Vasoactive substances produced by cultured rat brain endothelial cells

Kis, Béla; Szabó, Csilla; Pataricza, János; Krizbai, István A.; Mezei, Zsófia; Gecse, A; Telegdy, Gyula; Papp, Julius Gyula; Deli, Mária A.

doi:10.1016/s0014-2999(99)00024-2

Cited by 41 publications

(21 citation statements)

References 27 publications

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“…All animal experiments were approved by the Animal Care and Use Committee of Wake Forest University Health Sciences. Primary rat CECs were isolated and cultured in collagen type IV and fibronectin-coated 35-mm dishes as described previously (Kis et al, 1999). Culture medium consisted of Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine plasmaderived serum (Animal Technologies Inc., Tyler, TX), 2 mM glutamine, 1 ng/ml basic fibroblast growth factor, 50 g/ml endothelial cell growth supplement (BD Biosciences, Bedford, MA), 100 g/ml heparin, 5 g/ml vitamin C, and antibiotics.…”

Section: Methodsmentioning

confidence: 99%

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Cloning and Characterization of Cyclooxygenase-1b (Putative Cyclooxygenase-3) in Rat

Snipes¹,

Kis²,

Shelness³

et al. 2005

J Pharmacol Exp Ther

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A splice variant of cyclooxygenase-1 (COX-1), COX-1b (previously termed as COX-3), has been identified in canine tissues as an acetaminophen-sensitive isoform, but the sequence of COX-1b mRNA and the encoded protein are not known in rats. We cloned and sequenced rat COX-1b mRNA from cerebral endothelial cells. Sequence analysis indicated that the 98-base pair intron-1 of COX-1 gene remains unprocessed in the COX-1b mRNA, causing a frameshift mutation and a 127-amino acid open reading frame with no sequence similarity with known cyclooxygenases. Transient and permanent transfection of COS-7 cells with a vector containing the rat COX-1b cDNA resulted in synthesis of a protein of the expected size. We generated an affinity-purified polyclonal antibody against the rat COX-1b protein. Western blot analysis of rat tissues using this antibody demonstrated the likely existence of rat COX-1b protein in vivo with the highest expression in heart, kidney, and neuronal tissues. Our results on both stable and on transiently transfected COS-7 cells suggest that rat COX-1b does not have cyclooxygenase activity and does not have any effect on the inhibition of prostaglandin production by acetaminophen. Because this protein has a completely different amino acid sequence than COX-1 and COX-2 and it does not have cyclooxygenase activity, we suggest a name cyclooxygenase variant protein to distinguish it from the known prostaglandin-synthesizing cyclooxygenase isoforms.

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Section: Methodsmentioning

confidence: 99%

“…The pellet containing the microvessels was washed twice with phosphate-buffered saline and stored on dry ice until total RNA isolation. After further processing, this preparation leads to viable CECs that can be used for culturing (Kis et al, 1999).…”

Section: Methodsmentioning

confidence: 99%

Cloning and Characterization of Cyclooxygenase-1b (Putative Cyclooxygenase-3) in Rat

Snipes¹,

Kis²,

Shelness³

et al. 2005

J Pharmacol Exp Ther

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“…In vitro models of the BBB have been developed from cocultures between bovine, porcine, rodent, or human BMECs with rodent or human astrocytes (Cecchelli et al, 1999;Gaillard et al, 2001;Kis et al, 1999;Mégard et al, 2002). However, a number of drawbacks still limit the extensive use of these models in basic research as well as in drug-screening processes: (1) Their use is time consuming and expensive, which may hamper their routine use in nonexpert laboratories, (2) BMECs rapidly de-differentiate in vitro, losing the characteristics of BBB endothelial cells after a few passages in culture, which limits their use for biochemical or pharmacological studies, (3) In a primary culture, it is difficult to eliminate all nonendothelial cell contaminants (pericytes, leptomeningeal cells, smooth muscle cells).…”

Section: Introductionmentioning

confidence: 99%

Rat Brain Endothelial Cell Lines for the Study of Blood–Brain Barrier Permeability and Transport Functions

2005

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(1) In vitro models of the BBB have been developed from cocultures between bovine, porcine, rodent or human brain capillary endothelial cells with rodent or human astrocytes. Since most in vivo BBB studies have been performed with small laboratory animals, especially rats, it is important to establish a rat brain endothelial (RBE) cell culture system that will allow correlations between in vitro and in vivo results. The present review will constitute a brief description of the best characterized RBE cell lines (RBE4, GP8/3.9, GPNT, RBEC1, TR-BBBs and rBCEC4 cell lines) and will summarize their recent and important contribution to our current knowledge of the BBB transport functions and permeability to blood-borne solutes, drugs, and cells. (2) In most cases, primary cultures of RBE cells were transduced with an immortalizing gene (SV40 or polyoma virus large T-antigen or adenovirus E1A), either by transfection of plasmid DNA or by infection using retroviral vectors. In one case however, the conditionally immortalized TR-BBB cell line was derived from primary cultures of brain endothelial cells of SV40-T-expressing transgenic rats. (3) All cell lines appear to have an endothelial morphology. The absence of foci formation would mean that the cells are not transformed. The endothelial origin is shown by the expression of Factor VIII-related antigen. Immortalized RBE cells express all the enzymes and transporters that are considered as specific for the blood-brain barrier endothelium, with similar characteristics to those expected from in vivo analyses, but at a significantly lower level. Some RBE cell lines are responsive to astroglial factors, such as RBE4 cells, rBEC4, and TR-BBB cells. None of the immortalized RBE cell lines appear to generate the necessary restrictive paracellular barrier properties that would allow to use them in transendothelial permeability screening. (4) RBE cell lines have been used to demonstrate that transporters such as organic cation transporter/carnitine transporter, serotonin transporter, and the ATA2 system A isoform are expressed in rat brain endothelium. When the transporter is shown to be expressed with the same properties in the immortalized RBE cells as in vivo, regulation studies may be initiated even if the transporter is down-regulated. Pharmacological applications have been proposed with well-characterized transporters such as monocarboxylic acid transporter-1, large neutral amino acid tansporter-1, nucleoside carrier systems, and P-glycoprotein. RBE cell monolayers have also been used to investigate the mechanism of the transendothelial transport of large molecules, such as immunoliposomes or nanoparticles, potentially useful as drug delivery vectors to the brain. (5) RBE4 and GP8 cell lines have been extensively used to demonstrate that intercellular adhesion molecule-1 (ICAM-1) engagement in brain endothelial cells triggers multiple signal transduction pathways. Using functional assays, it was established that ICAM-1 signaling is intimately and actively involved in facili...

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“…4) as well as vasoactive mediators such as endothelin-1 (8), NO (9), angiotensin peptides (10), AM (6), and eicosanoids (11,12). Endothelial scavenger receptors participate in the metabolism of low density lipoproteins (13).…”

Section: The Blood-brain Barrier-an Overviewmentioning

confidence: 99%