Bioactive and Immunoactive ACTH in the Rat Pituitary: Influence of Stress and Adrenalectomy C. MICHAEL MORIARTY AND GWEN ASSISTANCE OF EDNA R. MATTHEWS
C.
MORIARTY,
WITH THE TECHNICAL
Departments of Physiology-Biophysics and Anatomy, The University of Nebraska Medical Center, Omaha, Nebraska 68105 ABSTRACT. Tissue levels of bioactive and immunoactive ACTH were measured in both the anterior and neuro-intermediate lobes of the rat pituitary. Similar concentrations of bioactive (65 ng/mg) and immunoactive (83 ng/mg) ACTH were found in the anterior lobes from control rats. A 2-min ether stress had no effect on either bioactive or immunoactive ACTH levels in the anterior lobe. Twenty-four h after adrenalectomy the anterior lobe content of both bioactive and immunoactive ACTH decreased, only to return to supranormal levels 21 days after the operation. A 30-min neurogenic stress had no effect on anterior lobe bioactive ACTH content but reduced the immunoactive ACTH level to 50 ng/mg. Synthetic Oh17"39 ACTH was used in our radioimmunoassay in order to measure the C-terminal ACTH activity of the neuro-intermediate lobe. The concentration of such C-terminal activity in control rats (890 ng ah17"39 ACTH/mg) considerably exceeded the amount of bioactive ACTH (15 ng/mg). This is presumably due primarily to the presence of
T
HERE is little question that the neurointermediate lobe of the rat pituitary contains bioactive ACTH. A number of previous studies (1-10) all confirm its existence while disagreeing considerably regarding the amount. Immunocytochemical methods have localized this neuro-intermediate lobe activity exclusively to the pars intermedia (11-13). Recently, Scott and coworkers (14-17) have identified another peptide in the pars intermedia of several species, including the rat. Termed CLIP (corticotropin-like
Received June 12, 1974. Address reprint requests to C. Michael Moriarty, University of Nebraska Medical Center, Omaha, Nebraska 68105. Supported in part by NSF Grant GB 30318, a Nebraska Heart Association grant-in-aid and an N.I.H. Institutional Grant FR 5391.
the so-called corticotropin-like intermediate lobepeptide (CLIP). The amounts of bioactive or C-terminal immunoactive ACTH in the neurointermediate lobe were not affected by ether stress nor short term (24-h) or long term (21-day) adrenalectomy. Neuro-intermediate lobe bioactive ACTH decreased (to 8 ng/mg) only with the introduction of a 30-min neurogenic stress. Neurogenic stress had no effect on the concentration of CLIP, but when the stress was imposed 24 h after adrenalectomy, a significant reduction was observed. The data support the presence of bioactive ACTH in the intermediate lobe of the rat pituitary and suggest that such ACTH is preferentially released by neurogenic stress and not appreciably regulated by circulating levels of glucocorticoids. Until the biological function and/or target organ of CLIP is identified, the significance of the changes in tissue levels of C-terminal immunoactive ACTH will remain unknown. (Endocrinology 96: 1419, 1975)
intermediate lobe peptide), it has no N-terminal- ACTH immunoactivity, no ACTH bioactivity, and is indistinguishable from a18"39 ACTH. Its function and target organ, if any, remain unknown. The purpose of this study was to determine the variations in the levels of bioactive and C-terminal immunoactive ACTH in the neuro-intermediate lobe of the rat pituitary under the influence of stress and changing levels of plasma corticosterone. For comparison purposes similar measurements were also performed on the anterior lobe. Materials and Methods Male rats of the Sprague-Dawley strain (175-200 g) were used in all experiments. They were maintained in a temperature-controlled environment (22 C) and given food and water ad libitum. All rats were acclimatized in a regu-
1419
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MORIARTY AND MORIARTY
1420
Endo • 1975 Vol 96 • No 6
RAT ACTH 10
60 r
25.6
51.2
100
FIG. 1. Standard curves obtained with l25I-ap'-39ACTH competing with aMSH, a'-24ACTH, ah17-39ACTH and ap'-39ACTH for antibody binding. On the upper abscissa is shown the biological activity of an extract of rat pituitary. Assuming a potency of pure rat ACTH of 100 U/mg which is consistent with potencies of ACTH from other species (38) it is seen that rat and porcine ACTH react equally with the antiserum.
50
40
S
30
1 pg
10 pg
100 pg AMOUNT OF COMPETING ANTIGEN
lated light-dark environment (lights on 0600 h, off 2200 h) for at least 1 week prior to use. Adrenalectomies were performed by the dorsal approach with the rats maintained under light ether anesthesia. Each rat was bilaterally adrenalectomized within 2 - 3 min and subsequently maintained on 0.9% NaCl in lieu of water. Exposure of rats to ether fumes for 2 min was used as a systemic stress. Neurogenic stress consisted of simultaneous exposure to a loud buzzer and flashing stroboscopic light for a period of 30 min in a darkened room. All animals were killed by decapitation wil:hin 30 s after termination of the stress. All experiments were performed in the morning, with decapitations occurring between 10 and 11 AM. After decapitation, the pituitaries were quickly removed and the anterior and neurointermediate lobes gently separated. The entire procedure was accomplished within 30 s and with minimal loss of cells from the friable pars intermedia. The isolated, individual lobes were then weighed and quickly frozen. Immediately before use, they were thawed and their ACTH content was extracted according to the modified glacial acetic acid technique of Birmingham et al. (18). All lobes were extracted and measured individually without pooling. All dilutions were made with 0.15M NaCl containing 0.5% bovine serum albumin, pH 3.5. The in vitro adrenal cell dispersion technique of Sayers et al. (19) was used for the ACTH bioassay. In our hands, an additional 20 min dispersion was found to be desirable. The III
Ing
IWS ACTH was used as a standard.1 The standard curve was determined by duplicate measurements of 8-10 different ACTH concentrations. All unknowns were measured in duplicate at a minimum of 2 doses. The antiserum for radioimmunoassay was produced by multiple injections of porcine ACTH (Armour, 75 U/mg) into adult male rabbits, according to the protocol of Hum and Landon (20). Since these rabbits had previously received a single injection of synthetic a1"24 ACTH, the final antiserum contained 2 populations of antibodies reactive with ACTH. Figure 1 shows the sensitivity of aMSH and various fragments of the ACTH molecule for the antiserum. As might be expected, since a1"24 ACTH was the initial immunogen, the antiserum binds most effectively with it. The antiserum cross-reacted equally with rat and porcine ACTH, as evidenced by the parallelism of the standard curves. All measurements of the anterior ptiuitary used ap1-39 ACTH1 both for standards and for iodination, and all are expressed as ng ACTH/mg wet weight to facilitate comparisons with the neuro-intermediate lobe values. In contrast to what was observed in the anterior pituitary, we found that our radioim1 5 IU of porcine ACTH (III IWS) was supplied in ampoules containing "approximately 50 fig" of material. In order to express ACTH in gravimetric terms we have assumed each vial contained exactly 50 /ig. Conversion to units can be made by the equivalency of 100 U/mg.
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BIOACTIVE AND IMMUNOACTIVE ACTH
1421
DILUTION Of NEURO-INTERMEDIATE LOBE EXTRACT
l:10 S
l:10 4
l:10 3
l:10 2
40
FIG. 2. Parallelism of extract of the neuro-intermediate lobe with ahI7"39ACTH. Each competed with 125I-ahI7-39ACTH for the ACTH antibody. On the lower abscissa is the amount of ah17~39ACTH and on the upper abscissa are the dilutions of the neuro-intermediate lobe extract.
o z
I
30
| 2
20
S~
10 17-39
1 pg
10 pg
100 pg 7-39
1 ng
ACTH
munoassay using ap1 39 ACTH was not suitable for the neuro-intermediate lobe. A comparison of the standard curve generated by use of ap1"39 ACTH with that obtained by varying dilutions of neuro-intermediate lobe extract demonstrated a significant lack of parallelism. When we used ah17~39 ACTH for iodination and for standards, we obtained good cross reactivity with rat neuro-intermediate lobe extract (Fig. 2). For iodination, we used 125I obtained from the Iso-Serve Division, Cambridge Nuclear Corp., Billerica, Mass. The procedure of Rees et al. (21) was followed for both iodination and separation of bound from free antigen. All radioimmunoassay incubations were for 48 h at 4 C. All pituitaries were measured individually at 3 doses, each in duplicate. The results of each radioimmunoassay experiment were calculated with the aid of computer programs kindly supplied by David A. Rodbard.
renalectomy (45 ng/mg) and reached supranormal levels (159 ng/mg) after 21 days. Twenty-four hours after adrenalectomy we found that the immunoactive ACTH content in the anterior lobe was significantly less than the bioactive component. Immunoactive and bioactive ACTH values were comparable 21 days after adrenalectomy. A 2-min ether stress decreased the bioactive and immunoactive ACTH levels by 24% and 22% respectively. Neither decrease, however, was significant at the P < 0.05 level. When the ether stress was applied 24 h after bilateral adrenalectomy, the bioactive but not the immunoactive ACTH decreased significantly when compared to the effect of adrenalectomy alone (Table 1). Bioactive ACTH levels in the anterior Results lobe were unaffected by the application of Anterior lobe a neurogenic stress although the imThe ACTH concentration in control an- munoactive ACTH levels decreased. terior lobes (Table 1) was not significantly Neuro-intermediate lobe different whether measured by bioassay When expressed per mg of neuro(65 ng/mg) or radioimmunoassay (83 ng/ intermediate lobe tissue (pars nervosa + mg). As expected from previous studies pars intermedia) the bioactive ACTH was (22,23), the bioactive ACTH of the anterior 15 ng/mg (Table 2). There was no siglobe decreased 24 h after bilateral ad- nificant change in the bioactive ACTH
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Endo • 1975 Vol 96 • No 6
MORIARTY AND MORIARTY
1422
TABLE 1. Concentration of ACTH in anterior lobe 8
Saffran and Serially assay (25). The discrepancy may be partly attributable to Immunoactive Bioactive methodology. Fortier (26) has pointed out 65 (59-74)c 83 (69-97)c that pituitary ACTH concentrations measControl (9)d 24 h after ured with Saffran and Schally's assay are 45 (37-55) 25 (18-32) adrenalectomy (7) consistently higher than those obtained 21 day after with Sayers' in vivo adrenal ascorbic acid 159 (139-203) 133 (110-156) adrenalectomy (6) 65 (37-93) 49 (36-67) Ether stress (6) depletion technique (27). With the adrenal Ether stress 24 hour after ascorbic acid technique, Sayers (27) meas31 (18-44) 12 (7-17) adrenalectomy (6) ured pituitary ACTH concentrations, 50 (32-68) 76 (55-123) Neurogenic stress (8) Neurogenic stress 24 h which agree with those measured with the 39 (11-67) Not done after adrenalectomy (5) dispersed adrenal cell technique in this study and with the technique of Lipscomb • All values are expressed per mg of anterior lobe Average weight = 8.4 ± (SE) 0.3 mg (N = 47). and Nelson used by others (9,15). b 1 39 Expressed in ng of a, ' ACTH (100 U/mg). c As others have reported (28,29) a 2-min 95% confidence limits. d Number of animals. ether stress failed to lower the anterior pituitary ACTH levels in intact rats. Since content 24 h or 21 days after bilateral plasma ACTH levels are reported to be adrenalectomy, after ether stress, or when significantly elevated by this time (21, the ether stress was applied 24 h after 30,31), this might suggest that the ether adrenalectomy. However, the introduction stress increased ACTH synthesis either of a neurogenic stress significantly de- directly or indirectly through the increase pleted neuro-intermediate lobe bioactive in release. In the present study, however, ACTH (Table 2). In no case did any even in the absence of an increase in treatment alter the weight of the neuro- synthesis we would not expect to be able to intermediate lobe. detect the small depletion in pituitary The entire neuro-intermediate lobe con- ACTH necessary to elevate plasma ACTH tained 890 ng of C-terminal (ah17"39) ACTH to the reported levels. immunoactivity per mg tissue (Table 2). When the ether stress was introduced 24 Adrenalectomy and/or ether stress was in- h after bilateral adrenalectomy the pituitary effective in altering these values. The application of a neurogenic stress resulted TABLE 2. Concentration of "ACTH" in in an apparent 36% decrease, but owing to neuro-intermediate lobe8 a large rat-to-rat variation this was not Bioactive" Immunoactive0 statistically significant. When the neurogenic stress was applied 24 h after bilateral Control (8)e 15 (13-18)d 890 (540-1,240)" adrenalectomy, the levels of C-terminal 21 days after adrenalectomy (7) 13 (11-15) 870 (550-1,190) ACTH activity dropped significantly, to 21 days after about 25% of control values. adrenalectomy (5) 19 (13-27) 950 (420-1480) b
b
Discussion Bioactive ACTH in the anterior lobe The concentrations of bioactive ACTH in the pars distalis are in good agreement with those reported in several other recent studies (9,15,24). We find approximately 40 times less bioactive ACTH than reported by Kraicer et al. (10), who used the in vitro
Ether stress (6) Ether stress 24 h after adrenalectomy (6) Neurogenic stress (8) Neurogenic stress 24 h after adrenalectomy (5)
13 (8-20)
750 (370-1130)
11 (7-16) 8 (6-11)
800 (250-1350) 570 (240-900)
Not done
210 (100-340)
1 All values are expressed per mg of neuro-intermediate lobe. Average weight = 2.1 ± (SE) 0.1 mg (N = 45). b Bioactive ACTH expressed in ngof a p '- M ACTH (100 U/mg). c Immunoactive activity expressed as ng of Oh'7"89 ACTH/mg. d 95% confidence limits. e Number of animals.
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BIOACTIVE AND IMMUNOACTIVE ACTH levels of bioactive ACTH fell sharply. This is in conflict with previous reports (28,29). Rees et al. (21) have reported that when an ether stress is applied 24 h after adrenalectomy, plasma ACTH values are identical with those of similarly stressed intact rats. Dallman et al. (30) confirmed this and showed that shortly after adrenalectomy (1-2 hr) the imposition of a laparotomy stress markedly elevated plasma ACTH levels over those of nonadrenalectomized controls. However, in both of these studies immunoactive ACTH was measured and, as will be discussed in the next section, their observations are consistent with our immunoactive ACTH data. At this time we do not have a reasonable explanation for the low levels of bioactive ACTH found in the anterior pituitary when the rats are stressed with ether 24 h after adrenalectomy. The fate of this ACTH lost from the hypophysis is unknown; it is certainly not detected in the plasma (21,30). In accord with previous studies (2,3,5) when rats were subjected to a 30-min neurogenic stress there was no change in the anterior pituitary content of bioactive ACTH. At this time we have no direct evidence regarding plasma ACTH levels under these conditions. From the work on posterior lobectomized rats discussed below we suspect that the anterior lobe ACTH cells may be less affected by neurogenic stress than cells in the neurointermediate lobe. Immunoactive
ACTH in the anterior lobe
The amount of immunoactive ACTH in the control anterior pituitaries was of the same order as the amount of bioactive ACTH. This is in agreement with the findings of Shapiro et al. (9). Scott et al. (15), however, reported that in rats the level of bioactive ACTH is 2-10 times less than that of immunoactive a13~18 ACTH. Following adrenalectomy or ether stress the values of immunoactive ACTH paralleled the bioactive values, except in one instance. For reasons that are not clear
1423
there was a greater decrease in immunoactive ACTH 24 h after adrenalectomy. Anterior lobe immunoactive ACTH dropped to 30% of the control value under these conditions, a value also recently reported by Dallman et al. (30). Immunoactive ACTH levels did not change further when an ether stress was applied 24 h after adrenalectomy. The difficulty in making quantitative comparisons between bioactive and immunoactive data is appreciated. Depending on the antiserum, one may be comparing the relative activities of two different portions of the molecule. Indeed, even with antiserum directed toward the N-terminal portion of ACTH, Besser et al. (32) have found variations in the half-lives of bioactive and immunoactive ACTH. Neurogenic stress reduced the immunoactive ACTH level in the anterior pituitary without affecting the bioactive component. Without additional studies and, particularly, measurements of plasma ACTH, the significance of this decrease in immunoactive ACTH will remain obscure. The possibility exists that there are fragments of the ACTH molecule in the anterior pituitary which are selectively released and/or intracellularly degraded. We have preliminary evidence that there is a large molecular weight moiety in the rat anterior pituitary that exhibits ACTH immunoactivity. Gel filtration indicates that such material has an average molecular weight slightly less than 69,000 daltons. The changes in the amount of this material (both immunoactive and bioactive) following adrenalectomy and stress are not yet known. Bioactive ACTH in the lobe
neuro-intermediate
We found 15 ng of bioactive ACTH per mg of neuro-intermediate lobe. Recently, immunocytochemical studies of the neurointermediate lobe have shown that both C-terminal (10-13) and N-terminal (11) ACTH activities are confined exclusively
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1424
MORIARTY AND MORIARTY
to the pars intermedia. Since the pars intermedia and pars nervosa are approximately equal in weight (33,34), this is equivalent to 30 ng of ACTH per mg of pars intermedia. In contrast, Kraicer et al. (10) have reported values of bioactive ACTH in the pars intermedia 15 times greater than this. Our results agree quite well with the recent reports of Scott et al. (15) and Shapiro et al. (9). The levels of bioactive ACTH in the pars intermedia did not change after adrenalectomy or ether stress, conditions under which the ACTH levels in the anterior lobe changed appreciably. At this time we cannot say that ACTH release was also unchanged under these conditions or whether release and synthesis changed in parallel. However, there was little morphologic evidence of any increase in synthetic activity, e.g., no increase in rough endoplasmic reticulum (Moriarty, Halmi, and Moriarty, submitted for publication). In contrast with the anterior pituitary, the tissue levels of bioactive ACTH in the neuro-intermediate lobe decreased significantly after a 30-min neurogenic stress. This finding agrees with that of earlier workers (2,4,5) who suggested that the neuro-intermediate lobe was the source of the ACTH released following stresses of neurogenic origin. However, Miller et al. (35) have recently shown that the increase in plasma corticosterone following a 15-min neurogenic stress is the same for both posterior hypophysectomized and control rats. Kastin et al. (36) studied the effect of posterior lobectomy on plasma ACTH in rats adrenalectomized one month previously and in rats exposed to a 2-min ether stress. They concluded that the neuro-intermediate lobe did not participate in stress-induced ACTH release although they did not examine a neurogenic stress. Clarification of the physiological role of the pars intermedia in regulating plasma corticosterone levels requires more work. The level of bioactive ACTH in the pars intermedia certainly does not change after adrenalectomy. Whether this is due to the
Endo • 1975 Vol 96 • No 6
