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P M Hinkle

Publications and source records attributed to P M Hinkle.

At least 73 records · Page 4Linked to original sources
PubMed1985

Thyrotropin-releasing hormone rapidly stimulates a biphasic secretion of prolactin and growth hormone in GH4C1 rat pituitary tumor cells.

The characteristics of TRH-induced acute PRL and GH secretion were studied in GH4C1 cells, a clonal rat anterior pituitary tumor cell line which secretes PRL and GH. The experiments were carried out both in a flow system in which microcarrier (Cytodex)-attached cells were perifused at a constant rate and in a conventional static culture system. In both systems, cells responded to TRH in a qualitatively similar manner. TRH significantly stimulated PRL and GH secretion within 5 sec without a detectable lag period. The secretion rate was highest during the initial 1 min, declined sharply thereafter despite the continuous presence of TRH, and plateaued at a lower level. The maximum dose of TRH caused 250-700% of basal secretion during the early period (approximately 8 min; first phase) and about 150% of basal secretion thereafter (second phase). The sustained lower secretion (second phase) was maintained as long as cells were exposed to TRH (up to 2.5 h), and the secretion rate returned to the basal level within 30 min of removal of TRH from the medium. The half-maximal doses for the first and second phase secretion were 2-3 and 0.5-1 nM, respectively, in both the perifusion and static culture systems. Over a 2-day period, TRH stimulated PRL synthesis and inhibited GH synthesis. The dose-response curves for these long term effects on hormone synthesis were similar to the dose-response curves for the first phase of release. [N3-methyl-His2]TRH gave similar results, but was more potent than TRH. [N3-methyl-His2]TRH stimulated first phase release with an ED50 of 0.4-0.8 nM, second phase release with an ED50 of 0.1-0.2 nM, and hormone synthesis with an ED50 of 0.7-0.8 nM. Preincubation of the cells with Ca+2-free medium significantly depressed both first and second phase secretion. Preexposure of the cells to cycloheximide (10 micrograms/ml) had little effect on the first phase of secretion, but reduced second phase secretion. The acute effects of TRH on GH and PRL were identical, except that the secretory response tended to be greater for PRL. We conclude that 1) TRH causes hormone secretion very rapidly in a biphasic manner; 2) the first phase of secretion consists primarily of the release of stored hormone, whereas the second phase includes the release of newly synthesized hormone; 3) the dose-response curve of second phase secretion is shifted to the left compared with that of first phase secretion; and 4) both phases of secretion are at least partially dependent on extracellular Ca+2.

Animals↗
PubMed1985

Differential effects of thyrotropin-releasing hormone, vasoactive intestinal peptide, phorbol ester, and depolarization in GH4C1 rat pituitary cells.

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1984

Regulation of thyrotropin-releasing hormone binding by monovalent cations and guanyl nucleotides.

Binding of thyrotropin-releasing hormone (TRH) to specific receptors on membranes isolated from GH4C1 pituitary cells was inhibited by monovalent cations and guanyl nucleotides. NaCl and LiCl inhibited TRH binding by 70%, with half-maximal inhibition at 30 mM; RbCl and KCl inhibited only 10% at concentrations up to 150 mM. NaCl decreased both the apparent number and the affinity of TRH receptors and increased the rate of dissociation of TRH from both membrane and Triton X-100-solubilized receptors. Guanyl nucleotides inhibited TRH binding up to 80%, with guanyl-5'-yl imidodiphosphate (Gpp(NH)p) approximately GTP much greater than GDP approximately ATP greater than GMP. GTP and Gpp(NH)p exerted half-maximal effects at 0.3 microM and decreased receptor affinity to one-third of control but did not change receptor number. Gpp(NH)p accelerated the dissociation of TRH from membranes but not from solubilized receptors. The effects of NaCl were independent of temperature, while GTP and Gpp(NH)p were much more inhibitory at 22 degrees C (70%) than at 0 degrees C (10%). Inhibition by NaCl could be reversed by washing the membranes, and inhibition by GTP was reversed if membranes were chilled to 0 degrees C. The inhibitory effects of low concentrations of NaCl and Gpp(NH)p were additive. Neither monovalent cations nor GTP prevented the TRH-receptor complex from undergoing transformation from a state with rapid dissociation kinetics to a slower dissociating form. The results suggest that sodium ion regulates TRH binding by interacting with a site on the receptor, while guanyl nucleotides regulate TRH binding indirectly.

Animals↗
PubMed1984

Thyrotropin-releasing hormone stimulates GTP hydrolysis by membranes from GH4C1 rat pituitary tumor cells.

Thyrotropin-releasing hormone (TRH) stimulates prolactin production by GH4C1 rat pituitary tumor cells, which possess high-affinity membrane receptors for the peptide. TRH caused up to a 50% increase in the activity of a low-Km GTPase in membranes from GH4C1 cells. The TRH stimulatory effect was maximal at GTP concentrations of 1 microM or lower. TRH caused an increase in GTPase activity of between 0.2 and 20 pmol of GTP hydrolyzed per mg of protein per min, depending on GTP concentration, while TRH binding was 0.3 pmol/mg of protein. TRH did not stimulate GTPase activity in membranes from GH12C1, or GH-Y cells, two pituitary lines lacking TRH receptors. Stimulation of GTPase depended on occupancy of the TRH receptor; half-maximal increases in GTPase activity required 46 nM TRH and 25 nM [N3-methyl-His]TRH, but the TRH free acid was inactive. The apparent Kds of these peptides for receptors were similar when measured under the same conditions. The fact that TRH binding to receptors is regulated by guanyl nucleotides, together with the demonstration of TRH stimulation of low-Km GTPase activity, suggests that the TRH receptor is associated with a guanyl nucleotide regulatory protein in the lactotroph membrane.

Animals↗
PubMed1984

Characterization of thyroid hormone stimulation of uridine uptake by rat pituitary tumor cells.

T3 caused a dose-related increase in the rate of [3H]uridine uptake into GH4C1 rat pituitary tumor cells. T3 increased uridine uptake to 130-180% of the control value, with a half-maximal effect at approximately 1 nM. T3 exerted a half-maximal effect at 1 h and a maximal effect at 2 h. In contrast, epidermal growth factor also increased uridine uptake by 75%, with an ED50 of 0.6 ng/ml (0.1 nM), but a half-maximal response required 4 min and a maximal effect required 20 min. T3 increased the rate of uptake at all uridine concentrations from 30 nM to 130 microM. Equilibrium binding of [125I]T3 to nuclear receptors required from 15 min at 50 nM [125I]T3 to 1 h at 0.5 nM, indicating that occupancy of nuclear receptors precedes maximal stimulation of uridine uptake. T3 did not stimulate the rate of uridine uptake at 20 C, when binding to nuclear receptors does not occur. Various thyroid hormones caused an increase in uridine uptake, with the rank order of potency 3,3',5-triiodothyroacetic acid greater than T3 greater than L-T4 greater than D-T4 approximately equal to 3,3',5,5'-tetraiodothyroacetic acid; rT3 was inactive. This order parallels the affinities of these compounds for nuclear thyroid hormone receptors.

Animals↗
PubMed1983

Binding and internalization of epidermal growth factor by rat pituitary tumor cells.

Epidermal growth factor (EGF) regulates hormone synthesis and decreases the growth rate of clonal lines of rat pituitary tumor cells. [125I]iodo-EGF bound to specific receptors on GH4C1 rat pituitary cells with an apparent Kd of 0.34 nM. At 0 degrees C, 84% of specifically bound [125I]iodo-EGF appeared to be on the cell surface, while at 37 degrees C, 84% of bound [125I]iodo-EGF appeared to be internalized and could not be removed by treatment with an acidic high-salt buffer. Internalization of surface-bound [125I]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 [125I]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 degrees C, approx. 35% of cell-associated [125I]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 GH4C1 cells with unlabeled EGF caused a prolonged and dose-related decrease in the ability of the cells to bind [125I]iodo-EGF in a subsequent incubation, i.e. down-regulation. The pathway for internalization and degradation of the EGF-receptor complex in GH4C1 cells resembles that described for fibroblasts, in which the cellular response is different, but differs from the behavior of other hormone-receptor complexes in GH4C1 pituitary cells.

Animals↗
PubMed1982

Evidence for an active step in thyroid hormone transport to nuclei: drug inhibition of L-125I-triiodothyronine binding to nuclear receptors in rat pituitary tumor cells.

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.

Animals↗
PubMed1982

Regulation of thyrotropin-releasing hormone receptors and responses by L-triiodothyronine in dispersed rat pituitary cell cultures.

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.

Animals↗
PubMed1981

Purification of a stimulator of microtubule assembly from bovine thyroid.

A protein from bovine thyroid which stimulated the assembly of microtubules from phosphocellulose-purified tubulin was isolated. The purification scheme involved fractionation of bovine thyroid homogenates successively on phosphocellulose, DEAE-cellulose, and Sephadex G-200. Activity was determined as ability to stimulate phosphocellulose-purified tubulin assembly measured turbidimetrically. Purification of stimulatory activity resulted in the selective enrichment to near homogeneity of a 40,000-45,000-dalton protein. The molecular weight was estimated by Sephadex G-200 gel filtration and polyacrylamide gel electrophoresis. This protein stimulated both the rate and the extent of assembly of thyroid and brain tubulin. In the turbidity assay which contained 4 M glycerol, the threshold for assembly of brain tubulin (at 1.7 mg/ml) was 0.01 mg/ml for the thyroid stimulator. Brain and thyroid microtubules assembled with thyroid stimulator appeared normal in diameter and shape by electron microscopy. Thyroid stimulator activity was destroyed by trypsinization or boiling. Activity was maximal at 1 mM GTP and 4 mM MgCl2 and was blocked by colchicine. No stimulatory protein of similar molecular weight could be isolated from bovine brain by similar techniques.

Animals↗
PubMed1981

Direct visualization of receptors for thyrotropin-releasing hormone with a fluorescein-labeled analog.

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.

Animals↗
PubMed1981

Mechanism of thyroid hormone inhibition of thyrotropin-releasing hormone action.

The addition of thyroid hormone to cultures of GH3 or GH4C1 pituitary tumor cells maintained in medium with hypothyroid serum decreased the concentration of specific receptors for TRH. The relationship between thyroid hormone effects on TRH receptors and TRH responses was examined by testing the concentration dependence, time course, and specificity of these changes. The concentrations of T3 giving half-maximal decreases in [3H]TRH binding and inhibition of the PRL response to TRH were 0.20 and 0.24 nM, respectively. TRH stimulated the rate of [3H]uridine uptake by 50% in cultures incubated without added T3 but did not increase [3H]uridine uptake in cells incubated with thyroid hormone. The PRL response to TRH was substantially inhibited 12 h after the addition of T3, and the uridine uptake response was completely blocked in 8 h. Two other stimuli of PRL secretion, sodium butyrate and isobutylmethylxanthine, were effective in the presence or absence of T3. Thyroid hormone did not reduce the specific binding of either [125I-Tyr1]somatostatin or [125I]iodoepidermal growth factor. Somatostatin decreased the secretion of GH and PRL by pituitary tumor cells grown with or without T3. The data show that the effects of thyroid hormones on TRH receptors are specific and suggest that regulation of receptor concentrations may be the direct cause of thyroid hormone regulation of pituitary responsiveness to TRH.

Animals↗
PubMed1980

Relationships between thyroid hormone and glucocorticoid effects in GH3 pituitary cells.

The interrelationships between thyroid hormone and cortisol actions were investigated in GH3 pituitary tumor cells. When GH3 cells were grown in thyroid hormone-deficient medium, cortisol did not affect the concentration of TRH receptors. Both thyroid hormones and TRH normally decrease the number of TRH receptors, and cortisol inhibited down-regulation by both hormones. TRH caused a greater increase in PRL synthesis when TRH receptors were high in the presence of cortisol and T3 than when TRH receptors were low (T3 alone). In the presence of cortisol, higher concentrations of T3 were required to decrease TRH receptors, while lower concentrations were necessary to stimulate GH synthesis. Cortisol and T3 alone stimulated GH synthesis 6- and 10-fold, respectively, while together they caused an 830-fold increase. In contrast, T3 did not alter the inhibition of PRL synthesis by the glucocorticoid. Cortisol did not significantly affect the amount of [125I]T3 bound to nuclei from cells incubated in thyroid hormone-deficient or T3-supplemented medium (approximately 100 and approximately 25 fmol/mg cell protein). The data suggest that cortisol modifies thyroid hormone action at a step subsequent to T3 receptor binding.

Animals↗
PubMed1980

Thyrotropin-releasing hormone-receptor interaction in GH3 pituitary cells.

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.

Animals↗
PubMed1980

Evidence for a thyrotropin-releasing hormone inhibitor in the pineal gland.

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.

Animals↗