Comparison Between Folic Acid and gH625 Peptide-Based Functionalization of Fe3O4 Magnetic Nanoparticles for Enhanced Cell Internalization.

A versatile synthetic route based on magnetic Fe3O4 nanoparticle (MNP) prefunctionalization with a phosphonic acid monolayer has been used to covalently bind the gH625 peptide on the nanoparticle surface. gH625 is a membranotropic peptide capable of easily crossing the membranes of various cells including the typical human blood-brain barrier components. A similar synthetic route was used to prepare another class of MNPs having a functional coating based on PEG, rhodamine, and folic acid, a well-known target molecule, to compare the performance of the two cell-penetrating systems (i.e., gH625 and folic acid). Our results demonstrate that the uptake of gH625-decorated MNPs in immortalized human brain microvascular endothelial cells after 24 h is more evident compared to folic acid-functionalized MNPs as evidenced by confocal laser scanning microscopy. On the other hand, both functionalized systems proved capable of being internalized in a brain tumor cell line (i.e., glioblastoma A-172). These findings indicate that the functionalization of MNPs with gH625 improves their endothelial cell internalization, suggesting a viable strategy in designing functional nanostructures capable of first crossing the BBB and, then, of reaching specific tumor brain cells.

Blood-Brain Barrier in a Haemophilus influenzae Type a In Vitro Infection: Role of Adenosine Receptors A2A and A2B.

The blood-brain barrier (BBB) is mainly made up of tightly connected microvascular endothelial cells (BMECs), surrounded by pericytes (BMPCs) which regulate BBB tightness by providing soluble factors that control endothelial proliferation. Haemophilus influenzae type a (Hia) is able to reach the BBB, crossing it, thus causing meningitis. In this study, by using an in vitro model of BBB, performed with human BMECs and human BMPCs in co-culture, we demonstrated that, after Hia infection, the number of hBMPCs decreased whereas the number of hBMECs increased in comparison with non-infected cells. SEM and TEM images showed that Hia was able to enter hBMECs and reduce TEER and VE-cadherin expression. When the cells were infected in presence of SCH58261 and PSB603 but not DPCPX, an increase in TEER values was observed thus demonstrating that A2A and A2B adenosine receptors play a key role in BBB dysfunction. These results were confirmed by the use of adenosine receptor agonists CGS21680, CCPA, and NECA. In infected co-cultures cAMP and VEGF increased and TEER reduction was counter-balanced by VEGF-R1 or VEGF-R2 antibodies. Moreover, the phosphorylated CREB and Rho-A significantly increased in infected hBMECs and hBMPCs and the presence of SCH58261 and PSB603 significantly abrogated the phosphorylation. In conclusion, this study demonstrated that the infection stimulated A2A and A2B adenosine receptors in hBMECs and hBMPCs thus inducing the pericytes to release large amounts of VEGF. The latter could be responsible for both, pericyte detachment and endothelial cell proliferation, thus provoking BBB impairment.

Ferritin-supported lipid bilayers for triggering the endothelial cell response.

Hybrid nanoassemblies of ferritin and silica-supported lipid bilayers (ferritin-SLBs) have been prepared and tested for the adhesion, spreading and proliferation of retinal microvascular endothelial cells (ECs). Lipid membranes with varying surface charge were obtained by mixing cationic 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) with zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at increasing POPC/POEPC ratios. The supported bilayer formation and their subsequent interaction processes with ferritin were studied at the pH of 7.4 at different protein concentrations, by using the quartz crystal microbalance with dissipation monitoring and by atomic force microscopy. Both kinetics and viscoelastic parameters of the protein-lipid membrane interface were scrutinized, as well as surface coverage. Phase-contrast optical microscopy analyses of the ferritin-SLBs substrates after their interaction with endothelial cells evidenced the highest cell adhesion (2-4h of incubation time) and proliferation (from 24h to 5 days) for the membranes of POPC/POEPC (75:25 ratio). Moreover, ferritin increased both cell adhesion and proliferation in comparison to control glass (respectively 1.5- and 1.75-fold) as well as proliferation in comparison to bare POPC/POEPC (95:5 ratio) (2 fold). Results are very promising in the goal of modulating the endothelial cell response through the interplay of viscoelastic/charge properties of the solid-supported membranes and the SLB-conditioned ferritin activity.

