Rhodopsin and retinochrome in the retina of a marine gastropod,Conomulex luhuanus

Vision Res. Vol.

26. No. 5. pp. 691-705.

1986

W?-6989

CopyrIght

F’nnted in Great Bntain. All rights reserved

RHODOPSIN AND RETINOCHROME IN THE RETINA A MARINE GASTROPOD, CONOMULEX LUHUANUS KOKHI

of Biology,

OF

OZAKI, AKIHISA TERAKITA, REKO HARA and TOWYUKI

Department

86 S3.00 + 0.M) Press Ltd

(1 1986 Pcrgnmon

Faculty

of Science,

Osaka

(Received 30 September 1985; in

HARA University,

recked form

Toyonaka.

25 Nocember

Osaka

560. Japan

1985)

Abstract-Photopigments in the conch retina were examined with special attention given to the photic vesicles characteristic of gastropod photoreceptors. Three different fractions of visual cell fragments iterr prepared from the retina: the MV-fraction containing the rhabdomal microvilli. and the PVH- and PVL-fractions containing the photic vesicles located in the visual cell body. Rhodopsin was found in the MV-fraction (,&, = 474 nm). and yielded a photoequilibrium mixture with metarhodopsin (j.,,, = 512 nm) on irradiation with blue light. Retinochrome was found in both of the PVH- and PVL-fractions (A,,,,. = 5 5 IO nm). and was bleached into metaretinochrome by exposure to orange light. showing no marked shift of the absorption peak. Unlike the PVH-fraction. the PVL-fraction contains much aporetinochrome in addition to retinochrome, suggesting that the large mass of photic vesicles around the nucleus may serve as storage for retinal in retinochrome and for newly synthesized aporetinochrome. The total amount of retinochrome in the retina was several times higher than that of rhodopsin. distinguishing the gastropod eye from the cephalopod eye. Retinal

Retinol

Rhodopsin

Retinochrome

Microvilli

Photic

vesicles

Retina

Conch

Gastropod

respectively. In this experiment, however, these two photopigments were distinguished from each other only by a difference in reactivity to a reducing agent, sodium borohydride. We have therefore decided to examine gastropod photopigments in extracts in more detail, with special reference to the geometrical configuration of the retinal chromophores (1 I-cis in rhodopsin. but all-[runs in retinochrome). Extensive work on both the electrophysiology and electron microscopy of the eye of a marine gastropod. Srronlhlls (Gillary, 1974, 1983; Gillary and Gillary, 1979) has particularly stimulated us to investigate the photopigments in a closely-related conch, Conomules. This animal was more suitable for biochemical analyses than the slug. because it has larger eyes. In the present study, the chemical and photochemical properties of photopigments were studied spectroscopically, and their location in the retina was observed microscopically. The results reported here give the first confirmatory evidence that a dual system of rhodopsin and retinochrome exists in the gastropod retina. A preliminary account of some of the results has been presented elsewhere (Ozaki et al., 1984), and discussed at the 2nd Joint Meeting of BPS-ARVO held in Bristol in September 1983.

INTRODUCTION

The visual cells of cephalopods contain a pair of photopigments, rhodopsin and retinochrome, as has been shown in the Octopoda and the Decapoda (Hara and Hara, 1972, 1982). These photopigments are also present in extraocular photoreceptors, an example being the parolfactory vesicles that are well developed in deep-sea squids such as Todarodes (Hara and Hara, 1980). Since we observed a compact mass consisting of small vesicles [photic vesicles, named by Eakin and Brandenburger (1970)] in the somata of the sensory cell of the snail (Koshida et al., 1963), we have suggested that the gastropod eye may also possess retinochrome in this peculiar structure (Hara et al., 1967). We have been unable to verify this until recently, when we devised a method for histochemically identifying rhodopsin and retinochrome and a means for determining their location in various photoreceptive tissues using epifluorescence microscopy (Ozaki ef al., 1983a). One of our experiments suggested that the slug retina (Limauflauas) contains retinochrome in the photic vesicles in addition to rhodopsin in the microvilli of the visual cells, inside and outside of the layer of black pigment, 691

and the other sedimented in Ihr 61)“0 >~~ros~ iphotic vesicle-containing heaq fracricln. PVHRed-mouthed conches, Conomrt1e.r luh~~anr~s fraction). After washing once L\lth phospharf L. (shell length, about 4.5 cm), were captured at buffer, each fraction was mixed with 2 or 1”o depths of 3-5 m, in Tanabe Bay. Wakayama digitonin (Nakarai Chemicals Ltd. Kyoro) dlsPrefecture, on the Pacific coast of Japan, and solved in 67 rnbl phosphate buffer, pH 6.5. and maintained in a seawater tank at about 23-C extracted by gentle shaking for I hr. The superunder cycles of I2 hr light (6:0&18:00, 600 lx natant isolated from the initial homogenate was by fluorescent lamps) and 12 hr dark. At the end centrifuged further at a higher speed (40,000 of the dark period (occasionally. after further rpm) for 2 hr to precipitate fine particles (photic adaptation to the dark extending to 1 or 2 vesicle-containing light fraction, PVL-fraction). weeks), they were anesthetized on ice for 30 min, This fraction was very rich in retinochrome but and their eyes were cut off under dim red light minute in volume, so it ivas necessary to suspend it in 10% sucrose containing phosphate and immediately used for experiments. All the operations for preparing photopigments were buffer in order to use it in spectrophotometry. carried out at temperatures as low as 4’C except The three different fractions prepared were also some extractions with digitonin. used for analyses of the stereoisomeric forms of the retinal chromophore of photopigments, as Preparation of photopigment -containing shown later. fractions Irradiation and absorption measurement For each preparation, 2&80 eyes were \I.ATERI.-\LS

A>D

>lETHODS

A 100 W tungsten filament projector lamp shielded by an S-cm water cell was used to irradiate the photopigment samples. Orange light (> 560 nm) was created by using a Toshiba V-O 56 cutoff glass, and blue light (480 nm), by combination of a Toshiba KL-48 interference and a Toshiba V-Y 46 glass filter. Absorption measurement of the samples was carried out with a Hitachi model 320 recording spectrophotometer.

homogenized in 4 ml of 67mM phosphate buffer, pH 6.5, with a teflon Potter-Elvehjem homogenizer, filtered through a stainless mesh to remove undesirable tissue fragments, and centrifuged for 30 min at 15,000 rpm to obtain a precipitate and a supernatant (Fig. I). The precipitate was again homogenized in phosphate buffer, placed on superimposed layers of 40 and 60% sucrose, and centrifuged at 40,000 rpm (I 3 1,OOOg) for 2 hr so as to obtain two fractions of cell fragments. One fraction floated on the 40% sucrose (microvillus-containing fraction, simply termed MV-fraction)

Analysis The

conch

131,OOOxg.

I. Flow chart showing

rei:mouthed

\’

homogenization

J

filtration

( PVH the fractionation

heavy fraction.

of the photopig-

r-pm, 30 m

@

Zh

conch, Conomulex luhuanus.MV,

vesicle-containing

chromophore

b>, HPLC

4

( MV) Fin

retinal

chromophore

Eyes

15,000

homogenization

of the retinal

131,OOOxg,

1

( PVL

of photopigment rhabdomal

membranes

from

microvillus-containing

PVL. photic vesicle-containing

light fraction.

2h

1 the fresh eyes of the fraction.

PVH.

photic

See text for further details.

Fig. 2. Transmission

electron micrographs

of the retina of3 dark-adapted red-mouthed conch. C‘ono~,r&.\-

M~unrzus. (A) Sagittrtl section showing the presence of three types ofcells. photorscrptor This

(7) and :t supporting

cell (3). bar = IO jlrn.

area is largely occupied by numerous

photoreceptor cytoplasmic

cell,

bar = 2 lrm.

(C) Somal

microvil!i portion

region around the nucleus is dtnscly

693

(6) Rhabdomal

a photoreceptor

(m) that emanate from near the nucleus

[I). an c~ccssot~

portion of ;i photoreceptor cell. this distal

process (p) of

of a photoreceptor

packed with the phonic vesicles (t,). bar =

celi. The

I ,um.

Fig. 3. Scanning homogenate in diameter). photic

derived

vesicles

appeared dominant,

electron

micrographs

showing

ofa conch eye. (A) MV-fraction:

dark,

from

(about

the microvilli

50nm

contaminated

perhaps

descended frsction

of photoreceptor

in diameter) aith

black

mainly NE

the characteristics

are found pigment

from bright

of cell fragments

most of the fragments together

the photic

with

other

(C) PVL-fraction:

vesicles

located

x 30,000.

separated

in structure

cells. (B) PVH-fraction:

granules.

red in color.

are tubular

compact

cell fragments; small

woIlen

in the perinuclear

bar = 0.5 {cm.

iron1 the

(about

I)

I pm

masses ot

this

fractton

spheres art) regions.

thl,

Retinal photopigments

ment was extracted by the method of Groenendijk et al. (1979, 1980) with slight modifications. Both the MV- and PVH-fractions were suspended in phosphate buffer, pH 6.5 (PVLfraction in 20% sucrose containing phosphate buffer), and irradiated with blue or orange light, if needed. A 0.5-m! portion of each was mixed successively with 50 ~1 2 M NH?OH, l-3 ml 90% cold methanol and IO-30 ml cold nhexane. The mixture was vigorously shaken for I min, and centrifuged at 4000 rpm to form a hexane layer that included retinaloxime derived from retina!. The hexane extract was concentrated to about 100 ,u!, injected onto a Lichrosorb SI-60 column (4 x 300 mm, particle size 5 pm), which was preequilibrated with n-hexane containing 8% diethyl ether and 0.33% ethanol, and assayed for the isomeric form by highperformance liquid chromatography (HPLC), using the Hitachi 635 HPLC system. The isomers of retinaloxime were eluted at a constant flow rate of 1.1 m!/min with the same solvent in the following order: 1 I-cis syn, all-[runs syn, 13cis syn, I3-cis anti, 1I-cis anti, and the all-trans anti form. Absorbance at 360 nm was monitored with a JASCO UVIDEC 100-V detector, and the peak area was determined by integrating the absorbance. Each isomer was quantified from the peak area by provisional use of the following values for extinction coefficients: all-trans syn = 54,900, all-[runs anti = 5 1,600, I I-cis syn = 35,000, 1I-cis anti = 29,600 (cf. Groenendijk et al.,. 1979), 13-cis syn = 49,000, and 13-cis anti = 52,100 (Hamanaka ef nl., persona! communication). Electron microscopy For transmission electron microscopy (cf. Goto et al., 1984), a fresh eye was hemisected to remove its anterior part and lens, fixed with 2.5% glutaraldehyde in 0.1% cacodylate buffer (pH 7.4) containing 0.4 M sucrose for 2 hr, washed with the buffer, and postfixed with 1% osmium tetroxide in the same buffer for 1 hr at room temperature. After fixation it was dehydrated through graded concentrations of ethanol, transferred into propylene oxide, and embedded in epoxy resin. Sections were stained with alcoholic urany! acetate followed by lead citrate, and examined with a Hitachi H-500H electron microscope. Prior to biochemical experiments, cell_ fragments in the MV-, PVH- and PVL-fractions were inspected morphologically (cf. Ozaki et al., 1982). The pellets of each of the fragments were

