bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha‐1 adrenoceptor up‐regulation induced by prazosin but not KMD‐3213 or reserpine in rats
Authors: Zhang Li , Taniguchi Takanobu , Tanaka Takashi , Shinozuka Kazumasa , Kunitomo Masaru , Nishiyama Masahiko , Kamata Koji , Muramatsu Ikunobu
Published: January 2005

We have investigated the effects of chronic administration of prazosin (a subtype‐nonspecific alpha‐1 AR antagonist), KMD‐3213 (an alpha‐1A AR subtype‐specific antagonist) and reserpine (a catecholamine depletor) on the density of alpha‐1 AR subtypes in various rat tissues (liver, kidney, submaxillary gland, heart and spleen).

Administration of prazosin (2 mg kg−1 day−1, i.p.) for 2 weeks did not affect KD values for [3H]‐prazosin or [3H]‐KMD‐3213 of alpha‐1 ARs in five rat tissues tested. However, it caused 52% up‐regulation of alpha‐1B AR in the spleen, and 84% and 107% up‐regulation of alpha‐1A‐ and alpha‐1B ARs, respectively, in the heart. Although major subtypes of alpha‐1 AR are alpha‐1A AR in the submaxillary gland, alpha‐1B AR in the liver, and alpha‐1A and alpha‐1B ARs in the kidney, these tissues showed no up‐regulation. The mRNA levels of alpha‐1 AR subtypes were not affected by prazosin administration in any tissue tested.

Neither administration of KMD‐3213 (2 mg kg−1 day−1, i.p.) nor reserpine (0.5 – 1 mg kg−1 day−1, i.p.) for 2 weeks caused any change in either the binding affinity for [3H]‐prazosin or [3H]‐KMD‐3213 or the density of the alpha‐1 AR subtypes in the five rat tissues.

Neither prazosin nor KMD‐3213 treatment reduced the noradrenaline content in the five rat tissues, in contrast to reserpine treatment, which markedly reduced it.

The findings of the present study demonstrated that up‐regulation of alpha‐1 AR is selectively caused by prazosin treatment in some tissues but neither by KMD‐3213 treatment nor by chemical denervation with reserpine. These results suggest that up‐regulation of alpha‐1 ARs is not caused by a simple blockade of sympathetic tone.

British Journal of Pharmacology (2002) 135, 1757–1764; doi:10.1038/sj.bjp.0704639
Role of protein kinase C‐epsilon in the development of κ‐opioid receptor tolerance to U50,488H in rat ventricular myocytes
Authors: Zhou Jing‐Jun , Bian Jin‐Song , Pei Jian‐Ming , Wu Song , Li Hong‐Yu , Wong Tak‐Ming
Published: January 2005

The role of protein kinase C‐epsilon (PKC‐ε) in the development of κ‐opioid receptor (κ‐OR) tolerance to the effects of trans‐(±)‐3,4‐dichloro‐N‐methyl‐N‐(2‐[1‐pyrrolidinyl]cyclohexyl) (U50,488H), the selective agonist of κ‐OR, was determined in rat ventricular myocytes.

Incubation of ventricular myocytes with 1 μM U50,488H for 24 h significantly attenuated the inhibitory effects of 30 μM U50,488H on the electrically‐induced [Ca2+]i transient and forskolin‐stimulated cyclic AMP accumulation, indicating the development of tolerance to the κ‐OR agonist. Chronic treatment of ventricular myocytes with U50,488H also induced translocation of PKC‐ε to the particulate fraction. On the other hand, administration of 30 μM U50,488H for 10 min induced translocation of PKC‐α to the particulate fraction in naïve ventricular myocytes, but not in cells pretreated with 1 μM U50,488H for 24 h.

In ventricular myocytes incubated for 24 h with 1 μM U50,488H together with 1 μM chelerythrine or 1 μM GF109203X, PKC inhibitors, or 0.1 μM εV1‐2 peptide, a selective inhibitor of PKC‐ε, 30 μM U50,488H still produced the inhibitory effect on the electrically‐induced [Ca2+]i transient as it did in naïve ventricular myocytes. Chronic treatment of ventricular myocytes with U50,488H and chelerythrine also attenuated the development of tolerance to acute U50,488H on cyclic AMP accumulation. Cells exposed to chelerythrine, GF109203X, or εV1‐2 peptide alone did not show an altered [Ca2+]i response to U50,488H.

These results indicate that activation of PKC‐ε is a critical step in the development of tolerance in the κ‐OR.

British Journal of Pharmacology (2002) 135, 1675–1684; doi:10.1038/sj.bjp.0704640
Generalized loss of inhibitory innervation reverses serotonergic inhibition into excitation in a rabbit model of TNBS‐colitis
Authors: Depoortere Inge , Thijs Theo , Peeters Theo L
Published: January 2005

Inflammation may affect subpopulations of neurons of the myenteric plexus.

In the present study the effect of trinitrobenzene sulphonic acid (TNBS) induced colitis on nitrergic, purinergic and adrenergic inhibitory neurotransmission was studied as well as the consequences of the related changes on the response of 5‐HT agonists using these neurotransmitters to mediate their effect.

Strips from normal and colitis rabbits (135 mg kg−1 TNBS) were subjected to electrical field stimulation (EFS, 0.3 ms, 6V, 0.5 – 32 Hz, 10 s train). The response was measured isometrically in the absence or presence of L‐NAME, suramin, guanethidine, the 5‐HT agonists (5‐HT1/5A/7: 5‐carboxamidotryptamine (5‐CT), 5‐HT2: α‐methyl‐5‐HT, 5‐HT3: 2‐methyl‐5‐HT, 5‐HT4: 5‐methoxytryptamine (5‐MeOT)) or a combination.

In normal strips L‐NAME (1 – 32 Hz), suramin (0.5 – 2, 8 Hz) and guanethidine (4, 16, 32 Hz) increased the response to EFS. This effect was abolished in inflamed strips and was accompanied by a decrease in nNOS expression.

In normal strips all 5‐HT agonists induced pronounced (5‐CT, α‐methyl‐5‐HT) or small (2‐methyl‐5‐HT, 5‐MeOT) inhibitory neural responses. In inflamed strips this was reversed to cholinergic excitatory responses.

The effect of inflammation on the 5‐HT4 response was mimicked by preincubation of normal strips with L‐NAME or suramin. Accordingly, in inflamed strips L‐NAME or suramin did not affect the excitatory effects of 5‐MeOT.

TNBS‐colitis abolishes nitrergic, purinergic and adrenergic neurotransmission. This reverses serotonergic inhibition into excitation.

British Journal of Pharmacology (2002) 135, 2011–2019; doi:10.1038/sj.bjp.0704648
Second messenger‐dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness
Authors: Ghadessy Roxana S , Kelly Eamonn
Published: January 2005

The present study investigated the role of second messenger‐dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108‐15 mouse neuroblastoma×rat glioma hybrid cells.

In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12‐myristate 13‐acetate (PMA) or by purinoceptor stimulation using uridine 5′‐triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin‐stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid‐IP receptor agonist iloprost, or the A2adenosine receptor agonist 5′‐(N‐ethylcarboxamido) adenosine (NECA).

In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of Gs‐coupling components in NG108‐15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation.

Cells exposed to the PKA inhibitor H‐89, exhibited a time‐dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H‐89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time‐dependent increase in secretin receptor responsiveness.

Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108‐15 cells.

British Journal of Pharmacology(2002)135, 2020–2028; doi:10.1038/sj.bjp.0704655
Differential contributions of nitric oxide synthase isoforms at hippocampal formation to negative feedback regulation of penile erection in the rat
Authors: Chang Alice Y. W. , Chan Julie Y. H. , Chan Samuel H. H.
Published: January 2005

We established previously that a novel negative feedback mechanism for the regulation of penile erection, which is triggered by ascending sensory inputs initiated by tumescence of the penis, exists in the hippocampal formation (HF). This study further evaluated the participation of nitric oxide (NO) and the contribution of nitric oxide synthase (NOS) isoforms at the HF in this process.

Adult, male Sprague‐Dawley rats that were anaesthetized and maintained with chloral hydrate were used, and intracavernous pressure (ICP) recorded from the corpus cavernosum of the penis was employed as our experimental index for penile erection.

Microinjection bilaterally of a NO donor, S‐nitroso‐N‐acetylpenicillamine (0.25 or 1 nmoles), or the NO precursor, L‐arginine (1 or 5 nmoles), into the hippocampal CA1 or CA3 subfield or dentate gyrus elicited a significant reduction in baseline ICP.

Bilateral hippocampal application of a NO trapping agent, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (10 nmoles), significantly potentiated the elevation in ICP induced by intracavernous administration of papaverine (400 μg).

Microinjection bilaterally into the HF of equimolar doses (0.5 or 2.5 pmoles) of two selective neuronal NOS inhibitors, 7‐nitroindazole or Nω‐propyl‐L‐arginine; or equimolar doses (50 or 250 pmoles) of two selective inducible NOS inhibitors, aminoguanidine or S‐methylisothiourea, significantly enhanced the magnitude and/or duration of the papaverine‐induced elevation in ICP. In contrast, hippocampal application of a potent endothelial NOS inhibitor, N5‐(1‐iminoethyl)‐L‐ornithine (18 or 92 nmoles), was ineffective. Neither of these inhibitors, furthermore, affected baseline ICP.

These results suggest that NO generated via both neuronal and inducible NOS at the HF may participate in negative feedback regulation of penile erection.

British Journal of Pharmacology (2002) 136, 1–8; doi:10.1038/sj.bjp.0704657
Characterization of cocaine and antidepressant‐sensitive norepinephrine transporters in rat placental trophoblasts
Authors: Jayanthi Lankupalle D , Vargas Gricelly , DeFelice Louis J
Published: January 2005

This paper reports on a primary cell culture system that predominately expresses native norepinephrine (NE) transporters (NETs), and is amenable to biophysical as well as biochemical analyses.

Previous research has identified human and rat placentas as rich sources of NET. We have exploited this to develop primary cultures of rat placental trophoblasts. NE uptake in these cultures is about 10 times higher when compared to 5HT uptake. The presence of NET protein is revealed by immunoblot analysis, while there is no detectable SERT protein.

NE transport in rat trophoblasts is sensitive to NET‐specific antagonists, desipramine (DS) and nisoxetine (NX), but not to the dopamine‐transporter (DAT) specific antagonist, GBR12909 or to the serotonin (5HT) transporter (SERT) specific antagonist paroxetine (PX). Drugs of abuse such as cocaine and amphetamine also inhibit NE transport in these cells. Together these results suggest that rat placental trophoblasts predominately express NET over other monoamine transporters.

Patch‐clamp analysis reveals that NETs in intact rat trophoblasts are electrogenic. Comparison of NE uptake with NE‐induced currents suggests that these two modes of transporter activity are differentially regulated.

British Journal of Pharmacology (2002) 135, 1927–1934; doi:10.1038/sj.bjp.0704658
Effects of chronic administration of the novel endothelin antagonist J‐104132 on endothelial dysfunction in streptozotocin‐induced diabetic rat
Authors: Kanie Noriyasu , Kamata Katsuo
Published: January 2005

The biosynthesis of endothelin‐1 is increased in the diabetic state. So this peptide may cause diabetic vascular complications. We tested this possibility by chronically administering J‐104132, a potent orally active mixed antagonist of endothelin A and B (ETA/ETB) receptors to streptozotocin (STZ)‐induced diabetic rats and focusing on changes in endothelial function.

The acetylcholine (ACh)‐induced endothelium‐dependent relaxation was impaired in diabetic rats and this impairment was significantly attenuated following chronic administration of J‐104132 (10 mg kg−1, p.o., daily for 4 weeks).

In an in vitro experiment using aortae from diabetic rats, the ACh‐induced relaxation was not changed by the presence of J‐104132 (3×10−9 M).

The expression levels of the mRNA for endothelial nitric oxide synthase was comparable among aortae from the three groups (control, diabetic and chronically J‐104132‐treated diabetic).

The amount of superoxide anion was significantly greater in aortae from diabetic rats than in controls. Chronic J‐104132 treatment significantly decreased the level of superoxide anion in diabetic rats.

The expression of the p22phox mRNA for the NADH/NADPH oxidase subunit was significantly increased in STZ‐induced diabetic rats and this increase was completely prevented by chronic administration of J‐104132.

These results suggest that in STZ‐induced diabetic rats, ET‐1 may be directly involved in impairing endothelium‐dependent relaxation via increased superoxide‐anion production.

British Journal of Pharmacology (2002) 135, 1935–1942; doi:10.1038/sj.bjp.0704659
Receptor subtypes Y1 and Y5 mediate neuropeptide Y induced feeding in the guinea‐pig
Authors: Lecklin Anne , Lundell Ingrid , Paananen Leena , Wikberg Jarl E S , Männistö Pekka T , Larhammar Dan
Published: January 2005

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated which receptor subtype mediates this response. Initially Y1 was proposed, but later Y5 was announced as a ‘feeding’ receptor in rats and mice. Very little is known regarding other mammals. The present study attempts to characterize the role of NPY in feeding behaviour in the distantly related guinea‐pig. When infused intracerebroventricularly, NPY dose‐dependently increased food intake.

PYY, (Leu31,Pro34)NPY and NPY(2 – 36) stimulated feeding, whereas NPY(13 – 36) had no effect. These data suggest that either Y1 or Y5 receptors or both may mediate NPY induced food intake in guinea‐pigs.

The Y1 receptor antagonists, BIBO 3304 and H 409/22 displayed nanomolar affinity for the Y1 receptor (Ki values 1.1±0.2 nM and 5.6±0.9 nM, respectively), but low affinity for the Y2 or Y5 receptors. When guinea‐pigs were pretreated with BIBO 3304 and H 409/22, the response to NPY was inhibited.

The Y5 antagonist, CGP 71683A had high affinity for the Y5 receptor (Ki 1.3±0.05 nM) without having any significant activities at the Y1 and Y2 receptors. When CGP 71683A was infused into brain ventricles, the feeding response to NPY was attenuated.

The present study shows that NPY stimulates feeding in guinea‐pigs through Y1 and Y5 receptors. As the guinea‐pig is very distantly related to the rat and mouse, this suggests that both Y1 and Y5 receptors may mediate NPY‐induced hyperphagia also in other orders of mammals.

British Journal of Pharmacology (2002) 135, 2029–2037; doi:10.1038/sj.bjp.0704667
Molecular and pharmacological characterization of genes encoding urotensin‐II peptides and their cognate G‐protein‐coupled receptors from the mouse and monkey
Authors: Elshourbagy Nabil A. , Douglas Stephen A. , Shabon Usman , Harrison Stephen , Duddy Graham , Sechler Jan L. , Ao Zhaohui , Maleeff Beverly E. , Naselsky Diane , Disa Jyoti , Aiyar Nambi V.
Published: January 2005

Urotensin‐II (U‐II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U‐II/UT receptor system. The goal of this study was to clone mouse and primate preproU‐II and UT for pharmacological profiling.

Monkey and mouse preproU‐II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU‐II has four exons, while the UT receptor has one exon.

Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U‐II/UT receptor mRNA in extra‐vascular tissue including lung, pancreas, skeletal muscle, kidney and liver.

Ligand binding studies showed that [125I]h U‐II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654±154 and 214±65 pM and Bmax of 1011±125 and 497±68 fmol mg−1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U‐II isopeptides to these receptors (Ki=0.8 – 3 nM). Fluorescein isothiocyanate (FITC) labelled U‐II, bound specifically to HEK‐293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor.

Exposure of these cells to human U‐II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2±0.8 and 1.1±0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2±1.8 and 0.9±0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.

British Journal of Pharmacology (2002) 136, 9–22; doi:10.1038/sj.bjp.0704671
Pharmacology of the nitric oxide receptor, soluble guanylyl cyclase, in cerebellar cells
Authors: Bellamy Tomas C. , Garthwaite John
Published: January 2005

The nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), is commonly manipulated pharmacologically in two ways. Inhibition of activity is achieved using 1‐H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐l‐one (ODQ) which oxidizes the haem prosthetic group to which NO binds, while the compound 3‐(5‐hydroxymethyl‐2‐furyl)‐1‐benzylindazole (YC‐1) is considered an ‘allosteric’ activator. Knowledge of how these agents function and interact in a normal cellular environment is limited. These issues were addressed using rat cerebellar cells.

Inhibition by ODQ was not simply competitive with NO. The rate of onset was ODQ concentration‐dependent and developed in two kinetic phases. Recovery from inhibition occurred with a half‐time of ∼5 min.

YC‐1 slowed the rate at which sGC deactivated on removal of NO by 45 fold, consistent with YC‐1 increasing the potency of NO for sGC. YC‐1 also enhanced the maximal response to NO by 2 fold. Furthermore, when added to cells in which sGC was 90% desensitized, YC‐1 abruptly enhanced sGC activity to a degree that indicated partial reversal of desensitization.

After pre‐exposure to YC‐1, sGC became resistant to inhibition by ODQ. In addition, YC‐1 rapidly reversed inhibition by ODQ in cells and for purified sGC, suggesting that YC‐1 either increases the NO affinity of the oxidized sGC haem or reverses haem oxidation.

It is concluded that the actions of ODQ and YC‐1 on sGC are broadly similar in cells and purified preparations. Additionally, YC‐1 transiently reverses sGC desensitization in cells. It is hypothesized that YC‐1 has multiple actions on sGC, and thereby both modifies the NO binding site and enhances agonist efficacy.

British Journal of Pharmacology (2002) 136, 95–103; doi:10.1038/sj.bjp.0704687
Discrimination by SZL49 between contractions evoked by noradrenaline in longitudinal and circular muscle of human vas deferens
Authors: Amobi Nnaemeka I. B. , Guillebaud John , Kaisary A V. , Turner Eileen , Smith I Christopher H.
Published: January 2005

The effects of irreversible α1‐adrenoceptor antagonists, SZL‐49 (an alkylating analogue of prazosin), dibenamine and benextramine on contractions to noradrenaline (NA) in longitudinal and circular muscle of human epididymal vas deferens were investigated. Competitive α1‐adrenoceptor antagonists were also used to further characterize the α1‐adrenoceptor subtype stimulated by NA in longitudinal and circular muscle.

NA evoked concentration‐dependent contractions of both muscle types (pD2; 5.4 and 5.2 respectively). The contraction of circular muscle was comparatively more sensitive than that of longitudinal muscle to pretreatment with SZL‐49. In contrast, dibenamine or benextramine produced comparable effects in both muscle types.

The relationship between receptor occupancy and contraction in either longitudinal or circular muscle was nonlinear, with half‐maximal response requiring similar receptor occupancy (longitudinal muscle 14%, circular muscle 16%). Maximal response in both muscle types occurred with little or no receptor reserve (<10%).

The competitive α1‐adrenoceptor antagonists produced dextral shifts of the dose‐response curves to NA in longitudinal and circular muscle. The inhibitory potencies, estimated from the apparent pKB values were significantly different in longitudinal and circular muscle respectively for either WB 4101 (pKB, 8.6 and 9.5) or RS‐17053 (pKB, 7.1 and 9.0) but not for Rec 15/2739 (pKB, 9.2 and 9.8) or HV 723 (pKB, 8.3 and 8.4).

In conclusion, the potency profile of the competitive α1‐adrenoceptor antagonists and the lack of different receptor reserves for NA in the muscle types suggest that the discriminatory effects of SZL‐49 is primarily due to a predominance of the α1L‐adrenoceptor subtype in longitudinal muscle and α1A‐subtype in circular muscle.

British Journal of Pharmacology (2002) 136, 127–135; doi:10.1038/sj.bjp.0704689
Interaction of group I mGlu and NMDA receptor agonists within the dorsal horn of the spinal cord of the juvenile rat
Authors: Dang K , Naeem S , Walker K , Bowery N G , Urban L
Published: January 2005

The modulatory effects of mGlu receptors on NMDA‐induced potential changes in spinal motoneurones were studied in vitro.

Selective activation of mGlu5 receptors by 10 μM (RS)‐2‐Chloro‐5‐hydroxyphenylglycine (CHPG; EC50=280±24 μM) did not produce any change in the ventral root potential. However, the same concentration of CHPG (10 min perfusion) significantly attenuated the NMDA‐induced ventral root depolarization (VRD). The effect persisted for 10 min after washout. NMDA‐induced responses returned to control in 30 min. Brief co‐application of CHPG and NMDA did not alter the NMDA‐induced response indicating lack of direct receptor interaction.

The attenuating effect of CHPG on the NMDA‐induced VRD was inhibited by the mGluR5 receptor antagonist, 2‐methyl‐6‐phenyl‐ethynylpyridine (MPEP).

In the presence of CGP56433A, a GABAB receptor antagonist, the NMDA‐induced VRD was unchanged. However, NMDA‐induced responses were potentiated after 10 min co‐application of CHPG and CGP56433A.

(2R,4R)‐4‐aminopyrrolidine‐2,4‐dicarboxylate ((2R,4R)‐APDC), a group II mGlu receptor agonist did not attenuate the NMDA‐induced response.

Under normal physiological conditions group I mGlu receptor agonists activate at least two populations of neurones: (1) GABA‐ergic cells, which could release GABA and inhibit dorsal horn neurones, and (2) deep dorsal horn neurones/motoneurones which express NMDA receptors. Therefore, activation of mGlu5 receptors located on GABA‐ergic interneurones could influence any direct potentiating interaction between mGlu5 and NMDA receptors in spinal cord and result in depression of the VRD. In the presence of a GABAB receptor antagonist, the direct synergistic interaction is unmasked. These data suggest that group I mGlu receptors provide a complex modulation of spinal synaptic processes.

British Journal of Pharmacology (2002) 136, 248–254; doi:10.1038/sj.bjp.0704698
Regulation by PGE2 of the production of interleukin‐6, macrophage colony stimulating factor, and vascular endothelial growth factor in human synovial fibroblasts
Authors: Inoue Hideo , Takamori Makiko , Shimoyama Yoshihito , Ishibashi Hideaki , Yamamoto Seizo , Koshihara Yasuko
Published: January 2005

We examined the effects of endogenous prostaglandin E2 (PGE2) on the production of interleukin‐6 (IL‐6), macrophage colony stimulating factor (M‐CSF), and vascular endothelial growth factor (VEGF) by interleukin‐1β (IL‐1β)‐stimulated human synovial fibroblasts.

NS‐398 (1 μM), a cyclo‐oxygenase‐2 (COX‐2) inhibitor, inhibited IL‐6 and VEGF production (35±4% and 26±2%, respectively) but enhanced M‐CSF production (38±4%) by IL‐1β (1 ng ml−1) in synovial fibroblasts isolated from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Exogenous PGE2 completely abolished the effects of NS‐398 on the production of each mediator by OA fibroblasts stimulated with IL‐1β.

8‐Bromo cyclic AMP and dibutyryl cyclic AMP, cyclic AMP analogues, mimicked the effects of PGE2 on IL‐6, M‐CSF, and VEGF production by OA fibroblasts.

The EP2 selective receptor agonist ONO‐AE1‐259 (2 nM) and the EP4 selective receptor agonist ONO‐AE1‐329 (2 or 20 nM), but not the EP1 selective receptor agonist ONO‐DI‐004 (1 μM) and the EP3 selective receptor agonist ONO‐AE‐248 (1 μM), replaced the effects of PGE2 on IL‐6, M‐CSF, and VEGF production by OA and RA fibroblasts stimulated with IL‐1β in the presence of NS‐398.

Both OA and RA fibroblasts expressed mRNA encoding EP2 and EP4 but not EP1 receptors. In addition, up‐regulation of EP2 and EP4 receptor mRNAs was observed at 3 h after IL‐1β treatment.

These results suggest that endogenous PGE2 regulates the production of IL‐6, M‐CSF, and VEGF by IL‐1β‐stimulated human synovial fibroblasts through the activation of EP2 and EP4 receptors with increase in cyclic AMP.

British Journal of Pharmacology (2002) 136, 287–295; doi:10.1038/sj.bjp.0704705
[Nphe1,Arg14,Lys15]Nociceptin‐NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor
Authors: Calo Girolamo , Rizzi Anna , Rizzi Daniela , Bigoni Raffaella , Guerrini Remo , Marzola Giuliano , Marti Matteo , McDonald John , Morari Michele , Lambert David G , Salvadori Severo , Regoli Domenico
Published: January 2005

Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions by activating a specific G‐protein coupled receptor (NOP). Few molecules are available that selectively activate or block the NOP receptor. Here we describe the in vitro and in vivo pharmacological profile of a novel NOP receptor ligand, [Nphe1,Arg14,Lys15]N/OFQ‐NH2 (UFP‐101).

UFP‐101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high affinity (pKi 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP‐101 competitively antagonizes the effects of N/OFQ on GTPγ35S binding in CHOhNOP cell membranes (pA2 9.1) and on cyclic AMP accumulation in CHOhNOP cells (pA2 7.1), being per se inactive at concentrations up to 10 μM.

In isolated peripheral tissues of mice, rats and guinea‐pigs, and in rat cerebral cortex synaptosomes preloaded with [3H]‐5‐HT, UFP‐101 competitively antagonized the effects of N/OFQ with pA2 values in the range of 7.3–7.7. In the same preparations, the peptide was inactive alone and did not modify the effects of classical opioid receptor agonists.

UFP‐101 is also active in vivo where it prevented the depressant action on locomotor activity and the pronociceptive effect induced by 1 nmol N/OFQ i.c.v. in the mouse. In the tail withdrawal assay, UFP‐101 at 10 nmol produces per se a robust and long lasting antinociceptive effect.

UFP‐101 is a novel, potent and selective NOP receptor antagonist which appears to be a useful tool for future investigations of the N/OFQ‐NOP receptor system.

British Journal of Pharmacology (2002) 136, 303–311; doi:10.1038/sj.bjp.0704706
Modification of NMDA responses by tri‐n‐butyltin in rat brain neurons
Authors: Kanemoto Yumiko , Ishibashi Hitoshi , Matsuo Shinichiro , Oyama Yasuo , Akaike Norio
Published: January 2005

The effects of the organotin, tri‐n‐butyltin (TBT), on N‐methyl‐D‐aspartate (NMDA) induced membrane currents were investigated in order to evaluate possible neuronal actions of this toxic environmental pollutant. Experiments were conducted on neurons acutely dissociated from the rat dorsal motor nucleus of vagus (DMV) using the nystatin‐perforated patch clamp recording technique.

In Mg2+‐free physiological recording solutions, the application of NMDA to single DMV neurons held at a holding potential (VH) of −40 mV evoked an inward current which rapidly reached a peak before declining to a steady‐state inward current. This was followed, immediately after NMDA washout, by a transient outward current. TBT (100 nM) reversibly caused a slight reduction in the inward currents and greatly increased the amplitude of the outward currents.

The reversal potential of the NMDA‐induced outward current in the presence of TBT was −86.7 mV, close to the theoretical K+ equilibrium potential of −85.7 mV.

The NMDA‐induced outward current was completely blocked when the K+ in the internal solution was replaced with equimolar Cs+. Under these conditions, the NMDA induced current was more sustained and was unaffected by TBT.

The NMDA‐induced outward current was markedly inhibited by 5 mM tetraethylammonium chloride and 300 nM charybdotoxin, and it was abolished by removal of extracellular Ca2+, suggesting that the outward current was due to the activation of Ca2+‐activated K+ channels by Ca2+ influx through NMDA receptors.

In conclusion, in rat DMV neurons, TBT potentiates the Ca2+‐activated K+ current induced by NMDA application without having any direct effects on the NMDA‐induced inward current. Given the significant role of NMDA receptor mediated excitation in various physiological and pathological processes, the modulation of this response by TBT may have an important influence on neuronal function.

British Journal of Pharmacology (2002) 136, 201–206; doi:10.1038/sj.bjp.0704707
Involvement of 5‐HT1B and 5‐HT1D receptors in sumatriptan mediated vasocontractile response in rabbit common carotid artery
Authors: Akin Demet , Gurdal Hakan
Published: January 2005

In this study we examined the involvement of 5‐HT1B and 5‐HT1D receptors in the vasocontractile response induced by 5‐HT1B/D‐receptor agonist sumatriptan in rabbit common carotid artery (CCA).

Immunoblotting experiments using specific antisera against 5‐HT1B or 5‐HT1D receptors revealed the presence of one weak (at 93 kD for 5‐HT1B or at 105 kD for 5‐HT1D) and one strong band (at 46 kD for 5‐HT1B or at 52 kD for 5‐HT1D) in CCA.

Sumatriptan‐mediated vasocontractile response was antagonized by SB216641 with an apparent pKb value of 8.6, which was consistent with its affinity for 5‐HT1B receptor. Antagonism by BRL15572 was weak and calculated apparent pKb (6.0) value was consistent with its affinity for 5‐HT1B subtype (but not for 5‐HT1D subtype). This result indicates insignificant or no involvement of 5‐HT1D receptor in the vasocontractile response.

