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.

The labelling pattern of cellular phosphoinositides (PtdInsP(n)) was studied in Ehrlich ascites cells labelled in vivo for 24 h with myo-[2-(3)H]- or l-myo-[1-(3)H]inositol and exposed to anisotonic or isosmotic volume perturbations. In parallel experiments the cell volume ([(14)C]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 PtdInsP(2) showed a marked, transient decrease and the content of PtdInsP a small, transient increase. The changes in PtdInsP(2) 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 PtdInsP(2) or formation of PtdInsP(3). 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 PtdInsP(2) showed a marked increase and the content of PtdInsP a 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 PtdInsP(2) and PtdInsP remained constant. Our results show that PtdInsP(2) is not volume sensitive per se, and moreover that the regulatory volume adjustments in Ehrlich ascites cells are not mediated by PtdInsP(2) hydrolysis and its subsequent production of second messengers. The simplest interpretation of the observed effects would be that PtdInsP(2) 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 PtdInsP(2) might orchestrate this 'set-point' shift.

Aberrant 3H in Ehrlich mouse ascites tumor cell nucleotides after in vivo labeling with myo-[2-3H]- and L-myo-[1-3H]inositol: implications for measuring inositol phosphate signaling.

After in vivo radiolabeling of Ehrlich cells for 24h with conventional myo-[2-3H]inositol we previously demonstrated an aberrant 3H-labeling of ATP that interfered in the HPLC analysis of inositol trisphosphates. This aberrant 3H-labeling was accounted for by the extensive kidney catabolism of myo-[2-3H] inositol with delivery of 3H-labeled metabolites to extrarenal tissues. As expected, the aberrant labeling of ATP is markedly reduced with the use of 3H-myo-inositol labeled at L-C1 rather than at C2, reflecting that the 3H at L-C1 disappears in the first step of the myo-inositol catabolism: the oxidative conversion to D-glucuronate. In contrast, with the 3H at C2 of myo-inositol, the 3H-C2 passes into the pentose phosphate conversions with resulting labeling of nucleotides. The extent of catabolism to 3H-labeled water, the cellular accumulation of 3H-myo-inositol, the incorporation into cellular inositol phospholipids, and the labeling pattern of cellular phosphoinositides were all found to be similar for the two labeled myo-inositol moieties. With the use of L-myo-[1-3H]inositol an aberrant 3H-labeling at about 25% remained, for which a presumptive mechanism is proposed. L-myo-[1-3H]Inositol appears nevertheless to be a preferable alternative to myo-[2-3H]inositol for tracing the intact myo-inositol molecule after in vivo labeling, with minimized interference from aberrant 3H-labeling of nucleotides.