(Received 19 April 1977)

Transcription

1 J. Physiol. (1978), 278, pp With 12 text-ftgure8 Printed in Great Britain KINETICS OF BLEACHING AND REGENERATION OF RHODOPSIN IN ABNORMAL (RCS) AND NORMAL ALBINO RATS IN VIVO From the BY IDO PERLMAN* Vision Research Laboratory, University of Mfichigan, Ann Arbor, Michigan 4819, U.S.A. (Received 19 April 1977) SUMMARY 1. Rhodopsin concentration has been measured by the method of densitometry in retinae of rats with inherited retinal dystrophy (RCS) raised in darkness and compared with that of normal rats similarly reared. 2. In both RCS and normal rats the fraction of rhodopsin bleached is always directly proportional to the photon content of the light, I. t, where I is the light intensity in effective quanta (5 nm) cm-2 sec' and t is the duration of the bleaching exposure in seconds. 3. Rhodopsin photosensitivity for bleaching is slightly higher in RCS rats than in normals (2.3 (1)-16 cm2 chromophore-1 compared with 1*3 (1)-16 cm2 chromophore-1). 4. Rhodopsin regeneration in the dark in both RCS and normal rats cannot be described by the kinetics of a simple monomolecular chemical reaction. 5. Following 5 min bleaches, the regeneration rate becomes slower as the preceding bleach is made stronger. Regeneration in the dark is significantly faster in the RCS rats than in the normal ones. 6. In normal rats, after a full bleach, rhodopsin regenerates back to the darkadapted level within 3-4 hr. In RCS rats rhodopsin regenerates to reach a plateau level, below the previous dark-adapted level, that lasts for several hours. 7. The faction of total rhodopsin that can regenerate gradually declines with age until in 7 day old RCS rats no rhodopsin regeneration can be measured by the densitometer. However, total rhodopsin density (fully bleached-dark-adapted) is still close to normal. INTRODUCTION Inherited retinal dystrophy in the RCS rat, first described by Bourne, Campbell & Tansley (1938), is manifest as a progressive loss of photoreceptor cells. The RCS rats have a normal electroretinogram up to age 18 days. At that time the sensitivity, as measured by b-wave criterion, starts to decline gradually with age until at 6-8 days no b-wave can be elicited (Dowling & Sidman, 1962; Noell, 1963). The deterioration of the electroretinogram is accompanied by accumulation of outer segment-like lamellae between the tips of the rods and the pigment epithelium and by progressive degeneration of inner segments and photoreceptor cell nuclei (Dowling & Sidman, * Present address: Vision Research Laboratory, Hadassah University Hospital, Jerusalem, Israel.

2 142 I. PERLMAN 1962). The accumulated extracellular debris, derived from rod outer segment disks shed during the process of outer segment renewal (Herron, Riegel & Rubin, 1971; Bok & Hall 1971) is probably the source of the higher than normal levels of rhodopsin extracted from the eyes of young RCS rats (Dowling & Sidman, 1962; Noell, 1963). The failure of the pigment epithelial cells to phagocytize the shed outer segment disks is probably due to genetic defect in the pigment epithelium and not in the rods (Mullen & La Vail, 1976). Delmelle, Noell & Organisciak (1975) studied the kinetics of retinol during illumination of the dystrophic retina. They found that the partition of retinol between pigment epithelium and retina during illumination and the fraction of total rhodopsin extractable 6 hr after a strong bleach were abnormally low. These low values, which decreased with age, could be explained by assuming that only the rhodopsin contained within surviving and functioning rods could regenerate in the dark and contribute retinol to the pigment epithelium when bleached. In this report and in a subsequent one (Perlman, 1978) studies on the RCS rats are described. These studies were carried out in order to further characterize the degeneration process and to compare rhodopsin kinetics in the RCS rods with normal ones. Very little is known about kinetics of rhodopsin in vivo in animals. Rushton (1958) and Alpern (1971) showed respectively that the kinetics of cone and rod pigments in humans could be described by the kinetics of a simple monomolecular photochemical reaction, e.g. dp _ Ip 1-p dt Qe to*() In this equation p is the fraction of unbleached pigment present at time t; I is the light intensity and the constants Q-1 and to are respectively the photosensitivity and time constant of regeneration. In animals, on the other hand, there is no agreement between different studies concerning rhodopsin kinetics in vivo. Experiments in which rhodopsin was extracted from albino rats at various time intervals after cessation of bleaching supported a first order chemical reaction model for the regeneration process (Tansley, 1931; Dowling, 196, 1963). However, retinal density measurements in the rabbit (Hagins & Rushton. 1953; Rushton, Campbell, Hagins & Brindley, 1955; Hagins, 1957) and in the rat (Lewis, 1957b) showed that rhodopsin regeneration in these living but anaesthetized animals occurred at a constant rate until most of the pigment had regenerated. This was followed by a gradual decrease in regeneration rate until completion was attained in about 9 min in the rabbit and 4-6 hr in the rat. Bonds & MacLeod (1974) reported that neither bleaching nor regeneration of rhodopsin in the living cat could be described by the simple kinetic equation (eqn. (1)). The first two parts of the present report describe experiments designed to study kinetics of rhodopsin in vivo in normal albino rats. These results are in agreement with a simple photochemical model of the bleaching process but do not support the hypothesis that regeneration is also governed by the same general kinetic equation (eqn. (1)). The third part deals with rhodopsin kinetics in RCS rats comparing this pathological case with the normal one.

3 RHODOPSIN KINETICS IN RATS 143 METHODS Preparation. The regeneration of rhodopsin was followed in the left eye of both RCS and normal albino rats both raised in darkness since birth in order to slow down the degeneration process in the RCS retinae (Dowling & Sidman, 1962). Anaesthesia was induced by sodium pentobarbitone (5 mg/oo g) administered ii.p. Atropine sulphate (.4 mg/l kg) was injected s.c. and the trachea was cannulated to keep the airways clear. The eyelids were drawn back by silk sutures and the pupil was dilated by atropine sulphate. A clear plastic contact lens was applied to the eye with wetting solution (methylcellulose 2 %) in order to keep the cornea moist. All stages of the preparation were performed in deep red light to prevent bleaching. G1 FS1 L1 G2 ND IF D L Fig. 1. Diagram of the reflecting retinal densitometer. Two beams from source A, the reference one (R) and the measuring one (IF), were flickered at 19-5 Hz by the rotating disk D and were focused on the rat cornea. The light reflected by the fundus was collected by lens L2 and focused on the photomultiplier tube, PC. The wedge W was used to null the signal from the photomultiplier tube originating from the reference and measuring beams. A third beam originating from source B was used for bleaching. Instrumentation. The densitometer used, shown in Fig. 1, is a modification of the model of the reflexion retinal densitometer described by Hood & Rushton (1971). Two beams from source A are brought together at the glass mixing plate and focused on the cornea by a Maxwellian view lens, L1. The measuring beam is attenuated by one of several narrow band Baird-Atomic interference filters, IF. The reference beam is attenuated by an Ilford no. 68 filter, R (transmitting only for A > 6 nm). The two beams are alternated at 19-5 Hz by means of a rotating disk, D. The light reflected by the fundus is gathered by the lens, L2, and an image of the illuminated patch of retina is formed in the plane of the field stop, FS2, immediately in front of a photomultiplier tube (EMI 9558B). A third beam, originating from source B, is focused on the cornea by the Maxwellian view lens, L3, and is used for bleaching. The output of the photomultiplier tube consists of two signals. The reference signal which is due to red light to which the rhodopsin is transparent and the measuring signal which arises from the absorbed light beam. These signals are presented to a lock-in amplifier (PAR JB-5) tuned to the frequency of the rotating disk, in antiphase so the signals may be balanced by a neutral wedge, W, in the reference beam. The relations between the 'red' wedge transmittance, needed to null the lock-in amplifier, and rhodopsin density have been previously described (Alpern & Pugh, 1974). In brief, T- Td(2) 1s(1efA) 2 To

4 144 I. PERLMAN To - T 3 TO-Td Tt, To and Td are respectively the 'red' wedge transmittances needed to balance the measuring and reference beams at time t, after a full bleach and in the fully dark-adapted state; 8 is the fraction of the light reaching the photornultiplier tube which is stray light; /1A is the density of rhodopsin at wave-length A; and p is the fraction of unbleached rhodopsin present at time t. Eqn. (2) gives the difference spectrum of the pigment studied. Eqn. (3) is an approximation that holds only for very small laa. In the kinetic studies the wave-length of the measuring beam was fixed at 55 =m, far enough from the rhodopsin absorption peak to allow the use of eqn. (3) and to minimize contamination of the signal by photo-products absorbing at short wave-lengths. Procedure. The animal was secured to a specially designed holder which provided the freedom of movement needed to align the rat so that the light entered the eye axially. The experimenter, putting his eye in place of the photomultiplier tube, could see the image of the funds. This image was then optimized and centred by fine adjustments of the lens, L,2. A dark-adapted base line was determined by readings taken after the animal had been in total darkness for at least 3 min. In kinetic studies, the eye was then partially or fully bleached and rhodopsin regeneration in the dark was followed with a 55 rm filter in the measuring beam. Difference spectra were obtained from rats that died during the initial preparations, because the apparatus in its present form did not permit measurements to be made during bleaching. The dead rat was left for more than 1 hr to allow stabilization of the eye media. Then 'red' wedge transmittances required to balance the measuring light attenuated by eleven interference filters that transmitted maximally at different wave-lengths spanning the visible spectrum were measured before and after a full bleach. RESULTS I. Does the densitometer measure rhodopsin? Difference spectra were obtained by measuring the 'red' wedge transmittance needed to balance the A and red signals of the photomultiplier tube before (Td) and after (T.) the retina was fully bleached. In Fig. 2 the open circles describe the mean of (To- Td)/To as a function of wave-number for five albino rats. An action spectrum was also obtained by measuring the light intensity needed to evoke a 3 /ZV b-wave in the electroretinogram of a dark-adapted rat. The filled circles in Fig. 2 describe the mean of the reciprocal of this intensity (sensitivity) as a function of wave-number for four normal albino rats. The curve drawn in Fig. 2 is the absorption spectrum for a pigment absorbing maximally at 5 nm computed from the Dartnall nomogram (Wyszecki & Stiles, 1967). The good agreement between the difference spectrum (open circles), the action spectrum (filled circles) and the absorption spectrum (curve) indicates that the densitometer measures rhodopsin, the visual pigment that determines the scotopic e.r.g. responses. Further support for this conclusion was obtained by comparing pigment regeneration curves from six normal rats with the recovery of the electroretinogram sensitivity in three normal rats after a full bleach. The electroretinogram sensitivity was defined as the light intensity needed to evoke a 3 1tV b-wave (I3). In Fig. 3 the relation between the elevation of log I3 above the fully dark-adapted level and the fraction of pigment present during the course of rod dark-adaptation is given. The straight line, obtained by a linear regression procedure, has the equation logit= Pt, Ida

5 RHODOPSIN KINETICS IN RATS where It and Ida are the light intensities needed to evoke a 3 /tv b-wave at time t and in fully dark-adapted rat respectively and Pt is the fraction of unbleached pigment present at time t. The excellent agreement between the line and the data points (r = - 987) confirms previous findings for humans' rhodopsin (Rushton, 1961) and rats (Dowling, 196, 1963) and further supports the conclusion that the densitometer is measuring the pigment used for rod vision, namely rhodopsin. Wave-length (nm) I I ~ I 145 '.. wf 1 1* I-.1 II _ 5~~~~~~~~~~~~~~~~~~~~~~~~\ *s I 1. =. C Z._ X 1 1 k 1 23,6 2,4 17,2 22, 18,8 Wave-number (cm 1) 15,6 Fig. 2. Average difference spectrum measured in the densitometer (ordinate scale to the left) from five normal rats (open circles) and average action spectrum using an electroretinogram criterion of 3 /tv b-wave (ordinate scale to the right) from three normal rats (filled circle). The continuous curve was plotted from a Dartnall nomogram for a pigment with = Amar 5 nm, confirming that both the difference spectrum and the action spectrum were determined by rhodopsin. II. Rhodopsin kinetics in normal rats In Fig. 4 the results of a regeneration experiment are shown. Plotted on the ordinate in this figure is the fraction of rhodopsin, (To- - Tt)/(To Td), present at time t. The arrows at the top of the figure indicate partial or full bleaching exposures lasting 5 min. Rhodopsin regeneration after a full bleach was followed to completion in six normal rats. In three rats the regeneration curve followed a single exponential time course (one of these is shown in Fig. 4) while in other cases rhodopsin regeneration proceeded in a constant rate for the first 2-4 min and then continued along

6 146 I. PERLMAN an exponential time course. However, the linear portion of the regeneration curves were not as pronounced as those reported before in the rabbit (Rushton et al. 1955), in the rat (Lewis, 1957b) and in the cat (Bonds & MacLeod, 1974). The mean time constant of the exponential part of the regeneration curves obtained after a full bleach was P3 min (S.E. of mean). This value represents a low limit estimate because in the cases where regeneration was initially linear the time constant V a. 5-H Q) V - Cu 'a ) 41-3H (V) U,. I.-_ 1) am 2- F 1 OF -J * I 5 1. Fraction of unbleached rhodopsin Fig. 3. The elevation of the log light intensity needed to evoke a 3 /WV e.r.g. response above the dark-adapted level as a function of rhodopsin content during the course of dark-adaptation following a full bleach in normal albino rats raised in cyclic light-dark conditions. The straight line, obtained by a linear regression procedure (r = ), has the equation, log It/Ida = Pt. where It and Ida are respectively the light intensities needed to evoke an electroretinogram criterion at time t in the dark and in the fully dark-adapted rat. Pt is the fraction of unbleached rhodopsin present at time t. calculated by fitting the entire regeneration curve to a single exponential time course was always longer than the one obtained for the tail part of the curve (covering about 8 % of the curve) that followed a single exponential time course. After low to moderate partial bleaches (less than about 6 %), rhodopsin regeneration in the dark followed a single exponential time course in all the rats studied. The regeneration data were fitted by a linear regression procedure on a semilogarithmic plot. The straight line thus obtained was extrapolated to t = to give the fraction of rhodopsin bleached by the light exposure. The time constant of regeneration