Role of cytosolic and calcium independent phospholipases A(2) in insulin secretion impairment of INS-1E cells infected by S. aureus.

Cytosolic PLA2 (cPLA2) and Ca(2+)-independent PLA2 (iPLA2) play a significant role in insulin ß-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

Asthenozoospermia and membrane remodeling enzymes: a new role for phospholipase A2.

Phosholipase A2 (PLA2 ) activity in the seminal plasma and in sperm heads is closely related to sperm motility and male fertility. Therefore, the purpose of this study was to investigate the possible involvement of different isoforms of phospholipase in asthenozoospermia. To accomplish this, cPLA2 , phospho-cPLA2 , iPLA2 , and sPLA2 were evaluated by immunofluorescence and immunoblot analyses in spermatozoa obtained from 22 normozoospermic men and 28 asthenozoospermic patients. We found significant differences in cPLA2 and its phosphorylated/activated form, iPLA2 , and sPLA2 content and distribution in normal and asthenozoospermic patients. cPLA2 was localized in heads, midpieces, and tails of all spermatozoa as constitutive enzyme, less expressed in the tail of spermatozoa with low progressive motility. While active phospho-cPLA2 distribution was homogeneous throughout the cell body of control-donor spermatozoa, lower levels were detected in the tails of asthenozoospermic patients, as opposed to its strong presence in heads. Low immunofluorescence signal for iPLA2 was found in astenozoospermic patients, whereas sPLA2 was significantly lower in the heads of asthenozoospermic patients. Spermatozoa with low progressive motility showed differences both in terms of total specific activity and of intracellular distribution. cPLA2 , iPLA2 , and sPLA2 specific activities correlated positively and in a significantly manner with sperm progressive motility both in normozoospermic men and asthenozoospermic patients. In conclusion, PLA2 s are expressed in different areas of human spermatozoa. Spermatozoa with low motility showed differences in total specific activity and enzyme distributions. We speculated that PLA2 expression and/or different distribution could be potential biomarkers of asthenozoospermia, one of the major causes of male factor infertility.

Klebsiella pneumoniae induces an inflammatory response in an in vitro model of blood-retinal barrier.

Klebsiella pneumoniae has become an important pathogen in recent years. Although most cases of K. pneumoniae endogenous endophthalmitis occur via hematogenous spread, it is not yet clear which microbial and host factors are responsible for the ability of K. pneumoniae to cross the blood-retinal barrier (BRB). In the present study, we show that in an in vitro model of BRB based on coculturing primary bovine retinal endothelial cells (BREC) and primary bovine retinal pericytes (BRPC), K. pneumoniae infection determines changes of transendothelial electrical resistance (TEER) and permeability to sodium fluorescein. In the coculture model, bacteria are able to stimulate the enzyme activities of endothelial cytosolic and Ca(2+)-independent phospholipase A2s (cPLA2 and iPLA2). These results were confirmed by the incremental expression of cPLA2, iPLA2, cyclo-oxygenase-1 (COX1), and COX2 in BREC, as well as by cPLA2 phosphorylation. In supernatants of K. pneumoniae-stimulated cocultures, increases in prostaglandin E2 (PGE2), interleukin-6 (IL-6), IL-8, and vascular endothelial growth factor (VEGF) production were found. Incubation with K. pneumoniae in the presence of arachidonoyl trifluoromethyl ketone (AACOCF3) or bromoenol lactone (BEL) caused decreased PGE2 and VEGF release. Scanning electron microscopy and transmission electron microscopy images of BREC and BRPC showed adhesion of K. pneumoniae to the cells, but no invasion occurred. K. pneumoniae infection also produced reductions in pericyte numbers; transfection of BREC cocultured with BRPC and of human retinal endothelial cells (HREC) cocultured with human retinal pericytes (HRPC) with small interfering RNAs (siRNAs) targeted to cPLA2 and iPLA2 restored the pericyte numbers and the TEER and permeability values. Our results show the proinflammatory effect of K. pneumoniae on BREC, suggest a possible mechanism by which BREC and BRPC react to the K. pneumoniae infection, and may provide physicians and patients with new ways of fighting blinding diseases.

