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night rhythm of vasopressin and oxytocin in rat retina, pineal and harderian gland
Peptides, Vol. 9, pp. 289-293. ©Pergamon Press plc, 1988. Printed in the U.S.A.
0196-9781/88 $3.00 + .00
A Day/Night Rhythm of Vasopressin and Oxytocin in Rat Retina, Pineal and Harderian Gland G U I L L E M E T T E G A U Q U E L I N , .1 C L A U D E G H A R I B , .2 F A R Z A M G H A E M M A G H A M I , * ANNE-MARIE ALLEVARD,* FARID CHERBAL,t GHISLAINE GEELEN,* FATHIA BOUZEGHRANEt AND JEAN-JACQUES LEGROS$
*Laboratoire de Physiologie, Facult# de M#decine Grange-Blanche 8, Avenue Rockefeller, 69373, Lyon C#dex 08 France tLaboratoire de Neurobiologie USTBH, BP 139 Darelbeida, Algiers, Algeria ~Laboratoire de Radioimmunologie Neuroendocrinologie, Universit# de Likge Sart-Tilman CHU 23, B 4000, Liege Belgique R e c e i v e d 24 S e p t e m b e r 1986 GAUQUELIN, G., C. GHARIB, F. GHAEMMAGHAMI, A.-M. ALLEVARD, F. CHERBAL, G. GEELEN, F. BOUZEGHRANE AND J.-J. LEGROS. A day~nightrhythm of vasopressin and oxytocin in rat retina, pineal and harderiangland.PEPTIDES 9(2) 289-293, 1988.--Arginine vasopressin (AVP), oxytocin (OT) and neurophysins (Np) have been found in the pineal gland and the retina of the rat. Because the retina, pineal gland and Harderian gland (HG) serve analogous functions, we undertook a study to determine the presence of these peptides in these three organs of rats. They were detected by two specific methods: HPLC and specific radioimmunoassays. For Np, total neurophysins (NpT) were measured. To determine a 24 hr rhythm, the animals were maintained under a light/dark cycle of 12 hr/12 hr for 3 weeks. The pineal glands, retinae and HG were collected. Day/night rhythms of AVP, OT and NpT were demonstrated in the retina and HG; but the pineal gland had only AVP rhythm. A significant decrease in the rhythms at 4 a.m. was demonstrated in the retina and HG. The 24 hr variation of AVP in the retina seemed parallel to that of the HG. Vasopressin
Oxytocin
Retina
Pineal gland
Harderian gland
T H E Harderian glands (HG) are found in all vertebrates (with the exception of the higher primates) and are located in the orbits [26]. Their functions have not been proved although they are supposedly involved in the lubrication of the eyes, in sexual behavior or aggression [29]. But several arguments are in favor of another function: the HG could be an extraretinal photoreceptor, since the removal of the gland abolishes the pineal serotonin response to light in blinded animals, and porphyrins of the glands show fluctuations under different lighting conditions [31]. The presence of melatonin is also demonstrated in all species studies. Moreover, the H G synthesize hundreds of times more methoxyindoles than the pineal or the retina whether it be a day or annual rhythmicity [4]. Existing data confirm the analogy among the 3 organs. Since the presence of the neuropeptides arginine vasopressin (AVP), oxytocin (OT) and their central nervous system-associated peptides neurophysins (Np) have been demonstrated in the pineal [8, 9, 21, 23] and the retina [8], the present study was undertaken to demonstrate (a) the presence of these neuropeptides in the HG and (b) the existence of a 24 hr rhythmicity in the 3 organs.
METHOD Two different experiments were carried out.
Determination of Neuropeptides and Neurophysin Direct determination. Twenty-four adult male Wistar rats (Iffa Credo) were decapitated at 9 a.m. The HG were rapidly removed, then frozen at -80°C until assay. After thawing, two HG were homogenized in 1 ml cold water p H 5.5 and centrifuged at 4°C at 11,000 rpm for 30 min. The resulting clear supernatant was divided into aliquots and stored at -20°C until analyzed for their AVP, OT and Np content. The radioimmunoassay of AVP, OT and Np has been described previously for pineal gland [7,9] and retina [8].
High performance liquid chromatography (HPLC). HPLC coupled with radioimmunoassay (RIA) was applied to 20 pooled HG. Extraction of tissues was accomplished using a modification of the method of Dorsa and Bottemiller [5]. The HPLC system was a Kontron Instrument equipped with a C-18 (5 microns) reversed phase Kontron column (25 cm x 4.6). Peptide elution was recorded at 206 nm. Peptides were separated by a solvent system consisting of 8 ( ~ 0.025
1G. Gauquelin is Fellow of CNES. 2Requests for reprints should be addressed to C. Gharib.
289
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FIG. 1. Absorption (top) and immunoreactivity (middle) after injection of a mixture of synthetic peptides. Immunoreactive AVP and OT content (bottom) of fractions collected after injection of an extract of 20 rat Harderian glands. M sodium phosphate pH 7.0 and 20% acetonitrile. The column was washed with standard buffer between each run. In addition, control injections were performed and tested for peptide by RIA. After injection of an HG sample, a mixture of synthetic AVP and OT (500 ng each) was injected to determine the recovery of the column. The separation and recovery of both AVP and OT were visualized on a recorder. Column elutions were collected in tubes at 1 min intervals using a fraction collector, then lyophilized and reconstituted with assay buffer for peptide determination by RIA, as previously described [9].
Rhythmicity of Peptide Secretion One hundred and eight adult male Wistar rats were used in this experiment. They were kept under light:dark conditions of 12:12. Twelve rats were sacrificed in February, at 10 a.m., 2 p.m., 6 p.m., 10 p.m., 2 a.m., 4 a.m., 7 a.m., 10 a.m. and 2 p.m.. After decapitation, the pineal, the retinae and the Harderian glands were quickly removed under a 25 W red light. The organs were frozen in liquid nitrogen and kept at -80°C. AVP, OT and Np were measured. After thawing, two retinae, and two Harderian glands were homogenized in 1 ml cold water pH 5.5 (the pineal gland in 100/zl) and centrifuged at 4°C at 11,000 rpm for 30 min. The supernatant was divided into aliquots and stored at 20°C until analyzed for AVP, OT and Np content. The proteins were measured by the method of Lowry [19].
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The mean was calculated and a one-way analysis of variance was performed, followed by a Newman and Keuls' test. The correlation coefficients (r) between OT, AVP, NpT were established by a simple correlation test. The calculation of periodic regression was done by a mathematical model leading to a symmetrical sine curve. The length of the fundamental period and the acrophase 0 (the maximum of the best least-squares fit sine wave) were estimated by the least squares method, with a fine step of adjustment. A cosine function Y = M + A cos (wt + 0) was used as a model of rhythm. Y is the value at a time t of the periodic function of angular frequency w : 2/t, A and 0. The amplitude A is measured by the difference between crest and
D A Y / N I G H T R H Y T H M O F V A S O P R E S S I N A N D OXYTOCIN
291
TABLE 1 MESOR, ACROPHASEAND AMPLITUDE FOR AVP, OT AND NpT IN THE PINEAL, THE HARDERIANGLAND AND THE RETINA (MEAN -+ SEM) Mesor
Acrophase Hour/Min
Amplitude
40 -+ 5 16.5 -+ 3 14 .+ 0.1
22hr40-+ 53 4 h r 4 .+ 73 10 hr 40
27.7 -+ 20 10.9 -+ 5 0.06 _+ 0.05
Pineal fmol/gland
AVP OT NpT
HG pmol/mg gland
AVP OT NpT
120 490 12
.+ 2 _+ 7 .+ 2
22hr 13-+ 1 2 hr 10 _+ 3 21hr40- 1
80 210 11
Retina fmol/mg prot
AVP OT NpT
254 132 50
.+ 43 .+ 12 .+ 0.7
17 hr 19 .+ 1 21 hr 19 _+ 45 21 hr 19 .+ 9
132.8 _+ 36 43.3 19 _+ 0.03
mesor of a best fitting cosine. The mesor M is the midway value between the peak and trough of the function used to approximate the biological rhythm [2]. RESULTS The results were expressed per mg of proteins for retina and HG. The presence of AVP (Range: 0.05 to 0.58 pM/mg prot.), OT (0.1 to 1 pM/mg prot.) and Np (5 to 19 fM/mg prot.) was demonstrated in 24 HG. The RIAs used were sensitive from 2.5 pg AVP/tube, 5 pg OT/tube and 0.2 ng Np/tube. AVP and OT standard curves and the dilution curves from the HG showed considerable parallelism. The Spearman correlation between Np and AVP was 0.6, and between Np and OT, 0.58. Over a 4 and 6-min period, the HPLC system reliably resolved A V P and OT respectively (Fig. 1). When eluates of the mixture and HG were collected and assayed by RIA, AVP and OT were detected only in those eluates shown to contain peptides by the 206 nm detector. HPLC recovery was 90% for 500 pmol and 60% for 50 pmol. Figure 2 shows the rhythms of AVP, OT and Np in pineal gland, retina and HG of rat. In the pineal gland, analysis of variance showed a significant variation over 24 hours (F=2.5, p<0.05) for AVP. Pineal AVP levels reached a peak during the dark phase. The t-test revealed that at 10 p.m. the mean A V P level increased significantly in comparison to 10 a.m., 2 p.m. and 4 a.m. levels. There was no significant variation for OT ( F = 1.9) and Np ( F = I . 1 ) . The correlation between Np and OT was rs=0.49 (p <0.01) and between AVP-OT and Np was rs=0.31 (p<0.05). In the retina, analysis of variance disclosed a significant variation over 24 hours for AVP (F=6.9, p<0.001), for OT ( F = 3 , p<0.01) and for Np (F=2.2, p<0.05). The t-test showed a significant decrease at 2 a.m., 4 a.m. and 7 a.m. for AVP, OT and Np, as compared with 10 a.m. In the HG, analysis of variance showed a significant variation over 24 hours for AVP (F=6.5, p<0.001), for OT (F=5.2, p<0.001) and for Np (F=5.4, p<0.001). The t-test revealed a significant decrease at 4 a.m. and 7 a.m. for A V P and OT, and an increase at 4 a.m. for Np. The correlation between retina and HG for AVP is rs=0.3 (p<0.01). Table 1 shows the times at which maximum levels occurred. In the HG, AVP and NpT had identical acrophases.
-+ 2 .+ 10 -+ 1
OT also had approximately the same acrophase as NpT. In the retina, we found the same acrophase for OT and NpT. Our results show that AVP and OT are not simultaneously synchronized with NpT in most of the cases, but the NpT are the precursors of AVP and OT. The antiserum used cross-reacted equally with Np linked to AVP and Np linked to OT, so it is difficult to have the same acrophase for AVP and/or OT and NpT. In these structures the pattern of AVP, OT and NpT is almost identical with a marked decrease in concentration at 4 a.m. This shows that the concentration of AVP, OT and NpT in the retina followed a circadian rhythm. An ultradian rhythm (5 hr period) was detected for OT in the pineal and Harderian gland and for NpT in the retina. DISCUSSION Our results clearly demonstrate the presence of AVP, OT and Np in the Harderian glands of the rat. For AVP and OT, the presence was confirmed by RIA measurements on HPLC fractions. These results are not surprising since AVP and OT have been found in several organs: pineal and retina [8], which are closely related to central nervous system, and also in the adrenal [3,22], ovary [17, 27, 30], placenta [6], testis [15,16] and thymus [10]. The presence of neurophysins, which are associated in the hypothalamic-neurohypophyseal system with AVP and OT, further indicates that they are either synthesized in the HG or originate in sympathetic nerves, since immunoreactive OT, AVP and Np have been described [14,28] in the intermediolateral column of the human spinal cord. The origin of these peptides in the spinal cord is probably the hypothalamus. Although we have no proof of a synthesizing activity of the cells of the intermediolateral column, the gland is innervated by the sympathetic cervical ganglion [26] and a vasopressin-like peptide [12] or other neuropeptides are widely distributed in the sympathetic nervous system [18]. It is likely that the neuropeptides are synthesized in the HG since there is more evidence of the capacity of the HG gland to synthesize an hormonal agent, melatonin. Melatonin is also found in large amounts in the retina. Thus, it is tempting to compare the 3 organs: eyes, pineal and HG, which contain melatonin, and the two neuropeptides, A V P and OT. Although the existence of a day/night rhythm for melatonin in the 3 organs is well documented [4, 24, 25], there is no such
292
GAUQUELIN ET AL.
documentation for AVP, OT and Np. However, day/night rhythm in rat was clearly demonstrated for AVP in cerebrospinal fluid [20]. The existence of a circadian rhythm of OT in C S F seems to depend on the species [1]. Our data are to be compared with those of Reiter [25] where retinal and HG melatonin content exhibit the same rhythm. The pineal shows a different rhythm. The significance (and function) of AVP and OT fluctuation in the Harderian gland are yet to be studied. Our data confirm other experiments which suggest HG as a possible extraretinal photoreceptor. For example, the removal of the HG abolishes the serotonin response to light in blinded
animals [31]. However, this does not exclude other possible roles for HG, such as thermal regulation or osmoregulation [13]. In another experiment [11] we have demonstrated a similar variation in AVP content in the pineal and Harderian glands. This variation seemed to be linked to the same thermal load. ACKNOWLEDGEMENTS This work was supported by grants from Facult6 de Mrdecine Grange-Blanche, Digital Equipment, DRET (Grant 87.056) Centre Jacques Cartier (Lyon), CNES and Universit6 CI. Bernard (Physiologie de l'Environnement).
REFERENCES 1. Amico, J., R. Tenicela, J. Johnston and A. G. Robinson. A time-dependent peak of oxytocin exists in cerebrospinal fluid but not in plasma of humans. J Clin Endocrinol Metab 57: 947951, 1983. 2. Anderson, T. W. Time Series Analysis. New York: J. Wiley and Sons, 1971, pp. 72-164. 3. Ang, V. T. Y. and J. S. Jenkins. Neurohypophysial hormones in the adrenal medulla. J Clin Endocrinol Metab 58: 688-691, 1984. 4. Balemans, M. G. M., P. Prvet, J. Van Benthem, C. HaldarMisra, I. Smith and H. Hendriks. Day/night rhythmicity in the methylating capacities for different 5-hydroxyindoles in the pineal, the retina and the Harderian gland of the golden hamster (Mesocricetus Auratus) during the annual seasons. J Neural Transm 56: 53-72, 1983. 5. Dorsa, D. M. and L. A. Bottemiller. Vasopressin-like peptides in the rat brain: immunologic and chromatographic behavior and their response to water deprivation. Regul Pept 6: 393--403, 1983. 6. Fields, Ph, A., R. K. Eldridge, A. R. Fuchs, R. F. Roberts and M. J. Fields. Human placenta and bovine corpora luteal oxytocin. Endocrinology 112: 1544-1546, 1983. 7. Gauquelin, G., G. Geelen, A. M. Allevard-Burguburu, M. Cellier, B. Sempore, F. Louis, J. J. Legros and C. Gharib. Presence of neurophysins I and II in the human pineal gland: Comparison with the content of neurohypophyseal hormones. Peptides 3: 805-809, 1982. 8. Gauquelin, G., G. Geelen, F. Louis, A, M. Allevard, C. Meunier, G. Cuisinaud, S. Benjanet, N. G. Seidah, M. Chretien, J. J. Legros and C. Gharib. Presence of vasopressin, oxytocin and neurophysin in the retina of mammals, effect of light and darkness, comparison with the neuropeptide content of the neurohypophysis and the pineal gland. Peptides 4: 50%515, 1983. 9. Geelen, G,, A. M. Allevard-Burguburu, G. Gauquelin, Y. Z. Xiao, J. Frutoso, C. Gharib, B. Sempore, C. Meunier and G. Augoyard. Radioimmunoassay of arginine vasopressin, oxytocin and arginine vasotocin-like material in the human pineal gland. Peptides 2: 459-466, 1981. 10. Geenen, V., J. J. Legros, P. Franchimont, M. Baudrihaye, M. P. Defresne and J. Boniver. The neuroendocrine thymus: coexistence of oxytocin and neurophysin in the human thymus. Science 232: 508--511, 1986. 11. Ghaemmaghami, F., G. Gauquelin, C. Gharib, D. Yoccoz, D. Desplanches, R. Favier and A. M. Allevard. Effects of treadmill running and swimming on plasma and brain vasopressin levels in rats. Eur J Appl Physiol 56: 1-6, 1987. 12. Hanley, M, R., H. P. Benton, S. L. Lightman, K. Todd, E. A. Bones, P. Fretten, S. Palmer, C. J. Kirk and R. H. Mitchell. A vasopressin like peptide in the mammalian sympathetic nervous system. Nature 309: 258-261, 1984. 13. Harriman, A. E. and D. E. L. Thiessen. Removal of Harderian exudates by sandbathing contributes to osmotic balance in mongolian gerbils. Physiol Behav 31: 317-323, 1983.
