Growth hormone and prolactin secretion in cultured somatomammotroph cells

A somatomammotropic cell line (P0) derived from adult rat pituitaries has been maintained in culture for 2 yr. Secretion of GH and PRL by this cell line has been studied in response to hypophysiotropic peptides known to affect the release of both hormones as well as agents that affect second messenger systems in an attempt to characterize the stimulus-secretion mechanisms used by these cells. GH and PRL release during short term (4 h) incubations of P0 cells and primary cultures of dispersed rat pituitary cells was initially measured in response to GRF, TRH, vasoactive intestinal peptide (VIP), and SRIF. In P0 cells, the minimal effective dose of each of the hypophysiotropic peptides was comparable with respect to GH and PRL secretion. The effects of TRH and VIP were similar to those in freshly dispersed cells with respect to PRL release, whereas those of GRF and SRIF were less potent with respect to GH release. The stimulation of GH and PRL release in P0 cells by adenylate cyclase-related agents ((Bu)2 cAMP and forskolin) was comparable to that for GH secretion in mature somatotrophs but much greater than that of PRL release in mature lactotrophs. Stimulation of GH and PRL release in P0 cells by protein kinase C-related agents (diacylglycerol and phorbol ester) was also similar to that observed for GH release from mature pituitary cells, whereas minimal or undetectable effects were observed on PRL release from mature cells. The results indicate that the P0 somatomammotropic cell line possesses receptors, second messenger systems, and secretory characteristics of both somatotrophs and lactotrophs, although where differences exist, there is more resemblance to somatotrophs. They also demonstrate that the responses to each of the agents studied are bihormonal and appear to be regulated by a common mechanism.     

Adenosine inhibits prolactin and growth hormone secretion in a clonal pituitary cell line

Although purine nucleosides have been shown to regulate the secretion of several peptide and steroid hormones, effects on pituitary hormone release have not been reported. We show here that in the clonal GH4C1 pituitary cell line maximal concentrations of adenosine (greater than or equal to 50 microM) inhibited PRL and GH secretion by 40%. Adenosine deaminase abolished the inhibitory effect of adenosine but not that of SRIF or (-)N6(R-2-phenylisopropyl)adenosine (PIA), a nonhydrolyzable adenosine analog. Furthermore, this enzyme increased basal secretion by 50%, and analysis of the incubation medium by HPLC demonstrated that the cells secreted biologically effective concentrations of adenosine. These results indicate that adenosine produced in culture tonically inhibits hormone release. In other target cells, adenosine inhibition is mediated by two types of binding sites: an extracellular Ri-site requiring an intact ribose moiety or an intracellular P-site requiring an intact purine ring. Four lines of evidence indicate that in GH4C1 cells, adenosine acts at an Ri-site. PIA, an Ri-site-specific agonist, was a potent inhibitor of hormone release (ED50 = 30 nM). Theophylline, an Ri-site antagonist, competitively inhibited the action of PIA (Ki = 2.4 microM). 3) 2'5'-Dideoxyadenosine, a P-site-specific agonist, did not inhibit PRL release even at a concentration of 1 mM. 4) Dipyridamole, an adenosine uptake inhibitor, did not reduce adenosine inhibition. In addition to its effect on basal secretion, PIA inhibited stimulation of hormone release by vasoactive intestinal peptide and TRH. PIA also reduced vasoactive intestinal peptide-stimulated cAMP accumulation by 75%, consistent with its action to inhibit adenylate cyclase via Ri receptors in other targets. Since PIA inhibition of PRL release and cAMP accumulation was not additive with the effects of SRIF and carbamyl choline, these inhibitors may act via a common rate-limiting step. Our results demonstrate that adenosine activates an Ri-type of adenosine receptor in GH4C1 cells and that the production of adenosine under normal culture conditions causes autocrine inhibition of secretion.     

Growth hormone secretion by cultured rat anterior pituitary cells. Effects of culture conditions and dexamethasone

Optimal conditions were sought for the study of GH secretion by cultured normal pituitary cells. Dispersed rat pituitary cells were cultured for 1 week in four different media supplemented with 10% fetal calf serum. Minimal essential medium resulted in high GH content and secretion during a 4-h incubation period, whereas GH secretion was lower (P less than 0.05) for cells cultured in medium 199, Ham's F-10, and RPMI-1640. GH secretion/24 h declined gradually with time. After 2 weeks in culture hormone secretion amounted to 30% of secretion on day 1, but after 3 weeks GH secretion was still measurable. GH recovery during the 3-weeks culture period was more than 600% of the amount initially plated. GH secretion was positively correlated with the bicarbonate concentration between 0.85 and 2.2 g/liter NaHCO3. When pituitary cells were cultured in concentrations varying from 0.5 X 10(5) to 10 X 10(5) cells per dish, GH secretion and content per cell were constant, suggesting that no direct autofeedback occurred in cultures with high cell densities and thus high medium GH. Dexamethasone stimulated GH secretion and content in a dose-dependent way (0.1 nM-10 microM). The stimulatory effect of 100 nM dexamethasone occurred within 24-48 h. After 7 days of treatment with 100 nM dexamethasone, GH secretion had increased to 190% and GH content to 230% of control. In contrast to the effects on GH, dexamethasone suppressed PRL secretion in a dose-dependent way, but this effect was seen only after 7 days of treatment and not after 4 days of treatment. Cycloheximide and actinomycin D prevented the stimulatory effect of dexamethasone on GH secretion. However, 24 h after cessation of cycloheximide treatment GH secretion was stimulated by dexamethasone. Four days of treatment with 100 nM dexamethasone did not affect the GH response to somatostatin, prostaglandin E1, and theophylline, nor the PRL response to dopamine, TRH, and theophylline. Thus, culture conditions may affect GH production, and dexamethasone can be used to culture somatotrophs for longer periods with steady GH production and normal responsiveness.     

Growth hormone secretion during long-term thiopental anesthesia in the adult male rat

To study the secretory pattern of growth hormone (GH) in the adult male rat in a continuous sleep state, the GH concentration was measured in blood sampled at 10-min intervals through an intracardiac cannula for 9-10 h long-term anesthesia with thiopental sodium. The cortical EEG was monitored to maintain stable EEG sleep throughout the sampling period. In 41 animals subjected to anesthesia, 32 showed an apparent fluctuation in GH levels. The analysis of the time series of the GH concentration in blood by the power spectrum and least-squares method revealed that 27 animals had periodicities within the mean +/- 2SD range obtained for the unanesthetized, but similarly sampled, animals from 2.40 to 3.85 h. The mean (+/- SE) of the periodicities for 27 animals was 3.01 +/- 0.06 h, similar to that obtained for the unanesthetized animals, approximately 3.0 h. The results indicate that, although the GH-controlling mechanism in the rat is facilitated under sleep, it has a 3.0-hour period ultradian rhythm mechanism that is independent from that for sleep.     

Growth hormone and prolactin secretion by dispersed cell cultures of a normal human pituitary: effects of thyrotrophin releasing hormone, theophylline, somatostatin, and 2-bromo-alpha-ergocryptine

Growth hormone (GH) and prolactin (Prl) secretion by a normal human pituitary in dispersed cell culture has been investigated. Prl secretion was significantly stimulated after 0.5, 1, 2 and 4 h exposure to 1, 10, 100 and 1000 ng/ml thyrotrophin releasing hormone (TRH). Maximal effects were obtained with 10 ng/ml TRH at 2 h, higher doses being less effective. GH secretion was unchanged with the exception that 1 ng/ml TRH produced a small decrease at 4 h. GH and Prl secretion was significantly inhibited by incubation with 0.01, 0.1, 1 or 10 micrograms/ml 2-bromo-alpha-ergocryptine (bromocriptine). The inhibition persisted for a further 24 h after removal of bromocriptine. Theophylline (10(-2) M) significantly increased GH and Prl secretion during a 4 h incubation and this effect was blocked by co-incubation with 10 ng/ml somatostatin (SRIF). SRIF also inhibited basal GH and Prl secretion during 4 h and removal of SRIF and incubation for at further 4 h led to a rebound in GH and Prl secretion to levels greater than control. It is concluded that cell culture techniques previously applied to the study of hormone secretion by pituitary adenomas can be equally applied to the normal human pituitary.     

