Sexual dimorphism of somatostatin and growth hormone-releasing factor signaling in the control of pulsatile growth hormone secretion in the rat.

A striking sexual dimorphism exists in the pattern of GH secretion and rate of somatic growth; however, the mechanism(s) mediating this sex difference is unknown. To elucidate the physiological roles of the hypothalamic neuropeptides, somatostatin (SRIF) and GRF, and their interrelation, in generating the sexually dimorphic GH secretory pattern we examined: 1) GH responsiveness to exogenous GRF and 2) the effects of immunoneutralization of endogenous SRIF and GRF on GH secretory dynamics, in free-moving male and female rats. In males, the GH response to 1 microgram rat(r)GRF(1-29)NH2 iv was significantly greater at peak compared to trough times of GH secretion (925.2 +/- 250.8 vs. 95.6 +/- 27.8 ng/ml; P less than 0.02), the latter known to be due to antagonization by the cyclic increased release of endogenous SRIF. In contrast, females failed to exhibit a time-dependent difference in GH responsiveness to GRF. Passive immunization with a specific antiserum to SRIF in males resulted in significant elevation of GH nadir levels but had no effect on GH peak amplitude. In contrast, immunoneutralization of endogenous SRIF in females caused a marked augmentation of plasma GH levels at all time points; there was a significant increase in GH peak amplitude (171.3 +/- 39.9 vs. 67.5 +/- 11.3 ng/ml; P less than 0.05), GH nadir (18.3 +/- 2.7 vs. 5.8 +/- 1.1 ng/ml; P less than 0.01) and mean 6-h plasma GH level (78.7 +/- 4.1 vs. 33.1 +/- 5.8 ng/ml; P less than 0.001), compared to normal sheep serum-treated controls. These results indicate that the pattern of hypothalamic SRIF secretion in females does not follow the male-like ultradian rhythm. Passive immunization with a specific antiserum to GRF obliterated spontaneous GH pulses in both sexes. Moreover, in females, anti-GRF serum attenuated GH nadir levels (4.3 +/- 1.7 vs. 21.4 +/- 3.5 ng/ml; P less than 0.01) indicating a physiological role for GRF in maintaining the elevated basal GH level of females, in addition to its important role in generating the episodic GH pulses. Taken together, these findings provide support for the hypothesis that, in female rats, the pattern of hypothalamic SRIF secretion into hypophyseal portal blood is continuous, rather than cyclical, as in the male; whereas in the case of GRF secretion, in addition to steady-state release which occurs at a higher level in females than males, there is also episodic GRF bursting which does not follow a specific rhythm, as in the male.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sexual differentiation of growth hormone feedback effects on hypothalamic growth hormone-releasing hormone and somatostatin

To investigate possible sex differences in the feedback regulation of growth hormone (GH) secretion, concentrations of immunoreactive GH-releasing hormone (GRF) and somatostatin (SS) were measured in the median eminence (ME) and the hypothalamus of male and female rats bearing the MtTW15 tumor, which secretes high amounts of GH and prolactin (PRL). Four weeks after tumor implantation in male rats, the GRF concentration in the whole hypothalamus, including the ME, was decreased by 37% (0.29 +/- 0.02 vs. 0.46 +/- 0.02 ng/mg protein in intact male controls; p less than 0.001) and the concentration of SS was increased by 40% (11.5 +/- 0.7 vs. 8.1 +/- 0.3 ng/mg protein in male controls; p less than 0.01). In female rats, the presence of tumor for 4 weeks caused a smaller (18%) reduction in GRF concentrations (0.27 +/- 0.02 vs. 0.33 +/- 0.03 ng/mg protein in intact female controls; p less than 0.05) and no significant change in SS concentrations (10.2 +/- 0.08 vs. 9.7 +/- 0.8 ng/mg protein in female controls). Tumor-related changes in GRF and SS concentrations were also more pronounced in male rats than in females, when determined separately in the microdissected ME and in the remaining hypothalamus. These differences occurred despite similar increases in serum GH, PRL and insulin-like growth factor I concentrations in male and female tumor-bearing rats. To assess which hormone (GH or PRL) was responsible for these changes, intact male rats were treated for 10 days with 2 daily s.c. injections of rat GH (rGH; 100 and 250 micrograms/day), rat PRL (100 and 250 micrograms/day) or vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogenesis of growth hormone-releasing hormone neurons in the rat hypothalamus

The ontogenesis of growth hormone releasing hormone (GH-RH) containing neurons in the rat hypothalamus has been studied by immunohistochemistry, using a specific anti-rat GH-RH serum. Immunoreactive fibers were first detected in the prospective median eminence on day 18 of gestation. During the subsequent 3 days, they rapidly increased in distribution and intensity of staining within this structure. On day 21, positive fibers were also visible in a plexus within the arcuate nucleus. In 1-day-old rats treated with colchicine, positive perikarya were distributed in several hypothalamic nuclei, including the arcuate nucleus, dorsomedial nucleus, basal lateral hypothalamus, and perifornical region. The distribution was similar to that previously described in adult rats. The intensity of staining in the various hypothalamic regions increased during early postnatal life to levels nearly comparable to those in adult rats by 30 days. These findings showing the early appearance of GH-RH-positive terminals in the median eminence and the wide distribution of the perikarya at an early stage of postnatal life support the view that hypothalamic GH-RH serves an important role in the regulation of growth hormone secretion during late prenatal and early neonatal periods.     

Sexual dimorphism of growth hormone in the hypothalamus: regulation by estradiol

GH is best known as an anterior pituitary hormone fundamental in regulating growth, differentiation, and metabolism. GH peptide and mRNA are also present in brain, in which their functions are less well known. Here we describe the distribution of GH neurons and fibers and sex differences in Gh mRNA in adult mouse brain. Cell bodies exhibiting GH immunoreactivity are distributed in many brain regions, particularly in the hypothalamus in which retrograde labeling suggests that some of these cells project to the median eminence. To determine whether Gh mRNA is sexual dimorphic, we carried out quantitative RT-PCR on microdissected brain nuclei. Ovary-intact mice had elevated Gh mRNA in the arcuate nucleus and medial preoptic area (MPOA) compared with gonad-intact males. In males, castration increased Gh mRNA in the MPOA, whereas ovariectomy decreased Gh mRNA in both regions. When gonadectomized adults of both sexes were treated with estradiol Gh mRNA increased in females but had no effect in castrated males. Tamoxifen was able to blunt the rise in Gh mRNA in response to estradiol in females. In addition, we found that estrogen receptor-α is coexpressed in GH neurons in the MPOA and arcuate nucleus. In summary, the findings reveal sexual dimorphisms in Gh gene expression in areas of the brain associated with reproduction and behavior. Interestingly, estradiol enhances Gh mRNA in females only, suggesting that multiple factors orchestrate this sexual dimorphism.     

Chronic food-restriction alters the expression of somatostatin and growth hormone-releasing hormone in the ovariectomised ewe.

Changes in the secretion of GH induced by long-term alterations in nutritional status are thought to result from alterations in somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) at the level of the hypothalamus. To date however, the effect of nutrition on the gene expression of SRIF and GHRH in a species where GH secretion is increased by food restriction, as is the case for the sheep and human, remains unknown. We determined the effect of under-nutrition on the expression of SRIF and GHRH in the hypothalamus of sheep. Ovariectomised ewes were randomly divided into two groups and either fed an ad lib diet (n=6) or a restricted diet of 500 g lucerne chaff per day (food-restricted; n=5) for 7 months. In situ hybridisation was used to study hypothalamic gene expression for GHRH, SRIF and galanin (GAL). The food-restricted animals had elevated plasma concentrations of GH; this was associated with an increase in GHRH mRNA levels in the arcuate nucleus (ARC) and reduced SRIF in the rostral periventricular nucleus and ventromedial hypothalamic nucleus. The level of gene expression of GAL in the ARC and SRIF in the caudal periventricular nucleus was similar in ad lib and food-restricted animals. In conclusion, the effect of chronic food-restriction on the secretion of GH reflects increased GHRH and reduced SRIF synthesis in the hypothalamus.     

Interaction of growth hormone-releasing hormone with the insulin-like growth-factors during prenatal development in the rat.

