Abstract
Abstract: :
Purpose. To determine if lysophosphatidic acid (LPA) or sphingosine–1–phosphate (S1P), two members of the phospholipid growth factor (PLGF) family, affect the tight junctional resistance across cultured rabbit corneal epithelial and/or endothelial cells. Methods. Electric cell–substrate impedance sensing (ECIS) was used to measure electrical resistance across cultured rabbit corneal endothelial and epithelial monolayers. In the ECIS system, gold film electrodes are set into the bottom of 8 well tissue culture dishes, and an AC current (usually at 40 KHz) is applied between 10 small active electrodes and a large counter electrode using tissue culture medium as the electrolyte. A computer controls the application of the AC current and records the voltage across the small electrode and converts this to an impedance value. When cells attach and spread upon the electrode, the impedance increases as the insulating cell membranes force current into the narrow spaces beneath the cell and through the intercellular junctions. Because the instrument also follows the phase of the voltage, the impedance can be broken down into resistance and capacitance, treating the system as a series RC circuit. A one hour equilibration time was allowed for all cultures, and then different concentrations of LPA or S1P were added to each well (along with a BSA control), which were followed for an additional four hours, with data points taken every 20 min. For cells significantly affected by LPA or S1P, pertussis toxin (PTX) was added to the media in separate experiments to determine if Gαi/o are involved in the response. Results. LPA significantly increased the resistance of both the epithelial and endothelial cells, while S1P increased the resistance in only the endothelial cells. Higher concentrations of S1P appeared to be toxic to the epithelial cells. PTX blocked both the LPA– and S1P–induced resistance increases. Conclusions. We conclude that PLGFs significantly increase the transcellular resistance in both epithelial (LPA) and endothelial cells (LPA and S1P), and that this effect is at least in part signaled through Gαi/o.
Keywords: cornea: endothelium • cornea: epithelium • cell–cell communication