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A study of ACTH and CRF in chickens
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
A Study M. H. M. SALEM,
14, 270-280 (1970)
of ACTH
and
H. W. NORTON,
CRF in Chickens AND
A. V. NALBANDOVl
Department of Animal Science-Genetics, University of Illinois, Urbana 61801 Received July 5, 1969 Previous work had demonstrated that chicken pituitaries contained a substance which was able to cause corticosterone synthesis by adrenals in vitro. In the present study chicken pituitary glands were assayed by the adrenal ascorbic acid-depletion method in hypophysectomized rats. It was found that perfect parallelism existed between the doses of 1.5-96.0 &g/100 g body weight of chicken adenohypophyses and 0.4-10.0 mU of a standard mammalian ACTH. The data presented show that chicken pituitary glands contain relatively more ACTII than do those of rats. When assayed on hypophysectomized rats, perfect parallelism was demonstrated between extracts of 10, 20, and 40 chicken hypothalamic fragments and 0.4, 2.0, and 10.0 mU of the ACTH standard. Similar data were obtained on intact blocked rats. These results led to the conclusion that chicken hypothalami contain ACTH or an ACTH-like substance. No activity was present in extracts of chicken cerebral cortices which were used as control test substances.
chickens (Frankel et al., 1967b). This dose of dexamethasone had no effect upon the capacity of the avian adrenal to synthesize corticosterone in vitro, and this was explained on the basis that in birds (Frankel et al., 1967b) and in mammals (Corbin et al., 1965) this substance acts at the hypothalamic and/or a higher level of the CNS. Furthermore, autotransplantation of the adenohypophysis to the kidney capsule in chickens resulted in reduction of corticosterone concentration in the adrenal venous plasma to a level not significantly different from that of hypophysectomized birds (Resko et al., 1964). This suggests that the integrity of the hypophyseal portal circulation is essential for transmission of stimuli to anterior pituitary cells, presumably by means of a humoral substance(s) that is carried from the median emience (ME) to the adenohypophysis. The nature of this substance has remained unknown, but it has been shown by neurohypophysectomy (Shirley et al., 1956) and by hypothalamic lesions (Frankel et al., 1967a) or hormone injec-
Little is known about neuroendocrine control mechanisms in birds and certainly much less than is known in mammals. The possible hypothalamic control of anterior pituitary secretion of adrenocorticotropic hormone (ACTH), which is an accepted fact in mammals (see, for instance, Mangili et aZ., 1966), is largely uninvestigated in birds. Peczely and Zboray (1967) suggest that a corticotropin-releasing factor (CRF) is present in the hypothalamus of birds, and indirect evidence suggests that the central nervous system (CNS) of birds regulates ACTH secretion in a manner similar to that of mammals. Hypothalamic lesions caused a significant reduction in corticosterone output in adrenal vein plasma of intact chickens (Frankel et al., 1967a). A small dose of dexamethasone phosphate (100 ,pg/kg body weight) was effective in causing complete inhibition of adrenal corticosterone output 66 hr after being injected into intact ‘Partial support acknowledged.
by NIH
Grant
HD 3043 is 270
A
STUDY
OF
ACTH
AND
tion into intact chickens (Frankel et al., 1967b) to be different from arginine vasotocin, the major neurohypophyseal hormone of the bird (Munsick, 1964). Pharmacological doses of vasopressin, however, cause a release of ACTH (Resko et al., 1964) comparable to that seen in mammals (see, for instance, McCann et al., 1959). There was a need to confirm earlier equivocal reports of Resko et al. (1964) and of De Roos et al. (1964) which suggested the presence of ACTH in the chicken anterior pituitary. It was felt that this is an essential step, since there is no reason for an animal to have a “releasing factor” for a hormone which does not exist! Furthermore, bird adrenals are unique in retaining partial capacity for steroidogenesis after the anterior pituitary gland has been removed (Frankel et al., 1967a,b; Resko et al., 1964; Nagra et al., 1963). On this basis some investigators (for instance, Brown et al., :1958; Newcomer, 1959) suggested that the avian adrenal gland may function independently from hormonal regulation. This concept of avian adrenal “autonomy” is not in complete agreement with concepts requiring the need for a releasing factor for ACTH in chickens. More ,. recent observations from this laboratory (Frankel et al., 1967a,b; Resko et al., 1964; Ma et al., 1963) suggested the possibility of the existence of an extrahypophyseal source of ACTH-like substance(s) from the hypothalamus and/or higher level of the CNS which supports adrenal function of ! the hypophysectomized birds. The present paper is concerned with the study of ACTH activity in chicken pituitary and chicken hypothalamic extracts.
t
MATERIALS AND METHODS of tissues.Hypothalami were collected from White Leghorn laying hens at a chicken slaughterhouse. Within 20 min after death, the top of the skull was chopped above the eyes, the brain was lifted out exposing the hypothalamus. The hypothalamus was dissected free from brain tissues with a curved scissors and quickly frozen in small vials on dry ice. The vials were kept on dry ice until they were transferred to the laboratory where they were stored at -20”. Small Collection
CRF
IN
CHICKENS
271
pieces of brain cortices were collected and similarly treated. Chicken pituitaries were collected from laying hens reared in the laboratory. The gland ~89 carefully removed within 2 min after death. The posterior lobe was discarded, then the two portions of the anterior lobe were frozen and stored. Blood was taken from White Leghorn adenohypophysectomized cockerels, weighing between 1 and 1.4 kg by cardiac puncture using a heparinized syringe. The blood was centrifuged at 5” for 20 min at 1700s and the plasma was decanted, frozen, and stored at -20” until used. Preparation of tissues for assay. On the morning of an assay, the hypothalami and brain cortices to be tested were homogenized in 0.1 N HCl in a ground-glass homogenizer. The homogenate was centrifuged at 5” for 1 hr at 15,00&, and the pH of the supernatant fluid was adjusted to pH 4.0 + 0.2 with a concentrated solution of NaHCOa. The adjusted supernatant fluid was diluted to the desired concentration (number of hypothalami per milliliter, or an equivalent amount, by weight, of brain cortex) and volume with the control injection solution. The control injection solution was made up from 0.1 N HCl which was adjusted in the same manner to pH 4.0. Chicken pituitary glands were homogenized directly in the control injection solution. These homogenates were neither centrifuged nor was the pH adjusted eince the desired concentrations (ag/ml) in this case were very small. Bioassays Assay for ACTH or ACTH-like activity. The adrenal ascorbic acid depletion (AAAD) method was used for assay of ACTH, the ascorbic acid content of the adrenals of hypophysectomized rate 18-24 hr post-hypophysectomy being taken as an index for ACTH or ACTH-like activity. The method of Sayers as modified by Munson et al. (1948) was used except that the two adrenals were analyzed separately for their ascorbic acid content, and the mean specific activity (SA, pg ascorbic acid/100 mg) of the two adrenals was taken as an index of response to a certain treatment. Sprague-Dawley female rats, hypophysectomized by the parapharyngeal approach were purchased from the Hormone Assay Laboratories, Chicago, Illinois, and the completeness of hypophysectomy was confirmed at the end of the assay by direct visual inspection of the pituitary sac. The weights of the rats, the range of the weights in each experiment, and the numbers as well as the random assignment of each rat to a certain treatment group (control, standards, and
