bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphodiesterase isoenzyme families in human osteoarthritis chondrocytes – functional importance of phosphodiesterase 4
Authors: Tenor Hermann , Hedbom Erik , Häuselmann Hans‐Jörg , Schudt Christian , Hatzelmann Armin
Published: January 2005

We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL‐1β‐induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized.

Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL‐1β‐induced chondrocyte products (nitric oxide and prostaglandin E2) were measured in culture supernatants after 48 h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A‐D proteins were detected by immunoblotting.

The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL‐1β‐induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL‐1β‐induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E2 (PGE2) enabled PDE4 inhibitors to reduce IL‐1β‐induced NO in this experimental setting.

Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL‐1β and cyclic AMP‐mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein.

Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects.

British Journal of Pharmacology (2002) 135, 609–618; doi:10.1038/sj.bjp.0704480
Characterization of the non‐nitrergic NANC relaxation responses in the rabbit vaginal wall
Authors: Ziessen Tom , Moncada Salvador , Cellek Selim
Published: January 2005

Electrical field stimulation (EFS)‐induced non‐adrenergic non‐cholinergic (NANC) relaxation responses in the rabbit vaginal wall were investigated.

These NANC responses were partially inhibited with the nitric oxide synthase (NOS) inhibitors NG‐nitro‐L‐arginine methyl ester (L‐NAME; 500 μM), NG‐nitro‐L‐arginine (300 μM) or N‐iminoethyl‐L‐ornithine (500 μM) or the selective soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ, 10 μM). Application of L‐NAME and ODQ concomitantly did not increase the degree of inhibition.

L‐NAME or ODQ were observed to be more effective at low frequencies. The resistant part of the responses was more pronounced at higher frequencies and was completely inhibited by tetrodotoxin (1 μM).

Exogenous application of the peptides vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP‐27 and PACAP‐38), peptide histidine methionine (PHM), peptide histidine valine (PHV), helospectin‐I or ‐II induced a relaxation response. Calcitonin gene‐related peptide or substance P did not cause any relaxation.

The peptidase α‐chymotrypsin (type II; 2 units ml−1) did not affect non‐nitrergic NANC responses, although it did inhibit relaxation responses elicited by exogenous VIP, PACAP‐27, PACAP‐38, PHM, PHV, helospectin‐I or ‐II.

K+ channel inhibitors apamin (1 μM) or charybdotoxin (100 nM) when used alone or in conjunction did not affect non‐nitrergic NANC responses.

The non‐nitrergic NANC responses were not associated with any increase in intracellular cyclic adenosine‐3′, 5′‐monophosphate (cyclic AMP) or cyclic guanosine‐3′, 5′‐monophosphate (cyclic GMP) concentrations. The peptide‐induced relaxations were all associated with increases in cyclic AMP concentrations.

These results suggest that a neuronal factor elicits non‐nitrergic NANC responses in the rabbit vaginal wall. The identity of this factor remains to be established.

British Journal of Pharmacology (2002) 135, 546–554; doi:10.1038/sj.bjp.0704481
Uraemic toxins induce proximal tubular injury via organic anion transporter 1‐mediated uptake
Authors: Motojima Masaru , Hosokawa Atsuko , Yamato Hideyuki , Muraki Takamura , Yoshioka Toshimasa
Published: January 2005

A direct effect of uraemic toxins in promoting progression of chronic renal disease has not been established. In this study, we investigated the toxic effects of organic anions which characteristically appeared in the patients with progressive renal disease on renal proximal tubular cells expressing human organic anion transporter (hOAT) 1.

A renal proximal tubular cell line, opossum kidney (OK) cells, was transformed with hOAT1. Among the organic anions examined, hippuric acid, para‐hydroxyhippuric acid, ortho‐hydroxyhippuric acid, indoxyl sulphate and indoleacetic acid showed a high affinity for hOAT1 expressed in the OK cells. Indoxyl sulphate and indoleacetic acid concentration‐dependently inhibited proliferation of the hOAT1‐transformed cells. The h.p.l.c. analysis demonstrated that cellular uptake of these organic anions was significantly elevated in hOAT1‐transformed cells.

These organic anions also concentration‐dependently stimulated cellular free radical production. The degrees of inhibition of cell proliferation and the stimulation of free radical production induced by the organic anions were significantly higher in the hOAT1‐transformed cells than vector‐transformed cells. The stimulatory effect of indoxyl sulphate on free radical production was abolished by anti‐oxidants and probenecid.

Less free radical production was observed in the hOAT1‐transformed cells treated with p‐hydroxyhippuric acid, o‐hydroxyhippuric acid compared with indoxyl sulphate and indoleacetic acid. Hippuric acid had little effect on free radical production.

Organic anions present in the serum of patients with progressive renal disease may cause proximal tubular injury via hOAT1‐mediated uptake. The mechanism of cellular toxicity by these uraemic toxins involves free radical production. Thus, some uraemic toxins may directly promote progression of chronic renal disease.

British Journal of Pharmacology (2002) 135, 555–563; doi:10.1038/sj.bjp.0704482
Pharmacological actions of a novel NO‐independent guanylyl cyclase stimulator, BAY 41‐8543: in vitro studies
Authors: Stasch Johannes‐Peter , Alonso‐Alija Cristina , Apeler Heiner , Dembowsky Klaus , Feurer Achim , Minuth Torsten , Perzborn Elisabeth , Schramm Matthias , Straub Alexander
Published: January 2005

BAY 41‐8543 is a novel, highly specific and so far the most potent NO‐independent stimulator of sGC. Here we report the effects of BAY 41‐8543 on the isolated enzyme, endothelial cells, platelets, isolated vessels and Langendorff heart preparation.

