Mel Silverman

The MDR1 gene product, P-glycoprotein, mediates the transport of the cardiac glycoside, digoxin.

Digoxin, a widely used cardiac glycoside with a low therapeutic index, is known to interact with a large and diverse group of co-administered drugs, frequently leading to toxic accumulation of the glycoside. Establishing the mechanism(s) of these interactions, therefore, has potential clinical significance. The present studies implicate P-glycoprotein, the MDR1 gene product overexpressed in multidrug resistant cells, as the apical membrane protein responsible for the renal secretion of digoxin and provide an explanation for the occurrence of digoxin toxicity in the presence of certain co-administered medications. Since digoxin is considered a prototype for endogenous digitalis-like glycosides, the results also allow for speculation that endogenous digitalis-like glycosides may be the natural substrates for P-gp.

Interaction of phlorizin and sodium with the renal brush-border membraned-glucose transporter: Stoichiometry and order of binding

SummaryThe order and stoichiometry of the binding of phlorizin and sodium to the renal brush-border membraned-glucose transporter are studied. The experimental results are consistent with a random-binding sites is one-to-one. When the kinetics of phlorizin binding are measured as a function of increasing sodium concentration no significant variation is found in the apparent number of binding sites; however, the apparent binding constant for phlorizin decreases rapidly from approximately 16 μm at [Na]=0 to 0.1 μm at [Na]=100mm and approaches 0.05 μm as [Na]→∞. The experimental data are fit to a random carrier-type model of the coupled transport of sodium andd-glucose. A complete parameterization of the phlorizin binding properties of this model under sodium equilibrium conditions is given.

PKC regulates turnover rate of rabbit intestinal Na+-glucose transporter expressed in COS-7 cells.

We have used the recombinant NH2-terminal myc-tagged rabbit Na+-glucose transporter (SGLT1) to study the regulation of this carrier expressed in COS-7 cells. Treatment of cells with a protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), caused a significant decrease (38.03 ± 0.05%) in methyl α-d-glucopyranoside transport activity that could not be emulated by 4α-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKC inhibitor bisindolylmaleimide I. The maximal rate of Na+-glucose cotransport activity ( V max) was decreased from 1.29 ± 0.09 to 0.85 ± 0.04 nmol ⋅ min-1 ⋅ mg protein-1 after PMA exposure. However, measurement of high-affinity Na+-dependent phloridzin binding revealed that there was no difference in the number of cell surface transporters after PMA treatment; maximal binding capacities were 1.54 ± 0.34 and 1.64 ± 0.21 pmol/mg protein for untreated and treated cells, respectively. The apparent sugar binding affinity (Michaelis-Menten constant) and phloridzin binding affinity (dissociation constant) were not affected by PMA. Because PKC reduced V max without affecting the number of cell surface SGLT1 transporters, we conclude that PKC has a direct effect on the carrier, resulting in a lowering of the transporter turnover rate by a factor of two.

Sugar uptake into brush border vesicles from dog kidney. I. Specificity

Abstract This study describes the specificity of uptake of sugars into osmotically active vesicles derived from a purified membrane fraction from dog kidney cortex. [3H]Phlorizin binding experiments have also been carried out in vesiculated and non-vesiculated membrane preparations. 1. 1. We demonstrate the existence of a sodium-dependent, phlorizin-sensitive d -glucose transport system in the vesiculated membrane preparation. 2. 2. This transport system has similar specificity characteristics to those observed in vivo for the glucose transporter in the brush border membrane of the proximal tubule. 3. 3. We also observe a sodium-dependent, glucose-sensitive phlorizin receptor in the same preparation with a K d for phlorizin ⋍0.3 μ M and K I for glucose ⋍3 mM at 37°C, pH 7.4, 100 mM NaCl. 4. 4. Detailed results relating to the specificity of inhibition of high affinity phlorizin binding are obtained using non-vesiculated brush border membrane fragments in the presence of d -glucose, α-methyl- d -glucoside, d -galactose, β-methyl- d -galactoside, 2-deoxy- d -glucose and d -mannose. 5. 5. Uptake studies using vesiculated membrane fragments from newborn dog kidney indicate that the brush border d -glucose transporter is already at an advanced stage of development at birth. Our results demonstrate that the d -glucose transport system from the dog proximal tubule brush border membrane together with its phlorizin receptor moiety is preserved intact in our membrane vesicle preparation.

Trichostatin A-histone deacetylase inhibitor with clinical therapeutic potential-is also a selective and potent inhibitor of gelatinase A expression.

Modulation of histone acetylation is currently being explored as a therapeutic strategy in treatment of cancer. Specifically, inhibition of histone deacetylase by trichostatin A (TSA) has been shown to prevent tumorigenesis and metastasis. In the present paper we demonstrate that increased histone acetylation by TSA-treated 3T3 cells decreases mRNA as well as zymographic activity of gelatinase A, a matrix metalloproteinase, which is itself, implicated in tumorigenesis and metastasis. Furthermore, TSA inhibits cytochalasin D-induced activation of gelatinase A, but TSA does not affect other members of the gelatinase A activation complex, MT1-MMP and TIMP-2. Thus, TSA is a selective and potent inhibitor of expression and activation of gelatinase A. This finding not only strengthens the rationale for continuing to investigate the therapeutic utility of TSA in cancer, but also, provides evidence that TSA inhibition of gelatinase A expression and activation can be used as a biological marker to monitor and determine end-points of clinical trials involving TSA.

Cysteine Scanning Mutagenesis of the Segment between Putative Transmembrane Helices IV and V of the High Affinity Na+/Glucose Cotransporter SGLT1 EVIDENCE THAT THIS REGION PARTICIPATES IN THE Na+AND VOLTAGE DEPENDENCE OF THE TRANSPORTER

Site-directed mutagenesis and chemical modification of specific cysteine amino acid side chains by methanethiosulfonate (MTS) derivatives were combined to elucidate structure/function relationships of the cloned rabbit Na+/glucose cotransporter, SGLT1. Each amino acid in the region (residues 162–173) between putative transmembrane helices IV and V of SGLT1 was replaced individually with Cys. Mutant proteins were expressed in Xenopus laevis oocytes and studied using the two-electrode voltage clamp method. At certain key positions, Cys substitution resulted in 1) a change in the apparent affinity for sugar, 2) an alteration in the voltage dependence of the transient currents, and 3) a sensitivity to inhibition by either the ethylamine (MTSEA) or the ethylsulfonate MTS derivatives. For the three Cys mutants inhibited by MTSEA (F163C, A166C, and L173C), inhibition of steady state transport is related to changes in membrane potential-dependent transitions within the Na+/glucose transport cycle. MTSEA shifted the transient currents of these Cys mutants toward more negative membrane potentials (ΔV 0.5 = −18 mV for F163C and A166C, −12 mV for L173C). When the mutations were combined to produce double and triple Cys mutants, the degree to which the transient currents were shifted along the membrane potential axis by MTSEA correlated with the number of cysteines. In this way it was possible to manipulate the voltage dependence of the transient currents over a range spanning 91 mV. Examination of the Na+ dependence of the transient currents indicates that a 91-mV shift is equivalent to that caused by a 10-fold reduction in the external Na+ concentration. We conclude that this region has a role in determining the Na+binding- and voltage-sensing properties of SGLT1 and that it forms an α-helix with one surface possibly lining a Na+ pore within SGLT1.

Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals

Disruption of actin filaments affects multiple cell functions including motility, signal transduction and cell division, ultimately culminating in cell death. Although this is the usual sequence of events, we have made the interesting observation that disruption of actin filaments by the potent toxin cytochalasin D (Cyto D) causes one cell type, mouse mesangial cells (MMC), to undergo apoptosis, while in another cell type (NIH 3T3), it has the opposite effect, resulting in production of survival signals. The purpose of this study was to investigate the molecular basis for these observed differences. In the present communication, we demonstrate that exposure to Cyto D induces the pro-apoptotic pathways, p38 and stress-activated protein kinase (SAPK)/jun amino-terminal kinase (JNK), in both cell types. However, in 3T3, but not MMC, the extracellular signal regulated kinase (ERK) 1/2 pathway is protected from inhibition following treatment with Cyto D-leading to phosphorylation of Bclxi/Bcl 2-associated death promoter (BAD). Inhibition of Cyto D-induced secretion and activation of gelatinase A in 3T3 cells reverses the production of survival signals by Cyto-D. To investigate this effect further we employed CS-1 cells, a well-characterized melanoma cell line that lacks integrin beta3, and also does not secrete gelatinase A. Co-transfection of CS-1 cells with integrin beta3 and a gelatinase A transgene, which enables the cells to secrete constituitively active gelatinase A, enhances CS-1 cell survival signals. Together, our findings suggest that extracellularly activated gelatinase A, through interaction with integrin alphaVbeta3, elicits survival signals mediated through ERK 1/2 that override activation of p38 and SAPK/JNK stress pathways.

