CONTROL OF CA2-RELEASING-HORMONE( ENTRY INTO RAT LACTOTROPHS BY THYROTROPIN)

1. Lactotrophs are adenohypophysial cells that synthesize and secrete prolactin (PRL), a hormone principally involved in mammalian milk prod uction. An increase in the intracellular Ca2+ concentration ([Ca2+](i) ) is an important signal for PRL secretion. Thyrotrophin-releasing hor mone (TRH) generates Ca2+ signals derived from both the release of Ca2 + from intracellular stores and the entry of extracellular Ca2+, the l atter being particularly important for PRL secretion. The identity of this TRH-sensitive Ca2+ entry pathway is unknown and therefore the sub ject of the present study. 2. [Ca2+](i) was measured by video imaging of fura-2 loaded into single rat anterior pituitary cells. Ca2+ influx was detected by quenching of fura-2 fluorescence by external Mn2+. Al l data are from lactotrophs isolated from lactating female rats. Indiv idual lactotrophs were identified by postexperimental immunofluorescen t detection of PRL in fixed cells. 3. TRH induced the release of Ca2from intracellular stores and also stimulated Mn2+-permeable Ca2+ infl ux. U73122 (1 mu M), a phospholipase C inhibitor, prevented the Ca2+-m obilizing actions of TRH. The chemically similar but inactive analogue , U73343 (1 mu M), had no effect on TRH responses. U73122 did not act as a global G protein inhibitor because the reduction of basal [Ca2+]( i) by dopamine (1 mu M, and G protein-mediated event) was not affected . 4. TRH-stimulated Mn2+ influx occurred either immediately after addi tion of TRH (early entry) or after a delay of about 130 a (late entry) . There were no statistically significant differences in the magnitude or temporal characteristics of the Ca2+ signals evoked from cells sho wing early or late Mn2+ entry. 5. The identity of Ca2+ channels permea ble to Mn2+ was investigated. Cell depolarization with 50 mM KCl stimu lated Ca2+/Mn2+ influx and was prevented by nifedipine (1 mu M). Bay K 8644 (1 mu M) also stimulated Mn2+ influx. Thus, the presence of Mn2-permeable L-type voltage-operated Ca2+ channels is likely. A second M n2+-permeable pathway was present in lactotrophs. Depletion of Ca2+ st ores by thapsigargin (1 mu M) stimulated a Ca2+ signal and Mn2+ influx . This 'capacitative entry pathway' was insensitive to nifedipine (1 m u M), indicating that putative L-type Ca2+ channels were not activated . 6. TRH-stimulated Mn2+ influx was not prevented by nifedipine (1 mu M). TRH added during KCl-induced Mn2+ influx reduced the quench rate w ithin the time frame of the TRH-induced Ca2+ spike. TRH may therefore inhibit putative L-type Ca2+ channels. 7. Addition of thapsigargin in Ca2+-free medium transiently increased [Ca2+](i) and prevented subsequ ent Ca2+ responses to TRH. The release of stored Ca2+ by TRH was there fore entirely sensitive to thapsigargin. This indicates that the Ca2pool released by TRH and thapsigargin was the same. In the presence of extracellular Ca2+, TRH-induced responses were completely abolished 1 5 min after the treatment with thapsigargin. TRH therefore stimulated a Ca2+ influx component sensitive to thapsigargin. 8. In conclusion, t he data suggest that TRH-generated Ca2+ signals are composed of the re lease of Ca2+ from thapsigargin-sensitive stores and the consequent in flux of Ca2+ via a capacitative entry pathway. Voltage-operated Ca2+ c hannels (probably L-type) are inhibited by TRH and do not contribute g reatly to the response.