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On the Presence of Neuropeptides in the Mammalian Pineal Gland and Subcommissural Organ
465
On the Presence of Neuropeptides in the Mammalian Pineal Gland and Subcommissural Organ J. DOGTEROM 1 , F.G.M. SNIJDEWINT l , P. PEVET
,2
and R.M. BUIJS
Netherlands Institute for Brain Research, Amsterdam, and 2 Department of Anatomy and Embryology, University of Amsterdam, Amsterdam (The Netherlands)
INTRODUCTION The presence of the neurohypophysial hormones arginine-8-vasopressin (AVP) and oxytocin (OX) is not confined to their production sites in the hypothalamus (suprachiasmatic, paraventricular and supraoptic nucleus), the hypothalamo-neurohypophysialtract and the neurohypophysis. AVP- and OX-containing fibres were visualized by immunocytochemical methods also in a number of rat brain nuclei outside the hypothalamus (Buijs, 1978; Buijs et al., 1978). These observations were confirmed by radioimmunoassay (RIA) measurements of both hormones in punched samples of rat brain (Dogterom et al., 1978a). In addition, several circumventricular organs, such as the subfornical organ (SFO), the organum vasculosum laminae terminalis (OVLT) and the subcommissural organ (SCO), appeared to contain one or more neurohypophysial hormones (Buijs, 1978; Buijs et al., 1978; Dogterom et al., 1978a; Summy-Long et al., (1978). Rare immunoreactive AVP-containing fibres were also observed in the rat pineal stalk (Buijs, unpublished observation). Numerous reports, mostly based on bioassay (Pavel, 1965, 1971), but also on chemical identification (Cheesman, 1970), described that arginine-8-vasotocin (AVT) - a neurohypophysial hormone of the lower vertebrates - is present in the pineal gland of several mammalian species. Also by RIA, AVT was demonstrated in the SCO of rabbit and in the pineal of rat and human fetuses but this was done with antisera that were raised against AVP and OX (Rosenbloom and Fisher, 1975a, b; Legros et al., 1976). Since peptidergic compounds like AVP, OX and AVT might be involved in one or more central nervous system functions like sleep regulation (Pavel et al., 1977), storage and retrieval of information (Bohus et al., 1978), water balance (Summy-Long et al., 1978) and synchronization of the reproduction cycle (Vaughan et al., 1978) the specific measurement of these hormones in well-defined brain regions under various conditions seems important. The present report describes the measurement of these hormones in pineal glands and SCO by 3 specific RIA’S employing sensitive AVP, OX and AVT antisera. MATERIAL AND METHODS All peptides used in this study were synthetic products generously supplied by Dr. H.M. Greven (Organon, Oss, The Netherlands), except mesotocin which was generously given by Dr. F. Vandesande, University of Ghent, Belgium. The following were used: AVP (for
466
immunization: 180 IU/mg, for standard curves and labelled hormone: 509 IU/mg), AVT (234 IU/mg) and OX (509 IU/mg). The peptides were stored in stock solutions of suitable concentration in 0.25% acetic acid at -20°C. Radwimmunoassays The procedure of raising antibodies, preparation and purification of labelled hormones, preparation of the standard curves and extraction of the hormones with activated Vycor glass powder from the samples have been described for the RIA of AVP and OX (Dogterom et al., 1977, 1978b). The RIA of AVT is similar to the RIA of OX. In order to control the specificity of the RIA’S and the recovery of the extraction procedure, standard amounts of AVP, OX and AVT were added to tubes containing the same solutions in which tissue samples were sonified. Routinely 6 tubes were included in each extraction experiment containing the following: 0.5 ml 0.1 N HC1, 50 pl 1 N NaOH, 1.5 ml veronal buffer (pH 7.0); two tubes contained in addition 128 pg AVT, 128 