Evidence that cobalt ion inhibition of prolactin secretion occurs at an intracellular locus.

Cobalt inhibition of stimulated prolactin secretion has been interpreted as demonstrating an essential role for enhanced calcium influx in the action of thyrotropin-releasing hormone (TRH) in GH3 cells. However, this interpretation is based on the assumption that cobalt ion (Co2+) binds to the external surface of cells to antagonize calcium-mediated processes only by blocking influx of extracellular calcium ion (Ca2+). In this report, we present evidence that Co2+ acts at an intracellular locus (or loci) to inhibit prolactin secretion.

Ca2+ ionophores affect phosphoinositide metabolism differently than thyrotropin-releasing hormone in GH3 pituitary cells.

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.

Arachidonic acid mobilizes calcium and stimulates prolactin secretion from GH3 cells.

Because arachidonic acid and/or its metabolites may be intracellular effectors of calcium-mediated secretion, we studied whether arachidonic acid added exogenously mobilizes calcium and stimulates prolactin secretion from GH3 cells, cloned rat pituitary cells. Arachidonic acid caused efflux of 45Ca from preloaded cells and stimulated prolactin secretion. The concentration dependencies of these effects were similar; stimulation was attained with 3 microM arachidonic acid.

Thyrotropin (TSH)-releasing hormone decreases phosphatidylinositol and increases unesterified arachidonic acid in thyrotropic cells: possible early events in stimulation of TSH secretion.

TRH stimulated the metabolism of lipids of the phosphatidylinositol (PI)-phosphatidic acid (PA) cycle and caused an increase in the level of free or unesterified arachidonic acid in mouse pituitary thyrotropic tumor (TtT) cells. In cells labeled with [32P]orthophosphate for 45 min, TRH caused a rapid specific increase in [32P]PA to 190 +/- 8% (+/- SE) of the control value at 15 sec (P less than 0.005) and in [32P]PI to 158 +/- 8% at 2 min (P less than 0.

Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Evidence for an early Ca2+-independent action.

Thyrotropin-releasing hormone (TRH; thyroliberin) stimulated rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phosphodiesterase (phospholipase C) in GH3 cells, a prolactin-secreting rat pituitary tumour cell line. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 60% of control and stimulated a marked transient increase in inositol 1,4,5-trisphosphate, the unique product of phosphodiesteratic hydrolysis of PtdIns(4,5)P2, to a peak of 410% of control at 15 s. TRH also caused decreases in phosphatidylinositol 4-monophosphate (PtdIns4P) and phosphatidylinositol (PtdIns) to 65% and 93% of control at 15 s respectively.

Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells.

TRH stimulation of prolactin secretion is thought to be mediated by an elevation of free cytoplasmic Ca2+. However, whether TRH-induced influx of extracellular Ca2+ is required to elevate cytoplasmic Ca2+ remains controversial. We measured cytoplasmic free Ca2+ concentration in GH3 cells with an intracellularly trapped fluorescent indicator, Quin 2.

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells.

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions.

Tetracaine, propranolol and trifluoperazine inhibit thyrotropin releasing hormone-induced prolactin secretion from GH3 cells by displacing membrane calcium: further evidence that TRH acts to mobilize cellular calcium.

TRH stimulation of prolactin release from GH3 cells is associated with loss of cellular Ca2+. Chlortetracycline (CTC), a fluorescent probe of Ca2+ in biological membranes, was previously employed to monitor indirectly changes in membrane Ca2+ in GH3 cells. Tetracaine, propranolol and trifluoperazine, agents that are known to displace Ca2+ from biological membranes, were utilized to demonstrate more rigorously that TRH affects cellular membrane Ca2+ in GH3 cells.

TRH mobilizes membrane calcium in thyrotropic cells as monitored by chlortetracycline.

Chlortetracycline (CTC), a probe of membrane-bound divalent cations, was used to study the action of thyrotropin-releasing hormone (TRH) in mouse pituitary thyrotropic tumor (TtT) cells in culture. Cellular fluorescence of CTC was caused by both Ca2+- and Mg2+-CTC complexes and was influenced by the concentration of these cations in the incubation medium. TRH, but not other peptides, caused a rapid, transient, and concentration-dependent decrease in the CTC fluorescence intensity; half-maximal effect occurred with 10--30 nM TRH.

Thyrotropin-releasing hormone causes loss of cellular calcium without calcium uptake by rat pituitary cells in culture. Studies using arsenazo III for direct measurement of calcium.

Thyrotropin-releasing hormone (TRH) may act to stimulate prolactin secretion by increasing the intracellular free Ca2+ concentration. This notion is supported by the finding that TRH acutely enhances 45Ca2+ efflux from pituitary cells which may reflect alterations in Ca2+ influx or efflux, or both. To differentiate among these possibilities, we measured loss and uptake of nonradioactive Ca2+ by GH3 cells, a cloned strain of rat pituitary cells that produce prolactin, during TRH action using the metallochromic indicator arsenazo III.

Evidence that TRH stimulates secretion of TSH by two calcium-mediated mechanisms.

Thyrotropin-released hormone (TRH) stimulation of thyrotropin (TSH) release from mouse thyrotropic tumor (TtT) cells is dependent on Ca2+. We demonstrate that TRH action in TtT cells does not require extracellular Ca2+ but that Ca2+ influx induced by TRH can augment TSH secretion. TRH caused a 46% increase in 45Ca2+ uptake by TtT cells in medium with 100 micro M Ca2+.

Thyrotropin-releasing hormone stimulation of prolactin release from clonal rat pituitary cells: evidence for action independent of extracellular calcium.

Thyrotropin-releasing hormone (TRH) stimulates prolactin release and (45)Ca(2+) efflux from GH(3) cells, a clonal strain of rat pituitary cells. Elevation of extracellular K(+) also induces prolactin release and increases (45)Ca(2+) efflux from these cells. In this report, we distinguish between TRH and high K(+) as secretagogues and show that TRH-induced release of prolactin and (45)Ca(2+) is independent of the extracellular Ca(2+) concentration, but the effect of high K(+) on prolactin release and (45)Ca(2+) efflux is dependent on the concentration of Ca(2+) in the medium.

Thyrotropin-releasing hormone (TRH) action in mouse thyrotropic tumor cells in culture: evidence against a role for adenosine 3',5'-monophosphate as a mediator of TRH-stimulated thyrotropin release.

It has been suggested that TRH stimulation of TSH release is mediated by the adenylate cyclase-cAMP system. To determine whether cAMP is a necessary intracellular messenger for TRH stimulation of TSH release, we have performed detailed studies of the TRH effect employing a nearly homogeneous population of mouse thyrotropic tumor cells in culture. Dibutyryl cAMP, methylisobutylxanthine, and cholera toxin caused an increase in TSH release which was additive to that of TRH.

Thyrotropin-releasing hormone stimulation of adrenocorticotropin production by mouse pituitary tumor cells in culture: possible model for anomalous release of adrenocorticotropin by thyrotropin-releasing hormone in some patients with Cushing's disease and Nelson's syndrome.

ACTH-producing mouse pituitary tumor cells in culture (AtT-20/NYU-1 cells) were found to have binding sites for thyrotropin-releasing hormone (TRH). These putative receptors bound TRH with high affinity; the apparent equilibrium dissociation constant was 3.7 nM.

Differential regulation of thyrotropin releasing hormone receptors in neoplastic rodent mammotropic, adrenocorticotropic and thyrotropic pituitary cells in culture.

Modulation of the concentration of receptors for thyrotropin releasing hormone (TRH) is different in neoplastic mammotropic, adrenocorticotropic, and thyrotropic pituitary cells in culture. Hydrocortisone increases the TRH receptor concentration in mammotropic and adrenocorticotropic cells but has no effect in thyrotropic cells. B-estradiol increases the TRH receptor number in mammotropic cells but has no effect in adrenocorticotropic and thyrotropic cells.

Thyrotropin releasing hormone stimulation of prolactin release. Evidence for a membrane potential-independent, Ca2+-dependent mechanism of action.

Thyrotropin releasing hormone (TRH) and high extracellular K+ induce prolactin release from rat pituitary cells (GH3 cells) in culture. TRH, as well as high K+ (50 mM), was shown to increase 45Ca2+ efflux from these cells. The effects of TRH and high K+ on membrane polarization was determined indirectly using the lipophilic cation, triphenylmethyl phosphonium ion (TPMP+).

Receptor affinity and biological potency of thyroid hormones in thyrotropic cells.

The nuclear receptor affinity for L-triiodothyronine (L-T3), L-thyroxine (L-T4), L-triiodothyroacetic acid (triac), and D-triiodothyronine (D-T3) was compared to the potency of these thyroid hormone analogues in regulating thyrotropin (TSH) production and the number of membrane receptors for thyrotropin-releasing hormone (TRH) in mouse thyrotropic tumor cells in culture. L-T3 and triac were equally potent and D-T3 was one-sixth to one-fifth as potent in binding to the receptor and in regulating TSH production and TRH receptor number. L-T4 was the least potent analogue in each instance, but its relative receptor-binding affinity, measured after 3 h, was significantly less than its somewhat variable relative biological potency, measured after 48 h.

Estrogens increase the number of thyrotropin-releasing hormone receptors on mammotropic cells in culture.

The number of plasma membrane receptors for TRH on tumor-derived mammotropic cells in culture, GH3 and GC cells, but not their affinity for TRH, was increased by estrogens. For GH3 cells, exposure to 10 nM 17 beta-estradiol for 48 h increased the receptor level from 54,000 to 90,000 sites/cell, while for GC cells, the number of receptors increased from 29,000 to 46,000 after 28 h. PRL accumulation in the medium was also increased by 17 beta-estradiol.

Bihormonal regulation of the thyrotropin-releasing hormone receptor in mouse pituitary thyrotropic tumor cells in culture.

Receptors for thyrotropin-releasing hormone (TRH) are present on mouse pituitary thyrotropic tumor cells. Incubation of thyrotropes with 100 nM TRH or 4 nM L-triiodothyronine (T3) for 48 h decreased the number of TRH receptors to approximately equal to 50 and 20% of control, respectively. There was no effect on the equilibrium dissociation constant which was 3-5 nM.