**1. Introduction**

Phosphoinositides are membrane phospholipids with the six-member cyclic polyol *myo*-inositol, (CHOH)6, or O-phosphorylated forms of inositol, as their headgroup (Figure 1). The phosphorylated forms are of low abundance in eukaryotic cells of all types, generally comprising 1% or less of total phospholipid. Nevertheless, they play critical roles in cellular regulation, and defects in their synthesis and regulation lead to devastating diseases [1–6]. The retina is clearly no exception to this generality, but surprisingly few details have been worked out in what is arguably one of the most extensively studied tissues in our bodies, about the regulation and regulatory roles of retinal phosphoinositides. Recent technological advances make it possible to make substantial advances in this field in the next few years.

**Figure 1.** Structures of the cellular phosphoinositides and enzymes responsible for their synthesis and interconversion. Phosphoinositides (PI) species are shown with the acyl chains most commonly found on phosphatidylinositol, which is the starting point for all the others, arachidonic acid (20:4) and stearic acid (C18). Relative font sizes correlate with relative abundance.

### **2. Chemical Structures of Phosphoinositides**

Phosphoinositides (abbreviated here as "PI") include phosphatidylinositol (PtdIns), in which the <sup>1</sup>-position of the inositol ring is attached via a phosphodiester bond to the *sn*-3 position of (1, 2) diacyl glycerol, and derivatives of PtdIns with one, two, or three phosphates attached in various combinations to the 4, 5 or 3 hydroxyls of PtdIns. Including PtdIns, a total of eight different PI head groups are commonly found in eukaryotic cells (Figure 1) [4,5].

### **3. Phosphatidylinositol Content in the Retina and RPE**

PtdIns is a relatively substantial component of the membranes of most cells in metazoans, with mole fractions ranging from ~4%–20% of total phospholipid [4]. Early reports stated that retina phospholipids contained 4.4%–6.4% PtdIns across six different mammalian species [7–9]. Interestingly, rod outer segmen<sup>t</sup> (ROS) membranes were found to have much lower levels, 1.5% to 2.5% in bovine and 2.1% in frog [10–13]. Retinal pigment epithelial cells (RPE), in contrast, have higher levels of PtdIns, at 6.5% of total phospholipid [14], while ER membranes isolated from bovine retinas contain 9.6% PtdIns [10]. The general picture that emerges is that total PtdIns content in total retinal membranes outside of outer segments and that of RPE are more or less "normal" as compared to other tissues and cell types, consistent with the notion that widespread roles of PI are likely to be conserved in many retinal cell types. In contrast, ROS have an unusually low PtdIns content, suggesting different roles

for this lipid class in that organelle. This idea is consistent with findings discussed below, indicating di fferent lipid compositions in plasma membranes as compared to ciliary membranes.

### **4. Content of Minor Phosphoinositides in the Retina and RPE**

As a negatively charged lipid, which typically contains one polyunsaturated side chain fatty acid, such as arachidonic acid [8], PtdIns has a major impact on the physical properties of the membrane domains containing it. In contrast, the phosphorylated forms are much less abundant, and exert their influence on cell physiology largely through interactions with proteins with high-a ffinity and high-specificity PI-binding domains [15–17]. There have been few measurements of phosphorylated PI in retina or cells isolated from retina, although there have been several papers addressing the turnover of PtdIns and other PI, or activity of enzymes involved in PI metabolism [18–21]. The likely reason for the absence of information on PI levels is their low abundance and the lack of sensitivity provided by conventional methods for lipid analysis. More sensitive techniques have been developed recently, including one based on recombinant phosphoinositide-binding domains fused to an epitope tag, allowing sensitive detection by enzyme-linked immunosorbent assays (ELISAs) and measurement of chemiluminescence [22]. This technique was used to quantify PI(3)P and PI(3,4,5)P3 in preparations of rod cells that contain both outer segments and fragments of the inner segments and demonstrated levels of PI(3)P at 0.0035 mol% of total phospholipid under illumination conditions that yielded the highest levels of that lipid, and at least 10-fold lower (i.e., undetectable) levels of PI(3,4,5)P3. PI(4)P and PI(4,5)P2 are, in general, found at much higher levels than the 3-phosphorylated PI, but even those appear to be present in rods at very low levels, which are only about 10-fold higher than the levels of PI(3)P, i.e., on the order of 0.04 mol% (He and Wensel, unpublished observations).

### *Comparison to Other Tissues and Cell Types*

These numbers are comparable to those found in other eukaryotic cells, reported as PtdIns3P, 0.002% of phospholipid mass; PtdIns4P, 0.05%; PtdIns5P, 0.002%; PtdIns(4,5)P, 0.05%; PtdIns(3,4)P, 0.0001%; PtdIns(3,5)P, 0.0001% [17], see also references in [23]. A more recent mass spectrometry study reported PI(3,4,5)3 levels as 50-fold or more lower than those of the more common PIs in mammalian U87MG cells [24]. An interesting observation derived from measurements of PI levels in cultured cells is that, generally, they do not change greatly upon activation with extracellular stimuli; for the more common forms, PtdIns, PI(4)P, PI(4,5)P2 and PI(3)P, the change is generally less than 30% [17]. The implication, for PI(4,5)P2, which is rapidly degraded to form InsP3 and diacylglycerol upon activation of phospholipase C isozymes by G-protein-coupled receptors or growth factor receptors [25,26], is that homeostatic mechanisms are in place to regenerate rapidly the pools of both PI(4,5)P2 and its precursor PI(4)P upon PLC activation. In contrast, PI(3,4,5)P3 level changes due to receptor stimulation are generally too low to have a major impact on levels of its precursor, PI(4,5)P2.
