Background The fluorochrome‐labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308–313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. Methods Apoptosis of human MCF‐7, U‐937, or HL‐60 cells was induced by camptothecin (CPT) or tumor necrosis factor‐α (TNF‐α) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA‐specific fluorochromes was measured by LSC. Results Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF‐7 cells treated with CPT. FLICA binding was largely (>90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. Conclusions The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation. Cytometry 44:73–82, 2001. © 2001 Wiley‐Liss, Inc.
/ ajplung.00312.2003.-We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-dependent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone. nitric oxide synthase; angiotensin type 2 receptor; pulmonary endothelium THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays a major role in the control of cardiovascular, renal, and adrenal functions (15). The main effector peptide molecule of the RAS, angiotensin II (ANG II), and its metabolites elicit cellular responses through at least three receptor subtypes (15,33,45): type 1 (AT 1 ), type 2 (AT 2 ), and type 4 (AT 4 ). Signaling pathways mediated via the AT 1 receptor include stimulation of phospholipases, protein kinases, and gene transcription; calcium mobilization; and inhibition of adenylate cyclase (15). Although the AT 2 receptor has been linked to inhibition of cell growth, neuronal differentiation, apoptosis, and regulation of blood pressure, there are conflicting data regarding the specific signaling pathways linked to this receptor (15, 45). For example, it has been reported that activation of the AT 2 receptor can lead to both an increase (5) and decrease (27) in protein phosphatase activity. Moreover, depending on experimental conditions, the p42/p44 MAPK pathway can be either activated (19) or inhibited (24) via the AT 2 receptor. Whereas cloning of the AT 1 and AT 2 receptors reveal...
© 2 0 0 2 L a n d e s B i o s c i e n c e . N o t f o r d i s t r i b u t i o n . ABSTRACTAnalogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect-while TLCK had no effect-on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISPreactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase-vs FLISP-reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.
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