bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective modifiers of glutathione biosynthesis and ‘repriming’ of vascular smooth muscle photorelaxation
Authors: Megson I L , Holmes Sally A , Magid K S , Pritchard Rebecca J , Flitney F W
Published: January 2005

Photorelaxation of vascular smooth muscle (VSM) is caused by the release of nitric oxide (NO) from a finite molecular store that can be depleted by irradiating pre‐contracted arteries with visible light. The ability of an ‘exhausted’ vessel to respond to a further period of illumination is lost temporarily but then recovers slowly as the photosensitive store is reconstituted in the dark. The recovery process, termed repriming, displays an absolute requirement for endothelium‐derived NO and is inhibited by pre‐treating arteries with ethacrynic acid, a thiol‐alkylating agent. Here we demonstrate that agents that up‐ or down‐regulate glutathione (GSH) biosynthesis influence the extent to which the store is regenerated in the dark.

Isolated rat tail arteries (RTAs) were perfused internally with Krebs solution containing phenylephrine (PE; mean [PE]±s.e.mean: 5.78±0.46 μM) and periodically exposed to laser light (λ=514.5 nm, 6.3 mW cm−2 for 6 min). Photorelaxations of control RTAs were compared with those from either (a) vessels taken from animals previously injected i.p. with buthionine sulphoximine (BSO), an inhibitor of γ‐glutamylcysteine synthetase (three injections, 100 mg kg−1 at 8 h intervals); or (b) isolated RTAs that were perfused ex vivo with oxothiazolidine (OXO), a precursor of cysteine (10−4 M OXO for 60 min). RTAs from BSO‐treated animals exhibited attenuated photorelaxations: the mean (±s.e.mean) amplitude of the response recorded after 72 min recovery in the dark was 12.4±1.6% versus 21.4±2.9% for control arteries (n=5; P<0.01). Conversely RTAs treated with OXO and allowed to recover for a similar period showed enhanced photorelaxations, 32.6±6.3% as compared to 21.4±2.9% for control arteries (n=5; P<0.01). A hyperbolic curve fit to repriming curves for BSO‐treated and control arteries returned asymptote values (maximum photorelaxations) of (mean±s.e.mean) 24.2±3.2% and 55.2±8.5%, respectively.

The level of GSH in RTA extracts was measured by high‐pressure liquid chromatography (HPLC). Injecting animals with BSO decreased GSH to 85% of control levels (P<0.05) while treatment of isolated vessels with OXO resulted in a 31% increase above control levels (P<0.05). Thus, drug‐induced changes in RTA GSH levels were positively correlated with altered photorelaxations.

The results lead us to postulate that the photosensitive store in VSM is generated, at least in part, from intracellular GSH which becomes converted to S‐nitrosoglutathione (GSNO) by nitrosating species that are formed ultimately from endothelium‐derived NO. The possible physiological significance of a photolabile store of NO in VSM is discussed briefly.

British Journal of Pharmacology (2000) 130, 1575–1580; doi:10.1038/sj.bjp.0703499
Adrenomedullin inhibits spontaneous and bradykinin‐induced but not oxytocin‐ or prostaglandin F2α‐induced periodic contraction of rat uterus
Authors: Yanagita Toshihiko , Yamamoto Ryuichi , Sugano Takashi , Kobayashi Hideyuki , Uezono Yasuhito , Yokoo Hiroki , Shiraishi Seiji , Minami Shin‐ichi , Wada Akihiko
Published: January 2005

In isolated rat uterine strips, adrenomedullin (AM) inhibited the spontaneous periodic contraction in a concentration‐dependent manner (IC50=22.3±0.7 nM). The inhibitory effect of AM was prevented by either AM22–52, a putative antagonist for AM receptors, or calcitonin gene‐related peptide (CGRP)8–37, a putative antagonist for CGRP receptors. AM also attenuated bradykinin (BK)‐induced periodic uterine contraction, which was blocked by AM22–52 or CGRP8–37, whereas AM had no effect on the periodic contraction caused by oxytocin or prostaglandin F2α (PGF2α). RT–PCR analysis showed that mRNAs for calcitonin receptor‐like receptor (CRLR), receptor‐activity‐modifying protein (RAMP)1, RAMP2 and RAMP3 were expressed in the rat uterus. These results demonstrate that AM selectively inhibits spontaneous and BK‐induced periodic contraction via activating receptors for AM and CGRP.

British Journal of Pharmacology (2000) 130, 1727–1730; doi:10.1038/sj.bjp.0703500
Abrogation of lung inflammation in sensitized Stat6‐deficient mice is dependent on the allergen inhalation procedure
Authors: Trifilieff Alexandre , El‐Hasim Ahmed , Corteling Randolph , Owen Charles E
Published: January 2005

Conflicting results have been reported about the role of Stat6 in allergen‐induced airway inflammation. We have studied the influence of the allergen inhalation procedure on the inflammatory response using wild‐type and Stat6‐deficient mice generated on a C57BL/6 background.

Animals were immunized i.p. on day 0 and 7 with ovalbumin (OVA) and then received aerosolized OVA or phosphate buffer saline challenge (acute on day 14; chronic on day 14, 15, 16, 17 and 18) before being sacrificed at different time points.

Following an acute challenge, Stat6‐deficiency fully abrogated the increase in serum IgE levels and the development of lung inflammation (inflammatory cell infiltration, IL‐4 and IL‐5 release, and increase in plasma leakage).

Following chronic challenge, despite the absence of IgE, IL‐4 and IL‐5, Stat6‐deficient mice develop a characteristic lung inflammation, although the intensity was smaller when compared with the wild‐type mice.

OVA‐induced early bronchoconstriction was observed in wild‐type mice only after chronic challenge, and this was totally abrogated in the Stat6‐deficient animals.

These results suggest that Stat6 signalling is essential for the development of allergic airway inflammation following an acute allergen exposure. However, in a more chronic situation, the airway inflammatory response seems to be only partially mediated by Stat6.

