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K L Audus

Publications and source records attributed to K L Audus.

87 records · Page 5Linked to original sources
PubMed1988

Carrier-mediated transport of baclofen across monolayers of bovine brain endothelial cells in primary culture.

The mechanism of transport of baclofen, a centrally acting muscle relaxant, across the blood-brain barrier (BBB) was studied using an in vitro model. The model consisted of a monolayer of bovine brain endothelial cells grown in primary culture on a porous regenerated cellulose membrane. The transport of baclofen across the monolayer expressed time and concentration dependency and was saturable. Transport data were corrected for diffusion and fitted to the Michaelis-Menten Vmax model: Km = 58.5 microM, Vmax = 0.23 nmol/min. The results validate the use of the in vitro BBB model as described and support the hypothesis that baclofen penetrates the BBB by means of a carrier-mediated transport system.

Animals↗
PubMed1988

Aluminum effects on growth of brain microvessel endothelial cells in primary culture.

Aluminum salts have been shown to stimulate 3H-thymidine incorporation in primary cultures of bovine brain microvessel endothelial cells (BMECs). Aluminum chloride or sulfate salts in concentrations between 0.01 to 100 microM were, in general, most effective in stimulation of thymidine uptake by BMECs with maximal effects observed after a 24 hour exposure to the metal. Concentrations of aluminum salts greater than 100 microM inhibited thymidine incorporation. Cell numbers were not affected by exposure to concentrations of the aluminum salts less than approximately 100 microM. Concentrations producing half-maximal stimulation of BMEC thymidine incorporation were approximately, 0.3 microM and 0.5 microM, for aluminum chloride and aluminum sulfate, respectively. These findings indicate that BMECs are sensitive to lower concentrations of aluminum salts than other mammalian cell types. Hydroxyurea completely inhibited thymidine incorporation into BMECs in the presence and absence of aluminum suggesting that thymidine incorporation into BMECs is representative of DNA synthesis. Endothelial cell growth factor (ECGF) stimulated both increased DNA synthesis and BMEC cell numbers in the primary culture system. Aluminum had only slight effects on DNA synthesis in endothelial cell growth factor stimulated BMECs. In contrast to ECGF, aluminum then, appears to provide a stimulus for DNA synthesis but not subsequent mitosis in BMECs. Results from this study are consistent with previous studies in other cell types and with current knowledge of the effects of aluminum on the blood-brain barrier in vivo.

Aluminum↗
PubMed1987

Characteristics of aminopeptidase activity from bovine brain microvessel endothelium.

Blood-brain barrier (BBB) aminopeptidase activity was investigated using an in vitro model consisting of primary cultures of brain microvessel endothelium. Using two different substrates, both membrane-bound and soluble aminopeptidases were found to be associated with brain endothelium. That the enzyme activity was aminopeptidase activity was confirmed with the competitive inhibition of substrate degradation by typical aminopeptidase inhibitors puromycin and bestatin. The aminopeptidase activity was also competitively inhibited by enkephalin, met-enkephalin, and leu-enkephalin. Results from parallel experiments with cerebral gray matter and kidney confirm assay conditions. This report supports previous suggestions that aminopeptidases of the enzymatic BBB may play a role in regulating levels of circulating neuropeptides in the cerebrovasculature.

Aminopeptidases↗
PubMed1987

Bovine brain microvessel endothelial cell monolayers as a model system for the blood-brain barrier.

Investigation of blood-brain barrier permeability and metabolic processes, and their regulation by endogenous or exogenous factors, will be important for development of efficient and selective delivery of therapeutic agents to the central nervous system. Primary cultures of brain microvessel endothelial cells offer a potentially powerful tool for studying at the cellular level the biochemical mechanisms regulating BBB function. Using this in vitro model, our studies are directed at characterization of the BBB processes that might be exploited as new schemes for drug delivery to the central nervous system.

Animals↗
PubMed1986

Characteristics of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers.

The large neutral amino acid (LNAA) transport system, or L (leucine) system, in primary cultures of bovine brain microvessel endothelial cell monolayers has been characterized. The transendothelial transport of leucine in this in vitro blood-brain barrier (BBB) model was determined to be bidirectional and time, temperature, and concentration dependent. Leucine transport was saturable, and the apparent Km and Vmax were determined to be 0.18 mM and 6.3 nmol/mg/min, respectively. Transendothelial transport of leucine was resistant to inhibition by ouabain and sodium azide. Other LNAAs, including the centrally acting drugs alpha-methyl-3,4-dihydroxyphenylalanine (alpha-methyldopa), L-3,4-dihydroxyphenylalanine (L-DOPA), alpha-methyltyrosine, and baclofen, inhibited leucine transport. The leucine carrier system was also found to be stereospecific. Sucrose, used as a diffusion marker, moved across the monolayers slowly, and its concentration was not significant for at least 30 min.

Animals↗
PubMed1986

Catecholamine-metabolizing enzymes of bovine brain microvessel endothelial cell monolayers.

The activities of monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), phenol sulfotransferase (PST), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GT), and angiotensin converting enzyme (ACE) were quantitated in primary cultures of bovine brain microvessel endothelial cell monolayers and cerebral gray matter. Significant MAO-A and -B, cytosolic and membrane-bound COMT, PST, AP, GT, and ACE activities are demonstrated in bovine gray matter. By comparison, enzyme activities of the monolayers vary with the age of the monolayer and are generally higher in complete monolayers. Relative to gray matter enzyme activities, the monolayers are enriched with AP, GT, and ACE, enzymes considered to be markers for brain endothelium. Results also indicate that the activities of MAO-A and PST in the monolayers approach those found in the gray matter. Conversely, cytosolic COMT and MAO-B activities in the monolayers are negligible and much lower, respectively, compared to activities in gray matter. Additional studies with both tissues suggest that the PST of both tissues is the thermostable form of the enzyme.

