1,25-DIHYDROXYVITAMIN D-3 ATTENUATES ADENYLYL-CYCLASE ACTIVITY IN RAT-THYROID CELLS - REDUCTION OF THYROTROPIN RECEPTOR NUMBER AND INCREASEIN GUANINE-NUCLEOTIDE-BINDING PROTEIN G(I-2)ALPHA

1,25-Dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] is the most potent of the naturally occurring vitamin D metabolites. In rat thyroid FRTL-5 cells , 1,25-(OH)(2)D-3 attenuated the increase in TSH-stimulated adenylyl c yclase activity obtained by removing TSH from the culture medium. When cells were incubated with 1,25-(OH)(2)D-3 (10 nmol/liter; 4 days), th e binding capacity for specific [I-125]TSH binding decreased from 20.1 +/- 1.8 to 8.8 +/- 1.6 fmol/10(6) cells (mean +/- SEM; n = 4; P < 0.0 1) compared to that in control cells. The K-d did not change (mean +/- SEM, 0.46 +/- 0.09 vs. 0.25 +/- 0.07 nmol/liter; n = 4; P = NS). West ern blotting revealed no change in the membrane content of the adenyly l cyclase (AC) stimulatory guanine nucleotide-binding protein (G-prote in) alpha-subunit (G(s) alpha) during 1,25-(OH)(2)D-3 treatment. Simil arly, levels of the AC inhibitory G-protein G(i.3)alpha- and G-protein beta-subunits were not altered by 1,25-(OH)(2)D-3. However, Western b lotting with antibodies recognizing both G(i-1)alpha and G(i-2)alpha w as augmented 4-fold, presumably representing an increase in G(i.2)alph a only, as G(i-1)alpha messenger RNA (mRNA) was not detected in FRTL-5 cells. 1,25-(OH)(2)D-3 (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 mu mol/liter)-stimulated direct activa tion of AC was inhibited by 39%. The TSH receptor mRNA level correlate d to the beta-actin mRNA was 2-fold higher in control cells compared t o that in 1,25-(OH)(2)D-3-treated cells 12 h after TSH removal. Only m inor alterations in the G(i-3)alpha mRNA/beta-actin mRNA and G(i-3)alp ha mRNA/beta-actin mRNA ratios were observed during 1,25-(OH)(2)D-3 tr eatment, whereas G(i-2)alpha mRNA increased 9-fold compared to that in control cells. No change in the resting intracellular Ca2+ concentrat ion could be detected after 4 days of 1,25-(OH)(2)D-3 treatment. Our s tudies show that 1,25-(OH)(2)D-3 attenuates AC activity by reducing th e TSH receptor number and increasing the level of the AC inhibitory G- protein G(i-2)alpha in FRTL-5 cells.