bluuebunny/crossref_metadata_2025 huggingface dataset:
Somatostatin receptors in Neuro2A neuroblastoma cells: operational characteristics
Authors: Koenig J A , Edwardson J M , Humphrey P P A
Published: August 2006

We have used somatostatin (SRIF) receptor subtype‐selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF1‐receptor selective ligand, BIM‐23027, was able to displace completely the specific binding of radioiodinated somatostatin, [125I]‐Tyr11‐SRIF‐14, with a pIC50 of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF1 receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM‐23027, indicate that the majority of receptors in Neuro2A cells are of the sst2 subtype.

The stable radioligand, [125I]‐BIM‐23027, bound with high affinity (Kd=13 pm, Bmax=0.2 pmol mg−1 protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4°C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15°C and 89% of the ligand dissociated at 37°C.

Equilibrium binding of [125I]‐BIM‐23027 was partially (25%) inhibited by 10 μm GTP, and by 120 mm NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [125I]‐BIM‐23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 μm) and sodium chloride (120 mm). Under these conditions 67% of the ligand dissociated at 4°C, 81% at 15°C and 93% at 37°C. Binding was totally inhibited by pretreatment of cells with pertussis toxin.

Functionally, BIM‐23027 inhibited forskolin‐stimulated cyclic AMP accumulation in a concentration‐dependent manner with an IC50 of 1.0 nm and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst2 receptor, no rise in intracellular calcium concentration was observed with SRIF‐14.

We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst2 type. In addition, we have found that activation of the receptor is associated with ligand‐receptor internalisation.

British Journal of Pharmacology (1997) 120, 45–51; doi:10.1038/sj.bjp.0700858
Inhibition by the immunosuppressive agent FK‐506 of antigen‐induced airways eosinophilia and bronchial hyperreactivity in mice
Authors: Eum Seok‐Yong , Zuany‐Amorium Claudia , Lefort Jean , Pretolani Marina , Vargaftig B Boris
Published: August 2006

The effect of the immunosuppressive agent, FK‐506, on allergen‐induced airways eosinophilia and bronchial hyperreactivity (BHR) in hyper IgE mice (BP2 selection) was investigated.

Administration of FK‐506 at 2 mg kg−1 s.c., 1 h before and 5 h after the first of four ovalbumin challenges, reduced the recruitment of eosinophils into the bronchoalveolar lavage fluid (BALF) from 1.36±0.22×105 to 0.53±0.24×105 cells ml−1 (n=5–6, P<0.05; 60% inhibition), inhibited by 80% BHR in response to i.v. 5‐HT and practically suppressed BHR in response to inhaled methacholine.

The antigen‐induced interleukin (IL)‐5 formation in the BALF and serum was inhibited by FK‐506 by 75% in both instances.

FK‐506 failed to modify the bronchoconstriction in BP2 mice, suggesting that different mechanisms are involved in acute bronchoconstriction and BHR.

The increased number of CD4+, CD8+, CD3+ T lymphocytes in the BALF of antigen‐challenged mice was unaffected by FK‐506.

These findings indicate that antigen‐induced in vivo IL‐5 release and eosinophil, but not T‐cell, infiltration into the bronchial lumen of sensitized BP2 mice are targets for the anti‐allergic activities of FK‐506.

British Journal of Pharmacology (1997) 120, 130–136; doi:10.1038/sj.bjp.0700860
Lack of haemodynamic effects of nitric oxide on post‐capillary pulmonary hypertension induced by acute sino‐aortic denervation
Authors: Galinier Michel , Rougé Pierre , Fourcade Joëlle , Senard Jean‐Michel , Albenque Jean‐Paul , Balanescu Serban , Doazan Jean‐Philippe , Montastruc Jean‐Louis , Bounhoure Jean‐Paul , Montastruc Paul
Published: August 2006

The aims of the present experiments were to define a new experimental model of pulmonary hypertension induced by a post‐capillary mechanism and to assess the haemodynamic effects of nitric oxide on post‐capillary pulmonary hypertension.

Cardiopulmonary variables of 28 male beagle dogs, anaesthetized with chloralose, 16 spontaneous breathing and 12 with assisted ventilation, were studied before and after sino‐aortic denervation (SAD). The haemodynamic effects of inhaled nitric oxide (25 p.p.m., 10 min), Nω‐nitro‐l‐arginine methyl ester (20 mg kg−1, i.v.), urapidil (0.5 mg kg−1, i.v.) and propranolol (300 μg kg−1, i.v.) were studied after SAD.

SAD induced an acute and transient pulmonary hypertension, more marked in spontaneous breathing dogs. This pulmonary hypertension involved a post‐capillary mechanism, secondary to the left ventricular haemodynamic effects of the acute increase of left ventricular after‐load induced by systemic hypertension. In fact, the increase of mean pulmonary arterial pressure after SAD and the decrease of this parameter after urapidil or propranolol were strongly correlated with the variations of pulmonary capillary wedge pressure. Furthermore, no significant change in pulmonary vascular resistances was found after SAD or administration of α or β‐adrenoceptor antagonists.

Inhaled nitric oxide did not reverse pulmonary hypertension induced by SAD. Nω‐nitro‐l‐arginine methyl ester had no significant haemodynamic effect on pulmonary circulation.

In conclusion, the lack of effect of inhaled nitric oxide and nitric oxide synthase inhibitor on pulmonary circulation parameters after SAD suggests that endothelium‐derived nitric oxide is not involved in the mechanisms leading to post‐capillary pulmonary hypertension.

