bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple effects and stimulation of insulin secretion by the tyrosine kinase inhibitor genistein in normal mouse islets
Authors: Jonas J.C. , Plant T.D. , Gilon P. , Detimary P. , Nenquin M. , Henquin J.C.
Published: July 2012

Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus‐secretion coupling in pancreatic β‐cells.

Genistein produced a concentration‐dependent (10–100 μm), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non‐metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 μm genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels.

Although genistein slightly inhibited ATP‐sensitive K+ channels, as shown by 86Rb efflux and patch‐clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP‐sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP‐sensitive K+ channels were opened by diazoxide and the β‐cell membrane depolarized by 30 mm K, but ineffective in the presence of diazoxide and normal extracellular K.

Genistein paradoxically decreased Ca2+ influx in β‐cells, as shown by the inhibition of glucose‐induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura‐2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in β‐cells.

Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way. Three other tyrosine kinase inhibitors, tyrphostin A47, herbimycin A and an analogue of erbstatin variably affected insulin secretion.

Genistein exerts a number of heretofore unrecognized effects. The unusual mechanisms, by which genistein increases insulin release in spite of a decrease in β‐cell [Ca2+]i and without activating known signalling pathways, do not seem to result from an inhibition of tyrosine kinases.
Acute pro‐inflammatory actions of endothelin‐1 in the guinea‐pig lung: involvement of ETA and ETB receptors
Authors: Filep János G. , Fournier Alain , Földes‐Filep Éva
Published: July 2012

Although recent observations suggest that endothelin‐1 (ET‐1) may play a role in the pathogenesis of asthma, to date little is known about the effects of ET‐1 on parameters other than bronchoconstriction. The objectives of the present experiments were to study whether intravenously administered ET‐1 could exert pro‐inflammatory actions in the guinea‐pig lung and to assess the involvement of endothelin ETA and ETB receptors in these events by using the ETA receptor‐selective antagonist, FR 139317, the novel ETA/ETB receptor antagonist, bosentan and the ETB receptor‐selective agonist, IRL 1620.

Bolus i.v. injection of ET‐1 (0.1‐1 nmol kg−1) to anaesthetized guinea‐pigs evoked dose‐dependent increases in mean arterial blood pressure which lasted for 6‐12min. This was accompanied by a dose‐dependent haemoconcentration (8–15% plasma volume losses) and increases (up to 546%) in albumin extravasation in the trachea, upper and lower bronchi, but not in the pulmonary parenchyma. Qualitatively similar changes were observed following i.v. injection of the ETB receptor agonist, IRL 1620 (0.3 and 1 nmol kg−1), although IRL 1620 appeared to be about 3 times less potent than ET‐1. The ETA receptor‐selective antagonist, FR 139317 (2.5mg kg−1) inhibited the ET‐1 (1 nmol kg−1)‐induced pressor response, haemoconcentration and albumin extravasation by 75, 77 and 60–70%, respectively, whereas it did not attenuate IRL 1620 (1 nmol kg−1)‐induced changes. The ETA/ETB receptor antagonist, bosentan (10 mg kg−1) almost completely inhibited the pressor, haemoconcentration and permeability effects of both ET‐1 and IRL 1620.

ET‐1, but not IRL 1620 (0.1‐1 nmol kg−1), produced a dose‐dependent neutropenia with relative lymphocytosis and monocytosis, but did not induce influx of neutrophil granulocytes into pulmonary tissues or the bronchoalveolar space. ET‐1 (1 nmol kg−1)‐induced neutropenia was prevented by pretreatment of the animals with FR 139317 (2.5 mg kg−1), bosentan (10 mg kg−1) or adrenaline (90 nmol kg−1), indicating that ET‐1 caused intravascular sequestration of neutrophil granulocytes.

ET‐1 or IRL 1620 (10−1‐10−6 m) alone did not activate alveolar macrophages in vitro, whereas at a concentration of 10−8m, ET‐1, but not IRL 1620, markedly potentiated superoxide production in response to f‐Met‐Leu‐Phe (10−9‐10−7 m) and platelet‐activating factor (PAF, 10−9‐10−7 m), but not to phorbol 12‐myristate 13‐acetate (10−9m). ET‐1 did not affect f‐Met‐Leu‐Phe‐ or PAF‐induced increases in intracellular free calcium concentration. This potentiating effect of ET‐1 was abolished by FR 139317 (1.5 × 10−7m).

We conclude that, in addition to evoking airway contractions, ET‐1 exerts pro‐inflammatory actions via activation of the ETA and to a lesser extent the ETB receptors, and therefore, might contribute to the airway inflammation present in asthma. These findings also suggest the therapeutic potential of ETA/ETB receptor and perhaps ETA receptor‐selective antagonists in this disease.
Mechanism of block of a human cardiac potassium channel by terfenadine racemate and enantiomers
Authors: Yang Tao , Prakash Chandra , Roden Dan M. , Snyders Dirk J.
Published: July 2012

The cardiac toxicity of racemic terfenadine (marked QT prolongation and polymorphic ventricular arrhythmias) is probably due to potassium channel blockade. To test whether one of its enantiomers would be a less efficient potassium channel blocker, we compared the mechanism of action of the racemate with that of the individual enantiomers.

We synthesized the individual enantiomers of terfenadine and examined under whole cell voltage‐clamp conditions the mechanism of action of the racemate, both enantiomers and a major metabolite on a cloned human cardiac potassium channel, hKvl.5. This delayed rectifier is sensitive to quinidine, clofilium and other ‘class III’ antiarrhythmic drugs at clinically relevant concentrations.

Upon depolarization, racemic terfenadine and its enantiomers induced a fast decline of hKvl.5 current towards a reduced steady state current level. During subsequent repolarization the tail currents deactivated more slowly than the control, resulting in a ‘crossover’ phenomenon.

The voltage‐dependence of block was biphasic with a steep increase in block over the voltage range of channel opening (−30 to 0 mV), and a more shallow phase positive to O mV (where the channel is fully open). The latter was consistent with a binding reaction sensing 21% of the transmembrane electrical field (with reference to the cell interior).

The EC50 for hKvl.5 block by racemic terfenadine was 0.88 μm, while the values for R‐ and S‐terfenadine were 1.19 μm and 1.16 μm, respectively. In contrast, the acid metabolite reduced hKvl.5 current by only 5% at a concentration of 50 μm.

These findings suggest that terfenadine blocks the hKvl.5 channel after it opens by entering into the internal mouth of the channel. We have previously shown that quinidine blocks hKvl.5 in a similar manner but with an apparent affinity of ∼6 μm. Thus, terfenadine and its enantiomers are approximately equipotent open state blockers of this human K+ channel and about 6 times more potent than quinidine. The similar state‐, time‐, and voltage‐dependence of hKvl.5 block by both enantiomers also indicates that the chiral centre does not significantly constrain the orientation of critical binding determinants of terfenadine with respect to the receptor site.
Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT‐20 cells
Authors: McFerran B.W. , MacEwan D.J. , Guild S.B.
Published: July 2012

The mouse AtT‐20/D16‐16 anterior pituitary tumour cell line was used as a model system for the study of protein kinase C (PKC)‐mediated enhancement of calcium‐ and guanine nucleotide‐evoked adrenocorticotrophin (ACTH) secretion.

A profile of the PKC isozymes present in AtT‐20 cells was obtained by Western blotting analysis and it was found that AtT‐20 cells express the α, β, ∍ and ζ isoforms of PKC.

PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically‐permeabilized cells. Phorbol 12‐myristate 13‐acetate (PMA, EC50 = 1 ± 0.05 nM, which activates all isozymes of PKC, except the ζ isozyme), thymeleatoxin (TMX, EC50 = 10 ± 0.5 nM, which activates the α, β and γ isozymes) and 12‐deoxyphorbol 13‐phenylacetate 20‐acetate (dPPA, EC50 = 3 ± 0.5 nm, α β1 selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration‐dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA‐stimulated ACTH secretion was only 60–70% of that obtained in response to PMA or TMX.

Calcium stimulated ACTH secretion from electrically‐permeabilized cells over the concentration‐range of 100 nM to 10 μm. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated. PMA (100 nM) and TMX (100 nM) significantly enhanced ACTH secretion in the effective absence of calcium (i.e. where the free calcium concentration is 1 nM).

GTP‐γ‐S stimulated ACTH secretion from permeabilized cells in a concentration‐dependent manner with a threshold of 1 μm. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) increased the amount of ACTH secretion evoked by every concentration of GTP‐γ‐S investigated.

The PKC inhibitor, chelerythrine chloride (10 μm), blocked the PMA (100nM)‐evoked enhancement of calcium‐ and GTP‐γ‐S‐stimulated ACTH secretion but did not significantly alter calcium‐ or GTP‐γ‐S‐evoked secretion itself.

The present paper indicates that AtT‐20 cells express multiple isoforms of PKC and that these act at different sites in the secretory pathway for ACTH secretion. The α and ∍ isozymes of PKC can act distal to calcium entry to modulate the ability of increased cytosolic calcium concentrations to stimulate ACTH secretion. This site of action is either at the level of, or at some stage distal to, a GTP‐binding protein which mediates the effects of calcium upon ACTH secretion. The β isozyme of PKC may act at a stage early in the secretory pathway to regulate the cytosolic calcium concentration.
Actions of the insecticide fipronil, on dieldrin‐sensitive and ‐resistant GABA receptors of Drosophila melanogaster
Authors: Hosie Alastair M. , Baylis Howard A. , Buckingham Steven D. , Sattelle David B.
Published: July 2012

Blocking actions of the novel insecticide, fipronil, were examined on GABA responses recorded from Xenopus oocytes expressing either wild type (dieldrin‐sensitive) or mutant (dieldrin‐resistant) forms of the Drosphila melanogaster GABA‐gated chloride channel homo‐oligomer, RDL (the product of the resistance to dieldrin locus: Rdl).

In the case of the wild type receptor, fipronil blocked GABA‐induced currents inducing both a shift to the right in the GABA dose‐response curve and depressing the maximum amplitude of responses to GABA. The potency of fipronil was dependent on the GABA concentration but was unaffected by membrane potential.

Mutant RDL GABA‐receptors, which have a naturally occurring amino acid substitution (A302→SS) in the putative ion‐channel lining region, conferring resistance to dieldrin and picrotoxinin, were markedly less sensitive to fipronil than the wild‐type receptors.

Fipronil antagonism is qualitatively similar to that produced by the structurally distinct compound, picrotoxinin. As the mutation A302→S reduces the potency of both fipronil and picrotoxinin, homo‐oligomeric RDL receptors should facilitate detailed studies of the molecular basis of convulsant/ insecticide antagonist actions on GABA receptors.
Phosphorus‐containing peptides as mixed inhibitors of endopeptidase 3.4.24.15 and 3.4.24.16: effect on neurotensin degradation in vitro and in vivo
Authors: Vincent Bruno , Dive Vincent , Yiotakis Athanasios , Smadja Claire , Maldonado Raphaël , Vincent Jean‐Pierre , Checler * Frederic
Published: July 2012

We have examined several phosphorus‐containing peptides as potential mixed inhibitors of two neurotensin‐degrading zinc metallopeptidases, endopeptidase 3.4.24.15 and endopeptidase 3.4.24.16.

Among a series of 13 phosphonamide peptides, N‐(2‐(2‐naphtyl)ethylphosphonyl‐glycyl‐prolyl‐norleucine (phosphodiepryl 08) was found to inhibit potently the hydrolysis of neurotensin by purified endopeptidase 3.4.24.15 and 3.4.24.16 with an identical Ki value of 0.4 nM.

Phosphodiepryl 08 displayed a strong selectivity towards the two peptidases since it failed to inhibit several other zinc‐containing peptidases such as endopeptidase 3.4.24.11, angiotensin‐converting enzyme, aminopeptidase M, leucine aminopeptidase and carboxypeptidases A and B.

The protective effect of phosphodiepryl 08 on neurotensin degradation was examined in vitro and in vivo in central and peripheral bioassays.

Phosphodiepryl 08 virtually abolished neurotensin degradation by 4‐day‐old plated pure cultured neurones from mouse embryos and greatly potentiated neurotensin‐induced antinociception in the mouse hot plate test.

In the periphery, phosphodiepryl 08 inhibited neurotensin degradation by membranes prepared from isolated longitudinal smooth muscle of guinea‐pig ileum and greatly potentiated the neurotensin‐induced contraction of the same longitudinal smooth muscle preparation.

Our study indicates that phosphodiepryl 08 behaves as a potent and selective mixed inhibitor of endopeptidase 3.4.24.15 and 3.4.24.16 and can be used as a powerful agent to prevent neurotensin degradation, in vitro and in vivo, in central and peripheral assays.
Interaction between a selective 5‐HT1Areceptor antagonist and an SSRI in vivo: effects on 5‐HT cell firing and extracellular 5‐HT
Authors: Gartside S.E. , Umbers V. , Hajós M. , Sharp T.
Published: July 2012

The acute inhibitory effect of selective 5‐hydroxytryptamine (serotonin) reuptake inhibitors (SSRIs) on 5‐HT neuronal activity may offset their ability to increase synaptic 5‐HT in the forebrain. 2Here, we determined the effects of the SSRI, paroxetine, and a novel selective 5‐HT1Areceptor antagonist, WAY 100635, on 5‐HT cell firing in the dorsal raphe nucleus (DRN), and on extracellular 5‐HT in both the DRN and the frontal cortex (FCx). Extracellular electrophysiological recording and brain microdialysis were used in parallel experiments, in anaesthetized rats.

Paroxetine dose‐dependently inhibited the firing of 5‐HT neurones in the DRN, with a maximally effective dose of approximately 0.8 mg kg−1, i.v. WAY 100635 (0.1 mg kg−1, i.v.) both reversed the inhibitory effect of paroxetine and, when used as a pretreatment, caused a pronounced shift to the right of the paroxetine dose‐response curve.

Paroxetine (0.8 mg kg−1, i.v.), doubled extracellular 5‐HT in the DRN, but did not alter extracellular 5‐HT in the FCx. A higher dose of paroxetine (2.4 mg kg−1, i.v.) did increase extracellular 5‐HT in the FCx, but to a lesser extent than in the DRN. Whereas 0.8 mg kg−1, i.v. paroxetine alone had no effect on extracellular 5‐HT in the FCx, in rats pretreated with WAY 100635 (0.1 mg kg−1), paroxetine (0.8 mg kg−1, i.v.) markedly increased extracellular 5‐HT in the FCx.

In conclusion, pretreatment with the selective 5‐HT1Areceptor antagonist, WAY 100635, blocked the inhibitory effect of paroxetine on 5‐HT neuronal activity in the DRN and, at the same time, markedly enhanced the effect of paroxetine on extracellular 5‐HT in the FCx. These results may be relevant to recent clinical observations that 5‐HT1Areceptor antagonists in combination with SSRIs have a rapid onset of antidepressant effect.
The in vitro pharmacology of ZM 241385, a potent, non‐xanthine, A2a selective adenosine receptor antagonist
Authors: Poucher S.M. , Keddie J.R. , Singh P. , Stoggall S.M. , Caulkett P.W.R. , Jones G. , Collis M.G.
Published: July 2012

This paper describes the in vitro pharmacology of ZM 241385 (4‐(2‐[7‐amino‐2‐(2‐furyl) [1,2,4]‐triazolo[2,3‐a][1,3,5]triazin‐5‐yl amino]ethyl) phenol), a novel non‐xanthine adenosine receptor antagonist with selectivity for the A2a receptor subtype.

ZM 241385 had high affinity for A2a receptors. In rat phaeochromocytoma cell membranes, ZM 241385 displaced binding of tritiated 5′‐N‐ethylcarboxamidoadenosine (NECA) with a pIC50 of 9.52, (95% confidence limits, c.l., 9.02–10.02). In guinea‐pig isolated Langendorff hearts, ZM 241385 antagonized vasodilatation of the coronary bed produced by 2‐chloroadenosine (2‐CADO) and 2‐[p‐(2‐carboxyethyl) phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS21680) with pA2 values of 8.57 (c.l., 8.45‐8.68) and 9.02 (c.l., 8.79‐9.24) respectively 3 ZM 241385 had low potency at A2b receptors and antagonized the relaxant effects of adenosine in the guinea‐pig aorta with a pA2 of 7.06, (c.l., 6.92‐7.19).

ZM 241385 had a low affinity at A1 receptors. In rat cerebral cortex membranes it displaced tritiated R‐phenylisopropyladenosine (R‐PIA) with a pIC50 of 5.69 (c.l., 5.57‐5.81). ZM 241385 antagonized the bradycardie action of 2‐CADO in guinea‐pig atria with a pA2 of 5.95 (c.l., 5.72‐6.18).

ZM 241385 had low affinity for A3 receptors. At cloned rat A3 receptors expressed in Chinese hamster ovary cells, it displaced iodinated aminobenzyl‐5′‐N‐methylcarboxamido adenosine (AB‐MECA) with a pIC50 of 3.82 (c.l., 3.67‐4.06).

