bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of three non‐peptide endothelin receptor ligands using human cloned ETA and ETB receptors
Authors: Buchan Kevin W. , Alldus Carl , Christodoulou Chris , Clark Kenneth L. , Dykes Colin W. , Sumner Michael J. , Wallace Donald M. , White David G. , Watts Ian S. 
Published: July 2012

A number of putative endothelin (ET) receptor ligands were synthesized with a view to assessing their relative affinity for human recombinant ET receptors.
Human (h) and endothelin ETA and ETB receptor open reading frames were cloned by reverse transcription‐polymerase chain reaction into the mammalian expression vector pcDNA1 and stable cell lines were created by transfection of Chinese hamster ovary cells.
Scatchard analyses of saturation isotherms for the specific binding of [125I]‐endothelin‐1 ([125I]‐ET‐1) to membranes, prepared from Chinese hamster ovary cells transfected with hETA or hETB receptors, yielded values for equilibrium dissociation constants (Kd) of 20.5 ± 1.8 pM and 25.5 ± 5.5 pM, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogeneous, non‐interacting receptor populations.
Pharmacological characterization of the transfected hETA and hETB receptors was undertaken by measuring the relative abilities of ETA and ETB receptor‐selective peptide ligands to inhibit binding of [125I]‐ET‐1. For interaction with hETA receptors, the relative order of potency was ET‐1 > ET‐3 = FR139317 = BQ123 > [Ala1,3,11,15]‐ET‐1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hETB receptors, was ET‐1 = ET‐3 = [Ala1,3,11,15]‐ET‐1 = S6c >> FR139317 = BQ123.
The novel non‐peptide ligands, Ro 46–2005, SB 209670 and BMS 182874, were found to inhibit [125I]‐ET‐1 binding to human recombinant ETA and ETB receptors. At hETA receptors, the calculated pIC50 values were 6.7 (Ro 46–2005), 8.7 (SB 209670) and 5.8 (BMS 182874), while at hETB receptors, the corresponding pIC50 values were 6.8, 7.5 and < 5, respectively.
In conclusion, we have characterized the pharmacology of human cloned ETA and ETB receptors and used these in membrane binding assays to determine the affinity and selectivity of three structurally‐diverse non‐peptide ET receptor ligands. SB 209670 is, to date, the highest affinity non‐peptide ligand to be described for ET receptors. As such, it may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of endothelins.
Cyclo‐oxygenase and nitric oxide synthase isoforms in rat carrageenin‐induced pleurisy
Authors: Tomlinson Annette , Appleton Ian , Moore Adrian R. , Gilroy Derek W. , Willis Dean , Mitchell Jane A. , Willoughby Derek A. 
Published: July 2012

The profiles of cyclo‐oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin‐induced pleurisy model of acute inflammation.
The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates.
COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6‐keto prostaglandin F1α) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out.
NOS activity was based on the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS).
Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced.
Western blot analysis demonstrated that the inducible isoform of COX (COX‐2), was the predominant enzyme at all time points. Low levels of COX‐2 were seen in PBLs. In pleural exudate cell pellets maximal COX‐2 protein levels were seen at 2h.
Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX‐2. In cell pellet smears from carrageenin‐induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX‐2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX‐2.
Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6h and greatly reduced by 24 h. This activity was solely attributable to iNOS.
The present results illustrated a similar profile of COX and NOS activity in the carrageenin‐induced pleurisy model of acute inflammation. It was demonstrated that COX‐2 and iNOS were the predominant isoforms of their respective enzymes.
The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites
Authors: Szabó Csaba , Southan Garry J. , Thiemermann Christoph , Vane John R. 
Published: July 2012

We have recently found that in the presence, but not in the absence, of foetal calf serum, spermine inhibits the production of nitric oxide (NO) in cultured J774.2 macrophages stimulated with bacterial endotoxin (lipopolysaccharide; LPS) or with 7‐interferon (IFN), showing that polyamines may act as suppressants of NO‐mediated immune functions. Here, we have studied the mechanisms and the specificity of this inhibitory action.
Other polyamines, as well as spermine, inhibit the formation of NO in cultured J774.2 macrophages, with the order of potency being spermine > spermidine > > putrescine = cadaverine. This inhibition of NO formation is not due to any cytotoxic effect of these agents for they neither reduced mitochondrial respiration nor increased the release of lactate dehydrogenase into the supernatant.
Spermine is not a direct inhibitor of the activity of iNOS in induced J774.2 cells as measured by its lack of effect on the conversion of L‐arginine to L‐citrulline in homogenates. Neither spermine, nor its metabolites, interfere with the production of nitrite from NO or act as scavengers of NO. Thus, spermine is an inhibitor of the induction of iNOS.
Spermine inhibits nitrite formation in the presence of foetal, newborn or adult bovine serum, but not rat or human serum.
The effect of sper mine on nitrite production can be prevented by isoniazid, hydrazine or hydroxy‐lamine, inhibitors of spermine oxidase, as well as by phenylhydrazine, an aldehyde inhibitor. We have, therefore, tested the effects of spermine dialdehyde or malon dialdehyde on the induction of iNOS. Spermine dialdehyde (SDA, 10−5m) inhibits nitrite formation by IFN‐activated J774.2 cells in the absence of serum when given as a pretreatment but not when given 6 h after stimulation. In contrast, malon dialdehyde was ineffective. Thus, aldehyde metabolites of spermine, such as SDA, account for the inhibitory effect of polyamines on the induction of NOS in vitro.
The inhibitory effect of polyamines on iNOS induction appears to be fairly specific to iNOS, for spermine does not inhibit LPS‐induced production of prostaglandin F2α or tumour necrosis factor.
Contribution of intra‐ and extracellular Ca2+ to noradrenaline exocytosis induced by ouabain and monensin from guinea‐pig vas deferens
Authors: Katsuragi Takeshi , Ogawa Shusuke , Furukawa Tatsuo 
Published: July 2012

Contributions of intra‐ and extracellular Ca2+ to noradrenaline (NA) release evoked by increasing intracellular Na+ concentrations (ouabain plus monensin) from adrenergic nerves of guinea‐pig vas deferens were evaluated under conditions eliminating carrier‐mediated NA release (with 100 μm cocaine).
Ouabain (100 μm) plus monensin (10 μm), unlike 100 mm KCl, produced a marked NA release which was unchanged by Ca2+‐removal.
In normal solution but not in Ca2+‐free solution, the release of NA evoked by ouabain plus monensin was reduced by adenosine, clonidine and neuropeptide Y, and by Ca2+‐channel blockers such as ω‐conotoxin GVIA and nifedipine. The release of NA was also decreased by cromakalim in a glibenclamide‐sensitive fashion.
In contrast, in the absence but not in the presence of Ca2+, the drug‐evoked NA release was inhibited by mitochondrial inhibitors (carbonylcyanide‐m‐chlorophenylhydrazone and olivomycin) and further by immobilizers of intracellular Ca2+ (TMB‐8 and BAPTA‐AM) and calmodulin antagonists (W‐7 and trifluoperazine).
These findings suggest that the release of NA evoked by elevation of [Na+1 from adrenergic nerves in the presence and absence of Ca2+ involves, in part, exocytotic processes which are triggered by depolarization‐induced Ca2+ influx and by utilization of Ca2+ from intracellular Ca2+ store sites such as mitochondria, respectively.
Evidence for P2‐purinoceptor‐mediated inhibition of noradrenaline release in rat brain cortex
Authors: von Kügelgen Ivar , Späth Leni , Starke Klaus 
Published: July 2012

Some postganglionic sympathetic axons possess P2Y‐like P2‐purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]‐noradrenaline, then superfused with medium containing desipramine (1 μm) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz.
The selective adenosine A1‐receptor agonist, N6‐cyclopentyl‐adenosine (CPA; 0.03‐3 μm) as well as the non‐subtype‐selective agonist 5′‐N‐ethylcarboxamido‐adenosine (NECA; 0.3‐3 μm) reduced the evoked overflow of tritium, whereas the adenosine A2a‐receptor agonist, 2‐p‐(2‐carbonylethyl)‐phenethylamino‐5′‐N‐ethylcarboxamido‐adenosine (CGS‐21680; 0.003–30 μm) and the adenosine A3‐receptor agonist N6‐2‐(4‐aminophenyl)ethyl‐adenosine (APNEA; 0.03‐3 μm) caused no change. Of the nucleotides tested, ATP (30–300 μm), adenosine‐5′‐0‐(3‐thiotriphosphate) (ATP7S; 30–300 μm), adenosine‐5′‐0‐(2‐thiodiphosphate) (ADPγS; 30–300 μm), P1, P4‐di(adenosine‐5−)‐tetraphosphate (Ap4A; 30–300 μm) and the preferential P2Y‐purinoceptor agonist, 2‐methylthio‐ATP (300 μm) decreased the evoked overflow of tritium. The P2X‐purinoceptor agonist, α,β‐methylene‐ATP (3–300 μm) caused no change.
The A1‐selective antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 10 nm) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP7S (apparent pKB 9.2) and ADPβS (apparent pKB 8.7). CGS‐21680 and APNEA were ineffective also in the presence of DPCPX. The A2‐selective antagonist 1,3‐dipropyl‐8‐(3,4‐dimethoxystyryl)‐7‐methylxanthine (KF‐17837) reduced the effects of CPA, NECA and ATPγS only when given at a concentration of 300 nm but not at 10 nm.
The P2‐purinoceptor antagonists, suramin (300 μm), reactive blue 2 (30 μm) and cibacron blue 3GA (30 μm) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration‐response curve of ATPγS to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPγS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration‐response curves were not shifted to the right. There was no antagonistic effect of suramin against ATP and ADPβS.
The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the known adenosine A1‐receptor, a separate purinoceptor for nucleotides (P2) which, in contrast to the A1‐receptor, is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATPγS activate both receptors. Inconsistencies in antagonist effects against nucleotides are probably due to this activation of two receptors. The presynaptic P2‐purinoceptor is P2Y‐like, as it is in the peripheral sympathetic nervous system.
Effects of dihydropyridine calcium antagonists on rat midbrain dopaminergic neurones
Authors: Mercuri Nicola B. , Bond Antonello , Calabresi Paolo , Stratta Francesca , Stefani Alessandro , Bernardi Giorgio 
Published: July 2012

The effects of the dihydropyridine calcium channel antagonists, nifedipine and nimodipine (300 nm‐30 μm) were tested in vitro on intracellularly recorded dopaminergic neurones in the rat ventral mesencephalon.
Bath applied nifedipine and nimodipine inhibited in a concentration‐dependent manner the spontaneous firing discharge of the action potentials, whereas, the dihydropyridine calcium channel agonist, Bay K 8644 increased the firing rate.
Pacemaker oscillations and bursts of action potentials were produced by loading the cells with caesium. Nifedipine and nimodipine reduced the rate and the duration of the caesium‐induced membrane oscillations and decreased the number of action potentials in a burst. During the blockade of potassium currents the dopaminergic neurones often developed a prolonged (100–800 ms) afterdepolarization that was also inhibited by dihydropyridines.
The spontaneous discharge of calcium spikes was also inhibited by both dihydropyridine calcium antagonists. The apparent input resistance and the level of membrane potential were not affected by the dihydropyridine calcium antagonists.
If the action potential duration was less than 150 ms the shape of the spike was not clearly influenced by both calcium antagonists. However, when the duration of the action potential was longer than 150–200 ms due to the intracellular injection of caesium ions plus the extracellular application of tetraethylammonium (10–50 μm), both nifedipine and nimodipine reversibly shortened the plateau potential.
It is suggested that nifedipine and nimodipine depress the rhythmic and bursting activity of the dopaminergic cells and shorten the calcium action potential by blocking dihydropyridine‐sensitive high‐threshold calcium currents.
Characterization of P2‐purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses
Authors: Evans R.J. , Kennedy C. 
Published: July 2012