British Journal of Pharmacology (2000) 130, 1581–1588; doi:10.1038/sj.bjp.0703501
Human urotensin‐II is an endothelium‐dependent vasodilator in rat small arteries
Authors: Bottrill Fiona E , Douglas Stephen A , Hiley C Robin , White Richard
Published: January 2005

The possible role of the endothelium in modulating responses to human urotensin‐II (U‐II) was investigated using isolated segments of rat thoracic aorta, small mesenteric artery, left anterior descending coronary artery and basilar artery.

Human U‐II was a potent vasoconstrictor of endothelium‐intact isolated rat thoracic aorta (EC50=3.5±1.1 nM, Rmax=103±10% of control contraction induced by 60 mM KCl and 1 μM noradrenaline). However the contractile response was not significantly altered by removal of the endothelium or inhibition of nitric oxide synthesis with L‐NAME (100 μM). Human U‐II did not cause relaxation of noradrenaline‐precontracted, endothelium‐intact rat aortae.

Human U‐II contracted endothelium‐intact rat isolated left anterior descending coronary arteries (EC50=1.3±0.8 nM, Rmax=20.1±4.9% of control contraction induced by 10 μM 5‐HT). The contractile response was significantly enhanced by removal of the endothelium (Rmax=55.4±16.1%). Moreover, human U‐II caused concentration‐dependent relaxation of 5‐HT‐precontracted arteries, which was abolished by L‐NAME or removal of the endothelium.

No contractile effects of human U‐II were found in rat small mesenteric arteries. However the peptide caused potent, concentration‐ and endothelium‐dependent relaxations of methoxamine‐precontracted vessels. The relaxant responses were potentiated by L‐NAME (300 μM) but abolished in the additional presence of 25 mM KCl (which inhibits the actions of endothelium‐derived hyperpolarizing factor).

The present study is the first to show that human U‐II is a potent endothelium‐dependent vasodilator in some rat resistance vessels, and acts through release of EDHF as well as nitric oxide. Our findings have also highlighted clear anatomical differences in the responses of different vascular beds to human U‐II which are likely to be important in determining the overall cardiovascular activity of this peptide.

British Journal of Pharmacology (2000) 130, 1865–1870; doi:10.1038/sj.bjp.0703513
Sphingosine‐1‐phosphate reduces rat renal and mesenteric blood flow in vivo in a pertussis toxin‐sensitive manner
Authors: Bischoff Angela , Czyborra Peter , Meyer zu Heringdorf Dagmar , Jakobs Karl H , Michel Martin C
Published: January 2005

Sphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine constrict isolated rat intrarenal and mesenteric microvessels in vitro. The present study investigates their effects on the cardiovascular system in vivo in anaesthetized rats.

The animals were given intravenous or intrarenal arterial bolus injections of sphingolipids (0.1–100 μg kg−1) with subsequent measurements of mean arterial pressure, heart rate and renal and mesenteric blood flows (RBF, MBF) using a pressure transducer and electromagnetic flow probes, respectively.

Intravenous injection of SPP rapidly (within 30 s), transiently and dose‐dependently reduced RBF (maximally −4.0±0.3 ml min−1) and MBF (maximally −1.4±0.2 ml min−1), without affecting mean arterial pressure or heart rate. Other sphingolipids had no significant effect.

Intrarenal arterial SPP administration caused greater blood flow reductions (maximally −6.4±0.3 ml min−1) than systemic administration. Upon intrarenal administration, sphingosylphos‐ phorylcholine also lowered RBF (maximally −2.8±0.6 ml min−1), while the other sphingolipids remained without effect.

Pretreatment with pertussis toxin (PTX, 10 μg kg−1) 3 days before the acute experiment abolished the SPP‐induced reductions of RBF and MBF.

These data demonstrate, that SPP is a potent vasoconstrictor in vivo, particularly in the renal vasculature, while the other structurally related sphingolipids had little if any effects. The PTX‐sensitivity strongly suggests that the effects of SPP on renal and mesenteric blood flow are mediated by receptors coupled to Gi‐type G‐proteins.

British Journal of Pharmacology (2000) 130, 1878–1883; doi:10.1038/sj.bjp.0703516
ADP is not an agonist at P2X1 receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets
Authors: Mahaut‐Smith Martyn P , Ennion Steven J , Rolf Michael G , Evans Richard J
Published: January 2005

ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X1, P2Y1 and P2TAC. Given the low potency of ADP at P2X1 receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re‐examined the activation of P2X1 receptors by ADP following HPLC and enzymatic purification.

Native P2X1 receptor currents in megakaryocytes were activated by α,β‐meATP (10 μM) and commercial samples of ADP (10 μM), but not by purified ADP (10–100 μM).

Purified ADP (up to 1 mM) was also inactive at recombinant human P2X1 receptors expressed in Xenopus oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca2+ mobilization in rat megakaryocytes.

In human platelets, P2X1 and P2Y receptor‐mediated [Ca2+]i responses were distinguished by their different kinetics at 13°C. In 1 mM Ca2+ saline, α,β‐meATP (10 μM) and commercial ADP (40 μM) activated a rapid [Ca2+]i increase (lag time 0.5 s) through the activation of P2X1 receptors. Hexokinase treatment of ADP shifted the lag time by ∼2 s, indicating loss of the P2X1 receptor‐mediated response.

A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X1 receptors, whereas ADP is a selective agonist at metabotropic (P2Y1 and P2TAC) receptors.

British Journal of Pharmacology (2000) 131, 108–114; doi:10.1038/sj.bjp.0703517
The novel Na+/Ca2+ exchange inhibitor KB‐R7943 also blocks native and expressed neuronal nicotinic receptors
Authors: Pintado Antonio J , Herrero Carlos J , García Antonio G , Montiel Carmen
Published: January 2005

We studied the effects of the novel Na+/Ca2+ exchange inhibitor KB‐R7943, 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain α3β4 and α7 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes.

