bluuebunny/crossref_metadata_2025 huggingface dataset:
Behavioural and physiological effects induced by an infusion of antisense to α2D‐adrenoceptors in the rat
Authors: Robinson E S J , Nutt D J , Jackson H C , Hudson A L
Published: January 2005

The aim of this study was to investigate the behavioural and physiological effects of an i.c.v. infusion of antisense oligonucleotide to the α2D‐adrenoceptor subtype. Behavioural and physiological parameters were monitored for 2 days before the infusion, throughout the 3‐day infusion period and for 3 days following the end of the infusion.

The antisense infusion resulted in a significant increase in behavioural activity characterized by increased locomotion and grooming scores. Behavioural activity scores of rats treated with antisense to α2D‐adrenoceptors were significantly higher than those of rats treated with vehicle (H2O) or the mismatch toxicity control on day 4 and day 5 and, significantly higher than vehicle controls on day 6.

Body weight gain was significantly reduced in the antisense‐treated rats at the end of the study compared to the vehicle (34%) and mismatch groups (30%), although daily food and water intakes were not significantly different at any time point.

Pupil diameters of rats infused with antisense to α2D‐adrenoceptors were significantly greater than those of animals treated either with vehicle or mismatch oligonucleotide on day 5 of the study. On day 6, the pupil diameters of these animals were still significantly greater than the mismatch group.

In conclusion, an i.c.v. infusion of antisense to the α2D‐adrenoceptor induced behavioural activation in rats, increased pupil diameter and reduced total weight gain. These effects were specific to the antisense‐treated group and were fully reversed post‐infusion.

British Journal of Pharmacology (2000) 130, 153–159; doi:10.1038/sj.bjp.0703296
Modulation of the dinucleotide receptor present in rat midbrain synaptosomes by adenosine and ATP
Authors: Díaz‐Hernández Miguel , Pintor Jesús , Miras‐Portugal M Teresa
Published: January 2005

Diadenosine polyphosphates activate dinucleotide receptors in rat midbrain synaptic terminals. The agonist with highest affinity at this receptor, diadenosine pentaphosphate (Ap5A), elicits Ca2+ transients at concentrations ranging from 10−7 to 10−3 M with a single‐phase curve and an EC50 value of 56.21±1.82 μM.

Treatment of synaptosomal preparations with alkaline phosphatase (AP) changes the dose‐response control curve into a biphasic one presenting two EC50 values of 6.47±1.25 nM and 11.16±0.83 μM respectively.

The adenosine A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) reversed the biphasic concentration‐response for Ap5A curve in the presence of AP, to a monophasic one with an EC50 value of 76.05±7.51 μM.

The application of adenosine deaminase produced the same effect as DPCPX, the EC50 value for Ap5A, in the presence of AP being 18.62±4.03 μM.

Activation of the adenosine A1 receptor by means of cyclohexyladenosine (CHA) shifted the dose response curve for Ap5A to the left, resulting in a monophasic curve with an EC50 of 5.01±0.02 pM.

The destruction of extrasynaptosomal nucleotides by AP or the addition of pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), a broad P2 antagonist compound, enhance maximal effect of the Ap5A up to 55.6% on the dose response curve, thus suggesting a negative modulation by P2 receptors.

In a summary, ATP and adenosine present at the extra‐synaptosomal space, are relevant natural modulators of the dinucleotide receptor, via P2 and adenosine A1 receptors respectively.

British Journal of Pharmacology (2000) 130, 434–440; doi:10.1038/sj.bjp.0703300
Pentaerythrityl tetranitrate attenuates structural changes in conduit arteries evoked by long‐term NO‐synthase inhibition
Authors: Kristek František
Published: January 2005

The aim of the study was to determine whether the pentaerythrityl tetranitrate (PETN), a tolerance devoid exogenous NO donor could prevent morphological changes in the cardiovascular system evoked by long‐term NO‐synthase inhibition.

Three groups of 10‐week‐old Wistar rats were used: (1) controls, (2) treated by L‐NG‐nitroarginine methyl ester (L‐NAME) in water (50 mg kg−1), and (3) treated by L‐NAME (50 mg kg−1 in water)+PETN (2×50 mg kg−1, using gavage). Blood pressure (BP) was measured by the tail plethysmographic method.

After sacrificing the animals were perfused (120 mmHg) by glutaraldehyde fixative and processed according to standard electron microscopy procedure. Wall thickness (WT), cross sectional area (CSA), inner diameter (ID) of thoracic aorta (TA), carotid (CA) and septal branch of the left descending coronary artery (RS) were measured in light microscopy.

After 6 weeks, the BP was increased to 172±1.7 mmHg (P<0.01) in the L‐NAME group, compared to 127±1.4 mmHg in controls. In L‐NAME+PETN‐treated rats, BP was 163±0.9 mmHg (P<0.01), and significantly lower (P<0.01) in comparison to L‐NAME‐treated rats. Heart weight and heart/body weight ratio was not significantly changed.

In L‐NAME‐treated rats, both WT and CSA were increased in all three arteries (P<0.01). ID was increased only in TA (P<0.01). Wall/diameter ratio (WD) was increased in TA (P<0.01) and CA (P<0.01). In L‐NAME+PETN treated rats, WT was found to be increased only in TA (P<0.01). In comparison to the L‐NAME treated group, WT was decreased in TA (P<0.01), in CA (P<0.01), in RS (P<0.05). CSA was increased only in TA (P<0.01), yet in comparison to the L‐NAME group it was decreased in CA (P<0.01). ID was increased in comparison to both control and L‐NAME treated rats only in TA (P<0.01). WD did not differ from the control value. In comparison to L‐NAME‐treated rats, it was decreased in both TA and CA (P<0.01).

These data suggest that the changes in the cardiovascular system evoked by long‐term NO‐synthase inhibition were attenuated by simultaneous administration of the exogenous donor of nitric oxide–PETN.

