Evidence for the involvement of protein kinase C isoforms in alpha-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells.
BACKGROUND
FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of alpha-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 mumol/L) was used as an alpha-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo-1. 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-alpha, -beta I, -beta II, -gamma, delta, -epsilon, -zeta, and -eta isoforms were identified. Norepinephrine induced translocation of PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon isoforms but not -zeta and -eta 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-alpha.


CONCLUSIONS
These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in alpha-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-alpha translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-alpha, to regulate AA generation is an intriguing question that remains to be clarified.
Wang
X D
XD
Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
Kiang
J G
JG
Atwa
M A
MA
Smallridge
R C
RC
D000818:Animals; D016718:Arachidonic Acid / Q000502:physiology; D015220:Calcium Channels / Q000187:drug effects; D002460:Cell Line / Q000201:enzymology; D010741:Phospholipases A / Q000502:physiology*; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D011916:Rats, Inbred F344; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects*; D015290:Second Messenger Systems / Q000502:physiology; D013961:Thyroid Gland / Q000166:cytology* / Q000201:enzymology*
D015220:Calcium Channels; D018340:Receptors, Adrenergic, alpha-1; D016718:Arachidonic Acid; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2