Unité de Recherche de Diabétologie et Radio-Immunologiques des Hormones Protéiques, U. 55 (Institut National de la Santé et de la Recherche Médicale), Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, Paris 12 d'Etudes

VASOACTIVE INTESTINAL PEPTIDE (VIP): TISSUE DISTRIBUTION IN THE RAT AS MEASURED BY RADIOIMMUNOASSAY AND BY RADIORECEPTORASSAY

By

J. Besson, M. Laburthe, D. Bataille, C. Dupont and G. Rosselin

ABSTRACT This work was undertaken to study the distribution of VIP in the digestive tract. VIP was measured both by radioimmunoassay and by radioreceptorassay in order to determine whether immunoreactive VIP is related to a biologically active component. The effect of digestive extracts in inhibiting the binding of porcine [1251]VIP to the antibody (RIA) and to the rat liver plasma membranes (RRA) paralleled that of porcine VIP used as the standard. VIP was found throughout the digestive tract with especially high concentrations between the duodenum (1676 \m=+-\186 ng/g) and the colon (1214 \m=+-\214 ng/g); the maximal quantity occurred in the jejuno-ileum (11 698 \m=+-\687 ng/g). Less than 1 % of VIP was found in the epithelium whereas almost all VIP (> 99%) was localized in the mucosal muscular tissue of the jejunoileum. VIP concentration in the pancreas was 180 \m=+-\26 ng/g of tissue. The VIP contents of the digestive tract were similar when measured either by radioimmunoassay or by radioreceptorassay. Immunoreactive VIP was found in the brain (155 \m=+-\7 ng/g of tissue) and brain extracts competitively inhibited the binding of [125I]VIP (purified from gut) to

liver plasma membranes. These results show that: 1) VIP from the overall gastrointestinal tract is a biological active molecule: 2) VIP from brain binds to receptors for intestinal VIP in liver.

799

The vasoactive intestinal peptide was originally isolated from porcine duo¬ denum by Said 8c Mutt (1970). If is a 28 residue-pepfide (Said 8c Mutt 1972; Mutt 8c Said glucagon and

1974) which is slructurally and biologically related to secrefin, gastric inhibitory peptide (GIP). By immunological techniques including radioimmunoassay and immunohistochemistry, immunoreactive VIP (IR-VIP) was found in different parts of the intestinal tract (Polak et al. 1974; Bloom et al. 1975; Said 8c Rosenberg 1976), in the brain (Said 8c Rosenberg 1976; Giachetti et al. 1976) and in Ihe peripheral nervous system (Larsson et al. 19766). Since VIP is a widely disfributed peptide of endocrine (Polak et al. 1974) and/or neural (Larsson et al. 1976a) origin, it was of special in¬ terest to investigate whether the IR-VIP is related to biologically active com¬ ponents, whatever the tissue from which it originates. In this study we have compared the VIP-like activity extracted from different parts of the rat dige¬ stive tract and from the brain, in two different systems: a radioimmunoassay described in this paper and a radioreceptorassay (Bataille et al. 1974; Laburthe et al. 1977a) which allows of the measurement of the biologically active mole¬ cules. MATERIALS AND METHODS

Materials The following were used as standards and for iodination: highly purified porcine VIP (lot 24/10/74) was a gift from V. Mutt (Karolinska Institute, Stockholm, Sweden). Porcine pancreatic glucagon (lot B 66 K 1070) was a gift from R. J. Schlichtkrull (The Novo Research Institute, Copenhagen, Denmark). Synthetic secretin (S 19) was a gift from E. Wünsch (Max Planck Institute, München, Germany). GIP was a gift from J. C. Brown (University of British Columbia, Vancouver, Canada). Synthetic fragments of VIP: TFA VIP 1-6 (CyIV9), VIP 14-28 (YK IV 68), VIP 18-28 (YKII2) were gifts from M. Bodanszky (Case Western Reserve University, Cleveland, Ohio, USA). Carrier free Na 125I (IMS 300, 600-800 mCi/ml) was purchased from the Radiochemical

Centre, Amersham, England. Other chemicals: bovine serum albumin (BSA, fraction V) was purchased from Miles (USA), bacitracin from Calbiochem (USA), Kallikrein inhibitor (Trasylol) from Bayer (Germany), peptidase inhibitor (fniprol) from Choay (France), talc powder (mean particle size 10 //m) from Lamotte-Coiffard (France). All other chemicals were of reagent grade.

Methods Animals. Wistar rats were obtained from our breeding colony. Male rats weighing about 500 g were housed under diurnal lighting conditions and were given food and water ad libitum. They were killed by decapitation and the organs were immediately removed, weighed, boiled for 3 min in deionised water and stored at -20°C. Before —

weighing, duodenum, jejuno-ileum

and colon were washed by injecting running water into the lumen while the stomach and caecum were cut and washed under running water. Boiling was performed in order to destroy the proteolytic enzymes and coa¬ gulate the bulk of the proteins (Mutt 1959).

800

yield

Extraction

Table 1. of VIP from the different organs. Extraction

Organ

yield

(%)

Oesophagus Stomach

36.2 1 3.5* 38.3 1 1.6

Duodenum

31.9 1 2.4

Jejuno-ileum

53.2 1 1.9 43.1 1 2.1 44.5 1 2.0

Caecum Colon Rectum Pancreas

29.8 1 0.8 49.5 1 4.1 45.7 1 1.0

Brain

Mean ±

sem

of four determinations.

Extraction procedure. Immediately before extraction the organs were thawed, then extracted in 0.5 M acetic acid as described previously (Laburthe et al. 1977a) except that the supernatant of the first centrifugation was brought to pH 7.5 with diluted NH4OH. VIP was then partially purified by adsorption on talc powder. The extraction yield was monitored with [125I]VIP. It ranged from 29.8 ± 0.8% (rectum) to 53.2 ± f.9% (jejuno-ileum) and was reproducible for each type of organ (Table 1). All results were corrected by the extraction yield. A modification, described for somatostatin, of the preceding method (Ghirlanda et al., in press) was performed on each organ for comparison. VIP was also extracted from the pancreas by a modification (Rançon et al. 1974) of the acid-alcohol procedure (Kenny 1955) used for insulin and glucagon. -

Preparation of epithelium from jejuno-ileum mucosa. The epithelium was separated by a minor modification of the methods described previously (Mitjavila et al. 1972; Laburthe et al. 19776): the everted jejuno-ileum was vigourously shaken by hand during 10 min in 100 ml of 14 %o NaCl, EDTA 2.5 mM, pH 7.5 at 4°C. The everted bowel was removed from the cell suspension and is referred to as mucosal-muscular tissue. The cell suspension is centrifuged at 2000 x g for 5 min and the resulting pellet is referred to as mucosal epithelium. -

Analytical procedures was prepared as described previously (Laburthe et al. modification of the initial method (Bataille et al. 1974) using chloramine T and purification by adsorption on silicate. Specific activities ranged from 250 to 300 ,/zCi/ug i.e., a mean of 0.4-0.5 atom of 125I per molecule. ["25I]VIP was stored in the lyophilized form at -80°C. It was purified again by adsorption on silicate (Laburthe et al. 1977a) before each assay procedure.

1) lodination. 1977a) according

[125I]VIP

-

to

a

801 Acta endocr. 87, 4

2) Radioimmunoassay. a) Production of antibodies: Random bred rabbits were a partially purified fraction from porcine jejuno-ileum (Bataille et al. 1975) containing 200 «g of VIP per mg powder, as measured by radioreceptorassay. For each immunization, the rabbits were injected with 200 pig of VIP, according to the method previously described by Said 8c Faloona (1975). Sera were collected on sodium heparinate from the external ear vein. b) Incubation conditions: Incubations were performed, at 4°C, in 250 pil of 0.05 M veronal buffer pH 8.4 containing 5 °/oo bovine serum albumin, Iniprol 2-10" U/l and Trasylol 10-5 U/l. [125I]VIP was used at 50 pg/ml and anti-VIP antiserum (Z05) at 1/10 000 final dilution. Equilibrium for binding was reached after 40 h. [12ÍTJVIP degradation, as measured by talc adsorption, was less than 4 % up to 4 days of in¬ immunized with

-

cubation.

c) Separation of tively adsorbed by

bound from free VIP: After incubation the free peptide was selec¬ 7.5 mg of talc powder suspended in 200//l of 0.05 m veronal buffer, in the presence of 100 jtl of human plasma. After centrifugation at 2000 x g for 15 min, the talc pellets were counted for radioactivity. Control consisted of [125I]VIP in¬ cubated in the same buffer with an aliquot of the samples to be assayed. The [i25I]VIP adsorbed to silicate under these conditions was not significantly different from the [1251] VIP which was adsorbed without samples suggesting the absence of degradation of [l25I]VIP during incubation with the samples.

The radioreceptorassay of VIP was carried out with 3) Radioreceptorassay. [1251] VIP and purified liver plasma membranes (Bataille et al. 1974) with the fol¬ lowing modification: incubations were performed for 30 min at 30°C in Krebs-Ringer phosphate buffer, pH 7.5 that contained 37.5 mM Tris-HCl, 24 mg/ml BSA and 450 ,//g/ml bacitracin as an inhibitor of VIP degradation in a final volume of 0.5 ml. Each tube contained 150-200 pg/ml of [,25I]VIP and 0.2-0.3 mg of plasma membrane proteins per ml of incubation medium. Membrane-bound [125I]VIP was isolated as described previously (Rodbell et al. 1971; Freychet et al. 1971). -

Expression of

results

Results of both binding assays were expressed as the percentage of initial binding (B./Bo x 100, where Bo represents the binding of [l25I]VIP in the absence of unlabelled VIP). Mathematical transformation of the results and statistical analyses were per¬ formed as described elsewhere (Laburthe et al. 1975), with the help of a desk computer fitted with a digital plotter: Linearization of the standard curves and of the sample dilutions

was

, obtained by plotting Ln

B/Bo

100-(B/Bo)

as

a

function of Ln fVIPl.

-

RESULTS

I

Radioimmunoassay of

VIP

-

At the dilution of the antibody Z03 used (1/10 000) 45% of ihe [i25I]VIP added was bound lo the antibody. This [I25I] VIP bound represents Bo in Fig. 1. The binding of [125I]VIP to the antibody was inhibited by increasing amounts of unlabelled VIP (Fig. 1 A). At 60 pg of unlabelled VIP per ml of incubation, the inhibition of the binding of [i25I]VIP to the antibody was significant 802

100-,

24

S

504 £13

£d



CD

0

-2

[VIP] 0

I/ml

,

0.6

0.3

[VIP]

,

[VIP]

Ln

Ln

nM

[VIP]

,

,

2

ng /ml nM

Fig. 1. Radioimmunoassay of VIP. A represents the mean ± sem of six standard curves of porcine VIP performed during a one-month interval; each point of each curve cor¬ responds to the mean of duplicate determination. B represents the linearization of the standard curve obtained by the logit-log transformation (see Methods).

(jP

Vasoactive intestinal peptide (VIP): tissue distribution in the rat as measured by radioimmunoassay and by radioreceptorassay.

Unité de Recherche de Diabétologie et Radio-Immunologiques des Hormones Protéiques, U. 55 (Institut National de la Santé et de la Recherche Médicale),...
689KB Sizes 0 Downloads 0 Views