Biomedical subjects

P M Hinkle

Publications and source records attributed to P M Hinkle.

At least 55 records · Page 3Linked to original sources

PubMed1989

Attenuation of thyroid hormone action by 1,25-dihydroxyvitamin D3 in pituitary cells.

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.

Animals↗

PubMed1989

Pituitary thyrotropin-releasing hormone (TRH) receptors: effects of TRH, drugs mimicking TRH action, and chlordiazepoxide.

Binding of TRH to specific cell surface receptors on clonal GH4C1 cells is followed within 10 min by receptor sequestration and over 24 h by receptor down-regulation. These experiments were designed to determine if TRH-activated second messenger systems are responsible for changes in receptor localization or number. BAY K8644 and A23187, which increase intracellular calcium, alone or together with 12-O-tetradecanoyl phorbol acetate (TPA), which activates protein kinase C, did not appear to internalize TRH receptors. Drug treatment did not alter the rate of [3H]MeTRH association or internalization, determined by resistance to an acid/salt wash, or the amount of [3H]MeTRH able to bind at 0 C, where only surface receptors are accessible. TPA (0-100 nM) alone or in combination with BAY K8644 or A23187, also failed to change receptor number or affinity after 48 h when TRH caused a 75% decrease in the density of specific binding sites. Chlordiazepoxide has been reported antagonize TRH binding and TRH-induced phospholipid breakdown. Chlordiazepoxide shifted the dose-response curves for TRH stimulation of PRL release and synthesis to the right, and did not change PRL release alone. The affinity of receptors for chlordiazepoxide was not affected by a nonhydrolyzable analog of GTP whereas affinity for TRH was decreased; these properties are consistent with the classification of chlordiazepoxide as a competitive antagonist. Several experiments tested whether chlordiazepoxide would cause receptor internalization and down-regulation. Chlordiazepoxide did not appear to internalize TRH receptors, because TRH-binding sites became available rapidly and at the same rate after they had been saturated with chlordiazepoxide at 0 or 37 C.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1989

Pituitary thyrotropin-releasing hormone receptors: local anesthetic effects on binding and responses.

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthetics, Local↗

PubMed1989

Solubilization and characterization of pituitary thyrotropin-releasing hormone receptors.

Thyrotropin-releasing hormone (TRH) receptors were solubilized from a rat pituitary tumor cell line, GH4C1, with digitonin. Convenient assays were developed based on the ability of hydroxylapatite and polyethyleneimine-soaked glass fiber filters to adsorb the solubilized [3H]methyl-TRH-receptor complex but not free [3H]methyl-TRH. The kinetics of [3H]methyl-TRH binding to solubilized receptors were extremely temperature dependent. Binding reached equilibrium at 10-20 nM [3H]methyl-TRH in 30 min at 23 degrees and 6 hr at 0 degree. The half-times for dissociation were less than 5 min at 23 degrees and 7.6 hr at 0 degree. Equilibrium binding experiments yielded linear Scatchard plots at 0 degree with Kd = 3 nM, whereas the Kd was greater than 20 nM at 23 degrees. A series of TRH congeners displaced [3H]methyl-TRH with the rank order reported for membrane receptors, N3-methyl-HisTRH greater than or equal to TRH greater than pGlu-N3-methyl-HisProNH(CH2)6NH2 greater than or equal to pGluHisProNH(CH2)6NH2 greater than pGluHisTyrNH2 much greater than TRH free acid. The antagonist chlordiazepoxide exhibited an IC50 of 10 microM. [3H]methyl-TRH binding to solubilized receptors displayed a broad pH optimum, from 6.5 to 7.5. The solubilized receptor could be obtained from cultured GH4C1 cells and in much larger quantities from GH4C1 tumors. Tumors from 12 rats yielded greater than 700 pmol of specific soluble TRH binding activity (1 g of protein). The solubilized receptor could be purified 10-20-fold by chromatography on wheat germ agglutinin columns and could be concentrated by adsorption on either DEAE-Sephadex or hydroxylapatite. The procedures outlined allow the solubilization of pituitary TRH receptors from a rich and abundant source, the rapid and reproducible assay of [3H]methyl-TRH binding, and substantial enrichment of receptor activity. These findings should be valuable for the purification and identification of the TRH receptor protein.

Animals↗

PubMed1988

Prolactin synthesis in cultured pituitary cells is chloride-dependent.

Release of prolactin from both normal pituitary cells and rat pituitary tumor (GH) cells is an osmotic process that is dependent upon chloride. The long term growth rate of GH-cells in medium in which chloride was exchanged with isethionate was completely normal, but, by 48 h, isethionate substitution resulted in a 70% decrease in the concentration of internal and secreted prolactin. Isethionate caused a much smaller reduction in growth hormone production (less than 20%). These results suggest that exchange of chloride with isethionate is inhibiting the synthesis of prolactin. Reduction of intracellular levels of prolactin in cells grown in isethionate-containing medium was evident by 30 h, and the level of prolactin was reduced 92% at 96 h. This reduction in the internal concentrations of prolactin was reversed when the cells were returned to normal medium containing chloride with a t1/2 of 48 h. Addition of epidermal growth factor and the calcium channel agonist BAY K 8644 to cells in medium containing chloride increased internal prolactin by 400%, and isethionate exchange reduced the response by 85%. To confirm that isethionate exchange was inhibiting the synthesis of prolactin, mRNA concentrations for prolactin and actin were determined. Both basal and hormone-stimulated levels of prolactin mRNA were reduced 70 to 90% by isethionate exchange, while actin mRNA levels did not change. To determine whether the effect of isethionate was at the level of gene transcription, GH-cells were transfected with a prolactin-chloramphenicol acetyltransferase fusion gene and chloramphenicol acetyltransferase expression was assessed using cells in chloride and isethionate-containing media. Both basal and hormone-stimulated synthesis of chloramphenicol acetyltransferase driven by the prolactin promoter was inhibited by isethionate exchange. These studies demonstrate that exchange of medium chloride with isethionate inhibits the synthesis of prolactin at the level of transcription.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1988

Osmotic regulation of prolactin secretion. Possible role of chloride.

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.

Animals↗

PubMed1988

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor.

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.

Animals↗

PubMed1988

Transient dopaminergic inhibition of prolactin release from hybrid cells derived by fusion of normal rat pituitary and GH4C1 tumor cells.

The clonal rat pituitary tumor cell line GH4C1 secretes PRL but does not respond to dopamine, a physiological inhibitor of PRL. In an attempt to generate a dopamine-responsive cell line, GH4C1 cells, which lack the enzyme hypoxanthine-guanine phosphoribosyltransferase, were fused with cells from the pituitary glands of lactating rats to generate cell hybrids. The GH4C1 cells were fused with dispersed normal pituitary cells by either chemical fusion in 40% polyethylene glycol or electrofusion. The fused cells were grown in medium with hypoxanthine, aminopterin, and thymidine (HAT) for 4 weeks to select for hybrid cells. Control fusions between GH4C1 cells only or normal cells only did not produce viable colonies. Of 36 HAT-selected colonies, 3 responded to 10 nM bromocryptine (BCR) with inhibition of TRH-stimulated PRL release. These hybrid colonies had an inhibitory response similar to that of normal pituitary cells in culture. Both TRH- and vasoactive intestinal peptide-stimulated PRL release were inhibited to basal levels by 10 nM BCR, with an IC50 for BCR of approximately 0.25 nM. Basal hormone release was not inhibited by BCR. The BCR-sensitive hybrid cells grew more slowly than the parental GH4C1 line both in culture and when passaged in female Wistar-Furth rats. The response of the hybrid cells to the dopamine agonist and the characteristic of slow growth were lost after 9 months of continuous culture and after freezing cells. The parental GH4C1 cells were grown in female Wistar-Furth rats, the resulting tumors were dissociated, and the cells were grown in culture. This resulted in a brief establishment of the dopamine response. Stimulated PRL and GH release from freshly dispersed GH4C1 tumor cells was inhibited by BCR at concentrations from 0.1-10 nM, and spiroperidol reversed the inhibition. The inhibitory response to the dopaminergic agonist was lost quickly as the cells were carried in culture. These results demonstrate that GH4C1 cells may have the genetic information necessary for dopaminergic inhibition of PRL synthesis, but that the dopamine response is not observed under standard tissue culture conditions.

