bluuebunny/crossref_metadata_2025 huggingface dataset:
Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists
Authors: Maemoto Takuya , Finlayson Keith , Olverman Henry J. , Akahane Atsushi , Horton Roger W. , Butcher Steven P.
Published: January 2005

The pharmacological profile of adenosine A1 receptors in human, guinea‐pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]‐8‐cyclopentyl‐1,3‐dipropylxanthine ([3H]‐DPCPX).

The affinity of [3H]‐DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea‐pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea‐pig or human cortical membranes.

The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5′‐N‐ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5–26.2 fold higher in rat cortical membranes than in human and guinea‐pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 μM) reduced 2‐chloro‐N6‐cyclopentyladenosine (CCPA) affinity 7–13.9 fold, whereas the affinity of DPCPX was unaffected.

The affinity of six xanthine‐based adenosine receptor antagonists was 2.2–15.9 fold higher in rat cortical membranes compared with human or guinea‐pig membranes. The rank order of potency was species‐independent. In contrast, three pyrazolopyridine derivatives, (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐2‐piperidine ethanol (FK453), (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐piperidin‐2‐yl acetic acid (FK352) and 6‐oxo‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl)‐1(6H)‐pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea‐pig, rat and mouse brain membranes. pKi values (M) for [3H]‐DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively.

Drug affinity for adenosine A2A receptors was determined in a [3H]‐2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine ([3H]‐CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]‐DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]‐DPCPX binding sites in human, rat, mouse and guinea‐pig brain membranes.

British Journal of Pharmacology (1997) 122, 1202–1208; doi:10.1038/sj.bjp.0701465
Heterologous expression of rat epitope‐tagged histamine H2 receptors in insect Sf9 cells
Authors: Beukers M. W. , Klaassen C. H. W. , De Grip W. J. , Verzijl D. , Timmerman H. , Leurs R.
Published: January 2005

Rat histamine H2 receptors were epitope‐tagged with six histidine residues at the C‐terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope‐tagged H2 receptor were prepared and were used to infect insect Sf9 cells.

The His‐tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]‐aminopotentidine (APT) binding studies.

In Sf9 cells expressing the His‐tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3).

The expression of the His‐tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg−1 protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×106 copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol‐β‐cyclodextrin complex resulted in an increase of [125I]‐APT binding up to 169±5% (mean±s.e.mean, n=3).

The addition of 0.03 mM cholesterol‐β‐cyclodextrin complex did not affect histamine‐induced cyclic AMP production. The EC50 value of histamine was 3.1±1.7 μM in the absence of cholesterol‐β‐cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol‐β‐cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/106 cells in the absence and 81±11 pmol/106 cells in the presence of 0.03 mM cholesterol‐β‐cyclodextrin complex (mean±s.e.mean, n=3).

Immunofluorescence studies with an antibody against the His‐tag revealed that the majority of the His‐tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well.

These experiments demonstrate the successful expression of His‐tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His‐tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.

British Journal of Pharmacology (1997) 122, 867–874; doi:10.1038/sj.bjp.0701466
Contrasting effects of carbachol, McN‐A‐343 and AHR‐602 on Ca2+‐mobilization and Ca2+‐influx pathways in taenia caeci
Authors: Hishinuma Shigeru , Hongo Ikuko , Matsumoto Yukio , Narita Fumihiro , Kurokawa Masanori
Published: January 2005

We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN‐A‐343 and AHR‐602 with those of carbachol in smooth muscle of guinea‐pig taenia caeci.

McN‐A‐343 and AHR‐602, as well as carbachol, completely displaced the atropine‐sensitive binding of [3H]‐quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol>McN‐A‐343>4AHR‐602.

In the presence of 2.2 mM extracellular Ca2+, McN‐A‐343 and AHR‐602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4‐DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF‐DX 116 (M2 selective). These contractions were inhibited by an L‐type Ca2+ channel blocker, verapamil.

In Ca2+‐free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN‐A‐343 and AHR‐602. Application of McN‐A‐343 or AHR‐602 inhibited the carbachol‐induced contraction in Ca2+‐free solution, and this inhibition was surmounted by a higher concentration of carbachol.

The EC50 value for carbachol‐induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+.

The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor‐mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN‐A‐343 and AHR‐602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.

British Journal of Pharmacology (1997) 122, 985–992; doi:10.1038/sj.bjp.0701467
Effect of lacidipine on cholesterol esterification: in vivo and in vitro studies
Authors: Bernini F. , Canavesi M. , Bernardini E. , Scurati N. , Bellosta S. , Fumagalli R.
Published: January 2005

Cholesterol esterification and accumulation in the arterial wall is a hallmark of atherogenesis. Several preclinical studies suggest that calcium antagonists may exert antiatherosclerotic activity by directly affecting atherogenesis in the arterial wall. We investigated the effect of the second generation dihydropyridine calcium antagonist lacidipine on cholesterol metabolism in vivo in the aortic arch of cholesterol fed rabbits, and in vitro in mouse cultured peritoneal macrophages.

Treatment of cholesterol‐fed rabbits with 1, 3 and 10 mg kg−1 day−1 of lacidipine for two months reduced, in a dose‐dependent manner, cholesterol esterification in the aortic arch: 24±6, 30±12, and 41±8% inhibition, respectively (P<0.001 vs HC control). Concomitantly, drug treatment reduced total cholesterol content of the vessel wall. Lacidipine 3 and 10 mg kg−1 day−1 reduced cholesterolaemia (∼20%); no effect was observed at the lowest dose used (1 mg kg−1 day−1). These results suggest that the action of lacidipine on cholesterol esterification in the arterial wall involves, at least in part, a direct effect on cellular cholesterol metabolism. Inhibition of cholesterol esterification in the arterial wall was observed also in a reference group of animals treated with the specific ACAT inhibitor CI‐976.

To evaluate the action of lacidipine on intracellular cholesterol metabolism we performed in vitro experiments with murine macrophages, the main cell type that accumulates cholesterol in the arterial wall. Lacidipine almost completely inhibited cholesterol esterification in cholesterol loaded macrophages in culture. The effect was observed independently of esterification stimuli and in cell free homogenates. The drug modified intracellular cholesterol distribution, doubling the free‐ to esterified sterol ratio, but did not influence the cellular rate of cholesteryl ester hydrolysis in the cell. All together these results indicate an inhibitory effect of lacidipine on cholesterol esterification catalyzed by the enzyme ACAT in murine macrophages.

We concluded that lacidipine influences cellular cholesterol metabolism. This effect may contribute to the potential antiatherosclerotic activity of this drug.

British Journal of Pharmacology (1997) 122, 1209–1215; doi:10.1038/sj.bjp.0701469
Morphological heterogeneity with normal expression but altered function of G proteins in porcine cultured regenerated coronary endothelial cells
Authors: Borg‐Capra Catherine , Fournet‐Bourguignon Marie‐Pierre , Janiak Philip , Villeneuve Nicole , Bidouard Jean‐Pierre , Vilaine Jean‐Paul , Vanhoutte Paul M.
Published: January 2005

Experiments were designed to investigate whether the pertussis toxin‐dependent endothelial dysfunction following balloon injury is due to a reduced expression or an insufficient function of G‐proteins.

Endothelium‐dependent responses of porcine coronary arteries were examined in vitro by use of conventional organ chambers. Morphological analysis was performed by isolating and culturing the endothelial cells from these arteries. The expression of Gi‐proteins in regenerated endothelial cells was measured by Western blots and immunolabelling. The function of G‐proteins was assessed by measuring the GTPase activity of cultured endothelial cells.

Eight days following denudation, endothelial regrowth was confirmed by histological examination and by demonstrating the presence of endothelium‐dependent relaxations to bradykinin and 5‐hydroxytryptamine (5‐HT). In primary culture, the regenerated endothelial cells displayed a ‘cobblestone’ pattern as seen with native endothelial cells.

Twenty eight days after denudation, the endothelium‐dependent relaxations induced by 5‐HT were impaired, but those to bradykinin were maintained. However, the latter were reduced when endothelium‐dependent hyperpolarization was prevented.

Twenty eight days after denudation, multinucleated giant cells were present in the regenerated but not in the native cultured endothelial cell populations. These regenerated endothelial cells incorporated less tritiated thymidine than native endothelial cells.

The intensities of the bands on the immunoblot of the regenerated endothelial cells, when several antibodies against Giα1/α2/α3 were used, were the same as those obtained in native endothelial cells. The immunolabelling with the same antibodies was similar between the giant cells and the regenerated endothelial cells of normal size. The hydrolysis of GTP was lower in regenerated than in native endothelial cell membranes.

In conclusion, endothelium‐dependent relaxations mediated by Gi‐proteins are impaired in balloon denuded coronary arteries. This dysfunction following regeneration cannot be explained by a reduced expression of Gi proteins but rather reflects an abnormal function of the G‐proteins in the regenerated endothelium.

British Journal of Pharmacology (1997) 122, 999–1008; doi:10.1038/sj.bjp.0701475
Cardiac inotropes inhibit the oedema caused by nifedipine in rabbit skin
Authors: Taherzadeh Morteza , Warren John B.
Published: January 2005

We have shown previously that exposing the rat or rabbit microcirculation to nifedipine increases the permeability of the post‐capillary venule, the segment of microcirculation that is known to control inflammatory oedema.

In the present study modulation by the inotropes isoprenaline, dopexamine and dobutamine of nifedipine‐induced oedema was examined in the rabbit skin microcirculation by measuring the localised leakage of 125I‐radiolabelled albumin after the i.d. injection of agents.

Coinjection of isoprenaline (10−11 moles per site), dopexamine (10−10 moles per site) or dobutamine (10−10 moles per site) suppressed significantly (P<0.05) the oedema response to nifedipine (10−7.2 moles per site) in the rabbit dorsal skin microcirculation.

To confirm the oedema suppresser effect of the inotropes, dopexamine or dobutamine were also coinjected with histamine 10−8 +PGE2 10−10 moles per site, or bradykinin 10−10+PGE2 10−10 moles per site. Both inotropes at 10−10 moles per site reduced significantly (P<0.05) the leakage of albumin caused by bradykinin+PGE2 and histamine+PGE2.

When measured by laser Doppler, basal local skin blood flow increased at 30 min by 57±14% with nifedipine 10−7.2 moles per site and 15±11% with isoprenaline 10−11 moles per site. Isoprenaline did not suppress the blood flow response to nifedipine, the response to coinjection being 68±11%.

Oedema caused by nifedipine can be suppressed by low concentrations of β‐adrenergic agonists that do not suppress the blood flow response to nifedipine. This suggests that cardiac inotropes can influence non‐inflammatory changes in microvascular permeability.

British Journal of Pharmacology (1997) 122, 1160–1164; doi:10.1038/sj.bjp.0701480
Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor‐β1
Authors: Giulio Romanelli Roberto , Caligiuri Alessandra , Carloni Vinicio , DeFranco Raffaella , Montalto Paolo , Ceni Elisabetta , Casini Alessandro , Gentilini Paolo , Pinzani Massimo
Published: January 2005

Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver.

Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor‐β1 (TGF‐β1) for different time periods in the presence or absence of PTF. TGF‐β1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3–6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9–12 h after the addition of TGF‐β1. Co‐incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell‐associated expression of the corresponding protein. Conversely, PTF dose‐dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells.

Pulse‐chase experiments employing L‐[3H]‐proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP‐1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP‐1) mRNA. Accordingly incubation with PTF increased the levels of ‘activated MMP‐1’ in cell supernatants in both basal and stimulated conditions.

These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.

British Journal of Pharmacology (1997) 122, 1047–1054; doi:10.1038/sj.bjp.0701484
Quantification and distribution of α1‐adrenoceptor subtype mRNAs in human vas deferens: comparison with those of epididymal and pelvic portions
Authors: Moriyama Nobuo , Nasu Kimio , Takeuchi Takumi , Akiyama Katsuyoshi , Murata Satoshi , Nishimatsu Hiroaki , Yano Junichi , Tsujimoto Gozoh , Kawabe Kazuki
Published: January 2005

This study was intended to quantify the amounts of the α1‐adrenoceptor subtype mRNAs in human vas deferens, and demonstrate the receptor subtype responsible for the vas contraction.

The RNase protection assay showed that the mean total amount of α1a mRNA was 7.4±2.2 pg/5 μg of poly (A)+ RNA (97.0% of the total α1 mRNA) in the epididymal portion (E‐vas) and 4.9±0.8 pg/5 μg of poly (A)+ RNA (96.3% of the total) in the pelvic portion (P‐vas). The E‐vas showed a tendency to have a greater α1a mRNA abundance than the P‐vas (P=0.11). The α1b and α1d mRNAs were absent or of extremely low abundance.

By an in situ hybridization, the α1a and α1d mRNAs were recognized in the smooth muscle cells of the E‐vas and the P‐vas, and the distribution pattern the same in both tissues. The α1b mRNA positive site was scarcely detectable in both vas portions.

In a functional study, l‐phenylephrine produced concentration‐dependent contraction in the E‐vas (Emax=2.24±0.70 g; pD2=5.32±0.09) and the P‐vas (Emax=2.46±0.46 g; pD2=5.07±0.12). KMD‐3213, a novel α1A‐adrenoceptor‐selective antagonist, caused parallel rightward shifts of the concentration–response curves for l‐phenylephrine. Apparent pKB values were 9.90±0.16 for the E‐vas and 9.71±0.17 for the P‐vas. There was no significant difference in Emax, pD2 or pKB estimates between the two portions.

We have found that α1a mRNA is predominant in the human vas deferens, and confirmed that contraction of this organ is mediated by the α1A‐adrenoceptor.

British Journal of Pharmacology (1997) 122, 1009–1014; doi:10.1038/sj.bjp.0701485
Tranilast inhibits the proliferation, chemotaxis and tube formation of human microvascular endothelial cells in vitro and angiogenesis in vivo
Authors: Isaji Masayuki , Miyata Hiroshi , Ajisawa Yoshiyuki , Takehana Yasuo , Yoshimura Nagahisa
Published: January 2005

First developed as an antiallergic drug, tranilast inhibits chemical mediator release from mast cells. In the present study, we examine the effects of tranilast on angiogenesis in vitro and in vivo and discuss the application of tranilast for angiogenic diseases.

Tranilast inhibited significantly the proliferation (IC50: 136 μM, 95% confidence limits: 124–137 μM) and vascular endothelium growth factor (VEGF)‐induced chemotaxis (IC50: 135 μM, 95% confidence limits: 124–147 μM) of human dermal microvascular endothelial cells (HDMECs) at concentrations greater than 25 μg ml−1. No toxicity to HDMECs measuring by LDH release and no inhibitory effects on metalloproteinase (MMP)‐2 and MMP‐9 activity were observed even at 100 μg ml−1 (306 μM).

Tube formation of HDMECs cultured on the matrigel as an in vitro angiogenesis model was inhibited by tranilast in a concentration‐dependent manner. The IC50 value and 95% confidence limits were 175 μM and 151–204 μM, respectively.

In vivo angiogenesis was induced in mice by the subcutaneous injection of matrigel containing 30 ng ml−1 VEGF and 64 μg ml−1 heparin. Tranilast was administered orally twice a day for 3 days. Tranilast dose‐dependently suppressed angiogenesis in the matrigel and a significant change was observed at a dose of 300 mg kg−1.

These results indicate that tranilast is an angiogenesis inhibitor which may be beneficial for the improvement of angiogenic diseases such as proliferative diabetic retinopathy, age‐related macular degeneration, tumour invasion and rheumatoid arthritis.

British Journal of Pharmacology (1997) 122, 1061–1066; doi:10.1038/sj.bjp.0701493
Acute effects of nitric oxide blockade with L‐NAME on arterial haemodynamics in the rat
Authors: Hu Cheng‐Tao , Chang Kuo‐Chu , Wu Chia‐Yen , Chen Hsing I.
Published: January 2005

We employed the technique of impedance spectral analysis to investigate the role of endogenous nitric oxide (NO) in the regulation of steady and pulsatile haemodynamics in Wistar Kyoto rat (WKY).

A total of 12 WKYs was anaesthetized with pentobarbitol sodium (40 mg kg−1, i.p.) and artificially ventilated with an animal respirator. The aortic pressure wave was monitored with a high fidelity Millar sensor, and aortic flow wave with an electromagnetic flow probe. The pressure and flow waves were subjected to Fourier transform for the analysis of impedance spectra.

The baseline cardiovascular parameters were mean arterial pressure (APm) 95±9 mmHg, heart rate (HR) 338±9 b.p.m., stroke volume (SV) 0.23±0.01 ml, cardiac output (CO) 77.8±1.6 ml min−1, total peripheral resistance (TPR) 98±11 (×103) dyne  s  cm−5, characteristic impedance (Zc) 2046±141 dyne  s  cm−5, arterial compliance at mean AP (Cm) 3.78±0.22 μl mmHg−1 and backward pulse wave (Pb) 12.9±0.6 mmHg.

An NO synthase inhibitor, NG‐nitro‐L‐arginine monomethyl ester (L‐NAME) was administered at graded intravenous doses. This agent caused dose‐dependent increases in AP and TPR with decreases in HR. At an accumulative dose of 10 mg kg−1, APm was increased by 29±3 mmHg (+31%) and TPR by 49±6 (×103) dyne  s  cm−5 (+50%), while HR was reduced by 37±5 b.p.m. (−11%) and CO by 10.4±0.8 ml min−1 (−14%). The pulsatile haemodynamics including Zc and Pb were slightly increased by 14–15%. Cm was decreased by 1.09 μl mmHg−1 (−29%). L‐NAME also did not significantly affect the ventricular work including the steady, oscillatory and total work.

Aminoguanidine, a specific inhibitor for inducible NO synthase (iNOS), in dose 10–60 mg kg−1 i.v. did not alter the AP, HR and other parameters. The result indicated that blockade of constitutive NOS, but not iNOS is involved in these changes.

Angiotensin II (Ang) in various infusion doses was used to produce a profile of AP increase similar to that caused by L‐NAME. Ang remarkably increased Zc, while TPR was moderately elevated. The pattern of haemodynamic changes was different from that following L‐NAME.

The results suggest that blockade of the endogenous NO affects predominantly the arterial pressure and peripheral resistance. The Windkessel functions such as arterial impedance and pulse wave reflection are slightly increased. Ventricular works are not significantly altered.

British Journal of Pharmacology (1997) 122, 1237–1243; doi:10.1038/sj.bjp.0701496
GR144053, a fibrinogen receptor antagonist, enhances the suppression of neointima formation by losartan, an angiotensin II receptor antagonist, in the injured carotid artery of hamster
Authors: Matsuno Hiroyuki , Kozawa Osamu , Niwa Masayuki , Kaida Takehiro , Hayashi Hideharu , Uematsu Toshihiko
Published: January 2005

The present study investigated the inhibitory effect of losartan, a type 1 angiotensin II receptor (AT1) antagonist, and of combined treatment with losartan and GR144053, a fibrinogen receptor (GPIIb/IIIa) antagonist, on neointima formation subsequent to vascular injury in the hamster carotid artery. Vascular injury was achieved by a roughened‐tip 2F catheter and the neointimal area was measured up to 2 weeeks inducing the injury.

Compared to non‐treated hamsters (intimal area (IA)/internal elastic laminal area (IELA) ratio = 60.3±5.9%, n=12), losartan dissolved in drinking water (1, 3 and 10 mg kg−1 per day, n=8 each) reduced neointimal area dose‐dependently, a significant decrease (IA/IELA=39.7±5.6%) being attained with the highest dose when it was administered from 1 day before injury. However, neointima formation was not prevented even with the highest dose of losartan when the administration was started after injury.

When the administration of GR144053 (1.0 mg kg−1 per hour) via an implanted osmotic pump was started 30 min before the injury and continued for the next 2 weeks, no suppression of neointima formation was observed, although platelet aggregation evoked ex vivo by adenosine diphosphate (ADP) at the end of treatment period was efficiently inhibited.

In separate experiments in which 5‐bromo‐2‐deoxy‐Uridine (BrdU) was used to test smooth muscle cell (SMC) proliferation 1 and 7 days after injury, the ratio of SMC proliferation in the injured area was only slightly decreased by losartan when its administration was started after the injury, despite the marked reduction of SMC proliferation when treatment was started before the injury. Treatment with GR144053 as indicated above also significantly decreased the SMC proliferating index 1 day after the injury.

To examine the potential benefit of the coadministration of the GPIIb/IIIa antagonist with the AT1 receptor antagonist, GR144053 (1.0 mg kg−1 per hour) was combined with post‐injury treatment with losartan (10 mg kg−1 per day). This markedly reduced the proliferation of SMCs and significantly decreased the neointimal area (IA/IELA=31.2±4.6%) measured 2 weeks following the catheterization.

According to the results of a time‐dependent study in which GR144053 was given in combination with post injury treatment with losartan for 1, 3, 7 or 14 days, neointima formation could be reduced by treatment with GR144053 for just 7 days.

In conclusion, GR144053, a fibrinogen receptor antagonist, enhanced the inhibitory effect of losartan, an AT1 receptor antagonist, on neointima formation in the damaged carotid artery of hamsters.

British Journal of Pharmacology (1997) 122, 1099–1104; doi:10.1038/sj.bjp.0701500
Electrophysiological basis for the antiarrhythmic action and positive inotropy of HA‐7, a furoquinoline alkaloid derivative, in rat heart
Authors: Su Ming‐Jai , Chang Gwo‐Jyh , Wu Mei‐Hwan , Kuo Sheng‐Chu
Published: January 2005

HA‐7, a new synthetic derivative of furoquinoline alkaloid, increased the contractile force of right ventricular strips and effectively suppressed the ischaemia‐reperfusion induced polymorphic ventricular tachyrhythmias in adult rat heart (EC50=2.8 μM).

In rat ventricular myocytes, HA‐7 concentration‐dependently prolonged the action potential duration (APD) and decreased the maximal rate of rise of the action potential upstroke (V̇max). The action potential amplitude and resting membrane potential were also reduced, but to a smaller extent. The prolongation of APD by HA‐7 was prevented by pretreating the cells with 1 mM 4‐AP.

Voltage clamp experiments revealed that HA‐7 decreased the maximal current amplitude of INa (IC50=4.1 μM) and caused a negative shift of its steady‐state inactivation curve and slowed its rate of recovery from inactivation. The use‐dependent inhibition of INa by HA‐7 was enhanced at a higher stimulation rate. The L‐type Ca2+ current (ICa) was also reduced, but to a lesser degree (IC50=5.3 μM, maximal inhibition=31.8%).

This agent also influenced the time‐ and voltage‐dependent K+ currents. The prolongation of APD was associated with an inhibition of a 4‐AP sensitive transient outward K+ current (Ito) (IC50=2.9 μM) and a slowly inactivating, steady‐state outward current (Iss) (IC50=2.5 μM). The inhibition of Ito by HA‐7 was associated with an acceleration of its time constant of inactivation. HA‐7 suppressed Ito in a time‐dependent manner and caused a significant negative shift of the voltage‐dependent steady‐state inactivation curve but did not affect its rate of recovery from inactivation.

At higher concentrations, the inward rectifier K+ current (IK1) was also inhibited but to a less extent. Its slope conductance after 3, 10 and 30 μM HA‐7 was decreased by 24±4%, 41±5% and 54±8%, respectively.

We conclude that HA‐7 predominantly blocks Ito and Na+ channels and that it also weakly blocks Ca2+ and IK1 channels. These changes alter the electrophysiological properties of the heart and terminate the ischaemia reperfusion induced ventricular arrhythmia. The significant Ito inhibition and minimal ICa suppression may afford an opportunity to develop an effective antiarrhythmic agent linked with positive inotropy.
Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10
Authors: Russ Ulrich , Metzger Friedrich , Kickenweiz Elisabeth , Hambrock Annette , Krippeit‐Drews Peter , Quast Ulrich
Published: January 2005

The ATP‐sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]‐P1075, and by electrophysiological techniques.

Saturation binding experiments gave a KD value of 9.2±5.2 nM and a binding capacity (BMax) of 140±40 fmol mg−1 protein for [3H]‐P1075 binding to A10 cells; from the BMax value a density of binding sites of 5–10 per μm2 plasmalemma was estimated.

KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]‐P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]‐P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta.

Resting membrane potential, recorded with microelectrodes, was −51±1 mV. P1075 and levcromakalim produced a concentration‐dependent hyperpolarization by up to −25 mV with EC50 values of 170±40 nM and 870±190 nM, respectively. The hyperpolarization induced by levcromakalim (3 μM) was completely reversed by glibenclamide with an IC50 value of 86±17 nM.

Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 μM) induced a current which reversed around −80 mV; the current‐voltage relationship showed considerable outward rectification. Glibenclamide (3 μM) abolished the effect of levcromakalim.

Analysis of the noise of the levcromakalim (10 μM)‐induced current at −40 and −20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 μm−2, 8.8 pS and 0.39, respectively.

The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]‐P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.

British Journal of Pharmacology (1997) 122, 1119–1126; doi:10.1038/sj.bjp.0701514
Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells
Authors: Khiroug L. , Giniatullin R. , Sokolova Elena , Talantova Maria , Nistri A.
Published: January 2005

The possible role of intracellular Ca2+ levels ([Ca2+]i) in desensitization of nicotinic acetylcholine receptors (AChRs) was investigated in rat cultured chromaffin cells by use of combined whole‐cell patch clamping and confocal laser scanning microscopy with the fluorescent dye fluo‐3.

On cells held at −70 mV, pressure‐application of nicotine elicited inward currents with associated [Ca2+]i rises mainly due to influx through nicotinic AChRs. These responses were blocked by (+)‐tubocurarine (10 μM) but were insensitive to α‐bungarotoxin (1 μM) or Cd2+ (0.1 mM).

Pressure applications of 1 mM nicotine for 2 s (conditioning pulse) evoked inward currents which faded biexponentially to a steady state level due to receptor desensitization and were accompanied by a sustained increase in [Ca2+]i. Inward currents evoked by subsequent application of brief test pulses of nicotine were depressed but recovered with a time course reciprocal to the decay of the [Ca2+]i transient induced by the conditioning pulse.

Omission of intracellular Ca2+ chelators or use of high extracellular Ca2+ solution (10 mM) lengthened recovery of nicotinic AChRs from desensitization while adding BAPTA or EGTA intracellularly had the opposite effect. When the patch pipette contained fluo‐3 or no chelators, after establishing whole cell conditions the rate of recovery became progressively longer presumably due to dialysis of endogenous Ca2+ buffers. None of these manipulations of external or internal Ca2+ had any effect on onset or steady state level of desensitization.

High spatial resolution imaging of [Ca2+]i in intact cells (in the presence of 0.1 mM Cd2+) showed that its level in the immediate submembrane area decayed at the same rate as in the rest of the cell, indicating that Ca2+ was in a strategic location to modulate (directly or indirectly) AChR desensitization.

The present data suggest that desensitized nicotinic AChRs are stabilized in their conformation by raised [Ca2+]i and that this phenomenon retards their recovery to full activity.
Influence of flupirtine on a G‐protein coupled inwardly rectifying potassium current in hippocampal neurones
Authors: Jakob Regina , Krieglstein Josef
Published: January 2005

Previous studies have shown that flupirtine, a centrally acting, non‐opioid analgesic agent, also exhibits neuroprotective activity in focal cerebral ischaemia in mice and reduces apoptosis induced by NMDA, gp 120 of HIV, prior protein fragment or lead acetate as well as necrosis induced by glutamate or NMDA in cell culture. To study the potential mechanism of the neuroprotective action of flupirtine, we investigated whether flupirtine is able to modulate potassium or NMDA‐induced currents in rat cultured hippocampal neurones by use of the whole‐cell configuration of the patch‐clamp technique.

We demonstrated that 1 μM flupirtine activated an inwardly rectifying potassium current (Kir) in hippocampal neurones (ΔI=−39±18 pA at −130 mV; n=10). This effect was dose‐dependent (EC50=0.6 μM). The reversal potential for Kir was in agreement with the potassium equilibrium potential predicted from the Nernst equation showing that Kir was predominantly carried by K+. Furthermore, the induced current was blocked completely by Ba2+ (1 mM), an effect typical for Kir.

The activation of Kir by flupirtine was largely prevented by pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX‐sensitive G‐protein in the transduction mechanism (ΔI=−3±6 pA at −130 mV; n=8). Inclusion of cyclic AMP in the intracellular solution completely abolished the activation of Kir (n=7).

The selective α2‐adrenoceptor antagonist SKF‐86466 (10 μM), the selective 5‐HT1A antagonist NAN 190 as well as the selective GABAB antagonist 2‐hydroxysaclofen (10 μM) failed to block the flupirtine effect on the inward rectifier.

Flupirtine (1 μM) could not change the current induced by 50 μM NMDA.

These results show that in cultured hippocampal neurones flupirtine activates an inwardly rectifying potassium current and that a PTX‐sensitive G‐protein is involved in the transduction mechanism.
Assessment of the role of α2‐adrenoceptor subtypes in the antinociceptive, sedative and hypothermic action of dexmedetomidine in transgenic mice
Authors: Hunter J. C. , Fontana D. J. , Hedley L. R. , Jasper J. R. , Lewis R. , Link R. E. , Secchi R. , Sutton J. , Eglen R. M.
Published: January 2005

The role of α2‐adrenoceptor (AR) subtypes in the modulation of acute nociception, motor behaviour and body temperature, has been investigated by determining the activity of the α2AR selective agonist dexmedetomidine (Dex) in mice devoid of individual α2AR subtypes through either a point (α2A) or null (α2B/α2C) mutation (‘knock‐out’).

In a rodent model of acute thermal nociception, the mouse tail immersion test, Dex, in wild type (WT) control animals, produced a dose‐dependent increase in the threshold for tail withdrawal from a 52°C water bath with mean ED50 values of 99.9±14.5 (α2A), 94.6±17.8 (α2B) and 116.0±17.1 (α2C) μg kg−1, i.p.

In comparison to the WT controls, Dex (100–1000 μg kg−1, i.p.), was completely ineffective as an antinociceptive agent in the tail immersion test in the α2AAR D79N mutant animals. Conversely, in the α2BAR and α2CAR knock‐outs, Dex produced a dose‐dependent antinociceptive effect that was not significantly different from that observed in WT controls, with ED50 values of 85.9±15.0 (P>0.05 vs WT control) and 226.0±62.7 (P>0.05 vs WT control) μg kg−1 i.p., respectively.

