Non-thyrotropic pituitary cells in culture convert T4 to T3.

Although intrapituitary conversion of T4 to T3 has been shown in rat pituitary homogenates, this deiodination has not been demonstrated in non-thyrotropic pituitary cells. GH3 cells, which produce growth hormone and prolactin, were used to demonstrate formation of T3 from T4 in monolayer culture. During a 1 hr incubation, the amount of T3 generated increased with increasing T4 substrate concentrations, reaching a plateau after 0.9 μM T₄. When dithiothreitol was added to the medium, production of T3 doubled at all concentrations of T4. T3 production in the presence of 2 μM T4 increased from .23 nM after 0.5 hr incubation to .8 nM after 3 hr incubation. Deiodination by this nonthyrotropic rat pituitary tumor cell is markedly enhanced by a thiol-protective agent.     

Hormonal regulation of prolactin storage in a clonal strain of rat pituitary tumor cells

GH4C1 cells (GH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. GH cells have been used to study hormone secretion, but they store relatively little prolactin compared to normal prolactin-secreting cells. They are not suitable, therefore, for studying some aspects of pituitary function. We have found that the amount of prolactin GH cells store can be regulated. When GH cells were plated at 10(6) cells/well and treated for six days with 180 nM insulin or 1 nM estradiol, there was a 60 percent increase in prolactin storage compared to control cells. Insulin and estradiol in combination acted synergistically to cause a 190 percent increase in prolactin storage. In contrast, they were additive in increasing extracellular prolactin; there was a 40 percent increase in extracellular prolactin after insulin, a 20 percent increase after estradiol, and a 50 percent increase after insulin plus estradiol. The increases in prolactin storage were always greater than the increases in extracellular prolactin. The increases in prolactin storage were dose-dependent and reached maximal levels after four days of treatment with 180 nM insulin plus 1 nM estradiol. Reducing the plating density to 10(3) cells/well increased the response to insulin and estradiol to nineteenfold. Epidermal growth factor (10 nM) acted synergistically with estradiol and insulin in combination to increase prolactin storage 27-fold. The insulin- and estradiol-induced increase in extracellular prolactin was caused by a specific increase in the rate of prolactin synthesis. The fractional increase in prolactin storage above the increase in prolactin production could not be explained by an increase in prolactin synthesis, an increase in intracellular transit time, or a change in the cell-cycle distribution of the population. Hormone storage can, therefore, be regulated independently from other processes which control hormone production. The prolactin stored in response to insulin and estradiol was releasable by potassium depolarization. Following depletion of intracellular prolactin by depolarization, the cells retained their increased capacity for prolactin storage. The ability to increase prolactin storage will make GH cells a more useful system in which to study pituitary function.     

In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish

We studied the in vitro and in vivo effects of octanoylated goldfish ghrelin peptides (gGRL-19 and gGRL-12) on luteinizing hormone (LH) and growth hormone (GH) release in goldfish. gGRL-19 and gGRL-12 at picomolar doses stimulated LH and GH release from dispersed goldfish pituitary cells in perifusion and static incubation. Incubation of pituitary cells for 2 h with 10 nM gGRL-12 and 1 or 10 nM gGRL-19 increased LH-beta mRNA expression, whereas only 10 nM gGRL-19 increased GH mRNA expression. Somatostatin-14 abolished the stimulatory effects of ghrelin on GH release from dispersed pituitary cells in perifusion and static culture. The GH secretagogue receptor antagonist d-Lys(3)-GHRP-6 inhibited the ghrelin-induced LH release, whereas no effects were found on stimulation of GH release by ghrelin. Intracerebroventricular injection of 1 ng/g body wt of gGRL-19 or intraperitoneal injection of 100 ng/g body wt of gGRL-19 increased serum LH levels at 60 min after injection, whereas significant increases in GH levels were found at 15 and 30 min after these treatments. Our results indicate that, in addition to its potent stimulatory actions on GH release, goldfish ghrelin peptides have the novel function of stimulating LH release in goldfish.     

