These data provide evidence that FP receptor activation leads to an increase in the secretion and activation of MMP-2 through PKC- and ERK1/2-dependent pathways. FP-agonist-induced activation of ERK1/2 was blocked by PKC inhibitors, indicating that PKC activation is required for ERK1/2 activation and MMP-2 secretion from HCM cells. In the ciliary muscle, the functional responses to ERK1/2 activation include secretion of MMP-2, supporting the hypothesis that increases in uveoscleral outflow facility induced by PG administration involves the secretion and activation of MMP-2.
Objective-The biosynthesis of endothelin-1 (ET-1), the most potent vasoconstrictor with mitogenic properties, involves the processing of intermediate protein big ET-1 by a unique metalloprotease, endothelin-converting enzyme-1 (ECE-1). ECE-1 has 4 subisoforms that possess the same catalytic properties but different localization patterns on the plasma membrane and cytosol. We investigated the trafficking of ECE-1 subisoforms using green fluorescent protein-tagged recombinant enzymes in target and nontarget cells. Methods and Results-ECE-1 localization was studied using confocal microscopy, which provides evidence for the first time that both ET-1 and ECE-1a are also found in the nuclear compartment in transiently transfected cells as well as in native endothelial cells that endogenously possess the ET system. In cells maintained in high-glucose medium, ECE-1a-specific staining shifted from plasma membrane to intracellular compartments. ECE-1b subisoform, however, is mainly in the cytosolic compartment, indicating a subisoform specificity for nuclear localization. Conclusions-Our findings define a novel localization pattern for the ET system, which may be differentially regulated under pathophysiological conditions. Key Words: endothelin Ⅲ subcellular localization Ⅲ high glucose T he family of endothelins (ETs) consists of 3 related vasoactive peptides, ET-1, ET-2, and ET-3, which play an important role in cardiovascular pathophysiology and embryonic development. [1][2][3] Because ETs mediate a wide spectrum of physiological functions via autocrine and paracrine mechanisms, ET biosynthesis is tightly regulated and involves a 2-step proteolytic maturation process. 4 -6 Preproendothelins (PPETs) are first processed at conserved multibasic sites by furin or a furin-like enzyme to generate big ET, which is additionally processed by endothelinconverting enzyme (ECE) to release the biologically active peptide. 4 During the release of ET-1, unprocessed big ET-1 is also secreted and cleaved to ET-1 in circulation, indicating the presence of the processing enzyme in the extracellular space.ECE is a type II membrane protein of the neutral endopeptidase family with a single transmembrane domain, a short N-terminal cytosolic tail, and a large extracellular domain that contains the catalytic site. 7,8 There are 3 isoforms, and ECE-1 has a broader tissue distribution and higher expression levels than ECE-2. ECE-3, isolated from bovine iris, has specificity for big The targeted disruption of the ECE-1 gene results in a lethal phenotype, whereas ECE-2 knockout model does not exhibit significant modification of the mouse phenotype. 3,10 ECE-1 is expressed in the endothelium of all organs as well as in nonvascular cells of many tissues, including brain and neuroendocrine tissues. 11 There are 4 subisoforms of ECE-1, ECE-1a through d, that are generated by alternative splicing. 12-14 These subisoforms share the same catalytic and transmembrane domains and only differ in the cytosolic N terminus, with no difference in their cat...
Purified anionic and cationic isoforms of horseradish peroxidase (HRP) immobilized by coupling the amino acid side‐chain amino groups and/or carbohydrate moieties to Sepharose have been studied for their resistance to denaturation. The isoforms were treated with periodate followed by ethylenediamine to generate additional amino groups in the glycosyl residues. The immobilized preparations were: Preparation I (Sp‐aHRP, Sp‐cHRP), in which HRP was covalently immobilized via side‐chain amino groups exclusively; Preparation II (Sp‐NHaHRP, Sp‐NHcHRP), in which periodate and ethylenediamine‐treated HRP was covalently immobilized via side‐chain amino groups and amino groups incorporated into glycosyl residues. HRP isoforms in preparation II lacked about 33‐55 % carbohydrate. Both strategies of immobilization induced significant stabilization against denaturation. Inclusion of 2 mM calcium enhanced isoenzyme stability significantly.
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