bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial dysfunction and metabolic control in streptozotocin‐induced diabetic rats
Authors: Rodríguez‐Mañas Leocadio , Angulo Javier , Peiró Concepción , Llergo José L. , Sánchez‐Ferrer Alberto , López‐Dóriga Pedro , Sánchez‐Ferrer Carlos F.
Published: January 2005

The aim of this work was to study the influence of the metabolic control, estimated by the levels of glycosylated haemoglobin in total blood samples (HbA1c), in developing vascular endothelial dysfunction in streptozotocin‐induced diabetic rats. Four groups of animals with different levels of insulin treatment were established, by determining HbA1c values in 5.5 to 7.4%, 7.5 to 9.4%, 9.5 to 12% and >12%, respectively.

The parameters analysed were: (1) the endothelium‐dependent relaxations to acetylcholine (ACh) in isolated aorta and mesenteric microvessels; (2) the vasodilator responses to exogenous nitric oxide (NO) in aorta; and (3) the existence of oxidative stress by studying the influence of the free radical scavenger superoxide dismutase (SOD) on the vasodilator responses to both ACh and NO.

In both isolated aortic segments and mesenteric microvessels, the endothelium‐mediated concentration‐dependent relaxant responses elicited by ACh were significantly decreased when the vessels were obtained from diabetic animals but only with HbA1c values higher than 7.5%. There was a high correlation between HbA1c levels and the impairment of ACh‐induced relaxations, measured by pD2 values.

The concentration‐dependent vasorelaxant responses to NO in endothelium‐denuded aortic segments were significantly reduced only in vessels from diabetic animals with HbA1c values higher than 7.5%. Again, a very high correlation was found between the HbA1c values and pD2 for NO‐evoked responses.

In the presence of SOD, the responses to ACh or NO were only increased in the segments from diabetic rats with HbA1c levels higher than 7.5%, but not in those from non‐diabetic or diabetic rats with a good metabolic control (HbA1c levels <7.5%).

These results suggest the existence of: (1) a close relation between the degree of endothelial dysfunction and the metabolic control of diabetes, estimated by the levels of HbA1c; and (2) an increased production of superoxide anions in the vascular wall of the diabetic rats, which is also related to the metabolic control of the disease.

British Journal of Pharmacology (1998) 123, 1495–1502; doi:10.1038/sj.bjp.0701749
The role of central melanocortin receptors in the activation of the hypothalamus‐pituitary‐adrenal‐axis and the induction of excessive grooming
Authors: Von Frijtag Josefien C. , Croiset Gerda , Hendrik Gispen Willem , Adan Roger A. H. , Wiegant Victor M.
Published: January 2005

In accord with previous studies intracerebroventricular (i.c.v.) injections of ACTH1‐24 (1 μg) induced a display of excessive grooming, and increased the plasma concentrations of ACTH and corticosterone. Pituitary‐adrenal activation was blocked by pretreatment with dexamethasone, indicating that the effect of the (i.c.v.) injected peptide was not caused by a peripheral effect on the adrenal cortex.

Doses of 1 and 3 μg of a non‐selective melanocortin‐3/4‐receptor antagonist (SHU 9119), or of 5 and 10 μg of a selective melanocortin‐4‐receptor antagonist ([D‐Arg8]ACTH4‐10), coadministered (i.c.v.) with 1 μg ACTH1‐24, inhibited the ACTH1‐24‐induced activation of the hypothalamus‐pituitary‐adrenal‐axis and excessive grooming.

In addition, several doses of the selective melanocortin‐3‐receptor agonist Lys‐γ2‐MSH were centrally administered, but neither neuroendocrine, nor excessive grooming responses were observed.

These results imply that the melanocortin‐4‐receptor, and not the melanocortin‐3‐receptor, is involved in the ACTH1‐24‐induced rise in plasma levels of ACTH and corticosterone, and excessive grooming.

British Journal of Pharmacology (1998) 123, 1503–1508; doi:10.1038/sj.bjp.0701750
Acetylcholine modulation of high‐voltage‐activated calcium channels in the neurones acutely dissociated from rat paratracheal ganglia
Authors: Murai Yoshinaka , Ishibashi Hitoshi , Akaike Norio , Ito Yushi
Published: January 2005

The modulation of high‐voltage‐activated (HVA) Ca2+ channels by acetylcholine (ACh) was studied in the paratracheal ganglion cells acutely dissociated from 2‐week‐old Wistar rats by use of the nystatin perforated patch recording configuration under voltage‐clamp conditions.

ACh inhibited the HVA Ca2+ currents in a concentration‐ and voltage‐dependent manner.

The inhibition was mimicked by a muscarinic agonist, oxotremorine. Pirenzepine and methoctramine produced parallel shifts to the right in the ACh concentration‐response curves. Schild analysis of the ACh concentration‐ratios yield pA2 values for pirenzepine and methoctramine of 6.85 and 8.57, respectively, suggesting the involvement of an M2 receptor.

Nifedipine, ω‐conotoxin‐GVIA and ω‐conotoxin‐MVIIC reduced the HVA ICa by 16.8, 59.2 and 6.3%, respectively. A current insensitive to all of these Ca2+ antagonists, namely ‘R‐type’, was also observed. The results indicated the existence of L‐, N‐, P/Q‐, and R‐type Ca2+ channels.

The ACh‐sensitive current component was markedly reduced in the presence of ω‐conotoxin‐GVIA, but not with both nifedipine and ω‐conotoxin‐MVIIC. ACh also inhibited the R‐type HVA ICa remaining in saturating concentrations of nifedipine, ω‐conotoxin‐GVIA and ω‐conotoxin‐MVIIC.

The inhibitory effect of ACh was prevented by pretreatment with pertussis toxin.

It was concluded that ACh selectively reduces both the N‐ and R‐type Ca2+ channels, by activating pertussis toxin sensitive G‐protein through the M2 muscarinic receptor in paratracheal ganglion cells.

British Journal of Pharmacology (1998) 123, 1441–1449; doi:10.1038/sj.bjp.0701753
Evidence for the existence of [3H]‐trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore
Authors: Morin Didier , Elimadi Aziz , Sapena Rosa , Crevat Aimé , Carrupt Pierre‐Alain , Testa Bernard , Tillement Jean‐Paul
Published: January 2005

Trimetazidine is an anti‐ischaemic drug effective in different experimental models but its mechanism of action is not fully understood. Data indicate that mitochondria could be the main target of this drug. The aim of this work was to investigate the binding of [3H]‐trimetazidine on a purified preparation of rat liver mitochondria.

[3H]‐trimetazidine binds to two populations of mitochondrial binding sites with Kd values of 0.96 and 84 μm. The total concentration of binding sites is 113 pmol mg−1 protein. Trimetazidine binding sites are differently distributed. The high‐affinity ones are located on the outer membranes and represent only a small part (4%) of total binding sites, whereas the low‐affinity ones are located on the inner membranes and are more abundant (96%) with a Bmax=108 pmol mg−1 protein.

Drug displacement studies with pharmacological markers for different mitochondrial targets showed that [3H]‐trimetazidine binding sites are different from previously described mitochondrial sites.

The possible involvement of [3H]‐trimetazidine binding sites in the regulation of the mitochondrial permeability transition pore (MTP), a voltage‐dependent channel sensitive to cyclosporin A, was investigated with mitochondrial swelling experiments. Trimetazidine inhibited the mitochondrial swelling induced by Ca2+ plus tert‐butylhydroperoxide (t‐BH). This effect was concentration‐dependent with an IC50 value of 200 μm.

Assuming that trimetazidine effectiveness may be related to its structure as an amphiphilic cation, we compared it with other compounds exhibiting the same chemical characteristic both for their ability to inhibit MTP opening and to displace [3H]‐trimetazidine bound to mitochondria. Selected compounds were drugs known to interact with various biological membranes.

A strong correlation between swelling inhibition potency and low‐affinity [3H]‐trimetazidine binding sites was observed: r=0.907 (n=24; P<0.001).

These data suggest that mitochondrial sites labelled with [3H]‐trimetazidine may be involved in the MTP inhibiton.

British Journal of Pharmacology (1998) 123, 1385–1394; doi:10.1038/sj.bjp.0701755
Binding of KATP channel modulators in rat cardiac membranes
Authors: Löffler‐Walz Cornelia , Quast Ulrich
Published: January 2005

The binding of [3H]‐P1075, a potent opener of adenosine‐5′‐triphosphate‐(ATP)‐sensitive K+ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.

Binding required MgATP. In the presence of an ATP‐regenerating system, MgATP supported [3H]‐P1075 binding with an EC50 value of 100 μm and a Hill coefficient of 1.4.

In saturation experiments [3H]‐P1075 binding was homogeneous with a KD value of 6±1 nm and a binding capacity (Bmax) of 33±3 fmol mg−1 protein.

Upon addition of an excess of unlabelled P1075, the [3H]‐P1075‐receptor complex dissociated in a mono‐exponential manner with a dissociation rate constant of 0.13±0.01 min−1. If a bi‐molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nm−1 min−1. From the kinetic experiments the KD value was calculated as 4.7±0.6 nm.

Openers of the ATP‐sensitive K+ channel belonging to different structural classes inhibited specific [3H]‐P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.

Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea‐related carboxylate, AZ‐DF 265, inhibited [3H]‐P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC50 value of glibenclamide being ∼amp;90 nm; affinities for the low affinity component were in the μm concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC50 value of 6 μm, a maximum inhibition of 94% and a Hill coefficient of 1.5.

It is concluded that binding studies with [3H]‐P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP‐regenerating system. The pharmacological profile of the [3H]‐P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR2A) and vascular smooth muscle cells (SUR2B), differs from that expected for SUR2A and SUR2B.

British Journal of Pharmacology (1998) 123, 1395–1402; doi:10.1038/sj.bjp.0701756
The adrenergic, cholinergic and NANC nerve‐mediated contractions of the female rabbit bladder neck and proximal, medial and distal urethra
Authors: Deplanne Valérie , Palea Stéfano , Angel Itzchak
Published: January 2005

The nerve‐mediated contraction of the female rabbit bladder neck and different portions of the urethra (proximal, medial and distal) was studied in vitro by electrical stimulation (50 V, 30 Hz, 0.05 ms width, trains of 5 s every 5 min) by use of a superfusion system.

The amplitude (Emax) and the duration (Dmax) of the stimulated contraction were studied in the four tissues. The Emax value was significantly higher in distal urethra (2.07±0.15 g) compared to the bladder neck (1.08±0.10 g), proximal urethra (0.73±0.07 g) and medial urethra (0.87±0.07 g). In contrast, the Dmax value appeared slightly but significantly lower (P<0.05) in distal urethra (68.5±2.3 s) than in bladder neck (76.7±6.0 s), proximal urethra (84.5±5.0 s) and medial urethra (81.3±3.5 s).

