bluuebunny/crossref_metadata_2025 huggingface dataset:
ETA receptors are the primary mediators of myofilament calcium sensitization induced by ET‐1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway
Authors: Evans A Mark , Cobban Hannah J , Nixon Graeme F
Published: January 2005

We have investigated the possibility that ET‐1 can induce an increase in myofilament calcium sensitivity in pulmonary artery smooth muscle. Arterial rings were permeabilized using α‐toxin (120 μg ml−1), in the presence of A23187 (10 μM) to ‘knock out’ Ca2+ stores, and pre‐constricted with pCa 6.8 (buffered with 10 mM EGTA). In the presence of this fixed Ca2+ concentration, 1 μM ET‐1 induced a sustained, reversible constriction of 0.15 mN.

Pulmonary arterial rings were freeze‐clamped at the peak of the induced constriction (time matched). Subsequent densitometric analysis revealed that ET‐1 (1 μM) increased the level of phosphorylated myosin light chains by 34% compared to an 11% increase in the presence of pCa 6.8 alone.

In contrast to ET‐1, the selective ETB receptor agonist Sarafotoxin S6C (100 nM) failed to induce a significant constriction.

The constriction induced by 1 μM ET‐1 was reversibly inhibited when the preparation was pre‐incubated (15 min) with the ETA receptor antagonist BQ 123 (100 μM). The constriction measured 0.13 mN in the absence and 0.07 mN in the presence of 100 μM BQ 123.

In contrast, the constriction induced by 1 μM ET‐1 measured 0.19 mN in the absence and 0.175 mN following a 15 min pre‐incubation with the ETB antagonist BQ 788 (100 μM).

The constriction induced by 1 μM ET‐1 measured 0.14 mN in the presence and 0.13 mN following pre‐incubation with the tyrosine kinase inhibitor Tyrphostin A23 (100 μM).

We conclude that ET‐1 induced an increase in myofilament calcium sensitivity in rat pulmonary arteries via the activation of ETA receptors and by a mechanism(s) independent of tyrosine kinase.

British Journal of Pharmacology (1999) 127, 153–160; doi:10.1038/sj.bjp.0702548
Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury
Authors: Parmentier Sophie , Böhme Georg Andrees , Lerouet Dominique , Damour Dominique , Stutzmann Jean‐Marie , Margaill Isabelle , Plotkine Michel
Published: January 2005

The aim of this study was to investigate the effect of N‐(3‐(aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of inducible calcium‐independent nitric oxide synthase (iNOS), on the functional and histopathological outcomes of experimental transient focal cerebral ischaemia in rats.

Transient ischaemia was produced by the occlusion for 2 h of both the left middle cerebral artery and common carotid artery. Treatments with 1400W (20 mg kg−1) or vehicle were started 18 h after occlusion of the arteries and consisted in seven subcutaneous injections at 8 h interval. Ischaemic outcomes and NOS activities (constitutive and calcium‐independent NOS) were evaluated 3 days after ischaemia.

1400W significantly reduced ischaemic lesion volume by 31%, and attenuated weight loss and neurological dysfunction.

1400W attenuated the calcium‐independent NOS activity in the infarct by 36% without affecting the constitutive NOS activity.

These findings suggest that iNOS activation contributes to tissue damage and that selective inhibitors of this isoform may be of interest for the treatment of stroke.

British Journal of Pharmacology (1999) 127, 546–552; doi:10.1038/sj.bjp.0702549
Comparative pharmacology of recombinant human M3 and M5 muscarinic receptors expressed in CHO‐K1 cells
Authors: Watson Nikki , Daniels Donald V , Ford Anthony P D W , Eglen Richard M , Hegde Sharath S
Published: January 2005

Affinity estimates were obtained for several muscarinic antagonists against carbachol‐stimulated [3H]‐inositol phosphates accumulation in Chinese hamster ovary (CHO‐K1) cells stably expressing either human muscarinic M3 or M5 receptor subtypes. The rationale for these studies was to generate a functional antagonist affinity profile for the M5 receptor subtype and compare this with that of the M3 receptor, in order to identify compounds which discriminate between these two subtypes.

The rank order of antagonist apparent affinities (pKB) at the muscarinic M5 receptor was atropine (8.7)tolterodine (8.6)=4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP, 8.6)>darifenacin (7.7)zamifenacin (7.6)>oxybutynin (6.6)=para‐fluorohexahydrosiladifenidol (p‐F‐HHSiD, 6.6)>pirenzepine (6.4)methoctramine (6.3)=himbacine (6.3)>AQ‐RA 741 (6.1).

Antagonist apparent affinities for both receptor subtypes compare well with published binding affinity estimates. No antagonist displayed greater selectivity for the muscarinic M5 subtype over the M3 subtype, but himbacine, AQ‐RA 741, p‐F‐HHSiD, darifenacin and oxybutynin displayed between 9‐ and 60 fold greater selectivity for the muscarinic M3 over the M5 subtype.

This study highlights the similarity in pharmacological profiles of M3 and M5 receptor subtypes and identifies five antagonists that may represent useful tools for discriminating between these two subtypes. Collectively, these data show that in the absence of a high affinity M5 selective antagonist, affinity data for a large range of antagonists is critical to define operationally the M5 receptor subtype.

British Journal of Pharmacology (1999) 127, 590–596; doi:10.1038/sj.bjp.0702551
Nitric oxide synthase inhibition by dimaprit and dimaprit analogues
Authors: Paquay Jos B G , 't Hoen Peter A Chr , Voss Hans‐Peter , Bast Aalt , Timmerman Henk , Haenen Guido R M M
Published: January 2005

The similarity in molecular structure between the histamine H2‐agonist dimaprit (3‐dimethylamino‐propyl‐isothiourea) and the endogenous nitric oxide synthase (NOS) substrate L‐arginine prompted us to study the effect of dimaprit and some dimaprit analogues on NOS activity. Dimaprit and some of its analogues were tested in an in vitro assay which measures the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline. Dimaprit inhibits rat brain NOS (nNOS) concentration dependently with an IC50 of 49±14 μM.

