In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 and P2Y 12 family members while among P2X receptors P2X 4 , P2X 5 and P2X 7 were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca 2+ ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X 5 play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y 2 -dependent Ca 2+ signaling, while the mRNA expression of P2Y 2 receptor reminds still on the high level. Moreover, P2X 2 receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern.Abbreviations: 2MeSADP -2-methylthio-ADP; BzATP -3 0 -0(4-benzoyl)benzoyl ATP; [Ca 2+ ]i -intracellular Ca 2+ concentration; DMEM -Dulbecco's modified essential medium; ERK -Ras/extracellular signal-regulated kinase; FCS -fetal calf serum; GAPDH -glycerol 3-phosphate dehydrogenase; HS -horse serum; InsP 3 -inositol 1,4,5-trisphosphate; PLCphospholipase C; PPADS -pirydoxal-phosphate-6-azophenyl-2 0 , 4 0 -disulphonic acid

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y 1 , P2Y 2 and P2Y 12 . Two of them, P2Y 1 and P2Y 2 , directly stimulate calcium response. The protein level of the P2Y 2 receptor did not change during the serum starvation, while P2Y 1 protein level fell dramatically. Observed changes in the calcium response generated by P2Y 1 are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y 12 , did not directly evoke calcium response, however it is activated by the same ligand as P2Y 1 . The experiments with AR-C69941MX, the P2Y 12 -specific antagonist, indicated that in control and serumstarved cells, calcium response evoked by P2Y 1 receptor is potentiated by the activity of P2Y 12 -dependent signaling pathways. This potentiation may be mediated by P2Y 12 inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y 1 and P2Y 12 receptor activity directly depends on the capacitative calcium entrance mechanism.

The focus of this study is glioma C6 cells, which are chemically transformed glial cells derived from adult rat brain. They have oligodendrocytic and astrocytic progenitor properties and are often used as a biochemical model system for studies related to astrocytes [1]. They are also used as a model for studying glioma biology [2], e.g. glioblastoma multiforme, which is pathologically characterized by a marked increase in cell growth and proliferation. C6 cells can be easily implanted into rat brains in vivo to test invasiveness. To date, there is no better model for examining this process. The increased invasiveness of these cells was found to be associated with the constitutive up-regulation of phosphatidylinositol 3-kinase (PI3-K) ⁄ protein We characterized the expression and functional properties of the ADPsensitive P2Y 1 and P2Y 12 nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1 ⁄ 2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y 12 receptor relative to P2Y 1 was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y 1 receptor was low, and the P2Y 12 receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y 12 receptor activated ERK1 ⁄ 2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y 12 receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y 1 receptor, indicating the inhibitory role of P2Y 1 in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y 1 to P2Y 12 would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.Abbreviations Akt, protein kinase B ⁄ Akt kinase; DIC, differential interference contrast; ERK, Ras extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; IRM, interference reflection microscopy; MEM, minimum essential medium; 2MeSADP, 2-methylthio-ADP; NaCl ⁄ P i , phosphate-buffered saline; PI3-K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog.