bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ser203 as well as Ser204 and Ser207 in fifth transmembrane domain of the human β2‐adrenoceptor contributes to agonist binding and receptor activation
Authors: Sato Takayuki , Kobayashi Hiroyuki , Nagao Taku , Kurose Hitoshi 
Published: January 2005

We examined the contribution of Ser203 of the human β2‐adrenoceptor (β2‐AR) to the interaction with isoprenaline. The affinity of (−)‐isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (−)‐isoprenaline derivative with only one hydroxyl group, at the meta‐position, showed reduced affinity for wild‐type β2‐AR and S207A‐β2‐AR and even lower affinities for S203A‐β2‐AR and S204A‐β2‐AR. By contrast, an (−)‐isoprenaline derivative with only a para‐hydroxyl group showed reduced affinity for wild‐type β2‐AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (−)‐isoprenaline was increased, by about 120 fold, for each alanine‐substituted β2‐AR mutant. These results suggest that Ser203 of the human β2‐AR is important for both ligand binding and receptor activation.British Journal of Pharmacology (1999) 128, 272–274; doi:10.1038/sj.bjp.0702813
Constitutive activity of the δ‐opioid receptor expressed in C6 glioma cells: identification of non‐peptide δ‐inverse agonists
Authors: Neilan Claire L , Akil Huda , Woods James H , Traynor John R 
Published: January 2005

G‐protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned δ‐(OP1) receptor (C6δ), or the cloned μ‐(OP3) opioid receptor (C6μ).
Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]‐GTPγS binding that could be inhibited by pre‐treatment with pertussis toxin (PTX). In C6δ cells the level of basal [35S]‐GTPγS binding was reduced by 51.9±6.1 fmols mg−1 protein, whereas in C6μ and C6 wild‐type cells treatment with PTX reduced basal [35S]‐GTPγS binding by only 10.0±3.5 and 8.6±3.1 fmols mg−1 protein respectively.
The δ‐antagonists N,N‐diallyl‐Tyr‐Aib‐Aib‐Phe‐Leu‐OH (ICI 174,864), 7‐benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C‐CAM), acted as δ‐opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]‐GTPγS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]‐GTPγS binding in C6μ or C6 wild‐type cell membranes.
Competition binding assays in C6δ cell membranes revealed a leftward shift in the displacement curve of [3H]‐naltrindole by ICI 174,864 and C‐CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions.
These data confirm the presence of constitutive activity associated with the δ‐opioid receptor and identify three novel, non‐peptide, δ‐opioid inverse agonists.

British Journal of Pharmacology (1999) 128, 556–562; doi:10.1038/sj.bjp.0702816
Thapsigargin‐induced endothelium‐dependent triphasic regulation of vascular tone in the porcine renal artery
Authors: Ihara Eikichi , Hirano Katsuya , Nishimura Junji , Nawata Hajime , Kanaide Hideo 
Published: January 2005

To elucidate the role of thapsigargin‐induced Ca2+ entry in endothelial cells in the regulation of vascular tone, changes in Ca2+ and force of smooth muscle were simultaneously monitored in fura‐2‐loaded strips of porcine renal artery.
During phenylephrine‐induced sustained contraction, thapsigargin caused an endothelium‐dependent triphasic response; an initial relaxation, a subsequent transient contraction, and a sustained relaxation. The initial relaxation and the contraction were associated with a decrease and an increase in [Ca2+]i, respectively. There was no apparent [Ca2+]i decrease during the sustained relaxation. Thapsigargin‐induced responses were observed at 10−8 M and higher concentrations, with the maximum response observed at 10−6 M.
The transient contraction was inhibited by a cyclo‐oxygenase inhibitor (10−5 M indomethacin), a thromboxane A2 (TXA2)/prostagrandin H2 (PGH2) receptor antagonist (10−5 M ONO‐3708), and a TXA2 synthase inhibitor (10−5 M OKY‐046).
During the phenylephrine‐induced contraction in the presence of indomethacin, thapsigargin caused an initial, but not a sustained relaxation, in the presence of Nω‐nitro‐L‐arginine methylester (L‐NAME). During the contraction induced by phenylephrine plus 40 mM K+‐depolarization in the presence of indomethacin, thapsigargin induced both a transient and a sustained relaxation. However, these relaxations were completely abolished in the presence of L‐NAME.
Thapsigargin caused a large Ca2+ elevation in cultured endothelial cells of the renal artery. The concentration‐response relation was thus similar to that for force development in the arterial strips.
In conclusion, thapsigargin‐induced Ca2+ entry in endothelial cells led to triphasic changes in the tone of the porcine renal artery. The endothelium‐dependent contraction was mediated mainly by TXA2. Nitric oxide and hyperpolarizing factor are both involved in the initial relaxation. However, a sustained relaxation was observed which mainly depended on nitric oxide.

British Journal of Pharmacology (1999) 128, 689–699; doi:10.1038/sj.bjp.0702821
Autoradiographical and behavioural effects of a chronic infusion of antisense to the α2D‐adrenoceptor in the rat
Authors: Robinson E S J , Nutt D J , Hall L , Jackson H C , Hudson A L 
Published: January 2005

The aims of this study were, firstly to use receptor autoradiography to investigate the effect of antisense oligonucleotides to the α2D‐adrenoceptor on receptor binding and, secondly to measure behavioural and physiological parameters to determine whether the chronic antisense infusion had any effect on α2‐adrenoceptor function in vivo.
A 3 day infusion of antisense to the α2D‐adrenoceptor significantly reduced specific [3H]‐RX821002 binding in the septum (20–30%) and anterior hypothalamic area (20–30%). β‐Adrenoceptor expression was unaffected in those brain areas examined, indicating the antisense knockdown was specific to the α2‐adrenoceptors.
On the second day of the infusion, the hypothermic response to UK 14,304 was significantly attenuated in the antisense‐treated group compared with both vehicle and mismatch controls. The effect was fully reversible and a similar decrease in body temperature was observed in all the treatment groups 4 days after the end of infusion.
During the second day of the infusion, the effects of UK 14,304 on behaviour were reduced in the antisense‐treated rats, but were not significantly lower than those of the vehicle and mismatch, UK 14,304 controls. These trends were not observed 4 days after the end of the infusion.
In conclusion, antisense has been shown to selectively knockdown α2‐adrenoceptor expression in specific brain areas. The consequence of this knockdown is a significant attenuation of UK 14,304‐induced hypothermia and a reduction in its sedative actions. These changes were fully reversed 4 days after the end of the infusion.

British Journal of Pharmacology (1999) 128, 515–522; doi:10.1038/sj.bjp.0702823
Catechol‐O‐methyltransferase activity in CHO cells expressing norepinephrine transporter
Authors: Percy Elodie , Kaye David M , Lambert Gavin W , Gruskin Sara , Esler Murray D , Du Xiao‐Jun 
Published: January 2005

We examined the existence of catecholamine metabolizing enzymes (catechol‐O‐methyltransferase, COMT, and monoamine oxidase, MAO) in CHO cells transfected with norepinephrine (NE) transporter (NET) cDNA.
NET activity was studied by incubating cells with [3H]‐NE (0.5 μCi ml−1, 20 min) in a Na+ containing medium. Incubation with [3H]‐NE lead to [3H] accumulation at 47797±4864 d.p.m. per well. Specific inhibitors of NET abolished this uptake.
During post‐uptake incubation, [3H] leaked rapidly from cells and the extracellular phase comprised 89% of total radioactivity within 40 min. Both [3H] retention and [3H]‘leakage’ were largely unaffected by inhibitors for MAO. In contrast, COMT inhibitors, U‐0521 and Ro 41‐0960, dose‐dependently increased intracellular [3H]‐NE retention with a maximal increase of 4.5 fold. The EC50 for Ro 41‐0960 was 139‐times lower than that of U‐0521. U‐0521 largely inhibited [3H]‘leakage’ and doubled the apparent Vmax for [3H]‐NE uptake.
Addition of U‐0521 during uptake incubation increased intracellular NE content by 8 fold. Normetanephrine, the COMT‐dependent metabolite of NE, was formed in large quantities during post‐uptake incubation. U‐0521 significantly inhibited the formation of NMN with an equal preservation of intracellular NE.
CHO cells expressing NET possess COMT activity, which is responsible for the metabolism of NE to form lipophilic metabolite normetanephrine. The apparent ‘properties’ of the NET function expressed in CHO cells changed, after inhibition of COMT, in such a way closer to that described in the native neuronal preparations.

British Journal of Pharmacology (1999) 128, 774–780; doi:10.1038/sj.bjp.0702831
On the role of 5‐HT1B/1D receptors in modulating transmission in a spinal reflex pathway in the decerebrated rabbit
Authors: Ogilvie J , Wigglesworth M , Appleby L , Kingston T O W , Clarke R W 
Published: January 2005

In decerebrated rabbits, the selective 5‐HT1B/1D receptor antagonist GR 127,935 had no significant effects on reflexes evoked in medial gastrocnemius motoneurones by electrical stimulation of the sural nerve, or on arterial blood pressure or heart rate when given by the intrathecal (up to 543 nmol cumulative) or intravenous (up to 1.8 μmol cumulative) routes.
In decerebrated, spinalized rabbits, intrathecal GR 127,935 in doses of up to 543 nmol, had no effect on the sural‐gastrocnemius reflex. Furthermore, this drug failed to alter enhancement of the sural‐gastrocnemius reflex induced by 8‐hydroxy‐2‐(di‐n‐propyl)aminotetralin (8‐OH‐DPAT), given at 300 nmol kg−1 i.v.
In decerebrated, spinalized rabbits, the selective 5‐HT1B/1D receptor agonists L‐694,247 (cumulative doses of 2–243 nmol kg−1 i.v.) and L‐741,604 (cumulative doses of 3–307 nmol kg−1 i.v.), each caused the sural‐gastrocnemius reflex to increase to 140% of pre‐drug levels, and arterial blood pressure to rise by about 10 mmHg. Subsequent administration of GR 127,935 at 0.9–1.8 μmol kg−1 reversed the pressor effect of the agonists but not the increase in reflexes. The 5‐HT1A receptor antagonist WAY‐100,635 (185 nmol kg−1 i.v.) also failed to reverse the increase in reflexes, but the 5‐HT1B/1D/5‐HT2/5‐HT7 ligand ritanserin (1.6 μmol kg−1 i.v.) restored reflexes to pre‐drug control values after L‐741,604 (it was not tested against L‐694,247).
These data indicate that 5‐HT1B/1D receptors do not significantly modulate transmission in the sural‐gastrocnemius reflex pathway, and that the enhancement of reflexes by 8‐OH‐DPAT and L‐741,604 is probably mediated by 5‐HT7 receptors.

British Journal of Pharmacology (1999) 128, 781–787; doi:10.1038/sj.bjp.0702832
Anti‐atherosclerotic effects of an angiotensin converting enzyme inhibitor and an angiotensin II antagonist in Cynomolgus monkeys fed a high‐cholesterol diet
Authors: Miyazaki Mizuo , Sakonjo Hiroshi , Takai Shinji 
Published: January 2005