As expected, KB‐R7943 blocked the Na+‐gradient dependent 45Ca2+ uptake into chromaffin cells (IC50 of 5.5 μM); but in addition, the compound also inhibited the 45Ca2+ entry and the increase of cytosolic Ca2+ concentration, [Ca2+]c, stimulated by 5 s pulses of ACh (IC50 of 6.5 and 1.7 μM, respectively).

In oocytes expressing α3β4 and α7 nicotinic AChRs, voltage‐clamped at −60 mV, inward currents elicited by 1 s pulses of 100 μM ACh (IACh) were blocked by KB‐R7943 with an IC50 of 0.4 μM and a Hill coefficient of 0.9.

Blockade of α3β4 currents by KB‐R7943 was noncompetitive; moreover, the blocker (0.3 μM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 μM and 1 mM ACh, respectively).

Inhibition of α3β4 currents by 0.3 μM KB‐R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 μM), the inhibition amounted to 33, 64 and 80% in oocytes voltage‐clamped at −40, −60 and −100 mV, respectively. The onset of blockade was faster and the recovery slower at −100 mV; the reverse was true at −40 mV.

In conclusion, KB‐R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open‐channel blocker at the rat brain α3β4 AChR. These results should be considered when KB‐R7943 is to be used to study Ca2+ homeostasis in cells expressing nicotinic AChRs and the Na+/Ca2+ exchanger.

British Journal of Pharmacology (2000) 130, 1893–1902; doi:10.1038/sj.bjp.0703519
Influence of cannabinoids on the delayed rectifier in freshly dissociated smooth muscle cells of the rat aorta
Authors: Van den Bossche I , Vanheel B
Published: January 2005

The influence of the cannabinoids anandamide, methanandamide and WIN 55212‐2 on the delayed rectifier K+ current (IK(V)) in rat arterial myocytes was investigated.

Anandamide caused a concentration‐dependent reduction of total peak and late K+ current (IK). The maximal effect (about 50% inhibition of IK) was reached with 3 μM, and half‐maximal current block was observed at 0.6 μM. Blockade was voltage‐independent. Inhibition of IK by the cannabinoid was associated with a characteristic increase in the rate of current relaxation.

Methanandamide (10 μM), a metabolically more stable analogue of anandamide, decreased IK with a similar time course. Current traces in the presence of the drug also showed an acceleration of inactivation.

The presence of TEA did not impair the inhibition by anandamide or methanandamide, but inhibition was prevented by pre‐exposure to 4‐AP, showing that both cannabinoids inhibited IK(V) while having no influence on Ca2+‐dependent K+ current (IK(Ca)).

The CB1 receptor antagonist SR141716A (10 μM) did not influence the action of anandamide or methanandamide.

Arachidonic acid (1 μM) increased IK considerably. However, in the presence of TEA it caused a decrease of IK(V) with a characteristic increase in the rate of current relaxation.

WIN 55212‐2 (20 μM) caused similar inhibition of IK.

Internally applied anandamide (10 μM) or methanandamide (10 μM) was ineffective at influencing IK. In the dialyzed cells, the additional external application of a cannabinoid promptly initiated inhibition.

The results show that anandamide, methanandamide and WIN 55212‐2 affect IK(V) in a cannabinoid receptor‐independent way similar to that of arachidonic acid, which, unlike the cannabinoids, additionally increases a Ca2+‐activated K+ current. It is suggested that cannabinoids might bind to an external site on or near the Kv channel of the vascular smooth muscle cells.

British Journal of Pharmacology (2000) 131, 85–93; doi:10.1038/sj.bjp.0703521
Characterization of prostanoid receptors mediating actions of the isoprostanes, 8‐iso‐PGE2 and 8‐iso‐PGF2α, in some isolated smooth muscle preparations
Authors: Sametz Wolfgang , Hennerbichler Simone , Glaser Sonja , Wintersteiger Reinhold , Juan Heinz
Published: January 2005

We investigated the contracting actions of the isoprostanes (isoPs), 8‐iso‐prostaglandin (PG) F2α and 8‐iso‐PGE2, in comparison to the effects of the thromboxane (TX) A2‐mimetic U 46619 and the traditional prostaglandin PGE2 in the isolated rat aorta, isolated rat gastric fundus and the isolated guinea‐pig ileum.

U 46619 and 8‐iso‐PGF2α caused contractions in the rat aorta and rat gastric fundus in a concentration‐dependent manner, whereas these agonists showed no effects in the guinea‐pig ileum. However, 8‐iso‐PGE2 and PGE2 caused contractions in all isolated organs used.

The prostanoid TP‐receptor antagonist SQ 29,548 (10 nM) significantly antagonized vasoconstrictions induced by the agonists used in the rat aorta. SQ 29,548 at a final concentration of 3 μM, but not at lower concentrations, significantly inhibited contractions induced by U 46619, 8‐iso‐PGF2α and 8‐iso‐PGE2 in the rat fundus. Responses to PGE2 were unchanged. The prostanoid EP1‐receptor antagonist SC 51089 (3 μM) significantly inhibited contractions induced by 8‐iso‐PGE2 and PGE2 in the rat fundus and in the guinea‐pig ileum. SC 51089 had no effect on responses to any of the agonists tested.

Our results show that 8‐iso‐PGE2, in contrast to 8‐iso‐PGF2α, can also cause contractions by activation of the EP1‐receptors in the rat gastric fundus and the guinea‐pig ileum. The findings of the present study do not support the existence of a unique isoP‐receptor in the tissues used.

British Journal of Pharmacology (2000) 130, 1903–1910; doi:10.1038/sj.bjp.0703522
Gabapentin inhibits excitatory synaptic transmission in the hyperalgesic spinal cord
Authors: Patel Manoj K , Gonzalez M Isabel , Bramwell Steven , Pinnock Robert D , Lee Kevin
Published: January 2005

In the present study we tested the effects of the antihyperalgesic compound gabapentin on dorsal horn neurones in adult spinal cord. Slices were taken from control and hyperalgesic animals suffering from streptozocin‐induced diabetic neuropathy. At concentrations up to 100 μM, bath application failed to affect the resting membrane properties of dorsal horn neurones taken from both groups of animal. In contrast, bath application of gabapentin dramatically reduced the magnitude of the excitatory postsynaptic current (EPSC) in neurones taken from hyperalgesic animals without altering the magnitude of the EPSC in control animals. Using a paired pulse stimulation protocol, together with analysis of miniature EPSC's, it was possible to demonstrate that gabapentin mediated these effects via a pre‐synaptic site of action.

British Journal of Pharmacology (2000) 130, 1731–1734; doi:10.1038/sj.bjp.0703530
Modulation of acetylcholine release in the guinea‐pig trachea by the nitric oxide donor, S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP)
Authors: Mang Christian F , Kilbinger Heinz
Published: January 2005

The effects of the nitric oxide (NO) donor S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) and the NO synthase inhibitor L‐NG‐nitroarginine (L‐NOARG) on the electrically evoked [3H]‐acetylcholine release were studied in an epithelium‐free preparation of guinea‐pig trachea that had been preincubated with [3H]‐choline.

SNAP (100 and 300 μM) caused small but significant increases of the electrically evoked [3H]‐acetylcholine release (121±4% and 124±10% of control). Resting outflow of [3H]‐ACh was not affected by SNAP. The increase by SNAP was abolished by the specific inhibitor of soluble guanylyl cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐α]quinoxalin‐1‐one (ODQ, 1 μM).

The facilitatory effect of SNAP (100 and 300 μM) was reversed into inhibition of release (to 74±4% and to 78±2%) after pretreatment of the trachea with capsaicin (3 μM). ODQ prevented the inhibition. Capsaicin pretreatment alone did not significantly alter the release of [3H]‐acetylcholine. A significant inhibition by SNAP (100 μM) of [3H]‐acetylcholine release (78±3%) was also seen in the presence of the NK2 receptor antagonist SR 48968 (30 nM).

L‐NOARG (10 and 100 μM) significantly enhanced the electrically‐evoked smooth muscle contractions, but caused no significant increases of the evoked release from capsaicin pretreated trachea strips. This might indicate that the inhibitory effect of endogenous NO on acetylcholine release is too small to be detected by overflow studies.

It is concluded that NO has dual effects on the evoked acetylcholine release. NO enhances release in the absence of modifying drugs, but NO inhibits acetylcholine release after blockade of the NK2 receptor or after sensory nerve depletion with capsaicin. This suggests that NO and endogenous tachykinins act in series to produce an increase in acetylcholine release.

British Journal of Pharmacology (2000) 131, 94–98; doi:10.1038/sj.bjp.0703531
Human urotensin‐II is a potent spasmogen of primate airway smooth muscle
Authors: Hay Douglas W P , Luttmann Mark A , Douglas Stephen A
Published: January 2005

The contractile profile of human urotensin‐II (hU‐II) was examined in primate airway and pulmonary vascular tissues. hU‐II contracted tissues from different airway regions with similar potencies (pD2s from 8.6 to 9.2). However, there were regional differences in the efficacy of hU‐II, with a progressive increase in the maximum contraction from trachea to smaller airway regions (from 9 to 41% of the contraction to 10 μM carbachol). hU‐II potently contracted pulmonary artery tissues from different regions with similar potencies and efficacies: pD2s=8.7 to 9.3 and maximal contractions=79 to 86% of 60 mM KCl. hU‐II potently contracted pulmonary vein preparations taken proximal to the atria, but had no effect in tissues from distal to the atria. This is the first report describing the contractile activity of hU‐II in airways and suggests that the potential pathophysiological role of this peptide in lung diseases warrants investigation.

British Journal of Pharmacology (2000) 131, 10–12; doi:10.1038/sj.bjp.0703533
Non‐competitive antagonism of β2‐agonist‐mediated cyclic AMP accumulation by ICI 118551 in BC3H1 cells endogenously expressing constitutively active β2‐adrenoceptors
Authors: Hopkinson H E , Latif M L , Hill S J
Published: January 2005

Constitutive activity of the β2‐adrenoceptor, which is sensitive to inhibition by an inverse agonist such as ICI 118551, has been readily demonstrated in recombinant systems expressing constitutively‐active mutant receptors or over‐expressing the wild‐type β2‐adrenoceptor. Here we demonstrate the presence of constitutive β2‐adrenoceptor activity in BC3H1 cells which endogenously express this receptor.

In BC3H1 cells, only ICI 118551 behaved as an inverse agonist at β2‐adrenoceptors, while propranolol, ICI 118551, atenolol and, to a lesser extent, alprenolol exhibited inverse agonism in CHO‐β24 cells transfected with cDNA for the human β2‐adrenoceptor (310 fmol.mg protein−1). The level of expression of β2‐adrenoceptors in BC3H1 cells was not high (78 fmol.mg protein−1) and the efficiency of receptor–effector coupling in this cell line was much lower than in the recombinant CHO‐β24 cells (as judged by the partial agonist nature of both salbutamol and clenbuterol).

ICI 118551 (log KD−9.73±0.07) and propranolol (log KD−9.25±0.12) both behaved as conventional competitive antagonists of isoprenaline‐stimulated cyclic AMP accumulation in high expressing CHO‐β24 cells. In contrast, ICI 118551 appeared to act as a non‐competitive antagonist in BC3H1 cells and in low expressing CHO‐β26 cells (50 fmol.mg protein−1).

This non‐competitive effect of ICI 118551 in BC3H1 cells was also observed when either salbutamol was used as agonist, or the incubation period with isoprenaline was extended to 30 min.

The possibility that these effects of ICI 118551 are due to an interaction with different affinity states (R, R* and R′) of the receptor is discussed.

