bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological characterization of muscarinic receptors in mouse isolated urinary bladder smooth muscle
Authors: Choppin A , Eglen R M
Published: January 2005

The pharmacological characteristics of muscarinic receptors in the male mice urinary bladder smooth muscle were studied.

(+)‐Cis‐dioxolane, oxotremorine‐M, acetylcholine, carbachol and pilocarpine induced concentration‐dependent contractions of the urinary bladder smooth muscle (pEC50=6.6±0.1, 6.9±0.1, 6.7±0.1, 5.8±0.1 and 5.8±0.1, EMax=3.2±0.8 g, 2.7±0.4 g, 1.0±0.1 g, 2.7±0.3 and 0.9±0.2 g, respectively, n=4). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pKB values): atropine (9.22±0.09), pirenzepine (6.85±0.08), 4‐DAMP (8.42±0.14), methoctramine (5.96±0.05), p‐F‐HHSiD (7.48±0.09), tolterodine (8.89±0.13), AQ‐RA 741 (7.04±0.12), s‐secoverine (8.21±0.09), zamifenacin (8.30±0.17) and darifenacin (8.70±0.09).

In this tissue, the pKB values correlated most favourably with pKi values for these compounds at human recombinant muscarinic M3 receptors. A significant correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between M3 and m5 receptors.

In recontraction studies, in which the muscarinic M3 receptor population was decreased, and conditions optimized to study M2 receptor activation, methoctramine exhibited an affinity estimate consistent with muscarinic M3 receptors (pKB=6.23±0.14; pA2=6.16±0.03).

Overall, these data study suggest that muscarinic M3 receptors are the predominant, if not the exclusive, subtype mediating contractile responses to muscarinic agonists in male mouse urinary bladder smooth muscle.

British Journal of Pharmacology (2001) 133, 1035–1040; doi:10.1038/sj.bjp.0704165
Combination of miglitol, an anti‐diabetic drug, and nicorandil markedly reduces myocardial infarct size through opening the mitochondrial KATP channels in rabbits
Authors: Minatoguchi Shinya , Wang Ningyuan , Uno Yoshihiro , Arai Masazumi , Hashimoto Kazuaki , Hashimoto Yasuko , Chen Xue‐Hai , Takemura Genzou , Fujiwara Hisayoshi
Published: January 2005

The anti‐diabetic drug miglitol, an α‐glucosidase inhibitor, which is currently used clinically, reduces myocardial infarct size by reducing the glycogenolytic rate through inhibition of the α‐1,6‐glucosidase of glycogen‐debranching enzyme in the heart. Nicorandil, a KATP channel opener with a nitrate‐like effect, which is also currently used clinically, also reduces the infarct size. Therefore, we hypothesized that combination of nicorandil and submaximal dose of miglitol could markedly reduce myocardial infarct size more than miglitol or nicorandil alone, and investigated the mechanism for the infarct size‐reducing effect.

Japanese white rabbits without collateral circulation were subjected to 30 min coronary occlusion followed by 48 h reperfusion. Pre‐ischaemic treatment with submaximal dose of miglitol (5 mg kg−1, i.v.) and nicorandil alone (100 μg kg−1 min−1 5 min) moderately reduced the infarct size as a percentage of area at risk (24±4 and 25±4%, respectively), and 10 mg kg−1 of miglitol markedly reduced the infarct size (15±2%) compared with the controls (42±2%). Combination of 5 mg kg−1 of miglitol and nicorandil (100 μg kg−1 min−1 5 min), and 10 mg kg−1 of miglitol and nicorandil (100 μg kg−1 min−1 5 min) significantly reduced the infarct size (13±4 and 12±3%, respectively) more than miglitol or nicorandil alone.

Pretreatment with 5HD completely abolished the infarct size‐reducing effect of 10 mg kg−1 of miglitol alone (36±7%) and that of combination of 5 mg kg−1 of miglitol and nicorandil (46±2%).

Combination of nicorandil and submaximal dose of miglitol markedly reduced the myocardial infarct size more than miglitol or nicorandil alone. This effect was suggested to be related to the opening of mitochondrial KATP channels.

British Journal of Pharmacology (2001) 133, 1041–1046; doi:10.1038/sj.bjp.0704166
A comparison of allergen and polycation induced cutaneous responses in the rabbit
Authors: Jones Helen , Paul William , Page Clive P
Published: January 2005

Allergic inflammatory responses contribute to the symptoms of a number of diseases including atopic dermatitis, asthma and rhinitis. Cationic proteins are released from inflammatory cells and levels are known to be raised in disease states.

Using an in vivo model of acute inflammation, we investigated the characteristics of cutaneous responses to antigen (Alternaria tenuis, AT) and poly‐L‐lysine (PLL, used as a paradigm for cationic proteins). We aimed to compare the inflammatory profile of cationic polypeptides and the allergic response and to identify similarities and differences between these responses.

Responses to intradermal injection of the polycation, PLL and antigen were compared using radiolabelled protein (125I‐bovine serum albumin, BSA) and cells (111In‐neutrophils, PMN) to study plasma exudation (PE) and PMN accumulation (PMNA) in the skin of AT sensitized rabbits.

Both PLL and antigen caused dose‐related increases in PE and PMNA. PE (and PMNA) responses to PLL were prolonged (up to 3 h), as were those to antigen. This is in contrast to PE responses to fMLP which were maximal at 45 min.

In immunized animals, treated with colchicine (1 mg kg−1 i.v.), PE responses to the directly acting mediator, bradykinin (BK), were not affected, whereas PE responses to the neutrophil dependent mediator, f‐met‐leu‐phe (fMLP), were significantly (P<0.01) reduced. Antigen‐induced PE responses were significantly (50, 500 (P<0.05); 200 (P<0.01) p.n.u. site−1) inhibited by colchicine, but PLL‐induced responses were not significantly affected.

We conclude that although PLL‐induced responses had a similar time course to those of antigen, some differences were observed between responses, which indicate that although polycations may contribute to allergic responses, these two responses are produced by distinct mechanisms.

British Journal of Pharmacology (2001) 133, 1181–1189; doi:10.1038/sj.bjp.0704172
Rho‐dependent inhibition of the induction of connective tissue growth factor (CTGF) by HMG CoA reductase inhibitors (statins)
Authors: Eberlein Michael , Heusinger‐Ribeiro Juliane , Goppelt‐Struebe Margarete
Published: January 2005

It was supposed that inhibitors of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG CoA) reductase (statins) might inhibit the expression of the fibrosis‐related factor CTGF (connective tissue growth factor) by interfering with the isoprenylation of Rho proteins.

The human renal fibroblast cell line TK173 was used as an in vitro model system to study the statin‐mediated modulation of the structure of the actin cytoskeleton and of the expression of CTGF mRNA.

Incubation of the cells with simvastatin or lovastatin time‐dependently and reversibly changed cell morphology and the actin cytoskeleton with maximal effects observed after about 18 h.

Within the same time period, statins reduced the basal expression of CTGF and interfered with CTGF induction by lysophosphatidic acid (LPA) or transforming growth factor beta. Simvastatin and lovastatin proved to be much more potent than pravastatin (IC50 1–3 μM compared to 500 μM).

The inhibition of CTGF expression was prevented when the cells were incubated with mevalonate or geranylgeranylpyrophosphate (GGPP) but not by farnesylpyrophosphate (FPP). Specific inhibition of geranylgeranyltransferase‐I by GTI‐286 inhibited LPA‐mediated CTGF expression whereas an inhibitor of farnesyltransferases FTI‐276 was ineffective.

Simvastatin reduced the binding of the small GTPase RhoA to cellular membranes. The effect was prevented by mevalonate and GGPP, but not FPP.

These data are in agreement with the hypothesis that interference of statins with the expression of CTGF mRNA is primarily due to interference with the isoprenylation of RhoA, in line with previous studies, which have shown that RhoA is an essential mediator of CTGF induction.

The direct interference of statins with the synthesis of CTGF, a protein functionally related to the development of fibrosis, may thus be a novel mechanism underlying the beneficial effects of statins observed in renal diseases.

British Journal of Pharmacology (2001) 133, 1172–1180; doi:10.1038/sj.bjp.0704173
Haemodynamic effects of the bacterial quorum sensing signal molecule, N‐(3‐oxododecanoyl)‐L‐homoserine lactone, in conscious, normal and endotoxaemic rats
Authors: Gardiner S M , Chhabra S R , Harty C , Williams P , Pritchard D I , Bycroft B W , Bennett T
Published: January 2005

N‐acylhomoserine lactones (AHLs) are small, diffusible signalling molecules, employed by Gram‐negative bacteria to coordinate gene expression with cell population density. Recent in vitro findings indicate that AHLs may function as virulence determinants per se, through modification of cytokine production by eukaryotic cells, and by stimulating the relaxation of blood vessels.

In the present study, we assessed the influence of AHLs on cardiovascular function in conscious rats, and draw attention to the ability of the N‐(3‐oxododecanoyl)‐L‐homoserine lactone (3‐oxo‐C12‐HSL), a signal molecule produced by P. aeruginosa, to cause marked bradycardia. This bradycardic effect was blocked by atropine and atenolol, and did not occur in vitro. Furthermore, modification of the acyl side chain length resulted in the loss of activity, whereas removal of the homoserine lactone ring, did not. The bradycardic effect of 3‐oxo‐C12‐HSL was also observed in endotoxaemic animals, albeit attenuated.

In normal rats, 3‐oxo‐C12‐HSL caused initial mesenteric and hindquarters vasoconstriction, but only slight, and delayed signs of vasodilatation in the renal and mesenteric vascular beds. Furthermore, administration of 3‐oxo‐C12‐HSL (pre‐treatment or 2 h post‐treatment) together with LPS, did not modify the established regional haemodynamic effects of the LPS, 6 h after the onset of its infusion.

Our observations do not provide any clear evidence for an ability of 3‐oxo‐C12‐HSL to modify the haemodynamic responses to LPS infusion. However, they are not inconsistent with the hypothesis that some of the cardiovascular sequelae of bacterial infection may be modulated by an influence of bacterial quorum sensing signalling molecules on the host.

British Journal of Pharmacology (2001) 133, 1047–1054; doi:10.1038/sj.bjp.0704174
‘THE MAN WHO NEVER WAS–Walter Ernest Dixon FRS’
Authors: Cuthbert Alan W
Published: January 2005

[Originally published: Volume vol 133(7), pages 945–950]

British Journal of Pharmacology (2001) 133, 945–950; doi:10.1038/sj.bjp.0704175
Treatment of hippocampal neurons with cyclosporin A results in calcium overload and apoptosis which are independent on NMDA receptor activation
Authors: Kaminska Bozena , Figiel Izabela , Pyrzynska Beata , Czajkowski Rafal , Mosieniak Grazyna
Published: January 2005

Calcineurin is a ubiquitous calcium/calmodulin dependent protein phosphatase that has been shown to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In the present study we show that CsA, a specific inhibitor of calcineurin, affects the survival of cultures developed from hippocampal dentate gyrus. Mixed neuronal‐glial cultures exposed to 8–40 μM CsA undergo cell death characterized by apoptotic changes in cellular and nuclear morphology.

TUNEL‐positive staining was observed only in neurons that developed pyknotic morphology after treatment with 8 μM CsA for 24–72 h.

Immunocytochemical staining with an anti‐GFAP monoclonal antibody revealed that astrocytes from mixed neuronal/glial cultures were unaffected by exposure to CsA at doses toxic for neurons and all TUNEL‐positive cells were neurons.

MK‐801, a noncompetitive inhibitor of glutamate receptor, does not inhibit the appearance of TUNEL‐positive neurons and apoptotic changes in nuclear morphology.

Preincubation of cells with 8 μM CsA increased basal intracellular calcium level in time dependent manner and decreased relative calcium response to glutamate. Application of 1 μM MK‐801 had no effect on CsA‐induced changes in Ca2+ level.

Our findings suggest that the neuronal death after CsA treatment is not a result of glutamate excitotoxicity and the increase in intracellular calcium concentration in neurons is not dependent on calcium influx via NMDA channel.

