Labeling Of Polyphosphoinositides Research Articles

C-Fos activates and physically interacts with specific enzymes of the pathway of synthesis of polyphosphoinositides

The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II β was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.

Phospholipid metabolism under muscarinic cholinergic stimulation exhibits brain asymmetry

In order to characterize some of the lateralized biochemical events promoted in brain upon massive neurotransmitter release, the labeling of lipids under specific stimulation of the muscarinic acetylcholine receptor (mAChR) has been studied in synaptosomes obtained from right and left cerebral cortex (RCC and LCC respectively). Synaptosomes were incubated with [32P]phosphate in the absence and in the presence of the cholinergic agonist carbamoylcholine and the muscarinic antagonist atropine. Binding of the agonist to the mAChR promoted an enhanced labeling of polyphosphoinositides, such effect being considerably more pronounced in the LCC than in the RCC. The differences observed could be due to a higher mAChR-elicited activity of phospholipase C in the RCC than in the LCC. The results show that mAChR stimulation activates the turnover of inositol lipids to a different extent in the two hemispheres, indicating either an uneven distribution of the receptor in brain and/or dissimilarities in the degree of coupling of the mAChR with its corresponding transmembrane signaling system in each hemicortex.

Phosphoinositides in giant barnacle muscle fibers: A quantitative analysis at rest and following electrical stimulation

Quantitative data are presented on the composition of the major phospholipids in isolated giant barnacle muscle fibers. It is shown, using internal perfusion techniques, that the high specific activity of labeling of polyphosphoinositides in vivo is attained by the activities of specific kinases. Electrical stimulation causes a reduction in the specific activity of labeling of PtdInsP 2. This phospholipid, which is the immediate precursor for the release of InsP 3, is found at a significant concentration (40 nmol/g wet weight) in single barnacle muscle fibers, sufficient to support a role as precursor of second messengers. The rapid catabolization of PtdInsP 2 in the absence of external Ca 2+ suggests that E-C coupling in barnacle muscle may be associated with a voltage-dependent, Ca 2+-independent, activation of the breakdown of polyphosphoinositides.

Light stimulates in vivo inositol lipid turnover in frog retinal pigment epithelial cells at the onset of shedding and phagocytosis of photoreceptor membranes

We have developed an experimental model to study in vivo inositol lipid metabolism in frog retinal pigment epithelial (RPE) cells, including the effect of light on phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. RPE cells were rapidly isolated after either brief light or dark periods. Light and electron microscopy showed complete detachment of the retina from the RPE cells, and that the RPE cell suspensions were devoid of photoreceptor cell outer segments. Frog tissues were labeled in vivo for 20 hr by intravitreal injection of [ 3H]inositol (4 μCi, 4μl per eye) within a 24-hr constant illumination period. Following 1 hr of darkness (priming period), frogs were intravitreally injected with LiCl (0·5 m, 4μl per eye) 15 min before the onset of either 30-min light stimulation or an additional 30 min of darkness (controls). In order to preserve endogenous inositol phosphate pools present after dark and light exposure, the RPE cells were harvested in the shortest time possible, at low temperatures (18–20°C), and in the presence of 10 m m LiCl. Total [ 3H]inositol-labeled water-soluble products (inositol plus inositol phosphates) were increased by 86% after 30 min of light. Inositol trisphosphate (IP 3) showed the highest accumulation (a 5·5-fold increase), followed by inositol bisphosphate (1·9-fold increase) and inositol monophosphate (1·4-fold increase). Free [ 3H]inositol also accumulated (2·8-fold increase), reflecting only a partial inhibition of phosphomonoesterase by LiCl. These changes were paralleled by a 12% decrease in 3H-labeled phosphatidylinositol with no significant difference in the labeling of polyphosphoinositides. This is the first time that IP 3 accumulation, correlated with a specific function in a cell type, has been demonstrated in vivo.

Phosphatidic acid and polyphosphoinositide metabolism in rod outer segments. Differential role of soluble and peripheral proteins

