bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharmacological characterization of the human melatonin Mel1a receptor following stable transfection into NIH3T3 cells
Authors: Nonno Romolo , Lucini Valeria , Pannacci Marilou , Mazzucchelli Cristina , Angeloni Deborah , Fraschini Franco , Stankov Bojidar M 
Published: January 2005

Mouse fibroblasts (NIH3T3) transfected with the full‐length coding region of the Mel1a melatonin receptor stably expressed the receptor, coupled to a pertussis toxin‐sensitive G‐protein(s) and exhibiting high affinity and adequate pharmacological profile.
The receptor protein had the tendency of a strong coupling to the G‐protein and therefore low‐affinity state was induced by uncoupling the receptor from its G‐protein in presence of high concentrations of NaCl (500–700 mM) and/or GTPγS (100 μM). Thereafter, the affinity of a series of melatonin analogues was determined to both, high‐ and low‐affinity receptor states, thus providing a basis for the prediction of their efficacy, according to the ternary complex model.
The cells were subsequently used to study the agonist‐induced G‐protein activation, determined by calculating the rate of GDP‐GTP exchange measured in presence of 35S‐labelled GTPγS. The natural ligand melatonin induced a significant increase in the GDP‐GTP exchange rate, the presence of GDP and NaCl being necessary to observe this effect.
The full agonists 2‐phenylmelatonin, 2‐bromomelatonin and 6‐chloromelatonin equally induced an increase of the GDP‐GTP exchange. 5‐Hydroxy‐N‐acetyltryptamine activated the GTP‐GDP exchange to a much lesser extent (53%) than melatonin, thus behaving as a partial agonist. As predicted by the model, the melatonin antagonist (N‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide) was without effect on basal G protein activation. Coincubation of this compound with melatonin induced a dose‐dependent rightward shift in the melatonin concentration‐effect curve, thus exhibiting the behaviour of a competitive and surmountable antagonist.
Using the equation proposed by Venter (1997) we were able to determine that there were no ‘spare’ receptors in the system. Therefore, the approach proposed in the present work can be successfully used for the determination of ‘drug action’ at the level of the human Mel1a melatonin receptor and evaluation of the efficacy of new selective melatonin analogues.
Effects of tumour necrosis factor‐α on the coronary circulation of the rat isolated perfused heart: a potential role for thromboxane A2 and sphingosine
Authors: Edmunds N J , Woodward B 
Published: January 2005

The actions of tumour necrosis factor‐α (TNF‐α) on the coronary circulation were investigated in the rat isolated heart, perfused under constant flow, recirculating conditions.
An early increase in coronary perfusion pressure (CPP) was observed upon treatment with TNF‐α (increase in CPP 10 min after TNF‐α treatment: 45±12 mmHg vs control: 15±4 mmHg, P<0.05). The role of sphingosine, prostanoids and endothelins, in this coronary constrictor action, was investigated with the use of pharmacological inhibitors and antagonists.
The TNF‐α induced increase in coronary tone was blocked by indomethacin, 10 μM (increase in CPP after 10 min: 13±4 mmHg vs TNF‐α alone, P<0.05).
The thromboxane receptor antagonist GR32191, 10 μM, attenuated the TNF‐α induced coronary constriction (12±2 mmHg vs TNF‐α alone, P<0.05), as did the joint thromboxane A2 synthesis inhibitor and receptor antagonist ZD1542, 10 μM (8±1 mmHg vs TNF‐α alone, P<0.05).
The ceramidase inhibitor N‐oleoylethanolamine (NOE), 1 μM, also blocked the TNF‐α induced response (8±4 mmHg vs TNF‐α alone, P<0.05).
In contrast, the coronary constrictor action of TNF‐α was not inhibited by the endothelinA/B receptor antagonist bosentan, 3 μM (38±9 mmHg vs TNF‐α, P=NS).
These data indicated that the early coronary vasoconstriction induced by TNF‐α was mediated by both thromboxane A2 and sphingosine, suggesting an interaction between both the sphingomyelinase and phospholipase A2 metabolic pathways.
Endothelin receptor subtypes and their functional relevance in human small coronary arteries
Authors: Pierre Lisa N , Davenport Anthony P 
Published: January 2005

