bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of nitric oxide‐ and vasoactive intestinal polypeptide‐containing neurones in human gastric fundus strip relaxations
Authors: Tonini M , De Giorgio R , De Ponti F , Sternini C , Spelta V , Dionigi P , Barbara G , Stanghellini V , Corinaldesi R
Published: January 2005

The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.

By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)‐diaphorase and specific rabbit polyclonal NO‐synthase (NOS) and VIP antisera, NOS‐ and VIP‐containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS‐immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP‐IR. The majority of myenteric neurones containing VIP coexpressed NADPH‐diaphorase.

Electrical stimulation of fundus strips caused frequency‐dependent NANC relaxations. NG‐nitro‐L‐arginine (L‐NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L‐NOARG‐resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.

Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L‐NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).

In the human gastric fundus, NOS‐ and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO‐mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.

British Journal of Pharmacology (2000) 129, 12–20; doi:10.1038/sj.bjp.0702977
α2‐Adrenoceptor and NPY receptor‐mediated contractions of porcine isolated blood vessels: evidence for involvement of the vascular endothelium
Authors: Roberts R E , Kendall D A , Wilson V G
Published: January 2005

Enhanced contractions to the α2‐adrenoceptor agonist UK14304 and neuropeptide Y (NPY) in the porcine ear artery can be uncovered by pharmacological manipulation. The aim of this study was to determine whether similar pharmacological manipulation can uncover enhanced contractions in the porcine splenic artery, and to determine whether the endothelium modulates these responses.

UK14304 (0.3 μM) and NPY (0.1 μM) produced small contractions of the porcine splenic artery. After pre‐contraction of the tissue with U46619, followed by relaxation with forskolin, the responses to both UK14304 and NPY were enhanced. Enhanced contractions to both UK14304 and NPY were also obtained after relaxation with SNP. These results demonstrate that, as in the porcine ear artery, α2‐adrenoceptors and NPY receptors are able to produce enhanced contractile responses through both adenylyl cyclase‐dependent and ‐independent signal transduction pathways.

Removal of the endothelium had no significant effect on responses to UK14304 either alone or in the presence of U46619 and forskolin in the porcine splenic artery. On the other hand, responses to UK14304 after relaxation with SNP were reduced after endothelium‐denudation in both the porcine splenic artery and ear artery. Similar results were obtained with NPY in the porcine ear artery.

In conclusion, enhanced contractile responses to UK14304 and NPY in the porcine splenic artery can be uncovered using methods similar to those employed in the porcine ear artery. Under certain conditions the responses to both agents are modulated by the endothelium. These data highlight further the similarities in the signal transduction pathways used by both α2‐adrenoceptors and NPY receptors to induce vasoconstriction.

British Journal of Pharmacology (1999) 128, 1705–1712; doi:10.1038/sj.bjp.0702979
Functional group I metabotropic glutamate receptors in submucous plexus of guinea‐pig ileum
Authors: Hu Hong‐Zhen , Ren Jun , Liu Sumei , Gao Chuanyun , Xia Yun , Wood Jackie D
Published: January 2005

Intracellular recording methods and immunostaining revealed the existence of functional group I metabotropic glutamate receptors (mGluRs) in submucous plexus neurons of guinea‐pig ileum. Selective group I, but not groups II or III metabotropic glutamate receptor agonists induced concentration‐dependent, slowly‐activating depolarizing responses. Group I metabotropic glutamate receptor antagonism observed with (S)‐4‐carboxyphenylglycine (S‐4‐CPG) (100–600 μM) was competitive as determined by Schild analysis (pA2=3.81±0.02). Neither the group II and III metabotropic nor ionotropic glutamate receptor antagonists altered responses evoked by group I receptor agonists. Immunoreactivities for metabotropic glutamate 1α and 5 receptors were found to locate exclusively in neurons in the submucous plexus of guinea‐pig ileum with the highest density around the cell bodies. The results suggest that group I metabotropic glutamate receptors are functionally expressed in the submucous plexus of guinea‐pig small intestine.

British Journal of Pharmacology (1999) 128, 1631–1635; doi:10.1038/sj.bjp.0702980
Stimulation of the ventral tegmental area enhances the effect of vasopressin on blood pressure in conscious rats
Authors: Van Den Buuse Maarten , Catanzariti Renaldo
Published: January 2005

The mesolimbic dopamine system projects to a large number of forebrain areas and plays an important role in the regulation of locomotor activity, cognition and reward. We previously found evidence for a functional interaction between the mesolimbic dopamine system and circulating vasopressin and the present study was performed to test the hypothesis that mesolimbic dopamine stimulation modulates the cardiovascular effects of vasopressin.

Sprague‐Dawley rats were stereotaxically implanted with a guide cannula into the region of origin of the mesolimbic system, the ventral tegmental area, and instrumented with catheters into the abdominal aorta and jugular vein. One week later, separate groups of conscious rats were injected intravenously with 1, 3 or 10 ng kg−1of arginine‐vasopressin or other vasopressor drugs before and after intra‐ventral tegmental area injection of 10 nmol of neurotensin.

Intra‐ventral tegmental area injections of neurotensin had no significant effect on mean arterial pressure and heart rate but significantly potentiated the pressor response to intravenous administration of vasopressin when compared to saline‐injections. However, the vasopressin‐induced bradycardia was unaffected. Intravenous pretreatment with raclopride blocked the ability of neurotensin, injected into the ventral tegmental area, to potentiate the vasopressin‐induced pressor response. Intra ventral tegmental area injections of neurotensin had no effect on the pressor response and bradycardia induced by intravenous angiotensin II or methoxamine.

In conclusion, these results suggest that the mesolimbic dopamine system, in addition to its well‐known role in the regulation of behaviour, modulates cardiovascular control by potentiating the effects of vasopressin on mean arterial pressure.

British Journal of Pharmacology(2000)129, 29–36; doi:10.1038/sj.bjp.0702982
[125I]‐GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors
Authors: Dumont Yvan , Quirion Rémi
Published: January 2005

GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method.

[125I]‐GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h.

Saturation binding experiments showed that [125I]‐GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes.

Competition binding experiments revealed that specific [125I]‐GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA.

Autoradiographic studies demonstrated that [125I]‐GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]‐GR231118/BIBO3304‐insensitive binding sites were detected in few areas. These [125I]‐GR231118/BIBO3304‐insensitive binding sites likely represent labelling to the Y4 receptor subtype.

In summary, [125I]‐GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]‐GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype.

British Journal of Pharmacology (2000) 129, 37–46; doi:10.1038/sj.bjp.0702983
Individual sympathetic varicosities possess different sensitivities to alpha 2 and P2 receptor agonists and antagonists in mouse vas deferens
Authors: O'Connor S C , Brain K L , Bennett M R
Published: January 2005

The diversity of α2 and purinergic autoreceptor actions on action potential evoked calcium transients in single varicosities has been investigated using the calcium indicator Oregon Green 488 BAPTA‐1.

During long trains of impulses (10 Hz for 30 s), the change in calcium concentration in varicosities from its resting level (Δ[Ca2+]v) increased in many varicosities during the first 3 s of stimulation before reaching a plateau.

The α2 adrenoceptor agonist clonidine (1 μM) decreased Δ[Ca2+]v by over 40% during short trains (five impulses at 5 Hz) in most varicosities, although some were unaffected. The α2 adrenoceptor antagonist idazoxan (2 μM) increased the Δ[Ca2+]v plateau following long trains in most varicosities. Hence, most varicosities possess α2 adrenoceptors which are activated when noradrenaline accumulates extracellularly.

During long trains of impulses, the P2y‐purinergic receptor agonist 2‐methyl‐thio‐ATP (100 μM) decreased Δ[Ca2+]v plateau by about 50% in most varicosities; α,β‐methylene ATP (100 μM) decreased it by about 50% in a minority of varicosities; adenosine (200 μM) had no significant effect. Suramin (100 μM) increased the Δ[Ca2+]v during all stimulus protocols in most varicosities, suggesting that ambient ATP modulates Δ[Ca2+]v responses. The P2y receptor antagonist reactive blue (100 μM) affected a minority of varicosities. Given that most varicosities respond to suramin, other P2 receptor subtypes are probably present.

The ATP ectoenzyme antagonist ARL67157 (50 μM) decreased the plateau Δ[Ca2+]v during long trains in complete strings of varicosities but not in others.

The present technique indicates that varicosities have diverse autoreceptor utilization.

British Journal of Pharmacology (1999) 128, 1739–1753; doi:10.1038/sj.bjp.0702984
Regional haemodynamic responses to infusion of lipopolysaccharide in conscious rats: effects of pre‐ or post‐treatment with glibenclamide
Authors: Gardiner S M , Kemp P A , March J E , Bennett T
Published: January 2005

To determine the putative contribution of KATP‐channels to the haemodynamic sequelae of endotoxaemia, three experiments were carried out in different groups of conscious, chronically‐instrumented, unrestrained, male Long Evans rats.

In the first experiment, pretreatment with the KATP‐channel antagonist, glibenclamide, abolished the initial hypotension, but not the renal vasodilatation caused by LPS infusion. Subsequently, however, in the presence of glibenclamide and LPS there was a significant increase in mean arterial blood pressure, and a bradycardia, in contrast to the fall in mean arterial blood pressure and the tachycardia seen in the presence of vehicle and LPS. The pressor and bradycardic changes in the presence of glibenclamide and LPS were accompanied by significant reductions in hindquarters flow and vascular conductance, and these were significantly greater than those seen in the presence of vehicle and LPS, or glibenclamide and saline.

Administration of glibenclamide 6 h after the onset of saline and LPS infusion, or 6 h after the onset of saline and LPS infusion in the presence of the AT1‐receptor antagonist, losartan, and the ETA‐, ETB‐ receptor antagonist, SB 209670, in the absence or presence of dexamethasone, caused a significant increase in mean arterial blood pressure and reductions in renal, mesenteric and hindquarters conductances, although the latter was the only vascular bed in which there was a reduction in flow.

The results are consistent with a contribution from KATP‐channels to the vasodilatation caused by LPS, particularly in the hindquarters vascular bed.

British Journal of Pharmacology (1999) 128, 1772–1778; doi:10.1038/sj.bjp.0702985
(−)1‐(Benzofuran‐2‐yl)‐2‐propylaminopentane, [(−)BPAP], a selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain
Authors: Knoll Joseph , Yoneda Fumio , Knoll Berta , Ohde Hironori , Miklya Ildikó
Published: January 2005

The brain constituents β‐phenylethylamine (PEA) and tryptamine enhance the impulse propagation mediated transmitter release (exocytosis) from the catecholaminergic and serotoninergic neurons in the brain (‘catecholaminergic/serotoninergic activity enhancer, CAE/SAE, effect’). (−)Deprenyl (Selegiline) and (−)1‐phenyl‐2‐propylaminopentane [(−)PPAP] are amphetamine derived CAE substances devoid of the catecholamine releasing property.

By changing the aromatic ring in PPAP we developed highly potent and selective CAE/SAE substances, structurally unrelated to the amphetamines. Out of 65 newly synthetized compounds, a tryptamine derived structure, (−)1‐(benzofuran‐2‐yl)‐2‐propylaminopentane [(−)BPAP] was selected as a potential follower of (−)deprenyl in the clinic and as a reference compound for further analysis of the CAE/SAE mechanism in the mammalian brain.

(−)BPAP significantly enhanced in 0.18 μmol 1−1 concentration the impulse propagation mediated release of [3H]‐noradrenaline and [3H]‐dopamine and in 36 nmol 1−1 concentration the release of [3H]‐serotonin from the isolated brain stem of rats. The amount of catecholamines and serotonin released from isolated discrete rat brain regions (dopamine from the striatum, substantia nigra and tuberculum olfactorium, noradrenaline from the locus coeruleus and serotonin from the raphe) enhanced significantly in the presence of 10−12–10−14 M (−)BPAP. BPAP protected cultured hippocampal neurons from the neurotoxic effect of β‐amyloid in 10−14 M concentration. In rats (−)BPAP significantly enhanced the activity of the catecholaminergic and serotoninergic neurons in the brain 30 min after acute injection of 0.1 μg kg−1 s.c. In the shuttle box, (−)BPAP in rats was about 130 times more potent than (−)deprenyl in antagonizing tetrabenazine induced inhibition of performance.

British Journal of Pharmacology (1999) 128, 1723–1732; doi:10.1038/sj.bjp.0702995
P‐glycoprotein‐ and mrp2‐mediated octreotide transport in renal proximal tubule
Authors: Gutmann Heike , Miller David S , Droulle Agathe , Drewe Jürgen , Fahr Alfred , Fricker Gert
Published: January 2005

Transepithelial transport of a fluorescent derivative of octreotide (NBD‐octreotide) was studied in freshly isolated, functionally intact renal proximal tubules from killifish (Fundulus heteroclitus).

Drug accumulation in the tubular lumen was visualized by means of confocal microscopy and was measured by image analysis. Secretion of NBD‐octreotide into the tubular lumen was demonstrated and exhibited the all characteristics of specific and energy‐dependent transport. Steady state luminal fluorescence averaged about five times cellular fluorescence and was reduced to cellular levels when metabolism was inhibited by NaCN.

NBD‐octreotide secretion was inhibited in a concentration‐dependent manner by unlabelled octreotide, verapamil and leukotriene C4 (LTC4). Conversely, unlabelled octreotide reduced in a concentration dependent manner the p‐glycoprotein (Pgp)‐mediated secretion of a fluorescent cyclosporin A derivative (NBDL‐CS) and the mrp2‐mediated secretion of fluorescein methotrexate (FL‐MTX).

This inhibition was not due to impaired metabolism or toxicity since octreotide had no influence on the active transport of fluorescein (FL), a substrate for the classical renal organic anion transport system.

The data are consistent with octreotide being transported across the brush border membrane of proximal kidney tubules by both Pgp and mrp2.

British Journal of Pharmacology (2000) 129, 251–256; doi:10.1038/sj.bjp.0703003
Effects of nicotine and chlorisondamine on cerebral glucose utilization in immobilized and freely‐moving rats
Authors: Marenco T , Bernstein S , Cumming P , Clarke P B S
Published: January 2005

Chlorisondamine blocks central nicotinic receptors for many weeks via an unknown mechanism. Intracerebroventricular administration of [3H]‐chlorisondamine in rats results in an anatomically restricted and persistent intracellular accumulation of radioactivity. The initial aim of the present study was to test whether nicotinic receptor antagonism by chlorisondamine is also anatomically restricted.

Male adult rats were pretreated several times with nicotine to avoid the disruptive effects of the drug seen in drug‐naïve animals. They then received chlorisondamine (10 μg i.c.v.) or saline, and local cerebral glucose utilization (LCGU) was measured 4 weeks later after acute nicotine (0.4 mg kg−1 s.c.) or saline administration. During testing, rats were partially immobilized. Nicotine significantly increased LCGU in the anteroventral thalamus and in superior colliculus. Chlorisondamine completely blocked the first of these effects. Chlorisondamine significantly reduced LCGU in the lateral habenula, substantia nigra pars compacta, ventral tegmental area, and cerebellar granular layer.

The second experiment was of similar design, but the rats were not pre‐exposed to nicotine, and were tested whilst freely‐moving. Acute nicotine significantly increased LCGU in anteroventral thalamus, superior colliculus, medial habenula and dorsal lateral geniculate. Overall, however, nicotine significantly decreased LCGU. Most or all of the central effects of nicotine on LCGU were reversed by chlorisondamine given 4 weeks beforehand.

These findings suggest that chlorisondamine blocks nicotinic effects widely within the brain. They also indicate that in freely‐moving rats, nicotine can reduce or stimulate cerebral glucose utilization, depending on the brain area.

British Journal of Pharmacology (2000) 129, 147–155; doi:10.1038/sj.bjp.0703005
Contraction to big endothelin‐1, big endothelin‐2 and big endothelin‐3, and endothelin‐converting enzyme inhibition in human isolated bronchi
Authors: Yap E Y S , Battistini B , McKay K O
Published: January 2005

All three endothelin precursor peptides, i.e. big endothelin‐1 (big ET‐1), big endothelin‐2 (big ET‐2) and big endothelin‐3 (big ET‐3), produced contractile responses in human isolated bronchi, demonstrating the presence of functional endothelin‐converting enzyme (ECE) in this tissue.

The maximal contractile responses were equal to 108.4±8.0% (0.1 μM big ET‐1; n=4), 85.2±11.8% (0.1 μM big ET‐2; n=7) and 43.0±7.2% (0.1 μM big ET‐3; n=5) of the reference response to acetylcholine (1 mM).

The response to big ET‐1 (0.1 μM), but not endothelin‐1 (ET‐1, 0.1 μM), was diminished after overnight storage of the tissue at 4°C, demonstrating instability of the enzyme.

The responses to all three big‐endothelins were significantly inhibited, by the ECE inhibitors CGS 26393 and CGS 26303, in a concentration‐related manner.

The responses to the mature peptides ET‐1, endothelin‐2 (ET‐2), and endothelin‐3 (ET‐3) were unaffected by CGS 26393 and CGS 26303.

Phosphoramidon (10 μM) also produced an inhibition of the response to big ET‐1 that was equivalent to that produced by CGS 26393 (10 μM). Combination of CGS 26393 (10 μM) and phosphoramidon (10 μM) did not produce an additive inhibition.

These results demonstrate the presence of functional ECE for all three big endothelins in human bronchus and inhibition of the enzyme by newly developed orally active ECE inhibitors, as well as phosphoramidon.

British Journal of Pharmacology (2000) 129, 170–176; doi:10.1038/sj.bjp.0703006
Role of gap junctions in the responses to EDHF in rat and guinea‐pig small arteries
Authors: Edwards G , Félétou M , Gardener M J , Thollon C , Vanhoutte P M , Weston A H
Published: January 2005

In guinea‐pig internal carotid arteries with an intact endothelium, acetylcholine (10 μM) and levcromakalim (10 μM) each hyperpolarized the smooth muscle whereas a 5 mM elevation of extracellular K+ was without effect.

Incubation of the carotid artery with the gap junction inhibitors carbenoxolone (100 μM) or gap 27 (500 μM) essentially abolished the hyperpolarization to acetylcholine but it was without effect on that to levcromakalim. Carbenoxolone had no effect on the acetylcholine‐induced endothelial cell hyperpolarization but inhibited the smooth muscle hyperpolarization induced by the endothelial cell K+ channel opener, 1‐ethyl‐2‐benzimidazolinone (600 μM).

In rat hepatic and mesenteric arteries with endothelium, carbenoxolone (100 or 500 μM) depolarized the smooth muscle but did not modify hyperpolarizations induced by KCl or levcromakalim. In the mesenteric (but not the hepatic) artery, the acetylcholine‐induced hyperpolarization was inhibited by carbenoxolone.

Phenylephrine (1 μM) depolarized the smooth muscle cells of intact hepatic and mesenteric arteries, an effect enhanced by carbenoxolone. Gap 27 did not have a depolarizing action. In the presence of phenylephrine, acetylcholine‐induced hyperpolarization of both hepatic and mesenteric artery myocytes was partially inhibited by each of the gap junction inhibitors.

Collectively, the data suggest that gap junctions play some role in the EDHF (endothelium‐derived hyperpolarizing factor) response in rat hepatic and mesenteric arteries. However, in the guinea‐pig internal carotid artery, electrotonic propagation of endothelial cell hyperpolarizations via gap junctions may be the sole mechanism underlying the response previously attributed to EDHF.

British Journal of Pharmacology (1999) 128, 1788–1794; doi:10.1038/sj.bjp.0703009
The peripheral antinociceptive effect induced by morphine is associated with ATP‐sensitive K+ channels
Authors: Rodrigues A R A , Duarte I D G
Published: January 2005

The effect of several K+ channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium (TEA), 4‐aminopyridine (4‐AP) and cesium on the peripheral antinociceptive effect of morphine was evaluated by the paw pressure test in Wistar rats.

The intraplantar administration of a carrageenan suspension (250 μg) resulted in an acute inflammatory response and a decreased threshold to noxious pressure. Morphine administered locally into the paw (25, 50, 100 and 200 μg) elicited a dose‐dependent antinociceptive effect which was demonstrated to be mediated by a peripheral site up to the 100 μg dose.

The selective blockers of ATP‐sensitive K+ channels glibenclamide (20, 40 and 80 μg paw−1) and tolbutamide (40, 80 and 160 μg paw−1) antagonized the peripheral antinociception induced by morphine (100 μg paw−1).

This effect was unaffected by ChTX (0.5, 1.0 and 2.0 μg paw−1), a large conductance Ca2+‐activated K+ channel blocker, or by apamin (2.5, 5.0 and 10.0 μg paw−1), a selective blocker of a small conductance Ca2+‐activated K+ channel.

Intraplantar administration of the non‐specific K+ channel blockers TEA (160, 320 and 640 μg), 4‐AP (10, 50 and 100 μg) and cesium (125, 250 and 500 μg) also did not modify the peripheral antinociceptive effect of morphine.

These results suggest that the peripheral antinociceptive effect of morphine may result from activation of ATP‐sensitive K+ channels, which may cause a hyperpolarization of peripheral terminals of primary afferents, leading to a decrease in action potential generation. In contrast, large conductance Ca2+‐activated K+ channels, small conductance Ca2+‐activated K+ channels as well as voltage‐dependent K+ channels appear not to be involved in this transduction pathway.

British Journal of Pharmacology (2000) 129, 110–114; doi:10.1038/sj.bjp.0703038
Nitric oxide, prostanoid and non‐NO, non‐prostanoid involvement in acetylcholine relaxation of isolated human small arteries
Authors: Buus Niels H , Simonsen Ulf , Pilegaard Hans K , Mulvany Michael J
Published: January 2005

The main purpose of the study was to clarify to which extent nitric oxide (NO) contributes to acetylcholine (ACh) induced relaxation of human subcutaneous small arteries.

Arterial segments were mounted in myographs for recording of isometric tension, NO concentration and smooth muscle membrane potential.

In noradrenaline‐contracted arteries, ACh induced endothelium‐dependent relaxations. The NO synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG) had a small significant effect on the concentration‐response curves for ACh, and in the presence of L‐NOARG, indomethacin only caused a small additional rightward shift in the ACh relaxation.

The NO scavenger, oxyhaemoglobin attenuated relaxations for ACh and for the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP). Inhibition of guanylyl cyclase with 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ), and inhibition of protein kinase G with β‐phenyl‐1, N2‐etheno‐8‐bromoguanosine‐ 3′, 5′‐ cyclic monophosphorothioate, Rp‐isomer, slightly attenuated ACh relaxation, but abolished SNAP induced relaxation.

ACh induced relaxation without increases in the free NO concentration. In contrast, for equivalent relaxation, SNAP increased the NO concentration 32±8 nM.

ACh hyperpolarized the arterial smooth muscle cells with 11.4±1.3 mV and 10.5±1.3 mV in the absence and presence of L‐NOARG, respectively. SNAP only elicited a hyperpolarization of 1.6±0.9 mV.

In the presence of indomethacin and L‐NOARG, ACh relaxation was almost unaffected by lipoxygenase inhibition with nordihydroguaiaretic acid, or cytochrome P450 inhibition with 17‐octadecynoic acid or econazole. ACh relaxation was strongly reduced by the combination of charybdotoxin and apamin, but small increments in the extracellular potassium concentration induced no relaxations.

The study demonstrates that the NO/L‐arginine pathway is present in human subcutaneous small arteries and to a limited extent is involved in ACh induced relaxation. The study also suggests a small contribution of arachidonic acid metabolites. However, ACh relaxation is mainly dependent on a non‐NO, non‐prostanoid endothelium dependent hyperpolarization.

British Journal of Pharmacology (2000) 129, 184–192; doi:10.1038/sj.bjp.0703041
The metabolism of clomethiazole in gerbils and the neuroprotective and sedative activity of the metabolites
Authors: Green A R , Murray T K , Misra A , Snape M F , Jones J A , Cross A J
Published: January 2005

A single dose of clomethiazole (600 μmol kg−1 i.p.) has previously been shown to be neuroprotective in the gerbil model of global ischaemia.

In gerbils, clomethiazole (600 μmol kg−1) injection produced a rapid appearance (peak within 5 min) of drug in plasma and brain and similar clearance (plasma t1/2: 40 min) from both tissues. The peak brain concentration (226±56 nmol g−1) was 40% higher than plasma.

One major metabolite, 5‐(1‐hydroxyethyl‐2‐chloro)‐4‐methylthiazole (NLA‐715) and two minor metabolites 5‐(1‐hydroxyethyl)‐4‐methylthiazole (NLA‐272) and 5‐acetyl‐4‐methylthiazole (NLA‐511) were detected in plasma and brain.

Evidence suggested that clomethiazole is metabolized directly to both NLA‐715 and NLA‐272.

Injection of NLA‐715, NLA‐272 or NLA‐511 (each at 600 μmol kg−1) produced brain concentrations respectively 2.2, 38 and 92 times greater than seen after clomethiazole (600 μmol kg−1).

Clomethiazole (600 μmol kg−1) injected 60 min after a 5 min bilateral carotid artery occlusion in gerbils attenuated the ischaemia‐induced degeneration of the hippocampus by approximately 70%. The metabolites were not neuroprotective at this dose.

In mice, clomethiazole (600 μmol kg−1) produced peak plasma and brain concentrations approximately 100% higher than in gerbils, drug concentrations in several brain regions were similar but 35% higher than plasma. Clomethiazole (ED50: 180 μmol kg−1) and NLA‐715 (ED50: 240 μmol kg−1) inhibited spontaneous locomotor activity. The other metabolites were not sedative (ED50 >600 μmol kg−1).

These data suggest that the neuroprotective action of clomethiazole results from an action of the parent compound and that NLA‐715 contributes to the sedative activity of the drug.

British Journal of Pharmacology (2000) 129, 95–100; doi:10.1038/sj.bjp.0703042
Inhibition by troglitazone of the antigen‐induced production of leukotrienes in immunoglobulin E‐sensitized RBL‐2H3 cells
Authors: Yamashita Masamichi , Kushihara Mikie , Hirasawa Noriyasu , Takasaki Wataru , Takahagi Hidekuni , Takayanagi Motoaki , Ohuchi Kazuo
Published: January 2005

The effect of troglitazone, an anti‐diabetic drug with insulin‐sensitizing action, on antigen‐induced production of leukotriene (LT) B4, C4 and E4 and prostaglandin D2 (PGD2) was examined in dinitrophenol (DNP)‐specific immunoglobulin E (IgE)‐sensitized RBL‐2H3 mast cells following stimulation by the antigen, DNP‐conjugated human serum albumin. Levels of LTB4, C4 and E4 and PGD2 in the conditioned medium were enzyme‐immunoassayed.

Troglitazone inhibited the antigen‐induced production of LTB4, C4 and E4 and the potency of the inhibition was comparable to that of zileuton, a specific inhibitor of 5‐lipoxygenase (5‐LOX) and a clinically used anti‐asthmatic drug. Neither troglitazone nor zileuton affected antigen‐induced production of PGD2, arachidonic acid release from membrane phospholipids and degranulation.

Troglitazone inhibited LTB4 production by the supernatant fraction of RBL‐2H3 cell lysate with similar potency to zileuton, suggesting that troglitazone inhibits LT production by direct inhibition of 5‐LOX activity.

Furthermore, it was shown that troglitazone as well as zileuton inhibited LTB4 production in A23187‐stimulated rat peritoneal neutrophils.

These findings suggest that troglitazone inhibits antigen‐induced LT production in the IgE‐sensitized RBL‐2H3 cells and A23187‐stimulated rat peritoneal neutrophils by direct inhibition of 5‐LOX activity.

British Journal of Pharmacology (2000) 129, 367–373; doi:10.1038/sj.bjp.0703044
Effects of spinally administered P2X receptor agonists and antagonists on the responses of dorsal horn neurones recorded in normal, carrageenan‐inflamed and neuropathic rats
Authors: Stanfa Louise C , Kontinen Vesa K , Dickenson Anthony H
Published: January 2005

The function and role of P2X receptors in the spinal transmission of nociception was investigated using the selective P2X receptor agonists, α,β‐methylene ATP (α,β‐me ATP) and β,γ‐methylene‐L‐ATP (β,γ‐me‐L‐ATP) and the P2X receptor antagonists pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonate (PPADS) and suramin.

Intrathecal administration of 5 and 50 μg of β,γ‐me‐L‐ATP produced a significant facilitation of the C‐fibre evoked response and a tendency towards increased excitability of the post‐discharge, but not Aβ‐fibre evoked response of dorsal horn neurones recorded in normal animals. Administration of similar doses of α,β‐me ATP did not produce an overall change in the response of the neuronal population.

Peripheral administration of 20 μg of these agonists into the paw of the rat evoked firing in the dorsal horn neurones.

Intrathecal administration of the antagonists, suramin (50 and 500 μg) and PPADS (5, 50 and 500 μg), to normal animals and to animals with a model of neuropathy induced by spinal nerve ligation did not alter the evoked neuronal responses. In contrast, intrathecal administration of 500 μg of suramin to animals 3 h after the induction of carrageenan inflammation produced a significant inhibition of the C‐fibre evoked response of the neurones. Similar inhibitions were also seen following high doses of intrathecal PPADS, although this did not reach significance.

These results suggest that spinal P2X receptors may play a role in the modulation of spinal nociceptive transmission following the development of inflammation, but that these receptors play at most a minor role in spinal nociceptive processing in normal and neuropathic animals.

British Journal of Pharmacology (2000) 129, 351–359; doi:10.1038/sj.bjp.0703047
Stereoselective modulatory actions of oleamide on GABAA receptors and voltage‐gated Na+ channels in vitro: a putative endogenous ligand for depressant drug sites in CNS
Authors: Verdon Bernard , Zheng Jian , Nicholson Russell A , Ganelli C Robin , Lees George
Published: January 2005

cis‐9,10‐octadecenoamide (‘oleamide’) accumulates in CSF on sleep deprivation. It induces sleep in animals (the trans form is inactive) but its cellular actions are poorly characterized. We have used electrophysiology in cultures from embryonic rat cortex and biochemical studies in mouse nerve preparations to address these issues.

Twenty μM cis‐oleamide (but not trans) reversibly enhanced GABAA currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks.

cis‐oleamide stereoselectively blocked veratridine‐induced (but not K+‐induced) depolarisation of mouse synaptoneurosomes (IC50, 13.9 μM).

The cis isomer stereoselectively blocked veratridine‐induced (but not K+‐induced) [3H]‐GABA release from mouse synaptosomes (IC50, 4.6 μM).

At 20 μM cis‐oleamide, but not trans, produced a marked inhibition of Na+ channel‐dependent rises in intrasynaptosomal Ca2+.

The physiological significance of these observations was examined by isolating Na+ spikes in cultured pyramidal neurones. Sixty‐four μM cis‐oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz).

cis‐Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC50 of 4.1 μM suggesting a frequency‐ or state‐dependent block of voltage‐gated Na+ channels.

Oleamide is a stereoselective modulator of both postsynaptic GABAA receptors and presynaptic or somatic voltage‐gated Na+ channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics.

Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal.

British Journal of Pharmacology (2000) 129, 283–290; doi:10.1038/sj.bjp.0703051
An indirect influence of phenylephrine on the release of endothelium‐derived vasodilators in rat small mesenteric artery
Authors: Dora K A , Hinton J M , Walker S D , Garland C J
Published: January 2005

The possibility that stimulation of smooth muscle α1‐adrenoceptors modulates contraction via the endothelium was examined in rat small mesenteric arteries.

Nω‐nitro‐L‐arginine methyl ester, (L‐NAME, 100 μM to inhibit NO synthase) increased contraction to single concentrations of phenylephrine (1–3 μM) by approximately 2 fold (from a control level of 14.2±3.0 to 34.1±4.2% of the maximum contraction of the artery, n=20). The action of L‐NAME was abolished by disrupting the endothelium.

The subsequent addition of apamin (to inhibit small conductance Ca2+‐activated K+ channels, 50 nM) further augmented phenylephrine contractions, in an endothelium‐dependent manner, to more than 3 fold above control (50.4±5.3% of the maximum contraction, n=11).

Charybdotoxin (non‐selective inhibitor of large conductance Ca2+‐activated K+ channels, BKCa, 50 nM) plus L‐NAME augmented the level of phenylephrine contraction to 4–5‐fold above control (64.1±3.1%, n=5), but this effect was independent of the endothelium. The potentiation of contraction by charybdotoxin could be mimicked with the selective BKCa inhibitor, iberiotoxin,.

Apamin together with L‐NAME and charybdotoxin further significantly increased the phenylephrine contraction by 5–6‐fold, to 79.9±3.5% of the maximum contraction of the artery (n=13).

Phenylephrine failed directly to increase the intracellular Ca2+ concentration in endothelial cells freshly isolated from the small mesenteric artery.

Stimulation of smooth muscle α1‐adrenoceptors in the mesenteric artery induces contraction that is markedly suppressed by the endothelium. The attenuation of contraction appears to reflect both the release of NO from the endothelium and the efflux of K+ from both endothelial and smooth muscle cells. This suggests that the release of NO and endothelium‐derived hyperpolarizing factor can be evoked indirectly by agents which act only on the smooth muscle cells.

British Journal of Pharmacology (2000) 129, 381–387; doi:10.1038/sj.bjp.0703052
Effects of P1 and P2 receptor antagonists on β,γ‐methyleneATP‐ and CGS21680‐induced cyclic AMP formation in NG108‐15 cells
Authors: Ohkubo Satoko , Kimura Junko , Nakanishi Hironori , Matsuoka Isao
Published: January 2005

We have previously shown that ATP increased cyclic AMP in NG108‐15 cells, which was inhibited by P1 receptor antagonist methylxanthines. In the present study, we examined the effects of P1 and P2 receptor antagonists on cyclic AMP formation induced by β,γ‐methyleneATP (β,γ‐MeATP) and CGS21680, an A2A adenosine receptor agonist, in NG108‐15 cells.

β,γ‐MeATP and CGS21680 increased intracellular cyclic AMP with EC50 values of 8.0±0.98 μM (n=4) and 42±7.5 nM (n=4), respectively.

Several P1 receptor antagonists inhibited both β,γ‐MeATP‐ and CGS21680‐induced cyclic AMP increase with a similar rank order of potency; ZM241385>CGS15943>XAC>DPCPX. However, the pKi values of these antagonists for β,γ‐MeATP were larger than those for CGS21680.

Alloxazine, a P1 receptor antagonist, and several P2 receptor antagonists (PPADS, iPPADS, reactive blue‐2) inhibited β,γ‐MeATP‐induced response, while these antagonists little affected CGS21680‐induced one. Suramin was effective only for β,γ‐MeATP‐induced response at 1 mM.

2‐chloroadenosine (2CADO) and 2‐chloroATP (2ClATP) increased cyclic AMP with similar potencies. The effects of these agonists were both inhibited by ZM241385, but only 2ClATP‐induced response was inhibited by PPADS.

ATP‐ and β,γ‐MeATP‐induced responses were little affected by α,β‐methyleneADP, a 5′‐nucleotidase inhibitor.

These results clearly demonstrate that ATP‐stimulated cyclic AMP formation can be distinguished from the A2A receptor agonist‐induced one by using the several P1 and P2 receptor antagonists.

British Journal of Pharmacology (2000) 129, 291–298; doi:10.1038/sj.bjp.0703053
Different role of IL‐4 in the onset of hapten‐induced contact hypersensitivity in BALB/c and C57BL/6 mice
Authors: Nagai Hiroichi , Ueda Yoshifumi , Ochi Takashi , Hirano Yosuke , Tanaka Hiroyuki , Inagaki Naoki , Kawada Kenji
Published: January 2005

To study the role of interleukin (IL)‐4 in the onset of contact hypersensitivity (CH) in mice, the effect of IL‐4 gene‐depletion and anti‐IL‐4 monoclonal antibody treatment on dinitrofluorobenzene (DNFB)‐induced CH was examined. Simultaneously, to clarify the effect of background gene, DNFB‐induced CH in BALB/c and C57BL/6 mice was compared.

Five repeated topical applications of DNFB to the ears of mice resulted in CH of the ears in terms of increases in ear thickness and histopathological changes. The magnitude of ear thickness increase in BALB/c mice was almost three times greater than that in C57BL/6 mice.

The CH in BALB/c mice was significantly suppressed by IL‐4 gene‐depletion and anti‐IL‐4 monoclonal antibody treatment. In contrast, the symptoms of dermatitis in C57BL/6 mice were slightly affected by the same treatment. These changes corresponded well to the production of specific IgE antibody.

Total IgE antibody production and the expression of productive Cε mRNA were dramatically suppressed by IL‐4 gene‐depletion and anti‐IL‐4 treatment in BALB/c and C57BL/6 mice. Neither total IgG nor IgM levels in either strain of mice was altered by depletion of IL‐4.

The expression of IFN‐γ in the skin lesion was dramatically suppressed by IL‐4 gene‐depletion in BALB/c mice, but not in C57BL/6 mice.

These findings indicate that IL‐4 plays an important role in the onset of DNFB‐induced CH in BALB/c mice, but not in C57BL/6 mice.

British Journal of Pharmacology (2000) 129, 299–306; doi:10.1038/sj.bjp.0703054
Lipopolysaccharide activates nuclear factor κB in rat intestine: role of endogenous platelet‐activating factor and tumour necrosis factor
Authors: De Plaen Isabelle G , Tan Xiao‐Di , Chang Hong , Wang Liya , Remick Daniel G , Hsueh Wei
Published: January 2005

We examined the effect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor κB (NF‐κB) activation in the intestine and the roles of endogenous platelet‐activating factor (PAF), tumour necrosis factor‐α (TNF) and neutrophils. We also compared the time course of NF‐κB activation in response to PAF and LPS.

Ileal nuclear extracts from LPS (8 mg kg−1, IV)‐injected rats were assayed for NF‐κB‐DNA‐binding activity and identification of the subunits. Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti‐TNF antibody, or anti‐neutrophil antiserum. NF‐κB p65 was localized by immunohistochemistry. An additional group was challenged with PAF (2 μg kg−1, IV) for comparison.

