bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a role for nitric oxide in relaxation of the frog oesophageal body to electrical field stimulation
Authors: Williams S. J. , Parsons M. E.
Published: January 2005

Electrical field stimulation (EFS) (1–10 Hz, 30 V, 2 ms) of frog oesophageal body strips resulted in frequency‐dependent non‐adrenergic, non‐cholinergic (NANC) relaxations.

Tetrodotoxin (TTX) (10−6–10−5 M) had no effect on EFS evoked relaxations with a 2 ms pulse width. At a pulse width of 0.5 ms only the responses to the highest frequency (10 Hz) were significantly inhibited by TTX at 10−5 M. Relaxations at the 2 ms pulse width were unaffected by ω‐conotoxin (10−6 M), nifedipine (10−6 M) or cobalt (5×10−4 M).

NG‐nitro‐L‐arginine (L‐NOARG) (10−6–10−4 M), a nitric oxide synthase (NOS) inhibitor, caused a concentration‐dependent inhibition of the EFS‐induced NANC relaxant responses. The inhibitory effect of L‐NOARG was both prevented and reversed by L‐arginine but not D‐arginine (5×10−3 M).

The phosphodiesterase type V inhibitor (PDE V), SK&F 96231 (10−7–10−4 M), caused a concentration‐dependent potentiation of both the percentage relaxation and the duration of the relaxant responses to EFS.

ODQ (10−7–10−5 M), a guanylate cyclase inhibitor, produced a concentration‐dependent inhibition of EFS‐evoked NANC relaxations.

Oxyhaemoglobin (10−6 M), which binds nitric oxide (NO), inhibited NANC relaxations to EFS.

The NO donor sodium nitroprusside (SNP) (10−8–10−4 M) produced a concentration‐dependent inhibition of evoked tone. L‐NOARG (10−4 M) had no effect on the SNP evoked relaxations. Preincubation with oxyhaemoglobin (10−6 M) caused a reduction in the SNP (10−6–10−5 M) induced relaxations.

These results suggest NO is the relaxant transmitter of the frog oesophageal body and the source of NO may be non‐neuronal.

British Journal of Pharmacology (1997) 122, 179–185; doi:10.1038/sj.bjp.0701298
Involvement of cyclic nucleotide‐dependent protein kinases in cyclic AMP‐mediated vasorelaxation
Authors: Eckly‐Michel Anita , Martin Viviane , Lugnier Claire
Published: January 2005

The involvement of cyclic AMP‐dependent protein kinase (PKA) and cyclic GMP‐dependent protein kinase (PKG) in the effects of cyclic AMP‐elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent‐protein kinase activities and ATP‐induced calcium signalling ([Ca2+]i) was studied in rat aorta. Cyclic AMP‐elevating agents used were a β‐adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram).

In rat intact aorta, the relaxant effect induced by isoprenaline (0.01–0.3 μM) was decreased by a specific inhibitor of PKA, H‐89, whereas a specific inhibitor of PKG, Rp‐8‐Br‐cyclic GMPS, was without effect. No significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 μM was used. At the same concentration, isoprenaline did not modify ATP‐induced changes in [Ca2+]i in smooth muscle cells. Neither H‐89 nor Rp‐8‐Br‐cyclic GMPS modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01–0.3 μM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved.

The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp‐8‐Br‐cyclic GMPS with no significant effect of H‐89. Neither SK&F 94120, nor rolipram at 30 μM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP‐induced transient increase in [Ca2+]i. This decrease was abolished by Rp‐8‐Br‐cyclic GMPS whereas H‐89 had no significant effect. These results suggest that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross‐activation of PKG by cyclic AMP in rat aorta.

The combination of 5 μM SK&F 94120 with rolipram markedly potentiated the relaxant effect of rolipram. This relaxation was decreased by H‐89 and not significantly modified by Rp‐8‐Br‐cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PKA rather than PKG is involved in this type of vasorelaxation. The differences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are differently localized in the cell.

These findings support a role for both PKA and PKG in cyclic AMP‐mediated relaxation in rat aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.

British Journal of Pharmacology (1997) 122, 158–164; doi:10.1038/sj.bjp.0701339
Central role of intracellular calcium stores in acute flow‐ and agonist‐evoked endothelial nitric oxide release
Authors: Hutcheson Iain R. , Griffith Tudor M.
Published: January 2005

We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of Ca2+ release from endothelial intracellular stores to nitric oxide (NO) production evoked by increases in shear stress and by acetylcholine in rabbit aorta.

Experiments were performed before and following incubation with either the endoplasmic reticulum Ca2+‐ATPase inhibitors cyclopiazonic acid (CPA, 10 μM) and thapsigargin (TSG, 1 μM) or ryanodine (30, 100 μM) which binds to a specific endoplasmic reticulum Ca2+‐release channel.

In cascade bioassay all three agents induced relaxations of the recipient ring (CPA, 24.4±3.8%; TSG, 51.5±10.6%; ryanodine, 17.4±1.6%) which were significantly attenuated by preincubation of the donor with 100 μM NG‐nitro‐L‐arginine methyl ester (L‐NAME). However, in isolated rings, only CPA and TSG induced L‐NAME‐sensitive relaxations (CPA 52.7±6.5%; TSG 61.3±7%).

Addition of superoxide dismutase (SOD) to the donor perfusate evoked relaxations of the recipient ring in cascade bioassay (13.3±1.4%, n=22). Prior administration of SOD attenuated relaxations to TSG (23.2±3.8%, n=4) and ryanodine (1.7±0.8%, n=4), and pre‐incubation with TSG and ryanodine blunted SOD‐induced responses (4±1.5%, n=4 and 8.9±1.1%, n=4, respectively). By contrast, no interaction was observed between the relaxations evoked by SOD and CPA. In isolated rings, SOD exerted no direct relaxant action and did not modulate relaxations to CPA, TSG or ryanodine.

