bluuebunny/crossref_metadata_2025_split huggingface dataset:
ATP, a partial agonist for the P2Z receptor of human lymphocytes
Authors: Gargett Caroline E. , Cornish Jean E. , Wiley James S. 
Published: January 2005

Although extracellular adenosine 5′‐triphosphate (ATP) is the natural ligand for the P2Z receptor of human lymphocytes it is less potent than 3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP) in opening the associated ion channel, which conducts a range of permeants including Ba2+ and ethidium+. We have quantified the influx of ethidium+ into lymphocytes produced by BzATP, ATP, 2‐methylthio‐ATP (2MeSATP) and ATPγS, studied competition between ATP and BzATP and investigated the effects of KN‐62, a new and potent inhibitor of the P2Z receptor.
BzATP and ATP stimulated ethidium+ influx with EC50 values of 15.4±1.4 μM (n=5) and 85.6±8.8 μM (n=5), respectively. The maximal response to ATP was only 69.8±1.9% of that for BzATP. Hill analysis gave nH of 3.17±0.24 (n=3) and 2.09±0.45 (n=4) for BzATP and ATP, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z receptor‐operated ion channel.
A rank order of agonist potency of BzATP>ATP=2MeSATP>ATPγS was observed for agonist‐stimulated ethidium+ influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATPγS.
Preincubation with 30–50 μM oxidized ATP (ox‐ATP), an irreversible P2Z inhibitor, reduced the maximal response but did not change the steepness of the Ba2+ influx‐response curve produced by BzATP (nH 3.2 and 2.9 for 30 and 50 μM ox‐ATP, respectively (n=2)).
ATP (300–1000 μM) added simultaneously with 30 μM BzATP (EC90) inhibited both ethidium+ and Ba2+ fluxes to a maximum of 30–40% relative to the values observed with BzATP alone. Moreover, ATP (300 μM) shifted the concentration‐response curve to the right for BzATP‐stimulated Ba2+ influx, confirming competition between ATP and BzATP.
KN‐62, a new and powerful inhibitor of the lymphocyte P2Z receptor, showed less potency in antagonizing BzATP‐mediated fluxes than ATP‐induced fluxes when maximal concentrations of both agonists (BzATP, 50 μM; ATP, 500 μM) were used.
These data suggest that the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a more efficacious agonist. Moreover the competitive studies show that only a single class of P2‐receptor (P2Z class) is expressed on human leukaemic lymphocytes.

British Journal of Pharmacology (1997) 122, 911–917; doi:10.1038/sj.bjp.0701447
Stimulation of insulin release from the MIN6 cell line by a new imidazoline compound, S‐21663: evidence for the existence of a novel imidazoline site in β cells
Authors: Le Brigand Laurence , Virsolvy Anne , Peyrollier Karine , Manechez Dominique , Godfroid Jean‐Jacques , Guardiola‐Lemaître Béatrice , Bataille Dominique 
Published: January 2005

The MIN6 cell line derived from in vivo immortalized insulin‐secreting pancreatic β cells was used to study the insulin‐releasing capacity and the cellular mode of action of S‐21663, a newly synthesized imadizoline compound known for its antidiabetic effect in vivo and its ability to release insulin from perfused pancreas.
S‐21663, at concentrations ranging from 10−5 M to 10−3 M was able to release insulin from MIN6 cells; its activity peaked at 10−4 M, a drop in the stimulant factor being noted between 10−4 and 10−3 M. Its efficacy, which did not differ whatever the glucose concentration (stimulant or not), was higher than that of the other secretagogues tested, glucose, sulphonylureas or the peptide tGLP‐1.
In contrast to tGLP‐1, S‐21663 did not change the cyclic AMP content, whereas it increased Ca2+ influx via verapamil‐ and nifedipine‐sensitive voltage‐dependent calcium channels, the insulin release being a direct consequence of this Ca2+ entry. The S‐21663‐induced Ca2+ influx appears to be essentially the consequence of closure of K+ channels which differ from the ATP‐dependent K+ (K‐ATP) channels as determined by measurement of 86Rb efflux and use of a K‐ATP channel opener.
Comparison of the effects of S‐21663 to that of efaroxan, another imidazoline compound shown to act on insulin release in a glucose‐dependent way via binding sites distinct from the imidazoline I1 and I2 sites, suggested that S‐21663 acts through a novel site which displays a remarkably stable expression along the cell culture.
It is concluded that S‐21663 is a very efficient, glucose‐independent insulin secretagogue acting through a novel imidazoline site, linked to K+ channels, distinct from the I1, I2 and ‘efaroxan’ binding sites. In vitro and in vivo features of S‐21663 indicate that this compound, or new drugs drived from it, might be the basis for a new pharmacological approach to the mangement of type II (non insulin‐dependent) diabetes.

British Journal of Pharmacology (1997) 122, 786–791; doi:10.1038/sj.bjp.0701449
Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen‐activated protein kinase
Authors: Albert Jennifer L. , Boyle John P. , Roberts Jonathan A. , John Challiss R. A. , Gubby Sharon E. , Boarder Michael R. 
Published: January 2005

The blood‐brain barrier is formed by capillary endothelial cells and is regulated by cell‐surface receptors, such as the G protein‐coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen‐activated protein kinases (MAPK).
Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2‐methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+‐response, but resulted in a more rapid decline to basal. There was no response to α,β‐MethylATP (α,βMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage.
ATP (log EC50 −5.1±0.2) also caused an increase in the total [3H]‐inositol (poly)phosphates ([3H]‐InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and α,βMeATP giving no detectable response.
Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3.
None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPγS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 μM forskolin.
Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen‐activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration‐dependency consistent with activation at P2Y2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP.
These results are consistent with brain endothelial regulation by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1‐like (2MeSATP‐sensitive) receptors which are linked to Ca2+ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P3 levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

British Journal of Pharmacology (1997) 122, 935–941; doi:10.1038/sj.bjp.0701453
Inhibitory effect of locally administered heparin on leukocyte rolling and chemoattractant‐induced firm adhesion in rat mesenteric venules in vivo
Authors: Xie X. , Thorlacius H. , Raud J. , Hedqvist P. , Lindbom L. 
Published: January 2005

Anti‐inflammatory actions of heparin and related glycosaminoglycans have been described in the literature. Here, we used intravital microscopy of the rat mesentery microcirculation to examine effects of locally administered heparin on leukocyte rolling and chemoattractant‐induced firm adhesion.
It was found that topical application of heparin reduced N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced leukocyte adhesion. Notably, the inhibitory action of heparin was not dose‐dependent, but rather a biphasic dose‐response was found, i.e. low (2 and 20 iu ml−1) and high (1000 iu ml−1) concentrations of heparin significantly reduced adhesion, whereas an intermediate dose (200 iu ml−1) was less effective.
Heparin, 2 and 20 iu ml−1, decreased rolling leukocyte flux, while having no effect on blood flow or total leukocyte flux. By contrast, heparin, 200 and 1000 iu ml−1, increased total leukocyte flux in parallel with a rise in volume blood flow resulting in recovery of the rolling leukocyte flux at these doses. Thus, the biphasic inhibitory action of heparin on fMLP‐induced firm adhesion could in part be attributed to changes in leukocyte delivery (i.e. blood flow) and rolling leukocyte flux induced by heparin.
When compensating for the influence of different rolling levels on fMLP‐evoked adhesion, a dose‐related inhibitory effect of heparin on the firm adhesive response per se was revealed. Similar results were obtained in a static adhesion assay in vitro where heparin 200 and 1000 iu ml−1 (but not 2 and 20 iu ml−1) significantly inhibited fMLP‐induced leukocyte adhesion in the absence of any modulatory influence on changes in rolling.
Our data show that locally administered heparin inhibits leukocyte rolling as well as chemoattractant‐induced firm adhesion in vivo which thus may contribute to the postulated anti‐inflammatory activity of this compound. However, because of interference with several microvascular functions, strict dose‐dependent responses to heparin treatment were not found, which illustrates the complex interplay between local blood flow, leukocyte rolling and chemoattractant‐induced adhesion as determinants of leukocyte recruitment to tissues in inflammation.

British Journal of Pharmacology (1997) 122, 906–910; doi:10.1038/sj.bjp.0701454
Enhanced α2A‐autoreceptor reserve for clonidine induced by reserpine and cholinomimetic agents in the rat vas deferens
Authors: Pineda Joseba , Ugedo Luisa , García‐Sevilla Jesús A. 
Published: January 2005

