Free Magnesium Concentration In Vertebrate Photoreceptor Outer Segments

Abstract

Abstract: : Purpose: To measure the free Mg₂₊ concentration in the outer segments of vertebrate rod and cone photoreceptors and examine the different mechanisms involved in Mg₂₊ homeostasis. Methods: Living, intact rod photoreceptors were isolated from larval tiger salamander (Ambystoma tigrinum) retinas. Isolated cells were loaded with the Mg₂₊-sensitive probe Furaptra by incubating with the acetoxymethyl ester form of the dye. Cells were placed in a chamber on a microscope stage and changes in fluorescence were measured with a digital CCD camera. The ratio F₃₄₀/F₃₈₀ of dye fluorescence excited at 340 and 380 nm (emission: 540 nm) is a measure of the free Mg₂₊ concentration. Fluorescence ratios were converted to free Mg₂₊ concentrations after calibrating intracellular dye with different Mg₂₊ concentrations. Results: Resting free Mg₂₊ concentrations in bleached rod and cone cell outer segments were 0.86±0.06 mM and 0.81±0.09 mM, respectively. The outer segment free Mg₂₊ concentration was not significantly affected by changes in extracellular pH, Ca₂₊ and Na₊, excluding a significant role for the respective exchangers in the regulation of Mg₂₊ homeostasis. The resting free Mg₂₊ concentration was also not significantly affected by exposure to 0 Mg₂₊, suggesting the lack of a significant Mg₂₊ influx. In cone cells, inhibition of the phosphodiesterase with 1 mM IBMX led to a significant increase in the Mg₂₊ concentration in the absence of Na₊ and Ca₂₊, but not in their presence, suggesting that significant Mg₂₊ influx through the cGMP-gated channels may occur only in the absence of other permeant ions, but not under physiological conditions. Conclusion: The free Mg₂₊ concentration is the same in bleached rod and cone outer segments. Mg₂₊ is known to regulate enzymes involved in phototransduction, like the guanylate cyclase, but it appears that there are no influx and efflux pathways that can significantly affect the Mg₂₊ outer segment concentration. Therefore, it is unlikely that Mg₂₊ plays a significant role in the dynamic modulation of phototransduction. Support: NIH grant EY11351.

Keywords: 517 photoreceptors • 431 imaging/image analysis: non-clinical