Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells

Wallace B Thoreson, Jennifer S. Ryan, Chanjuan Shi, Melanie E. Kelly, Eric J. Bryson, Myron Lee Toews, Tracy L. Ediger, David M. Chacko

Research output: Contribution to journalArticle

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Abstract

PURPOSE. Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K+ currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells. METHODS. Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays. RESULTS. LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to Gαi/o/t/z. LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-I, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca2+]i that persisted in Ca2+-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca2+]i increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca2+]i increases were blocked by PD98059 and by loading cells with antibodies to Gαi/o/t/z. RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA. CONCLUSIONS. LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca2+ from intracellular stores that appears independent of IP3 and ryanodine receptor activation.

Original languageEnglish (US)
Pages (from-to)2450-2461
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number7
StatePublished - Jul 9 2002

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Lysophosphatidic Acid Receptors
Retinal Pigments
Epithelial Cells
Cations
Phosphotransferases
RNA
Inositol 1,4,5-Trisphosphate Receptors
Ryanodine
Ryanodine Receptor Calcium Release Channel
Inositol Phosphates
Thapsigargin
Antibodies
Genistein
Mitogen-Activated Protein Kinase Kinases
Pertussis Toxin
Type C Phospholipases
Second Messenger Systems
Patch-Clamp Techniques
Chemotaxis
Protein Kinase Inhibitors

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells. / Thoreson, Wallace B; Ryan, Jennifer S.; Shi, Chanjuan; Kelly, Melanie E.; Bryson, Eric J.; Toews, Myron Lee; Ediger, Tracy L.; Chacko, David M.

In: Investigative Ophthalmology and Visual Science, Vol. 43, No. 7, 09.07.2002, p. 2450-2461.

Research output: Contribution to journalArticle

Thoreson, WB, Ryan, JS, Shi, C, Kelly, ME, Bryson, EJ, Toews, ML, Ediger, TL & Chacko, DM 2002, 'Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells', Investigative Ophthalmology and Visual Science, vol. 43, no. 7, pp. 2450-2461.
Thoreson, Wallace B ; Ryan, Jennifer S. ; Shi, Chanjuan ; Kelly, Melanie E. ; Bryson, Eric J. ; Toews, Myron Lee ; Ediger, Tracy L. ; Chacko, David M. / Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells. In: Investigative Ophthalmology and Visual Science. 2002 ; Vol. 43, No. 7. pp. 2450-2461.
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abstract = "PURPOSE. Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K+ currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells. METHODS. Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays. RESULTS. LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to Gαi/o/t/z. LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-I, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca2+]i that persisted in Ca2+-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca2+]i increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca2+]i increases were blocked by PD98059 and by loading cells with antibodies to Gαi/o/t/z. RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA. CONCLUSIONS. LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca2+ from intracellular stores that appears independent of IP3 and ryanodine receptor activation.",
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T1 - Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells

AU - Thoreson, Wallace B

AU - Ryan, Jennifer S.

AU - Shi, Chanjuan

AU - Kelly, Melanie E.

AU - Bryson, Eric J.

AU - Toews, Myron Lee

AU - Ediger, Tracy L.

AU - Chacko, David M.

PY - 2002/7/9

Y1 - 2002/7/9

N2 - PURPOSE. Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K+ currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells. METHODS. Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays. RESULTS. LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to Gαi/o/t/z. LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-I, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca2+]i that persisted in Ca2+-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca2+]i increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca2+]i increases were blocked by PD98059 and by loading cells with antibodies to Gαi/o/t/z. RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA. CONCLUSIONS. LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca2+ from intracellular stores that appears independent of IP3 and ryanodine receptor activation.

AB - PURPOSE. Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K+ currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells. METHODS. Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays. RESULTS. LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to Gαi/o/t/z. LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-I, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca2+]i that persisted in Ca2+-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca2+]i increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca2+]i increases were blocked by PD98059 and by loading cells with antibodies to Gαi/o/t/z. RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA. CONCLUSIONS. LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca2+ from intracellular stores that appears independent of IP3 and ryanodine receptor activation.

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