bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological evaluation of IQM‐95,333, a highly selective CCKAreceptor antagonist with anxiolytic‐like activity in animal models
Authors: Ballaz Santiago , Barber Ana , Fortuño Ana , Del Río Joaquín , Martín‐Martínez Mercedes , Gómez‐Monterrey Isabel , Herranz Rosario , González‐Muñiz Rosario , García‐López Maria‐Teresa
Published: August 2006

The pyridopyrimidine derivative IQM‐95,333 ((4aS,5R)‐2‐benzyl‐5‐[Nα‐tert‐butoxicarbonyl)L‐tryptophyl]amino‐1,3dioxoperhydropyrido[1,2‐c]pyrimidine), a new non‐peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluatedin vitroandin vivoin comparison with typical CCKAand CCKBreceptor antagonists, such as devazepide, lorglumide, L‐365,260 and PD‐135,158.

IQM‐95,333 displaced [3H]‐CCK‐8S binding to CCKAreceptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKBreceptors was negligible (IC50>10 μM). IQM‐95,333 was a more selective CCKAreceptor ligand than devazepide and other CCKAreceptor antagonists.

Like devazepide, IQM‐95,333 was a more potent antagonist of CCK‐8S‐ than of CCK‐4‐induced contraction of the longitudinal muscle from guinea‐pig ileum, suggesting selective antagonism at CCKAreceptors.

IQM‐95,333 and devazepide were also potent inhibitors of CCK‐8S‐stimulated amylase release from isolated pancreatic acini, a CCKAreceptor‐mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates.

Low doses (50–100 μg kg−1, i.p.) of IQM‐95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK‐8S, two effects associated with stimulation of peripheral CCKAreceptors.

IQM‐95,333 showed an anxiolytic‐like profile in the light/dark exploration test in mice over a wide dose range (10–5,000 μg kg−1). Typical CCKAand CCKBantagonists, devazepide and L‐365,260 respectively, were only effective within a more limited dose range.

In a classical conflict paradigm for the study of anxiolytic drugs, the punished‐drinking test, IQM‐95,333, devazepide and L‐365,260 were effective within a narrow dose range. The dose‐response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses.

In conclusion, IQM‐95,333 is a potent and selective CCKAreceptor antagonist bothin vitroandin vivowith an anxiolytic‐like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

British Journal of Pharmacology(1997)121, 759–767; doi:10.1038/sj.bjp.0701186
The interaction of a new anti‐tumour drug, KAR‐2 with calmodulin
Authors: Orosz F , Vértessy B G , Salerno C , Crifo C , Capuozzo E , Ovádi J
Published: August 2006

KAR‐2 (3′′‐(β‐chloroethyl)‐2′′,4′′‐dioxo‐3,5′′‐spiro‐oxazolidino‐4‐deacetoxy‐vinblastine) is a semisynthetic bis‐indol derivative, with high anti‐microtubular and anti‐tumour activities but with low toxicity. KAR‐2, in contrast to other biologically active bis‐indols (e.g. vinblastine), did not show anti‐calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests).

Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR‐2 to calmodulin. The binding affinity of KAR‐2 to calmodulin (dissociation constant was about 5 μM) in the presence of Ca2+ was comparable to that of vinblastine.

KAR‐2 was able to interact with apo‐calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature.

The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR‐2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady‐state level.

Comparison of the data obtained with KAR‐2 and bis‐indols used in chemotherapy suggests that the lack of anti‐calmodulin potency resides on the spiro‐oxazolidino portion of KAR‐2. This character of KAR‐2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti‐tumour test.
α1‐Adrenoceptor vasoconstriction in the tail artery during ageing
Authors: Vila Elisabet , Vivas Nuria M , Tabernero Antonia , Giraldo Jesús , Arribas Silvia M
Published: August 2006

We have studied the α1‐adrenoceptor‐mediated responses in intact tail artery rings from 3–4 and 20–22 months old Sprague‐Dawley rats, focusing on possible endothelial alterations. The influence of nitric oxide released by the endothelium, the number of α1‐adrenoceptors and the functional receptor reserve were evaluated to determine their contribution to the contractile response mediated by this receptor. The state of the endothelial layer was assessed by confocal microscopy.

Noradrenaline (1 nM–100 μM) induced concentration‐dependent vasoconstriction. The maximum contractions to noradrenaline (P<0.05) and to 75 mM KCl (P<0.01) were higher in young than in old animals.

The density (Bmax) of α1‐adrenoceptors and the dissociation constant (KD) obtained in [3H]‐prazosin binding experiments were unchanged by age.

The apparent affinity (pKA) and the percentage of functional receptors (qx100) remaining after phenoxybenzamine (0.03 μM) were similar in both age groups.

After partial α1‐adrenoceptor inactivation with phenoxybenzamine, NG‐nitro‐L‐arginine methylester (30 μM) significantly potentiated the E/[A] curve to noradrenaline in young rats. However, only responses to 0.1 to 1 μM noradrenaline were significantly potentiated in old animals. In addition, 94% of the vessels from young, but only 52% from old rats were relaxed by 80–100% of the noradrenaline (0.03 μM) contraction, with 1 μM acetylcholine.

No modifications in the area (μm2) or in the number of endothelial nuclei (per mm2) were observed between age groups. An elongation of the nuclei of endothelial cells was observed in the old animals.

These data suggest that the noradrenaline‐induced contraction is decreased in old rats probably due to differences in either the contractile machinery or postreceptor mechanisms. These alterations may be accompanied by an impairment of the release or production of NO from endothelial cells.
Effects of an orally active non‐peptide bradykinin B2 receptor antagonist, FR173657, on plasma exudation in rat carrageenin‐induced pleurisy
Authors: Majima Masataka , Kawashima Noriko , Hiroshi Ito , Katori Makoto
Published: August 2006

Effects of an orally active non‐peptide (BK) B2 receptor antagonist, FR173657 ((E)‐3‐(6‐acetamido‐3‐pyridyl)‐ N‐[N‐[2,4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl)oxymethyl]phenyl]‐N‐methylaminocarbonylmethyl] acrylamide) on the plasma exudation in rat carrageenin‐induced pleurisy were investigated.

Plasma exudation induced by intrapleural injection of bradykinin (BK, 3 nmol per rat) into male SD strain rats (SPF, 8 weeks old) were significantly inhibited by oral administration of novel B2 receptor antagonist FR173657 (3–30 mg kg−1, 1 h before BK injection) in a dose‐dependent manner, whereas that induced by histamine was not.

The inhibitory effect of 30 mg kg−1 FR173657 persisted for more than 4 h.

Intrapleural injection of λ‐carrageenin (2% (w/v), 0.1 ml per rat) caused marked plasma exudation and accumulation of exudates from 1 h after carrageenin injection. The maximum plasma exudation response was observed 5 h after carrageenin. The oral administration of FR173657 to rats (30 mg kg−1, 1 h before carrageenin) significantly (by 50–77%) blunted the plasma exudation 1, 3, 5, and 7 h after carrageenin, causing a significant parallel reduction (by 42–57%) in the volume of exudates.

The anti‐inflammatory effect of FR173657 on rat carrageenin‐induced pleurisy was almost equipotent with that of the peptide B2 antagonist Hoe140 (1 mg kg−1, i.v.), a plasma kallikrein inhibitor, soy bean trypsin inhibitor (0.3 mg per rat, intrapleural injection) and bromelain (10 mg kg−1, i.v.).

In pleurisy induced by intrapleural injection of a histamine releaser, compound 48/80, the plasma exudation was observed only within 20 min after the injection. This plasma exudation was not affected by FR173657, although it was completely inhibited by a mixture of pyrilamine (5 mg kg−1, i.v.) and methysergide (3 mg kg−1, i.v.).

These results indicate that FR173657 is an orally active, promising anti‐inflammatory agent for kinin‐dependent inflammation.

British Journal of Pharmacology (1997) 121, 723–730; doi:10.1038/sj.bjp.0701194
Renal α2a/d‐adrenoceptor subtype function: Wistar as compared to spontaneously hypertensive rats
Authors: Intengan H D , Smyth D D
Published: August 2006

The α2a/d‐adrenoceptor subtype in the rat kidney modulates solute excretion (osmolar clearance). Since the kidney plays a role in chronic regulation of blood pressure, altered renal function may be implicated in the development of hypertension. A second alteration ‐ that of the α2a/d‐adrenoceptor subtype gene ‐ has also been correlated with hypertension in rats and man.

We hypothesized that as a consequence of the altered α2a/d‐adrenoceptor subtype gene previously shown in spontaneously hypertensive (SH) rats, the increase in osmolar clearance following stimulation of the renal α2a/d‐subtype would be attenuated in SH rats as compared to normotensive Wistar rats. In contrast, based on the theory that such functional unresponsiveness of the α2a/d‐subtype would be genetically determined, we further hypothesized that in one kidney‐one clip (1K‐1C) rats, the response to stimulation of the renal α2a/d‐subtype would be intact as compared to the normotensive Wistar 1K‐sham rats.

Male rats were unilaterally nephrectomized under ether anaesthesia. In the 1K‐1C rats, a silver clip (diameter 0.254 mm) was also placed around the left renal artery. On the experimental day, rats were administered pentobarbitone (50.0 mg kg−1, i.p.). The carotid artery and jugular vein were cannulated for blood pressure monitoring and saline infusion. The ureter was catheterized for urine collection. A 31 gauge needle was advanced into the renal artery for infusion of the α2a/d‐selective agonist, guanfacine (vehicle, 1.0, 3.0 and 10.0 nmol kg−1 min−1 in Wistar and SH rats; vehicle and 10.0 nmol kg−1 min−1 in Wistar 1K‐sham and 1K‐1C rats).

In Wistar rats, guanfacine dose‐dependently increased urine flow and sodium excretion. An increase in osmolar clearance but not free water clearance was also observed. However, in SH rats guanfacine failed to alter urine flow, sodium excretion, osmolar and free water clearance. In contrast, in both Wistar 1K‐sham and 1K‐1C rats, guanfacine increased urine flow rate. Again, this response was due solely to an increase in osmolar clearance. At these doses, guanfacine did not alter blood pressure or creatinine clearance during the experiment.

In summary, the ability of the α2a/d‐adrenoceptor subtype to mediate an increase in osmolar clearance was absent in a genetic model of hypertension, the SH rats. This effect was intact in an acquired model of hypertension (1K‐1C rats). This suggested a defective modulation of solute excretion in SH rats which was probably due to alteration of the α2a/d‐subtype gene and not secondary to the elevated blood pressure. The altered α2a/d‐subtype gene and function may therefore play a causal role in the pathogenesis of hypertension.
Evidence that cyclic AMP phosphodiesterase inhibitors suppress interleukin‐2 release from murine splenocytes by interacting with a ‘low‐affinity’ phosphodiesterase 4 conformer
Authors: Souness John E. , Houghton Clare , Sardar Nughat , Withnall Michael T.
Published: August 2006

We have investigated the suppressive effects of rolipram, RP 73401 (piclamilast) and other structurally diverse inhibitors of cyclic AMP‐specific phosphodiesterase 4 (PDE4) on interleukin (IL)‐2 generation from Balb/c mouse splenocytes exposed to the superantigen, Staphylococcocal enterotoxin‐A (Staph. A). The purpose was to determine whether their potencies are more closely correlated with inhibition of PDE4 from CTLL cells, against which rolipram displays weak potency (low‐affinity PDE4), or displacement of [3H]‐(±)‐rolipram from its high‐affinity binding site (HARBS) in mouse brain cytosol.

RP 73401 (IC50 0.46±0.07 nM, n=4) was a very potent inhibitor of Staph. A‐induced IL‐2 release from Balb/c mouse splenocytes, being >1100 fold more potent than (±)‐rolipram (IC50 540±67 nM, n=3).

A close correlation (r=0.95) was observed between suppression of IL‐2 release by PDE inhibitors and inhibition of PDE4. In contrast, little correlation (r=0.39) was observed between suppression of IL‐2 release and their affinities for the high‐affinity rolipram binding site (HARBS).

RP 73401 only inhibited partially (30–40%) Staph. A‐induced incorporation of [3H]‐thymidine into splenocyte DNA. The PDE3 inhibitor, siguazodan (10 μM), had little or no effect on IL‐2 release or DNA synthesis. This concentration of siguazodan did not enhance the inhibitory action of RP 73401 on IL‐2 release but potentiated its effect on DNA synthesis, increasing potency and efficacy.

Staph. A‐induced DNA synthesis was only partially inhibited by anti‐IL‐2 neutralizing antibody, whereas dexamethazone (100 nM) and cyclosporine A (100 nM) completely blocked the response.

RP 73401 (IC50 6.3±1.9 nM, n=4) was 140 fold more potent than rolipram (IC50 900±300 nM, n=3) in inhibiting Staph. A‐induced [3H]‐thymidine incorporation into splenocyte DNA.

The results implicate a low‐affinity form of PDE4 in the suppression of Staph. A‐induced IL‐2 release from murine splenocytes by PDE inhibitors. The data also indicate that mitogenic factors other than IL‐2, whose elaboration or responses to which are regulated by PDE3 as well as PDE4, contribute to the superantigen‐induced DNA synthesis.

British Journal of Pharmacology (1997) 121, 743–750; doi:10.1038/sj.bjp.0701200
Impairment of endothelium‐dependent but not of endothelium‐independent dilatation in guinea‐pig aorta rings incubated in the presence of elevated glucose
Authors: Dorigo P , Fraccarollo D , Santostasi G , Maragno I
Published: August 2006

Purine compounds such as ATP and adenosine, respectively endothelium‐dependent and‐independent vasodilators, are largely involved in the control of vascular tone and vascular reactivity to contracting stimuli. We investigated the relaxing activity of ATP and adenosine in guinea‐pig aorta rings exposed for 6 h to elevated glucose concentration (50 mM), in order to mimic hyperglycaemic conditions. Guinea‐pigs were reserpine‐treated (2 mg kg−1, i.p., 48 and 24 h before death).

Rings of aortae incubated in 50 mM glucose, contracted submaximally by 1 μM noradrenaline, lost endothelium‐dependent relaxation in response to acetylcholine (10 nM to 10 μM). Aortae incubated with 50 mM mannose, as a hyperosmotic control, relaxed to acetylcholine normally. Rings of aortae incubated in 50 mM glucose, contracted submaximally by 3 mM 4‐aminopyridine, lost endothelium‐dependent relaxation in response to ATP (30 μM) whereas endothelium‐independent relaxation in response to adenosine (0.3 mM) was well preserved.

The relaxation induced by A23187 or sodium nitroprusside (10 nM to 0.1 μM) did not differ between rings exposed to control (5.5 mM) or elevated glucose (50 mM) and contracted submaximally by 3 mM 4‐aminopyridine.

When incubated with aortic tissue in the presence of elevated glucose, the cyclo‐oxygenase inhibitors, indomethacin (10 μM) and mefenamic acid (30 μM), or the scavenger of superoxide anions, superoxide dismutase (150 u ml−1), prevented the impairment of ATP‐mediated relaxation.

The present results indicate that endothelium‐dependent, receptor‐induced relaxation in response to acetylcholine and ATP is impaired in guinea‐pig aorta rings exposed to elevated glucose. The endothelial dysfunction caused by glucose might be located at a step between receptor activation and intracellular calcium increase, and might be related to an increased metabolism of arachidonic acid coupled to an increased production, or to a reduced inactivation of superoxide anions.
Modulatory role of 1,25 dihydroxyvitamin D3 on pancreatic islet insulin release via the cyclic AMP pathway in the rat
Authors: Bourlon P.‐M. , Faure‐Dussert A. , Billaudel B.
Published: August 2006

Previous studies have shown that vitamin D3 deficiency impairs the insulin response to glucose via an alteration of signal transduction pathways, such as Ca2+ handling and the phosphoinositide pathway. In the present study the adenylyl cyclase pathway was examined in islets from 3 independent groups: normal rats, 4 weeks‐vitamin D3 deficient rats and one week‐1,25 dihydroxyvitamin D3 (1,25(OH)2D3) treated rats.

We found that the very low rate of insulin release observed in vitamin D3 deficient rats could be restored in vitamin D3 deficient islets only with high concentrations of dioctanoyl‐cyclic AMP (DO‐cyclic AMP), whereas 1,25(OH)2D3 improved the sensitivity of the islets to this exogenous cyclic AMP analogue.

The beneficial effect of 1,25(OH)2D3 observed with or without DO‐cyclic AMP was protein kinase A‐dependent, since the addition of N‐[2‐(p‐bromocinnamylamino) ethyl]‐5‐isoquinolinesulphonamide (H‐89), a specific inhibitor of cyclic AMP‐dependent protein kinases, decreased the insulin release of treated rats back to the level seen in vitamin D3 deficient islets.

