bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of fentanyl on 5‐HT efflux involves both opioid and 5‐HT1Areceptors
Authors: Tao Rui , Karnik Meghana , Ma Zhiyuan , Auerbach Sidney B
Published: August 2003

Fentanyl is aμ‐opioid analgesic that might disinhibit 5‐HT neurons and thus increase 5‐HT efflux. However, fentanyl also binds to 5‐HT1Areceptors, and if it activates 5‐HT1Asomatodendritic autoreceptors, the resultant inhibition might offset opioid‐mediated increases in 5‐HT efflux. To test this hypothesis, we used microdialysis to study effects of fentanyl on extracellular 5‐HT in the dorsal raphe nucleus (DRN) of unanesthetized rats.

Systemic administration of fentanyl (0.01–0.2 mg kg−1, s.c.) increased 5‐HT efflux in the DRN. An intermediate dose of fentanyl (0.05 mg kg−1) produced the maximum increase in 5‐HT to ∼180% of baseline levels in the DRN. Naltrexone (10 mg kg−1, s.c.) blocked the increase in response to systemic fentanyl (0.05 mg kg−1).

In contrast, during infusion into the DRN, fentanyl (10–1000μM) induced a dose‐dependent decrease in 5‐HT. Naltrexone andnor‐binaltorphimine failed to block the decrease suggesting thatμ‐ andκ‐opioid receptors did not mediate this effect.

Systemic (−)‐pindolol (8 mg kg−1, s.c.) or infusion of WAY‐100635 (100μM) into the DRN blocked the decrease, and instead 5‐HT increased in response to local infusion of fentanyl (100μM). WAY‐100635 (0.3 mg kg−1, s.c.) also potentiated the effect of systemic fentanyl (0.2 mg kg−1, s.c.). (−)‐Pindolol and WAY‐100635 block 5HT1Areceptors, indicating that inhibition of 5‐HT neuronal activity resulting from fentanyl binding to somatodendritic autoreceptors attenuated opioid‐mediated increases in 5‐HT efflux.

These results provide novel evidence that besides stimulatingμ‐opioid receptors, fentanyl is a 5‐HT1Areceptor agonist. Possibly, this contributes to lethality of fentanyl overdose.

British Journal of Pharmacology(2003)139, 1498–1504. doi:10.1038/sj.bjp.0705378
Action potential changes associated with a slowed inactivation of cardiac voltage‐gated sodium channels by KB130015
Authors: Macianskiene R , Bito V , Raeymaekers L , Brandts B , Sipido K R , Mubagwa K
Published: August 2003

We have studied the acute cardiac electrophysiological effects of KB130015 (KB), a drug structurally related to amiodarone. Membrane currents and action potentials were measured at room temperature or at 37°C during whole‐cell patch‐clamp recording in ventricular myocytes. Action potentials were also measured at 37°C in multicellular ventricular preparations.

The effects of KB were compared with those of anemone toxin II (ATX‐II). Both KB and ATX‐II slowed the inactivation of the voltage‐gated Na+current (INa). While KB shifted the steady‐state voltage‐dependent inactivation to more negative potentials, ATX‐II shifted it to more positive potentials. In addition, while inactivation proceeded to completion with KB, a noninactivating current was induced by ATX‐II.

KB had no effect onIK1but decreasedICa‐LThe drug also did not changeItoin mouse myocytes.

The action potential duration (APD) in pig myocytes or multicellular preparations was not prolonged but often shortened by KB, while marked APD prolongation was obtained with ATX‐II. Short APDs in mouse were markedly prolonged by KB, which frequently induced early afterdepolarizations.

A computer simulation confirmed that long action potentials with high plateau are relatively less sensitive to a mere slowing ofINainactivation, not associated with a persisting, noninactivating current. In contrast, simulated short action potentials with marked phase‐1 repolarization were markedly modified by slowingINainactivation.

It is suggested that a prolongation of short action potentials by drugs or mutations that only slowINainactivation does not necessarily imply identical changes in other species or in different myocardial regions.

British Journal of Pharmacology(2003)139, 1469–1479. doi:10.1038/sj.bjp.0705379
Extracellular L‐arginine is required for optimal NO synthesis by eNOS and iNOS in the rat mesenteric artery wall
Authors: MacKenzie Andrew , Wadsworth Roger M
Published: August 2003

The formation of NO from endothelial nitric oxide synthase (eNOS) in rat superior mesenteric artery rings was dependent on extracellular L‐arginine, and was optimal at a concentration of L‐arginine close to the plasma level (carbachol‐stimulated NO: control 15.7±0.9, L‐arginine 100 μM 22.8±1.3 nM).

Enhancement of NO output by L‐arginine was stereospecific, required the cationic amino‐acid transporter and was dependent on caveolin.

Induction of inducible nitric oxide synthase (iNOS) impaired the stimulated NO synthesis from eNOS (100 nM carbachol‐stimulated NO: control 5.7±0.6, iNOS 0.3±0.3 nM).

The interaction between iNOS and eNOS was reversed by the superoxide scavenger MnTMPyP. Impairment of eNOS by iNOS was also prevented by L‐arginine 100 μM administered simultaneously with carbachol, but not by L‐arginine administered during incubation with lipopolysaccharide.

These data provide functional evidence that supplementing L‐arginine from the extracellular medium optimises the formation of NO from eNOS and suggests that the impairment of eNOS by iNOS is caused by excess formation of superoxide by NO synthase, which can be prevented by L‐arginine. These results provide an explanation for the observations that extracellular L‐arginine can enhance endothelium function only when the endothelium is impaired or when iNOS has been induced.

British Journal of Pharmacology (2003) 139, 1487–1497. doi:10.1038/sj.bjp.0705380
LF 16‐0687 Ms, a bradykinin B2 receptor antagonist, reduces ischemic brain injury in a murine model of transient focal cerebral ischemia
Authors: Ding‐Zhou Li , Margaill Isabelle , Palmier Bruno , Pruneau Didier , Plotkine Michel , Marchand‐Verrecchia Catherine
Published: August 2003

Bradykinin promotes neuronal damage and brain edema through the activation of the B2 receptor. The neuroprotective effect of LF 16‐0687 Ms, a B2 receptor antagonist, has been described when given prior to induction of transient focal cerebral ischemia in rat, but there are no data regarding the consequence of a treatment when given after injury. Therefore, in a murine model of transient middle cerebral artery occlusion (MCAO), we evaluated the effect of LF 16‐0687 Ms given prior to and/or after the onset of ischemia on neurological deficit, infarct volume and inflammatory responses including cerebral edema, blood–brain barrier (BBB) disruption and neutrophil accumulation.

LF 16‐0687 Ms (1, 2 and 4 mg kg−1) administered 0.5 h before and, 1.25 and 6 h after MCAO, decreased the infarct volume by a maximum of 33% and significantly improved the neurological recovery.

When given at 0.25 and 6.25 h after MCAO, LF 16‐0687 Ms (1.5, 3 and 6 mg kg−1) decreased the infarct volume by a maximum of 25% and improved the neurological score.

Post‐treatment with LF 16‐0687 Ms (1.5 mg kg−1) significantly decreased brain edema (−28%), BBB disruption (−60%) and neutrophil accumulation (−65%) induced by ischemia. Physiological parameters were not modified by LF 16‐0687 Ms.

These data emphasize the role of bradykinin B2 receptor in the development of infarct lesion, neurological deficit and inflammatory responses resulting from transient focal cerebral ischemia. Therefore, B2 receptor antagonist might represent a new therapeutic approach in the pharmacological treatment of stroke.

British Journal of Pharmacology (2003) 139, 1539–1547. doi:10.1038/sj.bjp.0705385
Involvement of extracellular Ca2+ influx and epidermal growth factor receptor tyrosine kinase transactivation in endothelin‐1‐induced arachidonic acid release
Authors: Kawanabe Yoshifumi , Nozaki Kazuhiko , Hashimoto Nobuo , Masaki Tomoh
Published: August 2003

Endothelin‐1 (ET‐1) activates two types of Ca2+‐permeable nonselective cation channels (designated NSCC‐1 and NSCC‐2) and a store‐operated Ca2+ channel (SOCC) in vascular smooth muscle cells (VSMCs). These channels can be distinguished by their sensitivity to Ca2+‐channel blockers, SK&F 96365 and LOE 908. LOE 908 is sensitive to NSCC‐1 and NSCC‐2, and SK&F 96365 is sensitive to NSCC‐2 and SOCC. Moreover, these channels play essential roles in ET‐1‐induced epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) transactivation. The main purpose of the present study was to demonstrate the involvement of EGFR PTK transactivation in ET‐1‐induced arachidonic acid release in VSMCs.

Both SK&F 96365 and LOE 908 inhibited ET‐1‐induced arachidonic acid release with the IC50 values correlated to those of ET‐1‐induced Ca2+ influx. Moreover, combined treatment with these blockers abolished ET‐1‐induced arachidonic acid release.

AG1478, a specific inhibitor of EGFR PTK, inhibited ET‐1‐induced arachidonic acid release and extracellular signal‐regulated kinase 1 and 2 (ERK1/2). The IC50 values of AG1478 for ET‐1‐induced arachidonic acid release and ERK1/2 correlated well with those for ET‐1‐induced EGFR PTK transactivation.

Mitogen‐activated protein kinase kinase inhibitor, PD 98059, inhibited ET‐1‐induced arachidonic acid release. The IC50 values of PD 98059 for ET‐1‐induced arachidonic acid release were similar to those for ET‐1‐induced ERK1/2 activity. In contrast, PD 98059 failed to inhibit ET‐1‐induced EGFR PTK transactivation.

These results indicate that (1) extracellular Ca2+ influx through NSCCs and SOCC plays important roles for ET‐1‐induced arachidonic acid release, (2) EGFR PTK transactivation/ERK1/2 pathways are involved in ET‐1‐induced arachidonic acid release.

British Journal of Pharmacology (2003) 139, 1516–1522. doi:10.1038/sj.bjp.0705386
Halogenation of a capsaicin analogue leads to novel vanilloid TRPV1 receptor antagonists
Authors: Appendino Giovanni , Harrison Selena , De Petrocellis Luciano , Daddario Nives , Bianchi Federica , Schiano Moriello Aniello , Trevisani Marcello , Benvenuti Francesca , Geppetti Pierangelo , Di Marzo Vincenzo
Published: August 2003

The C‐5 halogenation of the vanillyl moiety of resiniferatoxin, an ultrapotent agonist of vanilloid TRPV1 receptors, results in a potent antagonist for these receptors. Here, we have synthesized a series of halogenated derivatives of ‘synthetic capsaicin’ (nonanoyl vanillamide=nordihydrocapsaicin) differing for the nature (iodine, bromine–chlorine) and the regiochemistry (C‐5, C‐6) of the halogenation.

The activity of these compounds was investigated on recombinant human TRPV1 receptors overexpressed in HEK‐293 cells. None of the six compounds exerted any significant agonist activity, as assessed by measuring their effect on TRPV1‐mediated calcium mobilization. Instead, all compounds antagonized, to various extents, the effect of capsaicin in this assay.

All 6‐halo‐nordihydrocapsaicins behaved as competitive antagonists against human TRPV1 according to the corresponding Schild's plots, and were more potent than the corresponding 5‐halogenated analogues. The iodo‐derivatives were more potent than the bromo‐ and chloro‐derivatives.

Using human recombinant TRPV1, 6‐iodo‐nordihydrocapsaicin (IC50=10 nM against 100 nM capsaicin) was about four times more potent than the prototypical TRPV1 antagonist, capsazepine, and was tested against capsaicin also on native TRPV1 in: (i) rat dorsal root ganglion neurons in culture; (ii) guinea‐pig urinary bladder; and (iii) guinea‐pig bronchi. In all cases, except for the guinea‐pig bronchi, the compound was significantly more potent than capsazepine as a TRPV1 antagonist.

In conclusion, 6‐iodo‐nordihydrocapsaicin, a stable and easily prepared compound, is a potent TRPV1 antagonist and a convenient replacement for capsazepine in most of the in vitro preparations currently used to assess the activity of putative vanilloid receptor agonists.

British Journal of Pharmacology (2003) 139, 1417–1424. doi:10.1038/sj.bjp.0705387
Dopaminergic mechanisms underlying bladder hyperactivity in rats with a unilateral 6‐hydroxydopamine (6‐OHDA) lesion of the nigrostriatal pathway
Authors: Yoshimura Naoki , Kuno Sadako , Chancellor Michael B , de Groat William C , Seki Satoshi
Published: August 2003

This study was undertaken to elucidate dopaminergic mechanisms underlying bladder hyperactivity in a rat model of Parkinson's disease (PD) induced by a unilateral 6‐OHDA injection into the substantia nigra pars compacta.

In 6‐OHDA‐lesioned rats, voided volume per micturition (0.41±0.04 ml, mean±s.e.m.) measured during 24 h in a metabolic cage was significantly smaller than in sham‐operated rats (0.67±0.07 ml).

Cystrometrograms (CMG) in conscious animals revealed significantly smaller bladder capacity (BC) (0.46±0.03 ml) in 6‐OHDA‐lesioned rats than in sham rats (0.72±0.06 ml).

SKF38393 (D1/D5 receptor agonist, i.v.) significantly increased BC in 6‐OHDA rats without apparent effects in sham rats. SKF38393 applied intracerebroventricularly (i.c.v.) under urethane anesthesia also increased BC in 6‐OHDA‐lesioned rats and by a smaller increment in sham rats.

In contrast, quinpirole (D2/D3/D4 receptor agonist, i.v.) significantly reduced BC in sham and 6‐OHDA‐lesioned rats. Intrathecal injection of quinpirole similarly reduced BC in sham and 6‐OHDA‐lesioned rats.

PD128907 (D3‐receptor agonist) did not have significant effects on BC in 6‐OHDA‐lesioned rats.

These results indicate that a rat model of PD exhibited bladder hyperactivity as observed in patients with PD, and that stimulation of D1/D5 dopamine receptors at a supraspinal site can suppress bladder hyperactivity in PD, whereas stimulation of D2/D4, but not D3, dopamine receptors had the opposite effect to reduce bladder capacity. Thus, D1/D5 dopamine receptor agonists might be effective in treating neurogenic bladder hyperactivity in PD.

British Journal of Pharmacology (2003) 139, 1425–1432. doi:10.1038/sj.bjp.0705388
Agonists for neuropeptide Y receptors Y1 and Y5 stimulate different phases of feeding in guinea pigs
Authors: Lecklin Anne , Lundell Ingrid , Salmela Suvi , Männistö Pekka T , Beck‐Sickinger Annette G , Larhammar Dan
Published: August 2003

The stimulatory effect of neuropeptide Y (NPY) on food intake is well established but the roles of the receptor subtypes Y1 and Y5 have been difficult to define. We have studied the effects of two novel Y1‐preferring and two Y5‐preferring agonists on feeding in guinea pigs.

The Y1‐preferring receptor agonists [Arg6,Pro34]pNPY and [Phe7,Pro34]pNPY had high affinity for the Y1 receptor (Ki values 0.07 and 0.04 nM, respectively) and nanomolar affinity for the Y5 receptor. Administration of either compound into the third brain ventricle increased food intake equally to NPY.

The Y5 agonist [Ala31,Aib32]pNPY displayed a moderate affinity for the Y5 receptor (Ki 7.42 nM) and a low affinity for Y1 (Ki 1.7 μM). This compound had only a modest effect on feeding.

The other Y5‐preferring peptide [cPP1–7,NPY19–23,Ala31,Aib32,Gln34]hPP had a higher affinity at the Y5 receptor (Ki 1.32 nM) and also at the Y1 receptor (Ki 85 nM). It potently stimulated feeding: the food consumption after administration of this peptide was two‐fold compared to NPY.

Our results support the view that both the receptor subtypes Y1 and Y5 are involved in the stimulation of feeding. As the action profiles of the Y1 and Y5 agonists on feeding parameters were different, it seems that they influence different phases of eating.

