Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells

The role of 3,5,3′-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca2+ concentration ([Ca2+]i) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH3 cells in the absence or presence of extracellular Ca2+. When applied in Ca2+-free solution, T2 and T3 increased [Ca2+]i, in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by NG-nitro-l-arginine methyl ester and l- n5-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca2+]i increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca2+ stores did not interfere with the T2- and T3-induced [Ca2+]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-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca2+]i and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca2+]i increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na+/Ca2+ exchanger by CGP37157 significantly reduced the [Ca2+]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 [Ca2+]i and intracellular Na+ concentration; nimodipine reduced the [Ca2+]i increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na+/H+ pump by 5-( N-ethyl- N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na+/Ca2+ 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.