Cocaine (1 μM) significantly increased the basal Emax values in medial and distal urethra and the basal Dmax values in the four tissues.

Prazosin (1 μM) significantly reduced Emax value in proximal, medial and distal urethra and Dmax value in bladder neck and proximal urethra. Atropine (1 μM) also significantly reduced Emax values in bladder neck and proximal urethra and reduced Dmax value in bladder neck, but not in other tissues. Yohimbine (0.1 μM) was devoid of effect in the four tissues.

The association of prazosin (1 μM) and atropine (1 μM) did not modify the Emax and the Dmax values of the electrically‐induced contractions, except in proximal urethra and in bladder neck where an additive inhibitory effect (on Emax only) was observed compared to prazosin and atropine alone.

The residual contractile response after combined treatment with prazosin and atropine was significantly diminished by tetrodotoxin (TTX; 1 μM) but not completely abolished. These NANC contractions were insensitive to P2X‐purinoceptor desensitization by continuous tissue perfusion with α,β‐methylene ATP (30 μM).

These results demonstrate that bladder neck and proximal urethra are mainly innervated by the parasympathetic nervous system, whereas medial and distal urethras are to a greater extent under the control of the sympathetic innervation. The residual responses, insensitive to prazosin and atropine, may indicate a NANC innervation in the four tissues. However, the nature of the NANC neurotransmitter remains to be identified.

British Journal of Pharmacology (1998) 123, 1517–1524; doi:10.1038/sj.bjp.0701757
[3H]‐SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes
Authors: Varani Katia , Gessi Stefania , Dionisotti Silvio , Ongini Ennio , Andrea Borea Pier
Published: January 2005

The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo[4,3‐e]‐1,2,4 triazolo[1,5‐c]pyrimidine, ([3H]‐SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.

Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg−1 protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]‐SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.

Thermodynamic data indicated that [3H]‐SCH 58261 binding to human neutrophils is entropy and enthalpy‐driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors.

It was concluded that in human neutrophil membranes, [3H]‐SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]‐SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

British Journal of Pharmacology (1998) 123, 1723–1731; doi:10.1038/sj.bjp.0701758
Effect of melatonin in the rat tail artery: role of K+ channels and endothelial factors
Authors: Geary Greg G. , Duckles Sue P. , Krause Diana N.
Published: January 2005

The role of endothelial factors and potassium channels in the action of the pineal hormone melatonin to potentiate vasoconstrictor responses was investigated in the isolated perfused tail artery of the rat.

Melatonin (100 nM) potentiated contractile responses to both adrenergic nerve stimulation and α1‐adrenoceptor stimulation by phenylephrine. After removal of the endothelium, melatonin no longer caused potentiation.

The potentiating effect of melatonin was also lost when nitric oxide synthase was inhibited with L‐NAME (10 nM). Thus potentiating effects depend on the presence of nitric oxide released by the endothelium. However, melatonin did not affect relaxation responses to acetylcholine in endothelium‐intact arteries, nor did melatonin modulate relaxing responses to sodium nitroprusside in endothelium‐denuded arteries. While melatonin does not appear to modulate agonist‐induced release of nitric oxide nor its effect, melatonin may modulate nitric oxide production induced by flow and shear stress.

When the Ca2+‐activated K+ channel opener, NS 1619 (10 μM), was present, potentiating effects of melatonin were restored in endothelium‐denuded vessels. However, addition of the opener of ATP‐sensitive K+ channels, cromakalim (3 μM), did not have the same restorative effect. Furthermore, addition of a blocker of Ca2+‐activated K+ channels, tetraethylammonium (1 mM), significantly attenuated potentiating effects of melatonin. These findings support the hypothesis that melatonin inhibits the activity of large conductance Ca2+‐activated K+ channels to produce its potentiating effects.

Thus in the rat perfused tail artery, potentiation of constriction by melatonin depends on the activity of both endothelial factors and Ca2+‐activated K+ channels. Our findings suggest that melatonin inhibits endothelial K+ channels to decrease flow‐induced release of nitric oxide as well as block smooth muscle K+ channels to enhance vascular tone.

British Journal of Pharmacology (1998) 123, 1533–1540; doi:10.1038/sj.bjp.0701761
Effects of CGS 21680, a selective A2A adenosine receptor agonist, on cardiac output and vascular resistance in acute heart failure in the anaesthetized rat
Authors: Nekooeian Ali A. , Tabrizchi Reza
Published: January 2005

The effects of CGS 21680, a selective A2A adenosine receptor agonist, on cardiac output, blood pressure, mean circulatory filling pressure (Pmcf), arterial and venous resistances, heart rate and left ventricular end‐diastolic pressure were assessed in rats with acute heart failure by means of coronary artery occlusion.

Animals (n=6 in each group) were divided into five groups: group I, sham‐operated vehicle‐treated (0.9% saline; 0.018 mL min−1); groups II‐V, subject to coronary artery occlusion and treated with vehicle (0.9% saline; 0.018 ml min−1) and CGS 21680 (0.1, 0.3 and 1.0 μg kg−1 min−1), respectively. Haemodynamic measurements were taken one hour after completion of surgery, ninety minutes after coronary artery occlusion (except in group I), and fifteen minutes after infusion of saline or CGS 21680.

Baseline haemodynamic measurements before occlusion were found not to differ significantly between the different groups of animals. However, after occlusion, cardiac output, rate of rise in left ventricular pressure (+dP/dt) and blood pressure were significantly reduced when compared to corresponding values in sham‐operated animals. In addition, occlusion of the coronary artery resulted in a significant elevation in venous resistance, Pmcf and left ventricular end‐diastolic pressure as compared to corresponding values in sham‐operated animals.

Infusion with CGS 21680 at the highest dose significantly reduced blood pressure, arterial resistance and left ventricular end‐diastolic pressure when compared to occluded vehicle‐treated animals (group II). Administration of CGS 21680 at the highest dose also significantly increased cardiac output (28%) and heart rate (10%) in comparison to occluded vehicle‐treated animals. In addition, the highest dose of CGS 21680 significantly reduced Pmcf (9%) and venous resistance (62%) in comparison to occluded vehicle‐treated animals. Administration of CGS 21680 did not significantly affect +dP/dt when compared to occluded vehicle‐treated animals.

The results from the present investigation indicate that occlusion of the coronary artery in rats results in a state of heart failure characterized by reduced arterial pressure and cardiac output, and increased venous resistance, Pmcf and left ventricular end‐diastolic pressure. Administration of CGS 21680 to animals with acute heart failure resulted in increased cardiac output which was due to reduced venous resistance, as well as increased heart rate.

British Journal of Pharmacology (1998) 123, 1666–1672; doi:10.1038/sj.bjp.0701764
Agonistic properties of alniditan, sumatriptan and dihydroergotamine on human 5‐HT1B and 5‐HT1D receptors expressed in various mammalian cell lines
Authors: Lesage Anne S. , Wouters Ria , Van Gompel Paul , Heylen Lieve , Vanhoenacker Peter , Haegeman Guy , Luyten Walter H. M. L. , Leysen Josée E.
Published: January 2005

Alniditan, a novel migraine abortive agent, is a potent 5‐HT1B/5‐HT1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5‐HT1B receptor expressed at 200 fmol mg−1 protein (Bmax) in non‐induced L929sA cells, at 740 fmol mg−1 protein in HEK 293 and at 2300 fmol mg−1 protein in mIFNβ‐induced L929sA cells, and on the human cloned 5‐HT1D receptor expressed in C6 glioma cells (Bmax 780 fmol mg−1 protein).

Sodium butyrate treatment increased the expression level of human (h)5‐HT1B receptors in HEK 293 cells and h5‐HT1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [3H]‐5‐HT and [3H]‐alniditan were unaffected.

Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5‐HT1B receptor (IC50 values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5‐HT1D receptors (IC50 values of 1.3, 2.6 and 2.2 nM, respectively). At the h5‐HT1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).

This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5‐HT1B receptor, and twice as potent at the h5‐HT1D receptor. Dihydroergotamine was more potent an agonist at the h5‐HT1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5‐HT1D receptor.

British Journal of Pharmacology (1998) 123, 1655–1665; doi:10.1038/sj.bjp.0701766
Effect of the GABA uptake inhibitor tiagabine on sleep and EEG power spectra in the rat
Authors: Lancel Marike , Faulhaber Johannes , Deisz Rudolf A.
Published: January 2005

The sleep profiles induced by agonists and agonistic modulators of γ‐aminobutyric acidA (GABAA) receptors differ markedly. With regard to GABAA agonists, the effects may be due to the fact that these agents are poor substrates for uptake and are therefore likely to activate GABAA receptors tonically. To investigate this possibility, we assessed the sleep effects of two doses (2 and 10 mg kg−1) of the GABA re‐uptake inhibitor tiagabine, administered intraperitoneally at light onset in 8 rats. Electroencephalogram (EEG) and electromyogram were recorded during the first 8 h after the injection.

Compared with vehicle, tiagabine had minimal effects on the temporal pattern of non‐rapid eye movement sleep (non‐REMS) and on the total time spent therein. However, tiagabine dose‐dependently elevated EEG activity during non‐REMs, most prominently in the lower frequencies (1–8 Hz) and least pronounced in the frequencies between 11 and 16 Hz. During the first 2 h after the injection, 10 mg kg−1 tiagabine elicited repetitive episodes of hypersynchronous EEG waves during wakefulness and slightly suppressed REMS. Except for these effects, tiagabine hardly influenced the time spent in and EEG activity during wakefulness and REMS.

The effects of tiagabine on state‐specific EEG activity were qualitatively very similar to those elicited by GABAA agonists. These findings support the hypothesis that the influence of GABAA agonists on EEG signals may be caused by tonic stimulation of GABAA receptors.

British Journal of Pharmacology (1998) 123, 1471–1477; doi:10.1038/sj.bjp.0701769
Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery
Authors: McLaren G J , Burke K S , Buchanan K J , Sneddon P , Kennedy C
Published: January 2005

The site(s) at which P2‐receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2‐receptor antagonists and the extracellular ATPase inhibitor 6‐N,N‐diethyl‐D‐β,γ‐dibromomethyleneATP (ARL 67156).

Suramin (1 μM–1 mM) and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi‐effective concentrations of α,β‐methyleneATP (α,β‐meATP) (5 μM), 2‐methylthioATP (2‐meSATP) (100 μM) and adenosine 5′‐triphosphate (ATP) (1 mM) in a concentration‐dependent manner. Responses to α,β‐meATP and 2‐meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.

