Quinine sensitive changes in cellular Na+ and K+ homeostasis of COS-7 cells caused by a lipophilic phenol red impurity

Abstract

An impurity of phenol red (PRI) has been shown to markedly alter the intracellular Na+ and K+ homeostasis of several cell types. The effect of PRI seems to involve intracellular Ca(++)-dependent mechanisms. Using COS-7 cells as a model, we further characterized the mechanism of action of PRI by measuring cellular Na+/K+ contents and 86Rb+ efflux. Similar to human skin fibroblasts, in COS-7 cells calmodulin inhibition moderated the cationic transport effects of PRI. A TMB-8 dependent intracellular Ca++ pool does not seem to be involved in these transport events. We found no evidence for participation of the transcriptional-translational machinery in the effect of PRI. Both quinine and quinidine are able to prevent nearly all changes caused by PRI in the cellular Na+/K+ contents and 86Rb+ efflux. Although phenol red contained multiple impurities by high performance liquid chromatography (HPLC), phenolphthalein, a structurally close relative of phenol red, was free of any detectable contamination. Phenolphthalein elicited qualitatively similar transport changes to those observed during exposure to PRI. Regardless of the exact mechanism of action, we propose that the as yet unidentified substance is not a cellular toxin, rather it is a cationic transport modulator. Directly or indirectly, it may interact with the cellular Ca++/calmodulin system and activate some quinine/quinidine sensitive transport processes. This transport process is likely to be a Ca(++)-sensitive K+ channel but, due to the lack of specificity of quinine and quinidine, other transport mechanisms must be also considered. The chemical nature of PRI may be similar to phenolphthalein.

Regulation of 86Rb+ ion transport across polarized human colonocytes by bis‐phenolic compounds

“…This increased pump activity can be attributed to a higher Na + influx induced by phenolphthalein, as documented in other cell types. 5,6,8 In our experiments, there was a clear indication of increased Na + /K + -ATPase activity in the presence of phenolphthalein based on the decreased 86 Rb + accumulation in the presence of ouabain. Of note, the net 86 Rb + intracellular accumulation in the presence of phenolphthalein was lower than in controls, which is in apparent contrast with what may be expected with increased Na + /K + -ATPase activity.…”

“…In contrast, when cells were treated with phenolphthalein, ouabain treatment significantly reduced 86 Rb + accumulation, indicating that Na + /K + ‐ATPase activity was stimulated by phenolphthalein, resulting in increased basolateral uptake of 86 Rb + ions. This increased pump activity can be attributed to a higher Na + influx induced by phenolphthalein, as documented in other cell types 5,6,8 …”

“…However, it is likely that the total amount of K + in the cells falls significantly during the course of the experiment in response to the release of ions induced by the phenolphthalein, as has been documented for other cell types. 5,6,8 Under our experimental protocol, when the isotope loading and phenolphthalein exposure took place simultaneously, a large proportion of this net K + loss occurs before 86 Rb + is able to equilibrate across the basolateral membrane and, consequently, the effect of the pump stimulation (increased 86 Rb + accumulation) is not detected.…”

“…These results indicate an involvement of K + channels, as opposed to the Na + /K + ‐ATPase, in phenolphthalein‐stimulated 86 Rb + release from cells. Prior results with other cell types 8 have implicated Ca 2+ ‐sensitive K + channels in the release of 86 Rb + therefore, we examined the effect of a range of channel modulators on 86 Rb + release from T84 cells induced by phenolphthalein and 4,4(9‐fluorenylidene)diphenol. N ‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide (100 µmol/L), a calmodulin antagonist, 11 verapamil (50 µmol/L), a Ca 2+ channel blocker, 12 glibenclamide (50 µmol/L), an ATP‐dependent K + channel blocker, 13 and tetraethylammonium chloride (1 mmol/L), a general K + channel blocker, 12 had no significant effect on 86 Rb + ion release in the presence or absence of phenolphthalein or 4,4(9‐fluorenylidene)diphenol.…”

Nitric Oxide Does Not Mediate Promotion of Cellular Potassium Release by Phenolphthalein in Cos‐7 Cells

“…It has been demonstrated that phenolphthalein also stimulates 86 Rb + (a tracer for K + ) release from cells in a concentration‐dependent manner 13,14 . The in vivo laxative studies showed that phenolphthalein also stimulated K + release into the gut lumen and that this release could be inhibited by l ‐NAME, indicating that NO may also mediate K + release from cells 9 .…”