Interaction of carp growth hormone-releasing factor and somatostatin on in vitro release of growth hormone in rainbow trout (Oncorhynchus mykiss).

Possible antagonism between somatostatin (SS) and carp growth hormone-releasing factor (GRF) on growth hormone (GH) secretion was examined by radioimmunoassay in a dispersed rainbow trout pituitary cell culture system. SS (3 nM) significantly antagonized carp GRF(1-29; 1 nM, 10 nM)-induced GH secretion. The slope of the dose-response curve for carp GRF(1-29) with SS was statistically different from that of carp GRF(1-29) alone (p less than 0.05) suggesting a noncompetitive antagonism of SS to carp GRF. The carp GRF(1-29) was also indicated to be a noncompetitive antagonist to SS (p = 0.056). Carp GRF(1-29; 100 nM) was unable to restore the inhibitory effect of SS on GH release after pre-exposure of SS (30 nM) to the pituitary cells. We conclude that SS antagonizes carp GRF on GH release at the pituitary level in rainbow trout and this antagonism is noncompetitive. SS has a postantagonism to carp GRF which may implicate some important physiological adaptations in teleosts.     

Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.     

The interrelationship between the effects of somatostatin and human pancreatic growth hormone-releasing factor on growth hormone release by cultured pituitary tumor cells from patients with acromegaly

The effects of somatostatin (SRIF) and human pancreatic tumor GRF on GH release by cultured pituitary tumor cells obtained during transsphenoidal operation from 15 acromegalic patients were investigated. In a study of the sensitivity of pathological GH release to SRIF, 1-10 nM SRIF induced maximal inhibition of hormone release in 3 consecutive tumors. In 12 of 15 tumor cell cultures, 10 nM SRIF produced statistically significant inhibition of basal GH release by 39 +/- 3% (mean +/- SEM). In 2 of the 3 other tumors, SRIF inhibited GRF-stimulated GH release, while this was not investigated in the third tumor. A dose-response study of the effect of GRF on GH release by cultured pituitary tumor cells showed that doses of 0.1, 1, 10, and 100 nM induced similar maximal (35%) stimulation of hormone secretion. In four of five consecutive tumor cell suspensions, 1 and 10 nM GRF induced statistically significant GH stimulation by 18-300%. Preincubation of the tumor cells with 5 nM dexamethasone greatly increased the sensitivity and the maximal stimulation in response to GRF and made one tumor cell suspension, which did not react to GRF initially, sensitive to GRF. In the tumors of four patients, the interrelationship between the effects of SRIF and GRF on GH release were also studied. SRIF (10 nM) inhibited the stimulatory effects of GRF on GH release virtually completely. In conclusion, GH release by in vitro cell cultures of GH-secreting pituitary adenomas was inhibited by SRIF and stimulated by GRF. The interaction of GRF and SRIF on GH release by these pituitary tumor cells was similar to that in normal rat GH cells, as SRIF virtually completely overcame the GRF-induced GH release.     

Iso stimulation of GH and cAMP: comparison of beta-adrenergic- to GRF-stimulated GH release and cAMP accumulation in monolayer cultures of anterior pituitary cells in vitro

Growth hormone (GH) release and cAMP content were measured in monolayer cultures of anterior pituitary cells after beta-adrenergic and GH-releasing factor (GRF) receptor activation. Isoproterenol (Iso, ED50-20 nM) was less potent than GRF (ED50-20 pM) in stimulating GH release. Iso caused a rapid stimulation of GH release that was maximal after 15 min and declined thereafter, while GRF caused a more gradual increase in GH secretion that was maximal after 30 min and remained elevated after 3 h. Both Iso- and GRF-stimulated GH release were preceded by an increase in cAMP content in the pituitary cells. Further, the addition of 3-isobutyl-1-methylxanthine (IBMX) to the medium enhanced the GH-stimulatory and cAMP-accumulating effects of both secretagogues. Experiments performed with native catecholamines and synthetic catecholamine agonists and antagonists indicated that the GH-stimulatory effect of Iso was mediated by a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors. Additionally, experiments performed with cultured GH3 tumor cells, found that incubation with GRF, Iso, vasoactive intestinal polypeptide, forskolin, or cholera toxin caused an increase in cAMP content in the cells. However, compared to the responses observed in primary pituitary cultures the GH secretory response to these agents was comparatively small. Together, these studies suggest that a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors may act, at least in part, on somatotrophs in the anterior pituitary to stimulate GH release. Although both GRF and beta 2-adrenergic receptor agents affect GH release through a common second messenger system, their differing pharmacokinetic properties suggest distinct intracellular mechanisms.     

Stimulation of growth hormone release by vasoactive intestinal peptide and peptide PHI in rat anterior pituitary reaggregates. Permissive action of a glucocorticoid and inhibition by thyrotropin-releasing hormone

In superfused rat anterior pituitary cell reaggregates, cultured for 5 days in serum-free defined medium, vasoactive intestinal peptide (VIP) concentration-dependently stimulated prolactin (Prl) release but had only a marginal effect on growth hormone (GH) release. When reaggregates were cultured in the presence of 80 nM dexamethasone (Dex) VIP strongly stimulated GH release from a concentration as low as 0.1 nM. VIP did not stimulate LH release. Peptide PHI also stimulated GH release but thyrotropin-releasing hormone (TRH) or angiotensin II did not. In fact, TRH slightly but transiently inhibited basal GH release and strongly inhibited VIP-stimulated GH release. GH-releasing factor (GRF) stimulated GH more potently and with higher intrinsic activity than VIP but GRF did not increase Prl release. The present data indicate that under defined hormonal conditions VIP and PHI are capable of stimulating GH release and that TRH can antagonize this effect by a direct action on the pituitary.     

Actions of growth hormone-releasing factor and somatostatin on adenylate cyclase and growth hormone release in rat anterior pituitary

The interaction of growth hormone-releasing factor (GRF) and somatostatin (SRIF) on adenylate cyclase activity and growth hormone release was investigated in pituitary homogenates and 2-day cultured rat anterior pituitary cells. GRF stimulated growth hormone release by about 3-fold (ED50 1.6 X 10(-12) M) and caused a rapid 15-fold increase in cyclic AMP production (ED50 6.0 X 10(-12) M). The increase in cyclic AMP was due to direct stimulation of adenylate cyclase by GRF, which caused a 4-fold increase in the activity of the enzyme measured in anterior pituitary homogenates. GRF-induced cyclic AMP formation and GRF-stimulated adenylate cyclase activity were maximally inhibited to the extent of about 50% by 10(-8) M somatostatin. In contrast, GRF-stimulated growth hormone release was completely inhibited by somatostatin (ID50 3.2 X 10(-11) M), suggesting a second site of action of somatostatin. These studies demonstrate that GRF stimulates growth hormone release via activation of adenylate cyclase and a rise in intracellular cyclic AMP. In addition, these findings indicate that the inhibitory action of somatostatin on growth hormone release is exerted at two levels, one at the level of adenylate cyclase affecting the production of cyclic AMP, and the other beyond the formation of the nucleotide, at a site which modulates the release of growth hormone from the cell.     

Cellular mechanism of caprylic acid-induced growth hormone suppression

To determine the dynamic secretory pattern of growth hormone (GH) in the presence of free fatty acids (FFA), we studied the effect of caprylic acid on basal and rGRF(1-29)NH2 (GRF)-induced GH secretion in acutely dispersed and perifused rat pituitary cells. At a concentration of 3.0 mmol/L, caprylic acid inhibited both basal (P less than .05) and GRF-stimulated GH secretions (P less than .01), except when the maximal (near the EC100) GRF concentration of 100 pmol/L was used. Lower concentrations of caprylic acid such as 0.3 and 1.0 mmol/L significantly inhibited, in a concentration-dependent manner, GH secretion induced by a 6.25-pmol/L GRF. However, at a GRF concentration of 25 pmol/L, this inhibitory effect was abolished. The time-course of GH response to GRF was similar in both control and caprylic acid-treated cells. To elucidate the mechanism(s) of action of the caprylic acid-induced blockade of GH secretion, in 3-day cultured rat pituitary cells, the effect of caprylic acid on basal and GRF-stimulated GH and 3',5'-cyclic adenosine monophosphate (cAMP) release. We also tested its effect on the Ca2+ ionophore, A23187-induced GH release. Caprylic acid (0.3 to 3.0 mmol/L) significantly inhibited basal GH release and GRF- or A23187-induced GH secretion. Furthermore, it decreased both basal and GRF-stimulated cAMP release (P less than .05). In addition, the effect of caprylic acid on rGRF(1-29)NH2 affinity to GRF pituitary binding sites was determined using [125I-Tyr10]hGRF(1-44)NH2 as radioligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