ZM 241385 had no significant additional pharmacological effects on the isolated tissues used in these studies at concentrations three orders of magnitude greater than those which block A2a receptors. At 10 μm it displayed only minor inhibition of the bradycardic effects in guinea‐pig atria to some concentrations of carbachol. At 10 μm, ZM 241385 had a small inhibitory effect on relaxant effects of isoprenaline in guinea‐pig aortae but no effect on sodium nitrite‐induced relaxation. ZM 241385 (100 μm) was without effect on phenylephrine‐induced tone in guinea‐pig aortae.

ZM 241385 (10 μm) had no inhibitory effect on rat hepatocyte phosphodiesterase types I, II, III and IV but caused a small inhibition of the calcium calmodulin‐activated type I enzyme.

ZM 241385 is the most selective adenosine A2a receptor antagonist yet described and is therefore a useful tool for characterization of responses mediated by A2 adenosine receptors.
Coupling of an endogenous 5‐HT1B‐like receptor to increases in intracellular calcium through a pertussis toxin‐sensitive mechanism in CHO‐K1 cells
Authors: Dickenson John M. , Hill Stephen J.
Published: July 2012

Chinese hamster ovary cells (CHO‐K1) express an endogenous 5‐hydroxytryptamine (5‐HT)1B‐like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)‐sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO‐K1 cells (CHO‐A1) has been shown to mobilize intracellular Ca2+ through a PTX‐sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5‐HT1B‐like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO‐A1 cells.

In agreement with previous studies using CHO cells, 5‐hydroxytryptamine (5‐HT) elicited a concentration‐dependent inhibition of forskolin‐stimulated [3H]‐cyclic AMP production in CHO‐A1 cells (p[EC50] = 7.73±0.13). 5‐HT (1 μm) inhibited 47±5% of the [3H]‐cyclic AMP accumulation induced by 3 μm forskolin. Forskolin stimulated [3H]‐cyclic AMP accumulation was also inhibited by the 5‐HT1 receptor agonists (p[EC50] values) 5‐carboxyamidotryptamine (5‐CT; 8.07±0.08), RU 24969 (8.12 ±0.33) and sumatriptan (5.80 ±0.31).

5‐HT elicited a concentration‐dependent increase in [Ca2+]i in CHO‐A1 cells (p[EC50] = 8.07 ±0.05). In the presence of 2mM extracellular Ca2+, 5‐HT (1 μm) increased [Ca2+]i from 174±17nM to 376 ±22 nM. The 5‐HT1 receptor agonists (p[EC50] values), 5‐carboxyamidotryptamine (5‐CT; 7.9 ±0.02), RU 24969 (8.1 ±0.07) and sumatriptan (5.9 ±0.11) all elicited concentration‐dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5‐HT1A receptor agonist, 8‐OH‐DPAT (10 μm) did not stimulate increases in [Ca2+]i. 5‐HT (100 μm) and 5‐CT (10 μm) did not stimulate a measurable increase in [3H]‐inositol phosphate accumulation in CHO‐A1 cells.

5‐HT (1 μm)‐mediated increases in [Ca2+]i were insensitive to the 5‐HT receptor antagonist, ritanserin (5‐HT2; 100 nM), ketanserin (5‐HT2; 100 nM), LY‐278,584 (5‐HT3; 1 μm) and WAY 100635 (5‐HT1A; 1 μm). The response to 5‐HT (100 nM) was antagonized by the non‐selective 5‐HT1 antagonist, methiothepin (pKb = 8.90±0.09) and the 5‐HT1D antagonist GR 127935 (pKb= 10.44±0.06).

Pretreatment with PTX (200 ng ml−1 for 4 h) completely attenuated the Ca2+ response to 100 μm 5‐HT.

In untransfected CHO‐K1 cells, 5‐HT (1 μm), RU 24969 (1 μm), and 5‐CT (1 μm) elicited increases in [Ca2+]i similar to those observed in CHO‐A1 cells.

These data demonstrate that in CHO‐K1 cells the endogenously expressed 5‐HT1B‐like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX‐sensitive pathway, suggesting the involvement of Gi/Go protein(s).
The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor
Authors: Dickinson R. , Lieb W.R. , Franks N.P.
Published: July 2012

Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two‐electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (≤200 nM).

Inhibition of the ACh‐induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)‐tubocurarine was studied as a function of temperature (over the range 4–25°C).

The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)‐tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)‐tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage‐dependence.

The van't Hoff plots, (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non‐linear for (+)‐tubocurarine. From these plots, values for the changes in the standard Gibbs free energy ΔG°water→AChR, enthalpy ΔH°water→.AChR, entropy ΔS°water→AChR and heat capacity ΔC°p water→AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding.

A comparison between the temperature‐dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature‐dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.
Comparative effects of angiotensin II and its degradation products angiotensin III and angiotensin IV in rat aorta
Authors: Li Q. , Zhang J. , Pfaffendorf M. , van Zwieten P.A.
Published: July 2012

In the present study, the contractile effects of angiotensin III (AIII) and angiotensin IV (AIV) compared with those of angiotensin II (All) were determined in rat aortic ring preparations.

All three peptides caused concentration‐dependent contractions with similar maximal responses. AIII proved approximately 4 times less potent than All, whereas AIV was about 1000 times less active than AII

The selective AT1‐receptor antagonist, losartan (10–300 nM) caused parallel rightward shifts of the concentration‐response curves (CRC) for all three peptides. The Schild plot slopes for the effect of losartan on AIII curves were significantly lower than unity (P<0.05). The selective AT2‐receptor antagonist, PD123177 did not influence the CRCs for All and AIV. However, the AIII curves were moderately shifted leftward in the presence of PD123177 (0.1 and 1 μm).

Destruction of the endothelium or incubation with the NO‐synthesis inhibitor NG‐monomethyl‐l‐arginine acetate (l‐NMMA) (0.1 mm) significantly enhanced the contractile responses to all three peptides.

Tachyphylaxis was investigated by constructing a second CRC for all three peptides, after an interval of 1 h. The presence of endothelium significantly enhanced the development of tachyphylaxis to all three peptides. However, in endothelium‐denuded preparations, the Emax value of the second curve elicted by All was about 50%, compared with the first one, whereas for AIII and AIV Emax values were as high as 90% and 100%, respectively.

Our results indicate that both AIII and AIV are less potent but similarly efficacious vasoconstrictor agents compared with AII Their contractile effects are also mediated by AT1receptors and probably modulated by endothelium. Tachyphylaxis induced by AIII and AIV proved weaker than that for AII Tachyphylaxis appears to be enhanced by the presence of an intact endothelium.
Dose‐response comparisons of five lung surfactant factor (LSF) preparations in an animal model of adult respiratory distress syndrome (ARDS)
Authors: Häfner Dietrich , Beume Rolf , Kilian Ulrich , Krasznai Georg , Lachmann Burkhard
Published: July 2012

We have examined the effects of five different lung surfactant factor (LSF) preparations in the rat lung lavage model. In this model repetitive lung lavage leads to lung injury with some similarities to adult respiratory distress syndrome with poor gas exchange and protein leakage into the alveolar spaces. These pathological sequelae can be reversed by LSF instillation soon after lavage.

The tested LSF preparations were: two bovine: Survanta and Alveofact: two synthetic: Exosurf and a protein‐free phospholipid based LSF (PL‐LSF) and one Recombinant LSF at doses of 25, 50 and 100 mg kg−1 body weight and an untreated control group.

Tracheotomized rats (10–12 per dose) were pressure‐controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths min−1, inspiration expiration ratio of 1: 2, peak inspiratory pressure (PIP) of 28 cmH2O at positive end‐expiratory pressure (PEEP) of 8 cmH2O. Two hours after LSF administration, PEEP and in parallel PIP was reduced from 8 to 6 (1st reduction), from 6 to 3 (2nd reduction) and from 3 to 0 cmH2O (3rd reduction).

Partial arterial oxygen pressure (Pao2, mmHg) at 5 min and 120 min after LSF administration and during the 2nd PEEP reduction (Pao2(PEEP23/3)) were used for statistical comparison. All LSF preparations caused a dose‐dependent increase for the Pao2(120′), whereas during the 2nd PEEP reduction only bovine and recombinant LSF exhibited dose‐dependency. Exosurf did not increase Pao2 after administration of the highest dose. At the highest dose Exosurf exerted no further improvement but rather a tendency to relapse. The bovine and the Recombinant LSF are superior to both synthetic LSF preparations.

In this animal model and under the described specific ventilatory settings, even between bovine LSF preparations there are detectable differences that are pronounced when compared to synthetic LSF without any surfactant proteins. We conclude that the difference between bovine and synthetic LSF preparations can be overcome by addition of the surfactant protein C.
Renal effects of infusion of rilmenidine and guanabenz in conscious dogs: contribution of peripheral and central nervous system α2–adrenoceptors
Authors: Evans Roger G. , Anderson Warwick P.
Published: July 2012

We tested the renal effects of the α2‐adrenoceptor agonists, rilmenidine and guanabenz and the antagonists, 2‐methoxyidazoxan and idazoxan, in conscious dogs. Our aim was to test the hypothesis that putative imidazoline (I) receptors influence renal function. We reasoned that since rilmenidine and guanabenz are selective for I1‐ and I2‐binding sites respectively, an influence of one of these receptive sites on renal function would be reflected in qualitative differences between the effects of these agents. Moreover, effects mediated by putative I‐receptors should be relatively resistant to antagonism by the selective α2‐adrenoceptor antagonist, 2‐methoxyidazoxan. Since the effects of these drugs on renal function could be mediated in the central nervous system or periphery, the dogs were studied under both normal and ganglion‐blocked conditions.

In dogs with intact autonomic reflexes, 2‐methoxyidazoxan (15 μg kg−1 plus 0.6 μg kg−1 min−1) produced effects consistent with a generalized increase in sympathetic drive, including increases in mean arterial pressure and plasma renin activity, and a reduction in sodium excretion. In ganglion‐blocked dogs, 2‐methoxyidazoxan reduced sodium excretion but had no discernible effect on systemic or renal haemodynamics. We conclude that an α2‐adrenoceptor‐mediated mechanism in the central nervous system tonically inhibits sympathetic drive in the conscious dog.

In ganglion‐blocked dogs idazoxan (3‐300 μg kg−1) dose‐dependently increased arterial pressure. This was not abolished by concomitant administration of 2‐methoxyidazoxan (0.3‐30 μg kg−1). The pressor effect of idazoxan is therefore probably mediated by an agonist action at α‐adrenoceptors.

The effects of infusions of rilmenidine (0.1‐1.0 mg kg−1) and guanabenz (10‐100 μg kg−1) were indistinguishable. They comprised dose‐dependent increases in mean arterial pressure, urine excretion, and glomerular filtration rate (the latter in ganglion blocked dogs only), and dose‐dependent reductions in heart rate, renal blood flow and sodium excretion (only in dogs with intact autonomic reflexes). All of these effects were antagonized by 2‐methoxyidazoxan.

We conclude that the renal effects of rilmenidine and guanabenz infusions in conscious dogs are predominantly, if not completely, attributable to activation of α2‐adrenoceptors. Our results do not support the hypothesis that putative I‐receptors contribute towards the renal effects of these agents.
Calcium channel subtypes for the sympathetic and parasympathetic nerves of guinea‐pig atria
Authors: Hong S.J. , Chang C.C.
Published: July 2012

The Ca2+ channel subtypes of the autonomic nerves of guinea‐pig atria were elucidated by monitoring the effects of specific Ca2+ channel blockers on the negative and positive inotropic responses associated respectively, with stimulation of the parasympathetic and sympathetic nerves.

