Involvement of Intracellular Ca2+ in the Regulatory Volume Decrease After Hyposmotic Swelling in MDCK Cells

Iida-Tanaka, Naoko; Namekata, Iyuki; Kaneko, Megumi; Tamura, M.; Kawanishi, Toru; Nakamura, Reinosuke; Shigenobu, Koki; Tanaka, Hikaru

doi:10.1254/jphs.sc0070024

Cited by 7 publications

(8 citation statements)

References 16 publications

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“…[25][26][27][28] Coverslips with cells were placed in a chamber on the stage of the inverted microscope and perfused with HEPES-Tyrode solution of the following composition (mM): NaCl 143, KCl 5.4, MgCl 2 1.8, CaCl 2 2, NaH 2 PO 4 0.33, glucose 5.5 and HEPES 20. The solution was gassed with 100% O 2 .…”

Section: )mentioning

confidence: 99%

Effect of Terfenadine and Pentamidine on the hERG Channel and Its Intracellular Trafficking: Combined Analysis with Automated Voltage Clamp and Confocal Microscopy

Tanaka

Takahashi

Hamaguchi

et al. 2014

Biological & Pharmaceutical Bulletin

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The effects of terfenadine and pentamidine on the human ether-a-go-go related gene (hERG) channel current and its intracellular trafficking were evaluated. Green fluorescent protein (GFP)-linked hERG channels were expressed in HEK293 cells, and the membrane current was measured by an automated whole cell voltage clamp system. To evaluate drug effects on channel trafficking to the cell membrane, the fraction of channel present on the cell membrane was quantified by current measurement after drug washout and confocal microscopy. Terfenadine directly blocked the hERG channel current but had no effect on trafficking of hERG channels to the cell membrane after application in culture medium for 2 d. In contrast, pentamidine had no direct effect on the hERG channel current but reduced trafficking of hERG channels. The two drugs inhibited hERG channel function through different mechanisms: terfenadine through direct channel blockade and pentamidine through inhibition of channel trafficking to the cell membrane. Combined use of automated voltage clamp and confocal microscopic analyses would provide insights into the mechanisms of drug-induced QT-prolongation and arrhythmogenesis.

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Section: )mentioning

confidence: 99%

Effect of Terfenadine and Pentamidine on the hERG Channel and Its Intracellular Trafficking: Combined Analysis with Automated Voltage Clamp and Confocal Microscopy

Tanaka

Takahashi

Hamaguchi

et al. 2014

Biological & Pharmaceutical Bulletin

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“…Such phenomenon is generally known as regulatory volume decrease, and is considered to be a mechanism to maintain cellular integrity under osmotic stress [5,6] . In the case of MDCK cells, the regulatory volume decrease was reported to be dependent on an increase in intracellular Ca 2+ concentration [14] . The low osmotic stimuli-induced increase in the volume of the cell reconstructed by z stacking of horizontal sections (x-y) correlated to a certain extent with the increases in the area of the x-z and y-z vertical sections and also with the cell height.…”

Section: Discussionmentioning

confidence: 99%

“…Confocal imaging was performed with LSM 510 (Carl Zeiss) or LSM 5 LIVE (Carl Zeiss) with procedures described in our previous studies [14,15] . The cells loaded with PKH67 were excited at 488nm with an argon laser and the emission at wavelength between 500nm and 530nm was detected by a photomultiplier.…”

Section: Methodsmentioning

confidence: 99%

Analysis of cellular morphology by confocal microscopy and a membrane-intercalating fluorescent dye PKH67

Hamaguchi

Kaneko

Tamura

et al. 2014

Int J Hum Cult Stud.

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Cells of the body are exposed to anisosmotic environments under various physiological and pathological conditions, which cause mobilization of water and changes in cellular volume and morphology; these changes provide an indication of the cellular environment changes and predict the subsequent cellular damage. We have constructed confocal microscopy-based methods to analyze osmotic stimuli-induced changes in cell dimension and morphology using a membrane-bound fluorescent probe, PKH67. Low osmolarity stimulation of the renal tube derived MDCK cells resulted in an initial increase in their cell volume, which was followed by a subsequent decrease towards initial value. Transient exposure to high osmolarity formamide solution of mouse ventricular cardiomyocytes resulted in complete loss of their transverse tubules (T-tubules). Confocal microscopy in combination with a membrane-intercalating dye was shown to be simple, accurate and free of cellular damage, and would be useful for further studies on the regulation of cellular volume and morphology.

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“…This process, known as the regulatory volume decrease, is considered to be the result of activation of various ion channels and transporters. [10][11][12][13][14] The membrane-labeled MDCK cells established in the present study would be a useful model for the study of such mechanisms. The changes in relative cell volume could be also demonstrated when the cell height was used as an index (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…[8][9][10] Coverslips with cells were placed in a chamber on the stage of the inverted microscope and perfused with the normal medium for confocal microscopy, which was made by adding 25 mM HEPES to the EMEM. The pH of this normal medium was adjusted to be 7.4 and the osmolarity was 300 mOsm as determined by a freezing point based osmometer.…”

Section: Preparation Of Mdck Cells Stably Expressingmentioning

confidence: 99%

Membrane-Labeled MDCK Cells and Confocal Microscopy for the Analyses of Cellular Volume and Morphology

Iida-Tanaka

Namekata

Tamura

et al. 2008

Biological & Pharmaceutical Bulletin

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A clone of Madin-Darby canine kidney (MDCK) cells whose cell membrane was stably labeled with expressed cyan fluorescent protein (CFP) was established, and changes in their volume and shape induced by hyposmotic stress were analyzed with confocal microscopy. The membrane-targeted CFP was present not only on the cell membrane but also in the endoplasmic reticulum and Golgi apparatus, but was excluded from the mitochondria and cell nucleus. During hyposmosis, the initial swelling and the following regulatory volume decrease could be accurately measured by summation of the cellular volume in every confocal slice crossing the cell at different heights. Changes in the cellular height roughly paralleled the changes in cellular volume when the mean value was compared, but dissociation as much as 30% was observed for individual cells due to changes in cell shape. The present experimental system, which enables direct measurement of cell volume and simultaneous observation of various intracellular phenomena, would be useful for further investigation of cellular volume regulation.

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Involvement of Intracellular Ca2+ in the Regulatory Volume Decrease After Hyposmotic Swelling in MDCK Cells

Cited by 7 publications

References 16 publications

Effect of Terfenadine and Pentamidine on the hERG Channel and Its Intracellular Trafficking: Combined Analysis with Automated Voltage Clamp and Confocal Microscopy

Effect of Terfenadine and Pentamidine on the hERG Channel and Its Intracellular Trafficking: Combined Analysis with Automated Voltage Clamp and Confocal Microscopy

Analysis of cellular morphology by confocal microscopy and a membrane-intercalating fluorescent dye PKH67

Membrane-Labeled MDCK Cells and Confocal Microscopy for the Analyses of Cellular Volume and Morphology

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