Removal of one or both of the methyl groups from the non‐isothiourea nitrogen of dimaprit improved nNOS inhibitory properties. Aminopropylisothiourea is the most potent compound (IC50=4.1±0.9 μM) of the series followed by methylaminopropylisothiourea (IC50=7.6± μM).

The observed effect of aminopropylisothiourea and methylaminopropyl‐isothiourea are probably not due to the compounds themselves but to the corresponding mercaptoalkylguanidines, rearrangement products formed in aqueous solutions. This hypothesis is strengthened by the finding that aminobutylisothiourea is not active since a rearrangement to mercaptobutylguanidine does not occur.

Remarkably, nitrosylation of the isothiourea group of dimaprit decreases nNOS inhibitory activity, while nitrosylation of the guanidine analogue of dimaprit increases the inhibition of nNOS activity.

The pharmacological profile of dimaprit includes inhibition of nNOS. The nNOS inhibitory activity occurs in the same concentration range as the H2‐agonist and H3‐agonist activity of this compound.

British Journal of Pharmacology (1999) 127, 331–334; doi:10.1038/sj.bjp.0702552
Effect of insulin treatment on smooth muscle contractility and endothelium‐dependent relaxation in rat aortae from established STZ‐induced diabetes
Authors: Kobayashi Tsuneo , Kamata Katsuo
Published: January 2005

This study involved the chronic administration of low or high insulin to rats with established streptozotocin (STZ)‐induced diabetes. We studied the effect of such treatment on smooth muscle contractility and endothelium‐dependent relaxation using aortic strips.

Aortae from diabetic rats, but not those from high‐insulin‐treated diabetic rats, showed an impaired endothelium‐dependent in response to acetylcholine (ACh) by comparison with untreated controls.

Isotonic high K+‐induced contractility was impaired in diabetic aortae. This impairment was prevented by high‐insulin treatment.

Noradrenaline (NA)‐induced contractility was enhanced in aortae from high‐insulin‐treated diabetic rats, but not in those from untreated diabetic or low‐insulin treated diabetic rats.

In the combined presence of the nitric oxide inhibitor NG‐nitro‐L‐arginine and the cyclo‐oxygenase inhibitor indomethacin, NA‐induced contractility was significantly greater in aortae from high‐insulin‐treated diabetic rats than in those from controls or untreated diabetic rats.

An increased expression of the mRNA for the α1D and α1B adrenergic receptors was found in aortae from high‐insulin‐treated diabetic rats.

These results demonstrate that in rats with established STZ‐induced diabetes, high‐insulin treatment prevents the development of an impaired endothelium‐dependent relaxation in the aorta, and that such treatment enhances NA‐induced contractility. This enhancement may be related to an upregulation in the expression of the mRNA for the α1B or α1D adrenergic receptor that is secondary to the hyperinsulinaemia.

British Journal of Pharmacology (1999) 127, 835–842; doi:10.1038/sj.bjp.0702554
NO synthase in cholinergic nerves and NO‐induced relaxation in the rat isolated corpus cavernosum
Authors: Hedlund Petter , Alm Per , Andersson Karl‐Erik
Published: January 2005

In the rat corpus cavernosum (CC), the distribution of immunoreactivity for neuronal and endothelial NO synthase (nNOS and eNOS), and the pattern of NOS‐immunoreactive (‐IR) nerves in relation to some other nerve populations, were investigated. Cholinergic nerves were specifically immunolabelled with antibodies to the vesicular acetylcholine transporter protein (VAChT).

In the smooth muscle septa surrounding the cavernous spaces, and around the central and helicine arteries, the numbers of PGP‐ and tyrosine hydroxylase (TH)‐IR terminals were large, whereas neuropeptide Y (NPY)‐, VAChT‐, nNOS‐, and vasoactive intestinal polypeptide (VIP)‐IR terminals were found in few to moderate numbers.

Double immunolabelling revealed that VAChT‐ and nNOS‐IR terminals, VAChT‐ and VIP‐IR terminals, nNOS‐IR and VIP‐IR terminals, and TH‐ and NPY‐IR terminals showed coinciding profiles, and co‐existence was verified by confocal laser scanning microscopy. TH immunoreactivity was not found in VAChT‐, nNOS‐, or VIP‐IR nerve fibres or terminals.

An isolated strip preparation of the rat CC was developed, and characterized. In this preparation, cumulative addition of NO to noradrenaline (NA)‐contracted strips, produced concentration‐dependent, rapid, and almost complete relaxations. Electrical field stimulation of endothelin‐1‐contracted preparations produced frequency‐dependent responses: a contractile twitch followed by a fast relaxant response. After cessation of stimulation, there was a slow relaxant phase. Inhibition of NO synthesis, or blockade of guanylate cyclase, abolished the first relaxant phase, whereas the second relaxation was unaffected.

The results suggest that in the rat CC, nNOS, VAChT‐, and VIP‐immunoreactivities can be found in the same parasympathetic cholinergic neurons. Inhibitory neurotransmission involves activation of the NO‐system, and the release of other, as yet unknown, transmitters.

British Journal of Pharmacology (1999) 127, 349–360; doi:10.1038/sj.bjp.0702556
Expression of P2Y receptors in cell lines derived from the human lung
Authors: Communi Didier , Paindavoine Pascale , Place Graham A , Parmentier Marc , Boeynaems Jean‐Marie
Published: January 2005

Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo− and 16HBE14o− epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU‐3 serous cells, the 6CFSMEo− submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC‐1. These experiments revealed that P2Y2 and P2Y6 mRNA are co‐expressed in the 1HAEo−, 16HBE14o− and A549 epithelial cell lines. The CFPAC‐1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors.

