Membrane Of Capillary Endothelial Cells Research Articles

Dynamic Regulation of Order Lipid Meso-domains in the Capillary Endothelial Cell Membrane

The structure and function of ion channels are sensitive to direct interactions with lipids of the membrane and to the biophysical properties of the membrane microenvironment, such that membrane fluidity, lateral pressure profile, and thickness can modulate the channels. Cholesterol plays a significant role in regulating membrane fluidity and is capable of phase separation where cholesterol will spontaneously separate into its own domain, which is partially responsible for developing two distinct domains within the membrane. These ordered domains are regions of high cholesterol and low fluidity, and the disordered domains are regions of low cholesterol and high fluidity. Here we examine and provide evidence of the presence of ordered cholesterol membrane domains in capillary endothelial cells. We will test the hypothesis that capillary endothelial cell ordered domains function as a signaling hub for different phospholipids regulating ion channel function. Using the isolated retinal vasculature, confocal microscopy, and fluorescent Bodipy-cholesterol, a marker of ordered lipid domains, we observed large regions approximately 2.3± 0.9 μm long of cholesterol ordered meso-domains within capillary endothelial cells and not the feeding arteriole. To determine the dynamic nature of these meso-domains, we used membrane-disordering drugs to disrupt the organization of membrane lipids. The ordered meso-domains display dynamic membrane disordering by ethanol and can re-cluster into the large ordered meso-domains. In addition, we observed that the critical signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) co-localized with the ordered meso-domains. To determine the cooperativity between these lipid molecules, we examined the effects of ethanol-dependent disordering of cholesterol on the membrane localization of PIP2. In addition, we examined the effects of PIP2 depletion with the G-protein couple receptor agonist, U46619, on the disordered state of the cholesterol meso-domains. These data suggest that ordered meso-domains might play a role as a signaling hub affecting the membrane fluidity and bioavailability of signaling lipids modulating ion channel function in capillary endothelial cell. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A case with anti-GPIHBP1 antibody positive autoimmune hyperchylomicronemia accompanied with idiopathic thrombocytopenic purpura

Background and Aims: GPIHBP1 is a glycolipid-modified anchor protein that resides on the capillary endothelial cell membrane and plays a role in transporting lipoprotein lipase (LPL), especially from the outside of capillaries to the lumen of blood vessels. We have experienced 46-year-old man with anti-GPIHBP1 antibody postive autoimmune hyperchylomicronemia acompanied with idiopathic thrombocytopenic purpura(ITP).

Effects of Ropivacaine Nanoparticles on the Apoptosis of Cerebral Vascular Endothelial Cells.

To investigate the protective effect of ropivacaine nanoparticles on endothelial cells in the blood brain barrier (BBB) during the development of ischemic brain edema, and its effects on endothelial cell death. Forty-two male Wistar rats weighing 250-300 g and aged 3-4 months were randomly divided into three groups: (1) ropivacaine nanoparticles, (2) saline control and (3) sham operation groups. The membrane of capillary endothelial cells in the animals treated with ropivacaine nanoparticles were intact, with reduced edema, and less severe brain injury when compared to the control. In the ropivacaine nanoparticle group, the number of apoptotic cells decreased at 6 h and 24 h after ischemia, while the number of apoptotic cells in the ischemic penumbra increased. The number of apoptotic cells in the ropivacaine nanoparticles group was significantly lower than in the saline treated control. Ropivacaine nanoparticles exert significant protective effects on the vascular endothelial cells and the BBB during cerebral ischemia.

Abstract 5207: Vascular remodeling is associated with increased permeability of experimental brain metastases of breast cancer

Abstract Approximately 1 in 8 women will develop breast cancer. Of these women, a subset develop metastatic breast cancer, of which 10-16% develop brain metastases. Treatment options are limited. The blood-brain barrier (BBB) serves as a physical barrier, limiting drug entry into the brain, as well as the active efflux of drugs by P-Glycoprotein, Breast Cancer Resistance Protein, and/or Multidrug Resistance-associated Protein located on the capillary endothelial cell membranes composing the BBB. Active remodeling occurs when cancer cells extravasate through the BBB and begin to metastasize forming fully colonized tumors, creating the Blood-Tumor Barrier (BTB). In order for active remodeling to occur, induction of angiogenesis must be initiated, resulting in formation of new blood vessels. These new blood vessels undergo active remodeling, and are associated with increased permeability when compared to intact BBB. By measuring vascular density, our goals were to understand permeability as it relates to vascular density, determine if increases in lesion permeability facilitate paclitaxel uptake into the lesion, and evaluate permeability as it relates to vascular remodeling. In this study, we utilize two experimental models (murine: 4T1-BR5; and human: MDA-MB-231-BR-Her2) to analyze permeability, drug uptake and vascular characteristics. In an analysis of over 2,000 metastatic lesions, we observed an increased permeability (a range over 30 fold) of nearly all lesions, with paclitaxel concentrations elevated (p< 0.05) in 85% of the lesions; however, only ∼10% of the lesions received enough paclitaxel to induce cytotoxicity (greater than 10-fold and 1000 ng/g) within the lesion. Analysis of the lesion vasculature revealed decreased vessel density (n = 86 for Her2 and n = 40 for 4T1), as well as the presence of tortuous and abnormal vessels, with nearly all blood vessels associated with the metastatic lesions showing structural changes to some extent. Furthermore, with immunofluorescent analysis, we observed a decreased (∼1.5 fold) expression of the vascular endothelial tight junction protein zonula occludens-1 (p<0.05), and increased expression of two vascular proteins: nestin (25% in permeable lesions compared with non-permeable) and plasmalemma vesicle associated protein-1 (∼18 ± 2% in permeable lesions compared to non-permeable lesions at ∼8.5 ± 2%, p<0.05). When plotting nestin expression versus fold increase in permeability, a correlation of r2 = 0.33, p<0.05 was observed. Increased expression of nestin and plasmalemma vesicle associated protein-1, and decreased expression of zonula occludes-1 correlate to increased permeability. We hypothesize the changes in these vascular proteins are a contributory cause to vascular destabilization and/or vascular remodeling in brain metastases of breast cancer, resulting in an increased permeability of the BTB. Citation Format: Kaci A. Bohn, Tori B. Terrell-Hall, Chris E. Adkins, Rajendar K. Mittapalli, Mohamed I. Nounou, Afroz S. Mohammad, Paul R. Lockman. Vascular remodeling is associated with increased permeability of experimental brain metastases of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2015-5207

Association of the Novel Non-AT1, Non-AT2 Angiotensin Binding Site with Neuronal Cell Death

We have discovered a non-AT(1), non-AT(2) angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.

Design of lipoprotein-adsorbed liposomes retaining Mn-porphyrins for SOD mimic delivery to brains

Drug delivery to brains is becoming more and more important but is severely restricted by the blood-brain barrier (BBB). In this study, we have prepared an antioxidant for brain targeting by apolipoprotein E (ApoE), which was suggested to mediate this drug transport across the BBB. Superoxide dismutase (SOD) catalyzing the reduction from superoxide radical anion (O 2 ·― ) to hydrogen peroxide (H 2 O 2 ) is the key enzyme for the protection from oxidative stress. The oxidative stress is considered to be implicated in the pathogenesis of a number of diseases, such as Alzheimer's disease. The SOD mimic manganese porphyrins (Mn-porphyrins) have been retained by a liposome composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine, sodium oleate, and Tween-80. The Tween-80 is reported to lead to the adsorption of ApoE. The resulting liposomes were incubated with serum proteins containing ApoE. After the incubation, the protein determination proved that the amount of adsorbed proteins on the liposome surface increased almost in proportion to the amount of the Tween-80 of the liposome. Subsequently, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the adsorbed proteins contained ApoE. Furthermore, the cellular uptake of the liposome was examined using cells expressing low-density lipoprotein receptors which recognize ApoE. The intracellular Mn-porphyrin retained by the liposome increased in proportion to the amount of the Tween-80 of the liposome. Moreover, the brain uptake of the Mn-porphyrin was observed in our preliminary in vivo experiment, where i.v. injection of the resulting liposome was carried out. These results suggest that the Mn-porphyrin SOD mimic can be delivered across the BBB, probably after interaction with lipoprotein receptors on the brain capillary endothelial cell membranes. Our system is considered to be a promising approach for protection of the brain under oxidative stress.

