bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that nitric oxide acts as an inhibitory neurotransmitter supplying taenia from the guinea‐pig caecum
Authors: Shuttleworth C W R , Sweeney K M , Sanders K M
Published: January 2005

Nitric oxide synthase‐containing nerve fibres are abundant within taenia of the guinea‐pig caecum, but there is little previous evidence supporting a direct role for nitric oxide (NO) in responses to enteric inhibitory nerve stimulation. In this study we have attempted to identify an NO‐dependent component of inhibitory transmission in isolated taenia coli.

Isometric tension was recorded in the presence of atropine and guanethidine (both 1 μM). Tone was raised with histamine (1 μM), and intrinsic inhibitory neurons stimulated using either a nicotinic agonist (1,1‐dimethyl‐4‐phenylpiperazinium iodide; DMPP) or electrical field stimulation (EFS).

DMPP (1–100 μM) produced concentration‐dependent biphasic relaxations, comprising an initial peak relaxation followed by a sustained relaxation. Responses to DMPP were antagonized by tetrodotoxin (1 μM) or apamin (0.3 μM) and abolished by hexamethonium (300 μM). L‐nitro‐arginine (L‐NOARG; 100 μM) and oxyhaemoglobin (2%) both significantly reduced sustained relaxations produced by DMPP.

EFS (5 Hz, 30 s) also produced biphasic relaxations. Both L‐NOARG and an inhibitor of soluble guanylate cyclase (ODQ, 1–10 μM) reduced the sustained component of EFS responses.

Two NO donors, sodium nitroprusside (SNP) and diethylenetriamine‐nitric oxide adduct (DENO), produced concentration‐dependent relaxations. Responses to SNP and DENO were antagonized by ODQ (1 μM) and by apamin (0.3 μM).

These results suggest that NO contributes directly to a component of inhibitory transmission in guinea‐pig taenia coli. The actions of NO appear to be mediated via cyclic GMP synthesis, and may involve activation of small conductance calcium activated K+ channels. A role for NO is most evident during sustained relaxations evoked by longer stimulus trains or chemical stimulation of intrinsic neurons.

British Journal of Pharmacology (1999) 127, 1495–1501; doi:10.1038/sj.bjp.0702674
Evaluation of α1‐adrenoceptors in the rabbit iris: pharmacological characterization and expression of mRNA
Authors: Nakamura Seigo , Taniguchi Takanobu , Suzuki Fumiko , Akagi Yoshio , Muramatsu Ikunobu
Published: January 2005

Subtypes of α1‐adrenoceptor in rabbit iris have been examined in functional, binding and molecular biological experiments.

In functional studies, exogenous and endogenous noradrenaline produced contractions of the iris dilator muscle. The contractile responses to noradrenaline were competitively antagonized by a range of α1‐adrenoceptor antagonists (pA2 values): prazosin (8.1), WB4101 (8.2), BMY7378 (5.9), YM617 (9.5), JTH‐601 (8.8), HV723 (7.8) and KMD‐3213 (9.8). The same order of inhibitory potency was seen in the adrenergic responses to electrical stimulation. This affinity profile corresponds well to that of the putative α1L‐adrenoceptor, which has been proposed in lower urinary tract tissues.

In binding studies on rabbit iris membrane however, prazosin, KMD‐3213 and WB4101 displayed high affinity (pKd or pKi: 9.6, 10.3, 9.6, respectively), and BMY7378 displayed low affinity (pKi: 6.9). These results show that the binding sites typically correspond to α1A‐adrenoceptor subtype in character, and we could not detect the significant amount of α1L‐adrenoceptor subtype.

The expression of the three distinct mRNAs that encode proteins of α1a‐, α1b‐ and α1d‐adrenoceptors was studied using reverse transcription‐polymerase chain reaction (RT–PCR). RT–PCR demonstrated the strongest expression of the α1a‐adrenoceptor, weak expression of the α1b‐ adrenoceptor and undetectable expression of the α1d‐adrenoceptor.

The present study suggests that α1A‐adrenoceptor is a major subtype detectable in binding and RT–PCR studies in rabbit iris, but that the adrenergic contractions of iris dilator muscle are mediated via activation of α1‐adrenoceptor subtype having low affinity for prazosin and WB4101.

British Journal of Pharmacology (1999) 127, 1367–1374; doi:10.1038/sj.bjp.0702675
Expression of immediate early genes, GATA‐4, and Nkx‐2.5 in adrenergic‐induced cardiac hypertrophy and during regression in adult mice
Authors: Saadane Nacéra , Alpert Lesley , Chalifour Lorraine E
Published: January 2005

Adrenoreceptor agonists induce a hypertrophic phenotype in vitro and in vivo. To investigate the molecular remodeling in chronic cardiac hypertrophy we infused adult male mice with vehicle, isoproterenol, phenylephrine or both agonists for 3, 7 or 14 days.

All drugs increased cardiac mass. After minipump removal cardiac mass regressed to control levels within 7 days after PE and ISO treatment whereas ISO+PE treated hearts were incompletely regressed.

ANF and β‐MHC, but not α‐MHC, expression were increased by agonists at all time points. GATA‐4, Nkx‐2.5, Egr‐1, c‐jun and c‐fos expression were increased after 3, 7 and 14 days of treatment. Expression was greatest after ISO+PE>>ISO>PE>vehicle infusion suggesting a synergistic effect of adrenoreceptor stimulation and indicating a greater effect of β‐ than α‐adrenergic action in vivo.

After PE or ISO drug withdrawal the HW/BW was normal and Egr‐1, c‐jun, c‐fos and GATA‐4, but not Nkx2.5, expression dropped to control levels. HW/BW regression was incomplete after ISO+PE and elevated levels of Egr‐1, c‐jun and Nkx2.5 expression remained.

A hydralazine‐mediated reduction in blood pressure had no effect on the agonist‐induced cardiac hypertrophy or gene expression.

In conclusion, we found that continued agonist stimulation, and not blood pressure, is responsible for the maintained increase in gene expression. Further, we found the decrease in gene expression in the regression after drug withdrawal was gene specific.

British Journal of Pharmacology (1999) 127, 1165–1176; doi:10.1038/sj.bjp.0702676
Protection by imidazol(ine) drugs and agmatine of glutamate‐induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor
Authors: Olmos Gabriel , DeGregorio‐Rocasolano Nuria , Regalado M Paz , Gasull Teresa , Boronat M Assumpció , Trullas Ramón , Villarroel Alvaro , Lerma Juan , García‐Sevilla Jesús A
Published: January 2005

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate‐induced necrosis and on apoptosis induced by low extracellular K+in cultured cerebellar granule cells.

Exposure (30 min) of energy deprived cells toL‐glutamate (1–100 μM) caused a concentration‐dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide (MTT) into a reduced formazan product.L‐glutamate‐induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK‐801 (dizocilpine).

Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM(EC100)L‐glutamate with the rank order (EC50in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2‐styryl‐2‐imidazoline] (54)>LSL 60101 [2‐(2‐benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2‐(1,4‐benzodioxan‐6‐yl)‐4,5‐dihydroimidazole] (101)>RX821002 (2‐methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors.

Imidazol(ine) drugs and agmatine fully inhibited [3H]‐(+)‐MK‐801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting againstL‐glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]‐(+)‐MK‐801 binding.

In HEK‐293 cells transfected to express the NR1‐1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage‐ and concentration‐dependent block of glutamate‐induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50of 10–12 μMat 0 mV.

It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate‐induced necrotic neuronal cell deathin vitroand that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.

British Journal of Pharmacology(1999)127, 1317–1326; doi:10.1038/sj.bjp.0702679
Presynaptic inhibition preferentially reduces the NMDA receptor‐mediated component of transmission in rat midbrain dopamine neurons
Authors: Wu Yan‐Na , Shen Ke‐Zhong , Johnson Steven W
Published: January 2005

We used patch pipettes to record whole‐cell currents from single dopamine neurons in slices of rat midbrain. Pharmacological methods were used to isolate EPSCs evoked by focal electrical stimulation.

Baclofen was significantly more potent for inhibiting NMDA receptor‐mediated EPSCs (IC50=0.24 μM) compared with inhibition of EPSCs mediated by AMPA receptors (IC50=1.72 μM). The increased potency of baclofen for inhibiting the NMDA component persisted in superfusate that contained zero Mg2+ and when postsynaptic K+ conductances were reduced by Cs+ and QX‐314. Effects of baclofen on EPSCs were blocked by the GABAB receptor antagonist CGP‐35348.

Adenosine was 20 fold more potent for reducing the NMDA component of transmission (IC50=31 μM) compared with inhibition of AMPA receptor‐mediated EPSCs (IC50=654 μM). Effects of adenosine on EPSCs were blocked by the A1 receptor antagonist DPCPX.

Both baclofen and adenosine significantly increased the ratio of EPSCs in paired‐pulse studies, suggesting presynaptic sites of action. Although adenosine (1 mM) did not reduce currents evoked by exogenous NMDA (10 μM), baclofen (1 μM) reduced NMDA currents by 29%. Neither baclofen nor adenosine altered currents evoked by exogenous AMPA (1 μM).

We conclude that adenosine acts at presynaptic A1 receptors to cause a preferential reduction in the NMDA component of synaptic transmission. In contrast, baclofen preferentially reduces NMDA EPSCs by acting at both pre‐ and postsynaptic GABAB receptors. By regulating NMDA receptor function, A1 and GABAB receptors may play important roles in regulating the excitability of dopamine neurons.

British Journal of Pharmacology (1999) 127, 1422–1430; doi:10.1038/sj.bjp.0702680
Pharmacological characterization of nicotine‐induced acetylcholine release in the rat hippocampus in vivo: evidence for a permissive dopamine synapse
Authors: Reid Richard T , Lloyd G Kenneth , Rao Tadimeti S
Published: January 2005

In this study, the mechanism of nicotine‐induced hippocampal acetylcholine (ACh) release in awake, freely moving rats was examined using in vivo microdialysis.

Systemic administration of nicotine (0.4 mg kg−1, s.c.) increased the levels of ACh in hippocampal dialysates.

The nicotine‐induced hippocampal ACh release was sensitive to the pretreatment of neuronal nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine (3.0 mg kg−1, s.c.) and dihydro‐β‐erythrodine (DHβE; 4.0 mg kg−1, s.c.) as well as systemic administration of the dopamine (DA) D1 receptor antagonist SCH‐23390 (R‐(+)‐7‐chloro‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐benzazepine; 0.3 mg kg−1, s.c.).

Local perfusion of mecamylamine (100 μM), DHβE (100 μM) or SCH‐23390 (10 μM) through microdialysis probe did not increase basal hippocampal ACh release.

Hippocampal ACh release elicited by systemic administration of nicotine (0.4 mg kg−1, s.c.) was antagonized by local perfusion of SCH‐23390 (10 μM), but not by MEC (100 μM) or DHβE (100 μM).

Direct perfusion of nicotine (1 mM, but not 0.1 mM) increased hippocampal ACh levels; however, this effect was relatively insensitive to blockade by co‐perfusion of either mecamylamine (100 μM) or SCH‐23390 (10 μM).

These results suggest that nicotine‐induced hippocampal ACh release occurs by two distinct mechanisms: (1) activation of nAChRs outside the hippocampus leading to DA release and subsequent ACh release involving a permissive DA synapse, and (2) direct action of nicotine within the hippocampus leading to ACh release via non‐DA‐ergic mechanism.

British Journal of Pharmacology (1999) 127, 1486–1494; doi:10.1038/sj.bjp.0702683
Alternative splicing generates two isoforms of the β3‐adrenoceptor which are differentially expressed in mouse tissues
Authors: Evans B A , Papaioannou M , Hamilton S , Summers R J
Published: January 2005

The β3‐adrenoceptor (AR) differs from the β1‐AR and β2‐ARs in having introns within and downstream of the coding block. This study demonstrates two splice variants of the mouse β3‐AR which differ within the coding region.

Reverse transcription/polymerase chain reaction with intron‐spanning primers was used to demonstrate the splice variant of the mouse β3‐adrenoceptor. The novel β3b‐AR has 17 amino acids encoded by exon 2 (SSLLREPRHLYTCLGYP) which differ from the 13 in the known β3a‐AR (RFDGYEGARPFPT).

β3b‐AR mRNA is differentially expressed in mouse tissues, with levels relative to β3a‐AR mRNA highest in hypothalamus, cortex and white adipose tissue, and lower in ileum smooth muscle and brown adipose tissue.

British Journal of Pharmacology (1999) 127, 1525–1531; doi:10.1038/sj.bjp.0702688
IL‐12 as a therapeutic target for pharmacological modulation in immune‐mediated and inflammatory diseases: regulation of T helper 1/T helper 2 responses
Authors: Haskó György , Szabó Csaba
Published: January 2005

Interleukin‐12 (IL‐12) is a pivotal cytokine in driving the immune system towards a T helper (Th)1 type response and preventing a Th2 type immune profile. Therefore, IL‐12 is indispensable in the defense against certain, mainly intracellular pathogens, but overproduction of this cytokine is crucially involved in the etiology of several inflammatory and autoimmune diseases.

Hence, IL‐12 is an ideal target for pharmacological intervention in the therapy of autoimmune and inflammatory diseases.

The production of IL‐12 and a resultant Th1 type immune response can be suppressed with several pharmacological approaches including modulation of intracellular cyclic AMP levels, glucocorticoids and nuclear factor‐κB inhibition. IL‐12 responsiveness may be inhibited using anti‐IL‐12 antibodies, soluble IL‐12 receptors or the IL‐12 p40 homodimer.

Exploitation of these approaches may provide novel means for the experimental therapy of a variety of pathophysiological states.

British Journal of Pharmacology (1999) 127, 1295–1304; doi:10.1038/sj.bjp.0702689
Differential blockade of rat α3β4 and α7 neuronal nicotinic receptors by ω‐conotoxin MVIIC, ω‐conotoxin GVIA and diltiazem
Authors: Herrero Carlos J , García‐Palomero Esther , Pintado Antonio J , García Antonio G , Montiel Carmen
Published: January 2005

Rat α3β4 or α7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non‐toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 μM) applied at regular intervals.

The N/P/Q‐type Ca2+ channel blocker ω‐conotoxin MVIIC inhibited α3β4 currents with an IC50 of 1.3 μM; the blockade was non‐competitive and reversible. The α7 currents were unaffected.

At 1 μM, ω‐conotoxin GVIA (N‐type Ca2+ channel blocker) inhibited by 24 and 20% α3β4 and α7 currents, respectively. At 1 μM, ω‐agatoxin IVA (a P/Q‐type Ca2+ channel blocker) did not affect α7 currents and inhibited α3β4 currents by only 15%.

L‐type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on α3β4 nicotinic AChRs.

The mechanism of α3β4 currents blockade by ω‐conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage‐dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while ω‐toxins did not.

These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.

British Journal of Pharmacology (1999) 127, 1375–1387; doi:10.1038/sj.bjp.0702692
Characterization of the muscarinic receptor in isolated uterus of sham operated and ovariectomized rats
Authors: Choppin A , Stepan G J , Loury D N , Watson N , Eglen R M
Published: January 2005

The pharmacological characteristics of muscarinic receptors in rat isolated uterus were studied in ovariectomized (ov.) and sham operated (sh.) animals.

Competition radioligand binding studies, using uterine membranes and [3H]‐NMS, were undertaken with several muscarinic receptor antagonists. Most of the antagonists indicated a one‐site fit with apparent affinity estimates (pKi) unchanged by ovariectomy. The selective M2 antagonist, tripitramine revealed high (representing 33±8 and 38±2%) and low (67±8 and 62±2%) affinity binding sites in both sh. and ov. rat uterus, respectively. These sites likely represented muscarinic M2 and M3 receptors and the proportions were not significantly different in the two conditions.

Carbachol induced concentration‐dependent contractions which were surmountably antagonized by several muscarinic receptor antagonists (pKB, sh.; ov.): zamifenacin (9.19; 9.18), p‐F‐HHSiD (8.50; 9.06), tripitramine (7.23; 7.54), himbacine (7.21; 7.41), methoctramine (6.79; 7.49), pirenzepine (6.48; 7.21), AF DX 116 (6.26; 6.61), MTx 3 (<7.00; <7.00) and PD 102807 (<7.00; <7.00).

The apparent affinity values obtained in functional studies using the uteri from both sh. and ov. animals correlated most closely with values reported at human recombinant muscarinic M3 receptors. This suggests that the muscarinic M3 receptor mediates contraction under both conditions.

Radioligand binding experiments indicate the presence of M2 receptors, in addition to M3 receptors, which probably explains the discrepancies between functional and binding affinities. These data further suggest that the pharmacological profile and proportions of the two populations of muscarinic receptors are unaffected by ovariectomy.

British Journal of Pharmacology (1999) 127, 1551–1558; doi:10.1038/sj.bjp.0702696
Central control of blood pressure by nitrergic mechanisms in organum vasculosum laminae terminalis of rat brain
Authors: Lin M ‐T , Pan S ‐P , Lin J ‐H , Yang Y ‐L
Published: January 2005

Experiments were carried out to explore the possible role played by the nitric oxide (NO) system in the organum vasculosum laminae terminalis (OVLT) of rat brain in arterial pressure regulation.

Intracerebroventricular (ICV) or intra‐OVLT administration of NO donors such as hydroxylamine, sodium nitro‐prusside or s‐nitro‐acetylpenicillamine caused an up to 55 mmHg decrease in blood pressure (BP) but an increase in NO release (measured by porphyrin/nafion coated carbon fibre electrodes in combination with voltammetry) in the OVLT. In contrast, ICV or intra‐OVLT administration of NG‐nitro‐L‐arginine methyl ester (L‐NAME; a constitutive NO synthase inhibitor) caused an up to 45 mmHg increase in BP but a fall in NO release in the OVLT.

Compared with the BP responses induced by ICV injection of NO donors or NO synthase inhibitors, the OVLT route of injection required a much lower dose of NO donors or NO synthase inhibitors to produce a similar BP effect.

The depressor effects induced by ICV or intra‐OVLT administration of NO donors were attenuated by pretreatment with intra‐OVLT injection of methylene blue (an inhibitor of guanylate cyclase), haemoglobin (a NO scavenger), L‐NAME or spinal transection. On the other hand, the L‐NAME‐induced pressor effects were attenuated by pretreatment with intra‐OVLT injection of L‐arginine or spinal transection.

The data suggest that activation of cyclic GMP‐dependent NO synthase in the OVLT of rat brain causes cyclic GMP‐dependent decreases in arterial pressure via inhibiting the sympathetic efferent activity.

British Journal of Pharmacology (1999) 127, 1511–1517; doi:10.1038/sj.bjp.0702699
KATP channels and ‘border zone’ arrhythmias: role of the repolarization dispersion between normal and ischaemic ventricular regions
Authors: Picard Sandra , Rouet René , Ducouret Pierre , Puddu Paolo Emilio , Flais Frédéric , Criniti Anna , Monti Francesco , Gérard Jean‐Louis
Published: January 2005

In order to investigate the role of KATP channel activation and repolarization dispersion on the ‘border zone’ arrhythmias induced by ischaemia‐reperfusion, the effects of glibenclamide and bimakalim, agents modifying action potential (AP) duration, were studied in an in vitro model of myocardial ‘border zone’.

The electrophysiological effects of 10 μM glibenclamide and 1 μM bimakalim (n=8 each), respectively KATP channel blocker and activator, were investigated on guinea‐pig ventricular strips submitted partly to normal conditions (normal zone, NZ) and partly to simulated ischaemic then reperfused conditions (altered zone, AZ).

By preventing the ischaemia‐induced AP shortening (P<0.0001), glibenclamide reduced the dispersion of AP duration 90% (APD90) between NZ and AZ (P<0.0001), and concomitantly inhibited the ‘border zone’ arrhythmias induced by an extrastimulus (ES), their absence being significantly related to the lessened APD90 dispersion (χ2=8.28, P<0.01).

Bimakalim, which also reduced the APD90 dispersion (P<0.005) due to differential AP shortening in normal and ischaemic tissues, decreased the incidence of myocardial conduction blocks (25% of preparations versus 83% in control, n=12, P<0.05) and favoured ‘border zone’ spontaneous arrhythmias (75% of preparations versus 25% in control, P<0.05).

During reperfusion, unlike bimakalim, glibenclamide inhibited the ES‐induced arrhythmias and reduced the incidence of the spontaneous ones (12% of preparations versus 92% in control, P<0.05), this latter effect being significantly related (χ2=6.13, P<0.02) to the lessened ischaemia‐induced AP shortening in the presence of glibenclamide (P<0.0001).

These results suggest that KATP blockade may protect the ischaemic‐reperfused myocardium from ‘border zone’ arrhythmias concomitantly with a reduction of APD90 dispersion between normal and ischaemic regions. Conversely, KATP channel activation may modify the incidence of conduction blocks and exacerbate the ischaemia‐induced ‘border zone’ arrhythmias.

British Journal of Pharmacology (1999) 127, 1687–1695; doi:10.1038/sj.bjp.0702704
Effects of melatonin on rat pial arteriolar diameter in vivo
Authors: Régrigny Olivier , Delagrange Philippe , Scalbert Elizabeth , Lartaud‐Idjouadiene Isabelle , Atkinson Jeffrey , Chillon Jean‐Marc
Published: January 2005

Based on our finding that melatonin decreased the lower limit of cerebral blood flow autoregulation in rat, we previously suggested that melatonin constricts cerebral arterioles. The goal of this study was to demonstrate this vasoconstrictor action and investigate the mechanisms involved.

The effects of cumulative doses of melatonin (10−10 to 10−6 M) were examined in cerebral arterioles (30–50 μM) of male Wistar rats using an open skull preparation. Cerebral arterioles were exposed to two doses of melatonin (3×10−9 and 3×10−8 M) in the absence and presence of the mt1 and/or MT2 receptor antagonist, luzindole (2×10−6 M) and the Ca2+‐activated K+ (BKCa) channel blocker, tetraethylammonium (TEA+, 10−4 M). The effect of L‐nitro arginine methyl ester (L‐NAME, 10−8 M) was examined on arterioles after TEA+ superfusion. Cerebral arterioles were also exposed to the BKCa activator, NS1619 (10−5 M), and to sodium nitroprusside (SNP, 10−8 M) in the absence and presence of melatonin (3×10−8 M).

Melatonin induced a dose‐dependent constriction with an EC50 of 3.0±0.1 nM and a maximal constriction of −15±1%. Luzindole abolished melatonin‐induced vasoconstriction. TEA+ induced significant vasoconstriction (−10±2%). No additional vasoconstriction was observed when melatonin was added to the aCSF in presence of TEA+, whereas L‐NAME still induced vasoconstriction (−10±1%). NS1619 induced vasodilatation (+11±1%) which was 50% less in presence of melatonin. Vasodilatation induced by SNP (+12±2%) was not diminished by melatonin.

Melatonin directly constricts small diameter cerebral arterioles in rats. This vasoconstrictor effect is mediated by inhibition of BKCa channels following activation of mt1 and/or MT2 receptors.

British Journal of Pharmacology (1999) 127, 1666–1670; doi:10.1038/sj.bjp.0702714
In vivo B1 kinin‐receptor upregulation. Evidence for involvement of protein kinases and nuclear factor κB pathways
Authors: Campos Maria M , Souza Glória E P , Calixto João B
Published: January 2005

Intradermal (i.d.) injection of cytokines, IL‐1β and TNFα (5 ng, 60 and 30 min prior) produces a rapid onset up‐regulation of des‐Arg9‐BK‐mediated rat paw oedema. Here we analyse the mechanisms involved in des‐Arg9‐BK‐induced oedema in animals pre‐treated with IL‐1β or TNFα.

Co‐injection of anti‐IL‐1β, anti‐TNFα and anti‐IL‐8 (50 ng) significantly inhibited des‐Arg9‐BK‐induced oedema in animals pre‐treated with IL‐1β (65, 37 and 42%) or TNFα (39, 64, 25%). IL‐1 receptor antagonist (IRA, 100 μg) or IL‐10 (10 ng) inhibited the oedema caused by des‐Arg9‐BK, in rats that had received either IL‐1β (67 and 63%) or TNFα (46 and 35%).

Co‐injection of the PKC inhibitors, staurosporine (10 nmol) or RO 318220 (30 nmol) inhibited des‐Arg9‐BK‐induced paw oedema (44 and 42% for IL‐1β and, 53 and 30% for TNFα, respectively). Genistein (tyrosine kinase inhibitor, 2.5 mg kg−1, s.c.) or PD 098059 (MAP‐kinase inhibitor, 30 nmol) produced marked inhibition of des‐Arg9‐BK‐induced oedema (58 and 39% for IL‐1β and 31 and 35% for TNFα respectively).

The NF‐κB inhibitors TLCK (2 mg kg−1, i.p.) and PDCT (100 mg kg−1, i.p.) significantly inhibited the oedema of des‐Arg9‐BK in IL‐1β (27 and 83%) or TNFα (28 and 80%) pre‐treated animals.

It is concluded that up‐regulation of B1 receptors modulated by IL‐1β or TNFα involves the release of other cytokines, activation of PKC and tyrosine kinase pathways, co‐ordinated with the activation of MAP‐kinase and nuclear factor κB, reinforcing the view that B1 receptors may exert a pivotal role in modulating chronic inflammatory processes.

British Journal of Pharmacology (1999) 127, 1851–1859; doi:10.1038/sj.bjp.0702715
Influence of CGRP (8–37), but not adrenomedullin (22–52), on the haemodynamic responses to lipopolysaccharide in conscious rats
Authors: Gardiner S M , March J E , Kemp P A , Bennett T
Published: January 2005

The functional involvement of the vasodilator peptides, adrenomedullin (ADM) and calcitonin gene‐related peptide (CGRP), in the haemodynamic sequelae of continuous infusion of lipopolysaccharide (LPS) was assessed in conscious, male, Long Evans rats, by the use of peptide antagonists.

It was demonstrated that ADM (22–52) at a dose of 500 nmol kg−1 h−1 caused significant inhibition of the effects of ADM (1 nmol kg−1), without affecting responses to CGRP (0.1 or 1 nmol kg−1).

Even when the regional vasodilator responses to LPS infusion were enhanced (by pre‐treatment with dexamethasone and the endothelin antagonist, SB 209670, or by pretreatment with SB 209670 and the AT1‐receptor antagonist, losartan), ADM (22–52) had no significant cardiovascular effects. In contrast, the CGRP1‐receptor antagonist, CGRP (8–37), caused small, but significant, inhibitions of the hypotensive and renal and mesenteric vasodilator effects of LPS, but only 6 h after onset of infusion in the presence of dexamethasone and SB 209670.

The results indicate that, in this model of endotoxaemia, the marked regional vasodilatations seen in the presence of dexamethasone and SB 209670 do not involve ADM, but do involve CGRP, albeit only to a small extent.

British Journal of Pharmacology (1999) 127, 1611–1618; doi:10.1038/sj.bjp.0702718
Analysis of the atypical characteristics of adenosine receptors mediating negative inotropic and chronotropic responses of guinea‐pig isolated atria and papillary muscles
Authors: Gardner Neil M , Broadley Kenneth J
Published: January 2005

Adenosine receptor(s) mediating negative inotropy of paced left atria, isoprenaline‐stimulated paced left atria and papillary muscles, and negative chronotropy of spontaneously beating right atria were characterized.

Isometric tension of guinea‐pig isolated paced left atria and left ventricular papillary muscles and rate of contraction of spontaneously beating right atria were recorded. Papillary muscles were pre‐stimulated with isoprenaline (1×10−8 M). Concentration‐response curves (CRCs) for tension or rate reduction by N6‐cyclopentyladenosine (CPA), the stereoisomers of N6‐(2‐phenylisopropyl)adenosine ((+)‐PIA and (−)‐PIA), 5′‐(N‐carboxamido)adenosine (NECA), N6‐2‐(4‐aminophenyl)ethyladenosine (APNEA) and N6‐(3‐iodobenzyl)adenosine‐5′‐N‐methyuromide (IB‐MECA) revealed a potency order of CPA=(−)‐PIA>NECA in right atria and papillary muscles, which is consistent with involvement of A1‐receptors. The potency order in left atria was CPA=NECA>(−)‐PIA>(+)‐PIA>APNEA, which is not typical of A1 adenosine receptors. Weak activity of APNEA and IB‐MECA discounts involvement of A3 receptors.

pA2 values for the antagonism of CPA by 8(p‐sulfophenyl)theophylline (8‐SPT) were calculated from Schild plots (log concentration‐ratio against log 8‐SPT concentration), the unity slopes of which indicated competitive antagonism. The pA2 value in the papillary muscles was significantly higher than for atrial preparations, indicating a possible difference in receptor characteristics between atrial and papillary muscle responses.

