bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneous mechanisms of endothelium‐dependent relaxation for thrombin and peptide activators of protease‐activated receptor‐1 in porcine isolated coronary artery
Authors: Hamilton Justin R , Cocks Thomas M
Published: January 2005

Mechanisms of protease‐activated receptor‐1 (PAR1)‐ and PAR2‐induced relaxation were investigated in pre‐contracted porcine coronary artery ring preparations.

Thrombin (0.01–0.3 u ml−1) and the PAR1‐activating peptide SFLLRN (0.1–10 μM) caused concentration‐ and endothelium‐dependent relaxation. pEC50s (−log u ml−1 for enzymes, −log M for peptides) and maximum relaxations (Rmax, %) for thrombin were 1.8±0.1 and 93.5±2.8% respectively, and for SFLLRN 6.8±0.1 and 90.8±1.3%. Similar concentration‐ and endothelium‐dependent relaxations occurred with trypsin (pEC50 2.3±0.2; Rmax 94.1±1.9%) and the PAR2‐activating peptide SLIGRL (pEC50 6.5±0.2; Rmax 92.4±1.6%).

Relaxations to thrombin, SFLLRN, trypsin and SLIGRL were significantly inhibited (P<0.05) to similar extents by the nitric oxide (NO) synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG; 100 μM) and the NO scavenger oxyhaemoglobin (20 μM), both separately and in combination.

In the presence of the L‐type voltage‐operated calcium channel (L‐VOCC) inhibitor nifedipine (0.3 μM), K+ (67 mM) abolished the L‐NOARG‐resistant relaxations to thrombin, SFLLRN, trypsin and SLIGRL. However, nifedipine alone significantly (P<0.05) reduced the pEC50 (1.5±0.1) and Rmax (77.5±7.0%) for thrombin but had no effect on relaxations to SFLLRN, trypsin or SLIGRL. Furthermore, L‐NOARG‐resistant relaxations to thrombin were abolished by nifedipine, whereas relaxations to SFLLRN, trypsin or SLIGRL were not further inhibited by combined treatment with nifedipine and L‐NOARG, than they were with L‐NOARG treatment alone.

Similar selective inhibition of the L‐NOARG‐resistant relaxation to thrombin, but not SFLLRN, occurred with verapamil (1 μM) and diltiazem (3 μM).

Our results suggest heterogeneous mechanisms in the NO‐independent relaxation to thrombin and peptide activators of PAR1 in the porcine coronary artery.

British Journal of Pharmacology (2000) 130, 181–188; doi:10.1038/sj.bjp.0703146
Oxidative stress and S‐nitrosylation of proteins in cells
Authors: Beltrán Belén , Orsi Antonia , Clementi Emilio , Moncada Salvador
Published: January 2005

The effect of prolonged exposure to nitric oxide on enzymes involved in cell metabolism was investigated in T lymphocyte‐derived Jurkat and L929 fibroblast human cell lines using a constant concentration of nitric oxide (1.5 μM) released by the nitric oxide donor DETA‐NO (0.5 mM).

Nitric oxide inhibited immediately the respiration of the cells acting reversibly at complex IV. With time, the inhibition became progressively persistent, i.e. not reversed by trapping of nitric oxide with oxyhaemoglobin, and was preceded by a decrease in the concentration of the intracellular reduced glutathione. This persistent effect of nitric oxide on respiration was due to inhibition of complex I activity which could be reversed by addition of reduced glutathione or by cold light, suggesting that it was due to S‐nitrosylation of thiols necessary for the activity of the enzyme.

The activity of other enzymes also known to be susceptible to inhibition by S‐nitrosylation, i.e. glyceraldehyde‐3‐phosphate dehydrogenase and glutathione reductase, was progressively decreased by exposure to nitric oxide with a similar time course to that observed for the inhibition of complex I. Furthermore, inhibition of these enzymes only occurred when the concentrations of reduced glutathione had previously fallen and could be prevented by increasing the intracellular concentrations of reduced glutathione.

Our results suggest that S‐nitrosylation of different enzymes by nitric oxide may occur only if the reducing potential of the cells is impaired.

British Journal of Pharmacology (2000) 129, 953–960; doi:10.1038/sj.bjp.0703147
Bioactive β‐bend structures for the antagonist hα CGRP8–37 at the CGRP1 receptor of the rat pulmonary artery
Authors: Wisskirchen F M , Doyle P M , Gough S L , Harris C J , Marshall I
Published: January 2005

The aim of this study was to determine β‐bend structures and the role of the N‐ and C‐terminus in the antagonist hα CGRP8–37 at the rat pulmonary artery CGRP receptor mediating hα CGRP relaxation.

Hα CGRP8–37 Pro16 (10−6 M), with a bend‐biasing residue (proline) at position 16, did not antagonize hα CGRP responses, while a structure‐conserving amino acid (alanine16) at the same position retained antagonist activity (apparent pKB 6.6±0.1; 10−6 M). Hα CGRP8–37 Pro19 (10−6 M), with proline at position 19 was an antagonist (apparent pKB 6.9±0.1).

Incorporation of a β‐bend forcing residue, BTD (beta‐turn dipeptide), at positions 19 and 20 in hα CGRP8–37 (10−6 M) antagonized hα CGRP responses (apparent pKB 7.2±0.2); and BTD at positions 19,20 and 33,34 within hα CGRP8–37 was a competitive antagonist (pA2 7.2; Schild plot slope 1.0±0.1).

Hα CGRP8–37 analogues, substituted at the N‐terminus by either glycine8 or des‐NH2 valine8 or proline8 were all antagonists (apparent pKB 6.9±0.1; (10−6 M), 7.0±0.1 (10−6 M), and pA2 7.0 (slope 1.0±0.2), respectively); while replacements by proline8 together with glutamic acid10,14 in hα CGRP8–37 (10−6 M) or alanine amide37 at the C‐terminus of hα CGRP8–37 (10−5 M) were both inactive compounds.