272
SALEM,
NORTON,
unknowns) were performed according to the general principles of ACTH assay (Fisher, 1962). In accord with the method used, the homogenate of each adrenal was filtered using a double acid-washed filter paper (Munktell, type OK). Five milliliters from the clear colorless filtrate was obtained and its ascorbic acid content was determined by the spectrophotometric method described by Mindlin and Butler (1937). Most experiments were evaluated statistically by the method of least squares using the SSUPAC multiple correlation program, or by analysis of variance using the IBM digital computer. Assay for ACTH-releasing activity. This method is basically the same as that described above for assay of ACTH activity. Rats were injected intraperitoneally with dexamethasone 21phosphate in a dose of 0.5 mg/lOOg body weight (Kastin et al., 1964). Two to 4 hr later, rats were anesthetized with ip injection of sodium pentobarbital (Nembutal) 44.5 mg/lOOg body weight. About 10 min after the pentobarbital injection the anesthetized rats were injected with the standard or the unknown solutions. One hour later, both adrenals were removed and treated the same as for AC’I’H assay. A complete correlation has been reported between the response to hypothalamic extracts, (or vasopressin), as measured by AAAD and the response as measured by the rise in plasma corticosterone concentrations in the rat with ME lesions (Dhariwal et al., 1966) or in morphineNembuta-blocked rats (Leeman et al., 1962). Hormones used. The adrenocorticotropin (ACTH) used was a purified mammalian-porcine preparation (Armour, Acthar, lot 878212). Vials used contain 40 USP units (IU) of sterile powdered lyophilized ACTH and about 9 mg of hydrolyzed gelatin. The hormone was dissolved in the control injection solution (pH 4.0) and made up in a concentration of 3 IU/ml. This stock solution was stored frozen in small vials, and vials, once thawed, were discarded. Dexamethasone al-phosphate (Decadron Phosphate, Merck) was diluted with physiological saline in a concentration of 0.5 mg/ml, and kept refrigerated. EXPERIMENTS
ACTH
AND
RESULTS
Activity
ACTH activity in chicken adenohypophy&s. Five dose levels of chicken adenohypophysis were assayed for ACTH activity in hypophysectomized rats. Since prelimi-
AND
NALBANDOV
nary experiments had shown a great variation in its ACTH concentration (mp/mg), two extra doses of pituitary were used to establish the range within which linear dose response will occur. The activity of the two highest doses (P, and P4) did not differ significantly, therefore the P, dose was eliminated from further analysis. The specific activities of adrenals of the rats given the lower three doses (P, to P,) as well as of those given the higher three doses (PZ to P,) were fitted (together with the three groups of standard) into a sixpoint multiple correlation program. In both cases, linear and parallel dose-response curves for standard and unknown were obtained (Figs. 1 and 2). The pooled slope in both cases was significantly different from zero (p < 901). The dose range of 1.5 to 24 pg of chicken adenohypophysis was used for estimation of its relative potency since the response to these doses did not differ significantly from response to ACTH standard. ACTH activity in chicken adenohypophysis was equivalent to 401.7 (21.22)’ mU per milligram of frozen tissue, with a confidence limit of 270.0-597.7. It is much higher than the potency estimate of ACTH in chicken adenohypophysis reported by De Roos and De Roos (1964)) which was between 65 and 100 mU of mammalian ACTH per mg of fresh tissue. This difference in ACTH concentration could be explained by differences in experimental conditions. In addition to the fact that ACTH concentration in present experiment was reported on the basis of frozen weight there were differences in assay method as well as in the preparation of tissues for assay. Furthermore, there were age and sex differences between the birds used as a source of pituitary tissues. The present experiment, however, demonstrated that chicken anterior pituitaries contain a high concentration of ACTH activity when assayed by AAAD. In addition to this high concentration, the response of the hypophysectomized rats was proportional to the dose of chicken anterior pitui‘Standard
error
expressed
as a fraction.
A
STUDY
OF
ACTH
AND
CRF
IN
273
CHICKENS
DOSE RESPONSE TO CHICKEN PITUITARY TISSUE AS ASSAYED BY AAAD OF HYPOPHYSECTOMIZED ‘RATS
moJ ACTH STAHDARlUdO.4
I
PlTlJlTAFtY TISSUE f&P)
1
2.0 6.0
1.S
10.0
24.0
DOSE
Dose response obtained from 1.5-24.0 pg of chicken pituitary tissue assayed for ascorbic acid depletion in hypophysectomised rate. ACTH standard not significantly different from unknown. Number of animals per point 6 to 8. Vertical lines in this and subsequent figures: standard errors of the mean. FIG.
i.
DOSE RESPONSE TO CHICKEN PITUITARY TISSUE AS ASSAYED BY AAAD OF HYPOPHYSECTOMIZED RATS
450
1Control
400
200
L ACTH STANOAA: PITUITARY TISSUE
ml)
64
1
24.0
6.0
66.0
DOSE
Fm. 2. Dose response obtained from 6.0-26.0 pg of chicken pituitary tissue assayed for ascorbic acid depletion in hypophysectomized rats. Number of animals per point 6 to 8. ACTH standard vs unknown. p < .Ol.
274
SALEM,
NOKTOX,
tary. These observations suggest that the activity of the chicken anterior pituitary may be due to specific hormonal activityavian ACTS. ACTH-like
activity
in
chicken
hypo-
thalamic and brain cortex extracts. Preliminary experiments were conducted to determine whether crude acid extracts of chicken hypothalamus and brain cortex were active in depleting the AAA of the hypophysectomized rats. Doses below 10 hypothalamic extracts (HE) per 100 g body weight, and an equal amount by weight of brain cortex were without significant effect. On the basis of these observations, extracts of chicken hypothalami were made up in concentrations of 10, 20, and 40 HE per milliliter and were assayed in the hypophysectomized rats. Results show that the depletion caused by standard ACTH and HE fell on linear and parallel dose-response curves (Fig. 3) and the pooled slope of the two curves was significantly different from zero (p < .OOl). Relative potency was equivalent to 1.40 (21.195) mU of standard ACTH per 10 HE with a confidence limit of 0.980 to 1.999.
I
DOSE
RESPONSE T
AND
KALBANDOV
In contrast to hypothalamic extracts, chicken brain cortex extracts in a dose up to the equivalent of 30 HE has no detectable ACTH or ACTH-like activity (Table 1). However, the possibility that some acTABLE EFFECTS ON
1
OF CHICKEN BRAIN CORTEX AAAD IN HYPOPHYSECTOMIZED
Treatment Control (C) Hypothalamus Cortex X1
EXTRACTS RATS
Dose/100 g body weight
Mean SA + SE
1 ml 30
422.18 f 12.10 (7) 259.16 f 32.00 (6)0
6~ 30
410.38 414.61
Hz
X2
+. 23.97
(5)b
If: 10.89 (5)
a Hypothalamus vs control: p < .005. * Cortex vs control: NS. c Equivalent (by weight) to the hypothalamus.
tivity could be detected by using 5t more sensitive assay is not ruled out. These experiments demonstrated the presence of a substance(s) in the hypothalamus, but not in the brain cortex, of chickens which is active in the AAAD assay. This activity was proportional to the dose within the range of 1040 HE.
TO CHICKEN HYPOTHALAMIC AAAD OF HYPOPHYSECTOMIZED
EXTRACTS RATS
AS ASSAYED
BY
4oo- P.Control
250-
225
ACTH STANDARD HE
I 2.0
(mu) D:4 IO
DOSE
20
I 10.0
40
FIG. 3. Dose response obtained from extracts of 1040 chicken hypothalami assayed for ascorbic acid depletion in hypophysectomieed rats. Number of animals per point 6 to 9. ACTH standard not significantly different from unknown. p of pooled slope < .OOl.
A
ACTH-Releasing
STUDY
OF
ACTH
AND
Activity
Assay in intact-blocked rats. Results presented in Fig. 4 show that there was a linear and parallel relationship between the
CRF
IN
tivity when given in doses equivalent and 10 HE (Table 2).
different
OF CHICKEN BRAIN CORTEX EXTRACTS AS ASSAYED BY AAAD IN THE INTACT-BLOCKED RATS
Dose/100 g body Mean SA + SE weight
from zero (p < .OOl) .
Potency estimates for the HE was equivalent to 5.12 (51.19) mU of standard ACTH per 10 HE, with a confidence limit of 3.616-7.250. The response of rats to the HE should be due to a direct effect on the adrenals by these extracts plus a possible release of endogenous ACTH. The direct effect of HE on the adrenals of hypophy-
sectomized
rats was shown earlier to be equivalent to 1.40 2 1.195 mU of standard ACTH per 10 HE and represents about 27% of the total estimated potency in the intact-blocked rats. Thus, in intact-blocked rats, more than 70% of the potency estimate of the HE may be due to the endogenous release of ACTH. In contrast to HE, extracts of chicken brain cortex had no ACTH-releasing ac-
DOSE 400.
to 5
TABLE 2 EFFECT
log of the dose of both standard and unknown of 2.5,5, or 10 HE. The pooled slope of the responses to both treatments was significantly
275
CHICKENS
Treatment
Control (C) ACTH Standard S, SZ Cortex X1 X*
1 ml 1.5 mU 6.0 mU 5.0 10.0
387.07 310.40 249.66 404.66
f + + +
17.21 15.13 16.18 16.18
(8) (8) (7) (7)
394.13
f
17.47
(6)
Pooled standard vs control: p < .005. Pooled cortex vs control: NS. Assay in both hypophysectomized and intact-blocked rats. The previous conclu-
sion that the higher potency estimates of the HE in the intact rats is due only to the release of endogenous ACTH and not due to any differences
in the experimental
ditions, was tested further. Intact rats were kept in individual
RESPONSE TO CHICKEN HYPOTHALAMIC BY AAAD OF INTACT-DEXAMETHASONE-BLOCKED
EXTRACTS
con-
cages
AS ASSAYED RATS
Control
P
250-
200’ ACTH STANDARDhad HE
D.4
20 6.0
1 2.6
I IOD
10.0
DOSE
FIQ. 4. Dose response obtained from extracts of 2.5-10 chicken hypothalami assayed for ascorbic acid depletion in intact, dexamethansone-blocked rats. Number of animals per point 5 to 8. ACTH standard not signscantly diierent from unknown. p of pooled slope < .OOl.
276
SALEM,
NORTON,
AND
under controlled temperature for 48 hr before the experiment. One-half of the rats was assigned at random to bc hypophysectomized by the transauricular approach (Falconi et OZ., 1964), 16-24 hr before the assay. Five different treatments were assigned at random to the hypophysectomized as well as to the intact rats. These treatments, which were similar in both types of animals, included two doses of standard ACTH, two doses of HE, and the control. For every two similar groups in the two types of animals, one solution was prepared and was injected with the same syringe. All rats (including the hypophysectomized rats) were injected ip with dexamethasone 24 hr prior to being anesthetized with Nembutal. Two experiments of this type were conducted and the results of the two assays were pooled after appropriate statistical analysis had revealed no significant differences between the two experiments. Results based on specific activity (SA) are presented in Table 3. Linear and parallel doseresponse curves for standard and unknown were obtained (Figs. 5 and 6). The pooled slope in both cases was significantly different from zero (p < .OOl). In addition, there were significant differences b’etween hypophysectomized and intact rats in response to ACTH standard and HE (Table 3). However, AAA response to standard and unknown in these experiments is more easily discussed on the basis
of percentage of depletion (Fig. 7). This eliminates differences in mean specific activities of controls between the two types of animals (Table 3). In intact rats, there is greater response to equal doses of chicken HE than in hypophysectomized rats (Fig. 6). This higher response of intact rats is not due to their higher sensitivity, since hypophysectomized rats respond even more (though not significantly), to equal doses of ACTH standard than do intact rats. Therefore, the greater response of the intact-blocked rats to chicken HE is attributed to the ability of these extracts to cause endogenous release of ACTH in these animals. Estimation of relative potencies of HE in both types of animals was conducted by plotting the middle value (of SA or percentage of depletion) on the appropriate standard curve (Fig. 7). Potency estimates of HE in the hypophysectomized rats was 33.7% (on the basis of SA) and 34.2% (on the basis of percentage of depletion) of that in the intact-blocked rats. Thus, the ACTH-like activity of chicken HE represents about 34%. In this case, 66% of the relative potency in the intact-blocked rats is due to the endogenous release of ACTH, which agrees with results obtained earlier. Activity in Plasma of Hypophysectomized Chickens Blood was collected by cardiac puncture at several intervals (up to 9 weeks) from
TABLE ACTIVITY
OF CHICKEN
HYPOTHALAMIC
KALBANDOV
3
EXTRACTS
ASSAYED
INTACT-BLOCKED
Specific Treatment Control ACTH 1.5 mU 6.0 mU Hypothalamus 5 Hypothalamic 10 Hypothalamic Control Intact
Intact
extract extract
vs treated vs hypophysectomized
p < .OOl. p < .OOl.
IN
HYPOPHYSECTOMIZED
OR
RATS activity
(Mean
k SE) Hypophysectjomieed
363.2
f
13.9
(13)
409.3
I!I
8.4
(12)
274.6 226.3
5 13.6 + 13.6
(11) (11)
313.7 236.1
+ 13.6 + 13.0
(11) (12)
259.1 211.4
f 13.6 & 18.4
(11) (6)
345.3 291.0
f 13.6 I!z 15.9
(11) (8)
A
400-1
STUDY
$
OF
ACTH
AND
CRF
IN
277
CHICKENS
ACTIVITY OF CH!CKEN HYPOTHALAMIC EXTRACTS AS ASSAYED BOTH HYPOPHSECTOMIZED AND INTACT- BLOCKED RATS [cmtro~ [ 1 , _- - - Hypophywtcmlr~d InlPct
350-
200
150
ACTH STANDM&
i.5
HE
FIG.
ascorbic
5. Comparison acid depletion
I 5.0
of dose-response lines in hypophysectomized
obtained from and in intact,
completely adenhypophysectomized cockerels. Plasmas were stored at -20” until used. Activity of plasma collected at 1, 2, 3, 5, 7, and 9 weeks after hypophysectomy in a dose of 3 ml (per 100 g body weight) ACTIVITY I
dJ3 IO.0 DOSE
extracts of chicken dexamethansone-blocked
hypothalami assayed rats. See text.
of intact-blocked rats, was not significantly different from saline. On the other hand, the response to standard ACTH was highly significant (p < ,005). Since negative results were obtained
OF CHICKEN HYPOTHALAMIC HYPOPHYSECTOMIZED AND
EXTRACTS INTACT-BLOCKED
AS ASSAYED RATS
IN BOTH
50 1
HypophYSWOttd~d -Intact IO
,
I
ACTH STANOfQ$l.5 Ill
FIG
tomiaed
6. Percentage of depletion and dexamethansoneblocked
of ascorbic rats.
for
I
acid by extracts See text.
of chicken
5.0
hypothalami
assayed
in hypophysec-
278
SALEM,
A-ORTON,
AND
RELATIVE POTENCY OF CHICKEN IN BOTH HYPOPHYSECTOMlZED
T
NALBANDOV
HYPOTHALAMIC EXTRACTS AS ASSAYED AND INTACT-BLOCKED RATS
\\ h. PII P\ \ \ SI.
