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P Iserovich

Publications and source records attributed to P Iserovich.

11 recordsLinked 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

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↗
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

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↗
PubMed1995

Predictive evidence for a porin-type beta-barrel fold in CHIP28 and other members of the MIP family. A restricted-pore model common to water channels and facilitators.

Water channels are the subject of much current attention, as they may be central for cell functions in a host of tissues. We have analyzed the possible field of facilitators and water channels of the MIP family based on structural predictions, on findings about the topology of CHIP28, and on the biophysical characteristics of water channels. We developed predictions for the following proteins: MIP26, NOD26, GLP, BIB, gamma-TIP, FA-CHIP, CHIP28k, WCH-CD1, and CHIP28. We utilized Kyte Doolittle hydrophobicity, Eisenberg's amphiphilicity, Chou-Fasman-Prevelige propensities, and our own Union algorithm. We found that hydrophobic amphiphilic segments likely to be transmembrane were consistently shorter than required for alpha-helical segments, but of the correct length for beta-strands. Turn propensity was high at frequent intervals, consistent with transmembrane beta-strands. We propose that these proteins fold as porin-like 16-stranded antiparallel beta-barrels. In water channels, from the size of molecules excluded, an extramembrane loop(s) would enter the pore and restrict it to a bottleneck with a width 4 A < or = w < or = 5 A. A similar but more mobile loop(s) would act as gate and binding site for the facilitators of the MIP family.

Amino Acid Sequence↗
PubMed1994

Are most transporters and channels beta barrels?

Given the sequence of transporters or channels of unknown secondary structure, it is usual to predict their putative transmembrane regions as alpha-helical. However, recent evidence for a facilitative glucose transporter (GLUT1) appears inconsistent with such predictions, which has led us to propose an alternative folding model for GLUTs based on the 16-stranded antiparallel beta-barrel of porins. Here we apply the same predictive algorithms we used for GLUTs to several other membrane proteins. For some of them, a high-resolution structure has been derived (beta-barrels: Rhodobacter capsulatus and Escherichia coli porins; multihelical: colicin A, bacteriorhodopsin, and reaction center L chain); we use them to test the prediction procedures. The other proteins we analyze (GLUT1, CHIP28, acetylcholine receptor alpha subunit, lac permease, Na(+)-glucose cotransporter, shaker K+ channel, sarcoplasmic reticulum Ca(2+)-ATPase) are representative of classes of similar membrane proteins. As with GLUTs, we find that the predicted transmembrane segments of these proteins are consistently shorter than expected for transmembrane spanning alpha-helices, but are of the correct length and number for the proteins to fold instead as porin-like beta-barrels.

Algorithms↗
PubMed1993

Evidence that facilitative glucose transporters may fold as beta-barrels.

A widely accepted model for the structure of the facilitative glucose transporters (GLUTs) predicts that they form 12 transmembrane alpha-helices and that the highly conserved sequence Ile-386-Ala-405 in GLUT1 is intracellular. We raised a polyclonal antibody against a synthetic peptide encompassing this conserved sequence and found that antibody treatment increased 2-deoxy-D-glucose (DOG) uptake in Xe-nopus oocytes expressing GLUT1, GLUT2, or GLUT4 only when applied to the extracellular side. This effect was dose dependent and was specifically blocked by competition with the peptide Ile-386-Ala-405; it was due to a decrease in the Km for the transport of DOG. To ascertain GLUT orientation, we raised anti-peptide antibodies against the last 21 and 25 C-terminal amino acids of GLUT1 and GLUT4, respectively, which were previously shown to be intracellular. These antibodies increased DOG uptake when injected into oocytes expressing GLUT1 and GLUT4, but not when added extracellularly. Prompted by the noted discrepancy, we found sequence similarity between GLUTs and porins, two of which are known from crystallography to form 16-stranded transmembrane antiparallel beta-barrels. Analysis of the hydrophobicity, amphiphilicity, and turn propensity of GLUT1 leads us to propose that GLUTs fold as porin-like transmembrane beta-barrels. This model is consistent with the results of the present antibody studies and also with previously published experimental evidence inconsistent with the 12-helix model.

Amino Acid Sequence↗
PubMed1993

Cultured bovine corneal endothelial cells express CHIP28 water channels.

Cultured bovine corneal endothelial cells (CBCEC) transport fluid from the basal to the apical surface. In this study, we examined whether the plasma membranes of these cells have water channels. We cultured BCEC on glass plates and monitored the intensity of the light scattered (IS) by the cells. We determined the kinetic constant (k) of the change in IS on exposure to a 10% hypoosmotic challenge to calculate the osmotic permeability (Pf) of the plasma membrane. At 37 degrees C, we found values of k = 0.68 +/- 0.07 s-1 and Pf = 93.3 +/- 33 microns/S (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced Pf by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (Ea) of Pf in the range 10-37 degrees C was 4.7 +/- 0.7 kcal/mol (n = 5). The high Pf, values, the inhibition by pCMBS, and the low Ea strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)+ RNA prepared from CBCEC into Xenopus laevis oocytes results in the expression of water channels. Four days after injection, we measured oocyte Pf values from the rate of volume increase on exposure to hypoosmotic medium. In control oocytes injected with 50 nl of water, Pf was 13.4 +/- 0.3 microns/S (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/oocyte in 50 nl water), Pf was 40.9 +/- 1.6 microns/S (n = 72).(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗
PubMed1993

Determination of volume and water permeability of plated cells from measurements of light scattering.

Measurements of cell membrane water osmotic permeabilities can be inaccurate because of the technical difficulties inherent to cell volume measurements and because of the presence of an unstirred water layer in contact with the cells. We detail here a method we have developed to quantify transient changes in cell volumes from the intensity of light scattered by cells. For this, we theorize how an unstirred layer originates in a perfusing chamber, and we calculate values for both cell membrane water osmotic permeability and unstirred layer thickness from time transient changes in scattered light. We apply a computer algorithm that finds the best correspondence between experimental data and estimated values. This is done by solving a differential equation governing cell volume changes by numerical integration (Runge-Kutta) and iterating the procedure varying the test values of osmotic permeability and unstirred layer thickness until the best fit is achieved. We exemplify this procedure with experimental results obtained in adherent cultured cells.

Animals↗