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Calcium-sensitive accumulation of norepinephrine in rat cerebral cortex
European Journal of Pharmacology, 69 (1981) 301--312 © Elsevier/North-Holland Biomedical Press
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CALCIUM-SENSITIVE ACCUMULATION OF NOREPINEPHRINE IN RAT CEREBRAL CORTEX G. H O W A R D
BURROWS
*, M I C H A E L M. M Y E R S , S C O T T R. W H I T T E M O R E
and E D I T H D. H E N D L E Y
The Department of Physiology and Biophysics, The University of Vermont Medical School, Burlington, Vermont 05405, U.S.A. Received 11 September 1980, accepted 28 October 1980
G.H. BURROWS, M.M. MYERS, S.R. WHITTEMORE and E.D. HENDLEY, Calcium-sensitive accumulation of norepinephrine in rat cerebral cortex, European J. Pharmacol. 69 (1981) 301--312. The accumulation of high and low concentrations of [3H]l-norepinephrine has been examined in a crude synaptosomal preparation of rat cerebral cortex in the presence and absence of uptake1 inhibitors. When uptake1 was blocked, [3H]l-norepinephrine accumulation exhibited very rapid initial rates. It was not inhibited by 10 mM normetanephrine, a potent inhibitor of peripheral uptake2, but it was inhibited by 10 mM metaraminol. This accumulation was markedly reduced when calcium ions were omitted from the incubation medium, and is named here 'calcium-sensitive accumulation' (CSA) to distinguish it functionally from the sodium-dependent, high affinity, uptake1 process. CSA may be localized in nerve endings since it was found predominantly in the synaptosomal fraction of homogenates subjected to density gradient centrifugation in sucrose or in Ficoll-in-sucrose. At high concentrations of [3H]l-norepinephrine (1.0 pM) and short incubation times, CSA accounted for most of the total accumulation of [3H]l-norepinephrine whereas uptake1 contributed only a small portion. Since extracellular concentrations of brain norepinephrine are thought to reach levels in excess of 1.0 p_M, CSA may be a significant factor in noradrenergic neuronal transmission. Norepinephrine
Uptake I
Uptake2
Calcium
1. Introduction A major mechanism for the inactivation of the released neurotransmitter, norepinephrine (NE), is thought to be its accumulation into nerve endings (Iversen, 1975). A sodium
second type of uptake process which is observed at higher concentrations of NE (Natali et al., 1975; Wong and Bymaster, 1976). Hendley et al. (1970) described a high capacity, low affinity transport of [3H]normetanephrine, the characteristics of which resembled those of uptake2 in peripheral organs (Iversen, 1965; Paton, 1976b). However, the characteristics of a possible high capacity transport process for NE has not yet been adequately described in brain using NE, the natural substrate. Since high concentrations of NE (greater than 10 -6 M) are thought to occur in the synaptic cleft of noradrenergic synapses in the central nervous system (Hamberger, 1967; Stj~irne, 1975), an uptake process of greater capacity than uptake1 may be of significance
302
in the regulation of brain noradrenergic function. Accordingly, we have studied the characteristics of the accumulation of high concentrations of [3HI 1-NE in the presence and absence of inhibitors of uptake1. Data are presented to show that the accumulation of NE in the presence of inhibitors of uptake1 contrasts with uptake~ in that it exhibits more rapid initial rates, and it is n o t dependent on external sodium. In addition, this process is shown to differ from the peripheral uptake2 process thought to be primarily extraneuronal, in that it is markedly inhibited b y the removal of calcium from t h e medium, it is not blocked b y 10 mM normetanephrine, and it appears to be localized in the nerve ending fraction of cerebral cortex. Preliminary accounts of this work have appeared (Burrows et al., 1977, 1978).
2. Materials and m e t h o d s 2.1. Animals Male, Sprague-Dawley rats (Canadian Breeding Labs., a subsidiary of Charles River Breeding Labs., Inc) were used exclusively in these studies. B o d y weights ranged from 175 to 250 g. Animals were maintained on a 12 h light,lark cycle, and were killed b e t w e e n 900 and 1400 h.
G.H. B U R R O W S ET AL,
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2.2. Preparation o f tissue Rats were stunned b y a blow to the neck and shoulders and then quickly decapitated. The brain was removed and dissected on a chilled wax surface. The cerebral cortex, caudal to the level of the anterior commissure, was removed according to the procedure of Glowinski and Iversen (1966) and placed in 0.32 M sucrose on ice. 2.2.1. Homogenates Cerebral cortex was cleared of excess white matter, weighed and homogenized in ten vol of isotonic sucrose (0.32 M), using six strokes
The incubation medium used in these studies was a modified Krebs-Ringer solution consisting of NaC1 (140 mM), KC1 (5.0 mM), MgSO4 (2.0 mM), CaC12 (1.3 mM), EDTA (0.05 mg/ml), ascorbic acid (0.2 mg/ml), dextrose (2.0 mg/ml) and [3H]I-NE at various concentrations. A Tris-phosphate buffer (23 mM Tris base and 15 mM phosphoric acid) was added to maintain the pH at 7.4. Nialamide (10/~M) was included to inhibit NE degradation b y monoamine oxidase. [ 3H ] 1-NE accumulation, using this medium, was similar to that using phosphate-buffered or bicarbonate-buffered Krebs-Ringer solutions. [3H] 1-NE
CALCIUM-SENSITIVE ACCUMULATION OF NOREPINEPHRINE was added to the media immediately prior to incubation. When sodium-free medium was used, choline was substituted for sodium to maintain isotonicity and ionic strength. Calcium-free medium was prepared b y the omission of calcium chloride without replacement.
2.4. Measurement accumulation
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[3H]l-norepinephrine
Incubations were carried o u t at 31°C in a D u b n o f f metabolic shaker in nylon centrifugation tubes containing 2.0 ml of incubation medium and tissue homogenate derived from 10 mg of original wet weight of cerebral cortex. A temperature of 31°C was chosen in order to slow d o w n the initial linear portion of the uptake rates of CSA, and yet also maintain a temperature above the transition point (30°C) at which there is a break in the Arrhenius plot for catecholamine uptake in synaptosomes (Holz and Coyle, 1974}. The incubation was started b y the addition of tissue to the warmed media. At the end o f the incubation time, the reaction was terminated rapidly b y adding 10 ml of 4°C isotonic saline {0.15 M NaC1) to each t u b e and immersing the tubes in an ice water bath. Tubes were centrifuged at 2 4 0 0 0 × g for 10 min in a refrigerated centrifuge, the supernatant fluid was discarded, and the pellets were rinsed with 10 ml of isotonic saline, centrifuged for 10 additional min at 24 000 × g and the washings discarded. The tubes were inverted over absorbent paper for a minimum of 15 min, then [3H] 1-NE was extracted from the washed pellets using 6 ml o f Triton-toluene phosphor (Triton X-100 in toluene, 1 : 4, v/v, containing PPO, 5.9 g/l, POPOP, 0.13 g/l} and distilled water, 0.2 ml. Tritium was measured in a Packard Tricarb liquid scintillation spectrometer (Model 3385, Packard Instruments Corp.) at 45% efficiency.
2.5. Thin layer chromatography Thin layer chromatography was used to determine whether the tritium accumulated in
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the tissues represented unmetabolized NE. Cellulose F2s4 plates (E2¢I. Labs., Inc. Merck and Co., Elmsford, N.Y.) were used with a solvent system of butanol • acetic acid : water, 84 : 21 : 35. Following incubation pel. leted tissue samples were homogenized in 70% ethanol in ground glass homogenizers. After centrifugation the supernatant fractions were evaporated to dryness under N2 gas and the residue was taken up in 70% ethanol for spotting on plates with 25 pg of authentic 1-NE. Strips of 0.5 cm height between the origin and the solvent front were counted in Triton phosphor; authentic NE had an R~ of 0.49.
2.6. Subcellular localization of accumulated [3HI l-norepinephrine Cerebral cortex was chopped into slices o f 1.0 × 1.0 X 2.0 mm using the McIlwain Tissue Chopper. The slices were incubated at 31°C for 10 min in standard medium containing 1.0 pM [3H] 1-NE, nialamide (10 #M), and in the presence or absence of cocaine (10 #M). Following incubation the slices were pelleted and homogenized in 0.32 M sucrose for preparation o f a crude synaptosomal pellet (P2). P2 was resuspended in isotonic sucrose, then layered over a continuous sucrose density gradient, 0.32--1.5 M, and centrifuged for one hour at 130 000 X g. A density gradient fractionator (Instrument Specialities Co.) was used to collect 0.8 ml fractions o f the gradient. These were counted in Trition-toluene phosphor.
2.7. Drugs and chemicals [3H]l-NE (2--22 Ci/mmol) was obtained from New England Nuclear Corp. (Boston, MA). Its purity was verified monthly using thin layer chromatography on cellulose F2s 4 plates and a solvent system of butanol : acetic acid : water, 60 : 15 : 25. The following firms generously donated these chemicals: cocaine HC1 and metaraminol bitartrate from Merck, Sharpe and Dohme Research Labs. (Rahway, NJ); des-
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methylimipramine HC1 from USV Pharmaceutical Corp. (Tuckahoe, NY); nialamide from Pfizer and Co. (Brooklyn, NY);1-Norepinephrine HC1, dl-normetanephrine HC1 and Ficoll were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals and reagents were of reagent grade.
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3. Results The sodium-dependent, high affinity uptake of NE (uptake1) was used throughout this study for purposes of comparison in describing a second accumulation process for NE that i s observed when uptake1 is completely blocked b y either cocaine (10 pM), DMI (0.1 pM) or by substitution of sodium with choline. Because of the marked calciumsensitivity of this second accumulation process, we have referred to it as CSA (calciumsensitive accumulation).
3.1. Time course of [3H]I-NE accumulation by uptake~ and by CSA Homogenates of rat cerebral cortex were incubated in 2.0 ml o f standard incubation medium at 31°C for varying time periods, with and w i t h o u t the addition of cocaine, 10 pM, a concentration which inhibits uptake~ b y more than 90% (data n o t shown). Uptake~ was calculated as total [3H]I-NE accumulation, measured w i t h o u t cocaine, minus that obtained in the presence of cocaine. CSA was taken directly as the accumulation in the presence of cocaine. The time course for uptake~ and for CSA was determined at low (0.025 p_M) and high (1.0 #M) concentrations o f [3H] 1-NE (fig. 1). At b o t h concentrations, uptake~ was linear for at least 5 min, however, CSA began to approach a steady state after 2 min. At the low concentration of [3H] 1-NE (fig. 1A) the relative amounts of NE accumulation due to uptake~ were greater than the amounts due to CSA, except at the earliest time period when t h e y were equal. In contrast, at the higher
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concentration (fig. 1B) accumulation due to CSA was greater than that due to uptake~ at all time periods measured.
3.2. Effects o f calcium on uptakel and CSA Cerebral cortical homogenates were incubated for various time periods with and witho u t cocaine (10 pM), in either standard incubation medium or in calcium-free medium. Uptake1 and CSA were determined at b o t h
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Fig. 2. Effect of calcium on the time course of accumulation of [3H]I-NE by uptakel in cerebral cortical homogenates. In (A) incubations were carried out in 0.025 #M and in (B) in 1.0 #M concentrations of [3H]I-NE at 31°C. Nialamide was present at 10 #M. Solid lines refer to accumulation in standard medium containing 1.3 mM calcium. Dashed lines refer to accumulation in medium in which calcium was omitted. Uptake1 was taken as total accumulation minus that in the presence of 10 #M cocaine. Each point is the mean -+ S.E. of 4 separate determinations, each in duplicate.
Fig. 3. Effect of calcium on the time course of calcium-sensitive accumulation (CSA) of [3H]I-NE at 31°C in cerebral cortical homogenates. Nialamide was present at 10 #M and cocaine at 10 #M. In (A) incubations were carried out in 0.025 #M and in (B) in 1.0 #M concentrations of [3H]I-NE. Solid lines refer to accumulation in standard medium containing 1.3 mM calcium. Dashed lines refer to accumulation in medium in which calcium was omitted. Each point is the mean -+ S.E. of 4 separate determinations, each in duplicate.
high (1.0 #M) and low (0.025 #M) concentrations of [3H] I-NE. As expected from numerous other studies (reviewed in Paton, 1976a), the omission of calcium had no effect on uptakel (fig.2). In contrast, C S A was markedly reduced in the absence of calcium (fig. 3). At one rain of incubation time the accumulation in the presence of cocaine and
0.025 pM [3H] 1-NE (fig. 3A) was inhibited by 65% when calcium was omitted, and at 1.0 #M [3H]I-NE (fig. 3B) by 72%. This calcium sensitivity was noted at all times tested and at both high and low concentrations of [SH]INE. The calcium dependency of CSA was also observed when the high affinity uptake of NE
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INCUBATION TIME (min) Fig. 4. Time course of d e s m e t h y l i m i p r a m i n e (DMI)resistant accumulation o f [3H ]l-norepinephrine, effect of calcium omission. Cerebral cortical h o m o g enates were incubated at 3 1 ° C in standard i n c u b a t i o n m e d i u m containing 1.0 pM [3H]I-NE, 10 pM nialamide and 0.1 pM DMI. The dashed line refers to the total a c c u m u l a t i o n in the absence of c a l c i u m ; the solid line refers to the a c c u m u l a t i o n in 1.3 m M calcium. Each p o i n t is the mean-+ S.E. o f 4 separate experiments, each in duplicate.
was blocked b y DMI rather than cocaine (fig. 4). At 0.1 pM, DMI is a specific, maximaUy effective inhibitor of uptake, in noradrenergic neurons of the rat brain (Koe, ! 9 7 6 ) , as was also confirmed in our labora-
tory (data not shown). The time course for uptake of 1.0 #M [3H] 1-NE in the presence of DMI was similar to that observed using 10 #M cocaine (fig. 3B), and calcium omission resulted in a similar decrease in [3H]I-NE accumulation. This was further demonstrated in table 1 in which the one min accumulation o f [3H] 1-NE b y CSA was decreased to approximately 50% of controls b y calcium omission, whether DMI, choline replacement for sodium, or both were used to define CSA. 3.3. Identification o f the radioactivity accumulated by CSA Thin layer chromatography (TLC) was used in order to determine whether the tritium accumulated b y CSA was associated with unmetabolized NE. Homogenates were incubated for 5 min at 31°C in standard medium containing [3H]I-NE (1.0 pM), cocaine (10 pM) and nialamide (10 pM). Following incubation the rinsed pellets were extracted in 70% ethanol and spotted on TLC plates as described in Section 2.5. Under these conditions for CSA, 96% of the total radioactivity in the tissue extracts migrated with authentic NE, indicating no appreciable metabolism of the NE accumulated b y CSA. When calcium
TABLE 1 Effects of calcium on initial rates of [3H]l-norepinephrine a c c u m u l a t i o n in cerebral cortical h o m o g e n a t e s where u p t a k e was b l o c k e d either by d e s m e t h y l i m i p r a m i n e or by sodium replacement. Incubation conditions I
0.1/~M DMI 2 No s o d i u m 3 No s o d i u m + 0.1/~M DMI
[ 3H ]l-Norepinephrine a c c u m u l a t i o n , pmol/g 1.3 mM calcium
No calcium
167 -+ 30.0 202 -+ 20.8 192 -+ 21.9
71.5 -+ 36.8 97.8 -+ 18.3 112 + 22.4
E f f e c t of calcium omission % decrease
57 52 42
: I n c u b a t i o n s were carried o u t for one rain at 31°C in standard m e d i u m in the presence of 1.0/~M [3H]I-NE and 10 ~M nialamide. Values are the m e a n + S.E. o f 4 experiments. In each e x p e r i m e n t the m e d i a n of triplicate determinations was used. 2 Conditions as in (1) e x c e p t 0.1/~M d e s m e t h y l i m i p r a m i n e was added to the m e d i u m . 3 Conditions as in (1) e x c e p t NaC1 was replaced by choline chloride. Analysis of variance indicated no difference a m o n g the 3 i n c u b a t i o n c o n d i t i o n s ; however, the inhibition o f a c c u m u l a t i o n due to calcium omission was highly significant for all c o n d i t i o n s (P < 0.001).
CALCIUM-SENSITIVE ACCUMULATIONOF NOREPINEPHRINE was omitted from the incubation medium and all other conditions remained the same, 83% of the tritium accumulated in the tissues in 5 min was associated with unchanged NE. The higher percentage of unmetabolized NE in the samples incubated with calcium may reflect inhibition of catechol-O-methyltransferase (COMT) by calcium (Weinshilboum and R a y m o n d , 1976).
3.4. Effects o f normetanephrine and metaraminol on CSA In the perfused rat heart, metaraminol was one of the most p o t e n t inhibitors of uptakel and one of the least potent inhibitors of uptake2. Conversely, normetanephrine was a powerful inhibitor o f uptake2 with little effect on uptake1 (Iversen, 1965). Accordingly, these compounds were tested as possible inhibitors of the initial rate of accumulation o f [3H] 1-NE by CSA. Homogenates were incubated for one min at 31°C in the presence of 0.4 #M [3H]I-NE, 10 pM nialamide and 10 pM cocaine (table 2). In contrast with uptake2 in heart, CSA in cortex was n o t affected by normetanephrine at concentrations as high as 10 mM, yet metaraminol, 10 mM, inhibited CSA by more than 80%. These data suggested
307
that while CSA exhibited some of the characteristics of uptake2 in rat heart, it differed markedly from uptake2 with respect to competition by these analogs o f phenethylamine. Further evidence o f differences from uptake2 was observed in preliminary experiments (data not shown) in which other compounds known to have high affinity for uptake2 had no effects on CSA. Some of these compounds included the steroids, corticosterone and estradiol, and the phenoxybenzamine analogs, SKF 625a, 784a and 550 (Iversen and Salt, 1970; Iversen et al., 1972).
3.5. Subcellular localization o f CSA in cerebral cortical tissue The subcellular localization of CSA was determined on continuous sucrose density gradients. Slices of cerebral cortex (1 X 1 × 2 mm) were incubated for 10 min at 31°C in standard incubation medium containing 6.0 Z
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nialamide and 10/~M cocaine. Values are the percent inhibition o f [3H]l-NE-accumulation + S.E. determinations, each in triplicate.
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SUCROSE MOLARITY Fig. 5. Subcellular distribution of [3H]I-NE in cerebral cortical homogenates after accumulation in the presence (dashed line) and absence (solid line) of. cocaine, 10 pM. Slices o f rat cerebral cortex were incubated for 10 min at 31°C in the presence o f 1.0/~M [3H]I-NE, 10 #M nialamide and with or without 10 pM cocaine. Tissue was then homogenized and resuspended P2 was layered over a continuous sucrose density gradient and centrifuged for one h at 130 000 g. Each point is the mean o f duplicates taken in each 0.8 ml fraction o f the gradient. These data are the results o f one o f three similar experiments.
