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PubMed1986

31P-NMR investigation of magnetically oriented rod outer segments. Spectral analysis and identification of individual phospholipids.

A 31P-NMR study of magnetically oriented bovine rod outer segments is presented. We demonstrate that carefully isolated bovine rod outer segments retain the capacity to orient in a magnetic field. Maximal orientation (85-90%) is achieved at field strengths over 4.7 T in the NMR spectrometer. The lineshape of the 'oriented spectra' is totally different from the 'bilayer lineshape' of randomly oriented photoreceptor membranes. The oriented spectra consist of two phospholipid peaks, a major low-field peak (75-80% of the total intensity) near 30 ppm, and a minor high-field peak near - 14 ppm as well as two sharp metabolite peaks around 0 ppm. The phospholipid peaks are a composite of three narrower partially resolved resonances assigned to the individual phospholipid classes phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine. Based on the morphology and magnetic anisotropy of the rod outer segment, the major phospholipid peak is attributed to the flat part of the disk membranes while the phospholipids of the plasma membrane are thought to contribute only to the minor peak. Disk rim phospholipids and non-oriented material contribute to the minor peak and, in addition, contribute some intensity to the middle part of the spectrum. The phospholipid class composition of the major peak is estimated by spectral simulation and is consistent with the phospholipid class composition of rod outer segment membranes. Hence, 31P analysis of oriented rod outer segments resolves the main phospholipids in at least two different membrane pools in the rod outer segment and allows the differential investigation of these pools. Most of the mobile phosphate metabolite intensity resides in the Pi peak at 3.5 ppm. A slight shift in the Pi resonance position indicates a 0.2 pH unit acidification upon illumination of rhodopsin. The absence of detectable nucleotide resonances, when compared with chemical analysis, indicates that the majority of the nucleotide population present is rather immobile and probably bound to the membranes.

Animals↗
PubMed1973

Microbial phospholipid synthesis as a marker for microbial protein synthesis in the rumen.

Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with (33)P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of (33)P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with (33)P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs.

Animal Feed↗
PubMed1985

Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opi1 mutant.

Phospholipid metabolism in the Saccharomyces cerevisiae opi1 mutant, which excretes inositol and is constitutive for the biosynthetic enzyme inositol-1-phosphate synthase (M. Greenberg, P. Goldwasser, and S. Henry, Mol. Gen. Genet. 186:157-163, 1982), was examined and compared to that of a wild-type strain. In wild-type S. cerevisiae, the phospholipid composition and the relative rates of synthesis of individual phospholipids change in response to the availability of exogenous supplies of soluble phospholipid precursors, particularly inositol. The opi1 mutant, in contrast, displays a relatively invariant phospholipid composition, and its pattern of phospholipid synthesis does not change in response to exogenous phospholipid precursors. Phosphatidylinositol synthase was not found to be regulated in either wild-type or opi1 cells. In wild-type cells, phosphatidylserine synthase and the phospholipid N-methyltransferases are coordinately repressed in response to a combination of inositol and choline. However, in opi1 cells these activities are expressed constitutively. These results suggest that the gene product of the OPI1 locus participates in the coordinate regulation of phospholipid synthesis.

Choline↗
PubMed1997

Phospholipid biosynthesis and solvent tolerance in Pseudomonas putida strains.

The role of the cell envelope in the solvent tolerance mechanisms of Pseudomonas putida was investigated. The responses of a solvent-tolerant strain, P. putida Idaho, and a solvent-sensitive strain, P. putida MW1200, were examined in terms of phospholipid content and composition and of phospholipid biosynthetic rate following exposure to a nonmetabolizable solvent, o-xylene. Following o-xylene exposure, P. putida MW1200 exhibited a decrease in total phospholipid content. In contrast, P. putida Idaho demonstrated an increase in phospholipid content 1 to 6 h after exposure. Analysis of phospholipid biosynthesis showed P. putida Idaho to have a higher basal rate of phospholipid synthesis than MW1200. This rate increased significantly following exposure to xylene. Both strains showed little significant turnover of phospholipid in the absence of xylene. In the presence of xylene, both strains showed increased phospholipid turnover. The rate of turnover was significantly greater in P. putida Idaho than in P. putida MW1200. These results suggest that P. putida Idaho has a greater ability than the solvent-sensitive strain MW1200 to repair damaged membranes through efficient turnover and increased phospholipid biosynthesis.

Culture Media↗
PubMed1987

Effect of dietary fish oil on myocardial phospholipids and myocardial ischemic damage.

The effect of dietary fish oil on myocardial phospholipids and ischemic damage to the heart was studied in the rat. Four weeks of feeding 5% (i.e., 12 energy percent) menhaden oil (MO) produced both profound changes in the fatty acyl composition of phospholipids in myocardial membranes and a significant reduction in the loss of creatine kinase following coronary artery ligation compared with feeding 5% (i.e., 12 energy percent) corn oil (CO). The MO diet did not change the content of either phospholipids or cholesterol in the heart. However, dietary MO resulted in significant elevations in the percent of fatty acids in the total phospholipids that were saturated, the n-3/n-6 ratio and the double-bond index. The changes in total phospholipids were not uniform for all phospholipid classes. Although the n-3/n-6 ratio was increased in each of the individual phospholipids examined, the predominant n-3 fatty acid incorporated (i.e., 20:5, 22:5, 22:6) differed among the major phospholipid classes. Also, the percent saturation was elevated in phosphatidylcholine with no change in double-bond index, whereas both the percent saturation and double-bond index were elevated in phosphatidylethanolamine. Thus dietary MO resulted in selective alterations in individual myocardial phospholipids. These membrane changes may be involved in the observed reduction of ischemic damage in the heart.

Animals↗
PubMed1994

Phospholipid analysis of mammalian optic nerve tissue: a 31P nuclear magnetic resonance spectroscopic study.

Phospholipids of optic nerve (n = 30) from 5.6-kg rabbits were analyzed by 31P nuclear magnetic resonance (NMR) spectroscopy. Phospholipid metabolites detected were as follows (mol %): phosphatidylcholine (PC; 25.82 +/- 0.12), PC plasmalogen/alkylacyl PC (2.07 +/- 0.13), sphingosylphosphorycholine (1.12 +/- 0.20), phosphatidylinositol (PI; 2.17 +/- 0.21), lyso PC (0.85 +/- 0.06), sphingomyelin (12.52 +/- 0.10); phosphatidylserine (PS; 14.38 +/- 0.11), phosphatidylethanolamine (8.98 +/- 0.11), ethanolamine plasmalogen (28.99 +/- 0.30), unidentified phospholipid (1.10 +/- 0.01), phosphatidic acid (PA; 1.72 +/- 0.06), and lyso PS (0.28 +/- 0.10). The bulk of the ethanolamine phosphatide is in the form of its plasmalogen, which is the major phospholipid detected. The choline plasmalogen, or a reduced derivative thereof, also is present; thus, a significant phospholipid biosynthetic pathway for optic nerve tissue involves the plasmalogen route, which is a pathway distinct from the PA route responsible for the synthesis of PS, PI, and PC. This new 31P NMR lipid analytical technique offers potential for studying optic nerve phospholipid metabolism and degenerating optic nerve tissue, since the technique can accurately quantitate (1) both plasmalogen and nonplasmalogen phospholipids, (2) minor phospholipid components, and (3) previously undetected phospholipids.

Animals↗
PubMed1996

Phospholipids in meibomian gland secretion.

The bulk of the lipid layer overlying the aqueous portion of the precorneal tear film is composed of polar and nonpolar components. The nonpolar lipids have been the subject of numerous studies; however, the polar lipids have remained relatively uncharacterized. The polar lipids are thought to contain surfactant phospholipids that are critical to the spreading of a lipid film over the aqueous layer, by providing an interface between this layer and the nonpolar lipids. The purpose of the present study is to identify and quantitate the phospholipid complement of meibomian gland secretion which provides the tear film with phospholipids. Meibomian gland secretion was collected from rabbits and phospholipids identified and quantitated by 31P nuclear magnetic resonance spectroscopy. Ten phospholipids were detected from meibomian gland secretion: diphosphatidylglycerol, dihydrosphingomyelin, ethanolamine plasmalogen, phosphatidylethanolamine (PE), phosphatidylserine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol, alkylacylphosphatidylcholine, and phosphatidylcholine (PC). The two major phospholipids were PC and PE, together comprising nearly 60% of the total phospholipid profile. The nature and relative concentrations of the meibomian gland secretion phospholipids are congruous with a surfactant role at the aqueous-lipid interface and, considering the physical chemistry of the tear film, suggest that the phospholipids should be organized in a very flat or planar configuration.

Animals↗
PubMed1999

Role of group II secretory phospholipase A2 in atherosclerosis: 2. Potential involvement of biologically active oxidized phospholipids.

Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.

Animals↗
PubMed2000

Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position.

We previously described 3 bioactive oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) containing oxovaleroyl (POVPC), glutaroyl (PGPC), and epoxyisoprostane (PEIPC) groups at the sn-2 position that were increased in minimally modified/oxidized low density lipoprotein (MM-LDL) and rabbit atherosclerotic lesions. We demonstrated specific and contrasting effects of POVPC and PGPC on leukocyte-endothelial interactions and described an effect of PEIPC on monocyte binding. The major purpose of the present study was to determine the effects of structural changes on the bioactivities of these 3 lipids. We demonstrate herein that the group at the sn-2 position determines the specific bioactivity and that the substitution of stearoyl for palmitoyl at the sn-1 position or ethanolamine for choline at the sn-3 position of the phospholipid did not alter bioactivity. Oxidized PAPC, oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, and oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylethanolamine stimulated monocyte binding and inhibited lipopolysaccharide-induced expression of the neutrophil-binding molecule E-selectin. Furthermore, all oxovaleroyl phospholipids but not the glutaroyl phospholipids induced monocyte binding without an increase in vascular cell adhesion molecule-1 (VCAM-1) expression and inhibited lipopolysaccharide-induced E-selectin expression. In contrast, glutaroyl phospholipids but not oxovaleroyl phospholipids stimulated E-selectin and VCAM-1 expression. We further demonstrate that all parts of the phospholipid molecules are required for these bioactivities. Hydrolysis with phospholipase (PL) A(1), PLA(2), and PLC strongly reduced the bioactivities of POVPC, PGPC, and mixed isomers of PEIPC. PLD had a smaller but still significant effect. The effects of POVPC and PEIPC could be abolished by sodium borohydride treatment, indicating the importance of the reducible groups (carbonyl and epoxide) in these molecules. In summary, these studies identify 6 new bioactive, oxidized phospholipids that are increased in MM-LDL and, where measured, in atherosclerotic lesions. They thus suggest that a family of phospholipid oxidation products containing oxovaleroyl, glutaroyl, and epoxyisoprostane at the sn-2 position play an important role in the regulation of leukocyte-endothelial interactions, bioactivity being in part controlled by several types of phospholipid hydrolases.

Animals↗
PubMed1974

The role of platelet membrane phospholipids in the platelet release reaction.

The structure and function of the platelet surface was probed by phospholipase C (Clostridium perfringens) which hydrolyzes membrane phospholipids, particularly phosphatidylcholine. Platelet phospholipids were susceptible to phospholipase C, and extent of hydrolysis was dependent on concentration of phospholipase C and Ca(++). Phospholipase C (0.15 U/ml) with Ca(++) (0.55 mM) hydrolyzed 15.6% phospholipids during 5 min. Phospholipase C released platelet serotonin (5HT), ADP, and platelet factor 4. Hydrolysis of 5% phospholipids resulted in release of 70% 5HT. Platelet 5HT release was rapid, occurring within 2 min. Phospholipase C (0.2 U/ml) with Ca(++) (0.55 mM) also released 10.35 nmol sotrage pool ADP/10(9) platelets and 63% platelet factor 4 during 3 min. Phospholipase C did not cause leakage of cytoplasmic metabolic pool ADP, since only 6.6% [(3)H]ADP was released. Ultrastructural analysis of phospholipase C-modified platelets showed that platelets were intact. After 2% phospholipid hydrolysis, centralization of granules and contraction of microtubules were evident. After 18% phospholipid hydrolysis, there were morphological indications of degranulation. Phospholipase C-induced phospholipid hydrolysis caused the release of ADP and 5HT since: (a) Phospholipase C purified by heating was shown to be free of protease and neuraminidase activity and capable of inducing the platelet release reaction. (b) Antitoxin (Cl. perfringens) neutralized phospholipase C-induced 5HT release which rules out a contaminant. (c) Phosphorylcholine, the hydrolysis product, did not induce platelet 5HT release. This study demonstrates that minimal hydrolysis of platelet phospholipids triggers the release reaction. Our hypothesis is that phospholipids, presumably phosphatidylcholine, are situated at or near active site or "receptor" on the platelet surface and function as the modulator for the release reaction.

Adenosine Diphosphate↗
PubMed2002

Phospholipids in amelogenesis and dentinogenesis.

Phospholipids have been identified in enamel and dentin. Before demineralization, a group of phospholipids extracted by lipid solvents was associated with cell membranes and is therefore closely related to cell growth and intracellular regulations. After demineralization, a second group of phospholipids, associated with the extracellular matrix, was extracted; this group is probably linked to the mineralized phase. Using imidazole-osmium tetroxide fixation of rat incisors, we stained cellular unsaturated fatty acids, so that we could visualize the membrane domains, coated pits, and endocytic inclusions. Filipin, a probe for cholesterol, varied in density along the plasma membrane of secretory ameloblasts, and allowed us to visualize membrane remnants inside the forming enamel. With respect to phospholipids located in the extracellular matrix, the malachite-green-glutaraldehyde (MGA) method or iodoplatinate (IP) reaction retains and visualizes enamel and dentin phospholipids. In predentin, aggregates appearing as granules and filaments, or liposome-like structures, were located in the spaces between collagen fibrils. In dentin, organic envelopes coating the crystals, also named "crystal-ghost" structures, outlined groups of collagen fibrils. Histochemical data provided evidence that phospholipids are co-distributed or interact with proteoglycans. Radioautography after IP reaction established that [3H] choline was detected in dentin as early as 30 min after the intravenous injection of the labeled precursor, before any labeling was seen in odontoblasts and predentin. This suggests that blood-serum-labeled phospholipids pass between odontoblasts, cross the distal permeable junctional complex, and diffuse in dentin prior to any cellular uptake and phospholipid synthesis. Pharmacologically and genetically induced pathology also supports the suggestion that phospholipids play an important role in the formation and mineralization of dental tissues.

Ameloblasts↗
PubMed1980

Surfactant phospholipid metabolism in 3-day and 3-day postmature rabbits in vivo.

Three-day-old normal and 3-day postmature rabbits (prepared by human chorionic gonadotropin treatment of the does) were given injections of the radiolabeled phospholipid precursors palmitic acid, choline, and glycerol. The specific activities as cpm/mumol phospholipid phosphate were measured for phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylethanolamine from lung and alveolar wash fraction lipid extracts of 3-day-old rabbits. Specific activities similarly were measured for phosphatidylcholine and disaturated phosphatidylcholine from postmature rabbits. Curves describing the appearance of labeled phospholipids into the alveolar wash fraction demonstrated that in both 3-day and postmature rabbits, phospholipid secretion was delayed for 3 to 4 hr. Thereafter, there was a rapid accumulation of labeled phospholipids until 12 to 16 hr after labeled precursor injection. The curves were similar for all measured phospholipids and all labeled precursors studied. The phospholipid compositions of lung and alveolar wash fraction lipid extracts were determined by analysis of the various phospholipids separated by two-dimensional thin-layer chromatography. Forty percent of lung samples and 35% of alveolar wash samples from postmature rabbits contained no detectable phosphatidylglycerol, whereas all 22 lung and surfactant samples from 3-day-old rabbits contained this phospholipid.

Age Factors↗
PubMed1996

Meibomian gland phospholipids.

The content of the meibomian gland lipid exprimate is known, but little is known about the phospholipids that comprise the glandular cells. The purpose of the present study is to identify and quantitate the phospholipid complement of the meibomian gland cells that produce the lipid secretion of meibomian oil and which is vital to tear film stability. Eyelids (n = 50) were excised from rabbits, and after surgical removal of surrounding tissues, the tarsal plates with and without expressing meibomian oil were extracted and phospholipids of the plates quantified by 31P nuclear magnetic resonance (NMR). Seventeen phospholipids were quantified from tarsal plates expressed of oil and tarsal plates containing meibomian oil: alkylacylphosphatidylcholine (AAPC), dihydrosphingomyelin (DHSM), dimethylphosphatidylethanolamine, diphosphatidylglycerol (cardiolipin), ethanolamine plasmalogen (EPLAS), lysoethanolamine plasmalogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidic acid, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin (SM), sphingosylphosphorylcholine. The six zwitterionic and neutral phospholipids, DHSM, EPLAS, PE, SM, AAPC, and PC together comprise 79.5% of the total meibomian gland phospholipid profile (in meibomian oil this value is 84.2%). The zwitterionic and neutral phospholipids dominate meibomian gland phospholipid profiles. Since the meibomian gland cells undergo holocrine secretion and form the meibomian glad secretion, such a composition is consistent with the hypothesis that a chemically stable lamellar surfactant layer phospholipids bind non-polar meibomian oil to the aqueous layer of the tear film.

Animals↗
PubMedDate not supplied

[Features of phospholipid composition of marine proteobacteria of Pseudoalteromonas species].

The study of the phospholipid composition of 14 type strains of marine proteobacteria of the genus Pseudoalteromonas showed that phospholipids are the main polar lipid constituents of membranes in these proteobacteria. The phospholipid patterns of the strains studied were found to be similar and involved five phospholipids typical of gram-negative bacteria, namely, phosphatidylethanolamine, phosphatidylglycerol, bisphosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid. The major phospholipids were phosphatidylethanolamine and phosphatidylglycerol, which add up to 89-97% of total phospholipids; bisphosphatidic acid was dominant among minor phospholipids. The prevalence of phosphatidylethanolamine (62-77% of total phospholipids) and the absence of diphosphatidylglycerol are the characteristic features of most bacteria of this genus. As in Escherichia coli, the phospholipid composition of the marine proteobacteria depended on the presence of magnesium in the medium.

Culture Media↗
PubMed1990

[Experimental studies on the changes of mitochondrial membrane phospholipids during cerebral ischemia and recirculation].

Changes of brain mitochondrial phospholipids during cerebral ischemia and recirculation were experimentally studied in a rat 4-vessel occlusion model, to explore the relation between changes of mitochondrial phospholipids and dysfunction of mitochondria. Respiratory functions, activities of respiratory enzymes (cytochrome c oxidase, F0F1-ATPase) were analyzed after 30 and 60 minutes of ischemia, and after 30 minutes of recirculation following each ischemic period. Activities of respiratory functions and respiratory enzymes decreased progressively during ischemia, which recovered completely after recirculation following 30 minutes of ischemia, while only partial recovery was observed after recirculation following 60 minutes of ischemia. In phospholipid analyses, contents of phospholipid classes tended to decrease time-dependently during ischemia, and compositions of polyunsaturated fatty acids (PUFA) such as arachidonic acid (20:4) and docosahexaenoic acid (22:6) were decreased preferentially. In recirculation, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) showed recovery of contents of phospholipids and compositions of PUFA after recirculation following 30 minutes of ischemia, while further decrease of contents of phospholipids and compositions of PUFA were observed after recirculation following 60 minutes of ischemia, especially in CL. On the other hand, progressive degradation of phospholipids occurred after recirculation following both 30 and 60 minutes of ischemia in phosphatidylserine and phosphatidylinositol. Changes of major phospholipid classes such as PC, PE, and CL correlated with the changes of mitochondrial respiratory functions and activities of respiratory enzymes. In conclusion, changes of mitochondrial membrane phospholipids appear to affect the integrity of cellular energy metabolism via mitochondrial dysfunction during cerebral ischemia and recirculation.

Animals↗
PubMed1988

Arachidonate released upon agonist stimulation preferentially originates from arachidonate most recently incorporated into nuclear membrane phospholipids.

When icosanoid-producing cells are stimulated by an agonist, 2-10% of total cellular arachidonate is released from phospholipids, and a variable percentage of the released arachidonate is subsequently converted into icosanoids. We used a mouse fibrosarcoma cell line (HSDM1C1) which synthesizes prostaglandin E2 in response to bradykinin stimulation to address the following questions: 1) upon cell stimulation is newly incorporated arachidonate preferentially released from phospholipids over previously incorporated arachidonate and 2) is there a corresponding change in phospholipid or membrane compartmentation of arachidonate to explain preferential release of newly incorporated arachidonate? To study changes in the availability of arachidonate for release from phospholipids, we incubated HSDM1C1 cells with 0.67 microM [14C]arachidonate for 15 min and chased the pulse of radiolabeled arachidonate with normal serum fatty acids. We found that of the [14C]arachidonate incorporated into phospholipids during the 15-min pulse, the percent released upon stimulation decreased nearly 3-fold from 8.9 +/- 0.5% at 5 min of chase to 3.6 +/- 0.2% (mean +/- S.E., n = 6, P less than 0.001) after only 60 min of chase. Percent release of arachidonate from nonpulsed controls was 3-4%. Although arachidonate release from phospholipids decreased significantly after 60 min of chase, the arachidonate which was released always originated predominantly from phosphatidylinositol. There was no decrease in the activities of enzymes required for arachidonate release during this time period. We also observed that throughout the period of the chase, the radiolabeled arachidonate remained esterified to the same phospholipid class into which it was initially incorporated (approximately 40% of [14C]arachidonate in diacyl phosphatidylcholine, 40% in phosphatidylinositol, and 15% in diacyl phosphatidylethanolamine. In cell fractionation experiments, we found that after 1-3 h of chase, [14C]arachidonate decreased in subcellular fractions containing nuclei, as it became progressively unavailable for release from phospholipids. Thus, our results indicate that 1) upon cell stimulation, the most recently incorporated pool of arachidonate, which is in high concentration in the nuclear membrane, is preferentially released and that 2) arachidonate rapidly moves out of the nuclear membrane into a less releasable pool while remaining esterified to the phospholipid moiety into which it was initially incorporated. This study indicates that the subcellular compartmentation of arachidonate has a marked influence on the cellular metabolism of arachidonate.

Animals↗
PubMed1987

Role of acidic phospholipids in tissue distribution of quinidine in rats.

The mechanism of interorgan variation in tissue distribution of quinidine was investigated from a viewpoint of binding characteristics to phospholipids and the composition of phospholipids in various tissues. The order of binding of quinidine to an individual standard phospholipid, expressed as a product of the association constant (K) and the number of binding sites (n), was: phosphatidyl ethanolamine (PhE) less than dipalmitoyl phosphatidyl choline (saturated PhC) less than or equal to phosphatidyl choline (unsaturated PhC) less than phosphatidyl inositol (PhI) less than phosphatidyl glycerol (PhG) less than phosphatidic acid (PhA) less than phosphatidyl serine (PhS). Thus, quinidine was found to bind preferentially to acid phospholipids such as PhS, PhA, PhG, and PhI. The greatest binding was obtained in PhS among the various phospholipids and was more than 300-fold that of neutral phospholipids such as PhC and PhE. The concentration of individual components of phospholipids in the lung, kidney, liver and heart was determined using a two dimensional thin-layer chromatography. The concentration of PhS, highly responsible for the quinidine binding to phospholipids in each tissue, was ranked in the following order: heart less than liver less kidney less than lung. The contribution of PhS to quinidine binding was more than 86% in all tissues. A good correlation between the concentration of PhS in each tissue and the Ct/Cp ratio in vivo was obtained (r = 0.984). Thus, it was concluded that the tissue distribution of quinidine in vivo depended on the composition of phospholipids in tissues and that a determinant of interorgan variation in the tissue distribution of quinidine was the concentration of PhS in the tissues.

Animals↗
PubMed1986

Role of phospholipids in activation of mitochondrial D(-)-beta-hydroxybutyrate dehydrogenase.

Although phosphatidylcholine (PC) has been shown to be the type of phospholipid required for activation of mitochondrial beta-hydroxybutyrate dehydrogenase (BDH), mixtures of phospholipids containing PC are more effective activators. This study shows that apo-BDH, purified from bovine-heart mitochondria, and phospholipid-reconstituted BDH appear to be polydisperse. Upon cross-linking with dimethylpimelimidate and acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the enzyme exhibited molecular weight forms from monomeric to heptameric BDH as well as higher molecular weight aggregates that did not much penetrate the gels. When different phospholipid mixtures containing PC were used to activate apo-BDH, and the reconstituted samples were subjected to cross-linking and SDS-gel electrophoresis, a direct relationship was found between the activating effect of the phospholipids used and BDH monomer concentration in the gels. The effectiveness order of phospholipids used was as follows: a mixture of PC, phosphatidylethanolamine and diphosphatidylglycerol in a molar ratio of 5:4:1 greater than bovine-heart mitochondrial phospholipids greater than Asolectin greater than PC. These results suggest the following. In addition to PC, which is required by BDH, other types of phospholipids play a role in activation of purified apo-BDH, possibly via enzyme disaggregation. The activity exhibited by purified, phospholipid-reconstituted BDH is associated mainly with the lower molecular aggregates of the enzyme, especially monomeric BDH.

Animals↗