From the * Department of Physiological Science, and Department of Ophthalmology, University of California, Los Angeles, Los Angeles, California 90095;
Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, England CB2 3EG; and § Department of Physiology, Boston University Medical School, Boston, Massachusetts 02118
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ABSTRACT |
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A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 ± 250 nM in a dark-adapted rod and 30 ± 10 nM during response saturation after exposure to bright light (mean ± SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 ± 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.
Key words: photoreceptor; retinal rod; bleaching adaptation; calcium ![]() |
INTRODUCTION |
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Exposure of isolated rod and cone photoreceptors to
light sufficiently intense to bleach a significant fraction
of the photopigment initially results in the complete
suppression of the circulating current, which is followed by a gradual but partial recovery to a level that is
reduced in comparison with the original dark current of the dark-adapted cell. This persistent suppression of
the circulating current is accompanied by long-lasting
changes in sensitivity and response waveform. In an isolated photoreceptor, these adaptational changes appear to persist indefinitely, but can be reversed rapidly by the regeneration of the photopigment by exogenous
11-cis-retinal (Jones et al., 1989; Cornwall et al., 1990
).
The persistent suppression of the circulating current
after bleaching results from a continuing excitation of
phototransduction (Cornwall and Fain, 1994
; Cornwall et al., 1995
) via excitation of the transduction cascade
(Matthews et al., 1996a
) by a long-lived photoproduct
of rhodopsin bleaching that may be free opsin (Jin et
al., 1993
).
Bleaching adaptation shows considerable similarity
with the adaptational changes induced by steady light
(Fain and Cornwall, 1993; Leibrock et al., 1994
; Jones
et al., 1996
). Such light adaptation is believed to be
largely, if not exclusively, mediated by a light-induced fall in Ca2+i, which results from the graded suppression
of the circulating current by the background, since if
these changes in Ca2+i are prevented by interfering
with the normal mechanism of Ca2+ homeostasis then
adaptation is abolished (Matthews et al., 1988
; Nakatani and Yau, 1988
; Fain et al., 1989
). Similar manipulations of Ca2+ homeostasis have also been shown to prevent these bleach-induced adaptational changes in
cone photoreceptors (Matthews et al., 1996c
), suggesting that Ca2+i may also play a similar role in bleaching
adaptation.
The fall in Ca2+i that immediately follows the suppression of the circulating current by light has been
measured in several recent studies, which demonstrates
that Ca2+i varies in a graded manner during steady illumination (Gray-Keller and Detwiler, 1994, 1996
; McCarthy et al., 1994
; Younger et al., 1996
). However, considerably less is known about the changes in Ca2+ that
may take place during the photopigment cycle. We
have therefore applied a laser spot confocal technique,
originally developed for measuring the release of Ca2+
from sarcoplasmic reticulum (Escobar et al., 1994
), to
the measurement of light-induced changes in Ca2+i
from salamander rod photoreceptors. In common with
previous studies using fluorescent probes to measure
changes in Ca2+i from single rods, the fluorescence excitation beam resulted in considerable stimulation of
the photoreceptor. Making a virtue of this necessity, we
have used the photopigment bleaching that takes place
during the Ca2+ measurement itself to follow the
changes in Ca2+i that accompany photopigment bleaching and regeneration in isolated rod photoreceptors.
Preliminary results from this study have been reported
to the Physiological Society (Matthews et al., 1996b
) and to the Association for Research in Vision and Ophthalmology (Sampath et al., 1997
).