High glucose and advanced glycation end products induce phospholipid hydrolysis and phospholipid enzyme inhibition in bovine retinal pericytes.

In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20-50 mM glucose or 2-20 microM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1-(14)C]arachidonate and [Me-(3)H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1-(14)C]arachidonate and [Me-(3)H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A(2) (cPLA(2)), acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A(2) (cPLA(2)) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 microM AGEs. Simultaneously, [Me-(3)H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.

Amyloid beta(1-42) and its beta(25-35) fragment induce in vitro phosphatidylcholine hydrolysis in bovine retina capillary pericytes.

We describe the inhibitory effect of full-length Abeta(1-42) and Abeta(25-35) fragment of amyloid-beta peptide on phosphatidylcholine (PtdCho) metabolism in bovine retina capillary pericytes. Cell cultures were incubated with Abetas for 24 h. Peroxidation indices (malondialdehyde and lactate dehydrogenase release) significantly increased after 20-50 microM Abeta(1-42) or Abeta(25-35) treatment. In addition, [Me-3H]choline incorporation into PtdCho strongly decreased while either 3H-choline or 14C-arachidonic acid release from prelabeled cells increased, indicating PtdCho hydrolysis. The effect was very likely due to prooxidant action of both Abeta peptides. Reversed-sequence Abeta(35-25) peptide did not depress 3H-choline incorporation nor stimulate PtdCho breakdown. With addition of Abetas at low concentrations (2-20 microM) to pericytes, marked ultrastructural changes, well connected to metabolic alterations, emerged including shrinkage of cell bodies, retraction of processes, disruption of the intracellular actin network. Cells treated with higher concentrations (50-200 microM) displayed characteristics of necrotic cell death. The data suggest that: (a) Abeta(1-42) and Abeta(25-35) peptides may modulate phospholipid turnover in microvessel pericytes; (b) together with endothelial cells, pericytes could be the target of vascular damage during processes involving amyloid accumulation.

t-Butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes.

Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25-400 microM), native LDL or oxLDL (3.4-340 nmol hydroperoxides/mg protein, 1-100 micro). LPS (1 microg/ml), t-BuOOH (200 microM), and oxLDL (100 microM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF(3), a potent cPLA2 inhibitor, and the antioxidant alpha-tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 microM) or oxLDL (100 microM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 microM t-BuOOH or 100 microM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA2 mediates AA liberation; (iii) as long-term regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries.

Amyloid beta but not bradykinin induces phosphatidylcholine hydrolysis in immortalized rat brain endothelial cells.

We describe the inhibitory effect of A beta (25-35) fragment of amyloid-beta peptide and bradykinin (BK) on phosphatidylcholine (PtdCho) metabolism in immortalized rat brain GP8.39 endothelial cells (EC). Cultures were incubated either with A beta for 24-48 h, or with BK for 30 min-4 h. The peroxidation indices (malondialdehyde, conjugated dienes) and lactate dehydrogenase (LDH) release significantly increased after A beta peptide (10-50 microM) treatment. The BK (10 microM) stimulation of cells brought about an increase in conjugated dienes and LDH release only after 4 h. Following 24 h treatment with 50 microM A beta peptide, the [Me-3H]choline incorporation into PtdCho strongly decreased while the [3H]choline release increased, indicating PtdCho hydrolysis. The effect was most likely due to peptide prooxidant effect. After 4 h preincubation with BK, the [Me-3H]choline incorporation into PtdCho strongly decreased, but no significant [3H]choline release was found. Following long-term treatment, the action of 50 microM A beta on [3H]choline release was not enhanced by 10 microM BK. Cell exposure to alpha-tocopherol (1 mM) prior to the addition of both agents did not abolish stimulated PtdCho breakdown. The data suggest that: (a) A beta peptide and BK may modulate phospholipid turnover in microvessel cells; (b) they could not synergistically interact in vascular EC damage during processes involving amyloid accumulation and inflammatory response.