British Journal of Pharmacology (2003) 139, 1433–1440. doi:10.1038/sj.bjp.0705389
Atypical cardiostimulant β‐adrenoceptor in the rat heart: stereoselective antagonism by bupranolol but lack of effect by some bupranolol analogues
Authors: Malinowska Barbara , Kieć‐Kononowicz Katarzyna , Flau Karsten , Godlewski Grzegorz , Kozłowska Hanna , Kathmann Markus , Schlicker Eberhard
Published: August 2003

Atypical β‐adrenoceptors resistant to propranolol, but blocked by bupranolol, increase contractile force and/or frequency of the heart in humans and rats. We compared the potencies of the enantiomers of bupranolol and examined the possible effects of seven bupranolol analogues including bevantolol (BEV) at this receptor in pithed and vagotomized rats.

CGP 12177, an agonist of the atypical β‐adrenoceptor, increased heart rate dose‐dependently. Its dose–response curve was shifted to the right by S‐(−)‐bupranolol 10 μmol kg−1 by a factor of 8.4, but not affected by the same dose of R‐(+)‐bupranolol.

Desmethylbupranolol and compounds BK‐21, BK‐22, BK‐23 and BK‐25 also increased heart rate dose‐dependently. The β1‐adrenoceptor antagonist CGP 20712 given in combination with the β2‐adrenoceptor antagonist ICI 118,551 (0.1 μmol kg−1 each) reduced the positive chronotropic action of the five bupranolol analogues without affecting that of CGP 12177. The potencies of the bupranolol analogues to increase heart rate were correlated (r=0.91, P<0.05) with their affinities for β1‐adrenoceptor binding sites in rat brain cortex membranes labelled with [3H]CGP 12177 (in the presence of ICI 118,551).

BK‐26 and BEV, 10 μmol kg−1 each, had only minor effects on heart rate by themselves and did not antagonize the effect of CGP 12177. However, at 1 μmol kg−1, they antagonized the increase in heart rate elicited by the β1‐adrenoceptor agonist prenalterol.

In conclusion, bupranolol is a stereoselective antagonist at the atypical cardiostimulant β‐adrenoceptor. The effects of the bupranolol analogues are related to the activation or blockade of β1‐adrenoceptors, but not of atypical β‐adrenoceptors.

British Journal of Pharmacology (2003) 139, 1548–1554. doi:10.1038/sj.bjp.0705390
Cyclooxygenase‐1 and cyclooxygenase‐2 in the mouse ductus arteriosus: individual activity and functional coupling with nitric oxide synthase
Authors: Baragatti B , Brizzi F , Ackerley C , Barogi S , Ballou L R , Coceani F
Published: August 2003

Prenatal patency of the ductus arteriosus is maintained by prostaglandin (PG) E2, conceivably in concert with nitric oxide (NO). Local PGE2 formation is sustained by cyclooxygenase‐1 (COX1) and cyclooxygenase‐2 (COX2), a possible exception being the mouse in which COX1, or both COXs, are reportedly absent. Here, we have examined the occurrence of functional COX isoforms in the near‐term mouse ductus and the possibility of COX deletion causing NO upregulation.

COX1 and COX2 were detected in smooth muscle cells by immunogold electronmicroscopy, both being located primarily in the perinuclear region. Cytosolic and microsomal PGE synthases (cPGES and mPGES) were also found, but they occurred diffusely across the cytosol. COX1 and, far more frequently, COX2 were colocalised with mPGES, while neither COX appeared to be colocalized with cPGES.

The isolated ductus from wild‐type and COX1−/− mice contracted promptly to indomethacin (2.8 μM). Conversely, the contraction of COX2−/− ductus to the same inhibitor started only after a delay and was slower.

NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) weakly contracted the isolated wild‐type ductus. Its effect, however, increased three‐ to four‐fold after deleting either COX, hence equalling that of indomethacin.

In vivo, the ductus was patent in all mice foetuses, whether wild‐type or COX‐deleted. Likewise, no genotype‐related difference was noted in its postnatal closure.

We conclude that the mouse ductus has a complete system for PGE2 synthesis comprising both COX1 and COX2. The two enzymes respond differently to indomethacin but, nevertheless, deletion of either one results in NO upregulation. PGE2 and NO can function synergistically in keeping the ductus patent.

British Journal of Pharmacology (2003) 139, 1505–1515. doi:10.1038/sj.bjp.0705391
Direct effect of propylthiouracil on progesterone release in rat granulosa cells
Authors: Chen Jiann‐Jong , Wang Shyi‐Wu , Chien Eileen‐Jea , Wang Paulus S
Published: August 2003

The present study was to investigate the direct effect and action mechanism of propylthiouracil (PTU), an antithyroid drug, on the production of progesterone in rat granulosa cells.

PTU (3–12 mM) decreased the basal and human chorionic gonadotropin (hCG)‐stimulated release of progesterone from rat granulosa cells.

PTU (3–12 mM) attenuated the stimulatory effects of forskolin and 8‐bromo‐cyclic 3′:5′‐adenosine monophosphate on progesterone release from rat granulosa cells.

PTU (12 mM) inhibited the activities of both the cytochrome P450 side‐chain cleavage enzyme (P450scc, conversion of 25‐hydroxyl cholesterol to pregnenolone) and the 3β‐hydroxysteroid dehydrogenase (conversion of pregnenolone to progesterone) in rat granulosa cells. PTU decreased the Vmax but increased the Km of P450scc.

PTU (12 mM) decreased the hCG‐increased amount of steroidogenic acute regulatory (StAR) protein in rat granulosa cells.

The present results suggest that PTU decreases the progesterone release by granulosa cells via a thyroid‐independent mechanism involving the inhibition of post‐cAMP pathway, and the activities of intracellular calcium, steroidogenic enzyme, and StAR protein functions.

British Journal of Pharmacology (2003) 139, 1564–1570. doi:10.1038/sj.bjp.0705392
Involvement of Rho‐kinase in contraction of guinea‐pig aorta induced by prostanoid EP3 receptor agonists
Authors: Shum Winnie W C , Le Geng‐yun , Jones Robert L , Gurney Alison M , Sasaki Yasuharu
Published: August 2003

The mechanism of contraction of guinea‐pig isolated aorta induced by the prostanoid EP3 receptor agonist sulprostone (0.1–300 nM) has been investigated. In 60% of the experiments, the sulprostone log concentration–response curve (maximum=15–40% of 100 nM U‐46619 response; low‐responders) was unaffected by the removal of extracellular Ca2+, blockade of L‐type Ca2+ channels with nifedipine and depletion of internal Ca2+ stores. In the remaining preparations (35–65% of 100 nM U‐46619 response; high‐responders), contractions to higher sulprostone concentrations showed a nifedipine‐sensitive component, which was enhanced by charybdotoxin.

In Ca2+‐free Krebs solution, established contractions to 300 nM sulprostone were abolished by the Rho‐kinase inhibitors H‐1152, Y‐27632 and HA‐1077 (IC50 values=190, 770 and 2030 nM). The PKA/Rho‐kinase inhibitor H‐89 (10 nM–10 μM) caused enhancement progressing to inhibition. The selective PKC inhibitor Ro 32‐0432 (3 μM) had no effect, while staurosporine, recently shown to be a potent Rho‐kinase inhibitor, abolished sulprostone responses (IC50 ∼47 nM), but its action was slow. The MAP kinase inhibitors SB 202190, SB 203580 and PD 80958 produced little inhibition.

In normal Krebs solution, H‐1152 and Y‐27632 abolished established contractions to 300 nM sulprostone and 1 μM phenylephrine, and partially inhibited 10 μM phenylephrine and 50 mM K+ responses.

The results are discussed in relation to the reported potencies of the protein kinase inhibitors in enzyme assays. Activation of the Rho‐kinase pathway appears to be a primary mechanism of contraction induced by EP3 receptor agonists in guinea‐pig aorta.

British Journal of Pharmacology (2003) 139, 1449–1461. doi:10.1038/sj.bjp.0705393
Receptor‐independent activation of Rho‐kinase‐mediated calcium sensitisation in smooth muscle
Authors: Ayman Sinem , Wallace Pat , Wayman Chris P , Gibson Alan , McFadzean Ian
Published: August 2003

The aim of this work was to determine whether Rho‐kinase‐mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor‐dependent or receptor‐independent mechanisms.

The Rho‐kinase inhibitor Y27632 produced concentration‐dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco‐endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC50 values against CCh and Tg of 8.4±3.3 (n=6) and 6.1±2.1 (n=7) μM, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mM external potassium (69±7% (n=4) inhibition using 10 μM Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light‐chain phosphatase, calyculin‐A.

In β‐escin‐permeabilised preparations, both CCh and Tg produced significant increases in tone over‐and‐above that produced by a combination of calcium (1 μM) and GTP (100 μM). These responses to CCh and Tg were inhibited by Y27632 (10 μM).

Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane.

These findings suggest that Rho‐kinase‐mediated calcium sensitisation is activated by both receptor‐dependent and receptor‐independent mechanisms in the mouse anococcygeus.

British Journal of Pharmacology (2003) 139, 1532–1538. doi:10.1038/sj.bjp.0705394
β‐adrenoceptor activation plays a role in the reverse rate‐dependency of effective refractory period lengthening by dofetilide in the guinea‐pig atrium, in vitro
Authors: Kovács Anikó , Magyar János , Bányász Tamás , Nánási Péter P , Szénási Gábor
Published: August 2003

Blockers of the rapid component of the delayed rectifier potassium current (IKr) prolong cardiac action potential duration (APD) and effective refractory period (ERP) in a reverse rate‐dependent manner. Since activation of β‐adrenoceptors attenuates prolongation of APD evoked by IKr blockers, rate‐dependent neuronal noradrenaline liberation in the myocardium may contribute to the reverse rate‐dependent nature of the effects of IKr blockers. In order to test this hypothesis, we studied the effects of dofetilide, a pure IKr blocker, on ERP after activation or blockade of β‐adrenoceptors and after catecholamine depletion in guinea‐pig left atrial myocardium paced at 3, 2 and 1 Hz, in vitro.

Dofetilide (100 nM) lengthened ERP in a reverse rate‐dependent manner in the left atrial myocardium of guinea‐pigs. Strong activation of β‐adrenoceptors using 10 nM isoproterenol abolished the dofetilide‐induced lengthening of ERP at all pacing rates.

Blockade of the β‐adrenoceptors with metoprolol (1 μM), atenolol (3 μM) or propranolol (300 nM) increased the dofetilide‐evoked prolongation of ERP at 3 and 2 Hz, but not at 1 Hz. As a consequence, metoprolol attenuated while propranolol and atenolol fully eliminated the reverse rate‐dependent nature of the dofetilide‐induced ERP lengthening. In catecholamine‐depleted atrial preparations of the guinea‐pig (24 h pretreatment with 5 mg kg−1 reserpine i.p.), the effect of dofetilide on ERP was not frequency dependent, and propranolol did not alter the effects of dofetilide.

In contrast to results obtained in guinea‐pig atrial preparations, propranolol failed to change the reverse rate‐dependent effect of dofetilide on ERP in the right ventricular papillary muscles of rabbits and guinea‐pigs.

As an indication of the functional consequences of rate‐dependent noradrenaline liberation, propranolol decreased twitch tension at 3 and 2 Hz but not at 1 Hz in the atrial myocardium of control guinea‐pigs, whereas no such effect was detected in catecholamine‐depleted atrial preparations. Propranolol failed to change contractility of ventricular myocardium in guinea‐pigs and rabbits.

It is concluded that rate‐dependent noradrenaline release and the ensuing β‐adrenoceptor activation contributed to the reverse rate‐dependent nature of ERP prolongation caused by IKr blockers in isolated guinea‐pig atrial myocardium.

British Journal of Pharmacology (2003) 139, 1555–1563. doi:10.1038/sj.bjp.0705395
Differences in the mechanisms that increase noradrenaline efflux after administration of d‐amphetamine: a dual‐probe microdialysis study in rat frontal cortex and hypothalamus
Authors: Géranton Sandrine M , Heal David J , Stanford S Clare
Published: August 2003

The extent to which impulse‐independent release of noradrenaline and/or inhibition of its reuptake contribute to the response to d‐amphetamine in vivo is unclear. Here, dual‐probe microdialysis was used to investigate this question in the rat frontal cortex and hypothalamus.

After systemic administration of d‐amphetamine (10 mg kg−1), or its local infusion (10 μM), the increase in noradrenaline efflux in the hypothalamus was greater than in the frontal cortex.

In contrast, during local infusion of the noradrenaline reuptake inhibitor, BTS 54 354 (50 μM), the noradrenaline response was similar in the frontal cortex and hypothalamus, even after systemic administration of the α2‐antagonist, atipamezole, to block presynaptic inhibition of transmitter release and neuronal firing.

In the frontal cortex, but not the hypothalamus, the noradrenaline response to 10 μM d‐amphetamine was constrained by activation of α2‐adrenoceptors. This suggests that, at this concentration, inhibition of reuptake of noradrenaline, following its impulse‐dependent release, is evident in the frontal cortex, but that the noradrenaline response in the hypothalamus derives mostly from impulse‐independent release (retrotransport).

Atipamezole did not affect the noradrenaline response to 100 μM d‐amphetamine in either brain region possibly because, at this higher concentration, retrotransport of noradrenaline masks any compensatory reduction in impulse‐evoked release.

It is concluded that inhibition of reuptake and retrotransport make different contributions to the noradrenaline response to d‐amphetamine in the frontal cortex and hypothalamus and that retrotransport increases with the concentration of d‐amphetamine.

British Journal of Pharmacology (2003) 139, 1441–1448. doi:10.1038/sj.bjp.0705396
Neuroprotection by caffeine and adenosine A2A receptor blockade of β‐amyloid neurotoxicity
Authors: Dall'Igna Oscar P , Porciúncula Lisiane O , Souza Diogo O , Cunha Rodrigo A , Lara Diogo R
Published: August 2003

British Journal of Pharmacology (2003) 139, 1571. doi:10.1038/sj.bjp.0705400
Bradykinin B2 receptor antagonism: a new direction for acute stroke therapy?
Authors: Sobey Christopher G
Published: August 2003

Stroke is responsible for 10% of all deaths worldwide, and there remains an urgent need for the development of clinically effective treatments for acute stroke. Stroke is now considered to be a disease characterized by an ongoing inflammatory process rather than simply acute neurodegeneration. Bradykinin has attracted recent interest as a potential mediator of brain injury following stroke, because it activates several mechanisms responsible for the early manifestations of inflammation, including arteriolar dilatation, increased vascular permeability and oedema formation. These actions of bradykinin occur via activation of B2 receptors. New evidence suggests that blocking bradykinin B2 receptors after experimental cerebral ischaemia reduces brain oedema, infarct volume and neuronal necrosis, and improves neurological outcome. Thus, B2 receptor antagonists may be a promising new class of compounds for clinical use after the onset of cerebral ischaemia.

British Journal of Pharmacology (2003) 139, 1369–1371. doi:10.1038/sj.bjp.0705415
Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway
Authors: MURTHY Karnam S. , ZHOU Huiping , GRIDER John R. , BRAUTIGAN David L. , ETO Masumi , MAKHLOUF Gabriel M.
Published: August 2003

Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gβγi3 with activation of phospholipase C-β3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Gαq/11 with activation of phospholipase C-β1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44±5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28±3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLC20 phosphorylation and contraction.
Characterization of the interaction between α2-macroglobulin and fibroblast growth factor-2: the role of hydrophobic interactions
Authors: MATHEW Smitha , ARANDJELOVIC Sanja , BEYER Wayne F. , GONIAS Steven L. , PIZZO Salvatore V.
Published: August 2003

Basic fibroblast growth factor (FGF-2) is important in development, wound healing and angiogenesis. The human plasma proteinase inhibitor α2-macroglobulin (α2M) binds to and regulates the biological activity of various growth factors, including FGF-2. FGF-2 binds specifically and saturably to native α2M and conformationally modified α2M (α2M∗); however, the KD for FGF-2 binding to α2M∗ is 10-fold lower. This study investigates the biochemical nature of the interaction between FGF-2 and α2M∗ and localizes a possible FGF-2 binding site in the α2M subunit. FGF-2 binding to α2M∗ was not affected by shifts in pH between 6.5 and 10; however, increasing temperature decreased the KD for this interaction. The binding affinity of FGF-2 for α2M∗ also increased with increasing ionic strength. These results are consistent with the hypothesis that hydrophobic interactions predominate in promoting FGF-2 association with α2M∗. Consistent with this hypothesis, FGF-2 bound to a glutathione S-transferase fusion protein containing amino acids 591–774 of the α2M subunit (FP3) and to a hydrophobic 16-amino-acid peptide (amino acids 718–733) within FP3. Specific binding of FGF-2 to the 16-amino-acid peptide was inhibited by excess transforming growth factor-β1. When the 16-amino-acid peptide was chemically modified to neutralize the only two charged amino acids, FGF-2-binding activity was unaffected, supporting the predominant role of hydrophobic interactions. FGF-2 presentation to signalling receptors is influenced by growth factor binding to heparan sulphate proteoglycans (HSPGs), which is electrostatic in nature. Our results demonstrate that the interactions of FGF-2 with α2M∗ and HSPGs are biochemically distinct, suggesting that different FGF-2 sequences are involved.
Requirements for distinct steps of phospholipase Cγ2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling
Authors: RODRIGUEZ Rosie , MATSUDA Miho , STOREY Amy , KATAN Matilda
Published: August 2003

Studies of PLCγ (phospholipase Cγ) have identified a number of regulatory components required for signalling; however, molecular mechanisms and the relationship between events leading to translocation and an increase of substrate hydrolysis have not been well defined. The addition of a membrane-targeting tag to many signal transducers results in constitutive activation, suggesting that these processes could be closely linked and difficult to dissect. The present study of PLCγ2 regulation by cross-linking of the BCR (B-cell antigen receptor) or H2O2 stress in DT40 B-cells, demonstrated that the membrane targeting is a separate step from further changes that result in enzyme activation and substrate hydrolysis. Furthermore, we have defined the roles of different domains of PLCγ2 and, using a panel of cell lines deficient in components linked to PLCγ2 regulation, the involvement of signalling molecules with respect to each of the steps. We have found that only the lipid-raft-targeted Lyn–PLCγ2 construct, unlike non-specific membrane targeting, overcame the requirement for the adapter protein BLNK (B-cell linker). The stable expression of Lyn–PLCγ2 was not accompanied by an increase in substrate hydrolysis in resting cells, which followed stimulation and specifically required the presence and/or activation of Syk, Btk, phosphoinositide 3-kinase but not BLNK, as established using deficient cell lines or specific inhibitors. Based on mutational analysis of the specific tyrosine residues [Tyr753→Phe (Y753F)/Y759F] and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn–PLCγ2, we found that Tyr753/Tyr759 were essential, whereas the PLCγ2 SH2 domains did not have an important role in the transient activation of Lyn–PLCγ2 but may serve to stabilize an activated form in sustained activation.
Phospholipase activity on N-acyl phosphatidylethanolamines is critically dependent on the N-acyl chain length
Authors: CARAMELO Julio J. , FLORIN-CHRISTENSEN Jorge , DELFINO José M.
Published: August 2003

We have recently shown that an endogenous phospholipase A2 from bovine erythrocytes does not hydrolyse NAPEs (N-acyl l-α-phosphatidylethanolamines), which accumulate remarkably in this system [Florin-Christensen, Suarez, Florin-Christensen, Wainszelbaum, Brown, McElwain and Palmer (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 7736–7741]. Here we investigate the causes underlying this resistance. N-acylation of PE (l-α-phosphatidylethanolamine) results in alteration of charge, head-group volume and conformation, the last two features depending on the N-acyl chain length. To evaluate each effect separately, we synthesized NAPEs with selected N-acyl chain length. We found that phospholipase A2 has considerable activity against N-acetyl PE, but is poorly active against N-butanoyl PE and only marginally active against N-hexanoyl PE, whereas the activity is completely lost when N-hexadecanoyl PE is presented as a substrate. On the other hand, N-hexanoyl PE does not inhibit phospholipase A2 activity, suggesting that this substrate fails to enter the hydrophobic channel. Phospholipase C presents a similar, but less sharp pattern. Molecular dynamics simulations of the polar head group of selected NAPEs reveal a substantially increased conformational variability as the N-acyl chain grows. This larger conformational space represents an increased impairment limiting the access of these molecules to the active site. Our data indicate that, whereas a change in charge contributes to diminished activity, the most relevant effects come from steric hindrance related to the growth of the N-acyl chain.
Muscarinic receptor-mediated activation of p70 S6 kinase 1 (S6K1) in 1321N1 astrocytoma cells: permissive role of phosphoinositide 3-kinase
Authors: TANG Xiuwen , WANG Lijun , PROUD Christopher G. , DOWNES C. Peter
Published: August 2003

In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10–20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), or by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor. Carbachol also stimulated the phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a second mTOR-dependent event, with similar potency to its effect on S6K1. This response was blocked by rapamycin, but was not markedly affected by 100 nM wortmannin, implying separate roles for mTOR and PI3K in S6K1 activation. Wortmannin abolished the carbachol-stimulated rise in PtdIns(3,4,5)P3 and greatly reduced unstimulated levels of this lipid. By contrast, an inhibitor of epidermal growth factor receptor kinase, AG1478, which prevents carbachol-stimulated ErbB3 transactivation, PI3K recruitment and protein kinase B activation in 1321N1 cells, reduced activation of S6K1 by no more than 30%. This effect was overcome by 10 nM insulin, which on its own did not stimulate S6K1, but increased cellular PtdIns(3,4,5)P3 concentrations comparably with carbachol alone. These observations distinguish obligatory roles for mTOR and PI3K in regulating S6K1, but imply that minimal PI3K activity is sufficient to permit stimulation of S6K1 by other activating factors such as increased cytosolic Ca2+ concentrations, which are essential to the muscarinic receptor-mediated response. Moreover, 4E-BP1 and hence, presumably, mTOR can be regulated independently of PI3K activation through these mechanisms.
The three isoenzymes of human inositol-1,4,5-trisphosphate 3-kinase show specific intracellular localization but comparable Ca2+ responses on transfection in COS-7 cells
Authors: DEWASTE Valérie , MOREAU Colette , De SMEDT Florence , BEX Françoise , De SMEDT Humbert , WUYTACK Frank , MISSIAEN Ludwig , ERNEUX Christophe
Published: August 2003

