Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98:2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5–20 mol%) but strongly depressed by the incorporation of stearylamine (10–40 mol%). Marked stimulation (2–4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2–40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2–10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phosphatidylcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of $\text{V}$. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to liposomal phosphatidylserine and phosphatidic acid

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.     

Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC0.08) or high (PC0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38 degrees C in buffer-containing (control) liposomes prepared from PC0.08, whereas it was slightly increased in those prepared from PC0.66. Encapsulated hemolysate had no effect in PC0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.     

Binding of GAPR-1 to negatively charged phospholipid membranes: Unusual binding characteristics to phosphatidylinositol

Abstract

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis-related proteins group 1 (PR-1). GAPR-1 strongly associates with lipid rafts at the cytosolic leaflet of the Golgi membrane. The myristoyl moiety at the N-terminus of GAPR-1 contributes to membrane binding but is not sufficient for stable membrane anchorage. GAPR-1 is positively charged at physiological pH, which allows for additional membrane interactions with proteins or lipids. To determine the potential contribution of lipids to membrane binding of GAPR-1, we used a liposome binding assay. Here we report that non-myristoylated GAPR-1 stably binds liposomes that contain the negatively charged lipids phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or phosphatidic acid. GAPR-1 displays the highest preference for phosphatidic acid-containing liposomes. In contrast, lysozyme, which contains a similar surface charge, did not bind to these liposomes, except for a weak membrane association with PA-containing liposomes. Interestingly, GAPR-1 binds to phosphatidylinositol with unusual characteristics. Denaturation or organic extraction of GAPR-1 does not result in dissociation of phosphatidylinositol from GAPR-1. The association of phosphatidylinositol with GAPR-1 results in a diffuse gel-shift in SDS-PAGE. Mass spectrometric analysis of gel-shifted GAPR-1 showed the association of up to 3 molecules of phosphatidylinositol with GAPR-1. These results suggest that the lipid composition contributes to the GAPR-1 binding to biological membranes.     

Modulation of the in vitro activity of lysosomal phospholipase A1 by membrane lipids

Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1相似文献   

Regulation of liver base-exchange activity by acidic phospholipids

Liver microsomes were enriched in liposomal acidic lipids by Ca²⁺-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.Abbreviations DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - SHB suerose-HEPES buffer (0.25M sucrose, 3mM HEPES, pH 7.4)     

Acetyl-l-carnitine influences the fluidity of brain microsomes and of liposomes made of rat brain microsomal lipid extracts

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) was measured in different preparations (bovine spinal cord phosphatidylserine liposomes, rat brain microsomes, liposomes made with rat brain microsomal lipid having different phospholipid:cholesterol ratios) at temperatures ranging from 10° to 55°C. Phosphatidylserine liposomes exhibited an exponential relationship of rversus temperature, whereas the relationship shown by microsomes and liposomes prepared with microsomal lipid extracts was a linear one. The removal of protein and high phospholipid:cholesterol ratios decreased the slope of the lines (fluidity increased), although the intercept was unaffected. This means that differences were better appreciated at high temperatures and were well evident at 37°C. Acetyl-l-carnitine decreased r in rat brain microsomes and in liposomes made with microsomal lipids with different phospholipid:cholesterol ratios. The fluidifying effect of acetyl-l-carnitine was mild but statistically significant and could explain, at least in part, the data reported in the literature of acetyl-l-carnitine acting on some parameters affected by ageing. Besides, acetyl-l-carnitine seemed to oppose the changes of viscosity due to lipid peroxidation, which has been reported to increase in ageing and dementia.l-carnitine shares the properties of its acetyl ester, but only in part.Abbreviations DPH diphenylhexatriene - HEPES 4-(2-hydroxyethyl-l-piperazineethansulfonic) acid - r fluorescence anisotropy - SHB sucrose-HEPES-buffer (0.32 M sucrose, 2 mM HEPES, pH 7.0)     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

Microsomal protein mediates a pH-dependent fusion of liposomes to rat brain microsomes

The fusion between rat brain microsomes and liposomes is investigated by measuring the release of octadecylrhodamine B (R18) fluorescence self-quenching. In the experimental conditions used in this work, the method allows a rapid and quantitative evaluation of the mixing of microsome and liposome lipid phases. The decrease of pH below 7 produces an extensive fusion between microsomes and acidic phospholipid liposomes. Microsomal protein is necessary for fusion, which is inactivated by exposure of microsomes to pronase. Therefore, H(+)-induced fusion differs from Ca(2+)-induced fusion since the latter does not require microsomal protein. The pretreatment of microsomes with trinitrobenzenesulfonic acid (TNBS) in nonpenetrating conditions does not affect the extent of fusion. On the other hand, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent able to react with carboxyl groups, causes an extensive inactivation of fusion. Therefore, the H(+)-induced fusion described here depends on some microsomal protein and may have physiological significance because it occurs at pH values present in the living cell. H(+)-dependent fusion can be also considered as a means to enrich membranes in some selected lipid.     

Phosphatidic acid, phosphatidylinositol, phosphatidylserine and cardiolipin in the course of early embryonic development. Fatty acid composition and content in whole toad embryos and in mitochondrial fractions

The fatty acid composition and content of phosphatidylinositol, phosphatidylserine and phosphatidic acid have been studied during the early development of toad embryos. Acidic phospholipids have been analyzed in whole oocytes and embryos and in the following subcellular fractions: yolk platelets, mitochondria and microsomes. Also cardiolipin, a mitochondrial phospholipid, has been analyzed. Gastrula stage embryos have shown, mainly in the mitochondrial fraction, an increase in the content of phosphatidic acid, phosphatidylserine and phosphatidylinositol with respect to unfertilized oocytes. Changes in the distribution of acyl groups of phosphatidic acid have been detected when different subcellular fractions are compared. On the other hand, the phosphatidylserine composition remains unmodified. Arachidonate and stearate are the principal components of phosphatidylinositol. Cardiolipin shows the same composition up to gastrulation and linoleate comprises about 50% of the total acyl groups.     

Effect of Long-Term Feeding with Acetyl-L-Carnitine on the Age-Related Changes in Rat Brain Lipid Composition: A Study by 31P NMR Spectroscopy

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.     

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.     

Cerebral ischemia induced quantitative changes in rat brain membrane lipids involved in phosphoinositide metabolism

The content and acyl group composition of phosphatidylinositol, poly-phosphoinositides, diacylglycerols, phosphatidic acids, and free fatty acids in rat brain homogenates of cerebral cortex and subcellular fractions were examined with respect to a 2 min post-decapitative ischemic treatment. With the exception of free fatty acids, these lipids are involved in the cyclic event associated with the receptor-mediated poly-phosphoinositide turnover. The ischemic treatment elicited a decrease in poly-phosphoinositide level in brain homogenates, synaptosomes, plasma membranes, and microsomes but not in myelin, and an increase in diacylglycerols, which was observed in brain homogenates and synaptosomes but not in other subcellular fractions. On the other hand, the level of phosphatidylinositol was not altered. The acyl groups of phosphoinositides are enriched in stearic and arachidonic acids. The diacylglycerols and free fatty acids that accumulated during the ischemic treatment are also enriched with the same fatty acids. There is a decrease in phosphatidic acid level after the ischemic treatment, but the change was only found in brain homogenates and synaptosomes. Therefore, the diacylglycerols increased during the ischemic treatment may be derived from hydrolysis of poly-phosphoinositides and phosphatidic acids. However, the amount of poly-phosphoinositides degraded is not enough to account for both diacylglycerols and free fatty acid increase.     

Microsomal Protein Mediates a pH-Dependent Fusion of Liposomes to Rat Brain Microsomes

The fusion between rat brain microsomes and liposomes is investigated by measuring the release of octadecylrhodamine B (R18) fluorescence self-quenching. In the experimental conditions used in this work, the method allows a rapid and quantitative evaluation of the mixing of microsome and liposome lipid phases. The decrease of pH below 7 produces an extensive fusion between microsomes and acidic phospholipid liposomes. Microsomal protein is necessary for fusion, which is inactivated by exposure of microsomes to pronase. Therefore, H⁺-induced fusion differs from Ca²⁺-induced fusion since the latter does not require microsomal protein. The pretreatment of microsomes with trinitrobenzenesulfonic acid (TNBS) in nonpenetrating conditions does not affect the extent of fusion. On the other hand, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent able to react with carboxyl groups, causes an extensive inactivation of fusion. Therefore, the H⁺-induced fusion described here depends on some microsomal protein and may have physiological significance because it occurs at pH values present in the living cell. H⁺-dependent fusion can be also considered as a means to enrich membranes in some selected lipid.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Effects of fatty acid deficiency on the lipid composition and physical properties of guinea pig rough endoplasmic reticulum

Twenty-six days of fat deficiency brought about a decrease of linoleic and an increase of oleic acid in rough endoplasmic reticulum (RER) of guinea pig liver. Arachidonic acid was only slightly decreased in some phospholipids whereas eicose-5,8,11-trienoic acid was not enhanced except in phosphatidyl-inositol. All these changes were relevant specifically in phosphatidylinositol molecules and less important in phosphatidylcholine and phosphatidylethanolamine. Fat deficiency did not modify the relative proportion of phospholipids and cholesterol. Therefore, fat deficient guinea pig microsomes are a good model to study the effect of unsaturated fatty acids on membrane properties. Fluorescent anisotropy of RER membranes, lipids and phospholipids labeled with diphenylhexatriene, was increased by the fat deficiency. The most important increase was observed in liposomes of a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin. A small change was found in phosphatidylcholine and phosphatidylethanolamine dispersions at 37°C. The modification of the lipid unsaturation evoked fluorescent anisotropy changes. Temperature-dependent fluorescent polarization curves of RER membranes labeled with trans-parinaric acid did not show inflections in the temperature range from 5 to 45°C but, RER lipids and phospholipids presented a phase separation at about 20°C. This inflection point was not modified by the fat deficient diet. In those liposomes prepared with a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin, the inflection point was produced at about 37°C.The author is member of the Carrera del Investigador Cientifico, Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina.     

Effects of Short- and Long-Term Protriptyline Treatment on Phospholipid Metabolism in Rat Brain

Wistar rats were injected intraperitoneally with 10 mg/kg of protriptyline according to one of the following schedules: a single dose or daily for 4 days (short-term), or daily for 2 or 13 weeks (long-term). Total lipid, total phospholipid, and individual phospholipid contents in the brain were determined. Further, the incorporation of 32P into individual phospholipids in vivo and the fatty acid composition of phosphatidylethanolamine in the brains of rats treated with protriptyline for 13 weeks were studied. Three alternative phases of changes of total and individual phospholipid contents in the brain during 13 weeks of experimentation were distinguished. An increase of phospholipid contents after 4 days, a decrease after 2 weeks, and a further increase after 13 weeks of protriptyline administration were found. However, phosphatidylinositol and phosphatidic acid levels after 13 weeks of protriptyline administration were diminished. The decrease of specific radioactivity of phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine and the increase of phosphatidylinositol, phosphatidic acid, and sphingomyelin in rats treated with the drug for a longer period of time were noted. No greater differences in fatty acid composition of phosphatidylethanolamine in the brains of the same group of rats were observed as compared to control. These results indicate that during long-term treatment with protriptyline the contents of lipids and phospholipids in rat brain are altered. The modification of the biological function of phospholipids in brain cell membranes is suggested.