Brain Capillary Permeability Research Articles

Dietary Spray-Dried Porcine Plasma Prevents Cognitive Decline in Senescent Mice and Reduces Neuroinflammation and Oxidative Stress

Aging is characterized by chronic, low-grade inflammation that correlates with cognitive decline. Dietary supplementation with spray-dried porcine plasma (SDP) reduces immune activation in rodent models of inflammation and aging. We investigated whether the anti-inflammatory properties of SDP could ameliorate age-related cognitive deterioration and preserve brain homeostasis in an aging mouse model of senescence. Male senescence-accelerated prone 8 (SAMP8) mice were used. In Experiment 1, cognitive performance (n=10-14 mice/group) was analyzed by the novel object recognition test in 2-mo-old mice (2M group) and in mice fed a control diet or a diet supplemented with 8% SDP for 2 (4M-CTL and 4M-SDP groups) and 4 mo (6M-CTL and 6M-SDP groups). In Experiment 2, the permeability of the blood-brain barrier and junctional proteins in brain tissue was assessed, as well as synaptic density, oxidative stress markers, and inflammatory genes and proteins in mice from the 2M, 6M-CTL, and 6M-SDP groups ( n=5-11). Statistical analyses included one-factor ANOVA followed by Fisher's posthoc test. 6M-SDP mice had better cognitive performance than 6M-CTL mice in both short-term (P=0.024) and long-term (P=0.017) memory tests. In brain tissue, 6M-SDP mice showed reduced brain capillary permeability (P=0.034) and increased ZO1 and E-cadherin expression (both P<0.04) compared with 6M-CTL mice. SDP also prevented the NFκB activation observed in 6M-CTL mice (P=0.002) and reduced Il6 expression and hydrogen peroxide concentration (both P<0.03) observed in 6M-CTL mice. SDP also increased the concentration of IL10 (P=0.027), an anti-inflammatory cytokine correlated with memory preservation. In senescent SAMP8 mice, dietary supplementation with SDP attenuated cognitive decline and prevented changes in brain markers of neuroinflammation and oxidative stress.

Analysis of the Effect of IL-1β on Blood-Brain Barrier Permeability in M6 Glioma Mouse Model Using Intravital Microscopy.

We studied the effect of IL-1β on the permeability of brain capillaries in healthy mice. Intravital microscopy demonstrated that parenteral administration of IL-1β was followed by an increase in vascular permeability ensuring passage of free Alexa488 fluorescent label through the capillary walls, but not sufficient for penetration of liposomes. In addition, experiments on mice with intracranial M6 glioma showed penetration of liposomes through the walls of tumor capillaries after parenteral administration of IL-1β in a concentration of 2 μg/ml. Thus, the use of proinflammatory cytokine IL-1β in the therapy of brain tumors can significantly increase the therapeutic efficacy of drug delivery systems, in particular, for drugs poorly crossing the blood-brain barrier.

Effects of levocetirizine and diphenhydramine on regional glucose metabolic changes and hemodynamic responses in the human prefrontal cortex during cognitive tasks.

ObjectiveAntihistamines often have sedative side effects. This was the first study to measure regional cerebral glucose (energy) consumption and hemodynamic responses in young adults during cognitive tests after antihistamine administration.MethodsIn this double‐blind, placebo‐controlled, three‐way crossover study, 18 healthy young Japanese men received single doses of levocetirizine 5 mg and diphenhydramine 50 mg at intervals of at least six days. Subjective feeling, task performances, and brain activity were evaluated during three cognitive tests (word fluency, two‐back, and Stroop). Regional cerebral glucose consumption changes were measured using positron emission tomography with [18F]fluorodeoxyglucose. Regional hemodynamic responses were measured using near‐infrared spectroscopy.ResultsEnergy consumption in prefrontal regions was significantly increased after antihistamine administration, especially diphenhydramine, whereas prefrontal hemodynamic responses, evaluated with oxygenated hemoglobin levels, were significantly lower with diphenhydramine treatment. Stroop test accuracy was significantly impaired by diphenhydramine, but not by levocetirizine. There was no significant difference in subjective sleepiness.ConclusionsPhysiological “coupling” between metabolism and perfusion in the healthy human brain may not be maintained under pharmacological influence due to antihistamines. This uncoupling may be caused by a combination of increased energy demands in the prefrontal regions and suppression of vascular permeability in brain capillaries after antihistamine treatment. Further research is needed to validate this hypothesis.

Optimization of an in vitro human blood–brain barrier model: Application to blood monocyte transmigration assays

The blood–brain barrier (BBB) is a selectively permeable barrier that separates the circulating blood from the extracellular fluid of the brain and is an essential component in brain homeostasis. In vitro BBB models are valuable supporting tools that can precede and complement animal and human studies of the development and progression of the central nervous system diseases. At present, mono-, co-, and tri-culture models that use porcine, murine, or human cells have been developed. We have optimized a two-dimensional model of the human BBB using primary human brain microvascular endothelial cells and normal human astrocytes. We have validated the effectiveness of our model with transmigration assays of human blood monocytes toward CCL19, a natural ligand of the chemokine receptor CCR7. This model offers the following advantages:•It is simple, convenient, and requires small quantities of material, reagents, and primary cells.•It can be used to monitor cell migration through the BBB.•It can be used to assess brain capillary permeability in the presence of xenobiotic, pro-inflammatory, or other substances.

Expression of histamine receptor genes Hrh3 and Hrh4 in rat brain endothelial cells

Brain vascular endothelial cells express histamine H1 and H2 receptors, which regulate brain capillary permeability. We investigated whether H3 and H4 receptors are also expressed in these cells and may thus play a role in permeability regulation. An immortalized rat brain endothelial cell line RBE4 was used to assess the presence of H3 and H4 receptors. Reverse transcription-PCR (RT-PCR) and sequencing were used to identify the receptor mRNAs. The receptors were stimulated with histamine and immepip, and specific inverse agonists/antagonists ciproxifan and JNJ 7777120 were used to block H3 and H4 receptors, respectively. RT-PCR of mRNA extracted from cultured immortalized RBE4 cells revealed two rat H4 receptor gene (Hrh4) transcripts, one full-length (coding sequence 1173 bp), and one with a 164 bp deletion. Also, two rat H3 receptor gene (Hrh3) isoform mRNAs were expressed in RBE4 cells, and sequencing showed they were the full-length H3 receptor and the 144 bp deletion form. Both histamine and immepip (H3 and H4 receptor agonists) activated the Erk1/2 MAPK pathway in the RBE4 cells and in vivo in brain blood vessels by activating H4 receptors, as the H4 receptor-specific inverse agonists/antagonist JNJ 7777120, but not ciproxifan, H3 receptor antagonist, dose-dependently blocked this effect in RBE4 cells. Both Hrh3 and Hrh4 receptors are expressed in rat brain endothelial cells, and activation of the histamine H4 receptor activates the Erk1/2 cascade. H3 and H4 receptors in endothelial cells are potentially important for regulation of blood-brain barrier permeability, including trafficking of immunocompetent cells.

中药及其有效成分对血脑屏障作用的研究进展

血脑屏障是大脑脉管系统维持脑组织环境和功能稳定的重要特性。脑毛细管内皮细胞间的紧密连接(TJ)是血脑屏障的结构基础，它由跨膜蛋白、胞浆粘附蛋白和连接黏附分子及细胞骨架蛋白等共同组成，维持脑毛细血管内皮机械屏障和通透性。中药对多种脑源性疾病具有良好的疗效，这与中药有效成分透过血脑屏障或改善血脑屏障的形态结构和功能有关。 The blood brain barrier (BBB) is the important characteristic of the cerebral vasculature for maintaining the homeostasis of the brain environment and its function. The tight junctions (TJs) closely connecting the brain capillary endothelial cells (TJ) are the structural basis of BBB, which are composed of a transmembrane protein, intracellular adhesion protein and junctional adhesion molecules and cytoskeletal proteins, maintaining the brain capillary endothe-lial barrier and permeability. Traditional Chinese medicine (TCM) has excellent curative effect on various brain diseases, which might owe to the permeance of their active ingredients through the BBB and their effects of improving the morphological structure and function of the BBB.

Absence of Glial α-Dystrobrevin Causes Abnormalities of the Blood-Brain Barrier and Progressive Brain Edema

The blood-brain barrier (BBB) plays a key role in maintaining brain functionality. Although mammalian BBB is formed by endothelial cells, its function requires interactions between endotheliocytes and glia. To understand the molecular mechanisms involved in these interactions is currently a major challenge. We show here that α-dystrobrevin (α-DB), a protein contributing to dystrophin-associated protein scaffolds in astrocytic endfeet, is essential for the formation and functioning of BBB. The absence of α-DB in null brains resulted in abnormal brain capillary permeability, progressively escalating brain edema, and damage of the neurovascular unit. Analyses in situ and in two-dimensional and three-dimensional in vitro models of BBB containing α-DB-null astrocytes demonstrated these abnormalities to be associated with loss of aquaporin-4 water and Kir4.1 potassium channels from glial endfeet, formation of intracellular vacuoles in α-DB-null astrocytes, and defects of the astrocyte-endothelial interactions. These caused deregulation of tight junction proteins in the endothelia. Importantly, α-DB but not dystrophins showed continuous expression throughout development in BBB models. Thus, α-DB emerges as a central organizer of dystrophin-associated protein in glial endfeet and a rare example of a glial protein with a role in maintaining BBB function. Its abnormalities might therefore lead to BBB dysfunction.

TNF-alpha induced NFκB signaling and p65 (RelA) overexpression repress Cldn5 promoter in mouse brain endothelial cells

Inflammatory cytokine TNFα enhances permeability of brain capillaries constituting blood brain barrier (BBB). In the monoculture endothelial models of BBB TNFα alters tight junction (TJ) structure and protein content. Claudin-5 (Cldn5) is a key TJ protein whose expression in the brain endothelial cells is critical to the function of BBB. TNFα reduces Cldn5 promoter activity and mRNA expression in mouse brain derived endothelial cells but the regulatory elements and signaling mechanism involved are not defined. Here we report that TNFα acts through NFκB signaling and requires a conserved promoter region for the down-regulation of Cldn5 expression. Overexpression of the NFκB subunit p65 (RelA) alone repressed Cldn5 promoter activity in mouse brain endothelial cells. We observed partial loss of Cldn5 protein expression after prolonged TNFα treatment in primary endothelial culture isolated from C56BL/6 mice brain. Taken together, our results confirm and extend previous observations of TNFα induced down-regulation of Cldn5 expression in mouse brain endothelial cells.

The effects of ulinastatin on brain capillary permeability in severe acute pancreatitis in rats

胰性脑病是重症急性胰腺炎(SAP)的严重并发症,与大脑的毛细血管渗漏紧密相关.occludin蛋白是脑组织重要的紧密连接蛋白,与大脑毛细血管通透性密切相关.乌司他丁(UTI)是一种广谱的蛋白酶抑制剂,已经广泛用于临床,但其对胰性脑病的治疗及机制研究甚少.本研究旨在探讨occludin在SAP大脑毛细血管渗漏中的作用及UTI治疗胰性脑病的作用机制。

Delivering Drugs Into the Brain: Barriers and Possibilities

Delivering Drugs Into the Brain: Barriers and Possibilities

186 13N-ammonia pet of acute hepatic encephalopathy in cirrhosis patients: Brain capillary permeability and metabolism of ammmonia

186 13N-ammonia pet of acute hepatic encephalopathy in cirrhosis patients: Brain capillary permeability and metabolism of ammmonia

Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood–brain barrier endothelial cells