absence of corticosterone feedback receptors in the pars intermedia, an equal balance between synthesis and release, or to the poor vascularization that characterizes this lobe, is not known. In contrast with the anterior pituitary, the stimulated release of ACTH from the isolated neuro-intermediate lobe is not blocked by dexamethasone (37). Similarly, the nature of the agents regulating ACTH release from the pars intermedia are unknown, although evidence indicates that acetylcholine, a crude hypothalarnic extract, and dopamine may all influence release (37). 1mmunoactive ACTH in the intermediate lobe
neuro-
With the use of synthetic «h17-39 ACTH we measured 890 ng of C-terminal activity per mg of neuro-intermediate lobe or a ratio of C-terminal activity to N-terminal activity (i.e., bioactive ACTH) of approximately 60:1. Scott et al. (17) report a value of 32:1 and Shapiro et al. (9), using antiserum toward the N-terminal portion of ACTH, reported approximately equal amounts of immunoactive and bioactive ACTH in the rat neuro-intermediate lobe. Scott and his colleagues have succeeded in purifying the major C-terminal immunoactive peptide and report that it is indistinguishable from a18"39 ACTH (16). They have termed this peptide corticotropin-like intermediate lobe peptide or CLIP and have localized it to the pars intermedia. The only condition that caused a decrease in neuro-intermediate lobe levels of C-terminal ACTH was the introduction of a neurogenic stress 24 h after bilateral adrenalectomy. Part of this decrease must represent the loss of a1"39 ACTH from the pars intermedia. In contrast to our findings in the anterior lobe, we could find no large molecular weight component which exhibited ACTH immunoactivity in extracts of neuro-intermediate lobe. Thus, the Cterminal ACTH immunoactivity does not
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BIOACTIVE AND IMMUNOACTIVE ACTH appear to be due to the presence of "big ACTH". Similarly, it does not appear to be an artifact of the extraction procedure, since neuro-intermediate lobes in organ culture release immunoactive ACTH with a C/N ratio considerably greater than 1 (17). Until the target organ (if any) for CLIP becomes known, the significance of this C-terminal activity is likely to remain obscure. Acknowledgments The authors would like to express their appreciation to the Data Management Branch of the Division of Computer Research and Technology, National Institutes of Health, the Reproductive Research Branch, National Institute of Child Health and Human Development and particularly to Dr. David A. Rodbard for the computer programs used for the radioimmunoassay calculations. The generous supply of III IWS ACTH (National Institute for Medical Research, Mill Hill, London) and partially purified porcine ACTH from Dr. J. D. Fischer, Armour Pharmaceuticals, Kankakee, Illinois is gratefully acknowledged. Dr. W. Rittel, CIBA, Basle, Switzerland kindly supplied aMSH and Oh17"39 ACTH. The assistance of Dr. K. D. Patil with the statistical evaluations is appreciated.
References 1. Mialhe-Voloss, C , CR Acad Sci (Paris) 235: 743, 1952. 2. , CR Acad Sci (Paris) 241: 105, 1955. 3. J Physiol (Paris) 47: 251, 1955. 4. , Ann Endocrinol 17: 104, 1956. 5. Rochefort, G. J., J. Rosenberger, and M. Saffran,./ Physiol (Lond) 146: 105, 1959. 6. DeWied, D., Endocrinology 68: 956, 1961. 7. Smelik, P. C , Ada Endocrinol (Kbh) 33: 437, 1960. 8. Itoh, S.Jap J Physiol 12: 257, 1962. 9. Shapiro, M., W. E. Nicholson, D. N. Orth, W. M. Mitchell, D. P. Island, and G. W. Liddle, Endocrinology 90: 249, 1972. 10. Kraicer, J., J. L. Gosbee, and S. A. Bencosme, Neuroendocrinology 11: 156, 1973. 11. Baker, B. L., and T. Drummond, Am J Anat 134: 395, 1972. 12. Moriarty, G. C , and N. S. Halmi, Z Zellforsch 132: 1, 1972. 13. Phifer, R. F., and S. S. Spicer, Lab Invest 23: 543, 1970.
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14. Scott, A. P., H. P. J. Bennett, P. J. Lowry, C. McMartin, and J. G. Ratcliffe, J Endocrinol 55: xxvi, 1972. 15. , G. M. Besser, and J. G. Ratcliffe, J Endocrinol 51: i, 1971. 16. , J. G. Ratcliffe, L. H. Rees, J. Landon, and H. P. J. Bennett, Nature New Biol 244: 65, 1973. 17. , L. H. Rees, J. G. Ratcliffe, and G. M. Besser, 7 Endocrinol 53: xxviii, 1972. 18. Birmingham, M. K., E. Kurlents, G. J. Rochefort, M. Saffran, and A. V. Schally, Endocrinology 59: 677, 1956. 19. Sayers, G., R. L. Swallow, and N. D. Giordano, Endocrinology 88: 1063, 1971. 20. Hum, B. A. L., and J. Landon, In Kirkham, K. E., and W. M. Hunter (eds.), Radioimmunoassay Methods, Churchill Livingston, Edinburgh 1971, p 132. 21. Rees, L. H., D. M. Cook, J. W. Kendall, C. F. Allen, R. M. Kramer, J. G. Ratcliffe, and R. A. Knight, Endocrinology 89: 254, 1971. 22. Gemzell, C. A., D. C. vanDyke, C. A. Tobias, and H. M. Evans, Endocrinology 49: 325, 1951. 23. Fortier, C , Proc Soc Exp Biol Med 100: 13, 1968. 24. Kastin, A. J., A. Arimura, S. Viosca, L. Barrett, and A. V. Schally, Neuroendocrinology 2: 200, 1967. 25. Saffran, M., and A. V. Schally, Endocrinology 56: 523, 1955. 26. Fortier, C , Proc Soc Exp Biol Med 99: 628, 1958. 27. Sayers, M. A., G. Sayers, and L. A. Woodbury, Endocrinology 42: 379, 1948. 28. Sydnor, K. L., and G. Sayers, Endocrinology 55: 621, 1954. 29. Vernikos-Danellis, J., Endocrinology 72: 574, 1963. 30. Dallman, M. F., M. T. Jones, J. Vernikos-Danellis, and W. F. Ganong, Endocrinology 91: 961, 1972. 31. Cook, C. M., J. W. Kendall, M. A. Greer, and R. M. Kramer, Endocrinology 93: 1019, 1973. 32. Besser, G. M., D. N. Orth, W. E. Nicholson, and J. Woodham, J Clin Endocrinol Metab 32: 595, 1971. 33. Carrillo, A. J., A. J. Kastin, J. D. Dunn, and A. V. Schally, Neuroendocrinology 12: 120, 1973. 34. Gosbee, J. L., J. Kraicer, A. J. Kastin, and A. V. Schally, Endocrinology 86: 560, 1970. 35. Miller, R. E., H. Yueh-Chien, M. K. Wiley, and R. Hewitt, Neuroendocrinology 14: 233, 1974. 36. Kastin, A. J., S. Viosca, and L. Debeljuk, Program of the 53rd Meeting of the Endocrine Society, 1971, p. A-211, Abstract 338. 37. Fischer, J. L., and C. M. Moriarty, Fed Proc 33: 206, 1974 (Abstract). 38. Ney, R. L., E. Ogata, N. Shimizu, W. E. Nicholson, and G. W. Liddle, Exerpta Med Intern Congr Ser83: 1184, 1964.
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C.
MORIARTY,
WITH THE TECHNICAL
Departments of Physiology-Biophysics and Anatomy, The University of Nebraska Medical Center, Omaha, Nebraska 68105 ABSTRACT. Tissue levels of bioactive and immunoactive ACTH were measured in both the anterior and neuro-intermediate lobes of the rat pituitary. Similar concentrations of bioactive (65 ng/mg) and immunoactive (83 ng/mg) ACTH were found in the anterior lobes from control rats. A 2-min ether stress had no effect on either bioactive or immunoactive ACTH levels in the anterior lobe. Twenty-four h after adrenalectomy the anterior lobe content of both bioactive and immunoactive ACTH decreased, only to return to supranormal levels 21 days after the operation. A 30-min neurogenic stress had no effect on anterior lobe bioactive ACTH content but reduced the immunoactive ACTH level to 50 ng/mg. Synthetic Oh17"39 ACTH was used in our radioimmunoassay in order to measure the C-terminal ACTH activity of the neuro-intermediate lobe. The concentration of such C-terminal activity in control rats (890 ng ah17"39 ACTH/mg) considerably exceeded the amount of bioactive ACTH (15 ng/mg). This is presumably due primarily to the presence of
T
HERE is little question that the neurointermediate lobe of the rat pituitary contains bioactive ACTH. A number of previous studies (1-10) all confirm its existence while disagreeing considerably regarding the amount. Immunocytochemical methods have localized this neuro-intermediate lobe activity exclusively to the pars intermedia (11-13). Recently, Scott and coworkers (14-17) have identified another peptide in the pars intermedia of several species, including the rat. Termed CLIP (corticotropin-like
Received June 12, 1974. Address reprint requests to C. Michael Moriarty, University of Nebraska Medical Center, Omaha, Nebraska 68105. Supported in part by NSF Grant GB 30318, a Nebraska Heart Association grant-in-aid and an N.I.H. Institutional Grant FR 5391.
the so-called corticotropin-like intermediate lobepeptide (CLIP). The amounts of bioactive or C-terminal immunoactive ACTH in the neurointermediate lobe were not affected by ether stress nor short term (24-h) or long term (21-day) adrenalectomy. Neuro-intermediate lobe bioactive ACTH decreased (to 8 ng/mg) only with the introduction of a 30-min neurogenic stress. Neurogenic stress had no effect on the concentration of CLIP, but when the stress was imposed 24 h after adrenalectomy, a significant reduction was observed. The data support the presence of bioactive ACTH in the intermediate lobe of the rat pituitary and suggest that such ACTH is preferentially released by neurogenic stress and not appreciably regulated by circulating levels of glucocorticoids. Until the biological function and/or target organ of CLIP is identified, the significance of the changes in tissue levels of C-terminal immunoactive ACTH will remain unknown. (Endocrinology 96: 1419, 1975)
intermediate lobe peptide), it has no N-terminal- ACTH immunoactivity, no ACTH bioactivity, and is indistinguishable from a18"39 ACTH. Its function and target organ, if any, remain unknown. The purpose of this study was to determine the variations in the levels of bioactive and C-terminal immunoactive ACTH in the neuro-intermediate lobe of the rat pituitary under the influence of stress and changing levels of plasma corticosterone. For comparison purposes similar measurements were also performed on the anterior lobe. Materials and Methods Male rats of the Sprague-Dawley strain (175-200 g) were used in all experiments. They were maintained in a temperature-controlled environment (22 C) and given food and water ad libitum. All rats were acclimatized in a regu-
1419
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MORIARTY AND MORIARTY
1420
Endo • 1975 Vol 96 • No 6
RAT ACTH 10
60 r
25.6
51.2
100
FIG. 1. Standard curves obtained with l25I-ap'-39ACTH competing with aMSH, a'-24ACTH, ah17-39ACTH and ap'-39ACTH for antibody binding. On the upper abscissa is shown the biological activity of an extract of rat pituitary. Assuming a potency of pure rat ACTH of 100 U/mg which is consistent with potencies of ACTH from other species (38) it is seen that rat and porcine ACTH react equally with the antiserum.
50
40
S
30
1 pg
10 pg
100 pg AMOUNT OF COMPETING ANTIGEN
lated light-dark environment (lights on 0600 h, off 2200 h) for at least 1 week prior to use. Adrenalectomies were performed by the dorsal approach with the rats maintained under light ether anesthesia. Each rat was bilaterally adrenalectomized within 2 - 3 min and subsequently maintained on 0.9% NaCl in lieu of water. Exposure of rats to ether fumes for 2 min was used as a systemic stress. Neurogenic stress consisted of simultaneous exposure to a loud buzzer and flashing stroboscopic light for a period of 30 min in a darkened room. All animals were killed by decapitation wil:hin 30 s after termination of the stress. All experiments were performed in the morning, with decapitations occurring between 10 and 11 AM. After decapitation, the pituitaries were quickly removed and the anterior and neurointermediate lobes gently separated. The entire procedure was accomplished within 30 s and with minimal loss of cells from the friable pars intermedia. The isolated, individual lobes were then weighed and quickly frozen. Immediately before use, they were thawed and their ACTH content was extracted according to the modified glacial acetic acid technique of Birmingham et al. (18). All lobes were extracted and measured individually without pooling. All dilutions were made with 0.15M NaCl containing 0.5% bovine serum albumin, pH 3.5. The in vitro adrenal cell dispersion technique of Sayers et al. (19) was used for the ACTH bioassay. In our hands, an additional 20 min dispersion was found to be desirable. The III
Ing
IWS ACTH was used as a standard.1 The standard curve was determined by duplicate measurements of 8-10 different ACTH concentrations. All unknowns were measured in duplicate at a minimum of 2 doses. The antiserum for radioimmunoassay was produced by multiple injections of porcine ACTH (Armour, 75 U/mg) into adult male rabbits, according to the protocol of Hum and Landon (20). Since these rabbits had previously received a single injection of synthetic a1"24 ACTH, the final antiserum contained 2 populations of antibodies reactive with ACTH. Figure 1 shows the sensitivity of aMSH and various fragments of the ACTH molecule for the antiserum. As might be expected, since a1"24 ACTH was the initial immunogen, the antiserum binds most effectively with it. The antiserum cross-reacted equally with rat and porcine ACTH, as evidenced by the parallelism of the standard curves. All measurements of the anterior ptiuitary used ap1-39 ACTH1 both for standards and for iodination, and all are expressed as ng ACTH/mg wet weight to facilitate comparisons with the neuro-intermediate lobe values. In contrast to what was observed in the anterior pituitary, we found that our radioim1 5 IU of porcine ACTH (III IWS) was supplied in ampoules containing "approximately 50 fig" of material. In order to express ACTH in gravimetric terms we have assumed each vial contained exactly 50 /ig. Conversion to units can be made by the equivalency of 100 U/mg.
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BIOACTIVE AND IMMUNOACTIVE ACTH
1421
DILUTION Of NEURO-INTERMEDIATE LOBE EXTRACT
l:10 S
l:10 4
l:10 3
l:10 2
40
FIG. 2. Parallelism of extract of the neuro-intermediate lobe with ahI7"39ACTH. Each competed with 125I-ahI7-39ACTH for the ACTH antibody. On the lower abscissa is the amount of ah17~39ACTH and on the upper abscissa are the dilutions of the neuro-intermediate lobe extract.
o z
I
30
| 2
20
S~
10 17-39
1 pg
10 pg
100 pg 7-39
1 ng
ACTH
munoassay using ap1 39 ACTH was not suitable for the neuro-intermediate lobe. A comparison of the standard curve generated by use of ap1"39 ACTH with that obtained by varying dilutions of neuro-intermediate lobe extract demonstrated a significant lack of parallelism. When we used ah17~39 ACTH for iodination and for standards, we obtained good cross reactivity with rat neuro-intermediate lobe extract (Fig. 2). For iodination, we used 125I obtained from the Iso-Serve Division, Cambridge Nuclear Corp., Billerica, Mass. The procedure of Rees et al. (21) was followed for both iodination and separation of bound from free antigen. All radioimmunoassay incubations were for 48 h at 4 C. All pituitaries were measured individually at 3 doses, each in duplicate. The results of each radioimmunoassay experiment were calculated with the aid of computer programs kindly supplied by David A. Rodbard.
renalectomy (45 ng/mg) and reached supranormal levels (159 ng/mg) after 21 days. Twenty-four hours after adrenalectomy we found that the immunoactive ACTH content in the anterior lobe was significantly less than the bioactive component. Immunoactive and bioactive ACTH values were comparable 21 days after adrenalectomy. A 2-min ether stress decreased the bioactive and immunoactive ACTH levels by 24% and 22% respectively. Neither decrease, however, was significant at the P < 0.05 level. When the ether stress was applied 24 h after bilateral adrenalectomy, the bioactive but not the immunoactive ACTH decreased significantly when compared to the effect of adrenalectomy alone (Table 1). Bioactive ACTH levels in the anterior Results lobe were unaffected by the application of Anterior lobe a neurogenic stress although the imThe ACTH concentration in control an- munoactive ACTH levels decreased. terior lobes (Table 1) was not significantly Neuro-intermediate lobe different whether measured by bioassay When expressed per mg of neuro(65 ng/mg) or radioimmunoassay (83 ng/ intermediate lobe tissue (pars nervosa + mg). As expected from previous studies pars intermedia) the bioactive ACTH was (22,23), the bioactive ACTH of the anterior 15 ng/mg (Table 2). There was no siglobe decreased 24 h after bilateral ad- nificant change in the bioactive ACTH
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Endo • 1975 Vol 96 • No 6
MORIARTY AND MORIARTY
1422
TABLE 1. Concentration of ACTH in anterior lobe 8
Saffran and Serially assay (25). The discrepancy may be partly attributable to Immunoactive Bioactive methodology. Fortier (26) has pointed out 65 (59-74)c 83 (69-97)c that pituitary ACTH concentrations measControl (9)d 24 h after ured with Saffran and Schally's assay are 45 (37-55) 25 (18-32) adrenalectomy (7) consistently higher than those obtained 21 day after with Sayers' in vivo adrenal ascorbic acid 159 (139-203) 133 (110-156) adrenalectomy (6) 65 (37-93) 49 (36-67) Ether stress (6) depletion technique (27). With the adrenal Ether stress 24 hour after ascorbic acid technique, Sayers (27) meas31 (18-44) 12 (7-17) adrenalectomy (6) ured pituitary ACTH concentrations, 50 (32-68) 76 (55-123) Neurogenic stress (8) Neurogenic stress 24 h which agree with those measured with the 39 (11-67) Not done after adrenalectomy (5) dispersed adrenal cell technique in this study and with the technique of Lipscomb • All values are expressed per mg of anterior lobe Average weight = 8.4 ± (SE) 0.3 mg (N = 47). and Nelson used by others (9,15). b 1 39 Expressed in ng of a, ' ACTH (100 U/mg). c As others have reported (28,29) a 2-min 95% confidence limits. d Number of animals. ether stress failed to lower the anterior pituitary ACTH levels in intact rats. Since content 24 h or 21 days after bilateral plasma ACTH levels are reported to be adrenalectomy, after ether stress, or when significantly elevated by this time (21, the ether stress was applied 24 h after 30,31), this might suggest that the ether adrenalectomy. However, the introduction stress increased ACTH synthesis either of a neurogenic stress significantly de- directly or indirectly through the increase pleted neuro-intermediate lobe bioactive in release. In the present study, however, ACTH (Table 2). In no case did any even in the absence of an increase in treatment alter the weight of the neuro- synthesis we would not expect to be able to intermediate lobe. detect the small depletion in pituitary The entire neuro-intermediate lobe con- ACTH necessary to elevate plasma ACTH tained 890 ng of C-terminal (ah17"39) ACTH to the reported levels. immunoactivity per mg tissue (Table 2). When the ether stress was introduced 24 Adrenalectomy and/or ether stress was in- h after bilateral adrenalectomy the pituitary effective in altering these values. The application of a neurogenic stress resulted TABLE 2. Concentration of "ACTH" in in an apparent 36% decrease, but owing to neuro-intermediate lobe8 a large rat-to-rat variation this was not Bioactive" Immunoactive0 statistically significant. When the neurogenic stress was applied 24 h after bilateral Control (8)e 15 (13-18)d 890 (540-1,240)" adrenalectomy, the levels of C-terminal 21 days after adrenalectomy (7) 13 (11-15) 870 (550-1,190) ACTH activity dropped significantly, to 21 days after about 25% of control values. adrenalectomy (5) 19 (13-27) 950 (420-1480) b
b
Discussion Bioactive ACTH in the anterior lobe The concentrations of bioactive ACTH in the pars distalis are in good agreement with those reported in several other recent studies (9,15,24). We find approximately 40 times less bioactive ACTH than reported by Kraicer et al. (10), who used the in vitro
Ether stress (6) Ether stress 24 h after adrenalectomy (6) Neurogenic stress (8) Neurogenic stress 24 h after adrenalectomy (5)
13 (8-20)
750 (370-1130)
11 (7-16) 8 (6-11)
800 (250-1350) 570 (240-900)
Not done
210 (100-340)
1 All values are expressed per mg of neuro-intermediate lobe. Average weight = 2.1 ± (SE) 0.1 mg (N = 45). b Bioactive ACTH expressed in ngof a p '- M ACTH (100 U/mg). c Immunoactive activity expressed as ng of Oh'7"89 ACTH/mg. d 95% confidence limits. e Number of animals.
