Physiology and enzymology of frog photoreceptor membranes

Exp. Eye Res. (1974) 18,253-269

Physiology and Enzymology of Frog Photoreceptor Membranes D. BOWNDS, A. BRODIE, W. E. ROBINSON, J. MILLER AND A. SHEDLOVSKY

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PALMER,

Laboratory of Molecular Biology, Department of Zoology, University of Wisconsin, Madison, Wis. 53706, U.S.A. We have developed a convenient and reproducible assay for observing visual excitation and dark adaptation in suspensions of isolated bullfrog rod outer segments. These outer segments can be separated from other contaminating membranes using gentle procedures which do not destroy their physiological activity. Rhodopsin phosphorylating activity and cyclic nucleotide phosphodiesterase remain with these outer segments; ATPase and adenyl cyclase generally thought to be associated with outer segments are not present. In the light-induced rhodopsin phosphorylation reaction low levels of illumination cause the incorporation of more than one phosphate per rhodopsin bleached. This is the only reaction yet characterized in this system which involves an obvious amplification mechanism.

1. Introduction Intense interest is now focused on vertebrate photoreceptor membranes becauseof the exciting prospect that they may be the first system in which the generation and regulation of a nerve signal are understood in molecular terms. Several factors combine to make these membranes an especially favorable preparation. (1) They can be harvested and purified by gentle procedures which do not destroy physiological activity (Bownds, Dawes and Miller, 1973). (2) They consist almost entirely of one protein component : the visual pigment, rhodopsin (Bownds, Gordon-Walker, GaideHuguenin and Robinson, 1971). (3) The retinaldehyde prosthetic group which binds to the opsin (protein) portion of rhodopsin provides a label which is useful in the isolation and study of opsin. (4) Knowledge of the photochemistry of rhodopsin allows precise quantitation of the light input which is ultimately transduced into a sodium conductance decrease. The experiments presented in this symposium paper focus on the photoreceptor membranescontained in bullfrog (Rana cutesbeiana)rod outer segments.These outer segmentsare large cylindrical organelles (approximately 8 pm in diameter and 60 pm in length) consisting of a plasmamembrane surrounding 1500-2000 rhodopsid containing disc membranes. The outer segmentsproject from the retina and can be detached by gentle shaking, allowing their isolation with minimal contamination from other retinal cells. Quantities sufficient for analyt’ical studies can be obtained with little difficulty; outer segments containing 0.4 mg of rhodopsin can be harvested from a singlelarge retina. Most important is the fact, demonstrated by experiments presented in this article, that these outer segmentscan routinely be made to function in vitro. performing not only excitation but also dark adaptation. This opens the way for studies of substrates, rate controlling molecules, and pharmacologically active agents which interact with the system. Our laboratory has concentrated on three complementary approaches to understanding outer segment function : analytical studies, characterization of light dependent chemistry, and in vitro physiological studies. The goal of the analytical studies has been the specification of protein components and enzyme activities which are intrinsic to outer segmentsand not associatedwith contaminating membranes. Those enzyme activities which are light sensitive warrant most attention, and our interest, 353

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has focused on a light-induced rhodopsin phosphorylation (Bowncls. Dawcs, Jlillc~ and Stahlman, 19’72) because it appears to involve an amplification mechanism. Upon bleaching only l”//o of the rhodopsin in an outer segment suspension, one can 01)srrv~ phosphorylation of as much as 50% of the total rhodopsin (assuming that’ only one phosphate binds to each rhodopsin). Finally, the role of light-dependent) chcnlistr~ can be determined only if one has an “assay” for the physiological function of t,he system. This need has been met by developing procedures for observing excita,tion and dark adaptation in crude and purified suspensions of outer segments.

2. Materials and Methods Large bullfrogs, 12-20 cm in length, were used in these experiments, They were obtained from Mogul-Ed., Oshkosh, Wis., and kept in tanks at 20-2577, with timed lights providing a normal photoperiod. Because some animals were weak or starved on arrival, we found it necessary to force-feed frogs for at least 2 weeks before use in experiments. Each animal received approximately 5 b of Gaines Dog Meal three times a week. This regimen restored both the rhodopsin content and physiological activities of the outer segments obtained from these frogs. In preparation for studies on enzyme activities associated with outer segments, the outer segments were gently shaken from dark-adapted frog retinas and then separated from contaminating material by Ficoll gradient centrifugation : Each retina was swirled gently for l-2 min to detach outer segments in 200-400 ~1 of Ringer’s solution [115 mM-NaCl, 2 mM-MgCl,, 2 mM-KCl, 10 mM-HEPES (CalBiochem), pH 7.51. The outer segment suspension was then made 40% (w/v) in Ficoll (polymerized sucrose, Pharmacia) and layered under a continuous linear gradient of 22-33% Ficoll. After centrifugation for 15 min at 36 000 rev/min in the Beckman SW 36 rotor, l-ml fractions were collected from the bottom of the tube. The distribution of outer segments (i.e. rhodopsin) in such a gradient is shown in Fig. 1. For reasons described in more detail elsewhere (Robinson, in preparation), we found that this non-equilibrium flotation procedure yielded more reproducible and effective separations than sedimentation or equilibrium techniques. (All operations were carried out under dim red light at 4’C.) The rod-shaped outer segments obtained by this gentle flotation procedure were indistinguishable, in the light microscope, from those freshly shaken from the retina, except that their average length was 10-30’7’ smaller. In spite of this fragmentation, physiological activity was retained (see Results). The protein components of gradient fractions were analyzed with sodium dodecyl sulfate (SDS) gel electrophoresis. After experimentation with several different systems, optimum sensitivity and resolution of minor components was obtained using a modification of the stacking-SDS gel system of Laemmli (1970). Outer segments, along with any other membrane components present in a Ficoll gradient fraction, were first washed by suspending in glass distilled water and centrifuging for 30 min at 36 000 rev/min in the Beckman 40 rotor. The sedimented membranes were then dissolved in 8% SDS (0.5 mg/ ml) by stirring overnight under nitrogen. This material was then made 596 in mercaptoethanol, 15% in glycerol, and 0.06 M in Tris Chloride, pH 6.8, 2-4 hr before application to gels. SDS concentration was 0.5% throughout, and 0.05 “/o tetramethylethylenediamine was used in the 7.5% acrylamide gels. After electrophoresis, gels were stained with Coomassie blue as previously described (Bownds et al., 1971) and the absorbance of stained bands measured using a Gilford spectrophotometer with gel scanning attachment. Each gradient fraction was assayed for several different enzyme activities. Adenyl cyclase was measured by incubating aliquots of gradient fractions with 15p.~-[~H]ATp in the presence of 10 mM-MgSO,, 0.5 mM-EDTA, 8 m;w-NaF, O*O37< triton X-100, 0.2 mM Squibb 20006 (a phosphodiesterase inhibitor), and an ATP regenerating system. CAMP produced from tritiated ATP was assayed using the procedure of Krishna, Weiss and Brodie (1968). Ficoll was removed from the gradient fractions by centrifugation before

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assay. Cyclic nucleotide phosphodiesterase was assayed in the presence of O-03:/, triton X-100 using the procedure of Huang and Kemp (1971). ATPase activity was measured using the luciferase assay (Bownds et al., 1971) in the presence of 0.03% triton X-100 after Ficoll had been removed. Cytochrome c oxidase was assayed according to Wharton and GrifIiths (1962) with O*Ol~/, triton X-100 present. Rhodopsin concentrations were measured as previously described (Bownds et al., 1971). Th ese assay procedures are described in more detail elsewhere (Robinson, in preparation).

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Fro. 1. Distribution of enzyme activities after Ficoll gradient centrifugation of crude outer segment preparations. All enzyme activities are expressed with respect to the total rhodopsin in the gradient; i.e. moles of product formed min-1 mol-1 rhodopain present. The two curves for adenyl oyolase activity represent two separate experiments. (a) Phosphodiest’erase, 0-O ; Rhodopsin, ::x : cytochrome c oxidase, A - - -A ; ATPase, q . . .IJ. (b) Rhodopsin. :<- x ; adenyl cyclase, 0 . . .O and n - _ - .I. (o) Rhodopsin, 0-O; phosphorylation, x _- - x

Light activated phosphorylation was assayed using outer segments prepared by a discontinuous flotation procedure (Bownds et al., 1971) which was more effective in removing contaminating membranes than the continuous gradient centrifugation described above [cf. Fig. 2(b) and (c)l. A crude outer segment suspension was prepared by gentle shaking as described above and six volumes of 33% Ficoll were added. The suspension was mixed thoroughly, placed in a centrifuge tube, and gently covered with lS.57o Ficoll. After centrifugation for 15 min at 36 000 rev/min in a Beckman SW 36 rotor the layer of outer segments which formed at the interface between the two Ficoll concentrations was collected in a volume of less than 2 ml, washed to remove Ficoll, and diluted with Ringer’s

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solution to bring the optical bleaching of rhodopsin was no. C53-67 filters) calibrated attenuated with calibrated Palo Alto, Calif.

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density of the rhodopsin present to 0.2 (at 500 nm). Controlled accomplished with an orange light (heat filters plus Corning to bleach l”/ of the rhodopsin present/minute. The light was neutral density filters obtained from Optics Technolopv Inc.,

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FIG. 2. SDS stacking gel eleotrophoresis. (a) Shows material from solubilized gradient fractions, 4, 5 and 6 and (b) gradient fraction 8. (c) Outer segments prepared by the discontinuous flotation procedure. Major mitochondrial protein components, if present, would migrate to the points shown by the vertical bars. The dotted line indicates components which incorporated s2P from Y[~~P]ATP during the light-activated phosphorylation reaction.

The phosphorylation reaction was studied by adding Y-[~~P]ATP of known specific activity to portions of this suspension in the dark and after discrete amounts of rhodopsin bleaching (Bownds et al., 1972). Portions (50 ~1) of these incubation mixtures were withdrawn at intervals, pipetted into 2 ml of 10% trichloroacetic acid (TCA) containing O-2 mg bovine serum albumin, and kept at 0°C for 20 min. The precipitate was collected and washed on millipore filters: 3X with 2 ml 10% TCA; 5X with 2 ml O-1 N-NaOH; 3X with 3 ml Ringer’s solution. The filters were then dissolved in 5 ml Aquasol (New England Nuclear) and counted. Control incubations, using unbleached rods, were included in each experimental condition. Background radioactivity levels were determined by counting filters containing the precipitate from 2 ml TCA plus bovine serum albumin to which an appropriate amount of Y-[~~P]ATP had been added, followed by filtering and

