bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of contraction induced by bradykinin in the rabbit saphenous vein
Authors: Eguchi Daihiko , Nishimura Junji , Kobayashi Sei , Komori Kimihiro , Sugimachi Keizo , Kanaide Hideo
Published: August 2006

By using fura‐PE3 fluorometry and receptor‐coupled permeabilization by α‐toxin, the mechanism of the bradykinin (BK)‐induced contraction was determined in the rabbit saphenous vein (RSV). The receptor subtype responsible for the BK‐induced contraction of RSV was determined by means of a pharmacological blocker study and reverse transcription polymerase chain reaction (RT‐PCR).

In the presence of extracellular Ca2+ (1.25 mM), BK (10−11‐3 × 10−7 m) induced increases in both the cytosolic Ca2+ concentration ([Ca2+]i) and force, in a concentration‐dependent manner. Both the release of Ca2+ from the store site and the influx of extracellular Ca2+ contribute to an increase in [Ca2+]i induced by BK.

In the absence of extracellular Ca2+, the application of 10−7 M BK induced transient elevations of [Ca2+]i and force, both of which thereafter declined to the levels observed before the application of BK. When extracellular Ca2+ was replenished (1.25 mM), [Ca2+]i and force increased to form a peak, followed by a sustained elevation in the presence of BK. When an RSV strip was pretreated with 10−5 M thapsigargin for 20 min, the BK‐induced transient increases in both [Ca2+]i and force were markedly inhibited.

These responses induced by BK were inhibited by Hoe 140 (D‐Arg‐[Hyp3, Thi5, D‐Tic7, Oic8] bradykinin), a highly specific bradykinin B2 receptor antagonist, in a concentration‐dependent manner. In RT‐PCR, B2‐receptor mRNA was expressed in the smooth muscle of RSV.

The [Ca2+]i‐force relationships, which were determined by cumulative applications of extracellular Ca2+ (0–5 mM) during 118 mM K+‐depolarization, shifted to the upper left in the presence of BK, thus indicating that BK induced a greater force than 118 mM K+‐depolarization for a given level of [Ca2+]i.

In α‐toxin‐permeabilized preparations of RSV, application of 10−7 M BK after a steady state contraction had been induced by a mixture of 5 × 10−7 M Ca2+, 10−6 M GTP and 10−6 M captopril caused an additional force development at a constant [Ca2+]i. However, treatment with 1 mM guanosine‐5′‐O‐(β‐thiodiphosphate) (GDPβS) for 5 min before and during the application of BK (10−7 m), abolished this BK‐induced additional contraction.

These results indicated that in RSV: (1) BK elicits vasoconstriction by increasing the Ca2+ influx from the extracellular space, Ca2+ release from intracellular thapsigargin‐sensitive storage sites and increasing the Ca2+ sensitivity of the contractile apparatus, (2) the BK‐induced increase in Ca2+ sensitivity is mediated by G‐protein, (3) the BK‐induced contractions are mediated via B2‐receptors and (4) the smooth muscle cells express B2‐receptor mRNA.
Effects of riluzole on rat cortical neurones: an in vitro electrophysiological study
Authors: Siniscalchi Antonio , Bonci Antonello , Mercuri Nicola B , Bernardi Giorgio
Published: August 2006

The electrophysiological effects of riluzole on rat prefrontal and frontal cortical neurones were investigated by using both extracellular (field) and intracellular recording techniques in brain slices.

Bath applied riluzole (3–200 μm) depressed the cortico‐cortical stimulus‐evoked field potential in a concentration‐related manner (EC50=29.5 μm).

Riluzole (3–100 μm) reduced the tonic firing of the neocortical neurones which was caused by intracellular current injection, while it did not have any effect on the resting membrane potential and apparent input resistance of these cells.

In the presence of tetrodotoxin (1 μm) and tetraethylammonium (30 mm), the injection of a depolarizing current step generated a calcium spike in the neocortical neurones. Riluzole (30 μm) abolished this calcium‐dependent action potential. However, when the amount of the depolarizing current was increased the calcium‐dependent regenerative potential was evoked again.

The depolarization of the membrane (10–20 mV) caused by brief (8–15 s) bath applications of glutamate (300 μm–1 mm) were not changed in the presence of riluzole (30 μm).

It is concluded that riluzole has direct actions on rat neocortical neurones: (a) it blocks the repetitive discharge of sodium action potentials and (b) it increases the threshold for the generation of the calcium spike. These two cellular mechanisms might at least in part account for the depression of the cortico‐cortical field potential caused by this drug.

British Journal of Pharmacology (1997) 120, 225–230; doi:10.1038/sj.bjp.0700905
The effects of tamsulosin, a high affinity antagonist at functional α1A‐ and α1D‐adrenoceptor subtypes
Authors: Noble A J , Chess‐Williams R , Couldwell C , Furukawa K , Uchyiuma T , Korstanje C , Chapple C R
Published: August 2006

The actions of the α1‐adrenoceptor antagonist tamsulosin have been examined at functional α1‐adrenoceptor subtypes and compared with those at the human prostate receptor.

At the α1D‐adrenoceptors of the rat aorta, tamsulosin acted as a competitive antagonist with a high affinity (pKB=10.1).

At the α1B‐adrenoceptor of the rat spleen and rabbit corpus cavernosum penis, tamsulosin again acted as a competitive antagonist but with a significantly lower affinity (pKB=8.9–9.2).

Tamsulosin acted as an unsurmountable antagonist of the α1A‐adrenoceptor‐mediated responses of the rat and human vas deferens, reducing maximal responses to phenylephrine by 20% and 50%, respectively, at an antagonist concentration of 1 nm. Responses of depolarized (100 mm KCl) rat vas deferens preparations were unaffected by 10 nm tamsulosin but this concentration reduced maximal responses to 5‐hydroxytryptamine (5‐HT) in this tissue.

When longer antagonist incubation periods (60 min) were used, tamsulosin behaved as a competitive antagonist on the human prostate with a significantly higher affinity (pKB=10.0) than obtained at the α1B‐adrenoceptor.

The data demonstrate that tamsulosin is a high affinity antagonist at functional α1‐adrenoceptors with a selectivity α1Dα1A>α1B. In some tissues the compound exhibits an additional unsurmountable antagonist action, the clinical significance of which is unknown.

British Journal of Pharmacology (1997) 120, 231–238; doi:10.1038/sj.bjp.0700907
Mechanisms involved in the effect of nitric oxide synthase inhibition on L‐arginine‐induced insulin secretion
Authors: Gross R. , Roye M. , Manteghetti M. , Broca C. , Hillaire‐Buys D. , Masiello P. , Ribes G.
Published: August 2006

A constitutive nitric oxide synthase (NOSc) pathway negatively controls L‐arginine‐stimulated insulin release by pancreatic β cells. We investigated the effect of glucose on this mechanism and whether it could be accounted for by nitric oxide production.

NOSc was inhibited by N∞‐nitro‐L‐arginine methyl ester (l‐NAME), and sodium nitroprusside (SNP) was used as a palliative NO donor to test whether the effects of L‐NAME resulted from decreased NO production.

In the rat isolated perfused pancreas, L‐NAME (5 mM) strongly potentiated L‐arginine (5 mM)‐induced insulin secretion at 5 mM glucose, but L‐arginine and L‐NAME exerted only additive effects at 8.3 mM glucose. At 11 mM glucose, L‐NAME significantly inhibited L‐arginine‐induced insulin secretion. Similar data were obtained in rat isolated islets.

At high concentrations (3 and 300 μm), SNP increased the potentiation of arginine‐induced insulin output by L‐NAME, but not at lower concentrations (3 or 30 nM).

L‐Arginine (5 mM) and L‐ornithine (5 mM) in the presence of 5 mM glucose induced monophasic β cell responses which were both significantly reduced by SNP at 3 nM but not at 30 nM; in contrast, the L‐ornithine effect was significantly increased by SNP at 3 μm.

Simultaneous treatment with L‐ornithine and L‐arginine provoked a biphasic insulin response.

At 5 mM glucose, L‐NAME (5 mM) did not affect the L‐ornithine secretory effect, but the amino acid strongly potentiated the alteration by L‐NAME of L‐arginine‐induced insulin secretion.

L‐Citrulline (5 mM) significantly reduced the second phase of the insulin response to L‐NAME (5 mM) + L‐arginine (5 mM) and to L‐NAME + L‐arginine + SNP 3 μm.

The intermediate in NO biosynthesis, NG‐hydroxy‐L‐arginine (150–300 μm) strongly counteracted the potentiation by L‐NAME of the secretory effect of L‐arginine at 5 mM glucose.

We conclude that the potentiation of L‐arginine‐induced insulin secretion resulting from the blockade of NOSc activity in the presence of a basal glucose concentration (1) is strongly modulated by higher glucose concentrations, (2) is not due to decreased NO production but (3) is probably accounted for by decreased levels of NG‐hydroxy‐L‐arginine or L‐citrulline, resulting in the attenuation of an inhibitory effect on arginase activity.
Pharmacological classification of α1‐adrenoceptors mediating contractions of rabbit isolated ear artery: comparison with rat isolated thoracic aorta
Authors: Fagura M S , Lydford S J , Dougall I G
Published: August 2006

The present study attempted to classify pharmacologically the α1‐adrenoceptor subtype(s) present in two isolated, vascular ring preparations, the rabbit ear artery and rat thoracic aorta.

In the ear artery, the agonist effects of phenylephrine were antagonized by 5‐methyl urapidil (pA2=7.90; Schild slope=0.85) and BMY 7378 (pA2=6.11; Schild slope=0.80) but not in a simple competitive manner. The shallow Schild slopes are consistent with the activation of a heterogeneous receptor population. Indeed the 5‐methyl urapidil data set could be fitted to a two‐receptor model yielding a high antagonist affinity (pKBH) estimate of 7.85 and a low affinity (pKBL) estimate of 6.03.

The effects of clonidine in the ear artery were competitively antagonised by 5‐methyl urapidil (pKB=7.91) and BMY 7378 (pKB=5.53). These data are consistent with contractions to clonidine being mediated by a single receptor subtype.

In the aorta, the effects of phenylephrine were antagonized by 5‐methyl urapidil (pA2=7.95; Schild slope=1.11) and BMY 7378 (pA2=9.08; Schild slope=0.73). Neither data set was consistent with a simple competitive interaction. The BMY 7378 data suggested again, that phenylephrine was acting at a heterogeneous receptor population. Subsequent analysis by the two‐receptor model yielded a high affinity (pKBH) estimate of 8.95 and a low affinity (pKBL) estimate of 7.00.

The alkylating agent, chloroethylclonidine (CEC) elicited concentration‐dependent contractions in the ear artery with a potency (p[A]50) of 5.57. Pretreatment of this tissue with CEC (5 μm, 30 min incubation) had no effect on subsequent responses to phenylephrine. In contrast, in the aorta, CEC demonstrated no agonism but pretreatment with this agent (5 μm, 15 min incubation) caused a rightward shift and depression of subsequent phenylephrine concentration‐effect curves.

The affinity of clonidine in the rabbit ear artery (pKA=6.17) was found to be significantly different to its affinity in the rat thoracic aorta (pKA=7.12) suggesting that this agonist activates different α1‐adrenoceptor subtypes in the two tissues.

These results suggest that heterogeneous populations of α1‐adrenoceptors are present in both tissues. In the ear artery, the profile of antagonist and agonist activity is most consistent with α1A‐adrenoceptors being the predominant receptor subtype. The second receptor population does not appear to correspond to any of the recognized α1‐adrenoceptor subtypes. In the aorta α1D‐adrenoceptors appear to predominate, with α1A‐adrenoceptors being the most likely candidate for the second receptor population.

British Journal of Pharmacology (1997) 120, 247–258; doi:10.1038/sj.bjp.0700917
Pharmacological characterization of the sulphonylurea receptor in rat isolated aorta
Authors: Löffler Cornelia , Quast Ulrich
Published: August 2006

The binding of the sulphonylurea [3H]‐glibenclamide, a blocker of adenosine 5′‐triphosphate (ATP)‐sensitive K+ channels (KATP channels), was studied in endothelium‐denuded rings from rat aorta.

[3H]‐glibenclamide labelled two classes of binding sites with KD values of 20 ± 5 nM and 32± 1 μM. The high affinity component, which comprised 17% of total binding at 1 nM [3H]‐glibenclamide, had an estimated binding capacity of 150 fmol mg3 wet weight.

Other sulphonylureas such as glipizide and glibornuride and the sulphonylurea‐related carboxylate, AZ‐DF 265, inhibited high affinity [3H]‐glibenclamide binding with the potencies expected from their K+ channel activity. At very high concentrations, AZ‐DF 265 and glipizide started to interact also with the low affinity component of [3H]‐glibenclamide binding.

Openers of the ATP‐sensitive K+ channel belonging to different structural groups inhibited only the high affinity [3H]‐glibenclamide binding; the potencies in this assay were similar to those obtained in functional (i.e. vasorelaxation) studies.

High affinity [3H]‐glibenclamide binding was abolished by prolonged hypoxia combined with metabolic inhibition.

The data indicate that the high affinity component of [3H]‐glibenclamide binding mediates the block of the KATP channel by the sulphonylureas in rat aorta; hence, it represents the sulphonylurea receptor in this vessel. The pharmacological properties of this binding site resemble those of the binding site for the openers of the KATP channel; present evidence suggests that these two classes of sites are negatively allosterically coupled.
Identification of 5‐hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes
Authors: Hirst Warren D. , Price Gary W. , Rattray Marcus , Wilkin Graham P.
Published: August 2006

5‐Hydroxytryptamine (5‐HT) elicited a dose‐dependent stimulation of intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 ± 0.08 (mean ± s.e.mean).

In order to characterize the 5‐HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5‐carboxamidotryptamine (5‐CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5‐CT (pEC50 = 7.81 ±+ 0.09)>5‐methoxytryptamine (5‐MeOT) (pEC50 = 6.86 ±0.36) > 5‐HT (pEC50 = 6.68± 0.08). The following compounds, at concentrations up to 10 μm, did not affect basal cyclic AMP levels 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was meth‐iothepin (p Ki = 7.98 ±0.25)>mesulergine (p Ki = 7.58 ± 0.18)>ritanserin (p Ki = 7.20 ±0.24) >clozapine (p Ki = 7.03±0.19)>mianserin (p Ki= 6.41 ±0.19). The following compounds, at concentrations up to 10 μM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5‐HT7 receptor subtype‐mediated effects.

The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 ± 75% and 572 ± 63% of basal levels for 5‐CT and 5‐HT, respectively) compared to brainstem (274 ± 51% and 318 ± 46%, respectively) and colliculus astrocytes (244 ± 15% and 301±24%, respectively). No significant differences in pEC50 (for either 5‐HT or 5‐CT) values were observed.

Reverse transcriptase‐polymerase chain reaction (RT‐PCR) with primers specific for the 5‐HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5‐HT6 receptor also amplified a cDNA fragment from the same samples.

From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5‐HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally‐dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5‐HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5‐HT6 and 5‐HT7 receptor messenger RNA.
Furosemide interactions with brain GABAA receptors
Authors: Korpi Esa R , Lüddens Hartmut
Published: August 2006

The loop diuretic furosemide is known to antagonize the function of γ‐aminobutyric acid type A (GABAA) receptors. The purpose of the present study was to examine the direct interaction of furosemide with the GABAA receptors by autoradiography and ligand binding studies with native rat and human receptors and with recombinant receptors composed of rat subunits.

Autoradiography with [35S]‐t‐butylbicyclophosphorothionate ([35S]‐TBPS) as a ligand indicated that furosemide (0.1–1 mm) reversed the 5 μm GABA‐induced inhibition of binding only in the cerebellar granule cell layer of rat brain sections. In all other regions studied, notably also in the hippocampal and thalamic areas, furosemide failed to antagonize GABA. Furosemide 1 mm decreased [35S]‐TBPS binding only in a limited number of brain regions, but facilitation of the GABA‐inhibition of the binding was much more widespread.

In well‐washed rat cerebellar, but not cerebrocortical, membranes, furosemide enhanced the [35S]‐TBPS binding over basal level in the absence of added GABA. The GABAA antagonist, SR 95531, and the convulsant, Ro 5–4864, blocked this furosemide‐induced increase. Both interactions with the furosemide enhancement are likely to be allosteric, since furosemide affected the binding of [3H]‐SR 95531 and [3H]‐Ro 5–4864 identically in the cerebellar and cerebrocortical membranes. Maximal GABA‐antagonism induced by furosemide in cerebellar membranes was further increased by SR 95531 but not by Ro 5–4864, indicating additive antagonism only for SR 95531. In human cerebellar receptors, only GABA antagonism by furosemide, but not the enhancement without added GABA, was observed.

In recombinant GABAA receptors, furosemide antagonism of GABA‐inhibition of [35S]‐TBPS binding depended only on the presence of α6 and β2/3 subunits, irrespective of the presence or absence of γ2 or δ subunits.

In α6β3γ2 receptors, clozapine reversed the enhancement of [35S]‐TBPS binding by furosemide in the absence of GABA. However, it failed to affect the GABA‐antagonism of furosemide, suggesting that the enhancement of basal binding and the GABA antagonism might represent two different allosteric actions of furosemide.

In conclusion, the present results indicate that furosemide is a subtype‐selective GABAA antagonist with a mode of action not shared by several other antagonists, which makes furosemide a unique compound for development of potential GABAA receptor subtype‐specific and ‐selective ligands.

British Journal of Pharmacology (1997) 120, 741–748; doi:10.1038/sj.bjp.0700922
Ischaemia‐induced loss or reversal of the effects of the class I antiarrhythmic drugs on vulnerability to fibrillation
Authors: Aupetit Jean‐François , Loufoua‐Moundanga Joseph , Faucon Georges , Timour Quadiri
Published: August 2006

In the last decade, a number of clinical observations have questioned the efficacy of certain class I antiarrhythmic drugs against ischaemia‐induced ventricular fibrillation. The effects of three drugs of this class, disopyramide (Ia), lignocaine (Ib) and flecainide (Ic) on the vulnerability to fibrillation during experimental ischaemia were investigated.

The study was carried out in anaesthetized, open‐chest pigs (n = 8 for each of the drugs, in addition to the control group, n = 6). Vulnerability to fibrillation was evaluated by measuring electrical fibrillation threshold (EFT) by means of stepwise increased intensity of wide (100 ms) diastolic impulses applied to the ischaemic tissue at a 180 beats min−1 rate. Monophasic action potential (MAP) duration and conduction time in the ischaemic region were also measured.

EFT determinations were performed before and during periods of ischaemia induced by complete occlusion of the left anterior descending coronary artery near its origin. Ischaemic periods of increasing duration (30, 60, 90, 120, 150 s) were induced to determine the electrophysiological changes, of EFT especially, leading to fibrillation.

In the absence of ischaemia, all three drugs, administered by intravenous route (1 mg kg−1 plus 0.04 mg kg−1 min−1) increased EFT to a similar extent (from approximately 7 to 10 mA), despite a 25% prolongation of conduction time.

During ischaemia, none of the drugs prevented the fall in EFT towards 0 mA, resulting in spontaneous fibrillation. After 30 s of ischaemia, they no longer had any capacity for raising EFT and, after 60, 90 and 120 s of ischaemia, the decrease in EFT was exacerbated. This accelerated reduction in EFT shortened the time to onset of fibrillation (after 120 s of ischaemia, 62.5% of fibrillations with fiecainide instead of 12.5 under control conditions, 75% instead of 25 with lignocaine and 50% instead of 25 with disopyramide). The reduction in MAP duration due to ischaemia was also significantly accelerated (at 60 s, 178 ± 5 ms instead of 192 ± 4 with fiecainide, 175 ± 3 ms instead of 194 ± 5 with lignocaine and 180 ± 5 ms instead of 196 ± 3 with disopyramide) and the slowing of conduction was made worse (prolongation of conduction time by 70% instead of 50).

In conclusion, the antifibrillatory properties normally manifested by these drugs are first suppressed, then inverted by ischaemia, depending on oxygen debt varying with severity and duration of ischaemia.
Evidence that mechanisms dependent and independent of nitric oxide mediate endothelium‐dependent relaxation to bradykinin in human small resistance‐like coronary arteries
Authors: Kemp B K , Cocks T M
Published: August 2006

The effects of the nitric oxide (NO) synthase inhibitor, NG‐nitro‐l‐arginine (l‐NOARG), the NO scavenger, oxyhaemoglobin (HbO) and high extracellular K+ upon endothelium‐dependent relaxation to bradykinin were investigated in human isolated small coronary arteries.

Endothelium‐dependent relaxations to bradykinin were compared in vessels contracted to ∼50% of their maximum contraction to 124 mm KCl Krebs solution, regardless of treatments, with the thromboxane A2 mimetic, U46619 and acetylcholine. All relaxations were expressed as percentage reversal of the initial level of active force.

l‐NOARG (100 μm) caused a small but significant, 12% (P<0.01), decrease in the maximum relaxation (Rmax: 91.5±5.4%) to bradykinin but did not significantly affect the sensitivity (pEC50: 8.08±0.17). Increasing the concentration of l‐NOARG to 300 μm had no further effect on the pEC50 or Rmax to bradykinin. HbO (20 μm) and a combination of HbO (20 μm) and l‐NOARG (100 μm) reduced Rmax to bradykinin by 58% (P<0.05) and 54% (P<0.05), respectively. HbO (20 μm) and l‐NOARG (100 μm, combined but not HbO (20 μm) alone, caused a significant 11 fold (P<0.05) decrease in sensitivitiy to bradykinin. HbO (20 μm) decreased the sensitivity to the endothelium‐independent NO donor, S‐nitroso‐N‐acetylpenicillamine (SNAP), approximately 17 fold (P<0.05).

Raising the extracellular concentration of K+ isotonically to 30 mm, reduced the Rmax to bradykinin from 96.6±3.1% to 43.9±10.1% (P<0.01) with no significant change in sensitivity. A combination of HbO, l‐NOARG and high K+ (30 mm) abolished the response to bradykinin. High K+ did not change either the sensitivity or maximum relaxation to SNAP.

In conclusion, l‐NOARG does not completely inhibit endothelial cell NO synthesis in human isolated small coronary arteries. By comparison, HbO appeared to block all the effects of NO in this tissue and revealed that most of the relaxation to bradykinin was due to NO. The non‐NO ‐dependent relaxation to bradykinin in the human isolated small coronary arteries appeared to be mediated by a K+‐sensitive vasodilator mechanism, possibly endothelium‐derived hyperpolarizing factor (EDHF).

British Journal of Pharmacology (1997) 120, 757–762; doi:10.1038/sj.bjp.0700928
Alterations of the benzodiazepine site of rat α6β2γ2‐GABAA receptor by replacement of several divergent amino‐terminal regions with the αl counterparts
Authors: Im Wha Bin , Pregenzer Jeffrey F. , Binder Jay A. , Alberts Glen L. , Im Haesook K.
Published: August 2006

The benzodiazepine site of the α6β2γ2 subtype of γ‐aminobutyric acidA (GABAA) receptors is distinguishable from that of the α1β2γ2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15–1788, which behaves as an antagonist in the α1β2γ2 subtype.

The point mutation of Arg 100 of the α6 subunit to histidine (the corresponding residue in α1) has been shown to enable the α6β2γ2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15–1788 to enhance GABA‐induced Cl− currents.

Here we identified the segment of P161 to L187 of α6 to contain the functional region responsible for the agonistic action of Ro 15–1788. Its replacement with the corresponding α1 sequence abolished the ability of Ro 15–1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U‐92330 and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others.

In addition we have learned that several residues in the N‐terminal region of α6, such as R100, V142 and N143, have the ability to influence GABA‐dependent Cl current induction probably by allosterically modulating low affinity GABA sites.
Hypoxic dilatation of porcine small coronary arteries: role of endothelium and KATP‐channels
Authors: Liu Qiang , Flavahan Nicholas A.
Published: August 2006

The aim of the present study was to determine the cellular mechanims and potential mediators involved in hypoxic dilatation of porcine small coronary arteries.

Small coronary arteries were isolated from a branch of the left anterior descending artery of porcine hearts, cannulated with glass micropipettes and studied in a perfusion myograph system. At a transmural pressure of 40 mmHg, the arteries had an internal diameter of 167.8 ± 6.6 μm (n = 37).

