bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of PACAP receptor in primary afferent fibre‐evoked responses of ventral roots in the neonatal rat spinal cord
Authors: Sakashita Yoshihiko , Kurihara Takashi , Uchida Daigaku , Tatsuno Ichiro , Yamamoto Tatsuo
Published: January 2005

The role of PACAP receptor in nociceptive transmission was investigated in vitro using maxadilan, a PACAP receptor selective agonist and max.d.4, a PACAP receptor selective antagonist.

Potentials, from a ventral root (L3 – L5) of an isolated spinal cord preparation or a spinal cord – saphenous nerve – skin preparation from 0 – 3‐day‐old rats, were recorded extracellularly.

In the isolated spinal cord preparation, single shock stimulation of a dorsal root at C‐fibre strength induced a slow depolarizing response lasting about 30 s (slow ventral root potential; slow VRP) in the ipsilateral ventral root of the same segment. Bath‐application of max.d.4 (0.01 – 3 μM) inhibited the slow VRP in a concentration‐dependent manner.

In the spinal cord – saphenous nerve – skin preparation, application of capsaicin (0.1 μM) to the skin evoked a depolarization of the ventral root. This response was also depressed by max.d.4 (1 μM).

Application of maxadilan evoked a long‐lasting depolarization in a concentration‐dependent manner in the spinal cord preparation. In the presence of max.d.4 (0.3 μM), the concentration response curve of maxadilan was shifted to the right.

Reverse transcription‐polymerase chain reaction (RT – PCR) experiments demonstrated the existence of PACAP receptor and VPAC2 receptor in the neonatal rat spinal cord and [125I]‐PACAP27 binding was displaced almost completely by maxadilan and max.d.4, but not by vasoactive intestinal peptide (VIP). These data indicate that PACAP receptor is dominantly distributed in the neonatal rat spinal cord.

The present study suggests that PACAP receptor may play an excitatory role in nociceptive transmission in the neonatal rat spinal cord.

British Journal of Pharmacology (2001) 132, 1769–1776; doi:10.1038/sj.bjp.0703980
5‐HT4 receptors mediating enhancement of contractility in canine stomach; an in vitro and in vivo study
Authors: Prins N H , Grijn A van der , Lefebvre R A , Akkermans L M A , Schuurkes J A J
Published: January 2005

We aimed to study 5‐HT4 receptors in canine stomach contractility both in vivo and in vitro.

In anaesthetized Beagle dogs, the selective 5‐HT4 receptor agonist prucalopride (i.v.) induced dose‐dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the selective 5‐HT4 receptor antagonist GR 125487 (10 μg kg−1, i.v.).

Electrical field stimulation (EFS) of corpus longitudinal muscle strips resulted in atropine‐ and tetrodotoxin‐sensitive contractions (L‐NOARG (0.1 mM) present in all organ bath solutions). Prucalopride increased these contractions (maximal response after single‐dose addition (0.3 μM): 165% of initial value, or after cumulative addition: 188%). In the presence of methysergide (3 μM), 5‐HT also increased EFS‐contractions (after single‐dose addition (0.3 μM): increase to 192%, after cumulative addition: 148%).

The selective 5‐HT4 receptor antagonists GR 113808 (0.1 μM) or GR 125487 (10 nM) antagonized the prucalopride (0.3 μM)‐induced contraction increments. When EFS‐induced contractions were blocked by atropine or tetrodotoxin, prucalopride was ineffective. In the presence of methysergide (3 μM), the contraction increases to 5‐HT (0.3 μM) were prevented by GR 113808 (0.1 μM).

The prucalopride curve (pEC50 7.9) was shifted in parallel to the right by GR 113808 3 nM (pA2 9.4). In the presence of methysergide (3 μM), the curve to 5‐HT (pEC50 8.1) was competitively antagonized by GR 113808, yielding a Schild slope of 0.8±0.2 (pKB of 9.1 with unit Schild slope).

In corpus circular muscle strips, the prucalopride (0.3 μM)‐induced augmentation of EFS‐contractions (258%) was also prevented by GR 113808 (0.1 μM) (124%).

In conclusion, the effects of 5‐HT4 receptor agonists on proximal stomach motor activity in vivo can be explained by an effect on 5‐HT4 receptors on cholinergic nerves within the gastric muscle wall.

British Journal of Pharmacology (2001) 132, 1941–1947; doi:10.1038/sj.bjp.0703985
Rat arterial smooth muscle devoid of ryanodine receptor function: effects on cellular Ca2+ handling
Authors: Dreja Karl , Nordström Ina , Hellstrand Per
Published: January 2005

The roles of intracellular Ca2+ stores and ryanodine (Ry) receptors for vascular Ca2+ homeostasis and viability were investigated in rat tail arterial segments kept in organ culture with Ry (10 – 100 μM) for up to 4 days.

Acute exposure to Ry or the non‐deactivating ryanodine analogue C10‐Oeq glycyl ryanodine (10 μM) eliminated Ca2+ release responses to caffeine (20 mM) and noradrenaline (NA, 10 μM), whereas responses to NA, but not caffeine, gradually returned to normal within 4 days of exposure to Ry.

Ry receptor protein was detected on Western blots in arteries cultured either with or without Ry.

Brief Ca2+ release events (sparks) were absent after culture with Ry, whereas Ca2+ waves still occurred. The propagation velocity of waves was equal (∼19 μm s−1) in tissue cultured either with or without Ry.

Inhibition of Ca2+ accumulation into the sarcoplasmic reticulum (SR) by culture with caffeine (5 mM), cyclopiazonic acid or thapsigargin (both 10 μM) decreased contractility due to Ca2+‐induced cell damage. In contrast, culture with Ry did not affect contractility.

Removal of Ca2+ from the cytosol following a Ca2+ load was retarded after Ry culture. Thapsigargin reduced the rate of Ca2+ removal in control cultured rings, but had no effect after Ry culture.

It is concluded that intracellular Ca2+ stores recover during chronic Ry treatment, while Ry receptors remain non‐functional. Ry receptor activity is required for Ca2+ sparks and for SR‐dependent recovery from a Ca2+ load, but not for Ca2+ waves or basal Ca2+ homeostasis.

British Journal of Pharmacology (2001) 132, 1957–1966; doi:10.1038/sj.bjp.0703986
Enhanced acetylcholine release in the hippocampus of cannabinoid CB1 receptor‐deficient mice
Authors: Kathmann Markus , Weber Bernd , Zimmer Andreas , Schlicker Eberhard
Published: January 2005

We examined whether acetylcholine release in the hippocampus and striatum and noradrenaline release in the hippocampus is altered in CB1 receptor‐deficient mice. The electrically evoked tritium overflow from hippocampal slices preincubated with [3H]‐choline was increased by about 100% in CB1−/− compared to CB1+/+ mice whereas the electrically evoked tritium overflow from striatal slices preincubated with [3H]‐choline and from hippocampal slices preincubated with [3H]‐noradrenaline did not differ. The cannabinoid receptor agonist, WIN 55,212‐2, inhibited, and the CB1 receptor antagonist, SR 141716, facilitated, the evoked tritium overflow from hippocampal slices (preincubated with [3H]‐choline) from CB1+/+ as opposed to CB1−/− mice. Both drugs did not affect the evoked tritium overflow from striatal slices (preincubated with [3H]‐choline) and from hippocampal slices (preincubated with [3H]‐noradrenaline) from CB1+/+ and CB1−/− mice. The selective increase in acetylcholine release in CB1−/− mice may indicate that the presynaptic CB1 receptors on the cholinergic neurones of the mouse hippocampus are tonically activated and/or constitutively active in vivo.

British Journal of Pharmacology (2001) 132, 1169–1173; doi:10.1038/sj.bjp.0703987
Dose‐related neuroprotective effects of chronic nicotine in 6‐hydroxydopamine treated rats, and loss of neuroprotection in α4 nicotinic receptor subunit knockout mice
Authors: Ryan R E , Ross S A , Drago J , Loiacono R E
Published: January 2005

The present study examined the effect of a range of doses of chronic nicotine (0.75, 1.5, 3.0 and 30.0 mg kg−1 day−1, s.c., 14 days) upon striatal dopaminergic nerve terminal survival following 6‐hydroxydopamine (6‐OHDA; 10 μg intrastriatal unilaterally) in rats; and the effects of acute nicotine (1 mg kg−1, s.c.) pretreatment upon striatal neurodegeneration induced by methamphetamine (5 mg kg−1, i.p., three doses at 2 h intervals) in wild‐type and α4 nicotinic receptor (nAChR) subunit knockout mice.

In both models of Parkinsonian‐like damage, loss of striatal dopaminergic nerve terminals was assessed by [3H]‐mazindol autoradiography.

In rats, chronic nicotine infusion delivered by osmotic minipump implanted subcutaneously 7 days prior to intrastriatal 6‐OHDA injection produced significant and dose‐related protection against 6‐OHDA‐induced neurodegeneration. Low (0.75 and 1.5 mg kg−1 day−1) but not high (3.0 and 30.0 mg kg−1 day−1) nicotine doses significantly inhibited 6‐OHDA‐induced degeneration.

In wild‐type mice, acute nicotine treatment produced significant inhibition of methamphetamine‐induced neurodegeneration. In α4 nAChR subunit knockout mice, acute nicotine treatment failed to inhibit methamphetamine‐induced neurodegeneration.

Nicotine is capable of protecting dopaminergic neurons against Parkinsonian‐like neurodegeneration in vivo. In rats, this neuroprotective effect is critically dependent upon nicotine dose and is consistent with the activation of nAChRs, as high, desensitizing doses of nicotine fail to be neuroprotective. Further, neuroprotection is absent in α4 nAChR subunit knockout mice. The current results therefore suggest that activation of α4 subunit containing nAChRs constitutes a major component of the neuroprotective effect of nicotine upon Parkinsonian‐like damage in vivo.

British Journal of Pharmacology (2001) 132, 1650–1656; doi:10.1038/sj.bjp.0703989
Characterization of β1‐selectivity, adrenoceptor‐Gs‐protein interaction and inverse agonism of nebivolol in human myocardium
Authors: Maack C , Tyroller S , Schnabel P , Cremers B , Dabew E , Südkamp M , Böhm M
Published: January 2005

Intrinsic activity and β1‐selectivity are important features of β‐blockers in the treatment of patients with coronary syndromes and heart failure. In human myocardium, intrinsic activity and β1‐selectivity of the novel β‐adrenoceptor antagonist nebivolol have not yet been determined.

The study examines intrinsic activity, β‐adrenoceptor‐G‐protein coupling and β1‐selectivity of nebivolol and bisoprolol in human ventricular myocardium. Furthermore, intrinsic activity of both compounds is compared to the one of bucindolol, carvedilol and metoprolol in human atrial myocardium.

Radioligand binding studies ([125I]‐lodocyanopindolol) were performed on membrane preparations of human failing and nonfailing myocardium and on COS‐7 cells transfected with human β1‐ and β2‐adrenoceptors, respectively. Functional experiments were carried out on isolated muscle preparations of human left ventricular and right atrial myocardium from failing and nonfailing hearts.

Radioligand binding studies reveal 3 – 4 fold β1‐selectivity for nebivolol and 16 – 20 fold β1‐selectivity for bisoprolol in human myocardium. In COS‐7‐cells, β1‐selectivity is 3 fold for nebivolol and 15 fold for bisoprolol.

Neither the binding of nebivolol nor of bisoprolol is affected by the presence of guanylylimidodiphosphate (Gpp(NH)p).

Nebivolol and bisoprolol exert similar inverse agonist activity in human ventricular as well as atrial myocardium. In atrial myocardium, inverse agonism of both compounds is higher compared to bucindolol, equal to carvedilol and lower compared to metoprolol.

Favourable haemodynamic effects of nebivolol in humans are not due to β1‐selectivity or partial agonist activity of this agent. Other mechanisms, i.e. the production of nitric oxide, may thus be responsible for its unique haemodynamic profile.

British Journal of Pharmacology (2001) 132, 1817–1826; doi:10.1038/sj.bjp.0703992
In vivo steady‐state pharmacokinetic outcome following clinical and toxic doses of racemic citalopram to rats
Authors: Kugelberg Fredrik C , Apelqvist Gustav , Carlsson Björn , Ahlner Johan , Bengtsson Finn
Published: January 2005

The thymoleptic drug citalopram (CIT) belongs to the selective serotonin reuptake inhibitors (SSRIs) and is today extensively used in psychiatry. Further clarification of the enantiomer‐selective distribution of racemic CIT in both clinical and toxic doses is highly warranted.

By a steady‐state in vivo paradigm, rats underwent chronic systemic exposure for 10 days by using osmotic pumps and the total as well as the individual distributions of the S‐ and R‐enantiomers of CIT, and its metabolites in serum and two different brain regions, were analysed.

In serum, the S/R ratios in the groups treated with 10, 20, or 100 mg kg−1 day−1 were 0.94, 0.83, and 0.34, respectively. The ratios were almost the same in the brain regions.

In the group treated with 100 mg kg−1 day−1, the serum and brain total CIT levels were found to be 20 times and 6 – 8 times higher than in the rats treated with 10 or 20 mg kg−1 day−1, respectively. In all groups, the CIT levels were higher in brain tissue as compared to serum.

In a spontaneous open‐field behavioural test, a correlation between clinical and toxic drug concentrations was observed.

In conclusion, the R‐enantiomer was present in an increased proportion compared with the S‐enantiomer when higher steady‐state CIT concentration was prevailing. This is of particular interest, since the S‐enantiomer is responsible for the inhibition of serotonin reuptake in vitro. The present data may be of importance, as full understanding on where different racemic or enantiomeric drug effects of CIT and its main metabolites are unravelled.

British Journal of Pharmacology (2001) 132, 1683–1690; doi:10.1038/sj.bjp.0704015
Allosteric modulation of [3H]‐CGP39653 binding through the glycine site of the NMDA receptor: further studies in rat and human brain
Authors: Mugnaini Manolo , Meoni Paolo , Bunnemann Bernd , Corsi Mauro , Bowery Norman G
Published: January 2005

Binding of D,L‐(E)‐2‐amino‐4‐[3H]‐propyl‐5‐phosphono‐3‐pentenoic acid ([3H]‐CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques.

In rat cerebral cortical membranes the glycine antagonist 3‐[2‐(Phenylaminocarbonyl)ethenyl]‐4,6‐dichloro‐indole‐2‐carboxylic acid sodium salt (GV150526A) did not change basal [3H]‐CGP39653 binding, but competitively reversed the high affinity component of [3H]‐CGP39653 binding inhibition by glycine, with a pKB value of 8.38, in line with its affinity for the glycine site (pKi=8.49 vs [3H]‐glycine). Glycine (10 μM) significantly decreased [3H]‐CGP39653 affinity for the NMDA receptor (with no change in the Bmax), whereas enhanced L‐glutamate affinity (P<0.05, paired‐samples Student's t‐test).

In rat brain sections the addition of GV150526A (30 μM) to the incubation medium increased [3H]‐CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two‐way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [3H]‐CGP39653, L‐glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation.

Whole brain autoradiography revealed an uneven distribution of [3H]‐CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [3H]‐CGP39653 binding was increased to 216% of control (mean between areas) by 30 μM GV150526A. The enhancement, however, did not present significant regional differences.

These results introduce GV150526A as a useful tool to identify NMDA receptor subtypes by means of receptor autoradiography; moreover, they demonstrate that the allosteric inhibition of [3H]‐CGP39653 binding by glycine parallels an increase in receptor affinity to the endogenous ligand L‐glutamate. Finally, this study provides the first detailed anatomical description of the regional distribution of [3H]‐CGP39653 binding sites in human brain.

British Journal of Pharmacology (2001) 132, 1883–1897; doi:10.1038/sj.bjp.0704017
Incorporation of sodium channel blocking and free radical scavenging activities into a single drug, AM‐36, results in profound inhibition of neuronal apoptosis
Authors: Callaway Jennifer K , Beart Philip M , Jarrott Bevyn , Giardina Sarah F
Published: January 2005

AM‐36 is a novel neuroprotective agent incorporating both antioxidant and Na+ channel blocking actions. In cerebral ischaemia, loss of cellular ion homeostasis due to Na+ channel activation, together with increased reactive oxygen species (ROS) production, are thought to contribute to neuronal death. Since neuronal death in the penumbra of the ischaemic lesion is suggested to occur by apoptosis, we investigated the ability of AM‐36, antioxidants and Na+ channel antagonists to inhibit toxicity induced by the neurotoxin, veratridine in cultured cerebellar granule cells (CGC's).

Veratridine (10 – 300 μM) concentration‐dependently reduced cell viability of cultured CGC's. Under the experimental conditions employed, cell death induced by veratridine (100 μM) possessed the characteristics of apoptosis as assessed by morphology, TUNEL staining and DNA laddering on agarose gels.

Neurotoxicity and apoptosis induced by veratridine (100 μM) were inhibited to a maximum of 50% by the antioxidants, U74500A (0.1 – 10 μM) and U83836E (0.03 – 10 μM), and to a maximum of 30% by the Na+ channel blocker, dibucaine (0.1 – 100 μM). In contrast, AM‐36 (0.01 – 10 μM) completely inhibited veratridine‐induced toxicity (IC50 1.7 (1.5 – 1.9) μM, 95% confidence intervals (CI) in parentheses) and concentration‐dependently inhibited apoptosis.

These findings suggest veratridine‐induced toxicity and apoptosis are partially mediated by generation of ROS. AM‐36, which combines both Na+ channel blocking and antioxidant activity, provided superior neuroprotection compared with agents possessing only one of these actions. This bifunctional profile of activity may underlie the potent neuroprotective effects of AM‐36 recently found in a stroke model in conscious rats.

British Journal of Pharmacology (2001) 132, 1691–1698; doi:10.1038/sj.bjp.0704018
Existence of different α1‐adrenoceptor subtypes in junctional and extrajunctional neurovascular regions in canine splenic arteries
Authors: Yang Xiao‐Ping , Chiba Shigetoshi
Published: January 2005

The present study attempted to characterize the α1‐adrenoceptor subtypes mediating vasoconstrictor responses to administered and nerve stimulation‐evoked noradrenaline (NA) release in the isolated and perfused canine splenic artery.

A previous study demonstrated that periarterial electrical nerve stimulation (30 s trains of pulses at a frequency of 1, 4 or 10 Hz) induced a double peaked vasoconstriction consisting of an initial transient, predominantly P2X‐purinoceptor‐mediated constriction followed by a prolonged, mainly α1‐adrenoceptor‐mediated response in the canine splenic artery.

The effects of α1‐adrenoceptor subtype antagonists on neuronally‐mediated second peaked vasoconstrictions were analysed. BMY 7378 (10 – 100 nM), a selective α1D‐adrenoceptor antagonist produced a dose‐dependent inhibition of the second peak responses at all frequencies used. BMY 7378 (100 nM) reduced these responses by approximately 30%. Exposure of tissues to chloroethylclonidine (CEC, 60 μM), a selective α1B‐adrenoceptor antagonist attenuated the second peak response by approximately 60%, even in the presence of BMY 7378 (100 nM). On the other hand, WB 4101 (100 nM), a selective α1A‐adrenoceptor antagonist potentiated nerve‐stimulation‐evoked double peaked vasoconstrictions, especially at low frequencies (1 and 4 Hz).

Vasoconstrictor responses to administered NA were dose‐dependently antagonized by WB 4101 (10 – 100 nM), but were not significantly affected by either BMY 7378 (10 – 100 nM) or by CEC (60 μM).

The present results indicate that NA released from sympathetic nerves may junctionally exert its vasoconstrictor effect via activation of postjunctional α1B‐ and in part α1D‐adrenoceptors, whereas exogenous NA extrajunctionally activates α1A‐adrenoceptors to produce its vascular action in canine splenic arteries.

British Journal of Pharmacology (2001) 132, 1852–1858; doi:10.1038/sj.bjp.0704020
Selective inhibition of human inducible nitric oxide synthase by S‐alkyl‐L‐isothiocitrulline‐containing dipeptides
Authors: Park Jung‐Min , Higuchi Tsunehiko , Kikuchi Kazuya , Urano Yasuteru , Hori Hiroyuki , Nishino Takeshi , Aoki Junken , Inoue Keizo , Nagano Tetsuo
Published: January 2005

The aim of this study was to investigate the structure‐activity relationship of S‐alkyl‐L‐isothiocitrulline‐containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine‐induced NO production by human DLD‐1 cells.

In an in vitro assay, S‐methyl‐L‐isothiocitrulline (L‐MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L‐amino acid (L‐Phe, L‐Leu or L‐Trp) with L‐MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L‐MIT‐L‐Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S‐alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS.

These results suggest that the human NOS isozymes have different‐sized cavities in the binding site near the position to which the C‐terminal of L‐arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L‐MIT‐D‐Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well‐defined direction from the α carbon atom.

NO production in cytokine‐stimulated human DLD‐1 cells was measured with a fluorescent indicator, DAF‐FM. MILF, L‐MIT‐L‐Trp(‐CHO) (MILW) and L‐MIT‐L‐Tyr (MILY) showed more potent activity than L‐MIT in this whole‐cell assay.

Thus, S‐alkyl‐L‐isothiocitrulline‐containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell‐based assay.

British Journal of Pharmacology (2001) 132, 1876–1882; doi:10.1038/sj.bjp.0704023
The polycationic aminoglycosides modulate the vasoconstrictive effects of endothelin: relevance to cerebral vasospasm
Authors: Wickman Grant , Nessim Mourad A , Cook David A , Vollrath Bozena
Published: January 2005

The vasoactive peptide endothelin (ET) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage. In these studies we investigated the involvement of protein kinase C (PKC) in sustained vasoconstriction induced by ET‐1 in canine cerebral arteries. We also examined the ability of the aminoglycoside antibiotics to reverse the effects mediated by ET‐1 in canine cerebrovascular smooth muscle cells (CVSMC).

The ETA receptor antagonist, BQ‐123, showed a competitive inhibition of the ET‐1 responses.

The vasoconstrictor action of both ET‐1 (0.5 nM) and phorbol myristate acetate (PMA) (160 nM) was reversed by a selective PKC inhibitor, Ro‐32‐0432.

In cerebral arteries precontracted with ET‐1 the aminoglycosides caused a concentration‐dependent relaxation. The EC50s for the relaxation were as follows: 0.54±0.05, 0.63±0.01, 1.88±0.46 and 2.3±0.92 mM for gentamicin, neomycin, streptomycin and kanamycin, respectively.

Gentamicin caused a concentration‐dependent decrease of the PMA‐induced responses in calcium free medium.

PKC activity was elevated in CVSMC exposed to ET‐1 (170%) and PMA (167%) for a period of time (60 min) corresponding to maximum tonic contraction induced by these agents in arterial rings.

The administration of the aminoglycosides to CVSMC, in concentrations corresponding to the EC50s from contractility studies, reduced the effects of both ET‐1 and PMA on PKC activity to the levels not different from controls.

