bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiotoxic effects of fenfluramine hydrochloride on isolated cardiac preparations and ventricular myocytes of guinea‐pigs
Authors: Rajamani Sridharan , Studenik Christian , Lemmens‐Gruber Rosa , Heistracher Peter
Published: January 2005

The cardiotoxic effects of fenfluramine hydrochloride on mechanical and electrical activity were studied in papillary muscles, Purkinje fibres, left atria and ventricular myocytes of guinea‐pigs.

Force of contraction (fc) was measured isometrically, action potentials and maximum rate of rise of the action potential (Vmax) were recorded by means of the intracellular microelectrode technique and the sodium current (INa) with patch‐clamp technique in the cell‐attached mode. For kinetic analysis (S)‐DPI‐201‐106‐modified Na+ channels from isolated guinea‐pig ventricular heart cells were used.

Fenfluramine (1–300 μM) produced negative chronotropic and inotropic effects; additional extracellular Ca2+ competitively antagonized the negative inotropic effect.

Fenfluramine concentration‐dependently reduced Vmax and showed tonic blockade of sodium channels, shortened the action potential duration in papillary muscles and Purkinje fibres.

In cell‐attached patches, fenfluramine decreased INa concentration‐dependently (10–100 μM), frequency‐independently (0.1–3 Hz; 30 μM). The h∞ curve was shifted towards hyperpolarizing direction. At 30 μM, fenfluramine blocked the sodium channel at all test potentials to the same degree, and neither changed the threshold and reversal potentials nor the peak of the curve.

No effect on single channel availability, but a significant decrease in mean open times and increase in mean closed times was observed.

Mean duration of the bursts decreased and number of openings per record increased with increasing drug concentration.

It is concluded that the effect on INa plays an important role in the cardiotoxicity of fenfluramine in addition to primary pulmonary hypertension and valvular disorders.

British Journal of Pharmacology (2000) 129, 843–852; doi:10.1038/sj.bjp.0703118
Time‐dependent block of the slowly activating delayed rectifier K+ current by chromanol 293B in guinea‐pig ventricular cells
Authors: Fujisawa Susumu , Ono Kyoichi , Iijima Toshihiko
Published: January 2005

The slowly activating delayed rectifier K+ current (IKs) was recorded in single myocytes dissociated from guinea‐pig ventricles and the mechanism underlying the block of IKs by a chromanol derivative, 293B, was investigated.

In the presence of 1–100 μM 293B, activation phase of IKs was followed by a slower decay during 10 s depolarizing pulses. Both the rate and extent of the decay were increased in a concentration‐dependent manner.

The relationship between the concentration of 293B and the block showed a Hill's coefficient of approximately 1. The half‐inhibitory concentration was approximately 3.0 μM and did not differ significantly at various membrane potentials from +20 to +80 mV.

A mathematical model for the 293B block was constructed on the basis of multiple closed and open states for the IKs channels, and the blocking rate was calculated by fitting the model to the original current traces. The blocking rate constant showed a linear function with the 293B concentration, indicating 1 : 1 binding stoichiometry. At +80 mV the blocking rate was 4×104 M−1 s−1 and the unblocking rate was 0.2 s−1.

The results indicate that 293B is an open channel blocker with relatively smaller blocking rate than those reported so far for time‐dependent blockade of various ionic channels.

British Journal of Pharmacology (2000) 129, 1007–1013; doi:10.1038/sj.bjp.0703126
ATP suppression of interleukin‐12 and tumour necrosis factor‐α release from macrophages
Authors: Haskó György , Kuhel David G , Salzman Andrew L , Szabó Csaba
Published: January 2005

Immune cell activation releases ATP into the extracellular space. ATP‐sensitive P2 purinergic receptors are expressed on immune cells and activation of these receptors alters immune cell function. Furthermore, ATP is metabolized by ectonucleotidases to adenosine, which has also been shown to alter cytokine production. In the present study, we investigated how extracellular ATP affects interleukin (IL)‐12 and tumour necrosis factor (TNF)‐α production in bacterial lipopolysaccharide (LPS)‐treated murine peritoneal macrophages and we also examined whether extracellular ATP alters the production of the T helper 1 cytokine interferon (IFN)‐γ.

Pretreatment of the peritoneal macrophages with ATP or various ATP analogues decreased both IL‐12 and TNF‐α production induced by LPS (10 μg ml−1). The effect of ATP was partially reversed by cotreatment with adenosine deaminase (0.1–1 u ml−1), suggesting that the suppressive effect of ATP on cytokine production is, in part, due to its degradation products.

Immunoneutralization with an anti‐IL‐10 antibody demonstrated that although ATP increases IL‐10 production, the inhibition of IL‐12 and TNF‐α production is independent of the increased IL‐10.

The effect of ATP was pretranslational, as it suppressed steady state levels of mRNAs for IL‐12 (both p35 and p40).

In spleen cells stimulated with either LPS (10 μg ml−1) or anti‐CD3 (2 μg ml−1) antibody, ATP suppressed, in a concentration‐dependent manner, the production of IFN‐γ.

These results suggest that extracellular ATP has multiple anti‐inflammatory effects and that release of ATP into the extracellular space may play a role in blunting the overactive immune response in autoimmune diseases.