BAY 41‐8543 stimulates the recombinant sGC concentration‐dependently from 0.0001 μM to 100 μM up to 92‐fold. In combination, BAY 41‐8543 and NO have synergistic effects over a wide range of concentrations. Similar results are shown in implying that BAY 41‐8543 stimulates the sGC directly and furthermore makes the enzyme more sensitive to its endogenous activator NO.

In vitro, BAY 41‐8543 is a potent relaxing agent of aortas, saphenous arteries, coronary arteries and veins with IC50‐values in the nM range.

In the rat heart Langendorff preparation, BAY 41‐8543 potently reduces coronary perfusion pressure from 10−9 to 10−6 g ml−1 without any effect on left ventricular pressure and heart rate.

BAY 41‐8543 is effective even under nitrate tolerance conditions proved by the same vasorelaxing effect on aortic rings taken either from normal or nitrate‐tolerant rats.

BAY 41‐8543 is a potent inhibitor of collagen‐mediated aggregation in washed human platelets (IC50=0.09 μM). In plasma, BAY 41‐8543 inhibits collagen‐mediated aggregation better than ADP‐induced aggregation, but has no effect on the thrombin pathway. BAY 41‐8543 is also a potent direct stimulator of the cyclic GMP/PKG/VASP pathway in platelets and synergizes with NO over a wide range of concentrations.

These results suggest that BAY 41‐8543 is on the one hand an invaluable tool for studying sGC signaling in vitro and on the other hand its unique profile may offer a novel approach for treating cardiovascular diseases.

British Journal of Pharmacology (2002) 135, 333–343; doi:10.1038/sj.bjp.0704484
Evidence of a role for NK1 and CGRP receptors in mediating neurogenic vasodilatation in the mouse ear
Authors: Grant Andrew D , Gerard Norma P , Brain Susan D
Published: January 2005

The aims of this study were to develop a technique to measure blood flow in the mouse ear and to investigate the nature of the vasodilator mediator(s) involved in the response to capsaicin.

The response to capsaicin, applied topically, was investigated in anaesthetized CD1 or Sv129+C57BL/6 wild‐type (+/+) or NK1 receptor knockout mice (−/−). Blood flow was assessed by laser Doppler flowmetry and oedema formation by 125I‐albumin accumulation.

Capsaicin induced significant increases in blood flow (0.2–200 μg in 20 μl) and oedema (2–200 μg in 20 μl). The oedema response was absent in NK1−/− mice and NK1+/+mice treated with the selective NK1 receptor antagonist SR140333 (480 nmol kg−1) as expected. Furthermore, the capsaicin‐evoked increase in blood flow was significantly potentiated in the knockout mice (203% of wild‐type response, P<0.05) and wild‐type mice treated with SR140333 (201%, P<0.05).

The CGRP receptor antagonist CGRP8–37 (400 nmol kg−1) had no effect on capsaicin‐induced blood flow in NK1+/+mice but abolished the increased blood flow to capsaicin in NK1−/−, and NK1+/+wild‐type mice pre‐treated with SR140333.

The results indicate that neurogenic vasodilatation can be measured in the mouse ear. The capsaicin‐induced increased blood flow involves activation of, and possible interactions between, both NK1 and CGRP1 receptors.

British Journal of Pharmacology (2002) 135, 356–362; doi:10.1038/sj.bjp.0704485
Role of nitric oxide/cyclic GMP in myocardial adenosine A1 receptor‐inotropic response
Authors: Sterin‐Borda Leonor , Gómez Ricardo M , Borda Enri
Published: January 2005

In this study we have determined the different signalling pathways involved in adenosine A1‐receptor (A1‐receptor)‐dependent inhibition of contractility in rat isolated atria.

N‐cyclopentyladenosine (CPA) stimulation of A1‐receptor exerts: negative inotropic response, inositol phosphates accumulation, stimulation of nitric oxide synthase (NOS), increased production of nitric oxide (NO) and cyclic GMP.

Inhibitors of phospholipase C (PLC), protein kinase C (PKC), calcium/calmodulin, NOS and guanylate cyclase shifted the dose‐response curve of CPA on contractility to the right. Those inhibitors also attenuated the A1‐receptor‐dependent increase in cyclic GMP and activation of NOS.

These results suggest that CPA activation of A1‐receptors exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving calcium/calmodulin and PKC, leading to activation of NOS and soluble guanylate cyclase.

British Journal of Pharmacology (2002) 135, 444–450; doi:10.1038/sj.bjp.0704487
Subeffective doses of dexketoprofen trometamol enhance the potency and duration of fentanyl antinociception
Authors: Gaitán Gema , Herrero Juan F
Published: January 2005

The combination of classic non‐steroidal antiinflammatory drugs (NSAIDs) with opiates induces more analgesia than the summed effect of each drug given separately. No studies have been performed using new generation NSAIDs and fentanyl nor on the duration of this effect.

We have studied the analgesic effect of fentanyl alone and after the administration of subeffective doses of dexketoprofen trometamol in rat nociceptive responses. The responses were evoked by noxious mechanical stimulation and were recorded as single motor units in male Wistar rats anaesthetized with α‐chloralose.

The effective dose 50 (ED50) observed with fentanyl was 22.4±1.5 μg kg−1 and full recovery was apparent 20 min later. The administration of a total dose of 40 μg kg−1 of dexketoprofen trometamol did not induce any significant effect on the nociceptive responses. In the presence of dexketoprofen trometamol, the ED50 for fentanyl was 5 fold lower than before: 3.8±1.1 μg kg−1 and no significant recovery was observed 45 min later. The opioid antagonist naloxone (200 μg kg−1) did not reverse the effect, although in control experiments the same dose was able to prevent any action of fentanyl given alone.

We conclude that the combination of fentanyl and subeffective doses of dexketoprofen trometamol induces a more potent and longer lasting analgesic effect than that observed with fentanyl alone, and that this is not an opioid mediated action.

