclase‐linked (NPRC) receptors. Natriuretic peptide receptors have previously been distinguished on the basis of their differential affinity for peptide fragments and analogues; however, most of the available ligands are not fully selective. We have used the specific guanylyl cyclase‐linked receptor antagonist, HS‐142‐1, to investigate the differential distribution of natriuretic peptide receptor subtypes in the human, bovine and rat kidney.

Specific, high affinity 3‐([125I]‐iodotyrosyl)‐rat‐ANP‐(1‐28)([125I]‐rANP1‐28) binding sites were identified in all three species, localized to glomeruli, inner medulla, intrarenal arteries and regions in the outer medulla corresponding to vasa recta bundles. Binding sites were also identified in the smooth muscle lining of the hilar region in the bovine and rat kidney.

In the rat, [125I]‐rANP1‐28 binding was inhibited by unlabelled peptide sequences with a rank order of potency (rANP1‐28 > pCNPl‐22 > C‐ANP4‐23). The glomeruli exhibited a heterogeneous population of binding sites, C‐ANP4‐23 and pCNP1‐22 producing a significantly better fit to a two component inhibition curve compared to the single component curve for rANP1‐28.

Competitive inhibition experiments with the receptor selective ligands, C‐ANP4‐23 and HS‐142‐1, suggested that, like the rat, human and bovine glomeruli possessed a heterogeneous population of binding sites, whilst those in the inner medulla and intrarenal arteries of all three species represented a homogeneous population. Rat glomeruli exhibited a high proportion (> 80%) of the NPRc receptor subtype whereas in human and bovine glomeruli this receptor represented less than 20% of the total population, the majority of binding sites being HS‐142‐1‐sensitive.

C‐ANP4‐23 exhibited a significantly higher inhibitory potency for binding sites in rat glomeruli compared to those in human and bovine kidney whilst HS‐142‐1 was significantly more potent in the rat and bovine kidney compared to man. No evidence was found to suggest the presence of a renal NPRB receptor subtype.

The relative density, affinity and proportion of natriuretic receptor subtypes in the kidney exhibit significant species differences. HS‐142‐1 may be a valuable tool in further elucidating the localization and function of these receptors, but heterogeneity between species should be considered when selecting experimental models.
Long‐lasting inhibitory activity of the hetrazepinic BN 50730 on exudation and cellular alterations evoked by PAF and LPS
Authors: Pires Ana L.A. , e. Silva Patricia M.R. , Pasquale Claudia , Castro‐Faria‐Neto Hugo C. , Bozza Patricia T. , Cordeiro Renato S.B. , Rae Giles A. , Braquet Pierre , Lagente Vincent , Martins Marco A.
Published: July 2012

Inhibitory effects of the hetrazepinic derivative BN 50730 on the rat pleural inflammatory response, triggered by PAF or lipopolysaccharides (LPS), were examined. The type of pharmacological blockade exerted by this antagonist in in vitro assays of eosinophil chemotaxis and platelet aggregation were also investigated.

Intrathoracic injection of PAF (1 μg per cavity) caused a 4 fold increase in the extravasated protein within 15min and led to a marked eosinophil accumulation 24 h post‐challenge. BN 50730 (0.5–10 μg per cavity) inhibited exudation by PAF dose‐dependently without modifying the response induced by histamine, bradykinin or 5‐hydroxytryptamine (5‐HT).

The kinetics of the inhibitory effect on exudation revealed that the actions of WEB 2086 and BN 52021 (10 μg per cavity) were over within 2 and 4h respectively, whereas BN 50730 (10 μg per cavity) retained 80% of its inhibitory activity for 4 days.

Oral treatment with BN 50730 (10–20 mg kg−1, 1 h beforehand) suppressed the leucocyte accumulation and late eosinophilia observed 6 and 24 h after PAF respectively, but did not modify the eosinophilia induced by leukotriene B4 (LTB4) or bradykinin. BN 50730 also failed to reduce the eosinophil accumulation induced by LPS but drastically inhibited the neutrophil influx.

The pre‐incubation of rat peritoneal eosinophils for 10 min with BN 50730 (30nM‐1 μm) dose‐dependently inhibited the chemotaxis induced by PAF (0.1 μm) in vitro. The IC50 values for BN 52021, WEB 2086 and BN 50730 in this system were 5, 5 and 0.05 μm respectively.

