Localization of triiodothyronine in nerv

Proc Nat Acad Sci USA Vol 73, No 3, pp 941-944, March 1976 Medical Sciences Localization of triiodothyronine in nerve ending fractions of rat brain (thyroid hormones/adrenergic nervous system/synaptosomes/neurotransmitters/catecholamines) MARY B DRATMAN*, FLOY L CRUTCHFIELD*, JULIUS AXELRODt, ROBERT W COLBURNt, AND NGUYEN THOAt * Medical Service, Veterans Administration Hospital, Philadelphia, Pennsylvania 19104, and Department of Medicine, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129; t National Institute of Mental Health, Bethesda, Maryland 20014 Contributed by Julius Axelrod, January 5,1976 ABSTRACT Radioactive triiodothyronine reaching the rat brain after intravenous administration is rapidly and selectively taken up in the nerve ending fraction A concentration gradient of radioactivity from brain cytosol to synaptosomes is observed at min, increases linearly over the first hour, and is maintained for at least 10 hr Radioactivity in the synaptosomes is due to triiodothyronine (90%) plus a single unidentified metabolite (10%) Approximately 85% of the synaptosomal radioactivity is released by osmotic disruption of the particles The process of selective uptake, concentration, and retention of triiodothyronine in nerve terminals of the rat brain may be related to the sympathomimetic and behavior-altering effects of the thyroid hormones viously surgically thyroidectomized (Zivic-Miller Laboratories) Animals were provided with 4% calcium lactate in their drinking water To maintain the euthyroid state, they were given thyroxine, 15 gg/kg body weight daily, up to but not including the day of termination of the experiment Some experiments were performed in intact rats; results were no different from those in thyroidectomized euthyroid animals (unpublished observations) Approximately 50 jiCi of [125IIT3 labeled in the phenolic ring [obtained from B J Green of Abbotts Laboratories, specific activity approximately 500 jiCi/jig in 50% (vol/vol) propylene glycol] or 50% propylene glycol was administered as a single dose intravenously and animals were decapitated 5, 20, 60, 180, and 600 later Blood was collected from the decapitation site and the serum was separated and analyzed for radioactivity and radioactive iodocompounds Subcellular fractions of whole brain minus cerebellum were prepared according to the method of Whittaker et al (10) Briefly, following 1000 X g centrifugation of the brain homogenate for 10 min, nuclei and cellular debris were discarded, and the supernatant phase (Sl fraction) was layered on a discontinuous sucrose density gradient consisting of 1.2, 0.8, and 0.32 M sucrose, and centrifuged in a swinging bucket rotor at 50,000 X g for hr Individual gradient fractions including myelin, synaptosomes, and mitochondria were separated (see diagram, Table 1A) and diluted with 10-18 volumes of isotonic Krebs buffer (11), and pellets were separated by centrifugation at 20,000 X g for 20 To determine the extent of translocation of labeled T3 during the fractionation procedure, brains of animals which received intravenous propylene glycol without isotope were homogenized at 40 in sucrose containing 0.05 jgCi of [125I]T3 Approximately 75% of added ['25I]T3 was recovered in the cytosol (plus microsomes); the remainder was distributed among the various subcellular organelles as shown in Table 1B All brain fractions labeled in vivo were corrected for in vitro uptake at 40 Radioactivity in individual subcellular fractions and in serum was studied by means of paper chromatography in three solvent systems: butanol:ethanol:0.5 M ammonia, 5:1:2; butanol:acetic acid:water, 4:1:1; and tertiary amyl alcohol:2 M ammonia:hexane, 5:6:1 Added carrier compounds were identified by means of ultraviolet light at 259 nm After development, the radioactivity in each cm segment of the chromatogram was counted for at least 10 Thyroid hormones exert marked central stimulating and peripheral sympathomimetic effects, which are not explained by increased catecholamine production or enhanced adrenergic receptor sensitivity On the contrary, circulating levels of catecholamines (1, 2), turnover rates of noradrenaline in a number of tissues (3-5), and sensitivity of at least some adrenergic receptors (6-8) are reported to be inversely correlated with the thyroid state To explain their sympathomimetic actions, we have proposed that iodothyronines, like tyrosine and other tyrosine analogues, may be transformed to adrenergic neurotransmitters (9) The metabolic pathway leading from precursor amino acid to adrenergic neurotransmitter occurs within the nerve terminal Therefore, if iodothyronines enter this pathway, their uptake and metabolism in normally innervated tissues should be altered by adrenergic denervation To test this experimentally, endogenous triiodothyronine (T3) concentration and uptake of [125I]labeled T3 were compared in innervated and denervated salivary gland after unilateral superior cervical ganglionectomy The results demonstrated that adrenergic denervation significantly reduced the uptake and retention of T3 in the rat submaxillary salivary gland (M B Dratman, F L Crutchfield, J Axelrod, R D Utiger, and H Menduke, manuscript submitted) This evidence, while consistent with uptake and retention of T3 in adrenergic nerve terminals, nevertheless provided no direct information regarding this process Methods for isolating nerve terminal elements in salivary gland and other peripheral tissues are not available However, since enriched nerve ending preparations (synaptosomes) can be obtained by means of subcellular fractionation of brain (10), this method was used to examine rat brains after intravenous administration of [125I]T3 The results demonstrate that T3 is selectively taken up and retained within synaptosomes RESULTS METHODS Experiments were performed in 250 g adult male rats pre- Following intravenous administration of [125IIT3 to rats, levels of radioactivity in the serum decreased over the 10 hr period, whereas, the concentration in the brain increased, reaching a plateau after the first hour (Fig 1) Discontin- Abbreviations: T3, triiodothyronine; S1, supernatant phase of first centrifugation (1000 X g for 10 min) of rat brain homogenate 941 942 Medical Sciences: Dratman et al Proc Nat Acad Sci USA 73 (1976) Table Radioactivity from ['25I]T3 in subeellular A B Time after intravenous [ 25 I ] T3 Number of animals - -Cytosol (containing 0.32 M 65.5 ± 1.20 microsomes) Subcellular particles - ~~~~~~~~-myelin p/s 9.5 ± 0.55 0.8 M 3.05 ± 0.32 1.5 p/s synaptosomes 0- ," p/s 1.2 M l 6.85 ± 0.22 1.7 ± 0.06 -post synaptosomes p/s -m mtochondria p/s 1.9 ± 0.11 (A) Distribution of subcellular components on discontinuous sucrose gradient; M refers to sucrose concentration (B) Animals received only intravenous propylene glycol; S1 was derived from brains after homogenization at 40 in 0.32 M sucrose containing approximately 0.05 UCi of [125I]T3 (C) Animals received intravenous ['251]T3 and were decapitated at intervals as indicated; all values were corrected for con- uous sucrose density gradient separation of the Si fraction of brain showed that radioactivity from [125I]T3 was taken up into all subcellular particles (Table 1C and Fig 2) However, within and throughout the first hour after [125I]Ts, radioactivity associated with the nerve endings was more than 2-fold greater than that exhibited by any other particulate subcellular component, and was still more than 50% greater at the end of the hr period (Fig 2) Concentration of radioactivity in synaptosomes was calculated (11) and compared with concentration in the cytosol (see legend, Fig 3); a ratio of synaptosomal to cytosol radioactivity greater than one was observed at min; the ratio increased linearly over the first hour, and was maintained for at least 10 hr (Fig 3) No corrections were made for the presence of microsomal particles in the cytosol However, such corrections would 4,000r have increased further the cytosol to synaptosome concentration gradient To verify that the organelle identified as synaptosomes did, in fact, exhibit functional properties of nerve ending preparations, brain fractions from untreated animals were incubated in the presence of 0.4 ,uM [3H]norepinephrine The synaptosomal component behaved as a nerve ending preparation, exhibiting a highly temperature-iependent uptake of norepinephrine (40-fold increase in uptake at 370) To determine the identity of the radioactive compounds in individual subcellular components, suspensions of myelin, mitochondria, microsomes, and synaptosomes were applied to the origin of paper strips and the chromatograms were Serum _ 3,000 E a 2,000 f 1,0001 cm 70C) - Brain E E UL 50C Hours 30C I Z Hou rs 10 FIG Radioactivity in serum and brain after a single intravedose of [125IT3 Vertical bars indicate SEM nous FIG Radioactivity in subcellular particles of Si fraction of rat brain at various time intervals after intravenous [125IT3; fractions were separated according to diagram, Table 1; data are expressed as mean SEM (vertical bar); synaptosomes: 0; myelin: 0-0; post-myelin: A - A; post synaptosomes: A - A, mitochondria: X - -X | Medical Sciences: Dratman et al Proc Nat Acad Sci USA 73 (1976) 943 particles of Si fraction of rat brain homogenate 50.1 2.9 ± ± 0.86 0.71 3.9 ± 0.20 8.6 ±0.27 11 1.9 0.24 2.0 0.42 ± ± C 20 60 180 45.3 0.72 42.5 1.17 39.2 0.71 36.6 0.57 ± ± 5.7 600 ± ± 1.11 8.7 0.29 4.5 10.4 0.85 5.2 0.20 4.8 0.61 4.4 0.40 4.8 0.33 10.2 0.19 12 12.75 0.46 13 13.6 0.95 10 13.3 1.13 2.2 0.25 2.6 0.49 1.9 0.13 1.75 0.38 2.0 0.32 0.85 0.08 2.15 0.25 1.7 0.07 5.0 ± 1.23 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± centration of [125I]T3 in vitro at 4° as in (B) Data are expressed as mean %4o SEM of total cpm/mg of brain applied to gradient; p/s of particle-bound to nonparticle-bound radioactivity in individual fractions separated on gradient developed in three separate solvent systems At both and hr after [125I]T3, labeled T3 accounted for virtually all of the radioactivity associated with subcellular particles other than synaptosomes A second small peak of radioactivity was observed in chromatograms of the synaptosomal fraction, 26 24 c, - i 'D Ii I ratio amounting to approximately 10%, as compared with approximately 90% Ts Synaptosomal membranes rupture when subjected to hypotonic conditions, resulting in release of contained precursor amino acids and neurotransmitter vesicles (12) To determine whether osmotic disruption would release synaptosomal radioactivity derived from T3, portions of the nerve ending fraction were collected on Millipore filters; radioactivity on the filters and in the filtrate was measured following washing with either isoosmotic buffer or with water Synaptosomes exposed to hypoosmotic conditions lost 85% of their radioactivity as compared with samples treated with buffer (Table 2) Thus, [1251]T3 appears to be contained within, rather than attached to, the membranes of the synaptosomal particles 0 DISCUSSION The results of the present experiments demonstrate that the hormone triiodothyronine reaching the brain by way of the systemic circulation is differentially distributed among all C.) I4 l.2 = 20 Minutes 60 10 Hours FIG Concentration of radioactivity in synaptosomes relative to brain cytosol after intravenous [1251]T3, calculated as follows: cpm in synaptosomes/mg brain cpm in cytosol/mg brain 0J84 0.8 where brain density = 0.8 ml/g and synaptosomal density = 0.184 ml/g, derived from data in Fig of ref 11 The concentration gradient relationship to time is described by means of a least squares straight line, the slope of which is + 0.0116 concentration gradient units/min (r = 0.93) The relationship is not linear after the first hour; differences between 1, 3, and 10 hr ratios are not significant = mean value at each time iii * = ratio in individual animals; terval Table Effect of osmotic disruption on retention of synaptosomal radioactivity Conditions Experiment n 4 Isoosmotic Hypoosmotic Difference 941 ±15 144±22 898±30 134±13 -85 -84 Rats were given intravenous [1251]T3 and decapitated hr later Brains were homogenized and SI fractions were separated Portions (0.2 ml) of the synaptosomal fractions were applied to Millipore filters, pore size 0.8 Am, and each pellet was washed three times with ml portions of either isoosmotic buffer or water Data are expressed as mean cpm retained on Millipore filter + SEM 944 Proc Nat Acad Sci USA 73 (1976) Medical Sciences: Dratman et al the subcellular organelles, but is selectively taken up into the nerve ending (synaptosomal) fraction Particle-bound radioactivity is virtually all accounted for by T3, except for an additional unidentified metabolite in the synaptosomal fraction A concentration gradient of radioactivity from cytosol to synaptosomes is evident at min, increases linearly over the first hour, and is maintained for at least 10 hr after administration of labeled hormone More than 80% of the radioactivity in the synaptosomes is released by osmotic disruption of the particles These observations provide evidence that T3 is taken up, concentrated, and retained within nerve endings The presence of a metabolite of T3 detected only in the synaptosomes suggests that the hormone may be transformed within nerve terminals Although evidence derived from experiments with salivary gland suggests a role for T3 in peripheral adrenergic nerves, there is no information available from the present experiments regarding the nature of the nerve endings which concentrate the hormone in brain The aromatic amino acids, tyrosine and phenylalanine, are actively taken up into nerve terminals (13) and form a variety of adrenergic neurotransmitters including norepinephrine, dopamine, epinephrine, and also octopamine, phenylethylamine, and phenylethanolamine (14) Tyrosine analogues (e.g., a-methyl-m-tyrosine) are also concentrated in nerve endings, where they are converted to false adrenergic neurotransmitters (15) The possibility that iodothyronines may undergo analogous metabolic transformations, suggested by their aromatic amino-acid structure, their potent central and peripheral adrenergic effects, and the susceptibility of these effects to modification by sympatholytic drugs, is now given additional support by evidence that T3 is taken up, concentrated, retained, and probably metabolized within nerve terminals of the rat brain Christensen, N J (1973) "Plasma noradrenaline and adrena- 10 11 12 13 We thank Dr Francis H Sterling for his many contributions to this work and Mses Effie Erlichman and Bonni Wisdow for their expert technical assistance This work was supported by funds from the Medical Research Service, Veterans Administration Hospital, Philadelphia, Pa and National Institutes of Health Grant no AM16420 14 15 line in patients with thyrotoxicosis and myxoedema," CGn Sci Mol Med 45, 163-171 Stoffer, S S., Jaiang, N., Gorman, C & Pikler, M (1973) "Plasma catecholamines in hypothyroidism and hyperthyroidism," J Clin Endocrinol 36, 587-589 Landsberg, L & Axelrod, J (1968) "Influence of pituitary, thyroid and adrenal hormones on norepinephrine turnover and metabolism in the rat 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tyrosine," J Theor Biol 46, 255-270 Whittaker, V P., Michaelson, I A & Kirkland, R J A (1964) "The separation of synaptic vesicles from nerve-ending particles ('Synaptosomes')," Biochem J 90, 293-303 Colburn, R W., Goodwin, F K., Murphy, D L., Bunney, W E., Jr & Davis, J M (1968) "Quantitative studies of norepinephrine uptake by synaptosomes," Biochem Pharmacol 17, 957-964 Whittaker, V P (1965) "The application of subcellular fractionation techniques to the study of brain function," Prog Biophys 15, 39-96 Guroff, G (1972) in Basic Neurochemistry, eds Albers, R W., Siegel, G J., Katzman, R & Agranoff, B W (Little Brown & Co., Boston, Mass.), pp 197-198 Axelrod, J & Saavedera, J M (1974) "Aromatic amino acids in the brain," in Ciba Found Symp 22 (new ser.) (American Elsevier, New York), pp 51-59 Kopin, I J (1968) "False adrenergic transmitters," Annu Rev Pharmacol 8, 377-394  ... aromatic amino acids, tyrosine and phenylalanine, are actively taken up into nerve terminals (13) and form a variety of adrenergic neurotransmitters including norepinephrine, dopamine, epinephrine,... temperature-iependent uptake of norepinephrine (40-fold increase in uptake at 370) To determine the identity of the radioactive compounds in individual subcellular components, suspensions of myelin, mitochondria,... centration of [125I]T3 in vitro at 4° as in (B) Data are expressed as mean %4o SEM of total cpm/mg of brain applied to gradient; p/s of particle-bound to nonparticle-bound radioactivity in individual