2', 3'-O-(4-benzoylbenzoyl)-ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y(2) nucleotide receptor.

In this study, we examined the response of glioma C6 cells to 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X(7) receptor activity. We show here that in the absence of extracellular Ca(2+), BzATP-generated increase in [Ca(2+)](i)via Ca(2+) release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca(2+) response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X(7) receptor, did not reduce any of the two components of the Ca(2+) response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca(2+) elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y(2) but not the P2X(7) receptors.

Expression and regulation of phospholipase D isoforms in sphingosine and phorbol ester-stimulated glioma C6 cells.

Previously we have reported that in glioma C6 cells, sphingosine stimulatory effect on phospholipase D (PLD) activity is independent of protein kinase C [Cell. Signal. 12 (2000) 399]. In this paper we have shown that this effect was also GTPgammaS independent and was completely inhibited by the plasma membrane methyl-beta-cyclodextrin cholesterol depletion what destroys caveolae structure. On the contrary, phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA)-mediated PLD activity was enhanced by GTPgammaS and was only partially decreased by methyl-beta-cyclodextrin. We have also shown that TPA significantly increased expression of PLD1a and PLD1b mRNAs and had lower effect on PLD2 mRNA. Sphingosine only slightly increased expression of PLD mRNA isoforms and did not cause synergistic effect when applied together with TPA. These results indicate that TPA, but not sphingosine, stimulates transcriptional activity of PLD isoforms. We also suggest that TPA stimulates primarily PLD1, while sphingosine affects PLD2 activity. This last process might occur at plasma membrane lipid microdomains.

Ca2+-dependent phosphatidylserine synthesis in immature and mature starfish oocytes.

We found that in starfish oocytes two different enzymes, phosphatidylserine synthase-1 (PSS1) and -2 (PSS2), which synthesize phosphatidylserine by a base-exchange reaction, are present. We studied phosphatidylserine synthesis in immature oocytes which still contain the nucleus (germinal vesicles) and in mature cells, in which the re-initiation of the meiotic cycle induced by the hormone 1-methyladenine led to structural changes in the endoplasmic reticulum, to the disappearance of the nuclear envelope and to the intermixing of the nucleoplasm with the cytoplasm. It was found that the levels of PSS1 and PSS2 transcripts were higher in immature and mature oocytes, respectively. The level of the expressed PSS2 protein, higher than that of PSS1, was not influenced by the maturation process, whereas the level of PSS1 protein was higher in immature than in mature oocytes. Serine incorporation into phosphatidylserine was enhanced in immature oocytes. The depletion of calcium stores by thapsigargin resulted in 50% lowering of phosphatidylserine synthesis. We suggest that changes in phosphatidylserine synthesis may be affected by the release of calcium stored in the nuclear envelope and in the endoplasmic reticulum, the membranes that undergo disintegration and fragmentation during meiosis. The reason for the greater synthesis of PS may be the higher level of expression of PSS1 in immature oocytes.