siRNA-mediated inhibition of Na^sup +^ -K^sup +^-2Cl^sup -^ cotransporter (NKCC1) and regulatory volume increase in the chondrocyte cell line C-20/A4

Qusous, A., Geewan, C., Greenwell, P. and Kerrigan, M.J.P. 2011. siRNA-mediated inhibition of Na^sup +^ -K^sup +^-2Cl^sup -^ cotransporter (NKCC1) and regulatory volume increase in the chondrocyte cell line C-20/A4. Journal of Membrane Biology. 243 (1-3), pp. 25-34.

TitlesiRNA-mediated inhibition of Na^sup +^ -K^sup +^-2Cl^sup -^ cotransporter (NKCC1) and regulatory volume increase in the chondrocyte cell line C-20/A4
AuthorsQusous, A., Geewan, C., Greenwell, P. and Kerrigan, M.J.P.
Abstract

The Na^sup +^-K^sup +^-2Cl^sup -^ cotransporter (NKCC1) is an essential membrane transporter and has been linked to the regulation of volume, matrix synthesis and bone growth in chondrocytes; the sole resident cell type of articular cartilage. Despite the integral nature of NKCC1, its regulation is currently poorly understood, and therefore here we describe a NKCC1 knockdown technique that will permit the easier study of this transporter. Small interfering RNA (siRNA), designed to knock down NKCC1, was transfected into the chondrocyte cell line C-20/A4 and the efficacy determined at the message, protein and functional levels. NKCC1 expression was analyzed by reverse-transcriptase polymerase chain reaction, where NKCC1 expression declined to 25.10 ± 1.08% after 12 h of transfection and did not show any rise in the following 36 h. The efficacy of the designed siRNA molecules was confirmed by both Western blot and immunocytochemistry. The effect of the knockdown on regulatory volume increase (RVI, a novel assay for NKCC1 function) was investigated by confocal laser scanning microscopy in response to a 43% hypertonic challenge, whereby control chondrocytes underwent a decrease in volume to 67.38 ± 1.70%, followed by volume restoration to 82.17 ± 2.23 at 20 min (t1⁄2 = 22.11 ± 3.23 min). Conversely, upon knockdown, chondrocytes exhibited a slower rate of RVI (t1⁄2 = 43.26 ± 5.64 min), thus suggesting that NKCC1 plays an important and yet partial role in RVI in C-20/A4 chondrocytes. Together, these data provide a robust protocol for the study of NKCC1 in chondrocytes and suggest a mechanism for C-20/A4 chondrocyte RVI.

JournalJournal of Membrane Biology
Journal citation243 (1-3), pp. 25-34
ISSN0022-2631
YearOct 2011
PublisherSpringer
Digital Object Identifier (DOI)doi:10.1007/s00232-011-9389-z
Publication dates
PublishedOct 2011

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Qusous, A., Hook, C.S.V., McRobb, L. and Kerrigan, M.J.P. 2006. siRNA mediated inhibition of regulatory volume increase in the chondrocytic cell line C-20/A4. Orthopaedic Research Society. Transactions of the Annual Meeting. 52nd Annual Meeting of the Orthopaedic Research Society. 31 (0291).

Advanced online learning to support student-centred active learning
Kerrigan, M.J.P., Mackenzie, J., Raymond, R. and Patterson, A. 2006. Advanced online learning to support student-centred active learning. University of Westminster Teaching & Learning Symposium. University of Westminster, London 2006 Association for Learning Technology.

Analysis of glycosidases activity in Chlamydia trachomatis L2 serotype
Greenwell, P., Kakourou, G. and Rughooputh, S. 2006. Analysis of glycosidases activity in Chlamydia trachomatis L2 serotype. Internet Journal of Medical Update. 1 (1).

Vaccination against cervical cancer
Greenwell, P. and Rughooputh, S. 2005. Vaccination against cervical cancer. Biomedical Scientist. 49 (6), p. 441.

Proteomics: more than just biochemistry
Greenwell, P. and Rughooputh, S. 2005. Proteomics: more than just biochemistry. Biomedical Scientist. 49 (6), pp. 459-460.

Confocal microscopy for the identification of glycosylation changes associated with metastatic colorectal cancer
Zeqiraj, E., Saint-Guirons, J., Kerrigan, M.J.P. and Dwek, M. 2005. Confocal microscopy for the identification of glycosylation changes associated with metastatic colorectal cancer. The Molecular Biology of Colorectal Cancer. UBHT Education Centre, Bristol, UK 10 - 11 Mar 2005

Trichomonas vaginalis: paradigm of a successful sexually transmitted organism
Rughooputh, S. and Greenwell, P. 2005. Trichomonas vaginalis: paradigm of a successful sexually transmitted organism. British Journal of Biomedical Science. 62 (4), pp. 193-200.

Stimulation of regulatory volume decrease (RVD) by isolated bovine articular chondrocytes following F-actin disruption using latrunculin B
Kerrigan, M.J.P. and Hall, A.C. 2005. Stimulation of regulatory volume decrease (RVD) by isolated bovine articular chondrocytes following F-actin disruption using latrunculin B. Biorheology. 42 (4), pp. 283-293.

Online, interactive technologies to support e-learning
Kerrigan, M.J.P. 2005. Online, interactive technologies to support e-learning. University of Westminster Teaching & Learning Symposium. University of Westminster, London 2005 Association for Learning Technology.

Application of recombinant phage display antibody system in study of Codakia orbicularis gill proteins
Gourdine, J.P., Greenwell, P. and Smith-Ravin, E.J. 2005. Application of recombinant phage display antibody system in study of Codakia orbicularis gill proteins. Applied Biochemistry and Biotechnology. 125 (1), pp. 41-52.

The Unani system of medicine: does it have a scientific basis?
Syeda, A., Rughooputh, S. and Greenwell, P. 2004. The Unani system of medicine: does it have a scientific basis? Biomedical Scientist. 48 (9), pp. 971-972.

Genetically modified food: good news but bad press
Greenwell, P. and Rughooputh, S. 2004. Genetically modified food: good news but bad press. Biomedical Scientist. 48 (8), pp. 845-846.

Bioinformatics: a useful tool for the molecular microbiologist?
Greenwell, P. and Rughooputh, S. 2004. Bioinformatics: a useful tool for the molecular microbiologist? Biomedical Scientist. 48 (5), pp. 494-495.

Detection of human papillomavirus from liquid-based cytology specimens by in-house PCR: a pilot study
Rughooputh, S., Parmar, K. and Greenwell, P. 2004. Detection of human papillomavirus from liquid-based cytology specimens by in-house PCR: a pilot study. British Journal of Biomedical Science. 61 (1), pp. 22-25.

Use of bioinformatics and PCR in the search for ABC transporter homology among various bacteria
Jauhangeer, B.R., Wren, M.W.D., MacDonald, R.A.C., Perry, D. and Greenwell, P. 2004. Use of bioinformatics and PCR in the search for ABC transporter homology among various bacteria. British Journal of Biomedical Science. 61 (4), pp. 182-185.

Trichomonas vaginalis: an irritating protozoan or an important viral co-factor
Greenwell, P. and Rughooputh, S. 2004. Trichomonas vaginalis: an irritating protozoan or an important viral co-factor. Biomedical Scientist. 48 (1), pp. 40-41.

Human papillomavirus: an overview
Rughooputh, S. and Greenwell, P. 2003. Human papillomavirus: an overview. Biomedical Scientist. 47, pp. 1070-1071.

Isolation of Neisseria gonorrhoeae-specific antibodies from a phage display library
Rughooputh, S. and Greenwell, P. 2003. Isolation of Neisseria gonorrhoeae-specific antibodies from a phage display library. Molecular and Cellular Proteomics. 2 (9), p. 758.

Bioinformatics in the identification of glycosidase genes in the Chlamydia genome
Greenwell, P. and Rughooputh, S. 2003. Bioinformatics in the identification of glycosidase genes in the Chlamydia genome. Molecular and Cellular Proteomics. 2 (9), p. 826.

Phage display antibody against gill proteins of the tropical clam
Gourdine, J.P., Smith-Ravin, E.J. and Greenwell, P. 2003. Phage display antibody against gill proteins of the tropical clam. Molecular and Cellular Proteomics. 2 (9), p. 759.

Incidence and prognostic significance of C-MPL expression in acute myeloid leukemia
Chelvatheebam, S., Langabeer, S., Hills, R., Greenwell, P. and Linch, D. 2003. Incidence and prognostic significance of C-MPL expression in acute myeloid leukemia. Leukemia Research. 27 (9), pp. 839-870.

Absence of regulatory volume increase (RVI) in isolated bovine articular chondrocytes
Kerrigan, M.J.P., Matskevitch, I., Flatman, P.W. and Hall, A.C. 2002. Absence of regulatory volume increase (RVI) in isolated bovine articular chondrocytes. European Journal of Physiology. 443, pp. S288-S289.

Differential mechanisms of intracellular pH (pHi) recovery following acid load to in situ bovine articular chondrocytes within different cartilage zones
Kerrigan, M.J.P., Hall, A.C. and O'Neill, J.R. 2002. Differential mechanisms of intracellular pH (pHi) recovery following acid load to in situ bovine articular chondrocytes within different cartilage zones. Journal of Physiology. 544P, p. 108P.

The role of the actin cytoskeleton in regulatory volume decrease (RVD) in bovine articular chondrocytes
Kerrigan, M.J.P. and Hall, A.C. 2002. The role of the actin cytoskeleton in regulatory volume decrease (RVD) in bovine articular chondrocytes. Journal of Physiology. 544P, p. S123.

Regulatory volume decrease (RVD) by articular chondrocytes isolated from normal and degenerate human articular cartilage
Hall, A.C., Kerrigan, M.J.P., Bush, P.G. and Dunlop, D. 2002. Regulatory volume decrease (RVD) by articular chondrocytes isolated from normal and degenerate human articular cartilage. European Journal of Physiology. 443, p. S236.

Beyond hybridoma technology: production of monoclonal antibodies by phage display
Greenwell, P. and Rughooputh, S. 2001. Beyond hybridoma technology: production of monoclonal antibodies by phage display. Biomedical Scientist. 45, pp. 574-575.

Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization
Mobasheri, A., Oukrif, D., Dawodu, S.P., Sinha, M., Greenwell, P., Stewart, D., Djamgoz, M.B.A., Foster, C.S., Martin-Vasallo, P. and Mobasheri, R. 2001. Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization. Histology and Histopathology. 16 (1), pp. 141-154.

Regulatory volume decrease (RVD) by avian articular chondrocytes following hypotonic challenge
Kerrigan, M.J.P., Edmonston, J.L. and Hall, A.C. 2001. Regulatory volume decrease (RVD) by avian articular chondrocytes following hypotonic challenge. Journal of Physiology. 535P, p. S015.

The role of [Ca2+](i) in mediating regulatory volume decrease in isolated bovine articular chondrocytes
Kerrigan, M.J.P. and Hall, A.C. 2000. The role of [Ca2+](i) in mediating regulatory volume decrease in isolated bovine articular chondrocytes. Journal of Physiology. 527P, pp. S42-S43.

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