0013-7227/78/1031-0281$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 1 Printed in U.S.A.
Gonadotropin-Releasing Hormone Release from the Rat Hypothalamus: Dependence on Membrane Depolarization and Calcium Influx* HOMAYOON BIGDELI AND PETER J. SNYDER Endocrine Section, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19174 ABSTRACT. Release of gonadotropin-releasing hormone (GnRH) was studied by incubating individual rat hypothalami for 60 min, after a 30-min preincubation period, and measuring GnRH in the medium by immunoassay. During the 1 h of incubation, endogenous GnRH release was linear and exogenous GnRH was not destroyed. Membrane depolarization produced by increasing the medium potassium concentration to 60 mM increased GnRH release to 200-500% of control. Membrane depolarization produced by adding 10~5 or 10~4 M ouabain increased GnRH release to 200% of control. Melatonin (10~7 M) and prostaglandin E2 (4 x 10~4 M)
G
ONADOTROPIN-RELEASING hormone (GnRH) is found in hypothalamic neurons, principally in the medial basal hypothalamus (1-3), and is secreted into the hypothalamic-hypophyseal portal circulation (4-6). Although GnRH can be measured in picogram quantities by immunoassay (7), the regulation of GnRH secretion is not well understood. The GnRH concentration in the portal circulation has been determined in the rat (4) and monkey (5, 6) and has been found to fluctuate widely and rapidly (5) and to increase after the intraventricular administration of prostaglandin E2 (PGE2) (4). GnRH release from perifused or incubated rat medial basal hypothalami has also been reported (8-10); release was stimulated by PGE2 (9) or by melatonin (10).
also stimulated GnRH release to 200% and 170% of control, respectively. Inhibition of calcium influx by omission of medium calcium and addition of 0.05 M EDTA reduced GnRH release to 50% of control. Both no calcium-EDTA medium and verapamil (10~5 M) prevented the stimulation of GnRH release by 60 mM potassium, 10~3 M melatonin, and 4 x 10~4 M prostaglandin E2. We conclude that hypothalamic GnRH release depends on membrane depolarization and calcium influx, as does the secretion of hormones from other endocrine tissues. (Endocrinology 103: 281, 1978)
in this report. The method is simple and requires only one hypothalamus per incubation tube, yet GnRH release is linear for 1 h and not only can be stimulated by PGE2, melatonin, and membrane depolarization, but also can be inhibited by blocking intracellular calcium influx. Materials and Methods Incubation of hypothalami
Male Sprague-Dawley rats (200-250 g) were sacrificed by decapitation, and the hypothalami were removed with microdissection scissors. The borders of the hypothalamus were the optic chiasm anteriorly, the mammillary bodies posteriorly, and the hypothalamic fissures laterally. The mean (±SE) weight of each piece of tissue removed was 43 ± 2 mg. Immediately after removal, the hypothalami A method for measuring GnRH release from were placed in the medium to be used for the preincubation period at 37 C. the incubated rat hypothalamus is described The control incubation medium contained 133 mM NaCl; 5.3 mM KC1; 2.8 mM CaCl; 1.3 mM KH2PO4; 1.3 mM MgSO4, 2.7 mM Na HCO3; 20 mM Received November 14, 1977. Address requests for reprints to: Dr. Peter J. Snyder, Hepes; 56 mM glucose, Eagle's amino acids, and University of Pennsylvania, School of Medicine, Endo- vitamins; and 2.0 mM L-glutamine. The pH was crine Section, Department of Medicine, 522 Johnson Paadjusted to 7.0. Other media were similar to the vilion G2, Philadelphia, Pennsylvania 19174. * This work was supported by USPHS Grant HD- control medium except for the following modifica08555. tions: high potassium medium, 78 mM NaCl, 60 mM 281
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BIGDELI AND SNYDER
KC1; no calcium medium, CaCU replaced by NaCl, with and without 0.05 M EDTA. The following additions were also made to the control medium, as indicated: 10"4-10~6 M verapamil (Knoll Pharmaceuticals, NY), 10~3 M melatonin and 1.3 X 10~2 M ascorbic acid, 7 x 10~5 M prostaglandin E2 and 4 X 10"4 M ETOH, 10"6-10~8 M ouabain, and 2 X 10~5 to 2 X 10~4 M phenytoin sodium. The hypothalami were preincubated in 2 ml medium in 100 X 15-mm tubes for 25-30 min in a Dubnoff incubator at 37 C in room air. After the preincubation, the medium was aspirated and the hypothalami were washed twice with 1 ml preincubation medium. One milliliter of incubation medium was then added and the hypothalami were incubated for 60 min. In experiments where samples were collected both at 30 min and 60 min, the hypothalami of two rats were incubated in 2 ml medium. Additions to the control medium that were expected to inhibit GnRH secretion were made to the preincubation medium as well as to the incubation medium, whereas additions that were expected to stimulate GnRH secretion were made only to the incubation medium. At the end of incubation period, the medium was aspirated and frozen until assay of GnRH. GnRH immunoassay GnRH was assayed by a modification of the method of Nett and Niswender (7), using antiGnRH generously provided by them. Synthetic GnRH for standards and iodination was provided by the NIAMDD. [125I]Iodo-GnRH was prepared by the lactoperoxidase method (11). [125I]IodoGnRH and GnRH standard were stable for 2 months if lyophilized immediately after preparation. Binding of the [125I]iodo-GnRH to the antiGnRH at pH 7.4 was much less (approximately 33%) of that at pH 7.0-7.2; assay buffers, therefore, were all titrated to pH 7.2. Bicarbonate also inhibited the binding of [125I]iodo-GnRH to antiGnRH; for this reason, Hepes was the principal buffer for the media in which the hypothalami were incubated. The lower limit of sensitivity of the assay was 2 pg/assay tube.