British Journal of Pharmacology (2000) 130, 450–456; doi:10.1038/sj.bjp.0703307
Troglitazone prevents and reverses dexamethasone induced insulin resistance on glycogen synthesis in 3T3 adipocytes
Authors: Anil Kumar K L , Marita A R
Published: January 2005

Troglitazone lowers blood glucose levels in Type II diabetic patients. To evaluate the insulin sensitizing action of troglitazone on glycogen synthesis we have used dexamethasone‐treated 3T3 adipocytes as an in vitro model.

Differentiated 3T3 adipocytes were incubated with 100 nM dexamethasone for 6 days. Troglitazone (1.0 μM) or metformin (1.0 mM) with or without 200 nM insulin was added during the last 4 days. At the end, insulin (100 nM) stimulated glycogen synthesis was determined using 14C‐glucose.

Dexamethasone caused a 50% reduction in glycogen synthesis. Troglitazone caused an approximately 3 fold increase in glycogen synthesis from 43.9±3.4 to 120±16.2 nmols h−1. Under identical conditions metformin had no significant effect.

When cells were incubated with troglitazone and dexamethasone simultaneously for 6 days, troglitazone but not metformin completely prevented dexamethasone‐induced insulin resistance. RU 486 (1.0 μM) also completely prevented the insulin resistance.

Chronic incubation with dexamethasone and insulin resulted in a 73% reduction in glycogen synthesis. In these adipocytes, troglitazone was partially active with glycogen synthesis rising from 23.1±3.0 to 44.4±4.5 nmol h−1, P<0.01 while metformin was inactive.

Troglitazone stimulated 2‐deoxyglucose uptake by 2–3 fold in dexamethasone‐treated adipocytes. Metformin also increased glucose uptake significantly. Troglitazone did not affect insulin binding while a 2 fold increase was observed in normal adipocytes where it exhibited a modest effect.

Since the effect of troglitazone was greater in dexamethasone‐treated adipocytes, troglitazone is likely to act by preventing dexamethasone‐induced alterations which may include (i) binding to glucocorticoid receptor and (ii) effect on glucose uptake.

These data demonstrate the direct insulin sensitizing action of troglitazone on glycogen synthesis and suggest a pharmacological profile different from metformin.

British Journal of Pharmacology (2000) 130, 351–358; doi:10.1038/sj.bjp.0703313
[3H]‐SB‐269970 – A selective antagonist radioligand for 5‐HT7 receptors
Authors: Thomas David R , Atkinson Peter J , Ho Michael , Bromidge Steven M , Lovell Peter J , Villani Anthony J , Hagan Jim J , Middlemiss Derek N , Price Gary W
Published: January 2005

Binding of the 5‐HT7 receptor antagonist radioligand [3H]‐SB‐269970 to human 5‐HT7(a) receptors expressed in HEK293 cell membranes (h5‐HT7(a)/293) and to guinea‐pig cerebral cortex membranes, was characterized and compared with [3H]‐5‐CT binding.

[3H]‐SB‐269970 (1 nM) showed full association with h5‐HT7(a)/293 membranes after 40 min. Specific binding at equilibrium represented >90% of total binding and was fully reversible by methiothepin (10 μM), full dissociation occurring by 100 min. The association (k+1) and dissociation (k−1) rate constants were 0.05 nM−1min−1 and 0.05 min−1 respectively, giving a KD (k−1/k+1) of 1.0 nM.

[3H]‐SB‐269970 bound saturably and apparently monophasically to both h5‐HT7(a)/293 and guinea‐pig cortex membranes, with KD values of 1.25±0.05 and 1.7±0.3 nM respectively. The Bmax for [3H]‐SB‐269970 to both h5‐HT7(a)/293 and guinea‐pig cortex membranes (5780±380 and 125±8.2 fmoles mg protein−1 respectively) was similar to that for [3H]‐5‐CT (6190±940 and 143±19 fmoles mg protein−1 respectively). These data suggest that, in each tissue, both radioligands labelled the same population of receptors, which appear to be present in an agonist high affinity state.

The profile of compound inhibition of [3H]‐SB‐269970 binding to h5‐HT7(a)/293 and guineapig cortex membranes correlated well (corr. coeff. 0.98) with those for [3H]‐5‐CT binding and were consistent with the profiles reported previously for the human 5‐HT7(a) and guinea‐pig cortex 5‐HT7 receptors using [3H]‐5‐CT. Hill slopes for inhibition of [3H]‐SB‐269970 and [3H]‐5‐CT binding were close to 1, consistent with binding to a single receptor population in both tissues.

[3H]‐SB‐269970 represents the first selective 5‐HT7 antagonist radioligand, which should aid further characterization of 5‐HT7 receptors in recombinant and native tissues and help establish their role in brain function.

British Journal of Pharmacology (2000) 130, 409–417; doi:10.1038/sj.bjp.0703318
Pharmacological characterization of the cardiovascular responses elicited by kinin B1 and B2 receptor agonists in the spinal cord of streptozotocin‐diabetic rats
Authors: Cloutier Frank , Couture Réjean
Published: January 2005

Kinin receptor agonists and antagonists at the B1 and B2 receptors were injected intrathecally (i.t., at T‐9 spinal cord level) to conscious unrestrained rats and their effects on mean arterial pressure (MAP) and heart rate (HR) were compared in streptozotocin (STZ)‐diabetic rats (65 mg kg−1 STZ, i.p. 3 weeks earlier) and aged‐matched control rats.

The B1 receptor agonist, des‐Arg9‐Bradykinin (BK) (3.2–32.5 nmol), evoked dose‐dependent increases in MAP and tachycardia during the first 10 min post‐injection in STZ‐diabetic rats only. The cardiovascular response to 6.5 nmol des‐Arg9‐BK was reversibly blocked by the prior i.t. injection of antagonists for the B1 receptor ([des‐Arg10]‐Hoe 140, 650 pmol or [Leu8]‐des‐Arg9‐BK, 65 nmol) and B2 receptor (Hoe 140, 81 pmol or FR173657, 81 pmol) or by indomethacin (5 mg kg−1, i.a.).