In conclusion, possible bioactive structures of hα CGRP8–37 include two β‐bends (at 18–21 and 32–35), which were mimicked by BTD incorporation. Within hα CGRP8–37, the N‐terminus is not essential for antagonism while the C‐terminus may interact directly with CGRP1 receptors in the rat pulmonary artery.

British Journal of Pharmacology (2000) 129, 1049–1055; doi:10.1038/sj.bjp.0703152
Regional modulation of cyclic nucleotides by endothelin‐1 in rat pulmonary arteries: direct activation of Gi2‐protein in the main pulmonary artery
Authors: Mullaney Ian , Vaughan Diane M , MacLean Margaret R
Published: January 2005

The ability of endothelin‐1 (ET‐1) to modulate the cyclic nucleotides, guanosine 3′ 5′ cyclic monophosphate (cyclic GMP) and adenosine 3′ 5′ cyclic monophosphate (cyclic AMP) was assessed in the main elastic pulmonary elastic artery (4–5 mm i.d.) and the small muscular pulmonary arteries (150–200 μm i.d.) of the rat.

ET‐1 caused an increase in cyclic GMP in the larger vessels but had no effect in the smaller arteries. The increase in cyclic GMP was not dependent on an intact endothelium and was inhibited by the ETA‐receptor antagonist FR139137 (1 μM).

ET‐1 caused a decrease in cyclic AMP in the main pulmonary arteries, an effect that was partially blocked by FR139317 but not influenced by the ETB‐receptor antagonist BQ‐788 (1 μM) or removal of the vascular endothelium. In contrast, ET‐1 caused an increase in cyclic AMP in the small vessels, an effect that was blocked by BQ‐788 but unaffected by FR139317.

In the main pulmonary arteries, ET‐1 caused enhanced incorporation of radiolabelled ADP‐ribose by cholera toxin into Gi2 in the main pulmonary artery, an indicator of its receptor‐mediated activation.

In summary, we have shown that in the small muscular pulmonary artery of the rat, (where ETB mediated vasoconstriction prevails), there is an ETB‐mediated increase in cyclic AMP with no net effect on cyclic GMP levels. In the large arteries, (where vasoconstriction is mediated via the ETA receptor), there is an ETA‐mediated increase in cyclic GMP (endothelium independent) and an ETA‐mediated (endothelium independent) decrease in cyclic AMP.

British Journal of Pharmacology (2000) 129, 1042–1048; doi:10.1038/sj.bjp.0703153
The effect of long‐term streptozotocin‐induced diabetes on contractile and relaxation responses of coronary arteries: selective attenuation of CGRP‐induced relaxations
Authors: Sheykhzade Majid , Dalsgaard Grethe T , Johansen Tue , Nyborg Niels C Berg
Published: January 2005

This study investigates the effect of partially metabolic controlled long‐term (34 weeks) streptozotocin (STZ)‐induced diabetes on relaxation and contractile responses of isolated coronary arteries to seven different vasoactive agents.

The average fasting and non‐fasting blood glucose concentrations (mM) were significantly elevated in STZ‐induced diabetic rats (P<0.0001; 10.4±0.4 and 16.6±1.1, n=15) compared to those (4.3±0.03 and 4.7±0.18, n=11) in age‐matched controls. The level of glycated haemoglobin (HbA1) was also significantly (P<0.0001) increased in STZ‐induced diabetic rats. In STZ‐induced diabetic rats, the HbA1 levels were significantly correlated with the non‐fasting blood glucose concentrations (r=0.76; P=0.003; n=13). In both groups, there was no significant correlation between the HbA1 levels and maximal responses or sensitivities to the vasoactive agents.

The maximal relaxation induced by rat‐αcalcitonin gene‐related peptide (rat‐αCGRP) was significantly attenuated in the coronary arteries of STZ‐induced diabetic rats (P<0.05; 40±7%, n=15) compared to that in age‐matched controls (63±3%, n=11). However, there was no significant difference in the sensitivity to rat‐αCGRP between the two groups.

There was no significant difference in either maximal response or sensitivity to any of the six other vasoactive agents between STZ‐ induced diabetic rats (n=15) and age‐matched controls (n=11).

Our results show that partially metabolic controlled long‐term (34 weeks) STZ‐induced diabetes causes a selective depression of rat‐αCGRP‐induced relaxation in the intramural coronary arteries of Wistar rats.

British Journal of Pharmacology (2000) 129, 1212–1218; doi:10.1038/sj.bjp.0703159
BIIE0246, a potent and highly selective non‐peptide neuropeptide Y Y2 receptor antagonist
Authors: Dumont Yvan , Cadieux Alain , Doods Henri , Pheng Leng Hong , Abounader Roger , Hamel Edith , Jacques Danielle , Regoli Domenico , Quirion Rémi
Published: January 2005

BIIE0246, a newly synthesized non‐peptide neuropeptide Y (NPY) Y2 receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [125I]PYY3–36 binding sites in HEK293 cells transfected with the rat Y2 receptor cDNA, and in rat brain and human frontal cortex membrane homogenates.

Interestingly, in rat brain homogenates while NPY, C2‐NPY and PYY3–36 inhibited all specific [125I]PYY3–36 labelling, BIIE0246 failed to compete for all specific binding suggesting that [125I]PYY3–36 recognized, in addition to the Y2 subtype, another population of specific NPY binding sites, most likely the Y5 receptor.

Quantitative receptor autoradiographic data confirmed the presence of [125I]PYY3–36/BIIE0246‐sensitive (Y2) and‐insensitive (Y5) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation.

In the rat vas deferens and dog saphenous vein (two prototypical Y2 bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration‐response curves with pA2 values of 8.1 and 8.6, respectively. In the rat colon (a Y2/Y4 bioassay), BIIE0246 (1 μM) completely blocked the contraction induced by PYY3–36, but not that of [Leu31,Pro34]NPY (a Y1, Y4 and Y5 agonist) and hPP (a Y4 and Y5 agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y1 in vitro bioassays.

Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y2 receptor subtype. It should prove most useful to establish further the functional role of the Y2 receptor in the organism.

British Journal of Pharmacology (2000) 129, 1075–1088; doi:10.1038/sj.bjp.0703162
Differential effects of 17β‐estradiol and testosterone on the contractile responses of porcine coronary arteries
Authors: Teoh Hwee , Quan Adrian , Leung Susan W S , Man Ricky Y K
Published: January 2005

We investigated the effects of short‐term exposure to physiological levels of 17β‐estradiol and testosterone on vasocontractile responses in porcine coronary artery rings.

Concentration‐response curves to endothelin‐1, 5‐hydroxytryptamine, the thromboxane analogue U46619 and KCl were constructed in endothelium‐intact and endothelium‐disrupted artery rings.

Thirty minutes exposure to 17β‐estradiol (1 and 30 nM) significantly attenuated vasoconstriction to endothelin‐1, 5‐hydroxytryptamine and U46619. Conversely, the same concentrations of testosterone significantly potentiated responses elicited by these contractile agents. These inhibitory effects of 17β‐estradiol and enhancing actions of testosterone on contractions were endothelium‐independent. KCl‐mediated contractions were unaffected by the presence of either sex hormones.

The oestrogen receptor antagonists, tamoxifen (10 μM) and ICI 182,780 (10 μM), were unable to reverse the inhibitory influence 1 nM 17β‐estradiol had on the agonist‐mediated contractile responses. Similarly, the androgen receptor antagonists, flutamide (10 μM) and cyproterone acetate (10 μM), failed to affect the potentiating activities of 1 nM testosterone.

The alteration in vasoconstrictive responses observed following acute exposure to either 1 nM 17β‐estradiol and 1 nM testosterone were apparent even in the presence of the protein synthesis inhibitor cycloheximide (10 μM) and the transcription inhibitor actinomycin D (10 μM).

In conclusion, we report a unique type of sex hormone action on the coronary vasculature. These events occur at low nanomolar concentrations of 17β‐estradiol and testosterone, are insensitive to conventional sex hormone receptor antagonists, are not blocked by de novo protein synthesis inhibitors and have rapid time‐courses that are uncharacteristic of classical genomic activities.

British Journal of Pharmacology (2000) 129, 1301–1308; doi:10.1038/sj.bjp.0703164
The role of lipopolysaccharide injected systemically in the reactivation of collagen‐induced arthritis in mice
Authors: Yoshino Shin , Ohsawa Motoyasu
Published: January 2005

We investigated the role of bacterial lipopolysaccharide (LPS) in the reactivation of autoimmune disease by using collagen‐induced arthritis (CIA) in mice in which autoimmunity to the joint cartilage component type II collagen (CII) was involved.

CIA was induced by immunization with CII emulsified with complete Freund's adjuvant at the base of the tail (day 0) followed by a booster injection on day 21. Varying doses of LPS from E. coli were i.p. injected on day 50.

Arthritis began to develop on day 25 after immunization with CII and reached a peak on day 35. Thereafter, arthritis subsided gradually but moderate joint inflammation was still observed on day 50. An i.p. injection of LPS on day 50 markedly reactivated arthritis on a dose‐related fashion. Histologically, on day 55, there were marked oedema of synovium which had proliferated by the day of LPS injection, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells. The reactivation of CIA by LPS was associated with increases in anti‐CII IgG and IgG2a antibodies as well as various cytokines including IL‐12, IFN‐γ, IL‐1β, and TNF‐α. LPS from S. enteritidis, S. typhimurium, and K. neumoniae and its component, lipid A from E. coli also reactivated the disease. Polymyxin B sulphate suppressed LPS‐ or lipid A‐induced reactivation of CIA.

These results suggest that LPS may play an important role in the reactivation of autoimmune joint inflammatory diseases such as rheumatoid arthritis in humans.

British Journal of Pharmacology (2000) 129, 1309–1314; doi:10.1038/sj.bjp.0703166
Enhancement of noradrenaline release by angiotensin II and bradykinin in mouse atria: evidence for cross‐talk between Gq/11 protein‐ and Gi/o protein‐coupled receptors
Authors: Cox S L , Schelb V , Trendelenburg A U , Starke K
Published: January 2005

The interaction between α2‐autoreceptors and receptors for angiotensin (AT1) and bradykinin (B2) was studied in mouse isolated atria. The preparations were labelled with [3H]‐noradrenaline and then superfused with desipramine‐containing medium and stimulated electrically.

Angiotensin II (10−11–10−7 M), angiotensin III (10−10–10−6 M) and bradykinin (10−11–10−7 M) enhanced the evoked overflow of tritium when preparations were stimulated with conditions that led to marked α2‐autoinhibition (120 pulses at 3 Hz), but not when stimulated with conditions that led to little α2‐autoinhibition (20 pulses at 50 Hz).

Blockade of α‐adrenoceptors by phentolamine (1 or 10 μM) reduced or abolished the effect of angiotensin II and bradykinin on the overflow response to 120 pulses at 3 Hz.

Addition of the δ‐opioid agonist [D‐Ser2]‐leucine enkephalin‐Thr (DSLET, 0.1 μM), or of neuropeptide Y (0.1 μM), together with phentolamine, restored the effect of angiotensin II and bradykinin.

The β‐adrenoceptor agonist terbutaline (10−9–10−4 M) enhanced the evoked overflow of tritium irrespective of the degree of autoinhibition.

The experiments show that (i) a marked prejunctional facilitatory effect of angiotensin and bradykinin in mouse isolated atria requires prejunctional α2‐autoinhibition; (ii) in the absence of α2‐autoinhibition, activation of other prejunctional Gi/o protein‐coupled reeptors, namely opioid and neuropeptide Y receptors, restores a marked effect of angiotensin II and bradykinin; and (iii) the facilitatory effect of terbutaline is not dependent upon the degree of α2‐autoinhibition. The findings indicate that the major part of the release‐enhancing effect elicited through prejunctional Gq/11 protein‐coupled receptors is due to disruption of an ongoing, α2‐autoreceptor‐triggered Gi/o protein mediated inhibition.

British Journal of Pharmacology (2000) 129, 1095–1102; doi:10.1038/sj.bjp.0703167
Characterization of [Nphe1]nociceptin(1‐13)NH2, a new selective nociceptin receptor antagonist
Authors: Calo' Girolamo , Guerrini Remo , Bigoni Raffaella , Rizzi Anna , Marzola Giuliano , Okawa Hirobumi , Bianchi Clementina , Lambert David G , Salvadori Severo , Regoli Domenico
Published: January 2005

Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a specific G‐protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identification and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe1]nociceptin(1‐13)NH2 acts as the first truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity.

[Nphe1]nociceptin(1‐13)NH2 binds selectively to recombinant nociceptin receptors expressed in Chinese hamster ovary (CHO) cells (pKi 8.4) and competitively antagonizes the inhibitory effects of nociceptin (i) on cyclic AMP accumulation in CHO cells (pA2 6.0) and (ii) on electrically evoked contractions in isolated tissues of the mouse, rat and guinea‐pig with pA2 values ranging from 6.0 to 6.4.

[Nphe1]nociceptin(1‐13)NH2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe1]nociceptin(1‐13)NH2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine‐induced analgesia.

Collectively our data indicate that [Nphe1]nociceptin(1‐13)NH2, acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

British Journal of Pharmacology (2000) 129, 1183–1193; doi:10.1038/sj.bjp.0703169
Neuroleptics ameliorate phencyclidine‐induced impairments of short‐term memory
Authors: Schroeder Ulrike , Schroeder Helmut , Schwegler Herbert , Sabel Bernhard A
Published: January 2005

Phencyclidine (PCP), a non‐competitive NMDA‐receptor antagonist, is able to induce schizophrenia‐like symptoms in animals and in humans. It is known that schizophrenic patients have deficits in memory processes.

Therefore, it was investigated whether subchronic pulsatile or continuous application of 5.0 mg kg−1 PCP over 5 days induce short‐term memory deficits in holeboard learning and the action of two different neuroleptics on this behavioural test.

First, an impairment in the holeboard task was described when the animals were tested 24 h after the last application but not after 15 min or 1 h after the last injection. Secondly, the influence of haloperidol and risperidone on the PCP‐induced short‐term memory changes was tested.

The combined application of PCP and risperidone led to a complete antagonism of the short‐term deficits, but the combined treatment with haloperidol was accompanied by a partial abolishment of the PCP‐induced deficits.

PCP led to an upregulation of the glutamate binding sites in striatum and nucleus accumbens whereas the D2 binding sites were reduced in striatum. The D1 binding sites seem to be unchanged. The receptor protein expression of glutamate receptors mGluR1, GluR2, GluR5/7 and NMDAR1 were not modified in response to PCP treatment.

The determination of a subpopulation of GABAergic interneurons shows a decrease of the cells within the CA3 of the hippocampal formation.

These findings indicate that PCP induced impairments in short term memory can be detected by holeboard learning and may provide an interesting tool for the search of new neuroleptics.

British Journal of Pharmacology (2000) 130, 33–40; doi:10.1038/sj.bjp.0703171
Role of endothelial cell hyperpolarization in EDHF‐mediated responses in the guinea‐pig carotid artery
Authors: Quignard J ‐F , Félétou M , Edwards G , Duhault J , Weston A H , Vanhoutte P M
Published: January 2005

Experiments were performed to identify the potassium channels involved in the acetylcholine‐induced endothelium‐dependent hyperpolarization of the guinea‐pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly‐dissociated smooth muscle cells using patch clamp techniques.

In single myocytes, iberiotoxin (0.1 μM)‐, charybdotoxin (0.1 μM)‐, apamin (0.5 μM)‐ and 4‐aminopyridine (5 mM)‐sensitive potassium currents were identified indicating the presence of large‐ and small‐conductance calcium‐sensitive potassium channels (BKCa and SKCa) as well as voltage‐dependent potassium channels (KV). Charybdotoxin and iberiotoxin inhibited the same population of BKCa but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4‐aminopyridine (0.1–25 mM) induced a concentration‐dependent inhibition of KV without affecting the iberiotoxin‐ or the apamin‐sensitive currents.

In isolated arteries, both the endothelium‐dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4‐aminopyridine.

These results indicate that in the vascular smooth muscle cells of the guinea‐pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4‐aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium‐dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required.

British Journal of Pharmacology (2000) 129, 1103–1112; doi:10.1038/sj.bjp.0703175
Influence of bilirubin and other antioxidants on nitrergic relaxation in the pig gastric fundus
Authors: Colpaert E E , Lefebvre R A
Published: January 2005

The influence of several antioxidants (bilirubin, urate, ascorbate, α‐tocopherol, glutathione (GSH), Cu/Zn superoxide dismutase (SOD) and the manganese SOD mimic EUK‐8) on nitrergic relaxations induced by either exogenous nitric oxide (NO; 10−5 M) or electrical field stimulation (4 Hz; 10 s and 3 min) was studied in the pig gastric fundus.

Ascorbate (5×10−4 M), α‐tocopherol (4×10−4 M), SOD (300–1000 u ml−1) and EUK‐8 (3×10−4 M) did not influence the relaxations to exogenous NO. In the presence of GSH (5×10−4 M), the short‐lasting relaxation to NO became biphasic, potentiated and prolonged. Urate (4×10−4 M) and bilirubin (2×10−4 M) also potentiated the relaxant effect of NO. None of the antioxidants influenced the electrically evoked relaxations.

