Eye Hrs. (1989) 48. 589-603
Movements in the Frog Retinal Pigment Dependence of Pigment Migration on Na+ and Ca2+
RICARDO Ilepartamento Xvurocicncins.
de Biologia Per&o
and Departawbento de Fisiologia, Biofisica y y de EstudioP Bvanzados de1 IPN3 Apartado Postal 1&7/cO. Mexico D.F. 07000, Jferico
I.988 and nccPptrd %-II,r.yG.spdfopm
19 December 1988)
The ionic dependence of the screening-pigment migrations in the frog retinal epithelium (RPE) was quantitatively studied with eyecups incubated in media of different compositions. Typical migrations in response to light and darkness. equivalent to those observed in the intact animal, were fully accomplished and maintained for up to 6 hr by the isolated organ bathed in Ringer
solution rich with 0,. Pigment migration in either dire&on was completed under the appropriate illumination conditions at any time during the day, indicating that circadian influences, if present in the intact animal, can be overridden in tjhe isolated organ by light or darkness alone. Pigment aggregation toward the dark-adapted position was inhibited by : (a) low external Cal+, (b) high external Na+. and (c) drugs expected to increase the cytoplasmic levels of either Na+, or Ca”+. like ouabain, caffeine and the ionophore A23187. However, the inhibition caused by low Ca2+ did not occur if Na+ was also reduced in the incubation medium. On the other hand, an increase in the concentration of external Ca2+ or the addition of C!02+to the normal Ringer facilitated pigment aggregation in the dark. Pigment dispersion to the light-adapted position was unaffected by any of the above conditions. This is the first report of full and stable pigment responses in the RPE of vertebrate eyes incubated under simple physiological conditions. The results seem to conciliate a discrepancy of previous reports on the Ca”+ dependence of RPE movements, and are compatible with current views on ionic mechanisms in analogous systems of intracellular transport. h’py words; retinal pigment epithelium ; screening-pigment migrations : adaptation : motility ; intracsellular transport; frog.
1. Introduction The retinal pigment epithelium (RPE) of the lower vertebrates participates in light and dark adapt’ation through ample movements of the screening-pigment granules contained in the epithelial cells. Under bright illumination the pigment is dispersed along the narrow cell projections that interdigitate with the outer segments of the photoreceptors, and therefore absorbs much of the light that hits upon the retina. In the dark the pigment aggregates close to the basal ends of the epithelial cells, thereby fully exposing the outer segments of the photoreceptors, with the corresponding increase in visual sensitivity. Since its independent discovery by Kiihne (1877) and Boll (1877). the phenomenon has been the focus of numerous investigations attempting t,o characterize its physiological role, and understand the mechanisms involved in both the transport, of the pigment granules and its regulation (for historical reviews see Parker. 1932; Ali, 1971). rnfortunately, the analysis of the mechanisms of pigment migration in the RPE has been hindered by difficulties in obtaining a fully functional in vitro preparation. In clont,rast to other highly specialized structural responses of the retina in lower * To whom all correspondence and reprint requests should be addressed at Departamento de Biologia (leiular, Biofisira $ Neurosciencias. (‘entro de Investigation y de Estudios Avanzados del IPN, Apartado Postal 14-710. Mexico D.F. 07000, Mexico t~l4-~~835/89/O,iO589+
0 1989 Academic Press Limit,ed El3R
vertebrates to light and darkness. like photjoreceptor disc shedding and phagocytosis by RPE cells. or changes of photoreceptor length, which have been successfully induced in eyecups of Srnopus Znc7Gs incubated in cult’ure media prepared with standard c-onstit,uent’s (Besharse. Terrill and Dunis. 1980; Besharse. Dunis and Burnside. 1988; Besharsc and Dunis. 1983). the transloention of pigment granules within RPE cells has been found less easy for a similar experiment,al approach. Since pigment migration does not, occur in the RPE of intact X. lae& (see Besharse and Dunis, 1983), this particular response cannot, be studied in such otherwise ideally suited preparation which clearly sustains several light,-dependent morphological reactions of the retinal cells. On the ot*her hand, attempts with isolat’ed eyes of species that do display pigment movements in their RE’E have produced mixed or even contradictory results. The basic problem has consisted in an apparent) reluctance of the pigment to undergo complete and stable migrations in isolated eyes under simple incubation conditions. Thus, early efforts to react’ivate retinomotor movements in enucleated eyes of the salmon were frustrated as the pigment remained arrested in an int,ermediate position, irrespective of the presence or absence of light (Ali, 1962). Later on, Snyder and Zadunaisky (1976) succeeded in inducing complete pigment migrations with frog eyecups incubat,ed in plain Ringer medium, but the darkadapted position could not be maintained and spontaneous pigment dispersion to the light-adapt,ed posibion occurred within 1 hr in darkness. In more recent st,udies. pigment aggregat’ion in retinas excised from teleost fish required the presence of high levels of cyclic adenosine-3’,5’-monophosphate plus inhibitors of phosphodiesterase ether) activity (Burnside and Basinger. 