bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms underlying ACh induced modulation of neurogenic and applied ATP constrictions in the submucosal arterioles of the guinea‐pig small intestine
Authors: Kotecha N
Published: January 2005

Role of the vascular endothelium in acetylcholine (ACh) induced modulation of neurogenic and applied ATP (adenosine 5′‐triphosphate) constrictions of intestinal submucosal arterioles was investigated.

Arteriole constrictions, induced either by exogenous ATP or evoked by perivascular nerve stimulation, were attenuated in the presence of ACh. 100 nM ACh almost completely abolished neurogenic constrictions whereas up to 10 μM ACh reduced constrictions to exogenous ATP by only about 60%.

Treatment of the arterioles with 100 μM Nω‐nitro‐L‐arginine (NOLA) and 5 μM indomethacin, to block respectively nitric oxide (NO) and prostanoid release from the endothelium, had no effect on the ACh induced inhibition of neurogenic constrictions but significantly attenuated the inhibitory effects of ACh on constrictions to exogenous ATP.

Disruption of the vascular endothelium had no effect on the ACh induced inhibition of neurogenic constrictions but attenuated the inhibitory effects of ACh on applied ATP constrictions to the same extent as after treatment with NOLA and indomethacin. In comparison, endothelial disruption completely abolished the inhibitory effect of substance P (SP) on exogenously applied ATP constrictions.

50 nM ACh significantly attenuated the amplitude of neurally evoked excitatory junction potentials (ejps) recorded from the vascular smooth muscle without altering the time constant of decay (τdecay) of the ejps.

It is concluded that ACh inhibits neurogenic constrictions by prejunctional modulation of transmitter release from the perivascular sympathetic nerves with no major role for endothelial paracrine factors.

Endothelial NO and/or prostanoids mediate some of the ACh induced inhibition of constrictions to exogenous ATP whereas the endothelium independent inhibitory effects of ACh are attributed to a direct action of ACh on the vascular smooth muscle. However, an indirect effect resulting from activation of vasodilator nerves cannot be ruled out.

British Journal of Pharmacology (1999) 126, 1625–1633; doi:10.1038/sj.bjp.0702461
Adenosine mediates relaxation of human small resistance‐like coronary arteries via A2B receptors
Authors: Kemp B K , Cocks T M
Published: January 2005

The receptor subtype and mechanisms underlying relaxation to adenosine were examined in human isolated small coronary arteries contracted with the thromboxane A2 mimetic, 1,5,5‐hydroxy‐11α, 9α‐(epoxymethano)prosta‐5Z, 13E‐dienoic acid (U46619) to approximately 50% of their maximum contraction to K+ (125 mM) depolarization (Fmax). Relaxations were normalized as percentages of the 50% Fmax contraction.

Adenosine caused concentration‐dependent relaxations (pEC50, 5.95±0.20; maximum relaxation (Rmax), 96.7±1.4%) that were unaffected by either combined treatment with the nitric oxide inhibitors, NG‐nitro‐L‐arginine (L‐NOARG; 100 μM) and oxyhaemoglobin (HbO; 20 μM) or the ATP‐dependent K+ channel (KATP) inhibitor, glibenclamide (10 μM). The pEC50 but not Rmax to adenosine was significantly reduced by high extracellular K+ (30 mM). Relaxations to the adenylate cyclase activator, forskolin, however, were unaffected by high K+ (30 mM).

Adenosine and a range of adenosine analogues, adenosine, 2‐chloroadenosine (2‐CADO), 5′‐N‐ethyl‐carboxamidoadenosine (NECA), R(−)‐N6‐(2‐phenylisopropyl)‐adenosine (R‐PIA), S(+)‐N6‐(2‐phenylisopropyl)‐adenosine (S‐PIA), N6‐cyclopentyladenosine (CPA), 1‐deoxy‐1‐[6‐[[(3‐iodophenyl)methyl]amino]‐9H‐purin‐9‐yl]‐N‐methyl‐β‐D‐ribofuranuronamide (IB‐MECA), 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamido adenosine hydrochloride (CGS 21680), relaxed arteries with a rank order of potency of NECA=2‐CADO>adenosine=IB‐MECA=R‐PIA= CPA>S‐PIA)>CGS 21680.

Sensitivity but not Rmax to adenosine was significantly reduced approximately 80 and 20 fold by the non‐selective adenosine receptor antagonist, 8‐(p‐sulphophenyl)theophylline (8‐SPT) and the A2 receptor antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX). By contrast, the A1‐selective antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) had no effect on pEC50 or Rmax to adenosine.

These results suggest that A2B receptors mediate relaxation to adenosine in human small coronary arteries which is independent of NO but dependent in part on a K+‐sensitive mechanism.

British Journal of Pharmacology (1999) 126, 1796–1800; doi:10.1038/sj.bjp.0702462
Complex relationship between Ins(1,4,5)P3 accumulation and Ca2+‐signalling in a human neuroblastoma revealed by cellular differentiation
Authors: Martin Amanda K , Nahorski Stefan R , Willars Gary B
Published: January 2005

Differentiation of SH‐SY5Y neuroblastoma cells induces morphological and biochemical changes consistent with a more neuronal phenotype. These cells may therefore provide a model for studying phenomena such as signal transduction in a neuronal context whilst retaining the advantages of a homogenous cell population expressing a well characterized array of G‐protein coupled receptors.

This study examined the effects of differentiating SH‐SY5Y cells on muscarinic‐ and bradykinin‐receptor‐mediated phosphoinositide and Ca2+ signalling. Retinoic acid (10 μM, 6 days) along with a lowered serum concentration produced phenotypic changes consistent with differentiation including reduced proliferation and increased neurite outgrowth.

Differentiation increased the magnitude and potency of rapid Ins(1,4,5)P3 responses to a full muscarinic receptor agonist. Bradykinin receptor‐mediated Ins(1,4,5)P3 signalling was also potentiated following differentiation. Determination of agonist‐evoked accumulation of [3H]‐inositol phosphates under lithium‐block demonstrated these changes reflected enhanced phospholipase C activity which is consistent with observed increases in the expression of muscarinic and bradykinin receptors.

Despite the marked alterations in Ins(1,4,5)P3 signalling following differentiation, elevations of intracellular [Ca2+] were totally unaltered. Thus, in SH‐SY5Y cells, the relationship between the elevations of Ins(1,4,5)P3 and intracellular [Ca2+] is agonist dependent and affected by the state of differentiation. This demonstrates that mechanisms other than the measured increase in Ins(1,4,5)P3 regulate the elevation of intracellular [Ca2+].

British Journal of Pharmacology (1999) 126, 1559–1566; doi:10.1038/sj.bjp.0702464
Effects of anti‐epileptic drugs on glutamine synthetase activity in mouse brain
Authors: Fraser Caroline M , Sills Graeme J , Forrest Gerard , Thompson George G , Brodie Martin J
Published: January 2005

Glutamine synthetase (GS) is a key enzyme in the regulation of glutamate neurotransmission in the central nervous system. It is responsible for the conversion of glutamate to glutamine, and for the detoxification of ammonia.

We have investigated the effects of single and repeated intraperitoneal administration of a range of established and new anti‐epileptic drugs on GS activity in mouse brain.

Four hours after the final dose, animals were sacrificed and the brains removed for analysis of GS activity.

Both single and repeated doses of phenytoin and carbamazepine were found to reduce enzyme activity (P<0.05).

Single doses of phenobarbitone, felbamate and topiramate were without effect, however repeated administration of these drugs dose‐dependently reduced GS activity (P<0.05).

Single and repeated doses of sodium valproate, vigabatrin, lamotrigine, gabapentin, tiagabine, levetiracetam and desglycinyl‐remacemide were found to have no effect on GS activity.

The reduction in enzyme activity demonstrated is unlikely to be related to the anti‐epileptic actions of these drugs, but may contribute to their toxicity.

British Journal of Pharmacology (1999) 126, 1634–1638; doi:10.1038/sj.bjp.0702472
Modification of membrane currents in mouse neuroblastoma cells following infection with rabies virus
Authors: Iwata M , Komori S , Unno T , Minamoto N , Ohashi H
Published: January 2005

The effect on membrane currents of infection of mouse neuroblastoma NA cells with rabies virus was studied by using the whole‐cell patch clamp technique.

Three types of membrane currents, namely voltage‐dependent Na+ current (INa), delayed rectifier K+ current (IK‐DR) and inward rectifier K+ current (IK‐IR) were elicited in uninfected cells.

In cells 3 days after infection with the virus, no detectable change was observed in morphology and membrane capacitance, but INa and IK‐IR were significantly decreased in amplitude without any appreciable difference in the time course of current activation and inactivation. The voltage‐dependence of INa activation was significantly shifted in the positive direction along the voltage axis with a decreased slope. IK‐DR remained almost unaltered after the viral infection.

The resting membrane potential, measured with a physiological K+ gradient across the cell membrane, was decreased (depolarized) after the viral infection. The depolarization was associated with the decreased amplitude of IK‐IR.

These results suggest that infection of mouse neuroblastoma NA cells with rabies virus causes reduction of functional expression of ion channels responsible for INa and IK‐IR, and provide evidence for possible involvement of the change in membrane properties in the pathogenesis of rabies disease.

British Journal of Pharmacology (1999) 126, 1691–1698; doi:10.1038/sj.bjp.0702473
Mechanism of verapamil block of a neuronal delayed rectifier K channel: active form of the blocker and location of its binding domain
Authors: Catacuzzeno L , Trequattrini C , Petris A , Franciolini F
Published: January 2005

The mechanism of verapamil block of the delayed rectifier K currents (IK(DR)) in chick dorsal root ganglion (DRG) neurons was investigated using the whole‐cell patch clamp configuration. In particular we focused on the location of the blocking site, and the active form (neutral or charged) of verapamil using the permanently charged verapamil analogue D890.

Block by D890 displayed similar characteristics to that of verapamil, indicating the same state‐dependent nature of block. In contrast with verapamil, D890 was effective only when applied internally, and its block was voltage dependent (136 mV/e‐fold change of the on rate). Given that verapamil block is insensitive to voltage (Trequattrini et al., 1998), these observations indicate that verapamil reaches its binding site in the uncharged form, and accesses the binding domain from the cytoplasm.

In external K and saturating verapamil we recorded tail currents that did not decay monotonically but showed an initial increase (hook). As these currents can only be observed if verapamil unblock is significantly voltage dependent, it has been suggested (DeCoursey, 1995) that neutral drug is protonated upon binding. We tested this hypothesis by assessing the voltage dependence of the unblock rate from the hooked tail currents for verapamil and D890.

The voltage dependence of the off rate of D890, but not of verapamil, was well described by adopting the classical Woodhull (1973) model for ionic blockage of Na channels. The voltage dependence of verapamil off rate was consistent with a kinetic scheme where the bound drug can be protonated with rapid equilibrium, and both charged and neutral verapamil can unbind from the site, but with distinct kinetics and voltage dependencies.

British Journal of Pharmacology (1999) 126, 1699–1706; doi:10.1038/sj.bjp.0702477
Effects of the endogeneous cannabinoid, anandamide, on neuronal activity in rat hippocampal slices
Authors: Ameri Angela , Wilhelm Alwina , Simmet Thomas
Published: January 2005

The arachidonic acid derivative arachidonylethanolamide (anandamide) is an endogeneous ligand of cannabinoid receptors that induces pharmacological actions similar to those of cannabinoids such as Δ9‐tetrahydrocannabinol (THC). We examined whether anandamide can influence excessive neuronal activity by investigating stimulation‐induced population spikes and epileptiform activity in rat hippocampal slices. For this purpose, the effects of anandamide were compared with those of the synthetic cannabinoid agonist WIN 55,212‐2 and its inactive S(−)‐enantiomer WIN 55,212‐3.