Arachidonate transport through the blood-retina and blood-brain barrier of the rat after reperfusion of varying duration following complete cerebral ischemia.

The permeability-surface area product (PS) of [1-14C]arachidonate at the blood-retina and blood-brain barrier was determined by short carotid perfusion in young Wistar rats 1 or 6 h after recovery period following complete cerebral ischemia induced by temporary cardiac arrest. For the retina and structures of visual system, hypothalamus and olfactory bulb there was no significant difference over sham-operated rats among mean PSs. For cortex, hippocampus and striatum, significant increases were found at both time intervals of recovery after cardiac arrest. The ischemia-reperfusion model was characterized by a significant increase in tissue conjugated diene in the hippocampus and microsomal lysophosphatidylcholine acyltransferase activity in the cortex. Consistent with these findings, we also show ultrastructural evidence mainly represented by partial opening of interendothelial junctions and mild signs of tissue edema in surrounding neuropil, suggesting barrier leakiness predominantly in the cortex, hippocampus and striatum but almost absent in the retina microvessels. Our results indicate that ischemia-reperfusion does affect influex through blood-brain barrier into regional structures of rat central nervous system of arachidonate, a metabolic substrate and lipid mediator rapidly incorporated into microcapillary and brain lipids. The data also suggested that: (i) reactive oxyradicals were moderately generated during the early phase of ischemic-reperfusion process in the rat; (ii) after reperfusion, in vitro susceptibility of different brain regions to iron-induced peroxidation was highest in the hippocampus and lowest in the cortex and striatum; (iii) membrane phospholipid repair mechanisms were activated at the same time.

Phosphatidylcholine synthesis-related enzyme activities of bovine brain microvessels exhibit susceptibility to peroxidation.

In microvessels isolated from bovine brain, microsomal enzyme activities involved in phosphatidylcholine biosynthesis and degradation were determined. The microvessels possessed acyl-CoA:1-acyl-sn-glycero-3-phosphocholine (AT) and glycerophosphocholine phosphodiesterase (GroPChoPDE) activity at a higher level compared with bovine and rat brain or rat liver microsomes whereas they expressed CTP:phosphocholine cytidylyltransferase (CT) and choline phosphotransferase (CPT) activity at a lower level. Each enzyme has been characterized in terms of response to inhibitors or activators revealing properties very similar to those in brain and liver microsomes. In the homogenate prepared from t-butylhydroperoxide-treated microvessels (10 min exposure to 10 microM up to 1 mM concentrations), AT and CPT activities exhibited a significant dose-dependent inhibition. In contrast, GroPChoPDE activity was unaffected. CT was inhibited only at 1 mM concentration. Short treatment of microvessels with Fe2+ (20 microM)-ascorbate (0.25 mM) or 100 microM linoleate hydroperoxide did not have any effect on the activity of the four enzymes. Strong inhibition of all enzymes was noted when the linoleate hydroperoxide system was fortified by Fe2+ ions (100 microM). AT inactivation was also found when oxidized low density lipoprotein was preincubated with microvessels. On the other hand, oxidized LDL left unchanged CPT and GroPChoPDE activities whereas it promoted a slight stimulation of cytidylyltransferase activity. Overall, the results suggest a link between oxygen radical generation and the perturbation of the microvessel membrane structure in which the four enzymes are incorporated, coupled to a direct sulfhydryl protein modification.

