bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice
Authors: Pannirselvam Malarvannan , Simon Valerie , Verma Subodh , Anderson Todd , Triggle Chris R
Published: October 2003

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)‐induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg kg−1 day−1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA.

Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside‐induced relaxation, was improved with sepiapterin supplementation in db/db mice.

BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment.

Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment.

These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

British Journal of Pharmacology (2003) 140, 701–706. doi:10.1038/sj.bjp.0705476
Regulation of leptin distribution between plasma and cerebrospinal fluid by cholecystokinin receptors
Authors: Cano Victoria , Ezquerra Laura , Ramos M Pilar , Ruiz‐Gayo Mariano
Published: October 2003

Cholecystokinin (CCK) is a postprandial hormone that elicits a satiating effect and regulates feeding behaviour. CCK has been shown to enhance the effect of leptin in several experimental paradigms.

The goal of this work was to characterize the effect of endogenous CCK on plasma leptin content by using CCK receptor antagonists. Therefore, we administered SR‐27,897, a selective CCK1 receptor antagonist, and L‐365,260, a selective CCK2 receptor antagonist, to fed and food‐deprived rats and determined plasma leptin concentration by enzyme immunoassay. Plasma insulin and glucose concentration as well as food intake were also determined.

Under our conditions, SR‐27,897 increased plasma concentration of leptin both in fed and food‐deprived rats. It also increased food intake as well as plasma concentration of insulin in fed animals. L‐365,260 increased plasma leptin concentration only in fed rats.

In animals receiving exogenous leptin, CCK‐8 increased the ratio between the concentration of leptin in cerebrospinal fluid and plasma.

These results show that CCK receptor antagonists increases plasma concentration of leptin and suggest that endogenous CCK may facilitate the uptake of plasma leptin to the cerebrospinal fluid.

British Journal of Pharmacology (2003) 140, 647–652. doi:10.1038/sj.bjp.0705477
Expression of Rho‐kinase and its functional role in the contractile activity of the mouse vas deferens
Authors: Büyükafşar Kansu , Levent Adnan , Ark Mustafa
Published: October 2003

The effects of two Rho‐kinase inhibitors, Y‐27632 and fasudil, were investigated on the contractions produced by electrical field stimulation (EFS, 40 V, 1 mS, 2, 4, 8 and 16 Hz, for 20 s), KCl (30 – 60 mM), phenylephrine (Phe) (10−5 – 10−4 M), adenosine‐3′, 5′‐triphosphate (ATP) (10−4 – 10−3 M) and α,β‐methylene ATP (10−5 M).

EFS produced frequency‐dependent reproducible contractile activity, which was almost abolished by guanethidine (10−5 M, for 1 h). This contraction consisted of two components (a phasic initial contraction followed by a tonic one), and it was inhibited by Y‐27632 and fasudil (both at 10−5 M). However, these inhibitors had no effect on resting tension of the tissue.

Contractions elicited by KCl (30 – 60 mM) were insensitive to guanethidine (10−5 M, for 1 h), but suppressed by Y‐27632 (10−5 M) and fasudil (10−5 M). In addition, the contractions induced by Phe (an α1‐adrenoceptor agonist) and ATP (a purinergic agent) were inhibited significantly by Y‐27632 (10−5 M). Phasic contractions evoked by the selective P2X purinoceptor agonist α,β‐methylene ATP were also suppressed by Y‐27632 (10−5 M).

Western blot analysis revealed that the mouse vas deferens expresses Rho‐kinase (ROKα, ROCK‐2 isoform) protein with a molecular weight of approximately 160 kDa. As a positive control, the presence of this protein was also shown in homogenates of smooth muscle from the rat mesenteric artery.

In conclusion, Rho‐kinase protein is expressed in the mouse vas deferens, and it mediates neurogenic contractile activity as well as the contractions induced by KCl, Phe, ATP and α,β‐methylene ATP. Owing to the suppressive effects of Rho‐kinase inhibitors on the contractile activity of the vas deferens, the possibility that these compounds might impair ejaculation must be taken into account when considering them as potential agents in the treatment of erectile dysfunction.

British Journal of Pharmacology (2003) 140, 743–749. doi:10.1038/sj.bjp.0705479
Enhanced noradrenergic transmission in the spontaneously hypertensive rat anococcygeus muscle
Authors: Jiménez‐Altayó Francesc , Giraldo Jesús , McGrath John C , Vila Elisabet
Published: October 2003

There is a long‐known hyper‐responsiveness of vascular adrenergic transmission in the spontaneously hypertensive rat (SHR) that is uncovered specifically in the presence of cocaine and attributed to blockade of the neuronal monoamine transporter. We have now used the rat anococcygeus muscle to investigate whether this phenomenon is generic to sympathetic transmission to smooth muscle rather than a purely vascular phenomenon. We sought the origin of the effect by successively blocking the buffering effects of the neuronal monoamine transporter, prejunctional α2‐adrenoceptors and NO from nitrergic nerves with desipramine (0.1 μM), rauwolscine (0.01 μM) and L‐NG‐nitro‐arginine (100 μM).

In the presence of desipramine, contractile responses to electrical field stimulation but not to noradrenaline (1 nM–100 μM) were greater in SHR than in Wistar–Kyoto (WKY). Neither inhibition of prejunctional α2‐adrenoceptors nor the blockade of neuronal nitric oxide synthase (nNOS) accounted for the differential enhancement of response in SHR. The enhanced effectiveness of motor neurotransmission in SHR becomes most apparent when all known major buffering mechanisms are removed.

When nitrergic responses were isolated pharmacologically (phentolamine 1 μM and guanethidine 30 μM; tone raised with carbachol 50 μM), they were not different between SHR and WKY.

Western blots showed that both nNOS and tyrosine hydroxylase are expressed to a similar extent in anococcygeus muscle from SHR and WKY, suggesting similar adrenergic and nitrergic innervations in the two strains. This suggests that enhanced motor transmission is due to increased transmitter release per varicosity rather than there being normal transmission from a greater number of sites.

We conclude that there is a generic enhancement of sympathetic transmission in SHR rather than this being a vascular phenomenon.

British Journal of Pharmacology (2003) 140, 773–779. doi:10.1038/sj.bjp.0705480
Inhibition of cardiac sodium currents by toluene exposure
Authors: Cruz Silvia L , Orta‐Salazar Gerardo , Gauthereau Marcia Y , Millan‐Perez Peña Lourdes , Salinas‐Stefanón Eduardo M
Published: October 2003

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes.

In oocytes, toluene inhibited sodium currents (INa+) in a concentration‐dependent manner, with an IC50 of 274 μM (confidence limits: 141–407μM). The inhibition was complete, voltage‐independent, and slowly reversible.

Toluene had no effect on: (i) the shape of the I–V curves; (ii) the reversal potential of Na+; and (iii) the steady‐state inactivation.

The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged.

Block of INa+ by toluene was use‐ and frequency‐dependent.

In rat cardiac myocytes, 300 μM toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent.

These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state.

The use‐ and frequency‐dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect.

British Journal of Pharmacology (2003) 140, 653–660. doi:10.1038/sj.bjp.0705481
Vasoconstrictor responses, and underlying mechanisms, to isoprostanes in human and porcine bronchial arterial smooth muscle
Authors: Tazzeo Tracy , Miller John , Janssen Luke J
Published: October 2003

We investigated the effects of five different isoprostanes (8‐isoPGE1, 8‐isoPGE2, 8‐isoPGF1α, 8‐isoPGF2αand 8‐isoPGF2β) on vasomotor tone in human and porcine bronchial arterial tissues.

In the human bronchial arteries, 8‐isoPGE2and 8‐isoPGF2αevoked powerful constrictions (magnitudes several fold greater than the responses to high millimolar KCl) with negative log concentration causing 50% excitation (EC50) values of 6.8 and 6.5, respectively; 8‐isoPGE1was less potent (EC50not calculated, since a clear peak contraction was not obtained), while the other isoprostanes were largely ineffective. In the porcine arteries, on the other hand, all three F‐ring isoprostanes as well as 8‐isoPGE2evoked constrictor responses, although the peak magnitudes were approximately 50% of the KCl‐evoked response; 8‐isoPGE2and 8‐isoPGF2αwere the most potent, with negative log EC50values of 6.5.

We next sought to characterize the signaling pathways underlying the vasoconstrictor responses to 8‐isoPGE2, since this was the most potent of the isoprostanes we tested. These responses were largely reversed by the thromboxane A2‐selective (TP) prostanoid receptor antagonist ICI 192605 (10−8M; 4(Z)‐6‐[(2,4,5cis)2‐(2‐chlorophenyl)‐4‐(2‐hydroxy phenyl)1,3‐dioxan‐5‐yl]hexenoic acid) as well as by the nonspecific tyrosine kinase inhibitor genistein (10−5and 10−4M), and were reversed approximately 50% by the Rho‐kinase inhibitor Y27632 (10−5M; (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(pyridyl) cyclohexanecarboxamide dihydrochloride).

We conclude, therefore, that 8‐isoPGE2constricts bronchial vasculature through the activation of TP receptors, which in turn trigger tyrosine kinase and Rho‐kinase activities, resulting in powerful vasoconstriction. These findings are highly relevant to lung transplantation and to exercise‐induced asthma.

British Journal of Pharmacology(2003)140, 759–763. doi:10.1038/sj.bjp.0705482
Glycosylated human oxyhaemoglobin activates nuclear factor‐κB and activator protein‐1 in cultured human aortic smooth muscle
Authors: Peiró Concepción , Matesanz Nuria , Nevado Julián , Lafuente Nuria , Cercas Elena , Azcutia Verónica , Vallejo Susana , Rodríguez‐Mañas Leocadio , Sánchez‐Ferrer Carlos F
Published: October 2003

Diabetic vessels undergo structural changes that are linked to a high incidence of cardiovascular diseases. Reactive oxygen species (ROS) mediate cell signalling in the vasculature, where they can promote cell growth and activate redox‐regulated transcription factors, like activator protein‐1 (AP‐1) or nuclear factor‐κB (NF‐κB), which are involved in remodelling and inflammation processes. Amadori adducts, formed through nonenzymatic glycosylation, can contribute to ROS formation in diabetes.

In this study, we analysed whether Amadori‐modified human oxyhaemoglobin, glycosylated at either normal (N‐Hb) or elevated (E‐Hb) levels, can induce cell growth and activate AP‐1 and NF‐κB in cultured human aortic smooth muscle cells (HASMC).

E‐Hb (1 nM–1 μM), but not N‐Hb, promoted a concentration‐dependent increase in cell size from nanomolar concentrations, although it failed to stimulate HASMC proliferation. At 10 nM, E‐Hb stimulated both AP‐1 and NF‐κB activity, as assessed by transient transfection, electromobility shift assays or immunofluorescence staining. The effects of E‐Hb resembled those of the proinflammatory cytokine tumour necrosis factor‐α (TNF‐α). E‐Hb enhanced intracellular superoxide anions content and its effects on HASMC were abolished by different ROS scavengers.

In conclusion, E‐Hb stimulates growth and activates AP‐1 and NF‐κB in human vascular smooth muscle by redox‐sensitive pathways, thus suggesting a possible direct role for Amadori adducts in diabetic vasculopathy.

British Journal of Pharmacology (2003) 140, 681–690. doi:10.1038/sj.bjp.0705483
Pharmacological characterization of a 7‐benzylidenenaltrexone‐preferring opioid receptor in porcine ileal submucosa
Authors: Townsend De Wayne , Brown David R
Published: October 2003

In the intestine, opioids produce antidiarrhoeal and constipating actions that are mediated by enteric neurones. Through interactions with opioid receptors (ORs) on submucosal neurones, opioids suppress active ion transport evoked by transmural electrical stimulation (TES) in mucosa–submucosa sheets from the porcine ileum. In this study, we examined the pharmacological characteristics of the previously described OR, which is sensitive to theδ1‐OR antagonist 7‐benzylidenenaltrexone and modulates neurogenic transepithelial ion transport in this tissue preparation.

Increases in short‐circuit current (Isc, a measure of active anion transport) evoked by TES in ileal mucosa–submucosa sheets were inhibited by opioid agonists possessing high selectivity for eitherδ‐ orμ‐ORs including [D‐Pen2,5]enkephalin (DPDPE), [D‐Ala2, Glu4]deltorphin II, and [D‐Ala2,N‐Me‐Phe4, Gly5‐ol]enkephalin (DAMGO).

As determined by the Schild analysis, the actions of these agonists were competitively inhibited by 7‐benzylidenenaltrexone. The nonequilibriumμ‐OR antagonistβ‐funaltrexamine inhibited the actions of DAMGO only at a high concentration (1μM) but did not alter DPDPE or deltorphin II action. At concentrations up to 10μM, the nonequilibriumδ‐OR antagonist naltrindole 5′‐isothiocyanate did not alter the actions ofδ‐ orμ‐OR agonists.

Radioligand binding analyses of neuronal homogenates from the ileal submucosa revealed that the nonselective OR ligand [3H]diprenorphine bound to two populations of specific binding sites. One of these sites possessed binding characteristics similar to theδ‐OR.

In summary, neurogenic ion transport in the porcine intestine is modulated by an OR which shares pharmacological characteristics of bothμ‐ andδ‐ORs and may represent a novel receptor entity.

British Journal of Pharmacology(2003)140, 691–700. doi:10.1038/sj.bjp.0705485
Vanilloid type 1 receptors (VR1) on trigeminal sensory nerve fibres play a minor role in neurogenic dural vasodilatation, and are involved in capsaicin‐induced dural dilation
Authors: Akerman S , Kaube H , Goadsby P J
Published: October 2003

Capsaicin, the active substance in chilli peppers, activates the vanilloid type 1 receptor (VR1) rather than the vanilloid‐like receptor (VRL1) in the trigeminal ganglion and nucleus of small and medium C‐ and Aδ‐fibres.

Capsaicin induces calcitonin gene‐related peptide (CGRP) release when VR1 receptors are activated, and this can be reversed by both the VR1 receptor antagonist capsazepine and the CGRP blocker αCGRP8–37 in vitro.

In this study we used intravital microscopy to look at the possible role of the VR1 receptor in the trigeminovascular system in producing dilation of dural blood vessels. Capsazepine (3 mg kg−1) was given to study the effect of the VR1 receptor in dural vessel dilation produced by either electrical stimulation, CGRP (1 μg kg−1) or capsaicin (7 μg kg−1) bolus injection. We also looked at the effect of the CGRP blocker αCGRP8–37 (300 μg kg−1) on capsaicin‐induced dilation so that we could see if the results found in vitro could also be found in vivo.

Electrical stimulation of the dura mater produced a robust vasodilator response between 130 and 137% of baseline diameter that was no different across four repeat stimuli (F3,18=0.6, P=0.61). CGRP similarly produced a dilatation of 99–111% that was no different across four baseline infusions (F3,15=2.4, P=0.113). Capsaicin also produced a consistent dilation of between 112 and 120% of baseline across three injections (F2,10=0.6, P=0.567).

Capsazepine did not inhibit the dilation brought about by either electrical stimulation or CGRP injection, while it was able to inhibit the dilation brought about by capsaicin (t5=3.4, P<0.05). αCGRP8–37 also inhibited the capsaicin‐induced dilation (t5=7.4, P<0.05) probably inhibiting the action of released CGRP at the CGRP receptor.

The study demonstrates that capsaicin can repeatedly induce dural vessel dilation in vivo, presumably through inducing CGRP release from trigeminal sensory nerve fibres, while C‐fibres may have been desensitised. The data imply that the VR1 receptor plays only a minor role in trigeminovascular‐induced dural vessel dilation.

British Journal of Pharmacology (2003) 140, 718–724. doi:10.1038/sj.bjp.0705486
Adenosine‐induced IL‐6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK
Authors: Rees D Aled , Lewis B Mary , Lewis Mark D , Francis Karen , Scanlon Maurice F , Ham Jack
Published: October 2003

Activation of adenosine receptors in folliculostellate (FS) cells of the pituitary gland leads to the secretion of IL‐6 and vascular endothelial growth factor (VEGF).

We investigated the action of adenosine A2 receptor agonists on IL‐6 and VEGF secretion in two murine FS cell lines (TtT/GF and Tpit/F1), and demonstrated a rank order of potency, 5′‐N‐ethylcarboxamidoadenosine (NECA)>2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine>adenosine, suggesting mediation via the A2b receptor.

NECA‐mediated IL‐6 release was inhibited by the PLC inhibitor 1‐[6‐((17β‐3‐methoxyestra‐1,3,5(10)‐tiene‐17‐yl)amino)hexyl]‐1H‐pyrrole‐2,5‐dione, the PI3 kinase inhibitor wortmannin and the PKC inhibitors bisindolylmaleimide 1 and bisindolymaleimide X1 HCl (Ro‐32‐0432).

NECA‐mediated IL‐6 release was attenuated (<50%) by the extracellular signal‐regulated kinase MAPK inhibitor 2′‐amino‐3′‐methoxyflavone, and completely (>95%) inhibited by the p38 MAPK inhibitor 4‐(4‐fluorophenyl)‐2‐(4‐methylsulphinylphenyl)‐5‐(4‐pyridyl)1H‐imidazole.

NECA stimulates p38 MAPK phosphorylation that is inhibited by Ro‐32‐0432 but not by wortmannin.

Dexamethasone inhibits NECA‐stimulated IL‐6 and VEGF secretion.

These findings indicate that adenosine can stimulate IL‐6 secretion in FS cells via the A2b receptor coupled principally to PLC/PKC and p38 MAPK; such an action may be important in the modulation of inflammatory response processes in the pituitary gland.

British Journal of Pharmacology (2003) 140, 764–772. doi:10.1038/sj.bjp.0705488
Pharmacological profile of the 5‐HT‐induced inhibition of cardioaccelerator sympathetic outflow in pithed rats: correlation with 5‐HT1 and putative 5‐ht5A/5B receptors
Authors: Sánchez‐López Araceli , Centurión David , Vázquez Erika , Arulmani Udayasankar , Saxena Pramod R , Villalón Carlos M
Published: October 2003

Continuous infusions of 5‐hydroxytryptamine (5‐HT) inhibit the tachycardiac responses to preganglionic (C7‐T1) sympathetic stimulation in pithed rats pretreated with desipramine. The present study identified the pharmacological profile of this inhibitory action of 5‐HT.

The inhibition induced by intravenous (i.v.) continuous infusions of 5‐HT (5.6 μg kg−1 min−1) on sympathetically induced tachycardiac responses remained unaltered after i.v. treatment with saline or the antagonists GR 127935 (5‐HT1B/1D), the combination of WAY 100635 (5‐HT1A) plus GR 127935, ritanserin (5‐HT2), tropisetron (5‐HT3/4), LY215840 (5‐HT7) or a cocktail of antagonists/inhibitors consisting of yohimbine (α2), prazosin (α1), ritanserin, GR 127935, WAY 100635 and indomethacin (cyclooxygenase), but was abolished by methiothepin (5‐HT1/2/6/7 and recombinant 5‐ht5A/5B). These drugs, used in doses high enough to block their respective receptors/mechanisms, did not modify the sympathetically induced tachycardiac responses per se.

I.v. continuous infusions of the agonists 5‐carboxamidotryptamine (5‐CT; 5‐HT1/7 and recombinant 5‐ht5A/5B), CP 93,129 (r5‐HT1B), sumatriptan (5‐HT1B/1D), PNU‐142633 (5‐HT1D) and ergotamine (5‐HT1B/1D and recombinant 5‐ht5A/5B) mimicked the above sympatho‐inhibition to 5‐HT. In contrast, the agonists indorenate (5‐HT1A) and LY344864 (5‐ht1F) were inactive. Interestingly, 5‐CT‐induced cardiac sympatho‐inhibition was abolished by methiothepin, the cocktail of antagonists/inhibitors, GR 127935 or the combination of SB224289 (5‐HT1B) plus BRL15572 (5‐HT1D), but remained unchanged when SB224289 or BRL15572 were given separately.

Therefore, 5‐HT‐induced cardiac sympatho‐inhibition, being unrelated to 5‐HT2, 5‐HT3, 5‐HT4, 5‐ht6, 5‐HT7 receptors, α1/2‐adrenoceptor or prostaglandin synthesis, seems to be primarily mediated by (i) 5‐HT1 (probably 5‐HT1B/1D) receptors and (ii) a novel mechanism antagonized by methiothepin that, most likely, involves putative 5‐ht5A/5B receptors.

British Journal of Pharmacology (2003) 140, 725–735. doi:10.1038/sj.bjp.0705489
Ginsenoside Rg3 inhibits phenylephrine‐induced vascular contraction through induction of nitric oxide synthase
Authors: Kim Nak Doo , Kim Eun Mi , Kang Keon Wook , Cho Min Kyung , Choi So Yeon , Kim Sang Geon
Published: October 2003

Ginsenoside Rg3 (Rg3) isolated from Panax ginseng relaxes vessels and exerts a cytoprotective effect. In view of the fact that nitric oxide (NO) is involved in vascular hyporeactivity and immunostimulation, the effects of total ginsenosides (GS) and Rg3 on the vascular responses and the expression of inducible nitric oxide synthase (iNOS) were investigated.

Vasocontraction of endothelium‐denuded aortic ring was induced by phenylephrine with or without GS or Rg3. The expression of iNOS was assessed by Western blot and RT–PCR analyses. NF‐κB activation was monitored by gel shift, immunoblot and immunocytochemical analyses.

Incubation of the endothelium‐denuded aortic ring with GS or Rg3 inhibited phenylephrine‐induced vasocontraction, which was abrogated by NOS inhibition. GS or Rg3 increased NO production in aortic rings, but Rb1, Rc, Re and Rg1 had no effect.

Aortic rings obtained from rats treated with GS or Rg3 responded to phenylnephrine to a lesser extent, while producing NO to a larger extent, than those from control animals. GS or Rg3 induced iNOS in vascular smooth muscle.

Rg3 induced iNOS with increase in NO production in Raw264.7 cells. Rg3 increased NF‐κB DNA binding, whose band was supershifted with anti‐p65 and anti‐p50 antibodies, and elicited p65 nuclear translocation, which was accompanied by phosphorylation and degradation of I‐κBα. PKC regulated iNOS induction by Rg3.

In conclusion, Rg3 relaxes vessels as a consequence of NO production, to which iNOS induction contributes, and iNOS induction by Rg3 accompanied NF‐κB activation, which involves phosphorylation and degradation of I‐κBα and nuclear translocation of p65.

British Journal of Pharmacology (2003) 140, 661–670. doi:10.1038/sj.bjp.0705490
Myoblast‐mediated gene transfer for therapeutic angiogenesis and arteriogenesis
Authors: Von Degenfeld Georges , Banfi Andrea , Springer Matthew L , Blau Helen M
Published: October 2003

Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of ‘growth’ factors with ‘maturation’ factors like PDGF‐BB, angiopoietin‐1 or TGF‐β. Myoblast‐mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long‐lasting expression, and can be considered as a relatively rapid form of ‘adult transgenesis’ in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different ‘growth’ and ‘maturation’ factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline‐inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell‐based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors.

British Journal of Pharmacology (2003) 140, 620–626. doi:10.1038/sj.bjp.0705492
Testing clinical therapeutic angiogenesis using basic fibroblast growth factor (FGF‐2)
Authors: Aviles Ronnier J , Annex Brian H , Lederman Robert J
Published: October 2003

Therapeutic angiogenesis represents an attempt to relieve inadequate blood flow by the directed growth and proliferation of blood vessels. Neovascularization is a complex process involving multiple growth factors, receptors, extracellular matrix glycoproteins, intracellular and extracellular signaling pathways, and local and bone‐marrow‐derived constituent cells, all responding to a symphonic arrangement of temporal and spatial cues. In cardiovascular disease, patients with refractory angina and lower extremity intermittent claudication seem most amenable to early tests of therapeutic angiogenesis. Monotherapy with the recombinant protein basic fibroblast growth factor (FGF‐2) has been tested in six human trials. These have shown provisional safety, and two have provided ‘proof of concept’ for the strategy of therapeutic angiogenesis. One large randomized phase II trial failed to show significant efficacy in coronary artery disease. Another showed significant efficacy in peripheral artery disease, although the magnitude of benefit was disappointing at the dose tested. This overview details the suitable clinical trial design and further steps toward the clinical development of FGF‐2.

British Journal of Pharmacology (2003) 140, 637–646. doi:10.1038/sj.bjp.0705493
Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells
Authors: Gerritsen Mary E , Tomlinson James E , Zlot Constance , Ziman Michael , Hwang Stuart
Published: October 2003

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high‐density oligonucleotide arrays, representing approximately 22,000 genes. Notably, the genes significantly up‐ and downregulated by VEGF versus HGF exhibited very little overlap, indicating distinct signal transduction pathways. The combination of HGF and VEGF markedly increased the number of significantly up‐ and downregulated genes. At 4 h, the combination of the two growth factors induced a number of chemokine and cytokines and their receptors (IL‐8, IL‐6, IL‐11, CCR6, CXCR1,CXC1 and IL17RC), numerous genes involved in growth factor signal transduction (egr‐1, fosB, grb10, grb14,MAP2K3,MAP3K8, MAPKAP2,MPK3, DUSP4 and DUSP6), as well as a number of other growth factors (PDGFA, BMP2, Hb‐EGF, FGF16, heuregulin beta 1, c‐kit ligand, angiopoietin 2 and angiopoietin 4 and VEGFC). In addition, the VEGF receptors neuropilin‐1 and flt‐1 were also upregulated. At 24 h, a clear ‘cell cycle’ signature is noted, with the upregulated expression of various cell cycle control proteins and gene involved in the regulation of mitosis and mitotic spindle assembly. The receptor for HGF, c‐met, is also upregulated. These data are consistent with the hypothesis that the combination of HGF and VEGF results in the cooperative upregulation of a number of different molecular pathways leading to a more robust proliferative response, that is, growth factor(s), receptors, molecules involved in growth factor signal transduction, as well as, at later time points, upregulation of the necessary cellular proteins required for cells to escape cell cycle arrest and enter the cell cycle.

British Journal of Pharmacology (2003) 140, 595–610. doi:10.1038/sj.bjp.0705494
Emerging concept in angiogenesis: specification of arterial and venous endothelial cells
Authors: Sato Thomas N
Published: October 2003

British Journal of Pharmacology (2003) 140, 611–613. doi:10.1038/sj.bjp.0705495
Fishing for novel angiogenic therapies
Authors: Kidd Kameha R , Weinstein Brant M
Published: October 2003

The zebrafish has recently emerged as an important model for the study of vascular embryogenesis. Its genetic accessibility, external development, and optically clear embryo are just a few of the features that set the zebrafish apart as a particularly well‐suited model for studying vascular development. However, there is little precedent for its use as a tool for the experimental study of therapeutic angiogenesis. Here, we review the use of the zebrafish for studying vascular development and patterning, and discuss how the zebrafish might be used more directly as a model for developing and testing effective therapeutic angiogenesis approaches.

British Journal of Pharmacology (2003) 140, 585–594. doi:10.1038/sj.bjp.0705496
In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism
Authors: Ottonello Luciano , Arduino Nicoletta , Bertolotto Maria , Dapino Patrizia , Mancini Marina , Dallegri Franco
Published: October 2003

Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases.

This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha‐1‐antitrypsin (A1AT) inactivation by stimulated neutrophils and possibly to look into the mechanisms underlying this event.

Ambroxol inhibited the production of superoxide anion by activated neutrophils, whereas bromhexine had no inhibitory effect.

Ambroxol decreased the production of hypochlorous acid (HOCl) from activated neutrophils with high efficiency, whereas bromhexine had a modest activity.

Ambroxol and bromhexine were capable of limiting the chlorination of monochlorodimedon by HOCl, displaying the capacity of directly scavenging the oxidant.

Ambroxol decreased the release of elastase and myeloperoxidase from activated neutrophils, whereas bromhexine was ineffective.

Ambroxol prevented the A1AT inactivation by neutrophils, whereas bromhexine was completely ineffective.

Among drugs currently available for in vivo use in humans, ambroxol is unique by virtue of its ability to prevent neutrophil‐mediated A1AT inactivation via inhibition of HOCl production as well as HOCl scavenging. Also taking into account its capacity for curbing elastase release, the drug displays the potential to lessen the burden of oxidants/proteases and to increase the antiprotease shield at the site of inflammation. Thus, ambroxol appears to be a good candidate for raising attempts to develop new therapeutic histoprotective approaches to inflammatory bronchopulmonary diseases.

British Journal of Pharmacology (2003) 140, 736–742. doi:10.1038/sj.bjp.0705497
Mechanisms involved in the early increase of serotonin contraction evoked by endotoxin in rat middle cerebral arteries
Authors: Hernanz Raquel , Alonso María J , Briones Ana M , Vila Elisabet , Simonsen Ulf , Salaices Mercedes
Published: October 2003

The present study investigated the mechanisms involved in the increased 5‐hydroxytryptamine (5‐HT) vasoconstriction observed in rat middle cerebral arteries exposed in vitro to lipopolysaccharide (LPS, 10 μg ml−1) for 1–5 h. Functional, immunohistochemical and Western blot analysis and superoxide anion measurements by ethidium fluorescence were performed.

LPS exposure increased 5‐HT (10 μM) vasoconstriction only during the first 4 h. In contrast to control tissue, indomethacin (10 μM), the COX‐2 inhibitor NS 398 (10 μM), the TXA2/PGH2 receptor antagonist SQ 29,548 (1 μM) and the TXA2 synthase inhibitor furegrelate (1 μM) reduced 5‐HT contraction of LPS‐treated arteries from hour one. The iNOS inhibitor aminoguanidine (0.1 mM) increased 5‐HT contraction from hour three of LPS incubation.

The superoxide anion scavenger superoxide dismutase (SOD, 100 U ml−1) and the H2O2 scavenger catalase (1000 U ml−1), as well as the respective inhibitors of NAD(P)H oxidase and xanthine oxidase, apocynin (0.3 mM) and allopurinol (0.3 mM), reduced 5‐HT contraction after LPS incubation. LPS induced an increase in superoxide anion levels that was abolished by PEG‐SOD.

Subthreshold concentrations of the TXA2 analogue U 46619, xanthine/xanthine oxidase and H2O2 potentiated, whereas those of sodium nitroprusside inhibited, the 5‐HT contraction.

COX‐2 expression was increased at 1 and 5 h of LPS incubation, while that of iNOS, Cu/Zn‐SOD and Mn‐SOD was only increased after 5 h. All the three vascular layers expressed COX‐2 and Cu/Zn‐SOD. iNOS expression was detected in the endothelium and adventitia after LPS.

In conclusion, increased production of TXA2 from COX‐2, superoxide anion and H2O2 enhanced vasoconstriction to 5‐HT during the first few hours of LPS exposure; iNOS and SOD expression counteracted that increase at 5 h. These changes can contribute to the disturbance of cerebral blood flow in endotoxic shock.

British Journal of Pharmacology (2003) 140, 671–680. doi:10.1038/sj.bjp.0705501
The Influence of Probe Fiber Distance on Laser Doppler Perfusion Monitoring Measurements
Authors: MORALES FERNANDO , GRAAFF REINDERT , SMIT ANDRIES J. , GUSH RODNEY , RAKHORST GERHARD
Published: October 2003

Laser Doppler perfusion monitoring (LDPM) is a noninvasive technique for monitoring skin microcirculation. The aim of this article was to investigate the influence of fiber separation on clinical LDPM measurements. A dual‐channel LDPM system was used in combination with a probe that consists of two sets of detection fibers, at 0.2 and 1.0 mm from the illuminating fiber. Measurements were performed at the big toe of 8 healthy subjects and 11 subjects who had vascular disorders. In most cases, fluxes detected at both fiber distances showed very similar fluctuations. For each fiber separation, flux values of healthy subjects and patients were not significantly different. Furthermore, skin temperature (range: 22–34°C) influenced the toe's pulp microcirculation markedly, increasing similarly at both probe separations, with a higher flux at a separation of 1.0 mm than at 0.2 mm. The flux ratio signal, obtained by dividing the flux at 0.2 mm by the flux at 1.0 mm, was significantly different between the two groups (p < 0.05). In conclusion, the flux detected in vivo by means of LDPM, is influenced by the distance between the optical fibers. Use of the flux ratio with a multiseparation probe deserves attention as it is a possible marker for discriminating normal tissue perfusion from pathological skin tissue perfusion, independently from tissue temperature.
Hepatic Microcirculatory Dysfunction During Cholestatic Liver Injury in Rats
Authors: ITO YOSHIYA , BETHEA NANCY W. , BAKER GREGORY L. , MCCUSKEY MARGARET K. , URBASCHEK RENATE , MCCUSKEY ROBERT S.
Published: October 2003

Objective: The present study was conducted to elucidate the sequential alterations in the hepatic microvascular inflammatory response to extrahepatic biliary obstruction.

Methods: The hepatic microvasculature in anesthetized Sprague‐Dawley rats was studied by in vivo microscopy 3, 7, and 14 days after bile duct ligation (BDL) or sham operation.

Results: The numbers of adhering leukocytes and swollen sinusoidal endothelial cells were significantly increased at 3, 7, and 14 days after BDL when compared with sham‐operated controls. Concomitantly, the numbers of sinusoids containing blood flow were significantly and progressively decreased by up to 30%. The phagocytic activity of hepatic macrophages was significantly elevated during the development of biliary cholestasis. In particular, centrilobular phagocytosis at 14 days after BDL was significantly increased 1.4‐ to 2.0‐fold when compared with that at 3 and 7 days after BDL. Electron microscopy also revealed evidence of activated Kupffer cells reflected by numerous filopodia and ruffles.

Conclusions: These results suggest that hepatic microcirculatory dysfunction subsequent to BDL contributes to cholestatic liver injury.
A method for simultaneous determination of plasma and erythrocyte antioxidant status. Evaluation of the antioxidant activity of vitamin E in healthy volunteers
Authors: ABELLA ANNIE , MESSAOUDI CHERIF , LAURENT DENIS , MAROT DIDIER , CHALAS JACQUELINE , BREUX JOCELYNE , CLAISE CATHERINE , LINDENBAUM ALBERT
Published: October 2003

1Since oxygen free radicals are directly involved in a variety of pathologies such as atherosclerosis, diabetes mellitus, inflammation and/or when a deficit of defences of the organism against radicals occurs, we developed a suitable and simple method to determine both the erythrocyte sensitivity to an oxidative stress and plasma antioxidant protective capacity.

2This test is based on the introduction at 37° C of a radical initiator, 2,2′‐azobis (2‐amidinopropane) dihydrochloride (AAPH), within an erythrocyte suspension leading to a membrane alteration and ultimately to haemolysis. The latter can be quantified by determining the lacticodeshydrogenase activity released in the medium. The erythrocyte sensitivity to haemolysis and the volume of plasma inhibiting 50% of the haemolysis were determined.

3Intra‐assay CVs were 1.9 % for erythrocyte sensitivity to oxidative stress and 3.4% for inhibitory 50% plasma volume. Inter‐assay CVs for both erythrocyte sensitivity and inhibitory 50% plasma volume were 4%.

4The reliability of this method was assessed and applied to test the protective effect of vitamin E, a well known antioxidant agent, in six healthy volunteers. Two weeks after daily administration of 500 mg of vitamin E, the mean plasma vitamin E concentration increased by 41% from 10.7±2.0 mg l−1 before treatment (P<0.05). As the vitamin E concentration increased, the mean inhibitory 50% plasma volume and the percentage of haemolysed erythrocytes decreased respectively by 29% from 3.35±0.5 μl (P<0.05) and 18% from 71.5±3.8% (P<0.05). No significative variation of these parameters was observed in six adult men without vitamin E supplementation.

5Thus, this global and simple test permits an antioxidant status evaluation of a patient. It can be applied to various pathologies and allows the potency of new antioxidant molecules to be evaluated.
Comparison of the effects of clonidine on tyramine‐ and methoxamine‐evoked mydriasis in man
Authors: BITSIOS P. , LANGLEY R. W. , SZABADI E. , BRADSHAW C. M.
Published: October 2003

1 It has been reported previously that clonidine can potentiate tyramine‐evoked mydriasis on the pain‐free side of cluster headache patients. We examined whether a single oral dose of clonidine (200 μg) can also potentiate tyramine‐evoked mydriasis in healthy subjects, using mydriasis to methoxamine, a directly acting sympathomimetic amine, as a control.

2 Eight healthy male volunteers participated in four weekly sessions. In the first two sessions (Experiment 1) the effect of clonidine or placebo on the mydriasis to tyramine hydrochloride eyedrops (75 mm; 2×10 μl), and in the last two sessions (Experiment 2) the effect of clonidine or placebo on the mydriasis to methoxamine hydrochloride eyedrops (20 mm; 2×10 μl) was examined. In both experiments subjects were allocated to drugs and sessions according to a double‐blind balanced design. In both experiments, pupil diameter of both the treated and the untreated eyes was recorded in standard ambient light and in the dark, before, and 2 h after clonidine/placebo, via binocular infrared television pupillometry. Salivation (dental roll technique), systolic and diastolic blood pressure (sitting), heart rate, and self‐ratings of mood and feelings (visual analogue scales), were also measured before, and 2 h after the ingestion of clonidine or placebo.

3 Both tyramine and methoxamine produced a significant mydriasis, which was more prominent in the light condition (change in resting pupil size; mm±s.e.mean: tyramine/light 1.05±0.28; tyramine/dark: 0.73±0.15; methoxamine/light: 1.65±0.28; methoxamine/dark: 0.85±0.15). Clonidine produced a significant miosis in the untreated eye which was more prominent in the light condition (change in resting pupil size; mm±s.e.mean: Experiment 1, light: −1.34±0.19; Experiment 1, dark: −0.46±0.1; Experiment 2, light −0.97±0.18; Experiment 2, dark: −0.29±0.17). Clonidine had no significant effect on either tyramine‐ or methoxamine‐evoked mydriasis.

4 In agreement with previous reports, clonidine significantly reduced salivation (g, mean±s.e.mean; Experiment 1: −0.84±0.22; Experiment 2: −0.55±0.11), systolic blood pressure (mm Hg; Experiment 1: −17.5±3.76; Experiment 2: −23.38±4.67), diastolic blood pressure (mm Hg; Experiment 2: −12.38±2.05), alertness (mm; Experiment 2: −24.19±5.40), and anxiety (mm; Experiment 1: −13.82±4.60), indicating the presence of pharmacodynamically effective tissue levels of the drug.

5 These results show that a single oral dose (200 μg) of clonidine causes significant miosis in human subjects, and fails to potentiate tyramine‐evoked mydriasis. This indicates that the pupil on the asymptomatic side of cluster headache patients is affected differently from the pupils of healthy volunteers by tyramine and/or clonidine.
8‐Epi PGF2α: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo
Authors: DELANTY N. , REILLY M. , PRATICO D. , FITZGERALD D. J. , LAWSON J. A. , FITZGERALD G. A.
Published: October 2003

1Excessive free radical generation is thought to contribute to tissue injury in a broad spectrum of diseases. A particular constraint in addressing this hypothesis has been the inability to assess free radical generation in vivo and the lack of information on drugs or vitamins which act as effective antioxidants in vivo.

2Traditional approaches have relied upon measures of substrate oxidizability or spin trapping of free radical adducts ex vivo. It is unknown how these measurements might relate, in a quantitative fashion, to the generation of reactive oxygen species in vivo. Isoeicosanoids are free radical catalyzed products of arachidonic acid. One of these compounds, 8‐epi prostaglandin F2α (8‐epi PGF2α) exhibits biological activity and may function as an autacoid. Specific analysis of this 8‐epi PGF2α isomer indicates that it is elevated in certain syndromes thought to be associated with oxidant stress. These include vascular reperfusion, paracetamol poisoning and liver cirrhosis. Apparently healthy individuals who smoke cigarettes or consume alcohol exhibit dose dependent increments in excretion of 8‐epi PGF2α. Excretion is depressed by antioxidant vitamins, although not by the nonspecific cyclooxygenase (COX) inhibitor, aspirin, even though 8‐epi PGF2α may be formed by either COX‐1 or COX‐2.

3Specific analysis of this and other isoeicosanoids may afford an opportunity to evaluate the effects of antioxidant interventions in human diseases characterized by excessive lipid peroxidation in vivo.
Cholesterol lowering therapy inhibits the low‐flow mediated vasoconstriction of the brachial artery in hypercholesterolaemic subjects
Authors: MEGNIEN JEAN LOUIS , SIMON ALAIN , ANDRIANI ANTONIA , SEGOND PATRICK , JEANNIN SOPHIE , LEVENSON JAIME
Published: October 2003

1We tested whether lipid lowering treatment with HMG CoA reductase inhibitor modified the flow mediated large artery reactivity in primary pure hypercholesterolaemia.

2Abnormalities in arterial reactivity have been described in the presence of high blood cholesterol, in particular an enhanced constriction of the brachial artery in response to acute induction of a low flow state.

3Using pulsed‐Doppler, we measured brachial artery diameter and flow velocity at rest and their changes induced by wrist occlusion before and after 3 months of double‐blind treatment by pravastatin (40 mg orally) in 13 subjects and placebo in 15 others.

4The significant decrease (P<0.01) in diameter induced by wrist occlusion before (0.34±0.08 mm) placebo and pravastatin (0.39±0.10 mm) persisted after placebo (0.26±0.07 mm) but was abolished after pravastatin (0.07±0.05 mm). The absolute change in diameter induced by wrist occlusion was lower after than before pravastatin (P<0.01) and lower after pravastin than after placebo (P<0.05). Diameter during wrist occlusion was higher after pravastatin than after placebo (4.35±0.16 vs 3.89±0.09 mm); P<0.01).

5These findings indicate that the lipid changes induced by pravastatin and/or some unknown but direct mechanism of the drug itself inhibit low‐flow‐mediated vasoconstriction associated with hypercholesterolaemia. Such effects may have important implications for the treatment of vasospasm often seen in the presence of high blood cholesterol.
Taste loss to terbinafine: a case‐control study of potential risk factors
Authors: STRICKER B. H. Ch. , VAN RIEMSDIJK M. M. , STURKENBOOM M. C. J. M. , OTTERVANGER J. P.
Published: October 2003

1To identify risk factors associated with taste loss to terbinafine, we performed a case‐control study of 87 reports of probable terbinafine‐induced taste loss and 362 controls on terbinafine without taste loss, who had filled prescriptions from the same pharmacy and GP. Data on general health, diet, alcohol, smoking, drug use and medical history were collected by means of a self‐administered questionnaire.

2The mean latent period between first intake of terbinafine and taste loss was 35 days. Most patients recovered within 4 months after discontinuation. Cases were significantly older than controls. The odds ratio of taste loss in patients of 65 years and older was 4.4 in comparison with persons younger than 35 years of age (95% CI: 1.4–16.1). The risk in persons with a body mass index (BMI) below 21 kg m−2 was 4.4 times higher than in those with a BMI of more than 27 kg m−2 (95% CI: 1.6–14.2). The risk of taste loss in patients of 55 years and older with a BMI below 21 kg m−2 was 12.8 times higher than that in patients below 35 years of age (95% CI: 1.9–88.6).

3A low BMI, a history of taste loss, and ageing are risk factors for developing taste loss to terbinafine. Prescription of this drug to elderly patients with low BMI and low daily intake of nutrients requires careful follow‐up.