Abstract
The cerebellum of young rats contains significant 5′-deiodinase (5′-D) activity, but technical difficulties have made it impossible to identify the enzyme in cultured cerebellar astrocytes. We have developed a culture method which allows cerebellar astrocytes from 6-day-old rats to grow and develop 5′-D activity. Astrocytes cultured for 2 weeks in medium containing 3.25 µM reduced glutathione (GSH) and 0.21 µM vitamin E (VitE) as α-tocopherol had 5′-D activity which was stimulated by 1 mM dibutyryl cyclic adenosine monophosphate (dBcAMP) given 16 hours before measuring enzyme activity. Cells cultured without GSH and VitE showed little 5′-D activity, which was not stimulated by dBcAMP. Primary cultures of cerebellar astrocytes were cultured for four weeks with or without GSH + VitE, and stimulated by dBcAMP had high 5′-D activity, but were also sometimes contaminated with fibroblasts. The effect of such contamination on the astrocyte 5′-D activity was assessed by preparing primary cultures of fibroblasts from the meninges surrounding 6-day-old rat cerebella. They were grown in the same media and under the same conditions as the astrocytes. The cultured fibroblasts had 5′-D activity independent of GSH+VitE or culture time. The 5′-D activity of both cell populations could be type II 5′-deiodinase (5′-DII) because it was not inhibited by 6-n-propylthiouracil (PTU). Thus, cerebellar astrocytes cultured for 2 weeks in medium containing GSH and VitE have 5′-DII activity. Prolonged cultures favor enzyme activity, but also enhance contamination with fibroblasts, which may also show 5′-DII activity.
Key words
5′-Deiodinase - Astrocytes - Fibroblasts - Cerebellum - Neonate Rats
