bluuebunny/crossref_metadata_2025 huggingface dataset:
A study of the mechanisms involved in the neurotoxic action of 3,4‐methylenedioxymethamphetamine (MDMA, ‘ecstasy’) on dopamine neurones in mouse brain
Authors: Colado M Isabel , Camarero Jorge , Mechan Annis O , Sanchez Veronica , Esteban Blanca , Elliott J Martin , Green A Richard
Published: January 2005

Administration of 3,4‐methylenedioxymethamphetamine (MDMA, ‘ecstasy’) to mice produces acute hyperthermia and long‐term degeneration of striatal dopamine nerve terminals. Attenuation of the hyperthermia decreases the neurodegeneration. We have investigated the mechanisms involved in producing the neurotoxic loss of striatal dopamine.

MDMA produced a dose‐dependent loss in striatal dopamine concentration 7 days later with 3 doses of 25 mg kg−1 (3 h apart) producing a 70% loss.

Pretreatment 30 min before each MDMA dose with either of the N‐methyl‐D‐aspartate antagonists AR‐R15896AR (20, 5, 5 mg kg−1) or MK‐801 (0.5 mg kg−1×3) failed to provide neuroprotection.

Pretreatment with clomethiazole (50 mg kg−1×3) was similarly ineffective in protecting against MDMA‐induced dopamine loss.

The free radical trapping compound PBN (150 mg kg−1×3) was neuroprotective, but it proved impossible to separate neuroprotection from a hypothermic effect on body temperature.

Pretreatment with the nitric oxide synthase (NOS) inhibitor 7‐NI (50 mg kg−1×3) produced neuroprotection, but also significant hypothermia. Two other NOS inhibitors, S‐methyl‐L‐thiocitrulline (10 mg kg−1×3) and AR‐R17477AR (5 mg kg−1×3), provided significant neuroprotection and had little effect on MDMA‐induced hyperthermia.

MDMA (20 mg kg−1) increased 2,3‐dihydroxybenzoic acid formation from salicylic acid perfused through a microdialysis tube implanted in the striatum, indicating increased free radical formation. This increase was prevented by AR‐R17477AR administration. Since AR‐R17477AR was also found to have no radical trapping activity this result suggests that MDMA‐induced neurotoxicity results from MDMA or dopamine metabolites producing radicals that combine with NO to form tissue‐damaging peroxynitrites.

British Journal of Pharmacology (2001) 134, 1711–1723; doi:10.1038/sj.bjp.0704435
Comparison of the contractile and calcium‐increasing properties of platelet‐activating factor and endothelin‐1 in the rat mesenteric artery and vein
Authors: Claing Audrey , Shbaklo Hadia , Plante Mirco , Bkaily Ghassan , D'Orléans‐Juste Pedro
Published: January 2005

In the present study, the properties of endothelin‐1 (ET‐1) and platelet‐activating factor (PAF) in inducing contraction and increased intracellular‐free calcium level in rat mesenteric arteries and veins were studied. Furthermore, measurements of cytosolic ([Ca]c) and nuclear ([Ca]n) Ca2+ were performed by confocal microscopy.

PAF, at a concentration of 1 μM, and the selective ETB agonists, IRL‐1620 and sarafotoxin S6C (100 nM), induced a marked constriction and increase in [Ca]i in the mesenteric vein but not in the artery. On the other hand, endothelin‐1 (1–100 nM) induced a significant concentration‐dependent nifedipine‐insensitive increase in tension and [Ca]i in both arteries and veins.

Those responses to endothelin‐1 were significantly reduced by the ETA receptor antagonist, BQ‐123 (10−6 M), on both types of vessels, whereas the selective ETB receptor antagonist, BQ‐788, inhibited only the venous responses. The mixed ETA/ETB receptor antagonist, SB 209670, reduced the ET‐1‐induced venous responses to the same level of that found in presence of BQ‐123 or BQ‐788. However, concomitant applications of BQ‐123 and BQ‐788 reduced the vasoconstriction below to that induced by ETA or ETB blockade without further affecting [Ca]i.

PAF and the selective ETB agonists IRL‐1620, induced a sustained increase of [Ca]c and [Ca]n solely in venous cells and ET‐1 in both arterial and venous smooth muscle cells.

Thus, PAF increases total intracellular calcium concentration and tension on the smooth muscle cells from venous origin only. Furthermore, ET‐1‐induced vasoactive as well as [Ca]i and [Ca]n increasing effects are mediated by distinct receptors on venous and arterial smooth muscles.

British Journal of Pharmacology (2002) 135, 433–443; doi:10.1038/sj.bjp.0704441
Comparison of the affinity of β‐blockers for two states of the β1‐adrenoceptor in ferret ventricular myocardium
Authors: Lowe Martin D , Lynham James A , Grace Andrew A , Kaumann Alberto J
Published: January 2005

We compared the potency of 11 clinically available β‐blockers as antagonists of the positive inotropic effects of (−)‐isoprenaline and CGP12177 on ferret ventricular myocardium.

(−)‐CGP12177, (−)‐pindolol and (−)‐alprenolol were non‐conventional partial agonists with intrinsic activity of 0.7, 0.2 and 0.1 respectively.

All β‐blockers antagonized in a concentration‐dependent and surmountable manner the positive inotropic effects of both (−)‐isoprenaline and CGP12177. The potency of each β‐blocker was consistently higher against (−)‐isoprenaline than against CGP12177. Two groups of β‐blockers were identified. In one group the difference between the pKB values of blockade against (−)‐isoprenaline and CGP12177 was 1.1–1.6 log units ((−)‐alprenolol, (−)‐pindolol, (−)‐bupranolol, nadolol and carvedilol). In the other group the pKB difference was of 2.1–3.0 log units ((−)‐atenolol, metoprolol, bisoprolol, sotalol, (−)‐propranolol and (−)‐timolol).

The β‐blockers competed with (−)‐[125I]‐cyanopindolol for binding to ventricular β1‐adrenoceptors. The binding affinities correlated with the corresponding blocking potencies against (−)‐isoprenaline. On average the pKi values were 0.5 log units smaller than the pKB values against (−)‐isoprenaline but 1.6 log units greater than the pKB values against CGP12177.