The placenta is a chimeric organ that produces all the components of the hypothalamic-pituitary GH axis. We propose that placental GH-releasing hormone (GHRH) stimulates placental GH-like hormones which in turn stimulate production of the insulin-like growth factors (IGFs), IGF-I and IGF-II, and these placental IGFs are important for growth and development of the placenta as well as the fetus. To test this hypothesis, pregnant rats were given either GHRH antisera or preimmune sera ip from days 7-19 of gestation. Fetuses were killed on day 19, and IGF-I and IGF-II tissue and serum concentrations in the mother and fetus were measured by RIA. IGF-II receptor content was measured by Western analysis. IGF-I and IGF-II messenger (m) RNA levels were measured in the placentas as well as in the fetal livers. The GHRH antibody titer was highest at day 19 of gestation but continued to be present through day 20 of postnatal development. Although placental weights did not differ, antibody-treated animals had higher placental IGF-I and IGF-II levels (I, 108 +/- 6 (SD); II, 126 +/- 5 ng/g, respectively) vs. control animals (I, 88 +/- 2.5 (SD); II, 48 +/- 11 ng/g) in pooled specimens. The IGF-II receptor was also up-regulated in placentas from antibody-treated mothers. The fetuses of antibody-treated (A) mothers were larger than the controls (C) (A, 2.615 g; C, 2.49 g, P less than 0.05). Levels of both IGFs were significantly increased in livers of antibody treated fetuses (IGF-I: A, 15 +/- 1 (SD); C, 12 +/- 0.8 ng/g; and IGF-II: A, 295 +/- 10 (SD); C, 233 +/- 10 (SD) ng/g). In addition, the concentration of the IGF-II receptor in liver of antibody-treated fetuses was also increased. Further, pooled fetal sera from antibody-treated fetuses had higher levels of IGF-II than controls (A, 950 ng/ml; C, 700 ng/ml), and the circulating IGF-II receptor was increased as measured by Western analysis. In the liver, IGF-II mRNA levels of antibody-treated fetuses were increased to 117% of controls, whereas IGF-I mRNA levels were undetectable. The placenta showed no increase in placental lactogen or GH mRNA, whereas IGF-II and GHRH mRNA were slightly increased in antibody-treated animals. In conclusion, these data suggest that GHRH may interact with the IGFs in a different fashion during prenatal development then during postnatal development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of growth hormone secretagogues and growth hormone-releasing hormone/somatostatin

The class of novel synthetic compounds termed growth hormone secretagogues (GHSs) act in the hypothalamus through, as yet, unknown pathways. We performed physiologic and histochemical studies to further understand how the GHS system interacts with the well-established somatostatin (SRIF)/growth hormone-releasing hormone (GHRH) neuroendocrine system for regulating pulsatile GH secretion. Comparison of the GH-releasing activities of the hexapeptide growth hormone-releasing peptide-6 (GHRP-6) and GHRH administered intravenously to conscious adult male rats showed that the pattern of GH responsiveness to GHRP-6 was markedly time-dependent, similar to that observed with GHRH. Immunoneutralization of endogenous SRIF reversed the blunted GH response to GHRP-6 at trough times, suggesting that GHRP-6 neither disrupts nor inhibits the cyclical release of endogenous hypothalamic SRIF. By striking contrast, passive immunization with anti-GHRH serum virtually obliterated the GH responses to GHRP-6, irrespective of the time of administration. These findings suggest that the GHSs do not act by altering SRIF release but, rather, stimulate GH release via GHRH-dependent pathways. Our dual chromogenic and autoradiographic in situ hybridization experiments revealed that a subpopulation of GHRH mRNA-containing neurons in the arcuate (Arc) nucleus and ventromedial nucleus (VMN) of the hypothalamus expressed the GHS receptor (GHS-R) gene. These results provide strong anatomic evidence that GHSs may directly stimulate GHRH release into hypophyseal portal blood, and thereby influence GH secretion, through interaction with the GHS-R on GHRH- containing neurons. Altogether, these findings support the notion that an additional neuroendocrine pathway may exist to regulate pulsatile GH secretion, possibly through the influence of the newly discovered GHS natural peptide, ghrelin.     

Calcium and calmodulin mediation of growth hormone-releasing hormone release from the rat hypothalamus in vitro.

A major role for Ca2+ and calmodulin in stimulus-secretion coupling has been suggested for several neuropeptides; however, the cellular mechanisms of GH-releasing hormone (GHRH) release have been little investigated so far. We have used a previously validated acute rat hypothalamic explant system in order to elucidate whether Ca2+ acts as a second messenger in the regulation of GHRH release, and whether calmodulin-dependent pathways are involved. Calcium dependence of somatostatin (SRIH) release was assessed in the same experiments. Calmodulin dependence of SRIH was not investigated in detail, as it has been established previously. The calcium-entry antagonist, verapamil, antagonized K(+)-stimulated GHRH and SRIH release in a dose-dependent manner, with maximal inhibition shown at 10(-4) M. The calmodulin antagonist W7 also blocked K(+)-evoked GHRH release in a dose-dependent manner, with significant inhibition in the dose range 5 X 10(-5) M to 2 X 10(-4) M; similarly, a more specific calmodulin inhibitor, the W7 derivative 5-iodo-C8 (W8), reversed K(+)-stimulated GHRH release, showing slightly higher potency than W7. W7 also reversed GHRH release in response to the calcium-ionophore A23187, although verapamil had no effect on A23187-evoked GHRH or SRIH release. Thapsigargin, which increases the efflux of Ca2+ from calciosomes, did not affect either GHRH or SRIH release at 10(-5) M or 10(-4) M. The basal release of GHRH was clearly suppressed by W7 and W8 (10(-4) M), whereas verapamil had no effect. We conclude that calcium influx is crucial for depolarization-induced GHRH and SRIH release. Calcium entrance in response to A23187 appears to be independent of verapamil-sensitive calcium channels. The lack of effect of thapsigargin suggests that increased intracellular Ca2+ from intracellular stores is not equivalent to an increase in Ca2+ influx. Both basal and depolarization-induced release of GHRH in this system are calmodulin dependent.     

Effect of hypophysectomy and growth hormone administration on somatostatin content in the rat hypothalamus.

The effect of hypophysectomy and bovine GH administration on somatostatin (SRIF) content in the rat hypothalamus was investigated. SRIF content was determined by a specific radioimmunoassay method described elsewhere. The total SRIF content of the rat hypothalamus as well as its content per milligram wet weight had decreased 4 weeks after hypophysectomy but was restored significantly in those rats which were subjected to bovine GH administration for 7 days 3 weeks after hypophysectomy. Furthermore, in nonoperated rats, increase of hypothalamic SRIF content was observed after 7 days GH administration. These results indicate that growth hormone may influence the SRIF content of hypothalamus and it seems likely that a feedback mechanism between pituitary GH and hypothalamic SRIF exists.     

Reduction in adiposity affects the extent of afferent projections to growth hormone-releasing hormone and somatostatin neurons and the degree of colocalization of neuropeptides in growth hormone-releasing hormone and somatostatin cells of the ovine hypothalamus

Various neuropeptides and neurotransmitters affect GH secretion by acting on GHRH and somatostatin (SRIF) cells. GH secretion is also affected by alteration in adiposity, which could be via modulation of GHRH and SRIF cells. We quantified colocalization of neuropeptides in GHRH and SRIF cells and afferent projections to these cells in lean (food restricted) and normally fed sheep (n=4/group). The number of GHRH-immunoreactive (IR) cells in the arcuate nucleus was higher in lean animals, but the number of SRIF-IR cells in the periventricular nucleus was similar in the two groups. A subpopulation of GHRH-IR cells colocalized neuropeptide Y in lean animals, but this was not seen in normally fed animals. GHRH/galanin (GAL) colocalization was higher in lean animals with no difference in numbers of GHRH/tyrosine hydroxylase or GHRH/GAL-like peptide cells. SRIF/enkephalin colocalization was lower in lean animals. The percentage of GHRH neurons receiving SRIF input was similar in lean and normally fed animals, but more GHRH cells received input from enkephalin afferents in normally fed animals. The percentage of SRIF cells receiving GHRH, neuropeptide Y, GAL, and orexin afferents was higher in lean animals. These findings provide an anatomical evidence of central mechanism(s) by which appetite-regulating peptides and dopamine could regulate GH secretion. Increased input to SRIF cells in lean animals may be inhibitory and permissive of increased GH. The appearance of NPY in GHRH cells of lean animals may be a mechanism for regulation of increasing GH secretion with reduced adiposity.     

Age-related changes in growth hormone releasing factor and somatostatin in the rat hypothalamus

Distribution and staining intensities of growth hormone releasing factor (GR (GRF) and somatostatin (SRIF) were examined in young (3 months of age) and old (24 months of age) male rats of Sprague-Dawley strain, using the PAP immunocytochemical procedure. Some animals of each age group were intraventricularly injected with colchicine to demonstrate immunoreactive neuronal perikarya. GRF-immunoreactive intensities of old rats were markedly reduced in the median eminence as compared with those of young rats. No remarkable difference could be detected between SRIF immunoreactivities in the young and old animals, since intensive SRIF immunoreactivities were found in the external layer of the median eminence of both groups of animals. Between two age groups injected with colchicine, we also found no difference in the distribution and staining intensities of immunoreactive perikarya of GRF and SRIF in the hypothalamus and also detected no significant difference in total neuron numbers of each peptide. These findings suggest that the synthesis and/or release of GRF in GRF-containing neurons are decreased, though GRF-containing neurons themselves remain alive and have the capacity to synthesize GRF.     

Growth hormone-releasing factor and fibroblast growth factor regulate somatostatin gene expression

A multiple peptide-synthesizing clonal rat cell line was used to study the effect(s) of GRF and basic fibroblast growth factor (bFGF) on the synthesis and secretion of somatostatin (SS). The presence of SS-specific mRNA in 44-2C cells was shown morphologically by in situ hybridization. The release and cellular content of SS increased significantly after treatment with rat hypothalamic GRF (rGRF), the ED50 for rGRF stimulation of intracellular SS was 1.9 X 10(-11) M. GRF stimulated SS production in serum-supplemented and serum-free cultures. Results obtained after incubation of 44-2C cells with 125I-labeled rGRF indicated uptake and nuclear localization of rGRF by 44-2C cells. FGF stimulated the secretion and cellular content of SS. We propose that bFGF regulates the short term secretion and accumulation of SS and mediates rGRF-stimulated SS expression.     

Sexual dimorphism of pituitary gonadotropes during postnatal development in the rat

In the present study both the reverse hemolytic plaque assay for detecting luteinizing hormone (LH) secretion from single cells and LH immunocytochemistry (ICC) were applied to conduct quantitative studies on sexual differences in the gonadotrope population during postnatal development. Pituitary glands from both sexes at different ages were monodispersed with 0.1% trypsin. Freshly dispersed cells were incubated in Cunningham chambers in the presence of 10(-7) M gonadotropin-releasing hormone (GnRH) for measurement of the fraction of plaque-forming cells and the mean size of plaque formed, or attached to glass slides for measurement of the fraction of cells staining for LH by ICC. The percentage of immunostained LH cells increased with age in both sexes from about 5% of the total pituitary cell population at 5 days of age to a plateau of about 10% by 15 days and then fell to the adult level of about 5%. There were no significant sexual differences except at 30 and 40 days of age. In female rats the fraction of LH-secreting cells detected by plaque assay matched closely with that of LH-containing cells detected by ICC. However, there were significant sexual differences in the percentage of LH-secreting cells at day 15 through day 40. The mean LH output from individual cells of both sexes as indicated by the mean size of plaques also increased with age and reached a peak around 50 days. The sexual differences were first seen around 30 days of age with greater amounts in the female than in the male.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal transduction systems in growth hormone-releasing hormone and somatostatin release from perifused rat hypothalamic fragments.

The role of signal transduction systems was examined in the secretion of GH-releasing hormone (GHRH) and somatostatin (SS) from perifused rat hypothalamic fragments. Forskolin, an adenylate cyclase activator, stimulated the release of GHRH and SS in a concentration-dependent manner (10-100 microM) with greatest stimulation for GHRH at 100 microM (mean +/- SE, 249 +/- 14%) and for SS at 30 microM (172 +/- 18%). (Bu)2cAMP also augmented GHRH and SS release. The protein kinase-C activator phorbol 12-myristate 13-acetate did not significantly stimulate basal GHRH or SS release at concentrations of 10 nM to 1 microM. The calcium ionophore A23187 enhanced the release of GHRH and SS in a concentration-dependent manner (2-20 microM), with the greatest responses of 282 +/- 50% at 10 microM and 189 +/- 24% at 20 microM, respectively. Potentiation by phorbol 12-myristate 13-acetate of forskolin-stimulated GHRH and SS release was observed. A23187 at 10 microM did not enhance forskolin-stimulated GHRH release, but did potentiate forskolin-stimulated SS release in a more than additive response. We conclude that there is 1) cAMP stimulation of hypothalamic GHRH and SS release, 2) a modulating role of protein kinase-C on cAMP-stimulated release of GHRH and SS, 3) a stimulatory role of the calcium messenger system for GHRH and SS release, 4) interaction of the signal pathways with differences in net GHRH and SS responses, and 5) a modulatory effect of protein kinase-C in perifused hypothalamic fragments which differs from the stimulation of basal GHRH and SS release reported in fetal-derived hypothalamic cell cultures. Our observations suggest an important regulatory role of interacting signal transduction systems in the hypothalamic secretion of GHRH and SS.     

Sexual dimorphism in the control of growth hormone secretion

The secretory pattern of GH in the mature rat is sexually differentiated. In male rats GH is secreted in pulses occurring at regular 3- to 4-h intervals. In females the pulses are lower and plasma GH levels between the pulses are higher than in males. The continuous presence of testosterone appears to be necessary to maintain low basal GH levels in adult male rats. Neonatal, but not prepubertal, gonadectomy decreases GH pulse height in adult male rats to female levels. Administration of testosterone neonatally to castrated animals returns GH pulse height to normal suggesting that neonatal testicular androgen secretion is one determinant for GH pulse height in adult male rats. Administration of testosterone neonatally or during adult life to neonatally ovariectomized rats also produces higher GH pulses. In contrast to testosterone, estrogens elevate basal plasma GH levels and suppress the GH pulses under some conditions. Estrogens may stimulate basal GH secretion by acting directly on the pituitary. The physiological significance of the secretory pattern of GH has been investigated in hypophysectomized rats by simulating different plasma patterns of GH. The results suggest that high, infrequent GH pulses with low plasma GH levels in between (i.e. a masculine plasma GH pattern) promotes growth more effectively than an intermediate, rather constant level of plasma GH (i.e. a feminine plasma GH pattern). Since male sex steroids masculinize the secretory pattern of GH and have only minor growth-promoting effects in hypophysectomized animals it appears that the growth promoting effect of androgens is indirect and is due to an altered secretory pattern of GH. Presumably, neonatal androgen secretion stimulates body growth during adult life by irreversibly masculinizing the secretory pattern of GH. In contrast, estrogens appear to influence body growth by mechanisms that are mainly independent of the secretory pattern of GH. Evidence is accumulating that the secretory pattern of GH in the rat also affects various sexually differentiated hepatic characteristics such as steroid metabolism and prolactin receptor concentration. Thus, a feminization of the liver develops after continuous, but not intermittent, administration of GH to hypophysectomized rats. GH secretion is predominantly regulated by two hypothalamic peptides; GRF, and the GH-release-inhibiting factor, somatostatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the subsynaptosomal localization of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the rat hypothalamus.

In the current investigation, subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH, TRH, and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes, and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal particles which banded at sucrose densities between 0.6-1.0 M. Electron micorscopic analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. Norepinephrine was found in two pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with dense-cored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles.     

Localization of growth hormone-releasing hormone in the human hypothalamus and pituitary stalk

The localization of the recently identified GH-releasing hormone (GHRH) in the human hypothalamus and pituitary stalk was determined by microdissection techniques and a specific RIA for GHRH. The highest concentrations of GHRH immunoreactivity (IR-GHRH) in the hypothalamus were found in the area of the infundibular nucleus (83 +/- 4 ng/mg protein; average +/- range). Lower quantities were found in other hypothalamic regions. Very high concentrations of IR-GHRH were present in the upper portion of the pituitary stalk (1454 +/- 48 ng/mg protein), and they decreased gradually toward the distal end of the stalk (21 +/- 3 ng/mg). This concentration gradient suggests that the peptide reaches the anterior pituitary mainly by way of the long portal vessels. Somatostatin, the second neuropeptide involved in the regulation of GH secretion from the anterior pituitary, had a pattern of distribution along the pituitary stalk very similar to that of IR-GHRH.     

Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus

The distribution of GRF-immunoreactive structures in the rat hypothalamus was studied after colchicine treatment with peroxidase-antiperoxidase immunocytochemistry in vibratome sections. The majority of the GRF-immunoreactive cell bodies were found in the arcuate nucleus and the medial perifornical region of the lateral hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Fibers from the perifornical cell bodies formed a fan-like projection to the median eminence, where a dense accumulation of GRF-containing processes and terminals was found. GRF terminals were located in the central regions of the median eminence. The localization of GRF-immunoreactive structures in the hypothalamus and median eminence reinforces the view that GRF plays a physiological role in the regulation of pituitary function.