bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional analysis of desensitization of the β‐adrenoceptor signalling pathway in rat cardiac tissues following chronic isoprenaline infusion
Authors: McMartin L , Summers R J
Published: January 2005

This study examined β‐adrenoceptor signalling in cardiac tissues following infusion of isoprenaline (400 μg kg−1 h−1) or vehicle to rats for 14 days.

Isoprenaline infusion caused marked hypertrophy of atria and ventricles and reduced the resting rate of spontaneously beating right atria and the basal force of left atrial contraction.

In spontaneously beating right atria, concentration‐response curves to isoprenaline and forskolin were shifted 7.9 and 3.2 fold to the right following treatment whereas responses to the cyclic AMP analogue 5,6‐dichloro‐1‐ß‐D‐ribofuranosylbenzimidazole‐3′, 5′‐cyclic monophosphorothioate were unchanged.

In electrically driven left atria, concentration‐response curves to isoprenaline and forskolin were shifted 4 fold to the right and maximum responses reduced. Responses to dibutyryl cyclic AMP were shifted 3.2 fold to the right but those to Ca2+ were unchanged.

Inotropic responses of left and right ventricular papillary muscles to isoprenaline were abolished and markedly reduced respectively by isoprenaline treatment. Responses to forskolin were shifted 5 fold to the right. Responses to dibutyryl cyclic AMP were shifted to the right 3.2 and 2 fold in left and right ventricular papillary muscles. Responses to isobutyl methyl xanthine were shifted to the right 15.8 and 6.3 fold in left and right papillary muscles whereas those to Ca2+ were not significantly altered.

This study indicates differences in β‐adrenoceptor desensitization in different regions of the heart following chronic infusion of isoprenaline. Chronotropic responses showed impaired signalling between the receptor and adenylate cyclase whereas inotropic responses exhibited additional desensitization at the level of cyclic AMP dependent protein kinase.

British Journal of Pharmacology (1999) 127, 1012–1020; doi:10.1038/sj.bjp.0702618
Induction of prostanoid, nitric oxide, and cytokine formation in rat bone marrow derived macrophages by activin A
Authors: Nüsing Rolf M , Barsig Johannes
Published: January 2005

In this study we describe that activin A, a transforming growth factor (TGF) β‐like polypeptide affects the expression of inflammatory response genes and their products.

In rat bone marrow derived macrophages 15 nM activin A caused the stimulation of prostaglandin (PG) E2 and thromboxane (TX) A2 formation, production of nitrite as a marker for nitric oxide (NO) and the release of the cytokines tumour necrosis factor (TNF) α and interleukin (IL) ‐1β. As shown by mRNA analysis induction of cyclo‐oxygenase‐2 and inducible nitric oxide synthase by activin A gave rise to the enhanced release of prostanoids and NO.

Costimulation of bone marrow derived macrophages with 15 nM activin A and 100 nM 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) potentiated the synthesis of prostanoids in a synergistic manner. With respect to NO formation the effect of activin A and TPA was additive.

In contrast to the nitrite production activin A induced PGE2 synthesis was susceptible to tyrosine kinase inhibition by genistein and tyrphostin 46 (IC50 was 10 and 20 μM, respectively). This observed inhibition was caused by the selective suppression of activin A induced cyclo‐oxygenase‐2 mRNA expression. Further, the release of TNFα in the presence of activin A was potentiated by tyrosine kinase inhibition.

In summary, we report that activin A exerts proinflammatory activity which results in the formation of prostanoids, NO and cytokines in rat bone marrow derived macrophages. Tyrosine kinase dependent and independent signalling pathways are involved leading to the increased synthesis of these metabolites. Based upon these results, we speculate that activin A may be considered as a possible component of inflammatory processes affecting at least the haematopoietic system.

British Journal of Pharmacology (1999) 127, 919–926; doi:10.1038/sj.bjp.0702626
The structural requirements for phorbol esters to enhance serotonin and acetylcholine release from rat brain cortex
Authors: Iannazzo L , Kotsonis P , Majewski H
Published: January 2005

The effects of various phorbol‐based protein kinase C (PKC) activators on the electrical stimulation‐induced (S‐I) release of serotonin and acetylcholine was studied in rat brain cortical slices pre‐incubated with [3H]‐serotonin or [3H]‐choline to investigate possible structure‐activity relationships.

4β‐Phorbol 12,13‐dibutyrate (4βPDB, 0.1–3.0 μM), enhanced S‐I release of serotonin in a concentration‐dependent manner whereas the structurally related inactive isomer 4α‐phorbol 12, 13‐dibutyrate (4αPDB) and phorbol 13‐acetate (PA) were without effect. Another group of phorbol esters containing a common 13‐ester substituent (phorbol 12,13‐diacetate, PDA; phorbol 12‐myristate 13‐acetate, PMA; phorbol 12‐methylaminobenzoate 13‐acetate, PMBA) also enhanced S‐I serotonin release with PMA being least potent.

The deoxyphorbol monoesters, 12‐deoxyphorbol 13‐acetate (dPA), 12‐deoxyphorbol 13‐angelate (dPAng), 12‐deoxyphorbol 13‐phenylacetate (dPPhen) and 12‐deoxyphorbol 13‐isobutyrate (dPiB) enhanced S‐I serotonin release but 12‐deoxyphorbol 13‐tetradecanoate (dPT) was without effect. The 20‐acetate derivatives of dPPhen and dPAng were less effective in enhancing S‐I serotonin release compared to the parent compounds.

With acetylcholine release all phorbol esters tested had a far lesser effect when compared to their facilitatory action on serotonin release with only 4βPDB, PDA, dPA, dPAng and dPiB having significant effects.

The effects of the phorbol esters on serotonin release were not correlated with their reported in vitro affinity and isozyme selectivity for PKC. A comparison across three transmitter systems (noradrenaline, dopamine, serotonin) suggests basic similarities in the structural requirements of phorbol esters to enhance transmitter release with short chain substituted mono‐ and diesters of phorbol being more potent facilitators of release than the long chain esters. Some compounds notably PDA, PMBA, dPPhen, dPPhenA had different potencies across noradrenaline, dopamine and serotonin.

British Journal of Pharmacology (1999) 127, 1177–1189; doi:10.1038/sj.bjp.0702627
Role of tachykinin NK2‐receptor activation in the allergen‐induced late asthmatic reaction, airway hyperreactivity and airway inflammatory cell influx in conscious, unrestrained guinea‐pigs
Authors: Schuiling Martin , Zuidhof Annet B , Meurs Herman , Zaagsma Johan
Published: January 2005

In a guinea‐pig model of allergic asthma, we investigated the involvement of the tachykinin NK2 receptors in allergen‐induced early (EAR) and late (LAR) asthmatic reactions, airway hyperreactivity (AHR) after these reactions and inflammatory cell influx in the airways, using the selective non‐peptide NK2 receptor antagonist SR48968.

On two different occasions, separated by a 1 week interval, ovalbumin (OA)‐sensitized guinea‐pigs inhaled either vehicle (3 min) or SR48968 (100 nM, 3 min) at 30 min before as well as at 5.5 h after OA provocation (between the EAR and LAR) in a random crossover design.

SR48968 had no significant effect on the EAR, but significantly attenuated the LAR by 44.2±16.4% (P<0.05) compared to saline control.

The NK2 receptor antagonist did not affect the OA‐induced AHR to histamine after the EAR at 5 h after OA challenge (3.59±0.59 fold increase in histamine reactivity vs 3.79±0.61 fold increase in the controls, NS), but significantly reduced the AHR after the LAR at 23 h after OA challenge (1.59±0.24 fold increase vs 1.93±0.15 fold increase, respectively, P<0.05).

Bronchoalveolar lavage studies performed at 25 h after the second OA provocation showed that SR48968 significantly inhibited the allergen‐induced infiltration of neutrophils (P<0.05) and lymphocytes (P<0.01) in the airways.

These results indicate that NK2 receptor activation is importantly involved in the development of the allergen‐induced late (but not early) asthmatic reaction and late (but not early) AHR to histamine, and that NK2 receptor‐mediated infiltration of neutrophils and lymphocytes in the airways may contribute to these effects.

British Journal of Pharmacology (1999) 127, 1030–1038; doi:10.1038/sj.bjp.0702628
Role of N‐type calcium channels in autonomic neurotransmission in guineapig isolated left atria
Authors: Serone Adrian P , Angus James A
Published: January 2005

Calcium entry via neuronal calcium channels is essential for the process of neurotransmission. We investigated the calcium channel subtypes involved in the operation of cardiac autonomic neurotransmission by examining the effects of selective calcium channel blockers on the inotropic responses to electrical field stimulation (EFS) of driven (4 Hz) guineapig isolated left atria. In this tissue, a previous report (Hong & Chang, 1995) found no evidence for N‐type channels involved in the vagal negative inotropic response and only weak involvement in sympathetic responses.

The effects of cumulative concentrations of the selective N‐type calcium channel blocker, ω‐conotoxin GVIA (GVIA; 0.1–10 nM) and the nonselective N‐, P/Q‐type calcium channel blocker, ω‐conotoxin MVIIC (MVIIC; 0.01–10 nM) were examined on the positive (with atropine, 1 μM present) and negative (with propranolol, 1 μM and clonidine, 1 μM present) inotropic responses to EFS (eight trains, each train four pulses per punctate stimulus).

GVIA caused complete inhibition of both cardiac vagal and sympathetic inotropic responses to EFS. GVIA was equipotent at inhibiting positive (pIC50 9.29±0.08) and negative (pIC50 9.13±0.17) inotropic responses. MVIIC also mediated complete inhibition of inotropic responses to EFS and was 160 and 85 fold less potent than GVIA at inhibiting positive (pIC50 7.08±0.10) and negative (pIC50 7.20±0.14) inotropic responses, respectively. MVIIC was also equipotent at inhibiting both sympathetic and vagal responses.

Our data demonstrates that N‐type calcium channels account for all the calcium current required for cardiac autonomic neurotransmission in the guinea‐pig isolated left atrium.

British Journal of Pharmacology (1999) 127, 927–934; doi:10.1038/sj.bjp.0702629
Naloxone blocks endomorphin‐1 but not endomorphin‐2 induced inhibition of tachykinergic contractions of guinea‐pig isolated bronchus
Authors: Fischer Axel , Undem Bradley J
Published: January 2005

The recently identified endogenous agonists on the μ‐opioid‐receptor (μOR), endomorphin‐1 (EM‐1) and endomorphin‐2 (EM‐2), induce a concentration dependent inhibition of electrical field stimulation (EFS)‐induced tachykinin‐mediated contractions of the guinea‐pig bronchus (ED50s <10 nM for both compounds). Surprisingly, only endomorphin‐1 effects could be blocked by naloxone (10 μM), whereas endomorphin‐2 effects were not affected by specific antagonists for the μ‐, κ‐, and δ‐opioid‐receptor.

British Journal of Pharmacology (1999) 127, 605–608; doi:10.1038/sj.bjp.0702630
RS‐127445: a selective, high affinity, orally bioavailable 5‐HT2B receptor antagonist
Authors: Bonhaus Douglas W , Flippin Lee A , Greenhouse Robert J , Jaime Saul , Rocha Cindy , Dawson Mark , Van Natta Kristine , Chang L K , Pulido‐Rios Tess , Webber Andrea , Leung Edward , Eglen Richard M , Martin Graeme R
Published: January 2005

Efforts to define precisely the role of 5‐HT2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this deficiency, we developed a series of naphthylpyrimidines as potentially useful 5‐HT2B receptor antagonists.

RS‐127445 (2‐amino‐4‐(4‐fluoronaphth‐1‐yl)‐6‐isopropylpyrimidine) was found to have nanomolar affinity for the 5‐HT2B receptor (pKi=9.5±0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites.

In cells expressing human recombinant 5‐HT2B receptors, RS‐127445 potently antagonized 5‐HT‐evoked formation of inositol phosphates (pKB=9.5±0.1) and 5‐HT‐evoked increases in intracellular calcium (pIC50=10.4±0.1). RS‐127445 also blocked 5‐HT‐evoked contraction of rat isolated stomach fundus (pA2=9.5±1.1) and (±)α‐methyl‐5‐HT‐mediated relaxation of the rat jugular vein (pA2=9.9±0.3). RS‐127445 had no detectable intrinsic activity in these assays.

In rats, the fraction of RS‐127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS‐127445 (5 mg kg−1) produced plasma concentrations predicted to fully saturate accessible 5‐HT2B receptors for at least 4 h.

In conclusion, RS‐127445 is a selective, high affinity 5‐HT2B receptor antagonist suitable for use in vivo. The therapeutic potential of this molecule is being further evaluated.

British Journal of Pharmacology (1999) 127, 1075–1082; doi:10.1038/sj.bjp.0702632
Characterization of the inflammatory response to proteinase‐activated receptor‐2 (PAR2)‐activating peptides in the rat paw
Authors: Vergnolle Nathalie , Hollenberg Morley D , Sharkey Keith A , Wallace John L
Published: January 2005

In the present study, we have observed the development of an inflammatory reaction in the rat hindpaw, following the injection of specific agonists of PAR2 (two PAR2 activating peptides). This inflammation was characterized by oedema and granulocyte infiltration.

Two selective PAR2 activating peptides, SLGRL‐NH2 and trans‐cinnamoyl‐LIGRLO‐NH2 induced significant oedema in the rat hindpaw from 1–6 h following subplantar injection. Six hours after the PAR2‐activating peptide injection, the paw tissues showed a complete disruption of tissue architecture along with an inflammatory cell infiltrate.

In the inflamed paw, PAR2‐immunoreactivity was expressed on endothelial cells as well as on the infiltrating inflammatory cells.

The oedema induced by the injection of the two PAR2 activating peptides was slightly reduced in rats pre‐treated with compound 48/80, but was not modified by pre‐treatment of rats with cromolyn, a mast cell stabilizer. Pre‐treatment of rats with a cyclo‐oxygenase inhibitor (indomethacin) or a nitric oxide synthase inhibitor (L‐Nω‐nitro‐L‐arginine methyl ester) had no effect on the oedema induced by the PAR2‐activating peptides.

These results demonstrate that the administration of PAR2‐activating peptides into the rat paw induced an acute inflammatory response characterized by a persistent oedema (at least 6 h) and granulocyte infiltration. The PAR2‐induced inflammatory response occurred through a mechanism largely independent of mast cell activation, and of the production of prostanoids and nitric oxide.

British Journal of Pharmacology (1999) 127, 1083–1090; doi:10.1038/sj.bjp.0702634
The antiplatelet activity of PMC, a potent α‐tocopherol analogue, is mediated through inhibition of cyclo‐oxygenase
Authors: Sheu Joen R , Lee Cheng R , Lin Chang C , Kan Ya C , Lin Chien H , Hung Wei C , Lee Yen M , Yen Mao H
Published: January 2005

PMC, a potent α‐tocopherol derivative, dose‐dependently (5–25 μM) inhibited the ATP‐release reaction and platelet aggregation in washed human platelets stimulated by agonists (collagen and ADP).

PMC also dose‐dependently inhibited the intracellular Ca2+ mobilization, whereas it did not inhibit phosphoinositide breakdown in human platelets stimulated by collagen.

PMC (10 and 25 μM) significantly inhibited collagen‐stimulated thromboxane A2 (TxA2) formation in human platelets. On the other hand, PMC (25 and 100 μM) did not increase the formation of cyclic AMP or cyclic GMP in platelets. Moreover, PMC (25, 100, and 200 μM) did not affect the thromboxane synthetase activity of aspirin‐treated platelet microsomes.

PMC (10 and 25 μM) markedly inhibited the exogenous arachidonic acid (100 μM)‐induced prostaglandin E2 (PGE2) formation in the presence of imidazole (600 μM) in washed human platelets, indicating that PMC inhibits cyclo‐oxygenase activity.

We conclude that PMC may exert its anti‐platelet aggregation activity by inhibiting cyclo‐oxygenase activity, which leads to reduced prostaglandin formation; this, in turn, is followed by a reduction of TxA2 formation, and finally inhibition of [Ca2+]i mobilization and ATP‐release.

British Journal of Pharmacology (1999) 127, 1206–1212; doi:10.1038/sj.bjp.0702637
Blockade by N‐3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells
Authors: Singleton C B , Valenzuela S M , Walker B D , Tie H , Wyse K R , Bursill J A , Qiu M R , Breit S N , Campbell T J
Published: January 2005

The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias.

Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n‐3 polyunsaturated fatty acids (n‐3 PUFA) present in fish oils.

We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito.

The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8±0.2 and 5.3±0.4 ms for activation and 20.0±1.2 and 96.6±6.7 ms for inactivation at +45 mV at 23°C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation=−6.7±2.5 mV, V1/2 of steady state inactivation=−51.3±0.2 mV at 23°C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37°C) compared to room temperature (23°C).

The n‐3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 μmol L−1. Blockade of the transient outward current may be an important mechanism by which n‐3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.

British Journal of Pharmacology (1999) 127, 941–948; doi:10.1038/sj.bjp.0702638
Dual mode of stimulation by the β‐carboline ZK 91085 of recombinant GABAA receptor currents: molecular determinants affecting its action
Authors: Thomet Urs , Baur Roland , Scholze Petra , Sieghart Werner , Sigel Erwin
Published: January 2005

In electrophysiological measurements the β‐carboline ethyl 6‐benzyloxy‐β‐carboline‐3‐carboxylate (ZK 91085) acts as a positive allosteric modulator on rat recombinant α1β2γ2 GABAA receptors and binds with high affinity (IC50=1.5 nM) to the [3H]‐flunitrazepam site. Flumazenil was able to partially counteract the current modulation. These observations indicate an action of ZK 91085 at the benzodiazepine binding site.

At the dual subunit combination α1β2, which lacks the γ subunit required for benzodiazepine modulation, we still observed a potentiation of GABA currents. Thus ZK 91085 acts via an additional site on the channel. At the subunit combination α1β1, ZK 91085 potentiation is strongly reduced as compared to α1β2. In binding studies, ZK 91085 was able to decrease [35S]‐TBPS binding in α1β2γ2 and α1β2 but not in α1β1. This selectivity of ZK 91085 for receptors containing the β2 isoform over those containing the β1 isoform is reminiscent of the action of loreclezole.

To identify amino acid residues important for the second type of modulation, we functionally compared wild type α1β2 and mutant receptors for stimulation by ZK 91085. The mutation β2N265S, that abolishes loreclezole effects, also abolishes ZK 91085 stimulation. The mutation β2Y62L increased stimulation by ZK 91085 3–4 fold, locating an influencing entity of the second type of action of ZK 91085 at an α/β subunit interface.

Structural intermediates of ZK 91085 and the β‐carboline abecarnil, the latter of which only slightly potentiated GABA currents in α1β2, were analysed to determine structural requirements for modulation.

ZK 91085 thus allosterically stimulates the GABAA receptor through two sites of action: the benzodiazepine site and the loreclezole site in contrast to classical β‐carbolines, that confer negative allosteric modulation through the benzodiazepine site.

British Journal of Pharmacology (1999) 127, 1231–1239; doi:10.1038/sj.bjp.0702639
Treatment with inverse agonists enhances baseline atrial contractility in transgenic mice with chronic beta2‐adrenoceptor activation
Authors: Nagaraja Savitha , Iyer Srinivas , Liu Xiushi , Eichberg Joseph , Bond Richard A
Published: January 2005

In this study, we investigate whether chronic treatment with β‐adrenoceptor (βAR) ligands with inverse agonist activity enhances myocardial β2AR‐mediated atrial tension more than neutral antagonists in transgenic mice (TG35). These mice exhibit chronic adrenoceptor activation because they possess a greater number of constitutively active receptors than wild type mice due to cardiac‐specific overexpression of human β2ARs. TG35 and wild type mice were chronically treated for 90 h with three inverse agonists, ICI‐118,551, propranolol, and carvedilol, and one neutral antagonist, alprenolol. After 96 h, we compared the basal and isoprenaline‐stimulated (10 μM) increase in atrial tension in treated or untreated TG35 mice and wild type mice. In parallel, to determine the effect of chronic βAR ligand treatment on the amounts of G protein receptor kinase‐2 (GRK‐2) and G proteins, we performed Western blotting on myocardial cytosolic and membrane proteins.

Atria from the TG35 mice treated with inverse agonists showed increases in the baseline tension compared to those from alprenolol/vehicle‐treated mice. ICI‐118,551 and propranolol treatment restored the elevated myocardial G‐inhibitory protein (Giα) levels to that of wild type. Also, treatment with inverse agonists upregulated G‐stimulatory protein (Gsα) levels and GRK2 above those levels in vehicle‐treated TG35 or wild type mice. The increased baseline atrial tension was reversed by the addition of ICI‐118,551.

