J. Phygiol. (1977), 271, pp. 747-759 With 6 text-ftgurea Printed in Great Britain
747
REGENERATION OF RHODOPSIN IN FROG ROD OUTER SEGMENTS
BY K. AZUMA*, MASAMI AZUMAt AND WERNER SICKEL From the Department of Physiology, University of Cologne, Cologne 41, Germany
(Received 16 February 1977) SUMMARY
1. Bleaching/regeneration cycles were performed in perfused frog retina while the optical transmittance at suitable wave-lengths was measured continuously. Rhodopsin was identified from its spectral absorbance, its photosensitivity and from the kinetics of its regeneration. 2. In the absence of the pigment epithelium regeneration was complete when not more than 2-5 % of the rhodopsin initially present had been bleached. However, the cycles could be repeated to a total of regenerated rhodopsin exceeding that explicable on the utilization of stored chromophores. The rate of regeneration was fast, with 0-12 min-1 rate constant, following first order reaction kinetics. Under these conditions the cycle does not seem to involve stages beyond metarhodopsin II. With the moderate bleaching intensities used, half-time 53 min, the Bunsen-Roscoe law was obeyed up to 15 min, indicating a capacity for the photoproducts to be accommodated in situ for subsequent regeneration. 3. It is concluded that only substantial bleaches, which exceed that capacity, result in hydrolysed chromophores. These surplus chromophores become esterified and are temporarily taken up by the pigment epithelium to be re-entered into the visual cycle as fast as they can be processed by the regenerative machinery of the rod outer segments. INTRODUCTION
Rhodopsin, the visual pigment of the outer segments of retinal rods, mediates twilight vision. It is used up in the process and, for maintained light sensitivity, has to be regenerated. Regeneration of rhodopsin appears to involve the participation of the pigment epithelium, for little, if any, * Present address: Department of Biology, Osaka Medical College, Takatsuki, Osaka, Japan.
t Present address: Department of Health Science, Osaka Kyoiku Univ., Tennoji-ku, Osaka, Japan.
K. AZUMA, M. AZUMA AND W. SICKEL regeneration is observed in its absence (Ewald & Kuhne, 1878). Lack of regeneration after substantial bleaching in a retina detached from its pigment epithelium has been noted by many, including the present, authors (Baumann, 1972b). However, two observations suggest that regneration may be quite significant within the retina proper: (i) the delay of the rod-mediated off-effect, the e-wave of the electroretinogram, becomes shorter after bleaching (Crescitelli & Sickel, 1968), but returns to its former delayed position in subsequent darkness, with a time course different from that of the b-wave recovery but plausible for pigment regeneration; (ii) if regeneration is energy dependent (Zewi, 1939), it should be possible to specify the extra-energy expenditure involved. Extra oxygen uptake following and in proportion to a previous light exposure had been measured and tentatively been attributed to the powering of the regeneration process (Sickel, 1973). Again, the putative electrical and metabolic manifestations were absent after strong light exposure. Therefore, regeneration was investigated in perfused frog retina after small bleaches (Cramer & Sickel, 1975). The extraction technique employed permitted the conclusion that regeneration, both thermal and photic, does occur under these conditions and to identify the regenerated product as 1 -cis-rhodopsin. But the scatter of data prevented us from determining the precise extent and the kinetics of the process. The present paper provides these data. They were obtained from measurements in perfused frog retina whose optical transmittance at suitable wave-lengths was measured continuously during and following moderate bleaching. Regeneration was fast (0-12 min- first order rate constant) and complete if less than 5 % of the pigment was bleached. The regenerative capacity of viable rod outer segments seems sufficient to process larger quantities of bleached pigment at the rate the chromophores are returned from the pigment epithelium (Bridges, 1976). 748
METHODS
Frogs (Rana eaculenta) were used throughout in this study. They were kept in a big vat at 5 0C in cooled running water. Before dissection they were dark adapted for at least 3 h in a tank at tap water temperature. Experiments were performed at a temperature of 20 0C. In dim red light the retinas were isolated from the eye cups in standard experimental solution and mounted in the perfusion chamber. Details of the procedure have been given earlier (Sickel, 1972); the perfusate contained (in
m-mole/l.): NaCl 80; KCl 2; CaCl2 0-15; MgCl2 0-1; phosphate buffer pH 7.7, 15;
glucose 5; oxygen 0.25. The chamber was positioned in the spectrophotometer, as shown in Fig. 1. The spectrophotometer was designed to permit continuous measurements of weak analysing lights of various wave-lengths transmitted through the retina, while the retina was being exposed to a second light source. The electrical responses to such
REGENERATION OF RHODOPSIN
749
exposure could be recorded. It was essentially that used earlier to measure pyridine nucleotide oxidation/reduction at a fixed wave-length (Sickel, 1965), with the bandpass filter of the stimulus beam and the guard filter in front of the photomultiplier replaced by a rotating slotted disk. The rotating sector wheel D, Fig. 1, interrupted the bleaching light in two places, (i) near N on the path from L2 to the retina, and (ii) near R, on the path from the retina to the photomultiplier. Since interruptions (i) and (ii) alternated, the bleaching light was always excluded from the photomultiplier, which only received the analyzing beam from L1. This was also chopped, but at a frequency so high (900 Hz) that the signal could easily be smoothed out by an RC-filter while still providing acceptable time resolution. Straightforward DC-amplification was employed. The output of the photomultiplier was backed-off and only small changes of the photocurrent were displayed. A calibration signal corresponding to a 1% transmittance change could be produced by tilting by 8.10 a fine wire mesh inserted in the analysing beam.
----------
LI
L2
NF
Sh1 ASh2 D
PM
S2;1
A
IC
A2 Fig. 1. The optical and electrical recording system. An isolated retina (R) is mounted in the measuring cell and perfused by a nutrient medium. The analyzing beam is derived from a tungsten source (L1), run from a stabilized power supply. The wave-lengths are selected by a monochromator (G, grating; SL and S2, entrance and exit slits). The fraction of light transmitted through the retina is received by the photomultiplier (PM). The photocurrent is zeroed and small changes are amplified (Al) and recorded. A fine wire mesh (C) may be tilted for calibration. The bleaching beam from a microscope lamp (L2) is limited at the short wave-lengths by a Schott filter GG 05 or OG 5. It may be attenuated by neutral filters (N). A rotating sector wheel (D) intersects at 900 Hz the light beams so that only the chopped analyzing beam is admitted to the photomultiplier. Electromagnetic shutters in either beam (Sh) permit flashes of the analyzing and bleaching lights, respectively, to be delivered to the retina. The responses to such stimulation, electroretinograms, are led off the perfusate by silver/silver chloride electrodes, amplified (A2) and recorded.