British Journal of Pharmacology (2000) 129, 909–914; doi:10.1038/sj.bjp.0703134
Role of nitric oxide and septide‐insensitive NK1 receptors in bronchoconstriction induced by aerosolised neurokinin A in guinea‐pigs
Authors: Ricciardolo Fabio L M , Trevisani Marcello , Geppetti Pierangelo , Nadel Jay A , Amadesi Silvia , Bertrand Claude
Published: January 2005

The tachykinin, neurokinin A (NKA), contracts guinea‐pig airways both in vitro and in vivo, preferentially activating smooth muscle NK2 receptors, although smooth muscle NK1 receptors may also contribute. In vitro evidence suggests that NKA activates epithelial NK1 receptors, inducing the release of nitric oxide (NO) and subsequent smooth muscle relaxation. A number of selective NK1 receptor agonists have been reported to activate both smooth muscle and epithelial NK1 receptors, however septide appears only to activate smooth muscle NK1 receptors.

The aim of the present study was to investigate whether NKA‐induced bronchoconstriction in guinea‐pigs in vivo may be limited by NO release via NK1 receptor activation, and whether selective NK1 receptor agonists may activate this mechanism differently.

Aerosolized NKA caused an increase in total pulmonary resistance (RL) that was markedly reduced by the NK2 receptor antagonist, SR 48968, and abolished by the combination of SR 48968 and the NK1 receptor antagonist, CP‐99,994. The increase in RL evoked by NKA was potentiated by pretreatment with the NO synthase (NOs) inhibitor, L‐NAME, but not by the inactive enantiomer D‐NAME. Potentiation by L‐NAME of NKA‐induced increase in RL was reversed by L‐Arginine, but not by D‐Arginine.

Pretreatment with L‐NAME did not affect the increase in RL induced by the selective NK2 receptor agonist, [β‐Ala8]NKA(4‐10), and by the selective NK1 receptor agonist, septide, whereas it markedly potentiated the increase in RL caused by a different NK1 selective agonist, [Sar9,Met(O2)11]SP. Dose‐response curves showed that septide was a more potent bronchoconstrictor than [Sar9,Met(O2)11]SP to cause bronchoconstriction.

Pretreatment with the NK1 receptor antagonist, CP‐96,994, abolished the ability of L‐NAME to increase bronchoconstriction to aerosolized NKA. Bronchoconstriction to aerosolized NKA was increased by L‐NAME, after pretreatment with the NK3 receptor antagonist, SR 142801.

The present study shows that in vivo bronchoconstriction in response to the aerosolized naturally occurring tachykinin, NKA, is limited by its own ability to release relaxant NO via NK1 receptor activation. This receptor is apparently insensitive to septide, thus justifying, at least in part, the high potency of septide to cause bronchoconstriction in guinea‐pigs.

British Journal of Pharmacology (2000) 129, 915–920; doi:10.1038/sj.bjp.0703135
Effect of shear stress on the release of soluble ecto‐enzymes ATPase and 5′‐nucleotidase along with endogenous ATP from vascular endothelial cells
Authors: Yegutkin Gennady , Bodin Philippe , Burnstock Geoffrey
Published: January 2005

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5′‐nucleotidases).

The activity of soluble ATPase was progressively increased from 1.62±0.27 to 12.7±1.0 pmoles ml−1 h−1 after 60 min of stimulation by shear stress.

The rate of [3H]‐ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate‐6‐azophenyl‐2′4′‐disulphonic acid, and remained insensitive to the classic inhibitors of ion‐pumping and intracellular ATPases.

Shear stress also increased the activity of 5′‐nucleotidase in the medium from 2.0±0.5 to 27.2±2.8 pmoles ml−1 h−1. When shear stress was applied after removal of ecto‐5′‐nucleotidase by phosphatidylinositol‐specific phospholipase C, the release of 5′‐nucleotidase was drastically reduced.

These results show that soluble ATPase and 5′‐nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.

British Journal of Pharmacology (2000) 129, 921–926; doi:10.1038/sj.bjp.0703136
Nitroblue tetrazolium blocks BK channels in cerebrovascular smooth muscle cell membranes
Authors: Ye D , Pospisilik J A , Mathers D A
Published: January 2005

The effects of p‐nitroblue tetrazolium (NBT) on large conductance, calcium‐activated potassium channels (BK channels) in enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs) were examined.

Patch clamp methods were employed to record single BK channel currents from inside‐out patches of CVMC membrane maintained at 21–23°C.

When applied to the cytoplasmic face of inside‐out membrane patches (internally applied NBT), micromolar concentrations of NBT reversible reduced the mean open time of BK channels, without changing channel conductance.

NBT altered the frequency distribution of BK channel open times from a two exponential to a single exponential form.

In the absence of NBT, mean channel open time increased on membrane depolarization. In the presence of internally applied NBT, mean channel open became essentially independent of membrane potential.

Internally applied NBT also reduced the mean closed time of BK channels when measured at membrane potentials in the range −80 mV to +20 mV.

The combined effects of internal NBT on mean open and closed times resulted in the suppression of BK channel open probability when measured at positive membrane potentials.

When applied to the external membrane face, micromolar concentrations of NBT reduced mean channel open time progressively as the membrane was hyperpolarized, and also reduced open probability at negative membrane potentials.

A model is proposed in which NBT alters channel gating by binding to a site at or near to the cytoplasmic membrane face.

Externally applied NBT suppressed BK channel open probability at concentrations which also inhibit nitric oxide synthase (NOS). Therefore, the potential role of potassium channel block in NBT actions previously attributed to NOS inhibition is discussed.

British Journal of Pharmacology (2000) 129, 1035–1041; doi:10.1038/sj.bjp.0703143
Preservation of mitochondrial function by diazoxide during sustained ischaemia in the rat heart
Authors: Iwai Takeshi , Tanonaka Kouichi , Koshimizu Miki , Takeo Satoshi
Published: January 2005

A possible mechanism for the action of the KATP channel opener diazoxide on the improvement of energy metabolism of ischaemic/reperfused hearts was examined.

Isolated, perfused rat hearts were subjected to 40 min ischaemia followed by 60 min reperfusion. Diazoxide at concentrations of 3 to 30 μM was present in the perfusion buffer for the last 15 min of pre‐ischaemia.

Treatment of the perfused heart with diazoxide enhanced the post‐ischaemic recovery of rate‐pressure product, attenuated the post‐ischaemic rise in left ventricular end‐diastolic pressure, and suppressed the release of creatine kinase and purine nucleosides and bases from the reperfused heart. Treatment of the heart with diazoxide also restored myocardial ATP and creatine phosphate and attenuated the decrease in mitochondrial oxygen consumption rate after reperfusion. This attenuation was maintained at the end of ischaemia as well as at the end of reperfusion.

In another set of experiments, myocardial skinned bundles were incubated for 30 min under hypoxic conditions in the presence and absence of diazoxide, and then the mitochondrial oxygen consumption rate was determined. Hypoxia induced a decrease in the mitochondrial oxygen consumption rate of the skinned bundles to approximately 40% of the pre‐hypoxic value. In contrast, treatment of the bundles with 30 μM diazoxide preserved the normal mitochondrial oxygen consumption rate during hypoxia. This effect was abolished concentration‐dependently by the combined treatment with either the KATP channel blocker glibenclamide or 5‐hydroxydecanoate.

These results suggest that diazoxide is capable of attenuating ischaemia/reperfusion injury of isolated perfused hearts due to preservation of mitochondrial function during ischaemia.

British Journal of Pharmacology (2000) 129, 1219–1227; doi:10.1038/sj.bjp.0703148
Peroxisome proliferator‐activated receptors in the cardiovascular system
Authors: Bishop‐Bailey David
Published: January 2005

Peroxisome proliferator‐activated receptor (PPAR)s are a family of three nuclear hormone receptors, PPARα, ‐δ, and ‐γ, which are members of the steriod receptor superfamily. The first member of the family (PPARα) was originally discovered as the mediator by which a number of xenobiotic drugs cause peroxisome proliferation in the liver. Defined functions for all these receptors, until recently, mainly concerned their ability to regulate energy balance, with PPARα being involved in β‐oxidation pathways, and PPARγ in the differentiation of adipocytes. Little is known about the functions of PPARδ, though it is the most ubiquitously expressed. Since their discovery, PPARs have been shown to be expressed in monocytes/macrophages, the heart, vascular smooth muscle cells, endothelial cells, and in atherosclerotic lesions. Furthermore, PPARs can be activated by a vast number of compounds including synthetic drugs, of the clofibrate, and anti‐diabetic thiazoldinedione classes, polyunsaturated fatty acids, and a number of eicosanoids, including prostaglandins, lipoxygenase products, and oxidized low density lipoprotein. This review will aim to introduce the field of PPAR nuclear hormone receptors, and discuss the discovery and actions of PPARs in the cardiovascular system, as well as the source of potential ligands.

British Journal of Pharmacology (2000) 129, 823–834; doi:10.1038/sj.bjp.0703149
Release of GM‐CSF and G‐CSF by human arterial and venous smooth muscle cells: differential regulation by COX‐2
Authors: Stanford Salome J , Pepper John R , Mitchell Jane A
Published: January 2005

In addition to their traditional contractile function, vascular smooth muscle cells can be stimulated under inflammatory conditions to release a range of potent biological mediators. Indeed, we and others have shown that human vascular smooth muscle release the colony stimulating factors (CSF) granulocyte macrophage‐CSF (GM‐CSF) and granulocyte‐CSF (G‐CSF) as well as large amounts of prostaglandins following the induction of cyclo‐oxygenase‐2 (COX‐2), when stimulated with cytokines. Here we demonstrate, for the first time, that co‐induced COX‐2 activity simultaneously suppresses GM‐CSF release and potentiates G‐CSF release by human vascular cells. Moreover, the differential regulation of GM‐CSF and G‐CSF release by COX‐2 was mimicked by the prostacyclin (PGI2) mimetic, cicaprost. These observations suggest that PGI2, released following the induction of COX‐2, differentially regulates the release of GM‐CSF (suppresses) and G‐CSF (potentiates) from human vascular cells.

British Journal of Pharmacology (2000) 129, 835–838; doi:10.1038/sj.bjp.0703151
Nitric oxide‐dependent vasodilatation of rabbit femoral artery by β2‐adrenergic stimulation or cyclic AMP elevation in vivo
Authors: Xu Biao , Li Ji , Gao Lin , Ferro Albert
Published: January 2005

Some studies suggest that β‐adrenoceptor‐mediated vasorelaxation is in part mediated through nitric oxide (NO) release. We wished to determine the contribution of the L‐arginine / NO system to vasodilatation in response to β‐adrenoceptor stimulation with isoprenaline or cyclic adenosine‐3′,5′‐monophosphate (cyclic AMP) elevation with forskolin and dibutyryl cyclic AMP in vivo, using a rabbit femoral artery constant perfusion model.

Baseline femoral artery pressure was similar in rabbits receiving isoprenaline, forskolin or dibutyryl cyclic AMP. Isoprenaline, forskolin and dibutyryl cyclic AMP each decreased femoral artery pressure in a dose‐dependent manner. The doses (mol kg−1) of isoprenaline, forskolin and dibutyryl cyclic AMP which decreased pressure by 10% from baseline, expressed as a negative logarithm (−log ED10) were: 10.0±0.2, 9.5±0.1 and 4.9±0.1 respectively (P<0.0001 for each). Use of β‐adrenoceptor subtype‐selective antagonists showed that the vascular response to isoprenaline was purely due to stimulation of the β2‐adrenoceptor subtype.

