Jorge Fischbarg

A single amino acid residue can determine the sensitivity of SERCAs to artemisinins

Artemisinins are the most important class of antimalarial drugs. They specifically inhibit PfATP6, a SERCA-type ATPase of Plasmodium falciparum. Here we show that a single amino acid in transmembrane segment 3 of SERCAs can determine susceptibility to artemisinin. An L263E replacement of a malarial by a mammalian residue abolishes inhibition by artemisinins. Introducing residues found in other Plasmodium spp. also modulates artemisinin sensitivity, suggesting that artemisinins interact with the thapsigargin-binding cleft of susceptible SERCAs.

Autosomal Dominant Glut-1 Deficiency Syndrome and Familial Epilepsy

Glut‐1 deficiency syndrome was first described in 1991 as a sporadic clinical condition, later shown to be the result of haploinsufficiency. We now report a family with Glut‐1 deficiency syndrome affecting 5 members over 3 generations. The syndrome behaves as an autosomal dominant condition. Affected family members manifested mild to severe seizures, developmental delay, ataxia, hypoglycorrhachia, and decreased erythrocyte 3‐O‐methyl‐D‐glucose uptake. Seizure frequency and severity were aggravated by fasting, and responded to a carbohydrate load. Glut‐1 immunoreactivity in erythrocyte membranes was normal. A heterozygous R126H missense mutation was identified in the 3 patients available for testing, 2 brothers (Generation 3) and their mother (Generation 2). The sister and her father were clinically and genotypically normal. In vitro mutagenesis studies in Xenopus laevis oocytes demonstrated significant decreases in the transport of 3‐O‐methyl‐D‐glucose and dehydroascorbic acid. Xenopus oocyte membranes expressed high amounts of the R126H mutant Glut‐1. Kinetic analysis indicated that replacement of arginine‐126 by histidine in the mutant Glut‐1 resulted in a lower Vmax. These studies demonstrate the pathogenicity of the R126H missense mutation and transmission of Glut‐1 deficiency syndrome as an autosomal dominant trait.

Adenosine stimulation of fluid transport across rabbit corneal endothelium

SummaryThe rate of fluid transport across rabbit corneal endothelium has been measured with an automatic volumetric method. The present resolution of the procedure is 1–3 nanoliters, and intervals of measurement can be made as small as seconds. In the presence of glucose, oxidized glutathione (GSSG), and adenosine, the maximal rates were 6.2±1.0 μl/hr cm2, and 8.2±0.8 μl/hr cm2 if a large portion of the stroma was dissected away. In the presence of glucose and GSSG only, the rates were lower, namely 3.7±0.5 μl/hr cm2. The rates consistently increased or decreased when adenosine was added or deleted, respectively, during given experiments. The stimulation of fluid transport by adenosine was in the order of 40–50%. The results raise the possibility that this transport mechanism might be subject to metabolic control.

Defective glucose transport across brain tissue barriers: a newly recognized neurological syndrome.

Impaired glucose transport across brain tissue barriers causes infantile seizures, developmental delay and acquired microcephaly. Since the first report in 1991 (De Vivo et al, NEJM, 1991) 17 patients have been identified with the glucose transporter protein syndrome (GTPS). The diagnostic feature of the syndrome is an unexplained hypoglycorrhachia in the clinical setting of an infantile epileptic encephalopathy. We review our clinical experience by highlighting one illustrative case: a 6-year old girl who presented at age 2 months with infantile seizures and hypoglycorrhachia. The CSF/blood glucose ratio was 0.33. DNA sequencing identified a missense mutation in exon 7 (C1108T). Erythrocyte GLUT1 immunoreactivity was normal. The time course of 3-0-methyl-glucose (3OMG) uptake by erythrocytes of the patient was 46% that of mother and father. The apparent Km was similar in all cases (2–4 mmol/L), but the apparent Vmax in the patient was only 28% that of the parents (500 versus 1,766 fmol/s/106RBC; p < 0.004). In addition, a 3-month trial of oral thioctic acid also benefited the patient and increased the Vmax to 935 fmol/s/106 RBC (p < 3 × 10−7). Uptake of dehydroascorbic acid by erythrocytes of the patient was impaired to the same degree as that of 3OMG (Vmax was 38% of that of the mothers), which supports previous observations of GLUT1 being multifunctional. These studies confirm the molecular basis of the GTPS and the multifunctional role of GLUT1. The need for more effective treatment is compelling.

Active and passive properties of the rabbit corneal endothelium.

Some of the physiological properties of rabbit corneal endothelium were investigated by studying: (a) fluid transport; (b) osmotic water flow; (c) electrical potential difference; and (d) electrical impedance, all of them across the endothelial layer; (e) biochemical parameters (ATP level and Na++K+-activated ATPase activity) of endothelial cells, and (f) morphological (light and electronmicroscopic) appearance of the endothelium. Several experimental manipulations which decreased or a bolished fluid transport (total replacement of 43 mm bicarbonate by Cl, of 5 mm K by Na and of 150 mm Na by Li; treatment with 5 × 10−5 m ouabain and with 20 μ/ml cytochalasin B) also decreased or abolished the electrical potential difference. The electrical resistance of fresh preparations was 41 ± 5 Ωcm2 (s.e.m., n = 14; range: 16–81 Ωcm2); scraping off the endothelium and treating it with Ca-free media decreased the resistance to that of the supporting stromal layer (3–10 Ωcm2) Cytochalasin B (20 μg/ml) markedly decreased the resistance, while ouabain did not affect it. Omission of all metabolites normally present in the perfusion medium (reduced glutathione, adenosine, and glucose) decreased the endothelial ATP level markedly, while addition of reduced glutathione spared ATP from depletion. Since the transport ceased even in the presence of near normal ATP levels and (Na++K+)-activated ATPase activity, other yet undefined factors are also probably involved in pump function. Osmotically induced water flows across the endothelium were not linear but followed a square root function of the driving force. Cytochalasin B induced profound changes in the shape of the endothelial cells, causing some of them to clump together. The values of electrical resistance, capacitance and water flows experimentally measured agree reasonably well with the numerical values calculated from a model. The results are consistent with the possibility that the fluid transport across the endothelium would be due to an electrogenic ionic pump, and that the intercellular spaces and their gap junctions would constitute the dominant “shunt” pathway for water and electrolytes.

