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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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Concentration-dependent effect of lysophosphatidic acid on intracellular calcium concentration in C2C12 skeletal muscle cells. (A): Real time recording of [Ca2+]i in C2C12 cells. B: Quantified data of the LPAinduced increases in [Ca2+]i in C2C12 cells. Values are means ± S.E.M. of six different experiments. Cultured cells were trypsinized using 2 ml 0.25 % trypsin-1 mM ethylenediaminetetraacetic acid (EDTA) and washed twice with HEPES buffer. The cells were then incubated with 10 μM fura-2/AM in HEPES buffer for 40 min and then washed twice with HEPES uffer to remove any extracellular dye. The fluorescence was monitored by a SLM DMX-1100 dual wavelength pectrofluorometer. * P<0.05 versus control value. # P < 0.05 versus 1 μM LPA. ¶P < 0.05 versus 10 μM LPA. LPA: lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
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fig01: Concentration-dependent effect of lysophosphatidic acid on intracellular calcium concentration in C2C12 skeletal muscle cells. (A): Real time recording of [Ca2+]i in C2C12 cells. B: Quantified data of the LPAinduced increases in [Ca2+]i in C2C12 cells. Values are means ± S.E.M. of six different experiments. Cultured cells were trypsinized using 2 ml 0.25 % trypsin-1 mM ethylenediaminetetraacetic acid (EDTA) and washed twice with HEPES buffer. The cells were then incubated with 10 μM fura-2/AM in HEPES buffer for 40 min and then washed twice with HEPES uffer to remove any extracellular dye. The fluorescence was monitored by a SLM DMX-1100 dual wavelength pectrofluorometer. * P<0.05 versus control value. # P < 0.05 versus 1 μM LPA. ¶P < 0.05 versus 10 μM LPA. LPA: lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.

Mentions: We have previously reported that LPA increases [Ca2+]i in A10 vascular smooth muscle cells (VSMCs) [26, 27]. Likewise, LPA (1–50 μM) induced a significant concentration-dependent increase in [Ca2+]i, with the maximum response at 30 sec that declined back to basal levels at 2 min (Fig. 1A). A maximum response was achieved at 50 μM LPA (Fig. 1A and B). A concentration of 10 μM LPA was subsequently used for the investigation of the mechanisms of the LPA-induced increase in [Ca2+]i. This was also based on other studies that have employed this concentration of LPA [21, 26, 27]. It should be noted 20 μl water served as a control and exerted no significant effect on basal [Ca2+]i (data not shown).


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)

Concentration-dependent effect of lysophosphatidic acid on intracellular calcium concentration in C2C12 skeletal muscle cells. (A): Real time recording of [Ca2+]i in C2C12 cells. B: Quantified data of the LPAinduced increases in [Ca2+]i in C2C12 cells. Values are means ± S.E.M. of six different experiments. Cultured cells were trypsinized using 2 ml 0.25 % trypsin-1 mM ethylenediaminetetraacetic acid (EDTA) and washed twice with HEPES buffer. The cells were then incubated with 10 μM fura-2/AM in HEPES buffer for 40 min and then washed twice with HEPES uffer to remove any extracellular dye. The fluorescence was monitored by a SLM DMX-1100 dual wavelength pectrofluorometer. * P<0.05 versus control value. # P < 0.05 versus 1 μM LPA. ¶P < 0.05 versus 10 μM LPA. LPA: lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
© Copyright Policy
Related In: Results  -  Collection

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

fig01: Concentration-dependent effect of lysophosphatidic acid on intracellular calcium concentration in C2C12 skeletal muscle cells. (A): Real time recording of [Ca2+]i in C2C12 cells. B: Quantified data of the LPAinduced increases in [Ca2+]i in C2C12 cells. Values are means ± S.E.M. of six different experiments. Cultured cells were trypsinized using 2 ml 0.25 % trypsin-1 mM ethylenediaminetetraacetic acid (EDTA) and washed twice with HEPES buffer. The cells were then incubated with 10 μM fura-2/AM in HEPES buffer for 40 min and then washed twice with HEPES uffer to remove any extracellular dye. The fluorescence was monitored by a SLM DMX-1100 dual wavelength pectrofluorometer. * P<0.05 versus control value. # P < 0.05 versus 1 μM LPA. ¶P < 0.05 versus 10 μM LPA. LPA: lysophosphatidic acid, [Ca2+]i: intracellular calcium concentration.
Mentions: We have previously reported that LPA increases [Ca2+]i in A10 vascular smooth muscle cells (VSMCs) [26, 27]. Likewise, LPA (1–50 μM) induced a significant concentration-dependent increase in [Ca2+]i, with the maximum response at 30 sec that declined back to basal levels at 2 min (Fig. 1A). A maximum response was achieved at 50 μM LPA (Fig. 1A and B). A concentration of 10 μM LPA was subsequently used for the investigation of the mechanisms of the LPA-induced increase in [Ca2+]i. This was also based on other studies that have employed this concentration of LPA [21, 26, 27]. It should be noted 20 μl water served as a control and exerted no significant effect on basal [Ca2+]i (data not shown).

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