Michel Finet

The positive inotropic action of the nifedipine analogue, Bay K 8644, in guinea-pig and rat isolated cardiac preparations

1 The inotropic effect of Bay K 8644 has been studied in rat and guinea‐pig atria and ventricular strips stimulated at 1 Hz, in a medium containing CaCl2 1.8 mM. The positive inotropic effect at maximal effective concentrations of Bay K 8644 was in the following order: guinea‐pig ventricle > rat ventricle > guinea‐pig atria >> rat atria. 2 In rat preparations, the tension recorded at maximum effective concentrations of Bay K 8644 was similar at three different calcium concentrations (0.7, 1.8, 3.0 mM). The amplitude of the positive inotropic effect evoked by Bay K 8644 increased when atrial and ventricular contractions were reduced by lowering the external calcium concentration. 3 The contractile tension reached in the presence of maximum effective concentrations of Bay K 8644 (3 × 10−7 − 1 × 10−6 M) was greater than that produced by the maximum effective concentration of external calcium (3 mM) in rat ventricles but not in rat atria. 4 High doses of nifedipine (3 × 10−7 − 1 × 10−6 M) depressed the contraction of rat atria more than the contraction of rat ventricles. 5 In rat ventricles, nifedipine shifted to the right the inotropic dose‐effect curve of Bay K 8644. 6 It is concluded that the interaction between nifedipine and Bay K 8644 occurred at the same binding sites. These sites have some characteristics of the low affinity binding sites of nifedipine and other related dihydropyridines.

Cyclo-oxygenase and lipoxygenase pathways in mast cell dependent-neurogenic inflammation induced by electrical stimulation of the rat saphenous nerve

We investigated the role of arachidonic acid metabolism and assessed the participation of mast cells and leukocytes in neurogenic inflammation in rat paw skin. We compared the effect of lipoxygenase (LOX) and cyclo‐oxygenase (COX) inhibitors on oedema induced by saphenous nerve stimulation, substance P (SP), and compound 48/80. Intravenous (i.v.) pre‐treatment with a dual COX/LOX inhibitor (RWJ 63556), a dual LOX inhibitor/cysteinyl‐leukotriene (CysLt) receptor antagonist (Rev 5901), a LOX inhibitor (AA 861), a five‐lipoxygenase activating factor (FLAP) inhibitor (MK 886), or a glutathione S‐transferase inhibitor (ethacrynic acid) significantly inhibited (40 to 60%) the development of neurogenic oedema, but did not affect cutaneous blood flow. Intradermal (i.d.) injection of LOX inhibitors reduced SP‐induced oedema (up to 50% for RWJ 63556 and MK 886), whereas ethacrynic acid had a potentiating effect. Indomethacin and rofecoxib, a highly selective COX‐2 inhibitor, did not affect neurogenic and SP‐induced oedema. Surprisingly, the structurally related COX‐2 inhibitors, NS 398 and nimesulide, significantly reduced both neurogenic and SP‐induced oedema (70% and 42% for neurogenic oedema, respectively; 49% and 46% for SP‐induced oedema, respectively). COX‐2 mRNA was undetectable in saphenous nerves and paw skin biopsy samples, before and after saphenous nerve stimulation. A mast cell stabilizer, cromolyn, and a H1 receptor antagonist, mepyramine, significantly inhibited neurogenic (51% and 43%, respectively) and SP‐induced oedema (67% and 63%, respectively). The co‐injection of LOX inhibitors and compound 48/80 did not alter the effects of compound 48/80. Conversely, ethacrynic acid had a significant potentiating effect. The pharmacological profile of the effect of COX inhibitors on compound 48/80‐induced oedema was similar to that of neurogenic and SP‐induced oedema. The polysaccharide, fucoidan (an inhibitor of leukocyte rolling) did not affect neurogenic or SP‐induced oedema. Thus, (i) SP‐induced leukotriene synthesis is involved in the development of neurogenic oedema in rat paw skin; (ii) this leukotriene‐mediated plasma extravasation might be independent of mast cell activation and/or of the adhesion of leukocytes to the endothelium; (iii) COX did not appear to play a significant role in this process.

The inotropic effect of ouabain and its antagonism by dihydroouabain in rat isolated atria and ventricles in relation to specific binding sites.

1 The inotropic effect of ouabain has been studied in rat ventricles and atria. 2 The concentration‐effect curve of ouabain may be fitted by a model assuming the existence of two saturable components. The component with the higher sensitivity to ouabain accounted for 30% of the maximal increase in systolic tension in ventricles and for only 5% in atria. Increase in diastolic tension was only apparent at ouabain concentrations required to observe the low sensitivity component. 3 [3H]‐ouabain binding has been examined in microsomes prepared from atria and ventricles. High and low affinity binding sites have been observed. The ratio of high and low affinity ouabain binding sites was 4 fold lower in microsomes from rat atria than from rat ventricles. This could account for the difference in the response of these two tissues to the inotropic action of ouabain. 4 In ventricular strips the high sensitivity component was much less apparent in the presence of dihydroouabain than with ouabain. 5 When ventricular strips were preincubated in the presence of dihydroouabain 3 μm, the increase in systolic tension evoked by ouabain 1 μm was significantly reduced. Cumulative concentration‐effect curve studies showed dihydroouabain antagonism to the high sensitivity component.

L-arginine and NG-nitro-L-arginine methyl ester cause macromolecule extravasation in the microcirculation of awake hamsters

We investigated the effects of L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) on macromolecule extravasation in the microcirculation of awake hamsters by computer-assisted image analysis of the distribution of FITC (fluorescein isothiocyanate)-dextran fluorescence in dorsal fold skin preparations. This analysis made it possible to simultaneously study the time course of local (skin) and general (all irrigated organs) extravasation in 180-min experiments. Bolus injection of 30 or 150 mg/kg (i.v.) L-arginine induced immediate local and general macromolecule leakage and delayed venule dilation beginning 1 h later. Injection of 20 or 100 mg/kg (i.v.) L-NAME caused rapid venule constriction followed by local and general extravasation beginning 45-60 min later. These effects of L-arginine and L-NAME were not mimicked by their biologically inactive isomers, D-arginine and D-NAME. Simultaneous bolus injection of 20 mg/kg L-NAME and 150 mg/kg L-arginine caused no significant change in fluorescence distribution or venule diameter. L-arginine effects on macromolecule extravasation were mimicked by sodium nitroprusside (10 microg/kg, i.v.) and by 8-bromo-cGMP (1 mg/kg, i.v.). Sodium nitroprusside was ineffective on venule diameter. The effects of both L-arginine and sodium nitroprusside on FITC-dextran extravasation were prevented by simultaneous injection (10 microg/kg, i.v.) of the specific inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). This dose of ODQ mimicked the effects of L-NAME on macromolecule extravasation and venule diameter. Taken together, these results suggest that activation or inhibition of basal NO synthesis might induce macromolecule leakage in the microcirculation of awake hamsters via temporally distinct cGMP-dependent mechanisms.

Histamine-induced biphasic macromolecular leakage in the microcirculation of the conscious hamster: evidence for a delayed nitric oxide-dependent leakage

1 Late effects (up to 3 h) of intravenously‐injected histamine on FITC‐dextran extravasation were investigated in the conscious hamster, by use of computer‐assisted image analysis of fluorescence distribution in a microscopic window of dorsal skin fold preparations. This analysis allowed measurement of local (skin) and general (all organs) extravasations caused by a bolus injection of histamine (1 mg kg−1, i.v.) 2 Histamine doses higher than 0.01 mg kg−1 caused biphasic local and general extravasations. Initial phases developed fully within 15 min (for local) and 60 min (for general) and were followed by late phases beginning 90 min after histamine injection. Although the initial and late phases of histamine‐induced extravasations had differential apparent reactivities to the autacoid, all the effects of histamine on the microcirculation (1 mg kg−1) were inhibited by pyrilamine (1 mg kg−1, i.v.) but not by cimetidine (1 mg kg−1, i.v.). 3 Pretreatment with NG‐monomethyl‐L‐arginine (L‐NMMA, 30 mg kg−1, i.v.) or NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 mg kg−1, i.v.) did not affect the initial phases but did prevent the late phases of local and general extravasations triggered by 1 mg kg−1 histamine. The inhibitory effects of L‐NAME were reversed by L‐arginine (30 mg kg−1) but not by D‐arginine (30 mg kg−1) according to the enantioselectivity of nitric oxide synthase (NOS). A late NO‐mediated venular dilatation occurred in response to plasma histamine. 4 A low dose of aminoguanidine (1 mg kg−1, i.v.), a selective inhibitor of the inducible isoform of NOS (iNOS), mimicked the inhibitory effects of L‐NAME on the late phases of histamine‐induced macromolecular extravasations and venular dilatation. 5 Pretreatment with dexamethasone (1 mg kg−1, i.v.) prevented both the initial and late phases of histamine‐induced extravasations. Fucoidan (1 or 25 mg kg−1, i.v.) prevented the late phases without affecting initial phases, consistent with a role for leukocytes adhesion in the development of the late NO‐mediated effects of histamine. 6 We conclude that intravenous injection of histamine triggers a biphasic inflammatory cascade via initial activation of H1 receptors which induces a late NO‐mediated PMN‐dependent extravasation process.

