Inhibitory effects of calcitonin on adenylate cyclase activity in different rat brain areas

Life Sciences, Vol. 39, pp. 2253-2262 Printed in the U.S.A.

Pergamon Journals

INHIBITORY EFFECTS OF CALCITBNIN ON ADENYLATE CYCLASE ACTIVITY IN DIFFERENT RAI BRAIN AREAS

S. Nicosia, F. Guidobono*, M. Musanti and A. Pecile* Institute of Pharmacology and Pharmacognosy and *Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, I t a l y (Received in final form September 8, 1986)

Su~ms~ We have investigated the effect of calcitonin (CT) on adenylate cyclase in membranes From different rat brain areas. Salmon calcitonin (sCl) dose-dependently inhibited the enzyme activity in midbrain, hypothalamus, medulla, ports and caudate nucleus, but was ineffective in adenohypophysis, lhe inhibitory effect was enhanced by GIP. Comparison of calcitonins of different origin indicated that sCT was the most potent in inhibiting the enzyme in hypothalamic membranes, eel C] (eCI) was slightly less potent, and human CI (hCI) was ineffective. Chronic I.C.V. pretreatment with sCT did not modify the subsequent in vitro sensitivity of adenylate cyclase to sCT. It is concluded that some of CNS actions of CI might involve modulation of intrace]lular cAMP levels. lhe primary action of caleitonin (CI) so far recognized is the regulation of calcium hofaeostasis by inhibition of bone resorption (i) and increasing renal clearance of some electrolytes (2,3). High affinity receptors for CT have been documented in several tissues, including bone and kidney, and in human cancer cell lines (4-6), in which studies of the mechanism of CI action focused on characterization of hormone-receptor interaction demonstrated that the peptide stimulates adenylate cyclase and cyclic AMP-dependent kinase (7,8). In addition to its activities in bone and kidney, several types of evidence have suggested that CT might also function in the central nervous system. Cl-]ike molecules have been detected in the nervous systems of most vertebrates (9) and in the brains of cyciostomes and protochordates (10) and specific and saturable binding sites For CT have been demonstrated in the CNS of rats and humans (ll,12). It is possible that CT had an original function as a neurotransmitter or neuromodulator; thus, some earlier function of CT or perhaps some neuroactive CT-like peptide that evolved might explain the behavioral effects seen when CT is injected intracerebroventricuiarly (I.C.V.) into vertebrates (13,14).

0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.

2254

Calcitonin and CNS Adenylate Cyclase

Vol. 39, No. 23, [986

Since many neucopeptides have been shown to act by modulating the formation of a second messenger i n t r a c e l l u l a r l y , we have studied whether Cf-receptoc i n t e r a c t i o n in v i t r o in the CNS a f f e c t s the a c t i v i t y off adenylate cyclase in membranes firom d i f f e r e n t areas of r a t brain and whether t h i s i n t e r a c t i o n can be modified by previous I.C.V. treatment of animals with Cf in ViVO.

Materials

and Methods

M a t e r i a l s . [8-14C1ATP and L8-3HJcAMP were purchased firom Amersham (Amecsham, U.K.). ATP, cAMP, GTP, c r e a [ i n e phosphate, c r e a t i n e phosphokinase, i s o b u t y l methytxanthine, E6TA and bacitPacin were From Sigma Chemical Co. (St. l o u i s , NO, U.S.A.). Salmon CT (sCT), human CT (hCf) and eel CT (eCf) were g i f t s firom Sandoz (Basel, S w i t z e r l a n d ) , Ciba geigy (Ociggio, I t a l y ) and [ s t i t u t o Sclavo (Siena, I t a l y ) . Animals. Male Sptague-Dawley caLs (180-200 g) were used. They were kept under c o n t r o l l e d c o n d i t i o n s (22 + 2°C and l i g h t from 6 a.m. to 8 p.m.) and given a standard cat d i e t and waker ad l i b i t u m . Polyethylene cannulae were implanted by the technique of A l t a f f e r et a l . (15). i n t o the l e f t l a t e r a l v e n t r i c l e s of the b r a i n f o r I.C.V. a d m i n i s t r a t i o n of sCf. Treatment. sCI dissolved in i0 ul of saline was administered I.C.V. at the dose of 250 ng/rat, for I0 days or fop 14 days, or as a single administration. The animals were killed 24 h after the last treatment. Controls were given saline I.C.V.. Membrane p r e p a r a t i o n . Rats were k i l l e d by d e c a p i t a t i o n and the brains removed. [he d i f f e r e n t areas were dissected out and homogenized in 50 mM T r i s - C l , pH 7.4, c o n t a i n i n g 1 mM EGTA (1:20, w:v) in a g l a s s - t e f i l o n Pottec-Elvejhem homogenizer. 300 mM sucrose was included in the homogenization off the adenohypophysis, which otherwise y i e l d e d very low basal a c t i v i t i e s . The homogenates were c e n t r i f u g e d at 20000 x g tier tO min at 4°C, and the p e l l e t s were resuspended in 50 mM f r i s - C l , pH 7.4, to a p r o t e i n c o n c e n t r a t i o n of 1-2 mg/mt. These membrane f r a c t i o n s were immediately assayed flop adenylate cyclase activity. Adenyiate c~clase assay. Ihe assay mixture contained 50 mM f r i s - C l , pH 7.4; 0.15 mM [8-1BC]ATP (50 dpm/pmoI); 0.16 mM [8-H]cAMP (360 dpm/nmot); 10 mM creatine phosphate; 27 U/ml cmeatine phosphokinase; 2 mM MgCI2; 0.05 mg/ml bacitmacin; 3 ,uM GTP (except for adenohypophysis, where GTP was 0.3 ,uM), and CI at the i n & c a r e d concentrations, in a final volume of O.l ml. {he incubation was started by addition of 20 ,ul of membrane suspension and was carried out at 30°6 For 8 min i n p o l y s t y r e ne [ub.es ~n ord~£ 5o avoid absorption of Ci to glass. I s o l a t i o n and measurement of [8-#~, 8-I~CJcAMP was performed according to Salomon et a l . (16). I n c l u s i o n of [ 8 - ~cAHP Jn the assay mixture allowed c o r r e c t i o n f o r the p o s s i b l e e f f e c t off phosphodiesterases (17), whz~h in any case was almost n e g l i g i b l e , except in caudate nucleus, to which 10 M i s o b u t y l - m e t h y l - x a n t h i n e was added.

Vol. 39, No. 23, 1986

Calcitonin and CNS Adenylate Cyclase

Proteins were assayed by the method of Bradford sham, U.K.

2255

(18), using a kit from Amec-

ResulEs The membranes min, and inhibited

time-course study showed that the adenylate cyclase activity of from rat hypothalamus increased linearly with time for at least415 over the entire time interval the concentration of sCT used (10- M) the enzyme (Fig. i).

o

300__

O.

E 0

EO. M sCT

=E t~

oF 0

5 1() T i m e , rain

1'5

FIG. 1 Time-course of basal (O-O) and sCT-inhibited (Q-Q) adenylate eyclase in rat hypothalamus. Data are means + S.D. for triplicate determinations. sCf-induced inhibition of adeny]ate cyclase was not limited to the hypothalamus, but was also present in other areas of the brain. ]he inhibitory effect was marked and dose-dependent in the hypothalamus, midbcain, pons, medulla oblungata and caudate nucleus. It was low in hippocampus and negligible (statistically not significant, p > O . 0 5 ) in the adenohypophysis (Fig. 2 and fabte I). As demonstrated for inhibition of adeny]ate cyclase in other tissues by a number of agents (19), maxima] inhibition was 40-50%, never 100%,

2256

Calcitonin and CNS Adenylate Cyclase

Vol. 39, No. 23, 1986

the apparent affinity of sCT (IC50 , concentration eliciting 50% of the maximal inhibitory effect attainable) was not .~arked]y different in the various areas, in fact, it was approximate]y 2xlO- M in mi~brain and medulla oblongata, while in shypothalamus it was higher than 4 x 10- M and in the ports higher than 3 x lO- M. In these latter areas the 1C50 could not be eva]uated precise]y because a clear p]ateau had not been reached at the highest concentration tested. Lower concentrations of sCf (10- -lO-6M) did not have any significant effects. fhe inhibitory effect or sCI was compared with those of caicitonins From o t h e r s p e c i e s , l a b l e I I i n d i c a t e s t h a t t h e p e p t i d e from e e l ( e C f ) was a b l e to inhibit a d e n y ] a t e c y c ] a s e i n r a t h y p o t h a l a m u s membranes a l t h o u g h i t was l e s s p o t e n t t h a n s C f . On t h e c o n t r a r y , human Cf ( h C f ) d i d n o t s i g n i f i c a n t l y affect t h e enzyme a c t i v i t y .

We also tested the activity of sCT which had been partially inactivated by b o i l i n g i t f o r 5 min, and w h i c h p r o v e d t o be a l m o s t u n e f f e c k i v e in the

A

MIDBRAIN

IO0 m m

90_

~'0

80_

#

70_

I-.>"

60_

~¢.)

50_

'2

uJ

I

o~

I

I

I

I

PONS

HYPOTHALAMUS 100_

o )o

90_

L~J

~

80_

.,,,J >.

z

70_

Q <{

60_

UJ

50I

10 -6

i

1 0 ~5

F

I

10 - 4

10 -6

I

10 -5

i

10 4

sCT, M

FIG. 2 Dose-dependent inhibition of adenylate cyclase by aCT in various rat brain areas. Data are means + S.D. of triplicate determinations.

Vo].

39, No.

23,

1986

Calcitonin

and CNS A d e n y l a t e

Cyclase

2257

TABLE I In vitro effects

of sCI on adenylate various

sCI

Caudate

brain

nucleus

cyc]ase

activity

in

areas

hippocampus

adeno-hypophysis

(M) 10 -5

96+2

81

3xlO -5

82+2

79 + 10

89 + 20

10 -4

39+3

72 +

81 + 19

Data

are

means

triplicate,

and

+

S.D.

are

which was 254 + 60; for caudate nucleus,

for

Comparison

1

99 +

i

2 experiments,

expressed

as percent

each

8

performed

of basal

of effects cyclase

in

activity,

280 + 8; 22.3 + 5.2 pmol/mg prot. hyppocampus and adenohypophysis.

IABLE

on adeny]ate

+

x min

II

of various activity

calcitonins

in hypothalamic

membranes Calcitonin

cAMP pmol/mg

% inhibition

prot.

p

x min

352.3 + 15.1 sCT

208.4 +

7.i

41%

< 0.001

eCT

268.1 + 10./4

24%

< O. 005

hCT

334.8 + 24.1

314.4 + sdl inact.

5%

n.s.

2.78

2?5.3 + 16.0

12.4%

0.05

-4 Each caleitonin was 10 M. n.s. = not statistically significant, sCT inact~ = partially inactivated sCT: see text for details. Data are means + S.D. for triplicate determinations.

2258

Calcitonin and CNS Adenylate Cyclase

Vol. 39, No. 23, 1986

hot-plate analgesia test. As shown in Table IT, such inactivated sCI displayed a much lower activity than that of native sC[ in inhibiting adenylate cyclase activity of hypothalamic membranes. This demonstrated that the inhibitory activity was specifically elicited by the native peptide.

Since the receptor-mediated modulation of adenylate cyclase requices a GTP-binding protein and GIP itself (19,20), we investigated the GIP requirements for the inhibitory effects of sCT. As shown in Fig. 3, GTP only slightly decreased basal activity, but it markedly potentiated sCT-induced inhibition in a dose-dependent fashion. The effects of chronic I.C.V. treatment with sCT on sCT-sensitive adeny]ate cyclase were investigated. Tab]e Ill shows that pretreatment in vivo for i0 days with the peptide (250 ng/rat) did not affect the subsequent in vitro inhibition of the enzyme. The same was true when the chronic treatment was prolonged for 14 days and after a single I.C.V. administration (data not shown).

600 7

I T t-

•

50o ×

2 E i

0

E

o.

400

o

300

i

I

r t iil[

i

r

F

I i I fl I

10-7 GTP, M

10-8

I

I

10 -6

FIG. 3 Effects

of

GTP

on

hypothalamus.

basal Data

~0-0)

and

are

means

sCT-inhibited + S.D.

for

(0-0) triplicate

adenylate

cyelase

determinations.

in

rat

Vol.

39, No.

23,

1986

Calcitonin

and CNS Adenylate

Cyclase

2259

Discussion We

report

areas of r a t

here

that

sCI

inhibits

basal

adenylate

cyclase

in

selected

brain.

C I - s e n s i t i v e adenylate cyclase has been demonstrated in v a r i o u s t i s s u e s or c e l l s , such as kidney ( 2 1 ) , bone c e l l s (22), c a r t i l a g e (23), v a r i o u s cancer c e l l l i n e s ( 8 , 2 4 ) , g l i a (25). In a l l these systems, however, the p e p t i d e s t i m u l a t e s r a t h e r than i n h i b i t s the enzyme. Yet our r e s u l t s do agree w i t h those in the i n i t i a l r e p o r t by Rizzo and goltzman (26), who d e s c r i b e d the i n h i b i t i o n off adenylate eyclase by sCf in r a t whole b r a i n and hypothatamus. Furthermore, the i n h i b i t o r y e f f e c t of the p e p t i d e on t h i s enzyme i s not l i m i t e d to the c e n t r a l nervous system, because i t has a l s o been d e s c r i b e d in human mononuclear c e l l s (27). TABLE I I I Effects

of chronic

I.C.V.

pretreatment

with

sCl

on s C l - s e n s i t i v e adeny]at e cyclase

Hypothalamus

Pons

Medulla

sCI

(M)

controls

pre-

controls

treated

controls

pre-

pre-

treated

treated

10 - 5

90 + 3

82 + 4

92 + i

79 + 7

75+8

83+6

3xlO -5

79 + 8

68 + 3

74 + 4

74 + 2

72+3

64+9

10 -4

55 + 5

53 + i

57 + 2

50 + 4

55+4

52+5

Data are means ~ S.D.

for 3 experiments

performed

expressed

of basal

as percent

activity,

in triplicate,

which was:

718 + l l l

and are

(controls)

and 606 + 132 (pretreated); 395 + 124 (controls) and 323 + 125 (pretreated); 332 ~ 88 (controls) and 380 ~ i14 (pretreated) pmol/mg prot. x min in hypothalamus, pons and medulla, sCT (250 ng/rat) was administered I.C.V. for ten days and after the last administration. Control

the animals were killed 24 hrs animals were given saline I.C.V.

Adenylate cyclase was assayed in vitro, as described in Materials Methods, with or without the indicated sCT concentrations.

and

One must t h e r e f o r e conclude t h a t there are two d i f f e r e n t classes of CI r e c e p t o r s t h a t can be coupled to adenylate cyclase e i t h e r f o r s t i m u l a t i o n or i n h i b i t i o n . The dual e f f e c t on adenylate cyclase i s not a p e c u l i a r i t y of CI; i n f a c t , s i m i l a r s i t u a t i o n s , in which the same agent can modulate the enzyme a c t i v i t y in d i f f e r e n t ways, depending on the type of r e c e p t o r i n v o l v e d , have a l r e a d y been d e s c r i b e d f o r dopamine ( 2 8 ) , adenosine ( 2 9 ) , n o r a d r e n a l i n e (30)

2260

Calcitonin and CNS Adenylate Cyclase

Vol. 39, No. 23, 1986

and p o s s i b l y p r o s t a g l a n d i n s (31). Ihe p o s s i b l e e x i s t e n c e of m u l t i p l e r e c e p t o r s f o r CT had already been suggested by Koida et a l . (32) on the basis of b i n d i n g s t u d i e s w i t h b r a i n homogenates. The c o n c e n t r a t i o n s of sCI needed to e l i c i t adenylate cyclase i n h i b i t i o n in our hands are somewhat high (10 M). I t i s a genera] f i n d i n g t h a t to have e f f e c t s on adenylate cyclase in b r a i n f a i r l y high c o n c e n t r a t i o n s of agents are u s u a l l y needed and t h a t when the enzyme a c t i v i t y i s assayed in membranes, even h i g h e r e f f e c t o r c o n c e n t r a t i o n s are necessary (see f o r instance Ref. 33). However, in our case, i t i s also p o s s i b l e t h a t the s t i m u l a t o r y e f f e c t of Cf on the g l i a ] c e l t t h a t has been r e p o r t e d (23) might p a r t l y mask the i n h i b i t o r y e f f e c t s on other c e l l s in a mixed c e l l p o p u l a t i o n such as b r a i n homogenates. A l f i e r n a t i v e l y , the p o s s i b i l i t y t h a t CI a c t i v i t y on adeny]ate cyclase might be mediated by r e c e p t o r s s p e c i f i c f o r the novel p e p t i d e encoded by CI gene, c a l c i t o n i n gene r e l a t e d p e p t i d e (CGRP), might be considered. In f a c t , b i n d i n g s i t e s s p e c i f i c f o r CGRP, w i t h which CI i n t e r a c t s , have been i d e n t i f i e d in the CNS (34). However, d i s t i n c t b i n d i n g s i t e s f o r the two p e p t i d e s e x i s t and f u r t h e r m o r e Goltzman and M i t c h e l l demonstrated t h a t CGRP r e c e p t o r s are not coupled to adeny]ate cyclase (34). I h e r e f o r e i t i s u n l i k e l y t h a t CI acts on adenylate cyc]ase through CGRP r e c e p t o r s .

Despite the high sCI concentrations required for the inhibition of adenylate cyclase, compared to those used in binding studies (11,26), there is some evidence suggesting that our findings could be of biological importance. First, it is very likely to be a receptor-mediated process, since it both requires and is amplified by GIP (20). Second, there is marked inhibition of adenylate cyclase in the h y p o ~ l a m u s , the midbrain, the pons and the medulla, in which specific binding of - I-sCI is maximal (i1,26,35). In the pituitary, the enzyme activity was not altered, which is consistent with the lack of specific binding sites reported by Rizzo and Go]tzman (26). In a d d i t i o n , the potency of Cts of d i f f e r e n t o r i g i n , salmon, eel and human, in i n h i b i t i n g adenylate cyc]ase r e f l e c t s the order of potency f o r the b i o l o g i c a l a c t i v i t i e s of the p e p t i d e s in the CNS, such as a n a l g e s i a and i n h i b i t i o n of food i n t a k e , w i t h f i s h Cls more a c t i v e than mammalian Cls ( 3 6 , 3 7 ) . In b i n d i n g s t u d i e s in the r a t CNS, sCl had higher a f f i n i t y than hCl ( 2 6 , 3 8 ) . We were i n t e r e s t e d to see t h a t in b r a i n areas from c h r o n i c a l l y p r e t r e a t e d animals we obtained the same degree of adenylate cyclase i n h i b i t i o n as in areas from non t r e a t e d animals. I b i s f i n d i n g i s in agreement w i t h p r e v i o u s data t h a t showed t h a t a f t e r c h r o n i c t r e a t m e n t the a n a l g e s i c a c t i v i t y off sCl p e r s i s t s . On the c o n t r a r y , the a n o r e c t i c e f f e c t develops t o l e r a n c e and the d e n s i t y of b i n d i n g s i t e s d i s t r i b u t i o n i s reduced only in s p e c i f i c areas (39). l h i s evidence, taken t o g e t h e r w i t h the present r e s u l t s , suggests t h a t m u l t i p l e r e c e p t o r p o p u l a t i o n s f o r sCI e x i s t in the CNS. One might hypothesize t h a t only one of these p o p u l a t i o n s might be coupled to adenylate cyciase. In c o n c l u s i o n , our data suggest t h a t at l e a s t some of the a c t i o n s of sCl i n the CNS might i n v o l v e modulation of i n t r a c e l l u l a r cAMP l e v e l s .

Vol. 39, No. 23, 1986

Calcitonin and CNS Adenylate Cyclase

2261

Acknowledgemenls This work was supported by M.P.I. and by C.N.R. "SottoprogetLo C o n t r o ] ] o del D o l o r e " . We are g r a t e f u l to Sandoz (Basel), Ciba Geigy ( I t a l y ) and l s t i t u to Sclavo (Siena, I t a l y ) Far the g i r t of c a l c i t o n i n s .

