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

1. Lactotrophs are adenohypophysial cells that synthesize and secrete prolactin (PRL), a hormone principally involved in mammalian milk production. An increase in the intracellular Ca2+ concentration ([Ca2+](i)) is an important signal for PRL secretion. Thyrotrophin-releasing hormone (TRH) generates Ca2+ signals derived from both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+, the latter being particularly important for PRL secretion. The identity of this TRH-sensitive Ca2+ entry pathway is unknown and therefore the subject 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+. All data are from lactotrophs isolated from lactating female rats. Individual lactotrophs were identified by postexperimental immunofluorescent detection of PRL in fixed cells. 3. TRH induced the release of Ca2+ from intracellular stores and also stimulated Mn2+-permeable Ca2+ influx. U73122 (1 mu M), a phospholipase C inhibitor, prevented the Ca2+-mobilizing 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 addition 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 showing early or late Mn2+ entry. 5. The identity of Ca2+ channels permeable to Mn2+ was investigated. Cell depolarization with 50 mM KCl stimulated 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 Mn2+-permeable pathway was present in lactotrophs. Depletion of Ca2+ stores by thapsigargin (1 mu M) stimulated a Ca2+ signal and Mn2+ influx. This 'capacitative entry pathway' was insensitive to nifedipine (1 mu 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 within 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 subsequent Ca2+ responses to TRH. The release of stored Ca2+ by TRH was therefore entirely sensitive to thapsigargin. This indicates that the Ca2+ pool released by TRH and thapsigargin was the same. In the presence of extracellular Ca2+, TRH-induced responses were completely abolished 15 min after the treatment with thapsigargin. TRH therefore stimulated a Ca2+ influx component sensitive to thapsigargin. 8. In conclusion, the data suggest that TRH-generated Ca2+ signals are composed of the release of Ca2+ from thapsigargin-sensitive stores and the consequent influx of Ca2+ via a capacitative entry pathway. Voltage-operated Ca2+ channels (probably L-type) are inhibited by TRH and do not contribute greatly to the response.