Determination of the intracellular free chloride concentration in rat brain synaptoneurosomes using a chloride-sensitive fluorescent indicator.
The chloride-sensitive fluorescent indicator MQAE (N-(6-methoxyquinolyl) acetoacetyl ester) has been used for determination of the intracellular free chloride concentration in rat brain synaptoneurosomes. Loading of the synaptoneurosomes with MQAE occurs by transmembrane diffusion. Calibration of the intracellular MQAE was done by determining the correlation between fluorescence intensity and intrasynaptoneurosomal Cl- concentration in the presence of the Cl-/OH- exchanger tributyltin and the K+/H+ exchanger nigericin, starting from zero Cl- concentration. The total quenchable signal of MQAE was determined by adding KSCN in the presence of the K+ ionophore valinomycin. The correlation between the reciprocal of the fluorescence intensity and the chloride concentration was linear at least up to 50 mM Cl-. The fluorescence of freshly prepared synaptoneurosomes was then measured and the obtained value was plotted into the calibration curve and the corresponding Cl- was read. The mean intrasynaptoneurosomal chloride concentration was 14 +/- 4 mM. We also quantitatively estimated the Cl- flux after addition of the barbiturate, pentobarbitone that opens GABAA receptor-Cl(-)-channels, to the synaptoneurosomes. An addition of 1 mM pentobarbitone corresponded to an approx. 0.59 mM change in the intrasynaptoneurosomal free chloride concentration. The results show that the chloride-sensitive fluorescent indicator MQAE is a useful tool when determining intracellular chloride activity, and in quantitative determination of chloride fluxes in living cells and subcellular preparations.
Engblom
A C
AC
Department of Biochemistry and Pharmacy, Abo Akademi University, Finland.
Akerman
K E
KE
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002458:Cell Fractionation; D002712:Chlorides / Q000378:metabolism*; D005456:Fluorescent Dyes; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D009550:Nigericin / Q000494:pharmacology; D010424:Pentobarbital / Q000494:pharmacology; D011804:Quinolines; D051381:Rats; D013050:Spectrometry, Fluorescence; D013569:Synapses / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D013861:Thiocyanates / Q000494:pharmacology
D002712:Chlorides; D005456:Fluorescent Dyes; D011804:Quinolines; D013861:Thiocyanates; C078229:N-(6-methoxyquinolyl)acetoethyl ester; D010424:Pentobarbital; D009550:Nigericin; C009941:potassium thiocyanate