14. Jenkins, J. S., V. T. Y. Ang, J. Hawthorn, M. N. Rossor and L. L. lversen. Vasopressin, oxytocin and neurophysins in the human brain and spinal cord. Brain Res 291:111-117, 1984. 15. Kasson, B. G. and A. J. W. Hsueh. Arginine vasopressin as an intragonadal hormone in Brattleboro rats: presence of a testicular vasopressin like peptide and functional vasopressin receptors. Endocrinology 118: 23-31, 1986. 16, Kasson, B. G., R. Meidan and A. J. W. Hsueh. Identification and characterization of arginine vasopressin-like substances in the rat testis. J Biol Chem 260: 5302-5307, 1985. 17. Khan-Dawood, F. S., E. L. Marut and M. Y. Dawood. Oxytocin in the corpus luteum of the cynomolgus monkey (Macaca fascicularis). Endocrinology 115: 570-574, 1984. 18. Kondo, H. Immunohistochemical analysis of the localization of neuropeptides in the adrenal gland. Arch HistolJpn 48: 453-481, 1985. 19. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951. 20. Mens, W. B. J., A. D. Andringa-Bakker and T. B. Van Wimersma Greidanus. Changes in cerebrospinal fluid levels of vasopressin and oxytocin of the rat during various light-dark regimes. Neurosci Lett 34: 51-56, 1982. 21. Milqu, S. M., S. Pavel and C. Neacsu. Biological and chromatographic characterization of a polypeptide with pressor and oxytocic activities isolated from the bovine pineal gland. Endocrinology 72: 563-566, 1963. 22. Nussey, S. S., V. T. Y. Ang, J. S. Jenkins, H. S, Chowdrey and G. W. Bisset. Brattleboro rat adrenal contains vasopressin. Nature 310: 64-66, 1984. 23. Prvet, P. The different classes of proteic and peptidic substances present in the pineal gland. In: The Pineal Gland and its Endocrine Role, edited by J. Axelrod, F. Fraschini and G. P. Velo. New York: Plenum Publishing Corporation, 1983, pp. 113-149. 24. Reiter, R. J., B. A. Richardson, S. A. Matthews, S. J. Lane and B. N. Ferguson. Rhythms in immunoreactive melatonin in the retina and Harderian gland of rats; persistence after pinealectomy. Life Sci 32: 122%1236, 1983. 25. Reiter, R. J., B. A. Richardson and E. C. Hurlbut. Pineal, retinal and Harderian gland melatonin in a diurnal species, the Richardson's ground squirrel (Spermophilus Richardsonii). Neurosci Lett 22: 285-288, 1981. 26. Sakai, T. The mammalian Harderian gland: morphology, biochemistry, function and phylogeny. Arch Histol Jpn 44: 299-333, 1981. 27. Schams, D., E. Schallenberger and J. J. Legros. Evidence for the secretion of immunoreactive neurophysin I in addition to oxytocin from the ovary in cattle. J Reprod Fertil 73: 165-171, 1985.
DAY/NIGHT RHYTHM OF VASOPRESSIN AND OXYTOCIN 28. Sofroniev, M. V., A. Weindl, U. Schrell and R. Wetzstein. lmmunochemistry of vasopressin, oxytocin and neurophysin in the hypothalamus and extrahypothalamic regions of the human and primate brain. Acta Histochem [Suppl] (Jena) XXIV: 79--95. 1981. 29. Thiessen, D. D. and A. E. Harriman. Harderian gland exudates in the male Meriones unguiculatus regulate female proceptive behavior, aggression, and investigation. J Comp Psycho! 100: 85-87, 1986.
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30. Wathes, D. C., R. W. Swarm and B. T. Picketing. Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy. J Reprod Fertil 71: 551-557, 1984. 31. Wetterberg, L., E. GeUer and A. Yuwiler. Harderian gland: an extraretinal photoreceptor influencing the pineal gland in neonatal rats? Science 167: 884-885, 1970.
0196-9781/88 $3.00 + .00
A Day/Night Rhythm of Vasopressin and Oxytocin in Rat Retina, Pineal and Harderian Gland G U I L L E M E T T E G A U Q U E L I N , .1 C L A U D E G H A R I B , .2 F A R Z A M G H A E M M A G H A M I , * ANNE-MARIE ALLEVARD,* FARID CHERBAL,t GHISLAINE GEELEN,* FATHIA BOUZEGHRANEt AND JEAN-JACQUES LEGROS$
*Laboratoire de Physiologie, Facult# de M#decine Grange-Blanche 8, Avenue Rockefeller, 69373, Lyon C#dex 08 France tLaboratoire de Neurobiologie USTBH, BP 139 Darelbeida, Algiers, Algeria ~Laboratoire de Radioimmunologie Neuroendocrinologie, Universit# de Likge Sart-Tilman CHU 23, B 4000, Liege Belgique R e c e i v e d 24 S e p t e m b e r 1986 GAUQUELIN, G., C. GHARIB, F. GHAEMMAGHAMI, A.-M. ALLEVARD, F. CHERBAL, G. GEELEN, F. BOUZEGHRANE AND J.-J. LEGROS. A day~nightrhythm of vasopressin and oxytocin in rat retina, pineal and harderiangland.PEPTIDES 9(2) 289-293, 1988.--Arginine vasopressin (AVP), oxytocin (OT) and neurophysins (Np) have been found in the pineal gland and the retina of the rat. Because the retina, pineal gland and Harderian gland (HG) serve analogous functions, we undertook a study to determine the presence of these peptides in these three organs of rats. They were detected by two specific methods: HPLC and specific radioimmunoassays. For Np, total neurophysins (NpT) were measured. To determine a 24 hr rhythm, the animals were maintained under a light/dark cycle of 12 hr/12 hr for 3 weeks. The pineal glands, retinae and HG were collected. Day/night rhythms of AVP, OT and NpT were demonstrated in the retina and HG; but the pineal gland had only AVP rhythm. A significant decrease in the rhythms at 4 a.m. was demonstrated in the retina and HG. The 24 hr variation of AVP in the retina seemed parallel to that of the HG. Vasopressin
Oxytocin
Retina
Pineal gland
Harderian gland
T H E Harderian glands (HG) are found in all vertebrates (with the exception of the higher primates) and are located in the orbits [26]. Their functions have not been proved although they are supposedly involved in the lubrication of the eyes, in sexual behavior or aggression [29]. But several arguments are in favor of another function: the HG could be an extraretinal photoreceptor, since the removal of the gland abolishes the pineal serotonin response to light in blinded animals, and porphyrins of the glands show fluctuations under different lighting conditions [31]. The presence of melatonin is also demonstrated in all species studies. Moreover, the H G synthesize hundreds of times more methoxyindoles than the pineal or the retina whether it be a day or annual rhythmicity [4]. Existing data confirm the analogy among the 3 organs. Since the presence of the neuropeptides arginine vasopressin (AVP), oxytocin (OT) and their central nervous system-associated peptides neurophysins (Np) have been demonstrated in the pineal [8, 9, 21, 23] and the retina [8], the present study was undertaken to demonstrate (a) the presence of these neuropeptides in the HG and (b) the existence of a 24 hr rhythmicity in the 3 organs.
METHOD Two different experiments were carried out.
Determination of Neuropeptides and Neurophysin Direct determination. Twenty-four adult male Wistar rats (Iffa Credo) were decapitated at 9 a.m. The HG were rapidly removed, then frozen at -80°C until assay. After thawing, two HG were homogenized in 1 ml cold water p H 5.5 and centrifuged at 4°C at 11,000 rpm for 30 min. The resulting clear supernatant was divided into aliquots and stored at -20°C until analyzed for their AVP, OT and Np content. The radioimmunoassay of AVP, OT and Np has been described previously for pineal gland [7,9] and retina [8].
High performance liquid chromatography (HPLC). HPLC coupled with radioimmunoassay (RIA) was applied to 20 pooled HG. Extraction of tissues was accomplished using a modification of the method of Dorsa and Bottemiller [5]. The HPLC system was a Kontron Instrument equipped with a C-18 (5 microns) reversed phase Kontron column (25 cm x 4.6). Peptide elution was recorded at 206 nm. Peptides were separated by a solvent system consisting of 8 ( ~ 0.025
1G. Gauquelin is Fellow of CNES. 2Requests for reprints should be addressed to C. Gharib.
289
G A U Q U E L I N ET AL.
290
E (O
STANDARD
r-
4
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mm
FIG. 1. Absorption (top) and immunoreactivity (middle) after injection of a mixture of synthetic peptides. Immunoreactive AVP and OT content (bottom) of fractions collected after injection of an extract of 20 rat Harderian glands. M sodium phosphate pH 7.0 and 20% acetonitrile. The column was washed with standard buffer between each run. In addition, control injections were performed and tested for peptide by RIA. After injection of an HG sample, a mixture of synthetic AVP and OT (500 ng each) was injected to determine the recovery of the column. The separation and recovery of both AVP and OT were visualized on a recorder. Column elutions were collected in tubes at 1 min intervals using a fraction collector, then lyophilized and reconstituted with assay buffer for peptide determination by RIA, as previously described [9].