In separate assays, rat peritoneal eosinophils and rabbit washed platelets were preincubated with BN 50730 or WEB 2086 (1 μm) then subjected to a series of at least two consecutive washings in order to remove the antagonist from the receptor environment. Under such conditions, only the cells pretreated with WEB 2086 recovered the sensitivity to the lipid.

We conclude that BN 50730 is a potent, specific and long‐acting PAF antagonist and its effect seems to result from a high affinity and non‐competitive interaction of the drug with the PAF receptor.
Effect of cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca2+‐ATPase, on the frequency‐dependence of the contraction‐relaxation cycle of the guinea‐pig isolated atrium
Authors: Yard Nicola J. , Chiesi Michele , Ball Howard A.
Published: July 2012

The relevance of a functional sarcoplasmic reticulum (SR) membrane system to the contraction‐relaxation cycle and to the force‐frequency relationship of guinea‐pig atrial tissue was investigated. Cyclopiazonic acid (CPA) was used to inhibit selectively the activity of the SR Ca2+‐ATPase. IQo values of 0.2 μm or 1.0 μm were measured in guinea‐pig isolated SR membranes in the absence or presence of millimolar ATP, respectively. CPA (0.3–30 μm) did not inhibit the activity of the sarcolemmal Na+‐Ca2+‐exchanger as measured in isolated cardiac cell membrane preparations.

In guinea‐pig isolated left atrium paced at 2.5 Hz (30°C), CPA (1–100 μm) produced a concentration‐dependent reduction in developed tension and a fall in the maximum rate of tension increase (+ dT/dtmax) and decrease (‐dT/dtmax). The twitch duration was markedly increased due to a prolongation of the time to peak tension, and in particular, the relaxation phase.

The contraction‐relaxation cycle of the left atrium showed a marked dependence on the frequency of stimulation. The developed tension and +dT/dtmax showed a progressive increase from 0.5 Hz, reaching peak values at a stimulation rate of 1.5‐2.5 Hz, the positive staircase phenomenon. Higher frequencies of stimulation caused a fall in these parameters. Resting tension was unaffected. The time‐course of the contraction‐relaxation cycle was also frequency‐dependent, with both time to peak tension and relaxation time showing a progressive fall from 2.0–3.5 Hz.

The addition of CPA (30 μm) caused marked alterations in the frequency‐dependence of the contraction‐relaxation cycle. The frequency‐dependence of developed tension, + dT/dtmax, was shifted downwards, particularly at higher frequencies, and the frequency at which peak values of + dT/dtmax and — dT/dtmax were reached was shifted leftwards. The resting tension of the tissues in the presence of 30 μm CPA was increased markedly at frequencies greater than 2 Hz. The time‐course of the contraction‐relaxation cycle was markedly prolonged between 1.0 and 3.5 Hz, due to an effect on both time to peak tension and relaxation time.

In conclusion, these results show that CPA is a highly selective inhibitor of the cardiac SR Ca2+‐ATPase, without effect on the sarcolemmal Na+‐Ca2+‐exchanger, and suggest that a functional SR Ca2+‐ATPase is necessary for the normal contraction‐relaxation cycle of guinea‐pig cardiac tissue. Additionally, the results suggest an increasing dependence of tension development on SR Ca2+‐ATPase with increasing frequency, which may reflect either a frequency‐dependent activation of this enzyme or the diminished contribution of the Na+‐Ca2+ exchanger. These results also provide novel support for the mechanism of the depressed force‐frequency relation found in cardiac tissue of heart failure patients, in which there is a reduced expression of Ca2+‐ATPase.
FPL 66096: a novel, highly potent and selective antagonist at human platelet P2T‐purinoceptors
Authors: Humphries R.G. , Tomlinson W. , Ingall A.H. , Cage P.A. , Leff P.
Published: July 2012

ADP‐dependent platelet aggregation is mediated by the P2T‐purinoceptor and is specifically inhibited by ATP, which is a competitive P2T‐purinoceptor antagonist. However, ATP functions as an agonist at other P2‐purinoceptor subtypes in other tissues and is, therefore, non‐selective. This paper describes the effects of the novel ATP analogue, FPL 66096 (2‐propylthio‐D‐β,γ‐difluoromethylene ATP), on ADP‐induced and ADP‐independent aggregation of human washed platelets and in standard preparations containing P2X‐ (rabbit ear artery) and P2Y‐purinoceptors (guinea‐pig aorta).

In suspensions of human washed platelets, FPL 66096 (1‐100nM) produced concentration‐dependent rightward displacement of concentration‐effect (E/[A]) curves obtained for ADP‐induced platelet aggregation. Logistic fitting of E/[A] data indicated that the effect of FPL 66096 was consistent with simple competition with a pKB value of 8.66. FPL 66096 (10–1000 nm) had no effect on aggregation produced by the thromboxane A2‐mimetic, U46619 (0.1–10 μm) when the response to this agent was rendered ADP‐independent by inclusion of the non‐selective P2‐purinoceptor antagonist, suramin (100 μ).

The antiaggregatory potency of FPL 66096 was not influenced by increasing the incubation time from 2 to 15min nor by inclusion of the P1‐purinoceptor antagonist 8‐sulphophenyltheophylline at a concentration (300 μm) that produced a 68 fold rightward displacement of the anti‐aggregatory E/[A] curve for the P1‐purinoceptor agonist, 5′‐N‐ethylcarboxamidoadenosine (0.1–1000 μm).

FLP 66096 behaved as a weak (pA50 3.68) but full P2x‐purinoceptor agonist in preparations of the rabbit isolated ear artery and as a weak, competitive antagonist (apparent pKB 4.71) at P2Y‐purinoceptors in the guinea‐pig isolated aorta, indicating a selectivity of at least 9000 fold for the P2T‐subtype. In the latter preparation, non‐specific relaxations were produced by concentrations of FPL 66096 ≥ 10 μm.

These results indicate that FPL 66096 is a P2T‐purinoceptor antagonist of unprecedented potency and selectivity and that its effects are consistent with simple competition at the P2T‐purinoceptor. Therefore, FPL 66096 represents a novel pharmacological tool in the classification of P2‐purinoceptors and in the elucidation of the mechanisms involved in activation of platelets by ADP.
Blockade of noradrenaline‐induced constrictions by yohimbine and prazosin differs between consecutive segments of cutaneous arteries in guinea‐pig ears
Authors: Morris J.L.
Published: July 2012

The study has examined the receptors mediating constriction produced by brief local application of noradrenaline (NA) to cutaneous arteries and arterioles in the ear vasculature of anaesthetized guinea‐pigs. The innervation of the corresponding vascular segments has been examined immunohis‐tochemically at the conclusion of the pharmacological experiments.

Small arteries of branch order 4 (4°, 40–110 μm internal diameter) ‐were more sensitive to the vasoconstrictor action of NA than were more proximal arteries of branch order 3 (3°, 60–150 μm internal diameter), or more distal arteries and arterioles of branch orders 5 to 7 (5°‐7°, 18‐ 85 μm internal diameter). This higher sensitivity of 4° arteries was maintained after blockade of neuronal uptake with desipramine (1 μm), and after blockade of β‐adrenoceptors with propranolol (1 μm).

NA‐induced vasoconstrictions of distal arterioles (5°‐7°) were abolished or greatly reduced by yohimbine (1 μm). The blockade by yohimbine decreased progressively with increasing vessel diameter of proximal arteries, while the blockade by prazosin (1 μm) increased progressively in arteries >40 μm diameter.

In 3° and 4° arteries, a substantial component (approximately 50%) of NA‐induced vasoconstrictions remained after combined treatment with yohimbine and prazosin, in the presence or absence of desipramine. These constrictions were not further reduced by benextramine (1–10 μm), but were abolished by dihydroergotamine (1–10 μm). Constrictions induced by ATP (0.1‐1 μm) were not affected by dihydroergotamine.

5‐Hydroxytryptamine (3–100 μm) had a variable effect on 3° and 4° arteries including: concentration‐dependent constrictions (n = 3); small constrictions at some concentrations, and dilatations or no change in diameter at other concentrations (n = 6); concentration‐dependent dilatations only (n = 3). The 5‐HT2 receptor antagonist, ketanserin (0.1‐0.3 μm), did not affect NA‐induced constrictions.

In 16 arterial segments ranging from 3° arteries to 6° arterioles, there was a significant correlation between the presence of neuropeptide Y‐immunoreactive (NPY‐IR) sympathetic axons and the degree of blockade of NA‐induced constrictions by yohimbine, prazosin and dihydroergotamine.

These results demonstrate marked differences in the postsynaptic adrenoceptors mediating vasoconstriction to a bolus of NA applied briefly to the adventitial surface of different segments of the cutaneous vasculature of the guinea‐pig ear. Furthermore, the presence or absence of adrenoceptors sensitive to blockade by yohimbine or prazosin is related to the proportion of sympathetic axons innervating each vascular segment which contain NPY‐IR.
Dissociation of castor oil‐induced diarrhoea and intestinal mucosal injury in rat: effect of NG‐nitro‐L‐arginine methyl ester
Authors: Capasso Francesco , Mascolo Nicola , Izzo Angelo A. , Gaginella Timothy S.
Published: July 2012

Castor oil (2 ml orally) produced diarrhoea in rats 1–7 h after challenge, which was associated with gross damage to the duodenal and jejunal mucosa.

The injury was accompanied by release of acid phosphatase into the gut lumen, indicating cellular injury.

Intraperitoneal injection of the nitric oxide (NO) synthase inhibitor NG ‐nitro‐L‐arginine methyl ester (1‐NAME, 2.5–50 mg kg−1 twice), prevented the diarrhoea. The dose of 1‐NAME (50mg kg−1) completely blocked the diarrhoea but increased the release of acid phosphatase and worsened the gross damage.

The NO donating compound, isosorbide‐5‐mononitrate (IMN, 150 mg kg−1 twice) reversed the effects of 1‐NAME (50 mg kg1) on castor oil‐induced diarrhoea, gross damage and acid phosphatase release.

The apparent dissociation of the diarrhoeal and intestinal mucosal damaging effects of castor oil suggest that NO has a protective effect on the rat duodenal and jejunal mucosa, but that NO mediates, in part, the diarrhoea effect of this laxative.
Attenuation of the antiarrhythmic effects of ischaemic preconditioning by blockade of bradykinin B2 receptors
Authors: Vegh Agnes , Papp Julius Gy , Parratt James
Published: July 2012

The possibility that bradykinin is involved in the pronounced antiarrhythmic effects of ischaemic preconditioning in anaesthetized mongrel dogs was examined with the use of the selective B2 antagonist, icatibant (Hoe‐140).

Preconditioning, achieved by two 5 min occlusions of the left anterior descending coronary artery, followed 20 min later by a 25 min occlusion of the same artery resulted, during this prolonged occlusion, in less severe arrhythmias (VF 0% versus 47% in control non‐preconditioned dogs), reductions in the incidence and number of episodes of ventricular tachycardia (VT) and in the number of ventricular premature beats and increased survival following reperfusion (50% versus 0% in the controls).

Hoe‐140 was given in a dose of 300μg kg−1 either before the preconditioning procedure or after preconditioning but before the prolonged occlusion. This dose of Hoe‐140 had insignificant haemodynamic effects and failed to modify the increase in coronary blood flow that occurred in the circumflex coronary artery when the anterior descending branch was occluded.

It was difficult to precondition dogs in the presence of Hoe–140. There were more ventricular arrhythmias during the initial 5 min occlusion (44 ± 12 versus 10 ± 3) and a higher incidence of ventricular fibrillation (50% versus 21%) during the preconditioning procedure. There was also a more pronounced ST‐elevation (recorded from epicardial electrodes) during the preconditioning occlusions in those dogs given Hoe‐140.

In those dogs that survived to the long (25 min) occlusion, Hoe‐140 prevented the antiarrhythmic effects of preconditioning (reduction in ventricular premature beats and in VT) although all the dogs survived the occlusion period. However on reperfusion, survival in the preconditioned dogs given Hoe‐140 was less than in those dogs preconditioned without the B2 antagonist.

Changes in the degree of inhomogeneity of conduction within the ischaemic area, which were markedly suppressed by preconditioning, were attenuated in those dogs preconditioned in the presence of Hoe–140.

These results suggest that bradykinin acts as both a ‘trigger’ for preconditioning and as one of the mediator protective (antiarrhythmic) substances generated by the myocardium under these conditions. Since the protection afforded both by preconditioning and by local intracoronary infusions of bradykinin is markedly attenuated by an inhibitor of the 1‐arginine nitric oxide pathway, we suggest that much of the protection afforded by ischaemic preconditioning results from the generation of nitric oxide, and that bradykinin, released early during ischaemia, acts as a stimulant for this generation.
The relative importance of nitric oxide and nitric oxide‐independent mechanisms in acetylcholine‐evoked dilatation of the rat mesenteric bed
Authors: Parsons Sarah J.W. , Hill Anthony , Waldron Gareth J. , Plane Trances , Garland Christopher J.
Published: July 2012

The relative contribution of nitric oxide (NO) to acetylcholine‐induced smooth muscle relaxation was investigated in the rat perfused mesenteric vasculature and in isolated segments of second, third and fourth order arterial branches.

The EC50 values and maximal relaxation to acetylcholine were not significantly different in the sequential arterial branches, being approximately 0.05 μm and 85%, respectively.

The NO synthase inhibitor L‐NG‐nitro‐L‐arginine methyl ester (L‐NAME; 100 μm) reduced acetylcholine‐evoked endothelium‐dependent dilatation and relaxation in the perfused mesenteric bed and in isolated arterial segments. The maximum response to acetylcholine in both preparations was reduced by between 35% to 40% while the EC50 values were increased by 5–6 fold. L‐NAME had no effect on basal smooth muscle tone in either case.

In contrast, endothelium‐dependent dilatation of the perfused mesenteric bed evoked by A23187 (0.002–20 nmol), was unaffected by exposure to L‐NAME. The EC50 values and maximal responses elicited by A23187 (20 nmol) before and after exposure to L‐NAME were 0.96 ± 0.5 nmol and 67.0 ± 7.0% (n = 4), and 0.7 ± 0.4 nmol and 70.0 ± 5.0% (n = 4; P > 0.01), respectively.

Perfusion of the isolated mesenteric bed with raised K+‐Krebs buffer (25 μm) had no effect on basal tone, but reduced the amplitude of both acetylcholine‐ and A23187‐evoked dilatation. The maximum responses to acetylcholine (2 μmol) and A23187 (20 nmol) were reduced from 67.5 ± 7.3% and 65.4 ± 8.2% to 18.9 ± 11.0% (n = 5; P < 0.01) and 13.5 ± 12.0% (n = 4; P < 0.01), respectively.

Exposure of the mesenteric bed to L‐NAME in the presence of raised K+‐Krebs further reduced the maximal response elicited by acetylcholine to only 8.9 ± 2.8% (n = 4; P < 0.01).

These results indicate that acetylcholine‐evoked vasodilatation of the rat mesenteric vasculature is mediated by both NO‐dependent and ‐independent mechanisms. The relative contribution made by these mechanisms does not appear to differ in sequential branches of the mesenteric artery. In contrast, A23187‐evoked vasodilatation appears to be mediated predominantly by a NO‐independent mechanism which is sensitive to increases in the extracellular potassium concentration and may reflect the action of endothelium‐derived hyperpolarizing factor (EDHF).
[125I]‐PD151242: a selective ligand for endothelin ETA receptors in human kidney which localizes to renal vasculature
Authors: Davenport Anthony P. , Kuc Rhoda E. , Hoskins Sarah L. , Karet Fiona E. , Fitzgerald Fiona
Published: July 2012

The linear tetrapeptide radioligand, [125I]‐PD151242 was used to characterize ETA receptors in human kidney which is an ETB‐rich tissue. Saturation binding assays with [125I]‐PD151242 revealed a single population of high affinity endothelin receptors: KD = 0.75 ± 0.07 nm and Bmax = 48.4 ± 1.6 fmol mg−1 protein (n = 3 individuals ± s.e.mean). Hill slopes were close to unity and a one site fit was preferred to a two site model.

ETA‐receptor‐selective ligands competed for [125I]‐PD151242 binding with sub‐nanomolar affinity: BQ123 KD = 0.43 ± 0.10 nm, Bmax = 46.6 ± 7.9 fmol mg−1 protein; FR139317, KD = 0.37 ± 0.06nM, Bmax = 39.5 ± 6.5 fmol mg−1 protein (n = 3 individuals ± s.e.mean). In each case, monophasic inhibition curves were obtained and a one site fit was preferred to a two site model. The ETB‐selective agonist, BQ3020 at the highest concentration tested (10 μm) inhibited binding by only 50%. The non‐selective RO462005 competed for the binding of [125I]‐PD151242: KD = 1.31 ± 1.38 μm, Bmax = 33.0 ± 9.7 fmol mg−1 protein. Endothelin‐2 and sarafotoxin S6B inhibited [125I]‐PD151242 binding to renal tissue whereas ET‐3 and sarafotoxin S6C were less effective. Non‐endothelin and non‐sarafotoxin peptides did not compete.

No degradation of [125I]‐PD151242 was detected following incubation of the ligand with renal tissue under the conditions of the binding assay.

Polymerase chain reaction products corresponding to the expected size for mRNA encoding ETA and ETB receptor sub‐types were detected in cortex and medulla in each of the five individuals examined.

Autoradiographical studies showed that ETA receptors visualised with [125I]‐PD151242 were mainly localized to blood vessels including interlobular and arcuate arteries, arterioles and adjacent arcuate veins. ETB receptors localized with [125I]‐BQ3020 were concentrated in the medulla and the density of binding to vessels was low.

These data suggest [125I]‐PD151242 is selective for ETA receptors in human kidney and this sub‐type is mainly localized to the renal vasculature. The results provide further evidence that the human vasculature mainly expresses the ETA receptor.
Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung
Authors: Walsh David A. , Salmon Michael , Featherstone Roland , Wharton John , Church Martin K. , Polak Julia M.
Published: July 2012

The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]‐Bolton Hunter‐labelled substance P ([125I]‐BH‐SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung.

[125I]‐BH‐SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung.

Specific binding of [125I]‐BH‐SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150–800 μm and was less dense in trachea than in main bronchi.

[125I]‐BH‐SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125]‐BH‐SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors.

In both human and guinea pig lung, [125I]‐BH‐SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]‐BH‐SP binding sites in both guinea pig arteries and bronchi.

Similar distributions, densities and characteristics of [125I]‐BH‐SP binding sites were demonstrated in ovalbumin‐sensitized and ‐challenged guinea‐pig lung and in naive animals.

Differences in the distribution and characteristics of NK1 binding sites labelled with [125I]‐BH‐SP between guinea pig and human lung suggest limitations in the use of guinea pig models for studying roles of tachykinins in pulmonary disease. However, the similar microvascular distributions of NKj binding sites in human and guinea pig lung suggest that the selective tachykinin receptor antagonists FK888 and FK224 may be useful in the management of airway inflammation in man.
Dexamethasone‐induced selective inhibition of the central μ opioid receptor: functional in vivo and in vitro evidence in rodents
Authors: Pieretti S. , Di Giannuario A. , Domenici M.R. , Sagratella S. , Capasso A. , Sorrentino L. , Loizzo A.
Published: July 2012

Endogenous corticosteroids and opioids are involved in many functions of the organism, including analgesia, cerebral excitability, stress and others. Therefore, we considered it important to gain information on the functional interaction between corticosteroids and specific opioid receptor subpopulations.

We have found that systemic administration (i.p.) of the potent synthetic corticosteroid, dexamethasone, reduced the antinociception induced by the highly selective μ agonist, DAMGO or by less selective μ agonists morphine and β‐endorphin administered i.e.v., On the contrary dexamethasone exerted little or no influence on the antinociception induced by a δ1 agonist, DPDPE and a δ2 agonist deltorphin II. Dexamethasone potentiated the antinociception induced by the k agonist, U50,488.

In experiments performed in an in vitro model of cerebral excitability in the rat hippocampal slice, dexamethasone strongly prevented both the increase of the duration of the field potential recorded in CA1, and the appearance and number of additional population spikes induced by μ receptor agonists.

In both models pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the antagonism by dexamethasone of responses to the μ opioid agonists.

Our data indicate that in the rodent brain there is an important functional interaction between the corticosteroid and the opioid systems at least at the μ receptor level, while δ and k receptors are modulated in different ways.