The low rate of insulin release could not be consistently related to an alteration in cyclic AMP content of the islets. Indeed, low insulin response to a barium + theophylline stimulus observed in vitamin D3 deficient islets was paradoxically associated with a supranormal cyclic AMP content in the islets.

This paradoxical increase in cyclic AMP observed in these conditions could not be attributed to a lower total phosphodiesterase (PDE) activity, although the portion of Ca2+‐calmodulin‐independent PDE was predominant in islets from vitamin D3 deficient rats.

On the other hand, the higher cyclic AMP content of vitamin D3 deficient islets could be related to an increase in glucagon‐induced cyclic AMP synthesis in relation to the hyperglucagonaemia previously observed in vitamin D3 deficient rats. Since higher concentrations of exogenous glucagon and higher endogenous cyclic AMP concentrations were required in vitro to restore insulin release to normal values, the cyclic AMP‐dependent pathways that usually potentiate insulin secretion appeared to be less efficient in relation to an alteration in the post cyclic AMP effector system.

1,25(OH)2D3 exerted a stimulating effect on insulin release via protein kinase A activation but reduced the supranormal cyclic AMP synthesis, thus exerting a differential modulatory influence on biochemical disturbances in islets induced by vitamin D3 deficiency.

British Journal of Pharmacology (1997) 121, 751–758; doi:10.1038/sj.bjp.0701204
Identification and pharmacological characterization of somatostatin receptors in rat lung
Authors: Schloos Jürgen , Raulf Friedrich , Hoyer Daniel , Bruns Christian
Published: August 2006

[125I]‐[LTT]SRIF‐28 and [125I]‐SMS 201‐995 were used to identify and characterize somatostatin (SRIF) receptors localized in rat lung tissue. In vitro autoradiography of rat lung tissue sections showed the existence of specific, high affinity binding sites for [125I]‐[LTT]SRIF‐28 without any significant specific binding of the sst2/sst5‐receptor selective ligand [125I]‐SMS 201‐995.

In radioligand binding studies, specific binding of [125I]‐[LTT]SRIF‐28 to membranes of rat lung was linearly related to the concentration of membrane protein used with only a small portion of nonspecific binding. With [125I]‐SMS 201‐995 no specific binding could be observed up to a membrane concentration of 0.1 mg of protein/assay tube.

[125I]‐[LTT]SRIF‐28 bound rapidly to rat lung membranes with an apparent association rate constant (kapp) of 1.8±0.1 h−1 (n=3). The equilibrium of specific binding was reached after an incubation period of approximately 90 min at room temperature and remained constant for the next 3 h. The association rate constant (k1) was calculated to be 3.7×1010 M−1 h−1. The dissociation reaction followed first order kinetics with a dissociation rate constant (k−1)=0.44±0.07 h−1 corresponding to a half‐time of 95±15 min (n=3). From these kinetic experiments an equilibrium dissociation constant (KD) for the binding of [125I]‐[LTT]SRIF‐28 was calculated to be 11.9 pM.

Saturation binding of [125I]‐[LTT]SRIF‐28 revealed an equilibrium dissociation constant (KD) of 50.1 pM (pKD=10.3±0.1; n=3) and a receptor density (Bmax) of 78±3 fmol mg−1 protein. A Hill coefficient not significantly different from 1 indicated saturable binding to a single class of high affinity binding sites.

Specific binding of [125I]‐[LTT]SRIF‐28 to rat lung membranes was inhibited by SRIF‐14, SRIF‐28 and different SRIF analogues. SRIF and different synthetic short chain SRIF analogues exhibited the following rank order of potency: SRIF‐28>SRIF‐14>CGP 23996>>RC 160>BIM 23014>SMS 201‐995>BIM 23056>MK 678.

The binding affinities for SRIF and the various SRIF analogues determined using rat lung tissue were in close correlation to those obtained with Chinese hamster ovary (CHO) cells stably expressing sst1 (r=0.92) and sst4(r=0.95) receptors, respectively.

Reverse transcriptase ‐ polymerase chain reaction (RT‐PCR) showed the predominant expression of mRNA specific for sst4 receptors as well as some weak sst1 mRNA expression.

The findings suggest that sst4 receptor expression is the predominant form of the somatostatin receptors identified in rat lung tissue. In this study we demonstrated for the first time the existence of sst4 receptors in mammalian tissue.
Combined administration of 5‐HT2 and thromboxane A2 antagonists: effects on platelet aggregation and isolated cardiac muscle
Authors: Shaw Linda A , Batey Andrew J , Coker Susan J
Published: August 2006

To investigate possible mechanisms underlying the ability of combined administration of a 5‐hydroxytryptamine2 (5‐HT2) antagonist and a thromboxane A2 antagonist to reduce reperfusion‐induced arrhythmias, the effects of these drugs alone and in combination on platelet aggregation and on cardiac muscle were determined.

Platelet aggregation was measured in whole blood obtained from anaesthetized rats. Concentrations of 5‐HT (10 μM) and the thromboxane A2 mimetic U46619 (1 μM) which did not cause aggregation themselves, enhanced the responses to ADP (0.1 μM) and to collagen (1 μg ml−1). For example, the response of 1.0±0.5 Ω to ADP alone was increased significantly to 6.4±1.0 Ω by 5‐HT, 15.5±2.8 Ω by U46619, and 17.3±1.3 Ω when U46619, 5‐HT and ADP were added together.

In further experiments blood was obtained from rats which had received either the 5‐HT2 antagonist, ICI 170,809 (1 mg kg−1), or the thromboxane A2 antagonist, ICI 192,605 (1 mg kg−1 min−1), or both in combination. When ADP was used as the primary aggregating agent, the ability of U46619 alone, or together with 5‐HT, to enhance responses was reduced significantly by ICI 192,605 alone and in combination with ICI 170,809. Similar results were obtained with lower doses of ICI 170,809 (0.3 mg kg−1) and ICI 192,605 (0.3 mg kg−1 min−1).

When collagen was used as the primary aggregating agent ICI 170,809 (1 mg kg−1) reduced the response to 5‐HT (5.0±0.8 Ω versus 10.9±1.2 Ω in controls), and ICI 192,605 (1 mg kg−1 min−1) reduced the response to U46619 (6.8±2.5 Ω versus 11.2±2.2 Ω in control). The greatest reduction of platelet aggregation was seen in blood from rats which had received both antagonists, with the response to U46619 plus 5‐HT plus collagen being 2.7± 0.6 Ω compared to 14.2±1.7 Ω in controls. In contrast, there was no significant attenuation of platelet aggregation in blood from rats which had received the lower doses of each antagonist alone. Only the combination of ICI 170,809 (0.3 mg kg−1) and ICI 192,605 (0.3 mg kg−1 min−1) reduced the response to U46619 plus 5‐HT plus collagen (7.6±1.4 Ω versus 15.0±0.5 Ω in controls).

In rat isolated ventricular muscle preparations, ICI 170,809 increased the effective refractory period; e.g. from 39±4 to 86±18 ms, 10 min after adding 30 μM to left papillary muscles. ICI 192,605 did not increase the effective refractory period itself and did not alter the ability of ICI 170,809 to prolong the effective refractory period. In the presence of 100 μM ICI 192,605, ICI 170,809 (30 μM) increased the effective refractory period from 38±7 to 100±30 ms.

These results indicate that the previously observed antiarrhythmic activity of combined administration of the higher doses of ICI 170,809 and ICI 192,605 is unlikely to be due to direct effects on cardiac muscle but could be a consequence of reduced platelet aggregation.
Role of lipocortin‐1 in the anti‐hyperalgesic actions of dexamethasone
Authors: Ferreira S H , Cunha F Q , Lorenzetti B B , Michelin M A , Perretti M , Flower R J , Poole S
Published: August 2006

The effect of dexamethasone, lipocorton‐12–26 and an antiserum to lipocortin‐12–26 (LCPS1) upon the hyperalgesic activities in rats of carrageenin, bradykinin, tumour necrosis factor α (TNFα), interleukin‐12, interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin Eβ (PGE2) and dopamine were investigated in a model of mechanical hyperalgesia.

Hyperalgesic responses to intraplantar (i.pl.) injections of carrageenin (100 μg), bradykinin (500 ng), TNFα (2.5 pg), IL‐1β (0.5 pg), and IL‐6 (1.0 ng), but not responses to IL‐8 (0.1 ng), PGE2 (100 ng) and dopamine (10 μg), were inhibited by pretreatment with dexamethasone (0.5 mg kg−1, subcutaneously, s.c., or 0.04–5.0 μg/paw).

Inhibition of hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng) and IL‐1β (0.5 pg) by dexamethasone (0.5 mg kg−1, s.c.) was reversed by LCPS1 (0.5 ml kg−1, injected s.c., 24 h and 1 h before hyperalgesic substances) and hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng), TNFα (2.5 pg) and IL‐1β (0.5 pg), but not responses to PGE2 (100 ng), were inhibited by pretreatment with lipocortin‐12–26 (100 μg/paw). Also, lipocortin‐12–26 (30 and 100 μg ml−1) and dexamethasone (10 μg ml−1) inhibited TNFα release by cells of the J774 (murine macrophage‐like) cell‐line stimulated with LPS (3 μg ml−1), and LCPS1 partially reversed the inhibition by dexamethasone. These data are consistent with an important role for endogenous lipocortin‐12–26 in mediating the anti‐hyperalgesic effect of dexamethasone, with inhibiton of TNFα production by lipocortin‐12–26 contributing, in part, to this role.

Although arachidonic acid by itself was not hyperalgesic, the hyperalgesic response to IL‐1β (0.25 pg, i.pl.) was potentiated by arachidonic acid (50 μg) and the potentiated response was inhibited by dexamethasone (50 μg, i.pl.) and lipocortin‐12–26 (100 μg, i.pl.). Also, lipocortin‐12–26 (30 and 100 μg ml−1) inhibited/abolished PGE2 release by J774 cells stimulated with LPS (3 μg ml−1). These data suggest that, in inflammatory hyperalgesia, inhibition of the induction of cyclo‐oxygenase 2 (COX‐2), rather than phospholipase A2, by dexamethasone and lipocortin‐12–26 accounts for the anti‐hyperalgesic effects of these agents.

The above data support the notion that induction of lipocortin by dexamethasone plays a major role in the inhibition by dexamethasone of inflammatory hyperalgesia evoked by carrageenin, bradykinin and the cytokines TNFα, IL‐1β and IL‐6, and provides additional evidence that the biological activity of lipocortin resides within the peptide lipocortin‐12–26. Further, the data suggest that inhibition of lipocortin‐12–26 of eicosanoid production by COX‐2 also contributes to the anti‐hyperalgesic effect of lipocortin‐1.
Carbon monoxide‐induced vasorelaxation and the underlying mechanisms
Authors: Wang Rui , Wang Zunzhe , Wu Lingyun
Published: August 2006

Carbon monoxide (CO) induced a concentration‐dependent relaxation of isolated rat tail artery tissues which were precontracted with phenylephrine or U‐46619. This vasorelaxing effect of CO was independent of the presence of the intact endothelium.

The CO‐induced vasorelaxation was partially inhibited by the blockade of either the cyclicGMP pathway or big‐conductance calcium‐activated K (KCa) channels. When both the cyclicGMP pathway and KCa channels were blocked, the CO‐induced vasorelaxation was completely abolished.

Incubation of vascular tissues with hemin, in order to enhance the endogenous production of CO, suppressed the phenylephrine‐induced vasocontraction in a time‐ and concentration‐dependent manner. The hemin‐induced suppression of the vascular contractile response to phenylephrine was abolished after the vascular tissues were co‐incubated with either oxyhaemoglobin or zinc protoporphyrin‐IX, suggesting an induced endogenous generation of CO from vascular tissues.

The effect of hemin incubation on vascular contractility did not involve the endogenous generation of nitric oxide.

Our results suggest that CO may activate both a cyclicGMP signalling pathway and KCa channels in the same vascular tissues, and that the endogenously generated CO may significantly affect the vascular contractile responses.
Renal effects of intrathecally injected tachykinins in the conscious saline‐loaded rat: receptor and mechanism of action
Authors: Ding Yuan Yi , Couture Réjean
Published: August 2006

The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [β‐Ala8]NKA (4–10) and [MePhe7]neurokinin B (NKB) at T13 thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline‐loaded rat. Antagonists selective for NK1 (RP 67580), NK2 (SR 48968) and NK3 (R 820; 3‐indolylcarbonyl‐Hyp‐Phg‐N(Me)‐Bzl) receptors were used to characterize the spinal effect of SP on renal function.

Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9±0.8; SP value 0.7±0.3μmolmin−1, P<0.01) as well as the renal excretion of water (aCSF value 48.9±5.8; SP value 14.5±4.0μlmin−1, P<0.01) and potassium (aCSF value 4.8±0.6; SP value 1.5±0.6μmolmin−1, P<0.01) at 30min post‐injection. SP had no significant effect on urinary osmolality. The SP‐induced renal inhibitory effects during the first 30min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5min earlier with 6.5nmol RP 67580. In contrast, the co‐injection of SR 48968 and R 820 (6.5nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (−18.8±3.8mmHg at 1min and +6.3±2.4mmHg at 5min post‐injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes.

NKA (6.5nmol but not 0.65nmol) produced a transient drop in renal excretion of water (aCSF value 31.2±5.1; NKA value 11.3±4.2μlmin−1, P<0.05), sodium (aCSF value 1.7±0.8; NKA value 0.4±0.2μmolmin−1, P<0.05) and potassium (aCSF value 4.1±0.7; NKA value 1.5±0.4μmolmin−1, P<0.05) at 15min post‐injection. However, the same doses (6.5nmol) of selective agonists for tachykinin NK2 ([β‐Ala8]NKA(4–10)) and NK3 ([MePhe7]NKB) receptors were devoid of renal effects.

This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK1 receptors and the sympathetic renal nerve.
Voltage and pH dependent block of cloned N‐type Ca2+ channels by amlodipine
Authors: Furukawa Taiji , Nukada Toshihide , Suzuki Kazuyuki , Fujita Yoshihiko , Mori Yasuo , Nishimura Masao , Yamanaka Masami
Published: August 2006

Two types of Ca2+ channel α1‐subunits were co‐expressed in Xenopus oocytes with the Ca2+ channel α2‐ and β1‐subunits. The Ba2+ current through the α1Cα2β and the α1Bα2β channels had electrophysiological and pharmacological properties of L‐ and N‐type Ca2+ channels, respectively.

Amlodipine had a strong blocking action on both the L‐type and N‐type Ca2+ channels expressed in the oocyte. The potency of the amlodipine block on the N‐type Ca2+ channel was comparable to that on the L‐type Ca2+ channel. At −100mV holding potential, the IC50 values for amlodipine block on the L‐type and N‐type Ca2+ channel were 2.4 and 5.8μm, respectively.

The blocking action of amlodipine on the N‐type Ca2+ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L‐type Ca2+ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine.

The time course of block development by amlodipine was similar for L‐type and N‐type Ca2+ channels. However, it was slower than the time course of block development by nifedipine for the L‐type Ca2+ channel.
Down‐regulation of the hepatic cytochrome P450 by an acute inflammatory reaction: implication of mediators in human and animal serum and in the liver
Authors: El‐Kadi Ayman O S , Maurice Hélène , Ong Huy , du Souich Patrick
Published: August 2006

Infection and inflammation trigger a cascade of mediators that eventually will down‐regulate the hepatic cytochrome P450 (P450). The present study aimed to characterize the mediators contained in the serum of rabbits with an acute inflammatory reaction (AIR) induced by the s.c. injection of turpentine (5ml), and in the serum of humans with an acute upper respiratory tract viral infection.

Hepatocytes from control (HCONT) rabbits and rabbits with an AIR (HINFLA) were isolated and cultured. Compared with HCONT in HINFLA the production of theophylline metabolites, 3‐methylxanthine (3MX), 1‐methyluric acid (1MU), and 1,3‐dimethyluric acid (1,3DMU) was reduced as was the amount of total P450, while lipid peroxidation was increased. Incubation of HINFLA with serum of rabbits with an AIR (RSINFLA) for 4h further reduced the formation of the metabolites of theophylline as well as the amount of P450, and enhanced the lipid peroxidation. RSINFLA obtained 6, 12 and 24h after the injection of turpentine showed the same ability to down‐regulate hepatic P450 as the serum obtained at 48h.

The efficacy (Emax) of RSINFLA to inhibit the formation of theophylline metabolites differed, i.e. 1,3DMU>1MU>3MX, and the potency of serum mediators (IC50) was similar for 3MX and 1MU, but lower for 1,3DMU.

Incubation of serum of human volunteers (HSINFLA) with a viral infection with HCONT or HINFLA reduced the production of theophylline metabolites, as well as the amount of P450, and increased the lipid peroxidation. HSINFLA depressed 1,3DMU more efficiently than 3MX and 1MU. HSINFLA reduced 3MX with greater efficacy than did RSINFLA. Potency was very variable but not different from rabbits.

It is concluded that the serum of rabbits with an AIR or of humans with a viral infection contain several mediators that inhibit noncompetitively various isoenzymes of the hepatic P450. The decrease in P450 induced by HSINFLA or RSINFLA is closely associated with the increase in lipid peroxidation (r2=0.8870) suggesting that lipid peroxidation could directly or indirectly be involved in the P450 down‐regulation.
Pharmacological characterization of type 1α metabotropic glutamate receptor‐stimulated [35S]‐GTPγS binding
Authors: Akam Elizabeth C , Carruthers Alan M , Nahorski Stefan R , John Challiss R A
Published: August 2006

The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [35S]‐guanosine 5′‐[γ‐thio]triphosphate ([35S]‐GTPγS) binding assay.

l‐Glutamate increased the rate of [35S]‐GTPγS binding in a concentration‐dependent manner (−logEC50 (m) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60min incubation at 30°C with 70pm [35S]‐GTPγS, 1μmGDP, 10mmMgCl2, 100mm NaCl and 100μg membrane proteinml−1. The l‐glutamate (100μm)‐stimulated increase in [35S]‐GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)‐4‐carboxy‐3‐hydroxyphenylglycine (300μm).

Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of l‐glutamate revealed an equilibrium dissociation constant (KD) for [35S]‐GTPγS binding of 0.76±0.20nm and a maximal number of GTPγS‐liganded G proteins (Bmax) of 361±30fmol mg−1 protein.

Metabotropic glutamate receptor agonists, quisqualate (−logEC50 (m) 6.74±0.06), 1S,3R‐ACPD (4.64±0.08) and (S)‐3,5‐dihydroxyphenylglycine (5.16±0.23) also increased [35S]‐GTPγS binding in a concentration‐dependent manner, with the latter two agents behaving as partial agonists.

(+)‐α‐Methylcarboxyphenylglycine (300μm) caused a parallel rightward shift of the l‐glutamate concentration‐effect curve for [35S]‐GTPγS binding, allowing an antagonist equilibrium dissociation constant (KD) of 34.0±7.8μm to be calculated for this mGluR antagonist.

Pretreatment of BHK‐mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100ngml−1, 24h) before membrane preparation resulted in a marked decrease in agonist‐stimulated [35S]‐GTPγS binding (by 66.0±0.9%), and an altered concentration‐effect relationship for agonist‐stimulated [35S]‐GTPγS binding by the residual PTX‐insensitive G‐protein population.

The modulation of [35S]‐GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX‐sensitive and ‐insensitive G proteins.
Inducible nitric oxide synthase after sensitization and allergen challenge of Brown Norway rat lung
Authors: Fang Liu Shu , Haddad El‐Bdaoui , Adcock Ian , Salmon Michael , Koto Hiro , Gilbey Tom , Barnes Peter J , Fan Chung K
Published: August 2006

We studied the effects of ovalbumin (OA) sensitization and challenge on inducible nitric oxide synthase (iNOS) gene and protein expression in Brown‐Norway rats in vivo.

By use of Northern analysis, a 4.4‐kb iNOS mRNA transcript was weakly observed in control rat lung but there was a 3 fold increase in lungs sensitized to OA alone (P<0.05). In sensitized rats, four hours after exposure to OA aerosol, there was a 6 fold increase in iNOS mRNA transcript (P<0.05), which returned to baseline at 24 h.

Immunostaining with an anti‐mouse iNOS antibody revealed some patchy staining of airway epithelium in naïve rats. There were no changes in sensitized rats exposed to saline, but sensitized and OA‐exposed rats showed increased expression in iNOS staining in macrophages.

Electrophoretic mobility shift assays of lung nuclear extracts showed a marked increase in nuclear factor‐κB (NF‐κB)‐binding activity at 2 h after allergen exposure with return to baseline at 6, 12 and 24 h.

We concluded that there is increased iNOS gene and protein expression associated with increased NF‐κB DNA‐binding in lungs of sensitized and challenged rats. The increase in iNOS expression may underlie the increase in exhaled NO found after allergen challenge and may contribute to the development of allergen‐induced airway hyperresponsiveness.
Inherent tone of human bronchus: role of eicosanoids and the epithelium
Authors: Watson N , Magnussen H , Rabe K F
Published: August 2006

Airway preparations of different species possess varying degrees of inherent tone which is the result of different metabolites of arachidonic acid in different species. In human bronchial smooth muscle in vitro we have investigated the effects of 5‐lipoxygenase inhibition (zileuton, 10μm), cyclo‐oxygenase inhibition (indomethacin, 1μm) and mechanical epithelium removal on inherent tone. The shunting of arachidonic acid by inhibition of one or other of these enzymes, as a possible explanation for the effects observed, has also been investigated.

Zileuton caused a significant fall in tone either alone (−107±33mg) or after cyclo‐oxygenase inhibition (−203±48mg) and this effect was not significantly altered by epithelial removal (−191±43mg alone; −333±88mg after indomethacin). Indomethacin increased tone when applied alone (160±94mg), but this effect only reached statistical significance after 5‐lipoxygenase inhibition, (210±81mg; P<0.05). Epithelial removal did not alter the effect of indomethacin when applied alone (213±97mg), but significantly reduced the effect of indomethacin after 5‐lipoxygenase inhibition (34±23mg; P<0.05).

These data suggest that inherent tone in human bronchus is largely the result of contractile 5‐lipoxygenase products. However, the involvement of cyclo‐oxygenase products cannot entirely be discounted, since in the presence of 5‐lipoxygenase inhibition contractile and relaxant eicosanoids originating from the bronchial epithelium appear to influence significantly inherent tone.
Effects of dopamine on L‐type Ca2+ current in single atrial and ventricular myocytes of the rat
Authors: Zhao H , Matsuoka S , Fujioka Y , Noma A
Published: August 2006

The effects of dopamine on the L‐type Ca2+ current (ICa,L) of both atrial and ventricular single myocytes and on the force of contraction of atrial trabeculae in rat heart were investigated.

