Use of MQAE for measurement of intracellular [Cl-] in cultured aortic smooth muscle cells.
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.
Koncz
C
C
Department of Medicine, University of Illinois College of Medicine at Chicago.
Daugirdas
J T
JT
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic; D002478:Cells, Cultured; D002712:Chlorides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D005665:Furosemide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000737:chemistry*; D009994:Osmolar Concentration; D011806:Quinolinium Compounds / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014783:Viscosity
D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C085493:N-(ethoxycarbonylmethyl)-6-methoxyquinolinium; D005665:Furosemide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid