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K L Audus

Publications and source records attributed to K L Audus.

At least 73 records · Page 4Linked to original sources
PubMed1992

The effect of protein binding on ivermectin uptake by bovine brain microvessel endothelial cells.

The effect of albumin binding on ivermectin uptake and transfer across the endothelial component of the blood-brain barrier (BBB) was determined with an in vitro model comprised of bovine brain microvessel endothelial cell (BMEC) monolayers. Cellular uptake of ivermectin was limited in the absence of albumin and 90% inhibited in the presence of 10% albumin. Cell membrane association of ivermectin, as followed by fluorescent probe labelling, was observed only at high (micromolar) concentrations of the drug. Membrane association was about 75% inhibited in the presence of albumin. Similarly, transfer across BMEC monolayers was restricted, equivalent to that of BBB impermeant markers. Unlike the uptake studies, however, albumin had little effect on the transfer of ivermectin across BMEC monolayers. These results support recent in vivo findings on the distribution of ivermectin into the brain and suggest that ivermectin has only a limited affinity for the endothelial component of the normal BBB.

Animals↗
PubMed1992

Chlorhexidine effects on membrane lipid domains of human buccal epithelial cells.

The effect of chlorhexidine gluconate on the adherence of Candida albicans to human buccal epithelial cells (BEC) and drug-induced alterations in BEC membrane-lipid packing order were examined. Treatment of BEC with attached yeasts with 0.1 and 0.2% chlorhexidine resulted in significant yeast detachment after 90 and 60 min, respectively. Following pre-treatment of BEC with greater than 0.1% chlorhexidine, yeast adherence was inhibited by greater than 80%. In parallel experiments, the fluorescence anisotropy of BEC labeled with fluorescent membrane probes--diphenylhexatriene (DPH) and trimethylammonium DPH--was assessed following exposure to chlorhexidine. The fluorescence anisotropy decreased with increasing concentrations of chlorhexidine, which indicated that the drug decreased epithelial-cell membrane-lipid packing order. Chlorhexidine concentrations that altered epithelial-cell membrane-lipid packing order, particularly in superficial regions, were similar to those drug concentrations required for detachment of adherent yeasts. Similar results were obtained with a second antifungal, nystatin A. While the effects of chlorhexidine on the buccal-cell membrane-lipid packing order were not reversed by multiple washings, the opposite situation occurred with nystatin A. The results suggest that chlorhexidine-induced alterations of BEC membrane-lipid order may be involved in the antifungal actions of the drug.

Antifungal Agents↗
PubMed1991

Evidence for 21-aminosteroid association with the hydrophobic domains of brain microvessel endothelial cells.

Studies were conducted to demonstrate 21-aminosteroid distribution into the hydrophobic or lipid domains of biological membranes, a presumed site at which these compounds inhibit lipid peroxidation. Bovine brain microvessel endothelial cells (BMECs) were labeled with diphenylhexatriene fluorophores and interactions with cell membranes characterized with fluorescence anisotropy and lifetimes. Two 21-aminosteroids (U-74500A and U74006F) were shown to preferentially alter the fluorescence anisotropy and lifetime parameters of the diphenylhexatriene probe distributing into membranes throughout the BMECs. Little or no effect of the compounds was observed on the fluorescence parameters of the probe localized on the surface of BMEC plasma membranes. By contrast, cholesterol used as a positive control substantially altered the fluorescence parameters of BMECs labeled with either diphenylhexatriene probe. Results suggest 21-aminosteroid-induced changes in the molecular packing order and drug: fluorescent probe interactions in membrane hydrophobic (or lipid) domains throughout the BMEC. Concentrations of 21-aminosteroids altering the fluorescence parameters of diphenylhexatriene labeled BMECs correspond to those concentrations of 21-aminosteroids effective in vitro in inhibition of lipid peroxidation.

Animals↗
PubMed1991

Blood-brain barrier: transport studies in isolated brain capillaries and in cultured brain endothelial cells.

The development of in vitro BBB models consisting of isolated brain capillaries and cultured brain microvessel endothelial cells has made possible the study of BBB transport phenomena at the cellular level. Basic characteristics of BBB transport of endogenous and exogenous solutes and their biochemical, pharmacological, ontogenic, and pathological regulation mechanisms have been investigated. This information has led not only to a better understanding of BBB transport but also to the construction of strategies for improving drug delivery to the CNS for diagnosis and therapeutics. To elucidate the complexity of BBB transport, in vivo studies are always necessary at some point; however, in vitro systems can be useful complements to the in vivo systems. The tissue culture systems seem to be especially important in the clarification of cellular, biochemical and molecular features of BBB transport. Appropriate systems should be selected or combined, depending on the purpose of the investigation.

Animals↗
PubMed1991

Angiotensin peptide regulation of bovine brain microvessel endothelial cell monolayer permeability.

The passage of fluid-phase endocytosis markers--Lucifer yellow and a fluorescein isothiocyanate-conjugated dextran--across confluent primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers was characterized in the presence of angiotensin II (Ang II) peptides. Exposure to nanomolar concentrations of saralasin, an Ang II agonist, attenuated the passage of the fluorophores across the monolayers by 50-75%. In contrast, exposure to sarathrin, an Ang II antagonist, had no effect on passage of the fluorophores across the BMEC monolayers. Sarathrin, however, did inhibit the saralasin-induced reduction in transendothelial monolayer permeability and suggested specific Ang II binding site mediation. Other experiments performed suggest that saralasin-induced changes in BMEC monolayer permeability can be blocked by pretreatment with inhibitors of prostaglandin synthesis and that substantial saralasin-induced changes in BMEC monolayer permeability occurred only following exposure on the apical side of the endothelial cells. Results were consistent with previous studies demonstrating the existence of specific Ang II binding sites on BMECs and Ang II regulation of fluid-phase endocytosis. In addition, these studies support the role of angiotensin peptides in modulating permeability properties of the blood-brain barrier.

Animals↗
PubMed1991

A comparison of aminopeptidases from excised human buccal epithelium and primary cultures of hamster pouch buccal epithelium.

Aminopeptidase activity associated with human buccal tissue and primary cultures of hamster buccal epithelium homogenates was assayed fluorometrically using 4-methoxy-2-naphthylamides of leucine, alanine, and arginine. Kinetic parameters, Km and Vmax, for all substrates were estimated. Aminopeptidase parameters for human tissue were similar to those for the in-vitro system and reported literature values for rodent buccal tissue aminopeptidases. Aminopeptidases of both tissues were also found to be similarly sensitive to typical inhibitors, bestatin and puromycin. Overall results suggest that appropriate in-vitro systems derived from animal tissues may be useful in assessing the role and localization of peptidases associated with buccal tissue.

2-Naphthylamine↗
PubMed1990

Fluid-phase endocytosis by primary cultures of bovine brain microvessel endothelial cell monolayers.

Blood-brain barrier (BBB) fluid-phase endocytosis was examined in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. By fluorescence spectroscopy. Lucifer yellow (LY, a fluorescent, soluble molecule used as a marker for pinocytosis) accumulation by BMEC was observed to be linear over a concentration range of 0.05 to 1.0 mg/ml. Time-dependent uptake of LY exhibited curvilinear kinetics composed of an initially rapid uptake rate of 1338 ng of LY/mg protein per hour at 0.5 mg/ml LY. Within 20 min, the rate of LY accumulation slowed to a steady-state rate of 23 ng of LY/mg protein per hour. Accumulation of LY was inhibited in the presence of metabolic inhibitors, potassium cyanide or 2-deoxyglucose, and was decreased, but not completely inhibited, at 4 degrees. Pulse-chase experiments revealed that efflux of LY was very rapid with at least 80% of the accumulated LY being lost within 2 min and was not sensitive to low temperature. Only 3-5% of the LY initially accumulated by BMEC remained cell-associated after a 30-min chase. The calculated turnover of the endocytic compartment's total volume (per hour) is 8- to 20-fold less than values for fibroblasts and macrophages, respectively. We have interpreted these data to suggest that the efflux of most of the LY involves loss from a rapidly recycled compartment of finite volume, possibly caveolae, that had sequestered marker during accumulation and suggest that these results are consistent with the present understanding of BBB pinocytosis in vivo.

Animals↗
PubMed1990

The use of cultured epithelial and endothelial cells for drug transport and metabolism studies.

In an effort to develop novel strategies for delivery of drug candidates arising from rational drug design and recombinant DNA technology, pharmaceutical scientists have begun to employ the techniques of cell culture to study drug transport and metabolism at specific biological barriers. This review describes some of the general factors that should be considered in developing a cell culture model for transport studies and metabolism studies. In addition, we review in detail the recent progress that has been made in establishing, validating, and using cell cultures of epithelial barriers (e.g., cells that constitute the intestinal, rectal, buccal, sublingual, nasal, and ophthalmic mucosa as well as the epidermis of the skin) and the endothelial barriers (e.g., brain microvessel endothelial cells).

Animals↗
PubMed1990

Angiotensin peptide regulation of fluid-phase endocytosis in brain microvessel endothelial cell monolayers.

An in vitro model comprised of primary cultures of brain microvessel endothelial cells was used to investigate angiotensin II (Ang II) effects on blood-brain barrier fluid-phase endocytosis. The effects of Ang II, saralasin, sarathrin, bradykinin (BK), and phorbol myristate acetate (PMA) on brain microvessel endothelial cell fluid-phase endocytosis were determined using the fluorescent marker, Lucifer yellow. Nanomolar concentrations of saralasin (a partial Ang II agonist) stimulated brain microvessel endothelial cell endocytosis by 30% whereas Ang II treatment enhanced Lucifer yellow uptake by 20%. Sarathrin (an Ang II antagonist) had no effect on Lucifer yellow uptake. Nanomolar concentrations of BK and PMA also stimulated Lucifer yellow uptake by the brain microvessel endothelial cell by 40 and 95%, respectively. Stimulatory effects of Ang II and saralasin on Lucifer yellow uptake by brain microvessel endothelial cells could be completely blocked by pretreatment with either sarathrin or indomethacin (an inhibitor of prostaglandin synthesis). In contrast, the effects of neither BK nor PMA on brain microvessel endothelial cell uptake of Lucifer yellow ere altered by indomethacin pretreatment. Results indicated that Ang II, saralasin, BK, and PMA produce similar stimulatory effects on brain microvessel endothelial cell fluid-phase endocytosis with only Ang II and saralasin, producing increases in brain microvessel endothelial cell fluid-phase endocytosis that appeared to be mediated by prostaglandins.

Angiotensin II↗
PubMed1990

Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers.

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t1/2 values for RCAI binding, internalization and efflux were 5, 18 and 13-14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 degrees C became localized within more caveolae within 2.5 min of warming to 37 degrees C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.

Adsorption↗
PubMed1989

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 microM were used as substrates in PST assays of cytosol extracts. Endogenous catecholamines, epinephrine, norepinephrine, and dopamine, exhibited no detectable activity as substrates (500 microM) compared to 500 microM p-nitrophenol as substrate (1.8 pmol/mg/min specific activity) for the PST. In contrast, 500 microM of either deaminated or 3-O-methylated metabolites of catecholamines exhibited intermediate (approximately 1.0 pmol/mg/min specific activity) to low (approximately 0.2 pmol/mg/min specific activity) activity, respectively, as substrates compared to p-nitrophenol as substrate for the PST. Additionally, 500 microM of metabolites of catecholamines that were both deaminated and 3-O-methylated exhibited high activity (greater than 3.0 pmol/mg/min specific activity) as substrates compared to p-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 endogeneous catecholamines between the blood and the brain.

Animals↗
PubMed1989

The application of bovine brain microvessel endothelial-cell monolayers grown onto polycarbonate membranes in vitro to estimate the potential permeability of solutes through the blood-brain barrier.

Previously our laboratory (Rim et al., Int. J. Pharm. 32:79-84. 1986) described an in vitro blood-brain barrier (BBB) model consisting of cultured bovine brain microvessel endothelial cells (BMECs) grown onto regenerated cellulose acetate membranes. However, the utility of this in vitro BBB model system was limited because the regenerated cellulose acetate membrane and not the monolayer of bovine BMECs was rate limiting for the permeability of very lipophilic compounds. Therefore, in this study we have evaluated polycarbonate membranes as supports for growing bovine BMECs and for conducting in vitro drug permeability studies. Bovine BMECs were cultured on collagen-coated polycarbonate membranes (13-mm diameter, 12-microns pore size) which were then mounted into side-by-side diffusion cells for transport studies. The permeabilities of a series of solutes of varying lipophilicity (progesterone, estrone, testosterone, haloperidol, propranolol, antipyrine, caffeine, urea, acyclovir, ganciclovir, ribavirin, and glycerol) were determined and an excellent correlation (r = 0.97) was established between the permeability coefficients of the solutes and their log partition coefficients (PC)/(MW)1/2. These results suggest that bovine BMECs cultured onto polycarbonate membranes can be used as an in vitro model system for estimating the potential permeability of a solute through the BBB in vivo.

Animals↗
PubMed1989

Cultured buccal epithelium: an in vitro model derived from the hamster pouch for studying drug transport and metabolism.

Hamster pouch buccal epithelium (HPBE) was isolated and grown in primary cultures on rat-tail collagen-coated growth surfaces. The cultured pouch buccal epithelium (CPBE) was characterized morphologically with electron microscopy as stratified multilayers of epithelial cells with well-developed tonofibrillar-desmosomal complexes. Only the superficial layer of the cultured cells exhibited evidence of terminal differentiation. Alkaline phosphatase, alcohol dehydrogenase, and aminopeptidase activities in the primary cultured cells were determined by biochemical assays and found to be similar to those of homogenates of freshly excised hamster pouch epithelium. In addition, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), keratins and total proteins associated with the cultured cells were similar to those of freshly excised HPBE. The permeability characteristics of the cultured cells was determined by placing cultured cells grown on permeable polycarbonate disks in a Side-Bi-Side diffusion apparatus and quantitating the transcellular flux of tritium-labeled water, fluorescein, and fluorescein isothiocyanate dextrans (MW 3800 to 150,000). The cultured cells were least permeable on the third day of culture and were not permeable to substances with a MW greater than about 18,000. Our results indicate that primary cultures of hamster pouch epithelium exhibit biochemical properties similar to those of the excised hamster pouch epithelium from which they were derived. The morphological and permeability characteristics of cultured hamster epithelium were similar to those of nonkeratinized stratified oral epithelia typical of buccal mucosa in man, rabbit, and other species. CPBE, as described here, represents a potentially useful tool for in vitro drug transport, metabolism, pharmacology, and toxicology studies.

Animals↗
PubMed1989

Demonstration of acid hydrolase activity in primary cultures of bovine brain microvessel endothelium.

The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood-brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and beta-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69-77%). The majority of beta-galactosidase (approximately 48%) and total sulfatase (approximately 58%) activity was associated with the lysosome fraction of the BMECs. In contrast, approximately 52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.

Acid Phosphatase↗
PubMed1989

In-vitro characterization of blood-brain barrier permeability to delta sleep-inducing peptide.

The diffusion of delta sleep-inducing peptide (DSIP) across the blood-brain barrier (BBB) has been investigated with an in-vitro model comprised of primary cultures of brain microvessel endothelial cell (BMEC) monolayers. The BMEC monolayers were mounted in a side-by-side diffusion apparatus and the transendothelial flux of DSIP analysed by HPLC with UV detection at 280 nm. The transendothelial flux of the peptide was linear with time and increasing concentrations of DSIP (non-saturable), but was not altered by reduced temperature. The apparent permeability coefficient for DSIP penetration of BMEC monolayers was in a range similar to water-soluble substances (e.g. fluorescein, fluorescein isothiocyanate dextrans) that penetrate the blood-brain barrier to a limited degree based on molecular weight. DSIP flux across the BMEC monolayers was also found to be bidirectional, insensitive to metabolic inhibitors, and not altered by high concentrations of tryptophan. Little degradation (apparent t1/2 about 10 h) of DSIP to major metabolites, tryptophan (trp) and des-trp DSIP, occurred over the time of the diffusion experiments. The results of these studies support and confirm observations in-vivo indicating that intact DSIP crosses the BBB by simple transmembrane diffusion.

Animals↗