Brain Research, 509 (1990) 293-298 Elsevier
293
BRES 15207
Effect of intermittent infusions of somatostatin on growth hormone secretion in unrestrained male rats with hypothalamic deafferentation Shiro Minami ~, Ichiji Wakabayashi 1, Hitoshi Sugihara I , Jun Kamegai 1, Osamu Hasegawa 1, Taiko Kitamura 2 and Jinzo Yamada 2 Departments of IMedicine and eAnatomy, Nippon Medical School, Tokyo (Japan) (Accepted 18 July 1989)
Key words: Pulsatile growth hormone secretion; Growth hormone-releasing factor; fl-Endorphin
The effect of intermittent infusions of somatostatin (SS) on growth hormone (GH) secretion was studied in unrestrained adult male rats deprived largely of SS influence on the medial basal hypothalamus by anterolateral deafferentation (AL-cut). In addition, the influence of hypothalamic surgery on the plasma GH response to fl-endorphin (fl-END) was observed. In sham-operated rats, high-amplitude GH pulses separated by low baseline levels occurred at 185 min intervals. In rats with AL-cut, GH pulses were difficult to identify upon visual appraisal and baseline plasma GH levels became significantly higher than those of sham-operated rats. When AL-cut was performed unilaterally (half-AL-cut), low amplitude GH pulses separated by elevated baseline GH levels occurred at frequent intervals. The amount of GH secreted during 6 h was significantly reduced in rats with AL-cut or half-AL-cut as compared to that of sham-operated rats. The plasma GH response to intracerebroventricular injection of fl-END (4 ~g) was abolished in AL-cut rats, and the response was significantly reduced in half-AL-cut rats as compared to that of sham-operated rats. When AL-cut rats were subjected to repeated infusions of SS (30/~g/kg b. wt./h, 150 rain) separated by 30 min control periods, a large rebound of GH secretion was observed after removal and the amount of GH secreted during 6 h became comparablc to that of sham-operated rats. The results suggest that SS plays important roles in the dynamic secretion of GH.
INTRODUCTION
neuronal system by d eaf f er en t at i o n of M B H were subjected to r e p e a t e d infusions of SS. It is considered that
A l t h o u g h the stimulation of growth h o r m o n e ( G H ) secretion from the pituitary is driven by hypothalamic G H - r e l e a s i n g factor ( G R F ) 34, it is well recognized that the consistent effect of G R F on G H secretion cannot be e x p e c t e d unless it is a c c o m p a n ie d by a reduced inhibitory
exogenous SS acts on the hypothalamus and modulates the release of G R F 4'24'3°. Also included in the present study was the effect of hypothalamic deafferentation of M B H on G H secretion induced by fl-endorphin (fiE N D ) . This was done in part to define the hypothalamic
tone m e d i a t e d by hypothalamic somatostatin (SS) 3~" 35,36.38 Several lines of investigation suggest that the
surgery.
release of hypothalamic G R F is under the direct control
has been shown to require the presence of not only G R F 23"37 but also SS 9.
of SS. I m m u n o c y t o c h e m i c a l studies have shown that the
Th e
stimulatory effect of m o r p h i n e
or the
M e t - e n k e p h a l i n analogue, FK 33-824, on G H secretion
synaptic ends containing SS attached to the soma of G R F neurons in the arcuate nucleus 7 and the immunoreactivity of G R F
containing
cell bodies
increased
after
r e m o v a l of SS influence by the deafferentation of the medial basal hypothalamus ( M B H ) 6. A l t h o u g h physiological pulsatile bursts of G H secretion were abolished by i m m u n o n e u t r a l i z a t i o n of G R F 34, an identical p h e n o m e non was o b s e r v e d following the removal of SS influence either by i m m u n o n e u t r a l i z a t i o n of SS 2~ or by the deafferentation of the M B H I6. Th e present
MATERIALS AND METHODS
the
study was conducted to examine the
regulatory role of SS on the release of G R F in rats. To this end, rats deprived largely of SS influence on the G R F
Animals and surgery Adult male Wistar rats weighing 270-290 g were used. Each rat was placed in an individual cage and housed in an air-conditioned animal quarter with a lighting schedule of 08.00-20.(10 h and was fed food and water ad libitum. Two weeks prior to the study, anterolateral deafferentation (AL-cut) of the MBH was performed with a Halasz type knife ~l having a radius and height of 1.2 mm and 1.8 mm, respectively. Unilateral anterolateral deafferentation of the MBH (half-AL-cut) was made in another group of rats. The animal was fixed in the stereotaxic apparatus under ether anesthesia. The upper incisor bar was set 5 mm below the interauricular line (IAL) and the knife was lowered to the midline of the brain 5.8 mm rostral to IAL with the
Correspondence." I. Wakabayashi, Department of Medicine, Nippon Medical School, Sendagi 1-l-5, Bunkyoku, Tokyo 113, Japan. 0006-8993/90/$03.51i © 19911 Elsevier Science Publishers B.V. (Biomedical Division)
294 tip of the blade in front, to an initial position at the base of the skull It was then rotated 90 ° to one side and moved caudally in a horizontal plane for 2.0 mm. The knife was then returned on the same course to its initial position. In the rat with a half-AL-cut the knife was withdrawn, and in the rat with an AL-cut the whole procedure was repeated on the other side. The sham-operation consisted of lowering theknife to the initial mid-line position to the base of the skull and withdrawing without rotation. In some rats, a stainless-steel cannula for r - E N D injection was inserted into the third ventricle simultaneously. Four days prior to the study, rats were provided with indwelling right atrial cannulae under ether anesthesia for undisturbed blood collection. Some AL-cut rats were provided with two indwelling cannulae 4 days before use; one in the right atrium for undisturbed blood sampling and the other in the inferior vena cava for vehicle or SS infusion as described previously L29'3°.
developed and described by Merriam and Wachter:-'. Wc estimated the intraassay standard deviation at each GH standard and calculated the assay standard deviation as a function of the dose: SD(y) = [7.55y + 39.20]/100, where y was the individual plasma GH value. To set criteria for the identification of a GH pulse, individual cutoff values, G(n), were determined with respect to assay noise. A GH pulse was identified when a single GH plasma value was 3.8 S.D. [G(1)] above the baseline GH level, 2.6 S.D. for two consecutive GH samples [G(2)], 1.9 S.D. for 3 consecutive GIt samples [G(3)], 1.5 S.D. for 4 consecutive GH samples [G(4)l , and 1.2 S.D. for 5 consecutive GH samples above the baseline level. Because of the uncertainty of whether the PULSAR-identified GH peaks which occurred at either the first or last sample of GH secretory profile were true GH peaks, these data were eliminated from the analysis. When the PULSAR-identified smaller spikes were within a large peak (dips separating spikes were not deep enough to bring values down to the baseline), the complexes were
Peptides Synthetic rat r - E N D (Peninsula Labs, Belmont, CA, U.S.A.) and synthetic SS consisting of 14 amino acids (Peptide Institute, Osaka, Japan) were used. r-END was dissolved in 0.9% NaCI. SS was dissolved in 0.01 M acetic acid and then diluted with 0.9% NaCI immediately before use.
A
$ -endorphin
I ALcut
~o0
4~g Icy
~
Experimental procedures Serial blood specimens were obtained through the cannula placed in the right atrium at the times indicated. All blood specimens were immediately centrifuged and plasma was stored at -20 °C until assayed for GH. Red blood cells suspended in warm saline were returned to maintain circulation. In the first study, the effect of hypothalamic surgery on the plasma GH secretory profile and the plasma GH response to/3-END was observed. Plasma GH levels were monitored every 20 min from 10.00 to 16.00 h in 3 groups of rats, sham-operation, half-AL-cut and AL-cut. Immediately following the last blood sampling, 4/~g of r - E N D (4/A) was slowly administered through the stainless-steel cannula placed in the third ventricle at 16.00 h and serial blood specimens were collected. In the second study, the effect of intermittent infusions of SS on plasma GH secretory response was observed in a separate group of AL-cut rats. To ascertain the effect of hypothalamic surgery on GH secretion, each AL-cut rat was subjected to repeated infusions of 0.9% NaCt on one day, and was challenged with repeated infusions of SS for 150 min separated by 30 min control periods 2 days after the control study. Serial blood specimens were obtained through the cannula placed in the right atrium. Immediately after collecting the first blood sample, the cannula placed in the inferior vena cava was filled with SS solution or 0.9% NaCI and the free end of the cannula was connected to an infusion pump which was set to deliver 10 ~l/min. SS was infused continuously at a dose of 30/~g/kg b. wt./h through the cannula placed in the inferior vena cava. Each infusion of SS was preceded by an i.v. bolus injection of SS (100/~g/kg b. wt.). The dose and duration of infusion were chosen such that they altered the GH secretory profile of adult female rats to that of adult male rats 3°.
Histological examinations After the experiments, the brains of hypothalamic deafferented rats were fixed with 10% formaldehyde in 0.01 M phosphate buffer. Serial coronal frozen sections of the hypothalamus, 50 pm thick~ were made and stained with Cresyl violet in order to study the locations of cuts and lesions.
GH assay and data analysis GH concentrations were measured by double-antibody radioimmunoassay using materials supplied by NIADDK, NIH. All values are expressed as ng/ml in terms of the NIADDK reference preparation rat GH-RP-1. To identify the GH pulses and the trough periods, data obtained from each rat were subjected to the PULSAR computer program
o
B 200
half ALcut #-endorphin
4.g ic.
E
1!'
I00
x
¢-
/
:~ ,~
!i ',
i
31 LD 0
E
to o
C
sham 300
8 -endor phin 41Jg icy t
200 1
/
1 j/
I00
1
,
I
,o'oo
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doo
i
J,
'\
,3'oo ,4'o0 ,~oo Time of Day
l '\ ,6'0o
doo
Fig. 1. Effect of hypothalamic deafferentation on plasma GH profile and the response to fl-endorphin (~-END). A: AL-cut, anterolateral deafferentation. B: half-AL-cut, unilateral anterolateral deafferentation. C: sham-operation. Stars indicate GH peaks identified by 'PULSAR' computer program. Four/~g of rat r - E N D in 4/~1 0.9% NaCI was administered slowly through a stainless-steel cannula placed in the third ventricle at 16.00 h.
295 TABLE I Comparison of G H secretory pattern parameters using the PULSAR computer program
Values are mean +- S.E.M. ; numbers in parentheses indicate the sample size. Treatment groups
Peaks~6 h
Peak amplitude (ng/ml)
lnterpeak interval (min)
Baseline levels" (ng/ml)
Area under the curve (ug/ml x rain)
Sham-operation (n = 8) Half-AL-cut (n = 6) AL-cut (n = 14)
2.0 + 0.0 3.3 + 0.5* 1.0 +_0.2*'**
253.7 +_44.6 74.4 +- 11.1" -
185.0 +_5.0 107.5 +_ 14.6" -
15.0 +_ 1.4 24.9+_ 1.7" 42.6 +- 1.1"**
26.3 + 2.9 14.1 +_ 1.2" 16.5 +_0.9*
* Values significantly different from those of sham-operated rats. ** Values significantly different from those of half-AL-cut rats. regarded as one peak and the higher spike was used for the analysis. The area under the curve depicting the plasma GH concentrations from 10.(30 to 16.00 h for each rat was measured using a planimeter and expressed in an arbitrary unit:/~g/ml x rain. The data were analyzed by one-way analysis of variance, and comparisons between data were performed by Duncan's multiplerange test. When variance was not uniform, comparisons between data were performed by Student's t-test or the Cochran-Cox test. The former was used when variance was equal, and the latter used when variance was different. RESULTS A r e p r e s e n t a t i v e p l a s m a G H secretory pattern after s h a m - o p e r a t i o n , half-AL-cut or A L - c u t is shown in Fig. 1. In s h a m - o p e r a t e d rats, two large episodes of G H secretion with an i n t e r p e a k interval of 185 min and amplitudes ranging from 77.8 to 669.1 ng/mi were o b s e r v e d during a 6 h observation period (Table I). G H peaks were also identified by the P U L S A R p r o g r a m in some A L - c u t rats; no p e a k was identified in 4, 1 p e a k in 7, 2 p e a k s in 2 and 3 peaks in 1 rat during the 6 h period, In A L - c u t rats, the p e a k amplitude of G H pulses never e x c e e d e d 62.4 ng/ml and the baseline levels; G H levels b e t w e e n the episodic secretion were 3 times higher as c o m p a r e d to those of s h a m - o p e r a t e d rats (Table I). Thus, P U L S A R - i d e n t i f i e d peaks among A L - c u t rats were difficult to recognize as peaks on visual appraisal. In half-AL-cut rats, G H pulses occurred more frequently than in s h a m - o p e r a t e d rats. Peak amplitudes and the baseline levels were i n t e r m e d i a t e values between those of the s h a m - o p e r a t e d animals and the A L - c u t rats (Table I). The total a m o u n t of G H secreted during 6 h was significantly r e d u c e d in rats with A L - c u t or half-AL-cut as c o m p a r e d to that of s h a m - o p e r a t e d rats (Table I). f l - E N D was injected centrally at 16.00 h (Figs. 1 and 2). It was expected that plasma G H levels were at trough levels in s h a m - o p e r a t e d rats. In s h a m - o p e r a t e d rats, central injection of f l - E N D resulted in significant rises in p l a s m a G H levels. A m o n g rats with A L - c u t , plasma G H levels did not increase at all in response to central injections o f f l - E N D . In half-AL-cut rats, an intermediate response to f l - E N D was o b s e r v e d (Fig. 2).
Plasma G H profiles in A L - c u t rats subjected to r e p e a t e d infusions of SS are shown in Fig. 3. Plasma G H levels during SS infusion were significantly lower than those of A L - c u t rats which received 0.9% NaC1. A large r e b o u n d of G H secretion occurred r e p e a t e d l y following the cessation of SS infusion. The a m o u n t of G H secreted during 6 h as expressed area u n d e r the curve of plasma G H ~ g / m l x rain) (34.1 + 5.3 (n = 7)) was significantly larger ( P < 0.05) in A L - c u t rats which received r e p e a t e d infusions of SS as c o m p a r e d to that of A L - c u t rats which received 0.9% NaCI (19.2 + 1.5 (n = 7)). U p o n histological e x a m i n a t i o n , the rostral limits of AL-cut were placed on the p o s t e r i o r b o r d e r of the optic chiasma and the traces of the cut were p u r s u e d as gliosis and bleeding lesions to posterolatera[ly. T h e y passed through the lateral b o r d e r of the v e n t r o m e d i a l nucleus of the h y p o t h a l a m u s in most rats, and in some, through the mid portion of the V M H . It then reached the p r e m a m millary region. The p a r a v e n t r i c u l a r nuclei were placed
65~
.2~ t t,~,
• ALcut (n=14) 300
~---~
8 - e n d o r p h i n J' /
,.,cv l/ I!
hQIf ALcut ( n - 6 ) sham (n=8)
E 200
I o
E I O0
I000
I100
1200
1300
Time
1400
of
I~,00
1600
I?00
Day
Fig. 2. Plasma OH profiles and the responses to fl-END in hypothalamic deafferented rats. © ©, sham-operation; A---iX, half-AL-cut; I~-----Q, AL-cut. Four ~g of rat fl-END in 4 ,ul 0.9% NaCI was administered slowly through a stainless-steel cannula placed in the third ventricle at 16.00 h. Each point represents the mean and the vertical bar indicates S.E.M. Numbers in parentheses indicate the sample size.
296 tion of SS tone. When AL-cut was performed, baseline G H levels were further raised and the identification of pulsatile GH secretion became difficult upon visual appraisal. The findings complement previous studies by
SS I00 #g/kg
~i
,
S$ ?O.,/kQ.h
~u
700
I 600
others,6,17.
I 1 [
E 500 400
"r (.9 300 0 E
200
O [")
I O0
o Jobo
.6o
~o
~bo
~bo
eb~
~o
Time o f Day Fig. 3. Plasma GH profiles during intermittent infusions of somatostatin (SS) in unrestrained male rats with anterolateral hypothalamic deafferentation. Rats with AL-cut were subjected to repeated infusions indicated by horizontal bars of 0.9% NaC1 or SS at 30/~g/kg b. wt./h separated by 30 min control periods. Each infusion of SS was preceded by a bolus i.v. injection of SS (100/~g/kg b. wt.) as shown by the arrow. Each point represents the mean and the vertical bar indicates S.E.M. Numbers in parentheses indicate the sample size. Solid line indicates GH profile of rats which received SS, while dotted line indicates GH profile of rats which received 0.9% NaCI. just anterior to the cut in all rats. AL-cut spared the arcuate nuclei, the median eminence and the pituitary stalk in 21 rats with AL-cut and in 6 rats with half-AL-cut used in the analysis. Another 3 rats with AL-cut or half-AL-cut were excluded from the analysis because parts of the arcuate nucleus and median eminence appeared to be destroyed. DISCUSSION
G H secretion is controlled by defined hypothalamic regions that have either an inhibitory or a stimulatory influence. SS containing neuronal cell bodies are located in the anterior hypothalamic periventricular area 8'~8"2°, while GRF-containing neuronal cell bodies have been demonstrated in arcuate nuclei and ventromedial nuclei of the MBH 2'14'21'33. The removal of SS influence on the M B H either by half-AL-cut or AL-cut caused significant alterations in the G H secretory pattern in a dosedependent manner. After performing half-AL-cut, plasma G H secretory pattern closely resembled that of adult female rats as characterized by high frequency, low amplitude G H pulses separated by elevated baseline levels ~5. The inhibitory tone provided by SS is considered to be lower in female rats than that in male rats Ll°. Therefore, the alteration of G H secretory pattern after half-AL-cut appears to be causally related to the reduc-
We demonstrated that a significant reduction of SS influence on the MBH significantly reduced the amount of G H secreted which was restored by intermittent infusions of SS. Elevated baseline GH levels with little pulsatility after AL-cut have been suggested to reflect a continuous release of G R F 17. The pattern of G R F secretion may induce partial desensitization of the G R F receptors. This could offer an explanation for the reductions in G H secretion and the synthesis of G H in the pituitary after AL-cut ~3. At any rate, our data suggest that SS modulates somatotroph responsiveness to GRF. Previous investigators have demonstrated that the attenuation of the G H response to G R F through receptor effect is either partially reversed 5 or restored by SS 26. A number of explanations are available for the post-SS rebound in G H secretion 19'27. Exogenous SS can act to block temporarily G H release from the pituitary, while allowing the accumulation of G H in a releasable pool 27. Kraicer et al. 19 have assumed from in vitro studies that the major determinant of rebound G H secretion in vivo is the magnitude of the pool of G H available for immediate release upon disinhibition, and the size of this pool is related to the preceding influence of GRF, before the termination of SS infusion. However, studies examining the nature of post-SS rebound in G H secretion in vivo have suggested that exogenous SS acts on the hypothalamus and inhibits the release of GRF, while the sudden removal of SS triggers the secretion 4'z4"3~. Therefore, repeated infusions of SS would allow endogenous G R F to bring about a large rebound release of G H so that the amount of G H secreted in AL-cut rats became comparable to that of control rats. Although the stimulation of G H secretion by morphine or opioid peptide analogue requires the presence of G R F 23'37, fl-END-induced G H secretion was completely abolished in AL-cut rats, and the response was significantly reduced in half-AL-cut rats as compared to that of the sham-operated animals. The data are consistent with the interpretation that the stimulation of G H release by fl-END requires neural input from the anterolateral direction to G R F neurons in the MBH a2. We previously suggested that the stimulation of G H by the metenkephalin analogue, FK 33-824, accompanied the reduction of inhibitory tone mediated by SS 32. Frohman et al. 9 observed that the stimulation of G H by morphine sulfate was abolished in rats administered antiserum to SS. Taken together, our data support that the stimulation of G H release by opiates is mediated through inhibition
297 of SS release which in turn disinhibits the release of GRF.
by e n d o g e n o u s G R F was p r e s e r v e d after the surgery.
The
T h e r e f o r e , we consider that the knife cut was p e r f o r m e d
previous
observations
that
central
injection
of
f l - E N D stimulate G H secretion in rats treated with antiserum to SS is not inconsistent with our data 3. It may
successfully without destroying the M B H .
be that i m m u n o n e u t r a l i z a t i o n by antiserum to SS was
tion of pulsatile G H secretion by a-methyl-p-tyrosine is
i n a d e q u a t e in the previous studies.
exerted through the modulation of the phasic release of
Finally, we have previously suggested that the inhibi-
It was difficult to verify A L - c u t histologically, because
SS 29. O u r present data further strengthen that the phasic
the gliosis and bleeding lesions along the cut lines were
SS secretion plays important roles in the release and
not always clear. We considered it necessary to observe
synthesis of G H m e d i a t e d through G R F .
the alterations in G H secretory pattern and the inhibition of G H response t o / 3 - E N D after two types of hypothalamic surgery. Th e changes in secretory pattern and the response to f l - E N D were related to the size of hypothalamic deafferentation, A L - c u t and half-AL-cut, while the post-SS r e b o u n d in G H secretion, a response m e d i a t e d REFERENCES 1 Akira, S., Wakabayashi, I., Sugihara, H., Minami, S., Takahashi, F. and Motoyama, A., Effect of testosterone on growth hormone secretion in female rats during a continuous infusion of growth hormone releasing factor, Neuroendocrinology, 47 (1988) 116-124. 2 Bloch, B., Brazeau, P., Ling, N., Bohlen, P,, Esch, F., Wehrenberg, W.B., Benoit, R., Bloom, F. and Guillemin, R., Immunohistochemical detection of growth hormone-releasing factor in brain, Nature (Lond.), 301 (1983) 607-608. 3 Chihara, K., Arimura, A., Coy, D.H. and Schally, A.V., Studies on the interaction of endorphins, substance P, and endogenous somatostatin in growth hormone and prolactin release in rats, Endocrinology, 102 (1978) 281-290. 4 Clark, R.G., Carlsson, L.M.S., Rafferty, B. and Robinson, I.C.A.F., The rebound release of growth hermone (GH) following somatostatin infusion in rats involves hypothalamic GH-releasing factor release, J. Endocrinol., 119 (1988) 397-404. 5 Clayton, R.N. and Bailey, L.C., Somatostatin partially reverses desensitization of somatotrophs induced by growth hormonereleasing factor, J. Endocrinol., 112 (1987) 69-76. 6 Daikoku, S., Kawano, H., Noguchi, M., Nakanishi, J., Tokuzen, M., Chihara, K. and Nagatsu, I., GRF neurons in the rat hypothalamus, Brain Research, 399 (1986) 250-261. 7 Daikoku, S., Hisano, S., Kawano, H., Chikamori-Aoyama, M., Kagotani, Y., Zhang, R. and Chihara, K., Ultrastructural evidence for neuronal regulation of growth hormone secretion, Neuroendocrinology. 47 (1988) 405-415. 8 Epelbaum, J., Arancibia, L.T., Herman, J.P., Kordon, C. and Palkovits, M., Topography of median eminence somatostatinergic innervation, Brain Research, 230 (1981) 412-416. 9 Frohman, L.A., Downs, T.R., Katakami, H. and Jansson, J.-O., The interaction of growth hormone-releasing hormone and somatostatin in the regulation of growth hormone secretion, In O. Isaksson, C. Binder, K. Hall and B. H6kfelt (Eds.), Growth Hormone: Basic and C[inical Aspects, Proceedings of the 1st Nordisk Insulin Syrnnposiurn. Excerpta Medica, Amsterdam, 1987, pp.63-77, 10 Gross, D.S., Role of somatostatin in the modulation of hypophysiai growth hormone production by gonadal steroids, Am. J. Anat., 158 (1980) 507-519. ll Halasz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area, Endocrinology, 77 11965) 553-562. 12 Halasz, B., Nagy, G., Molnar, J., Marton, J., Banky, Z., Lukats, O. and Vizi, E.S., On the site and mode of action of enkephalins and enkephalin analogues on anterior pituitary
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26
27
28
29
30
31
mone (GHRH) infusions on the subsequent growth hormone (GH) response to GHRH, Neuroendocrinology. 45 (1987) 118-122. Soya, H, and Suzuki, M., Somatostatin rapidly restores rat growth hormone (GH) release response attenuated by prior exposure to human GH-releasing factor in vitro, Endocrinology, 122 (1988) 2492-2498. Stachura, M.E., Influence of synthetic somatostatin upon growth hormone release from perifused rat pituitaries, Endocrinology, 99 (1976) 678-683. Steiner, R.A., Stewart, J.K., Barber, J., Koerker, D., Goodner, C.J., Brown, A., Illner, P. and Gale, C.C., Somatostatin: a physiological role in the regulation of growth hormone secretion in the adolescent male baboon, Endocrinology, 102 (1978) 1587-1594. Sugihara, H., Wakabayashi, I., Minami, S., Takahashi, F., Shibasaki, T, and Ling, N., Effect of a-methyl-p-tyrosine and an antiserum to rat growth hormone (GH)-releasing factor (GRF) on plasma GH secretory profile during a continuous infusion of human GRF in rats, Brain Research, 475 (1988) 128-133. Sugihara, H., Minami, S. and Wakabayashi, 1.. Post-somato statin rebound secretion of growth hormone is dependent on growth hormone-releasing factor in unrestrained female rats, 3. Endocrinol., 122 (1989) 583-591. Tannenbaum, G.S. and Ling, N., The interrelationship of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion, Endocrinology, 115 (1984) 1952-1957.
32 Tonegawa, Y., Wakabayashi, 1., lhara, I., Hattori, M., Shibasaki, T. and Ling, N., Does opioid peptide stimulate growth hormone (GH) secretion via GH-releasing factor (GRF)?,
Program of the 7th International Congress of Endocrinology. 33
34
35
36
37
38
Abstr. 2311, Quebec City, 1984. VandePol, C.J., Leidy, Jr., J.W., Finger, T.E. and Robbins, R.J., Immunohistochemical localization of GRF-containing neurons in rat brain, Neuroendocrinology, 42 (1986) 143-147. Wehrenberg, W.B., Brazeau. P., Luben, R., Bohlen, P. and Guillemin, R., Inhibition of the pulsatile secretion of growth hormone by monoelonal antibodies to the hypothalamic growth hormone releasing factor (GRF), Endocrinology, 111 (1982) 2147-2148. Wehrenberg, W.B., Ling, N., Bohlen, P., Esch, E, Brazeau, P. and Guillemin, R., Physiological roles of somatocrinin and somatostatin in the regulation of growth hormone secretion. Biochem. Biophys. Res. Commun., 109 (1982) 562-567. Wehrenberg, W.B., Brazeau, P., Ling, N., Textor, G. and Guillemin, R., Pituitary growth hormone response in rats during a 24-hour infusion of growth hormone-releasing factor, Endocrinology, 114 (1984) 1613-1616. Wehrenberg, W.B., Bloch, B. and Ling, N., Pituitary secretion of growth hormone in response to opioid peptides and opiates is mediated through growth hormone-releasing factor, Neuroendocrinology, 41 (1985) 13-16. Wehrenberg, W.B., Continuous infusion of growth hormonereleasing factor: effects on pulsatile growth hormone secretion in normal rats, Neuroendocrinology, 43 (1986) 391-396.
