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Calcium dependency of potassium-stimulated thyrotropin-releasing hormone secretion from rat neurohypophysis in vitro
Neuroscience Letters, 27 (1981) 177-181
177
Elsevier/North-Holland Scientific Publishers Ltd.
CALCIUM DEPENDENCY OF POTASSIUM-STIMULATED THYROTROPIN-RELEASING HORMONE SECRETION FROM RAT NEUROHYPOPHYSIS IN VITRO
A N D R E W L A C K O F F and IVOR M.D. JACKSON*
Division of Endocrinology, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111 (U.S.A.) (Received August 5th, 1981; Revised version received September 2nd, 1981; Accepted September 14th, 1981)
Key words: thyrotropin-releasing h o r m o n e - neurophypophysis - secretion - calcium - potassium
The role of Ca 2÷ on the K÷-stimulated release of thyrotropin-releasing h o r m o n e (TRH) from rat neurohypophysis, stalk median eminence (SME) and hypothalamic fragments in vitro was studied. High K ÷ (50 mM) caused a significant increase (2-6-fold) in T R H release from all three tissues, (P < 0.05-0.01), and this effect was inhibited (by 6 2 - 8 0 % ) on removing Ca 2÷ (1.8 mM) from the medium (P < 0.01). The calcium dependency of the T R H response to a membrane depolarizing stimulus (high K ÷) is consistent with T R H release occurring by a neurosecretory process from the neurophypophysis, as well as from hypothalamic tissue.
The tripeptide, amide, p G l u - H i s - P r o N H 2, originally identified in the mammalian hypothalamus as the pituitary thyrotropin-releasing hormone (TRH), is widely distributed throughout the central nervous system (CNS) wherein it may function as a neurotransmitter or neuromodulator [6, 9, 19]. The highest neuronal concentrations of T R H are found in the stalk median eminence (SME) region of the hypothalamus, localized to nerve terminals from which it is secreted into the pituitary portal circulation for transport to the anterior pituitary [9]. Outside the hypothalamus, the highest concentration of T R H in the rat brain is found in the pars nervosa which contains levels of T R H 20 times that found in the adenohypophysis [8]. The source of neural lobe TRH is not known for sure, but immunohistochemical studies have revealed networks of TRH-positive fibers extending into the neurophypophysis of the rat suggesting the presence of a new hypothalamo-hypophysial system [4]. The classical studies of Douglas and Poisner [3] demonstrating the release of vasopressin from the neurophypophysis by a stimulus-secretion coupling mechanism established the usefulness of this tissue for *To w h o m all correspondence should be addressed. 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
178
the study of neural peptide secretion in vitro. Recently, the hypothalamic release inhibitory hormone, somatostatin, has been shown to be released from the neurohypophysis by depolarizing concentrations of K* in the presence of Ca 2 ÷ [13] similar to that described for vasopressin [3]. While depolarizing concentrations of K ÷ consistently bring about the release of T R H from hypothalamic fragments [10, 12], the effect on synaptosomal preparations is unclear, T R H responses having been observed in some [15, 18] but not in other [1, 10] studies. Since there have been no previous reports of T R H secretion from neurohypophysial tissue, we have examined that rat pars nervosa as a model for studying the in vitro release of T R H from nerve terminals and compared the responses with that obtained from hypothalamic and SME fragments. Male S p r a g u e - D a w l e y rats (200-250 g) obtained from Charles River (CD r) were decapitated and pools of 4 neural lobes, two SME and two hypothalami minus SME were placed in 1 or 2 ml (for hypothalami) of a modified Krebs-bicarbonate solution containing NaC1 127 mM, KC1 3.8 mM and CaC12 1.8 mM [5] at room temperature under 95O7o 02-5°7o CO 2. The medium for SME and hypothalamus, but not neurohypophysis, contained bacitracin (Sigma), 30 ~g/ml, to prevent T R H breakdown [11] since preliminary studies revealed no effect of bacitracin on the levels of T R H in neural lobe tissue following incubation. After pre-incubation for 30 min, the medium was removed and replaced with fresh solution and the tissues were then incubated for 60 min at 37 °C under 95°7o 02--5070 C O 2. In the first experiment, the effect of high K ÷ (KC1 50 mM substituted for NaC1) on T R H release was compared with normal Krebs-bicarbonate. In the second experiment, the role of Ca 2* on the high K~-stimulated T R H release was studied by replacing Ca 2~ with equimolar concentrations of Mn 2., a calcium antagonist in secretory processes [13], and comparing the response with high K ÷ in the presence of Ca 2~ . In each experiment, 5 or 6 pools of both control and test tissues were incubated. At the completion of the study, the individual media were drawn off and added to 10 vol. of 1 N acetic acid, centrifuged and the supernatant lyophilized. The tissue pools were homogenized in 1-2 ml of 1-N acetic acid and the extracts lyophilized. The dried media and tissue extracts were subsequently reconstituted in phospho-buffered saline (PBS) for T R H radioimmunoassay [7], which is currently sensitive to less than 1 pg. The levels of T R H found in the neurophyophysis, SME and hypothalamus are presented in Table 1. The high K ÷ stimulus caused a significant increase in the quantity of T R H released into the media taken from all three tissues (Fig. IA), though no significant tissue depletion was observed (Table 1). During 60 min incubation in the presence of Krebs-bicarbonate, 2.5°7o of the neural lobe tissue content of T R H was released into the medium compared with 0.3°7o and 0.6°7o for SME and hypothalamus respectively. Altering the K ÷ concentration to 50 mM, with a commensurate reduction in Na ÷, caused a marked increase in the amount of T R H released for all three tissues, almost 2-fold for the neurohypophysis
179 It}t
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B
Neural lobe
Stalk median eminence (SMF') Hypothalamus
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o
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High
K+ (Test)
2"/, 2"/, Y/, "'" r//
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NN
~
Normal
Ca ~+ High K + (Control)
A b s e n t Ca ~ High K + (Test)
Fig. 1. The effect of high K ÷ (50 mM), in the presence and absence of Ca 2 +, on the release of T R H from pools of 4 neural lobes, two SME and two hypothalamic fragments. Tissues (5 or 6 pools per group) taken from male rats (200-250 g) were incubated in a modified Krebs-bicarbonate under 95°7o 02-50/0 CO2 for 60 min and the medium then examined for immunoreactive (1R)-TRH following extraction in 1 N acetic acid. Each bar depicts the mean _+ S.E.M. of the responses. A: high K ÷ (50 mM) is compared to normal K ÷ (3.8 mM) both in the presence of Ca 2÷. The IR-TRH released into the medium of the tissues exposed to the normal K ÷ (control) is designated as 100%. B: the effect of replacing Ca 2 ÷ with Mn 2 ÷ on the high K ÷ stimulation of T R H . The IR-TRH released into the medium of tissues exposed to normal Ca 2÷ (1.8 mM) and high K ÷ (control in this experiment) is expressed as 10007o.
(P < 0.05), 6-fold for the SME (P < 0.01) and nearly 4-fold for the hypothalamus (P < 0.01) (Fig. IA). However, when Ca 2+ was omitted from the incubation medium, the response of the tissues to high K + was inhibited by 63°7o for neurohypophysis, 80°7o for SME and 71070 for hypothalamus (P < 0.01 for all three tissues) (Fig. 1B). It is likely that Ca 2+ is also necessary for basal release since the absolute levels of T R H released in the high K +, absent Ca 2 ÷ exposed tissues were in all instances less than the amount secreted from the control tissues incubated with normal K ÷ (3.8 mM) in the presence of Ca 2÷ (Fig. 1A). The absolute levels of T R H in the neurohypophysis (163 + 17 pg/lobe; mean + S.E.M.) are virtually identical to that which we reported previously [8]. This quantity is about one-fifth the somatostatin content, which in turn is one order of magnitude lower than that of vasopressin and oxytocin [13]. The mechanism of T R H release from the neurohypophysis, SME and the hypothalamus is consistent with a neurosecretory mechanism as evidenced by the response to a membrane depolarization stimulus (high K ÷) and the dependency of this process on Ca 2+ in the extracellular fluid [3]. When expressed as a percentage of
180 TABLE 1 TISSUE CONTENTS OF TRH After i n c u b a t i o n in K r e b s - b i c a r b o n a t e c o n t a i n i n g 3.8 m M K* (control) or 50 m M K ' (high K ~ ), the tissues were extracted in 1 N acetic acid. T R H means _+ S.E.M. Control Neurohypophysis a (4 lobes) SME (2 f r a g m e n t s ) Hypothalamus {2 f r a g m e n t s )
652 ±
values are in p g / t i s s u e pool. n :
6. Values are
High K +
68
573 +
81b
4038 ± 590
3462 ± 342 b
7333 + 994
7482 + 525 b
aThe pars i n t e r m e d i a tissue was not s e p a r a t e d from the n e u r o h y p o p h y s i s so that m o r e correctly neuroi n t e r m e d i a t e lobe tissue was studied. H o w e v e r , previous studies ( J a c k s o n and Reichlin, u n p u b l i s h e d ) have s h o w n only sparse levels of T R H in the rat pars intermedia. bThe differences are not significant.
the tissue content, the quantity of TRH release from the neurohypophysis into the medium (in both basal and K ~-stimulated states) was much greater than from SME or hypothalamus. Comparable findings for somatostatin release from the neurohypophysis relative to the ME were reported by Zingg et al. [20]. These workers speculated that the ME tissue incubates caused enhanced breakdown of somatostatin, or released some inhibitor substance which reduced the amount of somatostatin secreted, or a fraction of the neural peptide within the ME was not localized at nerve terminals. Each of these possibilities could play a role in the hypothalamic and SME secretory process for TRH in this study, but definitive evidence in favor of any of these hypotheses is not available. However, the enhanced release of T R H from the neurohypophysis, which has reduced degradative activity for the tripeptide, as well as the ready accessibility and uniformity of this tissue, make the pars nervosa an attractive neural organ for the study of TRH secretion. The functional significance of T R H located in the neurohypophysis has not been established. T R H has been reported to influence vasopressin secretion in rat [16] and man [17] and could, therefore, have a physiologic role in the regulation of vasopressin secretion. Additionally, it should be noted that T R H is found in high concentration in the pituitary complex of lower vertebrates [7], and there is evidence (reviewed by Sawyer [14] that in the bony fish the neurohypophysis may be a homologue of the SME in higher animals. It is thus possible that neurohypophysial TRH, which could reach the adenohypophysis via the short portal vessels [2], might regulate anterior pituitary secretion, as has been established for the tripeptide located in the hypothalamus and SME. We thank Mrs. Faye Soo-Hoo for technical assistance. This work was supported in part by NIH Grant AM 21863.
181 1 Bennett, G.W., Edwardson, J.A., Holland, D., Jeffcoate, S.L. and White, N., Release of immunoreactive luteinizing hormone-releasing hormone and thyrotrophin-releasing hormone from hypothalamic synaptosomes, Nature (Lond.), 257 (1975) 323-325. 2 Bergland, R.M. and Page, R.B., Pituitary-brain vascular relations: a new paradigm, Science, 204 (1979) 18-24. 3 Douglas, W.W. and Poisner, A.M., Stimulus-secretion coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis, J. Physiol. (Lond.), 172 (1964) 1-18. 4 HOkfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord, Neurosci. Lett., 1 0975) 133-139. 5 Iversen, L.L., Iversen, S.D., Bloom, F., Douglas, C., Brown, M. and Vale, W., Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro, Nature (Lond.), 273 (1978) 161-163. 6 Jackson, I.M.D., TRH in the rat nervous system: identity with synthetic TRH on high performance liquid chromatography following affinity chromatography, Brain Res., 201 (1980) 245-248. 7 Jackson, I.M.D. and Reichlin, S. Thyrotropin-releasing hormone (TRH): distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates, Endocrinology, 95 (1974) 854-862. 8 Jackson, I.M.D. and Reichlin, S., Brain thyrotrophin-releasing hormone is independent of the hypothalamus, Nature (Lond.), 267 (1977) 853-854. 9 Jackson, I.M.D. and Reichlin, S., Distribution and biosynthesis of TRH in the nervous system. In R. Collu, A. Barbeau, J.R. Ducharme and J.-G. Rochefort (Eds.), Central Nervous System Effects of Hypothalamic Hormones and Other Peptides, Raven Press, New York, 1979, pp. 3-54. l0 Joseph-Bravo, P., Charli, J.L., Palacios, J.M. and Kordon, C., Effect of neurotransmitters on the in vitro release of immunoreactive thyrotropin-releasing hormone from rat mediobasal hypothalamus, Endocrinology, 104 (1979) 801-806. l I McKelvy, J.F., Leblanc, P., Loudes, C., Petrie, G., Grimm-J6rgensen, Y., and Kordon, C., The use of bacitracin as an inhibitor of the degradation of thyrotropin releasing factor and luteinizing hormone releasing factor, Biochem. Biophys. res. Commun., 73 (1976) 507-515. 12 Maeda, K. and Frohman, L.A., Release of somatostatin and thyrotropin-releasing hormone from rat hypothalamic fragments in vitro, Endocrinology, 106 (1980) 1837-1842. 13 Patel, Y.C., Zingg, H.H. and Dreifuss, J.J., Calcium-dependent somatostatin secretion from rat neurohypophysis in vitro, Nature (Lond.), 267 (1977) 852. 14 Sawyer, W.H., Vertebrate neurohypophysial principles, Endocrinology, 75 (1964) 981-990. 15 Schaeffer, J.M., Axelrod, J. and Brownstein, M.J., Regional differences in dopamine-mediated release of TRH-like material from synaptosomes, Brain Res., 138 (1977) 571-574. 16 Skowsky, R. and Swan, L., Effect of hypothalamic releasing hormones on neurohypophyseal arginine vasopressin (AVP) secretion, Clin. Res., 24 (1976) 101. 17 Sowers, J.R., Hershman, J.M., Skowsky, W.R. and Carlson, H.E., Effect of TRH on serum arginine vasopressin in euthyroid and hypothyroid subjects, Horm. Res., 7 (1976) 232-237. 18 Warberg, J., Eskay, R.L., Barnea, A., Reynolds, R.C. and Porter, J.C., Release of luteinizing hormone releasing hormone and thyrotropin releasing hormone from a synaptosome-enriched fraction of hypothalamic homogenates, Endocrinology, 100 (1977) 814-825. 19 Wilber, J.F., Montoya, E., PIotnikoff, N.P., White, W.F., Gendrich, R., Renaud, L. and Martin, J.B., Gonadotropin-releasing hormone and thyrotropin-releasing hormone: distribution and effects in the central nervous system, Rec. Progr. Horm. Res., 32 (1976) 117-159. 20 Zingg, H.H., Dreifuss, J.J. and Patel, Y.C., Secretion de somatostatine a partir de l'eminence mediane et de la posthypophyse in vitro, Bull. Schweiz. Akad. Med. Wiss., 34 (1978) 145-153.
177
Elsevier/North-Holland Scientific Publishers Ltd.
CALCIUM DEPENDENCY OF POTASSIUM-STIMULATED THYROTROPIN-RELEASING HORMONE SECRETION FROM RAT NEUROHYPOPHYSIS IN VITRO
A N D R E W L A C K O F F and IVOR M.D. JACKSON*
Division of Endocrinology, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111 (U.S.A.) (Received August 5th, 1981; Revised version received September 2nd, 1981; Accepted September 14th, 1981)
Key words: thyrotropin-releasing h o r m o n e - neurophypophysis - secretion - calcium - potassium
The role of Ca 2÷ on the K÷-stimulated release of thyrotropin-releasing h o r m o n e (TRH) from rat neurohypophysis, stalk median eminence (SME) and hypothalamic fragments in vitro was studied. High K ÷ (50 mM) caused a significant increase (2-6-fold) in T R H release from all three tissues, (P < 0.05-0.01), and this effect was inhibited (by 6 2 - 8 0 % ) on removing Ca 2÷ (1.8 mM) from the medium (P < 0.01). The calcium dependency of the T R H response to a membrane depolarizing stimulus (high K ÷) is consistent with T R H release occurring by a neurosecretory process from the neurophypophysis, as well as from hypothalamic tissue.
The tripeptide, amide, p G l u - H i s - P r o N H 2, originally identified in the mammalian hypothalamus as the pituitary thyrotropin-releasing hormone (TRH), is widely distributed throughout the central nervous system (CNS) wherein it may function as a neurotransmitter or neuromodulator [6, 9, 19]. The highest neuronal concentrations of T R H are found in the stalk median eminence (SME) region of the hypothalamus, localized to nerve terminals from which it is secreted into the pituitary portal circulation for transport to the anterior pituitary [9]. Outside the hypothalamus, the highest concentration of T R H in the rat brain is found in the pars nervosa which contains levels of T R H 20 times that found in the adenohypophysis [8]. The source of neural lobe TRH is not known for sure, but immunohistochemical studies have revealed networks of TRH-positive fibers extending into the neurophypophysis of the rat suggesting the presence of a new hypothalamo-hypophysial system [4]. The classical studies of Douglas and Poisner [3] demonstrating the release of vasopressin from the neurophypophysis by a stimulus-secretion coupling mechanism established the usefulness of this tissue for *To w h o m all correspondence should be addressed. 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
178
the study of neural peptide secretion in vitro. Recently, the hypothalamic release inhibitory hormone, somatostatin, has been shown to be released from the neurohypophysis by depolarizing concentrations of K* in the presence of Ca 2 ÷ [13] similar to that described for vasopressin [3]. While depolarizing concentrations of K ÷ consistently bring about the release of T R H from hypothalamic fragments [10, 12], the effect on synaptosomal preparations is unclear, T R H responses having been observed in some [15, 18] but not in other [1, 10] studies. Since there have been no previous reports of T R H secretion from neurohypophysial tissue, we have examined that rat pars nervosa as a model for studying the in vitro release of T R H from nerve terminals and compared the responses with that obtained from hypothalamic and SME fragments. Male S p r a g u e - D a w l e y rats (200-250 g) obtained from Charles River (CD r) were decapitated and pools of 4 neural lobes, two SME and two hypothalami minus SME were placed in 1 or 2 ml (for hypothalami) of a modified Krebs-bicarbonate solution containing NaC1 127 mM, KC1 3.8 mM and CaC12 1.8 mM [5] at room temperature under 95O7o 02-5°7o CO 2. The medium for SME and hypothalamus, but not neurohypophysis, contained bacitracin (Sigma), 30 ~g/ml, to prevent T R H breakdown [11] since preliminary studies revealed no effect of bacitracin on the levels of T R H in neural lobe tissue following incubation. After pre-incubation for 30 min, the medium was removed and replaced with fresh solution and the tissues were then incubated for 60 min at 37 °C under 95°7o 02--5070 C O 2. In the first experiment, the effect of high K ÷ (KC1 50 mM substituted for NaC1) on T R H release was compared with normal Krebs-bicarbonate. In the second experiment, the role of Ca 2* on the high K~-stimulated T R H release was studied by replacing Ca 2~ with equimolar concentrations of Mn 2., a calcium antagonist in secretory processes [13], and comparing the response with high K ÷ in the presence of Ca 2~ . In each experiment, 5 or 6 pools of both control and test tissues were incubated. At the completion of the study, the individual media were drawn off and added to 10 vol. of 1 N acetic acid, centrifuged and the supernatant lyophilized. The tissue pools were homogenized in 1-2 ml of 1-N acetic acid and the extracts lyophilized. The dried media and tissue extracts were subsequently reconstituted in phospho-buffered saline (PBS) for T R H radioimmunoassay [7], which is currently sensitive to less than 1 pg. The levels of T R H found in the neurophyophysis, SME and hypothalamus are presented in Table 1. The high K ÷ stimulus caused a significant increase in the quantity of T R H released into the media taken from all three tissues (Fig. IA), though no significant tissue depletion was observed (Table 1). During 60 min incubation in the presence of Krebs-bicarbonate, 2.5°7o of the neural lobe tissue content of T R H was released into the medium compared with 0.3°7o and 0.6°7o for SME and hypothalamus respectively. Altering the K ÷ concentration to 50 mM, with a commensurate reduction in Na ÷, caused a marked increase in the amount of T R H released for all three tissues, almost 2-fold for the neurohypophysis
179 It}t
A 8
[~]
B
Neural lobe
Stalk median eminence (SMF') Hypothalamus
6oo
cO
o
's
,oo
5oo
N p
~
~
p
Nit
400
80
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Q)
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N
Q:: I 200 nI.tOO
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High
K+ (Test)
2"/, 2"/, Y/, "'" r//
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N~
NN
~
Normal
Ca ~+ High K + (Control)
A b s e n t Ca ~ High K + (Test)
Fig. 1. The effect of high K ÷ (50 mM), in the presence and absence of Ca 2 +, on the release of T R H from pools of 4 neural lobes, two SME and two hypothalamic fragments. Tissues (5 or 6 pools per group) taken from male rats (200-250 g) were incubated in a modified Krebs-bicarbonate under 95°7o 02-50/0 CO2 for 60 min and the medium then examined for immunoreactive (1R)-TRH following extraction in 1 N acetic acid. Each bar depicts the mean _+ S.E.M. of the responses. A: high K ÷ (50 mM) is compared to normal K ÷ (3.8 mM) both in the presence of Ca 2÷. The IR-TRH released into the medium of the tissues exposed to the normal K ÷ (control) is designated as 100%. B: the effect of replacing Ca 2 ÷ with Mn 2 ÷ on the high K ÷ stimulation of T R H . The IR-TRH released into the medium of tissues exposed to normal Ca 2÷ (1.8 mM) and high K ÷ (control in this experiment) is expressed as 10007o.
(P < 0.05), 6-fold for the SME (P < 0.01) and nearly 4-fold for the hypothalamus (P < 0.01) (Fig. IA). However, when Ca 2+ was omitted from the incubation medium, the response of the tissues to high K + was inhibited by 63°7o for neurohypophysis, 80°7o for SME and 71070 for hypothalamus (P < 0.01 for all three tissues) (Fig. 1B). It is likely that Ca 2+ is also necessary for basal release since the absolute levels of T R H released in the high K +, absent Ca 2 ÷ exposed tissues were in all instances less than the amount secreted from the control tissues incubated with normal K ÷ (3.8 mM) in the presence of Ca 2÷ (Fig. 1A). The absolute levels of T R H in the neurohypophysis (163 + 17 pg/lobe; mean + S.E.M.) are virtually identical to that which we reported previously [8]. This quantity is about one-fifth the somatostatin content, which in turn is one order of magnitude lower than that of vasopressin and oxytocin [13]. The mechanism of T R H release from the neurohypophysis, SME and the hypothalamus is consistent with a neurosecretory mechanism as evidenced by the response to a membrane depolarization stimulus (high K ÷) and the dependency of this process on Ca 2+ in the extracellular fluid [3]. When expressed as a percentage of
180 TABLE 1 TISSUE CONTENTS OF TRH After i n c u b a t i o n in K r e b s - b i c a r b o n a t e c o n t a i n i n g 3.8 m M K* (control) or 50 m M K ' (high K ~ ), the tissues were extracted in 1 N acetic acid. T R H means _+ S.E.M. Control Neurohypophysis a (4 lobes) SME (2 f r a g m e n t s ) Hypothalamus {2 f r a g m e n t s )
652 ±
values are in p g / t i s s u e pool. n :
6. Values are
High K +
68
573 +
81b
4038 ± 590
3462 ± 342 b
7333 + 994
7482 + 525 b
aThe pars i n t e r m e d i a tissue was not s e p a r a t e d from the n e u r o h y p o p h y s i s so that m o r e correctly neuroi n t e r m e d i a t e lobe tissue was studied. H o w e v e r , previous studies ( J a c k s o n and Reichlin, u n p u b l i s h e d ) have s h o w n only sparse levels of T R H in the rat pars intermedia. bThe differences are not significant.
the tissue content, the quantity of TRH release from the neurohypophysis into the medium (in both basal and K ~-stimulated states) was much greater than from SME or hypothalamus. Comparable findings for somatostatin release from the neurohypophysis relative to the ME were reported by Zingg et al. [20]. These workers speculated that the ME tissue incubates caused enhanced breakdown of somatostatin, or released some inhibitor substance which reduced the amount of somatostatin secreted, or a fraction of the neural peptide within the ME was not localized at nerve terminals. Each of these possibilities could play a role in the hypothalamic and SME secretory process for TRH in this study, but definitive evidence in favor of any of these hypotheses is not available. However, the enhanced release of T R H from the neurohypophysis, which has reduced degradative activity for the tripeptide, as well as the ready accessibility and uniformity of this tissue, make the pars nervosa an attractive neural organ for the study of TRH secretion. The functional significance of T R H located in the neurohypophysis has not been established. T R H has been reported to influence vasopressin secretion in rat [16] and man [17] and could, therefore, have a physiologic role in the regulation of vasopressin secretion. Additionally, it should be noted that T R H is found in high concentration in the pituitary complex of lower vertebrates [7], and there is evidence (reviewed by Sawyer [14] that in the bony fish the neurohypophysis may be a homologue of the SME in higher animals. It is thus possible that neurohypophysial TRH, which could reach the adenohypophysis via the short portal vessels [2], might regulate anterior pituitary secretion, as has been established for the tripeptide located in the hypothalamus and SME. We thank Mrs. Faye Soo-Hoo for technical assistance. This work was supported in part by NIH Grant AM 21863.
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