Thyrotropin-releasing hormone and epidermal growth factor stimulate prolactin synthesis by a pathway(s) that differs from that used by phorbol esters: dissociation of actions by calcium dependency and additivity.
TRH, epidermal growth factor (EGF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulate PRL synthesis by GH4C1 rat pituitary cells. Recent evidence suggests that TPA activates directly phospholipid- and calcium-dependent protein kinase C in other cell types and that TRH might act analogously by altering phospholipid metabolism in GH4C1 cells. To examine the pathways by which these three agents stimulate PRL synthesis, we determined their calcium dependencies as well as their combined effects on PRL production. By equilibration of GH4C1 cells in a protein-free medium for 24 h, the free cytosolic calcium concentration ([Ca2+]i) was found to increase (from 90 to 360 nM) when the extracellular calcium concentration ([Ca2+]e) was varied from 15 to 800 microM. Basal PRL production increased in parallel (from 1 to 4 micrograms/ml X 24 h). TPA-stimulated PRL production was highly calcium dependent and required 180 nM [Ca2+]i for maximal enhancement. TRH-stimulated PRL production was constant between 10 and 660 microM [Ca2+]e, whereas EGF stimulated PRL production to a similar extent as TRH at 10 microM [Ca2+]e, but continued to enhance production with increasing [Ca2+]e. TRH elevated [Ca2+]i acutely, and at [Ca2+]e greater than 100 microM caused both a burst and a plateau phase in elevated [Ca2+]i. At lower [Ca2+]e, at which TRH still caused a maximal stimulation of PRL production, only the burst phase of [Ca2+]i occurred. When cultures were treated with any combination of maximally effective concentrations of TPA, TRH, or EGF, PRL production was increased by additive increments. The additive actions of TPA and TRH could not be explained by a calcium-promoted increase in TPA-stimulated PRL production. We conclude that TPA stimulates PRL production by a highly calcium-dependent pathway and that TRH and EGF stimulate PRL production by a different pathway(s) requiring lower [Ca2+]i.
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000502:physiology; D010704:Phorbols / Q000494:pharmacology*; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism; D011388:Prolactin / Q000096:biosynthesis*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D007295:Inositol Phosphates; D010704:Phorbols; D013973:Thyrotropin-Releasing Hormone; D004815:Epidermal Growth Factor; D011388:Prolactin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium