During
stroke, cells in the
infarct core exhibit rapid failure of their permeability barriers, which releases
ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra). Plasma membrane degradation is key to penumbral spread and is mediated by
matrix metalloproteinases (
MMPs), which are released via vesicular exocytosis into the extracellular fluid in response to stress.
DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic
solution: IS) and we asked whether this action was mediated via inhibition of
MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular
proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active
MMP isoform expression increased 2-22 fold.
MMP-mediated extracellular gelatinolytic activity increased ∼20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital
dyes and >60% of plasma membranes were fragmented or blebbed).
DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active
MMPs.
DIDS similarly inhibited extracellular
MMP expression and cellular detachment induced by the pro-apoptotic agent
staurosporine or the general
proteinase agonist
4-aminophenylmercuric acetate (APMA). Conversely,
DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of
proMMP-2 to the active form, suggesting
DIDS interferes with the vesicular extrusion of
MMPs rather than directly inhibiting
proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular
ATPase also inhibited extracellular
MMP expression to a similar degree as
DIDS. In addition, in a
proteinase-independent model of vesicular exocytosis,
DIDS prevented stimulus-evoked release of
von Willebrand Factor from human umbilical vein endothelial cells. We conclude that
DIDS inhibits
MMP exocytosis and through this mechanism preserves neuronal membrane integrity during pathological stress.