Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron.

In many models, a protective role for heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, has been demonstrated. Also, HO-1 null mice (KO) are more susceptible to inflammation and hypoxia and transplant rejection. Nonetheless, their response to hyperoxia (> 95% O(2)) has not yet been evaluated. Surprisingly, after acute hyperoxic exposure, KO had significantly decreased markers of lung oxidative injury and survived chronic hyperoxia as well as wild-type (WT) controls. Disrupted HO-1 expression was associated with decreased lung reactive iron and iron-associated proteins, decreased NADPH cytochrome cp450 reductase activity, and decreased lung peroxidase activity compared to WT. Injection of tin protoporphyrin, an inhibitor of HO, in the WT decreased acute hyperoxic lung injury, whereas transduction of human HO-1 in the KO reversed the relative protection of the KO to acute injury and worsened hyperoxic survival. This suggests that disruption of HO-1 protects against hyperoxia by diminishing the generation of toxic reactive intermediates in the lung via iron and H(2)O(2).

Activation of mitogen activated protein kinase in human platelets by genistein.

Genistein, a putative tyrosine kinase inhibitor, stimulated human platelet mitogen activated protein kinase (MAPK) activity in a dose- and time-dependent manner. When MAPK was maximally stimulated by phorbol 12-myristate 13-acetate (PMA), genistein still elicited the increase in MAPK activity. Staurosporine (50 nm), significantly decreased the PMA-induced MAPK activity, but had little inhibitory effect on the genistein-induced MAPK activity. Both these observations indicated a protein kinase C (PKC) independent pathway for the genistein-stimulated MAPK activity. When other tyrosine kinase inhibitors (methyl-2,5-dihydroxycinnamate, and tyrphostin) were employed, similar increases in the MAPK activity were observed. Addition of genistein to cytosolic fraction of platelets had no effect on the MAPK activity and indicated that this effect is not due to direct physical interaction between genistein and MAPK and that intact platelets are required for it. MAPK activity of platelets from rabbit and pig was also stimulated by genistein. This effect of genistein was not observed in other cell types tested (BNLCL2, HEL and U937 cells). Forskolin, which increases cyclic AMP had little effect on the basal platelet MAPK activity or the genistein activated MAPK, while it decreased by half the PMA-induced MAPK activity. The inactive analog of genistein, daidzein, which does not inhibit tyrosine kinase had little effect on MAPK. Genistein caused a decrease in basal tyrosine phosphorylation of pp60(c-src) protein as detected with anti-phosphotyrosine (anti-PTyr) Ab. Thus, inhibition of basal tyrosine kinase results in an increase in MAPK activity. This study demonstrates for the first time a novel mechanism for regulation of MAPK in platelets in which inhibition of tyrosine kinase results in activation of MAPK, independent of PKC and cAMP pathways.

Morphological evidence for the antiatherogenic effect of scoparone in hyperlipidaemic diabetic rabbits.

OBJECTIVE: Scoparone (6,7-dimethoxycoumarin), a coumarin isolated from a hypolipidaemic Chinese herb Artemisia scoparia, has vasodilator and antiproliferative activities and possesses free radical scavenging properties in vitro. The aim of the study was to investigate the morphological effects of scoparone in the antiatherogenic process in vivo by using hyperlipidaemic diabetic rabbits as an animal model. METHODS: Male New Zealand White rabbits were divided into three groups: control (normal), hyperlipidaemic diabetic, and scoparone treated hyperlipidaemic diabetic. The plasma concentration of total cholesterol and triglycerides were determined. The thickness of the tunica intima was measured on paraffin sections of the aortas stained with Movat's pentachrome. The aortic samples were also processed for scanning and transmission electron microscopy. RESULTS: Neither the lipid profile in the plasma nor the structures of the aortic wall from the control group showed abnormalities. In contrast, the aortas from the hyperlipidaemic diabetic group showed prominent atherosclerotic plaques. Large numbers of monocytes were found adherent to the luminal surface and a markedly thickened intima filled with many lipid laden foam cells was clearly observed. By comparison, the scoparone treated group showed less advanced atherosclerosis with a lower plasma cholesterol. In the scoparone treated rabbits, the proportion of the aortic surface area covered with macroscopic plaques was 30%, and the thickness of the tunica intima 17%, of that of the non-scoparone treated hyperlipidaemic diabetic rabbits. CONCLUSIONS: Scoparone has an antiatherogenic action in hyperlipidaemic diabetic rabbits.

Protection by scoparone against the alterations of plasma lipoproteins, vascular morphology and vascular reactivity in hyperlipidaemic diabetic rabbit.

1. The in vivo pharmacological effects of scoparone (6,7-dimethoxycoumarin) in a hyperlipidaemic diabetic rabbit model were investigated. 2. Three groups of rabbits were studied: (1) normal, (2) hyperlipidaemic and diabetic-untreated and (3) hyperlipidaemic and diabetic-scoparone treated. The hyperlipidaemic diabetic rabbits were fed with 1% cholesterol and treated with alloxan, a diabetogenic agent. The plasma levels of total cholesterol, total triglyceride, very low-density lipoprotein (VLDL) cholesterol, low density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol were markedly increased as soon as the rabbit became diabetic at the second week. Scoparone-treatment (5 mg kg-1 day-1, s.c.) significantly reduced the plasma lipid and lipoprotein cholesterol levels of the hyperlipidaemic diabetic rabbit to 73.3% of total cholesterol, 48.3% of total triglyceride, 66.0% of VLDL cholesterol, 55.7% of LDL cholesterol and 79.5% of HDL cholesterol. 3. Six weeks after cholesterol-feeding, the aortic arch and thoracic aorta were dissected for morphological and functional studies. In vascular rings from the untreated hyperlipidaemic diabetic rabbit, there was intimal thickening with accumulation of fatty streaks, foam cells and migration of smooth muscle cells to the intima. In the rabbits treated with scoparone, there were fewer pathological morphology changes found in vascular segments than in the untreated hyperlipidaemic diabetic rabbits. 4. In the vascular reactivity experiments, the phenylephrine-induced contraction and nitroprusside induced dilatation did not differ significantly among the three rabbit groups, except that the contraction was enhanced in the thoracic aorta of hyperlipidaemic diabetic rabbits either untreated or treated withscoparone, as compared to the normal group, and the sensitivity to nitroprusside was increased in the thoracic aorta of the scoparone-treated group as compared to the untreated group.5. The endothelium-dependent dilatation induced by acetylcholine was significantly attenuated in both the aortic arch and thoracic aorta from the hyperlipidaemic diabetic rabbits as compared to the normal rabbits. This attenuation was partially prevented, when scoparone (5 mg kg-1) was administered daily.6. These results suggest that scoparone protects against some alterations of plasma lipoproteins,vascular morphology and vascular reactivity in the hyperlipidaemic diabetic rabbit. These protective effects of scoparone may be partly related to its free radical scavenging property.

Vasodilator effect of scoparone (6,7-dimethoxycoumarin) from a Chinese herb.

A possible mechanism of the vasodilator effect of scoparone was investigated. Scoparone (10(-6)-3 x 10(-5) M) dilated rat aortic rings precontracted with phenylephrine in a dose-dependent manner. The presence of endothelium facilitated the vasodilator effect. Scoparone depressed the contractile responses to phenylephrine and serotonin, but not that to potassium chloride. Both the vasoconstriction and O2- production induced by alloxan, a diabetogenic compound, were depressed by scoparone. It appears that scoparone exhibited a free radical scavenger-like effect. The dilatation elicited by acetylcholine was potentiated by scoparone. The dilator activity of scoparone was markedly inhibited by methylene blue and hemoglobin, guanylate cyclase inhibitors. Furthermore, the basal guanosine 3',5'-cyclic monophosphate (cGMP) level was elevated in the presence of scoparone. The dilator activity of scoparone was also inhibited by quinacrine (inhibitor of phospholipase A2) and indomethacin (inhibitor of cyclooxygenase). Our results showed further that the output of 6-keto-prostaglandin F1 alpha, a stable metabolite of prostacyclin, was enhanced by scoparone. It is suggested that the vasodilator effect of scoparone in rat aorta may be mediated through the enhancement of prostacyclin release, protecting against EDRF inactivation, and activating guanylate cyclase.