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washing as described above. Activity of the rods was expressed as the ratio of moles phosphate rendered TCA precipitable to moles rhodopsin in the sample. The activity of rods was variable from one preparation to another, but a given preparation was stable for several hours at 0°C. Outer segments frozen in liquid nitrogen and then stored at -20°C retained activity over a period of 3-4 weeks. 3. Results Enzyme studies In searching for enzymes relevant to outer segment function one needs to ask whether the activities found are in fact associated with outer segments or with other membrane fractions. One approach to determining this is shown by the experiments of Fig. 1. Crude outer segment suspensions are subjected to gradient centrifugation and enzyme assays performed on gradient fractions. It is clear that fractions containing outer segments (rhodopsin) also contain rhodopsin phosphorylating activity and cyclic nucleotide phosphodiesterase. The cyclic nucleotide phosphodiesterase activity has previously been associated with outer segments by Pannbacker, Fleishman and Reed (1972). The phosphorylating activity is highest on the left shoulder of the outer segment peak which probably contains relatively more fragmented and disrupted outer segments than the right shoulder. (Experiments in the following section demonstrate that disruption of outer segment structure stimulates the reaction.) The presence of a cyclic nucleotide phosphodiesterase and the observation that CAMP stimulates dark adaptation (Bownds et al., 1973) together raise the possibility t’hat an adenyl cyclase enzyme might also function in this system. Although Bitensky, Gorman and Miller (1971) have in fact reported a light inactivated adenyl cyclase associated with outer segments, we have been unable to confirm their data. The distribution of adenyl cyclase activity in two separate experiments is shown in Fig. l(b). In five such gradient experiments cyclase activity has been found mainly in fractions l-6. The total activity is less than 5% of that reported by Bitensky et al. (1971). Electrophoretic analysis [Fig. 2(a)] indicates that gradient fractions with adenyl cyclase [fractions 4, 5 and 6 of Fig. l(b)] contain membrane material not derived from outer segments. This material is also present in the separated outer segments [Fig. 2(b)], for most of the protein peaks seen in Fig. 2(a) are also seen, at much lower levels, in Fig. 2(b). This contamination varies from one preparation to another and is frequently less pronounced [Fig. l(c)] in outer segments prepared by the discontinuous flotation procedure described in the Methods section. The electrophoresis pattern obtained from the contaminating membranes is different than that obtained from frog retinal mitochondria. The location of the largest mitochondrial protein peaks is shown by the dark vertical bars in Fig. 2(c). The three major bands seen in Fig. 2(c) are the monomer, dimer and trimer of opsin. A major disadvantage of the SDS stacking gel electrophoresis system used here (Laemmli, 1970) is that it promotes formation of these rhodopsin polymers. The procedure, however, offers excellent resolution and detection of minor protein components at the level of 0.5-1-O o/o of the rhodopsin present (0.2-O-4 pg). It has allowed us, for example, to conclude that there is firm evidence for only one major protein component besides rhodopsin associated with outer segments : a high molecular weight species accounting for approximately 4% of the total staining material and having an apparent molecular weight of 220 000. Details relevant to these electrophoretic studies are discussed more fully elsewhere (Robinson, in preparation).

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Figure 1 also demonstrates that the distribution of ATPase, an enzyme usually thought to be associated with outer segments, does not peak with the outer segments: rather this act’ivity is uniform and low throughout the gradient. Its distribution most closely parallels that of mitochondrial cytochrome c oxidase. In a number of experiments utilizing both intact and disrupted outer segments, we have failed to find the ouabain inhibition of ATPase activity which would be expected if Na-K activated ATPase were present. The observed activity (approximately 1 mol ATP hydrolyzed mol rhodopsin-l present min-I) is large enough to account for known “ATPase” reactions such as the light-induced phosphorylation, but is insufficient to generate the dark current observed in vertebrate photoreceptors (Hagins, 1972). The important point brought out by these experiments is that outer segments can be effectively separated from adenyl cyclase and ATPase activities using procedures which do not destroy their physiological activity. (One cannot, of course, exclude the possibility that the low levels remaining with outer segments might play a physiological role.) Having shown that rhodopsin phosphorylating activity and cyclic nucleotide phosphodiesterase can be associated with outer segments, it would seem appropriate now to expand these experiments to include other enzyme activities suspected of playing a role in outer segment function. Light-dependent chemistry Those interested in photoreceptor membranes have focused many of their studies on light-dependent chemistry. The series of colored intermediates which form and decay after photon absorption by rhodopsin, as well as the oxidation-reduction cycle of the retinaldehyde prosthetic group, have been characterized in great detail (Wald, 1968). In experiments originally designed to explain effects of ATP on dark adaptation, we have come upon another example of light-dependent chemistry in this system: a light-induced phosphorylation of rhodopsin (Bownds et al., 1972). Independent work in Dreyer’s laboratory has shown a similar reaction in cattle outer segments (Kiihn and Dreyer, 1972). Upon bleaching small amounts of rhodopsin in a frog outer segment suspension, followed by subsequent incubation in the dark with ATP, one observes incorporation of more than one phosphate per rhodopsin bleached. Figure 3 shows the results from three experiments in which maximum phosphate incorporation is presented as a function of rhodopsin bleaching. Here the “quantum yield” of the reaction (moles phosphate incorporated/mole rhodopsin bleached) changes from 10 to 15 when less than 5% of the pigment is bleached, to less than one for bleaches of lo-100%. [It seems likely that in studying bovine outer segments (Kiihn and Dreyer, 1972; Frank, Cavanagh and Kenyon, 1973) only the second part of the curve (lo-100% bleach) of Fig. 3 is observed since bovine retinas obtained under slaughterhouse conditions are always partially bleached. The frog outer segments used in t’hese experiments contained their full rhodopsin complement.] Maximum phosphate incorporation in bullfrog outer segments (up to 4 mol phosphate/m01 rhodopsin) is observed in suspensions 2 x 10-6-2 x 1O-5 M in rhodopsin and 8 lllM in y-32P-labeled ATP suspended in the Ringer’s solution described in Methods. Little incorporation is observed in the absence of light. The reaction is apparently not limited by the rate of uptake of ATP by outer segments, for preincubation of outer segments with Y-[~~P]ATP before illumination has little or no effect on maximum 32P-incorporation. The reaction does not vary greatly with changes in pH between pH 5 and pH 8. Disruption of outer segment structure by