Endo • 1978 Vol 103 • No 1
ported that the incubated rat medial basal hypothalmus destroyed synthetic GnRH (8), the recovery of synthetic GnRH incubated with rat hypothalami was determined (Table 1). The amount of GnRH recovered in the incubation medium when synthetic GnRH was incubated for 1 h with single hypothalami was virtually the same as the sum of the GnRH recovered when GnRH and hypothalami were incubated separately. Hypothalami thus do not appear to destroy GnRH under the present incubation conditions. The time course of GnRH release into the incubation medium after a 30-min preincubation was determined. As in all experiments reported here, the preincubation medium was discarded before addition of the incubation medium, and zero time refers to when the incubation medium was added. GnRH release into the control incubation medium (Table 2) was similar during two 30-min incubation periods. In subsequent experiments, therefore, GnRH release was studied during a 60-min incubation period. Incubation under 100% O2 did not affect GnRH release compared to incubation under room air. Room air was therefore used in all experiments. Effect of membrane depolarization on GnRH release High medium potassium concentration and addition of ouabain to the incubation medium were used to cause membrane depolarization. When hypothalami were incubated in 60 mM K+ (Table 3), twice as much GnRH was released as into the basal medium. Pieces of frontal cortex released no detectable GnRH into either control or 60 mM K+ media. TABLE 1. Recovery of GnRH in the presence and absence of hypothalamic tissue
Analysis Statistical analyses were performed with the t test on nonpaired samples (12).
Results
n GnRH alone (50 pg/tube) Hypothalamus alone Hypothalamus + GnRH (50 pg/tube)
8 7 7
GnRH recovered after 1 Mpg) 43.9 ± 3.7 33.6 ± 4.7 76.0 ± 4.7
Because the rat hypothalamus is known to Synthetic GnRH was incubated in the presence and contain a peptidase which rapidly destroys absence of rat hypothalami, one per tube. Results are GnRH (13), and because Rotsztejn et al. re- expressed as means ± SE.
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283
GnRH RELEASE FROM RAT HYPOTHALAMUS TABLE 2. Time course of GnRH release
GnRH release (pg/hypothalamus/30 min)
First 30 min
Second 30 min
21.6 ± 1.4
24.8 ± 2.0
40
1
30
Two hypothalami were incubated per tube for two consecutive 30-min periods after a 30-min preincubation period (n = 4 for each group). Results are expressed as means ± SE.
T J.
20 10
TABLE 3. GnRH release by incubated hypothalami and frontal cortex in control medium and in high potassium (60 DIM) medium GnRH release (pg/h)
Control medium
K+ (60 mM)
Hypothalami 23.2 ± 1.8 52.5 ± 11.1° Frontal cortex Weights of pieces of frontal cortex were similar to weights of hypothalami (n = 8 for both groups). a P< 0.02 vs. control medium.
1. I, 1 1
1
...
n=8
Ouoboir IO" 6 M
Ouobo IO' 5 M
IO'"M
FIG. 1. Stimulation of GnRH release from incubated hypothalami by ouabain. Values are means ± SE. *, P < 0.05; f, P < 0.01, compared to control group. 50 Control 40
30
Ouabain, which is known to inhibit Na-KATPase and thereby cause membrane depolarization (14,15), was added to the incubation medium at 10~6-10~4 M. At 10~5 or 10~4 M, ouabain significantly increased the release of GnRH (Fig. 1). Phenytoin sodium, which stimulates Na-KATPase and thereby causes membrane hyperpolarization, was added to the medium at 2 X 10~5 to 2 X 10~4 M, but had no effect on GnRH release. Effect of inhibiting calcium uptake on GnRH release
20
0
30
60
Time (min)
FIG. 2. GnRH release by hypothalami incubated in control medium and in media from which Ca ++ was omitted, with and without the addition of 0.05 M EDTA. Statistical analysis for 60-min values compared to control values: no Ca ++ , P < 0.02; no Ca ++ -EDTA, P < 0.001. 100
Hypothalamic calcium uptake was inhibited 80 by omitting Ca++ from the incubation medium and by adding 0.05 M EDTA or verapamil to 60 the medium. Figure 2 illustrates that when calcium was omitted from the incubation me40 dium GnRH release was less than control, though still linear for 60 min. When Ca++ was 20 omitted and EDTA was added to chelate Ca++ released by the tissue, GnRH release was even 60mM K* 60mM K less. Omission of Ca++ and addition of EDTA NOCo inhibited not only basal release of GnRH, but also GnRH release stimulated by 60 mM K+ FIG. 3. Stimulation of GnRH release from incubated hy(Fig. 3), 10"3 M melatonin (Fig. 4), and 7 X pothalami by 60 mM potassium and inhibition of potassium-stimulated GnRH release by omitting calcium from, 10"5 M PGE2 (Fig. 5). and adding EDTA (0.05 M) to, the medium. Values are Verapamil, which inhibits the uptake of means ± SE. *, P < 0.001 vs. both control group and high Ca++ by cells (16, 17), had no effect on basal potassium-no calcium-EDTA group. +
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BIGDELI AND SNYDER
284 60r
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i
o
o 40
i l
Q.