Testing carrier models of cotransport using the binding kinetics of non-transported competitive inhibitors

The kinetic equations representing the binding of a non-transported competitive inhibitor are derived from three variations of the carrier model of cotransport. These are (a) the model in which the binding sequence of activator and substrate is random (random bi-bi); (b) the model in which activator must bind before substrate (ordered bi-bi, activator essential), and (c) the model in which substrate must bind before activator (ordered bi-bi, activator non-essential). In general it is found that the kinetic equations for inhibitor binding are considerably simpler and easier to test than the corresponding transport equations. The effect of trans-inhibitor, transported substrate, activator concentration and membrane potential on inhibitor binding are examined in some detail. The use of these results to test and characterize the three transport models is emphasized. Applications to transport mechanisms which are not of the mobile carrier type are also discussed. A summary of relevant experimental data interpreted in terms of the theoretical models concludes the paper.

Differential effects of trichostatin A on gelatinase A expression in 3T3 fibroblasts and HT-1080 fibrosarcoma cells: implications for use of TSA in cancer therapy

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor with potential in cancer therapeutics. In a recent communication, we demonstrated that TSA is a selective, potent inhibitor of gelatinase A in 3T3 fibroblasts. In the present study, we extend these observations and examine the effects of TSA in 3T3 fibroblasts compared to HT-1080 fibrosarcoma cells with respect to gelatinase A expression, cell viability, and apoptosis. We find that while expression of gelatinase A in 3T3 fibroblasts is exquisitely sensitive to inhibition by TSA, expression of this enzyme in HT-1080 cells is minimally affected by this compound. Moreover, we show that TSA is pro-apoptotic in HT-1080 cells, but is anti-apoptotic in 3T3 cells. We propose a two-pronged model for the therapeutic action of TSA. On the one hand TSA selectively decreases cancer cell viability, while enhancing the viability of stromal cells. On the other hand, by selectively decreasing gelatinase A expression in stromal but not cancer cells, TSA acts to control metastatic potential by reducing the ability of metastatic cells to recruit stromal cells to secrete gelatinase A.

Diacylglycerol‐Activated Hmunc13 Serves as an Effector of the GTPase Rab34

Hmunc13 is a cytosolic diacylglycerol (DAG)‐binding protein, which is upregulated in renal cortical tubule and mesangial cells by hyperglycemia. In response to DAG activation, hmunc13 translocates to the Golgi. To investigate how this may relate to its function, we used a bacterial two‐hybrid screen to look for hmunc13‐interacting proteins. Full‐length Rab34 was specifically isolated from a human kidney cDNA library. Co‐expression of the two proteins confirmed Rab34 as a Golgi‐associated protein, which was immunoprecipitated from cell lysates by hmunc13. Glutathione S‐transferase fusion proteins of WT, Q111L (GTP bound), and T66N (GDP bound) mutants were created, and their GTP‐binding activity verified by radioactive overlay assay. Binding of hmunc13 was observed with Q111L, barely detectable with T66N and enhanced with Rab34WT loaded with GTPγS compared with GDP loaded. Deletion of munc homolgy domain (MHD)‐2, eliminated the hmunc13/Rab34 interaction. The Q111L mutant localized to the Golgi apparatus, but T66N was cytosolic. Localization of both mutants and Rab34WT was unchanged by DAG activation. The data suggest that DAG activation of hmunc13 causes it to be translocated to the Golgi, where it binds to GTP‐bound Rab34 via MHD‐2. Because Rab34 is known to regulate intracellular lysosome positioning, we propose that hmunc13 serves as an effector of Rab34, mediating lysosome–Golgi trafficking.