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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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Desensitization of  P2U receptors inhibits calcium waves in UMR cells.  Ratio imaging was performed on fura-2–loaded  UMR cell monolayers. Top  panels: left, 1 mM ATP-elicited calcium transients; center, subsequent mechanical  stimulation failed to induce  intercellular calcium waves;  right, subsequent challenge  with 20 U/ml thrombin revealed that calcium stores  were not depleted. Middle  panels: left, thrombin-induced  calcium transients; center,  subsequent mechanical stimulation induced a calcium  wave; right, subsequent addition of ATP induced calcium  transients. Bottom panels:  propagated calcium waves  (left) frequently desensitized  a local area to subsequent  addition of ATP (right).
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Figure 7: Desensitization of P2U receptors inhibits calcium waves in UMR cells. Ratio imaging was performed on fura-2–loaded UMR cell monolayers. Top panels: left, 1 mM ATP-elicited calcium transients; center, subsequent mechanical stimulation failed to induce intercellular calcium waves; right, subsequent challenge with 20 U/ml thrombin revealed that calcium stores were not depleted. Middle panels: left, thrombin-induced calcium transients; center, subsequent mechanical stimulation induced a calcium wave; right, subsequent addition of ATP induced calcium transients. Bottom panels: propagated calcium waves (left) frequently desensitized a local area to subsequent addition of ATP (right).

Mentions: The above experiments suggested that intercellular calcium waves in UMR cells were mediated by activation of purinergic receptors. P2U purinergic receptors, like many other G protein–coupled receptors, undergo prolonged desensitization after stimulation with ligand. After exposure to 10 μM or 1 mM ATP and change of medium, UMR cells failed to respond to subsequent addition of ATP for at least 15 min (not shown). The failure to respond to subsequent challenge with ATP was not due to depletion of intracellular calcium stores, because subsequent challenge with thrombin (20 U/ml), which activates another G protein–coupled receptor in these cells (1), resulted in cytosolic calcium transients. Depletion of intracellular calcium stores with 50 nM thapsigargin for 30 min inhibited thrombin-induced calcium transients (20 U/ml thrombin) in UMR cells, demonstrating that thrombin- induced calcium transients required release of intracellular calcium stores as did ATP-mediated responses (not shown). We therefore assessed the effect of P2U receptor desensitization on calcium waves in UMR cells as follows (n = 4, Fig. 7). Cells were loaded with 5 μM fura-2, and calcium waves were demonstrated. Next, 10 μM ATP was added, which resulted in a calcium transient in all cells. After 2 min, [Ca2+]i returned to resting levels, and single cells were mechanically stimulated. This resulted in a rise in [Ca2+]i in the stimulated cell, but no calcium wave was initiated or propagated in the monolayers. Finally, 20 U/ml thrombin was added, resulting in an increase in [Ca2+]i, demonstrating that there remained releasable calcium stores. The same results were obtained using 1 mM ATP before mechanical stimulation.


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

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

Desensitization of  P2U receptors inhibits calcium waves in UMR cells.  Ratio imaging was performed on fura-2–loaded  UMR cell monolayers. Top  panels: left, 1 mM ATP-elicited calcium transients; center, subsequent mechanical  stimulation failed to induce  intercellular calcium waves;  right, subsequent challenge  with 20 U/ml thrombin revealed that calcium stores  were not depleted. Middle  panels: left, thrombin-induced  calcium transients; center,  subsequent mechanical stimulation induced a calcium  wave; right, subsequent addition of ATP induced calcium  transients. Bottom panels:  propagated calcium waves  (left) frequently desensitized  a local area to subsequent  addition of ATP (right).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139805&req=5

Figure 7: Desensitization of P2U receptors inhibits calcium waves in UMR cells. Ratio imaging was performed on fura-2–loaded UMR cell monolayers. Top panels: left, 1 mM ATP-elicited calcium transients; center, subsequent mechanical stimulation failed to induce intercellular calcium waves; right, subsequent challenge with 20 U/ml thrombin revealed that calcium stores were not depleted. Middle panels: left, thrombin-induced calcium transients; center, subsequent mechanical stimulation induced a calcium wave; right, subsequent addition of ATP induced calcium transients. Bottom panels: propagated calcium waves (left) frequently desensitized a local area to subsequent addition of ATP (right).
Mentions: The above experiments suggested that intercellular calcium waves in UMR cells were mediated by activation of purinergic receptors. P2U purinergic receptors, like many other G protein–coupled receptors, undergo prolonged desensitization after stimulation with ligand. After exposure to 10 μM or 1 mM ATP and change of medium, UMR cells failed to respond to subsequent addition of ATP for at least 15 min (not shown). The failure to respond to subsequent challenge with ATP was not due to depletion of intracellular calcium stores, because subsequent challenge with thrombin (20 U/ml), which activates another G protein–coupled receptor in these cells (1), resulted in cytosolic calcium transients. Depletion of intracellular calcium stores with 50 nM thapsigargin for 30 min inhibited thrombin-induced calcium transients (20 U/ml thrombin) in UMR cells, demonstrating that thrombin- induced calcium transients required release of intracellular calcium stores as did ATP-mediated responses (not shown). We therefore assessed the effect of P2U receptor desensitization on calcium waves in UMR cells as follows (n = 4, Fig. 7). Cells were loaded with 5 μM fura-2, and calcium waves were demonstrated. Next, 10 μM ATP was added, which resulted in a calcium transient in all cells. After 2 min, [Ca2+]i returned to resting levels, and single cells were mechanically stimulated. This resulted in a rise in [Ca2+]i in the stimulated cell, but no calcium wave was initiated or propagated in the monolayers. Finally, 20 U/ml thrombin was added, resulting in an increase in [Ca2+]i, demonstrating that there remained releasable calcium stores. The same results were obtained using 1 mM ATP before mechanical stimulation.

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