British Journal of Pharmacology (2000) 131, 124–130; doi:10.1038/sj.bjp.0703535
The stable pyrimidines UDPβS and UTPγS discriminate between the P2 receptors that mediate vascular contraction and relaxation of the rat mesenteric artery
Authors: Malmsjö Malin , Adner Mikael , Harden T Kendall , Pendergast William , Edvinsson Lars , Erlinge David
Published: January 2005

The contractile and relaxant effects of the different P2 receptors were characterized in the rat isolated mesenteric artery by use of extracellular nucleotides, including the stable pyrimidines uridine 5′‐O‐thiodiphosphate (UDPβS) and uridine 5′‐O‐3‐thiotriphosphate (UTPγS).

The selective P2X receptor agonist, αβ‐methylene‐adenosine triphosphate (αβ‐MeATP) stimulated a potent (pEC50=6.0) but relatively weak contraction (Emax=57% of 60 mM K+). The contractile concentration‐response curve of adenosine triphosphate (ATP) was biphasic when added in single concentrations. The first part of the response could be desensitized by αβ‐MeATP, indicating involvement of P2X receptors, while the second part might be mediated by P2Y receptors.

The contractile P2Y receptors were further characterized after P2X receptor desensitization with 10 μM αβ‐MeATP. Uridine diphosphate (UDP), uridine triphosphate (UTP) and ATP stimulated contraction only in high concentrations (1–10 mM). The selective P2Y6 agonist, UDPβS, and the P2Y2/P2Y4‐receptor agonists UTPγS and adenosine 5′‐O‐3‐thiotriphosphate (ATPγS) were considerably more potent and efficacious (Emax∼250% of 60 mM K+). Adenosine 5′‐O‐thiodiphosphate (ADPβS) was inactive, excluding contractile P2Y1 receptors.

After precontraction with 1 μM noradrenaline, UTP, ADP and ATP induced relaxations with similar potencies (pEC50∼5.0). UTPγS, ADPβS and ATPγS were approximately one log unit more potent indicating the presence of endothelial P2Y1 and P2Y2/P2Y4 receptors. The P2Y6 receptor agonist, UDPβS, had no effect.

UDPβS and UTPγS are useful tools when studying P2 receptors in tissue preparations with ectonucleotidase activity. Contractile responses can be elicited by stimulation of P2Y6 and, slightly less potently, P2Y2/P2Y4 receptors. The P2X response was relatively weak, and there was no P2Y1 response. Stimulation of P2Y1 and P2Y2/P2Y4 receptors elicited relaxation, while P2Y6 did not contribute.

British Journal of Pharmacology (2000) 131, 51–56; doi:10.1038/sj.bjp.0703536
Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines
Authors: Cartwright Judith E , Johnstone Alan P , Whitley Guy St J
Published: January 2005

Proliferation of endothelial cells is a vital component of vascular repair and angiogenesis. The endothelial cell mediator, nitric oxide (NO) has been reported both to inhibit and to promote endothelial cell proliferation. In this study we have generated cell lines which constitutively express antisense RNA to a region of inducible nitric oxide synthase (iNOS) from a murine endothelial cell line, sEnd‐1.

In response to stimulation with lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) these antisense cells had no detectable RNA for endogenous iNOS, barely detectable iNOS protein and produced 82% less NO than did the control transfected line.

Stimulation of the control transfected line caused significant NO production and inhibition of cell growth whereas for the antisense line, producing little NO in response to stimulation, proliferation remained the same as for unstimulated cells. No differences in cell death were observed between unstimulated and LPS/IFN‐γ stimulated cells.

The data presented in this study directly demonstrate that NO derived endogenously from iNOS inhibits proliferation of endothelial cells. This approach overcomes problems in other studies where NO donors or non‐isoform specific inhibitors of NO synthase have been used.

British Journal of Pharmacology (2000) 131, 131–137; doi:10.1038/sj.bjp.0703539
Pharmacokinetics and metabolism of thioridazine during co‐administration of tricyclic antidepressants
Authors: Daniel Wladyslawa A , Syrek Maciej , Haduch Anna , Wójcikowski Jacek
Published: January 2005

Because of serious side‐effects of thioridazine and tricyclic antidepressants (cardiotoxicity), a possible influence of imipramine and amitriptyline on the pharmacokinetics and metabolism of thioridazine was investigated in a steady state (2‐week treatment) in rats.

Imipramine and amitriptyline (5 and 10 mg kg−1 i.p., respectively) elevated 30 and 20 fold, respectively, the concentration of thioridazine (10 mg kg−1 i.p.) and its metabolites (N‐desmethylthioridazine, 2‐sulphoxide, 2‐sulphone, 5‐sulphoxide) in blood plasma. Similar, yet weaker increases in the thioridazine concentration were found in the brain. Moreover, an elevation of thioridazine/metabolite ratios was observed.

Imipramine and amitriptyline added to control liver microsomes in vitro inhibited the metabolism of thioridazine via N‐demethylation (an increase in Km), mono‐2‐sulphoxidation (an increase in Km and a decrease in Vmax) and 5‐sulphoxidation (mainly a decrease in Vmax). Amitriptyline was a more potent inhibitor than imipramine of the thioridazine metabolism.

The varying concentration ratios of antidepressant/thioridazine in vivo appear to be more important to the final result of the pharmacokinetic interactions than are relative direct inhibitory effects of the antidepressants on thioridazine metabolism observed in vitro.

Besides direct inhibition of the thioridazine metabolism, the decreased activity of cytochrome P‐450 towards 5‐sulphoxidation, produced by chronic joint administration of thioridazine and the antidepressants, seems to be relevant to the observed in vivo interaction.

The obtained results may also point to inhibition of another, not yet investigated, metabolic pathway of thioridazine, which may be inferred from the simultaneous elevation of concentrations of both thioridazine and the measured metabolites.

British Journal of Pharmacology (2000) 131, 287–295; doi:10.1038/sj.bjp.0703540
Expression and function of MIP‐2 are reduced by dexamethasone treatment in vivo
Authors: Schramm Rene , Liu Qing , Thorlacius Henrik
Published: January 2005

The objective of this study was to examine the effect of dexamethasone on tumour necrosis factor‐α (TNF‐α)‐induced expression and function of macrophage inflammatory protein‐2 (MIP‐2) and neutrophil recruitment. For this purpose, we used air pouches raised on the dorsal skin of C57/B16 mice.

