From the * Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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The retinal analogue -ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors
were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a
steady state, and then were exposed to
-ionone. Our experiments show that in bleach-adapted rods
-ionone
causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the
rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in
bleach-adapted rods
-ionone activates phototransduction in the dark. Control experiments showed no effect of
-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of
-ionone
with the free opsin produced by bleaching. We speculate that
-ionone binds specifically in the chromophore
pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that
a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a
decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin
and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin.
We show that binding of
-ionone to rod opsin produces effects opposite to its previously described effects on
cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone
opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors.
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INTRODUCTION |
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The visual cycle is the series of reactions occurring in
the eye through which visual pigment proceeds from
photoactivation to regeneration. Absorption of a photon by the pigment of a photoreceptor triggers within
milliseconds a series of reactions leading to a cell response. This sequence of reactions, collectively termed the phototransduction cascade, has been extensively
studied and is now well characterized (for reviews, see
Lagnado and Baylor, 1992; Stryer, 1992
; Pugh and
Lamb, 1993
; Yau, 1994
). According to this scheme,
photoisomerization of the chromophore from the 11-cis to the all-trans conformation (see Fig. 1, A and B)
ultimately results in the closure of cation channels and
the hyperpolarization of the cell. Another consequence
of the photon absorption is the eventual dissociation of
the chromophore from the protein moiety of the visual
pigment to form all-trans retinol and free opsin. The visual pigment is then regenerated through a series of reactions requiring less than a minute in cones and tens
of minutes in rods (Wald et al., 1955
). In the vertebrate
retina, this is a complex process that involves both photoreceptors and the pigment epithelium (for reviews,
see Rando, 1992
; Crouch et al., 1996
). Reduction of all-trans retinal to all-trans retinol must occur in the
bleached outer segment of photoreceptors before the
opsin binding site becomes accessible for regeneration.
All-trans retinol is then removed from the outer segment and translocated to the pigment epithelium.
There it is converted back to 11-cis retinal, and then returned to the receptor outer segment where it combines with opsin to form visual pigment.
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Aside from the ultimate recovery of visual sensitivity
(Pepperberg et al., 1978; Jones et al., 1989
; Cornwall et
al., 1990), very little is known about the physiological
consequences of the reactions involved in the reconstitution of the visual pigment within photoreceptors. An
important initial step in the recovery process is likely to
be the binding of 11-cis retinal, supplied from the pigment epithelium, into the chromophore pocket of opsin (Matsumoto and Yoshizawa, 1975
). The effects on
phototransduction and regeneration of a whole host of
retinal analogues have been studied with biochemical
as well as with physiological methods (for reviews, see
Corson and Crouch, 1996
; Nakanishi and Crouch, 1996
). Treating bleached pigment from rods with derivatives of retinal having shortened polyene chains
produces a catalytically active complex that promotes
phosphorylation of opsin by rhodopsin kinase (Buczylko et al., 1996). Competition studies with 11-cis retinal suggest that these compounds exert their effect by
binding in the chromophore pocket of opsin (Matsumoto and Yoshizawa, 1975
; Crouch et al., 1982
). These
retinoids, including
-ionone (Fig. 1 C), all-trans C17
aldehyde, and 9-cis C17 aldehyde do not form a covalent link with opsin via a Schiff base (Towner et al.,
1981
) because the shorter length of their side chain apparently does not allow them to span from the ring-binding site to the corresponding lysine of opsin (Daemen, 1978
; Crouch et al., 1982
). The fact that these
compounds do not form a covalent bond with the protein suggests that occupancy of the chromophore binding site of rod opsin alone is sufficient for catalytic activation of the complex. Thus, these biochemical experiments indicate that, in rods, retinoids with shortened
polyene chains act as agonists, activating the photoreceptor. On the other hand, physiological studies on intact isolated cone photoreceptor cells (Jin et al., 1993
;
Cornwall et al., 1995
) show that in these cells short-chain retinal analogues produce an opposite effect:
treatment of bleached cones with
-ionone or with 9-cis
C17 aldehyde results in downregulation of transduction, characterized by slowed flash responses, increased
sensitivity, and increased dark current. Thus, in cones,
short-chain retinal analogues act as reverse agonists, inactivating the photoreceptor. These latter studies were
interpreted as demonstrating that the noncovalent
binding of retinal in the chromophore pocket of cone opsin might be an important initial step in dark adaptation.
The present study was undertaken to resolve the contradictory findings regarding the role retinoids play in
regulating the activity of rod and cone photoreceptors.
The experiments described here also address the question of whether the occupancy of the chromophore
pocket of rod opsin by a retinoid produces physiological consequences that are separate from those due to
the consequent formation of the Schiff base linkage.
The retinal analogue chosen to address these questions
was -ionone because of its capability of binding in the
chromophore pocket of opsin without forming a covalent bond. Furthermore, as we demonstrate here, its
binding is rapidly and totally reversible. The use of
-ionone enabled us to study specifically the physiological
effect of the noncovalent binding of retinal in the
chromophore pocket of opsin without the interfering
effects of the covalent bond between retinal and opsin.
In addition, treating bleach-adapted rods with
-ionone allowed us to compare directly the effect of its
binding in the chromophore pocket of rod opsin with
its previously described effect in cones (Jin et al., 1993
;
Cornwall et al., 1995
). We present evidence that, in
bleach-adapted rods, occupancy of the chromophore
binding site of opsin by
-ionone causes activation of
the transduction cascade and desensitization of the
cell. This observation is opposite to the expected downregulation of transduction during dark adaptation,
when 11-cis retinal occupies the chromophore pocket.
It is also opposite to the effect of
-ionone observed in bleach-adapted cone photoreceptors. This newly discovered distinction between rods and cones reconciles
the conflict between biochemical and physiological results, and implies a fundamental difference in the interaction of rod and cone opsins with retinal.
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MATERIALS AND METHODS |
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Single rod photoreceptors from the retina of the larval tiger salamander (Ambystoma tigrinum) were isolated in physiological solution as previously described (Cornwall et al., 1990). In brief, animals were dark-adapted overnight. They were decapitated and pithed in dim red light, following which the eyes were removed, hemisected, and placed in ~1.5 ml saline solution. The retina was torn free of the pigment epithelium and chopped into small pieces with a razor blade. A fraction of the resulting suspension was transferred to a recording chamber located on the stage of an inverted microscope (Invertascope D; Carl Zeiss, Inc.). The remaining portion of the suspension was stored at 4°C in the
dark and remained viable for several hours. The preparation was
viewed via an infrared television camera (WV 5410; Panasonic)
fitted to the inverted microscope and connected to a video monitor. Membrane currents were recorded from solitary rods with a
suction microelectrode using methods similar to those reported
previously (Baylor et al., 1979
; Cornwall et al., 1990
). The inner
segment of the rod was drawn into the pipette so that the outer
segment could be rapidly superfused with test solutions. The tip
of the electrode was heat-polished to a diameter of ~10 µm. The
current recorded from the cell was converted to voltage and amplified using a patch clamp amplifier (EPC-7; List Electronic),
and low-pass filtered with an active eight-pole filter at 20 Hz cutoff frequency (902LPF; Frequency Devices Inc.). The data were
digitized at 250 Hz, stored on a computer, and subsequently analyzed using the pCLAMP 6 data acquisition and analysis software
(Axon Instruments) and the Origin 4 graphics and data analysis
software (Microcal Software, Inc.).
Light Stimulation
A dual-beam optical stimulator provided test flashes as well as
bleaching and background lights (Cornwall et al., 1990). The light source was calibrated at the beginning of each experiment with a photometer (80×; United Detector Technology). The absolute intensity of the test flash/bleaching beam was 1.36 × 107
photons µm
2 s
1 (500 nm); the absolute intensity for the background beam was 1.28 × 107 photons µm
2 s
1 (520 nm). Light
intensity for each beam was attenuated with a series of calibrated
neutral density filters. The wavelength was set with narrow band
interference filters (10 nm bandwidth at 1/2 transmission; Corion Optics). A stimulus spot 1 mm in diameter was focused at
the plane of the preparation by a 0.25 NA × 10 achromat objective located above the stage of the inverted microscope. Test
flash duration was 20 ms. The fraction of bleached pigment was
calculated according to the relation:
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(1) |
where F is the fraction of bleached pigment, I is the light intensity in photons µm2 s
1, and t is the duration of light exposure
in seconds. The value used for the photosensitivity of the cell P
was 6.2 × 10
9 µm2 (Jones, 1995
).
Solutions
The cell was perfused with saline solution that contained 110 mM NaCl, 2.5 mM KCl, 1.6 mM MgCl2, 1.0 mM CaCl2, 10 mM
dextrose, 10 mM HEPES, pH 7.8, and bovine serum albumin
(100 mg/liter1). The solution in which the guanylyl cyclase activity was measured was made by adding 3-isobutyl-1-methylxanthine (IBMX)1 to normal saline solution to a final concentration
of 500 µM (IBMX solution). The solution in which phosphodiesterase activity was measured (Li+ solution) was made by replacing NaCl in the saline solution with equimolar LiCl.
-Ionone was
purchased from Sigma Chemical Co. and was repurified by double
distillation. It was dissolved to 20 mM in EtOH and then to its final
concentration with saline solution (final EtOH concentration
<0.1%).
-Ionone concentration was measured with a spectrophotometer (
= 8,700 M
1 cm
1). Despite all attempts to minimize
the binding of
-ionone to the plastic tubing by replacing most of
it with glass, the concentration of
-ionone at the outflow of the
microperfusion system in the recording bath was significantly reduced. For this reason, the concentration of
-ionone to which the
cell was exposed was determined from samples collected from the
outflow of the microperfusion tubing after each experiment.
The outer segment of the cell was exposed to -ionone and test
solutions using a double-barrel microperfusion system driven by
a stepping motor (Cornwall et al., 1995
). This system allowed for
quick change of the solution perfusing the outer segment. The
time required for a complete solution change, estimated from the junction current recorded while the cell was exposed to
bright light, was ~25 ms. Test solutions for estimation of rate
constants of guanylyl cyclase and phosphodiesterase (to be referred to as rate of guanylyl cyclase and rate of phosphodiesterase) were delivered through one of the barrels of the microperfusion system using a multiple-way valve. The time for removing the dead volume in the system was ~45 s. This allowed
for effective estimation of both cyclase and phosphodiesterase
rates within 1 min.
-Ionone was delivered to the cell through
the other barrel of the microperfusion system. Initially,
-ionone
flow was turned off so that jumps could be performed from saline
solution into test solution (Li+ and IBMX). Subsequently,
-ionone flow was turned on and its effect on cyclase and phosphodiesterase rates was estimated by jumping the cell from
-ionone
into the test solution. The lack of
-ionone in the test solutions
had only a negligible effect on the measurements of cyclase and
phosphodiesterase rates.
The flow rates of the -ionone and test solutions were regulated by injecting phenol red solution into the experimental
chamber before each experiment. The sharp interface between
the bath solution and the solutions flowing through the solution
changer allowed us to observe and adjust the flow of both
-ionone and test solutions. The cells were not exposed to phenol red
because in control experiments we observed that phenol red activates phototransduction in bleach-adapted rods.