CTP: phosphocholine cytidylyltransferase is both a nuclear and cytoplasmic protein in primary hepatocytes.

CTP:phosphocholine cytidylyltransferase (CT) has recently been reported to be a predominantly intranuclear enzyme in several cell lines (Wang et al., J. Biol. Chem. 268, 5899-5904 (1993)). This contrasts with previous reports that CT was a cytosolic protein that translocated to the endoplasmic reticulum upon activation. The aim of the present study was to compare the localization of CT in CHO cells and in primary rat hepatocytes. Indirect immunofluorescence of CHO cells revealed a largely nuclear localization of the CT. On the other hand, immunogold electron microscopy and biochemical studies showed a similar density of distribution of CT between the nucleus and cytoplasm. In primary rat hepatocytes immunofluorescence studies indicated that CT was largely cytoplasmic. Studies by immunogold electron microscopy of rat hepatocytes demonstrated that the enzyme was homogeneously distributed throughout all cytoplasmic regions and the nucleoplasm. This result was confirmed by biochemical studies using digitonin and streptolysin O, which permeabilizes the plasma membrane of cells. Enucleation studies indicated that in CHO cells 76% of the CT activity was in the nuclear fraction, whereas in hepatocytes only 32% was recovered in this fraction. The data indicate that CT is found both in nuclear and cytoplasmic fractions of primary hepatocytes and is not predominantly a nuclear enzyme.

Evolutionary comparison of enzyme activities of phosphatidylcholine metabolism in the nervous system of an invertebrate (Loligo pealei), lower vertebrate (Mustelus canis) and the rat.

While steady-state kinetic parameters (metabolite pools, Km and activation energies) are partially known for the enzymes involved in phosphatidylcholine synthesis and degradation in mammalian brain, they are not available for the nervous system of lower vertebrates or invertebrates. Since the extent of evolutionary development of an enzyme is not known a priori, we evaluated the kinetic and thermodynamic parameters of choline kinase, CTP:phosphocholine cytidylyltransferase, choline phosphotransferase and glycerophosphorylcholine phosphodiesterase in squid (Loligo pealei) optic lobe, dogfish (Mustelus canis) and rat brain. For all these enzyme activities, basic similarities in Km and inhibitor effect were found. The same was true for the activation energies Ea, with the exception of squid choline kinase and dogfish cytidylyltransferase. Treatment of microsomal membranes with phospholipase C sharply inhibited cytidylyltransferase activity in all three animal species. In dogfish brain, glycerophosphorylcholine phosphodiesterase activity was undetectable. Our results are consistent with the notion that the kinetic properties of the enzyme activities leading to the preservation of the phosphatidylcholine membranous pool may have appeared early in metazoan evolution and been fully conserved in mammals.

Lipid peroxidation inhibits acyl-CoA:-1-acyl-sn-glycero-3-phosphocholine O-acyltransferase but not CTP: phosphocholine cytidylyltransferase activity in rat brain membranes.

In brain tissue in vivo peroxidized according to three model systems, we determined two microsomal enzyme activities involved in phospholipid biosynthesis. The first, short-term model, was based on the i.v. administration to normal rats, twice a day, for a period of 1 week, of a sonicated emulsion of a peroxidized mixture of phospholipids and linoleate (4:1, w/w; 500 mg/day; hydroperoxides: 200-250 nmol/mg lipid). The half-life time of the injected toxic lipid species in the blood circulation was about 1 h. At the end of the week's treatment, brain and liver malondialdehyde, conjugated diene and lipid hydroperoxide levels were significantly higher in treated rats than in the controls. The second model consisted of the acute injection of aqueous Fe2+ solution (50 mM) into lateral ventricles, and the collection of brain tissue 2 h later. The third model was based on two consecutive injections of hydroperoxylinoleate (1 mg each) into lateral ventricles over a period of 18 h, and the collection of brain tissue 2 h after the second administration. In brain microsomal membranes prepared from peroxide- or iron-treated rats, lysophosphatidylcholine acyltransferase activity exhibited a significant inhibition. On the contrary, in microsomal preparations derived from the short-term model, CTP:phosphocholine cytidylyltransferase activity was slightly stimulated. Intraventricular injection of linoleate or linoleic acid hydroperoxide left this enzyme activity unchanged. The effect of in vitro membrane peroxidation on both microsomal enzyme activities was investigated. By using an Fe2+ (20 microM)-ascorbate (0.25 mM) peroxidation system, the residual acyltransferase and cytidylyltransferase activities were 80 and 72% of initial activity respectively. Significant dose-dependent inactivation of acyltransferase (maximum loss of 45% of initial activity) was seen when 0.1-10 mumol of photooxidized phospholipids were preincubated with 100 micrograms of microsomal membranes. Unoxidized or photooxidized phospholipids (1 mM) promoted a slight stimulation of cytidylyltransferase activity. Altogether, the results suggest a link between oxygen radical generation and the perturbation of the membrane structure in which the enzymes are located.