British Journal of Pharmacology (1997) 120, 7–12; doi:10.1038/sj.bjp.0700864
Antagonist inhibition curves and the measurement of dissociation constants
Authors: Barlow R B , Bond Susan M , Bream Elizabeth , Macfarlane Lisa , McQueen D S
Published: August 2006

Experiments carried out on guinea‐pig isolated ileum with carbachol as agonist and diphenyl‐ acetoxyethyl‐ dimethyl‐ethyl‐ ammonium (DADMEA) bromide as antagonist gave results which fit the theoretical relation between fractional inhibition (Q) of the effects of an agonist ([A]) and the concentration of a competitive antagonist ([B]): this also involves the Hill coefficient (logistic slope factor, P) for the agonist concentration‐response curve and the degree of agonist stimulation, [A]/[A]50, where [A]50 produces a half‐maximum response.

Values of IC50 and an exponent, P′, can be obtained by fitting Q to [B] using a logistic approximation to the relation. Both P′ and IC50 should be greater with higher agonist stimulation but the increase in P′ may be masked by errors in extreme values of Q. Estimates of IC50, however, invariably increased with higher agonist stimulation but with a steep concentration‐response curve (P>1) and low agonist stimulation ([A]/[A]50 <1), IC50 can be less than KD.

K
D was calculated from the results in three ways: (i) by a least‐squares fit of Q to [B] using the values of P and [A]/[A]50 calculated from the control concentration‐response curve; (ii) from the value of IC50 for each line and the values of P and [A]/[A]50 and (iii) by using the agonist concentration‐response curve to calculate the dose‐ratio and estimate of KD for each response in the presence of the antagonist. The methods gave similar results (nm: 11 experiments), 12.4±1.1 (i), 11.7±0.9 (ii), 14.8±1.6 (iii) but there are advantages in using methods (i) or (ii) rather than (iii).

The method by which KD is calculated is less important than the experimental design: the plan used in this work, with alternative small and large responses from the tissue, is very suitable for estimating KD with low concentrations of antagonists and small dose‐ratios. Although it is not a sensitive test for competitive behaviour because only a small range of concentrations of antagonist is tested, the estimate of affinity should be free from complications involved in the use of higher concentrations of antagonist (and agonist) and the nature of the antagonism can always be checked by doing further experiments in the presence of a known competitive antagonist.

British Journal of Pharmacology (1997) 120, 13–18; doi:10.1038/sj.bjp.0700865
Depressant effects of hypoxia and hypoglycaemia on neuro‐effector transmission of guinea‐pig intestine studied in vitro with a pharmacological model
Authors: Corbett A D , Lees G M
Published: August 2006

Since intermittent ischaemia may play an important role in the ætiology of Inflammatory Bowel Disease, particularly Crohn's Disease, a pharmacological model of neuronal ischaemia was applied to guinea‐pig isolated intestinal preparations to mimic the acute effects of reduced blood flow on intestinal motility.

Neuro‐effector transmission and smooth muscle performance were examined in myenteric plexus‐longitudinal muscle preparations of guinea‐pig ileum exposed to sodium cyanide (NaCN), in order to inhibit oxidative phosphorylation, or to iodoacetic acid (IAA), to block glycolysis. Comparisons were made with the effects due to simple deprivation of oxygen or glucose.

Depressions of cholinergic neuro‐effector transmission induced by hypoxia or NaCN (effective concentration range 0.1–3 mm), given as separate treatments, singly or repetitively over 60–90 min, were apparent within 30 s and were reversible. The maximum inhibition was 90% and the IC50 for NaCN was 0.3 mm. A conspicuous component of these inhibitions was prejunctional.

Non‐cholinergic neuro‐effector contractions were inhibited by up to 90% by anoxia or NaCN but recovery was incomplete and slower than with cholinergic contractions.

Glucose‐free solutions also caused a reversible failure of cholinergic neuro‐effector transmission but of slower onset. In contrast, IAA (0.06–1 mm) abolished contractions irreversibly, apparently by a direct depressant effect on smooth muscle contraction. Unlike NaCN, IAA caused an initial potentiation of electrically‐induced contractions, partly by a prejunctional potentiation of cholinergic neuro‐effector transmission.

It is concluded that a disruption of intestinal activity in pathological conditions associated with intestinal ischaemia may result from disturbances in the function of enteric neurones.

British Journal of Pharmacology (1997) 120, 107–115; doi:10.1038/sj.bjp.0700870
Electrophysiological effects of CI‐980, a tubulin binding agent, on guinea‐pig papillary muscles
Authors: Pérez Onésima , Valenzuela Carmen , Delpón Eva , Tamargo Juan
Published: August 2006

The electrophysiological effects of CI‐980, a new tubulin‐binding agent that inhibits assembly of cytoplasmic microtubules, on transmembrane action potential characteristics were studied in right ventricular papillary muscles from guinea‐pig hearts.

In papillary muscles driven at 1 Hz, CI‐980 at concentrations 10−5 m produced a concentration‐dependent increase in the maximum upstroke velocity (Vmax) and a lengthening of the action potential duration at 50% (APD50) and 90% (APD90) of repolarization without affecting the resting membrane potential. Prolongation of the APD90 was accompanied by a parallel lengthening of the effective refractory period (ERP) so that the ERP/APD90 ratio remained unaltered at all drug concentrations tested.

CI‐980 exhibits a reverse use‐dependent effect on APD90 values, that is, drug‐induced APD90 prolongation become exagerated at slow rates and attenuated at fast rates.

CI‐980 at concentrations 10−6 m lengthened the APD of the slow action potentials elicited by isoprenaline in papillary muscles depolarized by high K+ (27 mm) solution.

At 10−5 m, CI‐980 produced a small tonic Vmax block. However, in muscles driven at rates between 0.5 and 3 Hz it produced an exponential decline in Vmax (use‐dependent Vmax block) which was augmented at higher rates of stimulation. At 3 Hz the onset kinetics of the use‐dependent Vmax block was fitted by a monoexponential function with a K value 0.07±0.01 per AP. The recovery time constant (τre) from the use‐dependent Vmax block was prolonged from 21.6±2.6 ms to 3.5±0.2 s.