^a. ^n a> o •s Q: 20
Tt Tt 1
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FIG. 4. Stimulation of GnRH release by melatonin (10 3 M) and inhibition of melatonin-stimulated GnRH release by no Ca++-0.05 M EDTA medium and by verapamil (10~5 M). Values are means ± SE. *, P < 0.01 vs. control; f, P < 0.001 vs. melatonin. 60r
£
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i
50-
40-
30-
2
20-
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n=8
FIG. 5. Stimulation of GnRH release by PGE2 (7 x 10~5 M) and inhibition of PGE2-stimulated GnRH release by no Ca++-0.05 M EDTA and by verapamil (10~5 M). Values are means ± SE. *, P < 0.01 vs. control; f, P < 0.001 vs. PGE2.
GnRH release at 10"3-10"5 M (not shown), but at 10~5 M inhibited melatonin-stimulated (Fig. 4) and PGE2-stimulated (Fig. 5) GnRH release. Neither 4 X 10~4 M ethanol, used to dissolve PGE2, nor 1.3 x 10~2 M ascorbic acid, used to dissolve melatonin, influenced GnRH release, as shown in separate experiments. Discussion Membrane depolarization has been shown to cause hormonal release from several endo-
Endo • 1978 Vol 103 • No 1
crine tissues. A high medium potassium concentration (>30 mM), which produces membrane depolarization, stimulates the release of insulin from rabbit pancreatic slices (18), vasopressin from the isolated rat posterior pituitary gland (19), and catecholamines from the perfused cat adrenal gland (20). More recently, GnRH release from the incubated (8) or perifused (9, 10) rat medial basal hypothalamus has been increased by a high medium potassium concentration. In the experiments reported here, a high medium potassium concentration likewise increased GnRH release (Table 3). Ouabain, which also produces membrane depolarization, by inhibiting sodium-potassium ATPase (14, 15), stimulated insulin release from slices of rabbit pancreas (18) and stimulated GnRH release in th present experiments (Fig. 1). Phenytoin sodium, which stimulates (Na-K)-ATPase and thereby inhibits membrane depolarization, inhibited insulin release from isolated islets of Langerhans (21) but, in the present experiments, did not inhibit basal or stimulated GnRH release. Depolarization of the cell membrane is followed by calcium influx (19). The release of several hormones from various endocrine tissues seems to be dependent on this intracellular entry of calcium. Omission of calcium from the incubation medium or perfusate inhibits the secretion of insulin (22, 23), vasopressin from isolated guinea pig posterior pituitary glands (24), and catecholamines from perfused cat adrenal glands (25). The dependence of hormonal secretion on calcium influx has also been demonstrated by the use of verapamil, a drug which inhibits the intracellular entry of calcium (16, 17). Verapamil inhibits insulin release from the isolated perfused rat pancreas (26), and a methoxy derivative of verapamil, called D600, inhibits the release of vasopressin and neurophysin from ox posterior pituitary slices (27). Both basal and high potassium-stimulated GnRH release from the incubated (8) or perifused (9) rat hypothalamus and rat hypothalamic synaptosomes (28) have been shown to be inhibited by omission of calcium from the medium. In the experiments described here, the dependence of GnRH release on calcium influx was demonstrated indirectly in several ways.
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GnRH RELEASE FROM RAT HYPOTHALAMUS Omission of calcium from the incubation medium and the addition of EDTA, to chelate any calcium released by the tissue, inhibited both basal release of GnRH (Fig. 2) and also GnRH release stimulated by a depolarizing concentration of potassium (Fig. 3) and by melatonin (Fig. 4) and PGE2 (Fig. 5). Addition of verapamil to the medium also inhibited melatonin- and PGE2-stimulated GnRH release (Figs. 4 and 5). The data presented here, therefore, provide new evidence that hypothalamic GnRH release depends on membrane depolarization and calcium influx. The demonstration of this dependence by the hypothalamic preparation described here is evidence that this preparation behaves, at least to some extent, physiologically and might therefore be used to study other factors that influence GnRH release. As basal as well as stimulated GnRH release from a single hypothalamus in 1 h was readily detectable and was inhibited by omission of calcium from the incubation medium, this system seems to be suitable for studying inhibitory as well as stimulatory factors. Acknowledgments The authors thank Drs. Terry M. Nett and Gordon D. Niswender for a generous gift of anti-GnRH serum, the NIAMDD for synthetic GnRH, Knoll Pharmaceuticals for verapamil, find Ms. Elaine Paolini for preparation of the manuscript.