Dopamine increased atrial ICa,L at concentrations higher than 1 μM, but had little or no effect on ICa,L at lower concentrations. The increase in ICa,L at high concentrations was reversed by propranolol and acetylcholine, but not by phentolamine. Activation and inactivation kinetics of ICa,L were not altered by dopamine.

In rat ventricular myocytes in which the D4 receptor mRNA does not express, dopamine (20–100 μM) also increased the ICa,L amplitude and propranolol reversed this effect.

Clozapine, a potent D4 receptor antagonist, blocked the augmenting effect of dopamine on ICa,L. However, this effect could be explained by β‐antagonism, since clozapine also inhibited the isoprenaline effect.

In the atrial trabeculae, the increase in contraction by dopamine (1 to 30 μM) was reversed by 1 μM propranolol, but not by 2 μM phentolamine. Low doses of dopamine (0.01 to 0.3 μM) did not affect the contraction in the controls or during a modest stimulation of the β‐adrenoceptor with 0.01 μM isoprenaline.

These results indicate that the positive inotropic action of dopamine is mediated through direct stimulation of the β‐adrenoceptor in both atrial and ventricular myocytes. Involvement of D4 receptor appears unlikely in the regulation of the atrial contraction.
Effects of the wasp venom peptide, mastoparan, on GTP hydrolysis in rat brain membranes
Authors: Odagaki Y , Nishi N , Koyama T
Published: August 2006

The effects of mastoparan, a wasp venom toxin, on GTP hydrolyzing activity were examined in rat brain membranes.

Mastoparan inhibited the low‐affinity GTPase activity, defined as the amount of 32Pi released from 0.3 μM [γ‐32P]‐GTP in the presence of 100 μM unlabelled GTP, in a concentration‐dependent manner. This inhibitory effect of mastoparan on low‐affinity GTPase activity was diminished by increasing concentrations of UDP and was completely attenuated at 20 mM, indicating that activation of nucleoside diphosphokinase (NDPK) is inolved in the phenomenon.

In the presence of 20 mM UDP, mastoparan stimulated the high‐affinity GTPase activity by increasing the Vmax value without affecting the apparent KM for GTP. Mastoparan‐stimulated high‐affinity GTPase activity was apparent at concentrations higher than 1 μM, in a concentration‐dependent manner, but without saturation even at 100 μM.

Mastoparan‐induced high‐affinity GTPase activity showed a characteristic sensitivity to MgCl2, quite different from that seen in L‐glutamate‐stimulated activity, a representative of receptor‐mediated G‐protein activation.

There appeared to be a simple additive interaction between mastoparan‐ and L‐glutamate‐stimulated high‐affinity GTPase activities, indicting that distinct pools of G‐proteins are involved in receptor‐independent and receptor‐mediated G‐protein activation.

These results suggest that G‐proteins in brain membranes are functionally altered by mastoparan through multiple mechanisms of action and that the mastoparan‐induced, direct G‐protein activating process lacks a synergistic or antagonistic interaction with an agonist‐induced, receptor‐mediated activation of G‐proteins.
Involvement of protein kinase C in reduced relaxant responses to the NO/cyclic GMP pathway in piglet pulmonary arteries contracted by the thromboxane A2‐mimetic U46619
Authors: Pérez‐Vizcaíno Francisco , Villamor Eduardo , Duarte Juan , Tamargo Juan
Published: August 2006

Impairment of nitric oxide (NO)/cyclic GMP production and/or increased activities of thromboxane A2 (TXA2) and endothelin‐1 (ET‐1) have been associated with pulmonary hypertension. We have analysed the interactions of noradrenaline (NA), the TXA2‐mimetic U46619 and ET‐1 with the relaxation induced via cyclic GMP in isolated piglet intrapulmonary arteries.

The contractions induced by NA were augmented by endothelium removal or by methylene blue and pre‐contracted rings were fully relaxed by acetylcholine, sodium nitroprusside (SNP), atrial natriuretic peptide and 8‐bromo‐cyclic GMP. In contrast, U46619‐ and ET‐1 induced contractions were endothelium‐independent and only partially relaxed by the latter vasodilators. Whereas the reduced responses to SNP in arteries contracted by U46619 were independent of the U46619‐induced tone, a higher concentration of ET‐1 (tone higher than that induced by NA) was required to reduce the vasodilator responses to SNP. NA, U46619 and ET‐1 had no effect on the SNP‐induced increases in cyclic GMP.

The reduced relaxant responses to SNP in arteries pre‐contracted by U46619 were specific for piglet pulmonary arteries since they were not observed in piglet mesenteric or coronary arteries or in rat pulmonary arteries. Furthermore, there were no differences in the relaxant response to the adenylate cyclase activator forskolin in piglet pulmonary arteries pre‐contracted by either NA, U46619 or ET‐1.

SNP‐induced relaxation was inhibited by thapsigargin (but not by inhibition of the membrane Na+/K+ ATPase nor K+ channels) indicating a role for Ca2+ sequestration by the Ca2+ ATPase in the effects of SNP.

The phorbol ester 12‐myristate, 13‐acetate inhibited the relaxant response to SNP. The inhibitory effect of U46619 on SNP‐induced relaxation was abolished by the protein kinase C inhibitor (PKC) staurosporine suggesting that PKC may be a part of the signal transduction mechanism.

In summary, piglet pulmonary arteries when activated by a TXA2‐mimetic show abnormally reduced relaxant responses to the NO/cyclicGMP pathway. This effect appears to be mediated by activation of PKC.