Protonophore- and pH-insensitive glucose and sucrose accumulation detected by FRET nanosensors in Arabidopsis root tips

Although soil contains only traces of soluble carbohydrates, plant roots take up glucose and sucrose efficiently when supplied in artificial media. Soluble carbohydrates and other small metabolites found in soil are in part products from exudation from plant roots. The molecular nature of the transporters for uptake and exudation is unknown. Here, fluorescence resonance energy transfer (FRET) glucose and sucrose sensors were used to characterize accumulation and elimination of glucose and sucrose in Arabidopsis roots tips. Using an improved image acquisition set-up, FRET responses to perfusion with carbohydrates were detectable in roots within less than 10 sec and over a wide concentration range. Accumulation was fully reversible within 10-180 sec after glucose or sucrose had been withdrawn; elimination may be caused by metabolism and/or efflux. The rate of elimination was unaffected by pre-incubation with high concentrations of glucose, suggesting that elimination is not due to accumulation in a short-term buffer such as the vacuole. Glucose and sucrose accumulation was insensitive to protonophores, was comparable in media differing in potassium levels, and was similar at pH 5.8, 6.8 and 7.8, suggesting that both influx and efflux may be mediated by proton-independent transport systems. High-resolution expression mapping in root tips showed that only a few proton-dependent transport of the STP (Sugar Transport Protein) and SUT/SUC (Sucrose Transporter/Carrier) families are expressed in the external cell layers of root tips. The root expression maps may help to pinpoint candidate genes for uptake and release of carbohydrates from roots.     

Effect of toxic Fe2+ levels on the biological characteristics of rice root border cells

Root border cells are a population of rhizosphere cells surrounding the root tips but separated from them. The root tip is a major target of Fe²⁺ toxicity; thus, it was hypothesized that the border cells might protect or exacerbate Fe²⁺ toxicity. To explore the effects of excess Fe²⁺ on the border cells in rice (Oryza sativa L.), experiments were carried out using the border cells in vitro (Shanyou No. 10). The border cells were precultured under ??hanging in the air?? and detached from the root tips. The shape, numbers, and viability of border cells were examined during exposure to toxic levels of Fe²⁺. When the root was 1 mm long, there were 205 border cells on average. With the growth of the root, more border cells were observed. When the root grew to 25 mm long, the total number of border cells reached a maximum, while the maximum activity of border cells appeared when the root was 20 mm long. The pectin methyl esterase (PME) activity of the root cap peaked at a root length of 2 mm. Border cell development was related to PME activity in rice. Excessive Fe²⁺ was toxic to detached border cells. After treatment with 200 ??M Fe²⁺ solution for 48 h, cell viability decreased by 72.70%. However, when treated with 400 ??M Fe²⁺ solution, the number of viable cells was actually higher, suggesting the induction of a cellular self-protection response. The activity of PME first increased under high concentrations of Fe²⁺ and then decreased. These results indicate that toxic levels of Fe²⁺ modulate PME activity and border cell survival.     

Normalization of glucose entry under the high glucose condition by phlorizin attenuates the high glucose-induced morphological and functional changes of cultured bovine retinal pericytes

We previously reported that sodium-dependent glucose uptake is present in bovine retinal pericytes and that phlorizin normalizes its glucose consumption under high glucose conditions. To clarify the effect of phlorizin on morphological and functional change of retinal pericytes under high glucose conditions, retinal pericytes were incubated in media with 5 mM glucose, 30 mM glucose, and 30 mM glucose plus 0.2 mM phlorizin for 7 days. The diameter of cells in the concentrations of glucose more than 10 mM were significantly larger than those in 5 mM glucose and 30 mM glucose plus phlorizin. Glucose, sorbitol and fructose contents of the cells in 30 mM glucose were significantly increased compared with those in 5 mM glucose, and were normalized by phlorizin. Thymidine uptake in the concentrations of glucose more than 20 mM was significantly decreased compared with that in 5 mM glucose. Myoinositol uptake, and DNA in 30 mM glucose were significantly reduced, and were normalized with phlorizin. Myoinositol content in 30 mM glucose was the same as that in 5 mM glucose, but was significantly decreased by phlorizin. The ratios of glucose to sorbitol or fructose in 30 mM glucose were significantly decreased, compared with those in 5 mM glucose and 30 mM glucose plus phlorizin. Therefore, the cellular enlargement and decreased DNA synthesis in cultured bovine retinal pericytes with abnormal glucose metabolism under high glucose conditions are attenuated by phlorizin, independent of the cellular myoinositol content.     

Inhibition of sodium for sodium exchange by phlorizin in frog sartorius muscle

The effects of phlorizin (2 X 10(-3) mol X l-1) on the Na transport of frog (Rana esculenta) sartorius muscle were investigated in glucose-free medium. Phlorizin decreased the rate coefficient of 24Na efflux by about 40%. The degree of inhibition was comparable to that caused by ouabain (10(-4) mol X l-1). Phlorizin could evoke a further reduction in the 24Na efflux also in the presence of ouabain. The intracellular Na content of the phlorizin-treated muscles remained unchanged, in contrast to a 60% increase induced by ouabain. 42K uptake was not affected by phlorizin. Data indicate that the ouabain-sensitive Na-K pump was not involved in the action of phlorizin. At the same time, phlorizin failed to alter the residual 24Na efflux measured in Li-Ringer solution containing ouabain. When Na: Na exchange was restored by replacing Na into the washout solution in the presence of ouabain, the increase of 24Na efflux was significantly diminished by phlorizin. Phlorizin reduced the 24Na uptake into a compartment with a half time of 6 min by about 40% without affecting the intracellular compartment. The results suggest that phlorizin inhibits the ouabain-insensitive Na: Na exchange in a superficial Na compartment.     

Endothelial Na+-D-glucose cotransporter: no role in insulin-mediated glucose uptake.

A recent report indicates that the Na+-D-glucose cotransporter SGLT1 is present in capillaries of skeletal muscle and is required for insulin-mediated glucose uptake in myocytes. This result is based on the complete inhibition of insulin-mediated muscle glucose uptake by phlorizin, an inhibitor of SGLT1. Using the pump-perfused rat hind limb, we measured glucose uptake, lactate efflux, and radioactive 2-deoxyglucose uptake into individual muscles with saline (control), phlorizin, insulin, and insulin plus phlorizin, as well as with saline and insulin using normal and low Na+ perfusion buffer. Insulin-mediated glucose uptake was not inhibited after correction for phlorizin interference in the glucose assay. Lactate efflux and 2-deoxyglucose uptake by individual muscles were unaffected by phlorizin. Low Na+ buffer did not affect insulin-mediated glucose uptake, lactate efflux, or 2-deoxyglucose uptake. We conclude that endothelial SGLT1 exerts no barrier for glucose delivery to myocytes.     

Kinetics of myo-inositol transport in corneal endothelial cells: diverse effects of sugars and implications in corneal deutergensence [corrected

Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied. Confluent corneal endothelial cells accumulated 3H-MI in a time dependent and saturable process. At a narrow range of external concentrations of 3H-MI (4-50 microM), the Na(+)-dependent MI uptake followed saturation kinetics. The apparent Km value was 20 microM with a maximum velocity (Vmax) of 16 pmol/20 min/micrograms DNA. At low external 3H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na(+)-independent fraction of MI uptake significantly increased. The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na+/H+ exchanger). The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of 3H-MI increased. Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake. Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 microM 3H-MI, was 6.6% +/- 0.8 or 35% +/- 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively. Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose. Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or alpha-methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of 3H-MI. However, glucose at lower levels (less than or equal to 10 mM) slightly increased MI uptake. These studies indicated that the uptake of MI into corneal endothelial cells was an Na(+)-dependent active process at a narrow range of external radioactive MI concentrations. Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s). Glucose influenced the uptake of MI in a complex manner. The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence. High glucose-induced inhibition of Na(+)-dependent MI uptake may be in part due to glucose regulation of Na+ fluxes and cell volume.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trapping of lead (Pb) by corn and pea root border cells

Aims

Most plants produce a root tip extracellular matrix that includes viable border cell populations programmed to disperse into soil. Like neutrophils, border cells export structures that trap pathogens and prevent root tip infection. Border cells also trap metals. The goal of this study was to determine if border cells trap Pb.

Methods

Border cell responses to Pb were observed microscopically. Border cell impact on Pb-induced injury to roots was assessed using root growth assays. Pb removal from solution was measured using inductively coupled plasma mass spectrometry (ICP-MS). Speciation of Pb associated with border cells was evaluated by synchrotron X-ray absorption spectroscopy (XAS).

Results

Increased border cell trap size and number occurred within minutes in response to Pb but not silicon (Si). Transient immersion of root tips into Pb after border cells were removed resulted in growth inhibition. Immersion of root tips and border cells into Pb solution resulted in significant removal of Pb. Si levels in the presence of root tips remained unchanged. The Pb speciation, measured with Pb L_III XAS, altered when reacted with border cells, indicating that direct binding by extracellular traps occurred.

Conclusions

Border cells can trap Pb and prevent damage to the root tip.

     

A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.     

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.     

A hydrolase-related transport system is not required to explain the intestinal uptake of glucose liberated from phlorizin

The fate of [³H]glucose released from a wide range of [³H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na⁺-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2–2.0 mM phlorizin, the [³H]glucose uptake was a constant 11–12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [³H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [¹⁴C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [¹⁴C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na⁺-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medium by virtue of the position of the site where it is formed, i.e. inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.