3H-normetanephrine uptake in rat brain slices. Relationship to extraneuronal accumulation of norepinephrine

EUROPEAN JOURNAL OF PHARMACOLOGY 12 (1970) 167-179. NORTH-HOLLAND PUBLISHING COMPANY

3 H - N O R M E T A N E P H R I N E U P T A K E IN RAT B R A I N SLICES. RELATIONSHIP TO E X T R A N E U R O N A L ACCUMULATION OF NOREPINEPHRINE * Edith D. HENDLEY, Kenneth M. TAYLOR and Solomon H. SNYDER Departments o f Pharmacology and Experimental Therapeutics and Psychiatry and the Behavtoral Scwnces, The Johns Hopkins University School o f Medwme, Baltimore, Maryland 21205, USA

Recewed 13 March 1970

Accepted 29 May 1970

E.D. HENDLEY, K.M. TAYLOR and S.H. SNYDER, 3H-normetanephrme uptake in rat bra:n shces Relatzonshtp to extraneuronal accumulatton o f norepmephrme, European J. Pharmacol. 12 (1970) 167-179. 3H-Normetanephrme accumulation m shces of rat brain was characterized and contrasted with 3H-norepmephrme accumulation. Imttal rates of normetanephrme uptake were very rapid; accumulated amine was not bound to particulate fracUons and was easdy washed out of the tissues. Unhke norepmephrme uptake, normetanephrme accumulation was unaffected by reserpine, was not stereospeclftc and was less affected by omission of glucose or sodmm than was norepmephnne uptake. The cerebral cortex was the region of greatest nonnetanephrme accumulation whereas the corpus stnatum accumulated norepmephnne to highest ussae: medmm ratios. Several features of normetanephrine uptake m brain resembled both Uptake 2 of norepmephrme in heart and the extraneuronal uptake of norepmephnne m cocaamzed hearts. In rat brains in which the catecholamme nerve terminals were destroyed by intraventncularly administered 6-hydroxydopamme, endogenous norepmephrine was markedly reduced; aH-notepmephrme accumulation by shces was decreased, and displayed characteristics more like normetanephrme uptake than ltke norepinephnne uptake m control animals. Normetanephnne

Exttaneuronal uptake

1. INTRODUCTION Extraneuronal binding of norepinephrme (NE) has been demonstrated m denervated rat salivary glands (Fischer et al., 1965), in some organs of immunosympathectomized rats and mice (Iversen et al., l966) and in the cocainized perfused rat heart (Eisenfeld et al., 1967a, b, c; Slmmonds and Gilhs, 1968). This extraneuronal uptake is characterized by the following: quantitatively it is lower than and is usually masked by the uptake of NE into intra• This research was supported by Public Health Servtce Grants 1-R01-NB-07275, 1-POI-GM-16492, by a Justice Department Contract J-6835, and by National Institute of Mental Health Research Scientist Development Award K3-MH-33128 to Solomon H. Snyder.

Norepmephrme

6-Hydroxydopamme

neuronal binding sites; extraneuronally accumulated amine is easily washed out in the tissues in contrast to the more firmly bound neuronal NE; it is inhibited by normetanephnne (NMN), metanephrine and similar analogs that are very poor inhibitors of intraneuronal NE uptake, whereas it is poorly inhibited by metaraminol, the most potent inhibitor of intraneuronal NE uptake; and it is inhibited by adrenerglc blocking agents. Simmonds and Gillis (1968) compared the uptakes of a H-normetanephrine (aH-NMN) and aH-norepinephnne (aH-NE) in perfused cocainized rat hearts and suggested that NMN was accumulated into the same transport system that takes up e x t r a neuronal NE, and that the cocaimzed heart extubited a ten-fold greater affinity for 3H-NMN than for 3 H-NE.

