Impaired endothelium-dependent relaxation occurs in diabetic rabbit aorta and normal aorta exposed to elevated concentrations of glucose and is prevented by cyclooxygenase inhibitors. The role of free radicals in the endothelial cell impairment was examined with free radical scavengers and in aortas from rabbits fed with probucol (1% wt/wt, a lipid-soluble antioxidant). Rings of aorta suspended for measurement of isometric tension were incubated for 6 h in control (5.5 mM) or elevated (44 mM) glucose. Impairment of endothelium-dependent relaxation to acetylcholine caused by exposure to elevated glucose was prevented by superoxide dismutase, catalase, deferoxamine, or allopurinol and did not occur in aortas from probucol-fed rabbits. Similarly, impairment of acetylcholine relaxations in aortas from alloxan-induced diabetic rabbits was restored to normal by superoxide dismutase. Oxygen-derived free radicals generated by xanthine oxidase also caused impaired acetylcholine relaxations. Exposure of aortic segments to elevated glucose or to xanthine oxidase caused a significant increase in release of immunoreactive prostanoids. These data indicate that the endothelial cell dysfunction caused by elevated glucose is mediated by free radicals that are likely generated through the increased cyclooxygenase catalysis occurring in the endothelium. Treatment with antioxidants protects against impaired endothelium-dependent relaxations caused by elevated glucose.

A possible relationship between protein kinase C activation and impaired receptor-mediated endothelium-dependent relaxation in diabetes mellitus was examined in isolated aorta from normal rabbit exposed to elevated glucose. Aorta treated for 10 min with 4-phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, showed decreased relaxations to the endothelium-dependent vasodilator, acetylcholine, similar to normal aorta exposed to elevated glucose (22 and 44 mM) for 6 h. Relaxations to the receptor-independent endothelium-dependent vasodilator, A23187, and those caused by the direct smooth muscle vasodilator, sodium nitroprusside, were unaffected by treatment with PMA or exposure to elevated glucose. Indomethacin increased relaxations to acetylcholine of aorta treated with PMA indicating a role for vasoconstrictor prostanoids. PMA caused a significant increase in basal and acetylcholine-stimulated release of vasoconstrictor prostanoids including thromboxane A2 from aortic segments with, but not without endothelium. Protein kinase C inhibitors, H-7 or sphingosine, restored the abnormal acetylcholine-induced relaxations as well as suppressed the abnormal release of prostanoids in aorta exposed to elevated glucose. These findings suggest that the dysfunction ofreceptor-mediated endotheliumdependent relaxation associated with exposure to elevated glucose is due to increased production of vasoconstrictor prostanoids by the endothelium as a consequence of protein kinase C activation. (J. Clin. Invest. 1991. 87:1643-1648

The effects of glucose on endothelium-dependent responses and vasoactive prostanoid production were determined by incubating isolated rabbit aortae in control (5.5 or 11 mM) or elevated (44 mM) glucose for 6 h to mimic euglycemic and hyperglycemic conditions. Rings of aortae incubated in elevated glucose, contracted submaximally by phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine compared with the aortae incubated in control glucose. Treatment with indomethacin, a cyclooxygenase inhibitor, or SQ29548, a prostaglandin H2/ thromboxane A2 receptor antagonist, restored acetylcholine relaxations of rings in elevated glucose to normal, while these agents had no effect on the relaxation of rings incubated in control glucose. Aortae incubated with mannose (44 mM) as a hyperosmotic control relaxed to acetylcholine normally. The relaxations in response to A23187 and sodium nitroprusside were not different between rings exposed to control and elevated glucose. Radioimmunoassay measurements showed a significant increase in acetylcholine-stimulated release of thromboxane A2 and prostaglandin F2, in aortae with, but not without endothelium incubated with elevated, but not with control glucose. Thus a possible mechanism for endothelium dysfunction in diabetes mellitus is the hyperglycemia-induced increased generation of endothelium-derived vasoconstrictor prostanoids. (J. Clin. Invest. 1990.85:929-932.) acetylcholine -cyclooxygenase products * endothelium -hyperglycemia

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Endothelium-dependent relaxations and vasoactive prostanoid production caused by acetylcholine were determined in the aortas of rabbits with diabetes mellitus induced by alloxan. Aortas of diabetic rabbits, contracted submaximally by phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine compared with the aortas of normal rabbits. Indomethacin, a cyclooxygenase inhibitor, and SQ 29548, a prostaglandin H2-thromboxane A2 (PGH2-TxA2) receptor antagonist, normalized the sensitivity of diabetic aortas to acetylcholine, whereas these agents had no effect on the response of normal aortas. The relaxations in response to a nonreceptor-mediated endothelium-dependent vasodilator, A23187, and an endothelium-independent vasodilator, sodium nitroprusside, were not different between normal and diabetic aortas. Acetylcholine also caused contractions of resting aortic rings with endothelium from diabetic, but not normal rabbits; these contractions were inhibited by indomethacin. Synthesis of TxA2, measured as immunoreactive TxB2, was significantly increased in diabetic aortic segments only when the endothelium was present. These results suggest that in the diabetic state, the endothelium releases a major vasoconstrictor cyclooxygenase product that either directly counteracts the relaxation caused by or selectively interferes with the release of endothelium-derived relaxing factor(s) induced by cholinergic receptor stimulation. The vasoconstrictor is most likely TxA2 or possibly its precursor, PGH2.