1-Acyl-2-lysophosphatidylcholine transport across the blood-retina and blood-brain barrier.

The transport of lysophospholipids through the rat blood-retina and blood-brain barrier was determined by using radioactive 1-palmitoyl-2-lysophosphatidylcholine (Pam-lysoPtdCho) and by measuring the uptake of this labeled compound into the retina and various brain regions after short in situ carotid perfusion. The transport was not affected by probenecid (0.25 mM), but it was inhibited, in a dose-dependent manner, by circulating albumin which is able to bind tightly to lysophosphatidylcholine and lowered the availability of the latter for tissue extraction. Radiotracer transfer in the retina was higher than in brain regions. The permeability-surface area products (PS) changed with the inclusion of unlabeled Pam-lysoPtdCho, showing that transport across retinal and brain microvessels is mainly saturable. The data provided an estimate of transport constants (Vmax, Km and non-saturable constant Kd). However, we could not distinguish whether this saturable process represents the saturation of a transport carrier or simple passive diffusion followed by the saturation of enzymatic reactions. In brain tissue lipid extract, 20 s after carotid injection, radiolabel was associated by 45% to unmetabolized Pam-lysoPtdCho. Partial acylation to phosphatidylcholine, as well as hydrolysis and redistribution of the fatty acyl moiety into main phospholipid classes also occurred. The present results, compared to our previous data, indicate that PamlysoPtdCho is transported faster and/or in greater amounts than unesterified fatty acids.

Lipid hydroperoxides induce changes in palmitate uptake across the rat blood-retina and blood-brain barrier.

The blood-retina and blood-brain transport of fatty acids was studied in control and lipid hydroperoxide-treated rats by measuring the permeability-surface area product (PS) to [1-14C]palmitate. An in situ carotid perfusion method was used. PS values were evaluated: (1) just after intracarotid injection of hydroperoxides; or (2) after a short-term systemic treatment for 1 week with sonicated emulsion of phospholipids-linoleate peroxidized mixture. Compared with saline-treated rats, PS remarkably decreased in the retina and most brain regions studied after acute, arterial injection of hydroperoxide preparations. On the contrary, the transport index significantly increased in the retina and almost all the brain areas after 7 days i.v. treatment with hydroperoxide emulsion. It is suggested that hydroperoxides acutely administered before transport radiotracer brought about a reinforcement of microvasculature junctional area or hampered substrate diffusion across endothelial membrane. On the other hand, upon short-term i.v. administration, hydroperoxides presumably triggered a lipid structure derangement of endothelial cell membranes and zonulae occludens due to their local accumulation and/or high capability of generating oxygen-free radicals.

Susceptibility of rat retina acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase and CTP:phosphocholine cytidylyltransferase activity to lipid peroxidation and hydroperoxide treatment.

