1 In view of the potential deleterious effects of high amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) in inflammation, the prevention of the expression of this enzyme represents an important therapeutic goal. In cytokine-stimulated cells, activation of nuclear factor KB (NF-KB) is crucial for the increase in iNOS gene expression. Since NF-KB activation appears to involve a redox-sensitive step, we have investigated whether three structurally unrelated antioxidants, 5,7-dihydroxyflavone (chrysin), 3,4-dichloroisocoumarin (DCI) and N-acetyl 5-hydroxytryptamine (Nacetylserotonin, NAS), affect iNOS expression in cultured RAW 264.7 monocyte/macrophages stimulated with bacterial lipopolysaccharide (LPS, 140 ng ml-') and interferon-y (IFNy, 5 u ml-').2 During a 6 h incubation period neither LPS nor IFNy alone exerted a significant effect but when combined, caused a prominent increase in nitrite formation, iNOS mRNA and protein abundance. Coincubation with chrysin (50 ,uM), DCI (50 gM) or NAS (1 mM) markedly attenuated this increase in iNOS gene expression. 3 DCI, but not chrysin or NAS, prevented the activation of NF-KB in cells exposed to LPS plus IFNy for 30 min. In contrast, all three antioxidants significantly blunted the DNA-binding activity of interferon regulatory factor 1 (IRF-1), which mediates the synergistic effect of IFNy on iNOS gene expression in cells treated for 2 h with LPS plus IFNy. 4 DCI thus appears to inhibit iNOS gene expression at the transcriptional level by preventing the activation of both NF-KB and IRF-1. The inhibitory effect of DCI on NF-KB activation, however, does not seem to be related to its antioxidative properties, since DCI, unlike chrysin or NAS, is a potent serine protease inihbitor which stabilizes the inactive NF-KB complex by protecting the inhibitory IKB-a subunit from proteolytic degradation. 5 The virtually identical inhibitory effect of chrysin, DCI and NAS on the activation of IRF-1 points to a redox-sensitive step in the activation of this transcription factor, which in contrast to NF-KB requires de novo protein synthesis. 6 Since iNOS gene expression in human cells and tissues usually requires the combination of several cytokines, antioxidants such as chrysin and NAS which do not interfere with the activation of NF-KB may be of therapeutic value for selectively inhibiting the enhanced expression of this enzyme in inflammation.

The mechanism of drug-induced inhibition of the transient outward current, Ito, has been investigated in rat ventricular myocytes using the whole cell patch clamp technique. Ito was activated by 300 ms depolarizing voltage clamp steps in 10 mV increments from -50 mV up to +40 mV. At +40 mV, Ito peaked after about 3 ms, and the time course of inactivation was appropriately described by two time constants, tau fast = 17 ms and tau slow = 203 ms. Verapamil, quinidine sulfate and nifedipine preferentially depressed Ito at the end of the 300 ms depolarizing voltage clamp step; the inactivation of Ito was accelerated by all drugs, whereas peak Ito was less affected. The time course of drug action at +40 mV was calculated by the fractional changes of Ito. Verapamil, quinidine sulfate and nifedipine exerted a block of Ito increasing during the depolarizing voltage clamp step. The onset of block in response to verapamil, quinidine sulfate and nifedipine (30 mumol/each) was appropriately described by monoexponential functions with time constants tau on = 9.3, 1.7 and 1.1 ms, respectively. Relief from block by verapamil, quinidine sulfate and nifedipine at -50 mV was assessed by comparison of the recovery process of peak Ito from inactivation with or without drugs. tau off amounted to 695 ms in the case of quinidine sulfate; verapamil and nifedipine did not significantly affect the recovery process so that the determination of the time course of relief from block was not possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of rats with cytokines has been associated with an increase in the circulating levels of endothelin 1 (ET-1). Here we show that administration of tumor necrosis factor a (TNF-a; 4 ,ugkg-1) to anesthetized rats caused within 15 min a strong elevation in the circulating levels of ET-1. This was associated with a striking coronary vasoconstriction in hearts from these animals when they were removed and perfused in vitro by the Langendorff technique. This vasoconstriction was largely overcome by treatment with either the endothelin type A (ETA) receptor antagonist FR 139317 or antibody against ET-1. Furthermore, it was mimicked by in vivo exposure to exogenous ET-1. Endogenously produced TNF-a may also cause such a coronary vasoconstriction, for treatment with interleukin 2 (600 ,.g-kg-1) produced an increase in coronary perfusion pressure that correlated with the increases in circulating TNF-a. This coronary vasoconstriction was substantially reversed by treatment either with antibody against TNF-a or with FR 139317. We suggest, therefore, that cytokine-driven changes in the production of ET-1 are key events in the development of vascular pathologies.Exogenously applied endothelin 1 (ET-1) acts via specific type A and B (ETA and ETB) receptors within the vasculature to produce potent and profound changes in vessel diameter, vessel permeability, and, in the longer term, vessel structure (see refs. 1 and 2). The importance of endogenously produced ET-1 in the regulation of blood vessel activity under physiological conditions is, however, not clear, although it may, for instance, regulate basal blood flow in humans (3). Nevertheless, there is much evidence implicating an increase in ET-1 release as a causative factor in numerous pathological states (see refs. 1 and 2), including myocardial dysfunctions in both animals and humans (4-7). However, the precise regulation of ET-1 biosynthesis and release, particularly in vivo, has yet to be fully elucidated, although it is generally believed to be a slowly responding system mediating chronic vasoconstrictor responses and/or resistance changes (1, 2, 8). Cytokines enhance the release of ET-1 both from endothelial cells (9) and within the circulation (10) and, interestingly, many of the pathological conditions in which there are elevations in the circulating levels of ET-1 are associated with increased production of cytokines. Here we show that there is a direct association between the endogenous production of cytokines and the release of ET-1 and that this rapidly leads to sustained vasoconstriction. MATERIALS AND METHODSBlood Pressure. Male Wistar rats (230-260 g; Glaxo) were anesthetized with sodium thiopentone (Intraval; 120 mg-kg-', i.p.). The trachea was cannulated to facilitate respiration and body temperature was maintained at 37°C by means of a rectal probe connected to a homeothermic blanket (Biosciences, Sheerness, Kent, U.K.). The right carotid artery was cannulated and connected to a pressure transducer (Elcomatic type 750) for measurement of a...

1 Inflammatory disease states predispose to myocardial infarction. Here we have investigated the effects of a systemic inflammatory response syndrome, i.e. lipopolysaccharide (LPS)-induced circulatory shock in rats, on coronary vascular tone. 2 Anaesthetized rats were given LPS (10 mg kg-', i.v.) and the hearts excised 180 min later for isolated perfusion at constant flow by the Langendorff technique. Once the ex vivo perfusion was started, the perfusion pressure strongly increased in these hearts compared to hearts from control rats (130+3 vs. 49+3 mmHg after 10 min). This increase in coronary resistance was not associated with a reduction in endothelial cell function, for the vasodilator responses to bradykinin were unchanged. 3 When hearts were removed 30 min after the injection of LPS, the LPS-induced rise in perfusion pressure was delayed. No changes in perfusion pressure were seen when the hearts were removed 15 min after the injection of LPS. Pre-treatment with cycloheximide or an anti-tumour necrosis factor-a (TNF-a) antibody or continuous infusion in vivo and in vitro of the endothelin ETA receptor selective antagonist FR 139317, greatly decreased the increase in coronary vascular resistance induced by LPS. 4 These data suggest that TNF-cx may induce the release of endothelin-1 (ET-1) and that this mediates at least part of the coronary vasoconstriction. This hypothesis is supported by the demonstration that LPS administration increased the circulating levels of both TNF-a and ET-1. 5 We conclude, therefore, that in inflammatory disease states, such as LPS-induced septic shock, there is the sequential release of TNF-a and endothelin-1 which leads to an increase in coronary vascular tone and so a predisposition to myocardial ischaemia. Inactivation of TNF-a by an antibody as well as ETA receptor blockade by a selective antagonist may effectively interfere with this pathway.

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1 Induction of the calcium-independent isoform of nitric oxide (NO) synthase (iNOS) in various cell types has been implicated in the circulatory failure in experimental models of septic shock. Tetrahydrobiopterin (BH4) appears to be an essential co-factor for NO formation and therefore an inhibition of its biosynthesis represents a feasible therapeutic target. We have investigated the effects of an inhibitor of BH4 synthesis, N-acetyl-5-hydroxytryptamine (N-acetylserotonin, NAS), on the expression of iNOS in cultured macrophages and smooth muscle cells in vitro, and on the hypotensive response to bacterial lipopolysaccharide (LPS) in the anaesthetized rat in vivo. 2 NAS (0.01-5 mM) caused a concentration-dependent inhibition of the accumulation of nitrite in the conditioned medium of LPS/interferon-y (IFNy)-stimulated RAW 264.7 macrophages and interleukin-lf (IL-lB)-activated vascular smooth muscle cells (VSMC). This effect was paralleled by a similar decrease in the iNOS protein content of these cells, as determined by immunoblot analysis.3 Pretreatment of RAW 264.7 macrophages with the BH4 precursor, dihydrobiopterin (BH2, 0.1 mM)did not restore nitrite formation in the presence of NAS (1 mM).4 Intravenous administration of NAS (1 mg kg-' min-' for 30 min) in anaesthetized rats significantly reduced the fall in mean arterial blood pressure, restored the pressor response to noradrenaline (1 Mg kg-') and ameliorated the increase in plasma nitrite following exposure to LPS (10 mg kg-1).5 NAS pretreatment also attenuated iNOS activity in lung homogenates, as determined by the conversion of radiolabelled L-arginine to L-citrulline, and partially restored the constrictor effect of noradrenaline in aortic rings isolated from LPS-treated rats. Moreover, NAS significantly reduced the rise in the plasma concentration of tumour necrosis factor a (TNFa) in response to LPS. 6 These findings suggest that NAS inhibits the expression rather than the activity of iNOS in cultured macrophages and smooth muscle cells. This effect of NAS appears to be independent of the availability of BH4, but may be related to an attenuation of the release of TNFx following LPS administration, as shown in the anaesthetized rat. This mechanism may also account for the beneficial haemodynamic effect of NAS in our experimental model of endotoxaemia.

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