The protective action of R56865 against ouabain-induced intoxication in rat heart isolated atria and ventricles.

The action of R56865 has been examined on the contractile effects produced by ouabain concentrations interacting with high (3 microM) and low (300 microM) affinity digitalis receptors on electrically stimulated ventricular strips isolated from rat. R56865 1 microM reduced the increase in resting tension produced by ouabain 300 microM and left unalterated the inotropic effect evoked by ouabain 3 and 300 microM that was reduced by higher concentrations (3 and 6 microM) of R56865. The action of R56865 was also studied on ouabain-induced intoxication in electrically stimulated and spontaneously beating atria of rat. On electrically stimulated (3 Hz) whole atria, R56865 0.3 microM reduced the maximal increase in resting tension produced by ouabain 300 microM and delayed the time to onset of the ouabain-induced arrhythmias but did not affect ouabains inotropic effect. Higher concentrations of R56865 were required to reduce the inotropic effect of ouabain. The protective action of R56865 against ouabain-induced intoxication was most pronounced on spontaneously beating atria where it reduced spontaneous rate of beats. Experiments in electrically driven left atria indicated that only a part of the protective effect of R56865 could be related to its bradycardic action. The effect of R56865 was also examined on ouabain-induced inhibition of sodium pump in human red blood cells. R56865 6 microM did not modify the inhibition produced by ouabain (from 0.3 to 10 nM), this indicates that the protective action of R56865 against ouabain-induced intoxication is not due to an interaction with the inhibitory effect of ouabain on sodium pump.

Selective inhibition by ethylisopropylamiloride of the positive inotropic effect evoked by low concentrations of ouabain in rat isolated ventricles

1 The biphasic positive inotropic effect of ouabain has been studied in rat ventricular strips in the presence of ethylisopropylamiloride (EIPA) an inhibitor of Na+/H+ exchange. 2 EIPA (10–20 μM) depressed dose‐dependently the high affinity component of the ouabain inotropic effect, whereas it did not significantly modify the low affinity inotropic effect related to high concentrations of ouabain. 3 At the EIPA concentrations studied, there was no observable modification of the inotropic effect of Bay K 8644 (10 nM, 0.3 μM) or of isoprenaline (10 nM, 1 μM). 4 These results indicate that the inotropic effect of ouabain resulting from its interaction with high affinity sites is selectively sensitive to EIPA.

The cyclo-oxygenase-dependent regulation of rabbit vein contraction: evidence for a prostaglandin E2-mediated relaxation.

Arachidonic acid (0.01–1μM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1μM. Concentrations higher than 1μM were required to induce dose‐dependent contraction of vena cava and thoracic aorta from the same animals. Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3μM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta. Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration‐relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid‐induced relaxations were prevented by indomethacin (10μM) pretreatment. In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration‐relaxation curves of this tissue to arachidonic acid in a dose‐dependent manner. In the presence of 1μM arachidonic acid, venous rings produced 8–10 fold more PGE2 than did aorta whereas 6keto‐PGF1α and TXB2 productions remained comparable. Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L‐NAME (100μM) or indomethacin (10μM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L‐NAME. We conclude that stimulation of the cyclo‐oxygenase pathway by arachidonic acid induced endothelium‐dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187‐induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2.

Respective role of lipoxygenase and nitric oxide-synthase pathways in plasma histamine-induced macromolecular leakage inconscious hamsters

Intravital microscopy technique was used to determine the distribution of a fluorescent plasma marker (fluorescein‐isothiocyanate‐dextran, 150 kD; FD‐150) into venular and interstitial compartments of dorsal skin fold preparations in conscious hamsters. One mg kg−1 histamine (i.v.) caused a biphasic decrease in venular fluorescence due to FD‐150 extravasation in all organs (general extravasation). Immediately after injection, the venular fluorescence decreased and plateaued in 60 min. Ninety minutes after histamine injection, venular fluorescence further decreased until 180 min. Prior treatment with indomethacin (0.1 mg kg−1, i.v.) did not modify the time‐course of general extravasation but prevented histamine‐induced venule dilatation. Prior treatment with the 5‐lipoxygenase activating protein (FLAP) inhibitor, 3‐[1‐(p‐chlorobenzyl)‐5‐(isopropyl)‐3‐t‐butylthioindol‐2‐yl]‐2,2‐dimethyl‐propanoic acid sodium (MK‐886)(10 μg kg−1, i.v.), the leukotriene receptor antagonist, benzenemethanol a‐pentyl‐3‐(2‐quinolinylmethoxy) (REV‐5901)(1 mg kg−1, i.v.), or the glutathione‐S‐transferase inhibitor, ethacrynic acid (1 mg kg−1, i.v.), delayed by 60 min the onset of general extravasation caused by 1 mg kg−1 histamine. Prior treatment with lipoxygenase pathway inhibitors and NG‐nitro‐L‐arginine‐methylester (L‐NAME)(100 mg kg−1, i.v.) abolished the general extravasation and venule dilatation induced by 1 mg kg−1 histamine. Injection of 1 μg kg−1 (i.v.), of leukotriene‐C4 (LTC4) or ‐D4 (LTD4) induced immediate and sustained general extravasation and reduction in venule diameter, these effects being blocked by REV‐5901. Histamine (1 mg kg−1, i.v.) induced biphasic decline in mean arterial blood pressure (MAP). An initial phase (from 0 to 60 min) was followed by a late phase beginning 90 min after histamine injection. L‐NAME (100 mg kg−1, i.v.) and aminoguanidine (1 mg kg−1, i.v.) prevented the late phase of histamine‐induced hypotension. Thus, plasma histamine can trigger both an immediate cysteinyl‐leukotriene (Cys‐LT)‐dependent and a late nitric oxide (NO)‐mediated inflammatory cascade. Although the cyclo‐oxygenase (COX) pathway might account for histamine‐induced venule dilatation, it would not influence histamine‐induced extravasation.

Effect of novel adenosine A3 receptor antagonist SSR161421 in allergic sheep models

Sheep is a species that shows 85% overall identity of the adenosine A3 receptor with its human ortholog. In this study we demonstrate a role for the adenosine A3 receptor in ovine allergic airway responses. In isolated tracheal preparations taken from sheep naturally sensitive to Ascaris suum, antigen challenge evoked airway smooth muscle contraction. The smooth muscle contractile response was dependent on the applied Ascaris suum antigen dose. The response to a threshold dose of antigen was enhanced in the presence of an adenosine A3 agonist AB-MECA (10-6M), suggesting that adenosine A3 receptor function contributed to the enhanced allergen response. This was confirmed by showing that a selective adenosine A3 receptor antagonist SSR16142, inhibited the increased antigen response with EC50=2•10M. We then tested the effects of SSR161421, given intravenously, on antigen-induced responses in allergic sheep following inhalation of Ascaris suum, in-vivo. SSR161421 (0.3-3 mg/kg) provided dose-dependent inhibition of the antigen-induced late bronchoconstrictor responses and the post antigen-induced airway hyper responsiveness. This is the first demonstration that a selective adenosine A3 receptor antagonist inhibits antigen-induced responses in a species with an A3 receptor that bears significant similarity to its human counterpart. Correspondence to: Endre G Mikus, LabMagister Training and Science Ltd., H-1045 Budapest To street 1-5, Hungary; E-mail: endre.mikus@labmagister.com