The i.t. injection of BK (8.1–810 pmol) induced dose‐dependent increases in MAP which were accompanied either by tachycardiac (STZ‐diabetic rats) or bradycardiac (control rats) responses. The pressor response to BK was significantly greater in STZ‐diabetic rats. The cardiovascular response to 81 pmol BK was reversibly blocked by 81 pmol Hoe 140 or 81 pmol FR173657 but not by B1 receptor antagonists nor by indomethacin in STZ‐diabetic rats.

The data suggest that the activation of kinin B1 receptor in the spinal cord of STZ‐diabetic rats leads to cardiovascular changes through a prostaglandin mediated mechanism. Thus, this study affords an accessible model for studying the expression, the pharmacology and physiopathology of the B1 receptor in the central nervous system.

British Journal of Pharmacology (2000) 130, 375–385; doi:10.1038/sj.bjp.0703319
Localization of the nephron site of gentamicin‐induced hypercalciuria in the rat: a micropuncture study
Authors: Parsons P P , Garland H O , Harpur E S
Published: January 2005

In vivo renal micropuncture techniques were used to locate the nephron site of hypercalciuria induced by acute gentamicin infusion in anaesthetized Sprague Dawley rats. Three series of experiments were conducted. The effect of gentamicin on calcium reabsorption in the proximal tubule (Series I) and loop of Henle (Series II) was investigated using in vivo microperfusion whereas the effect on distal calcium handling (Series III) was studied using in vivo microinfusion.

In all three experimental series, acute systemic gentamicin infusion at 0.28 mg kg−1 min−1 caused significant hypercalciuria within 30 min of commencing drug infusion. Gentamicin had no effect on the rates of urine flow or sodium excretion.

Acute gentamicin infusion had no effect on unidirectional calcium reabsorption in the proximal tubule or loop of Henle despite a simultaneous and highly significant hypercalciuria at the whole kidney level. Net fluid reabsorption was also unaffected by the drug in these nephron segments.

Acute gentamicin infusion significantly increased the urinary recovery of calcium following microinfusion into early distal tubules, whereas urinary calcium recovery was decreased after microinfusion into late distal tubules.

We conclude that acute gentamicin‐induced hypercalciuria is mediated by a decrease in calcium reabsorption in the early distal tubule. Thus, the acute hypercalciuric effect of gentamicin occurs at a different nephron site to the nephrotoxic effects associated with longer‐term administration of the drug. It is, therefore, unlikely that gentamicin‐induced hypercalciuria is involved in the pathogenesis of subsequent proximal tubular cell injury.

British Journal of Pharmacology (2000) 130, 441–449; doi:10.1038/sj.bjp.0703329
Comparison of serum, cerebrospinal fluid and brain extracellular fluid pharmacokinetics of lamotrigine
Authors: Walker M C , Tong X , Perry H , Alavijeh M S , Patsalos P N
Published: January 2005

We investigated the rate of penetration into and the intra‐relationship between the serum, cerebrospinal fluid (CSF) and regional brain extracellular fluid (bECF) compartments following systemic administration of lamotrigine in rat.

The serum pharmacokinetics were biphasic with an initial distribution phase, (half‐life approximately 3 h), and then a prolonged elimination phase of over 30 h. The serum pharmacokinetics were linear over the range 10–40 mg kg−1.

Using direct sampling of CSF with concomitant serum sampling, the calculated penetration half‐time into CSF was 0.42±0.15 h. At equilibrium, the CSF to total serum concentration ratio (0.61±0.02) was greater than the free to total serum concentration (0.39±0.01).

Using in vivo recovery corrected microdialysis sampling in frontal cortex and hippocampus with concomitant serum sampling, the calculated penetration half‐time of lamotrigine into bECF, 0.51±0.11 h, was similar to that for CSF and was not area or dose dependent. At equilibrium, the bECF to total serum concentration ratio (0.40±0.04) was similar to the free to total serum concentration (0.39±0.01), and did not differ between hippocampus and frontal cortex.

The species specific serum kinetics can explain the prolonged action of lamotrigine in rat seizure models. Lamotrigine has a relatively slow penetration into both CSF and bECF compartments compared with antiepileptic drugs used in acute seizures. Furthermore, the free serum drug concentration is not the sole contributor to the CSF compartment, and the CSF concentration is an overestimate of the bECF concentration of lamotrigine.

British Journal of Pharmacology (2000) 130, 242–248; doi:10.1038/sj.bjp.0703337
Conditions permitting suppression of stretch‐induced and vasoconstrictor tone by basal nitric oxide activity in porcine cerebral artery
Authors: Wallis Sarah J , Martin William
Published: January 2005

This study examined the ability of basal nitric oxide activity to suppress intrinsic and vasoconstrictor tone in isolated rings of porcine cerebral artery.

Following stretch of approximately 1 g, NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) produced a rise in tone in endothelium‐containing but not endothelium‐denuded rings. Thus, intrinsic tone was present and was powerfully suppressed by basal nitric oxide activity.

Nevertheless, when concentration‐response curves were constructed to U46619 and 5‐hydroxytryptamine (5‐HT), no endothelium‐dependent depression of vasoconstriction was observed. It therefore appeared that basal nitric oxide activity was able to suppress intrinsic but not vasoconstrictor tone in these vessels.

Stretch‐tension curves generated following the application of stretch over the range 0–5.5 g on endothelium‐denuded rings showed that tension was stretch‐induced. Experiments conducted in the presence of L‐NAME (100 μM) revealed that the level of tone present in endothelium‐containing rings was substantially higher than in endothelium‐denuded rings across the entire range of stretch.

When endothelium‐containing and endothelium‐denuded rings were set at similar levels of stretch‐induced tone, rather than similar levels of stretch, the presence of the endothelium now depressed significantly vasoconstrictor responses to U46619 and 5‐HT.

