Biomedical subjects

B R Stevens

Publications and source records attributed to B R Stevens.

At least 37 records · Page 2Linked to original sources

PubMed1995

Amino acid metabolism and the vascular endothelium: regulation and disease implications.

Amino acid metabolism by the vascular endothelium is a complex process that often begins with the carrier-mediated uptake of circulating amino acids into the endothelial cytoplasm. Amino acids are essential for maintaining intact endothelial functions, which include cell proliferation, regulation of blood flow and vascular tone, coagulation and fibrinolysis, and metabolism of a variety of macromolecules. The disturbances in endothelial amino acid transport and metabolism that occur during infection and inflammation are due, in part, to changes in substrate availability and to the local and/or systemic elaboration of specific mediators. An improved understanding of endothelial amino acid metabolism will not only provide new knowledge regarding disease mechanisms and regulation, but may also lead to new treatment strategies that may include the clinical use of specific nutritional formulas.

Amino Acid Sequence↗

PubMed1995

Regulation of system y+ arginine transport capacity in differentiating human intestinal Caco-2 cells.

This study describes the ability of passaged human intestinal Caco-2 cells to regulate transport of L-arginine via system y+. Subconfluent and confluent cells possessed system y+ activity, as determined by the sodium independence of uptake and the pattern of inhibition by amino acid analogues or N-ethylmaleimide. Initial rates of arginine uptake via system y+ decreased as the cells advanced from the undifferentiated to the differentiated state following culture passaging. Furthermore, kinetic analysis of the leucine-insensitive portion of uptake indicated that the Caco-2 system y+ transport capacity decreased with cell age, dropping from a maximal velocity (Vmax) = 1,094 pmol.mg-1.min-1 [Michaelis constant (Km) = 41 microM] in undifferentiated cells 2 days postseeding to Vmax = 320 pmol.mg-1.min-1 (Km = 37 microM) in confluent cells 9 days postseeding (from cells of the same passage). Northern analysis indicated that the levels of a single 7.9-kb mCAT-1 mRNA species were relatively constant over the course of Caco-2 differentiation and therefore were unsynchronized with the system y+ relative transport activities. It is concluded that the Caco-2 capacity to transport arginine via system y+ may be downregulated by posttransitional modifications in confluent cells compared with newly passaged undifferentiated cells. These data serve as a well-defined in vitro model for further studies regarding regulation of arginine transport in epithelial cells.

Amino Acids↗

PubMed1994

Thermodynamics of symport and antiport catalyzed by cloned or native transporters.

Thermodynamic measurements are required to confirm whether cloned transport-associated proteins in a membrane truly constitute a functional transport system. Symport or antiport, catalyzed by native systems or by cloned proteins in membranes, can lead to steady-state intracellular accumulation of solute when the electrochemical potentials of activator ion and solute are energetically coupled. Secondary active transport can occur if an appropriate physical coupling mechanism exists in the membrane. Driving forces for secondary active transport are ultimately established by primary active transport or respiration. Steep steady-state gradients of solute can be maintained when the ion:solute coupling ratio is greater than one and/or when coupling includes an electrical component. Although the steady-state accumulation of substrate is independent of the exact physical mechanism of transport, non-equilibrium and equilibrium transport kinetics aid in interpreting the rate, direction (symport versus antiport) and control of ion-coupled flux across a membrane. In some cases, the activator ion's chemical gradient alone is energetically adequate to maintain steady-state intracellular accumulation of solute, as demonstrated in invertebrate epithelial cells. To interpret accumulation ratios accurately, it is necessary to measure the intracellular activity coefficients for ions. For example, liquid ion-exchange microelectrode measurements demonstrate that over 30% of intracellular Na+ can be sequestered in epithelial cells.

Animals↗

PubMed1993

Recent advances in mammalian amino acid transport.

During the last four decades, mammalian amino acid transport systems have been described at the cellular level through general properties such as ion-dependence, kinetics, substrate specificity, regulation of activity, and numerous other characteristics. These studies have allowed the definition of multiple transport systems for neutral, anionic, and cationic amino acids. Each system is distinct but exhibits overlapping substrate specificity. Direct measurement of transport has permitted a wealth of information to be accumulated regarding the regulation of overall activity, but the underlying molecular mechanisms have not been investigated because of a lack of the appropriate tools. Recent research designed to obtain these tools has proven fruitful, and the field of amino acid transport clearly is entering a new era. In the immediate future, transporter properties such as hormonal regulation, adaptive control, ion-dependence, and trans-effects will be studied at the molecular level by assaying mRNA or protein content and by analyzing results obtained with altered protein structures following site-directed mutagenesis. Identification of specific proteins associated with activities already well described will provide answers to heretofore untestable questions. For example, is Na(+)-independent transport mediated by the same proteins that mediate Na(+)-dependent uptake except that their function in this mode does not require sodium binding? What is the protein composition of amino acid transporters? As discussed above, emerging evidence suggests that transporter proteins have different molecular structure, 12 versus 1 transmembrane domains, or that they exist as heterodimers or heterotetramers. Identification of certain transporter proteins and cloning of the respective genes also will provide valuable information about a number of inheritable diseases that are thought to be caused by defects in transporter synthesis or function. The opportunity to ask these questions will certainly generate renewed interest in the field of amino acid transport and lead to exciting advances in our knowledge.

Amino Acid Transport Systems↗

PubMed1993

Porphyria cutanea tarda in the setting of renal failure. Response to renal transplantation.

BACKGROUND: Porphyria cutanea tarda (PCT) and similar vesiculobullous disorders have been described with increased frequency in patients with chronic renal failure undergoing long-term hemodialysis. Descriptions of over 20 cases of hemodialysis-related PCT have appeared in the literature. The precise cause of PCT in chronic renal failure is unknown, and the treatment of PCT occurring in this setting is complex and often ineffective. OBSERVATIONS: We describe a patient who developed hemodialysis-related PCT, which, in this patient, was refractory to various treatment modalities. This patient eventually went on to receive a renal transplant and all symptoms of PCT resolved with normalization of urinary porphyrin levels. CONCLUSIONS: We propose that in PCT refractory to the currently available treatment modalities occurring after the development of chronic renal failure, renal transplantation is indicated as a potentially efficacious therapy.

Adult↗

PubMed1993

Regulation of small intestinal glutamine transport by epidermal growth factor.

BACKGROUND: Epidermal growth factor (EGF) stimulates cell replication and increases DNA content of the small intestine, but its effects on mucosal amino acid transport are unknown. METHODS: To investigate these effects, we treated adult rats with vehicle or EGF (10 micrograms/100 gm body weight subcutaneously every 8 hours for three doses). Jejunal brush border membrane vesicles (BBMVs) from each group were prepared by Mg++ aggregation/differential centrifugation. BBMVs were enriched fifteen-fold in alkaline phosphatase, indicating BBMV purity. Transport of 3H-glutamine and 3H-alanine was studied by a rapid mixing filtration technique. Uptakes were primarily Na+ dependent, occurred in an osmotically active space, exhibited classic overshoots, and had similar 2-hour equilibrium values. RESULTS: Glutamine transport by BBMVs more than doubled in rats treated with EGF (16.4 +/- 0.1 pmol glutamine/mg protein/10 sec in EGF vs 7.1 +/- 0.5 pmol glutamine/mg protein/10 sec in controls; p < 0.001). Kinetic studies of the glutamine transporter showed that the increase in transport was the result of a 70% increase in maximal transport velocity (total maximum glutamine uptake = 193 +/- 8 pmol glutamine/mg protein/10 sec in EGF vs 114 +/- 7 pmol glutamine/mg protein/10 sec in controls; p < 0.0001 with no change in transporter affinity (transporter affinity = 224 +/- 6 mumol/L in EGF vs 242 +/- 37 mumol/L in controls; difference, not significant). Alanine uptake by BBMVs was also increased with EGF administration (10.2 +/- 2.0 pmol alanine/mg protein/10 sec in EGF vs 4.5 +/- 0.5 pmol alanine/mg protein/10 sec in controls; p < 0.005). Simultaneously, glucose transport was decreased by 50% in EGF-treated rats, indicating that the Na(+)-dependent glucose cotransporter is regulated independently from and opposite to amino acid transporters. CONCLUSIONS: We conclude that EGF up-regulates amino acid transport activity in jejunal BBMVs, an event that is most likely caused by an increase in de novo biosynthesis of transporter protein. The increase in amino acid uptake not only may support de novo protein synthesis but, in the case of glutamine, also may be required for energy production and nucleotide biosynthesis.

Alanine↗

PubMed1992

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.

Adenocarcinoma↗

PubMed1992

Vertebrate intestine apical membrane mechanisms of organic nutrient transport.

This paper presents the current understanding of comparative vertebrate intestine basic mechanisms of brush-border membrane transport. Animals control the uptake of monosaccharides and amino acids at three levels: 1) mucosal hyperplasia increases uptake nonselectively, 2) individual enterocytes increase the transport capacity of specific transporter systems, and 3) the transporters themselves are modulated by solute and ion electrochemical gradients. In light of the current literature, This paper summarizes the kinetics, thermodynamics, and the physical arrangement of one mode of transport, the prototype Na(+)-solute cotransporter. The model presented is experimentally consistent with "preferred random" kinetics, with Na+ binding preferentially before solute at the extracellular face. In the case of glucose, the cotransporter system may be physically arranged in the membrane as a tetramer comprising 73,000 Da subunits. All vertebrates may have evolved with a similar mechanism, with particular variations reflecting selected arrangements from a pool of polypeptide sequence blocks. The same fundamental transport mechanisms may be observed in the intestines of animals ranging from lower vertebrates through humans.

Amino Acids↗

PubMed1991

Adaptive regulation of brush-border amino acid transport in a chronic excluded jejunal limb.

We examined the alterations in brush-border glutamine transport that occurred in a surgically defunctionalized jejunal limb excluded from mucosal food contact. Dogs were surgically prepared with Roux-en-Y gastrojejunostomies to permit same-intestine comparisons of glutamine transport and glutaminase activity in jejunal segments that were in incontinuity or excluded for a 6-mo period. Transport of glutamine, alanine, and glucose was measured in brush-border membrane vesicles prepared from each intestinal section; membrane marker enzymes were enriched to the same degree in incontinuity and excluded portions. The Na(+)-dependent glutamine cotransport apparent Km was the same in the excluded (779 +/- 63 microM) and incontinuity (873 +/- 105 microM) limbs. However, the Jmax for Na(+)-independent glutamine transport in the incontinuity jejunum (158.7 +/- 15.7 pmol.mg protein-1.s-1) was double that in the excluded limb (71.2 +/- 4.6 pmol.mg protein-1.s-1). Na(+)-dependent carrier-mediated glutamine transport rates were lower than the Na(+)-dependent system, but Na(+)-independent kinetic parameters were not significantly different in incontinuity vs. excluded limbs (Jmax 7.9 +/- 0.6 pmol.mg protein-1.s-1; Km 140 +/- 20 microM). Similarly, the passive diffusion permeability coefficient was the same for both excluded and incontinuity jejunal limbs (22.7 +/- 0.9 nl.mg protein-1.s-1). Mucosal glutaminase enzyme activity was increased by 28% in the incontinuity limb (4.32 +/- 0.21 vs. 3.36 +/- 0.35 mumol.mg protein-1.h-1; P less than 0.02). Transport rates of alanine and glucose were also diminished in the excluded limb (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1991

High-moisture diet for laboratory rats: complete blood counts, serum biochemical values, and intestinal enzyme activity.