The novel somatostatin analog SOM230 is a potent inhibitor of hormone release by growth hormone- and prolactin-secreting pituitary adenomas in vitro

To determine the inhibitory profile of the novel somatostatin (SRIF) analog SOM230 with broad SRIF receptor binding, we compared the in vitro effects of SOM230, octreotide (OCT), and SRIF-14 on hormone release by cultures of different types of secreting pituitary adenomas. OCT (10 nM) significantly inhibited GH release in seven of nine GH-secreting pituitary adenoma cultures (range, -26 to -73%), SOM230 (10 nM) in eight of nine cultures (range, -22 to -68%), and SRIF-14 (10 nM) in six of six cultures (range, -30 to -75%). The sst analysis showed predominant but variable levels of somatostatin receptor (sst)(2) and sst(5) mRNA expression. In one culture completely resistant to OCT, SOM230 and SRIF-14 significantly inhibited GH release in a dose-dependent manner with an IC(50) value in the low nanomolar range. In the other cultures, SOM230 showed a lower potency of GH release inhibition (IC(50), 0.5 nM), compared with OCT (IC(50), 0.02 nM) and SRIF-14 (IC(50), 0.02 nM). A positive correlation was found between sst(2) but not sst(5) mRNA levels in the adenoma cells and the inhibitory potency of OCT on GH release in vivo and in vitro, and the effects of SOM230 and SRIF-14 in vitro. In three prolactinoma cultures, 10 nM OCT weakly inhibited prolactin (PRL) release in only one (-28%), whereas 10 nM SOM230 significantly inhibited PRL release in three of three cultures (-23, -51, and -64.0%). The inhibition of PRL release by SOM230 was related to the expression level of sst(5) but not sst(2) mRNA. Several conclusions were reached. First, SOM230 has a broad profile of inhibition of tumoral pituitary hormone release in the low nanomolar range, probably mediated via both sst(2) and sst(5) receptors. The higher number of responders of GH-secreting pituitary adenoma cultures to SOM230, compared with OCT, suggest that SOM230 has the potency to increase the number of acromegalic patients which can be biochemically controlled. Second, compared with OCT, SOM230 is more potent in inhibiting PRL release by mixed GH/PRL-secreting adenoma and prolactinoma cells.     

The synergistic effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on growth hormone (GH)-releasing factor-stimulated GH release and intracellular adenosine 3',5'-monophosphate accumulation in rat primary pituitary cell culture

His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) stimulated GH release from rat primary pituitary cells in a time- and dose-dependent manner. Stimulation was observed after a 15-min, but not a 4-h, incubation. The concentrations of GHRP-6 required for half-maximal and maximal stimulation were 7 x 10(-9) and 10(-7) M, respectively. GH release induced by GHRP-6 was not affected by the addition of either naloxone or the GRF antagonist [N-Ac-Tyr1,D-Arg2]GRF-(1-29)-NH2. The latter inhibited GRF-stimulated GH release by shifting the dose-response curve to the right. His-D-Trp-D-Lys-Trp-D-Phe-Lys-NH2, an analog of GHRP-6, inhibited GH release stimulated by GHRP-6 without affecting that induced by GRF. When present together at maximal concentrations, GHRP-6 and GRF produced a synergistic effect on GH release. GHRP-6 had no effect on intracellular cAMP levels, whereas GRF increased intracellular cAMP concentrations by 3-fold. Combined treatment of pituitary cells with GRF and GHRP-6 resulted in a potentiation of the GRF-induced increase in cAMP levels. Basal GH release was reduced by 30% after pretreatment with GHRP-6 (10(-7) M) for 1 h. Pretreatment with GHRP-6 also decreased the subsequent response to GHRP-6, but not GRF. In contrast, pretreatment with GRF for 1 h had no effect on the subsequent action of GHRP-6 or GRF on GH release. The desensitization induced by GHRP-6 was completely reversed within 1 h after removal of the peptide. Results from this study indicate that GHRP-6 and GRF stimulated GH release from somatotrophs via different receptors and through discrete mechanisms.     

Development and validation of a highly sensitive radioimmunoassay for chinook salmon (Oncorhynchus tshawytscha) growth hormone

This study describes the development of a highly specific and very sensitive radioimmunoassay for salmonid growth hormone. Antiserum raised against chinook (Oncorhynchus tshawytscha) GH2, which did not recognize 125I-sPRL and 125I-sGTH (at 1:1000 initial dilution), was able to inhibit growth when injected into rainbow trout (Oncorhynchus mykiss). 125I-sGH2, used as tracer, was not recognized by anti-sGTH or by anti-sPRL. Mammalian GH and ACTH and salmonid GTH, TSH, and PRL did not cross-react in the sGH assay. The inhibition curves for pituitary extracts and plasma from salmonids were parallel to the salmon GH standard, whereas those from carp, tilapia, and catfish showed no significant cross reactivity. The RIA ED90 and ED50 values were 0.2 and 1.5 ng/ml, respectively. Using this RIA for measuring GH release by cultured pituitary cell we observed a strong inhibiting effect of SRIF (10(-6) M) and a stimulatory effect of hGRF (10(-6) M). This RIA allowed us also to detect daily fluctuations in the plasma GH concentration in cannulated rainbow trout.     

Galanin secretion from anterior pituitary cells in vitro is regulated by dopamine, somatostatin, and thyrotropin-releasing hormone.

Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. Furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 10(5) cells/well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules.     

The effects of thyroid hormone deprivation in vivo and in vitro on growth hormone (GH) responses to human pancreatic (tumor) GH-releasing factor (1-40) by dispersed rat anterior pituitary cells

Anterior pituitary cells from euthyroid and hypothyroid male rats have been cultured as monolayers for 3 days with or without 5 nM T3 and stimulated with either human pancreatic GH-releasing factor 1-40 (hpGRF), TRH, or the Ca2+ channel ionophore A23187. Basal GH secretion was reduced in the hypothyroid cultures (P less than 0.001) and basal TSH secretion increased (P less than 0.001). Culture with T3 increased GH secretion and intracellular GH content in euthyroid and hypothyroid cultures but suppressed TSH secretion with no effect on intracellular TSH content in either euthyroid or hypothyroid cultures. hpGRF released more GH from euthyroid [3.52 +/- 0.2 (SE) micrograms/6 h X 10(5) cells] than hypothyroid cultures of (0.17 +/- 0.01 micrograms/6 h X 10(5) cells, P less than 0.001) without a change in ED50 (approximately 0.02 nM). The reduction in hpGRF-induced GH release remained significant when corrected for the reduced intracellular GH content in the hypothyroid cultures. hpGRF-induced GH release also declined relative to A23187-induced GH release in hypothyroid cultures. Culture with 5 nM T3 doubled maximum hpGRF-induced GH release in euthyroid cultures and increased maximum release 10-fold in hypothyroid cultures without altering the ED50 of hpGRF action. In contrast, T3 suppressed TRH-induced TSH release in euthyroid cultures but was without effect on TRH-induced TSH release in the hypothyroid cultures. T3 did not effect the ED50 of TRH action (2-5 nM). In summary, hypothyroid rat anterior pituitary cells in culture have a reduced maximal GH response to hpGRF, but the same ED50. hpGRF activity can be partially restored by physiological concentrations of T3 in vitro.     

In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I

Using a monolayer approach, we have examined the acute (3 h) effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on GH release from pituitary cells of male and female 70-, 100-, and 130-day-old fetuses and newborn lambs and of prepubertal male lambs. GRF stimulated basal GH release in a dose-dependent (10(-12)-10(-8) M) manner at each stage in development. There was no linear relationship between maximal response and increasing age of the donor animals. The ED50 values for GRF were similar in all groups, except in the pituitaries from male and female 130-day-old fetuses, where the ED50 values were significantly higher. SRIF elicited a dose-related (10(-10)-10(-6) M) inhibition of basal GH secretion at each stage of fetal life and in the prepubertal period; although the response was lower in the youngest fetal pituitaries, there was no significant change in maximal response during the fetal or prepubertal period. No effect of SRIF on basal GH secretion was observed in newborn lambs. However, SRIF (10(-7) M) was able to block GRF (10(-8) M)-stimulated GH release in 100- and 130-day-old fetal and prepubertal as well as newborn lamb pituitary cells. Plasma IGF-I concentrations increased from 15.0 +/- 0.7 (mean +/- SE) and 13.8 +/- 0.9 ng/ml for male and female animals, respectively, at 70 days gestation to 55.8 +/- 3.2 and 51.8 +/- 11.1 ng/ml at the time of birth. The increase was much more pronounced in prepubertal lambs, especially in male animals, where IGF-I levels reached 300.8 +/- 37.7 ng/ml. IGF-I (100 ng/ml) had no effect on basal GH release in 70- and 100-day-old fetal, newborn, and prepubertal lamb pituitary cultures, but significantly inhibited basal GH secretion from 130-day-old fetal cells. This dose of IGF-I had no effect on GRF (10(-9) M)-stimulated GH release at 70 days gestation. It significantly inhibited this effect at 100 days and in prepubertal lamb cells. In 130-day-old fetal and newborn lamb pituitary cultures, IGF-I completely blocked the GH response to GRF.(ABSTRACT TRUNCATED AT 400 WORDS)     

EFFECTS OF GRF(1–40) AND DOMPERIDONE ON GH SECRETION IN NORMAL MAN

In eight normal adult men pituitary secretion following GRF(1-40) was studied. GRF administration (50 micrograms i.v.) was followed by an increase in GH release with a peak value between the 15 and 60 min. No effects were noticed on LH, FSH, PRL, TSH and ACTH secretion. GH and PRL release was also studied after domperidone (DOM) (5 mg i.v./h), and GRF plus DOM. PRL increased significantly after DOM and GRF plus DOM. During GRF plus DOM a more marked GH release was observed in comparison with the hormone response to GRF alone at 15-45 and 120 min (P less than 0.05). This phenomenon was found in in six out of eight subjects studied. Mean peak and secretory area was greater (P less than 0.05) after GRF plus DOM than after GRF alone. These data suggest that GRF(1-40) at the dose used is a useful tool in the study of GH secretion. The GH pattern during GRF plus DOM seems to indicate that dopaminergic tone may play a direct inhibitory role on GH secretion in man.     

Association of 45Ca2+ mobilization with stimulation of growth hormone (GH) release by GH-releasing factor in dispersed normal male rat pituitary cells

Dispersed normal male rat anterior pituitary cells were prelabeled with 45Ca2+ and perifused to study the influence of GH-releasing factor (GRF) on fractional calcium efflux and GH release. The cells were exposed for 2 min to 0, 0.03, 0.1, 0.3, 1.0, or 10.0 nM GRF in separate perifusion columns, and the response to each concentration was determined by integration of the area under the curve. Concentrations of 0.1 nM GRF and higher produced a simultaneous and significant stimulation of calcium efflux and GH release. The increase in calcium efflux was proportional to GRF concentration and was maximally responsive at 1 nM GRF. The value for the entire integrated response of GH release increased continuously with GRF concentration, but GH released rapidly (0-4 min) in response to GRF achieved a maximal response at 1 nM GRF and was significantly correlated with calcium efflux. Somatostatin (100 nM) abolished the stimulation of GH release and calcium efflux due to 10 nM GRF. We conclude that GRF receptor activation is intimately associated with calcium mobilization, although the relative dependence upon intracellular or extracellular calcium sources has yet to be defined. This interaction occurs at a GRF concentration about 10 times lower than that observed to cause a measurable increase in intracellular biochemical messengers such as cAMP, phosphatidylinositol, or arachidonate. We postulate that GRF-stimulated calcium mobilization is a rapid and very sensitive event contributing to GRF-stimulated GH release.     

