Peptides,Vol. 12, pp. 963-973. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0196-9781/91 $3.00 + .00

Substance P and Neurokinin A Are Codistributed and Colocalized in the Porcine Gastrointestinal Tract P E T E R S C H M I D T , S T E E N S. P O U L S E N , T O R B E N N. R A S M U S S E N , M A U R I Z I O B E R S A N I A N D J E N S J. H O L S T 1

Departments of Medical Physiology C and Anatomy B, The Panum Institute and Department of Experimental Pathology, Rigshospitalet, University of Copenhagen, DK-2200 Copenhagen N, Denmark R e c e i v e d 8 April 1991 SCHMIDT, P., S. S. POULSEN, T. N. RASMUSSEN, M. BERSANI AND J. J. HOLST. Substance P and neurokinin A are codistributed and colocalized in the porcine gastrointestinal tract. PEPTIDES 12(5) 963-973, 1991.--Immunoreactive substance P and neurokinin A were measured with radioimmunoassay in extracts of different segments of porcine gastrointestinal tract using C-terminally directed antisera. In all segments, the concentrations of substance P and neurokinin A were similar. The largest concentrations of both peptides were found in the mid-colon. By gel chromatography and reversed-phase high pressure liquid chromatography the immunoreactivity in extracts from ileum eluted as homogenous peptides at the positions of synthetic substance P and neurokinin A, respectively. No neurokinin B was found. By immunohistochemistry of porcine duodenum, jejunum, ileum and mid-colon, identical localization patterns were found for substance P and neurokinin A, and the two peptides demonstrated by double immunofluorescence to be colocalized in the enteric nervous system of the ileum. We conclude that the tachykinins substance P and neurokinin A are codistributed and colocalized in the porcine gastrointestinal tract and suggest that the two peptides are produced from a common precursor, 13- and/or -/-preprotachykinin, in the same neurons. Gastrointestinal tract

Tachykinins

Coexistence

Radioimmunoassay

SUBSTANCE P and neurokinin A belong to a group of neuropeptides, called tachykinins, which share a common C-terminal sequence of amino acids (13). The group of mammalian tachykinins also includes neurokinin B and the two N-terminally extended forms of neurokinin A, neuropeptide K (22) and neuropeptide ~/(7). Substance P, neurokinin A, neuropeptide K and neuropeptide "y are all encoded by the same gene, which gives rise to or-, 13- or ",/-preprotachykinin A (PlaT A) as a result of alternative splicing of a common transcription product, ot-PPT A may give rise to substance P alone, whereas 13- and ~/-PPT A contain both substance P and neurokinin A (15). Neuropeptide K and ~/are present in the 13- and ~/-PPT A, respectively (7,8). Neurokinin B is encoded by another gene (15). In rat myenteric neurons, large amounts of 13- and ~/-preprotachykinin messenger ribonucleic acid (mRNA), whereas only small amounts of et-preprotachykinin mRNA, are found (19). Substance P-immunoreactive nerve fibers are found in all layers of the gut in most mammalian species, including man. Immunoreactive nerve cell bodies are also found in both the myenteric and submucosal plexus in most species [for review see (5)]. Substance P and neurokinin A coexist in sensory neurons in a large number of tissues (2,20) and the two peptides are found in identically sized synaptic vesicles from guinea pig myenteric neurons (4).

Immunohistochemistry

Pig

The present investigation was undertaken to compare the extracted amounts and the immunohistochemicai distribution of substance P and neurokinin A throughout the porcine gastrointestinai tract in order to determine if the enteric neurons generally express both products of the common tachykinin gene. METHOD

Tissue Samples Four fasted Danish Landrace/Yorkshire pigs of 15 kg were anesthetized with 2.5% haiothane-N20. The abdomen was opened through a midline incision and tissue samples for extraction were excised and immediately frozen on dry ice. Tissue for immunohistochemical investigations was obtained from another 3 animals after perfusion fixation. A catheter was inserted into the aorta, which was subsequently clamped proximally and distally to a segment that included the celiac trunk and the superior (anterior) mesenteric artery. The portal vein was cut open, and the tissue supplied by the catheterized segment of aorta was perfused with a freshly prepared solution of paraformaidehyde (No. 4005, Merck, Darmstadt, FRG), 4% in 0.1 M sodium phosphate, pH 7.4, 200(2, for 10 min. Specimens from duodenum, jejunum, ileum and mid-colon were taken out and postfixed in the

~Requests for reprints should be addressed to Jens J. Hoist, M.D., Department of Medical Physiology C, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, 2200 Copenhagen N, Denmark.

963

964

SCHMIDT ET AL

same solution for 1 hour and then transferred to a 70% ethanol solution.

pmol/g 8 7

Extraction

- ZZS:

6

The frozen tissue samples were immersed into 10 volumes of boiling water, allowed to boil for 20 min, and then immediately placed on ice to cool. The material was blended for 1 min and then centrifuged at 4000 rpm for 30 min. The supernatants were decanted into another tube. The precipitates were resuspended in 10 volumes of 1 M acetic acid and left at room temperature for 1 hour. The homogenates were centrifuged at 4000 rpm for 30 min and the supernatants combined with the first supernatants. The extracts were then freeze dried and reconstituted with assay buffer containing 40 mmol/1 sodium phosphate, pH 7.4, and human serum albumin (Novo-Nordisk, Copenhagen, Denmark) 0.1%, thiomersal (Sigma, St. Louis, MO) 0.6 mmol/l, NaC1 6 g/l, and ethylenediaminetetraacetic acid 20 mmol/1 (17). Recovery of tachykinins in the extraction procedure was studied by adding a known amount of peptide, in a volume corresponding to 10% of the total volume, to one-half of the tissue homogenate before boiling and acetic acid extraction. The amount of peptide added to homogenates before boiling was such as to increase the concentration in the extract by 5, 50 and 500 pmol/1. The other half of the extract served as control. After extraction, the following amounts of added peptide were recovered: 55---3% for substance P, 81 ---2% for neurokinin A, 9 8 ± 5 % for neurokinin B and 80 ± 4% for neuropeptide K.

Radioimmunoassays Production of antiserum. Antisera against synthetic substance P and neurokinin A (Peninsula Laboratories, Inc., San Carlos, CA) were obtained from Danish white rabbits after six and eight injections of 100 i~g of peptide covalently coupled to bovine serum albumin (Sigma Chemical Co., St. Louis, MO). Three mg of substance P/neurokinin A were dissolved in 200 ixl of 0.01 mol/1 HC1, diluted with 800 ILl of 40 mmol/1 sodium phosphate, pH 7.4, and mixed with 5 mg of bovine serum albumin. Three hundred fifty Ixl of a solution of (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma Chemical Co.) (5 mg/ml) were added dropwise. The mixture was incubated overnight with gentle stirring in the dark at room temperature. The solution was diluted with 0.9% NaC1 to a final volume of 16 ml. The rabbits were immunized at multiple sites on the back with 1.1 ml of an emulsion consisting of 0.5 ml of peptide solution and 0.6 ml of Freund's adjuvant (Statens Seruminstitut, Copenhagen, Den-

5 4 3

t 0

o_o ~

3

8

~ o~

-

-

~'~

FIG. 1. Concentrations of substance P and neurokinin A expressed in pmol per g wet tissue in the different regions of the porcine gastrointesfinal tract. Substance P is shown with white columns and neurokinin A with black columns. Mean plus one S.E.M. (n=4).

mark), with complete adjuvant for the first immunization and incomplete adjuvant for the subsequent immunizations. The selected antiserum for substance P (code 250-2) could be used in final dilution of 1:250,000 and for neurokinin A (code 304-4) in 1:150,000, both resulting in 35% binding of 10 fmol labeled peptide. The equilibrium constant, as determined from Scatchard analysis of equilibrium incubations of antisera with labeled and unlabeled peptide, was 1.5 × 1011 1/mol for the substance P antiserum and 8.5 x 101° l/mol for the neurokinin A antiserum. The antiserum for substance P did not cross-react with neurokinin A, neurokinin B, neuropeptide K, galanin (all obtained from Bachem, Bubendorf, Switzerland), neuropeptide % human calcitonin gene related peptide, somatostatin, gastrin releasing peptide and vasoactive intestinal polypeptide (all obtained from Peninsula Laboratories, Belmont, CA) in concentrations up to 10,000 pmol/1, but cross-reacted 57% with substance P sulphoxide. The selected neurokinin A antiserum cross-reacted 50% with neurokinin A sulphoxide, 1% with substance P, 15% with neu-

TABLE 1 DETAILSOF ANTISERA Cross-Reactivity Antigen SP (No. SP (No. NKA (No. NKA (No.

;=

RIA

PAP

lmmunofluorescence

With SP

With NKA

Source

Yes

Yes

No

--

No

Rabbit, this laboratory

No

No

Yes

--

No

Rat, AH Diagnostik

Yes

No

No

1%

--

Rabbit, this laboratory

No

Yes

Yes

No

--

Rabbit, Milab

250-2) 1021) 304-4) B55-100)

SP= substance P, NKA = neurokinin A, RIA = Radioimmunoassay, PAP= peroxidase-antiperoxidase technique.

T A C H Y K I N I N S IN P O R C I N E G A S T R O I N T E S T I N A L T R A C T

NPK

KD

o

o,2

i

i

o.4 ~ I

NPT

o,8 i

~

965

SP NKA.NKB

o.8~; i

lo i

123

pmol/I

8

45

67

8

91011

%B ,50

50O C

7

40 6 30

5

250

Sub~ltance P - I R

20

4 3

/

2

i/ /

1

/

Substance P-IR

10 0

i 10

I 20

i 30

i 40

I 50

i 60

i 70

0

Fraction

pmolll

%B 50

5OO

40 30 250 20

I t/

Neurokinin A-IR

/

A

^

I

I

I

10

20

30

10 0

I

I

I

I

40

50

60

70

Fraction

FIG. 2. (A and B) Gel filtration profiles of immunoreactive substance P (A) and neurokinin A (B) in extracts of the ileum. Elution positions of synthetic substance P, neurokinin A, neurokinin B, neuropeptide K and neuropeptide "t are indicated by arrows on top. The immunoreactivity in the eluate, expressed as percent of the total amount of immunoreactive material applied to the colunm, is plotted against the coefficient of distribution, K d. Mean plus one S.E.M. (n=4). (C and D) High pressure liquid chromatography profiles of immunoreactive substance P (C) and neurokinin A (D) in one ileum extract. Arrows at the top indicate elution positions of: (1) neurokinin A sulphoxide, (2 and 3) oxidized neuropeptide % (4) neurokinin A, (5) neuropeptide % (6) substance P sulphoxide, (7) substance P, (8) neurokinin B sulphoxide, (9) neuropeptide K sulpboxide, (10) neurokinin B and (11) neuropeptide K. By oxidation of neurokinin B two minor forms occur at 25.0 and 25.3 percent of acetonitrile (not shown).

rokinin B, 15% with neurokinin B sulphoxide, 54% with neuropeptide K, 24% with neuropeptide K sulphoxide, 100% with neuropeptide ~/and 84% with neuropeptide ~ sulphoxide, but not with h u m a n calcitonin gene related peptide, somatostatin, gastrin releasing peptide, vasoactive intestinal polypeptide and galanin. All the oxidized tachykinins were prepared as described by Minamino (16).

Preparation of tracers. Synthetic substance P and neurokinin A, labeled with ~2SI-Bolton-Hunter reagent according to the manufacturer's instruction ( A m e r s h a m International, Buckinghamshire, UK), were used as tracers. The labeled peptide was purified using a high pressure liquid chromatography system from L K B and a M N Nucleosil C18 5-300 cartridge. A linear gradient o f acetonitrile (Rathburn, Walkerburn, Scotland, cata-

TABLE 2 OCCURRENCE OF IMMUNOREACTIVE NERVES AND CELLS IN THE VARIOUS TISSUES Immunoreactive Structure Endocrine cells Cells in lamina propria Cells in submucous plexus Fibers in submucous plexus Fibers in circular muscle Cells in myenteric plexus Fibers in myenteric plexus Fibers in longitudinal muscle

Duodenum

Jejunum

0/0 + +/+ + +/+ +/+ +++/+++ 0/0 + +/+ + + +/+

0/0 + /+ + +/+ + /+ ++/+++ 0/0 + +/+ + + 0/+

Ileum 0/0 + + /+ + +/+ + +/+ +++/+++ 0/0 + +/+ + +/+

Colon 0/0 + /+ + /+ 0/0 ++/++ 0/0 + +/+ 0/0

0, The element was not found; + , a small number of subjects; + + , an intermediate number of subjects; + + + , a high density of subjects. Substance P immunoreactivity is shown left of the slash and neurokinin A right of the slash for each organ and structure.

966

SCHMIDT ET AL.

FIG. 3. Substance P immunoreactive varicose nerve fibers (arrows) in the lamina propria from porcine ileum. The outlines on the intestinal surface epithelium can be distinguished in the top and the bottom of the picture. Antibody diluted 1:2000. × 610.

log No. RH 1015)/water/trifluoroacetic acid (Pierce Chemical Co., Rockford, IL, catalog No. 28093) (A 0:99.9:0.1 and B 99.9:0: 0.1) reaching from 20 to 40% in 100 min was used. The specific activity was 70 MBq/nmol for both labeled peptides. Assay procedure. Three hundred la,1 of unknowns and standards were preincubated in duplicate for 24 hours at 4°C with 100 p,1 of antiserum (final dilution of substance P 1:250,000 and neurokinin A 1:150,000) and then incubated with 100 IM of tracer (4000 cpm) for another 24 hours. Synthetic substance P and neurokinin A were used as standards. Separation of bound and free peptides was performed by mixing the samples with 1.5 ml of plasma-coated charcoal (the charcoal was obtained from Merck, Darmstadt, FRG, and plasma was prepared from heparinated bovine blood) and placed at 4°C for 20 min. The tubes were centrifuged at 4°C for 20 min at 3000 rpm and the supernatants were then decanted into counting vials and counted in a scintillation counter for 5 min. All handling of samples outside the cold room was performed on ice. The substance P and neu-

rokinin A concentrations in unknowns were calculated by Ria/ Fia Calc (Pharmacia LKB, Uppsala, Sweden). The intraassay variation (duplicate determinations) was 3.8% for substance P and 5.6% for neurokinin A and the interassay variation was 9.6% for substance P and 9.5% for neurokinin A at a level of 32 pmol/l. Detection limit in assay buffer and the smallest detectable increment were, for both peptides,