6‐Anilino‐5,8‐quinolinedione (LY83583; 10−5 M) had no influence on nitrergic nerve stimulation but nearly abolished the relaxant response to exogenous NO. Urate and GSH completely prevented this inhibitory effect, while it was partially reversed by SOD and bilirubin. Ascorbate, α‐tocopherol and EUK‐8 were without effect.

Hydroquinone (10−4 M) did not affect the electrically induced nitrergic relaxations, but markedly reduced NO‐induced relaxations. The inhibition of exogenous NO by hydroquinone was completely prevented by urate and GSH. SOD and ascorbate afforded partial protection, while bilirubin, EUK‐8 and α‐tocopherol were ineffective.

Hydroxocobalamin (10−4 M) inhibited relaxations to NO by 50%, but not the electrically induced responses. Full protection versus this inhibitory effect was obtained with urate, GSH and α‐tocopherol.

These results strengthen the hypothesis that several endogenous antioxidant defense mechanisms, enzymatic as well as non‐enzymatic, might play a role in the nitrergic neurotransmission process.

British Journal of Pharmacology (2000) 129, 1201–1211; doi:10.1038/sj.bjp.0703176
Beta‐3 adrenergic stimulation of L‐Type Ca2+ channels in rat portal vein myocytes
Authors: Viard Patricia , Macrez Nathalie , Coussin Frédéric , Morel Jean‐Luc , Mironneau Jean
Published: January 2005

The effects of β3‐adrenergic stimulation were studied on the L‐type Ca2+ channel in single myocytes from rat portal vein using the whole‐cell mode of the patch‐clamp technique.

Reverse transcription‐polymerase chain reaction showed that β1‐, β2‐ and β3‐adrenoceptor subtypes were expressed in rat portal vein myocytes. Application of both propranolol (a non‐selective β1‐ and β2‐adrenoceptor antagonist) and SR59230A (a β3‐adrenoceptor antagonist) were needed to inhibit the isoprenaline‐induced increase in L‐type Ca2+ channel current.

L‐type Ca2+ channels were stimulated by CGP12177A (a β3‐adrenoceptor agonist with potent β1‐ and β2‐adrenoceptor antagonist property) in a manner similar to that of isoprenaline. The CGP12177A‐induced stimulation of Ca2+ channel current was blocked by SR59230A, cyclic AMP‐dependent protein kinase inhibitors, H‐89 and Rp 8‐Br‐cyclic AMPs, but was unaffected by protein kinase C inhibitors, GF109203X and 19‐31 peptide. This stimulation was mimicked by forskolin and 8‐Br‐cyclic AMP. In the presence of okadaic acid (a phosphatase inhibitor), the β3‐adrenoceptor‐induced stimulation was maintained after withdrawal of the agonist.

The β3‐adrenoceptor stimulation of L‐type Ca2+ channels was blocked by a pretreatment with cholera toxin and by the intracellular application of an anti‐Gαs antibody. This stimulation was unaffected by intracellular infusion of an anti‐Gβcom antibody and a βARK1 peptide.

These results show that activation of β3‐adrenoceptors stimulates L‐type Ca2+ channels in vascular myocytes through a Gαs‐induced stimulation of the cyclic AMP/protein kinase A pathway and the subsequent phosphorylation of the channels.

British Journal of Pharmacology (2000) 129, 1497–1505; doi:10.1038/sj.bjp.0703187
Rapid non‐genomic activation of cytosolic cyclic AMP‐dependent protein kinase activity and [Ca2+]i by 17β‐oestradiol in female rat distal colon
Authors: Doolan Christina M , Condliffe Steven B , Harvey Brian J
Published: January 2005

In this study, the effect of 17β‐oestradiol on adenosine 3′ : 5′‐cyclic monophosphate (cyclic AMP)‐dependent protein kinase (PKA) activity was investigated.

Rapid (within 15 min) activation of basal PKA activity was observed in cytosolic fractions by 17β‐oestradiol but not by 17α‐oestradiol, progesterone or testosterone. This stimulation was abolished by the specific PKA inhibitor PKI but not by the classical oestrogen receptor antagonist tamoxifen.

17β‐Oestradiol did not stimulate basal PKA activity in membrane fractions or in cytosolic fractions from male rats.

The increase in cytosolic PKA activity was indirect as (i) it was inhibited by the adenylyl cyclase inhibitor SQ22536, (ii) it was mimicked by forskolin and (iii) 17β‐oestradiol did not cause a stimulation of basal PKA activity in either type I or type II commercially available PKA holoenzymes.

Protein kinase Cδ (PKCδ) was directly activated by 17β‐oestradiol. The specific PKC inhibitor, bisindolylmaleimide I (GF 109203X), abolished the 17β‐oestradiol‐induced PKA activation.

17β‐Oestradiol stimulated an increase in free intracellular calcium ion concentration ([Ca2+]i) in isolated female but not male rat colonic crypts. This was inhibited by verapamil, nifedipine and zero extracellular [Ca2+] but unaffected by tamoxifen. 17α‐Oestradiol, testosterone and progesterone failed to increase [Ca2+]i.

PKC and PKA inhibitors abolished the 17β‐oestradiol‐induced increase in [Ca2+]i.

These results demonstrate the existence of a novel 17β‐oestradiol‐specific PKA and Ca2+ signalling pathway, which is both sex steroid‐ and gender‐specific, in rat distal colonic epithelium.

British Journal of Pharmacology (2000) 129, 1375–1386; doi:10.1038/sj.bjp.0703193
Characterization of a novel nonpeptide vasopressin V2‐agonist, OPC‐51803, in cells transfected human vasopressin receptor subtypes
Authors: Nakamura Shigeki , Yamamura Yoshitaka , Itoh Shuji , Hirano Takahiro , Tsujimae Kenji , Aoyama Masashi , Kondo Kazumi , Ogawa Hidenori , Shinohara Tomoichi , Kan Keizo , Tanada Yoshihisa , Teramoto Shuji , Sumida Takumi , Nakayama Sunao , Sekiguchi Kazuo , Kambe Toshimi , Tsujimoto Gozoh , Mori Toyoki , Tominaga Michiaki
Published: January 2005

We discovered the first nonpeptide arginine‐vasopressin (AVP) V2‐receptor agonist, OPC‐51803. Pharmacological properties of OPC‐51803 were elucidated using HeLa cells expressing human AVP receptor subtypes (V2, V1a and V1b) and compared with those of 1‐desamino‐8‐D‐arginine vasopressin (dDAVP), a peptide V2‐receptor agonist.

OPC‐51803 and dDAVP displaced [3H]‐AVP binding to human V2‐ and V1a‐receptors with Ki values of 91.9±10.8 nM (n=6) and 3.12±0.38 nM (n=6) for V2‐receptors, and 819±39 nM (n=6) and 41.5±9.9 nM (n=6) for V1a‐receptors, indicating that OPC‐51803 was about nine times more selective for V2‐receptors, similar to the selectivity of dDAVP. OPC‐51803 scarcely displaced [3H]‐AVP binding to human V1b‐receptors even at 10−4 M, while dDAVP showed potent affinity to human V1b‐receptors with the Ki value of 13.7±3.2 nM (n=4).

OPC‐51803 concentration‐dependently increased cyclic adenosine 3′, 5′‐monophosphate (cyclic AMP) production in HeLa cells expressing human V2‐receptors with an EC50 value of 189±14 nM (n=6). The concentration‐response curve for cyclic AMP production induced by OPC‐51803 was shifted to the right in the presence of a V2‐antagonist, OPC‐31260.

At 10−5 M, OPC‐51803 did not increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells expressing human V1a‐receptors. On the other hand, dDAVP increased [Ca2+]i in HeLa cells expressing human V1a‐ and V1b‐receptors in a concentration‐dependent fashion.

From these results, OPC‐51803 has been confirmed to be the first nonpeptide agonist for human AVP V2‐receptors without agonistic activities for V1a‐ and V1b‐receptors. OPC‐51803 may be useful for the treatment of AVP‐deficient pathophysiological states and as a tool for AVP researches.

British Journal of Pharmacology (2000) 129, 1700–1706; doi:10.1038/sj.bjp.0703221
Blockade of the HERG human cardiac K+ channel by the antidepressant drug amitriptyline
Authors: Jo Su‐Hyun , Youm Jae Boum , Lee Chin O , Earm Yung E , Ho Won‐Kyung
Published: January 2005

Amitriptyline has been known to induce QT prolongation and torsades de pointes which causes sudden death. We studied the effects of amitriptyline on the human ether‐a‐go‐go‐related gene (HERG) channel expressed in Xenopus oocytes and on the rapidly activating delayed rectifier K+ current (IKr) in rat atrial myocytes.

The amplitudes of steady‐state currents and tail currents of HERG were decreased by amitriptyline dose‐dependently. The decrease became more pronounced at more positive potential, suggesting that the block of HERG by amitriptyline is voltage dependent. IC50 for amitriptyline block of HERG current was progressively decreased according to depolarization: IC50 values at −30, −10, +10 and +30 mV were 23.0, 8.71, 5.96 and 4.66 μM, respectively.

Block of HERG by amitriptyline was use dependent: exhibiting a much faster block at higher activation frequency. Subsequent decrease in frequency after high activation frequency resulted in a partial relief of HERG blockade.

Steady‐state block by amitriptyline was obtained while depolarization to +20 mV for 0.5 s was applied at 0.5 Hz: IC50 was 3.26 μM in 2 mM [K+]o. It was increased to 4.78 μM in 4 mM [K+]o, suggesting that the affinity of amitriptyline on HERG was decreased by external K+.

In rat atrial myocytes bathed in 35°C, 5 μM amitriptyline blocked IKr by 55%. However, transient outward K+ current (Ito) was not significantly affected.

In summary, the data suggest that the block of HERG currents may contribute to arrhythmogenic side effects of amitriptyline.

British Journal of Pharmacology (2000) 129, 1474–1480; doi:10.1038/sj.bjp.0703222
Augmentation of SR Ca2+ release by rapamycin and FK506 causes K+‐channel activation and membrane hyperpolarization in bladder smooth muscle
Authors: Weidelt Th , Isenberg G
Published: January 2005

The immunosuppressants rapamycin and FK506 are known to relax smooth muscle despite facilitating Ca2+ release through ryanodine‐receptors of the sarcoplasmic reticulum (SR). The apparent contradiction was studied in isolated guinea‐pig urinary bladder myocytes.

Modulation of spontaneous SR Ca2+ release was monitored by means of spontaneous transient outward currents (or STOCs) in isolated smooth muscle cells voltage‐clamped to −20 mV. Rapamycin (10 μM, n=18) significantly increased amplitude (50±12%, mean±s.d.), life time (77±19%), and time integral of STOCs (113±22%), and it reduced the interval between STOCs (20±7%). FK506 (20 μM, n=24) increased amplitude (15±7%), life time (50±7%), time integral (104±26%). Cyclosporin A (20 μM, n=18) had no significant effects on STOCs.

The basal cytoplasmic Ca2+ concentration ([Ca2+]c) measured by Indo1‐fluorescence was insensitive to rapamycin or FK506. Pretreatment with rapamycin (20 μM, 2 min) did not impair the SR Ca2+ load as can be concluded from caffeine‐induced Ca2+‐transients.