Thus, when endothelium‐containing and endothelium‐denuded rings of porcine cerebral artery are set at similar points along their respective stretch‐tension curves, rather than at similar levels of stretch, basal nitric oxide activity can be seen to inhibit both stretch‐induced and vasoconstrictor tone.

British Journal of Pharmacology (2000) 130, 567–574; doi:10.1038/sj.bjp.0703351
Involvement of tyrosine phosphorylation in the positive inotropic effect produced by H1‐receptors with histamine in guinea‐pig left atrium
Authors: Akaishi Yasuhiro , Hattori Yuichi , Yoshimoto Kimihiro , Kitabatake Akira , Yasuda Keishu , Kanno Morio
Published: January 2005

We investigated the effect of stimulation of H1‐receptors with histamine on protein tyrosine phosphorylation levels in guinea‐pig left atrium and evaluated the influences of tyrosine kinase inhibitors on the positive inotropic effect mediated by H1‐receptors in this tissue.

Histamine induced an increase in tyrosine phosphorylation in four main clusters of proteins with apparent molecular weights of 25, 35, 65 and 150 kDa. Tyrosine phosphorylation of these proteins attained a peak around 2–3 min following histamine stimulation and then declined to or below basal levels. Histamine‐induced protein tyrosine phosphorylation was antagonized by the H1‐receptor antagonists mepyramine (1 μM) and chlorpheniramine (1 μM), but not by the H2‐receptor antagonist cimetidine (10 μM).

The positive inotropic effect of histamine was depressed in a concentration‐dependent manner by the tyrosine kinase inhibitors tyrphostin A25 (50 to 100 μM) and genistein (10 to 50 μM) but not by the inactive genistein analogue daidzein (50 μM). The positive inotropic effect of isoprenaline was unchanged by tyrphostin A25 and genistein.

At a concentration of 1 μM histamine produced a dual‐component positive inotropic response composed of an initial increasing phase and a second and late developing, greater positive inotropic phase. Treatment with tyrphostin A25 (100 μM) and genistein (50 μM), but not daidzein (50 μM), significantly attenuated the two components of the inotropic response, although genistein suppressed the initial component more markedly than the late component.

We conclude that increased protein tyrosine phosphorylation may play an important role in initiating at least some part of the positive inotropic effect of H1‐receptor stimulation in guinea‐pig left atrium.

British Journal of Pharmacology (2000) 130, 907–915; doi:10.1038/sj.bjp.0703355
Characterization of SB‐269970‐A, a selective 5‐HT7 receptor antagonist
Authors: Hagan Jim J , Price Gary W , Jeffrey Phillip , Deeks Nigel J , Stean Tania , Piper David , Smith Martin I , Upton Neil , Medhurst Andrew D , Middlemiss Derek N , Riley Graham J , Lovell Peter J , Bromidge Steven M , Thomas David R
Published: January 2005

The novel 5‐HT7 receptor antagonist, SB‐269970‐A, potently displaced [3H]‐5‐CT from human 5‐HT7(a) (pKi 8.9±0.1) and 5‐HT7 receptors in guinea‐pig cortex (pKi 8.3±0.2).

5‐CT stimulated adenylyl cyclase activity in 5‐HT7(a)/HEK293 membranes (pEC50 7.5±0.1) and SB‐269970‐A (0.03–1 μM) inhibited the 5‐CT concentration‐response with no significant alteration in the maximal response. The pA2 (8.5±0.2) for SB‐269970‐A agreed well with the pKi determined from [3H]‐5‐CT binding studies.

5‐CT‐stimulated adenylyl cyclase activity in guinea‐pig hippocampal membranes (pEC50 of 8.4±0.2) was inhibited by SB‐269970‐A (0.3 μM) with a pKB (8.3±0.1) in good agreement with its antagonist potency at the human cloned 5‐HT7(a) receptor and its binding affinity at guinea‐pig cortical membranes.

5‐HT7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis.

SB‐269970‐A was CNS penetrant (steady‐state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min−1 kg−1). Following a single dose (3 mg kg−1) SB‐269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea‐pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested.

5‐CT (0.3 mg kg−1 i.p.) induced hypothermia in guinea‐pigs was blocked by SB‐269970‐A (ED50 2.96 mg kg−1 i.p.) and the non‐selective 5‐HT7 receptor antagonist metergoline (0.3–3 mg kg−1 s.c.), suggesting a role for 5‐HT7 receptor stimulation in 5‐CT induced hypothermia in guinea‐pigs.

SB‐269970‐A (30 mg kg−1) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3  h of EEG recording in conscious rats.

British Journal of Pharmacology (2000) 130, 539–548; doi:10.1038/sj.bjp.0703357
Specific inhibition of stretch‐induced increase in L‐type calcium channel currents by herbimycin A in canine basilar arterial myocytes
Authors: Kimura Makoto , Obara Kazuo , Sasase Tomohiko , Ishikawa Tomohisa , Tanabe Yoshiyuki , Nakayama Koichi
Published: January 2005

The effects of protein‐tyrosine kinase (PTK) and protein‐tyrosine phosphatase (PTP) inhibitors on voltage‐activated barium currents (IBa) through L‐type calcium channels increased by hypotonic solution were investigated in canine basilar arterial myocytes by the whole‐cell patch‐clamp technique.

IBa was elicited by depolarizing step from a holding potential of −80 to +10 mV and identified by using an L‐type calcium channel agonist, Bay K 8644 (100 nM), and an L‐type calcium channel blocker, nicardipine (1 μM).

Hypotonic superfusate induced cell swelling and acted as a stretch stimulus, which reversibly increased peak IBa amplitude at +10 mV. IBa was also decreased by nicardipine (1 μM) under the hypotonic condition.

PTK inhibitors such as herbimycin A (30 nM), genistein (10 μM), and lavendustin A (10 μM) decreased IBa enhanced by hypotonic solution. Genistein also decreased IBa in a concentration‐dependent manner under the isotonic condition. The inactive genistein analogue daidzein (10 μM) had no effect on IBa under either the isotonic or hypotonic condition. By contrast, herbimycin A did not decrease IBa under the isotonic condition. Sodium orthovanadate (10 μM), a PTP inhibitor, increased IBa under both conditions.

The present results suggest that cell swelling by hypotonic solution increases the L‐type calcium channel currents in canine basilar artery and that herbimycin‐sensitive PTK activity is primarily involved in the enhancement of calcium channel currents.

British Journal of Pharmacology (2000) 130, 923–931; doi:10.1038/sj.bjp.0703360
Insulin modulation of intracellular free magnesium in heart: involvement of protein kinase C
Authors: Amano Tetsuya , Matsubara Tatsuaki , Watanabe Junji , Nakayama Shinsuke , Hotta Nigishi
Published: January 2005

In the present study of rat heart using 31P‐nuclear magnetic resonance, we examined the interaction between β‐adrenergic and insulin receptors in terms of the intracellular free Mg2+ concentration ([Mg2+]i) regulation.

[Mg2+]i was estimated from the separation of the chemical shifts of the α‐ and β‐adenosine triphosphate (ATP) peaks, using the dissociation constant of MgATP 87 μM (established recently). In normal (phosphate‐free Krebs‐Henseleit) solution, [Mg2+]i was approximately 1.02 mM.

Insulin at physiological and pathological concentrations increased [Mg2+]i and contractility in a dose‐dependent manner.

Insulin (more than 100 μu ml−1) suppressed the decrease in [Mg2+]i caused by isoprenaline (100 nM), and these effects of insulin on [Mg2+]i and contractility were blocked by LY333531 (macrocyclic bis (indolyl) maleimide, 100 nM), a protein kinase C (PKC) inhibitor.

The isoprenaline‐induced decrease in the concentrations of ATP ([ATP]) with insulin application was significantly smaller than that without insulin.

Insulin modulates [Mg2+]i and haemodynamics, presumably via activation of PKC, thereby antagonizing the reduction of [Mg2+]i induced by β‐adrenoceptor stimulation.

British Journal of Pharmacology (2000) 130, 731–738; doi:10.1038/sj.bjp.0703361
Flibanserin has anxiolytic effects without locomotor side effects in the infant rat ultrasonic vocalization model of anxiety
Authors: Podhorna J , Brown R E
Published: January 2005

This study compared the effects of flibanserin, a novel 5‐HT1A agonist/5‐HT2A antagonist; diazepam, a traditional anxiolytic; and imipramine, a traditional antidepressant, on separation‐induced ultrasonic vocalizations (USVs), locomotor behaviour, negative geotaxis and body temperature of 7–8‐day‐old rat pups.

Flibanserin (5, 10, 25 and 50 mg kg−1 s.c.) reduced USVs but had no effects on locomotor behaviour or negative geotaxis. Lower doses of flibanserin (0.5, 1, 2 and 4 mg kg−1 s.c.) had no effect on any behaviour. Diazepam (0.25, 0.5, 1 and 2 mg kg−1 s.c.) not only reduced the USVs but also increased rolling and increased the latency of the negative geotaxic response. Imipramine (10, 15, 20 and 30 mg kg−1 s.c.) reduced USVs, increased total locomotor activity and rolling but had no effect on negative geotaxis. None of the drugs altered body temperature.

Our data showed that flibanserin is as effective in reducing the USVs as diazepam and imipramine but has a lower incidence of motor side effects. This suggests that flibanserin might be effective for the treatment of mood disturbances such as anxiety.

British Journal of Pharmacology (2000) 130, 739–746; doi:10.1038/sj.bjp.0703364
Tissue angiotensin II and endothelin‐1 modulate differently the response to flow in mesenteric resistance arteries of normotensive and spontaneously hypertensive rats
Authors: Matrougui Khalid , Lévy Bernard I , Henrion Daniel
Published: January 2005

In resistance arteries pressure‐induced (myogenic) tone (MT) and flow (shear stress)‐induced dilation (FD) are potent determinant of vascular resistance. We investigated the role of angiotensin II and endothelin‐1 in FD and MT in resistance arteries and their potential change in hypertension.

Flow–diameter–pressure relationship was established in situ, under anaesthesia, in two daughter branches of a mesenteric resistance artery (180 μM, n=7 per group) from spontaneously hypertensive (SHR) or normotensive (WKY) rats. One artery was ligated distally, so that it was submitted to pressure only, while the other was submitted to pressure and flow. Drugs were added to the preparation and external diameter, pressure and flow measured continuously.

External diameter (with flow) ranged from 150±3 to 191±7 μM in WKY (n=28) rats and from 168±6 to 186±6 μM in SHR (n=28). Flow induced a dilation of the non‐ligated arteries which was lower in SHR (13±5–31±4 μM vs WKY: 5±5–44±4 μM). In the ligated artery, the diameter did not significantly change, due to MT.

In the vessels submitted to flow angiotensin converting enzyme inhibition (perindopril, 10 μmol L−1) increased the diameter in SHR (+11±2 μM) significantly more than in WKY (+2±1 μM). Angiotensin type 1 receptor (AT1R) blockade (losartan, 10 μmol L−1) increased the diameter in the vessels with flow in SHR only (+6±1 μM). Angiotensin type 2 receptor (AT2R) blockade (PD 123319, 1 μmol L−1) decreased arterial diameter in WKY only (9±2). Endothelin‐1 type A receptor (ETAR) blockade (LU135252, 0.1 μmol L−1) increased the diameter only in SHR in the artery submitted to flow (by 6±1 μM).

Thus FD was counteracted by a flow‐dependent AT1 and ETA receptors‐activation in SHR whereas in WKY FD AT2‐dependent dilation is involved.

British Journal of Pharmacology (2000) 130, 521–526; doi:10.1038/sj.bjp.0703371
Pyrimidinoceptor potentiation of macrophage PGE2 release involved in the induction of nitric oxide synthase
Authors: Chen Bing‐C , Lin Wan‐W
Published: January 2005

We have previously demonstrated that Ca2+/calmodulin‐dependent protein kinase (CaMK) mediates pyrimidinoceptor potentiation of LPS‐elicited inducible nitric oxide synthase (iNOS) induction in murine J774 macrophages. In the present paper, we have explored the role of cyclo‐oxygenase (COX)‐dependent prostaglandin E2 (PGE2) formation in this event.

In J774 macrophages predominantly expressing P2Y6 receptors, the simultaneous addition of UTP and lipopolysaccharide (LPS) resulted in potentiated increase in PGE2 release.

UTP‐induced increased PGE2 release was demonstrated by a concomitant increase in COX‐2 protein expression, and was decreased by inhibitors specific for phosphatidylinositide‐phospholipase C (PI‐PLC), CaMK, protein kinase C (PKC), nuclear factor‐kappa B (NF‐κB) or COX‐2.

NS‐398 (a selective COX‐2 inhibitor) reduced LPS plus UTP‐elicited iNOS induction and nitrite accumulation, supporting for the positive regulation of iNOS gene expression by endogenous PGE2.

Moreover, the cyclic AMP/PKA‐dependent up‐regulation of iNOS expression mediated by PGE2 was drawn from the inhibitory effects of 2′,5′‐dideoxyadenosine, KT5720 and H‐89. Exogenous PGE2 induced NF‐κB activation and potentiated nitrite accumulation in response to LPS.

In addition to COX‐2 induction, arachidonic acid (AA) release and steady‐state mRNA levels of type V secretory phospholipase A2 (sPLA2) and Ca2+‐independent PLA2 (iPLA2) were also increased in the presence of LPS and UTP; the LPS‐induced increase in iPLA2 activity was also potentiated by UTP.

Taken together, we conclude that UTP‐mediated COX‐2 and iPLA2 potentiation and PGE2 formation contribute to the iNOS induction, and that CaMK activation is the primary step in the UTP enhancement of COX‐2 induction.

British Journal of Pharmacology (2000) 130, 777–786; doi:10.1038/sj.bjp.0703375
Effects of chronic fluoxetine treatment in the presence and absence of (±)pindolol: a microdialysis study
Authors: Dawson L A , Nguyen H Q , Smith D I , Schechter L E
Published: January 2005

Using in vivo microdialysis in the frontal cortex of the freely moving rat we evaluated the effects of chronic treatment with the serotonin specific reuptake inhibitor (SSRI) fluoxetine in the presence and absence of the 5‐HT1A/β‐adrenergic antagonist (±)pindolol.

Chronic vehicle treated animals produced no significant response to a challenge with fluoxetine (10 mg kg−1) on day 8 and 15. Alternatively, a significant (P<0.05) decrease in extracellular 5‐HT was observed in control animals upon challenge with the 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT; 0.03 and 0.1 mg kg−1).

Conversely, animals treated with fluoxetine (10 mg kg−1 o.d.) for 7 and 14 days produced a significant (P<0.05) 2 fold increase in extracellular 5‐HT when challenged with fluoxetine (10 mg kg−1) on day 8 and 15. Moreover, no significant decrease in extracellular 5‐HT was observed upon challenge with either dose of 8‐OH‐DPAT.

Animals chronically treated with (±)pindolol (10 or 20 mg kg−1 b.i.d.) produced a significant dose‐related increase in extracellular 5‐HT upon challenge with fluoxetine on day 15 only. Furthermore, both doses produced a significantly blunted response to the low dose challenge of 8‐OH‐DPAT (0.03 mg kg−1). In addition, 20 mg kg−1 (±)pindolol treated animals also had no response to the higher 0.1 mg kg−1 dose of 8‐OH‐DPAT.

Animals treated for 14 days with a combination of (±)pindolol (10 or 20 mg kg−1) and fluoxetine were not significantly different from vehicle treated animals when challenged with fluoxetine or 8‐OH‐DPAT.

Taken together it would therefore appear that although (±)pindolol alone has sufficient intrinsic activity to produce a desensitization of the 5‐HT1A receptor, when given in combination with fluoxetine it is able to prevent the desensitization induced by not only fluoxetine but also itself. This may suggest that the clinical augmentation of antidepressant action by pindolol, when co‐administered with a SSRI, is via antagonism of the 5‐HT1A receptor.

British Journal of Pharmacology (2000) 130, 797–804; doi:10.1038/sj.bjp.0703378
Manganese increases L‐DOPA auto‐oxidation in the striatum of the freely moving rat: potential implications to L‐DOPA long‐term therapy of Parkinson's disease
Authors: Serra Pier Andrea , Esposito Giovanni , Enrico Paolo , Mura Maria A , Migheli Rossana , Delogu M Rosaria , Miele Maddalena , Desole Maria S , Grella Giuseppe , Miele Egidio
Published: January 2005

We have previously shown that manganese enhances L‐dihydroxyphenylanine (L‐DOPA) toxicity to PC12 cells in vitro. The supposed mechanism of manganese enhancing effect [an increase in L‐DOPA and dopamine (DA) auto‐oxidation] was studied using microdialysis in the striatum of freely moving rats.

Systemic L‐DOPA [25 mg kg−1 intraperitoneally (i.p.) twice in a 12 h interval] significantly increased baseline dialysate concentrations of L‐DOPA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and uric acid, compared to controls. Conversely, DA and ascorbic acid concentrations were significantly decreased.

A L‐DOPA oxidation product, presumptively identified as L‐DOPA semiquinone, was detected in the dialysate. The L‐DOPA semiquinone was detected also following intrastriatal infusion of L‐DOPA.

In rats given L‐DOPA i.p., intrastriatal infusion of N‐acetylcysteine (NAC) significantly increased DA and L‐DOPA dialysate concentrations and lowered those of L‐DOPA semiquinone; in addition, NAC decreased DOPAC+HVA and uric acid dialysate concentrations.

In rats given L‐DOPA either systemically or intrastriatally, intrastriatal infusion of manganese decreased L‐DOPA dialysate concentrations and greatly increased those of L‐DOPA semiquinone. These changes were inhibited by NAC infusion.

These findings demonstrate that auto‐oxidation of exogenous L‐DOPA occurs in vivo in the rat striatum. The consequent reactive oxygen species generation may account for the decrease in dialysate DA and ascorbic acid concentrations and increase in enzymatic oxidation of xanthine and DA. L‐DOPA auto‐oxidation is inhibited by NAC and enhanced by manganese. These results may be of relevance to the L‐DOPA long‐term therapy of Parkinson's disease.

British Journal of Pharmacology (2000) 130, 937–945; doi:10.1038/sj.bjp.0703379
Taurine blocks ATP‐sensitive potassium channels of rat skeletal muscle fibres interfering with the sulphonylurea receptor
Authors: Tricarico Domenico , Barbieri Mariagrazia , Camerino Diana Conte
Published: January 2005

Taurine is a sulphonic aminoacid present in high amounts in various tissues including cardiac and skeletal muscles showing different properties such as antioxidative, antimyotonic and anti‐schaemic effects. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction of the sulphonic aminoacid with several ion channels.

Using the patch‐clamp technique we studied the effects of taurine in rat skeletal muscle fibres on ATP‐sensitive K+ channel (KATP) immediately after excision and on channels that underwent rundown.

The cytoplasmic application of 20 mM of taurine reduced the KATP current; this effect was reverted by washout of the drug solution. In this experimental condition the IC50 was 20.1 mM. After rundown, taurine inhibited the KATP current with similar efficacy. Competition experiments showed that taurine shifted the dose‐response inhibition curve of glybenclamide to the left on the log‐dose axis without significantly affecting those of ATP or Ca2+ ion.

Single channel recording revealed that taurine affects the close state of the channel prolonging it and reducing the bursts duration.

Our data indicate that taurine inhibits the muscular KATP channel interfering with the glybenclamide site on the sulphonylurea receptor of the channel or on the site allosterically coupled to it. During ischaemia and hypoxia, the skeletal and heart muscles undergo several changes; for example, the activation of KATP channels and loss of the intracellular taurine content. The depletion of taurine during ischaemia would contribute to the early activation of KATP channels and salvage the intracellular ATP content.

British Journal of Pharmacology (2000) 130, 827–834; doi:10.1038/sj.bjp.0703385
Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology
Authors: Jerman J C , Brough S J , Prinjha R , Harries M H , Davis J B , Smart D
Published: January 2005

The vanilloid receptor (VR1) is a ligand‐gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken.

HEK293 cells stable expressing rVR1 (rVR1‐HEK293) were loaded with Fluo‐3AM and then incubated at 25°C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca2+]i) were monitored using FLIPR, before and after the addition of various agonists.

The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC50 values of 7.47±0.06, 7.16±0.06, 8.19±0.06 and 6.02±0.03 respectively at pH 7.4 vs 7.71±0.05, 7.58±0.14, 8.10±0.05 and 6.04±0.08 at pH 6.4).

Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)‐induced Ca2+ response in rVR1‐HEK293 cells, with pKB values of 7.52±0.08, 6.92±0.11 and 8.09±0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist‐like activity.

The removal of extracellular Ca2+ abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 μM) partially (∼20%) inhibited, the capsaicin (10 μM)‐induced Ca2+ response. However, tetrodotoxin (3 μM), nimodipine (10 μM), ω‐GVIA conotoxin (1 μM), thapsigargin (1 μM), U73122 (3 μM) or H‐89 (3 μM) had no effect on the capsaicin (100 nM)‐induced response.

In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand‐gated Ca2+ channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

British Journal of Pharmacology (2000) 130, 916–922; doi:10.1038/sj.bjp.0703390
Activation of protein kinase B by the A1‐adenosine receptor in DDT1MF‐2 cells
Authors: Germack Renée , Dickenson John M
Published: January 2005

In this study the effect of insulin and A1‐adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT1MF‐2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser473.

Insulin, a recognized activator of PKB, stimulated a concentration‐dependent increase in PKB phosphorylation in DDT1MF‐2 cells (EC50 5±1 pM).

The selective A1‐adenosine receptor agonist N6‐cyclopentyladenosine (CPA) stimulated time and concentration‐dependent increases in PKB phosphorylation in DDT1MF‐2 cells (EC50 1.3±0.5 nM). CPA‐mediated increases in PKB phosphorylation were antagonized by the A1‐adenosine receptor selective antagonist 1,3‐dipropylcyclopentylxanthine (DPCPX) yielding an apparent KD value of 2.3 nM.

Pre‐treatment of DDT1MF‐2 cells with pertussis toxin (PTX, 100 ng ml−1 for 16 h), to block Gi/Go‐dependent pathways, abolished CPA (1 μM) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre‐treatment.

The phosphatidylinositol 3‐kinase (PI‐3K) inhibitors wortmannin (IC50 10.3±0.6 nM) and LY 294002 (IC50 10.3±1.2 μM) attenuated the phosphorylation of PKB elicited by CPA (1 μM) in a concentration‐dependent manner. Wortmannin (30 nM) and LY 294002 (30 μM) also blocked responses to insulin (100 nM).

Removal of extracellular Ca2+ and chelation of intracellular Ca2+ with BAPTA had no significant effect on CPA‐induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31‐8220; 10 μM), tyrosine kinase (genistein; 100 μM), mitogen‐activated protein (MAP) kinase kinase (PD 98059; 50 μM) and p38 MAPK (SB 203580; 20 μM) had no significant effect on CPA‐induced PKB phosphorylation.

In conclusion, these data demonstrate that A1‐adenosine receptor stimulation in DDT1MF‐2 cells increases PKB phosphorylation through a PTX and PI‐3K‐sensitive pathway.

British Journal of Pharmacology (2000) 130, 867–874; doi:10.1038/sj.bjp.0703396
Critical dependence of the NO‐mediated component of cyclic AMP‐induced vasorelaxation on extracellular L‐arginine in pulmonary arteries of the rat
Authors: Hucks D , Ward J P T
Published: January 2005

A component of isoprenaline‐mediated vasorelaxation in pulmonary arteries is mediated by nitric oxide (NO). We examined the effects of physiological concentrations (400 μM) of L‐arginine on isoprenaline‐induced relaxation in rat pulmonary arteries, and following inhibition of L‐arginine uptake with L‐lysine. In addition, we examined the role of the endothelium, and whether L‐arginine affected acetylcholine (ACh)‐induced relaxation.

Isoprenaline‐induced relaxation was potentiated by 400 μM L‐arginine in pulmonary arteries; maximum relaxation was increased from 83±4% of initial tone to 94±4% (P<0.05). L‐lysine (10 mM) not only abolished the potentiation by L‐arginine, but suppressed relaxation compared to control (70±4%, P<0.05), even in the absence of L‐arginine added to the bath. Blockade of NO synthase with 100 μM L‐NMMA or removal of the endothelium inhibited isoprenaline‐induced relaxation to the same extent as L‐lysine, and under these conditions the presence or absence of 400 μM L‐arginine made no difference. L‐lysine had no additional effect when applied in combination with L‐NMMA.

The effect of extracellular L‐arginine was concentration dependent, with an apparent EC50 of ∼1–7 μM.

Relaxation to the membrane permeant cyclic AMP analogue CPT cyclic AMP was also potentiated by L‐arginine and inhibited by L‐lysine. There was however no difference in relaxation induced by acetylcholine (ACh) in the presence of L‐arginine or L‐lysine, and isoprenaline‐induced relaxation of mesenteric arteries was unaffected by L‐arginine or L‐lysine.

These results strongly suggest that extracellular L‐arginine is critically important for development of the NO‐ and endothelium‐dependent component of cyclic AMP‐induced vasorelaxation in rat pulmonary arteries, but is not required for ACh‐induced relaxation. As the apparent EC50 for this effect is in the low micromolar range it is likely to be fully activated in vivo, as plasma L‐arginine is >150 μM.

British Journal of Pharmacology (2000) 130, 997–1004; doi:10.1038/sj.bjp.0703399
Effect of acute nicotine administration on striatal dopamine output and metabolism in rats kept at different ambient temperatures
Authors: Seppä Tiina , Ruotsalainen Minna , Laakso Into , Tuominen Raimo , Ahtee Liisa
Published: January 2005

The effect of ambient temperature on the nicotine‐induced (0.3, 0.5 or 0.8 mg kg−1 s.c.) changes of the striatal concentrations of dopamine (DA) and its metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was studied in freely‐moving rats by in vivo microdialysis.

At the ambient temperature of 30–33°C, but not at 20–23°C, nicotine doses of 0.5 (P<0.01) and 0.8 mg kg−1 (P<0.05) significantly increased the extracellular DA concentration. The nicotine doses of 0.5 and 0.8 mg kg−1 increased the DA metabolite levels similarly at both ambient temperatures studied (P0.0001), but the dose of 0.3 mg kg−1 only at 30–33°C (DOPAC: P<0.05; HVA: P<0.01).

At 30–33°C, dihydro‐β‐erythroidine (DHβE 2.8 mg kg−1 i.p.) blocked the nicotine‐induced (0.5 or 0.8 mg kg−1) increases of extracellular DA concentration but only tended to antagonize the increases of DA metabolites. Mecamylamine (5.0 mg kg−1 i.p.) blocked the increase of DA output induced by 0.5 mg kg−1 but not that induced by 0.8 mg kg−1 of nicotine and fully prevented the nicotine‐induced elevations of DOPAC and HVA.

Elevation of ambient temperature did not affect the cerebral concentration of nicotine or the nicotine‐induced elevation of serum corticosteroids. Also, the rectal temperatures of rats given nicotine at either ambient temperature did not significantly change.

Our results show that the nicotine‐induced output of striatal DA is enhanced at high ambient temperature. Further, our findings suggest that the nicotinic cholinoceptors mediating the effects of nicotine on striatal DA release are different from those mediating nicotine's effects on DA metabolism.

British Journal of Pharmacology (2000) 130, 1147–1155; doi:10.1038/sj.bjp.0703402
Diinosine pentaphosphate: an antagonist which discriminates between recombinant P2X3 and P2X2/3 receptors and between two P2X receptors in rat sensory neurones
Authors: Dunn P M , Liu M , Zhong Y , King B F , Burnstock G
Published: January 2005

We have compared the antagonist activity of trinitrophenyl‐ATP (TNP‐ATP) and diinosine pentaphosphate (Ip5I) on recombinant P2X receptors expressed in Xenopus oocytes with their actions at native P2X receptors in sensory neurones from dorsal root and nodose ganglia.

Slowly‐desensitizing responses to α,β‐methylene ATP (α,β‐meATP) recorded from oocytes expressing P2X2/3 receptors were inhibited by TNP‐ATP at sub‐micromolar concentrations. However, Ip5I at concentrations up to 30 μM was without effect.

Nodose ganglion neurones responded to α,β‐meATP with slowly‐desensitizing inward currents. These were inhibited by TNP‐ATP (IC50, 20 nM), but not by Ip5I at concentrations up to 30 μM.

In DRG neurones that responded to ATP with a rapidly‐desensitizing inward current, the response was inhibited by TNP‐ATP with an IC50 of 0.8 nM. These responses were also inhibited by Ip5I with an IC50 of 0.1 μM. Both antagonists are known to inhibit homomeric P2X3 receptors.

Some DRG neurones responded to α,β‐meATP with a biphasic inward current, consisting of transient and sustained components. While the transient current was abolished by 1 μM Ip5I, the sustained component remained unaffected.

In conclusion, Ip5I is a potent antagonist at homomeric P2X3 receptors but not at heteromeric P2X2/3 receptors, and therefore should be a useful tool for elucidating the subunit composition of native P2X receptors.

British Journal of Pharmacology (2000) 130, 1378–1384; doi:10.1038/sj.bjp.0703404