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Characterization of calcitonin gene-related peptide receptors and adenylate cyclase response in the murine macrophage cell line P388 D1
Neuropeptides (1991) 19,43-49 @ Longman Group UK Ltd 1991
Characterization of Calcitonin Gene-Related Peptide Receptors and Adenylate Cyclase Response in the Murine Macrophage Cell Line P388 Dl J. ABELLO, D. KAISERLIAN”, J. C. CUBER, J. P. REVILLARD* and J. A. CHAYVIALLE lnserm U 45, Pavilion Hbis, H&p&at Edouard Herriot, F-63437 Lyon Ckdex 3, France *lnserm U 80 CNRS URA 1177 UCBL, Pavilion P, Hbpital Edouard Herriot, F-69437 Lyon CMex 3, France (Reprint requests to JAI
receptors for calcitonin gene-related peptide (CGRP) were identified and characterized on plasma membranes from the interleukin-1 secreting murine macrophage-like cells line P388 01. The binding of [‘251]-ratCGRP I was time-dependent, reversible and the rate of dissociation of [1251]-ratCGRP I increased in the presence of GTP. Scatchard analysis was consistent with a single class of binding sites, with an apparent dissociation constant of 1.76nM and a maximal binding capacity of 85.48 fmol/mg protein. In competitive displacement studies, rat CGRP I, human CGRP I and human CGRP II were equipotent to inhibit the binding of [‘251]-ratCGRP I (I&,0 = 4nM) while rat CGRP II and the synthetic analogue [tyr”]-human CGRP I were ten-fold less potent. Porcine calcitonin and VIP did not inhibit tracer binding. In the presence of GTP, CGRP stimulation of adenylate cyclase was dose-dependent and strongly correlated with receptor occupation. These results indicate that the P388 Dl macrophage-like cell line expresses CGRP specific receptors functionally coupled to adenylate cyclase, which may be involved in CGRP-mediated macrophage immune response.
Abstract-Specific
Introduction
a wide spectrum of biological actions, including vasodilatation, positive inotropic and chronotropic effects on the heart, amylase release and reduction of gastric acid secretion (6-9). Studies using radiolabelled ligands have demonstrated specific receptors for CGRP in central nervous system (lo), heart and spleen (7)) lung (II), liver (12) and pancreas (8). Cells involved in immune responses were also identified as targets of CGRP. The peptide was reported to inhibit the proliferative response of T lymphocytes to mitogens (13)
Calcitonin gene-related peptide (CGRP) is a 37amino acid peptide produced by an alternative processing of the calcitonin gene (1, 2). The peptide is widely distributed in both the central and peripheral nervous system (3,4), including the nerves of the gastrointestinal tract (5). CGRP has
Date received 21 January 1991 Date accepted 14 February 1991
43
44 and to inhibit macrophage activation (14). The identification of CGRP binding sites in mouse T lymphocytes was recently reported (15), but the mechanisms of action of CGRP on macrophages have not been delineated as yet. We report the presence of specific-binding sites for CGRP on the murine macrophage-like cell line P388 Dl and demonstrate that CGRP receptors are coupled to the membrane adenylate cyclase system. Materials and Methods Cell culture. P388 Dl mouse macrophage-like cells were grown in culture flasks with RPMI-1640 medium supplemented with 10% (V/V) heat-inactivated fetal calf serum, antibiotics (lOOU/ml penicillin + 50 p&ml streptomycin), 1 mM sodium pyruvate, 2mM glutamine and 1% non essential amino acid solution in Minimum Eagle Medium (MEM) in a humidified incubator containing 5% CO2 and 95% air at 37°C. Cells were transferred every 3 days in seven volumes of fresh medium. Membrane preparation. The cells were harvested from culture flasks with a rubber policeman and centrifuged at 1000 X g for 1Omin at 20°C. Pellet cells were lyzed in hypotonic 1 mM NaHCOs (pH 7.0) and dispersed in liquid nitrogen. After thawing, the lyzate was homogenized and centrifuged at 4°C for 10min at 2000 x g. The supernatant was further centrifuged for 15 min at 12000 x g. The resulting crude pellet was resuspended in 1mM NaHCOs to a final 0.5-l.Omg protein/ml concentration and immediately used for binding studies and adenylate cyclase assay. Protein concentration was measured according to Lowry et al. (16) using bovine serum albumin as standard. Radioiodination of rat CGRP I. Rat CGRP I was
labelled with 125 iodine by the chloramine-T technique. For this purpose, 5 pg of peptide dissolved in 5 l~l isotonic saline was mixed with 10~1 phosphate buffer (0.5M, pH 7.5). After addition of 1mCi (Na[1251] the mixture was incubated for 30s with lOl.~.l chloramine-T (2 mg/ml). The reaction was stopped by addition of lop.1 metabisulfite (8mg/ml). The purification of the labelled peptide was performed by reverse-phase high
NEUROPEPTIDES
performance liquid chromatography on a C-18 PBondapak column (Waters). The elution buffer was water: acetonitrile (69/31) containing 0.05% trifluoroacetic acid. The flow rate was 1.5mYmin with a back pressure of 1500psi. [1251]-rat CGRP I was stored with 1% bovine serum albumin at -20°C. Binding of [I”Z]-rat CGRP I. [1251]-rat CGRP I binding to P388 Dl macrophage membranes was carried out in 50mM Tris-maleate buffer (pH 7.4) containing 5mM MgCl2, 5mg/ml bacitracin, 100 kallikrein inhibitor units/ml, and 1% bovine serum albumin. Incubation was conducted in a final volume of 120~1 at 37°C in the presence of 80-100pM [‘251]-rat CGRP I, 20-30 u,g membrane protein, and increasing concentrations of unlabelled peptide. The reaction was stopped by adding 2ml of an ice-cold 50mM sodium phosphate buffer (pH 7.4) to each sample, followed by rapid filtration through glass fibre filters (GF/C, Whatman, Maidstone, Kent, UK) presoaked for 24h in 0.1% poly (ethyleneimine) in order to reduce non specific adsorption of tracer to the filter. The filters were further rinsed three times with 2ml of the same buffer and their radioactivity Non specific binding was was measured. determined in the presence of 1 ~J,Mrat CGRP I. Specific binding was defined as total binding minus non specific binding. Degradation of [‘251]-rat CGRP I (assessed after 10% trichloracetic acid precipitation) was less than 10% at the end of the incubation. Adenylate cycfase assay. Adenylate cyclase activity
was determined with minor modifications of the procedure of Salomon et al (17). Membrane protein (15-25 pg) was incubated for 15min at 37°C in a total volume of 60~1 containing 0.5mM [alpha-32P] ATP, 5mM MgC12, 0.5mM EGTA, 1mM cyclic AMP, 1 mM theophylline, 10mM 30 &ml pyruvate phospho(enol)pyruvate, kinase, and 30mM Tris-HCl at a final pH of 7.5. The reaction was initiated by addition of membranes and was stopped aftr a 15min incubation at 37°C by adding 0.5ml of a 0.5% sodium dodecylsulfate solution containing 0.5mM ATP, 0.5mM cyclic AMP and 200OOcpm [8-3H] cyclic AMP (for determination of cyclic nucleotide recovery).