The electrophysiological actions of the P2‐purinoceptor agonists, adenosine 5′‐triphosphate (ATP), 2‐methylthioATP (2‐meSATP), α,β‐methyleneATP (α,β‐meATP) and uridine 5′‐triphosphate (UTP) were studied under concentration and voltage‐clamp conditions in acutely dissociated rat tail artery smooth muscle cells. For comparison, their actions as vasoconstrictors were studied in intact ring preparations.
Rapid application of ATP (100 nm‐1 μm) via a U‐tube supervision system activated concentration‐dependent inward currents with a latency to onset of less than 3 ms. The inward current decayed by more than 95% during a 2 s application of 300 nm and 1 μm ATP.
2‐meSATP (100nM‐1 μm) and α,β‐meATP (100 nm‐1 μm) also evoked transient inward currents. The agonist order of potency was ATP = 2‐meSATP ≥ α,β‐meATP. UTP (300nM‐1μm) did not produce a change in the holding current.
A second application of ATP (300 nm and 1 μm) 10 min after the first, evoked currents which were one third of the initial amplitude. This decline was dependent upon activation of the P2‐purinoceptor. Similar results were seen with 2‐meSATP and α,β‐meATP (both 300 nm and 1 μm). Cross‐desensitization was seen between ATP and 2‐meSATP or α,β‐meATP.
Inward currents evoked by ATP, 2‐meSATP and α,β‐meATP (all 1 μm) were abolished by the P2‐purinoceptor antagonist suramin (100 μm).
αβ‐meATP (100 nm‐30 μm), 2‐meSATP (3 μm‐100 μm), ATP (3 μm‐ 1 μm) and UTP (3 μm‐ 1 μm) produced concentration‐dependent contractions of rat tail artery rings. When measured at a level equal to 50% of the maximum response to noradrenaline, the rank order of agonist potency was α,β,‐meATP > > 2‐meSATP >UTP >ATP.
This study shows that the rank order of agonist potency at the P2X‐purinoceptor which mediates contractions of the rat isolated tail artery is very different from the potency order for evoking the inward current which initiates the contractions. It is concluded that this difference may be due to the relative absence of breakdown of some of the agonists in the single cell system compared with artery rings.
Inhibition by SK&F 96365 of Ca2+ current, IL‐2 production and activation in T lymphocytes
Authors: Chung S. Clare , McDonald Thomas V. , Gardner Phyllis 
Published: July 2012

By use of whole cell patch‐clamp and Indo‐1 fluorescence studies of the Jurkat T leukaemic cell line, we show that the new organic antagonist of receptor‐mediated Ca2+ entry, SK&F 96365, inhibits the T cell Ca2+ current in a dose‐dependent fashion, with an IC50 of 12 μm.
SK&F 96365 also inhibits [3H]‐thymidine incorporation and interleukin‐2 (IL‐2) synthesis in peripheral blood lymphocytes.
SK&F 96365 has no effect on Ca2+ stores release or K+ channels.
This is the first account of an organic inhibitor of the T cell Ca2+ current. The ability of SK&F 96365 to inhibit IL‐2 synthesis and cell proliferation suggests that a new class of related Ca2+ channel blockers can be developed as immunosuppressive agents.
Presence of neuropeptide Y in the rat seminal vesicle and its effects on noradrenaline‐ and nerve‐induced contractions
Authors: Iravani M.M. , Zar M.A. 
Published: July 2012

Immunohistochemical and functional studies have been performed to localize and determine the effects of neuropeptide Y (NPY) in the rat seminal vesicle.
An abundant presence of NPY‐immunoreactive nerves, mainly concentrated in the smooth muscle layer of the seminal vesicle was found. Chronic 6‐hydroxydopamine treatment (four doses of 50 mg kg−1 i.p. on days 1, 2, 4 and 6; rats killed one week after the last injection) led to a large reduction but not abolition of the NPY‐immunoreactivity.
NPY (1‐250nM) did not affect the resting tone of the seminal vesicle.
The seminal vesicle was contracted by electrical field stimulation (EFS) and by exposure to 5 μm noradrenaline (NA). These contractions were abolished by phentolamine (1 μm). Tetrodotoxin (0.5 μm) abolished EFS‐evoked contractions but did not affect NA‐evoked contractions.
Seminal vesicles, from animals chronically‐treated with reserpine (5 mg kg−1 i.p. on days 1 and 2; rats killed on day 3), were contracted by NA but not by EFS.
NPY (0.25–250 nm), concentration‐dependently, inhibited EFS‐evoked contractions by up to 70% maximum inhibition. Contractions evoked by EFS with short trains of pulses were inhibited by NPY to a greater degree than those with longer trains.
NPY had no significant effect on NA‐evoked contractions.
These data provide strong evidence that the motor transmission in rat seminal vesicle is predominantly if not exclusively, adrenergic. It is further concluded that a rich NPY‐containing innervation is present in the smooth muscle layer of rat seminal vesicle. The primary effect of NPY is modulation of adrenergic motor transmission by a prejunctional inhibition of NA release.
Location of the sulphonylurea receptor at the cytoplasmic face of the β‐cell membrane
Authors: Schwanstecher Mathias , Schwanstecher Christina , Dickel Corinna , Chudziak Franz , Moshiri Ali , Panten Uwe 
Published: July 2012

