substance P (SP) degradation, SP binding and SP-induced Ca2l mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP.
Biochem. J. (1994) 299, 683-693
(Printed in Great Britain)
Biochem. J. (1994) 299, 683-693 (Printed
683
in Great Britain)
Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK,) receptor expressed in mammalian cells Annette OKAMOTO,* Michelle LOVETT,* Donald G. PAYANt and Nigel W. BUNNETT*t§ Departments of *Surgery, tMedicine and
lPhysiology,
University of California, San Francisco, 521 Parnassus Avenue, San Francisco, CA 94143-0660, U.S.A.
Interactions between neutral endopeptidase-24. 1 1 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2l 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 °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 +0.50% 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.
INTRODUCTION
gastrointestinal tract (Matsas et al., 1986; Pollard et al., 1989; Kummer and Fisher, 1991; Bunnett et al., 1993). In these tissues NEP inhibitors suppress the degradation of SP and thereby increase SP binding to its receptors and magnify its biological effects. However, SP may interact with NK1, NK2 or NK3 receptors, and it is not known whether NEP regulates the interaction of SP with each tachykinin receptor subtype. Consequently, our first aim was to examine SP degradation, SP binding and SP-induced Ca2+ mobilization in cells coexpressing NEP and the SPR (NK1 receptor). In addition, it is not known whether NEP and the SPR must be coexpressed in the same cell for NEP to modulate the interaction between SP and the SPR, or whether NEP may modulate this interaction even if it is expressed in a different cell from the receptor. Therefore our second aim was to compare SP binding and SP-induced Ca2+ mobilization in cells
Neutral endopeptidase-24. 11 (NEP; EC 3.4.24.11), also known as enkephalinase, common acute lymphoblastic leukaemia antigen or CD 10, is an ectoenzyme that degrades biologically active
peptides in the extracellular fluid. The amino acid sequence of NEP includes a single sequence of 20 hydrophobic residues near the N-terminus that anchors the enzyme to the plasma membrane (Devault et al., 1987; Malfroy et al., 1987). The bulk of the protein, including the active site, projects into the extracellular fluid where it degrades peptides at the cell-surface by hydrolysing bonds on the amino side of hydrophobic residues (Matsas et al., 1984). Although NEP degrades many biologically active peptides, substance P (SP) is one of the most kinetically favourable substrates (Km 32 1uM, kcat 5062/min) (Matsas et al., 1983, 1984). NEP degrades SP by hydrolysis of the Gln6-Phe7, Phe7-Phe8 and Gly9-Leu'0 bonds, generating fragments that are devoid of biological activity in most assays. SP is widely expressed in the central (Hokfelt et al., 1987) and peripheral (Schultzberg et al., 1980) nervous systems where it affects neurotransmission, smooth-muscle contraction, vascular permeability, exocrine gland secretion and immune cell function [see Otsuka and Yoshioka (1993) for a review]. It exerts many of its effects through a high-affinity interaction with the SP or NK1 receptor (SPR) (Kd 3.5 nM) (Hershey and Krause, 1990). However, SP also interacts with the low-affinity NK2 and NK3 receptors. The amino acid sequence of the SPR contains seven domains of hydrophobic residues that are presumed to anchor the receptor to the plasma membrane (Yokota et al., 1989; Hershey and Krause, 1990). The tachykinin receptors and NEP are coexpressed in many tissues, including the central nervous system, the airways and the -
coexpressing NEP and the SPR with binding and Ca2+ mobilization in mixtures of cells expressing NEP alone and SPR alone. The results show that expression of NEP in the same cells as the SPR reduces SP binding and attenuates SP-induced Ca2+ mobilization. In contrast, mixing cells expressing SPR alone with cells expressing NEP alone only slightly reduces SP binding and SPinduced Ca2+ mobilization.
EXPERIMENTAL Expression vectors and cells An epitope-labelled chimaera composed of the rat SPR and an N-terminal Flag peptide (DYKDDDDK) in a neomycin-resistant expression vector (pcDNAINeoFlag-SPR) was generated as described (Vigna et al., 1993). Rat NEP cDNA (Gorman et al., 1989) in the expression vector pCISrENK was from Genentech Inc., South San Francisco, CA, U.S.A. The neomycin-resistant
Abbreviations used: NEP, neutral endopeptidase-24.11; rhNEP, recombinant human neutral endopeptidase-24.11; SPR, substance P receptor; SP, substance P; HBSS, Hanks balanced-salt solution; TFA, trifluoroacetic acid; KNRK cells, Kirsten murine sarcoma virus-transformed rat kidney cells; MNA, 4-methoxyl-2-naphthylamine; PMSF, phenylmethanesulphonyl fluoride; DMEM, Dulbecco's modified Eagle's medium; G-418, geneticin sulphate. § To whom correspondence should be addressed.
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expression vector pRC/CMV and the hygromycin-resistant expression vector pCEP 4 were from Invitrogen, San Diego, CA, U.S.A. Kirsten murine sarcoma virus-transformed rat kidney cells (KNRK) and COS-7 cells were from American Type Tissue Culture Collection, Rockville, MD, U.S.A.
Antibodies The generation and characterization of a polyclonal rabbit antiserum (no. 11884-5) to a peptide (KTMTESSSFYSNMLA) corresponding to the C-terminal 15 amino acid residues of the rat SPR [SPR-(393-407)] have been described (Vigna et al., 1993). The generation and characterization of rabbit polyclonal antiserum (no. 20) to recombinant human NEP (rhNEP) have also been described (Terashima et al., 1992). A mouse monoclonal antibody (M2) to the Flag epitope was from International Biotechnologies, New Haven, CT, U.S.A. Affinity-purified rhodamine-conjugated goat anti-rabbit IgG and fluoresceinconjugated goat anti-mouse IgG were from Cappel Research Products, Durham, NC, U.S.A. Goat anti-rabbit IgG conjugated to horseradish peroxidase was from Amersham, Arlington Heights, IL, U.S.A.
selection medium. COS-7 cells were transfected with pCISrENK (5,g) or salmon sperm DNA (5 ,g, control) using DEAEdextran, as described (Sambrook et al., 1989).