Two enzyme activities involved in phospholipid metabolism in the rat retina were determined after in vivo and in vitro peroxidation according to several model systems. The in vivo models were based on: (i) intravenous administration of a sonicated emulsion of phospholipid and linoleate photooxidized mixture to normal rat for a period of one week; (ii) acute injection of Fe2+ solution (20 mM) or (iii) 0.5 mg of hydroperoxylinoleate into the vitreous body, and collection of retinal tissue 4 h or 4 days later, respectively. Oleoyl CoA:lysophosphatidylcholine acyltransferase activity was unchanged or exhibited significant inhibition. On the contrary, CTP:phosphocholine cytidylyltransferase activity was stimulated. By incubating in vitro the retina with: (i) Fe(2+)-ascorbate; (ii) photooxidized phospholipid mixture (0.1-5 mM) or individual phospholipid classes; (iii) hydroperoxylinoleate (0.25-2 mM), with or without Fe2+, a significant inactivation of acyltransferase (six-fold maximum loss of initial activity) and a slight stimulation of cytidylyltransferase were seen. Altogether, the results suggest that in situ oxygen radical generation by a variety of agents irreversibly perturbs enzymes and/or membrane structures in which the enzymes are inserted; these events may bea causal factor in retinal degeneration accompanying some ocular diseases.

Differential transport of docosahexaenoate and palmitate through the blood-retina and blood-brain barrier of the rat.

The permeability-surface area product (PS) of [1-14C]docosahexaenoate and [1-14C]palmitate at the blood-retina (BRB) and blood-brain barrier (BBB) was determined after in situ brain perfusion in Sprague-Dawley rats. The intracarotid injection procedure involved continuous infusion of albumin-bound fatty acids for up to 20 s. In the retina, and visual, parietal and frontal cortex, there was a significant decrease in mean PSs for docosahexaenoate compared to the palmitate group. For optic nerve and tract, superior colliculus, lateral geniculate, striatum, hippocampus and olfactory bulb, the comparison of PS values between the two fatty acid-injected groups of animals did not reveal any difference. It is suggested that the lower docosahexaenoate transport, compared to 16:0, across microvascular endothelium of the retina and other cortical regions might help explain the highest availability and selective retention of the essential 22:6(n-3) fatty acid in these nervous system structures.

Lipid peroxidation inhibits oleoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase in rat CNS axolemma-enriched fractions.

The effect of phospholipid peroxidation on the acylation of lysoPtdCho (lysophosphatidylcholine) by axolemma-enriched fraction prepared from rat brain stem was investigated. After two types of peroxidative treatments, the in vitro induction of malondialdehyde and conjugated dienes formation in axolemmal membranes correlated to a shift in the ratio of saturated/unsaturated fatty acids. By using an Fe2+ (20 microM)-ascorbate (0.25 mM) peroxidation system, the residual acyltransferase activity was 55% of the initial one. No change in Km value for either oleoyl-CoA or lysoPtdCho was found, whereas a loss of 24% in Vmax was observed. After 5 min preincubation with 150 mM t-BuOOH, 70% inactivation of the acylation reaction was observed. A near suppression of enzyme activity was reached with 400 mM. The apparent Km for oleoyl-CoA decreased sharply (from 6.6 microM in control preparations to 4.1 microM in t-BuOOH-treated membranes), indicating a 2-fold increase in the enzymatic affinity for this substrate. The apparent Km for lysoPtdCho increased markedly (from 1.56 microM in the control preparations to 5.88 microM in t-BuOOH-treated membranes) whereas a decrease of Vmax (from 1.65 to 0.80 nmol/min/mg protein) for the same substrate was observed. Significant enzyme inactivation (loss of 60% of initial activity) was seen when 10 mumol of photooxidized phospholipids were preincubated with axolemmal membranes. Significant dose-dependent enzyme inactivation was brought about by addition of 10-60 mumol of peroxidized PtdEtn/100 micrograms axolemmal protein. The percent enzyme inhibition by peroxidized PtdCho at equivalent amounts was lower than that by PtdEtn.(ABSTRACT TRUNCATED AT 250 WORDS)