bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased function of inhibitory neuronal M2 muscarinic receptors in trachea and ileum of diabetic rats
Authors: Coulson Fiona R , Jacoby David B , Fryer Allison D
Published: January 2005

Release of acetylcholine from parasympathetic nerves is inhibited by neuronal M2 muscarinic receptors. The effects of streptozotocin‐induced diabetes on prejunctional M2 and postjunctional M3 muscarinic receptor function in rat trachea and ileum were investigated in vitro.

Neuronal M2 muscarinic receptor function was tested by measuring the ability of an agonist, pilocarpine, to inhibit and an antagonist, methoctramine, to potentiate electrical field stimulation (EFS)‐induced contraction of trachea and ileum. Concentration‐response curves to pilocarpine and methoctramine were shifted to the left in both to a greater degree in diabetics than controls.

In trachea, post‐junctional M3 muscarinic receptor function was increased since maximum contractile responses to the muscarinic agonists acetylcholine and carbachol were greater in diabetics than controls. This increase offset the increased function of the inhibitory neuronal M2 muscarinic receptors since EFS‐induced, frequency‐dependent contraction was equal in control and diabetic rats.

In contrast, post‐junctional M3 muscarinic receptor function was unchanged by diabetes since concentration‐response curves to acetylcholine and carbachol were not different between groups. Thus, EFS‐induced contractions of the ileum were decreased in diabetics versus controls.

In conclusion, inhibitory M2 muscarinic receptors on parasympathetic nerves in the trachea and ileum are hyperfunctional in diabetic rats. The function of post‐junctional M3 muscarinic receptors in the trachea, but not the ileum, is also increased in diabetes.

The dysfunction of inhibitory, neuronal M2 muscarinic receptors in the airways may protect against hyperreactivity and in the ileum may contribute to gastrointestinal dysmotility associated with diabetes.

British Journal of Pharmacology (2002) 135, 1355–1362; doi:10.1038/sj.bjp.0704602
Differential sensitivity to tetrodotoxin and lack of effect of prostaglandin E2 on the pharmacology and physiology of propagated action potentials
Authors: Farrag K J , Costa S K P , Docherty R J
Published: January 2005

We have studied the effects of prostaglandin E2 (PGE2) on action potential propagation in the isolated, desheathed vagus and saphenous nerves of rats using an extracellular grease gap recording method.

PGE2 evoked a small depolarization of vagus nerves but had no effect on the stimulation threshold, size or latency of either the A wave (corresponding to conduction in A fibres) or the C wave (corresponding to conduction in C fibres) of the compound action potential (CAP) recorded from either vagus or saphenous nerves.

Lidocaine (0.01 – 10 mM) reduced all components of the CAP of both vagus and saphenous nerves. PGE2 had no significant effect on the sensitivity of any component of the CAP to lidocaine.

Tetrodotoxin (TTX, 10 μM) blocked completely both the A wave and the C wave of the CAP in either vagus or saphenous nerves.

In saphenous nerve preparations the A wave was blocked by lower concentrations of TTX than the C wave or any component of the CAP in vagus nerve preparations which suggests that somatosensory A fibres express a different sub‐type of TTX‐sensitive voltage‐gated sodium channel (VGSC) than somatosensory C‐fibres or visceral sensory fibres.

Chemical activation of VGSCs with veratridine (10 or 50 μM) induced a depolarization in either nerve. The depolarization induced by 50 μM veratridine was blocked by 10 μM TTX.

Although TTX‐insensitive VGSCs are expressed by some vagal and some somatosensory neurones they do not appear to be expressed functionally in the axons.

British Journal of Pharmacology (2002) 135, 1449–1456; doi:10.1038/sj.bjp.0704607
Anti‐secretory properties of non‐peptide somatostatin receptor agonists in isolated rat colon: luminal activity and possible interaction with p‐glycoprotein
Authors: Emery P T J , Higgs N B , Warhurst A C , Carlson G L , Warhurst G
Published: January 2005

The diverse physiological actions of somatostatin are mediated by a family of G‐protein coupled receptors (SSTRs). Several peptide analogues of somatostatin such as octreotide have been developed for therapeutic use, including treatment of gastrointestinal disorders such as secretory diarrhoea. However, their development as anti‐diarrhoeal agents has been limited by poor oral bioavailability, necessitating parenteral administration. This in vitro study investigated the anti‐secretory potential of a group of novel, non‐peptide, somatostatin‐receptor agonists that selectively activate specific SSTR subtypes to assess their potential for oral administration.

The ability of the agonists to inhibit forskolin‐stimulated chloride secretion was measured using a sensitive bioassay system in isolated rat colonic mucosa.

The SSTR‐2 selective agonist, L‐779,976 was 10‐times more potent than octreotide as an inhibitor of secretion when added to the basolateral surface of rat colon. Non‐peptide agonists selective for SSTR1 (L‐797,591), SSTR3 (L‐796,778), SSTR4 (L‐803,087) or SSTR5 (L‐817,818) showed little or no anti‐secretory activity in this preparation.

L‐779,976 was able to inhibit secretion when applied to the luminal surface at sub‐micromolar concentrations suggesting that it can cross the colonic epithelium. The anti‐secretory potency of luminal L‐779,976 was increased 3 fold in the presence of GF120918, a known inhibitor of P‐glycoprotein.

Non‐peptide somatostatin receptor agonists may provide a basis for the development of new, orally available anti‐diarrhoeal therapies.

