Measurement of adenylate cyclase activity in the minute bovine ciliary epithelial cells during the pharmacological stimulation of adrenergic and cholinergic receptors
✍ Scribed by Norifumi Sawada; Atsushi Sugiyama; Kenji Kashiwagi; Shigeo Tsukahara; Keitaro Hashimoto
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 87 KB
- Volume
- 13
- Category
- Article
- ISSN
- 0887-8013
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✦ Synopsis
Although essential to the secretion of aqueous humor, little is known about the signal transduction underlying postreceptor adrenergic and cholinergic processes in the ciliary epithelium. We adopted a highly sensitive fluorometric assay technique in order to examine adenylate cyclase activity in minute membrane preparations made from the bovine ciliary epithelial cells. The protein concentration of the preparation was 3-5 mg/ ml. Norepinephrine (10 -7 , 10 -6 and 10 -5 M) and carbachol (10 -7 and 10 -5 M) were incubated with 10 ml of membrane preparation to analyze the extent of the receptor-coupled influences on the adenylate cyclase activity. Meanwhile, forskolin (10 -5 M) was used to estimate the maximum adenylate cyclase activity. After the initial enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, the diester linkage of cyclic AMP was cleaved and then converted to ATP. The ATP was enzymatically amplified to about 10,000 times of fructose-6-phosphate. The NADPH, formed when the fructose-6-phosphate was converted to 6-phosphogluconolactone, was measured fluorometrically. Basal and forskolin-stimulated maximum adenylate cyclase activities (pmol/mg protein/min) were 29.6 ± 7.6 and 86.6 ± 7.2 (mean ± SE), respectively. Norepinephrine increased the adenylate cyclase activity in a dose-dependent manner, while carbachol hardly affected the activity. These results indicate that the adenylate cyclase activity can be measured in the minute ciliary epithelial cells and, moreover, that the current assay can be applied to assess the efficacy of newly available ophthalmic solutions or systemic drugs influencing adenylate cyclase activity in a discrete portion in the eye.