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In vivo tyrosine hydroxylation in the diabetic rat retina: Effect of tyrosine administration
Brain Research, 298 (1984) 167-170 Elsevier
167
BRE20133
In vivo tyrosine hydroxylation in the diabetic rat retina: effect of tyrosine administration MADELYN H. FERNSTROM, ETTA A. VOLK and JOHN D. FERNSTROM University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, Pittsburgh, PA 15213 (U.S.A.) (Accepted December 13th, 1983) Key words: diabetes - - dopamine- - retina - - streptozotocin- - tyrosine- - tyrosine hydroxylation
The in vivo rate of 3,4-dihydroxyphenylalanine(DOPA) accumulationwas measured in light-adapted retinas of normal and diabetic albino rats. DOPA accumulation was significantlybelow normal in diabetic retinas; tyrosine levels were also reduced. In vitro tyrosine hydroxylase activity was normal. The administration of tyrosine methylester (500 mg/kg, i.p.) to diabetic animals significantly raised retinal tyrosine levels and enhanced DOPA accumulation. Reduced tyrosine levels may therefore contribute to the observed reduction in in vivo tyrosine hydroxylationrate in the diabetic retina. Experimental diabetes produces significant alterations in the levels of a variety of amino acids in the brain2, 5. Among the changes are reductions in the level of tyrosine, phenylalanine and tryptophan. These amino acids are of interest in part because (1) they are monoamine precursors and (2) the rates at which they are converted to their respective monoamine depend to some extent on their concentration within the neuron 7. In particular, data are available showing that the reduction in brain tryptophan level in the diabetic rat brain is associated with reduced brain levels of the 5-hydroxyindoles and a diminished rate of serotonin synthesis4,6A6. This effect is thought to follow from a change in substrate level 4, rather than in enzyme activity22. For the catecholamines, which are synthesized from tyrosine (and possibly phenylalanine), very few data are available: a single report recently appeared showing a reduction in dopamine (DA) synthesis in the diabetic rat brain 21. Conceivably, this effect might follow from the reduced level of tyrosine in the diabetic brain, though this point was not explored, Diabetes also influences amino acid levels in another portion of the central nervous system, the retina. Though data are few, it appears at least that reti-
nal levels of tyrosine and phenylalanine are abnormally low in diabetic rats (tryptophan has not been measured) 9. Because of this, and the putative relationship of substrate supply to transmitter production in catecholamine neurons 7, it seemed reasonable to hypothesize that dopamine production in the retina might be diminished in diabetes. We have therefore measured endogenous tyrosine levels and the in vivo rate of D O P A formation in retinas from diabetic rats. Because both were found to be below normal, we also tested whether an injection of tyrosine into diabetic animals would restore D O P A production to normal values. Adult male Sprague-Dawley rats weighing 150-200 g (COBS, Charles River Breeding Lab., Wilmington, MA) were housed 6 per cage and given free access, to water and food (Wayne Lab. Blox). They were exposed to light from 07.00 h to 19.00 h daily; ambient temperature was 22 °C. Diabetes was induced with a single intracardiac injection of streptozotocin (65 mg/kg, Calbiochem, La Jolla, CA); diabetes was confirmed by polydipsia, "polyurea, and urine glucose concentrations of greater than 2% (Tes-tape, Eli Lilly). Animals were used in an experiment 2-3 weeks following the induction
Correspondence: M. H. Fernstrom, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, 3811 O'Hara Street, Pittsburgh, PA 15213, U.S.A. 0006-8993/84/$03.00(~) 1984Elsevier Science Publishers B.V.
168 TABLE I
TABLE II
Effect of tyrosine injection on L-DOPA accumulation in retinas of normal and diabetic rats
Effect of experimental diabetes on dopamine levels and tyrosine hydroxylase activity in rat retina
Groups of 6 rats were exposed to light for 3 h prior to injection. All animals received NSD-1015 (100 mg/kg, i.p.) 30 min prior to decapitation. Tyrosine methylester (250 mg/kg, i.p.) or vehicle was injected into diabetic rats twice, at 10 and 30 min prior to NSD-1015. Vehicle was administered to all control animals (both diabetic and normal). * P < 0.01 vs normal; ** P < 0.001 vs diabetic, *** P < 0.05 vs normal (analysis of variance and the Newman-Keuls test).
