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Gap junction channels coordinate the propagation of intercellular Ca2+ signals generated by P2Y receptor activation - PubMed

  • ️Thu Jan 01 2004

Gap junction channels coordinate the propagation of intercellular Ca2+ signals generated by P2Y receptor activation

S O Suadicani et al. Glia. 2004.

Abstract

Astrocytes express gap junction proteins and multiple types of P2Y receptors (P2YRs) that contribute to the propagation of intercellular Ca(2+) waves (ICW). To gain access to the role played by gap junctional communication in ICW propagation generated by P2YR activation, we selectively expressed P2Y(1,2,4)R subtypes and Cx43 in the human 1321N1 astrocytoma cell line, which lacks endogenous P2 receptors. Fluorescence recovery after photobleaching revealed that 1321N1 cells are poorly dye-coupled and do not propagate ICW. Forced expression of Cx43 in 1321N1 cells (which did not show functional hemichannels) increased dye coupling and allowed short-range ICW transmission that was mainly mediated by intercellular diffusion of Ca(2+) generated in the stimulated cells. Astrocytoma clones expressing each of the P2YR subtypes were also able to propagate ICWs that were likely dependent on IP(3) generation. These waves exhibited properties particular to each P2YR subtype. Co-expression of eGFP-hCx43 and P2Y(1)R modified the properties of P2Y(1)R-generated ICW to those characteristics of P2Y(2)R. Increased coupling in P2Y(4)R clones induced by expression of eGFP-hCx43 abolished the ICWs observed in uncoupled P2Y(4)R clones. No changes in the behavior of ICWs generated in P2Y(2)R clones were observed after forced expression of Cx43. These data indicate that in 1321N1 cells gap junctional communication provides intercellular integration of Ca(2+) signals generated by P2YR activation, thus coordinating the propagation of intercellular calcium waves.

(c) 2004 Wiley-Liss, Inc.

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Figures

Fig. 1
Fig. 1

Gap junctional communication in 1321N1 cells. A: Semiquantitative RT-PCR indicating the expression level of Cx43 mRNA endogenously expressed in 1321N1 parental cells. The 324bp band corresponds to 18S used as an invariable internal control. B: Representative Western blot showing the endogenous expression levels of Cx43 in parental 1321N1 astrocytoma cells. Note the presence of the nonphosphorylated (NP) and one of the phosphorylated (P1) forms of Cx43 in these cells. GAPDH was used as an invariable internal control. C: Cx43 immunofluorescence images obtained from 1321N1 parental cells indicating that Cx43 immunoreactivity (arrows) is mainly localized in intracellular compartments. D: Bar histogram showing the mean half-time values of dye transfer obtained from 4–8 independent fluorescence recovery after photobleaching (FRAP) experiments performed in primary cultures of astrocytes, 1321N1 parental, and in 1321N1 transfected with rCx43 and eGFP-hCx43. Inset: representative time course curves of FRAP obtained for astrocytes, 1321N1 parental cells and Cx43 transfectants. Astrocyte cultures were obtained as previously described (Scemes et al., 2000). (***P < 0.001, ANOVA, Tukey’s multiple comparison test).

Fig. 2
Fig. 2

Characterization of P2YR expressing 1321N1 cells. A: Semiquantitative RT-PCR showing the expression levels of P2Y1R, P2Y2R and P2Y4R obtained from parental (1321N1) and from astrocytoma clones expressing each of these P2YR subtypes. B: Representative dose-response curves to P2YR agonists (2-MeS-ATP, UTP, and ATP) obtained for P2Y1R, P2Y2R and P2Y4R clones showing proper functional expression of these purinergic receptors as indicated by the EC50 values.

Fig. 3
Fig. 3

Characteristics of intercellular calcium wave propagation mediated by P2YR activation in the presence and absence of gap junctional communication. A: Time-lapse images showing changes in Indo-1 fluorescence ratio (pseudo-colored scale) following mechanical stimulation (arrow) of a single parental cell (1321N1) and of cells expressing P2Y1R and P2Y1R plus eGFP-hCx43 (Cx43+P2Y1R). Note the low number of 1321N1 parental cells participating in ICW propagation compared with that between P2Y1R cells and also the change in the behavior of ICW transmission between P2Y1R when Cx43 was co-expressed in these cells. B—D: Graphic illustration of the changes in the profile of ICW propagation induced by forced expression of Cx43 in cells expressing P2Y1R (B), P2Y2R (C) and P2Y4R (D). The ICW profiles are displayed in two forms, as 3D graphs (Ca2+amplitude × time × distance from stimulated cells; left and middle graphs) and as bar histograms of the number of cells responding with increase in cytosolic calcium (efficacy) per tiers (right graphs). Note in part B that forced expression of Cx43 in P2Y1R clones altered the ICW profile from a “saltatory” to a decremented” wave and greatly increased the efficacy of ICW transmission per tiers of cells. Forced expression of Cx43 in P2Y2R clones (part C), however, did not greatly alter the profile of ICW transmission, while in cells expressing P2Y4R (D), it reduced the efficacy of ICW per tiers of cells and changed ICW profile from a “restricted” to a “nonexistent” wave.

Fig. 4
Fig. 4

Contribution of the extracellular and intracellular Ca2+ compartments to intercellular calcium wave propagation. Bar histograms showing the mean values of intracellular Ca2+ transients recorded in the mechanically stimulated cells (A) and of the efficacy of ICW propagation (B) between parental and cells expressing rCx43, P2Y1R, P2Y2R, P2Y4R, and cells co-expressing rCx43 and P2Y1R (Cx43+P2Y1R) when bathed in DPBS containing 1.0 mM Ca2+ (black bars) and in Ca2+ -free DPBS with 1 mM EDTA (gray bars). (*P < 0.05; ***P < 0.001; unpaired t-test).

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