7 RHODOPSIN KINETICS IN RATS 147 is the time needed to leave 1/e of the initial bleached rhodopsin, calculated as described above, in the bleached state. The solid curves in Fig. 4 describe a single exponential time course obtained as described above to fit best the regeneration data. Rhodopsin regeneration in the rat is quite slow (3-4 hr are needed for complete recovery after a full bleach); therefore, in some experiments the regeneration after 1, see '_ Time (min) Fig. 4. Bleaching and regeneration of rhodopsin in a normal rat (45 days old). The data points describe the change in the fraction of unbleached rhodopsin, p(t) = (To- T)/(TO- Td), with time. The arrows indicate the onset of 5 min exposures to light that bleached rhodopsin either partially (first and third arrows) or fully (second arrow). The solid curves were obtained by fitting the regeneration data to a single exponential time course. The dashed curve describes the expected time course of rhodopsin regeneration if the rat was left in the dark to allow complete regeneration. a moderate bleach was interrupted and a full bleach was applied in order to save time. The dashed curve in Fig. 4 describes the expected time course of regeneration if the rat was left in the dark to allow complete regeneration. In Fig. 5 the relation between the time constant of regeneration and the fraction of rhodopsin bleached by the bleaching exposure is given for ten normal rats indicated by different symbols. The points for more than 6 % bleach were obtained from the rats that exhibited single exponential time course for the entire regeneration curve obtained after a full bleach. The continuous line was drawn through the data points by eye to emphasize the gradual increase in the time constant (decrease in regeneration rate) with increase in the strength of bleaching. Such a behaviour is not predicted by the simple monomolecular chemical reaction model suggested for the regeneration process (eqn. (1). second term). As stated in the Methods section the densitometer used for this study could not

8 Co = I. PERLMAN be used to measure rhodopsin density during bleaching exposures. Therefore, the independence of bleaching and regeneration (eqn. (1)) found for the visual pigments in man (Rushton, 1958; Alpern, 1971) could not be demonstrated for the rat rhodopsin. However, the relatively slow time course of rhodopsin regeneration in the rat (time constant > 5 min for bleaches > 2 %) together with the assumption that light has no effect on the regeneration ensured that during the 5 min bleaching 5-4- E 22~~~~~ Fraction of rhodopsin bleached Fig. 5. The relationship between the strength of bleaching and the subsequent rate of rhodopsin regeneration in normal rats. The time constant of regeneration was obtained by fitting a single exponential time course to the data as described in the text. Different symbols describe results from different rats. The continuous line was drawn through the data points by eye, to emphasize the strong dependency of the regeneration rate on the strength of the preceding bleach. exposure regeneration was negligible. Therefore, the regeneration term in the general kinetic equation (eqn. (1)) can be dropped, leaving only the bleaching term dip _Ip dt Qe (4) Solving eqn. (4) with the initial condition that p = 1 when t = gives P. = e-itq e (5) where P is the fraction of unbleached rhodopsin present at the termination of the bleaching exposure; I and t are respectively the intensity and duration of the bleaching exposure and Qe is the bleaching energy that leaves 1/e of the darkadapted rhodopsin unbleached. In Fig. 6 the relationship between PO, calculated as

9 RHODOPSIN KINETICS IN RATS 149 described above by extrapolating the regeneration curves back to t = and the bleaching energy given in log effective quanta (5 nni) cm-2 of retina is plotted for ten normal rats. The curve is defined by eqn. (5) with log Qe = (S.E. of mean) which is in fair agreement with the values of 15-98, 15-84, 15-83, and found respectively for rabbit (Hagins, 1957), frog (Baumann, 1965), cat (Bond & MacLeod, 1974), rat (Cone, 1963) and skate (Dowling & Ripps, 1971). 1 *.J' Ad 8 - n6-6 * \ a) ~~~~~~~~.4 - C o LC*u Log light exposure (effective quanta (5 nm) cm-2) Fig. 6. Rhodopsin bleaching in normal rats with 5 min exposures to the bleaching energies given in the abscissa in log effective quanta (5 nm) cm-2 retina. The continuous curve has the equation, P. = e-it!qe With log Qe = 15-9 quanta (5 nm) cm-2 of retina, where PO is the fraction of unbleached rhodopsin present at the termination of the bleaching exposure; I and t are the intensity and duration of the exposure and Qe, the inverse of photosensitivity, is the energy needed to leave t/e of rhodopsin unbleached. The small filled circles represent calculated bleaching at 1 min after dim light was turned on, corrected as described in the text for cases where the time constant of regeneration was smaller than 5 min. After very small bleaches (less than 1 (JO ) the time constant of regeneration was smaller than 5 min as evident in Fig. 5 and therefore the assumption of negligible regeneration during the bleaching exposure could not be used. An estimate of the error introduced by applying eqns. (4) and (5) to small bleaches was obtained by solving the general kinetic equation (eqn. (1)) and assuming that after 5 min of bleaching the retina approached a steady state (bleaching rate = regeneration rate). The results illustrated in Fig. 5 indicate that the general kinetic equation (eqn. (1))

10 15 I. PERLMAN cannot be used to describe rhodopsin regeneration in the rat; nevertheless, it was used as a first approximation. The solution to eqn. (1) P = P+(1-P)e-t/toP (6) in which P is the value of p when t = oo and to is the time constant of regeneration (Alpern, 1971); eqn. (7) was used together with the values of P and to obtained from the regeneration data to calculate the fraction of unbleached rhodopsin present 1 min after the bleaching light was turned on. The small solid circles in Fig. 6 describe the bleaching by dim light corrected according to the above procedure. The agreement., 25 ~-6-. ~,4 - / 4 )m C. ) f~~~~~~~~~~~~~ ~~~~~~~~~ C Fg.2 7. ~~~ a nw r ON6 8oS 45 indicated on the right side of the figure. The curves were arbitrarily shifted along the time axis for clarity as indicated by the arrows at the top of the figure. The continuous curves have the general equation p = A-B e-'/'r, where p is the fraction of rhodopsin present at time t and A, B and r are the parameters obtained from a least-square analysis routine. between the corrected points and the theoretical curve indicate that even for small bleaches for which the time constant of regeneration is smaller than 5 min, eqns. (4) and (5) can be used. III. Rhodopsin kinetics in RCS rats Representative curves describing rhodopsin regeneration in the dark after full bleaches are shown in Fig. 7 for RCS rats of different ages. The curves have been shifted arbitrarily along the time axis for clarity. The arrows at the top of the figure represent from left to right the onset of regeneration in 25-, 35-, 45-, and 6-day old RCS rats respectively. All the regeneration curves obtained from RCS rats after a full bleach differed from those obtained from normal rats in two features: (a) a single exponential curve

11 RHODOPSIN KINETICS IN RATS 151 could describe the regeneration data satisfactorily (smooth curves in Fig. 7) and (b) regeneration was never complete, the pre-bleaching dark-adapted level of rhodopsin was not reached even after waiting in the dark for as long as 6 hr. In order to exclude any artifact as the source of the apparently incomplete regeneration the difference spectrum between the retinal reflectances during the plateau of the regeneration curves and the prebleached dark-adapted state was measured. The Wave-length (nm) I I I I _ *1 _- I I I li I I I 23,6 22, 2,4 18,8 17,2 15,6 Wave-number (cm-') Fig. 8. Average difference spectrum from seven RCS rats, 3-5 days old, obtained by measuring the 'red' wedge transmittance needed to balance the A and red signals before bleaching (Td) and after the plateau of regeneration curve had been reached (T.). The continuous curve was calculated from a Dartnall nomogram for a pigment with Am..s - 51 nm. 'red' wedge transmittances needed to balance the A and 'red' signals were recorded before bleaching the eye Td and after the plateau ofthe regeneration curve (measured after a full bleach) had been reached and had lasted for more than 1 hr, Tp. In Fig. 8 the mean + 2 S.E. of mean of (Tp- Td)/TP obtained from seven RCS rats 3-5 days old is plotted as a function of wave-number. The continuous curve calculated from a Dartnall nomogram (Wyszecki & Stiles 1967) for a pigment with Amax = 51 nm. is in fair agreement with the data points indicating that the phenomenon of incomplete regeneration is due to a loss of rhodopsin. The fraction of rhodopsin represented by the plateau of the regeneration curve,