We investigated the relationship between angiotensin II formation and the development of atherosclerotic lesions in the aorta of monkeys (Macaca fascicularis) fed a high‐cholesterol (4% cholesterol and 6% corn oil) diet for 6 months, and studied the effects of an angiotensin converting enzyme (ACE) inhibitor, trandolapril (10 mg kg−1 per day, p.o.), and an angiotensin II type 1 receptor antagonist, 2‐butyl‐4‐(methylthio)‐1‐[[2′[[[(propylamino)carbonyl]amino]sulfonyl](1,1′‐biphenyl)‐4‐yl]methyl]‐1H‐imidazole‐5‐carboxylate (HR 720; 20 mg kg−1 per day, p.o.).
The level of low‐density lipoprotein was significantly increased by the cholesterol diet, whereas that of high‐density lipoprotein was significantly decreased. The relative areas of the atherosclerotic lesions in the thoracic aorta in the normal and cholesterol‐diet groups were 1.3±0.3 and 64±10%, respectively.
Plasma renin and ACE activities showed no differences between the normal and cholesterol‐diet groups. ACE activity and the concentration of angiotensin II were significantly increased in the aorta of the cholesterol‐fed monkeys.
Trandolapril and HR 720 decreased significantly the area of the atherosclerotic lesions in the thoracic aorta of cholesterol‐fed monkeys, but not the mean blood pressure and the levels of low‐density and high‐density lipoproteins.
In plasma and aorta, trandolapril, but not HR 720, decreased significantly the ACE activities in the cholesterol‐fed monkeys, while both of these drugs decreased significantly the angiotensin II levels.
In conclusion, blockade of angiotensin II function in vascular tissues by trandolapril or HR 720 may play an important role in preventing the development of atherosclerotic lesions.

British Journal of Pharmacology (1999) 128, 523–529; doi:10.1038/sj.bjp.0702833
Characterization of chemokine CCR3 agonist‐mediated eosinophil recruitment in the Brown‐Norway rat
Authors: Kudlacz Elizabeth M , Whitney Carrie A , Andresen Catharine J , Umland John P , Cheng John B 
Published: January 2005

The ability of various C‐C chemokines to elicit tissue eosinophil infiltration following intradermal injection or peripheral blood eosinophilia following intravenous injection were compared in the Brown‐Norway rat.
Eotaxin (0.1–3 μg site−1) of human and murine origin produced equivalent, dose‐dependent increases in eosinophil peroxidase activity in rat dermis 4 h post‐injection.
Human eotaxin‐2 was equipotent with human eotaxin in terms of dermal eosinophil recruitment. Other human CCR3 agonists, such as MCP‐3, RANTES and MCP‐4 failed to increase dermal eosinophil peroxidase activity at doses up to 1 μg site−1 whereas the latter did produce a small effect at 3 μg site−1.
Consistent with observations in vivo, human eotaxin displaced [125I]‐eotaxin from rat spleen membranes more potently (IC50=2 nM) than did MCP‐4 (IC50=500 nM). RANTES did not compete with the radiolabelled chemokine at concentrations up to 1 μM.
Human eotaxin (5 μg) administered intravenously increased circulating eosinophils ∼3 fold whereas MCP‐4 (5 μg, i.v.) increased circulating monocytes ∼3 fold without affecting eosinophil numbers.
Dexamethasone pretreatment inhibited eotaxin‐induced dermal eosinophil influx only at a steroid dose (0.1 mg kg−1, s.c.) which significantly reduced circulating eosinophil numbers. The steroid also reduced eosinophilia in peripheral blood resulting from systemic eotaxin administration (5 μg, i.v.).
These data suggest differences in rat CCR3 relative to other species as surmised from a distinctive rank order of chemokine potency. In addition to its chemotactic effects eotaxin, but not MCP‐4, promotes eosinophil recruitment into the circulation. One of the mechanisms by which glucocorticoids, such as dexamethasone, acutely inhibits eotaxin‐induced dermal eosinophil influx is to diminish the circulating numbers of these cells available for tissue recruitment.

British Journal of Pharmacology (1999) 128, 788–794; doi:10.1038/sj.bjp.0702835
Stimulatory effect of exogenous diadenosine tetraphosphate on insulin and glucagon secretion in the perfused rat pancreas
Authors: Silvestre Ramona A , Rodríguez‐Gallardo Jovita , Egido Eva M , Marco José 
Published: January 2005

Diadenosine triphosphate (AP3A) and diadenosine tetraphosphate (AP4A) are released by various cells (e.g. platelets and chromaffin cells), and may act as extracellular messengers. In pancreatic B‐cells, AP3A and AP4A are inhibitors of the ATP‐regulated K+ channels, and glucose increases intracellular levels of both substances.
We have studied the effect of exogenous AP3A and AP4A on insulin and glucagon secretion by the perfused rat pancreas.
AP3A did not significantly modify insulin or glucagon release, whereas AP4A induced a prompt, short‐lived insulin response (∼4 fold higher than basal value; P<0.05) in pancreases perfused at different glucose concentrations (3.2, 5.5 or 9 mM). AP4A‐induced insulin release was abolished by somatostatin and by diazoxide. These two substances share the capacity to activate ATP‐dependent K+ channels, suggesting that these channels are a potential target for AP4A in the B‐cell.
AP4A stimulated glucagon release at both 3.2 and 5.5 mM glucose. This effect was abolished by somatostatin.
The results suggest that extracellular AP4A may play a physiological role in the control of insulin and glucagon secretion.

British Journal of Pharmacology (1999) 128, 795–801; doi:10.1038/sj.bjp.0702837
Effect of pre‐exposure to vasoconstrictors on isoprenaline‐induced relaxation in rat aorta: involvement of inducible nitric oxide synthase
Authors: Eckly‐Michel Anita , Keravis Thérèse , Boudjemaa Najette , Lugnier Claire 
Published: January 2005

The aim of this study was to determine whether a brief (30 min) episode of contractile receptor stimulation could affect the degree of a subsequent vasorelaxation. Therefore, concentration–relaxation curves of the rat aorta to isoprenaline were compared before and after exposure of the tissue to noradrenaline (100 μM) or prostaglandin F2α (PGF2α, 100 μM).
Exposure to noradrenaline enhanced the second maximal relaxant effect of isoprenaline (from 20–95% relaxation). This effect was not due to significant differences in precontraction levels and was not modified by the presence of the endothelium. Treatment with PGF2α mimicked the actions of noradrenaline on subsequent vasorelaxation to isoprenaline.
Before exposure to noradrenaline (100 μM), forskolin (10 μM) did not produce any significant relaxation of the rat aorta. After exposure to noradrenaline, forskolin caused a concentration‐dependent relaxation with a maximal effect of more than 90% in rings with and without endothelium suggesting that the change in vasorelaxation to isoprenaline occured downstream from the β‐adrenoceptor.
The increase in relaxation due to exposure to noradrenaline was markedly attenuated by treatment with a protein synthase inhibitor (cycloheximide), a nitric oxide (NO) synthase inhibitor (L‐NG‐nitroarginine methyl ester, L‐NAME) and an inhibitor of the activation of soluble guanylyl cyclase (methylene blue).
Western blot analysis showed an increase of inducible NO synthase (iNOS) in aortic rings exposed to noradrenaline or PGF2α.
Together, these findings suggest that pretreatment of rat aorta with noradrenaline or PGF2α could induce vascular NOS which would in turn result in an increase in isoprenaline‐induced vasorelaxation, this increase occuring downstream from receptor activation. Such a mechanism might participate in cardioprotection during preconditioning induced by noradrenaline.