Contractions evoked by uridine 5′‐triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).

Desensitization of the P2X1‐receptor by α,β‐meATP abolished contractions evoked by 2‐meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.

Responses to α,β‐meATP (5 μM) and 2‐meSATP (100 μM) were abolished when tissues were bathed in nominally calcium‐free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.

ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β‐meATP (5 μM), 2‐meSATP (100 μM) and ATP (1 mM) in a concentration‐dependent manner.

These results suggest that two populations of P2‐receptors are present in the rat tail artery; ligand‐gated P2X1‐receptors and G‐protein‐coupled P2Y‐receptors.
Ribavirin uptake by human erythrocytes and the involvement of nitrobenzylthioinosine‐sensitive (es)‐nucleoside transporters
Authors: Jarvis Simon M. , Thorn James A. , Glue Paul
Published: January 2005

The major toxicity associated with oral therapy with ribavirin is anaemia, which has been postulated to occur as a result of accumulation of ribavirin triphosphate interfering with erythrocyte respiration. The objective of this study was to determine the mechanism by which ribavirin enters into erythrocytes.

Entry into human erythrocytes was examined by measuring influx rates of [3H]‐ribavirin alone and with the inhibitor nitrobenzylthioinosine (NBMPR), and by investigating the inhibitory effects of nucleoside and nucleobase permeants on ribavirin transport, by use of inhibitor oil‐stop methods. Transport mechanisms were further characterized by assessment of substrates to cause countertransport of ribavirin in preloaded erythrocytes, and by measuring the effects of ribavirin on [3H]‐NBMPR binding to erythrocyte membranes.

Human erythrocytes had a saturable influx mechanism for ribavirin (Km at 22°C of 440±100 μM) which was inhibited by nanomolar concentrations of NBMPR (IC50 0.99±0.15 nM). Nucleosides also inhibited the influx of ribavirin (adenosine more effective than uridine) but the nucleobases hypoxanthine and adenine had no effect. In addition, uridine caused the countertransport of ribavirin in human erythrocytes. Entry of ribavirin into horse erythrocytes, a cell type that lacks the NBMPR‐sensitive (es) nucleoside transporter, proceeded slowly and via a pathway that was resistant to NBMPR inhibition. Ribavirin was a competitive inhibitor of adenosine influx (mean Ki 0.48±0.14 mM) and also inhibited NBMPR binding to erythrocyte membranes (mean Ki 2.2±0.39 mM).

These data indicate that ribavirin is a transported permeant for the es nucleoside transporter of human erythrocytes. There was no evidence for ribavirin entering cells via a nucleobase transporter.

British Journal of Pharmacology (1998) 123, 1587–1592; doi:10.1038/sj.bjp.0701775
Serine‐O‐sulphate transport by the human glutamate transporter, EAAT2
Authors: Vandenberg Robert J. , Mitrovic Ann D. , Johnston Graham A. R.
Published: January 2005

Expression of the recombinant human excitatory amino aid transporters, EAAT1 and EAAT2, in Xenopus laevis oocytes allows electrogenic transport to be studied under voltage clamp conditions.

We have investigated the transport of the pharmacological substrate, L‐serine‐O‐sulphate transport by EAAT1 and EAAT2. The EC50 values for L‐serine‐O‐sulphate transport by EAAT2 showed a steep voltage‐dependence, increasing from 152±11 μM at −100 mV to 1930±160 μM at 0 mV. In contrast to EAAT2, EC50 values for L‐serine‐O‐sulphate transport by EAAT1 were relatively constant over the membrane potential range of −100 mV to 0 mV. The EC50 values for L‐glutamate and D‐aspartate transport, by EAAT2, were also relatively constant over this membrane potential range.

Chloride ions modulated the voltage‐dependent changes in EC50 values for transport by EAAT2. This effect was most apparent for L‐serine‐O‐sulphate transport, and to a lesser extent for L‐glutamate and not at all for D‐aspartate transport by EAAT2.

Extracellular sodium and proton concentrations also modulated the voltage‐dependence of L‐serine‐O‐sulphate EC50 values for EAAT2.

We speculate that these different properties of L‐serine‐O‐sulphate transport by EAAT2 compared to other substrates may be due to the much stronger acidity of the sulphate group of L‐serine‐O‐sulphate compared to carboxyl groups of L‐glutamate or D‐aspartate.

These results highlight some of the differences in the way different glutamate transporter subtypes transport substrates. This may be used to understand further the transport process and develop subtype selective inhibitors of glutamate transport.

British Journal of Pharmacology (1998) 123, 1593–1600; doi:10.1038/sj.bjp.0701776
Endothelium‐dependent relaxation and hyperpolarization in guinea‐pig coronary artery: role of epoxyeicosatrienoic acid
Authors: Eckman D M , Hopkins N , McBride C , Keef K D
Published: January 2005

Acetylcholine (ACh) elicits an endothelium‐dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea‐pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial‐derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 μM Nω‐nitro‐L‐arginine and 10 μM indomethacin.

ACh, AA and 11,12‐EET each produced concentration‐dependent relaxations in arteries contracted with the H1‐receptor agonist AEP (2,2‐aminoethylpyridine). The AA‐induced relaxation was significantly enhanced in the presence of the cyclo‐oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 μM).

The cytochrome P450 inhibitors proadifen (10 μM) and clotrimazole (10 μM) inhibited ACh, lemakalim (LEM) and AA‐induced relaxation, whereas 17‐octadecynoic acid (100 μM) and 7‐ethoxyresorufin (10 μM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 μM) and LEM (1 μM)‐induced hyperpolarization.

The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12‐EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12‐EET. In contrast, 4‐aminopyridine (4‐AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12‐EET. Combined IBTX plus (4‐AP) inhibited the ACh‐induced relaxation to a greater extent than 4‐AP alone. Apamin (1 μM), glibenclamide (10 μM) and BaCl2 (50 μM) had no significant effect on responses to ACh, AA and 11,12‐EET.

IBTX (100 nM) significantly reduced both 11,12‐EET (33 μM) and AA (30 μM) hyperpolarization without affecting the ACh (1 μM)‐induced hyperpolarization. In contrast, 4‐AP significantly reduced the ACh‐induced hyperpolarization without affecting either AA or 11,12‐EET‐induced hyperpolarizations.

In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12‐EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12‐EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO‐ and prostaglandin‐independent hyperpolarization and relaxation in the GPCA.
Effect of trans‐resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function
Authors: Rotondo Serenella , Rajtar Grazyna , Manarini Stefano , Celardo Antonio , Rotilio Domenico , De Gaetano Giovanni , Evangelista Virgilio , Cerletti Chiara
Published: January 2005

Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD).

Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality.

The aim of the present study was to investigate the effects of trans‐resveratrol (3,4′,5‐trihydroxy‐trans‐stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation.

trans‐Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 μM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3±0.13 μM, mean±s.e.mean), as evaluated by luminol‐amplified chemiluminescence.

trans‐Resveratrol prevented the release of elastase and β‐glucuronidase by PMN stimulated with the receptor agonists fMLP (1 μM, IC50 18.4±1.8 and 31±1.8 μM), and C5a (0.1 μM, IC50 41.6±3.5 and 42±8.3 μM), and also inhibited elastase and β‐glucuronidase secretion (IC50 37.7±7 and 25.4±2.2 μM) and production of 5‐lipoxygenase metabolites leukotriene B4 (LTB4), 6‐trans‐LTB4 and 12‐trans‐epi‐LTB4 (IC50 48±7 μM) by PMN stimulated with the calcium ionophore A23187 (5 μM).

trans‐Resveratrol significantly reduced the expression and activation of the β2 integrin MAC‐1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti‐MAC‐1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation‐dependent epitope on MAC‐1. Consistently, PMN homotypic aggregation and formation of mixed cell‐conjugates between PMN and thrombin‐stimulated fixed platelets in a dynamic system were also prevented by trans‐resveratrol.

These results, indicating that trans‐resveratrol interferes with the release of inflammatory mediators by activated PMN and down‐regulates adhesion‐dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.

British Journal of Pharmacology (1998) 123, 1691–1699; doi:10.1038/sj.bjp.0701784
Neuroregulation of mucus secretion by opioid receptors and KATP and BKCa channels in ferret trachea in vitro
Authors: Ramnarine Sean I. , Liu Yu‐Chih , Rogers Duncan F.
Published: January 2005

Opioid agonists inhibit neurogenic mucus secretion in the airways. The mechanism of the inhibition is unknown but may be via opening of potassium (K+) channels. We studied the effect on neurogenic secretion in ferret trachea in vitro of the OP1 receptor (formerly known as δ opioid receptor) agonist [D‐Pen2,5]enkephalin (DPDPE), the OP2 receptor (formely κ) agonist U‐50,488H, the OP3 receptor (formerly μ) agonist [D‐Ala2, N‐Me‐Phe, Gly‐ol5]enkephalin (DAMGO), the ATP‐sensitive K+ (KATP) channel inhibitor glibenclamide, the large conductance calcium activated K+ (BKCa) channel blocker iberiotoxin, the small conductance KCa (SKCa) channel blocker apamin, the KATP channel opener levcromakalim, a putative KATP channel opener RS 91309, and the BKCa channel opener NS 1619. Secretion was quantified by use of 35SO4 as a mucus marker.

Electrical stimulation increased tracheal secretion by up to 40 fold above sham‐stimulated levels. DAMGO or DPDPE (10 μM each) significantly inhibited neurogenic secretion by 85% and 77%, respectively, effects which were reversed by naloxone. U‐50,488H had no significant inhibitory effect on neurogenic secretion, and none of the opioids had any effect on ACh‐induced or [Sar9]substance P‐induced secretion.

Inhibition of neurogenic secretion by DAMGO or DPDPE was reversed by iberiotoxin (3 μM) but not by either glibenclamide or apamin (0.1 μM each). Iberiotoxin alone did not affect the neurogenic secretory response.

Levcromakalim, RS 91309 or NS 1619 (3 nM–3 μM) inhibited neurogenic secretion with maximal inhibitions at 3 μM of 68%, 72% and 96%, respectively. Neither levcromakalim nor RS 91309 at any concentration tested significantly inhibited acetylcholine (ACh)‐induced secretion, whereas inhibition (60%) was achieved at the highest concentration of NS 1619, a response which was blocked by iberiotoxin.

Inhibition of neurogenic secretion by levcromakalim (3 μM) or RS 91309 (30 nM) was inhibited by glibenclamide but not by iberiotoxin. In contrast, inhibition by NS 1619 (30 nM and 3 μM) was blocked by iberiotoxin but not by glibenclamide.

We conclude that, in ferret trachea in vitro, OP1 or OP3 opioid receptors inhibit neurogenic mucus secretion at a prejunctional site and that the mechanism of the inhibition is via opening of BKCa channels. Direct opening of BKCa channels or KATP channels also inhibits neurogenic mucus secretion. In addition, opening of BKCa channels inhibits ACh‐evoked secretion of mucus. Drugs which open BKCa channels may have therapeutic anti‐secretory activity in bronchial diseases in which neurogenic mechanisms and mucus hypersecretion are implicated in pathophysiology, for example asthma and chronic bronchitis.

British Journal of Pharmacology (1998) 123, 1631–1638; doi:10.1038/sj.bjp.0701786
Endogenous orphanin FQ: evidence for a role in the modulation of electroacupuncture analgesia and the development of tolerance to analgesia produced by morphine and electroacupuncture
Authors: Tian Jin‐Hua , Zhang Wei , Fang Yuan , Xu Wei , Grandy David K , Han Ji‐Sheng
Published: January 2005

Our previous work has demonstrated that exogenously administered orphanin FQ (OFQ) antagonizes morphine analgesia and electroacupuncture analgesia (EAA) in the brain and potentiates morphine analgesia and EAA in the spinal cord of the rat. In the present study we evaluated the role of endogenously released OFQ in the development of tolerance to morphine and electroacupuncture (EA) and the analgesia produced by electroacupuncture, by use of the IgG fraction of an anti‐OFQ antibody (OFQ‐Ab) microinjected into the rat central nervous system (CNS).

EAA was produced by stimulating rats at a frequency of 100 Hz. Rats were classified as either high responders (HR) or low responders (LR) based on the analgesic effects of EA. LRs could be converted into HRs by the intracerebroventricular (i.c.v.) microinjection of OFQ‐Ab at both 1 : 1 and 1 : 10 dilutions but not 1 : 100. HRs could be changed into LRs by the intrathecal (i.t.) injection of OFQ‐Ab at both 1 : 1 and 1 : 10 dilutions, but not 1 : 100.

Acute morphine tolerance was induced in rats by repeated subcutaneous (s.c.) injections of morphine (5 mg kg, every 2 h) for 16 h. When injected i.c.v. the OFQ‐Ab (1 : 1 dilution) had no effect on the development of acute morphine tolerance.

Chronic morphine tolerance was produced in rats by repeated injection of morphine (5–60 mg kg, s.c., 3× a day) for 6 days. I.c.v. injection of OFQ‐Ab (1 : 1 dilution) reversed this type of morphine tolerance in rats by 50% (P<0.01).

Acute tolerance to the analgesia produced by EA developed after 6 h of continuous (100 Hz, 3mA) stimulation. This tolerance was almost completely reversed by the i.c.v. injection of OFQ‐Ab (1 : 1 dilution) (P<0.05).

Chronic tolerance to the analgesic effect of EA was produced by repeatedly administering increasing current (1, 2 and 3 mA, each lasting for 10 min, for a total of 30 min) at a frequency of 100 Hz once a day for 6 days. I.c.v. injection of OFQ‐Ab (1 : 1 dilution) reversed this kind of tolerance by 50% (P<0.01).

Together these results suggest that 100 Hz EA may enhance the release of endogenous OFQ in the CNS of the rat, which in turn may act to antagonize EA‐produced analgesia in the brain but potentiate EA produced analgesia in the spinal cord. Therefore, OFQ appears to play an important role in the development of tolerance to the analgesic effects produced by EA.

The mechanisms underlying the development of acute morphine tolerance and chronic morphine tolerance appear to be different. Central OFQ may play an important role in the development of tolerance after chronic morphine administration.
The receptor subtype mediating the action of angiotensin II on intracellular sodium in rat proximal tubules
Authors: Wong Patrick S K , Johns Edward J
Published: January 2005

An investigation was undertaken to explore the subtype of receptor involved in mediating the actions of angiotensin II on intracellular sodium content in suspensions of isolated proximal tubules of the rat.

Intracellular sodium content of the proximal tubules was measured with 23Na n.m.r. spectroscopy and under these conditions basal sodium content of the tubular cells was 69.04±1.73 nmol mg−1 dry weight and the ATP levels, at 8.3±0.9 nmol ATP mg−1 protein, were consistent with active respiration by the tissue.

In the presence of 10−4M PD123319, a selective non‐peptide AT2 receptor antagonist, intracellular sodium levels rose from steady state by 30% (P<0.01; n=7) within 10 min of exposure to angiotensin II 10−11M. Over the subsequent 30 min steady state levels were re‐established. Administration of angiotensin II 10−11M, in the presence of the selective AT1 receptor antagonist, losartan at either 10−6M (n=5) or 10−4M (n=6), was without effect on intracellular sodium levels, which were significantly different (P<0.001) from those observed when PD 123319 was present.

Angiotensin II 10−5M, administered to the tubular suspension in the presence of 10−4M PD123319, decreased (P<0.01, n=6) intracellular sodium content by 16% in the first 5 min, but in the following 25 min returned to steady state levels. However, in the presence of losartan 10−4M, angiotensin II 10−5M had no effect on intracellular sodium content which was markedly different (P<0.001) from that obtained in the presence of PD123319.

These findings show that at both the high and low concentrations of angiotensin II, its modulation of intracellular sodium levels within the proximal tubule cells is mediated via the activation of AT1 receptors. The intracellular mechanism underlying this effect remain to be investigated.
Modulation of spasmogen‐stimulated Ins(1,4,5)P3 generation and functional responses by selective inhibitors of types 3 and 4 phosphodiesterase in airways smooth muscle
Authors: Challiss R A John , Adams David , Mistry Rajendra , Nicholson C David
Published: January 2005

The effects of isoenzyme‐selective inhibitors of phosphodiesterases PDE3 and PDE4 on cyclic AMP concentration, two indices of phosphoinositide hydrolysis, and contractile responses to spasmogens have been investigated in bovine tracheal smooth muscle (BTSM).

Neither the PDE3‐selective inhibitor ORG 9935, nor the PDE4‐selective inhibitor rolipram increased cyclic AMP levels in BTSM. However, rolipram addition in the presence of PDE3 inhibition (ORG 9935; 1 μM) concentration‐dependently (−log EC50 (M), 6.55±0.15; n=3) increased cyclic AMP levels to about 70% of the maximal response to the β‐adrenoceptor agonist isoprenaline.

Rolipram per se inhibited histamine‐stimulated [3H]‐inositol (poly)phosphate ([3H]‐InsPX) accumulation by >80% (−log EC50 (M), 6.92±0.11; n=3). Although ORG 9935 (1 μM) had little effect on histamine‐stimulated [3H]‐InsPX accumulation alone it greatly facilitated the inhibitory action of rolipram (−log EC50 (M), 8.82±0.39; n=3). The effects of PDE3 and/or PDE4 inhibition on [3H]‐InsPX accumulation stimulated by muscarinic acetylcholine (mACh) receptor activation were less marked. However, combined PDE3/4 inhibition significantly decreased this response at a submaximal concentration of mACh receptor agonist (carbachol; 1 μM).

The greater‐than‐additive effect of combined PDE3/4 inhibition was also observed at the level of contractile responses to histamine and carbachol. In experiments designed to investigate the effects of PDE3 and/or 4 inhibitors on the carbachol‐mediated phasic contraction, additions of rolipram (10 μM) or ORG 9935 (1 μM) were without effect, whereas added together the inhibitors caused a significant (P<0.01) 40% reduction in the peak phasic contractile response.

The effect on contraction correlated with a substantial inhibitory effect of PDE3/4 inhibition on the initial increase in inositol 1,4,5‐trisphosphate (InsP3) accumulation stimulated by spasmogen. Thus, in the presence of ORG 9935 (1 μM) rolipram concentration‐dependently inhibited carbachol‐stimulated InsP3 accumulation by 50% (−log EC50 (M), 6.77±0.21; n=4).

Carbachol (100 μM) addition caused a rapid decrease (by 67% at 10 s) in BTSM cyclic AMP level in the presence of PDE3/4 inhibition. However, omission of Ca2+ from the incubation medium prevented the carbachol‐evoked decrease in cyclic AMP and this coincided with a greater inhibition (80%) of the carbachol‐stimulated InsP3 response.

These data indicate that combined PDE3 and PDE4 inhibition has greater‐than‐additive effects on second messenger and functional responses to spasmogens in BTSM. Furthermore, the ability of PDE3/4 inhibition significantly to attenuate mACh receptor‐mediated contractile responses, may be, at least in part, attributed to an effect exerted at the level of InsP3 generation.
Electrophysiological and neurochemical evidence that pindolol has agonist properties at the 5‐HT1A autoreceptor in vivo
Authors: Clifford Elizabeth M , Gartside Sarah E , Umbers Valerie , Cowen Philip J , Hajós Mihály , Sharp Trevor
Published: January 2005

It has been hypothesized that 5‐HT1A autoreceptor antagonists may enhance the therapeutic efficacy of SSRIs and other antidepressants. Although early clinical trials with the β‐adrenoceptor/5‐HT1 ligand, pindolol, were promising, the results of recent more extensive trials have been contradictory. Here we investigated the actions of pindolol at the 5‐HT1A autoreceptor by measuring its effect on 5‐HT neuronal activity and release in the anaesthetized rat.

Pindolol inhibited the electrical activity of 5‐HT neurones in the dorsal raphe nucleus (DRN). This effect was observed in the majority of neurones tested (10/16), was dose‐related (0.2–1.0 mg kg−1, i.v.), and was reversed by the 5‐HT1A receptor antagonist, WAY 100635 (0.1 mg kg−1, i.v.), in 6/7 cases tested.

Pindolol also inhibited 5‐HT neuronal activity when applied microiontophoretically into the DRN in 9/10 neurones tested. This effect of pindolol was current‐dependent and blocked by co‐application of WAY 100635 (3/3 neurones tested).

In microdialysis experiments, pindolol caused a dose‐related (0.8 and 4 mg kg−1, i.v.) fall in 5‐HT levels in dialysates from the frontal cortex (under conditions where the perfusion medium contained 1 μM citalopram). In rats pretreated with WAY 100635 (0.1 mg kg−1, i.v.), pindolol (4 mg kg−1, i.v.) did not decrease, but rather increased 5‐HT levels.

We conclude that, under the experimental conditions used in this study, pindolol displays agonist effects at the 5‐HT1A autoreceptor. These data are relevant to previous and ongoing clinical trials of pindolol in depression which are based on the rationale that the drug is an effective 5‐HT1A autoreceptor antagonist.