The mechanisms leading to stroke in stroke-prone spontaneously hypertensive rats (SHRSP) are not well understood. We tested the hypothesis that the endothelial tight junctions of the blood–brain barrier are altered in SHRSP prior to stroke. We investigated tight junctions in 13-week-old SHRSP, spontaneously hypertensive stroke-resistant rats (SHR) and age-matched Wistar–Kyoto rats (WKY) by electron microscopy and immunocytochemistry. Ultrathin sections showed no difference in junction structure of cerebral capillaries from SHRSP, SHR and WKY, respectively. However, using freeze-fracturing, we observed that the blood–brain barrier specific distribution of tight junction particles between P- and E-face in WKY (58.7±3.6%, P-face; 41.2±5.59%, E-face) and SHR (53.2±19.3%, P-face; 55.6±13.25%, E-face) was changed to an 89.4±9.9% predominant E-face association in cerebral capillaries from SHRSP. However, the expression of the tight junction molecules ZO-1, occludin, claudin-1 and claudin-5 was not changed in capillaries of SHRSP. Permeability of brain capillaries from SHRSP was not different compared to SHR and WKY using lanthanum nitrate as a tracer. In contrast, analysis of endothelial cell polarity by distribution of the glucose-1 transporter (Glut-1) revealed that its abluminal:luminal ratio was reduced from 4:1 in SHR and WKY to 1:1 in endothelial cells of cerebral capillaries of SHRSP. In summary, we demonstrate that early changes exist in cerebral capillaries from a genetic model of hypertension-associated stroke. We suggest that a disturbed fence function of the tight junctions in SHRSP blood–brain barrier endothelial cells may lead to subtle changes in polarity. These changes may contribute to the pathogenesis of stroke.

Treatment of brain edema with a nonpeptide arginine vasopressin V1 receptor antagonist OPC-21268 in rats.

Recent experimental evidence suggests that centrally released arginine vasopressin plays a significant role in brain capillary water permeability as well as in pathogenesis of vasogenic brain edema. The purpose of this study was to examine the effects of orally administered OPC-21268, a nonpeptide arginine vasopressin V1 receptor antagonist, on cold-induced brain edema in rats. Cold brain injury was induced for 1 minute in 140 rats. Treatment with OPC-21268, at dosages of 100 mg (n = 20), 200 mg (n = 20), and 300 mg/kg (n = 15), or with saline (n = 17) was started 1 hour after the induction of cold injury and was continued every 8 hours for 24 hours. Two percent Evans blue in saline (1 ml/kg) was administered intravenously before cold injury in another group of rats, 15 of which were saline-treated and 55 of which were OPC-21268-treated at the above dosages. After 24 hours, brain tissue water and electrolytes, brain tissue swelling, blood-brain barrier permeability to Evans blue, and plasma electrolytes and osmolality were determined. Compared with the saline-treated group, OPC-21268 treatment at the dosages of 200 and 300 mg/kg significantly reduced brain water content in both hemispheres (P<0.01). Swelling of the traumatized hemispheres was also significantly reduced at 200 and 300 mg/kg dosages (P<0.05). Brain tissue sodium content was significantly reduced at the dosage of 300 mg/kg (P<0.05). Blood-brain barrier permeability to Evans blue was significantly decreased in a dose-dependent manner compared with the saline-treated group (P<0.01). No significant changes were observed in other parameters. Our results indicate that OPC-21268 predominantly exerts a protective effect in areas where the maximum amount of blood-brain barrier breakdown occurs, and it is effective in the treatment of cold-induced vasogenic brain edema.

Changes in the permeability of the blood-brain barrier following sodium dodecyl sulphate administration in the rat.

