In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.

Previous studies from this laboratory have demonstrated that endothelin-1 (ET) stimulates phosphatidylinositol (PI) hydrolysis, activates dihydropyridine-insensitive Ca2+ channels, and promotes prostaglandin E2 (PGE2) accumulation in cultured rat renal medullary interstitial cells (RMIC). The mechanism whereby ET augments PGE2 production was explored in the current study. ET-evoked PGE2 accumulation proceeded independent of large increments in cytosolic free Ca2+ concentration ([Ca2+]i), derived from either extracellular or intracellular sources. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid eliminated ET-evoked PGE2 production, indicating that eicosanoid production was nonetheless a Ca(2+)-requiring process. Nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA) alone did not stimulate PGE2 production, nor did PMA alter ET-stimulated PGE2 accumulation. Furthermore, downregulation of protein kinase C (PKC) by prolonged exposure of cells to PMA did not mitigate ET-mediated PGE2 production, demonstrating that PKC stimulation was not required for PGE2 production. ET stimulated PGE2 accumulation despite PI-specific phospholipase C (PI-PLC) inhibition by nanomolar concentrations of PMA, indicating that eicosanoid production was not a downstream event of PI hydrolysis. ET stimulated arachidonic acid metabolite release in parallel with a loss of label from membrane phospholipids. Phosphatidylethanolamine was the preferred substrate for ET-mediated activation of phospholipase A2 (PLA2). Immunocytochemical studies including immunostaining, immunoblotting, and immunoprecipitation confirmed the presence of cytosolic PLA2 (cPLA2) in RMIC. In summary, ET stimulation of PGE2 production in RMIC is mediated via agonist activation of cPLA2 independent of activation of PI-PLC, suggesting direct coupling to the ET receptor. Constitutive levels of [Ca2+]i rather than abrupt increments in [Ca2+]i are sufficient for activation of this receptor-effector system, with no obligatory requirement for PKC.

Radiolabeled substrate degradation assays and gelatin zymography are routinely employed to assay 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) in biological fluids. Enzyme-linked immunosorbent assays (ELISA) have recently been developed for the quantitation of these matrix metalloproteinases (MMP). In this study, we have compared ELISA to standard substrate degradation assays for measurement of MMP-2 and MMP-9 in human plasma and tumor-conditioned media. Gelatin Sepharose chromatography and gel filtration chromatography were employed as partial purification procedures for MMP-2 and MMP-9. The ELISA data for MMP-2 and MMP-9 are linear on a log:log regression curve over a wide range of MMP concentrations and are specific for the designated gelatinase, with no overlap detected with related metalloproteinases. The minimum detectable concentrations of MMP-2 and MMP-9 were approximately 0.5 ng/ml and 0.2 ng/ml, respectively, in the ELISA as compared to 4 ng/ml and 3 ng/ml, respectively, in gelatin zymography. The [3H]gelatin degradation assay required a combination of > 50 ng/ml of MMP-2 and MMP-9 for detection. Although gelatin zymography was less sensitive than ELISA (primarily due to the smaller sample volume employed) and was more difficult to quantitate, this procedure offers the important advantage of being able to distinguish between latent and activated gelatinases.

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