Degradation of vasoactive intestinal polypeptide by rabbit gastric smooth muscle membranes

Peptides,Vol. 15, No. 2, pp. 323-332, 1994 Copyright© 1994 ElsevierScienceLtd Printed in the USA.All rightsreserved 0196-9781/94 $6.00 + .00

Pergamon

0196-9781(93)E@002-9

Degradation of Vasoactive Intestinal Polypeptide by Rabbit Gastric Smooth Muscle Membranes RYOKO

K O B A Y A S H I , * Y U L I N G C H E N , * T E R R Y D. L E E , t M. T. D A V I S , t O S A M U ITO~: A N D J. H. W A L S H *l

*Center for Ulcer Research and Education~Digestive Disease Center, Department of Medicine, University of California Los Angeles and Veterans Administration Wadsworth West Los Angeles, Los Angeles, CA 90073, #City of Hope Beckmann Research Institute, Duarte, CA 91010-0269, and ~cEisai Co. Research Laboratories, Tsukuba-shi, 300-26, Japan R e c e i v e d 5 M a y 1993 KOBAYASHI, R., Y. CHEN, T. D. LEE, M. T. DAVIS, O. ITO AND J. H. WALSH. Degradation of vasoactive intestinal polypeptideby rabbitgastricsmooth musclemembranes. PEPTIDES 15(2) 323-332, 1994.--Crude membrane fractions prepared from rabbit gastric fundic muscle degraded vasoactive intestinal polypeptide (VIP) with an average specific activity of 0.96 nmol/ min/mg protein at 37°C, pH 7.5, and at [S]0 = 0.05 mM. The relative activities towards [LeuS]enkephalin, substance P, VIP, and neurotensin were approximately 7.7, 2.0, 1.0, and 0.54, respectively. The VIP degradation was inhibited by metal chelators EDTA and o-phenanthroline. CaCI2 at 0.3-1.0 mM enhanced VIP degradation up to twofold. Phosphoramidon, captopril, and bestatin, the specific inhibitors for endopeptidase-24, l l, angiotensin-converting enzyme, and aminopeptidase M. respectively, did not affect VIP degradation significantly. However, the complex mixtures of VIP fragments generated implicates action of multiple peptidases including the aforementioned three peptidases and other unidentified peptidase(s). Vasoactive intestinal polypeptide

Rabbit gastric muscles

Membrane-associated peptidase

VASOACTIVE intestinal polypeptide (VIP) is a 28 amino acid residue neuropeptide originally isolated from porcine duodenum (33). The amino acid sequence of VIP is identical in several mammalian species except for guinea pig (32). Vasoactive intestinal polypeptide-containing nerve fibers are abundantly localized in the mammalian digestive tract, including the stomach, and are found in all layers of the gut (23,36). Vasoactive intestinal polypeptide is known to be an important physiological mediator for relaxation of gastric fundic smooth muscle. Field stimulation can cause dose-dependent relaxation in the gastric fundic muscle strips of guinea pig that is inhibited by VIP antagonists (19) and antisera to VIP (18). Vasoactive intestinal polypeptide directly causes relaxation of the isolated gastric smooth muscle cells that is accompanied by an increase of intracellular adenosine 3',5'monophosphate (1). The physiological process of inactivation of VIP in the gastrointestinal tissue has not been well characterized, but enzymatic degradation may be the primary pathway. Several peptidases, known as ectoenzymes, are expressed on cell surfaces as integral membrane proteins with their catalytic sites directed towards the extraceilular substrates (24). Some of the ectopeptidases, including neutral endopeptidase-24.11 (NEP, EC 3.4.24.11, also called enkephalinase) and aminopeptidase M (APM, EC

3.4.11.2), are believed to be physiologically important inactivators of neuropeptides in the central nervous system (34). Previously, we isolated some of the membrane-associated peptidases from the membrane fraction of gastric fundic muscle to examine their relevance to neuropeptide inactivation in the gastric wall; namely, endopeptidase-24.11 isolated from the porcine stomach and angiotensin-converting enzyme (ACE, EC 3.4.15.1) from the rabbit stomach (5,6,27). A major aminopeptidase activity, that degrades [LeuS]enkephalin efficiently, was partially purified. The purpose of the present study was to characterize the membrane-associated peptidase activity that degrades VIP in the rabbit gastric fundic muscle. METHOD

Materials The frozen rabbit stomachs (young, mixed sex) were purchased from Pel-Freez (Rogers, AK). Synthetic vasoactive intestinal polypeptide (human, porcine, and rat sequences in common) was synthesized as described (22), and also obtained from Peninsula Laboratories (Belmont, CA). Other peptides were purchased from Sigma Chemical Co. (St. Louis, MO). Captopril was a gift from Squibb Institute (Princeton, N J). Chymostatin

Requests for reprints should be addressed to John H. Walsh, M.D., Director, Center for Ulcer Research and Education/Digestive Disease Center, Building i 15, VA Wadsworth West, Los Angeles, CA 90073.

323

324 was obtained from Boehringer Mannheim Corporation (Indianapolis, IN). Other peptidase inhibitors were purchased from Sigma. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) was obtained from Sigma. Tetrafluoroacetic acid (TFA) was from Applied Biosystems (Foster City, CA). HPLC-grade acetonitrile was obtained from EM Science (Gibbstown, NJ). Reversed-phase C18 columns (Vydac 218TP5405, 0.46 x 5.0 cm, and 218TP54, 0.46 × 25.0 cm; particle size 5 #, pore size 300 A) were purchased from Separation Group (Hesperia, CA).

Membrane Preparation The crude membrane fraction was prepared as described (6,27). Frozen stomachs were thawed in cold 0.14 M NaCI/0.02 M sodium phosphate, pH 7.4/0.05% NAN3, and the mucosal layer was scraped off from the muscle layer. The muscle layers were refrozen in dry ice and stored at - 7 0 ° C until use. The frozen muscle layers were broken into pieces and homogenized in 10 v/w of tissue of ice-cold buffer 1, 10 m M Tris/HCl, pH 7.5/0.05% NAN3, then centrifuged at 500 X g for 7 rain at 4°C. The pellet was rehomogenized in 3 v/w of original tissue and centrifuged under the same conditions. The combined supernatant was ultracentrifuged at 120,000 × g for 40 rain and the pellet was rehomogenized in buffer 1 using a Potter-Elvehjem tissue grinder and ultracentrifuged. The resulting pellet was resuspended in buffer 2, which was 0.5 M NaC1 added to buffer 1, and ultracentrifuged under the same conditions. The final pellet was suspended in buffer 3, 20 m M Tris/HC1, pH 7.5/ 0.02% NaN3 (assay buffer). The protein concentration of the membrane suspension was determined using Bio-Rad protein assay reagent by the procedure recommended by the manufacturer.

Degradation of Peptides The digestion of peptides by the crude membrane fraction was carried out at pH 7.5 at 37°C. Typically, 5 or 10 nmol of a peptide was incubated with 0.01-0. I0 mg membrane protein in the assay buffer (buffer 3, above) in a final volume of 0.20 ml. The reaction was terminated by heating the reaction mixture in a boiling water bath for 4 rain. It was then acidified by addition of 0.5 ml of 0.1% trifluoroacetic acid and stored cold until HPLC analysis. When the effectors, such as CaC12 or inhibitors, were included in the assay the control experiments without peptide were also performed by the same procedure to compare the HPLC profiles. The routine analysis of the assay mixture was performed by HPLC with a C18 column (0.46 X 5.0 cm), which was equipped in a Shimadzu LC-6A system with an autoinjector (Shimadzu Scientific Instruments, Inc., Columbia, MD) and was eluted isocratically with a mixture of acetonitrile and 0.1% trifluoroacetic acid (TFA) in water. The concentrations of acetonitrile used for the determination of peptides were: 15% for [LeuS]enkephalin, 18.5% for neurotensin, 20% for substance P, and 24% for VIP.

Inhibitor Study To determine the effects of peptidase inhibitors, the reagents to be tested were preincubated in the assay media with an appropriate amount of the enzyme (membrane fraction) for 15 min in an ice bath, then the reaction was started by addition of the substrate peptide and incubation at 37°C. Stock solutions of the reagents that are not freely soluble or unstable in the assay buffer were prepared as follows: 0.5 M diisopropyl fluorophosphate (DFP) in isopropyl alcohol, 50 m M trans-epoxysuccinyl-

KOBAYASH| ET AL. L-leucylamido-(4-guanidino)-butane (E-64) in dimethyl sulfoxide, 1 m M pepstatin in ethanol, and 25 mM chymostatin in dimethyl sulfoxide. They were diluted with assay buffer to the desired concentration immediately before the experiments. The control experiments without inhibitors were also performed for those assays including the organic solvents.

Detergent Solubilization and Fractionation of the MembraneBound Peptidases A crude membrane preparation was solubilized using CHAPS. A membrane sample (237 mg protein) was suspended in 10 m M CHAPS/0.15 M KCI/50 m M Tris/HC1, pH 7.5, at the protein concentration of 0.94 mg/ml and gently stirred at 4°C for 2.5 h. The suspension was ultracentrifuged at 120,000 X g for 40 min and the supernatant was collected as solubilized fraction. It was then dialyzed against 1 m M CHAPS/10 mM Tris/HCl, pH 7.8, at 4°C. The protein concentration was determined using bovine serum albumin dissolved in the same buffer as standard. Fractionation of the detergent-solubilized membrane-bound peptidases was performed by DE-52 chromatography with stepwise elution of the adsorbed proteins. The column (1.5 × I 1.3 cm) was equilibrated and washed, after the sample application, with 1 mMCHAPS/10 m M Tris/HCl, pH 7.8, and the proteins were eluted successively with the same buffer containing 0.05, 0.10, 0.15, 0.20, 0.25, 0.40, and 0.80 M NaCI (approximately 3.4 X column volumes, each). Fractions (4 ml) were collected. The elution of the protein was monitored by absorbance at 280 nm and the peptidase activities were assayed as described.

Identification of the Peptide Fragments by LC/MS The VIP digests with or without 1 m M CaCI2 were prepared as described for the routine enzymatic assays. Liquid chromatography/mass spectroscopy (LC/MS) analyses were performed using a micro LC system interfaced to a Finnigan MAT TSQ 700 triple sector quadrupole mass spectrometer through an electrospray ion source. The samples were fractionated over a capillary column (0.25 X 200 mm) (10) at an average flow rate of 0.002 ml/min under a constant pressure and were detected by the absorbance at 200 nm. A gradient elution was achieved by mixing buffer A (0.1% TFA) and buffer B (90% acetonitrile/ 0.07% TFA; v/v) by increasing concentration of buffer B from 2% to 62% linearly over 40 min, unless otherwise described. The column effluent was mixed with an equivalent flow of 2-methoxyethanol to stabilize the spray throughout the gradient. Electrospray spectra were obtained over the mass range from 500 to 2000 ainu (m/z) using a scan time of 3 s. The similar analyses of five HPLC-isolated peptide fractions were performed using a slightly modified elution system for the microcapillary column.

Amino Acid Analysis of the Peptides To identify the cleavage sites of VIP by the membrane-bound peptidases, the VIP-derived peptides were isolated by HPLC using an analytical C18 column (0.46 X 25.0 cm) with an elution system of increasing concentration of acetonitrile from 2% to 33% in 0.1% trifluoroacetic acid. The dried peptide samples were hydrolyzed in the vapor of hydrochloric acid at 150°C for 20 h and analyzed by Beckman 6300 Amino Acid Analyzer using the ninhydrin system.

Amino-Terminal Sequence Analysis of the Peptides Selected peptide fractions isolated by HPLC were dried in vacuo then brought up in 50% acetonitrile in water and spotted

VIP D E G R A D A T I O N BY G A S T R I C M E M B R A N E S

i

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(hr)

FIG. 1. Time course study of the membrane-associated peptidase activities derived from the rabbit gastric muscle. The experiments were performed at 37°C at pH 7.5 in 20 mM Tris/HC1 (n = 3). The initial peptide concentrations, [S]o, were 0.05 mM and the membrane-associated protein was 0.02 mg per 0.2 ml of assay volume (see the Method section for details). The symbols used are: A, [LeuS]enkephalin; [], substance P; O vasoactive intestinal polypeptide; and ~7, neurotensin. The vertical bars represent the SD.

onto a P V D F membrane ( 1 m m × 1 cm) for the amino-terminal sequence analysis. Automated Edman degradation was performed on a gas-phase microsequencer built at the Beckman Institute of the City of Hope (20) that includes a continuous flow reactor (35). The phenylthiohydantoin-amino acid derivatives were identified by on-line reversed-phase H P L C as described earlier (35).

Chloride]

I

I

1

10 (mid)

FIG. 3. Calcium dose dependency of VIP degradation by the crude membrane fraction. Assays were carried out in the presence of varying concentrations of CaC12. The control degradation without calcium was 52.2% at 2.5-h incubation at 37°C, [S]o = 0.025 mM, and with the membrane protein of 0.04 mg per assay (n = 2). Other reaction conditions were the same as those for Fig. 1.

was 2.35 + 0.54 mg/g wet tissue of the starting rabbit gastric muscle. The time courses of degradation of VIP, substance P, [LeuS]enkephalin, and neurotensin by the m e m b r a n e preparation were compared at 37°C in 20 m M Tris/HC1 at p H 7.5 (Fig. 1). The specific activities (nmol peptide hydrolyzed/min/mg m e m b r a n e protein) were 0.38 for neurotensin (NT), 1.13 for VIP, 1.86 for substance P (SP), and 7.93 for [LeuS]enkephalin

RESULTS 100

Peptidase Activities in the Crude Membrane Preparations The average yield, expressed as membrane-associated protein, of the four membrane preparations used for the present study

BES

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100

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i 30

Time

i 60

i 90

L 120

(rain)

FIG. 2. The effect of calcium ion on VIP degradation by the crude membrane fraction. Time courses of VIP-degradation in the absence and presence of 0.3 mM CaC12 are compared. The initial VIP concentration of 0.05 mM and membrane protein of 0.04 mg per assay were used (n = 3). Other reaction conditions were the same as those for Fig. 1. The symbols used are: O, assay without calcium; Q, assay with 0.3 mM calcium.

FIG. 4. Effects of peptidase inhibitors on the peptide degradation by the membrane preparation. The assays were performed in the presence of the indicated reagents. They were preincubated for 15 min with the crude membranes, 0.02 mg of the membrane protein for VIP and substance P and 0.01 mg of that for [Leu~]enkephalin per assay. [S]0 = 0.025 mM for VIP (n = 2), and [S]0 = 0.05 mM for substance P and for [LeuS]enkephalin (n = 3). The reaction period at 37°C was chosen to give approximately 50-60% of the control degradation without an inhibitor. The percent inhibition as average of the duplicate or triplicate assays is shown for each substrate: (hatched bars) VIP, (solid bars) substance P, and (open bars) [LeuS]enkephalin. The symbols and concentrations of inhibitors are: PM, phosphoramidon (0.01 mM); BES, bestatin (0.1 mM); CP, captopril (0.01 mM); EDTA, (2 mM); PHE, o-phenanthroline (0.5 raM); PEP, pepstatin (0.001 mM); LP, leupeptin (0.01 raM); DFP, diisopropyl fluorophosphate ( 1 mM); and E-64 (0.1 mM).

326

KOBAYASHI ET AL.

a.

c,

M

M

(~) lmM Ca ~ VIP Degr.= 20%

( ~ Ca ~' VlP Dt~Jr. = 24%

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FIG. 5. HPLC elution profiles of VIP digests by the membrane fraction. The reactions were carried out using 0.025 mM VIP in 20 mM Tris/HC1 (pH 7.5)/0.02% NaN3 with or without 1 mM CaC12at 37° for 4 h. The amount of the membrane protein per assay were: 0.01 mg for (a) (20% degradation) and (c) (24% degradation), and 0.033 mg for (b) (63% degradation) and (d) (55% degradation). The reaction mixtures were treated as described in the Method section and analyzed by reversed-phase HPLC using a C 18 column (0.46 × 25 cm), eluted with a linear gradient of acetonitrile concentration in 0.1% TFA. The acetonitrile concentration in the elution program was: 1% (0-2 min), 2-10% (2-10 rain), 10-33% (10-79 min), 33% (79-84 min), 33-2% (84-90 min), and 2% (90-110 min). After acidification,0.2 ml each of the samples was analyzed (see the Method section). The peptides were detected by absorbance at 214 nm, and the peaks were alphabetically marked in the order ofelution time according to the result of LC/ MS analysis (Table 1).

(ENK). These assays were performed within 4-6 days after preparation of the membrane samples.

The Effect of Calcium The rate of degradation of VIP by the membrane-associated peptidases was increased by addition of CaC12 to the assay mixture (Fig. 2). The specific activities determined in this experiment in the absence and presence of 0.3 m M calcium ion were 0.73 and 1.26 nmol/min/mg protein, respectively. The effect of calcium ion was dose dependent (Fig. 3). A maximum of 52% increase in the rate of degradation was obtained at 1.0 mMCaCI2 compared to the control assay without calcium, where 57% of VIP was degraded. The highest rates of hydrolysis were observed at calcium concentrations in the range of 0.3-1.0 m M among the experiments with different membrane preparations. The maximum enhancement of the activity obtained was about twofold at 0.3 m M CaCI2. The presence of 10 m M calcium ion inhibited degradation by up to 51%.

The Effect of lnhibitors in the Absence of Calcium Various inhibitors for peptidases were incubated with the assay mixture to examine their effects on the peptide hydrolysis. A summary of representative results is shown in Fig. 4. EDTA

(2 mM) and o-phenanthroline (0.5 mM) were effective inhibitors of VIP degradation, but the extent of their inhibition varied among experiments (EDTA: 33-70%, phenanthroline: 20-78%). In some experiments EDTA was more effective than o-phenanthroline, and in others vice versa. EDTA (2 mM) strongly inhibited degradation of substance P but not of enkephalin. Diisoproyl fluorophosphate (I raM) and E-64 (0.1 mM) demonstrated mild inhibitions for VIP and substance P degradation. When combined with EDTA, neither DFP nor E-64 significantly increased the inhibitory effect of EDTA alone on the VIP degradation (data from a separate experiment, not shown). Vasoactive intestinal polypeptide degradation was not significantly inhibited by either phosphoramidon (0.01 mM), bestatin (0.1 mM), or captopril (0.01 mM), nor by combination of the three (8.9%, not shown). These reagents are the specific inhibitors for NEP, APM, and ACE, respectively (9,13,31). On the other hand, phosphoramidon strongly inhibited the degradation of substance P (47%) and neurotensin (65%, not shown). Bestatin inhibited the degradation of [LeuS]enkephalin extensively (73%), as was observed earlier (26). Captopril showed a weak inhibition of substance P degradation. Chymostatin (0.01 mM) produeced a minor inhibition (13%, not shown) of VIP degradation.

VIP DEGRADATION BY GASTRIC MEMBRANES

TABLE 1 MAJOR VIP FRAGMENTSIDENTIFIEDBY LC/MS IN THE PRESENCE AND ABSENCEOF CALCIUMION Peptide*

ElutionTime(min)~"

AB C+ D E F÷ G H I+ J KL+

30.15 31.47 45.06 49.97 51.92 52.75 54.91 56.29 59.01 59.71 67.05 70.92

M N-

73.37 74.49

ObservedMass

Residuesof VIP

1006.4 776.8 1824.3 2262.7 1191.8 2511.0 2380.9 1262.8 2987.9 2783.8 2339.6 2930.6 3341.9 3326.4 3102.2

1-9 1-7 1-15 5-22 19-28 1-21 1-20 18-28 1-25 3-25 10-28 5-28 ox$ 1-28 ox¢ 1-28 3-28

* The peptides are alphabeticallymarked in the order of their elution times from CI 8 capillary column in the LC/MS system (see the Method section). The superscripts plus (+) or minus (-) indicate those peptides that were found in higher yields in the VIP digests with or without 1 mM calcium chloride, respectively. t" The elution times shown are those from C18 analyticalcolumn (0.46 × 25.0 cm) in a representativerun of this series of HPLC (see legend for Fig. 5). :~ox, oxidized form.

Inhibitor Experiments in the Presence of Calcium

327

cium, peaks I and L were generated less in the early stage [Fig. 5(c)] and further attenuated by 55% digestion [Fig. 5(d)]. On the other hand, peak A [VIP(1-9)] gradually increased in the course of digestion. Peak K [VIP(10-28)] was slowly generated in the absence of calcium ion and became prominent at 55% digestion, but was hardly detected in the presence of calcium. Peak N [VIP(3-28)], eluted immediately after the intact VIP, was generated in higher yields in the absence of calcium ion. An example of the microcapillary column elution profile of the VIP digest without calcium and the corresponding mass analysis result are shown in Fig. 6 and Table 2. Comparison of the analysis results on the VIP digests with and without l mM calcium ion suggested that the differences of the peptide compositions generated were quantitative and not qualitative. The peptide samples for the amino acid analysis were isolated by HPLC using another series of VIP digests, and 12 VIP fragments were clearly identified (the peptides numbered as 1-8, 10, 1l, 14, and 16 in Table 3). Two peptide fractions (numbered 9 and 12) appeared to be mixtures of two homologous peptides with one-residue-long differences from their compositions. In addition, the mass spectral analyses of five HPLC-isolated peptide fractions confirmed their amino acid compositions; they were peak X [VIP(1-7)/(1-8), unseparated], peak I [VIP(1-25)], peak J [VIP(2-24) or VIP(3-25)], peak L' that eluted between peaks L and M [VIP(5-28)], and peak N [VIP(3-28)]. Peak J had a composition that can be accounted by either VIP)2-24) or VIP(3-25); therefore, it was further examined by amino-terminal microsequencing analysis, which proved the peptide to be VIP(325) (the analysis result not shown). From all the data seven peptides were identified by both the mass spectral and amino acid analyses (Table 4). These peptides, however, were not necessarily major products of digestion. Other VIP fragments identified by only one of the methods may represent either minor components, those that were difficult to be isolated, or those that were not

Similar experiments were performed on VIP degradation in the presence of 2 m M CaC12. The following inhibitions were obtained under the experimental condition in which 67% of VIP was degraded without any inhibitor: 18.8% with phosphoramidon (0.01 mM), 10.5% with bestatin (0.1 mM), 6.0% with captopril (0.01 raM), 22.0% with E-64 (0.1 mM), and 20.9% with leupeptin (0.01 raM).

A

Identification of the VIP Fragments Generated by the Membrane-Bound Peptidases

cE 30.

A series of VIP digests with or without 1 m M CaCI2 were analyzed by LC/MS for identification of the VIP-derived peptides. The same samples were also fractionated over a C18 analytical column to examine the elution profiles of the peptides. Representative chromatograms of the samples at the early and at the later stages of digestion in the presence and absence of 1 m M calcium are shown in Fig. 5. The 14 major absorbance peaks, which gave positive identification of VIP-derived peptides by LC/MS (Table 1), were alphabetically marked in the order of the elution time. Intact VIP [VIP(1-28), marked with M] was eluted at 73.4 min in a representative run of this system. In the presence of 1 m M calcium ion, the absorbance peak I, identified as VIP fragment 1-25 [VIP(1-25)], and the peak marked L [a mixture of oxidized VIP(1-28) and oxidized VIP(5-28)], eluted in front of intact VIP, were prominent among others when 20% of VIP was digested [Fig. 5(a)]. Of these, peak I increased slightly but peak L decreased when the digestion proceeded [Fig. 5(b)]. The small peak B at 31.5 min [VIP(1-7)] and peak F at 52.8 min [VIP(I-21)], and the two eluted between peaks F and I, increased when the digestion proceeded. In the absence of cal-

20 ,o

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FIG. 6. The elution profile of VIP digest without added calcium from the microcapillary column for LC/MS analysis. The sample (0.005 ml) was fractionated over a Cl8 capillary column (0.25 × 200 ram) under a constant pressure at an approximate flow rate of 0.002 ml/min. The peptide peaks were detected at 200 nm at a detector sensitivity of 0.05 AUFS. The starting material (peak 20) was off-scale under these conditions. The result of mass spectral analysis is shown in Table 2.

328

K O B A Y A S H I ET AL.

TABLE 2 VIP-DERIVED PEPT1DES IDENTIFIED BY MASS SPECTRAL ANALYSIS IN A REPRESENTATIVE CAPILLARY COLUMN HPLC

TABLE 3 vIP FRAGMENTS IDENTIFIED BY AMINO ACID ANALYSIS

Peak No.

Residuesof VIP

CalculatedMol.Wt.

ObservedMol.Wt.

Peptide

Elution Time (min)*

Residues of VIPt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18a 18b 19 20

1-9 1-7 23-28 1-16 1-15 2-15 1-14 5-22 1-21 3-21 1-20 1-22 4-23 1-25 2-24/3-25* 10-28 ox? 10-28 1-28 oxt 5-28 oxt 5-28 1-28

1005.0 775.8 671.8 1951.1 1823.0 1685.9 1694.8 2261.7 2508.9 2284.7 2380.7 2672.1 2445.9 2986.4 2762.2 2354.9 2338.9 3341.9 2931.5 2915.5 3325.9

1004.6 775.5 672.0 1951.0 1822.3 1685.6 1694.6 2261.6 2508.4 2284.0 2380.0 2671.2 2446.6 2986.9 2762.8 2354.3 2338.3 3341.9 2930.6 2914.8 3326.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

24.75 25.19 29.51 29.88 42.52 43.75 48.54 50.36 54.13 56.75 65.64 67.63 69.95 70.99 71.75 73.06

22-23 5-6 1-7 1-8 1-15 9-25 22-28 1-20 1-22/1-23 1-25 10-28 8-28/9-28 1-28 ox$ 5-28 1-28§ 3-28

3101.6

3101.9

21

3-28

Aliquots of 0.005 ml of the VIP digest without added calcium were analyzed as described under METHOD. The elution profile of the peptides from the microcapillary column is shown in Fig, 6. The peptides that were unambiguously assigned by the mass spectral analysis are shown. Additional products were detected that could not be unambiguously identified. * VIP(2-24) and VIP(3-25) have the identical mass and the amino acid composition, so this method cannot discriminate between these two peptides. t o x , oxidized form. detected by the mass analysis because of the range of the scan, namely V I P ( 5 - 6 ) a n d VIP(22-23). The overall results indicated that complex mixtures of peptides were generated in the VIP digests, a n d that each peptide in the mixture was present in relatively low yield at any stage of digestion up to 55% of VIP degradation, which made it difficult to deduce the primary cleavage sites of VIP molecule. Thus, the recoveries of VIP(3-28), the peptide recovered in the highest a m o u n t s based on the a m i n o acid analysis a n d increased by time of digestion b o t h in the absence a n d presence of calcium ion, were only 13% a n d 7%, respectively, at 55% digestion of VIP (not shown). Figure 7 illustrates a tentative assignment of m a j o r cleavage sites of VIP by the crude m e m b r a n e fraction of the gastric s m o o t h muscles that was based o n the recoveries of the peptides isolated a n d quantified by the a m i n o acid analysis. T h e additional cleavage sites identified, b o t h in the presence a n d absence of calcium, by either one or b o t h of the analytical methods are also indicated (small arrows on the top of the sequence, Fig. 7). The VIP fragments with relatively higher yields are shown u n d e r the a m i n o acid sequence o f VIP.

Detergent Solubilization of the Membrane-Bound Peptidases The m e m b r a n e s were solubilized using a neutral ionic detergent, CHAPS, 10 m M in 0.15 M NaCI/50 m M Tris/HC1, p H 7.5, at 4 ° C as described u n d e r the M e t h o d section. The m e m -

* The peptides identified by amino acid analysis are numbered in the order of their elution times from a C 18 analytical column. t The amino acid sequences of the isolated peptides were deduced from the amino acid compositions. The two sequences separated by / indicates that the analysis result of the fraction could be explained by a mixture of the two peptides. $ The major component in this fraction was probably an oxidized form of VIP, as the elution position was consistent with the authentic sample (not shown). § Intact VIP.

b r a n e preparation before solubilization had specific peptidase activities towards VIP, substance P, a n d [LeuS]enkephalin of 0.89, 2.2, a n d 4.7 n m o l / m i n / m g protein, respectively. After the solubilization a n d dialysis to remove the excess of detergent, 17% of the protein a n d 24% o f the VIP-degrading activity were recovered. The solubilized fraction was partially characterized for peptidase activity (Table 5). The specific activity of VIP degradation was 1.28 n m o l / m i n / m g protein, w h e n assayed in 1 m M C H A P S / 2 0 m M Tris/HC1, pH 7.5/0.02% NaN3 at the initial VIP concentration of 0.025 raM. The recoveries of [Leu 5]

TABLE 4 VIP-DERIVED PEPTIDES IDENTIFIED BY LC/MS AND AMINO ACID ANALYSIS Peptide* 3, B 4, X 5, C 8, G 10, I 11, K 16, N

Residue Number

Sequencer

1-7 1-8 1-15 1-20

HSDAVFT HSDAVFTD

1-25

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VIP DEGRADATION BY GASTRIC MEMBRANES

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<...................................... (1-20) ................................ > <(22-23)> FIG. 7. The cleavage sites of VIP by the membrane fraction of the rabbit gastric fundic muscle. The major cleavage sites of the peptide in the absence and presence of 1 mM calcium ion are shown by the arrows with numbers indicating the order of the rate of hydrolysis, which were deduced from the yields of the peptides based on their amino acid compositions. The additional cleavage sites, which were found both in the presence and absence of calcium ion, are indicated by smaller arrows without numbers over the amino acid sequence of VIP. The seven major VIP fragments isolated are also shown underneath the sequence.

enkephalin and substance P degrading activities were 73% and 38%, respectively. The activities towards VIP and substance P in the CHAPSsolubilized fraction were not stable. The VIP-degrading activity decreased over time; namely, the specific activities determined 2 and 5 days after the solubilization were 0.68 and 0.43 nmol/ min/mg, respectively. The activity of the solubilized peptidase was also sensitive to dilution. The activity towards VIP was enhanced by addition of 1 m M CaCI2 by up to 40%. The CHAPSsolubilized VIP-degrading activity had a similar inhibitor sensitivity as the starting membrane fraction. EDTA (2 mM) and o-phenanthroline (0.5 mM) produced substantial inhibition, 42% and 77%, respectively. Figure 8 shows the result of a preliminary fractionation of CHAPS-solubilized proteins over a DE-52 column by stepwise elution with increasing concentrations of NaC1 in CHAPS-containing buffer at pH 7.8. Major VIP-degrading activities were detected in two fractions that eluted at 0.15 M and 0.20 M NaCI (pool 1 and pool 2, respectively). Only the peak fractions were collected and partially characterized (Table 5). Pool 1 and pool 2 had similar activities as those of the sample before fractionation towards VIP, substance P, and [LeuS]enkephalin, but the specific activities of VIP and enkephalin degradation increased some extent in both pools. The VIP- and substance P-degrading activities in the two pools were unstable, as those observed in the sample before fractionation by DE-52. The VIP-degrading activities in pool 1 and pool 2 were different in the sensitivity to phosphoramidon; the activity in pool 2 was not inhibited at all but that in pool 1 was more sensitive to this inhibitor. The activities in the two pools were not enhanced by the presence of 1 m M calcium ion. DISCUSSION

