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M.A. Watsky, J. Wang; G-Protein Involvement in the Phospholipid Growth Factor-Mediated Activation of a Cl- Current in Corneal Keratocytes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3813.
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Purpose: Wound-activated rabbit keratocytes contain a Cl- channel (IClLPA) that is activated by lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) via a receptor-mediated pathway. We have recently shown that cultured αSMA positive rabbit keratocytes (myofibroblasts) can serve as a useful model to study this Cl- current. The purpose of this study was to use this model to determine if Gαi and/or Gα12 are involved in the activation of IClLPA by LPA and/or S1P. Methods: Rabbit keratocytes were cultured at low density in the presence of serum. IClLPA was examined using either the traditional or perforated-patch whole cell patch clamp configurations. Involvement of Gαi in the activation of IClLPA was examined in two ways: By culturing cells in the presence of pertussis toxin (PTX; 100 ng/ml) and measuring Gαi current density in these cells after treating them acutely with LPA or S1P; or by adding activated Gαi into the cells via whole cell electrodes and looking for spontaneous Gαi activity. The influence of Gα12 was examined by co-transfecting myofibroblasts with GFP and a constitutively active Gα12 mutant and measuring IClLPA current density after addition of LPA or S1P. Results: PTX inhibited activation of IClLPA by LPA, but not by S1P. Addition of activated Gαi to the cells resulted in significant spontaneous activation of IClLPA. Gα12 transfection of the cells did not result in spontaneous IClLPA activity. However, when cells were exposed to low concentrations of LPA or S1P, GFP/Gα12 co-transfected cells had higher current densities than their GFP transfected control counterparts. Conclusions: We conclude that LPA-mediated activation of IClLPA, but not S1P-mediated activation, is working through Gαi. We also conclude that Gα12 is not directly linked to IClLPA activation by LPA and S1P, but that it might play a modulatory role.
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