pg OX and 32 pg AVP; two tubes contained 32 pg AVT, 32 pg OX and 8 pg AVP; two tubes contained no hormone. pH values of these tubes were controlled and adjusted between 6 and 7. This was done also for the unknown samples. The RIA data are corrected for recovery. Rat samples Male Wistar rats and male rats of the Brattleboro strain (homozygous for hereditary diabetes insipidus: HoDI) weighing 150-200 g were used. The animals had free access to water and food. The animal house was illuminated from 7.00 h to 19.00 h. Rats were anesthetized with pentobarbitone and perfused with saline in order to remove the blood content of the various structures, before decapitation and removal of the pineal glands and SCO’s. The latter samples consisted of a macroscopical dissected piece of tissue containing the SCO and surrounding tissue. The tissue was homogenized after weighmg in 0.5 mlO.1 N HCl by ultrasonification and stored at -7OOC. These homogenates were neutralized with 50 pl 1 N NaOH and buffered with 1.5 ml veronal buffer (pH 7.0) immediately before starting the extraction procedure. Pineals of 19-day-old rat fetuses and of 1-day-old pups were isolated immediately after decapitation and sonified in 0.5 mlO.1 N HCl. Rabbit and bovine samples SCO preparations of rabbits and bovine pineals were obtained from Pel Freeze Biological Inc. (Rogers, Arkansas, U.S.A.). The tissue arrived in dry ice and was stored at -70°C until homogenized and extracted as described for the rat samples. RESULTS Vasotocin-radwimmunoassay The immunization procedure employed produced AVT-antisera of similar quality as the AVP- and OX-antisera, The AVT tracers had specific activities varying between 250 and 500 mCi/mg. The AVT-antiserum No. 5 (22-6) appeared to be highly specific; the crossreactivity with mesotocin was negligible while the cross-reactivity with OX and AVP decreased rapidly with increasing levels of AVT (Fig. 1). The recovery of AVT in the extraction procedure with Vycor glass powder was 49.5 f.4.5 (n = 25) over a range of 4-128 pg when standard AVT was added to Ho-DI plasma or assay buffer. In blank tubes containing
467
.
-301 ‘ a25
”
I
k
”
4
8
‘ ‘we
”’
50
’ 5w
k?
pIcWr0m.I
5.103
&
5.d
Fig. 1. Representative standard curve of vasotocin-antiserum “5” (22-6) 1 : 20.000. Cross-reactivity with related peptides is also shown: , 0 X X, oxytocin; A--, mesotocin.
b
5.d
$.
in final dilution of arginine-8-vasopressin;
(0-)
HCI, NaOH and the veronal assay buffer, non-specific values were not found in any of the 3 assays. The detection limit for AVP and AVT was 2 pg and for OX 4 pg per sample. Within assay variability for the AVT-RIA was 19.3% (8 identical samples containing 16 pg AVT in duplicate in one assay) and in-between assay variability was 19.6% (8 assays of identical samples containing 16 pg standard AVT in duplicate). Assay results Table I summarizes the results of hormone measurements after extraction of the different tissue homogenates. OX was present in all samples; in the rabbit SCO preparation even in
TABLE I NEUROHYPOPHYSIAL HORMONE CONTENT OF RAT AND BOVINE PINEAL GLAND AND OF RAT AND RABBIT SCO WITH SURROUNDING TISSUE Measurements were performed in samples containing 3 pineals or one K O . Tissue was sonified in 0.5 ml 0.1 N HCl, neutralized with 50 pl 1 N NaOH, buffered with veronal buffer (pH 7.0) and subsequently extracted with Vycor glass powder. Values are expressed in pg hormonelmg wet tissue; mean f SEM is shown; n = number of samples; und. = undetectable. ~
~~
~
~
AVT
Wistar rat SCO
3.2 * 0.9 (n = 3) und.
Brattleboro rat SCO
und.
Wistar rat pineal
und.
Brattleboro rat pineal
und.
Bovine pineal (Pel Freeze)
und.
Rabbit SCO (Pel Freeze)
~
ox
AVP
1077.4 f 236.8 (n = 3) 16.8 i 1.6 (n = 3) 13.2 f 2.0 (n = 3) 1.3 f 0.8 (n = 3) 8.7 f 3.7 (n = 3) 3.7 f 1.4 ( n = 16)
3316.3 (n = 3)
28.0
(n = 3) und.
2.7 (n = 3) und. 3.7
f
522.0
f
5.0
f
0.8
(n= 16)
0.7
468
nanogram amounts. AVP was also detected in all the samples except from the Ho-DI rats. AVT was not detectable with exception of the rabbit SCO, but this “AVT”-immunoreactivity can be explained by the high amounts of AVP present in this preparation (crossreaction). Furthermore, AVT was not detectable in pineals of 19-day-old rat fetuses, or in 1-day-old rat pups. DISCUSSION In addition to previoudy described RIA’S for AVP and OX we have available an AVTRIA, using an antiserum raised with thyroglobulincoupled AVT itself. This assay has features similar t o the A W - and OX-RIA, as regards binding capacity, the specific activity of labelled hormone, the sensitivity of the standard curve and the recovery of the extraction procedure (Dogterom et al., 1977, 1978a). Measurement of the 3 hormones from the same extracted sample became thus possible. Since the specificity of each antiserum is well-defined, crossreaction with closely related compounds can be estimated very well. All 3 antisera employed had such specificity that it was possible to discriminate between the 3 hormones in the same samples. With the AVT-RIA, the AVT content of the pituitaries of frogs, young chickens, ducklings and several other birds could be measured adequately (Dogterom et al., in preparation). In these species, the dilution curves of pituitary homogenates paralleled the standard curve of our synthetic AVT preparation as well as two other synthetic AVT preparations provided by Bachem Inc. (U.S.A.) and Dr. F. Vandesande (University of Ghent, Belgium). The results in the rat and rabbit SCO preparations as reported in Table I concern more exactly the SCO and the adjacent brain tissue, removed during the dissection. This adjacent tissue is very rich in AVP/OX fibres (Buijs, 1978). Only few AVP-containing fibres have been observed by immunocytochemistry in the SCO (Buijs, unpublished observation), and the AVP level in the SCO itself (punched samples) was not detectable by RIA (Dogterom et al., in preparation). Rare AVP-containing fibres were also observed by immunocytochemistry in the rat pineal stalk (Buijs, unpublished observation). Since the pineal stalk is removed with the pineal during the dissection, this observation could explain the presence of the neurohypophysial hormones demonstrated by RIA. Neurohypophysial hormone activity in the pineal stalk was also demonstrated by bioassay (Benson et al., 1976). AVP and OX were detected in the pineal of all mammals studied by RIA. Recently, the existence of two neurophysins in the pineal of beef has been reported (Reinharz and Valoton, 1977). It might be that the two neurophysins are those related t o A W and OX. No evidence for the presence of AVT in the SCO preparations has been obtained either by RIA or immunocytochemistry. Similarly, no evidence for the presence of AVT in the pineal gland was obtained by RIA. Using an immunocytochemical technique, Bowie and Herbert (1976) obtained staining in the pineal. However, using several antisera against AVT, we have observed immunoreactivity with only one antiserum, but this appeared to be the result of cross-reaction with an AVT4ike compound and not of staining of AVT itself (PCvet et al., to be published). From the present series of experiments, we conclude that an AVT-like compound could be present in mammalian pineals and/or SCOH but that this compound is different from AVT itself.
469
ACKNOWLEDGEMENTS The Foundation for Medical Research FUNGO is gratefully acknowledged for additional financial support. REFERENCES Benson, B., Mathews, M.J., Hadly, M.E., Powers, S. and Hruby, V.J. (1976) Differential localization of antigonadotropic and vasotocic activity in bovine and rat pineal. Life Sci., 19: 747-754. Bohus, B., Urban, I., Van Wimersma Greidanus, Tj.B. and De Wied, D. (1978) Opposite effects of oxytocin and vasopressin on avoidance behaviour and hippocampal theta rhythm in the rat. Neuropharmacology, 17: 239-247. Bowie, E.P. and Herbert, D.C. (1976) Immunocytochemical evidence for the presence of argininevasotocin in the rat pineal gland. Nature (Lond.). 261: 5555-5556. Buijs, R.M. (1978) Intra- and extra-hypothalamic vasopressin and oxytocin pathways in the rat: pathways to the limbic system, medulla oblongata and spinal cord. Cell Tiss. Res., 192: 423-435. Buijs, R.M., Swaab, D.F., Dogterom, J. and Van Leeuwen, F.W. (1978) Intra- and extra-hypothalamic vasopressin and oxytocin pathways in the rat. Cell Tiss. Res., 186: 423-433. Cheesman, D.W. (1970) Structure elucidation of a gonadotropin inhibiting substance from the bovine pineal gland. Biochim. biophys. Acta, 207: 247-253. Dogterom, J., Van Wimersma Greidanus, Tj.B. and Swaab, D.F. (1977) Evidence for the release of vasopressin and oxytocin into cerebrospinal fluid: measurements in plasma and CFS of intact and hypophysectomized rats. Neuroendocrinology, 24: 108-1 18. Dogterom, J., Snijdewint, F.G.M. and Buijs, R.M. (1978a) The distribution of vasopressin and oxytocin in the rat brain. Neurosci. Lett., 9: 341-346. Dogterom, J., Van Wimersma Greidanus, Tj.B. and De Wied, D. (1978b) Vasopressin in cerebrospinal fluid and plasma of man, dog and rat. Amer. J. PhysioZ., 235 (5): E463-467. Legros, J.J., Louis, F., Demoulin, A. and Franchimont, P. (1976) Immunoreactive neurophysins and vasotocin in human foetal pineal glands. J. Endocr., 69: 289-290. Pavel, S. (1965) Evidence for the presence of lysine-vasotocin in the pig pineal gland. Endocrinology, 77: 812-817. Pavel. S , (1971) Evidence for the ependymal origin of arginine vasotocin in the bovine pineal gland. Endocrinology, 89: 613-614. Pavel, S., Patta, D. and Goldstein, R. (1977) Slow wave sleep induced in cats by extremely small amounts of synthetic and pineal vasotocin injected into the third ventricle of the brain. Bruin Res. BUN., 2: 251-254. Reinharz, A.C. and Vallotton, M.B. (1977) Presence of two neurophysins in the human pineal gland. Endocrinology, 100: 994-1001. Rosenbloom, A.A. and Fisher, D.A. (1975a) Arginine vasotocin in the rabbit subcommissural organ. Endocrinology, 96: 1038- 1039. Rosenbloom, A.A. and Fisher, D.A. (1975b) Radioimmunoassayable AVT and AVP in adult mammalian brain tissue: comparison of normal and Brattleboro rats. Neuroendocrinology, 17: 354-361. Summy-Long, J.Y., Keil, L.C. and Severs, W.B. (1978) Identification of vasopressin in the subfornical organ region: effects of dehydration. Brain Res., 140: 241-250. Vaughan, M.K. and Blask, D.E. (1978) Arginine vasotocin - A search for its function in mammals in: R.J. Reiter and P.O. Hubmant (Eds.), The Pineal and Reproduction. Karger, Basel, pp. 90-1 15.
DISCUSSION S. PAVEL: My work demonstrating for the first time the presence of AVT by differential bioassay in the mammalian pineal gland has been confnmed by a number of authors using chemical analysis, radioimmunoassay and immunocytochemistry. AVT was also identified in the neurohypophysis of fetal mammals by differential bioassay, while its biosynthesis was demonstrated in the neurohypophysis of
410
human fetuses. Since AVT shows an extremely specific pharmacological profile, it is very difficult to assume, as proposed by you, that the pineal gland would contain a peptide having such a pharmacological profile, but not being AVT. Any change that would modify the molecular structure of AVT would induce the loss of its extremely specific biological activities. D.F. SWAAB: A number of anti-AVT sera do not give any positive staining in the pineal. This supports the idea that the pineal, indeed, contains an AVT-like compound and not AVT itself.
I. NIR: Why is there no positive control for AVT in the experiment on the rat pineal? J. DOGTEROM: The hormone content of the pineal was measured by radioimmunoassay after an extraction procedure. Each extraction included control samples to which vasotocin was added and on which the recovery calculation was based. The data are corrected for recovery. The control values were not shown on the slide. Moreover, we measured vasotocin directly in diluted homogenates of frog and chicken pituitaries and found 0.5 1 rglgland in frog and 1.19 &gland in chicken.
On the Presence of Neuropeptides in the Mammalian Pineal Gland and Subcommissural Organ J. DOGTEROM 1 , F.G.M. SNIJDEWINT l , P. PEVET
,2
and R.M. BUIJS
Netherlands Institute for Brain Research, Amsterdam, and 2 Department of Anatomy and Embryology, University of Amsterdam, Amsterdam (The Netherlands)
INTRODUCTION The presence of the neurohypophysial hormones arginine-8-vasopressin (AVP) and oxytocin (OX) is not confined to their production sites in the hypothalamus (suprachiasmatic, paraventricular and supraoptic nucleus), the hypothalamo-neurohypophysialtract and the neurohypophysis. AVP- and OX-containing fibres were visualized by immunocytochemical methods also in a number of rat brain nuclei outside the hypothalamus (Buijs, 1978; Buijs et al., 1978). These observations were confirmed by radioimmunoassay (RIA) measurements of both hormones in punched samples of rat brain (Dogterom et al., 1978a). In addition, several circumventricular organs, such as the subfornical organ (SFO), the organum vasculosum laminae terminalis (OVLT) and the subcommissural organ (SCO), appeared to contain one or more neurohypophysial hormones (Buijs, 1978; Buijs et al., 1978; Dogterom et al., 1978a; Summy-Long et al., (1978). Rare immunoreactive AVP-containing fibres were also observed in the rat pineal stalk (Buijs, unpublished observation). Numerous reports, mostly based on bioassay (Pavel, 1965, 1971), but also on chemical identification (Cheesman, 1970), described that arginine-8-vasotocin (AVT) - a neurohypophysial hormone of the lower vertebrates - is present in the pineal gland of several mammalian species. Also by RIA, AVT was demonstrated in the SCO of rabbit and in the pineal of rat and human fetuses but this was done with antisera that were raised against AVP and OX (Rosenbloom and Fisher, 1975a, b; Legros et al., 1976). Since peptidergic compounds like AVP, OX and AVT might be involved in one or more central nervous system functions like sleep regulation (Pavel et al., 1977), storage and retrieval of information (Bohus et al., 1978), water balance (Summy-Long et al., 1978) and synchronization of the reproduction cycle (Vaughan et al., 1978) the specific measurement of these hormones in well-defined brain regions under various conditions seems important. The present report describes the measurement of these hormones in pineal glands and SCO by 3 specific RIA’S employing sensitive AVP, OX and AVT antisera. MATERIAL AND METHODS All peptides used in this study were synthetic products generously supplied by Dr. H.M. Greven (Organon, Oss, The Netherlands), except mesotocin which was generously given by Dr. F. Vandesande, University of Ghent, Belgium. The following were used: AVP (for
466
immunization: 180 IU/mg, for standard curves and labelled hormone: 509 IU/mg), AVT (234 IU/mg) and OX (509 IU/mg). The peptides were stored in stock solutions of suitable concentration in 0.25% acetic acid at -20°C. Radwimmunoassays The procedure of raising antibodies, preparation and purification of labelled hormones, preparation of the standard curves and extraction of the hormones with activated Vycor glass powder from the samples have been described for the RIA of AVP and OX (Dogterom et al., 1977, 1978b). The RIA of AVT is similar to the RIA of OX. In order to control the specificity of the RIA’S and the recovery of the extraction procedure, standard amounts of AVP, OX and AVT were added to tubes containing the same solutions in which tissue samples were sonified. Routinely 6 tubes were included in each extraction experiment containing the following: 0.5 ml 0.1 N HC1, 50 pl 1 N NaOH, 1.5 ml veronal buffer (pH 7.0); two tubes contained in addition 128 pg AVT, 128 pg OX and 32 pg AVP; two tubes contained 32 pg AVT, 32 pg OX and 8 pg AVP; two tubes contained no hormone. pH values of these tubes were controlled and adjusted between 6 and 7. This was done also for the unknown samples. The RIA data are corrected for recovery. Rat samples Male Wistar rats and male rats of the Brattleboro strain (homozygous for hereditary diabetes insipidus: HoDI) weighing 150-200 g were used. The animals had free access to water and food. The animal house was illuminated from 7.00 h to 19.00 h. Rats were anesthetized with pentobarbitone and perfused with saline in order to remove the blood content of the various structures, before decapitation and removal of the pineal glands and SCO’s. The latter samples consisted of a macroscopical dissected piece of tissue containing the SCO and surrounding tissue. The tissue was homogenized after weighmg in 0.5 mlO.1 N HCl by ultrasonification and stored at -7OOC. These homogenates were neutralized with 50 pl 1 N NaOH and buffered with 1.5 ml veronal buffer (pH 7.0) immediately before starting the extraction procedure. Pineals of 19-day-old rat fetuses and of 1-day-old pups were isolated immediately after decapitation and sonified in 0.5 mlO.1 N HCl. Rabbit and bovine samples SCO preparations of rabbits and bovine pineals were obtained from Pel Freeze Biological Inc. (Rogers, Arkansas, U.S.A.). The tissue arrived in dry ice and was stored at -70°C until homogenized and extracted as described for the rat samples. RESULTS Vasotocin-radwimmunoassay The immunization procedure employed produced AVT-antisera of similar quality as the AVP- and OX-antisera, The AVT tracers had specific activities varying between 250 and 500 mCi/mg. The AVT-antiserum No. 5 (22-6) appeared to be highly specific; the crossreactivity with mesotocin was negligible while the cross-reactivity with OX and AVP decreased rapidly with increasing levels of AVT (Fig. 1). The recovery of AVT in the extraction procedure with Vycor glass powder was 49.5 f.4.5 (n = 25) over a range of 4-128 pg when standard AVT was added to Ho-DI plasma or assay buffer. In blank tubes containing
467
.
-301 ‘ a25
”
I
k
”
4
8
‘ ‘we
”’
50
’ 5w
k?
pIcWr0m.I
5.103
&
5.d
Fig. 1. Representative standard curve of vasotocin-antiserum “5” (22-6) 1 : 20.000. Cross-reactivity with related peptides is also shown: , 0 X X, oxytocin; A--, mesotocin.
b
5.d
$.
in final dilution of arginine-8-vasopressin;
(0-)
HCI, NaOH and the veronal assay buffer, non-specific values were not found in any of the 3 assays. The detection limit for AVP and AVT was 2 pg and for OX 4 pg per sample. Within assay variability for the AVT-RIA was 19.3% (8 identical samples containing 16 pg AVT in duplicate in one assay) and in-between assay variability was 19.6% (8 assays of identical samples containing 16 pg standard AVT in duplicate). Assay results Table I summarizes the results of hormone measurements after extraction of the different tissue homogenates. OX was present in all samples; in the rabbit SCO preparation even in
TABLE I NEUROHYPOPHYSIAL HORMONE CONTENT OF RAT AND BOVINE PINEAL GLAND AND OF RAT AND RABBIT SCO WITH SURROUNDING TISSUE Measurements were performed in samples containing 3 pineals or one K O . Tissue was sonified in 0.5 ml 0.1 N HCl, neutralized with 50 pl 1 N NaOH, buffered with veronal buffer (pH 7.0) and subsequently extracted with Vycor glass powder. Values are expressed in pg hormonelmg wet tissue; mean f SEM is shown; n = number of samples; und. = undetectable. ~
~~
~
~
AVT
Wistar rat SCO
3.2 * 0.9 (n = 3) und.
Brattleboro rat SCO
und.
Wistar rat pineal
und.
Brattleboro rat pineal
und.
Bovine pineal (Pel Freeze)
und.
Rabbit SCO (Pel Freeze)
~
ox
AVP
1077.4 f 236.8 (n = 3) 16.8 i 1.6 (n = 3) 13.2 f 2.0 (n = 3) 1.3 f 0.8 (n = 3) 8.7 f 3.7 (n = 3) 3.7 f 1.4 ( n = 16)
3316.3 (n = 3)
28.0
(n = 3) und.
2.7 (n = 3) und. 3.7
f
522.0
f
5.0
f
0.8
(n= 16)
0.7
468
nanogram amounts. AVP was also detected in all the samples except from the Ho-DI rats. AVT was not detectable with exception of the rabbit SCO, but this “AVT”-immunoreactivity can be explained by the high amounts of AVP present in this preparation (crossreaction). Furthermore, AVT was not detectable in pineals of 19-day-old rat fetuses, or in 1-day-old rat pups. DISCUSSION In addition to previoudy described RIA’S for AVP and OX we have available an AVTRIA, using an antiserum raised with thyroglobulincoupled AVT itself. This assay has features similar t o the A W - and OX-RIA, as regards binding capacity, the specific activity of labelled hormone, the sensitivity of the standard curve and the recovery of the extraction procedure (Dogterom et al., 1977, 1978a). Measurement of the 3 hormones from the same extracted sample became thus possible. Since the specificity of each antiserum is well-defined, crossreaction with closely related compounds can be estimated very well. All 3 antisera employed had such specificity that it was possible to discriminate between the 3 hormones in the same samples. With the AVT-RIA, the AVT content of the pituitaries of frogs, young chickens, ducklings and several other birds could be measured adequately (Dogterom et al., in preparation). In these species, the dilution curves of pituitary homogenates paralleled the standard curve of our synthetic AVT preparation as well as two other synthetic AVT preparations provided by Bachem Inc. (U.S.A.) and Dr. F. Vandesande (University of Ghent, Belgium). The results in the rat and rabbit SCO preparations as reported in Table I concern more exactly the SCO and the adjacent brain tissue, removed during the dissection. This adjacent tissue is very rich in AVP/OX fibres (Buijs, 1978). Only few AVP-containing fibres have been observed by immunocytochemistry in the SCO (Buijs, unpublished observation), and the AVP level in the SCO itself (punched samples) was not detectable by RIA (Dogterom et al., in preparation). Rare AVP-containing fibres were also observed by immunocytochemistry in the rat pineal stalk (Buijs, unpublished observation). Since the pineal stalk is removed with the pineal during the dissection, this observation could explain the presence of the neurohypophysial hormones demonstrated by RIA. Neurohypophysial hormone activity in the pineal stalk was also demonstrated by bioassay (Benson et al., 1976). AVP and OX were detected in the pineal of all mammals studied by RIA. Recently, the existence of two neurophysins in the pineal of beef has been reported (Reinharz and Valoton, 1977). It might be that the two neurophysins are those related t o A W and OX. No evidence for the presence of AVT in the SCO preparations has been obtained either by RIA or immunocytochemistry. Similarly, no evidence for the presence of AVT in the pineal gland was obtained by RIA. Using an immunocytochemical technique, Bowie and Herbert (1976) obtained staining in the pineal. However, using several antisera against AVT, we have observed immunoreactivity with only one antiserum, but this appeared to be the result of cross-reaction with an AVT4ike compound and not of staining of AVT itself (PCvet et al., to be published). From the present series of experiments, we conclude that an AVT-like compound could be present in mammalian pineals and/or SCOH but that this compound is different from AVT itself.
469
ACKNOWLEDGEMENTS The Foundation for Medical Research FUNGO is gratefully acknowledged for additional financial support. REFERENCES Benson, B., Mathews, M.J., Hadly, M.E., Powers, S. and Hruby, V.J. (1976) Differential localization of antigonadotropic and vasotocic activity in bovine and rat pineal. Life Sci., 19: 747-754. Bohus, B., Urban, I., Van Wimersma Greidanus, Tj.B. and De Wied, D. (1978) Opposite effects of oxytocin and vasopressin on avoidance behaviour and hippocampal theta rhythm in the rat. Neuropharmacology, 17: 239-247. Bowie, E.P. and Herbert, D.C. (1976) Immunocytochemical evidence for the presence of argininevasotocin in the rat pineal gland. Nature (Lond.). 261: 5555-5556. Buijs, R.M. (1978) Intra- and extra-hypothalamic vasopressin and oxytocin pathways in the rat: pathways to the limbic system, medulla oblongata and spinal cord. Cell Tiss. Res., 192: 423-435. Buijs, R.M., Swaab, D.F., Dogterom, J. and Van Leeuwen, F.W. (1978) Intra- and extra-hypothalamic vasopressin and oxytocin pathways in the rat. Cell Tiss. Res., 186: 423-433. Cheesman, D.W. (1970) Structure elucidation of a gonadotropin inhibiting substance from the bovine pineal gland. Biochim. biophys. Acta, 207: 247-253. Dogterom, J., Van Wimersma Greidanus, Tj.B. and Swaab, D.F. (1977) Evidence for the release of vasopressin and oxytocin into cerebrospinal fluid: measurements in plasma and CFS of intact and hypophysectomized rats. Neuroendocrinology, 24: 108-1 18. Dogterom, J., Snijdewint, F.G.M. and Buijs, R.M. (1978a) The distribution of vasopressin and oxytocin in the rat brain. Neurosci. Lett., 9: 341-346. Dogterom, J., Van Wimersma Greidanus, Tj.B. and De Wied, D. (1978b) Vasopressin in cerebrospinal fluid and plasma of man, dog and rat. Amer. J. PhysioZ., 235 (5): E463-467. Legros, J.J., Louis, F., Demoulin, A. and Franchimont, P. (1976) Immunoreactive neurophysins and vasotocin in human foetal pineal glands. J. Endocr., 69: 289-290. Pavel, S. (1965) Evidence for the presence of lysine-vasotocin in the pig pineal gland. Endocrinology, 77: 812-817. Pavel. S , (1971) Evidence for the ependymal origin of arginine vasotocin in the bovine pineal gland. Endocrinology, 89: 613-614. Pavel, S., Patta, D. and Goldstein, R. (1977) Slow wave sleep induced in cats by extremely small amounts of synthetic and pineal vasotocin injected into the third ventricle of the brain. Bruin Res. BUN., 2: 251-254. Reinharz, A.C. and Vallotton, M.B. (1977) Presence of two neurophysins in the human pineal gland. Endocrinology, 100: 994-1001. Rosenbloom, A.A. and Fisher, D.A. (1975a) Arginine vasotocin in the rabbit subcommissural organ. Endocrinology, 96: 1038- 1039. Rosenbloom, A.A. and Fisher, D.A. (1975b) Radioimmunoassayable AVT and AVP in adult mammalian brain tissue: comparison of normal and Brattleboro rats. Neuroendocrinology, 17: 354-361. Summy-Long, J.Y., Keil, L.C. and Severs, W.B. (1978) Identification of vasopressin in the subfornical organ region: effects of dehydration. Brain Res., 140: 241-250. Vaughan, M.K. and Blask, D.E. (1978) Arginine vasotocin - A search for its function in mammals in: R.J. Reiter and P.O. Hubmant (Eds.), The Pineal and Reproduction. Karger, Basel, pp. 90-1 15.
DISCUSSION S. PAVEL: My work demonstrating for the first time the presence of AVT by differential bioassay in the mammalian pineal gland has been confnmed by a number of authors using chemical analysis, radioimmunoassay and immunocytochemistry. AVT was also identified in the neurohypophysis of fetal mammals by differential bioassay, while its biosynthesis was demonstrated in the neurohypophysis of
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human fetuses. Since AVT shows an extremely specific pharmacological profile, it is very difficult to assume, as proposed by you, that the pineal gland would contain a peptide having such a pharmacological profile, but not being AVT. Any change that would modify the molecular structure of AVT would induce the loss of its extremely specific biological activities. D.F. SWAAB: A number of anti-AVT sera do not give any positive staining in the pineal. This supports the idea that the pineal, indeed, contains an AVT-like compound and not AVT itself.
I. NIR: Why is there no positive control for AVT in the experiment on the rat pineal? J. DOGTEROM: The hormone content of the pineal was measured by radioimmunoassay after an extraction procedure. Each extraction included control samples to which vasotocin was added and on which the recovery calculation was based. The data are corrected for recovery. The control values were not shown on the slide. Moreover, we measured vasotocin directly in diluted homogenates of frog and chicken pituitaries and found 0.5 1 rglgland in frog and 1.19 &gland in chicken.