bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of effect of a selective vasopressin V1A receptor antagonist, SR 49,059, on potentiation by vasopressin of adrenoceptor‐mediated pressor responses in the rat mesenteric arterial bed
Authors: Heinemann Akos , Horina Gabi , Stauber Rudolf E , Pertl Christof , Holzer Peter , Peskar Bernhard A
Published: January 2005

The vasopressin receptor subtype involved in the enhancement by vasopressin of adrenoceptor‐mediated vasoconstriction was investigated in rat isolated perfused mesenteric arteries.

[Arg8]vasopressin (1–10 nm) dose‐dependently increased the perfusion pressure and enhanced the pressor response to the adrenoceptor agonist methoxamine (40 nmol) or electrical stimulation of periarterial nerves (16 Hz), at the concentration of 10 nm of [Arg8]vasopressin up to 4 and 3 fold, respectively.

During prolonged exposure (45 min) the direct vasoconstrictor effect of [Arg8]vasopressin (10 nm) rapidly declined whereas the potentiation of methoxamine‐induced vasoconstriction was maintained.

The selective vasopressin V1A receptor antagonist SR 49,059 (1–3 nm) and the non‐selective V1A/B and oxytocin receptor antagonist [deamino‐Pen1,Tyr(Me)2,Arg8]vasopressin (15–45 nm) inhibited the direct vasoconstrictor action of [Arg8]vasopressin but had no effect on the enhancement of the pressor response to methoxamine or electrical stimulation.

The V1B receptor agonist [deamino‐Cys1,β‐(3‐pyridyl)‐D‐Ala2,Arg8]vasopressin (100–1000 nm) and the V2 receptor agonist [deamino‐Cys1,D‐Arg8]vasopressin (1–10 nm) were devoid of any pressor activity and did not potentiate methoxamine‐evoked vasoconstriction. In contrast, [1‐triglycyl,Lys8]vasopressin (100–1000 nm) potentiated the methoxamine responses without per se inducing vasoconstriction.

In arteries precontracted with methoxamine (7.5 μm) pressor responses to [Arg8]vasopressin (3–10 nm) were not inhibited by a dose of SR 49,059 (3 nm) which abolished the peptide's vasoconstrictor effect under control conditions.

These data show that the direct vasoconstrictor effect of [Arg8]vasopressin is mediated by V1A receptors while the enhancement of adrenoceptor‐mediated pressor responses is insensitive to V1A, V1B, and oxytocin receptor antagonists and is not mimicked by selective agonists of V1B and V2 receptors. In conclusion, an unusual interaction of vasopressin with V1A receptors, or even the existence of a novel receptor subtype, has to be considered.

British Journal of Pharmacology (1998) 125, 1120–1127; doi:10.1038/sj.bjp.0702167
Potentiation of adenosine A1 receptor‐mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells
Authors: Dickenson John M. , Hill Stephen J.
Published: January 2005

The effect of protein tyrosine kinase inhibitors on human adenosine A1 receptor‐mediated [3H]‐inositol phosphate ([3H]‐IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO‐A1) cells.

In agreement with our previous studies the selective adenosine A1 receptor agonist N6‐cyclopentyladenosine (CPA) stimulated the accumulation of [3H]‐IPs in CHO‐A1 cells. Pre‐treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [3H]‐IPs elicited by 1 μM CPA (280±32%).

Genistein (EC50=13.7±1.2 μM) and tyrphostin A47 (EC50=10.4±3.9 μM) potentiated the [3H]‐IP response to 1 μM CPA in a concentration‐dependent manner.

Pre‐incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [3H]‐IPs elicited by 1 μM CPA.

Genistein (100 μM) had no significant effect on the accumulation of [3H]‐IPs produced by the endogenous thrombin receptor (1 u ml−1; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [3H]‐IP response (148±13%).

Genistein (100 μM) had no effect on the [3H]‐IP response produced by activation of the endogenous Gq‐protein coupled CCKA receptor with the sulphated C‐terminal octapeptide of cholecystokinin (1 μM CCK‐8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK‐8 (113±3% of control).

The phosphatidylinositol 3‐kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [3H]‐IP responses produced by 1 μM CPA and 1 μM CCK‐8.

These observations suggest that a tyrosine kinase‐dependent pathway may be involved in the regulation of human adenosine A1 receptor mediated [3H]‐IP responses in CHO‐A1 cells.

British Journal of Pharmacology (1998) 125, 1049–1057; doi:10.1038/sj.bjp.0702170
Potentiating action of MKC‐242, a selective 5‐HT1A receptor agonist, on the photic entrainment of the circadian activity rhythm in hamsters
Authors: Moriya T , Yoshinobu Y , Ikeda M , Yokota S , Akiyama M , Shibata S
Published: January 2005

Serotonergic projections from the midbrain raphe nuclei to the suprachiasmatic nuclei (SCN) are known to regulate the photic entrainment of circadian clocks. However, it is not known which 5‐hydroxytryptamine (5‐HT) receptor subtypes are involved in the circadian regulation. In order to verify the role of 5‐HT1A receptors, we examined the effects of 5‐{3‐[((2S)‐1,4‐benzodioxan‐2‐ylmethyl)amino]propoxy}‐1,3‐benzodioxole HCl (MKC‐242), a selective 5‐HT1A receptor agonist, on photic entrainment of wheel‐running circadian rhythms of hamsters.

MKC‐242 (3 mg kg−1, i.p.) significantly accelerated the re‐entrainment of wheel‐running rhythms to a new 8 h delayed or advanced light‐dark cycle.

MKC‐242 (3 mg kg−1, i.p.) also potentiated the phase advance of the wheel‐running rhythm produced by low (5 lux) or high (60 lux) intensity light pulses. In contrast, 8‐hydroxy‐dipropylaminotetralin (8‐OH‐DPAT)(5 mg kg−1, i.p.), a well known 5‐HT1A/5‐HT7 receptor agonist, only suppressed low intensity (5 lux) light‐induced phase advances.

The potentiating actions of MKC‐242 on light pulse‐induced phase advances were observed even when injected 20 or 60 min after the light exposure.

The potentiating action of MKC‐242 was antagonized by WAY100635, a selective 5‐HT1A receptor blocker, but not by ritanserin, a 5‐HT2/5‐HT7 receptor blocker, indicating that MKC‐242 is activating 5‐HT1A receptors.

Light pulse‐induced c‐fos expression in the SCN and the intergeniculate leaflet (IGL) were unaffected by MKC‐242 (3 mg kg−1, i.p.).

HPLC analysis demonstrated that MKC‐242 (3 mg kg−1, i.p.) decreased the 5‐HIAA content in the SCN.

The present results suggest that presynaptic 5‐HT1A receptor activation may be involved in the potentiation of photic entrainment by MKC‐242 in hamsters.

British Journal of Pharmacology (1998) 125, 1281–1287; doi:10.1038/sj.bjp.0702176
Exogenous and endogenous catecholamines inhibit the production of macrophage inflammatory protein (MIP) 1α via a β adrenoceptor mediated mechanism
Authors: Haskó György , Shanley Thomas P , Egnaczyk Greg , Németh Zoltán H , Salzman Andrew L , Vizi E Sylvester , Szabó Csaba
Published: January 2005

Noradrenaline (NA) and adrenaline (Ad) are modulators of cytokine production. Here we investigated the role of these neurotransmitters in the regulation of macrophage inflammatory protein (MIP)‐1α expression.

Pretreatment of RAW 264.7 macrophages with NA or Ad decreased, in a concentration‐dependent manner (1 nm–100 μm), MIP‐1α release induced by bacterial lipopolysaccharide (LPS 10 ng ml−1 LPS). The effect of NA was reversed by the selective β‐adrenoceptor antagonist propranolol (10 μm), but not by the α‐adrenoceptor antagonist phentolamine (10 μm).

In the concentration range of 10 nm–10 μm, isoproterenol, a β‐adrenoceptor agonist, but not phenylephrine (a selective α1‐adrenoceptor agonist) or UK‐14304 (a selective α2‐adrenoceptor agonist) mimicked the inhibitory effects of catecholamines on MIP‐1α production. Increases in intracellular cyclic adenosine monophosphate, elicited either by the selective type IV phosphodiesterase inhibitor rolipram (0.1–10 μm), or by prostaglandin E2, (10 nm–10 μm) decreased MIP‐1α release, suggesting that increased cyclic AMP may contribute to the suppression of MIP‐1α release by β‐adrenoceptor stimulation.

Northern blot analysis demonstrated that NA (100 nm–10 μm), Ad, isoproterenol, as well as rolipram (100 nm–10 μm) decreased LPS‐induced MIP‐1α mRNA accumulation. NA and Ad (1–100 μm) also decreased MIP‐1α production in thioglycollate‐elicited murine peritoneal macrophages.

Pretreatment of mice with either isoproterenol (10 mg kg−1, i.p.) or rolipram (25 mg kg−1, i.p.) decreased LPS‐induced plasma levels of MIP‐1α, while propranolol (10 mg kg−1, i.p.) augmented the production of this chemokine, confirming the role of a β‐adrenoceptor mediated endogenous catecholamine action in the regulation of MIP‐1α production in vivo.

Thus, based on our data we conclude that catecholamines are important endogenous regulators of MIP‐1α expression in inflammation.

British Journal of Pharmacology (1998) 125, 1297–1303; doi:10.1038/sj.bjp.0702179
Adenosine A1 receptor‐mediated excitation of nociceptive afferents innervating the normal and arthritic rat knee joint
Authors: Dowd E , McQueen D S , Chessell I P , Humphrey P P A
Published: January 2005

We tested the hypothesis that adenosine excites nociceptive primary afferents innervating the knee joint.

Neuronal recordings were made from fine nerve filaments innervating the knee joint in rats anaesthetized with pentobarbitone. Drugs were injected close‐arterially (i.a.) or into the articular space (i.art.). We studied normal and chronically inflamed arthritic joints, the latter 14–21 days after a single intra‐articular injection of Freund's Complete Adjuvant, performed under halothane anaesthesia.

Adenosine injected i.a. caused delayed (approximately 10 s) excitation of the majority of polymodal C‐fibre afferents, and had similar effects when injected directly into the joint.

Adenosine triphosphate (ATP) had biphasic effects on discharge, a fast (<1 s) excitation was followed by a delayed increase similar to that seen with adenosine.

The adenosine A1 receptor agonists N6‐cyclopentyladenosine (CPA) and N‐[(1S,trans)‐2‐hydroxypentyl] adenosine (GR79236) also excited the C‐fibre afferents. The A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) antagonized the responses evoked by adenosine, CPA, and the delayed increase seen after ATP, indicating that excitation of the nociceptive afferents was mediated via adenosine A1 receptors.

Adenosine and ATP evoked delayed excitatory effects of similar magnitude, regardless of whether or not the knee joint was chronically inflamed. The increased basal discharge observed in arthritic joints was unaffected by DPCPX, which implies that the increase in spontaneous activity associated with arthritis is unlikely to involve tonically released adenosine.

The results support the hypothesis that adenosine excites primary afferent nociceptive nerve terminals in the rat knee joint, an effect mediated by adenosine A1 receptors. ATP, adenosine, and A1 receptors may play a role in generating the peripheral nociceptive (pain) signal.

British Journal of Pharmacology (1998) 125, 1267–1271; doi:10.1038/sj.bjp.0702185
Evidence that [Phe1ψ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2, a peripheral ORL‐1 receptor antagonist, acts as an agonist in the rat spinal cord
Authors: Carpenter Kate J. , Dickenson Anthony H.
Published: January 2005

[Phe1 ψ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2, a pseudopeptide analogue of nociceptin is an antagonist in peripheral assays. Here, using in vivo electrophysiological recordings of dorsal horn neurones, [Phe1 ψ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2 appears to have agonist activity after spinal administration. The noxious evoked activity of the neurones was inhibited by [Phe1 ψ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2, which was as potent as nociceptin itself.

British Journal of Pharmacology (1998) 125, 949–952; doi:10.1038/sj.bjp.0702188
The activation of nitric oxide synthase by copper ion is mediated by intracellular Ca2+ mobilization in human pulmonary arterial endothelial cells
Authors: Demura Yoshiki , Ishizaki Takeshi , Ameshima Shingo , Okamura Seitarou , Hayashi Takio , Matsukawa Shigeru , Miyamori Isamu
Published: January 2005

The aim of the study was to elucidate the vasodilatory mechanism due to Cu2+ by assessing nitric oxide (NO) production as determined by NOx (NO, NO2−, and NO3−) that is released from human pulmonary arterial endothelial cell (HPAEC) monolayers using a NO chemiluminescence analyzer, and also to assess Ca2+ movement using 45Ca and fura 2 in HPAEC.

Cu2+ (10−6–10−4 m) significantly increased NO production in a dose‐dependent manner when extracellular Ca2+ was present.

45Ca influx into the adherent cells was dose‐dependently enhanced by Cu2+ (10−6–10−4 m), but not by Mn2+, Zn2+ or Fe2+.

[Ca2+]i, measured by monitoring the fluorescence changes of fura 2, was significantly elevated in the presence of Cu2+.

The increase in [Ca2+]i induced by Cu2+ was inhibited by either diethyldithiocarbamate (DDC) or the depletion of extracellular Ca2+.

The dihydropyridine receptor agonist, BayK8644, significantly attenuated the Cu2+‐induced increase in [Ca2+]i in a dose dependent manner and nitrendipine or nifedipine, the dihydropyridine receptor antagonists, dose‐dependently inhibited a Cu2+‐induced increase in [Ca2+]i.

These results suggest that Cu2+ activates eNOS through the mechanism of [Ca2+]i elevation due to Ca2+ influx into HPAEC and that the Cu2+‐induced [Ca2+]i elevation in HPAEC is likely due to activation of the dihydropyridine‐like receptors.

British Journal of Pharmacology (1998) 125, 1180–1187; doi:10.1038/sj.bjp.0702197
A microdialysis study of the in vivo release of 5‐HT in the median raphe nucleus of the rat
Authors: Adell Albert , Artigas Francesc
Published: January 2005

The present study has examined several characteristics of the release of 5‐HT in the median raphe nucleus in terms of its dependence of nerve impulse, provenance of a vesicular storage fraction as well as the regulatory role played by 5‐HT1A receptors.

Tetrodotoxin (1 μm) and reserpine (5 mg kg−1, i.p.) virtually suppressed the output of 5‐HT.

The administration of EEDQ (10 mg kg−1, i.p.) did not alter the basal release of 5‐HT but abolished the reduction of 5‐HT release induced by 8‐OH‐DPAT (0.1 mg kg−1, s.c.).

The perfusion of 1–100 μm of 8‐OH‐DPAT or the novel 5‐HT1A agonist BAY×3702 decreased the efflux of 5‐HT, whereas the perfusion of the 5‐HT1A antagonist WAY‐100635 failed to alter 5‐HT release.

The decrease in dialysate 5‐HT induced by 100 μm 8‐OH‐DPAT was reversed by the concurrent perfusion of 100 μm WAY‐100635. Also, the perfusion of 100 μm WAY‐100635 for 2 h inhibited partly the reduction of 5‐HT release evoked by the systemic administration of 8‐OH‐DPAT (0.1 mg kg−1).

These results indicate that extracellular 5‐HT in the median raphe nucleus is stored in vesicles and released in an impulse‐dependent manner. Also, the basal release of 5‐HT in the median raphe nucleus does not appear to be under the tonic control of somatodendritic 5‐HT1A receptors by endogenous 5‐HT. Instead, this feedback mechanism seems to be triggered when an excess of the transmitter or a 5‐HT1A agonist is present in the extracellular space of the median raphe nucleus.

British Journal of Pharmacology (1998) 125, 1361–1367; doi:10.1038/sj.bjp.0702206
Effects of depolarizing stimuli on calcium homeostasis in cultured rat motoneurones
Authors: Hubert Jean‐Pierre , Burgevin Marie‐Claude , Terro Faraj , Hugon Jacques , Doble Adam
Published: January 2005

Intracellular calcium concentrations in individual rat motoneurones in enriched primary cultures were measured by Indo‐1 fluorimetry.

Motoneurones in the cultures were characterized morphometrically and by cholineacetyltransferase immunocytochemistry.

Depolarization of the cells with glutamic acid or veratridine increased intracellular calcium levels, which returned to baseline only slowly after removal of the depolarizing agent.

The use of selective agonists (N‐methyl‐d‐aspartic acid, AMPA, kainic acid, quisqualic acid and 1R‐3S‐ACPD) and antagonists (MK 801 and CNQX) showed that the excitatory amino acid‐evoked responses were mediated by AMPA/kainate receptors rather than by NMDA receptors.

Depolarization‐evoked calcium transients in motoneurones are blocked by the neuroprotective drug riluzole

Calcium transients reflected entry of calcium from without the cell, and their blockade by nitrendipine and lanthanum chloride suggested that this entry took place primarily through voltage‐dependent calcium channels.

These findings may be relevant for understanding the selective vulnerability of motoneurones to excitotoxicity in amyotrophic lateral sclerosis, and the therapeutic activity of riluzole in the treatment of this disease.

British Journal of Pharmacology (1998) 125, 1421–1428; doi:10.1038/sj.bjp.0702207
Mechanism of ETA‐receptor stimulation‐induced increases in intracellular Ca2+ in SK‐N‐MC cells
Authors: Heinroth‐Hoffmann Ingrid , Vogelsang Magdalene , Schiewe Pia , Morawietz Henning , Holtz Jürgen , Pönicke Klaus , Brodde Otto‐Erich
Published: January 2005

The mechanism underlying endothelin‐1 (ET‐1)‐induced increases in intracellular Ca2+ concentrations in the human neuroblastoma cell‐line SK‐N‐MC was investigated.

ET‐receptor agonists increased inositol phosphate (IP)‐formation (assessed as accumulation of total [3H]‐IPs in [3H]‐myo‐inositol prelabelled cells) and intracellular Ca2+ (assessed by the FURA‐2 method) with an order of potency: ET‐1>sarafotoxin 6b (S6b)>ET‐3=S6c; the ETA‐receptor antagonist BQ‐123 inhibited both responses with apparent pKi‐values of 8.3 and 8.6, respectively, while the ETB‐receptor antagonist BQ‐788 did not.

Pretreatment of the cells with pertussis toxin (PTX, 500 ng ml−1 overnight) reduced ET‐1‐induced Ca2+ increases by 46±5%, but rather enhanced ET‐1‐induced IP‐formation.

Chelation of extracellular Ca2+ by 5 mm EGTA did not affect ET‐1‐induced IP‐formation. However, in the presence of 5 mm EGTA or SKF 96365, an inhibitor of receptor mediated Ca2+ influx (1.0–3.0×10−5 m) ET‐1‐induced Ca2+ increases were inhibited in normal, but not in PTX‐treated cells.

[125I]‐ET‐1 binding studies as well as mRNA expression studies (by RT–PCR) detected only ETA‐receptors whereas expression of ETB‐receptor mRNA was marginal.

ET‐1 (10−8 m) inhibited isoprenaline‐evoked cyclic AMP increases; this was antagonized by BQ‐123, not affected by BQ‐788 and abolished by PTX‐treatment.

We conclude that SK‐N‐MC cells contain a homogeneous population of ETA‐receptors that couple to IP‐formation and inhibition of cyclic AMP formation. Stimulation of these ETA‐receptors increases intracellular Ca2+ by at least two mechanisms: a PTX‐insensitive IP‐mediated Ca2+ mobilization from intracellular stores and a PTX‐sensitive influx of extracellular Ca2+.

British Journal of Pharmacology (1998) 125, 1202–1211; doi:10.1038/sj.bjp.0702208
Brain sites involved in the antinociceptive effect of bradykinin in rats
Authors: Couto L B , Corrêa F M A , Pelá I R
Published: January 2005

The localization of brain sites where bradykinin (BK) induces its antinociceptive effect in rats, was studied using as index the threshold for the jaw‐opening reflex elicited by the dental pulp electrical stimulation test (DPEST).

The microinjection of BK into the lateral or fourth cerebral ventricles induced an antinociceptive effect, with Index of Antinociception (IA) of 0.51±0.03 and 0.68±0.05, respectively. However, microinjections of the peptide into the third ventricle induced a less marked antinociception (IA=0.28±0.08).

The brain sites where the microinjection of BK caused an antinociceptive effect were: locus coeruleus, principal nucleus, oral part of the spinal sensorial trigeminal nucleus, and the sensory root of the trigeminal nerve.

The antinociceptive effect was more intense when BK (4–16 nmol) was injected into the locus coeruleus. Microinjection of BK (4 nmol) into the fourth ventricle, but not into the locus coeruleus, induced an increase in blood pressure. The microinjection of the peptide into the nucleus tractus solitarius, a site that is also involved in the pressor effect of BK, did not induce an antinociceptive effect. These results indicate that the antinociceptive effect of BK is not related to blood pressure changes.

The microinjection of BK into some of the sites involved in the mechanisms of analgaesia, including the periaquenductal gray matter (dorsal, lateral and ventrolateral) and the dorsal raphe nucleus did not induce an antinociceptive effect.

The results suggest that the most likely brain sites involved in the antinociceptive effect of BK are the locus coeruleus and the principal sensory trigeminal nucleus. The present results did not exclude the involvement of other brain sites surrounding the lateral and the third ventricles.

British Journal of Pharmacology (1998) 125, 1578–1584; doi:10.1038/sj.bjp.0702209
Modulation of AT1 receptor‐mediated contraction of rat uterine artery by AT2 receptors
Authors: Zwart Adam S. , Davis Elizabeth A. , Widdop Robert E.
Published: January 2005

The aim of this study was to characterize the angiotensin II receptors in isolated uterine arteries from non pregnant and pregnant rats, since it has been reported from binding studies that ovine uterine arteries contain AT2 receptors.

Uterine arterial segments were obtained from virgin, non‐pregnant and late pregnant (18–21 days) Sprague‐Dawley rats and mounted in small vessel myographs. Concentration‐response curves were constructed to angiotensin II (1 nm–10 μm) in the absence and presence of various angiotensin II receptor subtype selective compounds. These included losartan (AT1 antagonist; 1, 10 and 100 nm), PD 123319 (AT2 antagonist; 1 μm) and CGP 42112 (AT2 agonist; 1 μm). Responses to angiotensin II were measured as increases in force (mN) and expressed as a per cent of the response to a K+ depolarizing solution.

Losartan (1, 10 and 100 nm) caused significant concentration‐dependent rightward shifts of the angiotensin II concentration‐response curve in uterine arteries from non‐pregnant and pregnant rats. The pA2 values calculated from these data were 9.8 and 9.2, respectively, although the slope of the Schild plot in the non‐pregnant group was less than unity.

