Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Lin, Wan-Wan; Chuang, De‐Maw

doi:10.1007/bf00966782

Cited by 24 publications

(19 citation statements)

References 36 publications

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“…The phosphorylation of the 85-kDa MARCKS protein was determined autoradiographically as described under "Materials and Methods." and ATP, which are known to be physiological agonists for C6 glioma cells (Lin et al, 1990(Lin et al, , 1992(Lin et al, , 1993 trigger rapid increases in [Ca 2ϩ ] i and that pretreating these cells with 10 nM endothelin-1 or 100 M ATP 2 min before adding TPA prevents the TPA-stimulated PKCs from phosphorylating MARCKS just as did Ca 2ϩ in the BALB/MK keratinocytes of our previous study and ionomycin in the glioma cells of the present study. Thus, Ca 2ϩ ⅐calmodulin appears to be a physiological modulator of MARCKS phosphorylation by PKCs.…”

Section: Discussionsupporting

confidence: 61%

Ca2+·Calmodulin Prevents Myristoylated Alanine-rich Kinase C Substrate Protein Phosphorylation by Protein Kinase Cs in C6 Rat Glioma Cells

Chakravarthy

Isaacs

Morley

et al. 1995

Journal of Biological Chemistry

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Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. but raising the Ca 2ϩ concentration above 1 mM stops proliferation and starts differentiation-related processes such as cornified envelope formation Falco et al., 1988;Moscat et al., 1989; Aaroson, 1983, 1985;Whitfield, 1995;Whitfield et al., 1992Whitfield et al., , 1995. The signal triggered by extracellular Ca 2ϩ in murine BALB/MK keratinocytes includes an internal Ca 2ϩ transient and a large burst of membrane-associated PKCs activity . Despite the large burst of PKCs activity, neither Moscat et al. (1989) nor we have observed an increased phosphorylation of the keratinocytes' 80 -85-kDa MARCKS protein, an ubiquitous substrate of the conventional and novel PKC isoforms (Blackshear, 1993;Heemskerk et al., 1993;Fujise et al., 1994).We have recently presented strong, but indirect, evidence for the failure of the Ca 2ϩ -treated BALB/MK keratinocyte's activated PKCs to phosphorylate their MARCKS protein substrate being due to Ca 2ϩ ⅐calmodulin complexes somehow blocking access of the PKCs to their substrate's phosphorylation site domain . However, this suggestion seems to be contradicted a priori by the known abilities of mitogenic factors such as bradykinin, bombesin, platelet-derived growth factor and vasopressin to stimulate an internal Ca 2ϩ transient, membrane-associated PKCs activity, and MARCKS phosphorylation (Rozengurt, 1986;Issandou and Rozengurt, 1990). The reason for this apparent contradiction may lie in the relative timing of the internal Ca 2ϩ transient (and the Ca 2ϩ ⅐calmodulin complexes it produces) and the burst of membrane-associated PKCs activity in the Ca 2ϩ -stimulated BALB/MK cells: in mitogen-stimulated cells the Ca 2ϩ and PKC signals are coincident (Rozengurt, 1986;Issandou and Rozengurt, 1990) while in Ca 2ϩ -treated BALB/MK keratinocytes PKCs activity peaked 7 min after the [Ca 2ϩ ] i surge . This suggested that the MARCKS substrate can be phosphorylated only when the membrane-associated PKCs activity peaks before or at the same time as [Ca 2ϩ ] i , and a resulting Ca 2ϩ ⅐calmodulin, surge. In this report we extend our observations, using C6 rat glioma cells and BALB/MK keratinocytes, to provide the first direct evidence for Ca 2ϩ ⅐calmodulin actually being the blocker of PKCs-mediated MARCKS phosphorylation in the cell and to confirm the suggestion arising from the previous report that the relative timing of surges of internal Ca 2ϩ and PKCs activity determines the extent of MARCKS protein phosphorylation. ] i , intracellular ca...

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Section: Discussionsupporting

confidence: 61%

Ca2+·Calmodulin Prevents Myristoylated Alanine-rich Kinase C Substrate Protein Phosphorylation by Protein Kinase Cs in C6 Rat Glioma Cells

Chakravarthy

Isaacs

Morley

et al. 1995

Journal of Biological Chemistry

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“…PI hydrolysis was measured as lithium-dependent accumulation of [3HI IPs in cells prelabelled with myo-[3H]inositol, as described previously (Lin and Chuang 1993a). Briefly, confluent cells on 35-mm Petri dishes were labeled with 2.5 gCi/dish of myo-[3H]inositol for 24 h. Labelled cells were then washed and preincubated at 37 ° C for 20 min in physiological saline solution (118 mM NaC1, 4.7 mM KC1, 1.8 mM CaC12, 1.2mM MgC12, 1.2 mM KH2PO4, 11 mM glucose and 20 mM HEPES, pH 7.4) containing 10 mM LiC1.…”

Section: Intact Cellsmentioning

confidence: 99%

“…ATP, NaF and A23187 stimulate PI-PLC via activation of P2u purinoceptors (Lin and Chuang 1993a), G proteins and Ca 2 + influx respectively. Therefore, the common mechanism responsible for the potentiating effects of CaM antagonists is possibly at the PLC level.…”

Section: Intact Cellsmentioning

confidence: 99%

Potentiation of stimulus-induced phosphoinositide breakdown by calmodulin antagonists in C6 glioma cells

Lin

1995

Naunyn-Schmiedeberg's Arch Pharmacol

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To investigate the role of calmodulin (CaM)-dependent pathways in agonist-induced phosphoinositide (PI) turnover, the influence of several CaM antagonists on PI-phospholipase C (PLC) activation in intact and permeabilized C6 glioma cells was examined. The extent of PI turnover was assessed by measuring the accumulation of inositol phosphates (IPs) in the presence of LiCl in C6 glioma cells prelabelled with myo-[3H]inositol. Trifluoperazine, N-(6-aminohexyl)-5-chloro-1- naphthalenesulphonamide (W-7), fendiline and calmidazolium themselves had no effect on basal IP formation, but concentration-dependently (1-30 microM) potentiated ATP-, NaF- and A23187-stimulated IP formation. The maximal response to ATP (1 mM) was increased by up to 50%, while the concentration for half-maximal effect (EC50, 60 microM) was unaffected by trifluoperazine. In digitonin-permeabilized C6 glioma cells, the concentration-dependent increase of PI-PLC activation elicited by free Ca2+ was potentiated by the GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), with an EC50 of 6 microM. Trifluoperazine (1-30 microM) enhanced the Ca(2+)-stimulated IP formation concentration dependently and this potentiation was counteracted by the addition of CaM. In the combined presence of each CaM antagonist studied and GTP gamma S, an additive increase in IP formation was observed. The results indicate that CaM antagonists enhance stimulus-induced IP formation in C6 glioma cells primarily by increasing the Ca(2+)-dependent activation of PI-PLC.

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“…Attempts have been made to identify the mechanism of action of ATP on cancer cells and the purinergic receptor subtypes involved. Decreases in cancer cell number may be attributable to an inhibition of cell proliferation mediated via P2Y receptors (Dubyak and De Young 1985;Lin and Chuang 1993;Maaser et al 2002), the stimulation of differentiation with the subsequent inhibition of proliferation via P2X 5 receptors (Cowen et al 1991;Popper and Batra 1993) or the induction of cell death (apoptosis) via P2X 7 receptors (Chueh and Kao 1993). P1 receptors have been described in the human melanoma A375 cell line (Merighi et al 2001), with evidence for all four subtypes of receptor being present, and have been postulated as a possible target for the treatment of this cancer (Merighi et al 2002).…”

Section: Introductionmentioning

confidence: 99%

Human melanomas express functional P2X7 receptors

2005

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Adenosine 5'-triphosphate is known to function as a potent extracellular messenger, producing its effects via a distinct family of cell surface receptors. Different receptor subtypes have been shown to modulate different cellular functions such as proliferation, differentiation and apoptosis. We have investigated the functional expression and apoptotic action of the P2 X (7) receptor in human malignant melanoma tissue and cells. Incubation of cells with the potent P2 X (7) receptor agonist 2'-3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate leads to a decrease in cell number, which is dose-dependent and reversible by the antagonist 1-N,O-bis-[5-isoquinoline-sulfonyl]-N-methyl-L-tyrosyl)-4-phenyl-piperazine. Synthesis of the P2 X(7) receptor by these cells has been established by reverse transcriptase-polymerase chain reaction, immunohistochemistry, immunocytochemistry and cellular accumulation of the fluorescent DNA-binding dye YO-PRO-1. The P2 X(7) receptors have been shown to mediate apoptotic actions of extracellular nucleotides and represent a novel target for melanoma therapy.

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Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Cited by 24 publications

References 36 publications

Ca2+·Calmodulin Prevents Myristoylated Alanine-rich Kinase C Substrate Protein Phosphorylation by Protein Kinase Cs in C6 Rat Glioma Cells

Ca2+·Calmodulin Prevents Myristoylated Alanine-rich Kinase C Substrate Protein Phosphorylation by Protein Kinase Cs in C6 Rat Glioma Cells

Potentiation of stimulus-induced phosphoinositide breakdown by calmodulin antagonists in C6 glioma cells

Human melanomas express functional P2X7 receptors

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