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Effect of substance P on thyrotropin secretion from the pituitary gland in the rat
Peptides, Vol. 10, pp. 763-766. © Pergamon Press plc, 1989. Printed in the U.S.A.
0196-9781/89 $3.00 + .00
Effect of Substance P on Thyrotropin Secretion From the Pituitary Gland in the Rat M A S A Y O S H I A R I S A W A , I G A R Y D. S N Y D E R 2 A N D S A M U E L M. M c C A N N .3
*Department of Physiology, Neuropeptide Division The University of Texas Southwestern Medical Center at Dallas 5323 Har D, Hines Boulevard, Dallas, TX 75235-9040 R e c e i v e d 15 M a r c h 1989
ARISAWA, M., G. D. SNYDER AND S. M. McCANN. Effectof substance P on thyrotropin secretionfrom the pituitary gland in the rat. PEPTIDES 10(4) 763-766, 1989.--The role of substance P (SP) on thyrotropin (TSH) secretion was investigated in ovariectomized (OVX) female, estrogen-primed OVX, and normal male rats. Third ventricular administration of SP induced a significant increase in plasma TSH levels when compared to control animals in E-primed OVX rats (p<0.001). The plasma TSH levels increased in a dose-related manner and reached maximum levels at 10 min after injection. In contrast, intraventricularly injected SP failed to alter plasma TSH levels in both OVX rats and normal male rats. Intravenous administration of SP dramatically stimulated TSH release in E-primed OVX rats (p<0.001 ), whereas SP had no effect on the release of TSH when injected in OVX rats and normal male rats. To investigate any direct action of SP on TSH release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats and normal male rats. SP, in the dose range between 10-s M and 10 - 6 M, failed to alter the release of TSH into the culture medium in vitro. These findings indicate that SP has a stimulatory role in the control of TSH release by an action on the hypothalamus but only in estrogen-primed rats. Substance P
Thyrotropin secretion
Pituitary gland
Ovariectomy
SUBSTANCE P (SP), originally isolated from bovine hypothalamus, has been shown to be distributed throughout the central nervous system and the gastrointestinal tract (3, 12, 13). Immunocytochemical studies revealed that SP-like immunoreactivity is also located in particular cell types of the anterior pituitary (16), including thyrotrophs (7). There is considerable evidence that the SP concentrations in the central nervous system and anterior pituitary are affected by the thyroid and gonadal status of the animals (4-6, 8, 9, 17). In view of these findings, it is reasonable to speculate that SP may have some physiological role in the regulation of the hypothalamo-pituitary thyroid axis. However, so far, little is known of the effect of SP on TSH secretion in vivo and in vitro, though SP has various effects on the release of other pituitary hormones (2, 10, 14, 19). For these reasons, we have studied the role of SP in the control of TSH secretion and the involvement of the gonadal steroid status of the animals in the effects.
Estrogen priming
group cages under conditions of controlled temperature (23-25°C) and lighting (lights on 0500-1900 hr) and provided rat chow and water ad lib. All female rats were ovariectomized (OVX) three to five weeks prior to experimentation while anesthetized with ether. Animals were divided into three groups for Experiments I and II. The first group consisted of OVX rats. The second group of OVX rats was estrogen-primed (E-primed) by injection of 50 i~g estradiol benzoate (Sigma) subcutaneously (SC) three days prior to experimentation. Normal male rats constituted the third group. Synthetic SP was obtained from Peninsula Laboratories, Inc.
In Vivo Experiments Experiment I. A 23-gauge stainless steel cannula (17 mm in length) was implanted into the third ventricle (3V) of the experimental animals 6-8 days prior to the experiment as described earlier (1). A mandril prevented obstruction of the cannula. The cannula was considered to be located in the 3V if cerebrospinal fluid flowed continuously from it. An indwelling catheter was placed in the external jugular vein 24 hr prior to the experiment (11). On the day of the experiment, an extension of polyethylene tubing was attached to the distal end of the jugular cannula and the
METHOD Adult male and female Sprague-Dawley rats (Simonsen, Co., Gilroy, CA) weighing 280-320 g were used. They were housed in
IPresent address: Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinano, Shinjuku-ku, Tokyo, Japan. 2Present address: Department of Obstetrics and Gynecology, University of Iowa, College of Medicine, Building MRF, Room 466, Iowa City, IA 52242. 3Requests for reprints should be addressed to Samuel M. McCann.
