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M J Rebecchi

Publications and source records attributed to M J Rebecchi.

28 records · Page 2Linked to original sources
PubMed1983

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. Inositol 1,4-bisphosphate was increased to a peak of 450% at 30 s; inositol 1-monophosphate and inositol were not elevated until 30 s and 1 min respectively after TRH addition. To study whether PtdIns(4,5)P2 hydrolysis may be caused by an elevation in cytosolic Ca2+ concentration, the changes induced by TRH in the levels of inositol sugars were compared with the effects of membrane depolarization by high extracellular [K+]. The elevation in cytosolic [Ca2+] induced by K+ depolarization did not change the level of inositol 1,4,5-trisphosphate. These data suggest that phosphodiesteratic hydrolysis of PtdIns(4,5)P2 may be the initial event in TRH stimulation of inositol lipid metabolism in GH3 cells and that PtdIns(4,5)P2 hydrolysis is not stimulated by an elevation in cytosolic Ca2+ concentration. The decreases in PtdIns4P and PtdIns may be due to enhanced conversion of PtdIns into PtdIns4P into PtdIns(4,5)P2 or to their direct hydrolysis by phosphomonoesterases and/or phosphodiesterases. These results are consistent with the hypothesis that TRH-stimulated PtdIns(4,5)P2 breakdown causes Ca2+ mobilization leading to prolactin secretion.

Animals↗
PubMed1982

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. Cells were perfused in medium containing 2.8 microM Ca2+ and nonradioactive Ca2+ was measured in the perfusion effluent. Under these conditions, there was a sustained loss of Ca2+ from the cells for at least 30 min. TRH caused a transient, marked increase in the amount of Ca2+ released into the medium which occurred in parallel with enhancement in 45Ca2+ efflux and stimulation of prolactin secretion. There was no measurable decrease in Ca2+ concentration in the medium at the onset of the TRH effect which would have been consistent with Ca2+ influx into the cells. Furthermore, an identical response to TRH was observed in cells perfused with medium containing 50 microM verapamil, an agent which blocks Ca2+ influx. In static incubations performed in parallel, TRH caused a decrease in total cellular Ca2+ of 23 +/- 5%. These data provide direct evidence that TRH causes loss of Ca2+ from GH3 cells without causing measurable Ca2+ uptake and support the contention that TRH acts by mobilizing Ca2+ from a sequestered cellular pool (or pools).

Animals↗
PubMed1981

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. The increment in (45)Ca(2+) efflux induced by 50 mM K(+) during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca(2+) from 1,500 to 0.02 muM (by adding EGTA), whereas 1 muM TRH enhanced (45)Ca(2+) efflux similarly over the entire range of extracellular Ca(2+) concentrations. Although 50 mM K(+) caused release of 150 ng prolactin from 40 x 10(6) GH(3) cells exposed to 1,500 muM Ca(2+) (control), reduction of extracellular Ca(2+) to 2.8 muM decreased prolactin release caused by high K(+) to <3% of controls and no prolactin release was detected after exposure to 50 mM K(+) in medium with 0.02 muM free Ca(2+). In contrast, TRH caused release of 64 ng of prolactin from 40 x 10(6) GH(3) cells exposed to medium with 1,500 muM Ca(2+), and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 muM Ca(2+), respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of (45)Ca(2+) from GH(3) cells in static incubations with 1,500 or 3.5 muM Ca(2+), hereby confirming that the enhanced (45)Ca(2+) efflux caused by TRH in both low and high Ca(2+) medium was not an artifact of the perifusion system.Data obtained with chlortetracycline (CTC), a probe of membrane-bound Ca(2+), were concordant with those obtained by measuring (45)Ca(2+) efflux. Cellular fluorescence of CTC varied with the extracellular Ca(2+) concentration and the duration of incubation. TRH decreased the fluorescence of cell-associated CTC in a manner strongly suggesting stimulus-induced mobilization of Ca(2+), and this effect was still demonstrable in GH(3) cells incubated in 50 mM K(+). These data suggest that TRH acts to mobilize sequestered cell-associated Ca(2+) reflected as a (45)Ca(2+) efflux which is independent of the extracellular Ca(2+) concentration. Mobilization of sequestered Ca(2+) into the cytoplasm may elevate free intracellular Ca(2+) and serve to couple stimulation by TRH to secretion of prolactin.

Animals↗
PubMed1980

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. The affinity of the receptors for a series of TRH analogues was similar to those previously reported for TRH-receptor interactions on thyrotropic and mammotropic cells in culture. Like some human pituitary tumors in situ, AtT-20/NYU-1 cells were found to produce the alpha subunit of the glycoprotein hormones (alpha). Alpha accumulation in the medium was constant (3.1 ng/mg cell protein per h) and was not affected by TRH. In contrast, TRH increased the amount of ACTH accumulated in the medium from AtT-20/NYU-1 cells to 190 and 420% of control at 1 and 24 h, respectively. TRH induced a dose-dependent increase in ACTH release during a 30-min incubation; half-maximal stimulation occurred at approximately 0.1 nM. TRH had no effect on ACTH release in vitro from anterior pituitary cells derived from normal rats. Because TRH stimulates release of ACTH in some untreated patients with Cushing's disease and Nelson's syndrome as well as pathological states associated with pituitary tumors (but not in normal subjects), AtT-20/NYU-1 cells may serve as an important in vitro model for human pituitary ACTH-secreting adenomas. Moreover, these findings suggest that the primary abnormality in Cushing's disease and Nelson's syndrome, allowing TRH stimulation of ACTH release, may be intrinsic to neoplastic adrenocorticotrophs rather than in neuroregulation of ACTH release.

Adrenocorticotropic Hormone↗
PubMed1980

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. TRH stimulated TSH release in a dose-dependent fashion; half-maximal stimulation occurred at approximately 0.6 nM but had no effect on total intracellular cAMP levels measured in the presence or absence of methylisobutylxanthine. There was no correlation between total intracellular cAMP levels and TSH release after 1 h. Moreover, there was no effect of TRH on protein kinase-bound or total intracellular cAMP levels at 1, 5, or 60 min of incubation. Lastly, TRH had no effect on adenylate cyclase activity in homogenates of thyrotropic cells in the presence or absence of guanylylimidodiphosphate. These results suggest that stimulation of TRH release by TRH from these cells does not involve cAMP as an intracellular messenger.

1-Methyl-3-isobutylxanthine↗
PubMed1979

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. The cells were shown to monodeiodinate L-[125I]T4 to L-[125I]T3 in a time-dependent manner, and the enhanced biological potency of L-T4 was ascribed to its conversion to L-T3. Thyroid hormones appear to regulate TSH production and the number of receptors for TRH in thyrotropic cells in culture through interaction with a nuclear receptor.

Animals↗