bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha‐2 adrenoceptors are present in rat aorta smooth muscle cells, and their action is mediated by ATP‐sensitive K+ channels
Authors: Fauaz Grasiele , Feres Teresa , Borges Antonio C R , Paiva Therezinha B
Published: January 2005

The role of α2‐adrenoceptors in the response of aorta smooth muscle rings to the α2‐adrenoceptors agonists UK 14,304 and clonidine was studied.

Stimulation by 1–10 nM UK 14,304 caused dose‐dependent relaxant responses in BaCl2‐contracted endothelium‐denuded aorta rings, and hyperpolarization in rings with or without endothelium, which were inhibited by yohimbine and glibenclamide, but not affected by prazosin, propranolol, apamin or iberiotoxin. At higher concentrations (10 nM–10 μM) UK 14,304 also induced a depolarizing effect which was potentiated by yohimbine and inhibited by prazosin. These results indicate that UK 14,304 acts on α2‐adrenoceptors at lower concentrations and on both α1‐ and α2‐adrenoceptors above 10 nM.

In rings, with or without endothelium, noradrenaline had a depolarizing effect which was inhibited by prazosin. Adrenaline did not affect the membrane potential but in the presence of prazosin caused hyperpolarization, which was inhibited by yohimbine and glibenclamide. These results indicate that noradrenaline is more selective for α1‐, whereas adrenaline has similar affinities for α1‐ and α2‐adrenoceptors.

In aortae with endothelium, L‐NNA caused a small depolarization but did not affect the hyperpolarization induced by UK 14,304, indicating that NO is not involved in that response.

Glibenclamide induced a small depolarization in aortae, with or without endothelium, indicating that ATP‐sensitive K+ channels may play a role in maintaining the smooth muscle's membrane potential.

Our results indicate that, in rat aorta, α2‐adrenoceptors are also present in the smooth muscle, and that these receptors act through small‐conductance ATP‐sensitive K+ channels.

British Journal of Pharmacology (2000) 131, 788–794; doi:10.1038/sj.bjp.0703630
Analysis of 3‐morpholinosydnonimine and sodium nitroprusside effects on dopamine release in the striatum of freely moving rats: role of nitric oxide, iron and ascorbic acid
Authors: Serra Pier Andrea , Esposito Giovanni , Delogu M Rosaria , Migheli Rossana , Rocchitta Gaia , Grella Giuseppe , Miele Egidio , Miele Maddalena , Desole Maria S
Published: January 2005

The effects of intrastriatal infusion of 3‐morpholinosydnonimine (SIN‐1) or sodium nitroprusside (SNP) on dopamine (DA), 3‐methoxytyramine (3‐MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), L‐dihydroxyphenylalanine (L‐DOPA), ascorbic acid and uric acid concentrations in dialysates from the striatum of freely moving rats were evaluated using microdialysis.

SIN‐1 (1 mM) infusion for 180 min increased microdialysate DA and 3‐MT concentrations, while L‐DOPA, DOPCA+HVA, ascorbic acid and uric acid levels were unaffected. Co‐infusion with ascorbic acid (0.1 mM) inhibited SIN‐1‐induced increases in DA and 3‐MT dialysate concentration.

SNP (1 mM) infusion for 180 min increased greatly the dialysate DA concentration to a peak (2950% of baseline) at the end of the infusion, while increases in 3‐MT were negligible. In addition, SNP decreased ascorbic acid and L‐DOPA but increased uric acid concentration in the dialysate. Co‐infusion with deferoxamine (0.2 mM) inhibited the late SNP‐induced increase in DA dialysate concentration, but did not affect the decrease in ascorbic acid and increase uric acid dialysate concentrations.

SNP (1 mM) infusion for 20 min moderately increased uric acid, DA and 3‐MT, but decreased L‐DOPA levels in the dialysate. Ascorbic acid concentration increased at the end of SNP infusion. Co‐infusion with ascorbic acid (0.1 mM) inhibited the SNP‐induced increase in DA and 3‐MT, but did not affect the decrease in L‐DOPA and increase in uric acid dialysate concentrations.

These results suggest that NO released from SIN‐1 may account for the increase in the dialysate DA concentration. NO released following decomposition of SNP may account for the early increase in dialysate DA, while late changes in microdialysate composition following SNP may result from an interaction between NO and the ferrocyanide moiety of SNP. Exogenous ascorbic acid inhibits the effect of exogenous NO on DA release probably by scavenging NO, suggesting that endogenous ascorbic acid may modulate the NO control of DA release from 300 striatal dopaminergic terminals.

British Journal of Pharmacology (2000) 131, 836–842; doi:10.1038/sj.bjp.0703635
Transforming growth factor‐α stimulates prostaglandin generation through cytosolic phospholipase A2 under the control of p11 in rat gastric epithelial cells
Authors: Akiba Satoshi , Hatazawa Ryo , Ono Kyoko , Hayama Misako , Matsui Hirofumi , Sato Takashi
Published: January 2005

The regulatory effects of transforming growth factor (TGF)‐α on phospholipase A2 (PLA2) isozymes contributing to prostaglandin generation in rat gastric epithelial RGM1 cells were examined.

Stimulation with TGF‐α for 24 h time‐dependently induced prostaglandin E2 generation with an increase in cyclo‐oxygenase‐2 protein. The TGF‐α‐induced prostaglandin E2 generation was suppressed by NS‐398, a cyclo‐oxygenase‐2 inhibitor.

TGF‐α stimulated the activity and the protein synthesis of cytosolic PLA2 (cPLA2). A time‐dependent increase in cPLA2 protein occurred in parallel with PGE2 generation, which was inhibited by methyl arachidonyl fluorophosphonate (MAFP), a cPLA2 inhibitor. However, no change in activity of secretory PLA2 or Ca+2‐independent PLA2 was observed in the TGF‐α‐stimulated cells.

