Uptake of 3-O-methyl-D-[U-14C]glucose into brain of rainbow trout: possible effects of melatonin

The influx of glucose into the brain and plasma glucose disappearance were estimated in rainbow trout (Oncorhynchus mykiss) intravenously injected (1 ml · kg⁻¹ body weight) with a single dose (15 μCi · kg⁻¹ body weight) of 3-O-methyl-D-[U-¹⁴C]glucose ([U-¹⁴C]-3-OMG) at different times (2–160 min), and after intravenous injection at 15 min of increased doses (10–60 μCi · kg⁻¹ body weight) of [U-¹⁴C]-3-OMG. Brain and plasma radiotracer concentrations were measured, and several kinetic parameters were calculated. The apparent brain glucose influx showed a maximum after 15–20 min of injection then decreased to a plateau after 80 min. Brain distribution space of 3-OMG increased from 2 min to 20 min reaching equilibrium from that time onwards at a value of 0.14 ml · g⁻¹. The unidirectional clearance of glucose from blood to brain (k₁) and the fractional clearance of glucose from brain to blood (k₂) were estimated to be 0.093 ml · min⁻¹ · g⁻¹, and 0.867 min⁻¹, respectively. A linear increase was observed in brain and plasma radiotracer concentrations when increased doses of [U-¹⁴C]-3-OMG were used. All these findings support a facilitative transport of glucose through the blood-brain barrier of rainbow trout with characteristics similar to those observed in mammals. The injection of different doses of melatonin (0.25–1.0 mg · kg⁻¹) significantly increased brain glucose influx suggesting a possible role for melatonin in the regulation of glucose transport into the brain. Accepted: 26 January 2000     

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.     

Identification of the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney

Summary. Glucocorticoid hormones enhance the reabsorptive capacity of filtered amino acids in rat kidney, as it was shown in previous in vivo clearance experiments. In the present study, the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney was investigated using in vivo micropuncture technique. Adult female Wistar rats were treated with dexamethasone (DEX), and fractional excretion of L-glutamine (L-Gln) and L-leucine (L-Leu) were determined and related to inulin after microinfusion into different nephron segments. DEX reduced fractional excretion of both neutral amino acids as a sign of enhanced reabsorptive capacity. The site of main DEX action on L-Leu reabsorption has been localized in the proximal straight tubule. However, in the case of L-Gln, the inhibition of γ-glutamyltranspeptidase (γ-GT) by administration of acivicin indicated the importance of this brush border enzyme in reduced L-Gln excretion. DEX enhanced γ-GT activity by tubular acidification. It can be presumed a DEX-inducible transport system for neutral amino acids mainly localized in proximal straight tubules of rat kidney. Received July 8, 1999     

Modulation of taurine uptake in the goldfish retina and axonal transport to the tectum¶Effect of crushing the optic nerve or axotomy

Summary. Although there are a great number of studies concerning the uptake of taurine in several tissues, the regulation of taurine transport has not been studied in the retina after lesioning the optic nerve. In the present study, isolated retinal cells of the goldfish retina were used either immediatly after cell suspension or in culture. The high-affinity transport system of [³H]taurine in these cells was sodium-, temperature- and energy-dependent, and was inhibited by hypotaurine and β-alanine, but not by γ-aminobutyric acid. There was a decrease in the maximal velocity (V_max) without modifications in the substrate affinity (K_m) after optic axotomy. These changes were mantained for up to 15 days after the lesion. The results might be the summation of mechanisms for providing extracellular taurine to be taken up by other retinal cells or eye structures, or regulation by the substrate taurine, which increases after lesioning the optic nerve. The in vivo accumulation of [³H]taurine in the retina after intraocular injection of [³H]taurine was affected by crushing the optic nerve or by axotomy. A progressive retinal decrease in taurine transport was observed after crushing the optic nerve, starting at 7 hours after surgery on the nerve. The uptake of [³H]taurine by the tectum was compensated in the animals that were subjected to crushing of the optic nerve, since the concentration of [³H]taurine was only different from the control value 24 hours after the lesion, indicating an efficient transport by the remaining axons. On the contrary, the low levels of [³H]taurine in the tectum after axotomy might be an index of the non-axonal origin of taurine in the tectum. Axonal transport was illustrated by the differential presence of [³H]taurine in the intact or crushed optic nerve. The uptake of [³H]taurine into retinal cells in culture in the absence or in the presence of taurine might indicate the existence of an adaptive regulation of taurine transport in this tissue, however taurine transport probably differentially occurs in specific populations of retinal cells. The use of a purified preparation of cells might be useful for future studies on the modulation of taurine transport by taurine in the retina and its role during regeneration. Received June 11, 1999/Accepted August 31, 1999     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000     

A mathematical model of solute coupled water transport in toad intestine incorporating recirculation of the actively transported solute

A mathematical model of an absorbing leaky epithelium is developed for analysis of solute coupled water transport. The non-charged driving solute diffuses into cells and is pumped from cells into the lateral intercellular space (lis). All membranes contain water channels with the solute passing those of tight junction and interspace basement membrane by convection-diffusion. With solute permeability of paracellular pathway large relative to paracellular water flow, the paracellular flux ratio of the solute (influx/outflux) is small (2-4) in agreement with experiments. The virtual solute concentration of fluid emerging from lis is then significantly larger than the concentration in lis. Thus, in absence of external driving forces the model generates isotonic transport provided a component of the solute flux emerging downstream lis is taken up by cells through the serosal membrane and pumped back into lis, i.e., the solute would have to be recirculated. With input variables from toad intestine (Nedergaard, S., E.H. Larsen, and H.H. Ussing, J. Membr. Biol. 168:241-251), computations predict that 60-80% of the pumped flux stems from serosal bath in agreement with the experimental estimate of the recirculation flux. Robust solutions are obtained with realistic concentrations and pressures of lis, and with the following features. Rate of fluid absorption is governed by the solute permeability of mucosal membrane. Maximum fluid flow is governed by density of pumps on lis-membranes. Energetic efficiency increases with hydraulic conductance of the pathway carrying water from mucosal solution into lis. Uphill water transport is accomplished, but with high hydraulic conductance of cell membranes strength of transport is obscured by water flow through cells. Anomalous solvent drag occurs when back flux of water through cells exceeds inward water flux between cells. Molecules moving along the paracellular pathway are driven by a translateral flow of water, i.e., the model generates pseudo-solvent drag. The associated flux-ratio equation is derived.     

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

Membrane Transport Generated by the Osmotic and Hydrostatic Pressure. Correlation Relation for Parameters L p, σ, and ω

Standard approach to membrane transport generated by osmotic andhydrostatic pressures, developed by Kedem and Katchalsky, is based onprinciples of thermodynamics of irreversible processes. In this paper wepropose an alternative technique. We derive transport equations from fewfairly natural assumptions and a mechanistic interpretation of the flows.In particular we postulate that a sieve-type membrane permeability isdetermined by the pore sizes and these are random within certain range.Assuming that an individual pore is either permeable or impermeable tosolute molecules, the membrane reflection coefficient depends on the ratioof permeable and impermeable pores. Considering flows through permeableand impermeable pores separately, we derive equations for the total volumeflux, solute flux and the solvent flux across the membrane. Comparing themechanistic equations to the Kedem-Katchalsky equations we find the formereasier to interpret physically. Based on the mechanistic equations we alsoderive a correlation relation for the membrane transport parameters L _p,, and . This relation eliminates the need for experimentaldetermination of all three phenomenological parameters, which in somecases met with considerable difficulties.