P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking

More cross-talk between purinergic receptors

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Functional interactions between P2X4 and P2X7 receptors from mouse salivary epithelia

Mouse parotid acinar cells express P2X4 and P2X7 receptors (mP2X4R and mP2X7R) whose physiological function remains undetermined. Here we show that mP2X4R expressed in HEK-293 cells do not allow the passage of tetraethylammonium (TEA+) and promote little, if any, ethidium bromide (EtBr) uptake when stimulated with ATP or BzATP. In contrast, mP2X7R generates slowly decaying TEA+ current, sustained Na+ current and promotes robust EtBr uptake. However, ATP-activated TEA+ current from acinar cells was unlike that generated by mP2X7R or mP2X4R. Functional interactions between mP2X4R and mP2X7R were investigated in HEK cells co-transfected with different mP2X4 : mP2X7 cDNA ratios and using solutions containing either TEA+ or Na+ ions. Co-expressed channels generated a TEA+ current that displayed faster decay during ATP stimulation than mP2X7R alone. Moreover, cells transfected with a 2 : 1 cDNA ratio displayed decaying kinetics similar to those observed in acinar cells. Concentration–response curves in Na+-containing solutions were constructed for heterologously expressed mP2X4R, mP2X7R and mP2X4R:mP2X7R co-expressions as well as acinar cells. The EC50 values determined were 11, 220, 434 and 442 μm, respectively. Na+ currents generated by expressing mP2X4R or mP2X7R alone were potentiated by ivermectin (IVM). In contrast, IVM potentiation in acinar cells and HEK cells co-expressing P2X4 and P2X7 (1 : 1 or 2 : 1 cDNA ratios) was seen only when the ATP concentration was lowered from 5 to 0.03 mm. Taken together our observations indicate a functional interaction between murine P2X7 and P2X4 receptors. Such interaction might occur in acinar cells to shape the response to extracellular ATP in salivary epithelia

Evaluation of boar sperm maturation after co-incubation with caput, corpus and cauda epididymal cultures - Evaluation of boar sperm maturation in vitro

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Erratum to "Evaluation of boar sperm maturation after co-incubation with caput, corpus and cauda epididymal cultures"

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