Adrenocortical stimulation by a cholecystokinin preparation in the rat

Life Sciences, Vol. 25, pp. 1725-1730 Printed in the U.S.A.

Pergamon Press

ADRENOCORTICAL STIMULATION BY A CHOLECYSTOKININ PREPARATION IN THE RAT Shinji Itoh, Ryoji Hirota, Goro Katsuura and Kunihiro Odaguchi Shionogi Research Laboratory, Fukushima-ku, Osaka 553, Japan (Received in final form October 2, 1979)

Summary The e f f e c t of a cholecystokinin (CCK) preparation on the secretion of corticosterone when injected i n t r a p e r i t o n e a l l y and i n t r a v e n t r i c u l a r l y was studied in the r a t . Both routes of injections produced pronounced elevation of plasma corticosterone l e v e l s , but the minimum e f f e c t i v e dose by i n t r a v e n t r i c u l a r injection was I0 mU/rat and that by intraperitoneal injection 2 U/IO0 g, or approximately 5 U / r a t . Although the effect was observed in vagotomized rats, CCK did not a f f e c t the p i t u i t a r y gland i t s e l f . I t was inferred that CCK acts d i r e c t l y or i n d i r e c t l y on CRF neurones in the brain. Since CCK preparation used in the present experiments was contaminated with m o t i l i n , the e f f e c t of synthetic m o t i l i n on the adrenocortical secretion was also examined. However, no stimulatory e f f e c t was found following i n t r a v e n t r i c u l a r injection of this peptide. Large amounts of cholecystokinin (CCK) were shown to be present in the brain of man, dog and rat (1-3) and in the cerebrospinal f l u i d of man (4). Although the highest concentration was found in the cerebral cortex, the hypothalamus contained appreciable amounts of this peptide. Since CCK was also detected in the p i t u i t a r y stalk (2), this peptide may a f f e c t p i t u i t a r y hormone secretion. In this connection, recently Vijayan et al. (5) reported that i n t r a v e n t r i c u l a r injection of CCK produced s i g n i f i c a n t changes in plasma levels of LH, PRL, GH and TSH, and suggested that the peptide may act as a transmitter or modulator of neuronal a c t i v i t y controlling the release of hypothalamic releasing and/or i n h i b i t i n g hormones. The present study was attempted to observe the e f f e c t of CCK on pituitary-adrenocortical a c t i v i t y in the r a t . Materials and Methods Male Wistar rats, weighing approximately 250 g, were used throughout the experiments. They were housed at a constant temperature of 25 ± 2°C with a 12 h l i g h t and 12 h dark cycle, the l i g h t period starting at 07:00, and r a t biscuits (Oriental Yeast Co.) with water ad l i b i t u m . Cholecystokinin (CCK) used in the present study was a commercial preparation manufactured by Boots Co. In most experiments CCK solution was injected i n t r a p e r i t o n e a l l y at I0:00 in the morning unless otherwise noted, then the rats were sacrificed by decapitation 30 min l a t e r . The trunk blood was collected and plasma was obtained by centrifugation. Corticosterone content was measured by the method of Zenker and Bernstein (6) with minor modifications. The dose of CCK preparation was expressed in terms of Harper unit. 0024-3205/79/201725-06502.00/0 Copyright (c) 1979 Pergamon Press Ltd

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For i n t r a v e n t r i c u l a r i n j e c t i o n , a guide cannula of s t a i n l e s s steel was f i r s t f i x e d w i t h dental cement to the s k u l l under sodium pentobarbital (Nembutal, Pitman-Moore, I n c . , 50 mg/kg i . p . ) anesthesia. The cannula was 0.7 mm in outer diameter and 14 mm in length w i t h a guard on the outside at 2 mm from the t i p bottom. Employing a s t e r e o t a x i c instrument the t i p of the cannula was placed 6.5 mm a n t e r i o r to the lambda and 1.3 mm l e f t of the s a g i t a l suture. A f t e r a 5 days recovery period, another 5 days were allowed to accustom the animals to i n s e r t i o n of the i n j e c t i o n cannula i n t o the l a t e r a l v e n t r i c l e . The i n j e c t i o n cannula, s t a i n l e s s steel w i t h an outer diameter of 0.3 mm, was i n serted i n t o the brain through the guide cannula to a distance of 4.5 mm under the guard; t h a t i s , 16.5 n~n from the top of guide cannula. For the experiment peptide or s a l i n e s o l u t i o n in an amount of 3 ~I was i n j e c t e d by means of a microsyringe. A f t e r t e r m i n a t i o n of the experiment, adequacy of placement of the cannula was checked by i n j e c t i n g 5 ~I of 1% Evans blue s o l u t i o n . The dye was d i s t r i b u t e d not only in the l a t e r a l v e n t r i c l e but also in the t h i r d v e n t r i cle in every case. A group of rats were subdiaphragmatically vagotomized under Nembutal anesthesia. Through an upper abdominal i n c i s i o n the esophagus was i s o l a t e d from i t s surrounding t i s s u e near the esophago-gastric j u n c t i o n and the vagal nerve trunks were removed. The operated rats were allowed 7 to I0 days f o r recovery from the surgery. The stomachs of these rats showed atonic d i s t e n sion. In order to pharmacologically block the a c t i v i t y of c o r t i c o t r o p i n - r e leasing f a c t o r (CRF), chlorpromazine (Shionogi & Co.), morphine hydrochloride (Takeda Chemical I n d u s t r i e s ) and Nembutal were used. In t h i s experiment chlorpromazine (1.0 mg/kg, s . c . ) was i n j e c t e d at I 0 : 0 0 , morphine (2.0 mg/kg, s . c . ) at 13:00, Nembutal (25 mg/kg, i . p . ) at 13:15, and the f i r s t blood sample was c o l l e c t e d from the j u g u l a r vein at 13:55. CCK or s a l i n e s o l u t i o n ( 0 . I ml/lO0 g, i . p . ) was i n j e c t e d at 14:00 and the second blood sample was obtained at 14:30. D i r e c t a c t i o n of CCK on the p i t u i t a r y t i s s u e was examined by the in v i t r o method. Four pieces of p i t u i t a r y quarters were preincubated in 2 ml KrebsRinger bicarbonate s o l u t i o n w i t h 1% bovine serum albumin at 37°C f o r 30 min, using 95% 02 and 5% CO2 as the gas phase in a shaking incubator. The medium was then renewed, 0.2 ml of t e s t sample was added and incubated f o r 60 min. The medium was c e n t r i f u g e d f o r 10 min at 9000 rpm, and the supernatant was a c i d i f i e d w i t h 25 ~I of 2 N HCI to adjust the pH at 3 and boiled f o r 6 min. Hypophysectomized rats were i n j e c t e d i . v . w i t h 0.2 ml of the e x t r a c t and 15 min l a t e r blood was c o l l e c t e d f o r the measurement of c o r t i c o s t e r o n e . Hypophysectomy was performed through the external a u d i t o r y canal. Other drugs used in t h i s experiment were s y n t h e t i c m o t i l i n (Calbiochem Co.) and ACTH (Armour Pharmaceutical Co.), and f o r s t a t i s t i c a l analysis Student's t - t e s t was used. Results In i n t a c t male rats i . p . i n j e c t i o n of CCK in the morning at 10:00 caused a marked increase in the plasma c o r t i c o s t e r o n e l e v e l s 30 min a f t e r the i n j e c t i o n . As shown in Table 1, the e l e v a t i o n f o l l o w i n g a d m i n i s t r a t i o n of 1 U/IO0 g was nearly s i g n i f i c a n t and 2 to 8 U/IO0 g produced pronounced i n crements in the plasma l e v e l s . This e f f e c t was dose-related. Significant increase was also observed in the evening at 18:00 when r e s t i n g level of plasma c o r t i c o s t e r o n e was high due to c i r c a d i a n r h y t h m i c i t y .

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TABLE 1 Elevation of Plasma Corticosterone Levels Following I n t r a p e r i t o n e a l I n j e c t i o n of Cholecystokinin (CCK) in Normal, Hypophysectomized and Vagotomized Male Rats

Group Normal

Dose per 100 g

Time of injection

No. of rats

Plasma c o r t i c o s t e r o n e pg/dl (mean ± SEM)

I0:00

i0

14.1 ± 4.70

8 i0 I0 I0

26.5 36.2 40.9 46.8

Saline CCK I U 2 4 8 Saline

18:00

Vagotomized

3.70 3.75 3.09 1.39

9

31.2 ± 2.16

i0

46.2 ± 3.81

3

2.9 ± 0.43

ACTH 0 . I mU 1.0

3 4

4.9 ± 0.23 26.5 ± 2.57

CCK 4 U

5

non-detectable

10

18.4 ± 2.10

I0

36.4 ± 4.31

CCK 4 U Hypophysectomized

± ± ± ±

Saline

10:00

Saline

09:30

CCK 4 U

Rats were k i l l e d 30 min a f t e r the i n j e c t i o n .

t-Test

#0.05 <0.01 <0.001 <0.01 <0.01

NS <0.001

<0.001

NS: not s i g n i f i c a n t .

Time course of the increase in plasma c o r t i c o s t e r o n e is shown in Table 2. A peak was observed 30 min a f t e r the i n j e c t i o n and the l e v e l showed a tendency to decrease a f t e r 60 min. Two hours a f t e r the i n j e c t i o n the c o r t i c o s t e r o n e level was restored to i t s normal low value. TABLE 2 Time Course of Plasma Corticosterone Elevation A f t e r I n t r a p e r i t o n e a l I n j e c t i o n of Cholecystokinin (CCK) in Normal Male Rats

Time

No. of rats

Before i n j e c t i o n 09:00 20 10:00 10 After injection 10:30 I0 Ii:00 5 12:00 5 19:00 4

Saline Corticosterone ~g/dl (mean ± SEM)

CCK (4 U/tO0 g) No. of Corticosterone rats ~g/dl (mean ± SEM)

t-Test

4.7 -+ 0.39 4.7 ± 0.55 14.1 8.4 5.0 22.4

CCK was i n j e c t e d at 10:00

+ + ± +

4.70 0.74 1.39 1.93

10 5 5 4

40.9 29.6 4.0 28.2

± + -+ ±

3.09 7.18 0.44 2.60

<0.001 <0.05 NS NS

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Vol. 25, No. 20, 1979

Since CCK was administered i . p . in the above experiment, the peptide may d i r e c t l y a f f e c t the adrenal gland to s t i m u l a t e the secretion of c o r t i c o s t e r o n e , and so to examine t h i s p o s s i b i l i t y hypophysectomized rats were used. Although i . p . i n j e c t i o n of ACTH in a dose of 0. i mU/lO0 g was i n e f f e c t i v e , i n j e c t i o n of 1.0 mU produced the increase as expected. However, a large dose of CCK of 4 U/IO0 g could not cause any increase (Table I ) . This r e s u l t i n d i c a t e s that CCK does not a f f e c t the adrenal gland i t s e l f . S t i m u l a t i o n of abdominal organs may cause central a c t i v a t i o n of the CRFACTH system through the a f f e r e n t pathway of the vagus. Therefore, to c l a r i f y t h i s p o s s i b i l i t y , subdiaphragmatically vagotomized rats were i n j e c t e d w i t h CCK. A high e l e v a t i o n of plasma c o r t i c o s t e r o n e concentration was found again in these operated rats (Table I ) . I t is l i k e l y t h a t CCK stimulates the central mechanism of ACTH s e c r e t i o n . -x~

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FIGURE Elevation of plasma c o r t i c o s t e r o n e l e v e l s f o l l o w i n g i n t r a v e n t r i c u l a r i n j e c t i o n of c h o l e c y s t o k i n i n (CCK) in normal male r a t s . CCK was i n jected at 08:30 and rats were k i l l e d 30 min l a t e r . * * * P
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shown in Table 3, crude median eminence e x t r a c t from r a t hypothalamus showed a marked s t i m u l a t i o n of ACTH release, but a d d i t i o n of CCK did not produce such e f f e c t on the ACTH release from the p i t u i t a r y t i s s u e i n t o the medium. A large amount of CCK, 5 U in 2 ml of medium caused a s l i g h t increase in the release, but t h i s e f f e c t was regarded as a pharmacological one, since i . p . i n j e c t i o n of 2 U/IO0 g and i n t r a v e n t r i c u l a r i n j e c t i o n of I0 mU/rat were potent enough to elevate the plasma c o r t i c o s t e r o n e l e v e l s . TABLE 3 E f f e c t of Cholecystokinin (CCK) on the Release of ACTH from P i t u i t a r y Tissue in v i t r o

Dose

No. of rats

12.3 12.9 10.3 10.4 10.2

4 3 4 4 4

5.3 9.3 16.9 36.7 43.6

± ÷ + ± ±

0.47 1.87 1.47 1.71 2.91

NS 0.001 0.001 0.001

14.6 12.0 14.1 12.1

5 5 5 5

5.2 5.3 4.5 7.9

± ± ± ±

0.58 0.87 0.42 0.80

NS NS 0.05

Median eminence e x t r a c t 0 0.02 ME 0.I 0.5 1.0 CCK

ACTH assay Corticosterone ug/dl (mean ± SEM)

Pituitary weight (mg)

0 0.05 U 0.5 5.0

t-Test

NS: not s i g n i f i c a n t Bioassay was used f o r the measurement of ACTH, i n d i c a t i n g the e l e v a t i o n of plasma c o r t i c o s t e r o n e l e v e l s in hypophysectomized r a t s . TABLE 4 E f f e c t of I n t r a v e n t r i c u l a r I n j e c t i o n of Synthetic M o t i l i n on Plasma Corticosterone Level in Normal Male Rats Treatment

Dose

No. of rats

Saline

(3 u l )

20

12.6 ± 1.18

5 ng

7

11.4 ± 2.04

NS

7

15.0 ± 2.17

NS

Motilin

10

Corticosterone ug/dl (mean ± S E M )

t-Test

NS: not s i g n i f i c a n t I t might be argued t h a t CCK preparation used in the present study is contaminated w i t h other substance(s) which s t i m u l a t e ( s ) the secretion of c o r t i c o sterone. The most probable contaminant is m o t i l i n (7,8) and so the e f f e c t of s y n t h e t i c m o t i l i n on the p i t u i t a r y - a d r e n o c o r t i c a l axis was examined. As seen in Table 4, i n t r a v e n t r i c u l a r i n j e c t i o n of m o t i l i n at 08:00 in doses of 5 and I0 ng did not cause any s i g n i f i c a n t increase in the plasma c o r t i c o s t e r o n e l e v e l when determined 30 min a f t e r the i n j e c t i o n .

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Discussion CCK has been shown to be present in the brain in r e l a t i v e l y large amounts and to produce a suppressive influence on the appetite ( c f . 9). According to Straus and Yalow ( I 0 ) , the cerebral cortex of g e n e t i c a l l y obese (ob/ob) mice with hyperphagia contained very small amounts of CCK octapeptide in the brain cortex, suggesting a causal r e l a t i o n between the diminished CCK content and the unrestrained appetite of obese mice. However, central action of CCK other than the suppressive e f f e c t on appetite remained to be elucidated. Since CCK is localized not only to the hypothalamus but also to the p i t u i t a r y s t a l k (2), CCK may have a potency to a l t e r the release of p i t u i t a r y hormones. In t h i s regard, Vijayan et a l . (5) reported that CCK lowered plasma l e v e l s of TSH and LH and elevated GH and PRL l e v e l s a f t e r i t s i n t r a v e n t r i c u l a r i n j e c t i o n in the r a t . Since CCK was i n e f f e c t i v e to modify hormone release from incubated p i t u i t a r y tissue in v i t r o , i t was assumed that the peptide has no d i r e c t action on the p i t u i t a r y . Our study demonstrated that i n t r a v e n t r i c u l a r i n j e c t i o n of caerulein had no e f f e c t on the adrenocortical secretion in spite of a pronounced s t i m u l a t i o n of corticosterone secretion following i . p . i n j e c t i o n in the r a t . Moreover, pentagastrin could not cause the elevation of plasma corticosterone level (unpublished data). On the other hand, the present study indicated that not only i . p . i n j e c t i o n but also i n t r a v e n t r i c u l a r administration of the CCK preparation e l i c i t e d a marked dose-dependent increase in the plasma c o r t i c o sterone concentration. The minimum e f f e c t i v e dose of i . p . i n j e c t i o n was almost 500 times greater than that of i n t r a v e n t r i c u l a r i n j e c t i o n . The observation suggests that the action s i t e of CCK is l o c a l i z e d in the central nervous system. Since in vivo as well as in v i t r o CRF assay experiments showed that CCK could not produce ACTH release, the peptide probably d i r e c t l y or ind i r e c t l y a f f e c t s CRF neurones. The r e s u l t is in harmony with the above mentioned findings by Vijayan et a l . (5). A serious question in t h i s study is the p o s s i b i l i t y that the a c t i v i t y may not be due to CCK i t s e l f but produced by other substance(s) contaminating the CCK preparation manufactured by the Boots Co. I t has been shown that the CCK preparation contains some amounts of m o t i l i n (7,8). In order to check the s t i m u l a t o r y e f f e c t of m o t i l i n on the p i t u i t a r y - a d r e n o c o r t i c a l function, the synthetic peptide was injected i n t r a v e n t r i c u l a r l y but i t did not cause elevat i o n of the plasma corticosterone l e v e l . Although existence of more s p e c i f i c a c t i v e factors in the CCK preparation could not be excluded, CCK is the most probable candidate. Further i n v e s t i g a t i o n is necessary to afford evidence of t h i s presumption. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

G. J. J. J. E. N. J. J. K. E.

J. DOCKRAY, Nature 264 568-570 (1976) F. REHFELD, Nature 271 771-773 (1978) F. REHFELD, J. B i o l . Chem. 253 4022-4030 (1978) F. REHFELD and C. KRUSE-LARSEN, Brain Res. 155 19-26 (1978) VIJAYAN, W. K. SAMSON and S. M. McCANN, Brain Res. 172 295-302 (1979) ZENKER and D. E. BERNSTEIN, J. B i o l . Chem. 231 695-70-~T (1958) C. BROWN, Gastroenterology 52 225-229 (1967) C. BROWN and C. O. PARKES, Ga~stroenterology 53 731-736 (1967) MUELLER and R. S. HSIAO, Neurosci. Biobehav. R--ev. 2 79-87 (1978 STRAUS and R. S. YALOW, Science 203 68-69 (1979) -