Animals↗

PubMed1988

Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium.

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)

Animals↗

PubMed1987

Cadmium uptake and toxicity via voltage-sensitive calcium channels.

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.

Animals↗

PubMed1987

Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells.

To determine whether hormone synthesis by the GH4C1 pituitary cell line could be regulated by specifically modulating the movement of Ca2+ through voltage-sensitive channels, we have compared the effects of the dihydropyridine Ca2+ channel agonist BAY K8644 and the antagonist nimodipine on hormone production and Ca2+ current in these cells. BAY K8644 elicited, after a 10-15-h lag, a dose-dependent increase in prolactin (PRL) production as determined by measurements of total intracellular and secreted hormone. Over a 72-h period, GH4C1 cells incubated with 300 nM BAY K8644 produced 2-3 times as much total PRL as control cells. The effect on PRL was specific, since BAY K8644 did not increase growth hormone production, cell growth rate, or total cell protein. Exposing GH4C1 cells to BAY K8644 for short periods, up to 90 min, did not induce the delayed increase in PRL production observed with longer incubations. The effects of nimodipine were opposite to those of the Ca2+ channel agonist. PRL production was reduced 85% during 48-h treatment with 200 nM nimodipine, whereas growth hormone production was decreased less than 15%, and cell growth and total protein were unaffected. The actions of these two drugs on PRL production were well correlated with their effects on GH4C1 Ca2+ currents as measured by whole-cell patch-clamp recordings. BAY K8644 enhanced the magnitude of the peak Ca2+ current and shifted the current-voltage relationship such that Ca2+ channels were activated at less depolarized potentials. Nimodipine potently inhibited Ca2+ movement through the non-inactivating channel, while it antagonized the increases elicited by BAY K8644. These results indicate that PRL synthesis by GH4C1 cells can be specifically regulated by agents that enhance or block the movement of Ca2+ through voltage-sensitive channels. They also suggest that hormone synthesis by a secretory cell may be coupled to electrical activity by the opening of Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1987

Pituitary Ca2+ channels: blockade by conventional and novel Ca2+ antagonists.

We have identified several new agents that block Ca2+ channels in the rat pituitary GH4C1 cell line. These drugs, which include the diphenylbutylpiperidine antipsychotic pimozide, the calmodulin antagonist calmidazolium, and the steroidal Na+ channel toxin veratridine, were compared with several conventional Ca2+ antagonist in 45Ca2+ uptake, prolactin secretion, and whole cell patch voltage-clamp experiments. Pimozide, the most potent of these novel Ca2+ antagonists, inhibited depolarization-dependent 45Ca2+ uptake and prolactin secretion half maximally at a concentration of 100 nM, whereas calmidazolium and veratridine produced 50% inhibition at concentrations of 500 nM and 1 microM. In comparison, the three organic Ca2+ antagonists nitrendipine, verapamil, and diltiazem blocked 45Ca2+ uptake half maximally at concentrations of 2.5 nM, 1 microM, and 2.5 microM, respectively. All of the antagonists inhibited Ca2+ uptake and prolactin secretion stimulated by the dihydropyridine Ca2+ agonist BAY-K 8644 less potently than KCl-stimulated responses. In patch-clamp experiments, pimozide, veratridine, and nitrendipine blocked Ca2+ current through the slowly inactivating Ca2+ channels of GH4C1 cells. These results demonstrate that Ca2+ channels in an endocrine cell line can be blocked by a variety of molecules including sodium channel toxins and calmodulin antagonists. The data extend the pharmacological similarity between Ca2+ channels in pituitary and other excitable cells and suggest a structural similarity among several cellular proteins.

Animals↗

PubMed1987

Epidermal growth factor decreases thyroid hormone receptors and attenuates thyroid hormone responses in GH4C1 cells.

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.

Animals↗

PubMed1987

Regulation of thyroid hormone receptors and responses by thyrotropin-releasing hormone in GH4C1 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.

Animals↗

PubMed1986

Thyroid hormone induction of an autocrine growth factor secreted by pituitary tumor cells.

Thyroid hormones stimulate the rate of cell division by poorly understood mechanisms. The possibility that thyroid hormones increase cell growth by stimulating secretion of a growth factor was investigated. Thyroid hormones are nearly an absolute requirement for the division of GH4C1 rat pituitary tumor cells plated at low density. Conditioned media from cells grown with or without L-triiodothyronine (T3) were treated with an ion exchange resin to remove T3 and were tested for ability to stimulate the division of GH4C1 cells. Conditioned medium from T3-treated cells was as active as thyroid hormone at promoting GH4C1 cell growth but did not elicit other thyroid hormone responses, induction of growth hormone, and down-regulation of thyrotropin-releasing hormone receptors, as effectively as T3 did. A substance or substances associated with T3-induced growth stimulatory activity migrated at high molecular weight at neutral pH and was different from known growth-promoting hormones induced by T3. The results demonstrate that thyroid hormones stimulate the division of GH4C1 pituitary cells by stimulating the secretion of an autocrine growth factor.

Animals↗

PubMed1986

Thyroliberin action in pituitary cells is not inhibited by pertussis toxin.

The effects of pertussis toxin on the responses of rat pituitary-tumour (GH) cells to thyrotropin-releasing hormone (thyroliberin, TRH) were examined. Treatment of cells with pertussis toxin did not alter the affinity or concentration of TRH receptors, or the sensitivity of the TRH receptor to inhibition by guanine nucleotides. TRH caused an increase in low-Km GTPase activity in membrane-containing fractions from both control and pertussis-toxin-treated cells. TRH stimulation of inositol phosphate formation was insensitive to pertussis toxin. TRH caused a biphasic increase in the concentrations of cytosolic free Ca2+ as monitored by intracellularly trapped Quin 2, and this increase was the same in control and toxin-treated cultures. The toxin did not alter the increase in prolactin and growth-hormone (somatotropin) release stimulated by TRH or shift the TRH dose-response curve, and it did not affect the TRH-induced rise in prolactin synthesis measured over 24 h. However, pertussis toxin did block the ability of somatostatin and muscarinic agonists to inhibit prolactin and growth-hormone secretion stimulated by vasoactive intestinal peptide when analysed under the same conditions as those in which the TRH system was unaffected. These data indicate that the guanine nucleotide effects on TRH binding and activity are not mediated by Ni, but possibly by another member of the family of guanine-nucleotide-dependent regulatory proteins.

Animals↗

PubMed1986

Interaction of dihydropyridine Ca2+ agonist Bay K 8644 with normal and transformed pituitary cells.

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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1985

Dihydropyridine Ca2+ antagonists: potent inhibitors of secretion from normal and transformed pituitary cells.

Three dihydropyridine (DHP) Ca2+ antagonists were compared with several other organic Ca2+ antagonists with respect to their ability to inhibit depolarization-dependent hormone secretion from the GH4C1 pituitary cell line and from normal rat pituitary cells. The three DHP, nimodipine, nisoldipine, and nifedipine, potently and specifically inhibited KCl-stimulated prolactin secretion from GH4C1 cells (estimated IC50 values: 1.8, 1.8, and 6.0 nM, respectively). Both basal and thyrotropin-releasing hormone-stimulated secretion from GH4C1 cells were much less sensitive to inhibition by the DHP. The inhibition by the DHP was reversible, and their potency was independent of depolarizing concentrations of KCl between 18.8 and 53.8 mM. Other organic antagonists, including verapamil, cinnarizine, and diltiazem, blocked secretion from GH4C1 cells but at much higher concentrations. The estimated IC50 values for these three were 1,000, 1,100, and 3,500 nM, respectively. Depolarization-stimulated prolactin secretion from normal pituitaries was inhibited by the DHP and verapamil at the same concentrations found effective in GH4C1 cells. KCl-stimulated 45Ca2+ uptake by GH4C1 cells was also blocked by DHP at concentrations that inhibited secretion. Since depolarization-stimulated secretion and 45Ca2+ uptake are probably triggered by Ca2+ entering through voltage-sensitive channels, the above results suggest that DHP antagonists potently block these channels in both normal and transformed pituitary cells. These Ca2+ channels appear to be identical in this respect. These findings further suggest a similarity between the Ca2+ channels of endocrine cells and those of smooth muscle and other excitable cells.

Animals↗