750
K. AZUMA, M. AZUMA AND W. SICKEL
Calibration and cakculation8 The output of the monochromator was too weak to be measured with the calibrated instruments available. Therefore, the intensities used for analyzing were matched, using ERG responses to 1 see stimuli from either beam. The 'white' bleaching beam, which at maximum intensity produced 10 lm/m2 at the preparation, had to be attenuated by 5 log units for the match. The intensity of the monochromatic analyzing light was thus estimated to be less than 10-11 W/Cm2. As a stimulus it resulted in 30 ,#V b-waves; as a background during the measurements it attenuated the responses to scotopic white test stimuli but little, and caused bleaching of rhodopsin of less than 0-1% per hour, which is below the resolution set by the stability of the technique. The bleaching light was used with and without cut-off filters to eliminate the short wave-lengths of the spectrum. The filters had 50% transmission at 450 nm (Schott GG 05) and 550 nm (Schott OG 5), respectively. Its bleaching effect was calculated from the intensity of 500 nm light received by the photomultiplier. If I = intensity of the incident light, measured without retina, I, = undetermined portion of scattered light, which, from constant ERG responses, was assumed to be constant during the experiment and independent of concentration (Dartnall, 1961), = 10 light passing the retina before bleach, It = light passing the retina at time t, 40 = light passing the retina after total bleach; then
the initial amount of rhodopsin,
Rho = log I -log I' -00
the amount of rhodopsin bleached, Rh* = log 1t
I10,
the fraction of rhodopsin
bleached, Bh
=
log
Itlog log
RESULTS
Because it was the objective of this study to investigate small changes of the rhodopsin concentration, weak bleaching lights were used. In order to find out whether these unusually low intensities resulted in peculiar effects, a series of experiments was performed in which retinas were exposed to the standard bleaching light, 10 lm/m2 cut-off at 450 nm, for different lengths of time while the optical transmittance at 500 nm was being recorded. At various times the recording was interrupted, the photomultiplier protected and the retina exposed to a strong white light from an auxiliary source (not shown in Fig. 1) for total bleaching. Afterwards the transmittance at 500 nm was again recorded for some time to determine the total loss of optical density, which generally was close to 0-3.
751 REGENERATION OF RHODOPSIN Fig. 2 shows the results of seven such experiments. Half-bleach required an exposure of 53 min. Until that time rhodopsin seemed to disappear nearly linearly (curve 1); however, the total experimental period is better covered by an exponential time course, as seen from the logarithmic plot (curve 2).
1
-100 C
a
80
0
-o 0~~~~~~ .
60
40
0-4 cc
-c~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
0) C
o E
08
20 20
40 Time (min)
60
Fig. 2. Time course of bleaching of rhodopsin in perfused retina. The relative amounts of rhodopsin bleached were determined in seven retinas exposed for different durations to the full intensity bleaching light, 10 lmrm2, using Schott GG 05 filter to eliminate short wave-lengths (curve 1, fitted by eye; left ordinate). The straight line (curve 2; right ordinate) connects the data points when plotted as -ln( - Rh*/Rho) . Half-bleach was effected by an exposure of 53 min, or 15-66 log equiv. quanta500 x sec.
Because the bleaching process could be described by simple first order reaction kinetics, little interference seemed to be caused by the formation of products which absorb at the analyzing wave-length, such as metarhodopsin I or III. Accordingly, the increased transmittance immediately at the ends of the exposures did not further change afterwards, as was the case with briefer, more intense bleaching (Baumann, 1965). The amount of light required for a given bleaching effect characterizes the light sensitive material. The photosensitivity of rhodopsin in solution has been determined as 9 x 10-17 cm2 (Dartnall, Goodeve & Lythgoe, 1936). This figure may be visualized as the effective cross-section of a chromophore. It corresponds to 15-96 log quanta of Amax needed for halfbleach. The photosensitivity is higher in perfused frog retina, the corresponding figure being 15x86 (Baumann, 1965). Converting 'white' light into equivalent monochromatic quanta, with 1 lm = 1-46 x 1015 Q500/sec, would give 15-66 log quanta in the present case. This is a conservative
752
K. AZUMA, M. AZUMA AND W. SICKEL estimate, because the exact spectral distribution of the source has not been taken into account. But even this figure would not leave an argument for regeneration occurring during the bleaching periods. Experiments similar to those just described, but without the final bleaching, showed that no regeneration occurred in subsequent darkness if exposure to the bleaching light had been for more than one hour. But an increasing percentage of the rhodopsin bleached could be recovered in 2
3
~~~~4
5
2 min
jK~
5
AT=Jl%
24 0
10
20min|
Fig. 3. Changes of optical transmittance at 500 nm of perfused retina caused by repeated light exposures. The bleaching intensity was 10 lm/m2, 50% attenuated at 450 nm (Schott GG 05), the duration of the exposure 2 min each, intervals 20-30 min. Increased optical transmittance upwards. Bottom: the five cycles superimposed. The 1% transmittance change corresponded to 2.73% of the rhodopsin initially present bleached/regenerated.
the dark as the duration of the exposure was reduced. Fig. 3 shows that after an exposure of 2 min to the bleaching light, which bleached approx. 2 5 % of the rhodopsin initially present, this amount of rhodopsin was regenerated completely within less than 30 min, as judged by the loss and subsequent restoration of the optical density at 500 nm. Moreover, this cycle of bleaching/regeneration could be repeated several times. Eventually (see 5th cycle) regeneration would be incomplete and finally fail, as would the ERG responses. The total amount of rhodopsin regenerated in subsequent cycles exceeded 10 %. But a 10 % bleach would not be followed by complete regeneration, instead a fraction of the pigment would stay bleached, a greater fraction with greater bleaching. 5 % was about the maximum that could be completely regenerated. The contention that it is rhodopsin which is measured by the transmittance changes at 500 nm is supported by the photosensitivity of the absorbing material. More direct evidence for the specificity was sought from analyzing at suitable other wave-lengths. In addition to 500 nm, the wave-length close to the maximum absorption of rhodopsin, 600 nm,
REGENERATION OF RHODOPSIN
753 where the absorption of rhodopsin is very small, was used as well as 380 nm, absorbed by the bleaching products metarhodopsin II and retinal, and 420 nm, where there should be an isosbestic point between the parent pigment and the longer-lived products. Experiments of the kind of Fig. 4 600 nm AT=1 %
500
/~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
420
380
2 min
Fig. 4. Changes of optical transmittance at different wave-lengths caused by exposure of the retina to weak bleaching light. The bleaching light was 50% attenuated at 550 nm (Schott OG 5; orange light), otherwise as in Fig. 3. The recordings were obtained from top to bottom, with 30 min intervals between exposures.
showed that the bleaching effects were maximum at 500 and 380 nm, with a decrease and increase of optical density, respectively, and a tendency to return to the pre-exposure level in subsequent darkness. The transmittance changes were, therefore, considered to reflect the formation, and subsequent reconversion to rhodopsin, of metarhodopsin II or retinal. Care was taken in these experiments to exclude any possibility of photoisomerization of the photoproducts by using Schott OG 3 as a cut-off filter. Fig. 5 shows bleaching/regeneration cycles performed with lights of different intensities (I), namely the unattenuated intensity, one tenth and one quarter of it. The durations (t) of the exposures were adjusted for
K. AZUMA, M. AZUMA AND W. SICKEL 754 I x t to be constant. Nearly equal amounts of the rhodopsin were bleached in each of the cycles and regenerated in the dark. The inset shows the different rates of bleaching and a fairly uniform time course of regeneration. Thus the Bunsen-Roscoe law is obeyed over a considerable span of time, confirming a finding in the earlier studies of the metabolic correlates of regeneration.
1 5 min
/0
15 min
6 min Lo/° 4 / 10~~~~~~~~~/
AT=1 %
.:1.5
10
20 min
6; 15
Fig. 5. Changes of optical transmittance at 500 nm during and following the exposure of the retina to lights of different bleaching rates. Continuous recording, as in Fig. 3. The intensities (I) and durations (t) of the exposures adjusted for I x t = constant. Bottom: superposition of the three cycles, lined up at the ends of the exposure periods.
A more precise determination of the time course of regeneration was obtained from a number of experiments, each done in a fresh preparation under identical conditions. In Fig. 6 the results from nineteen retinas were averaged and normalized according to the amount of rhodopsin bleached by an exposure to the full intensity light of 2 min. Observation period was 25 min, when regeneration was almost complete. Half-return time from the bleached state was 6-7 min. When the relative amount of rhodopsin in the bleached state is plotted on a natural log scale, the data points fall closely on a straight line, which means that regeneration follows first order reaction kinetics. The rate constant is 0-12 min'. This value compares with that found for rhodopsin regeneration in human eyes, 0-17 min-, which likewise follows first order kinetics (Rushton, 1961).
REGENERATION OF RHODOPSIN
755 3
110 0. 0
-0
82 ~
.~~~~~ 06
~
~
~
~
0)6
0
~~~~~~~~~~~
`01 0-4 C
0
E 0-2 0
0
10 Time (min)
20
Fig. 6. Time course of regeneration of rhodopsin in perfused retina. Nineteen retinas were exposed for 2 min to the full intensity bleaching light of 10 lmIm2, 50 % attenuated at 450 nm by Schott filter GG 05. The changes of optical transmittance at 500 nm were recorded during and following the exposure, and again after total bleaching. The relative amounts of rhodopsin in the bleached state at the ends of the 2 min bleaching periods (t = 0) were normalized. Vertical lines represent standard deviations. The connecting line (curve 1; left ordinate) was drawn by hand. The straight line (curve 2; right ordinate) connects the data points when plotted as -ln (Rh*IRho). First order rate constant of regeneration was 0 12 min-'. DISCUSSION
'It is generally assumed that, in vertebrates, regeneration (other than photoregeneration) does not take place before hydrolysis of visual pigment. This view is consistent with present experimental evidence, but it has not been critically tested' (Rodieck, 1973, p. 222). Hydrolysis, the detachment of the chromophore, is followed by its distribution even beyond the confines of the rod outer segment, into the pigment epithelium. Therefore, regeneration is greatly reduced in a retina detached from its pigment epithelium. What little is left, is sometimes considered immaterial. Proportionally, however, it may be quite substantial, provided a small fraction only of the visual pigment is bleached (Figs. 3-6). In that case the chromophore must still be held in place, i.e. the bleaching sequence be terminated before hydrolysis, at one of the metarhodopsin stages, at which the chromophore is known to be still orientated (Wald, Brown & Gibbons, 1962). Measuring small changes is technically more demanding, but can be
K. AZUMA, M. AZUMA AND W. SICKEL 756 achieved. A big advantage comes from continuous analysis, instead of in successive samples, a further advantage is provided by simultaneous recording of the ERG, the preparation's answer as to its functional state and stability and a safeguard against deterioration. From the total number of light quanta needed to bleach half of the rhodopsin, which was an upper estimate, the photosensitivity of the preparation was found to be higher than for rhodopsin in solution. This can be explained by the orientation of molecules and funnelling effects (Rushton, 1956). Considering that this latter effect may be reduced in a flat retina preparation, the sensitivity is not much below that of human rhodopsin in vivo (Alpern & Pugh, 1974). The experiments employing selected wave-lengths do not provide the complete spectrum. But the spectra of extracts obtained under comparable condition (Cramer & Sickel, 1975) were considered a sufficient supplement. The measurements at 600, 500, 420 and 380 nm indicated that of the light absorbing candidates (cf. Baumann, 1972a) only two were involved in the bleaching/regeneration cycles: rhodopsin absorbing maximally at 500 nm and a product absorbing at 380 nm, with an isosbestic point in between. Metarhodopsin III465 did not appear under the conditions. This would also follow from work of Donner & Hemili (1975) who investigated the longerlived photoproducts as a function of the amount of rhodopsin bleached. Therefore, preference was given to an interpretation of the ultra-violet absorbing material as metarhodopsin "1380 rather than retinal380, which could otherwise not be discriminated optically. The kinetics of both bleaching and regeneration, when measured by the optical transmittance at 500 nm, could be approximated by a simple exponential time course, with minor departures neglected at present. Thus, the analysis of small amounts of bleaching/regeneration seems to be quite straightforward, with little interference from other coloured products. Metarhodopsin '478 would not be expected to cause much disturbance because its generation and decay are too fast. Metarhodopsin H380, which was observed to build up and decay in pace with the disappearance and reformation of rhodopsin, absorbs sufficiently far away. Metarhodopsin I11465 is not generated to any measurable extent, and this seems true for species beyond that stage of the visual cycle. The speed of regeneration in perfused retina at 20 'C is remarkably high; it is higher in fact than has been found, on stronger bleaching, in eye cups or intact frog eyes, i.e. with the retina-pigment epithelium contact still preserved (for compiled data, see Baumann, 1972b). The quantity of rhodopsin that could be regenerated was limited. The precise limit was not determined. 10 % of the total complement of rhodopsin could be regenerated in four successive bleaching/regeneration cycles (and probably some
REGENERATION OF RHODOPSIN 757 more, had more than 2-5 %, namely up to 5 %, been bleached each time). 10% is considered a limit for 'autoregeneration', which has been explained as being fed from a store of corresponding size of chromophores in the outer segment (Donner & Hemili, 1975). But the actual measurements have meanwhile revealed that this store is much smaller, namely 2 %, and presumably serves to feed renewal rather than regeneration (Bridges, 1976). Neither is the store exhausted, but is replenished each time, and the amount of rhodopsin regenerated is not a trivial fraction, but all of what had been bleached before. Thus it is clear that rod outer segments have the capacity to handle small bleaching loads very efficiently. The mechanisms involved seem to be delicate, for regeneration can easily be impaired by oxygen lack (Zewi, 1939) or by the addition of cyanide to the perfusate, or even by an inadequate rate of perfusion. When measuring the extra oxygen uptake associated with regeneration it was noted that it occurred not during the illumination but only afterwards; a debt had accumulated (Sickel, 1972). It was concluded that regeneration did not take place until after the light had been removed. This same behaviour is now seen again in the direct measurements of Fig. 5. Although the limits ofthe validity ofthe Bunsen-Roscoe summation law were not further probed, it is obvious that the retina, within limits, can and does stock the bleaching products to be regenerated. With more intense bleaching lights the I x t law has been found to hold for up to 45 see in human rods (Campbell & Rushton, 1955), with measurable regeneration starting after that time. The metarhodopsin II pool is filled up under these conditions; it offers a signal of ongoing regeneration as it is depleted (Donner & Reuter, 1968). Thus, it is only the surplus chromophores that cannot immediately be taken care of by the regenerating machinery or be accommodated locally which are detached and leave the outer segments of the rods. From Bridges' (1976) results there seems to be a fairly indiscriminate traffic back and forth of 1 1-cis- and all-trans-isomers, facilitated by their esterification to fatty acids. The rate of transport Bridges found, 130 molecules per second per square ,tm of surface of the rod outer segment, is perfectly matched to the rate of regeneration reported here, 12 % per minute, to keep the rod outer segments busy regenerating at their maximum rate as long as there is supply. The rod outer segment-pigment epithelium 'shuttle' first described by Jansco6 & Jansco (1936) seems to make sense in that detached prosthetic groups are remotely stored for later use, but taken out of the danger zone where they might otherwise combine with the opsin at 'wrong' sites 27
PHY 271
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K. AZUMA, M. AZUMA AND W. SICKEL (Futterman & Andrews, 1964). Rate limitations on the detour of the chromophores might also explain extravagant time courses of regeneration reported in the literature, with lag periods and periods of linear regeneration (e.g. Peskin, 1942; Zewi, 1939). A frog in a natural habitat does not make excessive use of the shuttle. The authors are indebted to Professor W. A. H. Rushton for reading the manuscript and giving his valuable advice. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to W. Sickel. Research Fellowships were held by M. Azuma from the Ministry of Education, Japan, and by K. Azuma from the Alexander v. Humboldt Stiftung. REFERENCES ALPERN, M. & PUGH, E. N., JR. (1974). The density and photosensitivity of human rhodopsin in the living retina. J. Phy8iol. 237, 341-370. BAumANw, CH. (1965). Die Photosensitivitat des Sehpurpurs in der isolierten Netzhaut. Vieion Re8. 5, 425-434. BAumMANN, CH. (1972a). Kinetics of slow thermal reactions during the bleaching of rhodopsin in the perfused frog retina. J. Phy8iol. 222, 643-663. BAuMANN, CH. (1972b). The regeneration and renewal of visual pigment in vertebrates. In Handbook of Sensory Phy8iology, vol. vn/1, ed. H. J. A. DAUTNALL. Berlin, Heidelberg, New York: Springer Verlag. BRIDGES, C. D. B. (1976). Vitamin A and the role of the pigment epithelium during bleaching and regeneration of rhodopsin in the frog eye. Expl Eye Res. 22, 43455. CAMPBELL, F. W. & RUSHTON, W. A. H. (1955). Measurements of the scotopic pigment in the living human eye. J. Phy8iol. 130, 131-147. CRAMER, C. & SICKEL, W. (1975). Measuring regeneration of rhodopsin by an extraction technique in perfused vertebrate retina. Pfager8 Arch. ge8. Physiol. 355, RIIO. CREsCTrELLI, F. & SICKEL, E. (1968). Delayed off-responses recorded from the isolated frog retina. Viaion Re8. 8, 801-816. DARTNALL, H. J. A. (1961). Visual pigments before and after extraction from visual cells. Proc. R. Soc. B 154, 250-266. DARTNALL, H. J. A., GOODEVE, C. F. & LYTHGOE, R. J. (1936). The quantitative analysis of the photochemical bleaching of visual purple solutions in monochromatic light. Proc. R. Soc. A 156, 158-170. DOMNER, K. 0. & HEMILA, S. (1975). Kinetics of long-lived rhodopsin photoproducts in the frog retina as a function of the amount bleached. Vieion Res. 15, 985-995. DONNER, K. 0. & REUTER, T. (1968). Visual adaptation of the rhodopsin rods in the frog's retina. J. Phyaiol. 199, 59-87. EWALD, A. & KUHNE, W. (1878). Untersuchungen uber den Sehpurpur. II. Die Entstehung der Retinafarbe. Unter8. physiol. Inst. Heidelb. 1, 248-290. FUTTERMAN, S. & ANDREWs, J. S. (1964). The fatty acid composition of human retinal vitamin A ester and the lipids of human retinal tissue. Invest. Ophthal. 3, 441-444.
JANcs6, N. v. & JAwcs6, H. v. (1936). Fluoreszenzmikroskopische Beobachtungen der reversiblen Vitamin-A-Bildung in der Netzhaut wahrend des Sehaktes. Biochem. Z. 287, 289-290. PESKrN, J. C. (1942). The regeneration of visual purple in the living animal. J. gen. Phy8iol. 26, 27-47.
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RODIECK, R. W. (1973). The Vertebrate Retina. San Francisco: W. H. Freeman. RUSHTON, W. A. H. (1956). The difference spectrum and the photosensitivity of rhodopsin in the living human eye. J. Phygiol. 134, 11-29. RUSHTON, W. A. H. (1961). Rhodopsin measurement and dark-adaptation in a subject deficient in cone vision. J. Phyuiol. 156, 193-205. SICKEL, W. (1965). Respiratory and electrical responses to light stimulation in the retina of the frog. Science, N.Y. 148, 648-651. SICKEL, W. (1972). Retinal metabolism in dark and light. In Handbook of Sensory Physiology, vol. vI/2, ed. M. G. F. FuoRTEs, pp. 667-727. Berlin, Heidelberg, New York: Springer Verlag. SICKEL, W. (1973). Energy in vertebrate photoreceptor function. In Biochemistry and Physiology of Virgal Pigments, ed. H. LANGER. Berlin, Heidelberg, New York: Springer Verlag. WALD, G., BROWN, P. K. & GIBBONS, 1. R. (1962). Visual excitation. A chemoanatomical study. Symp. Soc. exp. Biol. 16, 32-57. ZEWI, M. (1939). On the regeneration of visual purple. Acta Soc. Sci. fenn. 2, 1-56.
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747
REGENERATION OF RHODOPSIN IN FROG ROD OUTER SEGMENTS
BY K. AZUMA*, MASAMI AZUMAt AND WERNER SICKEL From the Department of Physiology, University of Cologne, Cologne 41, Germany
(Received 16 February 1977) SUMMARY
1. Bleaching/regeneration cycles were performed in perfused frog retina while the optical transmittance at suitable wave-lengths was measured continuously. Rhodopsin was identified from its spectral absorbance, its photosensitivity and from the kinetics of its regeneration. 2. In the absence of the pigment epithelium regeneration was complete when not more than 2-5 % of the rhodopsin initially present had been bleached. However, the cycles could be repeated to a total of regenerated rhodopsin exceeding that explicable on the utilization of stored chromophores. The rate of regeneration was fast, with 0-12 min-1 rate constant, following first order reaction kinetics. Under these conditions the cycle does not seem to involve stages beyond metarhodopsin II. With the moderate bleaching intensities used, half-time 53 min, the Bunsen-Roscoe law was obeyed up to 15 min, indicating a capacity for the photoproducts to be accommodated in situ for subsequent regeneration. 3. It is concluded that only substantial bleaches, which exceed that capacity, result in hydrolysed chromophores. These surplus chromophores become esterified and are temporarily taken up by the pigment epithelium to be re-entered into the visual cycle as fast as they can be processed by the regenerative machinery of the rod outer segments. INTRODUCTION
Rhodopsin, the visual pigment of the outer segments of retinal rods, mediates twilight vision. It is used up in the process and, for maintained light sensitivity, has to be regenerated. Regeneration of rhodopsin appears to involve the participation of the pigment epithelium, for little, if any, * Present address: Department of Biology, Osaka Medical College, Takatsuki, Osaka, Japan.
t Present address: Department of Health Science, Osaka Kyoiku Univ., Tennoji-ku, Osaka, Japan.
K. AZUMA, M. AZUMA AND W. SICKEL regeneration is observed in its absence (Ewald & Kuhne, 1878). Lack of regeneration after substantial bleaching in a retina detached from its pigment epithelium has been noted by many, including the present, authors (Baumann, 1972b). However, two observations suggest that regneration may be quite significant within the retina proper: (i) the delay of the rod-mediated off-effect, the e-wave of the electroretinogram, becomes shorter after bleaching (Crescitelli & Sickel, 1968), but returns to its former delayed position in subsequent darkness, with a time course different from that of the b-wave recovery but plausible for pigment regeneration; (ii) if regeneration is energy dependent (Zewi, 1939), it should be possible to specify the extra-energy expenditure involved. Extra oxygen uptake following and in proportion to a previous light exposure had been measured and tentatively been attributed to the powering of the regeneration process (Sickel, 1973). Again, the putative electrical and metabolic manifestations were absent after strong light exposure. Therefore, regeneration was investigated in perfused frog retina after small bleaches (Cramer & Sickel, 1975). The extraction technique employed permitted the conclusion that regeneration, both thermal and photic, does occur under these conditions and to identify the regenerated product as 1 -cis-rhodopsin. But the scatter of data prevented us from determining the precise extent and the kinetics of the process. The present paper provides these data. They were obtained from measurements in perfused frog retina whose optical transmittance at suitable wave-lengths was measured continuously during and following moderate bleaching. Regeneration was fast (0-12 min- first order rate constant) and complete if less than 5 % of the pigment was bleached. The regenerative capacity of viable rod outer segments seems sufficient to process larger quantities of bleached pigment at the rate the chromophores are returned from the pigment epithelium (Bridges, 1976). 748
METHODS
Frogs (Rana eaculenta) were used throughout in this study. They were kept in a big vat at 5 0C in cooled running water. Before dissection they were dark adapted for at least 3 h in a tank at tap water temperature. Experiments were performed at a temperature of 20 0C. In dim red light the retinas were isolated from the eye cups in standard experimental solution and mounted in the perfusion chamber. Details of the procedure have been given earlier (Sickel, 1972); the perfusate contained (in
m-mole/l.): NaCl 80; KCl 2; CaCl2 0-15; MgCl2 0-1; phosphate buffer pH 7.7, 15;
glucose 5; oxygen 0.25. The chamber was positioned in the spectrophotometer, as shown in Fig. 1. The spectrophotometer was designed to permit continuous measurements of weak analysing lights of various wave-lengths transmitted through the retina, while the retina was being exposed to a second light source. The electrical responses to such
REGENERATION OF RHODOPSIN
749
exposure could be recorded. It was essentially that used earlier to measure pyridine nucleotide oxidation/reduction at a fixed wave-length (Sickel, 1965), with the bandpass filter of the stimulus beam and the guard filter in front of the photomultiplier replaced by a rotating slotted disk. The rotating sector wheel D, Fig. 1, interrupted the bleaching light in two places, (i) near N on the path from L2 to the retina, and (ii) near R, on the path from the retina to the photomultiplier. Since interruptions (i) and (ii) alternated, the bleaching light was always excluded from the photomultiplier, which only received the analyzing beam from L1. This was also chopped, but at a frequency so high (900 Hz) that the signal could easily be smoothed out by an RC-filter while still providing acceptable time resolution. Straightforward DC-amplification was employed. The output of the photomultiplier was backed-off and only small changes of the photocurrent were displayed. A calibration signal corresponding to a 1% transmittance change could be produced by tilting by 8.10 a fine wire mesh inserted in the analysing beam.
----------
LI
L2
NF
Sh1 ASh2 D
PM
S2;1
A
IC
A2 Fig. 1. The optical and electrical recording system. An isolated retina (R) is mounted in the measuring cell and perfused by a nutrient medium. The analyzing beam is derived from a tungsten source (L1), run from a stabilized power supply. The wave-lengths are selected by a monochromator (G, grating; SL and S2, entrance and exit slits). The fraction of light transmitted through the retina is received by the photomultiplier (PM). The photocurrent is zeroed and small changes are amplified (Al) and recorded. A fine wire mesh (C) may be tilted for calibration. The bleaching beam from a microscope lamp (L2) is limited at the short wave-lengths by a Schott filter GG 05 or OG 5. It may be attenuated by neutral filters (N). A rotating sector wheel (D) intersects at 900 Hz the light beams so that only the chopped analyzing beam is admitted to the photomultiplier. Electromagnetic shutters in either beam (Sh) permit flashes of the analyzing and bleaching lights, respectively, to be delivered to the retina. The responses to such stimulation, electroretinograms, are led off the perfusate by silver/silver chloride electrodes, amplified (A2) and recorded.
