Notes and Comments Arginine Vasotocin in the Rabbit Subcommissural Organ A. A. ROSENBLOOM AND D. A. FISHER Departments of Medicine and Pediatrics, UCLA School of Medicine, Harbor General Hospital Campus, Torrance, California 90509 ABSTRACT. Subcommissural organs of young and mature rabbits were analyzed for their content of arginine vasotocin by radioimmunoassay. Younger animals had significantly greater quantities of this peptide. There was no detectable arginine vasopressin or oxytocin in subcommissural organ ex-
T
HE subcommissural organ (SCO) occurs throughout chordate phylogeny and forms an ependymal projection into the third ventricle at the border between the diencephalon and mesencephalon just below the posterior commissure (1). Histologic investigation of the SCO in the hagfish has revealed the presence of apical round granules in the ependymal cells (2), and histochemical studies in the rat have shown that the SCO granular material appears remarkably similar to that stored in the neurohypophysis (3). These findings have been corroborated by histological and histochemical studies in SCO tissue from 5 fetal (1) and 9 adult humans (4). Histological similarity also has been noted between the pineal and the SCO (5), and this similarity led Pavel to investigate the possible occurrence of arginine vasotocin (AVT) in bovine SCO, since AVT already had been identified in bovine pineal (6,7). Indeed, the bovine SCO in adult and fetal animals contains AVT as measured by frog bladder bioassay (7). In this communication we confirm the presence of radioimmunoassayable AVT in the SCO of young and mature rabbits and describe an apparent decrease in SCO AVT content with increasing age.
Materials and Methods Rabbit SCO were obtained from Pel-Freez Biologicals, Inc., Rogers, Arkansas. The tissues were removed rapidly from the brain after decapitation and quickly frozen. The animals were of mixed breed and
tracts. It is concluded that the subcommissural organ represents, in addition to the pineal and the fetal neurohypophysis, another significant source of arginine vasotocin in the mammalian central nervous system. (Endocrinology 96: 1038, 1975)
sex; however, young (8-12 weeks) and mature (1-3 yr) samples were kept separate. Upon arrival in dry ice the tissues were extracted with acetone for 18 h and dehydrated in vacuo. After weighing, on the day of assay, the samples were homogenized in VA% normal rabbit serum in phosphate (50 mM) buffered normal saline, 1.0 ml per organ. The suspension was centrifuged at 4 C for 30 min at 1200 x g and 100 ^tl of supernatant was added to the radioimmunoassay (RIA) mixture. A few tissue specimens were extracted with Vt% acetic acid and then placed in boiling water for two minutes. The suspension was centrifuged as above and the supernatant was stored at 4 C. RIA for AVT was carried out as recently described (10). Briefly, our RIA system utilizes two antisera with differing specificities for AVT and arginine vasopressin (AVP) (11). Oxytocin (OT) interferes with the AVT determination only if the ratio of OT:AVT is greater than 4:1. Identification of AVT by paper chromatography in butanol: acetic acid: water (4:l:5::v:v:v) was carried out on acetic acid extracts of SCO as previously described (10).
Results Table 1 shows the results of the SCO analysis. Young rabbit SCO weighed less than SCO from older animals, but contained more AVT per mg wet weight, and more total AVT per organ. All differences between young and mature animals were statistically significant (P < 0.05). AVP was undetectable in all tissues examined. An acetic acid extract of a pool of mature and young TABLE 1. Analysis of rabbit subcommissural organs AVT content*
Received September 16, 1974. Supported in part by Public Health Service Grant HD-06335 from the National Institute of Child Health and Human Development of the National Institutes of Health, Bethesda, Maryland.
Wt. (mg)* Young Mature
4.52 ± 0.23 (14) 5.85 ± 0.44 (11)
LJ/ni|?
9.9 ± 2.4 (10) 5.7 i 1.6 (10)
/xU/organ 44.0 ± 10.2 (10) 29.9 ± 6.2 (10)
* Mean ± SEM; values in parentheses represent the number of organs.
1038
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1039
NOTES AND COMMENTS
45PEP"riDE
rabbit SCO was subjected to paper chromatography to investigate the possible presence of OT. Figure 1 depicts the pattern of elution of immunoreactive material from chromatograms of SCO, reference AVT and OT. All immunoreactive material in the tissue was present in the slow-moving fraction with an Rf comparable to that of AVT.
3
References 1. Olsson, R., Gen Comp Endocrinol 1: 117, 1961. 2. Afzelius, B. A., and R. Olsson, Z Zellforsch 46: 672, 1957. 3. Wislocki, G. B., and E. H. LeducJ Comp Neurol 101: 283, 1954.
15-
1 Urn
450300-
Discussion AVT is the parent molecule of the antidiuretic hormones and has classically been thought to occur only in submammalian vertebrates (12). However, recent studies have revealed that AVT occurs in neurohypophyses of fetal mammals (13) and that the ratio of AVT:AVP decreases throughout fetal life (14). In addition, the present study, as well as others (6,7), have revealed the presence of AVT in adult life persisting in the ependymal areas of the pineal gland and in the SCO. It has been postulated that AVT is a pineal antigonadotrophin which acts by way of the cerebrospinal fluid (CSF) (15). AVT has also been identified in mammalian CSF after thiopental administration (8) and after hypertonic saline (16) or melatonin (9) injection into the third ventricle. In view of the anatomic similarity and close proximity of the SCO to the pineal it may well be that these tissues act in concert. In any case, it appears that the adult SCO represents a significant pool of AVT in the mammalian central nervous system whatever its physiological role might be.
30-
1500.2
—r0.4
0.6
0.8
Rf FIG. 1. Paper chromatography of acetic acid extracts of rabbit subcommissural organs. A-elution pattern of rabbit SCO; B-elution pattern of synthetic AVT and
OT. 4. Gilbert, G. J., Neurology 10: 138, 1960. 5. Palkovits, M , G. Imke, and G. Lukacs, Endokrinologie 42: 194, 1962. 6. Milcu, S. M., S. Pavel, and C. Neascu, Endocrinology 72: 563, 1963. 7. Pavel, S., Endocrinology 89: 613, 1971. , / Clin Endocrinol Metab 31: 369, 1970. 8. 9. , Nature New Biol 246: 183, 1973. 10. Rosenbloom, A. A., and D. A. Fisher, Endocrinology 95: 1726, 1974. 11. Skowsky, W. R., A. A. Rosenbloom, and D. A. Fisher J Clin Endocrinol Metab 38: 278, 1974. 12. Sawyer, W. H., In Berde, B. (ed.), Neurohypophysial Hormones and Similar Peptides. Handbuch der Experimentellen Pharmakologie, Springer-Verlag, Berlin, 1968, p. 717. 13. Vizsolyi, E., and A. M. Perks, Nature 223: 1169, 1969. 14. Skowsky, W. R., and D. A. Fisher, Clin Res 21: 205, 1973. 15. Pavel, S., M. Petrescu, and N. Vicoleanu, Neuroendocrinology 11: 370, 1973. 16. , and M. Coculescu, Endocrinology 91: 825, 1972.
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T
HE subcommissural organ (SCO) occurs throughout chordate phylogeny and forms an ependymal projection into the third ventricle at the border between the diencephalon and mesencephalon just below the posterior commissure (1). Histologic investigation of the SCO in the hagfish has revealed the presence of apical round granules in the ependymal cells (2), and histochemical studies in the rat have shown that the SCO granular material appears remarkably similar to that stored in the neurohypophysis (3). These findings have been corroborated by histological and histochemical studies in SCO tissue from 5 fetal (1) and 9 adult humans (4). Histological similarity also has been noted between the pineal and the SCO (5), and this similarity led Pavel to investigate the possible occurrence of arginine vasotocin (AVT) in bovine SCO, since AVT already had been identified in bovine pineal (6,7). Indeed, the bovine SCO in adult and fetal animals contains AVT as measured by frog bladder bioassay (7). In this communication we confirm the presence of radioimmunoassayable AVT in the SCO of young and mature rabbits and describe an apparent decrease in SCO AVT content with increasing age.
Materials and Methods Rabbit SCO were obtained from Pel-Freez Biologicals, Inc., Rogers, Arkansas. The tissues were removed rapidly from the brain after decapitation and quickly frozen. The animals were of mixed breed and
tracts. It is concluded that the subcommissural organ represents, in addition to the pineal and the fetal neurohypophysis, another significant source of arginine vasotocin in the mammalian central nervous system. (Endocrinology 96: 1038, 1975)
sex; however, young (8-12 weeks) and mature (1-3 yr) samples were kept separate. Upon arrival in dry ice the tissues were extracted with acetone for 18 h and dehydrated in vacuo. After weighing, on the day of assay, the samples were homogenized in VA% normal rabbit serum in phosphate (50 mM) buffered normal saline, 1.0 ml per organ. The suspension was centrifuged at 4 C for 30 min at 1200 x g and 100 ^tl of supernatant was added to the radioimmunoassay (RIA) mixture. A few tissue specimens were extracted with Vt% acetic acid and then placed in boiling water for two minutes. The suspension was centrifuged as above and the supernatant was stored at 4 C. RIA for AVT was carried out as recently described (10). Briefly, our RIA system utilizes two antisera with differing specificities for AVT and arginine vasopressin (AVP) (11). Oxytocin (OT) interferes with the AVT determination only if the ratio of OT:AVT is greater than 4:1. Identification of AVT by paper chromatography in butanol: acetic acid: water (4:l:5::v:v:v) was carried out on acetic acid extracts of SCO as previously described (10).
Results Table 1 shows the results of the SCO analysis. Young rabbit SCO weighed less than SCO from older animals, but contained more AVT per mg wet weight, and more total AVT per organ. All differences between young and mature animals were statistically significant (P < 0.05). AVP was undetectable in all tissues examined. An acetic acid extract of a pool of mature and young TABLE 1. Analysis of rabbit subcommissural organs AVT content*
Received September 16, 1974. Supported in part by Public Health Service Grant HD-06335 from the National Institute of Child Health and Human Development of the National Institutes of Health, Bethesda, Maryland.
Wt. (mg)* Young Mature
4.52 ± 0.23 (14) 5.85 ± 0.44 (11)
LJ/ni|?
9.9 ± 2.4 (10) 5.7 i 1.6 (10)
/xU/organ 44.0 ± 10.2 (10) 29.9 ± 6.2 (10)
* Mean ± SEM; values in parentheses represent the number of organs.
1038
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1039
NOTES AND COMMENTS
45PEP"riDE
rabbit SCO was subjected to paper chromatography to investigate the possible presence of OT. Figure 1 depicts the pattern of elution of immunoreactive material from chromatograms of SCO, reference AVT and OT. All immunoreactive material in the tissue was present in the slow-moving fraction with an Rf comparable to that of AVT.
3
References 1. Olsson, R., Gen Comp Endocrinol 1: 117, 1961. 2. Afzelius, B. A., and R. Olsson, Z Zellforsch 46: 672, 1957. 3. Wislocki, G. B., and E. H. LeducJ Comp Neurol 101: 283, 1954.
15-
1 Urn
450300-
Discussion AVT is the parent molecule of the antidiuretic hormones and has classically been thought to occur only in submammalian vertebrates (12). However, recent studies have revealed that AVT occurs in neurohypophyses of fetal mammals (13) and that the ratio of AVT:AVP decreases throughout fetal life (14). In addition, the present study, as well as others (6,7), have revealed the presence of AVT in adult life persisting in the ependymal areas of the pineal gland and in the SCO. It has been postulated that AVT is a pineal antigonadotrophin which acts by way of the cerebrospinal fluid (CSF) (15). AVT has also been identified in mammalian CSF after thiopental administration (8) and after hypertonic saline (16) or melatonin (9) injection into the third ventricle. In view of the anatomic similarity and close proximity of the SCO to the pineal it may well be that these tissues act in concert. In any case, it appears that the adult SCO represents a significant pool of AVT in the mammalian central nervous system whatever its physiological role might be.
30-
1500.2
—r0.4
0.6
0.8
Rf FIG. 1. Paper chromatography of acetic acid extracts of rabbit subcommissural organs. A-elution pattern of rabbit SCO; B-elution pattern of synthetic AVT and
OT. 4. Gilbert, G. J., Neurology 10: 138, 1960. 5. Palkovits, M , G. Imke, and G. Lukacs, Endokrinologie 42: 194, 1962. 6. Milcu, S. M., S. Pavel, and C. Neascu, Endocrinology 72: 563, 1963. 7. Pavel, S., Endocrinology 89: 613, 1971. , / Clin Endocrinol Metab 31: 369, 1970. 8. 9. , Nature New Biol 246: 183, 1973. 10. Rosenbloom, A. A., and D. A. Fisher, Endocrinology 95: 1726, 1974. 11. Skowsky, W. R., A. A. Rosenbloom, and D. A. Fisher J Clin Endocrinol Metab 38: 278, 1974. 12. Sawyer, W. H., In Berde, B. (ed.), Neurohypophysial Hormones and Similar Peptides. Handbuch der Experimentellen Pharmakologie, Springer-Verlag, Berlin, 1968, p. 717. 13. Vizsolyi, E., and A. M. Perks, Nature 223: 1169, 1969. 14. Skowsky, W. R., and D. A. Fisher, Clin Res 21: 205, 1973. 15. Pavel, S., M. Petrescu, and N. Vicoleanu, Neuroendocrinology 11: 370, 1973. 16. , and M. Coculescu, Endocrinology 91: 825, 1972.
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