(Hypertension. 1995;26:230-235.)
© 1995 American Heart Association, Inc.
Articles |
Regulation and Differential Expression of Neutral Endopeptidase 24.11 in Human Endothelial Cells
Presented in part at the scientific sessions of the American Heart Association, 1992 and 1993, and published in abstract form (Circulation. 1992;86[suppl I]:I-275; Circulation. 1993;88[suppl I]:I-315).
From the Department of Internal Medicine/Cardiology and Angiology, Free University and German Heart Institute Berlin (K.G., M.G., W.A.-S., E.F.), and the Department of Clinical Immunology, Free University Berlin, Universitätsklinikum Rudolf Virchow (P.K., M.Z.) (Germany).
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Abstract |
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Abstract Neutral endopeptidase 24.11, a membrane-bound metallopeptidase, cleaves and degrades vasoactive peptides such as atrial natriuretic peptide, endothelin, angiotensin I, substance P, and bradykinin. Therefore, the presence of this metallopeptidase may contribute to the regulation of vascular tone and local inflammatory responses in the vascular endothelium and elsewhere. We determined neutral endopeptidase in cultured human endothelial cells from different vascular beds and studied its regulation by protein kinase C. Neutral endopeptidase was detected in all cultured endothelial cell types. Lowest concentrations were measured in human endothelial cells from umbilical veins (360±14 pg/mg protein), followed by pulmonary and coronary arteries; higher concentrations were found in endothelial cells from the cardiac microcirculation (1099±73 pg/mg protein). Neutral endopeptidase content increased during cell growth but was not affected by endothelial cell growth factor or modifications of the growth medium. Stimulation of protein kinase C with 1-oleoyl-2-acetyl-rac-glycerol (0.1 to 1 µmol/L) and phorbol 12-myristate 13-acetate (0.01 to 0.1 µmol/L) induced a time- and concentration-dependent increase of endothelial cells that was inhibited by cycloheximide (5 µmol/L), an inhibitor of protein synthesis. Incubation with phospholipase C (1 µmol/L) and thrombin (10 IU/mL) induced upregulation of neutral endopeptidase, resulting in 158±26% and 150±22% increases, respectively, compared with controls. The thrombin effect was inhibited by calphostin C (1 µmol/L), an inhibitor of protein kinase C. Endothelial neutral endopeptidase is constitutively expressed in endothelial cells from different origins and is inducible by thrombin via activation of the protein kinase C pathway.
Key Words: endothelium • enzymes • phorbols • protein kinases • thrombin
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Introduction |
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The membrane-bound metallopeptidase NEP (EC 3.4.24.11) was initially demonstrated in the renal brush border1 ; it was further detected in various organs and cells, such as lung, kidney, brain, thyroid, intestine, leukocytes, lymphoblastic cells, epithelial cells, vascular smooth muscle cells, and the vascular endothelium.2 3 4 NEP modulates the action of various peptide hormones by degrading them to inactive metabolites or converting them to active mediators.5 6 7 NEP has a high affinity for some potent inflammatory and vasoactive peptides such as substance P, bradykinin, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, atrial natriuretic peptide, and endothelin.6 7 8 9 10 11 In rats NEP converts circulating angiotensin I to its active metabolite angiotensin,1 2 3 4 5 6 7 which is an endothelium-dependent vasodilator.5 12
The regulation of NEP expression is not well characterized at the cellular level. In leukocytes and epithelial cells a decrease of NEP activity was observed after treatment with phorbol esters.13 14 An increase in NEP activity was found during the cell growth of transfected epithelial cells and human leukemic cells and after glucocorticoid treatment.15 16 Glucocorticoids also increased NEP gene transcription and enhanced NEP activity in vascular smooth muscle cells of rabbit renal cortex.4 15 The same authors reported an increase of NEP after serum deprivation of cultured smooth muscle cells, indicating a regulatory action of serum factors.
In the present study we investigated the expression of NEP protein and activity during endothelial cell growth and in cultured endothelial cells from different vascular regions, including adult and embryonic phenotypes. Adult endothelial cells were derived from the pulmonary arteries, the microvasculature, and the epicardial coronary arteries of the human heart. Furthermore, we studied the regulation of this peptidase by PKC to elucidate how NEP is regulated in endothelial cells.
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Methods |
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Cell Culture
HUVECs, HPAECs, and macrovascular HCECs were harvested after collagenase treatment.17 18 The cells were seeded into a 35-mm dish coated with 0.2% gelatin and incubated in culture medium at 37°C and 5% CO2. Microvascular HCECs were isolated as described previously in detail.19 Briefly, capillaries were enzymatically isolated from heart muscle segments and seeded in culture medium. Separation from nonendothelial cells was performed with magnetic tosyl-activated beads (Dynal) coupled to Ulex europaeus I lectin. Purification was 98% endothelial cells determined by flow cytometry (FACS, Becton Dickinson) with a factor VIII–related antigen antibody.19 Culture conditions for all endothelial cells were identical. The culture medium contained medium 199, 20% FCS, 100 IU/mL penicillin, 100 µg/mL streptomycin, 10 mmol/L HEPES, and 0.2 mol/L L-glutamine. ECGF (0.8 µmol/L) was added to the culture medium except in the experiments in which the effect of ECGF on NEP expression was investigated (see below). Macrovascular and microvascular HCECs were cultivated in culture medium containing 20% pooled human serum. All experiments were performed with cells of the second passage.
Measurement of NEP in Endothelial
Cells
All experiments were performed with confluent monolayers of
endothelial cells grown in 35-mm dishes in 1 mL culture
medium. Confluent monolayers were washed twice with phosphate-buffered
saline, and incubation medium and drugs then were added. NEP expression
at different time intervals (1 to 48 hours) in response to various
drugs was studied. The medium was routinely replaced every 24 hours.
During observation periods longer than 24 hours, stimulating drugs were
not continued in the exchanged medium. Afterwards, supernatants were
aspirated, and the cells were washed twice, scraped off the dish with a
rubber policeman, and suspended in 1.0 mL Tris buffer (0.1 mol/L, pH
7.4) containing 0.1% Tween 20. The cells were sonicated for 5 seconds
(20% output energy) by a Sonifier B-12 (Branson Sonic Power Co) on
ice. Samples were immediately frozen and stored at -35°C until
further use.
Effect of Culture Medium on Endothelial NEP
Expression
HUVECs were cultured under standard conditions until cells were
subconfluent. The incubation medium was changed to 1% or 20% FCS
containing RPMI medium (instead of medium 199) or standard culture
medium containing 20% FCS and medium 199. In one set of experiments
subconfluent cells were cultured in medium without ECGF as opposed to
standard culture medium containing ECGF (0.8 µmol/L) for 3 days. The
medium was replaced every 24 hours. After treatment for 3 days in each
set, cells were harvested as described above and NEP was
quantified.
ELISA for Measurement of NEP
NEP protein was measured by a sandwich ELISA with the use of
three monoclonal antibodies against human NEP, as described
previously.20 Recombinant NEP was used as standard
reference at concentrations between 40 and 2500 pg/mL. Microtiter
plates were coated with the rat anti-NEP monoclonal antibody AL2 for 12
hours; standard dilutions, buffer, or sample was applied in duplicate
and incubated for 1 hour. A mixture of two mouse monoclonal antibodies
both reacting with human NEP (ALB-1 and MEK 5) was used for the next
1.5-hour incubation phase. After final incubation with alkaline
phosphatase–conjugated goat anti-mouse IgG, the substrate
(p-nitrophenylphosphate) was added to the plates, and
the optical density was determined at 405 nm absorbance (Multiskan
MCC/340 MK, Flow Laboratories SA). Intra-assay and interassay
coefficients of variation were 3% and 12%, respectively. Protein
content in each sample was quantified following the method described by
Lowry et al.21
Assay for NEP Activity
The assay for NEP activity has been described in detail by
Florentin et al.22 Confluent monolayers of HUVECs were
washed with ice-cold 50 mmol Tris-HCl buffer, scraped off the dish with
a rubber policeman, and centrifuged at 400g for 4
minutes. The cell pellet of one dish was lysed with 1 mL ice-cold
distilled water and immediately frozen for later use. Samples (50 µL)
were incubated with 100 µL (50 µmol/L) DAGPNG in the presence of
phosphoramidon (0.1 µmol/L) or diluent (total volume,
190 µL) at pH 7.4 and 37°C. The increase of fluorogenic activity
was measured at 562 nm with a fluorescence spectrometer
(Pharmacia) over 6 hours. Enzymatic activity was defined as the amount
of substrate (DAGPNG) cleaved at 37°C per minute (nanomoles per
liter per minute per milligram protein=milliunit [international unit]
per milligram protein) and was calculated from the maximal slope of the
degradation curve. The activity inhibited by
phosphoramidon was assigned to NEP activity.
Materials
Tissue culture plastic ware was obtained from Becton Dickinson.
Medium 199, RPMI, phosphate-buffered saline, FCS, trypsin-EDTA
solution, HEPES, and antibiotics were from GIBCO.
Collagenase was purchased from Roth. ECGF was from
Boehringer. Gelatin, phospholipase C (from Clostridium
perfringens), OAG, PMA, thrombin, cycloheximide,
p-nitrophenylphosphate, bovine serum albumin,
dimethylglutaryl acid, Ulex europaeus agglutinin I lectin,
DAGPNG, phosphoramidon, and staurosporine
were purchased from Sigma Chemical Co. Calphostin C and H-7
[1-(5-isoquinolinesulfonyl)-2-methylpiperazine] were purchased from
Calbiochem. Tris, Tween 20, and dimethyl sulfoxide were obtained from
Serva AG. Fluorescence-conjugated low-density lipoprotein was
from Paesel & Lorei. The antibody against factor VIII–related antigen
was from Behring. Recombinant NEP and mouse anti-NEP monoclonal
antibody MEK 5 were a generous gift from Khepri Pharmaceuticals. The
rat anti-NEP monoclonal antibody AL2 was provided by Dunn. The mouse
anti-NEP monoclonal antibody ALB-1 and alkaline phosphatase–conjugated
goat anti-mouse IgG were purchased from Dianova. Calphostin C,
cycloheximide, and PMA were solubilized in dimethyl sulfoxide (stock
solution, 10 or 1 mmol/L) and diluted in medium 199. Phospholipase C
was solubilized in 10 mmol/L dimethylglutaryl acid, 0.1% bovine serum
albumin, and distilled water at pH 7.3.
Calculations and Statistics
Each experiment was done with triplicate dishes. Results are
shown as mean±SEM (n=number of experiments). Cell numbers were
separately counted in each experiment in one
representative dish. Statistical significance was
analyzed by one-way ANOVA and the Mann-Whitney U
test for unpaired samples as appropriate. Probability values less than
.05 were accepted as statistically significant.
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Results |
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NEP Expression in Cultured Endothelial Cells Derived From Different Blood Vessels
The results of NEP measurement are summarized in Fig 1. Lowest NEP concentrations were found in HUVECs. Significantly higher values for NEP were found in HCECs and HPAECs. Microvascular HCECs contained threefold higher concentrations of NEP than HUVECs (P<.01, Fig 1).
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Effect of Growth and Culture Conditions on
Endothelial NEP Expression
Fig 2 summarizes the results of NEP protein and NEP
activity measurements in growing HUVECs from days 3 to 6 after the
second passage. At day 3 cells were in a subconfluent state, reaching
confluence between days 4 and 5. Enzymatic activity, measured by the
DAGPNG assay,22 and NEP protein showed a parallel increase
between days 3 and 5 after seeding. At day 6 no further increase
in NEP protein and activity was observed (Fig 2).
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The growth of subconfluent HUVECs in different media (1% or 20% FCS containing RPMI medium or 20% FCS containing medium 199) for 3 days did not affect cellular NEP protein in endothelial cells (RPMI+1% FCS, 376±24 pg/mg protein; RPMI+20% FCS, 341±21 pg/mg protein; growth medium+20% FCS, 433±58 pg/mg protein [n=6]). These treatments did not affect the protein content per dish. ECGF did not affect the NEP content of the cells. Endothelial cells cultured with 10 ng/mL ECGF contained 378±49 pg/mg protein, and those grown in ECGF-free medium (for 3 days) contained 433±29 pg/mg protein (n=9). In contrast, the total protein content per dish was significantly reduced from 624±45 µg protein per dish in ECGF-treated endothelial cells to 397±28 µg protein per dish (P<.01, n=9) in cells cultured without ECGF supplement for 3 days.
Regulation of NEP Expression by PKC Activation
Incubation of confluent HUVEC monolayers with the PKC
activator PMA resulted in a time-dependent increase of
cellular NEP concentrations, beginning after 4 hours and reaching a
maximum at 48 hours (Fig 3). Maximal stimulation of NEP
expression was found after 0.1 µmol/L PMA for 48 hours, resulting in
a 123±13% increase. Combined incubation of
endothelial cells with PMA and cycloheximide, an
inhibitor of protein synthesis, inhibited the PMA-mediated
increase of NEP (Table). OAG, an analogue of
diacylglycerol, significantly increased NEP at concentrations from 0.1
to 1 µmol/L (Fig 4). The effect of OAG on NEP
concentrations in endothelial cells was observed after
6 hours of incubation, reached its maximum after 9 hours, and declined
thereafter. Increased NEP concentrations were still detected after 24
hours of incubation (Table). The OAG-induced increase of cellular NEP
showed a different time pattern compared with the stimulation of
endothelial NEP by PMA. The effect of both PKC
activators, PMA and OAG, was blocked in the presence of 1
µmol/L calphostin C or staurosporine (Table).
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Another activator of the PKC pathway is thrombin. Incubation with increasing concentrations of thrombin induced a concentration-dependent increase of cellular NEP concentrations after 24 hours of incubation, reaching a maximal effect at 10 IU/mL (Fig 5). When thrombin was incubated in the presence of calphostin C (1 to 10 µmol/L), a specific inhibitor of PKC, the thrombin-mediated effect was abolished (Fig 5). Incubation with 10 µmol/L calphostin C alone slightly affected cellular NEP concentrations (225±32 pg/mg protein, n=14), inducing a decrease of 38±8% compared with control, whereas 1 µmol/L calphostin C (409±43 pg/mg protein, n=10) did not change cellular NEP concentrations but also inhibited the thrombin-mediated increase of cellular NEP. In line with these observations, thrombin (10 IU) and PMA (0.1 µmol/L) also increased NEP expression in macrovascular HCECs after 24 hours of incubation (Fig 6). This effect was abolished in the presence of H-7, a PKC inhibitor. Incubation with varying concentrations of phospholipase C also led to NEP induction in endothelial cells, with a maximal effect observed after incubation with 1 µmol/L (158±26% from control, Table). OAG (0.1 µmol/L, 189±44%), thrombin (20 IU, 150±22%), and phospholipase C induced the strongest upregulation of NEP in all experiments performed in this study.
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To investigate the role of calcium in NEP regulation, we incubated HUVECs with the calcium ionophore A23187, which did not increase NEP expression (control, 422±56 pg/mg protein, n=12; 0.1 µmol/L A23187, 427±83 pg/mg protein, n=8; 0.3 µmol/L A23187, 331±66 pg/mg protein, n=8; and 1 µmol/L A23187, 301±38 pg/mg protein, n=8).
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Discussion |
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Using a sensitive ELISA for human NEP 24.1120 we could measure this metallopeptidase in homogenates of various endothelial cells derived from the human vasculature. Comparable NEP activities have been described in the particulate fraction of cultured endothelial cells from aorta and umbilical veins.23 In the present study the lowest concentration of NEP was found in HUVECs. In HCECs and HPAECs, NEP concentration was slightly higher. The highest NEP concentration was observed in microvascular HCECs, a cell type characterized previously in our laboratory.19 All cultures were grown under identical conditions. They were of high purity and showed a typical common pattern of endothelial cells. Differences between macrovascular and microvascular cells have been described regarding some antigenic properties.19 24 25 Microvascular HCECs contain a twofold to threefold higher NEP concentration than endothelial cells from embryonic and venous origins and 40% more NEP than macrovascular HCECs.
The growth experiments demonstrated that the quantities of NEP protein and NEP activity increase proportionally during cell growth. When endothelial cells reached confluence, the highest amounts of NEP protein and activity were detected. The parallel increase of NEP activity confirmed that measurement by the ELISA method recognizes a functionally active peptidase. The dependence on growth suggests a constitutive expression of NEP in endothelial cells. This could also be demonstrated during decelerated growth in the absence of ECGF, when reduced NEP concentration paralleled reduced protein content of the cultures. Indeed, the reduction in the FCS content of the growth medium did not affect the cellular NEP content. Dussaule et al4 reported that low serum concentrations in the culture medium increased NEP expression in rabbit smooth muscle cells. This could not be confirmed in subconfluent HUVECs.
Nevertheless, NEP expression in endothelial cells can be regulated by PKC stimulation. High concentrations of thrombin increased NEP expression in a concentration-dependent manner. PKC inhibition by calphostin C abolished the thrombin effect. OAG and PMA, two direct activators of intracellular PKC, also augmented cellular NEP expression, which was completely blocked in the presence of calphostin C, staurosporine, and H-7.
Stimulation of NEP expression could also be achieved by addition of extracellular phospholipase C to confluent HUVEC monolayers. The mechanism by which extracellular phospholipase C penetrates into endothelial cells and activates intracellular sites is still unclear. It has been reported that extracellular addition of phospholipase C increases intracellular diacylglycerol in cultured mammalian cells26 and imitates the effect of other PKC activators, such as phorbol esters, in cultured human endothelial cells.27 A similar observation was made in the present study. Activation of the intracellular phospholipase C pathway leads to the generation of diacylglycerols and to an inositol 1,3,5-triphosphate–mediated increase of intracellular calcium. The calcium ionophore A23187 also augments intracellular calcium concentrations. In the present study A23187 did not affect NEP expression, which suggests that the upregulation of NEP is not mediated by increased calcium influx or intracellular calcium mobilization.
Interestingly, the PKC activators showed a different time pattern of PKC stimulation. OAG, an analogue of diacylglycerol, increased cellular NEP concentrations after 9 to 12 hours, whereas the induction by PMA was slower and reached its maximal effects after 24 to 48 hours. This difference has been reported by other authors.28 29 The action of the physiological activators of PKC, diacylglycerol and its analogues, is transient and restricted because of enzymatic degradation by diacylglycerol kinase or diacylglycerol lipase.30 The increase of NEP induced by a phorbol ester and OAG was abolished by an inhibitor of protein synthesis, indicating an induction of de novo synthesis by PKC activation. Previous studies in human granulocytes and epithelial cells showed an inhibition of NEP activity by phorbol esters.13 14 Thus the regulation of NEP appears to be a cell type–specific event.
The physiological relevance of endothelial NEP remains speculative. NEP is a very active enzyme with high substrate specificity, especially to bradykinin, substance P, and angiotensin I.2 5 6 It is questionable whether upregulation of NEP affects local peptide concentrations and actions. However, the local capacity of this peptidase depends on its own quantity as well as on local substrate concentrations, which are difficult to determine. Nevertheless, NEP is constitutively expressed in all human endothelial cell types investigated, which suggests a systemic as well as a local regulatory role, eg, in the coronary circulation.1 2 3 We have reported that endothelial NEP is mainly located on the membranes of human endothelial cells and contributes to the degradation of extracellular bradykinin, especially when angiotensin-converting enzyme is inhibited.7 Since NEP is a potent inactivator of vasoactive and inflammatory peptides such as the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, bradykinin, atrial natriuretic peptide, angiotensin I, endothelin, and tachykinins,6 7 8 10 its expression and upregulation might influence local vasomotor and inflammatory responses in the macrovasculature as well in the microvasculature. Local stimulation of NEP expression by thrombin, which is generated in high concentrations during acute vascular ischemic events, could theoretically limit the local effects of proinflammatory and vasoactive peptides by induction of NEP.
The present study suggests that the distribution of this peptidase varies in different vascular regions and is higher in the microvasculature than in the macrovasculature. Furthermore, the present data indicate that besides a constitutive expression of NEP, which is maximal in confluent endothelial cells, induction of this peptidase is possible via phospholipase C and PKC activation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Parts of this work will be included in the MD thesis of Petra Koehne at the Free University of Berlin. The authors wish to thank Karen Vetter and Arthur O'Connor for excellent technical assistance.
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Footnotes |
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Reprint requests to Dr Kristof Graf, German Heart Institute Berlin, Department of Internal Medicine/Cardiology and Angiology, Augustenburger Platz 1, 13353 Berlin, FRG.
Received November 16, 1994; first decision January 3, 1995; accepted April 19, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Kerr MA, Kenny AJ. The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. Biochem J. 1974;134:477-488.
- Erdös EG, Skidgel RA. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J. 1989;3:145-151. [Abstract]
- Johnson AR, Ashton J, Schulz WW, Erdös EG. Neutral metallopeptidase in human lung tissue and cultured cells. Am Rev Respir Dis. 1985;132:564-568. [Medline] [Order article via Infotrieve]
- Dussaule JC, Stefanske A, Béa ML, Ronco P, Ardaillou R. Characterisation of neutral endopeptidase in vascular smooth muscle cells of rabbit renal cortex. Am J Physiol. 1993;264:45-52.
-
Yamamoto K, Chappell MC, Brosnihan KB, Ferrario CM.
In vivo metabolism of angiotensin I by
neutral endopeptidase (E.C. 3.4.24.11) in spontaneously
hypertensive rats. Hypertension. 1992;19:692-696.
[Abstract/Free Full Text] - Skidgel RA, Engelbrecht S, Johnson AR, Erdös EG. Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase. Peptides. 1984;5:769-776. [Medline] [Order article via Infotrieve]
- Graf K, Gräfe M, Bossaller C, Niehus J, Schulz KD, Auch-Schwelk W, Fleck E. Degradation of bradykinin by neutral endopeptidase 24.11 in cultured human endothelial cells. Eur J Clin Chem Clin Biochem. 1993;31:267-272. [Medline] [Order article via Infotrieve]
-
Abassi ZA, Tate JE, Golomb E, Keiser HR. Role
of neutral endopeptidase in the metabolism
of endothelin. Hypertension. 1992;20:89-95.
[Abstract/Free Full Text] -
Painter R, Dukes R, Sullivan J, Carter R, Erdös
E, Johnson A. Function of neutral endopeptidase
on the cell membrane of human neutrophils. J
Biol Chem. 1988;263:9456-9461.
[Abstract/Free Full Text] - Stephenson SL, Kenny AJ. The hydrolysis of a human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase 24.11. Biochem J. 1987;243:183-187. [Medline] [Order article via Infotrieve]
-
Vijayaraghavan J, Scicli AG, Carretero OA, Slaughter C,
Moomaw C, Hersh LB. The hydrolysis of endothelins by neutral
endopeptidase 24.11 (enkephalinase).
J Biol Chem. 1990;265:14150-14155.
[Abstract/Free Full Text] - Pörsti I, Bara AT, Busse R, Hecker M. Release of nitric oxide by angiotensin (1-7) from porcine coronary endothelium: implications for a novel angiotensin receptor. Br J Pharmacol. 1994;111:652-654. [Medline] [Order article via Infotrieve]
- Erdös E, Wagner B, Harbury C, Painter R, Skidgel R, Fa X. Downregulation and inactivation of neutral endopeptidase 24.11 (enkephalinase) in human neutrophils. J Biol Chem. 1989;264:1419-1423.
-
Werb Z, Clark EJ. Phorbol diesters regulate
expression of the membrane neutral endopeptidase (EC
3.4.24.11) in rabbit synovial fibroblasts and mammary epithelial
cells. J Biol Chem. 1988;264:9111-9113.
[Abstract/Free Full Text] - Borson D, Gruenert D. Glucocorticoids induce neutral endopeptidase in transformed human tracheal epithelial cells. Am J Physiol. 1991;280:L83-L89.
- Milhiet PE, Beaumont A, Garbay-Jaureguiberry C, Roques BP. Increase of neutral endopeptidase 24.11 with cellular density and enzyme modulation with an inhibitor on human REH6 cell line. Biochem Pharmacol. 1992;43:1711-1715. [Medline] [Order article via Infotrieve]
-
Gräfe M, Bossaller C, Graf K, Auch-Schwelk W,
Baumgarten C, Hildebrandt A, Fleck E. Effect of
angiotensin converting enzyme inhibition on bradykinin
metabolism by human vascular endothelial
cells. Am J Physiol. 1993;264:H1493-H1497.
[Abstract/Free Full Text] - Graf K, Gräfe M, Auch-Schwelk W, Baumgarten CR, Scheffer H, Hildebrandt A, Fleck E. Tissue kallikrein activity and kinin release in human endothelial cells. Eur J Clin Chem Clin Biochem. 1994;32:495-500. [Medline] [Order article via Infotrieve]
-
Gräfe M, Auch-Schwelk W, Graf K, Terbeek D,
Hertel H, Unkelbach M, Hildebrandt A, Fleck E. Isolation and
characterisation of macrovascular and microvascular
endothelial cells from human hearts. Am J
Physiol. 1994;267:H2138-H2148.
[Abstract/Free Full Text] - Zhang M, Niehus J, Schnellbacher T, Müller S, Graf K, Schultz KD, Baumgarten CR, Lucas C, Kunkel G. Elisa for the neuropeptide degrading endopeptidase 3.4.24.11 in human serum and leukocytes. Peptides. 1994;15:843-848. [Medline] [Order article via Infotrieve]
-
Lowry OH, Rosebrough NH, Farr AL, Randall RJ.
Protein measurement with Folin phenol reagent.
J Biol Chem. 1951;193:265-275.
[Free Full Text] - Florentin D, Sassi A, Roques BP. A highly sensitive fluorometric assay for `enkephalinase,' a neutral metalloendopeptidase that releases tyrosine-glycine-glycine from enkephalins. Anal Biochem. 1984;141:62-69. [Medline] [Order article via Infotrieve]
-
Llorens-Cortes C, Huang H, Vicart P, Gasc J-M, Paulin
D, Corvol P. Identification and characterisation of neutral
endopeptidase in endothelial cells from
venous or arterial origins. J Biol
Chem. 1992;267:14012-14018.
[Abstract/Free Full Text] - Gordon E, Danielson P, Nguyens T, Winn R. A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells. In Vitro Cell Dev Biol. 1991;27A:312-326.
- Belloni PN, Nicolson GL. Differential expression of cell surface glycoproteins on various organ-derived microvascular endothelia and endothelial cell cultures. J Cell Physiol. 1988;136:398-410. [Medline] [Order article via Infotrieve]
- Kent C, Evers A, Haun SSL. Diacylglycerol metabolism in phospholipase C treated mammalian cells. Arch Biochem Biophys. 1986;250:519-525. [Medline] [Order article via Infotrieve]
-
Kooistra T, Bosma PJ, Toet K, Cohen LH, Griffioen M,
van den Berg E, le Clercq L, van Hindsbergh VWM. Role of protein
kinase C and cAMP in the regulation of t-TPA, PAI-1, and PDGF mRNA
levels in human endothelial cells.
Arterioscler Thromb. 1991;11:1042-1052.
[Abstract/Free Full Text] - Cantwell ME, Foreman JC. Phorbol esters induce a slow, non cytosolic release of histamine from rat peritoneal mast cells. Agents Actions. 1987;20:165-168. [Medline] [Order article via Infotrieve]
-
Kolesnick RN, Clegg S. 1,2-diacylglycerols, but
not phorbol esters, activate a potential inhibitory
pathway of protein kinase C in GH3 pituitary cells.
J Biol Chem. 1988;263:6534-6537.
[Abstract/Free Full Text] - Wilkinson SE, Hallam TJ. Protein kinase C: is its pivotal role in cellular activation over stated? Trends Pharmacol Sci. 1994;15:53-57.[Medline] [Order article via Infotrieve]
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X. Zhang, F. A. Recchia, R. Bernstein, X. Xu, A. Nasjletti, and T. H. Hintze Kinin-Mediated Coronary Nitric Oxide Production Contributes to the Therapeutic Action of Angiotensin-Converting Enzyme and Neutral Endopeptidase Inhibitors and Amlodipine in the Treatment in Heart Failure J. Pharmacol. Exp. Ther., February 1, 1999; 288(2): 742 - 751. [Abstract] [Full Text]
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D. J. Campbell, F. Anastasopoulos, A.-M. Duncan, G. M. James, A. Kladis, and T. A. Briscoe Effects of Neutral Endopeptidase Inhibition and Combined Angiotensin Converting Enzyme and Neutral Endopeptidase Inhibition on Angiotensin and Bradykinin Peptides in Rats J. Pharmacol. Exp. Ther., November 1, 1998; 287(2): 567 - 577. [Abstract] [Full Text]
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 C. J. Ferro, J. C. Spratt, W. G. Haynes, and D. J. Webb Inhibition of Neutral Endopeptidase Causes Vasoconstriction of Human Resistance Vessels In Vivo Circulation, June 16, 1998; 97(23): 2323 - 2330. [Abstract] [Full Text] [PDF]
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 L. A Brown, R. A.D Rutherford, D. J.R Nunez, John Wharton, D. G Lowe, and M. R Wilkins Downregulation of natriuretic peptide C-receptor protein in the hypertrophied ventricle of the aortovenocaval fistula rat Cardiovasc Res, December 1, 1997; 36(3): 363 - 371. [Abstract] [Full Text] [PDF]
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