AGONIST-INDUCED CA2+ MOBILIZATION IN CULTURED BOVINE AND HUMAN CORNEAL ENDOTHELIAL-CELLS

We investigated the possibility that cultured corneal endothelial cells express receptors that are coupled to the phosphoinositide cycle/intracellular Ca2+ signaling pathway. Agonist-stimulated changes in intracellular calcium ([Ca2+]i) in single bovine and human corneal endothelial cells (BCEC and HCEC, respectively) derived from confluent cultures were measured by microspectrofluorimetry using the Ca2+-sensitive probe, fura-2. Total inositol phosphates accumulated during a 30 min incubation in the presence or absence of agonists was determined in Li+ containing medium with cells pre-labelled for 48 hrs with 10 muCi/ml H-3-Myo inositol. Histamine (HA), ADP and ATP induced a rapid increase in [Ca2+]i. Subsequently, [Ca2+)i decreased to either a stable, agonist-dependent sustained elevation, or fell back to baseline to begin oscillatory fluctuations. The initial rise in (Ca2+)i was insensitive to removal of extracellular calcium (Ca2+)o whereas the stable elevations in [Ca2+]i and the [Ca2+]i oscillations required Ca2+o. In contrast, bradykinin (BK) and endothelin-I (ET-1) elicited an initial rise in [Ca2+]i that returned to prestimulatory levels within 2 min despite the continued presence of agonist. The Ca2+-mobilizing agonists carbachol, phenylephrine, adenosine and substance P were all ineffective in elevating [Ca2+]i. Histamine-induced Ca2+ mobilization was inhibited by the H-1-receptor antagonist triprolidine, but triprolidine had no effect on either BK or ATP stimulation of Ca2+ mobilization. In BCEC, 100 muM HA significantly increased total inositol phosphate accumulation (18.8-fold over unstimulated controls) and was 90% inhibited by 0.5 muM triprolidine. BK and ATP also significantly increased formation of inositol phosphates in BCEC. These results indicate that the signal transduction pathway involving PI turnover and Ca2+ mobilization is expressed in BCEC and HCEC cultures and is activated by HA, BK, ATP, ADP and ET-1.