of gastropod

695

similarly fixed and postfixed with sucrose-free fixatives (glutaraldehyde and osmium tetroxide) each time for 2 hr at I’C, dehydrated through a graded series of alcohol, treated with iso-amylacetate, dried at the critical point of CO: with a Hitachi HCP-2-dryer, and coated with gold by spattering with an Eiko IB-3 ion-coater. Observations were carried out with a Hitachi S-430 scanning electron microscope. Fluorescence microscopy On addition of sodium borohydride (NaBH,), retinochrome (or denatured rhodopsin) is reduced to an N-retinyl protein (Hara and Hara, 1973), which emits a yellow-green fluorescence under near-ultraviolet (NUV) light. Based on this, the location of photopigments in the retina was examined by means of epifluorescence microscopy. Genera! procedures were similar to those described previously (Ozaki et al., 1983a). The excised eye was transferred into Bright Cryo-M-Bed embedding compound, immediately frozen at -3O”C, sectioned at IO /lrn at - 25”C, mounted on glass slides, and air-dried for about 4 hr at room temperature. One specimen was immersed in 0.2% aqueous NaBH, for 1 set at 4’C, rinsed gently with water, and photographed to locate the fluorescence of reduced retinochrome using an Olympus BHA-RF-A epifluorescence microscope. Another specimen was treated with 20% formaldehyde (HCOH) for I min and 100% methanol (CH,OH) for 2 min at 2O’C to denature rhodopsin, washed with water, and immersed in 0.2% NaBH, for 5 set in order to take a micrograph of the fluorescence due to reduced products of both rhodopsin and retinochrome. Microspectrophotometry of the fluorescence was performed by a new Olympus system RFSP-OU-FMSP epifluorescence microscope. After a frozen section was successively treated with HCOH, CH,OH and NaBH,, a small area of the section (50pm in diameter) was excited by 334- and 365-nm beams isolated from a 100 W high-pressure mercury lamp. It took about IO,sec to complete one measurement of the emission spectrum. Because N-retiny! proteins can be readily destroyed by a 20-set irradiation with intense NUV light, the system was set up to compute the difference spectrum before and after exposure to this intense light. The net emission spectrum was finally obtained after averaging the difference spectra over 15 trials repeated at different positions on 5 sections.

We also observed another t>pe tit‘ (~1; i,lrr>;ng the bundles of cilia that pro!~: betue for the purpose of biochemical examination i>f the photopigments. From a homogenate of the eyes. three different fractions ofcell fragments (MV-. PVHand PVL-fractions) were prepared by centrifugation, as described in Methods. The MVfraction was reddish-orange in color; the PVH-fraction appeared rather dark owing to black-pigment granules, whereas the PVLfraction was a bright red. According to scanning electron microscopy, the MV-fraction consisted essentially of tubular structures (about 0.1 pm in diameter) that were undoubtedly derived from the microvilli of the photoreceptor cells [Fig. 3(A)]. On the other hand, the PVHfraction included many masses of small v,esicles that were similar in size to the photic vesicles [Fig. 3(B)]. This fraction, hower,er, was not as homogeneous as the MV-fraction, contaminated with black-pigment granules and other large fragments. The PVL-fraction consisted only of the lightest particles. Those globular particles were of unev’en size. more or less influenced by osmotic svvelling during preparation [Fig, 3(C)]. They seemed to be derived from the

RESL L-I-S

.tforphological

ohsrrr~utions 011 tirr retina atld it7

cell jiirgrnents

The red-mouthed conch. Conomrr1e.y ili/rriunus. has a small camera-like eye, about 1.5 mm in diameter. at each tip of the pair of retractile tentacles that can extend up to about I cm in length. The general features of the eye and retina are quite similar to those described in Strowhts by Gillary and Gillary ( 1979). Under a light microscope, the cup-shaped retina appeared to consist of four layers. the rhabdomes. the black pigment, the cell nuclei and the nerve plexus. With electron microscopy, vve could readily distinguish three types of cells in the Conornzrlr.~ retina [Fig. 2(A)], according to the Gillarys’ findings in Strombus. The first type is a photoreceptor ceil. The distal process that protrudes forward from the cell body is decorated with numerous long microvilli to form the rhabdomal layer [Fig. 2(B)]. The cylindrical cell body contains a spherical nucleus behind the pigmented area, and the cytoplasm, especially in the perinuclear region, is densely packed with a great number of minute vesicles (about 50 nm in diameter), often called the photic vesicles [Fig. 2(C)]. The second type is probably an accessory photoreceptor cell, which has many short microvilli extending from the cell body into the rhabdomal layer. The third type, a supporting cell, is much narrower except in the pigmented area, and is characterized by a slender nucleus.

0.15

-

zz 2 b

-

0.1

l-

Extract

2-

irradiated,

3-

irradiated,

from

MV-fraction 480

nm,

Smin

>560nm,

5min

2 4 0.05

-

Wavelength Fig. 4. Photochemical MV-fraction with

was prepared

an equal volume

of absorption of a mixture

properttes

of rhodopsin shift

80 eyes extracted

of phosphate

was shifted

a backward

from

of a photopigment

to longer

buffer,

*ith

showing

nm

in the MV-fraction

0.6 ml 2% digitonin

pH 6.5 (spectrum

wavelengths

and metarhodopsin

in absorption.

in

found

of a Conomule.r for

I), On irradiation

with an increase in absorbance. (spectrum

2). Further

the photoregeneration

irradiation

I

hr at 4’C,

rrith

blut

light.

suggesting with orange

of rhodopsin

retina.

The

and diluted the peak

the formation light caused

(Spectrum

3).

Retinai photopi~ents

photic vesicles located in the perinuclear regions which are less associated with black pigment. Photopigments (rhodopsin)

contained

in the

of

gastropod

697

Table I. Configurational change in the retinal chromophore of conch rhodopsin on wradiation of the MV-fraction

Dark (%)

Irradiated 180 nm. IO min (%I

Irradiated > 560 nm. IO min (,90)

IO 87 3

45 49 6

116 69 5

MV-fraction Isomer All-IWIJ I I .cis 134s

The absorption spectrum at pH 6.5 of a digitonin extract from the MV-fraction is shown in Fig. 4. The peak of absorption appeared near 470 nm (spectrum 1). When the extract is irradiated with blue light for 5 min at 2O’C, the peak is markedly shifted toward longer wavelengths, accompanied by a slight increase in absorbance (spectrum 2). On further irradiation with orange light for 5 min. spectrum 2 is conversely changed into spectrum 3. The conversion between spectra 2 and 3 was completely photoreversible. The corresponding changes in the isomeric form of the retinal chromophore extracted from the MV-fraction ware presented in Table 1. The MV-fraction from 80 eyes was suspended in 1.6 ml phosphate buffer, and a 0.5 ml portion was withdrawn for each HPLC analysis. In an extract from the original (dark) MV-fraction, the 1I-cis isomer was dominant (87%), sug-

gesting that most of the pigment was rhodopsin. On irradiation of the same fraction rvith blue light for 10 min at 1O’C, a fairly large amount of all-trans isomer was detected, showing that a photoequilibrium mixture of rhodopsin (I I-cis) and metarhodopsin (all-trans) had beet formed

during irradiation. On further irradiation with orange light for 10 min, the proportion of I I-cis isomer again increased (69%), indicating that rhodopsin was partially regenerated from metarhodopsin by light. The following experiments were then aimed at elucidating the reactivity of conch rhodopsin and metarhodopsin to hydroxylamine (NH,OH) and at determining the absorption maximum (j.,,,) of each.

0.2 I-

hlixture

of Rhodopsin

and

hletarhodopsin

0.15

3-

irradiated,

?560nm,

8Omin

0.1

0.00

(

-0.01

Wavelength Fig. 5. Effect of hydroxylamine (NH>OH) mixture of rhodopsin and metarhodopsin I; equivalent

(metarhodopsinf

S- 2-3

(rhodopsin)

in nm

on conch rhodopsin

and mrtarhodopsin.

A photoequilibrium

was prepared by the same method as outlined in Fig. 4 (spectrum

and kept

to spectrum 2 in Fig. 4). One ml of the mixture was mixed with 25 ~1 2M NH,OH,

in the dark for 20 min at 15’C. During incubation, retinaloxime

il- l-2

(spectrum

metarhodopsin

2). When exposed to orange light for 80 min at 22 C. any remaining

was completely

bleached through a state of metarhodopsin

4 (spectra

was at 512 nm for metarhodopsin,

I-2)

slowly lost visible absorption

(spectrum

3). The i,,

of direrence

to form

rhodopsin spectrum

while that of spectrum S (spectra 2-3) was at 474 nm

for rhodopsin.

Squid rhodopsin 1s hardly affected by 0.2 .1I NH,OH in the dark. but slowI>’ decomposed through metarhodopsin in the light CSeki er ui.. 1980). Figure 5 shows the stability of conch rhodopsin and metarhodopsin in the presence of NH,OH. A photoequilibrium mixture of them was prepared by a 5-min exposure of rhodopsin to blue light (spectrum 1). When 1 ml of it was mixed with 25 ~1 of freshly neutralized 2M NH:OH (final cont. about 50 mM) and kept in the dark, the absorbance gradually decreased in the visible and increased in the NUV range. forming retinaloxime. The decomposition of metarhodopsin was complete after 20 min (spectrum 2), leaving rhodopsin which is stable in the presence of NH,OH. As shown by the difference spectrum between spectra I and 2 (spectrum 4), lay at 512 nm. The the L,,, of metarhodopsin remaining rhodopsin was then irradiated with orange light for 80 min, until the photoregenerable metarhodopsin was entirely destroyed by the NHzOH which was present (spectrum 3). The difference spectrum before and after irradiation (spectra 2-3) showed that the L,,,, of rhodopsin lies at 474 nm (spectrum 5). As can be estimated from the results shown in Fig. 4, conch metarhodopsin seemed to have a little higher (about I .5 times) extinction coefficient than conch rhodopsin. It is now clear that the microvilli of the conch retina contain

rhodopsln with the same photochsmic:G !x~,I\ ior as \ve ha\,e observed in crphalop~,!~ 1;~ Hara and Hara, 1977). contained