These results show that the aminoglycosides are able to inhibit sustained vasoconstriction induced by ET‐1, an effect which is due, at least in part, to the inhibition of PKC.

British Journal of Pharmacology (2001) 133, 5–12; doi:10.1038/sj.bjp.0704025
Endogenous opioids suppress activation of nociceptors by sub‐nanomolar nicotine
Authors: Miao Frederick J‐P , Benowitz Neal L , Levine Jon D
Published: January 2005

Nicotine can activate primary afferent nociceptors, one result of which is to increase neurogenic plasma extravasation. In this study we have demonstrated a novel proinflammatory effect of sub‐nanomolar nicotine, mediated by peripheral action at sensory neurons. This action is normally masked by adrenal medulla‐derived δ‐opioid receptor agonists.

While neurogenic plasma extravasation in the knee joint of the rat was not increased by intra‐articular perfusion of nicotine (10−8 M), perfusion of nicotine, at concentrations as low as 10−10 M, combined with naloxone to block opioid receptors (or naltrindole to block δ‐opioid receptors) was able to enhance bradykinin‐induced plasma extravasation. This pro‐inflammatory effect of intra‐articular nicotine was mimicked by subcutaneous nicotine which was abolished by intra‐articularly‐administered hexamethonium, a nicotinic receptor antagonist.

Following denervation of the adrenal medulla, intra‐articular nicotine, alone at 10−8 M, enhanced plasma extravasation, which was no longer enhanced by naloxone.

Destruction of primary afferents by neonatal treatment with capsaicin or blockade of sensory neurotransmitter by neurokinin‐1 receptor antagonist RP‐87,580 abolished the pro‐inflammatory effect of nicotine.

The effect of nicotine we describe in promoting inflammation is exerted at extremely low concentrations and therefore could have relevance to smokers, patients receiving medicinal nicotine as therapy and even second‐hand smokers. Since receptor mechanisms on peripheral terminals of nociceptors may also be present on central terminals, actions of the endogenous nicotinic agonist acetylcholine, at central terminals of primary afferents or at other sites in the central nervous system, may be similarly modulated by opioids.

British Journal of Pharmacology (2001) 133, 23–28; doi:10.1038/sj.bjp.0704031
Mechanical effects of liriodenine on the left ventricular‐arterial coupling in Wistar rats: pressure‐stroke volume analysis
Authors: Chang Kuo‐Chu , Su Ming‐Jai , Peng Ying‐I , Shao Ching‐Chih , Wu Yang‐Chang , Tseng Yung‐Zu
Published: January 2005

In a recent in vivo study, liriodenine, an aporphine alkaloid, has been identified as a prominent anti‐arrhythmic agent that can prevent rats' sudden deaths, even at the dose as low as 10−7 g kg−1. The aim of this study was to determine whether liriodenine at its effective anti‐arrhythmic dose of 10−7 g kg−1 had effects on the left ventricular (LV)‐arterial coupling in Wistar rats.

LV pressure and ascending aortic flow signals were recorded to construct the ventricular and arterial end‐systolic pressure‐stroke volume relationships to calculate LV end‐systolic elastance (Ees) and effective arterial volume elastance (Ea), respectively. The optimal afterload (Qload) determined by the ratio of Ea to Ees was used to measure the optimality of energy transmission from the left ventricle to the arterial system.

Liriodenine at the dose of 10−7 g kg−1 showed no significant changes in basal heart rate (HR), cardiac output (CO), LV end‐systolic pressure (Pes), Ea, Ees, and Qload.

By contrast, liriodenine at the dose of 10−6 g kg−1 produced a significant fall of 2.0% in HR and a significant rise of 5.8% in CO, but no significant change in Pes. Moreover, liriodenine administration of 10−6 g kg−1 to rats significantly decreased Ees by 8.5% and Ea by 10.6%, but did not change Qload.

We conclude that liriodenine at the dose of 10−7 g kg−1 has no effects on the mechanical properties of the heart and the vasculature and the matching condition for the left ventricle coupled to its vasculature in rats. Even at 10 times the effective anti‐arrhythmic dose, liriodenine shows no effects on the efficiency of energy transferred from the left ventricle to the arterial system.

British Journal of Pharmacology (2001) 133, 29–36; doi:10.1038/sj.bjp.0704036
Mutation screening of the muscarinic M2 and M3 receptor genes in normal and asthmatic subjects
Authors: Fenech A G , Ebejer M J , Felice A E , Ellul‐Micallef R , Hall I P
Published: January 2005

Muscarinic receptors are important in the development of airway hyperresponsiveness, and dysfunction of these receptors has been suggested to be present in asthma.

The human muscarinic M2 and M3 receptor genes were screened for polymorphic variation using single‐stranded conformation polymorphism (SSCP) analysis, complemented by direct fluorescent sequencing. Forty‐six random DNA samples and 46 respiratory physician diagnosed asthmatic samples were used as a template for analysis.

Within the muscarinic M2 receptor gene, we identified two degenerate single base substitutions (1197T→C, Thr→Thr and 976A→C, Arg→Arg) in one random and one asthmatic sample respectively. Analysis of the 3′ UTR region revealed an additional ‘A’ at bp 1793 (c.f. ATG). This was present in all of 49 samples analysed by sequencing or BsmI digest, suggesting that the published sequence (GenBank Accession No. M16404) is incorrect. A common 3′ UTR polymorphism (T→A) was found at bp 1696 (c.f. ATG) (allelic frequency=65%, n=60), but this does not alter transcription factor recognition sites.

We were unable to identify any polymorphic variation within the muscarinic M3 coding region or the flanking regions investigated, using the methods described.

The coding regions for the human muscarinic M2 and M3 receptor genes are both highly conserved. These data suggest that polymorphic variation within these coding sequences is unlikely to account for inter‐individual variability in response to methacholine or anticholinergic therapy. The potential functional significance of the muscarinic M2 receptor 3′ UTR polymorphism (bp 1696) remains to be determined.

British Journal of Pharmacology (2001) 133, 43–48; doi:10.1038/sj.bjp.0704039
The biphasic immunoregulation of pyrimidylpiperazine (Y‐40138) is IL‐10 sensitive and requires NF‐κB targeting in the alveolar epithelium
Authors: Haddad J J E , Safieh‐Garabedian B , Saadé N E , Land S C
Published: January 2005

Pyrimidylpiperazine (Y‐40138), a synthetic derivative of N‐[1‐(4‐{[4‐(pyrimidin‐2‐yl)piperazin‐1‐yl]methyl}phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro‐ and anti‐inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro.

In alveolar epithelial cells, pre‐treatment (30 min) with Y‐40138 reduced LPS‐induced biosynthesis of IL‐1β, IL‐6 and TNF‐α, an effect paralleled by up‐regulating an anti‐inflammatory counter‐loop mediated through IL‐10.

This differential regulation of pro‐ and anti‐inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF‐κB subunits, particularly NF‐κB1 (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c‐Rel (p75). In addition, Y‐40138 blockaded, in a dose‐dependent manner, the LPS‐induced nuclear activation of NF‐κB.

Analysis of the upstream pathway involved in Y‐40138‐dependent retardation of LPS‐induced NF‐κB translocation/activation revealed the involvement of an IκB‐α sensitive pathway. Pre‐treatment with Y‐40138 ameliorated LPS‐induced degradation of IκB‐α in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase.

Recombinant IL‐10 (0 – 10 ng ml−1) blockaded, in a dose‐dependent manner, LPS‐induced biosynthesis of IL‐1β, IL‐6 and TNF‐α. Furthermore, rhIL‐10 reduced the DNA binding activity of NF‐κB. Immunoneutralization of endogenous IL‐10 by a polyclonal αIL‐10 (5 μg ml−1) reversed the inhibitory effect of Y‐40138 on pro‐inflammatory cytokines and partially restored the DNA binding activity of NF‐κB.

These results indicate that Y‐40138 mediated dual immunoregulation of pro‐ and anti‐inflammatory cytokines is IL‐10 sensitive and mediated through the IκB‐α/NF‐κB signal transduction pathway.

British Journal of Pharmacology (2001) 133, 49–60; doi:10.1038/sj.bjp.0704041
NK‐104, a 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitor, reduces osteopontin expression by rat aortic smooth muscle cells
Authors: Takemoto Minoru , Kitahara Masaki , Yokote Koutaro , Asaumi Sunao , Take Ayako , Saito Yasushi , Mori Seijiro
Published: January 2005

It has been suggested that osteopontin promotes the development of atherosclerosis, especially under diabetic conditions.

In the present study, we found that NK‐104, a new potent synthetic inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, reduced osteopontin expression both at protein and mRNA levels in cultured rat aortic smooth muscle cells.

The inhibitory effect of NK‐104 was almost completely reversed by mevalonate, suggesting that mevalonate or its metabolites play important roles in the regulation of osteopontin expression.

Furthermore, oral administration of NK‐104 (3 mg kg−1 day−1 for 7 days) effectively suppressed abnormally upregulated expression of osteopontin mRNA in the aorta and kidney of streptozotocin‐induced diabetic rats.

These data support a notion that NK‐104 is a suitable drug for the treatment of diabetic patients with hypercholesterolaemia.

British Journal of Pharmacology (2001) 133, 83–88; doi:10.1038/sj.bjp.0704046
Protection against β‐amyloid peptide toxicity in vivo with long‐term administration of ferulic acid
Authors: Yan Ji‐Jing , Cho Jae‐Young , Kim Hee‐Sung , Kim Kyoung‐Li , Jung Jun‐Sub , Huh Sung‐Oh , Suh Hong‐Won , Kim Yung‐Hi , Song Dong‐Keun
Published: January 2005

β‐Amyloid peptide (Aβ), a 39 – 43 amino acid peptide, is believed to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease. Ferulic acid is an antioxidant and anti‐inflammatory agent derived from plants; therefore, the potential protective activity of ferulic acid against Aβ toxicity in vivo was examined.

Mice were allowed free access to drinking water (control) or water containing ferulic acid (0.006%). After 4 weeks, Aβ1‐42 (410 pmol) was administered via intracerebroventricular injection.

Injection of control mice with Aβ1‐42 impaired performance on the passive avoidance test (35% decrease in step‐through latency), the Y‐maze test (19% decrease in alternation behaviour), and the water maze test (32% decrease in percentage time in platform‐quadrant). In contrast, mice treated with ferulic acid prior to Aβ1‐42 administration were protected from these changes (9% decrease in step‐through latency; no decrease in alternation behaviour; 14% decrease in percentage time in platform‐quadrant). Aβ1‐42 induced 31% decrease in acetylcholine level in the cortex, which was tended to be ameliorated by ferulic acid.

In addition, Aβ1‐42 increased immunoreactivities of the astrocyte marker glial fibrillary acidic protein (GFAP) and interleukin‐1β (IL‐1β) in the hippocampus, effects also suppressed by pretreatment with ferulic acid.

Administration of ferulic acid per se unexpectedly induced a transient and slight increase in GFAP and IL‐1β immunoreactivity in the hippocampus on day 14, which returned to basal levels on day 28. A slight (8%) decrease in alternation behaviour was observed on day 14.

These results demonstrate that long‐term administration of ferulic acid induces resistance to Aβ1‐42 toxicity in the brain, and suggest that ferulic acid may be a useful chemopreventive agent against Alzheimer's disease.

British Journal of Pharmacology (2001) 133, 89–96; doi:10.1038/sj.bjp.0704047
Histamine H3 receptor‐mediated inhibition of depolarization‐induced, dopamine D1 receptor‐dependent release of [3H]‐γ‐aminobutyric acid from rat striatal slices
Authors: Arias‐Montaño J‐A , Floran B , Garcia M , Aceves J , Young J M
Published: January 2005

A study was made of the regulation of [3H]‐γ‐aminobutyric acid ([3H]‐GABA) release from slices of rat striatum by endogenous dopamine and exogenous histamine and a histamine H3‐agonist. Depolarization‐induced release of [3H]‐GABA was Ca2+‐dependent and was increased in the presence of the dopamine D2 receptor family antagonist, sulpiride (10 μM). The sulpiride‐potentiated release of [3H]‐GABA was strongly inhibited by the dopamine D1 receptor family antagonist, SCH 23390 (1 μM). Neither antagonist altered basal release.

The 15 mM K+‐induced release of [3H]‐GABA in the presence of sulpiride was inhibited by 100 μM histamine (mean inhibition 78±3%) and by the histamine H3 receptor‐selective agonist, immepip, 1 μM (mean inhibition 81±5%). The IC50 values for histamine and immepip were 1.3±0.2 μM and 16±2 nM, respectively. The inhibitory effects of histamine and immepip were reversed by the H3 receptor antagonist, thioperamide, 1 μM.

The inhibition of 15 mM K+‐induced [3H]‐GABA release by immepip was reversed by the H3 receptor antagonist, clobenpropit, Kd 0.11±0.04 nM. Clobenpropit alone had no effect on basal or stimulated release of [3H]‐GABA.

Elevated K+ caused little release of [3H]‐GABA from striatal slices from reserpinized rats, unless the D1 partial agonist, R(+)‐SKF 38393, 1 μM, was also present. The stimulated release in the presence of SKF 38393 was reduced by 1 μM immepip to the level obtained in the absence of SKF 38393.

These observations demonstrate that histamine H3 receptor activation strongly inhibits the dopamine D1 receptor‐dependent release of [3H]‐GABA from rat striatum; primarily through an interaction at the terminals of GABA neurones.

British Journal of Pharmacology (2001) 133, 165–171; doi:10.1038/sj.bjp.0704053
Enhancement of the effects of a complete inhibitor of enkephalin‐catabolizing enzymes, RB 101, by a cholecystokinin‐B receptor antagonist in diabetic rats
Authors: Coudoré‐Civiale Marie‐Ange , Méen Murielle , Fournié‐Zaluski Marie‐Claude , Boucher Michel , Roques Bernard P , Eschalier Alain
Published: January 2005

RB 101, a complete inhibitor of enkephalin‐catabolizing enzymes, has been previously shown to produce antinociception in normal rats after systemic administration. Moreover, its coadministration with a cholecystokinin‐B (CCK‐B) receptor antagonist has been shown to strongly enhance its antinociceptive effect in normal rats. In this work, we determined whether RB 101 was able to reduce hyperalgesia and allodynia in diabetic rats, a model of neuropathic pain. The type of opioid receptors (μ or δ) involved was determined using naloxone and naltrindole, respectively, and the interactions between endogenous enkephalins and CCK on nociception control was investigated using coadministration of RB 101 and the CCK‐B receptor antagonist CI‐988.

RB 101 suppressed mechanical hyperalgesia (paw pressure‐induced vocalization test), partially alleviated mechanical allodynia (von Frey hair test), and was ineffective in thermal allodynia (tail immersion test). The analgesic effect was completely cancelled by naloxone or naltrindole, suggesting that is requires the availability of μ‐ and/or δ‐opioid receptors.

The combination of an inactive dose of CI‐988 with the lowest effective dose of RB 101 resulted in a stronger increase in the vocalization threshold comparatively to RB 101 alone.

The present study demonstrates that the antinociception generated by RB 101 induced by elevation of extracellular levels of endogenous enkephalins, can be extended to neuropathic pain in diabetic rats and that blockade of CCK‐B receptors potentiated antinociceptive effects elicited by RB 101.

British Journal of Pharmacology (2001) 133, 179–185; doi:10.1038/sj.bjp.0704059
Effects of terbutaline on NGF formation in allergic inflammation of the rat
Authors: Amann Rainer , Peskar Bernhard A , Schuligoi Rufina
Published: January 2005

The aim of this study was to determine the effects of the beta adrenergic agonist terbutaline on NGF increase caused by allergic inflammation in rats.

Intraplantar antigen injection in sensitized rats increased paw volume and stimulated NGF biosynthesis in the skin of the injected paw as determined 3 and 6 h after injection. Treatment of rats with terbutaline (0.1 – 0.3 mg kg−1, s.c.) had no significant effect on the NGF concentration in non‐inflamed skin, but reduced oedema, and at 0.3 mg kg−1 also NGF mRNA and immunoreactive NGF in the skin of the inflamed paw in a propranolol‐reversible manner. In carrageenan‐induced inflammation, terbutaline did not significantly reduce the inflammation‐induced increase of NGF in paw skin.

Exposure of sensitized rats to aerosolized antigen (twice, 24 h interval) increased protein content, eosinophil leukocytes, and immunoreactive NGF in the bronchoalveolar lavage fluid (BAL, obtained 16 h after the second antigen exposure). Treatment of rats with terbutaline (0.3 mg kg−1, s.c. 30 min before the second antigen challenge) suppressed antigen‐induced elevation of protein and eosinophil leukocytes, and reduced the concentration of NGF in BAL to values similar to those found in non‐sensitized rats.

The present results demonstrate anti‐allergic properties of terbutaline in rats that were accompanied by a marked reduction of antigen‐induced NGF increase in skin and BAL, respectively. These results are compatible with the assumption that terbutaline primarily suppressed the immune response to antigen thereby attenuating the release of vasoactive mediators and the stimulation of NGF biosynthesis.