References 1. Palkovits, M., A. Arimura, M. Brownstein, A. V. Schally, and J. M. Saavedra, Luteinizing hormonereleasing hormone (LH-RH) content of the hypothalamic nuclei in rat, Endocrinology 95: 554, 1974. 2. Barry, J., M. P. Dubois, and B. Carett, Immunofluorescence study of the preopticoinfundibular LRF neurosecretory pathway in the normal, castrated or testosterone-treated male guinea pig, Endocrinology 95: 1416, 1974. 3. Wheaton, J. E., L. Krulich, and S. M. McCann, Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology 97: 30,1975. 4. Eskay, R. L., J. Warberg, R. S. Mical, and J. C. Porter, Prostaglandin E2-induced release of LHRH into hypophyseal portal blood, Endocrinology 97: 816,1975. 5. Carmel, P. W., S. Araki, and M. Ferin, Pituitary stalk portal blood collection in rhesus monkeys: evidence for pulsatile release of gonadotropin-releasing hormone (GnRH), Endocrinology 99: 243,1976.
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6. Neill, J. D., J. M. Patton, P. A. Daily, R. C. Tsau, and G. T. Tindall, Luteinizing hormone-releasing hormone in pituitary stalk blood of rhesus monkeys: relationship to level of LH release, Endocrinology 101: 430, 1977. 7. Nett, T. M., A. M. Akbar, A. D. Niswender, M. T. Hedlund, and W. F. White, A radioimmunoassay for gonadotropin-releasing hormone (GnRH) in serum, J Clin Endocrinol Metab 36: 880, 1973. 8. Rotsztejn, W. H., J. L. Charli, E. Dattou, J. Epelbaum, and C. Kardon, In vitro release of luteinizing hormone-releasing hormone (LHRH) from rat mediabasal hypothalamus: effects of potassium, calcium and dopamine, Endocrinology 99: 1663, 1976. 9. Gallardo, E., and V. D. Ramirez, A method for the superfusion of rat hypothalami: secretion of luteinizing hormone-releasing hormone (LH-RH), Proc Soc Exp BiolMed 195: 79, 1977. 10. Kao, L. W. L., and J. Weisz, Release of gonadotrophinreleasing hormone (Gn-RH) from isolated perifused medial-basal hypothalamus by melatonin, Endocrinology 100: 1723, 1977. 11. Thorell, J. J., and B. C. Johansson, Enzymatic iodination of polypeptides with I25I to high specific activity, Biochim Biophys Acta 251: 363, 1971. 12. Snedecor, G. W., and W. G. Cochran, Statistical Methods, ed. 3, Iowa State University Press, Ames, 1967, p. 91. 13. Griffiths, E. C, K. C. Hooper, and C. R. N. Hopkinson, Evidence for an enzymic component in the rat hypothalamus capable of inactivating luteinizing hormone releasing factor (LRF), Acta Endocrinol (Kbh) 74: 49, 1973. 14. Skou, J. C, Enzymatic basis for active transport of Na+ and K+ across cell membranes, Physiol Rev 45: 586, 1965. 15. Glynn, I. M., The action of cardiac glycosides on sodium and potassium movements in human red cells, J Physiol 136: 148, 1957. 16. Russell, J. T., and N. A. Thorn, Calcium and stimulussecretion coupling in the neurohypophysis: effects of lanthanum, a verapamil analogue (D600) and prenylamine on 45Ca transport and vasopressin release of isolated rat neurohypophyses, Acta Endocrinol 76: 471, 1974. 17. Dreifus, J. J., J. D. Grau, and J. J. Kardmann, Effects on the isolated neurohypophysis of agents which affect the membrane permeability to calcium, J Physiol 231: 96, 1973. 18. Hales, C. N., and R. D. G. Miller, The role of sodium and potassium in insulin secretion from rabbit pancreas, J Physiol 194: 725, 1968. 19. Douglas, W. W., and A. M. Poisner, Stimulus-secretory coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis, J Physiol 172: 1,1969. 20. Douglas, W. W., and A. M. Poisner, Stimulation of uptake of calcium-45 in the adrenal gland by acetylcholine, Nature 192: 1299, 1961. 21. Kizer, J. S., M. Vargas-Cordon, K. Brendal, and R. Bressler, The in vitro inhibition of insulin secretion
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by diphenylhydantoin, J Clin Invest 49: 1942, 1970. 22. Giodsky, G. M., and L. L. Bennett, Cation requirements for insulin secretion in the isolated perfused pancreas, Diabetes 15: 910, 1966. 23. Milner, R. D. G., and C. N. Hales, The role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro, Diabetologia 3: 47, 1967. 24. Haller, E. W., H. Sachs, N. Sparelakis, and L. Share, Release of vasopressin from isolated guinea pig posterior pituitaries, AmJPhysiol 209: 79, 1965. 25. Douglas, W. W., and R. P. Rubin, The role of calcium in the secretory response of adrenal medulla to ace-
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tylcholine, J Physiol 159: 40, 1961. 26. Somers, G., G. Devis, E. Van Obberghen, and W. J. Malaise, Calcium antagonists and islet function: interaction of theophylline and verapamil, Endocrinology 99: 114, 1976. 27. Robinson, I. C. A. F., J. T. Russel, and N. A. Thorn, Calcium and stimulus secretion coupling in the neurohypophysis, Ada Endocrinol 83: 36, 1976. 28. Warberg, J., R. L. Eskay, A. Barnea, R. C. Reynolds, arid J. C. Porter, Release of LHRH and TRH from a synaptosome enriched fraction of hypothalamic homogenates, Endocrinology 100: 814,1977.
