The impact of high-dose acetylcholine on bovine corneal epithelium.

PURPOSE

Ion channels are formed when acetylcholine (ACh) combines with specific receptors on the postsynaptic membrane. We are testing whether this occurs in vitro in corneal epithelial cells when exposed to ACh, using intracellular ascorbic acid (AA) as a tracer.

METHODS

The corneal epithelium was separated from Bowman's membrane as an intact sheet with thermolysin (TL). These cells were incubated in a medium containing phospholine iodide (Phi) to block acetylcholinesterase (AChE) activity, and then, five different groups of specimens were examined: the basic level of ACh and AA in the cells was tested in groups I and II, respectively. In groups III-V, the culture medium was supplemented with AA, ACh and ACh+AA, respectively, and following 2 hr of incubation the cells were tested for AA. AA was determined by high-performance liquid chromatography (HPLC) with UV detection, and ACh by LC-MS/MS.

RESULTS

The ACh concentration in the corneal epithelial cells was 23.7 ng/mg wet weight. AA values were as follows: preexposure 0.17 mg/g. After exposure to AA, ACh and ACh+AA, the values were 0.30, 0.12 and 0.21 mg/g, respectively. The AA concentration mechanism of the corneal epithelium was intact despite exposure to Phi and ACh.

CONCLUSION

The main observation is that the AA content of the corneal epithelium drops in response to ACh exposure. Various explanations are discussed, in particular the possibility that ACh exposure may cause cell membrane leakage through pores (nicotinic receptors). This would be similar to the moderate membrane leakage (depolarization) in neural synapses in response to ACh release. Interesting in vivo consequences may occur when considering that AChE is extremely UV sensitive, as this substance could be a target at which actinic radiation may influence the ACh-AChE complex in this epithelium.