bluuebunny/crossref_metadata_2025 huggingface dataset:
Potentiation by tumour necrosis factor‐α of calcium signals induced by bradykinin and carbachol in human tracheal smooth muscle cells
Authors: Amrani Yassine , Martinet Nadine , Bronner Christian
Published: July 2012

The effect of tumour necrosis factor‐α (TNFα) on the increase in cytosolic free calcium ([Ca2+])i induced by carbachol and bradykinin (BK) was investigated in human tracheal smooth muscle cells in culture (TSMC). BK (10−12–10−9m) and carbachol (10−7–10−3m) produced a concentration‐dependent increase in [Ca2+]i (pD2 = 10.73 ± 0.05 and 5.57 ± 0.03 respectively). The increase in [Ca2+]i was significantly enhanced for both agonists in TNFα‐treated cells (10 ng ml−1 for 24 h). However, pD2 values were not modified (10.78 ± 0.03 and 5.62 ± 0.04 for BK and carbachol, respectively) suggesting that no change in the apparent receptor affinity occured. Thus, TNFα induced‐alterations in Ca2+ homeostasis in human TSMC may be a key mechanism underlying airway hyperreactivity.
The role of nitric oxide in cardiac depression induced by interleukin‐1β and tumour necrosis factor‐α
Authors: Schulz Richard , Panas Donna L. , Catena Robert , Moncada Salvador , Olley Peter M. , Lopaschuk Gary D.
Published: July 2012

Myocardial dysfunction during septic shock is associated with enhanced production of cytokines such as interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α). These cytokines depress cardiac mechanical function by a mechanism which is not well defined.

Bacterial endotoxin or cytokines cause the expression of Ca2+‐independent nitric oxide (NO) synthase in cardiac myocytes, vascular endothelial cells and endocardial endothelial cells, causing enhanced production of NO. As NO has negative inotropic actions on cardiac muscle, we tested the sum effects of IL‐1β plus TNF‐α in the intact heart to determine whether enhanced expression of NO synthase activity in the cells that comprise the heart is involved in cardiac depression associated with cytokine stimulation.

Rat isolated working hearts perfused with IL‐1β plus TNF‐α showed a markedly greater depression in contractile function, measured as cardiac work, after 2 h of perfusion compared with time‐matched control hearts. The depressant action of IL‐1β plus TNF‐α was first apparent after 1 h of perfusion; no early (15 min) cardiac depressant actions were seen.

The competitive inhibitor of Ca2+‐dependent and Ca2+‐independent NO synthases, NG‐nitro‐l‐arginine methyl ester (l‐NAME, 3 μm) when given concurrently with IL‐1β plus TNF‐α prevented the loss in contractile function such that these hearts after 2h of perfusion had similar function to time‐matched controls. l‐NAME did not acutely reverse the loss of contractile function in hearts exposed for 2 h to IL‐1β plus TNF‐α. The protective action of l‐NAME in the presence of cytokines was concentration‐dependent and was not seen at a higher concentration (10 μm) due to the significant reduction in coronary flow observed at this concentration.

In contrast, when l‐NAME (3 μm) was given in the absence of IL‐1β plus TNF‐α it depressed contractile function over the 2h perfusion period by significantly reducing coronary flow.

Inhibition of protein synthesis with cycloheximide (Cx) abolished the loss in function that occurred over 2h in both control and IL‐1β plus TNF‐α‐treated hearts.

Inducible, Ca2+‐independent NO synthase activity was not observed in freshly isolated hearts but was observed in control hearts perfused for 2 h in vitro and was doubled in hearts perfused with IL‐1β plus TNF‐α. Cx prevented the expression of Ca2+‐independent NO synthase in both control and cytokine‐treated hearts.

In summary, these results suggest that the depression of myocardial function by IL‐1β plus TNF‐α is mediated, at least in part, by induction of Ca2+‐independent NO synthase activity in the heart.
Involvement of B2 receptors in the bradykinin‐induced relaxation of guinea‐pig isolated trachea
Authors: Da Silva A. , Amrani Y. , Trifilieff A. , Landry Y.
Published: July 2012

The aim of this study was to determine the receptor type and involvement of arachidonic acid metabolites in bradykinin‐induced relaxation of the guinea‐pig isolated trachea.

In the resting tracheal preparation, bradykinin (0.1 nm − 30 μm induced a concentration‐related contractile response (pD2 = 8.8 ± 0.3). The maximal tension (1056 ± 321 mg) was observed at 0.3 μm bradykinin. In contrast, when tracheal preparations were pre‐contracted with histamine (30 μm leading to a half‐maximum response), a concentration‐related relaxation was observed with bradykinin. At the highest concentration of bradykinin used (3 μm), a reversal of 63 ± 13% of the contractile response to histamine was observed. Both effects of bradykinin were inhibited by the cyclo‐oxygenase inhibitor, indomethacin (1 μm). In concentration‐response curves, melittin (10 nm − 1 μm), a direct activator of phospholipase A2, mimicked both effects of bradykinin. The highest concentration of melittin used (1 μm), induced a tension of 813 ± 120 mg and led to the reversal of 41 ± 8% of the contractile response to histamine. The contractile effect of melittin was inhibited in the presence of both indomethacin (1 μm) and AA861 (1 μm), a 5‐lipoxygenase inhibitor.

[Des Arg9]‐bradykinin (1 nm‐3 μm), a B1‐receptor agonist, was unable to relax precontracted guinea‐pig tracheal preparations. The relaxation induced by bradykinin was antagonized by the B2 receptor antagonists, Hoe 140 (d‐Arg0[Hyp3, Thi5, d‐Tic7, Oic8]bradykinin) and NPC 17761 (d‐Arg0[Hyp3, d‐HypE(trans‐thiophenyl)7, Oic8]bradykinin). Hoe 140 (0.1 μm to 0.6 μm) behaved as a non‐competitive antagonist with an apparent pA2 = 7.2 ± 0.4, whereas NPC 17761 (0.3 to 1 μm) competitively antagonized bradykinin‐induced relaxation with a pKB = 7.3 ± 0.2. The Schild regression slope did not differ from unity, 0.96 ± 0.20, P < 0.05.

These data demonstrate that bradykinin‐induced relaxation of guinea‐pig trachea occurs via the activation of bradykinin B2‐receptors. The stimulation of B2‐bradykinin receptors induces the activation of the cyclo‐oxygenase pathway, leading either to contraction or relaxation depending on the tone of the trachea.
The kinin B1 receptor antagonist des‐Arg9‐[Leu8]bradykinin: an antagonist of the angiotensin AT1 receptor which also binds to the AT2 receptor
Authors: Pruneau D. , Duvoid A. , Luccarini J.M. , Bélichard P. , Bonnafous J.C.
Published: July 2012

Agonists and antagonists of kinin B1 and B2 receptors were evaluated in vitro for their effects against angiotensin II (AII)‐induced contractile responses in the rabbit aorta and for their binding properties to angiotensin AT1 and AT2 receptors from purified membrane of rat liver and lamb uterus respectively.

In aortic rings, the kinin B1 receptor antagonist, des‐Arg9‐[Leu8]bradykinin (BK) (3–100 μm) caused a concentration‐dependent decrease in sensitivity and a depression of the maximum response to AII. Des‐Arg10‐[Leu9]kallidin (KD), des‐Arg9‐BK, des‐Arg10‐KD, BK or KD at 3 μm had no effect against All‐induced contractions.

Des‐Arg9‐[Leu8]BK (3 or 100 μm) did not affect contractions of aortic rings to histamine, potassium chloride, endothelin‐1, 5‐hydroxytryptamine, noradrenaline and the thromboxane A2‐mimetic, U46619.

Des‐Arg9‐[Leu8]BK displaced [125I]‐Sar1‐AII binding to the AT1 subtype in rat liver membranes with a Ki value of 1.1 ± 0.4 μm. Values of Ki for des‐Arg9‐BK and KD were 45 ± 13 μm and 25 ± 22 μm, respectively. The other kinin derivatives des‐Arg10‐KD, BK and des‐Arg10‐[Leu9]KD at concentrations up to 100 μm did not bind to the AT1 receptor.

All the kinin derivatives except BK bound to AT2 receptors in lamb uterus membranes. Values of Ki for des‐Arg9‐[Leu8]BK, des‐Arg10‐[Leu9]KD, des‐Arg9‐BK, des‐Arg10‐KD and KD were 0.3 ± 0.1, 0.7 ± 0.1, 1.2 ± 0.3, 1.5 ± 0.3 and 7.0 ± 1.6 μm, respectively.

In conclusion, des‐Arg9‐[Leu8]BK is an insurmountable antagonist of All‐induced contractions in the rabbit aorta and also binds with a relatively high affinity to AT1 and AT2 receptors in isolated membrane fractions. These additional properties of des‐Arg9‐[Leu8]BK should be considered when it is used as an antagonist to characterize kinin B1 receptors.
Differential effects of phosphoramidon on contractile responses to angiotensin II in rat blood vessels
Authors: Chen Lihua , McNeill J. Robert , Wilson Thomas W. , Gopalakrishnan Venkat
Published: July 2012

Cumulative concentration‐tension response (C‐R) curves to angiotensin II (AII), big endothelin‐1 (big ET‐1), ET‐1 and arginine vasopressin (AVP) were determined in endothelium intact‐ring preparations of aorta, mesenteric artery and tail artery isolated from adult male Sprague‐Dawley rats in the presence or absence of the neutral metalloprotease inhibitor, phosphoramidon.

The order of sensitivity of the three rat vascular smooth muscle preparations to AII, big ET‐1 and ET‐1 was aorta> mesenteric artery> tail artery whereas that for AVP was reversed, namely, tail artery> mesenteric artery> aorta.

Phosphoramidon blocked the responses to AII in a concentration‐dependent manner, whereas even very high concentrations of phosphoramidon (100 μm) failed to affect the tension responses evoked by ET‐1 and AVP in all three preparations. Low concentrations of phosphoramidon (10 μm) produced significant increases in EC50 values for AII in tail artery (P<0.01) and mesenteric artery (P<0.05) but not in aorta. The rank order of sensitivity to the inhibition by phosphoramidon was tail artery< mesenteric artery< aorta. Phosphoramidon‐evoked rightward shifts in the C‐R curves to AII were much higher than those to big ET‐1 in both mesenteric artery and tail artery.

In endothelium‐denuded preparations, AII failed to evoke any increases in tension in tail artery while the responsiveness of the mesenteric artery to AII was reduced significantly relative to endothelium‐intact tissues with a rightward shift in the C‐R curve and a decrease in the maximal response. On the other hand, the C‐R curve to AII was shifted to the left in aorta following removal of the endothelium. Importantly, ET‐1 and AVP evoked vasoconstrictor responses were unaffected by the inclusion of a high concentration of phosphoramidon (100 μm) in endothelium‐denuded aorta and mesenteric artery.

The results suggest that AII‐evoked tension responses of blood vessels such as tail artery are completely endothelium‐dependent; in relatively larger blood vessels such as mesenteric artery they are partially endothelium‐dependent while in much bigger conduit type blood vessel such as aorta, they are endothelium‐independent. It is concluded that the vasoconstrictor responses to AII in mesenteric artery and tail artery may be mediated by the release of endothelins from the endothelium by increased formation from big ET, an effect that is blocked by phosphoramidon.
The protective action of chlormethiazole against ischaemia‐induced neurodegeneration in gerbils when infused at doses having little sedative or anticonvulsant activity
Authors: Cross A.J. , Jones J.A. , Snares M. , Jostell K.‐G. , Bredberg U. , Green A.R.
Published: July 2012

The effect of chlormethiazole administration on delayed neuronal death in gerbil hippocampus following transient global ischaemia has been examined. Chlormethiazole was administered either intraperitoneally or by intravenous infusion with either the dose or the time of infusion varied.

Chlormethiazole (600 μmol kg−1, i.p.) given 60 min after ischaemia produced substantial (>60%) neuroprotection when damage was assessed 5, 14 or 21 days later, indicating the drug does not merely delay cell death.

Infusion protocols were developed which would result in sustained and defined plasma concentrations. Chlormethiazole (930 μmol kg−1) was then infused intravenously for 30min, 76.5 min or HOmin in ways resulting in sustained plasma concentrations of 200, 100 and 50 nmol ml−1 respectively. When treatment was initiated 30 min after the ischaemic episode all protocols provided effective neuroprotection. There was a dose‐dependent decline in protection when plasma chlormethiazole concentrations of 50, 30 and 10 nmol ml−1 were sustained for 110 min with no protection observed at 10 nmol ml−1.

In contrast, when a plasma concentration of 10 nmol ml−1 was sustained by infusion for 24 h, almost total neuroprotection against the ischaemic damage was achieved. This plasma concentration produced no sedative or anticonvulsant activity.

These data suggest that neuroprotection depends on both dose and duration of chlormethiazole administration and that excellent neuroprotection is possible in the absence of the sedative and anticonvulsant effects of the drug.
Inhibition of rabbit aortic smooth muscle cell proliferation by selective inhibitors of protein kinase C
Authors: Newby Andrew C. , Lim Karene , Evans Marilyn A. , Brindle Nicholas P.J. , Booth Robert F.G.
Published: July 2012

We studied the effect of two structurally‐related, selective inhibitors of protein kinase C, Ro 31–8220 and Ro 31–7549, on the reinitiation of proliferation in quiescent first passage rabbit aortic smooth muscle cells in response to (a) the direct activator of protein kinase C, phorbol dibutyrate (PDBu), (b) platelet‐derived growth factor (PDGF), (c) a combination of PDGF and 5‐hydroxytryptamine (5‐HT) or (d) serum.

Ro 31–8220 and Ro 31–7549 concentration‐dependently inhibited prliferation in response to each mitogen. The inhibitory potency (IC50) of Ro 31–8220 and Ro 31–7549, respectively, was similar against proliferation induced by PDBu (0.55 and 1.1 μm), PDGF (0.6 and 0.9 μm), PDGF and 5‐HT (0.68 and 1.1 μm), although slightly less against serum (1.7 and 5 μm). The effects of the protein kinase C inhibitors on proliferation could not be ascribed to cytotoxicity. Neither Ro 31–8220 nor Ro 31–7549 (0.3‐3 μm) inhibited PDGF receptor tyrosine phosphorylation.

The results show that Ro 31–8220 and Ro 31–7549 are potent inhibitors of smooth muscle cell proliferation in response to a direct activator of protein kinase C, the defined growth factors, PDGF and 5‐HT, and the complex mixture of mitogens in serum. Protein kinase C activation thus appears to be an important growth transducing mechanism for each of these agents.
Pharmacological analysis of the interaction between purinoceptor agonists and antagonists in the guinea‐pig taenia caecum
Authors: Prentice D.J. , Shankley N.P. , Black J.W.
Published: July 2012

In the absence of adenosine uptake inhibition, adenosine produced a concentration‐dependent (threshold 30 μm) relaxation of the 5‐methylfurmethide pre‐contracted guinea‐pig taenia caecum. The relaxation was not blocked by 8‐phenyltheophylline (8‐PT, 3 μm) or 1,3‐dipropyl, 8‐cyclopentylxanthine (DPCPX, 30 μm).

In the presence of the adenosine uptake inhibitor, dipyridamole (Dip, 3μm), a biphasic adenosine concentration‐effect curve was obtained (threshold 0.3 μm). The time course of the responses to adenosine in the absence of Dip was similar to that of the second phase responses in the presence of Dip and occurred over the same adenosine concentration‐range. 5′‐(N‐ethyl) carboxamido‐adenosine (NECA) concentration‐effect curves (in the absence of Dip) were also biphasic. Only the first phases of the concentration‐effect curves obtained with NECA and adenosine (plus Dip) were inhibited by 8‐PT. The pA2 values for 8‐PT of 6.7 and 7.0 versus adenosine and NECA, respectively, were consistent with actions at P1‐purinoceptors. There was a trend towards an increase in the upper asymptote of the first phase of the NECA curve in the presence of increasing concentrations of 8‐PT. The A1‐purinoceptor selective antagonist, DPCPX, also blocked only the first phase of the NECA concentration‐effect curve and produced a significant increase in the upper asymptote. The pA2 value (6.8) obtained was consistent with activation of A2‐subtype P1purinoceptors by the low concentrations of NECA.

There was no correlation between A1‐purinoceptor affinity and the propensity to cause the increase in the upper asymptote of the first phase of the NECA concentration‐effect curves amongst a series of 9‐methyl adenine analogues, suggesting that the amplification was not due to inhibition of an underlying A1purinoceptor‐mediated contractile response.

DPCPX (10 μm) produced a significant increase in the upper asymptote of the NECA concentration‐effect curve, but had no effect on isoprenaline curves whereas the phosphodiesterase inhibitor Ro 20–1724 (30 μm) produced a significant increase in the upper asymptote of both NECA and isoprenaline concentration‐effect curves. Therefore the amplification of the first phase responses by DPCPX did not appear to be due to phosphodiesterase inhibition.

It was not possible to conclude whether second phase responses to adenosine and NECA were mediated by intracellular or extracellular sites of action. However, if intracellular sites of action were involved then adenosine did not apparently gain access by the Dip‐sensitive uptake system.
Contractile actions of thrombin receptor‐derived polypeptides in human umbilical and placental vasculature: evidence for distinct receptor systems
Authors: Tay‐Uyboco Jean , Poon Man‐Chiu , Ahmad Sultan , Hollenberg Morley D.
Published: July 2012

We studied the structure‐activity profiles of four thrombin receptor‐derived polypeptides (TRPs) (P5, SFLLR; P5‐NH2, SFLLR‐NH2; P7, SFLLRNP; P7‐NH2, SFLLRN) in contractile human placental artery (PA), umbilical artery (UA) and umbilical vein (UV) preparations and in a human platelet aggregation assay.

The contractile actions of the TRPs in the two arterial preparations were endothelium‐independent, whereas in the UV tissue a contractile response was observed only in an endothelium‐denuded preparation; no endothelium‐mediated relaxation responses were observed in any of the vascular preparations.

In the three vascular preparations, the contractile responses required extracellular calcium and were attenuated by the tyrosine kinase inhibitor, genistein.

The relative contractile orders of potencies of the TRPs in the three vascular preparations were distinct from each other (PA: P7‐NH2>P7>P5‐NH2>P5; UA: P7‐NH2≥P5‐P72‐∼P7> >P5; UV: P5‐NH2 > > P7‐NH2 = P7 > > P5) and these were in turn distinct from the potency order observed in the platelet aggregation assay (P5‐NH2≥P7‐NH2>P7> >P5).

Despite the markedly dissimilar TRP potency orders in the placental artery and umbilical vein preparations, the cDNA sequences for the thrombin receptor obtained by polymerase chain reaction cloning of cDNA from the two tissue sources were identical.

We conclude that the four tissues studied possess functionally distinct thrombin receptor systems that interact in a distinct way with agonist peptides. In view of the identity of the thrombin receptor cDNA in the two tissues displaying the most dissimilar structure‐activity profiles, we suggest that in different tissues, differences in post‐translational receptor processing or differences in receptor‐effector coupling interactions may result in unique thrombin receptor systems that can display distinct structure‐activity profiles.
The pharmacology and distribution of human 5‐hydroxytryptamine2B (5‐HT2b) receptor gene products: comparison with 5‐HT2a and 5‐HT2c receptors
Authors: Bonhaus Douglas W. , Bach Chinh , DeSouza Andrea , Salazar F.H. Rick , Matsuoka Barbara D. , Zuppan Patricia , Chan Hardy W. , Eglen Richard M.
Published: July 2012

Full length clones of the human 5‐HT2B receptor were isolated from human liver, kidney and pancreas. The cloned human 5‐HT2B receptors had a high degree of homology (∼80%) with the rat and mouse 5‐HT2B receptors.

PCR amplification was used to determine the tissue distribution of human 5‐HT2B receptor mRNA. mRNA encoding the 5‐HT2B receptor was expressed with greatest abundance in human liver and kidney. Lower levels of expression were detected in cerebral cortex, whole brain, pancreas and spleen. Expression was not detected in heart.

Northern blot analysis confirmed the presence of 5‐HT2B receptor mRNA (a 2.4 kB sized band) in pancreas, liver and kidney. An additional 3.2 kB sized band of hybridization was detected in liver and kidney. This raises the possibility of a splice variant of the receptor or the presence of an additional homologous receptor.

The human 5‐HT2B receptor was expressed in Cos‐7 cells and its ligand binding characteristics were compared to similarly expressed human 5‐HT2A and 5‐HT2C receptors. The ligand specificity of the human 5‐HT2B receptor (5‐HT > ritanserin > SB 204741 > spiperone) was distinct from that of the human 5‐HT2A (ritanserin > spiperone > 5‐HT > SB 204741) and 5‐HT2C (ritanserin > 5‐HT > spiperone = SB 204741) receptors. On the basis of a higher affinity for ketanserin and a lower affinity for yohimbine the human 5‐HT2B receptor also appeared to differ from the rat 5‐HT2B receptor.

These findings confirm the sequence of the human 5‐HT2B receptor and they demonstrate that the receptor has a widespread tissue distribution. In addition, these data suggest that there are differences in ligand affinities between different species homologues of the receptor. Finally, the finding of two distinct bands on the Northern blots of liver and kidney raises the possibility of splice variants or subtypes of 5‐HT2B receptors, within these tissues.
Elevation of plasma noradrenaline levels in urethane‐anaesthetized rats by activation of central prostanoid EP3 receptors
Authors: Yokotani Kunihiko , Nishihara Makoto , Murakami Yoshinori , Hasegawa Takashi , Okuma Yasunobu , Osumi Yoshitsugu
Published: July 2012

We studied the effects of intracerebroventricular (i.c.v.) administration of prostaglandin E2 (PGE2) and its receptor subtype ligands on plasma levels of catecholamines in urethane‐anaesthetized rats.

Administration of PGE2 (0.15, 0.3 and 1.5 nmol per animal, i.c.v.) dose‐dependently elevated plasma levels of noradrenaline (NA), while the levels of adrenaline were not affected.

Administration of sulprostone (EP3/EP1 agonist) and misoprostol (EP3/EP2 agonist) effectively elevated plasma NA levels in a dose‐dependent manner (0.1, 0.3, and 1.0 nmol per animal). Butaprost (EP2 agonist) (0.3, 1.0 and 3.0 nmol per animal) was without effect. 17‐Phenyl‐ω‐trinor PGE2 (EP1/EP3 agonist) effectively elevated plasma NA levels only at its highest dose (1.0 nmol per animal), but this elevation was not attenuated by pretreatment with SC‐19220 (selective EP1 antagonist) (20 nmol per animal, i.c.v.).

The potency of these test agents in elevating plasma levels of NA was as follows; misoprostol > sulprostone > PGE2> > 17‐phenyl‐ω‐trinor PGE2 > > > butaprost. These results suggest that activation of central prostanoid EP3‐receptors induces central sympathetic outflow in rats.
Cytokine‐mediated inflammatory hyperalgesia limited by interleukin‐10
Authors: Poole S. , Cunha F.Q. , Selkirk S. , Lorenzetti B.B. , Ferreira S.H.
Published: July 2012

The effect of interleukin‐10 (IL‐10) upon the hyperalgesic activities in rats of bradykinin, tumor necrosis factor α (TNFα), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E2 (PGE2) and carrageenin were investigated in a model of mechanical hyperalgesia.

Hyperalgesic responses to bradykinin (1 μg) were inhibited in a dose‐dependent manner by prior treatment with IL‐10 (1–100 ng).

Hyperalgesic responses to TNFα (2.5 pg), IL‐1β (0.5 pg) and IL‐6 (1.0 ng) but not to IL‐8 (0.1 ng) and PGE2 (50 ng and 100 ng) were inhibited by prior treatment with IL‐10 (10 ng).

Hyperalgesic responses to carrageenin (100 μg) were inhibited by IL‐10 (10 ng) when this cytokine was injected before but not after the carrageenin.

A monoclonal antibody to mouse IL‐10 potentiated the hyperalgesic responses to carrageenin (10 μg) and TNFα (0.025 pg) but not that to IL‐8 (0.01 ng).

In in vitro experiments in human peripheral blood mononuclear cells (MNCs), IL‐10 (0.25‐4.0 ng ml−1) inhibited in a dose‐dependent manner PGE2 production by MNCs stimulated with IL‐1β (1–64 ng ml−1) or endotoxin (lipopolysaccharide, LPS, 1 iu=143 pg ml−1) but evoked only small increases in IL‐Ira production.

These data suggest that IL‐10 limits the inflammatory hyperalgesia evoked by carrageenin and bradykinin by two mechanisms: inhibition of cytokine production and inhibition of IL‐1β evoked PGE2 production. Our data suggest that the latter effect is not mediated via IL‐10 induced IL‐Ira and may result from suppression by IL‐10 of prostaglandin H synthase‐2 (COX‐2).
Gi proteins and the response to 5‐hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF
Authors: Day Nancy S. , Ge Tong , Codina Juan , Birnbaumer Lutz , Vanhoutte Paul M. , Boulanger Chantal M.
Published: July 2012

The receptor‐mediated release of endothelium‐derived relaxing factor(s) (EDRF) requires the presence of different functional G proteins in endothelial cells. Release of EDRF in response to 5‐hydroxytryptamine (5‐HT), which involves activation of pertussis toxin‐sensitive Gi proteins, is impaired in both regenerated endothelium of the coronary artery following balloon catheterization and in porcine cultured endothelial cells. This study used porcine cultered endothelial cells as a model of regenerated endothelium to determine if the abnormal release of EDRF in response to 5‐HT may be associated with the loss of functional pertussis toxin‐sensitive Gi proteins.

Binding studies on porcine cultured endothelial cells demonstrated specific binding sites for [3H]‐5‐HT. Scatchard analyses revealed a single binding site for [3H]‐5‐HT with Kd of 7.2 ±3.5 nM and maximal binding Bmax) of 121.4±51.3 fmol mg−1 protein. Binding of [3H]‐5‐HT was displaced by methiothepin (5‐HT1 and 5‐HT2 antagonist; Ki = 62 ±1.2 nM), but not by ketanserin (preferential 5‐HT2 antagonist).

Giα protein was expressed in cultured but not in native endothelial cells. Giα2 and Giα3 proteins were expressed to significant levels in porcine native and cultured endothelial cells, as detected by Northern and Western blot analysis.

In membranes from cultured endothelial cells, two bands of 40 and 41 kDa, which corresponded to the Giα2 and the combination of Giα3‐Giα1 proteins, respectively, were ADP‐ribosylated by pertussis toxin. The labelling intensity was Gia2>Giα3‐Giαl and the amount of ADP‐ribosylation was not different between porcine native and cultured endothelial cells. Stimulation of the cultured cells with 5‐HT (3 × 10−6 m; 4 min) decreased significantly further ADP‐ribosylation of Gia2 by pertussis toxin, but not that of Giα3 and/or Giαl.

The present results suggest that porcine endothelial cell culture may lead to the abnormal expression of Giαl protein and that the dysfunctional release of EDRF from cultured porcine endothelial cells in response to 5‐HT is not associated with the loss of Giα proteins or the absence of 5‐HT binding sites.
Endothelial function in spontaneously hypertensive rats: influence of quinapril treatment
Authors: Kähönen Mika , Mäkynen Heikki , Wu Xiumin , Arvola Pertti , Pörsti Ilkka
Published: July 2012

Angiotensin converting enzyme (ACE) inhibition has been shown to restore the impaired endothelial function in hypertension, but the mediators underlying the promoted endothelium‐dependent dilatation have not been fully characterized. Therefore, we investigated the effects of 10‐week‐long quinapril therapy (10 mg kg−1 day−1) on responses of mesenteric arterial rings in vitro from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto (WKY) rats.

Endothelium‐dependent relaxations of noradrenaline (NA)‐precontracted rings to acetylcholine (ACh) and adenosine 5′‐diphosphate (ADP) were similar in WKY rats and quinapril‐treated SHR and more pronounced than in untreated SHR. The nitric oxide (NO) synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) attenuated the relaxations in both WKY groups and quinapril‐treated SHR, and completely inhibited them in untreated SHR. When endothelium‐dependent hyperpolarization was prevented by precontraction of the preparations with potassium chloride (KC1), no differences were found in relaxations to ACh and ADP between the study groups. In addition, in NA‐precontracted rings the L‐NAME‐ and indomethacin‐resistant relaxations to ACh were partially prevented by apamin, an inhibitor of calcium‐activated potassium channels.

Interestingly, in quinapril‐treated SHR but not in the other groups, exogenous bradykinin potentiated the relaxations to ACh in both NA‐ and KCl‐precontracted arterial rings.

