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Hypothalamus-pituitary-thyroid axis interactions with pineal gland in the rat G. L. BRAMMER, J. E. MORLEY, E. GELLER, A. YUWILER, AND J. M. HERSHMAN BRAMMER, G. IL., J. E. MORLEY, E. GELLER, A. YUWILER, AND J. M. HERSHMAN. Hypothalamus-pituitarythyroid axis ilzteractions with pineal gland in the rat. Am. J. Physiol. 236(4); E416-E420, 1979 or Am. J. Physiol,: Endocrinol. Metab. Gastrointest. Physiol. 5(4): E416-E420, 1979.-We examined in the rat several possible relationships between the pineal gland and the hypothalamus-pituitary-thyroid axis. The pineal gland, the retina, and the hypothalamus exhibited a diurnal rhythm in thyrotropin-releasing hormone (TRH) content with peak values occurring around 1200 h. This rhythm in the hypothalamus was abolished by constant light but was not affected by pinealectomy. Nor did pinealectomy affect hypothalamic TRH content, pituitary content of thyroid-stimulating hormone (TSH) or prolactin; serum levels of (TSH), triiodothyronine (?I&), or thyroxine (T4), or serum free-thyroxine index; or freetriiodothyronine index. Melatonin did not affect TSH or prolactin release from the anterior pituitary or TRH release from the hypothalamus in vitro. Isoproterenol did not affect the TRH content of pineal glands in vitro; nor did TRH or T3 affect basal or stimulated activities of serotonin N-acetyltransferase, the presumed controlling enzyme in melatonin production. We found no evidence for significant interactions between the pineal gland and the hypothalamus-pituitary-thyroid axis. pineal gland of the rat follows a diurnal rhythm that may be synchronized by environmental lighting. The rhythm is suppressed in constant light, but is free running in constant dark. Under diurnal lighting conditions, the onset of dark is accompanied by sympathetic nerve activity resulting in an adrenergically mediated, increased activity of serotonin N-acetyltransferase (SNAT), the apparent controlling enzyme in the production of melatonin. The circadian periodicity of pituitary TSH in rat (7) and in man (1) is well recognized. Constant dark produces an increase in thyroid weight (19) and radioactive iodine uptake (30). The effects of constant dark on TSH and PBI are controversial (20, 25). Vriend et al. (30) have shown that blinding of hamsters leads to depression of the plasma free-thyroxine index and that this effect can be reversed by pinealectomy or bilateral superior cervical ganglionectomy. Because of the uncertainty in the literature concerning the relationship of the pineal gland to the H-P-T axis, we have undertaken a number of experiments in an attempt to delineate this relationship more clearly. The availability of radioimmunoassays for TRH and TSH, as well as thyrotropin-releasing hormone; triiodothyronine for the thyroid hormones, has enabled us to study the entire H-P-T axis. Further, tissue culture techniques have aIlowed us to look for an interaction between TRH and factors in the pineal gland influencing melatonin THE RELATION OF THE PINEAL GLAND to the hypothalamus-pituitary-thyroid (H-P-T) axis is unclear. In 1972 production and to examine the effect of melatonin both Relkin (20) postulated that the pineal gland had an on the release of pituitary TSH and on the release of inhibitory influence on thyrotropin-l’eleasing hormone hypothalamic TRH. We have particularly examined the (TRH) secretion. This postulate was based on his obser- following: 1) the effect of lighting conditions on the TRH vations that 3 days after pinealectomy in the rat there content of the hypothalamus, the pituitary, the retina, was an acute increase in serum thyroid-stimulating hor- and the pineal gland and on thyroid function; 2) the mone (TSH) and protein-bound iodine (PBI), which was effect of pinealectomy on the TRH content of the hypono longer present by day 6. More recently, in support of thalamus and on thyroid function; 3) the effect of phis postulate, Relkin (22) has reported decreased plasma noradrenergic stimulation on the TRH content and the TSH after intraventricular injection of melatonin in the effect of added TRH or T3 on basal and stimulated rat, Ishibashi et al. (8) found that pinealectomy increased SNAT activity in cultured pineal glands; and 4) the effect thyroxine secretion rate; Rowe et al, (25), however, ob- of melatonin on the release of TSH from acutely cultured served no effect of pinealectomy on plasma TSH or PBI anterior pituitary glands and on the release of TRH from acutely cultured hypothalami. levels. Environmental lighting influences both the pineal gland and the thyroid, which also suggests a pineal- METHODS thyroid relationship. The production of melatonin in the Male Sprague-Dawley rats were housed in a temperaE416 Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016 Neurobiochemistry Research Laboratory T-85, Veterans Administration Brentwood Hospital, Los Angeles 90073; Department of Psychiatry and Biobehauioral Sciences, School of Medicine, University of California, Los Angeles 90024; and Endocrinology Research Laboratory, Veterans Administration Wadsworth Hospital, Los Angeles, California 90073 THYROID AXIS INTERACTIONS WITH PINEAL E417 GLAND a gyratory water bath shaker at 37°C under 5% CO2 in 02. The preincubation medium was then replaced by 1 ml preoxygenated medium containing either no additions, melatonin (lo-’ M), TRH (1.4 x 10B8M), or melatonin with TRH. There were five flasks in each group. After 1 h of further incubation at 37”C, the mediums were colIected and stored frozen until assayed for TSH and prolactin at a 1:20 dilution. The wet weight of pituitaries in each flask was recorded. Hypothalami (excluding the mammillary bodies) were removed from male rats and halved, and the halves were divided between control and treated groups. Pairs of hypothalamus halves were incubated in heat-treated, bacitracin-containing medium under the conditions described above for pairs of pineal glands. The preparation of the hypothalamus halves took; approximately 15 min. The tissue pieces were preincubated for 15 min in medium already equilibrated to the culture conditions. After the preincubation period, tissue and support screenswere removed, blotted, and placed in equilibrated medium containing no additions or 10Y4M melatonin for a 30-min test period. The medium was then frozen for TRH assay and the wet; weight of the hypothalamus pairs recorded. Assays. TSH was measured by radioimmunoassay with the National Institute of Arthritis, Metabolic, and Digestive Diseases (NIAMDD) rat TSH RP-1 used as a reference preparation. Thyroxine (Td) and triiodothyronine (Tz) were measured by radioimmunoassay as described previously (l), The unsaturated binding capacity of serum proteins was assessedby a triiodothyronine uptake test (TTJJ) (15). The free-thyroxine index (FTJ) and free-triiodothyronine index (FTJ) were obtained by calculating the product of the Td or T3 and total TJJ values. TRH was measured by the method of Bassiri and Utiger (2) with antibody kindly supplied by Dr. Robert Utiger of the University of Pennsylvania. Prolactin was measured with a radioimmunoassay kit supplied by NIAMDD, Pineal gland SNAT activity was measured by the method of Deguchi and Axelrod (6) as modified by Pa&t et al. (13). Results reported in the text are means t SD (n). Tabular values are means t SD. The significance of differences between means was determined by Student’s t test. RESULTS The immunoreactive TRH contents of the pineal gland, retina, and hypothalamus were