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freezing and thawing or exposure to detergents stimulates the reaction approximately two-fold. This effect is seen with O*2o/oDigitonin or 04025 M-cetyltrimethyl ammonium chloride, but only in preparations which are initially relatively inactive. Higher concentrations of either detergent completely inhibit phosphate incorporation. Activity is marginally stimulated by addition of glutathione, mercaptoethanol, dithiothreitol, or saturating the incubation medium with argon. Increasing temperature from 20 to 35°C also stimulates phosphate incorporation. Calcium ions inhibit the reaction at levels of 2-10 mM. Neither the 220 000 mol. wt. protein component of outer segments nor contaminating membranes are phosphorylated [Fig. 2(c)]. Using either frozen or fresh outer segments with 1 mM or 8 mM levels of Y-[~~P]ATP, the rhodopsin peaks are the only components in gel electrophoresis patterns containing

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FIG. 3. Maximum light stimulated s2P-incorporation as a function of rhodopsin bleaching. In these experiments outer segments are first irradiated to bleach a known amount of rhodopsin and then incubated in the dark with y-x2P-labeled ATP.

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Figure 4 demonstrates that the time course of the light-induced phosphorylation is quite different at micromolar and millimolar levels of ATP. It is at the higher ATP levels that we have observed incorporation of more than one phosphate per rhodopsin molecule and as much as 50 phosphates for each rhodopsin bleached. Phosphate incorporated at high ATP levels is not lost, although the reaction in vivo presumabl) involves both phosphorylation and dephosphorylation. In contrast, the reaction which occurs at micromolar levels of ATP is readily reversed. We are now attempting to specify the components of the phosphorylation-dephosphorylation system observed at low ATP concentrations. These experiments have not yet touched on the central issue: What is t’he physiological role of rhodopsin phosphorylation ? The most straightforward idea is that phosphorylation is used to regulate an enzymic activity of rhodopsin relevant t*o excitation or adaptation. Because the reaction appears to occur over minutes, rather than milliseconds, we suspect that it plays a role in adaptation. In vitro physiology experiments of the sort described in the next section have shown that the reaction precedes the return of excitability which occurs during dark adaptation. Still, this information is only of limited usefulness because the role of rhodopsin is not yet’ understood. Is vitro physioloyy

The physiological significance of enzyme reactions discussedthus far, as well as the biochemical pathways they regulate, will be understood best when they can be studied in vitro using preparations of outer segments which retain physiological activity characteristic of living rod outer segments.With such preparations one could study the effects of pharmacologically active agents, cofactors, and substrates. Pathways might be discerned by inducing and then reversing specific pharmacological lesions.With these ideas in mind we have worked over the past 2 years to develop a routine and simple assayfor outer segment function. The work derives from the observation, made by Korenbrot and Cone (1972) that illumination influences the osmotic properties of isolated outer segments. The actual experiments and manipulations are best visualized if presented step by step : outer segmentsare first broken off from dark-adapted frog retinas by swirling the retinas in the dark for 1 min in 1 ml of Ringer’s solution, 10% in calf serum and 3 mM in EGTA [ethylene-glycol-bis-@- aminoethyl ether) N&V’-tetraacetic acid]. After the retinas are removed the resulting suspensioncontains outer segment fragments of many different sizes; most are near their original length, 50-60 pm. This suspension is divided into several portions: someare left in the dark while others are illuminated. The outer segmentsswell slowly over a period of l-3 hr; their overall structure and rod shape, as viewed in the light microscope, does not change. The basic observation is that dark outer segments swell more rapidly than illuminated outer segments. Thus, illumination appears to suppress a spontaneous swelling reaction

which occurs in isolated outer segments. The phenomenonis also observed in hyperosmotic solutions. The effect is of interest becausethe suppressionof swelling caused by light has the same quantum sensitivity and ion specificity as the in vivo receptor potential. Further, a process analogous to dark adaptation can be observed (see below). The experimental problem is to measurethe volumes of many outer segmentsand outer segment fragments and to observe how these volumes change over a period of time. Because the photomicrographic procedures which can accomplish this are

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extremely tedious, we have turned to using a particle sizing device, a Coulter-type counter interfaced with a PDP-8 M computer. Suspended outer segment fragments are drawn through a 120-p” orifice for 1 min at a rate of approximately 30 000 fragments (or 2-3 ml) per minute. The passage of each rod-shaped fragment through the orifice causes a conductance perturbation proportional to its volume. Each perturbation is stored as a voltage in the computer. Distributions like those of Fig. 5 O-l-2-3678L 709 742 775 811 848887 927 969 ICI,,!cmI ,cx1159-

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FIG. 5. Effect of illuminetion on the swelling of rod outer segments in hyper-osmotic medium.;Curves A and B, respectively, show the volume distribution of suspended outer segment fragments kept’ illuminated and dark for 100 min. Curve C indicates the volume distribution of outer segments left in isoosmotic medium in the dark.

are obtained; the ordinate indicates the number of particles in each of 64 vol. classes, and the abscissa indicates the volumes in cubic microns. The volume distributions stored by the computer are virtually identical to the actual volume distributions of the same suspension obtained by direct measurements of outer segment lengths and widths using photomicrographic techniques. Figure 5 illustrates the basic point: Dark outer segments (curve B) swell more rapidly than illuminated outer segments (curve A). These curves are the volume

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distributions obtained after outer segments suspensions have incubated for 100 111it1 in the dark or light. [In this experiment) and those shown in Fig. 6, swelling ww\ monitored in the Ringer’sserum-EGTA solution mentioned above, but made hyperosmotic with NaCl (215 lnM, 425 mOsmo1). Thus at the start of the experiment (and in experiments shown in Pig. 6) the outer segments shrank to approximately HO”,, of their original volume and then began to slowly swell. Curve C of Fig. 5 showy the volume distribution of an outer segment suspension left in an iso-osmotic medium.] Becauseone wants to monitor changesin volume asfunction of time, analysis of the data shown in Fig. 5 is carried one step further. The total volume of fragments in a given distribution is determined by a computer operation which mult’iplies the number of particles in each volume classby t,hat vohmle (in cubic microns) and then sums _ (0) x/x-

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FIG.:~. Swelling reaction used to monitor the effect of illumination on suspended frog rod outer segments, (a) Illumination which bleaches 1% of the rhodopsin presentjmin suppresses swelling ( x - : : dark; O-0, hy). (b) Swelling is maximally suppressed by illumination which bleaches 5 x lo2 rhodopsin outer segment-’ see-1 ( n--a) in the absence of EGTA (5 x 10’ hy outer segment-’ set-I, 0-O). (c) If 20% of the rhodopsin in an outer segment suspension The swelling reaction shows “dark adaptation”. is bleached, subsequent swelling in the dark is slowed for 20 min (n-n). Then, the dark swelling rate returns (n-n) and illumination can again suppress this swelling (0-n). (d) The dark-adaptation mechanism is labile. The experiment shown in (c) is repeated with another portion of the same outer segment suspension which has incubated for one hour in iso-osmotic medium in the dark. Suppression of swelling by illumination is still seen (0-O and @-a), but the outer segments fail to show dark adaptation (A-/J). (e) During dark adaptation sensitivity recovers more slowly than the swelling se-’ ( x--/. ). Outer reaction. Dark outer segments ( x . . . x ) respond t,o 5 x 10s hy outer segment-l are not responsive to this level of segments which are dark adapting after a 20% bleach (n-n) illuminat.ion (0-u) even though they regain the dark rate of swelling and the suppression of this swelling by saturating light (0-O) is the same as that seen in non-dark-adapting controls (0-O).

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the product for all 64 vol. classes. Thus, each distribution is reduced to a volume value, and changes in this value are plotted as a function of time as in Fig. 6. As an example, the experiment of Fig. 5 is shown in more detail in Fig. 6(a). Outer segments were shaken from frog retinas in 1 ml of iso-osmotic Ringer’s serum-EGTA solution. Portions (100 ~1) of this suspension were then added to separate beakers containing 25 ml of the same medium made hyper-osmotic with NaCl. One of these beakers was kept dark and another illuminated. The first point was taken immediately after hyper-osmotic shock and the 25 ml suspensions were sampled further at 20-30 min intervals with the particle sizing device. The same basic protocol was followed in the other experiments illustrated by Fig. 6. In Fig. 6(c), for example, outer segments from one animal were divided among six separate 25-ml suspensions and different conditions of illumination used for each (see below). Figure 6(b) demonstrates that the dark rate of swelling is completely suppressed by constant illumination which bleaches 500 rhodopsin molecules outer segment-l set-’ in the absence of EGTA. This sensitivity is similar to that observed in living rod cells in the frog, which show maximum response when only several hundred of the 3 x lo9 rhodopsin molecules in an outer segment are bleached each second. EGTA lowers the sensitivity to light; with the bleaching of approximately 5 x lo3 quanta outer sepment-1 set-l required to half suppress the dark swelling rate [Fig. 6(e)]. [The lightdependent swelling reaction is observed routinely only in the presence of EGTA and calf serum. While the effect of illumination can sometimes be seen in the absence of EGTA, as in Fig. 6(b), it is usually much smaller or absent. This and other conditions which influence the light-dependent swelling reacbion will be outlined more fully elsewhere (Bownds and Brodie, in preparation).] The light-dependent swelling reaction is most clearly observed if the major salt present is sodium chloride. Using EGTA-serum-Ringer’s solution in which NaCl is replaced by KCl, the effect of illumination on swelling is small or absent. Sodium sulfate cannot substitute for NaCl. Thus the sw?elling reaction is similar to the in vivo receptor potential in its ion requirements. The difference in swelling rate> bet,ween dark and illuminated outer segments is lo-SO% smaller in preparations which have been purified by Ficoll flotation. This procedure frequently breaks omer segments into smaller fragments and removes some mitochondrial contamination. We have not yet been able to determine whether the presence or absence of mitochondria influences the swelling reaction. Analysis of the volume distribution indicates that both large and small rod outer segment fragments perform the light-dependent swelling reaction, but it has not yet been possible to determine whether all of the fragments present are functional. If 5--5Oo/o of the rhodopsin in an outer segment suspension is bleached, subsequent swelling in the dark is slowed [Fig. 6(c)], the outer segment fragments behave as if they were being continuously illuminated. After varying periods of time (lo-60 min, depending on the preparation), the more rapid swelling rate characteristic of dark outer segments returns and one can again suppress this swelling with illumination. In this sense: the outer segment fragments “dark adapt”. This process can tjtl observed in both crude and purified outer segment preparations. The lability of t,hc! mechanism in one preparation is shown in Fig. 6(c) and (d). Fresh outer segments can perform dark adaptation [Fig. 6(c)], but if t’he preparation is left at room temperature for one hour it loses this ability [Fig. 6(d)]. although response to illumination is retained. Tn t#hese experiments, as in electrophysiological studies, two variables can be

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measured. The first is the maximum “response amplitude” elicited by saturating illumination, measured by the difference in swelling rate between dark and illuminat~ed outer segments. The second variable is sensitivity, measured by determining how much illumination is required to effect a maximal suppression of dark swelling. The experiment of Fig. 6(e) demonstrates that maximum amplit’ude and sensitivity recover at, different rates during dark adaptation in vitro. An outer segment suspension whose swelling can be half suppressed by 5 x lo3 quanta outer segmenttl set-r is illuminated to bleach 20% of the rhodopsin present. The dark rate of swelling recovers after 30 min and can be maximally suppressed by relatively intense illumination (5 x lo5 quanta outer segment-r set-l) just as before the 20% bleach. Maximum response amplitude has returned. However, these outer segments no longer respond to 5 x lo3 quanta outer segment-l sec- I; their sensitivity is still lowered and ret,urns more slowly. Recovery of sensitivity and maximum response amplitude varies from preparation to preparation, requiring from 5 to 60 min. These data suggest that outer segments can retain, for relatively long periotls of time, the substrates and cofactors required for regulating excitation and dark adaptation. Some decay of endogenous material apparently occurs, however. for we have found in these experiments (Bownds and Brodie. in preparation), as well as in previous more limited experiments (Bownds et al., 1973) that the addition of ATP and CAMP accelerates the rate of dark adaptation in vitro. Because the light-dependent swelling reaction has characteristics very similar to the in vivo receptor potential we have chosen it as a measure of physiological activity. However, one does not yet know whether it is related to the membrane conductance changes demonstrated by electrophysiological techniques. The most attractive current proposal, presented by Korenbrot and Cone (1972), is that the swelling in isolated outer segments is regulated by their sodium conductance. They suggest that dark outer segments expand after hyper-osmotic shock because they are leaky to sodium chloride. Illumination abolishes high sodium conductance and expansion. This argument is strengthened by their calculation that the sodium chloride flux needed to support the complete volume recovery observed within 30 set in hyper-osmotically shocked dark rods is the same as the in vivo flux or “dark current” (Hagins, 1972). On this last point the data in the present paper is quite different from that obtained by Korenbrot and Cone (1972). In very careful efforts to duplicate the conditions of their experiments we have not been able to observe rapid expansion of dark outer segments. Using their solutions (containing Mg or Ca both without serum or EGTA) the effect of illumination has been small or absent and outer segment fragmentation extensive. [In previous work (Bownds et al., 1973), we in fact assumed that volume changes were complete within 30 set as reported by Korenbrot and Cone (1972) and thus did not observe the changes reported here. We studied only the small init,ial differences in volume seen after 5-10 min.] 4. Discussion Although we now have many approaches to studying the system and a large store of phenomenology, the parts do not yet’ fit together. Studies of rhodopsin intermediates, rhodopsin phosphorylation, and enzymes such as adenyl cyclase provide informat,ion on what might be fragments of relevant pathways. However, the nature of t’hose pathways, particularly the small molecules involved, remains obscure. Figure 7 illustrates some of the reactions commonly thought to intervene between photon

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absorption and a sodium conductance decrease; reactions which might be subject to regulation by cyclic nucleotides and the rhodopsin phosphorylating system. A hypothetical transmitter, “X”, is generated soon after photon absorption and diffuses to t#he plasma membrane where it blocks or inactivates the sites at which sodium entry can occur. Hagins and his co-workers have suggested that the transmitter is calcium. and Cone (1972) has further proposed that rhodopsin is a light activated pore or carrier which allows escape of calcium stored inside of the discs. Alternatively, the transmitter might be bound to the disc membranes and dissociated by light, converted from an inactive to an active form, or actually synthesized de novo from a precursor. Each of these schemes must provide for the removal OI inactivation of X, as well as its reuptake, readsorption or resynthesis. Because the sodium conductance of the plasma membrane might be regulated by a relativel! small number ( k103) of sodium channels (Korenbrot and Cone, 1972) the number of transmitters released per photon absorption might also be small (50-100 X,/excited rhodopsin). hu

X’“- -

” x

NO s

\

thought

c

%a -,NO

hV-

Flu. 7. Reactions decrease.