British Journal of Pharmacology (1999) 128, 591–596; doi:10.1038/sj.bjp.0702840
Involvement of CYP3A‐derived arachidonic acid metabolite(s) in responses to endothelium‐derived K+ channel opening substance in monkey lingual artery
Authors: Ayajiki Kazuhide , Okamura Tomio , Fujioka Hideyuki , Imaoka Susumu , Funae Yoshihiko , Toda Noboru 
Published: January 2005

In monkey lingual artery strips partially contracted with prostaglandin F2α, acetylcholine‐induced, concentration‐related relaxations were abolished by removal of the endothelium. The response was not significantly influenced by indomethacin but attenuated by NG‐nitro‐L‐arginine (L‐NOARG); the effect of the nitric oxide (NO) synthase inhibitor was reversed by L‐arginine.
The response to acetylcholine resistant to L‐NOARG was suppressed in the strips exposed to high K+ media. Charybdotoxin partially inhibited the relaxation, and the remaining relaxation was abolished by additional treatment with apamin, whereas glibenclamide, iberiotoxin or apamin alone was without effect. Relaxations induced by sodium nitroprusside were not influenced by charybdotoxin.
The L‐NOARG‐resistant acetylcholine‐induced relaxation was inhibited by metyrapone, proadifen and 17‐octadecynoic acid, non‐selective cytochrome P450 mono‐oxygenase (CYP) inhibitors, and progesterone and ketoconazole, inhibitors selective to CYP3A. The inhibitors did not affect the nitroprusside‐induced relaxation. Selective inhibitors of other CYP isoforms, such as debrisoquine and lauric acid, did not reduce the response to acetylcholine.
Reaction mixture containing human liver microsome rich in CYPs, arachidonic acid and NADPH incubated at 37°C and filtrated relaxed endothelium‐denuded monkey lingual artery strips, used as bioassay tissues. This response was abolished in the strips exposed to high K+ media. The response was also suppressed by combined treatment of the assay tissue with charybdotoxin plus apamin, but was not affected by treatment with iberiotoxin. The reaction mixture co‐incubated with ketoconazole failed to relax the strips.
It is concluded that the monkey lingual arterial relaxation dependent on the endothelium is mediated by NO and also by a charybdotoxin plus apamin‐sensitive but iberiotoxin‐insensitive Ca2+‐activated K+ channel opening substance(s) that may be a CYP3A‐derived arachidonic acid metabolite(s).

British Journal of Pharmacology (1999) 128, 802–808; doi:10.1038/sj.bjp.0702843
Adrenergic and purinergic components in bisected vas deferens from spontaneously hypertensive rats
Authors: Guitart Mònica , Giraldo Jesús , Goñalons Eduard , Vila Elisabet , Badia Albert 
Published: January 2005

Purinergic and adrenergic components of the contractile response to electrical field stimulation (EFS) have been investigated in epididymal and prostatic portions of Wystar Kyoto (WKY) and spontaneously hypertensive rat (SHR) vas deferens.
In both halves of SHR and WKY vas deferens, EFS (40 V, 0.5 ms for 30 s, 0.5–32 Hz) evoked frequency‐related contractions. The neurogenic responses were biphasic, consisting of a rapid non‐adrenergic response, dominant in the prostatic portion, followed by a slow tonic adrenergic component, dominant in the epididymal half.
Phasic and tonic components of the frequency‐response curves evoked by EFS were significantly higher in the epididymal but not in the prostatic portion of vas deferens from SHR compared to WKY rats.
The α1‐adrenoceptor antagonist prazosin (0.1 μM) was more effective against both components of the contractile response in the epididymal end of SHR than in WKY rats.
Inhibition by α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP 3 and 30 μM) was higher in both components of the contractile responses in WKY preparations than in SHR.
Combined α1‐adrenoceptor and P2x‐purinoceptor antagonism virtually abolished the EFS‐evoked contractile response in both strains. The degree of inhibition by prazosin (0.1 μM) after P2x‐purinoceptor blockade was higher in SHR than in WKY rats.
These results demonstrate a modification in the purinergic and noradrenergic contribution to neurogenic responses in SHR and WKY animals besides a co‐participation of ATP and noradrenaline in both contractile components of the response to EFS.

British Journal of Pharmacology (1999) 128, 873–880; doi:10.1038/sj.bjp.0702845