bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the pharmacological properties of EDHF‐mediated vasorelaxation in guinea‐pig cerebral and mesenteric resistance vessels
Authors: Dong Hui , Jiang Yanfen , Cole William C , Triggle Christopher R
Published: January 2005

In the presence of L‐NNA (100 μM), indomethacin (10 μM) and ODQ (10 μM), acetylcholine induced a concentration‐dependent vasorelaxation of guinea‐pig mesenteric and middle cerebral arteries precontracted with cirazoline or histamine, but not with high K+, indicating the contribution of an endothelium‐derived hyperpolarizing factor (EDHF).

In cerebral arteries, charybdotoxin (ChTX; 0.1 μM) completely inhibited the indomethacin, L‐NNA and ODQ‐insensitive relaxation; iberiotoxin (IbTX, 0.1 μM), 4‐aminopyridine (4‐AP, 1 mM), or barium (30 μM) significantly reduced the response; in the mesenteric artery, ChTX and IbTX also reduced this relaxation. Glibenclamide (10 μM) had no affect in either the mesenteric or cerebral artery.

Neither clotrimazole (1 μM) nor 7‐ethoxyresorufin (3 μM) affected EDHF‐mediated relaxation in the mesenteric artery, but abolished or attenuated EDHF‐mediated relaxations in the cerebral artery. AM404 (30 μM), a selective anandamide transport inhibitor, did not affect the vasorelaxation response to acetylcholine in the cerebral artery, but in the mesenteric artery potentiated the vasorelaxation response to acetylcholine in an IbTX, and apamin‐sensitive, but SR 141816A‐insensitive manner. Ouabain (100 μM) almost abolished EDHF‐mediated relaxation in the mesenteric artery, but enhanced the relaxation in the cerebral artery whereas the addition of K+ (5–20 mM) to precontracted guinea‐pig cerebral or mesenteric artery induced further vasoconstriction.

These data suggest that in the guinea‐pig mesenteric and cerebral arteries different EDHFs mediate acetylcholine‐induced relaxation, however, EDHF is unlikely to be mediated by K+.

British Journal of Pharmacology (2000) 130, 1983–1991; doi:10.1038/sj.bjp.0703474
Smooth muscle and parasympathetic nerve terminals in the rat urinary bladder have different subtypes of α1 adrenoceptors
Authors: Széll E A , Yamamoto T , De Groat W C , Somogyi G T
Published: January 2005

Neurally evoked contractions and release of 3H‐ acetylcholine (ACh) during electrical field stimulation were measured in rat urinary bladder strips.

The α1 agonist phenylephrine (PE, 2–8 μM) increased the amplitude of neurally evoked contractions, facilitated the release of ACh and increased the baseline tone of the bladder strips.

The PE‐induced facilitation of the contractions did not significantly change during a prolonged exposure to PE (120 min), whereas the PE‐induced rise in baseline tone gradually decreased to 65% of the initial value.

Low concentrations of specific α1A antagonists, 5‐methyl urapidil (5‐MU), REC15/2739 and WB‐4101 competitively inhibited the facilitation of the neurally‐evoked contractions (pA2: 8.77; 9.59 and 9.62, respectively), whereas higher concentrations of 5‐MU (IC50: 48 nM) were required to suppress the PE‐rise in baseline. WB‐4101 (100 μM) inhibited the PE‐induced facilitation of ACh release.

The irreversible α1B antagonist chloroethyl‐clonidine (CEC, 10–50 μM) inhibited the PE‐evoked rise in base line tone, but did not affect the PE‐induced facilitation of the neurally evoked contractions nor the facilitation of ACh release. However, CEC increased the area and amplitude of the neurally‐evoked contractions by 261±33 and 47.2±8.4%, respectively. Atropine significantly inhibited the CEC evoked increase in area and amplitude of the electrically evoked contractions (76.5±4.8 and 40.8±3%, respectively) indicating that CEC facilitated the cholinergic responses of the electrically stimulated bladder strips.

It is concluded that α1A and CEC sensitive α1B and/or α1D adrenoceptors are expressed in the rat bladder in different locations. On the cholinergic nerve terminals α1A adrenoceptors mediate prejunctional facilitation, whereas postjunctional α1B/α1D adrenoceptors mediate smooth muscle contraction.

British Journal of Pharmacology (2000) 130, 1685–1691; doi:10.1038/sj.bjp.0703475
Effects of BDF 9198 on action potentials and ionic currents from guinea‐pig isolated ventricular myocytes
Authors: Yuill K H , Convery M K , Dooley P C , Doggrell S A , Hancox J C
Published: January 2005

BDF 9198 (a congener of DPI 201–106 and BDF 9148) was found to be a positive inotrope on guinea‐pig isolated ventricular muscle strips. The effects of BDF 9198 on action potentials and ionic currents from guinea‐pig isolated ventricular myocytes were studied using the whole cell patch clamp method.

In normal external solution, at 37°C, action potential duration at 50% repolarization (APD50) was 167.4±8.36 ms (n=37). BDF 9198 produced a concentration‐dependent increase in APD50 (no significant increase at 1×10−10 M; and APD50 values of 273.03±35.8 ms at 1×10−9 M; n=6, P<0.01 and 694.7±86.3 ms at 1×10−7 M; P<0.001, n=7). At higher concentrations in the range tested, BDF 9198 also induced early and delayed and after‐depolarizations.

Qualitative measurements of INa with physiological [Na]o showed prolongation of the current by BDF 9198, and the appearance of transient oscillatory inward currents at high concentrations.

Quantitative recording conditions for INa were established using low external [Na] and by making measurements at room temperature. The current–voltage relation, activation parameters and time‐course of INa were similar before and after a partial blocking dose of Tetrodotoxin (TTX, 1 μM), despite a 2 fold difference in current amplitude. This suggests that voltage‐clamp during flow of INa was adequately maintained under our conditions.

Selective measurements of INa at room temperature showed that BDF 9198 induced a concentration‐dependent, sustained component of INa (ILate) and caused a slight left‐ward shift in the current–voltage relation for peak current. The drug‐induced ILate showed a similar voltage dependence to peak current in the presence of BDF 9198. Both peak current and ILate were abolished by 30 μM TTX and were sensitive to external [Na].

Inactivation of control INa during a 200 ms test pulse to −30 mV followed a bi‐exponential time‐course. In addition to inducing a sustained current component, BDF 9198 left the magnitude of the fast inactivation time‐constant unchanged, but increased the magnitude of the slow inactivation time‐constant. Additional experiments with a longer pulse (1 s) raised the possibility that in the presence of BDF 9198, INa inactivation may be comprised of more than two phases.

No significant effects of 1×10−6 M BDF 9198 were observed on the L‐type calcium current, or delayed and inward rectifying potassium currents measured at 37°C.

It is concluded that the prolongation of APD50 by BDF 9198 resulted from selective modulation of INa. Reduced current inactivation induced a persistent INa, increasing the net depolarizing current during the action potential. This action of the drug indicates a potential for ‘QT prolongation’ of the ECG. The observation of after‐depolarizations suggests a potential for proarrhythmia at some drug concentrations.

British Journal of Pharmacology (2000) 130, 1753–1766; doi:10.1038/sj.bjp.0703476
Evidence against a role of inducible nitric oxide synthase in the endothelial protective effects of delayed preconditioning
Authors: Laude Karine , Richard Vincent , Henry Jean‐Paul , Lallemand Françoise , Thuillez Christian
Published: January 2005

Preconditioning the heart with brief periods of ischaemia induces delayed endothelial protection against reperfusion injury, but the precise mechanisms involved in this endogenous protein are still unclear. Induction of the type II‐nitric oxide synthase (iNOS) acts as a mediator of the preconditioning against myocardial infarction and stunning. The present study was designed to assess whether iNOS also contributes to the delayed endothelial protective effects of preconditioning.

Rats were subjected to 20 min ischaemia followed by 60 min reperfusion 24 h after sham surgery or preconditioning (one cycle or 2 min ischaemia/5 min reperfusion and two cycles of 5 min ischaemia/5 min reperfusion). At the end of the reperfusion, coronary segments were removed distal to the site of occlusion and mounted in wire myographs.

Ischaemia‐reperfusion (I/R) decreased the endothelium‐dependent relaxations to acetylcholine (maximal relaxations: sham, 66±5%; I/R, 40±1%; P<0.05) and this impairment was prevented by preconditioning (maximal relaxation: 61±6%).

Administration of N‐(3‐aminomethyl)benzyl)acetaminide (1400W), a highly selective inhibitor for iNOS, 10 min before prolonged ischaemia did not modify the beneficial effect of preconditioning (maximal relaxation: 66±5%).

These data show that preconditioning induces delayed protection against reperfusion‐injury. However, in contrast to the myocytes, these endothelial protective effects of delayed preconditioning do not involve iNOS.

British Journal of Pharmacology (2000) 130, 1547–1552; doi:10.1038/sj.bjp.0703477
Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine‐induced inflammation: the role of cytokines
Authors: Bleau Anne‐Marie , Levitchi Mihaela C , Maurice Hélène , Du Souich Patrick
Published: January 2005

Serum from humans with an acute upper respiratory viral infection and from rabbits with turpentine‐induced inflammation reduce the catalytic activity of hepatic cytochrome P450 (P450). The aim of this study was to identify the serum mediators responsible for the decrease in P450 activity.

Rabbit and human sera were fractionated by size exclusion chromatography and the fractions tested for their ability to reduce the activity and amount of P450 after 4 h of incubation with hepatocytes from turpentine‐treated rabbits (HINF). Rabbit and human sera decreased P450 activity by around 40% without any change in the amount of CYP1A1 and 1A2 apoproteins.

In rabbit serum, the fraction containing proteins of Mr 23–15 kDa decreased P450 content by 41%, but did not alter the amount of the apoproteins. Anti‐IL‐6 antibody added to the Mr 23–15 kDa fraction restored P450 content to 97% of control values, while anti‐IL‐1β, TNF‐α and IFN‐γ antibodies had no effect. Supporting the role of IL‐6, incubation of HINF in the presence of IL‐6 for 4 h reduced P450 content by 40%.

In human serum, the fraction containing proteins of Mr >95 kDa lowered P450 content by 43% without modifying the amounts of CYP1A1/2. Neutralization experiments showed that IFN‐γ, IL‐6, and IL‐1β contributed to the decrease in P450 content.

In conclusion, the present results demonstrate that IL‐6, and IFN‐γ, IL‐6 and IL‐1β are the serum mediators released in vivo by a turpentine‐induced inflammatory reaction in the rabbit and an upper respiratory viral infection in humans, respectively, inactivating hepatic P450.

British Journal of Pharmacology (2000) 130, 1777–1784; doi:10.1038/sj.bjp.0703486
Neurotoxic activity of venom from the Australian Eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating
Authors: Rash Lachlan D , Birinyi‐Strachan Liesl C , Nicholson Graham M , Hodgson Wayne C
Published: January 2005

Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 μl ml−1 failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 μl ml−1) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations.

In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 μl ml−1) caused rapid fasciculations and an increase in indirectly evoked twitches.

Male venom (0.02 and 0.05 μl ml−1) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 μl ml−1) were attenuated by (+)‐tubocurarine (100 μM) and by tetrodotoxin (TTX, 1 μM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml−1).

In the unstimulated rat vas deferens, male venom (0.05 μl ml−1) caused contractions which were inhibited by a combination of prazosin (0.3 μM) and P2X‐receptor desensitization (with α,β‐methylene ATP 10 μM). In the rat stimulated vas deferens, male venom (0.05 μl ml−1) augmented indirectly evoked twitches.

Male venom (0.1 μl ml−1) causes a slowing of inactivation of TTX‐sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons.

These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX‐sensitive sodium channel gating. This action is similar to that of the δ‐atracotoxins from Australian funnel‐web spiders.

British Journal of Pharmacology (2000) 130, 1817–1824; doi:10.1038/sj.bjp.0703494
Tryptophan scanning mutagenesis in TM2 of the GABAA receptor α subunit: effects on channel gating and regulation by ethanol
Authors: Ueno Susumu , Lin Audrey , Nikolaeva Natalia , Trudell James R , Mihic S John , Harris R Adron , Harrison Neil L
Published: January 2005

Each residue in the second transmembrane segment (TM2) of the human GABAA receptor α2 subunit was individually mutated to tryptophan. The wild‐type or mutant α2 subunits were expressed with the wild‐type human GABAA receptor β2 subunit in Xenopus oocytes, and the effects of these mutations were investigated using two‐electrode voltage‐clamp recording.

Four mutations (V257W, T262W, T265W and S270W) produced receptors which were active in the absence of agonist, and this spontaneous open channel activity was blocked by both picrotoxin and bicuculline, except in the α2(V257W)β2 mutant receptor, which was not sensitive to picrotoxin.

Six mutations (V257W, V260W, T262W, T267W, S270W and A273W) enhanced the agonist sensitivity of the receptor, by 10–100 times compared with the wild‐type α2β2 receptor. Other mutations (T261W, V263W, L269W, I271W and S272W) had little or no effect on the apparent affinity of the receptor to GABA. Eight of the tryptophan mutations (R255, T256, F258, G259, L264, T265, M266 or T268) resulted in undetectable GABA‐induced currents.

The S270W mutation eliminated potentiation of GABA by ethanol, whereas T261W markedly increased the action of ethanol. The T262W mutation produced direct activation (10% of maximal GABA response) by ethanol in the absence of GABA, while other mutations did not alter the action of ethanol significantly.

These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABAA receptor channel function, in which specific residues within TM2 are critical for the regulation of channel gating (S270, L264), while other residues (L269, I271 and S272) have little effect on these functions and may be non‐critical structural residues.

British Journal of Pharmacology (2000) 131, 296–302; doi:10.1038/sj.bjp.0703504
Intestinal anti‐inflammatory activity of UR‐12746, a novel 5‐ASA conjugate, on acute and chronic experimental colitis in the rat
Authors: Gálvez Julio , Garrido Margarita , Merlos Manuel , Torres Maria Isabel , Zarzuelo Antonio
Published: January 2005

The present study was undertaken to investigate the intestinal anti‐inflammatory effects of UR‐12746 on the acute and chronic stages of a trinitrobenzene sulphonic acid (TNBS) experimental model of inflammatory bowel disease (IBD) in the rat. UR‐12746 is a novel, locally‐acting compound which combines, through an azo bond, 5‐aminosalicylic (5‐ASA) and UR‐12715, a potent platelet activating factor (PAF)‐antagonist.

UR‐12746 oral pretreatment of colitic rats (50 and 100 mg kg−1) reduced acute colonic damage when evaluated 2 days after colonic insult.

Postreatment for 4 weeks with UR‐12746 (50 and 100 mg kg−1) resulted in a faster recovery of the damaged colonic mucosa, which was macroscopically significant from the third week.

The intestinal anti‐inflammatory effect of UR‐12746 was associated with a decrease in leukocyte infiltration in the colonic mucosa, which was evidenced both biochemically, by a reduction in myeloperoxidase activity, and histologically, by a lower leukocyte count after morphometric analysis. This effect was higher than that seen with sulphasalazine, when assayed at the same doses and in the same experimental conditions.

Several mechanisms can be involved in the beneficial effects showed by UR‐12746: inhibition of leukotriene B4 synthesis in the inflamed colon, improvement of the altered colonic oxidative status, and reduction of colonic interleukin‐1β production.

The results suggest that the intestinal anti‐inflammatory activity of UR‐12746 can be attributed to the additive effects exerted by 5‐ASA and UR‐12715, the PAF antagonist compound, that are released in the colonic lumen after reduction of the azo bond by the intestinal bacteria.

British Journal of Pharmacology (2000) 130, 1949–1959; doi:10.1038/sj.bjp.0703505
Ligand regulation of green fluorescent protein‐tagged forms of the human β1‐ and β2‐adrenoceptors; comparisons with the unmodified receptors
Authors: McLean Alison J , Milligan Graeme
Published: January 2005

Stable clones of HEK293 cells expressing either FLAGTM epitope‐tagged, wild type human β1‐ and β2‐adrenoceptors or C‐terminally green fluorescent protein (GFP)‐tagged forms of these receptors were established.

The binding affinity of [3H]‐dihydroalprenolol and other ligands was little affected by addition of GFP to the C‐terminal of either receptor.

Isoprenaline induced the internalisation of both β1‐adrenoceptor‐GFP and β2‐adrenoceptor‐GFP and following removal of the agonist both constructs were able to recycle to the cell surface.

The extent of internalisation of β2‐adrenoceptor‐GFP produced by isoprenaline was substantially greater than for β1‐adrenoceptor‐GFP.

C‐terminal addition of GFP slowed markedly the rate of internalization of both the β1‐adrenoceptor and the β2‐adrenoceptor in response to isoprenaline.

Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native β2‐adrenoceptor compared to β2‐adrenoceptor‐GFP although both were equally effectively removed from the plasma membrane.

Sustained exposure to isoprenaline resulted in a large fraction of β2‐adrenoceptor‐GFP becoming trapped in internal vesicles/lysosomes but not degraded.

Even after sustained exposure to isoprenaline a significant fraction of β1‐adrenoceptor‐GFP remained at the cell surface.

These results indicate that although GFP tagging of β‐adrenoceptors can provide qualitative visual patterns of agonist‐induced receptor trafficking and regulation in HEK293 cells the quantitative details vary markedly from those obtained with the unmodified receptors.

British Journal of Pharmacology (2000) 130, 1825–1832; doi:10.1038/sj.bjp.0703506
Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line
Authors: Grolleau Françoise , Sattelle David B
Published: January 2005

Single channel recordings were obtained from a Drosophila S2 cell line stably expressing the wild‐type RDLac Drosophila melanogaster homomer‐forming ionotropic GABA receptor subunit, a product of the resistance to dieldrin gene, Rdl. GABA (50 μM) was applied by pressure ejection to outside‐out patches from S2‐RDL cells at a holding potential of −60 mV. The resulting inward current was completely blocked by 100 μM picrotoxin (PTX). The unitary current‐voltage relationship was linear at negative potentials but showed slight inward rectification at potentials more positive than 0 mV. The reversal potential of the current (EGABA=−1.4 mV) was close to the calculated chloride equilibrium potential.

The single channel conductance elicited by GABA was 36 pS. A 71 pS conductance channel was also observed when the duration of the pulse, used to eject GABA, was longer than 80 ms. The mean open time distribution of the unitary events was fitted best by two exponential functions suggesting two open channel states.

When either 1 μM fipronil or 1 μM BIDN was present in the external saline, the GABA‐gated channels were completely blocked. When BIDN or fipronil was applied at a concentration close to the IC50 value for suppression of open probability (281 nM, BIDN; 240 nM, fipronil), the duration of channel openings was shortened. In addition, the blocking action of BIDN resulted in the appearance of a novel channel conductance (17 pS).

The effects of co‐application of BIDN and fipronil were examined. Co‐application of BIDN (300 nM) with various concentrations (100–1000 nM) of fipronil resulted in an additional BIDN‐induced dose‐dependent reduction of the maximum Po value.

Thus both BIDN and fipronil shorten the duration of wild‐type RDLac GABA receptor channel openings but appear to act at distinct sites.

British Journal of Pharmacology (2000) 130, 1833–1842; doi:10.1038/sj.bjp.0703507
Serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in human neocortex slices: involvement of 5‐HT2C and 5‐HT1A receptors
Authors: Maura Guido , Marcoli Manuela , Pepicelli Olimpia , Rosu Christian , Viola Concetta , Raiteri Maurizio
Published: January 2005

The NMDA receptor/nitric oxide (NO)/cyclic GMP pathway and its modulation by 5‐hydroxytryptamine (5‐HT) was studied in slices of neocortical samples obtained from patients undergoing neurosurgery.

The cyclic GMP elevation produced by 100 μM NMDA was blocked by 100 μM of the NO synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG) or by 10 μM of the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3,‐α] quinoxaline‐1‐one (ODQ).

The NMDA effect was prevented by 5‐HT or by the 5‐HT2 agonist (±)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane ((±)‐DOI; EC50=22 nM). The (±)‐DOI inhibition was insensitive to the 5‐HT2A receptor antagonist MDL 100907 or the 5‐HT2B antagonist rauwolscine; it was largely prevented by 1 μM of the non‐selective 5‐HT2C antagonists mesulergine (5‐HT2A,B,C), ketanserin (5‐HT2A,C) or SB 200646A (5‐HT2B,C); it was completely abolished by 0.1 μM of the selective 5‐HT2C receptor antagonist SB 242084.

The NMDA‐induced cyclic GMP elevation also was potently inhibited by the selective 5‐HT2C agonist RO 60‐0175 and by the antidepressant trazodone, both added at 1 μM, in an SB 242084‐sensitive manner.

Finally, the 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT; 1 μM) inhibited the NMDA‐evoked cyclic GMP response, an effect blocked by the selective 5‐HT1A receptor antagonist WAY 100635.

In conclusion, the NMDA receptor/NO/cyclic GMP pathway in human neocortex slices can be potently inhibited by activation of 5‐HT2C or 5‐HT1A receptors.

British Journal of Pharmacology (2000) 130, 1853–1858; doi:10.1038/sj.bjp.0703510
The organic cation transporter OCT2 mediates the uptake of β‐adrenoceptor antagonists across the apical membrane of renal LLC‐PK1 cell monolayers
Authors: Dudley Adam J , Bleasby Kelly , Brown Colin D A
Published: January 2005

Previous studies have shown that β‐adrenoceptor antagonists may be substrates of organic cation transporters in kidney and lung. In this study we examined the transport of the β‐adrenoreceptor antagonists propranolol and metoprolol, in renal LLC‐PK1 cell monolayers.

Experiments with BCECF (2′,7′‐bis(2carboxyethyl)‐5(6)‐carboxyfluorescein) loaded LLC‐PK1 cell monolayers demonstrated that metoprolol and propranolol flux across the basolateral membrane was consistent with non‐ionic diffusion. Flux across the apical membrane consisted of both non‐ionic diffusion and the uptake of the cationic form of the β‐adrenoceptor antagonists.