PD 123319 (1 μm) caused significant 6‐ and 3 fold leftward shifts of the angiotensin II concentration‐response curve in uterine arteries from non‐pregnant and pregnant rats, respectively. In vessels from pregnant rats, PD 123319 also significantly increased the maximum response to angiotensin II.

CGP 42112 (1 μm) attenuated the response to angiotensin II of uterine arteries from non‐pregnant rats. This was reflected by a 14 fold rightward shift of the angiotensin II concentration‐response curve and a decrease in the maximum response. In uterine arteries from pregnant rats, CGP 42112 (1 μm) caused a 3 fold rightward shift of the angiotensin II concentration‐response curve, but had no effect on the maximum response.

PD 123319 (1 μm) and CGP 42112 (1 μm) had no effect on the concentration‐response curves to phenylephrine (PE) of uterine arteries from non‐pregnant or pregnant rats. In addition, CGP 42112 (1 nm–1 mm) had no vasodilator effect on tissues precontracted with phenylephrine.

These results suggest that the contractile responses of the rat uterine artery are mediated by the AT1 receptor. Furthermore, in this vascular preparation, the AT2 receptor appears to inhibit the response mediated by the AT1 receptor, although, this is not uniform between the non‐pregnant and pregnant states.

British Journal of Pharmacology (1998) 125, 1429–1436; doi:10.1038/sj.bjp.0702210
Endothelium‐dependent contractile actions of proteinase‐activated receptor‐2‐activating peptides in human umbilical vein: release of a contracting factor via a novel receptor
Authors: Saifeddine Mahmoud , Roy Samir S. , Al‐Ani Bahjat , Triggle Chris R. , Hollenberg Morley D.
Published: January 2005

The contractile actions of the proteinase‐activated receptor‐2‐activating peptides (PAR2APs), SLIGRL‐NH2 (SL‐NH2), SLIGKV‐NH2 (KV‐NH2), trans‐cinnamoyl‐LIGRLO‐NH2 (tc‐NH2), and the PAR1‐AP, TFLLR‐NH2 (TF‐NH2) as well as trypsin and thrombin were studied in endothelium‐denuded and intact human umbilical vein (HUV) ring preparations.

In HUV rings with, but not without an intact endothelium, PAR2APs caused a concentration‐dependent contractile response, whereas LSIGRL‐NH2 trypsin and PAR1APs were inactive. The contractile response was not affected by the endothelin ETA receptor antagonist, BQ123, the cyclooxygenase inhibitor, indomethacin, the leukotriene synthesis inhibitor, MK886, or the epoxygenase/P450 inhibitor, SKF‐525A. Other pharmacological antagonists (prazosin, Losartan®) were similarly inactive.

The order of potencies of the PAR2APs to cause a contraction in the endothelium‐intact preparation was: SL‐NH2>>KV‐NH2tc‐NH2.

Using an endothelium‐free rat aorta ring as a reporter tissue, surrounded with endothelium‐intact HUV as a donor tissue in a ‘sandwich assay,’ we also monitored the ability of SL‐NH2, TF‐NH2, trypsin and thrombin to release either contractile (EDCF) or relaxant (EDRF) factors.

In the ‘sandwich assay’ done in the presence of L‐NAME (0.1 mm), the endothelium‐intact HUV tissue (but not endothelium‐denuded HUV) released a contractile factor (EDCF) in response to SL‐NH2 (50 μm) but not to trypsin or LSIGRL‐NH2. The SL‐NH2‐mediated release/action of the EDCF was not affected by BQ123, indomethacin, MK886 or SKF‐525A.

In the ‘sandwich assay’, trypsin (4–10 nm), SL‐NH2, KV‐NH2 and tc‐NH2 caused the release of a relaxant activity (EDRF) from the endothelium‐intact (but not the denuded) HUV preparation. The release of EDRF was blocked by 0.1 mm ωnitro‐L‐arginine‐methylester (L‐NAME). Neither thrombin (10 u ml−1, 100 nm) nor TF‐NH2 (50 μm) were active in this EDRF‐release assay.

The relative potencies of the PAR2 agonists for causing the release of EDRF in the HUV sandwich assay were: trypsin>>SL‐NH2>>tc‐NH2>KV‐NH2. This order of potencies differed from the one observed for the same agonists in the HUV contraction assay (above) and in an intracellular calcium signalling assay, conducted with cloned human PAR2 that was expressed in cultured rat kidney KNRK cells: trypsin>>SL‐NH2=tc‐NH2>KV‐NH2.

We conclude that PAR2APs (but not PAR1APs) via a receptor distinct from PAR2, can cause a contractile response in endothelium‐intact HUV tissue via the release of a diffusable EDCF, that is different from previously recognized smooth muscle agonists (e.g. prostanoid metabolites, endothelin, noradrenaline, angiotensin‐II, acetylcholine).

British Journal of Pharmacology (1998) 125, 1445–1454; doi:10.1038/sj.bjp.0702213
Persistent nicotinic blockade by chlorisondamine of noradrenergic neurons in rat brain and cultured PC12 cells
Authors: Reuben M , Louis M , Clarke P B S
Published: January 2005

Chlorisondamine (CHL) blocks behavioural responses to nicotine for several weeks or months in rats. Persistent blockade has also been demonstrated ex vivo, in assays of nicotine‐evoked striatal dopamine release. Central administration of [3H]‐CHL leads to long‐term retention of radiolabel in nigrostriatal dopaminergic neurons and in few other cell groups. We investigated whether an analogous blockade also occurs in noradrenergic neurons in the brain and in cultured pheochromocytoma (PC12) cells, which have a similar noradrenergic phenotype.

