KEY WORDS: Vasoactive intestinal peptide (VIP); thymic peptides. Bovine t hymic peptide ... peptide extract occurs through VIP receptors. Finally, no VIP-like ...
Bioscience Reports, Vol. 6, No. 6, 1986
Interaction of a Bovine Thymic Peptide Extract with Vasoactive Intestinal Peptide (VIP) Receptors J. M. Guerrero, 1 R. Goberna, P. Molinero, J. Jimenez and J. R. Caivo Received June 19, 1986
KEY WORDS: Vasoactiveintestinal peptide (VIP); thymic peptides.
Bovine t hymic peptide extract (1-100/~g/ml) is shown to completely inhibit the binding of [x25I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [~ 25I]VIP binding with a potency that is 4000-7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.
INTRODUCTION A growing body of evidence indicates that vasoactive intestinal peptide (VIP) might play a regulatory role in immune response. In fact, high affinity and specific binding sites for VIP (1-5), an adenylate cyclase system highly sensitive to VIP (1,2,6), as well as an AMP-dependent protein kinase activity stimulated by VIP (7,8), have been 1To whom correspondenceshould be addressed. Dept. of Biochemistry,School of Medicine, University of Seville, Avda. S/mchez Pizjufin, 4, 4100% SEVILLE, Spain. 579 0144-8463/86/0600-0579505.00/0 9 Plenum Publishing Corporation
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described in peripheral blood mononuclear cells, lymphocytes from mesenteric lymph nodes, subcutaneous lymph nodes, spleen and Peyer's patches. These cells, however, include different subpopulations such as T-lymphocytes, B-lymphocytes, monocytes and cells of uncertain origin (K-NK cells). The cell type that specifically binds VIP in this mixture of lymphocytes and monocytes appears to be the T-lymphocytes and/or the cells of the K-NK system (4,9). B-lymphocytes do not bind the peptide, and the data obtained with monocytes are unclear (2-4,10). Thymus cells, on the other hand, have few VIP binding sites (4), which suggests that the expression of VIP receptors on cells from the secondary lymphoid organs is related to the differentiation of T cells. In the present work we show how a thymic peptide extract inhibits the binding of VIP to lymphoid cells and liver plasma membranes.
MATERIALS AND METHODS Materiak Synthetic porcine VIP and VIP antiserum were purchased from Peninsula Laboratories (San Carlos, CA, USA); purified porcine monocomponent insulin from Novo (Copenhagen, Denmark); carrier-free Na[ 12Sl] (IMS-30) and 2,8-3H adenosine Y,5'-cyclic phosphate (TRK-498) from the Radiochemical Centre (Amersham, UK). [125I]VIP was prepared as described by Laburthe et al. (11) at a specific activity of about 250 Ci/g. [125I]insulin was prepared at a specific activity of about 300 Ci/g according to the method described by Freychet et al. (12). All other chemicals were reagent grade.
Bovine Thymic Peptide Extract Extraction from bovine thymus of peptides with molecular weights similar to that of VIP was performed as described by Laburthe et al. (11): the thymus was weighed and boiled for 5 rain in deionized water in order to destroy the proteolytic enzymes and coagulate the bulk of the proteins, then extracted with 0.5 M acetic acid (about 5 ml per g of tissue). The sample was homogenized by a mechanical method for 20 seconds, and by ultrasonic disintegration for 10 seconds. After centrifugation (15009 for 20 rain), the supernatant was collected; the pH was then brought to 7.5 with NH4OH. Adsorption of peptides on silicate was performed by adding talc (20 mg per ml). A talc pellet was obtained by centrifugation (1500 g for 20 rain). Peptides were eluted from the adsorbant by l ml of 1 M acetic acid/ethanol (v/v) per 20rag talc. After centrifugation (2500 g for 30 min), the acid-alcohol supernatant was removed, frozen at - 80~ and lyophilized. -['he extract was dissolved in 3 ml of 0.2 M acetic acid and applied on a 1.5 x 70 cm column of Sephadex G-25, equilibrated and eluted with 0.2 M acetic acid. The column was previously calibrated under the same conditions with [12 si] porcine VI P. Fractions of proteins emerging with just the [1 z5I]VIP were collected, frozen, lyophylized and used for experiments.
Thymic Extract Binds VIP Receptors
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Experimental Procedures Peripheral blood mononuclear cells (MNC), lymphoid cells of spleen, and liver plasma membranes were obtained from Wistar rats as described in (t3), C[4) and (I5), respectively. Studies of[125I]VIP binding to rat blood mononuclear cells and lymphoid cells of spleen were conducted as in (1). Binding of [125I]VIP to liver plasma membranes was conducted as in (16). Binding of [~25i] insulin to liver plasma membranes was studied as in (17). Adenylate cyclase activity was assayed as in (16), and radioimmunoassay of VIP as in (18). RESULTS Bovine thymic peptide extract inhibits the specific binding of [t25I]VIP to peripheral blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes (Fig. 1). The dose-response of peptide extract (1--100.ug/ml concentration ~
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Guerrero, Goberna, Molinero, Jimenez, and Calvo
range; EDso = 7-14 #g/ml) parallels that of VIP, suggesting a competitive inhibition of labelled VIP binding by a substance present in the peptide extract. At maximal peptide extract concentration, the binding of [-125I]VIP to the specific receptors is completely suppressed. On a weight basis, 4000-7000 times as much thymic peptide extract as pure porcine VIP are thus needed to achieve an identical effect. Rat liver plasma membranes were previously shown to exhibit specific insulin receptors in addition to VIP receptors (16,17). Bovine thymic peptide extract (1100 #g/ml) does not alter the specific binding of [125I]insulin to its receptors (data not shown), indicating the specificity of action of thymic peptide extract on V! P receptors. As shown in Fig. 2, the bovine thymic peptide extract is able to stimulate cyclic AMP production in rat liver plasma membranes. The dose-response of peptide extract in stimulating the cyclic AMP production (EDso= 7#g/ml) and in inhibiting [125I]VIP binding are very similar, suggesting that a substance present in the thymic peptide extract acts on the enzyme through its interaction with VIP receptors. This is further supported by the fact that bovine thymic peptide extract and VIP tested at maximally active doses do not elicit an additive stimulatory effect on cyclic AMP production (Table 1). Such data suggest that bovine thymic peptide extract contains a VIP-like substance that behaves as a VIP agonist. However, as tested using antibody raised against mammalian VIP, the peptide extract is devoid of VIP-like immunoreactivity (Fig. 3).
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Thymic Extract Binds VIP Receptors
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Table 1. Cyclic AMP production in rat liver plasma membranes Addition
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Fig. 3. Effect of bovine thymic peptide extract on the binding of [t25I]VIP to an antibody raised against mammalian VIP. Results are expressed as the percentage of initial binding of [12sI]VIP to the antibody. Each point is the mean of triplicate determinations. Another experiment gave similar results.
DISCUSSION
The present data indicate that the bovine thymic peptide extract contains a substance that interacts specifically with VIP receptor in both lymphoid cells and liver plasma membranes of the rat. This peptide extract appears to be a full agonist of VIP since it is as efficient as VIP in inhibiting binding of [12si]Vi P to VIP receptors and in stimulating adenylate cyclase. Since the peptide extract could contain some degradative activities, the question whether it interacts with VIP receptors by destroying them has to be considered. Our data, indicating that the peptide extract does not alter the binding of [125I] insulin to its specific receptors, make such a hypothesis very unlikely. The peptide extract may thus contain a VIP-like bioactive substance. The active substance is not yet identified, but the absence of VIP-like immunoreactivity in the peptide extract indicates that VIP agonist does not possess the same antigenic determinant(s) as VI P. The nature of the active substance remains to be
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Guerrero, Goberna, Molinero, Jimenez, and Caivo
d e t e r m i n e d , b u t it is p r o b a b l y a p e p t i d e with a m o l e c u l a r weight of 3000. M a n y peptides have been isolated a n d c h a r a c t e r i z e d in bovine t h y m i c extracts with t h a t m o l e c u l a r weight (19); i.e. t h y m o s i n el (mol. wt., 3108) (20), fla a n d f14 (mol. wt., 4982) (20,21), a n d t h y m i c h u m o r a l factor (tool. wt., 3000) (22). A m o n g these peptides, t h y m o s i n cq consists of 28 a m i n o acids residues a n d possesses 5 a m i n o acids in c o m m o n w i t h the V I P (20), suggesting t h a t this p e p t i d e c o u l d be t h a t responsible for i n t e r a c t i o n with V I P receptors. A definitive a n s w e r to t h a t question has to a w a i t the purification a n d s t r u c t u r a l analysis of this VIP-1ike b i o a c t i v e s u b s t a n c e o r the use of purified/synthetic t h y m i c peptides.
