SYNERGISTIC ACTIVATION OF TYROSINE PHOSPHORYLATION BY O-VANADATE PLUSCALCIUM IONOPHORE A23187 OR AROMATIC 1,2-DIOLS

We have shown previously that treatment of WB rat liver epithelial cel ls with the Ca2+ ionophore A23187 provokes a rapid increase in protein -tyrosine phosphorylation that faithfully reproduces the Ca2+ dependen t response seen with angiotensin II. In the presence of the tyrosine p hosphatase inhibitor o-vanadate (2.0-200 mu M), the tyrosine phosphory lation response to A23187 was increased >10-fold in magnitude. This sy nergistic effect of A23187 and vanadate is clearly distinct from the c ombined effect of angiotensin II and vanadate, which was merely additi ve. Chelation of either extracellular or intracellular Ca2+ abolished the synergistic response to ionophore and vanadate, indicating its Ca2 + dependence. That divergent pathways were involved in the angiotensin II and the A23187/vanadate responses was shown definitely by studies of GN4 cells, a transformed line derived from WB cells by carcinogen t reatment. GN4 cells are 2-3-fold more responsive than WB cells to angi otensin II-dependent tyrosine kinase activation, yet they completely l acked the synergistic tyrosine phosphorylation response to A23187/vana date. To test the role of arachidonic acid metabolites in the A23187/v anadate response, cells were pretreated with either indomethacin or no rdihydroguaiaretic acid (NDGA). Neither compound was inhibitory, but s urprisingly, NDGA plus vanadate closely mimicked the A23187/vanadate r esponse in WB cells and, like A23187/vanadate, was ineffective in GN4 cells. NDGA contains catechol nuclei (i.e., aromatic 1,2-diols) and th erein resembles the flavonoid anti-oxidant quercetin, another compound found to increase tyrosine phosphorylation synergistically with vanad ate. In our studies, flavonoids that contain 1,2-diols (quercetin, fis etin) were synergistic with vanadate in elevating P-Tyr (in WB but not GN4 cells) while those lacking 1,2-diols (apigenin, morin) were inact ive. Overnight pretreatment with a glutathione precursor, N-acetylcyst eine, inhibited the responses to A23187/vanadate and NDGA/vanadate. We postulate that combinations of catechols with vanadate, separately or as chelation complexes, may potentiate tyrosine phosphatase inhibitio n by vanadate or may delay the metabolic inactivation of vanadate [e.g ., reduction to V(IV) by glutathione]. Similarly, the sustained elevat ion of calcium caused by A23187 may change intracellular redox status, perhaps by the depletion of intracellular glutathione. Since tyrosine phosphtases are exquisitely sensitive to sulfhydryl oxidation and pot entially to the regulated oxidative state of o-vanadate, these results reinforce the concept that local regulation of intracellular redox st ate may be an important determinant of tyrosine phosphatase activities .