bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences between proximal and distal portions of the male rabbit posterior urethra in the physiological role of muscarinic cholinergic receptors
Authors: Nagahama Katsushi , Tsujii Toshihiko , Morita Takashi , Azuma Hiroshi , Oshima Hiroyuki
Published: January 2005

The aim of the present study was to elucidate functional differences between embryologically different portions of the posterior urethra of male rabbits in response to muscarinic acetylcholine receptor (mAChR) stimulation using in vitro isometric tension experiments and radioligand binding studies.

In the in vitro isometric tension experiments, carbachol, produced a dose‐dependent contraction of the proximal portion under the resting state, but did not change the basal tone of the distal portion. Contraction of the proximal portion by 10−5 m noradrenaline (NA) was dose‐dependently enhanced by carbachol either in the presence or absence of NG‐nitro‐l‐arginine (NOARG). In contrast, carbachol induced relaxation of the distal portion contracted by 10−5 m NA, which was reversed to dose‐dependent contraction in the presence of NOARG.

Both portions of the urethra had a similar number of [3H]‐quinuclidinyl benzilate ([3H]‐QNB) binding sites (195.3±74.1 fmols mg−1 protein for the proximal portion and 146.5±8.5 fmols mg−1 protein for the distal portion) with similar affinities (115.0±45.4 pM for the proximal portion and 79.9± 2.9 pM for the distal portion).

The concentration‐response curves to carbachol in both portions were shifted to the right in a parallel manner in the presence of pirenzepine (an M1 antagonist), 11‐[[2‐[(diethylamino)methyl]‐1‐piperidinyl] acetyl]‐5, 11‐dihydro‐6H‐pyrido‐2,3‐b)‐(1,4)‐benzodiazepin‐6‐one (AFDX‐116, an M2 antgonist) and 4‐diphenyl‐acetoxy‐n‐methyl‐piperidine (4‐DAMP, an M1/M3 antagonist). The pA2 values for pirenzepine, AFDX‐116 and 4‐DAMP were 7.5±0.1, 7.2±0.02 and 9.3±0.1 respectively for the contraction of the proximal portion, and 7.2±0.1, 7.1±0.2 and 9.1±0.2, respectively for the relaxation of the distal portion.

In conclusion mAChR subtypes distribute in a similar fashion throughout the length of the male rabbit posterior urethra with the discrepant responses to carbachol attributable to the differential involvement of the NO pathway in mAChR‐generated reactions.

British Journal of Pharmacology (1998) 124, 1175–1180; doi:10.1038/sj.bjp.0701952
Role of VIP1/PACAP receptors in postoperative ileus in rats
Authors: De Winter Benedicte Y. , Robberecht Patrick , Boeckxstaens Guy E. , De Man Joris G. , Moreels Tom G. , Herman Arnold G. , Pelckmans Paul A.
Published: January 2005

Vasoactive intestinal polypeptide (VIP) is an inhibitory neurotransmitter in the enteric nervous system. We investigated the role of VIP1/PACAP receptors in postoperative ileus in rats.

Different degrees of inhibition of the gastrointestinal transit, measured by the migration of Evans blue, were achieved by skin incision, laparotomy or laparotomy plus mechanical stimulation of the gut.

The transit after skin incision or laparotomy was not altered by the VIP1/PACAP receptor antagonist Ac‐Hisl,D‐Phe2, K15, R16, VIP(3–7), GRF(8–27)‐NH2 nor by the VIP1/PACAP receptor agonist K15, R16, VIP(1–7), GRF(8–27)‐NH2 and the VIP2/PACAP receptor agonist RO 25‐1553 (5 μg kg−1).

However, the transit after laparotomy plus mechanical stimulation was significantly enhanced by the VIP1/PACAP receptor antagonist, whereas it was further inhibited by the VIP1/PACAP receptor agonist. The combination of the VIP1/PACAP receptor agonist and antagonist counteracted the effect of both drugs alone. The VIP2/PACAP receptor agonist did not alter the effect of the VIP1/PACAP receptor antagonist.

The combination of the VIP1/PACAP receptor antagonist plus the nitric oxide (NO) synthase inhibitor l‐nitroarginine had no effect on the transit after laparotomy plus mechanical stimulation, while the transit after skin incision was significantly decreased.

These findings suggest the involvement of VIP1/PACAP receptors, next to NO, in the pathogenesis of postoperative ileus. However, the combination of the VIP1/PACAP antagonist and the NO synthase inhibitor abolished the beneficial effect of each drug alone, suggesting the need for one of the inhibitory neurotransmitters to enable normal gastrointestinal transit.

British Journal of Pharmacology (1998) 124, 1181–1186; doi:10.1038/sj.bjp.0701954
MS‐551 and KCB‐328, two class III drugs aggravated adrenaline‐induced arrhythmias
Authors: Xue YiXue , Yamada Chikaomi , Nu Aye Nu , Hashimoto Keitaro
Published: January 2005

We investigated the proarrhythmic effects of MS‐551 and KCB‐328, class III antiarrhythmic drugs using adrenaline‐induced arrhythmia models in halothane anaesthetized, closed‐chest dogs. In the control period, adrenaline, starting from a low dose of 0.25 to up to 1.0 μg/kg/50 s i.v., was injected to determine the arrhythmia inducing dose and the non‐inducing dose. After MS‐551 or KCB‐328 administration, the adrenaline injection was repeated and the interval between the injection and the occurrence of arrhythmia (latent interval), the changes in arrhythmic ratio (as calculated by dividing the number of ventricular premature contraction by the number of the total heart rate) and the severity of arrhythmia were observed.

MS‐551 infusion, 1 mg/kg/30 min, decreased the heart rate (HR) by 16% (P<0.01) and prolonged the QTc interval by 20% (P<0.01). During the 30 min of MS‐551 infusion, arrhythmias occurred in three out of seven dogs (torsades de pointes (TdP) type VT in one dog). After these arrhythmias disappeared, MS‐551 decreased the latent interval of the adrenaline arrhythmias produced by the inducing dose (30±2 s compared with 43±3 s of the control interval, P<0.05), increased the arrhythmic ratio (P<0.05) and induced arrhythmias by non‐inducing adrenaline doses (P<0.05).

Effect of a new class III drug KCB‐328 infusion, 0.3 mg/kg/30 min, was compared witih MS‐551 using the same model. KCB‐328 decreased the HR by 21% (P<0.01) and prolonged the QTc interval by 25% (P<0.01). During the 30 min of infusion, arrhythmias occurred in five out of seven dogs (TdP in two dogs). KCB‐328 also decreased the latent interval of the adrenaline arrhythmias produced by the inducing doses (31±3 s compared with 49±7 s of the control period, P<0.05), but did not significantly alter the arrhythmic ratio.

Adrenaline induced TdP only after MS‐551 or KCB‐328 was administered, i.e. after MS‐551, 1 mg/kg/30 min, 3/7 versus 0/7 in the control; KCB, 0.3 mg/kg/30 min, 3/7 versus 0/7 in the control.

To examine the direct arrhythmogenic effect of MS‐551 and whether an adrenergic mechanism plays some role on this arrhythmogenesis, a bolus injection of MS‐551, 3 mg/kg, was injected either without pre‐treatment or after pre‐treatment with propranolol 0.3 mg/kg. MS‐551 induced arrhythmias in five out of seven dogs (TdP in one dog). Also in the propranolol pre‐treated dogs, MS‐551 induced arrhythmias in five out of seven dogs (TdP in 1 dog).

In conclusion, these observations indicate that MS‐551 and KCB‐328 induced arrhythmias and intensified proarrhythmic effects of adrenaline, MS‐551 being stronger than KCB‐328 at the same QTc prolonging doses. The direct arrhythmogenic effect of MS‐551 was not influenced by β‐blocker treatment.

British Journal of Pharmacology (1998) 124, 1712–1718; doi:10.1038/sj.bjp.0701987
Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea‐pig common bile duct
Authors: Patacchini Riccardo , De Giorgio Roberto , Barthó Lorand , Barbara Giovanni , Corinaldesi Roberto , Alberto Maggi Carlo
Published: January 2005

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea‐pig isolated common bile duct pretreated with atropine (1 μM), produced a slowly‐developing contraction (`on' response) followed by a quick phasic `off ' contraction (`off peak' response) and a tonic response (`off late' response), averaging 16±2, 73±3 and 20±4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 μM; 15 min before) abolished the overall response to EFS (n=8).

Neither in vitro capsaicin pretreatment (10 μM for 15 min), nor guanethidine (3 μM, 60 min before) affected the excitatory response to EFS (n=5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nω‐nitro‐L‐arginine (L‐NOARG, 100 μM, 60 min before) or naloxone (10 μM, 30 min before) significantly enhanced the `on' response (294±56 and 205±25% increase, respectively; n=6–8, P<0.01) to EFS. The combined administration of L‐NOARG and naloxone produced additive enhancing effects (655±90% increase of the `on' component, n=6, P<0.05).

The tachykinin NK2 receptor‐selective antagonist MEN 11420 (1 μM) almost abolished both the `on' and `off late' responses (P<0.01; n=5 each) to EFS, and reduced the `off‐peak' contraction by 55±8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor‐selective antagonist GR 82334 (1 μM) and of the tachykinin NK3 receptor‐selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 μM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 μM, GR 82334 significantly reduced (by 68±9%, P<0.05, n=6) the `on' response to EFS.

The contractile `off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 μM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 30 μM). PPADS (30 μM) selectively blocked (75±9 and 50±7% inhibition, n=4 each) the contractile responses produced by 100 and 300 μM ATP.

Tachykinin‐containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed.

In conclusion, this study shows that in the guinea‐pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.

British Journal of Pharmacology (1998) 124, 1703–1711; doi:10.1038/sj.bjp.0701999
Somatostatin displayed on filamentous phage as a receptor‐specific agonist
Authors: Rousch Mat , Lutgerink Jan T , Coote James , de Bruïne Adriaan , Arends Jan‐Willem , Hoogenboom Hennie R
Published: January 2005

In search of methods to identify bio‐active ligands specific for G protein‐coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage‐based selection and functional screening method.

First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti‐somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO‐K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries.

Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor‐specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling.

The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio‐active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand.

British Journal of Pharmacology (1998) 125, 5–16; doi:10.1038/sj.bjp.0702011
Influence of aminoguanidine on parameters of liver injury and regeneration induced in rats by a necrogenic dose of thioacetamide
Authors: Díez‐Fernández Carmen , Sanz Nuria , Alvarez Alberto M , Zaragoza Asunción , Cascales María
Published: January 2005

When aminoguanidine, a nucleophilic hydrazine compound, was administered to rats (50 mg kg−1 body wt) 30 min before a necrogenic dose of thioacetamide (500 mg kg−1 body wt), significant changes related to liver injury and hepatocellular regeneration were observed.

The extent of necrosis was noticeably less pronounced, as detected by the peak of serum aspartate aminotransferase activity. Depletion of hepatic glutathione (GSH) and the increase in malondialdehyde concentration as markers of oxidative stress, produced by thioacetamide metabolism, were significantly diminished. However, the activity of microsomal FAD monooxygenase, the system responsible for thioacetamide oxidation, did not show significant alterations. Antioxidant enzyme systems involved in the glutathione redox cycle, such as glutathione reductase and glutathione peroxidase activities, slightly decreased following aminoguanidine pretreatment.

Primary cultures of peritoneal macrophages from control rats, when incubated in the presence of serum collected following thioacetamide intoxication, showed a significant decrease in nitric oxide (NO) release at 24 h, that was more pronounced in the group pretreated with aminoguanidine. However, the sharp and progressive increase in macrophage NO release, when incubated in the presence of serum obtained at 48, 72 and 96 h, were increased by aminoguanidine‐pretreatment.

The cell population involved in DNA synthesis sharply increased in both groups at 48 h of intoxication, although the values at 0, 24, 72 and 96 h were markedly higher in the group pre‐treated with aminoguanidine. Polyploidy at 72 and 96 h of intoxication was delayed by the effect of aminoguanidine and a progressive increase in the hypodiploid hepatocyte population, which reached 16% of the total at 96 h, was observed.

These results indicate that a single dose of aminoguanidine before thioacetamide administration, markedly diminished the severity of the liver injury by decreasing oxidative stress and lipoperoxidation, but hepatocellular regeneration was apparently unaffected probably due to an enhanced mitogenic activity.

British Journal of Pharmacology (1998) 125, 102–108; doi:10.1038/sj.bjp.0702014
Rilmenidine reveals differences in the pharmacological characteristics of prejunctional α2‐adrenoceptors in the guinea‐pig, rat and pig
Authors: Ali A , Cheng H‐Y , Ting K N , Wilson V G
Published: January 2005

The α2A and α2D‐adrenoceptor subtypes are thought to be species homologs most easily differentiated on the basis of the potency of antagonists. In the present study we have compared the effect of rilmenidine with two other selective α2‐adrenoceptor agonists, UK‐14304 (5‐bromo‐6‐ [2‐imidazolin‐2‐ylamino]‐quinoxaline) and clonidine, against electrically‐evoked contractions in five isolated preparations from the rat, guinea‐pig and pig, and, where possible, determined the receptor subtype involved.

UK‐14034, clonidine and rilmenidine produced concentration‐dependent inhibition of the electrically‐evoked contractions of the rat isolated vas deferens and tail artery and the guinea‐pig ileum. These inhibitory effects were reversed by the selective α2‐adrenoceptor antagonist, RX‐811058 (1 μM), except in the rat tail artery preparations where the remaining neurogenic response was inhibited; evidence for the involvement of ‘innervated’ α2‐adrenoceptors. Both clonidine and UK‐14304 produced concentration‐dependent inhibition of responses in the porcine isolated tail artery and urinary bladder but clonidine was markedly less efficacious in these preparations. In contrast, rilmenidine failed to inhibit the neurogenic contractions in either preparation.

Although rilmenidine failed to elicit a detectable response in either the porcine isolated tail artery or urinary bladder, it (10 μM and 30 μM, respectively) competitively antagonised the inhibitory effects of UK‐14304 with an estimated dissociation constant of (pKB) 5.82 and 5.93, respectively.

Prazosin (1 μM) failed to alter the effect of UK‐14304 against neurogenic contractions in the porcine isolated urinary bladder, while rauwolscine (pKB 8.87) was 10 fold more potent than phentolamine (pKB 7.56). On the other hand, phentolamine (pKB 8.42) was only marginally more potent than rauwolscine (pK 8.05) against clonidine‐induced inhibition of electrically‐evoked contractions of the guinea‐pig isolated ileum. This pharmacological evidence with antagonists supports the presence of α2D‐adrenoceptors in the rat and guinea‐pig and the α2A‐adrenoceptors in the pig.

We have demonstrated that rilmenidine, unlike clonidine and UK‐14304, is devoid of any agonist activity at prejunctional α2A‐adrenoceptors in the pig, but is an efficacious agonist at α2D‐adrenoceptors in the rat and guinea‐pig.

British Journal of Pharmacology (1998) 125, 127–135; doi:10.1038/sj.bjp.0702016
Kinin B2 receptor‐mediated contraction of tail arteries from normal or streptozotocin‐induced diabetic rats
Authors: Wang Zunzhe , Wu Lingyun , Wang Rui
Published: January 2005

The vasoactive effects of bradykinin (BK) are mediated by different subtypes of kinin receptors, of which the expression varies among different tissues. In rat tail artery tissues, BK elicited a concentration‐dependent vasoconstriction (EC50, 25.9±2.4 nM; Emax, 0.39±0.01 g; n=16). This effect of BK was endothelium independent and indomethacin insensitive. The BK‐induced contraction of tail artery tissues, however, depended on both membrane potential‐sensitive extracellular Ca2+ entry and thapsigargin‐sensitive intracellular Ca2+ release.

Kinin B1 receptor antagonist or agonist did not affect the basal tension or the BK‐induced contraction of tail artery tissues in the absence or presence of endothelium (P>0.05). In contrast, the BK‐induced vasoconstriction was inhibited by kinin B2 receptor antagonists. Pretreatment of vascular tissues with Hoe 140 (1 nM) significantly changed EC50 of the BK‐induced vasoconstriction from 25.5±7.4 nM to 82.6±16.8 nM (n=8, P<0.01) and Emax from 0.43±0.03 g to 0.16±0.01 g (n=8, P<0.01).

In the tail artery tissues from streptozotocin‐induced diabetic rats, the BK‐elicited vasoconstriction was significantly reduced (EC50, 67.8±11 nM; Emax, 0.19±0.01 g) compared to their counterparts from normal rats. The decreased vasoconstrictive effects of BK on diabetic arteries were endothelium independent and indomethacin insensitive.

Our study demonstrated that the contraction of rat tail arteries induced by BK was mediated by B2 receptors located on vascular smooth muscles. The altered B2 receptor‐mediated vascular activity may play an important role in the vascular complications of diabetes.

British Journal of Pharmacology (1998) 125, 143–151; doi:10.1038/sj.bjp.0702017
Biphasic effects of cyclosporin A on formyl‐methionyl‐leucyl‐phenylalanine stimulated responses in HL‐60 cells differentiated into neutrophils
Authors: Nguyen Nathalie S D , Pulido Silvia M , Rüegg Urs T
Published: January 2005

The immunosuppressive drug cyclosporin A (CsA) depresses neutrophil oxidative burst which may lead to an increased susceptibility to infection in transplant patients. Using specific CsA analogues we investigated the mechanism of inhibition of the oxidative burst and evaluated short and long‐term effects of CsA on dimethylsulphoxide‐differentiated HL‐60 neutrophils.

A biphasic pattern was observed: a 4 h pre‐treatment with CsA (1 μM) diminished the fMLP induced [Ca2+]c rise, reactive oxygen species (ROS) production, and β‐glucuronidase release by about 40%, whereas a 20 h pre‐treatment increased these responses by about 1.5 fold.

[MeVal4]CsA, which binds with high affinity to cyclophilin but inhibits the interaction of the CsA‐cyclophilin complex with calcineurin, blocked the stimulation observed with CsA after a 20 h incubation but did not alter the CsA effects after a 4 h pre‐treatment.

PSC 833 (1 μM), a potent multi drug resistance transporter (MDR) inhibitor, diminished ROS production to the same extent as a 4 h CsA incubation but was ineffective after a 20 h pre‐treatment. An involvement of MDR as a basis for CsA or PSC 833 action was ruled out based on the results of the calcein retention assay.

[3H]CsA uptake showed that CsA and [MeVal4]CsA, but not CsH or PSC 833 were strongly taken up and retained by the cells.

In conclusion, the reduction of the responses after 4 h appear to be due to a primary reduction of calcium signalling, while the enhanced responses after 20 h may be due to calcineurin inhibition.

British Journal of Pharmacology (1998) 124, 1774–1780; doi:10.1038/sj.bjp.0702020
Direct dopamine D2‐receptor‐mediated modulation of arachidonic acid release in transfected CHO cells without the concomitant administration of a Ca2+‐mobilizing agent
Authors: Nilsson Christer L , Hellstrand Monika , Ekman Agneta , Eriksson Elias
Published: January 2005

In CHO cells transfected with the rat dopamine D2 receptor (long isoform), administration of dopamine per se elicited a concentration‐dependent increase in arachidonic acid (AA) release. The maximal effect was 197% of controls (EC50=25 nM). The partial D2 receptor agonist, (−)‐(3‐hydroxyphenyl)‐N‐n‐propylpiperidine [(−)‐3‐PPP], also induced AA release, but with somewhat lower efficacy (maximal effect: 165%; EC50=91 nM).

The AA‐releasing effect of dopamine was counteracted by pertussis toxin, by the inhibitor of intracellular Ca2+ release, 8‐(N N‐diethylamino)octyl‐3,4,5‐trimethoxybenzoate (TMB‐8), by excluding calcium from the medium, by the phospholipase A2 (PLA2) inhibitor, quinacrine, and by long‐term pretreatment with the phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). In addition, it was antagonized by the D2 antagonists, raclopride and (−)‐sulpiride–but not by (+)‐sulpiride–and absent in sham‐transfected CHO cells devoid of D2 receptors.

The results obtained contrast to the previous notion that dopamine and other D2 receptor agonists require the concomitant administration of calcium‐mobilizing agents such as ATP, ionophore A‐23187 (calcimycin), thrombin, and TRH, to influence AA release from various cell lines.

British Journal of Pharmacology (1998) 124, 1651–1658; doi:10.1038/sj.bjp.0702025
ATP‐sensitive K+ channels in smooth muscle cells of guinea‐pig mesenteric lymphatics: role in nitric oxide and β‐adrenoceptor agonist‐induced hyperpolarizations
Authors: von der Weid Pierre‐Yves
Published: January 2005

Intracellular microelectrode recordings were performed to investigate the membrane K+ conductances involved in smooth muscle hyperpolarization of lymphatic vessels in the guinea‐pig mesentery.

Nitric oxide (NO), released either by the endothelium after acetylcholine (ACh; 10 μM) stimulation or by sodium nitroprusside (SNP; 50–100 μM), hyperpolarized lymphatic smooth muscle. These responses were inhibited with the guanylyl cyclase inhibitor 1H‐[1,2,4]oxadiazole [4,3‐a]quinoxalin‐1‐one (ODQ, 10 μM).

ACh and SNP‐induced hyperpolarizations were inhibited (by about 90%) upon application of the ATP‐sensitive K+(KATP) channel blocker, glibenclamide (10 μM), or with 4‐aminopyridine (2.5 mM), but were not affected by the Ca2+‐activated K+ channels blocker, penitrem A (100 nM).

Hyperpolarization caused by the K+ channel opener, cromakalim (0.1–10 μM), isoprenaline (0.1 μM) or forskolin (0.5 μM) were all significantly blocked by glibenclamide.

Hyperpolarization evoked by ACh and SNP were inhibited with N‐[2‐(p‐bromociannamylamino)‐ethyl]‐5‐isoquinolinesulfonamide‐dichloride (H89, 10 μM), suggesting the involvement of cyclic AMP dependent protein kinase (PKA).

These results suggest that KATP channels play a central role in lymphatic smooth muscle hyperpolarization evoked by a NO‐induced increase in cyclic GMP synthesis, as well as by β‐adrenoceptor‐mediated production of cyclic AMP. Interestingly, both pathways lead to KATP channels opening through the activation of PKA.

British Journal of Pharmacology (1998) 125, 17–22; doi:10.1038/sj.bjp.0702026
Citalopram‐induced hypophagia is enhanced by blockade of 5‐HT1A receptors: role of 5‐HT2C receptors
Authors: Grignaschi G , Invernizzi R W , Fanelli E , Fracasso C , Caccia S , Samanin R
Published: January 2005

The selective 5‐hydroxytryptamine reuptake inhibitor citalopram (10 and 20 mg kg−1, i.p.) significantly reduced food intake in male rats (CD‐COBS) habituated to eat their daily food during a 4‐h period.

The 5‐HT1A receptor antagonist WAY100635 (0.3 mg kg−1) administered systemically did not modify feeding but significantly potentiated the reduction in food intake caused by 10 mg kg−1 i.p. citalopram. The dose of 5 mg kg−1 i.p. citalopram was not active in animals pretreated with vehicle but significantly reduced feeding in animals pretreated with WAY100635.

WAY100635 (0.1 μg 0.5 μl−1) injected into the dorsal raphe significantly potentiated the hypophagic effect of 10 mg kg−1 citalopram.

WAY100635 (1.0 μg 0.5 μl−1) injected into the median raphe did not modify feeding or the hypophagic effect of 10 mg kg−1 citalopram.

The 5‐HT2B/2C receptor antagonist SB206553 (10 mg kg−1, p.o.) slightly reduced feeding by itself but partially antagonized the effect of WAY100635 administered systemically (0.3 mg kg−1, s.c.) or into the dorsal raphe (0.1 μg 0.5 μl−1) in combination with 10 mg kg−1 i.p. citalopram. The hypophagic effect of 10 mg kg−1 i.p. citalopram alone was not significantly modified by SB206553.

Brain concentrations of citalopram and its metabolite desmethylcitalopram in rats pretreated with SB206553, WAY100635 and their combination were comparable to those of vehicle‐pretreated rats, 90 min after citalopram injection.

The hypophagic effect of citalopram was potentiated by blocking 5‐HT1A receptors. Only the effect of the WAY100635/citalopram combination seemed to be partially mediated by central 5‐HT2C receptors.

British Journal of Pharmacology (1998) 124, 1781–1787; doi:10.1038/sj.bjp.0702028
Frequency dependence of muscarinic facilitation of transmitter release in urinary bladder strips from neurally intact or chronic spinal cord transected rats
Authors: Somogyi G T , Zernova G V , Yoshiyama M , Yamamoto T , de Groat W C
Published: January 2005

Electrical stimulation evoked release of 3H‐noradrenaline (NA) and 14C‐acetylcholine (ACh), as well as neurally evoked contractions were measured at various (1–40 Hz, 100 shocks) stimulation frequencies in bladder strips from neurally intact (NI) and spinal cord transected (SCT) rats.

The frequency response curves for ACh and NA release were shifted to the left in SCT bladder strips as compared to NI bladder strips.

Atropine (1 μM) depressed ACh release in NI bladder strips at high frequency stimulation (10 and 40 Hz) but not at low frequency stimulation (2–5 Hz). However, in SCT bladders, atropine depressed ACh release both at low and high frequencies of stimulation, indicating that muscarinic facilitation occurs at lower frequencies.

Atropine depressed the release of NA in NI bladders at only 40 Hz stimulation, but depressed release at all frequencies in SCT bladders.

The amplitude of neurally evoked contractions of bladder strips from NI rats was enhanced as the frequency of stimulation was increased from 1 to 40 Hz (80 shocks). The frequency response curve was shifted to the left in SCT bladders. Atropine blocked the neurally evoked contractions in SCT bladder strips to a greater extent than the contractions in NI strips indicating a cholinergic dominance in the SCT bladders.

Maximal contractile force of SCT bladder strips evoked by neural stimulation at 20 Hz 10 shocks and 80 shocks was significantly lower than that of NI bladder strips, whereas the release of ACh was significantly higher in SCT than NI bladders indicating a postjunctional defect in the SCT preparations.

It is suggested that presynaptic muscarinic facilitatory mechanisms are upregulated in the cholinergic and adrenergic nerve terminals in SCT bladders leading to a larger relative contractile response at lower frequencies of stimulation (2–5 Hz). Thus the hyperreflexic bladder occurring after spinal cord injury may be due in part to an enhancement of transmitter release at bladder postganglionic nerve terminals.

British Journal of Pharmacology (1998) 125, 241–246; doi:10.1038/sj.bjp.0702041
Inhibition by nitric oxide‐releasing compounds of prostacyclin production in human endothelial cells
Authors: Kosonen Outi , Kankaanranta Hannu , Malo‐Ranta Ulla , Ristimäki Ari , Moilanen Eeva
Published: January 2005

The effects of two chemically unrelated nitric oxide (NO)‐releasing compounds were studied on prostacyclin production in lipopolysaccharide (LPS)‐stimulated human umbilical vein endothelial cells (HUVECs). The cells expressed cyclooxygenase‐2 (COX‐2) protein and produced prostacyclin by NS‐398‐sensitive manner suggesting that prostacyclin production derives principally by COX‐2 pathway.

A novel NO‐releasing oxatriazole derivative GEA 3175 (1–30 μM) inhibited LPS‐induced production of prostacyclin in HUVECs in a dose‐dependent manner being more potent than the earlier known NO‐donor S‐nitroso‐N‐acetylpenicillamine (SNAP).

The effects of the two NO‐donors on prostacyclin synthesis were reversed when red blood cells were added into the culture indicating that the effects are due to NO released from the compounds.

Addition of exogenous arachidonic acid into the culture did not alter the inhibitory action of NO‐donors suggesting that phospholipases are not the target of action of NO.

The NO‐donors did not inhibit prostacyclin production in the presence of a selective COX‐2 inhibitor NS‐398. These data suggest that NO affects COX‐2 pathway rather than has an overall effect on cyclooxygenases.

NO‐releasing compounds did not alter the level of COX‐2 protein expression in LPS‐treated HUVECs as measured by Western blot analysis.

The results suggest that NO‐donors inhibit the activity of COX‐2 in human endothelial cells. A link between NO and the regulation of eicosanoid synthesis could represent an important mechanism in controlling vascular and inflammatory responses in pathophysiological states and during treatment with nitrovasodilators.

British Journal of Pharmacology (1998) 125, 247–254; doi:10.1038/sj.bjp.0702042
Inhibition of serum and transforming growth factor beta (TGF‐β1)‐induced DNA synthesis in confluent airway smooth muscle by heparin
Authors: Okona‐Mensah K B , Shittu E , Page C , Costello J , Kilfeather S A
Published: January 2005

Airway remodelling occurs in asthma and involves an increase in airway smooth muscle mass through cell proliferation and hypertrophy. Increased eosinophil density in the airways is a feature of asthma. Eosinophils exhibiting activation in the airways of asthmatics also exhibit increased expression of transforming growth factor beta (TGF‐β1). We have examined the capacity of TGF‐β1 and epidermal growth factor (EGF) to influence airway smooth muscle division and the effect of heparin on TGF‐β1, EGF and serum‐induced smooth muscle DNA synthesis in confluent airway smooth muscle cells (ASMC) as an indication of entry into S phase preceding mitogenesis.

ASMC were obtained from cell populations growing out from explanted bovine trachealis muscle sections. Cell division was monitored in sparse plated cells by direct cell counting following nuclear staining. Cell DNA synthesis in confluent cells was monitored by uptake of [3H]‐thymidine.

TGF‐β1 (100 pM) inhibited FBS (10%)‐induced smooth muscle division in sparsely plated cells (40%). TGF‐β1 (100 pM) increased cell DNA synthesis (200%) in confluent cells in the presence of bovine serum albumin (BSA, 0.25%). EGF (0.7 nM) also increased airway smooth muscle DNA synthesis (69%) in the presence of BSA (0.25%). The facilitatory effect of TGF‐β1 was observed between 1–100 pM, while that of EGF was observed between 20–200 pM.

Heparin inhibited serum and TGF‐β1‐induced DNA synthesis in confluent ASMC (55%), consistent with our previous observation of inhibition of division in sparsely populated ASMC (Kilfeather et al., 1995a). This action of heparin was observed between concentrations of 1–100 μg ml−1. Heparin did not inhibit DNA synthesis in response to EGF. An anti‐mitogenic effect of heparin was also observed against responses to combined exposure to TGF‐β1 and EGF.

There was a clear inhibitory effect of heparin in absolute terms against serum‐induced division in cells plated at 10, 20 and 45×103 cells cm−2. The inhibitory effect of heparin was also observed at a plating density of 45,000 cells cm−2 when responses to serum were expressed as fold‐stimulation of basal DNA synthesis.

These findings demonstrate a potential role of TGF‐β1, EGF and heparin‐related molecules in regulation of airway smooth muscle division.

British Journal of Pharmacology (1998) 125, 599–606; doi:10.1038/sj.bjp.0702046
Effects of mitogen‐activated protein kinase kinase inhibitor PD 098059 on antigen challenge of guinea‐pig airways in vitro
Authors: Tsang F , Koh A H M , Ting W L , Wong P T H , Wong W S F
Published: January 2005

It has been shown that activation of protein tyrosine kinases is the earliest detectable signalling response to FcεRI cross‐linking on mast cell. Following tyrosine kinase activation, a family of mitogen‐activated protein kinases (MAPKs) was found to be activated as well. The present study examined the role of MAPK signalling cascade in in vitro model of allergic asthma using a specific MAPK kinase inhibitor PD 098059.

Guinea‐pigs were passively sensitized with IgG antibody raised against ovalbumin (OA). Effects of PD 098059 on OA‐induced anaphylactic contraction of isolated bronchi and release of histamine and peptidoleukotrienes from chopped lung preparations were studied.

PD 098059 (10–50 μM) produced only minor reduction of maximal OA‐induced bronchial contraction. In contrast, the rate of relaxation of OA‐induced bronchial contraction was markedly faster in the presence of PD 098059 than the vehicle control in a concentration‐dependent manner.

These observations corroborate well with the inability of PD 098059 (5–50 μM) to substantially block the OA‐induced release of histamine and with marked inhibition of OA‐induced release of peptidoleukotrienes from lung fragments in the presence of PD 098059. Exogenous arachidonic acid‐induced release of peptidoleukotrienes from lung fragments was not blocked by PD 098059.

In immunoblotting study, we found that p42MAPK was constitutively expressed in guinea‐pig bronchi. However, treatment with OA, histamine or LTD4 did not cause activation of p42MAPK. These findings together with the lack of inhibitory effects of PD 098059 on bronchial contraction induced by histamine or LTD4 suggest that histamine‐ and LTD4‐induced bronchial contractions are not mediated by p42MAPK activation.