Rhythmicity of Peptide Secretion One hundred and eight adult male Wistar rats were used in this experiment. They were kept under light:dark conditions of 12:12. Twelve rats were sacrificed in February, at 10 a.m., 2 p.m., 6 p.m., 10 p.m., 2 a.m., 4 a.m., 7 a.m., 10 a.m. and 2 p.m.. After decapitation, the pineal, the retinae and the Harderian glands were quickly removed under a 25 W red light. The organs were frozen in liquid nitrogen and kept at -80°C. AVP, OT and Np were measured. After thawing, two retinae, and two Harderian glands were homogenized in 1 ml cold water pH 5.5 (the pineal gland in 100/zl) and centrifuged at 4°C at 11,000 rpm for 30 min. The supernatant was divided into aliquots and stored at 20°C until analyzed for AVP, OT and Np content. The proteins were measured by the method of Lowry [19].
,
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FIG. 2. Pineal, retinal and Harderian gland AVP, OT and Np content of rat. Dark bar represents dark phase of light:dark cycle (12/12). Each point represents the mean_+SEM.
The mean was calculated and a one-way analysis of variance was performed, followed by a Newman and Keuls' test. The correlation coefficients (r) between OT, AVP, NpT were established by a simple correlation test. The calculation of periodic regression was done by a mathematical model leading to a symmetrical sine curve. The length of the fundamental period and the acrophase 0 (the maximum of the best least-squares fit sine wave) were estimated by the least squares method, with a fine step of adjustment. A cosine function Y = M + A cos (wt + 0) was used as a model of rhythm. Y is the value at a time t of the periodic function of angular frequency w : 2/t, A and 0. The amplitude A is measured by the difference between crest and
D A Y / N I G H T R H Y T H M O F V A S O P R E S S I N A N D OXYTOCIN
291
TABLE 1 MESOR, ACROPHASEAND AMPLITUDE FOR AVP, OT AND NpT IN THE PINEAL, THE HARDERIANGLAND AND THE RETINA (MEAN -+ SEM) Mesor
Acrophase Hour/Min
Amplitude
40 -+ 5 16.5 -+ 3 14 .+ 0.1
22hr40-+ 53 4 h r 4 .+ 73 10 hr 40
27.7 -+ 20 10.9 -+ 5 0.06 _+ 0.05
Pineal fmol/gland
AVP OT NpT
HG pmol/mg gland
AVP OT NpT
120 490 12
.+ 2 _+ 7 .+ 2
22hr 13-+ 1 2 hr 10 _+ 3 21hr40- 1
80 210 11
Retina fmol/mg prot
AVP OT NpT
254 132 50
.+ 43 .+ 12 .+ 0.7
17 hr 19 .+ 1 21 hr 19 _+ 45 21 hr 19 .+ 9
132.8 _+ 36 43.3 19 _+ 0.03
mesor of a best fitting cosine. The mesor M is the midway value between the peak and trough of the function used to approximate the biological rhythm [2]. RESULTS The results were expressed per mg of proteins for retina and HG. The presence of AVP (Range: 0.05 to 0.58 pM/mg prot.), OT (0.1 to 1 pM/mg prot.) and Np (5 to 19 fM/mg prot.) was demonstrated in 24 HG. The RIAs used were sensitive from 2.5 pg AVP/tube, 5 pg OT/tube and 0.2 ng Np/tube. AVP and OT standard curves and the dilution curves from the HG showed considerable parallelism. The Spearman correlation between Np and AVP was 0.6, and between Np and OT, 0.58. Over a 4 and 6-min period, the HPLC system reliably resolved A V P and OT respectively (Fig. 1). When eluates of the mixture and HG were collected and assayed by RIA, AVP and OT were detected only in those eluates shown to contain peptides by the 206 nm detector. HPLC recovery was 90% for 500 pmol and 60% for 50 pmol. Figure 2 shows the rhythms of AVP, OT and Np in pineal gland, retina and HG of rat. In the pineal gland, analysis of variance showed a significant variation over 24 hours (F=2.5, p<0.05) for AVP. Pineal AVP levels reached a peak during the dark phase. The t-test revealed that at 10 p.m. the mean A V P level increased significantly in comparison to 10 a.m., 2 p.m. and 4 a.m. levels. There was no significant variation for OT ( F = 1.9) and Np ( F = I . 1 ) . The correlation between Np and OT was rs=0.49 (p <0.01) and between AVP-OT and Np was rs=0.31 (p<0.05). In the retina, analysis of variance disclosed a significant variation over 24 hours for AVP (F=6.9, p<0.001), for OT ( F = 3 , p<0.01) and for Np (F=2.2, p<0.05). The t-test showed a significant decrease at 2 a.m., 4 a.m. and 7 a.m. for AVP, OT and Np, as compared with 10 a.m. In the HG, analysis of variance showed a significant variation over 24 hours for AVP (F=6.5, p<0.001), for OT (F=5.2, p<0.001) and for Np (F=5.4, p<0.001). The t-test revealed a significant decrease at 4 a.m. and 7 a.m. for A V P and OT, and an increase at 4 a.m. for Np. The correlation between retina and HG for AVP is rs=0.3 (p<0.01). Table 1 shows the times at which maximum levels occurred. In the HG, AVP and NpT had identical acrophases.
-+ 2 .+ 10 -+ 1
OT also had approximately the same acrophase as NpT. In the retina, we found the same acrophase for OT and NpT. Our results show that AVP and OT are not simultaneously synchronized with NpT in most of the cases, but the NpT are the precursors of AVP and OT. The antiserum used cross-reacted equally with Np linked to AVP and Np linked to OT, so it is difficult to have the same acrophase for AVP and/or OT and NpT. In these structures the pattern of AVP, OT and NpT is almost identical with a marked decrease in concentration at 4 a.m. This shows that the concentration of AVP, OT and NpT in the retina followed a circadian rhythm. An ultradian rhythm (5 hr period) was detected for OT in the pineal and Harderian gland and for NpT in the retina. DISCUSSION Our results clearly demonstrate the presence of AVP, OT and Np in the Harderian glands of the rat. For AVP and OT, the presence was confirmed by RIA measurements on HPLC fractions. These results are not surprising since AVP and OT have been found in several organs: pineal and retina [8], which are closely related to central nervous system, and also in the adrenal [3,22], ovary [17, 27, 30], placenta [6], testis [15,16] and thymus [10]. The presence of neurophysins, which are associated in the hypothalamic-neurohypophyseal system with AVP and OT, further indicates that they are either synthesized in the HG or originate in sympathetic nerves, since immunoreactive OT, AVP and Np have been described [14,28] in the intermediolateral column of the human spinal cord. The origin of these peptides in the spinal cord is probably the hypothalamus. Although we have no proof of a synthesizing activity of the cells of the intermediolateral column, the gland is innervated by the sympathetic cervical ganglion [26] and a vasopressin-like peptide [12] or other neuropeptides are widely distributed in the sympathetic nervous system [18]. It is likely that the neuropeptides are synthesized in the HG since there is more evidence of the capacity of the HG gland to synthesize an hormonal agent, melatonin. Melatonin is also found in large amounts in the retina. Thus, it is tempting to compare the 3 organs: eyes, pineal and HG, which contain melatonin, and the two neuropeptides, A V P and OT. Although the existence of a day/night rhythm for melatonin in the 3 organs is well documented [4, 24, 25], there is no such
292
GAUQUELIN ET AL.
documentation for AVP, OT and Np. However, day/night rhythm in rat was clearly demonstrated for AVP in cerebrospinal fluid [20]. The existence of a circadian rhythm of OT in C S F seems to depend on the species [1]. Our data are to be compared with those of Reiter [25] where retinal and HG melatonin content exhibit the same rhythm. The pineal shows a different rhythm. The significance (and function) of AVP and OT fluctuation in the Harderian gland are yet to be studied. Our data confirm other experiments which suggest HG as a possible extraretinal photoreceptor. For example, the removal of the HG abolishes the serotonin response to light in blinded
animals [31]. However, this does not exclude other possible roles for HG, such as thermal regulation or osmoregulation [13]. In another experiment [11] we have demonstrated a similar variation in AVP content in the pineal and Harderian glands. This variation seemed to be linked to the same thermal load. ACKNOWLEDGEMENTS This work was supported by grants from Facult6 de Mrdecine Grange-Blanche, Digital Equipment, DRET (Grant 87.056) Centre Jacques Cartier (Lyon), CNES and Universit6 CI. Bernard (Physiologie de l'Environnement).
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