bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in the pharmacological properties of rat and chicken brain fatty acid amidohydrolase
Authors: Fowler Christopher J , Börjesson Magnus , Tiger Gunnar
Published: January 2005

The pharmacological properties of fatty acid amidohydrolase (FAAH) were investigated in brains of 35‐day‐old chickens, since nothing is known about the enzyme in avian species.

FAAH activity towards both [3H]‐palmitoylethanolamide (PEA) [KM=1.5 μM] and [3H]‐anandamide (AEA) [KM=5.4 μM] was demonstrated in the chicken brains. The chicken FAAH was inhibited by the substrate analogues oleyl trifluoromethylketone (OTMK) and diazomethylarachidonyl ketone (DAK) with similar potencies to the rat FAAH. However, in contrast to the rat brain, phenylmethylsulphonyl fluoride (PMSF) and the enantiomers of ibuprofen had very weak effects on chicken brain FAAH.

Indomethacin and niflumic acid were found to inhibit rat brain AEA hydrolysis. The inhibition by indomethacin was reversible and competitive, with a Ki value of 120 μM. Chicken FAAH was less sensitive to indomethacin than its rodent counterpart, but the inhibition was also competitive (Ki).

It is concluded that chicken FAAH activity has different pharmacological properties to its rodent counterpart.

British Journal of Pharmacology (2000) 131, 498–504; doi:10.1038/sj.bjp.0703569
Mechanisms underlying the chronic pravastatin treatment‐induced improvement in the impaired endothelium‐dependent aortic relaxation seen in streptozotocin‐induced diabetic rats
Authors: Kobayashi Tsuneo , Matsumoto Takayuki , Kamata Katsuo
Published: January 2005

We investigated the effects of chronic pravastatin treatment on the impaired endothelium‐dependent relaxation seen in aortae from established streptozotocin (STZ)‐induced diabetic rats. Starting at 6 weeks of diabetes, pravastatin (10 mg kg−1) was administered to STZ‐induced diabetic rats for 4 weeks.

The increased total cholesterol and low‐density lipoprotein (LDL) cholesterol levels seen in STZ‐induced diabetic rats were not restored to normal by pravastatin. Aortae from pravastatin‐treated diabetic rats did not show an impaired endothelium‐dependent relaxation to acetylcholine. The expression of the mRNA for endothelial nitric oxide synthase was unaffected by diabetes or pravastatin.

The enhanced level of malondialdehyde (MDA)‐modified LDL seen in STZ‐induced diabetic rats was normalized by pravastatin treatment. The resistance of LDL to oxidation was assessed by measuring the amount of MDA or conjugated dienes generated by incubation with copper ions. LDL isolated from diabetic rats, but not those from pravastatin‐treated diabetics, showed enhanced the susceptibility to oxidation, but incubation in vitro with pravastatin had no effect on LDL oxidation.

Following incubation of control aortae for 6 h with LDL (0.1 mg protein ml−1) isolated from diabetic rats, the endothelium‐dependent relaxation to acetylcholine or A23187 was impaired, but LDL isolated from control or pravastatin‐treated rats had no such effect. This inhibitory effect of diabetic LDL was prevented by superoxide dismutase (SOD), a superoxide scavenger.

These results suggest that pravastatin preserves endothelial function in aortae from STZ‐induced diabetic rats without lowering plasma cholesterol, and its effect may be due to decreased LDL oxidation.

British Journal of Pharmacology (2000) 131, 231–238; doi:10.1038/sj.bjp.0703572
Investigation into the involvement of phospholipases A2 and MAP kinases in modulation of AA release and cell growth in A549 cells
Authors: Choudhury Qamrul G , Mckay Diane T , Flower Roderick J , Croxtall Jamie D
Published: January 2005

We have investigated the contribution of specific PLA2s to eicosanoid release from A549 cells by using specific inhibitors of secretory PLA2 (ONO‐RS‐82 and oleyloxyethylphosphocholine), cytosolic PLA2 (AACOCF3 and MAFP) and calcium‐independent PLA2 (HELSS, MAFP and PACOCF3). Similarly, by using specific inhibitors of p38 MAPK (SB 203580), ERK1/2 MAPK (Apigenin) and MEK1/2 (PD 98059) we have further evaluated potential pathways of AA release in this cell line.

ONO‐RS‐82 and oleyloxyethylphosphocholine had no significant effect on EGF or IL‐1β stimulated 3H‐AA or PGE2 release or cell proliferation. AACOCF3, HELSS, MAFP and PACOCF3 significantly inhibited both EGF and IL‐1β stimulated 3H‐AA and PGE2 release as well as cell proliferation. Apigenin and PD 98509 significantly inhibited both EGF and IL‐1β stimulated 3H‐AA and PGE2 release and cell proliferation whereas, SB 203580 had no significant effect on EGF or IL‐1β stimulated 3H‐AA release, or cell proliferation but significantly suppressed EGF or IL‐1β stimulated PGE2 release.

These results confirm that the liberation of AA release, generation of PGE2 and cell proliferation is mediated largely through the actions of cPLA2 whereas, sPLA2 plays no significant role. We now also report a hitherto unsuspected contribution of iPLA2 to this process and demonstrate that the stimulating action of EGF and IL‐1β in AA release and cell proliferation is mediated in part via a MEK and ERK‐dependent pathway (but not through p38MAPK). We therefore propose that selective inhibitors of MEK and MAPK pathways may be useful in controlling AA release, eicosanoid production and cell proliferation.

British Journal of Pharmacology (2000) 131, 255–265; doi:10.1038/sj.bjp.0703573
Endotoxin causes up‐regulation of endothelin receptors in cultured hepatic stellate cellsvianitric oxide‐dependent and ‐independent mechanisms
Authors: Gandhi Chandrashekhar R , Uemura Tadahiro , Kuddus Ruhul
Published: January 2005

Hepatic stellate cells (HSC) and their transformed phenotype found in the chronically injured liver play important roles in hepatic physiology and pathology. HSC produce and react to a potent contractile peptide endothelin‐1 (ET‐1) and also synthesize a vasorelaxant nitric oxide (NO) upon stimulation with endotoxin. However, whether endotoxin affects ET‐1 system of HSC and if this is a mechanism of endotoxin‐induced hepatic injury is not known.

We characterized synthesis of ET‐1 and NO and ET‐1 receptors in cultured quiescent and transformed HSC subjected to endotoxin treatment. Endotoxin (1–1000 ng ml−1) stimulated synthesis of ET‐1 and NO and up‐regulated ET‐1 receptors in both cell types.

Inhibition of NO synthesis by NG‐monomethyl‐L‐homoarginine strongly inhibited endotoxin‐induced increase in ET‐1 receptors in transformed HSC but produced small additional increase in quiescent HSC. Inhibition of soluble guanylyl cyclase by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one blocked the effect of endotoxin on ET‐1 receptors in both cell types. Moreover, ET‐1 receptors were increased in both cell types during earlier time points (1–4 h) of endotoxin treatment in the absence of the stimulation of NO synthesis.

These results demonstrate that endotoxin up‐regulates ET‐1 receptors in HSC by NO‐dependent and ‐independent mechanisms. Such effects of endotoxin can be of importance in acute endotoxemia and during chronic injury of the liver.

British Journal of Pharmacology(2000)131, 319–327; doi:10.1038/sj.bjp.0703577
Contractile responses to sumatriptan and ergotamine in the rabbit saphenous vein: effect of selective 5‐HT1F receptor agonists and PGF2α
Authors: Cohen Marlene L , Schenck Kathryn
Published: January 2005

Contractile responses to ergotamine, sumatriptan and the novel 5‐HT1F receptor agonists, LY334370 and LY344864 were examined using the rabbit saphenous vein.

Ergotamine (pEC50=8.7±0.06) was 30 fold more potent than 5‐hydroxytryptamine (5‐HT) (pEC50=7.2±0.13) and 300 fold more potent than sumatriptan (pEC50=6.0±0.08) in contracting the rabbit saphenous vein in vitro. The selective 5‐HT1F receptor agonists, LY334370 or LY344864 (up to 10−4 M), did not contract the rabbit saphenous vein.

The contractile response to ergotamine in this tissue resulted from activation of both alpha1 and 5‐HT1B/1D receptors based on the observation that prazosin (10−6 M), an α‐adrenoceptor antagonist, and GR127935 (10−8 M) a 5‐HT1B/1D receptor antagonist, dextrally shifted the contractile response to ergotamine. In contrast, prazosin (10−6 M) did not alter contraction to sumatriptan whereas GR127935 (10−8 M) was a potent antagonist (−log KB=10.0) suggesting that sumatriptan‐induced contraction of the rabbit saphenous vein was mediated only by activation of receptors similar or identical to 5‐HT1B/1D receptors.

PGF2α (3×10−7 M) produced a modest increase (approximately 5.0–10.0% maximum PGF2α contraction) in saphenous vein force. Precontraction with PGF2α (3×10−7 M) dramatically augmented the potency and maximal contractile response to sumatriptan (pEC50=7.1) and modestly enhanced the contractile potency of ergotamine (pEC50=9.0) in the rabbit saphenous vein. However, PGF2α (3×10−7 M) only unmasked a contraction to the 5‐HT1F receptor agonists when concentrations exceeded 10−5 M, concentrations considerably higher than their 5‐HT1F receptor affinities.

LY334370 (10−6 M) pretreatment did not alter contraction to either sumatriptan or ergotamine and a higher concentration (10−5 M) of LY334370 or LY344864 inhibited contraction to sumatriptan.

Thus, activation of 5‐HT1F receptors will not induce vascular contraction (either alone or following modest tone with PGF2α) or augment contraction to other contractile agonists in the rabbit saphenous vein.

British Journal of Pharmacology (2000) 131, 562–568; doi:10.1038/sj.bjp.0703587
Nitric oxide and sodium nitroprusside‐induced relaxation of the human umbilical artery
Authors: Lovren Fina , Triggle Chris
Published: January 2005

In the human umbilical artery (HUA) pre‐contracted with the thromboxane mimetic U46619 or with 5‐hydroxytryptamine (5‐HT), (and pretreated with indomethacin (3 μM) to suppress the synthesis of prostanoids), authentic nitric oxide (NO) evoked concentration‐dependent relaxation (pEC50 7.05 and 5.99, respectively). In contrast, sodium nitroprusside (SNP) induced relaxation only in U46619 pre‐contracted HUA (pEC50 6.52).

At high (>300 mmHg) vs low (<55 mmHg) oxygen tension the dose‐response curves to NO‐ and SNP‐induced relaxations were biphasic and shifted leftward.

Preincubation of the arterial rings with the soluble guanylyl cyclase (sGC) inhibitor 1H[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 10 μM) shifted the concentration‐response curve to NO, reduced the maximal relaxation response to NO (Emax 71%) and to SNP (Emax 10%).

Pre‐exposure of HUA rings to high extracellular K+ (50 mM) reduced Emax relaxation responses to NO (36%) and SNP (1%).

Pretreatment of the HUA with the K+ channel inhibitors, tetraethylammonium (TEA, 1 mM), 4‐aminopyridine (4‐AP, 0.5 mM), charybdotoxin (0.1 μM) or iberiotoxin (0.1 μM) increased the pEC30 for NO and SNP and changed the shape of the dose‐response curves from biphasic to monophasic.

Pre‐incubation of HUA rings with TEA (1 mM), 4‐AP (0.5 mM) and ODQ (10 μM) significantly reduced the NO‐induced maximal relaxation (Emax 26%) but not the pEC50 (5.60).