Taken together, our findings show that inhibition of MAPK signalling cascade by PD 098059 significantly reduced the OA‐triggered release of peptidoleukotrienes leading to rapid relaxation of anaphylactic bronchial contraction. On the other hand, p42MAPK did not play a role in histamine‐ or LTD4‐induced bronchial smooth muscle contraction suggesting that PD 098059 exerts its inhibitory effects on OA‐induced bronchial contraction primarily through inhibition of peptidoleukotrienes release from mast cells.

British Journal of Pharmacology (1998) 125, 61–68; doi:10.1038/sj.bjp.0702049
Relaxant effects of L‐citrulline in rabbit vascular smooth muscle
Authors: Ruiz E , Tejerina T
Published: January 2005

Vascular endothelium plays a pivotal role in the control of vascular tone through the release of vasoactive factors such as EDRF (NO).

The aim of this study was to investigate whether the addition of exogenous L‐citrulline, the byproduct of the NO‐synthesis, could relax vascular smooth muscle.

L‐citrulline relaxed both endothelium‐denuded and endothelium‐intact rabbit aortic rings precontracted with noradrenaline 10−6 M (maximum relaxations induced by L‐citrulline 10−8 M were 74.1±5.2% vs 51.3±2.8% in endothelium‐denuded and endothelium‐intact arteries, respectively).

This relaxant effect was enhanced by zaprinast (a phosphodiesterase type 5 inhibitor) and inhibited by HS‐142‐1 (a particulate guanylate cyclase inhibitor) and by apamin (a KCa‐channel blocker).

L‐citrulline (10−13–10−8 M) increased cGMP levels in aortic rings (maximum value with L‐citrulline 10−8 M was 0.165±0.010 pmol cGMP mg−1 of tissue vs 0.038±0.009 pmol mg−1 of tissue in basal).

L‐citrulline as well as NO were released from endothelial cells in culture stimulated with ACh. The values were 6.50±0.50 μM vs 2.30±0.20 μM (stimulated with ACh and basal respectively) for L‐citrulline and 4.22±0.10 μM vs 0.87±0.26 μM (stimulated with ACh and basal respectively) for NO.

These results suggest that L‐citrulline could be released together with NO from endothelium and may have actions complementary to those of NO in the control of vascular smooth muscle relaxation.

British Journal of Pharmacology (1998) 125, 186–192; doi:10.1038/sj.bjp.0702051
Role of transforming growth factor‐β pathway in the mechanism of wound healing by saponin from Ginseng Radix rubra
Authors: Kanzaki Tetsuto , Morisaki Nobuhiro , Shiina Ritsuko , Saito Yasushi
Published: January 2005

The effects of saponin from Ginseng Radix rubra on extracellular matrix metabolism, the activation and synthesis of TGF‐β1, and the modification of TGF‐β receptor in fibroblasts were examined to elucidate the contribution of the TGF‐β pathway to the mechanism of wound healing by saponin.

Fibronectin synthesis was analysed by the immunoprecipitation method. Activation and synthesis of TGF‐β1 were measured by ELISA. The expressions of TGF‐β receptors in fibroblasts were examined at protein and mRNA levels by the cross‐linking method and Northern blot analysis, respectively.

The fibronectin synthesis increased 2.3‐ and 3.9‐fold at fibroblasts treated with 1 and 10 μg ml−1 of saponin, respectively, compared with that in non‐treated cells. Fibronectin synthesis stimulated with 10 μg ml−1 of saponin was inhibited with 69% by 5 μg ml−1 of an anti‐TGF‐β1 antibody. mRNA of TGF‐β type I receptor increased 4.8‐ and 4.4‐fold at fibroblasts treated with 1 and 10 μg ml−1 of saponin, respectively, and that of TGF‐β type II receptor also increased 3.4‐ and 3.2‐fold at fibroblasts treated with 1 and 10 μg ml−1 of saponin, respectively. The significant increases of TGF‐β type I and II receptors and of fibronectin synthesis were observed at the same concentrations of saponin. TGF‐β1 content increased 1.74‐ and 1.87‐fold at conditioned medium of fibroblasts treated with 100 and 250 μg ml−1 of saponin, respectively, higher concentrations than those which accelerated fibronectin synthesis. Furthermore, the active TGF‐β1 content was below 10% of total TGF‐β1 at each concentration of saponin.

These results indicate that saponin stimulates fibronectin synthesis through the changes of TGF‐β receptor expressions in fibroblasts.

British Journal of Pharmacology (1998) 125, 255–262; doi:10.1038/sj.bjp.0702052
Peripheral and/or central effects of racemic‐, S(+)‐ and R(−)‐flurbiprofen on inflammatory nociceptive processes: a c‐Fos protein study in the rat spinal cord
Authors: Buritova Jaroslava , Besson Jean‐Marie
Published: January 2005

We have evaluated the effects of intravenous or intraplantar racemic‐, S(+)‐ and R(−)‐flurbiprofen on both the carrageenan‐evoked peripheral oedema and spinal c‐Fos immunoreactivity, an indirect index of neurons involved in spinal nociceptive processes.

Three hours after intraplantar injection of carrageenan (6 mg in 150 μl of saline) in awake rats, a peripheral oedema and numerous c‐Fos protein‐like immunoreactive (c‐Fos‐LI) neurons in L4–L5 segments were observed. c‐Fos‐LI neurons were essentially located in the superficial (I–II) and deep (V–VI) laminae of the dorsal horn.

Intravenous racemic‐flurbiprofen (0.3, 3 and 9  mg kg−1) dose‐relatedly reduced the carrageenan‐evoked oedema and spinal c‐Fos expression (r=0.64, r=0.88 and r=0.84 for paw diameter, ankle diameter and number of c‐Fos‐LI neurons; P<0.05, P<0.001 and P<0.001 respectively).

Similar effects to those of intravenous racemic‐flurbiprofen were obtained with intravenous S(+)‐flurbiprofen (0.3, 3 and 9 mg kg−1) which dose‐relatedly reduced the number of c‐Fos‐LI neurons (r=0.69, P<0.01) and diameters of paw and ankle (r=0.56 and r=0.52 respectively, P<0.05 for both).

For the dose of 0.3 mg kg−1 i.v., R(−)‐flurbiprofen did not modify the number of c‐Fos‐LI neurons and produced a weak reduction of oedema at only the ankle level (23±12% reduction, P<0.05). However, a ten times higher dose of R(−)‐flurbiprofen (3 mg kg−1 i.v.) was necessary to obtain effects comparable to those of S(+)‐ or racemic‐flurbiprofen (0.3 mg kg−1 i.v.).

Intraplantar racemic‐flurbiprofen (1, 10 and 30 μg) dose‐relatedly reduced the carrageenan‐enhanced ankle diameter (r=0.81, P<0.001) and the number of c‐Fos‐LI neurons in L4–L5 segments (r=0.83, P<0.001), with a 60±3% reduction of the number of c‐Fos‐LI neurons (P<0.001), and 30±3 and 67±7% reduction of paw and ankle diameter respectively (P<0.001 for both) for the dose of 30 μg.

For intraplantar S(+)‐flurbiprofen (1, 10 and 30  μg) the dose‐related effects (r=0.77, r=0.60 and r=0.59 for c‐Fos‐LI neurons, paw and ankle diameters respectively, P<0.001, P<0.01 and P<0.01) were similar to those of racemic‐flurbiprofen. In contrast, intraplantar R(−)‐flurbiprofen (1, 10 and 30 μg) did not have detectable effects on all studied parameters.

The present study provides clear evidence for potent anti‐inflammatory and antinociceptive effects of both intravenous or intraplantar racemic‐ and S(+)‐flurbiprofen. These results further demonstrate marked anti‐inflammatory and antinociceptive effects of intravenous, but not intraplantar, R(−)‐flurbiprofen. These results suggest that the main site of action of racemic‐ and S(+)‐flurbiprofen is in the periphery and indicate that the site of action of R(−)‐flurbiprofen is mainly of central origin.

British Journal of Pharmacology (1998) 125, 87–101; doi:10.1038/sj.bjp.0702053
Bradykinin B2‐receptor‐mediated modulation of membrane currents in guinea‐pig cardiomyocytes
Authors: Sakamoto Naoya , Uemura Hiroko , Hara Yukio , Saito Toshihiro , Masuda Yoshiaki , Nakaya Haruaki
Published: January 2005

In order to define the electrophysiological mechanism(s) responsible for bradykinin (BK)‐induced positive inotropic and chronotropic responses in isolated guinea‐pig atria, effects of BK on the membrane currents were examined in isolated atrial cells using patch clamp techniques.

BK (0.1–1000 nM) increased the L‐type Ca2+ current (ICa), which was recorded from enzymatically‐dissociated atrial myocytes by the nystatin‐perforated patch method, in a concentration‐dependent fashion, and the calculated EC50 value for increasing ICa was 5.2 nM. In conventional ruptured patch experiments, BK inhibited the muscarinic acetylcholine receptor‐operated K+ current (IK.ACh) that was activated by the muscarinic agonist carbachol (1 μM) with an EC50 value of 0.57 nM. Both the increase in ICa and the decrease in IK.ACh were blocked by HOE140, a selective bradykinin B2 receptor antagonist.

The BK‐induced inhibition of IK.ACh was significantly attenuated by staurosporine and calphostin C, protein kinase C inhibitors. In addition, the IK.ACh inhibition by BK was also attenuated by the tyrosine kinase inhibitor genistein or tyrphostin but not by daidzein, an inactive analogue of genistein. However, neither protein kinase C inhibitor nor tyrosine kinase inhibitor affected the BK‐induced increase in ICa.

In the presence and absence of muscarinic stimulation, BK prolonged the action potential recorded from the atrial cells in the current clamp mode.

We conclude that BK increases ICa and decreases IK.ACh in atrial cells, resulting in positive inotropic and chronotropic responses in atrial preparations. Protein kinase C activation, and possibly tyrosine kinase activation, may be involved in the B2‐receptor‐mediated IK.ACh inhibition.

British Journal of Pharmacology (1998) 125, 283–292; doi:10.1038/sj.bjp.0702060
Increased vascular permeability by a specific agonist of protease‐activated receptor‐2 in rat hindpaw
Authors: Kawabata Atsufumi , Kuroda Ryotaro , Minami Takeshi , Kataoka Kazuo , Taneda Mamoru
Published: January 2005

The present study examined the effect of intraplantar (i.pl.) administration of a selective agonist of protease‐activated receptor (PAR)‐2, SLIGRL‐NH2(PP6‐NH2), on vascular permeability in rat hindpaw. PP6‐NH2, administered i.pl. at 10–100 nmol per paw, enhanced vascular permeability and caused oedema formation in rat hindpaw. SLIGRL (PP6‐OH) and trypsin, by i.pl. administration, also elicited an increase in vascular permeability, although i.pl. administration of the mixture of constituent amino acids of PP6‐OH at an equivalent dose did not. The PP6‐NH2‐induced increase in vascular permeability was abolished by repeated pretreatment with compound 48/80 to deplete bioactive amines in mast cells. These findings suggest that the activation of PAR‐2 induces acute inflammation, at least partially, via mast cell degranulation in rat hindpaw.

British Journal of Pharmacology (1998) 125, 419–422; doi:10.1038/sj.bjp.0702063