Control of the local concentration of neuropeptides by enzymatic degradation may be an important regulatory mechanism of the peptide action in the tissue. A fraction of the peptide molecules released from nerve endings may interact with their specific receptors located on the plasma membrane of the target cells, but the remaining peptides may largely be inactivated by

the action of cell-surface membrane-bound peptidases (29). This hypothesis was based on the studies in the murine central nervous systems, and the roles of NEP and APM as physiological inactivators of opioid peptides have been established (30,34). Such mechanisms may also be working for inactivation of neuropeptides in the gastrointestinal tract. We demonstrated earlier that porcine gastric NEP and rabbit gastric ACE, both isolated from the membrane fractions of the gastric fundic muscles, can degrade and inactivate various gastrointestinal neuropeptides (5,6,27), and that the major aminopeptidase activity in the membrane fraction degraded [LeuS]enkephalin efficiently (26). The present study demonstrated that VIP was degraded by the gastric muscle membrane-associated peptidases at a rate slower than that for the enkephalin degradation and at approximately one-half of the rate of substance P degradation. The complex mixture of VIP fragments generated due to the multiplesite cleavages of VIP molecule suggests that several enzymes contributed to the degradation process. The strong inhibition of the reaction by metal chelators indicates the primary action of metallo-enzymes. Neutral endopeptidase-24.11, APM, and ACE are well-characterized membrane-associated peptidases that require zinc for their enzymatic activity (24). However, the specific inhibitors for these peptidases, even in combination, failed to inhibit the degradation of VIP by the crude membrane fraction, which strongly suggests involvement of other peptidase(s). Of the major cleavage sites of the peptide bonds, deduced from the amino acid composition of the isolated fragments (Fig. 7), Ser2-Asp3 appeared to be unique and could not be accounted for by any one of the aforementioned peptidases. It is possible, however, that this bond was hydrolyzed by sequential removal of the amino-terminal residues by the aminopeptidase action, supported by identification ofVIP(2-15) by LC/MS, even though bestatin did not inhibit overall degradation of VIP by the membrane fraction. The cleavage of the peptide bonds Ala4-Val 5, Lys21-Tyrn, and Sel~5-Ile26 may be due to the action of NEPlike enzyme, as it was reported that these bonds were hydrolyzed by the human recombinant NEP, of which Ala4-Val 5 was a primary cleavage site, although VIP was not a favorable substrate for this enzyme (l 6). In our study phosphoramidon did not show significant inhibition on the overall VIP degradation in the crude

330

KOBAYASHI ET AL.

membrane fraction, but it inhibited the reaction by the partially purified enzyme fraction (pool 1) to some extent. This seems to be reasonable because NEP, or NEP-like enzyme, appeared to be an active component in the m e m b r a n e fraction as it was demonstrated by the substantial inhibition of the degradation of substance P and neurotensin by phosphoramidon. On the other hand, it is possible that the cleavages at the bonds Lys 2~Tyr~2 and Leu23-Asn24 were secondary due to ACE that could act on VIP(1-25) sequentially from its carboxyl-terrninus, which is supported by the isolation of VIP(22-23) fragment. Another unique cleavage site was the Asn9-TyP ° bond. It could be hydrolyzed by NEP but it was not reported to be hydrolyzed by recombinant NEP (16). Vasoactive intestinal polypeptide(3-28) and VIP(10-28) were generated in higher yields in the absence of calcium (Fig. 5). On the contrary, VIP(I-25) was produced more in the presence of calcium. In addition, V I P ( I - 15) and VIP( 1-21), which may be noted as the products of cleavage after double basic amino acid residues, were generated in higher yields in the presence of calcium. Further study is necessary to clarify whether an enzyme with such specificity is present in this membrane preparation. The different patterns of the peptide generation from VIP in the presence and absence of calcium ion, together with the inhibitory effect of EDTA, suggest an involvement of a peptidase(s) whose activity is modulated by calcium ion. The earlier studies using synthetic fragments of VIP (3,4,12,14,22) indicated importance of whole VIP sequence for its biological activity. Thus, any of the peptide bond cleavages identified in the present study will cause direct inactivation of VIP. In addition, the peptide VIP(10-28) was reported to have a potent antagonistic effect on VIP receptor binding in the gastrointestinal muscle (19) and the HT29 colonic adenocarcinoma cell (37). It has been suggested that the central to carboxyl-terminal portion of VIP molecule is able to form a-helical conformation that is important for receptor binding (11,17). Therefore, the cleavage of Asn9-Tyr j° may have additional effect in the tissue if the carboxyl-terminal fragment, VIP(10-28), is stable in that environment. A recent study on the enzymatic regulation of VIP-induced pulmonary relaxation in the guinea pig suggested a major role

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FIG. 8. Fractionation of CHAPS-solubilized membrane-associated peptidases by DE-52 chromatography. The CHAPS-solubilized membraneassociated proteins, after dialysis (see the Method section), were applied to a DE-52 column (1.5 × 11.3 cm) equilibrated with l m M CHAPS/ 10 m M Tris/HC] (pH 7.8) at 4°C. The column was washed with the equilibration buffer, then the adsorbed proteins were eluted by a stepwise manner with the same buffer containing 0.05, 0.10, 0.15, 0.20, 0.25, 0.40, and 0.80 M NaCI (approx. 3.4 × column volumes, each); 4-rnl fractions were collected. The elution of the protein was monitored by the absorbance at 280 nm (---) and the fractions were assayed for the

peptide degrading activities as described. Only the VlP-degrading activity is shown in an arbitrary scale.

of endogenous NEP and a trypsin-like enzyme, possibly the mast cell tryptase (28). In their study, combinations of the inhibitors specific to NEP and to trypsin-like enzymes substantially increased the recovery of V1P-like immunoreactivity in the tracheal perfusate in parallel to the reduction of the airway opening pressure. The VIP hydrolyses at Asp3-Ala4 and Ala4-Leu 5 in the perfusate were strongly inhibited by SCH 32615, an inhibitor of NEP, and at Arg~4-Lys'5 by aprotinin. Similar to our findings, each of these inhibitors, if used individually, or a combination

DE-52

Protein recovery VIP-degrading activity Specific activity*: VIP Substance P [Leu5]enkephalin Activity enhancement by 1 mM CaC12 Inhibitors EDTA Phenanthroline Phosphoramidon

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TABLE 5 CHAPS.SOLUBILIZED VIP-DEGRAD1NGPEPTIDASE ACTIVITY

Starting

125

10 mM-CHAPS-membranes DE-52Solubilized

Pool I

Pool 2

(100%) (100%)

17% 24%

1.2% 2.1%

2.6% 5.9%

0.89 2.21 4.70 Yes

1.28 4.86 20.3 Yes

1.64 4.37 >32.5 No

1.99 4.3 >30.4 No

m/s m w

m s w

s w/m w/m/s

m s w/n

Unit is nmol/min/mg protein. The effect on the VIP degradation. The concentration of the inhibitors are the same as those for Fig. 4. The extent of inhibitions are expressed as: n, no inhibition (<5%); w, weak inhibition (6-25%); m, medium inhibition (26-55%); and s, strong inhibition (>56%).

VIP D E G R A D A T I O N BY G A S T R I C M E M B R A N E S

331

of the inhibitors to NEP, APM, and ACE plus leupeptin, did not show a substantial effect on the recovery of VIP. Trypsinand NEP-like activities on VIP were also observed with h u m a n cultured T lymphocytes and with the cell membranes (15). We did not examine in our m e m b r a n e system the effect ofaprotinin or soybean trypsin inhibitor, which may inhibit the mast cell tryptase (7), but a combination of the serine-protease inhibitor DFP and metal chelators o-phenanthroline and E D T A resulted in only 53% inhibition of VIP degradation in one experiment (not shown). Unlike the studies referred to above, we could not find substantial differences in the yields of various VIP fragments generated by the gastric membrane fraction, which made it difficult to assign primary cleavage sites. This result was not necessarily unexpected because the m e m b r a n e preparation used is still in a crude state and VIP may be exposed in vitro to multiple peptidases without any biological control. In addition, the methods for quantification of the peptides were different in their studies where either radioimmunoassay or absorbance comparison for the expected major peptide products were used. The preliminary attempt of solubilization and purification of the peptidases in the rabbit gastric membranes using C H A P S

partially separated two VlP-degrading enzyme fractions with different sensitivity to phosphoramidon. Unfortunately, the activity was recovered in relatively low yield and was not stable after the detergent solubilization, which made the experiment difficult to continue. C H A P S was used in the present study because it has several advantageous properties for protein purification among other detergents (21). However, the useful concentration of C H A P S appeared to be limited because a bell-shaped curve of the recovery of the peptidase activity was observed in a preliminary experiment (not shown). On the other hand, Triton X100 was found to be more effective in solubilizing membranebounds peptidases (6,26,27), including the VIP-degrading activity [(25), unpublished data]. Establishment of the conditions for solubilization and stabilization of the VIP-degrading activity will be necessary for purification of the responsible enzymes for further characterization. ACKNOWLEDGEMENTS We thank Mr. James Sligar at Beckman Research Institute of the City of Hope, Duarte, CA, for the amino acid analysis and Ms. Katherine Wen for technical assistance. This work was supported by NIH Grant DK 35740.

REFERENCES 1. Bitar, K. N.; Makhlouf, G. M. Relaxation of isolated gastric smooth muscle cells by vasoactive intestinal peptide. Science 216:531-533; 1982. 2. Bodansky, M. Synthesis of vasoactive intestinal peptide and related peptides. Ann. NY Acad. Sci. 527:20-28; 1988. 3. Bodansky, M.; Henes, J. B.; Yiotakis, A. E.; Said, S. I. Synthesis and pharmacological properties of the N-terminal decapeptide of the vasoactive intestinal peptide (VIP). J. Med. Chem. 20:14611464; 1977. 4. Bodansky, M.; Klausner, Y. R.; Said, S. I. Biological activities of synthetic peptides corresponding to N-terminal fragments of and to the entire sequence of the vasoactive intestinal peptide. Proc. Natl. Acad. Sci. USA 70:382-384; 1973. 5. Bunnett, N. W.; Debas, H. T.; Turner, A. J.; Kobayashi, R.; Walsh, J. H. Metabolism and inactivation of gastrin and cholecystokinin by endopeptidase-24.11 (EC 3.4.24.11, "enkephalinase") isolated from pig stomach. Am. J. Physiol. 255:G676-G684; 1988. 6. Bunnett, N. W.; Turner, A~ J.; Hryszko, J.; Kobayashi, R.; Walsh, J.H. Isolation of endopeptidase-24.11 (EC 3.4.24.11, "enkephalinase") from the pig stomach: Hydrolysis of substance P, gastrinreleasing peptide 10, [LeuS]enkephalin and [MetS]enkephalin. Gastroenterology 95:952-957; 1988. 7. Caughey, G. H.; Leidig, F.; Viro, N. F. Nadel, J. A. Substance P and vasoactive intestinal peptide degradation by mast cell tryptase and chymase. J. Pharmacol. Exp. Ther. 244:133-137; 1988. 8. Couvineau, A.; Rouyer-Fessard, C.; Fournier, A.; St Pierre, S.; Pipkorn, R.; Laburthe, M. Structural requirements for VIP interaction with specific receptors in human and rat intestinal membranes: Effect of nine partial sequences. Biochem. Biophys. Res. Commun. 121: 493-498; 1984. 9. Cushman, D. W.; Ondetti, M. A. Inhibitors ofangiotensin-converting enzyme for treatment of hypertension. Biochem. Pharmacol. 29: 1871-1877; 1980. 10. Davis, M. T.; Lee, T. D. Analysis of peptide mixture by capillary high performance liquid chromatography: A practical guide to smallscale separations. Prot. Sci. 1:935-944; 1992. 11. Fournier, A.; Saunders, J. K.; Boulanger, Y.; St-Pierre, S. A. Conformational analysis of vasoactive intestinal peptide and related fragments. Ann. NY Acad. Sci. 527:51-67; 1988. 12. Fournier, A.; Saunders, K.; St-Pierre, S. Synthesis, conformational studies and biological activities of VIP and related fragments. Peptides 5:169-177; 1984. 13. Fulcher, I. S.; Matsas, R.; Turner, A. J.; Kenny, A. J. Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic mem-

14. 15. 16.

17.

18. 19.

20. 21. 22. 23.

24.

25.

branes show similar sensitivity to inhibitors. Biochem. J. 203:519522; 1982. Gespach, C.; Emami, S.; Lhiaubet, A.-M.; et al. Structure-activity relationship of vasoactive intestinal peptide fragments in the human gastric cancer cell line HGT-I. RCS Med. Sci. 12:724-725; 1984. Goetzl, E. J.; Kodama, K. T.; Turck, C. W.; Schogolev, S. A.; Sreedharan, S. P. Unique pattern of vasoactive intestinal peptide by human lymphocytes. Immunology 66:554-558; 1989. Goetzl, E. J.; Sreedharan, S. P.; Turck, C. W.; Bridenbaugh, R.; Malfroy, B. Preferential cleavage of amino- and carboxyl-terminal oligopeptides from vasoactive intestinal polypeptide by human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11). Biochem. Biophys. Res. Commun. 158:850-854: 1989. Goossens, J. F.: Pommery, N.; Lohez, M.; et al. Antagonistic effect of a vasoactive intestinal peptide fragment, vasoactive intestinal peptide( 1-11 ), on guinea pig trachea smooth muscle relaxation. Mol. Pharmacol. 41:104-109; 1992. Gilder, J. R.; Cable, M. B.; Said, S. I.; Makhlouf, G. M. Vasoactive intestinal peptide as a neural mediator of gastric relaxation. Am. J. Physiol. 248:G73-G78; 1984. Grider, J. R.; Rivier, J. R. Vasoactive intestinal peptide (VIP) as transmitter of inhibitory motor neurons of the gut: Evidence from the use of selective VIP antagonists and VIP antiserum. J. Pharmacol. Exp. Ther. 253:738-742; 1990. Hawke, D. H.; Harris, D. C.; Shively, J, E. Microsequence analysis ofpeptides and proteins. V. Design and performance of a novel gasliquid-solid phase instrument. Anal. Biochem. 147:315-330; 1985. Hjelmeland, L. M.; Chrambach, A. Solubilization of functional membrane proteins. In: Jacoby, W. B., ed. Methods in enzymology. New York: Academic Press; 1984:305-318. Ito, O.; Tachibana, S. Vasoactive intestinal polypeptide precursors have highly potent bronchodilatory activity. Peptides 12:131-137; 1991. Keast, J. R.: Furness, J. B; Costa, M. Distribution of certain peptidecontaining nerve fibres and endocrine cells in the gastrointestinal mucosa in five mammalian species. J. Comp. Neurol. 236:403-422; 1985. Kenny, A. J. Endopeptidase-24.11: An ectoenzyme capable of hydrolysing regulatory peptides at the surface of many different cell types. In: Kreutzberg, G. W.: Reddington, M.; Zimmermann, H., eds. Cellular biology ofectoenzymes. Berlin: Springer-Verlag; 1986: 257-271. Kobayashi, R.; Chen, Y.; Lee, T.; Davis, M. T.; Ito, O.; Walsh, J. H. Degradation ofvasoactive intestinal polypeptide by gastric membrane

332

26. 27. 28. 29. 30.

31.

K O B A Y A S H I ET AL. peptidases. Ninth International Symposium on Gastrointestinal Hormones. Leuven, Belgium; 1992. Kobayashi, R.; Sun, X.; Walsh, J. H. Enkephalin-degrading peptidases in the rabbit stomach wall. Characterization and partial purification. Biochem. Res. Suppl. 1:37; 1988 (abstract). Kobayashi, R.; Sun, X.; Walsh, J. H. Angiotensin-converting enzyme in the rabbit stomach wall. Identification in the membrane fraction by affinity purification. Gastroenterology 100:25-32; 1991. Lilly, C. M.; Martins, M. A; Drazen, J. M. Peptidase modulation of vasoactive intestinal peptide pulmonary relaxation in tracheal superfused guinea pig lungs. J. Clin. Invest. 91:235-243; 1993. Malfroy, B.; Swert, J. B.; Guyon, A.; Roques, B. P.; Scwartz, J. C. High affinity enkephalin-degrading peptidase in brain is increased after morphine. Nature 276:523-526: 1978. Mckelvy, J. F. Enzymatic degradation of brain peptides. In: Cowan, W. M.; Shooter, E. M.; Stevens, C. F.; Thompson, R. F., eds. Annual Review of Neuroscience, vol. 9. Palo Alto: Annual Review Inc.; 1986:415-434. Muller, W. E. G.; Schuster, D. K.; Leyhausen, G.; Sobel, C.: Umezawa, H. Cell surface-bound leucine aminopeptidase: Target of the

32. 33. 34.

35. 36. 37.

immunomodulator bestatin. In: Kreutzberg, G. W.; Reddington, M.; Zimmermann, H., eds. Cellular biology ofectoenzymes. Berlin: Springer-Verlag; 1986:285-293. Mutt, V. Vasoactive intestinal polypeptide and related peptides: Isolation and chemistry. Ann. NY Acad. Sci. 527:1-19; 1988. Said, S.; Mutt, V. Polypeptide with broad biological activity: Isolation from small intestine. Science 169:1217-1218; 1970. Schwartz, J.-C.; Gros, C.; Giros, B.; et al. Ectopeptidases responsible for the inactivation of enkephalins. In: Kreutzberg, G. W.; Reddington, M.; Zimmermann, H., eds. Cellular biology ofectoenzymes. Berlin: Springer-Verlag; 1986:272-283. Shively, J. E.; Miller, P.; Ronk, M. Microsequence analysis ofpeptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis. Anal. Biochem. 163:517-529; 1987. Sundler, F.; Ekblad, E.: Grunditz, R.; Hakanson, R.; Uddman, R. Vasoactive intestinal peptide in the peripheral nervous system. Ann. NY Acad. Sci. 527:143-167; 1988. Turner, J. T.; Jones, S. B.; Bylund, D. B. A fragment of vasoactive intestinal peptide, VIP(10-28), is an antagonist of VIP in the colon carcinoma cell line, HT29. Peptides 7:849-854; 1986.