Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
125
[ i]Calcitonin Gene-Related Peptide Binding in Membranes of the Ciliary Body-Iris Block Outi I. Malminiemi & Kimmo H. Malminiemi 125
To cite this article: Outi I. Malminiemi & Kimmo H. Malminiemi (1992) [ i]Calcitonin GeneRelated Peptide Binding in Membranes of the Ciliary Body-Iris Block, Current Eye Research, 11:11, 1079-1085, DOI: 10.3109/02713689209015079 To link to this article: http://dx.doi.org/10.3109/02713689209015079
Published online: 02 Jul 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [McMaster University]
Date: 14 April 2016, At: 23:15
Current Eye Research
Volume 11 number 11 1992, 1079-1085
[ 1251]Calcitoningene-related peptide binding in membranes of the ciliary body-iris block Outi LMalrniniemi and Kimmo H.Malminiemi
Downloaded by [McMaster University] at 23:15 14 April 2016
Leiras, PO Box 33, SF-33721 Tampere, Finland
ABSTRACT Calcitonin gene-related peptide (CGRP) is a mediator of intraocular inflammatory responses, but it may also affect aqueous humour dynamics. The aim of the present work was to characterize CGRP binding sites in the eyes of various mammals. The binding of radiolabelled human CGRP to membranes from the ciliary body-iris (c + i) block of porcine eye showed characteristics expected of an interaction with a receptor site: it was reversible, saturable and displaced by rat CGRP and calcitonin. Studies with CGRP fragments demonstrated the importance of rather long carboxyterminal sequences of the CGRP molecule for high-affinity binding to the receptor. Rat islet amyloid polypeptide (IAPP), which has about 5 0 % structural similarity to CGRP, displaced radioligand binding nearly as efficiently as CGRP, while human IAPP was about twenty-fold less potent. No displaceable CGRP binding could be reliably demonstrated by the present method in c + i membranes from cat, rabbit and bovine eyes, thus indicating differences in the number or localization of CGRP receptors between different mammalian species.
inflammatory reaction (3, 4 ) . In the rabbit, CGRP injected into the anterior chamber of the eye causes vasodilation, breakdown of the blood-aqueous barrier and an increase in the intraocular pressure (IOP)(3, 5 , 6). In the cat, however, exogenous CGRP lowers IOP, apparently by facilitating the outflow of aqueous humour (7). Also in the cynomolgus monkey CGRP increases outflow facility, although the effect is smaller than in cats ( 8 ) . While very little is known about the CGRP receptors mediating these effects in the eye, the aim of this study was to characterize the binding sites for CGRP in the eyes of various mammals. MATERIALS AND METHODS Human (2-['zsI]iodohistidyl") calcitonin gene-related peptide, specific activity 2000 Ci/mmol, was purchased from Amersham International, Amersham, England. Unlabelled rat and human CGRP(I), rat and human IAPP and salmon calcitonin were obtained from Peninsula Laboratories, Inc., Belmont, California, U.S.A. Human CGRP fragments 15 - 37, 28 - 37 and 15 - 24 were obtained from Multiple Peptide System ( M P S ) , California, U.S.A. Other CGRP fragments were synthesized by Dr. Ari Koskinen, University of Surrey in Gilford, England. Bacitracin and aprotinin were from Sigma Chemical Company, St. Louis, U.S.A. Bovine and porcine eyes were
INTRODUCTION Calcitonin gene-related peptide (CGRP) is first and foremost known for its potent vasodilatory effects, but this neuropeptide participates in the regulation of many other physiological and pathophysiological functions throughout the central and peripheral nervous systems (see l). In the anterior segment of the eyes of several species, CGRP is localized to sensory neurons originating in the trigeminal ganglion (2, 3). Together with substance P I also present in these neurons, CGRP mediates some aspects of the intraocular -~
~~
~
~~~~
~
~~
-
~~~
_
_
_
~
Received on July 6 1992; accepted on October 12, 1992
@ Oxford University Press
1079
~
Current
Eye Research
obtained from a local slaughterhouse, kept on ice and dissected within six hours of the death of the animals. New Zealand White rabbits were killed by an overdose of Mebunat"(i.v.). Cats received 0.3 ml Domitor" and 0.3 ml KetalarR i.m. and then 4 5 ml MebunatR intracardially. The ciliary body-iris blocks (c + i) were dissected immediately after the death of the animals. Dissected tissue pieces were washed i n ice-cold isotonic saline, frozen in liquid nitrogen and stored at -80°C. Rabbits and cats were maintained and handled in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 86-23). mnbrane ureDaration The frozen tissues were thawed, cut into small pieces and homogenized with a glassglass homogenizer in 20 vol of ice-cold 10 mM Tris-HC1 buffer, pH 7 . 4 , containing 0.32 M sucrose, and centrifuged at 1000 g for 10 minutes. The supernatant was further centrifuged at 48000 g for 30 minutes and the resulting pellet was washed by resuspension and centrifugation. After suspending the pellet in the same buffer the membranes were stored at -80°C. Bindins assay On the day of the binding assay, the thawed membrane suspension was diluted with the incubation buffer ( 5 0 mM Tris-HC1 supplemented with 1 % BSA, 0.1 mg/ml bacitracin and 0.05 mg/ml aprotinin, pH 7 . 4 ) and 0 . 5 - 0.8 mg (w.w.) of membranes were incubated with the modulator studied or with 0.5 pM rat CGRP (nonspecific binding) for 10 min in an ice-bath. The binding reaction was started with the addition of radiolabelled human CGRP (0.08 nM or as indicated). After incubating for the desired time, the binding reaction was stopped by centrifugation for 3 min at 11500 g at +4"C or by filtration
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1080
on Whatman GF/B filters pretreated with polyethyleneimine. The filters were washed twice with 3 ml of ice-cold 5 mM Tris-HC1 buffer, pH 7 . 4 . The radioactivities of the filters and centrifugation pellets were counted with a Wallac Compugamma counter with a 7 0 % counting efficiency. Nonspecific binding to membranes of porcine c + i and tubes or filters accounted for about 30 4 0 % of the total binding. In membranes from inonpigmented tissues such as lung or spleen nonspecific binding is lower, about 5 .- 20 % (data not shown). The binding experiments were performed in triplicate. Binding data were collected using a self-made PC program and analyzed with a nonlinear, least-squares curve-fitting program (LIGAND, 9 ) .
-
RESULTS The binding of 0.08 nM human [USI]CGRP to washed membranes of porcine ciliary bodyiris block reached equilibrium in about 90
8 0 0
s! *
6
L0 -
.-
4
0 c
.-
n
f
2
0
n
rn
0 0
610
120 Incubation time
180
240
min
Fig. 1 Displaceable [ '''I]CGRP hinding in ciliary body-iris membranes from the porcine eye as a function of incubation time in an ice-bath. At 2 h, 0.5 pM unlabelled rat CGRP(1) was added and the incubation stopped at the time points indicated. Nonspecific binding was 3083 f 462 cpm with 0.6 mg membrane w.w./tube (n=3, mean ? sd)
.
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Current Eye Research min in an ice-bath (Fig. 1). When 0.5 pM rat CGRP was added to the incubation mixture after 2 h incubation, the radiolabel dissociated relatively slowly from the membranes, and after 2 h the dissociation was still incomplete (Fig 1). Nonspecific binding remained the same throughout the incubation. Specific CGRP binding increased in a linear manner up to about 0.8 mg membrane wet weight per tube (Fig. 2 ) . Specific binding of ['%]CGRP to porcine c + i membranes in an ice-bath was displaced by unlabelled human and rat CGRP(1) with IC, values of 1.8 f 0.4 nM (mean f sd) and 2.9 f 0.01 nM, respectively (Fig. 3A). Also human CGRP 8 - 37 and CGRP 15 - 37 displaced CGRP binding in nanomolar concentrations, whereas CGRP 28 - 37 was considerably less potent (Fig. 3B). Several shorter CGRP fragments from the N-terminal tail and the middle part of the molecule did not show any displacing potency in up to 10 pM concentrations (Table 1). Specifically bound radioligand was displaced by salmon calcitonin in near
100
-e
-
80
u
60
C
0
x 0
40
a
0
m
20 0 -12
-6 moll1
-4
-10 -8 -6 log Dirplacrr mol/l
-4
-10 -8 log Displrcer
100
-
80
2 " c
-s
60
0
w 0
40
a
m
20 0 -12
-
100
-
80
-
60
-
40
-
20
-
C
0
L L
c
l2
-
r
0
w 0
a
m
0""""' -12
Fig. 3 I
0
t
0.5
I
I
1
1 .o
Membrane wet weight
1.5 mg/tube
Fig. 2 [1251]CGRPbinding in ciliary bodyiris membranes from the porcine eye (n=3, mean f sd): 0 displaceable binding, 0 nonspecific binding.
-8 log Dirplacer
-10
-6
-4
rnol/l
Displacement of specific
["IICGRP binding in ciliary body-iris
membranes from the porcine eye by structurally related peptides: A) 0 human CGRP(I), 0 rat CGRP(I), 0 salmon calcitonin; B) 0 human CGRP 8-37, 0 human CGRP 15-37, 0 human CGRP 28-37; C) 0 human IAPP, 0 rat IAPP. The curves represent individual displacement experiments performed in triplicate (mean f se).
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Current Eye Research Table 1 Displacement of ['ISI]CGRP binding in cili,arybody-iris membranes of the porcine eye by structurally relat.ed peptides and peptide fragments Diaplacer
nM
ICS,
slope
~Human CGRP Rat CGRP
-
37 CGRP 8 CGRP 15 -- 37 CGRP 28 37 Human IAPP Rat I A P P
--
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Salmon calcitonin
1.8 2.9 0.7 13.6 1076 68.0 3.8 716
i i i i f i i
0.67 0.71 0.73 0.69 0.98 0.98 0.68 0.87
0.4 0.01 0.2 3.8 210 7.2
0.3
f 196
i 0.10 i 0.01 k 0.07
i 0.08 f 0.13 i 0.04 k 0.01 f 0.14
The IC, and slope values are the means f sd of the! results from three to four independent displacement experiments performed in triplicate. I A P P = islet amyloid polypeptide of the pancreas. CGRP fragments 1 - 8 , 2 - 8 , 1 - 1 0 , 1 5 2 1 , 1 5 -. 2 2 , 1 5 - 24 and 1 6 22 did not displace radioligand binding i.n up to 1 0 pM concentrations.
-
micromolar concentrations (Fig. 3A, Table 1). Rat I A P P potently displaced specific CGRP binding to porcine c + i membranes, but human IAPP was about twenty-fold less efficient (Fig. 3C, Table 1 ) . The Hill alopes of the displacement curves were generally below unity, except for human IAPP the Slope value was near unity (Table 1 ) . When porcine c + i membranes were incubated with increasing concentrations of ["I]CGRP, specific binding reached a plateau at about 4 nM radioligand (Fig. 4A). Scatchard transformation of the data suggested the presence of one binding site population with the binding constants of 0 . 6 nM (K,) and 42 pmok/g membrane wet (Fig. 4 B ) . weight (B,) Displaceable binding of radiolabelled human CGRP could not be repeatedly demonstrated in c -+ i membranes from bovine or albino rabbit eyes, whereas the binding in c ii membranes from the cat eye was too low to be reliably characterized (data not shown).
-
DISCUSSION
The binding of iodinated human CGRP in membranes from t:he ciliary body-iris block of the porcine eye showed characteristics expected of an j-nteractionwith a receptor site: it was time-dependent, reversible, saturable and it. increased with increasing amounts of tissue. Specific ['251]CGRP binding was potently displaced by rat. C G R P ( 1 ) and less efficiently by salmon calcitonin, as reported by authors using membranes from other animal tissues ( 1 0 , 1 1 ) . Salmon calcitonin has about 3 0 % structural homology with CGRP. Apparently the conformations of these peptides resemb1.e each other allowing them to interact with one another's receptors, although at a thousandfold higher concentration ( 1 2 ) . Human CGRP 8 - 37 acts as an antagonist at CGRP receptors inhibiting the activation of adenylate cyclase by CGRP in rat liver It also antagonizes the membranes ( 1 3 ) . actions of CGRP in guinea pig atrial and ileal preparations (14). Dennis et al.
Current kye Research A 40
i 3' 0 g 30
v z 0 m
-
10
0
2
0
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Free
4 nmol/l
6
B ao
trations, as reported earlier by other. doing binding studies (15) or biological assays (11, 16). However, Uaggi et al. (17) reported that i.v. injected CGRP 1 15 and 1 22 produced hypotendon and tachycardia in anesthetized rats, but with at least a hundredfold lower potency and smaller maximal effect than intact CGRP. Further shortening of the carboxyterminal peptide fragment decreased its affinity for the CGRP receptor: CGRP 15 37 showed an affinity about eight times lower than that of the native molecule, and near micromolar concentrations of CGRP 28 - 37 were needed for displacement of the radioligand. Buran CGRP 12 37 has previously displaced CGRP binding in splenic membranes with an IC, of 38.4 f 9.7 nM, while the displacing potencies of human and rat CGRP(1) were 3.2 f 0.6 nM and 3.5 f 0.2 nM (ll), comparable to those found in this work. Peptides from the middle part of the CGRP molecule showed no displacing potency, thus indicating the importance of the carboxyterminal tail in the binding reaction. The islet amyloid polypeptide (IAPP) of the pancreas, or amylin, is a 37-arino acid peptide with an aminoterminal disulphide bridge ring. It has a 40 - 50 % structural homology with CGRP (see 18). Considering the rather high degree of structural dissimilarity between the peptides, the
r
-
-
-
-z P
20
m
0 0
20 40 Bound pmol/g membrane
60 W.W.
Fig. 4 A ) Saturation assay of ['sI]CGRP binding in ciliary body-iris membranes from the porcine eye (n=3, mean f se). B) Scatchard transformation of the data. K, = 0.6 nM, ,,B = 41 pmol/g membrane W.W.
-
(14) found the affinity of CGRP 8 37 for the CGRP receptor to be higher than that of the native peptide, as was also seen in this study, demonstrating that the aminoterminal disulphide bridge ring of the CGRP molecule is not essential for binding but in fact hinders it slightly. In accordance, short peptides from the aminoterminus of the human CGRP molecule did not show any activity in micromolar concen-
affinity of rat IAPP for CGRP receptors in porcine c + i membranes was unexpectedly high, only about two times lower than that of human CGRP. Previously, IAPP has been reported to displace radiolabelled rat {TyrOJCGRP binding to rat liver plasma membranes, but at 100000-fold higher concentrations compared to rat CGRP (19). When this manuscript was in preparation, Galeazza et al. (18) reported that IAPP
1083
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compete8 for binding t.o two populations of CGRP receptora in rat hrain, muscle and liver membranes. Human IAPP was 10 to .1000-fold lesa potent than rCGRP(1) in displacing radiolabeled CGRP, depending on the tiauue and the binding site. In this work, rat I M P and human IAPP were found to be about 2- and 4O-fold less effective in competing with radi olatbelledhuman CGRP for binding to porcine c + imembranes. There is a continuous sequence of nine amino acid reaidues (from 20 to 2 8 ) in rat I M P (ten reddues in the human peptide) that differs from CGRP. It is interesting that the diseimilarity between rat and human I M P , which have about R twenty-fold difference in affinity, i e mainly located in this
area * Saturation assay revealed the presence of one ~ ~ n d i site ~ ipopulation ~ only in c + i block, the ~ ~ r ~ of n parcine e s Scatchard transformation data fitting a straight line. Instead, the slope values from displacement studies were generally low unity, suggesting more than one type of binding sites. The R,, value found (0.6 nbfj
agrees well. with several reports on
CGRP binding e.g. in membranes of the
spinal cord, cerebellum, liver and spleen (18). However, atherr studies have reported two binding S I te p o p t x l a t ions with affinities fox CGHiP varying from l a w picamolar t o micromol~arvalues in various animal tissues ( s e e 1.81. To our knowledge there are no reports about CGRP binding studies u s i n q eye t L a s u e . Human IAPP dieplaced bound rad i o X .igand with a slope value near unity, suggesting that its t ht? receptor site ( s ) may that ~ ~ iof t h e other displacers.
1 nteraction iv 1 th
diffier
t
~
t
Oi- rtie animal s p e c i e s studied, only
~ e m b r ~ fa~ urnethe ~ c i l ary ~ body-iris block o f t h e porci rie eye shcrwed displaceable '"l: jC(;KP bzndinq, whereas binding could riot be demvnivt I a t e t i i n preparations fI om
rabbit, cat and bovine eyes in sufficiently high amounts to allow characterization. CGRP-immunoreactive nerve fibres have been demonstrated in the ciliary body and the iris of the rat, cat and monkey eyes (2). While there apparently are CGRP binding sites also in the ciliary body of the rabbit eye (the authors' unpublished autoradiographic studies), the preparation used in this study is a membrane fraction from the ciliary body-iris block and may be too crude to detect binding sites in low numbers when nonspecific binding is relatively high. In conclusion, membranes from the ciliary body-irns block of the porcine eye contain binding sites for CGRP with characteristics similar to CGRP receptors previously demonstrated in other peripheral tissues. The high affinities o f rat and human IAPP for these binding sites may help to elucidate the! structural requirements for high-affinity binding to the CGRP receptor. ACKNOWLEDGEMENTS The authors wish to thank Mrs. Sirkka Helin for excell.ent technical assistance and Mr. Kimmo Hetkala for the care and handling of the animals. CORRESPONDING AUTHOR Outi Malminiemi, Tampere University Hospital, Dept. of Clinical Chemistry, P.0.Box 2000, SF'-33721 Tampere, Finland. REFERENCES 1. Breimer, L.H., MacIntyre, I. and Zaidi, M. (1988) Peptides from the calcitonin genes: molecular genetics, structure and function. Biochem. J. E ,3 7 7 - 3 9 0 . 2 . Terenghi, G., Polak, J.M., Ghatei, M.A. , Mulderry, P.K . , Butler, J.M., Unger, W.G. and Bloom, S.R. ( 1 9 8 5 ) Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye. J . Comp. Neural. 2 3 3 , 506-516.
Current Eye Research 3. Unger, W.G., Terenghi, G., Ghatei,
4. 5.
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6.
7.
8.
9.
M.A., Ennis, K.W., Butler, J.M., Zhang, S.Q., Too, H.P., Polak, J.M. and Bloom, S.R. (1985) Calcitonin gene-related polypeptide as a mediator of the neurogenic ocular injury response. J. Ocular Pharmacol. 1, 189-199. Krootila, K. (1988) CGRP in relation to neurogenic inflammation and CAMP in the rabbit eye. Exp. Eye Res. 47, 307-316. Oksala, 0. (1988) Effects of calcitonin gene-related peptide and substance P on regional blood flow in the cat eye. Exp. Eye Res. 47, 283-289. Oksala, 0. and Stjernschantz, J. (1988a) Effects of calcitonin generelated peptide in the eye: A study in rabbits and cats. Invest. Ophthalmol. Vis. Sci. 29, 1006-1011. Oksala, 0. and Stjernschantz, J. (1988b) Increase in outflow facility of aqueous humour in cats induced by calcitonin gene-related peptide. Exp. Eye Res. 4 7 , 787-790. Almeghd, B. and Andersson, S.E. (1990) Outflow facility in the monkey eye: Effects of calcitonin gene-related peptide, cholecystokinin, galanin, substance P and capsaicin. Exp. Eye. Res. 5 l , 685-689. McPherson, G.A. (1985) Analysis of radioligand binding experiments: A collection of computer programs for the IBM PC. J. Pharmacol. Methods,
gene-related peptide receptor heterogeneity in brain and periphery. J. Pharmacol. Exp. Ther. m, 123-128. 15. Wimalawansa, S.J. and MacIntyre, I. (1988) Calcitonin gene-related peptide and its specific binding sites in the cardiovascular system of rat. Int. J. Cardiol. 20, 29-37. 16. Tippins, J.R., Di Marzo, V., Panico, M., Morris, H.R. and MacIntyre, I. (1986) Investigation of the structure/activity relationship of human calcitonin gene-related peptide (CGRP). Biochem. Biophys. Re.. Coerun. 134 , 1306-1311. 17. Maggi, C.A., Rovero, P., Giuliani, S.,
Evangelista, S . , Regoli, D. and Ileli, A. (1990) Biological activity of Nterminal fragments of calcitonin generelated peptide. Eur. J. Pharmacol. 179, 217-219. 18. a e a z z a , H.T. , O'Brien, T.D. , Johnson, K.H. and Seybold, V.S. (1991) Islet amyloid polypeptide ( I M P ) competes for two binding sites for CGRP. Peptides, 12, 585-591. 19. Grishita, T. , Yamaguchi, A . , Fujita, T. and Chiba, T. (1990) Activation of adenylate cyclase by islet amyloid polypeptide with COOH-terminal a.m.j.de via calcitonin gene-related peptide receptors on rat liver plasma membranes. Diabetes, 39, 875-877.
14, 213-228. 10. hkamuta, H. , Fukuda, Y. , Koida, M. ,
Fujii, N., Otaka, A., Funakoshi, S . , Yajima, H., Mitsuyasu, N. and Orlowski, R.C. (1986) Binding sites of calcitonin gene-related peptide (CGRP): Abundant occurrence in visceral organs. Japan J. Pharmacol. 42, 175-180. 11. Dennis, T., Fournier, A., St. Pierre, S . and Quirion, R. (1989) Structureactivity profile of calcitonin generelated peptide in peripheral and brain tissues. Evidence for receptor multiplicity. J. Pharmacol. Exp. Ther. 251, 718-725. 12. Z t z m a n n , D. and Mitchell, J. (19985)
Interaction of calcitonin and calcitonin gene-related peptide at receptor sites in target tissues. Science, 227, 1343-1345. 13. Chiba, C., Yamaguchi, A., Yamatani, T., Nakamura, A., Morishita, T., Inui, T., Fukase, M., Noda, T. and Fujita, T. (1989) Calcitonin gene-related peptide receptor antagonist human-(8-37). Am. J. Physiol. 256, E331-E335. 14. Dennis, T., Fournier, A., Cadieux, A., Pomerleau, F., Jolicoeur, F.B., St. Pierre, S . and Quirion, R. (1990) hCGRP8-37, a calcitonin gene-related peptide antagonist revealing calcitonin
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ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
125
[ i]Calcitonin Gene-Related Peptide Binding in Membranes of the Ciliary Body-Iris Block Outi I. Malminiemi & Kimmo H. Malminiemi 125
To cite this article: Outi I. Malminiemi & Kimmo H. Malminiemi (1992) [ i]Calcitonin GeneRelated Peptide Binding in Membranes of the Ciliary Body-Iris Block, Current Eye Research, 11:11, 1079-1085, DOI: 10.3109/02713689209015079 To link to this article: http://dx.doi.org/10.3109/02713689209015079
Published online: 02 Jul 2009.
