2.5 Nitric oxide measurements in cell culture
Human glioblastoma cell line U-87 MG was cultured in DMEM-F12 cell media, supplemented by 10% fetal bovine serum (FBS) (Cultilab, Campinas, Brazil) and 1% of antibiotic/antimycotic solution (Thermo-Fisher, Waltham, MA, USA) at 37°C. Rat neonatal cardiomyocytes were isolated, as previously reported (Guatimosim et al. , 2008). Rat nenonatal cardiomyocytes were maintained with DMEM-F12 cell media supplemented by 10% FBS and 1% of antibiotic/antimycotic solution at 37°C until usage. Mice ventricular adult cardiomyocytes were isolated by collagenase type-2 (Worthington, Columbus, OH, USA), as previously described (Rocha-Resende et al. , 2012) and kept in Tyrode media (composition: 140 mM NaCl; 4 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2; 10 mM Glucose; 5 mM HEPES; pH = 7.4) until used.
For NO quantification, cells were starved with Hank’s Balanced Salt Solution (HBSS) for 1 hour and then loaded with DAF-FM diacetate (Thermo-Fisher, Waltham, MA, USA) at a final concentration of 5 µM for 30 minutes. Cells were rinsed with HBSS and stimulated for 15 minutes with BK-(1-9), BK-(1-7) or BK-(1-5), at the final concentration of 100 nM , all diluted with sterile saline. Kinin receptors involvement in NO production by the tested peptides was assessed by selective antagonism of B1 and B2 receptors with Lys-(des-Arg9-Leu8-BK-(1-9) and HOE-140 (Tocris, Bristol, UK), respectively, at a final concentration of 100 nM, in sterile saline, prior to stimulation by BK-(1-9) or its fragments. A concentration-response curve was generated by stimulating male neonatal rat cardiac myocytes with BK-(1-9) and its fragments using concentrations of 1, 10 and 100 nM, in sterile saline, for 15 minutes. After stimulation under these experimental conditions, cells were fixed with a 4% paraformaldehyde solution for 15 minutes and then mounted. Fluorescence emitted from cells was captured by either a confocal microscope Zeiss LSM 5 LIVE (Zeiss, Oberkochen, Germany) or a Nikon Eclipse Ti (Nikon, Melville, NY, USA), with λEx = 495nm and λEx = 505nm. Fluorescence emitted by cells was quantified by ImageJ (Schneider et al. , 2012).