Endocytosis and degradation of ovine prolactin by Nb2 lymphoma cells: characterization and effects of agents known to alter prolactin-induced mitogenesis.

Rat Nb2 node lymphoma cells proliferate in response to lactogens, but the signal transduction mechanism involved remains unclear. Specific binding, internalization, and degradation of ovine PRL (oPRL) were examined under a variety of experimental conditions to characterize the metabolism of receptor-bound hormone by these cells. Stationary-phase cells were incubated with [125I]oPRL in Fischer's medium containing horse serum. Cell suspensions were centrifuged, and the cell pellets were assayed to determine specific cell-associated radioactivity. Internalized ligand was measured by exposing the cells to an acidic buffer before centrifugation to dissociate hormone from plasma membrane receptors, and cell-surface ligand was calculated by subtracting internalized hormone from the total [125I]oPRL bound by the cells. Hormone degradation was assessed by measuring the radioactivity in an acid-soluble fraction prepared from the incubation medium. Endocytosis of [125I]oPRL was observed within 30 min at 37 C, and the internalized component accounted for approximately 50% of the bound hormone under steady-state conditions. Hormone degradation was detectable within 1 h at 37 C and continued at a relatively linear rate thereafter; by 4 h, 8% of the added [125I]oPRL was acid soluble. Chloroquine (0.2 mM), methylamine (20 mM) and monensin (20 microM) prevented [125I]oPRL degradation and elevated both cell-surface and intracellular hormone 2-fold during a 4-h incubation. Leupeptin (0.2 mM) decreased degradation by only 15% under the same conditions. Phorbol 12-myristate 13-acetate (PMA; 20 nM), a comitogen for lactogen-stimulated Nb2 cells, increased cell-surface hormone by 20% and decreased intracellular hormone by a corresponding amount 1 h after administration. Calcium ionophore A23187 (1 microM) produced similar changes, and a synergistic effect was noted when cells were exposed to both agents for 4 h. Amiloride (125 microM), an inhibitor of Nb2 cell mitogenesis, decreased [125I]oPRL degradation by 25% during a 4-h incubation. This response was abolished when the cells were exposed simultaneously to PMA. These experiments demonstrate that receptor-bound oPRL is rapidly internalized and extensively degraded via the endosome-lysosome pathway when Nb2 cells are maintained at 37 C. The inhibitory effect of PMA on oPRL internalization may help to explain the comitogenic action of this phorbol on Nb2 cells. Since amiloride also produced major changes in oPRL metabolism, post-binding events in lactogen processing by target cells could play an important role in the mitogenic response elicited by such hormones.

Thyroid hormone effects on protein and RNA metabolism in the rat anterior pituitary.

Thyroidectomy reduced the incorporation of [3H]amino acids into total rat pituitary proteins as well as into electrophoretic fractions corresponding to growth hormone (GH) and prolactin. A single injection of thyroxine (200 microgram/kg) reversed the effects of thyroidectomy on the GH (12 h) and prolactin (36 h) fractions. Evidence for a general increase in the rate of pituitary protein synthesis following this treatment was less conclusive, but two injections of the hormone had a definite stimulatory effect. [3H]Uridine uptake by the pituitary and its incorporation into RNA were elevated for 2 weeks after thyroidectomy. Thyroxine failed to suppress these changes for at least 36 h after injection. Actinomycin C (400 microgram/kg) inhibited pituitary RNA synthesis and blocked the stimulatory effect of thyroxine on amino acid incorporation into the GH fraction. These results indicate that thyroid hormones: (1) promote pituitary protein synthesis, probably as a consequence of their effects on somatotrophs and lactotrophs; (2) exert a stimulatory effect on GH synthesis by affecting transcription.

Prolactin and growth hormone synthesis: effects of perphenazine, alpha-methyltyrosine and estrogen in different thyroid states.

The effects of thyroid hormones on prolactin (PRL) and growth hormone (GH) synthesis by the rat anterior pituitary gland were assessed in vitro. A marked reduction (84-87%) in the rate of H3-leucine incorporation into GH was evident 2-4 weeks after thyroidectomy, while incorporation into PRL was 52-71% less than that measured in glands from intact rats. A single injection of T4 (200 mug/kg) administered to thyroidectomized (THX) rats 48 hr before sacrifice significantly increased incorporation into both pituitary hormones, although the stimulation of GH synthesis was much more dramatic. Perphenazine, alpha-methyltyrosine and estrogen enhanced the rate of PRL synthesis in intact rats. Thyroid ablation did not affect the response to perphenazine, but significantly increased the response to alpha-methyltyrosine and estrogen. On the other hand, administration of T4 to THX rats receiving perphenazine, alpha-methyltyrosine or estrogen diminished the stimulatory influence of these treatments on PRL synthesis. Perphenazine, alpha-methyltyrosine and estrogen had no effect on the rate of GH synthesis in THX rats, nor did they alter the ability of T4 to restore GH synthesis in these animals. These results indicate that GH synthesis in the rat is dependent upon thyroid hormones and support the concept that these hormones exert their stimulatory effect directly on pituitary somatotrophs. Pituitary lactotrophs, however, appear to retain much of their capacity to synthesize PRL under conditions of thyroid deficiency. The changes in pituitary PRL levels and synthesis rate induced by thyroid ablation might reflect differences in the number rather than the activity of these cells.