750
K. AZUMA, M. AZUMA AND W. SICKEL
Calibration and cakculation8 The output of the monochromator was too weak to be measured with the calibrated instruments available. Therefore, the intensities used for analyzing were matched, using ERG responses to 1 see stimuli from either beam. The 'white' bleaching beam, which at maximum intensity produced 10 lm/m2 at the preparation, had to be attenuated by 5 log units for the match. The intensity of the monochromatic analyzing light was thus estimated to be less than 10-11 W/Cm2. As a stimulus it resulted in 30 ,#V b-waves; as a background during the measurements it attenuated the responses to scotopic white test stimuli but little, and caused bleaching of rhodopsin of less than 0-1% per hour, which is below the resolution set by the stability of the technique. The bleaching light was used with and without cut-off filters to eliminate the short wave-lengths of the spectrum. The filters had 50% transmission at 450 nm (Schott GG 05) and 550 nm (Schott OG 5), respectively. Its bleaching effect was calculated from the intensity of 500 nm light received by the photomultiplier. If I = intensity of the incident light, measured without retina, I, = undetermined portion of scattered light, which, from constant ERG responses, was assumed to be constant during the experiment and independent of concentration (Dartnall, 1961), = 10 light passing the retina before bleach, It = light passing the retina at time t, 40 = light passing the retina after total bleach; then
the initial amount of rhodopsin,
Rho = log I -log I' -00
the amount of rhodopsin bleached, Rh* = log 1t
I10,
the fraction of rhodopsin
bleached, Bh
=
log
Itlog log
RESULTS
Because it was the objective of this study to investigate small changes of the rhodopsin concentration, weak bleaching lights were used. In order to find out whether these unusually low intensities resulted in peculiar effects, a series of experiments was performed in which retinas were exposed to the standard bleaching light, 10 lm/m2 cut-off at 450 nm, for different lengths of time while the optical transmittance at 500 nm was being recorded. At various times the recording was interrupted, the photomultiplier protected and the retina exposed to a strong white light from an auxiliary source (not shown in Fig. 1) for total bleaching. Afterwards the transmittance at 500 nm was again recorded for some time to determine the total loss of optical density, which generally was close to 0-3.
751 REGENERATION OF RHODOPSIN Fig. 2 shows the results of seven such experiments. Half-bleach required an exposure of 53 min. Until that time rhodopsin seemed to disappear nearly linearly (curve 1); however, the total experimental period is better covered by an exponential time course, as seen from the logarithmic plot (curve 2).
1
-100 C
a
80
0
-o 0~~~~~~ .
60
40
0-4 cc
-c~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
0) C
o E
08
20 20
40 Time (min)
60
Fig. 2. Time course of bleaching of rhodopsin in perfused retina. The relative amounts of rhodopsin bleached were determined in seven retinas exposed for different durations to the full intensity bleaching light, 10 lmrm2, using Schott GG 05 filter to eliminate short wave-lengths (curve 1, fitted by eye; left ordinate). The straight line (curve 2; right ordinate) connects the data points when plotted as -ln( - Rh*/Rho) . Half-bleach was effected by an exposure of 53 min, or 15-66 log equiv. quanta500 x sec.
Because the bleaching process could be described by simple first order reaction kinetics, little interference seemed to be caused by the formation of products which absorb at the analyzing wave-length, such as metarhodopsin I or III. Accordingly, the increased transmittance immediately at the ends of the exposures did not further change afterwards, as was the case with briefer, more intense bleaching (Baumann, 1965). The amount of light required for a given bleaching effect characterizes the light sensitive material. The photosensitivity of rhodopsin in solution has been determined as 9 x 10-17 cm2 (Dartnall, Goodeve & Lythgoe, 1936). This figure may be visualized as the effective cross-section of a chromophore. It corresponds to 15-96 log quanta of Amax needed for halfbleach. The photosensitivity is higher in perfused frog retina, the corresponding figure being 15x86 (Baumann, 1965). Converting 'white' light into equivalent monochromatic quanta, with 1 lm = 1-46 x 1015 Q500/sec, would give 15-66 log quanta in the present case. This is a conservative
752
K. AZUMA, M. AZUMA AND W. SICKEL estimate, because the exact spectral distribution of the source has not been taken into account. But even this figure would not leave an argument for regeneration occurring during the bleaching periods. Experiments similar to those just described, but without the final bleaching, showed that no regeneration occurred in subsequent darkness if exposure to the bleaching light had been for more than one hour. But an increasing percentage of the rhodopsin bleached could be recovered in 2
3
~~~~4
5
2 min
jK~
5
AT=Jl%
24 0
10
20min|
Fig. 3. Changes of optical transmittance at 500 nm of perfused retina caused by repeated light exposures. The bleaching intensity was 10 lm/m2, 50% attenuated at 450 nm (Schott GG 05), the duration of the exposure 2 min each, intervals 20-30 min. Increased optical transmittance upwards. Bottom: the five cycles superimposed. The 1% transmittance change corresponded to 2.73% of the rhodopsin initially present bleached/regenerated.
the dark as the duration of the exposure was reduced. Fig. 3 shows that after an exposure of 2 min to the bleaching light, which bleached approx. 2 5 % of the rhodopsin initially present, this amount of rhodopsin was regenerated completely within less than 30 min, as judged by the loss and subsequent restoration of the optical density at 500 nm. Moreover, this cycle of bleaching/regeneration could be repeated several times. Eventually (see 5th cycle) regeneration would be incomplete and finally fail, as would the ERG responses. The total amount of rhodopsin regenerated in subsequent cycles exceeded 10 %. But a 10 % bleach would not be followed by complete regeneration, instead a fraction of the pigment would stay bleached, a greater fraction with greater bleaching. 5 % was about the maximum that could be completely regenerated. The contention that it is rhodopsin which is measured by the transmittance changes at 500 nm is supported by the photosensitivity of the absorbing material. More direct evidence for the specificity was sought from analyzing at suitable other wave-lengths. In addition to 500 nm, the wave-length close to the maximum absorption of rhodopsin, 600 nm,
REGENERATION OF RHODOPSIN
753 where the absorption of rhodopsin is very small, was used as well as 380 nm, absorbed by the bleaching products metarhodopsin II and retinal, and 420 nm, where there should be an isosbestic point between the parent pigment and the longer-lived products. Experiments of the kind of Fig. 4 600 nm AT=1 %
500
/~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
420
380
2 min
Fig. 4. Changes of optical transmittance at different wave-lengths caused by exposure of the retina to weak bleaching light. The bleaching light was 50% attenuated at 550 nm (Schott OG 5; orange light), otherwise as in Fig. 3. The recordings were obtained from top to bottom, with 30 min intervals between exposures.
showed that the bleaching effects were maximum at 500 and 380 nm, with a decrease and increase of optical density, respectively, and a tendency to return to the pre-exposure level in subsequent darkness. The transmittance changes were, therefore, considered to reflect the formation, and subsequent reconversion to rhodopsin, of metarhodopsin II or retinal. Care was taken in these experiments to exclude any possibility of photoisomerization of the photoproducts by using Schott OG 3 as a cut-off filter. Fig. 5 shows bleaching/regeneration cycles performed with lights of different intensities (I), namely the unattenuated intensity, one tenth and one quarter of it. The durations (t) of the exposures were adjusted for
K. AZUMA, M. AZUMA AND W. SICKEL 754 I x t to be constant. Nearly equal amounts of the rhodopsin were bleached in each of the cycles and regenerated in the dark. The inset shows the different rates of bleaching and a fairly uniform time course of regeneration. Thus the Bunsen-Roscoe law is obeyed over a considerable span of time, confirming a finding in the earlier studies of the metabolic correlates of regeneration.
1 5 min
/0
15 min
6 min Lo/° 4 / 10~~~~~~~~~/
AT=1 %
.:1.5
10
20 min
6; 15
Fig. 5. Changes of optical transmittance at 500 nm during and following the exposure of the retina to lights of different bleaching rates. Continuous recording, as in Fig. 3. The intensities (I) and durations (t) of the exposures adjusted for I x t = constant. Bottom: superposition of the three cycles, lined up at the ends of the exposure periods.
A more precise determination of the time course of regeneration was obtained from a number of experiments, each done in a fresh preparation under identical conditions. In Fig. 6 the results from nineteen retinas were averaged and normalized according to the amount of rhodopsin bleached by an exposure to the full intensity light of 2 min. Observation period was 25 min, when regeneration was almost complete. Half-return time from the bleached state was 6-7 min. When the relative amount of rhodopsin in the bleached state is plotted on a natural log scale, the data points fall closely on a straight line, which means that regeneration follows first order reaction kinetics. The rate constant is 0-12 min'. This value compares with that found for rhodopsin regeneration in human eyes, 0-17 min-, which likewise follows first order kinetics (Rushton, 1961).
REGENERATION OF RHODOPSIN
755 3
110 0. 0
-0
82 ~
.~~~~~ 06
~
~
~
~
0)6
0
~~~~~~~~~~~
`01 0-4 C
0
E 0-2 0
0
10 Time (min)
20
Fig. 6. Time course of regeneration of rhodopsin in perfused retina. Nineteen retinas were exposed for 2 min to the full intensity bleaching light of 10 lmIm2, 50 % attenuated at 450 nm by Schott filter GG 05. The changes of optical transmittance at 500 nm were recorded during and following the exposure, and again after total bleaching. The relative amounts of rhodopsin in the bleached state at the ends of the 2 min bleaching periods (t = 0) were normalized. Vertical lines represent standard deviations. The connecting line (curve 1; left ordinate) was drawn by hand. The straight line (curve 2; right ordinate) connects the data points when plotted as -ln (Rh*IRho). First order rate constant of regeneration was 0 12 min-'. DISCUSSION
'It is generally assumed that, in vertebrates, regeneration (other than photoregeneration) does not take place before hydrolysis of visual pigment. This view is consistent with present experimental evidence, but it has not been critically tested' (Rodieck, 1973, p. 222). Hydrolysis, the detachment of the chromophore, is followed by its distribution even beyond the confines of the rod outer segment, into the pigment epithelium. Therefore, regeneration is greatly reduced in a retina detached from its pigment epithelium. What little is left, is sometimes considered immaterial. Proportionally, however, it may be quite substantial, provided a small fraction only of the visual pigment is bleached (Figs. 3-6). In that case the chromophore must still be held in place, i.e. the bleaching sequence be terminated before hydrolysis, at one of the metarhodopsin stages, at which the chromophore is known to be still orientated (Wald, Brown & Gibbons, 1962). Measuring small changes is technically more demanding, but can be
K. AZUMA, M. AZUMA AND W. SICKEL 756 achieved. A big advantage comes from continuous analysis, instead of in successive samples, a further advantage is provided by simultaneous recording of the ERG, the preparation's answer as to its functional state and stability and a safeguard against deterioration. From the total number of light quanta needed to bleach half of the rhodopsin, which was an upper estimate, the photosensitivity of the preparation was found to be higher than for rhodopsin in solution. This can be explained by the orientation of molecules and funnelling effects (Rushton, 1956). Considering that this latter effect may be reduced in a flat retina preparation, the sensitivity is not much below that of human rhodopsin in vivo (Alpern & Pugh, 1974). The experiments employing selected wave-lengths do not provide the complete spectrum. But the spectra of extracts obtained under comparable condition (Cramer & Sickel, 1975) were considered a sufficient supplement. The measurements at 600, 500, 420 and 380 nm indicated that of the light absorbing candidates (cf. Baumann, 1972a) only two were involved in the bleaching/regeneration cycles: rhodopsin absorbing maximally at 500 nm and a product absorbing at 380 nm, with an isosbestic point in between. Metarhodopsin III465 did not appear under the conditions. This would also follow from work of Donner & Hemili (1975) who investigated the longerlived photoproducts as a function of the amount of rhodopsin bleached. Therefore, preference was given to an interpretation of the ultra-violet absorbing material as metarhodopsin "1380 rather than retinal380, which could otherwise not be discriminated optically. The kinetics of both bleaching and regeneration, when measured by the optical transmittance at 500 nm, could be approximated by a simple exponential time course, with minor departures neglected at present. Thus, the analysis of small amounts of bleaching/regeneration seems to be quite straightforward, with little interference from other coloured products. Metarhodopsin '478 would not be expected to cause much disturbance because its generation and decay are too fast. Metarhodopsin H380, which was observed to build up and decay in pace with the disappearance and reformation of rhodopsin, absorbs sufficiently far away. Metarhodopsin I11465 is not generated to any measurable extent, and this seems true for species beyond that stage of the visual cycle. The speed of regeneration in perfused retina at 20 'C is remarkably high; it is higher in fact than has been found, on stronger bleaching, in eye cups or intact frog eyes, i.e. with the retina-pigment epithelium contact still preserved (for compiled data, see Baumann, 1972b). The quantity of rhodopsin that could be regenerated was limited. The precise limit was not determined. 10 % of the total complement of rhodopsin could be regenerated in four successive bleaching/regeneration cycles (and probably some
