Volume 13Number 7

Reports 535

Cyclic nucleotide metabolism in humanphotoreceptors. RICHARD G. PANNBACKER.

Human photoreceptor outer segments were pre-pared by sucrose density centrifugation of homog-enized retinas. This preparation was found tocontain high levels of the enzymes catalyzingsynthesis and degradation of cyclic GMP and,to a lesser extent, cyclic AMP. Light and calciumion influenced cyclic GMP synthesis. Cyclic nu-cleotide-dependent protein kinase, the presumedsite of action of cyclic nucleotides, was also pres-ent at high leveb. This enzyme was stimulatedto the same extent by cyclic GMP and cyclic AMP,but was more sensitive to cyclic AMP. In all theparameters measured, the enzymes of the humanpreparation resembled those found in bovine rodouter segments.

High levels of the enzymes which synthesizeand degrade cyclic nucleotides are observed inthe rod outer segments of several animal species.1"3

In contrast to most other tissues, the photo-receptors exhibit higher activities of the enzymeswhich synthesize and degrade cyclic GMP(3',5'-cyclic guanosine monophosphate) than ofthose concerned with cyclic AMP (3',5'-cyclicadenosine monophosphate). The role of cyclicnucleotides in the photoreceptor is unknown, butthe observed regulation of cyclic nucleotide synth-esis by light1- 3 and the absence of the character-istic photoreceptor phosphodiesterase2 in the reti-nas of mice with inherited retinal degeneration4

suggest that these enzymes may be involved inretinal function and disease. In the present studies,we have examined human photoreceptor prepara-tions for enzymes concerned with cyclic nucleotidemetabolism and function.

Materials and methods. Human eyes were ob-tained from the Colorado Eye Bank one to threedays after enucleation, and had either been usedas donors in corneal transplantations or judgedunsuitable for that purpose. As the eyes had beensubjected to strong light during the surgicalprocedures, no attempts were usually made in ourmanipulations to exclude light. The retinas weregently dissected away from the pigmented epi-thelium and homogenized by hand in a loose-fitting Teflon-glass homogenizer. Lactated Ringers(Hartmann's solution) was the homogenizationmedium. The homogenate was layered over acontinuous sucrose density gradient (24 to 41per cent) and centrifuged for three hours at30,000 r.p.m. A narrow band containing photo-receptors (as determined by light microscopy)formed in the upper half of the gradient, andthis material was used in the subsequent ex-periments. Electron microscopy of this prepara-tion revealed typical photoreceptor structures(Fig. 1). Fixation was carried out by suspending

Table I. Enzymes of cyclic nucleotide metabolismand function in human and bovine photoreceptors.Bovine photoreceptors were prepared aspreviously described.2 Assay conditions aredescribed in the text. Protein kinase wasmeasured in the presence of 10~r'M cyclic AMP.

Enzyme

Human Bovine(nmoles./min./mg. of protein)

Guanylate cyclaseAdenylate cyclaseCyclic GMP phosphodiesteraseCyclic AMP phosphodiesteraseProtein kinase

0.410.20

42.310.34.60

0.970.10

70.022.6

4.10

the preparation in 2 per cent glutaraldehyde in0.1 M Sorenson's phosphate buffer for one hourin the cold, centrifuging and resuspending incold buffer for 15 minutes, followed by suspensionin cold 1 per cent osmium tetroxide in bufferfor 30 minutes. The preparation was dehydratedin graded acetone, then embedded in Epon. Sec-tions were cut with a diamond knife on a SorvallMT-1 microtome at a setting of 0.1 p and ex-amined on a Philips 200 electron microscope. Nomitochondria were observed in the preparation.

Adenylate and guanylate cyclase activities weremeasured as previously described3 using alpha-32P-labeled nucleoside triphosphates as substrates,and separating the products by ion-exchange thin-layer chromatography. Phosphodiesterase activitywas measured by following the hydrolysis of 3H-labeled cyclic AMP or cyclic GMP, again separat-ing the reaction products by thin-layer chroma-tography.- In this experiment the substrate con-centration was 0.1 mM cyclic AMP or cyclicGMP. Protein kinase was measured by incubating10 fi\ of the photoreceptor preparation (whichwas 0.1 per cent in the nonionic detergent LubrolPX) with 200 fi\ of a reagent containing Tris(hy-droxymethyl)aminomethane buffer, pH 7.6 (50mM), sodium fluoride (10 mM), magnesiumacetate (5 mM), dithiothreitol (5 mM), and 8 x10-r>M l-ethyl-4-(ethylthio)-lH-pyrazolo-(3,4b)pyridine-5-carboxylic acid, ethyl ester (SQ65442,a phosphodiesterase inhibitor kindly provided byDr. S. M. Hess of the Squibb Institute for Med-ical Research). To this mixture was added 10 /*1of 10 mg. per milliliter of Worthington mixedhistone and 30 fi\ of 0.9 mM ATP containing 2.40/iCi of gamma-82P-labeled ATP and 10 fi\ of acyclic nucleotide solution where indicated. Thetubes were incubated for 10 minutes at 37° C. andthe reaction stopped by the addition of 1 ml. ofcold 31 per cent trichloroacetic acid, 1 mM inATP. Controls were incubated without histone,which was then added prior to the acid. The

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536 Reports Investigative OphthalmologyJuly 1974

Fig. 1. Electron micrograph of the human photoreceptor preparation <xl2,192).

acid-precipitated material was trapped on Mil-lipore filters, washed with 20 per cent trichloro-acetic acid, and counted on a gas-flow Geiger-Muller counter. Protein was measured by themethod of Lowry.0 Radiochemicals were obtainedfrom New England Nuclear.

Results. Table I compares the activities ofadenylate cyclase, guanylate cyclase, phospho-diesterase, and cyclic nucleotide-dependent proteinkinase in human and bovine photoreceptor prep-arations. In each case the specific activity of theenzyme in the human preparation is comparableto that observed in the bovine preparation.

In animal retinas, the activity of the enzymethat synthesizes cyclic GMP is subject to regula-tion by light and calcium ion.3 Because of thebleached nature of the material that we wereable to obtain in most cases, we usually couldnot test the effect of light on the activity of thisenzyme. One preparation was partially dark-adapted, however (A=So/Anoo = 7.9),6 and bleach-ing this preparation by the method previouslydescribed3 inhibited the activity of guanylatecyclase 21 ± 4 per cent. Calcium ion (2 to 3mM) stimulates the activity of bovine photore-ceptor guanylate cyclase3; in the human prepara-

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Volume 13Number 7

Reports 537

1.0-

Z 0.8-oo.o> 0 .6 -

0 .4 -

0 . 2 -

|mM)

Fig. 2. The effect of calcium ion on photoreceptorguanylate cyclase activity. Calcium ion (0.54mM) was carried over from the buffer in whichphotoreceptors were prepared. Calcium chloridewas added to give the higher concentrationsshown.

tion stimulation of the activity of this enzymewas observed at 2 mM calcium ion, with inhibi-tion at higher concentrations (Fig. 2).

As has been observed in rod outer segmentsof animal retinas2- 4 the phosphodiesterase foundin human photoreceptors exhibits a high Michaelisconstant (K,,,) for cyclic AMP (4 mM) with noevidence for the presence of a lower Km form.

The site of action of cyclic nucleotides in othertissues is thought to be a cyclic nucleotide-de-pendent protein kinase.7 As shown in Fig. 3,both cyclic AMP and cyclic GMP stimulate theactivity of this enzyme in the human photore-ceptor. Half-maximal stimulation was observedat 10-7M cyclic AMP or 5 x 10"GM cyclicGMP. Both nucleotides stimulated the proteinkinase to the same extent, and addition of optimallevels of cyclic AMP to optimal levels of cyclicGMP did not increase stimulation significantly,indicating that both nucleotides were acting atthe same site. Similar effects were observed withbovine rod outer segment protein kinase.8

Discussion. It is apparent from the above re-sults that human photoreceptors contain enzymesinvolved in the synthesis, breakdown, and actionof cyclic nucleotides, and that these enzymes havespecific activities and properties which are com-parable to those of animal photoreceptors. Whilea role for cyclic nucleotides in the function(s) ofhuman photoreceptors remains to be defined, theeffect of light on cyclic GMP synthesis suggestsan involvement in visual excitation or adaptation.The effect of light on cyclic AMP synthesis thathas been reported1 is now attributed to an effecton the activity of phosphodiesterase in the outersegment9 (or elsewhere in the retina10). In theexperiments reported here, as well as in those

200-i

150-

100

8 7 6 5

—LOG [CYCLIC NUCLEOTIDE] (M)

Fig. 3. Stimulation of photoreceptor protein kinaseactivity by cyclic AMP and cyclic GMP.

that we have performed on bovine rod outer seg-ments,3 the rate of cyclic GMP synthesis wasmeasured under conditions in which phospho-diesterase activity was completely inhibited, in-dicating an effect of light on the activity ofguanylate cyclase. This effect may be mediatedby calcium ion: Hagins11 has postulated thatvisual excitation occurs by release of calcium ionfrom outer segment discs; such a redistributionof calcium ion within the photoreceptor couldaffect the activity of the calcium-sensitive guany-late cyclase. A similar intermediary role for cal-cium has been proposed to account for hormonaleffects on cyclic GMP synthesis in other tissues.12

In bovine photoreceptors, the onset of inhibitionof guanylate cyclase following exposure to lightoccurs over a period of several minutes,3 suggest-ing that the cyclic nucleotide could be involvedin an adaptive process13 in the photoreceptor.Understanding the mechanism of this processwill depend in part on the determination of thesite of action of cyclic GMP, e.g., the in vivosubstrate for the cyclic nucleotide-dependent pro-tein kinase.

I thank Dr. Philip Ellis for his advice and en-couragement, Don Schoch and Kathrine Littlejohnfor their excellent technical assistance, and PamelaEller for performing the electron microscopy.