REFERENCES 1. Guerrero, J. M., Prieto, J. C., Elorza, F. L., Ramirez, R., and Goberna, R. (1981). Mol. Cell. Endocrinol. 21:151-160. 2. Calvo, J. R., Guerrero, J. M., Molinero, P., Blasco, R., and Goberna, R. (1986). Gen. Pharmac. 17 : 185189. 3. Danek, A., O'Dorisio, M. S., O'Dorisio, T. M., and George, J. M. (1983). J. Immunol. 131 : 1173-1177. 4. Ottaway, C. A., Bernaerts, C., Chan, B., and Greenberg, R. (1983). J. Physiol. Pharmacol. 61:664-671. 5. Ottaway, C. A., and Greenberg, R. (1984). J. Immunol. 132:417-423. 6. O'Dorisio, M. S., Hermina, N. S., O'Dorisio, T. M., and Balcerzak, S. P. (1981). J. lmmunol. 127:25512556. 7. Guerrero, J. M., Prieto, J. C., Calvo, J. R., and Goberna, R. (1984). Peptides 5:371-373. 8. O'Dorisio, M. S., Wood, C. L., Wenger, G. D., and Vassalo, L. M. (1985). J. Immunol. 134:4078-4085. 9. Rola-Pleszczynki, M., Bolduc, D., and St-Pierre, S. (1985). J. Immunol. 135:2569 2573. 10. Wilk, P., Opstad, P. K., and Boyum, A. (1985). Regul Pept. 12:145-154. 11. Laburthe, M., Bataille, D., and Rosselin, G. (1977). Acta Endocrinol. 84:588-599. 12. Freychet, P., Roth, J., and Neville, D. M. (1971). Biochem. Biophys. Res. Commun. 43:40(008. 13. Boyum, A. (1968). Scan& J. Clin. Lab. Invest. 21:51-76. 14. Cartier, P. H. (1980). J. Biol. Chem. 255:45744582. 15. Neville, D. M. (1968). Biochim. Biophys. Acta 154:540-552. 16. Guerrero, J. M., Prieto, J. C., Ramirez, R., Calvo, J. R., and Goberna, R. (198l ). Rev. Esp. Fisiol. 37 : 1 8. 17. Herrera, M. T., Prieto, J. C., Guerrero, J. M., and Goberna, R. (1981). Horm. Metab. Res. 13:441-445. 18. Besson, J., Laburthe, M., Bataille, D., Dupont, C., and Rosselin, G. (1978). Acta Endocrinol. (Kbh.) 87 : 799-810. 19. Genevienne, S. I. (1983). Clin. Immunol. All. 3:95-117. 20. Low, T. L. K., and Goldstein, A. L. (1979). J. Biol. Chem. 254:987-993. 21. Low, T. L. K., Hu, S. K., and Goldstein, A. L. (1981). Proc. Natl. Acad. Sci. USA 78:1162 1166. 22. Trainin, N., Umiel, T., Klein, B., and Kleir, I. (1980). In: Polypeptide Hormones (Beers, R. F., and Bassett, E. G., eds) pp. 467-488, Raven Press, New York.
