bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
Differential A1‐adenosine receptor reserve for inhibition of cyclic AMP accumulation and G‐protein activation in DDT1 MF‐2 cells
Authors: Baker Stephen P , Scammells Peter J , Belardinelli Luiz 
Published: January 2005

The A1‐adenosine receptor (A1AdoR) reserve for N6‐cyclopentyladenosine (CPA) mediated inhibition of (−)isoprenaline stimulated cyclic AMP accumulation and stimulation of [35S]‐guanosine‐5′‐O‐(thiotriphosphate) (GTPγS) binding, a measure of guanine nucleotide binding protein (G‐protein) activation, was determined in DDT1 MF‐2 cells.
Inactivation of the A1AdoRs with the chemoreactive ligand 8‐cyclopentyl‐3‐[3‐[[4‐(fluorosulphonyl)benzoyl]oxy]propyl]‐1‐propylxanthine (FSCPX) caused a progressive rightward shift of the concentration‐response curves for CPA to inhibit cyclic AMP accumulation, with a maximum of 10 fold increase in the EC50 value. In contrast, inactivation of A1AdoR's caused only a 1.7 fold rightward shift in the CPA concentration‐response for stimulation of [35S]‐GTPγS binding.
The A1AdoR occupancy‐response relationship for CPA inhibition of cyclic AMP accumulation was hyperbolic with 43% receptor occupancy required to elicit the maximal response, i.e. a 57% A1AdoR reserve. In contrast, the A1AdoR occupancy‐response relationship for CPA mediated stimulation of [35S]‐GTPγS binding was linear indicating little or no receptor reserve for G‐protein activation. The relationship between CPA stimulation of [35S]‐GTPγS binding and cyclic AMP inhibition was also hyperbolic with 44% G‐protein activation sufficient to cause maximal inhibition.
The data suggest that the A1AdoR reserve for CPA mediated inhibition of cyclic AMP accumulation occurs at the level of G‐protein interaction with adenylyl cyclase. However, each A1AdoR appears to activate a constant fraction of the total G‐protein population suggesting signal amplification at the receptor‐G‐protein level which may also contribute to the receptor reserve for CPA.

British Journal of Pharmacology (2000) 130, 1156–1164; doi:10.1038/sj.bjp.0703405
Apoptosis in the vasculature: mechanisms and functional importance
Authors: Mallat Ziad , Tedgui Alain 
Published: January 2005

Apoptotic death has now been recognized in a number of common and threatening vascular diseases, including atherosclerosis. Interest in apoptosis research relates to the fact that apoptosis, in contrast to oncosis, is a highly regulated process of cell death which raises the hope for the development of specific therapeutic strategies to alter disease progression. This review summarizes the mechanisms involved in vascular endothelial and smooth muscle cell survival/apoptosis, and the potential roles of apoptotic death in atherosclerosis and restenosis. The potential effects of modulation of apoptosis in these diseases are also discussed. British Journal of Pharmacology (2000) 130, 947–962; doi:10.1038/sj.bjp.0703407
Cholinergic facilitation of neurotransmission to the smooth muscle of the guinea‐pig prostate gland
Authors: Lau W A K , Pennefather J N , Mitchelson F J 
Published: January 2005

Functional experiments have been conducted to assess the effects of acetylcholine and carbachol, and the receptors on which they act to facilitate neurotransmission to the stromal smooth muscle of the prostate gland of the guinea‐pig.
Acetylcholine and carbachol (0.1 μM–0.1 mM) enhanced contractions evoked by trains of electrical field stimulation (20 pulses of 0.5 ms at 10 Hz every 50 s with a dial setting of 60 V) of nerve terminals within the guinea‐pig isolated prostate. In these concentrations they had negligible effects on prostatic smooth muscle tone. The facilitatory effects of acetylcholine, but not those of carbachol, were further enhanced in the presence of physostigmine (10 μM).
The facilitatory effects of carbachol were unaffected by the neuropeptide Y Y1 receptor antagonist BIBP 3226 ((R)‐N2‐(diphenylacetyl)‐N‐[(4‐hydroxyphenyl)methyl]‐argininamide) (0.3 μM, n=3) or suramin (100 μM, n=5). Prazosin (0.1 μM, n=5) and guanethidine (10 μM, n=5) alone and in combination (n=4), reduced responses to field stimulation and produced rightward shifts of the log concentration‐response curves to carbachol.
The rank orders of potency of subtype‐preferring muscarinic receptor antagonists in inhibiting the facilitatory actions of acetylcholine and carbachol were: pirenzepine > HHSiD (hexahydrosiladifenidol) > pF‐HHSiD (para‐fluoro‐hexahydrosiladifenidol) 5 himbacine, and pirenzepine > HHSiD > himbacine 5 pF‐HHSiD, respectively. These profiles suggest that muscarinic receptors of the M1‐subtype mediate the facilitatory effects of acetylcholine and carbachol on neurotransmission to the smooth muscle of the guinea‐pig prostate.

British Journal of Pharmacology (2000) 130, 1013–1020; doi:10.1038/sj.bjp.0703409
The inhibition of cholera toxin‐induced 5‐HT release by the 5‐HT3 receptor antagonist, granisetron, in the rat
Authors: Turvill J L , Connor P , Farthing M J G 
Published: January 2005

The secretagogue 5‐hydroxytryptamine (5‐HT) is implicated in the pathophysiology of cholera. 5‐HT released from enterochromaffin cells after cholera toxin exposure is thought to activate non‐neuronally (5‐HT2 dependent) and neuronally (5‐HT3 dependent) mediated water and electrolyte secretion. CT‐secretion can be reduced by preventing the release of 5‐HT.
Enterochromaffin cells possess numerous receptors that, under basal conditions, modulate 5‐HT release. These include basolateral 5‐HT3 receptors, the activation of which is known to enhance 5‐HT release.
Until now, 5‐HT3 receptor antagonists (e.g. granisetron) have been thought to inhibit cholera toxin‐induced fluid secretion by blockading 5‐HT3 receptors on secretory enteric neurones. Instead we postulated that they act by inhibiting cholera toxin‐induced enterochromaffin cell degranulation.
Isolated intestinal segments in anaesthetized male Wistar rats, pre‐treated with granisetron 75 μg kg−1, lidoocaine 6 mg kg−1 or saline, were instilled with a supramaximal dose of cholera toxin or saline. Net fluid movement was determined by small intestinal perfusion or gravimetry and small intestinal and luminal fluid 5‐HT levels were determined by HPLC with fluorimetric detection.
Intraluminal 5‐HT release was proportional to the reduction in tissue 5‐HT levels and to the onset of water and electrolyte secretion, suggesting that luminal 5‐HT levels reflect enterochromaffin cell activity.
Both lidocaine and granisetron inhibited fluid secretion. However, granisetron alone, and proportionately, reduced 5‐HT release.
The simultaneous inhibition of 5‐HT release and fluid secretion by granisetron suggests that 5‐HT release from enterochromaffin cells is potentiated by endogenous 5‐HT3 receptors. The accentuated 5‐HT release promotes cholera toxin‐induced fluid secretion.

British Journal of Pharmacology (2000) 130, 1031–1036; doi:10.1038/sj.bjp.0703414
Inhibition of mouse neuromuscular transmission and contractile function by okadaic acid and cantharidin
Authors: Hong S J 
Published: January 2005

Phosphorylations of cellular proteins modulate biological activities. The effects of okadaic acid (0.1–10 μM) and cantharidin (1–100 μM), inhibitors of protein phosphatases, on the synaptic transmission at the mouse neuromuscular junction were explored.
Both inhibitors almost completely depressed twitch forces elicited by electrical stimulation of diaphragm muscles (the IC50s for okadaic acid and cantharidin were 1.1±0.2 and 13±1 μM, n=5, respectively) and suppressed contractures evoked by high K+ and ryanodine more than 70%. Contractures caused by cardiotoxin, which destroys the integrity of sarcolemma, were not depressed.
Both okadaic acid (10 μM) and cantharidin (100 μM) depolarized muscle membranes from ∼−80 to ∼−60 mV in a partially reversible and tetrodotoxin‐sensitive manner. The initial short‐term enhancement of twitch responses (up to ∼40%) was correlated with the inhibitors‐induced repetitive firings of muscle action potential.
Treatment with either agent resulted in nearly complete inhibitions of endplate potential (epp). The IC50s were 0.8±0.2 and 9±2 μM (n=5), respectively, for okadaic acid and cantharidin. On high frequency stimulation, the coefficient of epps was increased more than 10 fold and the extent of epp run‐down during stimulations intensified from ∼25 to ∼75%. Analyses of presynaptic quantal releases revealed decreases in epp quantal content and the immediately available vesicle pool.
The frequency of miniature epp was initially elevated up to 2 fold then suppressed down to ∼30%. The small reduction in the amplitude was antagonized when the membrane of endplate area was repolarized.
The data suggest that okadaic acid and cantharidin inhibit mobilizations of synaptic vesicles and depress Ca2+ release from sarcoplasmic reticulum and that protein phosphatases participate in the modulation of motor function.

British Journal of Pharmacology (2000) 130, 1211–1218; doi:10.1038/sj.bjp.0703418
Beneficial effects of n‐acetylcysteine on ischaemic brain injury
Authors: Cuzzocrea Salvatore , Mazzon Emanuela , Costantino Giuseppina , Serraino Ivana , Dugo Laura , Calabrò Giusy , Cucinotta Giovanni , De Sarro Angela , Caputi A P 
Published: January 2005

Nitric oxide (NO), peroxynitrite, formed from NO and superoxide anion, poly (ADP‐ribole) synthetase have been implicated as mediators of neuronal damage following focal ischaemia. Here we have investigated the effects of n‐acetylcysteine (NAC) treatment in Mongolian gerbils subjected to cerebral ischaemia.
Treatment of gerbils with NAC (20 mg kg−1 30 min before reperfusion and 1, 2 and 6 h after reperfusion) reduced the formation of post‐ischaemic brain oedema, evaluated by water content.
NAC also attenuated the increase in the brain levels of malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischaemia.
Positive staining for nitrotyrosine was found in the hippocampus in Mongolian gerbils subjected to cerebral ischaemia. Hippocampus tissue sections from Mongolian gerbils subjected to cerebral ischaemia also showed positive staining for poly (ADP‐ribose) synthetase (PARS). The degree of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received NAC.
NAC treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischaemia and reperfusion.
Histological observations of the pyramidal layer of CA1 showed a reduction of neuronal loss in animals that received NAC.
These results show that NAC improves brain injury induced by transient cerebral ischaemia.

British Journal of Pharmacology (2000) 130, 1219–1226; doi:10.1038/sj.bjp.0703421
Effects of riluzole on electrically evoked neurotransmitter release
Authors: Jehle T , Bauer J , Blauth E , Hummel A , Darstein M , Freiman T M , Feuerstein T J 
Published: January 2005

The main purpose of the present study was to investigate the effects of the neuroprotective agent riluzole on the electrically evoked release of [3H]‐glutamate ([3H]‐Glu) in mouse neocortical slices. The reported selectivity of riluzole for excitatory amino acids was tested in release experiments with further neurotransmitters. Also distinct species, mouse, rat and man were compared.
[3H]‐Glu was formed endogenously during incubation of slices with [3H]‐glutamine ([3H]‐Gln). Released [3H]‐Glu and tissue [3H]‐Glu was separated by anion exchange chromatography. Electrically evoked [3H]‐Glu release was strongly diminished by tetrodotoxin (TTX) and Ca2+‐withdrawal.
Riluzole (100 μM) depressed the release of [3H]‐Glu up to 77% (IC50=19.5 μM). Riluzole was also able to inhibit strongly the electrically evoked release of [3H]‐acetylcholine ([3H]‐ACh) (at 100 μM by 92%, IC50=3.3 μM, and [3H]‐dopamine ([3H]‐DA) (at 32 μM by 72%, IC50=6.8 μM). However, the release of [3H]‐serotonin ([3H]‐5‐HT) was less diminished (at 100 μM by 53%, IC50=39.8 μM). Riluzole up to 100 μM did not affect [3H]‐noradrenaline ([3H]‐NA) release.
Between species, i.e. in mouse, rat and human neocortex, no significant differences between the effects of riluzole could be observed.
The NMDA‐receptor blocker MK‐801 (1 μM) and the AMPA/Kainate‐receptor blocker NBQX (1 μM) did neither affect the electrically evoked [3H]‐ACh release nor its inhibition by riluzole, indicating that effects of riluzole on transmitter release were neither due to modulation of ionotropic Glu receptors, nor due to indirect inhibition of Glu release through these receptors.
Taken together, riluzole inhibits the release of distinct neurotransmitters differently, but is not selective for the excitatory amino acid Glu.

British Journal of Pharmacology (2000) 130, 1227–1234; doi:10.1038/sj.bjp.0703424