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ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells.

Jorgensen NR, Geist ST, Civitelli R, Steinberg TH - J. Cell Biol. (1997)

Bottom Line: ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores.These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3.These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.

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UMR cells, but not ROS cells express P2U receptor  mRNA. (A) Calcium transients elicited by different concentrations of ATP in UMR cells. Fura-2–loaded UMR cells were  placed in a stirred cuvette at 37°C, and 340:380 ratios were obtained. (B) Total cell RNA (10 μg) from ROS, UMR, and the  mouse macrophage cell line J774 was electrophoresed on agarose-formaldehyde gel, transferred to nylon, and hybridized with  a radiolabeled cDNA probe for the human P2U receptor. Both  J774 cells and UMR cells expressed a hybridizing band of the appropriate size for mouse and rat P2U, respectively, but no band  was detected in ROS cell RNA.
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Figure 1: UMR cells, but not ROS cells express P2U receptor mRNA. (A) Calcium transients elicited by different concentrations of ATP in UMR cells. Fura-2–loaded UMR cells were placed in a stirred cuvette at 37°C, and 340:380 ratios were obtained. (B) Total cell RNA (10 μg) from ROS, UMR, and the mouse macrophage cell line J774 was electrophoresed on agarose-formaldehyde gel, transferred to nylon, and hybridized with a radiolabeled cDNA probe for the human P2U receptor. Both J774 cells and UMR cells expressed a hybridizing band of the appropriate size for mouse and rat P2U, respectively, but no band was detected in ROS cell RNA.

Mentions: In these studies, we elicited intercellular calcium waves by mechanical stimulation of single cells in monolayers of two rat osteoblastic cell lines, ROS and UMR. We previously demonstrated that ROS cells express Cx43 on the cell surface and are well dye coupled, but that UMR cells express predominantly Cx45 on the plasma membrane and are poorly dye coupled (25). When Lucifer yellow was microinjected into single cells, it diffused into 9.1 cells per injected cell in ROS, and 1.4 cells per injected cell in UMR (25). Because ATP receptors as well as gap junctional communication have been implicated in calcium wave propagation, we measured the calcium response in UMR and ROS after the addition of 1 mM ATP or UTP. Both nucleotides caused a rise in [Ca2+]i in UMR, but not in ROS (not shown). Concentrations of ATP as low as 0.1 μM were able to elicit calcium transients in UMR cells (Fig. 1). Response to both ATP and UTP suggested that UMR cells might express receptors of the P2U (P2Y2) class, which appear to be the most commonly observed receptors of the P2Y class. We therefore performed RNA blots on total RNA from ROS and UMR, using as a probe human P2U cDNA (Fig. 1). UMR, but not ROS, expressed a 3-kbp hybridizing band, consistent with rat P2U (17). The mouse macrophagelike cell line J774, used as a control in these experiments, demonstrated a faster-migrating band of ∼2.4 kbp, consistent with mouse P2U (17). Thus ROS and UMR cells differ both in gap junctional communication and in expression of P2U receptors.


ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells.

Jorgensen NR, Geist ST, Civitelli R, Steinberg TH - J. Cell Biol. (1997)

UMR cells, but not ROS cells express P2U receptor  mRNA. (A) Calcium transients elicited by different concentrations of ATP in UMR cells. Fura-2–loaded UMR cells were  placed in a stirred cuvette at 37°C, and 340:380 ratios were obtained. (B) Total cell RNA (10 μg) from ROS, UMR, and the  mouse macrophage cell line J774 was electrophoresed on agarose-formaldehyde gel, transferred to nylon, and hybridized with  a radiolabeled cDNA probe for the human P2U receptor. Both  J774 cells and UMR cells expressed a hybridizing band of the appropriate size for mouse and rat P2U, respectively, but no band  was detected in ROS cell RNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139805&req=5

Figure 1: UMR cells, but not ROS cells express P2U receptor mRNA. (A) Calcium transients elicited by different concentrations of ATP in UMR cells. Fura-2–loaded UMR cells were placed in a stirred cuvette at 37°C, and 340:380 ratios were obtained. (B) Total cell RNA (10 μg) from ROS, UMR, and the mouse macrophage cell line J774 was electrophoresed on agarose-formaldehyde gel, transferred to nylon, and hybridized with a radiolabeled cDNA probe for the human P2U receptor. Both J774 cells and UMR cells expressed a hybridizing band of the appropriate size for mouse and rat P2U, respectively, but no band was detected in ROS cell RNA.
Mentions: In these studies, we elicited intercellular calcium waves by mechanical stimulation of single cells in monolayers of two rat osteoblastic cell lines, ROS and UMR. We previously demonstrated that ROS cells express Cx43 on the cell surface and are well dye coupled, but that UMR cells express predominantly Cx45 on the plasma membrane and are poorly dye coupled (25). When Lucifer yellow was microinjected into single cells, it diffused into 9.1 cells per injected cell in ROS, and 1.4 cells per injected cell in UMR (25). Because ATP receptors as well as gap junctional communication have been implicated in calcium wave propagation, we measured the calcium response in UMR and ROS after the addition of 1 mM ATP or UTP. Both nucleotides caused a rise in [Ca2+]i in UMR, but not in ROS (not shown). Concentrations of ATP as low as 0.1 μM were able to elicit calcium transients in UMR cells (Fig. 1). Response to both ATP and UTP suggested that UMR cells might express receptors of the P2U (P2Y2) class, which appear to be the most commonly observed receptors of the P2Y class. We therefore performed RNA blots on total RNA from ROS and UMR, using as a probe human P2U cDNA (Fig. 1). UMR, but not ROS, expressed a 3-kbp hybridizing band, consistent with rat P2U (17). The mouse macrophagelike cell line J774, used as a control in these experiments, demonstrated a faster-migrating band of ∼2.4 kbp, consistent with mouse P2U (17). Thus ROS and UMR cells differ both in gap junctional communication and in expression of P2U receptors.

Bottom Line: ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores.These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3.These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.

Show MeSH
Related in: MedlinePlus