Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

Life Sciences, Vol. 36, pp. 643-647 Printed in the U.S.A.

Pergamon Press

REDUCTION IN BRAIN IMMUNOP~EACTIVE CORTICOTROPIN~RELEASING FACTOR (CRF) IN SPONTANEOUSLY HYPERTENSIVE RATS Kozo Hashimoto, Teruhiko Hattori, Kazuharu Murakami, Shuso Suemaru, Yoshiro Kawada , Jingo Kageyama and Zensuke Ota Third Department of Internal Medicine, Okayama University Medical School° 2-5-1 Shikata-cho, Okayama, Japan (Received in final form November 28, 1984) Summar~ The brain CRF concentration of spontaneously hypertensive rats (SHF) and normotensive Wistar Kyoto rats ( ~ Y ) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male W1star rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neurointermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axls, autonomic response and behavior of SHR. Several brain peptides including vasopressin (AVP) (i, 2), oxytocin (3) and angiotensin ~ (4) were reported to have a role in controlling blood pressure. Several investlgators have reported that AVP and oxytocin content in the brain stem in spontaneously hypertensive rats (SHR) were markedly reduced (3, 5, 6). Recently Brown et al. (7) reported that an intraventricular admlnistration of corticotropin releasing factor (CRF) Increased blood pressure by stimulating the noradrenergic sympathetc nervous system. It may thus be speculated SHR brain and that it might be In this study here we examined normotensive Wistar Kyoto rats

that an abnormal CRF content might exist In the involved in the hypertension development of SHR. the CRF concentration in the brain of SHR and (WKY) using rat CRF radioimmunoassay (RIA).

Materials and Methods Tissue extraction: Male SHR and normotenslve WKY at 5 weeks of age were purchased from Charles River Japan Inc. and habituated in an animal room and received food and water ad libitum for a week. One day before the experiment the animals were weighed, and their blood pressure were measured by tail cuff plethysmography (Ueda Seisakusho, Tokyo). The animals were decapitated at 6 weeks of age and the brain and neurohypophysis were quickly removed. The major brain regions were dissected out and weighed. The tissue samples were homogenized in a 2 ml solution composed of 80% acetone and 20% 0.5 N HCI with an 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.

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ultrasound sonicator. After centrifugation at 4~000 g, petroleum ether (3 ml) was added to the supernatant. The samples were mixed and centrifuged at 1,200 g, and the lower layer was transferred to another tube and dried at 45aC under a stream of nitrogen. The dried extracts were stored at -20°C. The samples were reconstituted with 1 ml RIA buffer, and I00 - 200 N 1 samples were assayed in duplicate, When 2,5 ng rat CRF was added to the hypothalamus and extracted with this method, the recovery rate of CRF was 79.9 ± 4.2 (Mean ± SD)%, Using 125I-rat CRF, similar recovery rate (75,5 ! 4 ~ 1 % ) was obtained. For d11utlon curves of CRF immunoreactivity in major brain tissues, male Wistar rats ,Jelghing approximately 250 g were decapitated, and several brain regions were quickly removed, The brain regions of 4 - 14 rats were individually pooled and extracted by acid acetone-petroleum ether extraction. Each dried extract was resuspended with 1 ml RIA buffer and serially dlluted with the buffer and radioimmunoassayed. CRF RIA: The rat CRF a n t i s e r u m was developed in our laboratory. T w o m l l l i grams of synthetic rat CRF was conjugated to 6 mg of bovine serum albumin (Sigma) with glutaraldebyde. Japanese white rabbits were immunized as previously reported (8). After 15 weeks of immunization, the animals were anesthesized, and blood was collected from the juglar vein. A n t i s e r u m was used at a dilution of 1 : 5,000 for RIA. Synthetic rat CRF was used as tracer and standard. Iodination of rat CRF and subsequent RIA were carried out using the same procedure that we used for ovine CRF RIA (8). The range of the standard curve extended from 20 pg/tube to i0 ng/tube, and the llmits of detection was 20 pg/tube. The intraassay coefficient of variatlon was 2 . 1 % for 463.2 pg, and the [nterassay coefficient of variatlon derived from seven independent assays using rat hypothalamic extract was 7 . 1 % . Crossreactivlties with rat CRF (i-20), rat CRF (820), rat CRF (11-20), and rat CRF (27-41) were 0.3, 0, 0, and less than 0.003%, respectively. Crossreactivlty with ovine CRF was 12.8 % whereas there was no crossreactivlty w~th sauvagine, TRH, LH-RH, somatostatin, AVP, oxytocin, anglotensin K , bradykinin, vasoactive intestinal peptide, substance P, ACTH,~-lipotropin, B-endorphin, ~-MSH, ~-MSH, CLIP, met-enkephalzn, leu-enkephalin, rat GH, TSH or prolactin. Synthetic rat and ovine CRF were purchased from Bachem Co~ (Torrance, CA). Rat CRF fragments were kindly supplied by Dr, N, Yanaihara (Shizuoka, Japan). B - L i p o t r o p i n was a gift from Dr. C, H. Li (San Francisco, CA). Other neuropeptzdes were purchased from the Peptide institute, Inc. (Osaka, Japan). Statlstlcal

analysis

was conducted

using Student's

t-test.

Results The displacement of 125I-rat CRF by serlally diluted rat neurolntermedlate lobe, hypothalamus, thalamus, cerebral cortex, midbrain, ports, medullaoblongata, and cerebellum paralleled that of synthetic rat CRF. The anterior pituitary extract did not show a parallel response curve (Fig. i). There was no signlficant difference ~n the body welght of t~$Y (148.3 + 2.5 g, Mean + SEM) and SHR (144.9 + 2.8 g). The svstorlc blood pressure was significantly higher (P <.01) in S--HR (153.6 + 3.0 mmHg, Mean + SEM) than in WKY (117.9 + 3.2 mmHg). In both ~KY and SHR the hypothalamus and neurointermediate lobe showed the highest levels of CRF immunoreactivity. A considerable amount of CRF immunoreactivity was also detected in the thalamus, mldbrain, pons, medulla oblongata, cerebra] cortex and cerebellum (Table i). The CRF immunoreactivity of the SHR hypothalamus, neurolntermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly lower than in comparable areas of WKY rats.

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CRF Reduction

Wet tissue weight 0 2

in SHR Brain

645

QT~J)

0 4

1

2

4

I0

20

40

i00

l

,

!

I

I

I

I

I

lO0 " 80

. /~nterlor Pituitary

A ~

Ne...... termedl]t~e \~,

Thalamus

. . . .

X

6O

Mldbr(lin Cerebrol cortex

":~.~\~

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Pons CereObelium

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FIG.

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1

Displacement of 1251-rat CRF by serially diluted rat CRF and by extracts of brain regions of male Wistar rats.

TABLE 1 CRF content in major brain regions of WKY and SHR at 6 weeks of age (Mean ~ SEM) Region

CRF Immunoreactlvity WKY (n)

SHR (n)

(ng/lO0 mg wet tissue weight) Hypothalamus

7.63 + 0,54

(9)

5.28 + 0.29 (i0)*

Thalamus

1,48 + 0.13

(i0)

1.21 + 0.14

(9)

Midbraln

2.66 + 0.]I

(I0)

1.81 + 0.13

(i0)*

Pons

1.86 + 0.13

(i0)

2.00 + 0.12

(10)

Medulla oblongata

2.00 + 0.01

(I0)

1.60 + 0.12

(i0)*

Cerebral

2.21 + 0.25

(i0)

1.39 + 0.14

(]0)*

Cerebellum

2.01 + 0.09

(i0)

2.27 + 0,12

(I0)

Neurointermedlate lobe

0.40 + 0.03

cortex

(ng/neurointermediate (i0)

lobe)

0.26 + 0.03

(9)*

* P <.01 vs WKY Discussxon The widespread distribution of CRF-IIke immunoreactlvlty was already reported in sheep (9, I0), rabbit (ii) and rat (8, 12-16) in radioimmunoassay and

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immunohistochemlcal investigations wlth anti-ovine CRF serum, The distrlbution of CRF immunoreactivity seemed to be more limilted in the rabbit and s h e e p b r a l n (I0, ii) than in rat brain (8, 12), Our results here using more specific antirat CRF serum confirm that considerable CRF immunoreact~vity is present outside the hypothalamus in rats. The CRF levels in the present examination were generally higher than those reported by us previously (8), probably because anti-rat CRF serum crossreacts more with rat CRF than the anti-ovine CRF serum that was used in the previous examination. Our results here show that the CRF levels were lower in the SHR hypothalamus, midbrain, m e d u l l a oblongata, cerebral cortex and neurointermedlate lobe when compared with WKY. CRF stimulates ACTH and D - e n d o r p h i n secretion from the pituitary but also produces a potent behavioral abnormality in rats (17. 18), and an intraventricular administration of CRF elevated blood pressure via peripheral noradrenalin elevation (7, ]9). Swanson et al. (16) reported that the CRF immunoreactivity was located at least three distlnct systems of the rat brain. First, CRF stalned fibers were located in the external zone of the median eminence, which appeared to arlse in the paraventricular nucleus of the hypothalamus and presumably modulate the release of ACTH and fi-endorphin from the pituitary. Second, a series of cell groups and fibers were located in the basal telencephalon, hypothalamus and brain stem, which might play a role in the mediation of autonomic responses. Third, scattered CRF-stained cells were found throughout most areas of the cerebral cortex. Our results suggest that the CRF content was lower in all three of these systems in the h y p e r t e n s l v e rats. It has been reported that AVP and oxytocin contents were reduced in the hypothalamus, amygdala, septum and brain stem of spontaneously h y p e r t e n s i v e rats (3, 5, 6) and It was suggested that AVP and oxvtocin might play some role In the control of b a r o r e c e p t o r function as b a r o r e c e p t o r activlty was impaired in SHR (20, 21). As a change in tissue content may reflect a synthesis, storage, release or inactivation, it is difficult to interpret the m e c h a n i s m of these decreased changes. We recently found that SHR at 6 weeks of age had elevated levels of serum corticosterone and SHR anesthesized by c b l o r p r o m a z i n e - m o r p h i n - N e m b u t a l showed an excess serum ACTH response to AVP but a poorer ACTH response to CRF compared to control WKY (in preparation). As the poorer ACTH response to CRF in SHR might be ascribed either to the desensltization of corticotrophs to CRF due to the Increased secretion of endogenous CRF or to the feedback effect of elevated corticosterone, we assumed that CRF turnover was increased in SHRbrain. Causal relationships between reduced CRF and h y p e r t e n s i o n in SHR are difflcult to know from present results. However, our present results allow us to speculate that a CRF reduction in some brain regions might be implicated in the reported abnormalities in autonomic responses, the pituitary-adrenal system (22, 23) and behavior (24) of SHR, and it might at least be partly implicated in the development of hypertension, although there remains a possibility that reduced CRF immunoreactivity might be an effect of hypertension or the other noted dlsorders. Further investigations were needed to clarify the relationship between the CRF reduction and abnormalitles in SHR° Acknowled&ement We are greatly indebted to Dr. N. Yanalhara for a kind supply of rat CRF fragments, and Dr. C. H. Li for a gift of ~-lipotropin. This w o r k was supported in part by a grant from the Japanese Minlstry of Education, Science and Culture. References i.

J.T.

CROFTON,

L. SHARE,

R.E.

SHADE~

C. ALLEN

and D. TARNOWSKI,

Am. J.

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