The phosphorylation of endogenous diacylglycerol (DAG) and phosphoinositides by [ π- 32 P]ATP] was studied in bovine rod outer segments (ROS) selectively depleted of soluble or peripheral and soluble proteins by treatment with moderate (100 mM) or low (5 mM) ionic strength medium, respectively. DAG kinase activity was similar in bleached and non-bleached ROS extracted with 100 mM medium, and amounted to 70% of that observed in the corresponding non-extracted ROS. Phosphatidic acid (PtdH) labelling in ROS extracted in the dark with low ionic strength medium was markedly lower than in those extracted in light. Thus, even when a major proportion of DAG kinase was associated to the membrane, a soluble form also occurred. Most of the membrane-bound fraction behaved as a peripherally associated protein, its binding to the membrane being modified by light. Ir ROS extracted at moderate ionic strength the labelling of inositides was similar to that in non-extracted ROS. A marked enhancement in polyphosphoinositide labelling was observed in ROS extracted in the dark with low ionic strength. Alkaline treatment of ROS also produced inhibition of polyphosphoinositide phosphorylation. A peripheral form of type C phospholipase, or a peripheral protein-mediated activation of a particulate form thereof, is suggested. Labelled polyphosphoinositides were more actively hydrolyzed in the light and in the dark plus GTPπS than in the dark-incubated membranes. The results of phosphorylation experiments in membranes wheredifferential extraction of the α subunit of transducin was carried out suggest that α and βπ subunits may play opposite modulating roles in PtdH and polyphosphoinositide metabolism.

Effect of albumin-bound DHA on phosphoinositide phosphorylation in collagen stimulated human platelets

The effect of exogenous albumin-bound docosahexaenoic acid (22:6n-3) (DHA), arachidonic acid (20:4n-6) (AA), and eicosapentaenoic acid (20:5n-3) (EPA) on phosphoinositide metabolism following collagen stimulation was studied using [ 3h] inositol prelabelled platelets. Collagen stimulation (3 min, 1.8 μg/ml) increased the labelling of both phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP 2). Of the fatty acids tested, only pre-incubation (2 min) with DHA (20 μM) significantly attenuated the collagen-induced increased PIP and PIP 2 labelling; EPA was without effect, while AA enhanced PIP labelling. Forty μM DHA was less effective at attenuating the increased PIP and PIP 2 labelling even though this concentration of DHA resulted in greater inhibition of platelet aggregation. Neither concentration of DHA attenuated the increased polyphosphoinositide labelling resulting from stimulation by the endoperoxide analogue U46619, or the phorbol ester, PMA. These data suggest that the effect of DHA on attenuating the increased PIP and PIP 2 labelling following collagen stimulation likely occurs before thromboxane receptor occupancy, may not occur at the level of protein kinase C activation, and could be mediated in part via a lessened synthesis of thromboxane A 2.

Acetylcholine stimulates selective liberation and re-esterification of arachidonate and accumulation of inositol phosphates and glycerophosphoinositol in C62B glioma cells.

Glioma C62B cells, incubated for 18 h with either an unsaturated (arachidonate or oleate) or saturated (palmitate or stearate) radioactive fatty acid, incorporated label into most species of cellular glycerolipids. Treatment of prelabeled C62B cells with 1 mM acetylcholine (ACh) resulted in an accumulation of radioactive phosphatidate irrespective of which fatty acid was used as a label. However, only in cells prelabeled with unsaturated fatty acids were increases in radioactive fatty acids observed. When exogenous radioactive arachidonate was added to C62B cells in the presence of 1 mM ACh, there was a rapid, selective, and transiently enhanced incorporation of label (several times the control) into phosphatidylinositol (PI). The ACh-enhanced incorporation into PI was not preceded by enhanced incorporation of label into sn-1,2-diacylglycerol or phosphatidate but was followed by an increased labeling of polyphosphoinositides. Similarly, incorporation of oleate into PI was enhanced by ACh. In contrast, ACh did not enhance the incorporation of label into any glycerolipids when saturated fatty acids were used. C62B cells, incubated with [2-3H]inositol for 18 h selectively incorporated label into phosphoinositides. Stimulation of [2-3H]inositol-labeled cells with 1 mM ACh in the presence of 25 mM LiCl resulted in a rapid accumulation of radioactive inositol phosphates (mono-, bis-, and trisphosphates) and glycerophosphoinositol. The accumulation of inositol trisphosphates preceded that of inositol monophosphate and glycerophosphoinositol, while the accumulation of glycerophosphoinositol paralleled the time required for the ACh-stimulated esterification of arachidonate. These results suggest that ACh stimulates activation of a phospholipase C in C62B cells and release of 1,4,5-inositol trisphosphate. There is subsequent activation of phospholipase A2, which in turn liberates arachidonate from PI. The resulting lyso PI is either rapidly reesterified with unsaturated fatty acid to resynthesize PI, or further deacylated to yield glycerophosphoinositol.

Effects of chronic ethanol administration on rat brain phospholipid metabolism.

Alterations in brain phospholipid metabolism were observed after chronic ethanol administration for 16 days to developing rats. Animals were injected intraperitoneally with 32Pi 16 h prior to killing. Overall uptake of 32Pi by brain did not differ between the control and ethanol-treated groups, which were killed 2 h and 24 h after the last ethanol feeding. Except for an increase in the labeling of myelin after ethanol treatment, the amount of radioactivity recovered in the synaptosomal-mitochondrial and plasma membrane fractions of control and ethanol-treated groups was not different. Relative to the radioactivity of phosphatidylcholines, which indicated no change, there were increases (20-44%) in labeling of ethanolamine plasmalogens, phosphatidic acids, and phosphatidylinositols in cortical synaptosomes from the 2-h ethanol-treated group. In the plasma membrane fractions, however, increases (9-14%) in labeling of phosphatidylserines and phosphatidylinositols were observed in both 2- and 24-h ethanol-treated groups. In both membrane fractions, there was an obvious increase (44-86%) in labeling of polyphosphoinositides at 24 h after withdrawal from ethanol. Results thus indicate an adaptive increase in the biosynthesis of ethanolamine plasmalogen and brain acidic phospholipids due to chronic ethanol administration. Furthermore, the increase in labeling of polyphosphoinositides in the 24-h withdrawal group may reflect the hypoactivity associated with ethanol withdrawal.

The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes.

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.

The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes.

Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products of their breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a 'second signal' (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.

Polyphosphoinositide labelling in Chinese hamster ovary (CHO-K1) cells: stimulation by serum and reduction by calcium ionophore A23187

Polyphosphoinositide labelling in Chinese hamster ovary (CHO-K1) cells: stimulation by serum and reduction by calcium ionophore A23187

Lithium-induced reduction in intracellular inositol supply in cholinergically stimulated parotid gland

The effects of lithium and cholinergic stimulation on inositol phospholipid metabolism have been assessed using rat parotid gland slices and isolated acinar cells labelled with 32Pi. Cholinergic stimulation using carbachol caused substantial breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and enhanced labelling of phosphatidate (PA) and phosphatidylinositol (PtdIns). Lithium alone had little effect upon 32Pi incorporation, but in combination with carbachol it greatly reduced the PtdIns labelling response to the agonist. Instead the label accumulated in a lipid identified as cytidine monophosphorylphosphatidate. There was also an enhancement of the PA labelling response to carbachol. These lithium-induced alterations in agonist-stimulated phospholipid metabolism were reversed if 10-30 mM-inositol was included in the incubation medium. Despite reduced PtdIns synthesis, lithium had relatively little effect on polyphosphoinositide labelling in stimulated cells. Resynthesis of polyphosphoinositides was monitored in acinar cells that had been stimulated with carbachol and then treated with atropine to block muscarinic receptors. Treatment with lithium during the carbachol-stimulation phase reduced the rate of phosphatidylinositol 4-phosphate synthesis, but had no significant effect upon PtdInsP2. The results suggest that an active inositol phosphatase pathway is essential to maintain intracellular inositol levels, but that PtdInsP2 synthesis is not markedly reduced by a substantial fall in intracellular inositol. This implies a close control over the rates of PtdInsP2 breakdown and resynthesis during agonist stimulation.

Increased labelling of polyphosphoinositide in chemically transformed cell line C3H10T1/2 CL8

The effect of malignant transformation of cells on phosphatidylinositol metabolism was investigated using C3H10T1/2 cells and its chemically transformed cell line, MCA CL-16 cells. We found that incorporation of [ 32P]P i into polyphosphoinositide was greatly increased in the transformed cells. A similar tendency was observed when myo-[2- 3H]inositol was used as a labelling reagent. It is also observed that influx of labelled inorganic phosphate is enhanced 2-fold by the cell transformation. Therefore, promotion of polyphosphoinositide labelling in the transformed cell might be caused not only by the enhanced metabolism of phosphatidylinositol but also by the increased membrane permeability for radioactive labelling reagents.

Molecular events involved in the proaggregating effect of heparin on human platelets.

Molecular mechanisms underlying the ability of heparin to enhance the platelet-aggregating effect of various agonists were studied. Heparin potentiates the aggregating effect of adenosine diphosphate (ADP) and epinephrine, but it is uneffective on the aggregation induced by ristocetin and collagen. Heparin inhibits aggregation induced by thrombin in the presence of plasma, but it is uneffective, or sometimes stimulates aggregation, in the absence of plasma. The effects on the platelet-activating factor- (PAF-acether) induced aggregation are very variable. The late phase of the ADP-induced aggregation is sensitive to proteinase inhibitors, but heparin overcomes this inhibitory effect. Drugs which inhibit remodeling of membrane phospholipids abolish the potentiating effect of heparin, while cyclooxygenase inhibitors do not. The proaggregating effect of heparin subfractions correlates with the lipoprotein lipase activity and, slightly, with the molecular weight, but it does not correlate with the anticoagulant activity. Platelets prelabelled with phosphatidyl[U14C]inositol show a very rapid effect of heparin in triggering phosphatidylinositor breakdown and a cooperative effect with ADP, a known agonist of the 'phosphatidylinositol cycle'. Heparin is also effective in stimulating the labelling of polyphosphoinositides in platelets prelabelled with 32Pi. These results, together with the selective sensitivity to drugs, lead to the conclusion that a stimulatory effect on the very early events of remodeling of membrane phospholipids is involved in the platelet proaggregating effect of heparin.

Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones.

Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.

Decreased Phosphorylation of Polyphosphoinositides in the Erythrocyte Membrane of Spontaneously Hypertensive Rats

1. The distribution and metabolism of inositol phospholipids were studied in the erythrocyte membrane of spontaneously hypertensive and normotensive Wistar-Kyoto rats. The incorporation of 32P into these lipids was considered as an index of their turnover, and a reflection of their metabolism. 2. With [γ-32P]ATP as the source of label only the polyphosphoinositides (diphosphoinositide and triphosphoinositide) incorporated 32P. The levels of labelling were lower in hypertensive than in normotensive rats. Our data indicate that, in the hypertensive rats, the kinases responsible for the labelling of polyphosphoinositides exhibited decreased Vmax. for ATP. 3. These changes may be related to those described for calcium binding and transport in the erythrocyte of spontaneously hypertensive rats.

Metabolism of phosphoinositides in the rat erythrocyte membrane: A reappraisal of the effect of magnesium on the 32P incorporation into polyphosphoinositides

The metabolism of phosphoinositides was investigated in the red blood cell membrane of the rat by measuring 32 P-incorporation into phospholipids after incubation of membranes with [γ- 32P]ATP in a medium containing magnesium. A new chromatographic procedure has been developed which facilitates the separation of triphosphoinositide, diphosphoinositide and phosphatidylinositol from the phospholipids present in lipid extracts of incubated ‘ghost’ membranes. Under our experimental conditions only two phospholipids, diphosphoinositide and triphosphoinositide, were 32P-labelled. Furthermore, the results indicate that either di- or triphosphoinositide could be labelled preferentially, depending upon the magnesium concentration of the incubation medium. This clarifies some apparent discrepancies reported in the literature between the 32P labelling of polyphosphoinositides observed in intact erythrocytes and that observed with ‘ghost’ membranes. In addition, the enzymatic pathways involved in the phosphoinositide metabolism are discussed.

Sound Stimulates Labeling of Polyphosphoinositides in the Auditory Organ of the Noctuid Moth

Abstract: The Noctuid moth possesses a simple auditory structure suitable for the investigation of biochemical correlates of sound stimulation in vivo. Stimulation with pulsed tones increased 32P incorporation into polyphos‐phoinositides but not into ATP or other lipids. The effect was seen in the scoloparium (sensory structure) but hot in the nodular sclerite, an adjacent nonsensory tissue. It was also not seen when the stimulus was a continuous tone, leading to adaptation of the action potential.

Biochemistry of neomycin ototoxicity: inhibition of calcium binding and turnover of membrane polyphosphoinositides

In contrast to the well-established antibacterial mechanism of aminoglycoside antibiotics, the biochemical basis of their toxic effects towards mammalian cells remains unclear. The nature of these effects suggests that the primary site of action is at the membrane. Of the various membrane components the polyphosphoinositide lipids play a crucial role in the control of membrane permeability. To analyze these trace lipids, phospholipids of stria vascularis, spiral ligament, and ductus cochlearis were labeled with radioactive phosphate. After short incubation times, the most highly labeled lipids were phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Treatment of guinea pigs with neomycin decreased labeling of these polyphosphoinositides, but not of other lipids. In vitro experiments on the labeling and hydrolysis of polyphosphoinositides demonstrate that this effect is caused by an inhibition of polyphosphoinositide turnover. Neomycin was also shown as a competitive inhibitor of calcium binding in vitro. Evidence suggests that neomycin directly binds to the polyphosphoinositides, displacing calcium and rendering these lipids inaccessible for their enzymes. The toxic effects result from this interference with calcium homeostasis at the membrane and inhibition of the turnover of vital membrane components. [Supported by HIH-NIEHS Contract NO1-ES-2-2110 and NIH Program Project Grant NS 05785.]