Rats were fed an irradiated high-moisture diet (KSC-25) with or without access to a water bottle. Physiologic values were compared between these two groups and a group of rats fed a purified diet. Hematologic and serum biochemical values, urine specific gravity, and intestinal enzyme activities were determined from samples collected from the three groups of rats. Sprague Dawley rats (n = 32) fed the irradiated high-moisture diet with or without a water bottle were the test animals. Rats (n = 16) fed an irradiated purified diet and water provided via a water bottle were the control group. The purified diet formulation modified AIN-76A, is a commonly used purified diet for laboratory rodents. All rats remained alert and healthy throughout the study. A comparison of the physiologic values of rats in this study with reported normal values indicated that all of the rats in the study were in good health. Significant differences (P less than 0.05) of the physiologic values from each rat group are reported.

Animal Feed↗

PubMed1990

Dietary modulation of small intestinal glutamine transport in intestinal brush border membrane vesicles of rats.

The effects of a glutamine-enriched diet on the transport of glutamine across brush border membrane vesicles (BBMV) from the rat jejunum were studied to gain further insight into the effects of diet on regulating gut glutamine utilization. Following fasting, rats were randomized to one of three nutritionally complete elemental diets supplemented with glutamine, glutamate, or glycine (control). Brush border membrane vesicles were prepared by a Mg2+ aggregation/differential centrifugation technique and uptake of radioactive [3H]glutamine by the BBMV was studied using a rapid mixing/filtration technique. BBMVs from all test diet groups were enriched in alkaline phosphatase 14-fold. [3H]Glutamine uptake courses for all groups demonstrated sodium dependency, overshoots, and similar 2-hr equilibrium values. Vesicles from animals fed the glutamine-enriched diet had a 75% increase in glutamine uptake compared to those of the control diet and a 250% increase compared to those of the glutamate-enriched diet (P less than 0.05). alpha-Methylamino isobutyric acid and glycine did not significantly inhibit total [3H]glutamine uptake, whereas asparagine and glutamine inhibited total [3H]glutamine uptake compared to the mannitol control. The brush border appears to possess the glutamine selective System N transporter, the activity of which can be stimulated by providing dietary glutamine.

Amino Acids↗

PubMed1990

Receptors, phosphoinositol hydrolysis and plasticity of nerve cells.

Excitatory amino acid neurotransmission has been shown to be necessary but may not be sufficient, for the production of LTP and other prolonged changes in synaptic transmission. Excitatory neurotransmission may produce depolarization-induced increases in intracellular calcium that cause PI hydrolysis and synergistically potentiate receptor-G protein induced PI hydrolysis. This synergistic potentiation of phosphoinositide hydrolysis, and increased [Ca]i due to positive cross stimulation, may lead to depolarization block, a persistent increase in protein kinase activation, altered morphology, oncogene activity and other plasticity changes important in memory.

Animals↗

PubMed1990

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-135 degrees C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10(-19) J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na(+)-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na(+)-dependent cotransporter activity Jmax without affecting apparent Km. The size of the transporting functional unit was 290 +/- 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 +/- 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.

Animals↗

PubMed1989

Inhibition of the Na+/glucose cotransporter functional lysyl residues by 4-(4-methoxy-3-sulfophenyl)-2,6-bis(4-sulfophenyl)pyryl ium.

Pyrylium salts react exclusively with the epsilon-amino group of lysyl moieties and display a degree of selectivity exceeding all other known amino group reagents. This study represents the first use of a pyrylium salt in positively identifying the functional role of lysyl residues in a biologically active molecule, the Na+/glucose cotransporter protein. We synthesized a hydrophilic pyrylium salt, 4-(4-methoxy-3-sulfophenyl)-2,6-bis(4-sulfophenyl)pyryliu m perchlorate, which formed a stable pyridinium cation upon reaction with N alpha-acetyl-L-lysine methyl ester. This pyrylium inhibited Na+/glucose cotransporter activity in purified brush border membranes, in a concentration-dependent manner. The data unequivocally establish that critical lysyl residues are responsible for the activity of the membrane-bound glucose cotransporter protein.

