Effect of Light on Dopamine Turnover and Metabolism in Rabbit Retina

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1 384 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Morch 1983 Vol. 24 humor from eyes without inflammation (Group 1) contain functional C2, C6, and C7. The small ratios of aqueous humor to serum measurements of C2 (1/ 720), C6 (1/1,657), and Cl (1/1,536) suggest that there is relatively little of these complement components in normal aqueous humor when compared to serum. We were unable to detect Factor B in normal aqueous humor. The single radial immunodiffusion technique that we used, however, may not be sensitive enough to detect small concentrations of Factor B that may be present in normal aqueous humor and fall below the working range of the plates. The mean values of all complement components and IgG were higher in inflamed aqueous humor (Group 2) than in normal aqueous humor (Group 1). Complement and IgG levels in serum did not differ statistically between Groups 1 and 2, so that increased serum levels could not be used to explain the increased aqueous levels in Group 2. Moreover, the ratios of aqueous humor to serum measurements for each complement component and IgG were higher in Group 2 than in Group 1. A comparison of the ratios of IgG to each complement component in normal and inflamed aqueous humor suggested that levels of IgG and complement increased proportionately in inflamed aqueous humor. The altered vascular permeability associated with anterior uveitis is probably responsible for the elevated levels of complement and IgG in aqueous humor samples from Group 2. The results of this and a previous study 2 indicate that normal aqueous humor contains components of the classical complement pathway: Cl, C4, C2, C3, C5, C6, and C7. Moreover, levels of these complement components are increased in inflamed aqueous humor. Factor B, a component of the alternative pathway, could not be demonstrated in normal aqueous humor but was found in five of six samples of inflamed aqueous humor. Key words: Complement, aqueous humor, anterior uveitis, classical pathway, alternative pathway Acknowledgments. Statistical analysis of the data was performed by Robert P. Hirsch, PhD, University of Pittsburgh School of Medicine. Margaret C. Sharrer, MD, provided aqueous humor from patient 13 and John C. Stuart, MD, from patient 11. From the Department of Ophthalmology, University of Pittsburgh School of Medicine and Eye and Ear Hospital of Pittsburgh. Supported in part by a grant from the National Eye Institute, RO1 EY (Dr. Mondino). Submitted for publication June 28, Reprint requests: Bartly J. Mondino, MD, Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California References 1. Chandler JW, Leder R, Kaufman HE, and Caldwell JR: Quantitative determinations of complement components and immunoglobulins in tears and aqueous humor. Invest Ophthalmol 13:151, Mondino BJ and Rao H: Hemolytic complement activity in aqueous humor. Arch Ophthalmol, in press. 3. Borsos T and Rapp HJ: Immune hemolysis: a simplified method for the preparation of EAC'4 with guinea pig or with human complement. J Immunol 99:263, Mondino BJ and Hoffman DB: Hemolytic complement activity in normal human donor corneas. Arch Ophthalmol 98:2041, Mancini G, Carbonara AO, and Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Int J Immunochem 2:235, Effect of Light on Dopamine Turnover and Metabolism in Rabbit Retina David Parkinson and Robert R. Rando Some neurochemical responses of dopaminergic neurons in the rabbit retina have been measured during prolonged light or dark adaptation. Light adaptation produced small increases (20%) in dopamine levels but larger increases (50-100%) in the two metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Light also significantly increased tyrosine hydroxylase activity; the increase was more pronounced when activity was measured in vivo than in vitro. Dopamine turnover, was faster in the light than in the dark. All these data support the suggestion that light leads to the activation of dopaminergic neurons in the rabbit retina. Invest Ophthalmol Vis Sci 24: , 1983 Dopamine has been identified as the neurotransmitter of a subset of amacrine cells 1 ' 2 and work with the albino rat has suggested that light leads to the activation of these dopaminergic neurones. 3 We have observed light dependent activation of dopaminergic neurones in the chick retina also. 4 It would seem premature, however, to assume, a priori, that dopamine has the same function in all vertebrate retinae; especially since dopaminergic neurons in the Cebus monkey and teleost fish are interplexiform cells 5 rather than the amacrine cells of other vertebrate retinae /83/0300/384/$ 1.05 Association for Research in Vision and Ophthalmology

2 No. 3 Reports 085 These interplexiform cells appear to be presynaptic to GABAergic horizontal cells in the teleost retina, 5 ' 6 while there appears to be an inhibitory GABAergic influence presynaptic to the dopaminergic neurons in the albino rat. 7 In addition, the neuronal processes of dopaminergic amacrine cells stratify in a variety of patterns in the retinae of different species 2 providing further opportunity for species variation in neuronal interconnections. The rabbit retina is much studied by electrophysiologic and anatomical methods and so it seemed pertinent to establish whether light also activated dopaminergic neurones in this tissue. We have determined the effects of prolonged light or dark adaptation on parameters that are known to be sensitive to the level of activity in dopaminergic neurones. In particular, we have measured the amounts of the two major metabolites of dopamine, dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA). Part of this study was accomplished by the use of a-fluoromethyldopa (afmd), a highly specific irreversible inhibitor of aromatic amino acid decarboxylase. We have previously shown that afmd will rapidly and effectively block dopamine biosynthesis in the retina after a single intravitreal injection 8 so that the rate of disappearance of dopamine from the retina can be used as an index of transmitter turnover and hence release rate. Because afmd is effective after the local injection of a small dose, eg, 2 ng (~10 nmoles), it has advantages over the reversible drugs used to produce a similar effect (eg, a-methyl-p-tyrosine) but which require large systemic doses (eg, 250 mg/kg). In this respect, afmd is particularly useful for investigating dopamine metabolism in large animals. Its efficacy after only a local application circumvents any undesirable side effects resulting from systemic drug administration such as behavioral depression. Materials and methods. Weanling New Zealand rabbits ( g body weight) were obtained from Margarets Home Farm, Attleboro, MA. The animals were kept in total darkness or exposed to normal fluorescent room lighting (intensity of approximately 50 fc at center of cage) for 48 hrs before use. Animals were anesthetised with ether before intravitreal injections (30-gauge needle) in a final volume of 10 /A of 0.9% w/v NaCl. At the appropriate time the animal was killed by cervical dislocation, the eyes rapidly enucleated under ambient lighting (supplemented with a red safety light (Kodak No. 1) in the dark) and placed on ice. The eyes were then removed to an area with low lighting to allow dissection under a stereo microscope with the minimum illumination necessary. After dissection the retinae were placed in preweighed microcentrifuge tubes and stored at -20 C until required. The time from killing the animal to final dissection was less than 5 min. Dopamine, DOPA, and DOPAC were measured by high pressure liquid chromatography (HPLC) with electrochemical detection modified from the method of Mefford et al. 9 Retinal samples were homogenized by sonication for 3 sec in an ice cold solution containing 0.1 M perchloric acid, 0.2 mm disodium EDTA, and 0.1 mm sodium bisulphite (300 fa per retina) with 5 ng of dihydroxybenzylamine (DHBA) as internal standard. After centrifugation for 20 min at 2700 g, 150 fi\ of the supernatant was mixed with 10 mg of alumina and 1 ml of 0.5 M TRIS HC1 ph 8.6 and shaken for 20 min. Subsequent procedures were as described by Mefford et al. 9 Catechols were resolved with an Altex Ultrasphere-C 18-IP column (4.6 X 150 mm) through which was pumped 50 mm sodium phosphate ph 3.5 containing 3% (v/v) methanol, 0.1 mm disodium EDTA, and 0.1 g/1 sodium heptane sulphonic acid (as ion pair) at a rate of 1 ml/ min. A 3-cm guard column (Brownlee) fitted with a Spheri-5 RP-18 cartridge was placed before the analytical column. The detector consisted of an LC-3 potentiostat and TL-3 thin layer cell (Bioanalytical Systems). The working electrode was made from oilbased carbon paste and was operated at volts relative to a silver/silver chloride reference electrode. Retinal HVA was measured by the method of Hefti 10 except that the HPLC running buffer was 50 mm sodium phosphate ph 5.0 containing 5% (v/v) methanol and 0.1 mm disodium EDTA (pumped at 1 ml/ min). A 3-cm guard column (ODS-10, Brownlee) was placed before the Waters /ubondapak C 18 (3.9 X 300 mm) column. The working electrode was operated at volts. Tyrosine hydroxylase was estimated by measuring the production of DOPA from L-tyrosine in the presence of an inhibitor of aromatic amino acid decarboxylase. Retinae were homogenized in an ice cold solution containing 150 mm KC1, 10 mm 2-mercaptoethanol and 10 mm sodium phosphate ph 7.0 (500 ix\ per retina). The supernatant obtained after centrifugation at 27,000 g for 20 min was used for assay. The incubation mixture contained, in a final volume of 100 /A: 50 fa of homogenate, 0.8 mm DMPH 4 as co-factor, 0.2 mg/ml catalase (bovine liver, twice crystallized, Sigma), 10~ 4 M brocresine (NSD 1055), 0.1 M sodium acetate ph 6.5, and 0.2 M L-tyrosine. To account for nonenzymatic hydroxylation of tyrosine, blanks for each sample were prepared with D-tyrosine instead of the L-isomer. After incubation for 15 min at 37 C with shaking, the reaction was stopped by placing the tubes on ice and adding 100 /x\ of 0.1 M acetic acid that contained 5 ng of DHBA. The mixture was then transferred to

3 386 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1983 Vol. 24 Table 1. The effect of prolonged light or dark adaptation (48 h) on the levels of dopamine, its metabolites, and tyrosine hydroxylase in rabbit retina Dark Light % dark P (t-test) DA* DOPAC* HVA* Tyrosine hydroxylasef ± 8.0 ± 1.0 ± 1.5 ± 20.7 (7) ± ± ± 1.4 (5) ± 13.7 (7) < Units: * ng/g wet weight f pmole DOPA formed/mg protein/h a microfuge tube, extracted with alumina and the DOPA content measured by hplc/ecd as described above. The data from hplc/ecd was corrected for efficiency of recovery by the internal standard ratio method, relative to calibration standards carried through the extraction procedure. The liquid chromatograph consisted of a Waters M6000A pump, WISP 710B automatic injector, 40-cm standing column pulse dampner, and a Houston two-pen recorder. Protein was measured by the method of Lowry et al" with bovine serum albumin as standard. Significant difference between results was tested by Student's t-test. Results. Retinal levels of dopamine and its two metabolites, which were measured after 48 hrs of light exposure or total darkness, are given in Table 1. Levels of all these compounds were significantly inc 0 <0001 1= 50- o c 3. o Q <002 D L D Fig. 1. Effect of an intravitreal injection of afmd (10 nmoles, 2h) on the accumulation of DOPA (A) and the loss of dopamine (B) from the rabbit retina after exposure to light (L) or darkness (D) for 48 hours. Dopamine values have been expressed as a percentage of the value of the saline treated (contralateral) eye of each animal. Absolute values are given in the text. Data are the mean (±SEM) of results from four animals. The level of statistical significance is given above the lower value of each pair (Student's t- test). creased in the light. Dopamine itself was only increased by about 20%, while DOPAC levels went up nearly 50% and HVA levels doubled in the light. An increase of nearly 50% in the in vitro activity of tyrosine hydroxylase, the first and rate-limiting enzyme in neuronal dopamine biosynthesis, was also observed (Table 1). In a second experiment, rabbits were light or dark adapted for 48 hrs, and then received an intravitreal injection of afmd. The control (untreated) levels of retinal dopamine in this experiment were ± 19.6 ng/g tissue (mean SEM, n = 4) in the light and ± 10.5 ng/g (n = 4) in the dark. These values were not significantly different although there was a 20% increase in the light. Two hours after the administration of afmd, dopamine levels were reduced in both light- and dark-adapted retinae. In the light, however, only 20% of the dopamine stores remained, while in the dark, over 40% of the untreated dopamine content could still be detected. Since a significantly greater proportion of dopamine was lost in the light than dark after afmd (P < 0.02), this result indicates greater dopamine turnover in the light. At the same time, we measured retinal DOPA levels after afmd as a crude measure of tyrosine hydroxylase activity in vivo. A small peak with the same retention time as DOPA on the HPLC was observed in untreated retinae. This amounted to 3.7 ± 0.4 ng/g tissue (n = 4) in the light and 5.2 ± 1.1 (n = 4) in the dark. These values are close to the sensitivity limits of the assay for catechols (ie ^2 ng/g). Two hours after afmd there was significant DOPA accumulation in the retinae (Fig. 1), but this was nearly three times greater in the light than in the dark. Discussion. Nichols et al 12 observed that the retinal dopamine content in rats and rabbits increased upon light exposure relative to dark-adapted animals while Iuvone et al 3 first showed that light increased dopamine turnover in the dark-adapted retina. Since the turnover rate of a transmitter is probably linked directly to the level of neuronal activity, these results are taken to indicate that dopaminergic neurones in the rat retina are activated in the light more than in the dark. Dopamine turnover is usually estimated by

4 No. 3 Reports 387 following its disappearance with time after systemic administration of a-methyl-p-tyrosine, a drug that reversibly inhibits tyrosine hydroxylase and hence abolishes dopamine biosynthesis. The same result is achieved by afmd by inhibiting aromatic amino acid decarboxylase (DOPA decarboxylase), the next enzymatic step after tyrosine hydroxylase. Because afmd is potent and irreversible, dopamine biosynthesis can be abolished rapidly after a single intravitreal injection without the fear of unwanted systemic side effects. The data in Figure 1 A, suggest that light increases dopamine turnover in rabbit retina because a larger proportion of the retinal dopamine content was lost in light than in the dark after afmd. Approximations of the turnover rate can be made by assuming that afmd is effective within 30 min of administration 8 and that the decay is first order up to our 2-hr time point. Then the half lifes for dopamine are 40 min in the light and 72 min in the dark. The corresponding turnover rates are 3.0 ng/g tissue/ min and 1.4 ng/g tissue/min, respectively, indicating about twofold faster turnover in the light. These values should only be regarded as preliminary until a more detailed kinetic investigation can be carried out. In the CNS, conditions that are thought to result in the activation of adrenergic neurons, and in particular dopaminergic neurons, are accompanied by increases in tissue levels of the metabolites of the neurotransmitter, and also be activation of transmitter biosynthesis. 13 In regard to the former, we have also observed elevated levels of DOPAC and HVA, the major metabolites of dopamine. These results further support the notion of increased dopaminergic activity in rabbit retina in the light, particularly as we have observed similar light dependent increases in dopamine metabolites in chick 4 and pigmented rat retina (Parkinson, unpublished). By comparison, the increases in DOPAC and HVA, particularly the latter, would seem to be more reliable indicators of increased activity than the smaller increase in the levels of the transmitter itself. Although increases of about 20% in dopamine levels were observed in both experiments, only in the first was this statistically significant. An activation of dopamine biosynthesis might be indicated by the increased level of tyrosine hydroxylase (Table 1). Since the assay for this enzyme was performed with saturating concentrations of substrate and co-factor, the 45% increase in activity probably results from a proportional increase in active enzyme protein. If, however, dopamine turnover has increased by about twofold in the light (vide supra), then synthesis must increase by at least a similar amount to maintain the dopamine stores; Iuvone et al 14 have reported that the light-dependent activation of tyrosine hydroxylase in the rat retina occurs in two phases. An early phase, detectable within 15 min of light exposure increases enzyme activity by an allosteric mechanism involving increased affinity for the co-factor. After long light exposures (96 h) increased amounts of active enzyme protein are observable with no change in the enzyme properties. After 48 hrs, we are presumably observing the latter phase. If this is so, then some other factors must be contributing to a higher synthesis rate since the increased amount of tyrosine hydroxylase cannot alone account for the required increase. Since the neuronal content of the biopterin co-factor for tyrosine hydroxylase is probably insufficient to saturate the enzyme, an increased supply of co-factor may contribute to a higher in vivo activity. 15 We attempted to study this further by following DOPA accumulation after afmd treatment as a measure of tyrosine hydroxylase activity in vivo since this data was available after using afmd to study dopamine turnover. Weiner 16 has considered the problems associated with this sort of technique, and indeed, the results would tend to be underestimates of the true rate unless account is taken of the rate of concurrent efflux of DOPA from the retina. Nevertheless, our data qualitatively suggest that DOPA is formed at a much greater rate (two- to three-fold) in the light-exposed rabbit retina than in the dark. Similar results have been observed in rat retinae In conclusion, we have presented several pieces of evidence, ie, (1) increased metabolite levels, (2) increased dopamine turnover, and (3) increased dopamine synthesis, that all qualitatively support the hypothesis that light produces activation of the dopaminergic neurons in the rabbit retina. Key words: Rabbit retina, dopamine turnover, dopamine metabolites, tyrosine hydroxylase, light. Acknowledgments. (S)-a-Fluoromethyldopa was a gift from Dr. J. Kollonitsch of Merck, Sharp and Dohme Research Laboratories. David Parkinson gratefully acknowledges a Wellcome Trust Travel Grant. Mrs. Susan Vos is thanked for typing the manuscript. From the Department of Pharmacology, Harvard Medical School, Boston, Massachusetts. Supported by National Institutes of Health Grant EY David Parkinson was a recipient of a Wellcome Trust Travel Grant. David Parkinson's present address: Department of Anatomy, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada. Submitted for publication July 15, Reprint requests: Robert R. Rando, Department of Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts References 1. Haggendal J and Malmfors T: Identification and cellular localization of the catecholamines in the retina and the choroid of the rabbit. Acta Physiol Scand 64:58, 1965.

5 388 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1983 Vol Ehinger B: Biogenic monoamines as transmitters in the retina. In Transmitters in the Visual Process, Bonting SL, editor. Oxford, Pergamon Press, 1976, pp Iuvone PM, Galli CL, Garrison-Gund CK, and NeffNH: Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons. Science 202:901, Parkinson D and Rando RR: The effects of light on dopamine metabolism in chick retina. J Neurochem 40:39, Dowling JE and Ehinger B: The interplexiform cell system. I. Synapses of the dopaminergic neurones in the goldfish retina. Proc R Soc Lond [Biol] 201:7, Marc RE, Stell WK, Bok D, and Lam DMK: GABA-ergic pathways in the goldfish retina. J Comp Neurol 182:221, Morgan WW and Kamp CW: A GABAergic influence on the light-induced increase in dopamine turnover in the darkadapted rat retina in vivo. J Neurochem 34:1082, Parkinson D, Baughman R, Masland RH, and Rando RR: Dopamine metabolism following irreversible inactivation of aromatic amino acid decarboxylase in retina.. J Neurosci 1:1205, Mefford IN, Gilberg M, and Barchas JD: Simultaneous determination of catecholamines and unconjugated 3,4-dihydroxyphenylacetic acid in brain tissue by ion-pairing reversephase high-performance liquid chromatography with electrochemical detection. Anal Biochem 104:469, Hefti F: A simple, sensitive method for measuring 3,4-dihydroxyphenylacetic acid and homovanillic acid in rat brain tissue using high-performance liquid chromatography with electrochemical detection. Life Sci 25:775, Lowry OH, Rosebrough NJ, Fair AL, and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265, Nichols CW, Jacobowitz D, and Hottenstein M: The influence of light and dark on the catecholamine content of the retina and choroid. Invest Ophthalmol 6: 642, Westerink BHC: The effects of drugs on dopamine biosynthesis and metabolism in the brain. In The Neurobiology of Dopamine, Horn AS, Korf J, and Westerink BHC, editors. New York, Academic Press, 1979, pp Iuvone PM, Galli CL, and NeffNH: Retinal tyrosine hydroxylase: comparison of short-term and long-term stimulation by light. Mol Pharmacol 14: 1212, Abou-Donia MM and Viveros OH: Tetrahydrobiopterin increases in adrenal medulla and cortex: A factor in the regulation of tyrosine hydroxylase. Proc Natl Acad Sci USA 78:2703, Weiner N: A critical assessment of methods for the determination of monoamine synthesis turnover rates in vivo. Adv Biochem Psychopharmacol 12:143, DaPrada M: Dopamine content and synthesis in retina and nucleus accumbens septi: pharmacological and light-induced modifications. Adv Biochem Psychopharmacol 16:311, Proll MA, Kamp CW, and Morgan WW: Use of liquid chromatography with electrochemistry to measure effects of varying intensities of white light on DOPA accumulation in rat retinas. Life Sci 30:11, Myeloid Bodies in the Mammalian Retinal Pigment Epithelium Garerh A. Tabor and Steven K. Fisher In the retinal pigment epithelium (RPE) of a mammal, the Eastern gray squirrel, a type of cytoplasmic organelle, the so-called "myeloid body" that was previously thought to be restricted to the RPE of lower vertebrates was observed. In the squirrel, these organelles are continuous with the smooth endoplasmic reticulum (SER), lack an enclosing membrane, and in general exhibit all the morphologic criteria used to identify myeloid bodies. The presumptive myeloid bodies in the squirrel RPE are most prevalent in animals killed during the early hours of the dark period of a 12L:12D lighting cycle. They are rarely observed in animals killed just prior to or during the light period. Thus, these findings document for the first time the occurrence of myeloid bodies in the mammalian RPE, and indicate that their presence is influenced by a diurnal lighting cycle. Invest Ophthalmol Vis Sci 24: , 1983 During a study monitoring the pattern of photoreceptor disc shedding and phagocytosis in light-entrained gray squirrels, 1 we observed structures in the retinal pigment epithelium (RPE) that closely resemble the myeloid bodies found in the RPE cells of a wide range of nonmammalian species. Myeloid bodies are actually specialized regions of the smooth endoplasmic reticulum (SER), comprising a stack of flattened cisternae. 2 They are generally believed to occur in the RPE of amphibians, reptiles, and birds, but neither in the RPE of fish nor mammals. 2 3 Myeloid bodies in the amphibian RPE have been shown to undergo striking alterations in structure in response to a cyclic lighting regime. 4 In this report, we describe our observations on a type of cytoplasmic organelle present in the RPE of Eastern gray squirrels that resembles the myeloid bodies described in other species. In the squirrel, the presumptive myeloid bodies are prominent structures of the RPE cells of animals killed during the first 1-3 hrs of the dark period, but are rarely observed in lightkilled animals. The latter finding may provide an explanation for the common belief that myeloid bodies do not exist in the mammalian RPE. Materials and methods. Adult Eastern gray squirrels (Sciurus carolinensis) were entrained for several weeks to a 12L:12D lighting cycle. For this study, observations were obtained from animals killed at 30 mins, 1, 2, 3, 4, 5, 7, 8, and 11 hours after the onset of darkness. Details of the lighting regime and fixation /83/0300/388/$ 1.00 Association for Research in Vision and Ophthalmology