bluuebunny/crossref_metadata_2025 huggingface dataset:
Pravastatin suppresses the interleukin‐8 production induced by thrombin in human aortic endothelial cells cultured with high glucose by inhibiting the p44/42 mitogen activated protein kinase
Authors: Takata Michiyo , Urakaze Masaharu , Temaru Rie , Yamazaki Katsuya , Nakamura Norio , Nobata Yuhko , Kishida Mika , Sato Akira , Kobayashi Masashi
Published: January 2005

3‐Hydroxy‐3‐methylglutaryl co‐enzyme A reductase inhibitors (statins) prevent the progression of atherosclerosis by lowering cholesterol. However, the effect of statins on the synthesis of pro‐inflammatory cytokines from endothelial cells has not yet been fully investigated. Here, we examined the effect of pravastatin, one of the statins, on IL‐8 synthesis induced by thrombin in human aortic endothelial cells (AoEC) cultured with high glucose concentrations.

Pravastatin significantly decreased the IL‐8 synthesis induced by thrombin.

Pravastatin inhibited the p44/42 MAP kinase activity induced by thrombin, but did not inhibit the p38 MAP kinase activity.

Translocation of ras protein from the cytosol to plasma membrane was inhibited by pravastatin.

Pravastatin inhibit the activator protein‐1 activity, but did not inhibit the activation of IκB‐α.

Dominant negative ras inhibited the p44/42 MAP kinase activity induced by PMA.

Our results suggest that pravastatin inhibits IL‐8 synthesis by blocking the ras‐MAP (p44/42) kinase pathway rather than nuclear factor‐κB. Pravastatin may prevent atherosclerosis not only by lowering cholesterol levels, but also by suppressing IL‐8 synthesis in AoEC through the inhibition of p44/42 MAP kinase, and this may be more beneficial in diabetic patients than in non‐diabetics.

British Journal of Pharmacology (2001) 134, 753–762; doi:10.1038/sj.bjp.0704305
Effects of dantrolene and its derivatives on Ca2+ release from the sarcoplasmic reticulum of mouse skeletal muscle fibres
Authors: Ikemoto Takaaki , Hosoya Takamitsu , Aoyama Hiroshi , Kihara Yasutaka , Suzuki Masaaki , Endo Makoto
Published: January 2005

We analysed the effect of dantrolene (Dan) and five newly synthesized derivatives (GIFs) on Ca2+ release from the sarcoplasmic reticulum (SR) of mouse skeletal muscle.

In intact muscles, GIF‐0185 reduced the size of twitch contraction induced by electrical stimulation to the same extent as Dan. GIF‐0082, an azido‐functionalized Dan derivative, also inhibited twitch contraction, although the extent of inhibition was less than that of Dan and of GIF‐0185.

In skinned fibres, Dan inhibited Ca2+‐induced Ca2+ release (CICR) under Mg2+‐free conditions at room temperature. In contrast, GIF‐0082 and GIF‐0185 showed no inhibitory effect on CICR under the same conditions.

Dan‐induced inhibition of CICR was not affected by the presence of GIF‐0082, whereas it was diminished in the presence of GIF‐0185.

GIF‐0082 and GIF‐0185 significantly inhibited clofibric acid (Clof)‐induced Ca2+ release, as did Dan.

Several Dan derivatives other than GIF‐0082 and GIF‐0185 showed an inhibitory effect on twitch tension but not on the CICR mechanism. All of these derivatives inhibited Clof‐induced Ca2+ release.

The magnitudes of inhibition of Clof‐induced Ca2+ release by all Dan derivatives were well correlated with those of twitch inhibition. This supports the notion that the mode of Clof‐induced opening of the RyR‐Ca2+ release channel may be similar to that of physiological Ca2+ release (PCR).

These results indicate that the difference in opening modes of the RyR‐Ca2+ release channel is recognized by certain Dan derivatives.

British Journal of Pharmacology (2001) 134, 729–736; doi:10.1038/sj.bjp.0704307
α2‐adrenoceptor antagonist properties of OPC‐28326, a novel selective peripheral vasodilator
Authors: Orito Kensuke , Kishi Masami , Imaizumi Takashi , Nakazawa Toru , Hashimoto Ayako , Mori Toyoki , Kambe Toshimi
Published: January 2005

Antagonistic properties of OPC‐28326 ([4‐(N‐methyl‐2‐phenylethylamino)‐1‐(3,5‐dimethyl‐4‐propionyl‐aminobenzoyl)] piperidine hydrochloride monohydrate), a selective peripheral vasodilator, were investigated by analysing the data from functional studies in various tissues from the rat and binding studies of the drug to α2‐adrenoceptor subtypes.

Using a human recombinant receptor and rat kidney cortex, we found that OPC‐28326 displays affinities to α2A‐, α2B‐ and α2C‐adrenoceptors with Ki values of 2040, 285, and 55 nM, respectively. The Ki values of yohimbine for α2A‐, α2B‐, and α2C‐adrenoceptors were 3.0, 2.0 and 11.0 nM, respectively.

B‐HT 920, an α2‐adrenoceptor agonist, produced a pressor response via peripheral postsynaptic α2‐adrenoceptor stimulation (thought to be an α2B‐subtype) in a reserpine‐pretreated pithed rat preparation. OPC‐28326 (3 – 30 mg kg−1, i.v.) and yohimbine (0.3 – 3 mg kg−1, i.v.) caused dose‐dependent rightward shift in the pressor dose‐response curve induced by B‐HT 920. The apparent pA2 values were 1.55 (0.87 – 2.75, 95% confidence interval) and 0.11 (0.06 – 0.21) mg kg−1, respectively. The potency of OPC‐28326 was about 14 times less than that of yohimbine.

Clonidine inhibited the tension developed by electrical stimulation, of the rat vas deferens, by its peripheral presynaptic α2A/D‐adrenoceptor action. OPC‐28326 (1 – 100 μM) and yohimbine (10 – 1000 nM) caused a rightward shift in the concentration‐response curve of clonidine. The pA2 values were 5.73 (5.54 – 5.91) and 7.92 (7.84 – 8.01), respectively, providing evidence for a potency of OPC‐28326 of about 155 times less than that of yohimbine.

Mydriasis was induced by brimonidine via stimulation of central α2A/D‐adrenoceptors in anaesthetized rats. Intravenous OPC‐28326 had no effect on this action, even at a very high dose of 10 mg kg−1 i.v., while yohimbine (0.1 – 0.3 mg kg−1 i.v.) inhibited mydriasis in a dose‐dependent manner, indicating that OPC‐28326 was at least 100 times less potent than yohimbine in regard to the anti‐mydriatic effect.

These data suggest that OPC‐28326 preferentially exerts peripheral and postsynaptic antagonistic actions on the α2B‐ and α2C‐adrenoceptor subtypes.

British Journal of Pharmacology (2001) 134, 763–770; doi:10.1038/sj.bjp.0704309
The role of sensory nerve endings in nerve growth factor‐induced airway hyperresponsiveness to histamine in guinea‐pigs
Authors: Vries Annick de , Rijnsoever Carolien van , Engels Ferdi , Henricks Paul A J , Nijkamp Frans P
Published: January 2005

Nerve growth factor induces an airway hyperresponsiveness in vivo in guinea‐pigs, as we have shown previously. Since antagonizing the neurokinin‐1 (NK1) receptor can prevent this NGF‐induced airway hyperresponsiveness and since sensory nerves release tachykinins, we investigated the role of sensory nerves in the NGF‐induced airway hyperresponsiveness.

We used isolated tracheal rings from guinea‐pigs to measure tracheal contractility. In these rings sensory nerve endings are present, but these endings lack any contact with their cell bodies.

In this in vitro system, NGF dose‐dependently induced a tracheal hyperresponsiveness to histamine. The NK1 receptor antagonist SR140333 could block the induction of tracheal hyperresponsiveness.

To further investigate the involvement of sensory nerve endings we used the cannabinoid receptor 1 (CB1) agonist R‐methanandamide to inhibit excitatory events at the nerve terminal. The CB1 receptor agonist was capable of blocking the tracheal hyperresponsiveness to NGF in the isolated system, as well as the airway hyperresponsiveness to NGF in vivo.

This indicates that NGF can induce an increase in airway responsiveness in the absence of sensory nerve cell bodies. NGF may act by increasing substance P release from sensory nerve endings, without upregulation of substance P in the neurons. Substance P in its turn is responsible for the induction of the NGF‐induced airway hyperresponsiveness.

British Journal of Pharmacology (2001) 134, 771–776; doi:10.1038/sj.bjp.0704310
Pharmacological comparison of LTB4‐induced NADPH oxidase activation in adherent and non‐adherent guinea‐pig eosinophils
Authors: Lynch Oonagh T , Giembycz Mark A , Barnes Peter J , Lindsay Mark A
Published: January 2005

Leukotriene B4 (LTB4) stimulation of guinea‐pig peritoneal eosinophils, induced a biphasic activation of the NADPH oxidase composed of a rapid (<3 min) phase mediated by non‐adherent cells and a sustained (3 – 120 min) phase mediated by CD11b/CD18 adherent eosinophils. Studies were undertaken to compare the intracellular mechanism that mediate these responses.

SB 203580 and PP1, inhibitors of p38 mitogen‐activated protein (MAP) kinase and the src‐family protein tyrosine kinases, respectively caused concentration‐dependent attenuation of both the rapid (SB203580: pD2=−6.31; PP1: pD2=−5.50) and sustained (SB203580: pD2=−6.50; PP1: pD2=−5.73) phases. Similarly, the MAP kinase kinase‐1 inhibitor, PD098059 produced partial inhibition of the both phases of superoxide generation.

The protein kinase C (PKC) inhibitors Ro‐31 8220, GF 109203X and Gö 6976 attenuated the rapid NADPH oxidase response (pD2s=−6.10, −6.72, −6.15 respectively) and, to a lesser extent, (pD2s=−5.54, −6.02, −6.51 respectively) the sustained phase.

An inhibitor of phosphatidylinositol 3‐kinase (PtdIns 3‐kinase), wortmannin caused concentration dependent attenuation of the sustained (pD2=−8.68) but not rapid phase of superoxide generation. In contrast, the syk kinase inhibitor, piceatannol abolished the rapid (pD2=−6.43) but not sustained respiratory responses.

This study demonstrates that LTB4‐induced superoxide generation from adherent and non‐adherent eosinophils is mediated via both common (p38 MAP kinase, MEK‐1, PKC and the src kinases) and divergent intracellular pathways (syk kinases and PtdIns 3‐kinase). This suggests the possibility of therapeutic intervention to selective attenuate activation of adherent tissue eosinophils.

British Journal of Pharmacology (2001) 134, 797–806; doi:10.1038/sj.bjp.0704314
Clonidine‐induced nitric oxide‐dependent vasorelaxation mediated by endothelial α2‐adrenoceptor activation
Authors: Figueroa Xavier F , Poblete M Inés , Boric Mauricio P , Mendizábal Victoria E , Adler‐Graschinsky Edda , Huidobro‐Toro J Pablo
Published: January 2005

To assess the involvement of endothelial α2‐adrenoceptors in the clonidine‐induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min−1). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA.

In phenylephrine‐precontracted mesenteries, clonidine elicited concentration‐dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 μM Lω‐nitro‐L‐arginine antagonized the clonidine‐induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine‐induced vasorelaxation.

In non‐contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123±13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, Lω‐nitro‐L‐arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine‐induced surge of NO. Ten μM ODQ obliterated the clonidine‐induced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 – 100 nM sildenafil increased tissue cyclic GMP accumulation without altering the clonidine‐induced NO release.

α2‐Adrenergic blockers antagonized the clonidine‐induced rise in NO. Consistent with a preferential α2D‐adrenoceptor activation, the KBs for yohimbine, rauwolscine, phentolamine, WB‐4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively.

Rat pretreatment with 100 mg kg−1 6‐hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9±15.5 pmol NO. Perfusion with 1 μM guanethidine or 1 μM guanethidine plus 1 μM atropine did not modify the NO surge evoked by 100 nM clonidine.

Clonidine and congeners activate endothelial α2D‐adrenoceptors coupled to the L‐arginine pathway, suggesting that the antihypertensive action of clonidine involves an endothelial vasorelaxation mediated by NO release, in addition to presynaptic mechanisms.

British Journal of Pharmacology (2001) 134, 957–968; doi:10.1038/sj.bjp.0704320
Opposing influences of glucocorticoids and interleukin‐1β on the secretion of growth hormone and ACTH in the rat in vivo: role of hypothalamic annexin 1
Authors: Philip J G , John C D , Cover P O , Morris J F , Christian H C , Flower R J , Buckingham J C
Published: January 2005

This study exploited established immunoneutralization protocols and an N‐terminal annexin 1 peptide (annexin 1Ac2 – 26) to advance our knowledge of the role of annexin 1 as a mediator of acute glucocorticoid action in the rat neuroendocrine system in vivo.

Rats were treated with corticosterone (500 μg kg−1, i.p.) or annexin 1Ac2 – 26 (0.1 – 10 ng rat−1, i.c.v.) and 75 min later with interleukin 1β (IL‐1β, 10 ng rat−1, i.c.v. or 500 μg kg−1, i.p). Blood was collected 1 h later for hormone immunoassay. Where appropriate, anti‐annexin 1 polyclonal antiserum (pAb) was administered subcutaneously or centrally prior to the steroid challenge.

Corticosterone did not affect the resting plasma corticotrophin (ACTH) concentration but suppressed the hypersecretion of ACTH induced by IL‐1β (i.p. or i.c.v.). Its actions were quenched by anti‐annexin 1 pAb (s.c. or i.c.v) and mimicked by annexin 1Ac2 – 26.

By contrast, corticosterone provoked an increase in serum growth hormone (GH) which was ablated by central but not peripheral administration of anti‐annexin 1 pAb. IL‐1β (i.c.v. or i.p.) did not affect basal GH but, when given centrally but not peripherally, it abolished the corticosterone‐induced hypersecretion of GH. Annexin 1Ac2 – 26 (i.c.v.) also produced an increase in serum GH which was prevented by central injection of IL‐1β.

The results support the hypothesis that the acute regulatory actions of glucocorticoids on hypothalamo‐pituitary‐adrenocortical function require annexin 1. They also provide novel evidence that the positive influence of the steroids on GH secretion evident within this timeframe is effected centrally via an annexin 1‐dependent mechanism which is antagonized by IL‐1β.

British Journal of Pharmacology (2001) 134, 887–895; doi:10.1038/sj.bjp.0704324
Mechanisms of anandamide‐induced vasorelaxation in rat isolated coronary arteries
Authors: White Richard , Vanessa Ho W ‐S , Bottrill Fiona E , Ford William R , Hiley C Robin
Published: January 2005

The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration‐dependent relaxation of 5‐HT‐precontracted, myograph‐mounted, segments of rat left anterior descending coronary artery.

This relaxation was endothelium‐independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 μM), and mimicked by the non‐hydrolysable anandamide derivative, methanandamide.

Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 μM), but unaffected by AM 251 (1 μM) and AM 630 (1 μM), more selective antagonists of cannabinoid CB1 and CB2 receptors respectively. Palmitoylethanolamide, a selective CB2 receptor agonist, did not relax precontracted coronary arteries.

Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 μM) or blockade of vanilloid VR1 receptors with capsazepine (5 μM). Nevertheless capsaicin relaxed coronary arteries in a concentration‐dependent and capsazepine‐sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine‐precontracted rat small mesenteric arteries.

Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca2+‐activated K+ channels (BKCa). Gap junction inhibition with 18α‐glycyrrhetinic acid (100 μM) did not affect anandamide relaxations.

This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide‐induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB1 or CB2 receptors, but may involve activation of BKCa. Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.

British Journal of Pharmacology (2001) 134, 921–929; doi:10.1038/sj.bjp.0704333
PKC‐ and ERK‐dependent activation of IκB kinase by lipopolysaccharide in macrophages: enhancement by P2Y receptor‐mediated CaMK activation
Authors: Chen Bing‐C , Lin Wan‐W
Published: January 2005

Although accumulating studies have identified IκB kinase (IKK) to be essential for controlling NF‐κB activity in response to several cytokines, the upstream kinases that control IKK activity are still not completely known. We have previously reported that G protein‐coupled P2Y6 receptor activation by UTP potentiates lipopolysaccharide (LPS)‐induced IκB phosphorylation and degradation, and NF‐κB activation in J774 macrophages. In this study, we investigated the upstream kinases for IKK activation by UTP and LPS.

In murine J774 macrophages, LPS‐induced NF‐κB activation was inhibited by the presence of PDTC, D609, Ro 31‐8220, PD 098059 and SB 203580.

Accompanying NF‐κB activation, LPS induced IκB degradation and IKK activation were reduced by PDTC, D609, Ro 31‐8220 and PD 098059, but not by SB 203580.

Although UTP itself slightly induced IKK activation, this response was synergistic with LPS. BAPTA/AM and KN‐93 (a calcium/calmodulin‐dependent protein kinase (CaMK) inhibitor) attenuated UTP‐ but not LPS‐stimulated IKK activity. Synergistic IKK activation between LPS and thapsigargin was further demonstrated in peritoneal macrophages.

LPS and UTP co‐stimulation additively increased p65 NF‐κB phosphorylation. In vitro kinase assays revealed that LPS and UTP induced extracellular signal‐regulated protein kinase (ERK) and p38 mitogen‐activated protein kinase activation were respectively inhibited by PD098059 and SB 203580.

Taken together, we demonstration that Gq protein‐coupled P2Y6 receptor activation can potentiate LPS‐stimulated IKK activity. While PKC and ERK participate in IKK activation by LPS and UTP, the phosphatidylinositide‐phospholipase C‐dependent activation of CaMK plays a major role in UTP potentiation of the LPS response.

British Journal of Pharmacology (2001) 134, 1055–1065; doi:10.1038/sj.bjp.0704334
Release of non‐neuronal acetylcholine from the isolated human placenta is mediated by organic cation transporters
Authors: Wessler Ignaz , Roth Elisabeth , Deutsch Carola , Brockerhoff Peter , Bittinger Fernando , Kirkpatrick Charles James , Kilbinger Heinz
Published: January 2005

The release of acetylcholine was investigated in the human placenta villus, a useful model for the characterization of the non‐neuronal cholinergic system.

Quinine, an inhibitor of organic cation transporters (OCT), reduced acetylcholine release in a reversible and concentration‐dependent manner with an IC50 value of 5 μM. The maximal effect, inhibition by 99%, occurred at a concentration of 300 μM.

Procaine (100 μM), a sodium channel blocker, and vesamicol (10 μM), an inhibitor of the vesicular acetylcholine transporter, were ineffective.

Corticosterone, an inhibitor of OCT subtype 1, 2 and 3 reduced acetylcholine in a concentration‐dependent manner with an IC50 value of 2 μM.

Substrates of OCT subtype 1, 2 and 3 (amiloride, cimetidine, guanidine, noradrenaline, verapamil) inhibited acetylcholine release, whereas carnitine, a substrate of subtype OCTN2, exerted no effect.

Long term exposure (48 and 72 h) of villus strips to anti‐sense oligonucleotides (5 μM) directed against transcription of OCT1 and OCT3 reduced the release of acetylcholine, whereas OCT2 anti‐sense oliogonucleotides were ineffective.

It is concluded that the release of non‐neuronal acetylcholine from the human placenta is mediated via organic cation transporters of the OCT1 and OCT3 subtype.

British Journal of Pharmacology (2001) 134, 951–956; doi:10.1038/sj.bjp.0704335
Reciprocal regulation of cyclic GMP content by cyclic GMP‐phosphodiesterase and guanylate cyclase in SHR with CsA‐induced nephrotoxicity
Authors: Hosogai Naomi , Seki Jiro , Goto Toshio
Published: January 2005

The effect of the immunosuppressant drug, cyclosporin A (CsA), on the nitric oxide (NO)‐cyclic GMP pathway was examined in spontaneous hypertensive rats (SHR).

CsA (50 mg kg−1) treatment for 14 days induced typical CsA nephrotoxicity, which was characterized by morphological changes in the glomerulus and proximal tubule as well as an abnormality of creatinine clearance, FENa and BUN.

CsA significantly decreased both NOS activity in the kidney and NOx contents in urine, but significantly increased cyclic GMP content in the kidney.

A marked change in two kinds of enzyme, which contribute towards the increase in cyclic GMP in tissue, namely, a decrease in cyclic GMP‐phosphodiesterase activity and increase in guanylate cyclase activity, was observed in the kidney treated with CsA.

In the isolated perfused kidney, a decreased in perfusion pressure induced by SNP in the kidney isolated from CsA group was significantly greater than that of control.

There seem to exist a reciprocal mechanism to maintain cyclic GMP content via both a decrease in cyclic GMP degradation and an increase in synthesis of cyclic GMP in the kidney treated with CsA. This mechanism is likely to be playing an important role to regulate the homeostasis in the kidney with CsA nephrotoxicity.

British Journal of Pharmacology (2001) 134, 995–1002; doi:10.1038/sj.bjp.0704337
Quantitative aspects of the inhibition by NG‐monomethyl‐L‐arginine of responses to endothelium‐dependent vasodilators in human forearm vasculature
Authors: Dawes M , Chowienczyk P J , Ritter J M
Published: January 2005

NG‐monomethyl‐L‐arginine (L‐NMMA) constricts human forearm resistance vasculature and selectively attenuates vasodilator responses to endothelium‐dependent vasodilators. Incomplete inhibition of such responses could be due to an inadequate dose of L‐NMMA or to NO‐independent vasodilator mechanisms.

This study sought to determine doses of L‐NMMA that are maximally effective in reducing basal and stimulated forearm blood flow. Drugs were infused via the brachial artery in 32 healthy men. Acetylcholine (11 – 330 nmol min−1) was compared with albuterol (0.33 – 10 nmol min−1), and nitroprusside (1.7 – 20 nmol min−1).

The effect of L‐NMMA on basal flow approached maximum (53±2% reduction) at a dose of 16 μmol min−1. L‐NMMA (16 μmol min−1) did not significantly influence responses to nitroprusside, but antagonized acetylcholine and albuterol (each P<0.001, by repeated measures analysis of variance).

Inhibition of acetylcholine by L‐NMMA (16 μmol min−1) was strongly influenced by acetylcholine dose (73±7% inhibition at 11 nmol min−1, P<0.01; 4±11% inhibition at 330 nmol min−1, P=NS, Student's paired t‐test). Significant inhibition of albuterol was observed at all doses.

A higher dose of L‐NMMA (64 μmol min−1) did not significantly inhibit the response to acetylcholine (330 nmol min−1). Responses to this dose of acetylcholine were unaffected by a cyclo‐oxygenase (COX) inhibitor (indometacin) alone but combined COX and NO inhibition attenuated acetylcholine responses by 42±19%, implying that there is a compensatory increase in the contribution of prostaglandins or NO to acetylcholine‐induced dilatation when one or other pathway is inhibited.

British Journal of Pharmacology (2001) 134, 939–944; doi:10.1038/sj.bjp.0704338
Inhibitory effect of palmitoylethanolamide on gastrointestinal motility in mice
Authors: Capasso Raffaele , Izzo Angelo A , Fezza Filomena , Pinto Aldo , Capasso Francesco , Mascolo Nicola , Di Marzo Vincenzo
Published: January 2005

We have studied the effect of palmitoylethanolamide (PEA, 2.5 – 30 mg kg−1, i.p.) on upper gastrointestinal transit in control mice and in mice with chronic intestinal inflammation induced by croton oil.

PEA significantly and dose‐dependently decreased intestinal transit. The inhibitory effect of PEA (10 mg kg−1) was not modified by the cannabinoid CB1 receptor antagonist SR141716A (0.3 mg kg−1, i.p.), the cannabinoid CB2 receptor antagonist SR144528 (1 mg kg−1, i.p.), NG‐nitro‐L‐arginine methyl ester (L‐NAME, 25 mg kg−1, i.p.), yohimbine (1 mg kg−1, i.p.), naloxone (2 mg kg−1, i.p.) or hexamethonium (1 mg kg−1, i.p.).

PEA levels were significantly decreased in the small intestine of croton oil‐treated mice. In these animals, PEA also inhibited motility and this effect was not counteracted by SR141716A (0.3 mg kg−1), or SR144528 (1 mg kg−1).

Pre‐treatment of mice with the amidase inhibitor phenylmethyl sulphonil fluoride (PMSF, 30 mg kg−1, i.p.) did not modify the inhibitory effect of PEA, either in control or in mice with inflammation.

It is concluded that PEA inhibits intestinal motility with a peripheral mechanism independent from cannabinoid receptor activation. The decreased levels of PEA in croton oil‐treated might contribute, at least in part, to the exaggerated transit observed during chronic intestinal inflammation.

British Journal of Pharmacology (2001) 134, 945–950; doi:10.1038/sj.bjp.0704339
Anandamide‐induced relaxation of sheep coronary arteries: the role of the vascular endothelium, arachidonic acid metabolites and potassium channels
Authors: Grainger J , Boachie‐Ansah G
Published: January 2005

The effects of the endocannabinoid, anandamide, and its metabolically stable analogue, methanandamide, on induced tone were examined in sheep coronary artery rings in vitro.

In endothelium‐intact rings precontracted to the thromboxane A2 mimetic, U46619, anandamide (0.01 – 30 μM) induced slowly developing concentration‐dependent relaxations (pEC50 [negative log of EC50]=6.1±0.1; Rmax [maximum response]=81±4%). Endothelium denudation caused a 10 fold rightward shift of the anandamide concentration‐relaxation curve without modifying Rmax. Methanandamide was without effect on U46619‐induced tone.

The anandamide‐induced relaxation was unaffected by the cannabinoid receptor antagonist, SR 141716A (3 μM), the vanilloid receptor antagonist, capsazepine (3 and 10 μM) or the nitric oxide synthase inhibitor, L‐NAME (100 μM).

The cyclo‐oxygenase inhibitor, indomethacin (3 and 10 μM) and the anandamide amidohydrolase inhibitor, PMSF (70 and 200 μM), markedly attenuated the anandamide response. The anandamide transport inhibitor, AM 404 (10 and 30 μM), shifted the anandamide concentration‐response curve to the right.

Precontraction of endothelium‐intact rings with 25 mM KCl attenuated the anandamide‐induced relaxations (Rmax=7±7%), as did K+ channel blockade with tetraethylammonium (TEA; 3 μM) or iberiotoxin (100 nM). Blockade of small conductance, Ca2+‐activated K+ channels, delayed rectifier K+ channels, KATP channels or inward rectifier K+ channels was without effect.

These data suggest that the relaxant effects of anandamide in sheep coronary arteries are mediated in part via the endothelium and result from the cellular uptake and conversion of anandamide to a vasodilatory prostanoid. This, in turn, causes vasorelaxation, in part, by opening potassium channels.

British Journal of Pharmacology (2001) 134, 1003–1012; doi:10.1038/sj.bjp.0704340
Structure‐activity relationships of diadenosine polyphosphates (ApnAs), adenosine polyphospho guanosines (ApnGs) and guanosine polyphospho guanosines (GpnGs) at P2 receptors in the rat mesenteric arterial bed
Authors: Ralevic Vera , Jankowski Joachim , Schlüter Hartmut
Published: January 2005

Vascular effects of diadenosine polyphosphates (ApnAs), adenosine polyphospho guanosines (ApnGs) and guanosine polyphospho guanosines (GpnGs), novel families of naturally‐occurring signalling molecules, were investigated in methoxamine preconstricted rat isolated perfused mesenteric arterial beds.

Three different types of response were elicited by ApnAs and ApnGs. Those with a short polyphosphate chain (n=2 – 3) elicited vasorelaxation. Ap3A was more potent than Ap2A, and both were more potent than the corresponding ApnG. Relaxations to Ap3A and Ap3G, but not to Ap2A and Ap2G, were blocked by endothelium removal and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), a P2 receptor antagonist.

Longer polyphosphate chain ApnAs and ApnGs (n=4 – 6) elicited dose‐dependent vasoconstriction followed by prolonged vasorelaxation, with a potency order for both types of response of Ap5AAp6A>Ap4A. A similar order and potency was observed for ApnGs. Contractions and prolonged relaxations were blocked by PPADS and P2X1 receptor desensitization with α,β‐methylene ATP (α,β‐meATP), and were largely endothelium‐independent.

In the presence of α,β‐meATP rapid relaxations to contractile ApnAs and ApnGs (n=4 – 6) were revealed.

GpnGs were virtually inactive, except for Gp2G which elicited vasoconstriction via PPADS‐ and α,β‐meATP‐sensitive smooth muscle P2X1‐like receptors.

These data show that, as with ApnAs, the length of the polyphosphate chain (n) is an important determinant of the activity of ApnGs at P2 receptors in the rat mesenteric arterial bed. When the chain is short (n=2 – 3) the purines elicit rapid vasorelaxation, which for Ap3A and Ap3G is mediated via endothelial P2Y1‐like receptors. When the chain is long (n=4 – 6) ApnAs and ApnGs elicit vasoconstriction via P2X1‐like receptors, followed by prolonged endothelium‐independent vasorelaxation. Rapid relaxation to contractile dinucleotides (n=4 – 6) is revealed by block of vasoconstriction. Regarding the purine moiety, one adenine is crucial and sufficient for vasoactivity as GpnGs were largely inactive, and ApnAs and ApnGs approximately equipotent.

British Journal of Pharmacology (2001) 134, 1073–1083; doi:10.1038/sj.bjp.0704341
Protein kinase C involvement in aloe‐emodin‐ and emodin‐induced apoptosis in lung carcinoma cell
Authors: Lee Hong‐Zin
Published: January 2005

This study demonstrated aloe‐emodin‐ and emodin‐induced apoptosis in lung carcinoma cell lines CH27 (human lung squamous carcinoma cell) and H460 (human lung non‐small cell carcinoma cell). Aloe‐emodin‐ and emodin‐induced apoptosis was characterized by nuclear morphological changes and DNA fragmentation.

During apoptosis, an increase in cytochrome c of cytosolic fraction and activation of caspase‐3, identified by the cleavage of its proform, were observed.

To elucidate whether the expression of protein kinase C (PKC) isozymes are involved in aloe‐emodin‐ and emodin‐induced apoptosis, this study examined the changes of PKC isozymes by Western blotting techniques during aloe‐emodin‐ and emodin‐induced apoptosis.

The expression of PKC isozymes involved in aloe‐emodin‐ and emodin‐induced apoptosis of CH27 and H460 cells. In this study, aloe‐emodin and emodin induced the changes of each of PKC isozymes in CH27 and H460 cells.

The decrease in the expression of PKCδ and ε may play a critical role in aloe‐emodin‐ and emodin‐induced apoptosis in CH27 and H460 cells.

The present study also demonstrated that PKC stimulation occurs at a site downstream of caspase‐3 in the emodin‐mediated apoptotic pathway.

British Journal of Pharmacology (2001) 134, 1093–1103; doi:10.1038/sj.bjp.0704342
Role of von Willebrand factor in tumour cell‐induced platelet aggregation: differential regulation by NO and prostacyclin
Authors: Jurasz Paul , Stewart Michael W , Radomski Anna , Khadour Fadi , Duszyk Marek , Radomski Marek W
Published: January 2005

We have studied the effects of a novel agonist, solid‐phase von Willebrand Factor (sVWF), on tumour cell‐induced platelet aggregation (TCIPA).

Washed platelet suspensions were obtained from human blood and the effects of HT‐1080 human fibrosarcoma cells and sVWF on platelets were studied using aggregometry, phase‐contrast microscopy, and flow cytometry.

Incubation of platelets with sVWF (1.2 μg ml−1) and HT‐1080 cells (5×103 ml−1) resulted in a two‐phased reaction characterized first by the adhesion of platelets to sVWF, then by aggregation.

TCIPA in the presence of sVWF was inhibited by S‐nitroso‐glutathione (GSNO, 100 μM) and prostacyclin (PGI2, 30 nM).

Platelet activation in the presence of tumour cells and sVWF resulted in the decreased surface expression of platelet glycoprotein (GP)Ib and up‐regulation of GPIIb/IIIa receptors.

Pre‐incubation of platelets with PGI2 (30 nM) resulted in inhibition of sVWF‐tumour cell‐stimulated platelet surface expression of GPIIb/IIIa as measured by flow cytometry using antibodies directed against both non‐activated and activated receptor. In contrast, GSNO (100 μM) did not affect sVWF‐tumour cell‐stimulated platelet surface expression of GPIIb/IIIa.

Flow cytometry performed with PAC‐1 antibodies that bind only to the activated GPIIb/IIIa revealed that GSNO (100 μM) caused inhibition of activation of GPIIb/IIIa.

The inhibitors exerted no significant effects on TCIPA‐mediated changes in GPIb.

Thus, sVWF potentiates the platelet‐aggregatory activity of HT‐1080 cells and these effects appear to be mediated via up‐regulation of platelet GPIIb/IIIa.

Prostacyclin and NO inhibit TCIPA‐sVWF‐mediated platelet aggregation. The mechanisms of inhibition of this aggregation by PGI2 differ from those of NO.

British Journal of Pharmacology (2001) 134, 1104–1112; doi:10.1038/sj.bjp.0704343
Inhibition of acetylcholine‐activated K+ currents by U73122 is mediated by the inhibition of PIP2‐channel interaction
Authors: Cho Hana , Youm Jae Boum , Ryu Shin Young , Earm Yung E , Ho Won‐Kyung
Published: January 2005

We have investigated the effect of U73122, a specific inhibitor of phospholipase C (PLC), on acetylcholine‐activated K+ currents (IKACh) in mouse atrial myocytes.

In perforated patch clamp mode, IKACh was activated by 10 μM acetylcholine. When atrial myocytes were pretreated with U73122 or U73343, IKACh was inhibited dose‐dependently (half‐maximal inhibition at 0.12±0.0085 and 0.16±0.0176 μM, respectively). The current‐voltage relationships for IKACh in the absence and in the presence of U73122 showed that the inhibition occurred uniformly from −120 to +40 mV, indicating a voltage‐independent inhibition.

When U73122 was applied after IKACh reached steady‐state, a gradual decrease in IKACh was observed. The time course of the current decrease was well fitted to a single exponential, and the rate constant was proportional to the concentration of U73122.

When KACh channels were directly activated by adding 1 mM GTPγS to the bath solution in inside‐out patches, U73122 (1 μM) decreased the open probability significantly without change in mean open time. When KACh channels were activated independently of G‐protein activation by 20 mM Na+, open probability was also inhibited by U73122.

Voltage‐activated K+ currents and inward rectifying K+ currents were not affected by U73122.

These findings show that inhibition by U73122 and U73343 of KACh channels occurs at a level downstream of the action of Gβγ or Na+ on channel activation. The interference with phosphatidylinositol 4,5‐bisphosphate (PIP2)‐channel interaction can be suggested as a most plausible mechanism.

British Journal of Pharmacology (2001) 134, 1066–1072; doi:10.1038/sj.bjp.0704347
Cellular target of voltage and calcium‐dependent K+ channel blockers involved in EDHF‐mediated responses in rat superior mesenteric artery
Authors: Ghisdal Philippe , Morel Nicole
Published: January 2005

We have investigated the cellular target of K+ channel blockers responsible for the inhibition of the EDHF‐mediated relaxation in the rat mesenteric artery by studying their effects on tension, smooth muscle cell (SMC) membrane potential and endothelial cell Ca2+ signal ([Ca2+]endo).

In arteries contracted with prostaglandin F2α (2.5 – 10 μM), relaxation evoked by ACh (0.01 – 3 μM) was abolished by a combination of charybdotoxin (ChTX, 0.1 μM) plus apamin (Apa, 0.1 μM) and was inhibited by 68±6% (n=6) by 4‐aminopyridine (4‐AP, 5 mM).

ACh (0.001 – 3 μM) increased [Ca2+]endo and hyperpolarized SMCs with the same potency, the pD2 values were equal to 7.2±0.08 (n=4) and 7.2±0.07 (n=9), respectively. SMCs hyperpolarization to ACh (1 μM) was abolished by high K+ solution or by ChTX/Apa. It was decreased by 66±5% (n=6) by 4‐AP.

The increase in [Ca2+]endo evoked by ACh (1 μM) was insensitive to ChTX/Apa but was depressed by 58±16% (n=6) and 27±4% (n=7) by raising external K+ concentration and by 4‐AP, respectively.

The effect of 4‐AP on [Ca2+]endo was not affected by increasing external K+ concentration. In Ca‐free/EGTA solution, the transient increase in [Ca2+]endo evoked by ACh (1 μM) was abolished by thapsigargin (1 μM) and was decreased by 75±7% (n=5) by 4‐AP.

These results show that inhibition of EDHF‐evoked responses by 4‐AP may be attributed to a decrease in the Ca2+ release activated by ACh in endothelial cells. The abolition of SMCs hyperpolarization to ACh by ChTX/Apa is not related to an interaction with the [Ca2+]endo.

British Journal of Pharmacology (2001) 134, 1021–1028; doi:10.1038/sj.bjp.0704348
α1‐Adrenoceptor subtypes in the mouse mesenteric artery and abdominal aorta
Authors: Yamamoto Yoshihisa , Koike Katsuo
Published: January 2005

Subtypes of α1‐adrenoceptor‐mediated contractions to noradrenaline in mouse mesenteric artery and abdominal aorta were examined.

In mesenteric artery, BMY7378, 5‐methylurapidil, WB4101 and prazosin were inhibited contraction to noradrenaline The good correlation for pA2 values of antagonists in native α1D‐ (rat thoracic aorta) adrenoceptor subtype and pKi values in rat cloned α1d‐adrenoceptor with the pA2 values estimated in the mouse mesenteric artery was obtained. However, the pA2 value for BMY7378 is significantly lower than the accepted value against the α1D‐adrenoceptor subtype.

In the abdominal aorta, it was obtained the regional difference for the sensitivity for noradrenaline.

In the upper abdominal aorta, the good correlation for the pA2 values of the antagonists in the native α1D‐adrenoceptor subtype and pKi values in the cloned α1d‐adrenoceptor with the pA2 values estimated in the upper abdominal aorta was obtained, and regression line was close to the line of identity.

In the lower abdominal aorta, the good correlation for the reported pKi values in the cloned α1a‐adrenoceptor subtype with the pA2 values estimated in the mouse lower abdominal aorta was obtained, and regression line was close to the line of identity.

In conclusion, the present functional data in the mouse suggest that (1) α1D‐like adrenoceptors are present in the mesenteric artery, (2) there is the regional difference for the sensitivity for noradrenaline in the abdominal aorta and (3) noradrenaline evokes the contraction mediated through α1D‐adrenoceptor in the upper abdominal aorta, whereas there is α1A‐adrenoceptor‐mediated contraction in the lower abdominal aorta.

British Journal of Pharmacology (2001) 134, 1045–1054; doi:10.1038/sj.bjp.0704350
Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line
Authors: Merighi Stefania , Varani Katia , Gessi Stefania , Cattabriga Elena , Iannotta Valeria , Ulouglu Canan , Leung Edward , Borea Pier Andrea
Published: January 2005

The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line.

Adenosine receptors were detected by RT – PCR experiments. A1 receptors were characterized using [3H]‐DPCPX binding with a KD of 1.9±0.2 nM and Bmax of 23±7 fmol mg−1 of protein. A2A receptors were studied with [3H]‐SCH 58261 binding and revealed a KD of 5.1±0.2 nM and a Bmax of 220±7 fmol mg−1 of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]‐MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3±0.7 nM and Bmax of 291±50 fmol mg−1 of protein.

The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype.

Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy‐ and enthalpy‐driven.

In functional assays the high affinity A2A agonists HE‐NECA, CGS 21680 and A2A – A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl‐IB‐MECA, IB‐MECA and NECA were able to stimulate Ca2+ mobilization.

In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.

British Journal of Pharmacology (2001) 134, 1215–1226; doi:10.1038/sj.bjp.0704352
Influence of membrane cholesterol on modulation of the GABAA receptor by neuroactive steroids and other potentiators
Authors: Sooksawate T , Simmonds M A
Published: January 2005

The influence of membrane cholesterol on some pharmacological properties of the GABAA receptor was investigated in acutely dissociated rat hippocampal neurones with whole cell patch clamp recording. The cholesterol levels were varied between 56% and 235% control using methyl‐β‐cyclodextrin as the cholesterol carrier.

Enrichment of neurones with cholesterol increased the effects of the non‐steroidal GABA potentiators propofol, flunitrazepam and pentobarbitone. A similar result was obtained after pre‐incubation of neurones with epicholesterol, the 3α‐hydroxy isomer of cholesterol.

In contrast, the effects of the steroidal GABA potentiators pregnanolone and alfaxalone were reduced by cholesterol enrichment, but not by epicholesterol. Depletion of membrane cholesterol increased the potentiation of GABA by pregnanolone and alfaxalone but did not affect the non‐steroidal potentiators.

The steroidal antagonist of GABA, pregnenolone sulphate, reduced the maximum response to GABA. This effect, also, was diminished in cholesterol‐enriched neurones and enhanced in cholesterol‐depleted neurones.

The effects of the cholesterol manipulations that were selective for the steroidal modulators of GABA are suggested to arise from direct interactions between membrane cholesterol and the GABAA receptor. The separate effects on the non‐steroidal potentiators of GABA of cholesterol‐enrichment or addition of epicholesterol to the neurones are suggested to be due to changes in membrane fluidity.

In view of the likely physiological modulation of GABAA receptors by endogenous neuroactive steroids and evidence of the in vivo lability of membrane cholesterol, the present observations may have physiological as well as pharmacological relevance.

British Journal of Pharmacology (2001) 134, 1303–1311; doi:10.1038/sj.bjp.0704360
Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channels
Authors: Goudet Cyril , Ferrer Tania , Galàn Loipa , Artiles Adriana , Batista Cesar F V , Possani Lourival D , Alvarez Julio , Aneiros Abel , Tytgat Jan
Published: January 2005

Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins.

We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials.

The effect on Na+ currents in rat cardiomyocytes was studied using the patch‐clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively.

On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 μM), shift the steady‐state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials.

The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins.

We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady‐state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels.

British Journal of Pharmacology (2001) 134, 1195–1206; doi:10.1038/sj.bjp.0704361
The effects of dentate granule cell destruction on behavioural activity and Fos protein expression induced by systemic methamphetamine in rats
Authors: Tani Kunihiko , Iyo Masaomi , Matsumoto Hideo , Kawai Masayoshi , Suzuki Katsuaki , Iwata Yasuhide , Won Taketoshi , Tsukamoto Toshio , Sekine Yoshimoto , Sakanoue Masatsuna , Hashimoto Kenji , Ohashi Yutaka , Takei Nori , Mori Norio
Published: January 2005

We destroyed dentate granule cells unilaterally or bilaterally by means of intrahippocampal injection of colchicine in rats. Subsequently, we observed behavioural changes following the intraperitoneal injection of 2 mg kg−1 methamphetamine or saline, in addition to quantitatively assessing Fos protein expression in several brain regions, including the medial prefrontal cortex, cingulate cortex, piriform cortex, dorsal striatum, and nucleus accumbens.

Bilaterally lesioned animals, when administered saline, showed a marked increase in locomotor activity compared with those of non‐lesioned animals. With respect to the methamphetamine response, bilateral destruction resulted in a marked enhancement of locomotor activity, while the unilateral destruction led to a marked increase in rotation predominantly contralateral to the lesioned side, with no identifiable change in locomotor activity.

Bilaterally lesioned animals, when administered saline and having undergone an immunohistological examination, showed a marked increase in Fos expression in both sides of the nucleus accumbens. Bilaterally lesioned animals administered methamphetamine showed a marked increase in Fos expression in the right and left sides of all regions tested. Unilaterally lesioned animals administered methamphetamine showed a significant and bilateral enhancement in Fos expression in the medial prefrontal and cingulate cortices, and a marked and unilateral (ipsilateral to the lesioned side) enhancement of Fos protein in the piriform cortex, dorsal striatum, and nucleus accumbens.

The present findings suggest that dentate granule cells regulate methamphetamine‐associated behavioural changes through the function of widespread areas of the brain, mostly the nucleus accumbens.

British Journal of Pharmacology (2001) 134, 1411–1418; doi:10.1038/sj.bjp.0704370