\\
\
\
\
\\ .H
\
\
‘.6 1
\ IOJ , , 123I.5
3.60
6.0
FIG.
7. Relative pot,ency rats. See text.
of extracts
of chicken
1.3
hypothalami
when plasma was assayed in the intactblocked rats, it was concluded that plasma of adenohypophysectomized chickens has no ACTH-like or ACTH-releasing activity that can be detected by the AAAD method. Reports that CRF can passinto the general circulation of hypophysectomized mammals (Brodish et al., 1962; Eik-Nes et al., 1958)) or of hypophysectomized rats with pituitary transplants (Kendall et al., 1966a,b) are not confirmed by results obtained in birds. It is felt that the main reason for the failure to detect both hormones in the plasma of adenohypophysectomized chickens, is that the AAAD method may not be sensitive enough to detect these active substances in the blood. Plasma of hypophysectomized sheep was also without ACTHreleasing activity by the use of this assay. DISCUSSION
Avian ACTH. Direct evidence for the presence of ACTH in the avian pituitary is poorly documented in the literature. The only reports on the existence of avian ACTH was based on the ability of bird adenohypophyseal extracts to promote
380
w
DOSE (mu ACTH STANDARD)
blocked
I 1
6.0
DOSE (mu ACTH STANDARD)
when
assayed
in intact,
dexamethasone-
steroidogenesis by chicken adrenal tissue in vitro. The present study confirms these earlier reports and provides evidence for possible biological and structural similarities between avian and mammalian ACTH. We have shown that chicken pituitary ACTH is active in mammals, and that it is similar to mammalian ACTH in depleting AAA of the rat, in its response to boiling, and to chemicals (Salem et al., 1969). There was a disparity between our results and those reported by De Roos and De Roos (1964) in the concentration of ACTH in the anterior pituitary of chickens. However, greater differences have been reported in the anterior pituitary of the normal rat where the reported range is 12.0-76.2 mU of ACTH/mg of fresh tissue (Marks et al., 1963; Fortier et al., 1964). Differences in experimental conditions and in the physiological status of the donor animals may be reflected in the great differences in potency estimates of pituitary ACTH in the same species. Nevertheless, it appears that the concentration of ACTH is much higher in chicken anterior pituitary than in the rat anterior pituitary. Activity in chicken hypothalamus. Pres-
A STUDY
OF
ACTH
AND
ent results also demonstrated two kinds of activities in the hypothalamus (but not in the brain cortex) of chickens: one an ACTH-like activity, and the other an ACTH-releasing activity. The ACTH-like activity contributes between 27-34s of the total activity (ACTH-like activity plus ACTH-releasing activity) of the chicken HE, when assayed in hypophysectomized and intact-blocked rats respectively. It is interesting that corticosterone output in the adrenal vein plasma of the adenohypophysectomized chickens was shown to be about 37% of that of intact controls (Frankel et al., 1967b). These authors also showed that injection with dexamethasone phosphate eliminates corticosterone from adrenal vein plasma of both groups of chickens suggesting that the steroidogenic substances are of a hypothalamic and/or of a higher origin in the CNS. Present results, however, support the hypothalamus as the origin of these substances. Further experiments were conducted to differentiate between the two activities in the hypothalamus and between them and ACTH activity in chicken pituitary and will be the subject of another paper (Salem et al., 1969). Results have shown that each activity is due to different substances of hypothalamic origin and that neither is due to vasotocin or to ACTH. REFERENCES BRODISH, A., AND LONQ, C. N. H. (1962). ACTHreleasing hypothalamic neurohumor in peripheral blood. Endocrinology 71, 298-306. BROWN, K. I., BROWN, D. J., AND MEYER, R. K. (1958). Effect of surgical trauma, ACTH and adrenal cortical hormones on electrolytes, water balance and gluconeogenesis in male chickens. Am. J. Physiol. 192, 43-50. C~RBIN, A., MANGILI, G.. MOTTA, M., AND MARTINI, L. (1965). Effect of hypothaIamic and mesencephalic steroid implantations on ACTH feedback mechanisms. Endocrinology 76, 811818. DE ROOS, R. (1963). The physiology of the avian interrenal gland: A review. In Proc. Intern. Ornithological Congr. 13th (C. G. Sibley, ed.), Vol. 2, pp. 1041-1058. DE ROOS, R., AND DE Roos, C. C. (1964). Effects of mammalian corticotropin and chicken adeno-
CRF
IN
CHICKENS
279
hypophysial extracts on steroidogenesis by chicken adrenal tissue in vitro. Gen. Camp. Endocrinol. 4, 602-607. DHAFUWAL, A. P. S., ANTUMES-RJJDRIGUES, J., RISER, F., CHO~I~RS, I., AND MCCANN, S. M. (1966). Purification of hypothalamic corticotrophin-releasing factor (CRF) of ovine or&in. Proc. Sot. Exptl. Biol. Med. 121, 8-12. EIK-NES, K. B., AND BRIZZEE, K. R. (1958). Some aspects of corticotrophin secretion in the trained dog. Acta Endocrinol. 29, 219-223. FALCONI, G., AND ROSSI, G. L. (1964). Transauricular hypophysectomy in rats and mice. Endocrinology 74, 301-303. FISHER, J. D. (1962). Adrenocorticotropin. In “Methods in Hormone Research” (R. I. Dorfman, ed.), Vol. 2, pp. 641669. Academic Press, New York. FORTIER, C., AND DE GROOT, Z. (1964). Residual synthesis and release of ACTH following electrolytic destruction of the median eminence in the rat. In “Major Problems in Neuroendocrinology” (E. Bajusz and G. Jasmin, eds.), pp. 203-219. Karger, Basel. FRANKEL, A. I., GRABER, J. W., AND NALBANDOV, A. V. (1967a). The effect of hypothalamic lesions on adrenal function in intact and adenohypophysectomized cockerels. Gen. Comp. Endocrind. 8, 387-396. FRANKEL, A. I., GRABER, J. W., AND NALBANDOV, A. V. (1967b). Adrenal function in cockerels. Endocrinology 80, 1013-1019. KASTIN, A. J., AND Ross, G. T. (1964). Melanocyte-stimulating hormone (MSH) and ACTH activities of pituitary homografts in albino rats. Endocrinology 75, 187-191. KENDALL, J. W., AND ALLEN, C. (1966). Braindependent ACTH secretion from multiple heterotopic pituitaries. Proc. Sot. Exptl. Biol. Med. 122, 335-337. KENDALL, J. W., STOTT, A. K., ALLEN, C., AND GREER, M. A. (1966). Evidence for ACTH secretion and ACTH suppressibility in hypophysectomized rats with multiple heterotopic pituitaries. Endocrinology 78, 533-537. LEFIMAN, S. E., G~ENISTER, D. W., AND YATES, F. E. (1962). Characterization of a calf hypothalamic extract with adrenocorticotropin-releasing properties. Endocrinology 70, 249-262. MA, R. C. S, AND NA~ANDOV, A. V. (1963). The transplanted hypophysis: discussion. In “Advances in Neuroendocrinology” (A. V. Nalbandov, ed.), pp. 306311. Univ. of Illinois Press, Urbana, Illinois. MANGILI, G., MOTTA, M., AND MARTINI, L. (1966). Control of adrenocorticotropic hormone secretion. In “Neuroendocrinology” (L. Martini and
280
SALEM,
KORTON,
W. F. Ganong. eds.), Vol. 1. pp. 297-370. Academic Press, New York. MARKS, B. H., AND VERNIKOS-DANELLIS, J. (1963). Effect of acute stress on the pituitary gland: action of ethionine on stress-induced ACTH release. Endocrinology 72, 582-587. MCCANN, S. M.? AND HEBERLAND, P. (1959). Relative abundance of vasopressin and corticotrophin-releasing factor in neurohypophysial extracts. Proc. Sot. Exptl. Biol. Med. 102, 319325. MINDLIN, R. L., AND BUTLER, A. M. (1937). The determination of ascorbic acid in plasma: a macromethod and micromethod. J. Biol. Chem. 122, 673-686. MUNSICK, R. A. (1964). Neurohypophysial hormones of chickens and turkeys. Endocrinology 75, 104-l 12. MUNSON, P. L., BARRY, A. G., AND KOCH, F. C., cited by SAYERS, M., SAYERS, A. G., AND WOODBURY, L. A. (1948). The assay of adenocorticotrophic hormone by the adrenal ascorbic aciddepletion method. Endocrinology 42, 379-393. NAGRA, C. L., BIRNIL G. G., BAUM, G. J., AND
AND
NALBANDOV
MEYER. 11. h. (1963). The role of the pituitary in regulating steroid secretion by the avian adrenal. Gen. Camp. Endocrinol. 3, 274-280. NEIITOMER, W. S. (1959). Effects of hypophysectomy on some functional aspects of the adrenal gland of the chicken. Endocrinology 65, 133-135. P&ZELY. P.. .~XD ZBORAY, G. (1967). CRF and ACTH activit,y in the median eminence of the pigeon. Acta Physiol. Acnd. Scient. Hung. 32, 229-239. RESKO. J. A., NORTON, H. W., AND NALB.QNDOV, A. V. (1964). Endocrine control of the adrenal in chickens. Endocrinology 75, 192-200. SALEM, M. H. M., NORTON, H. W., AND NALBANDOV, A. V. (1969). The role of vasotocin and of CRJ? in ACTH-release in the chicken. Gen. Camp. Endocrinol. (submitted). SHAPIRO, S.. MARMORSTON, J., AND NOBEL, H. (1958). Mobilization of the antidiuretic hormone and the secrrtion of ACTH following cold stress. Endocrinology 62, 278-282. SHIRLEP, H. V.. .~ND NALBANDOV, A. V. (1956). Effects of neurohypophysectomy in dome&c chickens. Endocn‘nology 58, 477483.
AND
COMPARATIVE
ENDOCRINOLOGY
A Study M. H. M. SALEM,
14, 270-280 (1970)
of ACTH
and
H. W. NORTON,
CRF in Chickens AND
A. V. NALBANDOVl
Department of Animal Science-Genetics, University of Illinois, Urbana 61801 Received July 5, 1969 Previous work had demonstrated that chicken pituitaries contained a substance which was able to cause corticosterone synthesis by adrenals in vitro. In the present study chicken pituitary glands were assayed by the adrenal ascorbic acid-depletion method in hypophysectomized rats. It was found that perfect parallelism existed between the doses of 1.5-96.0 &g/100 g body weight of chicken adenohypophyses and 0.4-10.0 mU of a standard mammalian ACTH. The data presented show that chicken pituitary glands contain relatively more ACTII than do those of rats. When assayed on hypophysectomized rats, perfect parallelism was demonstrated between extracts of 10, 20, and 40 chicken hypothalamic fragments and 0.4, 2.0, and 10.0 mU of the ACTH standard. Similar data were obtained on intact blocked rats. These results led to the conclusion that chicken hypothalami contain ACTH or an ACTH-like substance. No activity was present in extracts of chicken cerebral cortices which were used as control test substances.
chickens (Frankel et al., 1967b). This dose of dexamethasone had no effect upon the capacity of the avian adrenal to synthesize corticosterone in vitro, and this was explained on the basis that in birds (Frankel et al., 1967b) and in mammals (Corbin et al., 1965) this substance acts at the hypothalamic and/or a higher level of the CNS. Furthermore, autotransplantation of the adenohypophysis to the kidney capsule in chickens resulted in reduction of corticosterone concentration in the adrenal venous plasma to a level not significantly different from that of hypophysectomized birds (Resko et al., 1964). This suggests that the integrity of the hypophyseal portal circulation is essential for transmission of stimuli to anterior pituitary cells, presumably by means of a humoral substance(s) that is carried from the median emience (ME) to the adenohypophysis. The nature of this substance has remained unknown, but it has been shown by neurohypophysectomy (Shirley et al., 1956) and by hypothalamic lesions (Frankel et al., 1967a) or hormone injec-
Little is known about neuroendocrine control mechanisms in birds and certainly much less than is known in mammals. The possible hypothalamic control of anterior pituitary secretion of adrenocorticotropic hormone (ACTH), which is an accepted fact in mammals (see, for instance, Mangili et aZ., 1966), is largely uninvestigated in birds. Peczely and Zboray (1967) suggest that a corticotropin-releasing factor (CRF) is present in the hypothalamus of birds, and indirect evidence suggests that the central nervous system (CNS) of birds regulates ACTH secretion in a manner similar to that of mammals. Hypothalamic lesions caused a significant reduction in corticosterone output in adrenal vein plasma of intact chickens (Frankel et al., 1967a). A small dose of dexamethasone phosphate (100 ,pg/kg body weight) was effective in causing complete inhibition of adrenal corticosterone output 66 hr after being injected into intact ‘Partial support acknowledged.
by NIH
Grant
HD 3043 is 270
A
STUDY
OF
ACTH
AND
tion into intact chickens (Frankel et al., 1967b) to be different from arginine vasotocin, the major neurohypophyseal hormone of the bird (Munsick, 1964). Pharmacological doses of vasopressin, however, cause a release of ACTH (Resko et al., 1964) comparable to that seen in mammals (see, for instance, McCann et al., 1959). There was a need to confirm earlier equivocal reports of Resko et al. (1964) and of De Roos et al. (1964) which suggested the presence of ACTH in the chicken anterior pituitary. It was felt that this is an essential step, since there is no reason for an animal to have a “releasing factor” for a hormone which does not exist! Furthermore, bird adrenals are unique in retaining partial capacity for steroidogenesis after the anterior pituitary gland has been removed (Frankel et al., 1967a,b; Resko et al., 1964; Nagra et al., 1963). On this basis some investigators (for instance, Brown et al., :1958; Newcomer, 1959) suggested that the avian adrenal gland may function independently from hormonal regulation. This concept of avian adrenal “autonomy” is not in complete agreement with concepts requiring the need for a releasing factor for ACTH in chickens. More ,. recent observations from this laboratory (Frankel et al., 1967a,b; Resko et al., 1964; Ma et al., 1963) suggested the possibility of the existence of an extrahypophyseal source of ACTH-like substance(s) from the hypothalamus and/or higher level of the CNS which supports adrenal function of ! the hypophysectomized birds. The present paper is concerned with the study of ACTH activity in chicken pituitary and chicken hypothalamic extracts.