British Journal of Pharmacology (2002) 135, 393–398; doi:10.1038/sj.bjp.0704491
Inducing the cell cycle arrest and apoptosis of oral KB carcinoma cells by hydroxychavicol: roles of glutathione and reactive oxygen species
Authors: Chang M C , Uang B J , Wu H L , Lee J J , Hahn L J , Jeng J H
Published: January 2005

Hydroxychavicol (HC; 10 – 50 μM), a betel leaf component, was found to suppress the 2% H2O2‐induced lucigenin chemiluminescence for 53 – 75%. HC (0.02 – 2 μM) was also able to trap superoxide radicals generated by a xanthine/xanthine oxidase system with 38 – 94% of inhibition. Hydroxyl radicals‐induced PUC18 plasmid DNA breaks was prevented by HC (1.6 – 16 μM).

A 24‐h exposure of KB cells to HC (0.5, 1 mM) resulted in 54 – 74% cell death as analysed by a 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay. HC (10, 50 μM) further suppressed the growth of KB cells (15 and 76%, respectively). Long‐term colony formation of KB cells was inhibited by 51% with 10 μM HC.

Pretreatment of KB cells with 100 μM HC inhibited the attachment of KB cells to type I collagen and fibronectin by 59 and 29%, respectively.

Exposure of KB cells to 0.1 mM HC for 24 h resulted in cell cycle arrest at late S and G2/M phase. Increasing the HC concentration to 0.25 and 0.5 mM led to apoptosis as revealed by detection of sub‐G0/G1 peaks with a concomitant decrease in the number of cells residing in late S and G2/M phase.

Inducing the apoptosis of KB cells by HC was accompanied by marked depletion in reduced form of GSH (>0.2 mM) and the increasing of reactive oxygen species production (>0.1 mM) as analysed by CMF‐ and DCF‐single cell fluorescence flow cytometry.

These results indicate that HC exerts antioxidant property at low concentration. HC also inhibits the growth, adhesion and cell cycle progression of KB cells, whereas its induction of KB cell apoptosis (HC>0.1 mM) was accompanied by cellular redox changes.

British Journal of Pharmacology (2002) 135, 619–630; doi:10.1038/sj.bjp.0704492
Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti‐inflammatory effect
Authors: Shen Yuh‐Chiang , Chen Chieh‐Fu , Chiou Wen‐Fei
Published: January 2005

We have reported that andrographolide (ANDRO), an active component of Andrographis paniculata, inhibits inflammatory responses by rat neutrophils. To further elucidate the possible mechanism(s) underlying the ANDRO's effect, N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced adhesion and transmigration of isolated peripheral human neutrophils were studied.

Pretreatment with ANDRO (0.1–10 μM) concentration‐dependently prevented fMLP‐induced neutrophil adhesion and transmigration. We further examined the up‐expression of surface Mac‐1 (CD11b/CD18), an essential integrin mediated in neutrophil adhesion and transmigration. ANDRO pretreatment significantly decreased fMLP‐induced up‐expression of both CD11b and CD18.

Accumulation of reactive oxygen species (ROS) as well as quick intracellular calcium ([Ca++]i) mobilization induced by fMLP displays two important signalling pathways in regulating the up‐expression of Mac‐1 by neutrophils. That ANDRO pretreatment diminished fMLP‐induced production of H2O2 and O2.−, but failed to block that of [Ca++]i mobilization suggested that the ROS but not [Ca++]i signalling could be modulated by ANDRO.

To clarify whether ROS production impeded by ANDRO could be an antagonism of fMLP binding, phorbol‐12‐myristate‐13‐acetate (PMA), a direct protein kinase C (PKC) activator, was introduced to activate ROS production. PMA triggered remarkable ROS production and adhesion, and were partially reversed by ANDRO. This indicated that a PKC‐dependent mechanism might be interfered by ANDRO.

We conclude that the prevention of ROS production through, at least in part, modulation of PKC‐dependent pathway could confer ANDRO the ability to down‐regulate Mac‐1 up‐expression that is essential for neutrophil adhesion and transmigration.

British Journal of Pharmacology (2002) 135, 399–406; doi:10.1038/sj.bjp.0704493
Pharmacological and functional characterization of the guinea‐pig B2 bradykinin receptor stably expressed in CHO‐K1 cell line
Authors: Robert C , Pruneau D , Paquet J ‐L
Published: January 2005

In the present study, pharmacological properties of a bradykinin B2 receptor amplified either from guinea‐pig ileum or lung and homologous to the previously reported sequence except two amino‐acid changes L124→P and N227→Y in the receptor protein were characterized.

Tritiated bradykinin ([3H]‐BK) specifically bound to the cloned guinea‐pig B2 bradykinin receptor stably expressed in Chinese hamster ovary cells (CHO‐K1) with a KD value of 0.29±0.07 nM. In competition experiments, bradykinin (BK) affinity constant value was 0.21±0.05 nM while the two specific kinin B1 ligands, des‐Arg9‐bradykinin (DBK) and des‐Arg9‐Leu8‐bradykinin (DLBK) were unable to compete with [3H]‐BK. As the specific peptide antagonist D‐Arg‐[Hyp3,Thi5,D‐Tic7,Oic8]‐bradykinin (HOE140), (E)‐3‐(6‐acetamido‐3‐pyridil)‐N‐[‐N‐[2,4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl)oxymethyl]phenyl]‐N‐methylaminocarbonylmethyl]acrylamide (FR173657) and 1‐[[3‐[2,4‐dimethylquinolin‐8‐yl)oxymethyl] ‐ 2,4 ‐ dichloro ‐ phenyl]sulfonyl] ‐ 2(S) ‐ [[4‐[4‐(aminoiminomethyl)‐phenylcarbonyl]piperazin‐1‐yl]carbonyl]pyrrolidine (LF16‐0335C) exhibited a high affinity for this receptor with Ki values of 7.34±2.45 nM and 8.54±1.55 nM respectively.

BK and kallidin (KD) increased inositol phosphates (IPs) levels with EC50 values of 0.44±0.12 nM and 6.88±0.28 nM, respectively. Neither DLBK nor DBK (0.01 nM to 10 μM) stimulated or inhibited IPs turnover and as expected HOE140 did not raise IPs production. HOE140 (0.1 μM) and LF 16‐0335c (1 μM) right shifted the BK response curve with pKB values of 9.2±0.4 and 8.4±0.3, respectively.

The results indicate that this cloned guinea‐pig receptor displayed typical pharmacological properties of a bradykinin B2 receptor and support the existence of a single B2 receptor in this species.

British Journal of Pharmacology (2002) 135, 462–468; doi:10.1038/sj.bjp.0704494
The β‐lactam antibiotics, penicillin‐G and cefoselis have different mechanisms and sites of action at GABAA receptors
Authors: Sugimoto Masahiro , Fukami Sakae , Kayakiri Hiroshi , Yamazaki Shunji , Matsuoka Nobuya , Uchida Ichiro , Mashimo Takashi
Published: January 2005

The action of the β‐lactam antibiotics, penicillin‐G (PCG) and cefoselis (CFSL) on GABAA receptors (GABAA‐R) was investigated using the two‐electrode voltage clamp technique and Xenopus oocyte expressed murine GABAA‐R.

Murine GABAA‐Rs were expressed in Xenopus oocytes by injecting cRNA that encoded for each subunit (α1, β2, and γ2) and the effects of PCG and CFSL on the α1β2γ2s subunit receptors were examined using two‐electrode voltage clamp. Using the α1β2γ2s GABAA‐R, PCG and CFSL inhibited GABA‐induced currents in a concentration‐dependent manner, with IC50s of 557.1±125.4 and 185.0±26.6 μM, respectively. The inhibitory action of PCG on GABA‐induced currents was non‐competitive whereas that of CFSL was competitive.

Mutation of tyrosine to phenylalanine at position 256 in the β2 subunit (β2Y256F), which is reported to abolish the inhibitory effect of picrotoxin, drastically reduced the potency of PCG (IC50=28.4±1.42 mM) for the α1β2Y256Fγ2s receptor without changing the IC50 of CFSL (189±26.6 μM).

These electrophysiological data indicate that PCG and CFSL inhibit GABAA‐R in a different manner, with PCG acting non‐competitively and CFSL competitively. The mutational study indicates that PCG might act on an identical or nearby site to that of picrotoxin in the channel pore of the GABAA‐R.

British Journal of Pharmacology (2001) 135, 427–432; doi:10.1038/sj.bjp.0704496
Interaction of nitric oxide with calcium in the mesenteric bed of bile duct‐ligated rats
Authors: Nadal F Javier A , Atucha Noemí M , Iyu David , García‐Estañ Joaquín
Published: January 2005

We have analysed the interaction of NO with calcium in order to study the molecular mechanisms responsible for the vascular hyporesponse of liver cirrhosis. The experiments have been performed in the isolated and perfused mesenteric arterial bed of rats with bile duct ligation (BDL) and their controls.

While perfusing the vessels with normal Krebs, methoxamine (MTX) or KCl produced a lower pressor response in the BDL mesenteries. The NO synthesis inhibitor Nw‐nitro‐L‐arginine (NNA) potentiated those responses and abolished the differences between groups.

The administration of MTX under perfusion with zero calcium‐Krebs, to analyse the intracellular release of calcium, also induced a lower response in the BDL mesenteries and NNA potentiated and normalized the response.

To investigate calcium entry, the vessels were perfused with zero‐calcium Krebs containing high potassium to open voltage‐dependent calcium channels. Then, the addition of calcium (10−1–3×10−3 M) produced a lower pressor response in the BDL vessels, that was corrected by NNA. To study calcium entry through receptor‐operated channels, the vessels were perfused with zero‐calcium Krebs containing MTX. The addition of calcium elevated the perfusion pressure less in the BDL mesenteries than in the control and NNA potentiated the responses and eliminated the between groups differences. When calcium entry through both voltage‐ and receptor‐operated channels was simultaneously analysed, similar results were obtained.

In the mesenteric bed of bile duct ligated rats, an excess of nitric oxide affects vascular calcium regulation through an interaction with both calcium entry and intracellular calcium release.

British Journal of Pharmacology (2002) 135, 489–495; doi:10.1038/sj.bjp.0704504
Effects of cardioselective KATP channel antagonism on basal, stimulated, and ischaemic myocardial function in in vivo failing canine heart
Authors: Saavedra Walter F , Paolocci Nazareno , Kass David A
Published: January 2005

Inhibition of cardiomyocyte‐specific ATP‐sensitive potassium (KATP) channels prolongs the action potential during intense ischaemia with attendant antiarrhythmic effects. However, this is accompanied by contractile depression in some models. These changes may be particularly troublesome in dilated cardiomyopathic hearts that display basal systolic dysfunction, limited energy reserve, and prolonged repolarization favouring arrhythmia. Mechanical effects of selective myocyte KATP channel blockade on basal, β‐adrenergic stimulated, and ischemic responses were therefore tested in dogs with cardiac failure induced by tachypacing.

Cardiovascular function was assessed by pressure – dimension relationships in 10 conscious, chronically instrumented dogs (sonomicrometry/micromanometry), with or without cardiac failure. Cardiomyocyte KATP channels were inhibited by HMR 1098, and data obtained under basal conditions, during epinephrine infusion to raise metabolic demand, during regional ischaemia, and with combined ischaemia+epinephrine.

HMR 1098 had no effect on baseline cardiac function nor did it induce arrhythmia in normal or failing hearts. Epinephrine raised cardiac work 65% and oxygen consumption 55%, yet HMR 1098 had no functional effect in either heart condition. Regional ischaemia with or without epinephrine co‐stimulation depressed regional and global function, yet both were also unaffected by HMR 1098. There was minimal arrhythmia without HMR 1098, and drug infusion did not alter this.

Thus, myocyte‐KATP channels play a negligible role modulating intact in vivo cardiac contraction or arrhythmia in normal and failing heart with and without increased metabolic demand and/or regional ischaemia. This supports the feasibility of administering such agents to depressed hearts, despite underlying contractile and electrophysiologic abnormalities.

British Journal of Pharmacology (2002) 135, 657–662; doi:10.1038/sj.bjp.0704510
Inhibition of adenylyl cyclase by neuronal P2Y receptors
Authors: Unterberger Ursula , Moskvina Eugenia , Scholze Thomas , Freissmuth Michael , Boehm Stefan
Published: January 2005

P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y12. Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment.

ADP and ATP (0.1 – 100 μM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A2A or recombinant β2 receptors. Forskolin‐dependent cyclic AMP production was reduced by 1 μM and enhanced by 10 – 100 μM ADP; this latter effect was turned into an inhibition when A2A receptors were blocked.

The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin.

The rank order of agonist potencies for the reduction of cyclic AMP was (IC50 values): 2‐methylthio‐ADP (0.12 nM)=2‐methylthio‐ATP (0.13 nM)>ADPβS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, or adenosine‐3′‐phosphate‐5′‐phosphate, but attenuated by reactive blue 2, ATPαS, and 2‐methylthio‐AMP.

RT – PCR demonstrated the expression of P2Y2, P2Y4, P2Y6, and P2Y12, but not P2Y1, receptors in PC12 cells. In Northern blots, only P2Y2 and P2Y12 were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged.

We conclude that P2Y12 receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.

British Journal of Pharmacology (2002) 135, 673–684; doi:10.1038/sj.bjp.0704514
Nociceptin/orphanin FQ inhibits capsaicin‐induced guinea‐pig airway contraction through an inward‐rectifier potassium channel
Authors: Jia Yanlin , Wang Xin , Aponte Sonia I , Rivelli Maria A , Yang Richard , Rizzo Charles A , Corboz Michel R , Priestley Tony , Hey John A
Published: January 2005

Nociceptin/orphanin FQ (N/OFQ), an endogenous opioid‐like orphan receptor (NOP receptor, previously termed ORL1 receptor) agonist, has been found to inhibit capsaicin‐induced bronchoconstriction in isolated guinea‐pig lungs and in vivo. The underlying mechanisms are not clear. In the present studies, we tested the effect of N/OFQ on VR1 channel function in isolated guinea‐pig nodose ganglia cells. Capsaicin increased intracellular Ca2+ concentration in these cells through activation of vanilloid receptors. Capsaicin‐induced Ca2+ responses were attenuated by pretreatment of nodose neurons with N/OFQ (1 μM).

N/OFQ inhibitory effect on the Ca2+ response in nodose ganglia cells was antagonized by tertiapin (0.5 μM), an inhibitor of inward‐rectifier K+ channels, but not by verapamil, a voltage gated Ca2+ channel blocker, indicating that an inward‐rectifier K+ channel is involved in N/OFQ inhibitory effect.

In isolated guinea‐pig bronchus, N/OFQ (1 μM) inhibited capsaicin‐induced airway contraction. Tertiapin (0.5 μM) abolished the N/OFQ inhibition of capsaicin‐induced bronchial contraction.

Capsaicin (10 μg) increased pulmonary inflation pressure in the isolated perfused guinea‐pig lungs. This response was significantly attenuated by pretreatment with N/OFQ (1 μM). Tertiapin also abolished the N/OFQ inhibitory effect on capsaicin‐induced bronchoconstriction in perfused lungs. Capsaicin increased the release of substance P and neurokinin A from isolated lungs. N/OFQ (1 μM) blocked the capsaicin‐induced tachykinin release.

These results indicate that N/OFQ‐induced hyperpolarization of tachykinin containing airway sensory nerves, through an inward‐rectifier K+ channel activation, accounts for the inhibition of capsaicin‐evoked broncoconstriction.

British Journal of Pharmacology (2002) 135, 764–770; doi:10.1038/sj.bjp.0704515
Aconitine facilitates spontaneous transmitter release at rat ventromedial hypothalamic neurons
Authors: Yamanaka Hisashi , Doi Atsushi , Ishibashi Hitoshi , Akaike Norio
Published: January 2005

The effects of aconitine, an Aconitum alkaloid, on spontaneous inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs respectively) were investigated in the mechanically dissociated rat ventromedial hypothalamic (VMH) neurons in which native presynaptic nerve terminals remained intact.

Under current‐clamp conditions, aconitine (3×10−6 M) depolarized the neuron with generating the action potentials. The aconitine‐induced depolarization was markedly suppressed in the presence of CNQX but it was facilitated in the presence of bicuculline, suggesting that release of excitatory and inhibitory neurotransmitters may be involved in the aconitine action in addition to its direct action on postsynaptic membrane.

Under the voltage‐clamp conditions, aconitine reversibly increased the frequency of spontaneous IPSC and EPSC frequency, but it did not alter their amplitude distribution.

Tetrodotoxin (TTX, 3×10−7 M) completely abolished the aconitine action on spontaneous IPSC frequency. Likewise removal of extracellular Na+ completely suppressed the aconitine action.

Both Ca2+‐free external solution or addition of 10−4 M Cd2+ to normal solutions eliminated the facilitatory effect of aconitine on the IPSC frequency.

Overall these results suggest that aconitine depolarizes the presynaptic membrane by activating voltage‐dependent Na+ channels. Increase of intraterminal Ca2+ concentration via an activation of voltage‐dependent Ca2+ channels in turn enhances the spontaneous transmitter release from presynaptic nerve terminals. The presynaptic action of aconitine may play a crucial role for membrane excitability of rat VMH neurons.

British Journal of Pharmacology (2002) 135, 816–822; doi:10.1038/sj.bjp.0704517
Pharmacological characterization of a non‐inactivating outward current observed in mouse cerebellar Purkinje neurones
Authors: Bushell Trevor , Clarke Catherine , Mathie Alistair , Robertson Brian
Published: January 2005

Whole‐cell patch clamp recordings were used to investigate the properties of a non‐inactivating outward current observed in mouse cerebellar Purkinje neurones at a holding potential of −20 mV.

Increasing the external potassium (K+) concentration from 3 mM to 20 mM produced a rightward shift in the observed reversal potential of ∼30 mV or ∼40 mV for a K+‐or a caesium (Cs+)‐based intracellular solution respectively, indicating the outward current was a K+ current.

The outward current was partially inhibited by the K+ channel blocker, tetraethylammonium (TEA; IC50=0.15 mM). Subsequently, the background or TEA‐insensitive current was measured in the presence of 1 mM TEA.

The background current was reversibly inhibited by barium (Ba2+; 300 μM, 50%) and potentiated by the application of arachidonic acid (AA; 1 mM, 62%).

The volatile anaesthetic, halothane (1 mM), and the neuroprotectant, riluzole (500 μM), both reversibly inhibited the background current by 54% and 36% respectively.

The background current was insensitive to changes in both intracellular and extracellular acidification.

The GABAB and μ‐opioid receptor agonists, baclofen and [D‐Ala2, N‐MePhe4‐Gly‐ol5] enkephalin (DAMGO) both reversibly potentiated the outward current by 42% and 26% respectively. In contrast, the metabotropic glutamate receptor and acetylcholine receptor agonists, (S)‐3,5‐dihydroxyphenylglycine (DHPG) and muscarine both reversibly inhibited the outward current by 48% and 42% respectively.

These data suggest that cerebellar Purkinje neurones possess a background current which shares several properties with recently cloned two‐pore K+ channels, particularly THIK‐1.

British Journal of Pharmacology (2002) 135, 705–712; doi:10.1038/sj.bjp.0704518
Lysophosphatidylcholine potentiates vascular contractile responses in rat aorta via activation of tyrosine kinase
Authors: Suenaga Hiroshi , Kamata Katsuo
Published: January 2005

We previously reported that while lysophosphatidylcholine (LPC) does not itself produce contraction, it significantly potentiates the contractile responses induced by high‐K+, UK14,304 (a selective α2‐adrenoceptor agonist) and phorbol ester in the endothelium‐denuded rat aorta. To further investigate this phenomenon, we examined the effects of genistein and tyrphostin B42 (both tyrosine kinase inhibitors) on the LPC‐induced potentiation of the contractile responses to high‐K+ and UK14,304 in the endothelium‐denuded rat aorta.

Although genistein (3×10−6 M, 10−5 M) did not affect the high‐K+‐induced contractile response, it selectively inhibited the potentiating effect of LPC on the contraction and it strongly inhibited the LPC‐induced augmentation of the associated increases in [Ca2+]i. Genistein also attenuated the LPC‐induced augmentation effects on both the increase in [Ca2+]i and contractile response induced by the UK14,304. In contrast, daidzein (10−5 M) did not inhibit the potentiating effect of LPC. Tyrphostin B42 (3×10−5 M) attenuated the potentiating effect of LPC on high K+‐induced contractions.

Western blot analysis showed that LPC increased the tyrosine phosphorylation of a number of proteins, including 42 and 44 kDa proteins and 53 – 64 kDa proteins. These protein phosphorylations were inhibited by genistein.

Sodium orthovanadate (10−4 M), a tyrosine phosphatase inhibitor, also markedly enhanced the high‐K+‐induced contractile responses. This enhancing effect was attenuated by genistein.

These results suggest that the LPC‐induced augmentation of contractile responses in the rat aorta is due to activation of tyrosine kinase, which in turn regulates Ca2+ influx.

British Journal of Pharmacology (2002) 135, 789–799; doi:10.1038/sj.bjp.0704525
Role of endothelin ETA‐ and ETB‐receptors in haemodynamic compensation following haemorrhage in anaesthetized rats
Authors: Palacios Beatriz , Lim Su Lin , Pang Catherine C Y
Published: January 2005

This study examined the role of endothelin ETA and ETB receptors on haemodynamic compensation following haemorrhage (−17.5 ml kg−1) in thiobutabarbitone‐anaesthetized rats. Rats were divided into four groups (n=6 each): time‐control, haemorrhage‐control, haemorrhage after treatment with FR 139317 (ETA‐receptor antagonist), and haemorrhage after treatment with BQ‐788 (ETB‐receptor antagonist).

In the time‐control rats, there were no significant changes in any haemodynamics for the duration of the experiments. Relative to the time‐control rats, rats given haemorrhage had reduced mean arterial pressure (MAP), cardiac output (CO) and mean circulatory filling pressure (MCFP), but increased systemic vascular resistance (RSV). Venous resistance (RV) was slightly (but insignificantly) reduced by haemorrhage. MAP, however, gradually returned towards baseline (−17±4 and −3±2 mmHg at 10 and 60 min after haemorrhage, respectively) as a result of a further increase in RSV.

Pre‐treatment with FR 139317 (i.v. 1 mg kg−1, followed by 1 mg kg−1 h−1) accentuated haemorrhage‐induced hypotension through abolition of the increase in RSV. FR 139317 did not modify haemorrhage‐induced changes in CO, MCFP and RV.

Pre‐treatment of BQ‐788 (3 mg kg−1) did not affect MAP or MCFP following haemorrhage; however, CO was lower, and RSV as well as RV were higher relative to the readings in the haemorrhaged‐control rats.