Stimulation with the Ca2+ ionophore A23187 for 10 min induced MAFP‐sensitive arachidonic acid liberation. Interestingly, preincubation with TGF‐α for 24 h diminished A23187‐stimulated arachidonic acid liberation despite the increase in cPLA2 protein.

Under the conditions, TGF‐α was found to increase p11, an endogenous cPLA2 suppressor, also known as annexin II light chain. The TGF‐α‐induced increase in p11 was suppressed by tyrphostin AG1478, an inhibitor of tyrosine kinase of epidermal growth factor receptor, which was also found to restore the inhibition by TGF‐α of A23187‐stimulated arachidonic acid liberation. However, TGF‐α did not alter protein levels of annexin II heavy chain.

These results suggest that TGF‐α stimulates prostaglandin generation through an increase in cPLA2, the hydrolytic action of which may be under the control of p11.

British Journal of Pharmacology (2000) 131, 1004–1010; doi:10.1038/sj.bjp.0703637
The antimalarial agent mefloquine inhibits ATP‐sensitive K‐channels
Authors: Gribble Fiona M , Davis Timothy M E , Higham Claire E , Clark Anne , Ashcroft Frances M
Published: January 2005

The aim of this study was to determine whether antimalarial agents inhibit ATP‐sensitive potassium (KATP) channels and thereby contribute to the observed side‐effects of these drugs.

Mefloquine (10–100 μM), but not artenusate (100 μM), stimulated insulin release from pancreatic islets in vitro.

Macroscopic KATP currents were studied in inside‐out patches excised from Xenopus oocytes expressing cloned KATP channels.

Mefloquine (IC50 ∼3 μM), quinine (IC50 ∼3 μM), and chloroquine inhibited the pancreatic β‐cell type of KATP channel Kir6.2/SUR1. Artenusate (100 μM) was without effect.

Mefloquine and quinine also blocked a truncated form of Kir6.2 (Kir6.2ΔC36) when expressed in the absence of SUR1. The extent of block was similar to that observed for Kir6.2/SUR1 currents.

Our results suggest that inhibition of the β‐cell KATP channel accounts for the ability of quinoline‐based antimalarial drugs to stimulate insulin secretion, and thereby produce hypoglycaemia.

The results also indicate that quinoline‐based antimalarial agents inhibit KATP channels by interaction with the Kir6.2 subunit. This subunit is common to β‐cell, neuronal, cardiac, skeletal muscle, and some smooth muscle KATP channels suggesting that KATP channel inhibition may contribute to the other side effects of these drugs, which include cardiac conduction abnormalities and neuropsychiatric disturbances.

British Journal of Pharmacology (2000) 131, 756–760; doi:10.1038/sj.bjp.0703638
Pharmacological characterization of the human 5‐HT4(d) receptor splice variant stably expressed in Chinese hamster ovary cells
Authors: Mialet Jeanne , Berque‐Bestel Isabelle , Sicsic Sames , Langlois Michel , Fischmeister Rodolphe , Lezoualc'h Frank
Published: January 2005

The recently identified C‐terminal splice variant of the human 5‐HT4 receptor, the h5‐HT4(d) receptor, was stably expressed in a CHO cell line at 493±25 fmol mg−1 protein. We analysed its pharmacological properties by measuring binding affinities and 5‐HT4 ligand‐induced cyclic AMP production.

The pharmacological binding profile determined in competition studies with the specific antagonist [3H]‐GR113808 revealed a rank order of affinity of 5‐HT4 ligands for the h5‐HT4(d) receptor that was consistent with those previously reported for other 5‐HT4 receptor isoforms.

In adenylyl cyclase functional assays, the h5‐HT4(d) receptor displayed equipotent coupling for all 5‐HT4 agonists tested (EC50 in the range of 1–6 nM). EC50 values were lower than those previously obtained with the 5‐HT4(e) receptor stably expressed in CHO cells indicating that the 5‐HT4(d) receptor was more efficiently coupled to its effector than the 5‐HT4(e) receptor isoform. Moreover, in terms of agonist efficacy (Emax), the benzamide derivative, renzapride displayed full agonist properties at the h5‐HT4(d) receptor (same Emax as 5‐HT) whereas it was previously shown to be a partial agonist at the h5‐HT4(e) receptor.

A constitutive activity of the h5‐HT4(d) receptor was observed in CHO cells in the absence of any 5‐HT4 ligand. Surprisingly, two 5‐HT4 ligands, SB204070 and RS39604 which are described as highly potent antagonists in various biological models, revealed partial agonist properties at the h5‐HT4(d) receptor.

We conclude that C‐terminal tails of 5‐HT4 receptor isoforms may directly influence their functional properties.

British Journal of Pharmacology (2000) 131, 827–835; doi:10.1038/sj.bjp.0703641
Cannabinoid receptor mediated inhibition of excitatory synaptic transmission in the rat hippocampal slice is developmentally regulated
Authors: Al‐Hayani Abdulmonem , Davies Stephen N
Published: January 2005

The cannabinoid (CB) receptor agonist WIN55,212‐2 (500 nM) had no effect on the first of a pair of population spikes evoked in the CA1 region of hippocampal slices prepared from young adult (4–6 weeks old) rats, despite powerfully reducing paired‐pulse depression. In contrast WIN55,212‐2 caused a substantial depression of the single population spike (reduced to 43% control) and the field EPSP (reduced to 72% of control) recorded in slices prepared from neonatal (10–13 days old) rats. This effect was stereoselective and blocked by the CB1 receptor antagonist AM281 (500 nM). The results indicate that activation of CB1 receptors inhibits excitatory synaptic transmission in neonatal, but not adult rat hippocampus. This developmental regulation of CB1 receptor mediated control of excitatory transmission may help explain some, but not all, of the previous discrepancies in the literature.

