Intracellular and plasma membrane-initiated pathways involved in the [Ca~(2+)]_i elevations induced by iodothyronines (T3 and T2) in pituitary GH_3 cells

摘要

The role of 3,5,3'-triiodo-L-thyronine (T3) and its metabolite 3,5-diiodo-L-thyronine (T2) in modulating the intracellular Ca~(2+) concentration ([Ca~(2+)]_i) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH3 cells in the absence or presence of extracellular Ca~(2+). When applied in Ca~(2+)-free solution, T2 and T3 increased [Ca~(2+)]_i, in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by N~G-nitro-L-arginine methyl ester and L-N~5-(l-iminoethy-l)ornithine hydrochloride significantly reduced the [Ca~(2+)]_i increase induced by T2 and T3. However, while depletion of inositol trispho-sphate-dependent Ca~(2+) stores did not interfere with the T2- and T3-induced [Ca~(2+)]_i increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluorome-thoxy)phenylhydrazone prevented the increases in both [Ca~(2+)]_i and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca~(2+)]_i increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na~+/Ca~(2+) exchanger by CGP37157 significantly reduced the [Ca~(2+)]_i increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca~(2+)]_i and intracellular Na~+ concentration; nimodipine reduced the [Ca~(2+)]_i increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na~+/H~+ pump by 5-(N-ethyl-iV-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na~+/Ca~(2+) exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short-term nongenomic effects on intracellular calcium and NO by modulating plasma membrane and mitochondrial pathways that differ between these iodothyronines.