Endothelial nuclear bulging: Morphological evidence for an intraglomerular perfusion regulating mechanism in the axolotl

Kidneys of the Axolotl (Ambystoma mexicanum: Urodela, Amphibia) have been investigated by light and electron microscopy as well as microvascular corrosion casting. Numerous glomerular endothelial nuclei bulge into the glomerular capillary lumen. It is concluded that these cells virtually stop the blood-flow in the respective vessel by blockage of erythrocyte passage. A proposal for an intraglomerular regulation mechanism of capillary perfusion (and thus filtration) by endothelial isometric contraction in the Axolotl is made on the basis of morphological methods.     

Blood–Brain Barrier Protein and Phosphorylation and Dephosphorylation

Capillaries in vertebrate brain have unique permeability properties that make up the blood-brain barrier (BBB). Although it is known that capillaries are innervated by nerve endings of intracerebral origin and that brain capillary function is likely acutely regulated by neuronal inputs, the possible mechanisms of neuronal regulation of capillary function are at present unknown. One possible mode of regulation is via the phosphorylation of brain capillary proteins. The present studies characterize, for the first time, the major phosphoproteins in the bovine brain capillary using both intact bovine brain capillaries and plasma membrane fractions from bovine brain capillaries. The patterns of endogenous phosphorylation of capillary proteins are compared to similar patterns obtained with synaptosomal (P2) fractions from bovine brain. The major findings of this study are: (a) The activity of protein phosphorylation in brain capillaries is localized almost exclusively to the capillary plasma membrane, and is nearly comparable to the activity of protein phosphorylation in synaptosomal membranes. (b) A major phosphoprotein doublet in the capillary fraction comigrates on a sodium dodecyl sulfate gel with a major phosphoprotein doublet of approximate molecular weight of 80K in the synaptosomal fraction, and the latter is presumed to be synapsin I; in dephosphorylation assays the synaptosomal 80K phosphoprotein doublet is not subject to measurable dephosphorylation, whereas the capillary 80K doublet is subject to rapid dephosphorylation, and is essentially completely dephosphorylated within 5 s at 0 degrees C. (c) A prominent triplet of phosphoproteins with molecular weight of 50-55K is present in the capillary fraction, and is not present in the synaptosomal fraction; thus, this 50-55K triplet of phosphoproteins appears specific for brain capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression cloning of TMEM16A as a calcium-activated chloride channel subunit

Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.     

Catecholamines stimulate the IFN-gamma-induced class II MHC expression on bovine brain capillary endothelial cells.

The brain has been considered for a long time as an immunologically privileged site because of the lack of a true lymphatic system and the existence of several barriers that isolate it from the periphery. In the last few years, it became evident that cells in the central nervous system (astrocytes, microglial cells, and brain capillary endothelial cells) can be induced to express class II MHC and present Ag to T lymphocytes. The brain capillary endothelial cells, which are strategically located at the interface between blood and brain, could be involved in the initiation of immune responses within the brain parenchyma. We have previously characterized bovine brain capillary endothelial cells in culture and shown that they maintain in vitro a fully differentiated phenotype associated with the blood-brain barrier endothelium. In order to assess the role of these cells in the development of immune responses in the brain, we initiated the present study on the regulation of their class II MHC surface expression. Our data indicate that this expression on bovine brain capillary endothelial cells is inducible by IFN-gamma and further stimulated by catecholamines through activation of beta-adrenergic receptors. However, this latter effect is not mimicked by forskolin, theophylline, or dibutyryl-cAMP, suggesting the involvement of a cAMP-independent mechanism.     

In vitro development of blastema cells in axolotl limb regeneration: effect of insulin and nerve extracts on cellular proliferation

For the purpose of investigating the nature of the nervous factor which controls cell proliferation in limb blastema of Newts, we have cultured primary mesenchymous cells from limb blastemas of Axolotl. The cultures were carried out in Petri dishes (Primaria, Falcon) with a basal medium with contained diluted MEM supplemented with hormones (insulin, somatotropin, hydrocortisone and thyroxine). In this medium, the cells disperse from the explant from the 4th day of culture and begin to divide from the 7th day; 3 weeks later the culture begins to decline. During the course of culture, beginning at the 8th day, differentiation of myotubes and chondrogenesis occur. The mitotic index, measured on the 16th day after 48 hr of colchicine treatment, is about 1.6%. Addition of foetal calf serum to the basal medium favours cell migration and survival and stimulates proliferation (mitotic: index 6%); beef embryo extract has no effect on cell migration and a small effect on proliferation (mitotic index: 2.3%). Addition to the basal medium of insulin or nerve extracts (brain and spinal cord of adult newts, brain of 12 days chick embryos) 6 days before we measure the mitotic index stimulates proliferation in proportion to the dose, up to 6 times the mitotic index in basal medium. These results are discussed with respect to the problem of cell proliferation control during limb regeneration.     

