Norepinephrine-induced elevation of acetyl coenzyme A in rat pineal glands in culture

Abstract

A micromethod for the determination of acetyl coenzyme A was developed and the relationship between the concentration of acetyl coenzyme A and the activity of rat pineal serotonin N-acetyltransferase was studied. Acetyl coenzyme A was determined by converting it into N-acetylserotonin using rat liver serotonin N-acetyltransferase. Subsequently, the hydroxy group of N-acetylserotonin was O-methylated by hydroxyindole- O-methyltransferase and S-[methyl- 3H]adenosyl- l-methionine to form [ 3H]melatonin, which was then conveniently separated from S-[methyl- 3H]adenosyl- l-methionine by thin-layer chromatography. The amount of radioactivity in melatonin is a measure of acetyl coenzyme A concentration. This method is sensitive and specific, since it can detect as low as 5 pmol of acetyl coenzyme A but not structurally related substances such as coenzyme A, adenosine diphosphate, cysteamine, d-panthothenic acid, or sodium acetate. After treating cultured rat pineal glands with l-norepinephrine (10 μM) for 6 hr, the concentration of acetyl coenzyme A was increased significantly from 3.26 ± 0.37 to 10.24 ± 0.93 pmol/gland, while the activity of serotonin N-acetyltransferase increased 68-fold. This result suggests that acetyl coenzyme A may play an important role in the norepinephrine-induced induction of serotonin N-acetyltransferase. Sensitivity and adaptability of this method can be utilized to measure acetyl coenzyme A in discrete regions of rat brain and in experimental conditions in which micromeasuremenf of acetyl coenzyme A may be required.