GENE-RELATED PEPTIDE RECEPTORS AND ADENYLATE
45
CYCLASE RESPONSE
TOTAL EINMNC
All other reagents were of the highest available grade. Results Identification
oF
0
~
NON~SI'
10
;
20
I
:
30
nME(mln) Fig. 1
Time-course of [ 1251]-ratCGRP I binding to murine P388 Dl macrophage membranes at 37°C. Membranes (15-25 pg) were incubated with 80-1OOpM [1251]-ratCGRP I. Specific binding represented the difference between total binding and non-specific binding observed in the presence of 1 KM unlabelled rat CGRP I. Results are the mean values of three experiments performed in duplicate.
Cyclic AMP was separated from ATP by two successive chromatography steps on Dowex 50-W-X8 and neautral alumina. Under these conditions adenylate cyclase activity remained linear for at least 30min. Chemicals. RPMI-1640 medium and glutamine were purchased from Vietech (St Bonnet-deMure, France), antibiotics from BiomCrieux (Lyon, France), amino acid solution and sodium pyruvate from Eurobio (Paris, France) and fetal calf serum from PBS Orgenics (Illkirch, France). Synthetic rat CGRPs I and II, human CGRPs I and II, [tyr”]-human CGRP I, [tyr’]-rat CGRP I (28-37) and VIP were from Peninsula (St Helens, UK). [Tyr“]-rat CGRP I (23-37) was purchased from Bachem (Bubendorf, Switzerland). Porcine calcitonin was from Rorer (Levallois, France). Carrier-free Na[‘251] and [8-3H] cyclic AMP (20-30 Ci/mmol) were purchased from Amersham International (Amersham, Bucks, UK). [Alpha-32P] ATP (30 Ci/mmol) was from New England Nuclear (Boston, Ma, USA). Bovine serum albumin (fraction V), bacitracin, phospho(eno1) pyruvate, pyruvate kinase, cyclic AMP, GTP, ATP and Gpp(NH)p were purchased from Sigma (St Louis, MO, USA). Kallikrein inhibitor (Antagosan) was from Behring (Puteaux, France).
of CGRP binding sites. [‘251]-rat CGRP I bound rapidly to P388 Dl macrophage membranes. Equilibrium was achieved after a lo-min incubation period at 37°C and remained stable for at least 30min (Fig. 1). Non specific binding, determined by incubating the tracer and membranes in the presence of 1 PM unlabelled rat CGRP I, represented less than 20% of total binding. Dissociation of specifically bound [125I] -rat CGRP I (about 5500 cpm/mg protein) was measured after a 15min incubation at 37°C (Fig.2). The addition of 10k.M GTP provoked a 15% release of bound tracer within 5min. After adding 1 PM rat CGRP I at equilibrium, the dissociation of the tracer was rapid, 50% of the tracer being released after 7min. Combination of 10 &M GTP and 1 PM rat CGRP I reducud that time to 3min. Scatchard analysis of specific [125I]
+10@4GTP
+lOpb¶cm and lp?d ICGRPI 0 (
‘
I
1
0
10
20
30
TIME Fig. 2
(min)
Time-course of dissociation of [l”I]-rat CGRP I bound to murine P388 Dl macrophage membranes. Membranes were pre-incubated with 80-1OOpM [‘251]-rat CGRP I for 15min at 37°C. The radioactivity bound to membranes was measured at serial times under control conditions and after addition of either 1OpM GTP, 1 PM unlabelled rat CGRP I or both 1OkM GTP and 1 pM rat CGRP I. The data are the mean values of two experiments performed in duplicate.
46
NEUROPEPTIDES
I&, (4nM) whereas rat CGRP II and [tyr’]-human CGRP I were lo-fold less potent to displace tracer binding. [Tyr’]-rat CGRP I (23-37) and [tyr”]-rat CGRP I (2837), i.e. CGRP fragments lacking the 22 and 27 N-terminal amino acids respectively, recognized CGRP receptors albeit lOOO-fold less efficiently than intact rat CGRP I. Neither VIP nor porcine calcitonin caused detectable inhibition of 60 0 40 SO 100 20 binding at concentrations up to 1 ~J,Mand 10kM -I respectively. Secretin, somatostatin, cholecystoBOUND ffmol . mg protein 1 kinin, neurotensin and substance P did not displace [1251]-rat CGRP I binding at concentraFig. 3 Scatchard analysis of specific binding of [1251]-rat CGRP I to murine P388 Dl macrophage membranes. Memtions of 10 PM (not shown). branes CGRP shown ments
were incubated with serial concentrations of [1251]-rat I from 1.6pM to 640pM for 15min at 37°C. Results are the representative data of three separate experiperformed in duplicate.
activity in P388 Dl macrophage membranes was low (12 + 2 pmol cyclic AMP formed. mini. mg protein-‘, mean f SEM of 7 determinations). GTP induced a moderate stimulation (+ 60%), and the guanine triphosphate nucleotide analogue Gpp(NH)p and NaF provoked 12- and lo-fold stimulations of activity respectively (not shown). In the presence of 10~M GTP, rat CGRP I, human CGRP I and human CGRP II stimulated adenylate cyclase activity with a similar EC50 of 6nM (Fig. 5). Rat CGRP II was less potent (EC50 = 40nM). Considering rat CGRP I as the reference peptide (intrinsic activity = l.O), the efficacy of human CGRPs I and II was 0.75. Addition of tyrosine in
-rat CGRP I binding revealed the presence of one class of binding sites, with a concentration required to occupy 50% of the sites (I&) of 1.76nM and a maximum binding capacity (Bmax) of 85.48fmoYmg protein (Fig. 3). Characteristics of CGRP binding sites. CGRP binding sites were studied by comparing competition curves between the tracer and rat CGRPs (Fig. 4, left panel) or human CGRPs (Fig. 4, right panel) after a 15min incubation period at 37°C. Rat CGRP I, human CGRP I and human CGRP II inhibited [‘251]-rat CGRP I binding with a similar
~‘~~~ 0
t
’
-10
5
I
.
-9
-8
1
-7
PEPTIDE (log M)
Activation of membrane adenylate cyclase through CGRP receptors. The basal adenylate cyclase
5rry
,x;,,
.
-6
-5
0
-10
-9
-7
-6
-5
PEPTIDE (log Ml
Fig. 4 Inhibition of [lZsI]-rat CGRP I binding to murine P388 Dl macrophage membranes by (left panel) rat CGRP I, rat CGRP II, [tyr’]-rat CGRP I (23-37), [tyr”]-rat CGRP I (28-37), and by (right panel) human CGRP I, human CGRP II, [tyr”]-human CGRP I, VIP and porcine calcitonin. Membranes were incubated for 15min at 37°C with [iZSI]-rat CGRP I and increasing concentrations of unlabelled peptide. The data are the mean values of three experiments performed in duplicate.
GENE-RELATED
PEFTIDE RECEPTORS AND ADENYLATE
47
CYCLASE RESPONSE 120
60
rCGRPl
-10
-9
-6
-7
-6
PEPTIDE (log MI
0
-10
-9
-8
-7
-6
-5
PEPTIDE (log M)
Fig. 5 Dose-effect curves of adenylate cyclase activation in murine P388 Dl macrophage membranes by (left panel) rat CGRP I, rat CGRP II, [tyr”]-rat CGRP I (23-37) [tyr’]-rat CGRP I (28-37) and by (right panel) human CGRP I, human CGRP II, [tyr”]-human CGRP I, VIP and porcine calcitonin. Adenylate cyclase activity was determined in the presence of lOl.rM GTP. The results are expressed as pmol cyclic AMP. min-‘. mg protein-i (after substraction of the basal unstimulated values) and are the mean values of three experiments performed in duplicate.
position 0 decreased the potency of human CGRP I (EC50 = 30nM) without affecting its efficacy. VIP, porcine calcitonin and the synthetic fragments [tyr’]-rat CGRP I (23-37) and [tyr’]-rat CGRP I (28-37) were devoid of detactable stimulatory effects. [Tyr’]-rat CGRP I (23-37) and [tyr”] -rat CGRP I (28-37) slightly shifted the rat CGRP I adenylate cyclase stimulation curve to the right with a similar inhibition constant (Ki) of 10 FM and without significant modification of maximal activation, affording presumed evidence for competitive inhibition (not shown). Discussion
study demonstrated for the first time the existence of CGRP receptors on membranes from a murine macrophage-like cell line. Binding of [12’I]-rat CGRP I was time-dependent and reversible. The specificity of binding sites was demonstrated by the relative abilities of CGRP from different species to displace [12’I]-rat CGRP I, whereas calcitonin and unrelated peptides were uneffective. From Scatchard analysis, CGRP binding sites were found to consist in a single class of high affinity sites. The CGRP receptors found on P388 Dl cells were coupled to the guaninenucleotide-binding regulatory protein Gs, as based on GTP stimulation of adenylate cyclase and
This
NEUR.
C
on the marked acceleration of [12’I]-rat CGRP I dissociation by GTP in the presence of ll,~M unlabelled rat CGRP I. In addition CGRP concentrations required for half-maximal adenylate cyclase stimulation were similar to those required for half-maximal inhibition of tracer binding, suggesting a close linkage between receptor occupation and adenylate cyclase stimulation. On the basis of binding studies (inhibition of [‘251]-rat CGRP I) and functional studies (direct stimulation of adenylate cyclase activity), the receptor recognized with decreasing potency rat CGRP I = human CGRPs I and II > rat CGRP II = [tyr’]-human CGRP I, whereas calcitonin was without effect on tracer binding and adcnylate cyclase activity. The profile of CGRP receptors on P388 Dl cells clearly differs from that of CGRP receptors in the brain and spinal cord membranes (18) that were not linked to the adenylate cyclaseCAMP system. They also differ from liver, spleen and lung CGRP receptors (7, 11, 12) that efficiently recognize calcitonin, from receptors on pancreatic acini (8) on the basis of the relative efficacy of rat CGRP, human CGRP and CGRP analogues to displace tracer binding, and from mouse T lymphocyte receptors that recognize CGRP with a higher affinity (15). Furthermore our results suggest that slight structural modification in the C-terminal or N-terminal parts of the
48 molecule, such as monosubstitution of an amino acid in position 35 or addition of a tyrosine residue to the N-terminal end of the peptide, reduced the affinity for the CGRP receptor. In contrast the C-terminal fragments lacking the ring structure, [tyr’]-rat CGRP I (23-37) and [tyr’]-rat CGRP I (28-37), showed moderate inhibition of tracer binding and were devoid of adenylate cyclase activity, indicating that the C-terminal part of CGRP may be implicated in receptor recognition while the N-terminal site including the disulphide bridge is essential for biological activity (19). In addition [tyr”]-rat CGRP I (23-37) and [tyr’]-rat CGRP I (28-37) produced a moderate right shift in the rat CGRP I-dose response curve of adenylate cyclase without reduction in the maximum response elicited by rat CGRP I. Such results suggest that these analogues exert a competitive with a weak intrinsic type of antagonism, anatagonist activity at 10 ~J,M. Neuropeptides have been shown to play an increasingly important role in immune regulation. Substance P and VIP modulate immunoglobulin production (20) and lymphocyte cytotoxic activity (21, 22). In addition substance P increases the lymphocyte proliferation whereas VIP, somatostatin and CGRP have inhibitory effects (13, 20). The activity of macrophages can also be affected by neuropeptides, such as substance P and neurotensin that enhance the phagocytic activity by interacting through specific receptors (23, 24). Recently CGRP was found to inhibit the ability of macrophages to produce Hz02 in response to interferon-gamma and to reduce their capacity to activate T lymphocytes (14). Taking additionally in account that macrophages produce several cytokines, such as interleukin-1, interleukind and tumor necrosis factor-alpha, and prostaglandins, the functional relationship between the occupation of CGRP receptors and some intracellular biochemical events remains to be investigated.
NEUROPEFTIDES
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Acknowledgements This work was supported by grant from the Comite des Aides a la Recherche Fournier-Dijon, France.
13.
References 1. Amara, S. G., Jonas, V., Rosenfeld, M. G., Ong, E. S. and
14.
Evans, R. M. (1982) Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298: 240-244. Rosenfeld, M. G., Mermod, J. J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, W. W. and Evans, R. M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304: 129-13.5. Skofitsch, G. and Jacobowitz, D. M. (1985) Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6: 721745. Mulderry, P. K., Ghatei, M. A., Spokes, R. A., Jones, P. M., Pierson, A. M., Hamid, Q. A., Kanse, S., Amara, S. G., Burrin, J. M., Legon, S., Polak, J. M. and Bloom, S. R. (1988) Differential expression of CGRP I and CGRP II by primary sensory neurons and enteric autonomic neurons of the rat. Neuroscience 25: 195-205. Sternini, C., Reeve, J. R. and Brecha, N. (1987) Distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal and capsaicin-treated rats. Gastroenterology 93: 852-862. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. and Macintyre, I. (1985) Calcitonin gene-related peptide is a potent vasodilator. Nature 313: 54-56. Sigrist, S., France-Cereceda, A., Muff, R., Henke, H., Lundberg, J. M. and Fischer, J. A. (1986) Specific receptors and cardiovascular effects of calcitonin generelated peptide. Endocrinology 119: 381-389. Zhou, Z. C., Villanueva, M. L., Noguchi, M., Jones, S. W., Gardner, J. D., and Jensen, R. T. (1986) Mechanism of action of calcitonin gene-related peptide in stimulating pancreatic enzyme secretion. American Journal of Physiology 251: G391-G397. Beglinger, C., Born, W., Hildebrand, P., Ensinck, J. W., Burkhardt, F., Fischer, J. A. and Gyr, K. (1988) Calcitonin gene-related peptides I and II and calcitonin: distinct effects on gastric acid secretion in humans. Gastroenterology 95: 958-965. Tschopp, F. A., Henke, H., Petermann, J. B., Tobler, P. H., Jansen, R., Hokfelt, T., Lundberg, J. M., Cuello, C. and Fischer, J. A. (1985) Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary. Proceedings of the National Academy of Sciences of the U.S.A. 82: 248-252. Mak, J. C. W. and Barnes, P. J. (1988) Autoradiographic localization of calcitonin gene-related peptide binding sites in human and guinea pig lung. Peptides 9: 957-963. Yamaguchi, A., Chiba, T., Okimura, Y., Yamatani, T., Morishita, T., Nakamura, A., Inui, T., Noda, T. and Fujita, T. (1988) Receptors for calcitonin gene-related peptide on the rat liver plasma membranes. Biochemical and Biophysical Research Communications 152: 383-391. Umeda, Y., Takamiya, M., Yoshizaki, H. and Arisawa, M. (1988) Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide. Biochemical and Biophysical Research Communications 154: 227-235. Nong, Y. H., Titus, R. G., Ribeiro, J. M. C. and Remold,
GENE-RELATED
15.
16.
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20.
PEPTIDE RECEPTORS AND ADENYLATE
H. G. (1989) Peptides encoded by the calcitonin gene inhibit macrophage function. Journal of Immunology 143: 45-49. Umeda, Y. and Arisawa, M. (1989) Characterizationof the calcitonin gene-related peptide receptor in mouse T lymphocytes. Neuropeptides 14: 237-242. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275. Salomon, Y., Londos, C. and Rodbell, M. (1974) A highly sensitive adenylate cyclase assay. Analytical Biochemistry 58: 541-548. Gohzman, D. and Mitchell, J. (1985) Interaction of calcitonin and calcitonin gene-related peptide at receptor sites in target tissues. Science 227: 1343-1345. Tjppins, J. R., Di Matzo, V., Panico, M., Morris, H. R. and Macintyre, I. (1986) Investigation of the structure/ activity relationship of human calcitonin gene-related peptide. Biochemical and Biophysical Research Communications 134: 1306-1311. Stanisz, A. J., Befus, D. and Bienenstock, J. (1986)
CYCLASE RESPONSE
21.
22.
23.
24.
49
Differential effects of vasoactive intestinal peptide, substance P and somatostatin on immunoglobulin synthesis and proliferation by lymphocytes from Peyer’s patches, mesenteric lymph nodes, and spleen. Journal of Immunology 136: 152-156. Bienenstock, J., Croitoru, K., Ernst, P. B., Stead, R. H. and Stanisz, A. (1989) Neuroendocrine regulation of mucosal immunity. Immunological Investigations 18: 69-76. Rola-Pleszczynski, M., Bolduc, D. and St-Pierre, S. (1985) The effects of vasoactive intestinal peptide on human natural killer cell function. Journal of Immunology 135: 2569-2573. Hartung, H. P., Wolters, K. and Toyka, K. V. (1986) Substance P: binding properties and studies on cellular responses in guinea pig macrophages. Journal of Immunology 136: 3856-3863. Bar-Shavit, Z., Terry, S., Blumberg, S. and Goldman, R. (1982) Neurotensin-macrophage interaction: specific binding and augmentation of phagocytosis. Neuropeptides 2: 325-335.
Characterization of Calcitonin Gene-Related Peptide Receptors and Adenylate Cyclase Response in the Murine Macrophage Cell Line P388 Dl J. ABELLO, D. KAISERLIAN”, J. C. CUBER, J. P. REVILLARD* and J. A. CHAYVIALLE lnserm U 45, Pavilion Hbis, H&p&at Edouard Herriot, F-63437 Lyon Ckdex 3, France *lnserm U 80 CNRS URA 1177 UCBL, Pavilion P, Hbpital Edouard Herriot, F-69437 Lyon CMex 3, France (Reprint requests to JAI
receptors for calcitonin gene-related peptide (CGRP) were identified and characterized on plasma membranes from the interleukin-1 secreting murine macrophage-like cells line P388 01. The binding of [‘251]-ratCGRP I was time-dependent, reversible and the rate of dissociation of [1251]-ratCGRP I increased in the presence of GTP. Scatchard analysis was consistent with a single class of binding sites, with an apparent dissociation constant of 1.76nM and a maximal binding capacity of 85.48 fmol/mg protein. In competitive displacement studies, rat CGRP I, human CGRP I and human CGRP II were equipotent to inhibit the binding of [‘251]-ratCGRP I (I&,0 = 4nM) while rat CGRP II and the synthetic analogue [tyr”]-human CGRP I were ten-fold less potent. Porcine calcitonin and VIP did not inhibit tracer binding. In the presence of GTP, CGRP stimulation of adenylate cyclase was dose-dependent and strongly correlated with receptor occupation. These results indicate that the P388 Dl macrophage-like cell line expresses CGRP specific receptors functionally coupled to adenylate cyclase, which may be involved in CGRP-mediated macrophage immune response.
Abstract-Specific
Introduction
a wide spectrum of biological actions, including vasodilatation, positive inotropic and chronotropic effects on the heart, amylase release and reduction of gastric acid secretion (6-9). Studies using radiolabelled ligands have demonstrated specific receptors for CGRP in central nervous system (lo), heart and spleen (7)) lung (II), liver (12) and pancreas (8). Cells involved in immune responses were also identified as targets of CGRP. The peptide was reported to inhibit the proliferative response of T lymphocytes to mitogens (13)
Calcitonin gene-related peptide (CGRP) is a 37amino acid peptide produced by an alternative processing of the calcitonin gene (1, 2). The peptide is widely distributed in both the central and peripheral nervous system (3,4), including the nerves of the gastrointestinal tract (5). CGRP has
Date received 21 January 1991 Date accepted 14 February 1991
43
44 and to inhibit macrophage activation (14). The identification of CGRP binding sites in mouse T lymphocytes was recently reported (15), but the mechanisms of action of CGRP on macrophages have not been delineated as yet. We report the presence of specific-binding sites for CGRP on the murine macrophage-like cell line P388 Dl and demonstrate that CGRP receptors are coupled to the membrane adenylate cyclase system. Materials and Methods Cell culture. P388 Dl mouse macrophage-like cells were grown in culture flasks with RPMI-1640 medium supplemented with 10% (V/V) heat-inactivated fetal calf serum, antibiotics (lOOU/ml penicillin + 50 p&ml streptomycin), 1 mM sodium pyruvate, 2mM glutamine and 1% non essential amino acid solution in Minimum Eagle Medium (MEM) in a humidified incubator containing 5% CO2 and 95% air at 37°C. Cells were transferred every 3 days in seven volumes of fresh medium. Membrane preparation. The cells were harvested from culture flasks with a rubber policeman and centrifuged at 1000 X g for 1Omin at 20°C. Pellet cells were lyzed in hypotonic 1 mM NaHCOs (pH 7.0) and dispersed in liquid nitrogen. After thawing, the lyzate was homogenized and centrifuged at 4°C for 10min at 2000 x g. The supernatant was further centrifuged for 15 min at 12000 x g. The resulting crude pellet was resuspended in 1mM NaHCOs to a final 0.5-l.Omg protein/ml concentration and immediately used for binding studies and adenylate cyclase assay. Protein concentration was measured according to Lowry et al. (16) using bovine serum albumin as standard. Radioiodination of rat CGRP I. Rat CGRP I was
labelled with 125 iodine by the chloramine-T technique. For this purpose, 5 pg of peptide dissolved in 5 l~l isotonic saline was mixed with 10~1 phosphate buffer (0.5M, pH 7.5). After addition of 1mCi (Na[1251] the mixture was incubated for 30s with lOl.~.l chloramine-T (2 mg/ml). The reaction was stopped by addition of lop.1 metabisulfite (8mg/ml). The purification of the labelled peptide was performed by reverse-phase high
NEUROPEPTIDES
performance liquid chromatography on a C-18 PBondapak column (Waters). The elution buffer was water: acetonitrile (69/31) containing 0.05% trifluoroacetic acid. The flow rate was 1.5mYmin with a back pressure of 1500psi. [1251]-rat CGRP I was stored with 1% bovine serum albumin at -20°C. Binding of [I”Z]-rat CGRP I. [1251]-rat CGRP I binding to P388 Dl macrophage membranes was carried out in 50mM Tris-maleate buffer (pH 7.4) containing 5mM MgCl2, 5mg/ml bacitracin, 100 kallikrein inhibitor units/ml, and 1% bovine serum albumin. Incubation was conducted in a final volume of 120~1 at 37°C in the presence of 80-100pM [‘251]-rat CGRP I, 20-30 u,g membrane protein, and increasing concentrations of unlabelled peptide. The reaction was stopped by adding 2ml of an ice-cold 50mM sodium phosphate buffer (pH 7.4) to each sample, followed by rapid filtration through glass fibre filters (GF/C, Whatman, Maidstone, Kent, UK) presoaked for 24h in 0.1% poly (ethyleneimine) in order to reduce non specific adsorption of tracer to the filter. The filters were further rinsed three times with 2ml of the same buffer and their radioactivity Non specific binding was was measured. determined in the presence of 1 ~J,Mrat CGRP I. Specific binding was defined as total binding minus non specific binding. Degradation of [‘251]-rat CGRP I (assessed after 10% trichloracetic acid precipitation) was less than 10% at the end of the incubation. Adenylate cycfase assay. Adenylate cyclase activity
was determined with minor modifications of the procedure of Salomon et al (17). Membrane protein (15-25 pg) was incubated for 15min at 37°C in a total volume of 60~1 containing 0.5mM [alpha-32P] ATP, 5mM MgC12, 0.5mM EGTA, 1mM cyclic AMP, 1 mM theophylline, 10mM 30 &ml pyruvate phospho(enol)pyruvate, kinase, and 30mM Tris-HCl at a final pH of 7.5. The reaction was initiated by addition of membranes and was stopped aftr a 15min incubation at 37°C by adding 0.5ml of a 0.5% sodium dodecylsulfate solution containing 0.5mM ATP, 0.5mM cyclic AMP and 200OOcpm [8-3H] cyclic AMP (for determination of cyclic nucleotide recovery).
GENE-RELATED PEPTIDE RECEPTORS AND ADENYLATE
45
CYCLASE RESPONSE
TOTAL EINMNC
All other reagents were of the highest available grade. Results Identification
oF
0
~
NON~SI'
10
;
20
I
:
30
nME(mln) Fig. 1
Time-course of [ 1251]-ratCGRP I binding to murine P388 Dl macrophage membranes at 37°C. Membranes (15-25 pg) were incubated with 80-1OOpM [1251]-ratCGRP I. Specific binding represented the difference between total binding and non-specific binding observed in the presence of 1 KM unlabelled rat CGRP I. Results are the mean values of three experiments performed in duplicate.
Cyclic AMP was separated from ATP by two successive chromatography steps on Dowex 50-W-X8 and neautral alumina. Under these conditions adenylate cyclase activity remained linear for at least 30min. Chemicals. RPMI-1640 medium and glutamine were purchased from Vietech (St Bonnet-deMure, France), antibiotics from BiomCrieux (Lyon, France), amino acid solution and sodium pyruvate from Eurobio (Paris, France) and fetal calf serum from PBS Orgenics (Illkirch, France). Synthetic rat CGRPs I and II, human CGRPs I and II, [tyr”]-human CGRP I, [tyr’]-rat CGRP I (28-37) and VIP were from Peninsula (St Helens, UK). [Tyr“]-rat CGRP I (23-37) was purchased from Bachem (Bubendorf, Switzerland). Porcine calcitonin was from Rorer (Levallois, France). Carrier-free Na[‘251] and [8-3H] cyclic AMP (20-30 Ci/mmol) were purchased from Amersham International (Amersham, Bucks, UK). [Alpha-32P] ATP (30 Ci/mmol) was from New England Nuclear (Boston, Ma, USA). Bovine serum albumin (fraction V), bacitracin, phospho(eno1) pyruvate, pyruvate kinase, cyclic AMP, GTP, ATP and Gpp(NH)p were purchased from Sigma (St Louis, MO, USA). Kallikrein inhibitor (Antagosan) was from Behring (Puteaux, France).
of CGRP binding sites. [‘251]-rat CGRP I bound rapidly to P388 Dl macrophage membranes. Equilibrium was achieved after a lo-min incubation period at 37°C and remained stable for at least 30min (Fig. 1). Non specific binding, determined by incubating the tracer and membranes in the presence of 1 PM unlabelled rat CGRP I, represented less than 20% of total binding. Dissociation of specifically bound [125I] -rat CGRP I (about 5500 cpm/mg protein) was measured after a 15min incubation at 37°C (Fig.2). The addition of 10k.M GTP provoked a 15% release of bound tracer within 5min. After adding 1 PM rat CGRP I at equilibrium, the dissociation of the tracer was rapid, 50% of the tracer being released after 7min. Combination of 10 &M GTP and 1 PM rat CGRP I reducud that time to 3min. Scatchard analysis of specific [125I]
+10@4GTP
+lOpb¶cm and lp?d ICGRPI 0 (
‘
I
1
0
10
20
30
TIME Fig. 2
(min)
Time-course of dissociation of [l”I]-rat CGRP I bound to murine P388 Dl macrophage membranes. Membranes were pre-incubated with 80-1OOpM [‘251]-rat CGRP I for 15min at 37°C. The radioactivity bound to membranes was measured at serial times under control conditions and after addition of either 1OpM GTP, 1 PM unlabelled rat CGRP I or both 1OkM GTP and 1 pM rat CGRP I. The data are the mean values of two experiments performed in duplicate.
46
NEUROPEPTIDES
I&, (4nM) whereas rat CGRP II and [tyr’]-human CGRP I were lo-fold less potent to displace tracer binding. [Tyr’]-rat CGRP I (23-37) and [tyr”]-rat CGRP I (2837), i.e. CGRP fragments lacking the 22 and 27 N-terminal amino acids respectively, recognized CGRP receptors albeit lOOO-fold less efficiently than intact rat CGRP I. Neither VIP nor porcine calcitonin caused detectable inhibition of 60 0 40 SO 100 20 binding at concentrations up to 1 ~J,Mand 10kM -I respectively. Secretin, somatostatin, cholecystoBOUND ffmol . mg protein 1 kinin, neurotensin and substance P did not displace [1251]-rat CGRP I binding at concentraFig. 3 Scatchard analysis of specific binding of [1251]-rat CGRP I to murine P388 Dl macrophage membranes. Memtions of 10 PM (not shown). branes CGRP shown ments
were incubated with serial concentrations of [1251]-rat I from 1.6pM to 640pM for 15min at 37°C. Results are the representative data of three separate experiperformed in duplicate.