Immunocytochemistry KNRK cells were rinsed in 100 mM PBS, pH 7.4, and fixed in 40 g/l paraformaldehyde in PBS, pH 7.4, for 10 min. Cells were washed three times in PBS containing 10 ml/l normal goat serum and 1 ml/l saponin. Cells were incubated with the primary antibodies (SPR antiserum no. 11884-5, 1: 1000; Flag M2 antibody, 10 ug/ml; NEP antiserum no. 20, 1: 1000) in the same buffer for 4 h at 37 'C. Cells were washed and incubated with fluorescein- and rhodamine-conjugated secondary antibodies (1: 200) for 2 h at room temperature. Controls included staining cells transfected with vector alone, preabsorption of the diluted primary antibodies with SPR-(393-407) (10 ,M), Flag peptide (10 4uM) or rhNEP (10 g/ml) overnight at 4 'C before staining, and replacement of the primary antibodies with normal rabbit serum. Cells were examined using a Zeiss Axioplan microscope with fluorescein (Zeiss 487910, excitation wavelength 450490 nm, emission wavelength 515-565 nm, chromatic beam splitter 510 nm) and rhodamine (Zeiss 487915, excitation 546 nm, emission > 590 nm, chromatic beam splitter 580 nm) filters.
Other reagents
Western blotting
Glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine (Glu-AlaAla-Phe-MNA) was from Enzyme Systems Products, Livermore, CA, U.S.A. 4-Methoxy-2-naphthylamine (MNA), thiorphan (DL-3-mercapto-2-benzylpropanoylglycine), amastatin, leupeptin, pepstatin A, bacitracin, 1,10-phenanthroline, EDTA and phenylmethanesulphonyl fluoride (PMSF) were from Sigma Chemical Co., St. Louis, MO, U.S.A. Captopril was a gift from the Squibb Institute for Medical Research (Princeton, NJ, U.S.A.). rhNEP was from Genentech. Bolton-Hunter-labelled I2'I-SP (2000 Ci/mmol) and an ECL Western Blotting Detection Kit were from Amersham. Dulbecco's modified Eagle's medium (DMEM), Lipofectin Reagent and geneticin sulphate (G-418) were from Gibco/BRL, Gaithersburg, MD, U.S.A. Hygromycin was from Calbiochem, San Diego, CA, U.S.A. SP was from Peninsula Laboratories, San Carlos, CA, U.S.A. Fura-2/AM was from Molecular Probes, Eugene, OR, U.S.A.
COS-7 cells and KNRK cells were homogenized in 50 mM Tris/HCl, pH 7.5, containing 1 mM PMSF and 1 uM pepstatin A at 4°C. Total protein in the homogenate was measured (Bradford, 1976). The homogenate (10-200,g of total protein) or rhNEP (1O ng) was separated by SDS/PAGE (7.5 % polyacrylamide) under denaturing and reducing conditions (Laemmli, 1970), and electrophoretically transferred to nitrocellulose (Burnette, 1981). The filter was incubated with NEP antiserum (no. 20, 1: 10000) overnight at 4 °C, washed and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1: 8000) for 2 h at room temperature. The bound antibody was visualized using a chemiluminescent detection kit.
Generation and characterization of cell lines KNRK cells were cultured in DMEM containing 10 % bovine calf serum, 100 units/ml penicillin and 100 ,g/ml streptomycin at 37 °C in 5 % C02/95 % air. KNRK cells were transfected by lipofection (Felgner et al., 1987) using Lipofectin Reagent with the following plasmids: (1) pcDNAINeoFlag-SPR (5 ,g of DNA); (2) pCISrENK (5,ug) plus the neomycin-resistant expression vector pRC/CMV (0.5 ,ug); or (3) pRC/CMV (5 ,g) as the vector control. To generate cells coexpressing Flag-SPR and NEP, KNRK cells expressing the Flag-SPR (clone no. 17) were transfected with pCISrENK (5 ,g of DNA) and the hygromycinresistant expression vector, pCEP 4 (0.5,g) using Lipofectin Reagent. Transfected cells were cultured for 48 h and then placed into antibiotic selection medium containing either 400 ,tg/ml G418 (single transfectants) or 400 ,g/ml G-418 and 300 ,g/ml hygromycin (double transfectants). After 10 days of selection, visible colonies were cloned into selection medium. Positive clones were identified by immunocytochemistry, enzyme assay and Ca2+-signal transduction and maintained in appropriate
NEP assay NEP enzyme activity of COS-7 cells, KNRK cells and membranes was determined fluorimetrically using Glu-Ala-Ala-Phe-MNA (40,M) as a substrate (Terashima et al., 1992). The enzyme activity was expressed in pmol of MNA generated/h per ,ug of protein or per cell number. When used, thiorphan (10-'-10 -6 M) was preincubated with the cells for 10 min before addition of the substrate. Only activity that was inhibited by 1 1uM thiorphan was attributed to NEP.
SP degradation by membranes Membranes were prepared from KNRK cells (Terashima et al., 1992). Membranes (1-10,tg of protein) or rhNEP (10 ng) were incubated with SP (5 nmol) in 250 ,l of 50 mM Tris/HCl, pH 7.5, for 5-60 min at 37 'C. When used, enzyme inhibitors were preincubated with the membranes for 10 min before addition of the SP. Degradation was stopped by boiling (5 min), the samples were centrifuged (14000 g, 2 min) and the supernatant (150,u) was acidified with 2 vol. of 1 ml/l trifluoroacetic acid (TFA) in water. Membranes and NEP were omitted from control incubations. Samples were analysed by reversed-phase h.p.l.c. using a C18 column (Vydac, 5 ,m, 4.6 mm x 250 mm; Separations Group, Hesperia, CA, U.S.A.). The column was equilibrated in
Interactions between neutral endopeptidase and substance P receptor 1 ml/I TFA in water and peptides were eluted with a linear gradient of 0-80% acetonitrile containing 1 ml/I TFA over 35 min with a flow rate of 1 ml/min. Absorbance of the eluatp was monitored at 214 nm, and peak areas were measured by integration. Activity was calculated from the loss of substrate compared with control incubations without enzyme.
SP degradation by cells The degradation of 125I-SP by intact KNRK cells was also examined. KNRK cells were plated (100000 cells/well) and cultured for 24 h before the experiment. On the day of the experiment there were approx. 200000 cells/well. Cells were washed with Hanks' balanced-salt solution containing 1 g/l BSA (HBSS/BSA) and then incubated with Bolton-Hunter-labelled 1251-SP (100 pM) plus unlabelled SP (400 pM) in 250 ,ul of HBSS/BSA for 1-30 min at 37 'C. In control experiments, 1251.. SP and unlabelled SP were incubated in HBSS/BSA without cells. When used, enzyme inhibitors were preincubated with the cells for 10 min before addition of the 1251-SP. After incubation, the supernatant was aspirated, boiled for 5 min, centrifuged (14000 g, 2 min) and acidified with 1 vol. of 5 ml/l TFA in water. Samples were analysed by h.p.l.c. as described. The radioactivity of the eluate was monitored using a flow-through liquid-scintillation counter, and peak areas were measured by integration.
151-SP binding to membranes Membranes (10,ug of protein) were incubated in 100 1l of HBSS/BSA containing 10 pM Bolton-Hunter-labelled 1251-SP for 2.5-30 min at 37 'C (Torrens et al., 1991). Non-specific binding was measured by incubating membranes with 1 ,uM unlabelled SP. After incubation, membranes were collected on polyethylenimine-treated GF/C filters (Whatman International Ltd., Maidstone, Kent, U.K.), washed in ice-cold HBSS/BSA and counted. When enzyme inhibitors were used, membranes were preincubated with them for 10 min before addition of the label.
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[Ca2+1] was calculated (Grynkiewicz et al., 1985). When used, enzyme inhibitors were added to buffers during Fura-2/AM loading and fluorescence measurements. Statisical analysis Results are expressed as means+S.E.M. Differences between multiple groups were examined by a one-way analysis of variance (ANOVA) and the Student-Newman-Keuls test. A P < 0.05 was considered statistically significant.
RESULTS Transfection Twenty clones transfected with pcDNAINeoFlag-SPR (designated KNRK Flag-SPR cells) were screened for expression of Flag-SPR by immunocytochemistry and Ca2+ mobilization, and 18 clones expressed the receptor (see below). Thirty-nine clones transfected with pCISrENK plus pRC/CMV (KNRK NEP cells) were screened for expression of NEP by immunocytochemistry and enzyme assay, and all clones expressed NEP. Thirty-one clones transfected with pcDNAINeoFlag-SPR, pCISrENK and pCEP 4 (KNRK Flag-SPR-NEP cells) were screened for coexpression of the Flag-SPR and NEP by doubleantibody immunocytochemistry, and 23 clones coexpressed'the Flag-SPR and NEP. Both the SPR and NEP were undetectable in KNRK cells transfected with pRC/CMV (KNRK CMV cells), assessed by immunocytochemistry, binding, Ca2+ mobilization, Western blotting and enzyme assays [below and Mitsuhashi et al. (1992) and Vigna et al. (1993)]. The clones KNRK Flag-SPR no. 17, KNRK NEP no. 29 and KNRK FlagSPR-NEP no. 41 were judged to express the highest level of the receptor and enzyme, and were used for all further experiments. The level of SPR expression remained stable over time but the expression of NEP tended to decline. Therefore cells expressing NEP were usually discarded after about ten passages.
Immunocytochemistry 1251-SP binding to cells KNRK cells were plated (100000 cells/well) and cultured for 24 h before the experiment. On the day of the experiment there were approx. 200000 cells/well. Cells were washed with HBSS/BSA and incubated in the same buffer for 1 h at 37 °C to reduce non-specific binding. Cells were incubated in 250 #1 of HBSS/BSA containing 10 pM Bolton-Hunter-labelled 1251-SP for 5-30 min at 37 °C (Vigna et al., 1993). Non-specific binding was measured by incubating membranes with 2.5 ,uM unlabelled SP. After incubation, the cells were washed three times in icecold PBS, lysed in 250 ,ul of 0.5 M NaOH for 30 min at room temperature, and counted. When enzyme inhibitors were used, cells were preincubated with them for 10 min before addition of the label.
SP-lnduced Ca2+ mobilization KNRK cells grown to confluence on coverslips were washed with HBSS/BSA and loaded with 2.5 ,uM Fura-2/AM in HBSS/BSA for 20 min at 37 °C (Vigna et al., 1993). Cells were washed with HBSS/BSA and fluorescence was measured in a fluorimeter (F-2000, Hitachi Instruments, Irvine, CA, U.S.A.) at 340 and 380 nm excitation and 510 nm emission. Cells were exposed to a single concentration of SP (10-11_10-6 M), lysed with digitonin (50 uM), and then exposed to EGTA (5 mM). The relative
To determine the cellular localization of NEP and SPR in transfected cells, KNRK cells were stained using antibodies to NEP, SPR and the Flag epitope. NEP antiserum no. 20 stained the plasma membrane of KNRK NEP cells (Figure la) and KNRK Flag-SPR-NEP cells (Figure Id) but not KNRK CMV cells (Figure Ib). Staining was abolished by preabsorption of the NEP antiserum with rhNEP (Figure Ic) and was absent when the antiserum was replaced with normal rabbit serum. SPR antiserum no. 11884-5 and the Flag M2 antibody stained the plasma membrane of KNRK Flag-SPR cells and KNRK FlagSPR-NEP cells but not KNRK CMV cells [Figures le and If, and Vigna et al. (1993)]. Staining was abolished by preabsorption of the SPR antiserum and the Flag antibody with SPR-(393-407) or the Flag peptide and was absent when the antibodies were replaced with normal rabbit serum. To confirm the coexpression of NEP and the Flag-SPR by transfected cells, KNRK Flag-SPR-NEP cells were examined by simultaneous double-antibody immunocytochemistry using the NEP antiserum (rabbit polyclonal) and the Flag M2 antibody (mouse monoclonal). Rhodamine-conjugated goat anti-rabbit IgG and fluorescein-conjugated goat anti-mouse IgG were used as secondary antibodies. Both the NEP antiserum and the Flag M2 antibody stained the plasma membranes of the same cells from clone no. 41 and all cells were stained with both antibodies (Figures ld and le).
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Figure 1 Localizaton of NEP and Flag-SPR by Indirect immunocytochemistry
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(a) KNRK NEP cells stained with NEP antiserum; (b) KNRK CMV cells stained with NEP antiserum; (c) KNRK NEP cells stained with NEP antiserum preabsorbed with rhNEP (10 csg/ml). (d) and (e) The same cells stained with a mixture of NEP antiserum (rabbit polyclonal) and Flag M2 antibody (mouse monoclonal), followed by rhodamine-conjugated goat anti-rabbit IgG plus fluorescein-conjugated goat anti-mouse IgG. (d) KNRK Flag-SPR-NEP cells observed using a rhodamine filter to detect immunoreactive NEP; (e) the same cells as shown in (d) but observed using a fluoerscein filter to detect immunoreactive Flag peptide. The arrowheads indicate cells expressing both Flag and NEP. (f) KNRK Flag-SPR cells stained with SPR antiserum. Scale bar = 20 ,tm.
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Western blotting Western blotting was used to confirm the expression of NEP by COS-7 cells and KNRK cells transfected with pCISrENK. The NEP antiserum no. 20 recognized rhNEP with an apparent molecular mass of about 90 kDa (Figure 2a, lane 1). The antiserum also recognized proteins with the same electrophoretic mobility as rhNEP in homogenates from COS-7 NEP cells (Figure 2a, lane 3) and KNRK NEP cells (Figure 2a, lane 5). NEP was not detected in homogenates of COS-7 control cells (Figure 2a, lane 2) and KNRK CMV cells (Figure 2a, lane 4).
NEP assay An enzyme assay, which measured the conversion of Glu-AlaAla-Phe-MNA into the fluorogenic product MNA, was used to quantify the NEP activity of the transfected cells (Figure 2b). The generation of MNA by COS-7 NEP cells, KNRK NEP cells and KNRK Flag-SPR-NEP cells was inhibited by thiorphan (1 #uM) by more than 95 %, and thiorphan-inhibited activity was attributed to NEP. The NEP activity in pmol of MNA/h per ,ug of protein was 1923 for COS-7 NEP cells, 140 for KNRK NEP cells and 570 for KNRK Flag-SPR-NEP cells. NEP activity was
Figure 2 Detecton of NEP In cells by Western blotting and enzyme assay (a) Western blot of rhNEP and homogenates prepared from COS-7 cells and KNRK cells probed with the NEP antiserum. The arrow indicates the major protein band (above 90 kDa) recognized by the NEP antiserum. Lane 1, rhNEP (1 0 ng); lane 2, COS-7 control cells (10 uzg of protein); lane 3, COS-7 NEP cells (10 ,g of protein); lane 4, KNRK CMV cells (200 /tg of protein); lane 5, KNRK NEP cells (200,ug of protein). Prestained standards: myosin, 205 kDa; figalactosidase, 116.5 kDa; BSA, 80 kDa; ovalbumin, 49.5 kDa. (b) NEP enzyme assay of various numbers of KNRK NEP (0, 0) and KNRK CMV (A, A) cells in the absence (0, A) or presence (0, A) of thiorphan (1 ,uM). (c) Effects of graded concentrations of thiorphan on NEP enzyme assay of KNRK NEP cells (30000 cells per tube).
undetectable in COS-7 control cells and KNRK CMV cells. The enzyme activity of KNRK NEP cells was inhibited by thiorphan in a concentration-dependent manner, with an IC50 of about 50 nM and maximal inhibition at 100 nM (Figure 2c). The NEP
Interactions between neutral endopeptidase and substance P receptor
When membranes (10 jtg of protein) from KNRK CMV cells were incubated with SP for 60 min, there was only 8.9+ 1.9 % degradation (three observations). Degradation was unaffected by thiorphan (1 ,tM), captopril (1 ,uM), a mixture of EDTA (0. 5 mM) and phenanthroline (0.5 mM), or a mixture ofpepstatin A (1 1tM), leupeptin (1 uM), PMSF (1 mM) and amastatin (10 ,uM). Membranes from KNRK Flag-SPR-NEP cells rapidly degraded SP. When membranes (10 /ug of protein) from KNRK Flag-SPR-NEP cells were incubated with SP for 60 min, there was 78.8 + 0.3 % degradation. Degradation was inhibited 98.3 + 1.0 % by thiorphan, 0 % by captopril (1 ,uM), 76.5 + 9.7 % by a mixture of EDTA (0.5 mM) and phenanthroline (0.5 mM), and 9.9 + 3.2 % by a mixture of pepstatin A (1 ,uM), leupeptin (1 ,uM), PMSF (1 mM) and amastatin (1O,uM). The elution times of the products of degradation of SP by rhNEP and membranes from KNRK Flag-SPR-NEP cells were the same. Although the degradation products were not unequivocally identified by amino acid analysis, the fragments SP(1-6), -(1-7) and -(1-9) and corresponding C-terminal peptides were tentatively identified by their elution times from the chromatography column (Bunnett et al., 1988).
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SP degradation by cells Bolton-Hunter-labelled 1251-SP was incubated with intact KNRK cells to determine the relative importance of NEP to the overall degradation of SP by whole cells. The time course of 1251_ SP degradation by KNRK CMV cells and KNRK FlagSPR-NEP cells is shown in Figure 3(b) and the separation of the metabolites by h.p.l.c. is shown in Figure 5. The proportion of radioactivity eluted from the h.p.l.c. column as intact 1251-SP slowly declined when the peptide was incubated with HBSS/BSA without cells (control) at 37 'C. The proportion of radioactivity recovered as intact 1251-SP was 84.5 + 1.60% (n = 3 observations) after 0 min incubation but had declined to 49.7+0.70% after 30 min incubation in HBSS/BSA at 37 'C. This may represent oxidation of SP. In control experiments, KNRK CMV cells slowly degraded 125I-SP. After 5 min incubation there was 3.1 + 3.5 % degradation and after 30 min incubation there was 41.9 + 5.6 0 degradation. Degradation was unaffected by thiorphan, but was inhibited 100% by bacitracin (1 g/l), 1000% by a mixture of EDTA (0.5 mM) and phenanthroline (0.5 mM), and 100 % by a mixture of captopril (1 ,uM), pepstatin A (1 ,tM), leupeptin (1 ,uM), PMSF (1 mM) and amastatin (1O,uM). KNRK Flag-SPR-NEP cells rapidly degraded 1251-SP. After 5 mn incubation, there was 59.8 + 0.9 % degradation and after 30 min incubation there was 99.5 + 0.50% degradation. Degradation was inhibited 78.1+12.70% by thiorphan (1 ,M). The products were eluted from the h.p.l.c. column earlier than intact 1251SP but were not identified.
of degradation of SP by membranes and cells
(a) Time course of SP degradation by membranes from KNRK CMV cells (0) and KRNK Flag-SPR-NEP cells (0). Membranes (10 #g protein) were incubated with SP (5 nmol) in 200 ,u1 of 50 mM Tris/HCI, pH 7.5, for 540 min at 37 °C, and degradation was assessed by h.p.l.c. (b) Time course of 1251-SP degradation by KNRK CMV (0) and KNRK Flat-SPR-NEP (0) cells. Cells (approx. 200000) were incubated with 1251-SP (100 pM) plus unlabelled SP (400 pM) in 250 ul of HBSS/BSA for 5-30 min at 37 °C, and degradation was assessed by h.p.l.c. (n = 3 observations for membranes and cells).
activity of membranes from KNRK Flag-SPR-NEP cells 395 pmol MNA/h per ,ug of protein.
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SP degradation by membranes The effects of NEP expression on the degradation of SP were examined by incubating the peptide with membranes prepared from KNRK cells. The time course of SP degradation by membranes prepared from KNRK CMV cells and KNRK Flag-SPR-NEP cells is shown in Figure 3(a) and the separation of the metabolites by h.p.l.c. is shown in Figure 4. SP degradation by rhNEP was inhibited by 92.6 + 2.2 % (n = 3 observations) by thiorphan (1 ,uM). In control experiments, membranes from KNRK CMV cells (control, transfected with vector alone) only slowly degraded SP.
'251-SP binding to membranes Binding of 1251-SP to membranes from KNRK Flag-SPR cells was specific and saturable (Figure 6a). When 10 ,tg of membranes was incubated with 1251-SP at 37 'C, specific binding reached saturation within 5-10 min and was 26.1 + 5.4 % (triplicate observations, n = 3 experiments) of total counts. At this time the non-specific binding was less than 5 % of total counts. Binding of 1251-SP to membranes from KNRK Flag-SPR cells was unaffected by thiorphan (1,uM) (25.9 + 6.30% of total counts at 10 min). Coexpression of NEP with the SPR markedly reduced specific
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FIgure 4 H.p.l.c. proflles of the metaboltes of SP degradation by KNRK CMV membranes (a), KNRK Flat-SPR-NEP membranes (b) and KNRK FlaSPR-NEP membranes with tMrphan (1 pM) (c) Membranes (1
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Figure 5 H.p.l.c. profiles of the metaboltes of 1251-SP degradation by KNRK CMV cells (a), KNRK Flat-SPR-NEP cells (b) and KNRK Flag-SPR-NEP cells with thlorphan (1 pM) (c) Cells (approx. 200000) were incubated with 1251-SP (100 pM) plus unlabelled SP (400 pM) in 250 ,ul of HBSS/BSA for 5 min at 37 °C. The arrow indicates the elution position of 1251-SP.
binding. Binding of 125I-SP to membranes from KNRK FlagSPR-NEP cells was 5.4 + 1.7 % of total counts at 10 min and binding was increased to 14.9 + 4.8% of total counts with thiorphan.
1251-SP binding to cells Binding of 1251-SP to KNRK Flag-SPR was specific, saturable, temperature-dependent and reversible, with an apparent Kd of 5.6 nM (Vigna et al., 1993). At 37 °C, specific binding was maximal at 15-20 min (28.3 + 2.1 % of total counts at 15 min, triplicate observations, n = 3 experiments). The non-specific binding at the time of the peak was less than 7 % of total binding. To determine whether coexpression of NEP in the same cell as the SPR affected binding, the time courses of 125I-SP binding to
KNRK Flag-SPR cells (controls) and KNRK Flag-SPR-NEP cells were compared in the presence and absence of thiorphan (1 ,uM; Figure 6b). Specific binding is expressed as a percentage of the maximal saturated binding (100%) with thiorphan to control for small differences in the number of cells between experiments. In KNRK Flag-SPR cells, binding at 20 min was 92.2 + 3.1 % of maximal in the absence of thiorphan and was maximal (100 + 0 %) in the presence of thiorphan. Coexpression of NEP with Flag-SPR significantly reduced the peak binding (Figure 6b). Thus binding to KNRK Flag-SPR-NEP cells at 20 min was 13.9 + 0.5 % of maximal in the absence of thiorphan, and this was increased by thiorphan to 82.7 + 2.4 % of maximal. The effect of thiorphan on the peak binding of 1251-SP to KNRK Flag-SPR-NEP cells was concentration-dependent (Figure 7a) and paralleled the inhibition of NEP activity (Figure
Interactions between neutral endopeptidase and substance P receptor 35
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Figure 6 Binding of I'll-SP to membranes and cells (a) Time course of 1251-SP binding to KNRK Flag-SPR membranes (e), KNRK Flat-SPR-NEP membranes (A) and KNRK Flag-SPR-NEP membranes with thiorphan (10,uM) (A). Membranes (10 ,ug of protein) were incubated with 1251-SP (10 pM) in 100 ,ul of HBSS/BSA for 2.5-30 min at 37 OC. Specific binding is expressed as a percentage of total radioactivity (triplicate observations, n = 3 experiments). (b) Time course of 1251-SP binding to KNRK Flag-SPR cells (0), KNRK Flag-SPR-NEP cells (A) and KNRK Flag-SPR-NEP cells with thiorphan (1 4uM) (A). Cells (approx. 200000) were incubated with 1251-SP (10 pM) in 250 ,ul of HBSS/BSA for 5-30 min at 37 OC. Specific binding is expressed as a percentage of the maximal saturated binding measured in cells treated with thiorphan (triplicate observations, n = 3 experiments). *P < 0.05 compared with KNRK Flag-SPR cells.
2c). The EC50 of the increase in specific binding was observed at about 50 nM thiorphan and the maximal effect was at 100 nM thiorphan. To determine whether the expression of NEP in different cells from the SPR affected binding, the time course of 1251-SP binding was examined in mixtures of KNRK Flag-SPR cells and KNRK NEP cells. At 24-48 h before the binding experiment, KNRK Flag-SPR cells were plated with KNRK CMV cells (control) or KNRK NEP cells. The KNRK NEP cells expressed only about 25 % of the NEP activity of the KNRK Flag-SPR-NEP cells (140 compared with 570 pmol of MNA/h per ,ug of protein). Therefore cells were cultured in ratios of one KNRK Flag-SPR cell to three, six, 12 or 24 KNRK CMV cells or KNRK NEP cells to vary the NEP activity of the cell mixtures. The time course of
20
Time (min)
Figure 7 Binding of 1'I-SP to cells (a) Effects of thiorphan on saturable binding of '251-SP to KNRK Flag-SPR-NEP cells. Cells (approx. 200000) were incubated with 1251-SP (10 pM) and graded concentrations of thiorphan in 250 czl of HBSS/BSA for 15 min at 37 0C (triplicate observations, n = 3 experiments). (b) Time course of 1251-SP binding to mixtures of KNRK Flag-SPR cells and KNRK CMV cells (0), mixtures of KNRK Flag-SPR cells and KNRK NEP cells (A), and mixtures of KNRK Flag-SPR cells and KNRK NEP cells with thiorphan (1 0 1M) (A). Cells were mixed in ratios of one KNRK Flag-SPR cell to three KNRK cells or KNRK NEP cells. Cells (approx. 200000) were incubated with 1251-SP (10 pM) in 250,l of HBSS/BSA for 5-30 min at 37 0C. Specific binding is expressed as a percentage of the maximal saturated binding measured in cells treated with thiorphan (triplicate observations, n = 3 experiments).
125I-SP binding to mixtures of cells plated at a 1: 3 ratio is shown in Figure 7(b). Mixing KNRK Flag-SPR cells with KNRK NEP cells in a ratio of 1:3 slightly reduced the binding of 1251-SP, compared with KNRK control cells or KNRK NEP cells in the presence of thiorphan, but this reduction was not statistically significant. The maximal binding measured at 20 min for KNRK FlagSPR-NEP cells and mixtures of KNRK Flag-SPR cells and KNRK NEP cells (1: 3 to 1: 24) in the absence of thiorphan was compared with binding of the same cells in the presence of thiorphan. In the absence of thiorphan, the maximal binding to mixtures of cells expressing SPR alone or NEP alone was 76.3 + 4.3 % for the 1:3 ratio of SPR to NEP expressing cells, 67.2+10.1% for the 1:6 ratio, 66.3+3.8% for the 1:12 ratio,
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Figure 8 Effects of SP (0.3, 0.5 and 1.0 nM added at arrow) on [Ca2+], measured using Fura-2/AM in KNRK Flag-SPR cells (a), KNRK Flag-SPR-NEP cells (b), and KNRK Flag-SPR-NEP cells with thiorphan (1 pM) (c)
and 58.9+9.80% for the 1:24 ratio, of that observed in the of thiorphan. In contrast, in the absence of thiorphan the maximal binding to cells coexpressing SPR and NEP was only 16.8 + 0.3 % of that observed with thiorphan. Therefore the reduction in binding observed in the mixtures of cells was small compared with that observed in cells expressing both NEP and SPR.
presence
SP-induced Ca2+ mobilization SP induced a prompt increase in [Ca2'], in KNRK Flag-SPR cells, with a threshold of 0.1 nM, a maximal response to 100 nM, and an apparent EC50 of 0.66 nM (Figure 8) (Vigna et al., 1993). In control experiments, SP-induced Ca2+ mobilization was measured in KNRK Flag-SPR cells treated with enzyme inhibitors. Thiorphan (1 1tM) had no effect on the Ca2' response to SP (0.3-100 nM). For example, 1 nM SP induced an increase in [Ca2+]1 in KNRK Flag-SPR cells of 163.4+ 15.7 nM (n = 5 observations) in the absence of thiorphan and 138.4 + 38.1 nM (n = 4) in the presence of thiorphan. The Ca2+ response to 0.5 nM
SP was not potentiated by bacitracin (1 g/l) or by a mixture of captopril (1 ,uM), pepstatin A (1 ,uM), leupeptin (1 ,uM), PMSF (1 mM) and amastatin (10 ,M). The results of the degradation experiments indicated that KNRK CMV cells slowly degraded 1251-SP (Figures 3b and 5). Therefore the possibility that SP was degraded during the Ca2+ assay was examined. KNRK Flag-SPR cells (confluent cells on a 1 cm x 2 cm glass coverslip used for measuring Ca2+ mobilization) were incubated with "25I-SP (10 pM) plus unlabelled SP (490 pM) in 2 ml of HBSS/BSA (assay volume for measurement of Ca2l mobilization) at 37 °C, and degradation was assessed by h.p.l.c. After a 1 min incubation (typical duration of Ca2" response to SP) only 7.0 + 4.4 % (four observations) of the 1251SP was degraded. To determine whether coexpression of NEP with Flag-SPR affected Ca2+ mobilization, SP-induced Ca2+ mobilization was examined in KNRK Flag-SPR-NEP cells in the presence and absence of thiorphan. Coexpression of NEP with SPR in KNRK Flag-SPR-NEP cells abolished to undetectable the Ca2` response to 0.3 nM and 0.5 nM SP and markedly attenuated the response
Interactions between neutral endopeptidase and substance P receptor Table 1 Effects of graded concentrations of SP on [Ca2+], measured In KNRK Flag-SPR cells and KNRK Flag-SPP-NEP cells Values represent the increase in [Ca2+]i above basal. Results are expressed as means + S.E.M. of three to seven observations. *P < 0.05 compared with KNRK Flag-SPR cells and KNRK Flag-SPR-NEP cells with thiorphan.
[Ca2+]i (nM) SP (nM)
KNRK FlagSPR cells
KNRK FlagSPR-NEP cells
KRNK FlagSPR-NEP cells +thiorphan (1 4uM)
0.3 0.5 1 3 100
30.4+ 6.2 49.5+13.0 163.4 +15.7 220.5 + 38.8 318.5 +10.7
0+0* 0+0* 52.3 +13.1* 126.5 + 28.0 303.1 +31.4
61.0+15.5 97.6+12.5 161 .4+ 25.1 251.8 + 37.1 379.6 + 72.9
to 1 nM and 3 nM SP (Figure 8 and Table 1). These effects were reversed by thiorphan. Thus 1 nM SP induced an increase in [Ca2+]i in KNRK Flag-SPR-NEP cells of 52.3 + 13.1 nM (n = 7) in the absence of thiorphan and of 161.4 + 25.1 nM (n = 7) in the presence of thiorphan (P < 0.05). The magnitude of the Ca2+ response to higher concentrations of SP (> 100 nM) was unaffected by coexpression of NEP and the SPR (Table 1). To examine whether the expression of NEP in different cells from the SPR affected SP-induced Ca2+ mobilization, the Ca2+ response was measured in mixtures of KNRK Flag-SPR cells and KNRK NEP cells. At 24-48 h before the experiment, KNRK Flag-SPR cells were plated with KNRK CMV cells (control) or KNRK NEP cells. Cells were cultured in ratios of one KNRK Flag-SPR cell to three or six KNRK CMV cells or KNRK NEP cells, as described in the binding experiments. Higher ratios could not be examined because the Ca2+ response became too small to measure reproducibly when the number of KNRK Flag-SPR cells was further reduced. The Ca2+ response of the mixtures to 0.5 nM SP was examined because the coexpression of NEP in the same cell as the SPR abolished the increase in [Ca2+], in response to this concentration of SP (Figure 8 and Table 1). SP-induced Ca2+ mobilization in KNRK Flag-SPR cells was unaffected by coculture with KNRK NEP cells at ratios of 1: 3. In control experiments, when KNRK Flag-SPR cells were cocultured with KNRK CMV cells in the ratio of 1:3, the increase in [Ca2+]1 in response to 0.5 nM SP was 61.5 + 5.6 nM (n = 9). When KNRK Flag-SPR cells were cocultured with KNRK NEP cells in the ratio of 1: 3, the increase in [Ca2+]i in response to 0.5 nM SP was 60.7 + 10.1 nM (98.7 +±16.4 % of the control response, n = 8) in the absence of thiorphan, and 78.2 + 14.3 nM (127.1 + 23.2 % of the control response, n = 9) in the presence of thiorphan. These differences were not statistically significant. The Ca2+ response to 0.5 nM SP was slightly reduced when KNRK Flag-SPR cells were cocultured with KNRK NEP cells in a ratio of 1: 6. Thus when KNRK Flag-SPR cells were cocultured with KNRK NEP cells in the ratio of 1:6, the increase in [Ca2+]i in response to 0.5 nM SP was 77.7 + 12.9% (n = 6) of that observed in control experiments. This was restored to 114.2 + 14.2 % (n = 9) of the control by addition of thiorphan. These differences were also not statistically significant. To examine whether the addition of NEP affected SP-induced Ca2+ mobilization, the Ca2+ response to 0.5 nM SP was measured in KNRK Flag-SPR cells in the presence of graded concentrations of rhNEP. The rhNEP was added to HBSS/BSA in the
691
fluorimeter cuvette about 60 s before the addition of SP. The increase in [Ca2+]1 to 0.5 nM SP was unaffected by addition of 50 or 500 ng/ml rhNEP. These amounts of NEP were equivalent to between 0.5- and 5-fold the amount of activity present in the cuvette when the KNRK Flag-SPR-NEP cells were examined. A concentration of rhNEP of 5000 ng/ml reduced the Ca21 response to 17.2+10.3 % of control (no NEP), and 25000 ng/ml reduced the response to 0.8 + 0.4 % of control (n = 4 observations). When the rhNEP was inactivated by boiling for 10 min before addition, the Ca2+ response was restored to control levels.
DISCUSSION In this investigation the coexpression of NEP in the same cell as the SPR accelerated SP degradation, markedly reduced SP binding and attenuated the intracellular Ca2+ response to graded concentrations of SP. In contrast, binding and Ca2+ mobilization in cells expressing the SPR alone were unaffected or slightly diminished by coculture with cells expressing NEP. This suggests that, in this system, NEP effectively down-regulates the interaction of SP with its receptor only when it is colocalized in the same cell as the SPR. KNRK cells transfected with cDNA encoding NEP expressed high levels of functional NEP, as assessed by enzyme assays. As expected, the enzyme was localized to the plasma membrane by immunocytochemistry of cells, and by enzyme assays and Western blots of membrane preparations. The expression of NEP in KNRK Flag-SPR cells markedly accelerated the degradation of SP by membrane preparations and by intact cells. SP was degraded very slowly by membranes prepared from KNRK CMV cells. In contrast, membranes from KNRK Flag-SPRNEP cells rapidly degraded SP, and this was strongly inhibited by thiorphan (98 %) and by metal ion chelators and is thus attributable to NEP (Bunnett et al., 1988). lodinated SP was slowly degraded by KNRK CMV cells, and this was unaffected by thiorphan but inhibited by a variety of enzyme inhibitors with broad specificity. It is unlikely that this degradation is due to ectoenzymes because membranes from KNRK CMV cells degraded SP very slowly. KNRK cells may secrete SP-degrading enzymes, but this was not examined. SP was rapidly degraded by KNRK Flag-SPR-NEP cells. This was strongly inhibited by thiorphan (78 %) and is thus due to NEP. KNRK cells expressing the SPR demonstrate receptor-mediated endocytosis of SP, which is then degraded intracellularly to fragments that are released into the extracellular fluid (N. W. Bunnett, unpublished work). This may have contributed to the thiorphan-resistant degradation of SP by KNRK Flag-SPR-NEP cells but could not account for degradation by KNRK CMV cells, which do not express detectable SPR. SP binding to cells and membranes was markedly reduced by expression of NEP in the same cells as the SPR. The specific binding of 1251-SP to KNRK Flag-SPR cells was approx. 4-fold higher than that to KNRK Flag-SPR-NEP cells (measured at 20 min), and binding to KNRK Flag-SPR-NEP cells was fully restored to control levels by thiorphan. Similarly, the specific binding of 1251-SP to membranes prepared from KNRK FlagSPR cells was about 3-fold higher than maximal binding to membranes from KNRK Flag-SPR-NEP cells (measured at 5 min), and binding to membranes from KNRK Flag-SPR-NEP cells was restored to 57 % of control levels by thiorphan. It is surprising that thiorphan did not fully restore this binding because thiorphan completely inhibited degradation of SP by membranes. However, degradation of SP by membranes was also inhibited by a mixture of pepstatin A, leupeptin, PMSF and amastatin, and enzymes susceptible to these inhibitors may also
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have contributed to degradation. The reduction in SP binding due to expression of NEP correlated well with the NEP-mediated degradation of SP by cells and membranes. After 20 min incubation, the degradation of '25I-SP by KNRK Flag-SPR-NEP cells was about 4-fold higher than the degradation by KNRK CMV cells. After 5 min incubation, the degradation of 1251I-SP by membranes prepared from KNRK Flag-SPR-NEP cells was approx. 8-fold higher than the degradation by membranes from KNRK CMV cells. SP-induced Ca2+ mobilization, like binding, was markedly reduced by expression of NEP in the same cell as the SPR. The Ca2+ response to low concentrations of SP (0.3 and 0.5 nM) was reduced to undetectable by expression of NEP with the SPR, and the response to higher concentrations of SP (1 and 3 nM) was substantially diminished. Thiorphan restored the Ca2+ responses to levels observed in cells expressing the SPR alone, confirming that the reduction in Ca2+ mobilization was due to NEP. The increase in [Ca2+], in response to near-maximal concentrations of SP (> 100 nM) was unaffected by coexpression of NEP. Presumably, these concentrations are sufficiently high that many SP molecules escape degradation by NEP and are able to interact with the receptor. Together, the results of the experiments to measure 1251-SP binding and SP-induced Ca2+ mobilization support the hypothesis that NEP attenuates the responsiveness of cells to SP by degrading the peptide in the immediate vicinity of its receptor. It is possible that high levels of NEP expression may sterically hinder the interaction of SP with its receptor and that this, rather than degradation, may account for the diminished binding and Ca + response. However, the observation that thiorphan restored SP binding and SP-induced Ca2+ mobilization to control levels in cells expressing both NEP and the SPR suggests that NEP enzyme activity, rather than steric interference, down-regulates the responsiveness of cells to SP. The possibility that NEP modulates the interaction between SP and its receptor when NEP and the SPR are expressed in different cells was examined by measuring 1251-SP binding and SP-induced Ca2+ mobilization in mixtures of cells expressing SPR alone and NEP alone. Binding and Ca2+ mobilization were minimally affected by mixing KNRK Flag-SPR cells with KNRK NEP cells. For example, the maximal binding of SP in mixtures of one KNRK Flag-SPR cell to three KNRK NEP cells was still 76 % of that observed in the same cell mixtures treated with thiorphan. In contrast, the maximal specific binding of 1251-SP to KNRK cells expressing both SPR and NEP was only 170% of maximal binding observed in thiorphan-treated cells. The Ca2+ response was similarly affected in the mixing experiments. For example, the Ca2+ response of mixtures of one KNRK Flag-SPR cell to three KNRK NEP cells to 0.5 nM SP was 78 % of that observed in mixtures of one KNRK Flag-SPR cell to three KNRK control cells. In contrast, there was no detectable Ca2+ response of cells expressing both SPR and NEP to 0.5 nM SP. These results indicate that, in this system, NEP markedly down-regulates the response of cells to SP only when it is coexpressed in the same cell as the receptor. This conclusion was strengthened by experiments which examined the effects of rhNEP on the Ca2+ response to SP, which was diminished only by addition of large amounts of rhNEP. The Ca2+ response of KNRK Flag-SPR cells to 0.5 nM SP was unaffected by addition of up to 500 ng/ml rhNEP, which represents a 5-fold higher NEP activity in the fluorimeter than when the KNRK Flag-SPR-NEP cells were studied. When 5000 ng/ml NEP was added, representing a 50-fold excess compared with the KNRK Flag-SPR-NEP cells, the response was reduced to 17 % of controls. Therefore NEP coexpressed in
the plasma membrane with the SPR down-regulates the response of cells to SP far more effectively than the soluble enzyme. There are many reports that NEP inhibitors potentiate the biological effects of SP in a variety of systems. Thus the NEP inhibitors phosphoramidon and thiorphan inhibit the degradation of SP by membranes prepared from the cortex of the brain, and from gastric and intestinal smooth muscle (Matsas et al., 1983; Bunnett et al., 1985; Nau et al., 1986). NEP inhibitors increase specific binding of SP to intestinal muscle (Iwamoto et al., 1988; Keefer and Mong, 1990) and also increase the potency with which SP stimulates contraction of strips of intestinal muscle (Djokic et al., 1989). In the airway, NEP inhibitors potentiate the effects of SP on smooth-muscle contraction (Sekizawa et al., 1987) and mucus secretion (Borson et al., 1987). However, in some of these systems it is not unequivocally established whether SP interacts with the NK1, NK2 or NK3 receptor, and whether NEP and the receptor are expressed in the same or different cells. The results of the present investigation show that NEP, coexpressed in the same cell as the high-affinity SPR (NK1), accelerates SP breakdown and markedly downregulates binding and Ca2+ mobilization. In contrast, NEP has minimal effects on the interaction of SP with its receptor when the enzyme and the receptor are expressed in different cells. Indeed, the affinity of SP for the SPR (Kd 3.5 nM) (Hershey and Krause, 1990) is almost 10000-fold greater than the affinity of SP for NEP (Km 32 ,uM) (Matsas et al., 1983, 1984). Together, these results support the hypothesis that NEP degrades SP at the cell surface in close proximity to the SPR. -
This work was supported by NIH grants DK 43207 (to N. W. B. and D. G. P.) and NS 21710 (to N. W. B. and D. G. P.).
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Interactions between neutral endopeptidase and substance P receptor Otsuka, M., and Yoshioka, K. (1993) Physiol. Rev. 73, 229-309 Pollard, H., Bouthenet, M. L., Moreau, J., Souil, E., Verroust, P., Ronco, P. and Schwartz, J.-C. (1989) Neuroscience 30, 339-376 Sambrook, J., Fritsh, E. F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Schultzberg, M., Hokfelt, T., Nilsson, G., Terenius, L., Rehfeld, J. F., Brown, M., Elde, R., Goldstein, M. and Said, S. (1980) Neuroscience 5, 689-744 Sekizawa, K., Tamaoki, J., Graf, P. D., Basbaum, C. B., Borson, D. B. and Nadel, J. A. (1987) J. Pharmacol. Exp. Ther. 243, 1211-1217 Received 5 July 1993/17 November 1993; accepted 23 November 1993
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Terashima, H., Okamoto, A., Goetzl, E. J., Menozzi, D. and Bunnett, N. W. (1992) Peptides 13, 741-748 Torrens, Y., Beaujouan, J.-C., Dietl, M., Saffroy, M., Petitet, F. and Glowinski, J. (1991) in Neuropeptide Technology: Gene Expression and Neuropeptide Receptors (Conn, P. M., ed.), pp. 243-267, Academic Press, London Vigna, S., Bowden, G., McDonald, D. M., Fisher, J., Okamoto, A., McVey, D. C., Payan, D. and Bunnett, N. W. (1994) J. Neurosci. 14, 834-845 Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K, Shigemoto, R., Kakizuka, A., Ohkubo, H. and Nakanishi, S. (1989) J. Biol. Chem. 264, 17649-17652