These data indicate that SNP‐induced relaxation in the HUA is primarily mediated via sGC‐cyclic GMP whereas NO‐induced relaxation also involves the activation of KV and KCa channels and a cyclic GMP/K+ channel‐independent mechanism(s).

British Journal of Pharmacology (2000) 131, 521–529; doi:10.1038/sj.bjp.0703588
The effect of Gi‐protein inactivation on basal, and β1‐ and β2AR‐stimulated contraction of myocytes from transgenic mice overexpressing the β2‐adrenoceptor
Authors: Gong Haibin , Adamson Dawn L , Ranu Hardeep K , Koch Walter J , Heubach Jürgen F , Ravens Ursula , Zolk Oliver , Harding Sian E
Published: January 2005

The atria and ventricles of transgenic mice (TGβ2) with cardiac overexpression of the human β2‐adrenoceptor (β2AR) were initially reported to show maximum contractility in the absence of β‐AR stimulation. However, we have previously observed a different phenotype in these mice, with myocytes showing normal contractility but reduced βAR responses. We have investigated the roles of cyclic AMP and Gi in basal and βAR function in these myocytes.

ICI 118,551 at inverse agonist concentrations decreased contraction by 32%. However, the cyclic AMP antagonist Rp‐cAMPS had no effect on contraction in TGβ2 myocytes, indicating that there was no tonic influence of raised cyclic AMP. These findings cannot be explained by the proposed model for inverse agonism, where the activated receptor (R*) raises cyclic AMP levels and so increases contraction in the absence of agonist.

After pertussis toxin (PTX) pretreatment to produce inactivation of Gi, the basal contraction in 1 mM Ca2+ was increased in TGβ2 mice (7.82±0.47%, n=23) compared to LM mice (3.60±0.59%, n=11) (P<0.001). The contraction amplitude of myocytes to the maximal concentration of isoprenaline was also increased significantly by PTX in TGβ2 mice (9.40±1.22%, n=8) and was no longer reduced compared to LM mice (8.93±1.50%, n=11). Both β1‐ and β2AR subtypes were affected both by the original desensitization and by the resensitization with PTX.

PTX treatment has therefore restored the original phenotype, with high basal contractility and little further effect of isoprenaline. We suggest that both β‐AR desensitization and lack of increased basal contraction in ventricular myocytes from our colony of TGβ2 mice were due to increased activity of PTX‐sensitive G‐proteins.

British Journal of Pharmacology (2000) 131, 594–600; doi:10.1038/sj.bjp.0703591
The involvement of cytokines in the second window of ischaemic preconditioning
Authors: Yamashita Nobushige , Hoshida Shiro , Otsu Kinya , Taniguchi Naoyuki , Kuzuya Tsunehiko , Hori Masatsugu
Published: January 2005

We utilized a rat model of myocardial infarction to investigate whether manganese superoxide dismutase (Mn‐SOD), an intrinsic radical scavenger, and tumour necrosis factor‐ α (TNF‐α) and/or interleukin‐1β (IL‐1β) are involved in the late phase of ischaemic preconditioning (IP).

IP was induced in anaesthetized rats by four 3‐min left coronary artery (LCA) occlusions, each separated by 10 min of reperfusion. Twenty‐four hours after the repetitive brief ischaemia, the LCA was occluded for 20 min followed by reperfusion for 48 h. IP reduced the infarct size by approximately 46% as determined after 48 h of reperfusion.

Antisense oligodeoxynucleotides to Mn‐SOD inhibited the increases in Mn‐SOD content and activity, and abolished the expected decrease in myocardial infarct size. Sense or scrambled oligodeoxynucleotides did not abolish either Mn‐SOD induction or tolerance to ischaemia/reperfusion.

The simultaneous administration of the antibodies to TNF‐α (0.5 ml) and IL‐1β (0.5 mg) prior to IP abolished the cardioprotection and the increase in Mn‐SOD activity induced by IP.

We conclude that the induction and activation of Mn‐SOD, mediated by TNF‐α and IL‐1β after IP, plays an important role in the acquisition of late‐phase cardioprotection against ischaemia/reperfusion injury in rats.

British Journal of Pharmacology (2000) 131, 415–422; doi:10.1038/sj.bjp.0703594
Gene expression of receptors and enzymes involved in GABAergic and glutamatergic neurotransmission in the CNS of rats behaviourally dependent on ethanol
Authors: Eravci Murat , Schulz Olaf , Grospietsch Thorsten , Pinna Graziano , Brödel Oliver , Meinhold Harald , Baumgartner Andreas
Published: January 2005

The steady state levels of the messenger RNA (mRNA) of eight GABAA receptor subunits, five glutamate receptor subunits and seven enzymes involved in the synthesis of glutamate and GABA were measured in eight regions of rat brain in a recently developed animal model of ‘behavioural dependence’ on ethanol.

‘Behavioural dependence’ including loss of control was induced by offering the rats the choice between ethanol and water over a 9‐month period (Group A). This group was compared with a group given the choice between ethanol and water for only 2 months (not yet ‘behaviourally dependent’, Group B), a group forced to consume ethanol as sole fluid over a 9‐month period (also not ‘behaviourally dependent’, Group C) and ethanol‐naive control rats (Group D). All groups were sacrificed 1 month after the ethanol was withdrawn.

The mRNA concentrations of all eight GABA receptor subunits, four out of the five subunits of different glutamate receptors and those of seven enzymes involved in GABA and glutamate production were reduced almost exclusively in the parieto‐occipital cortex in Groups A and B, but not Group C.

These data suggest that the synthesis of glutamate and GABA and the activities of their respective neurons are selectively impaired in the parieto‐occipital cortex in the groups having consumed ethanol in a free‐choice design, in which its rewarding properties can better take effect than after forced administration.

As the parieto‐occipital cortex is believed to contain emotional memory structures, it may be hypothesized that the glutamatergic and GABAergic neuronal systems in this area are involved in the development of memory for reward from ethanol. However, they are not specifically associated with ‘behavioural dependence’.

British Journal of Pharmacology (2000) 131, 423–432; doi:10.1038/sj.bjp.0703596
Cannabinoid receptor‐mediated inhibition of acetylcholine release from hippocampal and cortical synaptosomes
Authors: Gifford Andrew N , Bruneus Magalie , Gatley S John , Volkow Nora D
Published: January 2005

In previous studies cannabinoid agonists have been found to inhibit and cannabinoid antagonists to enhance electrically‐evoked [3H]‐acetycholine (ACh) release in hippocampal slices. The present study was undertaken to determine if similar cannabinoid effects could be observed in synaptosomes.

[3H]‐ACh release was evoked by two methods, both sensitive to presynaptic receptor effects. The first involved the addition of 1.3 mM calcium following perfusion with calcium‐free Krebs and the second the addition of 11 mM potassium following perfusion with normal Krebs.

In hippocampal synaptosomes the 1.3 mM calcium‐evoked release and the high potassium‐evoked [3H]‐ACh release were inhibited by the cannabinoid agonist, WIN 55212‐2, by 59 and 39%, respectively, and with an EC50 of approximately 1 nM. WIN 55212‐2 produced a similar, although less potent, inhibition of [3H]‐ACh release in cortical synaptosomes. No inhibitory effect of WIN 55212‐2 on [3H]‐ACh release in striatal synaptosomes was observed, supporting previous data collected in this area with brain slices.

The cannabinoid antagonist, SR 141716A, produced a robust enhancement of 1.3 mM calcium‐evoked [3H]‐ACh release in hippocampal synaptosomes (EC50<0.3 nM) but had no effect on potassium‐evoked release or on [3H]‐ACh release in the cortex or striatum.

In conclusion our data demonstrates the inhibitory effects of WIN 55212‐2 observed on ACh release in brain slices can be observed in hippocampal and cortex synaptosomes, suggesting a direct action of these compounds on the synaptic terminals. The SR 141716A‐induced enhancement of ACh release can similarly be observed in hippocampal synaptosomes and is probably due to an inverse agonist action at constitutively active receptors.

British Journal of Pharmacology (2000) 131, 645–650; doi:10.1038/sj.bjp.0703599
Further evidence for the role of the α2δ subunit of voltage dependent calcium channels in models of neuropathic pain
Authors: Field Mark J , Hughes John , Singh Lakhbir
Published: January 2005

Current analgesic therapy is dominated by NSAIDs and opiates, however these agents have limited efficacy in the treatment of neuropathic pain. The novel anticonvulsant agent gabapentin (Neurontin) has been shown to be an effective treatment for neuropathic pain in the clinic. Recent studies have demonstrated that gabapentin selectively interacts with the α2δ subunit of voltage dependent calcium channels (VDCCs) which may be important in its mechanism of action.

Previous studies have identified a gabapentin analogue, 3‐methyl gabapentin, that stereoselectively interacts with the α2δ subunit of VDCCs. Thus, whilst (1S,3R) 3‐methyl gabapentin binds to the α2δ protein with high affinity (IC50=42 nM), the corresponding (1R,3R) isomer is 300 times weaker (Bryans et al., 1998: J. Med. Chem., 41, 1838–1845). The present study examines the activity of diastereoisomers of 3‐methyl gabapentin in two rat models of neuropathic pain to assess the importance of an interaction with the α2δ subunit of VDCCs.

(1S,3R) 3‐methyl‐gabapentin dose‐dependently (10–100 mg kg−1, p.o.) blocked the maintenance of static allodynia in the rat streptozocin and Chung models of neuropathic pain with MEDs of 30 mg kg−1. This isomer also dose‐dependently blocked the maintenance of dynamic allodynia in both models with respective MEDs of 30 and 100 mg kg−1. In contrast, (1R,3R) 3‐methyl gabapentin (100 mg kg−1, p.o.) failed to block either static or dynamic allodynia in the streptozocin model.

It is concluded that these data further support the hypothesis that the α2δ subunit of VDCCs plays an important role in the maintenance of mechanical hypersensitivity in models of neuropathic pain.

British Journal of Pharmacology (2000) 131, 282–286; doi:10.1038/sj.bjp.0703604
Inhibition of small G proteins of the Rho family by statins or Clostridium difficile toxin B enhances cytokine‐mediated induction of NO synthase II
Authors: Hausding Michael , Witteck Andrea , Rodriguez‐Pascual Fernando , Von Eichel‐Streiber Christian , Förstermann Ulrich , Kleinert Hartmut
Published: January 2005

In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG‐CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho‐proteins directly.

Human A549/8‐ and DLD‐1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1–100 μM) or TcdB (0.01–10 ng ml−1). Then NOS II expression was induced by cytokines. NOS II mRNA was measured after 4–8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h.

Statins and TcdB markedly increased cytokine‐induced NOS II mRNA expression and NO production. Statin‐mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate. It was only slightly reduced by farnesylpyrophosphate. Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction.

In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine‐induced NOS II promoter activity. In contrast, statins had a considerable superinducing effect in DLD‐1 cells stably transfected with pNOS2(16)Luc.

In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine‐induced NOS II expression via inhibition of small G proteins of the Rho family. This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability.

British Journal of Pharmacology (2000) 131, 553–561; doi:10.1038/sj.bjp.0703607
Developmental regulation of hippocampal excitatory synaptic transmission by metabotropic glutamate receptors
Authors: Ross F M , Cassidy J , Wilson M , Davies S N
Published: January 2005

The aims of this study were, to use agonists selective for the 3 mGlu receptor groups to identify developmental changes in their effects, and to assess the usefulness of proposed selective antagonists as pharmacological tools.