t
MATERIALS AND METHODS of tissues.Hypothalami were collected from White Leghorn laying hens at a chicken slaughterhouse. Within 20 min after death, the top of the skull was chopped above the eyes, the brain was lifted out exposing the hypothalamus. The hypothalamus was dissected free from brain tissues with a curved scissors and quickly frozen in small vials on dry ice. The vials were kept on dry ice until they were transferred to the laboratory where they were stored at -20”. Small Collection
CRF
IN
CHICKENS
271
pieces of brain cortices were collected and similarly treated. Chicken pituitaries were collected from laying hens reared in the laboratory. The gland ~89 carefully removed within 2 min after death. The posterior lobe was discarded, then the two portions of the anterior lobe were frozen and stored. Blood was taken from White Leghorn adenohypophysectomized cockerels, weighing between 1 and 1.4 kg by cardiac puncture using a heparinized syringe. The blood was centrifuged at 5” for 20 min at 1700s and the plasma was decanted, frozen, and stored at -20” until used. Preparation of tissues for assay. On the morning of an assay, the hypothalami and brain cortices to be tested were homogenized in 0.1 N HCl in a ground-glass homogenizer. The homogenate was centrifuged at 5” for 1 hr at 15,00&, and the pH of the supernatant fluid was adjusted to pH 4.0 + 0.2 with a concentrated solution of NaHCOa. The adjusted supernatant fluid was diluted to the desired concentration (number of hypothalami per milliliter, or an equivalent amount, by weight, of brain cortex) and volume with the control injection solution. The control injection solution was made up from 0.1 N HCl which was adjusted in the same manner to pH 4.0. Chicken pituitary glands were homogenized directly in the control injection solution. These homogenates were neither centrifuged nor was the pH adjusted eince the desired concentrations (ag/ml) in this case were very small. Bioassays Assay for ACTH or ACTH-like activity. The adrenal ascorbic acid depletion (AAAD) method was used for assay of ACTH, the ascorbic acid content of the adrenals of hypophysectomized rate 18-24 hr post-hypophysectomy being taken as an index for ACTH or ACTH-like activity. The method of Sayers as modified by Munson et al. (1948) was used except that the two adrenals were analyzed separately for their ascorbic acid content, and the mean specific activity (SA, pg ascorbic acid/100 mg) of the two adrenals was taken as an index of response to a certain treatment. Sprague-Dawley female rats, hypophysectomized by the parapharyngeal approach were purchased from the Hormone Assay Laboratories, Chicago, Illinois, and the completeness of hypophysectomy was confirmed at the end of the assay by direct visual inspection of the pituitary sac. The weights of the rats, the range of the weights in each experiment, and the numbers as well as the random assignment of each rat to a certain treatment group (control, standards, and
272
SALEM,
NORTON,
unknowns) were performed according to the general principles of ACTH assay (Fisher, 1962). In accord with the method used, the homogenate of each adrenal was filtered using a double acid-washed filter paper (Munktell, type OK). Five milliliters from the clear colorless filtrate was obtained and its ascorbic acid content was determined by the spectrophotometric method described by Mindlin and Butler (1937). Most experiments were evaluated statistically by the method of least squares using the SSUPAC multiple correlation program, or by analysis of variance using the IBM digital computer. Assay for ACTH-releasing activity. This method is basically the same as that described above for assay of ACTH activity. Rats were injected intraperitoneally with dexamethasone 21phosphate in a dose of 0.5 mg/lOOg body weight (Kastin et al., 1964). Two to 4 hr later, rats were anesthetized with ip injection of sodium pentobarbital (Nembutal) 44.5 mg/lOOg body weight. About 10 min after the pentobarbital injection the anesthetized rats were injected with the standard or the unknown solutions. One hour later, both adrenals were removed and treated the same as for AC’I’H assay. A complete correlation has been reported between the response to hypothalamic extracts, (or vasopressin), as measured by AAAD and the response as measured by the rise in plasma corticosterone concentrations in the rat with ME lesions (Dhariwal et al., 1966) or in morphineNembuta-blocked rats (Leeman et al., 1962). Hormones used. The adrenocorticotropin (ACTH) used was a purified mammalian-porcine preparation (Armour, Acthar, lot 878212). Vials used contain 40 USP units (IU) of sterile powdered lyophilized ACTH and about 9 mg of hydrolyzed gelatin. The hormone was dissolved in the control injection solution (pH 4.0) and made up in a concentration of 3 IU/ml. This stock solution was stored frozen in small vials, and vials, once thawed, were discarded. Dexamethasone al-phosphate (Decadron Phosphate, Merck) was diluted with physiological saline in a concentration of 0.5 mg/ml, and kept refrigerated. EXPERIMENTS
ACTH
AND
RESULTS
Activity
ACTH activity in chicken adenohypophy&s. Five dose levels of chicken adenohypophysis were assayed for ACTH activity in hypophysectomized rats. Since prelimi-
AND
NALBANDOV
nary experiments had shown a great variation in its ACTH concentration (mp/mg), two extra doses of pituitary were used to establish the range within which linear dose response will occur. The activity of the two highest doses (P, and P4) did not differ significantly, therefore the P, dose was eliminated from further analysis. The specific activities of adrenals of the rats given the lower three doses (P, to P,) as well as of those given the higher three doses (PZ to P,) were fitted (together with the three groups of standard) into a sixpoint multiple correlation program. In both cases, linear and parallel dose-response curves for standard and unknown were obtained (Figs. 1 and 2). The pooled slope in both cases was significantly different from zero (p < 901). The dose range of 1.5 to 24 pg of chicken adenohypophysis was used for estimation of its relative potency since the response to these doses did not differ significantly from response to ACTH standard. ACTH activity in chicken adenohypophysis was equivalent to 401.7 (21.22)’ mU per milligram of frozen tissue, with a confidence limit of 270.0-597.7. It is much higher than the potency estimate of ACTH in chicken adenohypophysis reported by De Roos and De Roos (1964)) which was between 65 and 100 mU of mammalian ACTH per mg of fresh tissue. This difference in ACTH concentration could be explained by differences in experimental conditions. In addition to the fact that ACTH concentration in present experiment was reported on the basis of frozen weight there were differences in assay method as well as in the preparation of tissues for assay. Furthermore, there were age and sex differences between the birds used as a source of pituitary tissues. The present experiment, however, demonstrated that chicken anterior pituitaries contain a high concentration of ACTH activity when assayed by AAAD. In addition to this high concentration, the response of the hypophysectomized rats was proportional to the dose of chicken anterior pitui‘Standard
error
expressed
as a fraction.
A
STUDY
OF
ACTH
AND
CRF
IN
273
CHICKENS
DOSE RESPONSE TO CHICKEN PITUITARY TISSUE AS ASSAYED BY AAAD OF HYPOPHYSECTOMIZED ‘RATS
moJ ACTH STAHDARlUdO.4
I
PlTlJlTAFtY TISSUE f&P)
1
2.0 6.0
1.S
10.0
24.0
DOSE
Dose response obtained from 1.5-24.0 pg of chicken pituitary tissue assayed for ascorbic acid depletion in hypophysectomised rate. ACTH standard not significantly different from unknown. Number of animals per point 6 to 8. Vertical lines in this and subsequent figures: standard errors of the mean. FIG.
i.
DOSE RESPONSE TO CHICKEN PITUITARY TISSUE AS ASSAYED BY AAAD OF HYPOPHYSECTOMIZED RATS
450
1Control
400
200
L ACTH STANOAA: PITUITARY TISSUE
ml)
64
1
24.0
6.0
66.0
DOSE
Fm. 2. Dose response obtained from 6.0-26.0 pg of chicken pituitary tissue assayed for ascorbic acid depletion in hypophysectomized rats. Number of animals per point 6 to 8. ACTH standard vs unknown. p < .Ol.
274
SALEM,
NOKTOX,
tary. These observations suggest that the activity of the chicken anterior pituitary may be due to specific hormonal activityavian ACTS. ACTH-like
activity
in
chicken
hypo-
thalamic and brain cortex extracts. Preliminary experiments were conducted to determine whether crude acid extracts of chicken hypothalamus and brain cortex were active in depleting the AAA of the hypophysectomized rats. Doses below 10 hypothalamic extracts (HE) per 100 g body weight, and an equal amount by weight of brain cortex were without significant effect. On the basis of these observations, extracts of chicken hypothalami were made up in concentrations of 10, 20, and 40 HE per milliliter and were assayed in the hypophysectomized rats. Results show that the depletion caused by standard ACTH and HE fell on linear and parallel dose-response curves (Fig. 3) and the pooled slope of the two curves was significantly different from zero (p < .OOl). Relative potency was equivalent to 1.40 (21.195) mU of standard ACTH per 10 HE with a confidence limit of 0.980 to 1.999.
I
DOSE
RESPONSE T
AND
KALBANDOV
In contrast to hypothalamic extracts, chicken brain cortex extracts in a dose up to the equivalent of 30 HE has no detectable ACTH or ACTH-like activity (Table 1). However, the possibility that some acTABLE EFFECTS ON
1
OF CHICKEN BRAIN CORTEX AAAD IN HYPOPHYSECTOMIZED
Treatment Control (C) Hypothalamus Cortex X1
EXTRACTS RATS
Dose/100 g body weight
Mean SA + SE
1 ml 30
422.18 f 12.10 (7) 259.16 f 32.00 (6)0
6~ 30
410.38 414.61
Hz
X2
+. 23.97
(5)b
If: 10.89 (5)
a Hypothalamus vs control: p < .005. * Cortex vs control: NS. c Equivalent (by weight) to the hypothalamus.
tivity could be detected by using 5t more sensitive assay is not ruled out. These experiments demonstrated the presence of a substance(s) in the hypothalamus, but not in the brain cortex, of chickens which is active in the AAAD assay. This activity was proportional to the dose within the range of 1040 HE.
TO CHICKEN HYPOTHALAMIC AAAD OF HYPOPHYSECTOMIZED
EXTRACTS RATS
AS ASSAYED
BY
4oo- P.Control
250-
225
ACTH STANDARD HE
I 2.0
(mu) D:4 IO
DOSE
20
I 10.0
40
FIG. 3. Dose response obtained from extracts of 1040 chicken hypothalami assayed for ascorbic acid depletion in hypophysectomieed rats. Number of animals per point 6 to 9. ACTH standard not significantly different from unknown. p of pooled slope < .OOl.
A
ACTH-Releasing
STUDY
OF
ACTH
AND
Activity
Assay in intact-blocked rats. Results presented in Fig. 4 show that there was a linear and parallel relationship between the
CRF
IN
tivity when given in doses equivalent and 10 HE (Table 2).
different
OF CHICKEN BRAIN CORTEX EXTRACTS AS ASSAYED BY AAAD IN THE INTACT-BLOCKED RATS
Dose/100 g body Mean SA + SE weight
from zero (p < .OOl) .
Potency estimates for the HE was equivalent to 5.12 (51.19) mU of standard ACTH per 10 HE, with a confidence limit of 3.616-7.250. The response of rats to the HE should be due to a direct effect on the adrenals by these extracts plus a possible release of endogenous ACTH. The direct effect of HE on the adrenals of hypophy-
sectomized
rats was shown earlier to be equivalent to 1.40 2 1.195 mU of standard ACTH per 10 HE and represents about 27% of the total estimated potency in the intact-blocked rats. Thus, in intact-blocked rats, more than 70% of the potency estimate of the HE may be due to the endogenous release of ACTH. In contrast to HE, extracts of chicken brain cortex had no ACTH-releasing ac-
DOSE 400.
to 5
TABLE 2 EFFECT
log of the dose of both standard and unknown of 2.5,5, or 10 HE. The pooled slope of the responses to both treatments was significantly
275
CHICKENS
Treatment
Control (C) ACTH Standard S, SZ Cortex X1 X*
1 ml 1.5 mU 6.0 mU 5.0 10.0
387.07 310.40 249.66 404.66
f + + +
17.21 15.13 16.18 16.18
(8) (8) (7) (7)
394.13
f
17.47
(6)
Pooled standard vs control: p < .005. Pooled cortex vs control: NS. Assay in both hypophysectomized and intact-blocked rats. The previous conclu-
sion that the higher potency estimates of the HE in the intact rats is due only to the release of endogenous ACTH and not due to any differences
in the experimental
ditions, was tested further. Intact rats were kept in individual
RESPONSE TO CHICKEN HYPOTHALAMIC BY AAAD OF INTACT-DEXAMETHASONE-BLOCKED
EXTRACTS
con-
cages
AS ASSAYED RATS
Control
P
250-
200’ ACTH STANDARDhad HE
D.4
20 6.0
1 2.6
I IOD
10.0
DOSE
FIQ. 4. Dose response obtained from extracts of 2.5-10 chicken hypothalami assayed for ascorbic acid depletion in intact, dexamethansone-blocked rats. Number of animals per point 5 to 8. ACTH standard not signscantly diierent from unknown. p of pooled slope < .OOl.
276
SALEM,
NORTON,
AND
under controlled temperature for 48 hr before the experiment. One-half of the rats was assigned at random to bc hypophysectomized by the transauricular approach (Falconi et OZ., 1964), 16-24 hr before the assay. Five different treatments were assigned at random to the hypophysectomized as well as to the intact rats. These treatments, which were similar in both types of animals, included two doses of standard ACTH, two doses of HE, and the control. For every two similar groups in the two types of animals, one solution was prepared and was injected with the same syringe. All rats (including the hypophysectomized rats) were injected ip with dexamethasone 24 hr prior to being anesthetized with Nembutal. Two experiments of this type were conducted and the results of the two assays were pooled after appropriate statistical analysis had revealed no significant differences between the two experiments. Results based on specific activity (SA) are presented in Table 3. Linear and parallel doseresponse curves for standard and unknown were obtained (Figs. 5 and 6). The pooled slope in both cases was significantly different from zero (p < .OOl). In addition, there were significant differences b’etween hypophysectomized and intact rats in response to ACTH standard and HE (Table 3). However, AAA response to standard and unknown in these experiments is more easily discussed on the basis
of percentage of depletion (Fig. 7). This eliminates differences in mean specific activities of controls between the two types of animals (Table 3). In intact rats, there is greater response to equal doses of chicken HE than in hypophysectomized rats (Fig. 6). This higher response of intact rats is not due to their higher sensitivity, since hypophysectomized rats respond even more (though not significantly), to equal doses of ACTH standard than do intact rats. Therefore, the greater response of the intact-blocked rats to chicken HE is attributed to the ability of these extracts to cause endogenous release of ACTH in these animals. Estimation of relative potencies of HE in both types of animals was conducted by plotting the middle value (of SA or percentage of depletion) on the appropriate standard curve (Fig. 7). Potency estimates of HE in the hypophysectomized rats was 33.7% (on the basis of SA) and 34.2% (on the basis of percentage of depletion) of that in the intact-blocked rats. Thus, the ACTH-like activity of chicken HE represents about 34%. In this case, 66% of the relative potency in the intact-blocked rats is due to the endogenous release of ACTH, which agrees with results obtained earlier. Activity in Plasma of Hypophysectomized Chickens Blood was collected by cardiac puncture at several intervals (up to 9 weeks) from
TABLE ACTIVITY
OF CHICKEN
HYPOTHALAMIC
KALBANDOV
3
EXTRACTS
ASSAYED
INTACT-BLOCKED
Specific Treatment Control ACTH 1.5 mU 6.0 mU Hypothalamus 5 Hypothalamic 10 Hypothalamic Control Intact
Intact
extract extract
vs treated vs hypophysectomized
p < .OOl. p < .OOl.