These results show that following compensated haemorrhage, ET maintains arterial resistance and blood pressure via the activation of ETA but not ETB receptors.

British Journal of Pharmacology (2002) 135, 876–882; doi:10.1038/sj.bjp.0704530
Lack of critical involvement of endothelial nitric oxide synthase in vascular nitrate tolerance in mice
Authors: Wang Ellen Q , Lee Woo‐In , Fung Ho‐Leung
Published: January 2005

We examined the direct involvement of endothelial nitric oxide (eNOS) in nitrate tolerance using eNOS knockout (eNOS (−/−)) and wild‐type (eNOS (+/+)) mice. Animals were treated with either nitroglycerin (NTG, 20 mg kg−1s.c. 3×daily for 3 days) or vehicle (5% dextrose, D5W), and nitrate tolerance was assessed ex vivo in isolated aorta by vascular relaxation studies and cyclic GMP accumulation. Western blot was performed to determine NOS expression after NTG treatment. In both the eNOS (−/−) and (+/+) mice, the EC50 from NTG concentration‐response curve was increased by ∼3 fold, and vascular cyclic GMP accumulation was similarly decreased after NTG pretreatment. Vascular tolerance did not lead to changes in eNOS protein expression in eNOS (+/+) mice. These results indicate that vascular nitrate tolerance was similarly induced in eNOS (−/−) and (+/+) mice, suggesting that eNOS may not be critically involved in nitrate tolerance development in mice.

British Journal of Pharmacology (2002) 135, 299–302; doi:10.1038/sj.bjp.0704532
Stimulation of β3‐adrenoceptors causes phosphorylation of p38 mitogen‐activated protein kinase via a stimulatory G protein‐dependent pathway in 3T3‐L1 adipocytes
Authors: Mizuno Katsushige , Kanda Yasunari , Kuroki Yasutomi , Nishio Masahiro , Watanabe Yasuhiro
Published: January 2005

This study deals with phosphorylation and activation of p38 mitogen‐activated protein kinase (MAPK) via β3‐adrenoceptor (AR) and the signal transduction pathway in 3T3‐L1 adipocytes.

β3‐AR agonist BRL37344A (10 nM) caused phosphorylation and activation of p38 MAPK in 3T3‐L1 adipocytes but not in fibroblasts. BRL37344A and also the other β3‐AR agonists, CGP12177A and SR58611A, caused p38 MAPK phosphorylation in dose‐dependent manners.

The p38 MAPK phosphorylations by BRL37344A (10 nM), CGP12177A (100 nM), and SR58611A (10 nM) were not antagonized by β1‐ and β2‐ARs antagonist 1‐propranolol (100 nM) but blocked by β3‐AR antagonist SR59230A (10 μM), suggesting the phosphorylation was caused via β3‐AR.

The phosphorylations of p38 MAPK were completely abolished by treatment with cholera toxin (CTX) but not pertussis toxin (100 ng ml−1, 24 h). Activation of Gs by CTX (100 ng ml−1) and adenylyl cyclase by forskolin mimicked p38 MAPK phosphorylation.

p38 MAPK phosphorylation by BRL37344A was reduced to almost 50% by cyclic AMP‐dependent protein kinase (PKA) inhibitors such as H89 (10 μM) and PKI (10 μM). A src‐family tyrosine kinases inhibitor PP2 (1 μM) also halved the p38 MAPK phosphorylation. Combined use of H89 (10 μM) and PP2 (10 μM) did not bring about further inhibition.

These results suggest that β3‐AR caused phosphorylation of p38 MAPK via Gs protein and partly through a pathway involving PKA and src‐family kinase(s), although the contribution of the unidentified pathway remains to be clarified.

British Journal of Pharmacology (2002) 135, 951–960; doi:10.1038/sj.bjp.0704537
Involvement of caspases and calpains in cerebrocortical neuronal cell death is stimulus‐dependent
Authors: Moore Jonathan D , Rothwell Nancy J , Gibson Rosemary M
Published: January 2005

Caspases and calpains are mediators of apoptotic cell death. The objective of this study was to determine the role of caspases and calpains in primary cerebrocortical neuronal (CCN) death in response to a range of stimuli which reportedly induce neuronal apoptosis.

Cell death of primary cultures of rat CCN was induced by staurosporine (STS), C2‐ceramide (CER), camptothecin (CMT), hydrogen peroxide (H2O2) or N‐methyl‐D‐aspartate (NMDA). Caspase and calpain activity were assessed by cleavage of α‐fodrin or fluorogenic substrates. Cell death was analysed by lactate dehydrogenase (LDH) assay in the absence or presence of the pan‐caspase inhibitor Boc‐Asp‐(OMe)‐Fluoromethylketone (Baf) and/or the calpain inhibitor calpeptin (CP).

Cell death induced by STS, CER or CMT was accompanied by chromatin condensation and activation of multiple caspases, particularly caspase‐3‐type proteases. Hydrogen peroxide (H2O2) treatment was accompanied by activation of caspases −1, −6 and −8, but not −3, whereas none of the caspases tested were activated in response to NMDA.

With the exception of H2O2, when cell death was accompanied by caspase activation, it was significantly suppressed by Baf.

All stimuli also induced calpain activation, but calpeptin only suppressed cell death induced by H2O2. Furthermore, co‐treatment with Baf and calpeptin did not alter the cell death relative to either inhibitor alone.

These findings suggest the existence of stimulus‐dependent routes for the activation of caspases and calpains during death of cortical neurones and imply that although caspases and calpains are activated, their involvement in the execution of cell death varies with the stimulus.

British Journal of Pharmacology (2002) 135, 1069–1077; doi:10.1038/sj.bjp.0704538
Glucocorticoids reverse IL‐1β‐induced impairment of β‐adrenoceptor‐mediated relaxation and up‐regulation of G‐protein‐coupled receptor kinases
Authors: Mak Judith C W , Hisada Takeshi , Salmon Michael , Barnes Peter J , Chung K Fan
Published: January 2005

The aim of the present study was to examine the effects of glucocorticoid dexamethasone on airway responsiveness to albuterol after intratracheal instillation of saline or IL‐1β in Brown‐Norway rats in vivo and to elucidate the molecular mechanism of this effect.

IL‐1β caused a significant reduction in albuterol‐mediated relaxation to protect against MCh‐induced bronchoconstriction. Dexamethasone attenuated the IL‐1β‐induced impaired relaxation while alone had no effect when compared to rats treated identically with saline.

The density of β2‐adrenoceptors was significantly reduced in lung membranes harvested from IL‐1β‐treated rats, which was associated with impaired isoproterenol‐ and forskolin‐stimulated cyclic AMP accumulation and adenylyl cyclase (AC) activity ex vivo. Dexamethasone did not prevent IL‐1β‐induced down‐regulation of β2‐adrenoceptors but completely blocked IL‐1β‐induced impairment of cyclic AMP accumulation and AC activity stimulated by isoproterenol and forskolin.

The inhibitory G‐protein subtypes, Giα1, Giα2 and Giα3, were detected in lung membranes prepared from all groups of rats but the intensity of Giα1 and Giα2 was markedly increased in IL‐1β‐treated rats, which were not prevented by dexamethasone.

The activity of cytosolic GRK and the expression of GRK2 and GRK5 were elevated in the lung of IL‐1β‐treated rats, which were completely abolished by dexamethasone.

These results indicate that treatment of rats with IL‐1β results in desensitization of pulmonary β2‐adrenoceptors. In light of data obtained in this study, we propose that both the decrease in AC activity and the increase in GRK activity, which are reversed by dexamethasone, may underlie β2‐adrenoceptor desensitization.

British Journal of Pharmacology (2002) 135, 987–996; doi:10.1038/sj.bjp.0704545
Proarrhythmic potential of halofantrine, terfenadine and clofilium in a modified in vivo model of torsade de pointes
Authors: Batey Andrew J , Coker Susan J
Published: January 2005

This study was designed to compare the proarrhythmic activity of the antimalarial drug, halofantrine and the antihistamine, terfenadine, with that of clofilium a K+ channel blocking drug that can induce torsade de pointes.

Experiments were performed in pentobarbitone‐anaesthetized, open‐chest rabbits. Each rabbit received intermittent, rising dose i.v. infusions of the α‐adrenoceptor agonist phenylephrine. During these infusions rabbits also received increasing i.v. doses of clofilium (20, 60 and 200 nmol kg−1 min−1), terfenadine (75, 250 and 750 nmol kg−1 min−1), halofantrine (6, 20 and 60 μmol kg−1) or vehicle.

Clofilium and halofantrine caused dose‐dependent increases in the rate‐corrected QT interval (QTc), whereas terfenadine prolonged PR and QRS intervals rather than prolonging cardiac repolarization. Progressive bradycardia occurred in all groups. After administration of the highest dose of each drug halofantrine caused a modest decrease in blood pressure, but terfenadine had profound hypotensive effects resulting in death of most rabbits.

The total number of ventricular premature beats was highest in the clofilium group. Torsade de pointes occurred in 6 out of 8 clofilium‐treated rabbits and 4 out of 6 of those which received halofantrine, but was not seen in any of the seven terfenadine‐treated rabbits.

These results show that, like clofilium, halofantrine can cause torsade de pointes in a modified anaesthetized rabbit model whereas the primary adverse effect of terfenadine was cardiac contractile failure.

British Journal of Pharmacology (2002) 135, 1003–1012; doi:10.1038/sj.bjp.0704550
Influence of antioxidant depletion on nitrergic relaxation in the pig gastric fundus
Authors: Colpaert E E , Timmermans J ‐P , Lefebvre R A
Published: January 2005

The hypothesis that endogenous tissue antioxidants might explain the inability of the superoxide generators 6‐anilino‐5,8‐quinolinedione (LY83583) and hydroquinone (HQ) and of the NO‐scavengers hydroxocobalamin (HC) and 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO) to affect nitrergic neurotransmission in the porcine gastric fundus was tested by selective pharmacological depletion of respectively Cu/Zn superoxide dismutase (Cu/Zn SOD) and reduced glutathione (GSH) in circular smooth muscle preparations.

Diethyldithiocarbamate (DETCA; 3×10−3 M), which almost completely abolished tissue Cu/Zn SOD activity, had no effect per se on nitrergic relaxations induced by either electrical field stimulation (EFS; 4 Hz, 10 s) or exogenous nitric oxide (NO; 10−5 M). In these DETCA‐treated tissues however, electrically‐induced nitrergic relaxations became sensitive to inhibition by LY83583 (10−5 M) or HC (10−4 M), but not by HQ (10−4 M) or c‐PTIO (10−4 M); only for the combination of DETCA plus LY83583, this inhibition was partially reversed by exogenous Cu/Zn SOD (1000 u ml−1).

Immunohistochemical analysis of porcine gastric fundus revealed a 100% colocalization of Cu/Zn SOD and neuronal nitric oxide synthase (nNOS) in the intrinsic neurons of the myenteric plexus.

Buthionine sulphoximine (BSO; 10−3 M) in the absence or presence of LY83583 (10−5 M) or HC (10−4 M) did not alter nitrergic relaxations, although it reduced per se the tissue GSH content to 62% of control.

Pharmacological depletion studies, corroborated by immunohistochemical data, thus suggest a role for Cu/Zn SOD but not for GSH in nitrergic neurotransmission in the porcine gastric fundus.

British Journal of Pharmacology (2002) 135, 917–926; doi:10.1038/sj.bjp.0704553