HEMOGLOBIN AUGMENTATION OF INTERLEUKIN-1-BETA-INDUCED PRODUCTION OF NITRIC-OXIDE IN SMOOTH-MUSCLE CELLS

The roles of hemoglobin (Hb) in the pathogenesis of cerebral vasospasm remain a matter of discussion. Hemoglobin is known to be released from extravasated red blood cells in a variety of pathological conditions, including subarachnoid hemorrhage. These conditions are often accompanied by infiltration of inflammatory cells and an associated release of multiple cytokines. Certain of these cytokines, including interleukin-1 beta (IL-1 beta), are capable of increasing nitric oxide (NO) production via the inducible form of nitric oxide synthase (NOS), and excessive NO production under these conditions may contribute to cellular dysfunction. This study further examines these questions by investigating the effects of Hb on the induction of NOS by IL-1 beta. The effects of Hb on IL-1 beta-induced NO production were examined in cultured smooth-muscle cells of rat aorta (RA-SMC's). Production of NO was estimated from the accumulation of nitrite, an oxidative product of NO, in the culture medium. The synthesis of NO was induced by IL-1 beta in a concentration-dependent manner. This activation of NO production was inhibited by: 1) a general inhibitor of NOS (N-omega-nitro-L-arginine); 2) a protein synthesis inhibitor (cycloheximide); and 3) two selective inhibitors of the inducible form of NOS (hydrocortisone and aminoguanidine). These results suggest that IL-1 beta promotes the expression of the inducible form of NOS in RA-SMC's. The effects of Hb on NO production were tested by adding purified human Hb to the culture medium of the cells in both the presence and absence of IL-1 beta. Nitrite accumulation was slightly but significantly increased by Hb in the absence of IL-1 beta. In contrast, Hb markedly augmented nitrite accumulation induced by IL-1 beta. This augmentation persisted even after the removal of Hb from the culture medium. The number of cells was not affected by Hb or IL-1 beta. The findings demonstrate that Hb can modify cytokine-induced production of NO in RA-SMC's by increasing the inducible form of NOS. These observations suggest that Hb can also modify the action of inflammatory cells by facilitating NO production in target cells.