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three human isoenzymes of InsP3 3-kinase (A, B and C) have been reported previously [Choi, Kim, Lee, Moon, Sim, Kim, Chung and Rhee (1990) Science 248, 64–66; Dewaste, Pouillon, Moreau, Shears, Takazawa and Erneux (2000) Biochem. J. 352, 343–351; Dewaste, Roymans, Moreau and Erneux (2002) Biochem. Biophys. Res. Commun. 291, 400–405; Takazawa, Perret, Dumont and Erneux (1991) Biochem. Biophys. Res. Commun. 174, 529–535]. The localization of InsP3 3-kinase isoenzymes fused at their N-terminus to the green fluorescent protein has been studied by confocal microscopy. The A isoform appeared to associate with the cytoskeleton, whereas the C isoform was totally cytoplasmic. The B isoform had a more complex localization: it appeared in the plasma membrane, cytoskeleton and in the endoplasmic reticulum. The three human isoenzymes of InsP3 3-kinase can thus be distinguished by their N-terminal sequence, sensitivity to Ca2+/calmodulin and localization on transfection in COS-7 cells. We have compared the cytosolic Ca2+ responses induced by ATP in COS-7 cells transfected with the three isoenzymes. Cells expressing high levels of any of the three isoforms no longer respond to ATP, whereas cells expressing low levels of each enzyme showed a reduced response consisting of one to three Ca2+ spikes in response to 100 μM ATP. These effects were seen only in wild-type InsP3 3-kinase-transfected cells. 3-Kinase-dead mutant cells behaved as vector-transfected cells. The results highlight the potential role of the three isoforms of InsP3 3-kinase as direct InsP3 metabolizing enzymes and direct regulators of Ca2+ responses to extracellular signals.
Cobalamin (vitamin B12)-deficiency-induced changes in the proteome of rat cerebrospinal fluid
Authors: GIANAZZA Elisabetta , VEBER Daniela , EBERINI Ivano , BUCCELLATO Francesca R. , MUTTI Elena , SIRONI Luigi , SCALABRINO Giuseppe
Published: August 2003

We studied the changes in the proteome of CSF (cerebrospinal fluid) in two animal models of Cbl (cobalamin) deficiency: TGX (totally gastrectomized) rats and rats fed a Cbl-D (Cbl-deficient) diet. Two-dimensional PAGE was used to detect qualitative and quantitative variations in proteins in the CSF samples. The peak increase in total CSF protein concentration was observed 4 months after TG (total gastrectomy) and after 6 months of eating a Cbl-D diet. There is a specific increase 4 months after TG in the spots corresponding to α1-antitrypsin and the de novo presence of thiostatin and haptoglobin β. Cbl-replacement treatment in 4-month-TGX rats corrected these alterations in the CSF proteome. However, most of the CSF proteome alterations attenuated in Cbl-untreated 8-month-TGX rats and in rats fed a Cbl-D diet for 16 months. Transthyretin concentration varied slightly in the CSF of both types of Cbl-D rat, whereas the relative abundance of prostaglandin D synthase rose sharply in the CSF of the rats fed a Cbl-D diet for 16 months. We have demonstrated previously that the histological and ultrastructural CNS (central nervous system) damage in both types of Cbl-D rat appears within 2–3 months of Cbl deficiency, and thus appears to precede the alterations in the CSF proteome. The CSF proteome patterns of rats in which phlogosis was induced in or outside the CNS are quite different from those of the CSF of Cbl-D rats. All these findings demonstrate that the alterations in the CSF proteome of Cbl-D rats are specifically linked to Cbl deficiency.
The acetyltransferase 60 kDa trans-acting regulatory protein of HIV type 1-interacting protein (Tip60) interacts with the translocation E26 transforming-specific leukaemia gene (TEL) and functions as a transcriptional co-repressor
Authors: NORDENTOFT Iver , JØRGENSEN Poul
Published: August 2003

The translocation E26 transforming-specific (ETS) leukaemia (TEL), alias the ETS variant (ETV6), gene is expressed in most human tissues and encodes a transcriptional repressor. The TEL gene is involved in more than 40 different chromosomal translocations associated with haematological malignancies. As little is known about the function of intact TEL, we searched for TEL-interacting proteins by yeast two-hybrid screening. Among the interacting partners, we identified the histone acetyltransferase protein Tip60 [60 kDa trans-acting regulatory protein of HIV type 1 (Tat)-interacting protein]. The interaction was reproduced in vitro, and in mammalian cells we mapped the interaction regions in TEL to the ETS domain and those in Tip60 to the MYST (‘MOZ, Ybf2/Sas3, SAS2 and Tip60’, where MOZ stands for male absent on the first, SAS for something about silencing and Ybf2 for identical with SAS2) region. Detailed analysis of the Tip60 MYST domain by introduction of point mutations revealed that an N-terminal C2HC zinc finger was essential for interaction with TEL. Finally, we showed that Tip60 functions in a reporter system as a co-repressor in TEL-mediated transcription repression.