Data Analysis
To estimate the rates of cyclase and phosphodiesterase in the
cell, we have used a modification of the method initially described by Hodgkin and Nunn (1988) and subsequently used by
Cornwall and Fain (1994)
. The light-sensitive current in photoreceptors, i, is controlled by the concentration of cyclic GMP according to the relation (see Hodgkin and Nunn, 1988
; Pugh and
Lamb, 1990
):
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(2) |
where im is the maximum possible membrane current, K1/2 is the Michaelis constant for binding of cGMP to the light-sensitive channels, and N is the Hill coefficient, reflecting the cooperative binding of cGMP to the channels. The level of cGMP in the cell is regulated by the relative rates of guanylyl cyclase, responsible for cGMP synthesis, and cGMP phosphodiesterase, responsible for its hydrolysis. The change in free cGMP concentration in the cell is given by:
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(3) |
where is the buffering capacity of the cell for cGMP,
is the
rate constant of cyclase, and
is the rate constant of phosphodiesterase. Following Hodgkin and Nunn (1988; see also Cornwall
and Fain, 1994
), we assumed that the cGMP binding sites are of
high affinity and saturated (
= 1). Combining Eqs. 2 and 3, one
obtains a relation between the current and the rates of cyclase
and phosphodiesterase:
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(4) |
One can estimate the rate of cyclase or phosphodiesterase in the
intact photoreceptor in different conditions of adaptation or in
the presence of drugs by suddenly blocking one of these two enzymes. The rate at which the current changes immediately after
the block provides an estimate of the velocity at which cGMP is
being synthesized or hydrolyzed by the unaffected enzyme. Using
this scheme, the rate of cyclase can be estimated by treating the
cell with the phosphodiesterase inhibitor, IBMX. Sudden exposure of the cell to saline solution containing 500 µM IBMX substantially inhibits phosphodiesterase and the continued production of cGMP by cyclase results in current increase. The rate of
increase of the current provides an estimate of the rate of cGMP
synthesis by cyclase. Alternatively, saline solution in which Li+ is
substituted for Na+ can be used to estimate the rate of phosphodiesterase. Li+ permeates the light-sensitive channels but the presence of Li+ inside the cell blocks the Na+/Ca2+-K+ exchange
mechanism (Yau and Nakatani, 1984; Hodgkin et al., 1985
). As a
result, [Ca2+]i increases rapidly and inhibits cyclase. The rate of
the resulting decrease in current provides an estimate of the rate
of hydrolysis of cGMP by phosphodiesterase.
Derivation of a useful expression of the current as a function
of the enzymatic rates of cyclase or phosphodiesterase with the
help of test solutions requires several important assumptions that
were implicitly made in the original work of Hodgkin and Nunn
(1988). However, new information makes necessary the explicit reexamination of these assumptions. The simpler case of the Li+
jump requires the assumption that the cGMP channel characteristics (K1/2 and N), as well as im, do not change during or immediately after the solution change. Recent evidence has revealed
that of these three parameters only K1/2 is not constant but
rather varies as a function of the concentration of calcium (Hsu
and Molday, 1993
; Gordon et al., 1995
; Nakatani et al., 1995
;
Bauer, 1996
). As pointed out above, stepping into Li+ results in
an increase of [Ca2+]i. A decrease in [Ca2+]i within the physiological range can cause a significant decrease of K1/2. However,
an increase in [Ca2+]i beyond the physiological range, as in the
case of Li+ step, causes very little or no change of K1/2. Thus, the
proposition that K1/2, N, and im do not change during and immediately after the step in Li+ solution is valid. A second assumption
is that the current i, recorded with the suction electrode, is much
smaller than im (respective typical values, 40 pA [Baylor et al.,
1984
] and >1,000 pA [Cameron and Pugh, 1990
]). Thus, the ratio i/(im
i) in Eq. 4 can be replaced by the more simple i/im.
Using these assumptions for the case of blocked cyclase (
= 0),
Eq. 4 simplifies to a form that relates the rate of phosphodiesterase to the normalized current:
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(5) |
From Eq. 5, the relative change in the rate of phosphodiesterase compared with that of the dark-adapted cell will be given by:
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(6) |
After subtracting the junction current resulting from the solution
change, the normalized current was plotted on a semilogarithmic graph. was estimated from the slope of the straight line fitted to
the current trace, and then the ratio
/
D was calculated.
In the more complicated case of the IBMX jump, one again
has to assume that the cGMP channel characteristics (K1/2, N)
and im do not change during the solution jump. Since the step
into IBMX causes an increase in the inward current, the level of
[Ca2+]i should either stay the same or increase slightly. Thus, in addition to being valid for N and im, the above assumption still applies for K1/2. One potential problem, however, derives from the current increase during the step into IBMX solution. The assumption i << im, which is again required, may no longer hold.
To test the validity of this approximation, we calculated the rates
of cyclase from a set of data with and without this assumption and compared the results. The relative velocities of cyclase in the two
cases differed by <2%, leading us to ignore this factor in further
analysis. Using the same assumptions as made in the case of Li+,
Eq. 4 can be simplified for the case of blocked phosphodiesterase ( = 0). Then, dividing by Eq. 2 written for the dark-adapted condition, one gets:
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(7) |
where D indicates the parameters of the cell in its dark-adapted
state. Following Hodgkin and Nunn (1988), we have used
' as a
measure of the relative changes in the rate of cyclase (see also
Cornwall and Fain, 1994
). The expression for the relative rate of
cyclase derived from Eq. 7 is:
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(8) |
where we assumed N = 3 (Yau and Baylor, 1989). Another potential complication is that K1/2 is likely to be different in rods that
are dark-adapted, bleached, or chemically treated. As can be
seen from Eq. 8, in contrast to the Li+ method, calculation of the
relative rate of cyclase requires direct comparison of K1/2 in the
dark-adapted and in the bleached or otherwise manipulated cell.