Uptake of the cationic form of metoprolol across the apical membrane was Na+‐independent, electrogenic and sensitive to external pH. Furthermore, uptake was sensitive to inhibition by Decynium‐22 and the organic cations TEA (tetraethylammonium) and MPP+ (1‐methyl 4‐phenylpyridinium). These results, allied with the apical location of the uptake mechanism suggest that β‐adrenoceptor antagonists may be substrates for the organic cation transporter, OCT2.

To confirm β‐adrenoceptor antagonists as substrates for OCT2, we demonstrate, in cells transiently transfected with an epitope tagged version of hOCT2 (hOCT2‐V5):(1) Decynium‐22 sensitive [14C]‐propranolol uptake, (2) cis‐inhibition of OCT2 by a range of β‐adrenoceptor antagonists and (3) metoprolol induced intracellular acidification.

British Journal of Pharmacology (2000) 131, 71–79; doi:10.1038/sj.bjp.0703518
Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes
Authors: Mall Marcus , Wissner Andreas , Seydewitz Hans H , Hübner Martin , Kuehr Joachim , Brandis Matthias , Greger Rainer , Kunzelmann Karl
Published: January 2005

The flavonoid genistein has been shown to activate a Cl− conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivo tissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevis expressing CFTR.

In oocytes, both wtCFTR and ΔF508‐CFTR were activated by genistein while both cyclic AMP (KvLQT1) and Ca2+ (SK4) activated K+ channels were inhibited at high concentrations of genistein.

Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non‐CF) and CF patients and in the presence of amiloride (10 μmol l−1; mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non‐CF airway epithelia, genistein (50 μmol l−1; mucosal side) increased lumen negative Isc but had no additional effects on tissues pre‐stimulated with IBMX and forskolin (100 μmol l−1 and 1 μmol l−1; both sides).

In non‐CF rectal biopsies, in the presence of amiloride (10 μmol l−1; mucosal side) and indomethacin (10 μmol l−1; basolateral side), genistein increased lumen negative Isc and enabled cholinergic (carbachol; CCH, 100 μmol l−1; basolateral side) stimulation of Cl− secretion indicating activation of luminal CFTR Cl− channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated Isc. In CF airway and intestinal tissues genistein failed to induce Cl− secretion.

Thus, genistein is able to activate luminal CFTR Cl− conductance in non‐CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K+ conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

British Journal of Pharmacology (2000) 130, 1884–1892; doi:10.1038/sj.bjp.0703520
Insulin‐like effect of pinitol
Authors: Bates Sarah H , Jones Robert B , Bailey Clifford J
Published: January 2005

D‐pinitol (3‐O‐methyl‐chiroinositol), an active principle of the traditional antidiabetic plant Bougainvillea spectabilis, is claimed to exert insulin‐like effects. This study investigates the effect of D‐pinitol on glucose homeostasis in animal models of diabetes, and on glucose transport by cultured muscle cells.

Plasma glucose concentrations were measured in normal, obese‐diabetic (ob/ob) and streptozotocin (STZ)‐diabetic mice after oral (p.o.) and intraperitoneal (i.p.) administration of D‐pinitol. Glucose transport was measured in L6 rat muscle cells by 2‐deoxyglucose (2DG) uptake.

In STZ‐diabetic mice, 100 mg kg−1 p.o. D‐pinitol acutely decreased the hyperglycaemia (by 22% at 6 h). A similar decrease in plasma glucose (by 21%) was observed after 100 mg kg−1 i.p. D‐pinitol. Insulin concentrations and the rate of insulin‐induced (1 unit kg−1 actrapid i.p.) glucose disappearance were not altered by 100 mg kg−1 p.o. D‐pinitol. Chronic administration of D‐pinitol (100 mg kg−1 i.p. twice daily for 11 days) to STZ‐diabetic mice maintained a reduction in plasma glucose concentrations from about 14 to 10 mmol l−1.

In normal non‐diabetic and severely insulin resistant ob/ob mice, 100 mg kg−1 p.o. D‐pinitol did not significantly affect plasma glucose or insulin during acute studies.

Incubation of L6 muscle cells with D‐pinitol (10−3 M) increased basal 2DG uptake by 41% after 10 min and by 34% after 4 h. The effect of D‐pinitol was inhibited by the phosphatidylinositol 3‐kinase inhibitor LY294002. D‐pinitol did not increase insulin‐stimulated 2DG uptake by L6 cells.

The data support the view that D‐pinitol can exert an insulin‐like effect to improve glycaemic control in hypoinsulinaemic STZ‐diabetic mice. D‐pinitol may act via a post‐receptor pathway of insulin action affecting glucose uptake.

British Journal of Pharmacology (2000) 130, 1944–1948; doi:10.1038/sj.bjp.0703523
The progestin levonorgestrel induces endothelium‐independent relaxation of rabbit jugular vein via inhibition of calcium entry and protein kinase C: role of cyclic AMP
Authors: Herkert Olaf , Kuhl Herbert , Busse Rudi , Schini‐Kerth Valérie B
Published: January 2005

The progestin and oestrogen component of oral contraceptives have been involved in the development of venous thromboembolic events in women. In the present study we determined the vasoactive effects of sex steroids used in oral contraceptives in isolated preconstricted rabbit jugular veins in the presence of diclofenac and examined the underlying mechanisms.

The natural hormone progesterone, the synthetic progestins levonorgestrel, 3‐keto‐desogestrel, gestodene and chlormadinone acetate, and the synthetic estrogen 17 α‐ethinyloestradiol induced concentration‐dependent relaxations of endothelium‐intact veins constricted with U46619. Levonorgestrel also inhibited constrictions evoked by either a high potassium (K+) solution or phorbol myristate acetate (PMA) in the absence and presence of extracellular calcium (Ca2+). In addition, levonorgestrel depressed contractions evoked by Ca2+ and reduced 45Ca2+ influx in depolarized veins.

Relaxations to levonorgestrel in U46619‐constricted veins were neither affected by the presence of the endothelium nor by the inhibitor of soluble guanylyl cyclase, NS2028, but were significantly improved either by the selective cyclic AMP phosphodiesterase inhibitor rolipram or in the absence of diclofenac, and decreased by the protein kinase A inhibitor, Rp‐8‐CPT‐cAMPS. Rolipram also potentiated relaxations to levonorgestrel in PMA‐constricted veins in the presence, but not in the absence of extracellular Ca2+. Levonorgestrel increased levels of cyclic AMP and inhibited PMA‐induced activation of protein kinase C in veins.

These findings indicate that levonorgestrel caused endothelium‐independent relaxations of jugular veins via inhibition of Ca2+ entry and of protein kinase C activation. In addition, the cyclic AMP effector pathway contributes to the levonorgestrel‐induced relaxation possibly by depressing Ca2+ entry.

British Journal of Pharmacology (2000) 130, 1911–1918; doi:10.1038/sj.bjp.0703524
Inhibitory effect of amiodarone on Na+/Ca2+ exchange current in guinea‐pig cardiac myocytes
Authors: Watanabe Yasuhide , Kimura Junko
Published: January 2005

The effect of amiodarone on Na+/Ca2+ exchange current (INCX) was examined in single guinea‐pig ventricular myocytes using the whole‐cell voltage clamp technique.

INCX was recorded by ramp pulses from the holding potential of −60 mV in the presence of 140 mM Na+ and 2 mM Ca2+ in the external solution, and 20 mM Na+ and 398 nM free Ca2+ (19 mM Ca2+ and 30 mM BAPTA) in the internal solution.

External application of amiodarone suppressed INCX in a concentration‐dependent manner. The IC50 value was 3.3 μM with a Hill coefficient of 1.

Intracellular application of trypsin via the micropipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the cytoplasmic side of the molecule.

This inhibitory effect of amiodarone on the Na+/Ca2+ exchanger may contribute to the cardioprotective action of the drug.

British Journal of Pharmacology (2000) 131, 80–84; doi:10.1038/sj.bjp.0703527
Inducible nitric oxide synthase‐derived superoxide contributes to hypereactivity in small mesenteric arteries from a rat model of chronic heart failure
Authors: Miller Alyson A , Megson Ian L , Gray Gillian A
Published: January 2005

The aims of this study were to (a) determine whether inducible nitric oxide synthase (iNOS) is expressed in small mesenteric arteries from rats with chronic heart failure (CHF), (b) investigate the functional significance of this potential source of nitric oxide (NO) on vascular responsiveness and (c) investigate the role that superoxide plays in modulating vascular function in these arteries.

CHF was induced in male Wistar rats by coronary artery ligation (CAL). In sham‐operated rats the ligature was not tied but pulled under the artery. Six weeks after surgery CAL rats had left ventricular (LV) infarctions and elevated LV end‐diastolic pressures.

Immunoreactive iNOS was found in endothelial cells, vascular smooth muscle cells and in the adventitia of small mesenteric arteries from CAL rats but not those from sham‐operated rats.

Third order mesenteric arteries (300–350 μm) were mounted in a small vessel pressure myograph. Endothelium‐intact arteries from CAL rats were more responsive to phenylephrine (PE) than arteries from sham‐operated rats (pD2 value, CAL, 6.2±0.1; sham‐operated, 5.9±0.1, P<0.05).

Both the selective iNOS inhibitor, N‐(3‐(Aminomethyl) benzyl) acetamidine dihydrochloride (1400W; 10−6 M) and the superoxide dismutase mimetic, Mn [III] tetrakis [1‐methyl‐4‐pyridyl] porphyrin, (MnTMPyP; 10−4 M) reversed the hyperesponsiveness (pD2 values, 1400W, 5.9±0.1; MnTMPyP, 5.81±0.1, P<0.05). The NOS substrate, L‐arginine (10−3 M), reduced responsiveness of endothelium‐denuded small mesenteric arteries from CAL rats (P<0.01). None of these drugs altered responses to PE in arteries from sham‐operated rats.

In summary, this study demonstrates that iNOS is expressed in mesenteric arteries from rats with CHF. However, instead of generating large quantities of NO, iNOS appears to be generating superoxide, perhaps because of a deficiency in its substrate, L‐arginine. Increased superoxide generation from iNOS contributes to the hyperesponsive nature of endothelium‐intact small mesenteric arteries from rats with CHF.

British Journal of Pharmacology (2000) 131, 29–36; doi:10.1038/sj.bjp.0703528
Study of A2A adenosine receptor gene deficient mice reveals that adenosine analogue CGS 21680 possesses no A2A receptor‐unrelated lymphotoxicity
Authors: Apasov Sergey G , Chen Jiang‐Fan , Smith Patrick T , Schwarzschild Michael A , Fink J Stephen , Sitkovsky Michail V
Published: January 2005

Cell surface A2A adenosine receptor (A2AR) mediated signalling affects a variety of important processes and adenosine analogues possess promising pharmacological properties.

Demonstrating the receptor specificity of potentially lymphotoxic adenosine‐based drugs facilitates their development for clinical applications.

To distinguish between the receptor‐dependent and ‐independent lymphotoxicity and apoptotic activity of adenosine and its analogues we used lymphocytes from A2AR‐deficient mice.

Comparison of A2AR‐expressing (+/+) and A2AR‐deficient (−/−) cells in cyclic AMP accumulation assays confirmed that the A2AR agonist CGS 21680 is indeed selective for A2A receptors in T‐lymphocytes.

Incubation of A2AR‐expressing thymocytes with extracellular adenosine or CGS 21680 in vitro results in the death of about 7–15% of thymocytes. In contrast, no death was induced in parallel assays in cells from A2AR‐deficient mice, providing genetic evidence that CGS 21680 does not display adenosine receptor‐independent intracellular cytotoxicity.

The A2A receptor‐specific lymphotoxicity of CGS 21680 is also demonstrated in a long‐term (6‐day) in vitro model of thymocyte positive selection where addition of A2AR antagonist ZM 241,385 did block the effects of CGS 21680, allowing the survival of T cells.

The use of cells from adenosine receptor‐deficient animals is proposed as a part of the screening process for potential adenosine‐based drugs for their receptor‐independent cytotoxicity and lymphotoxicity.

British Journal of Pharmacology (2000) 131, 43–50; doi:10.1038/sj.bjp.0703532
Inhibition of sustained hypoxic vasoconstriction by Y‐27632 in isolated intrapulmonary arteries and perfused lung of the rat
Authors: Robertson Tom P , Dipp Michelle , Ward Jeremy P T , Aaronson Phil I , Evans A Mark
Published: January 2005

We have examined the effects of Y‐27632, a specific inhibitor of Rho‐activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y‐27632 (100 nM–3 μM) was found to cause a concentration‐dependent inhibition of acute sustained HPV in rat IPA. Application of Y‐27632 (10–600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration‐dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y‐27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho‐associated kinase may be a pivotal step in the generation of sustained HPV.

British Journal of Pharmacology (2000) 131, 5–9; doi:10.1038/sj.bjp.0703537
Prevention by dexrazoxane of down‐regulation of ryanodine receptor gene expression in anthracycline cardiomyopathy in the rat
Authors: Burke Briant E , Gambliel Hervé , Olson Richard D , Bauer Frederic K , Cusack Barry J
Published: January 2005

Anthracyclines can cause cumulative dose‐related cardiotoxicity characterized by changes in Ca2+ metabolism, including dysfunction of the sacroplasmic reticulum (SR) and decreased expression of Ca2+‐handling proteins, such as the ryanodine receptor (RyR2). In this study, we examined the effect of dexrazoxane (ICRF‐187), an iron chelator which prevents anthracycline cardiotoxicity, on RyR2 gene expression in rats treated chronically with daunorubicin. Daunorubicin (2.5 mg kg−1 i.v. weekly for 6 weeks) produced cardiotoxicity as demonstrated by histopathologic changes. The ryanodine receptor/glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA ratio was decreased by 38±3% (P<0.02) compared to values in control rats. Dexrazoxane pre‐treatment (50 mg kg−1; 1 h prior to each daunorubicin injection) prevented the decrease in RyR2/GAPDH mRNA ratio and histopathologic lesions in daunorubicin‐treated rats. This is the first report that a protective agent such as dexrazoxane can ameliorate the decreased expression of a specific gene involved in anthracycline‐induced cardiotoxicity.

British Journal of Pharmacology (2000) 131, 1–4; doi:10.1038/sj.bjp.0703538
YM‐53601, a novel squalene synthase inhibitor, reduces plasma cholesterol and triglyceride levels in several animal species
Authors: Ugawa Tohru , Kakuta Hirotoshi , Moritani Hirosh , Matsuda Koyo , Ishihara Tsukasa , Yamaguchi Motoko , Naganuma Shin , Iizumi Yuichi , Shikama Hisataka
Published: January 2005

The aim of this study was to evaluate the potency of YM‐53601 ((E)‐2‐[2‐fluoro‐2‐(quinuclidin‐3‐ylidene) ethoxy]‐9H‐carbazole monohydrochloride), a new inhibitor of squalene synthase, in reducing both plasma cholesterol and triglyceride levels, compared with 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor and fibrates, respectively.

YM‐53601 equally inhibited squalene synthase activities in hepatic microsomes prepared from several animal species and also suppressed cholesterol biosynthesis in rats (ED50, 32 mg kg−1).

In guinea‐pigs, YM‐53601 and pravastatin reduced plasma nonHDL‐C (=total cholesterol–high density lipoprotein cholesterol) by 47% (P<0.001) and 33% (P<0.001), respectively (100 mg kg−1, daily for 14 days). In rhesus monkeys, YM‐53601 decreased plasma nonHDL‐C by 37% (50 mg kg−1, twice daily for 21 days, P<0.01), whereas the HMG‐CoA reductase inhibitor, pravastatin, failed to do (25 mg kg−1, twice daily for 28 days).

YM‐53601 caused plasma triglyceride reduction in hamsters fed a normal diet (81% decrease at 50 mg kg−1, daily for 5 days, P<0.001). In hamsters fed a high‐fat diet, the ability of YM‐53601 to lower triglyceride (by 73%, P<0.001) was superior to that of fenofibrate (by 53%, P<0.001), the most potent fibrate (dosage of each drug: 100 mg kg−1, daily for 7 days).

This is the first report that a squalene synthase inhibitor is superior to an HMG‐CoA reductase inhibitor in lowering plasma nonHDL‐C level in rhesus monkeys and is superior to a fibrate in significantly lowering plasma triglyceride level. YM‐53601 may therefore prove useful in treating hypercholesterolemia and hypertriglyceridemia in humans.

British Journal of Pharmacology (2000) 131, 63–70; doi:10.1038/sj.bjp.0703545
Geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) mediated signal transduction has anti‐inflammatory effects and interacts with glucocorticoid receptor in vivo
Authors: Bucci Mariarosaria , Roviezzo Fiorentina , Cicala Carla , Sessa William C , Cirino Giuseppe
Published: January 2005

Histamine, vascular endothelial growth factor, acetylcholine, oestrogen as well as fluid shear stress activates a mechanism that recruits heat shock protein 90 to the endothelial nitric oxide synthase. The interaction between Hsp90 and eNOS enhances the activation of the enzyme in cells and in intact blood vessels leading to NO production.

Intraplantar administration of carrageenan (50 μl paw−1) to mice causes an oedema lasting 72 h. Geldanamycin (0.1, 0.3, 1 mg kg−1), a specific inhibitor of Hsp‐90, that inhibits endothelium‐dependent relaxations of the rat aorta, mesentery and middle artery inhibits carrageenan‐induced mouse paw oedema in a dose dependent manner.

Co‐administration to mice of dexamethasone (1 mg kg−1) with geldanamycin (0.3 mg kg−1) at anti‐inflammatory dose causes a loss of the total anti‐inflammatory effect of each agent alone.

RU 486 (10 mg kg−1), a well known glucocorticoid receptorial antagonist, does not inhibit oedema formation but prevents the anti‐inflammatory action of dexamethasone (1 mg kg−1). Similarly, RU 486 prevents the anti‐inflammatory action of geldanamycin (0.3 mg kg−1).

In conclusion we have described for the first time that geldanamycin, an inhibitor of Hsp90 dependent signal transduction, is anti‐inflammatory in vivo implying that Hsp90 is critical for pathways involved in carrageenan‐induced paw oedema. In addition the ability of GA to block NO release and reduce oedema formation suggests a therapeutic rationale for specific inhibitors of Hsp90 as potential anti‐inflammatory drugs.

British Journal of Pharmacology (2000) 131, 13–16; doi:10.1038/sj.bjp.0703549
Influence of hypertension on nitric oxide synthase expression and vascular effects of lipopolysaccharide in rat mesenteric arteries
Authors: Briones Ana M , Alonso María J , Marín Jesús , Balfagón Gloria , Salaices Mercedes
Published: January 2005

Experiments were designed to investigate the effects of the inducible nitric oxide synthase (iNOS) stimulator, lipopolysaccharide (LPS), on noradrenaline (NA) responses and on NOS activity and its expression in intact mesenteric resistance arteries (MRAs) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats.

In MRAs from WKY, LPS (10 μg ml−1; 1–5 h) reduced the vasoconstrictor responses to NA (0.1–30 μM) in the presence, but not in the absence of L‐arginine (L‐Arg, 10 μM). However, in SHR arteries, LPS induced an incubation‐time dependent reduction of NA responses in the absence, as well as the presence, of L‐Arg. The LPS inhibitory effect was reduced by the non‐specific NOS inhibitor L‐NG‐nitroarginine methyl ester (L‐NAME, 100 μM) and the selective iNOS inhibitor, aminoguanidine (100 μM).

L‐NAME alone similarly shifted the concentration‐response curve to NA leftward in arteries from both strains, while aminoguanidine had no effect. L‐Arg shifted the curve to NA rightward only in SHR MRAs.

Basal activity of both iNOS and constitutive NOS (conversion of [3H]‐L‐Arg to [3H]‐L‐citrulline) was similar in arteries from both strains. After 5 h incubation with LPS, only iNOS activity in arteries from SHR was increased.

Basal iNOS protein expression was undetectable; basal endothelial (eNOS) protein expression was similar in arteries from both strains, while neuronal (nNOS) was greater in arteries from SHR. LPS induced iNOS protein expression, that was higher in arteries from SHR than in those from WKY.

These results indicate that NO production, via iNOS induction, is greater than in those from MRAs from SHR to WKY.

British Journal of Pharmacology (2000) 131, 185–194; doi:10.1038/sj.bjp.0703552
On the in vitro vasoactivity of bile acids
Authors: Ljubuncic Predrag , Said Omar , Ehrlich Yaron , Meddings Jon B , Shaffer Eldon A , Bomzon Arieh
Published: January 2005

We compared the vasorelaxant action of nine different bile acids and correlated their vasorelaxant activity with their individual indices for hydrophobicity or lipophilicity.

Vasorelaxant activity correlated with the relative lipid solubility of bile acids with lipophilic bile acids exhibiting the greatest vasorelaxant activity with modest to no vasorelaxant activity exhibited by hydrophilic bile acids.

We also investigated whether bile acid‐induced vasorelaxation is mediated by antagonism of a prototypal contractile receptor, the α1‐adrenoceptor, by stimulation of a bile acid surface membrane receptor, by the release of endothelium‐derived relaxant factors, by promoting the generation of reactive oxygen species and increasing the extent of lipid peroxidation, or by modifying membrane fluidity.

Lipophilic bile acids induce vasorelaxation possibly by antagonizing α1‐adrenoceptors, a phenomenon that manifests itself as a lowering of the affinity of vascular α1‐adrenoceptors. Bile acid‐induced vasorelaxation was not dependent upon stimulation of a bile acid surface membrane receptor or the release of endothelium‐derived relaxant factors.

Lipophilic bile acids can also increase the extent of lipid peroxidation with a subtle reduction in the fluidity of rat vascular smooth muscle membranes not associated with loss of membrane cholesterol or phospholipid.

We have concluded that lipophilic bile acids are non‐selective vasorelaxants whose mechanism of action is a multifaceted process involving antagonism of contractile surface membrane receptors possibly effected by an increased extent of lipid peroxidation and/or membrane fluidity but occurs independent of the release of endothelial‐derived relaxant factors or stimulation of a surface membrane bile acid binding site.

British Journal of Pharmacology (2000) 131, 387–398; doi:10.1038/sj.bjp.0703554