István A. Krizbai

The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase itch.

Tight junctions create a highly selective diffusion barrier between epithelial and endothelial cells by preventing the free passage of molecules and ions across the paracellular pathway. Although the regulation of this barrier is still enigmatic, there is evidence that junctional transmembrane proteins are critically involved. Recent evidence confirms the notion thatoccludin, a four-pass integral plasma-membrane protein, is a functional component of the paracellular barrier. The overall hydrophilicity of occludin predicts two extracellular loops bounded by NH2- and COOH-terminal cytoplasmic domains. To date, the binding of the COOH terminus of occludin to intracellular proteins is well documented, but information concerning the function of the cytoplasmic NH2 terminus is still lacking. Using yeast two-hybrid screening we have identified a novel interaction between occludin and the E3 ubiquitin-protein ligase Itch, a member of the HECT domain-containing ubiquitin-protein ligases. We have found that the NH2-terminal portion of occludin binds specifically to a multidomain of Itch, consisting of four WW motifs. This interaction has been confirmed by our results from in vivoand in vitro co-immunoprecipitation experiments. In addition, we provide evidence that Itch is specifically involved in the ubiquitination of occludin in vivo, and that the degradation of occludin is sensitive to proteasome inhibition.

Systemic nitroglycerin increases nNOS levels in rat trigeminal nucleus caudalis.

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraineurs a delayed attack of unknown mechanisms. Subcutaneous nitroglycerin (10 mg/kg) produced a significant increase of nitric oxide synthase (NOS)- and c-fos-immunoreactive neurons in the cervical part of trigeminal nucleus caudalis in rats after 4 h. This effect was not observed in the thoracic dorsal horn. Similar increase of NOS and c-fos was obtained in the brain stem after a somatic nociceptive stimulus, i.e. on the side of the formalin injection in the lip. Nitric oxide is thus able to increase NOS availability in second order nociceptive trigeminal neurons, which may be relevant for central sensitization and the understanding of its effect in migraine.

Expression of glutamate receptors on cultured cerebral endothelial cells

Activation of glutamate receptors has been shown to mediate a large number of neuronal processes such as long‐term potentiation and ischemic damage. In addition to neurons and glia, glutamate receptors may occur on cerebral endothelial cells (CECs). The aim of the present study was to determine which glutamate receptors are expressed in CECs and to demonstrate the functional presence of such channels. By using reverse transcriptase‐polymerase chain reaction, we showed that primary cultures of rat CECs express N‐methyl‐D‐aspartate (NMDA) receptors (NR1 subunit, which is necessary for the formation of functional NMDA receptors, and NR2A–C subunits), 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐4‐isoxazolyl‐propionate (AMPA) receptors (GLUR1–4 subunits), and metabotropic receptors (mGLUR). Exposure of the cultures to 2 mM glutamate, a well‐established mediator of ischemic damage, for 30 min increased significantly the phosphorylation of calcium/calmodulin‐dependent protein kinase II even after 10‐ and 60‐min recovery times. This effect could be prevented by the NMDA blocker MK‐801. The presence of multiple glutamate receptor types may confer a finely tuned responsiveness of the cerebral endothelium to glutamate in physiological and pathological conditions. J. Neurosci. Res. 54:814–819, 1998.

Effect of oxidative stress on the junctional proteins of cultured cerebral endothelial cells

Summary1. There is increasing evidence that the cerebral endothelium and the blood–brain barrier (BBB) plays an important role in the oxidative stress-induced brain damage. The aim of the present study was to investigate the role of interendothelial junctional proteins in the BBB permeability increase induced by oxidative stress.2. For the experiments, we have used cultured cerebral endothelial cells exposed to hypoxia/reoxygenation or treated with the redox cycling quinone 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) in the presence or absence of glucose. The expression of junctional proteins and activation of mitogen activated protein kinases (MAPK) was followed by Western-blotting, the interaction of junctional proteins was investigated using coimmunoprecipitation.3. Oxidative stress induces a downregulation of the tight junction protein occludin expression which is more pronounced in the absence of glucose. Furthermore, oxidative stress leads to disruption of the cadherin-β-catenin complex and an activation of extracellular signal-regulated kinase (ERK1/2), which is more intense in the absence of glucose.4. We have shown that one of the causes of the BBB breakdown is probably the structural alteration of the junctional complex caused by oxidative stress, a process in which ERK1/2 may play an important role.

Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages

Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations, micronuclei, and apoptosis

There is evidence accumulating that brain microvasculature is involved critically in oxidative stress‐mediated brain damage. Cultured cerebral microvascular endothelial cells were used to demonstrate the cytotoxic and genotoxic effects elicited by hypoxia/reoxygenation and DMNQ treatment in vitro. In addition, the effect of glucose deprivation during oxidative insult was assessed. The parameters determined were: 1) chromosomal aberrations; 2) induction of micronuclei; and 3) apoptosis. Our results indicate that both the exposure of the cerebral endothelial cells to 24 hr of hypoxia followed by 4 hr of reoxygenation, and treatment with the redox cycling quinone DMNQ, increased markedly the occurrence of chromosomal aberrations and micronuclei. It was found that expression of p53 was induced by oxidative stress, particularly when glucose had been omitted from the culture medium. Aglycemic culture conditions in general exacerbated the cytotoxic effects of oxidative insults, as evidenced by the increase in apoptotic cells and the decrease in the mitotic index. Interestingly, neither an elevation of cell lysis nor an increase in necrosis has been observed during our experiments. In summary, our data indicate that oxidative stress exerts considerable genotoxic and cytotoxic effects on cerebral endothelial cells, which might contribute to the progression of tissue damage in the central nervous system.

Functional Expression of the Serotonin Transporter in Immortalized Rat Brain Microvessel Endothelial Cells

There is evidence from recent studies that the brain endothelium (of capillaries and/or larger vessels) may serve as a specific target for serotonin [5‐hydroxytryptamine (5‐HT)]. This neurotransmitter is expected to be involved in the regulation of the blood‐brain barrier (BBB) permeability and/or of the cerebral blood flow via receptor‐mediated mechanisms. Effective control of these processes depends on a speedy uptake and metabolism of released 5‐HT molecules. To realize this, a similar mechanism of 5‐HT uptake as in brain may exist at the BBB. In this study, we have demonstrated using RT‐PCR that 5‐HT transporter mRNA is present in the brain endothelium and that a saturable transport system for 5‐HT is functionally expressed in immortalized rat brain endothelial cells (RBE4 cells). These cells take up [3H]5‐HT by an active saturable process with a Km value of 397 ± 64 nmol/L and a transport capacity of 51.7 ± 3.5 pmol · g‐1· min‐1. The 5‐HT uptake depends on Na+, as indicated by the replacement of NaCl by LiCl. The 5‐HT uptake was sensitive to specific 5‐HT transport inhibitors such as paroxetine, clomipramine, fluoxetine, and citalopram but not to inhibitors of the vesicular amine transporter such as reserpine or tetrabenazine. Our results demonstrate that cerebral endothelial cells are able to participate actively in the removal and metabolism of the released 5‐HT, which supports the concept of direct serotoninergic regulation of the BBB function.

Vasoactive substances produced by cultured rat brain endothelial cells

The vasoactive substances synthesized by primary cultures of rat brain endothelial cells were investigated and compared to those from two, immortalized cell lines, RBE4 and GP8. The vasoactivity of endothelium-derived substances was measured on isolated canine coronary artery. Vascular tone was significantly decreased by both primary and GP8, but not by RBE4 cells. Indomethacin pretreatment of primary and GP8 cells turned vasorelaxation into contraction while N(omega)-nitro-L-arginine pretreatment decreased the vasorelaxation induced by primary, but not by GP8 cells. Eicosanoid production was determined after incubation with [14C]arachidonic acid. The predominant vasoactive eicosanoid was prostaglandin E2 in both primary and GP8 cells. RBE4 cells synthetized mainly prostaglandin E2 and thromboxane B2 and significantly less prostaglandin E2 than did either primary or GP8 cells. The capacity of cerebral endothelium to regulate vascular tone by production of dilator and constrictor substances can be preserved under certain circumstances in immortalized cell lines.

Expression of Protein Kinase C Family Members in the Cerebral Endothelial Cells

Abstract: The protein kinase C (PKC) family is composed of at least four conventional (α, βI, βII, and γ) and several related novel (δ, ε, η, and ζ) isoforms with different distribution and sensitivity to Ca2+ and phorbol esters. The enzyme is known to be present in cerebral endothelial cells. We have investigated the occurrence of seven isoforms (α, β, γ, δ, ε, η, and ζ) by using reverse transcriptase‐polymerase chain reaction in rat brain, in a freshly isolated brain microvessel fraction, in primary cultures of rat brain endothelial cells, in an immortalized rat brain endothelial cell line, and in aortic endothelial cell cultures. Brain tissue contained all seven investigated isoforms. A similar expression pattern was seen in freshly purified microvessels, but the PKC‐γ isoform could not be detected. Primary cultures of endothelial cells expressed PKC‐α, ‐β, ‐δ, ‐η, and ‐ε isoenzymes, whereas the immortalized cell line expressed PKC‐α, ‐δ, ‐ε, and ‐η. The rat aortic endothelium contained only PKC‐α and ‐δ isoforms. The variety of expression patterns of PKC family members in endothelial cells of different type may reflect differences in the functional responsiveness to environmental stimuli. Because PKC has been shown to be involved in the regulation of the blood‐brain barrier permeability, the presence of different isoforms may confer a sophisticated intracellular regulatory mechanism to the brain endothelial cells.

Regulation of cerebral endothelial cell morphology by extracellular calcium

Cerebral endothelial cells interconnected by tight and adherens junctions constitute the structural basis of the blood-brain barrier. Extracellular calcium ions have been reported to play an important role in the formation and maintenance of the junctional complex. However, little is known about the action of calcium depletion on the structural characteristics of cerebral endothelial cells. Using atomic force microscopy we analyzed the effect of calcium depletion and readdition on the shape and size of living brain endothelial cells. It was found that the removal of extracellular calcium from confluent cell cultures induced the dissociation of the cells from each other accompanied by an increase in their height. After readdition of calcium a gradual recovery was observed until total confluency was regained. We have also demonstrated that Rho-kinase plays an important role in the calcium-depletion-induced disassembly of endothelial tight and adherens junctions. The Rho-kinase inhibitor Y27632 could prevent the morphological changes induced by a lack of calcium as well. Our results suggest that calcium depletion induces Rho-kinase-dependent cytoskeletal changes that may be partly responsible for the disassembly of the junctional complex.