As pointed out above, K1/2 of the cGMP-gated channel is not constant, but rather varies as a function of the concentration of calcium. The decrease in [Ca2+]i produced by the bleach (Sampath
et al., 1998
) and probably by
-ionone can cause a significant decrease of K1/2 (Nakatani et al., 1995
). To improve the accuracy of
the calculation of the rate of cyclase, we have incorporated an estimate of the change of K1/2 caused by the bleaching of pigment
in our analysis. This was done in the following way. The time derivative of the current di/dt was calculated using the Savitzki-Golay method (Press et al., 1992
). Since the data were acquired
at 250 Hz (4 ms/point) and filtered at 20 Hz (50 ms/point), the
linear regression was performed over 13 points (13 × 4 ms = 52 ms, corresponding to 19 Hz). The change in K1/2 after the bleach
was calculated from the corresponding drop in the dark current
using the relation between current and [Ca2+]i in Nakatani et al.
(1995, see Fig 6). Using Eq. 6,
' was obtained in each experimental condition, and then the ratio
/
D (equal to
'/
'D) was
calculated.
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The measurements of the rates of cyclase and phosphodiesterase in this study were done pairwise in the same cell, under
the same conditions, and within 1 min of each other. To minimize possible side effects, the order of measurements was varied
from cell to cell. Thus, direct comparison between the two enzymatic rates should be much more accurate than when two different cells from two different animals are considered. Since all of
the measurements of the velocities of cyclase and phosphodiesterase were done in steady state, it would be expected that the
two relative velocities (see Eqs. 6 and 8) will be equal. Accounting for the change in K1/2 and modifying the analysis of IBMX
jumps originally devised by Hodgkin and Nunn (1988) and later
used by Cornwall and Fain (1994)
compensated for the mismatch between the rates of cyclase and phosphodiesterase observed in these studies. In our experience, the two rates were very
similar for activation of the phototransduction cascade by
bleaches of up to 40%. For even higher transduction levels
(higher bleaches), the Li+ method saturates due to the overlap
of the initial rapid influx of Li+ ions, caused by the higher permeability of the light-dependent channels to Li+ than to Na+
(Hodgkin et al., 1985
), and the decrease in cGMP-activated current, caused by the block of cyclase. In addition, after higher
bleaches, the dark current is significantly reduced, which makes
the rate of its decrease in Li+ solution more difficult to measure.
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RESULTS |
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Photocurrent and Light Sensitivity
The experiment illustrated in Fig. 2 shows the effect
that -ionone has on the dark current and sensitivity of
a bleach-adapted rod. The cell was stimulated with a series of test flashes of increasing intensity to monitor
changes in the amplitude of the current and in sensitivity. Fig. 2 A illustrates a series of superimposed flash responses recorded from a dark-adapted rod. After a
brief exposure to bright light that bleached 20% of the
visual pigment, the cell was allowed to recover for 35 min until its dark (light-suppressible) current reached
a new steady state level from which no further recovery
occurred. The bleach caused a steady reduction in the dark current, as evidenced by the decreased amplitude
of the saturating flash response (Fig. 2 B). When the
rod was then exposed to
-ionone, the dark current declined further within several seconds to a new, lower
level (Fig. 2 C), which persisted for as long as the cell
remained in
-ionone solution. After returning the rod
to normal saline solution, the dark current recovered
to its bleach-adapted level within 5 min (Fig. 2 D). The
amplitudes of the flash responses in each condition
were plotted against flash intensity to create the corresponding intensity-response curves (Fig. 2 E). Bleaching increased the test flash intensity required to elicit a
threshold response (Fig. 2 E,
) compared with the
dark-adapted state (
). Thus, the sensitivity of the cell
after the bleach was lower than that of its dark-adapted
state. Treatment of the bleached cell with
-ionone reduced further its sensitivity (Fig. 2 E,
), at the same
time decreasing the maximal response amplitude. After returning the rod to normal saline solution, both sensitivity and maximal response amplitude recovered to
their bleach-adapted levels (Fig. 2 E,
). Thus,
-ionone caused a reversible decrease in dark current and
sensitivity in bleach-adapted rods. When cells were exposed to a sufficiently bright light to totally suppress the dark current and treated with
-ionone, only a
small junction current was observed. Thus, the decreased amplitude of the responses elicited in
-ionone
solution appeared to result from a decrease in the light-sensitive conductance.
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The effect of -ionone on the dark current and the
sensitivity was tested in a total of 36 bleach-adapted
cells. All of them exhibited a decrease in both dark current and sensitivity in the presence of
-ionone. The
magnitude of pigment bleaching ranged from 3 to
80%.
-Ionone concentrations used varied from 1 to
20 µM. For low concentrations of
-ionone (up to 5 µM),
the decrease in the dark current was greater for larger
bleaches until it saturated at ~20% pigment bleach.
For higher concentrations of
-ionone, the decrease in
the dark current was also larger for larger bleaches, but
the effect did not saturate in the range of bleaches that were tested. Owing to their variable nature, it was not
possible to determine the exact relationship between
the decrease in the dark current and the magnitude of
the pigment bleach or the concentration of
-ionone.
For this reason, we chose to restrict our analysis to the
effect of 10 µM
-ionone in rods in which 20% of the
pigment had been bleached. In a total of 21 cells tested
under these conditions, the mean decrease in the dark
current caused by
-ionone was 51 ± 8% (SEM, n = 21). The corresponding decrease in sensitivity was 0.91 ± 0.08 log units (SEM, n = 10).
