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Binding sites of calcitonin gene related peptide (CGRP) to trout tissues
Neuropeprides (1991) 20, 181-186 0 Longman Group UK Ltd 1991
Binding Sites of Calcitonin Gene Related Peptide (CGRP) to Trout Tissues Y. ARLOT-BONNEMAINS”, M. S. MOUKHTAR*
M. FOUCHEREAU-PEFiONt,
*URA 174 CNRS, 27 rue de Chaligny, 75072 Paris, France; de France, Place de la Croix, 29900 Concarneau, France
A. JULLIENNE”,
tLaboratoire
G. MILHAUD*
de Biologic
Marine
and
du Collkge
Abstract-We localized specific binding sites for human calcitonin gene related peptide (hCGRP) in different organs of the trout using labelled human CGRP. Maximal binding was observed in gill and spleen membranes. The binding of 1251-hCGRP was time and temperature dependent. Scatchard analysis of binding data for the spleen and the gills disclosed two binding sites. The constants for the site of high affinity and low capacity (KAM-’ and B,,, (fmol/mg of proteins)) were 2.9 x 10' for the spleen and 70 and 3.5 x 10’ for the gill. Salmon calcitonin (sCT) inhibited the binding of ‘251-hCGRP to spleen membranes with the same order of potency as hCGRP. In contrast sCT was less effective than hCGRP in suppressing the specific binding of ‘251-hCGRP to gill membranes.
Introduction Calcitinon-gene-related-peptide (CGRP I or II) is a 37 amino acid neuropeptide which is co-encoded with CT on a single gene and expressed through tissue specific processing of mRNA (l), the processing proceeds in a tissue specific manner to synthesize the precursor of CGRP for the neuronal tissue and the precursor for CT in the thyroid ‘C’ cells (2-3). In vitro and autoradiographic CGRP binding studies have revealed binding sites in various tissues or organs: brain (4), heart (S), pancreas (6), peripheral arteries (7), lung (8,9), visceral organs Date received 13 June 1991 Date accepted 28 July 1991
(lo), bladder, kidney (11, 12) and smooth muscle (13). Calcitonin an&CGRP cross react with their specific receptors (4, 14) due to their structural similarity. Recently we have demonstrated that ir-CGRP is present in different organs of trout S&no gairdnerii (15) and that hCGRP increases CAMP formation in trout organs especially in gill (16). These results suggested that in fishes CGRP may be involved in several physiological functions. We report here the distribution of specific CGRP and calcitonin binding sites in different organs of the trout using labelled human CGRP (hCGRP) and salmon calcitonin (sCT). We also characterized these binding sites in gill and spleen, organs in which their concentration is the highest.
181
182
NEUROPEPTIDES
Materials and Methods Animals
rainbow trout Salmo obtained from a local hatchery. 200g
gairdnerii
were
Chemicals
Human CGRP (l-37) (hCGRP) and radiolabelled hCGRP (20OOWmmol) were obtained from Bachem and Amersham respectively. Synthetic salmon calcitonin (sCT) (20001U/mg) Lot 87F 04101 and calcitonin (hCT) was purchased from Sigma, sCT was labelled by the chloramine-T method (17) (4OOtKi/l~.g). Human parathyroid hormone (hPTH) was obtained from Sigma. All other chemicals were of reagent grade. Tissue preparation
Membranes from trout tissues were prepared as described previously (18). Organs were dissected and homogenized in ice cold standard buffer 50mM Tris Hepes, pH 7.4, containing 5mM MgClz, 2mM EGTA and 0.24M sucrose. The homogenate was centrifuged at 4000g for 10 min at 4°C; the resulting supernatant was further centrifuged for 30min at 2OOOOg,at 4°C and finally the pellet was resuspended in standard buffer and stored at -80°C until used. Binding studies
Binding experiments for CT and CGRP receptors were developed in the same manner. Crude membrane preparation from different trout tissues were incubated as described previously (19). 2OOt.~lof preparation (0.8mg/ml of proteins) were incubated at 4°C for 180min with lz5-I peptide (70pM) with or without increasing amount of unlabelled peptide (0.07 to 45.6nM) in 50mM Hepes-Tris pH 7.4, containing 1% BSA and lOOOUI/ml Antagosan. At the end of the incubation, bound and free ligand was separated by centrifugation and the pellet was washed three times with iced standard buffer incubation solution containing 10% sucrose. Data are expressed as specific binding that was obtained by subtracting from the total binding the amount of radioactivity associated to the membranes in the presence of ~/_LMCGRP.
Fig. 1 Distribution of CT receptors and of CGRP receptors in different tissues of the trout Salmo gairdnerii. For CT and the CGRP binding studies, specific binding represent numerical difference between binding of the radioactive tracer in the absence of unlabelled and the binding inhibited by an excess of unlabelled peptide. Results are expressed as specific binding. Each bar repesents the mean of five determinations.
The protein concentration was determined by the method of Lowry (20) using bovine serum albumin as the standard.
1. Distribution of CGRP and sCT binding in trout tissues
The distribution of specific binding of ‘251-labelled sCT in peripheral trout tissues is shown in Figure 1. Maximal binding of ‘251-labelled hCGRP was observed in spleen and gills membranes with respectively 4.3% and 3.85% of the total binding. Significant binding was also observed in brain and heart but specific binding was less important representing only 30% of the binding measured in the spleen. No CGRP binding was demonstrable in stomach, gut, kidney or liver. Maximal binding of ‘251-sCT occurred in the spleen and represented 2.1% of the total binding and in contrast significant sCT binding was also observed in the heart and in the brain accounting for respectively 1.5 band 1% of total binding. The specific binding observed in the gills and the stomach was low (~2.2 x 10-4fmoles/mg of proteins). Minimal or no ‘*‘I-sCT binding was observed in the liver, the gut and the kidney.
183
BINDING SITES OF CALCITONIN GENE RELATED PEPTIDE (CGRP) TO TROUT TISSUES
0.0
1
0
100
200
300 Time.
min
Time course of specific 1~51-labelledhCGRP binding to spleen (a) and gills (b) preparation expressed as % of total radioactivity. O.Zmg/ml of proteins preparation were incubated with 0.07nM of ‘2SI-CGRP at 4°C for different times indicated with or without 2 PM of unlabelled CGRP. Specific binding was expressed as % of total radioactivity.
Fig. 2
2. ‘251-hCGRP binding to membrane preparation and Scatchard analysis of the data
Specific binding of lz51-hCGRP to spleen trout membrane (Fig. 2a) was time dependent. At 5°C binding reached an apparent equilibrium after 120-180min of incubation. Kinetics of ‘251-hCGRP binding to gill membranes are reported in Figure 2b. Increasing concentrations of synthetic hCGRP inhibited ‘251-hCGRP binding to spleen and gill trout membranes in a dose dependent fashion (Fig. 3). The binding of ‘251-labelled CGRP was competitively inhibited by native hCGRP in the range of 0.07-50nM. Half inhibition of the initial 12’I-hCGRP binding (i.e., in the absence of the native CGRP) was observed in spleen membrane (Fig. 3a) and gills membrane (Fig. 3c) with respectively 1.62nM (N = 5) and 2.16nM (N = 3) Table
CGRP. The interaction between hCGRP and its specific binding sites were quantified. Human CGRP binding sites were saturable. At a hormone concentration sufficient for saturating the CGRP binding sites (45nM) the amount of binding sites correspond to 70fmoles/mg of proteins in the spleen membranes and to 53fmoles/mg of proteins in the gills membranes. When these data were analyzed by the method of Scatchard (21) a curvilinear graph was observed with the spleen and gill membranes suggesting the presence of two classes of binding sites or the occurrence of a negative cooperativity (22) (Fig. 3b, d). In these two tissues, the affinity constant for the site I - high affinity low capacity - were of the same order of magnitude (2 to 3 x lO’M_‘). The number of binding sites accounting for were also the same in the gills and in the spleen membrane (Table). The specificity of 1251-hCGRP binding was investigated using salmon CT and human parathyroid hormone (hPTH) (Fig. 4). Half maximal inhibition of specific binding of hCGRP was obtained in the spleen with 3.2nM of CGRP. 2.9nM of sCT produced an inhibition of 50% of the 1251-hCGRP binding to the spleen membranes. In contrast, hPTH was inactive on the binding of CGRP (Fig. 4a). A different effect was observed on gills membranes: sCT was less effective than hCGRP on the displacement of the binding of ‘251-hCGRP to gill membranes. 4nM of unlabelled hCGRP is necessary to displace the binding of L251-hCGRP to the gill membranes, the same effect was observed with a 10 fold higher dose of sCT that is 58.8nM (Fig. 4b). hPTH was inactive on the binding of ‘251-hCGRP to gill membranes. When a spleen membrane preparation was incubated in the presence ‘251-sCT, 1.6nM of sCT displaced 50% of the binding of ‘251-sCT to the membrane; 1.3nM of hCGRP was necessary to obtain the same inhibition (Fig. 5).
