Thyrotropin-releasing hormone-induced cytosolic calcium transients: characterisation of store refilling in bovine anterior pituitary cells

The intracellular calcium ion concentration (Ca2+]i) in individual bovine anterior pituitary cells was measured using fura-2 and ratiometric imaging. Addition of thyrotropin-releasing hormone (TRH) in the presence of external calcium ion (Ca2+]e; 1 mM) caused a rapid transient increase in Ca2+]i falling to a plateau which remained above pre-stimulation levels in the continued presence of TRH. Decreasing Ca2+]e to 0.1 microM decreased Ca2+]i. At 0.1 microM Ca2+]e, the first TRH addition caused the rapid transient rise in Ca2+]i but no plateau phase and a second addition of TRH did not cause a second transient rise. However, the second application of TRH in 0.1 microM Ca2+]e caused a rise in Ca2+]i if it was preceded by transient exposure of the cells to 2 mM Ca2+]e. The presence of nitrendipine, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBHQ), or TRH during the re-exposure to external calcium blocked this recovery of subsequent responses to TRH in the presence of only 0.1 microM Ca2+]e. We conclude that refilling of the calcium stores depleted by TRH occurred only after the removal of agonist, used a tBHQ-sensitive uptake mechanism, and was mainly sustained by voltage-gated calcium entry into the cells.