Injection of 1 μmol kg−1 NG‐nitro‐L‐arginine methyl ester (L‐NAME) did not alter baseline pressure. However, it abolished the pressure response to isoprenaline (P<0.0001), and significantly attenuated the pressure responses to forskolin and dibutyryl cyclic AMP: −log ED10 values for forskolin and dibutyryl cyclic AMP, in the presence of L‐NAME, were 7.9±0.1 and 3.5±0.3 respectively (P<0.0001 for each, as compared with values in the absence of L‐NAME).

These results indicate that β2‐adrenergic stimulation and cylic AMP elevation activate the L‐arginine/NO system in rabbit femoral artery in vivo, and that NO generation contributes importantly to the changes in vascular tone induced by agents which modulate β‐adrenoceptors or cyclic AMP.

British Journal of Pharmacology (2000) 129, 969–974; doi:10.1038/sj.bjp.0703155
Vasorelaxant and antiplatelet activity of 4,7‐dimethyl‐1,2,5‐oxadiazolo[3,4‐d]pyridazine 1,5,6‐trioxide: role of soluble guanylate cyclase, nitric oxide and thiols
Authors: Kots Alexander Ya , Grafov Mikhail A , Khropov Yuri V , Betin Vasily L , Belushkina Natalya N , Busygina Olga G , Yazykova Marina Yu , Ovchinnikov Igor V , Kulikov Alexander S , Makhova Nina N , Medvedeva Natalya A , Bulargina Tamara V , Severina Irina S
Published: January 2005

Certain heterocyclic N‐oxides are vasodilators and inhibitors of platelet aggregation. The pharmacological activity of the furoxan derivative condensed with pyridazine di‐N‐oxide 4,7‐dimethyl‐1,2,5‐oxadiazolo[3,4‐d]pyridazine 1,5,6‐trioxide (FPTO) and the corresponding furazan (FPDO) was studied.

FPTO reacted with thiols generating nitrite (NO), S‐nitrosoglutathione and hydroxylamine (nitroxyl) and converted oxyHb to metHb. FPDO did not generate detectable amounts of NO‐like species but reacted with thiols and oxyHb.

FPTO and FPDO haem‐dependently stimulated the activity of soluble guanylate cyclase (sGC) and this stimulation was inhibited by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and by 0.1 mM dithiothreitol.

FPTO relaxed noradrenaline‐precontracted aortic rings and its concentration‐response curve was biphasic (pIC50=9.03±0.13 and 5.85±0.06). FPDO was significantly less potent vasodilator (pIC50=5.19±0.14). The vasorelaxant activity of FPTO and FPDO was inhibited by ODQ. oxyHb significantly inhibited only FPTO‐dependent relaxation.

FPTO and FPDO were equipotent inhibitors of ADP‐induced platelet aggregation (IC50=0.63±0.15 and 0.49±0.05 μM, respectively). The antiplatelet activity of FPTO (but not FPDO) was partially suppressed by oxyHb. The antiaggregatory effects of FPTO and FPDO were only partially blocked by sGC inhibitors.

FPTO and FPDO (10–20 μM) significantly increased cyclic GMP levels in aortic rings and platelets and this increase was blocked by ODQ.

Thus, FPTO can generate NO and, like FPDO, reacts with thiols and haem. The vasorelaxant activity of FPTO and FPDO is sGC‐dependent and a predominant role is played by NO at FPTO concentrations below 1 μM. On the contrary, inhibition of platelet aggregation is only partially related to sGC activation.

British Journal of Pharmacology (2000) 129, 1163–1177; doi:10.1038/sj.bjp.0703156
Enhanced blood pressure sensitivity to DOCA‐salt treatment in endothelin ETB receptor‐deficient rats
Authors: Matsumura Yasuo , Kuro Toshihiko , Konishi Fumiko , Takaoka Masanori , Gariepy Cheryl E , Yanagisawa Masashi
Published: January 2005

The role of endothelin ETB receptor‐mediated action in the development and maintenance of deoxycorticosterone acetate (DOCA)‐salt‐induced hypertension was evaluated using the spotting‐lethal (sl) rat which carries a naturally occurring deletion in the ETB receptor gene. Homozygous (sl/sl) rats treated with DOCA‐salt for 1 week exhibited an earlier onset of hypertension than heterozygous (sl/+) and wild‐type (+/+) rats (systolic blood pressure, SBP; 156.7±3.4 versus 128.8±5.3 and 132.9±3.7 mmHg, respectively). Four weeks after the start of DOCA‐salt treatment, homozygous rats developed marked hypertension, with a SBP of 206.0±4.5 mmHg, compared with 184.8±10.7 mmHg in heterozygous and 164.3±4.8 mmHg in wild‐type rats. Cardiovascular hypertrophy and renal dysfunction observed after 4‐weeks treatment with DOCA‐salt were more severe in homozygous rats, compared to wild‐type and heterozygous animals. These evidences support strongly the view that ETB receptor‐mediated actions are a protective factor in the pathogenesis of DOCA‐salt‐induced hypertension.

British Journal of Pharmacology (2000) 129, 1060–1062; doi:10.1038/sj.bjp.0703157
β3‐Adrenoceptor regulation and relaxation responses in mouse ileum
Authors: Hutchinson Dana S , Evans Bronwyn A , Summers Roger J
Published: January 2005

This study examines the relationship between β3a‐ and β3b‐adrenoceptor (AR) mRNA levels, β3‐AR binding and changes in ileum responses in mice treated with the β3‐AR agonist (R,R)‐5‐[2[[2‐(3‐chlorophenyl)‐2‐hydroxyethyl]‐amino]‐propyl]1,3‐benzodioxole‐2,2‐dicarboxylate (CL316243), or the β3‐AR antagonist 3‐(2‐ethylphenoxy)‐1‐[(1S)‐1,2,3,4‐tetrahydronapth‐1‐ylamino]‐2S‐2‐propanol oxalate (SR59230A), or dexamethasone or forskolin.

Levels of β3a‐ and β3b‐AR mRNA and the maximum number of binding sites (Bmax) in ileum were unaffected following CL316243 treatment, although responses to CL316243 were reduced by 50% following 4 and 24 h treatment, indicating another desensitization mechanism not involving changes in receptor expression or number. β3a‐AR mRNA levels were reduced in both brown (BAT) and white adipose tissue (WAT) but β3b‐AR mRNA levels were significantly reduced only in WAT. Levels of β3a‐ and β3b‐mRNA returned towards normal with continued treatment.

SR59230A treatment markedly increased β3‐AR mRNA levels in ileum and BAT but not in WAT. The increase in β3‐AR mRNA levels in ileum was associated with increased Bmax levels in binding analysis and increased responses to CL316243, suggesting these as the cause of sensitization.

Treatment with forskolin (4 h) or dexamethasone (4 h) significantly reduced β3a‐AR mRNA levels in BAT and WAT but did not alter levels in ileum. Responses to CL316243 in ileum were unaffected by either treatment.

In summary, the β3‐AR is differently regulated in adipose tissue and ileum: Treatment with SR59230A increased β3‐AR number, mRNA and responsiveness in ileum, whereas treatment with CL316243 reduced responses without affecting β3‐AR number or mRNA levels.

British Journal of Pharmacology (2000) 129, 1251–1259; doi:10.1038/sj.bjp.0703160
Effect of type‐selective inhibitors on cyclic nucleotide phosphodiesterase activity and insulin secretion in the clonal insulin secreting cell line BRIN‐BD11
Authors: Ahmad M , Abdel‐Wahab Y H A , Tate R , Flatt P R , Pyne N J , Furman B L
Published: January 2005

The cyclic nucleotide phosphodiesterases (PDEs) present in an insulin secreting cell line, BRIN–BD11, were characterized using calcium/calmodulin, IGF‐1, isoenzyme‐selective PDE inhibitors and RT–PCR.

Calmodulin activated cyclic AMP or cyclic GMP PDE activity in pellet and was 3 fold (P=0.002) more potent in activating cyclic nucleotide hydrolysis in pellet compared with supernatant fractions.

The PDE1/PDE5 inhibitor zaprinast inhibited both cyclic AMP and cyclic GMP PDE activity in both pellet and supernatant fractions of cell homogenates by a maximum of around 25% (IC50 1–5 μM), while rolipram (PDE4 selective) inhibited only cyclic AMP hydrolysis.

The PDE3‐selective inhibitors Org 9935 (0.02–10 μM) and siguazodan (0.1–10 μM) inhibited cyclic AMP PDE activity in the pellet but not the supernatant fractions of cell homogenates, with a maximum inhibition of about 30%. IGF‐1 (2–7.5 ng ml−1) potently augmented this PDE activity.

RT–PCR using specific primers for PDE3B, but not for PDE3A, amplified, from BRIN–BD11 cell total RNA, a 351 base pair product that was >97% homologous with rat adipose tissue PDE3B.

IBMX, Org 9935, siguazodan and rolipram (1–50 μM), but not zaprinast, each augmented glucose‐induced insulin secretion in the presence of 16.7 mM but not 1 mM glucose.

These findings, in a clonal insulin secreting cell line, are consistent with an important role for PDE3B in regulating the pool of cyclic AMP relevant to the modulation of glucose‐induced insulin secretion.

British Journal of Pharmacology (2000) 129, 1228–1234; doi:10.1038/sj.bjp.0703165
Respiratory actions of tachykinins in the nucleus of the solitary tract: effect of neonatal capsaicin pretreatment
Authors: Mazzone Stuart B , Geraghty Dominic P
Published: January 2005

The respiratory response to microinjection of capsaicin and tachykinin receptor agonists into the commissural nucleus of the solitary tract (cNTS) was investigated in adult, urethane‐anaesthetized rats which had been pretreated with capsaicin (50 mg kg−1 s.c.) or vehicle (10% Tween 80, 10% ethanol in saline) as day 2 neonates.

Microinjection of capsaicin (1 nmol) into the cNTS of vehicle‐pretreated rats, significantly reduced respiratory frequency (59 breaths min−1, preinjection control, 106 breaths min−1) without affecting tidal volume (VT). In capsaicin‐pretreated rats, the capsaicin‐induced bradypnoea was markedly attenuated (minimum frequency, 88 breaths min−1; control, 106 breaths min−1).

In vehicle‐pretreated rats, microinjection of substance P (SP, 33 pmol), neurokinin A (NKA, 33 pmol) and NKB (330 pmol), and the selective NK1 tachykinin receptor agonists, [Sar9,Met(O2)11]‐SP (33 pmol) and septide (10 pmol), increased VT (maxima, 3.60–3.93 ml kg−1) compared with preinjection control (2.82 ml kg−1), without affecting frequency. The selective NK3 agonist senktide (10 pmol) also increased VT (3.93 ml kg−1) which was accompanied by a bradypnoea (−25 breaths min−1). The selective NK2 agonist, [Nle10]‐NKA(4‐10) (330 pmol) increased VT slightly but significantly decreased frequency (−12 breaths min−1). In capsaicin‐pretreated rats, VT responses to SP and [Sar9,Met(O2)11]‐SP were increased whereas the response to septide was abolished. Both the VT and bradypnoeic responses to senktide and [Nle10]‐NKA(4‐10) were significantly enhanced.

These results show that neonatal capsaicin administration markedly reduces the respiratory response to microinjection of capsaicin into the cNTS. The destruction of capsaicin‐sensitive afferents appears to sensitize the NTS to SP, NKB, [Sar9,Met(O2)11]‐SP, senktide and [Nle10]‐NKA(4‐10). Moreover, the loss of septide responsiveness in capsaicin‐pretreated rats, suggests that ‘septide‐sensitive’ NK1 receptors may be located on the central terminals of afferent neurons.

British Journal of Pharmacology (2000) 129, 1132–1139; doi:10.1038/sj.bjp.0703173
The role of adenosine receptors in the action of theophylline on human peripheral blood mononuclear cells from healthy and asthmatic subjects
Authors: Landells L J , Jensen M W , Orr L M , Spina D , O'Connor B J , Page C P
Published: January 2005

The aim of the present study was to investigate the role of adenosine A2b receptors in the anti‐proliferative action of theophylline in human peripheral blood mononuclear cells (HPBMC) from healthy and asthmatic subjects.

Theophylline significantly inhibited PHA‐induced proliferation of HPBMC from both healthy and asthmatic donors but only at relatively high concentrations at 1 mM (P<0.05). Enprophylline, a drug which also acts as a non‐selective phosphodiesterase (PDE) inhibitor and is a selective A2b receptor antagonist, had no significant effect on proliferation of cells from either group at concentrations up to 10 μM (P>0.05; n=6).

Adenosine deaminase (2 u ml−1), which metabolizes adenosine, had no significant effect on PHA‐induced HPBMC proliferation over a range of concentrations (0–8 μg ml−1) in cells from either healthy or asthmatic subjects.

The adenosine receptor agonists N6‐cyclopentyladenosine (CPA, A1‐selective) and 5′‐N‐ethylcarboxamidoadenosine (NECA, A1/A2) produced a small but significant inhibition of PHA‐induced proliferation of HPBMC from healthy and asthmatic subjects (10 μM, P<0.05; n=6). In contrast, 5′‐N‐ethylcarboxamido‐2‐[4‐(2‐]carboxyethyl)phenethyl]adenosine (CGS21680, A2a‐selective) was without significant effect (P>0.05; n=6).

The adenosine receptor antagonist alloxazine (A2b‐selective) had no significant effect, while 8(3‐chlorostyryl)caffeine,(CSC, A2a‐selective) significantly inhibited PHA‐induced proliferation of HPBMC from both groups (P<0.05; n=6).

Our results suggest that endogenous or exogenous adenosine has little effect on the proliferation of HPBMC obtained from healthy or asthmatic subjects. Thus it would appear that the effect of high concentrations of theophylline is not related to adenosine receptor antagonism.

British Journal of Pharmacology (2000) 129, 1140–1144; doi:10.1038/sj.bjp.0703177
Functional role of muscarinic M2 receptors in α,β‐methylene ATP induced, neurogenic contractions in guinea‐pig ileum
Authors: Sawyer Gregory W , Lambrecht Günter , Ehlert Frederick J
Published: January 2005

The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist α,β‐methylene ATP (mATP) were investigated in guinea‐pig ileum.

mATP (0.1–30 μM) elicited a concentration‐dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non‐selective muscarinic receptor antagonist N‐methylscopolamine (NMS).

The contractile response to mATP was pertussis toxin‐insensitive, irreversibly antagonized by N‐(2‐chloroethyl)‐4‐piperidinyl diphenylacetate (4‐DAMP mustard), and unaffected by the muscarinic M2/M4 receptor selective antagonist AF‐DX 116 (1 μM).

When measured in the presence of histamine and isoproterenol after treatment with 4‐DAMP mustard, mATP elicited a pertussis toxin‐sensitive contractile response potently antagonized by AF‐DX 116.

Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M3 receptor and an indirect contractile response through the muscarinic M2 receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.

British Journal of Pharmacology (2000) 129, 1458–1464; doi:10.1038/sj.bjp.0703180
Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K+ channel Kir6.2 combination expressed in HEK‐293 cells
Authors: Gopalakrishnan Murali , Molinari Eduardo J , Shieh Char‐Chang , Monteggia Lisa M , Roch Jean‐Marc , Whiteaker Kristi L , Scott Victoria E S , Sullivan James P , Brioni Jorge D
Published: January 2005

The pharmacological properties of KATP channels generated by stable co‐expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K+ channel Kir6.2 were characterized in HEK‐293 cells.

[3H]‐Glyburide (glibenclamide) bound to transfected cells with a Bmax value of 18.5 pmol mg−1 protein and with a KD value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain.

Functional activity of KATP channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 μM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC50=0.92 nM).

Metabolic inhibition by 2‐deoxyglucose and oligomycin treatment triggered 86Rb+ efflux from transfected cells that was sensitive to inhibition by glyburide (IC50=3.6 nM).

Diazoxide, but not levcromakalim, evoked concentration‐dependent decreases in DiBAC4(3) fluorescence responses with an EC50 value of 14.1 μM which were attenuated by the addition of glyburide. Diazoxide‐evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities.

In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co‐expressed in HEK‐293 cells resemble those typical of native KATP channels described in pancreatic and neuronal tissues.

British Journal of Pharmacology (2000) 129, 1323–1332; doi:10.1038/sj.bjp.0703181
P2Y2 receptor‐mediated proliferation of C6 glioma cells via activation of Ras/Raf/MEK/MAPK pathway
Authors: Tu Ming‐Tze , Luo Shue‐Fen , Wang Chuan‐Chawn , Chien Chin‐Sung , Chiu Chi‐Tso , Lin Chih‐Chung , Yang Chuen‐Mao
Published: January 2005

Extracellular purine and pyrimidine nucleotides have been implicated in the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C6 glioma cells responsible for stimulating cell proliferation associated with mitogen‐activated protein kinase (MAPK) activation.

UTP and ATP produced a similar effect on [3H]‐thymidine incorporation in a time‐ and concentration‐dependent manner, suggesting the involvement of P2Y2 receptor in mediating proliferation of C6 glioma cells.

In response to UTP, both p42 and p44 MAPK were activated in a time‐ and concentration‐dependent manner using Western blot analysis with an anti‐phospho‐p42/p44 MAPK antibody. The phosphorylation reached maximal levels after 5 min and declining by 30 min.

Pretreatment with pertussis toxin (PTX) did not change these responses to UTP. Both DNA synthesis and phosphorylation of MAPK in response to UTP were attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca2+ by addition of BAPTA/AM plus EGTA.

UTP‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore, we showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf‐301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by ATP and UTP.

These results conclude that the mitogenic effect of UTP is mediated through a P2Y2 receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP‐mediated MAPK activation was modulated by Ca2+, PKC, and tyrosine kinase associated with cell proliferation in cultured C6 glioma cells.

British Journal of Pharmacology (2000) 129, 1481–1489; doi:10.1038/sj.bjp.0703182
Characterization of human recombinant α2A‐adrenoceptors expressed in Chinese hamster lung cells using extracellular acidification rate changes
Authors: MacLennan S J , Reynen P H , Martin R S , Eglen R M , Martin G R
Published: January 2005

Human α2A‐adrenoceptors heterologously expressed in Chinese hamster lung (CHL) fibroblasts have been characterized pharmacologically using a cytosensor microphysiometer to measure ligand‐induced extracellular acidification rate changes.

In untransfected CHL cells, noradrenaline had no effect at concentrations up to 100 μM. In α2A‐adrenoceptor transfected cells the rank order of agonist potency was A‐54741 (mean pEC50=8.96)>dexmedetomidine (8.88)>UK‐14304 (8.42)>B‐HT 920 (7.05)>noradrenaline (6.92). A‐54741, UK‐14304 and noradrenaline had the same maximum response while dexmedetomidine and B‐HT 920 behaved as partial agonists.

The selective α2‐adrenoceptor ligand rauwolscine antagonized acidification rate changes with an affinity independent of the agonist used; the affinity (mean pKB) against noradrenaline was 8.43.

The selective α1‐adrenoceptor ligands prazosin and doxazosin (each 3 μM) had no effect on noradrenaline responses.

Acidification rate changes induced by each agonist were abolished by pre‐treatment of cells with pertussis toxin.

These data suggest that agonist‐induced acidification rate responses in CHL cells transfected with the human α2A‐adrenoceptor are mediated exclusively by the recombinant protein, via pertussis toxin sensitive Gi/o proteins.

British Journal of Pharmacology (2000) 129, 1333–1338; doi:10.1038/sj.bjp.0703183
The P2Y1 receptor closes the N‐type Ca2+ channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists
Authors: Filippov Alexander K , Brown David A , Barnard Eric A
Published: January 2005

The rat P2Y1 nucleotide receptor, the P2Y subtype abundant in the brain, was heterologously expressed in rat superior cervical ganglion neurones by micro‐injection of the receptor cRNA or cDNA. ADP inhibited the N‐type Ca2+ current by 64%, with EC50 8.2 nM, an action blocked competitively by the P2Y1 receptor antagonist adenosine 3′, 5′‐bis‐phosphate (Ki 0.7 μM). 2‐Methylthio‐ADP inhibited the Ca2+ current likewise, but with EC50 0.57 nM, giving the highest potency reported therewith for P2Y1. Significantly, ATP and 2‐methylthio‐ATP were also agonists, the latter again at a very high potency (EC50 2.5 nM). We propose that this neuronal receptor, when present in brain at a high density as at synapses, can respond to very low concentrations of ATP and ADP as agonists, and that this would result in inhibition of N‐type Ca2+ currents and hence can reduce transmitter release or increase neuronal excitability.

British Journal of Pharmacology (2000) 129, 1063–1066; doi:10.1038/sj.bjp.0703185
Effects of protein tyrosine kinase inhibitors on voltage‐operated calcium channel currents in vascular smooth muscle cells and pp60c‐src kinase activity
Authors: Wijetunge S , Lymn J S , Hughes A D
Published: January 2005

Tyrosine kinases have been proposed as regulators of voltage‐operated calcium channels. The effects of a range of structurally different inhibitors of protein tyrosine kinases (PTK) were examined on voltage‐operated calcium channel currents (IBa) and pp60c‐src kinase (c‐src) activity in vitro.

IBa was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage‐clamp techniques. The activity of purified human c‐src was measured in vitro using a non‐radioactive assay.

Bath application of tyrphostin‐23 and genistein (non‐selective PTK inhibitors), bistyrphostin (a receptor‐PTK‐selective inhibitor) and PP1 (a src family‐selective inhibitor) inhibited IBa in a concentration‐dependent manner over a range of test membrane potentials. Intracellular application of peptide‐A, a peptide inhibitor of c‐src also inhibited currents. Inhibitor potency series against IBa was PP1 > genistein > tyrphostin 23 > bistyrphostin.

Tyrphostin‐23, genistein, PP1, and peptide‐A shifted the steady‐state inactivation curves in a hyperpolarized direction without altering their slope. The inhibitors had no significant effects on IBa activation calculated from current‐voltage relationships.

The agents inhibited c‐src activity in a concentration‐dependent manner. The order of potency was PP1 > genistein > peptide‐A > tyrphostin‐23 > bistyrphostin. The IC50 for inhibition of c‐src activity was similar to the IC50 for inhibition of IBa in all cases.

Western blot analysis with a specific antibody to c‐src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells.

A range of structurally dissimilar inhibitors of PTKs inhibit IBa and c‐src activity with similar potency. These data provide further evidence implicating endogenous c‐src in the modulation of L‐type calcium channels in vascular smooth muscle cells.

British Journal of Pharmacology (2000) 129, 1347–1354; doi:10.1038/sj.bjp.0703186
Pharmacology of nociceptin and its receptor: a novel therapeutic target
Authors: Calo' Girolamo , Guerrini Remo , Rizzi Anna , Salvadori Severo , Regoli Domenico
Published: January 2005

Nociceptin (NC), alias Orphanin FQ, has been recently identified as the endogenous ligand of the opioid receptor‐like 1 receptor (OP4). This new NC/OP4 receptor system belongs to the opioid family and has been characterized pharmacologically with functional and binding assays on native (mouse, rat, guinea‐pig) and recombinant (human) receptors, by using specific and selective agonists (NC, NC(1–13)NH2) and a pure and competitive antagonist, [Nphe1]NC(1–13)NH2. The similar order of potency of agonists and affinity values of the antagonist indicate that the same receptor is present in the four species. OP4 is expressed in neurons, where it reduces activation of adenylyl cyclase and Ca2+ channels while activating K+ channels in a manner similar to opioids. In this way, OP4 mediates inhibitory effects in the autonomic nervous system, but its activities in the central nervous system can be either similar or opposite to those of opioids. In vivo experiments have demonstrated that NC modulates a variety of biological functions ranging from nociception to food intake, from memory processes to cardiovascular and renal functions, from spontaneous locomotor activity to gastrointestinal motility, from anxiety to the control of neurotransmitter release at peripheral and central sites. These actions have been demonstrated using NC and various pharmacological tools, as antisense oligonucleotides targeting OP4 or the peptide precursor genes, antibodies against NC, an OP4 receptor selective antagonist and with data obtained from animals in which the receptor or the peptide precursor genes were knocked out. These new advances have contributed to better understanding of the pathophysiological role of the NC/OP4 system, and ultimately will help to identify the therapeutic potential of new OP4 receptor ligands.

British Journal of Pharmacology (2000) 129, 1261–1283; doi:10.1038/sj.bjp.0703219
Effects of inhibitors of small‐ and intermediate‐conductance calcium‐activated potassium channels, inwardly‐rectifying potassium channels and Na+/K+ ATPase on EDHF relaxations in the rat hepatic artery
Authors: Andersson David A , Zygmunt Peter M , Movahed Pouya , Andersson Tomas L G , Högestätt Edward D
Published: January 2005

In the rat hepatic artery, the SKCa inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 μM) and d‐tubocurarine (100 μM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM).

The IKCa inhibitors clotrimazole (3 μM) and 2‐chlorophenyl‐bisphenyl‐methanol (3 μM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono‐oxygenase inhibitor ketoconazole (10 μM) had no effect in the presence of apamin.

Ciclazindol (10 μM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated 86Rb+ influx via the Gardos channel (IKCa) in human erythrocytes.

The Na+/K+ ATPase inhibitor ouabain (500 μM) and the KIR blocker Ba2+ (30 μM) neither alone nor in combination inhibited EDHF relaxations. Ba2+ was also without effect in the presence of either apamin or charybdotoxin.

In contrast to EDHF, an increase in extracellular [K+] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K+‐free physiological salt solution, re‐admission of K+ always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba2+.

The present study provides pharmacological evidence for the involvement of SKCa and IKCa in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K+ activating Na+/K+ ATPase and KIR in this blood vessel.

British Journal of Pharmacology (2000) 129, 1490–1496; doi:10.1038/sj.bjp.0703226
Biphasic effect of bimoclomol on calcium handling in mammalian ventricular myocardium
Authors: Nánási Péter P , Sárközi Sándor , Szigeti Gyula , Jóna István , Szegedi Csaba , Szabó Ágnes , Bányász Tamás , Magyar János , Szigligeti Péter , Körtvély Ágnes , Csernoch László , Kovács László , Jednákovits Andrea
Published: January 2005

Concentration‐dependent effects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendorff‐perfused guinea‐pig hearts loaded with the fluorescent calcium indicator dye Fura‐2. Bimoclomol had a biphasic effect on contractility: both peak left ventricular pressure and the rate of force development significantly increased at a concentration of 10 nM or higher. The maximal effect was observed between 0.1 and 1 μM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentration‐dependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10–100 nM) increased, whereas high concentrations (10 μM) decreased the amplitude of intracellular calcium transients.

Effects of bimoclomol on action potential configuration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaffected at intermediate (0.1–1 μM) and decreased at high (10–100 μM) concentrations of the drug.

In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into artificial lipid bilayer, bimoclomol significantly increased the open probability of the channel in the concentration range of 1–10 μM. The increased open probability was associated with increased mean open time. The effect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol.

Bimoclomol (10 μM–1 mM) had no significant effect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be affected by the drug.

In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium.

British Journal of Pharmacology (2000) 129, 1405–1412; doi:10.1038/sj.bjp.0703230