Initially, we examined the dose‐response (0.01–0.5 μg ml−1) and kinetics (0–24 h) of TNF‐α‐induced leukocyte accumulation. The cellular response was maximal at 0.1 μg ml−1 of TNF‐α and 4 h after challenge and comprised more than 90% neutrophils.

Intraperitoneal (i.p.) pretreatment with 10 mg kg−1 of dexamethasone for 2 h, but not 1 mg kg−1, reduced TNF‐α‐induced recruitment of neutrophils by 87%. Administration of dexamethasone had no effect on the expression of CD18 on neutrophils.

TNF‐α (0.1 μg ml−1) markedly increased the levels of MIP‐2 in the air pouches 1 h after challenge and after 4 h the MIP‐2 values returned to baseline. Notably, 2 h pretreatment with dexamethasone (10 mg kg−1, i.p.) reduced MIP‐2 expression by 65% in response to TNF‐α (0.1 μg ml−1). On the other hand, dexamethasone treatment did not change the levels of interleukin‐10 (IL‐10) in the pouch exudate.

Administration of recombinant MIP‐2 increased neutrophil accumulation at 0.5 and 1.0 μg ml−1 after 4 h of challenge. Dexamethasone pretreatment for 2 h (10 mg kg−1, i.p.) abolished the MIP‐2‐induced recruitment of neutrophils.

Taken together, our data demonstrate that dexamethasone may downregulate TNF‐α‐induced neutrophil recruitment by inhibiting both the expression and function of MIP‐2 in vivo.

British Journal of Pharmacology (2000) 131, 328–334; doi:10.1038/sj.bjp.0703543
Enhanced reduction of myocardial infarct size by combined ACE inhibition and AT1‐receptor antagonism
Authors: Weidenbach Roland , Schulz Rainer , Gres Petra , Behrends Matthias , Post Heiner , Heusch Gerd
Published: January 2005

The effects of the angiotensin‐converting‐enzyme inhibitor (ACEI) ramiprilat, the angiotensin II type 1 receptor antagonist (AT1A) candesartan, and the combination of both drugs on infarct size (IS) resulting from regional myocardial ischaemia were studied in pigs.

Both ACEI and AT1A reduce myocardial IS by a bradykinin‐mediated process. It is unclear, however, whether the combination of ACEI and AT1A produces a more pronounced IS reduction than each of these drugs alone.

Forty‐six enflurane‐anaesthetized pigs underwent 90 min low‐flow ischaemia and 120 min reperfusion. Systemic haemodynamics (micromanometer), subendocardial blood flow (ENDO, microspheres) and IS (TTC‐staining) were determined. The decreases in left ventricular peak pressure by ACEI (by 9±2 (s.e.mean) mmHg), AT1A (by 11±2 mmHg) or their combination (by 18±3 mmHg, P<0.05 vs ACEI and AT1A, respectively) were readjusted by aortic constriction prior to ischaemia. With placebo (n=10), IS averaged 20.0±3.3% of the area at risk. IS was reduced to 9.8±2.6% with ramiprilat (n=10) and 10.6±3.1% with candesartan (n=10). Combined ramiprilat and candesartan (n=10) reduced IS to 6.7±2.1%. Blockade of the bradykinin‐B2‐receptor with icatibant prior to ACEI and AT1A completely abolished the reduction of IS (n=6, 22.8±6.1%). The relationship between IS and ischaemic ENDO with placebo was shifted downwards by each ACEI and AT1A and further shifted downwards with their combination (P<0.05 vs all groups); icatibant again abolished such downward shift.

The combination of ACEI and AT1A enhances the reduction of IS following ischaemia/reperfusion compared to a monotherapy by either drug alone; this effect is mediated by bradykinin.

British Journal of Pharmacology (2000) 131, 138–144; doi:10.1038/sj.bjp.0703544
Formation of 8‐iso‐PGF2α and thromboxane A2 by stimulation with several activators of phospholipase A2 in the isolated human umbilical vein
Authors: Sametz Wolfgang , Hummer Klaudia , Butter Martina , Wintersteiger Reinhold , Juan Heinz
Published: January 2005

We investigated the effects of the phospholipase A2 (PLA2) activators calcium ionophore A 23187, hydrogen peroxide (H2O2), bradykinin (BK), histamine and noradrenaline (NA) on the 8‐iso‐prostaglandin (PG)F2α formation in the isolated human umbilical vein and the isolated rabbit ear. For comparison, the influence of these substances on the thromboxane A2 (TXA2) release was also investigated. The release of total (esterified as well as free) 8‐iso‐PGF2α, free 8‐iso‐PGF2α and TXB2, the stable metabolite of TXA2, was determined by specific enzyme immunoassays.

The results show that bolus injections of 5.4 mmol H2O2, 30 nmol A 23187, 10 nmol BK, 50 nmol histamine and 20 nmol NA caused an increased release of total 8‐iso‐PGF2α in the umbilical vein and the rabbit ear. A perfusion with H2O2 at a final concentration of 0.3 mM also increased the release of this isoprostane. Increased formation of free 8‐iso‐PGF2α was induced by A 23187 injection and by both modes of H2O2 administration, but not by the other treatments.

Bolus injections of A 23187, BK and histamine induced an increased release of TXB2 in both organs. Both modes of H2O2 administration and NA showed no releasing effects.

In conclusion, our results show that the substances used are able to stimulate the formation of 8‐iso‐PGF2α concurrently with the release of PGs. This effect might be of pathophysiological relevance in inflammatory and cardiovascular diseases in which an enhanced release of free radicals, BK, histamine or NA play an important role.

British Journal of Pharmacology (2000) 131, 145–151; doi:10.1038/sj.bjp.0703547
Oxidation of nitroxyl anion to nitric oxide by copper ions
Authors: Nelli Silvia , Hillen Mark , Buyukafsar Kansu , Martin William
Published: January 2005

This study made use of a nitric oxide‐sensitive electrode to examine possible means of generating nitric oxide from nitroxyl anion (NO−) released upon the decomposition of Angeli's salt.

Our results show that copper ions (from CuSO4) catalyze the rapid and efficient oxidation of nitroxyl to nitric oxide. Indeed, the concentrations of copper required to do so (0.1–100 μM) are roughly 100‐times lower than those required to generate equivalent amounts of nitric oxide from S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP).

Experiments with ascorbate (1 mM), which reduces Cu2+ ions to Cu+, and with the Cu2+ chelators, EDTA and cuprizone, and the Cu+ chelator, neocuproine, each at 1 mM, suggest that the oxidation is catalyzed by copper ions in both valency states.

Some compounds containing other transition metals, i.e. methaemoglobin, ferricytochrome c and Mn(III)TMPyP, were much less efficient than CuSO4 in catalyzing the formation of nitric oxide from nitroxyl, while FeSO4, FeCl3, MnCl2, and ZnSO4 were inactive.

Of the copper containing enzymes examined, Cu‐Zn superoxide dismutase and ceruloplasmin were weak generators of nitric oxide from nitroxyl, even at concentrations (2500 and 30 u ml−1, respectively) vastly greater than are present endogenously. Two others, ascorbate oxidase (10 u ml−1) and tyrosinase (250 u ml−1) were inactive.

Our findings suggest that a copper‐containing enzyme may be responsible for the rapid oxidation of nitroxyl to nitric oxide by cells, but the identity of such an enzyme remains elusive.

British Journal of Pharmacology (2000) 131, 356–362; doi:10.1038/sj.bjp.0703550
L‐NAME‐resistant bradykinin‐induced relaxation in porcine coronary arteries is NO‐dependent: effect of ACE inhibition
Authors: Danser A H Jan , Tom Beril , De Vries René , Saxena Pramod R
Published: January 2005

NO synthase (NOS) inhibitors partially block bradykinin (BK)‐mediated vasorelaxation. Here we investigated whether this is due to incomplete NOS inhibition and/or NO release from storage sites. We also studied the mechanism behind ACE inhibitor‐mediated BK potentiation.

Porcine coronary arteries (PCAs) were mounted in organ baths, preconstricted, and exposed to BK or the ACE‐resistant BK analogue Hyp3‐Tyr(Me)8‐BK (HT‐BK) with or without the NOS inhibitor L‐NAME (100 μM), the NO scavenger hydroxocobalamin (200 μM), the Ca2+‐dependent K+‐channel blockers charybdotoxin+apamin (both 100 nM), or the ACE inhibitor quinaprilat (10 μM).

BK and HT‐BK dose‐dependently relaxed preconstricted vessels (pEC50 8.0±0.1 and 8.5±0.2, respectively). pEC50's were &10 fold higher with quinaprilat, and &10 fold lower with L‐NAME or charybdotoxin+apamin. Complete blockade was obtained with hydroxocobalamin or L‐NAME+ charybdotoxin+apamin.

Repeated exposure to 100 nM BK or HT‐BK, to deplete NO storage sites, produced progressively smaller vasorelaxant responses. With L‐NAME, the decrease in response occurred much more rapidly. L‐Arginine (10 mM) reversed the effect of L‐NAME.

Adding quinaprilat to the bath following repeated exposure (with or without L‐NAME), at the time BK and HT‐BK no longer induced relaxation, fully restored vasorelaxation, while quinaprilat alone had no effect. Quinaprilat also relaxed vessels that, due to pretreatment with hydroxocobalamin or L‐NAME+charybdotoxin+apamin, previously had not responded to BK.

In conclusion, L‐NAME‐resistant BK‐induced relaxation in PCAs depends on NO from storage sites, and is mediated via stimulation of guanylyl cyclase and/or Ca2+‐dependent K+‐channels. ACE inhibitors potentiate BK independent of their effect on BK metabolism.

British Journal of Pharmacology (2000) 131, 195–202; doi:10.1038/sj.bjp.0703555
Mecamylamine but not the α7 receptor antagonist α‐bungarotoxin blocks sensitization to the locomotor stimulant effects of nicotine
Authors: Kempsill F E J , Pratt J A
Published: January 2005

The involvement of α7 receptors in the locomotor stimulant effects of nicotine has been examined by determining the ability of intracerebroventricular (i.c.v.) administration of the α7 receptor antagonist α‐bungarotoxin (α‐bgt) to modify sensitization to the locomotor activating effects of chronic nicotine.

Intracerebroventricular administration of α‐bgt (0.02–8 nmoles) produced a dose dependent increase in convulsive behaviour. At doses less than 1.0 nmole, minimal convulsive behaviour occurred but larger doses evoked convulsions in all rats which displayed a more rapid onset time as the dose increased.

The binding distribution of α7 receptors 20 min and 3 h following an i.c.v. administration of [125I]‐α‐bgt (0.02 nmoles) revealed clear binding in the hippocampus, cingulate cortex and hypothalamus which was more intense after 3 h.

Rats chronically treated with nicotine (0.4 mg kg−1) and exposed to the locomotor activity apparatus daily acquired an increase in locomotor activity relative to the control group after 3 days of treatment which reached a maximum after 7 days of treatment and was maintained for the 2 week treatment period.

Pre‐treatment with mecamylamine (1 mg kg−1) prevented the expression of the locomotor stimulant effects of nicotine but pre‐treatment with i.c.v. α‐bgt (0.02 nmoles) did not affect nicotine‐induced changes in locomotor activity.

The results of this study support the conclusion that nicotinic receptors of the α4β2 subtype rather than the α7 subtype are important in mediating the expression of the locomotor stimulant effects of nicotine.