British Journal of Pharmacology (2002) 135, 1443–1448; doi:10.1038/sj.bjp.0704614
Inhibition of LPS‐induced chemokine production in human lung endothelial cells by lipid conjugates anchored to the membrane
Authors: Beck G Ch , Yard B A , Schulte J , Oberacker R , Ackern K van , Woude F J van der , Krimsky M , Kaszkin M , Yedgar S
Published: January 2005

In acute respiratory distress syndrome (ARDS) induced by endotoxins, a high production of inflammatory mediators by microvascular lung endothelial cells (LMVEC) can be observed. Activation of cells by endotoxins may result in elevated secretion of phospholipase A2 (sPLA2) which is thought to contribute to tissue damage. The present study was undertaken to investigate the role of sPLA2 in chemokine production in human lung microvascular endothelial cells (LMVEC) stimulated with the endotoxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA). In particular, we investigated the effects of sPLA2 inhibitors, specifically, the extracellular PLA2 inhibitors (ExPLIs), composed of N‐derivatized phosphatidyl‐ethanolamine linked to polymeric carriers, and LY311727, a specific inhibitor of non‐pancreatic sPLA2.

ExPLIs markedly inhibited LPS and LTA induced production and mRNA expression of the neutrophile attracting chemokines IL‐8, Gro‐α and ENA‐78, as well as of the adhesion molecules ICAM‐1 and E‐selectin. Concomitantly, ExPLIs inhibited the LPS‐induced activation of NF‐κB by LPS but not its activation by TNF‐α or IL‐1.

Endotoxin mediated chemokine production in LMVEC seems not to involve PLA2 activity, since LPS stimulation was not associated with activation of intracellular or secreted PLA2. It therefore seems that the inhibitory effect of the ExPLIs was not due to their PLA2 inhibiting capacity. This was supported by the finding that the LPS‐induced chemokine production was not affected by the selective sPLA2 inhibitor LY311727.

It is proposed that the ExPLIs may be considered a prototype of potent suppressors of specific endotoxin‐induced inflammatory responses, with potential implications for the therapy of subsequent severe inflammation.

British Journal of Pharmacology (2002) 135, 1665–1674; doi:10.1038/sj.bjp.0704618
Free‐radical production triggered by hyperthermia contributes to heat stress‐induced cardioprotection in isolated rat hearts
Authors: Arnaud Claire , Joyeux Marie , Garrel Catherine , Godin‐Ribuot Diane , Demenge Pierre , Ribuot Christophe
Published: January 2005

Heat stress (HS) is known to protect the myocardium against ischaemic damage. It has been reported that reactive oxygen species (ROS) are abundantly produced during this stress. Since mechanisms triggering the HS‐induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective phenomenon.

Rats were divided into four groups (n=8 in each group), subjected to either hyperthermia (42°C internal temperature for 15 min) or sham anaesthesia and treated or not with N‐2‐mercaptopropionyl glycine (MPG), a synthetic antioxidant, 10 min before HS. Twenty‐four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30‐min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial Hsp 27 and 70 expression was assessed by Western blot analysis (n=4). Cardiac activities of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) were also examined (n=4).

Infarct‐to‐risk zone ratio was significantly reduced in HS (17±1.3%) compared to Sham (34.3±1.7%) hearts. This effect was abolished by MPG pretreatment (40.6±1.9% in HS+MPG vs 39.8±2.5% in Sham+MPG hearts). This cardioprotection was associated with an enhanced Hsp 27 and 70 expression, which was not modified by MPG pretreatment. Antioxidant enzyme activities was not modified by heat stress or MPG pretreatment.

Free radical production following hyperthermia appears to play a role in the heat stress induced cardioprotection, independently of Hsp levels. Antioxidant enzyme activities do not seem to be implicated in this cardioprotective mechanism.

British Journal of Pharmacology (2002) 135, 1776–1782; doi:10.1038/sj.bjp.0704619
Stereoselective transport of hydrophilic quaternary drugs by human MDR1 and rat Mdr1b P‐glycoproteins
Authors: Hooiveld Guido J E J , Heegsma Janette , Montfoort Jessica E van , Jansen Peter L M , Meijer Dirk K F , Müller Michael
Published: January 2005

The present study was performed to evaluate and compare the ability of human MDR1‐, and rat Mdr1b‐ and Mdr2‐P‐glycoproteins to transport hydrophilic monoquaternary drugs. Transport studies were performed with plasma membrane vesicles isolated from MDR1‐, Mdr1b‐, or Mdr2‐overexpressing insect cells.

As model substrates we used the N‐methylated derivatives of the diastereomers quinidine and quinine, the monoquaternary compounds N‐methylquinidine and N‐methylquinine. Vincristine, an established MDR1 substrate, was used as a reference.

We observed ATP‐dependent uptake of all drugs studied into MDR1‐ and Mdr1b‐expressing vesicles. Mdr2 was not able to transport these compounds. MDR1‐ and Mdr1b‐mediated transport was saturable, and could be inhibited by various drugs, including PSC‐833.

For both MDR1 and Mdr1b the Vmax/Km ratios (or clearance) of N‐methylquinidine were greater than those determined for N‐methylquinine. This stereoselective difference was also evident from differential inhibitory studies with the two isomers.

Comparison of normalized clearance indicated that human MDR1 was more effective in transporting the tested substrates than rat Mdr1b.

In conclusion, our results demonstrate that MDR1 and Mdr1b, but not Mdr2, are able to transport the monoquaternary model drugs; both MDR1 and Mdr1b display stereospecificity for these cations; and indicate human MDR1 is more efficient in transporting these cations than its rat orthologue Mdr1b.

British Journal of Pharmacology (2002) 135, 1685–1694; doi:10.1038/sj.bjp.0704620
Stimulation of Ca2+ influx through ATP receptors on rat brain synaptosomes: identification of functional P2X7 receptor subtypes
Authors: Lundy Paul M , Hamilton Murray G , Mi Lei , Gong Wenrong , Vair Cory , Sawyer Thomas W , Frew Robert
Published: January 2005

ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons.

In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca2+ influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2‐me‐S‐ATP and ATPγS were much less active. α,β‐me‐ ATP and β,γ‐me‐ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca2+ influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X7 receptors.

Influx was unaffected by the VSCC inhibitors ω‐CTx‐MVIIC and (−) 202 – 791, indicating that ATP induced Ca2+ influx occurred primarily through P2X receptors.

P2X7 receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX‐42 but contained greatly enriched amounts of syntaxin and SNAP 25.

The various pharmacological and biochemical studies were all consistent with the presence of functional P2X7 receptors.

British Journal of Pharmacology (2002) 135, 1616–1626; doi:10.1038/sj.bjp.0704624
Evidence for sigma‐1‐like receptors in isolated rat liver mitochondrial membranes
Authors: Klouz Anis , Sapena Rosa , Liu Jun , Maurice Tangui , Tillement Jean‐Paul , Papadopoulos Vassilios , Morin Didier
Published: January 2005

Sigma (σ) receptors have generated a great deal of interest on the basis of their possible roles in various pathologies, including cytoprotection. Although the exact function of sigma‐1 (σ1) receptors is not yet known, their role in the regulation of intracellular Ca2+ levels and sterol biosynthesis, functions that could be assigned to mitochondria, are the only mechanisms described.

Using preparations of purified rat liver and brain mitochondria we demonstrate herein the presence of σ‐like binding sites. [3H](+)‐pentazocine, a σ1 radioligand was used to label these sites.

In the liver, [3H](+)‐pentazocine labelled one class of binding sites with high affinity (Kd=3 nM), similar to that observed in liver microsomes and synaptic membranes. These sites were located on the outer mitochondrial membranes and displayed high affinity for other σ1 ligands namely, haloperidol, ifenprodil, carbetapentane or 1,3‐di(2‐tolyl)guanidine (DTG).

The presence of σ1 receptors on liver mitochondria was confirmed using double fluorescence immunostaining.

[3H](+)‐pentazocine binding sites were also found on brain mitochondria but they appeared pharmacologically distinct to the liver ones as [3H](+)‐pentazocine and typical σ1 ligands displayed lower affinities for these sites. Nevertheless, [3H](+)‐pentazocine binding on both liver and brain mitochondria was modulated by progesterone, a putative endogenous ligand for σ receptors.

Our data demonstrates the presence of [3H](+)‐pentazocine binding sites with pharmacological characteristics identical to σ1 receptors on rat liver mitochondrial membranes. The pharmacological significance of these sites and their role on mitochondrial function remain unknown.

British Journal of Pharmacology (2002) 135, 1607–1615; doi:10.1038/sj.bjp.0704626
Dextran sodium sulphate‐induced colitis perturbs muscarinic cholinergic control of colonic epithelial ion transport
Authors: Sayer Brooke , Lu Jun , Green Christina , Söderholm Johan D , Akhtar Mahmood , McKay Derek M
Published: January 2005

Neuronal cholinergic input is an important regulator of epithelial electrolyte transport and hence water movement in the gut.

In this study, colitis was induced by treating mice with 4% (w v−1) dextran sodium‐sulphate (DSS)‐water for 5 days followed by 3 days of normal water. Mid‐colonic segments were mounted in Ussing chambers and short‐circuit current (Isc, indicates net ion movement) responses to the cholinergic agonist, carbachol (CCh; 10−4 M)±tetrodotoxin, atropine (ATR), hexamethonium (HEX), naloxone or phenoxybenzamine were assessed.

Tissues from mice with DSS‐induced colitis displayed a drop in Isc in response to CCh (−11.3±3.3 μA/cm2), while those from control mice showed a transient increase in Isc (76.3±13.0 μA/cm2). The ΔIsc in colon from DSS‐treated mice was tetrodotoxin‐sensitive, atropine‐insensitive and was reversed by hexamethonium (HEX+CCh=16.7±7.8 μA/cm2), indicating involvement of a nicotinic receptor.

CCh induced a drop in Isc in tissues from controls only when they were pretreated with the cholinergic muscarinic receptor blocker, atropine: ATR+CCh=−21.3±7.0 μA/cm2. Nicotine elicited a drop in Isc in Ussing‐chambered colon from both control and DSS‐treated mice that was TTX‐sensitive.

The drop in Isc evoked by CCh challenge of colonic tissue from DSS‐treated mice or ATR+CCh challenge of control tissue was not significantly affected by blockade of opiate or α‐adrenergic receptors by naloxone or phenoxybenzamine, respectively.

The data indicate that DSS‐colitis reveals a nicotinic receptor that becomes important in cholinergic regulation of ion transport.

British Journal of Pharmacology (2002) 135, 1794–1800; doi:10.1038/sj.bjp.0704633
Block of Ca2+‐channels by alpha‐endosulphine inhibits insulin release
Authors: Virsolvy Anne , Smith Paul , Bertrand Gyslaine , Gros Laurent , Héron Lisa , Salazar Guillermo , Puech Raymond , Bataille Dominique
Published: January 2005

α‐Endosulphine, isolated as an endogenous equivalent for sulphonylureas, is a 121‐amino acids protein of 19 kDa apparent molecular mass, member of a cyclic AMP‐regulated phosphoprotein family. We have previously shown that α‐endosulphine inhibits sulphonylurea binding and KATP channel activity, thereby stimulating basal insulin secretion.

We now describe that in the perfused rat pancreas, no stimulation was detected and that α‐endosulphine inhibited glucose stimulated insulin release. This inhibition was dose‐dependent and affected both phases of insulin secretion.

This inhibitory effect of α‐endosulphine also occurred on MIN6 β‐cells when insulin release was stimulated either by glucose, sulphonylureas or a high K+ depolarization. Inhibition was concentration‐dependent with a half‐maximal inhibition at 0.5 μM and was mirrored by inhibition of calcium influx.

Electrophysiological experiments demonstrated, in comparison to the effects of the sulphonylurea tolbutamide, that these inhibitory effects were linked to a direct inhibition of L‐type Ca2+‐channels and were independent from a regulation of KATP channels.

Although α‐endosulphine is able to stimulate insulin release under specific conditions acting via modulation of KATP channel activity, the present study suggests that, under physiological conditions, the peptide mainly acts to block voltage‐gated Ca2+‐channels. This block leads to the inhibition of calcium influx and triggers inhibition of insulin release.

We conclude that α‐endosulphine is not exclusively an endogenous equivalent for sulphonylureas and not solely a KATP channel regulator.

British Journal of Pharmacology (2002) 135, 1810–1818; doi:10.1038/sj.bjp.0704635
Rolipram inhibits leukocyte‐endothelial cell interactions in vivo through P‐ and E‐selectin downregulation
Authors: Sanz María‐Jesús , Alvarez Angeles , Piqueras Laura , Cerdá Miguel , Issekutz Andrew C , Lobb Roy R , Cortijo Julio , Morcillo Esteban J
Published: January 2005

Rolipram, a selective phosphodiesterase (PDE) type 4 inhibitor, was used to characterize leukocyte recruitment mechanisms in models of acute and subacute inflammation. Intravital microscopy within the rat mesenteric microcirculation was employed.

Mesentery superfusion with PAF (0.1 μM) induced a significant increase in leukocyte rolling flux, adhesion and emigration at 60 min. Rolipram pretreatment, markedly inhibited these parameters by 100, 95 and 95% respectively.

Similar effects were observed when the mesentery was superfused with LPS (1 μg ml−1) for the same time period and these leukocyte parameters were nearly abrogated by rolipram pretreatment.

LPS exposure of the mesentery for 4 h caused a greater increase in leukocyte rolling flux, adhesion and emigration which were inhibited by rolipram administration by 51, 71 and 81% respectively.

Immunohistochemistry revealed a significant increase in P‐selectin expression after 60 min superfusion with PAF which was attenuated by rolipram.

LPS exposure of the mesentery for 4 h caused a significant increase in P‐ and E‐selectin, intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) expression. Rolipram pretreatment down‐regulated both P‐ and E‐selectin expression but had no effect on ICAM‐1 and VCAM‐1 expression.

Significant increases in plasma cyclic AMP levels were detected at 4.5 h after rolipram administration.

In conclusion, we have demonstrated that rolipram is a potent in vivo inhibitor of leukocyte‐endothelial cell interactions. The effects observed are mediated through endothelial P‐ and E‐selectin downregulation. Therefore, selective PDE‐4 inhibitors may be useful in the control of different inflammatory disorders.

British Journal of Pharmacology (2002) 135, 1872–1881; doi:10.1038/sj.bjp.0704644
NCX4016 (NO‐aspirin) reduces infarct size and suppresses arrhythmias following myocardial ischaemia/reperfusion in pigs
Authors: Wainwright Cherry L , Miller Ashley M , Work Lorraine M , Soldato Piero Del
Published: January 2005

The effect of the nitro‐derivative of aspirin, NCX4016, was assessed on ischaemic ventricular arrhythmias and myocardial infarct size in anaesthetized pigs in comparison to native aspirin.

Pigs were given aspirin (10 mg kg−1; n=6), low dose NCX4016 (18.4 mg kg−1; n=6) or high dose NCX4016 (60 mg kg−1; n=7) orally for 5 days prior to coronary occlusion and reperfusion. None of the interventions had any effect on baseline haemodynamics prior to coronary occlusion in comparison to control pigs (n=9). Aspirin and high dose NCX4016 both prevented the generation of thromboxane A2 from platelets activated ex vivo with A23187 (30 μM), whereas all three interventions markedly attenuated platelet aggregation in response to collagen in whole blood in comparison to controls.

None of the drug interventions had any effect on the incidence of ventricular fibrillation (VF) during myocardial ischaemia (100% in all groups). However, 60 mg kg−1 NCX4016 significantly attenuated the total number of premature ventricular beats (PVB's) (62±16 vs 273±40 in control pigs; P<0.05) during the first 30 min of occlusion. The higher dose of NCX4016 also significantly reduced myocardial infarct size (22.6±3.7% of area at risk vs 53.0±2.8% of area at risk in control pigs; P<0.05).

These results suggest that the nitro‐derivative of aspirin, NCX4016, is an effective antiplatelet agent, which unlike aspirin also reduces the extent of myocardial injury following ischaemia and reperfusion.

British Journal of Pharmacology (2002) 135, 1882–1888; doi:10.1038/sj.bjp.0704646
Tachykinin NK1 and NK2 receptors mediate inhibitory vs excitatory motor responses in human isolated corpus cavernosum and spongiosum
Authors: Patacchini Riccardo , Barbagli Guido , Palminteri Enzo , Lazzeri Massimo , Turini Damiano , Maggi Carlo Alberto
Published: January 2005

Motor effects produced by tachykinins were studied in human isolated corpus spongiosum and cavernosum. In quiescent preparations neurokinin A caused potent contractions (pD2=8.3 – 7.9 respectively) prevented by the NK2 receptor‐selective antagonist nepadutant, whereas [Sar9]SP sulfone and senktide (NK1 and NK3 receptor‐selective agonists) produced no effect or spare contractions. In KCl‐precontracted corpus spongiosum septide (pD2=7.1) and [Sar9]SP sulfone (pD2=7.7) produced tetrodotoxin‐resistant relaxations, abolished by the tachykinin NK1 receptor‐selective antagonist SR 140333. [Sar9]SP sulfone (1 μM) produced similar relaxations in precontracted corpus cavernosum. Electrical field stimulation (EFS) elicited tetrodotoxin‐sensitive relaxations, which were additive to those produced by [Sar9]SP sulfone. Nω‐nitro‐L‐arginine (L‐NOARG) totally prevented both [Sar9]SP sulfone‐ and EFS‐induced relaxations. These results show that tachykinin NK1 and NK2 receptors mediate opposite motor effects in human penile tissues, suggesting a possible modulatory role of tachykinins on smooth muscle tone in these organs.

