bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle α‐actin content and abolishes the platelet‐derived growth factor‐BB‐stimulated DNA synthesis
Authors: Sachinidis Agapios , Seul Claudia , Gouni‐Berthold Ioanna , Seewald Stefan , Ko Yon , Vetter Hans , Fingerle Jürgen , Hoppe Jürgen
Published: January 2005

The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet‐derived growth factor β‐receptor (PDGF‐Rβ).

The effect of PDGF‐BB on VSMCs growth was assessed by [3H]‐thymidine incorporation. Tyrosine phosphorylation of PDGF‐Rβ, PLC‐γ1, ERK1 and ERK2, p125FAK and paxillin as well as Sm α‐actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm α‐actin filaments, paxillin and PDGF‐Rβ was performed by immunfluorescence microscopy.

Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm α‐actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125FAK and paxillin but decreased the content of a Sm α‐actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF‐BB stimulated DNA‐synthesis although PDGF‐Rβ level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF‐BB‐induced tyrosine phosphorylation of the PDGF‐Rβ, PI 3′‐K, PLC‐γ1 and ERK1/2 indicating an action of cyclic AMP on PDGF‐β receptor.

We conclude that although cyclic AMP attenuates the PDGF‐Rβ mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF‐BB‐induced DNA synthesis in VSMCs.

British Journal of Pharmacology (2000) 130, 1561–1570; doi:10.1038/sj.bjp.0703480
Evidence that adenosine 5′‐triphosphate is the third inhibitory non‐adrenergic non‐cholinergic neurotransmitter in the rat gastric fundus
Authors: Jenkinson Karl M , Reid Julianne J
Published: January 2005

In the rat gastric fundus, non‐adrenergic, non‐cholinergic (NANC) relaxations are mediated by nitric oxide (NO), vasoactive intestinal polypeptide (VIP), and a third, as yet unidentified, neurotransmitter.

The possible involvement of adenosine 5′‐triphosphate (ATP) in the NANC relaxations was examined using pyridoxalphosphate‐6‐azophenyl‐2′,5′‐disulphonic acid (PPADS), apamin and desensitization to α,β‐methylene ATP. NANC responses were studied in the absence and presence of NG‐nitro‐L‐arginine methyl ester (NAME; 100 μM) and α‐chymotrypsin (1 u ml−1), to inhibit responses to NO and VIP, respectively.

PPADS (100 μM), apamin (1 μM) and desensitization to α,β‐methylene ATP (10 μM, three additions) all significantly (P<0.05) reduced NANC relaxations to electrical field stimulation (0.5–4 Hz, 30 s trains) in longitudinal strips of rat gastric fundus and almost abolished the residual relaxation remaining in the presence of NAME and α‐chymotrypsin.

PPADS had no effect on responses to the NO‐donor, sodium nitroprusside (SNP), or VIP. Apamin slightly reduced relaxations to SNP, but did not affect those to VIP, whereas desensitization to α,β‐methylene ATP markedly reduced responses to both SNP and VIP.

The effects of PPADS and apamin in this study provide strong evidence that the third inhibitory NANC neurotransmitter in the rat gastric fundus is ATP.

British Journal of Pharmacology (2000) 130, 1627–1631; doi:10.1038/sj.bjp.0703481
The P2‐purinoceptor antagonist suramin is a competitive antagonist at vasoactive intestinal peptide receptors in the rat gastric fundus
Authors: Jenkinson Karl M , Reid Julianne J
Published: January 2005

The P2‐purinoceptor antagonist, suramin, was used to investigate the possible involvement of adenosine 5′‐triphosphate (ATP) in the inhibitory non‐adrenergic non‐cholinergic (NANC) innervation of the rat gastric fundus.

ATP (1–30 μM) produced biphasic responses consisting of concentration‐dependent relaxations followed by concentration‐dependent contractions. Suramin (200 μM) significantly reduced relaxations and abolished contractions to ATP.

Under NANC conditions, electrical field stimulation (EFS) induced frequency‐dependent relaxations. Suramin (200 μM) and the peptidase α‐chymotrypsin (1 u ml−1) had the same effects on EFS‐induced relaxations: their duration was reduced, but their magnitude was unaffected.

Cumulative relaxations to vasoactive intestinal peptide (VIP; 0.1–100 nM), and to the VIP analogue pituitary adenylate cyclase activating peptide 1–27 (PACAP; 0.2–100 nM), were almost completely abolished by α‐chymotrypsin (1 u ml−1), and were inhibited by suramin (3–200 μM) in an apparently competitive manner. Schild plot analysis indicated that suramin had pA2 values of 5.1±0.2 (Hill slope=0.9±0.2) and 5.6±0.1 (Hill slope=1.0±0.1), against VIP and PACAP, respectively.

Concentration‐dependent relaxations to nitric oxide (1–30 μM) and cumulative relaxations to isoprenaline (0.1–300 nM) were not affected by suramin (200 μM).

No conclusions can be made regarding the possible involvement of ATP in EFS‐induced NANC relaxations. The results suggest that suramin acts as a competitive antagonist at VIP receptors in the rat gastric fundus.

British Journal of Pharmacology (2000) 130, 1632–1638; doi:10.1038/sj.bjp.0703482
Effects of a selective neuropeptide Y Y1 receptor antagonist BIBP 3226 on double peaked vasoconstrictor responses to periarterial nerve stimulation in canine splenic arteries
Authors: Yang Xiao‐Ping , Chiba Shigetoshi
Published: January 2005

The periarterial electrical nerve stimulation (30 s trains of pulses at a frequency of 1, 4 or 10 Hz) induced a double peaked vasoconstriction consisting of an initial transient constriction (first peak) followed by a prolonged response (second peak) in the isolated, perfused canine splenic artery.

At low frequencies (1 and 4 Hz), a neuropeptide Y (NPY) Y1 receptor antagonist BIBP 3226 (0.1–1 μM) produced a dose‐dependent inhibitory effect on the second peak, but did not modify the first peak. At a high frequency (10 Hz), 1 μM BIBP 3226 induced a slight, but significant inhibition on both the first and second peaked responses.

At a low frequency (1 Hz), the first peak was not influenced by blockade of α1‐adrenoceptors or NPY Y1 receptors with prazosin (0.1 μM) or BIBP 3226 (1 μM), respectively, but abolished by P2X receptor desensitization with α,β‐methylene ATP (αβ‐m ATP, 1 μM). At a high frequency (10 Hz), the first peak was mostly inhibited by αβ‐m ATP and partially by prazosin and BIBP 3226. On the other hand, the second peak at a low frequency was largely decreased by BIBP 3226 and partially by prazosin and αβ‐m ATP, whereas at a high frequency, it was largely attenuated by prazosin and partially by αβ‐m ATP and BIBP 3226.

The results suggest that at a low frequency, the firstly transient constriction of double peaked responses is mainly induced via an activation of P2X‐receptors, whereas at a high frequency, it is mostly mediated by the P2X‐receptors, and partially by α1‐receptors and NPY Y1‐receptors. The secondary prolonged vasoconstriction at frequencies used is predominantly mediated via both α1‐receptor and NPY Y1 receptor activations, and in part by P2X‐receptors. Furthermore, an activation of NPY Y1 receptors may play an important role in evoking the prolonged vasoconstrictor response to longer pulse trains of stimulation at a low frequency, whereas an α1‐adrenoceptor activation exerts a main vasomotor effect for the prolonged response at a high frequency.

British Journal of Pharmacology (2000) 130, 1699–1705; doi:10.1038/sj.bjp.0703484
Induction of reactive astrocytosis and prevention of motoneuron cell death by the I2‐imidazoline receptor ligand LSL 60101
Authors: Casanovas Anna , Olmos Gabriel , Ribera Joan , Assumpció Boronat M , Esquerda Josep E , García‐Sevilla Jesús A
Published: January 2005

I2‐imidazoline receptors are mainly expressed on glial cells in the rat brain. This study was designed to test the effect of treatment with the I2‐imidazoline selective receptor ligand LSL 60101 [2‐(2‐benzofuranyl)imidazole] on the morphology of astrocytes in the neonate and adult rat brain, and to explore the putative neuroprotective effects of this glial response.

Short‐term (3 days) or chronic (7–10 days) treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h) enhanced the area covered by astroglial cells in sections of facial motor nucleus from neonate rats processed for glial fibrillary acidic protein (GFAP) immunostaining. Facial motoneurons surrounded by positive glial cell processes were frequently observed in sections of LSL 60101‐treated rats. A similar glial response was observed in the parietal cortex of adult rats after chronic (10 days) treatment with LSL 60101 (10 mg kg−1, i.p. every 12 h).

Western‐blot detection of the specific astroglial glutamate transporter GLT‐1, indicated increased immunoreactivity after LSL 60101 treatment in the pons of neonate and in the parietoccipital cortex of adult rats.

In the facial motor nucleus of neonate rats, the glial response after LSL 60101 treatment was associated to a redistribution of the immunofluorescence of the basic fibroblast growth factor (FGF‐2) from the perinuclear area of motoneurons to cover most of their cytoplasm, suggesting a translocation of this mitogenic and neurotrophic factor towards secretion pathways.

The neuroprotective potential of the above effects of LSL 60101 treatment was tested after neonatal axotomy of facial motor nucleus. Treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h from day 0 to day 10 after birth) significantly reduced (38%) motoneuron death rate 7 days after facial nerve axotomy performed on day 3 after birth.

It is concluded that treatment with the I2‐imidazoline selective receptor ligand LSL 60101 provokes morphological/biochemical changes in astroglia that are neuroprotective after neonatal axotomy.

British Journal of Pharmacology (2000) 130, 1767–1776; doi:10.1038/sj.bjp.0703485
Lipopolysaccharide enhances bradykinin‐induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells
Authors: Luo Shue‐Fen , Wang Chuan‐Chwan , Chiu Chi‐Tso , Chien Chin‐Sung , Hsiao Li‐Der , Lin Chien‐Huang , Yang Chuen‐Mao
Published: January 2005

Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen‐activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)‐induced inositol phosphates (IPs) accumulation and Ca2+ mobilization in canine cultured tracheal smooth muscle cells (TSMCs).

LPS stimulated phosphorylation of p42/p44 MAPK in a time‐ and concentration‐dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min‐incubation and the maximal effect was achieved with 100 μg ml−1 LPS.

Pretreatment of TSMCs with LPS potentiated BK‐induced IPs accumulation and Ca2+ mobilization. However, there was no effect on the IPs response induced by endothelin‐1, 5‐hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF‐BB enhanced BK‐induced IPs response.

These enhancements by LPS and PDGF‐BB might be due to an increase in BK B2 receptor density (Bmax) in TSMCs, characterized by competitive inhibition of [3H]‐BK binding using B1 and B2 receptor‐selective reagents.

The enhancing effects of LPS and PDGF‐BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF‐BB in TSMCs.

Furthermore, overexpression of dominant negative mutants, H‐Ras‐15A and Raf‐N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF‐BB, indicating that Ras and Raf may be required for activation of these kinases.

These results suggest that the augmentation of BK‐induced responses produced by LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

British Journal of Pharmacology (2000) 130, 1799–1808; doi:10.1038/sj.bjp.0703489
Functional, biochemical and molecular biological evidence for a possible β3‐adrenoceptor in human near‐term myometrium
Authors: Bardou M , Loustalot C , Cortijo J , Simon B , Naline E , Dumas M , Esteve S , Croci T , Chalon P , Frydman R , Sagot P , Manara L , Morcillo E J , Advenier C
Published: January 2005

The possible existence of a β3‐adrenoceptor (β3‐AR) in human near‐term myometrium was investigated by in vitro functional and biochemical studies and analysis of mRNA expression.

SR 59119A and SR 59104A and CGP 12177 (two selective agonists and a partial agonist, respectively, of the β3‐AR), salbutamol and terbutaline (β2‐AR agonists) each produced a concentration‐dependent relaxation of the myometrial spontaneous contractions. There were no differences in pD2 values for the relaxing potencies of terbutaline, salbutamol, CGP 12177 and SR 59119A. The rank order for their relaxing efficacies was SR 59119A>SR 59104A>terbutaline∼salbutamol∼CGP 12177 (Emax=52±7%, 42±12% and ∼ 30% respectively).

Propranolol, a β1‐ and β2‐AR antagonist, and ICI 118551, a β2‐AR antagonist (both at 0.1 μM), did not affect the SR 59119A‐induced relaxation whereas SR 59230A, a selective β3‐AR antagonist (1 μM), significantly reduced the maximal relaxing effect of SR 59119A.

SR 59119A and salbutamol induced a significant increase in cyclic AMP levels that was antagonized by SR 59230A but not by propranolol for SR 59119A, and by propranolol but not by SR 59230A for salbutamol.

The β3‐AR mRNA was positively expressed in myometrium preparations in a reverse transcription polymerase chain assay.

The results presented provide the first evidence for the existence of the β3‐AR subtype in human near‐term myometrium and suggest that the effects of SR 59119A might be mediated through an increase in cyclic AMP level.

British Journal of Pharmacology (2000) 130, 1960–1966; doi:10.1038/sj.bjp.0703491
Pharmacological characterization of volume‐sensitive, taurine permeable anion channels in rat supraoptic glial cells
Authors: Brès Vanessa , Hurbin Amandine , Duvoid Anne , Orcel Hélène , Moos Françoise C , Rabié Alain , Hussy Nicolas
Published: January 2005

To characterize the volume‐sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [3H]‐taurine efflux from acutely isolated SON.

Taurine release induced by hypotonic stimulus (250 mosmol l−1) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter.

The osmodependent release of taurine was blocked by a variety of Cl− channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4‐bromophenacyl bromide, mibefradil) known to block volume‐activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels.

Although the molecular identity of volume‐sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC3, phospholemman and VDAC1, and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC2.

In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume‐sensitive anion channel, which is likely to be formed by yet unidentified proteins.

British Journal of Pharmacology (2000) 130, 1976–1982; doi:10.1038/sj.bjp.0703492
Dual interaction of agmatine with the rat α2D‐adrenoceptor: competitive antagonism and allosteric activation
Authors: Molderings G J , Menzel S , Kathmann M , Schlicker E , Göthert M
Published: January 2005

In segments of rat vena cava preincubated with [3H]‐noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [3H]‐noradrenaline release, the EP3 prostaglandin receptor‐mediated and the α2D‐adrenoceptor‐mediated inhibition of evoked [3H]‐noradrenaline release was investigated.

Agmatine (0.1–10 μM) by itself was without effect on evoked [3H]‐noradrenaline release. In the presence of 10 μM agmatine, the prostaglandin E2(PGE2)‐induced EP3‐receptor‐mediated inhibition of [3H]‐noradrenaline release was not modified, whereas the α2D‐adrenoceptor‐mediated inhibition of [3H]‐noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine‐induced inhibition of [3H]‐noradrenaline release.

Agmatine concentration‐dependently inhibited the binding of [3H]‐clonidine and [3H]‐rauwolscine to rat brain cortex membranes (Ki values 6 μM and 12 μM, respectively). In addition, 30 and 100 μM agmatine increased the rate of association and decreased the rate of dissociation of [3H]‐clonidine resulting in an increased affinity of the radioligand for the α2D‐adrenoceptors.

[14C]‐agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [14C]‐agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine.

In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the α2D‐adrenoceptor and enhances the effects of α2‐adrenoceptor agonists probably by binding to an allosteric binding site of the α2D‐adrenoceptor which seems to be labelled by [14C]‐agmatine.

British Journal of Pharmacology (2000) 130, 1706–1712; doi:10.1038/sj.bjp.0703495
Comparison of zofenopril and lisinopril to study the role of the sulfhydryl‐group in improvement of endothelial dysfunction with ACE‐inhibitors in experimental heart failure
Authors: Buikema H , Monnink S H J , Tio R A , Crijns H J G M , De Zeeuw D , Van Gilst W H
Published: January 2005

We evaluated the role of SH‐groups in improvement of endothelial dysfunction with ACE‐inhibitors in experimental heart failure. To this end, we compared the vasoprotective effect of chronic treatment with zofenopril (plus SH‐group) versus lisinopril (no SH‐group), or N‐acetylcysteine (only SH‐group) in myocardial infarcted (MI) heart failure rats.

After 11 weeks of treatment, aortas were obtained and studied as ring preparations for endothelium‐dependent and ‐independent dilatation in continuous presence of indomethacin to avoid interference of vasoactive prostanoids, and the selective presence of the NOS‐inhibitor L‐NMMA to determine NO‐contribution.

