Cell content of phosphatidylinositol (4,5)bisphosphate in Ehrlich mouse ascites tumour cells in response to cell volume perturbations in anisotonic and in isosmotic media

Abstract

The labelling pattern of cellular phosphoinositides (PtdInsPn) was studied in Ehrlich ascites cells labelled in vivo for 24 h with myo-[2-3H]- or L-myo-[1-3H]inositol and exposed to anisotonic or isosmotic volume perturbations. In parallel experiments the cell volume ([14C]3-OMG space) was monitored. In hypotonic media the cells initially swelled osmotically and subsequently as expected showed a regulatory volume decrease (RVD) response. Concurrently, the cell content of PtdInsP2 showed a marked, transient decrease and the content of PtdInsPa small, transient increase. The changes in PtdInsP2 and PtdInsP content increased progressively with the extent of hypotonicity (in the range 1.00-0.50 relative osmolarity). No evidence was found for either hydrolysis of PtdInsP2 or formation of PtdInsP3. In hypertonic medium (relative osmolarity 1.50), cells initially shrank osmotically and subsequently as expected showed a small regulatory volume increase (RVI) response. Concurrently, the cell content of PtdInsP2 showed a marked increase and the content of PtdInsPa small decrease, i.e. changes in the opposite direction of those seen in hypotonic media. In isosmotic media with high (100 mm) or low (0.8 mm) K+ concentration, cells slowly swelled or shrank due to uptake or loss of isosmotic KCl. Under these conditions, with largely unchanged intracellular ionic strength, the cell content of PtdInsP2 and PtdIns P remained constant. Our results show that PtdIns P 2 is not volume sensitive per se, and moreover that the regulatory volume adjustments in Ehrlich ascites cells are not mediated by PtdInsP2 hydrolysis and its subsequent production of second messengers. The simplest interpretation of the observed effects would be that PtdInsP2 is controlled by ionic strength, probably via activation/inhibition of phosphoinositide-specific phosphatases/kinases. In Ehrlich ascites cells, as shown previously, the opposing ion channels and transporters activated during RVD and RVI, respectively, are controlled with tight negative coordination by a common cell volume 'set-point' that is shifted in anisotonic media, but unchanged during cell swelling in isosmotic high K+ medium. We hypothesize that PtdInsP2 might orchestrate this 'set-point' shift. © 2007 The Authors. Journal compilation © 2007 The Physiological Society.