308
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1.0 pM [3H] 1-NE and 10 pM nialamide. In a single experiment slices were incubated b o t h with and without 10 pM cocaine. Following incubation homogenates were prepared and resuspended P2 was layered over continuous sucrose density gradients as described in Section 2. 6. The radioactivity in 0.8 ml fractions of the gradients is shown in fig. 5. It was found that in either the presence or absence of cocaine, a single peak of radioactivity appeared in that portion of the gradient (near 1.0 M sucrose) associated with synaptosomal markers (Coyle and Snyder, 1969). A synaptosomal site for CSA was further suggested b y comparing CSA and its calcium sensitivity in three different preparations of cerebral cortical tissue, in which the nerve endings were present in intact slices (0.2 × 0.2 × 0.4 mm), or enriched as in crude homogenates, or highly enriched as in purified synaptosomes isolated b y the m e t h o d of Morgan et al. (1971). Each t y p e of tissue preparation was incubated for one min at 31°C in standard incubation medium containing [3H]I-NE, 1.0 pM, nialamide, 10 pM and cocaine, 10 pM. In each experiment the incubations were carried o u t in b o t h the presence and absence of 1.3 mM calcium. The results in table 3 indicated that in the absence of cal-
cium, CSA, expressed per mg protein, was increased about 15-fold as the synaptosomal content of the preparation was enriched from intact slices to purified synaptosomes. In the presence of calcium, CSA increased over 90fold when the nerve ending content of the preparation increased. The effectiveness of added calcium in enhancing CSA increased from 34% in slices to nearly 600% above nocalcium controls in purified synaptosomes. These results support the previous findings that CSA in rat cerebral cortex is localized in the nerve endings, or synaptosomal fraction of the tissue.
4. Discussion The accumulation of NE in the cerebral cortex is the result of at least two processes. At low concentrations o f NE (at or below the apparent Kin), most of the accumulation is due to uptake, which can be blocked to the same maximal extent by 10 pM cocaine or 0.1 pM DMI, or when sodium is replaced by choline. At higher concentrations of NE, this high affinity, low capacity, sodium
TABLE 3 E n h a n c e m e n t o f CSA b y c a l c i u m a n d b y e n r i c h m e n t o f s y n a p t o s o m a l c o n t e n t . Slices, h o m o g e n a t e s o r p u r i f i e d s y n a p t o s o m e s o f r a t c e r e b r a l c o r t e x were i n c u b a t e d for o n e rain a t 31°C in s t a n d a r d i n c u b a t i o n m e d i a c o n t a i n i n g [3H]I-NE, 1.0 pM, n i a l a m i d e , 10 pM, a n d cocaine, 10/~M. I n each e x p e r i m e n t t h e m e d i a c o n t a i n e d e i t h e r 1.3 m M or zero c a l c i u m . P r o t e i n was d e t e r m i n e d b y t h e m e t h o d o f B r a d f o r d ( 1 9 7 6 ) . E a c h value is t h e m e a n -+ S.E. o r 3 d e t e r m i n a t i o n s , each in d u p l i c a t e . Tissue p r e p a r a t i o n
Slices 1 Homogenates 2 Purified synaptosomes 3
Calcium-sensitive a c c u m u l a t i o n o f [3H]I-NE ( p m o l / m g p r o t e i n ) No c a l c i u m
1.3 m M c a l c i u m
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1.1 -+ 0.11 17.0 + 1.00 97.0 + 32.0
Effect of calcium p e r c e n t increase
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I M c I l w a i n slices, 0.2 × 0.2 × 0.4 ram. 2 Tissue h o m o g e n i z e d in 0 . 3 2 M s u c r o s e a n d c e n t r i f u g e d for 10 rain at 1 0 0 0 X g; t h e p e l l e t was d i s c a r d e d a n d t h e supernatant was t a k e n as a c r u d e s y n a p t o s o m a l p r e p a r a t i o n . 3 P u r i f i e d s y n a p t o s o m e s o b t a i n e d f r o m t h e 12% Ficoll layer o f t h e d i s c o n t i n u o u s Ficoll-in-sucrose g r a d i e n t ( M o r g a n e t al., 1 9 7 1 ) .
CALCIUM-SENSITIVEACCUMULATIONOF NOREPINEPHRINE high capacity, calcium-sensitive accumulation. This paper describes for the first time some of the characteristics of CSA in the rat brain using the natural substrate, NE. Some of the characteristics of CSA are similar to those of uptake: in peripheral organs (summarized in Hendley, 1976), and to the accumulation of [3H] normetanephrine in cerebral cortex (Hendley et al., 1970). Thus, CSA has a high capacity and rapid time course, and lacks stereoselectivity towards the d- and 1-isomers of NE (Burrows, 1979). However, other characteristics of CSA differ markedly from those of uptake2 in peripheral organs. For example, initial accumulation rates for CSA are less sensitive to inhibition by normetanephrine than by metaraminol. In addition, CSA is not altered by phenoxybenzamine analogs, nor by the steroid hormones (Burrows, 1979). Furthermore, CSA appears to be associated with the nerve ending fraction of rat cerebral cortex, which suggests that CSA is intraneuronal in brain tissue as opposed to the presumed extraneuronal localization of uptake2 in rat heart (l~hinger and Sporrong, 1968). Regarding the proposed intraneuronal localization for CSA in rat brain, there is also evidence for an intraneuronal component of uptake2 in rat heart, despite the generally held view that uptake2 is extraneuronal (Clarke and Jones, 1969). For example, Iversen {1965) reported that uptake2 was decreased following immunosympathectomy of the rat heart. Later, Salt and Iversen (1973) provided further evidence for decreased uptake2 following chemical sympathectomy with 6-hydroxydopamine pretreatment. Accordingly, an intraneuronal localization for CSA in rat brain may have its peripheral counterpart in the sympathetic nerves of the rat heart as well. In the present study, the question of a possible glial localization for CSA cannot be excluded, as 'gliasomes' can be formed and carried along with synaptosomes during subcellular fractionation procedures using either sucrose or Ficoll gradient separation tech-
309
niques (Henn, 1976). The question concerning whether CSA is confined exclusively to noradrenergic nerve terminals or not has not been dealt with in this study. This question would best be answered using cerebral cortex from locus coeruleus lesioned animals. However, it does seem probable that CSA is located primarily in noradrenergic nerve terminals, in view of the histochemical studies of HSkfelt and Ljungdahl (1972), Hosli et al. (1975) and of Hosli and Hosli (1976). In each of these studies, rat brains were incubated in the presence of 1.0 p_M NE and the distribution of exogenous NE in the tissues was compared with the normal distribution of endogenous NE in untreated tissues. After exposure to exogenous NE for even as brief a time as 30 sec, at which time CSA should account for the majority of the NE accumulation, the NE fluorescence was localized uniquely in a limited number of small varicosities, in a distribution pattern similar to that expected for noradrenergic nerve terminals. Another unresolved question in the present study concerns the possibility that [3H] 1-NE accumulated by CSA represents homoexchange of tritiated NE with endogenous stores of unlabeled NE, and thus may not represent a net accumulation of amine. One way to test for homoexchange would be to prelabel the endogenous stores of synaptosomes with [3H]-NE in the presence of a monoamine oxidase inhibitor, and then expose the synaptosomes to a high concentration of unlabeled NE, in the presence of calcium and either cocaine or DMI, and to note whether the accumulation of unlabeled NE, presumably by the process of CSA, displaced any of the labeled NE into the medium. While this experiment was not performed in the present study, Raiteri et al. (1977) carried out a very similar experiment in synaptosomes of rat hypothalamus and reported no evidence at all of displacement of labeled NE by 10 pM unlabeled NE and 1.0 #M DMI. While these findings suggest that CSA involves a net accumulation and not a homoexchange, the experiment should be verified using a highly puri-
310
fied preparation of cerebral cortical synaptosomes. A striking characteristic of CSA in the central nervous system is that it is markedly reduced when calcium is omitted from the incubation medium. This sensitivity to calcium was observed when either cocaine, DMI, or sodium replacement with choline were used to inhibit uptake1. These findings contrast with those of uptake2 in the rabbit ear artery which was unaffected b y removal of calcium (Gillespie and Towart, 1973). On the other hand, in the cocainized rat heart perfused with 1.0 pM [3H] 1-NE, omission of calcium in the perfusate resulted in a significant reduction of uptake2 (Mekanontchai and Trendelenburg, 1979), suggesting a possible role for calcium ions in uptake2 in the rat heart as well as for CSA in rat cerebral cortex. The concentration of calcium ions in the extracellular fluid o f cat cerebral cortex has been measured b y calcium electrode and found to be decreased markedly, from a normal level of 1.3 mM to as low as 0.8 mM, as a result o f repetitive stimulation of the tissue (Heineman et al., 1977). This decrease could serve to inhibit the removal of NE from the synaptic cleft region via the process of CSA, and thereby prolong the time that released NE would remain in the cleft for activation of postsynaptic receptors under conditions of increased nerve activity. The mechanism b y which calcium increases CSA remains unexplored. We have noted, however, that omission of magnesium in the incubation medium failed to alter CSA, and furthermore, that magnesium ions failed to substitute for calcium in stimulating CSA in homogenates of cortex (Burrows, 1979), suggesting a specificity for calcium in this process. By virtue of its rapid rate and high capacity for transport o f NE, CSA may have a physiological role in removal o f NE from the synaptic cleft following its release. Direct measurements o f NE concentration in the cleft region immediately following NE release have not been reported. However, it can be assumed
G.H. B U R R O W S E T AL.
that the concentration is highest at the site of its release from nerve terminals. Phillips and Apps {1979) estimated that the concentration of NE in synaptic vesicles is millimolar or higher in magnitude, suggesting that during exocytosis the concentration of NE in the immediate region of the presynaptic nerve terminal far exceeds the capacity for removal b y uptake,. Accordingly, it may be hypothesized that a more rapid and higher capacity uptake process, such as CSA, is required for the efficient removal of the high concentrations o f NE presumed to occur locally in the extracellular space nearest the noradrenergic nerve terminal following release. Furthermore, concentrations of NE in the extracellular spaces more distant from the immediate site of release are estimated to be micromolar or more in concentration (Stj~irne, 1975; Hamberger, 1967), suggesting that removal of NE by CSA is also feasible at regions more distant from the site of release. Another possible role for CSA in the brain may be that of modifying the actions of some psychoactive drugs. For example, Y a m a m o t o et al. (1978) reported that morphine injection markedly altered calcium content and binding of calcium to synaptosomal plasma membranes in the rat brain. By altering calcium availability one might predict that morphine alters the rate of removal of NE during noradrenergic nerve stimulation. In addition, CSA may be involved in the pharmacological effects of amphetamine administration, in view of the findings of Cho et al. {1977) that parahydroxyamphetamine, a major metabolic of amphetamine, is accumulated in rat brain by means of a transport process that is strikingly similar to that of CSA for NE as described in this study. These similarities suggest that parahydroxyamphetamine may compete with NE for transport via CSA, although this compound has n o t been directly tested as a competitive inhibitor of NE for accumulation by CSA. The calcium
CALCIUM-SENSITIVE ACCUMULATION OF NOREPINEPHRINE cellular c o n c e n t r a t i o n s o f t h e s e c a t i o n s d u r i n g nerve s t i m u l a t i o n m a y regulate t h e relative p r o p o r t i o n s o f uptake1 a n d C S A m e c h a n i s m s used f r o m m o m e n t t o m o m e n t f o r r e m o v a l o f NE f r o m t h e s y n a p s e d u r i n g nerve stimulation.
Acknowledgement Supported by a grant from the US Public Health Service, MH-25811.
References Bradford, M.M., 1976, A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 71,248. Burrows, G.H., 1979, Comparison of cocaine-sensitive and cocaine-insensitive accumulation of norepinephrine in rat cerebral cortex, Ph.D. Thesis, Univ. of Vermont, Dissertation Abst. International, Sect. B 39, 4202. Burrows, G.H., M.M. Myers and E.D. Hendley, 1977, Cocaine~ensitive and cocaine-insensitive l-norepinephrine uptake in rat cerebral cortex,tFed. Proc. 36,381. Burrows, G.H., M.M. Myers and E.D. Hendley, 1978, Characteristics of cocaine-resistant accumulation in rat cerebral cortex, Neurosci. Abstr. 4,268. Cho, A.K., J.F. Fischer and J.C. Schaeffer, 1977, The accumulation of p-hydroxyamphetamine by brain homogenates and its role in the release of catecholamines, Biochem. Pharmacol. 26, 1367. Clarke, D.E. and C.J. Jones, 1969, Are adrenergic nerves required for uptake2 in the isolated perfused rat heart?, European J. Pharmacol. 7,121. Coyle, J.T. and S.H. Snyder, 1969, Catecholamine uptake by synaptosomes in homogenates of rat brain: Stereospecificity in different areas, J. Pharmacol. Exp. Ther. 170,221. Ehinger, B. and B. Sporrong, 1968, Neuronal and extraneuronai localization of noradrenaline in the rat heart after perfusion at high concentration, Experientia 24, 265. Gillespie, J.S. and R. Towart, 1973, Uptake kinetics and ion requirements for extraneuronai uptake of noradrenaline by arterial smooth muscle and collagen, Br. J. Pharmacol. 47,556. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain. I. The disposition of [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem. 13,655.
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Hamberger, B., 1967, Reserpine-resistant uptake of catecholamines in isolated tissues of the rat, Acta Physiol. Scand. Suppl. 295, 1. Heineman, U., H.D. Lux and M.J. Gutnick, 1977, Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat, Exp. Brain Res. 27,237. Hendley, E.D., 1976, The mechanism of extraneuronal transport of catecholamines in the central nervous system, in: The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines, ed. D.M. Paton (Raven Press, New York) p. 313. Hendley, E.D., K.M. Taylor and S.H. Snyder, 1970, 3H-Normetanephrine uptake in rat brain slices. Relationship to extraneuronal accumulation of norepinephrine, European J. Pharmacol. 12,167. Henn, F.A., 1976, Neurotransmission and glial cells: A functional relationship?, J. Neurosci. Res. 2, 271. HSkfelt, T. and A. Ljungdahl, 1972, Application of cytochemicai techniques to the study of suspected transmitter substance in the nervous system, in Adv. Biochem. Psychopharmacology, Vol. 6, eds. E. Costa, L.L. Iversen and R. Paoletti (Raven Press, New York) p. 1. Holz, R.W. and J.T. Coyle, 1974, The effects of various salts, temperature, and the alkaloids veratridine and batrachotoxin on the uptake of [ 3H ]dopamine into synaptosomes from rat striatum, Mol. • Pharmacol. 10,746. Hosli, E., U.M. Bucher and L. Hosli, 1975, Uptake of 3H-noradrenaline and SH-5-hydroxytryptamine in cultured rat brainstem, Experientia 31,354. Hosli, E. and L. Hosli, 1976, Autoradiographic studies on the uptake of 3H-noradrenaline and SHGABA in cultured rat cerebellum, Exp. Brain Res. 26,319. Iversen, L.L., 1965, The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: A novel catechol amine uptake process, Br. J. Pharmacol. 25, 18. Iversen, L.L., 1975, Uptake processes for biogenic amines, in: Handbook of Psychopharmacology, Vol. 3, eds. L.L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p. 381. Iversen, L.L. and P.J. Salt, 1970, Inhibition of catecholamine uptake2 by steroids in the isolated rat heart, Br. J. Pharmacol. 40, 528. Iversen, L.L., P.J. Salt and H.A. Wilson, 1972, Inhibition of catechol amine uptake in the isolated rat heart by haloaikylamines related to phenoxybenzamine, Br. J. Pharmacol. 46, 647. Koe, K., 1976, Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain, J. Pharmacol. Exp. Ther. 199,649. Mekanontchai, R. and U. Trendelenburg, 1979, The
312 neuronal and extraneuronal distribution of 3H-(--)noradrenaline in the perfused rat heart, NaunynSchmiedeb. Arch. Pharmacol. 308, 199. Morgan, I.G., L.S. Wolfe, P. Mandel and G. Gombos, 1971, Isolation of plasma membranes from rat brain, Biochim. Biophys. Acta 2 4 1 , 7 3 7 . Natali, J.P., P. Joanny and F. Blanquet, 1975, Cerebral uptake of noradrenaline in v i t r o ; o c c u r r e n c e of different uptake systems and effect of partial external sodium substitution, Experientia 3 1 , 6 3 2 . Paton, D.M., 1976a, Characteristics of uptake of noradrenaline by adrenergic neurons, in: The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines, ed. D.M. Paton (Raven Press, New York) p. 49. Paton, D.M., 1976b, ed. The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines (Raven Press, New York). Phillips, J.H. and D.K. Apps, 1979, Storage and secretion of catecholamines: The adrenal medulla, in: Physiological and Pharmacological Biochemistry, vol. 26, Int. Rev. Biochem., ed. K.F. Tipton (University Park Press, Baltimore) p. 121. Raiteri, M., R. del Carmine, A. Bertollini and G. Levi, 1977, Effect of desmethylimipramine on the
G.H. BURROWS ET AL. release of [~H]norepinephrine induced by various agents in hypothalamic synaptosomes, Mol. Pharmacol. 1 3 , 7 4 6 . Salt, P.J. and L.L. Iversen, 1973, Catecholamine uptake sites in the rat heart after 6-hydroxydopamine treatment and in a genetically hypertensive strain, Naunyn-Schmiedeb. Arch. Pharmacol. 279, 381. Stj~/rne, L., 1975, Basic mechanisms and local feedback control of secretion of adrenergic and cholinergic neurotransmitters, in: Handbook of Psychopharmacology, Vol. 6, eds. L.L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p, 179. Weinshilboum, R.M. and F.A. Raymond, 1976, Calcium inhibition of rat liver catechol-O-methyltransferase, Biochem. Pharmacol. 25,573. Wong, D.T. and F.P. Bymaster, 1976, Effect of nisoxetine on uptake of catecholamines in synaptosomes isolated from discrete regions of rat brain, Biochem. Pharmacol. 25, 1979. Yamamoto, H., R.A. Harris, H.H. Loh and E.L. Way, 1978, Effects of acute and chronic morphine treatments on calcium localization and binding in the brain, J. Pharmacol. Exp. Ther. 205, 255.
301
CALCIUM-SENSITIVE ACCUMULATION OF NOREPINEPHRINE IN RAT CEREBRAL CORTEX G. H O W A R D
BURROWS
*, M I C H A E L M. M Y E R S , S C O T T R. W H I T T E M O R E
and E D I T H D. H E N D L E Y
The Department of Physiology and Biophysics, The University of Vermont Medical School, Burlington, Vermont 05405, U.S.A. Received 11 September 1980, accepted 28 October 1980
G.H. BURROWS, M.M. MYERS, S.R. WHITTEMORE and E.D. HENDLEY, Calcium-sensitive accumulation of norepinephrine in rat cerebral cortex, European J. Pharmacol. 69 (1981) 301--312. The accumulation of high and low concentrations of [3H]l-norepinephrine has been examined in a crude synaptosomal preparation of rat cerebral cortex in the presence and absence of uptake1 inhibitors. When uptake1 was blocked, [3H]l-norepinephrine accumulation exhibited very rapid initial rates. It was not inhibited by 10 mM normetanephrine, a potent inhibitor of peripheral uptake2, but it was inhibited by 10 mM metaraminol. This accumulation was markedly reduced when calcium ions were omitted from the incubation medium, and is named here 'calcium-sensitive accumulation' (CSA) to distinguish it functionally from the sodium-dependent, high affinity, uptake1 process. CSA may be localized in nerve endings since it was found predominantly in the synaptosomal fraction of homogenates subjected to density gradient centrifugation in sucrose or in Ficoll-in-sucrose. At high concentrations of [3H]l-norepinephrine (1.0 pM) and short incubation times, CSA accounted for most of the total accumulation of [3H]l-norepinephrine whereas uptake1 contributed only a small portion. Since extracellular concentrations of brain norepinephrine are thought to reach levels in excess of 1.0 p_M, CSA may be a significant factor in noradrenergic neuronal transmission. Norepinephrine
Uptake I
Uptake2
Calcium
1. Introduction A major mechanism for the inactivation of the released neurotransmitter, norepinephrine (NE), is thought to be its accumulation into nerve endings (Iversen, 1975). A sodium
second type of uptake process which is observed at higher concentrations of NE (Natali et al., 1975; Wong and Bymaster, 1976). Hendley et al. (1970) described a high capacity, low affinity transport of [3H]normetanephrine, the characteristics of which resembled those of uptake2 in peripheral organs (Iversen, 1965; Paton, 1976b). However, the characteristics of a possible high capacity transport process for NE has not yet been adequately described in brain using NE, the natural substrate. Since high concentrations of NE (greater than 10 -6 M) are thought to occur in the synaptic cleft of noradrenergic synapses in the central nervous system (Hamberger, 1967; Stj~irne, 1975), an uptake process of greater capacity than uptake1 may be of significance
302
in the regulation of brain noradrenergic function. Accordingly, we have studied the characteristics of the accumulation of high concentrations of [3HI 1-NE in the presence and absence of inhibitors of uptake1. Data are presented to show that the accumulation of NE in the presence of inhibitors of uptake1 contrasts with uptake~ in that it exhibits more rapid initial rates, and it is n o t dependent on external sodium. In addition, this process is shown to differ from the peripheral uptake2 process thought to be primarily extraneuronal, in that it is markedly inhibited b y the removal of calcium from t h e medium, it is not blocked b y 10 mM normetanephrine, and it appears to be localized in the nerve ending fraction of cerebral cortex. Preliminary accounts of this work have appeared (Burrows et al., 1977, 1978).
2. Materials and m e t h o d s 2.1. Animals Male, Sprague-Dawley rats (Canadian Breeding Labs., a subsidiary of Charles River Breeding Labs., Inc) were used exclusively in these studies. B o d y weights ranged from 175 to 250 g. Animals were maintained on a 12 h light,lark cycle, and were killed b e t w e e n 900 and 1400 h.
G.H. B U R R O W S ET AL,
of a motor
2.2. Preparation o f tissue Rats were stunned b y a blow to the neck and shoulders and then quickly decapitated. The brain was removed and dissected on a chilled wax surface. The cerebral cortex, caudal to the level of the anterior commissure, was removed according to the procedure of Glowinski and Iversen (1966) and placed in 0.32 M sucrose on ice. 2.2.1. Homogenates Cerebral cortex was cleared of excess white matter, weighed and homogenized in ten vol of isotonic sucrose (0.32 M), using six strokes
The incubation medium used in these studies was a modified Krebs-Ringer solution consisting of NaC1 (140 mM), KC1 (5.0 mM), MgSO4 (2.0 mM), CaC12 (1.3 mM), EDTA (0.05 mg/ml), ascorbic acid (0.2 mg/ml), dextrose (2.0 mg/ml) and [3H]I-NE at various concentrations. A Tris-phosphate buffer (23 mM Tris base and 15 mM phosphoric acid) was added to maintain the pH at 7.4. Nialamide (10/~M) was included to inhibit NE degradation b y monoamine oxidase. [ 3H ] 1-NE accumulation, using this medium, was similar to that using phosphate-buffered or bicarbonate-buffered Krebs-Ringer solutions. [3H] 1-NE
CALCIUM-SENSITIVE ACCUMULATION OF NOREPINEPHRINE was added to the media immediately prior to incubation. When sodium-free medium was used, choline was substituted for sodium to maintain isotonicity and ionic strength. Calcium-free medium was prepared b y the omission of calcium chloride without replacement.
2.4. Measurement accumulation
of
[3H]l-norepinephrine
Incubations were carried o u t at 31°C in a D u b n o f f metabolic shaker in nylon centrifugation tubes containing 2.0 ml of incubation medium and tissue homogenate derived from 10 mg of original wet weight of cerebral cortex. A temperature of 31°C was chosen in order to slow d o w n the initial linear portion of the uptake rates of CSA, and yet also maintain a temperature above the transition point (30°C) at which there is a break in the Arrhenius plot for catecholamine uptake in synaptosomes (Holz and Coyle, 1974}. The incubation was started b y the addition of tissue to the warmed media. At the end o f the incubation time, the reaction was terminated rapidly b y adding 10 ml of 4°C isotonic saline {0.15 M NaC1) to each t u b e and immersing the tubes in an ice water bath. Tubes were centrifuged at 2 4 0 0 0 × g for 10 min in a refrigerated centrifuge, the supernatant fluid was discarded, and the pellets were rinsed with 10 ml of isotonic saline, centrifuged for 10 additional min at 24 000 × g and the washings discarded. The tubes were inverted over absorbent paper for a minimum of 15 min, then [3H] 1-NE was extracted from the washed pellets using 6 ml o f Triton-toluene phosphor (Triton X-100 in toluene, 1 : 4, v/v, containing PPO, 5.9 g/l, POPOP, 0.13 g/l} and distilled water, 0.2 ml. Tritium was measured in a Packard Tricarb liquid scintillation spectrometer (Model 3385, Packard Instruments Corp.) at 45% efficiency.
2.5. Thin layer chromatography Thin layer chromatography was used to determine whether the tritium accumulated in
303
the tissues represented unmetabolized NE. Cellulose F2s4 plates (E2¢I. Labs., Inc. Merck and Co., Elmsford, N.Y.) were used with a solvent system of butanol • acetic acid : water, 84 : 21 : 35. Following incubation pel. leted tissue samples were homogenized in 70% ethanol in ground glass homogenizers. After centrifugation the supernatant fractions were evaporated to dryness under N2 gas and the residue was taken up in 70% ethanol for spotting on plates with 25 pg of authentic 1-NE. Strips of 0.5 cm height between the origin and the solvent front were counted in Triton phosphor; authentic NE had an R~ of 0.49.
2.6. Subcellular localization of accumulated [3HI l-norepinephrine Cerebral cortex was chopped into slices o f 1.0 × 1.0 X 2.0 mm using the McIlwain Tissue Chopper. The slices were incubated at 31°C for 10 min in standard medium containing 1.0 pM [3H] 1-NE, nialamide (10 #M), and in the presence or absence of cocaine (10 #M). Following incubation the slices were pelleted and homogenized in 0.32 M sucrose for preparation o f a crude synaptosomal pellet (P2). P2 was resuspended in isotonic sucrose, then layered over a continuous sucrose density gradient, 0.32--1.5 M, and centrifuged for one hour at 130 000 X g. A density gradient fractionator (Instrument Specialities Co.) was used to collect 0.8 ml fractions o f the gradient. These were counted in Trition-toluene phosphor.
2.7. Drugs and chemicals [3H]l-NE (2--22 Ci/mmol) was obtained from New England Nuclear Corp. (Boston, MA). Its purity was verified monthly using thin layer chromatography on cellulose F2s 4 plates and a solvent system of butanol : acetic acid : water, 60 : 15 : 25. The following firms generously donated these chemicals: cocaine HC1 and metaraminol bitartrate from Merck, Sharpe and Dohme Research Labs. (Rahway, NJ); des-
304
methylimipramine HC1 from USV Pharmaceutical Corp. (Tuckahoe, NY); nialamide from Pfizer and Co. (Brooklyn, NY);1-Norepinephrine HC1, dl-normetanephrine HC1 and Ficoll were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals and reagents were of reagent grade.
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3. Results The sodium-dependent, high affinity uptake of NE (uptake1) was used throughout this study for purposes of comparison in describing a second accumulation process for NE that i s observed when uptake1 is completely blocked b y either cocaine (10 pM), DMI (0.1 pM) or by substitution of sodium with choline. Because of the marked calciumsensitivity of this second accumulation process, we have referred to it as CSA (calciumsensitive accumulation).
3.1. Time course of [3H]I-NE accumulation by uptake~ and by CSA Homogenates of rat cerebral cortex were incubated in 2.0 ml o f standard incubation medium at 31°C for varying time periods, with and w i t h o u t the addition of cocaine, 10 pM, a concentration which inhibits uptake~ b y more than 90% (data n o t shown). Uptake~ was calculated as total [3H]I-NE accumulation, measured w i t h o u t cocaine, minus that obtained in the presence of cocaine. CSA was taken directly as the accumulation in the presence of cocaine. The time course for uptake~ and for CSA was determined at low (0.025 p_M) and high (1.0 #M) concentrations o f [3H] 1-NE (fig. 1). At b o t h concentrations, uptake~ was linear for at least 5 min, however, CSA began to approach a steady state after 2 min. At the low concentration of [3H] 1-NE (fig. 1A) the relative amounts of NE accumulation due to uptake~ were greater than the amounts due to CSA, except at the earliest time period when t h e y were equal. In contrast, at the higher
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concentration (fig. 1B) accumulation due to CSA was greater than that due to uptake~ at all time periods measured.
3.2. Effects o f calcium on uptakel and CSA Cerebral cortical homogenates were incubated for various time periods with and witho u t cocaine (10 pM), in either standard incubation medium or in calcium-free medium. Uptake1 and CSA were determined at b o t h
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Fig. 2. Effect of calcium on the time course of accumulation of [3H]I-NE by uptakel in cerebral cortical homogenates. In (A) incubations were carried out in 0.025 #M and in (B) in 1.0 #M concentrations of [3H]I-NE at 31°C. Nialamide was present at 10 #M. Solid lines refer to accumulation in standard medium containing 1.3 mM calcium. Dashed lines refer to accumulation in medium in which calcium was omitted. Uptake1 was taken as total accumulation minus that in the presence of 10 #M cocaine. Each point is the mean -+ S.E. of 4 separate determinations, each in duplicate.
Fig. 3. Effect of calcium on the time course of calcium-sensitive accumulation (CSA) of [3H]I-NE at 31°C in cerebral cortical homogenates. Nialamide was present at 10 #M and cocaine at 10 #M. In (A) incubations were carried out in 0.025 #M and in (B) in 1.0 #M concentrations of [3H]I-NE. Solid lines refer to accumulation in standard medium containing 1.3 mM calcium. Dashed lines refer to accumulation in medium in which calcium was omitted. Each point is the mean -+ S.E. of 4 separate determinations, each in duplicate.
high (1.0 #M) and low (0.025 #M) concentrations of [3H] I-NE. As expected from numerous other studies (reviewed in Paton, 1976a), the omission of calcium had no effect on uptakel (fig.2). In contrast, C S A was markedly reduced in the absence of calcium (fig. 3). At one rain of incubation time the accumulation in the presence of cocaine and
0.025 pM [3H] 1-NE (fig. 3A) was inhibited by 65% when calcium was omitted, and at 1.0 #M [3H]I-NE (fig. 3B) by 72%. This calcium sensitivity was noted at all times tested and at both high and low concentrations of [SH]INE. The calcium dependency of CSA was also observed when the high affinity uptake of NE
306
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I0
INCUBATION TIME (min) Fig. 4. Time course of d e s m e t h y l i m i p r a m i n e (DMI)resistant accumulation o f [3H ]l-norepinephrine, effect of calcium omission. Cerebral cortical h o m o g enates were incubated at 3 1 ° C in standard i n c u b a t i o n m e d i u m containing 1.0 pM [3H]I-NE, 10 pM nialamide and 0.1 pM DMI. The dashed line refers to the total a c c u m u l a t i o n in the absence of c a l c i u m ; the solid line refers to the a c c u m u l a t i o n in 1.3 m M calcium. Each p o i n t is the mean-+ S.E. o f 4 separate experiments, each in duplicate.
was blocked b y DMI rather than cocaine (fig. 4). At 0.1 pM, DMI is a specific, maximaUy effective inhibitor of uptake, in noradrenergic neurons of the rat brain (Koe, ! 9 7 6 ) , as was also confirmed in our labora-
tory (data not shown). The time course for uptake of 1.0 #M [3H] 1-NE in the presence of DMI was similar to that observed using 10 #M cocaine (fig. 3B), and calcium omission resulted in a similar decrease in [3H]I-NE accumulation. This was further demonstrated in table 1 in which the one min accumulation o f [3H] 1-NE b y CSA was decreased to approximately 50% of controls b y calcium omission, whether DMI, choline replacement for sodium, or both were used to define CSA. 3.3. Identification o f the radioactivity accumulated by CSA Thin layer chromatography (TLC) was used in order to determine whether the tritium accumulated b y CSA was associated with unmetabolized NE. Homogenates were incubated for 5 min at 31°C in standard medium containing [3H]I-NE (1.0 pM), cocaine (10 pM) and nialamide (10 pM). Following incubation the rinsed pellets were extracted in 70% ethanol and spotted on TLC plates as described in Section 2.5. Under these conditions for CSA, 96% of the total radioactivity in the tissue extracts migrated with authentic NE, indicating no appreciable metabolism of the NE accumulated b y CSA. When calcium
TABLE 1 Effects of calcium on initial rates of [3H]l-norepinephrine a c c u m u l a t i o n in cerebral cortical h o m o g e n a t e s where u p t a k e was b l o c k e d either by d e s m e t h y l i m i p r a m i n e or by sodium replacement. Incubation conditions I
0.1/~M DMI 2 No s o d i u m 3 No s o d i u m + 0.1/~M DMI
[ 3H ]l-Norepinephrine a c c u m u l a t i o n , pmol/g 1.3 mM calcium
No calcium
167 -+ 30.0 202 -+ 20.8 192 -+ 21.9
71.5 -+ 36.8 97.8 -+ 18.3 112 + 22.4
E f f e c t of calcium omission % decrease
57 52 42
: I n c u b a t i o n s were carried o u t for one rain at 31°C in standard m e d i u m in the presence of 1.0/~M [3H]I-NE and 10 ~M nialamide. Values are the m e a n + S.E. o f 4 experiments. In each e x p e r i m e n t the m e d i a n of triplicate determinations was used. 2 Conditions as in (1) e x c e p t 0.1/~M d e s m e t h y l i m i p r a m i n e was added to the m e d i u m . 3 Conditions as in (1) e x c e p t NaC1 was replaced by choline chloride. Analysis of variance indicated no difference a m o n g the 3 i n c u b a t i o n c o n d i t i o n s ; however, the inhibition o f a c c u m u l a t i o n due to calcium omission was highly significant for all c o n d i t i o n s (P < 0.001).
CALCIUM-SENSITIVE ACCUMULATIONOF NOREPINEPHRINE was omitted from the incubation medium and all other conditions remained the same, 83% of the tritium accumulated in the tissues in 5 min was associated with unchanged NE. The higher percentage of unmetabolized NE in the samples incubated with calcium may reflect inhibition of catechol-O-methyltransferase (COMT) by calcium (Weinshilboum and R a y m o n d , 1976).
3.4. Effects o f normetanephrine and metaraminol on CSA In the perfused rat heart, metaraminol was one of the most p o t e n t inhibitors of uptakel and one of the least potent inhibitors of uptake2. Conversely, normetanephrine was a powerful inhibitor o f uptake2 with little effect on uptake1 (Iversen, 1965). Accordingly, these compounds were tested as possible inhibitors of the initial rate of accumulation o f [3H] 1-NE by CSA. Homogenates were incubated for one min at 31°C in the presence of 0.4 #M [3H]I-NE, 10 pM nialamide and 10 pM cocaine (table 2). In contrast with uptake2 in heart, CSA in cortex was n o t affected by normetanephrine at concentrations as high as 10 mM, yet metaraminol, 10 mM, inhibited CSA by more than 80%. These data suggested
307
that while CSA exhibited some of the characteristics of uptake2 in rat heart, it differed markedly from uptake2 with respect to competition by these analogs o f phenethylamine. Further evidence o f differences from uptake2 was observed in preliminary experiments (data not shown) in which other compounds known to have high affinity for uptake2 had no effects on CSA. Some of these compounds included the steroids, corticosterone and estradiol, and the phenoxybenzamine analogs, SKF 625a, 784a and 550 (Iversen and Salt, 1970; Iversen et al., 1972).
3.5. Subcellular localization o f CSA in cerebral cortical tissue The subcellular localization of CSA was determined on continuous sucrose density gradients. Slices of cerebral cortex (1 X 1 × 2 mm) were incubated for 10 min at 31°C in standard incubation medium containing 6.0 Z
A
I . - ¢~ ,~._~ ._1 -,-
5.0 4O
2~
TABLE 2 Effects of inhibitors of uptakel and uptake 2 on calcium-sensitive accumulation (CSA). Homogenates of cerebral cortex were incubated at 31°C for one min in the presence of 0.4pM [3H]I-NE, 10/.tM
nialamide and 10/~M cocaine. Values are the percent inhibition o f [3H]l-NE-accumulation + S.E. determinations, each in triplicate.
Compound
Normetanephrine 0.1 mM 10.0 mM
of
Percent inhibition o f C S A
1.7-+ 5.7 5.9 -+ 8.2
Metaraminol 0.1 mM
--9.5 + 15
10.0raM
82
I Student's t-test; P < 0.001.
-+ 1.8 I
4
<
z
'~
2.0 o
1.0 "r~
°.7,
I.II
1.30 1.50
SUCROSE MOLARITY Fig. 5. Subcellular distribution of [3H]I-NE in cerebral cortical homogenates after accumulation in the presence (dashed line) and absence (solid line) of. cocaine, 10 pM. Slices o f rat cerebral cortex were incubated for 10 min at 31°C in the presence o f 1.0/~M [3H]I-NE, 10 #M nialamide and with or without 10 pM cocaine. Tissue was then homogenized and resuspended P2 was layered over a continuous sucrose density gradient and centrifuged for one h at 130 000 g. Each point is the mean o f duplicates taken in each 0.8 ml fraction o f the gradient. These data are the results o f one o f three similar experiments.
308
G.H. B U R R O W S ET AL.
1.0 pM [3H] 1-NE and 10 pM nialamide. In a single experiment slices were incubated b o t h with and without 10 pM cocaine. Following incubation homogenates were prepared and resuspended P2 was layered over continuous sucrose density gradients as described in Section 2. 6. The radioactivity in 0.8 ml fractions of the gradients is shown in fig. 5. It was found that in either the presence or absence of cocaine, a single peak of radioactivity appeared in that portion of the gradient (near 1.0 M sucrose) associated with synaptosomal markers (Coyle and Snyder, 1969). A synaptosomal site for CSA was further suggested b y comparing CSA and its calcium sensitivity in three different preparations of cerebral cortical tissue, in which the nerve endings were present in intact slices (0.2 × 0.2 × 0.4 mm), or enriched as in crude homogenates, or highly enriched as in purified synaptosomes isolated b y the m e t h o d of Morgan et al. (1971). Each t y p e of tissue preparation was incubated for one min at 31°C in standard incubation medium containing [3H]I-NE, 1.0 pM, nialamide, 10 pM and cocaine, 10 pM. In each experiment the incubations were carried o u t in b o t h the presence and absence of 1.3 mM calcium. The results in table 3 indicated that in the absence of cal-
cium, CSA, expressed per mg protein, was increased about 15-fold as the synaptosomal content of the preparation was enriched from intact slices to purified synaptosomes. In the presence of calcium, CSA increased over 90fold when the nerve ending content of the preparation increased. The effectiveness of added calcium in enhancing CSA increased from 34% in slices to nearly 600% above nocalcium controls in purified synaptosomes. These results support the previous findings that CSA in rat cerebral cortex is localized in the nerve endings, or synaptosomal fraction of the tissue.
4. Discussion The accumulation of NE in the cerebral cortex is the result of at least two processes. At low concentrations o f NE (at or below the apparent Kin), most of the accumulation is due to uptake, which can be blocked to the same maximal extent by 10 pM cocaine or 0.1 pM DMI, or when sodium is replaced by choline. At higher concentrations of NE, this high affinity, low capacity, sodium
TABLE 3 E n h a n c e m e n t o f CSA b y c a l c i u m a n d b y e n r i c h m e n t o f s y n a p t o s o m a l c o n t e n t . Slices, h o m o g e n a t e s o r p u r i f i e d s y n a p t o s o m e s o f r a t c e r e b r a l c o r t e x were i n c u b a t e d for o n e rain a t 31°C in s t a n d a r d i n c u b a t i o n m e d i a c o n t a i n i n g [3H]I-NE, 1.0 pM, n i a l a m i d e , 10 pM, a n d cocaine, 10/~M. I n each e x p e r i m e n t t h e m e d i a c o n t a i n e d e i t h e r 1.3 m M or zero c a l c i u m . P r o t e i n was d e t e r m i n e d b y t h e m e t h o d o f B r a d f o r d ( 1 9 7 6 ) . E a c h value is t h e m e a n -+ S.E. o r 3 d e t e r m i n a t i o n s , each in d u p l i c a t e . Tissue p r e p a r a t i o n
Slices 1 Homogenates 2 Purified synaptosomes 3
Calcium-sensitive a c c u m u l a t i o n o f [3H]I-NE ( p m o l / m g p r o t e i n ) No c a l c i u m
1.3 m M c a l c i u m
0.8 + 0.14 6.4 + 1.5 14.0 + 4.3
1.1 -+ 0.11 17.0 + 1.00 97.0 + 32.0
Effect of calcium p e r c e n t increase
+34 +176 +580
I M c I l w a i n slices, 0.2 × 0.2 × 0.4 ram. 2 Tissue h o m o g e n i z e d in 0 . 3 2 M s u c r o s e a n d c e n t r i f u g e d for 10 rain at 1 0 0 0 X g; t h e p e l l e t was d i s c a r d e d a n d t h e supernatant was t a k e n as a c r u d e s y n a p t o s o m a l p r e p a r a t i o n . 3 P u r i f i e d s y n a p t o s o m e s o b t a i n e d f r o m t h e 12% Ficoll layer o f t h e d i s c o n t i n u o u s Ficoll-in-sucrose g r a d i e n t ( M o r g a n e t al., 1 9 7 1 ) .
CALCIUM-SENSITIVEACCUMULATIONOF NOREPINEPHRINE high capacity, calcium-sensitive accumulation. This paper describes for the first time some of the characteristics of CSA in the rat brain using the natural substrate, NE. Some of the characteristics of CSA are similar to those of uptake: in peripheral organs (summarized in Hendley, 1976), and to the accumulation of [3H] normetanephrine in cerebral cortex (Hendley et al., 1970). Thus, CSA has a high capacity and rapid time course, and lacks stereoselectivity towards the d- and 1-isomers of NE (Burrows, 1979). However, other characteristics of CSA differ markedly from those of uptake2 in peripheral organs. For example, initial accumulation rates for CSA are less sensitive to inhibition by normetanephrine than by metaraminol. In addition, CSA is not altered by phenoxybenzamine analogs, nor by the steroid hormones (Burrows, 1979). Furthermore, CSA appears to be associated with the nerve ending fraction of rat cerebral cortex, which suggests that CSA is intraneuronal in brain tissue as opposed to the presumed extraneuronal localization of uptake2 in rat heart (l~hinger and Sporrong, 1968). Regarding the proposed intraneuronal localization for CSA in rat brain, there is also evidence for an intraneuronal component of uptake2 in rat heart, despite the generally held view that uptake2 is extraneuronal (Clarke and Jones, 1969). For example, Iversen {1965) reported that uptake2 was decreased following immunosympathectomy of the rat heart. Later, Salt and Iversen (1973) provided further evidence for decreased uptake2 following chemical sympathectomy with 6-hydroxydopamine pretreatment. Accordingly, an intraneuronal localization for CSA in rat brain may have its peripheral counterpart in the sympathetic nerves of the rat heart as well. In the present study, the question of a possible glial localization for CSA cannot be excluded, as 'gliasomes' can be formed and carried along with synaptosomes during subcellular fractionation procedures using either sucrose or Ficoll gradient separation tech-
309
niques (Henn, 1976). The question concerning whether CSA is confined exclusively to noradrenergic nerve terminals or not has not been dealt with in this study. This question would best be answered using cerebral cortex from locus coeruleus lesioned animals. However, it does seem probable that CSA is located primarily in noradrenergic nerve terminals, in view of the histochemical studies of HSkfelt and Ljungdahl (1972), Hosli et al. (1975) and of Hosli and Hosli (1976). In each of these studies, rat brains were incubated in the presence of 1.0 p_M NE and the distribution of exogenous NE in the tissues was compared with the normal distribution of endogenous NE in untreated tissues. After exposure to exogenous NE for even as brief a time as 30 sec, at which time CSA should account for the majority of the NE accumulation, the NE fluorescence was localized uniquely in a limited number of small varicosities, in a distribution pattern similar to that expected for noradrenergic nerve terminals. Another unresolved question in the present study concerns the possibility that [3H] 1-NE accumulated by CSA represents homoexchange of tritiated NE with endogenous stores of unlabeled NE, and thus may not represent a net accumulation of amine. One way to test for homoexchange would be to prelabel the endogenous stores of synaptosomes with [3H]-NE in the presence of a monoamine oxidase inhibitor, and then expose the synaptosomes to a high concentration of unlabeled NE, in the presence of calcium and either cocaine or DMI, and to note whether the accumulation of unlabeled NE, presumably by the process of CSA, displaced any of the labeled NE into the medium. While this experiment was not performed in the present study, Raiteri et al. (1977) carried out a very similar experiment in synaptosomes of rat hypothalamus and reported no evidence at all of displacement of labeled NE by 10 pM unlabeled NE and 1.0 #M DMI. While these findings suggest that CSA involves a net accumulation and not a homoexchange, the experiment should be verified using a highly puri-
310
fied preparation of cerebral cortical synaptosomes. A striking characteristic of CSA in the central nervous system is that it is markedly reduced when calcium is omitted from the incubation medium. This sensitivity to calcium was observed when either cocaine, DMI, or sodium replacement with choline were used to inhibit uptake1. These findings contrast with those of uptake2 in the rabbit ear artery which was unaffected b y removal of calcium (Gillespie and Towart, 1973). On the other hand, in the cocainized rat heart perfused with 1.0 pM [3H] 1-NE, omission of calcium in the perfusate resulted in a significant reduction of uptake2 (Mekanontchai and Trendelenburg, 1979), suggesting a possible role for calcium ions in uptake2 in the rat heart as well as for CSA in rat cerebral cortex. The concentration of calcium ions in the extracellular fluid o f cat cerebral cortex has been measured b y calcium electrode and found to be decreased markedly, from a normal level of 1.3 mM to as low as 0.8 mM, as a result o f repetitive stimulation of the tissue (Heineman et al., 1977). This decrease could serve to inhibit the removal of NE from the synaptic cleft region via the process of CSA, and thereby prolong the time that released NE would remain in the cleft for activation of postsynaptic receptors under conditions of increased nerve activity. The mechanism b y which calcium increases CSA remains unexplored. We have noted, however, that omission of magnesium in the incubation medium failed to alter CSA, and furthermore, that magnesium ions failed to substitute for calcium in stimulating CSA in homogenates of cortex (Burrows, 1979), suggesting a specificity for calcium in this process. By virtue of its rapid rate and high capacity for transport o f NE, CSA may have a physiological role in removal o f NE from the synaptic cleft following its release. Direct measurements o f NE concentration in the cleft region immediately following NE release have not been reported. However, it can be assumed
G.H. B U R R O W S E T AL.
that the concentration is highest at the site of its release from nerve terminals. Phillips and Apps {1979) estimated that the concentration of NE in synaptic vesicles is millimolar or higher in magnitude, suggesting that during exocytosis the concentration of NE in the immediate region of the presynaptic nerve terminal far exceeds the capacity for removal b y uptake,. Accordingly, it may be hypothesized that a more rapid and higher capacity uptake process, such as CSA, is required for the efficient removal of the high concentrations o f NE presumed to occur locally in the extracellular space nearest the noradrenergic nerve terminal following release. Furthermore, concentrations of NE in the extracellular spaces more distant from the immediate site of release are estimated to be micromolar or more in concentration (Stj~irne, 1975; Hamberger, 1967), suggesting that removal of NE by CSA is also feasible at regions more distant from the site of release. Another possible role for CSA in the brain may be that of modifying the actions of some psychoactive drugs. For example, Y a m a m o t o et al. (1978) reported that morphine injection markedly altered calcium content and binding of calcium to synaptosomal plasma membranes in the rat brain. By altering calcium availability one might predict that morphine alters the rate of removal of NE during noradrenergic nerve stimulation. In addition, CSA may be involved in the pharmacological effects of amphetamine administration, in view of the findings of Cho et al. {1977) that parahydroxyamphetamine, a major metabolic of amphetamine, is accumulated in rat brain by means of a transport process that is strikingly similar to that of CSA for NE as described in this study. These similarities suggest that parahydroxyamphetamine may compete with NE for transport via CSA, although this compound has n o t been directly tested as a competitive inhibitor of NE for accumulation by CSA. The calcium
CALCIUM-SENSITIVE ACCUMULATION OF NOREPINEPHRINE cellular c o n c e n t r a t i o n s o f t h e s e c a t i o n s d u r i n g nerve s t i m u l a t i o n m a y regulate t h e relative p r o p o r t i o n s o f uptake1 a n d C S A m e c h a n i s m s used f r o m m o m e n t t o m o m e n t f o r r e m o v a l o f NE f r o m t h e s y n a p s e d u r i n g nerve stimulation.
Acknowledgement Supported by a grant from the US Public Health Service, MH-25811.
References Bradford, M.M., 1976, A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 71,248. Burrows, G.H., 1979, Comparison of cocaine-sensitive and cocaine-insensitive accumulation of norepinephrine in rat cerebral cortex, Ph.D. Thesis, Univ. of Vermont, Dissertation Abst. International, Sect. B 39, 4202. Burrows, G.H., M.M. Myers and E.D. Hendley, 1977, Cocaine~ensitive and cocaine-insensitive l-norepinephrine uptake in rat cerebral cortex,tFed. Proc. 36,381. Burrows, G.H., M.M. Myers and E.D. Hendley, 1978, Characteristics of cocaine-resistant accumulation in rat cerebral cortex, Neurosci. Abstr. 4,268. Cho, A.K., J.F. Fischer and J.C. Schaeffer, 1977, The accumulation of p-hydroxyamphetamine by brain homogenates and its role in the release of catecholamines, Biochem. Pharmacol. 26, 1367. Clarke, D.E. and C.J. Jones, 1969, Are adrenergic nerves required for uptake2 in the isolated perfused rat heart?, European J. Pharmacol. 7,121. Coyle, J.T. and S.H. Snyder, 1969, Catecholamine uptake by synaptosomes in homogenates of rat brain: Stereospecificity in different areas, J. Pharmacol. Exp. Ther. 170,221. Ehinger, B. and B. Sporrong, 1968, Neuronal and extraneuronai localization of noradrenaline in the rat heart after perfusion at high concentration, Experientia 24, 265. Gillespie, J.S. and R. Towart, 1973, Uptake kinetics and ion requirements for extraneuronai uptake of noradrenaline by arterial smooth muscle and collagen, Br. J. Pharmacol. 47,556. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain. I. The disposition of [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem. 13,655.
311
Hamberger, B., 1967, Reserpine-resistant uptake of catecholamines in isolated tissues of the rat, Acta Physiol. Scand. Suppl. 295, 1. Heineman, U., H.D. Lux and M.J. Gutnick, 1977, Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat, Exp. Brain Res. 27,237. Hendley, E.D., 1976, The mechanism of extraneuronal transport of catecholamines in the central nervous system, in: The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines, ed. D.M. Paton (Raven Press, New York) p. 313. Hendley, E.D., K.M. Taylor and S.H. Snyder, 1970, 3H-Normetanephrine uptake in rat brain slices. Relationship to extraneuronal accumulation of norepinephrine, European J. Pharmacol. 12,167. Henn, F.A., 1976, Neurotransmission and glial cells: A functional relationship?, J. Neurosci. Res. 2, 271. HSkfelt, T. and A. Ljungdahl, 1972, Application of cytochemicai techniques to the study of suspected transmitter substance in the nervous system, in Adv. Biochem. Psychopharmacology, Vol. 6, eds. E. Costa, L.L. Iversen and R. Paoletti (Raven Press, New York) p. 1. Holz, R.W. and J.T. Coyle, 1974, The effects of various salts, temperature, and the alkaloids veratridine and batrachotoxin on the uptake of [ 3H ]dopamine into synaptosomes from rat striatum, Mol. • Pharmacol. 10,746. Hosli, E., U.M. Bucher and L. Hosli, 1975, Uptake of 3H-noradrenaline and SH-5-hydroxytryptamine in cultured rat brainstem, Experientia 31,354. Hosli, E. and L. Hosli, 1976, Autoradiographic studies on the uptake of 3H-noradrenaline and SHGABA in cultured rat cerebellum, Exp. Brain Res. 26,319. Iversen, L.L., 1965, The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: A novel catechol amine uptake process, Br. J. Pharmacol. 25, 18. Iversen, L.L., 1975, Uptake processes for biogenic amines, in: Handbook of Psychopharmacology, Vol. 3, eds. L.L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p. 381. Iversen, L.L. and P.J. Salt, 1970, Inhibition of catecholamine uptake2 by steroids in the isolated rat heart, Br. J. Pharmacol. 40, 528. Iversen, L.L., P.J. Salt and H.A. Wilson, 1972, Inhibition of catechol amine uptake in the isolated rat heart by haloaikylamines related to phenoxybenzamine, Br. J. Pharmacol. 46, 647. Koe, K., 1976, Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain, J. Pharmacol. Exp. Ther. 199,649. Mekanontchai, R. and U. Trendelenburg, 1979, The
312 neuronal and extraneuronal distribution of 3H-(--)noradrenaline in the perfused rat heart, NaunynSchmiedeb. Arch. Pharmacol. 308, 199. Morgan, I.G., L.S. Wolfe, P. Mandel and G. Gombos, 1971, Isolation of plasma membranes from rat brain, Biochim. Biophys. Acta 2 4 1 , 7 3 7 . Natali, J.P., P. Joanny and F. Blanquet, 1975, Cerebral uptake of noradrenaline in v i t r o ; o c c u r r e n c e of different uptake systems and effect of partial external sodium substitution, Experientia 3 1 , 6 3 2 . Paton, D.M., 1976a, Characteristics of uptake of noradrenaline by adrenergic neurons, in: The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines, ed. D.M. Paton (Raven Press, New York) p. 49. Paton, D.M., 1976b, ed. The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines (Raven Press, New York). Phillips, J.H. and D.K. Apps, 1979, Storage and secretion of catecholamines: The adrenal medulla, in: Physiological and Pharmacological Biochemistry, vol. 26, Int. Rev. Biochem., ed. K.F. Tipton (University Park Press, Baltimore) p. 121. Raiteri, M., R. del Carmine, A. Bertollini and G. Levi, 1977, Effect of desmethylimipramine on the
G.H. BURROWS ET AL. release of [~H]norepinephrine induced by various agents in hypothalamic synaptosomes, Mol. Pharmacol. 1 3 , 7 4 6 . Salt, P.J. and L.L. Iversen, 1973, Catecholamine uptake sites in the rat heart after 6-hydroxydopamine treatment and in a genetically hypertensive strain, Naunyn-Schmiedeb. Arch. Pharmacol. 279, 381. Stj~/rne, L., 1975, Basic mechanisms and local feedback control of secretion of adrenergic and cholinergic neurotransmitters, in: Handbook of Psychopharmacology, Vol. 6, eds. L.L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p, 179. Weinshilboum, R.M. and F.A. Raymond, 1976, Calcium inhibition of rat liver catechol-O-methyltransferase, Biochem. Pharmacol. 25,573. Wong, D.T. and F.P. Bymaster, 1976, Effect of nisoxetine on uptake of catecholamines in synaptosomes isolated from discrete regions of rat brain, Biochem. Pharmacol. 25, 1979. Yamamoto, H., R.A. Harris, H.H. Loh and E.L. Way, 1978, Effects of acute and chronic morphine treatments on calcium localization and binding in the brain, J. Pharmacol. Exp. Ther. 205, 255.