Vascular A10 cell membranes contain an endothelin metabolizing neutral endopeptidase

doi:10.1016/0006-291X(91)90941-Y

Biochemical and Biophysical Research Communications

Volume 176, Issue 1, 15 April 1991, Pages 423-430

https://doi.org/10.1016/0006-291X(91)90941-Y Get rights and content

Abstract

We have investigated the possible presence of endothelin-metabolizing neutral endopeptidase (NEP, EC 3.4.24.11) on A10 cell membranes using [¹²⁵I]-ET-1 binding and direct measurements of NEP. NEP activity of A10 cell membranes has been compared to that of solubilized rat kidney brush border membranes (KNEP). Specific [¹²⁵I]-ET-1 (50 pM) binding (defined with 100 nM ET-1) to A10 cell membranes was increased in a concentration dependent manner by the selective NEP inhibitors thiorphan, phosphoramidon, and SQ 28,603 {(±)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine} with EC₅₀ values of 9.4, 28.4, and 5.7 nM respectively. At equilibrium (150 min), 70% more specific binding was apparent in the presence of these inhibitors. Phosphoramidon (2 μM) did not alter B_max values, but it decreased the apparent K_D for [¹²⁵I] ET-1 from 63 (±3) to 27 (±2) pM. Thiorphan, phosphoramidon, and SQ 28,603 inhibited A10 cell NEP activity with IC₅₀ values of 5.3, 36.5, and 6.0 nM respectively, which was similar to values obtained with KNEP (3.6, 22.6, and 3.5 nM). ET-1 inhibited A10 cell NEP, and KNEP with IC₅₀ values of 30 and 21.3 μM respectively. The order of inhibitory potencies: ET-3 > ET-1 = ET-2 ≥ sarafotoxin-6b was similar for both systems. These data suggest A10 cell membranes contain a NEP which has similar characteristics to NEP 24.11, and which actively metabolizes [¹²⁵I]-ET-1.

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Citation Excerpt :
These data were obtained with the non-selective NEP inhibitor thiorphan, which also blocks the enzyme responsible for the conversion of big ET-1 to ET-1, endothelin-converting enzyme. However, the latter requires micromolar concentrations of thiorphan (Trapani et al., 1993), as compared to the nanomolar concentrations that are sufficient to block NEP (Dickinson et al., 1991). LCZ696 (sacubitril/valsartan, brand name Entresto) is the first-in-class ARNI.
Increasing the degree of renin-angiotensin system (RAS) blockade by combining ≥2 RAS blockers marginally increases efficacy, but results in more side effects. Hence, interference with other systems is currently being investigated, like potentiation of natriuretic peptides with neprilysin inhibitors. However, the neprilysin inhibitor thiorphan was recently found to increase endothelin-1 when administered to TGR(mREN2)27 (Ren2) rats on top of RAS blockade. Here we investigated whether this effect is thiorphan-specific, by comparing the neprilysin inhibitors thiorphan and sacubitril, administered by osmotic minipumps at a low or high dose for 7 days, in Ren2 rats. Plasma and urinary levels of endothelin-1, atrial and brain natriuretic peptide (ANP, BNP) and their second messenger cyclic guanosine 3′5′ monophosphate (cGMP) were monitored. No significant differences were found in the plasma concentrations of endothelin-1, cGMP, ANP and BNP after treatment, although plasma ANP tended to be higher in the high-dose thiorphan treatment group and the low- and high-dose sacubitril treatment groups, compared with vehicle. Urinary endothelin-1 increased in the low-dose thiorphan and high-dose sacubitril groups, compared with baseline, although significance was reached for the former only. Urinary cGMP rose significantly in the high-dose sacubitril treatment group compared with baseline. Both urinary endothelin-1 and cGMP were significantly higher in the high-dose sacubitril group compared with the low-dose sacubitril group. In conclusion, endothelin-1 upregulation occurs with both thiorphan and sacubitril, and is particularly apparent in neprilysin-rich organs like the kidney. High renal neprilysin levels most likely also explain why sacubitril increased cGMP in urine only.
Production of soluble Neprilysin by endothelial cells
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The amount of fluorescence specifically due to soluble NEP was calculated by taking the difference in respective fluorescence values generated in the presence and absence of thiorphan. Despite the relative specificity of thiorphan for NEP with an IC50 in the low nanomolar range [17], some studies have shown its ability to inhibit ACE [18], an enzyme which can also cleave bradykinin [15]. Given that the sequence of our QFS substrate is based on bradykinin, all enzyme assays were conducted in the presence of ACE inhibitor captopril in order to accurately determine NEP activity.
A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC₅₀ values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.
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1998, American Journal of Hypertension
The endothelial cell regulates vessel tone by elaborating a number of vasoactive substances such as thromboxane and endothelin, both of which are highly vaosconstrictive, and prostacyclin and nitric oxide, both of which are vasodilatory. The current study examines the postulate that one of the mechanisms responsible for the increased vessel tone found in hypertension is the presence of substances in the sera of patients with this disorder that stimulates selectively the endothelial cell production of thromboxane and endothelin. Sera from ten patients with mild hypertension and from 11 age-matched controls were incubated with human umbilical arterial endothelial cells and the concentrations of endothelin, thromboxane, prostacyclin, and nitric oxide produced by the cells was measured in the supernatant. The results of the assays showed that the amounts of thromboxane and endothelin produced by endothelial cells in response to stimulation by hypertensive sera were significantly higher than the amounts produced in response to control sera; in comparison, the amounts of prostacyclin and nitric oxide produced by the cells in response to either hypertensive sera or control sera were not significantly different. The findings suggest that a mechanism that may be responsible for the increased vascular tone found in hypertension is the presence of substances in hypertensive sera that stimulate endothelial cells selectively to produce increased amounts of the vasoconstrictive hormones, endothelin and thromboxane.
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1996, American Journal of Hypertension
Sera from patients with hypertension were examined for their ability to influence endothelial cell function. Sera from 10 patients with hypertension and from 11 age-matched controls were incubated with endothelial cells and their effect on cell growth assessed by endothelial cell ³HH-thymidine uptake. Sera from patients with hypertension had a more pronounced stimulatory effect on endothelial cell proliferation than control sera. In addition, hypertensive sera were also examined for their capacity to stimulate endothelial cell production of the vasoactive peptide endothelin. Confluent monolayers of endothelial cells were incubated with sera and the amount of endothelin generated in the supernatant measured by an immunoassay. Amounts of endothelin produced by the endothelial cells in response to hypertensive sera were significantly higher compared with the amounts produced in response to normal sera. The results of the studies suggest that a serum factor(s) in hypertensive patients may be of importance in modulating endothelial cell function. Such a factor(s) may have a significant role in the pathogenesis of hypertension.
Endothelin degradation by vascular smooth muscle cells
1996, Regulatory Peptides
The mechanism(s) of degradation of the potent vasoconstrictor endothelin-1 (ET-1) by rat vascular smooth muscle A-10 cells, which possess the ET_A receptor subtype, was investigated by incubating [¹²⁵I]ET-1 (0.1 nM) with cells for 0–4 h at 37°C in the presence and absence of lysosomal enzyme inhibitors, NH₄Cl and chloroquine, and a neutral endopeptidase inhibitor, phosphoramidon. The assay buffer and cell extracts were analyzed by reverse-phase HPLC, and the radioactivity in the fractions was measured. In the absence of inhibitors, most of the radioactivity in the medium was in the form of [¹²⁵I]Tyr after a 4 h incubation. When [¹²⁵I]ET-1 was incubated with A-10 cells at 4°C, six radiolabeled peaks, including some [¹²⁵I]Tyr and about 30% of the original [¹²⁵I]ET-1, were present in the medium. In the presence of 5 μM chloroquine there was no [¹²⁵I]Tyr peak in the medium, indicating that internalization and putative lysosomal degradation of ET-1 were blocked. NH₄Cl (50 and 100 mM) also reduced the amount of [¹²⁵I]Tyr formed. The presence of ET-1 fragments indicated that, in addition to lysosomal degradation, some of the ligand is metabolized by enzymes located on the cell membrane; we demonstrated, however, that secreted proteases from A-10 cells are not involved in the degradation of ET-1. The neutral endopeptidase inhibitor, phosphoramidon, did not completely inhibit the metabolism of [¹²⁵I]ET-1 to [¹²⁵I]Tyr. These results establish that various cell-associated enzymes are capable of degrading ET-1 in A-10 cells. Moreover, analysis of the cell lysates indicated the presence of a relatively stable pool of ET-1-occupied receptors or compartmentalized ET-1, protected from cell proteases, which may contribute to the potent contractility of ET-1.
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Vascular A10 cell membranes contain an endothelin metabolizing neutral endopeptidase

Abstract

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

J. Biol. Chem

Nature

Trends Pharmacol Sci