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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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Calcium waves in  ROS/P2U transfectants and  HTE cells. ROS/P2U transfectants (left) and HTE cells  (right) were loaded with fluo-3  and images were taken after  a mechanical stimulation of a  single cell. Panels are images  taken at the indicated time  after subtraction of the prestimulation fluo-3 image.
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Figure 8: Calcium waves in ROS/P2U transfectants and HTE cells. ROS/P2U transfectants (left) and HTE cells (right) were loaded with fluo-3 and images were taken after a mechanical stimulation of a single cell. Panels are images taken at the indicated time after subtraction of the prestimulation fluo-3 image.

Mentions: The above studies demonstrated that mechanically induced intercellular calcium waves in the UMR cell line were propagated by activation of P2U purinergic receptors. Because UMR cells expressed P2U receptors but ROS cells did not, we then asked whether expression of P2U in ROS cells was sufficient to allow ROS cells to propagate UMRlike calcium waves. ROS cells were transfected with a construct containing the human P2U receptor cDNA. Unlike the parent ROS cells, ROS/P2U transfectants responded to addition of ATP with a rise in [Ca2+]i (not shown). Mechanical stimulation of ROS/P2U cells (n = 8) elicited intercellular calcium waves that were much faster than the waves in the parent ROS cells and slightly slower than UMR calcium waves (average conduction velocity = 7.4 μm/s), with a maximal extension of the wave to 8–38 cells (Fig. 8; Table I). Maximal extension of the wave was reached after 15–20 s. We confirmed that these fast calcium waves did not involve gap junctional communication by demonstrating that 3.5 mM heptanol had no effect on the speed or extent of propagation (15–44 cells per stimulated cell, n = 4). In contrast, desensitization with 1 mM ATP as described above inhibited the fast UMRlike waves, and uncovered the slow ROS waves with extension to 3–4 cells (n = 4). The combination of 3.5 mM heptanol and 1 mM ATP desensitization resulted in a complete block of wave propagation, where only the stimulated cell showed an increase in intracellular calcium in all experiments (n = 4). These results demonstrated that expression of the P2U receptor in ROS cells was sufficient to allow these cells to propagate gap junction–independent calcium waves via activation of purinergic receptors in addition to the gap junction–dependent calcium waves that these cells normally expressed. These experiments also demonstrated that ROS cells are capable of releasing intracellular calcium via an IP3-dependent mechanism, when such a mechanism is activated.


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

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

Calcium waves in  ROS/P2U transfectants and  HTE cells. ROS/P2U transfectants (left) and HTE cells  (right) were loaded with fluo-3  and images were taken after  a mechanical stimulation of a  single cell. Panels are images  taken at the indicated time  after subtraction of the prestimulation fluo-3 image.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139805&req=5

Figure 8: Calcium waves in ROS/P2U transfectants and HTE cells. ROS/P2U transfectants (left) and HTE cells (right) were loaded with fluo-3 and images were taken after a mechanical stimulation of a single cell. Panels are images taken at the indicated time after subtraction of the prestimulation fluo-3 image.
Mentions: The above studies demonstrated that mechanically induced intercellular calcium waves in the UMR cell line were propagated by activation of P2U purinergic receptors. Because UMR cells expressed P2U receptors but ROS cells did not, we then asked whether expression of P2U in ROS cells was sufficient to allow ROS cells to propagate UMRlike calcium waves. ROS cells were transfected with a construct containing the human P2U receptor cDNA. Unlike the parent ROS cells, ROS/P2U transfectants responded to addition of ATP with a rise in [Ca2+]i (not shown). Mechanical stimulation of ROS/P2U cells (n = 8) elicited intercellular calcium waves that were much faster than the waves in the parent ROS cells and slightly slower than UMR calcium waves (average conduction velocity = 7.4 μm/s), with a maximal extension of the wave to 8–38 cells (Fig. 8; Table I). Maximal extension of the wave was reached after 15–20 s. We confirmed that these fast calcium waves did not involve gap junctional communication by demonstrating that 3.5 mM heptanol had no effect on the speed or extent of propagation (15–44 cells per stimulated cell, n = 4). In contrast, desensitization with 1 mM ATP as described above inhibited the fast UMRlike waves, and uncovered the slow ROS waves with extension to 3–4 cells (n = 4). The combination of 3.5 mM heptanol and 1 mM ATP desensitization resulted in a complete block of wave propagation, where only the stimulated cell showed an increase in intracellular calcium in all experiments (n = 4). These results demonstrated that expression of the P2U receptor in ROS cells was sufficient to allow these cells to propagate gap junction–independent calcium waves via activation of purinergic receptors in addition to the gap junction–dependent calcium waves that these cells normally expressed. These experiments also demonstrated that ROS cells are capable of releasing intracellular calcium via an IP3-dependent mechanism, when such a mechanism is activated.

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