763
764
ARISAWA, SNYDER AND McCANN
TABLE 1 EFFECT OF INTRAVENTRICULAR INJECTION OF SYNTHETIC SP ON PLASMA TSH (ng/ml) IN OVX RATS Time After Injection (min) Groups
N
0
10
20
40
60
Control SP 1 lag 5 lag
7 8 7
265.7 +_- 20.8* 226.5 _+ 16.4 235.0 +- 20.0
249.7 -* 25.3 215.1 --- 18.9 252.9 --+ 26.5
232.6 +-- 19.6 232.8 ± 10.8 251.0 +-- 18.8
270.6 -+- 30.0 231.5 -+ 15.2 230.9 ± 272.2
258.3 -'- 23.4 226.6 + 11.4 200.3 _+_ 13.6
*Mean - SEM, N = number of rats.
animal was left u n d i s t u d ~ for more than 60 min. Various doses of synthetic SP dissolved in a volume of 3 0,1 of 0.9% NaCI or an equal volume of saline alone were injected into the 3V of OVX, E-primed OVX and male rats just after the first blood sampling. Blood samples (0.8 ml) were withdrawn at various time intervals thereafter. Subsequent sampling volumes were replaced with heparinized isotonic saline. Plasma was promptly separated and stored frozen at - 20°C until later radioimmunoassay. Experiment H. Twenty-four hours prior to the experiment, an indwelling catheter was inserted into the right jugular vein in OVX, E-primed OVX and normal male rats as described in Experiment I. On the following morning, various doses of synthetic SP dissolved in a volume of 0.2 ml of 0.9% NaCl or an equal volume of saline alone were injected into the jugular cannula after removing the initial blood sample. Blood samples were collected thereafter as described above.
In Vitro Experiment Two days prior to decapitation, 50 Ixg of estradiol benzoate was injected SC into OVX rats. Anterior pituitaries were removed from the E-primed O V X rats and normal male rats after decapitation and dispersed in the presence of 0.1% trypsin as previously described 0 8 ) . Cells were incubated overnight in medium 199 containing 20 mM N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer, Gibco) at 370C. Estradiol benzoate was added at a concentration of 200 pg/ml to the medium containing the cells of E-primed OVX rats. On the day of experimentation, overnight culture medium was removed. Cells were resuspended in either 1 ml medium 199 (0.1% bovine serum albumin, 100 units/ml penicillin- 100 Izg/ml streptomycin, 20 mM HEPES buffer) alone or l ml of the same medium containing synthetic SP after a 30-min preincubation period. After incubation for 60 min, reactions were terminated by centrifugation and media were stored
frozen at - 2 0 ° for later radioimmunoassay.
Hormone Assays and Statistics Plasma TSH was measured by specific radioimmunoassay using the kits supplied from the National Institutes of Diabetes, Digestive and Kidney Diseases (NIDDK). Results were expressed in terms of NIDDK-rat-TSH-RP-I standard. Differences between groups and initial values in a given group studied were analyzed using a two-way analysis of variance followed by the Student-Newman-Keuls multiple comparison test. RESULTS
Effect of Intraventricular Administration of Substance P Intraventricular administration of SP had no effect on TSH release in both OVX female rats (Table 1) and normal male rats (data not shown). In contrast, SP significantly increased plasma TSH levels in E-primed OVX rats as compared to values in control animals, as illustrated in Fig. 1 (p<0.001). Plasma TSH peaked at 10 min after injection in a dose-related manner and then gradually decreased.
Effect of Intravenous Administration of Substance P Intravenously administered SP increased plasma TSH levels slightly in OVX rats (Fig. 2), but the effect was not statistically significant. In contrast to the results of OVX rats, SP dramatically increased plasma TSH levels as compared to concentrations in control animals in E-primed OVX rats, as illustrated in Fig. 3 (p<0.001). The effect was observed 10 min after injection and levels remained elevated for 40 min. On the other hand, little change in TSH release was observed after intravenous injection of
TABLE 2 OF INTRAVENOUS INJECTION OF SYNTHETIC SP ON PLASMA TSH (ng/ml) IN NORMAL MALE RATS Time After Injection (min) Groups
N
Control 1 lag 10 I~g 50 I~g
9 8 8 8
0 224.6 241.6 213.6 229.1
± 45.6* ± 43.4 -+- 25.5 ± 35.3
10 234.8 244.0 211.9 268.3
*Mean ± SEM, N = number of rats.
+_- 44.4 _ 50.4 ± 33.0 -'- 36.0
20 217.3 238.4 160.3 219.4
~ 44.2 --- 39.7 --- 15.3 --* 24.9
40 223.1 191.9 138.0 150.4
-'- 55.5 -'- 31.5 --+ 18.0 ± 14.3
60 197.3 178.4 145.3 153.0
+-- 32.9 -+- 32.7 --- 18.2 4- 17.5
SUBSTANCE P AND TSH
765
SP I.V.T.
1oool 500
SPIN.
900 E
400
, t.t.t
•
• 50/=0 (N.9)
o
* lOp.g (N,9)
/ ~ / ~ [
800
I
700
l,""
,=---,, "tp.g (N=8,
N,'*
o---o
Con,ro, CN=81
\
600 -r
t-
3oo
0
E
I-o E u}
500 400 300
O.
200
200
12.
"1o0
e--.
5 V 9 (N=6)
omo
'I/~g (N-6)
II---II
O.'l~q
t00
(N=5)
I
I
I
~10 20
Time After
l
I
I
i
I0
20
40
60
Time After tnieetion (rain)
o-.-o Control ( N = 7 ) 0
I
0
I
I
40
60
FIG. 3. Effect of intravenously injected SP or physiological saline on TSH release in unanesthetized, freely moving, E-primed O'v'X rats. *p<0.05, **p<0.005, ***p<0.001 vs. control values within a time point, respectively, fp<0.05, i'?p<0.025, t'H'p<0.005, fi'fi'p<0.001, respectively.
Injection (rain)
FIG. 1. Effect of intraventricularly injected SP or physiological saline on TSH release is unanesthetized, freely moving, E-primed OVX rats. In this and subsequent figures, the values obtained are given as mean---SEM. *p<0.025, **p<0.001 vs. control values within a time point, respectively. :l:p<0.025 vs. zero time sampie. N = number of rats.
the same doses of SP in normal male rats (Table 2).
In Vitro Studies Table 3 demonstrates the results of incubation of the overnight cultured pituitary cells from E-primed OVX rats and normal male rats with various concentrations of synthetic SP. SP (10- 8 - 1 0 - 6 M) did not significantly affect the release of TSH into the culture medium in either case. These cells were highly responsive to TRH. DISCUSSION
The present study demonstrates that intraventricular adminis-
tration of SP can stimulate the release of TSH in unanesthetized, freely moving, E-primed OVX rats. It is of interest that SP failed to stimulate the release of TSH in OVX female and normal male rats when injected either intravenously or intraventricularly. These findings strongly suggest that priming with estrogen is necessary to bring out the effect of SP on TSH release. Since SP failed to alter TSH release from anterior pituitary cells harvested from E-primed OVX rats in vitro, it appears likely that SP acts on the hypothalamus to stimulate TSH release by a stimulation of the release of thyrotropin-releasing hormone (TRH). SP presumably gains access to the hypothaiamus via the circumventricular organs and may well act to stimulate TRH following its uptake from the median eminence and an action on the terminals of the TRH neurons. Alternatively, it may act on interneurons which in turn stimulate TRH release from the terminals and/or cell bodies of the TRH neurons. Further work will be necessary to determine the precise mechanism. Intravenously injected SP presumably acts via the median eminence or via other circumventricular organs; however, a possible effect via systemic
SP I.V.
4OO
TABLE 3 EFFECT OF SP AND TRH ON TSH RELEASE (ng/ml) FROM DISPERSED
ANTERIORPITUITARYCELLS HARVESTEDFROM E-PRIMEDOVX RATS AND NORMALMALE RATS ng TSH Release
= 2oo
"r o'J I-
Treatment
e--e 50M~g(N=9)
E-Primed OVX
Male
o - - o 10,u.g (N.IO)
~00
B---Ill IM.g (N=9) o - - o Control(N-9)
G
I
lo
20
I
6o
Time After Injection ( m i n ) FIG. 2. Effect of intravenously injected SP or physiological saline on T S H release in unanesthetized, freely moving, O V X rats.
Control 10 -6 M SP 10 - 7 M SP 10 -8 M SP 50 ng TRH
664.2 755.0 650.0 658.0 2565.5
-+- 78.1" --+ 29.0 +-- 10.2 +-- 63.2 +-- 531.0+
473.5 516.0 416.4 469.8 1452.0
*Mean +_ S E M (ng/ml). ?p<0.001 versus control, N = 5, 5.0 x I0- 5 cells/tube.
+-- 65.6 --- 54.6 -+- 65.6 - 37.5 --- 103.1"~
766
ARISAWA, SNYDER AND McCANN
changes induced by the peptide cannot be ruled out. There has been considerable evidence that SP contributes to the regulation of the secretion of various anterior pituitary, hormones (2, 3, 19). SP is known to exist in the hypothalamus and anterior pituitary as well (3, 8, 13). In the guinea pig, using an immunocytochemical technique, De Palatis et al. found that SP-like immunoreactivity is located in a subpopulation of thyrotrophs (7). Furthermore, recent findings indicate that the concentration of immunoreactive SP in the rat central nervous system and anterior pituitary is affected by the thyroid status of the animal (4, 5. 9, 17). Aronin et al. reported that thyroid hormones participate in the regulation of the synthesis and/or release of immunoreactive SP from the anterior pituitary (4). SP was also reported to increase in the central nervous system in hypothyroid animals and these changes in hypothyroid animals were completely reversed by T4 replacement therapy (9). In view of these recent findings, it is reasonable to speculate that SP has some physiological role in the regulation of the hypothalamo-pituitary thyroid axis. However, the role of SP in the control of TSH release is poorly understood. Intraventricular injection of SP failed to alter TSH release (19) and SP also did not affect the release of TSH from incubated pituitaries
in vitro in earlier experiments in rats which were not primed with estrogen (14,19). Mitsuma et al. (15) reported that SP decreased plasma TSH levels after intravenous injection in male rats, but we could not find a significant suppressive effect of this peptide in males. To date, there has been no report of a stimulator3, effect of SP on TSH release. In this study, we demonstrated that intraventricular administration of SP strongly stimulated TSH release in estrogen-primed OVX rats. Gonadal status is known to be physiologically important in the regulation of SP in both central nervous system and anterior pituitary (2, 6, 8). Therefore, we conclude that SP may have a physiologically significant stimulatory effect on TSH release when endogenous estrogen levels are elevated as at midcycle. ACKNOWLEDGEMENTS This research was supported by NIDDK40994. The authors would like to thank the NIDDK for supplying materials for the RIA. The authors are also grateful to Mr. John H. Johnson and Ms. Thelma Williams for their technical assistance. The secretarial assistance of Ms. Judy Scott is greatly appreciated.
REFERENCES I. Antunes-Rodrigues, J.; McCann, S. M. Water, sodium chloride and food intake induced by injections Of cholinergic and adrenergic drugs into the third ventricle of the rat brain. Prec. Soc. Biol. Med. 133:1464-1470; 1970. 2. Aronin, N.; Coslovsky, R.; Leeman, S. E. Substance P and neurotensin: Their roles in the regulation of anterior pituitary function. Annu. Rev. Physiol. 48:537-549; 1986. 3. Aronin, N.; DiFiglia, M.; Leeman, S. E. Substance P. In: Brownstein, M. J.; Martin. J. B., eds. Brain peptides. New York: Wiley: 1983:783-803. 4. Aronin, N.; Morency, K.; Leeman, S. E.; Braverman, L. E.; Coslovsky, R. Regulation by thyroid hormone of the concentration of substance P in the rat anterior pituitary. Endocrinology 114:21382141; 1984. 5. Coslovsky, R.; Braverman, L. E.; Leeman, S. E.; Aronin, N. The differential effects of thyroid and gonadal hormones on substance P content in the anterior pituitary of the prepuhertal rat. Endocrinology 117:2198-2202; 1985. 6. Coslovsky, R.; Evans, R. W.; Leeman, S. E.; Braverman, L.; Aronin, N. The effects of gonadal steroids on the content of substance P in the rat anteiror pituitary. Endocrinology 115:2285-2289; 1984. 7. De Palatis, L. R.; Fiorindo, R. P.; Ho, R. H. Substance P immunoreactivity in the anterior pituitary gland of the guinea pig. Endocrinology 110:282-284; 1981. 8. De Palatis, L. R.; Khorram, O.; McCann, S. M. Age-, Sex-, and gonadal steroid-related change in immunoreactive substance P in the rat anterior pituitary gland. Endocrinology 117:1368-1373; 1985. 9. Dupont, A.; Dussault, J. H.; Rouleau, D.; DiPaolo, T.; Coulombe, P.; Gagne, B.; Merand, Y.; Moore, S.; Bardin, S. Effect of neonatal thyroid deficiency on the catecholamine, substance P, and thyrotropinreleasing hormone contents of discrete rat brain nuclei. Endocrinology
108:2039-2045; 1981. 10. Fisher, G. H.; Humphries, J.; Folkers, K. Synthesis and some biological activities of substance P. J. Med. Chem. 17:843-846; 1974. 1I. Harms, P. G.; Ojeda, S. R. A rapid and simple proc~ure for chronic cannulation of the rat jugular vein. J. Appl. Physiol. 36:391-392; 1974. 12. Hokfelt, T.; Per'now, B.; Nilsson, G.; Wetterherg, L.; Goldstein, M.; Jeffocate, S. L. Dense plexus of substance P immunoreactive nerve terminals in eminentia medialis of the primate hypothalamus. Proc. Natl. Acad. Sci. USA 75:1013-1015; 1978. 13. Leeman, S. E.; Aronin, N.; Ferris, C. Substance P and neurotensin, Recent Prog. Horm. Res. 38:93-109; 1982. 14. Maeda, K.; Frohman, L. A. Dissociation of systemic and central effects of neurotensin on the secretion of growth hormone, prolactin, and thyrotropin. Endocrinology 103:1903-1909; 1978. 15. Mitsuma, T.; Nogimori, T. Effects of substance P, angiotensin It, oxotremorine and prostaglandin D2 on thyrotropin secretion in rats. Horm. Res. 19:176-184; 1984. 16. Morel, G.; Chayvialle, J. A.; Kerdelhue, B.; Dubois, P. M. Ultrastructural evidence for endogenous substance-P-like immunoreactivity in the rat pituitary gland. Neuroendocrinology 35:86-92; 1982. 17. Savard, P.; Merand, Y.; DiPaolo, T.; Dupont, A. Effects of thyroid state on serotonin, 5-hydroxyindoleacetic acid and substance P contents in discrete brain nuclei of adult rats. Neuroscience 10:13991404; 1983. 18. Snyder, G.; Hymer, W. C. A short method for the isolation of somatotrophs from the rat pituitary gland. Endocrinology 96:792-796; 1975. 19. Vijayan, E.; McCann, S. M. Effects of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro. Life Sci. 26:321-327; 1979.