British Journal of Pharmacology (2002) 135, 1351–1354; doi:10.1038/sj.bjp.0704650
The sleep hormone oleamide modulates inhibitory ionotropic receptors in mammalian CNS in vitro
Authors: Coyne Leanne , Lees George , Nicholson Russell A , Zheng Jian , Neufield Katherine D
Published: January 2005

We examine the sensitivity of GABAA and glycine receptors (same ionotropic superfamily) to oleamide. We address subunit‐dependence/modulatory mechanisms and analogies with depressant drugs.

Oleamide modulated human GABAA currents (α1β2γ2L) in oocytes (EC50, 28.94±s.e.mean of 1.4 μM; Maximum 216%±35 of control, n=4). Modulation of human α1 glycine homo‐oligomers (significant), was less marked, with a lower EC50 (P<0.05) than GABA receptors (EC50, 22.12±1.4 μM; Maximum 171%±30, n=11).

Only the hypnogenic cis geometric isomer enhanced glycine currents (without altering slope or maximal current, it reduced the glycine EC50 from 322 to 239 μM: P<0.001). Modulation was not voltage‐dependent or associated with a shift in Er.

β1 containing GABAA receptors (insensitive to many depressant drugs) were positively modulated by oleamide. Oleamide efficacy was circa 2× greater at α1β1γ2L than α1β2γ2L (P=0.007). Splice variation in γ subunits did not alter oleamide sensitivity.

cis‐9,10‐Octadecenoamide had no effect on the equilibrium binding of [3H]‐muscimol or [3H]‐EBOB to mouse brain membranes. It does not directly mimic GABA, or operate as a neurosteroid‐, benzodiazepine‐ or barbiturate‐like modulator of GABAA‐receptors.

The transport of [3H]‐GABA into mouse brain synaptoneurosomes was unaffected by high micromolar concentrations of cis‐9,10‐octadecenoamide. Oleamide does not enhance GABA‐ergic currents or prolong IPSCs by inhibiting GABA transport.

Oleamide is a non‐selective modulator of inhibitory ionotropic receptors. The sleep lipid exerts its effects indirectly, or at a novel recognition site on the GABAA complex.

British Journal of Pharmacology (2002) 135, 1977–1987; doi:10.1038/sj.bjp.0704651
A direct inhibitory action of prostaglandins upon ACTH secretion at the late stages of the secretory pathway of AtT‐20 cells
Authors: Wilson Mary L , Guild Simon B
Published: January 2005

The mouse AtT‐20/D16‐16 anterior pituitary tumour cell line was used as a model system for the study of the effects of prostaglandins upon the late stages of the adrenocorticotrophin (ACTH) secretory pathway.

Calcium (1 nM – 100 μM), guanosine‐5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S) (1 – 100 μM) and mastoparan (1 and 10 μM) all stimulated ACTH secretion from permeabilized AtT‐20 cells in a concentration‐dependent manner. GTP‐γ‐S and mastoparan stimulated ACTH secretion from permeabilized cells in the absence of calcium. Co‐incubation with prostaglandins E1 and E2 (PGE1, PGE2) (10 μM) but not prostaglandin F2α (PGF2α) (10 μM) significantly inhibited calcium‐, GTP‐γ‐S and mastoparan‐evoked secretion by 30 – 50%.

The effects of PGE1 and PGE2 upon GTP‐γ‐S (100 μM)‐, calcium (10 μM)‐ and mastoparan (10 μM)‐evoked secretion were concentration‐dependent. PGE1 significantly inhibited GTP‐γ‐S‐ and calcium‐evoked secretion at concentrations of PGE1 above 1 μM but mastoparan‐evoked secretion only at the highest concentration of PGE1 investigated (10 μM). PGE2 was much more potent than PGE1 and significantly inhibited GTP‐γ‐S‐ and calcium‐evoked secretion at 10 nM and above and mastoparan‐evoked secretion above 1 μM.

The inhibitory effects of PGE1 and PGE2 upon calcium‐, GTP‐γ‐S‐ and mastoparan‐stimulated ACTH secretion from permeabilized cells were pertussis toxin (PTX) sensitive.

In intact cells PGE1, PGE2 and PGF2α (1 nM – 10 μM) acting singly had little or no effect upon ACTH secretion. However, only PGE2 (1 nM – 10 μM) significantly inhibited corticotrophin‐releasing factor‐41 (CRF‐41) (100 nM)‐evoked secretion in a concentration dependent manner.

The present study finds that prostaglandins of the E series exert an inhibitory action, via a pertussis toxin‐sensitive GTP‐binding (G)‐protein, in the late stages of the ACTH secretory pathway distal to the G‐exocytosis (Ge)/calcium point of control.

British Journal of Pharmacology (2002) 135, 1851–1858; doi:10.1038/sj.bjp.0704652
Inhibition of spontaneous acetylcholine secretion by 2‐chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction
Authors: Hirsh Jody K , Silinsky Eugene M
Published: January 2005

Previous studies have reported discrepancies in the potencies of A1 adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) in mediating the inhibitory effects of A1 receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2‐chloroadenosine (CADO).

CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 μM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 μM) produced highly statistically‐significant reductions in spontaneous ACh release.

In the presence of the non‐selective protein kinase inhibitor, H7 (50 μM), the potency of CADO was increased such that 1 μM CADO now reduced spontaneous quantal ACh release to approximately 63% of control.

Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 μM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory effect of 1 μM CADO. A number of other non‐selective PKA inhibitors also failed to increase the potency of CADO.

The results suggest that an endogenous H7‐sensitive process modulates the sensitivity of the mouse A1 adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition.

British Journal of Pharmacology (2002) 135, 1897–1902; doi:10.1038/sj.bjp.0704653
Stimulation of protein kinase B and p70 S6 kinase by the histamine H1 receptor in DDT1MF‐2 smooth muscle cells
Authors: Dickenson John M
Published: January 2005

Previous studies have shown that the histamine H1 receptor activates p42/p44 mitogen‐activated protein kinases (MAPK) in DDT1MF‐2 smooth muscle cells via a phosphatidylinositol 3‐kinase (PI‐3K)‐dependent pathway. In this study the effect of histamine H1 receptor stimulation on protein kinase B (PKB) and p70 S6 kinase, both of which are downstream targets of PI‐3K, has been investigated. Increases in PKB and p70 S6 kinase activation were monitored by Western blotting using phospho‐specific PKB (Ser473) and p70 S6 kinase (Thr421/Ser424) antibodies.

Histamine stimulated time and concentration‐dependent increases in the phosphorylation of PKB and p70 S6 kinase in DDT1MF‐2 cells. Both responses were completely inhibited by the histamine H1 receptor antagonist mepyramine and following pre‐treatment with pertussis toxin, to block Gi/Go protein dependent pathways.

The PI‐3K inhibitors wortmannin (IC50 5.9±0.5 nM) and LY 294002 (IC50 6.9±0.8 μM) attenuated the increase in PKB phosphorylation induced by histamine (100 μM) in a concentration‐dependent manner.

Histamine‐induced increases in p70 S6 kinase phosphorylation were partially sensitive to rapamycin (20 nM; 68% inhibition) but completely blocked by wortmannin (100 nM), LY 294002 (30 μM) and the MAPK kinase inhibitor PD 98059 (50 μM).

In summary, these data demonstrate that the histamine H1 receptor stimulates PKB and p70 S6 kinase phosphorylation in DDT1MF‐2 smooth muscle cells. However, functional studies revealed that histamine does not stimulate DDT1MF‐2 cell proliferation or attenuate staurosporine‐induced caspase‐3 activity. The challenge for future research will be to link the stimulation of these kinase pathways with the physiological and pathophysiological roles of the histamine H1 receptor.

British Journal of Pharmacology (2002) 135, 1967–1976; doi:10.1038/sj.bjp.0704664
Oxygen‐derived free radicals mediate endothelium‐dependent contractions to acetylcholine in aortas from spontaneously hypertensive rats
Authors: Yang Di , Félétou Michel , Boulanger Chantal M. , Wu Heng‐Fang , Levens Nigel , Zhang Ji‐Nan , Vanhoutte Paul M.
Published: January 2005

Experiments were designed to investigate whether or not oxygen‐derived free radicals mediate endothelium‐dependent contractions to acetylcholine in the aorta of spontaneously hypertensive rat (SHR).

Isometric tension was measured in aortic rings taken from adult male SHR and Wistar‐Kyoto rat (WKY) in the presence of NG‐nitro‐L‐arginine.

Endothelium‐dependent contractions to acetylcholine were significantly greater in rings from SHR compared to WKY. Oxygen‐derived free radicals, generated from xanthine plus xanthine oxidase, induced contractions that were larger in aortas from SHR than from WKY. Contractions to acetylcholine and free radicals were abolished by a selective TP‐receptor antagonist, S 18886, and a preferential inhibitor of cyclo‐oxygenase‐1, valeryl salicylate, but not by a preferential inhibitor of cyclo‐oxygenase‐2, NS‐398.

Allopurinol, deferoxamine and the combination of superoxide dismutase plus catalase inhibited the contractions to oxygen‐derived free radicals but did not significantly affect those to acetylcholine. In contrast, diethyldithiocarbamic acid, an inhibitor of superoxide dismutase, or Tiron, a scavenger of superoxide anion, reduced endothelium‐dependent contractions to acetylcholine in aortas from SHR. The effect of these two drugs was additive.

In SHR chronically treated with dimethylthiourea endothelium‐dependent contractions to acetylcholine were decreased, and reduced further by acute in vitro exposure to deferoxamine or the combination of superoxide dismutase plus catalase.

These results suggest that in the SHR aorta acetylcholine‐induced endothelium‐dependent contractions involve endothelial superoxide anion production and the subsequent dismutation into hydroxyl radicals and/or hydrogen peroxide. The free radicals activate cyclo‐oxygenase‐1, most likely to produce endoperoxides. Activation of TP‐receptors is required to observe endothelium‐dependent contractions to acetylcholine or endothelium‐independent contractions in response to free radical generation.

British Journal of Pharmacology (2002) 136, 104–110; doi:10.1038/sj.bjp.0704669
Relaxation by urocortin of human saphenous veins
Authors: Sanz Elena , Monge Luis , Fernández Nuria , Martínez María Angeles , Martínez‐León Juan B. , Diéguez Godofredo , García‐Villalón Angel Luis
Published: January 2005

Urocortin, an endogenous peptide structurally related to corticotropin‐releasing factor (CRF), has potent cardiovascular effects, suggesting that it may be of significance in cardiovascular regulation. The objective of this study was to analyse the effects of urocortin and its action mechanisms on human blood vessels.

To this, 3 mm long segments from human saphenous veins were prepared for isometric tension recording in an organ bath.

In the segments at basal resting tone, urocortin did not produce any effect, but in the segments precontracted with endothelin‐1 (1 – 10 nM), urocortin (1 pM – 10 nM) produced concentration‐dependent relaxation.

This relaxation was not modified by the inhibitor of nitric oxide synthase NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM), but it was potentiated by the cyclo‐oxygenase inhibitor meclofenamate (10 μM) and it was reduced by the inhibitors of high‐conductance Ca2+‐dependent potassium channels tetraethylammonium (TEA, 10 mM) and charybdotoxin (100 nM).

These results indicate that human saphenous veins are very sensitive to urocortin, which produces vascular relaxation by a mechanism independent of nitric oxide and dependent of high‐conductance Ca2+‐dependent potassium channels, and that it may be opposed by the release of vasoconstrictor prostanoids. Therefore, urocortin may be of significance for regulation of the venous circulation in humans.

British Journal of Pharmacology (2002) 136, 90–94; doi:10.1038/sj.bjp.0704670
Thioridazine interacts with the membrane of mitochondria acquiring antioxidant activity toward apoptosis – potentially implicated mechanisms
Authors: Rodrigues Tiago , Santos Antonio C. , Pigoso Acácio A. , Mingatto Fábio E. , Uyemura Sérgio A. , Curti Carlos
Published: January 2005

We evaluated the effects of the phenothiazine derivative thioridazine on mechanisms of mitochondria potentially implicated in apoptosis, such as those involving reactive oxygen species (ROS) and cytochrome c release, as well as the involvement of drug interaction with mitochondrial membrane in these effects.

Within the 0 – 100 μM range thioridazine did not reduce the free radical 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) nor did it chelate iron.

However, at 10 μM thioridazine showed important antioxidant activity on mitochondria, characterized by inhibition of accumulation of mitochondria‐generated O2•−, assayed as lucigenin‐derived chemiluminescence, inhibition of Fe2+/citrate‐mediated lipid peroxidation of the mitochondrial membrane (LPO), assayed as malondialdehyde generation, and inhibition of Ca2+/t‐butyl hydroperoxide (t‐BOOH)‐induced mitochondrial permeability transition (MPT)/protein‐thiol oxidation, assayed as mitochondrial swelling.

Thioridazine respectively increased and decreased the fluorescence responses of mitochondria labelled with 1‐aniline‐8‐naphthalene sulfonate (ANS) and 1‐(4‐trimethylammonium phenyl)‐6 phenyl 1,3,5‐hexatriene (TMA‐DPH).

The inhibition of LPO and MPT onset correlated well with the inhibition of cytochrome c release from mitochondria.

We conclude that thioridazine interacts with the inner membrane of mitochondria, more likely close to its surface, acquiring antioxidant activity toward processes with potential implications in apoptosis such as O2•− accumulation, as well as LPO, MPT and associated release of cytochrome c.

British Journal of Pharmacology (2002) 136, 136–142; doi:10.1038/sj.bjp.0704672
2‐Chloro N6‐methyl‐(N)‐methanocarba‐2′‐deoxyadenosine‐3′,5′‐bisphosphate is a selective high affinity P2Y1 receptor antagonist
Authors: Boyer José L , Adams Mary , Ravi R Gnana , Jacobson Kenneth A , Harden T Kendall
Published: January 2005

We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y1 receptor and that modification of the ribose in these analogues is tolerated in the P2Y1 receptor binding pharmacophore.

Here we delineate the pharmacological activity of one such non‐nucleotide molecule, 2‐chloro N6‐methyl‐(N)‐methanocarba‐2′‐deoxyadenosine‐3′,5′‐bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)‐conformation by a cyclopropane moiety.

MRS2279 antagonized 2MeSADP‐stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pKB=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y1 receptor (pKB=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y1 receptor since MRS2279 had no effect on activation of the human P2Y2, P2Y4, P2Y6, or P2Y11 receptors by their cognate agonists.

MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation.

In contrast, the P2Y1 receptor is known to be obligatory in the process of ADP‐induced platelet aggregation, and MRS2279 competitively inhibited ADP‐promoted platelet aggregation with an apparent affnity (pKB=8.05) similar to that observed at the human P2Y1 receptor heterologously expressed in 1321N1 cells.

Taken together these results illustrate selective high affinity antagonism of the P2Y1 receptor by a non‐nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.

British Journal of Pharmacology (2002) 135, 2004–2010; doi:10.1038/sj.bjp.0704673
Regulation of human penile smooth muscle tone byprostanoid receptors
Authors: Angulo Javier , Cuevas Pedro , La Fuente Jose M. , Pomerol Jose M. , Ruiz‐Castañé Eduardo , Puigvert Ana , Gabancho Sonia , Fernández Argentina , Ney Peter , Sáenz de Tejada Iñigo
Published: January 2005

We have characterized the prostanoid receptors involved in the regulation of human penile arterial and trabecular smooth muscle tone.

Arachidonic acid induced relaxation of human corpus cavernosum strips (HCCS) that was blocked by the cyclo‐oxygenase inhibitor, indomethacin, and augmented by the thromboxane receptor (TP) antagonist, SQ29548, suggesting that endogenous production of prostanoids regulates penile smooth muscle tone.

TP‐receptors mediate contraction of HCCS and penile resistance arteries (HPRA), since the agonist of these receptors, U46619, potently contracted HCCS (EC50 8.3±2.8 nM) and HPRA (EC50 6.2±2.2 nM), and the contractions produced by prostaglandin F2α at high concentrations (EC50 6460±3220 nM in HCCS and 8900±6700 nM in HPRA) were inhibited by the selective TP‐receptor antagonist, SQ29548 (0.02 μM).

EP‐receptors are responsible for prostanoid‐induced relaxant effects in HCCS because only prostaglandin E1 (PGE1), prostaglandin E2 and the EP2/EP4‐receptor agonist, butaprost, produced consistent relaxation of this tissue (EC50 93.8±31.5, 16.3±3.8 and 1820±1284 nM, respectively). In HPRA, both prostacyclin and PGE1 (EC50 60.1±18.4 and 109.0±30.9 nM, respectively) as well as the selective IP receptor agonist, cicaprost, and butaprost (EC50 25.2±15.2 and 7050±6020 nM, respectively) caused relaxation, suggesting co‐existence of IP‐ and EP‐receptors (EP2 and/or EP4).

In summary, endogenous production of prostanoids may regulate penile smooth muscle contractility by way of specific receptors. TP‐receptors mediate contraction in HCCS and HPRA, while the relaxant effects of prostanoids are mediated by EP2‐ and/or EP4‐receptors in HCCS and by EP‐ and IP‐receptors in HPRA.

British Journal of Pharmacology (2002) 136, 23–30; doi:10.1038/sj.bjp.0704675
Atorvastatin inhibition of cytokine‐inducible nitric oxide synthase expression in native endothelial cellsin situ
Authors: Wagner Andreas H. , Schwabe Oliver , Hecker Markus
Published: January 2005

Animal experimental studies have demonstrated that inducible nitric oxide synthase (iNOS) expression correlates with neointima formation and is prevented by HMG‐CoA reductase inhibitors (statins). In the present study we have investigated the underlying mechanism of action of these drugs in isolated segments of the rat aorta.

Western blot analysis and immunohistochemistry revealed that tumour necrosis factor α (TNFα) plus interferon‐γ (IFNγ) synergistically induce iNOS gene expression in the endothelium but not in the smooth muscle of these segments while constitutive endothelial NO synthase (eNOS) abundance was markedly reduced.

Pre‐treatment with 1 – 10 μMatorvastatin, cerivastatin or pravastatin decreased TNFα plus IFNγ stimulated iNOS expression in the endothelium irrespective of the presence of the HMG‐CoA reductase product mevalonate (400 μM).

Electrophoretic mobility shift assay experiments confirmed that the combination of TNFα plus IFNγ causes activation of the transcription factors STAT‐1 and NF‐κB in native endothelial cells. Neutralization of these transcription factors by employing the corresponding decoy oligonucleotides confirmed their involvement in TNFα plus IFNγ stimulated iNOS expression. Translocation of both transcription factors was attenuated by atorvastatin, and this effect was insensitive to exogenous mevalonate.

The present findings thus demonstrate a specific HMG‐CoA reductase‐independent inhibitory effect of statins, namely atorvastatin, on cytokine‐stimulated transcription factor activation in native endothelial cellsin situand the subsequent expression of a gene product implicated in vascular inflammation. This effect may be therapeutically relevant and in addition provide an explanation for the reported rapid onset of action of these drugs in humans.

British Journal of Pharmacology(2002)136, 143–149; doi:10.1038/sj.bjp.0704678
Involvement of purinergic signalling in central mechanisms of body temperature regulation in rats
Authors: Gourine Alexander V , Melenchuk Ekaterina V , Poputnikov Dmitry M , Gourine Valery N , Spyer K Michael
Published: January 2005

P2 purinoreceptors are present in hypothalamic and brainstem nuclei that are involved in the regulation of body temperature (Tb). The role of ATP acting on these P2 receptors in thermoregulation was investigated by studying the effects of the stable ATP analogue α,β‐methyleneATP (α,β‐meATP) and P2 receptor antagonists suramin and pyridoxal‐5′‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on Tb when injected intracerebroventricularly (i.c.v.) via a pre‐implanted cannula in conscious rats at various ambient temperatures and during lipopolysaccharide (LPS)‐induced fever.

Depending on ambient temperature, α,β‐meATP (0.2 μmol, i.c.v.) induced a fall in Tb (−3.3°C, P<0.05), no changes in Tb when compared to pre‐injection levels, or an increase in Tb (∼1.0°C, P<0.05) in rats maintained at 10°C, 25°C and 30°C ambient temperature, respectively.

Suramin (7 nmol, i.c.v.) induced a lasting (up to 6 h) increase in Tb (on average 1.2°C, P<0.05) in rats kept at 25°C or 30°C, but failed to induce any rise in Tb in rats at 10°C ambient temperature. An increase in Tb was also observed in rats (25°C ambient temperature) treated with PPADS (0.2 μmol, i.c.v.).

α,β‐meATP (0.2 μmol) injected i.c.v. or directly into the anterior hypothalamus caused a profound fall in Tb (by 0.9°C and 1.0°C, respectively; P<0.05) during LPS (E.coli; 50 μg kg−1)‐induced fever in rats at 25°C ambient temperature. Fever was initiated more rapidly in rats treated with suramin (7 nmol) or PPADS (70 nmol), however its late phase was unaffected. Suramin (7 nmol) and PPADS (70 nmol) injected at the time when fever was already developed (2.5 h after LPS injections) did not alter febrile Tb.

These data indicate that purinergic signalling may play a significant role in central mechanisms of Tb regulation at various ambient temperatures and during fever.

British Journal of Pharmacology (2002) 135, 2047–2055; doi:10.1038/sj.bjp.0704679
Identification of the monomeric G‐protein, Rhes, as an efaroxan‐regulated protein in the pancreatic β‐cell
Authors: Chan Sue L. F. , Monks Lara K. , Gao Hongwei , Deaville Pamela , Morgan Noel G.
Published: January 2005

Efaroxan induces membrane depolarization by interaction with the pore forming subunit of the ATP‐sensitive potassium channel, Kir6.2. However, this effect is not responsible for its full secretory activity.

In this study we have used an anti‐idiotypic approach to generate antibodies that recognize additional proteins that may be regulated by efaroxan in pancreatic β‐cells.

Using these antisera in an expression cloning strategy we have identified a monomeric GTP‐binding protein, Rhes, as a potential target for regulation by imidazoline ligands. Rhes is shown to be expressed in β‐cells and its expression is regulated by efaroxan under conditions when a structurally related molecule, KU14R, is ineffective.

The results reveal that β‐cells express Rhes and suggest that changes in the expression of this molecule may regulate the sensitivity of β‐cells to imidazoline secretagogues.

British Journal of Pharmacology (2002) 136, 31–36; doi:10.1038/sj.bjp.0704680