significantly higher at 1200 h compared with 2400 h in rats housed under diurnal lighting conditions (Fig. 1). Serum Ts and TSH levels were also significantly higher at 1200 h, but no difference in pituitary TRH or serum Td was observed between the two time points. Constant light abolished the difference between measures of hypothalamic TRH and serum TSH and Ts on samples taken at 1200 h and 2400 h (Table I). Pinealectomy did not abolish the diurnal difference in hypothalamic TRH or serum T3 (Table 2), but did abolish the diurnal difference observed in serum TSH (Tables 2 and 3). A significant difference was observed in serum T3 levels between pinealectomized and sham-pinealec- Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016 ture- and light-controlled room (lights on 0500-1700 h) with food and water ad libitum. Photic experiments made use of rats from our colony weighing ZOO-250g. Pinealectomized and sham-pinealectomized rats were obtained commercially (Zivic-Miller, Allison Park, PA). Completeness of pin .ealectomy was confirmed at post mortem. Animals were decapitated and trunk blood was collected. Serum samples were stored frozen until assayed. The anterior pituitary glands were weighed and disrupted by sonication in 1 ml 50 mM phosphate-buffered saline (pH 7.45). The hypothalamus, the retina, and the pineal gland were dissected and extracted in methanol, as previously described (18). Photic experiments. a) Animals were killed at 1200 h and 2400 h. b) Animals were killed at 2400 h after 67 h constant light and at 1200 h the next day. Pinealectomy experiments. a) Pinealectomy was performed on animals weighing 150 g, Twelve days after surgery, pinealectomized and sham-pinealectomized animals were killed at 1200 h and pinealectomized animals only were killed at 2400 h. b) Pinealectomy and sham procedures were performed on 60- to 80-g rats. At 13 days after surgery, animals were lightly etherized at 1300 h and blood samples were withdrawn from the external jugular vein, At 60 days after surgery, the same animals were killed at 1300 h. In addition to the other tissues, thyroid glands were also dissected free and weighed. Pineal glands for culture experiments were taken from 45- to 60-day-old rats of either sex from our colony at 0900-1000 h, Culture conditions were essentially as described by Pa&t et al. (14) except that the final concentration of glutamine in the culture medium was 2 mM. In brief, pairs of pineal glands were supported on a nylon mesh screen in a 16-mm-diameter well containing 0.15 ml medium. Test compounds were added in volumes of 10 pl and incubations were carried out at 37OC in an atmosphere of 5% CO2 in 02. Because TRH is degraded by protease activity in many mediums containing serum fractions, in some experiments examining the release of TRH or the effects of TRH on SNAT activity complete medium was heated at 56°C for 30 min and bacitracin was then added to a level of 50 U/ml before the medium was used for culture. Pineal culture experiments. a) After a 30-min preincubation period in complete medium, pineals were transferred to medium with or without 2 x IO-” M DL-isoproterenol. After 3 h pineal glands were removed and frozen on solid CO:! in groups of 3 or 4 TRH assay. b) Pineal glands were placed in culture with somatostatin or TRH and with or without isoproterenol. After 6 h glands were individually frozen until assayed for SNAT activity. c) After a 50-min preincubation period in complete medium with or without T3, pineal glands were transferred to Lnorepinephrine- containing mediu m wi.th or witho ut T3 for 6 h and then were individually frozen until assayed for SNAT activity. Anterior pituitaries were removed from male rats and quartered, and the quarters were randomized. Three quarters were placed ii lo-ml stoppered Erlenmeyer flasks containing 1 ml modified Gey and Gey medium (4) that had been equilibrated to the culture conditions. The tissue portions were individually preincubated for 2 h in E418 BRAMMER I p<.Ol 100 AL, SNAT activity stimulated in cultured pineal glands by either lo-’ M or lOm6M isoproterenol (Fig. 2). Triiodothyronine at a concentration of 7.7 x 10wl’ M (50 ng/dl) had no effect on the dose-response curve of norepinephrine-stimulated SNAT activity in cultured pineal glands (Fig. 3). In anterior pituitary cultures, 10B5M melatonin, alone or in conjunction with 1.4 x IO-’ M TRH, had no A TRH (pg/mg) 200 ET 2 1 PINEAL HYPOTHALAMUS RETlNA PITUITARY 3. Effect ofpinealectomy and 60 days after surgery -. -- 13 days TABLE 8 SERUM 13 days Sham 60 days PX” PX* Sham 429 * 93 403 t 73 TRKpg/w FIG. 1. Diurnal rhythms in TRH content and measures of H-P-T axis, A: TRH content of different tissues. B: serum measures of H-P-T axis function. Open bars indicate samples taken at 1200 h; hatched bars, samples at 2408 h. Vertical lines indicate 1 SD; n = 12 for all groups. Pituitary TSH, mu/w Pituitary wmg Serum Serum 6.8 AI 3.6 prolactin, 376 TSH, &J/ml prolactin, 92 &41 88 -t 38 T3, ng/dl T+ pg/dl TT&J FTJ FTJ wt, mg wt, mg 59 -t 8 5.5 * 0.7 53 t 9 4.8 k 0.9 t 173 5.4 k 4.3 270 81 -t- 54 28 t 18 t, 223 90 t, 62 17k12 @ml TABLE 1. Effect of constant light ,---_ - -~--.-. .- - .--1200 h Hypothalamic TRH, Serum TSH, @J/ml Serum Ts, ng/dl Serum Tq, pg/dl Values are means pg/mg 275t 153k 55t -..---- 2400 h 28 94 6 299Ik 31 122 k 61 52 zk 14 6*0 * 1.0 5.9 k 1.9 2. Effect ofpinealectomy after surgery _ . _--_ .-.--- ^ _----.~------px* n= mg k6 k 1.0 t 0.04 IL 9.8 9.8 k 1.9 10.5 26.5 are means A SE; n = 11 for all groups. * 1.4 k 5.3 45t 5.0 t 1.78 t 80 k 8.9 t 10.5 * 25.5 k 11 1.2 0.04 18 2.0 1.4 4.2 * Pinealectomized. IO 12 days 1200 h Pituitary Serum Serum Serum Serum Values 47 5.6 1.74 82 t SD; n = 12. TABLE Hypothalamic .~ Serum Serum Serum Serum Serum Pituitary Thyroid TRH, pg/ TSH, mU/mg TSH, $J/ml prolactin, rig/ml Ts, ng/dl T4, pg/dl _ __-- --- - Values are meins -t- SD, pared with PX at 1200 h. 313-+ 8,2 t 82-+ 13.4 -+ 50tn 5.4 t 2400 h Sham 10 32 2.6 47 12 1.9 7.6 100 13,9 35 3.9 * Pinealectomized. t15 t 1.7 t48 t 5.8 *12t t 1.9 6 (8) px* n=9 322 W 0 n=9 161 7.6 74 7.6 39 4.4 AL sot t 1.1 t xk k k TRH @ TRH + ld8M GUPROTERENOL l TRH + 16% ISOPROTERENOL 0 SOMATOSTATIN 48 10 St 2.0 t P < 0.01 com- tomized animals at 1200 h 12 days after surgery in one experiment (Table Z), but not at 13 days or 60 days after surgery in another experiment (Table 3). No significant differences were observed in any other measures comparing pinealectomized animals at 1200 and 2400 h or comparing pinealectomized and sham-pinealectomized animals at 1200 h at 12 days or at 1300 h at 13 or 60 days after surgery (Tables 2 and 3). The several experiments in which pineal glands in culture were used yielded no significant treatment differences. Pineal glands cultured for 3 h with either no additions or lo-” M isoproterenol had TRH contents (pghg) of 2.8 t 2.2 (8) and 4.3 t 3.8 (8), respectively. Somatostatin or TRH, each tested uver a range of concentrations, had no effect on basal SNAT activity in cultured pineal glands, nor did TRH have any effect on NONE: -13-12 HORMONE -9 -8 -5 -4 (log M) FIG. 2. Effect of hormones on serotonin N-acetyltransferase in cultured pineal glands. Vertical lines indicate I SD, samples in parentheses; where not indicated, n = 4. activity Number of Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016 Hypothalamic THYROID AXIS INTERACTIONS PINEAL -5 E419 GLAND -4 NOREPINEPHRINE 3. Effect of T3 on norepinephrine-stimulated tyltransferase activity in cultured pineal glands, I SD; n. = 4 for all groups. FIG. (log M) serotonin N-aceVertical lines indicate 4. Effect of melatonin on release of TSH and prulactin in anterior pituitary cultures ~- .