The curve relating membrane potential and Vmax was shifted by CI‐980 (10−5 m) in the hyperpolarizing direction by 2.3±1.1 mV.

It is concluded that in guinea‐pig papillary muscles, CI‐980 produces a use‐dependent inhibition of Vmax and a reverse use‐dependent prolongation of the ventricular action potential, thus exhibiting class I and class III antiarrhythmic actions, respectively. From the onset and offset kinetics of use‐dependent Vmax block, CI‐980 can be considered to be an intermediate kinetics (IA) Na+ channel blocker.

British Journal of Pharmacology (1997) 120, 187–192; doi:10.1038/sj.bjp.0700878
Contribution of adrenal hormones to nicotine‐induced inhibition of synovial plasma extravasation in the rat
Authors: Jia‐Pei Miao Frederick , Benowitz Neal L , Heller Philip H , Levine Jon D
Published: August 2006

In this study, we examined the mechanism(s) by which s.c. nicotine inhibits synovial plasma extravasation. We found that nicotine dose‐dependently inhibited bradykinin (BK)‐ and platelet activating factor (PAF)‐induced plasma extravasation.

The effect of nicotine on both BK‐ and PAF‐induced plasma extravasation was attenuated by adrenal medullectomy. ICI‐118,551 (a selective β2‐adrenoceptor blocker) (30 μg ml−1, intra‐articularly) significantly attenuated the inhibitory action of high‐dose (1 mg kg−1) nicotine on BK‐induced plasma extravasation without affecting the inhibition by low‐ (0.01 μg kg−1) dose nicotine or that on PAF‐induced plasma extravasation by nicotine at any dose. This suggested that β2‐adrenoceptors mediate the inhibitory actions of high‐dose, but not low‐dose, nicotine. We also found that systemic naloxone (an opioid receptor antagonist) (two hourly injections of 1 mg kg−1, i.p.) attenuated the inhibitory action produced by all doses of nicotine on BK‐ or PAF‐induced plasma extravasation, suggesting the contribution of endogenous opioids.

RU‐38,486 (a glucocorticoid receptor antagonist) (30 mg kg−1, s.c.) and metyrapone (a glucocorticoid synthesis inhibitor) (two hourly injections of 100 mg kg−1, i.p.) both attenuated the action of high‐dose nicotine without affecting that of low‐dose nicotine.

Spinal mecamylamine (a nicotinic receptor antagonist) (0.025 mg kg−1, intrathecally, i.t.) attenuated the action of high‐dose, but not low‐dose, nicotine, suggesting that part of the action of high‐dose nicotine is mediated by spinal nicotinic receptors.

Combined treatment with ICI‐118,551, naloxone and RU‐38,486 attenuated the action of low‐dose nicotine by an amount similar to that produced by naloxone alone but produced significantly greater attenuation of the effect of high‐dose nicotine when compared to the action of any of the three antagonists alone.

British Journal of Pharmacology (1997) 120, 298–304; doi:10.1038/sj.bjp.0700884
Selective block of N‐methyl‐d‐aspartic acid (NMDA)‐evoked whole‐cell currents in mouse cultured spinal neurones by CGP 40116
Authors: D'Hooge R , Raes A , Van Bogaert P P , Geelhand M , De Deyn P P
Published: August 2006

CGP 40116 is the active (R)‐enantiomer of the most potent N‐methyl‐d‐aspartic acid (NMDA) receptor antagonist presently available: 2‐amino‐4‐methyl‐5‐phosphono‐3‐pentenoic acid (CGP 37849). In this study, we describe the effect of CGP 40116 on whole‐cell currents induced by excitatory amino acids in cultured mouse spinal cord cells by use of the whole‐cell patch‐clamp technique.

We found that application of CGP 40116 in the nm range, concentration‐dependently inhibited whole‐cell current evoked by 20 μm NMDA in mouse cultured spinal neurones (IC50±s.e.mean 48±8 nm CGP 40116).

The compound appeared to be highly selective for the NMDA current. At concentrations as high 1 μm, currents evoked by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) or kainic acid were not affected by CGP 40116. The threshold concentration for antagonism of NMDA‐induced responses was 10 nm suggesting a selectivity ratio of 100 fold for NMDA receptors versus AMPA or kainate receptors.

CGP 40116 produced a parallel rightward displacement of the NMDA log concentration‐current curve indicating competitive antagonism at the transmitter recognition site of the NMDA receptor complex. An apparent dissociation constant for the antagonist was calculated from the displacement of the agonist concentration‐current curve: 117±53 nm CGP 40116 (estimated Kd±s.e.). Like other competitive NMDA antagonists, CGP 40116 blocked NMDA‐evoked current in a voltage‐independent manner.

British Journal of Pharmacology (1997) 120, 211–214; doi:10.1038/sj.bjp.0700886
In vitro and in vivo effects of UP 269‐6, a new potent orally active nonpeptide angiotensin II receptor antagonist, on vascular smooth muscle cell proliferation
Authors: Virone‐Oddos A. , Desangle V. , Provost D. , Cazes M. , Caussade F. , Cloarec A.
Published: August 2006

The present studies were designed to measure the affinity of UP 269‐6, a newly developed angiotensin AT1 receptor antagonist, for vascular AT1 receptors from normotensive and hypertensive rats and to investigate in vitro, its effects on angiotensin II (AII)‐induced hyperplasia and hypertrophy of vascular smooth muscle cells (VSMC). In addition the in vivo effects of UP 269‐6 on neointimal proliferation in a carotid artery balloon injury in normotensive rats were also investigated.

UP 269‐6 selectively inhibited [125I]‐Sar1‐Ile8‐AII binding to vascular AT1 receptors present on VSMC derived from normotensive Wistar rat and from SHR (Ki= 16.6 ± 3.6 nM and 7.5 ± 2.0 nM, respectively). In comparison, losartan and its metabolite, EXP 3174, inhibited [125I‐Sar1‐Ile8‐AII binding to vascular AT1 receptors derived from both cell models with Ki values slightly lower (losartan) and higher (EXP 3174), respectively, than that of UP 269‐6.

AII (1 μM) induced a weak and variable hyperplastic response (4 to 32% increase in cell number) in Wistar rat VSMC after 96 h.

AII (1 μM) induced a time‐dependent increase in cell number in VSMC from SHR. UP 269‐6 inhibited concentration‐dependently this effect with an IC50 value of 159 ± 58 nM. Losartan was clearly less potent and EXP 3174 showed nearly the same inhibitory potency, compared to UP 269‐6. UP 269‐6 (1 μm) inhibited nearly completely the action of AII.

AII (500 nM) caused maximal stimulation of protein synthesis in Wistar rat VSMC (117 ± 36%). UP 269‐6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 28 ± 6 nM, 3504 ± 892 nM and 21±3 nM, respectively.

AII (50 nM) induced maximal stimulation of protein synthesis in SHR VSMC (237 ± 67%). UP 269‐6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 16 ± 3 nM, 282± 122 nM and 3.3 ± 1.0 nM, respectively.

UP 269‐6 (75 mg kg−1 day−1) administered orally in the diet for 20 days induced a 38% reduction in neointimal area and a 36% reduction in neointima/media ratio associated with the intimal thickening induced by carotid artery balloon injury.

In conclusion, UP 269‐6 was shown to be a potent antiproliferative agent both in vitro on AII‐induced hyperplasia and hypertrophy of VSMC derived from normotensive and hypertensive rats, and in vivo upon intimal thickening induced by carotid artery balloon injury in the rat.
Bradykinin‐induced contractions of canine saphenous veins: mediation by B2 receptors and involvement of eicosanoids
Authors: Marsault Robert , Illiano Stéphane , Vanhoutte Paul M
Published: August 2006

Experiments were designed to determine the subtype of kinin‐receptors mediating the contraction of venous smooth muscle to bradykinin and to investigate the involvement of metabolites of arachidonic acid in this response.

Bradykinin (10−9 to 10−6 m) caused concentration‐dependent contractions of the canine isolated saphenous vein without endothelium, which were potentiated by indomethacin (10−5 m, an inhibitor of cyclo‐oxygenase). The concentration‐response curve was biphasic, reaching an asymptote at 10−8 m and a secondary maximal response at 10−6 m.

Bradykinin (10−8 m to 3×10−6 m) caused a three fold stimulation in the release of the vasodilator prostaglandin E2 (PGE2) and a two fold stimulation of that of the vasodilator prostacyclin, measured by the production of 6‐keto‐PGF1α (its stable breakdown product).

Under control conditions, nordihydroguaiaretic acid (NDGA, 10−5 m), an inhibitor of lipoxygenase, did not affect the response to bradykinin. In the presence of indomethacin (10−5 m), NDGA reduced contractions to bradykinin, suggesting the involvement of lipoxygenase metabolites in the potentiation evoked by the inhibitor of cyclo‐oxygenase.

The selective B1 receptor agonist [des‐Arg9]‐bradykinin, in the concentration‐range 10−6 to 10−5 m, induced contractions, which were abolished by the B2 receptor antagonist d‐Arg‐[Hyp3,Thi5,d‐Tic7,Oic8]‐bradykinin (Hoe 140, 10−6 m). The selective B1 receptor antagonist [des‐Arg9,Leu8]‐bradykinin, (10−7 to 10−5 m) had no significant effect on bradykinin‐induced contractions.

The B2 receptor antagonists Hoe 140 (10−8 to 10−6 m) and d‐Arg[Hyp3,d‐Phe7]‐bradykinin (10−7 to 10−5 m) shifted the concentration‐response curve to bradykinin to the right in a concentration‐dependent manner.

These results indicate that, in the canine saphenous vein, bradykinin causes contraction by activating B2 receptors. This results in the production of metabolites of arachidonic acid, which play a key role in the contraction of canine saphenous venous smooth muscle.

British Journal of Pharmacology (1997) 120, 215–220; doi:10.1038/sj.bjp.0700898
The effects of recombinant human granulocyte‐colony stimulating factor on vascular dysfunction and splanchnic ischaemia‐reperfusion injury
Authors: Squadrito Francesco , Altavilla Domenica , Squadrito Giovanni , Campo Giuseppe M , Ioculano Mariapatrizia , Ammedolia Letteria , Arlotta Mariarita , Saitta Antonino , Caputi Achille P
Published: August 2006

The aim of our study was to investigate the effects of recombinant human granulocyte‐colony stimulating factor in a rat model of splanchnic ischaemia‐reperfusion injury.

Male anaesthetized rats were subjected to clamping of the splanchnic arteries for 45 min. This surgical procedure resulted in an irreversible state of shock (splanchnic artery occlusion shock; SAO shock). Sham operated animals were used as controls. Survival rate, serum tumour necrosis factor‐α (TNF‐α), neutrophil count, bone marrow myeloid precursor cells, myeloperoxidase activity (MPO; studied as a quantitative means to assess leukocyte accumulation), mean arterial blood pressure and the responsiveness of aortic rings to phenylephrine (PE, 1 nm–10 μm) were studied.