REGENERATION OF RHODOPSIN 757 more, had more than 2-5 %, namely up to 5 %, been bleached each time). 10% is considered a limit for 'autoregeneration', which has been explained as being fed from a store of corresponding size of chromophores in the outer segment (Donner & Hemili, 1975). But the actual measurements have meanwhile revealed that this store is much smaller, namely 2 %, and presumably serves to feed renewal rather than regeneration (Bridges, 1976). Neither is the store exhausted, but is replenished each time, and the amount of rhodopsin regenerated is not a trivial fraction, but all of what had been bleached before. Thus it is clear that rod outer segments have the capacity to handle small bleaching loads very efficiently. The mechanisms involved seem to be delicate, for regeneration can easily be impaired by oxygen lack (Zewi, 1939) or by the addition of cyanide to the perfusate, or even by an inadequate rate of perfusion. When measuring the extra oxygen uptake associated with regeneration it was noted that it occurred not during the illumination but only afterwards; a debt had accumulated (Sickel, 1972). It was concluded that regeneration did not take place until after the light had been removed. This same behaviour is now seen again in the direct measurements of Fig. 5. Although the limits ofthe validity ofthe Bunsen-Roscoe summation law were not further probed, it is obvious that the retina, within limits, can and does stock the bleaching products to be regenerated. With more intense bleaching lights the I x t law has been found to hold for up to 45 see in human rods (Campbell & Rushton, 1955), with measurable regeneration starting after that time. The metarhodopsin II pool is filled up under these conditions; it offers a signal of ongoing regeneration as it is depleted (Donner & Reuter, 1968). Thus, it is only the surplus chromophores that cannot immediately be taken care of by the regenerating machinery or be accommodated locally which are detached and leave the outer segments of the rods. From Bridges' (1976) results there seems to be a fairly indiscriminate traffic back and forth of 1 1-cis- and all-trans-isomers, facilitated by their esterification to fatty acids. The rate of transport Bridges found, 130 molecules per second per square ,tm of surface of the rod outer segment, is perfectly matched to the rate of regeneration reported here, 12 % per minute, to keep the rod outer segments busy regenerating at their maximum rate as long as there is supply. The rod outer segment-pigment epithelium 'shuttle' first described by Jansco6 & Jansco (1936) seems to make sense in that detached prosthetic groups are remotely stored for later use, but taken out of the danger zone where they might otherwise combine with the opsin at 'wrong' sites 27
PHY 271
758
K. AZUMA, M. AZUMA AND W. SICKEL (Futterman & Andrews, 1964). Rate limitations on the detour of the chromophores might also explain extravagant time courses of regeneration reported in the literature, with lag periods and periods of linear regeneration (e.g. Peskin, 1942; Zewi, 1939). A frog in a natural habitat does not make excessive use of the shuttle. The authors are indebted to Professor W. A. H. Rushton for reading the manuscript and giving his valuable advice. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to W. Sickel. Research Fellowships were held by M. Azuma from the Ministry of Education, Japan, and by K. Azuma from the Alexander v. Humboldt Stiftung. REFERENCES ALPERN, M. & PUGH, E. N., JR. (1974). The density and photosensitivity of human rhodopsin in the living retina. J. Phy8iol. 237, 341-370. BAumANw, CH. (1965). Die Photosensitivitat des Sehpurpurs in der isolierten Netzhaut. Vieion Re8. 5, 425-434. BAumMANN, CH. (1972a). Kinetics of slow thermal reactions during the bleaching of rhodopsin in the perfused frog retina. J. Phy8iol. 222, 643-663. BAuMANN, CH. (1972b). The regeneration and renewal of visual pigment in vertebrates. In Handbook of Sensory Phy8iology, vol. vn/1, ed. H. J. A. DAUTNALL. Berlin, Heidelberg, New York: Springer Verlag. BRIDGES, C. D. B. (1976). Vitamin A and the role of the pigment epithelium during bleaching and regeneration of rhodopsin in the frog eye. Expl Eye Res. 22, 43455. CAMPBELL, F. W. & RUSHTON, W. A. H. (1955). Measurements of the scotopic pigment in the living human eye. J. Phy8iol. 130, 131-147. CRAMER, C. & SICKEL, W. (1975). Measuring regeneration of rhodopsin by an extraction technique in perfused vertebrate retina. Pfager8 Arch. ge8. Physiol. 355, RIIO. CREsCTrELLI, F. & SICKEL, E. (1968). Delayed off-responses recorded from the isolated frog retina. Viaion Re8. 8, 801-816. DARTNALL, H. J. A. (1961). Visual pigments before and after extraction from visual cells. Proc. R. Soc. B 154, 250-266. DARTNALL, H. J. A., GOODEVE, C. F. & LYTHGOE, R. J. (1936). The quantitative analysis of the photochemical bleaching of visual purple solutions in monochromatic light. Proc. R. Soc. A 156, 158-170. DOMNER, K. 0. & HEMILA, S. (1975). Kinetics of long-lived rhodopsin photoproducts in the frog retina as a function of the amount bleached. Vieion Res. 15, 985-995. DONNER, K. 0. & REUTER, T. (1968). Visual adaptation of the rhodopsin rods in the frog's retina. J. Phyaiol. 199, 59-87. EWALD, A. & KUHNE, W. (1878). Untersuchungen uber den Sehpurpur. II. Die Entstehung der Retinafarbe. Unter8. physiol. Inst. Heidelb. 1, 248-290. FUTTERMAN, S. & ANDREWs, J. S. (1964). The fatty acid composition of human retinal vitamin A ester and the lipids of human retinal tissue. Invest. Ophthal. 3, 441-444.
JANcs6, N. v. & JAwcs6, H. v. (1936). Fluoreszenzmikroskopische Beobachtungen der reversiblen Vitamin-A-Bildung in der Netzhaut wahrend des Sehaktes. Biochem. Z. 287, 289-290. PESKrN, J. C. (1942). The regeneration of visual purple in the living animal. J. gen. Phy8iol. 26, 27-47.
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RODIECK, R. W. (1973). The Vertebrate Retina. San Francisco: W. H. Freeman. RUSHTON, W. A. H. (1956). The difference spectrum and the photosensitivity of rhodopsin in the living human eye. J. Phygiol. 134, 11-29. RUSHTON, W. A. H. (1961). Rhodopsin measurement and dark-adaptation in a subject deficient in cone vision. J. Phyuiol. 156, 193-205. SICKEL, W. (1965). Respiratory and electrical responses to light stimulation in the retina of the frog. Science, N.Y. 148, 648-651. SICKEL, W. (1972). Retinal metabolism in dark and light. In Handbook of Sensory Physiology, vol. vI/2, ed. M. G. F. FuoRTEs, pp. 667-727. Berlin, Heidelberg, New York: Springer Verlag. SICKEL, W. (1973). Energy in vertebrate photoreceptor function. In Biochemistry and Physiology of Virgal Pigments, ed. H. LANGER. Berlin, Heidelberg, New York: Springer Verlag. WALD, G., BROWN, P. K. & GIBBONS, 1. R. (1962). Visual excitation. A chemoanatomical study. Symp. Soc. exp. Biol. 16, 32-57. ZEWI, M. (1939). On the regeneration of visual purple. Acta Soc. Sci. fenn. 2, 1-56.
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