Dim Flash Kinetics
The response amplitude is a linear function of light intensity at the lower end of the intensity-response curve
for both rods and cones (Baylor and Hodgkin, 1973;
Kleinschmidt and Dowling, 1975
). This implies that for
dim flashes, the photoresponse is equal to the linear
sum of the responses to single photons. Thus, analysis
of the kinetics of dim flash responses allows for investigation of the kinetics of the phototransduction cascade. Activation of transduction by a bleach or by background light accelerates the dim flash response (Cornwall et al., 1989
, 1990
; Matthews et al., 1990
). To
investigate whether the effect of
-ionone on the dark current and photosensitivity is a result of activation of
phototransduction as opposed to a nonspecific effect,
we looked at its impact on the kinetics of dim flash responses. Fig. 3 shows normalized dim flash responses
elicited from a cell, first in the dark-adapted state, 40 min after 20% of the pigment had been bleached, and then during and after exposure to
-ionone. As expected, bleaching accelerated significantly the photoresponse. After bleaching, the time to peak of the dim
flash response was reduced by >25%. Treatment of the
bleached cell with
-ionone resulted in further acceleration of the kinetics of the response. The effect of
-ionone was completely reversible and several minutes
after returning the cell to normal saline solution, the
response slowed to its pretreatment, bleach-adapted
level. Thus,
-ionone caused a reversible acceleration
of the dim flash response in a bleach-adapted rod.
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Rate of Guanylyl Cyclase
To investigate the effect of -ionone on phototransduction in detail, we performed a series of experiments designed to measure directly the rates of cyclase and
phosphodiesterase and to evaluate their change when
the cell was exposed to
-ionone (see MATERIALS AND
METHODS). Fig. 4 shows typical results from one experiment in which cyclase was studied. Using the microperfusion system, the rod was quickly exposed to saline test
solution containing 500 µM IBMX in order to block
phosphodiesterase. Fig. 4 (left) shows current recordings during the solution change. Fig. 4 (right) plots
d(J 1/3)/dt as a function of time, where J = i/i D. The
time course of the solution change in both cases is
shown on the top of the figure. Since the derivative of
J 1/3 is proportional to the rate of change in cGMP concentration (see MATERIALS AND METHODS, Eq. 2), its
maximum represents a measure of the rate of synthesis
of cGMP by guanylyl cyclase. The rate of cyclase was
first measured in the dark-adapted cell (Fig. 4 A). Here,
cyclase had only a low basal activity. After a 20% bleach followed by a recovery period, the rate of cyclase was
measured once again (Fig. 4 B). The rate of current increase upon jumping into IBMX solution (Fig. 4 B,
left) was higher than in the dark-adapted state (Fig. 4
A, left). As shown previously (Cornwall and Fain, 1994
), the rate of cyclase increases as a result of a
bleach, as can be seen here from the greater than
threefold increase in the peak of the derivative (Fig. 4
B, right) as compared with that measured before bleaching (Fig. 4 A, right). Exposure of the bleached cell to
-ionone produced a twofold further acceleration of
cyclase (Fig. 4 C). The effect of
-ionone was completely reversed a few minutes after the cell was returned to normal saline solution (Fig. 4 D). Thus,
-ionone caused a reversible acceleration of guanylyl cyclase
in a bleach-adapted rod.
The effect of -ionone on the rate of cyclase was studied in a total of 30 bleach-adapted rods. In all of them,
-ionone caused acceleration of cyclase. The range of
pigment bleaches used was from 3 to 80%, and the
range of
-ionone concentrations was from 3 to 20 µM.
No clear quantitative correlation between the fraction of pigment bleached, or the concentration of
-ionone, and the corresponding acceleration of cyclase
could be observed. However, in general, for a given
bleached fraction of visual pigment, the relative rate of
cyclase, compared with the bleach-adapted state, was
higher for higher concentrations of
-ionone. On the
other hand, for a given concentration of
-ionone, the
relative acceleration of cyclase was higher for smaller
bleaches. As in the experiments described in the previous section, we studied in detail the effect of 10 µM
-ionone after a 20% pigment bleach. A total of 18 rods were studied under these conditions. The rate of cyclase in the presence of
-ionone was 2.5 ± 0.3 (SEM, n = 18) times higher than in the corresponding bleach-adapted state.
Rate of Phosphodiesterase
To substantiate further the conclusion that -ionone
activates phototransduction, the rate of phosphodiesterase was measured within 2 min of the cyclase rate
measurement in these same rods. The minimum time
between the two measurements was limited by the time
required for the new test solution to fill the dead space in the microperfusion tubing. Since the current was at
steady state while the cell was exposed to
-ionone, the
concentration of cGMP should be constant, and the
rates of its synthesis by cyclase and its hydrolysis by
phosphodiesterase should be equal. The activation of
cyclase as a result of treatment of the bleached cell with
-ionone then should be accompanied by a corresponding acceleration of phosphodiesterase.