Transport of ions across the blood-brain barrier

Capillaries in the brain are formed by a uniquely specialized endothelial cell that regulates the movement of substances between blood and brain. Although they provide an impermeable barrier to some solutes, brain capillary endothelial cells facilitate the transcapillary exchange of others. In addition, they contain specific enzymes that contribute to a metabolic blood-brain barrier by limiting the movement of compounds such as neurotransmitters across the capillary wall. Studies of sodium and potassium transport by brain capillaries indicate that the endothelial cell contains distinct types of ion transport systems on the two sides of the capillary wall, i.e., the luminal and antiluminal membranes of the endothelial cell. As a result, specific solutes can be pumped across the capillary against an electrochemical gradient. These transport systems are likely to play a role in the active secretion of fluid from blood to brain and in maintaining a constant concentration of ions in the brain's interstitial fluid. In this way, the brain capillary endothelium is structurally and functionally related to an epithelium.     

Activation by Nitric Oxide of Guanylate Cyclase in Endothelial Cells from Brain Capillaries

Endothelial cells (ECs) from brain microvessels respond to exogenous nitric oxide (NO) donor molecules (N-ethoxycarbonyl-3-morpholinosydnonimine and sodium nitroprusside) with large (greater than 15-fold) increases in cyclic GMP (cGMP) levels. Comparable actions of sodium nitroprusside were observed in vascular smooth muscle cells and in neuroblastoma cells. Coculturing brain capillary ECs in the presence of N1E-115 neuroblastoma cells increased their cGMP levels fourfold. A further increase was observed in the presence of 50 nM neurotensin, although brain capillary ECs lack receptor sites for neurotensin. The neuroblastoma cell-dependent formation of cGMP was suppressed by 0.1 mM L-NG-monomethylarginine, indicating that NO, produced by N1E-115 cells in response to neurotensin, activated guanylate cyclase in brain capillary ECs. Similarly, culturing brain capillary ECs in the presence of aortic ECs increased their cGMP content in a manner that was amplified by bradykinin and that was inhibited by L-NG-monomethylarginine. Bradykinin had no action in pure cultures of brain capillary ECs. It is concluded that brain capillary ECs express high levels of guanylate cyclase activity that could be activated by exogenous NO donor molecules and by NO produced by neuroblastoma cells and by aortic ECs in response to specific agonists. Brain capillary ECs are thus potential target cells for brain-derived NO.     

Oxygen Free-Radical Reduction of Brain Capillary Rubidium Uptake

Free radicals are proposed to play a role in the injury following cerebral ischemia in which cerebral edema is a prominent feature. To determine whether free radicals might alter the movement of ions and water across the blood-brain barrier, we examined their effect on brain capillary transport. Rat brain capillaries were isolated, incubated with a system that generates free radicals, and various capillary transport systems were studied. Rubidium uptake was reduced 74% whereas rubidium efflux, glucose transport, and capillary water space were unchanged. The results following the addition of radical scavengers indicated that hydrogen peroxide or a related free radical was the toxic species. These data suggest that free radicals can impair capillary endothelial cell mechanisms that help maintain homeostasis of electrolytes and water in brain.     