British Journal of Pharmacology (2001) 133, 186–192; doi:10.1038/sj.bjp.0704060
Neuromodulatory effect of propentofylline on rat brain under acute and long‐term hypoperfusion
Authors: Plaschke Konstanze , Grant Masilo , Weigand Markus A , Züchner Janet , Martin Eike , Bardenheuer Hubert J
Published: January 2005

The effects of propentofylline (PPF, 25 mg kg−1 body weight per day) on rat cerebral energy state and cytokine expression as well as on behaviour and histopathology were studied after acute and long‐term permanent bilateral common carotid artery occlusion (BCCAO).

In the absence of PPF, acute ischaemia led to a decrease in energy‐rich phosphates in parietotemporal cortex and hippocampus which correlated with an increase in AMP and adenosine concentrations measured by high‐performance liquid chromatography technique. The concentrations of cortical cytokines TNFα and IL1β were increased 12 and 19 fold, respectively.

PPF had a neuroprotective action after 20 min of BCCAO, reducing the deleterious effect of acute ischaemia on rat brain energy state and microglial reaction. Simultaneously, PPF treatment increased cyclic‐AMP 3 fold.

Three weeks of permanent BCCAO did not significantly disturb brain energy metabolism, microglial reaction or histopathology. However, a significant reduction of 30 – 50% in rat memory capacities and a locomotor hyperactivity were obtained.

Continuous PPF‐application, however, led to a marked increase in rat working memory and to reduced locomotor activity, which were returned nearly to control levels by 1 week after permanent BCCAO.

In summary, PPF showed a clear neuroprotective effect on cerebral energy state and pro‐inflammatory cytokines under conditions of acute global ischaemia. Continuous administration of PPF led to memory improvement during permanent BCCAO. These results underscore the benefit of treatment with PPF in clinical practice, particularly during stroke, but also in cerebrovascular and neurodegenerative disorders.

British Journal of Pharmacology (2001) 133, 107–116; doi:10.1038/sj.bjp.0704061
Effects of chemically modified tetracyclines (CMTs) in sensitive, multidrug resistant and apoptosis resistant leukaemia cell lines
Authors: Tolomeo Manlio , Grimaudo Stefania , Milano Salvatore , La Rosa Marzia , Ferlazzo Viviana , Di Bella Gloria , Barbera Caterina , Simoni Daniele , D'Agostino Pietro , Cillari Enrico
Published: January 2005

Recently discovered chemically modified tetracyclines (CMTs) have shown in vitro and in vivo anti‐proliferative and anti‐tumour activities. Here, we evaluated in vitro the anti‐proliferative and apoptotic activity of six different dedimethylamino chemically modified tetracyclines (CMT‐1, CMT‐3, CMT‐5, CMT‐6, CMT‐7 and CMT‐8) in sensitive and multidrug resistant myeloid leukaemia cells (HL60 and HL60R) in vitro.

Three of these compounds (CMT‐5, CMT‐6, CMT‐7) showed low cytotoxic activity both in sensitive and in resistant cells, CMT‐3 was endowed with a high anti‐proliferative activity only in sensitive cells and was moderately effective as apoptosis inducing agent, with an activity similar to that shown by doxycycline. On the contrary, CMT‐1 and CMT‐8 were very effective as programmed cell death inducing agents.

The apoptotic pathway activated by these compounds involved the activation of caspases, especially caspase‐9 and, for CMT‐1, also the activation of Fas.

Interestingly CMT‐8, but not CMT‐1, was able to induce apoptosis in multidrug resistant HL60R and in Fas‐ligand resistant HUT78B1 cell lines. These properties, together with others previously described (e.g. anti‐metastatic and anti‐osteolytic activities), suggest that CMT‐8 may have important applications in the clinical management of cancer.

The comparative analysis of structure‐activity relationship of CMT‐8 and doxycycline suggests that the C‐5 hydroxy moiety may play an important role in conferring activity in multidrug resistant cells. These findings appear to support the hypothesis that CMT‐8 may represent an interesting lead for the development of a new class of potent apoptosis inducer agents active in multidrug resistant and Fas‐ligand resistant malignancies.

British Journal of Pharmacology (2001) 133, 306–314; doi:10.1038/sj.bjp.0704068
Synthesis and characterization of a novel tritiated KATP channel opener with a benzopyran structure
Authors: Manley Paul W , Löffler‐Walz Cornelia , Russ Ulrich , Hambrock Annette , Moenius Thomas , Quast Ulrich
Published: January 2005

The synthesis of a tritiated benzopyran‐type opener of the ATP‐dependent K+ channel (KATP channel), [3H]‐PKF217 – 744 {(3S,4R)‐N‐[3,4‐dihydro‐2,2‐dimethyl‐3‐hydroxy‐6‐(2‐methyl‐4‐pyridinyl)‐2H‐1‐benzopyran‐4‐yl]‐3‐[2,6‐3H]pyridinecarboxamide} with a specific activity of 50 Ci mmol−1 is described. Binding of the ligand was studied in membranes from human embryonic kidney cells transfected with the sulphonylurea receptor isoforms, SUR2B and SUR2A, respectively.

PKF217 – 744 was confirmed as being a KATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular KATP channel, and to inhibit binding of the pinacidil analogue, [3H]‐P1075, to SUR2B (Ki=26 nM).

The kinetics of [3H]‐PKF217 – 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9×106 M−1 min−1 and 0.09 min−1, respectively.

Binding of [3H]‐PKF217 – 744 to SUR2B/2A was activated by MgATP (EC50∼3 μM) and inhibited (SUR2B) or enhanced (SUR2A) by MgADP.

Binding of [3H]‐PKF217 – 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. Ki values were identical with those obtained using the opener [3H]‐P1075 as the radioligand.

Glibenclamide accelerated dissociation of the SUR2B‐[3H]‐PKF217 – 744 complex.

The data show that the affinity of [3H]‐PKF217 – 744 binding to SUR2B is ∼6 times lower than that of [3H]‐P1075. This is due to a surprisingly slow association rate of the benzopyran‐type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological properties of the two opener radioligands are identical.

British Journal of Pharmacology (2001) 133, 275–285; doi:10.1038/sj.bjp.0704071