John S. Ellingson

The effect of chronic ethanol consumption on the fatty acid composition of phosphatidylinositol in rat liver microsomes as determined by gas chromatography and 1H-NMR.

Cell membranes and vesicles composed of extracted phospholipids isolated from rats chronically-fed ethanol develop a resistance to disordering by ethanol in vitro (membrane tolerance) and a decreased partitioning of ethanol into the membranes. The anionic lipid phosphatidylinositol (PtdIns) is the only microsomal phospholipid from the ethanol-fed rats that confers tolerance to vesicles of microsomal phospholipids from control rats in a paradigm where phospholipid classes are sequentially swapped. To investigate the molecular basis of this adaptation, the fatty acid content of microsomal PtdIns extracted from the livers of rats chronically fed ethanol for 5 weeks and their calorically-matched controls was analyzed by gas-liquid chromatography (GLC) and 1H-NMR spectroscopy. Chronic ethanol consumption caused an 8.4% decrease in arachidonic acid [20:4(n - 6)], a 20.0% increase in oleic acid [18: 1(n - 9)] and a 47.1% increase in the quantitatively minor fatty acid [20:3(n - 6)]. 1H-NMR was used to quantitatively assay compositional changes in the delta 5 olefinic moiety of the acyl chains in PtdIns, an approach that should be broadly applicable to other lipid systems. After chronic ethanol feeding PtdIns had decreased delta 5 unsaturates (-7.9% NMR, -8.2% GLC) and a corresponding increase in delta 5 saturates (+5.4% NMR, +5.3% GLC). In the other phospholipids, chronic ethanol feeding caused alterations in the fatty acid compositions specific for each phospholipid. PtdIns was the only microsomal phospholipid that exhibited a significant decrease in both the polyunsaturate pool and the ratio of the total olefinic content to the saturated fatty acid content. The major adaptive response in rat liver microsomal PtdIns to chronic ethanol administration involves a decrease in arachidonic acid [20:4 (n - 6)], which is partly compensated for by increases in oleic acid [18:1(n - 9)] and eicosatrienoic acid [20:3 (n - 6)], resulting in a depressed unsaturation and polyunsaturation index. The decreased unsaturation at the delta 5 position may have special functional relevance, due to the proximity of this position to the membrane surface, where ethanol is believed to reside. Whether these acyl changes are merely coincident with, or causative of, membrane tolerance requires further elucidation.

Membrane structural alterations caused by chronic ethanol consumption: the molecular basis of membrane tolerance.

The effects of chronic ethanol consumption on the central nervous system, the liver, and a variety of other organs may be related in part to its interaction with biological membranes (reviewed in REF. I ) . The acute presence of ethanol generally exerts a disordering (fluidizing) effect on the lipid bilayer of biological membranes. By contrast, chronic consumption of ethanol leads to altered membranes, which are resistant to this disordering (membrane tolerance).24 In our animal model, rats ingest 14-16 g of ethanol/kg body weight for 35 days in a nutritionally adequate diet, while pair-fed controls consume the same diet, except that carbohydrate isocalorically replaces ethanol. Our criteria for a good animal model to investigate the membrane alterations caused by chronic ethanol consumption are: 1) the mode of ethanol administration is similar to that in human alcoholism, 2) the structural and chemical alterations are produced by physiologically relevant amounts of ethanol, and 3) the effects of acute or chronic ethanol treatment, measured by electron spin resonance (ESR) are large, easily discernible (i.e. “all or nothing”), and reproducible. The animal model we use meets all of these criteria. In this investigation we examined the structural properties of red blood cells and liver microsomes obtained from rats that had been chronically administered ethanol for 35 days and then withdrawn from ethanol for 1-10 days. In addition, using electron spin resonance, high pressure liquid chromatography (HPLC), and membrane reconstitution techniques, we identified the membrane component(s) responsible for membrane tolerance in the microsomal membrane for the first time.

Temporal and quantitative expression of the myelin-associated lipids, ethanolamine plasmalogen, galactocerebroside, and sulfatide, in the differentiating CG-4 glial cell line

We determined the expression of three myelin-typical lipids in the continuous CG-4 glial cell line of oligodendrocyte progenitor cells, as the cells differentiated into oligodendrocytes. On 6 different days during the first 9 days of oligodendrocyte development, cells were labeled for 24 h with [3H]ethanolamine to label ethanolamine plasmalogens or with [3H]galactose to label the galactocerebroside and sulfogalactocerebroside; and the amount of labeled lipid expressed on each day was determined. Each labeled lipid was expressed with its own specific time course and in a defined amount on each day of differentiation. Increased labeling of plasmalogens and sulfogalactocerebroside started at early developmental stages, and increased labeling of galactocerebroside started at later stages. The results indicate that the differentiating CG-4 cell line provides a valuable system to investigate factors affecting the early time course of myelin-lipid expression and the amounts expressed.

Protein kinase C inhibitors counteract the ethanol effects on myelin basic protein expression in differentiating CG-4 oligodendrocytes.

Abnormal formation of myelin appears to be one defect contributing to the development of the neuropathology associated with the fetal alcohol syndrome. Using the CG-4 cell line we previously showed that 25-75 mM EtOH downregulates the expression of myelin basic protein (MBP) in differentiating oligodendrocytes (OLGs) without affecting morphological development (Dev. Brain Res. 128 (2001) 9). Here we showed that a relatively low concentration of 12-phorbol-13-myristate acetate (PMA) mimicked the EtOH-caused inhibition of MBP expression without affecting morphology. The inhibition of MBP expression by 100 mM EtOH or 1 nM PMA was completely counteracted by three inhibitors of protein kinase C (PKC), bisindolylmaleimide I, chelerythrine chloride, and calphostin C, indicating that EtOH downregulated MBP expression by activating PKC. We investigated whether the EtOH activation resulted, in part, from upregulation of the expression of PKC isozymes. Of 11 PKC isozymes examined, CG-4 OLGs expressed nine; PKCs alpha, beta1, beta2; delta, epsilon, eta; lambda, zeta; mu; while PKC isozymes gamma and theta were not detected. Only five PKC isozymes, alpha, beta1, beta2, eta, and mu, displayed developmental changes in expression. However, EtOH did not upregulate the early expression of any PKC isozyme during the first 2 days of differentiation, the developmental stage when it downregulates MBP expression in CG-4 cells. The similar effects of PMA and EtOH indicate that EtOH delays MBP expression by activating at least one phorbol ester-sensitive PKC isozyme in oligodendrocytes without upregulating its expression.

Effects of transient ethanol exposure on the incorporation of [3H]ethanolamine into plasmalogen in the differentiating CG-4 oligodendrocyte cell line

We investigated the potential teratogenic effects of ethanol (EtOH) on myelination by monitoring its effects on the labeling of the myelin-typical lipid, ethanolamine plasmalogen (EPl), in the CG-4 cell line of differentiating oligodendrocytes (OLGs). On 5 different days during the first 8 days of OLG development, cells were labeled for 24 hr with [(3)H]ethanolamine to label EPl and diacyl-ethanolamine phosphoglycerols (diacyl-EPG), and the amount of labeled lipid expressed on each day was determined in the presence and absence of 25-120 mM EtOH. At early stages of development, a lower amount of [(3)H]EPl per cell was found in cells exposed to EtOH. The ratio of [(3)H]EPl to [(3)H]diacyl-EPG in cells exposed to 25, 50, or 120 mM EtOH was decreased by 50% after 4 days of differentiation compared with that in control cells. By adding or withdrawing EtOH at specific days of differentiation, we showed that EtOH inhibited the increased labeling of EPl if it was present for the first 48 hr of differentiation, and subsequent withdrawal failed to relieve the inhibition. Addition of EtOH anytime after the first day of differentiation did not inhibit the increased labeling of EPl. The results show that the increased labeling of EPl in differentiating OLGs resulted from an EtOH-sensitive, developmentally programmed, transient process active only during the first 2 days of differentiation.

Ethanol alters the expressions of c-Fos and myelin basic protein in differentiating oligodendrocytes

Myelination occurs in the central nervous system of the human fetus, adolescents, and young adults. Ethanol interferes with myelination in part by altering the composition of the myelin sheath. Here we show that ethanol also affected the expression of the transcription factor c-Fos in differentiating oligodendrocytes (OLGs). Central glial-4 OLG progenitors were induced to differentiate in the absence and presence of 100 mM ethanol, and ethanol-caused changes in the levels of c-Fos and myelin basic protein (MBP) were determined by Western blot analysis at selected developmental stages. The relatively high c-Fos level in progenitors did not immediately decrease to a low level at the onset of differentiation but displayed a downregulation at a later developmental stage. Ethanol delayed the developmental c-Fos downregulation maintaining c-Fos at a 45% higher level at 2 days of differentiation (DoD). Ethanol also decreased the rate of the burst of MBP expression that occurred between 1 and 2 DoD, reducing the MBP level by 47% at 2 DoD. The ethanol-caused delays of c-Fos downregulation and MBP upregulation were both blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM). Likewise, treatment of OLGs with a low 5-nM concentration of the PKC activator by 12-O-tetradecanoylphorbol-13-acetate mimicked the ethanol effects on the expression of both proteins, effects that were also counteracted by BIM. The results indicate that ethanol-caused delays of the stage-specific c-Fos downregulation and the inhibition of MBP expression both occur through a PKC-mediated mechanism. The ethanol-caused delay in c-Fos downregulation may disrupt normal timing for expression of genes involved in OLG differentiation, and the inhibited MBP expression may alter the myelin sheath composition.

High-performance liquid chromatographic method for determination of the metabolism of polyunsaturated molecular species of phosphatidylserine labeled in the polar group

A reversed-phase HPLC method to monitor the incorporation of radiolabeled precursors into the polar group of individual polyunsaturated molecular species of phosphatidylserine (PS) is presented. PS labeled in the polar group was decarboxylated and subsequently converted to trinitrophenyl-phosphatidylethanolamine (Tnp-PE), which was separated into its molecular species by reversed-phase HPLC within 90 min, using a gradient of acetonitrile-methanol and ammonium acetate. A major feature of the method is the complete resolution of the stearoyl species, 18:0/20:4 and 18:0/22:6, at ambient temperature. By determining the amount of radioactivity incorporated into each fraction, the metabolism of individual molecular species of PS, and also of PE, labeled in the polar group can be investigated.

Rats withdrawn from ethanol rapidly re-acquire membrane tolerance after resumption of ethanol feeding

The time course for the re-acquisition of membrane tolerance to the disordering effects of ethanol in vitro has been determined for liver microsomes obtained from chronically ethanol-fed rats that were withdrawn from ethanol for 2-4 days (during which tolerance is lost) followed by resumption of ethanol feeding. Naive rats require 28-35 days of chronic ethanol feeding to develop membrane tolerance. Microsomal membranes regain partial sensitivity to ethanol disordering after 2-3 days of withdrawal and regain the complete sensitivity observed in membranes from untreated control rats after 4 days of withdrawal. The period of ethanol re-feeding required for the re-acquisition of membrane tolerance was dependent on the withdrawal period, with tolerance appearing sooner if the withdrawal period was shorter. The time course for the re-development of tolerance in previously tolerant animals was considerably faster (4-14 days) than in naive rats being administered the ethanol diet for the first time (35 days). Microsomes from rats that were withdrawn for 2 days (which retained partial tolerance) and then re-fed the ethanolic-diet required only 4 days to re-acquire membrane tolerance. Microsomes from rats withdrawn 3 days required 8 days and those withdrawn 4 days required 15 days for full tolerance to re-develop. The same time-course for the re-acquisition of membrane tolerance was observed in either intact microsomes or in liposomes prepared from extracted microsomal total phospholipids. Phosphatidylinositol (PI) has previously been reported to be responsible for conferring membrane tolerance to liver microsomes in ethanol-fed rats (Taraschi, T.F., Ellingson, J.S., Wu, A., Zimmerman, R. and Rubin, E. (1986) Proc. Natl. Acad. Sci. USA 83, 9398-9402). The time course for re-acquisition of membrane tolerance by liver microsomes following ethanol withdrawal and resumption of ethanol feeding correlated with the ability of PI to confer tolerance.