Deuterium NMR studies of cerebroside-phospholipid bilayers

2H-NMR was used to probe the interaction of non-hydroxy fatty acid cerebroside and 2-hydroxy fatty acid cerebroside with the polar head group and with the acyl chains of dipalmitoylphosphatidylcholine in unsonicated bilayers. It is shown that the interior of the bilayer exhibits uniformly increasing orientational order as the concentration of both types of cerebroside increases, whereas the surface of the bilayer, as reflected by the head group motion, becomes disordered. The extent of the disorder at the surface is dependent upon the type and concentration of the cerebroside. These results are discussed in terms of hydrogen-bonding interactions.     

Factors affecting surface expression of glycolipids: influence of lipid environment and ceramide composition on antibody recognition of cerebroside sulfate in liposomes

The reactivity of the acidic glycolipid cerebroside sulfate (CBS) with antibody was studied as a function of its lipid environment in vesicles and of its ceramide composition. The lipid environment was varied by using phosphatidylcholine of varying chain length with cholesterol in a phosphatidylcholine:cholesterol:cerebroside sulfate molar ratio to glycolipid of 1:0.75:0.1. The ceramide structure of CBS was varied by using synthetic forms containing palmitic acid, lignoceric acid, or the corresponding alpha-hydroxy fatty acids. Reactivity with antibody was determined by measuring complement-mediated lysis of the vesicles containing a spin-label marker, tempocholine chloride. The data were analyzed by a theoretical model which gives relative values for the dissociation constant and concentration of antibodies within the antiserum which are able to bind to the glycolipid. If the phosphatidylcholine chain length was increased, increasing the bilayer thickness, only a small population of high-affinity antibodies were able to bind to cerebroside sulfate, suggesting decreased surface exposure of the glycosyl head group. A larger population of lower affinity antibodies were able to bind to it in a shorter chain length phosphatidylcholine environment. However, if the chain length of the cerebroside sulfate was increased, it could be recognized by more antibodies of lower affinity than the short chain length form, suggesting that an increase in chain length of the glycolipid increased surface exposure. Hydroxylation of the fatty acid inhibited antibody binding; only a smaller population of higher affinity antibodies was able to bind to the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

A C-NMR study of 10,12-tricosadiynoic acid and the corresponding phospholipid and phospholipid polymer

Diacetylene phospholipids are presently being studied because of their potential to polymerise in vesicles, multilayers and natural biomembranes. ¹³C-NMR spectra and spin-lattice relaxation times have now been obtained of a diacetylene phospholipid present in a sonicated dispersion in water. Similar data have been obtained of a monoacetylene phospholipid and a saturated phospholipid. For further comparison the spectrum of a diacetylenic fatty acid in benzene-d₆ was also examined and relaxation data obtained. A comparison of the various relaxation data provides an indication of the restricted motion associated with the two conjugated triple bonds of the diacetylene phospholipid within the lipid bilayer structure. A proximity interaction between diacetylene groups occurs and a conformation for the diacetylene part of the lipid in the bilayer is deduced. The ¹³C-NMR spectrum of a soluble phospholipid polymer in C²HCl₃, obtained by ultraviolet irradiation of the diacetylene phospholipid, shows that the two conjugated triple bonds of the monomer is replaced in the polymer by an alternating double and triple bonded conjugated structure.     

2H and 13C nuclear magnetic resonance study of N-palmitoylgalactosylsphingosine (cerebroside)/cholesterol bilayers.

13C- and 2H-NMR experiments were used to examine the phase behavior and dynamic structures of N-palmitoylgalactosylsphingosine (NPGS) (cerebroside) and cholesterol (CHOL) in binary mixtures. 13C spectra of 13C=O-labeled and 2H spectra of [7,7-2H2] chain-labeled NPGS as well as 3 alpha-2H1 CHOL indicate that cerebroside and CHOL are immiscible in binary mixtures at temperatures less than 40 degrees C. In contrast, at 40 degrees C < t < or = T(C) (NPGS), up to 50 mol% CHOL can be incorporated into melted cerebroside bilayers. In addition, 13C and 2H spectra of melted NPGS/CHOL bilayers show a temperature and cholesterol concentration dependence. An analysis of spectra obtained from the melted 13C=O NPGS bilayer phase suggests that the planar NH-C=O group assumes an orientation tilted 40 degrees-55 degrees down from the bilayer interface. The similarity between the orientation of the amide group relative to the bilayer interface in melted bilayers and in the crystal structure of cerebroside suggests that the overall crystallographic conformation of cerebroside is preserved to a large degree in hydrated bilayers. Variation of temperature from 73 degrees to 86 degrees C and CHOL concentration from 0 to 51 mol% results in small changes in this general orientation of the amide group. 2H spectra of chain-labeled NPGS and labeled CHOL in NPGS/CHOL bilayer demonstrate that molecular exchange between the gel and liquid-gel (LG) phases is slow on the 2H time scale, and this facilitates the simulation of the two component 2H spectra of [7,7-2H2]NPGS/CHOL mixtures. Simulation parameters are used to quantitate the fractions of gel and LG cerebroside. The quadrupole splitting of [7,7-2H2]NPGS/CHOL mixtures and 2H simulations allows the LG phase bilayer fraction to be characterized as an equimolar mixture of cerebroside and CHOL.     

Bile formation and hepatic plasma membrane composition in guinea-pigs and rats

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl⁻, phosphate and Ca²⁺ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na⁺,K⁺ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs. Total fatty acid analysis of the membrane revealed that palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2, n-6) were the predominant fatty acids in guinea-pigs, while palmitic acid (C16:0), stearic acid (C18:0) and arachidonic acid (C20:4, n-6) were the most important in rats. Thus, high bile flow rate and BAIF in the guinea-pig may be attributed to the low bile acid concentration (below the critical micellar concentration), secretion of hypercholeretic bile acids (e.g. ketolithocholate) and high bicarbonate output.     

Molecular topography of the Ca2+ — ATPase of sarcoplasmic reticulum

Summary The Ca²⁺ binding site region of the Ca²⁺ — ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca²⁺ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca²⁺ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I^– indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca²⁺ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca²⁺ binding is shown to be near the membrane surface.     

Carbon-13 nuclear magnetic resonance studies of cerebroside derivatives and their properties in lecithin bilayers (1)

The ¹³C NMR chemical shifts and spin-lattice relaxation times of D-galactosylsphingosine derivatives in CDCl₃-CD₃OD and in egg-yolk lecithin vesicles in D₂O, and of N-acetylpsychosine micelles, are reported. Results with sonicated, unilamellar vesicles containing cerebroside and EYL^a show that (1) cerebrosides decrease the fluidity of the lecithin bilayer membrane and have the greatest effect on the glycerol backbone and choline methyl carbons. (2) N-acetylpsychosine experiences a greater freedom of motion in the galactose region than does cerebroside and does not reduce the fluidity of the lecithin as much as cerebroside. (3) Ac-Psy/EYL vesicles formed are permeable to Yb³⁺ but cerebroside/lecithin vesicles are not. (4) The choline groups on the inner bilayer surface are less mobile than those on the outer surface according to preliminary T₁ measurements of the Yb³⁺-separated resonances. (5) Yb³⁺-induced chemical shifts of choline methyl and choline $\text{C}\text{H}{}_2$OP peaks in mixed cerebroside-lecithin vesicle systems indicate a small preference for cerebroside in the outside monolayer. The data show that these molecules have significant effects on bilayer conformational mobilities, particularly near the surface, and thus demonstrate one mechanism for modulation of cell surface properties by glycosphingolipids.     

The fidelity of response by nitroxide spin probes to changes in membrane organization: The condensing effect of cholesterol

The response of doxyl fatty acid spin probes in egg lecithin bilayers to added cholesterol is compared with results from ²H-NMR. Large differences are found between the profiles of order parameter vs. label position and cholesterol concentration.At constant cholesterol content, the ESR spin probe order parameter decreases continuously as the label position is moved toward the terminal methyl region of the bilayer whereas an order parameter ‘plateau’ is observed for the upper region of the bilayer by ²H-NMR. In addition, the spin probe order parameters are smaller than those observed by ²H-NMR.Differences are also observed in the profiles of order parameter vs. cholesterol content for each label position. The spin probes detect a maximal response to added cholesterol for the central portion of the chains with much weaker responses near both ends of the chains. In contrast, the ²H-NMR results indicate a large, approximately constant response for the first ten positions in the chains with a decreasing response toward the terminal methyl group. For all the positions examined, the spin probes show a weaker response than that observed by ²H-NMR.A direct measure of the perturbing effect of a spin label is made by comparing the deuterium quadrupole splittings in egg lecithin-cholesterol bilayers for stearic acid with and without an attached doxyl moiety. The spin-labelled fatty acid has a much reduced quadrupole splitting and an opposite response to cholesterol addition.     

Effect of fatty acid chain length,fatty acid hydroxylation,and various cations on phase behavior of synthetic cerebroside sulfate

Synthetic species of CBS containing palmitic, stearic, lignoceric, D-2-hydroxy palmitic, or D-2-hydroxy stearic acid were prepared and their phase behavior in the presence of a number of mono- and divalent cations was studied by differential scanning calorimetry and the use of fatty acid spin labels. The results showed that both the non-hydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) forms of cerebroside sulfate (CBS) can occur in two different gel states, a metastable state and a lower entropy stable state. The phase behavior is more sensitive to the type and concentration of cation present than in the case with acidic phospholipids. The sensitivity of the transition temperature (T_m) to cation concentration reflects, in part, increased participation of the lipid in intermolecular hydrogen bonding interactions as the negative charge of the sulfate is shielded. The extra hydroxyl group on the HFA also contributes to the intermolecular hydrogen bonding network causing a significant increase in the T_m.The HFA has an even more significant effect in causing inhibition of formation of the stable state. Formation of the stable state is also inhibited by Li⁺ and divalent cations. A similar mechanism may be involved, i.e.; cross-linking of adjacent lipids or increased intermolecular interactions inhibit the molecular rearrangement necessary to form the stable state. This inhibition is counteracted by an increase in fatty acid chain length. The results suggest that the stable state may be interdigitated as a result of the unequal chain length between the sphingosine base and the fatty acid.     

Effects of copper and cadmium ions on the physicochemical properties of lipids of the marine bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IB28 at different growth temperatures

The phospholipid and fatty acid composition and thermotropic behavior of total lipids were studied in the metal-accumulating marine strain Pseudomonas putida IB28 grown in the presence of Cu²⁺ and Cd²⁺ at 4 and 24°C. Despite the changes in acidic lipid content, unsaturated/saturated fatty acid ratio, and cyclopropane fatty acid level, the temperature range of calorimetric phase transitions of bacterial total lipids was slightly altered under these factors. The suppressive action of heavy metals on bacterial growth is attributable to the phase separation of lipids and, as a consequence, to a sharp increase in the ion permeability of the lipid bilayer. The increase in acidic phospholipid level under the influence of Cu²⁺ and Cd²⁺, especially at 24°C, is likely to be indicative of their complexation with heavy metal ions.     

A P-NMR study of structural and functional aspects of phosphate and phosphonate distribution in Tetrahymena

³¹P-NMR has been used to study the chemical nature of cytoplasmic components of live Tetrahymena in a non-invasive manner. The technique has further been used to characterize the physical behaviour of lipids extracted from this organism. In particular, we have shown the presence of large quantities of pyrophosphate and of tripolyphosphate in acid extracts of the organism. These are not detectable in the live cell due to the motionally rigid nature of the storage granules. We have characterized the distribution of phosphonic acids in the organism and followed the phase behaviour of the extracted cell lipids. Aqueous dispersions of extracted lipid show both bilayer and non-bilayer behaviour in the range of the growth temperature. The phosphonolipid in Tetrahymena appears to play a role similar to that of phosphatidylethanolamine in regulating the phase behaviour of the membrane. The high degree of unsaturation in the fatty acids of Tetrahymena is most likely responsible for the polymorphic phase behaviour observed near the growth temperature.     

1H NMR Ganglioside Ceramide Resonance Region on the Differential Diagnosis of Low and High Malignancy of Brain Gliomas

1. The high-resolution ¹H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3–5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 2). The broad resonance was identified as ceramide, a sphingosine–fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3–5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal.2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity.3. The properties of tumor gangliosides include antigenic specificity and immunosuppresive activity and the ceramide, a sphingosine–fatty acid combination, noticeably influences the ganglioside immunosuppressive activity.4. The observation of ¹H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberrant gangliosides and ceramide precursors on the grade of malignancy and invasiveness.5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.     

Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from ²H-hyperfine interaction with D₂O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D₂O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D₂O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D₂O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D₂O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.     

Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from ²H-hyperfine interaction with D₂O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D₂O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D₂O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D₂O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D₂O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.     

High resolution proton magnetic resonance of sonicated phospholipids

We have recorded high resolution proton magnetic resonance spectra of sonicated phospholipid vesicles. The following lipids were used in separate experiments: phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine from egg yolk as well as dimyristoyl phosphatidylcholine. Mixed lipid vesicles were also investigated. Assignments of the peaks associated with the various protons of the different lipids are presented. It is shown that in favorable cases, it is possible to resolve the different phospholipid head groups of mixed lipid samples. Spin lattice relaxation times (T₁) of each peak were collected at 500 MHz and 90 MHz. The influence of the addition of a small concentration of spin labeled phospholipid on i) the linewidths ii) the spin lattice relaxation times, was determined. It is shown that nitroxide radicals selectively broaden the peaks associated with the protons localized at a comparable depth of the bilayer. On the other hand, T₁ are less selectively perturbed. Potential applicability of ¹H-NMR for the investigation of lipid-proton specificity in membranes is discussed.     

Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids,sterols and carotenoids

One- and two-dimensional¹H- and¹³C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by¹H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.author for correspondence     

Aberrant Ca(2+) signalling through acidic calcium stores in pancreatic acinar cells

Pancreatic acinar cells possess a very large Ca²⁺ store in the endoplasmic reticulum, but also have extensive acidic Ca²⁺ stores. Whereas the endoplasmic reticulum is principally located in the baso-lateral part of the cells, although with extensions into the granular area, the acidic stores are exclusively present in the apical part. The two types of stores can be differentiated pharmacologically because the endoplasmic reticulum accumulates Ca²⁺ via SERCA pumps, whereas the acidic pools require functional vacuolar H⁺ pumps in order to maintain a high intra-organellar Ca²⁺ concentration. The human disease acute pancreatitis is initiated by trypsinogen activation in the apical pole and this is mostly due to either complications arising from gall bladder stones or excessive alcohol consumption. Attention has therefore been focussed on assessing the acute effects of bile acids as well as alcohol metabolites. The evidence accumulated so far indicates that bile acids and fatty acid ethyl esters - the non-oxidative products of alcohol and fatty acids - exert their pathological effects primarily by excessive Ca²⁺ release from the acidic stores. This occurs by opening of the very same release channels that are also responsible for normal stimulus-secretion coupling, namely inositol trisphosphate and ryanodine receptors. The inositol trisphosphate receptors are of particular importance and the results of gene deletion experiments indicate that the fatty acid ethyl esters mainly utilize sub-types 2 and 3.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Arachidonic acid inhibits capacitative Ca2+ entry and activates non-capacitative Ca2+ entry in cultured astrocytes

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca²⁺ entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La³⁺; (ii) AA (10 μM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and γ-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca²⁺ influx, which is not inhibited by 2-APB or La³⁺, indicating that AA activates a second Ca²⁺ entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca]_i overload is to cause delayed cell death in astrocytes.     

Effect of fatty acids and monoglycerides on permeability of lipid bilayer

The effect of fatty acids and monoglycerides on barrier properties of liposomal membranes prepared from egg phosphatidylcholine was investigated. The incorporation of these lipids as liposomal membrane components induced the alteration of the permeability to less permeable liposomally entrapped drugs, sulfanilic acid and procainamide ethobromide (PAEB). Monoolein caused greatly increased permeability of both drugs and unsaturated fatty acids markedly enhanced the release rate of PAEB, while saturated fatty acids caused a small increase in the release rate.Electron spin resonance (ESR) investigation with 5-nitroxide stearic acid showed that fatty acids disordered the hydrophobic region of the lipid bilayer and the disordering effect of unsaturated fatty acids was greater than that of saturated ones. It was demonstrated that the incorporated fatty acids and monoglycerides interacted with the polar region of the membranes by ESR study with cholestane label and ¹H-NMR study. These results indicated that the increase in the membrane permeability caused by fatty acids and monoglycerides associated with the disorder in the membranes' interior and the interaction of the incorporated lipid with the polar head group of phospholipid.