The vasocontractile response induced by sumatriptan was highly sensitive to pertussis toxin treatment of CCA. Nicardipine, a calcium channel blocker, also potently antagonized vasocontractile response induced by sumatriptan.

5‐HT, but not sumatriptan, stimulated inositol phosphate accumulation in CCA.

These results indicate that stimulation of 5‐HT1B subtype activate a pertussis toxin (PTX) sensitive G protein (Go/Gi) and mediate vasocontraction, in which L‐type voltage dependent calcium channels are involved.

British Journal of Pharmacology (2002) 136, 177–182; doi:10.1038/sj.bjp.0704709
Agonist‐directed trafficking explaining the difference between response pattern of naratriptan and sumatriptan in rabbit common carotid artery
Authors: Akin Demet , Onaran H Ongun , Gurdal Hakan
Published: January 2005

Sumatriptan or eletriptan produced vasocontraction in common carotid artery (CCA) by stimulating 5HT1B receptors (see also Akin & Gurdal, this issue).

Naratriptan as a 5HT1B/D agonist, was unable to produce vasocontraction in this artery, but inhibited the vasocontractile response induced by sumatriptan or eletriptan.

All these agonists inhibited forskolin‐stimulated cyclic AMP production with comparable potencies and maximal responses. This inhibition was mediated by 5HT1B receptors: 5HT1B antagonist SB216641 (1 μM) completeley antagonized sumatriptan‐, eletriptan‐ or naratriptan‐induced cyclic AMP inhibition, but 5HT1D antagonist BRL15572 (1 μM) did not affect this response.

Naratriptan‐induced stimulation of 5‐HT1B receptors resulted only in adenylate cyclase inhibition, whereas stimulation of these receptors by sumatriptan or eletriptan produced vasocontraction as well. Hence, we concluded that the 5HT1B‐mediated inhibition of adenylate cyclase was not a sufficient condition to couple the receptor stimulation to vasocontraction.

We discussed agonist‐induced trafficking as a plausible mechanism for the observed phenomenon.

British Journal of Pharmacology (2002) 136, 171–176; doi:10.1038/sj.bjp.0704710
Arginine to lysine 108 substitution in recombinant CYP1A2 abolishes methoxyresorufin metabolism in lymphoblastoid cells
Authors: Hadjokas Nicholas E , Dai Renke , Friedman Fred K , Spence Michael J , Cusack Barry J , Vestal Robert E , Ma Yongsheng
Published: January 2005

Cytochrome P4501A2 (CYP1A2) activates a large number of procarcinogens to carcinogens. Phytochemicals such as flavones can inhibit CYP1A2 activity competitively, and hydroxylated derivatives of flavone (galangin) may be potent, selective inhibitors of CYP1A2 activity relative to CYP1A1 activity. Molecular modelling of the CYP1A2 interaction with hydroxylated derivatives of flavone suggests that a number of hydrophobic residues of the substrate‐binding domain engage in hydrogen bonding with such inhibitors.

We have tested this model using site‐directed mutagenesis of these residues in expression plasmids transfected into the human B‐lymphoblastoid cell line, AHH‐1 TK+/−.

Consistent with the molecular model's predicted placement in the active site, amino acid substitutions at the predicted residues abolished CYP1A2 enzymatic activity.

Transfected cell lines contained equal amounts of immunoreactive CYP1A2.

Our results support the molecular model's prediction of the critical amino acid residues present in the hydrophobic active site, residues that can hydrogen bond with CYP1A2 inhibitors and modify substrate binding and/or turnover.

British Journal of Pharmacology (2002) 136, 347–352; doi:10.1038/sj.bjp.0704711
Effects of adenosine A1 and A2A receptor activation on the evoked release of glutamate from rat cerebrocortical synaptosomes
Authors: Marchi Mario , Raiteri Luca , Risso Francesca , Vallarino Annalisa , Bonfanti Andrea , Monopoli Angela , Ongini Ennio , Raiteri Maurizio
Published: January 2005

The effects of adenosine A2A and A1 receptor activation on the release of glutamate were studied in rat cerebral cortex synaptosomes exposed in superfusion to adenosine receptor ligands.

Adenosine (0.1 μM) produced a significant potentiation of the Ca2+‐dependent K+(15 mM)‐evoked [3H]‐D‐aspartate overflow (20.4±3.5%), which was blocked by A2A blocker SCH58261 (0.1 μM). At higher concentrations (10 – 1000 μM) adenosine inhibited in a DPCPX‐sensitive manner the Ca2+‐dependent K+‐evoked [3H]‐D‐aspartate overflow. The inhibitory effect of adenosine at 1000 μM was significantly increased by SCH58261. This inhibition was antagonized by 1 μM DPCPX. Adenosine did not produce any effect on basal release.

The A2A receptor agonist CGS 21680 was ineffective on basal release, but stimulated the Ca2+‐dependent K+‐evoked overflow of [3H]‐D‐aspartate (EC50 ≃ 1 pM). The effect of 0.01 nM CGS 21680 was totally sensitive to the A2A receptor antagonist SCH58261 (IC50 ≃5 nM).

The A1 receptor agonist CCPA inhibited the Ca2+‐dependent K+‐evoked [3H]‐D‐aspartate overflow (EC50 ≃ 20 nM). The effect of 100 nM CCPA was abolished by 100 nM of the A1 receptor antagonist DPCPX.

The K+(15 mM)‐evoked overflow of endogenous glutamate was enhanced by CGS 21680 (0.01 nM) and inhibited by CCPA (0.1 μM). The effect of CGS 21680 was abolished by SCH58261 (0.1 μM) and that of CCPA by DPCPX (0.1 μM).

It is concluded that adenosine and adenosine receptor agonists modulate glutamate release by activating inhibitory A1 and excitatory A2A receptors present on glutamatergic terminals of the rat cerebral cortex.

