Evidence for the involvement of protein kinase C isoforms in α-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells

Xiao Dong Wang, Juliann Gong Kiang, Mohamed A. Atwa, Robert Christian Smallridge

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Background: FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of α-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2(PLA2). Methods: Norepinephrine (NE, 10 μmol/L) was used as an α-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits. Results: Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-α, -βI, -βII, -γ, δ, -ε, -ζ, and - η isoforms were identified. Norepinephrine induced translocation of PKC-α, -βI, -βII, -γ, -δ, and -ε isoforms but not -ζ and -η from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-α. Conclusions: These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in α-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-α translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-α, to regulate AA generation is an intriguing question that remains to be clarified.

Original languageEnglish (US)
Pages (from-to)566-574
Number of pages9
JournalJournal of Investigative Medicine
Volume44
Issue number9
StatePublished - 1996

Fingerprint

Phospholipases A2
Adrenergic Agents
Protein Kinase C
Arachidonic Acid
Thyroid Gland
Protein Isoforms
Chemical activation
Norepinephrine
Adrenergic Receptors
Cells
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
Cell Line
Ethane
Staurosporine
Phospholipases
Protein C Inhibitor
Type C Phospholipases
Second Messenger Systems
Protein Kinase Inhibitors
Chelating Agents

Keywords

  • Adrenergic receptor
  • Arachidonic acid
  • Calcium
  • Norepinephrine
  • Phospholipase A
  • Phospholipase C
  • Protein kinase C

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Evidence for the involvement of protein kinase C isoforms in α-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells. / Wang, Xiao Dong; Kiang, Juliann Gong; Atwa, Mohamed A.; Smallridge, Robert Christian.

In: Journal of Investigative Medicine, Vol. 44, No. 9, 1996, p. 566-574.

Research output: Contribution to journalArticle

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title = "Evidence for the involvement of protein kinase C isoforms in α-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells",
abstract = "Background: FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of α-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2(PLA2). Methods: Norepinephrine (NE, 10 μmol/L) was used as an α-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits. Results: Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-α, -βI, -βII, -γ, δ, -ε, -ζ, and - η isoforms were identified. Norepinephrine induced translocation of PKC-α, -βI, -βII, -γ, -δ, and -ε isoforms but not -ζ and -η from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-α. Conclusions: These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in α-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-α translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-α, to regulate AA generation is an intriguing question that remains to be clarified.",
keywords = "Adrenergic receptor, Arachidonic acid, Calcium, Norepinephrine, Phospholipase A, Phospholipase C, Protein kinase C",
author = "Wang, {Xiao Dong} and Kiang, {Juliann Gong} and Atwa, {Mohamed A.} and Smallridge, {Robert Christian}",
year = "1996",
language = "English (US)",
volume = "44",
pages = "566--574",
journal = "Journal of Investigative Medicine",
issn = "1081-5589",
publisher = "Lippincott Williams and Wilkins",
number = "9",

}

TY - JOUR

T1 - Evidence for the involvement of protein kinase C isoforms in α-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells

AU - Wang, Xiao Dong

AU - Kiang, Juliann Gong

AU - Atwa, Mohamed A.

AU - Smallridge, Robert Christian

PY - 1996

Y1 - 1996

N2 - Background: FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of α-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2(PLA2). Methods: Norepinephrine (NE, 10 μmol/L) was used as an α-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits. Results: Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-α, -βI, -βII, -γ, δ, -ε, -ζ, and - η isoforms were identified. Norepinephrine induced translocation of PKC-α, -βI, -βII, -γ, -δ, and -ε isoforms but not -ζ and -η from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-α. Conclusions: These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in α-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-α translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-α, to regulate AA generation is an intriguing question that remains to be clarified.

AB - Background: FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of α-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2(PLA2). Methods: Norepinephrine (NE, 10 μmol/L) was used as an α-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits. Results: Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-α, -βI, -βII, -γ, δ, -ε, -ζ, and - η isoforms were identified. Norepinephrine induced translocation of PKC-α, -βI, -βII, -γ, -δ, and -ε isoforms but not -ζ and -η from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-α. Conclusions: These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in α-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-α translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-α, to regulate AA generation is an intriguing question that remains to be clarified.

KW - Adrenergic receptor

KW - Arachidonic acid

KW - Calcium

KW - Norepinephrine

KW - Phospholipase A

KW - Phospholipase C

KW - Protein kinase C

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