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 (less than 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 Ca(2+)-free medium during this phase of response lowered the plateau concentration of [Ca2+]i.
Akerman
S N
SN
Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Babraham, Cambridge.
Zorec
R
R
Cheek
T R
TR
Moreton
R B
RB
Berridge
M J
MJ
Mason
W T
WT
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D004298:Dopamine / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006706:Homeostasis / Q000187:drug effects; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000008:administration & dosage / Q000494:pharmacology*
D005456:Fluorescent Dyes; D005576:Colforsin; D003035:Cobalt; D008345:Manganese; D004533:Egtazic Acid; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D002118:Calcium; D016257:Fura-2; D004298:Dopamine