Life Sciences, Vol. 39, pp. 1271-1280 Pergamon Journals Printed in the U.S.A.

A D R E N O C O R T I C O T R O P I N R E V E R S A L OF E X P E R I M E N T A L HEMORRHAGIC

SHOCK I S A N T A G O N I Z E D BY MORPHINE

Alfio Bertolini, Salvatore Guarini,

William Ferrari and Elena Rompianesi

Institute of Pharmacology, University of Modena,

Via G. Campi 287, 41100 Modena, Italy

(Received in final form June 30, 1986)

Summary

ACTH-(I-24) dose-dependently improved cardiovascular

function in rats and dogs subjected to experimental hem-

orrhagic shock, an intravenous dose of 160 and I00

/ug/kg, respectively, completely restoring arterial blood

pressure and pulse amplitude. All saline-treated animals

died within 30 min of bleeding, while all ACTH-treated

animals were still alive at the end of the observation

period (2 hr). The injection of ACTH-(I-24) also dramat-

ically improved the respiratory function. Morphine, i.v.

injected into rats at the dose of 2.5 mg/kg,antagonised

the effect of ACTH-(I-24) to a greater or lesser degree,

depending on the dose of peptide employed: at 160/ug/kg, antagonism was complete, at 320/ug/kg antagonism was

only partial, while at 480/ug/kg antagonism was almost

completely overcome. These-data further support the idea

that melanocortins are physiological antagonists of opi-

oids, and suggest that melanocortin peptides may prove

to be rational and effective drugs in the treatment of

hypovolemie shock.

In a recent preliminary report (I) we showed that ACTH-(I-24),

intravenously injected into intact or adrenalectomized rats that

had been bled until otherwise irreversibly fatal hemorrhagic shock

set in, dose-dependently improves cardiovascular function and sur-

vival.

We suggest that such ACTH-induced reversal of hemorrhagic shock

is due to antagonism against endogenous opioids, which rise dra-

matically during circulatory shock (2). That is, in our opinion

ACTH would behave in shock states similarly to naloxone,the thera-

peutic efficacy of which has been clearly and repeatedly demon- strated(3-8).

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

1272 ACTH and Morphine in Shock Vol. 39, No. 14, 1986

In the present study we have investigated this phenomenon more

thoroughly ,and show that morphine counteracts the anti-shock effect

of ACTH-(I-24).

Methods

Female Wistar rats (Nossan, Correzzano, Milano, Italy) weighing

250 to 280 g, and male Beagle dogs (S. Morini, S. Polo d'Enza,

Reggio nell'Emilia, Italy) weighing 10 to 12 kg were used. Fol-

lowing anesthetization (ethylurethane, 1.25 g/kg i.p. in rats;

pentobarbital, 30 mg/kg i.v. in dogs) and heparinization (heparin

sodium, 5 mg/kg i.v.) a common carotid artery and an iliac vein,

in the rats, and a femoral artery and vein, in the dogs, were can-

nulated. Arterial blood pressure was recorded by means of pressu-

re transducer (Statham P23 Db) connected to a polygraph (Batta-

glia-Rangoni, Bologna, Italy). In some rats, the trachea was can-

nulated and respiration was recorded by means of a transducer

(Statham 10272) connected to the same polygraph. Hypovolemic shock

was produced by intermittently withdrawing blood from the venous

catheter, over a period of 25-30 minutes, until mean arterial

pressure fell to 16-30 mm Hg. In rats, the volume of blood removed

was 2-2.5 ml per IOO g of body weight and approximated to 50

percent of the estimated total blood volume (9); in dogs, the

volume of blood removed was 50-60 ml per kg of body weight. Fol-

lowing bleeding and mean blood pressure stabilization in the range

of 16-30 mm Bg (5-10 minutes), animals were given intravenous

bolus injections of either ACTH-(I-24) (Ciba-Geigy, Basle, Switz-

erland) or naloxone. HCl (Endo, Brussels, Belgium). Control an-

imals were intravenously injected with the same volume of saline

(0.i ml/lO0 g and 0.2 ml/kg of body weight in rats and dogs re-

spectively). In one set of experiments, intact rats were i.v.

injected with morphine-HCl (S.A.L.A.R.S., Como, Italy) 2 mins

before bleeding was commenced.

The blood pressure was recorded for 2 hours after treatment.

Statistical significance was assessed by Student's t-test for

paired data.

Results

Figure I shows some representative recordings. Tables I and II

report the overall data obtained in this study. In rats, the in-

travenous injection of ACTH-(I-24) dose-dependently restored blood

pressure and pulse amplitude: the effect started after a few min-

utes, gradually increased, reached a maximum in 15-30 minutes and

remained almost stable during the 2 hours of observation. All

rats intravenously injected with the same volume of saline died

after 20.83 ~ 2.71 minutes. The dose of 160/ug/kg J. v. of

ACTH-(1-24) completely restored blood pressure, while the lowest

Vol. 39, No. 14, 1986 ACTH and Morphine in Shock 1273

a

mmHg I rain t

~ 15rain 20rain 25rain S

b

oJ ~ ' ' , ~ - ~ - ~ - 0 mmHg lmin ~ 15rain 25mln

C 20 rain 30 rain

C

m ~ k ~ ~ m

F 150 I

~0

~5min 20min 30min 35mi.

d

10 = e c C 5 m i n 10 m i n 15 rain i i

FIG. 1

Representative recordings showing the effect of saline

(S) and ACTH-(I-2~) (C) on blood pressure (a-c) and on

respiration (d) after hypovolemic shock induced by

bleeding. (a) rat: intravenous injection of saline; (b)

rat: intravenous injection of ACTH-(I-24), 80/ug/kg; (c)

dog: intravenous injection of ACTH-(I-24), 100/ug/kg;

(d) rat: intravenous injection of ACTH-(1-24),~ug/kg.

dose used (~O/ug/kg i.v.) increased mean arterial pressure by

20 mm Hg within 30 minutes. In the same conditions, a dose of

I mg/kg i.v. of naloxone.HCl almost completely restored blood

pressure within 30 minutes. Similar results were obtained in dogs,

an intravenous dose of 100/ug/kg of ACTH-(I-24) causing an in-

crease of 53 mm Hg within 70 minutes. The intravenous injection

of ACTH-(I-24) in normal rats had no effect on blood pressure.

ACTH-(I-24) also dramatically improved the respiratory function,

which was severely depressed after bleeding (Fig. I and Table I). Pulse pressure was almost completely restored by any effective

dose of ACTH-(I-24), both in rats and dogs. For example, in rats

(mean value before bleeding: 21 mm Hg; mean value after bleeding:

8 mm Hg ), the i.v. injection of 80/ug/kg of ACTH-(I-24) restored

pulse pressure to 36 mm Hg within 15-30 minutes (Fig.l and Table I).

T he i n t r a v e n o u s i n j e c t i o n o f m o r p h i n e ( 2 . 5 m g / k g 2 m i n b e f o r e

TABLE I

Effect

of ACTH-(I-24),

Naloxone

and Saline

Treatment

on Mean

Arterial

Pressure,

Pulse

Pressure,

Respiratory

Rate

and

Survival,

Following

Severe

Hypotension

Induced

by Bleeding.

Animals*

Treatment

(/ug/kg

i.v.

after

bleeding)

Mean

arterial

pressure

(mmHg;

m+SoE.)

No.of

deaths

Pulse

pressure

--

120 min

after

before

after

15-30

min

treatment

bleeding

bleeding

after

treatment

76

+8

2

02

3

~ 2

1+

2

Ra

ts

(12

) S

ali

ne

,O.1

m

l/lO

0

g --

--

1

2

40~3

9~2 ~

10

~2

8029

22+2 ~

7528

Rats

(12

) ACTH-(1-24),

160

--

0 4324

10

!2

~ 4023

76+8

19

22

~

5626

Ra

ts

(12

) A

CT

H-(

1-2

4),

8

0

--

O

4123

8~

2

~ 36~4

~

80+7

18+3 ~

38+6

Rats

(12

) ACTH-(1-24),

40

--

--

--

0 38!5

923 ~

2824

6925

22+4 ~

60+6

Rats

(12)

Naloxone-HCl,lO00

--

--

O 39~4

10

!2

~ 3525

~

82

+6

8

52

6

Ra

ts

(6)

• S

ali

ne

,O.1

m

l/lO

0

g --

0

3524

3724

81

+8

8325

Ra

ts

(6)

• A

CT

H-(

1-2

4),

1

60

--

0

36

25

3

82

4

106+9

24+5 ~

27+4

Dogs

(4)

Saline,O.l

ml/IOO

g --

--

--

4 5

12

6

1123 ~

1223

104+5

2023 ~

73

29

Dogs

(4)

ACTH-(1-24),

100

--

0 55~5

10+2 ~

46+6

~

Re

spir

ato

ry

rate

(b

rea

ths/

min

; m

~S

.E.)

:

Ra

ts

(6)

AC

TH

-(1

-24

),

80

1

15

16

4

52

4

~ 1

08

26

A

0

>

~Z

f~

~D

D"

Do

O

<

©

* In

p

are

nth

ese

s th

e

nu

mb

er

of

an

ima

ls

use

d.

• R

ats

n

ot

sub

jec

ted

to

b

lee

din

g.

~ P

~ 0

.00

1

ve

rsu

s

va

lue

b

efo

re

ble

ed

ing

a

nd

A

p

<

0.0

2,

at

lea

st,

ve

rsu

s v

alu

e

aft

er

ble

ed

ing

(S

tud

en

t's

t-te

st

for

pa

ire

d

da

ta).

O0

TABLE

II

Influence

of Morphine

on the Anti-Shock

Effect

of ACTH-(1-24).

Six Rats per Group.

Pretreatment

(i.v.,

2 min

before bleeding)

Treatment

(/ug/kg i.v.

after

bleeding)

Mean arterial

pressure

(mmHg;

m+S.E.)

Pulse pressure

before

after

15-30 min

bleeding

bleeding

after treatment

No.of deaths

120

min after

treatment

OO

o~

Sal

ine,

0.1

ml/100 g

Morphine - HCI,

2.5 mg/kg

Morphine - HCI,

2.5 mg/kg

Morphine • HCI,

2.5 mg/kg

Morphine - HCI,

2.5

mg/

kg

80+7

21+3 z~

73+5

ACTH-(I-24),

160

0 40+5

8+2 zx

36+3

74

+5

1

9+

2 A

2

1+

3

ACTH-( 1-24),

160

6 36

--+5

7_

+1 "

11

--+3

81

+9

2

0+

2 A

4

6+

2

A

AC

TH

- (1

-a4

),

3aO

O

3

5+

5

8+1

z~

26

+4

A

i

__

_

_

76_+

5 18

--+1

zx

70-+

6 A

CT

H-(

1-2

4),

4

80

0

~2-

+3

9+

1 ~

37

-+2

~

73-+

4 1

9+

3 A

20

-+2

Saline,O.1 ml/lOO

g 6

38

+6

7

+2

~

9+2

> C2

m ~D

zr

o

P<O.O01

versus value before

bleeding

and Ap<o.02,

at least,

versus value after bleeding

(Student's

t-test

for paired

data).

bO

Ln

1276 ACTH and Morphine in Shock Vol. 39, No. 14, 1986

starting bleeding) completely antagonized the anti-shock effect

of a dose of 160/ug/kg of ACTH-(I-24), all rats dying after 31.80~

7.43 minutes. The effect of morphine was dose-dependently overcome

by increasing the amount of ACTH-(1-24) to 320 and 480/ug/kg

(Table II).

Discussion

The results of this study confirm and extend our previous pre-

liminary finding (I), and show that ACTH-(I-24) dose-dependently

improves circulatory and respiratory function and reverses other-

wise fatal hypovolemic shock resulting from massive bleeding, in

rats and dogs. Moreover, they show that this effect of ACTH is

antagonized by morphine.

A growing body of evidence indicates that endogenous opioJd

systems are involved in central cardiovascular regulation and in

the pathophysiology of shock (i). Intracerebroventricular injec-

tion of morphine and ~-endorphin or direct injection of E-end-

orphin into the nucleus tractus solitarii results in naloxone-

reversible hypotension and bradycardia (10,11). Opiates have been

reported to act on the brain-stem and hypothalamic nuclei to in-

crease parasympathetic and decrease sympathetic tone, causing

bradycardia and hypotension (12). Furthermore, the opiate antag-

onist naloxone has been reported to reverse the hypotensive effect

of clonidine and ~-methyldopa in spontaneously hypertensive rats

(13), while clonidine and ~-methylnoradrenaline induce the release

of a ~-endorphin - like peptide from brain-stem slices of these

rats (14), again indicating a depressor role of opioids. Moreover,

intravenous or intracerebroventricular injection of naloxone con-

sistently improves cardiovascular function and survival in septic,

hemorrhagic, anaphylactic, and neurogenic shock models Jn rats and

in other animal species, including primates, suggesting that

opioids may contribute to the hypotension and may be involved in

the pathophysiology of shock (2-6,15-~7).Naloxone has no intrinsic

pressor activity in normotensive animals; therefore, naloxone does

not act directly as a pressor agent in shock but instead acts in-

directly to antagonize the depressor effects of endogenous opioids

on the circulation (2).

On the other hand, a considerable number of independent inves-

tigations seem to indicate that a reciprocal functional antagonism

exists between opioid and melanocortin (ACTH-MSH) peptides. For

example, opioids inhibit adenylate cyclase activity (18,19); in-

hibit uptake of Ca ++ into synaptosomes (20); reduce both eate-

cholamine and acetylcholine transmission (21) and aeetylcholine

turnover rate (22) in several brain areas; inhibit neuronal firing

(23-25); induce analgesia (26); depress sexual behavior (2?); usu-

ally have an amnesic effect (28); increase tone and segmental con-

tractions of the intestine (29); inhibit the corticotropic activity

Vol. 39, No. 14, 1986 ACTH and Morphine in Shock 1277

of ACTH by a direct, stereospecific, dose-dependent mechanism (30, 31). Conversely, ACTH, MSH and related peptides stimulate adenylate

cyclase activity (32,33); increase intracellular concentrations

of Ca ++ in target cells (34,35); increase catecholamine and ace-

tylcholine transmission (21) as well as acetylcholine turnover

rate (36); increase neuronal firing (37); antagonize morphine in-

hibition of spinal reflex activity, both in vivo (38) and in vitro

(39); delay extinction of learned avoidance responses (40); induce

recurrent episodes of penile erection and ejaculation in males(41),

stimulate lordosis behavior in females (42), and facilitate sexual

motivation (43); bind to opiate-receptor sites (44,45) and compete

with ~-endorphin for them (46); counteract morphine-induced anal-

gesia (47) and morphine-induced excitation (48); attenuate E-end-

orphin-induced catatonic (49); antagonize morphine-induced colonic

contractions (50).

Moreover, melanocortins and endorphins are synthesized and co-

released by the same cells and derive from a common precursor

(pro-opiomelanocortin, POMC (51-53), and many, functionally very

important, nervous centers are targets for both melanocortins and

endorphins (35). Finally, POMC neurons can process and release

melanocortins and endorphins in widely varying concentrations and

ratios, as active or inactive compounds, depending on different

physiological and pathological conditions, both in the CNS and at

the peripheral level (35). On the whole, these abundant data

support the hypothesis that melanocortins are the most likely and

important physiological antagonists of opioids, and that melano-

coffin and opioid peptides constitute a co-ordinated and balanced

system modulating many body functions (35, 54-59). Their functional

antagonism very probably involves a different, often opposite,

effect on some elementary and general neuronal function ( e. g.

neuronal firings transmitter release) or on ubiquitous second

messengers (Ca , cAMP, or both) (60).

The results of the present study, showing as they do that ACTH

reverses hemorrhagic shock, and that this effect is antagonized

by morphine, give further and strong experimental support to such

a hypothesis. Our data seem to indicate that the pathophysiology

of shock includes a massive derangement of the melanocortin-opi-

opid homeostasis with prevalence of the opioid component. This

does not exclude the possibility of other nueropeptides outside

the POMC system (thyrotropin-releasing hormone; cholecystokinin)

acting as endogenous anti-opioids (61,62): indeed, thyrotropin-

releasing hormone, at very high doses, improves cardiovascular

function in experimental endotoxic and hemorrhagic shock (63).

The fact that ACTH is notoriously devoid of acute toxicity,

along with its rapid onset of action, and the possibility that

hormonally-inactive fragments share the same anti-shock effect

(as suggested by the results previously obtained in adrenalecto-

1278 ACTH and Morphine in Shock Vol. 39, No. 14, 1986

mized rats (I) makes these peptides of obvious, potential thera-

peutic interest. However, these findings must be confirmed in

other species, including primates, before use of ACTH-peptides in

clinics can be considered.

Acknowledgements

We thank Simonetta Tagliavini and Giogio Noera for their as-

sistance. This work was supported in part by grants from the Mi-

nistero della Pubblica Istruzione (40% and 60%), Roma.

References

1. A. BERTOLINI, S. GUARINI and W. FERRARI, Eur. J. Pharmacol.,

in press (1986).

2. E.W. BERNTON, J.B. LONG and J.W. HOLADAY, Fed. Proc. 44 g90-

299 (1985).

3. J.W. HOLADAY and A.I. FADEN, Nature 275 450-451 (1978).

4. A.I. FADEN and J.W. HOLADAY, Science 205 317-318 (1979).

5. J.W. HOLADAY and A.I. FADEN, Brain Res. 189 295-300 (1980).

6. A.I. FADEN and J.W. HOLADAY, J. Pharmacol. Exp. Ther. 21g 441-

447 (1980). 7. A.I. FADEN and J.W. HOLADAY, J. Infect. Dis. 14g 229-238(1980).

8. J.W. HOLADAY, Annu. Rev. Pharmacol. Toxicol. 23 541-594 (1983).

9. J.A. COLLINS, A. BRAITBERG, H.W. MARGRAF and H.R. BUTCHER,

Arch. Surg. 99 484-488 (1969).

10. M.R. FELDBERG and E. WEI, J. Physiol. 280 18P (1978).

11. M.A. PETTY, W. DE JONG and D. DE WIED, Life Sci. 30 1835-1840

(1982). 12. M.R. FELDBERG and E. WEI, J. Physiol. 275 57P (1978).

13. C. FARSANG, M.D. RAMIREZ-GONZALEZ, L. MUCCI and G. KUNOS, J.

Pharmacol. Exp. Ther. 214 203-208 (1980).

14. G. KUNOS, C. FARSANG and M.D. RAMIREZ-GONZALEZ, Science 211

82-84 (1981).

15. D.G. REYNOLDS, N.J. GURLL, T. VARGISH, R. LECHNER, A.I. FADEN

and J.W. HOLADAY, Circ. Shock 7 39-48 (1980).

16. A.I. FADEN, T.P. JACOBS and J.W. HOLADAY, Science 211 493-494

(1981). 17. N.J. GURLL, Adv. Shock Res. i0, 63-71 (1983).

18. H.O.I. COLLIER and A.C. ROY, Nature 248 24-27(1974).

19. S.K. SHARMA, M. NIREMBERfi and W.A. KLEE, Proc. Natl. Acad. Sci.

U.S.A. 72 590-594 (1975). 20. F. GUERRERO-MUNOZ, M.L. GUERRERO, E.L. WAY and C.H. LI,Science

206 89-91 (1979).

21. D.H.G. VERSTEEG, Pharmacol. Ther. 11 535-557 (1980).

22. F. MORONI, D.L. CHENEY and E. COSTA, Neuropharmacology 17 191-

196 (1978). 23. B.P. BRADLEY, I. BRIGGS, R.J. GRAYTON and L.A. LAMBERT, Nature

261 425-426 (1976).

24. R.C.A. FREDERICKSON and F.H. NORRIS, Science 194 440-442 (1976)

Vol. 39, No. 14, 1986 ACTH and Morphine in Shock 1279

25. R.A. NICOLL, G.R. SIGGINS, N.LING, F.E. BLOOM and R. GUILLEMIN,

Proc. Natl. Acad. Sci. U.S.A. 74 2584-2588 ( 1 9 7 7 ) . 26 . A . F . BRADBURY, W.F. FELDBERG, D.G. SMYTH a n d C . R . SNELL, i n

O p i a t e s a n d E n d o g e n o u s O p i o i d P e p t i d e s (H.W. K o s t e r l i t z , e d . ) p p . 9 - 1 7 , N o r t h - H o l l a n d , A m s t e r d a m ( 1 9 7 6 ) .

27 . B . J . MEYERSONand L. TERENIUS, E u r . J . P h a r m a e o l . 42 1 9 1 - 1 9 2 ( 1 9 7 7 ) .

28 . I . IZQUIERDO, D.O. SONZA, M.A. CARRASCO, R.D. DIAS, M.L. PERRY, E. EISINGER, E. ELISABETSKY a n d D.A. VENDITE, Psychopharmaco logy 70 1 7 3 - 1 7 8 ( 1 9 8 0 ) .

29. M.G.C. GILLAN and D. POLLOCK, Br. J. Pharmacol. 57 444P-445P

(1976). 30. K.S. SZALAY and E. STARK, Life sci. 29 1355-1361 (1981). 31. J.P. HEYBACH and J. VERNIKOS, Eur. J. Pharmacol. 75 I-6 (1981). 32. L.D. fiARREN, G.N. GILL, H. MASUI and G.M. WALTON, Rec. Progr.

Borm. Res. 27 433-478 (1971) . 33. V.M. WIEGANT, A . J . DUNN, P . SCHOTMAN a n d W.H. GISPEN, B r a i n .

R e s . 168 5 6 5 - 5 8 4 ( 1 9 7 9 ) . 34 . R . R . NOVALES, B . J . NOVALES, S . H . ZINNER a n d J . H . STONER, Gen .

Comp. E n d o e r . 2 2 8 6 - 2 9 5 ( 1 9 6 2 ) . 35 . T . L . O'DONOHUE a n d D.M. DORSA, P e p t i d e s ! 353-395 ( 1 9 8 2 ) . 36. P . L . WOOD, D. MALTHE-SORENSSEN, D .L . CHENEY a n d E. COSTA, L i f e

S c i . 22 6 7 3 - 6 7 8 ( 1 9 7 8 ) . 37 . W.A. KRIVOY a n d E. ZIMMERMANN, E u r . J . P h a r m a c o l . 46 3 1 5 - 3 2 2

( 1 9 7 7 ) . 38 . W.A. KRIVOY, D. KROEGER, A.N. TAYLOR a n d E. ZIMMERMANN, E u r .

J . P h a r m a c o l . 27 339-345 ( 1 9 7 4 ) . 39 . E. ZIMMERMANN a n d W.A. KRIVOY, P r o e . S o e . Exp . B i o l . Med. 146

5 7 5 - 5 7 9 ( 1 9 7 4 ) . 40 . D. DE WIED, i n F r o n t i e r s i n N e u r o e n d o c r i n o l o g y (W.F . G a n o n g

a n d L. M a r t i n i , e d s . ) pp . 9 7 - 1 4 0 , O x f o r d U n i v e r s i t y P r e s s , O x f o r d ( 1 9 6 9 ) .

4 1 . A. BERTOLINI, W. VERGONI, G . L . GESSA a n d W. FERRARI, N a t u r e 221 6 6 7 - 6 6 9 ( 1 9 6 9 ) .

4 2 . C.H. SAWYER, D.M. BALDWIN and D.K. HAUN, in Sexual Behavior:

Pharmacology and Biochemistry (M. Sandler and G.L. Gessa,eds.)

pp. 259-268, Raven Press, New York (1975).

43. B. BOHUS, Pharmacology 18 113-122 (1979). 44. L. TERENIUS, J. Pharm. Pharmacol. 27 450-452 (1975). 45. L. TERENIUS, W.H. GISPEN and D. DE WIED, Eur. J. Pharmaeol.

33 395-399 (1975). 46. H.AKIL, W.A. HEWLETT, J.D. BARCHAS and C.H. LI, Eur. J. Phar-

macol. 64 69-77 (1980). 47. W.H. GISPEN, J. BUITELAAR, V.M. WIEGANT, L. TERENIUS and D.

DE WIED, Eur. J. Pharmacol. 39 393-397 (1976). 48. R.T. KATZ, Eur. J. Pharmacol. 53 383-385 (1979). 49. J.M. VAN REE, B. BOHUS, K.M. CSONTOS, W.H. GISPEN, H.M.

GREVEN, F.P. NIJKAMP, F.A. OPMEER, G.A. DE ROTTE, T.B. VAN WIMERSMA GREIDANUS, A. WITTER and D. DE WIED, Life Sci. 28

2 8 7 5 - 2 8 8 8 ( 1 9 8 1 ) .

1280 ACTH and Morphine in Shock Vol. 39, No. 14, 1986

50. M. CASTELLI, G.L. GESSA and A. BERTOLINI, Eur. J. Pharmacol.

108 213-214 (1985).

51. R.E. MAINS, B.A. EIPPER and N.LING, Proc. Natl. Acad. Sci.

U.S.A. 7_.~ 3014-3018 (1977) . 52. S.J. WATSON, H. AKIL, C.W. RICHARDS and J.D. BARCHAS, Nature

275 226-228 (1978) . 53. M. RUBINSTEIN, S. STEIN and S. UDENFRIEND, Proc. Natl. Acad.

Sci. U.S.A. 75 671-699 (1978). 54. W.H. fiISPEN, J.M. VAN REE and D. DE WIED, Int. Rev. Neurobiol.

20 209-250 (1977) . 55. Y.F. JACQUET, Science 201 1032-1034 (1978).

56. A. BERTOLINI, R. POGGIOLI and W. FERRARI, Experientia 35

1216-1217 (1979). 57. A. BERTOLINI, R. POGGIOLI and W. FERRARI, Adv. Biochem.

Psychopharmacol. 22 109-I17 (1980).

58. A. BERTOLINI and G.L. GESSA, J. Endocrinol. Invest. 4 241-251

(1981). 59. D. DE WIED and J. JOLLES, Physiol. Rev. 62, 976-1059 (1982) . 60. A. BERTOLINI, R. POGGIOLI, A.V. VERGONI, M. CASTELLI and S.

GUARINI,in Central Effects of ACTH-Related Peptides (D. de

Wied and W. Perrari, eds.), Springer-Verlag, Berlin, Heidel-

berg, in press (1985).

61. J.W. HOLADAY, L.P. TSENG, H.H. LOH and C.H. LI, Life Sci. 22

1537-1544 (1978) . 62. P.L. PARIS, B.R. KOMISARUK, L.R. WATKINS and D.J. MAYER,

Science 219 310-312 (1983).

63. J.W. HOLADAY, R.J. D'AMATO and A.I. FADEN, Science 213 216-

218 (1981) .