We studied the synthesis and release of nerve growth factor (NGF) in cultured rat iris with a two-site enzyme immunoassay by measuring the time course of NGF levels remaining in the iris and relased into the medium up to 72 h. For up to 3 h, the NGF levels in the iris did not change significantly. After that, they increased to a maximal level of 350 +/- 30 pg NGF/iris at 19 h, which is 200 times higher than the in vivo content. Between 20 and 72 h in culture, the NGF level decreased to 130 +/- 10 pg NGF/iris, whereas general protein synthesis did not change during that time period. Maximal rate of NGF production (203 pg NGF/h/iris) was seen between 9 and 12 h in culture. In the medium, NGF levels were first detectable after 6 h. Levels then increased with a time course similar to that seen within the iris, reaching a maximal level of 1,180 +/- 180 pg after 19 h in vitro, and then did not significantly change for up to 48 h. The NGF production of the densely sympathetically innervated dilator was three times higher than that of the predominantly cholinergically innervated sphincter. The NGF production was blocked by inhibitors of messenger RNA synthesis (actinomycin D) and of polyadenylation (9-beta-D-arabinofuranosyladenine) as well as by inhibitors of translation (cycloheximide). Monensin, which interferes with the transport of proteins through the Golgi apparatus, decreased NGF levels to 8-12% of controls in the medium, suggesting that the Golgi apparatus is involved in the intracellular processing of NGF.

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