LPS activates intestinal NF‐κB, both as p50‐p50 and p50‐p65 dimers within 15 min, and the effect peaks at 2 h. The effect is slower and more sustained than that of PAF, which peaks at 30 min. Activated NF‐κB was immunolocalized within epithelial and lamina propria cells. LPS effect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti‐TNF antibody, or anti‐neutrophil antiserum (P<0.05).

LPS activates intestinal NF‐κB in vivo and neutrophil activation is involved in its action. The LPS effect is mediated by both endogenous PAF and TNF.

British Journal of Pharmacology (2000) 129, 307–314; doi:10.1038/sj.bjp.0703055
Enhanced nociception by exogenous and endogenous substance P given into the spinal cord in mice lacking NR2A/ε1, an NMDA receptor subunit
Authors: Inoue Makoto , Mishina Masayoshi , Ueda Hiroshi
Published: January 2005

In capsaicin‐pretreated mice, the nociceptive responses induced by intrathecally (i.t.) administered substance P (SP) were enhanced by N‐methyl‐D‐aspartate (NMDA)‐type receptor antagonists, dizocilpine (MK801) and D‐2‐amino‐5‐phosphonopentanoate (D‐AP5) in a dose‐dependent manner. Similar enhancement of SP‐induced nociception was also observed in mice lacking the NMDA‐type glutamate receptor NR2A/ε1 subunit gene (GluRε1−/− mice). On the other hand, GluRε1−/− mice showed a marked enhancement of the peripheral nociceptive responses induced by intraplantar (i.pl.) injection of SP and bradykinin (BK). As the nociceptive responses to SP and BK (i.pl.) were both antagonized by CP‐99994, an neurokinin1 (NK1) antagonist (i.t.), these results suggest that GluRε1 receptor may play an inhibitory role in the downstream mechanisms of primary nociceptive SP neurones, possibly through activation of unidentified inhibitory neurones.

British Journal of Pharmacology (2000) 129, 239–241; doi:10.1038/sj.bjp.0703056
Differential actions of L‐cysteine on responses to nitric oxide, nitroxyl anions and EDRF in the rat aorta
Authors: Ellis Anthie , Guang Li Chun , Rand Michael J
Published: January 2005

The effects of L‐cysteine were tested in rat aortic rings on responses to nitric oxide free radical (NO•), nitroxyl (NO−) derived from Angeli's salt and endothelium‐derived relaxing factor (EDRF) activated by acetylcholine, ATP and the calcium ionophore A23187. Concentrations of 300 μM or less of L‐cysteine had no effect on responses.

Relaxations produced by exogenous NO• (0.25–2.5 μM) were markedly prolonged and relaxations produced by sodium nitroprusside (0.001–0.3 μM) were enhanced by 1 and 3 mM L‐cysteine. The enhancements by L‐cysteine of responses to NO• and sodium nitroprusside may be attributed to the formation of S‐nitrosocysteine.

Relaxations mediated by the nitroxyl anion (0.3 μM) donated from Angeli's salt were more prolonged than those produced by NO•, and nitroxyl‐induced relaxations were reduced by L‐cysteine (1 and 3 mM).

EDRF‐mediated relaxations produced by acetylcholine (0.01–10 μM), ATP (3–100 μM) and the calcium ionophore A23187 (0.1 μM) were significantly reduced by 3 mM L‐cysteine.

The similarity between the inhibitory effects of L‐cysteine on responses to EDRF and on those to nitroxyl suggests that a component of the response to EDRF may be mediated by nitroxyl anion.

British Journal of Pharmacology (2000) 129, 315–322; doi:10.1038/sj.bjp.0703058
Functional characterization of the P2X4 receptor orthologues
Authors: Jones C A , Chessell I P , Simon J , Barnard E A , Miller K J , Michel A D , Humphrey P P A
Published: January 2005

The aim of this study was to functionally characterize the recombinant mouse P2X4 receptor and to compare its pharmacological properties with those of the human and rat orthologues.

Whole cell recordings were made from rafts of HEK‐293 cells stably expressing recombinant mouse, rat or human P2X4 receptors, using Cs‐aspartate containing electrodes (3–8 MΩ) in a HEPES‐buffered extracellular medium.

The agonist potency of ATP at the three species orthologues was similar, with mean EC50 values of 2.3 μM, 1.4 μM and 5.5 μM, respectively.

Adenosine‐5′‐tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X4 receptors (EC50=2.6 and 3.0 μM), but was significantly less potent at the rat orthologue (EC50=20.0 μM). α,β‐methylene adenosine‐5′‐triphosphate (α,β‐meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X4 and 24% at the human P2X4 receptor.

In contrast to the other species orthologues, α,β‐meATP failed to elicit a significant agonist response at rat P2X4 receptors, and was found to act as an antagonist, with an IC50 of 4.6 μM, against 10 μM ATP.

Mouse P2X4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (IC50=10.5 μM), as were human P2X4 receptors (IC50=9.6 μM). The rat receptor however, showed a low sensitivity to PPADS (IC50>100 μM).

All three orthologues were relatively suramin‐insensitive (IC50>100 μM) and insensitive to 1‐[N,O‐Bis(5‐isoquinoline sulphonyl)benzyl]‐2‐(4‐phenylpiperazine)ethyl]‐5‐isoquinoline sulphonamide (KN‐62; IC50>3 μM).

Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X4 receptor, and differ markedly from the rat receptor.

British Journal of Pharmacology (2000) 129, 388–394; doi:10.1038/sj.bjp.0703059