In insulin‐secreting cells the location of the sulphonylurea receptor was examined by use of a sulphonylurea derivative representing the glibenclamide molecule devoid of its cyclohexyl moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH7.4 compound IV is only present in charged form.
Lipid solubility declined in the order tolbutamide > compound III > compound IV.
The dissociation constant (KD) for binding of compound IV to the sulphonylurea receptor in HIT‐cells (pancreatic β‐cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III.
In mouse pancreatic β‐cells, drug concentrations inhibiting adenosine 5′‐triphosphate‐sensitive K+ channels (KATP‐channels) half‐maximally (EC50) were determined by use of the patch‐clamp technique. When the drugs were applied to the extracellular side of outside‐out or the intracellular side of inside‐out membrane patches, the ratio of extracellular to intracellular EC50 values was 281 for compound IV, 25.5 for compound III and 1.2 for tolbutamide.
In mouse pancreatic β‐cells, measurement of KATP‐channel activity in cell‐attached patches and recording of insulin release displayed much higher EC50 values for compound IV than inside‐out patch experiments. A corresponding, but less pronounced difference in EC50 values was observed for compound III, whereas the EC50 values for tolbutamide did not differ significantly.
It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the β‐cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.
Regulation of nicotinic receptors in rat brain following quasi‐irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine
Authors: El‐Bizri H. , Clarke P.B.S. 
Published: July 2012

Chronic administration of nicotinic agonists in vivo increases the density of brain nicotinic binding sites. It has been proposed that this up‐regulation results from agonist‐induced functional blockade of nicotinic receptors. This hypothesis was tested by examining post mortem [3H]‐nicotine and [125I]‐α‐bungarotoxin ([125I]‐αBTX) binding following treatment in vivo with the quasi‐irreversible and insurmountable CNS nicotinic blocker chlorisondamine, given either alone or in combination with chronic nicotine administration.
In rats that had not received chlorisondamine pretreatment, chronic nicotine administration (0.6 mg kg−1 s.c, twice daily for 12 days) increased [3H]‐nicotine binding density (Bmax) in forebrain tissue sections by 19%, with no change in the apparent dissociation constant (KD). Chlorisondamine (10 mg kg−1, s.c), given once prior to the chronic treatment phase, neither increased [3H]‐nicotine binding by itself, nor altered the extent of nicotine‐induced up‐regulation. Nevertheless, chlorisondamine pretreatment resulted in a persistent blockade of CNS nicotinic receptors, as demonstrated by complete block of acute locomotor responses to nicotine.
In a second experiment, [3H]‐nicotine and [125I]‐αBTX binding was measured in tissue homogenates prepared from several brain regions. In the absence of chlorisondamine pretreatment, chronic nicotine administration (1 mg kg−1 s.c, twice daily for 12 days) increased the Bmax of [3H]‐nicotine binding in the cerebral cortex (by 34%), striatum (by 28%), midbrain (by 16%) and hippocampus (by 36%); KD was unchanged. As before, this up‐regulation was neither mimicked nor blocked by chlorisondamine pretreatment (10 mg kg−1, s.c, given twice), despite persistent blockade of acute locomotor responses to nicotine. Chronic nicotine treatment also increased the Bmax (but not KD) of [125I]‐αBTX binding in cerebral cortex (by 35%), hippocampus (by 46%) and midbrain (by 35%). Chlorisondamine altered neither Bmax nor KD when given alone, but significantly attenuated the nicotine‐induced up‐regulation of toxin binding sites in midbrain, with a similar trend in the other two regions.
The finding that chronic receptor blockade neither mimicked nor blocked the agonist‐induced up‐regulation of [3H]‐nicotine binding sites suggests that up‐regulation of these receptors is not determined by their functional status. In contrast, it appears that chronic nicotine‐induced up‐regulation of [125I]‐αBTX binding sites may result from receptor activation.