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Hypothalamic polypeptides releasing pituitary hormones
Hypothalamic Polypeptides Releasing Pituitary Hormones By
ROGElI GUILLEMIr\'
HE PUHPOSE of this paper is essentially to present the current status of our own research on the purification of hypothalamic factors involved in the secretion of ovulation hormone (LH), thyroid-stimulating hormone (TSH), and adrenocorticotropin (ACTH). Professor Godfrey Harris has reminded us in his opening remarks to this session of the rationale for our current concept of the neurohumoral nature of the hypothalamic control of adrcnohypophysinl function. This will not be further discussed here but simply will be accepted as the basis on which to justify our own investigations. My friend and former associate, Dr. Schally, has presented most of the data we obtained together on CRF in my laboratory at Baylor College of Medicine; thus I will limit myself to a few remarks about the nature of CRF of hypothalamic origin to present in somewhat greater details the results of our recent studies on LRF (LH-releasing factor) and TRF (TSH-releasing factor), I have discussed at great length in two reviews recently publishedt-" my present position regarding the problem of the nature of CRF and particularly regarding the significance of ,B-CRF, at-CHF, a;!-CHF, of neurohypophysial origin',3.4 as they were presented to us by Dr. Schally in his brilliant lecture. I personally consider that the possible physiologic significance of these substances which we have isolated from commercial extracts of posterior pituitary may well have to be re-evaluated now that we have available large quantities of hypothalamic tissues. 'Ve must now repeat our various purification and extraction procedures on extracts of hypothalamic tissues containing the median eminence, with or without the pituitary stalk, and compare the results that we will obtain with what we have reported earlier with starting materials of neurohypophysial origin. It is ~ncouraging that in a few preliminary experiments, done recently in Paris and also in collaboration with Dr. Schally when he was still in my laboratory at Baylor, we have observed the presence of CHF activity in extracts of median eminence and hypothalamus of ovine origin with partition coefficients in counter current distribution elosely related to those which we had reported earlier for a-CRF and ,B-CRF of neurohypophysial origin." Anyway, the question of the nature of hypothalamic CRF must be approached with an open mind as I think that our earlier results on neurohypophysial peptides have not necessarily answered it. I would like now to summarize our recent results on' LRF and TRF of hypothalamic origin. These studies were performed in collaboration with Dr. Sakiz, Dr. Yamazaki, Dr. Schiavi, and Dr. Jutisz, at College de France in Paris and more recently with Dr. Dhariwal and Dr. Ward in our laboratories at Baylor College of Medicine in Houston. The filtration on the gel Sephadex
T
From the Lahoratoirc de llIorphologic cxperinu.ntalc ct endocrinologic, College de France, Paris.
1206 ~[ETAlIOl.lS~I, VOL. 13,
No. 100l'AIIT 2 (O<..TOIIEII), HJ6·]
1207
I1Yl'OTHALA:o.UC POLYPEPTIDES RELEASIl"G PITUITARY nOR:o.I0:-iES
S EPH A DEX G 25
z
0 .1 II. PYRIDI N E AC.
o
IU
15 0 0
CRF TRF
....
o
z
LH
30 00
U
T SH
" 10 0 0 E
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E
AC i H
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~
~
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f::.... 2 000
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Z
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500
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:?>-
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10 0 0
~
~
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~
o
20
40
60
80
10 0
NUMB ER OF F RA CT IO NS
Fig. I.-Molecular sieving 011 the gel Sephadex C-2.:> of all acid extract of sheep hypothalamus. Location of various hormonal (ACTH, LH, TSII) and neurohormonal (vasopressin, oxytocin, a-MSII, ,B-},ISII, CHI', TRF, LHF) activities. The zone marked CHF contains also a considerable ACTH-Iike adrenocorticotropic activity.
G-25 of an acetic acid extract of sheep hypothalamus (fragments containing hypothalamus, median eminence and pituitary stalk) yields two zones of activity in the assay for LH-LRF. The assay used here is the ovarian ascorbic acid depletion test'':? using immature animals. One zone of activity corresponds to the non-retained material (fig. 1, tubes 1-10); this activity persists in the assay animal after hypophysectomy. Thus it corresponds to substances acting directly at the level of the ovary; this may be LH or LII-like substances present in the extract of hypothalamus-pituitary stalk tissues. The second zone of activity in the assay corresponds to substances strongly retarded on the gel (fig. 1, tubes 60-70). This material is not active in the assay animal after hypophysectomy in conditions in which we have ascertained that the sensitivity of the hypophysectomized animal to LH is adequate. Thus the activity of this material appears to be transhypophysial, i.e., its effectiveness in the LH assay requires the presence of the pituitary. \Ve concluded that this material releases endogenous LH and that it should correspond to the presumed LH-releasing factor (LRF). Figure 1 shows-also the position in the effluent of such a column of the biological activities corresponding to ,B-~ISH, a-MSII, arginine-vasopressin and oxytocin," The zone corresponding to the LII-releasing activity is distinct from that of vasopressin and oxytocin and also from that corr.esponding to fI-MSH and a-MSH. In other experiments" we were able to show that a-l\'ISH, ,B-MSII, arginine-vasopressin, lysine-vasopressin, and oxytocin' are inactive in the tests used for LII-releasing activity at doses much greater th::111 those encountered in the LHF zone from the effluent of the Sephadex G-25 filtration of hypothalamic extract. Thus we have concluded that LRF was a substance different from these peptides. Identical results to those reported here on extracts of sheep hypothalamus have recently been obtained with ex-
1208
ROGEn
GUILLEMI~
CMC FOLLOWING SEPHADEX G-25 Am . Ac . BUFFER GRADIENT TO 1.0 M pH 7.0
GRADI ENT TO 1.0 MpH 7.0
+ =l
>!: >
arC}- VP
E
o
2000t~ ct;=
CD N
ci
o:u
o
oct (1)0:
1000
:3 ~
g::;) oE (I)
ct
•
>
50
100 . 150 N UMBER OF FRACTIONS
200
•
Fig. 2.-Chromatography on carboxymethylcellulose of the LRF·concentratc p repared by gel filtration on Sephadex G-25 .
tracts of beef hypothalamus in experiments conducted at Baylor University College of Medicine in collaboration with Dr. Sakiz and Dr. Dhariwal. Hydrolysis of a preparation of LRF of sheep origin by trypsin of pepsin'? leads to the disappearance of the biological activity; these data are in favor of a polyp eptidic structure for LRF. All these conclusions about the nature of LRF or sheep or beef origin purified by the - filtration 'on Sephadex G-25 are in agreement with those reached by McCann and his collaborators with crude acid extracts of rat hypothalamus.t" ... The LRF concentrate prepared by' filtration on Sephadex G-25 has been further purified by chromatography on carboxymethylcellulose'? (fig. 2) . The Llf-releasing activity is well concentrated in a discrete zone corresponding to substances considerably retained on the exchanger and well separated from the peak of arginine-vasopressin. This vasopressin represents the small quantities of that peptide present in the LRF zone in the effiuent of the gel filtration of Sephadex G-25 and which are concentrated by chromatography on the exchanger. The materials so purified by carboxymethylcelluIose are active in releasing LH in the ovarian ascorbic acid depletion test at doses of 20-50 ILg. injected i.v, in .the assay animals. Partition chromatography on paper, using butanol-acetic acid-water as a' solvent system, of LRF purified by CMC following Sephadex filtration has shown that we are dealing with a highly purified material with two main spots upon staining with ninhydrin. In a similar experiment recently performed in Houston in collaboration with Dr. Ward, an apparently homogeneous material was obtained. Upon acid hydrolysis with 6 N HCI, this material has yielded the following amino acids with approxi mately twice the quantity _of the basic amino acid as compared with -the others: Lys, Arg, His, Asp, Thre, Ser, Glu, Gly, Ala. We propose, of course, to repeat these preliminary experiments in the hope of isolating large enough quantities of this hypothalamic LHF to determine its complete structure. Both LRF prepared by gel filtration on Sephadex .G-25 and further purified by chromatography on CMC are able to specifically stimulate ovulation and
HYPOTIIALA~IIC
POLYPEPTIDES RELEASING PlTUITAnY nORMO:-''ES
1209
subsequent formation of corpora lutea in rats rendered acyclic by a hypothalamic lesion." Figure 1 summarizes also our results in the purification from crude hypothalamic extract of sheep origin of a fraction (TRF, for thyrotropin releasing factor) stimulating the secretion of TSII.13 Filtration by molecular sieving on Sephadex G-25 of acetic acid extracts of sheep hypothalamus yields two zones of activity in the bioassay for TRF.H The first one corresponds to large nonretained protein molecules (tubes 1-10, fig. 1). This highly' potent activity persists in the bioassay after hypophysectomy; it corresponds most likely to TSH or TSH-like substances present in the hypopthalamic extract. The second zone of activity in the bioassay corresponds to the emergence of substances with a spatial size of the order of trideca to octadecapeptides (tubes 45-55, fig. 1). This fraction is not active after hypophysectomy under conditions where the response to TSH of the hypophysectomized animal has been' shown to be similar to that of the normal animal. It does not act by potentiating TSII at the level of the thyroid. It seems therefore to act by releasing TSH from the anterior pituitary. Its activity is highly dependent upon the concentration of circulating thyroxin. Large doses of thyroxin inhibit at the pituitary level the response to TRF. The activity is not destroyed completely by trypsin but disappears after pepsin or acid hydrolysis.l? It is heat stable in contradiction to TSH. It gives a linear response in the bioassay as a function in the log of the dose injected. Similar results have recently been' obtained at Baylor College of Medicine in collaboration with Dr. Sakiz and Dr. Dhariwal on acid extracts of beef hypothalamus. As in the case of LRF, we have been able to further purify by chromatography on carboxymethylcellulose the TRF concentrate prepared by gel filtration on Sephadex G_25. 1GIn several experiments the TRF activity was strongly retained on the exchanger and appeared to be distinct from a-~ISII and arginine-vasopressin. Inconsistent results have recently been obtained regarding the purification of TRF by carboxymethylceIIulose; it may be that this exchanger is not the ideal method for further purification of TRF. In a recent series of experiments, we were able to show that THF, both the crude material following filtration on Sephadex or the material further purified on CMC, stimulates the secretion of TSH in an in vitro system in which the pituitary is incubated in Krebs-Ringer bicarbonate-glucosc.l" In vitro as in vivo the action of the THF peptide is highly dependent on the concentration of thyroxin, About the pituitary hormone-like activities found in the extract of hypothalamic tissues and concentrated during filtration on Sephadex G-25: in the non-retained fraction (fig. 1), ACTII, LII, and TSH-like activities have been observed in these acid extracts of hypothalamic tissues. It would be tempting, of course, to implicate these substances of adenohypophysial origin in some aspect of the overall feedback mechanism between adenohypophysis and hypothalamus. Before embarking upon' such a hypothesis, we would have to ascertain the exact location of these activities in the hypothalamic fragments used to prepare the extracts. Histologic studies of a few of the fragments of stalk-median eminence used in some of these studies, by Dr. J. Perr>:, at Bay-
1210
HOGER GUlLLE;\lL.'<
lor College of Medicine have shown the constant presence of large bands of tissues of pars tuberalis. Thus it is quite likely that the ACTH-, LII-, and TSH-like activities that we have observed in these extracts of hypothalamusmedian eminence-pituitary stalk fragments originate from pars tuberalis. Further studies will be necessary to see if the hypothalamic tissue proper also contains anterior lobe hormonal activities. We thave discussed elsewhere':" various hypotheses that should then be considered to explain such an observation. Finally, with availability of purified hypothalamic hypophysiotropic mediators, we have recently attempted to produce multiple stimulation of the pituitary in the same animal: immature female rats were placed on a low iodine diet and injected with p31 and thyroxin as in the assay for TRF; they were also injected with PMS and lIce as for the LRF assay; on the day of the experiment they were injected with dexamethasone-21 POt and morphine as in the CRF assay. The CRF preparation was a concentrate of neurohypophysial origin called Fraction Dl1 prepared by partition chromatography. The preparations of LRF and TRF were the concentrate prepared by filtration on Sephadex G-25. Preliminary results have shown that we can at will induce the pituitary specifically to secrete one or several of its hormones by injecting materials of hypothalamic origin. ACKNOWLEDGMENTS For the current support of our research activities we would like to acknowledge research funds from the following sources: At College de France, from the Centre National de la Recherche Scientifique (CNRS), the US PHS (grant no. A-5,S34), the Population Council, Inc., Rockefeller Institute; at Baylor University College of Mcdicim-, from the USPIIS (grant no. A-2.5·1J).
