Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases

Peptides, Vol. 6, pp. 277-283, 1985. ©Ankho International Inc. Printed in the U.S.A.

0196-9781/85$3.00 + .00

Catabolism of Gastrin Releasing Peptide and Substance P by Gastric Membrane-Bound Peptidases N. W. B U N N E T T , * R. K O B A Y A S H I , ~ M. S. O R L O F F , ~ J. R. R E E V E , ~ A. J. T U R N E R t 1 A N D J. H. W A L S H ~

*Department of Animal Physiology and Nutrition and tMRC Membrane Peptidase Research Group Department o f Biochemistry, The University o f Leeds, Leeds LS2 9JT, U.K. ~Center for Ulcer Research and Education, Bldg. 115, VA Hospital Wadsworth, Los Angeles, CA 90073 R e c e i v e d 9 J a n u a r y 1985 BUNNETT, N. W., R. KOBAYASHI, M. S. ORLOFF, J. R. REEVE, A. J. TURNER AND J. H. WALSH. Catabolism of gastrin releasingpeptide and substanceP by gastricmembrane-boundpeptidases. PEPTIDES 6(2) 277-283, 1985.--The catabolism of two gastric neuropeptides, the C-terminal decapeptide of gastrin releasing peptide-27 (GRP10) and substance P (SP), by membrane-bound peptidases of the porcine gastric corpus and by porcine endopeptid-~e~24.II ("enkephalinas¢") has been investigated. GRPI0 was catabolized by gastric muscle peptidases (specific activity 1.8 nmol min-~ mg-1 protein) by hydrolysis of the HisS-Leu9 bond and catabolism was inhibited by pbosphoramidon (150approx. 10-s M), a specific inhibitor of endopel~.idas¢-24.11. The same bond in GRPI0 was cleaved by purified endopeptidase-24.11, and hydrolysis was equally sensitive to inhibition by phospboramidon. SP was catabolized by gastric muscle peptidases (specific activity 1.7 nmol min -1 mg-~ protein) by hydrolysis of the GIns-PhC, Pher-Phe a and Glya-Leu ~° bonds, which is identical to the cleavage of SP by purified endopeptidase-24.11. The C-terminal cleavage of GRP10 and SP would inactivate the peptides. It is concluded that a membrane-bound peptidase in the stomach wall catabolizes and inactivates GRPI0 and SP and that, in its specificity and sensitivity to pbospboramidon, this peptidase resembles endopeptidase-24.11. Gastrin releasing peptide Substance P Phosphoramidon Captopril

Endopeptidase-24.11

GASTRIN releasing peptide (GRP) and substance P (SP) are neuropeptides of the brain and alimentary tract, GRP was first isolated from non-antral gastric tissue as a 27 amino acid peptide [ 13]. Three molecular variants of GRP have recently been isolated from the canine small intestine [17] and the amino acid sequence of the smallest form (GRPI0), which is common to all forms of GRP and represents the C-terminal (18-27) sequence of GRP'27, is: Glyl-Asl:-His3-Trp4-AlaS-ValSGlyr-HisS-Leua-Met1°-NHz. SP was originally identified in crude extracts of canine brain and intestine as a stimulant of smooth muscle contraction [22,23] and has since been isolated from the brain [3] and intestine [20].as a molecule of 11 amino acfd residues with the sequence: Afg~-Pro2-Lysa-Pro4GlnS-GlnsLpbeT-PheS-Gly°-Leul°-Met~-NH2. SP and GRP have the identical C-terminal dipeptide sequence, -Leu-Met-NH2. By use of immunocytochemistry, GRP and SP have been localized to nerve fibers and cell bodies of the myenteric and submucosal nerve plexuses, to the muscle and to the mucosa of the stomach wall [4, 14, 15] where they are regarded as putative neurotransmitters. As such they may be inactivated locally by membrane-bound peptidases, Previously, neuropeptide catabolism has been studied in the brain using ~Requests for reprints should be addressed to A. J. Turner.

277

Angiotensin converting enzyme

preparations of synaptosomes and synaptic membranes [11,12], but the catabolism of neuropeptides in the alimentary tract has been a neglected area. Several membrane-bound peptidases have been isolated from n e m ~ and non-neural tissues that catabofize SP. These include: (I) endope~idase-24.11 ("enkephalinase," EC 3.4.24. I1) which hydrolyzes the Gin sPhC, PheLPhe s and Glyg-Leu~° bonds of SP [11,18] and is competitively inhibited by phosphormnidon (Iso--8 riM) [6]; (2) ang/otensin converting enzyme (ACE; peptidyl dipeptidase A, EC 3.4.15,1 ) which hydrolyzes the PheS-Gly9bond of SP [ 19,25] and is competitively inhibRed by captopril [1] and MK422 (enalapr/1 diacid) [7]; and (3) dipeptidyl peptidase IV, (EC 3.4.14.5) which hydrolyzes the Pro2-Lyss and Pro4-Glns bonds [9] and is irreversibly inhibited by di-isopropylfluorophosphate (DFP) [10]. The catabolism o f GRP has not been studied previously. The present investigation compares the catabolism of SP and GRPI0 by memlnane-bound peptidases prepared from the stomach wall of the pig and attempts to identify the enzymes involved by use of selective inhibitors. It is concluded that an enzyme resemblin~ endopeptidase24.11 may play a major role in the c a m ~ l i s m of both GRP and SP in the pig stomach.

B U N N E T T ET AL.

278 TABLE 1

GRP/Pig muscle

6

THE SUSCEPTIBILITY TO INHIBITORS OF GRP10 AND SP CATABOLISM BY MEMBRANE-BOUND PEPTIDASES OF PIG GASTRIC MUSCLE

Inhibitor

Specificity

Inhibition (%) GRP SP

Phosphoramidon (10-5 M) Endopeptidase-24.11

93

85

Captopril (10-5 M) MK 422 (10-5 M)

Angiotensin converting enzyme

<5 <5

<5 13

1-10 phenanthroline (10-3 M) EDTA (10-3 M)

Metallo-enzymes

100

100

84

50

DFP (10-3 M) PMSF (10-3 M)

Serine proteases, e.g., dipeptidyl peptidase IV

29 --

9 15

PCMB (10-3 M)

Thiol peptidases

11

<5

Pepstatin (10-6 M) Leupeptin (10-5 M)

Acid proteases

16 24

<5 <5

Bestatin (10-4 M)

Aminopeptidases

<5

<5

1

.

The results are the mean of duplicate observations. Substrate concentration, 2.5 x 10-5 M. EDTA, ethylenediaminetetra-acetate; PMSF, phenylmethylsulphonylfluoride; DFP, diisopropylfluorophosphate; PCMB, pchioromercuribenzoate.

METHOD

Materials SP was purchased from Sigma Chemical Co., St. Louis, MO. GRP10 was synthesized by B. Clark, Beckman Research Institute, City of Hope, Duarte, CA 91010. Phosphoramidon was purchased from Sigma Chemical Co., London. Captopril (SQ 14225, D-3-mercapto-2-methylpropanoyl-L-Pro) was a gift from the Squibb Institute for Medical Research, Princeton, NJ, and MK422 (N-[(S)-l-carboxy-3-phenylpropyl]-L-Ala-L-Pro) was a gift from Dr. L. L. Iversen, Merck, Sharpe and Dohme Research Laboratories, Hoddesdon, Hertfordshire, U.K. The other enzyme inhibitors were purchased from Sigma Chemical Co., St. Louis, MO.

Preparation of a Membrane Fraction Membrane fractions were prepared by a previously described technique [21]. The gastric corpus was removed from pigs within 30 min of death. The muscle was separated from the mucosa and submucosa and tissues were homogenized separately in Tris/HCl buffer (10 mM, pH 7.5, 4°C). The homogenate was first centrifuged at low speed (1,000 g, 5 min) and the sediment of unbroken cells and cell debris was discarded. The supernatant was re-centrifuged (100,000 g, 45 min). The membrane pellet was washed and recentrifuged once in Tris/HC1 buffer (10 mM, pH 7.5) and then twice with the Tris/HC1 containing 0.5 M NaC1. After centrifugation the final pellet was

I

0

5

I

10 TIME(min)

I

20

FIG. 1. HPLC elution profile of GRP10 incubated with membranebound peptidases of the porcine gastric corpus muscle. Conditions: GRP10, 1.25x 10-4 M; membranes, 50/zg protein; Tris/HCl buffer, 20 mM pH 7.4; 37"C for 20 min. Peaks were identified by amino acid analysis. 1: not a peptide peak; 2: [9-10] GRP10; 3: [7-10] GRP10; 4: not identified; 5: [1-8] GRP10; 6: intact GRPI0. Gradient: 0--40% acetonitrile, 20 min.

resuspended in Tris/HCl buffer (10 mM, pH 7.5) to a protein concentration of 5-10 mg.ml -~ as measured by the method of Petersen [ 16] using bovine serum albumin as standard. Aliquots of this preparation were stored at - 7 & C .

Endopeptidase-24.11 Endopeptidase-24.11 was purified to homogeneity from porcine kidney by immunoadsorbent chromatography using a monoclonal antibody as described elsewhere [6,18].

Peptide-Degradation Studies GRP10 or SP (5× 10-5 to 1.25× 10-4 M) were incubated in Tris/HC1 buffer (20 mM, pH 7.5,200/zl total volume) with 50 or 100/zg protein of the membrane preparation or with 2 p.g of pure endopeptidase-24.11 at 37°C. The reactions were stopped by boiling for 5 min, or by acidification with 1 volume of 1% trifluoroacetic acid (TFA), followed by centrifugation. The supernatant was stored at - 7 & C until analysis. The nature of the membrane-bound peptidases was examined by using selective inhibitors (Table 1). Competitive inhibitors were added to the mixture just before addition of the enzyme. The irreversible inhibitors were incubated with the enzyme preparation for 10 min before the addition of substrate.

Chromatography The products of hydrolysis were separated by reverse-phase

GASTRIC PEPTIDASES

279

100~

8O

"O'GRPI-lO

A

E E - 60

100

"e- GRPI-a

.oi\

.o

-m- (b)

60

60

(a)

qmm~

GRP/E-24.11

GRP/Pig muscle

GRP/Pig muscle

ol

*m

-= 4or

o n-

•~ 4 0

k

40

20

0

20

n_

0 L. . . . . . .

20

9 8 7 6 5 4

0

.--

•

*

*

•

•

|

9 8 7 6 5 4

-k:,jmpa~,q~Umrmmknon(M) 0

-

"

~

0

1"

10

I

20 30 Time(min)

I

40

FIG. 2. The time course of GRPI0 breakdown by membrane-bound peptidases of the porcine gastric corpus muscle. The results are mean values of duplicate observations. (a) and (b) represent unidentified products. Conditions: GRP10, 2.5x 10-5 M; membrane preparation, 50/~g protein; Tris/HCl buffer, 20 mM pH 7.4; 37"C.

high pressure liquid chromatography (HPLC) using a Hypersil or Vydac C-18 column (HPLC Technology, Cheshire, UK, 5 /~m, 25 cm x 0.4 cm) equilibrated in 0.1% TFA. The bound peptides were eluted using a linear gradient of acetonitrile and a flow rate of 1.5 ml.min -1. The absorbance of the eluant was monitored at 214 nm.

Amino Acid Analysis The peptides separated by HPLC were initially identified by amino acid analysis. Samples were dried and then hydrolyzed under vacuum in constant boiling HC1 for 24 hr at 110*C. The hydrolysate was analyzed on a Beckman 6300 analyzer with a Hewlett Packard 3390A integrator. RESULTS

Membrane Yields The yields of membranes prepared from gastric muscle and mucosa from pigs were 3.2 and 9.6 mg of protein, g-i wet tissue, respectively. Consistently greater yields were obtained from the mucosa.

GRP Catabolism Muscle. The principal products of GRP10 hydrolysis by membranes from the muscle of the pig gastric corpus were consistent with cleavage of the HisS-Leu9 bond and were [1-8] GRPI0 and [9--10] GRP10 (Fig. 1). Other products, such as

FIG. 3. The susceptibility of GRP10 catabolism by porcine gastric membranes and by porcine endopeptidase-24.11 to inhibition by phosphoramidon. In both studies the Isovalues were similar (approx. 10-a M). Conditions: GRP10, 5x 10-4 M; enzyme, 2 p.g; membrane preparation, 50/~g protein; Tris/HC1 buffer, 20 mM pH 7.4; 37°C for 30 min.

