H. Takagi et al., ENDOTHELIN-MEDIATED CELL SIGNALING AND PROLIFERATION IN CULTURED RABBIT CORNEAL EPITHELIAL-CELLS, Investigative ophthalmology & visual science, 35(1), 1994, pp. 134-142
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.