Ionization-Specific Prediction of Blood–Brain Permeability

This study presents a mechanistic QSAR analysis of passive blood–brain barrier permeability of drugs and drug-like compounds in rats and mice. The experimental data represented in vivo log PS (permeability-surface area product) from in situ perfusion, brain uptake index, and intravenous administration studies. A data set of 280 log PS values was compiled. A subset of 178 compounds was assumed to be driven by passive transport that is free of plasma protein binding and carrier-mediated effects. This subset was described in terms of nonlinear lipophilicity and ionization dependences, that account for multiple kinetic and thermodynamic effects: (i) drug's kinetic diffusion, (ii) ion-specific partitioning between plasma and brain capillary endothelial cell membranes, and (iii) hydrophobic entrapment in phospholipid bilayer. The obtained QSAR model provides both good statistical significance (RMSE < 0.5) and simple physicochemical interpretations (log P and pKa), thus providing a clear route towards property-based design of new CNS drugs. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:122–134, 2009

Drug resistance in epilepsy: Why is a simple explanation not enough?

Drug resistance in epilepsy: Why is a simple explanation not enough?

In Situ Localization of P-glycoprotein (ABCB1) in Human and Rat Brain

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.

Covalent Linkage of Apolipoprotein E to Albumin Nanoparticles Strongly Enhances Drug Transport into the Brain

Drug delivery to the brain is becoming more and more important but is severely restricted by the blood-brain barrier. Nanoparticles coated with polysorbates have previously been shown to enable the transport of several drugs across the blood-brain barrier, which under normal circumstances is impermeable to these compounds. Apolipoprotein E was suggested to mediate this drug transport across the blood-brain barrier. In the present study, apolipoprotein E was coupled by chemical methods to nanoparticles made of human serum albumin (HSA-NP). Loperamide, which does not cross the blood-brain barrier but exerts antinociceptive effects after direct injection into the brain, was used as model drug. Apolipoprotein E was chemically bound via linkers to loperamide-loaded HSA-NP. This preparation induced antinociceptive effects in the tail-flick test in ICR mice after i.v. injection. In contrast, nanoparticles linked to apolipoprotein E variants that do not recognize lipoprotein receptors failed to induce these effects. These results indicate that apolipoprotein E attached to the surface of nanoparticles facilitates transport of drugs across the blood-brain barrier, probably after interaction with lipoprotein receptors on the brain capillary endothelial cell membranes.