Both anandamide (1 and 10 μM) and WIN 55,212‐2 (0.1 and 1 μM) decreased the amplitude of the postsynaptic population spike and the slope of the field excitatory postsynaptic potential (field e.p.s.p.) without affecting the presynaptic fibre spike of the afferents. At a concentration of 1 μM, WIN 55,212‐2 completely suppressed the postsynaptic spike, whereas the S(−)‐enantiomer WIN 55,212‐3 produced only a slight depression. The CB1 receptor antagonist SR 141716 blocked the inhibition evoked by the cannabinoids. SR 141716 had a slight facilitatory effect on neuronal excitability by itself.

Anandamide shifted the input‐output curve of the postsynaptic spike and the field e.p.s.p. to the right and increased the magnitude of paired‐pulse facilitation indicating a presynaptic mechanism of action.

Anandamide and WIN 55,212‐2, but not WIN 55,212‐3, attenuated both stimulus‐triggered epileptiform activity in CA1 elicited by omission of Mg2+ and spontaneously occurring epileptiform activity in CA3 elicited by omission of Mg2+ and elevation of K+ to 8 mM. The antiepileptiform effect of these cannabinoids was blocked by SR 141716.

In conclusion, cannabinoid receptors of the CB1 type as well as their endogeneous ligand, anandamide, are involved in the control of neuronal excitability, thus reducing excitatory neurotransmission at a presynaptic site, a mechanism which might be involved in the prevention of excessive excitability leading to epileptiform activity.

British Journal of Pharmacology (1999) 126, 1831–1839; doi:10.1038/sj.bjp.0702478
Direct inhibition of the N‐methyl‐D‐aspartate receptor channel by dopamine and (+)‐SKF38393
Authors: Castro Newton G , De Mello Maria Christina F , De Mello Fernando G , Aracava Yasco
Published: January 2005

Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G‐protein‐coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors.

Ionic currents induced by fast application of N‐methyl‐D‐aspartate (NMDA) were recorded under whole‐cell patch‐clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 μM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms.

The D1 receptor agonist (+)‐SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 μM on the NMDA response, while the enantiomer (−)‐SKF38393 was ineffective at 50 μM. However, the D1 antagonist R(+)‐SCH23390 did not prevent the inhibitory effect of (+)‐SKF38393.

The degree of inhibition by dopamine and (+)‐SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage‐dependent block by dopamine was also observed in the presence of MgCl2 1 mM.

Single‐channel recordings showed that the open times of NMDA‐gated channels were shortened by (+)‐SKF38393.

These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1‐type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)‐SKF38393 are NMDA channel ligands.

British Journal of Pharmacology (1999) 126, 1847–1855; doi:10.1038/sj.bjp.0702479
Molecular analysis of the Na+ channel blocking actions of the novel class I anti‐arrhythmic agent RSD 921
Authors: Pugsley Michael K , Goldin Alan L
Published: January 2005

RSD 921 is a novel, structurally unique, class I Na+ channel blocking drug under development as a local anaesthetic agent and possibly for the treatment of cardiac arrhythmias. The effects of RSD 921 on wild‐type heart, skeletal muscle, neuronal and non‐inactivating IFMQ3 mutant neuronal Na+ channels expressed in Xenopus laevis oocytes were examined using a two‐electrode voltage clamp.

RSD 921 produced similarly potent tonic block of all three wild‐type channel isoforms, with EC50 values between 35 and 47 μM, whereas the EC50 for block of the IFMQ3 mutant channel was 110±5.5 μM.

Block of Na+ channels by RSD 921 was concentration and use‐dependent, with marked frequency‐dependent block of heart channels and mild frequency‐dependent block of skeletal muscle, wild‐type neuronal and IFMQ3 mutant channels.

RSD 921 produced a minimal hyperpolarizing shift in the steady‐state voltage‐dependence of inactivation of all three wild‐type channel isoforms.

Open channel block of the IFMQ3 mutant channel was best fit with a first order blocking scheme with kon equal to 0.11±0.012×106 M−1 s−1 and koff equal to 12.5±2.5 s−1, resulting in KD of 117±31 μM. Recovery from open channel block occurred with a time constant of 14±2.7 s−1.

These results suggest that RSD 921 preferentially interacts with the open state of the Na+ channel, and that the drug may produce potent local anaesthetic or anti‐arrhythmic action under conditions of shortened action potentials, such as during anoxia or ischaemia.

British Journal of Pharmacology (1999) 127, 9–18; doi:10.1038/sj.bjp.0702488
Pharmacological analysis of the novel, selective α1‐adrenoceptor antagonist, KMD‐3213, and its suitability as a tritiated radioligand
Authors: Murata Satoshi , Taniguchi Takanobu , Muramatsu Ikunobu
Published: January 2005

Pharmacological profiles of tritiated KMD‐3213, a new antagonist of α1‐adrenoceptor (AR), were examined in recombinant and native α1‐AR, and compared with those of prazosin (PZ) and tamsulosin (YM‐617).

In saturation experiments, [3H]‐KMD (10–2000 pM) showed high affinity for α1a‐AR (pKD=10.5). However, no significant binding to α1b‐AR and insufficient/unsaturated binding to α1d‐AR were observed at concentrations up to 2000 pM. In contrast, [3H]‐PZ and [3H]‐YM bound to all subtypes with high affinity (pKD>9). In competition experiments, KMD‐3213 also had higher affinity for α1a‐AR than for other two subtypes; pKi=10.4, 8.1 and 8.6 for α1a‐, α1b‐ and α1d‐AR, respectively.

[3H]‐KMD also bound to the native α1A‐AR (rat submaxillary gland) with high affinity, but not to α1B‐AR (rat liver). In rat kidney which expresses α1A‐ and α1B‐AR, [3H]‐KMD and [3H]‐PZ bound to a single high‐affinity site (pKD=10.8 and 10.1, respectively) with distinct amount of binding sites (Bmax=159 and 267 fmol mg−1 protein, respectively). [3H]‐PZ binding sites consisted of low‐ and high‐affinity sites for KMD‐3213 (pKi=7.6 and 10.7, respectively), for WB4101 (pKi=8.1 and 10.0) and for YM‐617 (pKi=8.7 and 10.8). The proportion of the high affinity site was approximately 60% in these drugs which was compatible to the ratio between Bmax of [3H]‐KMD and [3H]‐PZ. [3H]‐KMD binding sites consisted of a single site for these drugs with affinities which were similar to those of the high affinity sites in [3H]‐PZ binding.

In functional experiments, KMD‐3213 antagonized the contractile responses to NS‐49 or noradrenaline (NA) with higher affinity in functional α1A‐ (rat caudal artery, pA2=10.0 against NS‐49) and α1L‐AR (dog mesenteric artery, pA2=9.9 against NA) than in α1B‐ (dog carotid artery, pA2=7.7 against NA) and α1D‐AR (rat thoracic aorta, pA2=8.3 against NA).

These results confirm the α1A‐AR selectivity and high affinity of KMD‐3213, and indicate that [3H]‐KMD can label selectively α1A‐AR.

British Journal of Pharmacology (1999) 127, 19–26; doi:10.1038/sj.bjp.0702489
Effects of hydroxocobalamin and carboxy‐PTIO on nitrergic transmission in porcine anococcygeus and retractor penis muscles
Authors: Li C G , Rand M J
Published: January 2005

The effects of carboxy‐PTIO and hydroxocobalamin were studied on nitrergic transmission in anococcygeus and retractor penis muscles taken during post mortem examination from young male pigs.

In both muscles under resting conditions, electrical field stimulation (EFS) caused contractions that were sensitive to tetrodotoxin (1 μM) and were greatly inhibited by prazosin (1 μM) and guanethidine (10–30 μM), but were not significantly affected by atropine (1 μM). In the anococcygeus muscle, but not in the retractor penis muscle, guanethidine produced a prolonged contraction.

After tone was raised by guanethidine in the anococcygeus or by phenylephrine (1 μM) in the presence of guanethidine in the retractor penis, EFS caused tetrodotoxin‐sensitive relaxations. The EFS‐induced relaxations were abolished by the NO synthase inhibitor NG‐L‐nitro‐arginine methyl ester (L‐NAME; 100 μM) and its effect was partly overcome by L‐arginine (1 mM), indicating it was mediated by nitrergic nerves.

Carboxy‐PTIO (0.1–1 mM) had no significant effect in reducing stimulation‐induced nitrergic relaxations in either muscle. However, hydroxocobalamin (0.1–1 mM) caused concentration‐dependent reductions of nitrergic relaxations in both muscles. Relaxations to exogenous nitric oxide (1 μM) in both muscles were abolished by carboxy‐PTIO (0.3 mM) and hydroxocobalamin (0.1 mM).

There were no differences in reactivity to carboxy‐PTIO or hydroxocobalamin between anococcygeus and retractor penis muscles from the same species (pig). The finding also confirms earlier observations that the nitrergic transmitter is generally resistant to the NO‐scavenger carboxy‐PTIO.

British Journal of Pharmacology (1999) 127, 172–176; doi:10.1038/sj.bjp.0702496
Role of blood‐brain barrier P‐glycoprotein in limiting brain accumulation and sedative side‐effects of asimadoline, a peripherally acting analgaesic drug
Authors: Jonker J W , Wagenaar E , Van Deemter L , Gottschlich R , Bender H M , Dasenbrock J , Schinkel A H
Published: January 2005

Studies with knockout mice lacking mdr1a P‐glycoprotein (P‐gp) have previously shown that blood‐brain barrier P‐gp is important in preventing the accumulation of several drugs in the brain.

Asimadoline (EMD 61753) is a peripherally selective κ‐opioid receptor agonist which is under development as a therapeutic analgaesic. From the structural characteristics of this drug and its peripheral selectivity, we hypothesized that it is transported by P‐gp.

Using a pig‐kidney polarized epithelial cell line transfected with mdr cDNAs, we demonstrate that asimadoline is transported by the mouse mdr1a P‐gp and the human MDR1 P‐gp.

Furthermore, we show that in mdr1a/1b double knockout mice, the absence of P‐gp leads to a 9 fold increased accumulation of asimadoline in the brain. In line with this accumulation difference, mdr1a/1b (−/−) mice are at least 8 fold more sensitive to the sedative effect of asimadoline than wild‐type mice.

Interestingly, the oral uptake of asimadoline was not substantially altered in mdr1a/1b (−/−) mice.

Our results demonstrate that for some drugs, P‐gp in the blood‐brain barrier can have a therapeutically beneficial effect by limiting brain penetration, whereas at the same time intestinal P‐gp is not a significant impediment to oral uptake of the drug.

British Journal of Pharmacology (1999) 127, 43–50; doi:10.1038/sj.bjp.0702497
Effects of intrathecal administration of nitric oxide synthase inhibitors on carrageenan‐induced thermal hyperalgesia
Authors: Osborne Michael G , Coderre Terence J
Published: January 2005

We examined the effects of various nitric oxide synthase (NOS) inhibitors on carrageenan‐induced thermal hyperalgesia.

First, we determined the time point at which a subcutaneous plantar injection of carrageenan into the rat hindpaw produced maximum thermal hyperalgesia. Subsequently, we demonstrated that intrathecal administration of the non‐selective NOS inhibitor L‐NG‐nitro‐arginine methyl ester (L‐NAME) produces a dose‐dependent reduction of carrageenan‐induced thermal hyperalgesia.

Four relatively selective NOS inhibitors were then tested for their efficacy at reducing carrageenan‐induced thermal hyperalgesia. Initially, the effects of prolonged treatment with inhibitors of neuronal [7‐nitroindazole (7‐NI) and 3‐bromo‐7‐nitroindazole (3‐Br)] and inducible [aminoguanidine (AG) and 2‐amino‐5,6‐dihydro‐methylthiazine (AMT)] NOS were examined. All agents were injected three times intrathecally during the course of inflammation caused by the plantar injection of carrageenan, and thermal hyperalgesia was measured at 6 h post‐carrageenan using a plantar apparatus.

All inhibitors, except for 7‐NI, were effective at attenuating the carrageenan‐induced thermal hyperalgesia when compared with vehicle treatment.

Finally, the effects of early versus late administration of neuronal and inducible NOS inhibitors on carrageenan‐induced thermal hyperalgesia were examined. We found that neither 3‐Br nor AG significantly affected thermal hyperalgesia when administered during the early phase of carrageenan inflammation, while only AG was able to reduce thermal hyperalgesia when administered during the late phase of the injury.

Our results suggest that inducible NOS contributes to thermal hyperalgesia in only the late stages of the carrageenan‐induced inflammatory response, while neuronal NOS likely plays a role throughout the entire time course of the injury.

British Journal of Pharmacology (1999) 126, 1840–1846; doi:10.1038/sj.bjp.0702508
Activation of nitric oxide synthase by β2‐adrenoceptors in human umbilical vein endothelium in vitro
Authors: Ferro Albert , Queen Lindsay R , Priest Rachel M , Xu Biao , Ritter James M , Poston Lucilla , Ward Jeremy P T
Published: January 2005

Some animal studies suggest that β‐adrenoceptor‐mediated vasorelaxation is in part mediated through nitric oxide (NO) release. Furthermore, in humans, we have recently shown that forearm blood flow is increased by infusion of β2‐adrenergic agonists into the brachial artery, and the nitric oxide synthase (NOS) inhibitor NG‐monomethyl‐L‐arginine (L‐NMMA) inhibits this response.

The purpose of the present study was to determine whether stimulation of human umbilical vein endothelial β‐adrenoceptors causes vasorelaxation and nitric oxide generation, and whether this might be mediated by cyclic adenosine‐3′,5′‐monophosphate (cyclic AMP).

Vasorelaxant responses were determined in umbilical vein rings to the nonselective β‐adrenergic agonist isoprenaline and to the cyclic AMP analogue dibutyryl cyclic AMP, following precontraction with prostaglandin F2α.

NOS activity was measured in cultured human umbilical vein endothelial cells (HUVEC) by the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline, and adenylyl cyclase activity by the conversion of [α‐32P]‐ATP to [32P]‐cyclic AMP.

Isoprenaline relaxed umbilical vein rings, and this vasorelaxation was abolished by β2‐ (but not β1‐) adrenergic blockage, and by endothelium removal or 1 mM L‐NMMA. In addition, vasorelaxant responses to dibutyryl cyclic AMP were inhibited by 1 mM L‐NMMA, with a reduction in Emax from 90.0±9.3% to 50.5±9.9% (P<0.05).

Isoprenaline 1 μM increased NOS activity in HUVEC (34.0±5.9% above basal, P<0.001). Furthermore, isoprenaline increased adenylyl cyclase activity in a concentration‐dependent manner; this response was inhibited by β2 (but not β1‐) adrenergic blockade. Forskolin 1 μM and dibutyryl cyclic AMP 1 mM each increased NOS activity in HUVEC, to a degree similar to isoprenaline 1 μM. The increase in L‐arginine to L‐citrulline conversion observed with each agent was abolished by co‐incubation with NOS inhibitors.

These results indicate that endothelial β2‐adrenergic stimulation and cyclic AMP elevation activate the L‐arginine/NO system, and give rise to vasorelaxation, in human umbilical vein.

British Journal of Pharmacology (1999) 126, 1872–1880; doi:10.1038/sj.bjp.0702512
Enhancement of vascular permeability by specific activation of protease‐activated receptor‐1 in rat hindpaw: a protective role of endogenous and exogenous nitric oxide
Authors: Kawabata Atsufumi , Kuroda Ryotaro , Nishikawa Hiroyuki , Asai Toshiharu , Kataoka Kazuo , Taneda Mamoru
Published: January 2005

To clarify the role of the first thrombin receptor/protease‐activated receptor (PAR)‐1 in an inflammatory process, we tested and characterized the effect of intraplantar (i.pl.) administration of the highly specific PAR‐1 agonist TFLLR‐NH2 in rat hindpaw.

TFLLR‐NH2 administered i.pl. at 0.01–0.03 μmol per paw enhanced vascular permeability in the hindpaw and produced paw oedema in a dose‐dependent manner. This effect was almost completely abolished by repeated pretreatment with compound 48/80 to deplete inflammatory mediators in mast cells.

The NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester or N‐iminoethyl‐L‐ornithine, preadministered i.pl., stereospecifically potentiated the i.pl. TFLLR‐NH2‐induced permeability increase, while the NO donor sodium nitroprusside or NOC‐18, given i.pl., suppressed the effect of TFLLR‐NH2.

These findings demonstrate that specific activation of PAR‐1 produces increased vascular permeability accompanied by oedema formation in the rat hindpaw, predominantly via mast cell degranulation, and that endogenous and exogenous NO plays a protective role in the PAR‐1‐mediated inflammatory event.

British Journal of Pharmacology (1999) 126, 1856–1862; doi:10.1038/sj.bjp.0702513
Gi‐protein α‐subunit mRNA antisense oligonucleotide inhibition of Gi‐coupled receptor contractile activity in the epididymis of the guinea‐pig
Authors: Haynes John M , Selbie Lisa A , Hill Stephen J
Published: January 2005

We have used a reversible permeabilization method to facilitate the entry of Giα1, 2 and 3 G‐protein subunit mRNA antisense or mismatch oligonucleotides into intact tissue, to investigate the G‐protein α‐subunit coupling of α2‐adrenoceptors, neuropeptide Y (NPY) Y1, and A1 adenosine receptors in preparations of the epididymis of the guinea‐pig.

The α2‐adrenoceptor agonist, xylazine, elicited concentration dependent contractions from preparations of phenylephrine (3 μM)‐stimulated epididymis (pEC50 value 6.52±0.39, maximum response 236±41 mg force). Compared to respective mismatch controls the incubation of preparations with Giα2, but not with Giα1 or Giα3 mRNA antisense oligonucleotides (30 μM) reduced the maximal xylazine‐potentiation of phenylephrine (3 μM)‐stimulated contractility (to 51±12% of Giα2 mismatch control). The oligonucleotide incubations had no effect upon the pEC50 values of xylazine.

The A1 adenosine receptor agonist, cyclopentyladenosine (CPA) elicited concentration dependent contractions from preparations of phenylephrine (3 μM)‐stimulated epididymis (pEC50 value 7.66±0.57, maximum response 208±54 mg force). Incubation of preparations of epididymis with Giα1, but neither Giα2 nor Giα3 antisense oligonucleotides reduced the maximal CPA‐potentiation of phenylephrine (3 μM)‐stimulated contractions (to 55±17% of Giα1 mismatch control), pEC50 values were not affected.

The incubation of preparations with Giα2 antisense mRNA oligonucleotides reduced the maximal NPY‐potentiation of phenylephrine (3 μM)‐stimulated contractions (to 62±15% of Giα mismatch control). Compared with Giα2 mismatch controls, the incubation of preparations with Giα1 and Giα3 oligonucleotides also reduced the NPY‐potentiation of phenylephrine (3 μM)‐stimulated contractions.

These studies indicate that, in the guinea‐pig epididymis, α2‐adrenoceptors and A1 adenosine receptors preferentially couple to effectors through Giα2 and Giα1 subunits respectively. In contrast NPY receptors may elicit effects through either Giα1, 2 or 3 subunits.

British Journal of Pharmacology (1999) 127, 85–90; doi:10.1038/sj.bjp.0702515
Ex vivo assay to determine the cyclooxygenase selectivity of non‐steroidal anti‐inflammatory drugs
Authors: Giuliano Francesco , Warner Timothy D
Published: January 2005

In this study we describe experiments to establish ex vivo the selectivity of non‐steroidal anti‐inflammatory drugs (NSAIDs) given in vivo.

Anaesthetised (Inactin, 120 mg kg−1) male Wistar rats (220–250 g) received an i.v. dose of one of the following compounds (dose mg kg−1): aspirin (20), diclofenac (3), L‐745,337 (30), nimesulide (15), salicylate (20), sulindac (10). Blood samples were taken before and up to 6 h after dosing and the plasma obtained from it was tested for its ability to inhibit prostanoid formation in IL‐1β‐treated A549 cells (COX‐2 system) and human washed platelets (COX‐1 system). For control the same compounds were also added directly to the assay systems.

All drugs, except sodium salicylate, inhibited COX‐1 and COX‐2 when added directly to the test systems. Plasma from aspirin‐treated rats was without effect on either COX‐1 or COX‐2, consistent with the rapid in vivo metabolism to salicylate. Conversely, plasma from sulindac‐treated rats inhibited COX‐1 and COX‐2 with potencies according with in vivo metabolism to sulindac sulphide. Diclofenac was COX‐1/2 non‐selective when tested in vitro, but a slightly preferential inhibitor of COX‐2 when tested ex vivo. Nimesulide was confirmed as preferential inhibitor of COX‐2 both in vitro and ex vivo. L‐745,337 was a selective COX‐2 inhibitor when tested in vitro or ex vivo.

In conclusion, our experiments show clearly (a) NSAIDs inactivation, (b) activation of pro‐drugs, and (c) NSAIDs selectivity. Our assay provides useful information about the selectivity of NSAIDs that could be extended by the analysis of plasma samples taken from humans similarly treated with test drugs.

British Journal of Pharmacology (1999) 126, 1824–1830; doi:10.1038/sj.bjp.0702518
Heterogeneity of prejunctional NPY receptor‐mediated inhibition of cardiac neurotransmission
Authors: Serone Adrian P , Wright Christine E , Angus James A
Published: January 2005

Neuropeptide Y (NPY) has been proposed as the candidate inhibitory peptide mediating interactions between sympathetic and vagal neurotransmission in several species, including man. Here, we have defined the NPY receptors involved in modulation of cardiac autonomic neurotransmission using receptor‐selective agonists and antagonists in the rabbit and guinea‐pig isolated right atria.

In isolated atrial preparations, sympathetically‐mediated tachycardia (ST; with atropine 1 μM) or vagally‐mediated bradycardia (VB; with propranolol 0.1–1 μM) in response to electrical field stimulation (EFS, 1–4 pulses) were tested 0–30 min after incubation with single concentrations of vehicle, NPY (0.01–10 μM), the Y2 receptor agonist N‐Acetyl‐[Leu28,31]NPY(24–36) (termed N‐A[L]NPY(24–36)) or the Y1 receptor agonist [Leu31,Pro34]NPY (LP). The effect of NPY on the concentration‐chronotropic response curves to isoprenaline and bethanechol were also assessed.

Guinea‐pig atria: NPY and N‐A[L]NPY(24–36) caused concentration‐dependent inhibition of VB and ST to EFS. Both peptides caused maximal inhibition of VB and ST within 10 min incubation and this remained constant. LP caused a concentration‐dependent, transient inhibition of ST which was antagonized by the Y1‐receptor antagonist GR231118 (0.3 μM), with apparent competitive kinetics. Rabbit atria: NPY (1 or 10 μM) had no effect on VB at any time point, but both NPY and LP caused a transient (∼10 min) inhibition of sympathetic tachycardia. This inhibition could be prevented by 0.3 μM GR231118. N‐A[L]NPY(24–36) had no effect on ST. NPY had no effect on the response to β‐adrenoceptor stimulation by isoprenaline nor muscarinic‐receptor stimulation by bethanechol in either species.

Thus, in the guinea‐pig, NPY causes a stable inhibition of both VB and ST to EFS via Y2 receptors and transient inhibition of ST via Y1 receptors. In contrast in the rabbit, NPY has no effect on the cardiac vagus and prejunctional inhibition of ST is transient and mediated by a Y1‐like receptor (rather than Y2). Therefore it would be surprising if NPY plays a functional role in modulation of cardiac neurotransmission in the rabbit.

British Journal of Pharmacology (1999) 127, 99–108; doi:10.1038/sj.bjp.0702519
A comparison of the inhibitory activity of PDE4 inhibitors on leukocyte PDE4 activity in vitro and eosinophil trafficking in vivo
Authors: Cooper Nicola , Teixeira Mauro M , Warneck Julie , Miotla Jadwiga M , Wills Ruth E , Macari David M T , Gristwood Robert W , Hellewell Paul G
Published: January 2005

Phosphodiesterase (PDE) 4 inhibitors have been shown to inhibit eosinophil PDE4 activity in vitro and accumulation of eosinophils in experimental airways inflammation. However, direct effects on eosinophil trafficking have not been studied in detail and it is not known if activity in vitro translates into efficacy in vivo. In the present study, we compared the activity of five PDE4 inhibitors in vitro and against trafficking of 111In‐eosinophils in cutaneous inflammation in the guinea‐pig.

The rank order of potency for inhibition of PDE4 activity in guinea‐pig eosinophil, neutrophil and macrophage, and human neutrophil lysates was RP73401>SB207499>CDP840>rolipram>LAS31025. On TNFα production by human PBMC, all inhibitors with the exception of rolipram showed potency similar to their effect on neutrophil lysates.

In a brain cerebellum binding assay, the rank order of potency at displacing [3H]‐rolipram was RP73401>rolipram>SB207499>CDP840>LAS30125.

Trafficking of 111In‐eosinophils to skin sites injected with PAF, ZAP or antigen in sensitized sites was inhibited by oral administration of all PDE4 inhibitors. The rank order of potency was RP73401=rolipram>LAS31025>SB207499>CDP840.

With the exception was RP73401, which was the most potent compound in all assays, there was no clear relationship between activity of PDE4 inhibitors in vitro and capacity to inhibit eosinophil trafficking in vivo. Thus, we conclude that in vitro activity of PDE4 inhibitors does not predict in vivo efficacy in an experimental model of eosinophil trafficking.

British Journal of Pharmacology (1999) 126, 1863–1871; doi:10.1038/sj.bjp.0702520
Minor structural changes in nicotinoid insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors
Authors: Tomizawa Motohiro , Casida John E
Published: January 2005

The major nitroimine insecticide imidacloprid (IMI) and the nicotinic analgesics epibatidine and ABT‐594 contain the 6‐chloro‐3‐pyridinyl moiety important for high activity and/or selectivity. ABT‐594 has considerable nicotinic acetylcholine receptor (AChR) subtype specificity which might carry over to the chloropyridinyl insecticides. This study considers nine IMI analogues for selectivity in binding to immuno‐isolated α1, α3 and α7 containing nicotinic AChRs and to purported α4β2 nicotinic AChRs.

α1‐ and α3‐Containing nicotinic AChRs (both immuno‐isolated by mAb 35, from Torpedo and human neuroblastoma SH‐SY5Y cells, respectively) are between two and four times more sensitive to DN‐IMI than to (−)‐nicotine.

With immuno‐isolated α3 nicotinic AChRs, the tetrahydropyrimidine analogues of IMI with imine or nitromethylene substituents are 3–4 fold less active than (−)‐nicotine. The structure‐activity profile with α3 nicotinic AChRs from binding assays is faithfully reproduced in agonist potency as induction of 86rubidium ion efflux in intact cells.

α7‐Containing nicotinic AChRs of SH‐SY5Y cells (immuno‐isolated by mAb 306) and rat brain membranes show maximum sensitivity to the tetrahydropyrimidine analogue of IMI with the nitromethylene substituent.

The purported α4β2 nicotinic AChRs [mouse (Chao & Casida, 1997) and rat brain] are similar in sensitivity to DN‐IMI, the tetrahydropyrimidine nitromethylene and nicotine.

The commercial insecticides (IMI, acetamiprid and nitenpyram) have low to moderate potency at the α3 and purported α4β2 nicotinic AChRs and are essentially inactive at α1 and α7 nicotinic AChRs.

In conclusion, the toxicity of the analogues and metabolites of nicotinoid insecticides in mammals may involve action at multiple receptor subtypes with selectivity conferred by minor structural changes.

British Journal of Pharmacology (1999) 127, 115–122; doi:10.1038/sj.bjp.0702526
Tacrolimus suppresses tumour necrosis factor‐α and protects against splanchnic artery occlusion shock
Authors: Squadrito Francesco , Altavilla Domenica , Squadrito Giovanni , Ferlito Marcella , Campo Giuseppe M , Arlotta Mariarita , Grimaldi Serafino , Quartarone Cristina , Saitta Antonino , Caputi Achille P
Published: January 2005

Tumour necrosis factor (TNF‐α) is a pleiotropic cytokine which is deeply involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. Tacrolimus, formerly known as FK506, is a macrolide antibiotic, that blocks the transcription of several proinflammatory cytokines including TNF‐α.

Male anaesthetized rats were subjected to clamping of the splanchnic arteries for 45 min. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham operated animals were used as controls. SAO shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham shocked rats survived more than 4 h), enhanced serum TNF‐α concentrations (415±12 U ml−1), decreased mean arterial blood pressure (MAP), leukopenia and increased ileal leukocyte accumulation studied by means of myeloperoxidase activity (MPO=7.5±0.3 U g−1 tissue). Moreover aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM–10 μM), reduced responsiveness to acetylcholine (ACh, 10 nM–10 μM) and increased staining for intercellular adhesion molecule‐1 (ICAM‐1). Furthermore increased mRNA for TNF‐α was observed in peritoneal macrophages of SAO shocked rats.

Tacrolimus (100 μg kg−1, 5 min after splanchnic arteries occlusion) increased survival rate (SAO+Tacrolimus=100% at 4 h of reperfusion), reverted the marked hypotension, reduced serum TNF‐α (15±3 U ml−1), ameliorated leukopenia, reduced ileal MPO (0.9±0.01 U g−1 tissue), restored to control values the hyporeactivity to PE, improved the reduced responsiveness to ACh and blunted the enhanced immunostaining for ICAM‐1 in the aorta. Finally tacrolimus suppressed cytokine mRNA levels in peritoneal macrophages.

The data suggest that tacrolimus may represent a new therapeutic approach in circulatory shock.

British Journal of Pharmacology (1999) 127, 498–504; doi:10.1038/sj.bjp.0702528
Effects of prolonged cold storage on double peaked vasoconstrictor responses to periarterial nerve stimulation in isolated canine splenic arteries
Authors: Yang Xiao‐Ping , Chiba Shigetoshi
Published: January 2005

P2X‐Purinoceptors and α1‐adrenoceptors have previously been shown to involve in the double peaked vasoconstrictor responses to periarterial electrical nerve stimulation in the isolated and perfused canine splenic artery. The present study made an attempt to investigate effects of prolonged cold storage (7 days at 4°C) on vasoconstrictor responses to periarterial electrical nerve stimulation, tyramine, noradrenaline and adenosine 5′‐triphosphate (ATP) in the isolated canine splenic artery.

The periarterial nerve stimulation (1–10 Hz) readily causes a double peaked vasoconstriction in the non‐stored preparations. After cold stored for 7 days, the double peaked vasoconstriction was still recognized, although the response became significantly smaller. The first phase was decreased relatively greater than the second phase by the cold storage.

In the cold stored preparations, the dose‐response curve for tyramine was shifted to the right in a parallel manner. Prazosin almost completely inhibited tyramine‐induced vasoconstriction but α,β‐methylene ATP failed to influence the response to tyramine.

The vasoconstrictor responses to noradrenaline and ATP were not significantly modified by the prolonged cold storage.

From these results, it is concluded that the functions of sympathetic co‐transmission of purinergic components might be influenced more than that of adrenergic components in the cold storage canine splenic artery.

British Journal of Pharmacology (1999) 126, 1810–1814; doi:10.1038/sj.bjp.0702531
Further characterization of the ORL1 receptor‐mediated inhibition of noradrenaline release in the mouse brain in vitro
Authors: Werthwein Sven , Bauer Ulrich , Nakazi Michael , Kathmann Markus , Schlicker Eberhard
Published: January 2005

Mouse brain slices preincubated with [3H]‐noradrenaline or [3H]‐serotonin were superfused with medium containing naloxone 10 μM; we studied whether nociceptin (the endogenous ligand at ORL1 receptors) affects monoamine release. Furthermore, the affinities of ORL1 ligands were determined using [3H]‐nociceptin binding.

The electrically (0.3 Hz) evoked tritium overflow in mouse cortex slices preincubated with [3H]‐noradrenaline was inhibited by nociceptin and [Tyr14]‐nociceptin (maximally by 80%; pEC50 7.52 and 8.28) but not affected by [des‐Phe1]‐nociceptin (pEC50<6). The ORL1 antagonist naloxone benzoylhydrazone antagonized the effect of nociceptin and [Tyr14]‐nociceptin.

The effect of nociceptin did not desensitize, was not affected by blockade of NO synthase, cyclo‐oxygenase and P1‐purinoceptors and was decreased by the α2‐adrenoceptor agonist talipexole. Nociceptin also inhibited the evoked overflow in mouse cerebellar, hippocampal and hypothalamic slices in a manner sensitive to naloxone benzoylhydrazone.

The electrically (3 Hz) evoked tritium overflow in mouse cortex slices preincubated with [3H]‐serotonin was inhibited by nociceptin; naloxone benzoylhydrazone antagonized this effect.

The affinities (pKi) for [3H]‐nociceptin binding to mouse cortex membranes were: nociceptin, 8.71; [Tyr14]‐nociceptin, 9.82; [des‐Phe1]‐nociceptin, <5.5; naloxone benzoylhydrazone, 5.85; naloxone, <4.5.

In conclusion, nociceptin inhibits noradrenaline release in the mouse cortex via ORL1 receptors, which interact with presynaptic α2‐autoreceptors on noradrenergic neurones. The effect of nociceptin does not desensitize nor does it involve NO, prostanoids or adenosine. Nociceptin also attenuates noradrenaline release from several subcortical regions and serotonin release from cortical slices by a naloxone benzoylhydrazone‐sensitive mechanism.

British Journal of Pharmacology (1999) 127, 300–308; doi:10.1038/sj.bjp.0702534
Diurnal variation in 5‐HT1B autoreceptor function in the anterior hypothalamus in vivo: effect of chronic antidepressant drug treatment
Authors: Sayer Tamsin J O , Hannon Serina D , Redfern Peter H , Martin Keith F
Published: January 2005

Intracerebral microdialysis was used to examine the function of the terminal 5‐hydroxytryptamine (5‐HT) autoreceptor in the anterior hypothalamus of anaesthetized rats at two points in the light phase of the light–dark cycle.

Infusion of the 5‐HT1A/1B agonist 5‐methoxy‐3‐(1,2,3,6‐tetrahydro‐4‐pyridyl)‐1H‐indole (RU24969) 0.1, 1.0 and 10 μM through the microdialysis probe led to a concentration‐dependent decrease (49, 56 and 65% respectively) in 5‐HT output. The effect of RU24969 (1 and 5 μM) was prevented by concurrent infusion of methiothepin (1 and 10 μM) into the anterior hypothalamus via the microdialysis probe. Infusion of methiothepin alone (1.0 and 10 μM) increased (15 and 142% respectively) 5‐HT output.

Infusion of RU24969 (5 μM) through the probe at mid‐light and end‐light resulted in a quantitatively greater decrease in 5‐HT output at end‐light compared with mid‐light.

Following treatment with either paroxetine hydrochloride (10 mg kg−1 i.p.) or desipramine hydrochloride (10 mg kg−1 i.p.) for 21 days the function of the terminal 5‐HT1B autoreceptor was more markedly attenuated at end‐light.

The data show that, as defined by the response to RU24969, the function of the 5‐HT1B receptors that control 5‐HT output in the anterior hypothalamus is attenuated following chronic desipramine or paroxetine treatment in a time‐of‐day‐dependent manner.

British Journal of Pharmacology (1999) 126, 1777–1784; doi:10.1038/sj.bjp.0702535