1,25-Dihydroxyvitamin D3 attenuates adenylyl cyclase activity in rat thyroid cells: reduction of thyrotropin receptor number and increase in guanine nucleotide-binding protein Gi-2 alpha.
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the most potent of the naturally occurring vitamin D metabolites. In rat thyroid FRTL-5 cells, 1,25-(OH)2D3 attenuated the increase in TSH-stimulated adenylyl cyclase activity obtained by removing TSH from the culture medium. When cells were incubated with 1,25-(OH)2D3 (10 nmol/liter; 4 days), the binding capacity for specific [125I]TSH binding decreased from 20.1 +/- 1.8 to 8.8 +/- 1.6 fmol/10(6) cells (mean +/- SEM; n = 4; P < 0.01) compared to that in control cells. The Kd did not change (mean +/- SEM, 0.46 +/- 0.09 vs. 0.25 +/- 0.07 nmol/liter; n = 4; P = NS). Western blotting revealed no change in the membrane content of the adenylyl cyclase (AC) stimulatory guanine nucleotide-binding protein (G-protein) alpha-subunit (Gs alpha) during 1,25-(OH)2D3 treatment. Similarly, levels of the AC inhibitory G-protein Gi-3 alpha- and G-protein beta-subunits were not altered by 1,25-(OH)2D3. However, Western blotting with antibodies recognizing both Gi-1 alpha and Gi-2 alpha was augmented 4-fold, presumably representing an increase in Gi-2 alpha only, as Gi-1 alpha messenger RNA (mRNA) was not detected in FRTL-5 cells. 1,25-(OH)2D3 (10 nmol/liter; 4 days) reduced cholera toxin (10 nmol/liter)-stimulated AC activity to 85% of the control value (P < 0.05), whereas forskolin (100 mumol/liter)-stimulated direct activation of AC was inhibited by 39%. The TSH receptor mRNA level correlated to the beta-actin mRNA was 2-fold higher in control cells compared to that in 1,25-(OH)2D3-treated cells 12 h after TSH removal. Only minor alterations in the Gs alpha mRNA/beta-actin mRNA and Gi-3 alpha mRNA/beta-actin mRNA ratios were observed during 1,25-(OH)2D3 treatment, whereas Gi-2 alpha mRNA increased 3-fold compared to that in control cells. No change in the resting intracellular Ca2+ concentration could be detected after 4 days of 1,25-(OH)2D3 treatment. Our studies show that 1,25-(OH)2D3 attenuates AC activity by reducing the TSH receptor number and increasing the level of the AC inhibitory G-protein Gi-2 alpha in FRTL-5 cells.
Hormone Laboratory, Aker Hospital, Oslo, Norway.
J P
JP
Berg
J A
JA
Sandvik
A H
AH
Ree
G
G
Sørnes
T
T
Bjøro
P A
PA
Torjesen
J O
JO
Gordeladze
E
E
Haug
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003470:Culture Media; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007457:Iodine Radioisotopes; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism*; D013961:Thyroid Gland / Q000187:drug effects / Q000201:enzymology*; D013972:Thyrotropin / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology
D000199:Actins; D003470:Culture Media; D007457:Iodine Radioisotopes; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D005576:Colforsin; D013972:Thyrotropin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002117:Calcitriol