Administration of CHL (10 mg kg−1 s.c. or 10 μg i.c.v.), 21 days prior, resulted in a near‐total block of nicotine‐evoked release of hippocampal [3H]‐noradrenaline ([3H]‐NA) from superfused rat synaptosomes; NMDA‐evoked [3H]‐NA release was unaffected.

Three weeks after administration of [3H]‐CHL (10 μg i.c.v.), preferential accumulation of radiolabel was observed in the locus coeruleus, which provides the entire noradrenergic innervation to hippocampus, as well as in previously noted structures.

In rat pheochromocytoma (PC12) cells, nicotine evoked [3H]‐NA release (EC50 approximately 30 μm). This effect was blocked by co‐incubation with mecamylamine (10 μm) or CHL (1 μm) but was not affected by α‐bungarotoxin. As in the hippocampus, the nicotinic agonist cytisine was at least as efficacious as nicotine.

Acute exposure of PC12 cells to CHL 10 or 100 μm (but not 1 μm), followed by 90 min wash‐out, almost completely blocked release evoked by 30 μm nicotine. More prolonged (24 h) exposure to CHL 100 μm (but not 1 or 10 μm), followed by 3 days of wash‐out, partially inhibited release evoked by nicotine, leaving responses to high K+ unchanged. A significant (30%) reduction was also seen 5 days after exposure.

We conclude that persistent nicotinic blockade by CHL is neither restricted to mesostriatal dopamine neurons, nor to the CNS, nor to neurons possessing the same nicotinic receptor pharmacology. In addition, the persistent blockade does not appear to result from an acute blocking action, but may be dependent upon intracellular accumulation of the antagonist.

British Journal of Pharmacology (1998) 125, 1218–1227; doi:10.1038/sj.bjp.0702215
The role of nitric oxide and metalloproteinases in the pathogenesis of hyperoxia‐induced lung injury in newborn rats
Authors: Radomski Anna , Sawicki Grzegorz , Olson David M. , Radomski Marek W.
Published: January 2005

The effects of nitric oxide (NO) and metalloproteinases (MMP‐2 and MMP‐9) in the pathogenesis of hyperoxia‐induced lung damage in newborn rats were examined.

Three‐day‐old rat pups were subjected to hyperoxia (95% O2) or room air for 7 and 14 days.

Some animals were treated with NG‐L‐nitro‐L‐arginine methyl ester (L‐NAME, 10 mg kg−1, s.c., daily).

Histology, morphometry, oedema, Ca2+‐dependent and ‐independent NO synthase (NOS) activities, expression of NOS isoforms and the activities of MMP‐2 and MMP‐9 were measured in lungs of hyperoxic and control animals.

Exposure of rats to hyperoxia for 7 days resulted in alveolar sac injury characterized by the presence of cellular debris, red cell extravasation and inflammatory infiltration with mononuclear cells. Lung water content, epithelial, smooth muscle layers and total airway thickness was similar to controls.

In contrast, exposure of rats to hyperoxia for 14 days resulted in lung oedema, inflammation and epithelial proliferation.

Hyperoxia caused a decrease in Ca2+‐dependent NOS activity, an effect that was associated with increased expression of eNOS protein.

In control rats, Ca2+‐dependent NOS activity and expression of eNOS were reduced at 14 days.

Hyperoxia caused 10 fold increase in the activity of Ca2+‐independent NOS that remained significantly elevated after 14 days of exposure to hyperoxia. The activity of this enzyme was unchanged in control rats.

In lungs of hyperoxic rats, the immunoblot showed time‐dependent, biphasic expression (peak at 7 days) of iNOS. The profile of expression of iNOS in control rats was similar.

The activities of MMPs were increased in lungs of hyperoxic animals.

The L‐NAME treatment of hyperoxic animals reduced lung oedema and epithelial proliferation, but enhanced the activities of MMPs. L‐NAME exerted no significant effects in control rats.

It is concluded that increased generation of NO contributes to the pathogenesis of hyperoxia‐induced lung damage in newborn rats.

British Journal of Pharmacology (1998) 125, 1455–1462; doi:10.1038/sj.bjp.0702216
Priming and induction of eosinophil trafficking in guinea‐pig cutaneous inflammation by tumour necrosis factor α
Authors: Macari David M T , Teixeira Mauro M , Ansari Tareq , Jeffery Peter K , Hellewell Paul G
Published: January 2005

Tissue eosinophilia is a hallmark of allergic and parasitic diseases. Priming mechanisms may play an important role in mediating the process of eosinophil accumulation in these conditions. We have previously shown that blockade of tumour necrosis factor α (TNFα) inhibited the capacity of lipopolysaccharide to prime skin sites for chemoattractant‐induced eosinophil recruitment. The present study was carried out to investigate the capacity of TNFα to prime an inflammatory site for enhanced eosinophil accumulation.

Initial experiments investigated the capacity of TNFα itself to induce eosinophil accumulation. Intradermal injection of murine TNFα (10–300 ng per site) in the guinea‐pig induced significant accumulation of 111In‐eosinophils. Kinetic studies showed the response to be delayed in onset and inhibited by cycloheximide, consistent with a dependency on protein synthesis. Trafficking of 111In‐eosinophils to sites treated for 2 h with TNFα (10–100 ng per site) was inhibited by monoclonal antibodies (mAbs) against β2 or α4 integrins.

Intradermal injection of a low dose (3 ng) of TNFα (which by itself had no significant effect on eosinophil trafficking) prior to chemoattractants or antigen in sensitized skin sites, induced significant priming of eosinophil accumulation. Recruitment of both 111In‐eosinophils and endogenous eosinophils was enhanced. Trafficking to TNFα‐primed responses was dependent on protein synthesis and β2 integrins. In contrast, the α4 integrin mAb failed to inhibit the TNFα primed response.

Thus, TNFα can induce and also prime eosinophil recruitment in guinea‐pig skin. Our results provide further evidence that this cytokine may be an important mediator of allergic‐ or parasite‐induced eosinophilic inflammation.

British Journal of Pharmacology (1998) 125, 1228–1235; doi:10.1038/sj.bjp.0702224
Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships
Authors: Varani K. , Calo' G. , Rizzi A. , Merighi S. , Toth G. , Guerrini R. , Salvadori S. , Borea P. A. , Regoli D.
Published: January 2005

The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C‐terminal; the ligand, [3H]‐NC‐NH2, has a specific activity of 24.5 Ci mmol−1

Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nm and Bmax of 94 fmol mg−1 of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs=0.104 min−1; K1=0.034 min−1; T1/2=20 min; K+1=0.07 min nm−1.

Thermodynamic analyses indicated that [3H]‐NC‐NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(δ), OP2(κ) and OP3(μ).

Receptor affinities of several NC fragments and analogues, including the newly discovered ORL‐1 receptor antagonist [Phe1ψ(CH2‐NH)Gly2]NC(1–13)‐NH2 ([F/G]NC(1–13)‐NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC‐OH=NC‐NH2=NC(1–13)‐NH2 >> NC(1–12)‐NH2 > NC(1–13)‐OH >> NC(1–11)‐NH2, and for NC and NC(1–13)‐NH2 analogues: [Tyr1]NC‐NH2 [Leu1]NC(1–13)‐NH2 [Tyr1]NC(1–13)‐NH2 [F/G]NC(1–13)‐NH2 >> [Phe3]NC(1–13)‐NH2 > [DF/G]NC(1–13)‐NH2.

Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]‐NC‐NH2 binding when applied at concentrations up to 10 μm indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor.

British Journal of Pharmacology (1998) 125, 1485–1490; doi:10.1038/sj.bjp.0702226
Behavioural and biochemical evidence for signs of abstinence in mice chronically treated with Δ‐9‐tetrahydrocannabinol
Authors: Hutcheson Daniel M , Tzavara Eleni Th. , Smadja Claire , Valjent Emmanuel , Roques Bernard P , Hanoune Jacques , Maldonado Rafael
Published: January 2005

Tolerance and dependence induced by chronic Δ‐9‐tetrahydrocannabinol (THC) administration were investigated in mice. The effects on body weight, analgesia and hypothermia were measured during 6 days of treatment (10 or 20 mg kg−1 THC twice daily). A rapid tolerance to the acute effects was observed from the second THC administration.

The selective CB‐1 receptor antagonist SR 141716A (10 mg kg−1) was administered at the end of the treatment, and somatic and vegetative manifestations of abstinence were evaluated. SR 141716A administration precipitated several somatic signs that included wet dog shakes, frontpaw tremor, ataxia, hunched posture, tremor, ptosis, piloerection, decreased locomotor activity and mastication, which can be interpreted as being part of a withdrawal syndrome.

Brains were removed immediately after the behavioural measures and assayed for adenylyl cyclase activity. An increase in basal, forskolin and calcium/calmodulin stimulated adenylyl cyclase activities was specifically observed in the cerebellum of these mice.

The motivational effects of THC administration and withdrawal were evaluated by using the place conditioning paradigm. No conditioned change in preference to withdrawal associated environment was observed. In contrast, a conditioned place aversion was produced by the repeated pairing of THC (20 mg kg−1), without observing place preference at any of the doses used.

This study constitutes a clear behavioural and biochemical model of physical THC withdrawal with no motivational aversive consequences. This model permits an easy quantification of THC abstinence in mice and can be useful for the elucidation of the molecular mechanisms involved in cannabinoid dependence.

British Journal of Pharmacology (1998) 125, 1567–1577; doi:10.1038/sj.bjp.0702228
Pharmacological modulation of GABAA receptor‐mediated postsynaptic potentials in the CA1 region of the rat hippocampus
Authors: Manuel Nicholas A , Davies Ceri H
Published: January 2005

It is unclear whether GABAA receptor‐mediated hyperpolarizing and depolarizing synaptic potentials (IPSPAs and DPSPAs, respectively) are evoked by (a) the same populations of GABAergic interneurones and (b) exhibit similar regulation by allosteric modulators of GABAA receptor function. We have attempted to address these questions by investigating the effects of (a) known agonists for presynaptic receptors on GABAergic terminals, and (b) a range of GABAA receptor ligands, on each response.

The GABA uptake inhibitor NNC 05‐711 (10 μm) enhanced whereas bicuculline (10 μm) inhibited both IPSPAs and DPSPAs.

(−)‐Baclofen (5 μm), [D‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin (DAGO; 0.5 μm), and carbachol (10 μm) caused substantial depressions (up to 99%) of DPSPAs that were reversed by CGP 55845A (1 μm), naloxone (10 μm) and atropine (5 μm), respectively. In contrast, 2‐chloroadenosine (CADO; 10 μm) only slightly depressed DPSPAs. Quantitatively, the effect of each agonist was similar to that reported for IPSPAs.

The neurosteroid ORG 21465 (1–10 μm), the anaesthetic propofol (50–500 μm), the barbiturate pentobarbitone (100–300 μm) and zinc (50 μm) all enhanced DPSPAs and IPSPAs.

The benzodiazepine (BZ) agonist flunitrazepam (10–50 μm) and inverse agonist DMCM (1 μm) caused a respective enhancement and inhibition of both IPSPAs and DPSPAs. The BZω1 site agonist zolpidem (10–30 μm) produced similar effects to flunitrazepam.

The anticonvulsant loreclezole (1–100 μm) did not affect either response.

These data demonstrate that similar populations of inhibitory interneurones can generate both IPSPAs and DPSPAs by activating GABAA receptors that are subject to similar allosteric modulation.