British Journal of Pharmacology (2000) 131, 663–665; doi:10.1038/sj.bjp.0703642
Neuroprotection against hypoxic/hypoglycaemic injury after the insult by the group III metabotropic glutamate receptor agonist (R,S)‐4‐phosphonophenylglycine
Authors: Sabelhaus Clemens F , Schröder Ulrich H , Breder Jörg , Henrich‐Noack Petra , Reymann Klaus G
Published: January 2005

The role of group III metabotropic glutamate receptors (mGluR) in ischaemic neurodegeneration is still unsettled. In order to examine a possible modulatory effect of these receptors on ischaemia‐induced damage we tested the novel selective agonist (R,S)‐4‐phosphonophenylglycine [(R,S)‐PPG] after an hypoxic/hypoglycaemic insult in rat hippocampal slices. The recovery of population spike amplitudes in the CA1‐region was used as parameter for neuronal viability. (R,S)‐PPG significantly improved the recovery of synaptic transmission in the CA1‐region even when applied only during the recovery period. The results imply that presynaptic glutamate release after an insult contributes to neurodegeneration. Since agonists of group III mGluR reduce neurotransmitter release – probably via presynaptic autoreceptors – we interpret the results obtained in our in vitro model of hypoxia/hypoglycaemia as support of the hypothesis that group III mGluR agonists might be beneficial drugs against diseases where excitotoxicity is one of the dominant pathological mechanisms.

British Journal of Pharmacology (2000) 131, 655–658; doi:10.1038/sj.bjp.0703646
Tetrahydrobiopterin impairs the action of endothelial nitric oxide via superoxide derived from platelets
Authors: Tajima Masamichi , Sakagami Hiroshi
Published: January 2005

The mechanism by which exogenous tetrahydrobiopterin (BH4) impairs the action of endothelial nitric oxide (NO) in the presence of platelets was investigated.

The endothelial NO generated by shear stress was determined by the anti‐aggregating activity of indomethacin‐treated endothelial cells and the cyclic GMP concentration in platelets.

The inhibitory effect of exogenous BH4 was suppressed by superoxide dismutase (SOD), or diclofenac sodium at concentrations inhibiting O2− generation, but not by allopurinol, a xanthine oxidase inhibitor.

BH4 similarly inhibited the anti‐aggregatory effect of sodium nitroprusside (SNP), a NO donor. The inhibitory effect was suppressed by diphenyleneiodonium, a specific inhibitor of NADPH oxidase.

Six(S)‐BH4, an inactive diastereoisomer of 6(R)‐BH4, and the 5,6,7,8‐tetrahydropterin compounds inhibited the endothelial NO action, whereas sepiapterin and 7,8‐dihydrobiopterin (BH2), 5,6‐double bond pterins, were inactive.

These tetrahydropterins, but not sepiapterin and BH2, scavenged superoxide (O2−) generated by the hypoxanthine‐xanthine oxidase reaction, possibly due to electron transfer during oxidation to its quinonoid‐form.

BH4 markedly stimulated the O2− generation from platelets, in the presence of NADH, rather than that of NADPH.

These findings suggest that BH4 stimulates platelet NAD(P)H oxidase to generate O2−, and inhibits the anti‐aggregating effect of NO. SOD activity in the local environment may modify the effect of BH4 on the endothelial NO activity.

British Journal of Pharmacology (2000) 131, 958–964; doi:10.1038/sj.bjp.0703648
Effects of a selective 5‐HT1B/1D receptor agonist on spinal and trigeminal reflexes in the anaesthetized rabbit
Authors: Jenkins Sarah , Richardson Bryan , Clarke Rob W
Published: January 2005

The effects of the 5‐HT1B/1D receptor agonist L‐741,604 on a trigeminally‐mediated (jaw depressor) reflex and a spinally‐mediated (flexion withdrawal) reflex have been compared between spinalized and intact, anaesthetized rabbits.

L‐741,604 depressed the jaw depressor reflex dose‐dependently in all animals, to a median of 5% (inter‐quartile range, IQR, 3–28%, n=18) of pre‐drug levels after a cumulative dose of 3.1 μmol kg−1 i.v. This effect was reversed by the 5‐HT1B/1D antagonist GR 127,935 (1–2 μmol kg−1 i.v.).

The flexion withdrawal reflex was depressed by L‐741,604 in non‐spinalized animals, to a median of 22% (IQR 10–36%, n=10) of pre‐drug levels after the highest dose, an action that was reversed by GR 127,935.

In spinalized rabbits, L‐741,604 up to 0.3 μmol kg−1 i.v. cumulative increased the flexion reflex to a median of 189% (IQR 169–198%, n=8) of pre‐drug controls. With higher doses the reflex decreased, so that after 3.1 μmol kg−1 it was 75% (IQR 55–96%) of pre‐drug levels. Subsequent GR 127,935 increased reflexes to a median of 180% (IQR 136–219%) of controls.

L‐741,604 increased arterial blood pressure and decreased heart rate in both preparations, effects that were reversed by GR 127,935.

Thus, when the spinal cord was intact L‐741,604 inhibited spinal and trigeminal reflexes in the same way. Although spinalization enabled a non‐5‐HT1B/1D‐mediated excitatory effect of L‐741,604 on spinal reflexes, there was a clear inhibitory effect of the drug at high doses. These data suggest that L‐741,604 inhibits spinal reflexes by increasing descending inhibition and by a direct action in the cord. The same processes could apply to inhibition of trigeminally‐mediated events.

British Journal of Pharmacology (2000) 131, 974–980; doi:10.1038/sj.bjp.0703649
Chronic, but not acute, dosing of antipsychotic drugs alters neurotensin binding in rat brain regions
Authors: Holtom P E , Needham P L , Bennett G W , Aspley S
Published: January 2005

The present study compared high affinity neurotensin (NT) binding in rat brain following acute or chronic treatment with the classical antipsychotic, haloperidol, and the newer antipsychotic drugs, clozapine and zotepine.

Drugs were given orally, as an acute treatment (1 dose) or chronically (21 day dosing) and binding to the NT high affinity receptor was examined in three brain regions; striatum, nucleus accumbens/olfactory tubercle and frontal cortex.

Acute dosing with either vehicle, haloperidol, clozapine or zotepine produced no significant changes in NT binding from controls (naïve rats).

Chronic (21 day) dosing resulted in an increase in the KD and Bmax of high affinity receptors in the striatum following haloperidol, but not clozapine, zotepine or vehicles. In contrast, the newer antipsychotics, clozapine and zotepine but not haloperidol or vehicles, significantly altered NT binding in the nucleus accumbens/olfactory tubercle by decreasing the KD and Bmax.

Further differentiation between the two newer antipsychotic drugs occurred in the frontal cortex. Clozapine had no significant effect on NT binding, whereas zotepine significantly reduced the KD of the high affinity receptor with no alteration in Bmax.

The antipsychotic drugs tested did not interact directly with the NT high affinity receptor. Therefore, they must be acting indirectly via an alternative receptor mechanism to alter NT high affinity binding. In accordance with previously reported NT/dopamine receptor interactions, this would suggest cross‐talk between these systems.

Overall, these data demonstrate that chronic, but not acute, administration of antipsychotic drugs alters NT binding in the rat brain. In addition, anatomical differences in NT binding arise according to the antipsychotic drug under test. This may be predictive of drug side‐effect profile, antipsychotic efficacy or atypicality.

British Journal of Pharmacology (2000) 131, 990–996; doi:10.1038/sj.bjp.0703653
Inhibition of HERG1 K+ channels by the novel second‐generation antihistamine mizolastine
Authors: Taglialatela Maurizio , Pannaccione Anna , Castaldo Pasqualina , Giorgio Giovanna , Annunziato Lucio
Published: January 2005

Ventricular arrhythmias are rare but life‐threatening side effects of therapy with the second‐generation H1 receptor antagonists terfenadine and astemizole. Blockade of the K+ channels encoded by the Human Ether‐à‐go‐go‐Related Gene 1 (HERG1) K+ channels, which is the molecular basis of the cardiac repolarizing current IKr, by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds.

In the present study, the potential blocking ability of the novel second‐generation H1 receptor antagonist mizolastine of the HERG1 K+ channels heterologously expressed in Xenopus oocytes and in HEK 293 cells or constitutively present in SH‐SY5Y human neuroblastoma cells has been examined and compared to that of astemizole.

Mizolastine blocked HERG1 K+ channels expressed in Xenopus oocytes with an estimated IC50 of 3.4 μM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K+ channel were 72.4±11.9 s for 3 μM mizolastine, and 1361±306 s for 1 μM astemizole.

In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose‐related inhibitory action on IHERG1, with an IC50 of 350±76 nM. Furthermore, mizolastine dose‐dependently inhibited HERG1 K+ channels constitutively expressed in SH‐SY5Y human neuroblastoma clonal cells.

The results of the present study suggest that the novel second‐generation H1 receptor antagonist mizolastine, in concentrations higher than those achieved in vivo during standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K+ channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1‐inhibitory action.

British Journal of Pharmacology (2000) 131, 1081–1088; doi:10.1038/sj.bjp.0703654
An evaluation of potassium ions as endothelium‐derived hyperpolarizing factor in porcine coronary arteries
Authors: Bény Jean‐Louis , Schaad Olivier
Published: January 2005

In the rat hepatic artery, the endothelium‐derived hyperpolarizing factor (EDHF) was identified as potassium. Potassium hyperpolarizes the smooth muscles by gating inward rectified potassium channels and by activating the sodium‐potassium adenosine triphosphatase (Na+‐K+ATPase). Our goal was to examine whether potassium could explain the EDHF in porcine coronary arteries.

On coronary strips, the inhibition of calcium‐dependent potassium channels with 100 nM apamin plus 100 μM charibdotoxin inhibited the endothelium‐dependent relaxations, produced by 10 nM substance P and 300 nM bradykinin and resistant to nitro‐L‐arginine and indomethacin.

The scavenging of potassium with 2 mM Kryptofix 2.2.2 abolished the endothelium‐dependent relaxations produced by the kinins and resistant to nitro‐L‐arginine and indomethacin.

Forty μM 18α glycyrrethinic acid or 50 μM palmitoleic acid, both uncoupling agents, did not inhibit these kinin relaxations. Therefore, EDHF does not result from an electrotonic spreading of an endothelial hyperpolarization.

Barium (0.3 nM) did not inhibit the kinin relaxations resistant to nitro‐L‐arginine and indomethacin. Therefore, EDHF does not result from the activation of inward rectified potassium channels.

Five hundred nM ouabain abolished the endothelium‐dependent relaxations resistant to nitro‐L‐arginine and indomethacin without inhibiting the endothelium‐derived NO relaxation.

The perifusion of a medium supplemented with potassium depolarized and contracted a coronary strip; however, the short application of potassium hyperpolarized the smooth muscles.

These results are compatible with the concept that, in porcine coronary artery, the EDHF is potassium released by the endothelial cells and that this ion hyperpolarizes and relaxes the smooth muscles by activating the Na+‐K+ATPase.

British Journal of Pharmacology (2000) 131, 965–973; doi:10.1038/sj.bjp.0703658
Release of a soluble ATPase from the rabbit isolated vas deferens during nerve stimulation
Authors: Westfall T D , Menzies J R W , Liberman R , Waterston S , Ramphir N , Westfall D P , Sneddon P , Kennedy C
Published: January 2005

The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isolated vas deferens were investigated.

Superfusate collected during EFS rapidly metabolised exogenous ATP (100 μM) and 50% was broken down in 5.67±0.65 min. The main metabolite was ADP, virtually no AMP was produced and adenosine was absent. No enzyme activity was seen in samples collected in the absence of EFS.

