title: Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin-induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells.
abstract: BACKGROUND
  Monensin, a Na+ ionophore, can increase cytosolic Ca2+ ([Ca2+]i) by reversing the Na+/Ca2+ exchange mechanism. This study provided additional insights into the mechanism of this Na+ ionophore-induced increase in [Ca2+]i, and emphasized the critical role of phospholipase C (PLC) in amplifying Na+/Ca2+ exchange-induced Ca2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored.
  
  
  METHODS
  FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter.
  
  
  RESULTS
  In Hank's balanced salt solution with Ca2+ (HBSS+), monensin (100 mumol/L) induced a 2.3-fold sustained Ca2+ increase associated with IP3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca2+, or replacement of Na+ by choline chloride in the medium, reduced the [Ca2+]i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na+ channel blocker, or Na+/Ca2+ exchange inhibitors. In HBSS without Ca2+ (HBSS-), monensin induced a 1-fold transient [Ca2+]i increase but did not increase the AA. This Ca2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca2+]i, but reduced the sustained second phase of [Ca2+]i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A2 (PLA2) inhibitor blocked the monensin-induced AA release without affecting the [Ca2+]i increase. Inhibition of PKC prevented 87% to 94% of the monesin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor.
  
  
  CONCLUSIONS
  The results suggest that monensin initiates an increase in [Ca2+]i via a Na+/Ca2+ exchange mechanism that triggers more pronounced and sustained [Ca2+]i increase via activation of PLC and Ca2+ influx. The PLC activation, followed by sustained Ca2+ influx and PKC activation, is a prerequisite for PLA2-mediated processes in monensin-challenged FRTL-5 thyroid cells.
authors.affiliation: Department of Clinical Pharmacology, University of Illinois, Chicago College of Medicine at Peoria, USA.
authors.forename: X D
authors.initials: XD
authors.lastname: Wang
authors.forename: J G
authors.initials: JG
authors.lastname: Kiang
authors.forename: L W
authors.initials: LW
authors.lastname: Scheibel
authors.forename: R C
authors.initials: RC
authors.lastname: Smallridge
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D007476:Ionophores / Q000494:pharmacology*; D008985:Monensin / Q000494:pharmacology*; D051381:Rats; D011916:Rats, Inbred F344; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D007476:Ionophores; D019831:Sodium-Calcium Exchanger; D016718:Arachidonic Acid; D008985:Monensin; D010738:Type C Phospholipases; D002118:Calcium