Basis for phospholipid incorporation into peripheral nerve myelin.

To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23-24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P0 (Golgi) and myelin basic proteins (more direct).

Barbiturates inhibit intracellular Ca2+ rise induced by thrombin in rat platelets.

The effects of a number of barbiturates (anesthetic as well as anticonvulsant) on thrombin-induced calcium mobilization were tested in rat platelets using the fluorescent Ca2+ probe Fura-2. All drugs, except barbituric acid and Na-barbital, inhibited the thrombin-induced intracellular Ca2+ rise. Both the uptake of extracellular Ca2+ and the release of calcium from intracellular organelles were affected but influx was inhibited more strongly and at lower concentrations of the drugs (e.g. IC50 of thiopental was 0.83 mM for influx and 1.2 mM for intracellular release). Inhibitory potencies of the various barbiturates were markedly different. Thiopental was the most and barbital the least potent inhibitor. The order of inhibitory potency of the drugs appeared generally to follow their lipid solubility and the order of their hypnotic efficiency, with hexobarbital as the most conspicuous exception. Therefore, barbiturate treatment of cells perturbs agonist-induced calcium mobilization. This effect may be partially linked to their previously reported inhibitory action on two kinases, protein kinase C and phosphatidylinositol 4-phosphate kinase [1, 2].

Lipid activators of protein kinase C.

Among the many reported lipid activators of protein kinase C only those of high affinity can be considered true physiological effectors, at present the tumor promoters, e.g., phorbol esters; 1,2-diacyl-sn-glycerols; and phosphatidylinositol 4,5-bisphosphate. Many other compounds (including arachidonic acid) are activators at high, unphysiological concentrations only, and they seem to be sterically unsuited for bonding to the enzyme. Such pseudo-activators possibly act by scrambling the structure of the regulatory moiety of the kinase.

Effect of barbiturates on polyphosphoinositide biosynthesis and protein kinase C activity in synaptosomes.

Previously, it has been found that phenobarbital inhibited protein kinase C (PKC) and the enzymes of the metabolism of polyphosphoinositide, especially phosphatidylinositol 4-phosphate (PIP) kinase (PIP-kinase). As a continuation of these studies, a number of barbiturates (barbituric acid, barbital, butabarbital, pentobarbital, amobarbital, phenobarbital, secobarbital and hexobarbital) were tested for inhibition of these enzymes and also of phosphatidylinositol (PI) kinase (PI-kinase), in a synaptosomal preparation at pH 7.8 from the brain of rat. All compounds, except barbituric acid (and Na-barbital for PI-kinase) inhibited the three kinases. However, PKC was approximately 3-5 fold more sensitive to inhibition by the drugs (measured by Ki values) than PIP-kinase, which was 2- to 4-fold more sensitive than PI-kinase. The inhibitory potency of the drugs increased with their lipophilicity, although to a lesser degree than expected from the differences in partition coefficients; the largest deviation from a positive correlation (i.e. hexobarbital) may be the result of the blockade of an imide (-NH) group at one position of the barbituric ring. Concentrations of drugs (after correction for the greater than normal ionization (pH 7.8) of the drugs in the assays) necessary for half-maximal inhibition were well within, or smaller than, those reported necessary for in vitro blocking of nerves. The possibility, therefore, exists that the physiological effects of the barbiturates are, in part, the result of an inhibition of protein kinase C and PIP-kinase.

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C.

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.

Studies on the submicrosomal fractions of bovine oligodendroglia: lipid composition and glycolipid biosynthesis.

Oligodendroglia were isolated from bovine brain, and a "crude" microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+ plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.

Effect of phenobarbital on metabolism of polyphosphoinositides of rat brain synaptosomes.

Synthesis and degradation of polyphosphoinositides in a rat brain synaptosome preparation were depressed by phenobarbital. Phosphatidylinositol-4-phosphate kinase (PIP-kinase), the enzyme which synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2) was most strongly affected (50% inhibition at 3 mM phenobarbital); phosphatidylinositol (PI-kinase) followed (50% at 15 mM). The phosphoesterases were less sensitive: PIP-monoesterase (50% at 39 mM), PIP2-monoesterase (at 47 mM), and, least inhibited, PIP-diesterase (50% at 65 mM) and PIP2-diesterase (at 68 mM). Phenobarbital by inhibiting PIP-kinase may reduce the membrane concentration of PIP2 and thus dampen the stimulus-response which leads to the hydrolysis of PIP2 and the formation of the second messenger, inositol-1,4,5-trisphosphate (IP3), involved in mobilization of intracellular Ca2+.

Concentrations of vitamin E in various neuroanatomical regions and subcellular fractions, and the uptake of vitamin E by specific areas, of rat brain.

Vitamin E concentrations were determined by high-performance liquid chromatography in different anatomical regions of the brain from 3-month-old Fischer 344 rats. Gray matter from cerebellum and cervical spinal cord contained the lowest concentrations, while gray matter from the frontal cortex and thalamus had the highest concentrations of vitamin E. Radioactive alpha-tocopherol injected intravenously into the rat was readily taken up by brain although the level of uptake was very low compared with the liver. The ratios of brain-to-serum radioactivities ranged from 0.011 to 0.016 depending upon the brain region. Cerebellar gray matter is characterized by a low concentration of unlabeled alpha-tocopherol and a high level of uptake of radioactive alpha-tocopherol and thus is particularly active in the metabolism of vitamin E. Concentrations of unlabeled alpha-tocopherol were highest in microsomal and mitochondrial fractions and were the lowest in cytosol and nuclear fractions.

Mutual stimulation by phosphatidylinositol-4-phosphate and myelin basic protein of their phosphorylation by the kinases solubilized from rat brain myelin.

Myelin basic protein and phosphatidylinositol-4-phosphate are phosphorylated in vitro by ATP and solubilized rat brain myelin. When both substrates are present together, the rate of phosphorylation of each is increased about eight-fold. It appears likely that the phosphate turnover of myelin basic protein and of phosphatidylinositol-4-phosphate are coupled in vivo.

Polyphosphoinositide mono- and diphosphoesterases of three subfractions of rat brain myelin.

Phosphomonoesterase and diesterase that cleave phosphatidylinositol-4-phosphate (diphosphoinositide, DPI) and phosphatidylinositol-4,5-bisphosphate (triphosphoinositide, TPI) were detected in three subfractions of purified rat brain myelin, and some properties of the enzymes were studied. Monoesterase activity was stimulated by KCl, maximally at a concentration of 25 mM, and inhibited at KCl concentrations above 50 mM. Addition of boiled pH 5 supernatant of rat brain homogenate doubled the enzymic activity; EDTA was inhibitory. The specific activities were nearly equal in the "low density", "medium density", and "heavy density" myelin fractions but about 30% lower than in whole brain homogenate. The monophosphatase could be solubilized by extraction with 0.2% Triton X-100. The phosphodiesterase activity was inhibited by EDTA and EGTA and not stimulated by KCl or pH 5 supernatant. Specific activities were nearly equal in whole brain and myelin but were by about 60 percent elevated in the "heavy density" over the "low density" myelin fractions. These results show that hydrolases operative in the fast turnover of the inositide phosphate groups are distributed over the entire myelin structure.

Axonal transport of phosphatidylcholine and two synthetic analogs.

Sonicated emulsions of egg phosphatidylcholine containing either [(14)C]-dipalmitoyl phosphatidylcholine (diester-PC) or two metabolically inert analogs. [ (14)C]-1- octadecyl -2- hexadecyl -sn- glycero -3- phosphocholine (diether-PC) and [(3)H]-2-tetradecyloctadecano-(1)-phosphocholine (dialkyl-PC) were injected into the vitreous of the eye of adult rabbits. After 1-40 days, radioactivities were measured in the stations of the optical pathway, and the identities of the labelled lipids arrived at the superior colliculus were ascertained by thin-layer chromatography and treatment with phospholipase A(2), with the following results: (1) phosphatidylcholine and its analogs were taken up from the vitreous by the retina at similar rates: (2) all three lipids were transported in the optic nerve axons at similar rates ('fast'). They reached maximal concentration in the superior colliculus some 20 days after injection: (3) phosphatidylcholine travelled from vitreous to superior colliculus as the intact molecule: (4) maximal accumulation of the two analogs in the superior colliculus reached only about 1 per cent of that of phosphatidylcholine. The results suggest that the vehicles of fast axonal transport can pick up intact phospholipid molecules originating in the ganglionic cell plasma membrane (and, likely, from other cellular compartments). The packaging process is promoted by the presence of carboxyl ester groups in the phospholipid; this fact suggests the involvement of ganglionic phospholipid transfer protein with specificity for these groups.

Rapid incorporation in vivo of intracerebrally injected 32Pi into polyphosphoinositides of three subfractions of rat brain myelin.

At intervals ranging from 1 to 10 min after injection of 32Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphosphoinositides (PPI) was then determined. There was rapid incorporation of 32Pi into PPI, which contained 50-70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated 32Pi into total recovered PPI in the order of medium greater than heavy greater than light fraction; however, the order of relative specific radioactivities was heavy greater than light greater than medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phosphatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di- (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged 32Pi rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.

Toxicity and neuronal transport of stable liposomes and phospholipid in the nervous system.

When unilamellar "stable" liposomes composed of a dialkyl analog of phosphatidylcholine, tetradecyloctadec-11-eno(1)phosphocholine (dialkyl-PC), plus cholesterol at 1:1 molar ratio, and a trace of [3H]dialkyl-PC were injected into the vitreous of the rabbit eye, macrophage infiltration and phagocytosis of lipid were observed in retina including the epiretinal myelinated nerve fiber bundles, with no other neurotoxic effects. Little or no incorporation of [3h]dialkyl-PC was observed in the distal tissues of the optic system. With "labile" vesicles composed of egg lecithin, trace amounts of [3H]dialkyl-PC, and phosphatidic acid, no morphological changes occurred. After a lag of more than 7 days [3H]dialkyl-PC appeared in superior colliculus, indicating axonal transport of the lipid in an anterograde direction. Experiments with submandibular and parotid gland indicated retrograde transport of the lipid. The data do not suggest axonal transport of intact (stable) liposomes, but suggest that intact phospholipid molecules can be axonally transported.

Retention of a dialkylphosphatidylcholine in myelin and other membranes.

We describe an attempt to incorporate a metabolically inert phospholipid analog into animal membranes, especially myelin, in vivo, with the view of eventual long-term membrane modification or membrane engineering. A sonicated suspension of a mixture of [14C]phosphatidylcholine and its dialkyl analog, [3H] tetradecyloctadecano(1)phosphocholine, was injected into the brain of weanling rats. Samples were counted of whole brain, myelin, liver, and carcass, at intervals from 1 to 63 days, and the composition of the extracted lipid was determined by thin-layer chromatography. Both lipid labels were found to be cleared from the body at similar rates, but while phosphatidylcholine was metabolized within a day, with the label appearing mainly in the phosphatidylethanolamine fraction and in nonpolar lipids, the dialkylphosphatidylcholine remained intact, with retention in myelin of a small but almost constant amount for a month. Ways will have to be found to enhance uptake of the lipids by the brain.

Distribution of phosphoinositides among subfractions of rat brain myelin.

Rat brain myelin was separated into three subfractions, heavy, medium, and light, and the concentrations of phosphatidic acids (PA), phosphatidylinositol (PI), di- (DPI), and triphosphoinositide (TPI) in these fractions were determined. PI was evenly distributed among the fractions, and PA, DPI, and TPI occurred in highest concentrations in the "light" myelin. This result indicates that these fast metabolizing lipids play an important role in the tightly packed central lamellae of the myelin sheath.

Isolation and characterization of an enriched Golgi fraction from rat brain.

A fraction rich in membranes of the Golgi apparatus was isolated from rat brain by discontinuous density gradient centrifugation. The fraction sedimented at the characteristic Golgi density of 1.11--1.15 (g/cm3, 5 degrees C) and had specific activities of Golgi-marker enzymes (N-acetyllactosaminyl synthetase, glycoprotein (Fetuin) galactosyltransferase, thiamine pyrophosphatase), 6--7 times over those of the original homogenates. The recovery of the enzyme activities in this fraction ranged from 17 to 31 %. The incorporation [3H]fucose into glycoproteins was 3-fold higher than in homogenate. Recovery and relative specific activities of marker enzymes for other subcellular organelles were low. Electron microscopic analysis of the fraction revealed in the presence of Golgi structures, namely, large sacs or plates with attached tubules and "blebbing" of the tubules into the vesicles.

A rapid and sensitive radiochemical assay for phosphatidic acid phosphohydrolase activity.

A technique is described for the radiochemical assay of phosphatidic acid phosphohydrolase activity in rat brain. Radiochemically pure 32P-labeled phosphatidic acid of known specific radioactivity and structure, which was biosynthesized in vitro by the diacylglycerol kinase of E. coli, was used as the substrate. As little as 5 microgram of microsomal or mitochondrial protein can be used for the assay, and product formation in the picomole range can be determined accurately. This procedure should be useful in situations where only limited amounts of tissue are available.