0013-7227/92/1301-0008$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
The Early
Vol. 130, No. 1
Printed
Society
History
of the Releasing
in U.S.A.
Factors
the work of Verney that stresses of various sorts would release vasopressin and since lesions which produced diabetes insipidus, thereby eliminating vasopressin, were associated with a blockade of ACTH release, we postulated that vasopressin could be the ACTH-releasing hormone via its release into portal vessels and its delivery to the corticotrophs in high concentration. We later showed (1957) that if we used the animals 48 h after lesions, we had a more sensitive test animal which could respond to as little as 0.1 U iv vasopressin and failed to respond to the abovementioned other putative releasers. Furthermore, synthetic lysine vasopressin kindly donated to us by duvigneaud (Cornell University Medical School, New York, NY) was equally active indicating that the ACTH-releasing activity was caused by vasopressin itself and not a contaminant in Pitressin. Before that time we had tested acid hypothalamic extracts from beef and rat in the animals with chronic lesions and found them to be inactive. Consequently, we concluded that vasopressin was the ACTH-releasing peptide. Thus, vasopressin was the first releasing factor to be discovered. Saffran and Schally (1955) developed an in uitro system of pituitary incubation and published confirmation of our findings that pressor posterior pituitary preparations caused a release of ACTH, but only from glands which were also incubated in the presence of norepinephrine. In retrospect, it is now apparent why it was necessary to incubate with norepinephrine. It was because of the failure to use preincubation. Therefore, the ACTH released at the time of the initial cutting of the gland presented a very high background, and it was difficult to observe further stimulation. Shortly thereafter in collaboration with B. J. Benfey, Saffran and Schally reported that chromatography of purified vasopressin preparations separated on the chromatogram a zone which released ACTH and yet was devoid of vasopressin. They named this substance CRF. We were unable to confirm this activity by in uiuo bioassay in our animals with acute hypothalamic lesions. Independent of Saffran and Schally, Guillemin and Rosenberg (1955) used a hypothalamic-pituitary coculture system to demonstrate increased ACTH release in the presence of hypothalami which could not be accounted for by the small amount of vasopressin released. With Hearn, Guillemin reported that Pitressin would
The brilliant studies of Harris, in which he showed that intact portal venous connections between the hypothalamus and pituitary gland were essential for normal pituitary function, stimulated a search for the putative neurohumoral agents now termed releasing and inhibiting factors. The principal problem in the releasing factor field as with all fields in biology was the development of suitable bioassays for the putative neurohormones. A number of abortive attempts were made to establish CRFs by injecting substances into Nembutal-anesthetized rats. These were of little value since the rat responds to many stimuli by a release of ACTH. For example, Martini (1954) injected Pitressin into Nembutal-anesthetized rats and evoked ACTH release, but in view of the vasoactive properties of vasopressin, this could have been attributed to a stress response. Later he applied Pitressin directly to anterior pituitary grafts in the anterior chamber of the eye and obtained ACTH release (1956). McCann and Brobeck (1954) published the first experiments designed to evaluate ACTH-releasing substances in an animal in which the ubiquitous ACTH release from stress was abolished. McCann (1953) had earlier shown that median eminence lesions would block the ACTH release from stress and the response to epinephrine. Consequently, he ruled out epinephrine as a corticotropin-releasing factor. McCann and Brobeck showed that a reproducible blockade of the response to stress occurred in animals with severe diabetes insipidus. They used these as assay animals for putative ACTH-releasing substances. In these animals with chronic median eminence lesions, they demonstrated that a high dose of iv Pitressin, a commercially available, partially purified vasopressin, would release ACTH and that this ACTH-releasing action was not shared by oxytocin, the other known neurohypophyseal hormone, or by other putative releasing compounds, such as epinephrine, serotonin, substance P, and histamine. In these animals with chronic lesions, the dose of Pitressin required was very high (5 U), a concentration which we thought would never reach the pituitary via the systemic circulation. It was already known from Received August 27, 1991. “Remembrance” articles discuss people and events as remembered by the author. The opinion(s) expressed are solely those of the writer and do not reflect the view of the Journal or The Endocrine Society. 8