References 1. P.L. MUNSON, Handbook oF Physiology, vo]. 7, p. 443, Am. P h y s i o l . Sac., Washington (1976). 2. O.L.H. BIJVOET, J. VAN DER SLUYSVEER, H.R. DE VRIE and A . ] . J . VAN KOPPEN, New Eng]. J. Med. 284 681-685 (1971). 3. H.G. HAAS, M.A. DAMBACHER, J. GUNCAGA and f . LUFFENBURGER, O. C l i n . I n vest. 50 2689-2702 (1971). 4. D. GOLTZMAN, Endocrinology 106 510-518 (1980). 5. S.J. MARX and G.D. AURBACH, Endocrinology 97 448-453 (1975). 6. D.M. FINDLAY, M. DE LUISE, V.P. HICHELANGELI, M. ELLISON and T.3. MARLIN, Cancer Res. 40 1311-1317 (1980). 7. S.J. LAMP, D.M. FINDLAY, J.M. MOSELY and f . J . MARTIN, O. B i o l . Chem. 254 12269-12274 (1981). 8. J.D. ZAJAC, S.A. LIVESEY and f . J . MARTIN, Biochem. Biophys. Res. Commun. 122 1040-1046 (1984). 9. F. GALAN GALAN, R.M. ROGERS, S.I. GIRGIS and I. NaclNTYRE, Brain Res. 212 59-66 (1981). 10. 5.1. GIRGIS, F. GALAN GALAN, T.R. ARNETT, R.M. ROGERS, O. BONE, M. RAVARROLA and I . MaclN]YRE, J. E n d o c r i n o l . 87 375-382 (1980). 11. V.R. OLGIAII, F. GUIDOBONO, C. NEITI and A. PECILE, Brain Res. 265 209215 (1983). 12. J.A. FISCHER, P.H. TOBLER, M. KAUFMANN, W. BORN, H. HENKE, P.E. COOPER, S.M. SAGAR and J.B. MARTIN, Proc. N a t l . Acad. Set. U.S.A. 78 7801-7805 (1981). 13. A. PECILE, S. FERRI, P.C. BRAGA and V.R. OLGIATI, Experientia 31 322-333 (1975). 14. W.J. FREED, M.J. PERLON and R.J. WYATT, Science 206 850-852 (1979). 15. F.B. ALfAFFER, F. DE BALBIAN VERSIER, S. HALL, C.J. LONG and P. D'ENCARNACAO, Physiol. Behav. 5 119-121 (1970). 16. Y. SALOMON, C. LONDOS and M. RODBELL, Anal. Biochem. 58 541-548 (1974). 17. M.S. KAIZ, T.M. KELLY, M.A. PINEYRO and R . I . GREGERMAN, J. C y c l i c Nucleot i d e Res. 5 389-407 (1978). 18. M. BRADFORD, Anal. Bioehem. 72 248-254 (1976). 19. D.M.F. COOPER, FEBS LetL. 138 157-163 (1982). 20. M. RODBELL, Nature 284 17-22 (1980). 21. W.H. CHAO and L.F. FORTE, Endocrinology 111 252-259 (1982). 22. M. SHLOSSMAN, H. BROWN, E. SHAPIRO and R. DRIAK, C a t c i f . Tissue I n t . 34 190-196 (1982). 23. W. KREUSSER, R. WEINKAUFF, O. MEHLS and E. RITZ, Eur. 3. C l i n . I n v es t . 12 337-343 (1982). 24. V.P. MICHELANGELI, D.M. FINDLAY, 3.M. MOSELEY and T.J. MARTIN, J. Cyclic Nucl. Prot. Phosp. Res. 9 129-142 (1983). 25. F. LOFFLER, D. VAN CALKER and B. HAMPRECHI, EMBO J. 1 297-302 (1982). 26. A.J. RIZZO and D. GOLTZMAN, Endocrinology 108 1672-1677 (1981).

2262

Calcitonin and CNS Adenylate Cyclase

27. J.L.

STOCK

and J.A.

CODERRE,

Biochem.

Biophys.

Vol. 39, No. 23, 1986

Res. Commun.

109 935-942

(1982). 28. I. CREESE, D.R. SIBLEY, M.W. HAMBLIN, S.E. LEFT, Ann. Rev. Neurosci. 6 43-71 (1983). 29. C. LONDOS, O. WOLFF and D.M.F. COOPER in "Purinergic Receptors" (G. Burnstock Ed.), Chapman and Nail, p 288, London (1981). 30. K.H. JAKOBS and G. SCHUIIZ, Trends in Pharmaco]. Sci. I 331-333 (1980). 31. S. NICOSIA and R. PAOLEITI, in "Cell Regulation by Intracellular Signals" (C. SWILLENS and J.E. DUMONI Eds.) Plenum Press, New York and London, pp. 109-116 (1982). 32. M. KOIDA, Y. YAMAMOTO, H. NASKAMUSA, J. MATSUO, H. OKAMOIO and I. MORIMOIO, Jar. J. Pharmacol. 32 981-986 (1982). 33. I.E. COTE, C.W. GREWE and J.W. KEBABIAN, Endocrinology ]08 420-426 (1981). 34. D. GOLIZMAN and J. MIICHELL, Science 227 1343-1345 (1985). 35. A. FABBRI, F. FRAIOLI, C.B. PERT and A. PERT, Brain Res. 343 205-215 (1985). 36. A. PECILE, W.R. OLGIAII and V. SIBILIA~ in "Pharmacological basis of anaesthesiology" (M. Iiengo and M.J. Cousins Eds.) pp. 157-165, Raven Press, New York (1983). 37. M.J. TWERY, J.F. OBIE and C.W. COOPER~ Peptides 3 749-755 (1982). 38. J.A. FISCHER, S.M. SAgAR and J.B. MARTIN, LiFe Sci. 29 663-671 (1981). 39. F. GUIDOBONO, C. NEIII~ V. SIBILIA, A. ZAMBONI and A. PECILE, in "Calcitonin '84" (A. Peci]e Ed.)~ pp. 253-260, Elsevier Sci. Publ. B.V., Biomed. Div. Amsterdam (1985).