Photopigment (retinochrome)

in

tile

P?.-!i-oC!ioti

The absorption spectrum at pH6.5 of a digitonin extract from the PVH-fraction is presented in Fig. 6(A). It sholvs a shoulder of absorption around 520 nm without any distinct peak (spectrum 1). This pigment differed from rhodopsin in the MV-fraction in the following two ways: it was readily bleached by irradiation with blue light (spectrum 2). and showed no marked change in absorption on further irradiation with orange light (spectrum 3). The L,,,, oi the difference spectrum for the bleaching la> near 520 nm (spectrum 4). On the other hand. ;i good preparation of photopigment with far less contamination could be obtained from the PVLfraction. characterized by a distinct peak near 510 nm, as seen in Fig. 6(B). On irradiation, the pigment in the PVL-fraction also showed essentially the same photolytic behavior as that in the PVH-fraction. The corresponding changes in the isomeric form of the retinal chromophore estracted from the PVHand PVL-fractions of animals adapted to darkness for 2 weeks are summarized in Table 3. In both of the extracts fr-om the

A

0.2

I-

Extract

from 23-

J f 9

PVH-fraction 1180 nm,

>560nm,

4-

0.1

-

l-

Suspension

of

PVL-fraction

5 min

2-

480

nm,

sm,r,

5min

3-

>560

nm,

5 mtn

l-3

0.1

b z u

pH

6.5,

20°C

0.02

-

pH

6.5,

ISOC

0

Fig. 6. Photochemical propertles of photopigments found in the PVH-, and PVL-fractions of a Conumul~~ retina. (A) The PVH-fraction from 80 eyes was extracted with 0.6 ml 4% digitonin for I hr at 20 C. and diluted 2-fold with phosphate buffer (spectrum I). When irradiated with blue light for 5 min. it was bleached (spectrum 2). but showed no marked change on further irradiation with orange light (spectrum 3). (B) the PVL-fraction from 24 eyes of conches adapted to darkness for 2 weeks was suspended in 0.8 ml of 20% sucrose in phosphate buffer (spectrum I), and similarly irradiated with blue light (spectrum 2) and then with orange light (spectrum 3). The i.,,, of the difference spectrum before and after bleaching lay at 523 nm (spectrum 4).

Retinal Table ?. Configurational

of conch retinochrome

change in the retinal chromophore on irradiation of the PVH- and PVL-fractions

PVH-fraction

Isomer All-!ratzr

114s 13-c&

photopigments

PVL-fraction

Dark (%)

Irradiated 480 nm, 20 min (Oh)

Dark (70)

76 18 6

36 59 5

83 14 3

Irradiated 480 nm, IO min (Oh) 36 63

I

(dark) fractions, the all-(runs isomer was dominant (76 and 83%, respectively), suggesting that most of the photopigment was retinochrome. After irradiation of the fractions with blue light at 20°C much of the all-trans isomer was changed into the 1 I-cis form (59 and 63%, respectively), indicative of the formation of metaretinochrome (or more specifically a photoequilibrium mixture of retinochrome and metaretinochrome). On further irradiation with orange light, the results remained virtually unchanged. Considering the results described above, the photopigments contained in the PVH- and PVL-fractions seemed to be retinochromes of the same nature, irrespective of their location in the retina. Accordingly, the retinochrome-rich PVL-fraction was prefered for use in the subsequent experiments. As formerly shown in cephalopods, lightbleached retinochrome is regenerated back to original

of gastropod

699

the original retinochrome during incubation with all-rrans-retinal in the dark (Hara and Hara, 1968). Such regeneration was tested with conch retinochrome, as shown in Fig. 7. A 0.5-ml suspension of the PVL-fraction was bleached by irradiation with orange light to convert retinochrome to metaretinochrome (Spectrum 1). This sample is equivalent to that used in the experiment shown in Fig. 6(B). It was then mixed with 5 ,uI of concentrated alltrans-retinal in ethanol, and incubated in the dark at 2OC. During incubation, the absorbance of the mixture gradually increased in the visible range, until the change was complete after about I hr (spectrum 4). When irradiated with orange light, the mixture was readily bleached (spectrum 5). showing that the regenerated pigment was as light-sensitive as the original retinochrome. The i.,,, of the difference spectrum before and after bleaching was at 523 nm (spectrum 6) similar to that of spectrum 4 in Fig. 6(B). We also noticed, however, that the amount of photobleaching in the resynthesized retinochrome was about 1.8 times greater than that in the original retinochrome [spectrum 4 in Fig. 6(B)], indicating the presence of much aporetinochrome (chromophore-free) in the photic vesicles of the PVL-fraction. On addition of all-trans-retinal to the lightbleached extract of the PVH-fraction, absorption in the visible range only recovered until

I-4

wavelength

Mixture of Retinochrome and Metaretinochrome 2, 3- kept

dark

with

in nm

Fig. 7. Dark regeneration of conch retinochrome from conch metaretinochrome in the presence of all-frans-retinal. A suspension of the PVL-fraction in 20% sucrose was irradiated with orange light to form a mixture of retinochrome and metaretinochrome. When 0.5 ml of the sample thus prepared (spectrum I) was mixed with 5 ~1 of all-rrans-retinal in ethanol (optical density, about 5.0) and kept in the dark, the absorbance near 500 nm increased with a slight shift in the i.,,, to longer wavelengths. Spectra 2. 3 and 4 were measured after .5, 30 and 55 min, respectively. The increase in absorbance was completed about I hr after the mixing. The regenerated pigment was readily bleached on S-min irradiation with orange light (spectrum 5). showing a j_, at 523 nm in the difference spectrum (spectrum 6). See text for further details.

I- Retinochrome (ai

+ all-trans-retinal

: c z b $

2-

after

3min

3-

after

30 min

inrrrad. I

0.12 irradiated

0.08

4-

after

25 ruin

5-

after

40 min

(b)

a”tl -

irrad.

0.04

pH 6.5,

17OC

0 I

,

I

0

.

I

16

8

Time

in

I

I

24

min

Fig. 8. Light isomerization of all-rrans-retinal with conch retinochrome. (A) Spectral changes. A 0.5-ml suspension of the PVL-fraction from animals adapted to darkness for I week (spectrum I) was mixed with I5 ~1 of concentrated all-rrans-retinal. Spectrum 2 is a transient spectrum measured after 3 min. About 30 min after the mixing, a stable mixture of retinal and pigment was completed (spectrum 3). and then it was steadily irradiated with orange light, which can be absorbed by retinochrome. but not by retinal The absorbance of retinal in the NUV range decreased with time as the wans-to-cis photoisomerization advanced, as shown by spectra 4 and 5 measured after 25 and 40 min. respectively. (B) Chromatograms of the extracts obtained from the unirradiated (a) and 25-min irradiated (b) samples. Upon comparing the two, it is clear that a large amount of all-[runs-retinal was isomerized to the I I-US form during irradiation. See text for further details.

it overlapped the spectrum of initial retinochrome, suggesting the absence of aporetinochrome. In any case, these regeneration experiments provide direct evidence for the belief that the 1I-cis chromophore of conch metaretinochrome in the photic vesicles is interchangeable with all-trans-retinal for reforming retinochrome. The next experiment was then designed to elucidate how effectively conch retinochrome promotes the isomerization of all-trans-retinal to the cis form in the light (Fig. 8). When a suspension of the PVL-fraction (spectrum I) was mixed with concentrated all-[runs-retinal in the dark, the absorbance rapidly increased near 520 nm as additional retinochrome was formed (spectrum 2) and slowly increased near 460 nm as 465-nm pigment was formed (cf. Hara and Hara, 1984), until a stable mixture containing much of the free all-trans-retinal was completed about 30min later (spectrum 3). On irradiation with orange light, which is absorbed by retinochromes alone, the absorption peak near 390 nm was gradually lowered and slightly shifted toward shorter wavelengths, displaying a signature of trans-to-cis isomerization of retinal

(spectra 4 and 5). Examples of the chromatograms obtained by HPLC during isomerization are presented in Fig. 8(B) It is obvious that a large amount of all-trans-retinal present in the unirradiated mixture was changed into the I lcis form during the 25-min irradiation (consider a far lower extinction coefficient of I I-cirretinal!). The present photosiomerization was also practically indifferent to any isomer other than the all-trans. 114s and l3-ci~ forms. Changes of chromophore composition with time are shown in Table 3. The isomerization of all-rruns-retinal catalyzed by retinochrome in steady light was almost complete within 30 min, converting about 70% to the I I-c& form. One may suspect that this proportion of I I-ci.s is not

Table 3. Isomer analysis of the mixture produced by photoisomerization of all-(ran.7 retinal with conch retinochrome Irradiated. > 560 nm __~~__._..

Retinal Solution

0 min

25 mm

Isomer

(%)

(%)

All-rrans I I-ci.7 I3.ci.c

99 0

95 1 3

I

-.._..

10 min

100 min

(%)

(%1

(96)

25 69 6

23

19

71

7;

6

s

wavelength

Fig. 9. Fluorescence the light-adapted by NaBH,

micrographs

showing the location

of rotinochromc

conch, CJWWF~&.Y lukuur~s. (A) the yellow-green

appears in &he somni lsycr of photoreceptor

and rhodopsin

fluorescence due to reduced rhodopsin is seen in the rhadbomal along the internal

side of the layer of black pigment

difference in features was detected between light- and dark-adapted respectively.

rcduccd

cells, associated with large masses of photic vesicles

of miorovilli

measured at the somal layer (For

in the retina of

fluorescence of rctinochromc

(V). (El) Additional (Xl),

(ml

reduced rctinochrome)

eyes.

and rhabdomal

(P).

iafand

layer consisting mainly

x 160. bar = IOOpm.

So

(b) show emission spectra

layer (for reduced rhodopsin).

Both spectra are similar in shape to each other, showing peaks near 475 nm as for :S-retintl proteins.