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Vol. 103, No. 1 Printed in U.S.A.
Gonadotropin-Releasing Hormone Release from the Rat Hypothalamus: Dependence on Membrane Depolarization and Calcium Influx* HOMAYOON BIGDELI AND PETER J. SNYDER Endocrine Section, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19174 ABSTRACT. Release of gonadotropin-releasing hormone (GnRH) was studied by incubating individual rat hypothalami for 60 min, after a 30-min preincubation period, and measuring GnRH in the medium by immunoassay. During the 1 h of incubation, endogenous GnRH release was linear and exogenous GnRH was not destroyed. Membrane depolarization produced by increasing the medium potassium concentration to 60 mM increased GnRH release to 200-500% of control. Membrane depolarization produced by adding 10~5 or 10~4 M ouabain increased GnRH release to 200% of control. Melatonin (10~7 M) and prostaglandin E2 (4 x 10~4 M)
G
ONADOTROPIN-RELEASING hormone (GnRH) is found in hypothalamic neurons, principally in the medial basal hypothalamus (1-3), and is secreted into the hypothalamic-hypophyseal portal circulation (4-6). Although GnRH can be measured in picogram quantities by immunoassay (7), the regulation of GnRH secretion is not well understood. The GnRH concentration in the portal circulation has been determined in the rat (4) and monkey (5, 6) and has been found to fluctuate widely and rapidly (5) and to increase after the intraventricular administration of prostaglandin E2 (PGE2) (4). GnRH release from perifused or incubated rat medial basal hypothalami has also been reported (8-10); release was stimulated by PGE2 (9) or by melatonin (10).
also stimulated GnRH release to 200% and 170% of control, respectively. Inhibition of calcium influx by omission of medium calcium and addition of 0.05 M EDTA reduced GnRH release to 50% of control. Both no calcium-EDTA medium and verapamil (10~5 M) prevented the stimulation of GnRH release by 60 mM potassium, 10~3 M melatonin, and 4 x 10~4 M prostaglandin E2. We conclude that hypothalamic GnRH release depends on membrane depolarization and calcium influx, as does the secretion of hormones from other endocrine tissues. (Endocrinology 103: 281, 1978)
in this report. The method is simple and requires only one hypothalamus per incubation tube, yet GnRH release is linear for 1 h and not only can be stimulated by PGE2, melatonin, and membrane depolarization, but also can be inhibited by blocking intracellular calcium influx. Materials and Methods Incubation of hypothalami
Male Sprague-Dawley rats (200-250 g) were sacrificed by decapitation, and the hypothalami were removed with microdissection scissors. The borders of the hypothalamus were the optic chiasm anteriorly, the mammillary bodies posteriorly, and the hypothalamic fissures laterally. The mean (±SE) weight of each piece of tissue removed was 43 ± 2 mg. Immediately after removal, the hypothalami A method for measuring GnRH release from were placed in the medium to be used for the preincubation period at 37 C. the incubated rat hypothalamus is described The control incubation medium contained 133 mM NaCl; 5.3 mM KC1; 2.8 mM CaCl; 1.3 mM KH2PO4; 1.3 mM MgSO4, 2.7 mM Na HCO3; 20 mM Received November 14, 1977. Address requests for reprints to: Dr. Peter J. Snyder, Hepes; 56 mM glucose, Eagle's amino acids, and University of Pennsylvania, School of Medicine, Endo- vitamins; and 2.0 mM L-glutamine. The pH was crine Section, Department of Medicine, 522 Johnson Paadjusted to 7.0. Other media were similar to the vilion G2, Philadelphia, Pennsylvania 19174. * This work was supported by USPHS Grant HD- control medium except for the following modifica08555. tions: high potassium medium, 78 mM NaCl, 60 mM 281
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282
BIGDELI AND SNYDER
KC1; no calcium medium, CaCU replaced by NaCl, with and without 0.05 M EDTA. The following additions were also made to the control medium, as indicated: 10"4-10~6 M verapamil (Knoll Pharmaceuticals, NY), 10~3 M melatonin and 1.3 X 10~2 M ascorbic acid, 7 x 10~5 M prostaglandin E2 and 4 X 10"4 M ETOH, 10"6-10~8 M ouabain, and 2 X 10~5 to 2 X 10~4 M phenytoin sodium. The hypothalami were preincubated in 2 ml medium in 100 X 15-mm tubes for 25-30 min in a Dubnoff incubator at 37 C in room air. After the preincubation, the medium was aspirated and the hypothalami were washed twice with 1 ml preincubation medium. One milliliter of incubation medium was then added and the hypothalami were incubated for 60 min. In experiments where samples were collected both at 30 min and 60 min, the hypothalami of two rats were incubated in 2 ml medium. Additions to the control medium that were expected to inhibit GnRH secretion were made to the preincubation medium as well as to the incubation medium, whereas additions that were expected to stimulate GnRH secretion were made only to the incubation medium. At the end of incubation period, the medium was aspirated and frozen until assay of GnRH. GnRH immunoassay GnRH was assayed by a modification of the method of Nett and Niswender (7), using antiGnRH generously provided by them. Synthetic GnRH for standards and iodination was provided by the NIAMDD. [125I]Iodo-GnRH was prepared by the lactoperoxidase method (11). [125I]IodoGnRH and GnRH standard were stable for 2 months if lyophilized immediately after preparation. Binding of the [125I]iodo-GnRH to the antiGnRH at pH 7.4 was much less (approximately 33%) of that at pH 7.0-7.2; assay buffers, therefore, were all titrated to pH 7.2. Bicarbonate also inhibited the binding of [125I]iodo-GnRH to antiGnRH; for this reason, Hepes was the principal buffer for the media in which the hypothalami were incubated. The lower limit of sensitivity of the assay was 2 pg/assay tube.
