Anales RANF

Aida Menéndez Méndez, Juan Ignacio Díaz Hernández, Felipe Ortega, Rosa Gómez Villafuertes, Javier Gualix @Real Academia Nacional de Farmacia. Spain 192 Figure 1. Effect of LPS treatment in the expression of purinergic receptors and VNUT in microglia cultures . Q-PCR analysis of mRNA levels of VNUT (A), P2X7 receptor (B) and P2Y 2 receptor (C) after stimulation with LPS (1µg/ml) for 24h. Values are represented as a percentage of those obtained in untreated (control) cells, which were set as a 100%. (D) Western blot detection of VNUT and P2X7 proteins in cellular lysates of both LPS treated and untreated microglial cells. VNUT (E) and P2X7 receptor (F) protein levels were normalized with respect to β -actin. Values are represented as a percentage of those obtained in untreated cells, which were set as a 100%. Data are the mean ± SEM of three independent experiments. **p < 0.01 (Student’s t -test). 3.2. Effect of LPS treatment in the ATP release by the microglia The effect of LPS in the microglial culture was visualized by immunofluorescence techniques, using a specific marker of microglial cells, such as the Iba1 (ionized calcium-binding adapter molecule 1) protein (34). As can be observed in figure 2A, microglial cells acquire an amoeboid morphology that is characteristic of the reactive microglia after 24h of treatment with the lipopolysaccharide, which confirmed that the LPS treatment was effective. Regarding the distribution of the nucleotide vesicular transporter, this was mainly localized in the proximal zone to the cell nucleus (Figure 2A). The release of ATP after the treatment with LPS was measured by luminometric techniques. As can be observed in Figure 2B, the treatment with the lipopolysaccharide for 5 min induced a significant increase in the luminescence values, which means a higher liberation of the nucleotide to the extracellular medium. These results are in agreement with those obtained by other research groups (35). However, the stimulation with LPS for 24h did not significantly modify the recorded luminescence values (Figure 2C). 150 Control LPS 0 50 100 ** VNUT mRNA (%) 0 50 100 150 Control LPS P2X7 mRNA (%) ** 0 100 200 300 Control LPS P2Y₂ mRNA (%) P2X7 VNUT Control LPS 75KDa 60KDa 42KDa 0 50 100 150 P2X7 Protein (%) Control LPS ** 0 50 100 150 VNUT Protein (%) Control LPS ** A B C E D F