X-‘&a-

to intervene

/I V

X

between

photon

absorption

and the

sodium

conductance

In discussing Fig. 7 one needs to consider not only the initial saturation of the rod response which occurs at levels of 100-200 quanta/rod, but also the changes in maximum response amplitude and sensitivity [as in Fig. 6(e)], which occur during dark adaptation. The experimental observation, repeated in many different studies, is that the maximum response amplitude recovers more rapidly after large bleaches than sensitivity, (the amount of light required to elicit this maximum response) (see, for example, Dowling and Ripps, 1972). One plausible model would have maximum response determined by the number of open sodium channels available to the plasma membrane. If half of the channels were blocked by X, further illumination would affect only the remaining half, and the maximum response amplitude would be reduced by 50%. The more rapid return of response amplitude during dark adaptation would be accounted for by removal of transmitter from the sodium conductance sites. Sensitivity might then be regulated more slowly by any one of several mechanisms : The amount of transmitter generated might change as a function of illumination ; the sodium conductance mechanisms might become “desensitized” to interaction with X after strong illumination; or the reutilization of X might be prevented by its sequestration in an active form (X’) for a limited period of time. Any of these mechanisms could account for the reduced effectiveness of illumination (lower sensitivity) observed during dark adaptation.

2ti6

D.

BOW’NDS

ET

AL.

This simple discussion illustrates a,n important point. The light-dependent cherni,+tr~ discovered thus far could have many possible roles and considerable ingenuity will be required to distinguish between them. Many ideas must be tested, and one tllltst be wary of creating highly explicit molecular models before appropriate (1at.a is available. ACKNOWLEDGMEKTS

This work was supported by NIH grant 5-ROl-EY-00463 and a fellowship, l-F03-EY49504, held by Dr W. Robinson. We.are grateful for the help provided by Dr Anu Ball, Dr Joan Dawes, Dr Ann Gordon-Walker and Mr Mark Stahlman. REFERENCES Bitensky, M. W., Gorman, R. E. and Miller, W. H. (1971). Proc. Nat. Acad. Sci. U.S.A. 68,561. Bownds, D., Gordon-Walker, A., Gaide-Huguenin, A. and Robinson, W. (1971). J. Gen. Physiol. 58, 225. Bownds, D., Dawes, J., Miller, J. and Stahlman, 11. (1972). Nature New Biol. 237, 125. Bownds, D., Dawes, J. and Miller, J. (1973). In Biochemistry and Physiology of Visual Pigments (Ed. Langer, H.). Springer-Verlag, New York. Cone, R. A. (1972). Nature New Biol. 236, 39. Dowling, J. E. and Ripps, H. (1972). J. Gen. Physiol. 60, 698. Frank, R. N., Cavanagh, H. D. and Kenyon, K. R. (1973). J. Bid. Chem. 248,596. Hagins, W. A. (1972). Ann. Rev. Biophys. Bioeng. 1, 131. Huang, Y. C. and Kemp, R. G. (1971). Biodem. 10, 2278. Korenbrot, J. I. and Cone, R. A. (1972). J. Cen. Physiol. 60,20. Krishna, G., Weiss, B. and Brodie, B. B. (1968). J. Phurm. Exp. Ther. 163,379. Laemmli, U. K. (1970). Nature (Lcndon) 227, 680. Pannbacker, R. G., Fleishman, D. E. and Reed, D. W. (1972). Science (New York) 175, 757. Kuhn, H. and Dreyer, W. J. (1972). E%BS Letters 20, 1. Wald, G. (1968). Science (New York) 162, 230.

Discussion Dr Drab Please explain your lightpletely clear to me.

and dark-adaptation

experiments;

they weren’t

com-

Dr Bowlzds The material that I have presented deals only with dark-adaptation experiments. Let me put it as simply as possible. Dark outer segments swell more rapidly than illuminated outer segments. If 20-50% of the rhodopsin in a dark outer segment suspension is bleached by light and the outer segments are subsequently left in the dark, they swell slowly as if the light were still on. After 20-40 min in the dark, however, they begin swelling more rapidly-at the same rate that unbleached outer segments swell. When this more rapid swelling resumes one is again able to suppress the swelling with illumination. Thus the bleached rods have “dark adapted” in that their behavior is now again like the behavior of rods which have not been bleached. There is one important difference, however, which is illustrated by Fig. 6. The “darkadapted” rods are not as sensitive to illumination as rods which have not been previously bleached. Thus we see, as in Fig. 6(e) that “dark-adapted” outer segments do not respond to illumination which bleaches 5 x IO3 rhodopsin molecules outer segment-l second-l while the swelling of unbleached rods is half suppressed by the same level of illumination.

Dr Dratz At the lowest level of ATP you used (10 PM) you may be significantly under counting the number of phosphates per rhodopsin because of the endogenous pool of ATP that you imply exists in these preparations. Have you done isotope dilution experiments, at low ATP levels, to measure the endogenous pool. If so, what is the endogenous ATP concentration?