British Journal of Pharmacology (2001) 133, 997–1004; doi:10.1038/sj.bjp.0704177
Neuroprotective and neurorescuing effects of isoform‐specific nitric oxide synthase inhibitors, nitric oxide scavenger, and antioxidant against beta‐amyloid toxicity
Authors: Law A , Gauthier S , Quirion R
Published: January 2005

Beta amyloid (Aβ) is implicated in Alzheimer's disease (AD). Aβ1–42 (5, 10, or 20 μM) was able to increase NO release and decrease cellular viability in primary rat cortical mixed cultures.

L‐NOARG and SMTC (both at 10 or 100 μM) – type I NOS inhibitors – reduced cellular NO release in the absence of Aβ1–42. At 100 μM, both drugs decreased cell viability.

L‐NIL (10 or 100 μM), and 1400W (1 or 5 μM) – type II NOS inhibitors – reduced NO release and improved viability when either drug was administered up to 4 h post Aβ1–42 (10 μM) treatment. L‐NOARG and SMTC (both at 10 or 100 μM) were only able to decrease NO release. Carboxy‐PTIO or Trolox (both at 10 or 100 μM) – a NO scavenger and an antioxidant, respectively–increased viability when administered up to 1 h post Aβ1–42 treatment. Either L‐NIL (50 μM) or 1400W (3 μM) and Trolox (50 μM) showed synergistic actions.

Peroxynitrite (100 or 200 μM) reduced cell viability. Viabilities were improved by L‐NIL (100 μM), 1400W (5 μM), carboxy‐PTIO (10 or 100 μM), and Trolox (10 or 100 μM).

Hence, the data show that Aβ1–42 induced NO release in neurons and glial cells, and that Aβ neurotoxicity is, at least in part, mediated by NO. NO concentration modulating compounds and antioxidant may have therapeutic importance in neurological disorders where oxidative stress is likely involved such as in AD.

British Journal of Pharmacology (2001) 133, 1114–1124; doi:10.1038/sj.bjp.0704179
Voltage‐dependent acceleration of Cav1.2 channel current decay by (+)‐ and (−)‐isradipine
Authors: Berjukow S , Hering S
Published: January 2005

Inhibition of Cav1.2 by antagonist 1,4 dihydropyridines (DHPs) is associated with a drug‐induced acceleration of the calcium (Ca2+) channel current decay. This feature is contradictorily interpreted as open channel block or as drug‐induced inactivation. To elucidate the underlying molecular mechanism we investigated the effects of (+)‐ and (−)‐isradipine on Cav1.2 inactivation gating at different membrane potentials.

α11.2 Constructs were expressed together with α2‐δ‐ and β1a‐ subunits in Xenopus oocytes and drug‐induced changes in barium current (IBa) kinetics analysed with the two microelectrode voltage clamp technique. To study isradipine effects on IBa decay without contamination by intrinsic inactivation we expressed a mutant (V1504A) lacking fast voltage‐dependent inactivation.

At a subthreshold potential of −30 mV a 200‐times higher concentration of (−)‐isradipine was required to induce a comparable amount of inactivation as by (+)‐isradipine. At +20 mV the two enantiomers were equally efficient in accelerating the IBa decay.

Faster recovery from (−)‐ than from (+)‐isradipine‐induced inactivation at −80 mV in a Cav1.2 construct (τ(−)‐isr.(Cav1.2)=0.74 s<τ(+)‐isr.(Cav1.2)=2.85 s) and even more rapid recovery of V1504A (τ(−)‐isr.(V1504A)=0.39 s<τ(+)‐isr.(V1504A)=1.98 s) indicated that drug‐induced determinants and determinants of intrinsic inactivation (V1504) stabilize the DHP‐induced channel conformation in an additive manner.

In the voltage range between −25 and 20 mV where the channels inactivate predominantly from the open state the (+)‐ and (−)‐isradipine‐induced acceleration of the IBa decay in V1504A displayed similar voltage‐dependence as intrinsic fast inactivation of Cav1.2.

Our data suggest that the isradipine‐induced acceleration of the Cav1.2 current decay reflects enhanced fast voltage‐dependent inactivation and not open channel block.

British Journal of Pharmacology (2001) 133, 959–966; doi:10.1038/sj.bjp.0704181
Inhibition of mechanical activation of guinea‐pig airway afferent neurons by amiloride analogues
Authors: Carr Michael J , Gover Tony D , Weinreich Daniel , Undem Bradley J
Published: January 2005

The aim of this study was to investigate a role for Epithelial Sodium Channels (ENaCs) in the mechanical activation of low‐threshold vagal afferent nerve terminals in the guinea‐pig trachea/bronchus.

Using extracellular single‐unit recording techniques, we found that the ENaC blocker amiloride, and its analogues dimethylamiloride and benzamil caused a reduction in the mechanical activation of guinea‐pig airway afferent fibres.

Amiloride and it analogues also reduced the sensitivity of afferent fibres to electrical stimulation such that greater stimulation voltages were required to induce action potentials from their peripheral terminals within the trachea/bronchus.

The relative potencies of these compounds for inhibiting electrical excitability of afferent nerves were similar to that observed for inhibition of mechanical stimulation (dimethylamiloride∼#38;benzamil>amiloride). This rank order of potency is incompatible with the known rank order of potency for blockade of ENaCs (benzamil>amiloride>>dimethylamiloride).

As voltage‐gated sodium channels play an important role in determining the electrical excitability of neurons, we used whole‐cell patch recordings of nodose neuron cell bodies to investigate the possibility that amiloride analogues caused blockade of these channels. At the concentration required to inhibit mechanical activation of vagal nodose afferent fibres (100 μM), benzamil caused significant inhibition of voltage‐gated sodium currents in neuronal cell bodies acutely isolated from guinea‐pig nodose ganglia.

Combined, our findings suggest that amiloride and its analogues did not selectively block mechanotransduction in airway afferent neurons, but rather they reduced neuronal excitability, possibly by inhibiting voltage‐gated sodium currents.

