bluuebunny/crossref_metadata_2025_split huggingface dataset:
A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis
Authors: Bellucci Francesca , Meini Stefania , Cucchi Paola , Catalani Claudio , Reichert Wolfgang , Zappitelli Sabrina , Rotondaro Luigi , Quartara Laura , Giolitti Alessandro , Maggi Carlo Alberto 
Published: October 2003

Binding affinity at the [3H]‐BK binding site and activity as inositol phosphate (IP) production by the peptide bradykinin (BK) and the nonpeptide FR190997 were studied at wild‐type or point‐mutated human B2 receptors (hB2R) expressed in CHO cells.
The effect of the following mutations were analyzed: E47A (TM1), W86A and T89A (TM2), I110A, L114A and S117A (TM3), T158A, M165T and L166F (TM4), T197A and S211A (TM5), F252A, W256A and F259A (TM6), S291A, F292A, Y295A and Y295F (TM7), and the double mutation W256A/Y295F.
As the wild‐type receptor‐binding affinity of FR190997 was 40‐fold lower than BK, whereas their agonist potency was comparable, both agonists produced similar maximal effects (Emax). Mutations were evaluated as affecting the affinity and/or efficacy of FR190997 compared with BK.
Two mutations were found to impair the agonist affinity of both agonists drastically: W86A and F259A. BK agonist affinity (pEC50) was reduced by 1400‐ and 150‐fold, and that of FR190997 was reduced by 400‐ and 25‐fold, at the W86A and F259A mutant B2 receptors, respectively.
Contrary to BK, the affinity of FR190997 was selectively decreased at I110A, Y295A, and Y295F mutants (>103‐fold), and a different efficacy was measured at the Y295 mutants, FR190997 being devoid of the capability to trigger IP production at Y295A mutant.
L114A, F252A, and W256A selectively impaired the efficacy of FR190997, whereas its binding affinity was not affected. As a consequence, FR190997 behaved as a high‐affinity antagonist in blocking the IP production induced by BK.
The lack of capability of FR190997 to activate or to bind the double mutant W256A/Y295F suggests that these residues are part of the same binding site, which is also important for receptor activation by the nonpeptide ligand.
Overall, by means of mutational analysis, we indicate an hB2R recognition site for the nonpeptide agonist FR190997 (between TM3, 6, and 7), different from that of BK, and show that in the same binding crevice some mutations (L114, W256, and F252) are selectively responsible for the agonist properties of only FR190997.

British Journal of Pharmacology (2003) 140, 500–506. doi:10.1038/sj.bjp.0705454
Voltage‐dependent calcium channels are involved in neurogenic dural vasodilatation via a presynaptic transmitter release mechanism
Authors: Akerman S , Williamson D J , Goadsby P J 
Published: October 2003

A missense mutation of the CACNA1A gene that encodes the α1A subunit of the voltage‐dependent P/Q‐type calcium channel has been discovered in patients suffering from familial hemiplegic migraine. This suggested that calcium channelopathies may be involved in migraine more broadly, and established the importance of genetic mechanisms in migraine.
Channelopathies share many clinical characteristics with migraine, and thus exploring calcium channel functions in the trigeminovascular system may give insights into migraine pathophysiology. It is also known that drugs blocking the P/Q‐ and N‐type calcium channels have been successful in other animal models of trigeminovascular activation and head pain.
In the present study, we used intravital microscopy to examine the effects of specific calcium channel blockers on neurogenic dural vasodilatation and calcitonin gene‐related peptide (CGRP)‐induced dilation.
The L‐type voltage‐dependent calcium channel blocker calciseptine significantly attenuated (20 μg kg−1, n=7) the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation.
The P/Q‐type voltage‐dependent calcium channel blocker ω‐agatoxin‐IVA (20 μg kg−1, n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation.
The N‐type voltage‐dependent calcium channel blocker ω‐conotoxin‐GVIA (20 μg kg−1, n=8 and 40 μg kg−1, n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation.
It is thought that the P/Q‐, N‐ and L‐type calcium channels all exist presynaptically on trigeminovascular neurons, and blockade of these channels prevents CGRP release, and, therefore, dural blood vessel dilation. These data suggest that the P/Q‐, N‐ and L‐type calcium channels may be involved in trigeminovascular nociception.

British Journal of Pharmacology (2003) 140, 558–566. doi:10.1038/sj.bjp.0705456
P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages
Authors: Bowler Jonathan W , Jayne Bailey R , Alan North R , Surprenant Annmarie 
Published: October 2003

ATP receptors present on rat alveolar macrophages (NR8383 cells) were identified by recordings of membrane current, measurements of intracellular calcium, RT–PCR and immunocytochemistry.
In whole‐cell recordings with a sodium‐based internal solution, ATP evoked an inward current at −60 mV. This reversed at 0 mV. The EC50 for ATP was 18 μM in normal external solution (calcium 2 mM, magnesium 1 mM). The currents evoked by 2′,3‐O‐(4‐benzoyl)benzoyl‐ATP were about five‐fold smaller than those observed with ATP. ADP, UTP and αβ‐methylene‐ATP (αβmeATP) (up to 100 μM) had no effect. ATP‐evoked currents were potentiated up to ten‐fold by ivermectin and were unaffected by suramin (30–100 μM), pyridoxal‐phosphate‐6‐azophenyl‐(2,4‐sulphonic acid) (30–100 μM), and brilliant blue G (1 μM).
In whole‐cell recordings with a potassium‐based internal solution and low EGTA (0.01 mM), ATP evoked an inward current at −60 mV that was followed by larger outward current. ADP and UTP (1–100 μM) evoked only outward currents; these reversed polarity at the potassium equilibrium potential and were blocked by apamin (10 nM). Outward currents were also blocked by the phospholipase C inhibitor U73122 (1 μM), and they were not seen with higher intracellular EGTA (10 mM). Suramin (30 μM) blocked the outward currents evoked by ATP and UTP, but not that evoked by ADP. PPADS (10 μM) blocked the ADP‐evoked outward current without altering the ATP or UTP currents.
RT–PCR showed transcripts for P2X subunits 1, 4 and 7 (not 2, 3, 5, 6) and P2Y receptors 1, 2, 4 and 12 (not 6). Immunocytochemistry showed strong P2X4 receptor expression partly associated with the membrane, weak P2X7 staining that was not associated with the cell membrane, and no P2X1 receptor immunoreactivity.
We conclude that rat alveolar macrophages express (probably homomeric) P2X4 receptors, but find no evidence for other functional P2X subtypes. The P2Y receptors are most likely P2Y1 and P2Y2 and these couple through phospholipase C to an increase in intracellular calcium and the opening of SK type potassium channels.

British Journal of Pharmacology (2003) 140, 567–575. doi:10.1038/sj.bjp.0705459
Interactions of androgens, green tea catechins and the antiandrogen flutamide with the external glucose‐binding site of the human erythrocyte glucose transporter GLUT1
Authors: Naftalin Richard J , Afzal Iram , Cunningham Philip , Halai Mansur , Ross Clare , Salleh Naguib , Milligan Stuart R 
Published: October 2003

This study investigates the effects of androgens, the antiandrogen flutamide and green tea catechins on glucose transport inhibition in human erythrocytes. These effects may relate to the antidiabetogenic effects of green tea.
Testosterone, 4‐androstene‐3,17‐dione, dehydroepiandrosterone (DHEA) and DHEA‐3‐acetate inhibit glucose exit from human erythrocytes with half‐maximal inhibitions (Ki) of 39.2±8.9, 29.6±3.7, 48.1±10.2 and 4.8±0.98 μM, respectively. The antiandrogen flutamide competitively relieves these inhibitions and of phloretin. Dehydrotestosterone has no effect on glucose transport, indicating the differences between androgen interaction with GLUT1 and human androgen receptor (hAR).
Green tea catechins also inhibit glucose exit from erythrocytes. Epicatechin 3‐gallate (ECG) has a Ki ECG of 0.14±0.01 μM, and epigallocatechin 3‐gallate (EGCG) has a Ki EGCG of 0.97±0.13 μM. Flutamide reverses these effects.
Androgen‐screening tests show that the green tea catechins do not act genomically. The high affinities of ECG and EGCG for GLUT1 indicate that this might be their physiological site of action.
There are sequence homologies between GLUT1 and the ligand‐binding domain (LBD) of hAR containing the amino‐acid triads Arg 126, Thr 30 and Asn 288, and Arg 126, Thr 30 and Asn 29, with similar 3D topology to the polar groups binding 3‐keto and 17‐β OH steroid groups in hAR LBD. These triads are appropriately sited for competitive inhibition of glucose import at the external opening of the hydrophilic pore traversing GLUT1.