From the Division of Ophthalmology and theDepartment of Pharmacology, University ofColorado Medical Center, 4200 E. Ninth Ave.,Denver, Colo. 80220. This work was supported

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538 Reports Investigative OphthalmologyJuly 1974

in part by Grant No. EY01171 from the NationalInstitutes of Health and a grant-in-aid from Fightfor Sight, Inc., New York, N. Y. Submitted forpublication April 8, 1974.Key words: outer segments, cyclic AMP, cyclicGMP, phosphodiesterase, cyclase, protein kinase,adaptation.

REFERENCES1. Bitensky, M. W., Gorman, R. E., and Miller,

W. H.: Adenyl cyclase as a link betweenphoton capture and changes in membranepermeability of frog photoreceptors, Proc.Nat. Acad. Sci. 68: 561, 1971.

2. Pannbacker, R. G., Fleischman, D. E., andReed, D. W.: Cyclic nucleotide phospho-diesterase: high levels in a mammalian pho-toreceptor, Science 175: 757, 1972.

3. Pannbacker, R. G.: Control of guanylatecyclase activity in the rod outer segment,Science 182: 1138, 1973.

4. Schmidt, S. Y., and Lolley, R. N.: Cyclicnucleotide phosphodiesterase, an early de-fect in inherited retinal degeneration of C3Hmice, J. Cell. Biol. 57: 117, 1973.

5. Lowry, O. H., Rosebrough, N. J., Farr, A. L.,et al.: Protein measurement with the Folinphenol reagent, J. Biol. Chem. 193: 265,1951.

6. Ebrey, T. G.: The use of ammonyx LO inthe purification of rhodopsin and rod outersegments, Vision Res. 11: 1007, 1971.

7. Walsh, D. A., Perkins, J. P., and Krebs, E.G.: An adenosine 3',5'-monophosphate-de-pendent protein kinase from rabbit skeletalmuscle, T. Biol. Chem. 243: 3763. 1968.

8. Pannbacker, R. G.: Protein phosphorylationin retinal photoreceptors, Miami WinterSymposia 5: 307, 1973.

9. Bitensky, M. W., Miki, N., Marcus, F. R.,et al.: The role of cyclic nucleotides in visualexcitation, Life Sci. 13: 1451, 1973.

10. Goridis, C, and Virmaux, N.: Light-regulatedguanosine 3',5'-monophosphate phosphodies-terase of bovine retina, Nature 248: 57,1974.

11. Hagins, W. A.: The visual process: excita-tory mechanisms in the primary receptorcells, Ann. Rev. Biophys. Bioeng. 1: 131,1972.

12. Goldberg, N. D., O'Dea, R. F., and Haddox,M. K.: Cyclic GMP. Adv. Cyclic Nucl. Res.3: 155, 1973.

13. Dowling, J. E.: The site of visual adaptation,Science 155: 273, 1967.

The electroretinogram of children deprivedof pattern vision. U. YINON AND E.AUERBACH.

The electroretinogram (ERG) was studied withflash stimulation in 16 children who, at youngages, suffered from monocular and binocular visual

deprivation. No significant difference was foundbetween the b-wave amplitudes of normal and ofdeprived eyes (0.4 > p > 0.2). The slight increasein the retinal response (b-wave) seen after cataractremoval is due to changes in the optical propertiesof the eye and not as a result of neural changesin the retina. No direct relationship was foundbetween changes in the level of visual acuity andthe level of responsiveness expressed by theamplitude and the latency of the ERG. In addi-tion, wave-form complexity was the same in thenormal and in the visually deprived eyes. Thatthe visual acuity level was sharply decreased inall subjects, despite the abovementioned findingsin the ERG, indicates that the site of the depriva-tion effect in humans is higher up in the visualsystem.

Early in life, environmental conditions play arole in altering the structure and function of thevisual system. This is reflected, for example, bythe effects of early pattern deprivation on thevisual system, causing neural changes and as aconsequence to these a sharp decrease in patterndiscrimination.1-4 The question asked is whetherthe cortical effects found1- 2 are secondary toneural changes in the retina of the deprived eye.

The condition of pattern deprivation in humansis more suitable for investigation in comparisonto the situations produced in animals by suturingtechnique1 since it is caused by various types oflens and corneal opacities. However, recordingfrom single neurons in human retina, like inanimal experiments, is almost impossible; there-fore, the electroretinographic techniques usedenable us to examine the neural elements of thedeprived mammalian retina, i.e., the receptorlayer and the bipolar cells.5

We characterized the complexity of the electro-retinogram (ERG) wave-form of children de-prived of pattern vision, mainly due to cataracts.Also, the relationship between the decrease invisual acuity of visually deprived eyes and theelectrical changes in their retina was investigated.Furthermore, when the ocular media in thesechildren became clear after removal of the cata-ract, we have been able to examine whether undernormal conditions of light input to the retina theeffect produced is in the previously deprivedretina or higher up in the visual system.

Methods. Sixteen visually deprived childrenserved for the electrophysiologic recordings; nineof them had unilateral or bilateral congenitalcataracts, five had traumatic cataracts, and twohad leukoma. Ten children's ages ranged between2 months to 6.5 years and the other children'sages ranged between 7 and 12 years. Ten normaladult subjects served as comparisons. Refractionand visual acuity were measured concurrently.For several children, 2 to 4 electrophysiologic

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