Dual actions of 1,25-dihydroxycholecalciferol on intracellular Ca2+ in GH4C1 cells: evidence for effects on voltage-operated Ca2+ channels and Na+/Ca2+ exchange.
In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] amplifies the TRH-induced spike phase of increase in cytosolic free calcium ([Ca2+]i). In the present report we describe the results of investigations on the mechanisms of action of 1,25-(OH)2D3 on Ca2+ homeostasis in these cells. Pretreatment with 1 nM 1,25-(OH)2D3 for at least 24 h caused no change in basal uptake of 45Ca2+ compared with that in control cells or in 45Ca2+ uptake induced by the calcium channel agonist Bay K 8644. However, when the cells were depolarized with 50 mM K+, 1,25-(OH)2D3-treated cells showed an up to 90% enhancement of uptake (3-120 min) of 45Ca2+. An enhanced increase in [Ca2+]i was also observed in fura-2-loaded cells. The effect was specific and dose dependent for 1,25-(OH)2D3. The calcium channel antagonists nimodipine and verapamil inhibited completely the enhancing action of 1,25-(OH)2D3 as did the protein synthesis inhibitor cycloheximide. No enhanced uptake of 45Ca2+ into intracellular stores was detected when cells were incubated with 1,25-(OH)2D3. Na+/Ca2+ exchange was determined by measuring exchange of extracellular 45Ca2+ for intracellular Na+. Na+/Ca2+ exchange was dependent on intracellular Na+, was inactive when Li+ replaced Na+, was insensitive to calcium channel antagonists, and showed electrogenic properties. In cells incubated with 1,25-(OH)2D3 for at least 24 h, Na+/Ca2+ exchange was enhanced up to 54% compared with that in control cells. Enhanced exchange was dose dependent and specific for 1,25-(OH)2D3. Ca2+ channel antagonists were without effect while dichlorobenzamil inhibited partially the 1,25-(OH)2D3 enhancement of Na+/Ca2+ exchange. Cycloheximide abolished completely the action of 1,25-(OH)2D3 on Na+/Ca2+ exchange. We conclude that in GH4C1 cells, 1,25-(OH)2D3 enhances membrane calcium transport by modulating voltage-operated Ca2+ channels and activating Na+/Ca2+ exchange by mechanisms requiring new protein synthesis.
Tornquist
K
K
Laboratory of Toxicology, Harvard School of Public Health, Boston, Massachusetts.
Tashjian
A H
AH
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002762:Cholecalciferol / Q000494:pharmacology; D002999:Clone Cells; D003513:Cycloheximide / Q000494:pharmacology; D007700:Kinetics; D010902:Pituitary Gland; D051381:Rats; D019831:Sodium-Calcium Exchanger; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D015220:Calcium Channels; D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D002762:Cholecalciferol; D013973:Thyrotropin-Releasing Hormone; D003513:Cycloheximide; D002117:Calcitriol; D002118:Calcium