Secretion of a Neuropeptide-Metabolizing Enzyme Similar to Endopeptidase 22.19 by Glioma C6-Cells

Ferro, Emer S.; Tambourgi, Denise V.; Gobersztejn, F.; Gomes, Marcelo D.; Sucupira, Maria Cláudia Araripe; Armelin, M C S; Kipnis, Thereza Liberman; Camargo, Antônio Carlos Martins de

doi:10.1006/bbrc.1993.1213

Cited by 31 publications

(39 citation statements)

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“…Whereas some neuropeptidases, such as neutral endopeptidase or angiotensin-converting enzyme, are bound to the membrane via a hydrophobic transmembrane segment and contain ectodomains with catalytic activity, EP24.15 does not contain this membrane anchoring motif. Earlier studies using C6 glioma cells suggested that EP24.15 can be released into the medium (Ferro et al, 1993), but these cells do not represent a refined model of regulated secretion. Therefore, in the present study, we investigated the release of EP24.15 into the extracellular medium in a well-established cell culture system for examining secretion: AtT20 cells.…”

Section: Discussionmentioning

confidence: 99%

“…Cell fractionation studies show the EP24.15 enzyme is present in a minor form in the particulate subcellular fractions (Acker et al, 1987;Oliveira et al, 1990), but the reported amino acid sequences derived from cDNA clones isolated from sev-eral species do not contain a classical secretory signal for a secreted protein. Previous studies indicate calcium-independ ent secretion of EP24.15 from C6 glioma cells (Ferro et al, 1993).…”

Section: Introduction Ementioning

confidence: 88%

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Secretion of Metalloendopeptidase 24.15 (EC 3.4.24.15)

Ferro

Tullai

Glucksman

et al. 1999

DNA and Cell Biology

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The metalloendopeptidase EP24.15 (EC3.4.24.1 5) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways. Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stim ulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides. 781

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Section: Discussionmentioning

confidence: 99%

Section: Introduction Ementioning

confidence: 88%

Secretion of Metalloendopeptidase 24.15 (EC 3.4.24.15)

Ferro

Tullai

Glucksman

et al. 1999

DNA and Cell Biology

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“…Interleukin-1␤ secretion is blocked at 4ЊC and highly increased at 42ЊC, two properties that are reminiscent of those observed in the case of astrocytic endopeptidase 3.4.24.16 secretion. Similarly, endopeptidase 3.4.22.19, which also lacks a signal peptide, is released from the astrocyte-derived C6 glioma cell line (Ferro et al, 1993). Therefore, endopeptidase 3.4.24.16 is not the only example of proteolytic activity undergoing a secretion process without exhibiting the structural requirements generally associated with classical secretory proteins.…”

Section: Discussionmentioning

confidence: 99%

Distinct Properties of Neuronal and Astrocytic Endopeptidase 3.4.24.16: A Study on Differentiation, Subcellular Distribution, and Secretion Processes

et al. 1996

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Endopeptidase 3.4.24.16 belongs to the zinc-containing metalloprotease family and likely participates in the physiological inactivation of neurotensin.The peptidase displays distinct features in pure primary cultured neurons and astrocytes. Neuronal maturation leads to a decrease in the proportion of endopeptidase 3.4.24.16-bearing neurons and to a concomitant increase in endopeptidase 3.4.24.16 activity and mRNA content. By contrast, there is no change with time in endopeptidase 3.4.24.16 activity or content in astrocytes. Primary cultured neurons exhibit both soluble and membrane-associated endopeptidase 3.4.24.16 activity. The latter behaves as an ectopeptidase on intact plated neurons and resists treatments with 0.2% digitonin and Na 2 CO 3 . Further evidence for an association of the enzyme with plasma membranes was provided by cryoprotection experiments and electron microscopic analysis.The membrane-associated form of endopeptidase 3.4.24.16 increased during neuronal differentiation and appears to be mainly responsible for the overall augmentation of endopeptidase 3.4.24.16 activity observed during neuronal maturation. Unlike neurons, astrocytes only contain soluble endopeptidase 3.4.24.16. Astrocytes secrete the enzyme through monensin, brefeldin A, and forskolin-independent mechanisms. This indicates that endopeptidase 3.4.24.16 is not released by classical regulated or constitutive secreting processes. However, secretion is blocked at 4°C and by 8 bromo cAMP and is enhanced at 42°C, two properties reminiscent of that of other secreted proteins lacking a classical signal peptide. By contrast, neurons appear unable to secrete endopeptidase 3.4.24.16.

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“…The effects of the EP24.15 and EP24.16 inhibitors on QFS-cleaving activity in cytosolic and solubilized membrane fractions of EA.hy926 cells are shown in Fig. 2 [6][7][8][9] , although BK [1][2][3][4][5][6][7] and BK [8][9] were sometimes also observed, particularly in EA.hy926 extracts (Fig. 3A).…”

Section: Inhibition Of Recombinant Ep2415 and Ep2416 By Specific Inmentioning

confidence: 99%

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

Norman

Reeve

Dive

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

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Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-D-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation. peptidase; bradykinin; inhibitor; endothelium THE VASODILATORY PEPTIDE bradykinin (BK) is readily degraded in the circulation by a number of metallopeptidases, including angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), aminopeptidase P, carboxypeptidases, and possibly endothelin-converting enzyme-1 (ECE-1) (12, 25). Recent evidence suggests that a portion of the hypotensive and particularly the cardioprotective effects of specific inhibitors of ACE and NEP are due to enhanced BK activity (5,17,22,23). BK is also efficiently cleaved in vitro by the closely related peptidases EP24.15 (also known as thimet oligopeptidase) and EP24.16 (also known as neurolysin) (21). We have recently shown that hypotensive responses to BK are markedly potentiated by the administration of a stable inhibitor of these enzymes N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate, termed JA2 (26). Unlike its predecessor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), JA2 is resistant to proteolytic cleavage by NEP and does not form an ACE inhibitor within the circulation. These results suggested that EP24.15 and/or EP24.16 contribute to BK metabolism in the vasculature.Although both EP24.15 and EP24.16 are primarily soluble, cytosolic enzymes, membrane-associated forms have been demonstrated in a number of cell types. For example, Crack et al. (4) have detected immunoreactive EP24.15 on the extracellular membrane of intact corticotrophic tumor cells, whereas EP24.16 has been localized to the plasma membrane in neurons, both in vivo (9, 31) and in vitro (28), as well as in human kidney cells transfected with the peptidase (29). Given that neither enzyme possesses a transmembrane domain, the exact mechanism of association with the membrane is not understood. Furthermore, the expression and subcellular distribution of EP24.15 or EP24.16 in cells of the cardiovascular system has not been explored.In the present study, we have used specific inhibitors that can distinguish EP24.15 and EP24.16 to characterize their presence in cultured vascular endothelial cells. The results show that both enzymes are present, although EP24.16 activity is higher, particularly in the membrane fraction. Furthermore, we present evidence that EP24.16 is present on the extrac...

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Secretion of a Neuropeptide-Metabolizing Enzyme Similar to Endopeptidase 22.19 by Glioma C6-Cells

Cited by 31 publications

References 0 publications

Secretion of Metalloendopeptidase 24.15 (EC 3.4.24.15)

Secretion of Metalloendopeptidase 24.15 (EC 3.4.24.15)

Distinct Properties of Neuronal and Astrocytic Endopeptidase 3.4.24.16: A Study on Differentiation, Subcellular Distribution, and Secretion Processes

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

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