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J Fischbarg

Publications and source records attributed to J Fischbarg.

At least 37 records · Page 2Linked to original sources
PubMed1999

Fluoxetine inhibits K(+) transport pathways (K(+) efflux, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) pump) underlying volume regulation in corneal endothelial cells.

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K(+) efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 microM by 26%, and 50 microM totally. Fluoxetine had a triphasic effect on K(+) efflux; from 20 to 100 microM it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 microM by 25%, and 50 microM completely. Bumetanide (1 microM) inhibited RVI by 43%. In a Cl(-)-free medium, fluoxetine (50-500 microM) progressively inhibited bumetanide-insensitive K(+) influx. The inhibitions of RVI and K(+) influx induced by fluoxetine 20 to 50 microM were similar to those induced by 1 microM bumetanide and by Cl(-)-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K(+) channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K(+) efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 microM inhibits NKCC, and at 100-1000 microM inhibits the Na(+) pump. One possible explanation for these findings is that fluoxetine could interact with K(+)-selective sites in K(+) channels, the NKC cotransporter and the Na(+) pump.

Animals↗
PubMed1999

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-O-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/10(6)RBC; 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/10(6) RBC (p < 3 x 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 mother's), 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.

3-O-Methylglucose↗
PubMed1999

Transport of fluid by lens epithelium.

We report for the first time that cultured lens epithelial cell layers and rabbit lenses in vitro transport fluid. Layers of the alphaTN4 mouse cell line and bovine cell cultures were grown to confluence on permeable membrane inserts. Fluid movement across cultured layers and excised rabbit lenses was determined by volume clamp (37 degrees C). Cultured layers transported fluid from their basal to their apical sides against a pressure head of 3 cmH2O. Rates were (in microliter. h-1. cm-2) 3.3 +/- 0.3 for alphaTN4 cells (n = 27) and 4.7 +/- 1.0 for bovine layers (n = 6). Quinidine, a blocker of K+ channels, and p-chloromercuribenzenesulfonate and HgCl2, inhibitors of aquaporins, inhibited fluid transport. Rabbit lenses transported fluid from their anterior to their posterior sides against a 2.5-cmH2O pressure head at 10.3 +/- 0.62 microliter. h-1. lens-1 (n = 5) and along the same pressure head at 12.5 +/- 1.1 microliter. h-1. lens-1 (n = 6). We calculate that this flow could wash the lens extracellular space by convection about once every 2 h and therefore might contribute to lens homeostasis and transparency.

4-Chloromercuribenzenesulfonate↗
PubMed1999

GLUT1-deficiency: barbiturates potentiate haploinsufficiency in vitro.

Barbiturates are known to inhibit glucose transport mediated by the facilitative sugar transporter GLUTI. We have studied such inhibition in children with GLUT1-deficiency. Zero-trans influx of 14C-labeled 3-O-methyl glucose (3OMG) into erythrocytes of patients (n = 3) was 35% of controls (n = 6). Preincubation with 10 mM phenobarbital or pentobarbital reduced patients' 30MG influx to 17%. In patients and controls, preincubation with barbiturates significantly decreased Vmax in a dose-dependent manner (for pentobarbital, IC50 = 0.84 mM, patient 2). The apparent Km in individuals remained largely unchanged. Three-OMG influx without preincubation resulted in a stronger inhibition at lower barbiturate concentrations. The patients' data are discussed in the light of individual missense mutations (patient 1: R126L and K256V; patient 2: T310I; patient 3: S66F) in the GLUTI gene. In conclusion, in controls and patients with GLUT1-deficiency barbiturates interact with GLUT1, lowering its intrinsic activity. The use of barbiturates in this condition for anesthesia or as anticonvulsants could therefore potentially aggravate the existing glucose transport defect and may put these patients at increased risk.

Adolescent↗
PubMed1999

Molecular identification and immunolocalization of the water channel protein aquaporin 1 in CBCECs.

PURPOSE: Water channel proteins are important pathways for water movements across cell membranes, including those in the corneal endothelium that contribute to the fluid transport mechanism essential in maintaining corneal transparency. This study was conducted to identify and locate the water channel protein(s) in cultured bovine corneal endothelial cells (CBCECs). METHODS: Poly(A)+ RNA was isolated from CBCECs, and MMLV reverse transcriptase and random hexamer primers were used to generate a cDNA pool by reverse transcription-polymerase chain reaction (RT-PCR). Two specific degenerate primers were synthesized based on consensus sequences from the major intrinsic lens protein superfamily; a "touchdown" PCR protocol accommodated the degeneracy. Immunolocalization was performed by incubating sections of CBCECs with an antibody against human aquaporin 1 (AQP1). Cryosections (0.85 microm) of CBCECs were used for light microscopy, and 800-A ultrathin cryosections were used for electron microscopy (EM). RESULTS: A 372-bp fragment was isolated. Its encoded amino acid sequence was 100% identical with that of bovine AQP1 (AQP2_bovin). CBCECs reacted strongly with the anti-AQP1 antibody, and the labeling was selectively localized to the plasma membrane by light microscopy. Subcellular localization by EM revealed immunoreactivity with the inner leaflets of the plasma membrane. CONCLUSIONS: The identity of the aquaporin, its abundance, and its membrane location suggest that it is a major pathway for fluid flow across endothelial cell membranes. This is consistent with transcellular endothelial fluid transport.

Amino Acid Sequence↗
PubMed1999

Cultured bovine corneal epithelial cells express a functional aquaporin water channel.

PURPOSE: Given recent physiological and in situ hybridization evidence for the presence of a water channel in corneal epithelium, this study was conducted to investigate its expression and characteristics using cultured bovine corneal epithelial cells (CBCEPCs). METHODS: CBCEPCs were grown in DMEM containing 2 ng/ml fibroblast growth factor and 6% fetal bovine serum. To determine their osmotic permeability (Pf), cells were passaged onto rectangular glass coverslips, and anisotonically induced volume changes were monitored by light scattering. To investigate expression, poly(A+) RNA from CBCEPCs was injected into Xenopus laevis oocytes, and the Pf of the oocytes was determined. RESULTS: For CBCEPCs challenged with a 10% hypotonic solution at 37 degrees C, the kinetic constant of volume change was k=0.52+/-0.04 seconds(-1), and the calculated Pf 72+/-6 microm/sec (n=16). The Pf of oocytes injected with water was 14+/-1.8 microm/sec (n=4); injection with poly(A+) RNA from CBCEPCs increased Pf to 77+/-6 microm/sec (n=6). This increase in Pf was inhibited by 72% (reduced to 22+/-1 microm/sec) by 0.3 mM HgCl2 and was inhibited by 56% to 58% by coinjection with aquaporin (AQP)5 antisense oligonucleotide. CONCLUSIONS: The comparatively high Pf determined for CBCEPCs, the presence of mRNA encoding water channels, and sensitivity to mercurial agents are typical of the expression of functional water channels. The predominant message is for AQP5, although the evidence was consistent with the presence of additional water channels. These findings bring renewed support for the notion that the epithelium can contribute to corneal hydration homeostasis.

Animals↗
PubMed1998

Effects of ciprofloxacin, streptomycin, and gentamicin on rabbit corneal transendothelial electrical potential difference.

PURPOSE: A previous report suggested that high concentrations of ciprofloxacin in the anterior chamber may cause dose-dependent acute corneal decompensation. Therefore we evaluated the effect of varying concentrations of ciprofloxacin in the anterior chamber on the corneal endothelium and compared these effects with those of gentamicin and streptomycin. METHODS: We assessed endothelial transport function by determining transendothelial electrical potential differences (TEPDs) of rabbit corneas. Our control solution was bicarbonate-buffered balanced saline with glucose (BSG), to which we added ciprofloxacin (50, 100, 125, and 150 microg/ml), gentamicin (1,000 and 2,000 microg/ml), and streptomycin (196, 437, and 696 microg/ml). RESULTS: At high concentrations exceeding minimal inhibitory concentrations against 90% of common ocular isolates (MIC90), accelerated decay of TEPDs was seen with all three antibiotics. Adverse effects on TEPDs were noted at concentrations corresponding to >50 times MICs with ciprofloxacin and 40 x MICs with gentamicin, but only 2 times MICs with streptomycin. CONCLUSION: Our study shows that concentrations of ciprofloxacin, gentamicin, and streptomycin below or equal to their MIC90 levels do not adversely affect endothelial transport function in a rabbit model.

Animals↗
PubMed1998

Sodium, potassium, two chloride cotransport in corneal endothelium: characterization and possible role in volume regulation and fluid transport.

PURPOSE: To search for membrane transporter proteins that could contribute to volume regulation and fluid transport by corneal endothelium. As an initial step, the authors have focused on Na+-K+-2Cl- cotransporters. METHODS: Bovine corneal endothelial cells were cultured to confluence. 86Rubidium was used as a tracer for K+ uptake determinations; uptake values were normalized per milligram of cell protein. RESULTS: Three components of K+ uptake were characterized: ouabain (1 mM) sensitive, bumetanide (0.1 mM) sensitive, and ouabain-bumetanide insensitive. Both the ouabain-sensitive and bumetanide-sensitive components increased in the presence of 26.2 mM HCO3-; 0.5 mM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid abolished this increase. The bumetanide-sensitive component was completely inhibited in the absence of Na+ or Cl-. This component was increased 33% by a 33% hypertonic solution and was decreased 38% by a 33% hypotonic solution. The protein kinase C activator phorbol 12-myristate 13-acetate decreased the activity of the cotransporter, whereas forskolin, in the presence of isobutylmethylxanthine, decreased it. Calyculin A (100 nM), an inhibitor of phosphatases 1 and 2a, produced a large (97%) activation of this component. CONCLUSIONS: These results provided for the first time conclusive evidence for the presence of a Na+-K+-2Cl- cotransporter in corneal endothelium and of its possible involvement in volume-regulatory processes in these cells. Given the uptake values reported here, such cotransporter could contribute significantly to electrolyte transport and hence to fluid transport across this preparation.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release.

The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (tauvr) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+]i transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mM Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 microM cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 microM ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]i transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 microM ryanodine; 2.10 microM CPA and 100 microM ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.

Animals↗
PubMed1997

Solutions containing miotic agents: effects on corneal transendothelial electrical potential difference.

BACKGROUND: Anterior chamber miotic solutions are widely used during anterior chamber surgery. We examined the effects of solutions containing miotic agents such as carbachol and/or acetylcholine on corneal endothelial pumping activity. METHODS: We monitored, in vitro, the transendothelial electrical potential difference of isolated rabbit corneal endothelial preparations. As controls, we used solutions without miotics. RESULTS: We found that a solution containing 55 mM acetylcholine and minimal amounts of salts (Miochol E) maintains transendothelial electrical potential difference some 30% above control levels for up to 4 h. Two other solutions, one including balanced salts and 0.55 mM carbachol (Miostat), the other a mixture of 0.19 mM carbachol and 55 mM acetylcholine plus minimal salts, are adequate to maintain the potential difference at control levels. Lastly, a solution with acetylcholine but without any salts (Miochol) greatly decreases the potential difference, to 30% of the control level, in 100 min. CONCLUSION: Our results indicate that: (1) 55 mM (1%) acetylcholine stimulates the endothelial electrical potential difference; (2) addition of 0.19 mM (0.003%) carbachol negates the stimulatory effect of acetylcholine; and (3) absence of electrolytes severely depresses the endothelial electrical activity.

Administration, Topical↗
PubMed1997

Effects of acetylcholine, carbachol, and mannitol on rabbit corneal endothelial function as assessed by corneal deturgescence.

BACKGROUND: Anterior chamber miotic solutions are widely used in ophthalmic surgery to induce pupillary contraction. We investigated whether the acetylcholine, carbachol, or mannitol present in perfusing solutions can affect corneal endothelial function. METHODS: Freshly dissected deepithelized rabbit corneas were mounted in a Dikstein-Maurice chamber at 36 degrees C. The endothelial sides were perfused with six solutions: (A) 55 mM (1%) acetylcholine Cl plus modified balanced salts; (B) control for A, with acetylcholine Cl replaced by sucrose; (C) 0.55 mM (0.01%) carbachol Cl plus balanced salts; (D) balanced salts solution (BS; control for C); (E) 3% mannitol plus modified balanced salts; and (F) modified balanced salts (control for E, with mannitol replaced by sucrose). Corneal thickness was followed for 3 h in each experiment. The effect of solution E did not differ from that of solution F. RESULTS: The carbachol-containing solution produced a small increase in corneal thickness compared to the control solution, while the acetylcholine-containing solution resulted in corneal thickness lower than that in control preparations. CONCLUSION: From these data, acetylcholine is harmless to the endothelium, and may actually stimulate its fluid pump mechanism. Carbachol, on the other hand, appears to have a detrimental effect.

Acetylcholine↗
PubMed1997

Macular holes: migratory gaps and vitreous as obstacles to glial closure.

PURPOSE: Retinal glia may play an important role in the closure of macular holes. This in vitro study examines whether and how the specific pathoanatomy, including foveal eversion and foveal vitreous, may interfere with glial closure of macular holes. METHODS: Culture dishes used to grow glial cells were modified by the placement of slopes, vertical steps, and gaps to mimic the in vivo migratory surface in and surrounding macular holes. In separate experiments, defects were made in a rodent glial monolayer. These defects were exposed to hyaluronic acid (HA) and to rabbit (RV) and bovine (BV) vitreous gel. The migratory behavior and completeness of closure of defects were compared to controls. RESULTS: As expected, glial cells migrated further and in greater numbers on a smooth surface. Slopes and steps were moderate obstacles to migration; gaps in the surface were absolute obstacles. HA modified the pattern of adhesion of cells at the bottom of defects. Defects in the glial monolayer were repaired in 5-7 days. Compared to these controls, repair was inhibited by 11% (n.s.), 28% (P = 0.02), and 58% (P = 0.004) after direct exposure of defects to HA, RV and BV, respectively. CONCLUSION: The elevated and everted margins of macular holes represent slope, step, and gap-like obstacles to the migration of glial cells and hence to the healing of defects. The defect allows extension of extracellular matrix into it and the subretinal space. Our results indicate that gaps in the migratory surface caused and aggravated by eversion and the presence of vitreous present obstacles to glial migration and closure of macular holes.

Animals↗
PubMed1997

A novel method to determine the diffusional water permeability of oocyte plasma membranes.

Measurements of the cell membrane diffusional water permeability (Pd) are important to characterize water passage across water channels and across the lipid bilayer component of the membrane. Existing methods for those measurements are involved; however, we report here a simple procedure to estimate Pd in Xenopus laevis oocytes and similar large cells. Due to the different densities of H2O and D2O (heavy water), an oocyte transferred from normal medium to a D2O-based medium floats initially, but subsequently sinks when a certain amount of the water originally in them is replaced by the D2O that diffuses in. We describe how the 'flotation time' (time that oocytes float in a heavy water solution before they start sinking) yields the Pd of the plasma membrane. Determination of Pd by this procedure and by the rate of tritiated water (T2O) efflux give for Pd results which are very close: 2.2 +/- 0.2 (n = 8) and 2.0 +/- 0.1 (n = 6) microns/s, respectively (T = 10 degrees C). Furthermore, our method detects the increase in Pd elicited in oocytes by either expression of water channel proteins, or by treating them with the pore-forming antibiotic amphotericin B. This method appears useful to gauge the expression and function of pore-forming, water-permeable membrane proteins.

Amphotericin B↗