British Journal of Pharmacology (2002) 136, 434–440; doi:10.1038/sj.bjp.0704712
Enzymatic activation of endothelial protease‐activated receptors is dependent on artery diameter in human and porcine isolated coronary arteries
Authors: Hamilton Justin R , Moffatt James D , Tatoulis James , Cocks Thomas M
Published: January 2005

Protease‐activated receptor (PAR)‐mediated vascular relaxations have been compared in coronary arteries of different diameters isolated from both humans and pigs.

Thrombin, trypsin, and the PAR1‐activating peptide, TFLLR, all caused concentration‐dependent relaxation of both large (epicardial; ∼2 mm internal diameter) and small (intramyocardial; ∼200 μm internal diameter) human coronary arteries. EC50 values for thrombin (0.006 u ml−1 in epicardial, 1.69 u ml−1 in intramyocardial) and trypsin (0.02 u ml−1 in epicardial, 1.05 u ml−1 in intramyocardial) were significantly (P<0.01) greater in intramyocardial arteries. By contrast, EC50 values for TFLLR were not different between epicardial (0.35 μM) and intramyocardial (0.43 μM) arteries.

In porcine coronary arteries, EC50 values for relaxations to thrombin (0.03 u ml−1 in epicardial 0.17 u ml−1 in intramyocardial) were also significantly (P<0.01) greater in the smaller arteries. EC50 values for both TFLLR and the PAR2‐activating peptide, SLIGKV, were not different between the two different‐sized pig coronary arteries.

PAR1‐immunoreactivity was localized to the endothelium of human epicardial and intramyocardial arteries and both PAR1‐ and PAR2‐immunoreactivity was observed in endothelial cells of equivalent porcine arteries.

These findings indicate that enzymatic activation of endothelial cell PARs in human (PAR1) and porcine (PAR1 and PAR2) coronary arteries is markedly reduced in intramyocardial arteries when compared with epicardial arteries, suggesting increased regulation of PAR‐mediated vascular responses in resistance‐type arteries.

British Journal of Pharmacology (2002) 136, 492–501; doi:10.1038/sj.bjp.0704714
Carvedilol attenuates neuroleptic‐induced orofacial dyskinesia: possible antioxidant mechanisms
Authors: Naidu Pattipati S , Singh Amanpreet , Kulkarni Shrinivas K
Published: January 2005

Tardive dyskinesia (TD), a syndrome of potentially irreversible, involuntary hyperkinetic disorder occurring in 20–40% of the patient population undergoing chronic neuroleptic treatment is a major limitation of neuroleptic therapy.

Oxidative stress and products of lipid peroxidation are implicated in the pathophysiology of various neurological disorders including tardive dyskinesia.

Chronic treatment with neuroleptics leads to the development of abnormal oral movements in rats known as vacuous chewing movements (VCMs). Vacuous chewing movements in rats are widely accepted as an animal model of tardive dyskinesia.

All the antipsychotics were administered i.p. once daily for 21 days, whereas carvedilol (also i.p.) was administered twice daily. Rats chronically treated with haloperidol (1.0 mg kg−1) or chlorpromazine (5 mg kg−1) but not clozapine (2 mg kg−1) significantly developed vacuous chewing movements and tongue protrusions. Carvedilol dose dependently (0.5–2 mg kg−1) reduced the haloperidol or chlorpromazine‐induced vacuous chewing movements and tongue protrusions.

Biochemical analysis revealed that chronic haloperidol or chlorpromazine but not clozapine treatment significantly induced lipid peroxidation and decreased the glutathione (GSH) levels in the forebrains of rats. Chronic haloperidol or chlorpromazine but not clozapine treated rats showed decreased forebrain levels of antioxidant defence enzymes, superoxide dismutase (SOD) and catalase.

Co‐administration of carvedilol (0.5–2 mg kg−1) significantly reduced the lipid peroxidation and restored the decreased glutathione levels by chronic haloperidol or chlorpromazine treatment. Co‐administration of carvedilol (1–2 mg kg−1) significantly reversed the haloperidol or chlorpromazine‐induced decrease in forebrain SOD and catalase levels in rats. However, lower dose of carvedilol (0.5 mg kg−1) failed to reverse chronic haloperidol or chlorpromazine‐induced decrease in forebrain SOD and catalase levels.

The major findings of the present study suggest that oxidative stress might play a significant role in neuroleptic‐induced orofacial dyskinesia. In conclusion, carvedilol could be a useful drug for the treatment of neuroleptic‐induced orofacial dyskinesia.

British Journal of Pharmacology (2002) 136, 193–200; doi:10.1038/sj.bjp.0704717
Caprylic acid and medium‐chain triglycerides inhibit IL‐8 gene transcription in Caco‐2 cells: comparison with the potent histone deacetylase inhibitor trichostatin A
Authors: Hoshimoto Aihiro , Suzuki Yasuo , Katsuno Tatsuro , Nakajima Hiroshi , Saito Yasushi
Published: January 2005

Medium‐chain triglyceride (MCT) is often administered to patients with Crohn's disease (CD) or short‐bowel syndrome. However, little is known about the effects of medium‐chain fatty acids (MCFAs) and MCT on intestinal inflammation. In this study we examined whether caprylic acid, one of the MCFAs, and MCT suppress IL‐8 secretion by differentiated Caco‐2 cells.

We found for the first time that caprylic acid and MCT suppress IL‐8 secretion by Caco‐2 cells at the transcriptional level when precultured together for 24 h. We also tried to clarify the mechanism of IL‐8 gene inhibition by examining the activation of NF‐κB and other transcription factors by electrophoretic mobility shift assay (EMSA), and found that caprylic acid did not modulate their activation.

The result of dual‐luciferase assay using Caco‐2 cells transfected with IL‐8 promoter/luciferase reporter plasmid revealed that caprylic acid inhibited the activation of IL‐8 promoter.

Similar results were observed when cells were precultured with the well‐known potent histone deacetylase inhibitor trichostatin A (TSA).

