Intracellular Ca2+ and NO Imaging

To confirm the role of the intracellular Ca²⁺ and NO in the vascular response, we determined intracellular Ca²⁺ and NO levels in the vascular smooth muscle cell line A7r5 (ATCC CRL-1444) and in aortic ring slices (<1 mm), respectively. Intracellular Ca²⁺ determinations^{22 (link)} and for NO measurements^{23 (link)} were performed as previously described. Cells were cultured in coverslips and incubated with 10 μM Fluo-4 AM or preincubated aortic rings with 5 μM 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate (Thermo Fisher Scientific) in KRB for 30 min at 37 °C. Cells were placed on a 1 mL chamber in a Carl Zeiss LSM-5 Pascal 5 Axiovert 200 microscope, excited with 488 nm (500 nm for DAF-FM DA) and the emitted fluorescence monitored at 527 nm (515 nm for DAF-FM DA). Cells or tissues were pretreated with both compounds, Sn–I or Ox–Sn–I (10⁻⁵ M), or vehicle for 30 min. Images of 4–5 different experiments were collected every 1 s and analyzed frame-by-frame with ImageJ software (NIH).^{24 (link)} Intracellular Ca²⁺ levels are expressed as relative fluorescence, ΔF/F₀, where ΔF represents the difference between the experimental value F and the basal fluorescence value F₀.