Pituitary polysomes and adrenocorticotropin secretion

701

PRELIMINARY NOTES

dose of ACTH is completely abolished in the face of only a partial inhibition of amino acid incorporation. Assuming that the inhibitory effect of chloramphenicol on adrenal protein synthesis bears a causal relationship to the inhibition of the steroidogenic effect of ACTH, the present results would suggest that chloramphenicol preferentially inhibits that protein synthesis which is more directly concerned with the steroidogenic effect of ACTH. This preferential inhibition conceivably could be related either to the different susceptibilities of subcellular fractions to chloramphenicol block 1° or to a more selective effect of chloramphenicol on blocking protein synthesis which is associated with new messenger RNAS, s. This study was supported by Grants AM-o7376 and AM-o558I from the National Institutes of Health, U.S. Public Health Service. The excellent technical assistance of Mrs. M. Kouls and Mr. P. WILSON, Jr. is gratefully acknowledged.

Department o/ Medicine, Buffalo General Hospital, State University o/New York at Bu[[alo, Bu//alo, N. Y. (U.S.A.)

ROBERT V. ~ARESE

1 j . j . FERGUSON, JR., J. Biol. Chem., 238 (1963) 2754. 2 E. F. GALE AND J. P. FOLKES, Biochem. J., 53 (1953) 493. D. NATHANS AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 497. * F. E. HAHN AND A. D. WOLFE, Biochem. Biophys. Res. Cornmun., 6 (1962) 464 . A. S. WEISBERGER, S. ARMENTROUT AND S. WOLFE, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 86. 6 C. T. AMBROSE AND A. H. COONS, J. Exptl. Med., 117 (1963) lO75. 7 It{. SILBER, R. BUscH AND R. OSLAPAS, Clin. Chem., 4 (1958) 278. s ]c~. V. ]TARESE AND W. J. REDDY, Endocrinology, 73 (1963) 294. 9 F. E. HAHN, J. E. HAYES, C. L. WISSEMAN, JR., H. E. HoPPS AND J. E. SMADEL, Antibiot. Chemotherapy, 6 (1956) 531. 10 1~. RENDI, ]~xptl. Cell Res., 18 (1959) 187,

Received June 2nd, I964 Biochim. Biophys. Acta, 87 (I964) 7Ol-7O 3

PN

91019 Pituitary polysomes and odrenocorticotropin secretion

The rate of secretion of the adenohypophysial polypeptide hormone, ACTH, is mediated in part by a feed-back mechanism, the controlled variable being the level of adrenal cortical hormone in the blood 1. Thus the rate of secretion of ACTH is increased following bilateral adrenalectomy 2 and decreased following the chronic injection of hydrocortisonO. The synthetic mechanisms involved have received scant attention. Following bilateral adrenalectomy there is an increase in total pituitary RNA 4 and an increase in the incorporation of amino acid into ACTH in vitro ~. As part of a study intended to elucidate the mechanisms and control of the biosynthesis of ACTH, changes in pituitary ribonucleoprotein have been investigated under conditions of suppression and of stimulation of the secretion of ACTFI. Male rats of the Wistar strain, the controls weighing 200-225 g at the time of Biochim. Biophys. Acta, 87 (1964) 7o3-7o6

7o2

PRELIMINARY NOTES

sacrifice, were maintained at constant temperature (24.5:~I °) and natural illumination and randomly divided into control, adrenalectomized and hydrocortisonetreated groups. Bilateral adrenalectomy was performed via the dorsal approach under ether anaesthesia. The animals were maintained post-operatively on a commercial cube diet and a 1 % NaC1-5 % glucose drinking solution ad libitum, and sacrificed 64 days after operation. Daily subcutaneous injection of hydrocortisone, (Cortril Acetate, Pfizer, 25 mg/ml in aqueous suspension) 3 mg/Ioo g body weight, was administered for 16 days and the animals sacrificed 4 h after the last daily injection. Animals were isolated in single cages for 24 h prior to sacrifice by decapitation in the early morning. These precautions are necessary to allow basal levels of adrenal cortical secretion. Analysis of ribosomal particles by sucrose-gradient centrifugation was carried out following a procedure essentially that described by MUNRO, JACKSON AND KOR~ER6. All steps were carried oat at 0-3 °. Eight fresh pituitaries were pooled and homogenized in 0.6 ml of 0.25 M sucrose in buffered medium (io mM magnesium acetate, 4 ° mM NaCI, IOO mM KC1, 20 mM Tris buffer p H 7.6) with 5 strokes of a loose-fitting Teflon pestle in a Potter-Elvehjem type homogenizer. The homogenate was centrifuged at IO ooo ×g for IO rain. To nine volumes of the supernatant was added one volume of a freshly prepared IO °/o solution of sodium deoxycholate in Tris buffer p H 8.2. An aliquot of the deoxycholate-treated supernatant was treated with o.I/~g/ml of pancreatic ribonuclease (EC 2.7.7.16) for 5 min at 37 °. Seventy-,ul samples of the supernatants were layered onto lO-3 ° °/o linear gradients of sucrose (containing the buffered medium) which had been prepared in 2-ml quartz centrifuge tubes (outside diameter IO ram). Centrifugation was carried out in a Spinco Model L ultracentrifuge (rotor SW 39) for 9 ° rain at 32 ooo rev./min. The quartz tubes were then scanned directly for absorbaney at 26o m# using an automatic scanning apparatus 7 in a Cary 15 recording spectrophotometer. The spectrum of each peak was obtained to verify its ribonucleoprotein nature. Under our conditions of sucrose-gradient centrifugation, single ribosomes locate at 18.5 mm from the top of the tube (Fig. I). The distribution of pituitary ribo~ 0.~.