Overcoming MDR at the blood–brain barrier

The brain capillaries represent at major barrier preventing entry into the brain for xenobiotics and many drugs, which otherwise may be therapeutically active in the central nervous system. The ABC transporter, p-glycoprotein, which is predominantly located in the lumenal surface of capillary endothelial cells, is a key player in this barrier function. Thus, particular efforts have been made to overcome the barrier or to circumvent this efflux pump. At present various options are under consideration: (a) direct modulation of the transporter by specific inhibitors, (b) interference with the regulatory cascade of protein transcription, translation and function, and (c) encapsulation of substrates into delivery systems, which are able to by-pass p-glycoprotein. Among the inhibitors being under development to block p-glycoprotein, the non-immuno-suppressive cyclosporin A-analogue PSC-833 has gained most attention. In-vivo data in nude mice with an intracerebrally transplanted human glioblastoma demonstrated, that administration of the otherwise non-effective cytostatic Paclitaxel results in a dramatic decrease of tumor size after co-administration of PSC-833. Understanding the cascades underlying the regulation of p-glycoprotein expression and function, such as endothelin-receptor regulated activity or the influence of nuclear transcription factors such as pregnane X receptor, on p-glycoprotein expression may offer novel therapeutic options to control drug transport across the blood–brain barrier. Several strategies designed to by-pass p-glycoprotein without direct inhibition have been described. The applied systems use antibody-coupled immunoliposomes, or albumin-associated liposomes and nanoparticles, respectively, to move the encapsulated drug through the lumenal plasma membrane of capillary endothelial cells avoiding direct contact with p-glycoprotein. Within the past decade it became increasingly clear that the brain microvascular endothelium is a dynamic barrier, with p-glycoprotein being a central modulator of barrier function. Although rapid progress has been made in identifying multiple mechanisms that modulate p-glycoprotein action, still much more remains to be discovered, including practical aspects of inhibitor development, modification of regulatory pathways or development of specific delivery systems to lower the barrier function in brain capillaries.

Bilateral Oedema with Angiopathy in the Dorsointernal Area of the Cerebral Hemispheres of a Domestic Cat

This report describes a feline case of oedema and angiopathy associated with the sudden onset of pyrexia and opisthotonus, followed by death one day later. Histologically, oedema and vascular lesions were found bilaterally in the dorso-internal area of the cerebral hemispheres. The vascular lesions consisted of perivascular exudation of periodic acid-Schiff-positive eosinophilic material and hyaline degeneration of the vascular walls. Immunohistochemically, the perivascular material reacted with anti-fibrinogen antibody; weak labelling for laminin was occasionally seen in the basement membranes of affected capillaries, arterioles and venules, in contrast to the strong labelling seen in the normal brain. Ultrastructurally, serum protein was observed in the space between the basement membranes of capillary endothelial cells and astrocytes, and vascular basement membranes were occasionally thickened. This is, to our knowledge, the first report of feline vasogenic oedema with a bilateral distribution in the cerebrum.

The MDR1 C3435T polymorphism: effects on P-glycoprotein expression/function and clinical significance.

C3435T, have been investigated. 1-3 Originally identified in a German Caucasian population by Hoffmeyer et al., C3435T was found to correlate with P-gp expression in the duodenum as determined by Western blots and quantitative immunohistology (P=0.056). 1 Individuals with the CC genotype (n=6) had higher levels of P-gp expression, approximately 2-fold, compared with individuals with the TT genotype (n=5); heterozygotes had intermediate expression levels (n=10). The mechanism by which the T allele results in lower duodenal P-gp expression is unknown, because C3435T is a silent mutation and does not result in changes in the P-gp sequence. However, Hoffmeyer et al. hypothesize that C3435T may be linked to other variants in the MDR1 gene. 1 The MDR1 gene product P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter family. P-gp utilizes the energy derived from ATP hydrolysis to pump a wide range of compounds, including numerous clinically used drugs, out of cells; this activity has important pharmacokinetic and pharmacodynamic consequences. For example, P-gp is expressed within the apical membranes of intestinal, renal, and hepatic epithelial cells, where it affects the absorption and elimination of its substrates. P-gp is also located within the apical membranes of capillary endothelial cells of the brain, where it can limit the penetration of drugs to the CNS. In addition to the roles of P-gp in absorption, distribution, and elimination, the overexpression of P-gp is implicated in the development of the multi-drug resistance (MDR) phenotype of some tumor cells. Consequently, P-gp inhibitors are now being developed as MDR reversal agents. Hoffmeyer et al. only examined the effects of the MDR1 C3435T polymorphism on P-gp expression in the duodenum. However, because the MDR1 gene is expressed in many normal tissues and cell types, it is important to establish whether the mutation alters P-gp expression exclusively in the duodenum, thereby affecting only drug absorption, or whether expression is altered in other tissues as well, leading to changes in distribution, elimination, or both of these processes. Using a rhodamine efflux assay as a measure of P-gp activity, Hitzl et al. examined P-gp activity in CD56+ natural killer cells from healthy subjects with the different genotypes at the 3435 locus. 2 Rhodamine is a P-gp substrate, thus CD56+ cells with higher P-gp activity would be predicted to have lower intracellular rhodamine fluorescence. Hitzl et al. found that CD56+ cells from individuals with the CC genotype (n=10) had lower rhodamine fluorescence (51.1 ± 11.4%) compared with CD56+ cells from individuals with the TT genotype (n=11) (67.5 ± 9.5%), indicating that cells from CC carriers have higher P-gp activity compared with cells isolated from TT carriers. 2 Although this difference was statistically significant, the consequences of a functional difference of this magnitude are debatable. In addition to these functional studies, Hitzl et al. quantified MDR1 RNA transcript levels in leukocytes. 2 They did not find a correlation between RNA levels and genotype at position 3435. Hitzl et al. hypothesize that the lack of a correlation was due to their use of leukocytes as the RNA source; leukocytes are a heterogeneous pool of cells that include CD56+ cells, but also other cell types. Although the results of the RNA expression experiments of Hitzl et al. do not necessarily invalidate the results of their functional studies, further experiments examining P-gp transcript levels, and ideally P-gp protein levels, in CD56+ cells are needed to resolve this issue. Interestingly, a number of P-gp substrates, including digoxin and cyclosporin A, exhibit substantial interindividual variability in their pharmacokinetics. Some of this variability could be attributed to environmental factors, but it is also reasonable to predict that some of this variability arises due to genetic factors, including mutations in genes involved in drug metabolism and transport, such as MDR1. Understanding the functional and clinical consequences of MDR1 variants is important—if this variability could be assigned to a mutation in the MDR1 gene, patients could be screened and appropriate dose adjustments could be made on the basis of their MDR1 genotype. Furthermore, MDR1 variants could have important pharmacodynamic consequences: patients carrying null MDR1 alleles, if such alleles exist, might not respond to P-gp inhibitors used as MDR reversal agents in cancer treatment.

Glial Cell Line-Derived Neurotrophic Factor Induces Barrier Function of Endothelial Cells Forming the Blood–Brain Barrier

Since a deep involvement of astrocytes, a kind of glial cells, in differentiation of the blood–brain barrier (BBB) has been suggested, we examined the relation of glial cell line-derived neurotrophic factor (GDNF) to the BBB. First, immunohistochemical examination of the cerebral cortex of rats revealed that glial cell line-derived neurotrophic factor receptor (GFRα1) was preferentially expressed on the cell membranes of capillary endothelial cells. Second, to elucidate the effects of GDNF on the BBB, capillary endothelial cells isolated from the porcine cerebral cortex were cultured and then changes in tight junction function of the endothelial cells were examined after addition of GDNF, in terms of transendothelial electrical resistance (TER) and permeability. GDNF at concentrations of 0.1 and 1 ng/ml significantly activated the barrier function of the endothelial cells in the presence of cAMP. Since GDNF is secreted from astrocytes sheathing capillary endothelial cells in the brain cortex, our results strongly suggest that GDNF enhances the barrier function of tight junctions of the BBB on the one hand, and also supports the survival of neurons on the other hand.

Monocarboxylate transporter (MCT1) abundance in brains of suckling and adult rats: a quantitative electron microscopic immunogold study