Hippocampal slices (400 μm) were prepared from neonate (9–14 days) and young adult (5–7 weeks) Sprague‐Dawley rats. Field excitatory postsynaptic potentials (fEPSP) were recorded from CA1.

DHPG (100 μM), a group I agonist, produced a slowly developing enhancement of fEPSP slope in slices from adults. In slices from neonates, DHPG (75 μM) depressed fEPSP slope.

DCG‐IV (500 nM), a group II agonist, did not affect the fEPSP recorded from slices from adults whereas perfusion in neonate slices produced a sustained depression.

The group III agonist L‐AP4 (50 μM) was ineffective in adult slices but depressed fEPSP slope in slices prepared from neonates.

DHPG‐induced depression of fEPSP slope was inhibited by 4‐CPG (400 μM), a group I antagonist, but was unaffected by MCCG (500 μM) and MAP4 (500 μM), group II and III receptor antagonists respectively. MCCG but not MAP4 antagonized the effects of DCG‐IV with 4‐CPG producing variable effects. The effect of L‐AP4 was unaffected by MCCG, blocked by MAP4, and enhanced by 4‐CPG.

The results show that the effects of the agonists for all groups of mGlu receptors are developmentally regulated. Furthermore, MCCG and MAP4 behave as effective and selective antagonists for group II and group III mGlu receptors respectively, whereas the usefulness of 4‐CPG as a group I antagonist may be limited.

British Journal of Pharmacology (2000) 131, 453–464; doi:10.1038/sj.bjp.0703610
Characterization of the binding of [125I]‐human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor
Authors: Langmead Christopher J , Szekeres Philip G , Chambers Jonathan K , Ratcliffe Steven J , Jones Declan N C , Hirst Warren D , Price Gary W , Herdon Hugh J
Published: January 2005

GPR10 is a novel G‐protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor‐1 (UHR‐1). Human prolactin‐releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [125I]‐PrRP‐20 to HEK293 cells stably expressing GPR10 receptors.

Specific binding of [125I]‐PrRP‐20 was saturable, and analysis suggested evidence of both high and low affinity sites, with KD values of 0.026±0.006 and 0.57±0.14 nM respectively, and Bmax values of 3010±400 and 8570±2240 fmol mg protein−1 respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a KD of 0.012 nM.

Competition studies revealed that human and rat PrRP‐20 and PrRP‐31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF‐amide neuropeptide FF, demonstrated any affinity for GPR10.

Human PrRP‐20 failed to alter basal or forskolin‐stimulated levels of intracellular cyclic AMP in HEK293‐GPR10 cells, suggesting that GPR10 does not couple via either Gs or Gi.

Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP‐20 and PrRP‐31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca2+ stores.

These studies indicate that [125I]‐PrRP‐20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.

British Journal of Pharmacology (2000) 131, 683–688; doi:10.1038/sj.bjp.0703617
Delayed myocardial protection induced by endotoxin does not involve kinin B1‐receptors
Authors: Mazenot C , Gobeil F , Ribuot C , Regoli D , Godin‐Ribuot D
Published: January 2005

Endotoxin is known to confer a delayed protection against myocardial infarction. Lipopolysaccharide (LPS) treatment also induces the de novo synthesis of kinin B1‐receptors that are not present in normal conditions. The aim of this study was to evaluate whether LPS‐induced B1‐receptors are implicated in the reduction of infarct size brought about by LPS.

Rabbits were submitted to a 30‐min coronary artery occlusion and 3‐h reperfusion sequence. Six groups were studied: pretreated or not (control animals) with LPS (5 μg kg−1 i.v.) 24 h earlier and treated 15 min before and throughout ischaemia–reperfusion with either the B1‐antagonist R‐715 (1 mg kg−1 h−1), the B1‐agonist Sar‐[D‐Phe8]‐des‐Arg9‐bradykinin (15 μg kg−1 h−1) or vehicle (saline). Infarct size and area at risk were assessed by differential staining and planimetric analysis.

The presence of B1‐receptors in LPS‐pretreated animals was confirmed by a decrease in mean arterial pressure in response to B1 stimulation. LPS‐pretreatment significantly reduced infarct size (6.4±1.7%, of area at risk vs 24.1±2.5% in control animals, P<0.05). This protection was not modified by B1‐receptor antagonism (7.4±2.2%, NS) or stimulation (5.2±1.2%, NS). Neither antagonist nor agonist modified infarct size in control animals.

In conclusion, these data suggest that LPS‐induced myocardial protection in the rabbit is not related to concomitant de novo B1‐receptor induction.

British Journal of Pharmacology (2000) 131, 740–744; doi:10.1038/sj.bjp.0703619
Bicuculline, pentobarbital and diazepam modulate spontaneous GABAA channels in rat hippocampal neurons
Authors: Birnir Bryndis , Eghbali Mansoureh , Everitt Andrea B , Gage Peter W
Published: January 2005

Spontaneously opening, chloride‐selective channels that showed outward rectification were recorded in ripped‐off patches from rat cultured hippocampal neurons and in cell‐attached patches from rat hippocampal CA1 pyramidal neurons in slices.

In both preparations, channels had multiple conductance states and the most common single‐channel conductance varied. In the outside‐out patches it ranged from 12 to 70 pS (Vp=40 mV) whereas in the cell‐attached patches it ranged from 56 to 85 pS (−Vp=80 mV).

Application of GABA to a patch showing spontaneous channel activity evoked a rapid, synchronous activation of channels. During prolonged exposure to either 5 or 100 μM GABA, the open probability of channels decreased. Application of GABA appeared to have no immediate effect on single‐channel conductance.

Exposure of the patches to 100 μM bicuculline caused a gradual decrease on the single‐channel conductance of the spontaneous channels. The time for complete inhibition to take place was slower in the outside‐out than in the cell‐attached patches.

Application of 100 μM pentobarbital or 1 μM diazepam caused 2–4 fold increase in the maximum channel conductance of low conductance (<40 pS) spontaneously active channels.

The observation of spontaneously opening GABAA channels in cell‐attached patches on neurons in slices suggests that they may have a role in neurons in vivo and could be an important site of action for some drugs such as benzodiazepines, barbiturates and general anaesthetics.

British Journal of Pharmacology (2000) 131, 695–704; doi:10.1038/sj.bjp.0703621
Negative feedback regulation of reactive oxygen species on AT1 receptor gene expression
Authors: Nickenig Georg , Strehlow Kerstin , Bäumer Anselm T , Baudler Stefanie , Waßmann Sven , Sauer Heinrich , Böhm Michael
Published: January 2005

Free radicals as well as the AT1 receptor are involved in the pathogenesis of cardiovascular disease. Both the intracellular mechanisms of AT1 receptor regulation and the effect of free radicals on AT1 receptor expression are currently unknown. This study investigates the role of free radicals in the modulation of AT1 receptor expression and in the angiotensin II‐induced AT1 receptor regulation.

AT1 receptor mRNA was assessed by Northern blotting and AT1 receptor density by radioligand binding assays, respectively, in vascular smooth muscle cells (VSMC). Free radical release was measured by confocal laser scanning microscopy. AT1 receptor mRNA transcription rate was determined by nuclear run‐on assays and AT1 receptor mRNA half‐life was measured under transcriptional blockade.

Angiotensin II caused a time‐dependent decrease of AT1 receptor mRNA expression in rat VSMC in culture (30±6% at 4 h with 100 nM angiotensin II). This was followed by a consistent decrease in AT1 receptor density. Angiotensin II caused release of reactive oxygen species in VSMC which was abolished by preincubation with 100 μM diphenylene iodonium (DPI). DPI inhibited partially the down‐regulating effect of angiotensin II on the AT1 receptor. Incubation of VSMC with either hydrogen peroxide or xanthine/xanthine oxidase caused a dose‐dependent decrease in AT1 receptor mRNA expression which was not mediated by a decreased rate of transcription but rather through destabilization of AT1 receptor mRNA. Experiments which included preincubation of VSMC with various intracellular inhibitors suggested that free radicals caused AT1 receptor downregulation through activation of p38‐MAP kinase and intracellular release of calcium. However, angiotensin II‐induced AT1 receptor expression was not inhibited by blockade of p38‐MAP kinase activation or intracellular calcium release.

Free radicals may at least in part mediate angiotensin II‐induced AT1 receptor regulation through direct post‐transcriptional effects on AT1 receptor mRNA expression which involves intracellular release of calcium and activation of p38‐MAP kinase. These findings may help to clarify the intracellular mechanisms involved in AT1 receptor regulation and reveal a novel biological feature for reactive oxygen species.

British Journal of Pharmacology (2000) 131, 795–803; doi:10.1038/sj.bjp.0703623
Evidence for VIP1/PACAP receptors in the afferent pathway mediating surgery‐induced fundic relaxation in the rat
Authors: Boeckxstaens G E , Hollmann M , Heisterkamp S H , Robberecht P , De Jonge W J , Van Den Wijngaard R M J G J , Tytgat G N J , Blommaart P J E
Published: January 2005

We previously reported activation of an inhibitory adrenergic and a non‐adrenergic non‐cholinergic (NANC) pathway during abdominal surgery relaxing the rat gastric fundus. In the present study, we investigated the possible role of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in the NANC part of the surgery‐induced fundic relaxation.

The effect of the NO biosynthesis inhibitor NG‐nitro‐L‐arginine (L‐NOARG), the non‐selective VIP receptor antagonist [D‐p‐Cl‐Phe6,Leu17]‐VIP and the selective VIP1 receptor antagonist [Acetyl‐His1,D‐Phe2,Lys15,Arg16,Leu17]‐VIP was investigated on the non‐adrenergic fundic relaxation induced by manipulation of the small intestine followed by resection of the caecum.

Guanethidine partly reduced the manipulation‐induced fundic relaxation. Addition of L‐NOARG reduced this non‐adrenergic component, whereas the non‐selective VIP receptor antagonist had no significant effect. Combination of L‐NOARG and the non‐selective VIP antagonist however further reduced the relaxation to manipulation.

The selective VIP1 receptor antagonist reduced the mean and maximal relaxation induced by abdominal surgery in the presence of guanethidine. When combined with L‐NOARG, the relaxation of the gastric fundus was almost completely abolished. The VIP1 receptor antagonist alone had no significant effect on the mean and maximal relaxation, but enhanced recovery of fundic tone.

In conclusion, as VIP1 receptors are not present in the rat gastric fundus, these results suggest that the NANC inhibitory pathway activated during abdominal surgery involves VIP1 receptors, most likely in the afferent limb. The inhibitory neurotransmitters released at the level of the gastric fundus smooth muscle are NO and a substance different from VIP.

British Journal of Pharmacology (2000) 131, 705–710; doi:10.1038/sj.bjp.0703625
Characterization of prostanoid receptors mediating contraction of the gastric fundus and ileum: studies using mice deficient in prostanoid receptors
Authors: Okada Yuji , Hara Akiyoshi , Ma Hong , Xiao Chun‐Yang , Takahata Osamu , Kohgo Yutaka , Narumiya Shuh , Ushikubi Fumitaka
Published: January 2005

Receptors mediating prostanoid‐induced contractions of longitudinal sections of gastric fundus and ileum were characterized by using tissues obtained from mice deficient in each type and subtype of prostanoid receptors.

The fundus and ileum from mice deficient in either EP3 (EP3−/− mice), EP1 (EP1−/− mice) and FP (FP−/− mice) all showed decreased contraction to PGE2 compared to the tissues from wild‐type mice, whereas contraction of the fundus slightly increased in EP4−/− mice.