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METHODS |
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Preparation
Aquatic tiger salamanders (Ambystoma tigrinum) purchased from Kons Scientific Co. (Germantown, WI) or Charles Sullivan (Nashville, TN) were kept between 7 and 10°C on a 12-h light-dark cycle. Animals were dark-adapted overnight and killed in accordance with protocols approved by the Animal Research Council of the University of California, Los Angeles. Under dim red illumination, the animals were decapitated, pithed both rostrally and caudally, and the eyes enucleated. All subsequent dissection was carried out under infrared illumination. Eyes were hemisected and the eye cup cut into two pieces along the optic disc. Each piece of eye cup was then placed in a silicone rubber- coated dish (Sylgard 184; Dow Corning Corp., Midland, MI) containing amphibian Ringer solution (111 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1.6 mM MgCl2, 3 mM HEPES-NaOH, 10 µM EDTA-NaOH, 10 mM glucose, 0.1 mg/ml BSA, pH 7.7) and stored between 7 and 10°C. At the time of the experiment, the retina was peeled from the eye cup, placed photoreceptor side up on the Sylgard, and chopped lightly using a piece of razor blade. A fixed volume of the resulting cell suspension was collected with a transfer pipette and injected into the recording chamber. In the majority of experiments, the dissociated tissue was preincubated at room temperature (23°C) in amphibian Ringer solution, without glucose or BSA, containing 10 µM fluo-3 acetoxymethyl ester (fluo-3 AM; Molecular Probes, Inc., Eugene, OR) for 30 min before recording. After this period, excess dye was removed from the recording chamber via the bath perfusion. In a few experiments, fluo-3 AM loading was carried out for 70 min at 4°C to minimize dye compartmentalization. All experiments were performed at room temperature. Unless specified, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Ca2+ Measurement
Simultaneous measurements of photocurrent and Ca2+ indicator
fluorescence were made with a spot confocal technique (Escobar et al., 1994) illustrated schematically in Fig. 1. The beam from an
argon ion laser (Model 98; Lexel Laser Inc., Freemont, CA), tuned to 514 nm, illuminated the 50-µm pinhole of a spatial filter assembly (Newport Corp., Irvine, CA). The beam was recollimated by a 4× microscope objective (Nikon Inc., Melville, NY),
and entered the inverted microscope (Zeiss IM; Carl Zeiss, Inc.,
Oberkochen, Germany) through the epi-fluorescence port via a
dichroic mirror (525DRLP; Omega Optical, Brattleboro, VT). A
40× objective lens (1.3 NA oil immersion; Nikon Inc.) formed a
reduced image of the pinhole to illuminate a spot ~8 µm in diameter on the outer segment of a rod photoreceptor, whose inner segment was drawn into a suction pipette. The fluo-3 fluorescence evoked by laser illumination passed through a 530-nm
emission filter (530EFLP; Omega Optical) and was imaged onto
a small-diameter PIN photodiode (model HR008; United Detector Technologies, Hawthorne, CA) mounted on the headstage of
an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The analog signal from the diode was low pass filtered at 250 Hz with an active 8-pole Bessel filter (Frequency Devices, Haverhill, MA) and digitized at 1,000 Hz (PClamp 6; Axon Instruments).
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The unattenuated intensity of the laser was considerably
higher than could be used to make a Ca2+ measurement without
significantly bleaching the Ca2+ indicator fluo-3. Therefore, the
laser beam was typically attenuated by 3 log10 units using a reflective neutral density filter (Newport Corp.) to yield a flux of 1.7 × 1011 photon µm2 s
1 at the plane of the pinhole image. In some
experiments, the laser intensity was reduced further by substituting a 4 log10 unit neutral density filter (Melles Griot, Tustin, CA).
Since the wavelength of the laser is close to the wavelength of
peak absorption of rhodopsin, exposure to the laser will also
have bleached the vast majority of the photopigment within the
area of the laser spot. Fluorescence was evoked using a series of
brief laser exposures controlled by an electromagnetic shutter
(Vincent Associates, Rochester, NY) driven by the data acquisition system. Most experiments characterizing the time course of
Ca2+ decline after intense laser illumination used a single 7-s laser exposure. In experiments investigating bleaching adaptation,
laser exposure was minimized by instead presenting four brief laser pulses, each of 20-ms duration.
In Situ Calibration
To quantify how photoreceptor Ca2+ concentration changes during exposure to light, both the Kd of fluo-3 and the maximum
and minimum fluorescence that can be elicited from the rod
outer segment in high and low Ca2+ (Fmax and Fmin) are required
(Minta et al., 1989). The Kd of the Ca2+ indicator was determined
with a haemocytometer (100 µm path length) containing 100 µM fluo-3 free acid in pseudo-intracellular solutions of buffered
Ca2+ (World Precision Instruments, Sarasota, FL), whose precise
free Ca2+ concentrations were verified using a Ca2+ electrode
(Calcium Ionophore I
cocktail A; Fluka, Ronkonkoma, NY). The
sigmoidal variation of the fluorescence signal (F) with free Ca2+
concentration (Fig. 1, inset) could be fitted by the Michaelis-Menten equation for Ca2+ binding:
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(1) |
which yielded a value for Kd of 400 nM.
Fmax and Fmin were determined in situ using the four-laser pulse protocol described above while exposing the photoreceptor outer segment to solutions of low and high Ca2+ concentration containing 25 µM ionomycin or 40 mg/ml saponin. The low Ca2+i solution that was used to determine Fmin consisted of 111 mM NaCl, 2.5 mM KCl, 2.05 mM MgCl2, 3 mM HEPES, and 2 mM EGTA. The high Ca2+ solution that was used to determine Fmax contained 76.6 mM CaCl2, 2.5 mM KCl, and 3 mM HEPES. In both cases, the pH was titrated to 7.7 with NaOH. Values of Fmin and Fmax measured in this way were used in Eq. 1 to determine the free Ca2+ concentration in the same dark-adapted rod before and after exposure to saturating laser illumination. The value of Fmin determined in situ was a greater fraction of Fmax than was obtained from fluo-3 in the cuvette (see Fig. 1, inset); this discrepancy may reflect nonspecific binding of the dye within the rod outer segment (see DISCUSSION).
Fluo-3-free Acid
To ensure that the fluorescence signal measured with fluo-3 AM
was not contaminated by compartmentalization of the dye within the outer segment, Ca2+ measurements were also made with the
cell-impermeant fluo-3-free acid incorporated into the rod cytoplasm by whole-cell patch-clamp recording. Patch pipettes with
tip resistances between 5 and 12 M were filled with a pseudo-intracellular solution (92 mM KAspartate, 7 mM NaCl, 5 mM
MgCl2, 10 mM HEPES, 1 mM Na2ATP, 1 mM Na4GTP, pH 7.0)
that included 100 µM fluo-3 (pentapotassium salt). Gigaohm seals were formed on the rod inner segment, and the whole-cell recording configuration was established by either gentle suction or hyperpolarizing voltage clamp pulses so as to allow access of
the pipette solution to the cytoplasm by diffusion. The membrane potential in the whole-cell configuration was held at
40
mV (after correction for a 10-mV liquid junction potential).
Electrical Recording and Light Stimuli
Light-induced changes in the circulating current were recorded from the suction or patch pipette with an EPC-7 patch clamp amplifier (List Electronics, Darmstadt, Germany). This signal was low pass filtered at 20 Hz using an active 8-pole Bessel filter (Frequency Devices) and digitized either at 100 or 1,000 Hz (for simultaneous photocurrent and calcium indicator fluorescence measurements).
Light stimuli were delivered from a DC-driven tungsten halogen lamp through a 570-nm interference filter, and unpolarized; flashes of 20-ms duration were delivered through electromagnetic shutters (Vincent Associates, Rochester, NY). Light intensities were adjusted with neutral density filters (Fish-Schurman
Corp., New Rochelle, NY) and calibrated with a silicon photodiode (Graseby Optronics, Orlando, FL). These absolute photon
fluxes can be converted to rates of rhodopsin isomerization using
a collecting area of 7 µm2 at 570 nm, obtained by multiplying the
value of 20 µm2 (Lamb et al., 1986) at the 516-nm wavelength of
peak sensitivity (Harosi, 1975
) by the ratio of the spectral sensitivities of the rod photopigment at these two wavelengths (Cornwall
et al., 1984
).
The percentage of photopigment bleached by the 514 nm
light from the argon ion laser was estimated from the photosensitivity at 520 nm for vitamin A2-based pigments in free solution
(Dartnall, 1972), corrected for the difference in dichroism in
free solution and in disk membranes (6.2 × 10
9 µm2) (Jones et
al., 1993
). Both the continuous and the short pulse laser exposures that were used to measure the light-induced decline in
Ca2+i can be calculated from the photosensitivity to bleach >99%
of the photopigment within the area of the laser spot, although
this may have been reduced somewhat in practice by photoregeneration from bleaching intermediates, especially for the short
pulses (Williams, 1964
; Pugh, 1975
). Less extensive bleaching is
also likely to have taken place in the remainder of the outer segment due to scattered laser light.
Pigment Regeneration
Pigment regeneration was accomplished by bath application of
phospholipid vesicles containing the chromophore 11-cis-retinal. A 25-mg aliquot of phosphatidyl choline solution (Type V; Sigma Chemical Co.) was dried in a stream of 100% N2. To this was
added 10 ml of Ringer solution without glucose or BSA, and the
mixture was sonicated for 10 min (60 W, 50% duty cycle) with a
probe sonicator (Vibracell; Sonics Materials Inc., Danbury, CT).
The resulting vesicle suspension was then added to a small vessel
containing 11-cis-retinal dried on its wall from an ethanolic solution. Loading of the vesicles with the retinoid was facilitated by
further brief sonication in darkness. The concentration of 11-cis-retinal present in the vesicles was measured spectrophotometrically (UV-2101PC; Shimadzu Scientific Instruments, Columbia,
MD) using the molar extinction coefficient in ethanol of 25,000 M1 cm
1 at 380 nm (Hubbard et al., 1971
), and the vesicle suspension was diluted with Ringer solution to a final concentration
of 300 µM. The photopigment of bleached rods was regenerated
by adding a sufficient quantity of the vesicle suspension to the recording chamber to achieve a final 11-cis-retinal concentration of
100 µM.
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RESULTS |
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Kinetics of the Light-induced Decline in Ca2+i
Fig. 2 shows an example of an experiment in which
fluo-3 fluorescence and suction pipette current were recorded simultaneously. The dark-adapted rod was first
loaded with the dye by incubation with fluo-3 AM, and
then the outer segment was exposed to the laser spot to
excite fluo-3 fluorescence. The 7-s laser exposure completely suppressed the circulating current (Fig. 2 A),
typically holding it in saturation for 10 or more min
thereafter, reflecting the bleaching of more than 99%
of the photopigment in the region illuminated by the
laser spot. The onset of laser excitation in this previously dark-adapted rod evoked a high intensity of fluo-3 fluorescence (Fig. 2 B, 1), which then progressively declined to a much lower level. In contrast, the second
(Fig. 2 B, 2) and subsequent laser exposures evoked
only a low and maintained fluorescence signal. Furthermore, a similarly low level of fluorescence was also obtained if the laser spot was then displaced along the
outer segment region from which fluorescence had not
previously been excited, but which presumably was exposed to sufficient scattered laser light to completely
suppress the dark current. These observations suggest
that the decay in fluo-3 fluorescence that is seen when a
dark-adapted rod is first exposed to the laser spot represents the light-induced decline in Ca2+i that is known
to follow suppression of the dark current (Yau and Nakatani, 1985; McNaughton et al., 1986
; Ratto et al.,
1988
).
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The decline of fluo-3 fluorescence evoked by laser illumination of a dark-adapted rod was best fitted by the sum of two decaying exponentials of similar amplitude but with time constants differing by nearly an order of magnitude. Histograms of these short and long time constants from 86 cells incubated with fluo-3 AM are shown in Fig. 3 (filled columns). These have been fitted with Gaussian distributions with means of 260 and 2,200 ms.
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The fluo-3 fluorescence signal during the saturated light response was calibrated by artificially manipulating Ca2+i to determine the maximum and minimum fluorescence that could be elicited from the dye in the region of the laser spot. A typical calibration experiment is shown in Fig. 4; in each case, fluorescence was excited by a sequence of brief 20-ms laser pulses instead of a continuous exposure to ensure that dye bleaching was insignificant during the multiple laser exposures used for these measurements. Fig. 4, A and B illustrates the responses to the first two sequences of laser pulses. When the rod was first stimulated by the exciting beam, fluo-3 fluorescence declined to a reduced level that was then maintained for subsequent laser pulses, during which period the circulating current remained suppressed. After these measurements, the rod outer segment was superfused with solutions designed to greatly lower or elevate Ca2+i to levels that would minimize or saturate fluo-3 fluorescence. In Fig. 4 C, the outer segment was exposed to a solution containing nominally zero-Ca2+ and 25 µM ionomycin, and which included Na+ to support extrusion of Ca2+ via Na+/ Ca2+-K+ exchange. This manipulation led to a dramatic elevation of the circulating current, which had previously been completely suppressed in Ringer solution by the preceding laser exposures, suggesting that Ca2+i had been lowered further from its already reduced level during response saturation. After this solution change, the fluorescence evoked by each laser pulse decreased to a level slightly below that seen during saturating illumination (compare with Fig. 4 B), also consistent with a further reduction in Ca2+i. In Fig. 4 D, the outer segment was exposed to a solution containing isotonic Ca2+ and 25 µM ionomycin, from which Na+ had been omitted to prevent extrusion of Ca2+ via Na+/Ca2+-K+ exchange. This manipulation, which would be expected to elevate Ca2+i substantially, increased the fluorescence evoked by each laser pulse to a level well above the reduced levels seen while the circulating current was suppressed in Ringer solution, and somewhat greater than that evoked from the dark-adapted rod at the onset of laser illumination.
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The fluorescence values from Fig. 4, C and D, and similar measurements from other cells, were taken as approximating to Fmin and Fmax, respectively. These values were then used in conjunction with the fluo-3 dissociation constant (Kd = 400 nM) determined in the recording chamber (Fig. 1, inset) to convert the initial and steady state levels of dye fluorescence into the corresponding values for Ca2+i using the Michaelis-Menten relation (Eq. 1). This procedure gave values for Ca2+i of 670 ± 250 nM in a dark-adapted rod and 30 ± 10 nM after saturating laser illumination (Mean ± SD, 12 cells).