IN
HYPOPHYSECTOMIZED
OR
RATS activity
(Mean
k SE) Hypophysectjomieed
363.2
f
13.9
(13)
409.3
I!I
8.4
(12)
274.6 226.3
5 13.6 + 13.6
(11) (11)
313.7 236.1
+ 13.6 + 13.0
(11) (12)
259.1 211.4
f 13.6 & 18.4
(11) (6)
345.3 291.0
f 13.6 I!z 15.9
(11) (8)
A
400-1
STUDY
$
OF
ACTH
AND
CRF
IN
277
CHICKENS
ACTIVITY OF CH!CKEN HYPOTHALAMIC EXTRACTS AS ASSAYED BOTH HYPOPHSECTOMIZED AND INTACT- BLOCKED RATS [cmtro~ [ 1 , _- - - Hypophywtcmlr~d InlPct
350-
200
150
ACTH STANDM&
i.5
HE
FIG.
ascorbic
5. Comparison acid depletion
I 5.0
of dose-response lines in hypophysectomized
obtained from and in intact,
completely adenhypophysectomized cockerels. Plasmas were stored at -20” until used. Activity of plasma collected at 1, 2, 3, 5, 7, and 9 weeks after hypophysectomy in a dose of 3 ml (per 100 g body weight) ACTIVITY I
dJ3 IO.0 DOSE
extracts of chicken dexamethansone-blocked
hypothalami assayed rats. See text.
of intact-blocked rats, was not significantly different from saline. On the other hand, the response to standard ACTH was highly significant (p < ,005). Since negative results were obtained
OF CHICKEN HYPOTHALAMIC HYPOPHYSECTOMIZED AND
EXTRACTS INTACT-BLOCKED
AS ASSAYED RATS
IN BOTH
50 1
HypophYSWOttd~d -Intact IO
,
I
ACTH STANOfQ$l.5 Ill
FIG
tomiaed
6. Percentage of depletion and dexamethansoneblocked
of ascorbic rats.
for
I
acid by extracts See text.
of chicken
5.0
hypothalami
assayed
in hypophysec-
278
SALEM,
A-ORTON,
AND
RELATIVE POTENCY OF CHICKEN IN BOTH HYPOPHYSECTOMlZED
T
NALBANDOV
HYPOTHALAMIC EXTRACTS AS ASSAYED AND INTACT-BLOCKED RATS
\\ h. PII P\ \ \ SI.
\\
\
\
\
\\ .H
\
\
‘.6 1
\ IOJ , , 123I.5
3.60
6.0
FIG.
7. Relative pot,ency rats. See text.
of extracts
of chicken
1.3
hypothalami
when plasma was assayed in the intactblocked rats, it was concluded that plasma of adenohypophysectomized chickens has no ACTH-like or ACTH-releasing activity that can be detected by the AAAD method. Reports that CRF can passinto the general circulation of hypophysectomized mammals (Brodish et al., 1962; Eik-Nes et al., 1958)) or of hypophysectomized rats with pituitary transplants (Kendall et al., 1966a,b) are not confirmed by results obtained in birds. It is felt that the main reason for the failure to detect both hormones in the plasma of adenohypophysectomized chickens, is that the AAAD method may not be sensitive enough to detect these active substances in the blood. Plasma of hypophysectomized sheep was also without ACTHreleasing activity by the use of this assay. DISCUSSION
Avian ACTH. Direct evidence for the presence of ACTH in the avian pituitary is poorly documented in the literature. The only reports on the existence of avian ACTH was based on the ability of bird adenohypophyseal extracts to promote
380
w
DOSE (mu ACTH STANDARD)
blocked
I 1
6.0
DOSE (mu ACTH STANDARD)
when
assayed
in intact,
dexamethasone-
steroidogenesis by chicken adrenal tissue in vitro. The present study confirms these earlier reports and provides evidence for possible biological and structural similarities between avian and mammalian ACTH. We have shown that chicken pituitary ACTH is active in mammals, and that it is similar to mammalian ACTH in depleting AAA of the rat, in its response to boiling, and to chemicals (Salem et al., 1969). There was a disparity between our results and those reported by De Roos and De Roos (1964) in the concentration of ACTH in the anterior pituitary of chickens. However, greater differences have been reported in the anterior pituitary of the normal rat where the reported range is 12.0-76.2 mU of ACTH/mg of fresh tissue (Marks et al., 1963; Fortier et al., 1964). Differences in experimental conditions and in the physiological status of the donor animals may be reflected in the great differences in potency estimates of pituitary ACTH in the same species. Nevertheless, it appears that the concentration of ACTH is much higher in chicken anterior pituitary than in the rat anterior pituitary. Activity in chicken hypothalamus. Pres-
A STUDY
OF
ACTH
AND
ent results also demonstrated two kinds of activities in the hypothalamus (but not in the brain cortex) of chickens: one an ACTH-like activity, and the other an ACTH-releasing activity. The ACTH-like activity contributes between 27-34s of the total activity (ACTH-like activity plus ACTH-releasing activity) of the chicken HE, when assayed in hypophysectomized and intact-blocked rats respectively. It is interesting that corticosterone output in the adrenal vein plasma of the adenohypophysectomized chickens was shown to be about 37% of that of intact controls (Frankel et al., 1967b). These authors also showed that injection with dexamethasone phosphate eliminates corticosterone from adrenal vein plasma of both groups of chickens suggesting that the steroidogenic substances are of a hypothalamic and/or of a higher origin in the CNS. Present results, however, support the hypothalamus as the origin of these substances. Further experiments were conducted to differentiate between the two activities in the hypothalamus and between them and ACTH activity in chicken pituitary and will be the subject of another paper (Salem et al., 1969). Results have shown that each activity is due to different substances of hypothalamic origin and that neither is due to vasotocin or to ACTH. REFERENCES BRODISH, A., AND LONQ, C. N. H. (1962). ACTHreleasing hypothalamic neurohumor in peripheral blood. Endocrinology 71, 298-306. BROWN, K. I., BROWN, D. J., AND MEYER, R. K. (1958). Effect of surgical trauma, ACTH and adrenal cortical hormones on electrolytes, water balance and gluconeogenesis in male chickens. Am. J. Physiol. 192, 43-50. C~RBIN, A., MANGILI, G.. MOTTA, M., AND MARTINI, L. (1965). Effect of hypothaIamic and mesencephalic steroid implantations on ACTH feedback mechanisms. Endocrinology 76, 811818. DE ROOS, R. (1963). The physiology of the avian interrenal gland: A review. In Proc. Intern. Ornithological Congr. 13th (C. G. Sibley, ed.), Vol. 2, pp. 1041-1058. DE ROOS, R., AND DE Roos, C. C. (1964). Effects of mammalian corticotropin and chicken adeno-
CRF
IN
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279
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