Investigating the effect of arachidonate supplementation on the phosphoinositide content of MCF10a breast epithelial cells

Phosphoinositides in primary mammalian tissue are highly enriched in a stearoyl/arachidonyl (C38:4) diacylgycerol backbone. However, mammalian cells grown in culture typically contain more diverse molecular species of phosphoinositides, characterised by a reduction in arachidonyl content in the sn-2 position. We have analysed the phosphoinositide species in MCF10a cells grown in culture by mass spectrometry. Under either serum or serum starved conditions the most abundant species of PI, PIP, PIP(2) and PIP(3) had masses which corresponded to C36:2, C38:4, C38:3, C38:2 and C36:1 diacylglycerol backbones and the relative proportions of each molecular species were broadly similar between each phosphoinositide class (approx. 50%, 25%, 10%, 10% and 10% respectively, for the species listed above). Supplementing the culture medium with BSA-loaded arachidonic acid promoted a rapid increase in the proportion of the C38:4 species in all phosphoinositide classes (from approx. 25%–60% of total species within 24 h), but the total amount of all combined species for each class remained remarkably constant. Stimulation of cells, cultured in either normal or arachidonate-enriched conditions, with 2 ng/ml EGF for 90 s caused substantial activation of Class I PI3K and accumulation of PIP(3). Despite the increased proportion of C38:4 PIP(3) under the arachidonate-supplemented conditions, the total amount of all combined PIP(3) species accumulating in response to EGF was the same, with or without arachidonate supplementation; there were however small but significant preferences for the conversion of some PIP(2) species to PIP(3), with the polyunsaturated C38:4 and C38:3 species being more favoured over other species. These results suggest the enzymes which interconvert phosphoinositides are able to act on several different molecular species and homoeostatic mechanisms are in place to deliver similar phosphoinositide pool sizes under quite different conditions of arachidonate availability. They also suggest enzymes regulating PIP(3) levels downstream of growth factor stimulation (i.e. PI3Ks and PIP(3)-phosphatases) show some acyl selectivity and further work should be directed at assessing whether different acyl species of PIP(3) exhibit differing signalling potential.