In left atria paced at 2–4 Hz, the negative inotropic effect induced by field stimulation of parasympathetic nerves (in the presence of propranolol) was abolished by ω‐conotoxin MVIIC, a blocker of N‐type and OPQ subfamily Ca2+ channels. ω‐Conotoxin GVIA (an N‐type blocker), ω‐agatoxin IVA (a P‐type blocker), nifedipine (an L‐type blocker) and Ni2+ (a T‐ and R‐type blocker) were much less effective.

The positive inotropic response resulting from field stimulation of the sympathetic nerves (in the presence of atropine) was abolished by both ω‐conotoxins, while ω‐agatoxin IVA, nifedipine and Ni2+ were ineffective.

In the spontaneously beating right atria, the early negative inotropic effect produced by 1,1‐dimethyl‐4‐phenylpiperazinium was abolished by ω‐conotoxin MVIIC; whereas the late positive inotropic effect was partially reduced, but not abolished, by a high concentration of ω‐conotoxin GVIA.

None of the peptide toxins affected the chronotropic and the inotropic responses evoked by carbachol and isoprenaline.

These results suggested that, under physiological conditions, the release of acetylcholine from paraysmpathetic nerves is dominated by an OPQ subfamily Ca2+ channel while that of noradrenaline from sympathetic nerves is controlled by an N‐type Ca2+ channel. Ligand‐induced noradrenaline release appeared to recruit additional type(s) of Ca2+ channel.
Sequential induction of nitric oxide synthase by Corynebacterium parvum in different organs of the mouse
Authors: Rees D.D. , Cunha F.Q. , Assreuy J. , Herman A.G. , Moncada S.
Published: July 2012

The ability of Corynebacterium parvum (C. parvum) to induce nitric oxide (NO) synthase in the macrophage, spleen, liver, aorta, heart and brain, and to elevate plasma NO2−/NO3− in the mouse was investigated. In addition, the relationship between NO synthase activity and blood pressure was studied.

C. parvum (100 mg kg−1, i.p.) induced a time‐dependent expression of a Ca2+‐independent NO synthase in the macrophage, spleen, liver, aorta and heart. The time course of induction of the NO synthase varied such that the maximum enzyme activity was at day 8 in the macrophage and liver, day 12 in the spleen and heart and day 16 in the aorta.

There was no significant induction of a Ca2+‐independent NO synthase in the brain, nor was there any change in the Ca2+‐dependent enzyme in this organ, during the study period.

C. parvum produced a gradual decrease in blood pressure, with a maximum fall at day 16 (from 108 ± 1 mmHg to 79 ± 3 mmHg), which recovered gradually by day 28.

Plasma NO2−/NO3− was significantly elevated between days 8 and 24, with a maximum increase at day 12.

These results show that C. parvum induces a Ca2+‐independent NO synthase in a number of tissues and that this induction occurs initially in macrophages and the liver. This suggests that induction of the NO synthase in the other tissues is secondary and probably the result of activation of macrophages and some cells of the liver.

Furthermore, the decrease in blood pressure induced by C. parvum is associated with the induction of NO synthase in the vasculature, whereas the increased concentration of plasma NO2−/NO3− seems to result from the generation of NO by a number of tissues.
Adenosine A2 receptor‐induced inhibition of leukotriene B4 synthesis in whole blood ex vivo
Authors: Krump E. , Lemay G. , Borgeat P.
Published: July 2012

Engagement of adenosine A2 receptors suppresses several leukocyte functions. In the present study, we examined the effect of adenosine on the inhibition of leukotriene B4 (LTB4) synthesis in heparinized human whole blood, pretreated with lipopolysaccharide (LPS) and tumour necrosis factor α (TNF‐α) and stimulated with the chemotactic peptide, N‐formyl‐Met‐Leu‐Phe (FMLP).

The FMLP‐induced synthesis of LTB4 in whole blood pretreated with LPS and TNF‐α was dose‐dependently inhibited by adenosine analogues in the following order of potency; 5′(N‐ethyl)carbox‐ amidoadenosine (NECA) ≅ CGS 21680 > 2‐Cl‐adenosine > N6‐cyclopentyladenosine (CPA), indicating the involvement of the adenosine A2 receptor subtype. The IC50 values for NECA, CGS 21680, 2‐Cl‐ adenosine, and CPA were 6nM, 9nM, 180nM, and 990 nM, respectively.

Dipyridamole, an agent that blocks the cellular uptake of adenosine by red cells and causes its accumulation in plasma, also inhibited the synthesis of LTB4 in LPS and TNF‐α‐treated whole blood stimulated by FMLP; moreover, this inhibition was reversed upon addition of adenosine deaminase.

A highly selective antagonist of the adenosine A2 receptor, 8‐(3‐chlorostyryl)caffeine (CSC), reversed the inhibition of LTB4 synthesis by 2‐Cl‐adenosine and dipyridamole in LPS and TNF‐α‐treated whole blood, stimulated by FMLP.

LTB4 synthesis in whole blood originates predominantly from neutrophils and to a lesser extent from monocytes. 2‐Cl‐adenosine also inhibited the synthesis of LTB4 induced by FMLP in these isolated LPS and TNF‐α‐treated cells; however, 2‐Cl‐adenosine was a more potent inhibitor of LTB4 synthesis in neutrophils than monocytes.

The present data demonstrate that adenosine, acting through A2 receptors, exerts a potent inhibitory effect on the synthesis of LTB4 and thus contribute to the understanding of its anti‐inflammatory properties.
Some effects of nipradilol, a β‐antagonist possessing a nitroxy group, on smooth muscle of the pig coronary artery
Authors: Abe Shimako , Nakamura Motoomi , Kanaide Hideo
Published: July 2012

The effects of nipradilol, a β‐adrenoceptor antagonist which possesses a nitroxy group, on cytosolic Ca2+ concentration ([Ca2+]i) and on tension development were simultaneously measured by front‐surface fluorometry and fura‐2‐loaded strips in the proximal portion of pig coronary arteries.

Nipradilol reduced in a concentration‐dependent manner both the [Ca2+]i and tension, irrespective of whether the strips were unstimulated or exposed to either high K+ or histamine containing solutions. However, both in the case of contractions induced by high K+‐depolarization and histamine stimulation, for a given [Ca2+]i elevation the tension which developed in the presence of nipradilol was smaller than that generated in its absence, so that the [Ca2+]i‐tension curves during the contraction were shifted to the right.

In the absence of extracellular Ca2+, the [Ca2+]i elevation due to the release of Ca2+ from histamine‐sensitive store was inhibited by nipradilol. Nipradilol had no effect on the [Ca2+]i elevation due to the release of Ca2+ from caffeine‐sensitive stores; however, it did inhibit the caffeine‐induced increase in tension. A derivative of nipradilol, which lacked a nitroxy molecule (Nip(‐N)), had no effect on the [Ca2+]i and tension elevated by histamine or caffeine in the absence of extracellular Ca2+.

The β‐adrenoceptor agonist, isoprenaline, reduced [Ca2+]i and tension when applied to steady state contractions induced by high K+, or at the peak level of tension to histamine. The reduction of [Ca2+]i and tension induced by isoprenaline was inhibited by Nip(‐N) in a concentration‐dependent manner and nipradilol inhibited the isoprenaline‐induced relaxation with bell‐shaped concentration‐response curves. At lower concentrations, nipradilol acted as a β‐blocker, the IC50 value being smaller than that of Nip(‐N), and at higher concentrations, it acted as a nitrovasodilator.

Thus, it is suggested that, at lower concentrations, nipradilol, an antianginal drug, acts as a β‐adrenoceptor antagonist. At higher concentrations, it relaxes the proximal portion of the coronary artery by directly reducing [Ca2+]i and the Ca2+‐sensitivity of the myofilaments, apparently due to the presence of the nitroxy molecule.
The effect of nociceptin on Ca2+ channel current and intracellular Ca2+ in the SH‐SY5Y human neuroblastoma cell line
Authors: Connor Mark , Yoe Alison , Henderson Graeme
Published: July 2012

The human neuroblastoma cell line SH‐SY5Y expresses the ‘orphan’ opioid receptor (ORL1). We have demonstrated that nociceptin, the putative endogenous ligand for ORL1, produces a concentration‐dependent inhibition of the N‐type calcium channel current in these cells (IC50 42 nM). In addition, in the presence of carbachol, nociceptin increased the intracellular concentration of Ca2+ (EC50 60 nM). Both effects of nociceptin were blocked by pertussis toxin pretreatment but not by the opioid antagonists CTAP (1 μm), naltrindole (1 μm) and naloxone (10 μm).
Reversal by NPY, PYY and 3–36 molecular forms of NPY and PYY of intracisternal CRF‐induced inhibition of gastric acid secretion in rats
Authors: Gué M. , Junien J.L. , Reeve J.R. , Rivier J. , Grandt D. , Taché Y.
Published: July 2012

The Y receptor subtype involved in the antagonism by neuropeptide Y (NPY) of intracisternal corticotropin‐releasing factor (CRF)‐induced inhibition of gastric acid secretion was studied in urethaneanaesthetized rats by use of peptides with various selectivity for Y1, Y2 and Y3 subtypes: NPY, a Y1, Y2 and Y3 agonist, peptide YY (PYY), a Y1 and Y2 agonist, [Leu31, Pro34]‐NPY, a Y1 and Y3 agonist, NPY(3–36) and PYY(3–36), highly selective Y2 agonists and NPY(13–36) a weak Y2 and Y3 agonist. Peptides were injected intracisternally 10 min before intracisternal injection of CRF (10 μg) and gastric acid secretion was measured by the flushed technique for 1 h before and 2 h after pentagastrin(10 μg kg−1 h−1, i.v.) infusion which started 10 min after CRF injection.

Intracisternal injection of CRF (10 μg) inhibited by 56% gastric acid secretion stimulated by pentagastrin. Intracisternal injection of NPY and PYY (0.1‐0.5 μg) did not influence the acid response to pentagastrin but blocked CRF‐induced inhibition of pentagastrin‐stimulated acid secretion. NPY(3–36) (0.5 μg) and PYY(3–36) (0.25 and 0.5 μg) also completely blocked the inhibitory action of CRF on pentagastrin‐stimulated acid secretion.

[Leu31, Pro34]‐NPY (0.5‐5 μm) and NPY(13–36) (0.5‐5 μm) injected intracistemally did not modify gastric acid secretion induced by pentagastrin or CRF inhibitory action.

The sigma antagonist, BMY 14802 (1 mg kg−1, s.c.) did not influence the acid response to pentagastrin but prevented the antagonism by PYY(3–36) (0.5 μm) of the CRF antisecretory effect.

These results show that both PYY and NPY and the 3–36 forms of PYY and NPY are equipotent in blocking central CRF‐induced inhibition of pentagastrin‐stimulated gastric acid secretion. The structure‐activity profile suggests a mediation through Y2 receptor subtype and the involvement of sigma binding sites.
Coupling of metabotropic glutamate receptors to phosphoinositide mobilisation and inhibition of cyclic AMP generation in the guinea‐pig cerebellum
Authors: Neil Karen E. , Kendall David A. , Alexander Stephen P.H.
Published: July 2012

The effects of metabotropic glutamate receptor (mGluR) agonists on cyclic nucleotide and phosphoinositide turnover were investigated in adult guinea‐pig cerebellar slices by use of radioactive precursors.