activity in P388 Dl macrophage membranes was low (12 + 2 pmol cyclic AMP formed. mini. mg protein-‘, mean f SEM of 7 determinations). GTP induced a moderate stimulation (+ 60%), and the guanine triphosphate nucleotide analogue Gpp(NH)p and NaF provoked 12- and lo-fold stimulations of activity respectively (not shown). In the presence of 10~M GTP, rat CGRP I, human CGRP I and human CGRP II stimulated adenylate cyclase activity with a similar EC50 of 6nM (Fig. 5). Rat CGRP II was less potent (EC50 = 40nM). Considering rat CGRP I as the reference peptide (intrinsic activity = l.O), the efficacy of human CGRPs I and II was 0.75. Addition of tyrosine in
-rat CGRP I binding revealed the presence of one class of binding sites, with a concentration required to occupy 50% of the sites (I&) of 1.76nM and a maximum binding capacity (Bmax) of 85.48fmoYmg protein (Fig. 3). Characteristics of CGRP binding sites. CGRP binding sites were studied by comparing competition curves between the tracer and rat CGRPs (Fig. 4, left panel) or human CGRPs (Fig. 4, right panel) after a 15min incubation period at 37°C. Rat CGRP I, human CGRP I and human CGRP II inhibited [‘251]-rat CGRP I binding with a similar
~‘~~~ 0
t
’
-10
5
I
.
-9
-8
1
-7
PEPTIDE (log M)
Activation of membrane adenylate cyclase through CGRP receptors. The basal adenylate cyclase
5rry
,x;,,
.
-6
-5
0
-10
-9
-7
-6
-5
PEPTIDE (log Ml
Fig. 4 Inhibition of [lZsI]-rat CGRP I binding to murine P388 Dl macrophage membranes by (left panel) rat CGRP I, rat CGRP II, [tyr’]-rat CGRP I (23-37), [tyr”]-rat CGRP I (28-37), and by (right panel) human CGRP I, human CGRP II, [tyr”]-human CGRP I, VIP and porcine calcitonin. Membranes were incubated for 15min at 37°C with [iZSI]-rat CGRP I and increasing concentrations of unlabelled peptide. The data are the mean values of three experiments performed in duplicate.
GENE-RELATED
PEFTIDE RECEPTORS AND ADENYLATE
47
CYCLASE RESPONSE 120
60
rCGRPl
-10
-9
-6
-7
-6
PEPTIDE (log MI
0
-10
-9
-8
-7
-6
-5
PEPTIDE (log M)
Fig. 5 Dose-effect curves of adenylate cyclase activation in murine P388 Dl macrophage membranes by (left panel) rat CGRP I, rat CGRP II, [tyr”]-rat CGRP I (23-37) [tyr’]-rat CGRP I (28-37) and by (right panel) human CGRP I, human CGRP II, [tyr”]-human CGRP I, VIP and porcine calcitonin. Adenylate cyclase activity was determined in the presence of lOl.rM GTP. The results are expressed as pmol cyclic AMP. min-‘. mg protein-i (after substraction of the basal unstimulated values) and are the mean values of three experiments performed in duplicate.
position 0 decreased the potency of human CGRP I (EC50 = 30nM) without affecting its efficacy. VIP, porcine calcitonin and the synthetic fragments [tyr’]-rat CGRP I (23-37) and [tyr’]-rat CGRP I (28-37) were devoid of detactable stimulatory effects. [Tyr’]-rat CGRP I (23-37) and [tyr”] -rat CGRP I (28-37) slightly shifted the rat CGRP I adenylate cyclase stimulation curve to the right with a similar inhibition constant (Ki) of 10 FM and without significant modification of maximal activation, affording presumed evidence for competitive inhibition (not shown). Discussion
study demonstrated for the first time the existence of CGRP receptors on membranes from a murine macrophage-like cell line. Binding of [12’I]-rat CGRP I was time-dependent and reversible. The specificity of binding sites was demonstrated by the relative abilities of CGRP from different species to displace [12’I]-rat CGRP I, whereas calcitonin and unrelated peptides were uneffective. From Scatchard analysis, CGRP binding sites were found to consist in a single class of high affinity sites. The CGRP receptors found on P388 Dl cells were coupled to the guaninenucleotide-binding regulatory protein Gs, as based on GTP stimulation of adenylate cyclase and
This
NEUR.
C
on the marked acceleration of [12’I]-rat CGRP I dissociation by GTP in the presence of ll,~M unlabelled rat CGRP I. In addition CGRP concentrations required for half-maximal adenylate cyclase stimulation were similar to those required for half-maximal inhibition of tracer binding, suggesting a close linkage between receptor occupation and adenylate cyclase stimulation. On the basis of binding studies (inhibition of [‘251]-rat CGRP I) and functional studies (direct stimulation of adenylate cyclase activity), the receptor recognized with decreasing potency rat CGRP I = human CGRPs I and II > rat CGRP II = [tyr’]-human CGRP I, whereas calcitonin was without effect on tracer binding and adcnylate cyclase activity. The profile of CGRP receptors on P388 Dl cells clearly differs from that of CGRP receptors in the brain and spinal cord membranes (18) that were not linked to the adenylate cyclaseCAMP system. They also differ from liver, spleen and lung CGRP receptors (7, 11, 12) that efficiently recognize calcitonin, from receptors on pancreatic acini (8) on the basis of the relative efficacy of rat CGRP, human CGRP and CGRP analogues to displace tracer binding, and from mouse T lymphocyte receptors that recognize CGRP with a higher affinity (15). Furthermore our results suggest that slight structural modification in the C-terminal or N-terminal parts of the
48 molecule, such as monosubstitution of an amino acid in position 35 or addition of a tyrosine residue to the N-terminal end of the peptide, reduced the affinity for the CGRP receptor. In contrast the C-terminal fragments lacking the ring structure, [tyr’]-rat CGRP I (23-37) and [tyr’]-rat CGRP I (28-37), showed moderate inhibition of tracer binding and were devoid of adenylate cyclase activity, indicating that the C-terminal part of CGRP may be implicated in receptor recognition while the N-terminal site including the disulphide bridge is essential for biological activity (19). In addition [tyr”]-rat CGRP I (23-37) and [tyr’]-rat CGRP I (28-37) produced a moderate right shift in the rat CGRP I-dose response curve of adenylate cyclase without reduction in the maximum response elicited by rat CGRP I. Such results suggest that these analogues exert a competitive with a weak intrinsic type of antagonism, anatagonist activity at 10 ~J,M. Neuropeptides have been shown to play an increasingly important role in immune regulation. Substance P and VIP modulate immunoglobulin production (20) and lymphocyte cytotoxic activity (21, 22). In addition substance P increases the lymphocyte proliferation whereas VIP, somatostatin and CGRP have inhibitory effects (13, 20). The activity of macrophages can also be affected by neuropeptides, such as substance P and neurotensin that enhance the phagocytic activity by interacting through specific receptors (23, 24). Recently CGRP was found to inhibit the ability of macrophages to produce Hz02 in response to interferon-gamma and to reduce their capacity to activate T lymphocytes (14). Taking additionally in account that macrophages produce several cytokines, such as interleukin-1, interleukind and tumor necrosis factor-alpha, and prostaglandins, the functional relationship between the occupation of CGRP receptors and some intracellular biochemical events remains to be investigated.
NEUROPEFTIDES
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Acknowledgements This work was supported by grant from the Comite des Aides a la Recherche Fournier-Dijon, France.
13.
References 1. Amara, S. G., Jonas, V., Rosenfeld, M. G., Ong, E. S. and
14.
Evans, R. M. (1982) Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298: 240-244. Rosenfeld, M. G., Mermod, J. J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, W. W. and Evans, R. M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304: 129-13.5. Skofitsch, G. and Jacobowitz, D. M. (1985) Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6: 721745. Mulderry, P. K., Ghatei, M. A., Spokes, R. A., Jones, P. M., Pierson, A. M., Hamid, Q. A., Kanse, S., Amara, S. G., Burrin, J. M., Legon, S., Polak, J. M. and Bloom, S. R. (1988) Differential expression of CGRP I and CGRP II by primary sensory neurons and enteric autonomic neurons of the rat. Neuroscience 25: 195-205. Sternini, C., Reeve, J. R. and Brecha, N. (1987) Distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal and capsaicin-treated rats. Gastroenterology 93: 852-862. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. and Macintyre, I. (1985) Calcitonin gene-related peptide is a potent vasodilator. Nature 313: 54-56. Sigrist, S., France-Cereceda, A., Muff, R., Henke, H., Lundberg, J. M. and Fischer, J. A. (1986) Specific receptors and cardiovascular effects of calcitonin generelated peptide. Endocrinology 119: 381-389. Zhou, Z. C., Villanueva, M. L., Noguchi, M., Jones, S. W., Gardner, J. D., and Jensen, R. T. (1986) Mechanism of action of calcitonin gene-related peptide in stimulating pancreatic enzyme secretion. American Journal of Physiology 251: G391-G397. Beglinger, C., Born, W., Hildebrand, P., Ensinck, J. W., Burkhardt, F., Fischer, J. A. and Gyr, K. (1988) Calcitonin gene-related peptides I and II and calcitonin: distinct effects on gastric acid secretion in humans. Gastroenterology 95: 958-965. Tschopp, F. A., Henke, H., Petermann, J. B., Tobler, P. H., Jansen, R., Hokfelt, T., Lundberg, J. M., Cuello, C. and Fischer, J. A. (1985) Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary. Proceedings of the National Academy of Sciences of the U.S.A. 82: 248-252. Mak, J. C. W. and Barnes, P. J. (1988) Autoradiographic localization of calcitonin gene-related peptide binding sites in human and guinea pig lung. Peptides 9: 957-963. Yamaguchi, A., Chiba, T., Okimura, Y., Yamatani, T., Morishita, T., Nakamura, A., Inui, T., Noda, T. and Fujita, T. (1988) Receptors for calcitonin gene-related peptide on the rat liver plasma membranes. Biochemical and Biophysical Research Communications 152: 383-391. Umeda, Y., Takamiya, M., Yoshizaki, H. and Arisawa, M. (1988) Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide. Biochemical and Biophysical Research Communications 154: 227-235. Nong, Y. H., Titus, R. G., Ribeiro, J. M. C. and Remold,
GENE-RELATED
15.
16.
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19.
20.
PEPTIDE RECEPTORS AND ADENYLATE
H. G. (1989) Peptides encoded by the calcitonin gene inhibit macrophage function. Journal of Immunology 143: 45-49. Umeda, Y. and Arisawa, M. (1989) Characterizationof the calcitonin gene-related peptide receptor in mouse T lymphocytes. Neuropeptides 14: 237-242. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275. Salomon, Y., Londos, C. and Rodbell, M. (1974) A highly sensitive adenylate cyclase assay. Analytical Biochemistry 58: 541-548. Gohzman, D. and Mitchell, J. (1985) Interaction of calcitonin and calcitonin gene-related peptide at receptor sites in target tissues. Science 227: 1343-1345. Tjppins, J. R., Di Matzo, V., Panico, M., Morris, H. R. and Macintyre, I. (1986) Investigation of the structure/ activity relationship of human calcitonin gene-related peptide. Biochemical and Biophysical Research Communications 134: 1306-1311. Stanisz, A. J., Befus, D. and Bienenstock, J. (1986)
CYCLASE RESPONSE
21.
22.
23.
24.
49
Differential effects of vasoactive intestinal peptide, substance P and somatostatin on immunoglobulin synthesis and proliferation by lymphocytes from Peyer’s patches, mesenteric lymph nodes, and spleen. Journal of Immunology 136: 152-156. Bienenstock, J., Croitoru, K., Ernst, P. B., Stead, R. H. and Stanisz, A. (1989) Neuroendocrine regulation of mucosal immunity. Immunological Investigations 18: 69-76. Rola-Pleszczynski, M., Bolduc, D. and St-Pierre, S. (1985) The effects of vasoactive intestinal peptide on human natural killer cell function. Journal of Immunology 135: 2569-2573. Hartung, H. P., Wolters, K. and Toyka, K. V. (1986) Substance P: binding properties and studies on cellular responses in guinea pig macrophages. Journal of Immunology 136: 3856-3863. Bar-Shavit, Z., Terry, S., Blumberg, S. and Goldman, R. (1982) Neurotensin-macrophage interaction: specific binding and augmentation of phagocytosis. Neuropeptides 2: 325-335.