We have not done careful isotope dilution experiments. The endogenous ATP levels appear to be approximately 1O-5 M, so that the stoichiometry indicated in the dashed curve of Fig. 4 could easily be an error by a factor of 2. In this instance we are not trying to make a point of the stoichiometry of the reaction, but rather point out that its time course is different from that observed at higher ATP concentrations. Dr Kaplan Using fast resolution X-ray diffraction techniques, Chabre and Cavagionni have reported a 2% aqueous volume increase for in vivo rod outer segments subsequent to a brief light flash. The volume increase has a half time of about 25 min at room temperature. Would you care to comment upon how these data correlate with your results in isolated rod outer segments?

Dr Chabre and I have discussed this and have not been able to decide whether the volume increase which he observes is related to those which I have reported here. There may be relationship, but we just don’t understand it yet.

Do your bullfrog preparations contain much porphyropsinl As you know, Reuter and his colleagues demonstrated the presence of porphyropsin (often in considerable amounts) in the dorsal sector of the retina of this species, an observation I have confirmed in bullfrog but not in other frogs of the genera Rana and Hyla.

These preparations do indeed contain porphyropsin, and for several years we have monitored the rhodopsin/porphyropsin ratio and completely confirmed Reuter’s observations. Recently we have stopped actually measuring this ratio because the porphyropsin content of the retinas rarely exceeds 15%, and this is not enough to affect the quantitation of our data. Dr Bensinger At micro-molar ,4TP levels, the increased phosphate incorporation into rhodopsin may lower the availability of substrate ATP for the NA-K ATPase suggesting a relationship between swelling of rods, light-dark and sodium conductance. To test t,his have you tried varying ATP levels, varying bleaching to intermediate levels, and measuring ATP or recovery of dark swelling or use of ATPase inhibitor, e.g. ouabain, NaF, etc.?

268

D.

BOWNDS

ET

AL.

Dr Bownds I’m not sure I understand how you want’ to link together the phonphorylatioll reaction, Na-K ATPase, and the light-dependent swelling reaction. We think that ATP plays a role in the dark-adaptation process, for it,s addition can stimulate the rate of dark adaptation. However, little or no ATP seems to be required for the CMductance change in outer segments which have never been bleached or in those which have been bleached and have dark adapted. In such preparations, we have found no effect of varying ATP levels or of adding apyrase, an enzyme mixture which degrades ATP. We do not, think that it is likely that a sodium potassium activated ATPaae is directly involved in the light-dependent swelling reaction for the reaction is not significantly inhibited by the addition of 1O-5 nf-ouabain. Dr

Franli

Data from my own laboratory, as well as Dreyer? in Pasadena, and yours, as published in t’he past year, has suggest)ed that no more than 1 mol of phosphate can be incorporat’ed per mol rhodopsin. Are you now suggesting that in fact. there is more than one phosphorylation site on the rhodopsin molecule? Or do your dat,a mean that, at very low light levels, more than one molecule of rhodopsin (including molecules that have not absorbed a phot’on) is phosphorylated when only one molecule is bleached?

Our data now do suggest that there can be more than one phosphorylation site on t,he rhodopsin molecule, for we have observed that up to four molecules of phosphate can be incorporated per molecule of rhodopsin present after a lOOo/o bleach. Either every rhodopsin molecule is, on the average, binding four phosphate groups or a smaller number of rhodopsin molecules are binding more than three. At low light levels we do not observe more than 1 molecule of phosphate incorporated per molecule of rhodopsin present, and we find that 3-50 molecules of phosphate can be incorporated for every rhodopsin molecule actually bleached. This suggests either that unbleached rhodopsin molecules are phosphorylated or that t’hose rhodopsin molecules which are bleached are repeatedly phosphorylated at 3-50 sites. This latter possibility seems less likely. Dr Fcrank A somewhat divergent question deals with your gel electrophoresis results. You. as well as some other authors, have stated that higher molecular weight bands in retinal outer segment gels represent dimers and trimers of rhodopsin. My colleague. Dr Dwight Cavanagh, has presented evidence (Cavanagh, H.D., PhD thesis, Harvard University, 1970) that the higher molecular weight band which contains the retinaldehyde chromophore in gel electrophoretic runs of cattle outer segments is not a dimer, but rather represents a separate visual pigment. What is your evidence that these bands are dimers or trimers of rhodopsin? Dr Bow&s The apparent molecular weights of the supposed dimers and trimers of rhodopsin, calculated using the SDS gel technique, are even multiples of the monomer molecular

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weight. While the relative amount of dimer and trimer material can be varied by changing the conditions of solubilization and electrophoresis, the staining material seen in the monomer, dimer, and trimer peaks always accounts for the same fraction of total outer segment protein. Using the SDS technique we can never exclude the possibility that there are two visual pigments each having a monomeric molecular weight of approximately 40 000 and containing the retinal prosthetic group, one of which polymerizes to dimers and trimers under certain conditions. The supposed dimer and trimer peaks, on the other hand, cannot be visual pigments having single polypeptide chains with molecular weights of 80 000 and 120 000, because their appearance can be suppressed by mild changes in solubilization procedure which should not cause peptide bond cleavage.