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Evidence for the
Mechanism of Reduction of Retinal to Retinol in Intact Salamander Rods
Cornwall, Carter1,
Tsina, Efthymia1, Koutalos, Yiannis2,
Chen, C.2 and Crouch, Rosalie3
Boston University School of Medicine1
University of Colorado School of Medicine2
Medical University of South Carolina3
Abstract-
Our purpose was to examine the origin and pattern of fluorescence in
the rod outer segment arising following the bleaching of an intact rod
and to provide evidence that it is due to accumulation of all-trans
retinol. Rods were isolated from dark- and light-adapted retinae of
salamanders and maintained in Ringer's solution in a chamber on
the stage of a microscope equipped with a high sensitivity digital CCD
camera. The spatial distribution of fluorescence (excitation: 360 nm;
emission: 540 nm) was measured before and at various times after either
treatment with exogenous agents or a bright slit of light focused on
the outer segment (520 nm), calculated to bleach >99% of the visual
pigment in that region. We found that following the bleaching light,
a persistent fluorescence was observed in rod outer segments. When this
bleaching light was focused on the distal tip, fluorescence was restricted
to this region, extending only to a minor extent into unbleached regions.
Treatment of uniformly bleached cells for 30 min. with lipid vesicles
(0.33 mg/ml) reduced fluorescence by 40%; subsequent treatment with
1% bovine serum albumin further reduced fluorescence intensity by an
additional 25%. Administration of exogenous 100  M
all-trans retinal resulted in an approximate 22-fold increase
in total outer segment fluorescence after 1 hour. In intact cells, fluorescence
intensity was greatest at the base and least at the tip of the outer
segment. In broken outer segments, the increase was only modest, and
no significant gradient was observed. Treatment of intact cells with
all-trans retinol (100 M)
also resulted in an increase in fluorescence in the outer segment. However,
no gradient of intensity was observed. Exogenous treatment with the
lipophilic aldehyde decanal resulted only in a modest decrease of fluorescence.
We conclude that the fluorescence increase is attributed to the appearance
of all-trans retinol in rods. This fluorescence declines only
following treatment with agents expected to facilitate removal of lipophilic
substances from the cell. The gradient of fluorescence, greatest at
the base and least at the tip of the outer segment, suggests that the
ellipsoid region containing mitochondria may be the source of the reducing
equivalents that fuel this reduction.
Keywords: retinol,
rods , retina
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