In these calibration measurements, the ratio Fmax/
Fmin was consistently less than that obtained in the cuvette (see Fig. 1, inset). This observation suggested either that a residual "pedestal" of Ca2+-insensitive fluorescence contributed to Fmin, or alternatively that Fmax
was consistently underestimated in these experiments. To address the second possibility that superfusion with
high Ca2+ solution in the presence of ionomycin might
not have raised Ca2+i sufficiently to completely saturate
fluo-3 fluorescence, similar measurements were carried
out in which saponin was used instead of ionomycin to
disrupt the outer segment membrane more extensively. While there was little difference between the results obtained with saponin and ionomycin in zero-Ca2+ solution, superfusion with high Ca2+ solution led to a similar initial rise that was followed by a further delayed increase in fluorescence in the presence of saponin. This
second component of the fluorescence increase seems
most likely to have represented the release of fluo-3
compartmentalized or bound within the outer or inner
segments (Schnetkamp et al., 1991b), although the possibility that ionomycin and isotonic Ca2+ may have
failed to saturate dye fluorescence cannot be completely excluded. We therefore interpret the reduced
ratio Fmax/Fmin as representing a residual Ca2+- and
light-insensitive pedestal of fluorescence, which may
represent fluo-3 bound or compartmentalized within
the rod outer segment (McCarthy et al., 1994
).
To address the possibility that the light-induced decay of the fluo-3 fluorescence signal might have originated from membrane-permeant fluo-3 AM, which had
entered a subcellular compartment other than the cytoplasm, experiments were carried out in which the
membrane-impermeant fluo-3-free acid was incorporated into the cytoplasm from a patch pipette. Fig. 5
compares the fluorescence responses from rods loaded
by incubation with fluo-3 AM (Fig. 5 A), and by 4 min
of whole cell recording with a patch pipette containing
an artificial intracellular solution that included 100 µM
fluo-3 pentapotassium salt (Fig. 5 B). It can be seen that
the fluorescence signal declined in a similar manner
during the first laser exposure (Fig. 5, trace 1) and remained depressed for the second (Fig. 5, trace 2), irrespective of the means by which fluo-3 had been incorporated. In Fig. 5 B, a slight decay in fluorescence occurred on the second laser exposure (trace 2); the
origin of this residual relaxation is not clear. Nevertheless, after complete suppression of the circulating current, a comparable level of residual fluorescence remained in both cases, despite the fact that the membrane-impermeant form of fluo-3 would be expected to
be less readily compartmentalized. Furthermore, qualitatively similar results were obtained in experiments in
which incubation with fluo-3 AM was carried out at 4°C
instead of room temperature, a procedure that is believed to decrease dye compartmentalization. These observations suggest that the residual pedestal of fluorescence may have originated instead from nonspecific
binding of the dye within the outer segment cytoplasm (McCarthy et al., 1994).
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Two exponential time constants were fitted to the initial decline in fluorescence signal in the eight rods loaded with fluo-3-free acid. The values for these time constants are plotted in the histograms of Fig. 3 as the open columns, and can be seen to fall within the same range as those from rods loaded with fluo-3 AM (filled columns). These results suggest that the light-induced decline in fluo-3 fluorescence represents a fall in Ca2+i within the cytoplasm itself, rather than another compartment within the outer segment to which only fluo-3 AM would have access. Consequently, incubation with fluo-3 AM was used to incorporate the dye in all subsequent experiments.
Changes in Ca2+i Induced by Bleaching
The principal objective of this study was to investigate how Ca2+i changes in the rod outer segment after photopigment bleaching. This was achieved by making repeated measurements of fluo-3 fluorescence from a single salamander rod when dark adapted, after exposure to laser light sufficiently intense to bleach a substantial fraction of the photopigment, and after regeneration with 11-cis-retinal, as illustrated in Figs. 6 and 7. First, the sensitivity of the dark-adapted rod was measured with dim flashes. Then laser pulses were delivered to investigate the decline of Ca2+i from its dark-adapted level (Fig. 6 A). The corresponding fluorescence intensity evoked by the first laser pulse has been plotted in Fig. 7 C, together with the level to which it declined after the bleach-induced suppression of the dark current. These values reflect relative Ca2+i levels before and immediately after the bleaching laser exposure. These two sequences of laser illumination, each consisting of four 20-ms laser pulses, are calculated to have bleached >99% of the photopigment within the area of the laser spot (see METHODS), while scattered laser light is likely to have caused significant bleaching within the remainder of the outer segment.
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This substantial localized bleach reduced both the circulating current (Fig. 7 A) and the dim flash sensitivity (Fig. 7 B), which then progressively stabilized over the next 50 min until no further increases could be detected. At this point, a second dye fluorescence measurement was made (Fig. 6 B), which revealed that the peak fluo-3 fluorescence evoked by the first laser pulse was considerably reduced after bleaching, its magnitude falling to 56 ± 5% of its dark-adapted value (mean ± SEM, nine cells), a change that is significant at the 5% level (paired Student's t test). After these measurements from the bleached rod, the photopigment was regenerated by addition of vesicles containing the retinoid 11-cis-retinal to the bath solution, resulting in substantial restoration of the original sensitivity and dark current. Once sensitivity and circulating current had stabilized, a further dye fluorescence measurement from the same cells showed that peak fluo-3 fluorescence had been restored by 11-cis-retinal to 127 ± 20% of its original dark-adapted level before the bleaching laser exposures, a value that does not differ significantly from unity at the 5% level.
