Paweł Sabała

Two subtypes of G protein‐coupled nucleotide receptors, P2Y1 and P2Y2 are involved in calcium signalling in glioma C6 cells

In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP3‐sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca2+. The rank order of potencies of nucleotide analogues in stimulating [Ca2+]i was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. α,β‐Methylene ATP, adenosine and AMP were ineffective. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+]i increase. Similarly, cross‐desensitization between ATP and UTP but not between ADP and UTP occurred. Suramin, a non‐specific nucleotide receptors inhibitor, antagonized ATP‐, UTP‐ and ADP‐evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y1 receptor‐generated InsP3 accumulation, decreased ADP‐initiated Ca2+ response with no effect on ATP and UTP. Pertussis toxin (PTX) reduced ADP‐ and ATP‐induced Ca2+ increases. Short‐term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+]i. ADP inhibited isoproterenol‐induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. RT – PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y1 and P2Y2. It is concluded that both P2Y1 and P2Y2 receptors co‐exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).

Inhibition of phosphatidylserine synthesis by glutamate, acetylcholine, thapsigargin and ionophore A23187 in glioma C6 cells

Phosphatidylserine synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of agents which increase the level of [Ca2+]i. It was found that glutamate and acetylcholine inhibited this synthesis by up to 40%, whereas thapsigargin and the ionophore A23187 inhibited by up to 70%. The inhibitory effect of thapsigargin and the A23187 was observed in Ca(2+)-free medium. The data show that the inhibition of this synthesis is caused by the Ca(2+)-depletion from endoplasmic reticulum, suggesting that the synthesis of phosphatidylserine occurs on the luminal side of these structures and can be regulated by transmembrane signaling systems.

Role of the actin cytoskeleton in store-mediated calcium entry in glioma C6 cells

The effects of actin cytoskeleton disruption by cytochalasin D and latrunculin A on Ca2+ signals evoked by ADP, UTP or thapsigargin were investigated in glioma C6 cells. Despite the profound alterations of the actin cytoskeleton architecture and cell morphology, ADP and UTP still produced cytosolic calcium elevation in this cell line. However, calcium mobilization from internal stores and Ca2+ influx through store-operated Ca2+ channels induced by ADP and UTP were strongly reduced. Cytochalasin D and latrunculin A also diminished extracellular Ca2+ influx in unstimulated glioma C6 cells previously incubated in Ca2+ free buffer. In contrast, the disruption of the actin cytoskeleton had no effect on thapsigargin-induced Ca2+ influx in this cell line. Both agonist- and thapsigargin-generated Ca2+ entry was significantly decreased by the blocker of store-operated Ca2+ channels, 2-aminoethoxydiphenylborate. The data reveal that two agonists and thapsigargin activate store-operated Ca2+ channels but the mechanism of activation seems to be different. While the agonists evoke a store-mediated Ca2+ entry that is dependent on the actin cytoskeleton, thapsigargin apparently activates an additional mechanism, which is independent of the disruption of the cytoskeleton.

INTRACELLULAR Ca2+ SIGNALS INDUCED BY ATP AND THAPSIGARGIN IN GLIOMA C6 CELLS. CALCIUM POOLS SENSITIVE TO INOSITOL 1,4,5- TRISPHOSPHATE AND THAPSIGARGIN

In glioma C6 cells, extracellular ATP generates inositol 1,4,5-trisphosphate (InsP3), indicating the presence of purinergic receptors coupled to phosphoinositide turnover. To identify the effect of ATP (acting via InsP3) and thapsigargin (acting without InsP3 production as a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase) on intracellular Ca2+ pools we used video imaging of Fura-2 loaded into single, intact glioma C6 cells. It has been shown that ATP and thapsigargin initiate Ca2+ response consistent with the capacitative model of Ca2+ influx. When the cells were stimulated by increasing concentrations of ATP (1, 10, 50 and 100 microM) the graded, quantal Ca2+ response was observed. In the absence of extracellular Ca2+ thapsigargin and ionomycin-releasable Ca2+ pools are overlapping, demonstrating that Ca2+ stores are located mainly in the endoplasmic reticulum. After maximal Ca2+ mobilization by ATP, thapsigargin causes further increase in cytosolic Ca2+ concentration, whereas emptying of thapsigargin-sensitive intracellular stores prevents any further Ca2+ release by ATP. Thus, the thapsigargin-sensitive intracellular pool of Ca2+ in glioma C6 cells seems to be larger than that sensitive to InsP3. Two hypothesis to explain this result are proposed. One postulates a presence of two different Ca2+ pools, sensitive and insensitive to InsP3 and both discharged by thapsigargin, and the other, the same intracellular pool of Ca2+ completely emptying by thapsigargin and only partially by InsP3. These results may contribute to understanding the mechanism of Ca2+ signalling mediated by ATP, the most potent intracellular Ca2+ mobilizing agonist in all types of glial cells.

The Role of Ca2+ and Protein Kinase C in Regulation of Phosphatidylserine Synthesis in Glioma C6 Cells

In living organisms there are two different pathways for the biosynthesis of phosphatidylserine (PS). The first is typical for prokaryotes and occurs in the presence of CDP-diacylglycerols and L-serine with the release of CMP. The second pathway is a base exchange reaction in which serine is directly exchanged with the base moiety of preexisting phospholipids. In general, this pathway is typical for eukaryotic organisms. In animal cells, the synthesis of PS appears to occur solely by this reaction (1). It occurs mainly in the endoplasmic reticulum (ER), is independent on metabolic energy and is characterized by a requirement for relatively high (mM) concentration of Ca2+ (1,2).