Aimin Zhang¹, Bella T. Altura^2,3 and Burton M. Altura^1,2,3

Departments of ¹Physiology and ²Medicine, and ³The Center for Cardiovascular and Muscle Research, State University of New York, Health Science Center at Brooklyn, NY 11203

Received 5/15/97; Accepted 5/21/97; On-line 6/1/97

3. MATERIALS AND METHODS

Experiments were carried out using a human aortic endothelial cell line (No. AG09799A, 20 passages) obtained from the NIA Aging Cell Repository (Camden, NJ). Measurements of [Mg²⁺]_i in single cells were done according to previously established methods (16). Briefly, endothelial cells were cultured in 199 media (Sigma Chem. Co., St Louis, MO) with 15% fetal serum albumin (FSA) at 37°C in an humidified atmosphere composed of 95% air-5% CO₂. The cells were loaded with mag-fura-2 (Molecular Probes, Eugene, OR) by incubating them with 5 micromolar mag-fura-2/AM in the culture media for 60 min under 95% air-5% CO₂. Then, the coverslips were placed in a chamber on a thermostat-regulated stage of a Nikon fluorescence microscope and superfused with 1.2 mM Mg²⁺ HEPES buffer solutions (pH 7.4, 37°C) followed by 4.8 mM Mg²⁺ solutions (pH 7.4, 37°C). The ionic activities of Mg²⁺ in HEPES buffer solutions were monitored by unique Mg²⁺ ion selective electrodes (NOVA Biomedical Corp, Waltham, MA) and adjusted with MgSO₄ (21). The HEPES buffer solutions also contained (mM): NaCl 118, KCl 4.7, CaCl₂ 2.2, KH₂PO₄ 1.2, HEPES 5, and glucose 10. Measurement of [Mg²⁺]_i was performed using a TN8500 FluorPlex III Image Analyzer (Tracor Northern, Madison , WN). Images of mag-fura-2 fluorescence at 510 nm emission were obtained with 335 and 370 nm excitation wavelengths using a silicon intensified target (SIT) camera. The time interval of switching between these two wavelengths was 2 seconds. Background fluorescence for both excitation wavelengths were acquired from blanks for each experiment and subtracted from each pair of images separately before ratioing. Fluorescence intensity ratios (R_335/370) were obtained by dividing the fluorescence intensity at 335 nm by the intensity at 370 nm. No image misalignments occurred when these two ratiometric images were superimposed.

To obtain absolute values of [Mg²⁺]_i in single cells, a final concentration of 5 micromolar mag-fura-2 pentapotassium salt containing either 10.0 mM (max) or 0 mM MgSO₄ (min) was used for an in-vitro calibration. The calibration solutions also contained: KCl 115 mM, NaCl 20 mM, and HEPES 5 mM, buffered with NaOH to pH 7.1 under air, at 37°C. From these Mg-standard solutions, the maximum and minimum intensities of fluorescence were obtained at the 335 nm and 370 nm wavelengths and a ratio of (R_335/370) was generated. [Mg²⁺]_i was calculated according to the following equation (22):

and a K_d of 1.5 mM (22) was used for the mag-fura-2/ Mg²⁺ complex. B is the ratio of fluorescence intensity of free mag-fura-2 to Mg-bound mag-fura-2 at 370 nm.

Where appropriate, mean values ± S.E.M. were calculated and compared for statistical significance using paired t tests. A p-value less than 0.05 was considered significant.