In ferret ventricle the effects of (−)‐isoprenaline appear to be antagonized by β‐blockers through the state of the β1‐adrenoceptor for which (−)‐[125I]‐cyanopindolol and β‐blockers have high affinity. The cardiostimulant effects of CGP12177 appear to be mediated through a low‐affinity state of the β1‐adrenoceptor for which β‐blockers have low affinity.

British Journal of Pharmacology (2002) 135, 451–461; doi:10.1038/sj.bjp.0704450
Redox/ROS regulation of lipopolysaccharide‐induced mitogen‐activated protein kinase (MAPK) activation and MAPK‐mediated TNF‐α biosynthesis
Authors: Haddad J J , Land S C
Published: January 2005

Redox and ROS regulation of MAPK‐mediated TNF‐α biosynthesis is not well characterized. It was hypothesized that the involvement of the MAPK pathway in regulating LPS‐mediated TNF‐α secretion is redox‐dependent, NF‐κB‐sensitive and attenuated by N‐acetyl‐L‐cysteine (NAC) and other antioxidants.

In alveolar epithelial cells, LPS induced a time‐ and dose‐dependent phosphorylation of MAPKp38. This was associated with the activation of MAPK‐activated protein kinase, which phosphorylated the small heat‐shock protein, Hsp27.

MAPKp38 inhibition (SB‐203580) abrogated LPS‐induced TNF‐α production. MAPKERK blockade (PD‐98059) attenuated TNF‐α secretion, an effect synergistically amplified in the presence of SB‐203580.

Regulation of NF‐κB by selective inhibitors revealed that this pathway is partially involved in regulating LPS‐mediated TNF‐α secretion. Whereas the proteasome inhibitor, MG‐132, had no effect on LPS‐mediated TNF‐α production, CAPE, sulfasalazine and SN‐50, a cell‐permeant NF‐κB inhibitor, attenuated but did not abrogate TNF‐α biosynthesis.

LPS up‐regulated ROS, an effect abrogated by 4′‐hydroxy‐3′‐methoxy‐acetophenone and NAC, which reduced TNF‐α secretion, induced the accumulation of GSH, reduced the concentration of GSSG, and blockaded the phosphorylation/activation of MAPKp38 pathway.

ROS induced MAPKp38 phosphorylation and selective antioxidants, including the permeant GSH precursor, γ‐GCE, reduced ROS‐dependent MAPKp38 phosphorylation.

These results indicate that the MAPK pathway and MAPK‐mediated regulation of TNF‐α production is redox‐dependent, GSH‐mediated and requires, at least in part, a NF‐κB/ROS‐sensitive mechanism.

British Journal of Pharmacology (2002) 135, 520–536; doi:10.1038/sj.bjp.0704467
Pharmacological profiles of a novel opioid receptor‐like1 (ORL1) receptor antagonist, JTC‐801
Authors: Yamada Hideki , Nakamoto Hiromitsu , Suzuki Yasunori , Ito Takao , Aisaka Kazuo
Published: January 2005

Pharmacological effects of a novel opioid receptor‐like1 (ORL1) receptor antagonist, [N‐(4‐amino‐2‐methylquinolin‐6‐yl)‐2‐(4‐ethylphenoxymethyl) benzamide monohydrochloride] (JTC‐801), were examined in in vitro and in vivo.

JTC‐801 inhibited the binding of [3H]‐nociceptin to human ORL1 receptors expressed in HeLa cells with a Ki value of 44.5 nM.

JTC‐801 completely antagonized the suppression of nociceptin on forskolin‐induced accumulation of cyclic AMP (IC50 : 2.58 μM) using ORL1 receptor expressing HeLa cells in vitro.

In in vivo, when given intravenously at dosages of 0.01 mg kg−1 and above, or orally at dosages 1 mg kg−1 and above, JTC‐801 antagonized the nociceptin‐induced allodynia in mice.

Effects of JTC‐801 on various nociceptive models were examined. In mouse hot‐plate test, JTC‐801 prolonged escape response latency (ERL) to exposed heat stimulus with minimum effective doses (MED) of 0.01 mg kg−1 by i.v. or 1 mg kg−1 by p.o.

In the rat formalin test, JTC‐801 reduced both the first and second phases of the nociceptive response with MED of 0.01 mg kg−1 by i.v. administration or 1 mg kg−1 by p.o. administration. This anti‐nociceptive action of JTC‐801 was not inhibited by naloxone (10 mg kg−1, s.c.).

We have demonstrated that JTC‐801 antagonizes the ORL1 receptor response, and that JTC‐801 has efficacious and potent anti‐nociceptive effects in acute pain animal models not only by intravenous injection but also oral administration. These results suggest that JTC‐801 may represent a new class of analgesics.

British Journal of Pharmacology (2002) 135, 323–332; doi:10.1038/sj.bjp.0704478
Behavioural and biochemical evidence for interactions between Δ9‐tetrahydrocannabinol and nicotine
Authors: Valjent Emmanuel , Mitchell Jennifer M , Besson Marie‐Jo , Caboche Jocelyne , Maldonado Rafael
Published: January 2005

Behavioural and pharmacological effects of Δ9‐tetrahydrocannabinol (THC) and nicotine are well known. However, the possible interactions between these two drugs of abuse remain unclear in spite of the current association of cannabis and tobacco in humans.

The present study was designed to analyse the consequences of nicotine administration on THC‐induced acute behavioural and biochemical responses, tolerance and physical dependence.

Nicotine strongly facilitated hypothermia, antinociception and hypolocomotion induced by the acute administration of THC. Furthermore, the co‐administration of sub‐threshold doses of THC and nicotine produced an anxiolytic‐like response in the light–dark box and in the open‐field test as well as a significant conditioned place preference. Animals co‐treated with nicotine and THC displayed an attenuation in THC tolerance and an enhancement in the somatic expression of cannabinoid antagonist‐precipitated THC withdrawal.

THC and nicotine administration induced c‐Fos expression in several brain structures. Co‐administration of both compounds enhanced c‐Fos expression in the shell of the nucleus accumbens, central and basolateral nucleus of the amygdala, dorso‐lateral bed nucleus of the stria terminalis, cingular and piriform cortex, and paraventricular nucleus of the hypothalamus.

These results clearly demonstrate the existence of a functional interaction between THC and nicotine. The facilitation of THC‐induced acute pharmacological and biochemical responses, tolerance and physical dependence by nicotine could play an important role in the development of addictive processes.

British Journal of Pharmacology (2002) 135, 564–578; doi:10.1038/sj.bjp.0704479
Cardiovascular actions of a novel NO‐independent guanylyl cyclase stimulator, BAY 41‐8543: in vivo studies
Authors: Stasch Johannes‐Peter , Dembowsky Klaus , Perzborn Elisabeth , Stahl Elke , Schramm Matthias
Published: January 2005

BAY 41‐8543 is a novel non‐NO‐based stimulator of sGC. This study investigates the acute effects of BAY 41‐8543 on haemodynamics in anaesthetized rats and dogs, its long‐term effects in conscious hypertension rat models and its antiplatelet effects.

In anaesthetized dogs, intravenous injections of BAY 41‐8543 (3–100 μg kg−1) caused a dose‐dependent decrease in blood pressure and cardiac oxygen consumption as well as an increase in coronary blood flow and heart rate.

In anaesthetized normotensive rats, BAY 41‐8543 produced a dose‐dependent and long‐lasting blood pressure lowering effect after intravenous (3–300 μg kg−1) and oral (0.1–1 mg kg−1) administration. A dose‐dependent and long‐lasting decrease in blood pressure was also observed in conscious spontaneously hypertensive rats with a threshold dose of 0.1 mg kg−1 p.o. After 3 mg kg−1 the antihypertensive effect lasted for nearly 24 h. After multiple dosages, BAY 41‐8543 did not develop tachyphylaxis in SHR.

BAY 41‐8543 prolonged the rat tail bleeding time and reduced thrombosis in the FeCl3 thrombosis model after oral administration.

In a low NO, high renin rat model of hypertension, BAY 41‐8543 prevented the increase in blood pressure evoked by L‐NAME and reveals a kidney protective effect. In this model, the overall beneficial effects of BAY 41‐8543 manifested as both antiplatelet effect and vasodilatation were reflected in a significant reduction in mortality.

The pharmacological profile of BAY 41‐8543 suggests therefore that this compound has the potential to be an important research tool for in vivo investigations in the sGC/cGMP field and it also has the potential of being a unique clinical utility for treatment of cardiovascular diseases.

British Journal of Pharmacology (2002) 135, 344–355; doi:10.1038/sj.bjp.0704483
A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors
Authors: Vial Catherine , Rolf Michael G , Mahaut‐Smith Martyn P , Evans Richard J
Published: January 2005

We have examined the role of ATP‐dependent P2X1 receptors in megakaryocytes (MKs) and platelets using receptor‐deficient mice and selective agonists.

α,β‐meATP‐ and ATP‐ evoked ionotropic inward currents were absent in whole‐cell recordings from MKs of P2X1−/− mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X1 receptors.

P2X1 receptor deficiency had no effect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development.

P2Y receptor‐stimulated [Ca2+]i responses were unaffected in MKs from P2X1−/− mice, however the inward cation current associated with Ca2+ release was reduced by ∼50%, suggesting an interaction between the membrane conductances activated by P2X1 and P2Y receptors.

Interaction between P2X1 and P2Y receptors in human platelets was also examined using [Ca2+]i recordings from cell suspensions. α,β‐meATP (10 μM) evoked a rapid transient P2X1 receptor‐mediated increase in [Ca2+]i, whereas ADP‐(10 μM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co‐application of α,β‐meATP and ADP resulted in marked acceleration and amplification of the peak [Ca2+]i response.

We conclude that ionotropic P2X1 receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation.

British Journal of Pharmacology (2002) 135, 363–372; doi:10.1038/sj.bjp.0704486
Mechanisms of nitric oxide generation from nitroglycerin and endogenous sources during hypoxia in vivo
Authors: Agvald Per , Adding L Christofer , Artlich Andreas , Persson Magnus G , Gustafsson Lars E
Published: January 2005

Nitroglycerin (GTN), often used in conditions of cardiovascular ischaemia, acts through the liberation of nitric oxide (NO) and the local concentration of NO in the tissue is responsible for any biological effect. However, little is known about the way in which the concentration of NO from GTN and other NO‐donors is influenced by low oxygen tension in the target tissues.

To evaluate the impact of changes in oxygen tension in the metabolism of NO‐donors we measured exhaled NO in anaesthetized rabbits in vivo and expired NO and perfusate nitrite (NO2−) in buffer‐perfused lungs in situ. The impact of acute hypoxia on NO formation from GTN, isosorbide‐5‐mononitrate (ISMN), dissolved authentic NO, NO2− and NO generated from endogenous NO‐synthase (NOS) was studied in either model.

Acute hypoxia drastically increased exhaled NO concentrations from all NO‐donors studied, both in vivo and in the perfused lung. During similar conditions endogenous NO generation from NOS was strongly inhibited. The effects were most pronounced at less than 3% inspired oxygen.

The mechanisms for the increased NO‐formation during hypoxia seems to differ between GTN‐ and NO2−‐derived NO. The former phenomenon is likely due to diminished breakdown of NO.

In conclusion, hypoxic conditions preserve very high local NO concentrations generated from organic nitrates in vivo and we suggest that this might benefit preferential vasodilation in ischaemic tissue regions. Our findings point out the necessity to consider the influence of oxygen tension when studying the action of NO‐donors.

British Journal of Pharmacology (2002) 135, 373–382; doi:10.1038/sj.bjp.0704489
Histamine H3‐receptor‐mediated [35S]GTPγ[S] binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors
Authors: Rouleau A , Ligneau X , Tardivel‐Lacombe J , Morisset S , Gbahou F , Schwartz J ‐C , Arrang J ‐M
Published: January 2005

Constitutive activity of the recombinant and native rat and human H3 receptors (H3Rs) was studied using H3R‐mediated [35S]GTPγ[S] binding and [3H]‐arachidonic acid release.

Ciproxifan, an inverse agonist at the rat H3R (rH3R), decreased [3H]arachidonic acid release from CHO cells expressing moderate densities (∼200–300 fmol mg−1 protein) of the human H3R (hH3R). This effect occurred with the same magnitude than at the rH3R.

The expression of the hH3R was associated with an increase in [35S]GTPγ[S] binding to membranes of CHO cells. Ciproxifan decreased [35S]GTPγ[S] binding to membranes of CHO (hH3R) cells. Both effects were correlated to receptor density and revealed that constitutive activity of the hH3R, although lower than that of the rH3R in this assay, was again observed at physiological densities (<500 fmol mg−1 protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (Ki=45 nM), but also as an inverse agonist (EC50=15 nM).

Constitutive activity of the hH3R was also evidenced using inhibition of [35S]GTPγ[S] binding by unlabelled GTPγS. The expression of the hH3R generated a high affinity binding for GTPγS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist.

[35S]GTPγ[S] binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H3R, whose effects were blocked by proxyfan, a neutral antagonist. [35S]GTPγ[S] binding was also decreased by an A1‐adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D2/D3 dopamine, H1 and H2 histamine, α2‐adrenergic and δ opioid receptors.

In conclusion, the present study shows that the recombinant rat and human H3 receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H3Rs is one of the highest among G‐protein‐coupled receptors present in rat brain.

British Journal of Pharmacology (2002) 135, 383–392; doi:10.1038/sj.bjp.0704490
Disparate effects of non‐steroidal anti‐inflammatory drugs on apoptosis in guinea‐pig gastric mucous cells: inhibition of basal apoptosis by diclofenac
Authors: Ashton Miranda , Hanson Peter J
Published: January 2005

Non‐steroidal anti‐inflammatory drugs (NSAIDs) induce apoptosis in gastrointestinal cancer cell lines. Similar actions on normal gastric epithelial cells could contribute to NSAID gastropathy. The present work therefore compared the actions of diclofenac, ibuprofen, indomethacin, and the cyclo‐oxygenase‐2 selective inhibitor, NS‐398, on a primary culture of guinea‐pig gastric mucous epithelial cells.

Cell number was assessed by staining with crystal violet. Apoptotic activity was determined by condensation and fragmentation of nuclei and by assay of caspase‐3‐like activity. Necrosis was evaluated from release of cellular enzymes.

Ibuprofen (250 μM for 24 h) promoted cell loss, and apoptosis, under both basal conditions and when apoptosis was increased by 25 μM N‐Hexanoyl‐D‐sphingosine (C6‐ceramide).

Diclofenac (250 μM for 24 h) reduced the proportion of apoptotic nuclei from 5.2 to 2.1%, and caused inhibition of caspase‐3‐like activity, without causing necrosis under basal conditions. No such reduction in apoptotic activity was evident in the presence of 25 μM C6‐ceramide.

The inhibitory effect of diclofenac on basal caspase‐3‐like activity was also exhibited by the structurally similar mefenamic and flufenamic acids (1–250 μM), but not by niflumic acid.

Inhibition of superoxide production by the cells increased caspase‐3‐like activity, but the inhibitory action of diclofenac on caspase activity remained. Diclofenac did not affect superoxide production.

Diclofenac inhibited caspase‐3‐like activity in cell homogenates and also inhibited human recombinant caspase‐3.

In conclusion, NSAIDs vary in their effect on apoptotic activity in a primary culture of guinea‐pig gastric mucous epithelial cells, and the inhibitory effect of diclofenac on basal apoptosis could involve an action on caspase activity.

British Journal of Pharmacology (2002) 135, 407–416; doi:10.1038/sj.bjp.0704497
Transient relaxation of rat mesenteric microvessels by ceramides
Authors: Czyborra Peter , Saxe Miriam , Fetscher Charlotte , Meyer zu Heringdorf Dagmar , Herzig Stefan , Jakobs Karl H , Michel Martin C , Bischoff Angela
Published: January 2005

We have investigated the vasodilating effects of D‐erythro‐C2‐ceramide (C2‐ceramide) in methoxamine‐contracted rat mesenteric microvessels.

C2‐ceramide (10–100 μM) caused a concentration‐dependent, slowly developing relaxation which reached maximum values after ∼10 min and partially abated thereafter.

Endothelium removal or inhibitors of guanylyl cyclase (3 μM ODQ), protein kinase A (10 μM H7, 1 μM H89) and various types of K+ channels (10 μM BaCl2, 3 mM tetraethylammonium, 30 nM charybdotoxin, 30 nM iberiotoxin, 300 nM apamine, 10 μM glibenclamide) had only small if any inhibitory effects against C2‐ceramide‐induced vasodilation, but some of them attenuated vasodilation by sodium nitroprusside or isoprenaline. A combination of ODQ and charybdotoxin almost completely abolished C2‐ceramide‐induced vasodilation.

A second administration of C2‐ceramide caused a detectable but weaker relaxation. L‐threo‐C2‐ceramide (100 μM), which should not be a substrate to ceramide metabolism, had no biphasic time course. The ceramidase inhibitor (1S,2R)‐D‐erythro‐2‐(N‐myristoylamino)‐1‐phenyl‐1‐propanol (100 μM) alone caused some vasodilation, indicating vasodilation by endogenous ceramides, and also hastened relaxation by exogenous C2‐ceramide. The late‐developing reversal of C2‐ceramide‐induced vasodilation was absent when α‐adrenergic tone was removed by addition of 10 μM phentolamine.

We conclude that C2‐ceramide relaxes rat resistance vessels in an endothelium‐independent manner which is prevented only by combined inhibition of guanylyl cyclase and charybdotoxin‐sensitive K+ channels. The vasodilation abates with time partly due to desensitization of the ceramide response and partly due to metabolism of C2‐ceramide to an inactive metabolite.