Alkaline Phosphatase↗
PubMed1984

Effect of tricyclic antidepressant drugs on lymphocyte membrane structure.

Tricyclic antidepressant-induced perturbations of murine splenic lymphocyte membranes and cell surface concanavalin A receptor mobility have been investigated using the fluorescent probes diphenylhexatriene and fluorescein-conjugated concanavalin A. Results of these studies illustrate the possible relationship between tricyclic antidepressant-induced membrane perturbations and tricyclic antidepressant-induced suppression of the normal murine lymphocyte mitogen response. Tricyclic antidepressant effects on murine splenic lymphocyte membranes are dose-, time- and temperature- dependent. Murine lymphocyte concanavalin A cell surface receptor mobility is not apparently altered by the tricyclic antidepressants.

Animals↗
PubMed1982

Characteristics of tricyclic antidepressant binding sites associated with murine lymphocytes from spleen.

A single population of high affinity, saturable, tricyclic antidepressant (3-H-desipramine) binding sites has been identified on the membranes of murine lymphocytes. There are approximately 300 binding sites per cell and the apparent dissociation constant (Kd) for these binding sites is about 0.4 nM. Competition studies between the radiolabeled ligand in concentrations near the Kd and other members of the class of tricyclic antidepressants indicate that binding is competitive, reversible, and does not display interactions characteristic of cooperativity.

Animals↗
PubMed1982

Tricyclic antidepressant effects on the murine lymphocyte mitogen response.

Tricyclic antidepressant binding sites have recently been detected on the membranes of murine splenic lymphocytes (1). It is reported here that the mitogenic response of murine lymphocytes is altered in the presence of tricyclic antidepressants at concentrations of 10(-5) M or greater. The time-dependent effects of these drugs, when added to the cultures at various times up to 24 hours subsequent to addition of a mitogen (either concanavalin A or lipopolysaccharide B), are also reported.

Animals↗
PubMedDate not supplied

Rapid optimization of the post-column fluorogenic ninhydrin reaction for the HPLC-based determination of bradykinin and related fragments.

A flow injection analysis scheme is demonstrated for the rapid optimization of reagent concentrations, flow rates, delay time and temperature using the guanidino moiety specific fluorogenic ninhydrin reaction. Using the amino acid arginine, non-arginine containing peptides, and the arginine-containing peptides, bradykinin and related fragments, specificity is demonstrated. These results serve to extend previous descriptions of the post-column reaction by offering a time efficient approach for the optimization of newly assembled post-column reactors using this chemistry. The reactor is subsequently added to a gradient elution HPLC system with the separation of bradykinin and bradykinin fragments demonstrated. Detection sensitivity in the high femtomole-low picomole mass range was achieved for these substances.

Bradykinin↗
PubMedDate not supplied

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.

Amino Acid Sequence↗
PubMedDate not supplied

Permeability of the blood-brain barrier to peptides: an approach to the development of therapeutically useful analogs.

Peptides have been shown in both in vivo and in vitro systems to cross the blood-brain barrier (BBB) and so affect function on the side contralateral to their origin. Some peptides cross primarily by transmembrane diffusion, a nonsaturable mechanism largely dependent on the lipid solubility of the peptide. Other peptides are transported by saturable systems across the BBB. These transport systems can be in the CNS to blood direction, as in the cases of Tyr-MIF-1 and methionine enkephalin, in the blood to CNS direction, as in the case of peptide T, or bidirectional, as in the case of LHRH. Other factors that also affect the amount of peptide crossing the BBB include binding in blood, volume of distribution, enzymatic resistance, and half-time disappearance from the blood. An in vitro model of the BBB has been characterized and used to confirm that peptides can cross the BBB. Results with the model agree with those obtained in vivo and have been used to study the permeability of the BBB to peptides, the effect of peptides on BBB integrity, the cellular pathway peptides and proteins use to cross the BBB, and the ability of the BBB to degrade peptides. The in vivo and in vitro methods have been used together to develop halogenated enkephalin analogs that are enzymatically resistant, cross the BBB readily to accumulate in areas of the brain rich in opiate receptors, and are powerful analgesics.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells.

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Use of fluorescent probes to monitor propranolol effects on the murine splenic lymphocyte.

Recent studies indicate that propranolol suppresses the normal murine lymphocyte mitogen dose-response(1) and that propranolol has a high affinity for the beta-adrenergic binding site on the murine lymphocyte(2). Since concentrations which suppress the lymphocyte mitogen dose-response are much greater than the Kd for the binding site, it may be suggested that the suppression of the mitogen dose-response is a result of nonspecific effects of propranolol. Three fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescein conjugated concanavalin A (F-Con A), and 3,3'-dithio-diisopropyl-oxacarbocyanine iodide (diS-C3-(5)) were used to investigate possible mechanisms of the nonspecific effects of propranolol on the murine splenic lymphocyte. This report presents data which suggests a possible relationship between propranolol concentrations that alter lipid membrane dynamics, as monitored by the fluorescence polarization of DPH, and suppression of the mitogen dose-response in short term cell cultures as effected by propranolol. Also reported are effects of propranolol on the relative mobility of surface membrane lectin receptors as monitored by the fluorescence polarization of F-Con A, effects of propranolol on the transmembrane potential as monitored by fluorescence emission of diS-C3-(5), and the propranolol effect on fluorescence polarization temperature-dependence of DPH and F-Con A. Studies of the effects of sotalol, alprenolol, and metoprolol are included and used for the comparison of different drug effects.

Alprenolol↗