The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.

The presence of lipoproteins, apolipoproteins, and their receptors in the brain could provide a system for cholesterol homeostasis, as they do in other tissues. This study was undertaken to determine whether plasma low-density lipoprotein, the major carrier of cholesterol, is involved in the delivery of lipids through the blood-brain barrier. 125I-Labeled low-density lipoprotein bound to a specific receptor on the endothelium of brain capillaries when it was injected immediately postmortem into bovine brain circulation. In contrast, no specific binding of 125I-low density lipoprotein was found when the incubations were performed with isolated capillaries. Incubations of endothelial or basement membranes of brain capillaries with 125I-low density lipoprotein demonstrated a high-affinity association of low-density lipoprotein with the membranes of bovine cerebral endothelial cells. The specificity of the low-density lipoprotein binding was determined in several ways using a dot blot assay. This receptor shows the same characteristics as the low-density lipoprotein receptor on human fibroblasts. The molecular weight of the bovine brain capillary low-density lipoprotein receptor (132,000) was determined by ligand blotting. These results demonstrated the occurrence of a low-density lipoprotein receptor on the endothelial cells of brain capillaries.

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The presence of large amounts of long chain-polyunsaturated fatty acids (PUFA) in the brain implies an exogenous intake of unsaturated fatty acids, either as essential fatty acids, or in the form of higher homologues resulting from hepatic metabolism. To determine the influence of the diet upon the potential availability of polyunsaturated fatty acids to the brain, four different diets were used with comparable amounts of 18:2 n-6, but variable amounts of 18:3 n-3 (0.2, 1, 2 and 9%). These diets were administered to female rats from the day of mating and during the periods of gestation and lactation. Fifteen days after birth suckling animals were killed and the fatty acid distribution was studied in the serum in two lipoprotein classes (VLDL-LDL and HDL). On the whole, an increase in dietary 18:3 n-3 resulted in an increase of polyunsaturated fatty acids of the n-3 series and a decrease in fatty acids of the n-6 series. The modification chiefly concerned the terminal fatty acids in each series (22:5 n-6 and 22:6 n-3). It is noteworthy that the influence of exogenous 18:3 n-3 upon the 20:4 n-6 content of lipoproteins was not significant below 2% of 18:3 n-3 intake, a level that we have previously shown to be both necessary and sufficient to satisfy the requirements of the brain for fatty acids of the n-3 series. In the liver, the intermediary metabolism ensures an important release of long-chain polyunsaturated fatty acids, which may help to satisfy the lipid requirements of the brain.

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In isolated human platelets, exposure of subtraction 3 high-density lipoprotein (HDL,) binding sites to high concentrations of HDL, (1 mg/mL) causes rapid desensitization of HDL3 (50 jU,g/mL)-stimulated breakdown of phosphatidylcholine, as shown in approximately a 70% depression of the maximal 1,2-diacylglycerol release activity by phospholipase C. This desensitization is HDL, dose dependent (IC 50 , 150±20 ig/mL, n=6) and time dependent (t 1/2 , <30 seconds). It requires the binding of HDL,, as pretreatment of HDL, by tetranitromethane does not cause the desensitization of HDLj-induced phospholipase C activity. Permeabilization of human platelets with 10 /ig/mL digitonin, used to permit access of charged inhibitors to the cytosol, does not interfere with the pattern of HDLj (1 mg/mL) -induced desensitization of HDL, (50 /ig/mL)-stimulated phospholipase C. Inhibitors of protein kinase C (100 /umol/L H-7 and 10 /nmol/L staurosporine) markedly inhibit desensitization of HDL,-induced P revious studies have demonstrated that subtraction 3 high-density lipoprotein (HDL,) binding sites are present on platelet membranes. 13 Koller et al 3 have reported that the glycoprotein Ilb/IIIa complex, the inducible platelet fibrinogen receptor, is able to interact with lipoproteins. HDL3 was found to bind to either the 95-to 110-kD glycoprotein Ilia or to the 136-to 140-kD glycoprotein lib. Glycoprotein lib/ Ilia acts until now as a platelet HDL, binding site.Furthermore, HDL 3 binding sites are coupled to phospholipase C (PLC) through pertussis toxin-sensitive GTP binding proteins. 45 In human platelets, HDL 3 binding sites act via the PLC-mediated hydrolysis of phosphatidylcholine and the generation of 1,2-diacylglycerol, which activates the multifunctional enzyme protein kinase C (PKC). The HDL 3 -induced signaling pathway is recorded only when platelets are stimulated by low concentrations of HDL 3 (50 ixglmL). Higher HDL3 concentrations (1 mg/mL) are not able to trigger phosphatidylcholine hydrolysis, indicating a loss of responsiveness. 4 One major form of regulation of the activity of G protein-coupled receptors is desensitization, 6 -9 which is a general biological phenomenon whereby the response to a specific ligand wanes over time despite the continuous presence of the ligand. The impairment of receptor functions is linked to protein phosphorylation, which appears to be a key factor. Most often, homologous desensitization is accompanied by receptor phosphorylation, which triggers the process of functional uncoupling from G proteins. 1012 In addition, a rapid sequestration of the receptors away from the cell surface and a modulation of the expression of the receptor gene itself result in a net decrease in receptor number. 13 Whereas desensitization of the adrenergic system is rather well understood, less is known about receptors coupled to phosphatidylinositol or phosphatidylcholine turnover. 1417 Moreover, until now little has been known about HDL3 binding site regulation. Platelets therefore would seem to ...