REFERENCES 1. Guillemin, R.: j. Physiol. 55:7-14, 1963. 2. - : Rec. Progress Hormone Res. 20:89130, 1964. 3. Sehally, A. V., and Gntllcnun, H.: Proc. Soc. Exper, BioI. & Med, 112: 10141017, 1963. ;. - , Lipscomb, H. S., and Cuillemin, n.: I, Endocrinology 71: 164-173, 1962. 5. Guillemin, R., and Schally, A. V.: Texas Rep. BioI. & Med. 21:541-545, 1963. I 7 8 6. Parlow, A.: Fct. Proc. 1 :402, 195 . 7. Sakiz, E., and Guillemin, R.: Eudocrinology 72:804-812, 1963. . R ., G Ul'11 emm, . R ., J u tiISZ, 'I I' ., 8 . C ourner, ki E d A II' l' .: C omp. t a "IZ, '., an sc meim, S rend. Acad. sc. 253:922-927, 1961. 9. Guillemin, R., jutisz, },I., and Sakiz, E.: Compt. rend. Acad. sc. 256:504, 1962. 10. Jutisz, xr., Dc La Llosa, .1'., Sakiz, E., Yamazaki, E., and Guillemin, R.: Compt. rend. Soc. bioI. 157:9..35-237,
1963. 11. :-'lcCann, S. :-'1.: Am. J. Physiol. 202: 39.5~100, 1962. 12. Schiavi, R., jutisz, :-'1., Saklz, E., and Guillemin, R.: Proc. Soc. Exper. BioI. & Med. 114:426-429, 1963. 13 • G Ul'11 cmm, . l'1., v-• amaza ki'1, E " J U t'lSZ, "'I ., and Sakiz, E.: Compt. rend. Acad. sc. ,,-zoo: 1018-10"0 :.., In6" uoz, 14. Yamazaki, E., Sakiz, E., and Guillemin, n.: Experientla 19:'180~183, 1963. 15. jutisz, :-'1., Yamazaki, E., Berault, A., Sakiz, E., and Guillemin, n.: Compt. rend. Acad. sc. (Paris), 256:29252927, 1963. 16. Guillemin, R., Yamazaki, E., Gard, D. A., Jutisz, :-'l., and Sakiz, E.: Endocrinology .73:564-572 1963. 17. - , Hearn, W. R., Cheek, \V. n., and Housholder, D. E.: Endocrinology 60: 488-506, 1957.
ROGElI GUILLEMIr\'
HE PUHPOSE of this paper is essentially to present the current status of our own research on the purification of hypothalamic factors involved in the secretion of ovulation hormone (LH), thyroid-stimulating hormone (TSH), and adrenocorticotropin (ACTH). Professor Godfrey Harris has reminded us in his opening remarks to this session of the rationale for our current concept of the neurohumoral nature of the hypothalamic control of adrcnohypophysinl function. This will not be further discussed here but simply will be accepted as the basis on which to justify our own investigations. My friend and former associate, Dr. Schally, has presented most of the data we obtained together on CRF in my laboratory at Baylor College of Medicine; thus I will limit myself to a few remarks about the nature of CRF of hypothalamic origin to present in somewhat greater details the results of our recent studies on LRF (LH-releasing factor) and TRF (TSH-releasing factor), I have discussed at great length in two reviews recently publishedt-" my present position regarding the problem of the nature of CRF and particularly regarding the significance of ,B-CRF, at-CHF, a;!-CHF, of neurohypophysial origin',3.4 as they were presented to us by Dr. Schally in his brilliant lecture. I personally consider that the possible physiologic significance of these substances which we have isolated from commercial extracts of posterior pituitary may well have to be re-evaluated now that we have available large quantities of hypothalamic tissues. 'Ve must now repeat our various purification and extraction procedures on extracts of hypothalamic tissues containing the median eminence, with or without the pituitary stalk, and compare the results that we will obtain with what we have reported earlier with starting materials of neurohypophysial origin. It is ~ncouraging that in a few preliminary experiments, done recently in Paris and also in collaboration with Dr. Schally when he was still in my laboratory at Baylor, we have observed the presence of CHF activity in extracts of median eminence and hypothalamus of ovine origin with partition coefficients in counter current distribution elosely related to those which we had reported earlier for a-CRF and ,B-CRF of neurohypophysial origin." Anyway, the question of the nature of hypothalamic CRF must be approached with an open mind as I think that our earlier results on neurohypophysial peptides have not necessarily answered it. I would like now to summarize our recent results on' LRF and TRF of hypothalamic origin. These studies were performed in collaboration with Dr. Sakiz, Dr. Yamazaki, Dr. Schiavi, and Dr. Jutisz, at College de France in Paris and more recently with Dr. Dhariwal and Dr. Ward in our laboratories at Baylor College of Medicine in Houston. The filtration on the gel Sephadex
T
From the Lahoratoirc de llIorphologic cxperinu.ntalc ct endocrinologic, College de France, Paris.
1206 ~[ETAlIOl.lS~I, VOL. 13,
No. 100l'AIIT 2 (O<..TOIIEII), HJ6·]
1207
I1Yl'OTHALA:o.UC POLYPEPTIDES RELEASIl"G PITUITARY nOR:o.I0:-iES
S EPH A DEX G 25
z
0 .1 II. PYRIDI N E AC.
o
IU
15 0 0
CRF TRF
....
o
z
LH
30 00
U
T SH
" 10 0 0 E
0
E
AC i H
l:I
~
~
ci
f::.... 2 000
Ul
I-
Z
Z
III
;;)
t',
500
t;
:?>-
o d
z
;;)
l:! g;
10 0 0
~
~
Ul
~
o
20
40
60
80
10 0
NUMB ER OF F RA CT IO NS
Fig. I.-Molecular sieving 011 the gel Sephadex C-2.:> of all acid extract of sheep hypothalamus. Location of various hormonal (ACTH, LH, TSII) and neurohormonal (vasopressin, oxytocin, a-MSII, ,B-},ISII, CHI', TRF, LHF) activities. The zone marked CHF contains also a considerable ACTH-Iike adrenocorticotropic activity.
G-25 of an acetic acid extract of sheep hypothalamus (fragments containing hypothalamus, median eminence and pituitary stalk) yields two zones of activity in the assay for LH-LRF. The assay used here is the ovarian ascorbic acid depletion test'':? using immature animals. One zone of activity corresponds to the non-retained material (fig. 1, tubes 1-10); this activity persists in the assay animal after hypophysectomy. Thus it corresponds to substances acting directly at the level of the ovary; this may be LH or LII-like substances present in the extract of hypothalamus-pituitary stalk tissues. The second zone of activity in the assay corresponds to substances strongly retarded on the gel (fig. 1, tubes 60-70). This material is not active in the assay animal after hypophysectomy in conditions in which we have ascertained that the sensitivity of the hypophysectomized animal to LH is adequate. Thus the activity of this material appears to be transhypophysial, i.e., its effectiveness in the LH assay requires the presence of the pituitary. \Ve concluded that this material releases endogenous LH and that it should correspond to the presumed LH-releasing factor (LRF). Figure 1 shows-also the position in the effluent of such a column of the biological activities corresponding to ,B-~ISH, a-MSII, arginine-vasopressin and oxytocin," The zone corresponding to the LII-releasing activity is distinct from that of vasopressin and oxytocin and also from that corr.esponding to fI-MSH and a-MSH. In other experiments" we were able to show that a-l\'ISH, ,B-MSII, arginine-vasopressin, lysine-vasopressin, and oxytocin' are inactive in the tests used for LII-releasing activity at doses much greater th::111 those encountered in the LHF zone from the effluent of the Sephadex G-25 filtration of hypothalamic extract. Thus we have concluded that LRF was a substance different from these peptides. Identical results to those reported here on extracts of sheep hypothalamus have recently been obtained with ex-
1208
ROGEn
GUILLEMI~
CMC FOLLOWING SEPHADEX G-25 Am . Ac . BUFFER GRADIENT TO 1.0 M pH 7.0
GRADI ENT TO 1.0 MpH 7.0
+ =l
>!: >
arC}- VP
E
o
2000t~ ct;=
CD N
ci
o:u
o
oct (1)0:
1000
:3 ~
g::;) oE (I)
ct
•
>
50
100 . 150 N UMBER OF FRACTIONS
200
•
Fig. 2.-Chromatography on carboxymethylcellulose of the LRF·concentratc p repared by gel filtration on Sephadex G-25 .
tracts of beef hypothalamus in experiments conducted at Baylor University College of Medicine in collaboration with Dr. Sakiz and Dr. Dhariwal. Hydrolysis of a preparation of LRF of sheep origin by trypsin of pepsin'? leads to the disappearance of the biological activity; these data are in favor of a polyp eptidic structure for LRF. All these conclusions about the nature of LRF or sheep or beef origin purified by the - filtration 'on Sephadex G-25 are in agreement with those reached by McCann and his collaborators with crude acid extracts of rat hypothalamus.t" ... The LRF concentrate prepared by' filtration on Sephadex G-25 has been further purified by chromatography on carboxymethylcellulose'? (fig. 2) . The Llf-releasing activity is well concentrated in a discrete zone corresponding to substances considerably retained on the exchanger and well separated from the peak of arginine-vasopressin. This vasopressin represents the small quantities of that peptide present in the LRF zone in the effiuent of the gel filtration of Sephadex G-25 and which are concentrated by chromatography on the exchanger. The materials so purified by carboxymethylcelluIose are active in releasing LH in the ovarian ascorbic acid depletion test at doses of 20-50 ILg. injected i.v, in .the assay animals. Partition chromatography on paper, using butanol-acetic acid-water as a' solvent system, of LRF purified by CMC following Sephadex filtration has shown that we are dealing with a highly purified material with two main spots upon staining with ninhydrin. In a similar experiment recently performed in Houston in collaboration with Dr. Ward, an apparently homogeneous material was obtained. Upon acid hydrolysis with 6 N HCI, this material has yielded the following amino acids with approxi mately twice the quantity _of the basic amino acid as compared with -the others: Lys, Arg, His, Asp, Thre, Ser, Glu, Gly, Ala. We propose, of course, to repeat these preliminary experiments in the hope of isolating large enough quantities of this hypothalamic LHF to determine its complete structure. Both LRF prepared by gel filtration on Sephadex .G-25 and further purified by chromatography on CMC are able to specifically stimulate ovulation and
HYPOTIIALA~IIC
POLYPEPTIDES RELEASING PlTUITAnY nORMO:-''ES
1209
subsequent formation of corpora lutea in rats rendered acyclic by a hypothalamic lesion." Figure 1 summarizes also our results in the purification from crude hypothalamic extract of sheep origin of a fraction (TRF, for thyrotropin releasing factor) stimulating the secretion of TSII.13 Filtration by molecular sieving on Sephadex G-25 of acetic acid extracts of sheep hypothalamus yields two zones of activity in the bioassay for TRF.H The first one corresponds to large nonretained protein molecules (tubes 1-10, fig. 1). This highly' potent activity persists in the bioassay after hypophysectomy; it corresponds most likely to TSH or TSH-like substances present in the hypopthalamic extract. The second zone of activity in the bioassay corresponds to the emergence of substances with a spatial size of the order of trideca to octadecapeptides (tubes 45-55, fig. 1). This fraction is not active after hypophysectomy under conditions where the response to TSH of the hypophysectomized animal has been' shown to be similar to that of the normal animal. It does not act by potentiating TSII at the level of the thyroid. It seems therefore to act by releasing TSH from the anterior pituitary. Its activity is highly dependent upon the concentration of circulating thyroxin. Large doses of thyroxin inhibit at the pituitary level the response to TRF. The activity is not destroyed completely by trypsin but disappears after pepsin or acid hydrolysis.l? It is heat stable in contradiction to TSH. It gives a linear response in the bioassay as a function in the log of the dose injected. Similar results have recently been' obtained at Baylor College of Medicine in collaboration with Dr. Sakiz and Dr. Dhariwal on acid extracts of beef hypothalamus. As in the case of LRF, we have been able to further purify by chromatography on carboxymethylcellulose the TRF concentrate prepared by gel filtration on Sephadex G_25. 1GIn several experiments the TRF activity was strongly retained on the exchanger and appeared to be distinct from a-~ISII and arginine-vasopressin. Inconsistent results have recently been obtained regarding the purification of TRF by carboxymethylceIIulose; it may be that this exchanger is not the ideal method for further purification of TRF. In a recent series of experiments, we were able to show that THF, both the crude material following filtration on Sephadex or the material further purified on CMC, stimulates the secretion of TSH in an in vitro system in which the pituitary is incubated in Krebs-Ringer bicarbonate-glucosc.l" In vitro as in vivo the action of the THF peptide is highly dependent on the concentration of thyroxin, About the pituitary hormone-like activities found in the extract of hypothalamic tissues and concentrated during filtration on Sephadex G-25: in the non-retained fraction (fig. 1), ACTII, LII, and TSH-like activities have been observed in these acid extracts of hypothalamic tissues. It would be tempting, of course, to implicate these substances of adenohypophysial origin in some aspect of the overall feedback mechanism between adenohypophysis and hypothalamus. Before embarking upon' such a hypothesis, we would have to ascertain the exact location of these activities in the hypothalamic fragments used to prepare the extracts. Histologic studies of a few of the fragments of stalk-median eminence used in some of these studies, by Dr. J. Perr>:, at Bay-
1210
HOGER GUlLLE;\lL.'<
lor College of Medicine have shown the constant presence of large bands of tissues of pars tuberalis. Thus it is quite likely that the ACTH-, LII-, and TSH-like activities that we have observed in these extracts of hypothalamusmedian eminence-pituitary stalk fragments originate from pars tuberalis. Further studies will be necessary to see if the hypothalamic tissue proper also contains anterior lobe hormonal activities. We thave discussed elsewhere':" various hypotheses that should then be considered to explain such an observation. Finally, with availability of purified hypothalamic hypophysiotropic mediators, we have recently attempted to produce multiple stimulation of the pituitary in the same animal: immature female rats were placed on a low iodine diet and injected with p31 and thyroxin as in the assay for TRF; they were also injected with PMS and lIce as for the LRF assay; on the day of the experiment they were injected with dexamethasone-21 POt and morphine as in the CRF assay. The CRF preparation was a concentrate of neurohypophysial origin called Fraction Dl1 prepared by partition chromatography. The preparations of LRF and TRF were the concentrate prepared by filtration on Sephadex G-25. Preliminary results have shown that we can at will induce the pituitary specifically to secrete one or several of its hormones by injecting materials of hypothalamic origin. ACKNOWLEDGMENTS For the current support of our research activities we would like to acknowledge research funds from the following sources: At College de France, from the Centre National de la Recherche Scientifique (CNRS), the US PHS (grant no. A-5,S34), the Population Council, Inc., Rockefeller Institute; at Baylor University College of Mcdicim-, from the USPIIS (grant no. A-2.5·1J).
REFERENCES 1. Guillemin, R.: j. Physiol. 55:7-14, 1963. 2. - : Rec. Progress Hormone Res. 20:89130, 1964. 3. Sehally, A. V., and Gntllcnun, H.: Proc. Soc. Exper, BioI. & Med, 112: 10141017, 1963. ;. - , Lipscomb, H. S., and Cuillemin, n.: I, Endocrinology 71: 164-173, 1962. 5. Guillemin, R., and Schally, A. V.: Texas Rep. BioI. & Med. 21:541-545, 1963. I 7 8 6. Parlow, A.: Fct. Proc. 1 :402, 195 . 7. Sakiz, E., and Guillemin, R.: Eudocrinology 72:804-812, 1963. . R ., G Ul'11 emm, . R ., J u tiISZ, 'I I' ., 8 . C ourner, ki E d A II' l' .: C omp. t a "IZ, '., an sc meim, S rend. Acad. sc. 253:922-927, 1961. 9. Guillemin, R., jutisz, },I., and Sakiz, E.: Compt. rend. Acad. sc. 256:504, 1962. 10. Jutisz, xr., Dc La Llosa, .1'., Sakiz, E., Yamazaki, E., and Guillemin, R.: Compt. rend. Soc. bioI. 157:9..35-237,
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