Submit your article to this journal
Article views: 3
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [McMaster University]
Date: 14 April 2016, At: 23:15
Current Eye Research
Volume 11 number 11 1992, 1079-1085
[ 1251]Calcitoningene-related peptide binding in membranes of the ciliary body-iris block Outi LMalrniniemi and Kimmo H.Malminiemi
Downloaded by [McMaster University] at 23:15 14 April 2016
Leiras, PO Box 33, SF-33721 Tampere, Finland
ABSTRACT Calcitonin gene-related peptide (CGRP) is a mediator of intraocular inflammatory responses, but it may also affect aqueous humour dynamics. The aim of the present work was to characterize CGRP binding sites in the eyes of various mammals. The binding of radiolabelled human CGRP to membranes from the ciliary body-iris (c + i) block of porcine eye showed characteristics expected of an interaction with a receptor site: it was reversible, saturable and displaced by rat CGRP and calcitonin. Studies with CGRP fragments demonstrated the importance of rather long carboxyterminal sequences of the CGRP molecule for high-affinity binding to the receptor. Rat islet amyloid polypeptide (IAPP), which has about 5 0 % structural similarity to CGRP, displaced radioligand binding nearly as efficiently as CGRP, while human IAPP was about twenty-fold less potent. No displaceable CGRP binding could be reliably demonstrated by the present method in c + i membranes from cat, rabbit and bovine eyes, thus indicating differences in the number or localization of CGRP receptors between different mammalian species.
inflammatory reaction (3, 4 ) . In the rabbit, CGRP injected into the anterior chamber of the eye causes vasodilation, breakdown of the blood-aqueous barrier and an increase in the intraocular pressure (IOP)(3, 5 , 6). In the cat, however, exogenous CGRP lowers IOP, apparently by facilitating the outflow of aqueous humour (7). Also in the cynomolgus monkey CGRP increases outflow facility, although the effect is smaller than in cats ( 8 ) . While very little is known about the CGRP receptors mediating these effects in the eye, the aim of this study was to characterize the binding sites for CGRP in the eyes of various mammals. MATERIALS AND METHODS Human (2-['zsI]iodohistidyl") calcitonin gene-related peptide, specific activity 2000 Ci/mmol, was purchased from Amersham International, Amersham, England. Unlabelled rat and human CGRP(I), rat and human IAPP and salmon calcitonin were obtained from Peninsula Laboratories, Inc., Belmont, California, U.S.A. Human CGRP fragments 15 - 37, 28 - 37 and 15 - 24 were obtained from Multiple Peptide System ( M P S ) , California, U.S.A. Other CGRP fragments were synthesized by Dr. Ari Koskinen, University of Surrey in Gilford, England. Bacitracin and aprotinin were from Sigma Chemical Company, St. Louis, U.S.A. Bovine and porcine eyes were
INTRODUCTION Calcitonin gene-related peptide (CGRP) is first and foremost known for its potent vasodilatory effects, but this neuropeptide participates in the regulation of many other physiological and pathophysiological functions throughout the central and peripheral nervous systems (see l). In the anterior segment of the eyes of several species, CGRP is localized to sensory neurons originating in the trigeminal ganglion (2, 3). Together with substance P I also present in these neurons, CGRP mediates some aspects of the intraocular -~
~~
~
~~~~
~
~~
-
~~~
_
_
_
~
Received on July 6 1992; accepted on October 12, 1992
@ Oxford University Press
1079
~
Current
Eye Research
obtained from a local slaughterhouse, kept on ice and dissected within six hours of the death of the animals. New Zealand White rabbits were killed by an overdose of Mebunat"(i.v.). Cats received 0.3 ml Domitor" and 0.3 ml KetalarR i.m. and then 4 5 ml MebunatR intracardially. The ciliary body-iris blocks (c + i) were dissected immediately after the death of the animals. Dissected tissue pieces were washed i n ice-cold isotonic saline, frozen in liquid nitrogen and stored at -80°C. Rabbits and cats were maintained and handled in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 86-23). mnbrane ureDaration The frozen tissues were thawed, cut into small pieces and homogenized with a glassglass homogenizer in 20 vol of ice-cold 10 mM Tris-HC1 buffer, pH 7 . 4 , containing 0.32 M sucrose, and centrifuged at 1000 g for 10 minutes. The supernatant was further centrifuged at 48000 g for 30 minutes and the resulting pellet was washed by resuspension and centrifugation. After suspending the pellet in the same buffer the membranes were stored at -80°C. Bindins assay On the day of the binding assay, the thawed membrane suspension was diluted with the incubation buffer ( 5 0 mM Tris-HC1 supplemented with 1 % BSA, 0.1 mg/ml bacitracin and 0.05 mg/ml aprotinin, pH 7 . 4 ) and 0 . 5 - 0.8 mg (w.w.) of membranes were incubated with the modulator studied or with 0.5 pM rat CGRP (nonspecific binding) for 10 min in an ice-bath. The binding reaction was started with the addition of radiolabelled human CGRP (0.08 nM or as indicated). After incubating for the desired time, the binding reaction was stopped by centrifugation for 3 min at 11500 g at +4"C or by filtration
Downloaded by [McMaster University] at 23:15 14 April 2016
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1080
on Whatman GF/B filters pretreated with polyethyleneimine. The filters were washed twice with 3 ml of ice-cold 5 mM Tris-HC1 buffer, pH 7 . 4 . The radioactivities of the filters and centrifugation pellets were counted with a Wallac Compugamma counter with a 7 0 % counting efficiency. Nonspecific binding to membranes of porcine c + i and tubes or filters accounted for about 30 4 0 % of the total binding. In membranes from inonpigmented tissues such as lung or spleen nonspecific binding is lower, about 5 .- 20 % (data not shown). The binding experiments were performed in triplicate. Binding data were collected using a self-made PC program and analyzed with a nonlinear, least-squares curve-fitting program (LIGAND, 9 ) .
