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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 receptor operated Ca2+ channels (A) and Na+–Ca2+ exchanger inhibitors (B) on lysophosphatidic acid-induced increase in intracellular calcium concentration in C2C12 cells. C2C12 cells were pre-treated with 2- minoethoxydiphenyl borate (2-APB, 100 μM), SK&F 96365 (10 μM), Ni2+ (1 mM) or KB-R7943 (KBR, 30 μM) 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. LPA: lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
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fig04: Effect of receptor operated Ca2+ channels (A) and Na+–Ca2+ exchanger inhibitors (B) on lysophosphatidic acid-induced increase in intracellular calcium concentration in C2C12 cells. C2C12 cells were pre-treated with 2- minoethoxydiphenyl borate (2-APB, 100 μM), SK&F 96365 (10 μM), Ni2+ (1 mM) or KB-R7943 (KBR, 30 μM) 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. LPA: lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.

Mentions: In C2C12 cells, EGTA (1 mM) reduced both the basal as well as the peak [Ca2+]i response evoked by LPA, whereas verapamil and diltiazem had no significant effect on the basal and LPA-induced increase in [Ca2+]i (Table 1), Similarly, both Gd3+ and La3+, blockers of store operated Ca2+ channels (SOCs), did not exert any significant effect on LPAevoked increase in [Ca2+]i (Table 1). To test whether receptor operated Ca2+ channels (ROCs) play a role in LPA-induced changes in [Ca2+]i in C2C12 cells, cells were pre-treated with SK&F 96365 (10 μM, Biomol Research Laboratory, PA, USA), an inhibitor of ROCs [34] and with the transient receptor potential (TRP) C7 cation channel blocker, 2-aminoethoxydiphenyl borate (2-APB, 100 μM, Sigma-Aldrich, MO, USA) [35] for 10 min. Both these agents significantly attenuated the LPAinduced increase in [Ca2+]i by 50–60 % (Fig. 4A). The cells pre-treated with SK&F 96365 and 2-APB did not alter the basal [Ca2+]i (vehicle control 81 ± 8; SK&F 96365 81 ± 11; 2-APB 100 ± 12 nM). Pre-treatment with the Na+–Ca2+ exchanger inhibitors, Ni (1 mM) and KB-R7943 (30 μM, Tocris Biosciences, MO, USA) [26], resulted in a significant inhibition (40–55%) of the LPA-induced increase in [Ca2+]i in C2C12 cells (Fig. 4B). The pre-treatment of cells with Ni and KB-R7943 alone exerted no significant action on the basal [Ca2+]i (vehicle control: 83 ± 11 nM; Ni:81 ± 11 nM; KB-R7943: 91 ± 12 nM).


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 receptor operated Ca2+ channels (A) and Na+–Ca2+ exchanger inhibitors (B) on lysophosphatidic acid-induced increase in intracellular calcium concentration in C2C12 cells. C2C12 cells were pre-treated with 2- minoethoxydiphenyl borate (2-APB, 100 μM), SK&F 96365 (10 μM), Ni2+ (1 mM) or KB-R7943 (KBR, 30 μM) 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. 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

fig04: Effect of receptor operated Ca2+ channels (A) and Na+–Ca2+ exchanger inhibitors (B) on lysophosphatidic acid-induced increase in intracellular calcium concentration in C2C12 cells. C2C12 cells were pre-treated with 2- minoethoxydiphenyl borate (2-APB, 100 μM), SK&F 96365 (10 μM), Ni2+ (1 mM) or KB-R7943 (KBR, 30 μM) 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. LPA: lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
Mentions: In C2C12 cells, EGTA (1 mM) reduced both the basal as well as the peak [Ca2+]i response evoked by LPA, whereas verapamil and diltiazem had no significant effect on the basal and LPA-induced increase in [Ca2+]i (Table 1), Similarly, both Gd3+ and La3+, blockers of store operated Ca2+ channels (SOCs), did not exert any significant effect on LPAevoked increase in [Ca2+]i (Table 1). To test whether receptor operated Ca2+ channels (ROCs) play a role in LPA-induced changes in [Ca2+]i in C2C12 cells, cells were pre-treated with SK&F 96365 (10 μM, Biomol Research Laboratory, PA, USA), an inhibitor of ROCs [34] and with the transient receptor potential (TRP) C7 cation channel blocker, 2-aminoethoxydiphenyl borate (2-APB, 100 μM, Sigma-Aldrich, MO, USA) [35] for 10 min. Both these agents significantly attenuated the LPAinduced increase in [Ca2+]i by 50–60 % (Fig. 4A). The cells pre-treated with SK&F 96365 and 2-APB did not alter the basal [Ca2+]i (vehicle control 81 ± 8; SK&F 96365 81 ± 11; 2-APB 100 ± 12 nM). Pre-treatment with the Na+–Ca2+ exchanger inhibitors, Ni (1 mM) and KB-R7943 (30 μM, Tocris Biosciences, MO, USA) [26], resulted in a significant inhibition (40–55%) of the LPA-induced increase in [Ca2+]i in C2C12 cells (Fig. 4B). The pre-treatment of cells with Ni and KB-R7943 alone exerted no significant action on the basal [Ca2+]i (vehicle control: 83 ± 11 nM; Ni:81 ± 11 nM; KB-R7943: 91 ± 12 nM).

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