Interfacial activation of porcine pancreatic phospholipase A(2) studied with 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled lipids

The interfacial activation of porcine pancreatic phospholipase A(2) (PLA(2)) during the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes at different temperatures has been monitored by fluorescence changes of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) lipid derivatives 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (C(12)-NBD-PC) and 12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)]dodecanoic acid (C(12)-NBD-FA) inserted in the substrate vesicles. These long-chain monitors, in contrast to the previously used C(6)-NBD-PC, detect latency times of PLA(2) action, similar to those measured by the classic titrimetric, pH-stat method. Interestingly, hydrolysis of the host vesicles results in a decrease in fluorescence not only of C(12)-NBD-PC, a substrate analog, but also of product derivative C(12)-NBD-FA. Ultrafiltration experiments show that C(12)-NBD-FA does not migrate to the aqueous phase upon hydrolysis of the host liposomes. Besides, in a simulated hydrolysis experiment in which increasing proportions of palmitic acid and 1-palmitoyl-sn-glycero-3-phosphocholine were cosonicated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C(12)-NBD-PC fluorescence was insensitive to products, whereas C(12)-NBD-FA did show a decreased emission intensity as in the actual hydrolysis experiments. The phenomenon is triggered above a critical concentration of products (10 mol%) suggesting that cosegregation of NBD-FA (either added as such or generated by hydrolysis of C(12)-NBD-PC) and products may be related to the decrease in fluorescence. Phase separation should create microdomains of increased C(12)-NBD-FA surface density and cause concentration quenching. In addition, and taking into account that the NBD group may be located near the interfacial region, it is possible that in segregating with products, the fluorescent moiety of C(12)-NBD-FA becomes exposed to microenvironments of higher surface polarity, which further decreases its quantum yield.     

A facile method for direct determination of phospholipase A2 activity in intact cells

The fluorescent phospholipid analogue 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3- diazole)aminocaproylphosphatidylcholine (C6-NBD-PC), which incorporates into cell membranes, is employed as a substrate for phospholipase A2 (PLA2) in intact cells. The interaction of this substrate with the cells produces only one fluorescent product; the fatty acid C6-NBD-FA, which does not incorporate into other lipids, and is not further metabolized. The product, a hydrophilic fatty acid, is separated from the substrate by aqueous: organic solvent phase separation. Using this method, the fatty acid produced is fully recovered and its amount, as measured by its fluorescence intensity, is a direct measure of the cell membrane PLA2 activity.     

HPLC-fluorimetric assay of phospholipase A2. Application to biological samples with high protein content and various reaction conditions

Phospholipase A(2) (PLA(2)) quantitation in real-time, using (7-nitro-2-1,3-benzoxadiazol-4-yl)amino-derivatives of phosphatidylcholine (NBD-PCs) as substrates, is influenced by high protein content, color or turbidity. The purpose of the study was to overcome such limitations by a complementary reversed-phase HPLC step to separate the substrates from the products of the reaction. Plasma and bronchoalveolar lavage (BAL) fluid were applied as enzymic sources, while standard porcine PLA(2) and human plasma PAF-acetylhydrolase (PAF-AH) were employed as positive controls. The method was validated with a radiometric assay and compared with the real-time fluorimetric assay. Regarding PLA(2) and PAF-AH determination, the isocratic elution systems CH(3)OH-H(2)O (80:20, v/v) and CH(3)OH-H(2)O-CH(3)COOH (60:40:0.2, v/v/v) separated efficiently C(12)-NBD-FA/C(12)-NBD-PC and C(6)-NBD-FA/C(6)-NBD-PC, with 4.4 and 2.2 resolution, respectively. Analysis time was ～15 min/injection. The reproducibility, expressed as relative standard deviation, was ≤13% for PLA(2) and ≤16% for PAF-AH, respectively. The assay was linear for PLA(2) activities extending from 1 pmol up to at least 250 nmol FA/h/mL sample, similar to the radiometric assay. It was appropriate for samples with high protein content, where the real-time fluorimetric assay was insufficient. The HPLC method was also evaluated under elevated temperatures, various pH values and Ca(2+) concentrations.     

OKY-046 inhibits thromboxane synthesis with no effect on brain edema and neurological status in head traumatized rats

Head trauma (HT) was induced in the left hemisphere of rats by a weight drop device. Edema was maximal 24 h after HT in the injured zone, and PGE2, TXB2 and 6-keto-PGF1 alpha were elevated in both the injured and remote areas. The effect of a specific thromboxane synthetase inhibitor, OKY-046, on the outcome of HT was studied. OKY-046, 100 mg/kg, was given to rats immediately and 8 h after HT. The neurological severity score (NSS) was evaluated at 1 h after HT, and at 24 h, just prior to sacrifice. Specific gravity (SG) of both hemispheres was measured after decapitation. Prostaglandins (PGs) were extracted from the site of injury and from the frontal lobes, remote from the injury, and assayed by RIA. Basal levels of PGE2 and 6-keto-PGF1 alpha were not reduced by the drug while basal TXB2 levels were lowered. However, the increased production due to HT of all PGs, was inhibited by OKY-046, especially that of TXB2. The ratio of TXB2/6-keto-PGF1 alpha, known to affect vascular tone, was reduced by OKY-046 treatment as a result of TXA2 synthesis inhibition. Still, no effect was found on the neurological outcome (as evaluated by the NSS), or on edema formation (expressed by reduced SG). Thus, based on the present findings increased TXA2 synthesis cannot be implicated in the pathophysiology of cerebral edema or dysfunction following HT.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Spatial Synchronization of Brain Bioelectric Potentials Differs in Boys and Girls Aged 12–13 Years Reading Narrative Texts

The contribution of each cortical zone to the organization of spatial synchronization of brain bioelectric potential s (SSBP) was estimated using cross-correlation matrices of 20-channell EEG during reading in boys and girls aged 12–13 years. Differences in the topical distribution of the SSBP levels in young boys and girls were revealed both in the resting state and during reading. In boys, the SSBP level in the left temporal lobe was lower than that in girls, and in the right parietal lobe it was higher than that in girls in all the states studied. A decrease of SSBP in central cortex zones was revealed, indicating the activation of motor cortical areas in all subjects during reading. Enhancement of SSBP in the parietal regions of the left hemisphere and the occipital zones of both hemispheres is observed in boys during reading, while in girls SSBP is increased bilaterally in the frontotemporal areas with the involvement of the left frontal lobe. Moreover, during reading intrahemispheric correlations between EEGs of the central and temporoparietal regions with emphasis on the right hemisphere were more pronounced in boys, while both intra- and interhemispheric correlations of the left temporal regions were pronounced in girls.     

Prognostic value of cortically induced motor evoked activity by TMS in chronic stroke: Caveats from a revealing single clinical case

Background

We report the case of a chronic stroke patient (62?months after injury) showing total absence of motor activity evoked by transcranial magnetic stimulation (TMS) of spared regions of the left motor cortex, but near-to-complete recovery of motor abilities in the affected hand.

Case presentation

Multimodal investigations included detailed TMS based motor mapping, motor evoked potentials (MEP), and Cortical Silent period (CSP) as well as functional magnetic resonance imaging (fMRI) of motor activity, MRI based lesion analysis and Diffusion Tensor Imaging (DTI) Tractography of corticospinal tract (CST). Anatomical analysis revealed a left hemisphere subinsular lesion interrupting the descending left CST at the level of the internal capsule. The absence of MEPs after intense TMS pulses to the ipsilesional M1, and the reversible suppression of ongoing electromyographic (EMG) activity (indexed by CSP) demonstrate a weak modulation of subcortical systems by the ipsilesional left frontal cortex, but an inability to induce efficient descending volleys from those cortical locations to right hand and forearm muscles. Functional MRI recordings under grasping and finger tapping patterns involving the affected hand showed slight signs of subcortical recruitment, as compared to the unaffected hand and hemisphere, as well as the expected cortical activations.

Conclusions

The potential sources of motor voluntary activity for the affected hand in absence of MEPs are discussed. We conclude that multimodal analysis may contribute to a more accurate prognosis of stroke patients.     

Effects of fluorescent probe NBD-PC on the structure, dynamics and phase transition of DPPC. A molecular dynamics and differential scanning calorimetry study

We present a combined theoretical (molecular dynamics, MD) and experimental (differential scanning calorimetry, DSC) study of the effect of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl chain-labeled fluorescent phospholipid analogs (C6-NBD-PC and C12-NBD-PC) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. DSC measurements reveal that < 1 mol% of NBD-PC causes elimination of the pre-transition and a large loss of cooperativity of the main transition of DPPC. Labeling with C6-NBD-PC or C12-NBD-PC shifts the main transition temperature to lower or higher values, respectively. Following our recent report on the location and dynamics of these probes (BBA 1768 (2007) 467-478) in fluid phase DPPC, we present a detailed analysis of 100-ns MD simulations of systems containing either C6-NBD-PC or C12-NBD-PC, focused on their influence on several properties of the host bilayer. Whereas most monitored parameters are not severely affected for 1.6 mol% of probe, for the higher concentration studied (6.2 mol%) important differences are evident. In agreement with published reports, we observed that the average area per phospholipid molecule increases, whereas DPPC acyl chain order parameters decrease. Moreover, we predict that incorporation of NBD-PC should increase the electrostatic potential across the bilayer and, especially for C12-NBD-PC, slow lateral diffusion of DPPC molecules and rotational mobility of DPPC acyl chains.     

Spatial synchronization of potentials in the frontal zones of the brain during performance of nominative speech in children

Characteristics of spatial-temporal relations of the frontal zones potentials with those of other brain centres were studied in five- to six-year old children during naming of visually presented objects. EEG cross correlation analysis has shown that during visual perception of familiar objects the correlations of frontal areas with the interior pariental area of the left hemisphere are enhanced; the rhythms of the inferior parietal and occipital zones in the left hemisphere precede the potentials in the frontal area, while in the right hemispere synphasic relations are set up between the frontal and occipital zones. Naming the objects by the appropriate word as compared with rest and showing of the object, leads to enhancement of both intra- and interhemispheric correlations between potentials in the frontal zone and the inferior parietal and temporal zones. The frontal lobe rhythms begin to precede the inferior parietal and occipital potentials and form synphasic relations with the temporal and motor zone potentials in the left hemisphere.     

Perinatal Hypoxic-Ischemic Brain Injury Enhances Quisqualic Acid-Stimulated Phosphoinositide Turnover

In an experimental model of perinatal hypoxic-ischemic brain injury, we examined quisqualic acid (Quis)-stimulated phosphoinositide (PPI) turnover in hippocampus and striatum. To produce a unilateral forebrain lesion in 7-day-old rat pups, the right carotid artery was ligated and animals were then exposed to moderate hypoxia (8% oxygen) for 2.5 h. Pups were killed 24 h later and Quis-stimulated PPI turnover was assayed in tissue slices obtained from hippocampus and striatum, target regions for hypoxic-ischemic injury. The glutamate agonist Quis (10(-4) M) preferentially stimulated PPI hydrolysis in injured brain. In hippocampal slices of tissue derived from the right cerebral hemisphere, the addition of Quis stimulated accumulation of inositol phosphates by more than ninefold (1,053 +/- 237% of basal, mean +/- SEM, n = 9). In contrast, the addition of Quis stimulated accumulation of inositol phosphates by about fivefold in the contralateral hemisphere (588 +/- 134%) and by about sixfold in controls (631 +/- 177%, p less than 0.005, comparison of ischemic tissue with control). In striatal tissue, the corresponding values were 801 +/- 157%, 474 +/- 89%, and 506 +/- 115% (p less than 0.05). In contrast, stimulation of PPI turnover elicited by the cholinergic agonist carbamoylcholine, (10(-4) or 10(-2) M) was unaffected by hypoxia-ischemia. The results suggest that prior exposure to hypoxia-ischemia enhances coupling of excitatory amino acid receptors to phospholipase C activity. This activation may contribute to the pathogenesis of irreversible brain injury and/or to mechanisms of recovery.     

Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells

We have investigated the transport and canalicular enrichment of fluorescent phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and β-BODIPY-PC. Fluorescent PC was efficiently transported to the biliary canaliculus (BC) and became enriched on the lumenal side of the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recycling compartment (ARC) in polarized HepG2 cells as shown by colocalization with fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respectively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of PC analogs from the canalicular membrane were found. Evidence for nonvesicular transport included enrichment of the PC-analog β-BODIPY-PC in the BC (t_1/2 = 3.54 min) prior to its accumulation in the SAC/ARC (t_1/2 = 18.5 min) at 37 °C. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton and at 2 °C. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in hepatocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.     

NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.     

Transbilayer movement of phospholipids at the main phase transition of lipid membranes: implications for rapid flip-flop in biological membranes

The transbilayer movement of fluorescent phospholipid analogs in liposomes was studied at the lipid phase transition of phospholipid membranes. Two NBD-labeled analogs were used, one bearing the fluorescent moiety at a short fatty acid chain in the sn-2 position (C(6)-NBD-PC) and one headgroup-labeled analog having two long fatty acyl chains (N-NBD-PE). The transbilayer redistribution of the analogs was assessed by a dithionite-based assay. We observed a drastic increase of the transbilayer movement of both analogs at the lipid phase transition of DPPC (T(c) = 41 degrees C) and DMPC (T(c) = 23 degrees C). The flip-flop of analogs was fast at the T(c) of DPPC with a half-time (t(1/2)) of ~6-10 min and even faster at the T(c) of DMPC with t(1/2) on the order of <2 min, as shown for C(6)-NBD-PC. Suppressing the phase transition by the addition of cholesterol, the rapid transbilayer movement was abolished. Molecular packing defects at the phase transition are assumed to be responsible for the rapid transbilayer movement. The relevance of those defects for understanding of the activity of flippases is discussed.     

Functional regional asymmetry of the EEG intrahemispheric coherence in dogs during conditioning

Individual features of the regional interhemispheric relations in the brain were studied in dogs during alimentary conditioning. The electrical activity was recorded from symmetrical anterior (frontal and motor cortices) and posterior (visual and auditory cortices) areas of the neocortex. Comparison between the averaged left and right intrahemispheric EEG coherences revealed a dynamic character of interhemispheric relations dependent on the stage of conditioning. Individual features were shown. In a dog with strong type of the nervous system, in the anterior brain regions, the EEG coherence was higher in the left hemisphere than in the right one, whereas, on the contrary, in the posterior regions, the values were higher in the right than in the left hemisphere. In dogs with weak type of the nervous system, there was an inverse relationship. Thus, the spatial organization of the cortical electrical activity in the associative and projection brain areas was different.     

Hemispheric dominance of cortical activity evoked by focal electrogustatory stimuli.

Functional magnetic resonance imaging was used to observe cortical hemodynamic responses to electric taste stimuli applied separately to the right and left sides of the tongue tip. In 11 right-handed normal adults activation occurred primarily in the insular cortex, superior temporal lobe, inferior frontal lobe, including premotor regions, and in inferior parts of the postcentral gyrus. Unexpectedly, the location and laterality of activation were largely identical regardless of the side of the tongue stimulated. Activation in the superior insula, the presumed location of primary gustatory cortex, was predominantly, but not exclusively, in the right hemisphere, whereas central (more inferior) insular activations were more evenly bilateral. Right hemispheric dominance of activation also occurred in premotor regions (Brodmann areas 6 and 44), whereas left hemispheric dominance occurred only in the superior temporal cortex (Brodmann areas 22/42). The electric taste-evoked hemodynamic response pattern was more consistent with activation of the gustatory system than activation of somatosensory systems. The results suggest that the sites for cortical processing of electric taste information are dependent on hemispheric specialization.     

Development of Prolonged Focal Cerebral Edema and Regional Cation Changes Following Experimental Brain Injury in the Rat

The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid-percussion brain injury. Animals receiving fluid-percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid-percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p less than 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p less than 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p less than 0.05) and injured hippocampus (10 min post injury, p less than 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p less than 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24-h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid-percussion brain injury and that these changes may persist for a prolonged period after brain injury.     

Tau Reduction Diminishes Spatial Learning and Memory Deficits after Mild Repetitive Traumatic Brain Injury in Mice

Objective

Because reduction of the microtubule-associated protein Tau has beneficial effects in mouse models of Alzheimer''s disease and epilepsy, we wanted to determine whether this strategy can also improve the outcome of mild traumatic brain injury (TBI).

Methods

We adapted a mild frontal impact model of TBI for wildtype C57Bl/6J mice and characterized the behavioral deficits it causes in these animals. The Barnes maze, Y maze, contextual and cued fear conditioning, elevated plus maze, open field, balance beam, and forced swim test were used to assess different behavioral functions. Magnetic resonance imaging (MRI, 7 Tesla) and histological analysis of brain sections were used to look for neuropathological alterations. We also compared the functional effects of this TBI model and of controlled cortical impact in mice with two, one or no Tau alleles.

Results

Repeated (2-hit), but not single (1-hit), mild frontal impact impaired spatial learning and memory in wildtype mice as determined by testing of mice in the Barnes maze one month after the injury. Locomotor activity, anxiety, depression and fear related behaviors did not differ between injured and sham-injured mice. MRI imaging did not reveal focal injury or mass lesions shortly after the injury. Complete ablation or partial reduction of tau prevented deficits in spatial learning and memory after repeated mild frontal impact. Complete tau ablation also showed a trend towards protection after a single controlled cortical impact. Complete or partial reduction of tau also reduced the level of axonopathy in the corpus callosum after repeated mild frontal impact.

Interpretation

Tau promotes or enables the development of learning and memory deficits and of axonopathy after mild TBI, and tau reduction counteracts these adverse effects.     

A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway

The fluorescent phospholipid analog N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was inserted into the plasma membrane of Baby hamster kidney cells at low temperature (2 degrees C). The mobility characteristics of the analog--as revealed by fluorescence photobleaching recovery--were very similar to those of membrane-inserted 1-acyl-2[6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]caproyl] phosphatidylcholine (C6-NBD-PC). Upon warming to 37 degrees C, followed by a 1-h incubation, all N-Rh-PE was located intracellularly. By contrast, after the same time interval, approximately 10% of the cell-associated PC-derivative was found intracellularly. Furthermore, the analogs moved to different intracellular sites, as N-Rh-PE associates with perinuclear and peri-Golgi structures, whereas C6-NBD-PC appears mainly in the Golgi complex. Colocalization with organelle-specific probes and Percoll gradient analysis identified the N-Rh-PE-labeled structures as lysosomes. Temperature and energy-dependent experiments supported the endocytic pathway as the mechanism of N-Rh-PE internalization. The mechanism of N-Rh-PE internalization appears to differ from that of C6-NBD-PC. In conjunction with a difference in the efficiency of removal of the lipid derivatives from the plasma membrane, the results suggest that N-Rh-PE is selectively internalized, implying that sorting of the lipid analogs already occurs at the level of the plasma membrane. The distinct difference in physical appearance of the probes after membrane insertion, i.e., N-Rh-PE being present as small clusters and C6-NBD-PC as monomers, could explain the selective sorting and internalization of N-Rh-PE. The results demonstrate that N-Rh-PE may serve as a useful marker for studying membrane traffic during endocytosis.     

Differences in the Transbilayer and Lateral Motions of Fluorescent Analogs of Phosphatidylcholine and Phosphatidylethanolamine in the Apical Plasma Membrane of Bovine Aortic Endothelial Cells

In the plasma membrane of various eucaryotic cell types, in particular blood platelets and erythrocytes, it is known that phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer and that this transverse asymmetry is controlled by an aminophospholipid translocase activity. In this respect, it was of interest to check whether there are differential transbilayer movements between amino- and neutral phospholipids in the apical plasma membrane of vascular endothelial cells which form the inner nonthrombogenic lining of the large blood vessel. In the first step we compared the transbilayer localization and also the rate of lateral motion of two fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine, namely C6-NBD-PC and C6-NBD-PE, inserted into the apical plasma membrane of bovine aortic endothelial cells, in vitro. By the use of back-exchange experiments we have found that C6-NBD-PC could be removed from the cell membrane toward the culture medium regardless of the incubation conditions used, i.e., just after cell labeling at 0°C or even after further cell incubation for 1 h at 0 or 20°C. In contrast, C6-NBD-PE could be removed only when the cells were maintained at 0°C. After incubation for 1 h at 20°C, 85% of the probe molecules remained nonexchangeable, indicating probe translocation from the outer to the inner leaflet of the lipid bilayer. This "flip" process, which occurred at 20°C, was abolished when the endothelial cells were preincubated with N-ethylmaleimide, diamide, vanadate (VO^3-₄) and vanadyl (VO²⁺) ions, a set of substances which inhibit aminophospholipid translocase activity in various systems, and with a combination of sodium azide and 2-deoxyglucose which led to nearly complete ATP depletion in the cells. Fluorescence recovery after photobleaching experiments were also carried out to specify more precisely the localization and dynamics of the probes in the two leaflets of the plasma membrane lipid bilayer. They produced lateral diffusion coefficients D of 1.2 ± 0.05 × 10^-9 cm²/s for C6-NBD-PC and 2.8 ± 0.3 × 10^-9 cm²/s for C6-NBD-PE, when the two probes were located in the outer leaflet of the plasma membrane, just after cell labeling at 0°C. After cell incubation for one hour at 20°C, i.e., when C6-NBD-PC was still in the outer leaflet whereas C6-NBD-PE was translocated in the inner leaflet, D was observed to slightly increase for C6-NBD-PC (D = 1.9 ± 0.06 × 10^-9 cm²/s) and to greatly increase by at least a factor of 3 for C6-NBD-PE (D = 9.1 ± 0.9 × 10^-9 cm²/s). These results show that the plasma membrane of bovine aortic endothelial cells is equipped with a protein-dependent and energy-mediated phosphatidylethanolamine translocase activity and that the lateral diffusion rate of this phospholipid is much faster in the inner than in the outer leaflet of the lipid bilayer, thus indicating large differences in the fluidity of the two halves of this membrane.