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The interaction between TRH and GH3 pituitary tumor cells was studied in monolayer cultures or membrane-containing fractions. In intact cells, the apparent dissociation constant (Kd) was approximately 10 nM, and the total number of binding sites was approximately 1.4 pmol/mg protein at temperatures of 0-37 C. In broken cell preparations, the number of sites occupied at saturating TRH concentrations was reduced by half when the temperature was increased from 0 to 30 C. Linear Scatchard plots were obtained under all conditions. The rate of dissociation of TRH was temperature dependent, and first order plots were nonlinear. The half-times for dissociation at 0 C were over 240 min in cells and membranes, while at 37 C, the half-time values were 24 min (cells) and less than 0.5 min (membranes). Identical dissociation kinetics were obtained by dilution alone or dilution with excess unlabeled hormone. When cultures which had been incubated with TRH were lightly fixed with glutaraldehyde, dissociation at 37 C became immeasurably slow. However, TRH dissociated immediately when sodium dodecyl sulfate, ethanol, or acetone was added, indicating that the tripeptide was not covalently coupled to cell proteins. The data indicate that binding of TRH to high affinity GH3 receptors is not cooperative, and that the majority of TRH bound after short incubations is dissociable.

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.

The role of osmotic pressure in the exocytosis of prolactin from rat pituitary tumor (GH) cells in culture was investigated. Reducing the osmotic strength of the medium from 300 mosm to 150 mosm by removal of NaCl did not alter basal secretion of prolactin but inhibited secretion stimulated by thyrotropin-releasing hormone (TRH) and forskolin. Both basal and stimulated secretion of prolactin were inhibited by increasing the osmotic strength of the medium with NaCl (IC50 at approximately 500 mosm). The stimulated release of hormone from GH-cells was independent of sodium and unaffected by replacement of sodium ion with tetramethylammonium or choline, or by addition of 500 nM tetrodotoxin. Secretagogue-stimulated release was, however, dependent upon chloride. Exchange of medium chloride with benzoate or isethionate significantly inhibited the stimulated release of prolactin (IC50 at approximately 60 mM exchange) regardless of the secretagogue utilized (phorbol ester, forskolin, depolarization plus BAY K8644, or TRH). Exchange of medium chloride with either isethionate or benzoate reduced cell volume by 10% compared to 60% for sucrose and mannitol, suggesting that inhibition of secretion by isethionate exchange was not a result of increased intracellular osmotic pressure. Complete exchange of medium chloride with isethionate did not alter equilibrium [3H]methyl-TRH binding, resting internal [Ca2+], or the [Ca2+]i response to depolarization and TRH as measured with intracellularly trapped Fura 2. Chloride removal did not change resting internal pH and recovery from an acid load as measured by the intracellular pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The stimulated secretion of prolactin was also inhibited by exchange of chloride with isethionate in normal pituitary cells in primary culture and the ability of normal cells to respond to the dopamine agonist bromocryptine was not affected by the exchange. These results suggest that exocytosis of prolactin from GH-cells and normal pituitary cells in culture is an osmotically driven process that is chloride-dependent. Stimulated release is more chloride-dependent than constitutive release. The inhibitory effect of isethionate substitution occurs after signal transduction and is distinct from the site of dopamine inhibition of prolactin release.

Interactions between thyroid hormone and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were examined in a rat pituitary tumor cell line, GH4C1. Cells were incubated in thyroid hormone-depleted medium for 2 days, and specific nuclear binding of [125I]T3 was measured. 1,25-(OH)2D3 decreased nuclear [125I]T3 binding without changing total cellular uptake of [125I]T3. This 1,25-(OH)2D3 effect required 2-3 h to become evident and 24 h to reach a maximum (40-50% of control) and was reversible. Treatment with 1,25-(OH)2D3 for 8 h changed the maximal binding capacity for [125I]T3 from 80.2 +/- 2.9 to 50.3 +/- 6.3 fmol/10(6) cells, whereas Kd was not significantly altered. The decrease in [125I]T3 binding was dose dependent, with an IC50 for 1,25-(OH)2D3 of 1 nM in thyroid hormone-depleted medium. 1,25-(OH)2D3 caused little change in [125I]T3 binding to isolated nuclei, i.e. 1,25-(OH)2D3 does not compete directly with [125I]T3 for binding. It is unlikely that 1,25-(OH)2D3 decreased [125I]T3 binding by increasing the concentration of intracellular free calcium ([Ca2+]i), since 1,25-(OH)2D3 did not change [Ca2+]i in Indo-I-loaded GH4C1 cells. Two major species (6 and 2.6 kilobases) of mRNA for c-erb-A, which have been reported to encode nuclear thyroid hormone receptors, were found by Northern blot analysis, and both were decreased by treatment with 1,25-(OH)2D3 for 8 h. T3 (2 nM) caused a 3-fold increase in GH production over 72 h and 1,25-(OH)2D3 inhibited GH induction by T3, with an IC50 at approximately 1 nM. 1,25-(OH)2D3 stimulated PRL synthesis 5-fold when 10 nM T3 was present, but not when T3 was absent. In summary, 1,25-(OH)2D3 caused a dose-dependent down-regulation of nuclear thyroid hormone receptors at a pretranslational level and diminished GH induction by T3. These results suggest that 1,25-(OH)2D3 inhibits GH synthesis indirectly, at least partly, by attenuating endogenous thyroid hormone action.

A hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), causes an increase in the rate of synthesis of prolactin by GH3 cells, a clonal strain of rat pituitary cells grown in culture. This cell system has been used to study the initial interaction between TRH and prolactin-producing cells. Tritiated TRH binds to a limited number of sites on GH3 cells, but TRH does not bind to control cell strains which do not respond biologically to the neurohormone. Half-maximum binding of TRH to GH3 cells occurs at a TRH concentration of 11 nm, while a half-maximum biological response to TRH, an increase in prolactin production, is obtained at a concentration of 2 nm. A maximum of 1.1 pmoles of TRH is bound per mg of GH3 cell protein, equivalent to 130,000 TRH receptors per cell. Initially the binding of TRH to GH3 cells is reversible, but it becomes irreversible over the ensuing 24-hour period. Binding of TRH to receptors has been studied in GH3 cell homogenates using a filter assay technique. At 0°, the dissociation constant of the TRH-GH3 receptor complex is 25 nm, and there is 0.75 pmole of TRH receptors per mg of GH3 cell protein. The halftime for the dissociation of the TRH-GH3 receptor complex is 2 to 3 hours. The binding of TRH to the GH3 receptor has a pH optimum of 7.5, does not require the presence of divalent cations, and is inhibited by high concentrations of calcium ion. We conclude that the action of TRH on GH3 cells involves an initial binding of the tripeptide to specific receptors.

When added to intact GH4Cl or GH3 cells, cytochalasin b, dansylcadaverine, and chloroquine inhibited the binding of L-125I-triiodothyronine (T3) to nuclear receptors within 30 min. These drugs also reduced the amount of 125I-T3 in the post-nuclear supernatant fraction of cells lysed with Triton X-100. If 125I-T3 was added before the drugs, 125I-T3 that was already bound did not dissociate, but further binding was blocked. Inhibition due to 10 microM cytochalasin b or 150 microM dansylcadaverine was reversible within 2 h of drug removal, and inhibition due to 150 microM chloroquine was partially reversible. Drug inhibition was overcome as the T3 concentration was raised. These drugs did not significantly inhibit 125I-T3 binding to isolated nuclei, and nuclei isolated from drug-treated cultures bound as much 125I-T3 as control nuclei. The results suggest that thyroid hormones reach their nuclear binding sites by an active transport process which is inhibited by the drugs studied.

Chemical and enzymatic treatment of a high-molecular weight fraction from a frog brain extract resulted in formation of a “TRH-Like material” (TRH-i). Sequential treatment with trypsin and carboxypeptidases A and B, acetic acid and then chemical amidation generated a quantity of TRH-i equivalent to 25% of the endogenous TRH. TRH-i was similar to TRH (pGlu·His·ProNH2) as assessed by molecular weight estimations, radioimmunoassay and susceptibility to serum inactivation. TRH and TRH-i also competed with [3H]-TRH for binding to TRH receptors, stimulated prolactin synthesis and uridine uptake, and “down-regulated” TRH receptors in pituitary cells. These results suggest the possibility that TRH may be processed from a macromolecular precursor.

Extracts of bovine pineal glands were filtered through Sephadex G-25 and tested for their effect on TRH-induced TSH release in cell cultures of dispersed rat pituitary glands. Highly significant anti-TRH activity was found in material which was retarded on G-25. This material was purified sequentially on Sephadex G-10, Sephadex LH-20, and high pressure reverse phase liquid chromatography columns. The ability of partially purified material to inhibit TRH-induced TSH release by dispersed pituitary cells and to displace [3H]TRH from pituitary tumor cell membranes was interpreted as evidence for a TRH inhibitory factor (TRH-IF) in bovine pineal glands. Evidence for binding to TRH receptors included parallel competition displacement curves with synthetic TRH and data showing that excess TRH could overcome both the inhibition of TRH-induced TSH release and the inhibition of [3H]TRH binding by TRH-IF. Copurification of anti-TRH bioactivity (as tested in dispersed pituitary cell cultures) and anti-TRH-binding activity (as tested in membrane preparations) suggests that the inhibitory activity in pineal extracts results from the binding of a TRH antagonist to receptors. Reports by others of PRL release-inhibiting activity in pineal extracts were confirmed. PRL release-inhibiting activity copurified with TRH-IF.

The present study was undertaken to examine the effect of long term exposure to epidermal growth factor (EGF) on thyroid hormone responses as well as the concentration of specific nuclear thyroid hormone receptors in GH4C1 rat pituitary tumor cells. GH4C1 cells were first incubated for 48 h in medium with 5% fetal calf serum depleted of thyroid hormones by ion exchange resin. EGF had no effect on thyroid hormone receptors after 2 h, but decreased [125I]T3 binding to 56% of control values at 24 h and 68% at 48 h. L-T3 (0.5 nM) caused down-regulation of thyroid hormone receptors, and addition of EGF caused a further decrease. T3 alone (0.5 nM) caused a 2- to 3-fold induction of GH after 48 h, and GH induction was significantly inhibited by the addition of 10 nM EGF. Scatchard analysis of specific nuclear [125I]T3 binding showed that 48-h incubation with 10 nM EGF decreased T3 receptors from a Bmax of 2.35 to 1.26 pmol/mg DNA in thyroid hormone-depleted medium without affecting receptor affinity (Kd, 80 pM). The decrease in nuclear thyroid hormone receptors caused by EGF was dose dependent, with half-maximal inhibition at 0.10 nM EGF. EGF attenuated the GH response to T3 with similar dose-response characteristics. When cells were incubated for 48 h with different concentrations of T3, EGF (10 nM) decreased thyroid hormone receptors to 56-72% of control values regardless of the dose of T3, and EGF shifted the ED50 for T3 stimulation of GH from 0.1 to 1.2 nM. EGF also reduced from 5- to 1.8-fold the increase in cell number caused by thyroid hormone over 2 weeks. In contrast, EGF stimulation of PRL synthesis was changed only slightly by thyroid hormone at all times. In conclusion, we demonstrate that low concentrations of EGF decrease nuclear thyroid hormone receptors and thyroid hormone responses; this may be the mechanism by which EGF suppresses T3-induced GH production in GH4C1 cells.

Epidermal growth factor (EGF) regulates hormone synthesis and decreases the growth rate of clonal lines of rat pituitary tumor cells. [ 125 I]iodo-EGF bound to specific receptors on GH 4 C 1 rat pituitary cells with an apparent K d of 0.34 nM. At 0°C, 84% of specifically bound [ 125 I]iodo-EGF appeared to be on the cell surface, while at 37°C, 84% of bound [ 125 I]iodo-EGF appeared to be internalized and could not be removed by treatment with an acidic high-salt buffer. Internalization of surface-bound [ 125 I]iodo-EGF required 10 min to reach completion and was markedly temperature-dependent. Monodansylcadaverine, bacitracin, ammonium chloride, chloroquine, quinacrine and vinblastin did not inhibit internalization of [ 125 I]iodo-EGF, suggesting that transglutaminase is not involved; but these drugs did cause an increased accumulation of radioactivity, probably by inhibiting degradation. In 2 h at 37°C, approx. 35% of cell-associated [ 125 I]iodo-EGF was degraded as analyzed by gel filtration, and 72% of the material that dissociated from the cells migrated as low molecular weight material or iodo-Tyr. Incubation of GH 4 C 1 cells with unlabeled EGF caused a prolonged and dose-related decrease in the ability of the cells to bind [ 125 I]iodo-EGF in a subsequent incubation, i.e. down-regulation. The pathway for internalization and degradation of the EGF-receptor complex in GH 4 C 1 cells resembles that described for fibroblasts, in which the cellular response is different, but differs from the behavior of other hormone-receptor complexes in GH 4 C 1 pituitary cells.

The anterior pituitary gland of mammalian species secretes six major trophic hormones: adrenocorticotropic hormone, two gonadotropins, thyrotrophin, prolactin and growth hormone. Synthesis and secretion of these pituitary hormones is regulated in part by hypothalamic factors which reach the pituitary gland by a portal system and which act upon specific target cells to stimulate or inhibit hormone production. In addition, pituitary function is regulated by circulating hormones including steroids and peptides.

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

The sequence of PRL and GH release from GH4C1 cells was studied in perifusion and static culture systems. The secretory pattern elicited by TRH differed from those caused by depolarizing concentrations of KCl (Ca2+-initiated secretion), vasoactive intestinal peptide (VIP), 8-bromo-cAMP, and forskolin (cAMP-mediated secretion), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (protein kinase C activation). TRH, K+, VIP, and TPA all caused secretion within 1 min in the perifusion system but the peak response to TRH and depolarization occurred earlier than the peak responses to TPA and VIP. PRL and GH release in response to a pulsatile application of TRH (0.4-min pulse every 5 min for 25 min) did not decline with a low dose, indicating that acute desensitization does not occur, but did decrease with a high concentration. When cells in the perifusion system were subjected to continuous stimulation, TRH caused a biphasic response with a 2- to 3-min period of high secretion followed by a second phase in which GH and PRL secretion were 60-70% the rates in the first phase. KCl caused predominantly first-phase secretion, and TPA caused a biphasic secretory pattern with a delay in its peak of action. VIP caused a modest but prolonged response whether administered in a pulsatile or sustained manner. When GH-cells were exposed to 100 nM TRH for 2 days, [3H] [N3-methyl-His2]TRH binding was decreased (down-regulation), intracellular PRL was increased (170% of control), and intracellular GH was decreased (65% of control). In these down-regulated cells, baseline PRL and GH secretion were changed in proportion to the relative intracellular hormone content. The responsiveness to TRH, KCl, and TPA during the initial 10-min period (first phase) was reduced; however, the responsiveness to these substances in the subsequent 50-min period (second phase) was unchanged. The ED50 for TRH stimulation of hormone release was increased 2- to 4-fold in down-regulated cells, but the dose-response curves for other secretagogues were not shifted. These data suggest that the initial burst of hormone release caused by TRH is mediated by Ca2+, and that prolonged exposure to TRH causes homologous desensitization.

Thyrotropin releasing hormone (TRH) is the smallest of the hypothalmic releasing hormones, having the structure pGlu.His.Pro.NH 2 . Efforts directed toward an understanding of its biosynthesis have been limited because the peptide is found in extremely small quantities in the brain or pancreas. Recent evidence suggests the existence of a precursor(s) macromolecule present in frog brain which can be converted by chemical and enzymatic digestion into a compound (referred to as TRH-i) which has both biological and immunological properties similar to TRH. The TRH-i binds to receptors occupied by radiolabeled TRH and stimulate prolactin secretion (and synthesis). In addition, TRH-i shows competitive binding with respect to TRH when antibody prepared against the tripeptide is used in radioimmunoassays. The majority of the TRH-i activity appears to have a similar molecular weight to that of the authentic hormone.

The dihydropyridine (DHP) Ca2+ agonist Bay K 8644 produced a dose-dependent increase in 45Ca2+ uptake by GH4C1 rat pituitary tumor cells. For agonist concentrations between 10(-9) and 10(-5) M, the enhanced 45Ca2+ uptake was well correlated with simultaneous increases in prolactin (PRL) secretion. Bay K 8644 combined with depolarizing concentrations of KCl produced more than additive effects on net Ca2+ uptake and hormone release. Nisoldipine, a DHP Ca2+ antagonist, competitively blocked Bay K 8644-stimulated 45Ca2+ uptake. This drug also potently inhibited 45Ca2+ uptake triggered by depolarization with KCl (estimated half-maximal inhibiting concentration: 2 nM). Bay K 8644 enhanced PRL secretion from normal rat pituitaries in culture and in a perifusion system. These results indicate that Bay K 8644 is a potent modulator of voltage-sensitive Ca2+ channels of both normal and transformed pituitary cells. In this respect endocrine cell Ca2+ channels resemble those found in heart, smooth muscle, and neuronal cell bodies.

Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide, binds rapidly and reversibly to specific membrane receptors on GH3 cells, a clonal strain of rat pituitary cells grown in culture. GH3 cells were incubated for 1-72 hr with unlabeled TRH, washed, and then incubated for 1 hr with [3H]TRH. Under these conditions 80% of any bound, unlabeled TRH exchanges with [3H]TRH in the medium, and the amount of radioactivity bound to the cells gives a measure of the number of TRH receptors. In GH3 cells, the number of available TRH receptors decreased from 92% of control after 1 hr to 35% after 48 or 72 hr of incubation with unlabeled TRH. Binding of [3H]TRH to both intact control and TRH-treated cells was half-maximal at 8 nM [3H]TRH, but the maximum amount of [3H]TRH bound was decreased by 75% in cells previously incubated for 48 hr with unlabeled TRH. Equilibrium binding studies were performed using membrane fractions prepared from control cells and cells previously exposed to TRH for various periods. The dissociation constant of the TRH-receptor complex was the same in all cases, but the maximum amount of TRH bound decreased progressively in membrane fractions from cells incubated with TRH for 1-51 hr. TRH receptors were not found in cytoplasmic fractions of control or TRH-treated cells. The loss of TRH receptors was reversible within 4 days. In the continued presence of the tripeptide the number of receptors remained low for 12 days. After incubation for 2 days with different concentrations of TRH, the number of receptors was decreased to 33% of control at 100-300 nM TRH, and half of this decrease occurred at about 1 nM TRH; half-maximal biological responses occur at 2 nM TRH. The biologically active Ntau-methylhistidyl derivative of TRH also effected a loss of receptors, while three inactive analogs of TRH did not cause reductions in the number of TRH receptors. In cultures incubated for 40 hr with cycloheximide, protein synthesis was inhibited by 85%, but the number of TRH receptors was 76% of control suggesting that the receptor has a long half-life. When GH3 cells were incubated with cycloheximide plus TRH, the number of TRH receptors decreased by only 23% as compared to a decrease of 73% in cells incubated with TRH alone, suggesting that receptor loss is partially dependent on active protein synthesis. We conclude that in GH3 cells TRH regulates the number of its own receptors.

The endocrine regulation of the anterior pituitary gland is complex. Hypothalamic and circulating hormones control the rate of secretion of each of the adenohypophyseal hormones and also the rate of de novo hormone biosynthesis. A widely used model system for studying regulation of secretory endocrine cells is the GH3 and related cell lines, collectively referred to as GH cells. GH cells are clonal lines derived from a rat pituitary tumor and have the characteristics of a somatomammotrope, i. e., they synthesize and secrete both growth hormone and prolactin. GH cells respond to many of the stimuli that regulate anterior pituitary function in vivo and are a particularly attractive model because they afford the opportunity to study control of both the release and the synthesis of prolactin and growth hormone. Although this chapter summarizes work from this laboratory, the molecular mechanisms of regulation of GH cell function have been studied by many investigators and are the subject of a number of recent reviews (Gourdji et al. 1981; Gershengorn, 1986; Hinkle, 1988).

The effects of physiological concentrations of L-T3 (T3) were examined in dispersed cell cultures of pituitaries obtained from 10- to 12-day-old rats. T3 inhibited TSH secretion by 50% and blunted the TSH response to TRH. The PRL response to TRH was also inhibited by T3, and GH secretion was increased 2-fold. These responses were half-maximal at 0.1 nM added T3 in medium supplemented with 10% hypothyroid calf serum, corresponding to a free T3 concentration of 5 pM. In the presence or absence of added T3, TRH effects were half-maximal at 0.5-3 nM, and T3 suppression was not overcome by high concentrations of TRH (up to 1 microM). Maximal inhibition of TSH responses to TRH occurred when cultures were preincubated with thyroid hormone for 24 h; a significant effect was observed after 8 h. The specific binding of [3H]TRH to dispersed rat pituitary cells was decreased 55-70% by T3 in a dose-dependent manner. Inhibition of TSH secretion by T3 was reversible within 24 h, and the fraction of thyrotrophs in the cultures (0.22) was not altered by T3 over the course of the experiments. The results demonstrate that physiological concentrations of T3 regulate TSH and PRL responses to TRH and control TRH receptor levels by a direct action on normal rat pituitary cells.

The present study was undertaken to test the effects of TRH on thyroid hormone receptors and responses in GH4C1 rat pituitary tumor cells. TRH caused a loss of up to 32% of specific nuclear thyroid hormone binding sites with an ED50 of approximately 1 nM, and this loss was additive to the receptor down-regulation caused by T3 itself. Scatchard analysis of nuclear T3 binding revealed that 10 nM TRH decreased the concentration of T3 receptors from Bmax (femtomoles per mg protein) of 110 to 50 while receptor affinity in serum-free medium changed from dissociation constant (Kd) 110 to 50 pM with TRH. TRH lowered the GH response to 0.5 nM T3 from 215% to 127% of control. The concentrations of TRH required to decrease T3 receptors and T3 responses were similar and indicated that these TRH effects are mediated by the TRH receptor. In the absence of added thyroid hormone TRH had little effect on the rate of GH synthesis. TRH did not affect the binding of 0.5 nM [125I]T3 to receptors during the first 8 h but reduced T3 receptor occupancy up to 25-50% in different experiments after 24 h. TRH blocked the induction of GH by T3 only after 48 h or longer. When cells were incubated for 2 weeks with or without 2 nM T3 and 10 nM TRH, the stimulation of cell growth by T3 was decreased by TRH (2- vs. 5-fold increase in cell number) as was stimulation of GH by T3 (5- vs. 13-fold). As expected, T3 blunted the PRL response to TRH from 19- to 3-fold. The effects of TRH on the density of thyroid hormone receptors could be mimicked by the calcium channel agonist BAY K8644 plus a protein kinase C-activating phorbol ester which together caused a 53% reduction in thyroid hormone binding. The dose-response and temporal relationships suggest a causal relationship between the TRH-mediated decrease in thyroid hormone receptors and the decrease in thyroid hormone responses in GH4C1 cells. It has previously been shown that thyroid hormones decrease the concentration of TRH receptors and TRH responsivity in pituitary cells. The results shown here for GH4C1 cells suggest that TRH regulation of T3 responses may also be important in feedback control at the pituitary level.

Thyrotropin-releasing hormone (TRH) binds to specific receptors on GH4C1 pituitary tumor cells. A fluorescently labeled analog of TRH was synthesized by coupling pGlu-His-ProNH(CH2)6NH2 to fluorescein isothiocyanate. The fluorescein-labeled peptide (FL-TRH) stimulated prolactin synthesis and release by GH4C1 cultures and bound to TRH receptors with an apparent Kd of 400 nM. Binding of FL-TRH to unfixed, viable GH4C1 cells was followed by fluorescence microscopy. After incubation with 1.4 microM FL-TRH for 1 hr at 37 degrees C, the surface of all cells was fluorescent and patches of intense fluorescence were evident. Control cultures incubated with FL-TRH and excess TRH were not fluorescent, and a line of pituitary tumor cells which lacks TRH receptors displayed little fluorescence after incubation with FL-TRH. When GH4C1 cells were incubated with FL-TRH for 1 hr at 37 degrees C and then with excess TRH for an additional 1 hr, the fluorescence associated with the cells was diminished to control levels. The results demonstrate that the fluorescein-labeled peptide labels specific TRH receptors.

The intrinsic tryptophan fluorescence of membranes prepared from the GH3 strain of hormone-producing pituitary cells was monitored by spectrofluorometry. Membranes of GH3 cells have specific receptors which bind thyrotropin-releasing hormone (TRH). When TRH binds to GH3 membranes there is quenching of tryptophan fluorescence. The kinetics of the change in fluorescence of GH3 membranes and of TRH binding are similar. In addition, the concentration of TRH required to produce a half-maximum change in fluorescence is 10 nM, and that required for half-maximum binding of TRH to receptors is 11 nM. Inactive TRH analogs which do not bind to TRH receptors likewise do not alter GH3 membrane fluorescence, and a pituitary cell strain which lacks TRH receptors does not change membrane fluorescence on incubation with TRH. We conclude that the TRH-receptor interaction in GH3 membranes is associated with a change in membrane conformation that is readily measured by differential spectrofluorometry.

Five clonal strains of mouse tumor cells (HSDM1) synthesize and secrete large quantities (0.70-2.0 μg/mg cell protein/24 hr) of prostaglandin E2. Five lines of control cells did not synthesize significant amounts of prostaglandins. HSDM1 cells produce prostaglandin E2 during both the logarithmic and stationary phases of the cell growth cycle. Prostaglandin production was inhibited by aspirin-like drugs; for example, 50% inhibition was obtained with as little as 3 × 10−9 M indomethacin. We conclude that the HSDM1 cell system will serve as a useful model system to study prostaglandin synthesis and secretion.