Departments of 1 Surgery and 2 Physiology, University of California, San Francisco, California 94143; 3 Division of Gastroenterology, Beth Israel Deaconess Medical Center, and Pulmonary Division Ina Sue Pelmutter Laboratory, Children's Hospital, Harvard Medical School, Boston 02115; and 4 Department of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Toxin A (TxA) of Clostridium difficile induces acute inflammation of the intestine initiated by release of substance P (SP) and activation of the neurokinin-1 receptor. However, the mechanisms that terminate this response are unknown. We determined whether the SP-degrading enzyme neutral endopeptidase (NEP, EC 3.4.24.11) terminates TxA-induced enteritis. We used both genetic deletion and pharmacological inhibition of NEP to test this hypothesis. In wild-type mice, instillation of TxA (0.5-5 µg) into ileal loops for 3 h dose dependently increased ileal fluid secretion, stimulated granulocyte transmigration determined by myeloperoxidase activity, and caused histological damage characterized by depletion of enterocytes, edema, and neutrophil accumulation. Deletion of NEP reduced the threshold secretory and inflammatory dose of TxA and exacerbated the inflammatory responses by more than twofold. This exacerbated inflammation was prevented by pretreatment with recombinant NEP. Conversely, pretreatment of wild-type mice with the NEP inhibitor phosphoramidon exacerbated enteritis. Thus NEP terminates enteritis induced by C. difficile TxA, underlying the importance of SP degradation in limiting neurogenic inflammation.
substance P; neurokinin-1 receptor; neurogenic inflammation; colitis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ONE OF THE MOST COMMON FORMS of iatrogenic infection is diarrhea and intestinal inflammation induced by the bacterium Clostridium difficile (20). Toxin A (TxA) is a large molecular weight protein released from C. difficile that is responsible for diarrhea and acute intestinal inflammation (20). The mechanism of TxA-induced enteritis involves toxin binding to enterocyte receptors, leading to activation of sensory and enteric nerves that results in enhanced intestinal secretion and motility, degranulation of mast cells, and infiltration of the mucosa by neutrophils (19, 35). Substance P (SP) released from the endings of sensory neurons and its neurokinin-1 receptor (NK1R) are critical mediators of TxA-induced inflammation in experimental animals. Thus chemical and surgical ablation of sensory neurons (7, 26, 27), pharmacological NK1R antagonism (36), or genetic deletion of the NK1R (8) markedly abrogate TxA-induced enteritis. SP activates the NK1R on a variety of intestinal cell types, including enteric nerves, endothelial cells, lamina propria macrophages, and leukocytes to stimulate plasma extravasation, fluid secretion, mast cell degranulation, and generation of cytokines (6, 13, 27). Although it is well established that SP and NK1R play an essential role in the initiation and progress of the secretory and inflammatory responses to TxA, there is no information on the mechanisms that potentially terminate these TxA-induced intestinal responses.
Cellular responses to SP are tightly regulated. Termination of
responses to SP are usually considered from the viewpoint of the NK1R.
SP binding triggers translocation of G protein receptor kinases and
protein kinase C to the plasma membrane, where they phosphorylate the
NK1R to promote its interaction with -arrestins (1, 22, 30,
31, 38, 43).
-arrestins disrupt association of the NK1R with
heterotrimeric G proteins and serve as adaptors for clathrin-dependent
endocytosis of the NK1R, both of which contribute to desensitization of
signal transduction. However, mechanisms that limit the concentration
of SP in the extracellular fluid represent the earliest step in
restricting its proinflammatory effects before receptor activation can proceed.
The concentration of SP in interstitial fluid is a balance between release from nerves and removal by dilution and degradation. The cell-surface enzyme neutral endopeptidase (NEP; EC 3.4.24.11) degrades SP by hydrolyzing Gln6-Phe7, Phe7-Phe8, and Gly9-Leu10 bonds and plays a major role in terminating the biological effects of SP (28, 29). NEP is widely distributed throughout the intestine, where it is expressed by many cells that are regulated by SP, including enterocytes, endothelial cells, neurons, and myocytes (5). Coexpression of NEP with the NK1R markedly diminishes responses to SP (33), and NEP inhibitors magnify the effects of SP in cell lines, tissues, and intact animals (10, 23, 25, 33). Although SP release and subsequent activation of the NK1R initiate TxA-induced enteritis, the role of NEP in terminating this inflammation is unknown. An understanding of the role of NEP in TxA-induced enteritis is of considerable interest, because NEP is downregulated in the inflamed intestine, which could exacerbate inflammation (15).
The purpose of this investigation was to determine the role of NEP in
terminating TxA-induced enteritis. To do so, we used the TxA animal
model of intestinal secretion and inflammation, which closely resembles
the secretory and inflammatory changes seen in human C. difficile infection (20). Our aims were to 1) compare the inflammatory responses to TxA in NEP +/+ and
congenic NEP /
animals, 2) determine the effectiveness
of recombinant human NEP (rhNEP) as an anti-inflammatory agent, and
3) examine whether NEP inhibitors that are under development
as therapeutic agents have proinflammatory effects.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals.
All experimental protocols were approved by the Animal Care and Use
Committee of the University of California at San Francisco. NEP /
mice were back-crossed for seven generations into C57/BL6 mice
(Taconic, Germantown, NY) (24). Deletion of NEP was
confirmed by Southern blotting, as described previously
(24). For NEP +/+ mice, C57/BL6 mice of the same weight,
age, and sex as the knockout animals were used. Colonies were
maintained under climate- (22 ± 4°C) and light-controlled
(12:12-h light-dark cycle) conditions in a barrier facility.
NEP localization.
To localize immunoreactive NEP, NEP +/+ and NEP /
mice were
anesthetized with pentobarbital sodium (60 mg/kg ip) and transcardially perfused with 4% paraformaldehyde in 100 mM PBS, pH 7.4. Segments of
small intestine and kidney were processed to obtain frozen sections
(5). NEP was localized by indirect immunofluorescence using antibody 20, as described previously (5).
NEP enzymatic assays.
NEP enzymatic activity was measured in extracts of tissues from NEP +/+
and NEP /
mice by a fluorometric assay using
glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine as a substrate, as
described previously (5). Only activity that was
inhibited by 1 µM thiorphan or phosphoramidon (selective NEP
inhibitors) was attributed to NEP. Activity was expressed as picomoles
of 4-methoxy-2-naphthylamine (MNA) generated per micrograms of protein
per hour.
Induction of intestinal inflammation.
TxA from C. difficile was purified to homogeneity as
previously described (37). Male and female mice
(20-35 g) were fasted overnight but allowed access to water. Mice
were anesthetized with metaphane. Through a midline laparotomy, two
4-cm ileal loops were ligated and injected with either 0.1 ml of 50 mM
Tris · HCl buffer pH 7.4 (control) or buffer containing TxA
(0.5-5 µg) (8, 36). The abdomen was closed, and the
animals were allowed to regain consciousness. Mice became ambulatory
within 60-90 min of completion of the surgery. Three hours after
administration of TxA, mice were killed with pentobarbital sodium (200 mg/kg ip), and the intestinal loops were removed. The loop length,
weight, and fluid volume were recorded. A portion of the loop was
frozen in 50 mM KH2PO4 buffer, pH 6, containing
0.5% hexadecyltrimethyl ammonium bromide (Sigma Chemical, St. Louis,
MO) for measurement of myeloperoxidase (MPO) activity
(19). The remaining tissue was fixed in 10% formalin and
embedded in paraffin, and sections were stained with hematoxylin and
eosin for histological grading of ileal inflammation (8,
36). Some NEP /
mice were injected with rhNEP, BSA (DAKO,
Carpenteria, CA), or boiled rhNEP (controls) (all 3 mg/kg) in the tail
vein 5 min before surgery. rhNEP was a gift from Dr. Donald Payan
(Rigel, South San Francisco, CA). Similarly, some NEP +/+ mice received
an injection of the NEP inhibitor phosphoramidon (3 mg/kg, Sigma
Chemical) or saline vehicle.
MPO assay. Ileal tissue was homogenized and sonicated, and the homogenate was centrifuged (12,000 g, 15 min). MPO activity in the supernatant was quantified with a microtiter plate assay using 5-O-dianiside (Aldrich, Milwaukee, WI) as the substrate (19). Human neutrophil MPO (Calbiochem, San Diego, CA) was used to generate a standard curve. Supernatants were assayed in duplicate at three dilutions, and the values falling within the linear portion of the standard curve were used. Results are expressed as units of MPO per gram of wet tissue.
Histology. The severity of inflammation was scored in coded slides by a pathologist on a scale of 1 (mild) to 3 (severe) for epithelial damage, edema, and neutrophil infiltration as previously described (8, 36).
Statistics. Results are reported as means ± SE. Data were compared using ANOVA and multiple comparison testing with the Student-Newman-Keuls t-test or Student's t-test with P < 0.05 considered to be significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of NEP /
mice.
To confirm the absence of NEP in NEP
/
mice, we examined the
expression of NEP protein by immunofluorescence and of NEP activity
using an enzymatic assay. As previously reported (5), immunoreactive NEP was most abundant in the brush border of the renal
proximal tubule and the small intestine in NEP +/+ mice (Fig.
1A). NEP was also detected in
other tissues, such as the muscularis externa of NEP +/+ mice (not
shown). There was no specific staining in the kidney and small
intestine of NEP
/
animals (Fig. 1A). NEP activity was
very high in extracts of the kidney and small intestine, followed by
the trachea and lungs, skin, urinary bladder, stomach, pancreas, and
colon of NEP +/+ mice (Fig. 1B). Activity was at background
levels in NEP
/
animals (Fig. 1B). These results confirm
the absence of NEP protein and enzymatic activity in NEP
/
mice.
|
Deletion of NEP exacerbates TxA-induced fluid secretion and
granulocyte infiltration.
We compared enteritis to graded doses of TxA (0.5-5 µg) in NEP
+/+ and NEP /
mice using accumulation of fluid in the intestinal lumen and MPO activity in the intestinal wall as endpoints. Basal fluid
secretion (expressed as loop weight-to-length ratio, in mg/cm) in
control loops (no TxA) was similar in NEP +/+ and NEP
/
animals
(33 ± 2 and 36 ± 2 mg/cm, respectively) (Fig.
2A). In NEP +/+ mice, TxA
induced a dose-dependent increase in secretion that was statistically
different from basal to 1 µg and maximal to 5 µg TxA (Fig.
2A). In NEP
/
mice, the secretory response was more
robust for all doses of TxA. A significant increase in secretion above
basal was observed to 0.5 µg TxA. Compared with NEP +/+ mice,
secretion in NEP
/
animals was increased by ~200% at 0.5 µg
and 135% at 5 µg TxA (Fig. 2A). Basal granulocyte
infiltration (assessed by MPO activity) in control loops was similar in
NEP +/+ and NEP
/
animals (3.6 ± 2 and 1 ± 0.2 U/g,
respectively) (Fig. 2B). Compared with NEP +/+ mice, MPO
activity in NEP
/
animals was increased by ~270% at 0.5 µg and
~150% at 1 µg TxA (Fig. 2B). However, MPO activity was
the same in both groups at 2 µg TxA and was higher in NEP +/+ mice
for 5 µg TxA. Thus deletion of NEP reduced the threshold and
increased the magnitude of TxA-induced fluid accumulation and
granulocyte infiltration. This effect was especially pronounced when
submaximal doses of TxA were used. Therefore, we used the half-maximal
dose of TxA in NEP
/
mice (0.5 µg) for all subsequent
experiments. This dose of TxA induced reliable inflammation in NEP
/
mice but had a minor effect in wild-type animals.
|
Pretreatment with rhNEP attenuates exacerbated TxA-induced
enteritis in NEP /
mice.
To confirm that the exacerbated enteritis observed in NEP
/
mice was due to lack of NEP, we pretreated animals with rhNEP. Pretreatment with BSA or boiled, inactive rhNEP (3 mg/kg iv, controls) had no effect on basal secretion (Fig.
3A), e.g., basal secretion was
33 ± 7 mg/cm (n = 3) with boiled rhNEP and
36 ± 2 mg/cm (n = 23) with saline. When rhNEP (3 mg/kg iv) was administered, basal secretion was 54 ± 4 mg/cm
(n = 7). Pretreatment with BSA or boiled rhNEP had no
effect on TxA-induced intestinal secretion (Fig. 3A). In
marked contrast, pretreatment with rhNEP diminished TxA-induced intestinal secretion. In BSA-treated mice TxA stimulated secretion by
300%, whereas in NEP-treated mice TxA stimulated secretion by only
120% over basal (Fig. 3A). This reduction was observed despite the increase in basal fluid secretion in mice treated with
rhNEP. In a similar manner, MPO activity induced by TxA was not
affected by pretreatment with BSA, but was markedly inhibited by
pretreatment with rhNEP (Fig. 3B). Thus administration of
rhNEP reduces the exacerbated fluid secretion and granulocyte
infiltration observed in NEP
/
mice, confirming the role of NEP in
terminating TxA-induced enteritis.
|
Pretreatment with NEP inhibitor phosphoramidon exacerbates
TxA-induced enteritis in NEP +/+ mice.
NEP inhibitors are under development as therapeutic agents, and
therefore it is important to understand their proinflammatory effects.
Because NEP deletion exacerbated TxA-induced enteritis, we reasoned
that NEP inhibition would have a similar effect in NEP +/+ mice.
Pretreatment of NEP +/+ mice with phosphoramidon (3 mg/kg iv) increased
TxA-induced secretion by 180% and MPO activity by 260% compared with
TxA-treated control animals (Fig. 4).
Phosphoramidon had no effect on basal secretion or MPO activity in
loops filled with buffer. Thus inhibition of NEP exacerbated
TxA-induced enteritis.
|
Deletion and inhibition of NEP exacerbates TxA-induced histological
damage of intestinal mucosa.
Tissue sections were scored for epithelial damage, edema, and
neutrophil infiltration. In NEP +/+ mice, TxA induced a dose-dependent increase in histological damage of the mucosa (results not shown). Whereas 0.5 µg TxA did not induce microscopically detectable
epithelial damage in NEP +/+ mice, there was noticeable epithelial
damage in NEP /
mice (Table 1).
Similarly, TxA-induced edema and neutrophil infiltration were markedly
higher in NEP
/
than in NEP +/+ mice. The total histological score
in NEP
/
mice was approximately twofold greater than in NEP +/+
mice. Whereas pretreatment of NEP
/
mice with rhNEP decreased the
microscopic damage to that of NEP +/+ animals, pretreatment of NEP +/+
mice with phosphoramidon exacerbated the damage to near NEP
/
levels (Table 1).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results show that TxA-induced enteritis is dramatically
enhanced in mice lacking NEP and in wild-type animals treated with NEP
inhibitors. To our knowledge, this is the first study to directly
demonstrate the importance of NEP in the pathophysiology of intestinal
secretion and inflammation in response to a bacterial toxin. Because SP
is a principal mediator of TxA-induced enteritis (8, 36)
and it is the most favorable substrate for NEP (28, 29),
the exacerbated response in NEP-deficient animals is likely due to
diminished degradation of SP. Thus degradation of SP by NEP serves to
restrict the proinflammatory effects of SP and to terminate TxA-induced
enteritis. The observation that NEP deficiency exacerbates inflammation
may be of importance because NEP is downregulated in the inflamed
intestine (15), which may contribute to the inflammatory
response. The finding that rhNEP prevents inflammation in NEP /
mice suggests that rhNEP could be considered as a novel form of
treatment for SP-mediated inflammation in the gut. In contrast, the
marked proinflammatory effect caused by NEP inhibitors suggests that
these drugs may have important side effects.
NEP terminates neurogenic inflammation.
Our results show that genetic deletion or pharmacological inhibition of
NEP exacerbates TxA-induced enteritis and support an important role for
neuropeptides such as SP as proinflammatory agents in the intestine
(32, 34). In view of the major role of SP in TxA-induced
enteritis and the finding that SP is one of the most kinetically
favorable NEP substrates (assessed by turnover rate) (28,
29), it is highly likely that disruption of NEP exacerbates
inflammation due to the diminished degradation of SP. In support of our
results, deletion of NEP or administration of NEP inhibitors results in
spontaneous plasma extravasation in multiple tissues, including the
ileum (23), and exacerbates trinitrobenzene sulfonic
acid-induced colitis (41) and allergic dermatitis by
SP/NK1R-dependent mechanisms (39). NEP inhibitors also
magnify other effects of SP in many systems, including the gastrointestinal tract, airway, and skin (10, 16, 25, 42). Surprisingly, MPO activity was higher in NEP +/+ than NEP /
mice
receiving high doses of TxA. This result suggests that NEP may play a
protective role against excessive granulocyte infiltration and raises
the possibility that peptides that are NEP substrates may have both
proinflammatory and anti-inflammatory effects.
Importance of NEP in regulating inflammation. The observation that NEP terminates SP-mediated enteritis raises the possibility that diminished expression of NEP may exacerbate inflammation. NEP activity is diminished by 84-fold in the mucosa and circular muscle and by 12-fold in the longitudinal muscle layer in the inflamed intestine of rats infected with Trichinella spiralis, which results in a 2- to 6-fold reduction in the rate of SP degradation (15). These findings, together with the present observation that disruption of NEP exacerbated enteritis, suggest that downregulation of NEP contributes to intestinal inflammation in experimental animals. It remains to be determined whether downregulation of NEP contributes to inflammation in the human intestine.
Administration of rhNEP markedly suppressed the exaggerated inflammatory and secretory responses to TxA in NEP ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Donald Payan (Rigel Pharmaceuticals) for recombinant NEP.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52388 (E. F. Grady), DK-46285 (K. S. Kirkwood), DK-39957 (N. W. Bunnett), and DK-47343 (C. Pothoulakis) and the Crohn's and Colitis Foundation (N. W. Bunnett and I. Castagliolo).
Address for reprint requests and other correspondence: E. F. Grady, Dept. of Surgery, Univ. of California, Box 0660, 521 Parnassus Ave., San Francisco, CA 94143-0660 (E-mail: gradye{at}surgery.ucsf.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 January 2001; accepted in final form 16 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barak, LS,
Warabi K,
Feng X,
Caron MG,
and
Kwatra MM.
Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and beta-arrestin 2 during homologous desensitization of the substance P receptor.
J Biol Chem
274:
7565-7569,
1999
2.
Brain, SD,
and
Williams TJ.
Interactions between the tachykinins and calcitonin gene-related peptide lead to the modulation of oedema formation and blood flow in rat skin.
Br J Pharmacol
97:
77-82,
1989[Abstract].
3.
Brain, SD,
Williams TJ,
Tippins JR,
Morris HR,
and
MacIntyre I.
Calcitonin gene-related peptide is a potent vasodilator.
Nature
313:
54-56,
1985[ISI][Medline].
4.
Bunn, PA, Jr,
Helfrich BA,
Brenner DG,
Chan DC,
Dykes DJ,
Cohen AJ,
and
Miller YE.
Effects of recombinant neutral endopeptidase (EC 3.42411) on the growth of lung cancer cell lines in vitro and in vivo.
Clin Cancer Res
4:
2849-2858,
1998[Abstract].
5.
Bunnett, NW,
Wu V,
Sternini C,
Klinger J,
Shimomaya E,
Payan D,
Kobayashi R,
and
Walsh JH.
Distribution and abundance of neutral endopeptidase (EC 3.42411) in the alimentary tract of the rat.
Am J Physiol Gastrointest Liver Physiol
264:
G497-G508,
1993
6.
Castagliuolo, I,
Keates AC,
Qiu B,
Kelly CP,
Nikulasson S,
Leeman SE,
and
Pothoulakis C.
Increased substance P responses in dorsal root ganglia and intestinal macrophages during Clostridium difficile toxin A enteritis in rats.
Proc Natl Acad Sci USA
94:
4788-4793,
1997
7.
Castagliuolo, I,
LaMont JT,
Letourneau R,
Kelly C,
O'Keane JC,
Jaffer A,
Theoharides TC,
and
Pothoulakis C.
Neuronal involvement in the intestinal effects of Clostridium difficile toxin A and Vibrio cholerae enterotoxin in rat ileum.
Gastroenterology
107:
657-665,
1994[ISI][Medline].
8.
Castagliuolo, I,
Riegler M,
Pasha A,
Nikulasson S,
Lu B,
Gerard C,
Gerard NP,
and
Pothoulakis C.
Neurokinin-1 (NK-1) receptor is required in Clostridium difficile-induced enteritis.
J Clin Invest
101:
1547-1550,
1998
9.
Conner, DA,
Mathier MA,
Mortensen RM,
Christe M,
Vatner SF,
Seidman CE,
and
Seidman JG.
-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation.
Circ Res
81:
1021-1026,
1997
10.
Djokic, TD,
Sekizawa K,
Borson DB,
and
Nadel JA.
Neutral endopeptidase inhibitors potentiate substance P-induced contraction in gut smooth muscle.
Am J Physiol Gastrointest Liver Physiol
256:
G39-G43,
1989
11.
Emanueli, C,
Grady EF,
Madeddu P,
Figini M,
Bunnett NW,
Parisi D,
Regoli D,
and
Geppetti P.
Acute ACE inhibition causes plasma extravasation in mice that is mediated by bradykinin and substance P.
Hypertension
31:
1299-1304,
1998
12.
Farina, NK,
Johnston CI,
and
Burrell LM.
Reversal of cardiac hypertrophy and fibrosis by S21402, a dual inhibitor of neutral endopeptidase and angiotensin converting enzyme in SHRs.
J Hypertens
18:
749-755,
2000[ISI][Medline].
13.
Figini, M,
Emanueli C,
Grady EF,
Kirkwood K,
Payan DG,
Ansel J,
Gerard C,
Geppetti P,
and
Bunnett NW.
Substance P and bradykinin stimulate plasma extravasation in the mouse gastrointestinal tract and pancreas.
Am J Physiol Gastrointest Liver Physiol
272:
G785-G793,
1997
14.
Francis, GS.
Is there still a future for neutral endopeptidase inhibitors?
Am Heart J
138:
1007-1008,
1999[ISI][Medline].
15.
Hwang, L,
Leichter R,
Okamoto A,
Payan D,
Collins SM,
and
Bunnett NW.
Downregulation of neutral endopeptidase (EC 3.42411) in the inflamed rat intestine.
Am J Physiol Gastrointest Liver Physiol
264:
G735-G743,
1993
16.
Iwamoto, I,
and
Nadel JA.
Tachykinin receptor subtype that mediates the increase in vascular permeability in guinea pig skin.
Life Sci
44:
1089-1095,
1989[ISI][Medline].
17.
Katayama, M,
Nadel JA,
Bunnett NW,
Di Maria GU,
Haxhiu M,
and
Borson DB.
Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase.
Peptides
12:
563-567,
1991[ISI][Medline].
18.
Keates, AC,
Castagliuolo I,
Qiu B,
Nikulasson S,
Sengupta A,
and
Pothoulakis C.
CGRP upregulation in dorsal root ganglia and ileal mucosa during Clostridium difficile toxin A-induced enteritis.
Am J Physiol Gastrointest Liver Physiol
274:
G196-G202,
1998
19.
Kelly, CP,
Becker S,
Linevsky JK,
Joshi MA,
O'Keane JC,
Dickey BF,
LaMont JT,
and
Pothoulakis C.
Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.
J Clin Invest
93:
1257-1265,
1994[ISI][Medline].
20.
Kelly, CP,
Pothoulakis C,
and
LaMont JT.
Clostridium difficile colitis.
N Engl J Med
330:
257-262,
1994
21.
Koch, WJ,
Rockman HA,
Samama P,
Hamilton RA,
Bond RA,
Milano CA,
and
Lefkowitz RJ.
Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor.
Science
268:
1350-1353,
1995[ISI][Medline].
22.
Kwatra, MM,
Schwinn DA,
Schreurs J,
Blank JL,
Kim CM,
Benovic JL,
Krause JE,
Caron MG,
and
Lefkowitz RJ.
The substance P receptor, which couples to Gq/11, is a substrate of -adrenergic receptor kinase 1 and 2.
J Biol Chem
268:
9161-9164,
1993
23.
Lu, B,
Figini M,
Emanueli C,
Gepetti P,
Grady EF,
Gerard NP,
Ansel JC,
Payan DG,
Gerard C,
and
Bunnett NW.
The control of microvascular permeability and blood pressure by neutral endopeptidase.
Nat Med
3:
904-907,
1997[ISI][Medline].
24.
Lu, B,
Gerard NP,
Kolakowski LF,
Bozza M,
Zurakowski D,
Finco O,
Carroll MC,
and
Gerard C.
Neutral endopeptidase modulation of septic shock.
J Exp Med
181:
2271-2275,
1995[Abstract].
25.
Maa, J,
Grady EF,
Kim EH,
Yoshimi SK,
Hutter MM,
Bunnett NW,
and
Kirkwood KS.
NK-1 receptor desensitization and neutral endopeptidase terminate SP-induced pancreatic plasma extravasation.
Am J Physiol Gastrointest Liver Physiol
279:
G726-G732,
2000
26.
Mantyh, CR,
McVey DC,
and
Vigna SR.
Extrinsic surgical denervation inhibits Clostridium difficile toxin A-induced enteritis in rats.
Neurosci Lett
292:
95-98,
2000[ISI][Medline].
27.
Mantyh, CR,
Pappas TN,
Lapp JA,
Washington MK,
Neville LM,
Ghilardi JR,
Rogers SD,
Mantyh PW,
and
Vigna SR.
Substance P activation of enteric neurons in response to intraluminal Clostridium difficile toxin A in the rat ileum.
Gastroenterology
111:
1272-1280,
1996[ISI][Medline].
28.
Matsas, R,
Fulcher IS,
Kenny AJ,
and
Turner AJ.
Substance P and [Leu]enkephalin are hydrolyzed by an enzyme in pig caudate synaptic membranes that is identical with the endopeptidase of kidney microvilli.
Proc Natl Acad Sci USA
80:
3111-3115,
1983[Abstract].
29.
Matsas, R,
Kenny AJ,
and
Turner AJ.
The metabolism of neuropeptides. Hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-2411.
Biochem J
233:
433-440,
1984.
30.
McConalogue, K,
Corvera CU,
Gamp PD,
Grady EF,
and
Bunnett NW.
Desensitization of the neurokinin-1 receptor (NK1-R) in neurons: effects of substance P on the distribution of NK1-R, Galphaq/11, G-protein receptor kinase-2/3, and beta-arrestin-1/2.
Mol Biol Cell
9:
2305-2324,
1998
31.
McConalogue, K,
Dery O,
Lovett M,
Wong H,
Walsh JH,
Grady EF,
and
Bunnett NW.
Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor.
J Biol Chem
274:
16257-16268,
1999
32.
McDonald, DM,
Bowden JJ,
Baluk P,
and
Bunnett Neurogenic inflammation NW
A model for studying efferent actions of sensory nerves.
Adv Exp Med Biol
410:
453-462,
1996[Medline].
33.
Okamoto, A,
Lovett M,
Payan DG,
and
Bunnett NW.
Interactions between neutral endopeptidase (EC 3.42411) and the substance P (NK1) receptor expressed in mammalian cells.
Biochem J
299:
683-693,
1994[ISI][Medline].
34.
Otsuka, M,
and
Yoshioka K.
Neurotransmitter functions of mammalian tachykinins.
Physiol Rev
73:
229-308,
1993
35.
Pothoulakis, C,
Castagliuolo I,
and
LaMont JT.
Neurons and mast cells modulate secretory and inflammatory responses to enterotoxins.
News Physiol Sci
13:
58-63,
1998
36.
Pothoulakis, C,
Castagliuolo I,
LaMont JT,
Jaffer A,
O'Keane JC,
Snider RM,
and
Leeman SE.
CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.
Proc Natl Acad Sci USA
91:
947-951,
1994[Abstract].
37.
Pothoulakis, C,
LaMont JT,
Eglow R,
Gao N,
Rubins JB,
Theoharides TC,
and
Dickey BF.
Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.
J Clin Invest
88:
119-125,
1991[ISI][Medline].
38.
Roush, ED,
Warabi K,
and
Kwatra MM.
Characterization of differences between rapid agonist-dependent phosphorylation and phorbol ester-mediated phosphorylation of human substance P receptor in intact cells.
Mol Pharmacol
55:
855-862,
1999
39.
Scholzen, TE,
Steinhoff M,
Bonaccorsi P,
Klein R,
Amadesi S,
Geppetti P,
Lu B,
Gerard NP,
Olerud JE,
Luger TA,
Bunnett NW,
Grady EF,
Armstrong CA,
and
Ansel JC
Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis.
J Immunol
166:
1285-1291,
2001
40.
Stuhr, LE.
Effects of SCH 42495, a neutral endopeptidase inhibitor, on cardiac function and mass in rats after repeated hyperbaric exposures.
Scand J Clin Lab Invest
60:
141-148,
2000[ISI][Medline].
41.
Sturiale, S,
Barbara G,
Qiu B,
Figini M,
Geppetti P,
Gerard N,
Gerard C,
Grady EF,
Bunnett NW,
and
Collins SM.
Neutral endopeptidase (EC 3.42411) terminates colitis by degrading substance P.
Proc Natl Acad Sci USA
96:
11653-11658,
1999
42.
Umeno, E,
Nadel JA,
Huang H-T,
and
McDonald DM.
Inhibition of neutral endopeptidase potentiates neurogenic inflammation in the rat trachea.
J Appl Physiol
66:
2647-2652,
1988
43.
Vigna, SR.
Phosphorylation and desensitization of neurokinin-1 receptor expressed in epithelial cells.
J Neurochem
73:
1925-1932,
1999[ISI][Medline].