Binding parameters of CGRP binding in trout tissues Site I KA (nM)
Spleen Gills
3.2 zk 0.5 x lo9 2.9 + 0.4 x lo9
Site 2 Nlmg of proteins
27.2 f 0.9 x 10H 52.2 x 10R
KA (nM)
4.4 + 1.2 x lo* 2.2 + 0.2 x 108
Nlmg of proteins
220 + 11 x 108 349 x lo8
184
NELJROPEPTIDES
b
d
0
0.00 t-
f--T-Y--T-
0
50
100 CGRP. nglml
0
25 6. fmoleslmg
50 of protein
Fig. 3 Inhibition curve of the binding of ‘251-hCGRP binding to spleen (a) and gills (c) membrane preparation of the trout. 0.2mg/ml of proteins of each preparation were incubated with 0.07nM of ‘251-CGRP and increasing amount of unlabelled hCGRP (0.07 to 45.6nM). Non specific binding was assessed by addition of 2 )IM unlabelled synthetic hCGRP. On the right is the Scatchard analysis of the binding data: (c) spleen membrane; (d) gill membrane. For each experiment, each value was determined in triplicate and the results are the mean of four determinations.
Discussion
The results described above demonstrate that hCGRP binding sites are abundant in trout spleen and gill membrane preparations. In the other tissues of the trout studied the binding of hCGRP was much lower. The distribution of hCGRP binding sites in trout organs is similar to that observed in mammals where the presence of binding sites for hCGRP is maximal in the brain (19, 23) and peripheral tissues such as spleen (5, 10) and lung (9). Unlike the situation observed in mammals (13), the digestive organs in fish are free from hCGRP binding sites. As described previously (24), no sCT binding sites were measured in the trout kidney, maximal
binding for ‘*?-sCT was observed in spleen, brain and heart. The distribution of specific binding sites for respectively hCGRP or sCT is not correlated to the presence of immunoreactive molecules in trout tissues as for instance maximal binding for CGRP is observed in the spleen in which no ir-CGRP is measured (15). Likewise no hCGRP binding is observed in gut where the greatest amount of ir-CGRP was found (15). Saturation experiments and Scatchard analysis suggested the presence of two classes of binding sites in trout spleen and gill preparations. The presence of two classes of binding sites for hCGRP is also reported in mammals: in lung (9) gastrointestinal tract (13), guinea pig pancreatic acini (6) and T lymphocytes (25). In contrast, only one class
185
BINDING SITESOF CALCITONIN GENE RELATED PEPTIDE (CGRP) TO TROUTTISSUES
120
120
100
100
80
80
60
60
40
40
20
20
0
0 0
20
40
60
80
100120
Peptides.
I I
0 ng/ml
100
I
I
200
300
Peptides.
nglml
Fig. 4 Competitive inhibition of the binding of “‘I-CGRP to the spleen membrane (a) and the gill membrane (b) of the trout with unlabelied human CGRP, salmon CT, and human PTH. The conditions of incubation were the same as described above. Unlabelled CGRP (0). salmon calcitonin (+) and human PTH (m) were added to the membrane preparation at various concentrations
of binding site was measured in the rat spleen. Scatchard analysis also revealed that the value of the high affinity constant was of the same order of magnitude (3 x 109M-‘) as that observed in mammals. 14
1.2
10
08
06
04
Peptide
nglml
Fig. 5 Competitive inhibition of the binding of “51-salmon calcitonin to the spleen membrane. 0.2mg.ml of proteins of spleen membrane were incubated in the presence of 0.07nM of ‘25I-sCT in the presence or absence of 2 pM of unlabelled sCT and increasing amount of unlabelled sCT (0) or human CGRP (a) for 180min at 4°C.
The value of the affinity constant for the high affinity binding site in gill membranes was comparable to the approximate value (5.3nM) corresponding to the 50% effective dose of hCGRP on adenylate cyclase in the same tissue (16). This numerical similarity between the two values is consistent with a close linkage between hCGRP receptors and adenylate cyclase system. This relationship between binding of hCGRP and increased in CAMP formation is similar to the results described in gastric smooth muscle (13) and pancreatic acini (6). This relationship is more complex in the spleen where no activation of adenylate cyclase activity has been observed. Furthermore the binding studies demonstrate that sCT completely inhibit the saturable binding of hCGRP employed at the same concentration and vice versa. Same observations were reported in the rat brain where Sexton et al (26) described a subtype of CT/CGRP binding site that exhibited high affinity for both CT and CGRP. In conclusion we have established for the first time the presence of specific receptors for CGRP in the gills and spleen membrane of the trout. The results are consistent with a physiologica action of CGRP in branchial cells. The physiological role of
186 CGRP binding sites in trout spleen is not known although its ability to bind two peptides which are biochemically distinct is intriguing. Acknowledgements We are grateful to Professor Y. Le Gal for critical comments.
References 1. Amara, S. G., Arizza, J. L., Leff, S. E., Swanson, L. W.,
Evans, R. and Rosenfeld, M. G. (1985). Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Science 220: 1094-1097. 2. Rosenfeld, M. G., Mermod, J. J. and Amara, S. G. (1983). Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304: 129-135. 3. Morris, H. R., Panico, M., Etienne, T., Tippins, J., Girgis, S. I. and MacIntrye, I. (1984). Isolation and characterization of human calcitonin gene related peptide. Nature (London) 308: 746. 4. Goltzmann, D. and Mitchell, J. (1985). Interaction of calcitonin gene related peptide at receptor sites in target tissues. Science 227: 1343-1345. 5. Sigrist, S., France-cereceda, A., Muff, R., Henke, H., Lundberg, J. M. and Fisher, J. A. (1986). Specific receptor and cardiovascular effects of CGRP. Endocrinology 119: 1,381-388. 6. Zhou, Z. C., Villanueva, M. L., Noguchi, M. (1986). Mechanism of action of calcitonin gene related peptide in stimulating pancreatic enzyme secretion. American Journal of Physiology 251: G391-G397. 7. Hirata, Y., Takagi, Y., Takata, S., Fukuda, Y., Yoshimi, H. and Fujita, T. (1988). Calcitonin gene related receptor in cultured vascular smooth muscle and endothelial cells. Biochemical and Biophysical Research Communications 151: 3, 1113-1121. 8. Cartrais, J. R. (1987). Distribution of calcitonin gene related peptide receptors in the lung. European Journal of Pharmacology 140: 357-358. 9. Umeda, Y. and Arisawa, M. (1989). Characterization of Calcitonin Gene Related Peptide (CGRP) receptors in guinea pig lung. Japan Journal of Pharmacology 57: 377-384. 10 Nakamuta, H., Fukuda, Y. and Koida, M. (1986). Binding sites of Calcitonin Gene Related Peptide (CGRP): Abundant occurrence in visceral organs. Japan Journal of Pharmacology 42: 175180. 11 Nimmo, A. J., Morrison, J. F. B. and Whitaker, E. M. (1988). A comparison of the distribution of substance P and CGRP receptors in the rat bladder. Quarterly Journal of Experimental Physiology 73: 789-792. 12. Kurtz, A., Schurek, H. J., Jelkmann, W., Muff, R., Lipp, H. P., Heckmann, U., Eckardt, K. U., Scholz, H., Fisher, J. A. andBauer, C. (1989). Renal mesangiumis a target for calcitonin gene related peptide. Kidney International 36: 222-227.
NEUROPEPTIDES
13. Maton. P. N., Sutliff, V. E., Zhou, Z. C., Collins. S. M., Gardner, J. D. and Jansen, R. T. (1988). Characterization of receptors for calcitonin gene related peptide on gastric smooth muscle. American Journal of Physiology 254: G789-G794. 14. Wohlwend. A., Malmstrom, K., Henke, H., Murer, J. D.. Vassali, D. and Fischer, J. A. (1985). Calcitonin and calcitonin gene related peptide interact with the same receptor in cultured LLC-PKl kidney cells. Biochemical and Biophysical Research Communication 131: 537-541. 15. Fouchereau-Peron, M., Arlot-Bonnemains. Y _, Moukhtar, M. S. and Milhaud, G. (1990). Distribution of calcitonin gene related peptide and calcitonin like immunoreactivity in trout. Regulatory Peptides 27: 171179. 16. Fouchereau-Peron, M.. Arlot-Bonnemains, Y.. Milhaud. G. and Moukhtar, M. S. (1990). Calcitonin gene related peptide stimulates adenylate cyclase activity in trout gill cell membranes. Biochemical and Biophysical Research Communications 172: 2,582-587. 17. Hunter, W. M. W., Greenwood, F. F. and Glower, J. S. (1963). Preparation of iodine 131 labelled human growth hormone of high specific activity. Biochemical Journal 89: 114-123. 18. Seifert, H., Chesnut, J., De Souza, E., Rivier, J. and Vale, W. (1985). Binding sites for calcitonin gene related peptide in distinct areas of rat brain. Brain Research 346: 195-198. 19. Henke, H., Tschopp, F. A. and Fischer, J. A. (1985). Distinct binding sites for CGRP and salmon calcitonin in rat central nervous system. Brain Research 360: 165-171. 20. Lowry, 0. M., Rosenbourgh, W. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: 265-275. 21. Scatchard, G. D. (1949). The attraction of proteins for small molecules and ions. Annual New York Academy of Science 51: 660-672. 22. De Meyts, P., Roth, J., Neville, D. M., Gavin, J. R. and Lesniak, M. A. (1973). Insulin interactions with its receptors. Experimental evidence for negative cooperativity. Biochemical and Biophysical Research Communication 55: 154-161. 23. Sexton, P. M., McKenzie, J. S.. Mason, R. T.. Moseley, J. M., Martin and Mendelsohn, F. A. 0. (1986). Localization of binding sites for calcitonin gene related peptide in rat brain by in vitro autoradiography. Neuroscience 19: 4, 1235-1245. Y., Fouchereau-Peron, M., Moukh24. Arlot-Bonnemains, tar, M. S. and Milhaud, G. (1983). Characterization of target organs for calcitonin in lower and higher vertebrates. Comparative Biochemistry and Physiology 76A: no 2. 377-380. 25. Umeda, Y. and Arisawa. M. (1989). Characterization of the Calcitonin gene related peptide receptor in mouse T lymphocytes. Neuropeptides 14: 237-242. 26. Sexton, M. P., McKenzie, J. S. and Mendelsohn, F. A. 0. (1988). Evidence for a new subclass of calcitonin/calcitonin gene related peptide binding site in rat brain. Neurochemical International 12: 3,232-335.
Binding Sites of Calcitonin Gene Related Peptide (CGRP) to Trout Tissues Y. ARLOT-BONNEMAINS”, M. S. MOUKHTAR*
M. FOUCHEREAU-PEFiONt,
*URA 174 CNRS, 27 rue de Chaligny, 75072 Paris, France; de France, Place de la Croix, 29900 Concarneau, France
A. JULLIENNE”,
tLaboratoire
G. MILHAUD*
de Biologic
Marine
and
du Collkge
Abstract-We localized specific binding sites for human calcitonin gene related peptide (hCGRP) in different organs of the trout using labelled human CGRP. Maximal binding was observed in gill and spleen membranes. The binding of 1251-hCGRP was time and temperature dependent. Scatchard analysis of binding data for the spleen and the gills disclosed two binding sites. The constants for the site of high affinity and low capacity (KAM-’ and B,,, (fmol/mg of proteins)) were 2.9 x 10' for the spleen and 70 and 3.5 x 10’ for the gill. Salmon calcitonin (sCT) inhibited the binding of ‘251-hCGRP to spleen membranes with the same order of potency as hCGRP. In contrast sCT was less effective than hCGRP in suppressing the specific binding of ‘251-hCGRP to gill membranes.
Introduction Calcitinon-gene-related-peptide (CGRP I or II) is a 37 amino acid neuropeptide which is co-encoded with CT on a single gene and expressed through tissue specific processing of mRNA (l), the processing proceeds in a tissue specific manner to synthesize the precursor of CGRP for the neuronal tissue and the precursor for CT in the thyroid ‘C’ cells (2-3). In vitro and autoradiographic CGRP binding studies have revealed binding sites in various tissues or organs: brain (4), heart (S), pancreas (6), peripheral arteries (7), lung (8,9), visceral organs Date received 13 June 1991 Date accepted 28 July 1991
(lo), bladder, kidney (11, 12) and smooth muscle (13). Calcitonin an&CGRP cross react with their specific receptors (4, 14) due to their structural similarity. Recently we have demonstrated that ir-CGRP is present in different organs of trout S&no gairdnerii (15) and that hCGRP increases CAMP formation in trout organs especially in gill (16). These results suggested that in fishes CGRP may be involved in several physiological functions. We report here the distribution of specific CGRP and calcitonin binding sites in different organs of the trout using labelled human CGRP (hCGRP) and salmon calcitonin (sCT). We also characterized these binding sites in gill and spleen, organs in which their concentration is the highest.
181
182
NEUROPEPTIDES
Materials and Methods Animals
rainbow trout Salmo obtained from a local hatchery. 200g
gairdnerii
were
Chemicals
Human CGRP (l-37) (hCGRP) and radiolabelled hCGRP (20OOWmmol) were obtained from Bachem and Amersham respectively. Synthetic salmon calcitonin (sCT) (20001U/mg) Lot 87F 04101 and calcitonin (hCT) was purchased from Sigma, sCT was labelled by the chloramine-T method (17) (4OOtKi/l~.g). Human parathyroid hormone (hPTH) was obtained from Sigma. All other chemicals were of reagent grade. Tissue preparation
Membranes from trout tissues were prepared as described previously (18). Organs were dissected and homogenized in ice cold standard buffer 50mM Tris Hepes, pH 7.4, containing 5mM MgClz, 2mM EGTA and 0.24M sucrose. The homogenate was centrifuged at 4000g for 10 min at 4°C; the resulting supernatant was further centrifuged for 30min at 2OOOOg,at 4°C and finally the pellet was resuspended in standard buffer and stored at -80°C until used. Binding studies
Binding experiments for CT and CGRP receptors were developed in the same manner. Crude membrane preparation from different trout tissues were incubated as described previously (19). 2OOt.~lof preparation (0.8mg/ml of proteins) were incubated at 4°C for 180min with lz5-I peptide (70pM) with or without increasing amount of unlabelled peptide (0.07 to 45.6nM) in 50mM Hepes-Tris pH 7.4, containing 1% BSA and lOOOUI/ml Antagosan. At the end of the incubation, bound and free ligand was separated by centrifugation and the pellet was washed three times with iced standard buffer incubation solution containing 10% sucrose. Data are expressed as specific binding that was obtained by subtracting from the total binding the amount of radioactivity associated to the membranes in the presence of ~/_LMCGRP.
Fig. 1 Distribution of CT receptors and of CGRP receptors in different tissues of the trout Salmo gairdnerii. For CT and the CGRP binding studies, specific binding represent numerical difference between binding of the radioactive tracer in the absence of unlabelled and the binding inhibited by an excess of unlabelled peptide. Results are expressed as specific binding. Each bar repesents the mean of five determinations.
The protein concentration was determined by the method of Lowry (20) using bovine serum albumin as the standard.
1. Distribution of CGRP and sCT binding in trout tissues
The distribution of specific binding of ‘251-labelled sCT in peripheral trout tissues is shown in Figure 1. Maximal binding of ‘251-labelled hCGRP was observed in spleen and gills membranes with respectively 4.3% and 3.85% of the total binding. Significant binding was also observed in brain and heart but specific binding was less important representing only 30% of the binding measured in the spleen. No CGRP binding was demonstrable in stomach, gut, kidney or liver. Maximal binding of ‘251-sCT occurred in the spleen and represented 2.1% of the total binding and in contrast significant sCT binding was also observed in the heart and in the brain accounting for respectively 1.5 band 1% of total binding. The specific binding observed in the gills and the stomach was low (~2.2 x 10-4fmoles/mg of proteins). Minimal or no ‘*‘I-sCT binding was observed in the liver, the gut and the kidney.
183
BINDING SITES OF CALCITONIN GENE RELATED PEPTIDE (CGRP) TO TROUT TISSUES
0.0
1
0
100
200
300 Time.
min
Time course of specific 1~51-labelledhCGRP binding to spleen (a) and gills (b) preparation expressed as % of total radioactivity. O.Zmg/ml of proteins preparation were incubated with 0.07nM of ‘2SI-CGRP at 4°C for different times indicated with or without 2 PM of unlabelled CGRP. Specific binding was expressed as % of total radioactivity.
Fig. 2
2. ‘251-hCGRP binding to membrane preparation and Scatchard analysis of the data
Specific binding of lz51-hCGRP to spleen trout membrane (Fig. 2a) was time dependent. At 5°C binding reached an apparent equilibrium after 120-180min of incubation. Kinetics of ‘251-hCGRP binding to gill membranes are reported in Figure 2b. Increasing concentrations of synthetic hCGRP inhibited ‘251-hCGRP binding to spleen and gill trout membranes in a dose dependent fashion (Fig. 3). The binding of ‘251-labelled CGRP was competitively inhibited by native hCGRP in the range of 0.07-50nM. Half inhibition of the initial 12’I-hCGRP binding (i.e., in the absence of the native CGRP) was observed in spleen membrane (Fig. 3a) and gills membrane (Fig. 3c) with respectively 1.62nM (N = 5) and 2.16nM (N = 3) Table
CGRP. The interaction between hCGRP and its specific binding sites were quantified. Human CGRP binding sites were saturable. At a hormone concentration sufficient for saturating the CGRP binding sites (45nM) the amount of binding sites correspond to 70fmoles/mg of proteins in the spleen membranes and to 53fmoles/mg of proteins in the gills membranes. When these data were analyzed by the method of Scatchard (21) a curvilinear graph was observed with the spleen and gill membranes suggesting the presence of two classes of binding sites or the occurrence of a negative cooperativity (22) (Fig. 3b, d). In these two tissues, the affinity constant for the site I - high affinity low capacity - were of the same order of magnitude (2 to 3 x lO’M_‘). The number of binding sites accounting for were also the same in the gills and in the spleen membrane (Table). The specificity of 1251-hCGRP binding was investigated using salmon CT and human parathyroid hormone (hPTH) (Fig. 4). Half maximal inhibition of specific binding of hCGRP was obtained in the spleen with 3.2nM of CGRP. 2.9nM of sCT produced an inhibition of 50% of the 1251-hCGRP binding to the spleen membranes. In contrast, hPTH was inactive on the binding of CGRP (Fig. 4a). A different effect was observed on gills membranes: sCT was less effective than hCGRP on the displacement of the binding of ‘251-hCGRP to gill membranes. 4nM of unlabelled hCGRP is necessary to displace the binding of L251-hCGRP to the gill membranes, the same effect was observed with a 10 fold higher dose of sCT that is 58.8nM (Fig. 4b). hPTH was inactive on the binding of ‘251-hCGRP to gill membranes. When a spleen membrane preparation was incubated in the presence ‘251-sCT, 1.6nM of sCT displaced 50% of the binding of ‘251-sCT to the membrane; 1.3nM of hCGRP was necessary to obtain the same inhibition (Fig. 5).
Binding parameters of CGRP binding in trout tissues Site I KA (nM)
Spleen Gills
3.2 zk 0.5 x lo9 2.9 + 0.4 x lo9
Site 2 Nlmg of proteins
27.2 f 0.9 x 10H 52.2 x 10R
KA (nM)
4.4 + 1.2 x lo* 2.2 + 0.2 x 108
Nlmg of proteins
220 + 11 x 108 349 x lo8
184
NELJROPEPTIDES
b
d
0
0.00 t-
f--T-Y--T-
0
50
100 CGRP. nglml
0
25 6. fmoleslmg
50 of protein
Fig. 3 Inhibition curve of the binding of ‘251-hCGRP binding to spleen (a) and gills (c) membrane preparation of the trout. 0.2mg/ml of proteins of each preparation were incubated with 0.07nM of ‘251-CGRP and increasing amount of unlabelled hCGRP (0.07 to 45.6nM). Non specific binding was assessed by addition of 2 )IM unlabelled synthetic hCGRP. On the right is the Scatchard analysis of the binding data: (c) spleen membrane; (d) gill membrane. For each experiment, each value was determined in triplicate and the results are the mean of four determinations.
Discussion
The results described above demonstrate that hCGRP binding sites are abundant in trout spleen and gill membrane preparations. In the other tissues of the trout studied the binding of hCGRP was much lower. The distribution of hCGRP binding sites in trout organs is similar to that observed in mammals where the presence of binding sites for hCGRP is maximal in the brain (19, 23) and peripheral tissues such as spleen (5, 10) and lung (9). Unlike the situation observed in mammals (13), the digestive organs in fish are free from hCGRP binding sites. As described previously (24), no sCT binding sites were measured in the trout kidney, maximal
binding for ‘*?-sCT was observed in spleen, brain and heart. The distribution of specific binding sites for respectively hCGRP or sCT is not correlated to the presence of immunoreactive molecules in trout tissues as for instance maximal binding for CGRP is observed in the spleen in which no ir-CGRP is measured (15). Likewise no hCGRP binding is observed in gut where the greatest amount of ir-CGRP was found (15). Saturation experiments and Scatchard analysis suggested the presence of two classes of binding sites in trout spleen and gill preparations. The presence of two classes of binding sites for hCGRP is also reported in mammals: in lung (9) gastrointestinal tract (13), guinea pig pancreatic acini (6) and T lymphocytes (25). In contrast, only one class
185
BINDING SITESOF CALCITONIN GENE RELATED PEPTIDE (CGRP) TO TROUTTISSUES
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Fig. 4 Competitive inhibition of the binding of “‘I-CGRP to the spleen membrane (a) and the gill membrane (b) of the trout with unlabelied human CGRP, salmon CT, and human PTH. The conditions of incubation were the same as described above. Unlabelled CGRP (0). salmon calcitonin (+) and human PTH (m) were added to the membrane preparation at various concentrations
of binding site was measured in the rat spleen. Scatchard analysis also revealed that the value of the high affinity constant was of the same order of magnitude (3 x 109M-‘) as that observed in mammals. 14
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Fig. 5 Competitive inhibition of the binding of “51-salmon calcitonin to the spleen membrane. 0.2mg.ml of proteins of spleen membrane were incubated in the presence of 0.07nM of ‘25I-sCT in the presence or absence of 2 pM of unlabelled sCT and increasing amount of unlabelled sCT (0) or human CGRP (a) for 180min at 4°C.
The value of the affinity constant for the high affinity binding site in gill membranes was comparable to the approximate value (5.3nM) corresponding to the 50% effective dose of hCGRP on adenylate cyclase in the same tissue (16). This numerical similarity between the two values is consistent with a close linkage between hCGRP receptors and adenylate cyclase system. This relationship between binding of hCGRP and increased in CAMP formation is similar to the results described in gastric smooth muscle (13) and pancreatic acini (6). This relationship is more complex in the spleen where no activation of adenylate cyclase activity has been observed. Furthermore the binding studies demonstrate that sCT completely inhibit the saturable binding of hCGRP employed at the same concentration and vice versa. Same observations were reported in the rat brain where Sexton et al (26) described a subtype of CT/CGRP binding site that exhibited high affinity for both CT and CGRP. In conclusion we have established for the first time the presence of specific receptors for CGRP in the gills and spleen membrane of the trout. The results are consistent with a physiologica action of CGRP in branchial cells. The physiological role of
186 CGRP binding sites in trout spleen is not known although its ability to bind two peptides which are biochemically distinct is intriguing. Acknowledgements We are grateful to Professor Y. Le Gal for critical comments.
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