The Experts below are selected from a list of 171 Experts worldwide ranked by ideXlab platform

Yoshiki Shiba - One of the best experts on this subject based on the ideXlab platform.

  • Forskolin-induced clearance of the fluorescent dye sulforhodamine from rat parotid Intralobular Duct lumen: Visualization of the secretory function under a confocal laser scanning microscope
    The Journal of membrane biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Kazuhisa Takemoto, Yoshiki Shiba
    Abstract:

    Cyclic AMP evokes fluid secretion with bicarbonate in exocrine Ducts. Clearance of fluorescent dyes from rat parotid Intralobular Ducts by forskolin was visualized as a fluorescence change in the Duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the Ducts. When the isolated rat parotid Intralobular Duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the Duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the Duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the Ducts. Methazolamide inhibited a forskolin-induced sustained decrease in Duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in Duct fluorescence. These findings suggest that a major portion of the steady decrease in Duct fluorescence by forskolin was related to intracellular HCO3? proDuction, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from Duct space possibly due to fluid secretion in rat parotid Ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

  • Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate proDuction in rat parotid Intralobular Ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope.
    European journal of cell biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yoshiki Shiba, Kazuhisa Takemoto, Yasumasa Akagawa
    Abstract:

    Summary Fluid secretion is observed at the openings of Ducts in the exocrine gland. It remains unclear whether the Ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the Duct lumen by carbachol (CCh) in isolated parotid Intralobular Duct segments to clarify the ability of the Ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the Duct lumen were obtained under the optical sectioning at the level of the Duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the Duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated Duct lumen, suggesting that CCh might induce fluid secretion in the Duct, leading to the clearance of the dye and small stained clumps from the Duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca 2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl − , bumetanide or DIDS, suggesting that methazolamide-sensitive proDuction of HCO 3 − , but not the Cl − uptake mechanism, might contribute to the CCh-induced clearance of the dye from the Duct lumen. These results represent the first measurements of fluid movement in isolated Duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca 2+ -activated, DPC-sensitive anion channels with HCO 3 − secretion in the rat parotid Intralobular Ducts.

  • gramicidin perforated patch analysis on hco3 secretion through a forskolin activated anion channel in rat parotid Intralobular Duct cells
    The Journal of Membrane Biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

  • Gramicidin-Perforated Patch Analysis on HCO3 − Secretion Through a Forskolin-Activated Anion Channel in Rat Parotid Intralobular Duct Cells
    The Journal of membrane biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

Yasumasa Akagawa - One of the best experts on this subject based on the ideXlab platform.

  • Forskolin-induced clearance of the fluorescent dye sulforhodamine from rat parotid Intralobular Duct lumen: Visualization of the secretory function under a confocal laser scanning microscope
    The Journal of membrane biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Kazuhisa Takemoto, Yoshiki Shiba
    Abstract:

    Cyclic AMP evokes fluid secretion with bicarbonate in exocrine Ducts. Clearance of fluorescent dyes from rat parotid Intralobular Ducts by forskolin was visualized as a fluorescence change in the Duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the Ducts. When the isolated rat parotid Intralobular Duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the Duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the Duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the Ducts. Methazolamide inhibited a forskolin-induced sustained decrease in Duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in Duct fluorescence. These findings suggest that a major portion of the steady decrease in Duct fluorescence by forskolin was related to intracellular HCO3? proDuction, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from Duct space possibly due to fluid secretion in rat parotid Ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

  • Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate proDuction in rat parotid Intralobular Ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope.
    European journal of cell biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yoshiki Shiba, Kazuhisa Takemoto, Yasumasa Akagawa
    Abstract:

    Summary Fluid secretion is observed at the openings of Ducts in the exocrine gland. It remains unclear whether the Ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the Duct lumen by carbachol (CCh) in isolated parotid Intralobular Duct segments to clarify the ability of the Ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the Duct lumen were obtained under the optical sectioning at the level of the Duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the Duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated Duct lumen, suggesting that CCh might induce fluid secretion in the Duct, leading to the clearance of the dye and small stained clumps from the Duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca 2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl − , bumetanide or DIDS, suggesting that methazolamide-sensitive proDuction of HCO 3 − , but not the Cl − uptake mechanism, might contribute to the CCh-induced clearance of the dye from the Duct lumen. These results represent the first measurements of fluid movement in isolated Duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca 2+ -activated, DPC-sensitive anion channels with HCO 3 − secretion in the rat parotid Intralobular Ducts.