12 PERLMAN termed the fraction of 'regenerative' rhodopsin, decreased with the age of the RCS rats as described in Fig. 9 (filled circles). The open triangles represent data obtained by extracting rhodopsin from the eyes of RCS rats left for 6 hr in the dark after a strong bleach (Delmelle et al. 1975). Considering the differences between the extraction and retinal densitometry techniques, there is excellent agreement between the two sets of data (Fig. 9, triangles and circles). 1 U r._ CL In - T a) 81-6 (1) (3) A (6) 4) a, c ) a3) I- C4 4, (4)(6 { LU 2 k (3) * (3) l (3) (3). (4) 1 1 1~~ Age (days) Fig. 9. The fraction of total rhodopsin that regenerates after a full bleach in RCS rats of different ages. The filled circles are the mean + 2 S.E. of mean of the fraction of 'regenerative' rhodopsin obtained from a number of RCS rats as indicated above the data points. The open triangles represent data obtained by extracting rhodopsin from the eyes of RCS rats (Delmelle et al. 1975). In order to measure the absolute density of rhodopsin using eqn. (2), the fraction, s, of the light reaching the photomultiplier tube which is stray light has to be measured. An estimate of rhodopsin density at 55 nm can be obtained from ((To Td)/To)5s assuming that the stray light is small (Lewis, 1957a) and constant - between rats. In Fig. 1 the dependence on age of the estimated density at 55 nm of total rhodopsin, ((To Td)/To)55, in dark-adapted normal and RCS rats (filled - and open squares respectively) and of the 'regenerative' rhodopsin, ((T -Tp)/To)55o in RCS rats (circles) is plotted. The dashed curve, describing the build up of total rhodopsin with age in normal rats, was drawn through the solid squares using data

13 RHODOPSIN KINETICS IN RATS 153 from Dowling & Sidman (1962). The conversion was made by transforming Dowling and Sidman's data for the decadic molar extinction coefficient at 5 nm to the Napierian extinction coefficient at 55 nm (cc,) and then calculating a constant that would make their data for a 6 day old rat equal to the mean rhodopsin density (f55) at 6 days calculated from eqn. (2) (assuming s = ). This constant was then used with the values of a5m to calculate ((To - Td)/To)w from eqn. (2). The gradual decrease with age of the amount of rhodopsin that can regenerate in RCS rats is described by the straight line through the filled circles obtained (after excluding the 9 days point) by a linear regression procedure (r = ). 5 - a t^ to.-, 1- I-.. %-O I;- 4 k 3 F 2 P.11- To a a *3B i* e /M / 6 I' sol~ *61 Ek 9 S a Fig.. 1 II Age (days) The dependence of estimates of total rhodopsin density at 55 nm, ((To-!Td)/ T.)5,, on age of RCS and normal rats (open and filled squares respectively). The dashed curve was drawn through the filled squares using data on normal rats taken from Dowling & Sidman (1962). The estimates of the density at 55 nm of the 'regenerative' rhodopsin, ((T Tp)/T.)55, in RCS rats (filled circles) were fitted to the straight line - by a linear regression procedure (r = ). After the plateau of the regeneration curves had been reached, further partial or full bleaches of the 'regenerative' rhodopsin resulted in full recovery back to the plateau level provided that enough time was allowed in the dark. Therefore, the fraction of 'regenerative' rhodopsin was treated as the total rhodopsin located within I

14 PERLMAN surviving photoreceptors in the dystrophic retina that could regenerate. The same procedure as that described for normal rats was applied to the rhodopsin regeneration curves measured after partially bleaching the 'regenerative' rhodopsin in RCS rats to different levels. Figs. 11 and 12 describe respectively the dependence of the time constant of regeneration on the fraction of 'regenerative' rhodopsin bleached and the relationship between unbleached 'regenerative' rhodospin present at the 4 3 / / / T * I *+ a) c E -W a 2 2 C), E 1' I ~~ / * / I / la / /(1D I it * I/ I I I Fraction of regenerative rhodopsin bleached Fig. 11. Rate of rhodopsin regeneration after variable bleaches of RCS rats. The data points describe the time constant of regeneration, obtained by fitting an exponential curve to the regeneration data, as a function of the fraction of the 'regenerative' rhodopsin bleached by the light exposure. All the data points fall to the right of the dashed line which describes the regeneration rates in normal rats (replotted from Fig. 5) indicating a faster regeneration in RCS rats than in normal ones following identical bleaching strengths. termination of the bleaching exposure and the bleaching energy. The general pattern of the data points in Fig. 11 agrees qualitatively with the one found in normal rats (Fig. 5), namely a decrease in the regeneration rate constant (ti) with increase in bleaching. However, the deviation of the data points in Fig. 11 from the dashed line describing regeneration rates in normal rats (replotted from Fig. 5) indicates a faster regeneration rate of the 'regenerative' rhodopsin in RCS rats than in normals. Moreover, the mean time constant of rhodopsin regeneration after a full bleach measured in twenty-four RCS rats younger than 5 days old,