Effects of ambient bicarbonate, phosphate and carbonic anhydrase inhibitors on fluid transport across rabbit corneal endothelium

Bicarbonate has been long held to be indispensable for fluid pumping by the endothelium; however, such need has been disputed recently. We investigated this issue and found that: (1) the corneal endothelium pumps fluid equally well (at 6-8 microliters hr-1 cm-2) whether the bathing solution contains 43 mM bicarbonate or 10 mM phosphate, (2) if bicarbonate and most of the phosphate are absent, fluid pumping is noticeably lowered (2-4 microliters hr-1 cm-2), (3) carbonic anhydrase inhibitors (5 mM acetazolamide; 0.1, 0.2 and 0.3 mM ethoxzolamide) block this lowered fluid pumping, and (4) in the absence of external bicarbonate, 20 mM HEPES is insufficient to preserve adequate fluid pumping. These results are consistent with existing models for endothelial transport in which exogenous and endogenous CO2 are converted to HCO3- by carbonic anhydrase, with HCO3- fueling the transport mechanism and therefore the fluid pump.

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force

The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead.Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.

An Update on Corneal Hydration Control

An account is provided of developments in our understanding of the mechanism of corneal hydration control, particularly as regards the possibility of an active system for its regulation. Emphasis is given to issues that are contentious, such as the role of bicarbonate in the endothelial pump and the significance of water channels in both corneal limiting cell layers.

Disulfide stimulation of fluid transport and effect on ATP level in rabbit corneal endothelium

Abstract Transendothelial fluid transport in rabbit corneal preparations is known to be improved when perfusates are supplemented with reduced glutathione. Evidence is now presented showing that oxidized glutathione which arises by autoxidation of reduced glutathione under the conditions of perfusion is responsible for this effect. An optimal level of about 10−5 m -oxidized glutathione is necessary to support fluid transport while only 10−7 m oxidized glutathione will sustain endogenous endothelial adenosine triphosphate levels. Of the structurally related disulfides tested, cystine and to a lesser degree homocystine, can replace oxidized glutathione. Levels of adenosine triphosphate and activities of (Na++K+)-activated and Mg2+-activated adenosine triphosphatase were measured in individual endothelia after maximal thinning of preswollen corneal preparations had been achieved and the criteria for pump failure had been met. Data for these physiological and biochemical parameters after perfusion with basal salts, glucose or adenosine in the absence or presence of glutathione have been obtained. Adenosine triphosphate levels in the presence of basal salt perfusates are comparably elevated by supplementation with either glutathione or glucose and remain unchanged when both substrates are simultaneously available. Similarly, the elevated ATP levels that accompany adenosine supplementation are not affected by the additional presence of glutathione or glucose. Adenosine triphosphatase activities are somewhat depressed in the absence of all metabolites and are not improved by glutathione alone. These activities remain essentially normal when glucose or adenosine is present.

Glucose Transporter Type 1 Deficiency Syndrome (Glut1DS): Methylxanthines Potentiate GLUT1 Haploinsufficiency In Vitro

Methylxanthines such as caffeine and theophylline are known to inhibit glucose transport. We have studied such inhibition in the glucose transporter type 1 deficiency syndrome (Glut1DS) by erythrocyte glucose transport assays. Data from four patients with individual mutations in the GLUT1 gene are discussed: patient 1 (hemizygosity), 3 (S66F), 15 (368Ins23), and 17 (R333W). Zero-trans influx of 14C-labeled 3-O-methyl glucose (3-OMG) into erythrocytes of patients is reduced (patient 1, 51%; 3, 45%; 15, 31%; 17, 52%) compared with maternal controls. Inhibition studies on patients 1, 3, 17, and maternal controls show an IC50 for caffeine of approximately 1.5 mM both in controls (n = 3) and patients (n = 3) at 5 mM 3-OMG concentration. In the same two groups, kinetic studies show that 3 mM caffeine significantly decreases Vmax (p < 0.005), whereas the decrease in Km is significant (p < 0.01) only in the three controls and one patient (patient 3). Kinetic data from individual patients permit us to speculate that the interactions between caffeine and Glut1 are influenced by the mutation. Three mM caffeine also inhibits the transport of dehydroascorbic acid (DHA), another substrate for Glut1. The combined effects of caffeine (3 mM) and phenobarbital (10 mM) on glucose transport, as determined in patient 15 and the maternal control, show no additive or synergistic inhibition. These data indicate that caffeine and phenobarbital have similar Glut1 inhibitory properties in these two subjects. Our study suggests that Glut1DS patients may have a reduced safety margin for methylxanthines. Consumption of methylxanthine-containing products may aggravate the neurologic symptoms associated with the Glut1DS.