1983) or ethyleneglycol-bis-(P-amino-ethyl N,N-tet,ra-acetic acid (EGTA) buffers (Dearry and Burnside, 1984). but, it did not take place in the normal physiological solution. Therefore, many questions about t,he basic properties of the RPE photomechanical responses have remained unanswered or controversial because of the lack of a convenient experimental model. In particular. conflict,ing views are still unsolved with regard t,o the role of (la’+ in the control of the migration process low levels being considered as eit’her antagonistic (Snyder and Zadunaisky, 1976) or favorable (Dearry and Burnside, 1984) for the aggregated position of the pigment in the dark. We have tried, on Rana yipiens eyecups, incubation conditions that had proved successful for sustaining the ext,ensive migrations of screening-pigment granules in the photoreceptor cells of crustacean compound-eyes (Frixione. Arechiga and Tsutsumi, 1979; Frixione and Arichiga, 1981). Such conditions were found support,ive also of full pigment migrat,ions in the isolated retina-RPE-choroid-sclera unit, elicited by their natural stimuli, without’ t,he inconvenience of spontaneous dispersion of the granules in t’he dark. Thus, the preparation is useful for a detailed inspection of t,he RPE responses in both standard and altered media of chemically defined compositions. This paper describes the procedures that permit a satisfactory mot,ile activity of t,he frog RPE in vitro, and reports observations on the changes of pigment behaviour upon manipulations of the ionic balance in the external and intracellular milieus. The results are discussed in relation to previous work with isolated eyes of vert,ebrates, and t’o similar findings with analogous systems of intracellular transport,.
Aninbals and dissection Adult frogs (R. pipiens) were bought from specialized collectors and kept in outdoors aquaria provided with small terraces where they could feed ad libit,um from insects among the vegetation. The animals were decapitated. and their eyes were removed from the sockets and further dissected while immersed in Ringer (solution A\, Table I). Eyecups, i.e. the complete retina-RPE-choroid-sclera complexes freely exposed to the environment, were prepared by hemisection behind the ora serrata. elimination of the front part of the rye, and enucleation of the vitreous body. TABLE
of the solutions used for incubation
110 110 110 110
2 2 2 I 2 2 2 2
(B) (C) (D) (E) (F) (G) (H)
Low Ca2+ High Ca2+ cc?+ Low Na+ High Na+ Control high Na+ Loa Ca.2+.low Na’
9.0 1.8 1.8 1% 1.8
1156 112.0 [email protected]
125.6 115.6 170.6 170.6 1124
of the eyec’ups (rn:v)
110 55 110
Imidazole (JlH $4) 11) I0 10 10 10 10 10 I0
Incubation of eyecups Closed incubation chambers were prepared as previously described for similar experiments with isolated eyes of crustaceans (Frixione et al., 1979). The eyecups were fastened (concave side up) with stainless steel pins on the small face of large rubber stoppers, which were then fitted on wide-mouthed 100 ml glass vials containing 6 ml of incubation medium. The volume of liquid was selected so that the eyecups were just covered when the chambers were inverted and left, standing on the large face of their stoppers as the final position. *Just before each chamber was tightly closed, the vial was thoroughly flushed with 100 “/o 0,. The incubation chambers were then kept under light-proof conditions to induce pigment movement to the dark-adapted position. For experiments on light adaptation. after 3 hr in the dark the eyecups were irradiated with approximately 2500 lux of incandescent illumination. Mild manual agitation of the chambers at frequent intervals, rather than continuous shaking by mechanical means, was provided t’hroughout the incubation period. All experiments were carried out at 17°C. The test solutions were tried as follows: (i) for experiments on dark-adaptation, after dissection in Ringer under illumination, the eyecups were incubated in the test solution for 3 hr in the dark and then fixed in darkness; (ii) for experiments on light-adaptation, eyecups dissected under illumination were first pre-incubated for 3 hr in Ringer in the dark. t,hen placed in the desired solution while in darkness, and finally exposed to light for 3 hr before fixation. Controls were similarly manipulated, substituting fresh Ringer instead of a different solution. Exchange of test solutions could be done very easily, either in the light or in total darkness, by merely transferring the rubber stoppers with the attached eyecups to O,-flushed vials with the desired new media. Caffeine and ouabain were dissolved directly in Ringer at the specified concentrations. A23187 was first prepared as a stock solution in ethanol, and then diluted in Ringer to a final concentration of 16 ,UM ionophore and 1% ethanol. The drugs and the ionophore were obtained from Sigma Chemical Co. (St Louis, MO).
