Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK1) receptor expressed in mammalian cells

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR.

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Experimental Hypomagnesemia Induces Neurogenic Inflammation and Cardiac Dysfunction

Hearts ◽

10.3390/hearts1020011 ◽

2020 ◽

Vol 1 (2) ◽

pp. 99-116

Author(s):

Jay H. Kramer ◽

I. Tong Mak ◽

Joanna J. Chmielinska ◽

Christopher F. Spurney ◽

Terry M. Phillips ◽

...

Keyword(s):

Substance P ◽

Cardiac Dysfunction ◽

Neurogenic Inflammation ◽

Nitrosative Stress ◽

Disease Status ◽

Neutral Endopeptidase ◽

Receptor Blockade ◽

Mg Deficiency ◽

Substance P Receptor ◽

Experimental Findings

Hypomagnesemia occurs clinically as a result of restricted dietary intake, Mg-wasting drug therapies, chronic disease status and may be a risk factor in patients with cardiovascular disorders. Dietary restriction of magnesium (Mg deficiency) in animal models produced a pro-inflammatory/pro-oxidant condition, involving hematopoietic, neuronal, cardiovascular, renal and other systems. In Mg-deficient rodents, early elevations in circulating levels of the neuropeptide, substance P (SP) may trigger subsequent deleterious inflammatory/oxidative/nitrosative stress events. Evidence also suggests that activity of neutral endopeptidase (NEP, neprilysin), the major SP-degrading enzyme, may be impaired during later stages of Mg deficiency, and this may sustain the neurogenic inflammatory response. In this article, experimental findings using substance P receptor blockade, NEP inhibition, and N-methyl-D-aspartate (NMDA) receptor blockade demonstrated the connection between hypomagnesemia, neurogenic inflammation, oxidative stress and enhanced cardiac dysfunction. Proof of concept concerning neurogenic inflammation is provided using an isolated perfused rat heart model exposed to acute reductions in perfusate magnesium concentrations.

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Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells

Journal of Neuroscience ◽

10.1523/jneurosci.14-02-00834.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 834-845 ◽

Cited By ~ 177

Author(s):

SR Vigna ◽

JJ Bowden ◽

DM McDonald ◽

J Fisher ◽

A Okamoto ◽

...

Keyword(s):

Substance P ◽

Mammalian Cells ◽

Substance P Receptor

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Comparison of the spatial distribution of endopeptidase-24.11 with substance P, substance P receptor (NK-1r) and gastric efferent neurons in the dorsal vagal complex of the rat

Neuroscience Letters ◽

10.1016/s0304-3940(97)13337-7 ◽

1997 ◽

Vol 222 (1) ◽

pp. 41-44 ◽

Cited By ~ 3

Author(s):

Lance Ladic ◽

Alison Buchan

Keyword(s):

Spatial Distribution ◽

Substance P ◽

Dorsal Vagal Complex ◽

Endopeptidase 24.11 ◽

Efferent Neurons ◽

Substance P Receptor

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CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide- induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation

Blood ◽

10.1182/blood.v78.7.1834.1834 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1834-1841 ◽

Cited By ~ 103

Author(s):

MA Shipp ◽

GB Stefano ◽

SN Switzer ◽

JD Griffin ◽

EL Reinherz

Keyword(s):

Substance P ◽

Cell Surface ◽

Enzymatic Activity ◽

Inflammatory Responses ◽

Lymphoblastic Leukemia ◽

Neutral Endopeptidase ◽

Biologically Active ◽

Zinc Metalloprotease ◽

Endopeptidase 24.11 ◽

Induced Changes

Abstract The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu- phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

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CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide- induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation

Blood ◽

10.1182/blood.v78.7.1834.bloodjournal7871834 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1834-1841 ◽

Cited By ~ 4

Author(s):

MA Shipp ◽

GB Stefano ◽

SN Switzer ◽

JD Griffin ◽

EL Reinherz

Keyword(s):

Substance P ◽

Cell Surface ◽

Enzymatic Activity ◽

Inflammatory Responses ◽

Lymphoblastic Leukemia ◽

Neutral Endopeptidase ◽

Biologically Active ◽

Zinc Metalloprotease ◽

Endopeptidase 24.11 ◽

Induced Changes

The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu- phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

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The Type 2 CD10/Neutral Endopeptidase 24.11 Promoter: Functional Characterization and Tissue-Specific Regulation by CBF/NF-Y Isoforms

Blood ◽

10.1182/blood.v89.11.4136 ◽

1997 ◽

Vol 89 (11) ◽

pp. 4136-4145 ◽

Cited By ~ 28

Author(s):

Fumihiko Ishimaru ◽

Bernard Mari ◽

Margaret A. Shipp

Keyword(s):

Epithelial Cells ◽

Cell Lines ◽

Functional Characterization ◽

Neutral Endopeptidase ◽

Major Type ◽

Specific Expression ◽

Tissue Specific ◽

Endopeptidase 24.11 ◽

Region I

Abstract The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 ([NEP] neprilysin) functions as part of a regulatory loop to control local concentrations of peptide substrates and associated peptide-mediated signal transduction. The physiologic role of the enzyme depends on available substrates in specific organs and cell types. Although CD10/NEP is expressed on a restricted subset of normal and malignant lymphoid progenitors, the enzyme is also expressed by a variety of epithelial cells. To explore the mechanism of tissue-specific expression of this regulatory enzyme, we characterized the major (type 2) CD10/NEP promoter and identified three functionally active transcription factor binding sites (regions I to III). CBF/NF-Y binds to the inverted CCAAT box in region I, whereas a second positive and a third negative factor bind to regions II and III, respectively. Although region I is required for maximal CD10/NEP-driven luciferase activity in the examined epithelial cell lines, this region is not required for maximal activity in the evaluated lymphoid cell lines. The apparent tissue-specific differences in requirements for region I (and CBF/NF-Y) are of particular interest because lymphoid and epithelial cells express alternatively spliced versions of CBF/NF-Y that differ in biologic activity.

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