British Journal of Pharmacology (2003) 140, 487–499. doi:10.1038/sj.bjp.0705460
Norcantharidin‐induced apoptosis is via the extracellular signal‐regulated kinase and c‐Jun‐NH2‐terminal kinase signaling pathways in human hepatoma HepG2 cells
Authors: Chen Yan‐Nian , Cheng Chi‐Chih , Chen Jung‐Chou , Tsauer Wei , Hsu Shih‐Lan 
Published: October 2003

Norcantharidin (NCTD) is an anticancer drug routinely used against hepatoma in China. Previously, we reported that NCTD could induce mitotic arrest and apoptosis in human hepatoma HepG2 cells. However, the intracellular signaling pathways involved in NCTD‐induced apoptotic cell death are still obscure.
Caspase inhibitors were used to clarify the role of specific caspase in NCTD‐triggered apoptotic process. Results showed that activation of caspase‐9/caspase‐3 cascade is required for NCTD‐induced apoptotic death.
To decipher the upstream signals for NCTD‐induced apoptosis, we characterized the involvement of mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase (ERK), c‐Jun NH2‐terminal kinase (JNK), and p38MAPK. The role of their downstream targets, transcription factors activating protein‐1 (AP‐1), and nuclear factor κB (NF‐κB) in NCTD‐induced apoptosis was also analyzed.
Immunoblot analyses and in vitro kinase assay demonstrated that NCTD‐induced apoptosis was accompanied by the elevations of the levels of phosphorylated form and kinase activity of ERK and JNK, but not p38MAPK.
The inhibitor of ERK pathway (U0126 or PD98059) or JNK pathway (SP600125) markedly prevented kinase activation, and also greatly reduced NCTD‐induced apoptotic cell death.
Increased DNA‐binding activity of AP‐1 and NF‐κB was also observed after NCTD treatment.
Inhibition of NF‐κB activation by PDTC or inhibition of AP‐1 activation by curcumin drastically blocked NCTD‐induced cell death.
These results imply that activation of ERK and JNK, and modulation of downstream transcription factors NF‐κB and AP‐1, may be involved in NCTD‐induced apoptosis.

British Journal of Pharmacology (2003) 140, 461–470. doi:10.1038/sj.bjp.0705461
Delayed administration of interleukin‐1 receptor antagonist protects against transient cerebral ischaemia in the rat
Authors: Mulcahy Nicholas J , Ross Jerard , Rothwell Nancy J , Loddick Sarah A 
Published: October 2003

The cytokine interleukin‐1 (IL‐1) has been implicated in ischaemic, excitotoxic and traumatic brain damage in rodents. The naturally occurring IL‐1 receptor antagonist (IL‐1ra) markedly reduces neuronal injury in these conditions. However, the effects of IL‐1ra on focal, transient cerebral ischaemia in the rat, which is of major clinical relevance, have not been reported.
The objectives of this study were to test the effects of IL‐1ra on cell death after temporary cerebral ischaemia, and to investigate the therapeutic time window for IL‐1ra treatment.
Ischaemia was induced by temporary (60 min) occlusion of the middle cerebral artery (MCAO) in rats, via surgical insertion (and subsequent removal) of a thread into the internal carotid artery. Damage was quantified at various times after MCAO to investigate the temporal progression of damage and establish an appropriate time to assess the effects of IL‐1ra on cell death.
Cell death was complete 18–24 h after temporary MCAO. Intracerebroventricular injection of IL‐1ra (10 μg) at the time of MCAO and 60 min later reduced the lesion volume measured 24 h (57% reduction) or 48 h (52% reduction) after MCAO. Cell death was also significantly reduced when IL‐1ra (20 μg) was administered as a single injection, 1 h (47%), 2 h (57%) or 3 h (46%) after MCAO, when compared to vehicle.
These data show that IL‐1ra markedly reduces cell death even when administration is delayed until 3 h after induction of reversible, focal cerebral ischaemia in the rat, and support our proposal that IL‐1ra may be of therapeutic benefit in stroke.