Somatostatin desensitization in rat anterior pituitary cells

The possibility that desensitization to the inhibitory effects of somatostatin (SS) might develop following chronic exposure to this tetradecapeptide was examined in cultured rat anterior pituitary cells. Pretreatment with 1μM SS for 48 h caused a shift in the IC₅₀ of SS to inhibit 3-isobutyl-1-methylxanthine (IBMX) or growth hormone-releasing factor (GRF)-induced growth hormone (GH) and TRH + IBMX-induced thyroid-stimulating hormone (TSH) release by more than 2 orders of magnitude. Refractoriness developed after 12 h of exposure to doses of SS of 10 nM or more and became maximal at 48 h. Restoration of SS responsiveness followed a similar time-course upon removal of the pep tide. In superfused cells, 10 nM SS lowered GH secretion rates to < 5 ng/min within 2 h, but GH release began to rise after 16 h despite the continued presence of SS. However, when somatostatin was delivered in pulses, it remained fully effective for more than 36 h. Somatosvtatin-28 was also capable of inducing refractoriness in cultured pituitary cells. However, cells made refractory to either SS-14 or SS-28 were not made refractory to the same extent to the other form of somatostatin. These results indicate that the pituitary can become desensitized to the inhibitory actions of somatostatin just as it does to the stimulatory actions of the other hypothalamic releasing hormones.     

Physiological role of growth hormone (GH)-releasing factor and somatostatin in the dynamics of GH secretion in adult male rat

To investigate the physiological role of GRF and somatostatin (SRIF) in GH secretion in adult male rats, we prepared in vitro models using the perifusion system of cultured rat anterior pituitary cells exposed to various combinations of human GRF-(1-44)NH2 (hGRF) and SRIF. We studied the following three models on GRF secretion: 1) pulsatile GRF secreted at 1-h intervals, 2) pulsatile GRF secreted at 3-h intervals, and 3) GRF continuously secreted. When 5-min pulses of 20 nM GRF were delivered at 1-h intervals, the responses to GRF gradually declined. The addition of continuous 20 nM SRIF with short pauses prevented this attenuated response and produced high peaks of GH at 3-h intervals. When 5-min pulses of 20 nM GRF were delivered at 3-h intervals, three high peaks of GH were observed regardless of the addition of SRIF. Pretreatment with GRF pulses enhanced the peaks of GH during SRIF pauses. When 20 nM GRF was continuously delivered, a rapid attenuated response to GRF was observed. Although the addition of continuous 20 nM SRIF with short pauses produced three small peaks of GH, these results were caused by the post-SRIF rebound release. These observations suggest that preexposure to prolonged SRIF can prevent an attenuated response to repeated GRF pulses, and that pretreatment with GRF pulses enhances post-SRIF rebound release.     

Growth hormone (GH)-releasing factor does not regulate GH release or GH mRNA levels in GH3 cells

Previous studies have shown that GH-releasing factor (GRF) regulates both GH production and GH mRNA levels in primary cultures of rat pituitary cells. Investigations were carried out to ascertain the ability of GRF to regulate GH production or mRNA levels in a clonal strain of rat pituitary tumor (GH3) cells. Incubation of the cells with GRF at 1-1000 nM for 4 h to 10 days did not result in a stimulation of GH or PRL production, nor did it affect the cytoplasmic levels of the corresponding mRNAs. The lack of response to GRF was not affected by dexamethasone, T3, or serum. We conclude that GH3 cells do not provide a useful model system for studies of the mechanism(s) of action of GRF on either GH release or GH gene expression.     

In vitro and in vivo growth hormone responsiveness to growth hormone-releasing factor in male and female Zucker rats

In order to determine whether there is an anomaly in the pituitary responsiveness to growth hormone (GH)-releasing factor in the genetically obese rat, we examined the in vitro and in vivo effects of rGRF(1-29)NH2 (GRF) on GH release in male and female Zucker rats. The effect of increasing GRF concentrations (1.56, 6.25, 12.5 25 and 50 pM) was first tested on GH release from freshly perifused anterior pituitary cells. In both sexes, the GH response per one pituitary equivalent to each GRF concentration tested was reduced in the obese group. However, when GH release was expressed as a percent of initial cell GH content, there was no difference between the lean and the obese groups. Furthermore, under pentobarbital anesthesia, GRF was injected intravenously at two consecutive doses of 0.8 and 4.0 microgram/kg body weight in obese and lean animals. In both sexes, the GH response to each dose of GRF tested was decreased in the obese group. Basal serum GH concentrations were similar in male and female obese rats compared to their respective lean siblings. In conclusion, this study demonstrates a decrease of the in vitro and in vivo pituitary response to GRF in the obese Zucker rat, suggesting a possible secondary defect at the pituitary level.