Total dilatation after receptor‐dependent stimulation with acetylcholine (ACh) was attenuated (−49%, P<0.05) in untreated MI (n=11), compared to control rats with no‐MI (n=8). This was in part due to impaired NO‐contribution in MI (−50%, P<0.05 versus no‐MI). At the same time the capacity for generation of biologically active NO after receptor‐independent stimulation with A23187 remained intact.

Chronic treatment with n‐acetylcysteine (n=8) selectively restored NO‐contribution in total dilatation to ACh. In contrast, both ACE‐inhibitors fully normalized total dilatation to ACh, including the part mediated by NO (no significant differences between zofenopril (n=10) and lisinopril (n=8)).

Zofenopril, but not lisinopril, additionally potentiated the effect of endogenous NO after A23187‐induced release from the endothelium (+100%) as well as that of exogenous NO provided by nitroglycerin (+22%) and sodium nitrite (+36%) (for all P<0.05 versus no‐MI).

We conclude that ACE‐inhibition with a SH‐group has a potential advantage in improvement of endothelial dysfunction through increased activity of NO after release from the endothelium into the vessel wall. Furthermore, this is the first study demonstrating the selective normalizing effect of N‐actylcysteine on NO‐contribution to ACh‐induced dilatation in experimental heart failure.

British Journal of Pharmacology (2000) 130, 1999–2007; doi:10.1038/sj.bjp.0703498
A comparative study of the ability of calcitonin gene‐related peptide and adrenomedullin13–52 to modulate microvascular but not thermal hyperalgesia responses
Authors: Chu Duc Quyen , Choy ManKin , Foster Paul , Cao Thong , Brain Susan D
Published: January 2005

Calcitonin gene‐related peptide (CGRP), a neuropeptide, is a potent vasodilator. Adrenomedullin (ADM) is suggested to be produced by vascular cells in inflamed tissue. ADM shares some structural homology with CGRP. We have compared the ability of CGRP and ADM to modulate microvascular and thermal hyperalgesic responses in rat skin. Vasodilator activity was assessed by laser Doppler flowmetry, inflammatory oedema by the extravascular accumulation of intravenously‐injected labelled albumin, and neutrophil accumulation by tissue myeloperoxidase, in dorsal skin. Hyperalgesia was assessed by a thermal hyperalgesimeter in paw skin.

ADM (10–300 pmol) was 3 fold less potent than CGRP (3–100 pmol) as a direct vasodilator. CGRP (30 pmol) potentiated oedema formation induced by mediators of increased microvascular permeability, as expected (P<0.01). However, ADM (30–100 pmol) was without a potentiating effect, although ADM (300 pmol) was effective (P<0.01). By comparison ADM (100 pmol) potentiated neutrophil accumulation induced by interleukin‐1β (P<0.05), whereas CGRP (30 pmol) did not. No thermal hyperalgesia was observed to either CGRP or ADM, when given as single or repeated treatments.

Thus despite a dilator activity neither CGRP nor ADM appears to mediate hyperalgesic activity in the periphery. However ADM, like CGRP, has the ability to potentiate inflammatory oedema formation and, in addition, ADM can potentiate neutrophil accumulation.

ADM may, as suggested for CGRP, act as a modulator of the vascular phases of inflammation. The property of the two compounds of evoking differential microvascular responses and neutrophil accumulation may be due to differing mechanisms of action.

British Journal of Pharmacology (2000) 130, 1589–1596; doi:10.1038/sj.bjp.0703502
Localization and function of ET‐1 and ET receptors in small arteries post‐myocardial infarction: Upregulation of smooth muscle ETB receptors that modulate contraction
Authors: Gray Gillian A , Mickley Emma J , Webb David J , McEwan Pauline E
Published: January 2005

Endothelin‐1 (ET‐1) has been implicated as a mediator of increased vascular tone during development of heart failure post‐myocardial infarction (MI). In the present study, expression and pharmacology of ET‐1 and its receptors were studied in small mesenteric arteries from rats at 5 and 12 weeks after coronary artery ligation for induction of MI, or sham‐operation.

In vessels from sham‐operated and 5 week post‐MI rats preproET‐1mRNA, immunoreactive (ir) ET‐1, ETB receptor mRNA and irETB receptor were confined to the endothelium, while ETA receptor mRNA was distributed throughout the media. At 12 weeks post‐MI, preproET‐1 and irETA receptor localization was similar but ETB receptor mRNA and immunoreactivity were detectable in the media, as well as the endothelium.

The ET‐1 concentration‐response curve (CRC) was progressively shifted to the right in pressurized third generation mesenteric arteries from 5 and 12 week post‐MI rats relative to sham‐operated rats, with no change in the maximum. The ETA receptor antagonist BQ‐123 (10−6 M) induced a rightward shift of the ET‐1 CRC in all vessels. Desensitization of ETB receptors, by exposure to SRTX S6c (3×10−8 M), had no effect on the ET‐1 CRC in vessels from 5 week post‐MI or sham‐op rats but induced a leftward shift in vessels from 12 week post‐MI rats.

These results identify the endothelium as the primary site of ET‐1 synthesis in small arteries and the ETA receptor as mediating the effects of ET‐1 in these vessels. However, ETB receptor expression increases in vascular smooth muscle post‐MI and is linked to mechanisms that inhibit the contractile response to ET‐1.

British Journal of Pharmacology (2000) 130, 1735–1744; doi:10.1038/sj.bjp.0703503
Nitroparacetamol exhibits anti‐inflammatory and anti‐nociceptive activity
Authors: Al‐Swayeh O A , Futter L E , Clifford R H , Moore P K
Published: January 2005

Nitroparacetamol (NCX‐701) is a newly synthesized nitric oxide‐releasing derivative of paracetamol. Following i.p. administration, nitroparacetamol inhibits carrageenan‐induced hindpaw oedema formation (ED50, 169.4 μmol kg−1) and mechanical hyperalgesia (ED50, 156 μmol kg−1) in the rat. In contrast, the parent compound, paracetamol, exhibits no significant anti‐oedema activity in this model (ED50>1986 μmol kg−1, i.p.) and is markedly less potent than nitroparacetamol as an inhibitor of carrageenan‐mediated hyperalgesia (ED50, 411.6 μmol kg−1, i.p.). In a second model of nociception (inhibition of acetic acid induced abdominal constrictions in the mouse), nitroparacetamol administered orally (ED50, 24.8 μmol kg−1), was again considerably more potent than paracetamol (ED50, 506 μmol kg−1, p.o.). Thus, compared with paracetamol, nitroparacetamol not only exhibits augmented antinociceptive activity in both rat and mouse but, intriguingly, is also anti‐inflammatory over a similar dose range.

British Journal of Pharmacology (2000) 130, 1453–1456; doi:10.1038/sj.bjp.0703509
Ginsenoside‐induced relaxation of human bronchial smooth muscle via release of nitric oxide
Authors: Tamaoki J , Nakata J , Kawatani K , Tagaya E , Nagai A
Published: January 2005

Ginsenoside, an extract of Panax ginseng, is an essential constituent of anti‐asthmatic Chinese herbal medicine. To elucidate whether ginsenoside affects airway smooth muscle tone and, if so, what the mechanism of action is, we studied relaxant responses of human bronchial strips under isometric condition in vitro, and directly measured the release of nitric oxide (NO) by an amperometric sensor for this molecule.

Addition of ginsenoside relaxed the tissues precontracted with acetylcholine in a dose‐dependent manner, the maximal relaxation and the ginsenoside concentration required to produce 50% relaxation being 67±8% and 210±29 μg ml−1, respectively.

The relaxant responses to ginsenoside were inhibited by NG‐nitro‐L‐arginine methylester (L‐NAME) and removal of the epithelium, but not by NG‐nitro‐D‐arginine methylester (D‐NAME) or tetrodotoxin. This inhibitory effect of L‐NAME was reversed by L‐arginine but not by D‐arginine.

Addition of ginsenoside to the medium containing bronchial tissues dose‐dependently increased NO‐selective electrical current, and this effect was greatly attenuated by the epithelial removal or Ca2+‐free medium.

Ginsenoside also increased tissue cyclic GMP contents, an effect that was abolished in the presence of L‐NAME.

It is concluded that ginsenoside induces relaxation of human bronchial smooth muscle via stimulation of NO generation predominantly from airway epithelium and cyclic GMP synthesis. This action might account for the anti‐asthmatic effect of Panax ginseng.

British Journal of Pharmacology (2000) 130, 1859–1864; doi:10.1038/sj.bjp.0703511
No involvement of nicotinic receptors in the facilitation of acetylcholine outflow in mouse cortex in the presence of neostigmine and atropine
Authors: Iannazzo Lydia , Majewski Henryk
Published: January 2005

The role of nicotinic and muscarinic receptors in the modulation of acetylcholine release was studied using field stimulated mouse cortex slices incubated with [3H]‐choline.

Both acetylcholine (100 μM) and the cholinesterase inhibitor neostigmine (100 μM) inhibited the stimulation‐induced (S‐I) outflow of radioactivity but in the presence of atropine (0.3 μM) an enhancement was seen, which may be indicative of facilitatory nicotinic receptors.

Mecamylamine (100 μM) was unable to antagonize the enhancement seen in the presence of acetylcholine and atropine. The nicotinic agonist dimethylphenylpiperazinium (30 μM) did not facilitate S‐I outflow of radioactivity.

A range of nicotinic blockers had no effect on the enhancement seen in the presence of neostigmine and atropine, nor did indomethacin, the 5HT3 antagonist MDL 7222 nor the NMDA antagonist MK‐801.

The inability to block this effect suggests that nicotinic receptors are not involved. We postulate, at least for neostigmine, that the facilitation is an artefact because of the use of [3H]‐choline as a radiotracer whereby the efflux of radioactivity is enhanced because the radiolabelled acetylcholine is not metabolized to choline and therefore flows out of the tissue more readily.

British Journal of Pharmacology (2000) 130, 2008–2014; doi:10.1038/sj.bjp.0703512
Barnidipine block of L‐type Ca2+ channel currents in rat ventricular cardiomyocytes
Authors: Wegener Jörg W , Meyrer Hans , Rupp Johanna , Nawrath Hermann
Published: January 2005

The effects of barnidipine and nifedipine on L‐type Ca2+ current (ICa(L)) were investigated in ventricular cardiomyocytes from rats.

Both barnidipine and nifedipine reduced ICa(L) in a concentration and voltage dependent manner; the EC50 were 80 and 130 nM at a holding potential of −80 mV, respectively, and 18 and 6 nM at −40 mV, respectively.

Both drugs induced a leftward shift of the steady‐state inactivation curve of ICa(L).

Using a twin pulse protocol, the relationships between the amount of block of ICa(L) by either drug, seen during the second pulse, and the length of the first pulse were described by monoexponential functions reflecting onset of block, dependent on drug concentration. The onset of block by barnidipine was three times faster than that by nifedipine.

With both drugs, recovery of ICa(L) was 50 times slower than under control conditions and described by monoexponential functions reflecting offset of block (independent of drug concentration). The offset of block with barnidipine was three times slower than that with nifedipine.

The time constants of block and unblock of ICa(L) by both drugs were used to calculate binding and unbinding and to predict their effects at two frequencies.

It is suggested that barnidipine exhibits a higher affinity to the inactivated Ca2+ channel state as compared to nifedipine.

British Journal of Pharmacology (2000) 130, 2015–2023; doi:10.1038/sj.bjp.0703514
Sphingosine‐1‐phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro
Authors: Bischoff Angela , Czyborra Peter , Fetscher Charlotte , Meyer zu Heringdorf Dagmar , Jakobs Karl H , Michel Martin C
Published: January 2005

Sphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G‐protein‐coupled receptors, some of which were cloned and designated as Edg‐receptors.

Sphingolipid‐induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid‐induced elevations in intracellular Ca2+ concentration were measured in cultured rat aortic smooth muscle cells.

SPPC and SPP (0.1–100 μmol l−1) caused concentration‐dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC50 5.63±0.17 and Emax 49±3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC50 D‐erythro‐SPPC 5.69±0.08, L‐threo‐SPPC 5.31±0.06) and inhibited by pretreatment with pertussis toxin (Emax from 44±5 to 19±4%), by chelation of extracellular Ca2+ with EGTA and by nitrendipine (Emax from 40±6 to 6±1 and 29±6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (Emax from 87±3 to 70±4%).

SPP and SPPC caused transient increases in intracellular Ca2+ concentrations in rat aortic smooth muscle cells in a pertussis toxin‐sensitive manner.

Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca2+ concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin‐sensitive G‐proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

British Journal of Pharmacology (2000) 130, 1871–1877; doi:10.1038/sj.bjp.0703515
Pharmacological characterization of [3H]‐prostaglandin E2 binding to the cloned human EP4 prostanoid receptor
Authors: Davis T L , Sharif N A
Published: January 2005

Prostaglandin (PG) E2 (PGE2) is a potent prostanoid derived from arachidonic which can interact with EP1, EP2, EP3 and EP4 prostanoid receptor subtypes.

Recombinant human EP4 receptors expressed in human embryonic kidney (HEK‐293) cells were evaluated for their binding characteristics using [3H]‐PGE2 and a broad panel of natural and synthetic prostanoids in order to define their pharmacological properties.

[3H]‐PGE2 binding was optimal in 2‐[N‐Morpholino]ethanesulphonic acid (MES) buffer (pH 6.0) yielding 98±0.7% specific binding. The receptor displayed high affinity (Kd=0.72±0.12 nM; n=3) for [3H]‐PGE2 and interacted with a saturable number of binding sites (Bmax=6.21±0.84 pmol mg−1 protein).

In competition studies, PGE2 (Ki=0.75±0.03 nM; n=12) and PGE1 (Ki=1.45±0.24 nM; n=3) displayed high affinities, as did two derivatives of PGE1, namely 11‐deoxy‐PGE1 (Ki=1.36±0.34 nM) and 13,14‐dihydro‐PGE1 (Ki=3.07±0.29 nM).

Interestingly, synthetic DP receptor‐specific agonists such as BW245C (Ki=64.7±1.0 nM; n=3) and ZK118182 (Ki=425±42 nM; n=4), and the purported EP3 receptor‐specific ligand enprostil (Ki=43.1±4.4 nM), also displayed high affinity for the EP4 receptor.

Two known EP4 receptor antagonists were weak inhibitors of [3H]‐PGE2 binding akin to their known functional potencies, thus: AH23848 (Ki=2690±232 nM); AH22921 (Ki=31,800±4090 nM).

These studies have provided a detailed pharmacological characterization of the recombinant human EP4 receptor expressed in HEK‐293 cells.

British Journal of Pharmacology (2000) 130, 1919–1926; doi:10.1038/sj.bjp.0703525
The 5HT1B receptor agonist, CP‐93129, inhibits [3H]‐GABA release from rat globus pallidus slices and reverses akinesia following intrapallidal injection in the reserpine‐treated rat
Authors: Chadha Anita , Sur Cyrille , Atack John , Duty Susan
Published: January 2005

This study examined whether activation of 5HT1B receptors in the rodent globus pallidus (GP) could reduce GABA release in vitro and reverse reserpine‐induced akinesia in vivo.

Microdissected slices of GP from male Sprague Dawley rats (300–350 g) were preloaded with [3H]‐GABA. During subsequent superfusion, 4 min fractions were collected for analysis of release. The effects of the 5HT1B receptor agonist, 3‐(1,2,5,6‐tetrahydropyrid‐4‐yl)pyrrolo[3,2‐b]pyrid‐5‐one (CP‐93129), on 25 mM KCl‐evoked release were examined using a standard dual stimulation paradigm.

Male Sprague Dawley rats (270–290 g), stereotaxically cannulated above the GP, were rendered akinetic by injection of reserpine (5 mg kg−1 s.c.). Eighteen hours later, the rotational behaviour induced by unilateral injection of CP‐93129 was examined.

CP‐93129 (0.6–16.2 μM) produced a concentration‐dependent inhibition of 25 mM KCl‐evoked [3H]‐GABA release reaching a maximum inhibition of 52.5±4.5%. The effect of a submaximal concentration of CP‐93129 (5.4 μM) was fully inhibited by the 5HT1B receptor antagonist, isamoltane (10 μM).

Following intrapallidal injection, CP‐93129 (30–330 nmol in 0.5 μl) produced a dose‐dependent increase in net contraversive rotations reaching a maximum of 197±32 rotations in 240 min at 330 nmol. Pre‐treatment with isamoltane (10 nmol in 1 μl) inhibited the effects of a submaximal dose of CP‐93129 (220 nmol) by 84±6%.

These data suggest that at least some 5HT1B receptor function as heteroreceptors in the GP, reducing the release of GABA. Moreover, CP‐93129‐mediated activation of these receptors in the GP provides relief of akinesia in the reserpine‐treated rat model of PD.

British Journal of Pharmacology (2000) 130, 1927–1932; doi:10.1038/sj.bjp.0703526
Characterization of changes in mechanical responses to histamine in omental resistance arteries in pre‐eclampsia
Authors: Suzuki Yoshikatsu , Saitoh Michihiro , Suzumori Kaoru , Kajikuri Junko , Itoh Takeo
Published: January 2005

Changes in the effect of histamine on the smooth muscle of resistance arteries in pre‐eclampsia were investigated by measuring isometric contractions in endothelium‐denuded strips of omental resistance arteries from pre‐eclamptic and normotensive pregnant women (pregnancy‐term matched).

Histamine (0.03–1 μM) caused concentration‐dependent relaxation of the contraction induced by 9,11‐epithio‐11,12‐methano‐thromboxane A2 (STA2) in strips from both groups. Sensitivity (for pre‐eclampsia: pD2=6.66±0.04, n=5 and for normotensive pregnant women: pD2=7.07±0.03, n=10, P<0.001) was lower and the maximum response (90.6±0.6% vs 95.5±1.1%, P<0.05) was smaller in strips from pre‐eclamptic women.

Although 8‐bromoadenosine‐3′, 5′‐cyclic monophosphorothioate (Sp‐isomer: Sp‐8‐Br‐cAMPS, 0.1–0.3 mM), a phosphodiesterase (PDE)‐resistant activator of adenosine‐3′,5′‐cyclic monophosphate (cyclic AMP)‐dependent protein kinase, concentration‐dependently attenuated the contraction induced by STA2 in strips from both groups, the sensitivity (for pre‐eclampsia: pD2=3.68±0.04, n=5 and for normotensive pregnant women: 3.94±0.09, n=7, P=0.02) was lower and the maximum response (64.2±2.4% vs 74.9±4.4%, P<0.05) was smaller in pre‐eclampsia.

In β‐escin‐skinned strips, the pD2 value for the contraction‐inducing effect of Ca2+ did not differ significantly between the two groups (for pre‐eclampsia, n=6; for normotensive pregnant women, n=6).

Thus, omental resistance arteries from human subjects with pre‐eclampsia showed (i) a weaker H2‐receptor‐mediated relaxation to histamine and (ii) a weaker cyclic AMP‐analogue‐induced relaxation, suggesting that the reduced action of histamine may be partly due to a decreased effect of cyclic AMP.

British Journal of Pharmacology (2000) 131, 37–42; doi:10.1038/sj.bjp.0703529
The p38 MAP kinase inhibitor SB203580 enhances nuclear factor‐kappa B transcriptional activity by a non‐specific effect upon the ERK pathway
Authors: Birkenkamp Kim U , Tuyt Leonore M L , Lummen Chantal , Wierenga Albertus T J , Kruijer Wiebe , Vellenga Edo
Published: January 2005

In the present study we investigated a possible role for the p38 mitogen‐activated protein (MAP) kinase pathway in mediating nuclear factor‐kappa B (NF‐κB) transcriptional activity in the erythroleukaemic cell line TF‐1.

TF‐1 cells stimulated with the phosphatase inhibitor okadaic acid (OA) demonstrated enhanced NF‐κB and GAL4p65‐regulated transcriptional activity which was associated with elevated p38 phosphorylation. However, pretreatment with the p38 MAPK specific inhibitor SB203580 (1 μM) or overexpression of kinase‐deficient mutants of MKK3 or MKK6 did not affect OA‐enhanced NF‐κB transcriptional potency, as determined in transient transfection assays. In fact, 5 and 10 μM SB203580 enhanced rather than inhibited NF‐κB‐mediated promoter activity by 2 fold, which was independent of phosphorylation of the p65 subunit.

The SB203580‐mediated increase in NF‐κB transcriptional activity was associated with enhanced phosphorylation of extracellular signal‐regulated kinase (ERK)1/2 and c‐Jun N‐terminal kinase (JNK), but not p38 kinase.

Overexpression of kinase‐deficient mutants belonging to the ERK1/2, JNK, and p38 pathways showed that only dominant‐negative Raf‐1 abrogated SB203580‐enhanced NF‐κB activity. This would implicate the involvement of the ERK1/2 pathway in the enhancing effects of SB203580 on NF‐κB‐mediated gene transcription.

This study demonstrates that the p38 MAP kinase pathway is not involved in the OA‐induced activation of NF‐κB. SB203580 at higher concentrations activates the ERK pathway, which subsequently enhances NF‐κB transcriptional activity.

British Journal of Pharmacology (2000) 131, 99–107; doi:10.1038/sj.bjp.0703534
Probenecid interferes with renal oxidative metabolism: A potential pitfall in its use as an inhibitor of drug transport
Authors: Masereeuw Rosalinde , Van Pelt Ard P , Van Os Sandra H G , Willems Peter H G M , Smits Paul , Russel Frans G M
Published: January 2005

The anionic drug probenecid has been traditionally used as an inhibitor of renal organic anion transport. More recently the drug was found to inhibit organic cation transport as well, and it is used to retain intracellularly loaded fluorophores. In these investigations it is implicitly assumed that probenecid performs its activity through competition for transport. Here we studied the possibility that probenecid provokes its effect through inhibition of cellular oxidative metabolism.

Oxygen consumption was measured in isolated rat kidney cortex mitochondria. At concentrations of 1 mM or higher, probenecid increased the resting state (state 4) and decreased the ADP‐stimulated respiration (state 3). A complete loss in respiratory control was observed at 10 mM probenecid.

After incubating isolated rat kidney proximal tubular cells (PTC) for 30 min with probenecid a concentration‐dependent reduction in ATP content was observed, which was significant at concentrations of 1 mM and higher. Using digital image fluorescence microscopy the membrane potential in PTC was measured with bisoxonol. The mitochondrial effects of probenecid were paralleled by a depolarization of the plasma membrane, immediately after drug addition.

All events are likely to be a result of membrane disordering due to the lipophilic character of probenecid, and may explain, at least in part, the various inhibitory effects found for the drug. We recommend to be cautious with applying probenecid in cellular research.

British Journal of Pharmacology (2000) 131, 57–62; doi:10.1038/sj.bjp.0703541
Inhibition of neuronal M2 muscarinic receptor function in the lungs by extracellular nitric oxide
Authors: Golkar Laleh , Yarkony Kathryn A , Fryer Allison D
Published: January 2005

These experiments were carried out to test whether neuronal M2 muscarinic receptor function in the lungs is affected by nitric oxide (NO) and whether the source of the NO is epithelial or neuronal.

In pathogen free, anaesthetized guinea‐pigs, the muscarinic agonist pilocarpine inhibited vagally induced bronchoconstriction demonstrating functional neuronal M2 muscarinic receptors. In the presence of the NO donor, 3‐morpholino‐sydnonimine (SIN‐1), pilocarpine no longer inhibited vagally induced bronchoconstriction. In contrast, inhibiting endogenous NO with NG‐monomethyl‐L‐arginine methyl ester (L‐NMMA) did not affect the ability of pilocarpine to decrease vagally induced bronchoconstriction.

In isolated tracheas, pilocarpine inhibited contractions induced by electrical field stimulation demonstrating that neuronal M2 muscarinic receptors function in vitro. As in the anaesthetized guinea‐pigs, SIN‐1 shifted the pilocarpine dose response curve to the right, demonstrating decreased neuronal M2 receptor function. However, in vitro, L‐NMMA shifted the pilocarpine dose response curve to the left, demonstrating that endogenous NO was inhibiting the ability of the M2 receptors to decrease acetylcholine (ACh) release.

Both haemoglobin (Hb), which scavenges NO, and epithelial removal also shifted the pilocarpine dose response curve to the left, demonstrating that the NO inhibiting neuronal M2 receptor function was extracellular and probably of epithelial origin.

In conclusion, extracellular NO appears to inhibit the ability of the M2 receptors to decrease ACh release from the parasympathetic nerves in the lungs in vivo and in vitro in pathogen free guinea‐pigs. However, while the neuronal M2 receptors will respond to NO (from SIN‐1) in vivo, there does not appear to be an endogenous source of NO since L‐NMMA had no effect in vivo.

British Journal of Pharmacology (2000) 131, 312–318; doi:10.1038/sj.bjp.0703542
Mechanisms of the thapsigargin‐induced Ca2+ entry in in situ endothelial cells of the porcine aortic valve and the endothelium‐dependent relaxation in the porcine coronary artery
Authors: Kuroiwa‐Matsumoto Mari , Hirano Katsuya , Ahmed Abu , Kawasaki Junya , Nishimura Junji , Kanaide Hideo
Published: January 2005

The mechanisms of the thapsigargin (TG)‐induced capacitative Ca2+ entry in in situ endothelial cells and its role in the regulation of arterial tone were investigated using front‐surface fluorimetry and fura‐2‐loaded strips of porcine aortic valve and coronary artery.

In the presence of extracellular Ca2+, TG induced an initial rapid and a subsequent sustained elevation of cytosolic Ca2+ concentration ([Ca2+]i) in valvular strips. In the absence of extracellular Ca2+, TG induced only a transient increase in [Ca2+]i.

The TG‐induced sustained elevation of [Ca2+]i in endothelial cells was inhibited completely by 1 mM Ni2+ and partly by 10 μM econazole and 30 μM ML‐9, but not by 900 ng ml−1 pertussis toxin or 100 μM wortmannin. Therefore, cytochrome P450 and protein phosphorylation are suggested to be involved in the TG‐induced Ca2+ influx in in situ endothelial cells.

TG induced an endothelium‐dependent large relaxation consisting of an initial and a late sustained relaxation in coronary arterial strip precontracted with U46619 (a thromboxane A2 analogue). Indomethacin alone had no effect, while indomethacin plus Nω‐nitro‐L‐arginine (L‐NOARG) markedly inhibited the sustained phase and slightly inhibited the initial phase of the TG‐induced relaxation.

TG induced a smaller but sustained relaxation during the 40 mM K+‐induced precontraction than that seen during the U46619‐induced precontraction. This relaxation was completely abolished by the pretreatment with indomethacin plus L‐NOARG.

In conclusion, both nitric oxide (NO) and endothelium‐derived hyperpolarizing factor were suggested to mediate the TG‐induced relaxation, while NO plays a major role in the sustained relaxation. The TG‐induced sustained [Ca2+]i elevation in endothelial cells was thus suggested to be mainly linked to the sustained production of NO.

British Journal of Pharmacology (2000) 131, 115–123; doi:10.1038/sj.bjp.0703548