We have then performed RT–PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo−, 16HBE14o−, 6CFSMEo−, HASMSC1 and CFPAC‐1. P2Y1 mRNA was only detected in the CALU‐3 cell line.

Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo− and the 16HBE14o− airway epithelial cell lines which co‐express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo− submucosal cells expressed a UTP‐specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

British Journal of Pharmacology (1999) 127, 562–568; doi:10.1038/sj.bjp.0702560
Structural and ionic determinants of 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid block of the CFTR chloride channel
Authors: Walsh Kenneth B , Long Kathryn J , Shen Xufeng
Published: January 2005

The goals of this study were to identify the structural components required for arylaminobenzoate block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in drug binding.

Wild‐type and mutant CFTR chloride channels were expressed in Xenopus oocytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild‐type CFTR current by 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) occurred in a voltage‐dependent manner with preferential inhibition of the inward currents (Kd=166 μM at −90 mV).

Removal of the phenyl ring from the aliphatic chain of NPPB, with the compound 2‐butylamino‐5‐nitrobenzoic acid, caused only a small change in CFTR inhibition (Kd=243 μM), while addition of an extra phenyl ring at this position (5‐nitro‐2‐(3,3‐diphenylpropylamino)‐benzoic acid) increased drug potency (Kd=58 μM). In contrast, removal of the benzoate ring (2‐amino‐4‐phenylbutyric acid) or the 5‐nitro group (2‐(3‐phenylpropylamino)‐benzoic acid) of NPPB severely limited drug block of the wild‐type channel.

NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage‐dependent manner. However, the Kds for NPPB block were increased to 371 and 1573 μM, for the K335E and R347E mutants, respectively.

NPPB block of the inward wild‐type CFTR current was reduced in the presence of 10 mM of the permeant anion SCN−.

These studies present the first step in the development of high affinity probes to the CFTR channel.

British Journal of Pharmacology (1999) 127, 369–376; doi:10.1038/sj.bjp.0702562
Neuropeptide Y is a prejunctional inhibitor of vagal but not sympathetic inotropic responses in guinea‐pig isolated left atria
Authors: Serone Adrian P , Angus James A
Published: January 2005

The effects of NPY and related peptides were examined on basal contractile force and nerve‐mediated inotropic responses to electrical field stimulation of the guinea‐pig isolated left atrium.

Electrical field stimulus (EFS)‐inotropic response curves were constructed by applying 1–64 trains of four field pulses (200 Hz, 0.1 ms duration, 100 V) across isolated left atria (paced at 4 Hz, 2 ms, 1–4 V) within the atrial refractory period. Curves were constructed in presence of vehicle, propranolol (1 μM) or atropine (1 μM) to determine appropriate stimulus conditions.

The effects of PYY (1–10,000 nM), NPY (0.01–10 μM), N‐Ac‐[Leu28,31]NPY(24–36) (N‐A[L]NPY(24–36); 0.01–10 μM) and clonidine (0.1–1000 nM) were examined on the positive and negative inotropic responses to EFS (eight trains, four pulses per refractory period).

NPY‐related peptides had no effect on basal force of contraction nor on the inotropic concentration‐response curves to bethanechol or isoprenaline. All three peptides inhibited vagally‐mediated negative inotropic responses; rank order of potency PYY>NPYN‐A[L]NPY(24–36) was consistent with an action at prejunctional Y2‐receptors. Clonidine concentration‐dependently inhibited sympathetic inotropic responses. However, PYY, NPY and N‐A[L]NPY(24–36) failed to mediate any significant inhibition of the positive inotropic response to EFS.

These data demonstrate that NPY is an effective inhibitor of vagal but not sympathetically‐mediated inotropic responses in the guinea‐pig isolated left atria. This may suggest that endogenously co‐released NPY is important in mediating cross talk between efferent components of the autonomic nervous system modulating cardiac contractility, acting overall to sustain positive inotropic responses.

British Journal of Pharmacology (1999) 127, 383–390; doi:10.1038/sj.bjp.0702565
Hyperosmolarity reduces the relaxing potency of nitric oxide donors in guinea‐pig trachea
Authors: Hjoberg Josephine , Högman Marieann , Hedenstierna Göran
Published: January 2005

Non‐responders to inhaled nitric oxide treatment have been observed in various patient groups. The bronchodilatory effect of inhaled nitric oxide was attenuated when the airway lumen was rendered hyperosmolar in an in vivo study on rabbits. We used a guinea‐pig tracheal perfusion model to investigate the effects of increased osmolarity (450 mOsm, NaCl added) on the relaxing potency of the nitric oxide donors sodium nitroprusside (SNP) and (±)‐S‐nitroso‐N‐acetylpenicillamine (SNAP).

Under iso‐osmolar conditions SNP relaxed the carbachol (CCh, 1 μM) contracted trachea by 83±3%. After pretreatment with intraluminal hyperosmolarity SNP relaxed the CCh‐contracted trachea by only 31±7% (P<0.05). When the trachea was contracted to the same extent under untreated and hyperosmolar conditions, the untreated trachea was completely relaxed by SNP but, after hyperosmolar pretreatment, SNP could no longer relax the trachea.

SNAP relaxed the CCh contracted trachea by 27±5%. After pretreatment with intraluminal hyperosmolarity, SNAP relaxed the trachea by 11±4%, which was less than in the iso‐osmolar control (P<0.05).

Extraluminal hyperosmolarity did not affect carbachol elicited contraction, and SNP administered externally during extraluminal hyperosmolarity was able to relax the trachea (P<0.05).

The cell permeable guanosine 3′5′‐cyclic monophosphate analogue 8‐Br‐cGMP relaxed the CCh contracted trachea in both iso‐osmolar (P<0.05) and hyperosmolar conditions (P<0.05).

The relaxant effect of nitric oxide donors on tracheal smooth muscle is markedly reduced when the airway epithelium is exposed to hyperosmolar solution.