15 RHODOPSIN KINETICS IN RATS P3 min (S.E. of mean), is slightly smaller (P =.8) than the value ( ) obtained for normal rats. In Fig. 12 the continuous curve, describing the bleaching rate of 'regenerative' rhodopsin in RCS rats, has the equation PO = e-tiq (eqn. (5)). The value log Qe = 15*64 + *5 (S.E. of mean) is modestly smaller than the value measured for normal rats (P = 26). The small increase of rhodopsin photosensitivity (Qe-1) in RCS rats above normal ones is also emphasized by the deviation of most of the data points in Fig. 12 from the dashed curve describing rhodopsin bleaching in normal rats (replotted from Fig. 6) e 382 e * 1-24 ems *z8_ \\\ * 451 % 455* o O466E 516 o CD) > as AA 678A C 6 -* 1r_ ~~~~~~~~~.75 & = I ' 4 Co \ \ 2 _.* \ \.a I 9 % "~ _ le Log light exposure (quanta (5 nm) cm2) Fig. 12. Bleaching of the 'regenerative' rhodopsin in RCS rats by 5 min exposures to the energies given in the abscissa in log quanta (5nm) cm-2 of retina. The continuous curve has the equation P_ = e-itlqe with log Qe = quanta (5 nm) cm-2 of retina. The dashed curve, describing rhodopsin bleaching in normal rats, is replotted from Fig. 6 in order to emphasize the slight, but significant, increase in rhodopsin photosensitivity measured in RCS rats compared to normal ones. In the inset, the fraction of the total rhodopsin that can regenerate ('regenerative' rhodopsin) for each rat is presented opposite the appropriate symbol. DISCUSSION Regeneration. Not enough is known about the steps involved in the regeneration of rhodopsin in vivo and the reaction rates to construct a theoretical model for the regeneration process. In man, rhodopsin regeneration is described by the kinetics of a simple first-order chemical reaction (Campbell & Rushton, 1955;

16 156 I. PERLMAN Alpern, 1971). Rhodopsin regeneration in both normal and RCS rats cannot be described by the above model. The linear portion seen in the beginning of regeneration after a full bleach in some of the normal albino rats has been previously observed in the rat (Lewis, 1957b), in the rabbit (Hagins & Rushton, 1953; Rushton et al. 1955; Hagins, 1957) and in the cat (Bonds & MacLeod, 1974). The increase in the time constant of rhodopsin regeneration with bleaching strength found in both normal (Fig. 5) and RCS rats (Fig. 11) cannot be explained by a simple chemical reaction model of any order. Enzymes are expected to play a role in the chain of reactions leading to rhodopsin regeneration. Substrate inhibition of a rate-limiting enzymic reaction may account for the data illustrated in Figs. 5 and 11. As more substrate is made available by increasing the strength of the bleaching exposure (the substrate is a photoproduct of the bleaching reaction) the apparent time constant, measured from the exponential time course of rhodopsin regeneration, gets longer. The initial constant rate of regeneration observed occasionally in normal rats after a full bleach is then due to saturation of the enzyme. According to the above scheme the faster rhodopsin regeneration observed in RCS rats compared with normal ones (Fig. 11) might be due to the presence of a larger quantity of more efficient enzyme involved in the rate-limiting step of the rhodopsin regeneration process. The pigment epithelium has been supposed to play an essential role in rhodopsin regeneration (Weinstein, Hobson & Dowling, 1967). Since the regeneration rate measured in RCS rats is faster than in normal rats despite the thick membranous 'debris' accumulated between the pigment epithelium cells and the tips of the rods (Dowling & Sidman, 1962), diffusion cannot be a rate-limiting step in the regeneration process. It is possible that long processes from pigment epithelium cells traverse the entire 'debris' layer to approach surviving photoreceptors. Retinol and retinal may be exchanged between photoreceptors and pigment epithelium only via these processes, thus explaining the inability of retinol derived from bleaching the rhodopsin in the 'debris' to reach the pigment epithelium (Delmelle et al. 1975). Photosensitivity. The dependence of the fraction of rhodopsin bleached on the strength of the bleaching exposure could be described by P_ = e-it/q1 which is the solution of a photochemical reaction (eqn. (4)). Rhodopsin photosensitivity to bleaching, Q-1 (Alpern, 1971), measured in ten dark-reared normal rats had a mean of 1-3 (1)-16 cm2/chromophore. This value is in fair agreement with the values 1-5 (l)-16, 1-45 (l)-16, 1-2 (1)-16, 1-78 (l)-16, 1.48 (l)-16 CM2/chromophore found respectively for rabbit (Hagins, 1957), frog (Baumann, 1965), skate (Dowling & Ripps, 1971), rat (Cone, 1963) and cat (Bonds & MacLeod, 1974) but 2-3 times smaller than rhodopsin photosensitivity in humans (Alpern & Pugh, 1974). The mean photosensitivity of the 'regenerative' rhodopsin measured in thirteen RCS rats, 2-3 (1)-16cm2/chromophore, is slightly but significantly higher (P < 1) than the value measured in normal rats. This increase in photosensitivity, though small, may account for the higher than normal early receptor potential measured in young RCS rats (Arden & Ikeda, 1966) since its size is proportional to the fraction of rhodopsin bleached by the flash (Cone, 1964). Moreover, if, as has been suggested by Reading (197), retinol is toxic to the photoreceptors, then the increased photosensitivity would result in more retinol release with the same illumination in RCS rats which, in turn, would accelerate retinal degeneration. Studies

