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Bovine, non-pigmented, ciliary body epithelial cells were isolated and grown in culture to determine whether timolol maléate might affect the activity of their plasma membrane ATPases. The possible effects were tested in drug concentrations in a range of 5 x 1019 to 5 x 10'5 M over an incubation period of 30 min at 37°. Assays of specific activity showed that the drug significantly (p < .001 for most concentrations) inhibited both Na,K-ATPase and Mg-ATPase. However, the inhibition was partially reversed in concentrations greater than 106 M for Na,K-ATPase and 105 M for Mg-ATPase. The latter enzyme also indicated a second partial reversal in activity at concentrations between 10~12 and 10"9 M. These reversals in activity suggest that more than one binding site is involved in the inhibition of both enzymes. Since Na,K-ATPase in non-pigmented, ciliary body cells is responsible for the generation of aqueous fluid and the intraocular pressure (IOP), this inhibition demonstrates a possible mechanism for the pharmacological action of timolol maléate in lowering IOP.

Eight putative, low-molecular-weight peptides have been found in the media of bovine corneal tissue cocultures and in bovine aqueous fluid. By investigating amine fluid extracts from these sources by HPLC, possible amino acids, vitamins and urea were eliminated as the unknown peaks of interest. The molecular weights of these substances were equal to or less than 1,000 D. The peaks did not correspond to glutathione or bradykinin either which fall in this molecular weight range. As the cultures aged from 10 to 32 days, it was found that 5 of the 8 peptides varied in concentration. Five peptides increased in concentration to day 17 and slowly decreased afterwards. Efforts are now directed toward the identification of these peptides.