British Journal of Pharmacology (1999) 127, 391–396; doi:10.1038/sj.bjp.0702569
Pharmacological characterization of the muscarinic receptor antagonist, glycopyrrolate, in human and guinea‐pig airways
Authors: Haddad El‐Bdaoui , Patel Hema , Keeling Joelle E , Yacoub Magdi H , Barnes Peter J , Belvisi Maria G
Published: January 2005

In this study we have evaluated the pharmacological profile of the muscarinic antagonist glycopyrrolate in guinea‐pig and human airways in comparison with the commonly used antagonist ipratropium bromide.

Glycopyrrolate and ipratropium bromide inhibited EFS‐induced contraction of guinea‐pig trachea and human airways in a concentration‐dependent manner. Glycopyrrolate was more potent than ipratropium bromide.

The onset of action (time to attainment of 50% of maximum response) of glycopyrrolate was similar to that obtained with ipratropium bromide in both preparations. In guinea‐pig trachea, the offset of action (time taken for response to return to 50% recovery after wash out of the test antagonist) for glycopyrrolate (t1/2 [offset]=26.4±0.5 min) was less than that obtained with ipratropium bromide (81.2±3.7 min). In human airways, however, the duration of action of glycopyrrolate (t1/2 [offset]>96 min) was significantly more prolonged compared to ipratropium bromide (t1/2 [offset]=59.2±17.8 min).

In competition studies, glycopyrrolate and ipratropium bromide bind human peripheral lung and human airway smooth muscle (HASM) muscarinic receptors with affinities in the nanomolar range (Ki values 0.5–3.6 nM). Similar to ipratropium bromide, glycopyrrolate showed no selectivity in its binding to the M1–M3 receptors. Kinetics studies, however, showed that glycopyrrolate dissociates slowly from HASM muscarinic receptors (60% protection against [3H]‐NMS binding at 30 nM) compared to ipratropium bromide.

These results suggest that glycopyrrolate bind human and guinea‐pig airway muscarinic receptors with high affinity. Furthermore, we suggest that the slow dissociation profile of glycopyrrolate might be the underlying mechanism by which this drug accomplishes its long duration of action.

British Journal of Pharmacology (1999) 127, 413–420; doi:10.1038/sj.bjp.0702573
The effects of verapamil and diltiazem on N‐, P‐ and Q‐type calcium channels mediating dopamine release in rat striatum
Authors: Dobrev Dobromir , Milde Alexander S , Andreas Klaus , Ravens Ursula
Published: January 2005

The putative inhibitory effects of verapamil and diltiazem on neuronal non‐L‐type Ca2+ channels were studied by investigating their effects on either K+‐ or veratridine‐evoked [3H]‐dopamine ([3H]‐DA) release in rat striatal slices. Involvement of N‐, P‐ and Q‐type channels was identified by sensitivity of [3H]‐DA release to ω‐conotoxin GVIA (ω‐CTx‐GVIA), ω‐agatoxin IVA (ω‐Aga‐IVA) and ω‐conotoxin MVIIC (ω‐CTx‐MVIIC), respectively.

KCl (50 mM)‐evoked [3H]‐DA release was abolished in the absence of Ca2+, and was insensitive to dihydropyridines (up to 30 μM). It was significantly blocked by ω‐CTx‐GVIA (1 μM), ω‐Aga‐IVA (30 nM) and was confirmed to be abolished by ω‐CTx‐MVIIC (3 μM), indicating involvement of N‐, P‐ and Q‐type channel subtypes.

Verapamil and diltiazem inhibited K+‐evoked [3H]‐DA release in a concentration‐dependent manner. The inhibitory effects of verapamil or diltiazem (each 30 μM) were fully additive to the effect of ω‐CTx‐GVIA (1 μM), whereas co‐application with ω‐Aga‐IVA (30 nM) produced similar effects to those of ω‐Aga‐IVA alone.

As shown previously, veratridine‐evoked [3H]‐DA release in Ca2+ containing medium exclusively involves Q‐type Ca2+ channels. Here, diltiazem (30 μM) did not inhibit veratridine‐evoked [3H]‐DA release, whereas verapamil (30 μM) partially inhibited it, indicating possible involvement of Q‐type channels in verapamil‐induced inhibition. However, verapamil (30 μM) inhibited this release even in the absence of extracellular Ca2+, suggesting that Na+ rather than Q‐type Ca2+ channels are involved.

Taken together, our results suggest that verapamil can block P‐ and at higher concentrations possibly N‐ and Q‐type Ca2+ channels linked to [3H]‐DA release, whereas diltiazem appears to block P‐type Ca2+ channels only.

British Journal of Pharmacology (1999) 127, 576–582; doi:10.1038/sj.bjp.0702574
Role of potassium channels and nitric oxide in the relaxant effects elicited by β‐adrenoceptor agonists on hypoxic vasoconstriction in the isolated perfused lung of the rat
Authors: Dumas Jean‐Paul , Goirand Françoise , Bardou Marc , Dumas Monique , Rochette Luc , Advenier Charles , Giudicelli Jean‐François
Published: January 2005

The aims of this study were to compare, in the rat isolated perfused lung preparation, the antagonist effects of a nonselective β‐adrenoceptor agonist (isoprenaline), a selective β2‐adrenoceptor agonist (salbutamol) and a selective β3‐adrenoceptor agonist (SR 59104A) on the hypoxic pulmonary pressure response, and to investigate the role of K+ channels, endothelium derived relaxing factor and prostaglandins in these effects. K+ channels were inhibited by glibenclamide, charybdotoxin or apamin, NO synthase and cyclo‐oxygenase were inhibited by NG‐nitro‐L‐arginine methyl ester (L‐NAME) and indomethacin, respectively.

Hypoxic ventilation produced a significant increase in perfusion pressure (+65%, P<0.001) and L‐NAME significantly increased this response further (+123%, P<0.01). After apamin, L‐NAME, indomethacin, post‐hypoxic basal pressure did not return to baseline values (P<0.001).

Glibenclamide partially inhibited the relaxant effects of isoprenaline (P<0.05) and salbutamol (P<0.001) but not that of SR 59104A. In contrast, charybdotoxin and apamin partially inhibited the relaxant effects of SR 59104A (P=0.053 and <0.01, respectively) but did not modify the effects of isoprenaline and salbutamol. L‐NAME partially inhibited the dilator response of salbutamol (P<0.01) and SR 59104A (P<0.05) but not that of isoprenaline.

We conclude that (a) EDRF exerts a significant inhibition of the hypoxic pulmonary response, (b) SKCa channel activation, EDRF and prostaglandins contribute to the reversal of the hypoxic pressure response, (c) the vasodilation induced by isoprenaline is mediated in part by activation of KATP channels, that of salbutamol by activation of KATP channels and EDRF. In contrast, SR 59104A partly operates through BKCa, SKCa channels and EDRF activation, differing in this from the β1 and β2‐adrenoceptor agonists.

British Journal of Pharmacology (1999) 127, 421–428; doi:10.1038/sj.bjp.0702575
Interleukin‐6 production by activated human monocytic cells is enhanced by MK‐571, a specific inhibitor of the multi‐drug resistance protein‐1
Authors: Vellenga Edo , Tuyt Leonore , Wierenga Bart‐Jan , Müller Michael , Dokter Wim
Published: January 2005

The intracellular transport of leukotriene C4 (LTC4) in hematopoietic cells such as human monocytes is controlled by an ATP dependent carrier encoded by the multidrug resistance protein1 (MRP1) gene whose function can be blocked by the compound MK‐571. Since LTs play a major role in control of cytokine expression in monocytes, we questioned whether blocking of the MRP1 mediated function by MK‐571 might affect cytokine production.

MK‐571 strongly enhanced IL‐6 expression at mRNA and protein level in lipopolysaccharide (LPS) and interleukin‐1 (IL‐1) stimulated human monocytes giving rise to 2.0±0.4 (x±s.d.) and 5.7±3.5 fold induction of IL‐6 protein secretion. The increase in IL‐6 secretion was accompanied by an enhanced phosphorylation of p38 but not of c‐Jun‐N terminal kinase.

The involvement of the kinase signalling pathways was further analysed by using SB203580 and PD98059, specific inhibitors of the p38 and ERK1/2 signalling route. MK‐571 mediated upregulation of IL‐6 in the presence of IL‐1 was partially attenuated by SB203580 and PD98059. Electrophoretic mobility shift assays demonstrated that MK‐571 did not affect the IL‐1 induced DNA binding activity of Activator Protein‐1 and Nuclear Factor‐κB but rather enhanced the transactivational activity of an IL‐6 promoter construct. Finally it was shown that the MK‐571 mediated effects on IL‐6 secretion could not be inhibited by the LT synthesis inhibitor SB203347 or by the anti‐oxidant pyrrolidine dithiocarbamate (PDTC).

These results indicate that the membrane transporter MRP1 is involved in the regulation of IL‐6 expression in activated human peripheral blood monocytes.

British Journal of Pharmacology (1999) 127, 441–448; doi:10.1038/sj.bjp.0702577
Recovery of impaired K+ channels in mesenteric arteries from spontaneously hypertensive rats by prolonged treatment with cholecalciferol
Authors: Borges Antonio C R , Feres Teresa , Vianna Lucia M , Paiva Therezinha B
Published: January 2005

The mechanism responsible for blood pressure reduction in spontaneously hypertensive rats (SHR) after prolonged cholecalciferol treatment was studied. Two‐week treatment of SHR with 0.125 mg cholecalciferol kg−1 body weight per day orally caused significant reductions of systolic blood pressure and of the resting perfusion pressure of the mesenteric vascular bed at constant flow.

In addition, the treated animals presented a normalization of the maximum vasoconstriction response to noradrenaline and a reduction of the maximum effect of the adrenaline concentration‐response curves. This latter effect probably was due to recovery of the impaired Ca2+‐dependent K+ channels coupled to α2‐adrenoceptors since it was prevented by apamin.

The treatment with cholecalciferol also normalized the smooth muscle cell membrane potential of de‐endothelialized mesenteric arteries of SHR and their hyperpolarizing responses to α2‐adrenergic agonists, which were depressed in untreated SHR.

In mesenteric rings with endothelium, α2‐adrenergic agonists caused similar hyperpolarizing responses in the SHR and in normotensive Wistar (NWR) and Wistar Kyoto (WKY). In non cholecalciferol‐treated SHR the hyperpolarizing mediator involved in this effect was NO, while in NWR it was the endothelium‐derived hyperpolarizing factor (EDHF). After cholecalciferol treatment, the hyperpolarization induced by α2‐adrenergic agonists in SHR smooth muscle cells was mediated by EDHF, as in NWR.

Our results indicate that the hypotensive effect of cholecalciferol in the SHR is probably due to the normalization of vascular reactivity, by restoring the functioning of apamin‐ and ATP‐sensitive K+ channels located in the vascular smooth muscle cell membrane, which are impaired in the SHR.

British Journal of Pharmacology (1999) 127, 772–778; doi:10.1038/sj.bjp.0702581
Protease‐activated receptor‐2 turnover stimulated independently of receptor activation in porcine coronary endothelial cells
Authors: Hamilton Justin R , Chow Jean M , Cocks Thomas M
Published: January 2005

Protease‐activated receptors (PARs) are activated by an irreversible proteolytic mechanism which renders cleaved receptors unresponsive to subsequent challenges with activating enzymes. Non‐specific proteolysis of PARs downstream of the activation site also prevents subsequent enzymic activation. Therefore, we investigated the effects of non‐activating amino‐terminal proteolysis with the bacterial protease thermolysin on PAR‐mediated relaxation of porcine coronary artery ring preparations contracted with the thromboxane A2 mimetic U46619 (1–10 nM).

Treatment of contracted artery ring segments with thermolysin (0.01–1 u ml−1, 20 min) caused no response, but abolished endothelium‐dependent relaxations induced by the enzymic activators of PAR‐1 and PAR‐2, thrombin (0.01–0.3 u ml−1) and trypsin (0.003–0.1 u ml−1) respectively. The same treatment, however, did not affect similar responses to the proteolysis‐independent PAR‐1 and PAR‐2 activating peptides, SFLLRN‐NH2 and SLIGRL‐NH2 respectively (0.1–10 μM).

The inhibition of responsiveness to trypsin after thermolysin treatment recovered in a time‐dependent manner, with maximal recovery (77.3±8.0% of time controls) occurring 150 min after thermolysin treatment. No recovery of responsiveness to thrombin after thermolysin treatment was observed within this time, however, the thrombin response returned to control levels after 20 h.

The recovery of responsiveness to trypsin was inhibited by the translation inhibitor cycloheximide (100 μM; 17.3±4.7%) and the protein trafficking inhibitor brefeldin A (10 μM; 12.1±4.8%) but was unaffected by the transcription inhibitor actinomycin D (2 μM; 65.1±3.6%), which did, however, abolish upregulation of B1‐kinin receptors in this preparation.

In conclusion, our findings indicate that activation‐independent amino‐terminal proteolysis of PARs stimulates selective recovery of endothelial cell PAR‐2 responsiveness, which appears to be regulated by translation. Such a novel mechanism for the maintenance of responsiveness to enzymic PAR‐2 activators may imply that these receptors play important roles in vascular homeostasis.

British Journal of Pharmacology (1999) 127, 617–622; doi:10.1038/sj.bjp.0702583
Bidirectional effects of the neuroactive steroid tetrahydrodeoxycorticosterone on GABA‐activated Cl− currents in cultured rat hypothalamic neurons
Authors: Wetzel C H R , Vedder H , Holsboer F , Zieglgänsberger W , Deisz R A
Published: January 2005

The nongenomic effects of tetrahydrodeoxycorticosterone (THDOC; 5‐alpha‐pregnane‐3‐alpha, 21‐diol‐20‐one) were studied in cultured hypothalamic neurons of the rat.

The effects of THDOC (10 nM–1 μM) on responses to different concentrations of exogenously applied GABA and on spontaneous inhibitory postsynaptic currents (IPSCs) were measured with whole‐cell voltage clamp recordings.

Application of GABA induced inward currents with dose‐dependently increasing amplitudes (up to 3.9 nA at a holding potential of −20 mV). High doses of THDOC (100 nM–1 μM) induced small inward currents on its own (14±3 and 24±3 pA, respectively).

Simultaneous application of 10 μM GABA with 100 nM or 1 μM THDOC increased current amplitudes by 125 and 128%, respectively. At 10 nM THDOC exerted no consistent effects on GABA currents.

Responses to 1 μM of GABA were modulated in a bidirectional manner by different doses of THDOC: 10 nM THDOC reduced the amplitude of GABA responses to 80% (P=0.018, n=15), whereas 100 nM and 1 μM THDOC enhanced the GABA response to 115 and 180% (P=0.0007, n=15), respectively.

The time constant of decay of spontaneous inhibitory postsynaptic currents (IPSCs) was reversibly increased from 91±10 to 314±34 ms (n=3) by the application of THDOC (1 μM). The amplitudes of the IPSCs were not affected by THDOC.

These data indicate that THDOC modulates GABA responses of hypothalamic neurons in a bidirectional manner, resulting in a complex tuning of neuronal excitability in the hypothalamus.

British Journal of Pharmacology (1999) 127, 863–868; doi:10.1038/sj.bjp.0702597
The effect of deep pore mutations on the action of phenylalkylamines on the Kv1.3 potassium channel
Authors: Rauer Heiko , Grissmer Stephan
Published: January 2005

We investigated the action of the phenylalkylamines verapamil and N‐methyl‐verapamil on the Kv1.3 potassium channel using the whole‐cell configuration of the patch‐clamp technique. Our goal was to identify their binding as a prerequisite for using the phenylalkylamines as small, well‐defined molecular probes, not only to expand the structural findings made with peptide toxins or by crystallization, but also to use them as lead compounds for the generation of more potent and therefore more specific K+ channel modulators.

Competition experiments with charybdotoxin, known to interact with external residues of Kv1.3, showed no interaction with verapamil. The internal application of quarternary N‐methyl‐verapamil in combination with verapamil suggested competition for the same internal binding site.

Verapamil affinity was decreased 6 fold by a mutation (M395V) in a region of the internal pore which forms part of the internal tetraethylammonium (TEA+) binding site, although mutations at neighbouring residues (T396 and T397) were without effect.

Modification of C‐type inactivation by mutations in the internal pore suggest that this region participates in the inactivation process.

The action of phenylalkylamines and local anaesthetics on L‐type Ca2+ channels and Na+ channels, respectively, and verapamil on Kv1.3 indicate very similar blocking mechanisms. This might allow the use of these compounds as molecular probes to map the internal vestibule of all three channel types.

British Journal of Pharmacology (1999) 127, 1065–1074; doi:10.1038/sj.bjp.0702599
Halothane enhances exocytosis of [3H]‐acetylcholine without increasing calcium influx in rat brain cortical slices
Authors: Gomez R S , Prado M A M , Carazza F , Gomez M V
Published: January 2005

The effect of halothane on the release of [3H]‐acetylcholine ([3H]‐ACh) in rat brain cortical slices was investigated.

Halothane (0.018 mM) did not significantly affect the basal and the electrical field stimulation induced release of [3H]‐ACh. However, halothane (0.063 mM) significantly increased the basal release of [3H]‐ACh and this effect was additive with the electrical field stimulation induced release of [3H]‐ACh.

The release of [3H]‐ACh induced by 0.063 mM halothane was independent of the extracellular sodium and calcium ion concentration and was decreased by tetracaine, an inhibitor of Ca2+‐release from intracellular stores or dantrolene, an inhibitor of Ca2+‐release from ryanodine‐sensitive stores.

Using 2‐(4‐phenylpiperidino)‐cyclohexanol (vesamicol), a drug that blocks the storage of ACh in synaptic vesicles, we investigated whether exocytosis of this neurotransmitter is involved in the effect of halothane. Vesamicol significantly decreased the release of [3H]‐ACh evoked by halothane.

It is suggested that halothane may cause a Ca2+ release from intracellular stores that increases [3H]‐ACh exocytosis in rat brain cortical slices.

British Journal of Pharmacology (1999) 127, 679–684; doi:10.1038/sj.bjp.0702603
Microphysiometric analysis of human α1a‐adrenoceptor expressed in Chinese hamster ovary cells
Authors: Taniguchi Takanobu , Inagaki Rika , Murata Satoshi , Akiba Isamu , Muramatsu Ikunobu
Published: January 2005

The human recombinant α1a‐adrenoceptor (AR) has been stably expressed in Chinese hamster ovary cells. Four stable clones, aH4, aH5, aH6 and aH7, expressing 30, 370, 940 and 2900 fmol AR mg−1 protein, respectively, have been employed to characterize this AR subtype using radioligand binding and microphysiometry to measure extracellular acidification rates.

Noradrenaline (NA) gave concentration‐dependent responses in microphysiometry with increasing extracellular acidification rates. The potency of NA increased as the receptor density increased; pEC50 values of NA for the clones aH4, aH5, aH6 and aH7 were 6.9, 7.5, 7.8 and 8.1, respectively. This increase of potency according to receptor density indicates the presence of spare receptor for NA. Methoxamine, phenylephrine, oxymetazoline and clonidine also gave concentration‐dependent responses with various intrinsic activities.

Antagonists shifted concentration‐response curves for NA rightward in a concentration‐dependent manner. Schild analysis revealed that the affinity profile of this AR subtype to antagonists in the clone aH7 had a typical pattern for the α1a‐AR; high affinity for prazosin and WB 4101, and low affinity for BMY7378 (pA2=9.5, 9.8 and 7.3, respectively). This profile is similar in the case of the clone aH4. These affinities were in good agreement with those obtained in binding experiments.

These results have demonstrated that (1) classical receptor theory can be applied in microphysiometry, and (2) microphysiometry is a useful tool to investigate the pharmacological characterization of α1a‐AR.

British Journal of Pharmacology (1999) 127, 962–968; doi:10.1038/sj.bjp.0702609
A single amino acid confers barbiturate sensitivity upon the GABA ρ1 receptor
Authors: Belelli Delia , Pau Davide , Cabras Giulia , Peters John A , Lambert Jeremy J
Published: January 2005

Many structurally diverse general anaesthetics enhance inhibitory neurotransmission in the central nervous system by interacting with the GABAA receptor. By contrast, GABA receptors composed of the ρ1 subunit are anaesthetic‐insensitive. Here, we demonstrate that both δ‐hexachlorocyclohexane (δ‐HCH; 1–100 μM), a positive allosteric modulator of the GABAA receptor, and the anaesthetic pentobarbitone (10–600 μM) have no effect on GABA‐evoked currents mediated by wild‐type ρ1 recombinant receptors (expressed in Xenopus laevis oocytes). By contrast, these agents produce up to a 10 fold enhancement of GABA responses transduced by a ρ1 receptor in which a transmembrane located isoleucine residue is replaced by serine. However, not all general anaesthetics were similarly influenced by this mutation, because propofol and 5β‐pregnan‐3α‐ol‐20‐one (5β3α) remained ineffective. These data are discussed in relation to the specificity of general anaesthetic action.

British Journal of Pharmacology (1999) 127, 601–604; doi:10.1038/sj.bjp.0702611
Involvement of serotonin and calcium channels in the intestinal fluid secretion evoked by bile salt and cholera toxin
Authors: Peregrin Attila Timar , Ahlman Håkan , Jodal Mats , Lundgren Ove
Published: January 2005

The enteric nervous system (ENS) is activated when exposing the intestinal mucosa to cholera toxin or certain bile salts. Cholera toxin stimulates ENS, at least in part, by the release of 5‐hydroxytryptamine (5‐HT) from the enterochromaffin cells. Calcium channel blockers of the L‐type markedly attenuate the fluid secretion and the luminal release of 5‐HT caused by cholera toxin.

The objective of the present study was to elucidate if sodium deoxycholate activated ENS in a similar manner as cholera toxin. Furthermore, the effect of several calcium channel blockers was tested on the fluid secretion caused by cholera toxin or bile salt.

Sodium deoxycholate (4 mM) caused a release of 5‐HT into the intestinal lumen, which was inhibited by calcium channel blockade. Granisetron, a 5‐HT3 receptor blocker, partly inhibited the fluid secretion caused by bile salt.

The effects of nifedipine, felodipine, R‐felodipine, H186/86 (t‐butyl analogue of felodipine) on the fluid secretion caused by cholera toxin or sodium deoxycholate were studied. Both secretory states were markedly attenuated in a dose dependent manner by all calcium channel blockers tested regardless of their effects on arterial pressure.

It is concluded that both cholera toxin and bile salt activate ENS, at least in part, via a release of 5‐HT from the enterochromaffin cells. The antisecretory effect calcium channel blockers is partly explained by an inhibition of this release of 5‐HT.

British Journal of Pharmacology (1999) 127, 887–894; doi:10.1038/sj.bjp.0702615
Interactions between cholecystokinin and opioids in the isolated guinea‐pig ileum
Authors: Romanelli Luca , Amico Maria Carmela , Mattioli Francesca , Morrone Luigi Antonio , Valeri Pacifico
Published: January 2005

Although cholecystokinin octapeptide sulphate (CCK‐8) activates the opioid system of isolated guinea‐pig ileum (GPI) whether it activates the μ‐ or κ‐system, or both, remains unclear. Neither is it known whether CCK‐8 influences the withdrawal responses in GPI preparations briefly exposed to opioid agonists. This study was designed to clarify whether CCK‐8 activates μ‐ or κ‐opioid systems or both; and to investigate its effect on the withdrawal contractures in GPI exposed to μ‐ or κ‐agonists and on the development of tolerance to the withdrawal response.

In GPI exposed to CCK‐8, the selective κ‐antagonist nor‐binaltorphimine elicited contractile responses that were concentration‐related to CCK‐8 whereas the selective μ‐antagonist cyprodime did not.

In GPI preparations briefly exposed to the selective μ‐agonist, dermorphin, or the selective κ‐agonist, U‐50, 488H, and then challenged with naloxone, CCK‐8 strongly enhanced the withdrawal contractures.

During repeated opioid agonist/CCK‐8/opioid antagonist tests tolerance to opioid‐induced withdrawal responses did not develop.

These results show that CCK‐8 preferentially activates the GPI κ‐opioid system and antagonizes the mechanism(s) that control the expression of acute dependence in the GPI.

British Journal of Pharmacology (1999) 127, 909–918; doi:10.1038/sj.bjp.0702621
Role of superoxide dismutase in in vivo and in vitro nitrate tolerance
Authors: Münzel Thomas , Hink Ulrich , Yigit Hussein , Macharzina Roland , Harrison David G , Mülsch Alexander
Published: January 2005

We assessed whether pharmacological inhibition of CuZn‐superoxide dismutase (SOD) mimics the molecular mechanism of either in vitro or in vivo nitrovasodilator tolerance.

In endothelium‐intact aortic rings from in vivo tolerant rabbits the GTN‐ and acetylcholine (ACh)‐induced maximal relaxation was attenuated by 36 and 23%, respectively. In vitro treatment of control rings with GTN (1 h 10 μM) similarly attenuated the vasorelaxant response to GTN, but not to ACh.

Formation of superoxide radicals (•O2−) in endothelium‐intact rings (lucigenin‐chemiluminescence) increased 2.5 fold in in vivo tolerance, but significantly decreased in in vitro tolerance. The membrane associated NADH oxidase activity was increased 2.5 fold in homogenates of in vivo tolerant aortae, but was not changed in in vitro tolerant aorta.

Conversely, SOD activity and protein expression was halved in in vivo tolerance, but SOD activity was not altered by in vitro tolerance. The •O2− scavenger tiron (10 mM) effectively restored the vasorelaxant response to GTN in in vivo tolerant aortic rings, but not the reduced response to GTN in in vitro tolerant rings.

Pretreatment (1 h) of vessels with diethyldithiocarbamate (DETC; 10 mM) attenuated vasorelaxant responses to GTN and ACh, increased vascular •O2− production, and inhibited SOD activity in vessel homogenates to a similar degree as observed in in vivo tolerance. DETC‐treatment of in vivo‐tolerant vessels induced an additional increase in •O2− production.

Increased •O2− production in in vivo nitrate tolerant aorta is associated with activation of vascular NADH oxidase and inactivation of CuZnSOD. Therefore, in vivo tolerance can be mimicked by in vitro inhibition of CuZnSOD, but not by in vitro exposure to GTN, which does not affect vascular •O2− production, NADH oxidase and CuZnSOD.

British Journal of Pharmacology (1999) 127, 1224–1230; doi:10.1038/sj.bjp.0702622
Effects of endothelial impairment by saponin on the responses to vasodilators and nitrergic nerve stimulation in isolated canine corpus cavernosum
Authors: Okamura Tomio , Ayajiki Kazuhide , Fujioka Hideyuki , Toda Megumi , Fujimiya Mineko , Toda Noboru
Published: January 2005

Responsiveness to EDRF‐releasing substances and inhibitory nerve stimulation of canine isolated penile corpus cavernosum with and without saponin treatment were investigated.

Histological studies demonstrated that saponin did not detach endothelial cells from underlying tissues, but induced degenerative changes in the endothelial cells selectively.

In the cavernous strips contracted with phenylephrine, addition of acetylcholine, sodium nitroprusside, ATP and Ca2+ ionophore A23187 induced relaxations, but substance P and bradykinin did not change the muscle tone.

Acetylcholine‐induced relaxation was significantly attenuated but not abolished by NG‐nitro‐L‐arginine (L‐NOARG). L‐arginine restored the response inhibited by L‐NOARG. The L‐NOARG resistant relaxation was not influenced by 1H[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ) but was suppressed in the strips contracted with K+. Treatment with saponin abolished the relaxation elicited by acetylcholine and A23187 but did not influence the response to nitroprusside and ATP. The ATP‐induced relaxation was attenuated by aminophylline.

Transmural electrical stimulation at 2–20 Hz produced endothelium‐independent relaxations which were abolished by tetrodotoxin and L‐NOARG but unaffected by treatment with saponin. In saponin‐treated cavernous strips, the neurogenic relaxation was not affected by acetylcholine, physostigmine, atropine and vasoactive intestinal peptide (VIP) but was abolished by ODQ.

It is concluded that acetylcholine‐induced relaxations are endothelium‐dependent and mediated partly by NO and also by other substances from the endothelium. The endothelium‐independent relaxation to ATP is likely to be mediated by P1 purinoceptors. The function of nitrergic nerve does not seem to be prejunctionally modulated by acetylcholine and VIP.

British Journal of Pharmacology (1999) 127, 802–808; doi:10.1038/sj.bjp.0702623