Interferon-gamma inhibits stimulated adrenocorticotropin, prolactin, and growth hormone secretion in normal rat anterior pituitary cell cultures

Preincubation of rat anterior pituitary (AP) cells with homologous interferon-gamma (IFN-gamma) caused a dose-dependent inhibition of ACTH secretion stimulated by CRF. The effect was seen in both monolayer and aggregate AP cell cultures and was not due to cytotoxicity. In monolayer cultures IFN-gamma also inhibited PRL and GH release stimulated by various hypothalamic releasing factors. IFN-gamma did not affect the time kinetics of the ACTH response to CRF. The dose needed for half-maximal inhibition amounted to approximately 1 (antiviral) U/ml. The effect of IFN-gamma was abrogated by an IFN-gamma-neutralizing monoclonal antibody. Furthermore, ACTH secretion by the AP cells was not affected by the anti-IFN-gamma antibody added alone, indicating that in the culture system no endogenous IFN-gamma is operational in regulating the ACTH response studied. Of the other cytokines tested [interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-alpha/beta (IFN-alpha/beta)] only TNF-alpha and IL-6 were found to inhibit CRF-stimulated ACTH release, although this inhibition was less pronounced than that caused by IFN-gamma. Lipopolysaccharide, even at high doses, did not significantly inhibit the ACTH response to CRF. These results identify IFN-gamma as one of the inflammatory cytokines that, like IL-1, TNF-alpha, and IL-6, have the potential to regulate pituitary function.     

Effect of therapeutic doses of ionising radiation on the somatomammotroph pituitary cell line, GH3

Ionising radiation is used for the treatment of pituitary tumours as fractionated radiotherapy, where the total dose reaching the tumour area is in the range of 40-50 Gy, or during stereotactic radiosurgery, where the total dose reaching the tumour area during one session is in the range of 20-90 Gy. In this study, we investigated the effect of ionising radiation of (60)Co (dose rate of 3 Gy/min, similar to that used during gamma knife procedure) on the mode of cell death of the somatomammotroph pituitary cell line, GH3, an immortalized cell line derived from a rat pituitary adenoma. We found that the basic mechanism of cell death induced by irradiation of this GH3 cell line by gamma-rays was programmed cell death-apoptosis. Doses of 20-50 Gy were shown to inhibit proliferation in these cells. 24 hours after irradiation with a dose of 20 and 50 Gy, cells were shown to accumulate in the G(2)/M phase of cell cycle. This cell cycle arrest lasted for at least ten days. Apoptosis was detected 72 hours towards until the end of the study (10 days). However, a significant number of cells were still alive ten days following irradiation. We conclude that ionising radiation doses of 20 and 50 Gy induce pituitary GH3 cell apoptosis following cell cycle arrest in the G(2)/M phase.     

Growth hormone-releasing factor-induced growth hormone secretion from perifused rat anterior pituitary cells: lack of influence of glucose concentration, and normal responses in pituitary cells from diabetic animals

The mechanism responsible for the suppression of GH secretion in hyperglycaemia and hypoglycaemia in rats has been investigated using perifusion of anterior pituitary cells. When perifused with Krebs-Ringer bicarbonate containing normal (5 mmol/l), high (20 mmol/l) and low (1 mmol/l) concentrations of glucose, the GH responses to GH-releasing factor (GRF) were 85 +/- 5, 85.5 +/- 5.4 and 89 +/- 3.0 (S.E.M.)% respectively compared with the initial response to GRF at 5 mmol/l in each column. The mean GH response to GRF from anterior pituitary cells of normal rats was 6.58 +/- 0.88 micrograms/three pituitaries, which was not statistically different form that of cells from rats with streptozotocin-induced diabetes (5.40 +/- 0.68 micrograms/three pituitaries). It is concluded that GH suppression in diabetic rats and during hypoglycaemia is not mediated by changes in the GH response to GRF.     

Caffeine stimulates growth hormone secretion by cultured rat pituitary cells

To study the effect of caffeine on growth hormone secretion a culture system of dispersed rat anterior pituitary cells was employed. The cells were incubated overnight in medium 199 containing 10(-5) to 10(-1) M caffeine. The medium was then collected and assayed for rat growth hormone content. A dose dependent stimulatory effect of caffeine on growth hormone secretion into the culture medium was observed. It is concluded that caffeine, like other xanthine phosphodiesterase inhibitors stimulates growth hormone secretion by a direct effect on pituitary cells.     

Impaired generation of cyclic adenosine 3',5'-monophosphate in a somatomammotroph cell line derived from dwarf (dw) rat anterior pituitaries.

The dwarf (dw) mutation in rats results in 40-50% growth retardation associated with a selective reduction in pituitary somatotroph number, GH content, and GH mRNA levels and a decreased GH secretory response to GH-releasing factor (GRF). Recent studies in freshly dispersed pituitary cells have provided evidence for a defect in adenylate cyclase-linked GRF signal transduction in dw somatotrophs. To further examine this defect in a more specific cell population, we developed a somatomammotroph cell line (DP) derived from anterior pituitaries of male dw rats. A similar cell line from normal rats (Po) was used as control. We studied acute GH (4-h release) and cAMP (30-min intracellular accumulation) responses to GH secretagogues known to interact with the adenylate cyclase system. Basal GH release in both cell lines was 80-130% of the cell content, thus limiting the capacity for further GH responses. GRF (10(-8) M) produced a doubling of cAMP levels in Po and DP cells (P less than 0.01), but inconsistent effects on GH release. (Bu)2cAMP (5 x 10(-3) M) increased GH secretion by 50-100% in both groups (P less than 0.01). Cholera toxin (10(-9) M) increased GH release by 50% in both Po and DP (P less than 0.01), but the cAMP response in DP cells was only half that in Po cells (P less than 0.01). Forskolin (10(-5) M), a direct stimulator of adenylate cyclase, doubled GH release in both groups (P less than 0.01). However, cAMP generation was impaired in DP, with a maximal response to forskolin less than one third that in Po (P less than 0.01). In somatotrophs, cAMP mediates not only GRF-stimulated GH release, but also GH synthesis and mitogenesis. The impairment in maximal cAMP generation in DP cells, while not affecting acute GH release, may underlie the defect in somatotroph cell number and GH content in the dw pituitary gland.     

Bile acid synthesis by the HuH-7 cell line derived from a human hepatocellular carcinoma

Bile acids in conditioned media used for culture of the HuH-7 cell line, derived from a human hepatocellular carcinoma, were analyzed by gas chromatography and gas chromatography/mass spectrometry to ascertain the presence or absence of any abnormalities in bile acid metabolism. Cholic and chenodeoxycholic acids, the primary bile acids of humans, were found in the conditioned media, and more than half of the bile acids were conjugated with glycine but not with taurine or sulfuric acid. In addition, 3β-hydroxy-5-cholenoic, 3α,7α,12α-trihydroxy-5β-cholestanoic and 3α,7α-dihydroxy-5β-cholestanoic acids were found in the media, and most of the 3β- hydroxy-5-cholenoic acid was in a sulfated form, while all of the cholestanoic acids were unconjugated. Since the cells had been cultured in serum-free media, all these bile acids had to have been newly synthesized and conjugated. In this study, 3β-hydroxy-5-cholenoic acid, a presumed intermediate for the alternative pathway of chenodeoxycholic acid synthesis, was the major bile acid synthesized by the HuH-7 cells. These results suggest that HuH-7 cells may employ some unusual and unique metabolic pathways for the production of bile acid.     

Activin-A modulates growth hormone secretion from cultures of rat anterior pituitary cells

Activins, initially identified as FSH-releasing proteins, have now been shown to exert effects on other cell types of the anterior pituitary, including the somatotrophs. In the present study the inhibitory action of activin-A (beta A beta A) on GH secretion was characterized using primary cultures of rat anterior pituitary cells. Activin-A suppressed basal GH secretion for up to 72 h (the longest time tested). Immediately after the treatment period with activin-A, when the cells were thoroughly washed and further incubated with or without rat GH-releasing factor (rGRF), basal and stimulated GH secretion were partially inhibited as well. In parallel, activin-A pretreatment diminished rGRF-stimulated cAMP accumulation. The effects of activin-A were time- and concentration-dependent, with half-maximal inhibition occurring in the range of 20-30 pM activin-A. A minimum pretreatment time of 3 h was required for maximal effect, and when rGRF and activin-A were added simultaneously, no inhibition was evident. Secretory responses of activin-A-pretreated cells to rGRF were influenced by glucocorticoids. When cells were cultured in the presence of the synthetic glucocorticoid dexamethasone, pretreatment (72 h) with activin-A attenuated rGRF-stimulated GH secretion only during short (1-h), but not longer (3-h), exposure periods to the neuropeptide. In the absence of dexamethasone, rGRF-stimulated GH secretion was inhibited at all incubation times tested (up to 3 h). A 3-h exposure to the protein factor did not alter total (cellular plus secreted) immunoreactive GH levels, suggesting that the inhibition of secretion with the shorter treatment was not secondary to attenuated GH biosynthesis. However, longer (72-h) treatment with activin-A decreased total GH levels, indicating lower GH biosynthetic rates, as previously shown. Somatostatin is recognized as the primary negative modulator of GH secretion. Activin-A and SRIF inhibited GH secretion additively, suggesting distinct mechanisms of action for each. GH secretion in response to other secretagogues, such as 12-O-tetradecanoyl-phorbol-13-acetate, forskolin, cholera toxin, and 8-bromo-cAMP, was also suppressed after activin-A pretreatment. The presence of the RNA synthesis inhibitor actinomycin-D completely blocked the inhibitory effect of a 3-h activin-A pretreatment on subsequent rGRF-stimulated GH secretion. Pertussis toxin was only partially effective in preventing the inhibition by activin-A. The results of this study indicate that activin-A plays a crucial role as a modulator of somatotropic function, inhibiting GH secretion at the level of the secretory process and secondary to the inhibition of GH biosynthesis.     

Growth hormone secretion by individual somatotropes of the testicular feminized rat

To investigate the cellular mechanisms underlying the unique GH secretory apparatus of the androgen-resistant testicular feminized (Tfm) rat we employed a reverse hemolytic plaque assay to assess GH secretion by individual cells from normal male, normal female, and Tfm rats. Acutely dispersed pituitary cells were incubated for 90 min with GH anti-serum in the presence of medium alone, 0.01, 0.1, 1, 10, or 100 nM GHRH, or 3 microM forskolin after which hemolytic plaques were developed over an additional 30 min. Body weights of the Tfm rats [318 +/- 7 g (mean +/- SEM)] were intermediate between intact males (372 +/- 18 g) and females (218 +/- 7 g). The total number of cells recovered from dispersion of Tfm rat pituitaries [3.20 +/- 0.42 X 10(6) (mean +/- SEM)] was greater than that from males (1.43 +/- 0.12 X 10(6); P = 0.001), but not distinguishable from that from females (2.31 +/- 0.30 X 10(6); P = 0.06). However, the absolute population of recovered somatotropes from the Tfm animals (1.24 +/- 0.22 X 10(6) exceeded both male (0.56 +/- 0.10 X 10(6); P = 0.002) and female (0.80 +/- 0.14 X 10(6); P = 0.046) values. Mean basal and maximal GH plaque areas were greater for cells from male rats than for those from either female or Tfm rats (P less than 0.05) regardless of whether GHRH or forskolin was used as the secretagogue. Plaque areas from female and Tfm cells were indistinguishable under all study conditions. These data suggest that a deficiency of androgen receptors prevents establishment of the greater GH secretory capacity of individual somatotropes characteristic of the adult male rat. This androgen receptor-dependent modulation of GH secretory capacity appears to occur at a step distal to the GHRH receptor. The data also suggest that an increase in the absolute population of somatotropes is an additional consequence of androgen receptor deficiency. This combination of individual somatotropes, each possessing a GH secretory capacity similar to that of cells from normal females, but present in greater absolute numbers, may explain the intermediate values found during previous studies of the Tfm rat GH axis which were based on assessment of large mixed populations of pituitary cells.     

Pyridoxal phosphate inhibits pituitary cell proliferation and hormone secretion

Pyridoxal phosphate (PLP), a bioactive form of pyridoxine, dose-dependently (10-1000 microm) inhibited cell proliferation in rat pituitary MMQ and GH3 cells and in mouse AtT-20 cells. After 4 d, MMQ cell numbers were reduced by up to 81%, GH3 cell numbers were reduced by up to 64% (P < 0.05), and AtT-20 cell numbers were reduced by up to 90%. Cell proliferation rates recovered and dose-dependently reverted to control levels after PLP withdrawal. After 4 d, PLP (400 and 1000 microm) decreased [3H]thymidine incorporation by up to 71% (P < 0.05). PLP (400-1000 microm) reduced GH3 cell GH and prolactin secretion and AtT-20 cell ACTH secretion (adjusted for cell number) by approximately 70% after 2 d. The 100 microm PLP also inhibited prolactin secretion (65%, P < 0.05) in primary rat pituitary cells treated for 2 d. PLP decreased the percentage of AtT-20 and GH3 cells in S phase and increased those in G0-G1 phase. Furthermore, PLP induced AtT-20 and GH3 cell apoptosis (28 vs. 6, P < 0.05; 26 vs. 3, P < 0.05, respectively) and dose-dependently reduced content of the antiapoptosis gene Bcl-2. These results indicate that pharmacological doses of PLP inhibit pituitary cell proliferation and hormone secretion, in part mediated through PLP-induced cell-cycle arrest and apoptosis. Pyridoxine may therefore be appropriate for testing as a relatively safe drug for adjuvant treatment of hormone-secreting pituitary adenomas.     

Induction (or stimulation) of prolactin and growth hormone production in a rat pituitary tumor cell line by bromodeoxyuridine

Under basal conditions, a rat pituitary tumor cell line (C8 11RAP) does not secrete any detectable PrL, FSH, and LH, and secretes only minute amounts of GH (27.1 +/- 0.5 ng/10(6) cells.24 h), as evaluated by RIA. Bromodeoxyuridine (BrdUrd) added to the culture medium induced the accumulation of PRL into cells and medium, increased that of GH, but did not induce that of LH or FSH. The amount of radioimmunoassayable PRL and GH accumulated in the medium increased after a lag period of 15 days and was drug concentration dependent. Maximal accumulation was 232.9 +/- 36.8 and 493.6 +/- 41.5 ng/10(6) cells.24 h for PRL and GH, respectively, at 50 micrograms/ml BrdUrd. In the presence of BrdUrd (greater than or equal to 20 micrograms/ml), the cells grew more slowly and were more strongly attached to the flasks. All of the effects induced by BrdUrd were reversible. PRL and GH were characterized by three methods; 1) radiocompetition with increasing dilution of samples; 2) Sephadex chromatography, followed by RIAs; and 3) sodium dodecyl sulfate-polyacrylamide gel electrophoresis done on the immunoprecipitate of the proteins secreted by cells incubated with [3H]leucine. Chronic treatment with TRH (3 X 10(-6) M) of cells grown without BrdUrd was unable to increase the production of GH or to induce that of PRL. On the other hand, after the same treatment of cells cultured in the presence of BrdUrd, the amounts of PRL accumulated in the culture medium or cells were increased 2- to 7-fold over unstimulated levels; under the same conditions, GH accumulation in the medium was also increased, but this augmentation was less than that of PRL. These results indicate that BrdUrd simultaneously induces or stimulates the production of PRL and GH in C8 11RAP cells, and that TRH increases the production of both hormones only in BrdUrd-treated cells.     

Activin-A modulates gonadotropin-releasing hormone secretion from a gonadotropin-releasing hormone-secreting neuronal cell line.

The recent development of GnRH-secreting neuronal cell lines (GT1-1, GT1-3 and GT1-7 clones) has provided a model system for the study of the neural regulation of GnRH expression and secretion. We report here that activin-A stimulates GnRH secretion by GT1-7 cells in a dose-dependent manner, with an EC50 of approximately 2.5 ng/ml. The maximal response (50% stimulation) was achieved after 2 days of incubation with 20 ng/ml activin-A. Activin-A treatment increased total GnRH (secreted + cellular) in GT1-7 cells, possibly reflecting a stimulation of GnRH biosynthetic rates. The secretory effect of activin-A was also accompanied by a change in the cellular morphology to a more neuronal phenotype. The addition of TGF-beta (10 ng/ml), which is structurally related to activins, did not significantly increase secretion of GnRH by GT1-7 cells illustrating the specificity of the activin effect on this cell line. Although inhibin (20 ng/ml) alone did not directly affect the spontaneous secretion of GnRH, it was able to partially block the stimulatory effect of activin. The present study with the GT1-7 clonal cell line suggests that activin, and perhaps inhibin, might act at hypothalamic sites to regulate reproduction through the control of GnRH production and/or secretion.     

Growth hormone regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone

GH is first detectable in the fetal rat pituitary between gestational days 18 and 19. The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear. We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function. In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day. These cells were stimulated by GH-releasing hormone (GHRH), but not by T3 or the morphogen retinoic acid. The stimulatory effect of T3 (0.25 mM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures. GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-day-old neonatal rats. The presence or absence of T3 in the culture medium did not alter the response to GHRH. In contrast, only 2-fold induction of GH was observed in adult male pituitary cells during the same time course. Insulin-like growth factor-I (IGF-I; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses. IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions. The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted. Addition of T3 partially restored the responsiveness of these cells to IGF-I. The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary. Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance.