Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to examine the cycling kinetics of ferrotransferrin (Tf) and to provide evidence for a transcytotic pathway in vitro. Binding of 125I-Tf to BMECs grown on matrix-coated plastic was measured in the presence of saponin to calculate the total number of transferrin receptors (TfRs). Nonlinear regression analysis of the binding isotherm showed that there were 100,000 high-affinity receptors per cell and that expression was maximum at cell confluence. Binding of Tf at 4 degrees C indicated that there was a large intracellular receptor pool comprising 85-90% of the total cellular receptors. Accumulation of Tf at 37 degrees C, inhibited at low temperature and in the presence of metabolic poisons, occurred with an initial rate coefficient of 0.030 min-1 and this decreased by 83% after 60 min. Concomitant accumulation of 59Fe from Tf-59Fe was linear. In the absence of externally added ligand, 80% of the accumulated 125I-Tf was released into the medium with a rate coefficient of 0.017 min-1 and this was inhibited at low temperature. In the presence of the weak base primaquine, the accumulation of Tf and 59Fe and the efflux of Tf were decreased. Moreover, phorbol myristate acetate (PMA) caused a 30% increase in surface TfRs and an 82% increase in Tf accumulation, although the size of the recycling pool remained unchanged. Despite the low numbers of TfR expressed by post-confluent cells, filter-grown BMEC monolayers were used to measure transcytosis of Tf. A small portion of the Tf that was accumulated from the apical side entered a transcytotic pathway. Most of the Tf and all of an accumulated fluid-phase tracer were recycled towards the apical side. These results showed that cultured BMECs cycle Tf-TfR complexes slowly and vectorially and suggested that the large intracellular receptor pool may facilitate steady state accumulation and regulate transcellular transport of iron.     

Biochemical characteristics of primary and passaged cultures of primate brain microvessel endothelial cells

Rhesus macaque monkey brain microvessel endothelial cells (BMECs) were isolated and grown in culture in an effort to establish an appropriate primate in vitro model of the endothelial component of the blood-brain barrier. The presence of Factor VIII antigen, alkaline phosphatase, -glutamyl transpeptidase, lactate dehydrogenanse, total protein, and the passive permeability properties was documented for both primary and passaged cultures. Primate BMECs were shown to exhibit similar morphological and biochemical properties described for other BMEC culture systems derived from other species. In addition, the passaged primate BMECs were particularly notable for the changes in enzyme activities and total protein that parallel age-dependent changes in brain capillary endothelia. This study provides further support for the possible application of BMEC culture systems in investigations of blood-brain barrier functions under normal, aging, and diseased conditions.To whom to address reprint requests. Phone (913)864-3609; FAX (913)864-3578.     

Substrate specificity of phenol sulfotransferase from primary cultures of bovine brain microvessel endothelium

The substrate specificity of the thermostable phenol sulfotransferase (PST) from primary cultures of brain microvessel endothelial cell monolayers was characterized. Selected catecholamines, catecholamine metabolites, and p-nitrophenol at 5, 50, and 500 M were used as substrates in PST assays of cytosol extracts. Endogenous catecholamines, epinephrine, norepinephrine, and dopamine, exhibited no detectable activity as substrates (500 M) compared to 500 M p-nitrophenol as substrate (1.8 pmol/mg/min specific activity) for the PST. In contrast, 500 M of either deaminated or 3-O-methylated metabolites of catecholamines exhibited intermediate (1.0 pmol/mg/min specific activity) to low (0.2 pmol/mg/min specific activity) activity, respectively, as substrates compared to p-nitrophenol as substrate for the PST. Additionally, 500 M of metabolites of catecholamines that were both deaminated and 3-0-methylated exhibited high activity (>3.0 pmol/mg/min specific activity) as substrates compared top-nitrophenol as substrate for the PST. Qualitatively similar results were observed at lower substrate concentrations. Therefore, results from this study suggest a potential role for PST as part of the enzymatic blood-brain barrier in regulating transendothelial passage of endogenous catecholamines between the blood and the brain.     

Effects of selected vasoactive substances on adenylate cyclase activity in brain,isolated brain microvessels,and primary cultures of brain microvessel endothelial cells

The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6±2.1, 15.6±2.7, and 8.4±1.5 pmol cAMP/mg protein/min±SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.     

Permeability of bovine brain microvessel endothelial cells in vitro: barrier tightening by a factor released from astroglioma cells.

It has been shown both in vivo and in culture that astrocytes communicate with brain microvessel endothelial cells (BMECs) to induce many of the blood-brain barrier characteristics attributed to these unique cells. However, the results using cultured cells are conflicting as to whether this communication is dependent upon cell-cell contact. In this study we used primary cultures of bovine BMECs grown as monolayers on polycarbonate filters to study the formation of the barrier in vitro and examine its modulation by rat C6 glioma cells. Effects were examined by treating postconfluent BMEC monolayers with medium conditioned continually by C6 cells from the basolateral side to mimic the in vivo orientation. Cell monolayer integrity was assessed using electrical resistance and by measuring diffusion of uncharged molecules. BMEC monolayers form a functionally polarized and leaky barrier, with maximal resistance of 160 omega . cm2 and significant flux of molecules of molecular weight less than 350 Da. Treatment with rat or human astroglioma cells rather than pericytoma cells or transformed fibroblasts results in a concentration-dependent 200-440% increase in electrical resistance and a coincident 50% decrease in permeability to sucrose and dextran (70 kDa). The decrease in passive diffusion is most likely due to a change in tight junctions and not to transcellular vesicular traffic. The findings support that astroglioma cells release one or more signals that are required for cultured BMECs to express a "differentiated" phenotype associated with a tighter barrier, increased gamma-glutamyl transpeptidase activity, and decreased pinocytic activity. The relative ease and quickness of this culture system makes it amenable to studies on cell-cell interaction and regulation of barrier maintenance.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

Sodium and potassium uptake in primary cultures of proliferating rat astroglial cells induced by short-term exposure to an astroglial growth factor

Primary cultures of rat astroglial cells were maintained in a serum-free medium. After 8–10 days of cultivation the cells were exposed to an astroglial growth factor (AGF2) for short periods (1–120 min). Subsequently, uptake of²²Na⁺ and⁴²K⁺ into control and AGF2-pretreated cells was studied. Assay of the Na⁺ and K⁺ values in the cells was also performed by atomic absorption spectrometry. Treatment of rat astroglial cells with AGF2 resulted in a significant increase of the uptake of both Na⁺ and K⁺ depending on the duration of the exposure period. To reach the maximum increase of cation uptake, 6–10 min and 30 min of AGF2 pretreatment were needed for Na⁺ and K⁺, respectively. Amiloride blocked this increase of Na⁺ and K⁺ uptake elicited by AGF2 pretreatment, but the control cells were amiloride resistant. Treatment with AGF2 increased the ouabain sensitivity of the K⁺ uptake as that: 10^–4 M ouabain inhibited K⁺ uptake of the AGF2-treated cells to the same degree as 5×10^–3 M ouabain with the control cells. The Na⁺ uptake of AGF2-treated cells, however, exhibited no relevant changes in the presence of ouabain. A significant part of the AGF2-induced K⁺ uptake could be inhibited by both ouabain and amiloride, but a ouabain-resistant and amiloride-sensitive component also was revealed. The furosemide sensitivity of both Na⁺ and K⁺ uptake into cultured astroglial cells was also significantly increased by AGF2. Our findings suggest that short-term exposure of cultured glial cells to AGF2 induces these very early ionic events: 1) The appearance of a relevant amiloride-sensitive Na⁺/H⁺ exchange, and as a consequence of increased Na⁺ entry into the cells, secondary activation of the ouabain-sensitive K⁺ uptake via the Na⁺,K⁺-pump. 2) A direct effect of AGF2 on the Na⁺,K⁺-pump assembly in the membrane, resulting in increased Na⁺ sensitivity of the inner pump sites and enhanced ouabain sensitivity of the external K⁺-binding sites. 3) An increase of ouabain-resistant but amiloride- or furosemide-sensitive Na⁺ and K⁺ uptake.Some of the results reported here were presented as a lecture at a Symposium on Na⁺/H⁺ exchange of the Second European Congress on Cell Biology, Budapest, Hungary, 1986.     

Amino acid content in astroglial primary cultures from different brain regions during cultivation

Free amino acids in astroglial primary cultures obtained from newborn rat cerebral cortex, striatum and hippocampus were analyzed and compared during cultivation. Glutamate and taurine exhibited the highest concentrations. Aspartate and glutamate showed the highest values after 1 and 3 weeks of cultivation with lower values after 2 weeks in culture, while taurine, -alanine/hypotaurine and phosphoethanolamine showed the highest value after 2 weeks in culture. The non-neuroactive amino acids and -aminobutyric acid were present at a low level and the former showed the lowest concentration at 2 weeks of cultivation. Astrocytes from the different regions did generally not differ with respect to amino acid content. We conclude that the morphological and biochemical maturation of glia in culture is accompanied with marked quantitative changes in amino acid pattern.     

Some characteristics of specific angiotensin II binding sites on bovine brain microvessel endothelial cell monolayers

Angiotensin II (Ang II) binding sites were characterized in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. Binding of [3H]Ang II to BMECs was time dependent and saturable. Scatchard plot analysis of dose-dependent [3H]Ang II binding revealed a single population of binding sites (Kd = 3.1 nM, Bmax = 52 fmoles/mg protein). Sarathrin, an Ang II antagonist, and saralsin, a partial agonist, inhibited [3H]Ang II binding to BMEC monolayers, whereas two unrelated peptides, bradykinin and arginine-vasopressin, had no effect on the specific binding of [3H]Ang II. At 37 degrees C, [3H]Ang II was internalized in BMECs and this uptake appeared to be saturable. Nanomolar concentrations of Ang II and saralasin stimulated [3H]thymidine uptake in serum-free starved BMEC monolayers, corresponding to an increase in DNA synthesis. On the other hand, sarathrin had no effect on [3H]thymidine uptake. The affinity of the single population of Ang II of binding sites was consistent with the concentration range of Ang II actions demonstrated in several cell types including BMECs. The Ang II-mediated actions on DNA synthesis suggest that this peptide-hormone may possess growth regulating properties in BMECs through either surface or internal site interactions. Collective findings support the complex nature of Ang II in regulating vascular and nonvascular cell growth and permeability characteristics.     

Ethanol acutely decreases astroglial gap junction permeability in primary cultures from defined brain regions

The acute effect of hyperosmotic ethanol on gap junction permeability was examined in astroglial cells in primary culture from five different brain regions. Gap junction permeability was analyzed by measuring dye spreading from cell to cell with the low molecular weight dye Lucifer Yellow. Ethanol concentrations 25-300 mM significantly decreased dye spreading in cultures from the cerebral cortex in a dose-dependent but time-independent manner for up to 60 min. Besides cerebral cortex, exposure to 150 mM ethanol decreased dye spreading in astroglial cultures from the hippocampus and from the brain stem, while cultures from the olfactory bulb and from the hypothalamus were not significantly affected. The ethanol-induced decrease in dye spreading in cultures from the cerebral cortex was not mediated through changes in cell volume, osmolarity, protein kinase C (PKC) phosphorylation, intracellular pH, or intracellular calcium concentration ([Ca(2+)](i)). The decrease in dye spreading was abolished upon incubation in sodium-reduced buffer, and after blockage of the Na(+)/K(+)/2Cl(-) cotransporter with furosemide. The results presented here indicate that ethanol-mediated decrease in dye spreading is directly or indirectly dependent on sodium.     

Different phenotyes of cultured microvessel endothelial cells obtained from bovine corpus luteum

Summary Morphological heterogeneity has not been documented for cultured endothelial cells isolated from the microvascular bed of any organ. As the corpus luteum depends on a rich microvascularization, endothelial cells were dislodged from developing corpora lutea by mechanical dissection followed either by collagenase digestion or by no digestion. Cell separation was carried out by Percoll density centrifugation. Although the yield of intact cells was higher with collagenase treatment than without, successful endothelial cell cultures were only established when cells remained untreated. Viewed by light microscopy after an average lag phase of 10 days, five different phenotypes of endothelial cells were found under similar simple culture conditions: isomorphic epithelioid, polymorphic epithelioid, spindle-shaped, round, and phase-dense phenotypes. Monolayers appeared within 2–4 weeks. After an additional period of 2–4 weeks, tubular forms with a specific pattern were noted for types 1–3, the so-called pseudotubular forms for type 4, and none for type 5. Cell types differed in their cytochemical and immunocytochemical responses. Examined by SEM, type 1 displayed a more conspicuous surface anatomy than type 2. Types 3–5 demonstrated striking cell processes that were characteristic of each type. Tubular forms of types 1 and 2 showed cell borders and a marked increase in surface specializations, whereas tubular forms of type 3 lacked detectable cell borders in the absence of a striking surface anatomy. Pseudotubular forms of type 4 developed no particular spatial organization. Thus, for the first time, morphological evidence is provided that different endothelial cell types are obtained from diverse segments of the microvascular bed.     

Protein metabolism and electrophoretic profiles in astroglial primary cultures from different regions of newborn rat brain

Primary astroglial cultures (14 days of age) from cerebral cortex, striatum, and hippocampus of newborn rat brain contained similar amounts of soluble proteins. Uptake and incorporation of [³H]valine into soluble protein measured after 30 and 60 min of incubation, respectively, was on a similar level in the various cultures. [³H]valine labeling of protein bands from the cell soluble fractions and incubation media of hemisphere cultures, and which were separated by isoelectric focusing (IEF) or sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated that proteins are released to the extracellular medium after being synthesized within cultivated cells. Acidic and high molecular weight proteins were more heavily labelled in the incubation media than in the cell soluble fractions. Two-dimensional electrophoresis (IEF×SDS-PAGE) of soluble proteins from the different cultures showed similar patterns, which were quite different from the serum-free extracellular protein patterns. Both fractions were different from the pattern obtained from fetal calf-serum. In striatum and hippocapus culture media a spot was seen with Ip 6.0–6.8 and m.w. 105,000, and in the media from cerebellar cultures another spot was observed with Ip 5.2–5.6 and m.w. 55,000. The results show that the different cultures are similar in their protein synthetic capacity and protein composition. The specific differences observed in proteins obtained from the serum-free incubation media might indicate specific properties among astroglial cells from various brain regions.     

A novel method to establish primary cultures of bovine corneal endothelial cells

This paper presents a novel, safe and time-saving method to obtain uncontaminated primary cultures of corneal endothelial cells from calf eyes. These primary cultures can be used either for subcultivation or for preparation of extracellular matrix in vitro. The method eliminates the risk of deleterious mechanical or chemical influence on the cells.     

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells.

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.     

Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.     

Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells

The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes. © 1996 Wiley-Liss, Inc.     

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum.

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 microns for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 microns in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.