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Uday B. Kompella - One of the best experts on this subject based on the ideXlab platform.

  • pigmented MDCK p MDCK Cell Line with tunable melanin expression an in vitro model for the outer blood retinal barrier
    Molecular Pharmaceutics, 2012
    Co-Authors: Rajendra S. Kadam, Robert I. Scheinman, Uday B. Kompella
    Abstract:

    Retinal pigment epithelium, which forms the outer blood–retinal barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) Cell Line as a rapidly established in vitro model for the outer blood–retinal barrier to assess the influence of melanin pigment on solute permeability. A melanin synthesizing P-MDCK Cell Line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) Cells. Melanin content, tyrosinase activity (conversion of l-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK Cells was confirmed by confocal microscopy. Effect of l-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK Cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = −1.14) were investigated. P-MDCK Cells expres...

  • Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier
    Molecular pharmaceutics, 2012
    Co-Authors: Rajendra S. Kadam, Robert I. Scheinman, Uday B. Kompella
    Abstract:

    Retinal pigment epithelium, which forms the outer blood–retinal barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) Cell Line as a rapidly established in vitro model for the outer blood–retinal barrier to assess the influence of melanin pigment on solute permeability. A melanin synthesizing P-MDCK Cell Line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) Cells. Melanin content, tyrosinase activity (conversion of l-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK Cells was confirmed by confocal microscopy. Effect of l-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK Cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = −1.14) were investigated. P-MDCK Cells expres...

R.m. Warn - One of the best experts on this subject based on the ideXlab platform.

  • C-Met signalling in an HGF/SF-insensitive variant MDCK Cell Line with constitutive motile/invasive behaviour
    Journal of Cell Science, 1996
    Co-Authors: C.p. Webb, K. Lane, A.p. Dawson, G.f. Vande Woude, R.m. Warn
    Abstract:

    The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK Cell Line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall Cell surface expression and distribution of Met were found to be similar in parental MDCK Cells and the MDCK-1 Cell Line, p145met autophosphorylation (+/ HGF/SF) was significantly reduced in MDCK-1 Cells in vitro and in vivo when compared with parental MDCK Cells. In contrast, EGF induced Cell proliferation and EGF receptor autophosphorylation to similar levels in both Cell Lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 Cells when compared with parental MDCK Cells, including that of two prominent proteins with molecular masses of approximately 185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 Cells (+/-HGF/SF), but not parental MDCK Cells. In addition, Met immunocomplexes from MDCK-1 Cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK Cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 Cells with the tyrosine kinase inhibitor herbimycin A reverted the Cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 Cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 Cell behaviour.

  • c met signalling in an hgf sf insensitive variant MDCK Cell Line with constitutive motile invasive behaviour
    Journal of Cell Science, 1996
    Co-Authors: C.p. Webb, K. Lane, A.p. Dawson, G.f. Vande Woude, R.m. Warn
    Abstract:

    The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK Cell Line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall Cell surface expression and distribution of Met were found to be similar in parental MDCK Cells and the MDCK-1 Cell Line, p145met autophosphorylation (+/ HGF/SF) was significantly reduced in MDCK-1 Cells in vitro and in vivo when compared with parental MDCK Cells. In contrast, EGF induced Cell proliferation and EGF receptor autophosphorylation to similar levels in both Cell Lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 Cells when compared with parental MDCK Cells, including that of two prominent proteins with molecular masses of approximately 185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 Cells (+/-HGF/SF), but not parental MDCK Cells. In addition, Met immunocomplexes from MDCK-1 Cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK Cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 Cells with the tyrosine kinase inhibitor herbimycin A reverted the Cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 Cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 Cell behaviour.

Craig P. Smith - One of the best experts on this subject based on the ideXlab platform.

  • Acute regulation of mUT-A3 urea transporter expressed in a MDCK Cell Line
    American Journal of Physiology-Renal Physiology, 2007
    Co-Authors: Gavin Stewart, Sarah L. King, Elizabeth A. Potter, Craig P. Smith
    Abstract:

    Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral memb...

  • Acute regulation of mUT-A3 urea transporter expressed in a MDCK Cell Line
    American journal of physiology. Renal physiology, 2006
    Co-Authors: Gavin Stewart, Sarah L. King, Elizabeth A. Potter, Craig P. Smith
    Abstract:

    Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) Cells. In this study, we engineered a Madin-Darby canine kidney (MDCK) I Cell Line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A-targeted antibody ML446 detected a approximately 40-kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive (14)C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ Cells under basal conditions [not significant (NS), ANOVA]. However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea (P < 0.05, ANOVA) or phloretin (P < 0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intraCellular levels of cAMP, via forskolin (P < 0.05, ANOVA), or intraCellular calcium, via ATP (P < 0.05, ANOVA). Finally, 1-h preincubation with a specific PKA inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP (P < 0.05, ANOVA). In conclusion, we have produced the first renal Cell Line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.

Hans Oberleithner - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK Cell Line
    Pflugers Archiv : European journal of physiology, 1996
    Co-Authors: Bonnie L. Blazer-yost, Rae D. Record, Hans Oberleithner
    Abstract:

    The Madin-Darby canine kidney (MDCK) Cell Line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent Line forms an epithelium which expresses many of the characteristics of principal Cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal Cells and is an ideal model for studying hormonal modulation of Na+ transport.

  • characterization of hormone stimulated na transport in a high resistance clone of the MDCK Cell Line
    Pflügers Archiv: European Journal of Physiology, 1996
    Co-Authors: Bonnie L Blazeryost, Rae D. Record, Hans Oberleithner
    Abstract:

    The Madin-Darby canine kidney (MDCK) Cell Line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent Line forms an epithelium which expresses many of the characteristics of principal Cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF 1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal Cells and is an ideal model for studying hormonal modulation of Na+ transport.

Rajendra S. Kadam - One of the best experts on this subject based on the ideXlab platform.

  • pigmented MDCK p MDCK Cell Line with tunable melanin expression an in vitro model for the outer blood retinal barrier
    Molecular Pharmaceutics, 2012
    Co-Authors: Rajendra S. Kadam, Robert I. Scheinman, Uday B. Kompella
    Abstract:

    Retinal pigment epithelium, which forms the outer blood–retinal barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) Cell Line as a rapidly established in vitro model for the outer blood–retinal barrier to assess the influence of melanin pigment on solute permeability. A melanin synthesizing P-MDCK Cell Line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) Cells. Melanin content, tyrosinase activity (conversion of l-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK Cells was confirmed by confocal microscopy. Effect of l-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK Cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = −1.14) were investigated. P-MDCK Cells expres...

  • Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier
    Molecular pharmaceutics, 2012
    Co-Authors: Rajendra S. Kadam, Robert I. Scheinman, Uday B. Kompella
    Abstract:

    Retinal pigment epithelium, which forms the outer blood–retinal barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) Cell Line as a rapidly established in vitro model for the outer blood–retinal barrier to assess the influence of melanin pigment on solute permeability. A melanin synthesizing P-MDCK Cell Line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) Cells. Melanin content, tyrosinase activity (conversion of l-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK Cells was confirmed by confocal microscopy. Effect of l-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK Cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = −1.14) were investigated. P-MDCK Cells expres...