The adaptive changes in the functional parameters of the presynaptic α2A‐adrenoceptors in rat vas deferens were examined after treatments with the monoamine depleter reserpine or with the direct/indirect cholinomimetic agents pilocarpine and neostigmine.
For this purpose, we studied the inhibition induced by the α2‐adrenoceptor agonist clonidine on the twitch contraction of the vas deferens elicited by electrical field stimulation, in animals that had been treated with acute (single dose), short‐term (for 4 days) and chronic (for 11 days) regimens of reserpine (0.25 mg kg−1, s.c., every 48 h), pilocarpine (10 mg kg−1, i.p., every 12 h) or neostigmine (0.1 mg kg−1, i.p., every 12 h). The irreversible receptor alkylating agent N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ, 300 nM) was used to block partially the α2A‐adrenoceptor‐mediated effect of clonidine.
In control (untreated) animals, clonidine inhibited concentration‐dependently the twitch response of the vas deferens (pEC50=8.66) with a maximal effect near 100%. The apparent affinity constant for clonidine was estimated with the nested hyperbolic methodology (pKA=7.10). The analysis of the occupancy‐effect relation for clonidine revealed a large receptor reserve at α2A‐adrenoceptors.
Acute, short‐term and chronic treatments with reserpine increased the sensitivity of α2A‐adrenoceptors to clonidine (decreased the EC50) by about 3, 4 and 9 fold, respectively, and also increased the pool of receptor reserve for this agonist (decreased the KE) by 4, 10 and 10 fold, respectively. Receptor affinity values were not changed after treatments.
Short‐term and chronic, but not acute, treatments with pilocarpine and neostigmine increased the sensitivity of α2A‐adrenoceptors to clonidine (decreased the EC50) by about 3 and 2 fold, respectively, and also increased the pool of receptor reserve for this agonist (decreased the KE) by 2 and 3 fold, respectively. Receptor affinity values were not changed after these treatments.
These results indicate that an enhancement of the receptor reserve for clonidine might account for the supersensitivity of α2A‐adrenoceptors induced by reserpine, pilocarpine or neostigmine treatments in the rat vas deferens.

British Journal of Pharmacology (1997) 122, 833–840; doi:10.1038/sj.bjp.0701455
Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage‐sensitive calcium channels
Authors: Fontana Giovanni , Fedele Ernesto , Cossu Massimo , Munari Claudio , Raiteri Maurizio 
Published: January 2005

Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L‐[3H]‐arginine to L‐[3H]‐citrulline.
Elevation of extracellular K+ to 20, 35 or 60 mM concentration‐dependently augmented L‐[3H]‐citrulline production. The response to 35 mM KCl was abolished by NG‐nitro‐L‐arginine (100 μM) demonstrating NO synthase specific conversion of L‐arginine to L‐citrulline. Increasing extracellular MgCl2 concentration up to 10 mM also prevented the K+ (35 mM)‐induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity.
However, the effect of high K+ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK‐801), 6‐nitro‐7‐sulphamoylbenzo(f)quinoxaline‐2–3‐dione (NBQX) or L‐2‐amino‐3‐phosphonopropionic acid (L‐AP3) as well as to the nicotinic receptor antagonist, mecamylamine.
The 35 mM K+ response was insensitive to ω‐conotoxin GVIA (1 μM) and nifedipine (100 μM), but could be prevented in part by ω‐agatoxin IVA (0.1 and 1 μM). The inhibition caused by 0.1 μM ω‐agatoxin IVA (∼30%) was enhanced by adding ω‐conotoxin GVIA (1 μM) or nifedipine (100 μM). Further inhibition (up to above 70%) could be observed when the three Ca2+ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 μM) the K+‐evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 μM ω‐conotoxin GVIA plus 100 μM nifedipine. No further inhibition could be observed upon addition of MK‐801 or/and NBQX.
It was concluded that elevation of extracellular [K+] causes NO synthase activation by external Ca+ entering cells mainly through channels of the P/Q‐type. Other Ca2+ channels (L‐ and N‐type) appear to contribute when P/Q‐channels are blocked.

British Journal of Pharmacology (1997) 122, 930–934; doi:10.1038/sj.bjp.0701457
Activation of thromboxane receptors and the induction of vasomotion in the hamster cheek pouch microcirculation.
Authors: Verbeuren Tony J. , Vallez Marie‐Odile , Lavielle Gilbert , Bouskela Eliete 
Published: January 2005

The present study was designed to investigate a possible role of thromboxane A2 (TXA2) on arteriolar vasomotion (spontaneous rhythmic variations of the vessel diameter). Therefore the microcirculatory effects of the thromboxane‐receptor (TP‐receptor) agonist, U 46619, as well as the effects of the TP‐receptor antagonists S 17733 and Bay U3405 were evaluated in the hamster cheek pouch microcirculation. For comparison some effects of angiotensin II were also investigated.
For microcirculatory measurements, the cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. The TV monitor display was used to obtain arteriolar internal diameter measurements by means of an image shearing device.
Superfusion (0.1 nM to 1 μM) or bolus application (1 pmol to 10 nmol) of U 46619 concentration‐ or dose‐dependently decreased the arteriolar diameter and induced vasomotion in arterioles with a mean initial diameter of 24±2 μm. Both the vasoconstriction and the vasomotion induced by U 46619 were inhibited by the TP‐receptor antagonists S 17733 (100 mg kg−1, i.v.) and Bay U3405 (10 mg kg−1, i.v.).
Bolus applications of angiotensin II (0.1 pmol to 1 nmol) induced transient vasoconstriction followed by vasodilatation in the cheek pouch arterioles. The dilatation but not the constriction, was sensitive to treatment with the NO‐synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG; 100 μM). Angiotensin II did not induce vasomotion in control conditions or in the presence of L‐NOARG.
Bolus application of phenylephrine (10 pmol) induced vasoconstriction but no vasomotion in previously quiescent hamster cheek pouch arterioles.
These results indicate that activation of TP‐receptors causes vasomotion in the hamster cheek pouch arterioles. These spontaneous rhythmic variations in arteriolar diameter are not observed with equipotent doses of angiotensin II and phenylephrine. Thus, the vasoconstriction by itself cannot explain the occurrence of vasomotion observed with the TP‐receptor agonist.

British Journal of Pharmacology (1997) 122, 859–866; doi:10.1038/sj.bjp.0701464
Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists
Authors: Maemoto Takuya , Finlayson Keith , Olverman Henry J. , Akahane Atsushi , Horton Roger W. , Butcher Steven P. 
Published: January 2005

The pharmacological profile of adenosine A1 receptors in human, guinea‐pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]‐8‐cyclopentyl‐1,3‐dipropylxanthine ([3H]‐DPCPX).
The affinity of [3H]‐DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea‐pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea‐pig or human cortical membranes.
The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5′‐N‐ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5–26.2 fold higher in rat cortical membranes than in human and guinea‐pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 μM) reduced 2‐chloro‐N6‐cyclopentyladenosine (CCPA) affinity 7–13.9 fold, whereas the affinity of DPCPX was unaffected.
The affinity of six xanthine‐based adenosine receptor antagonists was 2.2–15.9 fold higher in rat cortical membranes compared with human or guinea‐pig membranes. The rank order of potency was species‐independent. In contrast, three pyrazolopyridine derivatives, (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐2‐piperidine ethanol (FK453), (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐piperidin‐2‐yl acetic acid (FK352) and 6‐oxo‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl)‐1(6H)‐pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea‐pig, rat and mouse brain membranes. pKi values (M) for [3H]‐DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively.
Drug affinity for adenosine A2A receptors was determined in a [3H]‐2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine ([3H]‐CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]‐DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]‐DPCPX binding sites in human, rat, mouse and guinea‐pig brain membranes.

British Journal of Pharmacology (1997) 122, 1202–1208; doi:10.1038/sj.bjp.0701465
Heterologous expression of rat epitope‐tagged histamine H2 receptors in insect Sf9 cells
Authors: Beukers M. W. , Klaassen C. H. W. , De Grip W. J. , Verzijl D. , Timmerman H. , Leurs R. 
Published: January 2005

Rat histamine H2 receptors were epitope‐tagged with six histidine residues at the C‐terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope‐tagged H2 receptor were prepared and were used to infect insect Sf9 cells.
The His‐tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]‐aminopotentidine (APT) binding studies.
In Sf9 cells expressing the His‐tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3).
The expression of the His‐tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg−1 protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×106 copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol‐β‐cyclodextrin complex resulted in an increase of [125I]‐APT binding up to 169±5% (mean±s.e.mean, n=3).
The addition of 0.03 mM cholesterol‐β‐cyclodextrin complex did not affect histamine‐induced cyclic AMP production. The EC50 value of histamine was 3.1±1.7 μM in the absence of cholesterol‐β‐cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol‐β‐cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/106 cells in the absence and 81±11 pmol/106 cells in the presence of 0.03 mM cholesterol‐β‐cyclodextrin complex (mean±s.e.mean, n=3).
Immunofluorescence studies with an antibody against the His‐tag revealed that the majority of the His‐tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well.
These experiments demonstrate the successful expression of His‐tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His‐tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.

British Journal of Pharmacology (1997) 122, 867–874; doi:10.1038/sj.bjp.0701466