RESUMO
We found that stimulation of P2Y2 receptor (P2Y2R), which is endogenously expressed in CHO-K1 cells, induced intracellular calcium ([Ca2+]i) oscillation with a low frequency of 11.4 +/- 2.7 mHz. When CHO-K1 cells expressing GFP-tagged kinase-negative gammaPKC (gammaPKC-KN-GFP), which is a neuron-specific subtype of PKC, were stimulated with UDP, gammaPKC-KN-GFP, but not wild-type gammaPKC (gammaPKC-GFP) showed an oscillatory translocation. The oscillatory translocation of gammaPKC-KN-GFP corresponded with [Ca2+]i oscillation, which was not observed in the cells expressing gammaPKC-GFP. We examined the mechanism ofP2Y2R-induced [Ca2+]i oscillation pharmacologically. gammaPKC-KN-GFP oscillation was stopped by an extracellular Ca2+ chelator, EGTA, an antagonist of P2Y2R, Suramin, and store-operated calcium channel (SOC) inhibitors, SKF96365 and 2-ABP. Taken together, P2Y2R-induced [Ca2+]i oscillation in CHO-K1 cells is related with Ca2+ influx through SOC, whose function may be negatively regulated by gammaPKC. This [Ca2]i oscillation was distinct from that induced by metabotropic glutamate receptor 5 (mGluR5) stimulation in the frequency (72.3 +/- 5.3 mHz) and in the regulatory mechanism.
Assuntos
Relógios Biológicos/fisiologia , Sinalização do Cálcio/fisiologia , Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Relógios Biológicos/efeitos dos fármacos , Células CHO , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Quelantes/farmacologia , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Proteína Quinase C/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2Y2 , Proteínas Recombinantes de Fusão/metabolismo , Suramina/farmacologia , Tripanossomicidas/farmacologiaRESUMO
Rac1 and Rac2 are capable of stimulating superoxide production in vitro, but their targeting and functional mechanisms are still unknown. In the present study, we found that Rac1, 2, and 3 all accumulate at the phagosome during Fc gammaR-mediated phagocytosis, and that the order of accumulation (Rac1 > Rac3 > Rac2) depends on the net positive charge in their polybasic (PB) regions (183-188 aa). Although all GFP-tagged prenylated PB regions of Rac isoforms (GFP-Rac(PB)) and GFP-tagged prenylated 6 Ala (GFP-6A) accumulated during phagocytosis, GFP-Rac2(PB) and GFP-6A showed weak accumulation at the phagosome through a linear structure connecting the phagosome and endomembranes. The PB region of Rac1 showed strong phospholipid interaction with PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and phosphatidic acid, however, that of Rac2 did not. Constitutively active Rac2, GFP-Rac2(Q61L), was predominantly localized at the endomembranes; these endomembranes fused to the phagosome through the linear structure during phagocytosis, and this accumulation mechanism did not depend on positive charge in the PB region. Our conclusion is that Rac1 directly targets to the phagosome using the positively charged PB region and this accumulation mechanism is likely enhanced by the phospholipids. In addition to this mechanism, Rac2 has a positive charge-independent mechanism in which Rac2 initially targets to endomembranes and then these endomembranes fuse to the phagosome.
