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A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells.

Bony C, Roche S, Shuichi U, Sasaki T, Crackower MA, Penninger J, Mano H, Pucéat M - J. Cell Biol. (2001)

Bottom Line: However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma.A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking.We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research, CNRS UPR1086 Montpellier 34293, France.

ABSTRACT
Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma. We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event. We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.

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PI3K is required for ATP-induced slowing of cell Ca2+ spiking rate. A fluo3-loaded cell was superfused in the absence (A) or presence (B) of LY294002 with 20 μM ATP. Fluo3 fluorescence was recorded every 30 ms with a photomultiplier. The figure is representative of 10 similar experiments performed using two different cell cultures. Data are compared in the bar graph shown in (C). ^Significantly increased. (p ≤ 0.01). ATP, ATP-stimulated cells; C, control.
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Figure 2: PI3K is required for ATP-induced slowing of cell Ca2+ spiking rate. A fluo3-loaded cell was superfused in the absence (A) or presence (B) of LY294002 with 20 μM ATP. Fluo3 fluorescence was recorded every 30 ms with a photomultiplier. The figure is representative of 10 similar experiments performed using two different cell cultures. Data are compared in the bar graph shown in (C). ^Significantly increased. (p ≤ 0.01). ATP, ATP-stimulated cells; C, control.

Mentions: IP3, generated by activation of purinergic P2Y receptors, is a crucial regulator of rhythmic Ca2+ spiking in neonatal rat cardiomyocytes plated at high density. After an intracellular Ca2+ release, IP3 slows or stops cell spontaneous Ca2+ oscillations (Jaconi et al. 2000). We tested the effect of PI3K inhibitors on purinergic effect in spontaneous Ca2+ spiking cells. Extracellular ATP, acting through P2Y receptors slowed the rate of Ca2+ spiking of fluo3-loaded cardiomyocytes after an intracellular Ca2+ release, revealed by a transient rise in diastolic Ca2+ (Fig. 2 A), as described previously (Jaconi et al. 2000). In contrast, the purinergic agonist had no effect or accelerated this rate in cardiomyocytes treated with wortmannin or LY294002 (Fig. 2B and Fig. c). Acceleration of the rate and sustained increase in diastolic Ca2+ in the presence of ATP can be attributed to a transient membrane depolarization after activation of the ATP-gated ion channel through P2X receptors, an effect that is unmasked when IP3 generation by P2Y receptors is blocked (Jaconi et al. 2000). Wortmannin and LY294002 added alone did not affect the rate of Ca2+ oscillations (Fig. 2 C).


A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells.

Bony C, Roche S, Shuichi U, Sasaki T, Crackower MA, Penninger J, Mano H, Pucéat M - J. Cell Biol. (2001)

PI3K is required for ATP-induced slowing of cell Ca2+ spiking rate. A fluo3-loaded cell was superfused in the absence (A) or presence (B) of LY294002 with 20 μM ATP. Fluo3 fluorescence was recorded every 30 ms with a photomultiplier. The figure is representative of 10 similar experiments performed using two different cell cultures. Data are compared in the bar graph shown in (C). ^Significantly increased. (p ≤ 0.01). ATP, ATP-stimulated cells; C, control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195768&req=5

Figure 2: PI3K is required for ATP-induced slowing of cell Ca2+ spiking rate. A fluo3-loaded cell was superfused in the absence (A) or presence (B) of LY294002 with 20 μM ATP. Fluo3 fluorescence was recorded every 30 ms with a photomultiplier. The figure is representative of 10 similar experiments performed using two different cell cultures. Data are compared in the bar graph shown in (C). ^Significantly increased. (p ≤ 0.01). ATP, ATP-stimulated cells; C, control.
Mentions: IP3, generated by activation of purinergic P2Y receptors, is a crucial regulator of rhythmic Ca2+ spiking in neonatal rat cardiomyocytes plated at high density. After an intracellular Ca2+ release, IP3 slows or stops cell spontaneous Ca2+ oscillations (Jaconi et al. 2000). We tested the effect of PI3K inhibitors on purinergic effect in spontaneous Ca2+ spiking cells. Extracellular ATP, acting through P2Y receptors slowed the rate of Ca2+ spiking of fluo3-loaded cardiomyocytes after an intracellular Ca2+ release, revealed by a transient rise in diastolic Ca2+ (Fig. 2 A), as described previously (Jaconi et al. 2000). In contrast, the purinergic agonist had no effect or accelerated this rate in cardiomyocytes treated with wortmannin or LY294002 (Fig. 2B and Fig. c). Acceleration of the rate and sustained increase in diastolic Ca2+ in the presence of ATP can be attributed to a transient membrane depolarization after activation of the ATP-gated ion channel through P2X receptors, an effect that is unmasked when IP3 generation by P2Y receptors is blocked (Jaconi et al. 2000). Wortmannin and LY294002 added alone did not affect the rate of Ca2+ oscillations (Fig. 2 C).

Bottom Line: However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma.A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking.We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research, CNRS UPR1086 Montpellier 34293, France.

ABSTRACT
Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma. We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event. We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.

Show MeSH
Related in: MedlinePlus