0196-9781/89 $3.00 + .00
Effect of Substance P on Thyrotropin Secretion From the Pituitary Gland in the Rat M A S A Y O S H I A R I S A W A , I G A R Y D. S N Y D E R 2 A N D S A M U E L M. M c C A N N .3
*Department of Physiology, Neuropeptide Division The University of Texas Southwestern Medical Center at Dallas 5323 Har D, Hines Boulevard, Dallas, TX 75235-9040 R e c e i v e d 15 M a r c h 1989
ARISAWA, M., G. D. SNYDER AND S. M. McCANN. Effectof substance P on thyrotropin secretionfrom the pituitary gland in the rat. PEPTIDES 10(4) 763-766, 1989.--The role of substance P (SP) on thyrotropin (TSH) secretion was investigated in ovariectomized (OVX) female, estrogen-primed OVX, and normal male rats. Third ventricular administration of SP induced a significant increase in plasma TSH levels when compared to control animals in E-primed OVX rats (p<0.001). The plasma TSH levels increased in a dose-related manner and reached maximum levels at 10 min after injection. In contrast, intraventricularly injected SP failed to alter plasma TSH levels in both OVX rats and normal male rats. Intravenous administration of SP dramatically stimulated TSH release in E-primed OVX rats (p<0.001 ), whereas SP had no effect on the release of TSH when injected in OVX rats and normal male rats. To investigate any direct action of SP on TSH release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats and normal male rats. SP, in the dose range between 10-s M and 10 - 6 M, failed to alter the release of TSH into the culture medium in vitro. These findings indicate that SP has a stimulatory role in the control of TSH release by an action on the hypothalamus but only in estrogen-primed rats. Substance P
Thyrotropin secretion
Pituitary gland
Ovariectomy
SUBSTANCE P (SP), originally isolated from bovine hypothalamus, has been shown to be distributed throughout the central nervous system and the gastrointestinal tract (3, 12, 13). Immunocytochemical studies revealed that SP-like immunoreactivity is also located in particular cell types of the anterior pituitary (16), including thyrotrophs (7). There is considerable evidence that the SP concentrations in the central nervous system and anterior pituitary are affected by the thyroid and gonadal status of the animals (4-6, 8, 9, 17). In view of these findings, it is reasonable to speculate that SP may have some physiological role in the regulation of the hypothalamo-pituitary thyroid axis. However, so far, little is known of the effect of SP on TSH secretion in vivo and in vitro, though SP has various effects on the release of other pituitary hormones (2, 10, 14, 19). For these reasons, we have studied the role of SP in the control of TSH secretion and the involvement of the gonadal steroid status of the animals in the effects.
Estrogen priming
group cages under conditions of controlled temperature (23-25°C) and lighting (lights on 0500-1900 hr) and provided rat chow and water ad lib. All female rats were ovariectomized (OVX) three to five weeks prior to experimentation while anesthetized with ether. Animals were divided into three groups for Experiments I and II. The first group consisted of OVX rats. The second group of OVX rats was estrogen-primed (E-primed) by injection of 50 i~g estradiol benzoate (Sigma) subcutaneously (SC) three days prior to experimentation. Normal male rats constituted the third group. Synthetic SP was obtained from Peninsula Laboratories, Inc.
In Vivo Experiments Experiment I. A 23-gauge stainless steel cannula (17 mm in length) was implanted into the third ventricle (3V) of the experimental animals 6-8 days prior to the experiment as described earlier (1). A mandril prevented obstruction of the cannula. The cannula was considered to be located in the 3V if cerebrospinal fluid flowed continuously from it. An indwelling catheter was placed in the external jugular vein 24 hr prior to the experiment (11). On the day of the experiment, an extension of polyethylene tubing was attached to the distal end of the jugular cannula and the
METHOD Adult male and female Sprague-Dawley rats (Simonsen, Co., Gilroy, CA) weighing 280-320 g were used. They were housed in
IPresent address: Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinano, Shinjuku-ku, Tokyo, Japan. 2Present address: Department of Obstetrics and Gynecology, University of Iowa, College of Medicine, Building MRF, Room 466, Iowa City, IA 52242. 3Requests for reprints should be addressed to Samuel M. McCann.
763
764
ARISAWA, SNYDER AND McCANN
TABLE 1 EFFECT OF INTRAVENTRICULAR INJECTION OF SYNTHETIC SP ON PLASMA TSH (ng/ml) IN OVX RATS Time After Injection (min) Groups
N
0
10
20
40
60
Control SP 1 lag 5 lag
7 8 7
265.7 +_- 20.8* 226.5 _+ 16.4 235.0 +- 20.0
249.7 -* 25.3 215.1 --- 18.9 252.9 --+ 26.5
232.6 +-- 19.6 232.8 ± 10.8 251.0 +-- 18.8
270.6 -+- 30.0 231.5 -+ 15.2 230.9 ± 272.2
258.3 -'- 23.4 226.6 + 11.4 200.3 _+_ 13.6
*Mean - SEM, N = number of rats.
animal was left u n d i s t u d ~ for more than 60 min. Various doses of synthetic SP dissolved in a volume of 3 0,1 of 0.9% NaCI or an equal volume of saline alone were injected into the 3V of OVX, E-primed OVX and male rats just after the first blood sampling. Blood samples (0.8 ml) were withdrawn at various time intervals thereafter. Subsequent sampling volumes were replaced with heparinized isotonic saline. Plasma was promptly separated and stored frozen at - 20°C until later radioimmunoassay. Experiment H. Twenty-four hours prior to the experiment, an indwelling catheter was inserted into the right jugular vein in OVX, E-primed OVX and normal male rats as described in Experiment I. On the following morning, various doses of synthetic SP dissolved in a volume of 0.2 ml of 0.9% NaCl or an equal volume of saline alone were injected into the jugular cannula after removing the initial blood sample. Blood samples were collected thereafter as described above.
In Vitro Experiment Two days prior to decapitation, 50 Ixg of estradiol benzoate was injected SC into OVX rats. Anterior pituitaries were removed from the E-primed O V X rats and normal male rats after decapitation and dispersed in the presence of 0.1% trypsin as previously described 0 8 ) . Cells were incubated overnight in medium 199 containing 20 mM N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer, Gibco) at 370C. Estradiol benzoate was added at a concentration of 200 pg/ml to the medium containing the cells of E-primed OVX rats. On the day of experimentation, overnight culture medium was removed. Cells were resuspended in either 1 ml medium 199 (0.1% bovine serum albumin, 100 units/ml penicillin- 100 Izg/ml streptomycin, 20 mM HEPES buffer) alone or l ml of the same medium containing synthetic SP after a 30-min preincubation period. After incubation for 60 min, reactions were terminated by centrifugation and media were stored
frozen at - 2 0 ° for later radioimmunoassay.
Hormone Assays and Statistics Plasma TSH was measured by specific radioimmunoassay using the kits supplied from the National Institutes of Diabetes, Digestive and Kidney Diseases (NIDDK). Results were expressed in terms of NIDDK-rat-TSH-RP-I standard. Differences between groups and initial values in a given group studied were analyzed using a two-way analysis of variance followed by the Student-Newman-Keuls multiple comparison test. RESULTS
Effect of Intraventricular Administration of Substance P Intraventricular administration of SP had no effect on TSH release in both OVX female rats (Table 1) and normal male rats (data not shown). In contrast, SP significantly increased plasma TSH levels in E-primed OVX rats as compared to values in control animals, as illustrated in Fig. 1 (p<0.001). Plasma TSH peaked at 10 min after injection in a dose-related manner and then gradually decreased.
Effect of Intravenous Administration of Substance P Intravenously administered SP increased plasma TSH levels slightly in OVX rats (Fig. 2), but the effect was not statistically significant. In contrast to the results of OVX rats, SP dramatically increased plasma TSH levels as compared to concentrations in control animals in E-primed OVX rats, as illustrated in Fig. 3 (p<0.001). The effect was observed 10 min after injection and levels remained elevated for 40 min. On the other hand, little change in TSH release was observed after intravenous injection of
TABLE 2 OF INTRAVENOUS INJECTION OF SYNTHETIC SP ON PLASMA TSH (ng/ml) IN NORMAL MALE RATS Time After Injection (min) Groups
N
Control 1 lag 10 I~g 50 I~g
9 8 8 8
0 224.6 241.6 213.6 229.1
± 45.6* ± 43.4 -+- 25.5 ± 35.3
10 234.8 244.0 211.9 268.3
*Mean ± SEM, N = number of rats.
+_- 44.4 _ 50.4 ± 33.0 -'- 36.0
20 217.3 238.4 160.3 219.4
~ 44.2 --- 39.7 --- 15.3 --* 24.9
40 223.1 191.9 138.0 150.4
-'- 55.5 -'- 31.5 --+ 18.0 ± 14.3
60 197.3 178.4 145.3 153.0
+-- 32.9 -+- 32.7 --- 18.2 4- 17.5
SUBSTANCE P AND TSH
765
SP I.V.T.
1oool 500
SPIN.
900 E
400
, t.t.t
•
• 50/=0 (N.9)
o
* lOp.g (N,9)
/ ~ / ~ [
800
I
700
l,""
,=---,, "tp.g (N=8,
N,'*
o---o
Con,ro, CN=81
\
600 -r
t-
3oo
0
E
I-o E u}
500 400 300
O.
200
200
12.
"1o0
e--.
5 V 9 (N=6)
omo
'I/~g (N-6)
II---II
O.'l~q
t00
(N=5)
I
I
I
~10 20
Time After
l
I
I
i
I0
20
40
60
Time After tnieetion (rain)
o-.-o Control ( N = 7 ) 0
I
0
I
I
40
60
FIG. 3. Effect of intravenously injected SP or physiological saline on TSH release in unanesthetized, freely moving, E-primed O'v'X rats. *p<0.05, **p<0.005, ***p<0.001 vs. control values within a time point, respectively, fp<0.05, i'?p<0.025, t'H'p<0.005, fi'fi'p<0.001, respectively.
Injection (rain)
FIG. 1. Effect of intraventricularly injected SP or physiological saline on TSH release is unanesthetized, freely moving, E-primed OVX rats. In this and subsequent figures, the values obtained are given as mean---SEM. *p<0.025, **p<0.001 vs. control values within a time point, respectively. :l:p<0.025 vs. zero time sampie. N = number of rats.
the same doses of SP in normal male rats (Table 2).
In Vitro Studies Table 3 demonstrates the results of incubation of the overnight cultured pituitary cells from E-primed OVX rats and normal male rats with various concentrations of synthetic SP. SP (10- 8 - 1 0 - 6 M) did not significantly affect the release of TSH into the culture medium in either case. These cells were highly responsive to TRH. DISCUSSION
The present study demonstrates that intraventricular adminis-
tration of SP can stimulate the release of TSH in unanesthetized, freely moving, E-primed OVX rats. It is of interest that SP failed to stimulate the release of TSH in OVX female and normal male rats when injected either intravenously or intraventricularly. These findings strongly suggest that priming with estrogen is necessary to bring out the effect of SP on TSH release. Since SP failed to alter TSH release from anterior pituitary cells harvested from E-primed OVX rats in vitro, it appears likely that SP acts on the hypothalamus to stimulate TSH release by a stimulation of the release of thyrotropin-releasing hormone (TRH). SP presumably gains access to the hypothaiamus via the circumventricular organs and may well act to stimulate TRH following its uptake from the median eminence and an action on the terminals of the TRH neurons. Alternatively, it may act on interneurons which in turn stimulate TRH release from the terminals and/or cell bodies of the TRH neurons. Further work will be necessary to determine the precise mechanism. Intravenously injected SP presumably acts via the median eminence or via other circumventricular organs; however, a possible effect via systemic
SP I.V.
4OO
TABLE 3 EFFECT OF SP AND TRH ON TSH RELEASE (ng/ml) FROM DISPERSED
ANTERIORPITUITARYCELLS HARVESTEDFROM E-PRIMEDOVX RATS AND NORMALMALE RATS ng TSH Release
= 2oo
"r o'J I-
Treatment
e--e 50M~g(N=9)
E-Primed OVX
Male
o - - o 10,u.g (N.IO)
~00
B---Ill IM.g (N=9) o - - o Control(N-9)
G
I
lo
20
I
6o
Time After Injection ( m i n ) FIG. 2. Effect of intravenously injected SP or physiological saline on T S H release in unanesthetized, freely moving, O V X rats.
Control 10 -6 M SP 10 - 7 M SP 10 -8 M SP 50 ng TRH
664.2 755.0 650.0 658.0 2565.5
-+- 78.1" --+ 29.0 +-- 10.2 +-- 63.2 +-- 531.0+
473.5 516.0 416.4 469.8 1452.0
*Mean +_ S E M (ng/ml). ?p<0.001 versus control, N = 5, 5.0 x I0- 5 cells/tube.
+-- 65.6 --- 54.6 -+- 65.6 - 37.5 --- 103.1"~
766
ARISAWA, SNYDER AND McCANN
changes induced by the peptide cannot be ruled out. There has been considerable evidence that SP contributes to the regulation of the secretion of various anterior pituitary, hormones (2, 3, 19). SP is known to exist in the hypothalamus and anterior pituitary as well (3, 8, 13). In the guinea pig, using an immunocytochemical technique, De Palatis et al. found that SP-like immunoreactivity is located in a subpopulation of thyrotrophs (7). Furthermore, recent findings indicate that the concentration of immunoreactive SP in the rat central nervous system and anterior pituitary is affected by the thyroid status of the animal (4, 5. 9, 17). Aronin et al. reported that thyroid hormones participate in the regulation of the synthesis and/or release of immunoreactive SP from the anterior pituitary (4). SP was also reported to increase in the central nervous system in hypothyroid animals and these changes in hypothyroid animals were completely reversed by T4 replacement therapy (9). In view of these recent findings, it is reasonable to speculate that SP has some physiological role in the regulation of the hypothalamo-pituitary thyroid axis. However, the role of SP in the control of TSH release is poorly understood. Intraventricular injection of SP failed to alter TSH release (19) and SP also did not affect the release of TSH from incubated pituitaries
in vitro in earlier experiments in rats which were not primed with estrogen (14,19). Mitsuma et al. (15) reported that SP decreased plasma TSH levels after intravenous injection in male rats, but we could not find a significant suppressive effect of this peptide in males. To date, there has been no report of a stimulator3, effect of SP on TSH release. In this study, we demonstrated that intraventricular administration of SP strongly stimulated TSH release in estrogen-primed OVX rats. Gonadal status is known to be physiologically important in the regulation of SP in both central nervous system and anterior pituitary (2, 6, 8). Therefore, we conclude that SP may have a physiologically significant stimulatory effect on TSH release when endogenous estrogen levels are elevated as at midcycle. ACKNOWLEDGEMENTS This research was supported by NIDDK40994. The authors would like to thank the NIDDK for supplying materials for the RIA. The authors are also grateful to Mr. John H. Johnson and Ms. Thelma Williams for their technical assistance. The secretarial assistance of Ms. Judy Scott is greatly appreciated.
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108:2039-2045; 1981. 10. Fisher, G. H.; Humphries, J.; Folkers, K. Synthesis and some biological activities of substance P. J. Med. Chem. 17:843-846; 1974. 1I. Harms, P. G.; Ojeda, S. R. A rapid and simple proc~ure for chronic cannulation of the rat jugular vein. J. Appl. Physiol. 36:391-392; 1974. 12. Hokfelt, T.; Per'now, B.; Nilsson, G.; Wetterherg, L.; Goldstein, M.; Jeffocate, S. L. Dense plexus of substance P immunoreactive nerve terminals in eminentia medialis of the primate hypothalamus. Proc. Natl. Acad. Sci. USA 75:1013-1015; 1978. 13. Leeman, S. E.; Aronin, N.; Ferris, C. Substance P and neurotensin, Recent Prog. Horm. Res. 38:93-109; 1982. 14. Maeda, K.; Frohman, L. A. Dissociation of systemic and central effects of neurotensin on the secretion of growth hormone, prolactin, and thyrotropin. Endocrinology 103:1903-1909; 1978. 15. Mitsuma, T.; Nogimori, T. Effects of substance P, angiotensin It, oxotremorine and prostaglandin D2 on thyrotropin secretion in rats. Horm. Res. 19:176-184; 1984. 16. Morel, G.; Chayvialle, J. A.; Kerdelhue, B.; Dubois, P. M. Ultrastructural evidence for endogenous substance-P-like immunoreactivity in the rat pituitary gland. Neuroendocrinology 35:86-92; 1982. 17. Savard, P.; Merand, Y.; DiPaolo, T.; Dupont, A. Effects of thyroid state on serotonin, 5-hydroxyindoleacetic acid and substance P contents in discrete brain nuclei of adult rats. Neuroscience 10:13991404; 1983. 18. Snyder, G.; Hymer, W. C. A short method for the isolation of somatotrophs from the rat pituitary gland. Endocrinology 96:792-796; 1975. 19. Vijayan, E.; McCann, S. M. Effects of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro. Life Sci. 26:321-327; 1979.