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

We investigated the possibility that cultured corneal endothelial cell s express receptors that are coupled to the phosphoinositide cycle/int racellular Ca2+ signaling pathway. Agonist-stimulated changes in intra cellular calcium ([Ca2+]i) in single bovine and human corneal endothel ial cells (BCEC and HCEC, respectively) derived from confluent culture s were measured by microspectrofluorimetry using the Ca2+-sensitive pr obe, fura-2. Total inositol phosphates accumulated during a 30 min inc ubation in the presence or absence of agonists was determined in Li+ c ontaining 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 i n [Ca2+]i. Subsequently, [Ca2+)i decreased to either a stable, agonist -dependent sustained elevation, or fell back to baseline to begin osci llatory fluctuations. The initial rise in (Ca2+)i was insensitive to r emoval of extracellular calcium (Ca2+)o whereas the stable elevations in [Ca2+]i and the [Ca2+]i oscillations required Ca2+o. In contrast, b radykinin (BK) and endothelin-I (ET-1) elicited an initial rise in [Ca 2+]i that returned to prestimulatory levels within 2 min despite the c ontinued presence of agonist. The Ca2+-mobilizing agonists carbachol, phenylephrine, adenosine and substance P were all ineffective in eleva ting [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 tr iprolidine. BK and ATP also significantly increased formation of inosi tol phosphates in BCEC. These results indicate that the signal transdu ction 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.