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Mechanisms of the lysophosphatidic acid-induced increase in [Ca(2+)](i) in skeletal muscle cells.

Xu YJ, Tappia PS, Goyal RK, Dhalla NS - J. Cell. Mol. Med. (2008)

Bottom Line: The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells.The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365.However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada.

ABSTRACT
Although lysophosphatidic acid (LPA) is known to increase intracellularfree calcium concentration ([Ca(2+)](i)) in different cell types, the effect of LPA on the skeletal muscle cells is not known. The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells. LPA induced a concentration and time dependent increase in [Ca(2+)](i), which was inhibited by VPC12249, VPC 32183 and dioctanoyl glycerol pyrophosphate, LPA1/3 receptor antagonists. Pertussis toxin, a G(i) protein inhibitor, also inhibited the LPA-induced increase in [Ca(2+)](i). Inhibition of tyrosine kinase activities with tyrphostin A9 and genistein also prevented the increase in [Ca(2+)](i) due to LPA. Likewise, wortmannin and LY 294002, phosphatidylinositol 3-kinase (PI3-K) inhibitors, inhibited [Ca(2+)](i) response to LPA. The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365. However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA. Our data suggest that the LPA-induced increase in [Ca(2+)](i) might occur through G(i)-protein coupled LPA(1/3) receptors that may be linked to tyrosine kinase and PI3-K, and may also involve the Na(+)-Ca(2+) exchanger as well as the ROC. In addition, LPA stimulated C2C12 cell proliferation via PI3-K. Thus, LPA may be an important phospholipid in the regulation of [Ca(2+)](i) and growth of skeletal muscle cells.

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Related in: MedlinePlus

Effect of lysophosphatidic acid on C2C12 cell proliferation in the absence and presence of phosphatidyli-nositol 3-kinase inhibition with wortmannin. The C2C12 cell proliferation was determined by the incorporation of [3H]-thymidine into C2C12 cells. 100 nM wortmannin (WOR) was added to the culture medium 10 min prior to the addition of LPA (10 μM). After a 4 hrs incubation with LPA, 1 μCi [3H]-thymidine was added. The reaction was terminated after 22 hrs. Values are means ± S.E.M. of six experiments. *P<0.05 versus vehicle control value; #P<0.05 versus LPA value in the absence of wortmannin.
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fig06: Effect of lysophosphatidic acid on C2C12 cell proliferation in the absence and presence of phosphatidyli-nositol 3-kinase inhibition with wortmannin. The C2C12 cell proliferation was determined by the incorporation of [3H]-thymidine into C2C12 cells. 100 nM wortmannin (WOR) was added to the culture medium 10 min prior to the addition of LPA (10 μM). After a 4 hrs incubation with LPA, 1 μCi [3H]-thymidine was added. The reaction was terminated after 22 hrs. Values are means ± S.E.M. of six experiments. *P<0.05 versus vehicle control value; #P<0.05 versus LPA value in the absence of wortmannin.

Mentions: Intracellular Ca2+ signalling plays an important role in gene expression and DNA replication [36]. Since LPA has been observed to increase Ca2+ in both the nucleus and cytoplasm, it is possible that LPA might also induce DNA synthesis in C2C12 cells. Accordingly, we determined the effect of LPA on DNA synthesis in C2C12 cells. It can be seen in Figure 6 that LPA induced an increase in DNA synthesis as evidenced by an increase in [3H]-thymidine incorporation in C2C12 cells, which was prevented by the PI3-K inhibitor, wortmannin (100 nM). Wortmannin alone had no effect on [3H]-thymidine incorporation in C2C12 cells.


Mechanisms of the lysophosphatidic acid-induced increase in [Ca(2+)](i) in skeletal muscle cells.

Xu YJ, Tappia PS, Goyal RK, Dhalla NS - J. Cell. Mol. Med. (2008)

Effect of lysophosphatidic acid on C2C12 cell proliferation in the absence and presence of phosphatidyli-nositol 3-kinase inhibition with wortmannin. The C2C12 cell proliferation was determined by the incorporation of [3H]-thymidine into C2C12 cells. 100 nM wortmannin (WOR) was added to the culture medium 10 min prior to the addition of LPA (10 μM). After a 4 hrs incubation with LPA, 1 μCi [3H]-thymidine was added. The reaction was terminated after 22 hrs. Values are means ± S.E.M. of six experiments. *P<0.05 versus vehicle control value; #P<0.05 versus LPA value in the absence of wortmannin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401138&req=5

fig06: Effect of lysophosphatidic acid on C2C12 cell proliferation in the absence and presence of phosphatidyli-nositol 3-kinase inhibition with wortmannin. The C2C12 cell proliferation was determined by the incorporation of [3H]-thymidine into C2C12 cells. 100 nM wortmannin (WOR) was added to the culture medium 10 min prior to the addition of LPA (10 μM). After a 4 hrs incubation with LPA, 1 μCi [3H]-thymidine was added. The reaction was terminated after 22 hrs. Values are means ± S.E.M. of six experiments. *P<0.05 versus vehicle control value; #P<0.05 versus LPA value in the absence of wortmannin.
Mentions: Intracellular Ca2+ signalling plays an important role in gene expression and DNA replication [36]. Since LPA has been observed to increase Ca2+ in both the nucleus and cytoplasm, it is possible that LPA might also induce DNA synthesis in C2C12 cells. Accordingly, we determined the effect of LPA on DNA synthesis in C2C12 cells. It can be seen in Figure 6 that LPA induced an increase in DNA synthesis as evidenced by an increase in [3H]-thymidine incorporation in C2C12 cells, which was prevented by the PI3-K inhibitor, wortmannin (100 nM). Wortmannin alone had no effect on [3H]-thymidine incorporation in C2C12 cells.

Bottom Line: The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells.The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365.However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada.

ABSTRACT
Although lysophosphatidic acid (LPA) is known to increase intracellularfree calcium concentration ([Ca(2+)](i)) in different cell types, the effect of LPA on the skeletal muscle cells is not known. The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells. LPA induced a concentration and time dependent increase in [Ca(2+)](i), which was inhibited by VPC12249, VPC 32183 and dioctanoyl glycerol pyrophosphate, LPA1/3 receptor antagonists. Pertussis toxin, a G(i) protein inhibitor, also inhibited the LPA-induced increase in [Ca(2+)](i). Inhibition of tyrosine kinase activities with tyrphostin A9 and genistein also prevented the increase in [Ca(2+)](i) due to LPA. Likewise, wortmannin and LY 294002, phosphatidylinositol 3-kinase (PI3-K) inhibitors, inhibited [Ca(2+)](i) response to LPA. The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365. However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA. Our data suggest that the LPA-induced increase in [Ca(2+)](i) might occur through G(i)-protein coupled LPA(1/3) receptors that may be linked to tyrosine kinase and PI3-K, and may also involve the Na(+)-Ca(2+) exchanger as well as the ROC. In addition, LPA stimulated C2C12 cell proliferation via PI3-K. Thus, LPA may be an important phospholipid in the regulation of [Ca(2+)](i) and growth of skeletal muscle cells.

Show MeSH
Related in: MedlinePlus