Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors

Chou Long Huang, Tadaomi Takenawa, Harlan E. Ives

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Abstract

Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+ i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2+-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (<5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+ i of approximately 200 nM. This change in Ca2+ i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10-7 M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in <5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We conclude that entry of extracellular Ca2+ induced by PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.

Original languageEnglish (US)
Pages (from-to)4045-4048
Number of pages4
JournalJournal of Biological Chemistry
Volume266
Issue number7
StatePublished - Mar 5 1991

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Inositol 1,4,5-Trisphosphate Receptors
Platelet-Derived Growth Factor
Phosphatidylinositols
Heparin
Antibodies
Cells
Platelet-Derived Growth Factor Receptors
Suramin
Nicardipine
Inositol 1,4,5-Trisphosphate
Chondroitin Sulfates
Fura-2
Microinjections
Vascular Smooth Muscle
Metabolism
Thrombin
Smooth Muscle Myocytes
Muscle
Rats
Intercellular Signaling Peptides and Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{c1c198e01a29436e9b1dd96381e2e2ce,
title = "Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors",
abstract = "Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+ i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2+-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (<5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+ i of approximately 200 nM. This change in Ca2+ i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10-7 M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in <5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We conclude that entry of extracellular Ca2+ induced by PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.",
author = "Huang, {Chou Long} and Tadaomi Takenawa and Ives, {Harlan E.}",
year = "1991",
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language = "English (US)",
volume = "266",
pages = "4045--4048",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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T1 - Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors

AU - Huang, Chou Long

AU - Takenawa, Tadaomi

AU - Ives, Harlan E.

PY - 1991/3/5

Y1 - 1991/3/5

N2 - Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+ i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2+-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (<5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+ i of approximately 200 nM. This change in Ca2+ i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10-7 M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in <5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We conclude that entry of extracellular Ca2+ induced by PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.

AB - Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+ i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2+-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (<5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+ i of approximately 200 nM. This change in Ca2+ i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10-7 M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in <5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We conclude that entry of extracellular Ca2+ induced by PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.

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