Physiological de novo thyroid hormone formation in primary culture of porcine thyroid follicles: adenosine 3',5'-monophosphate alone is sufficient for thyroid hormone formation.
We describe a method of culturing intact porcine thyroid follicles for physiological de novo thyroid hormone formation; the roles of cAMP and protein kinase-C in thyroid hormone formation were also studied. Thyroid follicles were obtained by digesting minced porcine thyroid tissue with 0.04% collagenase and cultured in Coon's Modified Ham's F-12 medium supplemented with 0.5% calf serum, 0.5 mU/ml TSH, other standard hormones, and 3 antibiotics (6H medium). On the fourth day of culture, 6000-8000 follicles/well were plated in 12-well culture dishes. On the sixth day, thyroid hormone formation was carried out by incubating thyroid follicles with 0.5 microM KI in the presence of 6H medium for 2 days in a 5% CO2-95% air incubator at 37 C. To examine the effects of cAMP and protein kinase-C on de novo thyroid hormone formation, follicles were incubated with KI in the presence of 1-2.5 mM (Bu)2cAMP, 10 microM forskolin, 2 microM prostaglandin E2 (PGE2), or 0.5-1 microM 12-O-tetradecanoylphorbol-13-acetate in TSH-free medium for 2 days. The amount of newly formed thyroid hormone was measured by RIA of T3 content in the Pronase digest of thyroid follicular cells. Thyroid follicles cultured in 6H medium had normal polarity of the membrane, determined by electron microscope, and thyroid cAMP was responsive to the alteration of TSH. In this culture system cAMP alone was sufficient to form thyroid hormone. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase-C stimulator, disrupted thyroid follicles and inhibited cAMP-mediated thyroid hormone formation. The integrity of follicular structure was also required for thyroid hormone formation in this culture system. This study introduces perhaps the most physiological culture system for de novo thyroid hormone formation. Our data provide direct evidence that thyroid hormone formation is linked to cAMP and that the protein kinase-C system acts as an inhibitor of thyroid hormone formation.
Murakami
S
S
Medical, Service, Wadsworth Veterans Administration Hospital, Los Angeles, California 90073.
Summer
C N
CN
Iida-Klein
A
A
Anderson
D G
DG
Sugawara
M
M
D000818:Animals; D046508:Culture Techniques; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D011188:Potassium / Q000097:blood; D011193:Potassium Iodide / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D013552:Swine; D013961:Thyroid Gland / Q000033:anatomy & histology / Q000378:metabolism* / Q000648:ultrastructure; D013963:Thyroid Hormones / Q000096:biosynthesis*; D013972:Thyrotropin / Q000494:pharmacology
D013963:Thyroid Hormones; D011193:Potassium Iodide; D013972:Thyrotropin; D000242:Cyclic AMP; D011493:Protein Kinase C; D011188:Potassium