In arteries contracted with acetylcholine (ACh), hypoxia (0% O2, 30 min) caused dilatation (86.9± 6.7% relaxation, n = 6) in vessels with endothelium but constriction in endothelium‐denuded vessels.

Hypoxic vasodilatation occurring in arteries with endothelium was abolished by the KAtp channel inhibitor, glibenclamide (0.44 μM), but was not affected by inhibition of nitric oxide synthase (l‐NAME, 44 μM) or cyclo‐oxygenase (indomethacin, 4.4 μM).

Bradykinin evoked endothelium‐dependent relaxation that was inhibited by l‐NAME (44 μM) but not glibenclamide 0.44 μM). Cromakalim (0.1–0.3μM), a KATP channel opener, caused relaxation that was inhibited by glibenclamide, but was not affected by l‐NAME (44 μm) and/or indomethacin (4.4 μm).

Endothelium‐removal inhibited vasodilatation evoked by cromakalim, but increased vasodilator responses to the NO donor, SIN‐1 (10−8 to 10−5 m).

These results indicate that hypoxia acted directly on vascular smooth muscle of small coronary arteries to cause contraction. However, this effect was overwhelmed by endothelium‐dependent relaxation in response to hypoxia. This relaxation was most likely mediated by release of an endothelium‐derived factor, distinct from nitric oxide or prostacyclin, that activated smooth muscle KATP‐channels.
Pharmacological and biochemical evidence for the simultaneous expression of CCKB/gastrin and CCKA receptors in the pig pancreas
Authors: Philippe C. , Lhoste E.F. , Dufresne M. , Moroder L. , Corring T. , Fourmy D.
Published: August 2006

In the pig, the secretory response of the pancreas is not inhibited by the antagonist MK329 suggesting that cholecystokininA (CCKA) receptors are not involved.

Membranes were isolated from the pancreas of 6 Large White pigs to characterize their CCK receptors.

The binding of [M125I]‐BH‐[Thr, Nle]CCK‐9 was dependent on pH, maximal after a 90 min incubation period, saturable and reversible. Saturation analysis of the binding demonstrated a single class of high affinity sites (Kd = 0.22 ± 0.02 nM) and a binding capacity, Bmax= 110.64±12.50 fmol mg−1 protein.

Competition binding by agonists and antagonists of CCKA and CCKB/gastrin receptors demonstrated the presence of two distinct binding components, sites presenting a high affinity for [Thr, Nle]CCK‐9, gastrin, PD 135158, L‐365,260 and a low affinity for MK329, SR 27897, and sites presenting a high affinity for [Thr, Nle]CCK‐9, MK329, SR 27897 and a low affinity for gastrin, PD 135158, L‐365,260.

These pharmacological data demonstrate the presence of both CCKA and CCKB/gastrin receptors in the pig pancreas, the latter being predominant.

Two distinct membrane proteins (50 and 85–100 kDa, respectively) display pharmacological features of CCKB/gastrin and CCKA receptors.

In pigs, as in calves and humans, CCKB/gastrin receptors are predominant in the pancreas.
Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone
Authors: Coceani F. , Kelsey L. , Seidlitz E. , Marks G.S. , McLaughlin B.E. , Vreman H.J. , Stevenson D.K. , Rabinovitch M. , Ackerley C.
Published: August 2006

We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction controlling the formation of endothelin‐1 (ET‐1). In the present study, we have examined whether CO is formed naturally in the vessel.

The CO‐forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO‐2) and inducible (HO‐1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO‐1 was localized to endothelial and muscle cells, while HO‐2 was found only in muscle cells. Inside the muscle cells, HO‐1 and HO‐2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO‐1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma.

CO was formed enzymatically from added substrate (hemin, 50 μm) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 μM).

ZnPP (10 μM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin‐treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2).

ZnPP (10 μm) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 μM)‐contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged.

We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.
Regulation of citrulline recycling in nitric oxide‐dependent neurotransmission in the murine proximal colon
Authors: Shuttleworth C.W.R. , Conlon S.B. , Sanders K.M.
Published: August 2006

We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis.

Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 μm). Electrical field stimulation (EFS, 0.3–20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component).

L‐Nitro‐arginine‐methyl ester (l‐NAME, 100 μM) selectively abolished the slow component of i.j.ps. The effects of L‐NAME were reversed by L‐arginine (0.2–2 mM) but not by D‐arginine (2 mM). Sodium nitroprusside (an NO donor, 1 μM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps.

L‐Citrulline (0.2 mM) also reversed the effects of L‐NAME, and this action was maintained during sustained exposures to L‐citrulline (0.2 mM). This may reflect intraneuronal recycling of L‐citrulline to L‐arginine.

Higher concentrations of L‐citrulline (e.g. 2 mM) had time‐dependent effects. Brief exposure (15 min) reversed the effects of L‐NAME, but during longer exposures (30 min) the effects of L‐NAME gradually returned. In the continued presence of L‐citrulline, L‐arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L‐citrulline, the ability to generate arginine from citrulline was reduced.

Aspartate (2 mM) had no effect on i.j.ps, the effects of L‐NAME, or the actions of L‐citrulline in the presence of L‐NAME. L‐Citrulline (0.2–2 mM) alone had no effect on i.j.ps under control conditions.

S‐methyl‐L‐thiocitrulline (10 μm), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L‐arginine (2 mM), but not by L‐citrulline (2 mM).

These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L‐citrulline to L‐arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L‐citrulline are elevated.
Involvement of tyrosine phosphorylation in endothelin‐1‐induced calcium‐sensitization in rat small mesenteric arteries
Authors: Ohanian Jacqueline , Ohanian Vasken , Shaw Linda , Bruce Christopher , Heagerty Anthony M.
Published: August 2006

We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist.

Endothelin‐1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration‐dependent (1‐100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α‐toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin‐1‐induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin Al.

In intact small arteries tyrphostin A23 inhibited the force developed to endothelin‐1 at all concentrations studied; at higher concentrations (10 and 100 nM) the profile of contraction was altered from a sustained to a transient response. Tyrphostin Al inhibited the contractile response to endothelin‐1 at all concentrations except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endothelin‐1 (100 nM) in the absence of extracellular calcium.

In rat α‐toxin permeabilized mesenteric arteries endothelin‐1 caused a concentration‐dependent increase in force in the presence of 10 μM GTP and low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin Al did not affect endothelin‐1‐induced calcium sensitization of force.

We conclude that increased tyrosine phosphorylation is important in the contractile response induced by endothelin‐1 in intact small mesenteric arteries. Furthermore our data implicate activation of this signalling pathway in the tonic phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.
Characterization of the contraction to 5‐HT in the canine colon longitudinal muscle
Authors: Prins Nicolaas H. , Briejer Michel R. , Schuurkes Jan A.J.
Published: August 2006

Although conscious dogs have often been used for colonic motility studies with 5‐hydroxytryptamine (5‐HT), the effects of 5‐HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5‐HT of the canine isolated colon longitudinal muscle.

Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5‐HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5‐HT re‐uptake, monoamine oxidase or blockade of α‐adrenoceptors. Tetrodotoxin (0.3 μm) did not affect the responses to 5‐HT, suggesting that smooth muscle 5‐HT receptors are involved. The selective 5‐HT4 receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5‐HT and therefore it was included in the organ bath solution in all further experiments. The 5‐HT1 and 5‐HT2 receptor antagonist methysergide (0.1 μm) depressed the curve to 5‐HT, but the selective 5‐HT3 receptor antagonist granisetron (0.3 μm) had no effect.

Besides 5‐HT, α‐methyl‐5‐HT (α‐Me‐5‐HT), 5‐methoxytryptamine (5‐MeOT), 2‐methyl‐5‐HT (2‐Me‐5‐HT) and 5‐carboxamidotryptamine (5‐CT) also induced contractions, with the following rank order of potency (pEC50 values in parentheses): 5‐HT (6.9) = α‐methyl‐5‐HT (6.9) >2‐Me‐5‐HT (5.8) = 5‐MeOT (5.7) = 5‐CT (5.6), indicative of 5‐HT2 receptor involvement. α‐Me‐5‐HT produced a bell‐shaped curve, which was not affected by α‐adrenoceptor blockade. 5‐HT, 5‐MeOT, 2‐Me‐5‐HT and 5‐CT produced a monophasic concentration‐response curve, consistent with an interaction with a single receptor site. 8‐Hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.

The selective 5‐HT2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5‐HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with p Kb values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μm) shifted the curve to 5‐MeOT rightward yielding an apparent pA2 of 7.1. Other antagonists at 5‐HT2A receptors also surmountably inhibited the contractions to 5‐HT (apparent pA2 value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo‐irreversible’ antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5‐HT2A receptors.

It was concluded that 5‐HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5‐HT2A receptors. The presence of inhibitory 5‐HT4 receptors cannot be ruled out.
The effect of big endothelin‐1 in the proximal tubule of the rat kidney
Authors: Beara‐Lasić Lada , Knotek Mladen , Čejvan Kenan , Jakšić Ozren , Lasić Zoran , Skorić Boško , Brkljačić Vera , Banfić Hrvoje
Published: August 2006

An obligatory step in the biosynthesis of endothelin‐1 (ET‐1) is the conversion of its inactive precursor, big ET‐1, into the mature form by the action of specific, phosphoramidon‐sensitive, endothelin converting enzyme(s) (ECE). Disparate effects of big ET‐1 and ET‐1 on renal tubule function suggest that big ET‐1 might directly influence renal tubule function. Therefore, the role of the enzymatic conversion of big ET‐1 into ET‐1 in eliciting the functional response (generation of 1,2‐diacylglycerol) to big ET‐1 was studied in the rat proximal tubules.

In renal cortical slices incubated with big ET‐1, pretreatment with phosphoramidon (an ECE inhibitor) reduced tissue immunoreactive ET‐1 to a level similar to that of cortical tissue not exposed to big ET‐1. This confirms the presence and effectiveness of ECE inhibition by phosphoramidon.