Animals↗

PubMed1989

Physiological constraint on feeding behavior: intestinal membrane disaccharidases of the starling.

Animals clearly choose what they eat and can even choose among chemically different sugars. The physiological and biochemical mechanisms that constrain feeding choices are largely unknown. In this study, European starlings (Sturnus vulgaris) preferred mixture solutions of D-glucose plus D-fructose to equimolar (double molar caloric value) solutions of sucrose. Intubation feeding of sucrose did not increase blood glucose levels. Sucrose is a useless energy source for these birds because they lack a single digestive enzyme (sucrase) on the small intestinal brush border membrane. However, the membranes possessed separate maltase and isomaltase disaccharidases. This expression pattern and expression patterns of membrane disaccharidases among mammals suggest a role for intestinal enzymes in the coevolutionary interactions between vertebrates and their plant food sources.

Animals↗

PubMed1989

Energetics of sodium-coupled active transport mechanisms in invertebrate epithelia.

The Na+ gradient has been implicated as the sole or primary energy source for accumulative transport of organic solutes (e.g., sugars, amino acids) across the mucosal membrane of a variety of epithelial cells. A basic question concerning the Na+-coupled transport process in epithelia is whether the energy available from the transmucosal Na+ electrochemical difference is sufficient to sustain an accumulated organic solute steady-state level. Measurements of Na+ activities, with Na+-sensitive microelectrodes, gave accurate estimates of the Na+ electrochemical potential difference across the mucosal membrane of Aplysia californica gut. The results suggest that the transmucosal Na+ gradient can furnish sufficient energy to sustain the observed intracellular levels of the cotransported species. Many other species sustain large intracellular-extracellular gradients (less than 10(6):1) of free solutes. Theoretical models suggest that secondary active transport mechanisms in these epithelia operate by energetic multiple coupling to the Na+ electrochemical gradient; coupling coefficients of approximately 3 may represent an evolutionary optimization of these epithelial cotransporters. To properly investigate the cotransport mechanisms and energetics in invertebrate membranes, prototype mammalian vesicle experiments should be extended to the invertebrate laboratory.

Animals↗

PubMed1988

Neonatal rat brain astroglial dipeptidyl peptidase II activity regulation by cations and anions.

Astrocytic glial cells from neonatal rat brains were grown in primary culture. Dipeptidyl peptidase II (DPP-II) enzyme activity was measured in cells disrupted by nonionic detergent. The rate of enzyme activity was measured in the presence of various ions, under isosmotic conditions adjusted using mannitol and NaCl. DPP-II activity was not affected by the candidate metal co-factors (2 mM) Co2+, Mg2+, and Mn2+, nor by the metal chelators EDTA and o-phenanthroline. However, selected cations (50 mM Cl- salts) significantly inhibited DPP-II activity compared to Na+ control; the relative inhibition ranking was Rb+ less than K+ less than Zn2+ less than Hg2+. Many test anions (50 mM Na+ salts) also inhibited DPP-II activity compared to Cl- control: SO4(2-) less than NO3- less than F- less than SO3(2-). Surprisingly, the anion S2O3(2-) was the only test agent which strongly stimulated activity. The data are consistent with the concept that specific ion species interact with the glial DPP-II enzyme to affect catalytic activity.

Animals↗

PubMed1988

Lanthanide-stimulated glucose and proline transport across rabbit intestinal brush-border membranes.

Trivalent cations of the lanthanide series (La3+----Yb3+) stimulated uptake of proline or glucose in rabbit small intestinal brush-border membrane vesicles. The lanthanides stimulated uptake to an extent greater than Al3+, choline, and in many cases, Na+. A time-course of Er3+-stimulated glucose uptake gave initial rates and overshoots greater than Na+ stimulation. The best activators were Sm3+, Eu3+ and Tm3+, which stimulated proline initial uptakes by 400-600%, and stimulated glucose uptake by 120-150%, compared to Na+. The best lanthanide cotransport activators possessed high third ionization potentials.

Animals↗