As noted above, a significant light- and Ca2+-insensitive pedestal of fluorescence remained after complete suppression of the circulating current. In some cells, this pedestal increased somewhat during the prolonged time course of the experiment. This may have reflected entry into the outer segment of dye previously compartmentalized in the inner segment, or slight movement of the outer segment relative to the laser spot. To attempt to compensate for this variability, the data were also analyzed by dividing the peak fluo-3 fluorescence for each cell by the corresponding value for this pedestal. When analyzed in this way, the normalized peak fluorescence fell significantly from 3.7 ± 0.4 times the pedestal value at the start of the experiment to 2.4 ± 0.3 after bleaching by the laser spot; a decrease of 35%. However, after regeneration of the photopigment with 11-cis-retinal, the normalized peak fluorescence rose again to 3.4 ± 0.4, a value not significantly different from the original level in darkness. These measurements indicate that when the dark current and sensitivity were depressed after bleaching, the fluo-3 fluorescence signal was depressed also, whereas regeneration of the photopigment with 11-cis-retinal restored all of these parameters to near their original dark-adapted values.
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DISCUSSION |
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Measurement of Ca2+i Using the Spot Confocal Technique
The spot confocal technique used in this study offers
several advantages when compared with other methods
for the measurement of Ca2+i. First, the use of a single
wavelength Ca2+ indicator allows high time resolution
since it is not necessary to change between excitation
or emission wavelengths, as would be the case for a ratiometric measurement (Grynkiewicz et al., 1985; Minta
et al., 1989
). Furthermore, the accuracy of the measurement is not compromised at values of Ca2+i much
lower than the dissociation constant, as would be the
case for a ratiometric Ca2+ indicator. Second, the confocally imaged laser spot can be used to excite and then
collect dye fluorescence from a well-defined region of
the outer segment (Escobar et al., 1994
). This is of particular importance in the intact rod since the ellipsoid
region of the inner segment loads heavily with fluo-3
AM and might otherwise contribute to the fluorescence
signal. Finally, this method can also be used for cells
with much smaller outer segments than the salamander
rods employed in this study, such as amphibian cones (Matthews et al., 1996b
) and mammalian rods.
A potential drawback of this single-wavelength measurement is that if significant fluo-3 bleaching were to take place during the laser exposure, the corresponding change in fluorescence would be indistinguishable from that caused by a real decrease in Ca2+i. However, several pieces of evidence indicate that significant dye bleaching is unlikely to have taken place during these measurements. First, if the total laser exposure is substantially reduced, either by attenuating the laser beam intensity or by presenting repeated brief laser pulses instead of a continuous exposure, then the time course of the subsequent decline in fluorescence is little affected. These approaches will each have reduced the laser exposure by at least a further order of magnitude. Second, if after the initial laser exposure the laser spot is displaced to a region of the outer segment that has not previously been excited directly, then fluo-3 fluorescence is depressed there also, even though the dye in this region has not previously been exposed to the laser spot. Third, repeated laser exposures show that fluo-3 fluorescence remains depressed after the first exposure of a dark-adapted rod to the laser and does not show a further relaxation within the time course of our measurements, consistent with the notion that Ca2+i has already reached its minimum level once the circulating current has been completely suppressed. Furthermore, the subsequent recovery of the fluorescence signal appears to take place in parallel with the gradual recovery of the dark current after photopigment bleaching. Finally, application of exogenous 11-cis-retinal restores the fluorescence signal along with circulating current and sensitivity. Taken together, these observations indicate that the decline in fluo-3 fluorescence during saturating illumination of a dark-adapted rod is a real physiological phenomenon and not an artifact of fluo-3 bleaching.
The excitation and emission wavelengths for fluo-3
fall relatively close to the peak of the absorbance spectrum for rhodopsin. Nonetheless, bleach-induced
changes in self-screening by rhodopsin or its photoproducts seems unlikely to have affected the intensity of fluo-3 fluorescence significantly for two reasons.
First, the absolute magnitude of such self-screening is
relatively small, corresponding to little over 10% for
randomly polarized transverse illumination of a dark-adapted salamander rod (Harosi, 1975). Second, the
exciting beam was sufficiently intense that the vast majority of the photopigment within the area of the laser
spot will have been bleached within a few milliseconds
of the initial laser exposure. Subsequent fluorescence
measurements taken a few seconds later once the circulating current was fully suppressed were made at a time
when meta III, the only photoproduct that absorbs at
wavelengths close to those of fluorescence excitation or
emission, will not yet have appeared (Baumann, 1972
;
Donner and Hemilä, 1975
). In contrast, tens of minutes later when Ca2+i was measured again once the responses of the bleached rod had stabilized, most of the
meta III produced by the initial laser exposure will subsequently have decayed, and therefore will not have
contributed to self-screening.
Light-induced Decline in Ca2+i
The decline in fluo-3 fluorescence that accompanies
the exposure of a dark-adapted rod to the laser spot is
consistent with the fall in Ca2+i known to take place
during the photoresponse (Yau and Nakatani, 1985;
McNaughton et al., 1986
; Ratto et al., 1988
; Gray-Keller and Detwiler, 1994
; McCarthy et al., 1994
). The fluorescence signal was best fitted by the sum of two exponential components of approximately equal amplitude but
with time constants differing by a factor of nearly 10. These observations agree well with the results obtained previously by others, who have also found that the decline in Ca2+i can be best fitted by two or three exponential components (Gray-Keller and Detwiler, 1994
;
McCarthy et al., 1996
). In contrast, the electrogenic current carried by Na+/Ca2+-K+ exchange can in many
cases be fitted adequately by just a single exponential
(Yau and Nakatani, 1985
). However, this discrepancy between the time courses of the fall in Ca2+i and the decline in the rate of Ca2+ efflux through the exchanger
may be more apparent than real. The fluorescent
probe measures the change in Ca2+i spatially averaged
over the volume of the outer segment encompassed by
the laser spot, whose diameter approaches that of the
outer segment. On the other hand, the exchange current is governed by Ca2+i near the plasma membrane.
The difference in their time courses may reflect the restricted radial diffusion and slowly equilibrating low affinity buffering of Ca2+ (McCarthy et al., 1996
). It is
also possible that the multiple components seen in the
decline in Ca2+i represent the presence of multiple
buffers of differing affinity and capacity within the rod
outer segment (Hodgkin et al., 1987
; Lagnado et al.,
1992
).
Calibration of these light-dependent fluorescence
signals by exposure of the outer segment to solutions
designed to raise or lower Ca2+i to levels much greater
than or much less than the Kd of fluo-3 indicated that
Ca2+i falls from ~670 nM in darkness to 30 nM during
response saturation. These values are broadly consistent with those obtained previously using other Ca2+
probes (McNaughton et al., 1986; Ratto et al., 1988
;
Gray-Keller and Detwiler, 1994
; McCarthy et al., 1994
;
Younger et al., 1996
). Since there was some uncertainty
in the determination of Fmax as a result of the difficulty
in reliably obtaining complete saturation of fluo-3 fluorescence when exposing the outer segment to ionomycin and high Ca2+, it seems possible that the value for
Ca2+i in darkness may have been overestimated somewhat in our study. In contrast, after complete suppression of the circulating current, fluo-3 fluorescence was
consistently greater than Fmin determined in situ by superfusing the outer segment with ionomycin and low Ca2+. Despite the bleach-induced saturation of the response by the preceding laser pulses, exposure to this
solution resulted in a substantial increase in circulating
current, indicating that Ca2+i must have been lowered
further from its already substantially reduced value after bleaching. This qualitative observation adds support
to our measurement of a value for Ca2+i significantly
greater than zero even when the circulating current was completely suppressed. This contrasts with the
much lower level of Ca2+i that would be expected if the
Na+/Ca2+-K+ exchanger were to attain thermodynamic
equilibrium (Cervetto et al., 1989
; Perry and McNaughton, 1993
). Therefore, these results suggest either that
the exchanger is inhibited before this equilibrium is attained (Schnetkamp et al., 1991a
), or that under these
conditions a residual Ca2+ influx, perhaps from the inner segment, may balance a slow exchange extrusion of
Ca2+.
Even when the outer segment was exposed to low
Ca2+ and ionomycin, considerable fluorescence could
still be excited, despite negligible fluo-3 fluorescence
under zero Ca2+ conditions in the cuvette (Fig. 1, inset).
It seems unlikely that this pedestal of fluorescence was
caused by compartmentalization of fluo-3 within the
outer segment (such as in the disks), since incubation
with fluo-3 AM at 4°C or incorporation of fluo-3-free acid, both of which would be expected to reduce dye
compartmentalization, produced a similar residual fluorescence after complete suppression of the circulating
current. It is also difficult to account for this pedestal in
terms of fluorescence from incompletely hydrolyzed dye, since fluo-3 exhibits negligible fluorescence in its
esterified form. Instead, it seems more probable that
this component of the total fluorescence may have
originated from nonspecific binding of fluo-3 within
the outer segment, perhaps to membrane phospholipids (McLaughlin and Brown, 1981) or to components
of the cytoskeleton. Broadly comparable effects have
been seen in previous studies that used fura-2 (McCarthy et al., 1994
) or indo-1 (Gray-Keller and Detwiler,
1994
). The pedestal of fluorescence that we observe appears to resemble most closely the slowly quenched
pool of fura-2 fluorescence (McCarthy et al., 1994
),
since we saw no sign of the artifactual increases in Ca2+i
that have been reported with free indo-1 (Gray-Keller
and Detwiler, 1994
). We therefore believe that once
this pedestal is excluded, the light- and Ca2+-dependent fluo-3 fluorescence that remains is likely to represent a "well-behaved" measure of Ca2+i, as was the case
for the rapidly quenched pool of fura-2 fluorescence (McCarthy et al., 1994
).
Changes in Ca2+i After Photopigment Bleaching
The primary goal of this study was to investigate
changes in rod outer segment Ca2+i after photopigment bleaching. Our measurements show that fluo-3 fluorescence is significantly depressed after bleaching,
but recovers to a level not significantly different from
the original dark-adapted value after regeneration of
the photopigment with 11-cis-retinal, which also restores circulating current, response kinetics, and sensitivity (Jones et al., 1989; Corson et al., 1990
). These systematic changes in fluorescence intensity indicate that
the bleach-induced suppression of circulating current
is accompanied by a persistent decrease in Ca2+i, which
is restored when the photopigment is regenerated.
Since bleach-induced changes in photoresponse sensitivity and kinetics are also abolished if this fall in Ca2+i
is prevented (Matthews et al., 1996c
), our observations
are consistent with the notion that the reduction in
Ca2+i after bleaching is responsible for the adaptation
that ensues, and that its restoration after the regeneration of the photopigment leads to the recovery of dark-adapted sensitivity.
Outer segment Ca2+i is believed to be controlled by
the dynamic balance between Ca2+ influx through the
light-sensitive channel (Yau and Nakatani, 1984; Hodgkin
et al., 1985
) and efflux via Na+/Ca2+-K+ exchange (Yau
and Nakatani, 1985
; Hodgkin et al., 1987
; Lagnado et
al., 1992
). Partial suppression of the circulating current by steady light is known to cause a graded decrease in
Ca2+i (Gray-Keller and Detwiler, 1994
, 1996
; Younger et
al., 1996
), which is believed to be largely, if not exclusively, responsible for light adaptation (Koutalos et al.,
1995
; Matthews, 1995
, 1996
; Koutalos and Yau, 1996
).
It is difficult in our experiments to make a quantitative
comparison between the magnitude of the fall in Ca2+i
and the degree of suppression of the circulating current by bleaching, since the laser spot excites fluorescence and bleaches pigment within only a small region
of the outer segment, while the suction pipette collects
a fixed fraction of the entire outer segment current.
The suppression of circulating current is believed to be
closely localized to the site of illumination or bleaching (Lamb et al., 1981
; Baylor and Lamb, 1982
; Cornwall et
al., 1990
); thus, it seems likely that the degree of current suppression in the area of the outer segment illuminated by the laser spot will have been rather greater
than indicated by the suction pipette recording. Nonetheless, the partial suppression of both circulating current and fluo-3 fluorescence by bleaching and their
near-complete restoration by 11-cis-retinal suggests that
they are modulated in a parallel fashion during the
photopigment cycle. This is consistent with the notion
that the persistent activation of the transduction cascade that follows bleaching (Cornwall and Fain, 1994
;
Cornwall et al., 1995
) leads, through partial suppression of the circulating current, to a reduction in Ca2+
influx and a corresponding fall in Ca2+i. This bleach-induced fall in Ca2+i will not only accelerate the rate of
cGMP production by guanylyl cyclase (Koch and Stryer,
1988
; Gorczyca et al., 1994
), but also act on the cyclic
nucleotide-gated channel (Hsu and Molday, 1993
; Nakatani et al., 1995
) and at other sites earlier in the
transduction cascade (Kawamura and Murakami, 1991
;
Kawamura, 1993
; Lagnado and Baylor, 1994
) to produce the characteristic adaptational changes in sensitivity and response waveform that accompany bleaching in the isolated rod, in much the same way as does
steady light.
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FOOTNOTES |
---|
Address correspondence to Dr. G.L. Fain, Department of Physiological Science, Life Sciences 3836, UCLA, Los Angeles, CA 90095. Fax: 310-825-4667; E-mail: gfain{at}ucla.edu
Received for publication 2 September 1997 and accepted in revised form 20 October 1997.
We thank Dr. J. Vergara for his assistance with early experiments and for generous advice with implementing the spot confocal technique, Mr. Z. Maler for construction of equipment, and Dr. R.C. Crouch for the provision of 11-cis-retinal.
This work was supported by grants EY-01844, EY-07026, and EY-01157 from the National Eye Institute of the National Institutes of Health, and by a grant from the Wellcome Trust.
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