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.
Nielsen
Doris K
DK
August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark.
Jensen
Annelie Kolbjørn
AK
Harbak
Henrik
H
Christensen
Søren C
SC
Simonsen
Lars Ole
LO
D000818:Animals; D001834:Body Water / Q000502:physiology; D002286:Carcinoma, Ehrlich Tumor / Q000473:pathology / Q000503:physiopathology*; D048429:Cell Size; D003470:Culture Media; D005260:Female; D007295:Inositol Phosphates / Q000502:physiology; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*
D003470:Culture Media; D007295:Inositol Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate