FURA-2 IMAGING OF THYROTROPIN-RELEASING-HORMONE AND DOPAMINE EFFECTS ON CALCIUM HOMEOSTASIS OF BOVINE LACTOTROPHS

Dual wavelength digital imaging microscopy to detect fura-2 has been employed to characterize in normal bovine PRL-secreting cells the effects of TRH and dopamine on the intracellular ionized calcium concentration ([Ca2+]i). Concentrations of TRH greater than 10 nM caused a rapid but transient increase in [Ca2+]i, arising mainly from intracellular calcium stores, since it was unaffected by lowering extracellular calcium with EGTA or blocking calcium channels with Co2+. The threshold for TRH action was close to 0.1 nM. TRH action was dose dependent, with lower concentrations (< 1-10 nM) slowing the time to peak [Ca2+]i response. The TRH-induced [Ca2+]i rise had a Q10 of about 2. TRH caused multiple transient increases in [Ca2+]i, but a recovery time of 10-15 min was required for full restoration of the TRH-induced response. In some cells the [Ca2+]i response to TRH was polarized to one region of the cell, suggesting the following possibilities, none of them exclusive: 1) Ca2+ release sites may be localized within the cell; or 2) an efficient local mechanism exists for lowering Ca2+ once it is liberated inside the cells; or 3) barriers may exist to diffusion of Ca2+ released within the cell. Extracellular application of Co2+, Mn2+, and EGTA under basal conditions resulted in lowering of [Ca2+]i within seconds, consistent with tonic Ca2+ influx under resting conditions which could contribute to the basal release of hormone. Dopamine, a PRL release-inhibiting factor, also lowered [Ca2+]i under basal conditions. However, the [Ca2+]i response of lactotrophs to TRH was unaffected by dopamine. This suggests that dopamine and TRH act via separate intracellular pathways to modulate hormone secretion. Applications of forskolin preceding the TRH-induced transient rise in [Ca2+]i resulted in a prolonged plateau rise in [Ca2+]i. This was mainly due to increased influx of Ca2+ since addition of Co2+ or EGTA-containing or Ca2+-free medium during this phase of response lowered the plateau concentration of [Ca2+]i.