Abstract
Abstract: :
Purpose: In another report we showed that VIP prepared the corneal endothelial (CE) cell to enter apoptosis, at the expense of the necrotic death pathway during subsequent lethal oxidative stress exposure. Since poly(ADP–ribose) polymerase (PARP)–1, through its consumption of intracellular ATP, is a molecular switch of the two cell death pathways, we studied PARP–1 and ATP levels in VIP pretreated and hydrogen peroxide injured CE cells in cornea cultures. Methods: Bovine corneoscleral explants were conditioned in Eagle's MEM/ 20 mM HEPES (1h, 37oC), transferred and incubated in medium containing VIP (0, 10–14–10–6 M, 15 min, 37oC), and then treated with 1.4 mM H2O2/PBS (15 min, 37 o C). CE cells were then scraped off cornea cups and homogenized in lysis buffers for Western blot analysis using antibodies against PARP–1 (Ab–2, Calbiochem) and actin (Ab–1, Calbiochem), and ATP level determination using a luciferase/luciferin assay kit (Molecular Probes). Results:VIP pretreatment of bovine corneoscleral explants resulted in a decreased level of PARP–1 in CE cells during the subsequent H2O2 treatment in a VIP concentration–dependent manner. While the effect was observed at VIP concentration as low as 10–12M, the maximal effect was observed at 1x 10–8 M VIP. The effect of VIP was antagonized by a VIP receptor antagonist, (SN)VIPhyb, in a concentration–dependent manner. On the other hand, Western blot analysis showed that VIP pretreatment preserved the actin levels in CE cells subjected to subsequent H2O2 treatment in a VIP concentration–dependent manner. VIP concentration–dependently helped the CE cells maintain their ATP levels during the subsequent oxidative stress; from ∼1 nmole/g protein to ∼4 nmole/g protein in the control and 10–6 M VIP– pretreated explants, respectively. Conclusions: By preserving ATP levels, through degradation of PARP–1 and prevention of actin degradation during subsequent lethal oxidative stress, VIP pretreatment of the corneoscleral explants can influence CE cells in their choice of the death pathways, in favor of the apoptosis. Since necrotic cell death, but not apoptosis, causes damage to the neighbors of dying cells and is associated with initiation of an inflammatory response, VIP, which is an autocrine of the CE cells and present in the aqueous humor, may play a role in the promotion of CE cell survival under oxidative stress.
Keywords: cell death/apoptosis • neuroprotection • cornea: endothelium