Effects of melittin on endothelium-dependent relaxation and cyclic GMP levels in rat aorta

The present study investigates the mechanism of endothelium-dependent relaxation of vascular smooth muscle. Melittin, a polypeptide found in honeybee venom and a known activator of phospholipase A₂, induced transient, endothelium-dependent relaxations of rat thoracic aortae contracted with norepinephrine. Higher concentrations of melittin induced relaxations followed by contractions. Prior incubation of melittin with trypsin abolished the changes in relaxation and contraction due to melittin. Melittin (10 μg/ml)-induced relaxations were associated with transiently elevated levels of cyclic GMP with a peak increase of 30-fold, which occurred 30 seconds after melittin exposure. Melittin (10 μg/ml) elevated cyclic AMP levels less than twofold and this effect was variable. A lower concentration of melittin (1 μg/ml) elevated cyclic GMP levels approximately twofold, while exposure to 1 μg/ml melittin in the presence of the cyclic GMP phosphodiesterase inhibitor, M&B 22948 (1 mM), increased cyclic GMP levels fivefold. Removal of the endothelium prevented the increased levels of cyclic GMP and cyclic AMP due to melittin. Exposure to the guanylate cyclase inhibitor, methylene blue, prevented the increased levels of cyclic GMP. Methylene blue, nordihydroguaiaretic acid, and the phospholipase A₂ inhibitor, parabromophenacyl bromide, inhibited melittin-induced relaxations, while the cyclooxygenase inhibitor, indomethacin, was without effect. Arachidonic acid increased cyclic AMP levels but had no effect on cyclic GMP levels in the presence or absence of indomethacin. Relaxations to melittin, and to the endothelium-dependent vasodilators acetylcholine, trypsin, histamine, and the Ca²⁺ ionophore A23187, and/or the associated increased cyclic GMP levels, were reduced following exposure to melittin. Prior exposure to polyarginine (10 μg/ml), which induced endothelium-dependent relaxations that were prevented by methylene blue, also inhibited relaxations to the endothelium-dependent vasodilators. In contrast, relaxations to sodium nitroprusside were potentiated in tissues previously exposed to melittin. Removal of the endothelium by rubbing the intimal surface also potentiated relaxations to sodium nitroprusside. Scanning electron micrographs of the intimal surface demonstrated that melittin and polyarginine greatly damaged the endothelial cells. The present results suggest that polycation containing peptides induce endothelium-dependent relaxation through elevation of cyclic GMP levels within the smooth muscle. Furthermore, these peptides may densenitize tissues to endothelium-dependent vasodilation by causing damage to the endothelial cells.