A novel fluorescent indicator, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide (MQAE), was used to measure intracellular chloride concentration ([Cl-]i) in primary cultures of rat aortic smooth muscle cells (VSMC). The hydrolytic and fluorescent properties of the dye were characterized. The intracellular Stern-Volmer constant was calculated to be 25 M-1. Cl- efflux curves were characteristic of saturation-type kinetics, with an apparent Michaelis-Menten constant value of 11 +/- 4.8 (SD) mM, a maximum velocity of 0.038 +/- 0.021 mM/s, and a half time (t1/2) of 9.0 +/- 3.7 min. The average efflux rate in the first 10 min (0.023 +/- 0.004 mM/s) was reduced in the presence of either 130 microM 4,4'-diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid (H2DIDS) (0.014 +/- 0.006, P = 0.02) or 40 microM furosemide (0.017 +/- 0.004, P = 0.04). Restoration of physiological extracellular chloride concentration ([Cl-]o) after zero Cl- resulted in net Cl- influx with a t1/2 of 3.6 +/- 1.0 min. The initial Cl- influx rate was reduced after exposure to furosemide, from 0.069 +/- 0.006 to 0.046 +/- 0.008 mM/s, P < 0.002, and was reduced after exposure to H2DIDS from 0.102 +/- 0.013 to 0.033 +/- 0.003 mM/s, P < 0.001. Furosemide reduced the steady-state [Cl-]i from 31.6 +/- 3.2 to 26.1 +/- 2.4 mM, P < 0.01, whereas H2DIDS had little effect on [Cl-]i. Our results demonstrate that MQAE can be used to measure [Cl-]i in primary cultures of VSMC.
Inhibitory effects of cnidium components on proliferation of mouse aortic smooth muscle cells in primary cultures
