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Figure 1 Impairment of the endothelium-dependent increase in coronary flow and its prevention by superoxide dismutase (SOD) and vitamin E. Diabetes was induced in rats by streptozotocin. After a diabetes duration of 16 weeks, the stimulation of coronary flow by 5-hydroxytryptamin was measured in the isolated heart preparation as described (29). The half-maximal concentration (EC-50) was determined and represents a measure for the sensitivity of endothelium to dilate the coronary vasculature. As can be seen, in diabetes (DB), the sensitivity of endothelium is impaired as compared with healthy controls (C), but perfusion with SOD (50 pU/mL) or pretreatment of the animals with a-tocopherol (1000 U/kg body weight) were able to improve or to restore the endothelium-dependent vasodilatation in diabetes. (From Ref. 23.)

Figure 1 Impairment of the endothelium-dependent increase in coronary flow and its prevention by superoxide dismutase (SOD) and vitamin E. Diabetes was induced in rats by streptozotocin. After a diabetes duration of 16 weeks, the stimulation of coronary flow by 5-hydroxytryptamin was measured in the isolated heart preparation as described (29). The half-maximal concentration (EC-50) was determined and represents a measure for the sensitivity of endothelium to dilate the coronary vasculature. As can be seen, in diabetes (DB), the sensitivity of endothelium is impaired as compared with healthy controls (C), but perfusion with SOD (50 pU/mL) or pretreatment of the animals with a-tocopherol (1000 U/kg body weight) were able to improve or to restore the endothelium-dependent vasodilatation in diabetes. (From Ref. 23.)

Such an interaction between superoxide anions with NO has already been described. In a diffusion controlled reaction, both compounds react with each other under the formation of peroxynitrite (32-34).

Direct evidence for this conclusion is derived from experiments using isolated aortas from streptozotocin diabetic rats. This model enables us to directly measure the formation of superoxide anions by standard techniques as the reduction of cytochrome c (35). When aortas from diabetic and control animals were perfused under normoglycemic conditions, vessels from diabetic rats released significantly more superoxide anions than those from controls. In addition, the generation of superoxide anions was stimulated in both types of aortas by hyperglycemic buffers (10-30 mM glucose). The increased generation of superoxide anions could be totally reduced to control values when the endothelium was removed from the intact aortas by mechanical disruption (Rosen 1998, unpublished data). It is interesting to note that an endothelial production of ROI has also been reported for vessels isolated from hypercho-

lesterolemic and hypertonic animals (13-15). Thus, the stimulus for activation of endothelium is different in these various pathophysiological conditions, but the consequences seem to be comparable.

These experimental observations lead to the conclusion that endothelium is an important source of ROI and identify hyperglycemia as a stimulus for the formation of superoxide anions. Furthermore, the disturbed endothelium-dependent vasomotion in diabetes is an immediate pathophysiological consequence of the release of superoxide anions by the vasculature.

II. WHICH MECHANISMS CONTRIBUTE TO THE ENDOTHELIAL FORMATION OF SUPEROXIDE ANIONS IN DIABETES?

To study the mechanisms of ROI generation induced by hyperglycemia in more detail, we used human umbilical vein endothelial cells (HUVECs). To identify the generation of ROI, HUVECs were loaded with dichlorodihy-drofluorescin ester (DCF) (36), which is taken up by the cells and then rapidly hydrolyzed. DCF reacts with superoxide anions but presumably also other ROI under the emission of fluorescence light so that the formation of ROI can be determined in a time- and concentration-dependent manner.

Incubation of DCF-loaded cells with increasing concentrations of glucose (5-30 mM) leads to a time- and glucose-dependent increase in fluorescence (Fig. 2). A comparable increase in fluorescence was also observed if the cells were incubated with 3-0-methyl-d-glucose (30 mM), a glucose derivative, which is taken by the cells but not metabolized by glycolysis. These data indicate that the formation of ROI is dependent on high glucose in the culture medium but not on the synthesis of diacyl-glycerol and a glucose-dependent activation of protein kinase C. In line with this conclusion, we did not observe an alteration in DCF fluorescence by treating the cells with an inhibitor bisindolylmaleimide (BIM) or activator phorbol 12-myristate 13-ace-tate (PhA) of protein kinase C (23).

The formation of ROI by endothelial cells incubated with high glucose was completely inhibited by antioxidants (a-tocopherol 10 (J.g/mL and thioctic acid 0.5 ^M) and by diphenyliodonium (DPI, 1 (iM), a selective inhibitor of flavoprotein containing NAD(P)H oxidases (37). The inhibitory effect of DPI is consistent with the assumption that NAD(P)H oxidases are the major source of ROI in HUVECs cultivated in hyperglycemic glucose. DPI was also reported to inhibit the NADH-dependent production of superoxide anions in bovine coronary endothelial (38).

Figure 2 Increase in the formation of ROIs by human endothelial cells in dependence of glucose. HUVECs were preloaded with the DCF (1 |iM) and dichlorodihy-drofluorescin (10 |iM) for 45 min. After washing, the cells were incubated with d-glucose (5-30 mM). For control, cells were incubated with mannitol and l-glucose (25 + 5 mM). After a 15-min incubation (37°C), the fluorescence intensity as a parameter of the ROI generation was analyzed by fluorescent microscopy and quantified.

Although cyclooxygenases and lipoxygenases may also be sources of superoxide anion generation in endothelium (35,38), our data do not link these enzymes to the production of superoxide anions induced by hyperglycemia, because indomethacin and nordihydroguaretic acid did not inhibit the release of superoxide anions. Similar observations have already been reported for porcine endothelial cells (35).

Surprisingly, the DCF fluorescence was also prevented by inhibitors of NO synthase (L-nitroarginine, 100 |iM) and a chelator of intracellular calcium 1,2-bis (2-Aminoprenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). These observations indicate that the mobilization of intracellular calcium and an activation of NO synthesis are necessary steps for the formation of DCF fluorescence by hypoglycemia. In line with this conclusion, the release of nitrite (as parameter of NO synthesis) by HUVECs was stimulated by glucose (Fig. 3).

Thus, under hyperglycemic conditions, both NAD(P)H oxidase and NO synthase become activated, and both steps are a precondition for the formation of DCF fluorescence by HUVECs in hyperglycemia. This synergistic actions of NAD(P)H oxidase and NO synthase suggest that DCF fluorescence does not

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