Studies by the applicant's research group indicate several mechanisms by which ethanol and cocaine could induce cerebral venular inflammatory states and strokes. The working hypothesis for this grant application is that both ethanol and cocaine induce cerebrovasospasm, leukocyte rolling-adhesion (probably mediated by P-selectins) and stroke via an initial reduction in cerebral vascular smooth muscle (CVSM) cell [Mg2+]i, triggering entry and release of [Ca2+]i followed by activation of the sphingomyelinase (SMase)-sphingolipid pathway and oxidation of membrane fatty acid double-bonds, leading to formation of PAF-like phospholipids and further entry of [Ca2+]i, which in turn activates certain PKC isoforms (alpha, epsilon) and NF-kappaB. The result is a sustained, localized inflammatory response characterized by leukocyte rolling-adhesion on venules, intense microvascular ischemia or venular rupture leading to focal hemorrhages. Thus, aim 1 of the proposal seeks to determine the brain ceramides synthesized and released in situ by these drugs and to determine their relationship to changes in brain [Mg2+]i. These experiments will exploit 31P-NMR and 1H-NMR spectroscopy and state-of-the-art methods used in lipid biochemistry. Finally, it will be important to determine whether ethanol/cocaine causes increased expression of P-selectin on the endothelial surface of brain postcapillary venules and whether this effect can be ameliorated by feeding animals high MG in their diets.
Aim 2 tests the hypothesis that both alcohol and cocaine activate certain PKC isoforms (alpha and epsilon) and the nuclear transcription factor NF-kappaB in primary cultured CVSM cells, leading to [Ca2+]i overload and membrane lipid oxidation (after an early decrease in [Mg2+]i) and some membrane entry of Ca2+.
Aim 3 will test the hypothesis that biologically active ceramides (proposed mediators of ethanol/cocaine induced stroke) will increase concentrations of [Ca2+]i and activate the same PKC isoforms and NF-kappaB in CVSM cells. If successful, the experiments will provide direct support for a major role of ceramides in stroke pathogenesis.
Aim 4 proposes to clarity the membrane changes associated with reductions in dietary intake of Mg, specifically the alterations of membrane lipids (and their lipid oxidation products) of brain CVSM cells. Progressive drops in brain [Mg2+]i after dietary deficiency of Mg, might be expected to exacerbate the membrane changes in phospholipid ratios, fatty acid components and the triggered transformation from an unsaturated to a saturated state, leading to additional membrane peroxidation. The information generated by this proposal will fill-in many of the still-elusive intermediate steps between membrane events of ethanol/cocaine-induced reduction in [Mg2+]i (and a reciprocal elevation in [Ca2+]) and activation of the cellular biochemical cascade leading to cerebrovasospasm, leukocyte-endothelial rolling-adhesion, microvessel rupture and hemorrhage. An understanding of how ethanol and cocaine induce strokes may ultimately suggest new targets for pharmacologic manipulation, and thus, improving the clinical outcome after """"""""binge- drinking"""""""" or cocaine intoxication.
Showing the most recent 10 out of 114 publications