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REMEMBRANCE release ACTH in uitro, but that synthetic vasopressin had no activity. In fact, the response to synthetic vasopressin was only slightly less than that to Pitressin, but not quite statistically significant. Probably if they had increased the dose of synthetic vasopressin slightly, they would have confirmed our in uiuo results. Because of this finding and the report of Saffran et al., they chromatographed posterior pituitary extracts using a different system than that employed by Saffran et al. and reported the separation of fraction D, which ran at the solvent front and contained no pressor activity but would release ACTH in vitro. We repeated this work using our in uiuo assay system and confirmed that fraction D, which was running at the solvent front, was well separated from the pressor portion of the chromatogram (Rf = 0.4), which released ACTH in animals with median eminence lesions. Fraction D was ineffective even at a lo-fold higher dose than the pressor fraction from the chromatogram. Guillemin provided us with his fraction D. It produced only a very small response at a dose of 0.5 mg. I elevated the dose to 1 mg, and it was ineffective. These doses were massive compared to the dose of vasopressin required. At this point, Hearn left Houston for a position at Iowa State, where he worked with a graduate student to purify posterior pituitary extracts. They reported that they could find nothing in such extracts which would release ACTH other than vasopressin. This was also reported later by Porter. In the meantime, Schally had joined Guillemin in Houston, and they went on to isolate and determine a proposed structure for ,&CRF which found its way into biochemistry textbooks for the next 15 years. They also isolated crl and a2 CRF which were purported to be related to MSH. In his lecture at the Laurentian Hormone Conference in 1963, Guillemin admitted that it was not clear whether there was any meaning to any of the postulated posterior pituitary CRFs. In discussion, Saffran reported that fractionation of 16 batches of posterior pituitary extract had revealed a CRF other than vasopressin only in 6 of the extracts, and he was reporting his CRF in terms of vasopressin units. The first important work on hypothalamic CRF was performed by Royce and Sayers (1958). They found that stalk median eminence extracts from beef would release ACTH in animals with acute median eminence lesions and went on to purify the active substance. They showed that it was a peptide and obtained preparations which were essentially free from vasopressin. We (1959) showed that even crude median eminence extracts from both rat and beef had CRF activity which could not be accounted for by vasopressin and confirmed the activity of the
9
purified CRF provided to us by Royce and Sayers. At this point in time, it was quite clear that there were at least two peptides with releasing factor activity, namely, vasopressin and CRF of unknown structure. Another advance was to realize that one could not use the posterior pituitary powders provided by Parke Davis for extraction of releasing factors since, as mentioned above, there was little CRF in posterior pituitaries, and what little was there was masked by the massive amounts of vasopressin, the other CRF. A number of other people purified hypothalamic CRF, including Porter, Schally, Guillemin, Leeman, and ourselves, but it was not isolated, and its structure was not determined. In retrospect, this failure was probably caused by the large size of the molecule (41 amino acids). Its structure was finally elucidated in 1981 by Vale’s group. Even in these early days (1958), we found that adrenal corticoids probably exerted part of their suppressive action on ACTH release by suppressing the release of vasopressin, since we were able to show that the stressinduced increase in blood vasopressin levels was decreased by treatment of the rats with cortisol. We also demonstrated in collaboration with Yates’s group that vasopressin would enhance CRF-induced ACTH release by direct pituitary action in the anesthetized rat (1968), a result confirmed in vitro nearly 10 years later by Gillies and Lowry (1978). Furthermore, animals with hereditary diabetes insipidus and complete loss of vasopressin had an impairment in ACTH secretion, albeit relatively small (1966). We postulated that vasopressin and CRF cooperated in control of ACTH release. Studies carried out after the elucidation of the structure of CRF over 20 years later have clearly demonstrated that this is indeed the case. Nonetheless, neither the role of vasopressin in control of ACTH secretion nor the releasing factor concept was generally accepted in the late 1950s; however, these early experiments led directly to the search for and rapid discovery of the other releasing and inhibiting hormones during the 1960s (see refs. in Ref. 1). Samuel M. McCann Department of Physiology Neuropeptide Division The University of Texas Southwestern Medical Center References 1. McCann SM 1988 Saga of the discovery of hypothalamic releasing and inhibiting hormones. In: McCann SM (ed) People and Ideas in Endocrinology. American Physiological Society, Bethesda, pp 41-62
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 March 2015. at 21:15 For personal use only. No other uses without permission. . All rights reserved.