Fix&ion and microscopy The eyecups were fixed in the light or in total darkness by substitution of the incubation medium with 4 ‘!! glut’araldehyde in @1 M phosphate buffer (pH 7.4). Fixation was c~arried out fnr 30 min at 17°C. followed by 60 min on ice. After three subsequent washes wit’h buffer. the eyecups were divided into narrow strips and post.tixed for I hr with cold 1% 0~0, in phosphate buffer (pH 7.4). These pieces of tissue were then rinsed in distilled wat,er. dehydrated in a series of ethanol and propylene oxide, and embedded in Kpurr or Araldite resin. Semi-thin sections (150-200 nm) from t,he blocks were cut on glass knives with a Reichert,&Jung ultramicrot,ome, mounted on glass slides, and st,ained with t.otuidine blur for observation and micrography with bright-field optics in a (‘arl Zeiss photo-mic.ros~c,pr. Thick sections (2-3 j”rn) from representative specimens were also made and left unsta~inrtl for inspection by Adeomicroseopy of asymmetric illumination caontrast (Kachar. 19X.5). using a KC-67M Dage-MT1 newvicon-tube camera coupled to a Reichert mi~rosc.ope. and a Panasonic WV-5350 high-resolution tnonitor. I’ideomicrographs were produc.ett by pt1ot.o graphy of t.he monitor screen on 35 mm Kodak Plus-S film with a (‘anon A-l (‘amera equipped with a ma(m) lens. Preparation of eyes light- or dark-adapted in Situ For dark-adapted specimens, frogs were left overnight in a photographic darkroom. and their eyes were dissected and fixed as described above. Dissection was carried out early in the morning under a deep-red safe-light (25 watt bulb through a Hansa No. 4C filter). Lightadapted eyes were prepared similarly. except that the procedures were performed under fluorescent laboratory lamps. with frogs previously exposed for I hr to bright incandescent illuminat,ion. Further processing for microscopy and micrography were the same as desrribed above for egecups adapted in inrubation. Assessment
The position of the pigment was measured with an eyepiece-micrometer installed in the microscope, by an adaptation of the met.hod previously described for crustacean eyes (Frixione et al.. 1979; Frixione and Arechiga, 1981 ; see also Burnside. Adler and O’Connor. 1983). The pigment index is obtained as a quotient of two measurements: (a) the distance from the Bruch’s membrane to the inner margin of the pigment mass. which varies depending upon the ext,ent of pigment. migration; and (h) the distance from the Bruch’s membrane to the outer limiting membrane of the retina. which is fixed for any given eye. Therefore, the index (n/b) expresses the fraction of the distance from the Rruch’s membrane to t.he outer limiting membrane being occupied by the pigment. Since the inner margin of the pigment mass may vary slightly from cell to cell in each epithelium. 5-10 measurements from every retina (different sections from different blocks obtained from the same eye) were averaged in t,he determination of the corresponding index. Index values from at least five eyes processed in different experiments were used to calculate the data shown on the figures.
3. Results Pigment
and in situ
The preparation and standard incubation conditions used in these experiments permitted full migrations of the screening-pigment to the light-adapted and t’o t’he dark-adapted positions in the frog RPE. Eyecups isolated from light-adapted animals, and then incubated in imidazole-buffered Ringer (solution A in Table I) for 1 hr or longer in the dark, showed a typiral aggregat.ion of t,he pigment granules within the basal ends of the epithelial cells [Figs l(a, b) ; for general anatomical reference and detailed morphological description see Nguyen-Legros, 19781. Similar results were obtained using Ringer buffered with sodium bicarbonate according to the composition described by Sillman, Ito and ‘I’omita (1969). Nevertheless, in order t.o have a buffering capacity independent, of a11 t.he ionir species and at,mosphere
BM Fro. 1. Micrographs of frog retinal pigment epithelium (RPE) after dark-adaptation (top) and lightadaptation (bottom) of eyecups in vitro. Micrographs on the left were obtained by videomicroscopy of asymmetric illumination contrast, whereas those on the right were made by standard bright-field optics. The width of the pigment mass is best appreciated in the thirker sections used for the videomicrographs (2-3 pm). while the familiar histological image can be seen in the common light-micrographs of st.ained semi-thin sections (200 nm). In the dark-adapted state (a. b) the screening-pigment is compactly aggregated at the basal ends of the epithelial cells. close to the Rruch’s membrane (RM). After lightadaptation (r, d) the pigment, granules are found fully dispersed along the cells, filling the apical processes that intcrdigitate with the outer segments of the photoreceptors and up to the level of the outer limiting membrane (OLM). OS. outer segments of photoreceptors: n. nuclei of photoreceptors. x 400
-BM FIG. 2. Micrographs of frog retinal pigment epithelium (RPE) after dark-adaptatio n (top) and lightadaptation (bottom) of whole eyes in situ. Micrographs on the left were obtained by vi deomicroscopy of asymmetric illumination contmst.. whereas those on the right were made by standard t right-field optics. The pigment positions are approximately t.he same as those observed in eyecups adq )ted in vitro (Fig. l), except for a less extreme span of migration. BM, Uruch’s membrane; OLM, outer lill niting membrane. x400