Lineweaver‐Burke analysis of the initial rates of ATP hydrolysis gave a KM of 40 μM and Vmax of 20.3 nmol ATP metabolized min−1 ml−1 superfusate. ATPase activity was unaffected by storage at room temperature for 24 h, but was abolished at pH4 or by heating at 80°C for 10 min.

ARL 67156 inhibited ATP breakdown in a concentration‐dependent manner (IC50=25 μM (95% confidence limits=22–27 μM), Hill slope=−1.06±0.04).

When EFS was applied three times at 30 min intervals, ATP metabolism was 20–30% less in superfusate collected during the second and third stimulation periods compared with the first. ATPase activity was released in a frequency‐dependent manner, with significantly greater activity seen after stimulation at 4 and 8 Hz than at 2 Hz.

In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens causes release of substantial ATPase, but little ADPase activity into the extracellular space. This contrasts with the guinea‐pig vas deferens, which releases enzymes that degrade ATP to adenosine. Thus, the complement of enzymes released by nerve stimulation is species‐dependent.

British Journal of Pharmacology (2000) 131, 909–914; doi:10.1038/sj.bjp.0703662
Extracellular signal‐regulated kinase 1/2 control Ca2+‐independent force development in histamine‐stimulated bovine tracheal smooth muscle
Authors: Koch Andrea , Nasuhara Yasuyuki , Barnes Peter J , Lindsay Mark A , Giembycz Mark A
Published: January 2005

The role of extracellular signal‐regulated kinase (ERK)‐1 and ERK‐2 in controlling histamine‐induced tone in bovine trachealis was investigated. PD 098059, an inhibitor of mitogen‐activated protein kinase kinase (MKK)‐1, had no effect on the histamine concentration‐response relationship that described contraction. However, in the presence of EGTA, PD 098059 produced a parallel 5 fold rightwards shift of the histamine concentration‐response curve without reducing the maximum response. The β2‐adrenoceptor agonist, procaterol, also displaced the histamine‐concentration response curve to the right but the effect was much greater than that evoked by PD 098059, non‐competitive and seen in the absence and presence of EGTA.

A low basal level of pERK‐1 and pERK‐2 was always detected in untreated trachealis, which was significantly higher in EGTA‐treated tissues and inhibited by PD 098059 and procaterol. Histamine markedly enhanced the phosphorylation of ERK‐1 and ERK‐2 by a mechanism that was also enhanced by EGTA and significantly attenuated by procaterol and PD 098059.

Neither cholera toxin nor Sp‐8‐Br‐cAMPS mimicked the ability of procaterol to dephosphorylate ERK. Similarly, neither pertussis toxin (PTX) nor Rp‐8‐Br‐cAMPS, an inhibitor of cyclic AMP‐dependent protein kinase (PKA), affected basal pERK levels or antagonized the inhibitory effect of procaterol.

These data implicate the MKK‐1/ERK signalling cascade in Ca2+‐independent, histamine‐induced contraction of bovine trachealis. In addition, the ability of procaterol to dephosphorylate ERK in an Rp‐8‐Br‐cAMPS‐ and PTX‐insensitive manner suggests that this may contribute to the anti‐spasmogenic activity of β2‐adrenoceptor agonists by activating a novel PKA‐independent pathway.

British Journal of Pharmacology (2000) 131, 981–989; doi:10.1038/sj.bjp.0703663
β‐Agonist‐induced constitutive β2‐adrenergic receptor activity in bovine tracheal smooth muscle
Authors: De Vries Berber , Meurs Herman , Roffel Ad F , Elzinga Carolina R S , Hoiting Ben H , De Vries Marleen M L , Zaagsma Johan
Published: January 2005

According to the two state receptor model, the β2‐adrenergic receptor (β2‐AR) isomerizes between an inactive state and a constitutively active state, which couples to the stimulatory G‐protein in the absence of agonist. In bovine tracheal smooth muscle (BTSM), we investigated the effect of short and long term β2‐AR activation by fenoterol on constitutive receptor activity.

Preincubation of the BTSM strips for 5 min, 30 min and 18 h with 10 μM fenoterol, followed by extensive washout (3 h, 37°C), caused a rapid and time‐dependent inhibition of KCl‐induced contraction, reaching 68±10, 51±6 and 46±4% of control, respectively, at 40 mM KCl (P<0.05 all). At all time points, the EC50 values to KCl were significantly reduced as well.

Preincubation of BTSM with 0.1, 1.0 and 10 μM fenoterol during 18 h caused a concentration‐dependent decrease of the 40 mM KCl response to 70±5, 47±12 and 43±9% of control, respectively (P<0.05 all).

The reduced KCl contractions were reversed in the presence of 1 μM timolol. Moreover, the sensitivity to KCl in the presence of timolol was enhanced after fenoterol incubation. Inverse agonism was also found for other β‐blockers, with a rank order of efficacy of pindolol timolol=propranolol>alprenololsotalol>labetalol.

At 25 mM KCl‐induced tone, the contraction induced by cumulative timolol administration was competitively antagonized by the less efficacious inverse agonist labetalol, indicating that the fenoterol‐induced effects cannot be explained by residual β‐agonist binding.

In conclusion, fenoterol treatment of BTSM causes a time‐ and concentration‐dependent development of constitutive β2‐AR activity, which can be reversed by various inverse agonists. The β‐agonist‐induced changes could represent a novel regulation mechanism of β2‐AR activity.

British Journal of Pharmacology (2000) 131, 915–920; doi:10.1038/sj.bjp.0703664
Endothelial nitric oxide synthase is a site of superoxide synthesis in endothelial cells treated with glyceryl trinitrate
Authors: Kaesemeyer Wayne H , Ogonowski Alison A , Jin Liming , Caldwell Ruth B , Caldwell Robert W
Published: January 2005

Tolerance to glyceryl trinitrate (GTN) involves superoxide (O2.−) production by endothelial cells. Nitric oxide synthase (NOS) produces O2.− when L‐arginine (L‐arg) is limited. The purpose of this study was to test the hypothesis that GTN stimulates NOS to increase O2.− synthesis in endothelial cells when L‐arg is limited.