Endo • 1978 Vol 103 • No 1
ported that the incubated rat medial basal hypothalmus destroyed synthetic GnRH (8), the recovery of synthetic GnRH incubated with rat hypothalami was determined (Table 1). The amount of GnRH recovered in the incubation medium when synthetic GnRH was incubated for 1 h with single hypothalami was virtually the same as the sum of the GnRH recovered when GnRH and hypothalami were incubated separately. Hypothalami thus do not appear to destroy GnRH under the present incubation conditions. The time course of GnRH release into the incubation medium after a 30-min preincubation was determined. As in all experiments reported here, the preincubation medium was discarded before addition of the incubation medium, and zero time refers to when the incubation medium was added. GnRH release into the control incubation medium (Table 2) was similar during two 30-min incubation periods. In subsequent experiments, therefore, GnRH release was studied during a 60-min incubation period. Incubation under 100% O2 did not affect GnRH release compared to incubation under room air. Room air was therefore used in all experiments. Effect of membrane depolarization on GnRH release High medium potassium concentration and addition of ouabain to the incubation medium were used to cause membrane depolarization. When hypothalami were incubated in 60 mM K+ (Table 3), twice as much GnRH was released as into the basal medium. Pieces of frontal cortex released no detectable GnRH into either control or 60 mM K+ media. TABLE 1. Recovery of GnRH in the presence and absence of hypothalamic tissue
Analysis Statistical analyses were performed with the t test on nonpaired samples (12).
Results
n GnRH alone (50 pg/tube) Hypothalamus alone Hypothalamus + GnRH (50 pg/tube)
8 7 7
GnRH recovered after 1 Mpg) 43.9 ± 3.7 33.6 ± 4.7 76.0 ± 4.7
Because the rat hypothalamus is known to Synthetic GnRH was incubated in the presence and contain a peptidase which rapidly destroys absence of rat hypothalami, one per tube. Results are GnRH (13), and because Rotsztejn et al. re- expressed as means ± SE.
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283
GnRH RELEASE FROM RAT HYPOTHALAMUS TABLE 2. Time course of GnRH release
GnRH release (pg/hypothalamus/30 min)
First 30 min
Second 30 min
21.6 ± 1.4
24.8 ± 2.0
40
1
30
Two hypothalami were incubated per tube for two consecutive 30-min periods after a 30-min preincubation period (n = 4 for each group). Results are expressed as means ± SE.
T J.
20 10
TABLE 3. GnRH release by incubated hypothalami and frontal cortex in control medium and in high potassium (60 DIM) medium GnRH release (pg/h)
Control medium
K+ (60 mM)
Hypothalami 23.2 ± 1.8 52.5 ± 11.1° Frontal cortex Weights of pieces of frontal cortex were similar to weights of hypothalami (n = 8 for both groups). a P< 0.02 vs. control medium.
1. I, 1 1
1
...
n=8
Ouoboir IO" 6 M
Ouobo IO' 5 M
IO'"M
FIG. 1. Stimulation of GnRH release from incubated hypothalami by ouabain. Values are means ± SE. *, P < 0.05; f, P < 0.01, compared to control group. 50 Control 40
30
Ouabain, which is known to inhibit Na-KATPase and thereby cause membrane depolarization (14,15), was added to the incubation medium at 10~6-10~4 M. At 10~5 or 10~4 M, ouabain significantly increased the release of GnRH (Fig. 1). Phenytoin sodium, which stimulates Na-KATPase and thereby causes membrane hyperpolarization, was added to the medium at 2 X 10~5 to 2 X 10~4 M, but had no effect on GnRH release. Effect of inhibiting calcium uptake on GnRH release
20
0
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60
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FIG. 2. GnRH release by hypothalami incubated in control medium and in media from which Ca ++ was omitted, with and without the addition of 0.05 M EDTA. Statistical analysis for 60-min values compared to control values: no Ca ++ , P < 0.02; no Ca ++ -EDTA, P < 0.001. 100
Hypothalamic calcium uptake was inhibited 80 by omitting Ca++ from the incubation medium and by adding 0.05 M EDTA or verapamil to 60 the medium. Figure 2 illustrates that when calcium was omitted from the incubation me40 dium GnRH release was less than control, though still linear for 60 min. When Ca++ was 20 omitted and EDTA was added to chelate Ca++ released by the tissue, GnRH release was even 60mM K* 60mM K less. Omission of Ca++ and addition of EDTA NOCo inhibited not only basal release of GnRH, but also GnRH release stimulated by 60 mM K+ FIG. 3. Stimulation of GnRH release from incubated hy(Fig. 3), 10"3 M melatonin (Fig. 4), and 7 X pothalami by 60 mM potassium and inhibition of potassium-stimulated GnRH release by omitting calcium from, 10"5 M PGE2 (Fig. 5). and adding EDTA (0.05 M) to, the medium. Values are Verapamil, which inhibits the uptake of means ± SE. *, P < 0.001 vs. both control group and high Ca++ by cells (16, 17), had no effect on basal potassium-no calcium-EDTA group. +
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BIGDELI AND SNYDER
284 60r
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FIG. 4. Stimulation of GnRH release by melatonin (10 3 M) and inhibition of melatonin-stimulated GnRH release by no Ca++-0.05 M EDTA medium and by verapamil (10~5 M). Values are means ± SE. *, P < 0.01 vs. control; f, P < 0.001 vs. melatonin. 60r
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FIG. 5. Stimulation of GnRH release by PGE2 (7 x 10~5 M) and inhibition of PGE2-stimulated GnRH release by no Ca++-0.05 M EDTA and by verapamil (10~5 M). Values are means ± SE. *, P < 0.01 vs. control; f, P < 0.001 vs. PGE2.
GnRH release at 10"3-10"5 M (not shown), but at 10~5 M inhibited melatonin-stimulated (Fig. 4) and PGE2-stimulated (Fig. 5) GnRH release. Neither 4 X 10~4 M ethanol, used to dissolve PGE2, nor 1.3 x 10~2 M ascorbic acid, used to dissolve melatonin, influenced GnRH release, as shown in separate experiments. Discussion Membrane depolarization has been shown to cause hormonal release from several endo-
Endo • 1978 Vol 103 • No 1
crine tissues. A high medium potassium concentration (>30 mM), which produces membrane depolarization, stimulates the release of insulin from rabbit pancreatic slices (18), vasopressin from the isolated rat posterior pituitary gland (19), and catecholamines from the perfused cat adrenal gland (20). More recently, GnRH release from the incubated (8) or perifused (9, 10) rat medial basal hypothalamus has been increased by a high medium potassium concentration. In the experiments reported here, a high medium potassium concentration likewise increased GnRH release (Table 3). Ouabain, which also produces membrane depolarization, by inhibiting sodium-potassium ATPase (14, 15), stimulated insulin release from slices of rabbit pancreas (18) and stimulated GnRH release in th present experiments (Fig. 1). Phenytoin sodium, which stimulates (Na-K)-ATPase and thereby inhibits membrane depolarization, inhibited insulin release from isolated islets of Langerhans (21) but, in the present experiments, did not inhibit basal or stimulated GnRH release. Depolarization of the cell membrane is followed by calcium influx (19). The release of several hormones from various endocrine tissues seems to be dependent on this intracellular entry of calcium. Omission of calcium from the incubation medium or perfusate inhibits the secretion of insulin (22, 23), vasopressin from isolated guinea pig posterior pituitary glands (24), and catecholamines from perfused cat adrenal glands (25). The dependence of hormonal secretion on calcium influx has also been demonstrated by the use of verapamil, a drug which inhibits the intracellular entry of calcium (16, 17). Verapamil inhibits insulin release from the isolated perfused rat pancreas (26), and a methoxy derivative of verapamil, called D600, inhibits the release of vasopressin and neurophysin from ox posterior pituitary slices (27). Both basal and high potassium-stimulated GnRH release from the incubated (8) or perifused (9) rat hypothalamus and rat hypothalamic synaptosomes (28) have been shown to be inhibited by omission of calcium from the medium. In the experiments described here, the dependence of GnRH release on calcium influx was demonstrated indirectly in several ways.
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GnRH RELEASE FROM RAT HYPOTHALAMUS Omission of calcium from the incubation medium and the addition of EDTA, to chelate any calcium released by the tissue, inhibited both basal release of GnRH (Fig. 2) and also GnRH release stimulated by a depolarizing concentration of potassium (Fig. 3) and by melatonin (Fig. 4) and PGE2 (Fig. 5). Addition of verapamil to the medium also inhibited melatonin- and PGE2-stimulated GnRH release (Figs. 4 and 5). The data presented here, therefore, provide new evidence that hypothalamic GnRH release depends on membrane depolarization and calcium influx. The demonstration of this dependence by the hypothalamic preparation described here is evidence that this preparation behaves, at least to some extent, physiologically and might therefore be used to study other factors that influence GnRH release. As basal as well as stimulated GnRH release from a single hypothalamus in 1 h was readily detectable and was inhibited by omission of calcium from the incubation medium, this system seems to be suitable for studying inhibitory as well as stimulatory factors. Acknowledgments The authors thank Drs. Terry M. Nett and Gordon D. Niswender for a generous gift of anti-GnRH serum, the NIAMDD for synthetic GnRH, Knoll Pharmaceuticals for verapamil, find Ms. Elaine Paolini for preparation of the manuscript.
References 1. Palkovits, M., A. Arimura, M. Brownstein, A. V. Schally, and J. M. Saavedra, Luteinizing hormonereleasing hormone (LH-RH) content of the hypothalamic nuclei in rat, Endocrinology 95: 554, 1974. 2. Barry, J., M. P. Dubois, and B. Carett, Immunofluorescence study of the preopticoinfundibular LRF neurosecretory pathway in the normal, castrated or testosterone-treated male guinea pig, Endocrinology 95: 1416, 1974. 3. Wheaton, J. E., L. Krulich, and S. M. McCann, Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology 97: 30,1975. 4. Eskay, R. L., J. Warberg, R. S. Mical, and J. C. Porter, Prostaglandin E2-induced release of LHRH into hypophyseal portal blood, Endocrinology 97: 816,1975. 5. Carmel, P. W., S. Araki, and M. Ferin, Pituitary stalk portal blood collection in rhesus monkeys: evidence for pulsatile release of gonadotropin-releasing hormone (GnRH), Endocrinology 99: 243,1976.
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6. Neill, J. D., J. M. Patton, P. A. Daily, R. C. Tsau, and G. T. Tindall, Luteinizing hormone-releasing hormone in pituitary stalk blood of rhesus monkeys: relationship to level of LH release, Endocrinology 101: 430, 1977. 7. Nett, T. M., A. M. Akbar, A. D. Niswender, M. T. Hedlund, and W. F. White, A radioimmunoassay for gonadotropin-releasing hormone (GnRH) in serum, J Clin Endocrinol Metab 36: 880, 1973. 8. Rotsztejn, W. H., J. L. Charli, E. Dattou, J. Epelbaum, and C. Kardon, In vitro release of luteinizing hormone-releasing hormone (LHRH) from rat mediabasal hypothalamus: effects of potassium, calcium and dopamine, Endocrinology 99: 1663, 1976. 9. Gallardo, E., and V. D. Ramirez, A method for the superfusion of rat hypothalami: secretion of luteinizing hormone-releasing hormone (LH-RH), Proc Soc Exp BiolMed 195: 79, 1977. 10. Kao, L. W. L., and J. Weisz, Release of gonadotrophinreleasing hormone (Gn-RH) from isolated perifused medial-basal hypothalamus by melatonin, Endocrinology 100: 1723, 1977. 11. Thorell, J. J., and B. C. Johansson, Enzymatic iodination of polypeptides with I25I to high specific activity, Biochim Biophys Acta 251: 363, 1971. 12. Snedecor, G. W., and W. G. Cochran, Statistical Methods, ed. 3, Iowa State University Press, Ames, 1967, p. 91. 13. Griffiths, E. C, K. C. Hooper, and C. R. N. Hopkinson, Evidence for an enzymic component in the rat hypothalamus capable of inactivating luteinizing hormone releasing factor (LRF), Acta Endocrinol (Kbh) 74: 49, 1973. 14. Skou, J. C, Enzymatic basis for active transport of Na+ and K+ across cell membranes, Physiol Rev 45: 586, 1965. 15. Glynn, I. M., The action of cardiac glycosides on sodium and potassium movements in human red cells, J Physiol 136: 148, 1957. 16. Russell, J. T., and N. A. Thorn, Calcium and stimulussecretion coupling in the neurohypophysis: effects of lanthanum, a verapamil analogue (D600) and prenylamine on 45Ca transport and vasopressin release of isolated rat neurohypophyses, Acta Endocrinol 76: 471, 1974. 17. Dreifus, J. J., J. D. Grau, and J. J. Kardmann, Effects on the isolated neurohypophysis of agents which affect the membrane permeability to calcium, J Physiol 231: 96, 1973. 18. Hales, C. N., and R. D. G. Miller, The role of sodium and potassium in insulin secretion from rabbit pancreas, J Physiol 194: 725, 1968. 19. Douglas, W. W., and A. M. Poisner, Stimulus-secretory coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis, J Physiol 172: 1,1969. 20. Douglas, W. W., and A. M. Poisner, Stimulation of uptake of calcium-45 in the adrenal gland by acetylcholine, Nature 192: 1299, 1961. 21. Kizer, J. S., M. Vargas-Cordon, K. Brendal, and R. Bressler, The in vitro inhibition of insulin secretion
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by diphenylhydantoin, J Clin Invest 49: 1942, 1970. 22. Giodsky, G. M., and L. L. Bennett, Cation requirements for insulin secretion in the isolated perfused pancreas, Diabetes 15: 910, 1966. 23. Milner, R. D. G., and C. N. Hales, The role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro, Diabetologia 3: 47, 1967. 24. Haller, E. W., H. Sachs, N. Sparelakis, and L. Share, Release of vasopressin from isolated guinea pig posterior pituitaries, AmJPhysiol 209: 79, 1965. 25. Douglas, W. W., and R. P. Rubin, The role of calcium in the secretory response of adrenal medulla to ace-
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tylcholine, J Physiol 159: 40, 1961. 26. Somers, G., G. Devis, E. Van Obberghen, and W. J. Malaise, Calcium antagonists and islet function: interaction of theophylline and verapamil, Endocrinology 99: 114, 1976. 27. Robinson, I. C. A. F., J. T. Russel, and N. A. Thorn, Calcium and stimulus secretion coupling in the neurohypophysis, Ada Endocrinol 83: 36, 1976. 28. Warberg, J., R. L. Eskay, A. Barnea, R. C. Reynolds, arid J. C. Porter, Release of LHRH and TRH from a synaptosome enriched fraction of hypothalamic homogenates, Endocrinology 100: 814,1977.
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