We examined the state of H4 acetylation in IL‐8 promoter using the technique known as chromatin immunoprecipitation (Chr‐IP). TSA rapidly induced H4 acetylation in IL‐8 promoter chromatin, whereas caprylic acid did not. These results suggest that the inhibition of IL‐8 gene transcription induced by caprylic acid and TSA does not necessarily require the marked suppression of transcription factors, and the mechanism of inhibition of IL‐8 gene transcription may be different between caprylic acid and TSA.

British Journal of Pharmacology (2002) 136, 280–286; doi:10.1038/sj.bjp.0704719
Protective effect of propranolol on mitochondrial function in the ischaemic heart
Authors: Iwai Takeshi , Tanonaka Kouichi , Kasahara Sayaka , Inoue Rie , Takeo Satoshi
Published: January 2005

The present study was aimed to determine whether propranolol improves contractile function of the ischaemic/reperfused heart through protection of the mitochondrial function during ischaemia.

Isolated perfused rat hearts were subjected to 35‐min ischaemia followed by 60‐min reperfusion. Pre‐treatment with propranolol at the concentrations of 10 to 100 μM for the final 3 min of pre‐ischaemia resulted in the improvement of ischaemia/reperfusion‐induced contractile dysfunction, release of creatine kinase (CK) into perfusate, and decrease in myocardial high‐energy phosphates. Propranolol also attenuated ischaemia‐induced accumulation in Na+, suggesting that cytosolic sodium overload during ischaemia was prevented by propranolol.

The mitochondrial oxygen consumption rate of skinned bundles from the perfused heart decreased at the end of ischaemia and it further decreased at the end of reperfusion. These decreases were cancelled by treatment with propranolol. A release of cytochrome c from the perfused heart was observed during ischaemia, and this release was suppressed by treatment with propranolol.

To elucidate the direct effect of propranolol on mitochondria, the mitochondria were isolated from normal hearts and their activities were determined in the presence of various concentrations of Na+ and propranolol. The addition of sodium lactate, which mimicked sodium overload in the ischaemic heart, reduced the state 3 respiration, whereas this reduction was not attenuated by the presence of propranolol.

These results suggest that cardioprotection of propranolol may be exerted via attenuating Na+ influx into cardiac cells followed by prevention of the mitochondrial dysfunction in the ischaemic heart, leading to improvement of energy production of the heart during reperfusion.

British Journal of Pharmacology (2002) 136, 472–480; doi:10.1038/sj.bjp.0704724
‘Entourage’ effects of N‐acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide‐induced vanilloid receptor activation and upon anandamide metabolism
Authors: Smart Darren , Jonsson Kent‐Olov , Vandevoorde Séverine , Lambert Didier M , Fowler Christopher J
Published: January 2005

The abilities of a series of saturated N‐acyl ethanolamines and related compounds to affect the ability of anandamide (AEA) to produce a Ca2+ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1‐HEK293 cells) has been investigated.

The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither affected basal Ca2+‐influx, nor the influx in response to a submaximal concentration of AEA (1 μM). In contrast, the C12:0, C17:0, C18:0 ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an augmentation of the response to AEA.

Lauroylethanolamide (C12:0) produced a leftward shift in the dose‐response curve for AEA. EC50 values for AEA to produce Ca2+ influx into hVR1‐HEK293 cells were 1.8, 1.5, 1.1 and 0.22 μM in the presence of 0, 1, 3 and 10 μM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose – response curves to capsaicin.

Palmitoylethylamide was synthesized and found to be a mixed‐type inhibitor (Ki(slope) 4.1 μM, Ki(intercept) 66 μM) of [3H]‐AEA metabolism by rat brain membranes.

The ‐amide, ‐ethylamide, ‐isopropylamide, ‐butylamide, ‐cyclohexamide and ‐trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca2+ influx response to 1 μM AEA.

There was no obvious relation between the abilities of the compounds to enhance the Ca2+ influx response to 1 μM AEA into hVR1‐HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes.

It is concluded that although palmitoylethanolamide has entourage‐like effects at VR1 receptors expressed on hVR1‐HEK293 cells, other N‐acyl ethanolamines have even more dramatic potentiating effects. It is possible that they may play an important role under conditions where their synthesis is increased, such as in severe inflammation.

British Journal of Pharmacology (2002) 136, 452–458; doi:10.1038/sj.bjp.0704732
Pharmacologically activated migration of aortic endothelial cells is mediated through p38 SAPK
Authors: Dénes László , Jednákovits Andrea , Hargitai Judit , Pénzes Zoltán , Balla András , Tálosi László , Krajcsi Péter , Csermely Péter
Published: January 2005

Impairment in endothelial cell (EC) function plays a central role in vascular diseases (e.g. atherosclerosis, restenosis, diabetic angiopathies, microvascular angina, peripheral arterial disease). BRX‐235 (a novel small molecule synthesized by Biorex, Hungary) has a potent vasculoprotective activity in different in vivo and in vitro studies. Since the importance of the p38 pathway in EC homeostasis and migration in particular is well documented, we have carried out studies to address the role of the p38 stress activated protein kinase (p38 SAPK) in the mode of action of BRX‐235. In this study, Bovine aortic endothelial cells were used in a wounding migration assay (WMA) and for Western‐blot analysis to study the effect and molecular mechanism of BRX‐235‐induced EC migration.

The bovine aortic endothelial (BAE) cells were shown to be good models for EC migration.

Both endothelial cell growth factor (ECGF)‐ and BRX‐235‐induced BAE cell migration were shown to be inhibited by SB 203580, a specific inhibitor of p38 SAPK.

It was also shown that, BRX‐235 induces phosphorylation of p38 SAPK without affecting p38 SAPK protein levels. Thus, BRX‐235 acts upstream of p38 SAPK.

In summary, we have shown that p38 SAPK is a potential pharmacological mediator for candidate drugs that target the endothelium.

British Journal of Pharmacology (2002) 136, 597–603; doi:10.1038/sj.bjp.0704738