The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries.
Small coronary arteries were mounted in a wire‐myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7±7.9 μm; Group B) were compared to those in larger arteries (mean 586.2±23.1 μm; Group A).
ET‐1 consistently and potently contracted arteries from Group A and B, with EC50 values of 1.7 (0.9–3.2) nM (n=15) and 2.3 (1.4–4.2) nM (n=14), respectively. No correlation was observed between ET‐1 potency and internal diameter. The response to ET‐1 was potently antagonized by the selective ETA receptor antagonist PD156707 in both Group A and Group B, yielding pA2 values of 8.60±0.12 (n=4–6) and 8.38±0.17 (n=4–6), respectively. Slopes from Schild regression were not significantly different from unity.
In contrast to ET‐1, individual responses to ET‐3 were variable. While all arteries from Group A responded to ET‐3 (EC50∼69 (23–210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 μM. ET‐1 was more potent than ET‐3 in all arteries tested. A biphasic ET‐3 response was observed in 8 arteries suggesting that a small ETB population was also present in some patients. The selective ETB receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4–6).
Responses to ET‐1 and ET‐3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ETB receptors on endothelial cells (n=5).
Using autoradiography, specific high density binding of the non‐selective, ETA/ETB ligand [125I]‐ET‐1 and selective ETA ligand [125I]‐PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ETB receptor ligand [125I]‐BQ3020 was observed (n=5). Similarly, [125I]‐ET‐1 binding to vascular smooth muscle was absent in the presence of the selective ETA receptor antagonist PD156707.
We conclude that human small epi‐ and intramyocardial coronary arteries express predominantly ETA receptors and it is these receptors which mediate ET‐induced contractions. A constrictor ETB receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET‐1 can be blocked fully by the selective ETA receptor antagonist PD156707.
Functional characterization of a mutated chicken α7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2
Authors: Buckingham Steven D , Adcock Charlotte , Sansom Mark S P , Sattelle David B , Baylis Howard A 
Published: January 2005

Site‐directed mutagenesis was used to create an altered form of the chicken α7 nicotinic acetylcholine (ACh) receptor subunit (α7x61) in which a leucine residue was inserted between residues Leu9′ and Ser10′ in transmembrane domain 2. The properties of α7x61 receptors are distinct from those of the wild‐type receptor.
Oocytes expressing wild‐type α7 receptors responded to 10 μM nicotine with rapid inward currents that desensitized with a time‐constant of 710±409 ms (mean±s.e.mean, n=5). However in α7x61 receptors 10 μM nicotine resulted in slower onset inward currents that desensitized with a time‐constant of 5684±3403 ms (mean±s.e.mean, n=4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild‐type receptors was observed. Dihydro‐β‐erythroidine (DHβE) acted as an antagonist on both receptors.
Molecular modelling of the α7x61 receptor channel pore formed by a bundle of M2 α‐helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10′ would be replaced by the leucine insertion, Val13′ and Phe14′ would be replaced, by Thr and Val, respectively.
When present in the LEV‐1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild‐type and α7x61 receptors. However, block was more dependent on membrane potential for the α7x61 receptors.
We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.
Effects of inhibitors of the activity of cyclo‐oxygenase‐2 on the hypotension and multiple organ dysfunction caused by endotoxin: A comparison with dexamethasone
Authors: Leach Mary , Hamilton Lorna C , Olbrich Antje , Wray Gillian M , Thiemermann Christoph 
Published: January 2005

Endotoxaemia is associated with the expression of the inducible isoform of cyclo‐oxygenase, cyclo‐oxygenase‐2 (COX‐2), and an overproduction of arachidonic acid (AA) metabolites. The role of the AA metabolites generated by COX‐2 in the circulatory failure and multiple organ dysfunction caused by endotoxin is unclear. Dexamethasone prevents the expression of COX‐2 and exerts beneficial effects in animal models of shock.
Here we compare the effects of two inhibitors of COX‐2 activity, namely NS‐398 (5 mg kg−1, i.p., n=7) and SC‐58635 (3 mg kg−1, i.p., n=9) with those of dexamethasone (3 mg kg−1, i.p., n=9) on the circulatory failure and organ dysfunction caused by lipopolysaccharide (LPS, E. coli, 6 mg kg−1, i.v., n=11) in the rat.
Endotoxaemia for 6 h caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of 6‐keto‐PGF1α (indicator of the induction of COX‐2) and nitrite/nitrate (indicator of the induction of iNOS).
Pretreatment of rats with dexamethasone attenuated the hypotension, the renal dysfunction, the hepatocellular and pancreatic injury and the induction of COX‐2 and iNOS caused by LPS. In contrast, inhibition of COX‐2 activity with SC‐58635 or NS‐398 neither attenuated the circulatory failure nor the multiple organ failure caused by endotoxin.
Thus, the prevention of the circulatory failure and the multiple organ injury/dysfunction caused by dexamethasone in the rat is not due to inhibition of the activity of COX‐2. Our results suggest that an enhanced formation of eicosanoids by COX‐2 does not contribute to the development of organ injury and/or dysfunction in rats with endotoxaemia.
Effect of the peripherally selective κ‐opioid agonist, asimadoline, on adjuvant arthritis
Authors: Binder Waltraud , Walker Judith S 
Published: January 2005

Opioids, though widely used as analgesics, have not been seriously considered as therapy for rheumatoid arthritis. The present study evaluated the dose‐effect and time‐dependence relationships of a new peripherally selective κ agonist, asimadoline, in rats with adjuvant arthritis.
The arthritis was assessed by a pooled severity index combining the comprehensive criteria of oedema, radiography and histological changes, in the hind limbs. Asimadoline was extremely effective in attenuating joint damage (by up to 80%) when administered parenterally (0.5 to 10 mg kg−1 day−1, i.p.) throughout the disease or during its early phase; treatment was less successful if confined to the latter stages. Ten fold higher doses were effective orally.
Equimolar doses of a peripherally‐selective antagonist, naloxone methiodide, and the κ‐selective antagonist, MR2266, fully reversed the peripheral anti‐arthritic effects of asimadoline (5 mg kg−1 day−1), indicating that asimadoline acts through peripheral κ‐opioid receptors. However, an equivalent dose of MR2266 did not fully reverse the anti‐arthritic effects of the highest dose of asimadoline (40 mg kg−1 day−1), suggesting a loss of κ‐selectivity at this dose.
Asimadoline also exhibited analgesic effects (mechanical nociceptive thresholds) in arthritic but not non‐arthritic rats, indicating that inflammation is necessary for asimadoline‐induced analgesia.
These data confirm our previous findings that κ‐opioids possess anti‐arthritic properties and that these effects are mediated via peripheral κ‐receptors. The present results are new in showing that the peripherally acting κ‐opioid agonist, asimadoline, is a potent anti‐arthritic agent. Such novel drugs, essentially lacking central side effects, herald new treatments for rheumatoid arthritis.
The involvement of the opioidergic system in the antinociceptive mechanism of action of antidepressant compounds
Authors: Gray A M , Spencer P S J , Sewell R D E 
Published: January 2005

Debate exists as to the nature of antidepressant‐induced antinociception. It is unclear whether antidepressants are inherently antinociceptive, are able to potentiate opioid antinociception or both. We have used the acetic acid induced abdominal constriction assay in mice to investigate antidepressant‐induced antinociception.
All the antidepressants tested (s.c.) produced dose‐dependent protection against acetic acid‐induced abdominal constriction. Similarly, morphine and aspirin were also effective antinociceptive agents in this nociceptive assay.
Opioid antagonists, naloxone (0.5 mg kg−1, s.c.) and naltrindole (1 mg kg−1, s.c.), shifted the dose‐response relationships to the right for each of the antidepressant agents (dothiepin, amitriptyline, sibutramine, (+)‐oxaprotiline and paroxetine). In this context the naloxone dose‐ratios were 1.95, 3.90, 2.32, 4.50 and 2.65, with naltrindole dose‐ratios of 4.36, 17.00, 4.28, 11.48 and 2.65 were obtained, respectively. Naloxone also shifted the morphine dose‐response relationship to the right, by a factor of 2.62, whilst naltrindole had no effect upon morphine antinociception. Aspirin antinociception remained unaffected by both opioid antagonists.
The enkephalin catabolism inhibitor acetorphan, by itself, produced no activity in this test at a dose of 10 mg kg−1 (s.c.). However, at higher doses, acetorphan produced a linear dose‐response relationship against acetic acid‐induced abdominal constriction.
When acetorphan was administered before either the antidepressants or morphine, there was a clear potentiation of the antidepressant‐ or morphine‐induced antinociception. However, acetorphan had no effect on aspirin antinociception.
Since neither of the opioid antagonists were able to attenuate, nor was acetorphan able to potentiate, aspirin antinociception, we concluded that the mechanism of antidepressant‐induced antinociception is different from that of the non‐steroidal anti‐inflammatory drugs.
These data are consistent with the view that antidepressants may induce endogenous opioid peptide release, as shown by the acetorphan study. In this context, the ability of naltrindole to displace the antidepressant dose‐response relationship to the right without affecting morphine antinociception, implicates the δ‐opioid receptor and endogenous opioid peptides in antidepressant‐induced antinociception.
Nitric oxide‐induced cytotoxicity attenuation by thiopentone sodium but not pentobarbitone sodium in primary brain cultures
Authors: Shibuta Satoshi , Kosaka Jun , Mashimo Takashi , Fukuda Yutaka , Yoshiya Ikuto 
Published: January 2005

We describe the effects of barbiturates on the neurotoxicity induced by nitric oxide (NO) on foetal rat cultured cortical and hippocampal neurones. Cessation of cerebral blood flow leads to an initiation of a neurotoxic cascade including NO and peroxynitrite. Barbiturates are often used to protect neurones against cerebrovascular disorders clinically. However, its neuroprotective mechanism remains unclear.
In the present experiment, we established a new in vitro model of brain injury mediated by NO with an NO‐donor, 1‐hydroxy‐2‐oxo‐3‐(3‐aminopropyl)‐3‐isopropyl‐1‐triazene (NOC‐5) on grid tissue culture wells. We also investigated the mechanisms of protection of CNS neurones from NO‐induced neurotoxicity by thiopentone sodium, which contains a sulphhydryl group (SH‐) in the medium, and pentobarbitone sodium, which does not contain SH‐.
Primary cultures of cortical and hippocampal neurones (prepared from 16‐day gestational rat foetuses) were used after 13–14 days in culture. The cells were exposed to NOC‐5 at the various concentrations for 24 h in the culture to evaluate a dose‐dependent effect of NOC‐5.
To evaluate the role of the barbiturates, neurones were exposed to 4, 40 and 400 μM of thiopentone sodium or pentobarbitone sodium with or without 30 μM NOC‐5. In addition, superoxide dismutase (SOD) at 1000 u ml−1 and 30 μM NOC‐5 were co‐administered for 24 h to evaluate the role of SOD.
Exposure to NOC‐5 induced neural cell death in a dose‐dependent manner in both cortical and hippocampal cultured neurones. Approximately 90% of the cultured neurones were killed by 100 μM NOC‐5.
This NOC‐5‐induced neurotoxicity was significantly attenuated by high concentrations of thiopentone sodium (40 and 400 μM) as well as SOD, but not by pentobarbitone sodium. The survival rates of the cortical neurones and hippocampal neurones that were exposed to 30 μM NOC‐5 were 11.2±4.2% and 37.2±3.0%, respectively, and in the presence of 400 μM thiopentone sodium, the survival rate increased to 65.3±3.5% in the cortical neurones and 74.6±2.2% in the hippocampal neurones.
These findings demonstrate that thiopentone sodium, which acts as a free radical scavenger, protects the CNS neurones against NO‐mediated cytotoxicity in vitro. In conclusion, thiopentone sodium is one of the best of the currently available pharmacological agents for protection of neurones against intraoperative cerebral ischaemia.
The relationship between the agonist‐induced activation and desensitization of the human tachykinin NK2 receptor expressed in Xenopus oocytes
Authors: Maudsley S , Gent J P , Findlay J B C , Donnelly D 
Published: January 2005

Repeated applications of neurokinin A (NKA) to oocytes injected with 25 ng wild‐type hNK2 receptor cRNA caused complete attenuation of second and subsequent NKA‐induced responses while analogous experiments using repeated applications of GR64349 and [Nle10]NKA(4–10) resulted in no such desensitization. This behaviour has been previously attributed to the ability of the different ligands to stabilize different active conformations of the receptor that have differing susceptibilities to receptor kinases (Nemeth & Chollet, 1995).
However, for Xenopus oocytes injected (into the nucleus) with 10 ng wild‐type hNK2 receptor cDNA, a single 100 nM concentration of any of the three ligands resulted in complete desensitization to further concentrations.
On the other hand, none of the ligands caused any desensitization in oocytes injected with 0.25 ng wild‐type hNK2 receptor cRNA, even at concentrations up to 10 μM.
The two N‐terminally truncated analogues of neurokinin A have a lower efficacy than NKA and it is likely that it is this property which causes the observed differences in desensitization, rather than the formation of alternative active states of the receptor.
The peak calcium‐dependent chloride current is not a reliable measure of maximal receptor stimulation and efficacy is better measured in this system by studying agonist‐induced desensitization.
The specific adenylyl cyclase inhibitor SQ22536 can enhance NKA and GR64349‐mediated desensitization which suggests that agonist‐induced desensitization involves the inhibition of adenylyl cyclase and the subsequent down‐regulation of the cyclic AMP‐dependent protein kinase, possibly by cross‐talk to a second signalling pathway.
5‐HT potentiation of the GABAA response in the rat sacral dorsal commissural neurones
Authors: Xu Tian‐Le , Pang Zhi‐Ping , Li Ji‐Shuo , Akaike Norio 
Published: January 2005

The modulatory effect of 5‐hydroxytryptamine (5‐HT) on the γ‐aminobutyric acidA (GABAA) response was investigated in the neurones freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage‐clamp conditions.
5‐HT potentiated GABA‐induced Cl− current (IGABA) without affecting the reversal potential of IGABA and the apparent affinity of GABA to its receptor.
α‐Methyl‐5‐HT mimicked the potentiation effect of 5‐HT on IGABA while ketanserine blocked it. 1‐Oleoyl‐2‐acetyl‐glycerol (OAG) potentiated IGABA, and the effect of 5‐HT on IGABA was occluded by OAG pretreatment. In the presence of chelerythrine, 5‐HT failed to potentiate IGABA, suggesting that protein kinase C (PKC) is involved in the pathway through which the activation of the 5‐HT2 receptor potentiates the IGABA.
The facilitatory effect of 5‐HT on IGABA remained in the presence of BAPTA‐AM. LiCl also had no effect on 5‐HT‐induced potentiation of IGABA.
H‐89, genistein, okadaic acid and pervanadate all had no effects on 5‐HT potentiation of IGABA. Pertussis toxin treatment for 6–8 h did not block the facilitatory effect of 5‐HT on IGABA.
The present results show that GABAA receptor in the rat SDCN could be modulated in situ by 5‐HT, one of the major transmitters involved in the supraspinal control of nociception, and that the phosphorylation of GABAA receptor by PKC may be sufficient to support such modulation. The results also strongly support the hypothesis that the cotransmission by 5‐HT and GABA has an important role in the spinal cord.
Novel inhibition of contractility by wortmannin in skeletal muscle
Authors: Hong S J. , Chang C C. 
Published: January 2005

The effects of wortmannin and 2‐(4‐morpholinyl)‐8‐phenyl‐1[4H]‐benzopyran‐4‐one (LY294002), inhibitors of phosphatidylinositol 3‐kinase, on the contractile responses of murine skeletal muscle were studied. Wortmannin (10–100 μM) suppressed twitch and tetanic contraction evoked by field stimulation of diaphragm without causing elevation of muscle tone. The inhibition was quasi‐irreversible with IC50∼15 μM. In contrast, LY294002 increased twitch responses and elevated muscle tone.
Wortmannin reversibly depressed the maximal slope of action potential upstroke by ∼40% and inhibited the membrane depolarization and spontaneous burst of action potential induced by crotamine, a polypeptide toxin that activates the Na+ channel of skeletal muscle.
Wortmannin inhibited contractures evoked by high K+, ryanodine and caffeine, but potentiated the contracture induced by rapamycin, which binds to myoplasmic FK506 binding protein, an immunophilin closely associated with the ryanodine receptor. The contractures elicited by cardiotoxin, which disrupts the integrity of sarcolemma and thereby elevates ‘myoplasmic’ Ca2+ level, were suppressed only slightly.
In placed left atrium and ventricular strip, wortmannin and LY294002 produced a positive inotropic effect.
The results suggest that, in addition to depressing the Ca2+ mobilization from sarcoplasmic reticulum, wortmannin exerts a novel inhibitory action on the excitation‐contraction coupling in skeletal muscle but not in cardiac muscle.