The blood-brain barrier (BBB) arises from epithelial-like tight junctions that virtually cement adjoining capillary endothelium together in the brain microvasculature. Several experimental manipulations have been shown able to increase the permeability of brain capillaries, by altering endothelial cell membrane integrity or activating specific biochemical pathways involved in regulation of BBB functionality. Because of its amphiphilic nature, sodium dodecyl sulphate (an anionic surfactant widely used as solubilizer or stabilizer in several pharmaceutical preparations; SDS) may enter into interactions with the major membrane components, which are lipids and proteins. The aim of the present study was to determine the effect of an intracarotid infusion of SDS (25, 50 and 100 microg/kg; infusion rate: 3 ml/min for 30 s) on the functionality of the BBB in the rat. An extensive, dose-dependent Evans blue extravasation was observed, in the ipsilateral brain hemisphere, 15 min following SDS infusion. These results were confirmed by the significant increase in [14C]alpha-aminoisobutyric acid ([14C]AIB) transport (evaluated by calculating a unidirectional transfer constant, Ki, for the tracer from blood to brain) measured in several ipsilateral brain regions 2 min after SDS infusion; this SDS-elicited BBB opening to [14C]AIB proved to be reversible. Since the BBB is created by the plasma membrane and tight junctions of the endothelial cells, the change in BBB permeability caused by SDS might be explained as a nonspecific surfactant-membrane interaction. Furthermore, SDS might affect the functional characteristics of brain vascular endothelial cells by an interaction with specific BBB proteins and/or biochemical pathways. In conclusion, one can suggest that intracarotid infusion of SDS might provide a useful clinical approach for the intentional introduction of different substances into the brain. On the other hand, these findings should call attention to possible dangerous consequences of using SDS as solubilizer in drug excipients.

Increased delivery of a new cisplatin analogue (254-S) in a rat brain tumor by an intracarotid infusion of bradykinin.

The intracarotid infusion of bradykinin has been shown to selectively increase capillary permeability in a brain tumor without affecting either normal brain capillary permeability or the systemic blood pressure. We examined whether the intracarotid infusion of bradykinin could selectively increase the delivery of a new watersoluble antitumor agent, cis-diammine glycolato-platinum (254-S, 303.2 mol. wt.), to transplanted RG2 glioma in rats. The platinum contents in the brain, tumor tissues and plasma were measured using an atomic absorption spectrophotometer. The transfer ratio of 254-S from plasma to the tissues was calculated and expressed as the volume of plasma containing platinum per g tissue (Dp, microliter g-1). Intracarotid bradykinin infusion at a rate of 20 micrograms kg-1 min-1 increased the delivery of 254-S in the tumor tissue by 1.3-fold when compared with intracarotid infusion of 0.9% saline (48.78 +/- 18.11 vs. 37.12 +/- 12.53; p < 0.05). In normal brain tissue including the ipsilateral cortex, the contralateral basal ganglia and the contralateral cortex, bradykinin did not significantly increase the delivery of 254-S in comparison with 0.9% saline (12.28 +/- 9.53 vs. 10.70 +/- 5.05, 4.96 +/- 3.54 vs 4.96 +/- 4.80, 7.64 +/- 4.10 vs. 13.07 +/- 11.38, respectively). These results indicate that the intracarotid infusion of bradykinin selectively increases the delivery of 254-S to the brain tumor without affecting the normal brain. This method may, therefore, enhance the antitumor effect of 254-S for the treatment of brain tumors and also reduce neurotoxicity in the normal brain.

Pharmacological blood-brain barrier modification for selective drug delivery.

Vasoactive agents have been identified through studies of peritumoral edema and effects on systemic capillaries. Abnormal blood-brain barrier or blood-tumor barrier can develop transient increases in permeability with the intraarterial delivery of vasoactive agents. Normal blood-brain barrier resists the effects of these compounds because of a biochemical barrier that may inactivate or become inert to vasoactive agents. Vasoactive compounds, including leukotrienes, bradykinin, and histamine appear to selectively increase permeability in abnormal brain capillaries. Intracarotid infusion of leukotrienes, bradykinin, and other vasoactive agents can increase drug delivery to diseased tissue.

Enzymatic barrier protects brain capillaries from leukotriene C4

Leukotriene C4 (LTC4) increases vascular permeability in systemic, brain tumor, and ischemic brain capillaries, but not in normal brain capillaries. This study examines whether the abundance of gamma-glutamyl transpeptidase (gamma-GTP) in normal brain capillaries might act as an enzymatic barrier to vasoactive leukotrienes in the brain. Blood-brain barrier (BBB) permeability was determined by quantitative autoradiography using 14C-aminoisobutyric acid. Ischemia was produced by occluding the middle cerebral artery. Seventy-two hours after occlusion, gamma-GTP activity in ischemic brain disappeared, and LTC4 (4-micrograms total dose), which was infused into the carotid artery ipsilateral to the occlusion, selectively increased permeability, Ki, approximately twofold within core ischemic tissue and adjacent tissue, compared to vehicle alone in seven brains (15.53 +/- 6.03 vs. 7.29 +/- 3.36, p < 0.05, and 8.76 +/- 4.02 vs. 4.32 +/- 2.65, p < 0.05, respectively). No effect on BBB was seen in nonischemic brain tissue. Twenty-four hours postocclusion, gamma-GTP activity was still present, and LTC4 infusion did not increase permeability within ischemic tissue. However, inhibition of gamma-GTP with acivicin allowed LTC4 to increase permeability even 24 hours after occlusion in ischemic core and adjacent tissue compared to vehicle alone in seven brains (17.21 +/- 16.32 vs. 8.23 +/- 6.58, p < 0.05, and 11.78 +/- 7.96 vs. 4.56 +/- 1.93, p < 0.01, respectively). Acivicin almost completely blocked both the histochemical activity of gamma-GTP in brain capillaries and the metabolism of LTC4 in isolated bovine capillaries. These findings suggest that gamma-GTP may help normal brain capillaries resist the vasoactive effects of LTC4. In contrast, gamma-GTP is lost in injured brain capillaries, which allows LTC4 (in combination with other factors) to increase vascular permeability in ischemic brain and brain tumors.

Intracarotid infusion of RMP-7, a bradykinin analog: a method for selective drug delivery to brain tumors

The bradykinin analog, RMP-7, was investigated for its ability to selectively increase uptake of molecular tracers in RG2 glial tumors. When infused in low doses (0.1 microgram/kg/min) through the intracarotid artery ipsilateral to RG2 gliomas in rats, RMP-7 significantly increased the permeability of tumor capillaries to methotrexate and to four other tracers of varying molecular weights, compared to intracarotid infusion of vehicle alone. Tracers used to examine permeability included radiolabeled alpha-aminoisobutyric acid (M(r) 103 D), sucrose (M(r) 342 D), methotrexate (M(r) 454.5 D), inulin (M(r) 5000 D), and dextran (M(r) 70,000 D). Permeability was expressed as the unidirectional transfer constant, Ki (microliters/gm/min). The permeability (Ki) of tumors in the RMP-7 group compared to the vehicle control group was as follows: alpha-aminoisobutyric acid, 35.3 +/- 9.11 versus 12.7 +/- 4.56 (p < 0.001); sucrose, 16.5 +/- 3.83 versus 9.28 +/- 3.12 (p < 0.05); methotrexate, 26.3 +/- 10.3 versus 8.98 +/- 6.78 (p < 0.005); inulin, 13.5 +/- 3.23 versus 6.55 +/- 4.32 (p < 0.005); dextran, 15.2 +/- 3.42 versus 1.47 +/- 1.24 (p < 0.001). The permeability of RG2 gliomas to high-molecular-weight dextran (70,000 D) was 10.3-fold higher in the RMP-7 group than in the vehicle control group. Intracarotid infusion of RMP-7 did not significantly increase the blood volume in tumor or brain tissue. The permeability of normal brain capillaries was unaffected by intracarotid infusion of 0.1 microgram/kg/min RMP-7 relative to that achieved in tumor. These data support the idea that intracarotid infusion of RMP-7 will be a useful technique for selective delivery of antitumor compounds to brain tumors.

Bradykinin selectively opens blood-tumor barrier in experimental brain tumors.

Bradykinin, infused in low doses (10 micrograms/kg/min) through the carotid artery ipsilateral to RG2 glioma in rats, significantly increased the permeability in tumor capillaries to six different tracers of varying molecular weights compared with intracarotid infusion of saline alone. Permeability in normal brain capillaries was not significantly increased by intracarotid bradykinin infusion. Tracers used to examined permeability included radiolabeled alpha-aminoisobutyric acid (AIB; MW 103), sucrose (MW 342.3), inulin (MW 5000), and dextran (MW 70,000), horseradish peroxidase (HRP) and Evans blue (EB). Permeability was expressed as the unidirectional transfer constant K(i) (microliter/g/min). The permeabilities (K(i)) of tumors in the bradykinin group versus the control saline group for AIB, sucrose, inulin, and dextran were 25.91 +/- 6.78 vs. 13.95 +/- 4.29 (p < 0.01), 17.90 +/- 2.65 vs. 10.75 +/- 4.55 (p < 0.01), 23.92 +/- 6.99 vs. 6.20 +/- 4.37 (p < 0.01), and 17.84 +/- 1.00 vs. 1.47 +/- 1.24 (p < 0.001), respectively (mean +/- SD). Permeability of RG2 gliomas to high molecular weight dextran (70,000) was 12-fold higher in the bradykinin group than in the saline infusion group. Intracarotid infusion of bradykinin did not significantly increase the blood volume in tumor or brain tissue despite its known vasodilative effect. The permeability of normal brain capillaries was unaffected by intracarotid bradykinin infusion. The increased permeability was reversed 20 min after stopping the intracarotid infusion. Electron microscopic and gross qualitative analysis was performed using HRP and EB. Intracarotid bradykinin infusion increased HRP and EB within tumor tissue but not normal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid Metabolism in Neural Microvascular Endothelial Cells after Exposure to Lead in Vitro

Lead (Pb 2+) is known to alter the permeability of brain capillaries. A possible mechanism for this alteration may be related to the ability of Pb 2+ to substitute for Ca 2+. Products derived from phospholipid metabolism, namely eicosanoids and diacylglycerol, control endothelial permeability, are partly regulated by intracellular Ca 2+, and thus may be sensitive to Pb 2+. We asked in this study whether Pb 2+ increased arachidonic acid release or stimulated phosphatidylcholine breakdown in an in vitro model of brain capillaries, namely cultured bovine retinal endothelial (BRE) cells. Pb 2+ stimulated arachidonic acid release and phosphatidylcholine and phosphatidylinositol metabolism in the presence of ionomycin, but not by itself. More arachidonic acid was released than phosphorylcholine in BRE cells stimulated with ionomycin and Pb 2+, but the magnitudes of these responses were similar in cells exposed to ionomycin plus Ca 2+. Ionomycin plus Pb 2+ or plus Ca 2+ resulted in the activation of phospholipase A 2, since an increase in lysophosphatidylcholine and arachidonic acid was observed. Protein kinase C was not required for arachidonic acid release because release was observed in cells with a down-regulated enzyme. Ionomycin plus other metals (La 3+, Cd 2+, or Mg 2+) did not result in arachidonic acid release, but Cd 2+ or Co 2+ inhibited arachidonic acid release by more than 80% when cells were exposed to ionomycin with either Pb 2+ or Ca 2+. Thapsigargin or maitotoxin plus Ca 2+ increased arachidonic acid release that was inhibited by the receptor-dependent calcium channel antagonist SK&F 96365 but not by the voltage-dependent calcium channel antagonist nifedipine. However, thapsigargin or maitotoxin plus Pb 2+ failed to stimulate arachidonic release. Since in this in vitro model Pb 2+ stimulated phospholipid metabolism solely in the presence of an ionophore, the increase in permeability observed in Pb 2+-exposed animals is probably not due to a release of metabolites of arachidonic acid.