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Chondrocyte channel transcriptomics: do microarray data fit with expression and functional data?

Lewis R, May H, Mobasheri A, Barrett-Jolley R - Channels (Austin) (2013)

Bottom Line: We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes.The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions.We discuss whether functional or protein data exist which support the microarray data.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Biology; Institute of Ageing and Chronic Disease; Faculty of Health & Life Sciences; University of Liverpool; Liverpool, UK; The D-BOARD European Consortium for Biomarker Discovery.

ABSTRACT
To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels.

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Related in: MedlinePlus

Figure 3. Water Permeability (Aquaporin) Assay. Water permeability can be calculated from the initial slope of the relative volume (V/Vo) against time curve. Where V is the volume at time t and Vo is the volume at time zero. This is the accepted physiological assay for aquaporin expression. (A) Permeability is 30±3% (p<0.05, n=4) greater in chondrocytes from dogs with osteoarthritis (OA). TEA (a blocker of AQP153,54), bumetanide54 (pIC50 5.17±0.11µM, n=6, “Bumex”, a blocker of AQP1 and 4), and mercuric chloride (HgCl2 a non-specific AQP blocker reversed by 2-mercaptoethanol) ME) are included to determine AQP type (B and C). Chondrocytes were harvested from canine clinical waste tissue with owner consent. Cells were placed in a “physiological saline” solution including 120 mM sucrose (osmolarity 300mOsm), then moved to an identical physiological saline without the sucrose. Cells at first swell as water enters the cell due to osmosis. Live cell imaging was achieved with a Nikon Diaphot microscope equipped with a Sony ICX098QB high sensitivity CCD. Images were analysed offline with ImageJVolume was calculated from the 2D surface area (A) of the cell disc by assuming the cell is approximately spherical as described previously,6 using the following equation:  (Equation 2). Except where stated, data are presented normalized for starting volume (V0) as V/V0, where V is the volume at time t. Visual data were analyzed with ImageJ and ANOVA performed with SPSS (SPSS Inc.). Note that canine tissue was harvested from clinical waste tissue with Local Ethical Approval, no dogs were harmed for the study.
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Figure 3: Figure 3. Water Permeability (Aquaporin) Assay. Water permeability can be calculated from the initial slope of the relative volume (V/Vo) against time curve. Where V is the volume at time t and Vo is the volume at time zero. This is the accepted physiological assay for aquaporin expression. (A) Permeability is 30±3% (p<0.05, n=4) greater in chondrocytes from dogs with osteoarthritis (OA). TEA (a blocker of AQP153,54), bumetanide54 (pIC50 5.17±0.11µM, n=6, “Bumex”, a blocker of AQP1 and 4), and mercuric chloride (HgCl2 a non-specific AQP blocker reversed by 2-mercaptoethanol) ME) are included to determine AQP type (B and C). Chondrocytes were harvested from canine clinical waste tissue with owner consent. Cells were placed in a “physiological saline” solution including 120 mM sucrose (osmolarity 300mOsm), then moved to an identical physiological saline without the sucrose. Cells at first swell as water enters the cell due to osmosis. Live cell imaging was achieved with a Nikon Diaphot microscope equipped with a Sony ICX098QB high sensitivity CCD. Images were analysed offline with ImageJVolume was calculated from the 2D surface area (A) of the cell disc by assuming the cell is approximately spherical as described previously,6 using the following equation: (Equation 2). Except where stated, data are presented normalized for starting volume (V0) as V/V0, where V is the volume at time t. Visual data were analyzed with ImageJ and ANOVA performed with SPSS (SPSS Inc.). Note that canine tissue was harvested from clinical waste tissue with Local Ethical Approval, no dogs were harmed for the study.

Mentions: The accepted assay for aquaporin expression is that developed by Preston et al.51 Cells are challenged with a hypotonic solution, causing them to swell, and the rate of swell can be measured to determine the aquaporin expression. The bioinformatics showed a 38-fold increase in AQP1 transcript abundance in OA and we see a significant increase in functional aquaporin expression in tissue from OA joints (Fig. 3A), although the increase is much smaller than the change in AQP1 transcript abundance. Current pharmacological tools do not allow categorical determination of aquaporin subtype, however, here, water permeability of chondrocytes was blocked by concentrations of bumetanide, TEA, and HgCl2 consistent with that expected for AQP153,54 (Fig. 3B and C).


Chondrocyte channel transcriptomics: do microarray data fit with expression and functional data?

Lewis R, May H, Mobasheri A, Barrett-Jolley R - Channels (Austin) (2013)

Figure 3. Water Permeability (Aquaporin) Assay. Water permeability can be calculated from the initial slope of the relative volume (V/Vo) against time curve. Where V is the volume at time t and Vo is the volume at time zero. This is the accepted physiological assay for aquaporin expression. (A) Permeability is 30±3% (p<0.05, n=4) greater in chondrocytes from dogs with osteoarthritis (OA). TEA (a blocker of AQP153,54), bumetanide54 (pIC50 5.17±0.11µM, n=6, “Bumex”, a blocker of AQP1 and 4), and mercuric chloride (HgCl2 a non-specific AQP blocker reversed by 2-mercaptoethanol) ME) are included to determine AQP type (B and C). Chondrocytes were harvested from canine clinical waste tissue with owner consent. Cells were placed in a “physiological saline” solution including 120 mM sucrose (osmolarity 300mOsm), then moved to an identical physiological saline without the sucrose. Cells at first swell as water enters the cell due to osmosis. Live cell imaging was achieved with a Nikon Diaphot microscope equipped with a Sony ICX098QB high sensitivity CCD. Images were analysed offline with ImageJVolume was calculated from the 2D surface area (A) of the cell disc by assuming the cell is approximately spherical as described previously,6 using the following equation:  (Equation 2). Except where stated, data are presented normalized for starting volume (V0) as V/V0, where V is the volume at time t. Visual data were analyzed with ImageJ and ANOVA performed with SPSS (SPSS Inc.). Note that canine tissue was harvested from clinical waste tissue with Local Ethical Approval, no dogs were harmed for the study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042480&req=5

Figure 3: Figure 3. Water Permeability (Aquaporin) Assay. Water permeability can be calculated from the initial slope of the relative volume (V/Vo) against time curve. Where V is the volume at time t and Vo is the volume at time zero. This is the accepted physiological assay for aquaporin expression. (A) Permeability is 30±3% (p<0.05, n=4) greater in chondrocytes from dogs with osteoarthritis (OA). TEA (a blocker of AQP153,54), bumetanide54 (pIC50 5.17±0.11µM, n=6, “Bumex”, a blocker of AQP1 and 4), and mercuric chloride (HgCl2 a non-specific AQP blocker reversed by 2-mercaptoethanol) ME) are included to determine AQP type (B and C). Chondrocytes were harvested from canine clinical waste tissue with owner consent. Cells were placed in a “physiological saline” solution including 120 mM sucrose (osmolarity 300mOsm), then moved to an identical physiological saline without the sucrose. Cells at first swell as water enters the cell due to osmosis. Live cell imaging was achieved with a Nikon Diaphot microscope equipped with a Sony ICX098QB high sensitivity CCD. Images were analysed offline with ImageJVolume was calculated from the 2D surface area (A) of the cell disc by assuming the cell is approximately spherical as described previously,6 using the following equation: (Equation 2). Except where stated, data are presented normalized for starting volume (V0) as V/V0, where V is the volume at time t. Visual data were analyzed with ImageJ and ANOVA performed with SPSS (SPSS Inc.). Note that canine tissue was harvested from clinical waste tissue with Local Ethical Approval, no dogs were harmed for the study.
Mentions: The accepted assay for aquaporin expression is that developed by Preston et al.51 Cells are challenged with a hypotonic solution, causing them to swell, and the rate of swell can be measured to determine the aquaporin expression. The bioinformatics showed a 38-fold increase in AQP1 transcript abundance in OA and we see a significant increase in functional aquaporin expression in tissue from OA joints (Fig. 3A), although the increase is much smaller than the change in AQP1 transcript abundance. Current pharmacological tools do not allow categorical determination of aquaporin subtype, however, here, water permeability of chondrocytes was blocked by concentrations of bumetanide, TEA, and HgCl2 consistent with that expected for AQP153,54 (Fig. 3B and C).

Bottom Line: We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes.The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions.We discuss whether functional or protein data exist which support the microarray data.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Biology; Institute of Ageing and Chronic Disease; Faculty of Health & Life Sciences; University of Liverpool; Liverpool, UK; The D-BOARD European Consortium for Biomarker Discovery.

ABSTRACT
To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels.

Show MeSH
Related in: MedlinePlus