Occludin binds to the SH3-hinge-GuK unit of zonula occludens protein 1: potential mechanism of tight junction regulation.

The interaction between tight junction proteins occludin and zona occludens protein 1 (ZO-1) was clarified. The sequence cc1 within the hinge region of ZO-1, connecting its SH3 and GuK domains, was identified as a new association site for the occludin C-terminus, core binding area GLRSSKRNLRKSR (mouse ZO-1(606-618)). Occludin also bound to the sequence H2 within GuK, core area HKLRKNNH (ZO-1(759-766)). In occludin, the binding core was ELSRLDKELDDYREESEEY (mouse occludin(455-473)). Helicity of the sequences was suggested by circular dichroism. Because basic residues in ZO-1, acidic residues in occludin (underlined), coiled-coil helix-forming leucine heptad motifs (bold) in occludin and, probably, in cc1 were essential, we conclude that interactions were both helical and ionic. Moreover, the GuK domain bound other GuK molecules, suggesting oligomerization of ZO-1. Generally, the assumption is supported that the SH3-hinge-GuK region represents a functional and regulatory unit in ZO-1 forming a multiprotein tight junction complex with occludin.

Use of surface plasmon resonance for real-time analysis of the interaction of ZO-1 and occludin.

Surface plasmon resonance (SPR) spectroscopy was applied to study in real time, the interaction between the tight junction proteins ZO-1 and occludin. To imitate the morphology of tight junctions, a cytosolic tail of mouse occludin was immobilised at the sensor and guanylate kinase-like domain (Guk) was allowed to pass over the modified chip surface. The Guk domain of ZO-1 (residues 644-812) was found to bind to the cytoplasmic, carboxy-terminal region of occludin (residues 378-521). This interaction was systematically characterised with respect to the concentrations of both proteins and the binding conditions. Under the given experimental conditions, association and dissociation showed saturation kinetics, with affinity in micromolar range: k(a) = 4.14 +/- 0.52 x 10(3) M(-1) s(-1), k(d) = 3.04 +/- 0.38 x 10(-3) s(-1), K(D) = 639 +/- 51 nM. The results support the hypothesis that the Guk domain of ZO-1 is involved in the recruitment of the transmembrane protein occludin at tight junctions by interacting with the cytosolic carboxy-terminal sequence of occludin, located far from the cell membrane. We demonstrate the use of SPR spectroscopy as an effective approach for characterisation of the interactions of junction proteins.

Protein kinase C regulates the phosphorylation and cellular localization of occludin.

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.

Thiol-induced nitric oxide release from 3-halogeno-3,4-dihydrodiazete 1,2-dioxides.

In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.

Nitric oxide protects blood-brain barrier in vitro from hypoxia/reoxygenation-mediated injury.

A cell culture model of blood-brain barrier (BBB, coculture of rat brain endothelial cells with rat astrocytes) was used to investigate the effect of nitric oxide (.NO) on the damage of the BBB induced by hypoxia/reoxygenation (H/R). Permeability coefficient of fluorescein across the endothelium was used as a marker of BBB tightness. The permeability coefficient increased 5.2 times after H/R indicating strong disruption of the BBB. The presence of the .NO donor S-nitroso-N-acetylpenicillamine (SNAP, 30 microM), authentic .NO (6 microM) or superoxide dismutase (50 units/ml) during H/R attenuated H/R-induced increase in permeability. 30 microM SNAP or 6 microM .NO did not influence the function of BBB during normoxia, however, severe disruption was observed using 150 microM of SNAP and more than 24 microM of .NO. After H/R of endothelial cells, the content of malondialdehyde (MDA) increased 2.3 times indicating radical-induced peroxidation of membrane lipids. 30 microM SNAP or 6 microM authentic .NO completely prevented MDA formation. The results show that .NO may effectively scavenge reactive oxygen species formed during H/R of brain capillary endothelial cells, affording protection of BBB at the molecular and functional level.

Synthesis and spin trapping applications of 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N.

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

[Studies of in vitro and in vivo derived 1,2-diazetine and nitronylnitroxide as donors and acceptors of nitric oxide]. / Issledovaniia in vitro i in vivo proizvodnykh 1,2-diazetina i nitronilnitroksida v kachestve donorov i aktseptorov oksida azota.

The series of nitronylnitroxyl radicals (NNR) were studied as paramagnetic scavengers of nitric oxide. These radicals react with NO with rate constants of (0.6-1.1).10(4) M-1.sec-1 forming stable iminonitroxyl radicals. They can be used to assay nitric oxide in solutions by EPR spectroscopy; the sensitivity of the method is 1 microM for the detection of NO concentration and 0.3 nM/sec for the measurements of the rates of NO generation for 1 h in 0.2 ml sample. To overcome fast reduction of the radicals in biological samples, charged NNR was incorporated into the inner volume of large unilamellar phosphatidylcholine liposomes thus decreasing the rates of NNR reduction about 1000-fold. The method was used for the NO synthase activity assay in rat cerebellum cytosol. NNR was used to study the kinetics of the decomposition of 3,4-dihydro-1,2-diazete 1,2-dioxides (DD). Several DD derivatives at 5-80 microM concentrations are very effective vasodilators in perfused rat tail artery. Intraperitoneal injection of several DD (40-200 micrograms/kg weight). in hereditary hypertensive rats (ISIAH-strain) significantly (by 30%) decreased systolic arterial blood pressure whereas similar effect of trinitroglycerin was detected at significantly higher dose (900 micrograms/kg weight).

Kinetics of nitric oxide liberation by 3,4-dihydro-1,2-diazete 1,2-dioxides and their vasodilatory properties in vitro and in vivo.

Derivatives of 3,4-dihydro-1,2-diazete 1,2-dioxides (DD) have been investigated as NO donors in vitro and in vivo. Using nitronylnitroxides as spin traps for NO, these compounds were shown to decompose in water solutions at physiological pH and temperature, producing two molecules of NO per one DD molecule. Rate constants of DD decomposition were found to be in the range from 10(-8) to 6.5 x 10(-7) c-1 in water and between 3 x 10(-7) and 1.6 x 10(-5) c-1 in dimethylsulfoxide. In vitro experiments performed with perfused rat tail artery showed that some of DD derivatives are highly effective vasodilators in concentrations from 5 to 80 microM while standard NO donor 3-(4-morpholino)-sydnonimine, SIN-1, does not lead to arterial vasodilation in these concentrations. Significant (up to 30%) decrease of systolic arterial blood pressure was observed in hereditary hypertensive rats (ISIAH-strain) when some of DD were injected intraperitoneally in doses 40-200 micrograms/kg b.w., while the same effect of trinitroglycerin, TNG, was found at much higher dose equal to 900 micrograms/kg b.w.

Spin trapping of nitric oxide by nitronylnitroxides: measurement of the activity of no synthase from rat cerebellum.

Nitric oxide (NO) has been shown to be an important mediator in vasodilation, neurotransmission and cellular cytotoxicity. We investigated a new series of nitronylnitroxyl radicals (NNR) as spin traps for NO. It was found these radicals react with NO with rate constants of about 10(4) M-1c-1 forming stable iminonitroxides with dramatic changes in EPR spectra. To overcome fast reduction of the radicals (a few seconds in rat cerebella cytosol), NNR with charged trimethylammoniophenyl group (Ib) was incorporated into the inner volume of large unilamellar phosphatidylcholine liposomes. In this case the reduction of the radical Ib in rat cerebella cytosol is slow (ca. 1% per min). The rate of NO production by NO synthase from rat cerebellum measured by NNR, Ib, is in a reasonable agreement with that obtained by spectrophotometric method.