17 RHODOPSIN KINETICS IN RATS 157 in vitro showed that solutions of rhodopsin extracted from either RCS or normal rats had the same absorption spectrum (Dowling & Sidman, 1962) and the same photosensitivity (Chaitin & Williams, 1976). Therefore, some structural differences such as coupling between rhodopsin molecules (increased quantum efficiency) or the ability of the photoreceptors to collect the incident light (funnelling) probably account for the measured increase in photosensitivity of rhodopsin in RCS rats. The increased rate of regeneration and the slightly elevated rhodopsin photosensitivity found in RCS rats compared with normals suggest that the rods in the dystrophic retina differ functionally from the normal rods. Whether those differences are due to the same gene that causes the failure of the pigment epithelium to phagocytize rod outer-segment disks shed during the renewal process, or due to strain differences remains to be tested by studying rats of the same strain as the RCS rats, but which do not possess the afflicted gene. Rhodopsin content. It has been shown that the eyes of young RCS rats, whether raised in darkness or in a cyclic light-dark environment contain higher than normal levels of rhodopsin (Dowling & Sidman, 1962; Noell, 1963; La Vail & Batelle, 1975; Demelle et al. 1975). In this study, on the other hand, the estimates of total rhodopsin density at 55 nm are similar for RCS and normal rats of all ages (Fig. 1). There are at least three ways to reconcile these findings. (a) The fraction of stray light, s, in the equation (To- Td)/To = (1- s)(1- e-2fi) may be bigger for RCS rats than for normal rats due to increased light scattering by the extracellular membranous 'debris'. (b) Dark-reared albino rats used in the present study may have more rhodopsin than those used in the other studies. (c) Rhodopsin may be more easily extracted from the extracellular 'debris' than from intact outer segments. In this study no attempt was made to extract rhodopsin or to measure the fraction of stray light which reached the photomultiplier tube in the retinal densitometer. Therefore, it could not be decided which one of the above three possibilities (or a combination of them) is responsible for the discrepancy between rhodopsin density data obtained from the retinal densitometer and those obtained by extraction techniques. From the extraction data (Dowling & Sidman, 1962; Noell, 1963; La Vail & Batelle, 1975) and the measured fractions of 'regenerative' rhodopsin (Fig. 9) the amount of rhodopsin present in the functioning rods in the RCS eyes was calculated. It was found that up to age 4 days RCS retinae contained more functional rhodopsin than their normal counterparts and thus visual sensitivity was expected to be at least the same (Perlman, 1976). However, electroretinographic measurements showed that sensitivity of RCS rats was at least 1-2 log units below normal level (Dowling & Sidman, 1962; Noell, 1963; Perlman, 1978) and therefore support the densitometric data on rhodopsin content of the RCS and normal eyes. The gradual decline with age in RCS rats of both the fraction of 'regenerative' rhodopsin and its density at 55 nm is obvious from Figs. 9 and 1. This finding, confirmed previously (Delmelle et al. 1975), is expected assuming that the 'debris' lacks the appropriate enzymic machinery needed for the regeneration of bleached rhodopsin. Delmelle et al. (1975) calculated the fraction of total rhodopsin contained within surviving and functioning rods assuming that (a) normal-looking photoreceptor cell nuclei represented still functioning photoreceptors, and (b) each surviving photoreceptor in RCS retina contained the same amount of rhodopsin as those

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