mixtures t’o be tested in these and future experiments, imidazole was selected for a good control of pH in routine work. The eyecups proved capable of maintaining a stable dark-adapted pigment position for up to 6 hr. When eyecups that had been incubated in the dark for 3 hr were subsequently illuminated for 1 hr or longer, the pigment was found dispersed throughout the epithehal cells. with many pigment granules dist’ributed along the apical processes that interdigitate with the outer segments of the photoreceptors [Figs l(c, d)]. The light-adapted position of the pigment was maintained for as long as the eyecups remained exposed to light before fixaCon. Except for a few specimens possibly damaged during dissection, which needs to be carefully gentle, t’hese responses were consistent.ly observed. DefecGve prrparat.ions invariably showed t.hc pigment in an int,ermediate or light-adapted position. Pigment migration in isolated eyes is often more pronounced than in the intact animal, particularly as regards the extent of dispersion in the light-adapted st’ate. Pigment granules reaching up to the out(er limiting tnembrane of the retina were commonly seen in light-adapted eyecups [Figs l(c, d)], which is seldom the case in the intact eye [Figs Z(c, d)]. This difference might be perhaps att,ributed to a more direct exposure of the retina to light - and therefore a higher level of photic excitation ~ in the bare eyecups than in whole eyes in the animal, since the adaptive value of the mobile screening-pigment seems t’o depend on a rel&ionship between light int)ensitl and ext,ent of pigment migration (for discussion see Parker, 1932; Ali. 1971). The pigment responses were also more uniform among cells of the same epithelium and among different, epithelia when t.he eyes were light -adapted or dark-adapt,ed in vitro. Mrasuremenm of average pigment indexes and S.D. for each of these conditions are shown on Fig. 3.
In situ Dark-adopted
In Situ Llqht
In Vitro -adopted
Fm. 3. Pigment positions in the RPE of the frog after 3 hr of dark-adaptation and light-adaptation in situ and in vitro. The pigment index represents the fraction of the distance between the Rruch’s membrane and the outer limiting membrane occupied by the pigment mass. Pigment index values were calculated from measurements in several sections obtained frotn at least five retinas, as described in Methods; vertical lines correspond to S.D. Eyecups adapted in vitro show a more uniform and slightly wider span of pigment migration, particularly as regards the extent of dispersion in the light-adapted state. Statistical analysis of the data by the Students’ t-test revealed that the difference between lightadapted eyes in vitro and in situ is significant at the P < 0901 level.
AND E. FKISIONF,
Our first experiments were carried out taking into account a possible circadian rhythmicity of the screening-pigment, migrations (see Levinson and Burnside. 1981). Incubation of isolated eyes in the dark was initially performed at night. whereas light’adaptation was tested with eyes excised in the morning under a red safe-light from frogs that had been kept in the dark since the previous evening. Nevertheless. we soon found out that pigment migration in either direct,ion can occur fully at any time of the day in the isolated preparation, provided that appropriat,e conditions of illumination and incubation are met. This offers the advant,ageous possibility of starting every experiment with light-adapted frogs. from which dark-adapted eyecups can be prepared when needed during the day. The inconvenience of dissecting under dim filtered illumination (for t.he preparation of dark-adapt.ed specimens used in light-adaptation studies) is thus avoided. Accordingly. most of t,he data reported herewith were obtained in experiments performed during the daytime. Our incubation setup permits that all changes of solutions, including fixat,ives, be easily made either in the light or in t,otal darkness as explained in the Methods section. The risk of triggering pa.rtial pigment dispersion because of complicated manipulations after dark-adaptation is thus ruled out. All incubations in altered media were carried out for 3 hr, long after the 60 min period required for the completion of the RPE retinomotor movements either in sit,u or in vitro (Snyder and Zadunaisky. 1976 ; Burnside et al., 1983), so it is reasonably safe to assume that the data from every experiment represent’ the result, at equilibrium for any given set of conditions. Ca2+ dependence of pigment
Ca*+ levels, both external and intracellular, appear to be critical for the control of pigment position (Fig. 4). Incubation of isolated eyes in Ringer prepared without Ca (solution B) was incompatible with dark adaptat,ion of the RPE ; pigment aggregat’ion was blocked or reversed under these conditions. Low external Ca2+ did not affect the dispersed position of the pigment in light-adapt,ed eyes kept in the light, but it prevented pigment. withdrawal upon light-deprivat,ion or caused pigment dispersion when applied to dark-adapted eyes which were then maintained for an additional 3 hr in the dark. On the other hand, a five-fold increase in the concentration of external Ca2+ (up to 9 mm, solution C) not only permit’s a complete pigment migration in eit,her direction, but in fact it seemed t’o facilitate aggregation in qualitative terms, i.e. it’ produced a somewhat ‘cleaner’ dark-adaptation. Interestingly, a similar appearance was seen after incubation in the dark with Ringer containing 5 mM Co%+(solut,ion D). Since Co2+ is a well known Ca 2+ channel antagonist in most, membranes, this finding suggests that, rather than a Ca-dependent effect, facilitation of pigment aggregation is unspecifically produced by the presence of relatively high levels of divalent cations in the external medium. Pigment migration to the light-adapt’ed position was insensitive to this condition; eyecups bathed in high Ca2+ medium following incubation for 3 hr in Ringer in the dark, showed a fully dispersed pigment after being re-exposed to illumination. The correlation between low external Ca2+ and inhibition of pigment aggregation is apparently inverted when attempts are made to modify the levels of intracellular Ca2+, i.e. an elevation of internal Ca2+ seems to oppose pigment aggregation. Thus, incubation in Ringer containing 16 ,BM of the Ca2+- selective ionophore A23187, which would be expected to render the plasma membranes of the cells permeable to Ca2+ (Pressman, 1976; Gomez-Puyou and Gdmez-Lojero, i977), thereby increasing its activity in the cytoplasm through influx from the bathing medium, abolished pigment
FIG. 4. Effects of conditions tending to modify the external and intracellular levels of Ca2+ upon darkadaptation of frog RPE in vitro. Pigment aggregation was not ohserved after incubating the eyecups for 3 hr in darkness with Ringer prepa& without (‘a’+ UT with drugs expwted to rise (‘a’+ activity in the rptoplasm.
aggregation. Similarly, Ringer wit)h 5 mM caffeine. a drug which might rise t,he concentration of Ca”+ in the cytoplasm (Chiarandini, Reuben. Brandt and Grundfest, 1970). also cancelled pigment’ aggregation in the dark. None of these compounds affected pigment migration to t’he light’-adapted posit,ion. Xa’ dependenw
Alterations in the concentration of external Nat produced effects opposite t,o those of changes in external Ca2+ (Fig. 5). The replacement of Na’ with choline in the otherwise normal Ringer (solution E) allowed a complete pigment migration to the dark-adapted position, while a 1.5 increase in the concent,rat’ion of external Na+ (solution F) arrested the pigment in the light-adapt,ed st.at,e. The lat.ter medium is h,vpertonir for frog t’issues, so there might, be a possibilit,y that’ its effect is the result of osmotic stress over the epithelial cells. However, a normal Ringer with choline chloride added t#o compensate for NaCl excess in solution F (solution G) was compatible wit,h a full aggregation of the screening-pigment to t’he dark-adapted position. Similar manipulations of external I;a+ did not’ interfere wit,h the full dispersion of t.he pigment in evecups exposed t,o illumination after subst,it.ut,ion of t,he inctubation media in the dark. Since the incubat,ion medium prepared by substitut’ion of Na+ with choline had opposite effects on dark-adapt,at’ion of the RPE than that in which no Ca2+ was added, we t,ried a combination of both conditions. Incubation in a medium prepared without Sat and (la*+ (solution H) permitted a normal pigment aggregation toward the darkadaptSed position. A typical dark-adapt&ion of the RPE was observed also in eyecups incubated in t,he dark with an imidazole-buffered 200 mM solution of sucrose. i.e. in the absence of all the regular ions of the Ringer solution. In order to explore the influence of intracellular Na’ on the pigment movements we tested the effect of ouabain. which would be expected to increase the activity of Na+ in the cytoplasm through depression of the ,“u’a+/K+ pump (see Stahl. 1986). The presence of #1 mM and O-.5m&l ouabain in t,he normal Ringer inhibiad pigment
0.5 mM ’
Fm. 5. Effects of conditions tending to modify the external and intracellular levrls of Na+ upon darkadaptation of frog RPE in vitro. Pigment aggregation was not observed after incubating the eyecups for 3 hr in darkness with Ringer containing 1.5 x the normal concentration of Sa+, or ouabain. A4 simultaneous reduction of Na+ and (‘a?+ m the incubation medium is compatible with a typical darkadapted position. The bar labeled as Choline control corresponds to Ringer plus Aoline chloride added (Solution G in Table I) to compensa.te osmotically for NaC’I escess in the high Na+ medium.
aggregation in the dark by about 30% and 450/o, respectively (Fig. 5). When eyecups similarly treated with ouabain were re-exposed to illumination before fixation, the pigment was found in a full light-adapted position. As a rule, incubation in altered media affected only the capacity for the RPE t.o attain a dark-adapted position. Eyecups exposed to illuminat’ion always showed a full light-adapted state, irrespective of the composition of t,he incubation medium.
4. Discussion Pigment
in isolated eyes
The results presented in this paper are the first’ report of full and stable pigment responses in t,he RPE of eyecups incubated under simple physiological conditions. Previous attempts had either been unsuccessful in reactivating pigment migration (Ali, 1962), or in maintaining a stable pigment aggregation in the dark (Snyder and Zadunaisky, 1976), or otherwise required incubation media of special compositions to produce complete migrations (Burnside and Basinger, 1983; Dearry and Burnside. 1984). These difficulties were perplexing because incubation of eyecups in culture media prepared with standard constituents had proved capable of sustaining other structural responses induced by light and darkness in the retina of the lower vertebrates, such as changes in photoreceptor length and photoreceptor disc shedding and phagocytosis by RPE cells (Besharse et, al., 1980, 1982; Besharse and Dunis. 1983). In this regard, the present preparation opens a new alternative for the in vitro study of retinomotor movements. Two factors may account for the satisfactory pigment behaviour observed in our experiments: the relatively mild agitation provided during incubation, and the strategy used for supplying oxygen to the isolated organs. The intensity and
frequency of agitation demand consideration in view of recent findings about regulation of retinomotor movements by dopamine and other neuromodulat80rs locally released in the vertebrate retina (Dearry and Burnside, 1986a.b). Since t’he eyecup preparation retains an intact relationship of the RPE with neural element)s of the ret.ina, a minimized washout of diffusible modulators which seem to favor the dark-adapted state. e.g. y-amino-n-butyric acid may facilitate pigment aggregation if it concurs with a depressed release of light-adapting modulators such as dopamine and 5-hydroxytryptamine in the dark. The so far unwieldy dark-adapted position of the scareening-pigment could be thus stabilized. The oxygenation procedure coould be anot,her helping fact,or because pigment migmtion in t,he RPE. particularly the aggregation phase. is probably as highly dependent upon oxidative metabolism as many other examples of active translocat,ion of intracellular components. Axoplasmic transport (Ochs and Hollingsworth, 1971). pigment aggregation toward the dark-adapted position in crayfish retinula cells (Frixione et al.. 1979) and pigment dispersion in fish rhromatophores (Saidel. 1977; Lub,v and Porter, 1980). are all either inhibit’ed or reversed by hypoxia, or b> trrat.ment wit.b metabolic blockers like cyanide and dinitrophenol. Slthough bhe importance of this point has been t’aken into account by other authors working w&h isolated eyes, in our experience the preservation of motility functions in retinal cells embedded deeply within the ocular tissues requires of special efforts t,o insure a very rich oxygenation of the incubation medium. Therefore, we used tight’ly closed chambers with a large ratio of 0,.at,mosphrre volume to Ringer volume, and a high surface-to-volume rat.io of liquid. This configurat.ion of the chambers, plus the slight. pressurization that results from fitting the rubber stopper to the mouth of the vials. ant1 a modest gain in 0, solubility in the saline by incubation at a temperat’ure several degrees cooler than those tried by previous authors. apparently secures the high tension of 0, needed to support a complete and stable pigment aggregaCon in vit#ro for rrlat,ively long periods. A similar st,rat.egy for intensive oxygenation, i.e. an excess of gaseous 0, in a sealed cont,ainer with a minimum of liquid surrounding the tissue. was chosen to sustain fast axoplasmic t,ransport in vitro (Lochs and Hollingsworth. 1971). The fact that pigment migrations elicited by the nat’ural stimuli in isolated eyes can be equivalent or even better than those observed in the intact animal. demonstrates t.hat. the processes involved are intrinsically independent from control mechanisms external to the eye itself. Nevertheless, these findings do not exclude the possibility t)hat the RPE behaviour could be modulated by hormonal factors or efferent activit’y through the optic nerve. as it has been shown for invertebrate eyes (Kass and Barlow, 1984). At least t,he circadian rhythmicity of retinomotor movements in some species is likely dependent upon modulations by chemical messengers (Dearry and Burnside. 1986a.b). Dependence
Our observations on the influence of calcium in the incubation medium over pigment position confirm previous findings by Snyder and Zadunaisky (1976), i.e. pigment aggregation in the dark is inhibited when calcium is eliminated from the Ringer composition. However, this is probably not t.he result of an induced decrease in the levels of cytoplasmic Claz+ m t,he RPE cells, as the above authors interpret,, because the inhibitory effect of a low-Ca2+ solution does not occur if Na+ is also suppressed from the medium. It could be argued that Na+-deficient solutions might
i<. hloNI)KL4C:tiN AND E. JI’kIxIo,lrTE
block the operation of a Na+/Ca’+ exchanger at the RPE plasma membranes, thereby helping the cells to retain sufficient Ca2+ to support pigment aggregat,ion. Yet, drugs that are expected to increase intracellular Ca ‘+, like the ionophore A23187 (Pressman, 1976; Gdmez-Puyou and Gomez-Lojero, 1977) and caffeine (Chiarandini et al.. 1970). also opposed pigment aggregation. Taken together these data suggest that low Ca2+ levels in the cytoplasm of retinal cells are permissive or conducive, rather than inhibitory, to dark-adaptation and pigment aggregation in the RPE of the frog, as concluded for the case of fish RPE (Burnside and Basinger. 1983). If so, the mechanisms controlling pigment posit’ion in the RPE might, be analogous to those found in arthropod retinula cells, where high intracellular Ca”+ has been associat,ed with pigment migration to t’he light-adapt,ed posit,ion (Olivo and Larsen. 1978; Kirschfeld and Vogt, 1980; Frixione and Arechiga, 1981; Frixione and Ruiz, 1988). The pigment position was found sensitive also t,o manipulations of Nat or Na+handling systems in the retinal tissue. Inhibition of pigment aggregation was observed under conditions expected to rise the intracellular levels of Na’. such as an abnormally high Na+ gradient across the plasma membranes or depression of the Na+/K+ pump by ouabain. The inhibition of pigment aggregation that results from as an indirect effect a diminution in extracellular Ca”+ might also be explained involving Na+. External Ca”+ has been shown to modulate Na+ conductance (Fulpius and Baumann, 1969; Brown and Pinto. 1974; Hagins and Yoshikami. 1975; Lipton, Ostroy and Dowling, 1977) and internal Na’ activity (Coles and Orkand, 1982) in various retinal cells. It’ is thus conceivable that a decrease in external Ca*+ could enhance the permeability of the plasma membranes of the cells to Na+. leading to an increase of Na’ activity in their cytoplasm. This view would explain the absence of inhibition by low Ca”+ when Na’ is also withdrawn from the medium. On t,he other both of which probably decrease Na+ hand, media containing high Cat2+ or Co’+, leakage into the cells (see Blausbein and Goldman, 1968; Fulpius and Baumann, 1969), would be expected to ease pigment aggregation as we have found. Moreover, if a Na+ increase in the cytoplasm of some retinal cells is indeed associated wit.h pigment dispersion, stimulation of the Na+/K+ pump at the plasma membrane with high external K+ should favor pigment aggregation, in particular when combined wit,h a simultaneous reduction of external Na+ and Ca*+, which might, collaborate for a more thorough Na+ deplet’ion from the cells through gradient, inversion and increased membrane permeability, respectively. Spontaneous pigment aggregation, i.e. despite constant’ illumination, has been in fact’ observed in fish retinas incubat,ed under such conditions (Dearry and Burnside, 1984). Possible effects of Na+ and C’a2+ expekments
on the RPE
Given the complexity of cellular interactions in t’he vertebrate retina, the results of treating whole eyecups with media of different caompositions cannot be analyzed, without additional evidence, at the subcellular level for a particular cell type. Therefore, discussion of the exact effects of Na+ and Ca*+ manipulations on the physiological mechanisms that control pigment migration in the RPE must await further research. Nevertheless, possible direct effects of t.he present experiments upon the RPE are worth brief consideration at this point, due to an obvious resemblance of the results with those of equivalent tests on analogous systems of intracellular transport. A Na+-induced Ca”+ -release from internal stores has been suggested t’o affect pigment migration in crustacean retinula cells (Frixione and Arechiga, 1981) and axoplasmic transport in mammalian nerve (Worth and Ochs, 1982). Recent
Nat AND (‘a’+ I)EPENDEX(‘E
OF RPE ,1IO\‘EJlESTS
findings of a drastic diminut,ion of 45Ca2+ uptake by the naked cytoplasm of the ret’inula cells in the presence of Na+ have strengt’hened this view. and led to the proposal that Na+-induced high levels of intracellular Ca*+ could directly inhibit’ the mechanism for pigment aggregation (Frixione and Ruiz, 1988). Similarly, fluct~uations of Na+ activity in the cytoplasm of the RPE cells could modulate internal (la” the pigment translocating apparatus. storage, which in turn might intluence Experimental conditions t,ending t’o elevate either Sa’ or Ca2+ in the cytoplasm of RPE cells could thus inhibit the dark-adapted position of the screening-pigment. Intracellular compartments presumptively involved in the regulation of Ca2+ within RPE cells are yet to be investigat,ed. However, the pigment granules themselves, which are known to constitute Ca deposits in many cells (Brown. Baur and Tuley. 1975; Krauhs. Sordahl and Brown. 1977; White and Michaud, 1980). could function as a Ca”+ reservoir in the RPE of vert,ebrates (Panessa and Zadunaisky. 1981). Alternatively, this role could be played mainly by the endoplasmir reticulum. which seems involved in Ca2+ regulation in many cells (see Somlyo. 1984). including retinal cells in both vertebrat,es (Ungar, Piscopo, Letizia and Holtjzman. 1984; Somlyo and Walz, 1985) and invertebrates (Perrelet and Bader, 1978; Walz. 1979. 1982aac; Frixione and Ruiz. 1988). In this regard. it’ is interesting that the smooth endoplasmic reticulum is particularly well developed and organized in RPE cells (Nguyen-Legros. 1978). ACKNOWLEDGMENTS We thank Francisco Garcia-Sierra for his generous help in the preparation of the videomicrographs, and MS Leonor Fierros for her assistance in typing the manuscript,. This work was supported in part by the research grant PCCBBNA-020479 provided by CONACYT (Mexico). During the execution of t)his investigation. R.M. was an undergraduate student of the Facultad de Estudios Superiores Cuautitlin. I’niversiclad National Autonoma de Mexico. REFEREXCES Ali. M. A. (1962). Retinal responses in enurleat,ed eyes of Atlantic salmon (Salmo s&r). Rw. Can. Biol. 21, 7-15. Ali. M. A. (1971). Les responses retinomotrices: caracteres et mecanismes. V&ion Res. 11. 1225-1288. Besharse, J. C. and Dunis, D. A. (1983). Rod photoreceptor disc shedding in eye cups: relationship to bicarbonate and amino acids. Exp. Eye Res. 36. 567-580. Besharse. J. C.. Dunis, D. A. and Burnside, B. (1982). Effects of cyclic adenosine 3’5. monophosphate on photoreceptor disc shedding and retinomotor movement. Inhibition of rod shedding and stimulation of cone elongation. J. Cl’en. Physiol. 79, 775-790. Besharse. J. C.. Terrill, R. 0. and Dunis, D. A. (1980). Light-evoked disc shedding by rod photoreceptors in vitro : relationship to medium bicarbonate concentration. Inwst. Ophthalmol. Vis. Sci. 19, 1512-1517. Blaustein, M. P. and Goldman, D. E. (1968). The action of certain polyvalent cations on the voltage-clamped lobster axon. J. Oen. Physiol. 51. 279291. Boll. F. (1877). Zur Anatomie und Physiologie der Retina. Arch. Amt. Physiol. 4. 783--787. Brown. A. M., Baur. P. S., Jr. and Tuley. F. H.,
Burnside, B. and Basinger. S. (1983). Retinomotor pigment migration in the teleost retinal pigment epithelium. TT. Cyclic-3’5.adenosine monophosphate induction of darkadaptive movement in vitro. Zn~esf. 0$thaln~ol. c’is. &+. 24, 16-23. C’hiarandini, D. J., Reuben.
Panessa, B. J. and Zadunaiskg. .J. A. (1981). Pigment granules: a calcium reservoir in the vertebrate eye. Exp. Eye Res. 32. 593-604. Parker, G. H. (1932). The movements of the retinal pigment. Ergeh. RioZ. IX. 239-291. Perrelet, A. and Bader, C.-R. (1978). Morphological evidence for (‘a++ stores in the retina of honeybee drones. J. Ultrastruct. Res. 63. 237-253. Pressman. B. (‘. (1976). Biological applications of ionophores. dnn. Rev. Riocheru. 45. 5OlL.530. Saidel. W. M. (1977). Metabolic energy requirements during teleost melanophore adaptations. Esperientia 33, 1573- 1571. Sillman. A. ,J.. Ito. H. and Tomita. T. (1969). Studies on the mass receptor potential. I. General properties of the response. Vision Res. 9, 14351441”. Snyder. W. Z. and Zadunaisky. .J. A. (1976). A role for calcium in the migration of rrt’inal screening pigment, in the frog. Esp. Eye Res. 22, 377.--388. &tuw (London) 309. 51%517. Somlyo. A. P. (1984). Cellular site of calcium regulation. Somlyo. A. 1’. and Walz. B. (1985). Elemental distribution in Ram pipiens retinal rods: quantitative electron probe analysis. J. Physiol. (London) 358. 183-196. of nervous tissue. A’~urochwu. Int. 8. 44S476. Stahl. W. 1~. (1986). The Ka. K-ATPase E. (1984). CTptake of calcium by the Vngar, F.. Piscopo. I.. Letizia. J. and Holtzman, rndoplasmic reticulum of the frog photoreceptor. J. f’rll Viol. 98. 164551655. Walz. B. (1979). Subcellular calcium localization and ATP-dependent Ca2+-uptake hy smooth endoplasmic-retirulum in an invrrtebratc phot,oreceptor cell. Ultrastruct~ural. cytochemical and S-ray micw~analytical study. Eur. J. (‘ell Kiol. 20, X3-91. Walz. B. (1982a). Ca2+-sequestering smooth endoplasmic reticulum in an invertebrate photoreceptor. I. Intracellular topography as revealed hy OsFeCh’ staining and in situ (‘a accumulation. J. Cell Biol. 93. 839-848. Walz. B. (198zb). C’a%aquestering smooth endoplasmic reticulum in an invertebrate photoreceptor. II. Its properties as revealed by miqrophotometric measurements. ,1. PPII Kiol. 93. 84!%859. Walz. B. (198%). Calcium-sequestering smooth endoplasmic reticulum in retinula cells of the tdow-fly. J. t%rastruct. Res. 81. 240-248. White, R. H. and Michaud. N. A. (1980). Calcium is a component of ommochrome pigment granules in insect eyes. C’omp. Hiochem. Physiol. A 65, 239-242. Worth. R. M. and Orbs. S. (1982). Dependence of batrachotoxin block of axoplasmictransport on sodium. J. ,%urohiol. 13. 537-549.