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Regulation of Cyclic Nucleotide Concentrations in Photoreceptors

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Regulation of Cyclic Nucleotide Concentrations in Photoreceptors 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%.
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