| Abstract: | Thyrotropin-releasing hormone stimulation of prolactin secretion from rat pituitary (GH3) cells is biphasic with a secretory burst (0-2 min) at a higher rate, followed by sustained secretion (beyond 2 min) at a lower rate. Based on the effects of calcium ionophores, K+ depolarization, and diacylglycerol (or phorbol esters), it was suggested that the secretory burst is dependent on elevation of cytoplasmic free calcium concentration ( Ca2+]i) whereas sustained secretion is mediated by lipid-activated protein phosphorylation. In this study, we pretreated GH3 cells with 0.03 mM arachidonic acid to abolish thyrotropin-releasing hormone-induced elevation of Ca2+]i (Kolesnick, R. N., and Gershengorn, M. C. (1985) J. Biol. Chem. 260, 707-713). In control cells, basal secretion was 0.7 +/- 0.2 ng/10(6) cells/min which increased to 8.3 +/- 0.8 between 0 and 2 min after TRH and remained elevated at 3.3 +/- 0.2 between 2-10 min. In cells pretreated with arachidonic acid, TRH stimulated prolactin secretion to only 2.6 +/- 0.3 ng/10(6) cells/min between 0 and 2 min and to 3.2 +/- 0.2 between 2 to 10 min; these values are not different from each other nor from the response between 2 and 10 min in control cells. K+ depolarization, which elevates Ca2+]i even in arachidonic acid-pretreated cells but does not affect lipid metabolism, caused only a secretory burst. Bovine serum albumin, which binds free arachidonic acid and reverses arachidonic acid inhibition of TRH-induced elevation of Ca2+]i, reversed the inhibition of the secretory burst stimulated by TRH. These studies present direct evidence that the burst of prolactin secretion stimulated by TRH is dependent on an elevation of Ca2+]i whereas the sustained phase of secretion is independent of such elevation. |
