bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of the putative endogenous imidazoline receptor ligand, clonidine‐displacing substance, on insulin secretion from rat and human islets of Langerhans
Authors: Chan Susan L F , Atlas Daphne , James Roger F L , Morgan Noel G
Published: August 2006

The effects of a rat brain extract containing clonidine‐displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated.

CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mm glucose, in a dose‐dependent manner. The magnitude of this effect was similar to that in response to the well‐characterized imidazoline secretagogue, efaroxan.

CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose‐induced insulin release, in both rat and human islets.

These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline‐mediated actions of CDS can be blocked by specific imidazoline antagonists.

The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri‐filtration through a 3,000 Da cut‐off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide.

Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor.

British Journal of Pharmacology (1997) 120, 926–932; doi:10.1038/sj.bjp.0700964
Effects of FR173657, a non‐peptide B2 antagonist, on kinin‐induced hypotension, visceral and peripheral oedema formation and bronchoconstriction
Authors: Griesbacher Thomas , Legat Franz J
Published: August 2006

Kinins are believed to play a key role in many inflammatory conditions. Therefore, bradykinin antagonists are being developed for potential therapeutic applications. In the present investigation we describe the pharmacology, in vivo, of (E)‐3‐(6‐ acetamido‐ 3‐pyridyl)‐ N‐[N‐ [2,4‐ dichloro‐ 3‐[(2‐ methyl‐8‐ quinolinyl)oxymethyl]phenyl]‐ N‐methylaminocarbonylmethyl]acrylamide (FR173657), a novel, non‐peptide bradykinin antagonist.

The hypotensive effects of i.v. injections of bradykinin (50 pmol) in captopril‐pre‐treated anaesthetized rats were significantly inhibited by 100 nmol kg−1 FR173657 s.c., and completely abolished by 300 nmol kg−1. The full inhibitory effect developed within 60 min and remained unchanged for at least 4 h. However, the effect was reversible, since 24 h after an injection of 300 nmol kg−1 FR173657 no inhibitory effect could be observed.

The plasma protein extravasation into the pancreas and duodenum induced by an i.v. infusion of bradykinin (11 nmol kg−1 within 20 min) in captopril‐treated anaesthetized rats was completely abolished by FR173657 at doses of 30 nmol kg−1 s.c. and above, given 60 min before bradykinin. FR173657 3 nmol kg−1 was ineffective, while a dose of 10 nmol kg−1 produced an intermediate effect.

The paw oedema induced by the subplantar injection of bradykinin (30 nmol) in anaesthetized rats was inhibited slightly by s.c. injection of FR173657 0.3 μmol kg−1, whereas 1 and 3 μmol kg−1 produced significant inhibition of the bradykinin‐induced oedema. The maximum inhibition amounted to about 50% and could not be increased even when the dose of FR173657 was increased to 30 μmol kg−1. FR173657 did not effect the oedema caused by histamine or 5‐hydroxytryptamine.

Bradykinin (20 nmol kg−1, i.v.) caused increases in pulmonary inflation pressure by 300–600 Pa in anaesthetized, respirated guinea‐pigs. The effect was reduced to 58±9% of the initial value 60 min after the s.c. injection of FR173657 1 μmol kg−1, whereas only 9±7% remained after 10 μmol kg−1. The bronchoconstrictor actions of histamine remained unaffected by FR173657.

In summary, FR173657 is a highly potent and selective bradykinin antagonist. The inhibitory action in vivo lasts for longer than 4 h but is fully reversible. FR173657, or similar compounds, will be a useful tool for the pharmacological investigation of pathophysiological states and may possess a therapeutic potential in diseases involving the endogenous release of kinins.

British Journal of Pharmacology (1997) 120, 933–939; doi:10.1038/sj.bjp.0700966
Nerve‐induced release of nitric oxide in the rabbit gastrointestinal tract as measured by in vivo microdialysis
Authors: Iversen Henrik H. , Celsing Fredrik , Leone Anna M. , Gustafsson Lars E. , Wiklund N. Peter
Published: August 2006

Nitric oxide (NO) has been suggested as a gastrointestinal neurotransmitter, mediating the gastric receptive relaxation and the relaxation in the peristaltic reflex. The aim of the present study was to measure nerve‐induced NO formation in vivo in the gastrointestinal tract.

Formation of the nitric oxide oxidation products nitrite and nitrate during vagal nerve stimulation were measured in the anaesthetized rabbit. Microdialysis probes were inserted into the wall of the stomach and proximal colon, and nitrite and nitrate in dialysate measured by capillary electrophoresis.

During bilateral vagal nerve stimulation there was an increase in nitrite and nitrate formation at the level of the stomach and in nitrite formation at the level of the colon. This increase was inhibited by intravenous administration of the NO synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (l‐NAME 30 mg kg−1). Furthermore, L‐NAME significantly increased nerve‐induced gastric and colonic contractions, as well as spontaneous colonic contractions.

In summary, we present a new methodological procedure for quantification of small changes in nitric oxide formation in vivo. This study provides evidence that nitric oxide is released in the stomach and colonic wall during vagal nerve activity, at concentrations able to cause inhibition of smooth muscle contractions in vivo.
Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils
Authors: Hsu Mei F , Raung Shue L , Tsao Lo T , Kuo Sheng C , Wang Jih P
Published: August 2006

The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated.

Abruquinone A caused an irreversible and a concentration‐dependent inhibition of formylmethionyl‐leucyl‐phenylalanine (fMLP) plus dihydrocytochalasin B (CB)‐ and phorbol 12‐myristate 13‐acetate (PMA)‐induced superoxide anion (O2.−) generation with IC50 values of 0.33±0.05 μg ml−1 and 0.49±0.04 μg ml−1, respectively.

Abruquinone A also inhibited O2 consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O2.− in xanthine‐xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation.

Abruquinone A inhibited both the transient elevation of [Ca2+]i in the absence of [Ca2+]o (IC50 7.8±0.2 μg ml−1) and the generation of inositol trisphosphate (IP3) (IC50 10.6±2.0 μg ml−1) in response to fMLP.

Abruquinone A did not affect the enzyme activities of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA).

Abruquinone A had no effect on intracellular guanosine 3′: 5′‐cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) levels.

The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/CB was inhibited by abruquinone A with IC50 values of 2.2±0.6 μg ml−1 and 2.5±0.3 μg ml−1, respectively. Abruquinone A did not inhibit the fMLP/CB‐induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73–78 kDa in activated neutrophils.

Abruquinone A inhibited both the O2.− generation in PMA‐activated neutrophil particulate NADPH oxidase (IC50 0.6±0.1 μg ml−1) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)‐activated cell‐free system (IC50 1.5±0.2 μg ml−1).

Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.

British Journal of Pharmacology (1997) 120, 917–925; doi:10.1038/sj.bjp.0700974
Effect of non‐toxic mercury, zinc or cadmium pretreatment on the capacity of human monocytes to undergo lipopolysaccharide‐induced activation
Authors: Koropatnick J , Zalups R K
Published: August 2006

Metal salts can inhibit cell activity through direct toxicity to critical cellular molecules and structures. On the other hand, they can also change cell behaviour by inducing specific genes (including genes encoding members of the metallothionein [MT] gene family). Therefore, transition metals may affect cell functions either by acting as a toxin, or by transmitting or influencing signals controlling gene expression.

To explore the latter possibility, we measured the ability of low, non‐toxic metal pretreatment to alter immune cell behaviour. We previously found that pretreatment of human monocytes with zinc induces metallothionein gene expression and alters their capacity to undergo a bacterial lipopolysaccharide‐induced respiratory burst. We showed here that cadmium and mercury salts, at concentrations that exert no discernible toxicity, inhibit activation of human monocytic leukemia (THP‐1) cells. CdCl2 1 μm, ZnCl2 20–40 μm or HgCl2 2 μm pretreatment for 20 h induced MT‐2 mRNA and total MT protein accumulation and had no effect on proliferation potential or metabolic activity, but significantly inhibited the ability of subsequent lipopolysaccharide treatment to induce the oxidative burst, increased adhesion to plastic, and MT‐2 and interleukin‐1β (IL‐1β) mRNA accumulation.

The phenomenon of metal‐induced suppression of monocyte activation, at metal concentrations that have no effect on cell viability, has important implications for assessment of acceptable levels of human exposure to cadmium, zinc and mercury.

British Journal of Pharmacology (1997) 120, 797–806; doi:10.1038/sj.bjp.0700975
Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells
Authors: Paul Andrew , Doherty Kirsten , Plevin Robin
Published: August 2006

In RAW 264.7 murine macrophages and rat aortic smooth muscle (RASM) cells lipopolysaccharide (LPS) alone or in combination with interferon γ (IFNγ) or forskolin, respectively, stimulated the expression of the 130 kDa inducible isoform of nitric oxide synthase (iNOS) in both a time‐ and concentration‐dependent manner.

Incubation with the direct activator of protein kinase C (PKC), phorbol 12‐myristate 13‐acetate (PMA) alone, did not result in detectable iNOS expression in either cell type.

Chronic PMA pretreatment resulted in significant down‐regulation of α, β and ε isoforms of PKC in RAW 264.7 macrophages and corresponded to a 20–30% reduction in LPS‐induced iNOS expression. In contrast, IFNγ alone or in combination with LPS stimulated an approximate 20% and 50% potentiation, respectively.

Pre‐incubation with PKC inhibitors (calphostin C and H‐7) showed similar effects upon stimulated induction of iNOS.

In RASM cells chronic PMA pretreatment resulted in down‐regulation of α and ε PKC isoforms and corresponded to potentiation of iNOS expression in response to LPS alone or in combination with forskolin.

Co‐incubation of RASM cells in the presence of PMA, angiotensin II (AII) or foetal calf serum (FCS) resulted in the inhibition of iNOS expression in response to LPS alone or in combination with forskolin.

Differential sensitivity to PKC inhibitors (calphostin C and H‐7) was observed in RASM cells and exhibited both negative and positive modulation of stimulated induction.

In addition the PKC inhibitor compound Ro‐31–8220 abolished stimulated induction in both cell types in response to all treatments.

These results suggest that PKC activation is required for induction of the 130 kDa isoform of NOS in both RAW 264.7 macrophages and RASM cells. However, individual PKC isoforms regulate iNOS expression in both a positive and negative manner.

British Journal of Pharmacology (1997) 120, 940–946; doi:10.1038/sj.bjp.0700976
Renal adenosine A1 receptor binding characteristics and mRNA levels during the development of acute renal failure in the rat
Authors: Gould J , Morton M J , Sivaprasadarao A , Bowmer C J , Yates M S
Published: August 2006

The binding characteristics and mRNA levels for renal adenosine A1 receptors were investigated in normal rats and rats with acute renal failure (ARF) induced by either glycerol or HgCl2.

Saturation isotherms determined from the binding of [3H]‐1,3‐dipropyl‐8‐cyclopentylxanthine ([3H]‐DPCPX), a selective adenosine A1 antagonist, to renal membranes of untreated rats gave values of 0.62 nm for the equilibrium dissociation constant (Kd) and 19.9 fmol mg−1 protein for the density of binding sites (Bmax). No saturable binding was observed with [3H]‐2‐(p‐(carboxylethyl)‐phenylethylamino)‐5′‐N‐ethylcarboxamido adenosine ([3H]‐CGS 21680), a selective adenosine A2a agonist.

By contrast to time‐matched controls, renal membranes obtained from rats 16 and 48 h following the induction of ARF with glycerol, showed statistically significant increases (2–4 fold) in both Bmax and Kd for the binding of [3H]‐DPCPX. No significant changes in the binding characteristics of [3H]‐DPCPX were noted with membranes from rats 48 h following the production of ARF with HgCl2.

Adenosine A1 receptor mRNA levels were significantly elevated 0.5, 16 and 48 h following induction of ARF with glycerol, whilst no change was noted in mRNA levels for β‐actin at the same time points. No statistically significant changes in adenosine A1 receptor or β‐actin mRNA levels were noted 48 h after the induction of ARF with HgCl2.

This study indicates that glycerol‐induced ARF in the rat is associated with an increase in renal adenosine A1 receptor density which appears to result from increased transcription of the gene for this receptor. An increase in adenosine A1 receptor density in renal resistance vessels may explain, at least in part, the enhanced renal vasoconstrictor response to adenosine in glycerol‐induced ARF that was noted in a previous study.

British Journal of Pharmacology (1997) 120, 947–953; doi:10.1038/sj.bjp.0700980
Inhibition of [3H]‐U69593 binding and the cardiac effects of U50,488H by calcium channel blockers in the rat heart
Authors: Zhang Wei‐Min , Wang Hong‐Xin , Xia Qiang , Wong Tak‐Ming
Published: August 2006

The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [3H]‐U69593 ((5a,7a,8b)‐(+)‐N‐methyl‐N‐(7‐[1‐pyrrolidinyl]‐1‐oxaspiro[4,5]dec‐8‐yl)‐benzeneacetamide), a specific κ‐opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [3H]‐U69593 (4 nm) in a dose‐dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine.

The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of κ‐receptor stimulation by a specific κ‐receptor agonist, U50,488H (trans‐(±‐3,4‐dichloro‐N‐methyl‐N‐(2‐[1‐pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80–800 nmol dose‐dependently induced arrhythmias, which were completely abolished by a selective κ‐receptor antagonist, nor‐BNI (nor‐binaltorphimine,17,17′‐(dicyclopropylmethyl)‐6,6′,7,7′‐6,6′‐imino‐7,7′‐binorphinan‐3,4′,14, 14′‐tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose‐dependent manner. The ED50 values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca2+ influx in the cardiac muscle.

Measurement of [Ca2+]i in the absence of free extracellular Ca2+ by a spectrofluorometric method, with fura‐2 as Ca2+ indicator, showed that U50,488H 5×10−5 m slowly increased [Ca2+]i in single ventricular myocytes and this effect was abolished by pretreatment with nor‐BNI (5 μm), or ryanodine (5 μm). Verapamil 1 and 10 μm abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 μm had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine‐induced Ca2+ transient.

The observations suggest that CCBs may inhibit the actions of κ‐receptor stimulation at the level of the κ‐receptor.

British Journal of Pharmacology (1997) 120, 827–832; doi:10.1038/sj.bjp.0700985
Nitric oxide modulation of transcellular biosynthesis of cys‐leukotrienes in rabbit leukocyte‐perfused heart
Authors: Buccellati Carola , Rossoni Giuseppe , Bonazzi Albino , Berti Ferruccio , Maclouf Jacques , Folco Giancarlo , Sala Angelo
Published: August 2006

We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys‐LT) generated upon neutrophil‐vascular wall interactions and their functional consequences, in the spontaneously beating, cell‐perfused, heart of the rabbit.

Hearts were perfused under recirculating conditions (50 ml) with 5×106 purified human neutrophils (PMNL), and challenged with 0.5 μm A‐23187 for 30 min. Coronary perfusion pressure (CPP) and left‐ventricular end‐diastolic pressure (LVEDP) were monitored. Cys‐LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL adhesion to the coronary endothelium.

Basal CPP and LVEDP values averaged 45±1.4 mmHg and 5±0.1 mmHg, respectively; A‐23187 triggered an increase in CPP (134±9 mmHg, at 30 min) which was significantly attenuated by pretreatment with l‐arginine, 100 μm (90±3 mmHg, at 30 min). Pretreatment with NG‐monomethyl‐l‐arginine, 10 μm (l‐NMMA), induced a marked increase in CPP (290±40 mmHg, at 20 min) and in LVEDP (47±16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by l‐arginine, 100 μm (CPP 115±10 mmHg, LVEDP 6±1.1 mmHg, at 30 min).

The increase in CPP was accompanied by the release of cys‐LT in the circulating buffer, which was reduced significantly by l‐arginine. Pretreatment with l‐NMMA, caused a marked rise in cys‐LT concentrations which was prevented by l‐arginine.

Neither l‐arginine nor l‐NMMA affected directly the A‐23187‐induced arachidonic acid (AA) metabolism in isolated PMNL alone.

Pretreatment with l‐NMMA caused a prompt drop in myeloperoxidase (MPO) activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by l‐arginine.

This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys‐LT by preventing PMNL adhesion to the coronary intima.

British Journal of Pharmacology (1997) 120, 1128–1134; doi:10.1038/sj.bjp.0700994
Differential influence of D1 and D2 dopamine receptors on acute opiate withdrawal in guinea‐pig isolated ileum
Authors: Capasso A , Sorrentino L
Published: August 2006

The effects exerted by D1 and D2 dopamine agonists and antagonists on the acute opiate withdrawal induced by μ‐ and κ‐receptor agonists were investigated in vitro.

Following a 4 min in vitro exposure to morphine (moderately selective μ‐agonist), [d‐Ala2, Me‐Phe4, Gly‐ol5]enkephalin (DAMGO, highly selective μ‐agonist) or U‐50488H (highly selective κ‐agonist) the guinea‐pig isolated ileum exhibited a strong contracture after the addition of naloxone.

The non‐selective dopamine receptor antagonist haloperidol when added before or after the opioid agonists, was able dose‐dependently to prevent or to reverse the naloxone‐induced contracture after exposure to μ‐ (morphine and DAMGO) and κ‐ (U‐50488H) opioid agonists. The non‐selective dopamine receptor agonist, apomorphine, was able to exert the same effects only at the highest concentration used.

The selective D2 dopamine receptor antagonist, sulpiride, was also able to reduce dose‐dependently both μ‐ and κ‐opioid withdrawal, whereas the D1‐receptor selective antagonist SCH 23390 did not affect either μ‐ or κ‐opioid withdrawal.

Bromocriptine, a D2 selective dopamine receptor agonist was able to increase significantly, and in a concentration‐dependent manner, the naloxone‐induced contracture by μ‐ and κ‐opioid agonists, whereas SKF 38393, a D1 selective dopamine receptor agonist, increased only the withdrawal after morphine or U50‐488H.

Our data indicate that both D1 and D2 dopamine agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the dopaminergic system and opioid withdrawal at both the μ‐ and κ‐receptor level.

Furthermore, the ability of sulpiride to block strongly opiate withdrawal when compared to SCH 23390, as well as the effect of bromocriptine to increase opiate withdrawal suggest that D2 dopamine receptors may be primarily involved in the control of opiate withdrawal.

British Journal of Pharmacology (1997) 120, 1001–1006; doi:10.1038/sj.bjp.0700995
Evidence for direct and indirect mechanisms in the potent modulatory action of interleukin‐2 on the release of acetylcholine in rat hippocampal slices
Authors: Seto David , Kar Satyabrata , Quirion Rémi
Published: August 2006

The biphasic nature of the potent modulatory action of interleukin‐2 (IL‐2) on hippocampal acetylcholine (ACh) release was investigated by use of brain slice superfusion.

Both the potentiating (10−13 m) and inhibitory (10−9 m) effects of IL‐2 on hippocampal ACh release were stimulation‐dependent and were blocked by a neutralizing IL‐2 receptor antibody, suggesting the activation of typical IL‐2 receptors in both cases.

Tetrodotoxin (TTX; 10 μm) failed to block the potentiation of ACh release induced by a very low concentration of IL‐2 (10−13m) suggesting a direct effect on cholinergic nerve terminals.

In contrast, the inhibitory effect seen at a higher concentration (10−9 m) was TTX‐sensitive, and hence indicative of an indirect action.

To establish the nature of this intermediate mediator, blockers of nitric oxide synthesis, and of opioid and γ‐aminobutyric acid (GABA) receptors were used. Only GABAA and GABAB receptor antagonists altered the inhibitory action of IL‐2, suggesting the participation of GABA as mediator.

Taken together, these results provide further evidence for the potent role of IL‐2 in the modulation of cholinergic function in the rat hippocampus.

British Journal of Pharmacology (1997) 120, 1151–1157; doi:10.1038/sj.bjp.0701002
The relative potencies of dendrotoxins as blockers of the cloned voltage‐gated K+ channel, mKv1.1 (MK‐1), when stably expressed in Chinese hamster ovary cells
Authors: Owen David G , Hall Adam , Stephens Gary , Stow John , Robertson Brian
Published: August 2006

The mKv1.1 voltage‐gated K+ channel has been expressed stably in Chinese hamster ovary cells and whole‐cell currents recorded by the patch‐clamp method.

A range of structurally related peptide toxins (dendrotoxins) from the venom of green mamba (Dendroaspis angusticeps) and black mamba (Dendroaspis polylepis polylepis) snakes were tested for mKv1.1 channel blocking activity. Their potencies were compared based on EC50s derived from their respective concentration‐inhibition relationships.

The rank order of potency, thus determined was: Toxin K>γ‐dendrotoxin(γ‐Dtx)>δ‐Dtx>Toxin I=α‐Dtx>β‐Dtx.

Block was independent of voltage and no effects of the toxins on the kinetics of activation were observed. These results are consistent with a mechanism involving the block of closed channels.

A wide range of activity was observed even between toxins with an extremely high degree of sequence homology. Toxin K, in particular was an exquisitely potent blocker of the mKv1.1 channel, having an EC50 of 30 pm compared with 1.8 nm for δ‐Dtx in spite of 95% sequence identity.

British Journal of Pharmacology (1997) 120, 1029–1034; doi:10.1038/sj.bjp.0701004
Effects of l‐NG‐nitro‐arginine on noradrenaline induced contraction in the rat anococcygeus muscle
Authors: Hoyo Yolanda , Giraldo Jesús , Vila Elisabet
Published: August 2006

The influence of l‐NG‐nitro‐arginine (l‐NOARG, 30 μm) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.

Noradrenaline (0.1–100 μm) contracted the muscle in a concentration‐dependent manner. l‐NOARG (30 μm) had no effect on noradrenaline responses.

Phenoxybenzamine (Pbz 0.1 μm) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC50 control: 6.92±0.09; pEC50 Pbz: 5.30±0.10; n=20).

The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.

Contractile responses to noradrenaline after partial α1‐adrenoceptor alkylation with phenoxybenzamine (0.1 μm) were clearly enhanced by l‐NOARG.

The potentiating effect of l‐NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μm) l‐arginine but not by (100 μm) d‐arginine.

These results indicate that spontaneous release of NO by nitrergic nerves can influence the α1‐adrenoceptor‐mediated response to exogenous noradrenaline.

British Journal of Pharmacology (1997) 120, 1035–1038; doi:10.1038/sj.bjp.0701005
Inhibition of synaptic transmission and epileptiform activity in central neurones by fluspirilene
Authors: Wang Su‐Jane , Lu Kwok‐Tung , Gean Po‐Wu
Published: August 2006

Recent studies have shown that fluspirilene, a dopamine D2 receptor antagonist which is a long‐acting neuroleptic useful in the maintenance therapy of schizophrenic patients, also displays Ca2+ channel blocking activity. In the present study, we have investigated the effect of fluspirilene on synaptic transmission and epileptiform activity induced in slices of hippocampus and amygdala.

Fluspirilene reversibly suppressed the field excitatory postsynaptic potential (f‐e.p.s.p) in a concentration‐dependent manner in the area CAl of the hippocampus without affecting the size and shape of fibre volley. Fluspirilene also inhibited the intracellularly recorded e.p.s.p. in amygdala neurones without affecting the resting membrane potential or neuronal input resistance.

Fluspirilene increased the ratio of paired‐pulse facilitation suggesting a presynaptic mode of action.

Epileptiform activity induced in the disinhibited slices was suppessed by fluspirilene in a concentration‐dependent manner. This antiepileptic effect was occluded in slices pretreated with the adenosine A1 receptor agonist, N6‐cyclopentyladenosine (CPA).

It is concluded that fluspirilene‐induced synaptic inhibition is probably due to a reduction in presynaptic Ca2+ currents. In clinical trials, the low incidence of seizures provoked by fluspirilene might be related to its intrinsic ability to inhibit synaptic transmission and epileptiform activity.

British Journal of Pharmacology (1997) 120, 1114–1118; doi:10.1038/sj.bjp.0701008
Endothelium‐independent relaxation of aortic rings by the nitric oxide synthase inhibitor diphenyleneiodonium
Authors: Dodd‐o Jeffrey M , Zheng Gemin , Silverman Howard S , Lakatta Edward G , Ziegelstein Roy C
Published: August 2006

The flavoprotein binder diphenyleneiodonium (DPI) is a potent, irreversible inhibitor of nitric oxide synthase (NOS), but produces only a transient pressor response following systemic administration to animals, despite evidence of persistent NOS inhibition. To characterize further the effects of DPI on vascular tone, isometric tension was recorded from rat isolated aortic rings mounted between steel wires in an organ bath.

The NOS inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 1 mm) initiated an additional contraction of prostaglandin F2α‐preconstricted rings with endothelium which was sustained throughout the period of l‐NAME exposure (+234±39% at 15 min). In contrast, addition of DPI (5 μm) to rings with endothelium produced a transient initial contraction (+111±27% at 2 min) followed by a more sustained relaxation (−27±19% at 15 min, P<0.001 vs l‐NAME).

The contraction to DPI was also observed in rings without endothelium, was abolished by l‐NAME pretreatment, and was unaffected by the α‐adrenoreceptor inhibitor prazosin. Relaxation in response to DPI was not inhibited by endothelium removal or by pretreatment with either l‐NAME or with the ATP‐sensitive potassium channel blocker glibenclamide.

The endothelium‐independent relaxation to DPI was inhibited at 23°C and its time course was delayed by pretreatment with the guanylate cyclase inhibitor methylene blue.

Thus, in addition to a transient initial contraction due to NOS inhibition, DPI produces an endothelium‐independent, temperature‐dependent relaxation which appears in part due to activation of guanylate cyclase. This relaxant effect of DPI may explain the transient nature of its pressor effect in vivo despite sustained NOS inhibition.

British Journal of Pharmacology (1997) 120, 857–864; doi:10.1038/sj.bjp.0701014
Effects of 1,25‐dihydroxyvitamin D3 and its 20‐epi analogues (MC 1288, MC 1301, KH 1060), on clonal keratinocyte growth: evidence for differentiation of keratinocyte stem cells and analysis of the modulatory effects of cytokines
Authors: Gniadecki Robert
Published: August 2006

Keratinocytes are functionally divided into stem cells, transit amplifying cells and terminally differentiated cells. In a hyperproliferative skin disease, psoriasis, increased mitotic activity of the stem cells is chiefly responsible for epidermal hyperplasia. The effects of 1,25dihydroxyvitamin D3 (1,25(OH)2D3) and potent vitamin D3 analogues (MC 1288: 20‐epi‐1,25(OH)2D3, MC 1301: 20‐epi‐24a‐homo‐26,27‐dimethyl‐1,25(OH)2D3, KH 1060: 20‐epi‐22‐oxa‐24a‐homo‐26,27‐dimethyl‐1,25(OH)2D3) on the stem cells were investigated.

Stem cells were identified retrospectively as those giving rise to large keratinocyte colonies in culture (holoclones). 1,25(OH)2D3 (10−8–10−6 m) suppressed formation of holoclones by stimulating the progenitor cell differentiation into the phenotype expressing differentiation markers (keratins K1/K10 and involucrin).

20‐Epi vitamin D3 analogues were more potent than 1,25(OH)2D3 in inhibiting the clonal keratinocyte growth. This activity correlated with the ability to induce cell differentiation (KH 1060>MC 1301>MC 1288>1,25(OH)2D3).

Cytokines modulated the effects of 1,25(OH)2D3 on clonal growth. One of the following cytokines (epidermal growth factor, transforming growth factor α, interleukin‐1α, interleukin‐1β, interleukin‐6, interleukin‐8) was required for 1,25(OH)2D3 to suppress clonal growth and induce cell differentiation. In contrast, keratinocyte growth factor and insulin‐like growth factor I attenuated the effects of 1,25(OH)2D3.

In conclusion, 1,25(OH)2D3 and 20‐epi vitamin D3 analogues suppress clonal growth by directly inducing the differentiation of progenitor cells. It is conceivable that stimulation of stem cells differentiation is a major mechanism of action of vitamin D3 compounds in psoriasis. Balance between different types of cytokines in psoriatic epidermis may be an important factor determining the clinical effect of vitamin D‐based therapy.

British Journal of Pharmacology (1997) 120, 1119–1127; doi:10.1038/sj.bjp.0701015
Negative modulation of nitric oxide production by neurotensin as a putative mechanism of the diuretic action of SR 48692 in rats
Authors: Croci Tiziano , Landi Marco , Gully Danielle , Maffrand Jean‐Pierre , Le Fur Gérard , Manara Luciano
Published: August 2006

We investigated the effect of the non‐peptide neurotensin (NT) antagonist SR 48692 on renal function in rats and the involvement of nitric oxide (NO) in the diuretic action of this compound.

In fed animals, SR 48692 dose‐dependently (0.5 to 12.5 mg kg−1, p.o., 0.03 to 1 mg kg−1, i.p. and 0.1 to 1 μg/rat, i.c.v.) increased urine output and urinary excretion of Na+, K+ and Cl− and reduced urine osmolality. The diuretic activity was also evident in water‐deprived, fasted animals and in fasted, water‐loaded rats.

NT (0.1 μg/rat, i.c.v.) had no effect on urine output in fed rats, but reduced the diuretic action of SR 48692 (1 μg/rat, i.c.v.). The opposite result was obtained in fasted, water‐loaded animals: NT dose‐dependently (0.01 and 0.1 μg/rat, i.c.v.) inhibited diuresis and this effect was significantly inhibited by i.c.v. SR 48692. In this experimental condition, SR 48692 did not further increase the on‐going diuresis.

The NO synthesis inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME; 30 mg kg−1, i.p.) alone had no effect on urine output in fed rats but prevented the diuretic action of i.c.v. or i.p. SR 48692; l‐arginine (1 g kg−1, i.p.) but not d‐arginine (1 g kg−1, i.p.) restored the SR 48692‐dependent increase in diuresis. l‐NAME had no effect on furosemide‐stimulated diuresis.

Systemically administered l‐NAME or i.c.v. NT in fasted, water‐loaded rats significantly reduced water diuresis but this effect was no longer seen in animals given i.p. l‐arginine. Rats receiving i.c.v. NT, whose diuresis was significantly reduced, also excreted less nitrates and nitrites in urine.

Increased diuresis after central or systemic administration of SR 48692 to fed rats was paralleled by increased urinary excretion of nitrates and nitrites, this being consistent with peripheral enhancement of NO production after NT‐receptor blockade by SR 48692. The increase in diuresis after furosemide also involved an increase of nitrates and nitrites in urine, but this effect was about half that attained with an equipotent diuretic dose of SR 48692.

In fed rats, the NO donor isosorbide‐dinitrate, reduced systolic blood pressure (unlike SR 48692 which did not affect blood pressure) but also dose‐dependently (1 and 5 mg kg−1, i.p.) stimulated urine output.

The overall effects of SR 48692 strongly support a link between the actions of endogenous NT, AVP and peripheral NO production in the modulation of renal excretion of water, Na+, K+ and Cl−.

British Journal of Pharmacology (1997) 120, 1312–1318; doi:10.1038/sj.bjp.0701016
Suppression of neointimal thickening by a newly developed HMG‐CoA reductase inhibitor, BAYw6228, and its inhibitory effect on vascular smooth muscle cell growth
Authors: Igarashi Masahiko , Takeda Yuki , Mori Seijiro , Ishibashi Naoko , Komatsu Eiichi , Takahashi Kentaro , Fuse Tsunekazu , Yamamura Mikako , Kubo Kazuki , Sugiyama Yasuo , Saito Yasushi
Published: August 2006

The aim of this study was to determine whether BAYw6228 (BAYw), a newly developed 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) reductase inhibitor, could suppress an atherogenic process such as intimal thickening by a mechanism other than lowering the level of serum cholesterol.

First, we evaluated the in vitro effect of BAYw on the proliferation of vascular smooth muscle cells (SMC) from various species: Sprague‐Dawley (SD) rats, New Zealand (NZ) white rabbits, intimal cells from Watanabe hereditary hyperlipidemic (WHHL) rabbit and SMC from the new‐born human aorta. The increasing rate of total protein content of these cells was inhibited by the addition of BAYw in a dose‐dependent fashion. In the presence of 2% foetal calf serum (FCS), the value of IC50 was 1.0 μm in SD rats. 2.1 μm in NZ white rabbits, and 0.3 μm in WHHL rabbits. With human SMC, the value was 0.02 μm in the presence of 10% FCS and 0.2 μm with a mixture of growth factors.

Based on these above in vitro findings, we next examined the in vivo effect of the agent to determine whether it could suppress rabbit intimal thickening induced by balloon catheterization. A balloon catheter was inserted from a peripheral branch of the left external carotid artery to the aorta to denude the endothelium of the left common carotid artery in Japanese white rabbits. After 12 days they were divided into control and BAYw groups. The former were subcutaneously injected with saline and the latter with BAYw 1 mg kg−1 day−1. Two days after the beginning of treatment, a second balloon injury was performed to the previously injured left common carotid artery in both groups. After another two weeks, the left common carotid artery was removed and variously stained. Although the total serum cholesterol in the BAYw group was significantly lower than in the control (P<0.05), the difference was not enough to affect intimal thickening. In addition, the BAYw group had a smaller intima/media ratio than the control group, decreasing to 45% of control (P<0.05). By anti‐α smooth muscle actin antibody staining, these intimal thickening areas were entirely occupied by SMCs, and their amount was attenuated by BAYw. By anti‐rabbit macrophage antibody (RAM 11) staining, the number of positive cells in the intimal thickening was markedly decreased in the BAYw group compared to control (P<0.01).

These results indicate that BAYw has an inhibitory effect on intimal thickening by attenuating intimal SMC proliferation and infiltration of macrophages, suggesting that BAYw could be effective in the prevention of the progression of atherosclerotic plaque‐like restenosis after angioplasty.

British Journal of Pharmacology (1997) 120, 1172–1178; doi:10.1038/sj.bjp.0701018
Inhibition of the constitutive activity of human 5‐HT1A receptors by the inverse agonist, spiperone but not the neutral antagonist, WAY 100,635
Authors: Newman‐Tancredi Adrian , Conte Caroline , Chaput Christine , Spedding Michael , Millan Mark J
Published: August 2006

At recombinant human 5‐hydroxytryptamine (5‐HT)5‐HT1A receptors expressed in Chinese hamster ovary cells (CHO‐5‐HT1A), 5‐carboxamidotryptamine (5‐CT), acted as a full agonist (relative to 5‐HT=100%) for stimulation of receptor‐mediated [35S]‐GTPγS (guanylyl 5′‐[γ‐thio]‐tryphosphate) binding. In contrast, spiperone inhibited basal [35S]‐GTPγS binding by 30.2% (IC50=55.5 nm) in CHO‐5‐HT1A membranes but not in control untransfected membranes. The antagonist, N‐{2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl}‐N‐(2‐pyridinyl)‐cyclohexane‐carboxamide (WAY 100,635), blocked both 5‐CT‐induced stimulation and spiperone‐induced inhibition of [35S]‐GTPγS binding without itself modifying [35S]‐GTPγS binding. It is concluded that, in this heterologous expression system, 5‐HT1A receptors display ‘constitutive’ activation of G‐proteins and that spiperone displays inverse agonist activity whereas WAY 100,635 acts as a ‘neutral’ antagonist at this site.

British Journal of Pharmacology (1997) 120, 737–739; doi:10.1038/sj.bjp.0701025
Receptor subtypes Y1 and Y5 are involved in the renal effects of neuropeptide Y
Authors: Bischoff Angela , Avramidis Prodromos , Erdbrügger Wilhelm , Münter Klaus , Michel Martin C.
Published: August 2006

Systemic infusion of neuropeptide Y (NPY) reduces renal blood flow and can concomitantly increase diuresis, natriuresis and calciuresis in anaesthetized rats. The present study was designed to investigate whether the apparently contradictory NPY effects on renal blood flow and urine formation and composition are mediated by distinct NPY receptor subtypes.

NPY and its analogues, peptide YY (PYY), [Leu31, Pro34]NPY and NPY1336, were infused in incremental doses of 0.3, 1 and 3 μg kg−1 min−1 for 45 min each and the results compared to those obtained in vehicle‐infused rats. Renal blood flow was monitored in 15 min intervals, while urine excretion and composition were determined in 15 min collection periods. Plasma renin activity and aldosterone concentrations were measured at the end of the final infusion period.

Relative to vehicle NPY, PYY and [Leu31, Pro34]NPY dose‐dependently reduced renal blood flow and increased diuresis, natriuresis and calciuresis with roughly similar potency; NPY1336 slightly but significantly increased renal blood flow but had no effect on diuresis, natriuresis and calciuresis. None of the peptides significantly affected endogenous creatinine clearance or kaliuresis.

Plasma renin activity was significantly reduced by PYY. Quantitatively similar reductions were observed with NPY and [Leu31, Pro34]NPY but failed to reach statistical significance with the given number of experiments. NPY1336 did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations.

In another series of experiments infusion of PYY336 (2 μg kg−1 min−1 for 120 min) did not reduce renal blood flow but significantly enhancd diuresis and natriuresis to a similar extent as the NPY 2 μg kg−1 min−1.

In a final series of experiments the Y1‐selective antagonist, BIBP 3226 (1 or 10 μg kg−1 min−1) dose‐dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.130 μg kg−1). BIBP 3226 (10 μg kg−1 min−1) also inhibited the effects of a 120 min infusion of NPY (2 μg kg−1 min−1) on renal blood flow but had only minor inhibitory effects on enhancements of diuresis and did not significantly affect enhancements of natriuresis.

We conclude that NPY reduces renal blood via a classical Y1 subtype of NPY receptor. In contrast enhancements of diuresis, natriuresis and calciuresis occur via a distinct subtype which resembles the receptor that mediates NPY‐induced enhancement of food intake.

British Journal of Pharmacology (1997) 120, 1335–1343; doi:10.1038/sj.bjp.0701028
Antiproliferative effects of PCA‐4230, a new antithrombotic drug, in vascular smooth muscle cells
Authors: Del Rio Marcela , Sunkel Carlos , Larcher Fernando , Pilar Ortega M.
Published: August 2006

In the present study we examined the effects of PCA‐4230, a novel antithrombotic agent, on the growth of cultured A10 vascular smooth muscle cells (rat aorta).

The action of PCA‐4230 on cell proliferation and on serum‐induced DNA synthesis was determined by measuring the cell number and the incorporation of the thymidine analogue 5‐bromo‐2′‐deoxyuridine (BrdU), respectively.

PCA‐4230 reversibly inhibited vascular smooth muscle cell proliferation. The increase in cell number was significantly reduced in the presence of 1 and 50 μm PCA‐4230.

DNA synthesis was concentration‐dependently inhibited by PCA‐4230 (0.5 to 50 μm) in A10 cells that were synchronized by 48 h serum starvation and then re‐stimulated by serum repletion, with an IC50 value of 13 μm. However, serum‐induced DNA synthesis in bovine aortic endothelial cells was not significantly affected by PCA‐4230. In addition, PCA‐4230 (50 μm) caused a significant drop in PDGF‐BB‐mediated BrdU incorporation in A10 cells.

The effect of PCA‐4230 on serum‐induced DNA synthesis was compared to that elicited by nifedipine, another dihydropyridine‐class inhibitor of vascular smooth muscle proliferation. PCA‐4230 (10 μm) elicited a degree of inhibition similar to that of nifedipine at equimolar concentration.

To define the nature of the cell proliferation inhibition, an evaluation of cell cycle progression was undertaken. Flow cytometry studies of DNA content in synchronized cells revealed a block of the serum‐inducible cell cycle progression. This inhibitory effect was markedly reduced when PCA‐4230 was added 2 h after serum repletion.

Accordingly, PCA‐4230 (50 μm) caused a 95 and 90% decrease in the elevation of c‐fos and c‐jun proto‐oncogenes expression as evaluated by Northern blot analysis of mRNA induced early after serum addition.

The present results indicate that PCA‐4230 inhibits vascular smooth muscle cell proliferation, in culture, by altering the cell cycle progression. Flow cytometric studies of DNA content and the down regulation of c‐fos and c‐jun proto‐oncogenes, suggest that the drug is acting at the early G0/G1 transition phase. PCA‐4230 may hold promising potential for the prevention of structural abnormalities of blood vessels associated with atherosclerosis and vascular diseases.

British Journal of Pharmacology (1997) 120, 1360–1366; doi:10.1038/sj.bjp.0701035
Functional and molecular evidence for β1‐, β2‐ and β3‐adrenoceptors in human colon
Authors: Roberts S. J. , Papaioannou M. , Evans B. A. , Summers R. J.
Published: August 2006

Relaxation of carbachol pre‐contracted human colonic muscle to (–)‐isoprenaline was examined in circular, longitudinal and taenia coli preparations to determine the β‐adrenoceptor subtypes involved. β1‐, β2‐ and β3‐Adrenoceptor mRNAs were also measured in colonic muscle and mucosa.

(–)‐Isoprenaline caused relaxation of longitudinal smooth muscle preparations with pEC50=7.39±0.12, and this response was inhibited by both propranolol (0.1 μm, pKB 8.55±0.12) and the selective β1‐antagonist, CGP 20712A (0.1 μm, pKB 8.80±0.20), while the selective β2‐antagonist, ICI 118551 (0.1 μm) failed to inhibit isoprenaline relaxation consistently.

(–)‐Isoprenaline caused relaxation of taenia coli with a pEC50 of 6.70±0.17. Propranolol (0.1 μm), CGP 20712A (0.1 μm) and ICI 118551 (0.1 μm) inhibited the isoprenaline response with similar low affinities (pKB values 7.93, 7.71 and 7.54, respectively). Carbachol pre‐contracted circular smooth muscle preparations failed to relax consistently to isoprenaline and these responses were not characterized.

β1‐ and β2‐Adrenoceptor mRNAs were present in circular/longitudinal muscle samples and taenia coli samples, and lower levels were detected in mucosa. β3‐mRNA was also present in both muscle preparations but was not detected in human colonic mucosa.

In summary, β1‐adrenoceptors are the predominant subtype mediating isoprenaline‐induced relaxation of the thin longitudinal smooth muscle of human colon, while β3‐receptors do not appear to be involved in these responses. However, β3‐adrenoceptors may play a role in relaxation of the taenia coli as conventional antagonist affinities are low. β3‐Adrenoceptor mRNA was present in taenia coli and circular/longitudinal smooth muscle but absent from human colonic mucosa.

British Journal of Pharmacology (1997) 120, 1527–1535; doi:10.1038/sj.bjp.0701056