bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of both G protein αs and βγ subunits in β‐adrenergic stimulation of vascular L‐type Ca2+ channels
Authors: Viard Patricia , Macrez Nathalie , Mironneau Chantal , Mironneau Jean
Published: January 2005

Previous data have shown that activation of β3‐adrenoceptors stimulates vascular L‐type Ca2+ channels through a Gαs‐induced stimulation of the cyclic AMP/PKA pathway. The present study investigated whether β‐adrenergic stimulation also uses the Gβγ/PI3K/PKC pathway to modulate L‐type Ca2+ channels in rat portal vein myocytes.

Peak Ba2+ current (IBa) measured using the whole‐cell patch clamp method was maximally increased by application of 10 μM isoprenaline after blockade of β3‐adrenoceptors by 1 μM SR59230A. Under these conditions, the isoprenaline‐induced stimulation of IBa was reversed by ICI‐118551 (a specific β2‐adrenoceptor antagonist) but not by atenolol (a specific β1‐adrenoceptor antagonist). The β2‐adrenoceptor agonist salbutamol increased IBa, an effect which was reversed by ICI‐118551 whereas the β1‐adrenoceptor agonist dobutamine had no effect on IBa.

Application of PKA inhibitors (H‐89 and Rp 8‐Br‐cyclic AMPs) or a PKC inhibitor (calphostin C) alone did not affect the β2‐adrenergic stimulation of IBa whereas simultaneous application of both PKA and PKC inhibitors completely blocked this stimulation.

The β2‐adrenergic stimulation of L‐type Ca2+ channels was blocked by a pre‐treatment with cholera toxin and by intracellular application of an anti‐Gαs antibody (directed against the carboxyl terminus of Gαs). In the presence of H‐89, intracellular infusion of an anti‐Gβcom antibody or a βARK1 peptide as well as a pre‐treatment with wortmannin (a PI3K inhibitor) blocked the β2‐adrenergic stimulation of IBa.

These results suggest that the β2‐adrenergic stimulation of vascular L‐type Ca2+ channels involves both Gαs and Gβγ subunits which exert their stimulatory effects through PKA and PI3K/PKC pathways, respectively.

British Journal of Pharmacology (2001) 132, 669–676; doi:10.1038/sj.bjp.0703864
Mechanism of prolonged vasorelaxation to ATP in the rat isolated mesenteric arterial bed
Authors: Ralevic Vera
Published: January 2005

This study investigated the mechanism of prolonged relaxation to ATP in the rat isolated perfused mesenteric arterial bed.

In methoxamine pre‐constricted preparations, ATP elicited dose‐dependent, endothelium‐dependent, rapid relaxation at 5 pmol – 0.05 μmol (Rmax 76±5.6%, pD2 9.2±0.2), and contraction, followed by prolonged endothelium‐independent vasorelaxation at 0.05, 0.5 and 5 μmol (56±3.0, 87±2.9 and 85±4.6%). Suramin (100 μM), attenuated rapid (pD2 7.8±0.1) and prolonged relaxation to ATP. The selective P2 receptor antagonist PPADS (10 μM) reduced prolonged, but not rapid relaxation. Neither phase of relaxation was affected by 8‐sulphophenyltheophylline (1 μM) or indomethacin (10 μM).

α,β‐methylene ATP (α,β‐meATP; 10 μM) attenuated prolonged relaxation to ATP (relaxations at 0.05 and 0.5 μmol were 25±8.3 and 48±9.0%, respectively). α,β‐meATP blocked contractions and revealed rapid relaxation to ATP at 0.05 – 5 μmol.

Capsaicin pre‐treatment did not affect either phase of vasorelaxation to ATP. α,β‐meATP (10 μM) had no effect on vasorelaxation mediated by electrical stimulation of capsaicin‐sensitive sensory nerves.

High K+ (25 mM) attenuated prolonged relaxation to ATP (21±2.6 and 64±5.8%, at 0.05 and 0.5 μmol, respectively), but had no effect on rapid relaxation. Ouabain (1 mM), an inhibitor of Na+/K+‐ATPase, and glibenclamide (10 μM), an inhibitor of KATP channels, also attenuated prolonged relaxation to ATP. Charybdotoxin (100 nM), a selective inhibitor of KCa channels, and tetraethylammonium (10 mM) had no effect on rapid or prolonged relaxations.

These results show that the prolonged phase of vasorelaxation to ATP in the rat isolated mesenteric arterial bed, which may be mediated by P2Y receptors, is endothelium‐independent, involves activation of Na+/K+‐ATPase and KATP channels, and is inhibited by α,β‐meATP. Neither prolonged nor rapid vasorelaxation to ATP involves capsaicin‐sensitive sensory nerves, adenosine P1 receptors, prostanoids or KCa channels.

British Journal of Pharmacology (2001) 132, 685–692; doi:10.1038/sj.bjp.0703868
Amantadine inhibits RANTES production by influenzavirus‐infected human bronchial epithelial cells
Authors: Asai Yasukiyo , Hashimoto Shu , Kujime Kousei , Gon Yasuhiro , Mizumura Kenji , Shimizu Kazufumi , Horie Takashi
Published: January 2005

Amantadine can prevent and decrease airway inflammation by inhibiting influenza virus (IV) replication; however, the effect of amantadine on RANTES production by human bronchial epithelial cells (BEC) has not been determined. In the present study, we examined the effect of amantadine on RANTES production and also analysed p38 mitogen‐activated protein (MAP) kinase and c‐Jun‐NH2‐terminal kinase (JNK) activation to clarify the mechanism in the effect of amantadine on RANTES production, since we have previously shown that p38 MAP kinase and JNK regulate RANTES production by IV‐infected BEC.

BEC that had been preincubated with amantadine were infected with IV and then p38 MAP kinase and JNK activation in the cells and RANTES concentrations in the culture supernatants were determined.

Amantadine‐induced inhibition of virus replication resulted in a decrease in p38 MAP kinase and JNK activity and decreased expression of RANTES in IV‐infected cells.

Amantadine did not inhibit p38 MAP kinase and JNK activation induced by tumour necrosis factor‐α (TNF‐α) as a non‐viral stimulus.

These results indicate that amantadine inhibits IV infection‐induced RANTES production by human BEC and that the inhibition by amantadine of RANTES production might result from an indirect inhibitory effect of amantadine on p38 MAP kinase and JNK activation via the inhibition of virus replication, and we emphasize that amantadine may produce a beneficial effect on controlling bronchial asthma exacerbation caused by IV infection.

British Journal of Pharmacology (2001) 132, 918–924; doi:10.1038/sj.bjp.0703870
Neuropeotide Y changes the excitability of fine afferent units in the rat knee joint
Authors: Just Stefan , Heppelmann Bernd
Published: January 2005

The aim of the present study was to examine the effects of the sympathetic co‐transmitter Neuropeotide Y on primary afferent nerve fibres of the rat knee joint. The responses to passive joint rotations at defined torque were recorded from 41 slowly conducting afferent nerve fibres (0.9 – 18.8 m s−1) innervating the knee joint capsule.

About 70% of the joint afferents were significantly affected in their mechanosensitivity by topical application of Neuropeptide Y. Significant effects occurred at a concentration of 10 nM.

Decreased mechanosensitivity was observed in about 40% of nerve fibres, whereas 30% of the units increased the mechanosensitivity. In addition, in about 35% of the fibres resting activity was induced or increased. Neither the conduction velocity nor the mechanical threshold of the units correlated with the described effects of Neuropeptide Y.

NPY(13 – 36), a specific Y2‐receptor agonist, only modulated the mechanosensitivity, with no effect on the resting activity. The effects on the mechanosensitivity were similar to Neuropeptide Y, i.e. increase and decrease of the response.

Studies with the Y1‐agonist (Leu31, Pro34)‐NPY showed that activation of the Y1‐receptor predominantly resulted in an enhanced mechanosensitivity and an induction or increase of a resting activity. The opposite effect was observed by application of BIBP 3226 BS, a Y1‐receptor antagonist.

In conclusion, these data indicate that Neuropeptide Y affects the excitability of sensory nerve fibre endings.

British Journal of Pharmacology (2001) 132, 703–708; doi:10.1038/sj.bjp.0703871
Involvement of protein kinase C in the presynaptic nicotinic modulation of [3H]‐dopamine release from rat striatal synaptosomes
Authors: Soliakov Lev , Wonnacott Susan
Published: January 2005

Presynaptic nicotinic ACh receptors modulate transmitter release in the brain. Here we report their interactions with protein kinase C (PKC) with respect to [3H]‐dopamine release from rat striatal synaptosomes, monitored by superfusion.

Two specific PKC inhibitors, Ro 31‐8220 (1 μM) and D‐erythro‐sphingosine (10 μM) significantly reduced (by 51 and 26% respectively) [3H]‐dopamine release stimulated by anatoxin‐a (AnTx), a potent and selective agonist of nicotinic ACh receptors. The inactive structural analogue of Ro 31‐8220, bisindolylmaleimide V (1 μM) had no effect.

Two phorbol esters, PDBu (1 μM) and PMA (1 μM) potentiated AnTx‐evoked [3H]‐dopamine release by 50 – 80%. This was Ca2+‐dependent and prevented by PKC inhibitors. In the absence of nicotinic agonist, phorbol esters enhanced basal release through a PKC‐independent mechanism.

A 86Rb+ efflux assay of nicotinic ACh receptor function confirmed that Ro 31‐8220 has no nonspecific effect on presynaptic nicotinic ACh receptors.

These results suggest that PKC is activated by nicotinic ACh receptor stimulation and mediates a component of AnTx‐evoked [3H]‐dopamine release. In addition, independent activation of PKC can further amplify the response, offering a potential mechanism for receptor crosstalk.

British Journal of Pharmacology (2001) 132, 785–791; doi:10.1038/sj.bjp.0703873
Effects of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246, on Y2 receptors at peripheral neuroeffector junctions
Authors: Smith‐White Margaret A , Hardy Todd A , Brock James A , Potter Erica K
Published: January 2005

This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2 receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea‐pig trachea and purinergic neuroeffector transmission in guinea‐pig vas deferens produced by the NPY Y2 receptor agonist, N‐acetyl [Leu28,31] NPY 24‐36.

In pentobarbitone anaesthetized rats, supramaximal stimulation every 30 s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100 ms. This response was attenuated by intravenous administration of N‐acetyl [Leu28,31] NPY 24‐36 (10 nmol kg−1).

Transmural stimulation of segments of guinea‐pig trachea at 1 min intervals with 5 s trains of stimuli at 0.5, 5, 10, 20 and 40 Hz evoked contractions which were reduced in force by N‐acetyl [Leu28,31] NPY 24‐36 (2 μM).

In guinea‐pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1 Hz was reduced in the presence of N‐acetyl [Leu28,31] NPY 24‐36 (1 μM).

In all preparations BIIE0246 attenuated the inhibitory effect of N‐acetyl [Leu28,31] NPY 24‐36 but had no effect when applied alone.

The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2 receptors which, when activated, reduce neurotransmitter release.

British Journal of Pharmacology (2001) 132, 861–868; doi:10.1038/sj.bjp.0703879
Anti‐apoptotic effect of benidipine, a long‐lasting vasodilating calcium antagonist, in ischaemic/reperfused myocardial cells
Authors: Gao Feng , Gong Bing , Christopher Theodore A , Lopez Bernard L , Karasawa Akira , Ma Xin L
Published: January 2005

Ischaemia/reperfusion causes intracellular calcium overloading in cardiac cells. Administration of calcium antagonists reduces myocardial infarct size. Recent in vitro studies have demonstrated that calcium plays a critical role in the signal transduction pathway leading to apoptosis. However, whether or not calcium antagonists may reduce myocardial apoptosis induced by ischaemia‐reperfusion, and thus decrease myocardial infarction, has not been directly investigated.

The present study investigated the effects of benidipine, an L‐type calcium channel blocker, on myocardial infarct size, apoptosis, necrosis and cardiac functional recovery in rabbits subjected to myocardial ischaemia/reperfusion (MI/R, 45 min/240 min). Ten minutes prior to coronary occlusion, rabbits were treated with vehicle or benidipine (10 μg kg−1 or 3 μg kg−1, i.v.).

In the vehicle‐treated group, MI/R caused cardiomyocyte apoptosis as evidenced by DNA ladder formation and TUNEL positive nuclear staining (12.2±1.1%). Treatment with 10 μg kg−1 benidipine lowered blood pressure, decreased myocardial apoptosis (6.2±0.8%, P<0.01 vs vehicle) and necrosis, reduced infarct size (20±2.3% vs 49±2.6%, P<0.01), and improved cardiac functional recovery after reperfusion. Administering benidipine at 3 μg kg−1, a dose at which no haemodynamic effect was observed, also exerted significant anti‐apoptosis effects, which were not significantly different from those observed with higher dose benidipine treatment. However, treatment with this low dose benidipine failed to reduce myocardial necrosis.

These results demonstrate that benidipine, a calcium antagonist, exerts significant anti‐apoptosis effects, which are independent of haemodynamic changes. Administration of benidipine at a higher dose produced favourable haemodynamic effects and provided additional protection against myocardial necrotic injury and further improved cardiac functional recovery.

British Journal of Pharmacology (2001) 132, 869–878; doi:10.1038/sj.bjp.0703881
Black tea extract, thearubigin fraction, counteract the effects of botulinum neurotoxins in mice
Authors: Satoh Eiki , Ishii Toshiaki , Shimizu Yoshio , Sawamura Shin‐ichi , Nishimura Masakazu
Published: January 2005

Botulinum neurotoxin type A (BoNT/A, 1.5 nM) completely inhibited indirectly evoked twitches in in vitro mouse phrenic nerve‐diaphragm preparations within 40 – 45 min. Black tea extract, thearubigin fraction (TRB), mixed with BoNT/A blocked the inhibitory effect of the toxin. The protective effect of TRB extended to botulinum neurotoxins types B and E (BoNT/B and BoNT/E) and tetanus toxin, but not to tetrodotoxin. TRB was also effective against oral toxicity of BoNT/A, B and E. Thus, TRB may be of potential benefit in protecting the paralytic actions of botulinum neurotoxins (BoNTs), but its use is limited by mixing with the toxin.

British Journal of Pharmacology (2001) 132, 797–798; doi:10.1038/sj.bjp.0703883
A1 adenosine receptors inhibit multiple voltage‐gated Ca2+ channel subtypes in acutely isolated rat basolateral amygdala neurons
Authors: McCool Brian A , Farroni Jeffery S
Published: January 2005

The anticonvulsant properties of 2‐chloroadenosine (CADO) in the basolateral amygdala rely on the activation of adenosine‐specific heptahelical receptors. We have utilized whole‐cell voltage‐clamp electrophysiology to examine the modulatory effects of CADO and other adenosine receptor agonists on voltage‐gated calcium channels in dissociated basolateral amygdala neurons.

CADO, adenosine, and the A1 subtype‐selective agonists N6‐(L‐2‐Phenylisopropyl)adenosine (R‐PIA) and 2‐chloro‐N6‐cyclopentyladenosine (CCPA) reversibly modulated whole cell Ba2+ currents in a concentration‐dependent fashion. CADO inhibition of barium currents was also sensitive to the A1 antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX).

The A2A‐selective agonist 4‐[2‐[[6‐Amino‐9‐(N‐ethyl‐β‐D‐ribofuranuronamidosyl)‐9H‐purin‐2‐yl]amino]ethyl]benzenepropanoic acid (CGS21680) was without effect.

CADO inhibition was predominantly voltage‐dependent and sensitive to the sulphydryl‐modifying reagent N‐ethylmaleimide, implicating a membrane‐delimited, Gi/o‐coupled signal transduction pathway in the channel regulation.

Using Ca2+ channel subtype‐selective antagonists, CADO inhibition appeared to target multiple channel subtypes, with the inhibition of ω‐conotoxin GVIA‐sensitive calcium channels being more prominent.

Our results indicate that the anti‐convulsant effects CADO in the basolateral amygdala may be mediated, in part, by the A1 receptor‐dependent inhibition of voltage gated calcium channels.

British Journal of Pharmacology (2001) 132, 879–888; doi:10.1038/sj.bjp.0703884
Analysis of S‐nitroso‐N‐acetylpenicillamine effects on dopamine release in the striatum of freely moving rats: role of endogenous ascorbic acid and oxidative stress
Authors: Serra Pier Andrea , Esposito Giovanni , Delogu M Rosaria , Migheli Rossana , Rocchitta Gaia , Miele Egidio , Desole Maria S , Miele Maddalena
Published: January 2005

We showed previously that interaction between NO and iron(II), both released following decomposition of sodium nitroprusside (SNP), accounted for the late SNP‐induced dopamine (DA) increase in dialysates from the striatum of freely moving rats.

In this study, intrastriatal infusion of the NO‐donor S‐nitroso‐N‐acetylpenicillamine (SNAP) (0.2 mM for 180 min) induced a moderate increase in dialysate DA and decreases in ascorbic acid dialysate concentrations; in contrast, SNAP 1 mM infusion induced a long‐lasting decrease in both DA and ascorbic acid dialysate concentrations. 3‐Methoxy‐tyramine (3‐MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and uric acid levels were unaffected.

Co‐infusion of ferrous sulphate [iron(II), 1 mM for 40 min] with SNAP either 1 or 0.2 mM (for 180 min), produced a significant increase in both DA and 3‐MT dialysate concentrations, but it did not affect decreases in dialysate ascorbic acid levels. All other dialysate neurochemicals were unaffected.

Co‐infusion of ascorbic acid (0.1 mM) with SNAP (1 mM) for 180 min did not modify SNAP‐induced decreases in dialysate DA levels. In contrast, co‐infusion of uric acid (1 mM) reversed SNAP‐induced decreases in dialysate DA; co‐infusion of a superoxide dismutase mimetic delayed SNAP‐induced DA decreases for a short period, while co‐infusion of the antioxidant N‐acetylcysteine (NAC, 0.1 mM) significantly increased dialysate DA.

The results of this study show that SNAP induces concentration‐related changes in DA dialysate levels. At higher concentrations, SNAP induces non‐enzymatic DA oxidation, which is inhibited by uric acid and NAC; ascorbic acid failed to protect dialysate DA from oxidation, probably owing to its promoting effect on SNAP decomposition; exogenous iron(II) may react with NO generated from SNAP decomposition, with a consequent increase in dialysate DA and 3‐MT, therefore mimicking SNP effects on striatal DA release.

British Journal of Pharmacology (2001) 132, 941–949; doi:10.1038/sj.bjp.0703887
Gq/11 and Gi/o activation profiles in CHO cells expressing human muscarinic acetylcholine receptors: dependence on agonist as well as receptor‐subtype
Authors: Akam Elizabeth C , Challiss R A John , Nahorski Stefan R
Published: January 2005

Profiles of G protein activation have been assessed using a [35S]‐GTPγS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M1, M2, M3 or M4 muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M1 and M3, or M2 and M4 receptors were approximately equal.

Maximal [35S]‐GTPγS binding to Gq/11α stimulated by M1/M3 receptors, or Gi1 – 3α stimulated by M2/M4 receptors occurred within approximately 2 min of agonist addition. The increases in Gq/11α‐[35S]‐GTPγS binding after M1 and M3 receptor stimulation differed substantially, with M1 receptors causing a 2 – 3 fold greater increase in [35S]‐GTPγS binding and requiring 5 fold lower concentrations of methacholine to stimulate a half‐maximal response.

Comparison of M2 and M4 receptor‐mediated Gi1 – 3α‐[35S]‐GTPγS binding also revealed differences, with M2 receptors causing a greater increase in Gi1 – 3α activation and requiring 10 fold lower concentrations of methacholine to stimulate a half‐maximal response.

Comparison of methacholine‐ and pilocarpine‐mediated effects revealed that the latter partial agonist is more effective in activating Gi3α compared to Gi1/2α for both M2 and M4 receptors. More marked agonist/partial agonist differences were observed with respect to M1/M3‐mediated stimulations of Gq/11α‐ and Gi1 – 3α‐[35S]‐GTPγS binding. Whereas coupling to these Gα subclasses decreased proportionately for M1 receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M3 receptors for Giα versus Gq/11α activation.

These data demonstrate that mACh receptor subtype and the nature of the agonist used govern the repertoire of G proteins activated. They also provide insights into how the diversity of coupling can be pharmacologically exploited, and provide a basis for a better understanding of how multiple receptor subtypes can be differentially regulated.

British Journal of Pharmacology (2001) 132, 950–958; doi:10.1038/sj.bjp.0703892
Cyclic AMP‐dependent inhibition of human neutrophil oxidative activity by substituted 2‐propynylcyclohexyl adenosine A2A receptor agonists
Authors: Sullivan Gail W , Rieger Jayson M , Michael Scheld W , Macdonald Timothy L , Linden Joel
Published: January 2005

Novel 2‐propynylcyclohexyl‐5′‐N‐ehtylcarboxamidoadenosines, trans‐substituted in the 4‐position of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors (ARs). Two esters, 4‐{3‐[6‐amino‐9‐(5‐ethylcarbamoyl‐3,4‐dihydroxy‐tetrahydro‐furan‐2‐yl)‐9H‐purin‐2‐yl]‐prop‐2‐ynyl}‐cyclohexanecarboxylic acid methyl ester (ATL146e) and acetic acid 4‐{3‐[6‐amino‐9‐(5‐ethylcarbamoyl‐3, 4‐dihydroxy‐tetrahydro‐furan ‐2‐yl)‐9H‐purin‐2‐yl] ‐prop‐2‐ynyl}‐cyclohexylmethyl ester (ATL193) were >50×more potent than 2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS21680) for human A2A AR binding. Human A2A AR affinity for substituted cyclohexyl‐propynyladenosine analogues was inversely correlated with the polarity of the cyclohexyl side chain. There was a comparable order of potency for A2A AR agonist stimulation of human neutrophil [cyclic AMP]i, and inhibition of the neutrophil oxidative burst. ATL146e and CGS21680 were ∼equipotent agonists of human A3 ARs.

We measured the effects of selective AR antagonists on agonist stimulated neutrophil [cyclic AMP]i and the effect of PKA inhibition on A2A AR agonist activity. ATL193‐stimulated neutrophil [cyclic AMP]i was blocked by antagonists with the potency order: ZM241385 (A2A‐selective)>MRS1220 (A3‐selective)>>N‐(4‐Cyano‐phenyl)‐2‐[4‐(2,6‐dioxo‐1,3‐dipropyl‐2,3,4,5,6,7‐hexahydro‐1H‐purin‐8‐yl)‐phenoxy]‐acetamide (MRS1754; A2B‐selective) ∼amp; 8‐(N‐methylisopropyl)amino‐N6‐(5′‐endohydroxy‐endonorbornyl)‐9‐methyladenine (WRC0571; A1‐selective). The type IV phosphodiesterase inhibitor, rolipram (100 nM) potentiated ATL193 inhibition of the oxidative burst, and inhibition by ATL193 was counteracted by the PKA inhibitor H‐89.

The data indicate that activation of A2AARs inhibits neutrophil oxidative activity by activating [cyclic AMP]i/PKA.

British Journal of Pharmacology (2001) 132, 1017–1026; doi:10.1038/sj.bjp.0703893
Clotrimazole analogues: effective blockers of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurones
Authors: Shah M M , Miscony Z , Javadzadeh‐Tabatabaie M , Ganellin C R , Haylett D G
Published: January 2005

The pharmacology of the slow afterhyperpolarization (sAHP) was studied in cultured rat hippocampal pyramidal neurones.

Clotrimazole, its in vivo metabolite, 2‐chlorophenyl‐bisphenyl‐methanol (CBM) and the novel analogues, UCL 1880 and UCL 2027, inhibited the sIAHP with similar IC50s (1 – 2 μM).

Clotrimazole and CBM also inhibited the high voltage‐activated (HVA) Ca2+ current in pyramidal neurones with IC50s of 4.7 μM and 2.2 μM respectively. UCL 1880 was a less effective Ca2+ channel blocker, reducing the HVA Ca2+ current by 50% at 10 μM. At concentrations up to 10 μM, UCL 2027 had no effect on the Ca2+ current, indicating that its effects on the sIAHP were independent of Ca2+ channel block.

Clotrimazole also inhibited both the outward holding current (IC50=2.8 μM) present at a potential of −50 mV and the apamin‐sensitive medium AHP (mAHP; IC50∼amp;10 μM). The other clotrimazole analogues tested had smaller effects on these two currents. The present work also shows that 100 nM UCL 1848, an inhibitor of apamin‐sensitive conductances, abolishes the mAHP.

Currents were recorded from HEK293 cells transfected with hSK1 and rSK2. The SK currents were very sensitive to inhibition by UCL 1848 but were not significantly reduced by the sIAHP inhibitor, UCL 2027 (10 μM). 10 μM UCL 1880 reduced the hSK1 current by 40%.

UCL 2027 appears to be the first relatively selective blocker of the sAHP to be described. Furthermore, the ability of UCL 2027 to block the sAHP with minimal effect on SK1 channel activity questions the role of this channel in the sAHP.

British Journal of Pharmacology (2001) 132, 889–898; doi:10.1038/sj.bjp.0703895
Ligand internalization and recycling by human recombinant somatostatin type 4 (h sst4) receptors expressed in CHO‐K1 cells
Authors: Smalley K S M , Koenig J A , Feniuk W , Humphrey P P A
Published: January 2005

There is controversy as to whether somatostatin sst4 receptors internalize. In this study, CHO‐K1 cells expressing human sst4 receptor (CHOsst4 cells) cells internalized [125I]‐[11Tyr]‐SRIF in a time‐dependent manner, reaching a steady state at 60 min (1.4±0.1×104 molecules internalized per cell). Internalization was blocked by hypertonic sucrose (0.5 M), ATP depletion or by decreasing the temperature to 4°C.

Internalization of [125I]‐[11Tyr]‐SRIF was also inhibited (pIC50 values) by increasing concentrations of SRIF (7.74), L‐362855 (6.27) and NNC‐296100 (6.50) with pIC50 values approximately 10 fold lower than those obtained for inhibition of [125I]‐[11Tyr]‐SRIF binding to membrane homogenates.

Internalized ligand recycled rapidly to the extracellular media (t1/2 3.9±0.7 min) with only 6.8±0.6% of internalized radioactivity remaining in the cell after 45 min.

Confocal microscopy of permeabilized, HA‐epitope tagged CHOsst4 cells labelled with a Cy‐3 conjugated antibody revealed little internal immunostaining after SRIF (1 μM) treatment, consistent with the small proportion of receptors (3.5%) estimated to be internalized by radioimmunoassay.

In summary, CHOsst4 cells internalized [125I]‐[11Tyr]‐SRIF in a clathrin‐ and ATP‐dependent manner with subsequent rapid recycling to the extracellular medium. Rapid receptor recycling and the consequent low proportion of receptors internalized at any one time may explain the inability to visualize internalized receptors by confocal microscopy. It seems unlikely therefore that the marked receptor desensitization observed in CHOsst4 cells following SRIF treatment can be accounted for by a decrease in cell surface receptor expression.

British Journal of Pharmacology (2001) 132, 1102–1110; doi:10.1038/sj.bjp.0703896
Potentiation of the antinociceptive effect of clomipramine by a 5‐ht1A antagonist in neuropathic pain in rats
Authors: Ardid D , Alloui A , Brousse G , Jourdan D , Picard P , Dubray C , Eschalier A
Published: January 2005

The benefit of antidepressant treatment in human neuropathic pain is now well documented, but the effect is limited and slow to appear. It has been demonstrated that the association of a 5‐HT1A antagonist and a serotoninergic antidepressant reduced the delay of action and increases the thymoanaleptic effect of the drug.

The purpose of this work was to evaluate the combination of an antidepressant and a 5‐HT1A antagonist in animal models of chronic neuropathic pain. We studied the antinociceptive effect of the co‐administration of clomipramine and a 5‐HT1A antagonist (WAY 100,635) in a pain test applied in normal rats and in two models of neurogenic sustained pain (mononeuropathic and diabetic rats).

The results show an increase in the antinociceptive effect of acutely injected clomipramine due to WAY 100,635 in these models, which is majored when the two drugs are repeatedly injected. The 5‐HT1A antagonist reduced the delay of onset and increased the maximal antinociceptive effect of clomipramine.

These new findings argue for using the combination of an antidepressant and a 5‐HT1A antagonist in human neuropathic pain therapy.

British Journal of Pharmacology (2001) 132, 1118–1126; doi:10.1038/sj.bjp.0703897
Glucuronide and glucoside conjugation of mycophenolic acid by human liver, kidney and intestinal microsomes
Authors: Shipkova M , Strassburg C P , Braun F , Streit F , Gröne H‐J , Armstrong V W , Tukey R H , Oellerich M , Wieland E
Published: January 2005

Mycophenolic acid (MPA) is primarily metabolized to a phenolic glucuronide (MPAG) as well as to two further minor metabolites: an acyl glucuronide (AcMPAG) and a phenolic glucoside (MPAG1s). This study presents investigations of the formation of these metabolites by human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as by recombinant UDP‐glucuronosyltransferases.

HLM (n=5), HKM (n=6), HIM (n=5) and recombinant UGTs were incubated in the presence of either UDP‐glucuronic acid or UDP‐glucose and various concentrations of MPA. Metabolite formation was followed by h.p.l.c.

All microsomes investigated formed both MPAG and AcMPAG. Whereas the efficiency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation efficiency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM.

HKM produced a second metabolite when incubated with MPA and UDP‐glucose, which was labile to alkaline treatment. Mass spectrometry of this metabolite in the negative ion mode revealed a molecular ion of m/z 481 compatible with an acyl glucoside conjugate of MPA.

All recombinant UGTs investigated were able to glucuronidate MPA with KM values ranging from 115.3 to 275.7 μM l−1 and Vmax values between 29 and 106 pM min−1 mg protein−1.

Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a significant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.

British Journal of Pharmacology (2001) 132, 1027–1034; doi:10.1038/sj.bjp.0703898
The effects of pH on the interaction between capsaicin and the vanilloid receptor in rat dorsal root ganglia neurons
Authors: McLatchie L M , Bevan S
Published: January 2005

The vanilloid receptor of sensory neurons is a polymodal nociceptor sensitive to capsaicin, protons, heat and anandamide. Although it is known that interaction occurs between these different mediators the mechanism by which this occurs is poorly understood. In this study capsaicin elicited currents were recorded from vanilloid receptors found in adult rat isolated dorsal root ganglia (DRG) neurons under conditions of varying pH and the mechanism whereby protons can modulate this capsaicin response investigated.

Under whole‐cell voltage clamp, modulating extracellular pH shifted the position of the capsaicin log(concentration)‐response curve. Acidification from pH 9.0 to pH 5.5 lowered the EC50 values from 1150±250 nM to 5±2 nM with coincident change in the mean apparent slope factor from 2.3±0.3 to 0.9±0.2 and no change in maximal response.

The magnitude of the potentiation seen on reducing extracellular pH was not significantly affected by changes in extracellular calcium and magnesium concentration.

The response to capsaicin was not potentiated by a reduction in intracellular pH suggesting a site of action more accessible from the extracellular than the intracellular side of the membrane.

Potentiation by low pH was voltage independent indicating a site of action outside the membrane electric field.

At the single channel level, reducing extracellular pH increased channel open probability but had no significant effect on single channel conductance or open time.

These results are consistent with a model in which, on reducing extracellular pH, the vanilloid receptor in rat DRG neurons, changes from a state with low affinity for capsaicin to one with high affinity, coincident with a loss of cooperativity. This effect, presumed to be proton mediated, appears to involve one or more sites with pKa value 7.4 – 7.9, outside the membrane electrical field on an extracellularly exposed region of the receptor protein.

British Journal of Pharmacology (2001) 132, 899–908; doi:10.1038/sj.bjp.0703900
Effects of KRN2391 on ionic currents in rabbit femoral arterial myocytes
Authors: Muraki Katsuhiko , Sasaoka Akiko , Watanabe Minoru , Imaizumi Yuji
Published: January 2005

The effects of KRN2391, an ATP‐sensitive K+ channel opener (KCO) which also acts as a nitrate, on ionic membrane currents in rabbit femoral arterial myocytes were examined.

Under whole‐cell clamp conditions where cells were superfused with physiological salts solution containing 5.9 mM K+, KRN2391 elicited an outward current at a holding potential of −30 mV. KRN2391‐induced current had a reversal potential of −78 mV and was abolished by glibenclamide (glib). KRN2391 was approximately 25 times more potent than nicorandil to activate an ATP‐sensitive K+ current (IKATP). On the other hand, 10 μM KRN2391 did not affect either voltage‐dependent Ca2+ or delayed rectifier K+ channel currents.

In the inside‐out patch configuration, KRN2391 activated 47 pS K+ channels in the presence of nucleotide diphosphates (NDPs) under the symmetrical 140 mM K+ conditions. Glib and intracellular ATP reversibly inhibited the activity of the 47 pS K+ channels.

The 47 pS K+ channels activated by KRN2391 are similar in their conductance and other properties to NDP‐sensitive K+ channels (KNDP channels) described in other smooth muscles and the cloned channels. KRN2391 is a potent activator of the 47 pS K+ channels and the activation can contribute to the KRN2391‐induced vasodilation in arterial muscles.

British Journal of Pharmacology (2001) 132, 1154–1160; doi:10.1038/sj.bjp.0703903
Alteration of excitation‐contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine
Authors: De Luca Annamaria , Pierno Sabata , Liantonio Antonella , Cetrone Michela , Camerino Claudia , Simonetti Simonetta , Papadia Francesco , Camerino Diana Conte
Published: January 2005

No clear data is available about functional alterations in the calcium‐dependent excitation‐contraction (e‐c) coupling mechanism of dystrophin‐deficient muscle of mdx mice.

By means of the intracellular microelectrode ‘point’ voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle fibres of dystrophic mdx mouse of two different ages: 8–12 weeks, during the active regeneration of hind limb muscles, and 6–8 months, when regeneration is complete.

The EDL muscle fibres of 8–12‐week‐old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction‐ and relaxation‐related calcium movements) with respect to control mice of 6–8 months. However, at both ages, the EDL muscle fibres of mdx mice contracted at more negative potentials with respect to age‐matched controls and had markedly slower time constants to reach the rheobase.

The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e‐c coupling, was without effect on 6–8‐month‐old wildtype EDL muscle, while it significantly ameliorated the MT of mdx mouse.

HPLC determination of taurine content at 6–8 months showed a significant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue difficulty in retaining appropriate levels of the amino acid.

The data is consistent with a permanent alteration of e‐c coupling in mdx EDL muscle fibres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid.

British Journal of Pharmacology (2001) 132, 1047–1054; doi:10.1038/sj.bjp.0703907
Effects of the oestrous cycle and gender on acute vasodilatory responses of isolated pressurized rat mesenteric arteries to 17 β‐oestradiol
Authors: Shaw Linda , Taggart Michael , Austin Clare
Published: January 2005

The influence of the oestrous cycle and gender on responses of isolated pressurized mesenteric arteries to acute 17 β‐oestradiol was investigated.

All vessels, pre‐contracted with 60 mM KCl or 10 μM U46619 (9,11 dideoxy‐11α, 9α‐epoxy methano‐prostaglandin), exhibited concentration‐dependent vasodilatory responses to 17 β‐oestradiol (3–30 μM). The largest responses were seen in vessels from female rats in pro‐oestrous (38.9±5.4% U46619 max and 63.1±4.0% KCl max for 30 μM oestradiol), the smallest from animals in di‐oestrous (20.1±3.7% U46619 and 50.1±4.5% KCL–both P<0.05 cf pro‐oestrous (all n=8)). Responses of vessels from male rats were similar to those from pro‐oestrous rats (41.5±9.1% U46619 (n=10) and 54.9±2.9% KCl (n =8)).

All responsees were unaffected by inhibition of nitric oxide synthase (NOS).

Female rats in pro‐oestrous had the highest plasma concentrations of 17 β‐oestradiol and testosterone (40.76±4.73 pg ml−1 and 0.29±0.05 ng ml−1 respectively (n=8)) while those in di‐oestrous had the lowest (15.24±3.94 pg ml−1 for oestradiol and 0.08±0.03 ng ml−1 for testosterone (n=8)). In male rats the concentration of oestrogen was 10.29±1.21 pg ml−1 (n=7) while that of testosterone was 3.15±0.36 ng ml−1 (n=7).

Incubation of arteries isolated from male rats and from female rats in pro‐oestrous and di‐oestrous with testosterone (1 μM, 3 h) significantly enhanced the subsequent vasodilatory responses to acute 17 β‐oestradiol. Following incubation, the responses to 17 β‐oestradiol were similar in all groups.

These observations suggest that gender and the oestrous cycle may influence the vascular responses to acute 17 β‐oestradiol administration.

British Journal of Pharmacology (2001) 132, 1055–1062; doi:10.1038/sj.bjp.0703908
Mitochondrial involvement in cocaine‐treated rat hepatocytes: effect of N‐acetylcysteine and deferoxamine
Authors: Zaragoza Asunción , Díez‐Fernández Carmen , Alvarez Alberto M , Andrés David , Cascales María
Published: January 2005

The cytotoxicity of cocaine (0–1000 μM), was studied on parameters related to the mitochondrial role and the cascade of events that lead to apoptosis in hepatocyte cultures from phenobarbitone (PB) pretreated rats.

Cytotoxicity was dose‐dependent and LDH leakage was significantly enhanced above 100 μM cocaine. Apoptosis was visualized by DNA fragmentation on agarose gel, and appeared at 50 and 100 μM cocaine. Cocaine induced biphasic changes in mitochondrial transmembrane potential and significantly increased the mitochondrial release of cytochrome c, the caspase‐3 like DEVDase activity and the level of 20 kDa subunit, a product of pro‐caspase‐3 cleavage.

The protective effect of N‐acetylcysteine (NAC) and deferoxamine (DFO) on all these parameters confirmed the involvement of oxygen radicals in cocaine‐induced necrosis/apoptosis.

We conclude: first, that the biphasic changes recorded in mitochondrial inner membrane potential by the effect of cocaine, were parallel to apoptosis; second, that caspase‐3 activity and cleavage to it p20 subunit increased sharply in parallel to the translocation of cytochrome c from mitochondria to cytosol; and third, that the antioxidants, NAC or DFO exerted a noticeable protective role in counteracting the cytotoxicity of cocaine, these effects being more pronounced in the case of DFO than NAC.

These findings demonstrate that cocaine cytotoxicity involves mitochondrial damage.

British Journal of Pharmacology (2001) 132, 1063–1070; doi:10.1038/sj.bjp.0703909
The GR127935‐sensitive 5‐HT1 receptors mediating canine internal carotid vasoconstriction: resemblance to the 5‐HT1B, but not to the 5‐HT1D or 5‐ht1F, receptor subtype
Authors: Centurión David , Sánchez‐López Araceli , De Vries Peter , Saxena Pramod R , Villalón Carlos M
Published: January 2005

This study has further investigated the pharmacological profile of the GR127935‐sensitive 5‐HT1 receptors mediating vasoconstriction in the internal carotid bed of anaesthetized vagosympathectomized dogs.

One‐minute intracarotid infusions of the agonists 5‐hydroxytryptamine (5‐HT; 0.1–10 μg min−1; endogenous ligand) and sumatriptan (0.3–10 μg min−1; 5‐HT1B/1D), but not PNU‐142633 (1–1000 μg min−1; 5‐HT1D) or LY344864 (1–1000 μg min−1; 5‐ht1F), produced dose‐dependent decreases in internal carotid blood flow without changing blood pressure or heart rate.

The responses to 5‐HT were apparently resistant to blockade by i.v. administration of the antagonists SB224289 (300 μg kg−1; 5‐HT1B), BRL15572 (300 μg kg−1; 5‐HT1D) or ritanserin (100 μg kg−1; 5‐HT2). In contrast, the responses to sumatriptan were antagonized by SB224289, but not by BRL15572.

In the animals receiving SB224289, but not those receiving BRL15572, the subsequent administration of ritanserin abolished the 5‐HT‐induced vasoconstriction and unmasked a vasodilator component. Similarly, in ritanserin‐treated animals, the subsequent administration of SB224289, but not BRL15572, completely blocked the 5‐HT‐induced vasoconstriction, revealing vasodilatation. In animals receiving initially BRL15572, the subsequent administration of SB224289 did not affect (except at 10 μg min−1) the vasoconstrictor responses to 5‐HT.

Notably, in animals pretreated with 1000 μg kg−1 of mesulergine, a 5‐HT2/7 receptor antagonist, 5‐HT produced a dose‐dependent vasoconstriction, which was practically abolished by SB224289. After BRL15572, no further blockade was produced and the subsequent administration of ritanserin was similarly inactive.

These results suggest that the GR127935‐sensitive 5‐HT1 receptors mediating canine internal carotid vasoconstriction resemble the 5‐HT1B but not the 5‐HT1D or 5‐ht1F, receptor subtype.

British Journal of Pharmacology (2001) 132, 991–998; doi:10.1038/sj.bjp.0703913
Evidence for 5‐HT1B/1D and 5‐HT2A receptors mediating constriction of the canine internal carotid circulation
Authors: Centurión David , Ortiz Mario I , Sánchez‐López Araceli , De Vries Peter , Saxena Pramod R , Villalón Carlos M
Published: January 2005

The present study has investigated the preliminary pharmacological profile of the receptors mediating vasoconstriction to 5‐hydroxytryptamine (5‐HT) in the internal carotid bed of vagosympathectomised dogs.

One minute intracarotid infusions of the agonists 5‐HT (0.1–10 μg min−1), sumatriptan (0.3–10 μg min−1; 5‐HT1B/1D), 5‐methoxytryptamine (1–100 μg min−1; 5‐HT1, 5‐HT2, 5‐HT4, 5‐ht6 and 5‐HT7) or DOI (0.31–10 μg min−1; 5‐HT2), but not 5‐carboxamidotryptamine (0.01–0.3 μg min−1; 5‐HT1, 5‐ht5A and 5‐HT7), 1‐(m‐chlorophenyl)‐biguanide (mCPBG; 1–1000 μg min−1; 5‐HT3) or cisapride (1–1000 μg min−1; 5‐HT4), resulted in dose‐dependent decreases in internal carotid blood flow, without changing blood pressure or heart rate.

The vasoconstrictor responses to 5‐HT, which remained unaffected after saline, were resistant to blockade by i.v. administration of the antagonists ritanserin (100 μg kg−1; 5‐HT2A/2B/2C) in combination with tropisetron (3000 μg kg−1; 5‐HT3/4) or the cyclo‐oxygenase inhibitor, indomethacin (5000 μg kg−1), but were abolished by the 5‐HT1B/1D receptor antagonist, GR127935 (30 μg kg−1). Interestingly, after administration of GR127935, the subsequent administration of ritanserin unmasked a dose‐dependent vasodilator component. GR127935 or saline did not practically modify the vasoconstrictor effects of 5‐MeO‐T. In animals receiving GR127935, the subsequent administration of ritanserin abolished the vasoconstrictor responses to 5‐MeO‐T unmasking a dose‐dependent vasodilator component.

The vasoconstriction induced by sumatriptan was antagonized by GR127935, but not by ritanserin. Furthermore, ritanserin (100 μg kg−1) or ketanserin (100 μg kg−1; 5‐HT2A), but not GR127935, abolished DOI‐induced vasoconstrictor responses.

The above results suggest that 5‐HT‐induced internal carotid vasoconstriction is predominantly mediated by 5‐HT1B/1D and 5‐HT2A receptors.

British Journal of Pharmacology (2001) 132, 983–990; doi:10.1038/sj.bjp.0703914
The role of several α1‐ and α2‐adrenoceptor subtypes mediating vasoconstriction in the canine external carotid circulation
Authors: Willems Edwin W , Valdivia Luis Felipe , Saxena Pramod R , Villalón Carlos M
Published: January 2005

It has recently been shown that both α1‐ and α2‐adrenoceptors mediate vasoconstriction in the canine external carotid circulation. The present study set out to identify the specific subtypes (α1A, α1B and α1D as well as α2A, α2B and α2C) mediating the above response.

Consecutive 1 min intracarotid infusions of phenylephrine (α1‐adrenoceptor agonist) and BHT933 (α2‐adrenoceptor agonist) produced dose‐dependent decreases in external carotid blood flow, without affecting mean arterial blood pressure or heart rate.

The responses to phenylephrine were selectively antagonized by the antagonists, 5‐methylurapidil (α1A) or BMY7378 (α1D), but not by L‐765,314 (α1B), BRL44408 (α2A), imiloxan (α2B) or MK912 (α2C). In contrast, only BRL44408 or MK912 affected the responses to BHT933.

The above results support our contention that mainly the α1A, α1D, α2A and α2C‐adrenoceptor subtypes mediate vasoconstriction in the canine external carotid circulation.

British Journal of Pharmacology (2001) 132, 1292–1298; doi:10.1038/sj.bjp.0703915
Interaction of stilbene disulphonates with cloned KATP channels
Authors: Proks Peter , Jones Phillippa , Ashcroft Frances M
Published: January 2005

In this study, we tested the effects of the stilbene disulphonates DIDS and SITS on three different types of cloned KATP channel (Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2ΔC) heterologously expressed in Xenopus oocytes, with the aim of identifying the part of the channel which is involved in mediating disulphonate inhibition.

We found that the inhibitory site(s) for these drugs lies within the Kir6.2 subunit of the channel, although its properties are further modulated by the sulphonylurea (SUR) subunit. In particular, SUR2A reduces both the rate and extent of block, by impairing the ability of DIDS binding to produce channel closure.

The disulphonate‐binding site interacts with the ATP inhibitory site on Kir6.2 because ATP is able to protect against irreversible channel inhibition by disulphonates. This effect is not mimicked by tolbutamide (at a concentration that interacts with Kir6.2) and is abolished by mutations that render the channel ATP insensitive.

A number of point mutations in both the N and C termini of Kir6.2 reduced the extent and reversibility of channel inhibition by SITS. The results are consistent with the idea that residue C42 of Kir6.2 is likely to be involved in covalently linking of SITS to the channel.

Other types of Kir channel (Kir1.1, Kir2.1 and Kir4.1) were also irreversibly blocked by DIDS, suggesting that these channels may share common binding sites for these stilbene disulphonates.

British Journal of Pharmacology (2001) 132, 973–982; doi:10.1038/sj.bjp.0703916