17‐phenyl‐PGE2 also showed decreased contraction of the fundus from EP3−/−, EP1−/− and FP−/− mice. Sulprostone showed decreased contraction of the fundus from EP3−/− and FP−/− mice, and decreased contraction of the ileum to this compound was seen in tissues from EP3−/−, EP1−/− and FP−/− mice. In DP−/− mice, sulprostone showed increased contraction.

DI‐004 and AE‐248 caused the small but concentration‐dependent contraction of both tissues, and these contractions were abolished in tissues obtained from EP1−/− and EP3−/− mice, respectively, but not affected in other mice.

Contractions of both fundus and ileum to PGF2α was absent at lower concentrations (10−9 to 10−7 M), and suppressed at higher concentrations (10−6 to 10−5 M) of the agonist in the FP−/− mice. Suppression of the contractions at the higher PGF2α concentrations was also seen in the fundus from EP3−/−, EP1−/− and TP−/− mice and in the ileum from EP3−/− and TP−/− mice.

Contraction of the fundus to PGD2 was significantly enhanced in DP−/− mice, and contractions of the fundus and ileum to this PG decreased in FP−/− and EP3−/− mice.

Contractions of both tissues to I‐BOP was absent at 10−9 to 10−7 M and much suppressed at higher concentrations in TP−/− mice. Slight suppression to this agonist was also observed in the tissues from EP3−/− mice.

PGI2 induced small relaxation of both tissues from wild‐type mice. These relaxation reactions were much potentiated in EP3−/− mice. On the other hand, significant contraction to PGI2 was observed in both tissues obtained from IP−/− mice.

These results show that contractions of the fundus and ileum induced by each prostanoid agonist are mediated by actions of this agonist on multiple types of prostanoid receptors and in some cases modified by its action on relaxant receptors.

British Journal of Pharmacology (2000) 131, 745–755; doi:10.1038/sj.bjp.0703627
Pharmacological evaluation of the role of cytochrome P450 in intracellular calcium signalling in rat pancreatic acinar cells
Authors: Bruce Jason I E , Elliott Austin C
Published: January 2005

We have investigated whether the cytochrome P450 system is involved in Ca2+ signalling in rat pancreatic acinar cells. Intracellular free [Ca2+] ([Ca2+]i) was measured in collagenase‐isolated cells using fura‐2 microspectrofluorimetry and imaging.

The imidazole P450 inhibitor ketoconazole (5–50 μM) inhibited [Ca2+]i oscillations induced by cholecystokinin octapeptide (CCK). However, ketoconazole also raised baseline [Ca2+]i when applied in the absence of CCK. These effects were mimicked by 5–50 μM SKF96365, an imidazole widely used as an inhibitor of Ca2+ entry.

The non‐imidazole P450 inhibitor proadifen (SKF525A) inhibited CCK‐induced [Ca2+]i oscillations at a concentration of 10–50 μM. Proadifen alone caused intracellular Ca2+ release at 25 or 50 μM, but not at 10 μM.

Octadecynoic acid and 1‐aminobenzotriazole, structurally‐unrelated non‐imidazole P450 inhibitors, did not alter baseline [Ca2+]i or CCK‐evoked oscillations.

We compared cumulative CCK dose‐response relationship in control cells and in cells where P450 had been induced by prior injection of animals with β‐naphthoflavone. Only minor differences were apparent, with induced cells showing some decrease in responsiveness at moderate and higher concentration of CCK (30 pM–3 nM).

Direct assessment of depletion‐activated Ca2+ entry showed no clear differences between control and induced cells.

In conclusion, we could find no compelling evidence for a role of P450 in controlling Ca2+ signalling generally, or Ca2+ entry in particular, in pancreatic acinar cells. Induction of P450 is therefore probably toxic to acinar cells via a Ca2+‐independent mechanism.

British Journal of Pharmacology (2000) 131, 761–771; doi:10.1038/sj.bjp.0703631
Sub‐family selective actions in the ability of Erk2 MAP kinase to phosphorylate and regulate the activity of PDE4 cyclic AMP‐specific phosphodiesterases
Authors: Baillie George S , MacKenzie Simon J , McPhee Ian , Houslay Miles D
Published: January 2005

Expressed in intact cells and in vitro, PDE4B and PDE4C isoenzymes of cyclic nucleotide phosphodiesterase (PDE), in common with PDE4D isoenzymes, are shown to provide substrates for C‐terminal catalytic unit phosphorylation by the extracellular signal‐regulated kinase Erk2 (p42MAPK).

In contrast, PDE4A isoenzymes do not provide substrates for C‐terminal catalytic unit phosphorylation by Erk2.

Mutant PDE4 enzymes were generated to show that Erk2 phosphorylation occurs at a single, cognate serine residue located within the C‐terminal portion of the PDE4 catalytic unit.

PDE4 long‐form isoenzymes were markedly inhibited by Erk2 phosphorylation.

The short‐form PDE4B2 isoenzyme was activated by Erk2 phosphorylation.

These functional changes in PDE activity were mimicked by mutation of the target serine for Erk2 phosphorylation to the negatively charged amino acid, aspartic acid.

Epidermal growth factor (EGF) challenge caused diametrically opposed changes in cyclic AMP levels in COS1 cells transfected to express the long PDE4B1 isoenzyme compared to cells expressing the short PDE4B2 isoenzyme.

We suggest that PDE4 enzymes may provide a pivotal point for integrating cyclic AMP and Erk signal transduction in cells with 4 genes encoding enzymes that are either insensitive to Erk2 action or may either be activated or inhibited. This indicates that PDE4 isoenzymes have distinct functional roles, giving credence to the notion that distinct therapeutic benefits may accrue using either PDE4 subfamily or isoenzyme‐selective inhibitors.

British Journal of Pharmacology (2000) 131, 811–819; doi:10.1038/sj.bjp.0703636
A comparison of agonist‐specific coupling of cloned human α2‐adrenoceptor subtypes
Authors: Rudling Jane E , Richardson Jo , Evans Peter D
Published: January 2005

The agonist‐specific coupling properties of the three cloned human α2‐adrenoceptor subtypes have been compared, when expressed at similar levels in Chinese hamster ovary (CHO) cell lines, using noradrenaline and (±)‐meta‐octopamine as agonists.

Noradrenaline can couple the receptor to both the inhibition and stimulation of forskolin‐stimulated cyclic AMP production in all three receptor subtypes, with the relative strength of the coupling to the pathways varying for each of the receptor subtypes.

meta‐Octopamine selectively couples the α2A‐adrenoceptor only to the inhibition of forskolin‐stimulated cyclic AMP production. However, meta‐octopamine couples the α2B‐ and α2C‐adrenoceptors to both the inhibition and stimulation of forskolin‐stimulated cyclic AMP production.

The relative potency of meta‐octopamine to noradrenaline varies between the different α2‐adrenoceptor subtypes. The effects of meta‐octopamine are around two orders of magnitude less potent than those of noradrenaline on both the α2A‐ and α2B‐adrenoceptor subtypes. In contrast, in the case of the α2C‐adrenoceptor, meta‐octopamine is only one order of magnitude less potent than noradrenaline in the stimulation of forskolin‐stimulated cyclic AMP production and, in addition, is equipotent with noradrenaline in the inhibition of forskolin‐stimulated cyclic AMP production and has an increased maximal response. This raises the possibility that meta‐octopamine may have physiologically important actions via α2C‐adrenoceptors in vivo.

The results show that the modulation of cyclic AMP production occurs in both a subtype‐ and agonist‐specific manner for α2A‐adrenoceptors and in a subtype specific manner for α2B‐ and α2C‐adrenoceptors.

British Journal of Pharmacology (2000) 131, 933–941; doi:10.1038/sj.bjp.0703644
Adenosine A1 receptor stimulation inhibits α1‐adrenergic activation of the cardiac sarcolemmal Na+/H+ exchanger
Authors: Avkiran Metin , Yokoyama Hiroyuki
Published: January 2005

Sarcolemmal Na+/H+ exchanger (NHE) activity is increased by stimulation of Gq protein‐coupled receptors (GqPCRs), but the roles of other GPCRs are largely unknown. We determined the effects of N‐[(1S,trans)‐2‐hydroxycyclopentyl]adenosine (GR79236), a selective agonist of the GiPCR adenosine A1 receptor, on sarcolemmal NHE activity in adult rat ventricular myocytes (n=8–10 per group). NHE activity was indexed by the H+ efflux rate after intracellular acidification, measured by microepifluorescence. GR79236 alone (0.01–10 μM) had no effect on NHE activity. However, co‐administration of GR79236 inhibited, in a concentration‐dependent manner, the stimulation of NHE activity by the α1‐adrenoceptor agonist phenylephrine (10 μM). The inhibitory effect of GR79236 (10 μM) was abolished by (1) the selective A1 antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (0.1 μM), confirming an A1 receptor‐mediated action, and (2) pre‐treatment with pertussis toxin (5 μg ml−1 for 60 min), indicating a Gi protein‐mediated mechanism. Our data suggest the existence of inhibitory crosstalk between the GiPCR adenosine A1 receptor and the GqPCR α1‐adrenoceptor in the regulation of sarcolemmal NHE activity.

British Journal of Pharmacology (2000) 131, 659–662; doi:10.1038/sj.bjp.0703647
Non‐competitive pharmacological antagonism at the rabbit B1 receptor
Authors: Larrivée Jean‐François , Gera Lajos , Houle Steeve , Bouthillier Johanne , Bachvarov Dimcho R , Stewart John M , Marceau François
Published: January 2005

The B1 receptor for kinins, stimulated by kinin metabolites without the C‐terminal Arg residue (e.g., des‐Arg9‐bradykinin (BK) and Lys‐des‐Arg9‐BK), is an increasingly recognized molecular target for the development of analgesic and anti‐inflammatory drugs. Recently developed antagonists of this receptor were compared to a conventional antagonist, Ac‐Lys‐[Leu8]‐des‐Arg9‐BK, in pharmacological assays based on the rabbit B1 receptor.

B‐9858 (Lys‐Lys‐[Hyp3, Igl5, D‐Igl7, Oic8]des‐Arg9‐BK) and three other analogues possessing the α‐2‐indanylglycine5 (Igl5) residue (order of potency B‐9858 ∼ B‐10146>B‐10148>B‐10050) were partially insurmountable antagonists of des‐Arg9‐BK in the contractility assay based on rabbit aortic rings. B‐9858‐induced depression of the maximal effect was more pronounced in tissues treated with the protein synthesis inhibitor cycloheximide to block the spontaneous increase of response attributed to the post‐isolation formation of B1 receptors, and only partly reversible on washing.

By comparison, Ac‐Lys‐[Leu8]des‐Arg9‐BK was a surmountable antagonist (pA2 7.5), even in cycloheximide‐treated tissues. B‐9958 (Lys‐[Hyp3, CpG5, D‐Tic7, CpG8]des‐Arg9‐BK) was also surmountable (pA2 8.5).

The binding of [3H]‐Lys‐des‐Arg9‐BK to recombinant rabbit B1 receptors expressed in COS‐1 cells was influenced by two of the antagonists: while Ac‐Lys‐[Leu8]des‐Arg9‐BK competed for the radioligand binding without affecting the Bmax, B‐9858 decreased the Bmax in a time‐dependent and washout‐resistant manner.

B‐9858 and analogues possessing Igl5 are the first reported non‐competitive, non‐equilibrium antagonists of the kinin B1 receptor.

British Journal of Pharmacology (2000) 131, 885–892; doi:10.1038/sj.bjp.0703656