Ca²⁺ Oscillations induced by fibroblast growth factor 2 in Xenopus oocytes expressing fibroblast growth factor receptors

Abstract

Double electrode voltage clamp technique was used to follow precisely the calcium signalling pathway activated by FGF receptors from a normal and a carcinogenous cell environment. Functional FGF receptors were expressed in Xenopus oocytes following either the injection of PFR1 cRNA from Pleurodeles, an homologue of the human FGFR1 mRNA, or breast cancer MCF7 cells total mRNA. Cytosolic calcium oscillations were monitored through the endogenous Ca²⁺-dependent Cl⁻¹ channel activity from both RNA injected systems, under FGF2 treatment. The Ca²⁺-dependent Cl⁻ channel was demonstrated using the Cl⁻ channel blocker SITS (250 μM) and by the determination of the reversal potential of the Cl⁻ ions close to - 20 mV. The FGF2-evoked Ca²⁺-dependent Cl⁻ current was abolished by external application of genistein (10 μM, tyrosine kinase inhibitor), neomycin (10 mM, phosphatidylinositol turnover inhibitor), caffeine (10 mM, inhibitor of Ins(1,4,5)P3-mediated release of intracellular calcium), and injection of BAPTA (50 μM, calcium chelator) or heparin (2 μg/ml, inhibitor of the binding of lns(1,4,5)P3). The recorded current was independent of extracellular Ca²⁺ but involved tyrosine kinase phosphorylation and intracellular Ins(1,4,5)P3 sensitive stores. External application of heparin enhanced the oscillatory Ca²⁺ rise, suggesting a role for the heparan sulfates in the regulatory mechanism of the FGF receptors. The similarities in the Ca⁺² -dependent Cl⁻ current obtained in PFR1 and total MCF7 FGF receptors expressing oocytes are discussed.

Key Words:

• FGF2
• FGF receptors
• Ca²⁺ mobilization
• Xenopus oocytes
• MCF-7 cell line