British Journal of Pharmacology (2003) 140, 471–476. doi:10.1038/sj.bjp.0705462
Ragaglitazar: a novel PPARα & PPARγ agonist with potent lipid‐lowering and insulin‐sensitizing efficacy in animal models
Authors: Chakrabarti Ranjan , Vikramadithyan Reeba K , Misra Parimal , Hiriyan Jagadheshan , Raichur Suryaprakash , Damarla Ravi K , Gershome Cynthia , Suresh Juluri , Rajagopalan Ramanujam 
Published: October 2003

Ragaglitazar [(−) DRF 2725; NNC 61‐0029] is a coligand of PPARα and PPARγ.
In ob/ob mice, ragaglitazar showed significant reduction in plasma glucose, triglyceride and insulin (ED50 values <0.03, 6.1 and <0.1 mg kg−1). These effects are three‐fold better than rosiglitazone and KRP‐297. In Zucker fa/fa rats, ragaglitazar showed dose‐dependent reduction in triglyceride and insulin, hepatic triglyceride secretion and triglyceride clearance kinetics (maximum of 74, 53, 32 and 50% at 3 mg kg−1), which are better than rosiglitazone and KRP‐297.
In a high‐fat‐fed hyperlipidaemic rat model, the compound showed an ED50 of 3.95, 3.78 mg kg−1 for triglyceride and cholesterol lowering, and 0.29 mg kg−1 for HDL‐C increase. It also showed improvement in clearance of plasma triglyceride and hepatic triglyceride secretion rate. All these effects are 3–10‐fold better than fenofibrate and KRP‐297.
Ragaglitazar treatment showed significant reduction in plasma Apo B and Apo CIII levels, and increase in liver CPT1 and CAT activity and ACO mRNA. Significant increase of both liver and fat LPL activity and fat aP2 mRNA was also observed.
In a high‐fat‐fed hamster model, ragaglitazar at 1 mg kg−1 showed 83 and 61% reduction in triglyceride and total cholesterol, and also 17% reduction in fat feed‐induced body weight increase. In these hyperlipidaemic animal models, PPARγ ligands failed to show any significant efficacy. Taken together, ragaglitazar shows better insulin‐sensitizing and lipid‐lowering potential, as compared to the standard compounds.

British Journal of Pharmacology (2003) 140, 527–537. doi:10.1038/sj.bjp.0705463
Tetrahydrocannabinol‐induced neurotoxicity depends on CB1 receptor‐mediated c‐Jun N‐terminal kinase activation in cultured cortical neurons
Authors: Downer Eric J , Fogarty Marie P , Campbell Veronica A 
Published: October 2003

Δ9‐Tetrahydrocannabinol (THC), the main psychoactive ingredient of marijuana, induces apoptosis in cultured cortical neurons. THC exerts its apoptotic effects in cortical neurons by binding to the CB1 cannabinoid receptor. The CB1 receptor has been shown to couple to the stress‐activated protein kinase, c‐Jun N‐terminal kinase (JNK). However, the involvement of specific JNK isoforms in the neurotoxic properties of THC remains to be established.
The present study involved treatment of rat cultured cortical neurons with THC (0.005–50 μM), and combinations of THC with the CB1 receptor antagonist, AM 251 (10 μM) and pertussis toxin (PTX; 200 ng ml−1). Antisense oligonucleotides (AS) were used to deplete neurons of JNK1 and JNK2 in order to elucidate their respective roles in THC signalling.
Here we report that THC induces the activation of JNK via the CB1 receptor and its associated G‐protein, Gi/o. Treatment of cultured cortical neurons with THC resulted in a differential timeframe of activation of the JNK1 and JNK2 isoforms.
Use of specific JNK1 and JNK2 AS identified activation of caspase‐3 and DNA fragmentation as downstream consequences of JNK1 and JNK2 activation.
The results from this study demonstrate that activation of the CB1 receptor induces JNK and caspase‐3 activation, an increase in Bax expression and DNA fragmentation. The data demonstrate that the activation of both JNK1 and JNK2 isoforms is central to the THC‐induced activation of the apoptotic pathway in cortical neurons.

British Journal of Pharmacology (2003) 140, 547–557. doi:10.1038/sj.bjp.0705464
Novel data point to a broader mechanism of action of oxidized ATP: the P2X7 receptor is not the only target
Authors: Di Virgilio Francesco 
Published: October 2003

Oxidized ATP (oATP) is a Schiff‐base‐forming reagent that has been used for some years as an antagonist at the P2X7 receptor (P2X7R). Preincubation of mononuclear phagocytes with this inhibitor leads to attenuation of several proinflammatory responses triggered by extracellular ATP as well as a few non‐nucleotide agonists. Novel data show that oATP reduces NFκB activation and IL‐8 release in cells lacking P2X7R, thus suggesting that some anti‐inflammatory effects of oATP may not be due to blockade of the P2X7R. This effect of oATP resembles the action of other natural or synthetic Schiff‐base‐forming reagents with immunomodulatory activity.British Journal of Pharmacology (2003) 140, 441–443. doi:10.1038/sj.bjp.0705469
Oxidized ATP (oATP) attenuates proinflammatory signaling via P2 receptor‐independent mechanisms
Authors: Beigi Reza D , Kertesy Sylvia B , Aquilina Gretchen , Dubyak George R 
Published: October 2003

Periodate‐oxidized ATP (oATP), which covalently modifies nucleotide‐binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X7 nucleotide receptor (P2X7R) is irreversibly antagonized by oATP, it is unclear whether anti‐inflammatory actions of oATP are predominantly mediated via its actions on P2X7R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X7R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes.
oATP decreased by 40–70% the secretion of interleukin (IL)‐8 stimulated by tumor necrosis factor‐α (TNF‐α) in all three cell types, by IL‐1β in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF‐α‐stimulated IL‐8 secretion by oATP was similar in wild‐type HEK cells or HEK cells stably expressing recombinant P2X7R.
oATP also attenuated cytokine‐stimulated expression of nuclear factor‐κB‐luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of IκB.
oATP had no effect on uridine triphosphate‐induced activation of native P2Y2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y1 receptors. However, inhibition of P2Y1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF‐α‐stimulated IL‐8 secretion.
Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto‐ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP.
In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.

British Journal of Pharmacology (2003) 140, 507–519. doi:10.1038/sj.bjp.0705470
CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22–52, CGRP8–37 and BIBN4096BS
Authors: Hay D L , Howitt S G , Conner A C , Schindler M , Smith D M , Poyner D R 
Published: October 2003

Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor‐like receptor (CL) and receptor activity‐modifying protein (RAMP) 2 or 3: We report the effects of the antagonist fragments of human AM and CGRP (AM22–52 and CGRP8–37) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells.
AM22–52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat 2), 7.00±0.05 (L6), 6.25±0.17 (rCL/hRAMP2)). CGRP8–37 (10 μM) resembled AM22–52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0.12 (L6)).
On CL/RAMP3 receptors, 10 μM CGRP8–37 was an effective antagonist at all combinations (apparent pA2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/hRAMP3)). However, 10 μM AM22–52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73±0.14).
BIBN4096BS (10 μM) did not antagonise AM at any of the receptors.
Where investigated (all‐rat and rat/human combinations), the agonist potency order on the CL/RAMP3 receptor was AM∼βCGRP>αCGRP.
rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence.
This study shows that on CL/RAMP complexes, AM22–52 has significant selectivity for the CL/RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP8–37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

British Journal of Pharmacology (2003) 140, 477–486. doi:10.1038/sj.bjp.0705472
Electrophysiological evidence for a gradient of G protein‐gated K+ current in adult mouse atria
Authors: Lomax Alan E , Rose Robert A , Giles Wayne R 
Published: October 2003

Whole cell current and voltage clamp techniques were used to examine the properties of acetylcholine‐sensitive K+ current (IKACh) in myocytes from adult mouse atrium. Superfusion of a maximal dose of carbachol (CCh; 10 μM) caused a substantial increase in K+ current in all myocytes examined.
The current–voltage (I–V) relation of maximally activated IKACh exhibited weak inward rectification. Consequently, CCh increased the amount of depolarising current necessary to evoke action potentials (APs), and APs evoked in CCh had significantly shorter durations than control APs (P<0.05).
The effects of CCh on K+ current and on AP properties were blocked by the muscarinic receptor antagonist methoctramine (1 μM). ACh (10 μM) activated a K+ current with identical properties to that activated by CCh, as did the A1 receptor agonist adenosine (100 μM).
Right atrial myocytes had significantly more IKACh than left atrial myocytes (P<0.05), regardless of whether IKACh was evoked by superfusion of muscarinic or A1 receptor agonists. IKACh current density was significantly higher in SA node myocytes than either right or left atrial myocytes.
These data identify a gradient of IKACh current density across the supraventricular structures of mouse heart. This gradient, combined with the heterogeneous distribution of parasympathetic innervation of the atria, may contribute to the proarrhythmic ability of vagal nerve stimulation to augment dispersion of atrial refractoriness.

British Journal of Pharmacology (2003) 140, 576–584. doi:10.1038/sj.bjp.0705474