  • gramicidin perforated patch analysis on hco3 secretion through a forskolin activated anion channel in rat parotid Intralobular Duct cells
    The Journal of Membrane Biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

  • Gramicidin-Perforated Patch Analysis on HCO3 − Secretion Through a Forskolin-Activated Anion Channel in Rat Parotid Intralobular Duct Cells
    The Journal of membrane biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

Tetsuji Nakamoto - One of the best experts on this subject based on the ideXlab platform.

  • Forskolin-induced clearance of the fluorescent dye sulforhodamine from rat parotid Intralobular Duct lumen: Visualization of the secretory function under a confocal laser scanning microscope
    The Journal of membrane biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Kazuhisa Takemoto, Yoshiki Shiba
    Abstract:

    Cyclic AMP evokes fluid secretion with bicarbonate in exocrine Ducts. Clearance of fluorescent dyes from rat parotid Intralobular Ducts by forskolin was visualized as a fluorescence change in the Duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the Ducts. When the isolated rat parotid Intralobular Duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the Duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the Duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the Ducts. Methazolamide inhibited a forskolin-induced sustained decrease in Duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in Duct fluorescence. These findings suggest that a major portion of the steady decrease in Duct fluorescence by forskolin was related to intracellular HCO3? proDuction, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from Duct space possibly due to fluid secretion in rat parotid Ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

  • Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate proDuction in rat parotid Intralobular Ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope.
    European journal of cell biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yoshiki Shiba, Kazuhisa Takemoto, Yasumasa Akagawa
    Abstract:

    Summary Fluid secretion is observed at the openings of Ducts in the exocrine gland. It remains unclear whether the Ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the Duct lumen by carbachol (CCh) in isolated parotid Intralobular Duct segments to clarify the ability of the Ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the Duct lumen were obtained under the optical sectioning at the level of the Duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the Duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated Duct lumen, suggesting that CCh might induce fluid secretion in the Duct, leading to the clearance of the dye and small stained clumps from the Duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca 2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl − , bumetanide or DIDS, suggesting that methazolamide-sensitive proDuction of HCO 3 − , but not the Cl − uptake mechanism, might contribute to the CCh-induced clearance of the dye from the Duct lumen. These results represent the first measurements of fluid movement in isolated Duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca 2+ -activated, DPC-sensitive anion channels with HCO 3 − secretion in the rat parotid Intralobular Ducts.

  • gramicidin perforated patch analysis on hco3 secretion through a forskolin activated anion channel in rat parotid Intralobular Duct cells
    The Journal of Membrane Biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

  • Gramicidin-Perforated Patch Analysis on HCO3 − Secretion Through a Forskolin-Activated Anion Channel in Rat Parotid Intralobular Duct Cells
    The Journal of membrane biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

Chikara Hirono - One of the best experts on this subject based on the ideXlab platform.

  • Forskolin-induced clearance of the fluorescent dye sulforhodamine from rat parotid Intralobular Duct lumen: Visualization of the secretory function under a confocal laser scanning microscope
    The Journal of membrane biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Kazuhisa Takemoto, Yoshiki Shiba
    Abstract:

    Cyclic AMP evokes fluid secretion with bicarbonate in exocrine Ducts. Clearance of fluorescent dyes from rat parotid Intralobular Ducts by forskolin was visualized as a fluorescence change in the Duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the Ducts. When the isolated rat parotid Intralobular Duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the Duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the Duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the Ducts. Methazolamide inhibited a forskolin-induced sustained decrease in Duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in Duct fluorescence. These findings suggest that a major portion of the steady decrease in Duct fluorescence by forskolin was related to intracellular HCO3? proDuction, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from Duct space possibly due to fluid secretion in rat parotid Ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

  • Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate proDuction in rat parotid Intralobular Ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope.
    European journal of cell biology, 2002
    Co-Authors: Tetsuji Nakamoto, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, Yoshiki Shiba, Kazuhisa Takemoto, Yasumasa Akagawa
    Abstract:

    Summary Fluid secretion is observed at the openings of Ducts in the exocrine gland. It remains unclear whether the Ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the Duct lumen by carbachol (CCh) in isolated parotid Intralobular Duct segments to clarify the ability of the Ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the Duct lumen were obtained under the optical sectioning at the level of the Duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the Duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated Duct lumen, suggesting that CCh might induce fluid secretion in the Duct, leading to the clearance of the dye and small stained clumps from the Duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca 2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl − , bumetanide or DIDS, suggesting that methazolamide-sensitive proDuction of HCO 3 − , but not the Cl − uptake mechanism, might contribute to the CCh-induced clearance of the dye from the Duct lumen. These results represent the first measurements of fluid movement in isolated Duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca 2+ -activated, DPC-sensitive anion channels with HCO 3 − secretion in the rat parotid Intralobular Ducts.

  • gramicidin perforated patch analysis on hco3 secretion through a forskolin activated anion channel in rat parotid Intralobular Duct cells
    The Journal of Membrane Biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

  • Gramicidin-Perforated Patch Analysis on HCO3 − Secretion Through a Forskolin-Activated Anion Channel in Rat Parotid Intralobular Duct Cells
    The Journal of membrane biology, 2001
    Co-Authors: Chikara Hirono, Tetsuji Nakamoto, Makoto Sugita, Yoshiko Iwasa, Yasumasa Akagawa, Yoshiki Shiba
    Abstract:

    Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3 − secretion in the Intralobular Duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conDuctance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3 −. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3 − significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and proDuction of cytosolic HCO3 − induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3 − exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3 − exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3 − produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid Duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

D I Cook - One of the best experts on this subject based on the ideXlab platform.

  • Ion channels in the basolateral membrane of Intralobular Duct cells of mouse mandibular glands
    Pflügers Archiv, 1994
    Co-Authors: Anuwat Dinudom, J. A. Young, D I Cook
    Abstract:

    We have used single-channel patch-clamp techniques to study the ion channels in the basolateral membranes of Intralobular Duct cells from the mouse mandibular gland. In 39% of cell-attached patches, we observed a K^+ channel that had an inwardly rectifying current/voltage ( I/V ) relation with a maximum slope conDuctance of 123±9 pS ( n =12) and a zero current potential of +49.4±3.4 mV ( n =5) relative to the resting cell potential. The selectivity sequence of this channel, as estimated by zero current potential measurements, was: K^+ (1) > Rb^+ (0.38) > NH _4 ^+ (

  • ion channels in the basolateral membrane of Intralobular Duct cells of mouse mandibular glands
    Pflügers Archiv: European Journal of Physiology, 1994
    Co-Authors: Anuwat Dinudom, J. A. Young, D I Cook
    Abstract:

    We have used single-channel patch-clamp techniques to study the ion channels in the basolateral membranes of Intralobular Duct cells from the mouse mandibular gland. In 39% of cell-attached patches, we observed a K+ channel that had an inwardly rectifying current/voltage (I/V) relation with a maximum slope conDuctance of 123±9 pS (n=12) and a zero current potential of +49.4±3.4 mV (n=5) relative to the resting cell potential. The selectivity sequence of this channel, as estimated by zero current potential measurements, was: K+ (1) > Rb+ (0.38) > NH4+( Na+ ( Rb+ ( NH4+( Na+ (<0.09). The activity of this channel type was not affected by changes in membrane potential. In 18% of excised patches, we also observed a non-selective cation channel that was not demonstrable in cell-attached patches. It had a slope conDuctance of 22±2 pS (n=6) and was blocked by the non-selective cation channel blocker, flufenamate (10 μmol/l). A fourth channel type, observed only in 5% of patches was a Cl− channel with a slope conDuctance of 40 pS and a linearI/V relation. The K+ channels observed in this study seem likely to underlie the K+ conDuctance described in the basolateral membrane of extralobular Ducts by in vitro perfusion studies. Our finding that they are inwardly rectifying suggests that they may not be the sole route of K+ transport across the basolateral membrane.

  • Ion channels in the basolateral membrane of Intralobular Duct cells of mouse mandibular glands.
    Pflugers Archiv : European journal of physiology, 1994
    Co-Authors: Anuwat Dinudom, J. A. Young, D I Cook
    Abstract:

    We have used single-channel patch-clamp techniques to study the ion channels in the basolateral membranes of Intralobular Duct cells from the mouse mandibular gland. In 39% of cell-attached patches, we observed a K+ channel that had an inwardly rectifying current/voltage (I/V) relation with a maximum slope conDuctance of 123 +/- 9 pS (n = 12) and a zero current potential of +49.4 +/- 3.4 mV (n = 5) relative to the resting cell potential. The selectivity sequence of this channel, as estimated by zero current potential measurements, was: K+ (1) > Rb+ (0.38) > NH4+ (< 0.34), Cs+ (< 0.16) > Na+ (< 0.028). The activity of the channel was not affected by changes in membrane potential, nor was it affected by changes in the free Ca2+ concentration on the cytosolic side of inside-out excised patches in the range 1 nmol/l to 1 mumol/l. In 38% of cell-attached patches we observed a second K+ channel type with a maximum slope conDuctance of 62 +/- 3 pS (n = 12) and an inwardly rectifying I/V relation. The selectivity sequence of this channel was K+ (1) > Rb+ (< 0.5) > NH4+ (< 0.2) > Na+ (< 0.09). The activity of this channel type was not affected by changes in membrane potential. In 18% of excised patches, we also observed a non-selective cation channel that was not demonstrable in cell-attached patches. It had a slope conDuctance of 22 +/- 2 pS (n = 6) and was blocked by the non-selective cation channel blocker, flufenamate (10 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

  • na and cl conDuctances are controlled by cytosolic cl concentration in the Intralobular Duct cells of mouse mandibular glands
    The Journal of Membrane Biology, 1993
    Co-Authors: Anuwat Dinudom, J. A. Young, D I Cook
    Abstract:

    Our previously published whole-cell patch-clamp studies on the cells of the Intralobular (granular) Ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na+ conDuctance in the plasma membrane. In this study we demonstrate the presence also of a Cl− conDuctance and we show that the sizes of both conDuctances vary with the Cl− concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl− concentration rises from 5 to 150 mmol/liter, the size of the inward Na+ current declines, the decline being half-maximal when the Cl− concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl− concentration increases, the inward Cl− current remains at a constant low level until the Cl− concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl− in the pipette solution was replaced by other anions indicate that the Na+ current is suppressed by intracellular Br-, Cl− and NO 3 - but not by intracellular I-, glutamate or gluconate. Our studies also show that the Cl− conDuctance allows passage of Cl− and Br- equally well, I-less well, and NO 3 - , glutamate and gluconate poorly, if at all. The findings with NO 3 - are of particular interest because they show that suppression of the Na+ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl− conDuctance. In mouse granular Duct cells there is, thus, a reciprocal regulation of Na+ and Cl− conDuctances by the cytosolic Cl− concentration. Since the cytosolic Cl− concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na+ and Cl− conDuctances may provide a mechanism by which Ductal Na+ and Cl transport rates are adjusted so as to maintain a stable cell volume.

  • na and cl conDuctances are controlled by cytosolic cl concentration in the Intralobular Duct cells of mouse mandibular glands
    The Journal of Membrane Biology, 1993
    Co-Authors: Anuwat Dinudom, J. A. Young, D I Cook
    Abstract:

    Our previously published whole-cell patch-clamp studies on the cells of the Intralobular (granular) Ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na+ conDuctance in the plasma membrane. In this study we demonstrate the presence also of a Cl- conDuctance and we show that the sizes of both conDuctances vary with the Cl- concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl- concentration rises from 5 to 150 mmol/liter, the size of the inward Na+ current declines, the decline being half-maximal when the Cl- concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl- concentration increases, the inward Cl- current remains at a constant low level until the Cl- concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl- in the pipette solution was replaced by other anions indicate that the Na+ current is suppressed by intracellular Br-, Cl- and NO3- but not by intracellular I-, glutamate or gluconate. Our studies also show that the Cl- conDuctance allows passage of Cl- and Br- equally well, I- less well, and NO3-, glutamate and gluconate poorly, if at all. The findings with NO3- are of particular interest because they show that suppression of the Na+ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl- conDuctance. In mouse granular Duct cells there is, thus, a reciprocal regulation of Na+ and Cl- conDuctances by the cytosolic Cl- concentration.(ABSTRACT TRUNCATED AT 250 WORDS)