Abstract
Abstract: :
Purpose: Retinitis pigmentosa refers to a group of degenerative retinal diseases that are often associated with mutations in genes encoding rod specific proteins. Although the mutations are expressed solely in rods, their death is almost invariably followed by a progressive loss of cones, leading to eventual blindness. We have postulated that dying rods transmit toxic substances to neighboring cones via the gap junctions that link rods and cones in all vertebrate species. The present study was designed to test whether gap junctions are able to mediate this form of bystander cell death in a cell line expressing a well characterized gap–junctional protein. Methods: BHK cells were stably transfected with connexin32, a protein that is known to form gap junctions in various tissues and expression systems. The presence of connexin32 in transfected BHK cells was established by immunocytochemistry, western blot analysis and RT–PCR, and cell coupling was confirmed by intercellular transfer of Lucifer yellow. Bystander killing was induced by scrape loading confluent cells with cytochrome C (cyC), a mitochondria–derived apoptotic agent. A region of the monolayer was cut and incubated with 1 mM cyC for 2 minutes at room temperature and thereafter with the growth medium for 1 hour at 37ºC. The spread of cell death was investigated by TUNEL staining, annexin V assay, and immunostaining for activated caspases; the different markers of apoptosis were detected by fluorescence microscopy. Results: Stably transfected BHK cells expressed very high levels of connexin32, as observed by RT–PCR, western blot analysis and immunocytochemistry; no Cx32 expression was seen in non–transfected cells. In the in vitro dye transfer assay, Lucifer yellow was transferred to Cx32–transfected BHK cells located several cell layers away from the scrape, and when scrape loaded with cyC, these cells exhibited fluorescent signals associated with the various assays for apoptosis. In contrast, BHK cells not transfected with connexin32 and scrape loaded with cyC did not show signs of apoptosis away from the cut. Conclusions: Our results indicate that the cyC–induced apoptotic signal is communicated via gap junctions in cells expressing Cx32. However, because cyC (MW = 12,327) is too large to traverse the ∼16 nm pores of gap–junctional channels, the death signal must be conveyed by a smaller molecule (molecular mass ≤ 1 Kd) that is produced as a result of exposure to cyC. Experiments in progress are aimed at determining whether the gap junctions that link rods and cones in the mammalian retina can transmit agents that induce "bystander" cell death.
Keywords: gap junctions/coupling • apoptosis/cell death • retinal degenerations: cell biology