British Journal of Pharmacology (2000) 131, 997–1003; doi:10.1038/sj.bjp.0703560
Abnormal activation of K+ channels in aortic smooth muscle of rats with endotoxic shock: electrophysiological and functional evidence
Authors: Chen Shiu‐Jen , Wu Chin‐Chen , Yang San‐Nan , Lin Cheng‐I , Yen Mao‐Hsiung
Published: January 2005

This study examined the role of K+ channels in vascular hyporeactivity of rats with endotoxic shock ex vivo.

At the end of the in vivo experiments, thoracic aortas were removed from endotoxaemic and control rats. After removal of the endothelium, aortic segments were mounted in myographs for recording of isometric tension and smooth muscle membrane potential.

Membrane potentials recorded from endotoxaemic rats were hyperpolarized compared to those of the controls. This hyperpolarization was partially reversed by tetraethylammonium, charybdotoxin or glibenclamide, but not significantly affected by apamin. The hyperpolarization was also partially attenuated by Nω‐nitro‐L‐arginine methyl ester (L‐NAME) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐l‐one (ODQ).

In phenylephrine‐contracted aortic rings, both agonists of K+ channels, NS1619 and pinacidil, induced greater relaxations and re‐polarizations in the preparations obtained from endotoxaemic rats. The NS1619‐induced relaxation and re‐polarization in arteries from endotoxaemic rats were partially inhibited by tetraethylammonium and completely inhibited by charybdotoxin, L‐NAME or ODQ, but not significantly affected by apamin. Similarly, the greater relaxation and re‐polarization induced by pinacidil in arteries from endotoxaemic rats were also inhibited by glibenclamide, L‐NAME or ODQ. However, these inhibitors had no significant effect on relaxations and re‐polarizations induced by NS1619 and pinacidil in arteries from controls.

This study provides the electrophysiological and functional evidence showing an abnormal activation of K+ channels in vascular smooth muscle in animals with endotoxic shock. Our observations suggest that overproduction of nitric oxide causes an activation of large conductance Ca2+‐activated K+ channels and ATP‐sensitive K+ channels which contributes to endotoxin‐mediated vascular hyporeactivity.

British Journal of Pharmacology (2000) 131, 213–222; doi:10.1038/sj.bjp.0703564
Low doses of 8‐OH‐DPAT prevent the impairment of spatial learning caused by intrahippocampal scopolamine through 5‐HT1A receptors in the dorsal raphe
Authors: Carli Mirjana , Balducci Claudia , Samanin Rosario
Published: January 2005

We studied the effects of low doses of 8‐OH‐DPAT, a 5‐HT1A receptor agonist, on the impairment of spatial learning caused by scopolamine injected into the CA1 region of the dorsal hippocampus of rats performing a two‐platform spatial discrimination task.

Bilateral injections of 4 μg (in 1 μl) of scopolamine into the CA1 region of the dorsal hippocampus 10 min before each training session impaired choice accuracy with no effect on choice latency and errors of omission.

Administered subcutaneously 20 min before each training session, 8‐OH‐DPAT 10 and 30 (but not 3) μg kg−1 did not modify choice accuracy but prevented the impairment by intrahippocampal scopolamine

Injection of 1.0 μg (in 0.5 μl) of WAY 100635, a 5‐HT1A receptor antagonist, into the dorsal raphe 5 min before scopolamine had no effect on choice accuracy and latency or errors of omission and did not modify the effect of scopolamine, but completely antagonized the effect of 10 and 30 μg kg−1 8‐OH‐DPAT on scopolamine‐induced impairment of choice accuracy

The results confirm previous findings that stimulation of presynaptic 5‐HT1A receptors in the dorsal raphe attenuates the deficit of spatial learning caused by blockade of cholinergic excitatory input on hippocampal pyramidal cells.

Drugs that stimulate presynaptic 5‐HT1A receptors such as 5‐HT1A receptor partial agonists may be useful in the symptomatic treatment of human memory disturbances associated with loss of cholinergic innervation to the hippocampus.

British Journal of Pharmacology (2000) 131, 375–381; doi:10.1038/sj.bjp.0703567
Vascular bed heterogeneity in age‐related endothelial dysfunction with respect to NO and eicosanoids
Authors: Matz Rachel L , De Sotomayor Maria Alvarez , Schott Christa , Stoclet Jean‐Claude , Andriantsitohaina Ramaroson
Published: January 2005

Endothelial dysfunction has been described with ageing but the mechanisms responsible have not been clearly elucidated and might be different from one vessel to the other. This study assesses the relative contribution of endothelial nitric oxide (NO) and cyclo‐oxygenase (COX) metabolites in relaxation to acetylcholine with ageing in the aorta and the small mesenteric artery of the rat.

In the aorta and branch II or III of superior mesenteric artery (SMA), endothelium‐dependent relaxation to acetylcholine was not different between 12–14 (adult) and 32‐week‐old rats whereas it was reduced at 70–100 (old) weeks of age.

Despite an increased endothelial NO‐synthase protein expression, the NO‐synthase inhibitor, NG‐nitro‐L‐arginine‐sensitive component of relaxation decreased with ageing.

In old rats, exposure to the COX inhibitor, indomethacin, but not the selective COX‐2 inhibitor, NS‐398, potentiated response to acetylcholine. The thromboxane A2/prostaglandin H2 receptor antagonist, GR 32191B enhanced relaxation to acetylcholine in aorta but it had no effect in SMA. Furthermore, acetylcholine increased thromboxane B2 production (enzymeimmunoassay) in aorta but not in SMA. Finally, Western blot analysis showed enhanced expression of COX‐1 and 2 in the two arteries with ageing.

These results suggest that the decrease in acetylcholine‐induced relaxation with ageing involves reduced NO‐mediated dilatation and increased generation of vasoconstrictor prostanoids most likely from COX‐1. They also point out vascular bed heterogeneity related to the nature of prostanoids involved between the aorta (i.e., thromboxane A2) and the SMA (unidentified) arteries even though increased expression of COX occurs in both vessels.

British Journal of Pharmacology (2000) 131, 303–311; doi:10.1038/sj.bjp.0703568