Ceramides modulate cell-surface acetylcholine receptor levels.

The effects of ceramides (Cer) on the trafficking of the nicotinic acetylcholine receptor (AChR) to the plasma membrane were studied in CHO-K1/A5 cells, a clonal cell line that heterologously expresses the adult murine form of the receptor. When cells were incubated with short- (C6-Cer) or long- (brain-Cer) chain Cer at low concentrations, an increase in the number of cell-surface AChRs was observed concomitant with a decrease in intracellular receptor levels. The alteration in AChR distribution by low Cer treatment does not appear to be a general mechanism since the surface expression of the green fluorescent protein derivative of the vesicular stomatitis virus protein (VSVG-GFP) was not affected. High Cer concentrations caused the opposite effects, decreasing the number of cell-surface AChRs, which exhibited higher affinity for [125I]-alpha-bungarotoxin, and increasing the intracellular pool, which colocalized with trans-Golgi/TGN specific markers. The generation of endogenous Cer by sphingomyelinase treatment also decreased cell-surface AChR levels. These effects do not involve protein kinase C zeta or protein phosphatase 2A activation. Taken together, the results indicate that Cer modulate trafficking of AChRs to and stability at the cell surface.

Lipid composition of purified transverse tubule membranes isolated from amphibian skeletal muscle.

The level and proportion of lipids and their fatty acid composition were analyzed in highly purified transverse tubule membranes of amphibian skeletal muscle. Tubule membranes show (a) a higher content of lipids, (b) a higher phospholipid/cholesterol ratio and (c) a different phospholipid composition from other subcellular fractions, such as the light and heavy membranes from sarcoplasmic reticulum, which are similar in lipid profile. Transverse tubule membranes are characterized by a high percentage of phosphatidylserine and sphingomyelin and a low proportion of phosphatidylcholine compared with the other membranes. All three show a high proportion of ethanolamine plasmalogens (50% of the total ethanolamine glycerophospholipid). Transverse tubule membrane lipids contain a high proportion of 20- and 22-carbon polyunsaturated fatty acids, predominantly 20:4, 20:5, 22:5 and 22:6. Arachidonate predominates in phosphatidylinositol, eicosapentaenoate and docosahexaenoate in ethanolamine and serine glycerophospholipids.

Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor.

The effects of metabolic inhibition of cholesterol biosynthesis on the trafficking of the nicotinic acetylcholine receptor (AChR) to the cell membrane were studied in living CHO-K1/A5, a Chinese hamster ovary clonal line that heterologously expresses adult alpha2betadeltaepsilon mouse AChR. To this end, we submitted CHO-K1/A5 cells to long-term cholesterol deprivation, elicited by Mevinolin, a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase and applied a combination of biochemical, pharmacological and fluorescence microscopy techniques to follow the fate of the AChR. When CHO-K1/A5 cells were grown for 48 h in lipid-deficient medium supplemented with 0.5 microM Mevinolin, total cholesterol was significantly reduced (40%). Concomitantly, the maximum number of binding sites (Bmax) of the cell-surface AChR for the competitive antagonist alpha-bungarotoxin was reduced from 647+/-30 to 352+/-34 fmol/mg protein, i.e. by 46%. The apparent dissociation constant (Kdapp) for alpha-bungarotoxin of the AChRs remaining at the cell surface was not modified by cholesterol depletion. Similarly, the half-concentration inhibiting the specific binding of the radioligand (IC50) for another competitive antagonist, d-tubocurarine, did not differ from that in control cells. The decrease in cell-surface AChR was paralleled by an increase in intracellular AChR levels, which rose from 44+/-2.1% in control cells to 74+/-3.3% in Mevinolin-treated cells. When analyzed by wide-field fluorescence microscopy, the fluorescence signal arising from alpha-bungarotoxin labeled cell-surface AChRs was reduced by approximately 70% in Mevinolin-treated cells. The distribution of intracellular AChR also changed: Alexa594-alpha-bungarotoxin-labeled AChR exhibited a highly compartmentalized pattern, concentrating at the perinuclear and Golgi-like regions. Temperature-arrest of protein trafficking magnified this effect, emphasizing the Golgi localization of the AChR. Colocalization studies using the transiently expressed fluorescent trans-Golgi/trans-Golgi network marker pEYFP/human beta1,4-galactosyltransferase and the trans-Golgi network marker syntaxin 6 provided additional support for the Golgi localization of intracellular AChRs. The low AChR cell-surface expression and the increase in intracellular AChR pools in cholesterol-depleted cells raise the possibility that cholesterol participates in the trafficking of the receptor protein to the plasmalemma and its stability at this surface location.

Myogenic differentiation of the muscle clonal cell line BC3H-1 is accompanied by changes in its lipid composition.

Phospholipid and neutral lipid composition was studied in the course of myogenic differentiation of the clonal cell line BC3H-1. Total phospholipid content increased during differentiation, predominantly in the major classes of choline and ethanolamine glycerophospholipids. The contents of other lipids, such as triacylglycerols, diminished more than 50% during this period. The content and distribution of fatty acids also underwent marked differentiation-dependent changes. The polyunsaturated (tetrapenta- and hexaenoic) fatty acid species of several phospholipid classes diminished during differentiation, especially those in choline, serine and inositol glycerophospholipids. Most noticeable were the changes in phosphatidylserine; long-chain fatty acids having 20 to 22 carbon atoms and 4 to 6 double bonds decreased from about 30 to about 10 mol%. Although increased levels of saturation in other phospholipid fatty acyl chains appear to accompany the myogenic changes of BC3H-1 cells, some unsaturated fatty acids, such as oleic acid (18:1), increased by as much as 80% during the same period, suggesting the activation of a delta 9 desaturase. Sphingomyelin contained only saturated and monoenoic fatty acids and exhibited a four- to five-fold decrease in its content of monoenoic acyl groups. Diacylglycerols became enriched in arachidonate and docosahexaenoate. The amount of cholesterol and its esters increased slightly during differentiation of BC3H-1 cells. The data show that several metabolic pathways change during myogenic differentiation of the BC3H-1 clonal cell line, particularly de novo biosynthetic pathways, elongation/desaturation reactions, and acyl chain turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Boundary lipids in the nicotinic acetylcholine receptor microenvironment.

The structural and functional properties of the nicotinic acetylcholine receptor (AChR), the archetype molecule in the superfamily of Cys-looped ligand-gated ion channels, are strongly dependent on the lipids in the vicinal microenvironment. The influence on receptor properties is mainly exerted by the AChR-vicinal ("shell" or "annular") lipids, which occur in the liquid-ordered phase as opposed to the more disordered and "fluid" bulk membrane lipids. Fluorescence studies from our laboratory have identified discrete sites for fatty acids, phospholipids, and cholesterol on the AChR protein, and electron-spin resonance spectroscopy has enabled the establishment of the stoichiometry and selectivity of the shell lipid for the AChR and the disclosure of lipid sites in the AChR transmembrane region. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids. I surmise that the outermost ring of M4 helices constitutes the boundary interface, most suitable to convey the signals from the lipid microenvironment to the rest of the transmembrane region, and to the channel inner ring in particular.

Brain asymmetry in phospholipid polar head group metabolism: parallel in vivo and in vitro studies.

Phospholipid content and 32P-incorporation have been studied in individual rat cerebral hemispheres. The total phospholipid content was 44.9 +/- 0.9 and 47.9 +/- 1.3 mumol lipid P/100 mg protein for the right and left hemispheres respectively. Individually, only sphingomyelin was significantly (about 30%) higher in the left hemisphere. Metabolic experiments have been conducted in vivo using i.p. injection of 32P and following its incorporation into total and individual phospholipids in each cerebral hemisphere. Higher incorporations were attained by phosphatidate and phosphatidylinositol-4,5-bisphosphate (PIP2) in the left cerebral hemisphere than in the right. In an attempt to determine whether phospholipid metabolism is also lateralized in specific subcellular compartments related with the neurotransmission process, we have studied in vitro the [32P] incorporation into phosphoglycerides of synaptosomal fractions obtained from each cerebral cortex. The precursor was taken up differently by the two cerebral cortex preparations, resulting in different profiles of distribution among lipids. In addition, the kinetics of lipid labeling showed higher rates of 32P-incorporation in fractions derived from the left cerebral cortex, mainly in PIP and PIP2. These results are interpreted to indicate that several enzymes involved in lipid metabolism are modulated to a different extent in the two hemispheres.

Phospholipid metabolism under muscarinic cholinergic stimulation exhibits brain asymmetry.

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

Down-regulation of brain muscarinic cholinergic receptor promoted by diacylglycerols and phorbol ester.

Sustained agonist stimulation induces an asymmetric down-regulation of brain muscarinic acetylcholine receptor (mAChR): 43 +/- 2% in the right and 26 +/- 2% in the left cerebral hemisphere, respectively (Ref. 1). In order to determine the possible involvement of endogenous diacylglycerols produced under muscarinic stimulation in the down-regulation phenomenon, here we have studied the effects of synthetic diacylglycerols and a phorbol ester on cells dissociated from rat cerebral cortex. Oleylacetylglycerol decreased the amount of cell-surface mAChR by 37 +/- 2% and 25 +/- 2% in right and left cerebral cortex, respectively. Long-term treatment with phorbol dibutyrate also produced internalization of the mAChR (25 +/- 1.5% and 33 +/- 2% in right and left cortical cells, respectively). These changes occurred without modification of the Kdapp for the selective antagonist pirenzepine. The action of calcium ions was also studied using incubation of cells with the ionophore A23187. No changes were observed in the amount of mAChR detected at the plasma membrane with the ionophore alone, but when used in combination with phorbol dibutyrate and the agonist carbamylcholine a sinergistic decrease in mAChR was apparent. It is concluded that long-term exposure to exogenously added diacyglycerols and phorbol ester significantly reduces the amount of mAChR detected at the plasma membrane and abolishes the asymmetry of the down-regulation phenomenon observed under specific muscarinic stimulation, suggesting that diacylglycerols may be one of the factors responsible for such asymmetry.

Free fatty acid content and release kinetics as manifestations of cerebral lateralization in mouse brain.

Free fatty acid (FFA) content was analyzed in mouse cerebral hemispheres and cerebellum under basal and postdecapitative ischemic conditions. Total FFA content immediately after decapitation (2 s) was about two-fold higher in the left hemisphere than in the right. Marked dissimilarities between hemispheres were also apparent when FFA levels were measured during short periods of ischemia. Whereas in the right side a significant FFA release took place as early as 10 s, no accumulation was detected in the left in the 2-20 s interval. The highest rates of total fatty acid release occurred in the 20-30 s interval in both hemispheres and decreased afterwards (3 min). Individual FFA, especially stearate and arachidonate, differed in their rates of production, the right cerebral hemisphere being more active in releasing arachidonic acid. In cerebellum, FFA levels were lower and accumulation was slower than in cerebrum in both intervals. When subjected to 3 min ischemia, the same difference in FFA levels between right and left hemispheres (50%) was observed in heads kept at 20 or 30 degrees C. The differences between hemispheres are interpreted as manifestations of an inherent lateralization in the regulation of acylation-deacylation reactions of complex lipids.

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain.

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.

Reduced labeling of brain phosphatidylinositol, triacylglycerols, and diacylglycerols by [1-14C]arachidonic acid after electroconvulsive shock: potentiation of the effect by adrenergic drugs and comparison with palmitic acid labeling.

The effect of electroconvulsive shock on the labeling of phospholipids and neutral lipids in mice brains was examined after intracerebral injection of [1-14C] arachidonic acid or [1-14C]palmitic acid. Electroconvulsive shock reduced greatly the removal of radiolabeled arachidonic acid from the free fatty acid pool. At the same time, the incorporation of arachidonic acid was partially inhibited in triacylglycerol, diacylglycerol, and phosphatidylinositol, whereas the incorporation of [1-14C]palmitic acid was not affected. Pretreatment with desipramine and pargyline potentiated the lipid effect of electroconvulsive shock in neutral glycerides. These electroconvulsive shock-induced changes reflect alterations in the metabolism of intracerebrally injected arachidonic acid, but not of similarly injected palmitic acid. From the available data whether decreased ATP, enzyme inhibition or other factors are involved cannot be ascertained. Moreover, the electroconvulsive shock-enhanced endogenous free arachidonic acid may possibly dilute the injected radiolabeled fatty acid, thus decreasing its availability for arachidonoyl-coenzyme A synthesis. Hence, a partial inhibition of the activation-acylation of these fatty acids, primarily arachidonic acid, also may be involved in the seizure-induced accumulation of free fatty acids in the brain.

Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex.

The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [3H]quinuclidinyl benzilate (QNB) and [3H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [3H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M1 and M2) detected with [3H]Pz. Under basal conditions the RCC had roughly 50% more M1 subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [3H]Pz, i.e. the M1 subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.

Diffusion of intracerebrally injected [1-14C]arachidonic acid and [2-3H]glycerol in the mouse brain. Effects of ischemia and electroconvulsive shock.

[2-3H]Glycerol and [1-14C]arachidonic acid were injected into the region of the frontal horn of the left ventricle of mice and were distributed rapidly throughout the brain. After 10 sec, most of the radioactive fatty acid was found in the hemisphere near the injection site; after 10 min, it was recovered in similar proportions in the cerebellum and brain stem. [2-3H]Glycerol showed a heterogeneous distribution, with most of the label remaining in the left hemisphere even after 10 min. On a fresh weight basis, cerebrum, cerebellum, and brain stem were found to contain similar amounts of labeled glycerol. However, the amount of [1-14C]arachidonate in cerebrum was only 50% of that recovered from cerebellum or brain stem. Brain ischemia or a single electroconvulsive shock reduced the spread of the label, producing an accumulation of radioactivity in the injected hemisphere, except for an increase in [2-3H]glycerol in the brain stem during ischemia. Despite the significant decrease in available precursor in the cerebellum and brain stem after electroshock, the amount of label incorporated into lipids was not altered in these areas and only slightly diminished in the cerebrum.

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor.

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.