The Early
Vol. 130, No. 1
Printed
Society
History
of the Releasing
in U.S.A.
Factors
the work of Verney that stresses of various sorts would release vasopressin and since lesions which produced diabetes insipidus, thereby eliminating vasopressin, were associated with a blockade of ACTH release, we postulated that vasopressin could be the ACTH-releasing hormone via its release into portal vessels and its delivery to the corticotrophs in high concentration. We later showed (1957) that if we used the animals 48 h after lesions, we had a more sensitive test animal which could respond to as little as 0.1 U iv vasopressin and failed to respond to the abovementioned other putative releasers. Furthermore, synthetic lysine vasopressin kindly donated to us by duvigneaud (Cornell University Medical School, New York, NY) was equally active indicating that the ACTH-releasing activity was caused by vasopressin itself and not a contaminant in Pitressin. Before that time we had tested acid hypothalamic extracts from beef and rat in the animals with chronic lesions and found them to be inactive. Consequently, we concluded that vasopressin was the ACTH-releasing peptide. Thus, vasopressin was the first releasing factor to be discovered. Saffran and Schally (1955) developed an in uitro system of pituitary incubation and published confirmation of our findings that pressor posterior pituitary preparations caused a release of ACTH, but only from glands which were also incubated in the presence of norepinephrine. In retrospect, it is now apparent why it was necessary to incubate with norepinephrine. It was because of the failure to use preincubation. Therefore, the ACTH released at the time of the initial cutting of the gland presented a very high background, and it was difficult to observe further stimulation. Shortly thereafter in collaboration with B. J. Benfey, Saffran and Schally reported that chromatography of purified vasopressin preparations separated on the chromatogram a zone which released ACTH and yet was devoid of vasopressin. They named this substance CRF. We were unable to confirm this activity by in uiuo bioassay in our animals with acute hypothalamic lesions. Independent of Saffran and Schally, Guillemin and Rosenberg (1955) used a hypothalamic-pituitary coculture system to demonstrate increased ACTH release in the presence of hypothalami which could not be accounted for by the small amount of vasopressin released. With Hearn, Guillemin reported that Pitressin would
The brilliant studies of Harris, in which he showed that intact portal venous connections between the hypothalamus and pituitary gland were essential for normal pituitary function, stimulated a search for the putative neurohumoral agents now termed releasing and inhibiting factors. The principal problem in the releasing factor field as with all fields in biology was the development of suitable bioassays for the putative neurohormones. A number of abortive attempts were made to establish CRFs by injecting substances into Nembutal-anesthetized rats. These were of little value since the rat responds to many stimuli by a release of ACTH. For example, Martini (1954) injected Pitressin into Nembutal-anesthetized rats and evoked ACTH release, but in view of the vasoactive properties of vasopressin, this could have been attributed to a stress response. Later he applied Pitressin directly to anterior pituitary grafts in the anterior chamber of the eye and obtained ACTH release (1956). McCann and Brobeck (1954) published the first experiments designed to evaluate ACTH-releasing substances in an animal in which the ubiquitous ACTH release from stress was abolished. McCann (1953) had earlier shown that median eminence lesions would block the ACTH release from stress and the response to epinephrine. Consequently, he ruled out epinephrine as a corticotropin-releasing factor. McCann and Brobeck showed that a reproducible blockade of the response to stress occurred in animals with severe diabetes insipidus. They used these as assay animals for putative ACTH-releasing substances. In these animals with chronic median eminence lesions, they demonstrated that a high dose of iv Pitressin, a commercially available, partially purified vasopressin, would release ACTH and that this ACTH-releasing action was not shared by oxytocin, the other known neurohypophyseal hormone, or by other putative releasing compounds, such as epinephrine, serotonin, substance P, and histamine. In these animals with chronic lesions, the dose of Pitressin required was very high (5 U), a concentration which we thought would never reach the pituitary via the systemic circulation. It was already known from Received August 27, 1991. “Remembrance” articles discuss people and events as remembered by the author. The opinion(s) expressed are solely those of the writer and do not reflect the view of the Journal or The Endocrine Society. 8
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 March 2015. at 21:15 For personal use only. No other uses without permission. . All rights reserved.
REMEMBRANCE release ACTH in uitro, but that synthetic vasopressin had no activity. In fact, the response to synthetic vasopressin was only slightly less than that to Pitressin, but not quite statistically significant. Probably if they had increased the dose of synthetic vasopressin slightly, they would have confirmed our in uiuo results. Because of this finding and the report of Saffran et al., they chromatographed posterior pituitary extracts using a different system than that employed by Saffran et al. and reported the separation of fraction D, which ran at the solvent front and contained no pressor activity but would release ACTH in vitro. We repeated this work using our in uiuo assay system and confirmed that fraction D, which was running at the solvent front, was well separated from the pressor portion of the chromatogram (Rf = 0.4), which released ACTH in animals with median eminence lesions. Fraction D was ineffective even at a lo-fold higher dose than the pressor fraction from the chromatogram. Guillemin provided us with his fraction D. It produced only a very small response at a dose of 0.5 mg. I elevated the dose to 1 mg, and it was ineffective. These doses were massive compared to the dose of vasopressin required. At this point, Hearn left Houston for a position at Iowa State, where he worked with a graduate student to purify posterior pituitary extracts. They reported that they could find nothing in such extracts which would release ACTH other than vasopressin. This was also reported later by Porter. In the meantime, Schally had joined Guillemin in Houston, and they went on to isolate and determine a proposed structure for ,&CRF which found its way into biochemistry textbooks for the next 15 years. They also isolated crl and a2 CRF which were purported to be related to MSH. In his lecture at the Laurentian Hormone Conference in 1963, Guillemin admitted that it was not clear whether there was any meaning to any of the postulated posterior pituitary CRFs. In discussion, Saffran reported that fractionation of 16 batches of posterior pituitary extract had revealed a CRF other than vasopressin only in 6 of the extracts, and he was reporting his CRF in terms of vasopressin units. The first important work on hypothalamic CRF was performed by Royce and Sayers (1958). They found that stalk median eminence extracts from beef would release ACTH in animals with acute median eminence lesions and went on to purify the active substance. They showed that it was a peptide and obtained preparations which were essentially free from vasopressin. We (1959) showed that even crude median eminence extracts from both rat and beef had CRF activity which could not be accounted for by vasopressin and confirmed the activity of the
9
purified CRF provided to us by Royce and Sayers. At this point in time, it was quite clear that there were at least two peptides with releasing factor activity, namely, vasopressin and CRF of unknown structure. Another advance was to realize that one could not use the posterior pituitary powders provided by Parke Davis for extraction of releasing factors since, as mentioned above, there was little CRF in posterior pituitaries, and what little was there was masked by the massive amounts of vasopressin, the other CRF. A number of other people purified hypothalamic CRF, including Porter, Schally, Guillemin, Leeman, and ourselves, but it was not isolated, and its structure was not determined. In retrospect, this failure was probably caused by the large size of the molecule (41 amino acids). Its structure was finally elucidated in 1981 by Vale’s group. Even in these early days (1958), we found that adrenal corticoids probably exerted part of their suppressive action on ACTH release by suppressing the release of vasopressin, since we were able to show that the stressinduced increase in blood vasopressin levels was decreased by treatment of the rats with cortisol. We also demonstrated in collaboration with Yates’s group that vasopressin would enhance CRF-induced ACTH release by direct pituitary action in the anesthetized rat (1968), a result confirmed in vitro nearly 10 years later by Gillies and Lowry (1978). Furthermore, animals with hereditary diabetes insipidus and complete loss of vasopressin had an impairment in ACTH secretion, albeit relatively small (1966). We postulated that vasopressin and CRF cooperated in control of ACTH release. Studies carried out after the elucidation of the structure of CRF over 20 years later have clearly demonstrated that this is indeed the case. Nonetheless, neither the role of vasopressin in control of ACTH secretion nor the releasing factor concept was generally accepted in the late 1950s; however, these early experiments led directly to the search for and rapid discovery of the other releasing and inhibiting hormones during the 1960s (see refs. in Ref. 1). Samuel M. McCann Department of Physiology Neuropeptide Division The University of Texas Southwestern Medical Center References 1. McCann SM 1988 Saga of the discovery of hypothalamic releasing and inhibiting hormones. In: McCann SM (ed) People and Ideas in Endocrinology. American Physiological Society, Bethesda, pp 41-62
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