Overall, our data suggests that inverse agonists enhance baseline atrial tension more than neutral antagonists. Based on this, we propose that upregulation of the active conformation of the β2ARs, Gsα protein and restoration of Giα as three possible mechanisms to explain this enhanced receptor activity.

Therefore, the favourable effects of some ligands used in pathological conditions involving chronic adrenoceptor activation may be due to the inverse agonist activity of the ligand.

British Journal of Pharmacology (1999) 127, 1099–1104; doi:10.1038/sj.bjp.0702645
Characterization of the [125I]‐neurokinin A binding site in the circular muscle of human colon
Authors: Warner Fiona J , Comis Alfio , Miller Robert C , Burcher Elizabeth
Published: January 2005

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]‐NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists.

125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]‐NKA was saturable (KD 0.47±0.05 nM), of high capacity (Bmax 2.1±0.1 fmol mg−1 wet weight tissue) and reversible (kinetically derived KD 0.36±0.07 nM).

The rank order of agonists competing for the [125I]‐NKA binding site was neuropeptide γ (NPγ)≥NKA≥[Lys5,MeLeu9,Nle10]NKA (4–10) (NK2 agonist)>>substance P (SP)>neurokinin B (NKB)≥[Pro9]SP (NK1 agonist)>>senktide (NK3 agonist), indicating binding to an NK2 site.

The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]‐NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968≥MEN11420>GR94800≥MEN10627>MEN10376≥R396. The NK1 antagonist SR140333 was a weak competitor.

The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 μM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected.

In conclusion, binding of the radioligand [125I]‐NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear.

British Journal of Pharmacology (1999) 127, 1105–1110; doi:10.1038/sj.bjp.0702648
Mechanisms of gastroprotection by transdermal nitroglycerin in the rat
Authors: Calatayud Sara , Sanz María‐Jesús , Canet Amparo , Bello Regina , De Rojas Francisco Díaz , Esplugues Juan V
Published: January 2005

Nitric oxide (NO) donors prevent experimentally‐induced gastric mucosal damage, but their clinical utility is limited by short duration of action or unsuitability of the pharmaceutical form employed. This study analyses the gastroprotection elicited by a clinically used mode of continuous administration of an NO donor, namely the nitroglycerin patch.

Application to rats of a transdermal patch that releases doses of nitroglycerin comparable to those used in man (40, 80, 160 and 400 ng min−1 rat−1) reduced gastric damage induced by indomethacin (25 mg kg−1, p.o. or s.c.). The nitroglycerin patch (160 ng  min−1 rat−1) also diminished damage by oral administration (1 ml) of acidified bile salts (100 mg kg−1 taurocholic acid in 150 mM HCl) or 50% ethanol.

Transdermal nitroglycerin (160 ng min−1 rat−1) did not influence basal gastric blood flow, as measured by lasser‐doppler flowmetry, but prevented its reduction by indomethacin.

Transdermal nitroglycerin (160 ng min−1 rat−1) prevented in vivo leukocyte rolling and adherence in the rat mesentery microvessels superfused with indomethacin, as evaluated by intravital microscopy.

The transdermal nitroglycerin patch protects the gastric mucosa from damage by mechanisms that involve maintenance of mucosal blood flow and reduction of leukocyte‐endothelial cell interaction.

British Journal of Pharmacology (1999) 127, 1111–1118; doi:10.1038/sj.bjp.0702649
Inhibition of gelatinase activity in human airway epithelial cells and fibroblasts by dexamethasone and beclomethasone
Authors: Carver Julia E , Galloway W Alan , Robinson Clive
Published: January 2005

The effects of dexamethasone and beclomethasone on gelatinase activity released from lung epithelial cells (A549, NCI‐H292 and Calu‐3 cell lines and NHBE primary cultures) and human lung fibroblasts (HLF) were investigated.

All cells spontaneously released gelatin‐degrading activity but the amounts were unaffected by treatment with glucocorticoids.

Phorbol myristate acetate (PMA) increased the amount of gelatinase activity in conditioned media prepared from all cell types examined. In epithelial cells, PMA induced the expression of gelatinase B, whereas in HLF the increased gelatinase activity resulted from increased activation of gelatinase A.

Dexamethasone and beclomethasone produced concentration‐dependent inhibition of PMA‐induced gelatinase activity in HLF and epithelial cell lines. In the epithelial cell lines, the inhibition of activity was associated with an attenuation of enzyme induction by PMA.

In contrast, primary cultures of human bronchial epithelial cells were unresponsive to dexamethasone at concentrations that were maximally effective at inhibiting gelatinase activity induced in other cells.

British Journal of Pharmacology (1999) 127, 1119–1128; doi:10.1038/sj.bjp.0702650
Comparative antiplatelet effects of aspirin, vapiprost and GR144053, a GPIIb/IIIa antagonist, with a special reference to the role of platelet microaggregates
Authors: Matsuno Hiroyuki , Kozawa Osamu , Nagashima Satoru , Kanamaru Mitutaka , Uematsu Toshihiko
Published: January 2005

Microthrombi produced have a potential to form larger thrombi, leading to vascular occlusions. Recently, a new device to easily detect microaggregates using laser‐light scattering (LS) has been developed. We adopted this device to comparatively evaluate the inhibitory effects of aspirin (1, 3 or 10 mg kg−1), vapiprost (0.3, 1 or 3 mg kg−1) or GR144053 (0.1, 0.3 or 1 mg kg−1) on ex vivo aggregation of hamster platelets in relation to their in vivo antithrombotic effects.

A transluminal thrombus was produced in the hamster femoral artery by the photochemical reaction. Each compound was injected i.v. as a bolus 10 min prior to the reaction, showing a dose‐dependent antithrombotic effect, i.e. they prolonged the time before the artery occluded. At that time cyclic flow reductions occurred more marked when aspirin or vapiprost was given.

At the end of experiments, blood was collected to evaluate the platelet aggregation using both the new LS device and the conventional optical density (OD) method. Many more small aggregates were still formed when the highest dose of aspirin or vapiprost was used as compared with that of GR144053, although suppression of the platelet aggregation using the OD method, prolongation of the occlusion time and the bleeding time were quite similar.

In conclusion, a GPIIb/IIIa antagonist markedly suppressed the microthrombi and reduced the cyclic flow reduction. This further indicates the importance of small aggregates as triggers of thrombosis and shows that prevention of their formation may result in improved vascular patency after thrombotic insult.

British Journal of Pharmacology (1999) 127, 1129–1134; doi:10.1038/sj.bjp.0702651
Pharmacological evidence that α1‐ and α2‐adrenoceptors mediate vasoconstriction of carotid arteriovenous anastomoses in anaesthetized pigs
Authors: Willems Edwin W , Trion Marjo , De Vries Peter , Heiligers Jan P C , Villalón Carlos M , Saxena Pramod R
Published: January 2005

Vasoconstriction of carotid arteriovenous anastomoses may be involved in the therapeutic action of acutely acting antimigraine agents, including the triptans and ergot alkaloids. While 5‐HT1B/1D receptors mediate the effect of triptans, ergotamine and dihydroergotamine also interact with α‐adrenoceptors. In the present study, we investigated the potential role of α1‐ and α2‐adrenoceptors in mediating vasoconstriction of carotid arteriovenous anastomoses in anaesthetized pigs.

Ten minute intracarotid infusions of phenylephrine (1, 3 and 10 μg kg−1 min−1) or BHT 933 (3, 10 and 30 μg kg−1 min−1) produced dose‐dependent decreases in total carotid and arteriovenous anastomotic conductances; no changes were observed in the capillary fraction.

The carotid vascular effects of phenylephrine and BHT 933 were selectively abolished by prazosin (100 μg kg−1, i.v.) and rauwolscine (300 μg kg−1, i.v.), respectively. The responses to phenylephrine and BHT 933 were not affected by the selective 5‐HT1B/1D receptor antagonist GR127935 (500 μg kg−1, i.v.).

These results show that both α1‐ and α2‐adrenoceptors can mediate vasoconstriction of carotid arteriovenous anastomoses in anaesthetized pigs. Since vasoconstrictor activity in this in vivo model is predictive of anti‐migraine activity, an agonist activity at particularly the α2‐adrenoceptor subtypes, in view of their less ubiquitous nature, could provide migraine abortive potential. Thus, the present results may aid further understanding of the mode of action of some current anti‐migraine agents and may eventually be helpful in the development of future treatment in migraine.

British Journal of Pharmacology (1999) 127, 1263–1271; doi:10.1038/sj.bjp.0702655
Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells
Authors: Coldwell M C , Boyfield I , Brown A M , Stemp G , Middlemiss D N
Published: January 2005

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer.

Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells.

The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues.

Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH‐232 and (−)3‐PPP were partial agonists in this assay system.

The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]‐iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities.

The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells.

British Journal of Pharmacology (1999) 127, 1135–1144; doi:10.1038/sj.bjp.0702657
Ligand efficacy and potency at recombinant human MT2 melatonin receptors: evidence for agonist activity of some mt1‐antagonists
Authors: Nonno Romolo , Pannacci Marilou , Lucini Valeria , Angeloni Deborah , Fraschini Franco , Stankov Bojidar M
Published: January 2005

NIH3T3 fibroblast cells transfected with the full‐length coding region of the MT2 human melatonin receptor stably expressed the receptor that is coupled to a pertussis toxin‐sensitive G protein and exhibits high affinity for melatonin (KI=261 pM).

The order of apparent affinity for selected compounds was: 4‐phenyl‐2‐propionamidotetralin (4P‐PDOT)>2‐phenylmelatonin>2‐iodomelatonin>2‐bromomelatonin>6‐chloromelatoninmelatonin>luzindole>N‐acetyl‐tryptamineN‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide (compound 6)>N‐acetylserotonin.

4P‐PDOT exhibited a very high selectivity (∼22,000 times) for the MT2 receptor with respect to the mt1 receptor subtype, as tested in comparative experiments with membrane preparations from NIH3T3 cells stably transfected with the human mt1 receptor.

MT2 melatonin receptors mediated incorporation of [35S]‐GTPγS into isolated membranes via receptor catalyzed exchange of [35S]‐GTPγS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]‐GTPγS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]‐GTPγS binding by 250% over basal (taken as 100%). Luzindole did not increase basal [35S]‐GTPγS binding but competitively inhibited melatonin‐stimulated [35S]‐GTPγS binding, thus exhibiting antagonist action.

The other two mt1 antagonists used here, 4P‐PDOT and N‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT2 subtype, with relative intrinsic activities of 0.37 and 0.39, respectively.

These findings show, for the first time, important differences in the intrinsic activity of analogues between the human mt1 and MT2 melatonin receptor subtypes.

British Journal of Pharmacology (1999) 127, 1288–1294; doi:10.1038/sj.bjp.0702658
Participation of protein kinases in staurosporine‐induced interleukin‐6 production by rat peritoneal macrophages
Authors: Yamaki Kouya , Ohuchi Kazuo
Published: January 2005

The incubation of rat peritoneal macrophages in the presence of staurosporine, a non‐specific protein kinase inhibitor, induced interleukin‐6 (IL‐6) production in a time‐ and concentration‐dependent manner at 6.3–63 nM, but at 210 nM, the stimulant effect on IL‐6 production was reduced.

The levels of IL‐6 mRNA as determined by a reverse transcription‐polymerase chain reaction were also increased by staurosporine in parallel with the ability to induce IL‐6 production.

Compounds structurally related to staurosporine including K‐252a (non‐specific protein kinase inhibitor) and KT‐5720 (inhibitor of cyclic AMP‐dependent protein kinase, PKA), did not increase IL‐6 production by peritoneal macrophages.

Staurosporine‐induced increases in IL‐6 production and expression of IL‐6 mRNA were decreased by the PKC inhibitors, H‐7 (2.7–27 μM), Ro 31–8425 (1–10 μM) and calphostin C (0.3–3 μM) and by the phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor LY294002 (30–100 μM), but were further increased by the protein tyrosine kinase (PTK) inhibitor, genistein (12–37 μM).

The staurosporine‐induced increase in IL‐6 production was not affected by the PKA inhibitor, H‐89 (0.1–3 μM).

These findings suggest that the induction of IL‐6 production by staurosporine is secondary to elevation of IL‐6 mRNA level, which, in turn, is positively regulated by the activation of PKC and PI 3‐kinase and negatively regulated by the activation of PTK. PKA does not appear to play a significant role.

British Journal of Pharmacology (1999) 127, 1309–1316; doi:10.1038/sj.bjp.0702659
Cross‐resistance with dieldrin of a novel tricyclic dinitrile GABA receptor antagonist
Authors: Matsuda Kazuhiko , Hosie Alastair M , Holyoke Caleb W , Rauh James J , Sattelle David B
Published: January 2005

A novel tricyclic dinitrile, KN244, blocked the wild‐type (dieldrin‐sensitive) homo‐oligomeric γ‐aminobutyric acid (GABA)‐gated chloride channel of Drosophila melanogaster expressed in Xenopus oocytes. Sensitivity to the block by KN244 of the response to 30 μM GABA (IC50=41.6 nM, wild‐type RDLac) was reduced abut 100 fold (IC50=4.5 μM) in the dieldrin‐resistant (RDLacA302S) form of RDL.

British Journal of Pharmacology (1999) 127, 1305–1307; doi:10.1038/sj.bjp.0702663
Endothelin generating pathway through endothelin1–31 in human cultured bronchial smooth muscle cells
Authors: Hayasaki‐Kajiwara Yoko , Naya Noriyuki , Shimamura Toshitake , Iwasaki Takanori , Nakajima Masatoshi
Published: January 2005

The effects of endothelin (ET)‐11–31 and ET‐21–31, human chymase products of the corresponding big ETs, on the intracellular free Ca2+ concentration ([Ca2+]i) and [125I]‐ET‐1 binding were investigated using human cultured bronchial smooth muscle cells (BSMC).

ET‐11–31 (10−8 M – 3×10−7 M) and ET‐21–31 (3×10−8 M–3×10−6 M) caused an increase in [Ca2+]i in a concentration‐dependent manner. Big ET‐1 (3×10−8 M – 10−6 M) also caused this increase, but not big ET‐2 at concentrations up to 10−6 M. The [Ca2+]i increase induced by ET‐1 was inhibited by both BQ123, an ETA‐receptor antagonist, and BQ788, an ETB‐receptor antagonist, whereas that induced by ET‐3 was inhibited by BQ788 but not by BQ123.

Increases in [Ca2+]i caused by ET‐11–31, big ET‐1 and ET‐21–31 were completely inhibited by 10−4 M phosphoramidon, a dual neutral endopeptidase (NEP)/endothelin‐converting enzyme (ECE) inhibitor, and 10−5 M thiorphan, a NEP inhibitor.

Scatchard plot analyses of the saturation curves of [125I]‐ET‐1 and [125I]‐ET‐3 showed that both ETA‐ and ETB‐ receptors at the ratio of 4 : 1 were expressed on BSMC. ET‐11–31, big ET‐1 and ET‐21–31 inhibited [125I]‐ET‐1 binding in a concentration‐dependent manner, and these effects were attenuated by treatment with thiorphan. On the other hand, big ET‐2 slightly inhibited the binding at a high concentration and this was not affected by thiorphan.

These results suggest that ET‐11–31, big ET‐1 and ET‐21–31 cause an increase in [Ca2+]i by being converted into the corresponding ET‐1 and ET‐2 by NEP, but this did not occur with big ET‐2 in human BSMC. ET‐21–31 produced by human chymase from big ET‐2 might be important for the generation of ET‐2 in human bronchial tissue.

British Journal of Pharmacology (1999) 127, 1415–1421; doi:10.1038/sj.bjp.0702664
Pharmacological characterization of 5‐HT4 receptors mediating relaxation of canine isolated rectum circular smooth muscle
Authors: Prins N H , Van Haselen J F W R , Lefebvre R A , Briejer M R , Akkermans L M A , Schuurkes J A J
Published: January 2005

This study aimed to characterize for the first time in vitro 5‐HT4 receptors in the canine gastrointestinal tract. For this purpose, we used circular muscle strips of the canine isolated rectum.

In the presence of methysergide (60 μM), 5‐HT induced relaxation of methacholine (1 μM)‐precontracted muscle strips, yielding a monophasic sigmoidal concentration‐relaxation curve (pEC50 7.2±0.07).

Tetrodotoxin (0.3 μM) did not affect the curve to 5‐HT, suggesting the inhibitory 5‐HT receptor is located on the smooth muscle. Granisetron (0.3 μM) did also not affect the curve to 5‐HT, which excludes the 5‐HT3 receptor mediating the relaxation to 5‐HT. The presence of methysergide rules out the involvement of 5‐HT1, 5‐HT2 or 5‐HT7 receptors.

5‐HT, the selective 5‐HT4 receptor agonists R076186, prucalopride (R093877) and SDZ HTF‐919 and the 5‐HT4 receptor agonists cisapride and 5‐MeOT relaxed the muscle strips with a rank order of potency R076186=5‐HT>cisapride>prucaloprideSDZ HTF‐919>5‐MeOT.

The selective 5‐HT4 receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5‐HT, yielding pKB estimates of 9.7, 7.9 and 9.1, respectively. The selective 5‐HT4 receptor antagonist SB 204070 shifted the curve to 5‐HT rightward and depressed the maximal response (apparent pA2 10.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5‐HT4 receptor agonists R076186 (pA2 8.8).

It is concluded that 5‐HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5‐HT4 receptors. For the first time, a non‐human species was shown to exhibit relaxant 5‐HT4 receptors in the large intestine.

British Journal of Pharmacology (1999) 127, 1431–1437; doi:10.1038/sj.bjp.0702665
Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats
Authors: Salmon Michael , Walsh David A , Huang Tung‐Jung , Barnes Peter J , Leonard Thomas B , Hay Douglas W P , Chung K Fan
Published: January 2005

Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin.

There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin‐exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline‐exposed rats (1.3%, 0.6–2.1; P<0.001).

Treatment with a 5‐lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect.

There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146.

A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146.

Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness.

These data indicate that the cysteinyl leukotrienes are important mediators in allergen‐induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure.

British Journal of Pharmacology (1999) 127, 1151–1158; doi:10.1038/sj.bjp.0702669
Effects of superoxide dismutase mimetics on the activity of nitric oxide in rat aorta
Authors: MacKenzie Andrew , Filippini Silvia , Martin William
Published: January 2005

A number of structurally distinct superoxide dismutase (SOD) mimetics were examined to determine if they shared the ability of authentic Cu/Zn SOD to produce endothelium‐dependent relaxation of rings of rat aorta by protecting basal nitric oxide from destruction by endogenously produced superoxide anion.

MnCl2 (10 nM–100 μM), CuSO4 (100 nM–1 mM) and CuDIPS (Cu [II]‐[diisopropylsalicylate]2; 100 nM–30 μM) each mimicked the ability of Cu/Zn SOD (0.1–300 u ml−1) to produce relaxation of phenylephrine‐precontracted aortic rings in a manner inhibited by endothelial removal or treatment with NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM).

In contrast, MnTMPyP (Mn [III] tetrakis [1‐methyl‐4‐pyridyl] porphyrin; 10 nM–30 μM) augmented phenylephrine‐induced contraction and this was blocked by endothelial removal or treatment with L‐NAME (100 μM), consistent with destruction rather than protection of basal nitric oxide activity. Pretreatment with Cu/Zn SOD (250 u ml−1) blocked this augmentation suggesting that it arose paradoxically through destruction of nitric oxide by superoxide anion.

The spin trap agents tiron (100 nM–1 mM), tempol (100 nM–1 mM) and PTIYO (4‐phenyl‐2,2,5,5‐tetramethyl imidazolin‐1‐yloxy‐5‐oxide; 100 nM–300 μM) all failed to promote endothelium‐dependent relaxation. In fact, the last two augmented phenylephrine‐induced tone and this was blocked by endothelial removal or treatment with L‐NAME (100 μM), consistent with destruction of basal nitric oxide activity. This destruction was unaffected by pretreatment with Cu/Zn SOD (250 u ml−1) and probably reflected the direct ability of tempol and PTIYO to destroy nitric oxide.

Thus, the ideal SOD mimetic for protection of nitric oxide activity in conditions of oxidant stress still awaits development.

British Journal of Pharmacology (1999) 127, 1159–1164; doi:10.1038/sj.bjp.0702670