Life Sciences, Vol . 24, pp . 549-556

Pergamon PressPrinted in the U.S .A .

STUDIES ON THE TISSUE DISTRIBUTION AND STABILZTSC ~ SIG" CAF(CORTICOTimPIN-RLIFASING FACTOR)

Naaki Yasuda and Monte A. Green

Department of Medicine, Division of EndocrinologyUniversity of Oregon Health Sciences Center

Portland, OR 97201

(Received in final form December 28, 1978)

Su~

Extracts of various bovine or rat neural tissues made with 0 .1 NHC1, 2N acetic acid or distilled water were fractionated onSephanlex G-100 column with 0.2 N acetic acid as the eluant . Adistinct peak of "big" CRF which elutes in the wid whims ofSephadex 6-100 was observed only with hypothalamic median en-inence and hypophyseal stalk . Human serum and extracts ofcerebral vertex, neurohypophysis and an ACTH~roducirg lungtuimr, had CRF activity which eluted fram Sephadex G-100 withdiffuse patterns without a distinct peak .

wig" CAF is stableduring storage at -20 C in water or at 4 C in acid, but p[ogres-sively disappeared when stored at -20 C in acid.

Although CAF was the first hypothalamic hypophysiotropic hormone describedby Saffran and Schally (1), and Guillemin and Rosenberq in 1955 (2), its isola-tion and characterization have been hanpered by a lads of sensitive and reproducible assays to measure CRF activity . However, there has been a major break-through in technology during the past several years. In vitro CAF assays withgreatly improved sensitivity and reliability (3-8) arenow available .

In 1975, Takebe et al . described a CIä~ assay system (5) in which ACTHsecreted from dispersédwltured rat adennhypophyseal cells is measured by aspecific radioimwnaassay.

The sensitivity of the assay is greatly enhanced bythe use of the adrenal.ectanized rats as pituitary donors (8) .

We have extensively studied the distribution of CAF activity within theoent[al nervous system and extraneural tissues. The most concentrated CRFactivity is in the hypophyseal stalk in a variety of manmialian species (9,10) .This ag[ees with the distribution of other hypothalamic hormones (TAH, Gn-RH)detected by direct radioimfo~uboassay (11,12) .

We have found 2 CAF peaks on fractionation of 0.1 N HC1 extracts of bovinehypaphyseal stalk on Sephadex G-100, one eluting in the wid whims (CRF-A,"big" CAF), the other eluting slightly in front of ACTH (CAF-ß, "little" CAF)(13) . CAF-B coincides with the estimation of molecular size of CRF reportedrecently from other laboratories (14,15) .

However, the CAF with high molecularweight which elutes in the wid wlutoe with Sephadez G-100 chramtography hasnot been described before. The present study was undertaken to determine thestability and tissue diet[ibution of this "big" CAF .

0300-9653/79/0205-054902 .00/0Copyright (c) 1979 Pergamon Press

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Distribution and Stability of "Big" CRF

Vol . 24, No . 6, 1979

Materials and Methods

Fresh bovine neural tissues were obtained from cows at a local slaughter-house and extracted with either 0 .1 N HC1, 2 N acetic acid or distilled wateraccording to methods previously described (9,16) . Tissues were dissectedwithin 1 hour of the death of animals . After the skull was opened, the en-tire brain was gently lifted up and the optic nerves were cvt with a scalpel .The hypophyseal stalk was transected at the level of pituitary diaphragm . Theentire brain was turned upside-down and the connection of the stalk to themedian eminence (ME) was severed and the stalk thus isolated . Median eminencewas then excised parallel to the base of the hypothalamus . Cerebral cortextissue was obtained from the frontal pole of the brain. The medial-posteriorportion of the neurohypophysis (without pans intermedia tissue) was alsodissected . The average weights of one stalk and median eminence were 70 and100 mg respectively . Two tenths ml of extraction vehicles were used per 10 m9of stalk or neurahypophysis and 20 m9 of median eminence or cerebral cortexrespectively . Rat tissues were obtained in our laboratory after rapid decapi-tation of intact, unstressed female Sprague-Dawley rats (250 g) . Combinedmedian eminence and stalk (MES) were excised as a unit.

Samples obtained as above were pooled fray several anüoals and imoecïiatelyplaced in a cold extraction vehicle. They were hariogenized at 4 C followed by4 C centrifugation at 3000 x g. The clear supernatant fluid was separated andfractionated on a Sep~hadex G~100 (fine grade) wlumn (0 .9 x 60 an) immediatelyor after storage at -20 C . All samples were eluted with 0 .2 N acetic acid at aflow rate of 4 ml/h and 1 ml fractions were collected . Aliquots of individualfractions were neutralized with 1 N NaOä and assayed for ACI9 and CRF . Thevoid volume and the salt whims of the column were determined with blue dextran2000 (Pharmacia Fine Chemicals, Uppsala, Sweden) and 125I- respectively .The wlumn was further calibrated with purified proteins, human transferrin,bw.ine serum albumin and C+valbumin (all purchased from Sigma Chemical Co.) and1251 A~'fH.

The elution positions of these marker substances were determinedby optical density at 280 or 254 rm or by radioactivity .

CRF assay was perfoomed with an in vitro system using cultured rat adeno-hypophyseal cells from adrenalectanizéd~orors and ACiB measurewent by radio-imaunoassay ewploying a specific antibody which reacts with the 11-24 segmentof ACTH (8) . ACPA contamination of the test substance and the basal secretionof ACTH in the control incubation were always subtracted from the AiCißconcentration in the test incubation for quantitation of net CRF activity ofthe test substance .

All samples from a given column fractionation wereanalyzed in the woos assays to avoid interassay variation .

Results

Elution

tterns of CRF activi

of fresh 0 .1 N HC1 extracts of various bovineor rat tissues on Steep aâex

Two

o

,non- cozen ~ .1 N äCl extracts of bovine stalk, median emi-nence, cerebral vertex, or rat median eminence-stalk (MES) ware fractionated onSephadex Cr100 .

As shown in Fig. 1, a distinct peak of "big" CRF (CAF-~1) inthe void whims of the wlumn was found in bovine stalk, bovine median emi-nence, and rat median eminence stalk.

Bovine cerebral vertex showed a diffuseelution pattern with a minimal CRF peak in the void volume .

Elution

henna of CRF activit of 1 month-frozen 0.1 N äC1 eztcacts of var-ious bovine tissues on

CrN äC extracts o

ne

" yseal stalk, posterior pituitary, orcerebral cortex were fractionated on Sephadex G-100 after 1 month storage at-20 C. As shown in Fig . 2, there were two praninent CRF peaks in the hypophyseal stalk, consistent with our previous observations (13), CRF-A and the

Vol . 24, No . 6, 1979

Distribution and Stability of "Big" CRF

551

OCI

~i i Zi

20

BOVINE STALK

10a

BOVINE ME

RAT MES

BOVINE CORTEX

10 PO 30 40 10 20 30 40FRACTION NUMBER

FIG. 1Net ACfH secretion from a~ltured aderohypophyseal cells induced by 0.4ml sample aliquots after Sephadex fractionation of fresh 0.1 N HClextracts of 100 mg of bovine hypophyseal stalk or cat median aninence-stalk (1~) or 200 mg of bovine median eminence (lam) or cerebralcortex on G-100. Adeno~hypophyseal cells frcm adrenalectcmized femalerats were cultured for 4 days and incubated with each fraction for 4 h .one basal ACIS secretion in control inwbations ranged from 0 .2-0.6ng/di.sh in this and all the other experiments. Sane Sephadex fractionsfran cat oc bovine median eminence or stalk contained ACTH . However,the ACTH content of the maximally contaminated fraction (#29) were<0 .3 ng/aliquot .

Elution positions of various marker substancesare inr3icated at the top of the figure by arrows in this and subsequentfiguresf Vo = Blue dextran, I = human transferrin II = bovine serumalbumin, III = C+valbumin, IV = 1-39 ACTH, Vt = 12~I-,

other (CAF-B) eluting between C+valbumin and ACTH . Although fresh extracts ofbovine hypophyseal stalk had some CAF activity in the elution position cor-responding to CAF-B (Fig . 1), only frozen extract showed a distinct CRF peak inthis position .

Neurohypophysis and cerebral cortex showed a diffuse elutionpattern without distinct CRF peaks (Fig. 2) .

Effect of extraction and stor e at -20 C with different solutions on theatabi t o bi CAF .

eavu>,e ypop ysea stalk was extracted with 0.1 N HC1 (pH 1.05), 2 N aceticacid (PH 2 .08), or distilled water (PH 6.1), and was fractionated on Sept~adexG-100 immediately or after storage at -20 C for 3 months .

In the case ofextracts prepared with water, one-ninth volume of 2 N acetic acid was slavlyadded to give adequate ionic strength to the extracts immediately before ap-plication to the G-100 column . After thawing the frozen 0.1 N HC1 extract,insoluble precipitates were noted which were removed by centrifugation. Asshown in Fig. 3, CIa~ activity of CAF-x4 was well-retained after 3 months storageat -20 C with the extract prepared with water but considerably reduced with the0 .1 N äC1 extract . The extract prepared with 2 N acetic acid showed an inter-mediate result .

This difference of stability of CI4?-A during frozen stor-age at -20 C is most likely due to the differences in pH between the solutions.

O u _

Effect Of scor

at low

at 4 C OC -20 C on the stabili

Of "bi " CRF .A re

. N

extract o

ne hypopTiyse

sta7Jc was rast ona

on

552

Distribution and Stability of "Big" CRF

Vol. 24, No. 6, 1979

PO

10

0

Pa

lo

0

FRACTION NUMBER

FIG. 2det ACPH secretion fraD cultured adenohypophyseal cells induced byale aliquots after Sephadex fractionation of 1-month-frozen 0 .1N HC1 extracts of 100 m9 of bovine hypophyseal stalk or posteriorpituitary, or 200 mg of cerebral cortex.

0.1 N NCI

P N ACETIC ACID

WATER

lo ~o lo ao loFRACTION NUMBER

FIG. 3Net ACTH secretion from cultured adenohypopl~yseal cells induced bysample aliquots after Sephadex fractionation of fresh (upper

1) or 3-months-frozen (laver panel) extracts of bovineyseal stalk prepared with 0 .1 N HCl (left column), 2 N

acetic acid (middle column), or distilled water (right column) .

j FRESH FRESH FRESN

1

FR02EN FROZE]V FRCZEN

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Distribution and Stability of "Big" CRF

553

Sephadex 6-100 and the fractions with CRF activity in the void volume werepooled.

Aliquots of this CRF-A pool in 0 .2 N acetic acid were rechramato-graphed on Sept~adex G-100 after various treatments . Three equal amounts ofaliquots were fortified with HC1 by slowly adding ~e-ninth volume of 1 N HC1while the samples were vigorously agitated on a Vortex mixer (pB 1 .0) .

one ofthe tiCl-fortified aliquots was rechrnmatographed immediately on Sephadex 6-100,while the other two aliquots were rechramatograp~hed after 1 month storageeither at 4 C oc -20 C . There were massive precipitates after thawing thesample stored at -20 C which were removed by centrifugation.

As shown in Fig .4, CIB? activity eluted only in the void volume when the C1~-A pool wasrechromatographed immediately. CAF activity of the sample stored at 4 C waswell-retained after 1 month storage and the major portion of the activityeluted in the void volume .

There was considerable reduction in CAF activitywith the sample stored at -20 C. With both of the samples stored at 4 C and-20 C, a small CAF peak was detected at the elution position of CAF-ß .

Theseresults indicate that the combination of law pe and storage at -20 C areresponsible for the disappearance of CAF-A and that there is a small butdetectable degree of conversion of CRF-A into CRF-$ during 1 month storage at 4C or -20 C .

FRACTION NUMBEA

FIG. 4Net ACfH secretion from cultured adenohypophyseal cells induced bysample aliquots after rechramatography of isolated CRFA on SephadexG-100 with or without 1-month-storage at 4 C or -20 C.

CRF-A wasfortified with HC1 before storage or rechrcBlatography.

Comparison of CRF of an ACiß-producing lung tumor to that of control lung tis-sue.

Tumr tissue and normal lung tissue were obtained at the autopsy of a pati-ent with oat cell lung cancer with ectopic production of ACTS. The basal plas-ma ACTH concentration before the patient died was in the range of 7-9 ng/ml,and the KRH content in the tumor was 55 ~/g . Both t1700C and the control lungtissue were extracted with 0.1 N HC1 and added to the CRF assay system eitherdirectly or after fractionation on Sephadex G-100.

Both the tumor and controllung showed weak, but significant, CRF activity .

7tiere were no significant

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Distribution and Stability of "Big" CRF

Vol . 24, No . 6, 1979

differences (p>0 .5, Student's t test) in CRF activity between the tumor andcontrol lung extract. The CRFelution patterns on 6-100 for both tissues werealso similar and showed a diffuse pattern comparable to that of cerebral cortexshown in Fig. 1 (data not shown) .

Elution pattern on Sephadex G-100 of the CRF activity of human peripheralserum.

We previously showed that rat and human peripheral blood contain same CRFactivity, as assessed bY cell culture assay, although it is less potent thanthe activity of rat hypothalamic extract (17) . Therefore, 2 ml of human serumobtained from a healthy volunteer was fractionated on Sephadex Gr100, and CRFactivity of each fraction was assessed . The CRF elution pattern was diffuseand comparable to that of cerebral cortex (data rot shown) .

Discussion

Our data indicate that Clä~-"A is the major CRF component in fresh extractsof bovine hypophyseal stalk and hypothalamic median eminence, but not ofcerebral vertex or peripheral blood.

Rat median eminence-stalk extract alsohas prominent CRF-A.

These data suggest that CRF

may play sane role in theregulation of ACTH secretion, but it is unknown whether it is the activehormone which stimulates the wrticotraphs in viw. With long-term storage ofextracts at low p~I at -20 C, CRF-B becomes ~Tie~aninant CRF .

Since CRF-A canclearly be converted to CRF-B by boiling at lav p8 (18), it is highly unlikelythat CAF-,A and CRF-B exist independently.

The dose-response slopes for CRF-,Aand CRF-B are parallel and distinctly different from that for cerebral cortex(13) . it is likely that CRFA is either the prohornrone or a carrier-bound formof CRF-B, as we pointeä out previously (13,18) .

CRF activity of CRF-,A was stable in distilled water, but progressivelydisappeared in 0 .1 N HC1 during prolonged storage at -20 C . More than 908 ofthe CkF activity of isolated CRF-A dis~peared after storage at low pti at -20 Cand only minimal CRF activity was detected at the CRF-

and CRF

positions onrechromatography . These data indicate that CRF-A is labile at -20 C in acidand that there is a limited conversion of A to B CRF under these conditions.Sire CRFA is stable at low pH when stored at 4 C, it is the eanbination oflow pH and storage at -20 C that is responsible for the disappearance of CRFA.However, it must be pointed out that massive precipitates were rated whenaliquots of CRFA pool stored at -20 C in acid were thawed,

it is possiblethat CRFA may be precipitated rather than destroyed during storage in acid at-20 C.

Prolonged freezing of hypothalamic extracts at low pä beforefractionation may be one reason why high molecular weight CRF has rot beenrecognized before .

It is also possible that previous studies (14,15) failed toreveal CRF-,A simply because Sephadex G-25 or G-50 instead of G-100 wereemployed in these studies.

With chromatography on Sephadex G-25, CRFA aryl Bcoelute in the void volume (13) .

Although there are same previous reports indicating an ectopic productionof CRF in same cases of ACPfi-producing tumors (19-22), we did rat find any dif-ference in CRF activity between the AL`fH-producing tumor and the axitrol lungtissues . CRF activity of both showed similarly diffuse elution patterns on6-100, comparable to that for cerebral cortex . CRF activity found in both tis-sues may be a ran-specific 'noise' of the assay. However, our data are ineorr~elusive relative to the presence of CRF in the tumor tissue since we studiedonl one case .

Only sane cases of ALBS-producing tumor were reported topuce CRF (21) .

Adcrawled eurent : Supported by grants from the NIA14flD, NIH. We are indebted toe a ion

ituitary Agency and the NIAl~D for the gift of ALTH antibody andto Susan Green for invaluable technical assistance .

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Distribution and Stability of "Big" CRF

555

References

1 . M. Saffron and A.V . Schally, Çanad. J. Biocta~n . Physiol. 33 408-415 (1955) .2 . R. C~illemin and B . Rosenberg, Enâoc~laly - 5

ÙT055) .3 . R. Portonova, D.R . Smith, aryl G. Sayers, Proc. Soc. Exp. Biol . Med. 133

573-576 (1970) .4 . A.F . Pearhautter, E. Rapino, and M. Saffron, Neuroendocrinology 15 106-119

(1974) .5. R. Takebe, N. Yasuda, and M.A. Green, Ercbcrinolo

97 1248-1255 (1975) .6 .

W. Vale, C . Rivier, M. Brown, L. Chan, N. L rg, a

~Rivier, in Hypo-thalamus and Endocrine Function, F. Labris, J . Meites, and G. Pelletier(eds .) 3 397-429 (1975) .

7 . P.J. Lowry, J . Endocr . 62 163-164 (1974) .8 .

N. Yasuda, K.Tacf~, arm M.A. Greec, Enäocrinology 98 717-721 (1976) .9 .

N. Yasuda, M.A. Green, S.E . Green, and P. Ponton, J.Brdocr . 75 293-303(1977) .

10 . N. Yasuda and M.A. Gceer, Proc . Soc. Ex . Biol . Med. _158, 421-425 (1978) .11 . E . Cloon and Y. IGoch, Nature

34 34

19712 . K.S . Estes, V. Padmana~ a~ E.M. Convey, Biol. Reproduc . 17 706-711

(1977) .13 . N. Yasuda, M.R. McClurg, and M.A. Green, Biodiem. Biophys . Res . Canin. 81

1187-1194 (1978) .14 . A.V. Schally, A. Animons, T.W. Bedding, R . Chihara, A. Gordin, W.-Y. Huang,

and M. Saffron, Program 59th Ann. Mtg. Endocrine Soc., June 8-10, p. 95(abstract) (1977) .

15 . W. Vale, and C. Rivier, ibid . p. 217 (abstract) (1977) .16 . N. Yasuda and M.A. Green, Erdocrinolo

99 944-948 (1976) .17 . N. Yasuda and M.A. Green, Acts En ocrinol: 84 1-10 (1977) .18 . N. Yasuda, M.R . McClurg,

M.A. Green,

in. ReB. 26 551A (abstract)(1978) .

19 . B.C . Birkenhâger, G.V. Upton, H .J . Seldenrath, D.T. Rrieger, and A.H .Tashjian, ACta Erdocrinol . 83 280-292 (1976) .

20 . G.V . Upton

T.T. Amatrudâ, N . E

. J. Med 285 419-,424 (1971) .21 . H. Yamamoto, Y. Hirata, S . Mats cura, H. Imurâ, M . Nakamura, and A. Tana-

ka, Acts Endocrinol. 82 183-192 (1976) .22 . T. S a, H. Demurs, R.Demurs, I. Wakabayashi, R. Navra, E . Odagiri, and

R. Shizume, J. Clin. Endocrinol . Metab. 44 440-446 (1977) .