December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Volume Regulated Anion Channels (VRAC) In Cultured Rabbit Corneal Keratocytes
Author Affiliations & Notes
  • H Liu
    School of Optometry
    Indiana University Bloomington IN
  • SP Srinivas
    Indiana University Bloomington IN
  • M Satpathy
    Indiana University Bloomington IN
  • R Mitra
    Indiana University Bloomington IN
  • MA Watsky
    Physiology University of Tennessee Health Science Center Memphis TN
  • Footnotes
    Commercial Relationships   H. Liu, None; S.P. Srinivas, None; M. Satpathy, None; R. Mitra, None; M.A. Watsky, None. Grant Identification: Support: EY11107 (SPS) and EY12821 (MW)
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1699. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      H Liu, SP Srinivas, M Satpathy, R Mitra, MA Watsky; Volume Regulated Anion Channels (VRAC) In Cultured Rabbit Corneal Keratocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1699.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

  • Supplements

Abstract: : Purpose: Blockers of VRAC inhibit proliferation of vascular endothelial cells and also prevent angiogenesis (Nilius et al., Gen Pharmacol, 27: 67-77, 1996). In this study, we have characterized VRAC in corneal keratocytes to facilitate identification of agents that might inhibit excessive stromal cell proliferation and potentially stromal/haze and/or scar formation during the corneal wound healing response. Methods: Cultured keratocytes were grown to confluence on glass coverslips. Dynamic fluorescence quenching (DFQ) (Srinivas and Bonanno. Am J Physiol. 272:C1405-14, 1997) was employed to measure changes in cell volume using the halide-sensitive fluorescent dye MQAE under Cl--free conditions; Cl- was replaced by NO3-. The same dye was employed to assess the relative permeability of I- and NO3- quantitatively under isosmotic and hyposmotic conditions (Srinivas et al., Biophys J. 75(1):115-23,1998). All experiments were conducted at 37°C under HCO3--free conditions. RT-PCR was employed to determine expression of CLC homologues, candidates for VRAC activity. Results: Hyposmotic and hyperosmotic shocks caused increase and decrease in MQAE fluorescence, respectively (n=8). Exposure to Gramicidin (1-2 µM) known to form Na+ channels, under hyposmotic conditions caused an increase in MQAE fluorescence indicating secondary cell swelling (n=4). RT-PCR results indicated positive bands for CLC-3 and CLC-5, but expression of CLC-2 and CFTR could not be detected. Under hyposmotic conditions (200-220 mosM), both inward and outward permeability of I- were significantly enhanced (≷ 200%; n = 4) relative to isosmotic conditions. Conclusion:(1). Rapid changes in cell volume of keratocytes can be measured using the DFQ method. (2). GC induced secondary swelling is consistent with Na+-NO3- influx; the cation influx being mediated by GC conductance for Na+ and that of the anion by VRAC activity, presumably CLC-3 which has been suggested to possess VRAC characteristics. (3) Increased I- permeability is consistent with selectivity of VRAC for their relatively higher permeability to I-.

Keywords: 374 cornea: stroma and keratocytes • 445 ion channels • 631 wound healing 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.