Tlzeromyzon tessulatum vitellin was identified as a lipoglycoprotein of 490 kDa. The insolubility of this molecule in low-ionic-strength media was used to extract it from the ovaries. Antiserum prepared against vitellin was shown to react with a coelomic fluid component of 520 kDa. This vitellin precursor, or vitellogenin, was purified by gel permeation and ion-exchange column chromatography. These two lipoglycoproteins were characterized by amino acid, carbohydrate and lipid analysis and subunit composition. In spite of differences in terms of native molecular mass, solubility and isoelectric point, the lipoglycoproteins isolated from the coelomic fluid and the ovary were similar in their subunit components (a single polypeptide of 165 kDa) and in their amino acid and carbohydrate compositions. However, vitellogenin was found to be more highly lipidated (31.8% by mass) than vitellin (24% by mass) and lipid analysis indicated a higher amount of sterols and phospholipids in vitellogenin. From these data, we conclude that vitellogenin and vitellin are probably dimers of two identical subunit polypeptides plus lipid and that, after vitellogenin is sequestered in the oocyte, part of its lipid component is stripped from the molecule to give vitellin. Furthermore, electrophoretic analysis seems to indicate that vitellogenin synthesis and secretion is initiated following the third and last blood meal of the animal but that vitellogenin significantly accumulates in the coelomic fluid before being incorporated in the oocytes suggesting a complex mode of vitellogenesis regulation.The production of egg yolk involves a massive synthesis of specific proteins which is under hormonal control. These proteins, termed vitellogenins, are generally synthesized externally to the site of accumulation. Depending upon the species, vitellogenins are secreted in the bloodstream or in the hemolymph, taken up by the developing oocytes and deposited as vitellins, the major yolk proteins [l, 21. Thus, in addition to its physiological importance for reproduction, vitellogenesis also provides a good system for the study of molecular mechanisms and hormonal regulation of egg maturation.Yolk proteins have been extensively investigated in oviparous vertebrates [I] and in insects [3, 41. In annelids, oocyte vitellin has been purified from nereid polychaetes [5, 61 and it has been shown that oocyte differentiation depends upon the incorporation of vitellogenin [7, 81. On the other hand, little is known for the two groups of clitellates: oligochaetes [9, 101 and hirudinae [I I]. In clitellates, eggs are generally of a small size and laid within a cocoon secreted by the clitellum and abundantly provided with reserve substances from which the embryo will develop, therefore making an accumulation of

The conversion of phosphatidylethanolamine (PE) into phosphatidylcholine (PC) by a sequence of three transmethylation reactions is shown to be stimulated by the apolipoprotein E-free subclass of high-density lipoprotein (HDL3) in isolated bovine brain capillary (BBC) membranes, HDL3-induced stimulation of BBC membranes pulsed with [methyl-14C]methionine causes a transient increase in each methylated phospholipid, i.e. phosphatidyl-N-monomethylethanolamine (PMME), phosphatidyl-NN-dimethylethanolamine (PDME) and PC. PC substrate arising from the activation of PE N-methyltransferase (PEMT) is hydrolysed by a phospholipase A2 (PLA2), as demonstrated by the accumulation of lysophosphatidylcholine (lyso-PC). When PE containing [14C]arachidonic acid in the sn-2 position ([14C]PAPE) is incorporated into BBC membranes, HDL3 stimulation induces the formation of PMME, PDME, PC and lyso-PC and the release of [14C]arachidonic acid, which correlates with the previous production of lyso-PC, suggesting that HDL3 stimulates a PLA2 that can release polyunsaturated fatty acids (PUFA). Both PEMT and PLA2 activities depend on a HDL3 concentration in the range 0-50 micrograms/ml and are strictly dependent on HDL3 binding, because HDL3 modified by tetranitromethane is no longer able to bind to specific receptors and to trigger PEMT and PLA2 activation. Moreover, HDL3 prelabelled with [14C]PAPE can stimulate PDME and lyso-PC synthesis in BBC membranes in the presence of S-adenosylmethionine, suggesting that HDL3 can supply BBC membranes in polyunsaturated PE and can activate enzymes involved in PE N-methylation and PUFA release. The results support the hypothesis of a close relationship between HDL3 binding, PE methylation and PUFA release, and suggest that the PC pool arising from PE could be used as a pathway for the supply of PUFA to the brain.