Fig. 5 shows one example of an experiment in which
the rate of phosphodiesterase was measured. In this
case, cyclase was blocked by suddenly exposing the cell
to a test solution in which Li+ was substituted for Na+
on an equimolar basis. The time course of the jump
into Li+ solution is shown at the top of the figure. Fig. 5
(left) shows the current recordings from the cell; Fig. 5
(right) shows semi-logarithmic plots of the normalized
current as a function of time. Here, the slope is proportional to the rate of the phosphodiesterase (Eq. 5). As
with cyclase, bleaching produced an acceleration of
phosphodiesterase (Fig. 5 B), and subsequent treatment with -ionone resulted in an even higher enzymatic rate (Fig. 5 C). Again, the effect of
-ionone was
completely reversible upon return of the cell to normal
saline solution (Fig. 5 D). Thus,
-ionone caused a reversible acceleration of cGMP phosphodiesterase in a
bleach-adapted rod.
|
The effect of -ionone on phosphodiesterase was
measured under the same bleaching conditions and
the same
-ionone concentrations as cyclase (see the
previous section) in a total of 30 cells. In 25 cells,
-ionone caused acceleration of phosphodiesterase and in
the remaining 5 either no effect or a small deceleration was observed. However, in 4 of these 5 cells the fraction
of the bleached pigment was 40% or higher. We interpret the inconsistency in these cells as being due to the
inability of the Li+ jump method to resolve the high
rate of phosphodiesterase after large bleaches (see MATERIALS AND METHODS). This speculation is supported
by the fact that in contrast to phosphodiesterase, cyclase was accelerated in these cells and there was a decrease in both the dark current and the sensitivity as a
result of the treatment with
-ionone. In the remaining
25 cells, the acceleration of phosphodiesterase was
higher after smaller bleaches for each
-ionone concentration. In 17 experiments similar to those presented in Fig. 5, we examined the effect of 10 µM
-ionone after a 20% pigment bleach. In these cells, the rate
of phosphodiesterase in the presence of
-ionone was
1.7 ± 0.2 (SEM, n = 17) times higher than in the corresponding bleach-adapted state.
Phototransduction in a Dark-adapted Rod in the Presence
of -Ionone
Experiments were designed to investigate the possible
site of action of -ionone on phototransduction. Specifically, we were interested in testing the hypothesis that
-ionone activates transduction by binding directly in
the chromophore pocket of opsin. To test this idea, we
compared the effect of
-ionone in a dark-adapted and
background light-adapted rod with its effect after a
bleach. As a first step, we examined the effect of
-ionone in dark-adapted cells. A total of 16 cells were tested.
In a small fraction of these, treatment with
-ionone resulted in a slight activation of phototransduction. The
decrease in the dark current, the loss of sensitivity, and
the acceleration of cyclase and phosphodiesterase produced by
-ionone in these dark-adapted rods, although significant, was small compared with the effect
of
-ionone seen after 20% or higher pigment bleaches.
The observed acceleration of phototransduction in
some of the control experiments could be due to the
fact that some of the cells were not completely dark
adapted and contained some small fraction of bleached
pigment. In accord with this notion, we observed that
-ionone activates phototransduction even after bleaches
as low as 3%. Because the cells were exposed to dim red and infrared light during preparation, it is possible that
in some cases a small but significant fraction of pigment was bleached. This idea is supported by the observation that the sensitivity of the cells slightly increased
during the first 30-60 min after the dissection (data not
shown), despite the fact that the animals were dark-adapted overnight. The interaction of the pigment
bleached during the dissection with
-ionone could
produce the observed activation of phototransduction.
To test this hypothesis directly, two dark-adapted
cells that showed some acceleration of the transduction
cascade during exposure to -ionone were treated with
11-cis retinal in an effort to regenerate any residual
bleached pigment. In both cells, about 1 h after addition of 11-cis retinal, the effect of
-ionone on sensitivity, dim flash kinetics, and cyclase and phosphodiesterase velocities was either significantly reduced or
absent. Thus, complete regeneration of the pigment
abolished the effect of
-ionone. Another argument,
consistent with this notion, is based on the following
observation: a hemisected eyecup was dark adapted for 24 h at 4°C after the dissection. Treatment of five cells
from this preparation with
-ionone produced no effect, presumably because the pigment bleached during
the dissection was regenerated in the intact retina during this additional dark period. Taken together, these
observations indicate that treatment with
-ionone produces a small effect on transduction in freshly dissected
dark-adapted cells and no effect in fully dark-adapted
cells where all of the pigment has been regenerated.
Rate of Guanylyl Cyclase and Phosphodiesterase in Bleached vs. Light-adapted Cell
As a second step in investigating the possible site of action of -ionone, we compared its effect in a background light-adapted rod with its effect after a bleach.
In both cases, the level of phototransduction activity is
higher than in the dark-adapted state. However, the
background light intensity required to produce activation comparable with that of a significant pigment
bleach is so low that it only photoactivates a negligible
fraction of the pigment. For instance, a steady background producing activation of cyclase (phosphodiesterase) equivalent to a 20% bleach (12.8 photons
µm
2 s
1, see Fig. 6) would bleach <0.03% of the pigment in 1 h. Thus, even though both bleaching and
background light produce elevation of the rate of phototransduction, the retinal binding pocket on opsin
may be free and available for binding only in the
bleached state.
In four rods, the effect of -ionone on cyclase in
background light was directly compared with its effect
on cyclase after a bleach. Each cell was first exposed to
a series of backgrounds of increasing intensity. For
each background, the rate of cyclase was measured in
both saline solution and in
-ionone solution. An example at one background is shown in Fig. 6, A (saline
solution) and B (
-ionone). The cell was then bleached
and allowed to recover to a steady state. The rate of cyclase was measured once again in saline solution (Fig. 6
C), and then in
-ionone (D). Comparison between
Fig. 6, A and C, shows that one of the backgrounds
induced exactly the same activation of cyclase as the
subsequent bleaching.
-Ionone failed to produce any
effect on the rate of cyclase in the background light-adapted state (Fig. 6 B), but in contrast increased significantly the rate of cyclase after the cell was bleached
(D). This result indicates that the activation of phototransduction is not sufficient to induce acceleration
of cyclase by
-ionone.
In an experiment similar to the one just described,
the effects of -ionone on the rate of phosphodiesterase in the light- and bleach-adapted states were
also compared. In the two tested rods,
-ionone only
accelerated phosphodiesterase in the bleach-adapted state, but did not affect the background light-adapted
state. This result complements the result for cyclase described in the previous paragraph. Taken together,
they indicate that
-ionone activates phototransduction
only in the presence of bleached pigment. This observation supports the hypothesis that
-ionone affects phototransduction by interacting selectively with bleached pigment.
Time Course of the Effect of -Ionone After a Bleach
The results illustrated in Figs. 7 and 8 are from an experiment designed to determine the time course of onset of transduction activation triggered by -ionone.
Since for the first few minutes after bleaching the rates
of both cyclase and phosphodiesterase are too high to
be measured reliably with a solution step, we chose the
amplitude of the dark current as an index of the level of transduction. The cell was exposed to saturating test
flashes to measure the dark current first in saline solution and then 25 s after exposure to
-ionone solution.
The fact that the effect of
-ionone is completely reversible allowed us to do repetitive measurements in
the course of recovery of the cell after the bleach. Current recordings from one such experiment are presented in Fig. 7. Fig. 7 A shows recordings from one rod
in its dark-adapted state and at different times after the
bleach. In the dark-adapted cell, a jump into
-ionone
caused only a small junction current shift, but not a
change in the amplitude of the saturating photoresponse. Thus, as described above,
-ionone did not affect the light-sensitive current in the dark-adapted cell.
After bleaching 20% of the pigment, the current was
initially completely suppressed, and then started to rapidly recover. During this period when the dark current
was increasing quickly, estimation of the effect of
-ionone was rendered unreliable by the fact that measurements of the current in saline solution and in
-ionone
were made 45 s apart. Several minutes after the bleach,
the recovery of the cell slowed down sufficiently to allow reliable comparison of the dark current in saline
solution and in
-ionone solution without any significant drift in the current during the 45 s between test
flashes. Jumping into
-ionone solution produced a
drop in the current whose total amplitude increased
with time after the bleach. The kinetics of the decrease in the current caused by
-ionone were carefully examined. The effect of
-ionone on the current at different
times after the bleach can be seen in Fig. 7 B. These recordings are from the same cell as Fig. 7 A, but are superimposed and plotted on a different time scale. For
each of the traces recorded after the bleach, the current decrease caused by
-ionone could be described
by a single exponential decay function. For the cell
shown on Fig. 7, the time constant of these exponential
functions changed from 12 s, measured 13 min after
the bleach, to 3.7 s 1-h later. The time constant of the
exponential current decay decreased with time after
the bleach in all nine rods tested. On average, the decrease of the current caused by
-ionone shortly after
the bleach was two times slower than the decrease in
the current recorded 1-h later.
|
|
Fig. 8 shows the time course of the effect of -ionone
on the dark current amplitude for the same cell as a
function of time after the bleach. Fig. 8 A presents the
amplitude of the current in saline solution and in
-ionone in the dark-adapted cell and at different time points
after the bleach. As pointed out above, the current immediately after the bleach was completely suppressed. The amplitude of the current in
-ionone 7 min after
the bleach was slightly higher than the corresponding
amplitude in saline solution measured 45 s earlier. As
discussed above, we interpret this observation not to be
a result of the treatment with
-ionone, but rather to
follow from the fast recovery of the cell during that period. 13 min after the bleach, the amplitude of the current in
-ionone was already clearly smaller than the
corresponding current in saline solution and with time
that difference became more pronounced. To estimate
the rate of the increase of this effect, we calculated the
relative decrease in the current caused by
-ionone as a
function of time after the bleach. Fig. 8 B shows the ratio of the amplitudes of the current in
-ionone and in saline solution from A, plotted against the time after
the bleach. The decrease of that ratio could be fit by a
single exponential decay function with a time constant
of 13 ± 1.1 (SEM) min. The average time constant for
all cells tested in this way was 13 ± 2.1 (SEM, n = 8)
min. This rate of increase of the effect of
-ionone as a
function of time after a bleach is of the same order as
the rate of decay of photoactivated pigment to free opsin and all-trans retinol derived from spectrophotometric experiments (Baumann, 1972
; Brin and Ripps, 1977
)
or with early receptor potential measurements (Cone
and Cobbs, 1969
). The fact that the amplitude of the
effect of
-ionone correlates with the concentration of free opsin in the cell provides another argument in
support of the notion that
-ionone exerts its effect by
interacting exclusively with free opsin.
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DISCUSSION |
---|
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---|
Effect of -Ionone on Phototransduction in a
Bleach-adapted Rod
Our experiments show that in bleach-adapted rods -ionone causes (a) a decrease in the dark current, (b) a decrease in the sensitivity, (c) an acceleration of the dim
flash photoresponse, and (d) an increase in the rates of
guanylyl cyclase and cGMP phosphodiesterase. Together,
these results indicate that in bleach-adapted rods
-ionone acts as an agonist and activates phototransduction in the dark.
-Ionone produces no effect in fully dark-adapted cells, where the basal level of transduction is
low and the chromophore pockets of the opsin molecules are still occupied by native 11-cis retinal. Furthermore,
-ionone produces no effect in background light-adapted cells, where the level of transduction is high
but still most of the chromophore pockets are occupied by 11-cis retinal. The rate at which the amplitude
of the observed effect increases in time after the bleach
is of the same order as the rate of decay of photoactivated rod pigment to free opsin. Based on these observations, we conclude that the effect of
-ionone is the
result of its direct interaction with the free opsin molecules produced by the bleaching light.
We suggest that -ionone exerts its physiological effect by binding directly in the chromophore pocket of
free opsin. Fig. 9 presents a model of the proposed
mechanism of activation of phototransduction by
-ionone in rods. The chromophore pocket of opsin is shown
schematically and the points of interaction of opsin
with retinal or with
-ionone are indicated by a star. For each intermediate state, the relative enzymatic activity is indicated by the amplitude of the vertical bar to
the right. In the dark-adapted state, 11-cis retinal is
bound in the chromophore pocket of opsin and is covalently attached to it via a Schiff base linkage (Fig. 9
A). The dark-adapted pigment is not capable of activating phototransduction beyond its basal level, as indicated by the absence of a vertical bar on the right. Absorption of a photon by the pigment molecule results
in the photoisomerization of retinal and the full activation of the complex (Fig. 9 B). Decay of this photoactivated rhodopsin produces all-trans retinol and free opsin. The bleached pigment exhibits low catalytic activity (Fig. 9 C), resulting in a level of transduction in the
bleach-adapted state higher than in the dark-adapted
state. Binding of
-ionone in the free chromophore
pocket of opsin produces a complex with catalytic activity that is higher than that of free opsin alone (Fig. 9
D). The upregulation of phototransduction caused by
this increase in the catalytic activity of the bleached pigment can be observed physiologically and is reported
here. As expected for the noncovalent type of interaction between
-ionone and opsin, the binding of
-ionone is reversible and its removal from the pocket results in restoration of the bleach-adapted catalytic activity of free opsin (Fig. 9 C).
|
Contrast between the Effect of -Ionone in Rods and Cones
The effect of -ionone in bleach-adapted salamander
cones has been studied previously (Jin et al., 1993
;
Cornwall et al., 1995
). In cones,
-ionone partially reverses the effect of bleaching and downregulates phototransduction. In other words, administration of
-ionone to a bleach-adapted cone restores some of the
large loss in sensitivity produced by the bleach and reverses the acceleration of the dim flash response. The
dark current recovers partially. Treatment of bleach-adapted cones with
-ionone also causes a decrease in
the rate of guanylyl cyclase. These results on cones are
in direct contrast with the results reported here for
rods. Thus, the interaction of
-ionone with the chromophore-binding pocket of rod and cone opsins leads
to opposite physiological effects. This observation resolves the current discrepancy between biochemical
and physiological data and suggests that the biochemical observation that opsin is activated by the binding of
retinoid in the chromophore pocket is correct but applies only to rod opsin rather than to both rod and
cone opsin.
The contrast between the chromophore pockets of
rods and cones is not limited to the interaction with
-ionone. Jones et al. (1989)
observed a significant decrease in the sensitivity of bleach-adapted rods treated
with 11-cis retinol. In the same study, 11-cis retinol caused recovery of sensitivity in bleach-adapted cones.
Together, these observations indicate that the contrast
between rods and cones outlined here may be a fundamental feature of the interaction of the two types of opsin with their native chromophore.
Nature of Bleaching Adaptation
The state of opsin that is responsible for bleaching adaptation is a matter of controversy. According to one
model, the free opsin produced after bleaching exhibits a low catalytic activity that sustains an elevated level
of transduction in the bleached cell (Cornwall and
Fain, 1994; Cornwall et al., 1995
). An alternative view is
that interaction of all-trans retinal with free opsin is the
factor causing bleaching adaptation (Fukada and
Yoshizawa, 1981
; Hofmann et al., 1992
; Palczewski et al.,
1994
). A strong argument for a free opsin mechanism
of bleaching adaptation deriving from our findings is
that the occupancy of the chromophore pocket by
-ionone produces opposite effects in rods and cones.
The noncovalent binding of all-trans retinal in the
pocket would also be expected to produce opposite effects in rods and cones and therefore could not account for the bleaching adaptation in both photoreceptor types. In contrast, the sustained catalytic activity
of free opsin could explain the elevated rate of phototransduction observed in both rods and cones after a
bleach. Thus, the results from our experiments make it
unlikely that bleaching adaptation is the result of the
interaction of all-trans retinal with the chromophore pocket of free opsin.
Finally, our observations of the effect of -ionone in
bleach-adapted rods may have important implications
for the physiology of regeneration of rhodopsin in the
intact retina. By analogy with the results reported here,
binding of the native 11-cis retinal in the chromophore
pocket of rod opsin might be expected to form an active noncovalent complex that results in transient upregulation of phototransduction and transient reduction in the sensitivity of the rod. In contrast, binding of
retinal in the chromophore pocket of cone opsin
might be expected to downregulate transduction, and
thus contribute to the partial recovery of the sensitivity of the cone even before the formation of the Schiff base
linkage and the regeneration of the pigment. Verification of this model will require further investigation.
![]() |
FOOTNOTES |
---|
Address correspondence to Vladimir Kefalov, Department of Physiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. Fax: 617-638-4273; E-mail: vkefalov{at}bu.edu
Original version received 22 October 1998 and accepted version received 24 December 1998.
Portions of this work were previously published in abstract form (Kefalov, V.J., M.C. Cornwall, and R.C. Crouch. 1998. Invest. Ophthalmol. Vis. Sci. 39:S1123).We thank Dr. Gregor Jones for helpful discussions on the analysis of data and Drs. Gordon Fain and David Cameron for comments on the manuscript.
This work was supported by National Institutes of Health grants EY01157 (M.C. Cornwall) and EY04939 (R.K. Crouch), and the Foundation to Prevent Blindness (R.K. Crouch).
![]() |
Abbreviation used in this paper |
---|
IBMX, 3-isobutyl-1-methylxanthine.
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