Effect of caerulein on plasma corticosterone concentration in the rat

Life Sciences, Vol. 27, pp. 2205-2210 Printed in the U.S.A.

Pergamon Press

EFFECT OF CAERULEIN ON PLASMA CORTICOSTERONE CONCENTRATION IN THE RAT Shinji Itoh, Goro Katsuura,

Ryoji Hirota and Kunihiro Odaguchi

Shionogi Research Laboratories Sagisu, Fukushima-ku, Osaka 553, Japan (Received in final form October i, 1980) Summary Intraperitoneal injection of caerulein produced a pronounced increase in plasma corticosterone levels in intact rats. Since this effect was not observed in hypophysectomized rats, it is assumed that the peptide does not affect the adrenal gland directly. Intracerebroventricular injection of caerulein was also ineffective in stimulating corticosterone secretion, and in vitro experiments for ACTH release indicated that caerulein could not affect pituitary tissue itself. The fact that the effect of caerulein disappeared after subdiaphragmatical vagotomy suggests that the action site is at a peripheral level, but not in the central nervous system. The presence of cholecystokinin (CCK)- and gastrin-like peptides in the brain of several species of animals has been demonstrated by a number of investigators (1-5) which suggests that these peptides may act as neurotransmitters or neuromodulators in the central nervous system. The peptides were found not only in the hypothalamus, but also in the hypophysis (4). Therefore, it could be assumed that the peptides play a role in regulating the secretion of pituitary hormones. Since caerulein is a decapeptide that mimics many actions of gastrin and CCK, it was thought of interest to observe its effect on pituitary secretion. The present study was undertaken to examine the possible action of caerulein on the pituitary-adrenal axis. Materials and Methods Male Wistar rats, weighing approximately 250 g, were housed at a constant temperature of 25 ± 2°C under a light:dark ratio of 12:12 hours with lights turned on at 07:00 h. Rat biscuits (Oriental Yeast Co.) and water were available ad libitum. Caerulein diethylamine (Montedison Pharmaceut. Co.) was diluted with isotonic saline solution in graded concentrations from 6 ng to 20 ~g/ml and 0.I ml of the solution per 100 g body weight was injected i.p. into normal, hypophysectomized and subdiaphragmatically vagotomized rats at i0:00 h. Thirty min after the injection the animals were sacrificed by decapitation and trunk blood was collected. In another experiment, blood samples were also obtained 60 and 120 min after the injection.

0024-3205/80/492205-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd.

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The method of intracerebroventricular (i.c.v.) injection was described in our previous paper (6). Briefly speaking, under Nembutal anesthesia a guide cannula was first fixed to the skull with dental cement, in a position adequate for the insertion of an injection cannula into the left lateral ventricle using a stereotaxic instrument. After a 5 days recovery period, another 5 days were allowed to accustom the animals to insertion of the injection cannula into the ventricle. For the experiment, a peptide or saline solution in an amount of 3 ~i was injected by means of a microsyringe. After termination of the experiment, adequacy of placement of the cannula was checked by injecting 5 ~i of i% Evans blue solution. In every case the dye was distributed not only in the lateral ventricle but also throughout the third ventricle. Hypophysectomy was performed through the external auditory canal approximately 24 h before the injection of caerulein, saline or ACTH (Armour). Subdiaphragmatic vagotomy was done under Nembutal anesthesia. Through an upper abdominal midline incision the esophagus was isolated from its surrounding tissue near the esophago-gastric junction and the vagal nerve trunks were completely removed. The vagotomized rats were used for experiments 7 to i0 days after the surgery. After the experiment vagotomy was verified anatomically using a dissecting microscope for each rat. For in vitro measurement of ACTH releasing activity, four pieces of rat pituitary quarters were preincubated in 2 ml Krebs-Ringer bicarbonate solution with 1% bovine serum albumin at 37°C for 30 min in a shaking incubator, using 95% CO 2 and 5% 02 as the gas phase. The medium was then renewed, 0.2 ml of test sample was added and incubated for 60 min. The medium was centrifuged, and the supernatant was acidified to adjust the pH at 3 and boiled for 6 min. Hypophysectomized rats were injected i.v. with 0.2 ml of the extract and 15 min later blood was collected for the measurement of corticosterone. The response was expressed in terms of plasma corticosterone levels after injection of ACTH released into the medium from i0 mg pituitary tissue. As reference preparation of rat median eminence (ME) extract, aceton powder obtained from normal male rats under resting condition was dissolved in 0.i N HCI and the solution was diluted with saline in order to adjust I/I0 and I/2 equivalent amounts of the ME extract from one rat. Plasma corticosterone was determined by the method of Zenker and Bernstein (7) with minor modifications. Student's t-test was used for the statistical analysis of the data. Results Changes in plasma corticosterone concentration of rats sacrificed 30 min after the i.p. injection of caerulein are shown in Table i. Significant dosedependent increases were observed following the injection of 3 to 200 ng/100 g. When 200 ng/lO0 g was injected, the elevation was sustained for 60 min, but the c o n c e n t r a t i o n s d e c r e a s e d to nearly control levels 120 min after the injection (Fig. i). In hypophysectomized rats injection of ACTH caused a marked elevation of corticosterone level. The plasma level rose from 2.9 ± 0.43 ~g/dl (n=3) to 26.5 ± 2.57 (n=4) 30 min after ACTH injection in a dose of 1 mU/lO0 g, while injection of caerulein in a dose of 800 ng/100 g did not produce any significant increase in the plasma corticosterone level (Table i). Plasma level of vagotomized rats, when measured in the morning, was slightly higher than that in normal rats, but caerulein in a dose of 200 ng/100 g had no effect and even 400 ng/100 g induced only a small and non-significant increase.

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TABLE 1 Changes in Plasma Corticosterone Levels 30 Min after Intraperitoneal Injection of Caerulein in Normal, Hypophysectomized and Vagotomized Male Rats.

Group Normal

Dose per i00 g

Treatment

No. of rats

Saline Caerulein

(0.i ml) 0.6 ng 3 16 80 200 400 800 2000

i0 i0 i0 i0 i0 5 15 5 i0

Hypophysectomized

Saline Caerulein ACTH

(0.i ml) 800 ng 1.0 mU

Vagotomized

Saline Caerulein

(0.i ml) 200 ng 400

Corticosterone Dg/dl (mean ÷ SEM) 14.1 17.3 25.1 27.5 34.3 37.6 38.2 37.7 43.6

4.14 3.39 3.13 2.87 5.09 5.83 3.26 4.99 3.60

NS <0.05 <0.05 <0.01 <0.01 <0.001 <0.01 <0.001

3 5 4

2.9 ± 0.43 3.5 ± 0.16 26.5 ± 2.57

NS <0.001

16 i0 8

18.4 i 2.10 16.8 ± 1.91 27.3 ± 5.83

~

± ± + ± ± + ± ÷ +

8

40 r

o 30 In o 1.2 "C o 20 u

lein

£ ~n 1 0 O

2O

10

I

I

g

Saline

10, t

Hour of

t-Test

-'-o s

I

I

11

12

day

FIGURE 1 Time course of elevation of plasma corticosterone levels following intraperitoneal injection of caerulein in a dose of 200 ng/100 g. Arrow indicates the time of injection (I0:00 h)° Numbers in the figure represent the number of rats, and vertical lines are standard errors.

NS NS

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=k

n

G-u No. of rats Dose

I7 0

5 0.5

6 5

6 50

6 200

Saline Caerulein (ng/rot) FIGURE 2 Plasma corticosterone levels 30 min after intraventricular injection of caerulein in normal male rats. * P < 0.05 Caerulein injected i.c.v, in doses from 0.5 to 50 ng/rat failed to cause any increase in the plasma corticosterone concentration, but a larger dose of 200 ng/rat produced a slight decrease (Fig. 2). Moreover, addition of caerulein in amounts from 20 to 2000 ng in 2 ml medium had no effect in stimulating the release of ACTH in our pituitary incubation system, while crude median eminence extract caused a pronounced stimulation of ACTH release from incubated pituitary tissue (Table 2). TABLE 2 Effect of Caerulein on the Release of ACTH from Pituitary Tissue in vitro. Bioassay was used for the Measurement of ACTH Released into Incubation Medium. Plasma Corticosterone Levels in Hypophysectomized Rats 15 Min after Intravenous Injection of Extract were Indicated in This Table.

Addition

Dose

No. of rats

Saline Crude extract Caerulein

ACTH bioassay Corticosterone ~g/dl (mean ± SEM)

(0.2 ml)

4

4.3 ± 0.38

0.I ME 0.5

4 4

16.4 ± 1.43 36.7 ± 1.64

5 5 5

5.3 ± 0.36 5.6 ± 0.19 5.0 ± 0.49

20 ng 200 2000

t-Test

<0.001 <0.001 NS NS NS

0.i and 0.5 ME = I/I0 and 1/2 equivalent amounts of median eminence extract obtained from one rat. NS = not significant. Discussion Recently we have found that a commercial CCK preparation manufactured by Boots Co., which contains not only CCK but also motilin and some amounts of other substances, has a potent action in stimulating adrenocortical secretion (6). Since caerulein possesses a similar structure to the C-terminal peptide of CCK and its actions resemble to those of CCK, it is postulated that caerulein may have a regulatory effect on adrenocortical secretion.

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In the present study i.p. injection of eaerulein was found to produce a marked dose-dependent elevation of plasma corticosterone levels in intact rats. However, since the peptide was injected i.p., the effect might be caused by direct stimulation on the adrenal gland and therefore large amounts of caerulein were injected i.p. into hypophysectomized rats. As indicated in Table i, the injection did not cause any significant elevation of the plasma corticosterone concentration. This result indicates that the peptide does not affect the adrenal gland directly. Caerulein may stimulate corticosterone secretion either by affecting hypothalamic CRF neurons or pituitary tissue. In this regard, there is a question whether caerulein can cross the blood-brain barrier or not. At the present time no evidence is available. However, CCK, a similar but larger molecule than caerulein, has been shown by many investigators (cf. 8) to affect hypothalamic nuclei inhibiting food intake. Accordingly, CCK is likely to be able to cross the blood-brain barrier. In any case, in order to clarify the above mentioned possibility, caerulein was injected into the lateral ventricle. It was found that the injection was completely ineffective to cause any increase in the plasma corticosterone (Fig. 2). Although very large doses of the peptide suppressed the plasma level of corticosterone, this result would be regarded as a pharmacological toxic effect. It is, therefore, apparent that the action site of caerulein is not at the hypothalamic level. Moreover, in in vitro experiment caerulein was shown not to affect pituitary tissue directly (Table 2). Consequently, the site of action of caerulein is assumed to be at the peripheral level, but not located in the central nervous system. This assumption was supported by the finding that the stimulatory effect was markedly reduced after subdiaphragmatic vagotomy (Table I). The observation suggests that caerulein is likely to affect visceral organs and secondarily activates the hypothalamo-hypophyseal system through afferent pathways in which case the effect is, at least in part, mediated through the vagus nerve. The effect of CCK in suppressing food intake and inducing other satietyrelated responses has been repeatedly documented and caerulein was shown to have the same effect (9, I0). Anika et al. (11) reported that the effectiveness of CCK and caerulein does not depend on the functional integrity of the vagal nerve, since food intake was significantly depressed by exogenous caerulein after subdiaphragmatic vagotomy. However, the effect of these peptides on the adrenocortical secretion might be influenced by the active participation of the nerve supply to the gastrointestinal tract. The peptides are well known to stimulate the contraction of gallbladder and secretion of pancreatic enzymes. In addition, they have significant effects on gastrointestinal secretion and movement. Therefore the stimulatory action of caerulein might be mediated via the vagus nerve to the hypothalamo-hypophyseal axis. References i. 2. 3. 4. 5. 6.

J. J. VANDERHAEGEN, J. C. SIGNEAU and W. GIPTS, Nature (Lond.) 257, 604605 (1975) G. J. DOCKRAY, Nature (Lond.) 264, 568-570 (1976) G. J. DOCKRAY, R. A. GREGORY, J. B. HUTCHINSON, J. I. HARRIS and M. J. RUNSWICK, Nature (Lond.) 274, 711-713 (1978) J. F. REHFELD, Nature (Lond.) 271, 771-773 (1978) R. B. INNIS, F. M. A. CORREA, G. R. UHL, B. SCHNEIDER and S. H. SNYDER, Proc. Natl. Acad. Sci. USA 76, 521-525 (1979) S. ITOH, R. HIROTA, G. KATSUURA and K. ODAGUCHI, Life Sci. 25, 1725-1730 (1979)

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