Calcitonin Inhibits Thyrotropin-Releasing Hormone Induced Increases in Cytosolic Ca2+in Isolated Rat Anterior Pituitary Cells*

Abstract

Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.