Transcellular transport of energy substrates across the vascular endothelial cells of the brain is accomplished by integral membrane carrier proteins, such as the glucose transporter GLUT1 and the monocarboxylic acid transporter MCT1. The abundance of these proteins may vary depending on age and nutritional status. In this study we compared the expression of MCT1 in cerebral cortex of suckling and adult rats to determine whether the former, which use considerably more monocarboxylates such as lactate and ketone bodies as fuel than do older rats, correspondingly express more MCT1 than adults. Using electron microscopic immunogold methods, we found that 17-day old suckling rat pups had 25 times more MCT1 labeling in the membranes of capillary endothelial cells than adults. This transporter was nearly equally distributed in luminal and abluminal membranes with less than 10% of the immunogold particles in the endothelial cytoplasmic compartment. The suckling rats also had 15 times more immunogold particles associated with pericyte membranes and 19 times heavier labeling of membranes associated with astrocytic end feet adjacent to microvessels. Neuropil and choroid plexus were lightly labeled. Some MCT1-positive astrocyte and neuron cell bodies were observed, suggesting active synthesis of MCT1 by these cells. The potential for regulation of expression of MCTs by dietary or other factors may have important consequences for the progression and treatment of cerebrovascular disorders and other diseases.

Capillaries demonstrate changes in membrane potential in response to pharmacological stimuli.

It has been proposed that capillaries can detect changes in tissue metabolites and generate signals that are communicated upstream to resistance vessels. The mechanism for this communication may involve changes in capillary endothelial cell membrane potentials which are then conducted to upstream arterioles. We have tested the capacity of capillary endothelial cells in vivo to respond to pharmacological stimuli. In a hamster cheek pouch preparation, capillary endothelial cells were labeled with the voltage-sensitive dye di-8-ANEPPS. Fluorescence from capillary segments (75-150 microns long) was excited at 475 nm and recorded at 560 and 620 nm with a dual-wavelength photomultiplier system. KCl was applied using pressure injection, and acetylcholine (ACh) and phenylephrine (PE) were applied iontophoretically to these capillaries. Changes in the ratio of the fluorescence emission at two emission wavelengths were used to estimate changes in the capillary endothelial membrane potential. Application of KCl resulted in depolarization, whereas application of the vehicle did not. Application of ACh and PE resulted in hyperpolarization and depolarization, respectively. The capillary responses could be blocked by including a receptor antagonist (atropine or prazosin, respectively) in the superfusate. We conclude that the capillary membrane potential is capable of responding to pharmacological stimuli. We hypothesize that capillaries can respond to changes in the milieu of surrounding tissue via changes in endothelial membrane potential.

The role of calcium ion in anoxia/reoxygenation damage of cultured brain capillary endothelial cells.

Capillary endothelial cells are critical targets in both ischemia and reperfusion of the brain. Arachidonic acids and oxygen free radicals have been shown to cause disruption of blood-brain barrier (BBB) by destruction of capillary endothelial cell membrane. However, the exact mechanism of BBB breakdown by cerebral ischemia/reperfusion remains undetermined. The aim of the present study is to clarify the mechanism of intracellular calcium ion ([Ca2+]i) change in brain capillary endothelial cells under anoxia/reoxygenation. Brains capillary endothelial cells were isolated from ten male Sprague-Dawley rats by a two step enzymatic process. [Ca2+]i was measured by means of a confocal laser scanning microscope using Indo 1-A/M as a calcium indicator. The endothelial cells were subjected to anoxia and reoxygenization under different conditions. [Ca2+]i increased gradually during anoxia and slightly decreased after reoxygenation. Indomethacin and SOD suppressed the elevation of [Ca2+]i during anoxia. NG-nitro-L-arginine methyl ester and catalase moderately suppressed the elevation, however nifedipine did not suppress it at all. In this model, rapid [Ca2+]i change was not observed during the reoxygenation phase. The results indicate that the anoxia induced elevation of [Ca2+]i in the brain capillary endothelial cells depends on superoxide and peroxynitrite generation.

Adenosine kinetics in canine coronary circulation.

Adenosine kinetics in the coronary circulation were investigated in anesthetized closed-chest dogs by analysis of multiple-indicator dilution experiments. During simultaneous intracoronary bolus injections of 125I-labeled albumin, [14C]sucrose, and [3H]adenosine, dilution curves were measured by automated sampling of coronary venous blood, using high-performance liquid chromatography and isotope detection techniques. Under control conditions, only 1% of the injected [3H]adenosine was detected in coronary venous samples, compared with the reference tracer, [14C]sucrose, and the peak of the adenosine dilution curve preceded those of the reference tracers by 2-4 s. Optimized model fits to the control adenosine curves required the combination of high ratios of the capillary endothelial cell membrane permeability-surface area product to flow (5.3) and endothelial cell consumption capacity to flow (23), in addition to a broad heterogeneity of flow. Dilution curves were measured under control conditions and during increased coronary flow (nitroglycerin), inhibition of the enzyme adenosine kinase (iodotubercidin), and blockade of membrane adenosine transport (dipyridamole). Reliability of the parameter estimates was confirmed using residual analysis, sensitivity function analysis, and Monte Carlo simulation techniques. Accuracy of the model was confirmed in separate experiments in which nontracer adenosine was infused into the coronary artery and the model prediction of coronary venous adenosine concentrations was compared with measured values. Using published measurements of coronary blood flow and arterial and coronary venous plasma adenosine concentrations in open-chest dogs, the estimated in vivo interstitial adenosine concentration is 100-220 nM.

In vitro evidence of neutrophil-mediated lung injury after intestinal reperfusion.

This study examines the hypothesis that neutrophils isolated from animals sustaining intestinal reperfusion (IIR) induce pulmonary microvascular dysfunction. Lungs were isolated from normal Sprague-Dawley rats and perfused with a physiologic buffer in vitro. Neutrophils (2 x 10(6)) isolated from animals sustaining IIR (n = 5) or sham operation (SHAM; n = 6) were infused into the isolated lung model. A third group of lungs underwent in vitro perfusion without exposure to neutrophils (n = 5). Lung injury was assessed by measuring wet to dry weight ratios and pulmonary artery pressure (PAP). Pulmonary ultrastructure was assessed by electron microscopy. The wet:dry ratio of lungs from animals sustaining IIR was greater than that of lungs exposed to SHAM neutrophils (p = .03) or perfusate alone (p = .02). The PAP of lungs exposed to IIR neutrophils was nearly 10 times greater than that of lungs exposed to SHAM neutrophils (p = .003) or buffer alone (p = .006). Ultrastructural examination of lungs exposed to IIR neutrophils demonstrated interstitial edema with occasional focal disruptions in the alveolar capillary endothelial cell membrane whereas lungs exposed to SHAM neutrophils were normal. These experiments provide important in vitro correlation of prior in vivo studies suggesting that neutrophils are important pathogenic mediators of IIR-induced lung injury.

Blood-brain barrier permeability is markedly decreased in cholestasis in the rat

The blood-brain-barrier plays an essential role in regulating the entrance of substances into the brain. To date, permeability of the blood-brain barrier has not been studied in models of cholestatic liver injury, although levels of substances known to enhance vascular permeability (bile acids, substance P, histamine) are elevated in cholestasis. Two rat models of cholestasis were studied: bile duct resection (5 days after surgery) and α-naphthylhisothiocyanate treatment (45 mg/kg/day for 7 days). The mean value for whole brain blood-to-brain transfer constant in bile duct resection rats was about 50% less than corresponding values in sham-operated and unoperated control rats (p ± 0.05, respectively). Reductions in blood-to-brain transfer constant of similar magnitude were found in the caudate nuclei, cortexes and hippocampi of bile duct-resected rats. Blood-to-brain transfer constant values in α-naphthylisothiocyanate-treated rats were also about 50% less in whole brain and specific brain regions than corresponding control values. A precedent for a decrease in blood-to-brain transfer constant is the dexamethasone-treated rat, in which the phenomenon has been attributed to a decrease in cerebral capillary endothelial cell membrane fluidity. We confirmed that blood-to-brain transfer constant values are reduced by about 50% in dexamethasone-treated rats. A decrease in membrane fluidity affords a rational explanation for a decrease in blood-to-brain transfer constant in cholestasis as a consequence of the dynamic equilibrium between elevated plasma levels of cholesterol in cholestasis and cell membranes exposed to the circulation. (HEPATOLOGY 1993;17:1103–1108.)