98th Annual Meeting DOG 2000

K 348

Nitric oxide production from porcine pigmented ciliary epithelial cells in culture

C. Erb1,3, Z. Callaerts-Végh2, W. E. Haefeli2, J. Flammer1, I. O. Haefliger1

Purpose: To evaluate whether porcine pigmented ciliary epithelial cells (PCEC) in culture produce nitric oxide (NO) and if this production can be modulated pharmacologically.

Methods: By trypsinisation PCEC were obtained from freshly enucleated porcine eyes. Cells were grown in culture in Dulbecco´s modified Eagle´s medium supplemented with glucose (4.5 g/l), fetal calf serum (10%), and L-arginine (2mM). Once the cells had reach confluence the medium was replaced. Then, 2, 6, 12, 24, 48, 72, and 96 hours later samples were taken from the medium and nitrate, a stable metabolite of NO, measured by high pressure liquid chromatography. Experiments were also conducted by comparing nitrate levels 2 hours and 24 hours after exposure to L-NAME (1 m M), acetylcholine (10 m M), or bradykinin (10m M).

Results: Between the second (8 ± 3 m M) and the twenty-fourth hour (19.1 ± 2.3 m M) nitrate production gradually increased to reach a plateau. In comparison to control experiments, nitrate production measured after 24 hours was inhibited by L-NAME (15.3 ± 0.6, p<0.001) or acetylcholine (12.9 ± 0.8, p<0.001) and increased by bradykinin (22 ± 1.1, p<0.01).

Discussion: These observations provide some evidence that porcine PCEC in culture are able to produce NO and that this production can be modulated by drugs such as acetylcholine or bradykinin.

1University Eye Clinic, 2Department of Internal Medicine, Division of Clinical Pharmacology, University Hospitals of Basel, Switzerland, 3Hannover Medical School, Dep. of Ophthalmology