-
RESULTS The binding of 0.08 nM human [USI]CGRP to washed membranes of porcine ciliary bodyiris block reached equilibrium in about 90
8 0 0
s! *
6
L0 -
.-
4
0 c
.-
n
f
2
0
n
rn
0 0
610
120 Incubation time
180
240
min
Fig. 1 Displaceable [ '''I]CGRP hinding in ciliary body-iris membranes from the porcine eye as a function of incubation time in an ice-bath. At 2 h, 0.5 pM unlabelled rat CGRP(1) was added and the incubation stopped at the time points indicated. Nonspecific binding was 3083 f 462 cpm with 0.6 mg membrane w.w./tube (n=3, mean ? sd)
.
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Current Eye Research min in an ice-bath (Fig. 1). When 0.5 pM rat CGRP was added to the incubation mixture after 2 h incubation, the radiolabel dissociated relatively slowly from the membranes, and after 2 h the dissociation was still incomplete (Fig 1). Nonspecific binding remained the same throughout the incubation. Specific CGRP binding increased in a linear manner up to about 0.8 mg membrane wet weight per tube (Fig. 2 ) . Specific binding of ['%]CGRP to porcine c + i membranes in an ice-bath was displaced by unlabelled human and rat CGRP(1) with IC, values of 1.8 f 0.4 nM (mean f sd) and 2.9 f 0.01 nM, respectively (Fig. 3A). Also human CGRP 8 - 37 and CGRP 15 - 37 displaced CGRP binding in nanomolar concentrations, whereas CGRP 28 - 37 was considerably less potent (Fig. 3B). Several shorter CGRP fragments from the N-terminal tail and the middle part of the molecule did not show any displacing potency in up to 10 pM concentrations (Table 1). Specifically bound radioligand was displaced by salmon calcitonin in near
100
-e
-
80
u
60
C
0
x 0
40
a
0
m
20 0 -12
-6 moll1
-4
-10 -8 -6 log Dirplacrr mol/l
-4
-10 -8 log Displrcer
100
-
80
2 " c
-s
60
0
w 0
40
a
m
20 0 -12
-
100
-
80
-
60
-
40
-
20
-
C
0
L L
c
l2
-
r
0
w 0
a
m
0""""' -12
Fig. 3 I
0
t
0.5
I
I
1
1 .o
Membrane wet weight
1.5 mg/tube
Fig. 2 [1251]CGRPbinding in ciliary bodyiris membranes from the porcine eye (n=3, mean f sd): 0 displaceable binding, 0 nonspecific binding.
-8 log Dirplacer
-10
-6
-4
rnol/l
Displacement of specific
["IICGRP binding in ciliary body-iris
membranes from the porcine eye by structurally related peptides: A) 0 human CGRP(I), 0 rat CGRP(I), 0 salmon calcitonin; B) 0 human CGRP 8-37, 0 human CGRP 15-37, 0 human CGRP 28-37; C) 0 human IAPP, 0 rat IAPP. The curves represent individual displacement experiments performed in triplicate (mean f se).
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Current Eye Research Table 1 Displacement of ['ISI]CGRP binding in cili,arybody-iris membranes of the porcine eye by structurally relat.ed peptides and peptide fragments Diaplacer
nM
ICS,
slope
~Human CGRP Rat CGRP
-
37 CGRP 8 CGRP 15 -- 37 CGRP 28 37 Human IAPP Rat I A P P
--
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Salmon calcitonin
1.8 2.9 0.7 13.6 1076 68.0 3.8 716
i i i i f i i
0.67 0.71 0.73 0.69 0.98 0.98 0.68 0.87
0.4 0.01 0.2 3.8 210 7.2
0.3
f 196
i 0.10 i 0.01 k 0.07
i 0.08 f 0.13 i 0.04 k 0.01 f 0.14
The IC, and slope values are the means f sd of the! results from three to four independent displacement experiments performed in triplicate. I A P P = islet amyloid polypeptide of the pancreas. CGRP fragments 1 - 8 , 2 - 8 , 1 - 1 0 , 1 5 2 1 , 1 5 -. 2 2 , 1 5 - 24 and 1 6 22 did not displace radioligand binding i.n up to 1 0 pM concentrations.
-
micromolar concentrations (Fig. 3A, Table 1). Rat I A P P potently displaced specific CGRP binding to porcine c + i membranes, but human IAPP was about twenty-fold less efficient (Fig. 3C, Table 1 ) . The Hill alopes of the displacement curves were generally below unity, except for human IAPP the Slope value was near unity (Table 1 ) . When porcine c + i membranes were incubated with increasing concentrations of ["I]CGRP, specific binding reached a plateau at about 4 nM radioligand (Fig. 4A). Scatchard transformation of the data suggested the presence of one binding site population with the binding constants of 0 . 6 nM (K,) and 42 pmok/g membrane wet (Fig. 4 B ) . weight (B,) Displaceable binding of radiolabelled human CGRP could not be repeatedly demonstrated in c -+ i membranes from bovine or albino rabbit eyes, whereas the binding in c ii membranes from the cat eye was too low to be reliably characterized (data not shown).
-
DISCUSSION
The binding of iodinated human CGRP in membranes from t:he ciliary body-iris block of the porcine eye showed characteristics expected of an j-nteractionwith a receptor site: it was time-dependent, reversible, saturable and it. increased with increasing amounts of tissue. Specific ['251]CGRP binding was potently displaced by rat. C G R P ( 1 ) and less efficiently by salmon calcitonin, as reported by authors using membranes from other animal tissues ( 1 0 , 1 1 ) . Salmon calcitonin has about 3 0 % structural homology with CGRP. Apparently the conformations of these peptides resemb1.e each other allowing them to interact with one another's receptors, although at a thousandfold higher concentration ( 1 2 ) . Human CGRP 8 - 37 acts as an antagonist at CGRP receptors inhibiting the activation of adenylate cyclase by CGRP in rat liver It also antagonizes the membranes ( 1 3 ) . actions of CGRP in guinea pig atrial and ileal preparations (14). Dennis et al.
Current kye Research A 40
i 3' 0 g 30
v z 0 m
-
10
0
2
0
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Free
4 nmol/l
6
B ao
trations, as reported earlier by other. doing binding studies (15) or biological assays (11, 16). However, Uaggi et al. (17) reported that i.v. injected CGRP 1 15 and 1 22 produced hypotendon and tachycardia in anesthetized rats, but with at least a hundredfold lower potency and smaller maximal effect than intact CGRP. Further shortening of the carboxyterminal peptide fragment decreased its affinity for the CGRP receptor: CGRP 15 37 showed an affinity about eight times lower than that of the native molecule, and near micromolar concentrations of CGRP 28 - 37 were needed for displacement of the radioligand. Buran CGRP 12 37 has previously displaced CGRP binding in splenic membranes with an IC, of 38.4 f 9.7 nM, while the displacing potencies of human and rat CGRP(1) were 3.2 f 0.6 nM and 3.5 f 0.2 nM (ll), comparable to those found in this work. Peptides from the middle part of the CGRP molecule showed no displacing potency, thus indicating the importance of the carboxyterminal tail in the binding reaction. The islet amyloid polypeptide (IAPP) of the pancreas, or amylin, is a 37-arino acid peptide with an aminoterminal disulphide bridge ring. It has a 40 - 50 % structural homology with CGRP (see 18). Considering the rather high degree of structural dissimilarity between the peptides, the
r
-
-
-
-z P
20
m
0 0
20 40 Bound pmol/g membrane
60 W.W.
Fig. 4 A ) Saturation assay of ['sI]CGRP binding in ciliary body-iris membranes from the porcine eye (n=3, mean f se). B) Scatchard transformation of the data. K, = 0.6 nM, ,,B = 41 pmol/g membrane W.W.
-
(14) found the affinity of CGRP 8 37 for the CGRP receptor to be higher than that of the native peptide, as was also seen in this study, demonstrating that the aminoterminal disulphide bridge ring of the CGRP molecule is not essential for binding but in fact hinders it slightly. In accordance, short peptides from the aminoterminus of the human CGRP molecule did not show any activity in micromolar concen-
affinity of rat IAPP for CGRP receptors in porcine c + i membranes was unexpectedly high, only about two times lower than that of human CGRP. Previously, IAPP has been reported to displace radiolabelled rat {TyrOJCGRP binding to rat liver plasma membranes, but at 100000-fold higher concentrations compared to rat CGRP (19). When this manuscript was in preparation, Galeazza et al. (18) reported that IAPP
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compete8 for binding t.o two populations of CGRP receptora in rat hrain, muscle and liver membranes. Human IAPP was 10 to .1000-fold lesa potent than rCGRP(1) in displacing radiolabeled CGRP, depending on the tiauue and the binding site. In this work, rat I M P and human IAPP were found to be about 2- and 4O-fold less effective in competing with radi olatbelledhuman CGRP for binding to porcine c + imembranes. There is a continuous sequence of nine amino acid reaidues (from 20 to 2 8 ) in rat I M P (ten reddues in the human peptide) that differs from CGRP. It is interesting that the diseimilarity between rat and human I M P , which have about R twenty-fold difference in affinity, i e mainly located in this
area * Saturation assay revealed the presence of one ~ ~ n d i site ~ ipopulation ~ only in c + i block, the ~ ~ r ~ of n parcine e s Scatchard transformation data fitting a straight line. Instead, the slope values from displacement studies were generally low unity, suggesting more than one type of binding sites. The R,, value found (0.6 nbfj
agrees well. with several reports on
CGRP binding e.g. in membranes of the
spinal cord, cerebellum, liver and spleen (18). However, atherr studies have reported two binding S I te p o p t x l a t ions with affinities fox CGHiP varying from l a w picamolar t o micromol~arvalues in various animal tissues ( s e e 1.81. To our knowledge there are no reports about CGRP binding studies u s i n q eye t L a s u e . Human IAPP dieplaced bound rad i o X .igand with a slope value near unity, suggesting that its t ht? receptor site ( s ) may that ~ ~ iof t h e other displacers.
1 nteraction iv 1 th
diffier
t
~
t
Oi- rtie animal s p e c i e s studied, only
~ e m b r ~ fa~ urnethe ~ c i l ary ~ body-iris block o f t h e porci rie eye shcrwed displaceable '"l: jC(;KP bzndinq, whereas binding could riot be demvnivt I a t e t i i n preparations fI om
rabbit, cat and bovine eyes in sufficiently high amounts to allow characterization. CGRP-immunoreactive nerve fibres have been demonstrated in the ciliary body and the iris of the rat, cat and monkey eyes (2). While there apparently are CGRP binding sites also in the ciliary body of the rabbit eye (the authors' unpublished autoradiographic studies), the preparation used in this study is a membrane fraction from the ciliary body-iris block and may be too crude to detect binding sites in low numbers when nonspecific binding is relatively high. In conclusion, membranes from the ciliary body-irns block of the porcine eye contain binding sites for CGRP with characteristics similar to CGRP receptors previously demonstrated in other peripheral tissues. The high affinities o f rat and human IAPP for these binding sites may help to elucidate the! structural requirements for high-affinity binding to the CGRP receptor. ACKNOWLEDGEMENTS The authors wish to thank Mrs. Sirkka Helin for excell.ent technical assistance and Mr. Kimmo Hetkala for the care and handling of the animals. CORRESPONDING AUTHOR Outi Malminiemi, Tampere University Hospital, Dept. of Clinical Chemistry, P.0.Box 2000, SF'-33721 Tampere, Finland. REFERENCES 1. Breimer, L.H., MacIntyre, I. and Zaidi, M. (1988) Peptides from the calcitonin genes: molecular genetics, structure and function. Biochem. J. E ,3 7 7 - 3 9 0 . 2 . Terenghi, G., Polak, J.M., Ghatei, M.A. , Mulderry, P.K . , Butler, J.M., Unger, W.G. and Bloom, S.R. ( 1 9 8 5 ) Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye. J . Comp. Neural. 2 3 3 , 506-516.
Current Eye Research 3. Unger, W.G., Terenghi, G., Ghatei,
4. 5.
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M.A., Ennis, K.W., Butler, J.M., Zhang, S.Q., Too, H.P., Polak, J.M. and Bloom, S.R. (1985) Calcitonin gene-related polypeptide as a mediator of the neurogenic ocular injury response. J. Ocular Pharmacol. 1, 189-199. Krootila, K. (1988) CGRP in relation to neurogenic inflammation and CAMP in the rabbit eye. Exp. Eye Res. 47, 307-316. Oksala, 0. (1988) Effects of calcitonin gene-related peptide and substance P on regional blood flow in the cat eye. Exp. Eye Res. 47, 283-289. Oksala, 0. and Stjernschantz, J. (1988a) Effects of calcitonin generelated peptide in the eye: A study in rabbits and cats. Invest. Ophthalmol. Vis. Sci. 29, 1006-1011. Oksala, 0. and Stjernschantz, J. (1988b) Increase in outflow facility of aqueous humour in cats induced by calcitonin gene-related peptide. Exp. Eye Res. 4 7 , 787-790. Almeghd, B. and Andersson, S.E. (1990) Outflow facility in the monkey eye: Effects of calcitonin gene-related peptide, cholecystokinin, galanin, substance P and capsaicin. Exp. Eye. Res. 5 l , 685-689. McPherson, G.A. (1985) Analysis of radioligand binding experiments: A collection of computer programs for the IBM PC. J. Pharmacol. Methods,
gene-related peptide receptor heterogeneity in brain and periphery. J. Pharmacol. Exp. Ther. m, 123-128. 15. Wimalawansa, S.J. and MacIntyre, I. (1988) Calcitonin gene-related peptide and its specific binding sites in the cardiovascular system of rat. Int. J. Cardiol. 20, 29-37. 16. Tippins, J.R., Di Marzo, V., Panico, M., Morris, H.R. and MacIntyre, I. (1986) Investigation of the structure/activity relationship of human calcitonin gene-related peptide (CGRP). Biochem. Biophys. Re.. Coerun. 134 , 1306-1311. 17. Maggi, C.A., Rovero, P., Giuliani, S.,
Evangelista, S . , Regoli, D. and Ileli, A. (1990) Biological activity of Nterminal fragments of calcitonin generelated peptide. Eur. J. Pharmacol. 179, 217-219. 18. a e a z z a , H.T. , O'Brien, T.D. , Johnson, K.H. and Seybold, V.S. (1991) Islet amyloid polypeptide ( I M P ) competes for two binding sites for CGRP. Peptides, 12, 585-591. 19. Grishita, T. , Yamaguchi, A . , Fujita, T. and Chiba, T. (1990) Activation of adenylate cyclase by islet amyloid polypeptide with COOH-terminal a.m.j.de via calcitonin gene-related peptide receptors on rat liver plasma membranes. Diabetes, 39, 875-877.
14, 213-228. 10. hkamuta, H. , Fukuda, Y. , Koida, M. ,
Fujii, N., Otaka, A., Funakoshi, S . , Yajima, H., Mitsuyasu, N. and Orlowski, R.C. (1986) Binding sites of calcitonin gene-related peptide (CGRP): Abundant occurrence in visceral organs. Japan J. Pharmacol. 42, 175-180. 11. Dennis, T., Fournier, A., St. Pierre, S . and Quirion, R. (1989) Structureactivity profile of calcitonin generelated peptide in peripheral and brain tissues. Evidence for receptor multiplicity. J. Pharmacol. Exp. Ther. 251, 718-725. 12. Z t z m a n n , D. and Mitchell, J. (19985)
Interaction of calcitonin and calcitonin gene-related peptide at receptor sites in target tissues. Science, 227, 1343-1345. 13. Chiba, C., Yamaguchi, A., Yamatani, T., Nakamura, A., Morishita, T., Inui, T., Fukase, M., Noda, T. and Fujita, T. (1989) Calcitonin gene-related peptide receptor antagonist human-(8-37). Am. J. Physiol. 256, E331-E335. 14. Dennis, T., Fournier, A., Cadieux, A., Pomerleau, F., Jolicoeur, F.B., St. Pierre, S . and Quirion, R. (1990) hCGRP8-37, a calcitonin gene-related peptide antagonist revealing calcitonin
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