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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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Effect of lysophosphatidic acid receptor antagonists and Gi protein inhibitor on LPA-induced changes in intracellular Ca2+ concentration in C2C12 cells. C2C12 cells were pre-treated with 10 μM of the LPA receptor antagonists, VPC 12249, VPC 32183 and DGPP (8.0), for 10 sec or with pertussis toxin (PTX, 100 ng/ml), a Gi protein inhibitor, for 10 min prior to the addition of LPA (10 μM). The [Ca2+]i was measured as described in the Materials and methods. Values are means ± S.E.M. of six different experiments. *P < 0.05 versus vehicle control value. DGPP: dioctanoyl glycerol pyrophosphate; LPA : lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
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fig02: Effect of lysophosphatidic acid receptor antagonists and Gi protein inhibitor on LPA-induced changes in intracellular Ca2+ concentration in C2C12 cells. C2C12 cells were pre-treated with 10 μM of the LPA receptor antagonists, VPC 12249, VPC 32183 and DGPP (8.0), for 10 sec or with pertussis toxin (PTX, 100 ng/ml), a Gi protein inhibitor, for 10 min prior to the addition of LPA (10 μM). The [Ca2+]i was measured as described in the Materials and methods. Values are means ± S.E.M. of six different experiments. *P < 0.05 versus vehicle control value. DGPP: dioctanoyl glycerol pyrophosphate; LPA : lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.

Mentions: In order to determine if the LPA-induced increase in [Ca2+]i was a LPA-receptor mediated response, C2C12 cells were pre-treated with LPA1/3 receptor antagonists, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 10 μM), VPC12249 (10 μM) or VPC 32183 (10 μM) [29–31] (Avanti Polar Lipids, Inc, Al, USA) for 10 sec before the addition of LPA. The pretreatment with these receptor antagonists had no significant effect on the basal [Ca2+]i (data not shown). However, these receptor blockers significantly inhibited the LPA-induced increase in [Ca2+]i (Fig. 2). Furthermore, to understand the LPA receptor signalling mechanisms, C2C12 cells were also pre-treated with a Gi protein inhibitor, pertussis toxin (PTX, 100 ng/ml, Sigma-Aldrich, MO, USA) [32] for 10 min. This time of pre-treatment with PTX is based on previous experience [21]. It can be seen in Figure 2 that the LPA-induced increase in [Ca2+]i in C2C12 cells was significantly inhibited.


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 receptor antagonists and Gi protein inhibitor on LPA-induced changes in intracellular Ca2+ concentration in C2C12 cells. C2C12 cells were pre-treated with 10 μM of the LPA receptor antagonists, VPC 12249, VPC 32183 and DGPP (8.0), for 10 sec or with pertussis toxin (PTX, 100 ng/ml), a Gi protein inhibitor, for 10 min prior to the addition of LPA (10 μM). The [Ca2+]i was measured as described in the Materials and methods. Values are means ± S.E.M. of six different experiments. *P < 0.05 versus vehicle control value. DGPP: dioctanoyl glycerol pyrophosphate; LPA : lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401138&req=5

fig02: Effect of lysophosphatidic acid receptor antagonists and Gi protein inhibitor on LPA-induced changes in intracellular Ca2+ concentration in C2C12 cells. C2C12 cells were pre-treated with 10 μM of the LPA receptor antagonists, VPC 12249, VPC 32183 and DGPP (8.0), for 10 sec or with pertussis toxin (PTX, 100 ng/ml), a Gi protein inhibitor, for 10 min prior to the addition of LPA (10 μM). The [Ca2+]i was measured as described in the Materials and methods. Values are means ± S.E.M. of six different experiments. *P < 0.05 versus vehicle control value. DGPP: dioctanoyl glycerol pyrophosphate; LPA : lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
Mentions: In order to determine if the LPA-induced increase in [Ca2+]i was a LPA-receptor mediated response, C2C12 cells were pre-treated with LPA1/3 receptor antagonists, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 10 μM), VPC12249 (10 μM) or VPC 32183 (10 μM) [29–31] (Avanti Polar Lipids, Inc, Al, USA) for 10 sec before the addition of LPA. The pretreatment with these receptor antagonists had no significant effect on the basal [Ca2+]i (data not shown). However, these receptor blockers significantly inhibited the LPA-induced increase in [Ca2+]i (Fig. 2). Furthermore, to understand the LPA receptor signalling mechanisms, C2C12 cells were also pre-treated with a Gi protein inhibitor, pertussis toxin (PTX, 100 ng/ml, Sigma-Aldrich, MO, USA) [32] for 10 min. This time of pre-treatment with PTX is based on previous experience [21]. It can be seen in Figure 2 that the LPA-induced increase in [Ca2+]i in C2C12 cells was significantly inhibited.

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