Effect of neuraminidase treatment on the topological distribution of phospholipids in chick brain microsomes.

The desialylation of chick brain microsomal membranes affects the transbilayer distribution of phospholipids. When intact microsomes were treated with neuraminidase, less phosphatidylcholine and sphingomyelin could be hydrolysed with phospholipase C under experimental conditions which allowed the hydrolysis of the phospholipids of the external leaflet only. In contrast, the accessibility of phosphatidylethanolamine and phosphatidylserine to the external probes (trinitrobenzene sulfonic acid or phospholipase C) was not affected. After neuraminidase treatment of a microsomal fraction, less phosphatidylcholine, newly synthesized through the cytidine pathway, could be hydrolysed by phospholipase C, whereas the reaction of newly synthesized phosphatidylethanolamine molecules with trinitrobenzene sulfonic acid was not affected. The results suggest that in biological membranes some choline phospholipid molecules may interact with the sialyl residue of sialocompounds. This interaction may contribute to the maintenance of phospholipid asymmetry in brain membranes.

Axon-myelin transfer of phospholipids and phospholipid precursors. Labeling of myelin phosphoinositides through axonal transport.

Previous studies have provided evidence for axon-to-myelin transfer of intact lipids and lipid precursors for reutilization by myelin enzymes. Several of the lipid constituents of myelin showed significant contralateral/ipsilateral ratios of incorporated radioactivity, indicative of axonal origin, whereas proteins and certain other lipids did not participate in this transfer-reutilization process. The present study will examine the labeling of myelin phosphoinositides by this pathway. Both 32PO4 and [3H]inositol were injected monocularly into 7-9-wk-old rabbits and myelin was isolated 7 or 21 days later from pooled optic tracts and superior colliculi. In total lipids 32P counts of the isolated myelin samples showed significant contralateral/ipsilateral ratios as well as increasing magnitude of contralateral-ipsilateral differences during the time interval. Thin-layer chromatographic isolation of the myelin phosphoinositides revealed significant 32P-labeling of these species, with PIP and PIP2 showing time-related increases. This resembled the labeling pattern of the major phospholipids from rabbit optic system myelin in a previous study and suggested incorporation of axon-derived phosphate by myelin-associated enzymes. The 32P label in PI, on the other hand, remained constant between 7 and 21 days, suggesting transfer of intact lipid. This was supported by the labeling pattern with [3H]inositol, which also showed no increase over time for PI. These results suggest axon-myelin transfer of intact PI followed by myelin-localized incorporation of axon-derived phosphate groups into PIP and PIP2. The general topic of axon-myelin transfer of phospholipids and phospholipid precursors is reviewed.

Phosphoinositide breakdown in isolated myelin is stimulated by GTP analogues and calcium.

Purified myelin from rat brainstem, prelabeled in vivo by intracerebral injection of [3H]myoinositol, showed enhanced breakdown of phosphoinositides on treatment with 5'-guanylylimidodiphosphate [Gpp-(NH)p] and Ca2+. Concentration variation of the former in the presence of Ca2+ showed a dose-dependent release of inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3), while inositol 1-phosphate (IP) release was erratic. Concentration-dependent release of IP2 and IP3 was also observed with Ca2+ as the variable in the presence of Gpp(NH)p. Carbachol, when present, did not enhance the stimulatory effect of Gpp(NH)p alone. Addition of diphosphoglycerate during incubation enhanced IP3 at the expense of IP2, suggesting the presence of IP3 phosphatase in myelin.

Detection of G proteins in purified bovine brain myelin.

Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 +/- 9 vs. 271 +/- 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5'-adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [alpha-32P]GTP bound to three bands in the 21-27-kDa range in a manner inhibited by GTP and GTP gamma S but not App(NH)p. ADP-ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholate extract of myelin subjected to chromatography on a column of phenyl-Sepharose gave at least three major peaks of [35S]GTP gamma S binding activity. SDS-PAGE and immunoblot analyses of peak I indicated the presence of Go alpha, Gi alpha, and Gs alpha. Further fractionation of peak II by diethyl-aminoethyl-Sephacel chromatography gave one [35S]GTP gamma S binding peak with the low-molecular-mass (21-27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of CDP-choline on hypocapnic neurons in culture.

Neuronal cultures from chick embryo cerebral hemispheres were protected against a hypocapnic injury by adding to their growth medium 10(-6)M CDP-choline before or after the injury. The protection obtained with CDP-choline was analyzed by a morphometric analysis and showed that pretreatment of neuronal cultures with CDP-choline maintained the number of cell aggregates and of primary neuronal processes at control values after hypocapnic shock. Various experiments showed that the intact molecule was responsible for the protective action, since pretreatment with different concentrations of various nucleosides and nucleotides (up to 10(-5) M), choline, and phosphorylcholine was without protective effect. The addition of CDP-choline after the hypocapnic injury resulted in a protection of the cultures as shown by morphological observation. Incubation of neurons with radioactive choline showed that hypocapnia increased the incorporation of the label into phospholipids whereas the presence of CDP-choline reduced it. The de novo synthesis of choline was affected by neither hypocapnia nor CDP-choline treatment. The results indicate that CDP-choline may have the capacity to protect neurons under conditions of basic pH and that cellular proliferation may be stimulated by the compound.

Topological biosynthesis of phosphatidylcholine in brain microsomes.

The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base-exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP-[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base-exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.