Vision Res. Vol. 32, No. 1, pp. 29-36, 1992 Printed in Great Britain. All rights reserved

0042-6989/92 $5.00 + 0.00 Copyright 0 1992 Pergamon Press plc

Cyclic GMP, Calcium and Photoreceptor Sensitivity in Mice Heterozygous for the Rod Dysplasia Gene Designated “rd” AL1 A. HUSSAIN,*t Received

NICHOLAS

J. WILLMOTT,*t

MARY J. VOADEN*$

15 August 1991; in revised form 8 March 1991

The rise in photoreceptor cGMP, induced by c l.OnM extracellular calcium, is delayed in retinas of mice heterozygous for the rod dysplasia gene (+lrd). The calcium ionophore A23187 reduces the delay, suggesting that +/rd outer segments contain more calcium than normal. In turn, this might explain the increased photosensitivity of the +/rd retina. During the response to low calcium there is no correlation in +/t-d retinas between the total concentration of cGMP and the photoresponse amplitude and its time to peak. The observations imply that either free cGh4P is abnormally independent of the bound pool in the +/t-d photoreceptor outer segment or that factors other than cGMP and its phosphodiesterase are modulating the rising phase of the response. The time-to-peak of PIII in a + lrd retina, incubated in a standard medium and stimulated with dim light, is abnormally delayed. Reduction of extracellular calcium induces an abnormal delay as well in responses to higher light levels. In addition to this, a second delay manifests slowly in both the normal and the + lrd retina. More studies are needed to explain these observations. + /rd mice

Photoreceptor

function

Calcium

ionophore

INTRODUCTION

PI11

cGMP

a lower affinity than normal for cGMP at its active centre (Doshi et al., 1985), and a 30% reduction in the capacity for cGMP binding at non-catalytic sites (Voaden & Willmott, 1990). The latter may explain the similar reduction in the total concentration of cGMP in +/rd retinas as compared to normal (Ferrendelli & Cohen, 1976; Doshi et al., 1985; Voaden & Willmott, 1990) as more than 95% of the cGMP present in outer segments may be bound to PDE (Pugh & Lamb, 1990) and most, perhaps more than 95% of retinal cGMP is present in photoreceptors (Cohen, 1984). In marked contrast to the overall reduction in cGMP concentration, it is possible that the higher #Z,,,of the +/rd enzyme will lead to an increase in the concentration of outer segment free cGMP (normally c 10% of the total) and a greater dark current (Pugh & Lamb, 1990). In turn, a higher dark current would lead to an increased intracellular level of calcium (Schnetkamp & Bownds, 1987; Pugh & Lamb, 1990), and calcium via its actions on the photoreceptor guanyl cyclase (Lolley dz Racz, 1982; Pepe, Boero, Vergani, Panfoli & Cugnoli, 1986), and PDE (Robinson, Kawamura, Abramson & Bownds, 1980; Kawamura & Bownds, 1981; Detwiler & Rispoli, 1989; Kawamura & Murakami, 1991) and/or rhodopsin kinase (Wagner, Ryba & Uhl, 1989) potentially modulates response recovery, rod sensitivity and light adaptation (Koch & Stryer, 1988; Matthews, Murphy, Fain & Lamb, 1988; Nakatani & Yau, 1988; Pugh

The mouse rod dysplasia gene, designated as rd, produces an abnormality in the b-subunit of the rod specific cGMP phosphodiesterase (cGMP PDE) (Bowes, Dantiger, Baxter, Applebury & Farber, 1990). Consequently, in mice homozygous for the gene, i.e. rd/rd, the normal PDE complex fails to form (Lee, Lieberman, Hurwitz & Lolley, 1985; Lee, Navon, Brown, Fung & Lolley, 1988) and enzymic activity is greatly reduced (Farber & Lolley, 1976). Diminished cGMP hydrolysis in the presence of a functional guanyl cyclase leads to the intracellular accumulation of cGMP and rod degeneration (Farber & Lolley, 1976), cells disappearing by the 30th day (CarterDawson, La Vail & Sidman, 1978). Adult heterozygotic (+ /rd) mice have normal retinal structure (Noell, 1958; Carter-Dawson et al., 1978) and a full complement of rhodopsin (Doshi, Voaden & Arden, 1985; Low, 1987a) but, again, exhibit abnormalities in metabolism and function that are attributable, at least in part, to defects in cGMP PDE. In particular, the enzyme has a higher K,,, of 409 vs 256 PM and, therefore, *Department of Visual Science, Institute of Ophthalmology, B.P.M.F., University of London, Judd Street, London WClH 9QS, England TFormerly of the Comparative Ophthalmology Unit, The Animal Health Trust, Lanwades Park, Kennett CB8 9PN, England. $To whom all correspondence should be addressed. VR3211-s

A23187

29

ALI A. HUSSAIN cf td

30

& Lamb, 1990). It is pertinent, therefore, that, of the above parameters investigated, termination of the photoresponse is slowed in +/rd mice (Arden & Low, 1980) and sensitivity is increased (Low, 1987b). At low light intensities, the photoresponse time-to-peak is also increased (Arden & Low, 1980). A reduction in extracellular calcium to a level of around 1.OnM rapidly activates the photore~ptor specific guanyl cyclase and, in dark-adapted retinas, leads to a several-fold increase in intracellular cGMP (Cohen, Hall & Ferrendelli, 1978; Woodruff & Fain, 1982). Here we show that the response in +/rd retinas is of normal magnitude but is delayed. Moreover, it is speeded up if the calcium ionophore A23 187 is included in the medium, thus providing indirect evidence for an increase in intracellular calcium in +/rd retinas. In contrast to normal, there is no correlation in the calcium-depleted +/rd retina between the total concentration of cGMP and the photoresponse amplitude and its time-to-peak. Preliminary reports of aspects of the present work have appeared (Willmott, Hussain & Voaden, 1988; Voaden, Willmott, Hussain & Al-Mahdawi, 1989; Voaden, Hussain & Willmott, 1991).

MATERIALS AND METHODS Retinas were obtained from 3-4 month old, pigmented mice of the normal C57BL (6)J (+/i-) strain or from congenic animals that were heterozygous ( + /rd.le) for the closely linked “retinal dystrophy” (rd) and “light ear” (le) recessive genes (see LaVail, 1981). When appropriate, animals were dark-adapted for at least 2 hr before use and manipulations done under dim red light. Retinas were isolated in Earle’s medium and all subsequent incubations and manipulations performed in standard or modified Eagle’s MEM (see below) which were pregassed with 95% 02/5% CO, to a pH of 7.2. Media modifications were as follows-(l) Eagle’s MEM plus 10 mM sodium glutamate, (2) as (1) but without calcium salts and with either 3.0 mM EGTA (to lower the concentration of extracellular calcium to c 10Ty M; Portzehl, Caldwell & Ruegg, 1964) and 3.0 mM EGTA and 10.0 FM of the calcium ionophore A23 187. The EGTA was initially dissolved in NaOH and neutralized with HCl before addition, leading to an overall change in sodium of about 12 mM. However, in control experiments, a similar increase in the sodium level had no effect on retinal cGMP, photoreceptor sensitivity or response waveform. measurement of transr~tinui potentials All manipulations were done in dim red light. In order to avoid loss of ROS during media changes, the darkadapted retina was mounted receptor side down onto a 3.0 x 3.0 mm nitrocellulose prewashed, moistened, membrane (Millipore type HA, 0.22 pm). The retina/ membrane preparation was then positioned between two

tellon supports that left a central, circular portr a tungsten-halogen lamp whose output was brought to the retina via a fibre optic light guide: the end of the guide was fixed perpendicular to the surface of the retina. The number of photons incident on the retina from the flash of highest intensity was calculated to be approx. 2 x 10” photons/mm’. As necessary, neutral density filters were interposed between the light source and the retina. Trans-retinal photoresponses were recorded with Ag/AgCl electrodes, connected to agar bridges, using a Medelec MS6 Mainframe. Both the amplitude and implicit time of each photoresponse were measured. PI11 amplitude was taken as the voltage difference between the pre-stimulus baseline and the trough of the photoresponse, and the implicit time as spanning stimulus onset and. again, the response trough. PI11 amplitude vs light intensity profiles were obtained for each retina. The data generated depicted a sigmoidal distribution of the type t’ = E’,., Ye”’ i-j -L 1f. where Vis response amplitude in pV, V,,l,,the saturating response amplitude, B the slope parameter of response at Z,, I is tog light intensity, and I,, the log light intensity required to elicit a half-maximal response. Retinas were initially maintained in standard MEM until the trans-retinal PI11 stabilized. The medium was then rapidly replaced at “zero” time with the relevant test solution and measurements continued.

cGMP and protein For comparison of the time course of the changes in the concentration of cGMP with those occurring in the photoresponse, dark-adapted retinas were incubated in identical media for corresponding periods and then analyzed for one or the other. Cyclic GMP was radioimmunoassayed (Doshi et al., 1985), using a kit supplied by Amersham International plc, and protein was measured by the method of Lowry, Rosebrough, Farr and Randall 1951) as modified by Miller (1959). Materials Ethylene glycol-bis (~-aminoethyl ether) N,N, N’, N’tetra-acetic acid (EGTA), ionophore A23 187, Earle’s medium and Eagle’s MEM were purchased from the Sigma Chemical Company Ltd.

RESULTS Dark-adapted retinas, incubated for 20min in standard Eagle’s MEM containing 10 mM glutamate, showed no significant change in the sensitivity or timeto-peak of the photoresponse induced by unattenuated flashes of white light [Fig. l(b) and (c)] and, after the initial few minutes of incubation, the endogenous con-

PHOTORECEPTOR

FUNCTION

31

IN +/rd MICE

100

(a) 0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4

(b)

0.2 0.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

(cl

0.0 0

2

4

6

8

10

Incubation

12

time

14

16

18

20

(mins)

FIGURE 1. The photoresponse and concentration of cGMP in dark-adapted retinas incubated in MEM. Retinal cGMP (a), PI11 amplitude (b), and PI11 time-to-peak (c) were measured in +/ + (0-O) and +/rd (+---0) mouse retinas, incubated in Eagle’s MEM, containing 10 mM glutamate, and stimulated with unattenuated flashes of white light (see Methods). PI11 amplitude and time-to-peak have been normalized relative to stable values initially obtained in Earle’s medium. Each point represents the mean of 4 determinations rl: SEM.

centration of cGMP also remained stable, exhibiting at all times a lower level of cGMP in +/rd retinas as compared with +/ + ones [Fig. l(a)]. Trans-retinal PIIIs were recorded over a range of light intensities, and amplitude and time-to-peak measurements for +/+ and +/rd genotypes are reproduced in Fig. 2. The maximum response amplitudes meaned at 241.Ok28.5 DV (n = 18) for +/+ mice and 244.4 f 29.8 PV (n = 15) for +/rd ones. In contrast, f, was reduced by 0.31 log units (P < 0.05) in heterozygotes, shifting the intensity-response curve towards greater sensitivity. Time-to-peak measurements of the trans-retinal PI11 from flash onset are plotted in Fig. 2(b). The data have an essentially sigmoidal relationship

and, with increasing stimulus intensity, time-to-peak of PI11 decreases. The timings of trans-retinal PI11 in the two genotypes superimposed at higher light levels (- 2.0 log units) but from -4.0 to - 3.0 log units, there is a deviation and +/rd responses are slower than normal. At -4.0 log units, the time-to-peak for the +/+ PI11 is 125 + 7 msec and for the + /rd PI11 150 * 7 msec (P < 0.05). In the following section, the time-dependent effects on PI11 and the level of cGMP following the reduction of extracellular calcium are discussed. For these studies, photoresponses were elicited every 30 set with 4.0 msec flashes of unattenuated white light, as used for the results shown in Fig. 1.

32

AL1 A. HUSSAIN er ul.

160.

140. iZOlQOEiO60 40 20 0

140120. loo607 60. 40. 20.

fb)

OS -5

0

Log stimulus

intensity

FIGURE 2. Photoreceptor sensitivity in dark-adapted + / + and + /rd mouse retin&. PIII amplitude (a) and time-to-peak (b) have been recorded for +/ + (O-0) and + jrd (e0) retinas, inaibated in MEM contai~ng 10 mM ~~~, and stimulated with light of varying intensity. Points reprwnt the mean of I8 estimations for i-/+ retinas and 15 for +/rd ones f SEM.

Depletion of medium calcium from 1.8 mM to < 1.OnM caused a rapid increase in the concentration of cGMP in the dark-adapted normal mouse retina, the peak (at approx. g-fold the basal level) being reached at about 2,Omin after the medium adjustment [Fig. 3(a)]. The amplitude of PI11 also increased, peaking within 2.0 min of the medium change at more than 3-fold the basal value [Fig. 3(b)]. In the heterozygotic retina the extent of the increase in cGMP was normal but its maximum concentration was not reached until 4.0-5.0 min after the medium change, In spite of this, the changes in PI11 followed a normal time course, at least from 2.0 min after exposure of the retinas to low calcium [Fig. 3(b)]. The other significant effects of calcium depletion were increases in time-to-peak of PIII. Two apparently inda pendent phenomena were observed Fig. 3(c)]: an abnormal delay in the +/rd response to the non-attenuated light and, superimposed on this, an additional delay that manifested slowly in both the normal and the +/rd retina. To assess whether the later rise in total cGMP in “ + /rd” retinas might be due to a high intra~llular starting level of calcium and thus to its slower depletion, A23187 was included in the medium. Although this caused a further increase in the maximum amplitude of

PI11 and in the ~on~ntration of CGMP in both genotypes, the rise and fall of the responses in the normal retina, and for PI11 in the f /rd tissue, occurred over a similar time course to those seen in the absence of the ionophore [Fig. 4(a) and (b)]. In contrast, the time taken for eGMP to reach its concentration peak in the “ + /rd” retina was reduced by 2.0 min [Fig. 4(b)].

In studies on the isolated retina it is common practice to block photor~eptor ~ansmission with 10 mM extracellular glutamate and then to use the extracellular potential gradient as a measure of the light sensitive current in photoreceptor outer limbs (Dowling, 1987). Secondary factors such as potassium f&x through Mueller cells in response to changes in extraceilular ~~~ (~~~, 19&4) and vol~~-~~ve changes in inner segment Na+ canductance (Fain, Quandt, Bastian & Gerschenfeld, 197Q are likely ro contribute to the overall response, but present evidence suggests that, in general, these are proportional to the light sensitive current and do not sign&an@ distort the ~la~onship between potential and ~on~~an~ changes. The incubation media employed in the present study were based on Eagle’s MEM, containing 10 mM

PHOTORECEPTOR

FUNCTION

IN +/rd

MICE

33

(b) 4.0 3.5 3.0 2.52.01.5-

4

6

6

10

Incubation

12

14

16

16

20

time (mins)

FIGURE 3. The photoresponse and concentration of cGMP in dark-adapted retinas incubated in MEM depleted of calcium. Retinal cGMP (a), PI11 amplitude (b) and PHI time-to-peak (c) were measured in +/+ (0-O) and +/rd (0-O) retinas, incubated from r = 0 in MEM without calcium salts and supplemented with 3.0 mM EGTA. PHI amplitude and time-to-peak have been normalized relative to stable values obtained initially in standard MEM. Each point represents the mean of at least 5 estimations + SEM.

glutamate, since we found that over a period of at least 1 hr this medium will maintain stable photoreceptor sensitivity, response waveform and cGMP homeostasis. Confirming the earlier observations of e.g. Cohen et al. (1978) and Woodruff and Fain (1982), on changing to a medium containing EGTA and greatly reduced in calcium, dramatic increases were seen in both PI11 and cGMP. Comparable effects on the photoresponse have been noted previously by e.g. Brown and Pinto (1974), Lipton, Ostroy and Dowling (1977) and Bastian and Fain (1982). The rise in cGMP is thought to be due to activation of guanyl cyclase and the fall to reflect the residual PDE activity that persists in a dark-adapted

retina (Cohen et al., 1978). As intracellular cGMP increases because of the reduction in intracellular calcium, the difference in resting levels of cGMP between + /+ and +/rd retinas is eliminated (Figs 3 and 4). This might occur because cyclase activity in the +/rd retina is normal (Doshi ef al., 1985). However, if PDE activity is responsible for the declining phase, we might expect to have seen a slower fall in the +/rd retina because of reduced turnover of the + /rd enzyme (Doshi et al., 1985; Voaden et al., 1991). There is evidence of this in Fig. 4(a) but not Fig. 3(a). It may be that the increased level of free substrate has saturated the low level of active enzyme present in a dark-adapted retina (Pugh & Lamb, 1990). If so capacity (I’,,,,,) rather than substrate affinity

AL1 A. HUSSAIN rr crl 700 600 500 400 300 200 100 0

P

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

(b)

6

2

4

G

8

Incubation

10

12

time

14

18

10

20

(mins)

FIGURE 4. Effect of A23187 on the response of retinal cGMP and PI11 to a medium depleted of calcium. Retinal cGMP (a) and PI11 amplitude (b) were measured in +/+ (0-O) and +/rd (a---0) retinas, incubated from t = 0 in MEM depleted of calcium and supplemented with 10.0 PM of ionophore A23187. PI11 amplitude has been normalized relative to stable values obtained initially in standard MEM. Each point represents the mean of at least 4 estimations + SEM.

will determine turnover, and PDE capacity is the same in the +/+ and +/rd genotypes (Doshi et al., 1985). In the normal retina the changes induced in total cGMP by exposure to a “low calcium” medium operate over a time scale similar to that of the rise and fall of PI11 (Fig. 3). This is not the case, however, for the heterozygotic tissue. For, whereas the peak in PI11 amplitude corresponds with normal and occurs within 2.0 min, the peak in cGMP is delayed to 5.0 min (Fig. 3). In contrast, when the divalent cation ionophore, A23 187, was included in a calcium-depleted medium, the increase in cGMP was speeded up. It is known that in conditions comparable to those used here A23 187 increases the rate of loss of rod outer segment internal calcium and releases over 90% of it (Schnetkamp & Bownds, 1987). Thus, since the kinetics of cyclase and its response to calcium withdrawal are normal in the +/rd mouse (Doshi et al., 1985), the observation supports the notion that the resting level of calcium is higher in “ + /rd” outer segments (see Introduction). The 0.31 log unit greater sensitivity found for +/rd animals in the present in vitro study is comparable to the 0.43 log unit greater sensitivity calculated from measurements of the ERG b-wave in vivo by Low (1987b). Similarly, the slower time-to-peak of +/rd PIIIs at low light intensities is consistent with the in vivo results, obtained by Low (1987b).

The greater sensitivity of +/rd retinas is not due to variation in the concentration of rhodopsin as this is normal (Doshi et al., 1985; Low, 1987a). However, it might arise from the combination of a higher dark current with a higher concentration of intracellular calcium (see Introduction), since in the presence of the latter the activity of rhodopsin kinase may be inhibited (Wagner et al., 1989; but cf. Binder, Biembaum & Bownds, 1990) and/or that of PDE increased (Robinson et al., 1980; Kawamura & Bownds, 1981; Detwiler & Rispoli, 1989; Kawamura & Murakami, 1991), leading to a greater gain per photon absorbed by rhodopsin. Below PI11 saturation, this might also contribute to the slower time-to-peak in the +/rd retina [Fig. 2(b)]. However, if the rise time of the photoresponse reflects cGMP hydrolysis (cf. Pugh & Lamb, 1990), reduced affinity of PDE for its substrate is also likely to be a factor-again, one, that would diminish as PI11 saturation is approached. Curiously, when calcium is reduced a delayed time to peak is also evident at higher flash intensity [Fig. 3(c)], suggesting a specific effect on the + /rd PDE. This merits further investigation as it may be that calcium modulation of PDE activity is dependent on the extent of nucleotide binding to non-catalytic sites. Figure 3(c) also shows that an additional delay in time-to-peak manifests

PHOTORECEPTOR

FUNCTION

slowly in both the +/+ and +/rd retinas. This also requires further investigation. Termination of fast PI11 is thought to be aided by the reduction in intracellular calcium that follows closure of the cGMP-gated channels in the plasma membrane, and the consequent stimulation of guanyl cyclase and, possibly, rhodopsin kinase (Pugh & Lamb, 1990). If the resting level of calcium in +/rd retinas is higher than normal, extrusion of calcium would be slowed and, as observed by Arden and Low (1980), termination of the photoresponse delayed. Perhaps the most intriguing observation in the present studies is that in +/rd photoreceptors, low in calcium, the overall endogenous level of cGMP is not in equilibrium with PI11 amplitude nor its time to peak (Fig. 3), although correspondence appears to exist in the +/+ retina. It is now clear that in some circumstances the relationship between the total measured cGMP concentration and the degree of suppression of the dark current can also vary in a normal photoreceptor (e.g. Woodruff & Fain, 1982; Cote, Nicol, Burke & Bownds, 1986, 1989). It may be, therefore, that free cGMP is abnormally independent of the bound pool in the +/rd outer segment because the locus of the PDE defect in the + /rd animal has a functional role in this regard. Alternatively, there may be factors independent of those related to cGMP and its PDE that can control the rising phase and amplitude of the photoresponse. Although the bulk of available evidence suggests that this is unlikely (Pugh & Lamb, 1990) Krapivinsky, Filatov, Filatova, Lyubarsky and Fesenko (1989) have reported that transducin can regulate the cGMP-dependent conductance in the plasma membrane of rod outer segments. Whatever the situation, it would appear that the +/rd retina will provide valuable information concerning not only homozygous vs heterozygous gene expression but also normal phototransduction. REFERENCES Arden, G. B. & Low, J. C. (1980). Altered kinetics of the photoresponse from retinas of mice heterozygous for the retinal degeneration gene. Journal of Physiology, London, 308, 8OP. Bastian, B. L. & Fain, G. L. (1982). The effects of low calcium and background light on the sensitivity of toad rods. Journal of Physiology, London, 330, 307-329. Binder, B. M., Biembaum, M. S. & Bownds, M. D. (1990). Light activation of one rhodopsin molecule causes the phosphorylation of hundreds of other. Journal of Biological Chemistry, 265, 15333-15340. Bowes, C., Li, T., Danciger, M., Baxter, L. C., Applebury, M. L. & Farber, D. B. (1990). Retinal degeneration in the rd mouse is caused by a defect in the /3 subunit of rod cGMP-phosphodiesterase. Nature, 347, 677680. Brown, J. E. & Pinto, L. H. (1974). Ionic mechanism for the photoreceptor potential of the retina of Bufo marinus. Journal of Physiology, London, 236, 575-59 1. Carter-Dawson, L. D., La Vail, M. M. & Sidman, R. L. (1978). Differential effect of the rd mutation on rods and cones in the mouse retina. Investigative Ophthalmology and Visual Science, I7, 489498. Cohen, A. I. (1984). Some contributions to the cell biology of photoreceptors. Investigative Ophthalmology and Visual Science, 25, 13541365.

IN

+/rd

MICE

35

Cohen, A. I., Hall, A. I. & Ferrendelli, J. A. (1978). Calcium and cyclic nucleotide regulation in incubated mouse retinas. Journal of General Physiology, 71, 595-612. Cote, R. H., Nicol, G. D., Burke, S. A. & Bownds, M. D. (1986). Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors. Journal of Biological Chemistry, 261, 12965-12975. Cote, R. H., Nicol, G. D., Burke, S. A. & Bownds, M. D. (1989). Cyclic GMP levels and membrane current during onset, recovery and light adaptation of the photoresponse of detached frog photoreceptors. Journal of Biological Chemistry, 264, 1538415391. Detwiler, P. B. & Rispoli, G. (1989). Phototransduction in detached rod outer segments: Calcium control of the cGMP economy. Investigative Ophthalmology and Visual Science (Suppl.), 30, 162. Doshi, M., Voaden, M. J. & Arden, G. B. (1985). Cyclic GMP in the retinas of normal mice and those heterozygous for early-onset photoreceptor dystrophy. Experimental Eye Research, 41, 6165. Dowling, J. E. (1987). The retina. Cambridge, Mass.: Belknap Press. Fain, G. L., Quandt, F. N., Bastian, B. L. & Gerschenfeld, H. M. (1978). Contribution of a caesium-sensitive conductance increase to the photo-response. Nature, 272, 467469. Farber, D. B. & Lolley, R. N. (1976). Enzymic basis for cyclic GMP accumulation in degenerative photoreceptor cells of mouse retina. Journal of Cyclic Nucleotide Research, 2, 139-148. Ferrendelli, J. A. & Cohen, A. I. (1976). The effects of light and dark adaptation on the levels of cyclic nucleotides in retinas of mice heterozygous for a gene for photoreceptor dystrophy. Biochemical and Biophysical Research Communications, 73, 421427. Kawamura, S. & Bownds, M. D. (1981). Light adaptation of the cyclic GMP phosphodiesterase of frog photoreceptor membranes mediated by ATP and calcium ions. Journal of General Physiology, 77, 571-591. Kawamura, S. & Murakami, M. (1991). Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods. Nature, 349, 420423. Koch, W.-H. & Stryer, L. (1988). Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions. Nature, 334, 6466. Krapivinsky, G. B., Filatov, G. N., Filatova, E. A., Lyubarsky, A. L. & Fesenko, E. E. (1989). Regulation of cGMP-dependent conductance in cytoplasmic membrane of rod outer segments by transducin. FEBS Letters, 247, 435437. LaVail, M. M. (1981). Analysis of neurological mutants with inherited retinal degeneration. Investigative Ophthalmology and Visual Science, 21, 638657. Lee, R. H., Lieberman, B. S., Hurwitz, R. L. & Lolley, R. N. (1985). Phosphodiesterase-probes show distinct defects in rd mice and Irish setter dog disorders. Investigative Ophthalmology and Visual Science, 26, 156991579. Lee, R. H., Navon, S. E., Brown, B. M., Fung, B. K.-K. & Lolley, R. N. (1988). Characterization of a phosphodiesterase-immunoreactive polypeptide from rod photoreceptors of developing rd mouse retinas. Ikvestigative Ophthalmology and Visual Science, 29, 1021-1027. Lipton, S., Ostroy, S. E. & Dowling, J. E. (1977). Electrical and adaptive properties of rod photoreceptors in Bufo marinus. I. Effects of altered extracellular Ca+ + levels. Journal of General Physiology, 70, 7477770. Lolley, R. N. & Racz, E. (1982). Calcium modulation of cyclic GMP synthesis in rat visual cells. Vision Research, 22, 1481-1486. Low, J. C. (1987a). Rhodopsin levels in the isolated normal and +/rd mouse retina. Ophthalmic Research, 19, 49-51. Low, J. C. (1987b) The cornea1 ERG of the heterozygous retinal degeneration mouse. Von Graefe’s Archives of Clinical and Experimental Ophthalmology, 225, 413417. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. Matthews, H. R., Murphy, R. L. W., Fain, G. L. & Lamb, T. D. (1988). Photoreceptor light adaptation is mediated by cytoplasmic calcium concentration. Nature, 334, 6769. Miller, G. L. (1959). Protein determination for large numbers of samples. Analytical Chemistry, 31, 964.

36

At1

A. HUSSAIN er ui.

Nakatani, K. & Yau, K.-W. (1988). Calcium and light adaptation in retinal rods and cones. Nature, 334, 69-71. Newman. E. A. (1984). Regional specialization of retinal glial cell membrane. Nnturt, 3@9$i S-157. N~e~l W. K. (f958). D~~eren~ation~ metabolic organization and viabifity of the visual ceil. Archives of Ophthalmology, 60. 702-733. Pepe, I. M., Boero, A., Verganl. L., Panfoli, I. & Cugnoli, C. (1986). Effect of Xight and calcium on cyclic GMP synthesis in rod outer segments of toad retina. Biockimica Biuphysica Acta, 889, 271-276. Portzehl, H., Caldwell, P. C. & Ruegg, J. C. (1964). The dependence of contraction and relaxation of muscle fibres from the crab &&rio ~q~~iff~Qon the internal concentration of freecalcium ions. Biochimica BiophJwica Acta, 79, 581-591.

Pugh, E. N, & Lamb, T. D. (1990). Cyclic GMP and calcium: The internal messengers of excitation and adaptation in vertebrate photoreceptors. vision Research, .3Q 1923-1948. Robinson, P. R,, Kawamura, S., Abramson, B. & Bownds, M. D. (tQ80), Control of the cyclic GMP pho~hod~este~se of frog photoreceptor membranes. Journal of Gene& Pkysioiogy, 76. 631-645.

Voaden, M. J., Hussam, A. A. & Willmott, N. J. (IQYI ). Abno~rnaht~e~ in cGMP phosphodiesterase in mice heterozygous for the rd gene In Anderson, R. E., Holtyfield, J. G. 8z LaYail, M. M. (Eds), Reti& ~~~ener~~~~ns(pp. $67 -172). New York: CRC Press Voaden, M. J,, Willmott, N. J.. Hussain, A. A. L Ai-~a~~daw~~ 5. (1989). Functional and biochemical abnormalities in the retinas of mice heterozygous for the rd gene. In LaVail. M. M.. Hollyfield. J. G. & Anderson, R. E. (Eds), Inketitedund em~ironmentd~~ induwd retinaf degenerations (pp. I83 -I 89). New York: Liss. Wagner, R., Ryba, N. & Uhl, R. (1989). Cat&urn regulates the rate of rhodopsin d&activation and primary arnpii~~at~~~nstep in fisuai transduction, FE&S Lptters, 24.2. 249,254. Willmott. N. J., Hussain, A. A. & Voaden, M. J. (1988). Biochemical and electrophysiological abnormalities in the photoreceptors of mice heterozygous for the rd gene. Biorkemicut Snciet), Transac~rionx. I6. 1074 1075.

Woodruff, M. L. & Fain, G. t. (iQ82) Ca’ ’ -dependent changes in cyclic GMP levels are not correlated with opening and closing of the ljght-de~nde~t permeability of toad photoreceptors, Journal q,i General Physiology, 80. 537~.555.

Schnetkamp, P. P. M. & Bownds, M. D. (1987). Na+- and cGMPinduced CaZf fluxes in frog rod photoreceptors. Jauvnai or General Physiology, 8% 48Ib500.

Voaden, M. f. & Willmott, N. J. (1990). Evidence for reduced binding of cychc GMP to cychc GMP phosph~j~teraae in photo~pto~ of mice heterozygous for the rd gene. furrmt Eye Researck, 9. 643-651.

A~k~~~~e~geme~t*~-We thank Dr R. Curtis for providing ERG facilities, the American Ret&&is Pj~entosa Foundation for financiaf support and the British Retinitis Figmemosa Society for a studentship for N.J.W.