SAO shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham shocked rats survived more than 4 h), increased serum levels of TNF‐α (201±10 u ml−1; sham shocked rats=undetectable), neutropenia, enhanced MPO activity in the ileum (0.11±0.06 u × 10−3 g−1 tissue; sham shocked rats=0.02±0.001 u × 10−3 g−1 tissue) and in the lung (1.5±0.2 u × 10−3 g−1 tissue; sham shocked rats=0.19±0.05 u × 10−3 g−1 tissue) and unchanged bone marrow myeloid precursor cells. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to PE.

Administration of recombinant human granulocyte colony stimulating factor (rh G‐CSF; 5, 10 and 20 μg kg−1 5 min following the release of occlusion) increased in a dose‐dependent manner survival rate (90% at 4 h of reperfusion with the dose of 20 u × 10−3 g kg−1), reduced serum TNF‐α (13±5 u ml−1) and MPO activity in the ileum (0.065±0.002 u × 10−3 g−1 tissue) and in the lung (0.7±0.03 μ g kg−1 tissue), improved neutropenia and mean arterial blood pressure but did not modify bone marrow myeloid progenitor cells. Furthermore rh G‐CSF, either in vivo or in vitro (200 nm for 1 h in the organ bath), restored to control values the hyporeactivity to PE. Finally rh G‐CSF potently inhibited the activity of inducible nitric oxide synthase in peritoneal macrophages activated with endotoxin.

Our results suggest that rh G‐CSF protects against splanchnic ischaemia reperfusion injury by a mechanism(s) that does not depend upon its haematopoietic effects.

British Journal of Pharmacology (1997) 120, 333–339; doi:10.1038/sj.bjp.0700904
The antihypertensive effect of orally administered nifedipine‐loaded nanoparticles in spontaneously hypertensive rats
Authors: Kim Young Il , Fluckiger Laurence , Hoffman Maurice , Lartaud‐Idjouadiene Isabelle , Atkinson Jeffrey , Maincent Thilippe
Published: August 2006

The therapeutic use of nifedipine is limited by the rapidity of the onset of its action and its short biological half‐life. In order to produce a form devoid of these disadvantages we made nanoparticles of nifedipine from three different polymers, poly‐ɛ‐caprolactone (PCL), polylactic and glycolic acid (1:1) copolymers (PLAGA), and Eudragit RL/RS (Eudragit). Nifedipine in polyethylene glycol 400 (PEG) solution was used as a control.

The average diameters of the nanoparticles ranged from 0.12 to 0.21 μm; the encapsulation ratio was 82% to 88%.

In spontaneously hypertensive rats (SHR), the initial rapid fall in systolic arterial blood pressure following oral administration of nifedipine in PEG solution (from 193 ± 3 to 102 ± 2 mmHg) was not seen following administration of the same dose in Eudragit nanoparticles (from 189 ± 2 to 156 ± 2 mmHg); with PCL and PLAGA nanoparticles the initial fall in blood pressure was significantly reduced (nadirs PCL 124 ± 2 and PLAGA 113 ± 2 mmHg). Ten hours following administration, blood pressure in rats administered the nifedipine/PEG preparation had returned to normal (183 ± 3 mmHg) whereas that of animals given nifedipine in nanoparticles (PCL 170 ±3, PLAGA 168 ± 2, Eudragit 160 ±3 mmHg) was still significantly reduced.

All of the nanoparticle dosage forms decreased Cmax and increased Tmax and the mean residence time (MRT) values. Relative bioavailability was significantly increased with Eudragit nanoparticles compared to the nifedipine/PEG solution.

There was an inverse linear correlation between the fall in blood pressure and plasma nifedipine concentration with all preparations.

The nanoparticle nifedipine preparations represent sustained release forms with increased bioavailability, a less pronounced initial antihypertensive effect and a long‐lasting action.
Effect of adrenergic and nitrergic blockade on experimental ileus in rats
Authors: De Winter Benedicte Y. , Boeckxstaens Guy E. , De Man Joris G. , Moreels Tom G. , Herman Arnold G. , Pelckmans Paul A.
Published: August 2006

In a rat model of experimental ileus, the effect of blockade of adrenergic and nitrergic neurotransmission was studied on the intestinal transit of Evans blue.

Ether anaesthesia and skin incision had no influence on the transit. Laparotomy significantly inhibited the transit of Evans blue. This inhibition was even more pronounced when the small intestine was manipulated.

Reserpine (5 mg kg−1), a drug that blocks adrenergic neurotransmission, completely reversed the inhibition of the transit induced by laparotomy but only partially reversed that induced by laparotomy with manipulation of the small intestine.

Nω‐nitro‐L‐arginine (L‐NOARG, 5 mg kg−1), a nitric oxide synthase inhibitor, completely reversed the reserpine‐resistant inhibition induced by laparotomy with manipulation of the small intestine. The effect of L‐NOARG was prevented by concomitant administration of L‐arginine. L‐Arginine itself slightly, but significantly enhanced the inhibition. S‐methylisothiourea and aminoguanidine, selective inhibitors of the inducible NO synthase, had no effect on the transit after the three operations.

Treatment of the rats with reserpine plus L‐NOARG had no additional effect on the transit after laparotomy as compared to reserpine alone. However, reserpine plus L‐NNA completely reversed the inhibition of the transit induced by laparotomy with manipulation of the small intestine.

These findings support the involvement of adrenergic pathways in the pathogenesis of ileus and suggest that the additional inhibitory effect of mechanical stimulation results from an enhanced release of NO by the constitutive NO synthase.
The NK1 receptor and its participation in the synergistic enhancement of corneal epithelial migration by substance P and insulin‐like growth factor‐1
Authors: Nakamura Masatsugu , Ofuji Keiko , Chikama Tai‐ichiro , Nishida Teruo
Published: August 2006

We have previously shown that substance P (SP) and insulin‐like growth factor‐1 (IGF‐1) act synergistically to enhance the migration of rabbit corneal epithelial cells in an organ culture model. The present study was designed to identify the epithelial cell SP receptor that participates in this synergistic effect.

Rabbit corneal blocks were incubated for 24 h, then the length of the path of epithelial migration was measured. Reagents tried in the TC‐199 culture medium, in the presence or absence of IGF‐1, were: SP, agonists of tachykinin receptors NK1; NK2or NK3 and antagonists of tachykinin receptors NK1 or NK2.

The binding characteristics of SP receptors were examined in rabbit cultured corneal epithelial cells by binding assays with [125I]‐SP in the presence or absence of excess unlabelled SP or ligands of NKl, NK2 or NK3 receptors.

As was demonstrated previously, SP and IGF‐1 stimulated epithelial migration when they were added to the culture medium together, but individually they had no effect. NK1 agonists had the same synergistic effect with IGF‐1 as did SP, but the NK2 and NK3 agonists did not. Furthermore, the NK1 antagonist abolished the synergistic effect of SP and IGF‐1, but the NK2 antagonist had no effect.

SP bound specifically to rabbit cultured corneal epithelial cells. The binding affinity was 0.44 nM and there were 2.43 × 104 binding sites per cell. The NK1 ligand competed, in a dose‐dependent fashion, with the binding of SP to corneal epithelial cells, but neither the NK2 nor NK3 ligand affected binding.

We conclude that the SP receptor in rabbit corneal epithelial cells is NK1 and that this receptor participates in the synergistic enhancement of corneal epithelial migration by SP and IGF‐1. The precise mechanism(s) of this interaction requires more study. These findings imply that both neural and humoral factors are essential for the maintenance and healing of corneal epithelium.
Subunit‐dependent interaction of the general anaesthetic etomidate with the γ‐aminobutyric acid type A receptor
Authors: Hill‐Venning Claire , Belelli Delia , Peters John A , Lambert Jeremy J
Published: August 2006

The GABA modulating and GABA‐mimetic actions of the general anaesthetic etomidate were examined in voltage‐clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ‐aminobutyric acidA (GABAA) receptor subunits.

Currents mediated by recombinant receptors with the ternary subunit composition αxβyγ2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC10, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms.

For the β2‐subunit containing receptors tested, the EC50 for the potentiation of GABA‐evoked currents by etomidate (range 0.6 to 1.2 μm) was little affected by the nature of the α subunit present within the hetero‐oligomeric complex. However, replacement of the β2 by the β1 subunit produced a 9–12 fold increase in the etomidate EC50 (6 to 11 μm) for all α‐isoforms tested.

For α1, α2 and α6, but not α3‐subunit containing receptors, the maximal potentiation of GABA‐evoked currents by etomidate was greater for β2‐ than for β1‐subunit containing receptors. This was most clearly exemplified by receptors composed of α6β1γ2L compared to α6β2γ2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC10 to 28±2% and 169±4% of the maximal GABA response, respectively.

For α1 subunit‐containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α‐pregnan‐3α‐ol‐20‐one was marginally higher for β1 rather than the β2 subunit‐containing receptor, although its maximal effect was similar at the two receptor isoforms.

The GABA‐mimetic action of etomidate was supported by β2‐ but not β1‐subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β2‐subunit containing receptors, both the agonist EC50 and the maximal current produced by etomidate were additionally influenced by the α isoform.

It is concluded that the subtype of β‐subunit influences the potency with which etomidate potentiates GABA‐evoked currents and that the β isoform is a crucial determinant of the GABA‐mimetic activity of this compound. The nature of the α‐subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

British Journal of Pharmacology (1997) 120, 749–756; doi:10.1038/sj.bjp.0700927
Protection by amylin of gastric erosions induced by indomethacin or ethanol in rats
Authors: Guidobono F. , Pagani F. , Ticozzi C. , Sibilia V. , Pecile A. , Netti C.
Published: August 2006

The effect of amylin on gastric ulcers induced by oral administration of indomethacin (Indo, 20 mg kg−1 at a dosing volume of 5 ml) or ethanol 50% (EtOH, 1 ml/rat) was investigated in conscious rats.

Amylin given intracerebroventricularly (0.22, 0.66 and 2.2 μg/rat, i.c.v.) demonstrated a dose‐dependent cytoprotective effect against both Indo and EtOH‐induced ulcers. In contrast, amylin, given subcutaneously at doses effective in inhibiting acid gastric secretion (2.5, 10 and 40 μg kg−1, s.c), did not show any cytoprotective effect.

The interaction between amylin and endogenous nitric oxide (NO) in the maintenance of gastric mucosal integrity was investigated by pretreating the rats with a selective inhibitor of NO‐synthesis, NG‐nitro‐L‐arginine methyl ester (l‐NAME, 25 and 70 mg kg−1, s.c). Administration of L‐NAME to rats did not significantly increase the degree of the Indo‐induced ulcer index and was not able to remove the protective effect of amylin on Indo‐induced ulcers, thus excluding a role for endogenous NO in mediating the protective effect of this peptide.

To determine whether the cytoprotective effect of amylin was mediated by endogenous prostaglandins, we studied the effect of amylin (2.2 μg/rat, i.c.v.) on EtOH‐induced ulcers in rats pretreated with Indo (10 mg kg−1, s.c.) to inhibit prostanoid biosynthesis; Indo was injected 30 min before amylin and EtOH after a further 30 min. Pretreatment with Indo did not significantly increase the ulcer index induced by EtOH but counteracted the ability of amylin to prevent the ulcer formation.

These findings suggest that amylin exerts a gastroprotective activity that is not strictly related to inhibition of acid gastric secretion and can be partly explained through a prostaglandin‐dependent mechanism mediated by receptors for the peptide in the brain. Amylin might be considered as a new brain‐gut peptide.
Role of N‐, P‐and Q‐type voltage‐gated calcium channels in transmitter release from sympathetic neurones in the mouse isolated vas deferens
Authors: Waterman Sally A.
Published: August 2006

N‐type voltage‐gated calcium channels are known to play an important role in transmitter release from autonomic neurones, and recent studies have demonstrated that non‐N‐type calcium channels are also involved. The calcium channels coupled to transmitter release from sympathetic neurones in the mouse isolated vas deferens were investigated in the present study.

Contractions of the mouse vas deferens were evoked by electrical stimulation at 1–50 Hz. The contractions were entirely nerve‐mediated, since they were abolished by tetrodotoxin, and were used as an indirect measure of transmitter release.

The N‐type calcium channel blocker, ω‐conotoxin GVIA, inhibited contractions in a concentration‐dependent manner, with a maximal effect at 30 nM. Contractions evoked by stimulation frequencies less than 10 Hz were abolished, and those evoked by 20 and by 50 Hz stimulation were decreased in amplitude by 51.3 ± 13.9% and 9.3±2.6%, respectively.

The N‐, P‐and Q‐type channel blocker, ω‐conotoxin MVIIC, inhibited contractions in a concentration‐dependent manner and caused greater maximum inhibition than ω‐conotoxin GVIA, suggesting an action on P‐and/or Q‐type channels, in addition to N‐type.

The P‐type channel blocker, ω‐agatoxin IVA, alone did not have a significant effect at concentrations up to 300 nM, but inhibited contractions in the presence of ω‐conotoxin GVIA. Subsequent addition of ω‐conotoxin MVIIC abolished the remaining contractions. Identical results were obtained when the three toxins were tested cumulatively on the purinergic and noradrenergic components of the contraction in the presence of 0.3 μM prazosin and following desensitization to 10μM μ,β‐methylene adenosine 5′‐triphosphate (α,β‐MeATP), respectively.

The results suggest that N‐, P‐and Q‐type channels are involved in the release of noradrenaline and ATP from sympathetic neurones in the mouse vas deferens.
The identification of an orally active, nonpeptide bradykinin B2 receptor antagonist, FR173657
Authors: Asano Masayuki , Inamura Noriaki , Hatori Chie , Sawai Hiroe , Fujiwara Tatsujiro , Katayama Akira , Kayakiri Hiroshi , Satoh Shigeki , Abe Yoshito , Inoue Takayuki , Sawada Yuki , Nakahara Kunio , Oku Teruo , Okuhara Masakuni
Published: August 2006

An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)‐3‐(6‐acetamido‐3‐pyridyl)‐N‐[N‐[2‐4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl) oxymethyl]phenyl]‐N‐methylaminocarbonyl‐methyl]acrylamide) has been identified.

This compound displaced [3H]‐BK binding to B2 receptors present in guinea‐pig ileum membranes with an IC50 of 5.6 × 10−10 M and in rat uterus with an IC50 of 1.5 × 10−9 M. It did not inhibit different specific radio‐ligand binding to other receptor sites.

In human lung fibroblast IMR‐90 cells, FR173657 displaced [3H]‐BK binding to B2 receptors with an IC50 of 2.9 × 10−9 M and a Ki of 3.6 × 10−10 M, but did not reduce [3H]‐des‐Arg10‐kallidin binding to B1, receptors.

In guinea‐pig isolated preparations, FR173657 antagonized BK‐induced contractions with an IC50 of 7.9 × 10−9 M, but did not antagonize acetylcholine or histamine‐induced contractions even at a concentration of 10−6 M. FR173657 caused parallel rightward shifts of the concentration‐response curves to BK at concentrations of 10−9 M and 3.2 × 10−9 M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration‐response curve at a concentration of 10−8 M. Analysis of the data yield a pA2 of 9.2 ± 0.2 (n = 5) and a slope of 1.5 ± 0.2 (n = 5).

In vivo, the oral administration of FR173657 inhibited BK‐induced bronchoconstriction dose‐dependently in guinea‐pigs with an ED50 of 0.075 mg kg−1, but did not inhibit histamine‐induced bronchoconstriction even at 1 mg kg−1. FR173657 also inhibited carrageenin‐induced paw oedema with an ED50 of 6.8 mg kg−1 2 h after the carrageenin injection in rats.

These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.
Localization of leukaemia inhibitory factor to airway epithelium and its amplification of contractile responses to tachykinins
Authors: Knight Darryl , McKay Karen , Wiggs Barry , Robert Schellenberg R , Bai Tony
Published: August 2006

In neural tissue, leukaemia inhibitory factor (LIF) is an important trophic cytokine. In this investigation, we determined if LIF was present in human and guinea‐pig airways and examined the role of this cytokine in modulating airway responses to endogenous and exogenous tachykinins as well as muscarinic receptor and β‐adrenoceptor stimulation.

The presence of LIF in both human and guinea‐pig airways was determined by immunohistochemistry. Guinea‐pig tracheal explants were incubated in CRML‐1066 media containing LIF (0.5, 5 or 50 ng ml−1) for periods of 3, 6, 24 and 48 h. Tracheal rings were then transferred to organ baths for measurement of isometric force in response to carbachol, capsaicin, the neurokinin1 (NK1) receptor agonist [Sar9,Met(O2)11]‐substance P (SP), the NK2 receptor agonist neurokinin A (NKA) and isoprenaline.

LIF immunoreactivity was observed primarily in basally situated cells in the airway epithelium of both large and small airways. Less intense immunoreactivity was observed in vascular endothelium and glandular epithelium.

Treatment with LIF (0.5 ng ml−1) for 3 and 6 h significantly increased contractile responses to capsaicin by 42% and 43%, respectively, compared to time controls, whereas higher concentrations of LIF (5 and 50 ng ml−1) enhanced capsaicin‐induced contractions only after 6 h. After 24 h, responses to capsaicin were not significantly different from 0 h control. Contractile responses to capsaicin following exposure to LIF at any concentration for 24 h were not significantly different from relative time control values.