As it was expected from the enhanced STOC activity, the non‐clamped membrane was hyperpolarized by rapamycin (15±2 mV) or by FK506 (15±3 mV).

The data are consistent with the idea that rapamycin and FK506 augment spontaneous SR Ca2+ release by removal of FK‐binding proteins from the RyR‐complex. Smooth muscle relaxation is interpreted as negative Ca2+ feedback: augmented Ca2+ activation of STOCs induces membrane hyperpolarization that reduces Ca2+ influx through voltage gated channels.

British Journal of Pharmacology (2000) 129, 1293–1300; doi:10.1038/sj.bjp.0703223
Inhibition by various antipsychotic drugs of the G‐protein‐activated inwardly rectifying K+ (GIRK) channels expressed in Xenopus oocytes
Authors: Kobayashi Toru , Ikeda Kazutaka , Kumanishi Toshiro
Published: January 2005

To investigate the effects of various chemical classes of antipsychotic drugs: haloperidol, thioridazine, pimozide and clozapine, on the G‐protein‐activated inwardly rectifying K+ (GIRK) channels, we carried out Xenopus oocyte functional assays with GIRK1 and GIRK2 mRNAs or GIRK1 and GIRK4 mRNAs.

In oocytes co‐injected with GIRK1 and GIRK2 mRNAs, application of each of the various antipsychotic drugs immediately caused a reduction of inward currents through the basally active GIRK channels. These responses were not observed in the presence of 3 mM Ba2+, which blocks the GIRK channels. In addition, in uninjected oocytes, none of the drugs tested produced any significant current response. These results indicate that all the antipsychotic drugs tested inhibited the brain‐type GIRK1/2 heteromultimeric channels. Furthermore, similar results were obtained in oocytes co‐injected with GIRK1 and GIRK4 mRNAs, indicating that the antipsychotic drugs also inhibited the cardiac‐type GIRK1/4 heteromultimeric channels.

All the drugs tested inhibited, in a concentration‐dependent manner, both types of GIRK channels with varying degrees of potency and effectiveness at micromolar concentrations. Only pimozide caused slight inhibition of these channels at nanomolar concentrations.

We conclude that the various antipsychotic drugs acted as inhibitors at the brain‐type and cardiac‐type GIRK channels. Our results suggest that inhibition of both types of GIRK channels by these drugs underlies some of the side effects, in particular seizures and sinus tachycardia, observed in clinical practice.

British Journal of Pharmacology (2000) 129, 1716–1722; doi:10.1038/sj.bjp.0703224
Pharmacological comparison of the effect of ibogaine and 18‐methoxycoronaridine on isolated smooth muscle from the rat and guinea‐pig
Authors: Mundey M K , Blaylock N A , Mason R , Glick S D , Maisonneuve I M , Wilson V G
Published: January 2005

Ibogaine and 18‐methoxycoronaridine are naturally occurring alkaloids reported to possess antiaddictive properties in several models of drug dependence. We have examined their effect at μ‐opioid receptors regulating neurogenic contractions of several smooth muscle preparations and also against spontaneous contractions of the rat isolated portal vein.

Ibogaine (pIC50 5.28) and 18‐methoxycoronaridine (pIC50 5.05) caused a concentration‐dependent inhibition of cholinergic contractions of the guinea‐pig ileum which was not affected by the opioid receptor antagonist naloxone (1 μM).

In the rat isolated vas deferens ibogaine and 18‐methoxycoronaridine caused a concentration‐dependent enhancement of purinergic contractions. Both agents (30 μM) caused a 3–5 fold rightward displacement of DAMGO‐induced inhibition of purinergic contractions, but similar effects were observed for ibogaine against α2‐adrenoceptor‐mediated inhibition of neurogenic responses.

In the guinea‐pig isolated bladder both ibogaine (10 μM) and 18‐methoxycoronaridine (10 μM) caused a 2 fold increase in the purinergic component of neurogenic contractions without significantly altering cholinergic contractions or responses to exogenous ATP. In contrast, ibogaine (1–30 μM), but not 18‐methoxycoronaridine, caused a concentration‐dependent enhancement of spontaneous contractions of the rat isolated portal vein.

In summary, while ibogaine and 18‐methoxycoronaridine modulated electrically‐evoked contractions in the three preparations examined, we have no evidence for a selective interaction with pre‐junctional μ‐opioid receptors. The pronounced enhancement of purinergic contractions produced by both agents is a novel finding and worthy of further investigation.

British Journal of Pharmacology (2000) 129, 1561–1568; doi:10.1038/sj.bjp.0703227
Compounds that block both intermediate‐conductance (IKCa) and small‐conductance (SKCa) calcium‐activated potassium channels
Authors: Malik‐Hall M , Ganellin C R , Galanakis D , Jenkinson D H
Published: January 2005

Nine bis‐quinolinyl and bis‐quinolinium compounds related to dequalinium, and previously shown to block apamin‐sensitive small conductance Ca2+‐activated K+ channels (SKCa), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca2+‐activated K+ channels (IKCa) in rabbit blood cells.

In most experiments, a K+‐sensitive electrode was employed to monitor the IKCa‐mediated net loss of cell K+ that followed the addition of the Ca2+ ionophore A23187 (2 μM) to red cells suspended at an haematocrit of 1% in a low K+ (0.12–0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 μM) to a very dilute suspension of red cells (haematocrit 0.02%).

Of the compounds tested, the most potent IKCa blocker was 1,12 bis[(2‐methylquinolin‐4‐yl)amino]dodecane (UCL 1407) which had an IC50 of 0.85±0.06 μM (mean±s.d.mean).

The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K+], and (iii) a time course of onset that suggested use‐dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IKCa blocking action resembles that of cetiedil rather than of clotrimazole.

Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1‐[N‐[1‐(3,5‐dimethoxybenzyl)‐2‐methylquinolinium‐4‐yl]amino]‐10‐[N′‐(2‐methylquinolinium‐4yl)amino] decane (trifluoroacetate) which had an IC50 of 1.8±0.1 μM. The corresponding bisquaternary UCL 1438 (1,10‐bis[N‐[1‐(3,5‐dimethoxybenzyl)‐2‐methylquinolinium‐4‐yl]amino] decane bis(trifluoroacetate) was almost as active (IC50 2.7±0.3 μM).

A bis‐aminoquinolium cyclophane (UCL 1684) had little IKCa blocking action despite its great potency at SKCa channels (IC50 4.1±0.2 nM).

The main outcome is the identification of new intermediate‐conductance Ca2+‐activated K+ channel blockers with a wide range of IKCa/SKCa selectivities.

British Journal of Pharmacology (2000) 129, 1431–1438; doi:10.1038/sj.bjp.0703233
Inhibition by adenosine A2A receptors of NMDA but not AMPA currents in rat neostriatal neurons
Authors: Wirkner Kerstin , Assmann Heike , Köles Laszlo , Gerevich Zoltan , Franke Heike , Nörenberg Wolfgang , Boehm Rudolf , Illes Peter
Published: January 2005

Whole‐cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A2A receptors in modulating N‐methyl‐D‐aspartate (NMDA)‐induced currents in rat striatal brain slices. The A2A receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) current in a subset of striatal neurons.

Lucifer yellow‐filled pipettes in combination with immunostaining of A2A receptors were used to identify CGS 21680‐sensitive cells as typical medium spiny striatal neurons.

Dibutyryl cyclic AMP and the protein kinase A activator Sp‐cyclic AMPs, but not the protein kinase A inhibitors Rp‐cyclic AMPS or PKI(14–24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U‐73122, but not the inactive structural analogue U‐73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12‐myristate 13‐acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1,4,5‐trisphosphate (InsP3) and a more efficient buffering of intracellular Ca2+ by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680‐induced inhibition.

The calmodulin antagonist W‐7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281–309) and KN‐93 but not the inactive structural analogue KN‐92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680.

It is suggested that the transduction mechanism of A2A receptors to inhibit NMDA receptor channels is the phospholipase C/InsP3/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.

British Journal of Pharmacology (2000) 130, 259–269; doi:10.1038/sj.bjp.0703234
Investigation of the role of intracellular Ca2+ stores in generation of the muscarinic agonist‐induced slow afterdepolarization (sADP) in guinea‐pig olfactory cortical neurones in vitro
Authors: Postlethwaite Michael , Constanti Andrew , Libri Vincenzo
Published: January 2005

Intracellular recordings were made from guinea‐pig olfactory cortical brain slice neurones to assess the possible role of intracellular Ca2+ stores in the generation of the slow post‐stimulus afterdepolarization (sADP) and its underlying tail current (IADP), induced by muscarinic receptor activation.

Caffeine or theophylline (0.5–3 mM) reduced the amplitude of the IADP (measured under ‘hybrid’ voltage clamp) induced in the presence of the muscarinic agonist oxotremorine‐M (OXO‐M, 10 μM) by up to 96%, without affecting membrane properties or muscarinic depolarization of these neurones.

The L‐type Ca2+ channel blocker nifedipine (1, 10 μM) also inhibited IADP (by up to 46%), while ryanodine (10 μM) (a blocker of Ca2+ release from internal stores) produced a small (∼10%) reduction in IADP amplitude; however, neither 10 μM dantrolene (another internal Ca2+ release blocker) nor the intracellular Ca2+ store re‐uptake inhibitors thapsigargin (3 μM) or cyclopiazonic acid (CPA, 15 μM) affected IADP amplitude.

IBMX (100 μM), a phosphodiesterase inhibitor, also had no effect on IADP. Furthermore, inhibition of IADP by caffeine was not reversed by co‐application of 100 μM adenosine.

Caffeine (3 mM) or nifedipine (10 μM) reduced the duration of presumed Ca2+ spikes revealed by intracellular Cs+ loading. When applied in combination, nifedipine and caffeine effects were occlusive, rather than additive, suggesting a common site of action on L‐type calcium channels.

We conclude that Ca2+‐induced Ca2+ release (CICR) from internal stores does not contribute significantly to muscarinic IADP generation in olfactory cortical neurones. However caffeine and theophylline, which enhance CICR in other systems, blocked IADP induction. We suggest that this action might involve a combination of L‐type voltage‐gated Ca2+ channel blockade, and a direct inhibitory action on the putative IADP K+ conductance.

British Journal of Pharmacology (2000) 129, 1447–1457; doi:10.1038/sj.bjp.0703236
Upregulation of bradykinin B1‐receptor expression after myocardial infarction
Authors: Tschöpe Carsten , Heringer‐Walther Silvia , Koch Matthias , Spillmann Frank , Wendorf Michael , Leitner Eszter , Schultheiss Heinz‐Peter , Walther Thomas
Published: January 2005

To determine the influence of the myocardial infarction (MI) on bradykinin B1‐receptor (B1R) regulation, we studied its expression in the left ventricle (LV) after MI. Rats were submitted to a permanent occlusion of the left coronary artery. Six hours, 24 h and 6 days after MI or after sham operation, left ventricular pressure (LVP) and dP/dtmax were measured. LV‐total RNA was extracted and B1R expression was analysed by a RNase‐protection assay (each group n=6). LVP and dP/dtmax were impaired at all time points after MI. Basal B1R expression was not detectable in controls. Six hours after MI, the B1R expression was upregulated and reached a maximum 24 h after MI (4 fold vs 6 h). Six days post‐MI, B1R expression returned to levels found 6 h after MI. These data are the first demonstration for an induced myocardial B1R expression in an in vivo model of MI.

British Journal of Pharmacology (2000) 129, 1537–1538; doi:10.1038/sj.bjp.0703239