L‐Glutamate, 1‐aminocyclopentane‐1S,3R‐dicarboxylate (1S,3R‐ACPD) and RS‐3,5‐dihydroxyphe‐nylglycine (DHPG) evoked concentration‐dependent increases in the accumulation of [3H]‐inositol phosphates with pEC50 values of 2.98±0.02, 4.45±0.06 and 4.47±0.07, respectively. Maximal responses to these agents were 43±8, 52±12 and 84±11% of the response to 1 mM histamine, respectively.

The phosphoinositide response to 1S,3R‐ACPD was antagonized in the presence of (+)‐α‐methyl‐4‐carboxyphenylglycine, with a calculated pKi value of 3.55±0.03.

Forskolin‐stimulated accumulation of [3H]‐cyclic AMP was not significantly altered in the presence of 10 μm DCG‐IV or 300 μm 1S,3R‐ACPD. Similarly, 300 μm 1S,3R‐ACPD failed to alter isoprenaline‐ (1 μm) or 2‐chloroadenosine (2‐CA, 30 μm)‐stimulated accumulation of [3H]‐cyclic AMP.

Forskolin‐stimulated accumulation of [3H]‐cyclic AMP was concentration‐dependently inhibited in the presence of L‐glutamate and L‐serine‐O‐phosphate (L‐SOP) with pIC50 values of 2.91±0.17 and 2.86±0.04 with maximal inhibitions of 47±2 and 92±3%, respectively. L‐2‐Amino‐4‐phosphonobutyrate (L‐AP4) inhibited the forskolin response without saturating, evoking an inhibition of 71±7% at 3 mM.

2‐CA‐evoked accumulation of [3H]‐cyclic AMP was also inhibited by L‐glutamate and L‐SOP with pIC50 values of 2.71±0.03 and 2.72±0.08 and maximal inhibitions of 51±5 and 99±0%, respectively. L‐AP4 inhibited the 2‐CA response without saturating, evoking an inhibition of 68±1% at 3 mM.

Isoprenaline‐evoked accumulation of [3H]‐cyclic AMP was inhibited by L‐glutamate and L‐SOP with pIC50 values of 3.21±0.01 and 2.96±0.08 and maximal inhibitions of 88±2 and 93±3%, respectively.

These results suggest that the guinea‐pig cerebellum expresses Group I and Group III mGluRs coupled to phosphoinositide turnover and inhibition of cyclic AMP generation, respectively.
The role of the endocardium in the facilitatory effect of bradykinin on electrically‐induced release of noradrenaline in rat cardiac ventricle
Authors: Vaz‐da‐Silva M. , Magina S. , Domingues‐Costa A. , Moura D. , Guimarães S.
Published: July 2012

The present investigation was undertaken to study the role of bradykinin in noradrenaline release from the ventricle of the rat induced by electrical stimulation. Slices of the left ventricle of adult Wistar rats with or without endocardium were previously loaded with 0.2 μm [3H]‐noradrenaline and washed out before electrical stimulation was applied.

Bradykinin (0.1–100 nM) concentration‐dependently increased tritium release evoked by electrical stimulation (EC50 = 3.5 (1.2–10.2) nM; n= 12). The angiotensin converting enzyme inhibitor, captopril (1 μm), which per se had no effect on tritium release, caused a marked enhancement of the bradykinin facilitatory effect, shifting the concentration‐response curve of bradykinin to the left by about one log unit. The compound Hoe 140, a selective inhibitor of B2‐bradykinin receptors, competitively antagonized the effect of bradykinin, indicating the involvement of these receptors in the action of bradykinin.

In endocardium‐free ventricle, bradykinin had no effect either in the absence or in the presence of captopril.

These results show that: (1) bradykinin is able to facilitate noradrenaline release evoked by electrical stimulation of the rat ventricle through activation of B2‐bradykinin receptors located on endocardial cells; (2) this action of bradykinin which is markedly potentiated by the inhibition of the angiotensin‐converting enzyme seems to be exerted through the release of some factor which is formed in the endocardium and diffuses into the myocardium where it acts.
Preservation of endothelium‐dependent relaxation in cholesterol‐fed and streptozotocin‐induced diabetic mice by the chronic administration of cholestyramine
Authors: Kamata Katsuo , Sugiura Makoto , Kojima Satoshi , Kasuya Yutaka
Published: July 2012

Experiments were designed to investigate the effects of the low density lipoprotein (LDL)‐lowering drugs cholestyramine on serum LDL levels and endothelium‐dependent relaxation to acetylcholine (ACh) in cholesterol‐fed or streptozotocin (STZ)‐induced diabetic mice.

In aortic rings from control mice, ACh or A23187 caused concentration‐dependent relaxation. The relaxations caused by ACh or A23187 were significantly attenuated in aortic rings from cholesterol‐fed and STZ‐diabetic mice. The attenuated vasodilatation in both cholesterol‐fed and diabetic mice was returned to normal by chronic administration of cholestyramine. The endothelium‐independent relaxations of aortic rings induced by sodium nitroprusside (SNP) were not significantly different between control, cholesterol‐fed and STZ‐induced diabetic mice.

The increased LDL levels in cholesterol‐fed and diabetic mice were returned to normal by the chronic administration of cholestyramine. Chronic administration of cholestyramine had no effects on serum glucose levels.

These results suggest that attenuated endothelium‐dependent vasodilatations in both cholesterol‐fed and STZ‐diabetic mice are improved by the chronic administration of cholestyramine, and these effects are, at least in part, due to lowering serum LDL levels.