701

Retinal photopigments

so high compared with the well-known value in the same type of experiment with squid retinochrome (>98%, Ozaki et al., 1983~). The reason for this low value is that the present experiment was intentionally designed to transform a moderately small amount of retinal to retinochrome in order to show interactions between the two throughout the experiment. Consequently, much of the all-rmns retinal was trapped by inactive sites of retinochrome and perhaps by lipids and proteins in membranes other than retinochrome, and was not accessible for photoisomerization. At any rate, gastropod retinochrome is also capable of reconstituting the II-c-is form of retinal in the light, which is required for maintaining the visual pigment in the retina. Lacntion of rhudo~s~~ and retinochrome gastropod ret&n

in the

Cephalopod retinochrome is readily reduced to a fluorescent product (N-retinyl protein) by addition of NaBH,, but cephalopod rhodopsin is not reduced until opsin has been denatured (Ozaki et al., 1983a). In the present study, conch retinochrome and rhodopsin were also discriminated from each other by this difference, and fluorograms were prepared in order to demonstrate the location of those photopigments in the retina. The frozen section of the retina was originally nonfluorescent, suggesting the absence of retinol and retinyl ester. However, when it was treated with NaBH,, a distinct band of fluorescence appeared in the somal layer just outside of the layer of black pigment [Fig. 9(A)]. Since this area is filled with photic vesicles as shown in Fig. 2(A), it is clear that the fluorescence due to reduced retinochrome is closely associated with those vesicles of the photoreceptor cells. At the layer of black pigment, the fluorescence due to the retinochrome might be masked by bock-pigment granules. When the section was reduced by NaBH, after denaturation with HCOH and CH,OH, an additional band of fluorescence was observed inside the layer of black pigment [Fig. 9(B)], indicating that the location of rhodopsin is restricted to within the microvilli in the rhabdomes. Figure 9(a) and (b) present the spectra of the yellowgreen fluorescence due to “reduced retinochrome” and “reduced rhodopsin”, which were measured at the rhabdomal and somal layers, respectively. Both of the emission spectra for it;‘-retinyl protein were similar in shape to each other, their peaks being at 47~8Onm. Al-

70;

of gastropod

though these two fluorescences were too alike in color to discriminate them under the microscope. the intensity of fluorescence was apparently stronger in the somal layer than in the rhabdomal layer, suggesting that the retina might contain much more retinochrome than rhodopsin. Eventually, all the results of microscopical observation were consistent with those obtained from photopi~ent extraction experiments. DISCUSSiON

Most biochemical studies on photopigments in invertebrate photoreceptors have been performed using the eyes of cephalopods, insects and crustaceans, with only a few using the eyes of gastropods. Although retinochrome has been known as a photopigment located at the myeloid bodies in cephalopod retinas (Hara and Hara, 1976, 1982) our previous histochemical work in the slug suggested that the gastropod retina might also possess two kinds of photopigments, rhodopsin in the rhabdomal microvilli and retinochrome in the photic vesicles (Ozaki er al., 1983a). The term photic vesicles was first introduced by Eakin and Brandenburger (1970) for the numerous vesicles of uniform size (about 800 8, in diameter) characteristic of the type I photosensory cell in the snail. They were postulated to function as transporters of any vitamin A-containing photopi~ent or its precursors within the cell, based upon experiments of vitamin A incorporation into the retina (Eakin and Brandenburger, 1968; Brandenburger and Eakin, 1970). Recently these vesicles have been especially recognized in relation to the problem of the turnover in microvillar membranes (cf. Eakin and Brandenburger, 1982). Our present investigation was particularly aimed at elucidating the biochemical components of the photic vesicles. As shown by the present studies, the microvilli of the conch photoreceptors contain a photopigment carrying the 1l-c& chromophore (rhodopsin), while the photic vesicles possess another photopigment carrying the ah-truns, that is, retinochrome. This is the first instance’ that rhodopsin and retinochrome have been identified biochemically in extracts of the gastropod retina. The i.,,,, of Conomulex rhodopsin in extract was at 474 nm. It is close to the absorption peak of Strombus visual pigment at about 485 nm, sensitivity estimated from ERG spectral (Gillary, 1974, 1983). On irradiation, conch rhodopsin yielded a photoequilibrium mixture

with its mctarhodopsin, which show
Throughout the present studies on the conch retina, no free retinol was detected b\ HPLC or microscopy, but plenty of retinochrome was found in the photic vesicles. where its content reached 7-3 times that of rhodopsin by rough estimation (neglecting aporetinochrome). Although the Tudurorirs retina contains an amount of retinochrome comparable to that of rhodopsin (Hara and Hara. 1976). the abundance of retinochrome in the conch further distinguishes gastropods from cephalopods. We know that the photic vesicles still retain much metaretinochrome at the end of a 1ZL 1ZD cycle, but that after adaptation of animals to darkness for 1 week or so, it is almost c*ompletely converted into retinochrome. For this reason, animals adapted to darkness for long periods have been appropriately used for the present experiments. An examination of the dynamic aspects in photopigments and fine structures during adaptation are therefore in progress. .-1cX_noIrledgemenrs-We wish to thank Mr Hiroshi Iguchi and Mr Takeshi Ogawa of the Marine Propagation Laboratory in Wakayama Prefecture for a steady supply of fresh Conomule~. We are indebted to Professor Shigeo Minami, Faculty of Engineering, Osaka University, and the research staff of Olympus Co. Ltd., for the construction of a new system combining the epifluorescence microscope with a spectrophotometer. Special thanks are also due to Professor Masao Yoshida, Ushimado Marine Laboratory, Okayama University, for his cooperative examinations in electron microscopy.

REFERENCES Brandenburger J. L. and Eakin R. M. (1970) Pathway of incorporation of vitamin A ‘Hz into photoreceptors of a snail, Helix aspersa. Vision Res. 10, 639-653. Eakin R. M. and Brandenburger J. L. (1968) Localization of vitamin A in the eye of a plumonate snail. Proc. nufn. Acad. Sci., U.S.A. 60, 140-145. Eakin R. M. and Brandenburger J. L. (1970) Osmic staining of amphibian and gastropod photoreceptors. J. L’lrroSITUCI. Res. 30, 619-641. Eakin R. M. and Brandenburger J. L. (1982) Pinocytosis in eyes of a snail, Helix asperse. J. L’lrrasrrucl. Res. 80, Z 14-229. Gillary H. L. (1974) Light-evoked electrical potentials from the eye and optic nerve of Slrombus: Response waveform and spectral sensitivity. J. e.rp. Biol. 60, 383-396. Gillary H. L. (1983) Electrical potentials from the regenerating eye of Slrombus. J. exp. Viol. 107, 293-310. Gillary H. L. and Gillary E. W. (1979) Ultrastructural features of the retina and optic nerve of Srrombus luhuanus, a marine gastropod. J. Xforphol. 159, 89-l 16. Goto T.. Takasu N. and Yoshida M. (1984) A unique photoreceptive structure in the arrowworms Sagirra crossu and Spodella schizoprera (Chaetognatha). Crll Tissue Res. 235, 47 l-175.

Retinal photopigments Groenendijk G. W. T., De Grip W. J. and Daemen F. J. M. (1979) Identification and characterization of syn- and anri-isomers of retinaloximes. Anal~f. Biochem. 99, 304310. Groenendijk G. W. T., De Grip W. J. and Daemen F. J. M. (1980) Quantitative determination of retinals with complete retention of their geometric configuration. Biochim. biophys. Acra 617, 430-438. Hamdorf K.. Paulsen R. and Schwemer J. (1973) Photoregeneration and sensitivity control of photoreceptors of invertebrates. In Biochemistry and Physiology uf Visual Pigments (Edited by Langer H.). pp. 155-166. Springer, Berlin. Hara R. and Hara T. (1984) Squid m-retinochrome. Two forms of metaretinochrome. Vision Res. 24, 1629-1640. Hara T. and Hara R. (1968) Regeneration of retinochrome. ~Vuturr. Lond. 219, 450-454. Hara T. and Hara R. (1972) Cephalopod retinochrome. In Handbook of Sensory Physio1og.v. Vol. VII, Part I, Photochemistry of Vision (Edited by Dartnall H. J. A.), pp. 7X-746. Springer, Berlin. Hara T. and Hara R. (1973) Biochemical properties of retinochrome. In Biochemistry and Physiology of Visual Pigmenrs (Edited by Langer H.), pp. 181-191. Springer, Berlin. Hara T. and Hara R. (1976) Distribution of rhodopsin and retinochrome in the squid retina. J. gen. Physiol. 67, 79 I-805.

Hara T. and Hara R. (1980) Retinochrome and rhodopsin in the extraocular photoreceptor of the squid, Todurodes. J. gen. Physiol. 75, I-19.

of gastropod

705

Hara T. and Hara R. (1982) Cephalopod retinochrome. In Merhods in En:_vmolog_v.Vol. 81. Biomembrunes. Part H: Visual Pigments and Purple membranes (Edited by Packer L.). pp. 190-197. Academic Press, New York. Hara T.. Hara R. and Takeuchi J. (1967) Rhodopsin and retinochrome in the octopus retina. Nuture. Lond. 214. 572-573.

Hubbard R. system of Koshida Y. properties

and St. George R. C. C. (1958) The rhodopsin the squid. J. gen. Physiol. 41, 501-528. Hara T. and Tanaka A. (1963) Histochemical and fine structures of gastropod eyes. Zool. Msg.. Tokyo 72. 3 15-3 16. Ozaki K.. Hara R. and Hara T. (1982) Dependency of absorption characteristics of retinochrome on pH and salts. E,rpl Eye Res. 34, 499-508. Ozaki K., Hara R. and Hara T. (1983a) Histochemical localization of retinochrome and rhodopsin studied by fluorescence microscopy. Cell Tissue Res. 233. 335-34s. Gzaki K., Hara R. and Hara T. (1983b) Retinal-binding protein found in the squid retina. Zool. Mug., Tokyo 92, 534. Ozaki K., Hara R., Hara T. and Kakitani T. (1983~) Squid retinochrome. Configurational changes of the retinal chromophore. Biophys. J. 44, 127-137. Ozaki K., Hara R. and Hara T. (1984) Examination of retinochrome and rhodopsin in the gastropod retina. Vision Res. 24, 1697.

Seki T., Hara R. and Hara T. (1980) Reconstitution of squid rhodopsin in rhabdomal membranes. Photochem. Phorobiol. 32, 469-179.