Dex (10–300 μg kg−1, i.p.) produced a dose‐dependent reduction in spontaneous locomotor activity in the α2A, α2B and α2CAR WT control animals with ED50 values of 30.1±9.0, 23.5±7.1 and 32.3±4.6 μg kg−1, i.p., respectively. Again, Dex (100–1000 μg kg−1, i.p.) was ineffective at modulating motor behaviour in the α2AAR D79N mutants. In the α2BAR and α2CAR knock‐out mice, Dex produced a dose‐dependent reduction in spontaneous locomotor activity with ED50 values of 29.1±6.4 (P>0.05 vs WT control) and 57.5±11.3 (P>0.05 vs WT control) μg kg−1, respectively.

Dex was also found to produce a dose‐dependent reduction in body temperature in the α2A, α2B and α2CAR WT control mice with ED50 values of 60.6±11.0, 16.2±2.5 and 47.2±9.1 μg kg−1, i.p., respectively. In the α2AAR D79N mutants, Dex had no effect on body temperature at a dose (100 μg kg−1, i.p.) that produced a significant reduction (−6.2±0.5°C; P<0.01 vs vehicle) in temperature in WT controls. However, higher doses of Dex (300 and 1000 μg kg−1, i.p) produced a small, but statistically significant decrease in temperature corresponding to −1.7±0.4°C and −2.4±0.3°C (both P<0.01 vs vehicle), respectively. In the α2BAR and α2CAR knock‐out mice, Dex produced a dose‐dependent reduction in body temperature with ED50 values of 28.4±4.8 (P>0.05 vs WT control) and 54.1±8.0 (P>0.05 vs WT control) μg kg−1, respectively.

In conclusion, the data are consistent with the α2AAR being the predominant subtype involved in the mediation of the antinociceptive, sedative and hypothermic actions of Dex. This profile would appear to indicate that an α2AAR subtype selective analgesic will have a narrow therapeutic window, particularly following systemic administration.
Influence of interleukin 1α on superoxide anion, platelet activating factor release and phospholipase A2 activity of naive and sensitized guinea‐pig alveolar macrophages
Authors: Mugnai Sabrina , Ciuffi Mario , Maurizi Manuela , Bindi Daniela , Franchi‐Micheli Sergio , Zilletti Lucilla
Published: January 2005

We studied the effect exerted by hr‐interleukin‐1α (IL‐1α) on responsiveness of alveolar macrophages (AM) from naive and sensitized guinea‐pigs, through O2‐. production (by ferricytochrome C reduction), platelet‐activating factor (PAF) release (by platelet aggregation), prostaglandin E2 (PGE2) release (by a radioimmunoassay), and cytosolic phospholipase A2 (cPLA2) activity (by hydrolysis of radioactive substrate).

In naive guinea‐pig AM, 0.06 nM hr‐IL‐1α pretreatment decreased by 65% O2‐. release stimulated with 10 nM fMLP. In contrast, O2‐. production was not affected in sensitized guinea‐pig AM.

O2‐. release elicited by fMLP stimulation in both cell groups was affected by PLA2 inhibitors (10 μM bromophenacyl bromide, BPB or 10 μM methylprednisolone, MP). In contrast, 10 μM arachidonyl trifluoromethyl ketone (AACOCF3), a cPLA2 inhibitor, was ineffective.

In naive AM, PAF release was elicited by hr‐IL‐1α pretreatment and by separate fMLP‐stimulation, but when the stimulus was added to hr‐IL‐1α‐pretreated cells inhibition of PAF release was observed. In sensitized AM, PAF release was lower than that found in naive guinea‐pig AM in both hr‐IL‐1α‐pretreated and fMLP‐stimulated cells.

PGE2 release was unaffected by hr‐IL‐1α pretreatment and it was decreased by fMLP in both naive and sensitized AMs. The latter released less PGE2 than naive cells in basal conditions and after fMLP treatment.

Sensitized AM showed a greater cPLA2 activity in all experimental conditions in comparison to naive cells. cPLA2 activity assayed in the cytosolic fraction was found to be enhanced by hr‐IL‐1α pretreatment and by fMLP stimulation in naive but not in sensitized AM. However, when the stimulus was added to hr‐IL‐1α‐pretreated cells we observed a decrease in cPLA2 activity in the cytosol and an increase in the membranes, thus suggesting a translocation of enzymatic activity.

In conclusion, hr‐IL‐1α can modulate the responsiveness of AM from naive and sensitized guinea‐pigs, as suggested by changes found in the release of PAF and O2− and in cPLA2 activity; therefore, sensitization itself may affect cellular responsiveness.
Lipolytic effects of conventional β3‐adrenoceptor agonists and of CGP 12,177 in rat and human fat cells: preliminary pharmacological evidence for a putative β4‐adrenoceptor
Authors: Galitzky Jean , Langin Dominique , Verwaerde Patrick , Montastruc Jean‐Louis , Lafontan Max , Berlan Michel
Published: January 2005

The nature of rat and human fat cell β3‐adrenoceptors was investigated by studying the effects of the new β3‐adrenoceptor selective antagonist, SR 59,230A, on lipolysis induced by the conventional β3‐adrenoceptor agonists, CL 316,243 and SR 58,611A, and by the non‐conventional partial β3‐adrenoceptor agonist CGP 12,177 (a potent β1‐ and β2‐adrenoceptor antagonist with partial β3‐adrenoceptor agonist property).

In rat fat cells, the rank order of potency of agonists was: CL 316,243>isoprenaline>SR 58,611A>CGP 12,177. The three former agents were full agonists whereas CGP 12,177 was a partial agonist (intrinsic activity of 0.70). In human fat cells, the lipolytic effect of CGP 12,177 reached 25 % of isoprenaline effect. CL 316,243 was a poor inducer of lipolysis and SR 58,611A was ineffective.

In rat fat cells, lipolysis induced by CL 316,243 and SR 58,611A was competitively antagonized by SR 59,230A. Schild plots were linear with pA2 values of 6.89 and 6.37, respectively. Conversely, 0.1, 0.5 and 1 μM SR 59,230A did not modify the concentration‐response curve of CGP 12,177. A rightward shift of the curve was however observed with 10 and 100 μM of SR 59,230A. The apparent pA2 value was 5.65. The non‐selective β‐adrenergic antagonist, bupranolol, competitively displaced the concentration‐response curve of CGP 12,177 and CL 316,243. Schild plots were linear with pA2 values of 6.70 and 7.59, respectively. CL316,243‐mediated lipolytic effect was not antagonized by CGP 20,712A. In human fat cells, CGP 12,177‐mediated lipolytic effect was antagonized by bupranolol and CGP 20,712A. SR 59,230A (0.1, 1 and 10 μM) did not modify the concentration‐response curve of CGP 12,177. A rightward shift was however observed at 100 μM leading to an apparent pA2 value of 4.32.

The results suggest that the non‐conventional partial agonist CGP 12,177 can activate lipolysis in fat cells through the interaction with a β‐adrenoceptor pharmacologically distinct from the β3‐adrenoceptor, i.e. through a putative β4‐adrenoceptor. They suggest that the two subtypes coexist in rat fat cells whereas only the putative β4‐adrenoceptor mediates lipolytic effect of CGP12,177 in human fat cells.

British Journal of Pharmacology (1997) 122, 1244–1250; doi:10.1038/sj.bjp.0701523
Activation of two sites by adenosine receptor agonists to cause relaxation in rat isolated mesenteric artery
Authors: Prentice D. J. , Payne S. L. , Hourani S. M. O.
Published: January 2005

In this study we have characterized the receptor(s) in the rat mesenteric artery mediating relaxant responses to adenosine and a number of adenosine analogues, N6 ‐R‐phenylisopropyladenosine (R‐PIA), N6‐cyclopentyladenosine (CPA), N6‐(3‐iodo‐benzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA) and 5′‐N‐ethylcarboxamidoadenosine (NECA), by use of the non‐selective antagonist 8‐sulphophenyltheophylline (8‐SPT) and the A2A selective ligands 2‐[p‐(2‐carbonylethyl)‐phenylethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) and 4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]‐triazolo[2,3‐a][1,3,5]‐triazin‐5‐ylamino]ethyl) phenol (ZM 241385). We have also studied the effects of endothelial removal and uptake inhibition by nitrobenzylthioinosine (NBTI) and the effects of the A3 receptor antagonist 1,3‐dipropyl‐8‐(4‐acrylate)phenylxanthine (BWA1433).

Adenosine, NECA, CPA and R‐PIA all elicited relaxant responses in tissues precontracted with phenylephrine (1 μM) with the following potency order: NECA>R‐PIA>adenosine=CPA. However, E/[A] curves to NECA were biphasic. CGS 21680 was inactive at concentrations up to 30 μM and IB‐MECA elicited relaxant responses which were resistant to blockade by 8‐SPT and BWA1433 (100 μM).

Removal of the endothelium produced a small but significant decrease in the asymptote of the high potency phase of E/[A] curves to NECA with no change in p[A]50. E/[A] curves to adenosine were not altered by removal of the endothelium. However, there were small rightward shifts of E/[A] curves to CPA and R‐PIA in the absence of endothelium.

Inhibition of uptake by NBTI (1 μM) had no effect on E/[A] curves to NECA, CPA or R‐PIA, but E/[A] curves to adenosine were significantly left‐shifted in the presence of NBTI.

8‐SPT (10–100 μM) caused significant rightward shifts of the high potency phase of the E/[A] curves to NECA (pA2=5.63±0.26). The second phase of the concentration‐response curve to NECA appeared to be resistant to blockade by 8‐SPT, as were E/[A] curves for adenosine, CPA or R‐PIA. However, in the presence of NBTI (1 μM), 8‐SPT (100 μM) gave significant rightward shifts of E/[A] curves to adenosine.

ZM 241385 (0.1–1 μM) produced significant rightward shifts of the high potency phase of NECA E/[A] curves (pA2=7.65±0.25 in the presence and 7.20±0.12 in the absence of endothelium), while curves to R‐PIA were not significantly shifted by 1 μM ZM 241385. In the presence of NBTI E/[A] curves to adenosine were significantly rightward shifted by ZM 241385 (0.1 μM, pA2=7.50±0.16).

In conclusion, the results suggest activation of A2B receptors located primarily on the smooth muscle by low concentrations of NECA and by adenosine under conditions of uptake blockade, and of another, as yet undefined site which may be intracellular, by higher concentrations of NECA, by CPA, R‐PIA and adenosine under conditions where uptake is operational.