. . . . .

Control

-g

_

Adr¢n~ll¢ctorny

.............

~ 0.3 o

_

Hydrocortison¢

0.2

0.1 "o
10 TOP

J5

20

25

30 mm BOTTOM

Fig. I. Sucrose-gradient analysis of polysome profile in control, adrenalectomized and hydrocortisone-treated groups. Curves have been superimposed to give equal absorbancy values at 3 ° mm.

nucleoprotein particles showed the major ribosomal peak at 18. 5 mm and two fasteimoving peaks at 21. 5 and 23.5 ram. Following bilateral adrenalectomy, there was a marked increase in the amount of ribonucleoprotein in the 23.5-mm peak. Following Biochim. Biophy.s. Acta, 87 (1964) 703 706

PRELIMINARY NOTES

703

the chronic administration of hydrocortisone, the ribonucleoprotein profile was similar to the control group. In all three groups, treatment with ribonuclease removed the two faster-moving peaks (Fig. 2). ,~

,' ',

.....

"~

..... RNAasetreated _ _ Adrenal¢ctomy

RNAase treated

0.2 0.1

~" 0.3

°1 0.2

l 02

~

..... RNAa$=treated

O.I

~

Control

"

_

1O 15 20 25 30ram TOP BOTTOM Fig. 2. Sucrose-gradient analysis of polysome profile in control, adrenalectomized and hydrocortisone-treated groups before and after treatment with ribonuclease, o.I/~g/ml at 37° for 5 min. Following bilateral adrenalectomy there is an increase in the amount of the fast-moving ribonucleoprotein fraction, this fraction being extremely labile to ribonuclease. This fast-moving component represents the polyribosomes, aggregates of ribosomes attached to a linear messenger :RNA molecule, actively engaged in the synthesis of polypeptides and protein s& That the chronic injection of hydrocortisone does not significantly alter the polyribosome profile is not unexpected, since the rate of secretion of ACTI-I in the control group is at a low level. Following chronic administration of hydrocortisone, the pituitary is still actively synthesizing other polypeptides and proteins. The increase in polyribosomes following bilateral adrenalectomy most probably indicates an increase in the content of messenger RNA (although increased affinity of the ribosomes for messenger RNA, however unlikely, has not as yet been ruled out). Further experiments will be carried out to ascertain whether the increase in messenger R N A is due to a predominant increase in its synthesis or, less likely due to an inhibition of its rate of breakdown. But whichever be the case, our results demonstrate a significant primary effect on the proteinsynthesizing unit, the polyribosome. A corticotropin-releasing factor of hypothalamic origin has been implicated in the immediate control of ACT}{ secretion 1°. Corticotropin-releasing factor could exert its effect b y controlling the synthesis of that messenger RNA which "codes" for the synthesis of ACTH. A similar mechanism has been suggested for the action of the anabolic hormones on protein synthesis n. The control of the synthesis of the Biochim. Biophys. Acta, 87 (1964) 703-706

704

PRELIMINARY NOTES

polypeptide and protein hormones could, as well, be mediated through the regulation of the synthesis of messenger RNA. The author expresses his gratitude to Professor J. BRACHET for sponsoring the project, to Professor H. CHANTRENNE for his encouragement and advise, and to Professors R. CORDIER and H. ]-[ERLANT for their interest and for providing the animals and the facilities for their care. This work was supported in part by EuratomULB contract o16-61-1o ABIB. The author is a Fellow of the Medical Research Council of Canada.

Laboratoire de Chimie Biologique, Facultd des Sciences de l'Universitd Libre de Bruxelles, Brussels (Belgium)

JACOB KRAICER*

1 F. E. YATES AND J. URQUHART, Physiol. Rev., 42 (1962) 359. C. FORTIER, Proe. Soc. Exptl. Biol. Med., IOO (I959) 13 . a C. FORTIER, Proe. Soc. Exptl. Biol. Med., IOO (1959) 16. 4 M. HESS, J. J. CORRIGAN, JR. AND J. A. HODAK, Proc, Soc. Exptl. Biol. ~Ied., lO6 (1961) 420. I. G. WOOL, R. SCHARFF AND N. MAGES, Am. J. Physiol., 2Ol (1961) 547. A. J. MUNRO, R. J. JACKSON AND A. KORNER, Biochem. J., in the press. 7 A. HERZOG, R. LOMBAERT AND R. HAMERS, unpublished. 8 IR. J. WARNER, A. RICH AND C. E. HALL, Science 138 (1962) 1399. 9 V. O. V~'ETTSTEIN, T. STAEHELIN ANn H. ~OLL, Nature, 197 (1963) 43 o. 10 G. W. HARRIS, Neural Control o/ the Pituitary Gland, E d w a r d Arnold Ltd., London, 1955. 11 A. KORNER, Biochem. Biophys. Res. Cornmun., 13 (1963) 386.

Received June 4th, 1964 * P r e s e n t address: D e p a r t m e n t of Physiology, Queen's University, Canada.

Kingston, Ontario,

Biochim. Biophys. Acta, 87 (~964) 7o3--7o6