In freshly isolated proximal tubule cells, big ET‐1 stimulated the generation of 1,2‐diacylglycerol (DAG) in a time‐and dose‐dependent manner. Neither phosphoramidon nor chymostatin, a chymase inhibitor, influenced the generation of DAG evoked by big ET‐1.

Big ET‐1‐dependent synthesis of DAG was found in the brush‐border membrane. It was unaffected by BQ123, an ETA receptor antagonist, but was blocked by bosentan, an ETA B‐nonselective endothelin receptor antagonist.

These results suggest that the proximal tubule is a site for the direct effect of big ET‐1 in the rat kidney. The effect of big ET‐1 is confined to the brush‐border membrane of the proximal tubule, which may be the site of big ET‐1‐sensitive receptors.
Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies
Authors: Jane Dixon C , Bowler Wayne B , Walsh Catherine A , Gallagher James A
Published: August 2006

Human osteoblasts responded to the application of extracellular nucleotides, acting at P2‐receptors, with increases in cytosolic free calcium concentration ([Ca2+]i).

In populations of human osteoblasts, adenosine 5′‐diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5′‐triphosphate (ATP).

ATP and uridine 5′‐triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested.

However, from the results of single cell studies with ADP (and 2‐methylthioATP (2‐meSATP)) two groups of cells were delineated: one group responded to ADP (or 2‐meSATP) with a rise in [Ca2+]i indistinguishable from that evoked by ATP; whereas the second group failed completely to respond to ADP (or 2‐meSATP).

Therefore heterogeneity of receptor expression exists within this population of human osteoblasts. The limited distribution of the ADP‐responsive receptor underlies the small response to ADP, compared with ATP, recorded in populations of human osteoblasts. This heterogeneity may reflect differences in the differentiation status of individual cells.

British Journal of Pharmacology (1997) 120, 777–780; doi:10.1038/sj.bjp.0700961
Induction of cyclo‐oxygenase‐2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type
Authors: Belvisi Maria G , Saunders Michael A , Haddad El‐Bdaoui , Hirst Stuart J , Yacoub Magdi H , Barnes Peter J , Mitchell Jane A
Published: August 2006

Cyclo‐oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H2 (PGH2) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX‐1 is expressed constitutively, whereas COX‐2 is expressed in response to pro‐inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined.

COX‐1 and COX‐2 protein were measured by Western blot analysis with specific antibodies for COX‐1 and COX‐2. COX‐2 mRNA levels were assessed by Northern blot analysis by use of a COX‐2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE2, thromboxane B2 or 6‐ketoPGF1α by radioimmunoassay.

Under control culture conditions HASM cells expressed COX‐1, but not COX‐2, protein. However, a mixture of cytokines (interleukin‐1β (IL‐1β), tumour necrosis factor α (TNFα) and interferon γ (IFNγ) each at 10 ng ml−1) induced COX‐2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 μm; added 30 min before the cytokines). Furthermore, COX‐2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 μm) and cyclohexamide (10 μg ml−1; added 30 min before the cytokines).

Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL‐1β, TNFα, IFNγ each at 10 ng ml−1 for 24 h) caused the accumulation of PGE2 and 6‐keto‐PGF1α.

In experiments where COX‐2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL‐1β in combination with TNFα caused a similar release of PGE2 to that when the three cytokines were given in combination.

In other experiments designed to measure COX‐2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 μm for 15 min) after which PGE2 was measured. IL‐1β and TNFα increased COX‐2 activity and an additional small increase was produced by the three cytokines in combination.

These findings suggest that the increased expression of COX‐2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro‐inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma.

British Journal of Pharmacology (1997) 120, 910–916; doi:10.1038/sj.bjp.0700963
Inhibition of delayed rectifier K+ channels by phenytoin in rat neuroblastoma cells
Authors: Nobile Mario , Vercellino Paolo
Published: August 2006

The action of the anticonvulsant drug phenytoin on K+ currents was investigated in neuroblastoma cells by whole‐cell voltage‐clamp recording.

Neuroblastoma cells expressed an outward K+ current with a voltage‐and time‐dependence which resembled the delayed‐rectifier K+ current found in other cells. When added to the standard external solution at concentrations ranging between 1 and 200 μM, phenytoin reduced the current (n = 65). Inhibition was concentration‐dependent with a half‐maximal inhibitory concentration of 30.9 + 0.8 μM.

The K+ current inhibition by phenytoin was voltage‐dependent with block by phenytoin being relieved by depolarization.

The times taken to reach steady‐state inhibition and complete recovery from inhibition were about 20 s. Neither the activation and inactivation rates of the K+ current nor the K+ channel availability were significantly altered by the blocking drug. A use‐dependent block was observed at phenytoin concentrations of 10, 25 and 50 μM.

These results suggest that phenytoin affects K+ currents and that this effect might lead to a reduction in neuronal excitability.