The blood-brain barrier: an alternative hypothesis

Brain blood vessels, unlike most vessels elsewhere in the body, exhibit a blood-brain barrier (BBB) to certain substances, e.g. trypan blue. Under some circumstances this barrier is no longer effective and the permeability of the vessels increases. Although capillarization is much less in the brain than in many other organs, e.g. heart muscle, total cerebral blood flow per minute is enormous. Consequently, to accommodate a large blood volume with a limited capillary bed, the velocity of blood through brain vessels must be extremely fast. The hypothesis presented in this paper is that this rapid flow results in a low or negative pressure on the endothelium, and plasma and trypan blue are prevented from passing through the wall. The tight junctions of cerebral endothelial cells may be able to withstand only a limited amount of pressure on their luminal surface. If the velocity of blood in brain capillaries decreases, pressure on the endothelium should increase, and brain vessels, like blood vessels elsewhere in the body, become permeable to vital dyes. Other conditions also increase capillary permeability, e.g. acute arterial hypertension or venous congestion. Although brain vessels can adapt to a moderate, gradual change in systemic pressure, when a significant rise in cerebral arterial pressure is abrupt, the compensatory changes in the postcapillary venous bed may be inadequate and consequently intracapillary pressure and vascular permeability are increased. Venous congestion increases intracapillary pressure by restricting capillary outflow as well as by reducing velocity through capillary beds. Under such conditions increased capillary permeability may be indicated by cerebral edema, and even, on occasion, by petechial hemorrhages. In short, if the flow is fast and unimpeded the BBB will be effective; if the velocity decreases, or intracapillary pressure increases for whatever reason, the permeability of the brain endothelium will be abnormally increased.     

An Easier, Reproducible, and Mass-Production Method to Study the Blood–Brain Barrier In Vitro

To provide an "in vitro" system for studying brain capillary function, we have developed a process of coculture that closely mimics the "in vivo" situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. Under these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood-brain barrier, including tight junctions and gamma-glutamyl transpeptidase activity. The average electric resistance for the monolayers was 661 omega cm2. The system is impermeable to inulin and sucrose but allows the transport of leucine. Arabinose treatment increases transcellular transport flux by 70%. The relative ease with which such monolayers can be produced in large quantities would facilitate the "in vitro" study of brain capillary functions.     

Scaling of brain metabolism and blood flow in relation to capillary and neural scaling

Brain is one of the most energy demanding organs in mammals, and its total metabolic rate scales with brain volume raised to a power of around 5/6. This value is significantly higher than the more common exponent 3/4 relating whole body resting metabolism with body mass and several other physiological variables in animals and plants. This article investigates the reasons for brain allometric distinction on a level of its microvessels. Based on collected empirical data it is found that regional cerebral blood flow CBF across gray matter scales with cortical volume V as CBF ~ V(-1/6), brain capillary diameter increases as V(1/12), and density of capillary length decreases as V(-1/6). It is predicted that velocity of capillary blood is almost invariant (~V(ε)), capillary transit time scales as V(1/6), capillary length increases as V(1/6+ε), and capillary number as V(2/3-ε), where ε is typically a small correction for medium and large brains, due to blood viscosity dependence on capillary radius. It is shown that the amount of capillary length and blood flow per cortical neuron are essentially conserved across mammals. These results indicate that geometry and dynamics of global neuro-vascular coupling have a proportionate character. Moreover, cerebral metabolic, hemodynamic, and microvascular variables scale with allometric exponents that are simple multiples of 1/6, rather than 1/4, which suggests that brain metabolism is more similar to the metabolism of aerobic than resting body. Relation of these findings to brain functional imaging studies involving the link between cerebral metabolism and blood flow is also discussed.     

Advances in cell biology of blood-brain barrier transport.

The blood-brain barrier (BBB) is present in the brain of all vertebrates, and arises from epithelial-like high resistance tight junctions that join virtually all capillary endothelium in brain. Recent advances in understanding the cell biology of BBB transport are extending prior physiologic models. For example, glucose transport through the BBB is mediated by a protein that is expressed by the GLUT-1 glucose transporter gene and is asymmetrically localized on lumenal and ablumenal membranes of brain endothelium. Other examples of polarized function at the BBB include asymmetric distribution of endothelial surface charge and ectoenzymes. The tissue-specific gene expression within the brain capillary endothelium is believed to be orchestrated by neighboring cells such as astrocytes, the foot process of which cover more than 95% of the brain microvascular endothelium.     

Expression of the neonatal Fc receptor (FcRn) at the blood-brain barrier

The blood-brain barrier (BBB) restricts transport of immunoglobulin G (IgG) in the blood to brain direction. However, IgG undergoes rapid efflux in the brain to blood direction via reverse transcytosis across the BBB after direct intracerebral injection. This BBB IgG transport system has the characteristics of an Fc receptor (FcR), but there is no molecular information on the putative BBB FcR. The present study uses confocal microscopy and an antibody to the rat neonatal FcR (FcRn), and demonstrates the expression of the FcRn at the brain microvasculature and choroid plexus epithelium. Co-localization with the Glut1 glucose transporter indicates the brain microvascular FcRn is expressed in the capillary endothelium. The capillary endothelial FcRn may mediate the 'reverse transcytosis' of IgG in the brain to blood direction.     

Synergistic effects of neurons and astrocytes on the differentiation of brain capillary endothelial cells in culture

Brain capillary endothelial cells form a functional barrier between blood and brain, based on the existence of tight junctions that limit paracellular permeability. Occludin is one of the major transmembrane proteins of tight junctions and its peripheral localization gives indication of tight junction formation. We previously reported that RBE4.B cells (brain capillary endothelial cells), cultured on collagen IV, synthesize occludin and correctly localize it at the cell periphery only when cocultured with neurons. In the present study, we describe a three-cell type-culture system that allowed us to analyze the combined effects of neurons and astrocytes on differentiation of brain capillary endothelial cells in culture. In particular, we found that, in the presence of astrocytes, the neuron-induced synthesis and localization of occludin is precocious as compared to cells cocultured with neurons only.     

The genes coding for 4 snRNAs of Drosophila melanogaster: localization and determination of gene numbers.

Four small nuclear RNAs (snRNAs) have been isolated from Drosophila melanogaster flies. They have been characterized by base analysis, fingerprinting, and injection into Axolotl oocytes. The size of the molecules and the modified base composition suggest that the following correlations can be made: snRNA1 approximately U2-snRNA; snRNA2 approximately U3-snRNA; snRNA3 approximately U4-snRNA; snRNA4 approximately U6-snRNA. The snRNAs injected into Axolotl oocytes move into the nuclei, where they are protected from degradation. The genes coding for these snRNAs have been localized by "in situ" hybridization of 125-I-snRNAs to salivary gland chromosomes. Most of the snRNAs hybridize to different regions of the genome: snRNA1 to the cytological regions 39B and 40AB; snRNA2 to 22A, 82E, and 95C; snRNA3 to 14B, 23D, 34A, 35EF, 39B, and 63A; snRNA4 to 96A. The estimated gene numbers (Southern-blot analysis) are: snRNA1:3; snRNA2:7; snRNA3:7; snRNA4:1-3. The gene numbers correspond to the number of sites labeled on the polytene salivary gland chromosomes.     

Immunohistochemical detection of dysbindin at the astroglial endfeet around the capillaries of mouse brain

Dysbindin was first identified by the yeast two hybrid assay as a binding partner of dystrobrevin which is a cytoplasmic member of dystrophin glycoprotein complex. Immunolocalization of dystrobrevin in the astrocyte endfeet and endothelial cells in the rat cerebellum was reported. Therefore, we were interested in the expression and localization of dystrobrevin binding protein dysbindin in the mouse brain capillary wall and its surrounding astroglial endfeet. We examined whether the dysbindin expression is present in astroglial endfeet and/or capillary endothelial cells at light and electron microscopic levels. Using brain samples from five normal mice (C57BL/6ScSn), we prepared the anti-dysbindin antibody stained brain samples with immunoperoxidase method at light microscopic level and with immunogold method at ultrastructural level. Immunohistochemistry showed that dysbindin was located in the brain capillary at light microscopic level. Immunogold electron microscopy revealed that dysbindin signal was observed at the inside surface of plasma membrane of glial endfeet which surrounded the brain capillary endothelial cells and pericytes.     

Method of quantitative assessment of the cerebral vascular bed in experimental morphologic studies

Cleared brain sections 20 mkm thick with injected vessels are photographed and then projected on the screen. Vascular contours are sketched on the paper, cut out and weighed. Vessel-capillary network/the whole section field ratio in percents is then calculated. Having separated the area of the vessels, the area occupied by the capillaries is divided into the mean capillary diameter, thus it is possible to estimate the capillary length per area unit and the volume of the brain substance.     

Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells

Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat brain capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat brain capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat brain capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat brain capillary fraction (per total mRNA) was 35.6-fold greater than that in whole brain, while the level of claudin-12 mRNA was only 13.9% of that in whole brain, suggesting that expression of claudin-12 mRNA is not restricted to brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell-cell boundaries. The permeability surface product of [(14)C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 x 10(-3) and 11.6 x 10(-3) cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in brain capillaries, and plays a key role in the appearance of barrier properties of brain capillary endothelial cells.