Colley A M, Cavanagh H D, Law M L
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322.
Metab Pediatr Syst Ophthalmol (1985). 1987;10(2):36-8.
Activities of adenylate and guanylate cyclases and cAMP and cGMP phosphodiesterases (cAPDE, cGPDE) were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine on enzyme activities in each fraction were evaluated. Activity of cyclases and phosphodiesterases was detectable in control incubations of homogenates, nuclei, the mitochondrial/lysosomal fraction, microsomes, and cytosol, although microsomal guanylate cyclase represented a very small proportion of the total cellular activity. In homogenates, carbamylcholine significantly elevated guanylate cyclase and cAPDE and reduced cGPDE activity. In mitochondria/lysosomes, guanylate cyclase was elevated and cGPDE reduced, but the drug did not alter cAPDE activity. In microsomes, carbamylcholine enhanced cAPDE but did not alter guanylate cyclase of cGPDE activity. In the soluble cytoplasmic fraction the drug reduced guanylate cyclase activity. The purified nuclear fraction exhibited substantial activity of cyclases and phosphodiesterases. Carbamylcholine significantly elevated activity of nuclear guanylate cyclase and cAPDE and significantly reduced nuclear cGPDE activity. The drug did not significantly alter adenylate cyclase in homogenates or in any cell fraction. The presence of activity of enzymes of cyclic nucleotide metabolism in the cell nucleus and the sensitivity of nuclear guanylate cyclase, cAPDE and cGPDE to carbamylcholine, which in the same concentration range enhances activity of DNA and RNA polymerases, suggested the hypothesis that effects on cyclic nucleotide-dependent phosphorylation of nuclear proteins might be among regulatory mechanisms by which the drug alters rates of replication and transcription in corneal epithelial cells.
在培养的兔角膜上皮细胞匀浆和亚细胞组分中检测了腺苷酸环化酶和鸟苷酸环化酶以及环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）磷酸二酯酶（cAPDE、cGPDE）的活性，并评估了氨甲酰胆碱对各组分中酶活性的影响。在匀浆、细胞核、线粒体/溶酶体组分、微粒体和胞质溶胶的对照孵育中可检测到环化酶和磷酸二酯酶的活性，尽管微粒体鸟苷酸环化酶在总细胞活性中所占比例非常小。在匀浆中，氨甲酰胆碱显著提高鸟苷酸环化酶和cAPDE的活性，并降低cGPDE的活性。在线粒体/溶酶体中，鸟苷酸环化酶活性升高，cGPDE活性降低，但该药物未改变cAPDE的活性。在微粒体中，氨甲酰胆碱增强了cAPDE的活性，但未改变鸟苷酸环化酶或cGPDE的活性。在可溶性细胞质组分中，该药物降低了鸟苷酸环化酶的活性。纯化的细胞核组分表现出显著的环化酶和磷酸二酯酶活性。氨甲酰胆碱显著提高了细胞核鸟苷酸环化酶和cAPDE的活性，并显著降低了细胞核cGPDE的活性。该药物在匀浆或任何细胞组分中均未显著改变腺苷酸环化酶的活性。细胞核中环核苷酸代谢酶活性的存在以及细胞核鸟苷酸环化酶、cAPDE和cGPDE对氨甲酰胆碱的敏感性，在相同浓度范围内可增强DNA和RNA聚合酶的活性，这提示了一种假说，即对细胞核蛋白环核苷酸依赖性磷酸化的影响可能是该药物改变角膜上皮细胞复制和转录速率的调节机制之一。
