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 C. Smallridge

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


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
Issue number9
StatePublished - 1996


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

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


Dive into the research topics of 'Evidence for the involvement of protein kinase C isoforms in α-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells'. Together they form a unique fingerprint.

Cite this