Effect of increasing glucose concentrations and protein phosphorylation on intercellular communication in cultured rat retinal pigment epithelial cells.

PURPOSE

The intercellular communication between cultured rat retinal pigment epithelial (RPE) cells grown in increasing glucose concentrations or after modulation of the protein kinase C-induced protein phosphorylation was investigated by studying the conduction of the [Ca2+]i wave elicited by mechanical stimulation and by analyzing the fluorescence recovery after photobleaching (FRAP).

METHODS

Subconfluent monolayers of RPE cells isolated from neonatal Long Evans rats were cultured in growth medium with various glucose levels and analyzed using the fluorescent dye fluo-3 for measurements of intracellular Ca2+ after mechanical stimulation and using 6-carboxyfluorescein diacetate to investigate the intercellular communication with FRAP.

RESULTS

Mechanical stimulation in 5 or 12 mM glucose resulted in a Ca2+ wave that spread centrifugally through the neighboring cells. An inhibition of the propagation of this wave, similar to that induced by halothane, could be observed in cells grown for 72 hours in 14-mM or higher concentrations of glucose. This inhibitory effect was not caused by a hyperosmotic effect, in that results of experiments on cells cultured in growth medium supplemented with mannitol instead of glucose did not differ from those of experiments in the control medium. Activation of protein kinase C by incubation of the cells for 30 minutes with phorbol 12-myristate 13-acetate (PMA) resulted in a strong inhibition of [Ca2+]i-wave propagation. This inhibition did not depend on the oxidizing effects of PMA because the addition of glaucine, a known antioxidant, did not prevent the inhibition. Cells grown for 72 hours in glucose-rich medium (25 or 50 mM) and in which all protein kinase C activity was downregulated by a previous 72-hour exposure to 1 microM PMA, did not display the inhibitory effect on the propagation of the Ca2+ wave that is normally induced by this elevated glucose level. Stimulation or inhibition of protein kinase A activity by incubating RPE cells with Sp-cyclic adenosine monophosphate or Rp-cyclic adenosine monophosphate respectively, or inhibition of tyrosine kinase activity with herbimycin A did not alter the intercellular communication after mechanical stimulation. To determine whether the observed changes were caused by alterations in gap junction conductance (GJC), FRAP experiments were performed in control conditions, after a 30-minute incubation with PMA, and in cells cultured in 50 mM glucose in the presence and in the absence of 1 microM PMA. The measured GJC was consistent with the inhibitory effect on propagation of an intercellular Ca2+ wave in all tested conditions.

CONCLUSIONS

In RPE cells, a glucose concentration of 14 mM (224 mg/dl) or higher inhibits Ca(2+)-wave propagation and intercellular GJC. This effect may be mediated by protein kinase C activity.