Thyroliberin is rapidly transferred to the nucleus of GH3 pituitary cells at both 4°C and 37°C

The time-course of thyroliberin transfer to the nucleus of GH3/B6 rat pituitary prolactin cells was studied by both autoradiography and cell fractionation of intact cells exposed to [³H]thyroliberin at 4°C or 37°C. It was previously shown that thyroliberin is not degraded in these conditions. It is found by autoradiography that [³H]-thyroliberin is transferred to the nucleus of GH3/B6 cells within 5 min at least at both 37° C and 4°C. Consistent results are obtained by fractionation of cells exposed to [³H]thyroliberin at 37°C. However after binding at 4°C 50% of the cell radioactivity is extractible by glutaraldehyde and after fractionation the isolated nuclei retain only 1–1.5% of the cell radioactivity. This suggests the existence of both tightly bound and loosely bound internalized thyroliberin molecules.     

Characterization of bombesin receptors in a rat pituitary cell line

Bombesin is a tetradecapeptide which stimulates prolactin secretion in rats and man and in cultures of GH4C1 cells, a clonal strain of rat pituitary tumor cells. We have utilized [125I-Tyr4]bombesin to identify and characterize specific high affinity receptors in GH4C1 cells. Scatchard analysis of equilibrium binding data at 4 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (RT = 3600 +/- 500 sites/cell). The value for the equilibrium dissociation constant (Kd = 1.2 +/- 0.4 nM) agreed closely with the ED50 (0.5 nM) for bombesin stimulation of prolactin release. [125I-Tyr4]Bombesin binding at steady state at 37 degrees C was inhibited by increasing concentrations of unlabeled bombesin in a dose-dependent manner, with an ID50 = 1.4 +/- 0.2 nM. However, binding of [125I-Tyr4] bombesin was not inhibited by 100 nM thyrotropin-releasing hormone, vasoactive intestinal peptide, epidermal growth factor, or somatostatin. Therefore, [125I-Tyr4]bombesin binds to a receptor distinct from the receptors for other peptides which regulate hormone secretion by GH4C1 cells. The analog specificity for high affinity binding showed that the receptors for bombesin recognize the COOH-terminal octapeptide sequence in the molecule. Among five pituitary cell strains tested, two which contained saturable binding sites for [125I-Tyr4]bombesin (GH4C1 and GH3) had previously been shown to respond to bombesin with increased hormone secretion, whereas three which lacked receptors (GC, F4C1, and AtT20/D16v) were unresponsive. Therefore, the [125I-Tyr4]bombesin binding sites appear to be necessary for the biological actions of bombesin. Examination of the processing and metabolism of receptor-bound peptide demonstrated that at 4 degrees C [125I-Tyr4]bombesin binds to receptors on the surface of GH4C1 cells. At 37 degrees C, receptor-bound peptide is rapidly internalized and subsequently degraded in lysosomes. In summary, we have characterized for the first time specific, high affinity pituitary bombesin receptors which are necessary for the biological action of bombesin.     

Effects of intraventricular injection of gastrin on release of LH,prolactin, TSH and GH in conscious ovariectomized rats

Conscious ovariectomized (OVX) rats bearing a cannula implanted in the 3rd ventricle were injected with 2 μl of 0.9% NaCl containing varying doses of synthetic gastrin and plasma gonadotropin, GH and TSH levels were measured by RIA in jugular blood samples drawn through an indwelling silastic catheter. Control injections of saline iv or into the 3rd ventricle did not modify plasma hormone levels. Intraventricular injection of 1 or 5 μg gastrin produced significant suppression of plasma LH and prolactin (Prl) levels within 5 min of injection. Injection of 1 μg gastrin had no effect on plasma GH, but increasing the dose to 5 μg induced a progressive elevation, which reached peak levels at 60 min. By contrast, TSH levels were lowered by both doses of gastrin within 5 min of injection and the lowering persisted for 60 min. Intravenous injection of gastrin had no effect on plasma gonadotropin, GH and TSH, but induced an elevation in Prl levels. $\text{In}$$\text{vitro}$ incubation of hemipituitaries with gastrin failed to modify gonadotropin, GH or Prl but slightly inhibited TSH release at the highest dose of 5 μg gastrin. The results indicate that synthetic gastrin can alter pituitary hormone release in unrestrained OVX rats and implicate a hypothalamic site of action for the peptide to alter release of a gonadotropin, Prl and GH. Its effect on TSH release may be mediated both via hypothalamic neurons and by a direct action on pituitary thyrotrophs.     

Differential effects of dopamine antagonists on prolactin secretion from cultured rat pituitary cells.

Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing $\text{in vitro}$ H³-haloperidol binding in rat striatum reported by others (10). Concentration-effect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above 25nM. When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an $\text{in vitro}$ test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.     

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH₃, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH₃ on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with ¹²⁵I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available ¹²⁵I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH₃ cultured in the serum-supplemented and the defined medium.     

Intracellular location of newly synthesized growth hormone

The current model for the synthesis and secretion of protein hormones by cells of the anterior pituitary leads to a prediction that newly synthesized GH should be membrane-associated. Experiments were performed to test this prediction. Rat pituitary tumor cells (GH₃) were pulsed with ³H-leucine for 2 min, and chased with cold leucine for min. Analysis of subcellular fractions by SDS polyacrylamide gel electrophoresis and by immunoprecipitation of GH demonstrated that most (86%) of the newly synthesized GH is membrane-associated.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Control of the production of two protein hormones by rat pituitary cells in culture

Summary A clonal strain of rat pituitary tumor cells (GH₃) has continued to produce the pituitary protein hormones growth hormone (GH) and prolactin during 5 years of continued growth in monolayer culture. Studies of the effects of external stimuli have indicated that, in spite of the physical similarity of these protein hormones (each is a single polypeptide of molecular weight ≈23,000), their production is controlled by different mechanisms. Addition of hydrocortisone (HC) (3×10⁻⁶ m) to the growth medium leads, after a lag of 12 to 24 hr, to an increased relative rate (rate in experimental cells divided by rate in control cells) of GH production. The relative rate reaches a maximum of 5 to 8 at 30 to 100 hr. Stimulation by HC of GH production is observed in cells growing in either the stationary or the exponential phase of growth. Indirect estimates indicate that, in exponentially growing cells, GH represents about 2% and 14% of the total protein synthesized by control and fully stimulated cells, respectively. Maintenance of the stimulated state requires HC. HC decreases both the growth rate of GH₃ cells and their incorporation of amino acids into acid-insoluble material. At the same time that HC stimulates GH production it decreases the relative rate of prolactin production to about 0.2 to 0.3. On the other hand, addition of acid extracts of bovine hypothalamus, cerebral cortex, kidney, or liver (0.3 to 1.0 mg of protein per ml) to the medium leads to an increase of the relative rate of prolactin production to 6 to 9, while decreasing the relative rate of GH production to about 0.5. Chromatographic fractionation of simple extracts of bovine liver has yielded a macromolecular, heatlabile fraction exhibiting these effects at a concentration as low as 20 μg per ml. GH₃ cells which have been adapted to growth in suspension culture produce both GH and prolactin. HC is observed to stimulate GH production and suppress prolactin production by cells growing in this state, without affecting the growth rate of the cells. This investigation was supported in part by Research Grant AM 11011 from the National Institute of Arthritis and Metabolic Diseases.     

The calcium agonist Bay K 8644 stimulates secretion from a pituitary cell line

The dihydropyridine calcium agonist Bay K 8644 acts in a dose-dependent manner to increase prolactin secretion from the GH₄C₁ pituitary cell line. Enhanced secretion was observed at agonist concentrations as low as 10 nM. In the continued presence of Bay K 8644 secretion remained elevated for at least 30 min. The effect of the agonist was Ca²⁺-dependent and competitively antagonized by dihydropyridine antagonists. Apparently Bay K 8644 acts at the dihydropyridine binding site associated with GH₄C₁ Ca²⁺ channels to enhance Ca²⁺ influx and stimulate secretion from these cells. This is the first report demonstrating that the newly discovered Ca²⁺ agonist can, by itself, stimulate secretion from a cell.     

Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis

Melanin-concentrating hormone (MCH), a 19-amino acid orexigenic (appetite-stimulating) hypothalamic peptide, is an important regulator of energy homeostasis. It is cleaved from its precursor prepro-MCH (ppMCH) along with several other neuropeptides whose roles are not fully defined. Because pituitary hormones such as growth hormone (GH), ACTH, and thyroid-stimulating hormone affect body weight and composition, appetite, insulin sensitivity, and lipoprotein metabolism, we investigated whether MCH exerts direct effects on the human pituitary to regulate energy balance using dispersed human fetal pituitaries (21-22 wk gestation) and cultured GH-secreting adenomas. We found that MCH receptor-1 (MCH-R1), but not MCH receptor-2, is expressed in both normal (fetal and adult) human pituitary tissues and in GH cell adenomas. MCH (10 nM) stimulated GH release from human fetal pituitary cultures by up to 62% during a 4-h incubation (P < 0.05). Interestingly, neuropeptide EI (10 nM), which is also cleaved from ppMCH, increased human GH secretion by up to 124% in fetal pituitaries. A milder, albeit significant, induction of GH secretion by MCH (20%) was seen in cultured GH-secreting pituitary adenomas. A comparable stimulation of GH secretion was seen when cultured mouse pituitary cells were treated with MCH. Treatment of cultured GH adenoma cells with MCH (100 nM) induced extracellular signal-regulated kinases 1 and 2 phosphorylation, suggesting activation of MCH-R1. In aggregate, these data suggest that MCH may regulate pituitary GH secretion and imply a potential cross-talk mechanism between appetite-regulating neuropeptides and pituitary hormones.     

Somatostatin prevents the desensitizing action of growth hormone-releasing factor on growth hormone release

We have investigated the effect of prior exposure to somatostatin (SRIF) alone or in combination with growth hormone-releasing factor (GRF) on the subsequent cyclic AMP and GH responses to GRF in rat anterior pituitary cells in primary culture. The maximal 4.5-fold stimulation of GH release induced by a 3-hr incubation with GRF is reduced by 60% following a prior 3-hr exposure to 30 nM GRF. A 3-hr preincubation with GRF in the presence of 30 nM SRIF doubles spontaneous GH release while the maximal amount of GH released during a subsequent 3-hr exposure to GRF is similar to that measured in cells pretreated with control medium, thus completely preventing the loss of GH responsiveness induced by prior exposure to GRF. The prevention by SRIF of the desensitizing action of GRF on GH release is not observed on the cyclic AMP response which remains almost completely inhibited in GRF-pretreated cells. Similar protective effects are obtained when SRIF is incubated with prostaglandin E2 (PGE2), thus completely preventing the desensitizing action of PGE2 on GH release. Prior treatment with pertussis toxin completely prevents the protective action of SRIF on GH responsiveness. Pretreatment with GRF + SRIF increases by 85 and 60% the maximal amount of GH release induced by cholera toxin and 8-bromoadenosine 3',5'-monophosphate, respectively. The post-SRIF rebound effect on GH release occurs mainly during the first 30 min following withdrawal of the tetradecapeptide. The present data demonstrate that simultaneous preincubation with SRIF and GRF prevents the marked inhibition of GH release during subsequent exposure to GRF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Denovo synthesis of prolactin by human decidua

Relatively large amounts of immunoreactive prolactin were measured in homogenates of human decidual tissue obtained immediately after delivery of normal term pregnancies. In order to study the release and possible synthesis of prolactin by this tissue, explants of decidua were incubated for 24 hours at 37°C in oxygenated Gey's buffer containing 20% fetal calf serum. When cycloheximide was added to the medium in concentrations sufficient to prevent $\text{in}$$\text{vitro}$ protein synthesis, 85–90% of the prolactin present in the tissue was released into the medium during the first 3 hours of incubation. No additional prolactin accumulated in either the medium or the tissue during the remainder of the incubation period. In the absence of cycloheximide, the prolactin concentration in the medium increased progressively during incubation, so that after 24 hours the total amount of hormone present in the tissue and medium was significantly greater than that in the tissue and medium prior to incubation (37.6 ± 9.6 ng/ml at 0 time vs 82.2 ± 7.7 ng/ml at 24 hours). When ³H-1-leucine (100 u Ci) was supplied during incubation, radioactive proteins were detected in the medium at 24 hr, 14–20% of which were specifically precipitated by antiserum to human pituitary prolactin. When aliquots of this medium were chromatographed on Sephadex G-100, 80–95% of the ³H-proteins precipitated by antiserum to pituitary prolactin eluted in the same position as did purified, iodinated pituitary prolactin. These data indicate that a species of prolactin which is identical to pituitary prolactin by the criteria of immunoprecipitation and gel chromatography is synthesized by human decidual tissue $\text{in}$$\text{vitro}$.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.     

Effect of central injection of bradykinin and bradykinin potentiating factor upon release of anterior pituitary hormones in ovariectomized female rats

The effects of third ventricular (IVT) injection of 25 μg of bradykinin (BK) upon plasma levels of LH, FSH, TSH, GH and prolactin were investigated in conscious ovariectomized female rats bearing indwelling jugular cannulae. Some animals were pretreated with bradykinin potentiating factor (BPF). Intravenous administration of BK had no effect upon hormone levels. IVT injection of BK significantly depressed plasma prolactin levels at 15 and 30 min post-drug, with levels returning to control values by 60 min. Pretreatment of animals with BPF (75 μg/3 μl) prolonged the prolactin suppression induced by BK for up to two hours. Plasma LH, FSH, TSH and GH levels in BK-rats were not significantly different from those of saline-injected animals at any time point measured. Neither BPF alone nor in conjunction with BK had any effects upon plasma levels of TSH; however, BK plus BPF suppressed FSH concentrations at 75 min post-BPF, while BPF alone appeared to increase GH levels at 45 min. In vitro incubation of hemipituitaries with 0.083, 0.83 or 8.33 μg/ml BK had no effect upon the release of LH, TSH or prolactin compared to control values. However, the secretion of GH and FSH was suppressed by the lowest dose of BK tested. These results suggest that BK may play a physiological inhibitory role in the regulation of prolactin, which can be augmented by preventing its degradation, i.e. via BPF. The effect of the peptide seems to be mediated by the CNS since neither intravenous injection of BK nor in vitro incubation of pituitaries with the peptide modified prolactin release.     

Effects of osmotic pressure on prolactin and growth hormone secretion from organ-cultured eel pituitary

Summary Effects of medium osmotic pressure on the release of prolactin (PRL) and growth hormone (GH) from the pituitary of the Japanese eel, Anguilla japonica, were examined during long-term organ culture in a defined medium. Prolactin and GH release, as measured by homologous radioimmunoassays, increased gradually for 7 days during incubation in isosmotic medium (295 mOsmolal). On day 7, 3 to 5 times more PRL and GH were released than on day 1. The amount of GH released was about 100 times greater than that of PRL. Electron microscopic observation revealed that both PRL and GH cells were in good condition after 7 days incubation. The reduction of medium osmotic pressure from 295 (isosmotic) to 235 or 260 mOsmolal significantly stimulated PRL release for 4 days. By contrast, an increase in medium osmolality from 295 to 360 mOsmolal was without effect. These treatments produced no significant alterations in GH release. The stimulatory effect of hyposmotic medium (235 mOsmolal) was no longer evident by 12 h after the pituitaries were returned to isosmotic medium. The isosmotic but low-sodium medium, prepared by adding mannitol to the hyposmotic medium, did not stimulate PRL release from the pituitary. These results indicate that plasma osmolality may be an important physiological factor controlling PRL release during freshwater adaptation of the eel.Abbreviations GH growth hormone - OAPBS PBS with 1% ovalbumin - PAGE polyacrylamide gel electrophoresis - PBS phosphatebuffered saline - PRL prolactin - rER rough endoplasmic reticulum     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.