Groups of 6 normal and diabetic rats were killed around 10.00 h (3 h after lights on) and retinas were removed for assay of dopamine content or tyrosine hydroxylase activity. By Student's ttest, no significant effects were observed.
Group
DO PA (ng/mg Retinal tyrosine Serum tyrosine protein) (nmol/mg protein) (nmol/ml)
Normal 2.39 ___0.08 2.24 + 0.17 Diabetic 1.64 ± 0.13" 1.62 ± 0.11"** Diabetic + tyrosine 2.70 + 0.26** 15.85 + 3.50**
121.5 _+5.5 140.9 ± 6.6 576.8 ± 87.3**
of diabetes. All other drugs were injected intraperitoneally, m-Hydroxybenzylhydrazine (NSD-1015) (100 mg/kg) was purchased from Sigma Chemicals (St. Louis, MO). Tyrosine methyl ester (500 mg/kg) was purchased from Aldrich Chemicals (Milwaukee, WI). Drugs were dissolved in water; control animals received water only. Animals were sacrificed by decapitation and trunk blood was collected. Eyes were removed and hemisected; the vitreous body was then removed, the retina dissected from the posterior portion of the eye TMand homogenized in 0.1 N perchloric acid. Samples were centrifuged in the cold for 10 min at 1000 g and aliquots of the clear supernatant taken for further extraction, and then H P L C analysis of D O P A 20, dopamine 10,I7 and tyrosineS was executed. The resultant pellet was resuspended in 1 N N a O H and assayed for total protein content 15. Tyrosine hydroxylase activity was also assayed in retina by the method of Acheson et al. 1. This procedure gives values for hydroxylase activity in retina very similar to those reported by other investigators ~3.14. Blood was allowed to clot on ice and then serum harvested by centrifugation at 2000 rpm at 4 °C. Serum tyrosine was assayed fluorimetrically23. Data were analyzed using analysis of variance followed by the Newman-Keuls test24, or by Student's t-test. Each study was repeated at least 3 times, with similar results. L - D O P A accumulation in retina 30 min after NSD1015 injection was significantly lower in diabetic rats
Group
Dopamine (ng/mg protein)
Tyrosine hydroxylase activity (pmol/min/mg protein)
Normal Diabetic
2.59 + 0.18 2.77 _+0.22
3.65 + 0.34 3.11 + 0.22
than in control animals (P < 0.01; Table I). Tyrosine levels in the diabetic retinas were also significantly below normal (P < 0.05), though serum tyrosine concentrations were not (Table I). When diabetic rats were injected with tyrosine methylester (250 mg/kg i.p., twice) just prior to NSD-1015 administration, L - D O P A accumulation was restored to normal levels. Serum tyrosine levels rose 4-fold following tyrosine administration to diabetic rats, while retinal tyrosine concentrations increased almost 8-fold (Table I). Retinal dopamine levels, and the activity of tyrosine hydroxylase in retinal homogenates, were normal in diabetic rats (Table II). These data show that D O P A accumulation (an index of in vivo tyrosine hydroxylation rate3) in the diabetic rat retina is below normal and that the acute administration of tyrosine can restore it to normal. There are at least two straight-forward reasons why D O P A accumulation might be below normal in the diabetic retina. First, the amount and/or actiVity of tyrosine hydroxylase, the enzyme catalyzing the rate-limiting step in dopamine biosynthesis, might be abnormally low (for any of a number of reasons). And second, the amount of precursor in the diabetic retina might be low enough to unsaturate the hydroxylase sufficiently to reduce tyrosine hydroxylation rate, despite normal amounts of enzyme. The second possibility seems most attractive, based on the present data. First, in vitro tyrosine hydroxylase activity was normal in diabetic rat retina (Table H). Second, retinal tyrosine levels are reduced below normal in the diabetic retina. At normal retinal tyrosine levels (2.2 × 10-4 M; Table I), tyrosine hydroxylase should be almost fully saturated: its Km for tyrosine is about
169 1.3 x 10-4 Ml3. Because the diabetic rat retina begins with a lower tyrosine level (about 1.6 x 10 -4 M; Table I), the enzyme should be only about half-saturated. Consequently, an increase in retinal tyrosine level might be expected to increase enzyme saturation sufficiently to elicit a m e a s u r a b l e rise in hydroxylation rate. Third, some indirect d a t a have already been offered that local tyrosine levels m a y influence d o p a m i n e turnover in the normal rat retina 1~. A n d fourth, tyrosine administration did stimulate D O P A accumulation in the diabetic rat retina (Table I). Tyrosine levels in b l o o d were normal, but retinal tyrosine concentrations were below n o r m a l in diabetic rats. This result does not diminish the likelihood that the source of the fall in retinal tyrosine was a diminished u p t a k e from blood. M o r e likely, it p r o b a b l y means that, as for brain, tyrosine u p t a k e into retina does not simply reflect b l o o d tyrosine levels. Instead, the blood-retinal barrier p r o b a b l y has a competitive transport carrier for large neutral amino acids~2,19,
1 Acheson, A. L., Kapatos, G. and Zigmond, M. J., The effects of phosphorylating conditions on tyrosine hydroxylase activity are influenced by assay conditions and brain region, Life Sci., 28 (1981) 1407-1420. 2 Brosnan, J. T., Man, K. W., Hall, D. E., Colbourne, S. A. and Brosnan, M. E., Interorgan metabolism of amino acids in streptozotocin-diabetic ketoacidotic rat, Amer. J. Physiol., 244 (1983) E151-E158. 3 Carlsson, A., Kehr, W., Lindqvist, M., Magnusson, T. and Atack, C. V., Regulation of monoamine metabolism in the central nervous system, Pharmacol. Rev., 24 (1972) 371-384. 4 Crandall, E. A., Gillis, M. A. and Fernstrom, J, D., Reduction in brain serotonin synthesis rate in streptozotocin-diabetic rats, Endocrinology, 109 (1981) 310-312. 5 Crandall, E. A. and Fernstrom, J. D., Effect of experimental diabetes on the levels of aromatic and branched-chain amino acids in rat blood and brain, Diabetes, 32 (1983) 222-230. 6 Curzon, G. and Fernando, J. C. R., Drugs altering insulin secretion: effects on plasma and brain concentrations of aromatic amino acids and on brain 5-hydroxytryptamine turnover, Brit. J. Pharmacol., 60 (1977) 401-408. 7 Fernstrom, J. D., The role of precursor availability in the control of monoamine biosynthesis in the brain, Physiol. Rev., 63 (1983) 484-545. 8 Fernstrom, M. H. and Fernstrom, J. D., Rapid measurement of free amino acids in serum and CSF using high-performance liquid chromatography, Life Sci., 29 (1981) 211%2130. 9 Frayser, R. and Buse, M. G., Branched chain amino acid metabolism in the retina of diabetic rats, Diabetologia, 14 (1978) 171-176.
just as does the brain. A s a consequence, retinal tyrosine levels are p r o b a b l y r e d u c e d for the same reason that brain tyrosine levels are below normalS: viz., the blood levels of 3 of tyrosine's transport competitors, leucine, isoleucine, and valine are very high, owing to the absence of insulin secretion. Such high levels of these competitors p r o b a b l y inhibit tyrosine t r a n s p o r t across the b l o o d - r e t i n a l barrier. In conclusion, these results d e m o n s t r a t e that in vivo D O P A synthesis is below n o r m a l in the diabetic rat retina; this effect m a y be related to diminished precursor levels in this tissue.
These studies were s u p p o r t e d by a grant from the National Eye Institute (EY04980). The expert technical assistance of Ms. Trish G r u b b is greatly appreciated. W e thank Dr. Michael J. Z i g m o n d and Ms. Midge Heil for teaching us the tyrosine hydroxylase assay.
10 Gauchy, C., Tassin, J. P., Glowinski, J. and Cheramy, A., Isolation and radioenzymatic estimation of picogram quantities of dopamine and norepinephrine in biological sampies, J. Neurochem., 26 (1976) 471-480. 11 Gibson, C. J., Watkins, C. J. and Wurtman, R. J., Tyrosine administration enhances dopamine synthesis and release in light-activated rat retina, J. neural Trans., 56 (1983) 153--160. 12 Hjelle, J. T., Baird-Lambert, J., Cardinale, G., Spector, S. and Udenfriend, S., Isolated microvessels: the blood-brain barrier in vitro, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 4544-4548. 13 Iuvone, P. M., Galli, C. L. and Neff, N. H., Retinal tyrosine hydroxylase: comparison of the short-term and longterm stimulation by light, Molec. Pharmacol., 14 (1978) 1212-1219. 14 Iuvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine cells, Science, 202 (1978) 901-902. 15 Lowry, O., Rosebrough, M., Farr, A. and Randall, R., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 16 MacKenzie, R. G. and Trulson, M. E., Effects of insulin and streptozotocin-induced diabetes on brain tryptophan and serotonin metabolism in rats, J. Neurochem., 30 (1978) 205-211. 17 Moyer, T. P. and Jiang, N. S,, Optimized isocratic conditions for analysis of catecholamines by high-performance reversed-phase paired-ion chromatography with amperometric detection, J. Chromatogr., 153 (1978) 365-372. 18 Oraedu, A. C. I., Voaden, M. J. and Marshall, J., Photochemical damage in the albino rat retina: morphological
170 changes and endogenous amino acids, J. Neurochem.. 35 (1980) 1361-1369. 19 Rapoport, S., Sites and functions of the blood-aqueous and the blood-vitreous barriers of the eye. In Blood-Brain Barrier in Physiology and Medicine, Raven, New York, 1976, pp. 207-232, 20 Sved, A. F. and Fernstrom, J. D., Tyrosine availability and dopamine synthesis in the striatum: studies with gamma-butyrolactone, Life Sci., 29 (1981) 743-748. 21 Trulson, M. E. and Himmel, C. D., Decreased brain dopamine synthesis rate and increased [3H]spiroperidol binding
in streptozotocin-diabetic rats. J. Neurochem., 4() (1983) 1456-1459. 22 Trulson, M. E. and MacKenzie, R. G., Increased tryptophan hydroxylase activity may compensate for decreased brain tryptophan levels in streptozotocin-diabetic rats. J. Pharmacol, exp. Ther., 212 (1980) 269-273. 23 Waalkes, T. P. and Udenfriend, S.. A fluorimetric method for the estimation of tyrosine in plasma and tissues, J. Lab. clin. Med., 50 (1957) 733-736. 24 Zivin, J. A. and Bartko, J. J., Statistics for disinterested scientists, Life Sci., 18 (1976) 15-26.
167
BRE20133
In vivo tyrosine hydroxylation in the diabetic rat retina: effect of tyrosine administration MADELYN H. FERNSTROM, ETTA A. VOLK and JOHN D. FERNSTROM University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, Pittsburgh, PA 15213 (U.S.A.) (Accepted December 13th, 1983) Key words: diabetes - - dopamine- - retina - - streptozotocin- - tyrosine- - tyrosine hydroxylation
The in vivo rate of 3,4-dihydroxyphenylalanine(DOPA) accumulationwas measured in light-adapted retinas of normal and diabetic albino rats. DOPA accumulation was significantlybelow normal in diabetic retinas; tyrosine levels were also reduced. In vitro tyrosine hydroxylase activity was normal. The administration of tyrosine methylester (500 mg/kg, i.p.) to diabetic animals significantly raised retinal tyrosine levels and enhanced DOPA accumulation. Reduced tyrosine levels may therefore contribute to the observed reduction in in vivo tyrosine hydroxylationrate in the diabetic retina. Experimental diabetes produces significant alterations in the levels of a variety of amino acids in the brain2, 5. Among the changes are reductions in the level of tyrosine, phenylalanine and tryptophan. These amino acids are of interest in part because (1) they are monoamine precursors and (2) the rates at which they are converted to their respective monoamine depend to some extent on their concentration within the neuron 7. In particular, data are available showing that the reduction in brain tryptophan level in the diabetic rat brain is associated with reduced brain levels of the 5-hydroxyindoles and a diminished rate of serotonin synthesis4,6A6. This effect is thought to follow from a change in substrate level 4, rather than in enzyme activity22. For the catecholamines, which are synthesized from tyrosine (and possibly phenylalanine), very few data are available: a single report recently appeared showing a reduction in dopamine (DA) synthesis in the diabetic rat brain 21. Conceivably, this effect might follow from the reduced level of tyrosine in the diabetic brain, though this point was not explored, Diabetes also influences amino acid levels in another portion of the central nervous system, the retina. Though data are few, it appears at least that reti-
nal levels of tyrosine and phenylalanine are abnormally low in diabetic rats (tryptophan has not been measured) 9. Because of this, and the putative relationship of substrate supply to transmitter production in catecholamine neurons 7, it seemed reasonable to hypothesize that dopamine production in the retina might be diminished in diabetes. We have therefore measured endogenous tyrosine levels and the in vivo rate of D O P A formation in retinas from diabetic rats. Because both were found to be below normal, we also tested whether an injection of tyrosine into diabetic animals would restore D O P A production to normal values. Adult male Sprague-Dawley rats weighing 150-200 g (COBS, Charles River Breeding Lab., Wilmington, MA) were housed 6 per cage and given free access, to water and food (Wayne Lab. Blox). They were exposed to light from 07.00 h to 19.00 h daily; ambient temperature was 22 °C. Diabetes was induced with a single intracardiac injection of streptozotocin (65 mg/kg, Calbiochem, La Jolla, CA); diabetes was confirmed by polydipsia, "polyurea, and urine glucose concentrations of greater than 2% (Tes-tape, Eli Lilly). Animals were used in an experiment 2-3 weeks following the induction
Correspondence: M. H. Fernstrom, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, 3811 O'Hara Street, Pittsburgh, PA 15213, U.S.A. 0006-8993/84/$03.00(~) 1984Elsevier Science Publishers B.V.
168 TABLE I
TABLE II
Effect of tyrosine injection on L-DOPA accumulation in retinas of normal and diabetic rats
Effect of experimental diabetes on dopamine levels and tyrosine hydroxylase activity in rat retina
Groups of 6 rats were exposed to light for 3 h prior to injection. All animals received NSD-1015 (100 mg/kg, i.p.) 30 min prior to decapitation. Tyrosine methylester (250 mg/kg, i.p.) or vehicle was injected into diabetic rats twice, at 10 and 30 min prior to NSD-1015. Vehicle was administered to all control animals (both diabetic and normal). * P < 0.01 vs normal; ** P < 0.001 vs diabetic, *** P < 0.05 vs normal (analysis of variance and the Newman-Keuls test).
Groups of 6 normal and diabetic rats were killed around 10.00 h (3 h after lights on) and retinas were removed for assay of dopamine content or tyrosine hydroxylase activity. By Student's ttest, no significant effects were observed.
Group
DO PA (ng/mg Retinal tyrosine Serum tyrosine protein) (nmol/mg protein) (nmol/ml)
Normal 2.39 ___0.08 2.24 + 0.17 Diabetic 1.64 ± 0.13" 1.62 ± 0.11"** Diabetic + tyrosine 2.70 + 0.26** 15.85 + 3.50**
121.5 _+5.5 140.9 ± 6.6 576.8 ± 87.3**
of diabetes. All other drugs were injected intraperitoneally, m-Hydroxybenzylhydrazine (NSD-1015) (100 mg/kg) was purchased from Sigma Chemicals (St. Louis, MO). Tyrosine methyl ester (500 mg/kg) was purchased from Aldrich Chemicals (Milwaukee, WI). Drugs were dissolved in water; control animals received water only. Animals were sacrificed by decapitation and trunk blood was collected. Eyes were removed and hemisected; the vitreous body was then removed, the retina dissected from the posterior portion of the eye TMand homogenized in 0.1 N perchloric acid. Samples were centrifuged in the cold for 10 min at 1000 g and aliquots of the clear supernatant taken for further extraction, and then H P L C analysis of D O P A 20, dopamine 10,I7 and tyrosineS was executed. The resultant pellet was resuspended in 1 N N a O H and assayed for total protein content 15. Tyrosine hydroxylase activity was also assayed in retina by the method of Acheson et al. 1. This procedure gives values for hydroxylase activity in retina very similar to those reported by other investigators ~3.14. Blood was allowed to clot on ice and then serum harvested by centrifugation at 2000 rpm at 4 °C. Serum tyrosine was assayed fluorimetrically23. Data were analyzed using analysis of variance followed by the Newman-Keuls test24, or by Student's t-test. Each study was repeated at least 3 times, with similar results. L - D O P A accumulation in retina 30 min after NSD1015 injection was significantly lower in diabetic rats
Group
Dopamine (ng/mg protein)
Tyrosine hydroxylase activity (pmol/min/mg protein)
Normal Diabetic
2.59 + 0.18 2.77 _+0.22
3.65 + 0.34 3.11 + 0.22
than in control animals (P < 0.01; Table I). Tyrosine levels in the diabetic retinas were also significantly below normal (P < 0.05), though serum tyrosine concentrations were not (Table I). When diabetic rats were injected with tyrosine methylester (250 mg/kg i.p., twice) just prior to NSD-1015 administration, L - D O P A accumulation was restored to normal levels. Serum tyrosine levels rose 4-fold following tyrosine administration to diabetic rats, while retinal tyrosine concentrations increased almost 8-fold (Table I). Retinal dopamine levels, and the activity of tyrosine hydroxylase in retinal homogenates, were normal in diabetic rats (Table II). These data show that D O P A accumulation (an index of in vivo tyrosine hydroxylation rate3) in the diabetic rat retina is below normal and that the acute administration of tyrosine can restore it to normal. There are at least two straight-forward reasons why D O P A accumulation might be below normal in the diabetic retina. First, the amount and/or actiVity of tyrosine hydroxylase, the enzyme catalyzing the rate-limiting step in dopamine biosynthesis, might be abnormally low (for any of a number of reasons). And second, the amount of precursor in the diabetic retina might be low enough to unsaturate the hydroxylase sufficiently to reduce tyrosine hydroxylation rate, despite normal amounts of enzyme. The second possibility seems most attractive, based on the present data. First, in vitro tyrosine hydroxylase activity was normal in diabetic rat retina (Table H). Second, retinal tyrosine levels are reduced below normal in the diabetic retina. At normal retinal tyrosine levels (2.2 × 10-4 M; Table I), tyrosine hydroxylase should be almost fully saturated: its Km for tyrosine is about
169 1.3 x 10-4 Ml3. Because the diabetic rat retina begins with a lower tyrosine level (about 1.6 x 10 -4 M; Table I), the enzyme should be only about half-saturated. Consequently, an increase in retinal tyrosine level might be expected to increase enzyme saturation sufficiently to elicit a m e a s u r a b l e rise in hydroxylation rate. Third, some indirect d a t a have already been offered that local tyrosine levels m a y influence d o p a m i n e turnover in the normal rat retina 1~. A n d fourth, tyrosine administration did stimulate D O P A accumulation in the diabetic rat retina (Table I). Tyrosine levels in b l o o d were normal, but retinal tyrosine concentrations were below n o r m a l in diabetic rats. This result does not diminish the likelihood that the source of the fall in retinal tyrosine was a diminished u p t a k e from blood. M o r e likely, it p r o b a b l y means that, as for brain, tyrosine u p t a k e into retina does not simply reflect b l o o d tyrosine levels. Instead, the blood-retinal barrier p r o b a b l y has a competitive transport carrier for large neutral amino acids~2,19,
1 Acheson, A. L., Kapatos, G. and Zigmond, M. J., The effects of phosphorylating conditions on tyrosine hydroxylase activity are influenced by assay conditions and brain region, Life Sci., 28 (1981) 1407-1420. 2 Brosnan, J. T., Man, K. W., Hall, D. E., Colbourne, S. A. and Brosnan, M. E., Interorgan metabolism of amino acids in streptozotocin-diabetic ketoacidotic rat, Amer. J. Physiol., 244 (1983) E151-E158. 3 Carlsson, A., Kehr, W., Lindqvist, M., Magnusson, T. and Atack, C. V., Regulation of monoamine metabolism in the central nervous system, Pharmacol. Rev., 24 (1972) 371-384. 4 Crandall, E. A., Gillis, M. A. and Fernstrom, J, D., Reduction in brain serotonin synthesis rate in streptozotocin-diabetic rats, Endocrinology, 109 (1981) 310-312. 5 Crandall, E. A. and Fernstrom, J. D., Effect of experimental diabetes on the levels of aromatic and branched-chain amino acids in rat blood and brain, Diabetes, 32 (1983) 222-230. 6 Curzon, G. and Fernando, J. C. R., Drugs altering insulin secretion: effects on plasma and brain concentrations of aromatic amino acids and on brain 5-hydroxytryptamine turnover, Brit. J. Pharmacol., 60 (1977) 401-408. 7 Fernstrom, J. D., The role of precursor availability in the control of monoamine biosynthesis in the brain, Physiol. Rev., 63 (1983) 484-545. 8 Fernstrom, M. H. and Fernstrom, J. D., Rapid measurement of free amino acids in serum and CSF using high-performance liquid chromatography, Life Sci., 29 (1981) 211%2130. 9 Frayser, R. and Buse, M. G., Branched chain amino acid metabolism in the retina of diabetic rats, Diabetologia, 14 (1978) 171-176.
just as does the brain. A s a consequence, retinal tyrosine levels are p r o b a b l y r e d u c e d for the same reason that brain tyrosine levels are below normalS: viz., the blood levels of 3 of tyrosine's transport competitors, leucine, isoleucine, and valine are very high, owing to the absence of insulin secretion. Such high levels of these competitors p r o b a b l y inhibit tyrosine t r a n s p o r t across the b l o o d - r e t i n a l barrier. In conclusion, these results d e m o n s t r a t e that in vivo D O P A synthesis is below n o r m a l in the diabetic rat retina; this effect m a y be related to diminished precursor levels in this tissue.
These studies were s u p p o r t e d by a grant from the National Eye Institute (EY04980). The expert technical assistance of Ms. Trish G r u b b is greatly appreciated. W e thank Dr. Michael J. Z i g m o n d and Ms. Midge Heil for teaching us the tyrosine hydroxylase assay.
10 Gauchy, C., Tassin, J. P., Glowinski, J. and Cheramy, A., Isolation and radioenzymatic estimation of picogram quantities of dopamine and norepinephrine in biological sampies, J. Neurochem., 26 (1976) 471-480. 11 Gibson, C. J., Watkins, C. J. and Wurtman, R. J., Tyrosine administration enhances dopamine synthesis and release in light-activated rat retina, J. neural Trans., 56 (1983) 153--160. 12 Hjelle, J. T., Baird-Lambert, J., Cardinale, G., Spector, S. and Udenfriend, S., Isolated microvessels: the blood-brain barrier in vitro, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 4544-4548. 13 Iuvone, P. M., Galli, C. L. and Neff, N. H., Retinal tyrosine hydroxylase: comparison of the short-term and longterm stimulation by light, Molec. Pharmacol., 14 (1978) 1212-1219. 14 Iuvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine cells, Science, 202 (1978) 901-902. 15 Lowry, O., Rosebrough, M., Farr, A. and Randall, R., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 16 MacKenzie, R. G. and Trulson, M. E., Effects of insulin and streptozotocin-induced diabetes on brain tryptophan and serotonin metabolism in rats, J. Neurochem., 30 (1978) 205-211. 17 Moyer, T. P. and Jiang, N. S,, Optimized isocratic conditions for analysis of catecholamines by high-performance reversed-phase paired-ion chromatography with amperometric detection, J. Chromatogr., 153 (1978) 365-372. 18 Oraedu, A. C. I., Voaden, M. J. and Marshall, J., Photochemical damage in the albino rat retina: morphological
170 changes and endogenous amino acids, J. Neurochem.. 35 (1980) 1361-1369. 19 Rapoport, S., Sites and functions of the blood-aqueous and the blood-vitreous barriers of the eye. In Blood-Brain Barrier in Physiology and Medicine, Raven, New York, 1976, pp. 207-232, 20 Sved, A. F. and Fernstrom, J. D., Tyrosine availability and dopamine synthesis in the striatum: studies with gamma-butyrolactone, Life Sci., 29 (1981) 743-748. 21 Trulson, M. E. and Himmel, C. D., Decreased brain dopamine synthesis rate and increased [3H]spiroperidol binding
in streptozotocin-diabetic rats. J. Neurochem., 4() (1983) 1456-1459. 22 Trulson, M. E. and MacKenzie, R. G., Increased tryptophan hydroxylase activity may compensate for decreased brain tryptophan levels in streptozotocin-diabetic rats. J. Pharmacol, exp. Ther., 212 (1980) 269-273. 23 Waalkes, T. P. and Udenfriend, S.. A fluorimetric method for the estimation of tyrosine in plasma and tissues, J. Lab. clin. Med., 50 (1957) 733-736. 24 Zivin, J. A. and Bartko, J. J., Statistics for disinterested scientists, Life Sci., 18 (1976) 15-26.