Bruce R. Stevens

Physiological Correlates of Preference and Aversion for Sugars in Three Species of Birds

We studied preferences of red-winged blackbirds, common grackles, and European starlings for the disaccharide sucrose and the monosaccharides glucose and fructose. We also tested the hypothesis that sucrose preference is correlated with activity of sucrase, the intestinal enzyme that hydrolyzes sucrose. All species preferred a 1:1 mixture of glucose and fructose over water at concentrations > 0.75 M. Starlings rejected concentrated solutions of sucrose and lacked sucrase activity. Red-winged blackbirds preferred sucrose solutions at intermediate concentrations (0.35 M) but rejected concentrated ones, whereas common grackles preferred sucrose at all the concentrations offered. Sucrase activity was higher in grackles than in red-winged blackbirds. Blood glucose levels were measured after a meal of a mixture of glucose and fructose or of sucrose. Blood glucose concentration increased in all species after the monosaccharide meal and peaked after about 30 min. In starlings the concentration of glucose in blood remained constant after a sucrose meal, whereas in the other species there was an increase similar to that observed after a monosaccharide meal. Because starlings are mainly insectivorous and blackbirds and grackles are granivorous, we hypothesize that relative activity of intestinal sucrase and hence the preference for sucrose are related to the proportion of complex carbohydrates in a birds diet.

Double-stranded RNA signals antiviral and inflammatory programs and dysfunctional glutamate transport in TLR3-expressing astrocytes

Astrocyte inflammation, reactive oxygen species (ROS) formation, and dysfunction form a common denominator shared by all the major neurodegenerative disorders. Viral infections are emerging as important events in the etiology of CNS damage involving astrocytes, but molecular understanding is incomplete. Double‐stranded RNA (dsRNA) is a byproduct of viral replication and serves as the signature molecule for viral infection via Toll‐like receptor 3 (TLR3) largely restricted to circulating peripheral dendritic cells. However, astrocytes are strategically located at the blood‐brain barrier (BBB) and throughout brain tissues, making these cells ideal candidates as innate immunity sentinels within the CNS. We hypothesized that extracellular dsRNA, mimicked by polyinosinic‐polycytidylic acid (Poly(I:C); PIC), initiates signaling of the double‐edged sword of antiviral plus pathophysiological events in astrocytes. Using Western blot analysis and real‐time qPCR, we determined that neonatal rat astrocyte cultures constitutively express TLR3 mRNA and protein, and that PIC dsRNA induced phosphorylation of eIF2α, as well as mRNA type I interferon (α/β IFN)‐response genes Mx1, PKR, and TLR3. Astrocyte TLR3 protein was downregulated after PIC treatment, however. PIC signaled degradation of IκBα with the consequence of upregulating iNOS, TNF‐α, and IL‐1β mRNAs and proteins. In addition to antiviral protection events, dsRNA induced astrocyte dysfunction, evidenced by inhibiting EAAT1/GLAST transporter gene expression and attenuating L‐glutamate uptake via sodium‐dependent transport system X  AG− , as well as inducing cytotoxicity. Anti‐TLR3 blocking antibody attenuated PIC upregulation of TNF‐α mRNA and iNOS activity. Extracellular PIC‐induced events were prevented by 2‐aminopurine, implicating PKR as an important downstream player in astrocyte dsRNA sensing pathways. The effects of plasma membrane impermeable poly(I:C) were dose‐dependent (0–50 μM). In concert, these data provide evidence that dsRNA/TLR3‐activated astrocytes initiate a battery of rapid innate pathogen‐associated molecular pattern (PAMP) immune responses that are important for mounting antiviral defense in the CNS, yet also lead to pathophysiological events associated with the glutamate neurotoxicity of neurodegenerative diseases.

Resistance exercise prevents plantar flexor deconditioning during bed rest

Because resistance exercise (REX) and unloading induce opposing neuromuscular adaptations, we tested the efficacy of REX against the effects of 14 d of bed rest unloading (BRU) on the plantar flexor muscle group. Sixteen men were randomly assigned to no exercise (NOE, N = 8) or REX (N = 8). REX performed 5 sets x 6-10 repetitions to failure of constant resistance concentric/eccentric plantar flexion every other day during BRU. One-repetition maximum (1RM) strength was tested on the training device. The angle-specific torque-velocity relationship across 5 velocities (0, 0.52, 1.05, 1.75, and 2.97 rad.s-1) and the full range-of-motion power-velocity relationship were assessed on a dynamometer. Torque-position analyses identified strength changes at shortened, neutral, and stretched muscle lengths. Concentric and eccentric contractile work were measured across ten repetitions at 1.05 rad.s-1. Maximal neural activation was measured by surface electromyography (EMG). 1RM decreased 9% in NOE and improved 11% in REX (P < 0.05). Concentric (0.52 and 1.05 rad.s-1), eccentric (0.52 and 2.97 rad.s-1), and isometric angle-specific torques decreased (P < 0.05) in NOE, averaging 18%, 17%, and 13%, respectively. Power dropped (P < 0.05) in NOE at three eccentric (21%) and two concentric (14%) velocities. REX protected angle-specific torque and average power at all velocities. Concentric and eccentric strength decreased at stretched (16%) and neutral (17%) muscle lengths (P < 0.05) in NOE while REX maintained or improved strength at all joint positions. Concentric (15%) and eccentric (11%) contractile work fell in NOE (P < 0.05) but not in REX. Maximal plantar flexor EMG did not change in either group. In summary, constant resistance concentric/eccentric REX completely prevented plantar flexor performance deconditioning induced by BRU. The reported benefits of REX should prove useful in prescribing exercise for astronauts in microgravity and for patients susceptible to functional decline during bed- or chair-bound hospital stays.

Amino Acid Transport in Intestine

During the past 30 years many of the universal principles of membrane transport have been established or confirmed using the readily accessible and testable small intestine. Historically, amino acid transport studies have been conducted in a variety of species at various stages of ontogenetic development. The intact epithelium, isolated enterocytes, and isolated brush border and basolateral membranes have each been used to investigate the physiologic, thermodynamic and kinetic events of absorption. Recently, our laboratory (Pan et al., 1991) and others (Hidalgo & Borchardt, 1990) have used cultured intestinal cell lines to study amino acid transport regulation in monolayers that emulate the in vivo epithelium (Zweibaum et al., 1991).

A novel electrogenic amino acid transporter is activated by K+ or Na+, is alkaline pH-dependent, and is Cl--independent.

A new eukaryotic nutrient amino acid transporter has been cloned from an epithelium that is exposed to high voltages and alkaline pH. The full-length cDNA encoding this novel CAATCH1 (cation-anion-activated Amino acidtransporter/channel) was isolated using a polymerase chain reaction-based strategy, and its expression product inXenopus oocytes displayed a combination of several unique, unanticipated functional properties. CAATCH1 electrophysiological properties resembled those of Na+,Cl−-coupled neurotransmitter amine transporters, although CAATCH1 was cloned from a gut absorptive epithelium rather than from an excitable tissue. Amino acids such as l-proline, l-threonine, andl-methionine elicited complex current-voltage relationships in alkaline pH-dependent CAATCH1 that were reminiscent of the behavior of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, NET) in the presence of their substrates and pharmacological inhibitors such as cocaine or antidepressants. These I-V relationships indicated a combination of substrate-associated carrier current plus an independent CAATCH1-associated leakage current that could be blocked by certain amino acids. However, unlike all structurally related proteins, CAATCH1 activity is absolutely independent of Cl−. Unlike related KAAT1, CAATCH1 possesses a methionine-inhibitable constitutive leakage current and is able to switch its narrow substrate selectivity, preferring threonine in the presence of K+ but preferring proline in the presence of Na+.

Are intact peptides absorbed from the healthy gut in the adult human

For over 100 years it was believed that dietary protein must be completely hydrolysed before its constituent amino acids could be absorbed via specific amino acid transport systems. It is now known that the uptake of di- and tripeptides into the enterocyte is considerable, being transported across the intestinal endothelium by the PepT1 H+/peptide co-transporter. There is also evidence that some di- and tripeptides may survive cytosolic hydrolysis and be transported intact across the basolateral membrane. However, other than antigen sampling, the transport of larger intact macromolecules across the intestinal endothelium of the healthy adult human remains a controversial issue as there is little unequivocal in vivo evidence to support this postulation. The aim of the present review was to critically evaluate the scientific evidence that peptides/proteins are absorbed by healthy intestinal epithelia and pass intact into the hepatic portal system. The question of the absorption of oliogopeptides is paramount to the emerging science of food-derived bioactive peptides, their mode of action and physiological effects. Overall, we conclude that there is little unequivocal evidence that dietary bioactive peptides, other than di- and tripeptides, can cross the gut wall intact and enter the hepatic portal system in physiologically relevant concentrations.

Protein kinase C-dependent regulation of l-arginine transport activity in Caco-2 intestinal cells

The regulation of plasma membrane L-arginine transport activity was investigated in differentiated and undifferentiated states of the human intestinal cell line, Caco-2. The sodium-independent, leucine-insensitive uptake of L-arginine measured in this study has been assigned by us previously to system y+ in Caco-2 cells. Treatment of cells with serum-free media containing epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated system y+ arginine transport activity in Caco-2 cells. Transport upregulation by these growth factors or by TPA was blocked by cycloheximide or the PKC inhibitor chelerythrine. Arginine uptake was diminished during the course of differentiation, attributable to a reduction in the transport system y+ capacity (Vmax) with no change in apparent affinity (Km). TPA stimulated arginine uptake required at least 3 h of continual exposure, and increased the membranes transport capacity (Vmax) in both undifferentiated and differentiated cells. TPA elevated the diminished transport Vmax of differentiated cells TPA to the elevated Vmax value associated with undifferentiated cells. We conclude that upregulation of arginine transport is part of a pleiotropic response to EGF/TGF alpha, and that this involves PKC and de novo synthesis of polypeptides associated with system y+ transport activity.

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.

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.

Human intestinal brush border angiotensin-converting enzyme activity and its inhibition by antihypertensive Ramipril

Angiotensin-converting enzyme (ACE) has been identified as a prominent brush border membrane-bound enzyme of human jejunum. In this study, we purified brush border membrane vesicles enriched in ACE, and characterized the ACE with regard to (a) its stability in the membrane, (b) substrate hydrolysis kinetics compared with pulmonary endothelial ACE, and (c) pharmacologic interaction with Ramipril. These investigations resulted in the following findings. The uninhibited enzyme is stable in native membranes in vitro, with a half-life of 195 +/- 7 h. Kinetic analysis of ACE hydrolysis activity revealed the presence of a single enzyme species, which yielded a high Vmax and displayed a Km similar to purified ACE from lung endothelium. Brush border ACE was inhibited by Ramipril, one of the most specific and potent orally administered ACE inhibitors indicated for hypertension. We determined the brush border ACE value of IC50 = 3 X 10(-9) M Ramipril-diacid, which is the same value for serum and lung ACE. Brush border ACE remains 100% inhibited by 10 microM Ramipril during at least 8 days in vitro. The data indicate that ACE is a prominent jejunal brush border enzyme that behaves pharmacologically and kinetically like its peripheral circulation counterpart. This study suggests that high doses of orally administered ACE inhibitors may affect intestinal epithelial function.