Measurement of MLT in the culture medium

Cells were seeded at a density of 2x10⁵ cells/well. Before the stimulation, the cells were washed several times with HBSS to suppress extracellular MLT. Then, the cells were incubated for 1 h at 37°C with HBSS supplemented with 50 μM L-ascorbic acid and various compounds. The pharmacological agents used were forskolin (adenylyl cyclase catalytic activator, 1 μM), isoproterenol (β-adrenergic receptor agonist, 1 μM), propranolol (β-adrenergic receptor antagonist, 1 μM), phenylephrine (α1-adrenergic receptor agonist, 1 μM), Prazosin (α1- adrenergic receptor antagonist, 1 μM) and luzindole (MLT receptor antagonist, 1 μM). All the supernatants were collected and stored at -80°C.

The MLT concentration was measured in the supernatants by a direct radioimmunoassay (RIA) using 2-[^125I]-MLT as reported previously [35–37]. Briefly, 100 μl of culture supernatants were incubated with 300 μl of 2-[^125I]-MLT (20,000 c.p.m/tube, 21 pM, specific activity: 2,200 Ci.mmol^-1) and 100 μl of rabbit polyclonal anti-MLT diluted at 1:400,000 [37]. After 18 h of incubation at 4°C, 1 ml of sheep anti-rabbit whole serum diluted at 1:300 was added for 1 h at 4°C. All reactions were stopped by centrifugation at 2,800 x g for 30 min at 4°C and radioactivity was measured using a gamma counter. The sensitivity of the assay was 4 pg/ml and the mean intra- assay coefficient of variation was 11.3%.