Richard J. Naftalin

Mechanisms of glucose transport at the blood-brain barrier: an in vitro study.

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood-brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood-brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood-brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood-brain barrier model of approximately 0.85. Under conditions where [glucose](lumen) was higher than [glucose](ablumen), influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.

Docking Studies Show That D-Glucose and Quercetin Slide through the Transporter GLUT1

On a three-dimensional templated model of GLUT1 (Protein Data Bank code 1SUK), a molecular recognition program, AUTODOCK 3, reveals nine hexose-binding clusters spanning the entire “hydrophilic” channel. Five of these cluster sites are within 3-5 Å of 10 glucose transporter deficiency syndrome missense mutations. Another three sites are within 8 Å of two other missense mutations. d-Glucose binds to five sites in the external channel opening, with increasing affinity toward the pore center and then passes via a narrow channel into an internal vestibule containing four lower affinity sites. An external site, not adjacent to any mutation, also binding phloretin but recognizing neither d-fructose nor l-glucose, may be the main threading site for glucose uptake. Glucose exit from human erythrocytes is inhibited by quercetin (Ki = 2.4 μm) but not anionic quercetin-semiquinone. Quercetin influx is retarded by extracellular d-glucose (50 mm) but not by phloretin and accelerated by intracellular d-glucose. Quercetin docking sites are absent from the external opening but fill the entire pore center. In the inner vestibule, Glu254 and Lys256 hydrogen-bond quercetin (Ki ≈ 10 μm) but not quercetin-semiquinone. Consistent with the kinetics, this site also binds d-glucose, so quercetin displacement by glucose could accelerate quercetin influx, whereas quercetin binding here will competitively inhibit glucose efflux. β-d-Hexoses dock twice as frequently as their α-anomers to the 23 aromatic residues in the transport pathway, suggesting that endocyclic hexose hydrogens, as with maltosaccharides in maltoporins, form π-bonds with aromatic rings and slide between sites instead of being translocated via a single alternating site.

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

The secretion of uterine luminal fluid initially provides a transport and support medium for spermatozoa and unimplanted embryos, while the absorption of uterine luminal fluid in early pregnancy results in the closure of the lumen and allows blastocysts to establish intimate contact with the uterine epithelium. We have established an in vivo perfusion technique of the lumen to study the hormonal control of the events in the peri-implantation period. Fluorescein-labelled dextran was included in the perfusion medium to monitor fluid movements and the concentrations of Na+ and CI− ions in the effluent were monitored. Using an established regimen of steroid treatment of ovariectomized rats mimicking early pregnancy, oestradiol caused fluid secretion, while progesterone resulted in an amiloride-sensitive fluid absorption. Fluid absorption peaked at about the expected time of implantation. The effect of progesterone could be inhibited by treatment with a high dose of oestradiol, by the anti-progestin RU486, and by the presence of an intra-uterine contraceptive device. Studies of expression of Na+ and CI− channels (ENaC, CFTR) indicated that these channels were subject to tissue-specific regulation within the uterus, but more work is required to determine their role and the factors controlling their abundance and localization in early pregnancy.

Regional crypt function in rat large intestine in relation to fluid absorption and growth of the pericryptal sheath.

1 Confocal microscopic studies of rat colonic mucosa showed that the pericryptal sheath surrounding distal colonic crypts is an effective barrier both to dextran and NaCl movement, whereas no such structure surrounds the caecal crypts. 2 The distal colonic pericryptal barrier was functionally demonstrated by accumulation of Sodium Green within the pericryptal space. After exposure to benzamil, Sodium Green accumulation was decreased. Fluorescein isocyanate‐labelled dextran (FITC dextran; molecular mass 10000 Da) was accumulated in the crypt lumens and pericryptal spaces. Both dextran and Sodium Green accumulation were absent from the pericryptal zone surrounding caecal crypts. 3 Low dietary Na+ intake raised rat plasma aldosterone and stimulated distal pericryptal sheath growth and adhesiveness as shown by increased amounts of F‐actin, smooth muscle actin, β‐catenin and E‐cadherins in the pericryptal zone. It also raised the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. This linkage indicates that trophic effects on the colon resulting from a low Na+ diet are not confined solely to effects on transepithelial Na+ transport, but are observed in the pericryptal sheath. 4 A computer model of crypt function confirms that a pericryptal sheath with low permeability to NaCl is an essential component of the crypt dehydrating mechanism.

Interactions of androgens, green tea catechins and the antiandrogen flutamide with the external glucose-binding site of the human erythrocyte glucose transporter GLUT1

This study investigates the effects of androgens, the antiandrogen flutamide and green tea catechins on glucose transport inhibition in human erythrocytes. These effects may relate to the antidiabetogenic effects of green tea. Testosterone, 4‐androstene‐3,17‐dione, dehydroepiandrosterone (DHEA) and DHEA‐3‐acetate inhibit glucose exit from human erythrocytes with half‐maximal inhibitions (Ki) of 39.2±8.9, 29.6±3.7, 48.1±10.2 and 4.8±0.98 μM, respectively. The antiandrogen flutamide competitively relieves these inhibitions and of phloretin. Dehydrotestosterone has no effect on glucose transport, indicating the differences between androgen interaction with GLUT1 and human androgen receptor (hAR). Green tea catechins also inhibit glucose exit from erythrocytes. Epicatechin 3‐gallate (ECG) has a Ki ECG of 0.14±0.01 μM, and epigallocatechin 3‐gallate (EGCG) has a Ki EGCG of 0.97±0.13 μM. Flutamide reverses these effects. Androgen‐screening tests show that the green tea catechins do not act genomically. The high affinities of ECG and EGCG for GLUT1 indicate that this might be their physiological site of action. There are sequence homologies between GLUT1 and the ligand‐binding domain (LBD) of hAR containing the amino‐acid triads Arg 126, Thr 30 and Asn 288, and Arg 126, Thr 30 and Asn 29, with similar 3D topology to the polar groups binding 3‐keto and 17‐β OH steroid groups in hAR LBD. These triads are appropriately sited for competitive inhibition of glucose import at the external opening of the hydrophilic pore traversing GLUT1.

Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1.

17 beta-Oestradiol (ED when subscript to K) and the phytoestrogen isoflavone genistein (GEN) inhibit glucose transport in human erythrocytes and erythrocyte ghosts. The selective oestrogen receptor modulators or anti-oestrogens, faslodex (ICI 182780) (FAS) and tamoxifen (TAM), competitively antagonize oestradiol inhibition of glucose exit from erythrocytes (K(i(ED/FAS))=2.84+/-0.16 microM and K(i(ED/TAM))=100+/-2 nM). Faslodex has no significant inhibitory effect on glucose exit, but tamoxifen alone inhibits glucose exit (K(i(TAM))=300+/-100 nM). In ghosts, ATP (1-4 mM) competitively antagonizes oestradiol, genistein and cytochalasin B (CB)-dependent inhibitions of glucose exit, (K(i(ATP/ED))=2.5+/-0.23 mM, K(i(ATP/GEN))=0.99+/-0.17 mM and K(i(ATP/CB))=0.76+/-0.08 mM). Tamoxifen and faslodex reverse oestradiol-dependent inhibition of glucose exit with ATP>1 mM (K(i(ED/TAM))=130+/-5 nM and K(i(ED/FAS))=2.7+/-0.9 microM). The cytoplasmic surface of the glucose transporter (GLUT)1 contains four sequences with close homologies to sequences in the ligand-binding domain of human oestrogen receptor beta (hesr-2). One homology is adjacent to the Walker ATP-binding motif II (GLUT1, residues 225-229) in the large cytoplasmic segment linking transmembrane helices 6 and 7; another GLUT (residues 421-423) contains the Walker ATP-binding motif III. Mapping of these regions on to a three-dimensional template of GLUT indicates that a possible oestrogen-binding site lies between His(337), Arg(349) and Glu(249) at the cytoplasmic entrance to the hydrophilic pore spanning GLUT, which have a similar topology to His(475), Glu(305) and Arg(346) in hesr-2 that anchor the head and tail hydroxy groups of oestradiol and genistein, and thus are suitably placed to provide an ATP-sensitive oestrogen binding site that could modulate glucose export.

Evidence from fluorescence microscopy and comparative studies that rat, ovine and bovine colonic crypts are absorptive.

1. To test whether colonic crypts are secretory or absorptive interstitial [Na+] in rat descending colonic mucosa is determined using video‐enhanced imaging of the impermeant acid form of the fluorescent Na+ probe SBFI (Molecular Probes) and intracellular [Na+] is monitored with SBFI (AM form). In rat descending colonic mucosa perifused with isotonic Tyrode solution interstitial [Na+] = 500‐650 mM. Following exposure to Tyrode solution containing theophylline (10 mM) interstitial [Na+] falls by 300‐450 mM within 1 min. Exposure to amiloride (0.2 mM) reduces the intracellular [Na+] from ca 25 to 12 mM within 15 min and concurrently decreases [Na+] in the interstitial fluid surrounding the crypts at the mucosal surface by approximately 200 mM. 2. The route of fluid inflow across the rat colonic mucosa is directly traced by perifusing with Tyrode solution containing the impermeant fluorescent dye, fluorescein disulphonate (FS). FS accumulates rapidly within crypt lumens of control tissues to a 2‐fold higher concentration than in the external bathing solution, but FS does not accumulate in crypts of tissues treated with azide (2 mM). The increment in FS accumulation within the crypt lumen above the bulk solution decreases by 80% within 1 min following exposure to theophylline (10 mM), indicating that fluid absorption into crypts is reduced. Estimates of the total fluid influx from the rate and extent of FS concentration polarization within crypts indicate that it is sufficient to account for the entire transcolonic fluid absorption. 3. Comparative studies of isolated bovine and ovine colon were also undertaken to investigate the failure of bovine colon to generate a hypertonic absorbate and hence its incapacity to produce hard faeces. The interstitial fluid surrounding ovine colonic crypts is hypertonic to the bulk solution, whereas the interstitial fluid surrounding bovine colonic crypts is nearly isotonic with the bathing solution. Additionally, fluorescein disulphonate accumulates within ovine colonic crypt lumens by concentration polarization, whereas no concentration of FS occurs within bovine colonic crypt lumens. This corroborates the view that a hypertonic interstitial fluid is absent from bovine colon mainly because of a high rate of transepithelial leakage of low molecular weight solutes via paracellular routes.

Radiation induced cytochrome c release causes loss of rat colonic fluid absorption by damage to crypts and pericryptal myofibroblasts

BACKGROUND Therapeutic or accidental exposure to radiation commonly causes gastrointestinal disturbances, including diarrhoea. Rats subjected to whole body ionising radiation at a dose of 8 Gy lose their capacity to absorb fluid via the descending colon after four days. After seven days, fluid absorption recovers to control levels. AIMS To investigate the effect of ionising radiation on colonic permeability together with its effect on mitochondria dependent apoptotic signals and intercellular adhesion molecules. METHODS Rats were irradiated with doses of 0–12 Gy. Colonic permeability was measured by accumulation of fluorescein isothiocyanate (FITC) dextran in crypt lumens. Changes in levels of cytochrome c, caspase 3, E and OB cadherin, β-catenin smooth muscle actin, and collagen IV were assessed using immunocytochemistry with confocal microscopy. RESULTS Cytosolic cytochrome c increased after 8 Gy (t1/2 1.4 (0.6) hours) and peaked at approximately six hours. Caspase 3 increased more slowly, particularly in crypt epithelial cells (t1/2 57 (14.5) hours). Pericryptal myofibroblasts disintegrated within 24 hours as was evident from loss of OB cadherin and smooth muscle actin. This coincided with increased crypt permeability to dextran. Intercellular adhesion between crypt luminal cells was not lost until day 4 when both β-catenin and E-cadherin were minimal. The half maximal dose-response for these effects was in the range 2–4 Gy. Recovery of colonic transport was concurrent with recovery of pericryptal smooth muscle actin and OB cadherin. The pan caspase inhibitor Z-Val-Ala-Asp.fluoromethylketone (1 mg/kg per day) had a small effect in conserving the pericryptal sheath myofibroblasts and sheath permeability but had no systemic therapeutic effects. CONCLUSIONS These data suggest that radiation damage to the colon may be initiated by mitochondrial events. Loss of crypt fluid absorption and increased permeability coincided with decreased intercellular adhesion between crypt epithelial cells and loss of pericryptal sheath barrier function.

Regional differences in rat large intestinal crypt function in relation to dehydrating capacity in vivo

1 Rat descending colon absorbed fluid against a large hydraulic resistance, imposed by 10% agarose (w/v) gel plugs inserted in the lumen, by raising the tonicity of the absorbate from the gel to 880 ± 54 mosmol kg−1; the tonicity of the absorbate from 2.5% gels was 352 ± 38 mosmol kg−1. The hypertonic absorbate generated an osmotic pressure which created a fluid tension in the crypt lumen. This was monitored as a suction tension in colonic luminal gels of 45.3 ± 3 cmH2O with 2.5% gels and 725 ± 145 cmH2O with 10% gels. The caecum was unable to absorb fluid against a significant hydraulic resistance. 2 Fluorescein isothiocyanate‐labelled dextran (FITC dextran; molecular mass 10000 Da) accumulated within descending colonic crypt lumens by concentration polarization. Maximal accumulation at a depth of 20–40 μm below the mucosal surface was 5.68 ± 0.2‐fold above control levels. Caecal crypts accumulated dextran to a maximum of 1.8 ± 0.17‐fold above control levels. 3 The relationship between crypt luminal tension and suction tension of the distal colon was also demonstrated using paraffin, which occluded the crypt lumens with microscopic droplets and completely inhibited fluid absorption from high resistance luminal gels. 4 Reduction in dietary Na+ intake raised plasma aldosterone and the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. The caecum did not respond in this way to varied Na+ intake.

Osmotic Water Transport with Glucose in GLUT2 and SGLT

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na+-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na+-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.