Measurement of adenylate cyclase activity in the minute bovine ciliary epithelial cells during the pharmacological stimulation of adrenergic and cholinergic receptors

Abstract

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.

Measurement of adenylate cyclase activity in the right ventricular endomyocardial biopsy samples from patients with chronic congestive heart failure

“…The results from this study also demonstrate that while basal adenylate cyclase activities were similar to those reported in the previous studies using larger tissue samples (1)(2)(3)(4)(5)(6), the extent of the stimulation by norepinephrine as well as forskolin was not so great (1,4,5,9,10). The most straightforward explanation for this attenuation of adenylate cyclase stimulation by the β-receptor pathway is down regulation of the receptor-effector pathway.…”

“…The results from this study demonstrate that it is feasible with the new fluorometric method to measure adenylate cyclase activity in a single endomyocardial biopsy sample. While others have used radioimmunoassay and the Salomon method to measure adenylate cyclase activity, these are not sensitive or flexible enough to measure adenylate cyclase activity in a small amount of tissue as currently obtained from biopsy samples of the heart (7)(8)(9)(10)(11)(12). Measurement of adenylate cyclase activity from samples weighing < 1 mg with the new approach may provide new insights into individualizing patient care.…”

“…The adenylate cyclase activity of the membrane preparation was measured with an enzymatic fluorometric assay technique that was essentially the same as those previously described (7)(8)(9)(10). Each reaction is summarized in Fig.…”

“…To begin to overcome this methodological barrier, we adopted a recently developed, highly sensitive, nonradioactive enzymatic fluorometric assay technique (7)(8)(9)(10), and ex-amined the effects of pharmacological interventions on the adenylate cyclase activities in minute membrane preparation made from human right ventricular biopsy samples. In this study, norepinephrine, adenosine, and forskolin were incubated with the membrane preparation to analyze the extent of the receptor-coupled influences on the adenylate cyclase activity.…”