In cascade bioassay studies time‐averaged shear stress was manipulated with dextran (1–4% w/v, 80000 MW) to increase perfusate viscosity. NO‐dependent relaxation of the recipient ring induced by increased perfusate viscosity was significantly attenuated by CPA (P<0.01; n=6) and TSG (P<0.05; n=7), but not by ryanodine (n=6).

Endothelium‐dependent relaxations to acetylcholine (0.1–30 μM) in cascade bioassay and in isolated aortic ring preparations were markedly attenuated by pretreatment with CPA and TSG, but were unaffected by ryanodine. Ryanodine and CPA caused only a small attenuation of endothelium‐independent relaxations to sodium nitroprusside (0.001–10 μM), whereas TSG had no effect.

We conclude that release of Ca2+ from CPA‐ and TSG‐sensitive endothelial stores is necessary for NO release evoked by acute flow changes and agonists in rabbit abdominal aorta. Ca2+‐induced Ca2+ release via the ryanodine‐sensitive release channel plays no direct role in these responses. Free radical interactions may complicate the interpretation of findings in cascade bioassay compared with isolated ring preparations.

British Journal of Pharmacology (1997) 122, 117–125; doi:10.1038/sj.bjp.0701340
Blockade of N‐type Ca2+ current by cilnidipine (FRC‐8653) in acutely dissociated rat sympathetic neurones
Authors: Uneyama Hisayuki , Takahara Akira , Dohmoto Hideki , Yoshimoto Ryota , Inoue Kazuhide , Akaike Norio
Published: January 2005

The inhibitory effects of cilnidipine (FRC‐8653) and various organic Ca2+ channel blockers on high voltage‐activated Ba2+ currents (HVA IBa) in rat sympathetic neurones were examined by means of the conventional whole‐cell patch‐clamp recording mode under voltage‐clamped conditions.

HVA IBa was classified into three different current components with subtype selective peptide Ca2+ channel blockers. No ω‐Agatoxin IVA‐sensitive (P‐type) or ω‐conotoxin MVIIC‐sensitive (Q‐type) current components were observed. Most (>85%) IBa was found to consist of ω‐conotoxin GVIA‐sensitive N‐type components.

The application of cilnidipine inhibited HVA IBa in a concentration‐dependent manner. The Kd value for cilnidipine was 0.8 μM. Cilnidipine did not shift the current‐voltage (I‐V) relationship for HVA IBa, as regards the threshold potential and peak potential where the amplitude reached a maximum.

High concentrations of three hypotensive Ca2+ channel blockers, nifedipine, diltiazem and verapamil, all inhibited HVA IBa in a concentration‐dependent manner. The Kd values for nifedipine, diltiazem and verapamil were 131, 151 and 47 μM, respectively. A piperazine‐type Ca2+ channel blocker, flunarizine, showed a relatively potent blocking action on IBa. The Kd value was about 3 μM.

These results thus show that cilnidipine potently inhibits the sympathetic Ca2+ channels which predominantly consist of an ω‐Cg‐GVIA‐sensitive component. This blockade of the N‐type Ca2+ channel, as well as the L‐type Ca2+ channel by cilnidipine suggests that it could be used therapeutically for treatment of hypersensitive sympathetic disorders associated with hypertension.

British Journal of Pharmacology (1997) 122, 37–42; doi:10.1038/sj.bjp.0701342
Effect of adenosine and some of its structural analogues on the conductance of NMDA receptor channels in a subset of rat neostriatal neurones
Authors: Nörenberg W. , Wirkner K. , Illes P.
Published: January 2005

In order to investigate the modulatory effects of adenosine on excitatory amino acid projections onto striatal medium spiny neurones, whole‐cell patch clamp experiments were carried out in rat brain slices. The effects of various agonists for P1 (adenosine) and P2 (ATP) purinoceptors and their antagonists were investigated. The A2A receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 0.1 μM), the A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA; 10 μM) and the non‐selective P1 purinoceptor antagonist 8‐(p‐sulphophenyl)‐theophylline (8‐SPT; 100 μM) did not alter the resting membrane potential, the threshold current necessary to elicit an action potential, the amplitude of spikes, their rise time, the amplitude of the afterhyperpolarization (AHP) and the time to peak of the AHP.

N‐methyl‐D‐aspartate (NMDA; 1–1000 μM) caused a concentration‐dependent inward current which was larger in the absence than in the presence of Mg2+ (1.3 mM). In a subset of striatal neurones, the current response to NMDA (10 μM) and the accompanying increase in conductance were both inhibited by CGS 21680 (0.01–1 μM). The effect of CGS 21680 (0.1 μM) persisted in the presence of tetrodotoxin (0.5 μM) or in a Ca2+‐free medium, under conditions when synaptically mediated influences may be negligible.

The A3 receptor agonist N6‐2‐(4‐aminophenyl)ethyladenosine (APNEA; 0.1–10 μM) also diminished the effect of NMDA (10 μM), while the A1 receptor agonists CCPA (0.01–10 μM) and (2S)‐N6‐[2‐endo‐norbornyl]adenosine [S(−)‐ENBA; 10 μM] as well as the endogenous, non‐selective P1 purinoceptor agonist adenosine (100 μM) were inactive. The endogenous non‐selective P2 purinoceptor agonist ATP (1000 μM) also failed to alter the current response to NMDA (10 μM). Adenosine (100 μM), but not ATP (1000 μM) became inhibitory after blockade of nucleoside uptake by S(4‐nitrobenzyl)‐6‐thioguanosine (NBTG; 30 μM).

8‐(p‐Sulphophenyl)‐theophylline (8‐SPT; 100 μM), as well as the A2A receptor antagonist 8‐(3‐chlorostyryl)caffeine (CSC; 1 μM) and the A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) at 0.03, but not 0.003 μM abolished the inhibitory action of CGS 21680 (0.1 μM). None of these compounds altered the effect of NMDA (10 μM) by itself. DPCPX (0.03 μM) prevented the inhibition by APNEA (10 μM).

There was no effect of CGS 21680 (0.1 μM), when guanosine 5′‐O‐(3‐thiodiphosphate (GDP‐β‐S; 300 μM) was included in the pipette solution in order to block G protein‐mediated reactions.

In conclusion, adenosine receptors, probably of the A2A‐subtype, inhibit the conductance of NMDA receptor channels in a subset of medium spiny neurones of the rat striatum by a transduction mechanism which involves a G protein.

British Journal of Pharmacology (1997) 122, 71–80; doi:10.1038/sj.bjp.0701347
Development of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo
Authors: Paolini Moreno , Pozzetti Laura , Sapone Andrea , Luigi Biagi Gian , Cantelli‐Forti Giorgio
Published: January 2005

The sensitivity of the developing embryo to xenobiotics is highly dependent on the expression of metabolizing enzymes including cytochromes P450 (CYP). In the present study, therefore, the ontogeny of the CYP‐dependent system in the chick was investigated with testosterone hydroxylase activity as a marker of CYP expression.

Chicken embryo livers were assayed for basal and phenobarbitone (PB)‐induced regio‐ and stereo‐selective testosterone hydroxylase activity, from the first appearance of the liver as a discrete organ at 5 days of incubation through day 10 posthatching. In addition, whole embryo preparations were assayed at 3 and 4 days of incubation.

Whereas testosterone 16β‐hydroxylase and androst‐4‐ene‐3,17‐dione‐linked activities were expressed during all stages of embryonic development, testosterone 6α‐, 6β‐, 7α‐ and 16α‐hydroxylase activities were observed only in basal embryos from 8 days of incubation. Furthermore, testosterone 2α‐ and 2β‐ hydroxylase activities were detected exclusively from 10 days of incubation onward. All activities increased steadily throughout development as did the responsiveness of the embryonic liver to PB induction.

A typical pattern of development with a higher activity from 10 to 14 days of incubation (testosterone 16α‐, 7α‐, 6α‐ and 2β‐hydroxylase activities; up to 4.1±0.3 pmol mg−1 protein min−1 at 13 days of incubation for testosterone 7α‐hydroxylase) or shifted to 14 to 18 days of incubation (testosterone 6β‐, 2α‐ and 16β‐hydroxylase activities: up to 56.6±1.4 pmol mg−1 protein min−1 at 16 days of incubation for testosterone 6β‐hydroxylase) was observed. There was a tendency towards an increased activity for all activities around hatching, specifically from 19 days of incubation to 4 days posthatching (up to 1,759.3±179.4 pmol mg−1 protein min−1 at 1 day posthatching for androst‐4‐ene‐3,17‐dione‐linked activity).

The highest level of PB‐induced enzyme activity was observed for testosterone 2α‐hydroxylase activity (95.14±7.35 and 660.19±45.27 pmol mg−1 protein min−1) at 12 days of incubation and day 3 posthatching, respectively. Except for testosterone 2α‐ and 2β‐hydroxylase activities at 3 to 4 days of incubation, all metabolites were detectable during the first period of organogenesis in the presence of PB.

The use of highly specific substrates, studies on the immunoinhibition of metabolism by polyclonal antibodies raised against highly purified rat CYPs, and the use of selective inhibitors seemed to reveal a wide pleiotropic response with the posssible presence in liver of PB‐treated chickens of CYP1A together with CYP2H1/H2, CYP2E and CYP3A.

British Journal of Pharmacology (1997) 122, 344–350; doi:10.1038/sj.bjp.0701352
The interaction between methylene blue and the cholinergic system
Authors: Pfaffendorf M. , Bruning T. A. , Batink H. D. , Van Zwieten P. A.
Published: January 2005

The inhibitory effects of methylene blue (MB) on different types of cholinesterases and [3H]‐N‐methylscopolamine ([3H]‐NMS) binding to muscarinic receptors were studied.

Human plasma from young healthy male volunteers, purified human pseudocholinesterase and purified bovine true acetylcholinesterase were incubated with acetylcholine and increasing concentrations of MB (0.1–100 μmol l−1) in the presence of the pH‐indicator m‐nitrophenol for 30 min at 25°C. The amount of acetic acid produced by the enzymatic hydrolysis of acetylcholine was determined photometrically.

Rat cardiac left ventricle homogenate was incubated with [3H]‐NMS and with increasing concentrations of MB (0.1 nmol l−1–100 μmol l−1) at 37°C for 20 min. The binding of [3H]‐NMS to the homogenate was quantified by a standard liquid scintillation technique.

MB inhibited the esterase activity of human plasma, human pseudocholinesterase and bovine acetylcholinesterase concentration‐dependently with IC50 values of 1.05±0.05 μmol l−1, 5.32±0.36 μmol l−1 and 0.42±0.09 μmol l−1, respectively. MB induced complete inhibition of the esterase activity of human plasma and human pseudocholinesterase, whereas bovine acetylcholinesterase was maximally inhibited by 73±3.3%.

MB was able to inhibit specific [3H]‐NMS binding to rat cardiac left ventricle homogenate completely with an IC50 value of 0.77±0.03 μmol l−1, which resulted in a Ki value for MB of 0.58±0.02 μmol l−1.

In conclusion, MB may be considered as a cholinesterase inhibitor with additional, relevant affinity for muscarinic binding sites at concentrations at which MB is used for investigations into the endothelial system. In our opinion these interactions between MB and the cholinergic system invalidate the use of MB as a tool for the investigation of the L‐arginine‐NO‐pathway, in particular when muscarinic receptor stimulation is involved.

British Journal of Pharmacology (1997) 122, 95–98; doi:10.1038/sj.bjp.0701355
Acute nociception mediated by hindpaw P2X receptor activation in the rat
Authors: Bland‐Ward P. A. , Humphrey P. P. A.
Published: January 2005

The functional consequences of P2X receptor activation on peripheral sensory neurones have been investigated in vivo. Behavioural indices of acute nociception were monitored in the conscious rat following subplantar injection of adenosine 5′‐triphosphate (ATP), α,β‐methylene ATP, adenosine 5′‐diphosphate (ADP) and adenosine.

Signs of overt nociception, i.e. hindpaw lifting and licking, were apparent in animals injected subplantar with the P2X receptor agonist, α,β‐methylene ATP. Nociceptive behaviours continued for 15 min following administration of α,β‐methylene ATP (200 nmol) and were dose‐related (0–5 min hindpaw lifting times after injection of α,β‐methylene ATP 100 nmol and 1000 nmol were 89±26 s and 232±11 s, respectively). Subplantar ATP evoked a modest response only at the highest dose tested (1000 nmol; 0–5 min hindpaw lifting time 66±19 s) whilst ADP or adenosine (both 600 nmol) elicited negligible spontaneous nociceptive activity.

Morphine (3 mg kg−1, i.v.) abolished hindpaw licking behaviour induced by subplantar injection of either α,β‐methylene ATP (600 nmol) or bradykinin (1 nmol) and substantially reduced (88±5%) paw licking in formalin (0.5%, 0.1 ml) injected animals. In contrast, hindpaw lifting was only modestly inhibited (34±11%) in morphine‐pretreated animals that had received subplantar bradykinin and was unaffected in rats in which the noxious stimulus was either subplantar α,β‐methylene ATP or formalin. Pretreatment of hindpaws with subplantar bupivacaine (1% w/v, 0.1 ml) abolished α,β‐methylene ATP‐evoked nociceptive behaviours.

Hindpaw lifting and licking mediated by α,β‐methylene ATP (600 nmol, subplantar) were inhibited (72±15% and 95±5%, respectively) by 30 min local pretreatment with 600 nmol α,β‐methylene ATP. Subplantar α,β‐methylene ATP pretreatment did not inhibit behaviour stimulated by subsequent bradykinin (1 nmol) or formalin (0.5%, 0.1 ml) injection into the hindpaw.

Desensitization of small diameter sensory neurones with a single subplantar injection of capsaicin (100 μg) abolished all behaviours indicative of spontaneous nociceptive sensation in animals subsequently injected with α,β‐methylene ATP (600 nmol), bradykinin (1 nmol) or formalin (0.5%, 0.1 ml).

We conclude that activation of P2X receptors present on small diameter (capsaicin‐sensitive) primary afferent neurones in the rat hindpaw mediates behaviour indicative of acute nociception.

British Journal of Pharmacology (1997) 122, 365–371; doi:10.1038/sj.bjp.0701371
Stable expression of human kinin B1 receptor in 293 cells: pharmacological and functional characterization
Authors: Bastian S. , Loillier B. , Paquet J. L. , Pruneau D.
Published: January 2005

We compared the binding properties of [3H]‐desArg10‐[Leu9]‐kallidin, a radiolabelled kinin B1 receptor antagonist, to membranes from IMR‐90 human embryonic fibroblasts and from 293 cells transiently or stably transfected with the human B1 receptor.

The dissociation constant (KD) of [3H]‐desArg10‐[Leu9]‐kallidin and the affinity of several kinin receptor agonists and antagonists were similar between the native and cloned receptor, either transiently or stably expressed in 293 cells. In IMR‐90 cells, the rank order of potency was that expected for a kinin B1 receptor.

The receptors transiently or stably expressed in 293 cells were fully functional with respect to their signalling properties. Phosphoinositide hydrolysis was increased in a concentration‐dependent manner by the B1 receptor agonist, desArg10‐kallidin. Functional coupling to the calcium pathway was also demonstrated for the native and stably expressed human B1 receptor.

In conclusion, the established stable and functional 293 cell clone may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of the kinin B1 receptor.

British Journal of Pharmacology (1997) 122, 393–399; doi:10.1038/sj.bjp.0701380
The contrasting effects of dendrotoxins and other potassium channel blockers in the CA1 and dentate gyrus regions of rat hippocampal slices
Authors: Southan A. P. , Owen D. G.
Published: January 2005

The effects of potassium channel blocking compounds on synaptic transmission in the CA1 and dentate gyrus regions of the rat hippocampus were examined by means of simultaneous field potential recording techniques in brain slices.

4‐Aminopyridine (4‐AP) enhanced the excitatory postsynaptic potential (e.p.s.p.) and induced multiple population spike responses in both regions. EC50 values were 6.7 μM in the CA1 (n=5) and 161.7 μM (n=5) in the dentate gyrus.

Tetraethylammonium (TEA) increased the amplitude and induced broadening of the population spike in both regions. In the dentate gyrus (n=5) a single slow spike response was introduced (EC50 12.8 mM) and in the CA1 region (n=5) the response was transformed into two wide spikes (EC50 2.6 mM).

In the CA1 region all of the dendrotoxins (toxin I, toxin K, α‐Dtx and δ‐Dtx) induced multiple population spikes and enlarged e.p.s.p. responses. Potentials recorded simultaneously in the dentate gyrus exhibited comparatively minor enhancements. The EC50 value for toxin I in the CA1 was calculated to be 237 nM (n=4). Estimated EC50 values were obtained for α‐Dtx (1.1 μM, n=3), toxin K (411 nM, n=4) and δ‐Dtx (176 nM, n=3).

