ENDOTHELIN-MEDIATED CELL SIGNALING AND PROLIFERATION IN CULTURED RABBIT CORNEAL EPITHELIAL-CELLS

Purpose. To determine if there is endothelin-mediated regulation of ce ll signaling and proliferation in rabbit corneal epithelium. Methods. Endothelin-1 (ET-1) gene and protein expression by the rabbit corneal epithelial (RCE) cells were analyzed by polymerase chain reaction, seq uence analysis, and enzyme immunoassay. DNA synthesis was characterize d by [H-3]-thymidine uptake. Endothelin receptor linkage to cell signa ling pathways was determined based on measurements of the dose depende nt effects of ET-1, ET-2, and ET-3 on intracellular Ca2+ concentration ([Ca2+](i)) transients in fura-2-loaded cells, and of ET-1 on phospho inositide turnover and cAMP accumulation in the isolated rabbit cornea l epithelium. Results. The authors detected the mRNA for prepro ET-1 i n RCE cells, and ET-like immunoreactivity was identified in conditione d culture medium. ET-1 (1 nM) maximally stimulated [H-3]thymidine upta ke by twofold (EC(50) = 0.3 nM). Endothelins elicited transient increa ses in [Ca2+](i) with a rank order of potency of ET-1 greater than or equal to ET-2 much greater than ET-3. These increases consisted of bot h intracellular Ca2+ mobilization and influx of Ca2+ from the bathing solution. Intracellular mobilization was linked to increases in IP3 tu rnover because 1 mu M ET-1 increased IP3 content by 48% from its contr ol value (EC(50) = 23 nM), whereas Ca2+ influx occurred through a non- L-type Ca2+ channel because preexposure to 1 mu M nicardipine did not affect either the height or the duration of a [Ca2+](i) transient. One micromolar of ET-1 was required to elicit a significant increase in c AMP accumulation of 69% from its control value. This increase was depe ndent on the presence of Ca2+ in the bathing solution and was comparab le to and nonadditive with that of the Ca2+ ionophore, A23187 (1 mu M) . Conclusion. These data suggest that endothelin production by primary cultures of RCE cells can mediate an increase in cell proliferation t hrough an ET(A) receptor subtype. This receptor subtype appears to be involved based on the rank order of potency of ETs to elicit [Ca2+](i) transients, increases in phosphoinositide turnover, and cAMP accumula tion.