Intracellular transport of 3,5,3'-triiodo-L-thyronine in rat skeletal myoblasts.
Since in skeletal muscle circulating L-T3 is the only source for the hormone bound to nuclei, we investigated the intracellular and intranuclear transport of L-T3 in L6E9 rat skeletal muscle cells. The characteristics of this process were assessed by analyzing the nuclear bound L-T3 as a marker of the internalized hormone and by determining the initial rate of L-T3 uptake. [125I]L-T3 cellular uptake at 37 C reached a plateau at 2 h when the nuclear uptake, after an initial lag phase, was still increasing and represented 4.7% of the cellular uptake. Incubation at 4 C caused [125I]L-T3 cellular uptake to decrease by 77% and nuclear uptake to be abolished. A similar effect on [125I]L-T3 nuclear uptake was obtained after myoblasts were incubated at 37 C with a 1000-fold excess of unlabeled L-T3. The addition of various inhibitors of ATP production, cytoskeleton integrity, endocytosis, and Na+, K+-ATPase that did not interfere with [125I]L-T3 binding to the cell surface or to isolated nuclei caused a dose-dependent reduction of both extranuclear and nuclear uptake, ranging from 34-85%. Scatchard analysis revealed the presence on the myoblast surface of L-T3 high affinity (Ka = 1.6 X 10(9) M-1) and low affinity (Ka = 5.4 X 10(6) M-1) binding sites; other iodothyronines exhibited lower affinity for both sites. Kinetic analysis of L-T3 transport after 60-sec incubation at 23 C defined a process with a Km of 17 +/- 5.6 nM and a maximum velocity of 83 +/- 35 pmol/mg DNA. These results indicate the existence in rat myoblasts of a temperature-dependent, energy-requiring, saturable, and stereospecific L-T3 uptake mechanism, probably mediated through an endocytotic pathway, located on the myoblast plasma membrane, that may regulate L-T3 action in skeletal muscle.
Pontecorvi
A
A
Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Lakshmanan
M
M
Robbins
J
J
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000378:metabolism*; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism*
D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine