Proc. Nat. Acad. Sci. USA Vol. 70. No. 12, Part II, pp. 3820-3824, December 1973

Regulation of Cyclic Nucleotide Concentrations in Photoreceptors: An ATP-Dependent Stimulation of Cyclic Nucleotide Phosphodiesterase by Light (cyclic AMP/cyclic GMP/adenylate cyclase/rhodopsin/retinal rods)

NAOMASA MIKI, JAMES J. KEIRNS, FREDERICK R. MARCUS, JENNY FREEMAN, AND MARK W. BITENSKY Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, Connecticut 06510 Communicated by Lewis Thomas, August 20, 1973

ABSTRACT Regulation of cyclic nucleotide concentra- in photoreceptor organelles as a function of illumination. The tions in rod outer segments (Ranapipiens) has been further examined. The present studies show that illumination sensitivity of phosphodiesterase to an ATP-dependent light- markedly diminishes the concentration of cyclic nucleo- generated activator appears unique to the photoreceptor sys- tides in suspensions of photoreceptor membranes, but the tem. The present paper describes some properties of this locus of regulation is cyclic nucleotide phosphodiesterase light-sensitive phosphodiesterase and explains properties (EC 3.1.4.c) (light-stimulated) and not adenylate cyclase. previously attributed to photoreceptor cyclase. Furthermore, There is a marked disproportionality between bleaching of rhodopsin and stimulation of phosphodiesterase. it examines some possible mechanisms of phosphodiesterase Bleaching only 0.6% of the rhodopsin produces half the activation by light and ATP, including the involvement of stimulation produced by bleaching 100% of the rhodopsin. calcium or a protein kinase. The process of activation of phosphodiesterase by light is in two steps, a light-dependent step followed by an ATP- MATERIALS AND METHODS dependent step. Illumination (in the absence of ATP) pro- duces a trypsin-resistant, heat-labile, macromolecular All dark procedures (dissection, dark assay, etc.) were done stimulator. In the presence of 0.75 mM ATP (GTP or ITP) under near-infrared light with a Metascope No. 9902E infra- this stimulator produces a greater than 5-fold increase in red image converter (Varo, Inc., Garland, Texas). Infrared the V.. of photoreceptor phosphodiesterase without sources were constructed from tungsten lamps and CS#7-56 changing the Km. At physiological substrate concentra- tions (10-7 M) the rate of hydrolysis of cyclic GMP is 23 filters (Corning Glass Co., Corning, N.Y.). Rana pipiens were times greater than that ofcyclic AMP. The light-produced dark-adapted for 12 hr and killed; the eyes were dissected. stimulator appears unique to the photoreceptor mem- Rod outer segments were obtained by flotation on 47% su- branes and does not activate phosphodiesterase in other crose (6). tissues. The photoreceptor membranes prepared in this way and The role of cyclic nucleotides in visual excitation has been a examined by electron microscopy immediately after flotation subject of investigation since 1970. Earlier data had suggested consist of intact outer segments (judged to be more than 95% that light regulates cyclic nucleotide concentrations in photo- of the membranes). After the harvesting of these outer seg- receptor outer-segment membranes (1-5). The present ments and their dispersion in a hypotonic medium, the pre- studies (6) confirm our earlier findings but provide a new dominant morphological element is a vesicle smaller than the locus for the effects of illumination. Although light regulates intact disc. the amounts of retrievable newly synthesized cyclic nucleo- The phosphodiesterase activity was assayed by incubating tides in suspensions of photoreceptor membranes, the mecha- tritiated cAMP with the photoreceptor membranes (20-40 nism of this regulation is a stimulation of cyclic nucleotide ,ug of protein per tube) at pH 7.4 and 32°, using a reaction phosphodiesterase (EC 3.1.4.c) by light rather than an inhibi- volume of 20 ,ul and an incubation time of 1 min. The buffer tion of adenylate cyclase. In our discussion of these earlier for phosphodiesterase assay is 50 mM Tris * HCl, 3 mM MgCl2, findings, the term "light-mediated adenylate cyclase inactiva- and 5 mM [8-8H]cAMP (diluted to 3.5 Ci/mol, Schwartz- tion" should more appropriately be replaced by "light- Mann). The reaction was stopped by boiling. Adenylate mediated phosphodiesterase activation." cyclase was assayed with 100-150 1Ag of protein per tube and Earlier studies failed to detect light activation of phospho- an incubation time of 5 min in a reaction mixture containing diesterase because this activation depends on an unantici- 50 mM Tris . HCl buffer, 3 mM MgCl2, 0.1 mM papaverine pated requirement for the presence of ATP. The absence of hydrochloride, 1 mM cAMP, 0.5 mM (2,8-3H]ATP (diluted ATP from earlier phosphodiesterase reaction mixtures re- to 500 Ci/mol, New England Nuclear Corp.), 2.5 mM theo- flects the fact that it is not required for phosphodiesterase phylline, and a regenerating system (2 mg/ml of creatine assay and, in fact, can competitively inhibit phosphodiester- phosphokinase and 35 mM phosphocreatine, Tris salt). ase. We now find that ATP renders photoreceptor phospho- Labeled cAMP was isolated by sequential chromatography diesterase sensitive to a greater than 5-fold stimulation by in two solvents on polyethyleneimine-cellulose thin layers light. This fact accounts entirely for changes in cyclic AMP (6, 7). Phosphodiesterase activity with cGMP as substrate (cAMP) concentrations, which we have previously recorded was measured in exactly the same way except for the sub- stitution of cGMP for cAMP. After development with the Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; first solvent, cGMP (RF = 0.35) was easily separated from cGMP, guanosine 3': 5'-cyclic monophosphate; EGTA, ethylene GTP, GDP, and GMP (RF all less than 0.08). In the sec- glycol bis(,3-amino ethyl ether)-NN'-tetraacetic acid. ond solvent, cyclic GMP moved less than 0.5 cm while gua- 3820 Proc. Nat. Acad. Sci. USA 70 (1978) Light-Stimulated 3': 5'-Cyclic Nucleotide Phosphodiesterase 3821

TABLE 1. Apparent inhibition of adenylate cyclase by light and 5 recovery of cyclic AMP in adenylate cyclase assay 4- Dark Light 20

Apparent adenylate cyclase activity a 3 0 cAMP formed per 10 min per mg of ID protein) a 168 pmol 24 pmol 2 Recovery of cyclic AMP (1 mM) ._ Adenylate cyclase reaction mixture s0 0. 0 excluding ATP 85% 86% 0 Adenylate cyclase reaction mixture 85% 10% 0~ -I Phosphodiesterase reaction mixture 30% 32% Phosphodiesterase reaction mixture 0 0.01 0.1 1.0 10.0 +0.5mMATP 29% 5% [ATP] (mM) FIG. 1. Requirement for nucleoside triphosphate in light stim- Adenylate cyclase was assayed as described in the text. The cy- ulation of phosphodiesterase. Photoreceptor phosphodiesterase clic AMP was and purified by thin-layer chromatography counted activity of illuminated (0) or unilluminated (-) rod outer-segment (6, 7). For recovery, 1 mM was incu- cyclic [3H]AMP (3 Ci/mol) membranes as a function of ATP concentration. The values bated for 0 or 5 min in the buffers for or cyclase phosphodiesterase shown are the means of three determinations. The maximum assay. The labeled cAMP was and counted Per- purified (6, 7). range of values for a given point was 6%. A unit of phosphodiester- cent recovery is (cAMP counts after 5 min)/(cAMP counts after ase activity is 1 Mmole of cAMP hydrolyzed per min per mg of pro- 0 X 100. The table the means for min) shows six determinations tein. The experiment was repeated three times with the same under each condition. The range of values for a given entry was result. no more than 7%. ments for nucleoside triphosphate in the light-dependent nine, guanosine, and xanthosine all moved to a final position stimulation of phosphodiesterase. Phosphodiesterase activity more than 10 cm ahead of cGMP. in light and dark is shown as a function of ATP concentration were determined the of Protein concentrations by method in Fig. 1. Half-maximal activation is seen at an ATP con- Lowry (8), and ATP concentrations by the luciferase method centration of 0.2 mM. Inhibition (probably competitive) of were measured after dilution of (9). Rhodopsin spectra photo- phosphodiesterase activity is seen at an ATP concentration receptor times materials 5-10 with 1% cetyltrimethylammo- of 2.5 mM. GTP or ITP (0.75 mM) are as effective as ATP nium bromide. Protein kinase was assayed by the method of in the light-dependent phosphodiesterase activation reaction. Kuo and Greengard (10). SQ20009 was a gift from Dr. Sidney ADP and AMP are without effect. Hess (Squibb). Purified (11) bovine-heart protein kinase An aliquot of photoreceptor material was illuminated in was kindly provided by Dr. Paul Greengard. Purified (12) the absence of ATP and then added (in the ratio of 2% illu- rabbit-thyroid protein kinase was a gift from Dr. Steve minated to 98% unilluminated) to unilluminated material. soy Spaulding. Trypsin, trypsin inhibitor, and phos- If no ATP was present during assay the phosphodiesterase was were 1- pholipase-C obtained from Sigma Chemical Co. not activated. If ATP was added to the unilluminated ma- Methyl, 3-isobutylxanthine was from Aldrich Chemical Co. terial (98% by volume) the illuminated material (2% by RESULTS volume) produced full activation. If, on the other hand, 0.3 Evidence for Increased cAMP Destruction in the Presence of mM ATP was added to unilluminated photoreceptor material ATP and Illuminated Photoreceptor Membranes. Our previous which was then bleached and added to the unilluminated interpretation of the data arose from the impression that re- TABLE 2. Light-dependent and ATP-dependent steps in covery of cyclic nucleotides in adenylate cyclase assay of phosphodiesterase activation photoreceptor membrane preparations was adequate. Mea- surements of phosphodiesterase activity in illuminated mem- Phosphodiesterase branes using either a phosphodiesterase reaction mixture or activity (,smol of an adenylate cyclase reaction mixture from which ATP had cAMP destroyed been excluded, showed that recovery of labeled cAMP was per min per mg of better than 85%. Under these conditions the apparent light- protein) dark difference in cyclase activity was about 7:1 (Table 1). No ATP present 0.8 4 0.1 However, using an adenylate cyclase reaction mixture which 0. 3 mM ATP present during bleaching included unlabeled ATP, we have now observed the following: but diluted to 0. 006 mM before assay 0.8 4 0.1 the recovery of cAMP in the dark is better than 85%. How- 0.3 mM ATP present only during assay 4. 2 4 0. 2 ever, under that unique condition where ATP and light are 0. 3 mM ATP present during both bleach- present together, we observe a marked stimulation of phos- ing and assay 4.2 4 0.2 phodiesterase activity so that only 5-10% of newly syn- thesized or added cAMP was recovered in spite of the Phosphodiesterase activity is measured in a mixture of 2% presence of papaverine, theophylline, and 1 mM unlabeled illuminated-98% unilluminated photoreceptor membranes. The cAMP (Table 1). specific activity of illuminated material was 4.6 0.2; that of the unilluminated material was 0.8 4 0.1. The table shows the mean Nucleoside Triphosphate Requirements for the Activation of and standard error for three determinations. The experiment was Phosphodiesterase by Light. We then examined the require- repeated four times with the same results. 3822 Biochemistry: Miki et al. Proc. Nat. Acad. Sci. USA 70 (1978)

TABLE 3. Effect of phosphodiesterase inhibitors on the A I--, photoreceptor enzyme 8

Phosphodiesterase :f activity (% of light or ax dark value without inhibitors) a) I-~~~~~~~~ Inhibitor Dark Light 0 None 100 100 Theophylline (0.05mM) 85 95 Isobutylmethylxanthine (0.05 mM) 35 35 Dipyridamol (Persantine) (0.05 mM) 65 52 -0.25 0 0.25 0.5 0.75 1.0 1.25 1.5 Squibb 20009 35 45 1 mM / [CAMP] Papaverine 56 56 B Phosphodiesterase activity was assayed by using the reaction 86 mixture in the text plus 0.75 mM ATP. With no inhibitor specific as 0~~~~ activity in the dark is 0.6 .mol of cAMP destroyed per min per mg of protein, and in the light it is 3.5 jsmol of cAMP/min per mg 0s14 of protein. The percentages shown are the means for three deter- DO 2 minations. The maximum range of a given value was 7%. The ex- 0 periment was repeated twice with similar results.

-1 0 1 2 3 4 5 6 7 material in the usual 2:98 ratio (thus effectively diluting the 0.1 mM / [c GMP] ATP to below 10 MuM), no phosphodiesterase activation was FIG. 3. Lineweaver-Burke plots of cyclic nucleotide phospho- achieved unless additional ATP was added (Table 2). diesterase activity (with 0.75 mM ATP) in illuminated (0) and unilluminated (@) photoreceptor membranes. (A) cAMP as sub- Relationship Between Phosphodiesterase Activation and strate; (B) cGMP as substrate. Rhodopsin Bleaching. The effects of illumination on phospho- diesterase were quantitated with mixing experiments, which 0.6% bleached material to 99.4% unbleached (Fig. 2). If the had been useful in attempting to establish a relationship be- relationship between bleaching and phosphodiesterase ac- tween rhodopsin bleaching and cyclase activity (6). A portion tivity is studied by bleaching for various time intervals and of the photoreceptor membranes was maximally bleached then ascertaining the percent rhodopsin bleached, similar with room fluorescent light. Phosphodiesterase in activity results are obtained. Light exposures that bleach about 20, various mixtures of illuminated and unilluminated materials 5, 50, or 100% of the rhodopsin all gave maximal phosphodiester- was then measured. We found that half-maximal stimulation ase activation. of phosphodiesterase was achieved with a mixture of only Steady-State Kinetic Parameters and Substrate Preference for Phosphodiesterase. Phosphodiesterase activity as a func- 4 tion of cAMP concentration was examined in both light and dark. These experiments revealed that the Km for cAMP in 4._ both light and dark was 8 mM. However the Vm,,, was in- C.) 0 3 TABLE 4. Effects of homogenization and p-mercuribenzoic 0 acid on light- and ATP-dependent 0 to 2 10I? stimulation of phosphodiesterase

0

a Phosphodiesterase activity 0 1 (pmol of cAMP hydrolyzed per i. min per mg of protein)

Dark Light Light 0 0.04 0.1 0.2 0.6 1.0 2.0 4.0 100 % admixture of bleached enzyme Treatment of with 0.75 without 0.75 with 0.75 photoreceptor material mM ATP mM ATP mM ATP FIG. 2. Relationship between rhodopsin bleaching and phos- phodiesterase activity. The indicated percentage of illuminated None 0.7 0.7 3.5 photoreceptor material (5 min in room fluorescent light) was p-Mercuribenzoic acid added to a complementary percentage of unilluminated material. (0.1 mM) 0.3 0.3 0.3 Phosphodiesterase activity of the mixture was measured in the Homogenization 3.4 3.3 3.5 presence of 0.75 mM ATP. The values shown are the means of EGTA (1 mM) 0.7 0.7 3.4 three determinations. The maximum range of values for a given CaCl2 (1 mM) 0.7 0.6 3.4 point was 8%. A unit of phosphodiesterase activity is 1 Mmol of cAMP hydrolyzed per min per mg of protein. The experiment was Each entry represents the mean of three determinations. The repeated four times with the same results. variation from the mean was less than 6%. Proc. Nat. Acad. Sci. USA 70 (1973) Light-Stimulated 3':5'-Cyclic Nucleotide Phosphodiesterase 3823 TABLE 5. Sedimentation ofrod outer-segment phosphodiesterase TABLE 6. Some properties of the phosphodiesterase stimulator Phosphodiesterase activity (% of light or Phosphodiesterase dark activity in un- activity (Amol of fractionated material) Treatment of illuminated cAMP destroyed photoreceptor material per min per mg of Centrifugal fraction Dark Light before mixing protein) Unfractionated 100& lOOb None 3.1 Pellet (66,000 X g, 30 min) 60 70 Trypsin digestion 3.3 Pellet (60,000 X g, 30 min) resus- Phospholipase-C digestion 3.0 pended and pelleted twice 1 1 Heating (900, 10 min) 0.7 Pellet (200,000 X g, 60 min) 83 80 Dialysis against 0.5 mM EGTA 3.0 Supernatant (200,000 X g, 60 min) 1 1 Phosphodiesterase activity is measured in a mixture of 2% il- Phosphodiesterase activity was assayed with the reaction mix- luminated/98% unilluminated photoreceptor membranes in the ture described in the text plus 0.75 mM ATP. a Specific activity, reaction mixture described in the text plus 0.75 mM ATP. The 0.64 Amol/min per mg of protein. b Specific activity, 3.2 Mmol/ specific activity of unilluminated membranes is 0.7; that of il- min per mg of protein. The percentages shown are the means of luminated membranes is 3.7. The values shown are the means of four experiments. The maimum range of values for a given entry three determinations. The maximum range of values for a given was 6%. entry was 8%. creased more than 5-fold as a result of illumination (Fig. 3A). Phosphodiesterase activity with cGMP as a substrate has also quential centrifugation at 60,000 and 200,000 X g, most of been examined. Light increased the Vmas for the hydrolysis the phosphodiesterase is in the 60,000 X g pellet and essen- of cGMP by phosphodiesterase in a manner comparable to tially none in the 200,000 X g supernatant (Table 5). that of cAMP. The Km for cGMP is 0.16 mM both in light Characteristics of the Light-Produced Stimulator of Photo- and dark (Fig. 3B). At physiological substrate concentrations receptor Phosphodiesterase. Stimulator was prepared by illumi- (10-7 M) (13), the ratio of activities for the substrates nation of rod outer segments and assayed by addition to dark (cGMP/cAMP) is 23/1. photoreceptor membranes (2% light, 98% dark). Heating of Inhibitors of Phosphodiesterase Activity. The photoreceptor the illuminated material to 900 for 10 min resulted in de- phosphodiesterase was examined for its sensitivity to struction of the light-generated activator. However, treat- these phosphodiesterase inhibitors: isobutylmethylxanthine, ment of the material with trypsin (0.12 mg/ml) for 1 hr at Squibb 20009, papaverine, dipyridamol, and theophylline. 250 followed by addition of soy bean inhibitor (0.24 mg/ml), All of the inhibitors were examined at a concentration of or treatment with phospholipase-C (0.12 mg/ml) for 1 hr 0.05 mM. Isobutylmethylxanthine was most effective (Table followed by dilation, had no effect on the stimulator. Exten- 3). sive dialysis of the illuminated photoreceptor material against 0.5 mM EGTA did not affect its ability to stimulate phos- Effects of Aging, Homogenization, p-Mercuribenzoic Acid, phodiesterase (Table 6). and Ca++ on Photoreceptor Phosphodiesterase. At 40 the photoreceptor membrane phosphodiesterase remained stable Effects of Protein Kinase on the Activation of Photoreceptor for about 3 days, after which it exhibited a gradual declne Phosphodiesterase. Two different protein kinases (from bovine (Fig. 4). In addition, the ability of the material to be activated myocardium and from rabbit thyroid) were evaluated for by light significantly diminished by the fourth day (Fig. 4). The material was stable and light sensitive for at least 1 month at -20°. The effects of mechanical trauma on photo- receptor phosphodiesterase were examined by homogenizing i- 4 the material (microhomogenizer, glass on glass, 30 strokes by U- hand) in the dark and then examining activity in the dark 03 ATP. These and in the light in the presence and absence of 0 revealed that obviated the re- experiments homogenization 0 quirement for light and ATP, producing a fully activated en phosphodiesterase (Table 4). Significantly, addition of 1 mM 0 ._ calcium or 0.5 mM EGTA to the light or dark photoreceptor a, material did not influence phosphodiesterase activity. Addi- 0 cl tion of 1 mM p-mercuribenzoic acid before illumination A I blocked the stimulation of phosphodiesterase activity by ATP W 2 3 4 and light (Table 4). Days at 40 FIG. 4. Effects of storage at 40 on phosphodiesterase activity. Effects of Washing or Sedimentation. The photoreceptor Unilluminated material was stored at 40 in the dark and assayed membranes were washed by sedimentation at 60,000 X g and with 0.75 mM ATP either before (0) or after (0) illumination. resuspension in buffer twice. After this procedure, we found Values shown are the means of three determinations (maximum that the phosphodiesterase was absent from the 60,000 X g range 10%). A unit of phosphodiesterase activity is 1 umol of pellet. If the photoreceptor material is fractionated by a se- cAMP hydrolyzed per min per mg of protein. 3824 Biochemistry: Miki et al. Proc. Nat. Acad. Sci. USA 70 (1973) their ability to mimic light in the activation of photoreceptor drolysis and its 23-fold greater velocity at physiological cyclic phosphodiesterase. Neither of the kinases was able to mimic nucleotide concentrations suggest that cGMP participates the effects of illumination when added to the unilluminated in the regulation of photoreceptor function at least in Rana photoreceptor material. Neither kinase stimulated phospho- pipiens. Our data do not permit us to distinguish whether we diesterase in lighted photoreceptor material, and neither have been dealing with two or one phosphodiesterase, but kinase exhibited measurable phosphodiesterase activity by the data clearly show that photoreceptor phosphodiesterase itself. is light-activated with either cAMP or cGMP as substrate. Effects of Phosphodiesterase Stimulator on Other Tissues. Although extrinsic protein kinases do not activate photo- The effect of the stimulator generated by illumination of receptor phosphodiesterase, a role for an intrinsic kinase in photoreceptor membranes on phosphodiesterase in rat liver, the activation sequence is not excluded. myocardium, and brain was studied. In no instance was there Three questions remain in the elucidation of the role of a stimulation of these phosphodiesterases with or without cyclic nucleotides in photoreceptor physiology. The first is ATP. The phosphodiesterase activity of the mixture was the the identity of the link between photon capture and the sum of the phosphodiesterase activities of the photoreceptor generation of the activator; the second is the biochemical membranes and the other tissue. identity of the activator; and the third is its mode of action. The answers to these questions will depend on the purification DISCUSSION of photoreceptor phosphodiesterase and on isolation of the These data demonstrate that light regulates the amount of activator. cyclic nucleotides retrieved from photoreceptor membranes by The importance of cyclic nucleotides in the regulation of controlling the rate of hydrolysis rather than the rate of syn- photoreceptor function is supported by these findings, which thesis. All previous conditions that have been established for confirm the earlier demonstration that light regulates their the preparation of light-sensitive photoreceptor cyclase are concentrations in the rod outer segment. On the basis of these actually necessary for the preparation of light-stimulated data, the locus of this regulation is the phosphodiesterase phosphodiesterase (6). The observation that washing removed whose activation by light is dependent on the presence of light-inactivation of cyclase is actually explained by the fact nucleoside triphosphate and whose preferred substrate (in that washing removes phosphodiesterase. Similarly, ATP Rana pipiens) is cyclic GMP. concentration requirements, the effects of light leaks, the This work was supported by USPHS Grants AM15016 and effects of homogenization, and the effects of aging (6) are all CA13444, by American Cancer Society Grant BC-106C and by the grant from the Jane Coffin Childs Memorial Fund for Medi- explained in terms of a light-activated phosphodiesterase. cal Research. J.J.K. is a fellow of the Jane Coffin Childs Memorial The properties that had previously been attributed to an Fund for Medical Research. inhibitor of cyclase now appear to reside in an activator of 1. Bitensky, M. W., Gorman, R. E. & Miller, W. H. (1971) phosphodiesterase. These include resistance to trypsin and Proc. Nat. Acad. Sci. USA 68, 561-562. phospholipase-C, heat lability, nondialyzability, and inacti- 2. Miller, W. H., Gorman, R. E. & Bitensky, M. W. (1971) vation by p-mercuribenzoic acid. In view of the fact that Science 174, 295-297. small amounts of activator can transform large quantities of 3. Bitensky, M. W., Gorman, R. E. & Miller, W. H. (1972) Science 175, 1363-1364. phosphodiesterase to a fully active form, the process is prob- 4. Bitensky, M. W., Miller, W. H., Gorman, R. E., Neufeld, ably enzymic. Furthermore, the sensitivity of this process to A. H. & Robison, R. (1972) in Advances in Cyclic Nucleotide minute quantities of light favors its physiological significance. Research, eds. Greengard, P., Paoletti, R. & Robinson, G. A. The stimulation sequence can be divided into two steps. (Raven Press, New York), Vol. 1, pp. 317-335. The first is light-sensitive and ATP-independent; the second 4. Bitensky, M. W., Keirns, J. J. & Wagner, R. C. (1973) in Biochemistry and Physiology of Visual Pigments, ed. Langer, involves an interaction between ATP, phosphodiesterase, H. (Springer Verlag, Berlin), pp. 335-340. and the activator generated in step one. The necessity to 6. Miki, N., Keirns, J. J., Marcus, F. R. & Bitensky, M. W. regard the activation sequence as a two-step process derives (1973) Exp. Eye Res., in press. from the observation that nucleotide triphosphates need not 7. Kreiner, P. W., Keirns, J. J. & Bitensky, M. W. (1973) is Proc. Nat. Acad. Sci. USA 70, 1785-1789. be present during illumination. Their ultimate addition 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, necessary, however, in the absence of visible light. R. J. (1951) J. Biol. Chem. 193, 265-275. The work of Yoshikami and Hagins (14) has suggested that 9. Stanley, P. R. & Williams, S. G. (1969) Anal. Biochem. 29, calcium is involved in the regulation of sodium conductance in 381-392. et shown that 10. Kuo, J. F. & Greengard, P. (1970) J. Biol. Chem. 245, 2493- the photoreceptor system. Neufeld al. (15) have 2498. ATP can stimulate the binding of calcium to bovine disc 11. Miyamoto, E., Kuo, J. F. & Greengard, P. (1969) J. Biol. membranes. Szuts (18) has found that illumination can lower Chem. 244,6395-6402. the calcium content of photoreceptor disc membranes. We 12. Spaulding, S. W. & Burrow, G. N. (1972) Endocrinology 91, have been unable, however, to show any connection between i343-1349. 13. Steiner, A. L., Parker, C. W. & Kipnis, D. M. (1970) J. Clin. calcium and the light stimulation of phosphodiesterase in Invest. 49, 43abstr. photoreceptor membranes. The photoreceptor stimulator 14. Yoshikami, S. & Hagins, W. A. (1973) in Biochemistry and differs in several respects from the brain phosphodiesterase Physiology of Visual Pigments, ed. Langer, H. (Springer activator described by Cheung (16) and by Kakiuchi (17). Verlag, Berlin), pp. 245-255. is rather 15. Neufeld, A. H., Miller, W. H. & Bitensky, M. W. (1972) The brain phosphodiesterase stimulator calcium- Biochim. Biophys. Acta 266, 67-71. than ATP-dependent as well as trypsin-sensitive and heat- 16. Cheung, W. Y. (1970) Biochem. Biophys. Res. Commun. 38, stable. It should be noted that the stimulator of photoreceptor 533-538. phosphodiesterase does not stimulate the phosphodiesterase 17. Kakiuchi, S., Yamazaki, R. & Teshima, Y. (1972) in Ad- in other tissues. vances in Cyclic Nucleotide Research, eds. Greengard, P., Paoletti, R. & Robison, G. A. (Raven Press, New York), The presence of a light-sensitive, ATP-dependent phos- Vol. 1, pp. 455-477. phodiesterase stimulation is a unique marker for the photo- 18. Szuts, E. cited by Poo, M. & Cone, R. A. (1973) Exp. Eye receptor membrane system. The lower Km for the cGMP hy- Res., in press.