British Journal of Pharmacology (2001) 133, 1255–1262; doi:10.1038/sj.bjp.0704197
Bradykinin increases Na+‐K+ pump activity in cultured guinea‐pig tracheal smooth muscle cells
Authors: Dodson Alicia M , Rhoden Kerry J
Published: January 2005

The effect of bradykinin on the Na+‐K+ pump of airway smooth muscle was investigated by measuring ouabain‐sensitive 86Rb+ uptake in cultured guinea‐pig tracheal smooth muscle cells.

Bradykinin induced a concentration‐dependent increase in ouabain‐sensitive 86Rb+ uptake, with an EC50 of 3 nM (pD2=8.50±0.10). Stimulation was not affected by indomethacin (1 μM) suggesting that it is not mediated by cycloxygenase products of arachidonic acid.

The B1 receptor agonists Lys‐des‐Arg9‐bradykinin and des‐Arg9‐bradykinin had no effect on ouabain‐sensitive 86Rb+ uptake. In contrast, the B1 and B2 receptor agonist Lys‐bradykinin induced a concentration‐dependent increase in ouabain‐sensitive 86Rb+ uptake with an EC50 of 6 nM (pD2=8.21±0.20).

The B1 receptor antagonist des‐Arg10‐HOE 140 (1 μM) had no effect on bradykinin‐stimulated ouabain‐sensitive 86Rb+ uptake. The B2 receptor antagonists HOE 140 and WIN 64338 antagonized bradykinin‐stimulated ouabain‐sensitive 86Rb+ uptake with pKB values (−log M) of 8.20±0.08 and 8.11±0.20 respectively.

Reducing extracellular Na+ from 146 mM to 11 mM caused a 53.5% decrease in basal ouabain‐sensitive 86Rb+ uptake and abolished bradykinin‐induced uptake. Two inhibitors of the Na+‐H+ exchanger, methylisobutyl‐amiloride (MIA; 1–100 μM) and ethylisopropyl‐amiloride (EIPA; 0.1–10 μM), inhibited bradykinin‐stimulated ouabain‐sensitive 86Rb+ uptake without affecting basal uptake.

These results suggest that bradykinin increases Na+‐K+ pump activity of guinea‐pig tracheal smooth muscle via stimulation of B2 receptors and activation of the Na+‐H+ exchanger.

British Journal of Pharmacology (2001) 133, 1339–1345; doi:10.1038/sj.bjp.0704198
Activation of the p38 and p42/p44 mitogen‐activated protein kinase families by the histamine H1 receptor in DDT1MF‐2 cells
Authors: Robinson Alex J , Dickenson John M
Published: January 2005

The mitogen‐activated protein kinases (MAPKs) consist of the p42/p44 MAPKs and the stress‐activated protein kinases, c‐Jun N‐terminal kinase (JNK) and p38 MAPK. In this study we have examined the effect of histamine H1 receptor activation on MAPK pathway activation in the smooth muscle cell line DDT1MF‐2.

Histamine stimulated time and concentration‐dependent increases in p42/p44 MAPK activation in DDT1MF‐2 cells. Responses to histamine were inhibited by the histamine H1 receptor antagonist mepyramine (KD 3.5 nM) and following pre‐treatment with pertussis toxin (PTX; 57% inhibition).

Histamine‐induced increases in p42/p44 MAPK activation were blocked by inhibitors of MAPK kinase 1 (PD 98059), tyrosine kinase (genistein and tyrphostin A47), phosphatidylinositol 3‐kinase (wortmannin and LY 294002) and protein kinase C (Ro 31‐8220; 10 μM; 41% inhibition). Inhibitors of Src tyrosine kinase (PP2) and the epidermal growth factor tyrosine kinase (AG1478) were without effect. Removal of extracellular Ca2+, chelation of intracellular Ca2+ with BAPTA and inhibition of focal adhesion assembly (cytochalasin D) had no significant effect on histamine‐induced p42/p44 MAPK activation.

Histamine stimulated time and concentration‐dependent increases in p38 MAPK activation in DDT1MF‐2 cells but had no effect on JNK activation. Histamine‐induced p38 MAPK activation was inhibited by pertussis toxin (74% inhibition) and the p38 MAPK inhibitor SB 203580 (95% inhibition).

In summary, we have shown the histamine H1 receptor activates p42/p44 MAPK and p38 MAPK signalling pathways in DDT1MF‐2 smooth muscle cells. Interestingly, signalling to both pathways appears to involve histamine H1 receptor coupling to Gi/Go‐proteins.

British Journal of Pharmacology (2001) 133, 1378–1386; doi:10.1038/sj.bjp.0704200
High level of α2‐adrenoceptor in rat foetal liver and placenta is due to α2B‐subtype expression in haematopoietic cells of the erythrocyte lineage
Authors: Cussac Daniel , Schaak Stéphane , Denis Colette , Flordellis Christodoulos , Calise Denis , Paris Hervé
Published: January 2005

Rat foetal liver contains large amounts of α2‐adrenoceptors. The present work aimed to identify the receptor subtype and the cell type accounting for high expression and to clarify the mechanisms responsible for the sharp decrease in hepatic receptivity occurring during the late stage of foetal development.

Binding experiments indicated that the density of α2‐adrenoceptors in the foetal liver (embryonic day 18; 615±155 fmol mg−1 of protein) is 18 fold higher than in newborn or adult (35.2±4.3 fmol mg−1). A high amount of receptor is also found in the placenta (443±53 fmol mg−1). In both tissues, the rank order of antagonists to inhibit radioligand binding matched the pharmacological profile of the α2B‐adrenoceptor and exclusively RNG transcripts were detected by RNase protection assays.

Isolation of cell fractions from foetal liver showed that α2B‐adrenoceptor is primarily expressed by haematopoietic cells. Consistent with this view, the receptor is found to be abundant in foetal blood, carried by reticulocytes. The expression in blood gradually declines to zero at 3 weeks of age and it is not recovered following induction of reticulocytosis in adults.

In foetal reticulocytes, a low proportion of the receptor population is coupled to G‐protein. The α2‐agonist UK14304 has a marginal effect on cyclic AMP level but significantly increases arachidonic acid release. The function of the receptor remains to be elucidated. However, together with observations on α2B‐knockout mice, the current finding strongly suggests a role for α2B‐adrenoceptor during foetal haematopoiesis in rodents.

British Journal of Pharmacology (2001) 133, 1387–1395; doi:10.1038/sj.bjp.0704203
Estimation of anti‐platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method
Authors: Sudo Toshiki , Ito Hideki , Ozeki Yasushi , Kimura Yukio
Published: January 2005

The effects of anti‐platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method.

The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time‐dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter.

Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre‐incubation (60 min), compared with short pre‐incubation (2 min). Such enhancement was only observed with ADP‐ and not collagen‐induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP‐induced platelet aggregation was more potent with PRP than whole blood (PATI200=3.80±0.95 μM for whole blood; 2.04±0.61 μM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes.

These results demonstrate that the SFP aggregometer can sensitively detect anti‐platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti‐platelet drugs.

British Journal of Pharmacology (2001) 133, 1396–1404; doi:10.1038/sj.bjp.0704204
Differential effects of chronic drug treatment on α3* and α7 nicotinic receptor binding sites, in hippocampal neurones and SH‐SY5Y cells
Authors: Ridley Diana L , Rogers Adrian , Wonnacott Susan
Published: January 2005

The aim of this study was to compare the effects of chronic treatment (for 4 or 7 days) with nicotinic drugs and 20 mM KCl on numbers of surface α7 nicotinic AChR, identified by [125I]‐α bungarotoxin (α‐Bgt) binding, in primary hippocampal cultures and SH‐SY5Y cells. Numbers of α3* nicotinic AChR were also examined in SH‐SY5Y cells, using [3H]‐epibatidine, which is predicted to label the total cellular population of predominantly α3β2* nicotinic AChR under the conditions used.

All the nicotinic agonists examined, the antagonists d‐tubocurarine and methyllycaconitine, and KCl, upregulated [125I]‐α Bgt binding sites by 20–60% in hippocampal neurones and, where examined, SH‐SY5Y cells.

Upregulation of [125I]‐α‐Bgt binding sites by KCl was prevented by co‐incubation with the L‐type Ca2+ channel blocker verapamil or the Ca2+‐calmodulin dependent kinase II (CaM‐kinase II) inhibitor KN‐62. Upregulation of [125I]‐α‐Bgt binding sites by nicotine or 3,[(4‐dimethylamino) cinnamylidene] anabaseine maleate (DMAC) was insensitive to these agents.

[3H]‐Epibatidine binding sites in SH‐SY5Y cells were not affected by KCl but were upregulated in a verapamil‐insensitive manner by nicotine and DMAC. KN‐62 itself provoked a 2 fold increase in [3H]‐epibatidine binding. The inactive analogue KN‐04 had no effect, suggesting that CaM‐kinase II plays a role in regulating numbers of α3* nicotinic AChR.

These data indicate that numbers of α3* and α7 nicotinic AChR are modulated differently. Nicotinic agonists and KCl upregulate α7 nicotinic AChR through distinct cellular mechanisms, the latter involving L‐type Ca2+ channels and CaM‐kinase II. In contrast, α3* nicotinic AChR are not upregulated by KCl. This difference may reflect the distinct physiological roles proposed for α7 nicotinic AChR.

British Journal of Pharmacology (2001) 133, 1286–1295; doi:10.1038/sj.bjp.0704207
Proinflammatory characteristics of a nonpeptide bradykinin mimic, FR190997, in vivo
Authors: Hayashi Izumi , Ishihara Keiko , Kumagai Yuji , Majima Masataka
Published: January 2005

Proinflammatory potency of the nonpeptide bradykinin (BK) B2 receptor agonist FR190997 (8‐[2,6‐dichloro‐3‐[N‐[(E)‐4‐(N‐methylcarbamoyl)cinnamidoacetyl]‐N‐methylamino]benzyloxy]‐2‐methyl‐4‐(2‐pyridylmethoxy)quinoline) was investigated.

Intradermal injection of FR190997 (0.03–3 nmol site−1) into dorsal skin of rats increased vascular permeability in a dose‐dependent manner. The effect was less than that of BK, but it was long‐acting and was inhibited by treatment with FR173657 (3 mg kg−1, p.o.). Captopril (10 mg kg−1, i.p.) did not enhance the plasma extravasation by FR190997 (0.3 nmol site−1) in the presence of soybean trypsin inhibitor (SBTI, 30 μg site−1).

Subcutaneous injection of FR190997 (3 nmol site−1) into the hindpaw of mice markedly induced paw swelling. The oedema lasted up to 3 h after the injection. Administration of indomethacin or NS‐398 (10 mg kg−1, i.p.) significantly reduced it at 3 h after the injection.

Simultaneous i.p. injection of prostaglandin (PG) E2 (1 nmol site−1) or beraprost sodium (0.5 nmol site−1) with FR190997 (5 nmol site−1) greatly enhanced frequency of writhing reactions in mice.

FR190997 (0.3–30 nmol kg−1, i.v.) showed less increase in airway opening pressure (Pao) in the guinea‐pig after i.v. injection. Furthermore, FR190997 (0.03–30 nmol) resulted in a very weak contraction of tracheal ring strips and lung parenchymal sections in vitro.

In mice sponge implants, topical application of FR190997 increased angiogenesis and granulation with enhanced expressions of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) mRNAs.

These results indicate that FR190997 has proinflammatory long‐lasting characteristics and it might be ‘a stable tool’ for studying the role of BK B2 receptor in vivo.

British Journal of Pharmacology (2001) 133, 1296–1306; doi:10.1038/sj.bjp.0704208
CGRP receptors mediating CGRP‐, adrenomedullin‐ and amylin‐induced relaxation in porcine coronary arteries. Characterization with ‘Compound 1’ (WO98/11128), a non‐peptide antagonist
Authors: Hasbak Philip , Sams Anette , Schifter Søren , Longmore Jenny , Edvinsson Lars
Published: January 2005

Calcitonin gene‐related peptide (CGRP), amylin and adrenomedullin (AM) belong to the same family of peptides. Accumulating evidence indicate that the calcitonin (CT) receptor, the CT receptor‐like receptor (CRLR) and receptor‐activity‐modifying proteins (RAMPs) form the basis of all the receptors in this family of peptides.

Using reverse transcriptase–polymerase chain reaction the presence of mRNA sequences encoding the CRLR, RAMP1 and RAMP2 were demonstrated in porcine left anterior descending (LAD) coronary arteries, whereas porcine calcitonin (CT) receptor mRNA was not present. The partial porcine mRNA sequences shared 82–92% nucleotide identity with human sequences.

The human peptides αCGRP, βCGRP, AM and amylin induced relaxation with pEC50 values of 8.1, 8.1, 6.7 and 6.1 M respectively.

The antagonistic properties of a novel non‐peptide CGRP antagonist ‘Compound 1’ (WO98/11128), βCGRP8–37 and the proposed AM receptor antagonist AM22–52 were compared to the well‐known CGRP1 receptor antagonist αCGRP8–37.

The αCGRP8–37 and βCGRP8–37 induced concentration‐dependent (10−7–10−5 M) rightward shift of both the αCGRP and βCGRP concentration‐response curves. βCGRP8–37 (10−6 M) had the same effect as αCGRP8–37 (10−6 M), but with less potent rightward shift of the concentration‐response curves for αCGRP, AM and amylin.

Preincubation with ‘Compound 1’ (10−7–10−5 M) and AM22–52 (10−6 M) had no significant antagonistic effect.

In conclusion, the building blocks forming CGRP and AM receptors were present in the porcine LAD, whereas those of the amylin receptor were not. αCGRP, βCGRP, AM and amylin mediated vasorelaxation via the CGRP receptors. No functional response was detected to adrenomedullin via the adrenomedullin receptor.

British Journal of Pharmacology (2001) 133, 1405–1413; doi:10.1038/sj.bjp.0704210
In vivo evidence that protease‐activated receptors 1 and 2 modulate gastrointestinal transit in the mouse
Authors: Kawabata Atsufumi , Kuroda Ryotaro , Nagata Nanae , Kawao Naoyuki , Masuko Takashi , Nishikawa Hiroyuki , Kawai Kenzo
Published: January 2005

Protease‐activated receptors (PARs) 1 and 2 modulate the gastric and intestinal smooth muscle motility in vitro. In the present study, we examined if activation of PAR‐2 and PAR‐1 could alter gastrointestinal transit in mice.

Intraperitoneal administration of the PAR‐2‐activating peptide SLIGRL‐NH2, but not the inactive control LSIGRL‐NH2, at 1–5 μmol kg−1, in combination with the aminopeptidase inhibitor amastatin at 2.5 μmol kg−1, facilitated gastrointestinal transit in a dose‐dependent manner. The human PAR‐1‐derived peptide SFLLR‐NH2 and the specific PAR‐1 agonist TFLLR‐NH2, but not the inactive control FSLLR‐NH2, at 2.5–10 μmol kg−1, in combination with amastatin, also promoted gastrointestinal transit.

The Ca2+‐activated, small conductance K+ channel inhibitor apamin at 0.01 μmol kg−1 significantly potentiated the actions of SLIGRL‐NH2 and TFLLR‐NH2 at subeffective doses.

The increased gastrointestinal transit exerted by either SLIGRL‐NH2 at 5 μmol kg−1 or TFLLR‐NH2 at 10 μmol kg−1 was completely abolished by the L‐type Ca2+ channel inhibitor verapamil at 61.6 μmol kg−1. In contrast, the tyrosine kinase inhibitor genistein at 18.5 μmol kg−1 failed to modify the effects of the agonists for PAR‐2 or PAR‐1.

These findings demonstrate that PAR‐1 and PAR‐2 modulate gastrointestinal transit in mice in vivo. Our data also suggest that the PAR‐1‐and PAR‐2‐mediated effects are modulated by apamin‐sensitive K+ channels and are dependent on activation of L‐type Ca2+ channels, but independent of tyrosine kinase. Our study thus provides novel evidence for the physiological and/or pathophysiological roles of PARs 1 and 2 in the digestive systems, most probably during inflammation.

British Journal of Pharmacology (2001) 133, 1213–1218; doi:10.1038/sj.bjp.0704211
Ca2+‐sensitizing effect is involved in the positive inotropic effect of troglitazone
Authors: Furuse Yoshiyuki , Ogino Kazuhide , Shimoyama Masaki , Sasaki Norihito , Hisatome Ichiro
Published: January 2005

Troglitazone, an insulin sensitizing agent, has a direct positive inotropic effect. However, the mechanism of this effect remains unclear. Thus, we examined the inotropic effect of troglitazone while focusing on intracellular Ca2+ handling.

Troglitazone significantly increased peak isovolumic left ventricular pressure (LVPmax), peak rate of rise of LVP (dP/dtmax), peak rate of fall of LVP (dP/dtmin) in isolated rat hearts perfused at a constant coronary flow and heart rate. This inotropic effect of troglitazone was not inhibited by pretreatment with carbachol (muscarine receptor agonist), H89 (protein kinase A inhibitor), U73122 (phospholipase C inhibitor), H7 (protein kinase C inhibitor), verapamil (L‐type Ca2+ channel antagonist), thapsigargin (Ca2+‐adenosine triphosphatase inhibitor) or ryanodine (ryanodine receptor opener).

Radioimmunoassay showed that the cyclic adenosine monophosphate concentration in the left ventricle was not increased by troglitazone.

Whole‐cell patch clamp analysis revealed that troglitazone had no effect on inward Ca2+ currents in cardiomyocytes.

In fura‐2 loaded perfused rat hearts, troglitazone exerted its positive inotropic effect without increasing Ca2+ concentration.

These results suggest that neither the inward Ca2+ currents nor Ca2+ handling in the sarcoplasmic reticulum was involved in the inotropic effect of troglitazone. Furthermore, troglitazone exerted its positive inotropic effect without affecting the intracellular concentration of Ca2+.

In conclusion, the positive inotropic effect of troglitazone is mediated by a sensitization of Ca2+.

British Journal of Pharmacology (2001) 133, 1307–1313; doi:10.1038/sj.bjp.0704212
Protein kinase A‐independent responses to β‐adrenoceptor agonists
Authors: Dent Gordon
Published: January 2005

British Journal of Pharmacology (2001) 133, 1199–1200; doi:10.1038/sj.bjp.0704214
Effect of M40403 treatment of diabetic rats on endoneurial blood flow, motor nerve conduction velocity and vascular function of epineurial arterioles of the sciatic nerve
Authors: Coppey Lawrence J , Gellett Jill S , Davidson Eric P , Dunlap Joyce A , Lund Donald D , Salvemini Daniela , Yorek Mark A
Published: January 2005

To further explore the effect of antioxidants in preventing diabetes‐induced vascular and neural dysfunction we treated streptozotocin‐induced diabetic rats daily with subcutaneous injections of 10 mg kg−1 of M40403 (n=11) and compared the results obtained from 17 control rats and 14 untreated diabetic rats. M40403 is a manganese(II) complex with a bis(cyclo‐hexylpyridine)‐substituted macrocyclic ligand that was designed to be a selective functional mimetic of superoxide dismutase. Thus, M40403 provides a useful tool to evaluate the roles of superoxide in disease states.

Treatment with M40403 significantly improved diabetes‐induced decrease in endoneurial blood flow, acetylcholine‐mediated vascular relaxation in arterioles that provide circulation to the region of the sciatic nerve, and motor nerve conduction velocity (P<0.05). M40403 treatment also reduced the appearance of superoxide in the aorta and epineurial vessels and peroxynitrite in epineurial vessels. Treating diabetic rats with M40403 reduced the diabetes‐induced increase in thiobarbituric acid reactive substances in serum but did not prevent the decrease in lens glutathione level. Treating diabetic rats with M40403 did not improve sciatic nerve Na+/K+ ATPase activity or the sorbitol, fructose or myo‐inositol content of the sciatic nerve.

These studies provide additional evidence that diabetes‐induced oxidative stress and the generation of superoxide and perhaps peroxynitrite may be partially responsible for the development of diabetic vascular and neural complications.

British Journal of Pharmacology (2001) 134, 21–29; doi:10.1038/sj.bjp.0704216
Pharmacological characterization of adenosine receptors in PGT‐β mouse pineal gland tumour cells
Authors: Suh Byung‐Chang , Kim Tae‐Don , Lee Jung‐Uek , Seong Je‐Kyung , Kim Kyong‐Tai
Published: January 2005

The adenosine receptor in mouse pinealocytes was identified and characterized using pharmacological and physiological approaches.

Expression of the two adenosine receptor subtypes A2B and A3 was detected in mouse pineal glands and PGT‐β cells by polymerase chain reaction and nucleotide sequencing.

Adenosine and 5′‐N‐ethylcarboxamidoadenosine (NECA) evoked cyclic AMP generation but the A2A‐selective agonist 2‐(4‐(2‐carboxyethyl)phenylethylamino)adenosine‐5′‐N‐ethylcarboxamideadenosine (CGS 21680) and the A1‐specific agonists R‐N6‐(2‐phenylisopropyl)adenosine (R‐PIA) and N6‐cyclopentyladenosine (CPA) had little effect on intracellular cyclic AMP levels. The A2B receptor selective antagonists alloxazine and enprofylline completely blocked NECA‐mediated cyclic AMP accumulation.

Treatment of cells with the A3‐selective agonist N6‐(3‐iodobenzyl)‐5′‐(N‐methylcarbamoyl)adenosine (IB‐MECA) inhibited the elevation of the cyclic AMP level induced by NECA or isoproterenol in a concentration‐dependent manner with maximal inhibition of 40 – 50%. These responses were blocked by the specific A3 adenosine receptor antagonist MRS 1191. Pretreatment of the cells with pertussis toxin attenuated the IB‐MECA‐induced responses, suggesting that this effect occurred via the pertussis toxin‐sensitive inhibitory G proteins.

IB‐MECA also caused a concentration‐dependent elevation in [Ca2+]i and IP3 content. Both the responses induced by IB‐MECA were attenuated by treatment with U73122 or phorbol 12‐myristate 13‐acetate.

These data suggest the presence of both A2B and A3 adenosine receptors in mouse pineal tumour cells and that the A2B receptor is positively coupled to adenylyl cyclase whereas the A3 receptor is negatively coupled to adenylyl cyclase and also coupled to phospholipase C.

British Journal of Pharmacology (2001) 134, 132–142; doi:10.1038/sj.bjp.0704218
Differential effects of anandamide on acetylcholine release in the guinea‐pig ileum mediated via vanilloid and non‐CB1 cannabinoid receptors
Authors: Mang Christian F , Erbelding Doris , Kilbinger Heinz
Published: January 2005

The effects of anandamide on [3H]‐acetylcholine release and muscle contraction were studied on the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig ileum preincubated with [3H]‐choline.

Anandamide increased both basal [3H]‐acetylcholine release (pEC50 6.3) and muscle tone (pEC50 6.3). The concentration‐response curves for anandamide were shifted to the right by 1 μM capsazepine (pKB 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 μM). The CB1 and CB2 receptor antagonists, SR141716A (1 μM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide.

Anandamide inhibited the electrically‐evoked release of [3H]‐acetylcholine (pEC50 5.8) and contractions (pEC50 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 μM anandamide.

The inhibitory effects of anandamide were not changed by either capsazepine (1 μM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 μM). SR141716A (1 μM) produced rightward shifts in the inhibitory concentration‐response curves for anandamide yielding pKB values of 6.6 and 6.2.

CP55940 inhibited the evoked [3H]‐acetylcholine release and contractions, and SR141716A (0.1 μM) shifted the concentration‐response curves of CP55940 to the right with pKB values of 8.4 and 8.9.

The experiments confirm the existence of release‐inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.

British Journal of Pharmacology (2001) 134, 161–167; doi:10.1038/sj.bjp.0704220
Mechanism of action of angiotensin II in human isolated subcutaneous resistance arteries
Authors: Garcha Robinder S , Sever Peter S , Hughes Alun D
Published: January 2005

Human isolated subcutaneous arteries were mounted in a myograph and isometric tension measured. In some experiments, intracellular calcium [Ca2+]i was also measured using fura‐2.

Angiotensin II (100 pM – 1 μM) increased [Ca2+]i and tone in a concentration‐dependent manner. The effects of angiotensin II (100 nM) were inhibited by an AT1‐receptor antagonist, candesartan (100 pM).

Ryanodine (10 μM), had no effect on angiotensin II‐induced responses, but removal of extracellular Ca2+ abolished angiotensin II‐induced rise in [Ca2+]i and tone. Inhibition of Ca2+ entry by Ni2+ (2 mM), also inhibited angiotensin II responses. The dihydropyridine, L‐type calcium channel antagonist, amlodipine (10 μM), only partially attenuated angiotensin II responses.

Inhibition of protein kinase C (PKC) by chelerythrine (1 μM), or by overnight exposure to a phorbol ester (PDBu; 500 nM) had no effect on angiotensin II‐induced contraction.

Genistein (10 μM), a tyrosine kinase inhibitor, inhibited angiotensin II‐induced contraction, but did not inhibit the rise in [Ca2+]i, suggesting that at this concentration it affected the calcium sensitivity of the contractile apparatus. Genistein did not affect responses to norepinephrine (NE) or high potassium (KPSS).

A selective MEK inhibitor, PD98059 (30 μM), inhibited both the angiotensin II‐induced contraction and rise in [Ca2+]i, but had no effect on responses to NE or KPSS.

AT1 activation causes Ca2+ influx via L‐type calcium channels and a dihydropyridine‐insensitive route, but does not release Ca2+ from intracellular sites. Activation of tyrosine kinase(s) and the ERK 1/2 pathway, but not classical or novel PKC, also play a role in angiotensin II‐induced contraction in human subcutaneous resistance arteries.

British Journal of Pharmacology (2001) 134, 188–196; doi:10.1038/sj.bjp.0704222
A possible role of lipoxygenase in the activation of vanilloid receptors by anandamide in the guinea‐pig bronchus
Authors: Craib Susan J , Ellington Heather C , Pertwee Roger G , Ross Ruth A
Published: January 2005

In the absence of indomethacin, anandamide did not contract the guinea‐pig bronchus at concentrations up to 100 μM. In the presence of indomethacin (10 μM), anandamide induced concentration‐related contractions with a pEC50 value of 5.18±0.11. It was significantly less potent than capsaicin (pEC50 7.01±0.1). The anandamide uptake inhibitor AM404, produced only a 14.1±3.22% contraction at 100 μM. All experiments were conducted in the presence of PMSF (20 μM).

The vanilloid receptor antagonist, capsazepine (10 μM), significantly attenuated the contractile effect of anandamide, the response to 100 μM anandamide being 40.53±7.04% in the presence of vehicle and 1.57±8.93% in the presence of 10 μM capsazepine. The contractile actions of anandamide and AM404 were markedly enhanced by the peptidase inhibitor thiorphan.

The log concentration‐response curve of anandamide was unaltered by the CB1 receptor antagonist, SR141716A. The pEC50 values for anandamide were 4.88±0.08 and 5.17±0.19 in the presence of vehicle and SR141716A (1 μM) respectively.

The lipoxygenase inhibitors 5,8,11,14‐eicosatetraynoic acid (ETYA) and 5,8,11 eicosatriynoic acid (ETI) reduced the effect of 100 μM anandamide from 34.7±1.9% (vehicle) to 7.7±5% (ETYA, 10 μM) and from 41.85±4.25% (n=6) (vehicle) to 10.31±3.54 (n=6) (ETI, 20 μM). Neither inhibitor significantly affected contraction of the tissue by substance P.

This study provides evidence that anandamide acts on vanilloid receptors in the guinea‐pig isolated bronchus. These data raise the possibility that the contractile action of anandamide may be due, at least in part, to lipoxygenase metabolites of this fatty acid amide that are vanilloid receptor agonists.

British Journal of Pharmacology (2001) 134, 30–37; doi:10.1038/sj.bjp.0704223
Angiotensin II reduces infarct size and has no effect on post‐ischaemic contractile dysfunction in isolated rat hearts
Authors: Ford William R , Clanachan Alexander S , Robin Hiley C , Jugdutt Bodh I
Published: January 2005

In order to test the hypothesis that angiotensin II exacerbates myocardial ischaemia‐reperfusion (IR) injury, we examined the effects of graded angiotension II concentrations of angiotensin II on IR injury in both working and non‐working (Langendorff) isolated rat hearts.

Non‐working hearts were subjected to 30 min aerobic perfusion (baseline) then 25 min of global, no‐flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=7) or presence of 1 (n=6) or 10 nM (n=5) angiotensin II). Recoveries of LV developed pressure and coronary flow after 30 min reperfusion in control hearts (58±9 and 40±8% of baseline levels, respectively) were no different from hearts treated with 1 or 10 nM angiotensin II. Infarct size (determined at the end of reperfusion by triphenyltetrazolium chloride staining) was reduced by angiotensin II in a concentration‐dependent manner (from a control value of 27±3 to 18±4% and 9±3% of the LV, respectively).

Working hearts were subjected to 50 min pre‐ischaemic (pre‐I) aerobic perfusion then 30 min of global, no‐flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=14) or presence of 1 (n=8), 10 (n=7) or 100 nM (n=7) angiotensin II). In controls, post‐ischaemic (post‐I) left ventricular (LV) work and efficiency of oxygen consumption were depressed (43±9 and 42±10% of pre‐I levels, respectively). The presence of angiotensin II throughout IR had no effect on LV work compared with control.

Thus, angiotensin II reduces infarct size in a concentration‐dependent manner but has no effect on contractile stunning associated with IR in isolated rat hearts.

British Journal of Pharmacology (2001) 134, 38–45; doi:10.1038/sj.bjp.0704225