293
BRES 15207
Effect of intermittent infusions of somatostatin on growth hormone secretion in unrestrained male rats with hypothalamic deafferentation Shiro Minami ~, Ichiji Wakabayashi 1, Hitoshi Sugihara I , Jun Kamegai 1, Osamu Hasegawa 1, Taiko Kitamura 2 and Jinzo Yamada 2 Departments of IMedicine and eAnatomy, Nippon Medical School, Tokyo (Japan) (Accepted 18 July 1989)
Key words: Pulsatile growth hormone secretion; Growth hormone-releasing factor; fl-Endorphin
The effect of intermittent infusions of somatostatin (SS) on growth hormone (GH) secretion was studied in unrestrained adult male rats deprived largely of SS influence on the medial basal hypothalamus by anterolateral deafferentation (AL-cut). In addition, the influence of hypothalamic surgery on the plasma GH response to fl-endorphin (fl-END) was observed. In sham-operated rats, high-amplitude GH pulses separated by low baseline levels occurred at 185 min intervals. In rats with AL-cut, GH pulses were difficult to identify upon visual appraisal and baseline plasma GH levels became significantly higher than those of sham-operated rats. When AL-cut was performed unilaterally (half-AL-cut), low amplitude GH pulses separated by elevated baseline GH levels occurred at frequent intervals. The amount of GH secreted during 6 h was significantly reduced in rats with AL-cut or half-AL-cut as compared to that of sham-operated rats. The plasma GH response to intracerebroventricular injection of fl-END (4 ~g) was abolished in AL-cut rats, and the response was significantly reduced in half-AL-cut rats as compared to that of sham-operated rats. When AL-cut rats were subjected to repeated infusions of SS (30/~g/kg b. wt./h, 150 rain) separated by 30 min control periods, a large rebound of GH secretion was observed after removal and the amount of GH secreted during 6 h became comparablc to that of sham-operated rats. The results suggest that SS plays important roles in the dynamic secretion of GH.
INTRODUCTION
neuronal system by d eaf f er en t at i o n of M B H were subjected to r e p e a t e d infusions of SS. It is considered that
A l t h o u g h the stimulation of growth h o r m o n e ( G H ) secretion from the pituitary is driven by hypothalamic G H - r e l e a s i n g factor ( G R F ) 34, it is well recognized that the consistent effect of G R F on G H secretion cannot be e x p e c t e d unless it is a c c o m p a n ie d by a reduced inhibitory
exogenous SS acts on the hypothalamus and modulates the release of G R F 4'24'3°. Also included in the present study was the effect of hypothalamic deafferentation of M B H on G H secretion induced by fl-endorphin (fiE N D ) . This was done in part to define the hypothalamic
tone m e d i a t e d by hypothalamic somatostatin (SS) 3~" 35,36.38 Several lines of investigation suggest that the
surgery.
release of hypothalamic G R F is under the direct control
has been shown to require the presence of not only G R F 23"37 but also SS 9.
of SS. I m m u n o c y t o c h e m i c a l studies have shown that the
Th e
stimulatory effect of m o r p h i n e
or the
M e t - e n k e p h a l i n analogue, FK 33-824, on G H secretion
synaptic ends containing SS attached to the soma of G R F neurons in the arcuate nucleus 7 and the immunoreactivity of G R F
containing
cell bodies
increased
after
r e m o v a l of SS influence by the deafferentation of the medial basal hypothalamus ( M B H ) 6. A l t h o u g h physiological pulsatile bursts of G H secretion were abolished by i m m u n o n e u t r a l i z a t i o n of G R F 34, an identical p h e n o m e non was o b s e r v e d following the removal of SS influence either by i m m u n o n e u t r a l i z a t i o n of SS 2~ or by the deafferentation of the M B H I6. Th e present
MATERIALS AND METHODS
the
study was conducted to examine the
regulatory role of SS on the release of G R F in rats. To this end, rats deprived largely of SS influence on the G R F
Animals and surgery Adult male Wistar rats weighing 270-290 g were used. Each rat was placed in an individual cage and housed in an air-conditioned animal quarter with a lighting schedule of 08.00-20.(10 h and was fed food and water ad libitum. Two weeks prior to the study, anterolateral deafferentation (AL-cut) of the MBH was performed with a Halasz type knife ~l having a radius and height of 1.2 mm and 1.8 mm, respectively. Unilateral anterolateral deafferentation of the MBH (half-AL-cut) was made in another group of rats. The animal was fixed in the stereotaxic apparatus under ether anesthesia. The upper incisor bar was set 5 mm below the interauricular line (IAL) and the knife was lowered to the midline of the brain 5.8 mm rostral to IAL with the
Correspondence." I. Wakabayashi, Department of Medicine, Nippon Medical School, Sendagi 1-l-5, Bunkyoku, Tokyo 113, Japan. 0006-8993/90/$03.51i © 19911 Elsevier Science Publishers B.V. (Biomedical Division)
294 tip of the blade in front, to an initial position at the base of the skull It was then rotated 90 ° to one side and moved caudally in a horizontal plane for 2.0 mm. The knife was then returned on the same course to its initial position. In the rat with a half-AL-cut the knife was withdrawn, and in the rat with an AL-cut the whole procedure was repeated on the other side. The sham-operation consisted of lowering theknife to the initial mid-line position to the base of the skull and withdrawing without rotation. In some rats, a stainless-steel cannula for r - E N D injection was inserted into the third ventricle simultaneously. Four days prior to the study, rats were provided with indwelling right atrial cannulae under ether anesthesia for undisturbed blood collection. Some AL-cut rats were provided with two indwelling cannulae 4 days before use; one in the right atrium for undisturbed blood sampling and the other in the inferior vena cava for vehicle or SS infusion as described previously L29'3°.
developed and described by Merriam and Wachter:-'. Wc estimated the intraassay standard deviation at each GH standard and calculated the assay standard deviation as a function of the dose: SD(y) = [7.55y + 39.20]/100, where y was the individual plasma GH value. To set criteria for the identification of a GH pulse, individual cutoff values, G(n), were determined with respect to assay noise. A GH pulse was identified when a single GH plasma value was 3.8 S.D. [G(1)] above the baseline GH level, 2.6 S.D. for two consecutive GH samples [G(2)], 1.9 S.D. for 3 consecutive GIt samples [G(3)], 1.5 S.D. for 4 consecutive GH samples [G(4)l , and 1.2 S.D. for 5 consecutive GH samples above the baseline level. Because of the uncertainty of whether the PULSAR-identified GH peaks which occurred at either the first or last sample of GH secretory profile were true GH peaks, these data were eliminated from the analysis. When the PULSAR-identified smaller spikes were within a large peak (dips separating spikes were not deep enough to bring values down to the baseline), the complexes were
Peptides Synthetic rat r - E N D (Peninsula Labs, Belmont, CA, U.S.A.) and synthetic SS consisting of 14 amino acids (Peptide Institute, Osaka, Japan) were used. r-END was dissolved in 0.9% NaCI. SS was dissolved in 0.01 M acetic acid and then diluted with 0.9% NaCI immediately before use.
A
$ -endorphin
I ALcut
~o0
4~g Icy
~
Experimental procedures Serial blood specimens were obtained through the cannula placed in the right atrium at the times indicated. All blood specimens were immediately centrifuged and plasma was stored at -20 °C until assayed for GH. Red blood cells suspended in warm saline were returned to maintain circulation. In the first study, the effect of hypothalamic surgery on the plasma GH secretory profile and the plasma GH response to/3-END was observed. Plasma GH levels were monitored every 20 min from 10.00 to 16.00 h in 3 groups of rats, sham-operation, half-AL-cut and AL-cut. Immediately following the last blood sampling, 4/~g of r - E N D (4/A) was slowly administered through the stainless-steel cannula placed in the third ventricle at 16.00 h and serial blood specimens were collected. In the second study, the effect of intermittent infusions of SS on plasma GH secretory response was observed in a separate group of AL-cut rats. To ascertain the effect of hypothalamic surgery on GH secretion, each AL-cut rat was subjected to repeated infusions of 0.9% NaCt on one day, and was challenged with repeated infusions of SS for 150 min separated by 30 min control periods 2 days after the control study. Serial blood specimens were obtained through the cannula placed in the right atrium. Immediately after collecting the first blood sample, the cannula placed in the inferior vena cava was filled with SS solution or 0.9% NaCI and the free end of the cannula was connected to an infusion pump which was set to deliver 10 ~l/min. SS was infused continuously at a dose of 30/~g/kg b. wt./h through the cannula placed in the inferior vena cava. Each infusion of SS was preceded by an i.v. bolus injection of SS (100/~g/kg b. wt.). The dose and duration of infusion were chosen such that they altered the GH secretory profile of adult female rats to that of adult male rats 3°.
Histological examinations After the experiments, the brains of hypothalamic deafferented rats were fixed with 10% formaldehyde in 0.01 M phosphate buffer. Serial coronal frozen sections of the hypothalamus, 50 pm thick~ were made and stained with Cresyl violet in order to study the locations of cuts and lesions.
GH assay and data analysis GH concentrations were measured by double-antibody radioimmunoassay using materials supplied by NIADDK, NIH. All values are expressed as ng/ml in terms of the NIADDK reference preparation rat GH-RP-1. To identify the GH pulses and the trough periods, data obtained from each rat were subjected to the PULSAR computer program
o
B 200
half ALcut #-endorphin
4.g ic.
E
1!'
I00
x
¢-
/
:~ ,~
!i ',
i
31 LD 0
E
to o
C
sham 300
8 -endor phin 41Jg icy t
200 1
/
1 j/
I00
1
,
I
,o'oo
,,bo
doo
i
J,
'\
,3'oo ,4'o0 ,~oo Time of Day
l '\ ,6'0o
doo
Fig. 1. Effect of hypothalamic deafferentation on plasma GH profile and the response to fl-endorphin (~-END). A: AL-cut, anterolateral deafferentation. B: half-AL-cut, unilateral anterolateral deafferentation. C: sham-operation. Stars indicate GH peaks identified by 'PULSAR' computer program. Four/~g of rat r - E N D in 4/~1 0.9% NaCI was administered slowly through a stainless-steel cannula placed in the third ventricle at 16.00 h.
295 TABLE I Comparison of G H secretory pattern parameters using the PULSAR computer program
Values are mean +- S.E.M. ; numbers in parentheses indicate the sample size. Treatment groups
Peaks~6 h
Peak amplitude (ng/ml)
lnterpeak interval (min)
Baseline levels" (ng/ml)
Area under the curve (ug/ml x rain)
Sham-operation (n = 8) Half-AL-cut (n = 6) AL-cut (n = 14)
2.0 + 0.0 3.3 + 0.5* 1.0 +_0.2*'**
253.7 +_44.6 74.4 +- 11.1" -
185.0 +_5.0 107.5 +_ 14.6" -
15.0 +_ 1.4 24.9+_ 1.7" 42.6 +- 1.1"**
26.3 + 2.9 14.1 +_ 1.2" 16.5 +_0.9*
* Values significantly different from those of sham-operated rats. ** Values significantly different from those of half-AL-cut rats. regarded as one peak and the higher spike was used for the analysis. The area under the curve depicting the plasma GH concentrations from 10.(30 to 16.00 h for each rat was measured using a planimeter and expressed in an arbitrary unit:/~g/ml x rain. The data were analyzed by one-way analysis of variance, and comparisons between data were performed by Duncan's multiplerange test. When variance was not uniform, comparisons between data were performed by Student's t-test or the Cochran-Cox test. The former was used when variance was equal, and the latter used when variance was different. RESULTS A r e p r e s e n t a t i v e p l a s m a G H secretory pattern after s h a m - o p e r a t i o n , half-AL-cut or A L - c u t is shown in Fig. 1. In s h a m - o p e r a t e d rats, two large episodes of G H secretion with an i n t e r p e a k interval of 185 min and amplitudes ranging from 77.8 to 669.1 ng/mi were o b s e r v e d during a 6 h observation period (Table I). G H peaks were also identified by the P U L S A R p r o g r a m in some A L - c u t rats; no p e a k was identified in 4, 1 p e a k in 7, 2 p e a k s in 2 and 3 peaks in 1 rat during the 6 h period, In A L - c u t rats, the p e a k amplitude of G H pulses never e x c e e d e d 62.4 ng/ml and the baseline levels; G H levels b e t w e e n the episodic secretion were 3 times higher as c o m p a r e d to those of s h a m - o p e r a t e d rats (Table I). Thus, P U L S A R - i d e n t i f i e d peaks among A L - c u t rats were difficult to recognize as peaks on visual appraisal. In half-AL-cut rats, G H pulses occurred more frequently than in s h a m - o p e r a t e d rats. Peak amplitudes and the baseline levels were i n t e r m e d i a t e values between those of the s h a m - o p e r a t e d animals and the A L - c u t rats (Table I). The total a m o u n t of G H secreted during 6 h was significantly r e d u c e d in rats with A L - c u t or half-AL-cut as c o m p a r e d to that of s h a m - o p e r a t e d rats (Table I). f l - E N D was injected centrally at 16.00 h (Figs. 1 and 2). It was expected that plasma G H levels were at trough levels in s h a m - o p e r a t e d rats. In s h a m - o p e r a t e d rats, central injection of f l - E N D resulted in significant rises in p l a s m a G H levels. A m o n g rats with A L - c u t , plasma G H levels did not increase at all in response to central injections o f f l - E N D . In half-AL-cut rats, an intermediate response to f l - E N D was o b s e r v e d (Fig. 2).
Plasma G H profiles in A L - c u t rats subjected to r e p e a t e d infusions of SS are shown in Fig. 3. Plasma G H levels during SS infusion were significantly lower than those of A L - c u t rats which received 0.9% NaC1. A large r e b o u n d of G H secretion occurred r e p e a t e d l y following the cessation of SS infusion. The a m o u n t of G H secreted during 6 h as expressed area u n d e r the curve of plasma G H ~ g / m l x rain) (34.1 + 5.3 (n = 7)) was significantly larger ( P < 0.05) in A L - c u t rats which received r e p e a t e d infusions of SS as c o m p a r e d to that of A L - c u t rats which received 0.9% NaCI (19.2 + 1.5 (n = 7)). U p o n histological e x a m i n a t i o n , the rostral limits of AL-cut were placed on the p o s t e r i o r b o r d e r of the optic chiasma and the traces of the cut were p u r s u e d as gliosis and bleeding lesions to posterolatera[ly. T h e y passed through the lateral b o r d e r of the v e n t r o m e d i a l nucleus of the h y p o t h a l a m u s in most rats, and in some, through the mid portion of the V M H . It then reached the p r e m a m millary region. The p a r a v e n t r i c u l a r nuclei were placed
65~
.2~ t t,~,
• ALcut (n=14) 300
~---~
8 - e n d o r p h i n J' /
,.,cv l/ I!
hQIf ALcut ( n - 6 ) sham (n=8)
E 200
I o
E I O0
I000
I100
1200
1300
Time
1400
of
I~,00
1600
I?00
Day
Fig. 2. Plasma OH profiles and the responses to fl-END in hypothalamic deafferented rats. © ©, sham-operation; A---iX, half-AL-cut; I~-----Q, AL-cut. Four ~g of rat fl-END in 4 ,ul 0.9% NaCI was administered slowly through a stainless-steel cannula placed in the third ventricle at 16.00 h. Each point represents the mean and the vertical bar indicates S.E.M. Numbers in parentheses indicate the sample size.
296 tion of SS tone. When AL-cut was performed, baseline G H levels were further raised and the identification of pulsatile GH secretion became difficult upon visual appraisal. The findings complement previous studies by
SS I00 #g/kg
~i
,
S$ ?O.,/kQ.h
~u
700
I 600
others,6,17.
I 1 [
E 500 400
"r (.9 300 0 E
200
O [")
I O0
o Jobo
.6o
~o
~bo
~bo
eb~
~o
Time o f Day Fig. 3. Plasma GH profiles during intermittent infusions of somatostatin (SS) in unrestrained male rats with anterolateral hypothalamic deafferentation. Rats with AL-cut were subjected to repeated infusions indicated by horizontal bars of 0.9% NaC1 or SS at 30/~g/kg b. wt./h separated by 30 min control periods. Each infusion of SS was preceded by a bolus i.v. injection of SS (100/~g/kg b. wt.) as shown by the arrow. Each point represents the mean and the vertical bar indicates S.E.M. Numbers in parentheses indicate the sample size. Solid line indicates GH profile of rats which received SS, while dotted line indicates GH profile of rats which received 0.9% NaCI. just anterior to the cut in all rats. AL-cut spared the arcuate nuclei, the median eminence and the pituitary stalk in 21 rats with AL-cut and in 6 rats with half-AL-cut used in the analysis. Another 3 rats with AL-cut or half-AL-cut were excluded from the analysis because parts of the arcuate nucleus and median eminence appeared to be destroyed. DISCUSSION
G H secretion is controlled by defined hypothalamic regions that have either an inhibitory or a stimulatory influence. SS containing neuronal cell bodies are located in the anterior hypothalamic periventricular area 8'~8"2°, while GRF-containing neuronal cell bodies have been demonstrated in arcuate nuclei and ventromedial nuclei of the MBH 2'14'21'33. The removal of SS influence on the M B H either by half-AL-cut or AL-cut caused significant alterations in the G H secretory pattern in a dosedependent manner. After performing half-AL-cut, plasma G H secretory pattern closely resembled that of adult female rats as characterized by high frequency, low amplitude G H pulses separated by elevated baseline levels ~5. The inhibitory tone provided by SS is considered to be lower in female rats than that in male rats Ll°. Therefore, the alteration of G H secretory pattern after half-AL-cut appears to be causally related to the reduc-
We demonstrated that a significant reduction of SS influence on the MBH significantly reduced the amount of G H secreted which was restored by intermittent infusions of SS. Elevated baseline GH levels with little pulsatility after AL-cut have been suggested to reflect a continuous release of G R F 17. The pattern of G R F secretion may induce partial desensitization of the G R F receptors. This could offer an explanation for the reductions in G H secretion and the synthesis of G H in the pituitary after AL-cut ~3. At any rate, our data suggest that SS modulates somatotroph responsiveness to GRF. Previous investigators have demonstrated that the attenuation of the G H response to G R F through receptor effect is either partially reversed 5 or restored by SS 26. A number of explanations are available for the post-SS rebound in G H secretion 19'27. Exogenous SS can act to block temporarily G H release from the pituitary, while allowing the accumulation of G H in a releasable pool 27. Kraicer et al. 19 have assumed from in vitro studies that the major determinant of rebound G H secretion in vivo is the magnitude of the pool of G H available for immediate release upon disinhibition, and the size of this pool is related to the preceding influence of GRF, before the termination of SS infusion. However, studies examining the nature of post-SS rebound in G H secretion in vivo have suggested that exogenous SS acts on the hypothalamus and inhibits the release of GRF, while the sudden removal of SS triggers the secretion 4'z4"3~. Therefore, repeated infusions of SS would allow endogenous G R F to bring about a large rebound release of G H so that the amount of G H secreted in AL-cut rats became comparable to that of control rats. Although the stimulation of G H secretion by morphine or opioid peptide analogue requires the presence of G R F 23'37, fl-END-induced G H secretion was completely abolished in AL-cut rats, and the response was significantly reduced in half-AL-cut rats as compared to that of the sham-operated animals. The data are consistent with the interpretation that the stimulation of G H release by fl-END requires neural input from the anterolateral direction to G R F neurons in the MBH a2. We previously suggested that the stimulation of G H by the metenkephalin analogue, FK 33-824, accompanied the reduction of inhibitory tone mediated by SS 32. Frohman et al. 9 observed that the stimulation of G H by morphine sulfate was abolished in rats administered antiserum to SS. Taken together, our data support that the stimulation of G H release by opiates is mediated through inhibition
297 of SS release which in turn disinhibits the release of GRF.
by e n d o g e n o u s G R F was p r e s e r v e d after the surgery.
The
T h e r e f o r e , we consider that the knife cut was p e r f o r m e d
previous
observations
that
central
injection
of
f l - E N D stimulate G H secretion in rats treated with antiserum to SS is not inconsistent with our data 3. It may
successfully without destroying the M B H .
be that i m m u n o n e u t r a l i z a t i o n by antiserum to SS was
tion of pulsatile G H secretion by a-methyl-p-tyrosine is
i n a d e q u a t e in the previous studies.
exerted through the modulation of the phasic release of
Finally, we have previously suggested that the inhibi-
It was difficult to verify A L - c u t histologically, because
SS 29. O u r present data further strengthen that the phasic
the gliosis and bleeding lesions along the cut lines were
SS secretion plays important roles in the release and
not always clear. We considered it necessary to observe
synthesis of G H m e d i a t e d through G R F .
the alterations in G H secretory pattern and the inhibition of G H response t o / 3 - E N D after two types of hypothalamic surgery. Th e changes in secretory pattern and the response to f l - E N D were related to the size of hypothalamic deafferentation, A L - c u t and half-AL-cut, while the post-SS r e b o u n d in G H secretion, a response m e d i a t e d REFERENCES 1 Akira, S., Wakabayashi, I., Sugihara, H., Minami, S., Takahashi, F. and Motoyama, A., Effect of testosterone on growth hormone secretion in female rats during a continuous infusion of growth hormone releasing factor, Neuroendocrinology, 47 (1988) 116-124. 2 Bloch, B., Brazeau, P., Ling, N., Bohlen, P,, Esch, F., Wehrenberg, W.B., Benoit, R., Bloom, F. and Guillemin, R., Immunohistochemical detection of growth hormone-releasing factor in brain, Nature (Lond.), 301 (1983) 607-608. 3 Chihara, K., Arimura, A., Coy, D.H. and Schally, A.V., Studies on the interaction of endorphins, substance P, and endogenous somatostatin in growth hormone and prolactin release in rats, Endocrinology, 102 (1978) 281-290. 4 Clark, R.G., Carlsson, L.M.S., Rafferty, B. and Robinson, I.C.A.F., The rebound release of growth hermone (GH) following somatostatin infusion in rats involves hypothalamic GH-releasing factor release, J. Endocrinol., 119 (1988) 397-404. 5 Clayton, R.N. and Bailey, L.C., Somatostatin partially reverses desensitization of somatotrophs induced by growth hormonereleasing factor, J. Endocrinol., 112 (1987) 69-76. 6 Daikoku, S., Kawano, H., Noguchi, M., Nakanishi, J., Tokuzen, M., Chihara, K. and Nagatsu, I., GRF neurons in the rat hypothalamus, Brain Research, 399 (1986) 250-261. 7 Daikoku, S., Hisano, S., Kawano, H., Chikamori-Aoyama, M., Kagotani, Y., Zhang, R. and Chihara, K., Ultrastructural evidence for neuronal regulation of growth hormone secretion, Neuroendocrinology. 47 (1988) 405-415. 8 Epelbaum, J., Arancibia, L.T., Herman, J.P., Kordon, C. and Palkovits, M., Topography of median eminence somatostatinergic innervation, Brain Research, 230 (1981) 412-416. 9 Frohman, L.A., Downs, T.R., Katakami, H. and Jansson, J.-O., The interaction of growth hormone-releasing hormone and somatostatin in the regulation of growth hormone secretion, In O. Isaksson, C. Binder, K. Hall and B. H6kfelt (Eds.), Growth Hormone: Basic and C[inical Aspects, Proceedings of the 1st Nordisk Insulin Syrnnposiurn. Excerpta Medica, Amsterdam, 1987, pp.63-77, 10 Gross, D.S., Role of somatostatin in the modulation of hypophysiai growth hormone production by gonadal steroids, Am. J. Anat., 158 (1980) 507-519. ll Halasz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area, Endocrinology, 77 11965) 553-562. 12 Halasz, B., Nagy, G., Molnar, J., Marton, J., Banky, Z., Lukats, O. and Vizi, E.S., On the site and mode of action of enkephalins and enkephalin analogues on anterior pituitary
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mone (GHRH) infusions on the subsequent growth hormone (GH) response to GHRH, Neuroendocrinology. 45 (1987) 118-122. Soya, H, and Suzuki, M., Somatostatin rapidly restores rat growth hormone (GH) release response attenuated by prior exposure to human GH-releasing factor in vitro, Endocrinology, 122 (1988) 2492-2498. Stachura, M.E., Influence of synthetic somatostatin upon growth hormone release from perifused rat pituitaries, Endocrinology, 99 (1976) 678-683. Steiner, R.A., Stewart, J.K., Barber, J., Koerker, D., Goodner, C.J., Brown, A., Illner, P. and Gale, C.C., Somatostatin: a physiological role in the regulation of growth hormone secretion in the adolescent male baboon, Endocrinology, 102 (1978) 1587-1594. Sugihara, H., Wakabayashi, I., Minami, S., Takahashi, F., Shibasaki, T, and Ling, N., Effect of a-methyl-p-tyrosine and an antiserum to rat growth hormone (GH)-releasing factor (GRF) on plasma GH secretory profile during a continuous infusion of human GRF in rats, Brain Research, 475 (1988) 128-133. Sugihara, H., Minami, S. and Wakabayashi, 1.. Post-somato statin rebound secretion of growth hormone is dependent on growth hormone-releasing factor in unrestrained female rats, 3. Endocrinol., 122 (1989) 583-591. Tannenbaum, G.S. and Ling, N., The interrelationship of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion, Endocrinology, 115 (1984) 1952-1957.
32 Tonegawa, Y., Wakabayashi, 1., lhara, I., Hattori, M., Shibasaki, T. and Ling, N., Does opioid peptide stimulate growth hormone (GH) secretion via GH-releasing factor (GRF)?,
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Abstr. 2311, Quebec City, 1984. VandePol, C.J., Leidy, Jr., J.W., Finger, T.E. and Robbins, R.J., Immunohistochemical localization of GRF-containing neurons in rat brain, Neuroendocrinology, 42 (1986) 143-147. Wehrenberg, W.B., Brazeau. P., Luben, R., Bohlen, P. and Guillemin, R., Inhibition of the pulsatile secretion of growth hormone by monoelonal antibodies to the hypothalamic growth hormone releasing factor (GRF), Endocrinology, 111 (1982) 2147-2148. Wehrenberg, W.B., Ling, N., Bohlen, P., Esch, E, Brazeau, P. and Guillemin, R., Physiological roles of somatocrinin and somatostatin in the regulation of growth hormone secretion. Biochem. Biophys. Res. Commun., 109 (1982) 562-567. Wehrenberg, W.B., Brazeau, P., Ling, N., Textor, G. and Guillemin, R., Pituitary growth hormone response in rats during a 24-hour infusion of growth hormone-releasing factor, Endocrinology, 114 (1984) 1613-1616. Wehrenberg, W.B., Bloch, B. and Ling, N., Pituitary secretion of growth hormone in response to opioid peptides and opiates is mediated through growth hormone-releasing factor, Neuroendocrinology, 41 (1985) 13-16. Wehrenberg, W.B., Continuous infusion of growth hormonereleasing factor: effects on pulsatile growth hormone secretion in normal rats, Neuroendocrinology, 43 (1986) 391-396.