British Journal of Pharmacology (2002) 135, 417–426; doi:10.1038/sj.bjp.0704498
Spontaneous phasic activity of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators
Authors: Buckner Steven A , Milicic Ivan , Daza Anthony V , Coghlan Michael J , Gopalakrishnan Murali
Published: January 2005

A hallmark for unstable bladder contractions is hyperexcitability and changes in the nature of spontaneous phasic activity of the bladder smooth muscle. In this study, we have characterized the spontaneous activity of the urinary bladder smooth muscle from the pig, a widely used model for studying human bladder function.

Our studies demonstrate that phasic activity of the pig detrusor is myogenic and is influenced by the presence of urothelium. Denuded strips exhibit robust spontaneous activity measured as mean area under the contraction curve (AUC=188.9±15.63 mNs) compared to intact strips (AUC=7.3±1.94 mNs).

Spontaneous phasic activity, particularly the amplitude, is dependent on both calcium entry through voltage‐dependent calcium channels and release from ryanodine receptors as shown by inhibition of spontaneous activity by nifedipine and ryanodine respectively.

Inhibition of BKCa channels by iberiotoxin (100 nM) resulted in an increase in contraction amplitude (89.1±20.4%) and frequency (92.5±31.0%). The SKCa channel blocker apamin (100 nM) also increased contraction amplitude (69.1±24.3%) and frequency (53.5±13.6%) demonstrating that these mechanisms are critical to the regulation of phasic spontaneous activity.

Inhibition of KATP channels by glyburide (10 μM) did not significantly alter myogenic contractions (AUC=18.5±12.3%). However, KATP channel openers (KCOs) showed an exquisite sensitivity for suppression of spontaneous myogenic activity. KCOs were generally 15 fold more potent in suppressing spontaneous activity compared to contractions evoked by electrical field‐stimulation. These studies suggest that potassium channel modulation, particularly KATP channels, may offer a unique mechanism for controlling spontaneous myogenic activity especially those associated with the hyperexcitability occurring in unstable bladders.

British Journal of Pharmacology (2002) 135, 639–648; doi:10.1038/sj.bjp.0704499
Alterations in phenylephrine‐induced contractions and the vascular expression of Na+,K+‐ATPase in ouabain‐induced hypertension
Authors: Rossoni Luciana V , Salaices Mercedes , Marín Jesús , Vassallo Dalton V , Alonso María J
Published: January 2005

Hypertension development, phenylephrine‐induced contraction and Na+,K+‐ATPase functional activity and protein expression in aorta (AO), tail (TA) and superior mesenteric (SMA) arteries from ouabain‐ (25 μg day−1, s.c., 5 weeks) and vehicle‐treated rats were evaluated.

Ouabain treatment increased systolic blood pressure (127±1 vs 160±2 mmHg, n=24, 35; P<0.001) while the maximum response to phenylephrine was reduced (P<0.01) in AO (102.8±3.9 vs 67.1±10.1% of KCl response, n=12, 9) and SMA (82.5±7.5 vs 52.2±5.8%, n=12, 9).

Endothelium removal potentiated the phenylephrine response to a greater extent in segments from ouabain‐treated rats. Thus, differences of area under the concentration‐response curves (dAUC) in endothelium‐denuded and intact segments for control and ouabain‐treated rats were, respectively: AO, 56.6±9.6 vs 198.3±18.3 (n=9, 7); SMA, 85.5±15.4 vs 165.4±24.8 (n=6, 6); TA, 13.0±6.1 vs 39.5±10.4% of the corresponding control AUC (n=6, 6); P<0.05.

The relaxation to KCl (1 – 10 mM) was similar in segments from both groups. Compared to controls, the inhibition of 0.1 mM ouabain on KCl relaxation was greater in AO (dAUC: 64.8±4.6 vs 84.0±5.1%, n=11, 14; P<0.05), similar in SMA (dAUC: 39.1±3.9 vs 43.3±7.8%, n=6, 7; P>0.05) and smaller in TA (dAUC: 62.1±5.5 vs 41.4±8.2%, n=12, 13; P<0.05) in ouabain‐treated rats.

Protein expression of both α1 and α2 isoforms of Na+,K+‐ATPase was augmented in AO, unmodified in SMA and reduced in TA from ouabain‐treated rats.

These results suggest that chronic administration of ouabain induces hypertension and regional vascular alterations, the latter possibly as a consequence of the hypertension.

British Journal of Pharmacology (2002) 135, 771–781; doi:10.1038/sj.bjp.0704501
The effect of 3,4‐methylenedioxymethamphetamine (MDMA, ?ecstasy?) and its metabolites on neurohypophysial hormone release from the isolated rat hypothalamus
Authors: Forsling Mary L , Fallon John K , Shah Darshna , Tilbrook Gary S , Cowan David A , Kicman Andrew T , Hutt Andrew J
Published: January 2005

Methylenedioxymethamphetamine (MDMA, “ecstasy”), widely used as a recreational drug, can produce hyponatraemia. The possibility that this could result from stimulation of vasopressin by MDMA or one of its metabolites has been investigated in vitro.

Release of both oxytocin and vasopressin from isolated hypothalami obtained from male Wistar rats was determined under basal conditions and following potassium (40 mM) stimulation. The results were compared with those obtained for basal and stimulated release in the presence of MDMA or metabolites in the dose range 1 μM to 100 pM (n=5 – 8) using Student's t‐test with Dunnett's correction for multiple comparisons.

All compounds tested affected neurohypophysial hormone release, HMMA (4‐hydroxy‐3‐methoxymethamphetamine) and DHA (3,4‐dihydroxyamphetamine) being more active than MDMA, and DHMA (3,4‐dihydroxymethamphetamine) being the least active. The effect on vasopressin release was greater than that on oxytocin. In the presence of HMMA the ratio test:control for basal release increased for vasopressin from 1.1±0.16 to 2.7±0.44 (s.e.m., P<0.05) at 10 nM and for oxytocin from 1.0±0.05 to 1.6±0.12 in the same hypothalami. For MDMA the ratio increased to 1.5±0.27 for vasopressin and to 1.28±0.04 for oxytocin for 10 nM.

MDMA and its metabolites can stimulate both oxytocin and vasopressin release in vitro, the response being dose dependent for each drug with HMMA being the most potent.

British Journal of Pharmacology (2002) 135, 649–656; doi:10.1038/sj.bjp.0704502
The effects of heparin and related molecules on vascular permeability and neutrophil accumulation in rabbit skin
Authors: Jones Helen , Paul William , Page Clive P
Published: January 2005

Unfractionated heparin (UH) has been shown to possess a wide range of properties which are potentially anti‐inflammatory. Many of these studies, including effects of heparin on adhesion of inflammatory cells to endothelium, have been carried out in vitro. In the present study, we have used radioisotopic techniques to study the effect of UH, and related molecules, on in vivo inflammatory responses (plasma exudation (PE) and PMN accumulation) in rabbit skin induced by cationic proteins, mediators and antigen.

Intradermal (i.d.) pretreatment with UH dose‐dependently inhibited poly‐L‐lysine (PLL)‐induced responses. The same treatment had no effect on antigen (extract of Alternaria tenuis, AT)‐, formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐ or leukotriene (LT) B4‐induced responses, although i.d. dextran sulphate (DS) significantly inhibited responses to all of these mediators.

High dose (10,000 u kg−1) intravenous UH significantly decreased cutaneous responses to fMLP and LTB4. By comparison, the selectin inhibitor, fucoidin, and DS, were very effective inhibitors of these responses, and of responses to AT and PLL.

In contrast to the weak effect in the in vivo studies, UH significantly inhibited in vitro homotypic aggregation of rabbit PMNs, showing that it can modify PMN function.

Our data with i.d. UH confirm the important ability of this molecule to interact with and neutralize polycationic peptides in vivo, suggesting that this is a prime role of endogenous heparin. The lack of effect of exogenous heparin on acute inflammatory responses induced by allergen, suggests that cationic proteins are unlikely to be primary mediators of the allergen‐induced PE or PMN accumulation.

British Journal of Pharmacology (2002) 135, 469–479; doi:10.1038/sj.bjp.0704505
Effects of chronic drug treatments on increases in intracellular calcium mediated by nicotinic acetylcholine receptors in SH‐SY5Y cells
Authors: Ridley Diana L , Pakkanen Jukka , Wonnacott Susan
Published: January 2005

SH‐SY5Y cells express α7 and α3* subtypes of nicotinic acetylcholine receptors (AChR). Numbers of these receptors are upregulated by chronic treatment with nicotinic agonists or KCl. In this study we have examined the functional consequences of these drug treatments on nicotine‐ or KCl‐evoked increases in [Ca2+]i, in SH‐SY5Y cells.

In untreated cells, nicotine increased [Ca2+]i (EC50 7.5 μM). Responses to 10 μM nicotine were abolished by the non‐selective nicotinic antagonist mecamylamine and were partially blocked by α7‐selective antagonists, the α3β2*‐selective antagonist α‐conotoxin‐MII, and by cadmium and verapamil.

After treatment for 4 days with nicotinic agonists, nicotine‐evoked increases in [Ca2+]i were significantly decreased by about 25%. Nicotine‐evoked responses were paradoxically increased in the presence of acute methyllycaconitine (MLA; an α7‐selective antagonist) although other α7‐selective antagonists were without effect, while α‐conotoxin‐MII gave a partial inhibition. The increase observed with MLA was abolished by mecamylamine but not by α‐conotoxin‐MII and was still observed 24 h after chronic nicotine treatment.

After treatment for 4 days with KCl, nicotine‐evoked increases in [Ca2+]i were also decreased by 25%, but acute MLA was without effect. Responses to 20 mM KCl were unchanged by prior treatment with nicotine or KCl. Treatment for 4 days with 5 μM verapamil reduced responses to both nicotine and KCl by about 50%.

Multiple nicotinic AChR subtypes contribute to nicotine‐evoked increases in [Ca2+]i in SH‐SY5Y cells. Responses to acute nicotine are reduced after chronic nicotine or KCl treatment, with loss of the component attributed to the α7 subtype. However, in nicotine‐treated cells this effect is reversed when nicotine stimulation is applied in the presence of acute MLA. The antagonist may assist in converting a non‐functional α7 nicotinic AChR to a conducting state.

British Journal of Pharmacology (2002) 135, 1051–1059; doi:10.1038/sj.bjp.0704508
α2‐Adrenoceptors in the enteric nervous system: a study in α2A‐adrenoceptor‐deficient mice
Authors: Scheibner Jens , Trendelenburg Anne‐Ulrike , Hein Lutz , Starke Klaus , Blandizzi Corrado
Published: January 2005

Mammals possess three types of α2‐adrenoceptor, α2A, α2B and α2C. Our aim was to determine the type of α2‐adrenoceptor involved in the control of gastrointestinal motility.

In transmitter overflow experiments, myenteric plexus longitudinal muscle (MPLM) preparations of the ileum were preincubated with [3H]‐choline and then superfused. The α2‐adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium from preparations taken from wild type but not α2A‐adrenoceptor‐knockout mice.

In a second series of overflow experiments, MPLM preparations were preincubated with [3H]‐noradrenaline and then superfused. Again medetomidine reduced the electrically evoked overflow of tritium from wild type but not α2A‐knockout preparations.

In organ bath experiments, medetomidine reduced electrically evoked contractions of segments of the ileum from wild type but not α2A‐knockout mice.

In each of these three series, phentolamine antagonized the effect of medetomidine in wild‐type preparations with greater potency than rauwolscine.

In conscious mice, gastrointestinal transit was assessed by means of an intragastric charcoal bolus. In α2A‐knockout mice, the speed of gastrointestinal transit was doubled compared to wild‐type. Medetomidine, injected intraperitoneally, slowed gastrointestinal transit in wild type but not α2A‐knockout mice.

We conclude that the cholinergic motor neurons of the enteric nervous system of mice possess α2‐heteroreceptors which mediate inhibition of acetylcholine release, of neurogenic contractions and of gastrointestinal transit. The noradrenergic axons innervating the intestine possess α2‐autoreceptors. Both hetero‐ and autoreceptors are exclusively α2A. It is the α2A‐adrenoceptor which in vivo mediates the inhibition of intestinal motility by the sympathetic nervous system.

British Journal of Pharmacology (2002) 135, 697–704; doi:10.1038/sj.bjp.0704512
Agmatine, an endogenous ligand at imidazoline binding sites, does not antagonize the clonidine‐mediated blood pressure reaction
Authors: Raasch Walter , Schäfer Ulrich , Qadri Fatimunnisa , Dominiak Peter
Published: January 2005

Since agmatine has been identified as a clonidine displacing substance (CDS), the aim of this study was to investigate whether agmatine can mimic CDS‐induced cardiovascular reactions in organ bath experiments, pithed spontaneously hypertensive rats (SHR) and anaesthetized SHR.

Intravenously‐administered agmatine significantly reduced the blood pressure and heart rate of anaesthetized SHR at doses higher than 1 and 3 mg kg−1, respectively. These effects are probably mediated via central mechanisms, since there was an approximate 8 fold rightward shift of the dose‐response curve in the pithed SHR (indicating a weakened cardiovascular effect). Moreover, in organ bath experiments, agmatine failed to alter the contractility of intact or endothelium‐denuded aortal rings. When agmatine was administered i.c.v. to anaesthetized SHR, blood pressure was increased without any alteration of heart rate, whereas blood pressure was unchanged and heart rate was increased after injection into the 4th brain ventricle. This suggests that haemodynamic reaction patterns after central application are related to distinct influences on central cardiovascular mechanisms.

Agmatine reduces noradrenaline release in pithed SHR while α2‐adrenoceptors are irreversibly blocked with phenoxybenzamine, but not while I1‐binding sites are selectively blocked with AGN192403. This suggests that agmatine may modulate noradrenaline release in the same way that clonidine does, i.e. via imidazoline binding sites; this involves a reduction in sympathetic tone which in turn reduces blood pressure and heart rate.

Finally, CDS‐like cardiovascular activity appears not to be due to agmatine, since (i) blood pressure in anaesthetized SHR is decreased by agmatine and clonidine, and (ii) agmatine did not antagonize the blood pressure reaction to clonidine in pithed or anaesthetized SHR.

British Journal of Pharmacology (2002) 135, 663–672; doi:10.1038/sj.bjp.0704513
Evidence for an atypical receptor mediating the augmented bronchoconstrictor response to adenosine induced by allergen challenge in actively sensitized Brown Norway rats
Authors: Hannon J P , Tigani B , Wolber C , Williams I , Mazzoni L , Howes C , Fozard J R
Published: January 2005

The bronchoconstrictor response to adenosine is markedly and selectively increased following ovalbumin (OA) challenge in actively sensitized, Brown Norway rats. We present a pharmacological analysis of the receptor mediating this response.

Like adenosine, the broad‐spectrum adenosine receptor agonist, NECA, induced dose‐related bronchoconstriction in actively sensitized, OA‐challenged animals. In contrast, CPA, CGS 21680 and 2‐Cl‐IB‐MECA, agonists selective for A1 A2A and A3 receptors, respectively, induced no, or minimal, bronchoconstriction.

Neither the selective A1 receptor antagonist, DPCPX, nor the selective A2A receptor antagonist, ZM 241385, blocked the bronchoconstrictor response to adenosine.

MRS 1754, which has similar affinity for rat A2B and A1 receptors, failed to block the bronchoconstrictor response to adenosine despite blockade of the A1 receptor‐mediated bradycardia induced by NECA.

8‐SPT and CGS 15943, antagonists at A1, A2A, and A2B but not A3 receptors, inhibited the bronchoconstrictor response to adenosine. However, the degree of blockade (approximately 3 fold) did not reflect the plasma concentrations, which were 139 and 21 times greater than the KB value at the rat A2B receptor, respectively.

Adenosine and NECA, but not CPA, CGS 21680 or 2‐Cl‐IB‐MECA, induced contraction of parenchymal strip preparations from actively sensitized OA‐challenged animals. Responses to adenosine could not be antagonized by 8‐SPT or MRS 1754 at concentrations >50 times their affinities at the rat A2B receptor.

The receptor mediating the bronchoconstrictor response to adenosine augmented following allergen challenge in actively sensitized BN rats cannot be categorized as one of the four recognized adenosine receptor subtypes.

British Journal of Pharmacology (2002) 135, 685–696; doi:10.1038/sj.bjp.0704516
Caffeine‐induced immobilization of gating charges in isolated guinea‐pig ventricular heart cells
Authors: Leroy Jérôme , Lignon Jacques M , Gannier François , Argibay Jorge A , Malécot Claire O
Published: January 2005

The effects of 10 mM caffeine (CAF) on intramembrane charge movements (ICM) were studied in isolated guinea‐pig ventricular heart cells with the whole‐cell patch‐clamp technique.

In the presence of CAF, the properties (voltage dependence, maximum QON[Qmax], availability with voltage) of QON charge activated from −110 mV were barely affected. Following a 100 ms prepulse to −50 mV to decrease the participation of charges originating from Na channels, the voltage dependence of QON was shifted by 5 mV (negative component) and by 10 mV (positive component) towards negative potentials, and Qmax was depressed by 16.5%.

CAF drastically reduced in a time‐ and voltage‐dependent manner QOFF on repolarization to −50 mV, the effects being greater at positive potentials.

CAF‐induced QOFF immobilization could be almost entirely removed by repolarization to voltages as negative as −170 mV. In these conditions, the voltage‐dependence of QOFF (repolarization to +30 to −170 mV) was shifted by 17 mV (negative component) and 30 mV (positive component) towards negative potentials, suggesting an interconversion into charge 2.

Most of CAF effects were suppressed when the sarcoplasmic reticulum (SR) was not functional or when the cells were loaded with BAPTA‐AM.

We conclude that CAF effects on ICM are likely due to Ca2+ ions released from the SR, and which accumulate in the subsarcolemmal fuzzy spaces in the vicinity of the Ca channels. Because CAF effects were more pronounced on QOFF than on QON the channels have likely to open before Ca2+ ions could affect their gating properties. It is speculated that such an effect on gating charges might contribute to the Ca‐induced inactivation of the Ca current.

British Journal of Pharmacology (2002) 135, 721–734; doi:10.1038/sj.bjp.0704520
A single subcutaneous bolus of erythropoietin normalizes cerebral blood flow autoregulation after subarachnoid haemorrhage in rats
Authors: Springborg Jacob Bertram , Ma XiaoDong , Rochat Per , Knudsen Gitte Moos , Amtorp Ole , Paulson Olaf B , Juhler Marianne , Olsen Niels Vidiendal
Published: January 2005

Systemic administration of recombinant erythropoietin (EPO) has been demonstrated to mediate neuroprotection. This effect of EPO may in part rely on a beneficial effect on cerebrovascular dysfunction leading to ischaemic neuronal damage. We investigated the in vivo effects of subcutaneously administered recombinant EPO on impaired cerebral blood flow (CBF) autoregulation after experimental subarachnoid haemorrhage (SAH).

Four groups of male Sprague‐Dawley rats were studied: group A, sham operation plus vehicle; group B, sham operation plus EPO; group C, SAH plus vehicle; group D, SAH plus EPO. SAH was induced by injection of 0.07 ml of autologous blood into the cisterna magna. EPO (400 iu kg−1 s.c.) or vehicle was given immediately after the subarachnoid injection of blood or saline. Forty‐eight hours after the induction of SAH, CBF autoregulatory function was evaluated using the intracarotid 133Xe method.

CBF autoregulation was preserved in both sham‐operated groups (lower limits of mean arterial blood pressure: 91±3 and 98±3 mmHg in groups A and B, respectively). In the vehicle treated SAH‐group, autoregulation was abolished and the relationship between CBF and blood pressure was best described by a single linear regression line. A subcutaneous injection of EPO given immediately after the induction of SAH normalized autoregulation of CBF (lower limit in group D: 93±4 mmHg, NS compared with groups A and B).

Early activation of endothelial EPO receptors may represent a potential therapeutic strategy in the treatment of cerebrovascular perturbations after SAH.

British Journal of Pharmacology (2002) 135, 823–829; doi:10.1038/sj.bjp.0704521
P2 receptors: new potential players in atherosclerosis
Authors: Virgilio Francesco Di , Solini Anna
Published: January 2005

Atherosclerosis is a focal inflammatory disease of the arterial wall. It starts with the formation of fatty streaks on the arterial wall that evolve to form a raised plaque made of smooth muscle cells (SMCs), and infiltrating leukocytes surrounding a necrotic core. The pathogenesis of the atherosclerotic lesion is incompletely understood, but it is clear that a dysfunction of the endothelium, recruitment and activation of inflammatory cells and SMC proliferation have a pivotal role. Over recent years receptors for extracellular nucleotides, the P2 receptors, have been recognized as fundamental modulators of leukocytes, platelets, SMCs and endothelial cells. P2 receptors mediate chemotaxis, cytokine secretion, NO generation, platelet aggregation and cell proliferation in response to accumulation of nucleotides into the extracellular milieu. Clinical trials have shown the benefit of antagonists of the ADP platelet receptor(s) in the prevention of vascular accidents in patients with atherosclerosis. Therefore, we anticipate that a deeper understanding of the involvement of P2 receptors in atheroma formation will open new avenues for drug design and therapeutic intervention.

British Journal of Pharmacology (2002) 135, 831–842; doi:10.1038/sj.bjp.0704524
5‐HT1B but not 5‐HT6 or 5‐HT7 receptors mediate depression of spinal nociceptive reflexes in vitro
Authors: Hedo G , Lopez‐Garcia J A
Published: January 2005

The identity of the serotonin (5‐HT) receptors modulating the transmission of segmental C‐fibre mediated signals was studied using an in vitro preparation of the hemisected spinal cord from rat pups.

Responses to trains of stimuli delivered to a lumbar dorsal root were recorded from the corresponding ventral root. The resulting cumulative depolarization (CD) mediated by unmyelinated fibres was quantified in terms of integrated area. The amplitude of the mono‐synaptic reflex was also measured. Serotonergic agents were superfused at known concentrations and their effects on the reflexes evaluated.

5‐HT had depressant effects on the CD (EC50 34 μM). The rank order of potency of agonists for the depression of the CD was 5‐carboxamidotryptamine (5‐CT)>α‐methylserotonin (α‐met‐5‐HT) ∼5‐HT>42‐methylserotonin (2‐met‐5‐HT)∼8‐OH‐DPAT.

All the agonists including 2‐met‐5‐HT and 8‐OH‐DPAT had strong depressant effects on the mono‐synaptic reflex with the following order of potency: 5‐CT>48‐OH‐DPAT>4α‐met‐5‐HT ∼5‐HT∼2‐met‐5‐HT.

The inhibitory effects of 5‐HT, α‐met‐5‐HT and 5‐CT were attenuated by the non‐specific 5‐HT antagonist methiothepin (1 μM) and by the 5‐HT1A/1B antagonist SDZ 21009 (100 nM) but not by the selective 5‐HT1A antagonist WAY 100135 (1 μM).

Other antagonists known to block 5‐HT2, 5‐HT6 and/or 5‐HT7 receptors (ketanserin, RO 04‐6790, ritanserin and clozapine) did not change the effect of the agonists.

The data suggest an important contribution of 5‐HT1B receptors to the inhibition of spinal C‐fibre mediated nociceptive reflexes but no experimental support was found for the intervention of 5‐HT2, 5‐HT6 or 5‐HT7 receptors in this in vitro model.

British Journal of Pharmacology (2002) 135, 935–942; doi:10.1038/sj.bjp.0704526