--------.. TABLE TSR, pU/mg Control Melatonin (10s5 M) TRW (1.4 x lO+ M) Melatonin + TRH Values are means per ml 493 t 213 874 t 725 760 & 261 676 +- 411 Praiactin, ng/mg per ml t 196 -4 207 t 176 k 156 33 32 64 25 * SD; 12 = 5 for all groups. significant effect upon the (Table 4). In hypothalamic had no significant effect on into the medium (control, treated, 17.3 t 6.6 (4)). release of TSH or prolactin cultures, 10B4 M melatonin the amount of TRH released 25.5 -t 8.5 (6) pg/mg per ml; DISCUSSION The hypothalamic trophic hormones, TRH, luteinizing hormone-releasing hormone (LHRH), and somatostatin, have all been shown to be present in the pineal of a variety of mammalian species (31). Our results show the presence of a diurnal rhythm of immunoreactive TRH in the pineal of the rat. In addition, we confirm that there is a diurnal rhythm of TRH in the retina (26) and the hypothalamus (5). The diurnal rhythm in hypothalamic TRH content and serum T3 level was abolished by constant light but was not affected by pinealectomy. Diurnal lighting, but not diurnal melatonin production, seems correlated with the diurnal rhythm in both hypothalamic TRH content and serum T3 level. The diurnal rhythm in serum TSH level was abolished by both constant light and pinealectomy. The TRH content of the hypothalamus, peaking during the light period, is phase reversed from pineal melatonin production, peaking during the dark period. If the reduced hypothalamic TRH content is a reflection of functional release of TRH, then the phase reversal of TRH content and melatonin production seems inconsistent with the postulate that melatonin inhibits TRH release from the hypothalamus. Compared with sham-operated controls, pinealectomy had no effect on hypothalamic TRH content, TSH levels, or measures of thyroid function. The difference in serum T3 levels between sham-operated and pinealectomized animals observed in one experiment (Table 2) was not observed in another experiment (Table 3). These differing results may be due to chance, but we cannot exclude an effect of pinealectomy on T3 production. Thus our data confirm and extend the work of Rowe et al. (25), which showed no effect of pinealectomy on plasma TSH or PBI levels. We conclude that it is unlikely that the pineal gland plays a physiological role in the regulation of thyroid function. TRH has been reported to be distributed throughout the brain (9), the retina (%), the gastrointestinal tract (12), and the placenta (27). It has been suggested that TRH is a ubiquitous neurotransmitter (9, 12) that has been co-opted by the pituitary to provide a stimulus to maintain basal TSH levels. Our observation of a diurnal rhythm in rat pineal gland TRH content that is phase reversed from the adrenergically regulated diurnal rhythm in melatonin production and reports of interactions between constituents of the H-P-T axis and adrenergically induced responses suggested to us the possibility of an interaction between H-P-T constituents and the regulation of melatonin production. We found no evidence for an interaction between pineal TRH content or exogenous TRH or T3 and the adrenergic SNAT regulatory system in the rat pineal gland. The lack of an inhibitory effect of TRH on norepinephrine stimulation of pineal SNAT activity is contrary to the report of Tsang and Martin (28) indicating an inhibitory effect of TRH on norepinephrine stimulation of cyclic AMP levels in the rat pineal gland because increased cyclic AMP seems to mediate the increase of SNAT activity. A number of studies have shown that pinealectomy depresses basal prolactin levels in rats (21,24) and blocks the prolactin rise that occurs in constant darkness (23). Pineal extracts (3), melatonin (11), and arginine vasototin (29), a polypeptide recently identified in the pineal gland of rats (17) and man (16), are all capable of elevating prolactin in vivo. Although mean prolactin levels were lower in our pinealectomized rats compared with the sham-operated controls, at no time were the levels significantly different. Similarly there was no significant difference in pituitary prolactin content, Peak prolactin levels have been shown to occur between 0400 and 0500 h (23) and it is possible that a significant difference could have been observed at that time. In addition, the “basal” prolactin levels reported by Ronnekleiv and McCann (24) were very high (between 40 and 100 rig/ml) and this could have represented an effect of pinealectomy on decreasing the stress-related prolactin rise. Melatonin Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016 -6 WITH E420 produced no effect on prolactin or TSH release from the anterior pituitary in vitro. This result is in keeping with earlier studies suggesting that the action of melatonin on prolactin release is at the hypothalamic rather than at the pituitary level (11). Our results suggest that the pineal gland does not play a major physiological role in the control of prolactin. BRAMMER The skilled technical assistance of J. Briggs, A. Reed is gratefully acknowledged. This study was supported by the Veterans worth Medical Research Service, the Veterans wood Research and Development Service, and Health Grant HD-71-81. Received 15 August 1978; accepted in final form E. Martin, ET J. Park, AL. and Administration WadsAdministration BrentNational Institutes of 22 November 1978. REFERENCES Biosynthesis of a vasotocin-like peptide in cell cultures from pineal glands of human fetuses. 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Plasma thyrotropin, thyroxine and triiodothyronine relationships in man. J, CZin. EndocrinoZ. Metab. 43: 533-542, 1976. 2. BASSIRI, R. M., AND R. D. UTIGER. The preparation and specificity of antibody to thyrotropin releasing hormone. Endocrinology 90: 722-727, 1972. 3. BLASK, D. E., M. K. VAUGHAN, R. J. REITER, L. Y. JOHNSON, AND G. M. VAUGHAN, Prolactin-releasing and release-inhibiting factor activities in the bovine, rat and human pineal gland: in vitro and in vivo studies. Endocrinology 99: 152-162, 1976. 4. CARLSON, H. E., I. K. MARIZ, J. E. BRIGGS, AND W. H. DAUGHADAY. Altered responsiveness to hypophysiotropic hormones of perfused rat pituitary tumors. Acta EndocrinoZ. 84: 512-522, 1977. 5, COLLER, R., P. Du RUISSEAU, Y. TACHE, AND J. R. DUCHARME. Thyrotropin-releasing hormone in rat brain: nyctohemeral variations. Endocrinology 100: 1391-1393, 1977. 6. DEGUCHI, T., AND J. AXELROD, Sensitive assay for serotonin Nacetyltransferase activity in rat pineal. AnaL. 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Thyrotropin-releasing hormone in the gastrointestinal tract. Bio&em. Biophys. Res. Commun. 79: 314-318, 1977. 13. PARFITT, A., J, L. WELLER, D. C. KLEIN, K. K, SAKAI, AND B. H. MARKS. Blockade by ouabain or elevated potassium ion concentration of the adrenergic and adenosine cyclic 3’,5’-monophosphateinduced stimulation of pineal serotonin N-acetyltransferase activity. MOL. PharmacoZ. II: 241-255, 1975. 14. P~RFITT, A., J. L. WELLER, AND D. C. KLEIN. Beta adrenergicblockers decrease adrenergically stimulated IV-acetyltransferase activity in pineal glands in organ culture. Neuropharmacology 15: 353-358, 1976. 15. PARSLOW, M. E., T. H. UDDIE, AND D. A. FISHER. Evaluation of serum triiodothyronine and adjusted triiodothyronine (free triiodothyronine index) in pregnancy. Ch. Chem. 23: 490-492, 1977. 16. PAVEL, S., M. DORCESCU, R. PETRESCU-HOLBON, AND E. GHINEA.