Pretreatment with 1,25-dihydroxycholecalciferol enhances thyrotropin-releasing hormone- and inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ in permeabilized GH4C1 pituitary cells

Abstract

In GH4C1 cells 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] has been shown to enhance the TRH- and bombesin-induced increase in intracellular Ca2+ ([Ca2+]i). The aim of the present study was to investigate whether this increase in [Ca2+]i could be due to enhanced release of sequestered Ca2+ in cells pretreated with 1,25-(OH)2D3. In digitonin-permeabilized cells, the addition of 10 microM inositol 1,4,5-trisphosphate (IP3) rapidly increased free Ca2+ ([Ca2+]) to 50 +/- 10 nM (mean +/- SE) in cells pretreated with 1 nM 1,25-(OH)2D3 for 24 h, compared with 25 +/- 5 in control cells (P < 0.05). Furthermore, stimulating permeabilized cells with TRH increased [Ca2+]. The increase in control cells was 20 +/- 2, compared with 55 +/- 11 in cells pretreated with 1,25-(OH)2D3 (P < 0.05). Repeated additions of IP3 resulted in an attenuation of the response of [Ca2+] in both control cells and cells pretreated with 1,25-(OH)2D3. However, only the first addition of IP3 resulted in an enhanced increase in [Ca2+] in cells pretreated with 1,25-(OH)2D3 compared with control cells. If the cells were stimulated first with TRH and then with IP3, no difference in the [Ca2+] response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Furthermore, if cells were stimulated with IP3 and then with TRH, no difference in the [Ca2+] response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Stimulating the permeabilized cells with thapsigargin resulted in an increase in [Ca2+]. However, no difference in the response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Addition of GTP or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) had no effect on [Ca2+]. The results suggest that 1,25-(OH)2D3 has a modulatory effect on an IP3-sensitive intracellular Ca2+ pool in GH4C1 cells.