[1-6] GRPI0 and [%10] GRP10, formed after prolonged incubation (data not shown) but [1-8] GRP10 appeared to be rather stable. The time course of GRP10 catabolism by membrane preparations is shown in Fig. 2. The specific activity of GRP10 breakdown was calculated to be 1.8 nmol.min-lmg -~ protein under the experimental conditions. The susceptibility of GRP10 catabolism to a range of inhibitors is summarized in Table 1. Of the competitive inhibitors tested only phosphoramidon (10-5 M) strongly inhibited GRP breakdown and an I~0 value of approx. 10- s M was estimated (Fig. 3). However, even at higher concentrations of phosphoramidon (10-4 M) the inhibition was not complete. A residual phosphoramidoninsensitive activity (< 10% of the total)was observed, suggesting that other peptidases may be involved in the catabolism of GRP. At low substrate concentrations (2.5 x 10-5 M) ACE inhibitors were without effect. At higher substrate concentrations (5 x 10-4 M GRP10), the ACE inhibitors captopril (10-5 M) and MK422 (10-5 M) produced some inhibition of GRP hydrolysis (18% and 16% respectively). These f'mdings suggest that ACE may have the potential to hydrolyze GRP at high concentrations of substrate. Of the other inhibitors tested, EDTA ( 10-3 M) and 1,10-phenanthroline (10-3 M) both blocked GRP catabolism, implicating a metailo-enzyme. DFP, an inhibitor of serine proteases, inhibited GRP breakdown by 29%. Mucosa. The same catabolic products from the digestion of GRP10 by muscle peptidases were also produced by membrane preparations from gastric mucosa. Endopeptidase-24.11. GRP10 was hydrolyzed by endo-

280

B U N N E T T ET AL. GRP/E-24.11

GRP/E-24.11/phosphoramidon

GRPIO

GRPIO

1-8

E c

~,,

r, 0

z
,<

!

0

10

I

TIMElmin) 0

!

10

FIG. 4. HPLC elution profile of GRPI0 incubated with homogeneous porcine endopeptidase-24.11 in the absence and presence of phosphoramidon, (10-8 M) conditions: GRPI0, 5× 10-4 M; enzyme, 2/zg; Tris/HCl buffer (20 mM, pH 7.4); 37°C for 30 min. Peaks were identified by amino acid analysis. Gradient: 4.8--50% acetonitrile, 20 min.

peptidase-24.11 by cleavage of the HisS-Leu 9 bond to form [1--8] GRP10 and [9-10] GRP10 (Fig. 4). With prolonged incubation additional products were formed which may represent hydrolysis at the His3-Trp4 bond, but insufficient amounts were obtained for amino acid analysis. Hydrolysis was completely inhibited by phosphoramidon (10 -9 M) and the I50 value for inhibition by phosphoramidon was 7 × 10-9 M, comparable to that obtained for the gastric muscle membrane preparation (Fig. 3). SP Catabolism Muscle. The principal products of SP hydrolysis by membrane preparations from the muscle of the pig gastric corpus were consistent with cleavage of the Glne-Phe7, Pher.Phe s and Glyg-Leu1° bonds and were [1-6] SP, [1-7] SP, [1-9] SP, [7-11] SP, [8-11] SP and [10-11] SP (Fig. 5). The time course of SP catabolism by membrane preparations is shown in Fig. 6. The specific activity of SP breakdown under the experimental conditions was calculated to be 1.7 nmol.min-X.mg -1 protein. The susceptibility of SP catabolism to various enzymic inhibitors is summarized in Table 1. The response to inhibitors was similar for the catabolism of GRP10 and SP. Of the competitive inhibitors, phosphoramidon alone (10-s M) strongly inhibited SP breakdown with an 15ovalue of approx. 10-2 M. At low concentrations of substrate (2.5 x 10-s M SP) captopril (10-5 M) and MK422 (10-5 M) were without effect. However, when the substrate concentration was increased 10-fold (5× 10-4 M SP)

captopril (10-5 M) and MK422 (10-s M) inhibited the catabolism of SP by 22% and 24% respectively. Of the other inhibitors, EDTA (10-a M) and 1,10-phenanthroline (10-2 M) strongly inhibited SP catabolism, implicating a metaUo-enzyme. DFP (10-a M) and PMSF (10 -2 M) were less potent, suggesting some role for dipeptidyl peptidase IV. The other inhibitors used had no detectable effect (Table 1). Mucosa. SP was catabolized by mucosal membranes to the same products identified after digestion by the muscle enzymes. In contrast to the muscle, SP catabolism was pepstatin-sensitive (1 ~M) suggesting contamination of the mucosal membranes with digestive peptidases such as pepsin. Indeed, pepsin efficiently degraded SP to produce the same catabolites as endopeptidase-24.11, and this was completely inhibited by pepstatin (10-6 M). GRP was resistant to pepsin. Pepstatin had no inhibitory effect on the muscle membrane preparation. Endopeptidase-24.11. SP was catabolized by endopeptidase-24.11 by cleavage of the Glna-Phe r, Phe7-Phe a and and Glyg-Leu 1° bonds, as previously described [ 11,18]. Degradation was inhibited completely by phosphoramidon (10 ttM) and the I5o value for inhibition by phosphoramidon was 6x 10-9 M, comparable to that of the muscle membranes. DISCUSSION Previously, the catabolism of neuropeptides by membrane-bound peptidases has been examined in the cen-

GASTRIC PEPTIDASES

_

281

Substance P/Pig muscle

Substance P/Pig muscle

8 100

"0" SP1-11 •0" S P l - r

E C

-i- SP1-6

8O

"D" S P l - e "~" SPa-11

.f=

U.I

(J z < m nO (t) m

im

e

3:60 (W q) n_

1

A


23

0

4O 5

10

I

20

TIME(Din) FIG. 5. HPLC elution profile of SP incubated with membrane-bound peptidases of the porcine gastric corpus muscle. Conditions: SP, 25x 10-5 M; membranes, 50 p.g; Tris/HCl buffer, 20 mM pH 7.4; 37°C for 20 rain. Peaks were identified by amino acid analysis. 1: not a peptide peak; 2: [1-6] SP; 3: [1-4] SP + phe-like; 4: [8-11] SP; 5: [1-7] SP; 6: [1-9] SP; 7: unidentified; 8: intact SP. Gradient: 0-40% acetonitrile, 20 min.

tral nervous system using preparations o f synaptosomes or synaptic membranes [11,12]. In the present investigation the catabolism of two neuropeptides, GRP and SP, by membrane-bound peptidases prepared from the muscle and mucosa of the gastric corpus has been examined. The results show that gastric membrane peptidases hydrolyze GRP10 by cleavage of Hisa-Leu 9 bond (and the Vale-Gly7 bond after prolonged incubation) and hydrolyze SP by cleavage of the Glne-Phe r, PheLPhe 8 and Glya-Leu 1° bonds and suggest that an enzyme resembling endopeptidase-24.11 is responsible for these cleavages (Fig. 7). The evidence for this is that: (1) the elution profiles of the peptides resolved by HPLC after hydrolysis of GRP and SP by gastric membrane-bound peptidases were similar to those observed after hydrolysis with purified endopeptidase-24.11; (2) phosphoramidon, a selective inhibitor of endopeptidase-24.11, inhibited the catabolism of both peptides by the gastric membranes and by the purified porcine enzyme with a potency which was similar in all cases; and (3) the sensitivity of the gastric peptidases to the metal chelators EDTA and 1,10-phenanthroline resembles that of the purified enzyme [6, 11, 18]. Previously, endopeptidase-24.11 was implicated in the catabolism of enkephalin and SP by caudate synaptic membranes prepared from the pig brain because the patterns of catabolism resembled those of pure endopeptidase-24.11 and breakdown was inhibited by phosphoramidon [11]. However, positive identification of the membrane-bound peptidase in the stomach wall as endopeptidase-24.11 will require its isolation

0

0

20 Time(rain)

40

FIG. 6. The time course of SP breakdown by membrane-bound peptidases of the porcine gastric corpus muscle. The results are mean values of duplicate observations. Conditions: SP, 5 × 10-5 M; membranes, 50 /zg; Tris/HC! buffer, 20 mM pH 7.4; 37°C. Gradient: 0-40% acetonitrile, 20 min.

Endopepttdase-24.11: membrane-bound

peptldases:

0

i

1 2 3 4 5 6 7 8=9 I0 GIy-As F-HI s-Tr p-Al a-Va l-Cly-Hi s~t~eu-Me t-NH 2

GRPIO i

i

Ar g -pr o-Ly s - l ~ o - - ~ n - G ~ P h e~Ph e-G ~y~Le u-He t -NH2 , i

t I

SP

i i

FIG. 7. The proposed pathway of GRPI0 and SP catabolism by membrane-bound peptidases of the muscle of the porcine gastric corpus and by porcine kidney endopeptidase-24.11. The major cleavage sites are indicated.

282

BUNNETT ET AL.

and biochemical characterization. Indeed, an enzyme which cross-reacts with a monoclonal antibody to porcine endopeptidase-24.11 can be measured by an immunoradiometric assay for the enzyme in crude homogenates and in membrane fractions prepared from the muscle and mucosa of the porcine gastric corpus and antrum. Furthermore, the enzyme has been localized by immunocytochemistry to nerve fiber bundles of the myenteric and submucosal nerve plexuses of the pig stomach, which are known to be innervated by peptidergic nerves (Bunnett and Turner, unpublished observations). Work is in progress to ascertain the location of the peptidase in the pig stomach and to correlate the distribution of endopeptidase-24.11 in the stomach wall with the distribution o f particular neuropeptides. Captopril and MK422, specific inhibitors of ACE, partially inhibited catabolism of SP and GRP by the gastric membranes. Thus ACE may play a minor role in the catabolism of these neuropeptides in the pig stomach. These observations support the contention [19,25] that ACE can function as an endopeptidase in hydrolyzing certain C-terminally amidated peptides such as SP, and suggest that GRP may be a substrate for ACE. The catabolism of SP and GRP10 by gastric membrane peptidases was inhibited by DFP, an inhibitor of serine peptidases. In the case of SP the serine enzyme responsible may be dipeptidyl peptidase IV, which cleaves the dipeptides Arg-Pro and Lys-Pro successively from the amino-terminus of SP [9]. The lack of inhibition by pepstatin and leupeptin suggests that acid proteases are not involved in GRP or SP catabolism by membrane fractions from stomach muscle, although they may play a minor role in the catabolism of GRP10. To be of physiological importance a peptidase must inactivate neuropeptides by cleaving in the biologically active region. The biologically active region of GRP and SP is the C-terminal sequence. Testing a series of synthetic fragments

of GRP on a variety of in vitro smooth muscle systems revealed that the C-terminal nonapeptide sequence was required for full activity [2] although the octapeptide and heptapeptide retained some activity. Similar results were found for the effects of these fragments on gastrin release [5]. The minimal sequence of SP that is required for biological activity depends somewhat on the biological system under investigation but the C-terminal octapeptide [4--11] SP, is probably the minimal sequence needed for full activity in the guinea pig ileum assay, although [5--11] SP and [6-11] SP possess about 30% of full activity [8,24]. Amino-terminal fragments of SP are capable of displacing SP from receptors in the brain but their biological significance is unknown [8,24]. In the present investigation cleavage of the His-Leu bond of GRP and the Glnr-Phe 7, PheT-Phe s and GlyS-LeuTM bonds of SP by gastric membrane peptidases and by endopeptidase-24.11 are o f significance because they inactivate the peptides. Whether phosphoramidon, a specific and non-toxic inhibitor of endopeptidase-24.11 prolongs the biological effects of these peptides in vivo by preventing their biological inactivation has not yet been investigated but is one criterion which must be met if a physiological role is to be ascribed to this enzyme. In conclusion, a membrane-bound peptidase from the gastric muscle of the pig stomach catabolizes and inactivates SP and GRP which, in its sensitivity to a variety of inhibitors and in its pattern of hydrolysis, resembles endopeptidase24.11.

ACKNOWLEDGEMENTS Supported by the Medical Research Council (U.K.), NIH grants AM 17294 and AM 17328 the Veterans Administration, and a NATO Exchange Grant. We thank Mr. N. S. Gee for performing the immunoradiometric assay.

REFERENCES

1. Abrams, W. B., R. D. Davies and R. K. Ferguson. Overview: the role of angiotensin converting enzyme inhibitors in cardiovascular therapy. Fed Proc 43: 1314--1321, 1984. 2. Broccardo, M., G. Falconieri, V. Erspamer, P. Melchiorri, L. Negri and R. deCastiglioni. Relative potency of bombesin-like peptides. Br J Pharmacol 55: 221-227, 1975. 3. Chang, M. M., S. E. Leeman and H. D. Niall. Amino-acid sequence of substance P. Nature (New Biol) 233: 86--87, 1971. 4. Dockray, G. J., C. Vaillant and J. H. Walsh. The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract. Neuroscience 4: 1561-1568, 1979. 5. Erspamer, V. and P. Melchiorri. Active polypeptides of the amphibian skin and their synthetic analogues. Pure Appl Chem 35: 463--494, 1973. 6. Gee, N. S., R. Matsas and A. J. Kenny. A monoclonal antibody to endopeptidase-24.11. Its application in immunoadsorbent purification of the enzyme and immunofluorescent microscopy of kidney and intestine. Biochem J 214: 377-386, 1983. 7. Gross, D. M., C. S. Sweet, E. H. Ulm, E. P. Backlund, A. A. Morris, D. Weitz, D. L. Bohn, H. C. Wenger, T. C. Vassil and C. A. Stone. Effects of N-[(S)-l-Carboxy-3-phenylpropyl]-L-AlaL-Pro and its ethyl ester (MK421) on angiotensin converting enzyme in vitro and angiotensin pressor responses in vivo. J Pharmacol Exp Ther 216: 552-557, 1981.

8. Iversen, L. L., M. R. Hanley, B. E. B. Sandberg, C. M. Lee, R. D. Pinnock and S. P. Watson. Substance P receptors in the nervous system and possible receptor subtypes. In: Substance P in the Nervous System. Ciba Foundation Symposium 91,

edited by R. Porter and M. O'Connor. London: Pitman Books Ltd., 1982, pp. 186-205. 9. Kato, T., T. Nagatsu, K. Fukasawa, M. Harando, I. Nagatsu and S. Sakakibara. Successive cleavage of N-terminal Argl-Pro2 and Lysa-Pro4from substance P but no release of Argl-Pro2from bradykinin, by X-Pro dipeptidyl aminopeptidase. Biochim Biophys Acta 525: 417-422, 1978. 10. Macnair, R. D. C. and A. J. Kenny. Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV. Biochem J 179: 379--395, 1979. 11. Matsas, R., I. S. Fulcher, A. J. Kenny and A. J. Turner. 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. 12. McDermott, J. R., A. I. Smith, P. R. Dodd, J. A. Hardy and J. A. Edwardson. Mechanism of degradation of LH-RH and neurotensin by synaptosomal peptidases. Peptides 4: 25-30, 1983.

GASTRIC PEPTIDASES

13. McDonald, T. J., H. Jornvall, G. Nilsson, M. Vagne, M. Ghatei, S. R. Bloom and V. Mutt. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissues. Biochem Biophys Res Commun 90: 227-233, 1979. 14. Minagawa, H., S. Shiosaka, S. Inoue, H. Hayashi, A. Kasahara, T. Kamata, M. Tohyama and Y. Shiotani. Origins and three dimensional distribution of substance P containing structures of the rat stomach using whole-mount tissue. Gastroenterology 86: 51-59, 1984. 15. Nilsson, G. and E. Brodin. Tissue distribution of substance P-like immunoreactivity in dog, cat, rat and mouse. In: Substance P, edited by U. S. von Euler. New York: Raven Press, 1977, pp. 49-54. 16. Peterson, G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83: 346-356, 1977. 17. Reeve, J. R., J. H. Walsh, P. Chew, B. Clark and J. E. Shively. Amino acid sequences of three bombesin-like peptides from canine intestinal extracts. J Biol Chem 258: 5582-5588, 1983. 18. Relton, J. M., N. S. Gee, R. Matsas, A. J. Turner and A. J. Kenny. Purification of endopeptidase-24.11 ('enkephalinase') from pig brain by immunoadsorbent chromatography. Biochem J 215: 519-523, 1983.

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19. Skidgel, R., S. Engelbrecht, A. R. Johnson and E. Erdos. Hydrolysis of substance P and neurotensin by conve~ing enzyme and neutral endopeptidase. Peptides 5: 769-776, 1984. 20. Studen, R. O., A. Trzeciak and W. Lergier. Isoliernng und Aminosauresequenz yon substanz P aus Pferdedarm. Helv Chim Acta 56: 860-867, 1973. 21. Turner, A. J. and M. J. Dowdall. The metabolism of neuropeptides. Both phosphoramidon-sensitive and Captopril-sensitive metallopeptidases are present in the electric organ of Torpedo Marmorata. Biochem J 222: 255-259, 1984. 22. Von Euler, U. S. Preparation of substance P. Scand Arch Physiol 73: 142-144, 1936. 23. Von Euler, U. S. and J. H. Gaddum. An unidentified depressor substance in certain tissue extracts. J Physiol (Lond) 72: 74-87, 1931. 24. Watson, S. P. Are the proposed substance P receptor sub-types, substance P receptors7 Life Sci 25: 797-808, 1984. 25. Yokasawa, H., S. Endo, Y. Ogura and S.-I. Ishii. A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P. Biochem Biophys Res Commun 116: 735-741, 1983.