Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2 receptor.

The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys-LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys-LTs by RP-HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A(4) into LTC(4) were studied by repeated incubations with LTA(4) and immunogold electron microscopy. The CysLT(2) receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration-response curves in HUVECs and in transfected COS-7 cells were used to characterize a novel specific CysLT(2) receptor antagonist (pA(2) of 8.33 and 6.79 against CysLT(2) and CysLT(1) receptors, respectively). The results obtained provide evidence that the mGST-II synthesizing LTC(4) in HUVECs is pharmacologically distinguishable from the LTC(4)-synthase (IC(50) of MK886 <5 µM for LTC(4)-synthase and >30 µM for mGST-II), is not suicide-inactivated and is strategically located on endothelial transport vesicles. The CysLT(2) receptor is responsible for the increase in intracellular Ca(2+) following exposure of HUVECs to cys-LTs and is coupled to a pertussis toxin-insensitive G(q) protein. The synthesis of cys-LTs from LTA(4) by endothelial cells is directly associated with the activation of the CysLT(2) receptor (EC(50) 0.64 µM) in a typical autocrine fashion.

Molecular anatomy of the cerebral microvessels in the isolated guinea-pig brain.

Isolated organ preparations represent valuable models for biomedical research, provided that the functional and morphological integrity of vascular and parenchymal compartments is preserved. In this investigation, we have studied the molecular organization of the cerebral microvessels in the isolated guinea-pig brain maintained in vitro by arterial perfusion, a preparation previously proposed as a model of blood-brain barrier (BBB). Using lectin cytochemistry and immunohistochemistry, we examined the microvasculature of the cerebral cortex after 5 h in vitro to assess: (a) the structure of the endothelial glycocalyx at microscopical and ultrastructural level; (b) the distribution of the junctional molecules occludin, ZO-1, PECAM-1 and vinculin; (c) the distribution of basal lamina molecules, such as collagen type IV, laminin and heparan sulfate proteoglycan. All these components of microvessel wall have been previously shown to be vulnerable to ischemic conditions and their organization could be altered in consequence of the transient hypoxia associated with the brain isolation procedure. Our observations demonstrate that the distribution pattern of the molecules considered (i) is comparable to that shown in the cerebral microvasculature of other mammals and (ii) is similar in brains maintained in vitro and in control brains perfused in situ with fixative. The complex of our observation indicates that the molecular organization of the cerebral microvessels is preserved in isolated guinea-pig brain, thus indicating that these preparations can be used to study the cerebrovascular structure and blood-brain barrier function in a variety of experimental conditions.

Differential expression of several molecules of the extracellular matrix in functionally and developmentally distinct regions of rat spinal cord.

We have examined the regional distribution of several chondroitin sulfate proteoglycans (neurocan, brevican, versican, aggrecan, phosphacan), of their glycosaminoglycan moieties, and of tenascin-R in the spinal cord of adult rat. The relationships of these molecules with glial and neuronal populations, identified with appropriate markers, were investigated by using multiple fluorescence labeling combined with confocal microscopy. The results showed that the distribution of the examined molecules was similar at all spinal cord levels but displayed area-specific differences along the dorso-ventral axis, delimiting functionally and developmentally distinct areas. In the gray matter, laminae I and II lacked perineuronal nets (PNNs) of extracellular matrix and contained low levels of chondroitin sulfate glycosaminoglycans (CS-GAGs), brevican, and tenascin-R, possibly favoring the maintenance of local neuroplastic properties. Conversely, CS-GAGs, brevican, and phosphacan were abundant, with numerous thick PNNs, in laminae III-VIII and X. Motor neurons (lamina IX) were surrounded by PNNs that contained all molecules investigated but displayed various amounts of CS-GAGs. Double-labeling experiments showed that the presence of PNNs could not be unequivocally related to specific classes of neurons, such as motor neurons or interneurons identified by their expression of calcium-binding proteins (parvalbumin, calbindin, calretinin). However, a good correlation was found between PNNs rich in CS-GAGs and the neuronal expression of the Kv3.1b subunit of the potassium channel, a marker of fast-firing neurons. This observation confirms the correlation between the electrophysiological properties of these neurons and the specific composition of their microenvironment.

Mutation of SOD1 in ALS: a gain of a loss of function.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by motoneuron loss. Some familial cases (fALS) are linked to mutations of superoxide dismutase type-1 (SOD1), an antioxidant enzyme whose activity is preserved in most mutant forms. Owing to the similarities in sporadic and fALS forms, mutant SOD1 animal and cellular models are a useful tool to study the disease. In transgenic mice expressing either wild-type (wt) human SOD1 or mutant G93A-SOD1, we found that wtSOD1 was present in cytoplasm and in nuclei of motoneurons, whereas mutant SOD1 was mainly cytoplasmic. Similar results were obtained in immortalized motoneurons (NSC34 cells) expressing either wtSOD1 or G93A-SOD1. Analyzing the proteasome activity, responsible for misfolded protein clearance, in the two subcellular compartments, we found proteasome impairment only in the cytoplasm. The effect of G93A-SOD1 exclusion from nuclei was then analyzed after oxidative stress. Cells expressing G93A-SOD1 showed a higher DNA damage compared with those expressing wtSOD1, possibly because of a loss of nuclear protection. The toxicity of mutant SOD1 might, therefore, arise from an initial misfolding (gain of function) reducing nuclear protection from the active enzyme (loss of function in the nuclei), a process that may be involved in ALS pathogenesis.

Distribution of Aquaporin 4 in rodent spinal cord: relationship with astrocyte markers and chondroitin sulfate proteoglycans.

Water balance between cells and extracellular compartments is essential for proper functioning of the central nervous system, as demonstrated by its perturbations in pathological conditions. Aquaporin 4 (AQP4) is the predominant water channel in brain and spinal cord, where it is present mainly on astrocytic endfeet contacting vessels. A role in water homeostasis control has been proposed also for the extracellular matrix, that in brain consists mainly of chondroitin sulfate proteoglycans (CSPGs). Using cytochemical and immunocytochemical techniques, we investigated their distribution in rodent spinal cord, to better understand the role of these two classes of molecules. The results show that in spinal gray matter AQP4 labeling is intense in all perivascular profiles and (1) displays a marked dorsoventral gradient in the neuropil; and (2) coexists extensively with glial glutamate transporter-1 (GLT-1) but scarcely with glial fibrillary acidic protein (GFAP). In white matter the overlap between AQP4, GLT-1, and GFAP is almost complete. Ultrastructural examination shows that AQP4-labeled astrocytic processes surround blood vessels, neuronal perikarya and processes, and both asymmetric and symmetric synapses, indicating that the protein may be involved in the regulation of water fluxes around both inhibitory and excitatory synapses. CSPGs, visualized by labeling with Wisteria floribunda agglutinin, show a distribution complementary to that of AQP4, being absent or weekly expressed in AQP4-enriched areas. These findings suggest that different mechanisms may contribute to the regulation of water homeostasis in different spinal cord regions.