Phospholipase C activation by Na⁺/Ca²⁺ exchange is essential for monensin-induced Ca²⁺ influx and arachidonic acid release in FRTL-5 thyroid cells

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 μmol/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 monensin-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 PLA,-mediated processes in monensin-challenged FRTL-5 thyroid cells.