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BIOACTIVE AND IMMUNOACTIVE ACTH levels of bioactive ACTH fell sharply. This is in conflict with previous reports (28,29). Rees et al. (21) have reported that when an ether stress is applied 24 h after adrenalectomy, plasma ACTH values are identical with those of similarly stressed intact rats. Dallman et al. (30) confirmed this and showed that shortly after adrenalectomy (1-2 hr) the imposition of a laparotomy stress markedly elevated plasma ACTH levels over those of nonadrenalectomized controls. However, in both of these studies immunoactive ACTH was measured and, as will be discussed in the next section, their observations are consistent with our immunoactive ACTH data. At this time we do not have a reasonable explanation for the low levels of bioactive ACTH found in the anterior pituitary when the rats are stressed with ether 24 h after adrenalectomy. The fate of this ACTH lost from the hypophysis is unknown; it is certainly not detected in the plasma (21,30). In accord with previous studies (2,3,5) when rats were subjected to a 30-min neurogenic stress there was no change in the anterior pituitary content of bioactive ACTH. At this time we have no direct evidence regarding plasma ACTH levels under these conditions. From the work on posterior lobectomized rats discussed below we suspect that the anterior lobe ACTH cells may be less affected by neurogenic stress than cells in the neurointermediate lobe. Immunoactive
ACTH in the anterior lobe
The amount of immunoactive ACTH in the control anterior pituitaries was of the same order as the amount of bioactive ACTH. This is in agreement with the findings of Shapiro et al. (9). Scott et al. (15), however, reported that in rats the level of bioactive ACTH is 2-10 times less than that of immunoactive a13~18 ACTH. Following adrenalectomy or ether stress the values of immunoactive ACTH paralleled the bioactive values, except in one instance. For reasons that are not clear
1423
there was a greater decrease in immunoactive ACTH 24 h after adrenalectomy. Anterior lobe immunoactive ACTH dropped to 30% of the control value under these conditions, a value also recently reported by Dallman et al. (30). Immunoactive ACTH levels did not change further when an ether stress was applied 24 h after adrenalectomy. The difficulty in making quantitative comparisons between bioactive and immunoactive data is appreciated. Depending on the antiserum, one may be comparing the relative activities of two different portions of the molecule. Indeed, even with antiserum directed toward the N-terminal portion of ACTH, Besser et al. (32) have found variations in the half-lives of bioactive and immunoactive ACTH. Neurogenic stress reduced the immunoactive ACTH level in the anterior pituitary without affecting the bioactive component. Without additional studies and, particularly, measurements of plasma ACTH, the significance of this decrease in immunoactive ACTH will remain obscure. The possibility exists that there are fragments of the ACTH molecule in the anterior pituitary which are selectively released and/or intracellularly degraded. We have preliminary evidence that there is a large molecular weight moiety in the rat anterior pituitary that exhibits ACTH immunoactivity. Gel filtration indicates that such material has an average molecular weight slightly less than 69,000 daltons. The changes in the amount of this material (both immunoactive and bioactive) following adrenalectomy and stress are not yet known. Bioactive ACTH in the lobe
neuro-intermediate
We found 15 ng of bioactive ACTH per mg of neuro-intermediate lobe. Recently, immunocytochemical studies of the neurointermediate lobe have shown that both C-terminal (10-13) and N-terminal (11) ACTH activities are confined exclusively
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1424
MORIARTY AND MORIARTY
to the pars intermedia. Since the pars intermedia and pars nervosa are approximately equal in weight (33,34), this is equivalent to 30 ng of ACTH per mg of pars intermedia. In contrast, Kraicer et al. (10) have reported values of bioactive ACTH in the pars intermedia 15 times greater than this. Our results agree quite well with the recent reports of Scott et al. (15) and Shapiro et al. (9). The levels of bioactive ACTH in the pars intermedia did not change after adrenalectomy or ether stress, conditions under which the ACTH levels in the anterior lobe changed appreciably. At this time we cannot say that ACTH release was also unchanged under these conditions or whether release and synthesis changed in parallel. However, there was little morphologic evidence of any increase in synthetic activity, e.g., no increase in rough endoplasmic reticulum (Moriarty, Halmi, and Moriarty, submitted for publication). In contrast with the anterior pituitary, the tissue levels of bioactive ACTH in the neuro-intermediate lobe decreased significantly after a 30-min neurogenic stress. This finding agrees with that of earlier workers (2,4,5) who suggested that the neuro-intermediate lobe was the source of the ACTH released following stresses of neurogenic origin. However, Miller et al. (35) have recently shown that the increase in plasma corticosterone following a 15-min neurogenic stress is the same for both posterior hypophysectomized and control rats. Kastin et al. (36) studied the effect of posterior lobectomy on plasma ACTH in rats adrenalectomized one month previously and in rats exposed to a 2-min ether stress. They concluded that the neuro-intermediate lobe did not participate in stress-induced ACTH release although they did not examine a neurogenic stress. Clarification of the physiological role of the pars intermedia in regulating plasma corticosterone levels requires more work. The level of bioactive ACTH in the pars intermedia certainly does not change after adrenalectomy. Whether this is due to the
Endo • 1975 Vol 96 • No 6
absence of corticosterone feedback receptors in the pars intermedia, an equal balance between synthesis and release, or to the poor vascularization that characterizes this lobe, is not known. In contrast with the anterior pituitary, the stimulated release of ACTH from the isolated neuro-intermediate lobe is not blocked by dexamethasone (37). Similarly, the nature of the agents regulating ACTH release from the pars intermedia are unknown, although evidence indicates that acetylcholine, a crude hypothalarnic extract, and dopamine may all influence release (37). 1mmunoactive ACTH in the intermediate lobe
neuro-
With the use of synthetic «h17-39 ACTH we measured 890 ng of C-terminal activity per mg of neuro-intermediate lobe or a ratio of C-terminal activity to N-terminal activity (i.e., bioactive ACTH) of approximately 60:1. Scott et al. (17) report a value of 32:1 and Shapiro et al. (9), using antiserum toward the N-terminal portion of ACTH, reported approximately equal amounts of immunoactive and bioactive ACTH in the rat neuro-intermediate lobe. Scott and his colleagues have succeeded in purifying the major C-terminal immunoactive peptide and report that it is indistinguishable from a18"39 ACTH (16). They have termed this peptide corticotropin-like intermediate lobe peptide or CLIP and have localized it to the pars intermedia. The only condition that caused a decrease in neuro-intermediate lobe levels of C-terminal ACTH was the introduction of a neurogenic stress 24 h after bilateral adrenalectomy. Part of this decrease must represent the loss of a1"39 ACTH from the pars intermedia. In contrast to our findings in the anterior lobe, we could find no large molecular weight component which exhibited ACTH immunoactivity in extracts of neuro-intermediate lobe. Thus, the Cterminal ACTH immunoactivity does not
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BIOACTIVE AND IMMUNOACTIVE ACTH appear to be due to the presence of "big ACTH". Similarly, it does not appear to be an artifact of the extraction procedure, since neuro-intermediate lobes in organ culture release immunoactive ACTH with a C/N ratio considerably greater than 1 (17). Until the target organ (if any) for CLIP becomes known, the significance of this C-terminal activity is likely to remain obscure. Acknowledgments The authors would like to express their appreciation to the Data Management Branch of the Division of Computer Research and Technology, National Institutes of Health, the Reproductive Research Branch, National Institute of Child Health and Human Development and particularly to Dr. David A. Rodbard for the computer programs used for the radioimmunoassay calculations. The generous supply of III IWS ACTH (National Institute for Medical Research, Mill Hill, London) and partially purified porcine ACTH from Dr. J. D. Fischer, Armour Pharmaceuticals, Kankakee, Illinois is gratefully acknowledged. Dr. W. Rittel, CIBA, Basle, Switzerland kindly supplied aMSH and Oh17"39 ACTH. The assistance of Dr. K. D. Patil with the statistical evaluations is appreciated.
References 1. Mialhe-Voloss, C , CR Acad Sci (Paris) 235: 743, 1952. 2. , CR Acad Sci (Paris) 241: 105, 1955. 3. J Physiol (Paris) 47: 251, 1955. 4. , Ann Endocrinol 17: 104, 1956. 5. Rochefort, G. J., J. Rosenberger, and M. Saffran,./ Physiol (Lond) 146: 105, 1959. 6. DeWied, D., Endocrinology 68: 956, 1961. 7. Smelik, P. C , Ada Endocrinol (Kbh) 33: 437, 1960. 8. Itoh, S.Jap J Physiol 12: 257, 1962. 9. Shapiro, M., W. E. Nicholson, D. N. Orth, W. M. Mitchell, D. P. Island, and G. W. Liddle, Endocrinology 90: 249, 1972. 10. Kraicer, J., J. L. Gosbee, and S. A. Bencosme, Neuroendocrinology 11: 156, 1973. 11. Baker, B. L., and T. Drummond, Am J Anat 134: 395, 1972. 12. Moriarty, G. C , and N. S. Halmi, Z Zellforsch 132: 1, 1972. 13. Phifer, R. F., and S. S. Spicer, Lab Invest 23: 543, 1970.
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14. Scott, A. P., H. P. J. Bennett, P. J. Lowry, C. McMartin, and J. G. Ratcliffe, J Endocrinol 55: xxvi, 1972. 15. , G. M. Besser, and J. G. Ratcliffe, J Endocrinol 51: i, 1971. 16. , J. G. Ratcliffe, L. H. Rees, J. Landon, and H. P. J. Bennett, Nature New Biol 244: 65, 1973. 17. , L. H. Rees, J. G. Ratcliffe, and G. M. Besser, 7 Endocrinol 53: xxviii, 1972. 18. Birmingham, M. K., E. Kurlents, G. J. Rochefort, M. Saffran, and A. V. Schally, Endocrinology 59: 677, 1956. 19. Sayers, G., R. L. Swallow, and N. D. Giordano, Endocrinology 88: 1063, 1971. 20. Hum, B. A. L., and J. Landon, In Kirkham, K. E., and W. M. Hunter (eds.), Radioimmunoassay Methods, Churchill Livingston, Edinburgh 1971, p 132. 21. Rees, L. H., D. M. Cook, J. W. Kendall, C. F. Allen, R. M. Kramer, J. G. Ratcliffe, and R. A. Knight, Endocrinology 89: 254, 1971. 22. Gemzell, C. A., D. C. vanDyke, C. A. Tobias, and H. M. Evans, Endocrinology 49: 325, 1951. 23. Fortier, C , Proc Soc Exp Biol Med 100: 13, 1968. 24. Kastin, A. J., A. Arimura, S. Viosca, L. Barrett, and A. V. Schally, Neuroendocrinology 2: 200, 1967. 25. Saffran, M., and A. V. Schally, Endocrinology 56: 523, 1955. 26. Fortier, C , Proc Soc Exp Biol Med 99: 628, 1958. 27. Sayers, M. A., G. Sayers, and L. A. Woodbury, Endocrinology 42: 379, 1948. 28. Sydnor, K. L., and G. Sayers, Endocrinology 55: 621, 1954. 29. Vernikos-Danellis, J., Endocrinology 72: 574, 1963. 30. Dallman, M. F., M. T. Jones, J. Vernikos-Danellis, and W. F. Ganong, Endocrinology 91: 961, 1972. 31. Cook, C. M., J. W. Kendall, M. A. Greer, and R. M. Kramer, Endocrinology 93: 1019, 1973. 32. Besser, G. M., D. N. Orth, W. E. Nicholson, and J. Woodham, J Clin Endocrinol Metab 32: 595, 1971. 33. Carrillo, A. J., A. J. Kastin, J. D. Dunn, and A. V. Schally, Neuroendocrinology 12: 120, 1973. 34. Gosbee, J. L., J. Kraicer, A. J. Kastin, and A. V. Schally, Endocrinology 86: 560, 1970. 35. Miller, R. E., H. Yueh-Chien, M. K. Wiley, and R. Hewitt, Neuroendocrinology 14: 233, 1974. 36. Kastin, A. J., S. Viosca, and L. Debeljuk, Program of the 53rd Meeting of the Endocrine Society, 1971, p. A-211, Abstract 338. 37. Fischer, J. L., and C. M. Moriarty, Fed Proc 33: 206, 1974 (Abstract). 38. Ney, R. L., E. Ogata, N. Shimizu, W. E. Nicholson, and G. W. Liddle, Exerpta Med Intern Congr Ser83: 1184, 1964.
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