Contractile sensitivity of endothelium‐intact rings to NA was reduced in SHR by quinapril, and was more effectively increased by L‐NAME in quinapril‐treated than untreated SHR.

In conclusion, since the relaxations to ACh and ADP in quinapril‐treated SHR were augmented in the absence and presence of NO synthesis inhibition but not under conditions which prevented hyperpolarization, enhanced endothelium‐dependent relaxation after long‐term ACE inhibition can be attributed to increased endothelium‐dependent hyperpolarization. However, the potentiation of the response to ACh by exogenous bradykinin in quinapril‐treated SHR, as well as the increased attenuating effect of the endothelium on NA‐induced contractions in these animals appear to result from enhanced endothelium‐derived NO release.
Absence of a losartan interaction with renal lithium excretion in the rat
Authors: Barthelmebs Mariette , Alt‐Tebacher Martine , Madonna Olivier , Grima Michèle , Imbs Jean‐Louis
Published: July 2012

The interaction of losartan, a non‐peptide specific AT1 receptor antagonist with the renal handling of lithium was analysed in conscious normotensive Wistar rats and compared with the known increase in renal tubular lithium reabsorption induced by the non‐steroidal anti‐inflammatory drug, indomethacin.

The rats were treated for five days with losartan (10 mg kg−1 day−1, orally), indomethacin (2.5 mg kg−1 day−1, intramuscularly) or their solvents. Lithium chloride (16.7 mg kg−1, i.p.) was given as a single dose on the fifth day; renal functions were then measured.

Indomethacin, in the absence of any effect on creatinine clearance, increased renal fractional lithium reabsorption and led to an increase in plasma lithium levels.

Losartan did not modify renal lithium handling and its plasma level. No change was observed in renal lithium clearance, the quantity of filtered lithium or the fractional reabsorption of the metal. As expected, losartan had no effect on systolic blood pressure in normotensive rats.

In conclusion, our results indicate that losartan, when given orally in the rat at a dose of 10 mg kg−1 day−1 over five days, does not modify renal lithium handling. They suggest that blockade of the angiotensin II receptors does not interfere with renal lithium reabsorption, which occurs mainly at a proximal tubular site.
Pituitary adenylate cyclase‐activating polypeptide, helospectin, and vasoactive intestinal polypeptide in human corpus cavernosum
Authors: Hedlund P. , Alm P. , Ekström P. , Fahrenkrug J. , Hannibal J. , Hedlund H. , Larsson B. , Andersson K.‐E.
Published: July 2012

The distribution and effects of pituitary adenylate cyclase‐activating polypeptide (PACAP‐27 and −38), helospectin (Hel‐l and Hel‐2), and vasoactive intestinal polypeptide (VIP), were investigated in isolated preparations of human corpus cavernosum (CC).

Immunohistochemistry revealed coinciding profiles of nerve structures that showed immunoreactivities for VIP and PACAP, and VIP and Hel. Confocal microscopy showed the co‐existence of VIP‐ and PACAP‐immunoreactivities, and VIP‐ and Hel‐immunoreactivities in most (90%) varicose nerve structures.

As determined by radioimmunoassay, the amounts of VIP, PACAP‐27, and PACAP‐38 in the preparations were 61.7 ±11.6, 0.1 ±0.05, and 3.7±0.5 pmol g−1 wet weight of tissue (pmol g−1 wet wt.), respectively. In tissue from patients with diabetes, the content of VIP was lower (13.7 ±0.5 pmol g−1 wet wt.), whereas that of PACAP (−27 and −38) was unchanged.

Cyclic nucleotide levels were determined in preparations exposed to PACAP‐27, PACAP‐38, Hel‐1, Hel‐2, and VIP. All the peptides, but Hel‐2, significantly increased the concentrations of cyclic AMP, whereas the levels of cyclic GMP were unchanged.

The peptides concentration‐dependently relaxed noradrenaline‐contracted preparations. The order of potency was VIP>PACAP 27>Hel‐1>Hel‐2>PACAP‐38.

Hel‐1, VIP and PACAP‐27 effectively counteracted electrically induced contractions. At 10−6 m, the highest peptide concentration used, the inhibitory effects obtained reached 96±3%, 87±6%, and 80±3%, respectively. 7 The results suggest that PACAP and Hel‐1 are co‐localized with VIP in nerve structures within the human cavernous tissue, and that the peptides are effective relaxants of CC preparations in vitro. The role of the investigated peptides for penile erection remains to be established.
Failure of the putative neuropeptide Y antagonists, benextramine and PYX‐2, to inhibit Y2 receptors in rat isolated prostatic vas deferens
Authors: Palea S. , Corsi M. , Rimland J.M. , Trist D.G. , Ratti E.
Published: July 2012

The pharmacological activity of neuropeptide Y (NPY) and some analogues in inhibiting the twitch contractions induced by electrical stimulation (single pulses at 25 V, 0.15 Hz, 1 ms) in the prostatic portion of the rat isolated vas deferens was investigated. The rank order of agonist potency was: PYY > NPY2–36 > NPY >> NPY13–36 >> NPY>8–36 >>> [Leu31, Pro34]NPY = hPP, which is consistent with the activation of a Y2 receptor.

The putative Y1 and Y2 antagonist, benextramine (BXT), incubated at 100 μm for 10 or 60 min, was ineffective against PYY‐induced inhibition of the twitch response, suggesting that the prejunctional Y2 receptor in this tissue is different from the postjunctional one reported in the literature to be sensitive to BXT blockade.

The putative NPY antagonist, PYX‐2, incubated at 1 μm for 20 min, was completely ineffective in antagonizing PYY‐induced inhibition of twitches.

The twitch response was totally inhibited by suramin (100 μm) but was little affected by prazosin (1 μm). Futhermore, NPY was without effect on the dose‐response curve to ATP in resting conditions. Taken together, these results suggest that in our paradigm, NPY inhibits the release of a purinergic neurotransmitter which mediates contraction of the prostatic portion of the rat vas deferens.
Role of nitric oxide (NO) in ocular inflammation
Authors: Wang Zun‐Yi , Håkanson Rolf
Published: July 2012

The actions of nitric oxide (NO) have been investigated in the rabbit eye, with particular emphasis on the relationship between NO and C‐fibres and on those effects of NO that may be of importance in the inflammatory response to C‐fibre stimulation.

The NO synthase inhibitor, NG‐nitro‐L‐arginine (L‐NAME; 10–200 mg kg−1), but not the inactive analogue D‐NAME (200 mg kg−1), was found to block the inflammatory response induced by infrared irradiation of the iris in a dose‐dependent manner. The inhibitory effects of L‐NAME (200 mg kg−1) were partially reversed by L‐arginine (500 mg kg−1), but not by D‐arginine (500 mg kg−1).

L‐NAME (200 mg kg−1) virtually abolished the ocular effects of intravitreal injection of calcitonin gene‐related peptide (CGRP) (0.3 nmol).

The concentration of CGRP in aqueous humour from untreated rabbit eyes was 0.1 +0.001 nmol 1−1. Irradiation of the iris raised the CGRP concentration to 8.9±1.5 nmol 1−1. L‐NAME (200 mg kg−1) greatly suppressed the irradiation‐evoked release of CGRP, the concentration in the aqueous humour being 1.2 ± 0.2 nmol 1−1 (P<0.001). L‐Arginine reversed the L‐NAME‐induced inhibition of release of CGRP, the concentration of CGRP in the aqueous humour being 9.7 ± 0.6 nmol 1−1.

In addition, a NO donor, sodium nitroprusside (0.9 μmol), was found to raise the concentration of CGRP in the aqueous humour (14.8 ± 0.8 nmol 1−1) and to induce symptoms of ocular inflammation. The elevation in concentration of CGRP induced by sodium nitroprusside was not affected by L‐NAME (200 mg kg−1) (14.5 ± 1.2 nmol 1−1). Ocular responses were not inhibited by L‐NAME. 6 Our findings suggest that NO plays an important role in ocular inflammation by activating C‐fibres (directly or indirectly) and by mediating CGRP‐induced responses.
Effects of besipirdine at the voltage‐dependent sodium channel
Authors: Tang L. , Smith C.P. , Huger F.P. , Kongsamut S.
Published: July 2012

Besipirdine (HP 749) is a compound undergoing clinical trials for efficacy in treating Alzheimer's disease. Among other pharmacological effects, besipirdine inhibits voltage‐dependent sodium and potassium channels. This paper presents a pharmacological study of the interaction of besipirdine with voltage‐dependent sodium channels.

Besipirdine inhibited [3H]‐batrachotoxin binding (IC50 = 5.5±0.2 μm) in a rat brain vesicular preparation and concentration‐dependently inhibited veratridine (25 μm)‐stimulated increases in intracellular free sodium ([Na+]i) and calcium ([Ca2+]i) in primary cultured cortical neurones of rat.

Besipirdine (30–100 μm) concentration‐dependently inhibited (up to 100%) veratridine‐stimulated release of [3H]‐noradrenaline (NA) from rat cortical slices.

When examined in greater detail, besipirdine was found to inhibit [3H]‐batrachotoxin binding in vesicular membranes competitively. However, when examined in rat brain synaptosomes, we found that the antagonism by besipirdine was not competitive; that is, the maximal stimulation of [Ca2+]i induced by veratridine decreased with increasing concentrations of besipirdine.

These results show that besipirdine is an inhibitor of voltage‐sensitive sodium channels and appears to bind to a site close to the batrachotoxin/veratridine binding site.
Intraneuronal accumulation and persistence of radiolabel in rat brain following in vivo administration of [3H]‐chlorisondamine
Authors: El‐Bizri H. , Rigdon M.G. , Clarke P.B.S.
Published: July 2012

Chlorisondamine (CHL), a bisquaternary amine, produces a remarkably long‐lasting blockade of central responses to nicotine. The mechanism underlying this blockade is not known. The main aim of this study was to test for possible accumulation of [3H]‐CHL in rat brain during the period of chronic blockade.

Rats received CHL, either systemically (10mg kg−1) or centrally (10 μg i.c.v.). Seven days later, striatal synaptosomes prepared from these animals were tested for nicotine‐induced [3H]‐dopamine release. This experiment showed that i.c.v. administration of CHL was as effective as systemic administration in producing ex vivo blockade of central nicotinic receptors.

Rats received bilateral i.c.v. infusions of [3H]‐CHL (10 μg) and radioactivity was subsequently quantified in dissected cerebral cortex, striatum, hippocampus, midbrain and cerebellum. Radiolabel was detected at all three survival times (1, 7, and 21 days). Regional heterogeneity was apparent at 7 and 21 days survival. Radiolabel was almost exclusively confined to the insoluble subcellular fraction in all areas sampled.

The anatomical distribution of radiolabel was also visualized in brain sections. Rats received bilateral i.c.v. infusions of [3H]‐CHL (10 μg) and were killed at 1, 7, 21 or 84 days. Immediately before they were killed, all rats were tested behaviourally, and central nicotinic blockade was demonstrated at 1, 7 and 21 days; partial recovery was observed at 84 days. Particularly at longer survival times, tritium was found to be heavily concentrated in the substantia nigra pars compacta, ventral tegmental area, dorsal raphe nucleus, and the granular layer of the cerebellum.

The possibility of retrograde axonal transport of radiolabel was then examined. Rats received a unilateral intrastriatal infusion of [3H]‐CHL (0.34 or 0.034 μg) one week before they were killed. Autoradiographic labelling was largely confined to the site of infusion and to the ipsilateral substantia nigra pars compacta and dorsal raphe nucleus.