British Journal of Pharmacology (1998) 125, 1529–1542; doi:10.1038/sj.bjp.0702237
Orphan G‐protein‐coupled receptors: the next generation of drug targets?
Authors: Wilson Shelagh , Bergsma Derk J. , Chambers Jon K. , Muir Alison I. , Fantom Kenneth G. M. , Ellis Catherine , Murdock Paul R. , Herrity Nicole C. , Stadel Jeffrey M.
Published: January 2005

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein‐coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these ‘orphan’ receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.

British Journal of Pharmacology (1998) 125, 1387–1392; doi:10.1038/sj.bjp.0702238
The antinociceptive effect of combined systemic administration of morphine and the glycine/NMDA receptor antagonist, (+)‐HA966 in a rat model of peripheral neuropathy
Authors: Christensen Dennis , Idänpään‐Heikkilä Juhana J. , Guilbaud Gisèle , Kayser Valérie
Published: January 2005

We evaluated the ability of the functional antagonist at the glycine site of the N‐methyl‐D‐aspartate (NMDA) receptor complex, (+)‐(1‐Hydroxy‐3‐aminopyrrolodine‐2‐one) ((+)‐HA966), to modulate the antinociceptive action of systemic morphine in a rat model of neuropathic pain produced by chronic constriction injury to the sciatic nerve. Mechanical (vocalization threshold to hindpaw pressure) and thermal (struggle latency to hindpaw immersion into a water bath) stimuli were used.

In the mechanical test, morphine (0.05, 0.1 and 0.3 mg kg−1, i.v.) alone produced dose‐dependent effects in both neuropathic and uninjured rats. Likewise, morphine (0.1, 0.3 and 1 mg kg−1, i.v.) dose‐dependently increased struggle latencies of the nerve‐injured hindpaw in the hot noxious (46°C) test but was ineffective in the non‐noxious warm (44°C) and cold (10°C) test.

Pretreatment with (+)‐HA966 (2.5 mg kg−1, s.c.) dose‐dependently enhanced the effect of morphine in the mechanical test with the relative potency being nerve‐injured hindpaw>contralateral hindpaw>uninjured rat.

Likewise, (+)‐HA966 dose‐dependently enhanced the effect of morphine against a hot (46°C) stimulus and produced, in combination with morphine, a dose‐dependent effect against a warm (44°C) stimulus. In the cold (10°C) test, (+)‐HA966 reversed the ineffectiveness of the highest dose of morphine.

Naloxone blocked the effect of the combination of (+)‐HA966 with morphine in all tests. The drug combination produced no motor deficits in animals using the rotarod test.

These findings suggest that combined administration of antagonists, acting at the glycine site of the NMDA receptor complex and morphine may be a promising approach in the treatment of neuropathic and acute pain.
Tyrosine phosphorylation as a convergent pathway of heterotrimeric G protein‐ and rho protein‐mediated Ca2+ sensitization of smooth muscle of rabbit mesenteric artery
Authors: Sasaki Motoi , Hattori Yuichi , Tomita Fumishi , Moriishi Kohji , Kanno Morio , Kohya Tetsuro , Oguma Keiji , Kitabatake Akira
Published: January 2005

The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and receptor agonists on β‐escin‐skinned smooth muscle of rabbit mesenteric artery.

In the homogenate of the β‐escin‐skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [32P]‐ADP‐ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti‐rho p21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTPγS‐induced Ca2+ sensitization, although the effect of GTPγS at higher concentrations (30 μM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5′‐triphosphate, of the Ca2+‐induced contraction was not affected by C3 pretreatment.

The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13‐dibutyrate of the Ca2+‐induced contraction. However, these PKC inhibitors had no effect on GTPγS‐ and receptor agonist‐induced Ca2+ sensitization.

The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTPγS of the Ca2+‐induced contraction without affecting this Ca2+ contraction. The inactive genistein analogue daidzein did not modify the effect of GTPγS. The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors.

These results suggest that rho p21 predominantly mediates GTPγS‐induced Ca2+ sensitization of β‐escin‐skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein‐coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein‐ and heterotrimeric G protein‐mediated Ca2+ sensitization mechanisms.
Differential effects of wortmannin on the release of substance P and amino acids from the isolated spinal cord of the neonatal rat
Authors: Suzuki Hidenori , Yoshioka Koichi , Maehara Taketoshi , Guo Jiang‐Zhong , Nonomura Yoshiaki , Otsuka Masanori
Published: January 2005

Effects of wortmannin, an inhibitor of myosin light chain kinase, on the release of substance P and amino acids, GABA and glutamate, were investigated in the isolated spinal cord preparation of the neonatal rat.

Wortmannin at 0.5–10 μM depressed the release of substance P evoked by high‐K+ (90 mM) medium from the spinal cord (IC50=1.1 μM). Wortmannin also depressed the high‐K+ (70 mM)‐evoked release of substance P from cultured dorsal root ganglion neurons of neonatal rats. In contrast, the high‐K+ (90 mM)‐evoked release of GABA and glutamate from the spinal cord was not affected by wortmannin (0.1–10 μM).

Upon stimulation of a dorsal root, a monosynaptic reflex and a subsequent slow ventral root depolarization were evoked in the ipsilateral ventral root of the same segment in the isolated spinal cord preparation. The magnitude of the slow ventral root depolarization was depressed gradually to about 70% of the control during the course of 30 min under wortmannin (1 μM). In contrast, the monosynaptic reflex was unaffected by wortmannin.

Immunofluorescent staining revealed that immunoreactivities of substance P and myosin II were colocalized at presynaptic terminals in the dorsal horn of the neonatal rat spinal cord.

The present results suggest that myosin phosphorylation by myosin light chain kinase may play a crucial role in the release of substance P, but not in the release of GABA and glutamate in the neonatal rat spinal cord. This may reflect a difference in the exocytic mechanisms of substance P‐containing large dense core vesicles and amino acid‐containing small clear vesicles.