The cffcet of ma8ncsium (ME)-dcficicnt culture on endothelial cell susceptibility to oxidativc stress was examined. Bovine endothelial eclls were cultured in tither control sufficient (0.8 mM) or deficient (0.4 mM) levuls of MgC13. Oxygen radicals wcrc produeed cxtmecllularly by the addition of dihydroxyfumaratc and Fe'+-ADP. Isolated Mg-deficient cndothclial eclls produced 2-to 3.fold higher levels of thiobarbituric acid (TBA)-reactive materials when incubated with this free radical syslem. Additional studies were pcrformcd using digitized video microscopy and 2',7'-dichlorofluoresoin diacetatc (DCFDA) as an intracellular indicator foroxidativc events at the sin&? ccl1 level. In rcsponx 10 theexogenousoxidativc stress, cndothelial eclls exhibited a time-dependent increase in fluoresccncc, sug8estivc of intracellular lipid peroxidation. The incrcasc in ecllular fluoresecnee began within 1 min of free radical addition; the Mg-dcficicnt cells exhibited a mote rapid increase in Ruorcsecnee than tbat of Mg-sufficient eells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicnls, but the loss of cellular viability began to occur only after 15 min of free radical exposure, lag& behind the detection of intracellular oxidation products. These results sugScst that increased oxidative cndothelial eel1 injury may eontributc to vaseuktr injury during Mg deficiency.

We have developed two rodent models of diet-induced magnesium-deficiency in which histologically defined cardiac lesions can be induced within two to three weeks. During the development of these lesions, the magnesium-deficient animals exhibit circulating cytokine levels which are indicative of a generalized inflammatory state. Dramatic elevations of the macrophage-derived cytokines, IL-1, IL-6, and TNF-alpha together with significantly elevated levels of the endothelial cell-derived cytokine, endothelin, were detected in the plasma of these animals. We believe that the pathophysiological effects caused by the action of these cytokines may play a role in the promotion of cardiovascular pathology associated with magnesium deficiency.

During the progression of Mg deficiency in a rodent model, we have observed dramatic increases in serum levels of inflammatory cytokines [interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha)] after 3 wk on a Mg-deficient diet. Sequential analyses of these cytokine changes in the serum of rats revealed an initial rise at day 12, followed by a major elevation in all three cytokine levels by day 21. Of greater interest was an early peak in the serum level of the neuropeptide substance P after only 5 days on the diet. This "neuronal" tachykinin is thought to be released from neural tissues, and it is known to stimulate production of certain cytokines, including IL-1, IL-6, and TNF-alpha. In addition, there was a concomitant increase in histamine levels, which may have resulted from stimulation and degranulation of mast cells by substance P. Thus we hypothesize that the release of substance P may be the earliest pathophysiological event leading to stimulation of the inflammatory cytokines, which may then stimulate the free radical mechanisms of injury previously confirmed by our work.