Responses to [Sar9,Met(O2)11]‐SP, carbachol and isoprenaline were not influenced by time in culture or by exposure to LIF for up to 48 h. Contractile responses induced by NKA were not influenced by 3 or 6 h exposure to LIF, but at 24 and 48 h the mean maximum contractile responses to NKA were significantly increased by 33% and 35%, respectively, compared to control.

These results demonstrate that LIF is present in guinea‐pig and human airway epithelium, and modulates airway responses to tachykinins. In the acute setting LIF augments the capsaicin‐induced release of endogenous tachykinins, whilst in the longer term (>24 h), LIF increases airway smooth muscle responses to tachykinins via an NK2 receptor selective mechanism. We conclude that LIF may be an important effector molecule in the response of airways to injury or inflammation.

British Journal of Pharmacology (1997) 120, 883–891; doi:10.1038/sj.bjp.0700965
Visual detection of transport‐P in peptidergic neurones
Authors: Al‐Damluji Saad , Porter Denise , Krsmanovic Lazar Z , Knutson Jay R , Kopin Irwin J
Published: August 2006

Hypothalamic peptidergic neurones possess an uptake process for amines (transport‐P), for which prazosin is a substrate. It is characterized by a paradoxical increase in the accumulation of [3H]‐prazosin when the concentration of unlabelled prazosin is increased above 10−7 m. This increase is due to activation of a proton‐dependent, vacuolar‐type ATPase‐linked pump that is blocked by tricyclic antidepressants. This study utilized a fluorescence method to detect amine uptake in individual cells.

Prazosin is fluorescent but most of its emission spectrum is in the ultraviolet range. We therefore used an analogue of prazosin in which the furan ring had been substituted with a fluorescent group, BODIPY FL. This compound's emission maximum is in the green part of the visible spectrum.

BODIPY FL prazosin accumulated in immortalised peptidergic neurones and the characteristic emission spectrum of the compound was evident in these cells. Accumulation of BODIPY FL prazosin was saturable and was inhibited by the tricyclic antidepressant desipramine and by unlabelled prazosin. As previously described for prazosin, uptake of BODIPY FL prazosin was blocked by cold temperature and by the organic base chloroquine. Thus, prazosin and BODIPY FL prazosin were accumulated by the same uptake process.

BODIPY FL prazosin accumulated in a granular distribution, which is compatible with storage in intracellular vesicles.

Hypothalamic cells from foetal rats in primary culture also accumulated BODIPY FL prazosin by a desipramine‐sensitive process. Uptake was predominantly in neurones and glial cells did not accumulate the amine.

Fluorescent detection provides visual evidence for amine uptake in peptidergic neurones and should enable detailed study of the distribution of this process in the brain.

British Journal of Pharmacology (1997) 120, 876–882; doi:10.1038/sj.bjp.0700970
Dose‐dependent effects of chronic ethanol on mouse adipose tissue lipase activity and cyclic AMP accumulation
Authors: Shih Mei‐Fen , Taberner Peter V.
Published: August 2006

The effects of two chronic ethanol treatment schedules, which produce different plasma ethanol concentrations, on the specific activities of adipose tissue lipoprotein lipase (LPL) and hormone‐sensitive lipase (HSL) have been investigated in brown and white fat.

Mice provided with 20% ethanol solution as sole drinking fluid for 28 days consumed between 13 and 15 g ethanol kg−1 body weight day−1 over days 22–28. The mean plasma ethanol concentration was 4.94± 1.4 mM (n = 8) at 09 h 00 min on day 28 when the lipase assays were performed. Mice given ethanol in a liquid diet for 7 days consumed between 15 and 18 g ethanol day−1 over days 3–7. The mean plasma ethanol concentration was 15.9 ± 4.7 mM (n = 8) at 09 h 00 min on day 7. These concentrations of ethanol had no effect on the activity of either LPL or HSL in vitro.

LPL activity in white and brown fat (expressed as nmol fatty acids released h−1. mg−1 acetone powder) was unaltered 60 min following an acute injection of ethanol (2.5 g kg−1, i.p.) which produced a mean blood ethanol level of 37.5 ± 6.7 mM. HSL activity in white fat (expressed as nmol fatty acid released h−1 mg−1 protein) was also unaffected by this acute dose of ethanol, but the activity in brown fat was significantly reduced: 3.07 ± 0.30 (n = 8) after ethanol compared to 4.36 + 0.25 (n = 12) in controls (P<0.01).

LPL activity in white fat was little altered by either of the chronic ethanol treatment schedules whilst LPL activity in the brown fat from the same animals was significantly increased compared to the respective control values: 0.27±0.03 (ethanol drinking), control: 0.16±0.01; 0.79±0.14 (ethanol liquid diet), control: 0.39 ±0.05.

HSL activity in white fat was significantly increased by the chronic drinking treatment (7.7 ± 0.5; control: 3.78 ± 0.17, n = 8) at the same time that the activity in brown fat was reduced (3.76 ± 0.2; control: 4.74 ± 0.16). The ethanol liquid diet also reduced HSL activity in brown fat but had negligible effect in white fat.

The effects of the two chronic ethanol treatments on adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) accumulation in brown and white fat were very similar, both qualitatively and quantitatively, to the effects on HSL.

It has been shown that brown and white adipose tissues respond differently to the presence of chronic ethanol and that the response is dependent both upon the concentration of ethanol and the nature of the diet with which the ethanol is administered. The effects of ethanol on adipose tissue HSL activity appear to be mediated via changes in the tissue cyclic AMP level and, in this respect, brown fat is more sensitive to ethanol than white fat.