In the presence of toxin I, DL‐2‐amino‐5‐phosphonovaleric acid (APV) induced slight reduction of the late e.p.s.p. phase (n=3). 6‐Cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) abolished all population spikes leaving a late slow positive waveform (n=3). Co‐application of APV and CNQX abolished all postsynaptic responses.

Charybdotoxin (CbTx) was significantly less potent than the dendrotoxins and had mixed actions in the CA1 region (n=3). Again the dentate gyrus exhibited reduced sensitivity (n=3).

In the presence of mast cell degranulating peptide (MCDP), enhancement of the CA1 field potential response (n=5) was greater than that observed in the dentate gyrus (n=5).

The results show that some potassium channel modulators can profoundly enhance CA1 region synaptic responses in the absence of notable changes in dentate gyrus excitability. Selective enhancement of defined synaptic pathways by potassium channel modulators may prove to have considerable therapeutic potential.

British Journal of Pharmacology (1997) 122, 335–343; doi:10.1038/sj.bjp.0701392
Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea‐pig ileum
Authors: López‐Redondo F. , Lees G. M. , Pertwee R. G.
Published: January 2005

The effect of cannabinoid receptor agonists was studied in guinea‐pig myenteric neurones in vitro by use of conventional intracellular recording techniques.

Exposure of myenteric neurones of the S‐cell type to the cannabinoid receptor agonists WIN 55,212‐2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively.

The depressant effect of WIN 55,212‐2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude‐time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect.

The inhibitory effect of WIN 55,212‐2 appeared to be concentration‐dependent over the range 1–100 nM. WIN 55,212‐3, its (−)‐enantiomer (100 nM), was inactive.

The cannabinoid CB1 receptor antagonist, SR141716A (1 μM), reversed the inhibitory effects of WIN 55,212‐2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)‐induced depolarizations in 42% of neurones tested (5/12).

When tested on its own, SR141716A (1 μM) caused a 40–50% reduction in the amplitude of fast e.p.s.ps (n=9).

WIN 55,212‐2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 μM).

It is concluded that cannabinoid‐induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB1 receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S‐neurones and their synapsing cholinergic and non‐cholinergic, non‐adrenergic terminals are not endowed with cannabinoid receptors.

British Journal of Pharmacology (1997) 122, 330–334; doi:10.1038/sj.bjp.0701393
κ1‐ and κ2‐opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum
Authors: Schoffelmeer Anton N. M. , Hogenboom Francois , Mulder Arie H.
Published: January 2005

The effects of selective opioid receptor agonists and antagonists on N‐methyl‐D‐aspartate (NMDA, 10 μM)‐induced release of [3H]‐dopamine and [14C]‐acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional κ‐opioid receptor subtypes in rat brain.

The κ receptor agonists (−)‐ethylketocyclazocine ((−)‐EKC), U69593 and the endogenous opioid peptide dynorphin A1–13 caused a naloxone‐reversible inhibition of NMDA‐induced [3H]‐dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the μ agonist Tyr‐D‐Ala‐Gly‐(NMe)Phe‐Gly‐ol (DAMGO) and theδ agonist D‐Pen2‐D‐Pen5‐enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1–13, U69593 and (−)‐EKC on NMDA‐induced [3H]‐dopamine release were not changed by the δ1/δ2‐opioid receptor antagonist naltrindole (up to a concentration of 1 μM), but reversed by the κ receptor antagonist nor‐binaltorphimine (nor‐BNI), with an IC50 as low as 0.02 nM, indicating the involvement of U69593‐sensitive κ1‐opioid receptors.

NMDA‐induced [14C]‐ACh release was reduced in a naloxone‐reversible manner by DPDPE (pD2 about 7.2), dynorphin A1–13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1–13 and (−)‐EKC, on NMDA‐induced [14C]‐ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of δ‐opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1–13 and (−)‐EKC on [14C]‐ACh release were readily antagonized by nor‐BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor‐BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593‐insensitive κ2‐opioid receptors in the inhibitory effects of dynorphin A1–13 and (−)‐EKC.

Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative κ3‐opioid receptor, antagonized the inhibitory effects of dynorphin A1–13 and (−)‐EKC on [3H]‐dopamine and [14C]‐ACh release as well as that of U69593 on [3H]‐dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case.

In conclusion, whereas activation of κ1‐opioid receptors causes presynaptic inhibition of NMDA‐induced dopamine release, κ2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct κ‐opioid receptor subtypes in the brain.

British Journal of Pharmacology (1997) 122, 520–524; doi:10.1038/sj.bjp.0701394
RPR 106541, a novel, airways‐selective glucocorticoid: effects against antigen‐induced CD4+T lymphocyte accumulation and cytokine gene expression in the Brown Norway rat lung
Authors: Underwood S. L. , Raeburn D. , Lawrence C. , Foster M. , Webber S. , Karlsson J. A.
Published: January 2005

The effects of a novel 17‐thiosteroid, RPR 106541, were investigated in a rat model of allergic airway inflammation.

In sensitized Brown Norway rats, challenge with inhaled antigen (ovalbumin) caused an influx of eosinophils and neutrophils into the lung tissue and airway lumen. In the lung tissue there was also an accumulation of CD4+ T lymphocytes and increased expression of mRNA for interleukin‐4 (IL‐4) and IL‐5, but not interferon‐γ (IFN‐γ). These findings are consistent with an eosinophilia orchestrated by activated Th2‐type cells.

RPR 106541 (10–300 μg kg−1), administered by intratracheal instillation into the airways 24 h and 1 h before antigen challenge, dose‐dependently inhibited cell influx into the airway lumen. RPR 106541 (100 μg kg−1) caused a significant (P<0.01) (98%) inhibition of eosinophil influx and a significant (P<0.01) (100%) inhibition of neutrophil influx. RPR 106541 was approximately 7 times and 4 times more potent than budesonide and fluticasone propionate, respectively.

When tested at a single dose (300 μg kg−1), RPR 106541 and fluticasone each caused a significant (P<0.01) (100%) inhibition of CD4+ T cell accumulation in lung tissue. Budesonide (300 μg kg−1) had no significant effect. RPR 106541 and fluticasone (300 μg kg−1), but not budesonide (300 μg kg−1), significantly (P<0.05) inhibited the expression within lung tissue of mRNA for IL‐4. RPR 106541 (300 μg kg−1) also significantly (P<0.05) inhibited expression of mRNA for IL‐5.

The high topical potency of RPR 106541 in this model, which mimics important aspects of airway inflammation in human allergic asthmatics, suggests that this glucocorticoid may be useful in the treatment of bronchial asthma.

British Journal of Pharmacology (1997) 122, 439–446; doi:10.1038/sj.bjp.0701398
Comparison of the acute cardiotoxicity of the antimalarial drug halofantrine in vitro and in vivo in anaesthetized guinea‐pigs
Authors: Batey Andrew J. , Lightbown Ian D. , Lambert James P. , Edwards Geoffrey , Coker Susan J.
Published: January 2005

Several unrelated drugs have pro‐arrhythmic activity associated with an ability to prolong the QT interval of the ECG. The aim of this work was to examine the effects of the antimalarial drug halofantrine in vivo and in vitro.

In anaesthetized guinea‐pigs consecutive bolus doses of halofantrine (0.3, 1, 3, 10 and 30 mg kg−1, i.v.) at 25 min intervals caused dose‐dependent prolongation of the rate corrected QTc interval and bradycardia. The change in heart rate became significant after administration of 10 mg kg−1 halofantrine (−23±9 beats min−1) whereas the increase in QTc was significant with only 1 mg kg−1 halofantrine (22±10 ms). It was only with the highest dose of halofantrine that the PR interval was increased (from 52±3 to 67±4 ms) and second degree atrioventricular (AV) block (type 1 Mobitz) occurred in all animals. No changes were observed in any parameters in a separate group of guinea‐pigs which received vehicle (dimethylacetamide 60% propylene glycol 40%) at equivalent time points.

The blood concentrations of halofantrine ranged from 0.26±0.17 μM after administration of 0.3 mg kg−1 to 2.79±0.87 μM after 30 mg kg−1, i.v. There was a significant correlation between the blood concentrations of halofantrine and the changes in QTc interval.

In guinea‐pig left papillary muscles the effective refractory period was increased significantly 60 min after addition of halofantrine; from 161±4 to 173±6 ms with 10 μM, 156±8 to 174±6 ms with 30 μM and 165±6 to 179±5 ms with 100 μM halofantrine. However, the vehicle (0.1% Tween 80 in DMSO; final concentration of vehicle in Krebs, 1%) also increased the effective refractory period from 164±5 to 173±6 ms. Similar results were obtained in right ventricular strips but left atrial effective refractory periods were not altered by either the vehicle or halofantrine.

The results of these experiments suggest that any direct effects that halofantrine may have had on the effective refractory period of cardiac muscle cannot be separated from those of the vehicle. The prolongation of QTc and consistent observation of AV block with halofantrine in anaesthetized guinea‐pigs suggest that in vivo models may be more useful for further studies investigating the mechanisms underlying the cardiotoxicity of halofantrine.

British Journal of Pharmacology (1997) 122, 563–569; doi:10.1038/sj.bjp.0701402
Nitric oxide effects on cell growth: GMP‐dependent stimulation of the AP‐1 transcription complex and cyclic GMP‐independent slowing of cell cycling
Authors: Sciorati Clara , Nisticò Giuseppe , Meldolesi Jacopo , Clementi Emilio
Published: January 2005

The role of nitric oxide (NO) in the control of cell growth is controversial since both stimulation and (more often) inhibition have been demonstrated in various cell types. In order to reinvestigate the problem and identify the sites of NO action, we have employed murine NIH‐3T3 fibroblasts overexpressing epidermal growth factor (EGF) receptors.

The effects of four structurally‐unrelated NO donors: S‐nitroso‐N‐acetyl penicillamine, S‐nitroso‐L‐glutathione, 3‐morpholinosydnonimine and isosorbide dinitrate (0.01–3 mM) on EGF (10 nM)‐stimulated cell growth were estimated by both thymidine incorporation and the colorimetric MTT assay, while those of a messenger generated in response to NO, cyclic GMP, were revealed by the use of 8‐Br cyclic GMP (0.01–3 mM) as well as of blockers of guanylyl cyclase and cyclic GMP‐dependent kinase I.

Studies were focused on: (i) multiple signalling events, including receptor‐induced tyrosine phosphorylations, phosphorylation of mitogen‐activated protein kinase, activation of the AP‐1 transcription complex and deoxyribonucleotide synthesis; (ii) the progression through the cell cycle, dissected out by the use of staurosporine (1 nM), lovastatin (10 μM), mimosine (200 μM), hydroxyurea (1 mM) and nocodazole (1.5 μM).

NO was found to have no effects on the phosphorylation events of the growth factor cascade. In contrast, later processes were modified by the messenger but with opposite effects.

A cyclic GMP‐dependent stimulation of growth was shown to be sustained in part by the activation of the AP‐1 transcription complex, while a predominant, cyclic GMP‐independent inhibition was found to be mediated by both the negative regulation of ribonucleotide reductase and the marked slowing down of the cell cycle occurring at early and late G1 and during the S phase.

Although multiple and apparently conflicting, the effects of NO here described could work coordinately in a general programme of cell growth regulation. In particular, the cyclic GMP‐dependent actions might function as rapid modulatory events, while the effects on cell cycle might operate collectively as a multi‐switch process whenever growth inhibition is required.

British Journal of Pharmacology (1997) 122, 687–697; doi:10.1038/sj.bjp.0701413
Effects of ibuprofen enantiomers and its coenzyme A thioesters on human prostaglandin endoperoxide synthases
Authors: Neupert Werner , Brugger Roland , Euchenhofer Christian , Brune Kay , Geisslinger Gerd
Published: January 2005

Ibuprofen enantiomers and their respective coenzyme A thioesters were tested in human platelets and blood monocytes to determine their selectivity and potency as inhibitors of cyclo‐oxygenase activity of prostaglandin endoperoxide synthase‐1 (PGHS‐1) and PGHS‐2.

Human blood from volunteers was drawn and allowed to clot at 37°C for 1 h in the presence of increasing concentrations of the test compounds (R‐ibuprofen, S‐ibuprofen, R‐ibuprofenoyl‐CoA, S‐ibuprofenoyl‐CoA, NS‐398). Immunoreactive (ir) thromboxane B2 (TXB2) concentrations in serum were determined by a specific EIA assay as an index of the cyclo‐oxygenase activity of platelet PGHS‐1.

Heparin‐treated blood from the same donors was incubated at 37°C for 24 h with the same concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 μg ml−1). The contribution of PGHS‐1 was suppressed by pretreatment of the volunteers with aspirin (500 mg; 48 h before venepuncture). As a measure of LPS induced PGHS‐2 activity immunoreactive prostaglandin E2 (irPGE2) plasma concentrations were determined by a specific EIA assay.

S‐ibuprofen inhibited the activity of PGHS‐1 (IC50 2.1 μM) and PGHS‐2 (IC50 1.6 μM) equally. R‐ibuprofen inhibited PGHS‐1 (IC50 34.9) less potently than S‐ibuprofen and showed no inhibition of PGHS‐2 up to 250 μM. By contrast R‐ibuprofenoyl‐CoA thioester inhibited PGE2 production from LPS‐stimulated monocytes almost two orders of magnitude more potently than the generation of TXB2 (IC50 5.6 vs 219 μM).

Western blotting of PGHS‐2 after LPS induction of blood monocytes showed a concentration‐dependent inhibition of PGHS‐2 protein expression by ibuprofenoyl‐CoA thioesters.

These data confirm that S‐ibuprofen represents the active entity in the racemate with respect to cyclo‐oxygenase activity. More importantly the data suggest a contribution of the R‐enantiomer to therapeutic effects not only by chiral inversion to S‐ibuprofen but also via inhibition of induction of PGHS‐2 mediated by R‐ibuprofenoyl‐CoA thioester.

The data may explain why racemic ibuprofen is ranked as one of the safest non‐steroidal anti‐inflammatory drugs (NSAIDs) so far determined in epidemiological studies.

British Journal of Pharmacology (1997) 122, 487–492; doi:10.1038/sj.bjp.0701415
Localization of Fos‐like immunoreactivity induced by the NK3 tachykinin receptor agonist, senktide, in the guinea‐pig brain
Authors: Yip Jane , Chahl Loris A.
Published: January 2005

The effects of intracerebroventricular (i.c.v.) administration of the NK3 tachykinin receptor agonist, senktide (10 nmol each side), in guinea‐pigs pretreated with the selective NK3 tachykinin receptor antagonist, SR142801 (3 mg kg−1 subcutaneous, s.c., 30 min before senktide), or its less active enantiomer, SR142806 (3 mg kg−1 s.c. 30 min before senktide), on behaviour and on the distribution of Fos‐like immunoreactivity (Fos‐LI) in central neurones were investigated. Guinea‐pigs were chosen for the study since they possess NK3 tachykinin receptors with pharmacological characteristics similar to those in man.

Wet‐dog shakes, but not locomotor activity, elicited by senktide i.c.v. were significantly reduced by SR142801 but not by SR142806, confirming the involvement of NK3 tachykinin receptors in wet‐dog shake behaviour.

Senktide induced increased numbers of Fos‐LI neurones in the following brain areas: frontal, parietal and piriform cortex, the lateral septum, the CA1, CA2, subiculum and dentate gyrus of the hippocampus, most areas in the amygdala, thalamus and hypothalamus, medial geniculate nucleus and the ventral cochlear nucleus. Pretreatment with SR142801, but not with SR142806, before administration of senktide inhibited Fos‐LI expression in the cingulate cortex, dentate gyrus of the hippocampus, some regions of the thalamus, hypothalamus and amygdala and the ventral cochlear nucleus.

The present results are the first demonstration that senktide induces Fos‐LI in widespread areas of the guinea‐pig brain. It is proposed that NK3 tachykinin receptors may play a more extensive role in the control of diverse brain functions, including cortical processing, learning and memory, neuroendocrine and behavioural regulation, than is currently recognized.

British Journal of Pharmacology (1997) 122, 715–725; doi:10.1038/sj.bjp.0701416
Modulation of a recombinant invertebrate γ‐aminobutyric acid receptor‐chloride channel complex by isoflurane: effects of a point mutation in the M2 domain
Authors: Edwards Michelle D. , Lees George
Published: January 2005

Inhalational anaesthetics modulate ligand‐gated ion channels at clinical concentrations. In this paper we address submolecular mechanisms for γ‐aminobutyric acid (GABA) receptor modulation by isoflurane.

Wild‐type Drosophila melanogaster homo‐oligomeric GABA receptors were characterized and compared with an ion‐channel mutant (alanine substituted to a serine in M2) by means of two‐electrode voltage‐clamp in membrane‐invariant Xenopus oocytes.

Both channel receptor isoforms generated outwardly rectifying, bicuculline‐insensitive currents with reversal potentials characteristic of a chloride current.

As previously shown, the point mutation in the M2 domain conferred a profound resistance to the blocking action of 10 μM picrotoxinin (PTX): circa 7 fold reduction at the GABA EC20.

Isoflurane, 195–389 μM, enhanced GABA conductance in both receptor variants by significantly increasing the affinity of the agonist for its receptor without changing Hill slope or maximal response. Relative potencies were statistically indistinguishable.

Isoflurane concentration‐response curves (on circa GABA EC25) demonstrated that enhancement was effected at around 100–195 μM for both receptor subtypes, but a dramatic divergence was evident at concentrations above 400 μM: wild‐type receptors exhibited concentration‐dependent block, whilst mutant conductances continued to increase over the same concentration range, showing no tendency to saturate (up to 3330 μM).

The above divergence was not attributable to differential desensitization: neither wild‐type nor mutant conductance desensitized significantly (P>0.05) in the absence or presence of anaesthetic.

This work demonstrates that modulatory sites for anaesthetic are present on a relatively primitive insect ion channel.

The depression of GABA response at high isoflurane concentrations, in WT receptors, (typical of a variety of anaesthetic agents) may reflect low affinity channel block via the PTX site.

The non‐saturable enhancement of chloride conductances, when the PTX site is mutated, is not consistent with topical proposals that inhalational anaesthetics (stereoselectively) occupy a finite number of sites on these membrane spanning proteins.

British Journal of Pharmacology (1997) 122, 726–732; doi:10.1038/sj.bjp.0701417
Effect of L‐lysine on nitric oxide overproduction in endotoxic shock
Authors: Liaudet Lucas , Gnaegi Alex , Rosselet Anne , Markert Michèle , Boulat Olivier , Perret Claude , Feihl François
Published: January 2005

An enhanced production of nitric oxide (NO) from L‐arginine, related to the diffuse expression of an inducible NO synthase (iNOS), contributes to the pathogenesis of endotoxic shock. Since iNOS activity depends on extracellular L‐arginine, we hypothesized that limiting cellular L‐arginine uptake would reduce NO production in endotoxic shock. We investigated the effects of L‐lysine, an inhibitor of L‐arginine uptake through system y+, on NO production, multiple organ dysfunction and lactate levels, in normal and endotoxaemic rats.

Anaesthetized rats challenged with intravenous lipopolysaccharide (LPS, 10 mg kg−1) received a 5 h infusion of either L‐lysine (500 μmol kg−1 h−1, n=12) or isotonic saline (2 ml kg−1 h−1, n=11). In rats treated with saline, LPS produced a large increase in plasma nitrate and L‐citrulline concentrations at 5 h, both markers of enhanced NO production. LPS also caused severe hypotension, low cardiac output and marked hyperlactataemia. All these changes were significantly reduced byL‐lysine administration.

Endotoxaemia also caused a significant rise in the plasma levels of alanine aminotransferase (ALAT), lipase, urea and creatinine, and hence, liver, pancreatic and renal dysfunction. These changes tended to be less pronounced in rats treated with L‐lysine, although the differences did not reach statistical significance.

Similar experiments were conducted in 10 rats challenged with LPS vehicle in place of LPS and then treated with L‐lysine (500 μmol kg−1  h−1, n=5) or saline (2 ml kg−1  h−1, n=5) for 5 h. In these animals, all the haemodynamic and metabolic variables remained stable and not statistically different between both treatment groups, except for a slight rise in ALAT, which was comparable in L‐lysine and saline‐treated rats.

In conclusion, L‐lysine, an inhibitor of cellular L‐arginine uptake, reduces NO production and exerts beneficial haemodynamic effects in endotoxaemic rats. L‐lysine also reduces hyperlactataemia and tends to blunt the development of organ injury in these animals. Contrastingly, L‐lysine has no effects in the absence of endotoxin and thus appears to act as a selective modulator of iNOS activity.

British Journal of Pharmacology (1997) 122, 742–748; doi:10.1038/sj.bjp.0701419
Differential regulation of cyclo‐oxygenase‐2 and 5‐lipoxygenase‐activating protein (FLAP) expression by glucocorticoids in monocytic cells
Authors: Goppelt‐Struebe M. , Schaefer D. , Habenicht A. J. R.
Published: January 2005

The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo‐oxygenase‐2 (COX‐2) and 5‐lipoxygenase activating protein (FLAP). The human monocytic cell line THP‐1 was used as a model system. mRNA and protein levels of COX‐2 and FLAP were determined by Northern and Western blot analyses, respectively.

Low levels of COX‐2 and FLAP mRNA were expressed in undifferentiated THP‐1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days.

Coincubation of the undifferentiated cells with dexamethasone (10−9–10−6 M) and phorbol ester prevented induction of COX‐2 mRNA, but did not affect the induction of FLAP mRNA.

Dexamethasone downregulated COX‐2 mRNA and protein in differentiated, monocyte‐like THP‐1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP‐1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5‐lipoxygenase mRNA expression.

Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP‐1 cells by ELISA. Calcium ionophore‐dependent PGE2 synthesis was associated with COX‐2 expression, whereas COX‐1 and COX‐2 seemed to participate in arachidonic acid‐dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP‐1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release.

These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid‐mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5‐lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

British Journal of Pharmacology (1997) 122, 619–624; doi:10.1038/sj.bjp.0701425