Production of O2.− by bovine aortic endothelial cells (BAEC, passages 3–5) was determined by spectrophotometrically measuring superoxide dismutase‐inhibited reduction of ferricytochrome C to ferrocytochrome C. Cells were incubated in buffer without L‐arg. O2.− production was measured using BAEC either untreated or treated with L‐NAME or L‐arg alone or following treatment with GTN (10−9 to 10−6 M) for 30 min or DPTA NONOate (10−7 and 10−6 M) alone or with GTN or DPTA NONOate after pretreatment with nitro‐L‐arginine methyl ester (L‐NAME), L‐arg or their inactive enantiomers, D‐NAME or D‐arg (all 5×10−4 M) (n=6–7/group).

L‐NAME alone produced a 69% reduction in O2.− levels. Treatment with L‐arg alone had no effect. Cells treated with GTN alone exhibited an increase in O2.−. This effect was prevented by pretreatment with either L‐NAME or L‐arg, and was unaffected by D‐NAME or D‐arg. We observed a dose‐response relationship in O2.− production to GTN over a range of 10−9 to 10−7 M.

The NO donor, DPTA‐NONOate, unlike GTN, did not have a significant effect on O2.− production.

In conclusion, endothelial NOS is a site of O2.− synthesis in endothelial cells activated by GTN.

British Journal of Pharmacology (2000) 131, 1019–1023; doi:10.1038/sj.bjp.0703665
Effects of nimesulide and its reduced metabolite on mitochondria
Authors: Mingatto Fábio Erminio , Dos Santos Antonio Cardozo , Rodrigues Tiago , Pigoso Acácio Antonio , Uyemura Sérgio Akira , Curti Carlos
Published: January 2005

We investigated the effects of nimesulide, a recently developed non‐steroidal anti‐inflammatory drug, and of a metabolite resulting from reduction of the nitro group to an amine derivative, on succinate‐energized isolated rat liver mitochondria incubated in the absence or presence of 20 μM Ca2+, 1 μM cyclosporin A (CsA) or 5 μM ruthenium red.

Nimesulide uncoupled mitochondria through a protonophoretic mechanism and oxidized mitochondrial NAD(P)H, both effects presenting an EC50 of approximately 5 μM.

Within the same concentration range nimesulide induced mitochondrial Ca2+ efflux in a partly ruthenium red‐sensitive manner, and induced mitochondrial permeability transition (MPT) when ruthenium red was added after Ca2+ uptake by mitochondria. Nimesulide induced MPT even in de‐energized mitochondria incubated with 0.5 mM Ca2+.

Both Ca2+ efflux and MPT were prevented to a similar extent by CsA, Mg2+, ADP, ATP and butylhydroxytoluene, whereas dithiothreitol and N‐ethylmaleimide, which markedly prevented MPT, had only a partial or no effect on Ca2+ efflux, respectively.

The reduction of the nitro group of nimesulide to an amine derivative completely suppressed the above mitochondrial responses, indicating that the nitro group determines both the protonophoretic and NAD(P)H oxidant properties of the drug.

The nimesulide reduction product demonstrated a partial protective effect against accumulation of reactive oxygen species derived from mitochondria under conditions of oxidative stress like those resulting from the presence of t‐butyl hydroperoxide.

The main conclusion is that nimesulide, on account of its nitro group, acts as a potent protonophoretic uncoupler and NAD(P)H oxidant on isolated rat liver mitochondria, inducing Ca2+ efflux or MPT within a concentration range which can be reached in vivo, thus presenting the potential ability to interfere with the energy and Ca2+ homeostasis in the liver cell.

British Journal of Pharmacology (2000) 131, 1154–1160; doi:10.1038/sj.bjp.0703667
Characterization of endothelial factors involved in the vasodilatory effect of simvastatin in aorta and small mesenteric artery of the rat
Authors: Álvarez De Sotomayor Maria , Herrera Maria Dolores , Marhuenda Elisa , Andriantsitohaina Ramaroson
Published: January 2005

Vascular effects of the 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase inhibitor, simvastatin, were studied in conductance (aorta) and resistance vessels (branch II or III of superior mesenteric artery, SMA) of the rat (12–14 weeks old).

Simvastatin produced relaxation of both aorta and SMA, with and without functional endothelium. These responses were inhibited by the product of HMG‐CoA reductase, mevalonate (1 mmol l−1).

In vessels with functional endothelium, the NO‐synthase inhibitor, L‐NG‐nitroarginine (L‐NOARG, 30 μmol l−1), inhibited simvastatin‐induced relaxation. In the presence of L‐NOARG, relaxation to simvastatin was lower in vessels with endothelium than in endothelium‐denuded arteries without L‐NOARG.

The cyclo‐oxygenase inhibitor, indomethacin (10 μmol l−1), abolished endothelium‐dependent component of the response to simvastatin in both arteries. The combination of L‐NOARG plus indomethacin did not produce further inhibition. The Tp receptor antagonist, GR 32191B (3 μmol l−1), did not affect relaxation in aorta but it reduced response to low concentrations of simvastatin in SMA. However, the inhibitory effect of L‐NOARG was less marked in the presence of GR 32191B in aorta but not in SMA.

The endothelium‐dependent relaxation to simvastatin was inhibited by the superoxide dismutase (SOD, 100 u ml−1) or by the tyrosine kinase inhibitor, genistein (30 μmol l−1) in the two arteries.

The present study shows that simvastatin produces relaxation of conductance and small arteries through mevalonate‐sensitive pathway. The endothelium‐dependent relaxation to simvastatin involves both NO and vasodilator eicosanoids by a mechanism sensitive to SOD, and to genistein. Also, the results highlighted participation in the aorta of endothelial vasoconstrictor eicosanoids acting on the Tp receptor after blockage of NO synthase only.

British Journal of Pharmacology (2000) 131, 1179–1187; doi:10.1038/sj.bjp.0703668
Magnolol stimulates steroidogenesis in rat adrenal cells
Authors: Wang Seu‐Mei , Lee Li‐Jen , Huang Yu‐Tsung , Chen Jian‐Jiun , Chen Yuh‐Lien
Published: January 2005

This study investigated the effect of magnolol, a compound purified from Magnolia officinalis, on glucocorticoid production by primary adrenal cell culture.

Magnolol increased corticosterone secretion in a dose‐dependent manner, this effect being maximal at 40 μM. A similar effect was seen in a minced adrenal gland system.

In magnolol‐treated cells, the number and total area of cytoplasmic lipid droplets were reduced, suggesting a high utilization rate of cholesterol esters stored in lipid droplets. In control cells, the capsule of the lipid droplet was clearly delineated by immunostaining with antibody A2, whereas capsular staining was discontinuous or undetectable following magnolol treatment. The percentage of decapsulated cells increased significantly from 20% in the control group to 80% in the magnolol‐treated group.

Magnolol‐induced steroidogenesis was not mediated either via the traditional ACTH‐cyclic AMP‐protein kinase A pathway or by protein kinase C, since the intracellular cyclic AMP level did not change and inhibition of protein kinase A or C did not block the action of magnolol. Furthermore, calcium/calmodulin‐dependent protein kinase II was not involved in magnolol‐induced steroidogenesis.

The stimulatory effect of magnolol on steroidogenesis apparently requires new protein synthesis, since cycloheximide inhibited magnolol‐induced corticosterone production by 50%.

Although other studies have shown that high concentrations of magnolol inhibit acyl‐CoA: cholesterol acyltransferase and 11β‐hydroxysteroid dehydrogenase in a cell‐free system, our data show that, in adrenal cell cultures, low concentrations of magnolol have a stimulatory effect on steroidogenesis, and the glucocorticoid produced may explain the effective control of asthma by Magnolia officinalis.

British Journal of Pharmacology (2000) 131, 1172–1178; doi:10.1038/sj.bjp.0703669
Chondroitin sulphate inhibits connective tissue mast cells
Authors: Theoharides T C , Patra P , Boucher W , Letourneau R , Kempuraj D , Chiang G , Jeudy S , Hesse Leah , Athanasiou A
Published: January 2005

Mast cells derive from the bone marrow and are responsible for the development of allergic and possibly inflammatory reactions. Mast cells are stimulated by immunoglobulin E (IgE) and specific antigen, but also by a number of neuropeptides such as neurotensin (NT), somatostatin or substance P (SP), to secrete numerous pro‐inflammatory molecules that include histamine, cytokines and proteolytic enzymes.

Chondroitin sulphate, a major constituent of connective tissues and of mast cell secretory granules, had a dose‐dependent inhibitory effect on rat peritoneal mast cell release of histamine induced by the mast cell secretagogue compound 48/80 (48/80). This inhibition was stronger than that of the clinically available mast cell ‘stabilizer’ disodium cromoglycate (cromolyn). Inhibition by chondroitin sulphate increased with the length of preincubation and persisted after the drug was washed off, while the effect of cromolyn was limited by rapid tachyphylaxis.

Immunologic stimulation of histamine secretion from rat connective tissue mast cells (CTMC) was also inhibited, but this effect was weaker in umbilical cord‐derived human mast cells and was absent in rat basophilic leukemia (RBL) cells which are considered homologous to mucosal mast cells (MMC). Oligo‐ and monosaccharides were not as effective as the polysaccharides.

Inhibition, documented by light and electron microscopy, involved a decrease of intracellular calcium ion levels shown by confocal microscopy and image analysis. Autoradiography at the ultrastructural level showed that chondroitin sulphate was mostly associated with plasma and perigranular membranes.

Chondroitin sulphate appears to be a potent mast cell inhibitor of allergic and nonimmune stimulation with potential clinical implications.

British Journal of Pharmacology (2000) 131, 1039–1049; doi:10.1038/sj.bjp.0703672
The pharmacology of nucleotide receptors on primary rat brain endothelial cells grown on a biological extracellular matrix: effects on intracellular calcium concentration
Authors: Sipos Ildikó , Dömötör Eszter , Abbott N Joan , Adam‐Vizi Vera
Published: January 2005

Brain capillary endothelial cells express a variety of nucleotide receptors, but differences have been reported between culture models. This study reports examination of nucleotide receptors on primary cultured rat brain capillary endothelial cells (RBCEC) grown on a biological extracellular matrix (ECM) to produce a more differentiated phenotype.

Fura‐2 fluorescence ratio imaging was used to monitor intracellular free calcium concentration [Ca2+]i. ATP, UTP, and 2‐methylthioATP (2‐MeSATP) increased [Ca2+]i to similar levels, while 2‐MeSADP, ADP and adenosine gave smaller responses.

Removal of extracellular calcium caused no significant change in the [Ca2+]i response to 2‐MeSATP, evidence that the response was mediated by a metabotropic (P2Y) receptor.

All cells tested responded to ATP, UTP, 2‐MeSATP and ADP, while 63% responded to adenosine and 50% to 2‐MeSADP. No cells responded to α,β‐methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform [Ca2+]i responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile.

The [Ca2+]i response to the P2Y1‐selective agonist 2‐MeSADP was abolished in the presence of the subtype‐selective antagonist adenosine 3′‐phosphate 5′‐phosphosulphate (PAPS).

The P2Y2 antagonist suramin completely blocked the response to ATP and inhibited the response to UTP by 66%.

The A1 subtype‐selective adenosine receptor agonist N6‐Cyclopentyladenosine (CPA) gave a small but characteristic [Ca2+]i response, while A2A and A2B subtype‐selective agonists failed to generate [Ca2+]i changes.

The results are consistent with the presence on RBCEC of a P2Y2‐like receptor coupled to phospholipase C, and a P2Y1‐like receptor mobilizing intracellular Ca2+. The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.

British Journal of Pharmacology (2000) 131, 1195–1203; doi:10.1038/sj.bjp.0703675
Effects of acidosis and NO on nicorandil‐activated KATP channels in guinea‐pig ventricular myocytes
Authors: Moncada Gustavo A , Kishi Yukio , Numano Fujio , Hiraoka Masayasu , Sawanobori Tohru
Published: January 2005

Nicorandil is a hybrid compound of K+ channel opener and nitrate. We investigated a possible interaction of acidosis and nitric oxide (NO)‐donors on the nicorandil‐activated ATP‐sensitive K+ channel (KATP) in guinea‐pig ventricular myocytes using the patch‐clamp technique.

In whole‐cell recordings, external application of 300 μM nicorandil activated KATP in the presence of 2 mM intracellular ATP concentration ([ATP]i) at external pH (pHo) 7.4, but the activated current was decreased by reducing pHo to 6.5–6.0.

Single‐channel recordings of inside‐out patches revealed decreased open‐state probability (Po) of KATP activated by nicorandil with reducing internal pH (pHi) from 7.2 to 6.0, whilst the channel activity increased at low pHi in the absence of nicorandil.

Application of NO donors, 1 mM‐sodium nitroprusside (SNP) or ‐NOR‐3 to the membrane cytoplasmic side at pHi 7.2 increased the channel activity but decreased it at pHi 6.5–6.0. Neither removal of the drugs nor application of NO‐scavengers reversed depression of channel activity induced by NO‐donors.

We conclude that an increase in pHo and pHi depresses rather than stimulates the nicorandil‐activated KATP. Since NO‐donors at low pHi exhibited a similar trend, involvement of H+ and NO interaction can be considered as a mechanism of decreased KATP activated by nicorandil.

British Journal of Pharmacology (2000) 131, 1097–1104; doi:10.1038/sj.bjp.0703678
Effect of neutral endopeptidase inhibitor on endogenous atrial natriuretic peptide as a paracrine factor in cultured cardiac fibroblasts
Authors: Maki Toshiyuki , Horio Takeshi , Yoshihara Fumiki , Suga Shin‐ichi , Takeo Satoshi , Matsuo Hisayuki , Kangawa Kenji
Published: January 2005

Cardiac remodelling is a fundamental response to hypertension, myocardial infarction and chronic heart failure, and involves cardiac fibroblast proliferation and production of extracellular matrix components such as collagen. The present study was performed to examine the role of endogenous atrial natriuretic peptide (ANP) as a possible paracrine factor for cardiac fibroblasts, and to examine the effects of three neutral endopeptidase (NEP) inhibitors, thiorphan, phosphoramidon and ONO‐BB‐039‐02 (ONO‐BB) on endogenous ANP‐induced changes in collagen synthesis by cultured neonatal rat cardiac fibroblasts.

Each NEP inhibitor singly had no significant effect on collagen synthesis by cardiac fibroblasts, except for maximum concentration (10−3 M) of thiorphan.

Exogenous ANP inhibited collagen synthesis in a concentration‐dependent manner (10−8–10−6 M). Thiorphan (10−4 and 10−3 M) and phosphoramidon (10−5 and 10−4 M) enhanced the ANP (10−7 M)‐induced decrease in collagen synthesis. ONO‐BB (10−5 and 10−4 M) slightly enhanced the ANP‐induced decrease in collagen synthesis.

Myocyte‐conditioned medium (MC‐CM), as well as exogenous ANP, inhibited collagen synthesis dose‐dependently. The decrease in collagen synthesis at 100% MC‐CM was augmented by thiorphan (10−3 M), phosphoramidon (10−4 M) and ONO‐BB (10−4 M).

HS‐142‐1, a natriuretic peptide receptor antagonist, significantly reduced the MC‐CM plus thiorphan‐ and MC‐CM plus ONO‐BB‐induced decrease in collagen synthesis, by 92 and 62%, respectively and showed a tendency to attenuate the MC‐CM plus phosphoramidon‐induced decrease in collagen synthesis by 40%.

Our observations suggested that endogenous ANP released from cardiomyocytes inhibited collagen synthesis as a paracrine factor and that NEP inhibitors enhanced the activity of this peptide in cardiac fibroblasts.

British Journal of Pharmacology (2000) 131, 1204–1210; doi:10.1038/sj.bjp.0703679
Direct interaction of Na‐azide with the KATP channel
Authors: Trapp Stefan , Ashcroft Frances M
Published: January 2005

The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from Xenopus oocytes expressing cloned ATP‐sensitive potassium (KATP) channels of the Kir6.2/SUR1 type.

In inside‐out patches from oocytes expressing Kir6.2ΔC36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na‐azide inhibited macroscopic currents with an IC50 of 11 mM. The inhibitory effect of Na‐azide was mimicked by the same concentration of NaCl, but not by sucrose.

Na‐azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively. Inhibition was abolished in the absence of intracellular Mg2+. In contrast, Kir6.2ΔC36 currents were inhibited by Na‐azide both in the presence or absence of intracellular Mg2+.

Kir6.2/SUR1 currents were less sensitive to 3 mM Na‐azide in the presence of MgATP. This apparent reduction in sensitivity is caused by a small activatory effect of Na‐azide conferred by SUR.

We conclude that, in addition to its well‐established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na‐azide has direct effects on the KATP channel. Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR. There is also a small, ATP‐dependent, stimulatory effect of Na‐azide mediated by the SUR subunit. The direct effects of 3 mM Na‐azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na‐azide concentration.

British Journal of Pharmacology (2000) 131, 1105–1112; doi:10.1038/sj.bjp.0703680