Thus, after i.c.v. administration, CHL (and/or centrally‐formed derivatives) is initially widely distributed within the brain and is then selectively retained within a few brain areas. A persistent accumulation occurs within putative dopaminergic and 5‐hydroxytryptaminergic neurones, at least partly through uptake by terminals and/or axons followed by retrograde transport. This persistent and anatomically‐selective intraneuronal accumulation possibly underlies the long‐term central nicotinic blockade associated with chlorisondamine.
Application of intracerebral microdialysis to study regional distribution kinetics of drugs in rat brain
Authors: de Lange Elizabeth C.M. , Bouw M. Rene , Mandema Jaap W. , Danhof Meindert , de Boer Albertus G. , Breimer Douwe D.
Published: July 2012

The purpose of the present study was to determine whether intracerebral microdialysis can be used for the assessment of local differences in drug concentrations within the brain.

Two transversal microdialysis probes were implanted in parallel into the frontal cortex of male Wistar rats, and used as a local infusion and detection device respectively. Within one rat, three different concentrations of atenolol or acetaminophen were infused in randomized order. By means of the detection probe, concentration‐time profiles of the drug in the brain were measured at interprobe distances between 1 and 2 mm.

Drug concentrations were found to be dependent on the drug as well as on the interprobe distance. It was found that the outflow concentration from the detection probe decreased with increasing lateral spacing between the probes and this decay was much steeper for acetaminophen than for atenolol. A model was developed which allows estimation of kbp/Deff (transfer coefficient from brain to blood/ effective diffusion coefficient in brain extracellular fluid), which was considerably larger for the more lipohilic drug, acetaminophen. In addition, in vivo recovery values for both drugs were determined.

The results show that intracerebral microdialysis is able to detect local differences in drug concentrations following infusion into the brain. Furthermore, the potential use of intracerebral microdialysis to obtain pharmacokinetic parameters of drug distribution in brain by means of monitoring local concentrations of drugs in time is demonstrated.
Characterization of U‐97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes
Authors: Im Haesook K. , Im Wha Bin , Pregenzer Jeff F. , Carter Donald B. , Jacobsen E. Jon , Hamilton Beverly J.
Published: July 2012

U‐97775 (tert‐butyl 7‐chloro‐4,5‐dihydro‐5‐[(1‐(3,4,5‐trimethyl)piperazino)carbonyl]‐imidazo[1,5‐a])quinoxaline‐3‐carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells.

The drug produced a bell‐shaped dose‐response profile in the α1β2γ2 receptor subtype as monitored with GABA‐induced C1− currents in the whole cell patch‐clamp technique. At low concentrations (<0.5 μM), U‐97775 enhanced the currents with a maximal increase of 120% as normalized to 5 μM GABA response (control). An agonist interaction of U‐97775 with the benzodiazepine site is suggested, because Ro 15–1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]‐flunitrazepam binding was inhibited by U‐97775 with a Ki of 1.2 nM.

The enhancement of GABA currents progressively disappeared as the U‐97775 concentration was raised above 1 μM, and the current amplitude was reduced to 40% below the control at 10 μM U‐97775. The current inhibition by U‐97775 (10 μM) was not affected by Ro 15–1788. It appears that U‐97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents.

U‐97775 at low concentrations reduced and at high concentrations enhanced [35S]‐TBPS binding. Ro 15–1788 selectively blocked the effect of U‐97775 at low concentrations. Analysis of the binding data in the presence of Ro 15–1788 yielded a single low affinity site with an estimated Kd of 407 nM.

In other αβγ receptor subtypes, U‐97775 at low concentrations enhanced C1− currents in the α3β2γ2 but not in the α6β2γ2 subtype. On the other hand, U‐97775 at high concentrations reduced C1− currents in all the receptor subtypes we examined, including those of two subunits, α1β2, β2γ2 and α1gamma;2 subtypes.

Therapeutically, U‐97775 could be unique among benzodiazepine ligands because of its ability to limit its own agonistic activity such that, at high doses the appearance of agonistic activity would be delayed until occupancy of its second site wanes. This property should make the total agonistic activity of U‐97775 relatively constant over a wide range of drug doses, and may minimize its liability to abuse.
Blockade by ONO‐NT‐012, a unique prostanoid analogue, of prostaglandin E2‐induced allodynia in conscious mice
Authors: Minami Toshiaki , Nishihara Isao , Sakamoto Kazuichi , Ito Seiji , Hyodo Masayoshi , Hayaishi Osamu
Published: July 2012

Intrathecal (i.t.) administration of prostaglandin E2 (PGE2) to conscious mice was reported to induce allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli through prostaglandin E receptor subtype EP1 and hyperalgesia through prostaglandin E receptor subtypes EP2 and/or EP3. In the present study, we investigated the effects of an EP1 antagonist on these sensory disorders by use of ONO‐NT‐012 or AH6809.

ONO‐NT‐012 dose‐dependently antagonized the PGE2‐induced allodynia but had no effect on the PGE2‐induced hyperalgesia by the hot plate test. On the other hand, AH6809 blocked the PGE2‐induced hyperalgesia at the highest dose examined (50 μ kg−1) but had no effect on the PGE2‐induced allodynia. The i.t. injection of AH6809 or ONO‐NT‐012 alone did not have any effect on the response to noxious or innocuous stimuli.

Increasing doses (5 pg kg−1‐500 ng kg−1) of ONO‐NT‐012 produced parallel shifts to the right of the dose‐response curves to PGE2. The Schild plot regression line was linear and the slope was close to unity. The pA2 value against PGE2 was calculated to be 9.96.

The present study demonstrates that i.t. administration of PGE2 exerts allodynia through EP1 in the mouse spinal cord and that ONO‐NT‐012 is a highly potent, simple competitive antagonist for the PGE2‐induced allodynia.