Phospholipid Pools Research Articles

Impaired ABCA1-dependent Lipid Efflux and Hypoalphalipoproteinemia in Human Niemann-Pick type C Disease

The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.

Effect of surfactant protein A on granular pneumocyte surfactant secretion in vitro.

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.

Hormone-sensitive lipase activity and triacylglycerol hydrolysis are decreased in rat soleus muscle by cyclopiazonic acid

Cyclopiazonic acid (CPA) is a sarcoplasmic reticulum Ca2+-ATPase inhibitor that increases intracellular calcium. The role of CPA in regulating the oxidation and esterification of palmitate, the hydrolysis of intramuscular lipids, and the activation of hormone-sensitive lipase (HSL) was examined in isolated rat soleus muscles at rest. CPA (40 micro M) was added to the incubation medium to levels that resulted in subcontraction increases in muscle tension, and lipid metabolism was monitored using the previously described pulse-chase procedure. CPA did not alter the cellular energy state, as reflected by similar muscle contents of ATP, phosphocreatine, free AMP, and free ADP. CPA increased total palmitate uptake into soleus muscle (11%, P < 0.05) and was without effect on palmitate oxidation. This resulted in greater esterification of exogenous palmitate into the triacylglycerol (18%, P < 0.05) and phospholipid (89%, P < 0.05) pools. CPA decreased (P < 0.05) intramuscular lipid hydrolysis, and this occurred as a result of reduced HSL activity (20%, P < 0.05). Incubation of muscles with 3 mM caffeine, which is also known to increase Ca2+ without affecting the cellular energy state, reduced HSL activity (24%, P < 0.05). KN-93, a calcium/calmodulin-dependent kinase inhibitor (CaMKII), blocked the effects of CPA and caffeine, and HSL activity returned to preincubation values. The results of the present study demonstrate that CPA simultaneously decreases intramuscular triacylglycerol (IMTG) hydrolysis and promotes lipid storage in isolated, intact soleus muscle. The decreased IMTG hydrolysis is likely mediated by reduced HSL activity, possibly via the CaMKII pathway. These responses are not consistent with the increased hydrolysis and decreased esterification observed in contracting muscle when substrate availability and the hormonal milieu are tightly controlled. It is possible that more powerful signals or a higher [Ca2+] may override the lipid-storage effect of the CPA-mediated effects during muscular contractions.

Pneumonitis and Emphysema in sp-C Gene Targeted Mice

SP-C-deficient (SP-C -/-) mice developed a severe pulmonary disorder associated with emphysema, monocytic infiltrates, epithelial cell dysplasia, and atypical accumulations of intracellular lipids in type II epithelial cells and alveolar macrophages. Whereas alveolar and tissue surfactant phospholipid pools were increased, levels of other surfactant proteins were not altered (SP-B) or were modestly increased (SP-A and SP-D). Analysis of pressure-volume curves and forced oscillatory dynamics demonstrated abnormal respiratory mechanics typical of emphysema. Lung disease was progressive, causing weight loss and cardiomegaly. Extensive alveolar remodeling was accompanied by type II cell hyperplasia, obliteration of pulmonary capillaries, and widespread expression of alpha-smooth muscle actin, indicating myofibroblast transformation in the lung parenchyma. Dysplastic epithelial cells lining conducting airways stained intensely for the mucin, MUC5A/C. Tissue concentrations of proinflammatory cytokines were not substantially altered in the SP-C (-/-) mice. Production of matrix metalloproteinases (MMP-2 and MMP-9) was increased in alveolar macrophages from SP-C (-/-) mice. Absence of SP-C caused a severe progressive pulmonary disorder with histologic features consistent with interstitial pneumonitis.

(n-3) Polyunsaturated Fatty Acids Promote Activation-Induced Cell Death in Murine T Lymphocytes

Previous studies showing dietary (n-3) polyunsaturated fatty acids (PUFA) attenuate T cell immune-mediated inflammatory diseases led us to hypothesize that (n-3) PUFA promote activation-induced cell death (AICD) in T cells. Because T cell subsets display a differential resistance to AICD, we compared the effects of (n-3) PUFA feeding on T cells stimulated in vitro to express different cytokine profiles. Mice were fed either diets lacking (n-3) PUFA (control) or (n-3) PUFA-containing diets for 14 d. Splenic T cells were stimulated with alphaCD3/alphaCD28, phorbol myristate acetate (PMA)/Ionomycin or alphaCD3/PMA for 48 h, followed by reactivation with the same stimuli for 5 h. Apoptosis was measured using Annexin V/propidium iodide. (n-3) PUFA were selectively incorporated into membrane phospholipid pools. Cytokine analyses revealed that (n-3) PUFA enhanced AICD only in T cells expressing a T helper cell (Th)1-like cytokine profile after stimulation with PMA/Ionomycin compared to mice fed the (n-6) PUFA control diet (P = 0.0008). In contrast, no increase in apoptosis was seen in T cells stimulated with alphaCD3/PMA, which exhibited a Th2 cytokine profile. These data demonstrate that the ability of (n-3) PUFA to promote AICD is dependent on the activation stimulus. In conclusion, we have identified a novel mechanism by which (n-3) PUFA modulate T cell-mediated immunity by selective deletion of Th1-like cells while maintaining or enhancing the Th2-mediated humoral immune response.

Residual cftr Expression Varies with Age in cftrtm1Hgu Cystic Fibrosis Mice: Impact on Morphology and Physiology

Mouse models for cystic fibrosis (CF) mimic intestinal manifestations of the human disease, but the lung disease phenotypes are lacking in most strains. In this work, the issue was addressed whether aging of the respiratory tract leads to lung pathophysiology in the exon 10 insertional mutant cftr^tm1Hgu mouse. Weight gain, body weight and life-span of cftr^tm1Hgu mice were significantly reduced compared with control mice. cftr^tm1Hgu mice expressed 20, 21 or 37% (median) of wild-type cystic fibrosis conductance transmembrane regulator (cftr) mRNA transcript in lungs, intestine and kidney. Wild-type cftr mRNA in renal and respiratory epithelia varied with age from levels similar to Ztm:MF1 controls at the age of 2 and 4 months to levels seen in patients with CFTR splice mutations beyond the age of 6 months. The morphology of the bronchi and more distal airways was apparently normal in cftr^tm1Hgu mice during their first year of life. The alveolar surfactant phospholipid pool was increased in cftr^tm1Hgu mice by 1.5- to 2-fold compared with Ztm:MF1 controls. Alveolar clearance of γ-labelled scandium oxide – the first report of lung clearance measurement in living mice – was reduced in cftr^tm1Hgu mice compared with littermate controls. Although no progressive lung pathology was seen in the cftr expression of cftr^tm1Hgu mice, surfactant phospholipid homeostasis, and alveolar and mucociliary clearance were abnormal. Therefore, the described model is useful for studying the initial CF lung pathophysiology.

Activin/TGF-beta induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP.

Members of the transforming growth factor beta (TGF-beta) family regulate fundamental physiological processes, such as cell growth, differentiation and apoptosis, in almost all cell types. As a result, defects in TGF-beta signalling pathways have been linked to uncontrolled cellular proliferation and carcinogenesis. Here, we explored the signal transduction mechanisms downstream of the activin/TGF-beta receptors that result in cell growth arrest and apoptosis. We show that in haematopoietic cells, TGF-beta family members regulate apoptosis through expression of the inositol phosphatase SHIP (Src homology 2 (SH2) domain-containing 5' inositol phosphatase), a central regulator of phospholipid metabolism. We also demonstrated that the Smad pathway is required in the transcriptional regulation of the SHIP gene. Activin/TGF-beta-induced expression of SHIP results in intracellular changes in the pool of phospholipids, as well as in inhibition of both Akt/PKB (protein kinase B) phosphorylation and cell survival. Our results link phospholipid metabolism to activin/TGF-beta-mediated apoptosis and define TGF-beta family members as potent inducers of SHIP expression.

Reversibility of Pulmonary Abnormalities by Conditional Replacement of Surfactant Protein D (SP-D) in Vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.

DNA-bound lipids of normal and tumor cells: retrospective and outlooks for functional genomics

By very soft phenol method, the high-molecular-mass natural DNA complexes (10 8–10 9 Da), which contain 1–3% specific lipids, were isolated from different eukaryotic and prokaryotic cells. Two pools of DNA-bound lipids were isolated: loosely bound (extracted with 35% ethanol) and tightly bound lipids (extracted after additional treatment DNAse I). The composition of these two lipid pools of different sources (rat thymus, liver, regenerating liver, loach sperm, pigeon erythrocytes, Zajdel ascites hepatoma, Ehrlich ascites carcinoma, sarcoma 37, Escherichia coli B, T2 phage) was studied. The DNA-bound lipid pools consist of neutral lipids (NL) and phospholipids (PL), moreover NL is always in a few fold more than PL. The composition of these lipid pools of eukaryotes distinguishes between themselves, mainly, by free cholesterol (minor fraction), cardiolipin (major fraction), and by phosphatidylcholine. Only the tightly bound lipid pool was present in T2 phage DNA. The dramatic redistribution effect between all fractions of NL pools (free and ester cholesterol, free fatty acids, diglycerides) was observed in DNA synthesis phase of cell cycle on the background of the unchanged composition of PL pools. Comparative analysis of DNA-bound lipid pools of normal and cancer cells was carried out. The DNA-bound lipid pools of transformed cells significantly differ from the same normal cells both by PL composition (cardiolipin) and by the presence of additional fractions (mono- and triglycerides) as well. The possible functions of DNA-bound lipid pools, especially of cardiolipin and cholesterol at the attachment of DNA loops to the nuclear matrix, DNA replicon organization, replication, and transcription are discussed.

Oleate and Linoleate Enhance the Growth-promoting Effects of Insulin-like Growth Factor-I through a Phospholipase D-dependent Pathway in Arterial Smooth Muscle Cells

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.

Glutathione Replacement Preserves the Functional Surfactant Phospholipid Pool Size and Decreases Sepsis‐Mediated Lung Dysfunction in Ethanol‐Fed Rats

Background Alcohol abuse increases the incidence of acute respiratory distress syndrome (ARDS). Previous evidence from our laboratory links ethanol‐mediated glutathione depletion to impaired surfactant production by alveolar epithelial cells in vitro and to endotoxin‐mediated edematous injury in isolated lungs ex vivo. ARDS patients have an imbalance between the inactive small aggregate (SA) and the bioactive large aggregate (LA) forms of surfactant phospholipid (as reflected by increased SA/LA ratios). Therefore, we hypothesized that ethanol ingestion, via glutathione depletion, could alter surfactant phospholipid distribution between LA and SA forms and thereby exacerbate sepsis‐mediated lung dysfunction in vivo.Methods Rats fed an isocaloric diet with or without ethanol (36% total calories) for 6 weeks were made septic via cecal ligation and perforation. Some ethanol‐fed rats had their diets supplemented with the glutathione precursor procysteine (l‐2‐oxothiaxolidine‐4‐carboxylate). Sepsis physiology was assessed by determining respiratory rates, arterial blood pressures, and plasma lactate levels, and lung dysfunction was assessed by determining lung lavage fluid protein levels (index of alveolar endothelial/epithelial barrier disruption), arterial hypoxemia (index of impaired gas exchange) and surfactant phospholipid SA and LA fractions (index of the alveolar epithelium's ability to maintain a functional surfactant pool during sepsis).Results Ethanol ingestion decreased (p &lt; 0.05) lung lavage fluid glutathione levels, and this defect was prevented by procysteine. Although ethanol ingestion had no effect (p &lt; 0.05) on any of the indices of sepsis, it increased (p &lt; 0.05) lung lavage fluid protein levels, worsened hypoxemia, and decreased the functional (LA) surfactant phospholipid pool after sepsis, all of which was prevented by procysteine.Conclusions Ethanol ingestion, via glutathione depletion, increased sepsis‐mediated lung dysfunction, and these effects could contribute to the increased risk of ARDS seen in alcoholic patients.

Glutathione Replacement Preserves the Functional Surfactant Phospholipid Pool Size and Decreases Sepsis-Mediated Lung Dysfunction in Ethanol-Fed Rats

Background Alcohol abuse increases the incidence of acute respiratory distress syndrome (ARDS). Previous evidence from our laboratory links ethanol-mediated glutathione depletion to impaired surfactant production by alveolar epithelial cells in vitro and to endotoxin-mediated edematous injury in isolated lungs ex vivo. ARDS patients have an imbalance between the inactive small aggregate (SA) and the bioactive large aggregate (LA) forms of surfactant phospholipid (as reflected by increased SA/LA ratios). Therefore, we hypothesized that ethanol ingestion, via glutathione depletion, could alter surfactant phospholipid distribution between LA and SA forms and thereby exacerbate sepsis-mediated lung dysfunction in vivo. Methods Rats fed an isocaloric diet with or without ethanol (36% total calories) for 6 weeks were made septic via cecal ligation and perforation. Some ethanol-fed rats had their diets supplemented with the glutathione precursor procysteine (l-2-oxothiaxolidine-4-carboxylate). Sepsis physiology was assessed by determining respiratory rates, arterial blood pressures, and plasma lactate levels, and lung dysfunction was assessed by determining lung lavage fluid protein levels (index of alveolar endothelial/epithelial barrier disruption), arterial hypoxemia (index of impaired gas exchange) and surfactant phospholipid SA and LA fractions (index of the alveolar epithelium's ability to maintain a functional surfactant pool during sepsis). Results Ethanol ingestion decreased (p < 0.05) lung lavage fluid glutathione levels, and this defect was prevented by procysteine. Although ethanol ingestion had no effect (p < 0.05) on any of the indices of sepsis, it increased (p < 0.05) lung lavage fluid protein levels, worsened hypoxemia, and decreased the functional (LA) surfactant phospholipid pool after sepsis, all of which was prevented by procysteine. Conclusions Ethanol ingestion, via glutathione depletion, increased sepsis-mediated lung dysfunction, and these effects could contribute to the increased risk of ARDS seen in alcoholic patients.

In vitro mimicry of essential fatty acid deficiency in human endothelial cells by TNFα impact of ω-3 versus ω-6 fatty acids

Severe endothelial abnormalities are a prominent feature in sepsis with cytokines such as tumor necrosis factor (TNF)alpha being implicated in the pathogenesis. As mimic to inflammation, human umbilical vascular endothelial cells (HUVEC) were incubated with TNFalpha for 22 h, in the absence or presence of the omega-6 fatty acid (FA), arachidonic acid (AA), or the alternative omega-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). TNFalpha caused marked alterations in the PUFA profile and long chain PUFA content of total phospholipids (PL) decreased. In contrast, there was a compensatory increase in mead acid [MA, 20:3(omega-9)], the hallmark acid of the essential fatty acid deficiency (EFAD) syndrome. Corresponding changes were noted in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but not in the sphingomyelin fraction. Supplementation with AA, EPA, or DHA markedly increased the respective FA contents in the PL pools, suppressed the increase in MA, and resulted in a shift either toward further predominance of omega-6 or predominance of omega-3 FA. We conclude that short-term TNFalpha incubation of HUVEC causes an EFAD state hitherto only described for long-term malnutrition, and that endothelial cells are susceptible to differential influence by omega-3 versus omega-6 FA supplementation under these conditions.

Lorenzo's oil, adrenoleukodystrophy, and the blood– brain barrier

Adrenoleukodystrophy is a rapid, progressive demyelinating disease affecting the CNS that is characterized by large increases in plasma and tissue very long saturated fatty acids (VLCFA). Lorenzo's oil (LO), consisting of erucic (22:1 n‐9) and oleic (18:1 n‐9) acid in a triglyceride form, is a dietary therapy effective in reducing plasma and tissue VLCFA. Despite the decreased VLCFA, clinical studies indicated that LO failed to stop the progressive demyelination, suggesting that erucic acid, the active component of LO, did not cross the BBB. We addressed this question by infusing [14‐14C] 22:1 n‐9 (170 μCi/kg) into male rats using two different infusion paradigms. The radiotracer was infused (i.v.) into awake, adult male rats over a 10‐min period or infused (i.c.v.) into the fourth ventricle over a 7‐day period using an osmotic mini‐pump. Brains were removed from the cranium, frozen in liquid nitrogen, lipids extracted, and separated using standard techniques. [1‐14C] 20:4 n‐6 was infused (i.v.) and used as a positive control. Following i.v. infusion, 0.011% of the erucic acid was extracted by the brain, compared to 0.055% of the arachidonic acid. About 60% of the brain erucic acid was found in the aqueous fraction compared to 30% for arachidonic acid. Further, erucic acid was targeted to cholesteryl ester and triacylglyceride pools, whereas arachidonic acid was targeted to phospholipid pools. In animals infused i.c.v., 0.078% of the dose was taken up and about 60% of the erucic acid was targeted to phospholipid pools. These results clearly demonstrate that erucic acid crosses the BBB, similar to arachidonic acid, and is incorporated into specific lipid pools.Acknowledgements: This work was supported by The Myelin Project.

Lipid metabolism during aging of high-α-linolenate-phenotype potato tubers

Previous studies demonstrated that high levels of α-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-α-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-α-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-α-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-α-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-α-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-α-linolenate tubers. These age- and high-α-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-α-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-α-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-α-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Δ5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.

Glutamate signalling and secretory phospholipase A2 modulate the release of arachidonic acid from neuronal membranes.

The lipid mediators generated by phospholipases A(2) (PLA(2)), free arachidonic acid (AA), eicosanoids, and platelet-activating factor, modulate neuronal activity; when overproduced, some of them become potent neurotoxins. We have shown, using primary cortical neuron cultures, that glutamate and secretory PLA(2) (sPLA(2)) from bee venom (bv sPLA(2)) and Taipan snake venom (OS2) elicit synergy in inducing neuronal cell death. Low concentrations of sPLA(2) are selective ligands of cell-surface sPLA(2) receptors. We investigated which neuronal arachidonoyl phospholipids are targeted by glutamate-activated cytosolic calcium-dependent PLA(2) (cPLA(2)) and by sPLA(2). Treatment of (3)H-AA-labeled cortical neurons with mildly toxic concentrations of sPLA(2) (25 ng/ml, 1.78 nM) for 45 min resulted in a two- to threefold higher loss of (3)H-AA from phosphatidylcholine (PC) than from phosphatidylethanolamine (PE) and in minor changes in other phospholipids. A similar profile, although of greater magnitude, was observed 20 hr posttreatment. Glutamate (80 microM) induced much less mobilization of (3)H-AA than did sPLA(2) and resulted in a threefold greater degradation of (3)H-AA PE than of (3)H-AA PC by 20 hr posttreatment. Combining sPLA(2) and glutamate resulted in a greater degradation of PC and PE, and the N-methyl-D-aspartate receptor antagonist MK-801 only blocked glutamate effects. Thus, activation of the arachidonate cascade induced by glutamate and sPLA(2) under experimental conditions that lead to neuronal cell death involves the hydrolysis of different (perhaps partially overlapping) cellular phospholipid pools.

Effects of Enrichment of Fibroblasts with Unesterified Cholesterol on the Efflux of Cellular Lipids to Apolipoprotein A-I

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.

Dietary n-3 PUFA alter colonocyte mitochondrial membrane composition and function.

There is experimental evidence that dietary fish oil, which contains the n-3 fatty acid family, i.e., EPA and DHA, protects against colon tumor development, in part by increasing apoptosis. Since mitochondria can act as central executioners of apoptosis, we hypothesized that EPA and DHA incorporation into colonocyte mitochondrial membranes, owing to their high degree of unsaturation, would enhance susceptibility to damage by reactive oxygen species (ROS) generated via oxidative phosphorylation. This, in turn, would compromise mitochondrial function, thereby initiating apoptosis. To test this hypothesis, colonic crypts were isolated from rats fed either fish oil, purified n-3 fatty acid ethyl esters, or corn oil (control). Dietary lipid source had no effect on colonic mitochondrial phospholipid class mole percentages, although incorporation of EPA and DHA was associated with a reduction in n-6 fatty acids known to enhance colon tumor development, i.e., linoleic acid LNA) and its metabolic product, arachidonic acid (ARA). Select compositional changes in major phospholipid pools were correlated to alterations in mitochondrial function as assessed by confocal microscopy. The mol% sum of LNA plus ARA in cardiolipin was inversely correlated with ROS (P = 0.024). Ethanolamine glycerophospholipid ARA (P = 0.046) and choline glycerophospholipid LNA (P = 0.033) levels were positively correlated to mitochondrial membrane potential. In contrast, ethanolamine glycerophospholipid EPA (P = 0.042) and DHA (P = 0.024) levels were negatively correlated to mitochondrial membrane potential. Additionally, EPA and DHA levels in choline glycerophospholipids (P = 0.026) were positively correlated with caspase 3 activity. These data provide evidence in vivo indicating that dietary EPA and DHA induce compositional changes in colonic mitochondrial membrane phospholipids that facilitate apoptosis.

Pulmonary surfactant and inflammation in septic adult mice: role of surfactant protein A.

Surfactant alterations, alveolar cytokine changes, and the role of surfactant protein (SP)-A in septic mice were investigated. Sepsis was induced via cecal ligation and perforation (CLP). Septic and sham mice were euthanized at 0, 3, 6, 9, 12, 15, and 18 h after surgery. Mice deficient in SP-A and mice that overexpressed SP-A were euthanized 18 h after surgery. In wild-type, sham-operated mice, surfactant pool sizes were similar at all time points, whereas in the CLP groups there was a significant decrease in small-aggregate surfactant pool sizes beginning 6 h after CLP. Interleukin-6 concentrations in bronchoalveolar lavage fluid from septic animals increased from 6 to 18 h after surgery. Identical surfactant alterations and concentrations of cytokines were observed in septic mice that were SP-A deficient or that overexpressed SP-A. In conclusion, alterations of pulmonary surfactant and alveolar cytokines occur simultaneously, 6 h after a systemic insult. In addition, we did not detect a role for SP-A in regulating surfactant phospholipid pool sizes or pulmonary inflammation in septic mice.

Dual coupling of MT 1 and MT 2 melatonin receptors to cyclic AMP and phosphoinositide signal transduction cascades and their regulation following melatonin exposure

In this investigation, we wanted to determine whether MT 1 or MT 2 melatonin receptors are capable of coupling to the phosphoinositide (PI) signal transduction cascade. In addition, we wanted to assess the effects of chronic melatonin exposure on MT 1 and MT 2 melatonin receptor-mediated stimulation of PI hydrolysis. We also assessed the effects of chronic melatonin exposure on other parameters of the MT 2 melatonin receptor function including total specific 2-[ 125 I ]-iodomelatonin binding, the affinity of melatonin for the receptor, and melatonin (1 nM)-mediated inhibition of cyclic 3′,5′-adenosine monophosphate (cAMP) accumulation. Investigation of the PI signal transduction cascade activated by either the MT 1 or MT 2 melatonin receptor expressed in Chinese hamster ovary (CHO) cells showed that melatonin (1 pM to 1 mM) was able to stimulate the formation of PIs to ∼40–60% over basal [ ec 50: MT 1=29 nM (2–300 nM) and MT 2=1.1 nM (0.32–3.5 nM), N=5]. This response was mediated via receptors based upon the findings that melatonin did not stimulate the formation of PIs in CHO cells devoid of receptor and that antagonism of MT 2 melatonin receptors by 4P-PDOT (AH 024; 4-phenyl-2-propionamidotetralin) attenuated melatonin-mediated stimulation of PI hydrolysis in CHO cells expressing the MT 2 melatonin receptor. The consequence of chronic melatonin exposure on MT 1 and MT 2 receptor function was also examined. Pretreatment of either MT 1- or MT 2-CHO cells with melatonin (1 μM for 5 hr) resulted in: (a) a complete loss of melatonin-mediated stimulation of PI hydrolysis, and (b) an attenuation of melatonin (1 nM)-mediated inhibition of forskolin-induced cAMP accumulation by ∼20–40%. The desensitization of the PI hydrolysis signal transduction cascades coupled to either MT 1 or MT 2 melatonin receptors following chronic melatonin exposure was not due to depleted phospholipid pools, to elevated basal levels, or to decreases in receptor affinity and density. This dual coupling of melatonin receptors to different signal transduction cascades may contribute to the diversity of melatonin receptor function in vivo.