In left and right atria there was a limit to the displacement of the CPA CRCs at higher concentrations of 8‐SPT. The 8‐SPT‐resistant component of the response is suggested to arise from duality of coupling of a common A1 receptor through either different G proteins or G protein subunits to independent transduction pathways. The results with papillary muscles can be explained by a typical A1 receptor coupled to a single transduction pathway.

British Journal of Pharmacology (1999) 127, 1619–1626; doi:10.1038/sj.bjp.0702719
Action of AT1 receptor antagonists on angiotensin II‐induced tone in human isolated subcutaneous resistance arteries
Authors: Garcha Robinder S , Sever Peter S , Hughes Alun D
Published: January 2005

Human isolated subcutaneous arteries were studied under isometric conditions in a myograph.

Addition of angiotensin II (AII) induced a concentration‐dependent increase in tone in isolated arteries. The active metabolite of candesartan (CV 11974), losartan and the active metabolite of losartan, E‐3174 antagonized AII‐induced tone in a non‐competitive manner, but the AT2 selective antagonist, PD123319, was without effect on responses to AII. The effects of candesartan, losartan and E‐3174 were analysed using a classical model of non‐competitive antagonism and a two‐state receptor model.

Mechanical removal of the endothelium; pre‐incubation with Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME); pre‐incubation with indomethacin, a cyclo‐oxygenase inhibitor; or pre‐incubation with BQ 485, an endothelin antagonist; had no significant effect on contractions induced by AII.

Our results suggest AII contracts human isolated resistance arteries by an action on AT1 receptors and does not involve release of endothelial factors. Use of a two‐state receptor model successfully described the action of the AT1 antagonists without sacrificing assumptions regarding the competitive nature of binding of these antagonists.

British Journal of Pharmacology (1999) 127, 1876–1882; doi:10.1038/sj.bjp.0702722
Pregnancy reduces brain sigma receptor function
Authors: Bergeron Richard , De Montigny Claude , Debonnel Guy
Published: January 2005

Sigma (σ) receptors have recently been cloned, though their endogenous ligand(s) remain unidentified. However, some neuroactive steroids, such as progesterone, have a high affinity for these receptors. Some σ ligands, such as DTG, (+)‐pentazocine and DHEA, act as σ ‘agonists’ by potentiating the neuronal response to NMDA. Others, such as haloperidol, NE‐100 and progesterone, act as σ ‘antagonists’ by reversing the potentiations induced by σ ‘agonists’.

We compared the effects of σ ‘agonists’ in four series of female rats: in controls, at day 18 of pregnancy, at day 5 post‐partum, and in ovariectomized rats following a 3‐week treatment with a high dose of progesterone.

In pregnant rats and following a 3‐week treatment with progesterone, 10 fold higher doses of DTG, (+)‐pentazocine and DHEA were required to elicit a selective potentiation of the NMDA response comparable to that obtained in control females. Conversely, at day 5 post‐partum and following the 3‐week treatment with a progesterone and after a 5‐day washout, the potentiation of the NMDA response induced by the σ ‘agonist’ DTG was greater than in control females.

The present data suggest that endogenous progesterone acts as an ‘antagonist’ at σ receptors. The resulting changes in the function of σ receptors during pregnancy and post‐partum may be implicated in emotional phenomena occurring during these periods.

British Journal of Pharmacology (1999) 127, 1769–1776; doi:10.1038/sj.bjp.0702724
Triggering role of nitric oxide in the delayed protective effect of monophosphoryl lipid A in rat heart
Authors: György Katalin , Muller Bernard , Végh Agnes , Kleschyov Andrei L , Stoclet Jean‐Claud
Published: January 2005

The main objective of the present study was to further evaluate the role of nitric oxide (NO) in delayed cardiac protection against ischaemia‐reperfusion injury induced by monophosphoryl lipid A (MLA).

For this purpose, rats were administered with either 0.5 or 2.5 mg kg−1 MLA (i.p.). Eight or 24 h later, in vivo NO production in the heart was analysed by electron paramagnetic resonance (EPR) spin trapping technique. In parallel experiments, hearts were removed and perfused according to Langendorff. Functional ventricular parameters and incidence of ventricular fibrillation (VF) were determined after 30 min global ischaemic insult (37°C) followed by 30 min reperfusion. Vascular reactivity of aortic rings was also assessed.

Hearts from rats pretreated with 2.5 mg kg−1 MLA for 24 h (but not those from rats treated with 0.5 mg kg−1 MLA for 8 and 24 h, or with 2.5 mg kg−1 MLA for 8 h) exhibited preservation of ventricular function (LVDP, ±dP/dtmax) and a reduced incidence of VF (25% vs 87.5% in vehicle control) during reperfusion. At the cardioprotective dose of 2.5 mg kg−1 (for 8 or 24 h), MLA did not produce alterations of the contractile response of aortic rings to noradrenaline.

An increased formation of NO was detected in hearts removed from rats pretreated with 2.5 mg kg−1 MLA for 8 h, but not in those from rats treated for 24 h (or with 0.5 mg kg−1 MLA).

Pretreatment of the animals with the inhibitors of inducible NO‐synthase, aminoguanidine (2×300 mg kg−1) or L‐N6‐(1‐Iminoethyl)‐lysine (L‐NIL, 10 mg kg−1) abolished both MLA (2.5 mg kg−1)‐induced rise of NO production (observed 8 h after MLA) and cardioprotection (observed 24 h after MLA). However MLA‐induced cardioprotection was not attenuated when the hearts were perfused with aminoguanidine (150 μM) for 30 min before the ischaemic insult.

Altogether, the present data suggest that NO acts as a trigger rather then a direct mediator of the delayed cardioprotective effect of MLA in rat heart.

British Journal of Pharmacology (1999) 127, 1892–1898; doi:10.1038/sj.bjp.0702725
PKCβI mediates the inhibition of P2Y receptor‐induced inositol phosphate formation in endothelial cells
Authors: Chen Bing‐C , Lin Wan‐W
Published: January 2005

Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C (PLC)‐linked P2Y1 and P2Y2 receptors, for them 2‐methylthio‐ATP (2MeSATP) and UTP are respective agonists. Here, we have investigated the particular protein kinase C (PKC) isoform(s) responsible for the inhibition of P2Y1 and P2Y2 receptor‐evoked inositol phosphate (IP) formation by phorbol 12‐myristate 13‐acetate (PMA).

Although short‐term (20 min) pretreatment of cells with PMA attenuated 2MeSATP‐ and UTP‐induced phosphoinositide (PI) breakdown, this inhibition was lost after 15 h. Preincubation with PMA for 24 h, on the contrary, potentiated 2MeSATP and UTP responses. The IP formation stimulated by NaF was unaltered by PMA pretreatment.

Western blot analysis showed that treatment of CPAE with PMA resulted in a rapid translocation of PKC isoform βI, ε and μ, but not λ, from the cytosol to the membrane fraction.

Pretreatment of the selective PKC inhibitor Ro 31–8220 attenuated the inhibitory effect of PMA on IP formation. Go 6976 (an inhibitor of conventional PKCα, β and γ) and LY 379196 (a selective PKCβ inhibitor) also dose‐dependently inhibited the PMA‐mediated desensitization.

Transfection of PKCβ‐specific antisense oligonucleotide reduced PKCβI protein level and inhibited PMA‐mediated PI reduction.

RT–PCR analysis showed that PMA treatment for 4–24 h up‐regulated P2Y1 and P2Y2 receptors at the mRNA levels.

These results suggest that PKCβI may exert a negative feedback regulation on endothelial P2Y1 and P2Y2 receptor‐mediated PI turnover. The down‐regulation of PKCβI and enhanced P2Y receptor expression together might contribute to the late PI enhancing effect of PMA.

British Journal of Pharmacology (1999) 127, 1908–1914; doi:10.1038/sj.bjp.0702727
Antisense down‐regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5‐FUdR, 5‐FU and Tomudex in HeLa cells
Authors: Ferguson Peter J , Collins Olga , Dean Nicholas M , DeMoor Janice , Sha‐Li Chen , Vincent Mark D , Koropatnick James
Published: January 2005

Thymidylate synthase (TS), the key enzyme in de novo synthesis of thymidine, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide down‐regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyrimidine analogues 5‐fluorouracil (5‐FU) and 5‐fluorodeoxyuridine (5‐FUdR), and the folate analogue Tomudex (ICI D1694; N‐(5‐[N‐(3,4‐dihydro‐2‐methyl‐4‐oxoquinazolin‐6‐ylmethyl)‐N‐methylamino]‐2‐theonyl‐L‐glutamic acid).

2′‐Methoxyethoxylated, phosphorothioated 20‐mer oligodeoxynucleotides (ODNs), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomized order, against which all the biological effects were compared. Following a 6‐h transfection of HeLa cells using polycationic liposome at 3 μg ml−1, ODN 83 (50 nM), complementary to a region in the 3′‐untranslated region of the TS mRNA, decreased TS mRNA levels by approximately 70% within 24 h. ODN 83 also decreased TS enzyme activity, as measured by binding of TS to radiolabelled 5‐fluorodeoxyuridine monophosphate. In addition to inhibiting proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxicity of Tomudex or 5‐FU, added 1 day following transfection, by 50–60%. ODN 83 also enhanced sensitivity to 5‐FUdR by 70%, but did not affect the toxicity of cisplatin, chlorambucil, melphalan, doxorubicin, ionizing radiation, paclitaxel, or irinotecan.

These data indicate that antisense ODN down‐regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS‐targeted drugs.

British Journal of Pharmacology (1999) 127, 1777–1786; doi:10.1038/sj.bjp.0702728
Effects of indoramin in rat vas deferens and aorta: concomitant α1‐adrenoceptor and neuronal uptake blockade
Authors: Pupo André S , Cavenaghi Daniela L C , Campos Marcelo , Morais Paola de Lucena , Jurkiewicz Neide H , Jurkiewicz Aron
Published: January 2005

The actions of the α1‐adrenoceptor antagonist indoramin have been examined against the contractions induced by noradrenaline in the rat vas deferens and aorta taking into account a putative neuronal uptake blocking activity of this antagonist which could result in self‐cancelling actions.

Indoramin behaved as a simple competitive antagonist of the contractions induced by noradrenaline in the vas deferens and aorta yielding pA2 values of 7.38±0.05 (slope=0.98±0.03) and 6.78±0.14 (slope=1.08±0.06), respectively.

When the experiments were repeated in the presence of cocaine (6 μM) the potency (pA2) of indoramin in antagonizing the contractions of the vas deferens to noradrenaline was increased to 8.72±0.07 (slope=1.10±0.05) while its potency remained unchanged in the aorta (pA2=6.69±0.12; slope=1.04±0.05).

In denervated vas deferens, indoramin antagonized the contractions to noradrenaline with a potency similar to that found in the presence of cocaine (8.79±0.07; slope=1.09±0.06).

It is suggested that indoramin blocks α1‐adrenoceptors and neuronal uptake in rat vas deferens resulting in Schild plots with slopes not different from unity even in the absence of selective inhibition of neuronal uptake. As a major consequence of this double mechanism of action, the pA2 values for this antagonist are underestimated when calculated in situations where the neuronal uptake is active, yielding spurious pKB values.

British Journal of Pharmacology (1999) 127, 1832–1836; doi:10.1038/sj.bjp.0702735
Eukaliuric diuresis and natriuresis in response to the KATP channel blocker U37883A: Micropuncture studies on the tubular site of action
Authors: Huang Dan Y , Osswald Hartmut , Vallon Volker
Published: January 2005

Systemic application of U37883A, a blocker of ATP sensitive potassium (KATP) channels, elicits diuresis and natriuresis without significantly altering urinary potassium excretion.

To elucidate tubular sites of action upstream to the distal nephron, micropuncture experiments were performed in nephrons with superficial glomeruli of anaesthetized Munich‐Wistar‐Frömter rats during systemic application of U37883A (1, 5 or 15 mg kg−1 i.v.).

The observed eukaliuric diuresis and natriuresis in response to U37883A at 15 mg kg−1 was accompanied by an increase in early distal tubular flow rate (VED) from 10–18 nl min−1 reflecting a reduction in fractional reabsorption of fluid up to this site (FR‐fluid) of 13%. The latter proposed an effect on water‐permeable segments such as the proximal tubule which could fully account for the observed reduction in fractional reabsorption of Na+ up to the early distal tubule (FR‐Na+) of 8% and the increase in early distal tubular Na+ concentration ([Na+]ED) from 35–51 mM whereas [K+]ED was left unaltered.

In comparison, furosemide (3 mg kg−1 i.v.), which acts in the water‐impermeable thick ascending limb, elicited diuresis, natriuresis and kaliuresis which were associated with a fall in FR‐Na+ of 10% with no change in FR‐fluid, and a rise in [Na+]ED from 42–117 mM and [K+]ED from 1.2–5.7 mM with no change in VED.

Direct late proximal tubular fluid collections confirmed a significant inhibition of fluid reabsorption in proximal convoluted tubule in response to systemic application of U37883A.

These findings suggest that the diuretic and natriuretic effect upstream to the distal tubule in response to systemic application of U37883A involves actions on water‐permeable segments such as the proximal convoluted tubule.

British Journal of Pharmacology (1999) 127, 1811–1818; doi:10.1038/sj.bjp.0702742
Stereoselective interaction of thiopentone enantiomers with the GABAA receptor
Authors: Cordato D J , Chebib M , Mather L E , Herkes G K , Johnston G A R
Published: January 2005

As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the ∼2 fold greater potency for CNS effects of (−)‐S‐ over (+)‐R‐thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABAA receptor, the primary receptor for barbiturate hypnotic effects.

Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABAA receptor subtype α1β2γ2 to determine relative differences in potentiation of the GABA response by rac‐, (+)‐R‐ and (−)‐S‐thiopentone, and rac‐pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated.

With 3 μM GABA, the EC50 values were (−)‐S‐thiopentone (mean 26.0±s.e.mean 3.2 μM, n=9 cells) >rac‐thiopentone (35.9±4.2 μM, n=6, P=0.1) >(+)‐R‐thiopentone (52.5±5.0 μM, n=8, P<0.02) >rac‐pentobarbitone (97.0±11.2 μM, n=11, P<0.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC50 values for (+)‐R‐ or (−)‐S‐thiopentone.

Uninjected oocytes responded to >100 μM (−)‐S‐ and R‐thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N1,N1‐tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (−)‐S‐thiopentone (20.6±3.2 μM, n=8) <(+)‐R‐thiopentone (36.2±3.2 μM, n=9, P<0.005).

(−)‐S‐thiopentone was found to be ∼2 fold more potent than (+)‐R‐thiopentone in the potentiation of GABA at GABAA receptors expressed on Xenopus oocytes. This is consistent with the differences in potency for CNS depressant effects found in vivo.

British Journal of Pharmacology (1999) 128, 77–82; doi:10.1038/sj.bjp.0702744
Enhancement by galactosamine of lipopolysaccharide(LPS)‐induced tumour necrosis factor production and lethality: its suppression by LPS pretreatment
Authors: Endo Yasuo , Shibazaki Masahiko , Yamaguchi Kouji , Kai Kenzou , Sugawara Shunji , Takada Haruhiko , Kikuchi Hideaki , Kumagai Katsuo
Published: January 2005

D‐Galactosamine (GalN) depletes UTP primarily in the liver, resulting in decreased RNA synthesis in hepatocytes. Co‐injection of GalN and lipopolysaccharide (LPS) into mice produces fulminant hepatitis with severe hepatic congestion, resulting in rapid death. Although the underlying mechanism is uncertain, GalN enhances the sensitivity to tumour necrosis factor (TNF). Administration of uridine (a precursor of UTP) prior injection of either LPS itself or interleukin‐1 (IL‐1) reduces the lethality of GalN+LPS. The present study focused on the effects of these agents on TNF production.

Intraperitoneal injection of GalN+LPS into mice greatly elevated serum TNF. Although large doses of LPS alone also greatly elevated serum TNF, LPS itself induced neither hepatic congestion nor rapid death. Administration of a macrophage depletor, liposomes encapsulated with dichloromethylene bisphosphonate, reduced both the TNF production and mortality induced by GalN+LPS.

Uridine, when injected 0.5 h after the injection of GalN+LPS, reduced the production of TNF. Prior injection of LPS, but not of IL‐1, also reduced this TNF production.

Serum from LPS‐injected mice reduced the TNF production induced by GalN+LPS, but it was less effective at reducing the lethality. Its ability to reduce TNF production was abolished by heat‐treatment.

We hypothesize that a factor inhibiting TNF production by macrophages is produced by hepatocytes in response to LPS. Possibly, production of this hepatocyte‐derived TNF‐down‐regulator (TNF‐DRh) may be: (i) inhibited by GalN, causing over‐production of TNF by macrophages and (ii) stimulated by LPS‐pretreatment (and restored by uridine), causing reduced TNF production.

British Journal of Pharmacology (1999) 128, 5–12; doi:10.1038/sj.bjp.0702747