168

lz: D Hendley. K.M. Taylor, S.H Snyder. Normetanephrme uptake tn brain

lversen (1965a) described a novel system i or carecholamme accumulation by the isolated perfused heart that appeared to operate at faMy high concentratlons of NE and which was called Uptake2 to distinguish it from Uptake i which represented tile mtraneuronal accumulation of NE (lversen, 1967). Farnebo and Malmfors (1969) confirmed by hlstochemical fluorescence studies that low concentrations of NE (0.02 to 0.2/ag/ml) perfused tn rat hearts were taken up exclusively into adrenergic nerves whereas high concentrations (5 to 20/ag/ml) increased fluorescence of the muscle cells and certain connective tissue cells particularly in the atria. Recently Lightman and lversen (1969) found that Uptake: accounts for a substantial proportion of NE accumulated by the perfused heart even at low medium concentratlons of NE. Since NE accumulation in the perfused heart by Uptake~ is unaffected by 6-hydroxydopamine (6-HD) treatment (Clarke and Jones, 1969), It appears to be an extraneuronal uptake system. Since the pattern of affinity of a variety of drugs for Uptake: (Burgen and lversen, 1965) and for the "extraneuronal" uptake of N E m the cocamized heart (Eisenfeld et at., 1967) is the same, it is likely that Uptake: represents accumulation of NE by this extraneuronal NE uptake system. Although the neuronal uptake of NE by brain tissue has been shown to resemble this process in the periphery (Glowinskl et al., 1965; Dengler et al., 1962; Snyder et al., 1968a), uptake systems analogous to peripheral extraneuronal uptake of NE or NMN have not heretofore been examined m the brain. In the present study the uptake of a H-NMN in rat brain slices was characterized and contrasted with 3H-NE uptake. In some experiments the uptake of 3H-NE was measured in cerebral cortex slices from rats pretreated with mtraventncular injections of 6-1tD, an agent that causes a marked and long lasting depletion of catecholamines from rat brain (Uretsky and Iversen, 1970) presumably as a result of degeneration of catecholanune-contammg nerve endings in brain (Bloom et al., 1969) sinular to that reported m the peripheral tissues (Tranzer and Thoenen, 1968).

2. METItODS Male Sprague-Dawley rats (150--200 g) were killed

by cervical fracture and brams were rapidly removed and chilled. Individual areas were d~ssected as described earlier (Glowmski and Iversen, 1966, Snyder et al., 1968a), weighed and sliced to 0.1 × 0 1 × 0.5 mm pieces using a tissue chopper (Mcllwam and Rodmght, 1962). The chopped tissue pieces were suspended m 20 volumes of ice-cold Krebs-Henseleit bicarbonate medmm with glucose (Krebs and Henselelt, 1932) modified to contain ethylenediammetetraacetic acid (0.05 mg/ml), ascorblc acid (0.2 mg/ ml) and one-half the CaC12 concentration. Fwe or 7.5 mg aliquots (0.1 or 0.15 ml) of well-stirred t~ssue suspension were added to 20 ml incubation beakers containing 2 ml of modified Krebs-Henseleit medium, malamlde (1.25 × 10-s M), a monoamme oxidase mhibltor that does not inhibit the brain NE uptake system (Hendley and Snyder, 1968) and a gas phase of 95% o x y g e n - 5% carbon dioxide. Under these conditions of incubation and using initial rates of uptake, the accumulation of 3H-NE or of 3H-NMN, 0.1/aM, was found to be linear with tissue concentratlons up to at least 10 mg. Beakers were mcubated at 37°C with shaking in a metabolic incubator in the presence of test drugs for a minimum of 2 minutes after which 3It-NMN or 3 H-NE, O. 1/aM, was added and the incubation contmued for varying times. The incubation was terminated by pouring the tissue onto Whatman #540 filter paper discs moistened with 0.9% saline m Gooch crucibles mounted m a manifold vacuum assembly of 18 small Buchner tunnels, a procedure sHmlar to that described by lversen and Neal (1968). Ten ml of ice-cold saline were used to rinse out the beakers and to wash the tissue pieces and filter discs of radioactive medium Each washed filter disc containing tissue was extracted with 3 rnl absolute ethanol m a counting vial for at least 10 rmn, after which 10 ml of toluene phosphor were added and the tritium content was assayed in a liquid scintillation spectrophotometer (Nuclear-Chicago). The small amount of radioactwe medium adhering to the f'dter discs was estunated m each experiment and subtracted from the 3H concentratlon of each tissue sample. When 3H-NE was used carrier dI-NE, 2 mg% m saline, was included m the tissue rinse as it lessened the amount of radioactive NE from the medium adhering to the filter paper discs due to adsorption of catechols on cellulose. It was not necessary to add carrier NMN to the saline

E.D.Hendley, K.M. Taylor, S H.Snyder, Normetanephrine uptake in brain rinse when 3H-NMN was used since the amount of radioactiwty adhering to the filter paper was very low with 3H.NMN. Usually 10% o f the total counts were subtracted as fdter paper blanks. The radioactivity of the incubating medmm was determined by sampling 0.1 ml in 3 ml absolute ethanol and 10 ml toluene phosphor, In all experiments the uptake of 3H-NMN or of aH-NE was determined from the concentration of tritium accumulated m the slices since nearly all of the radioactivity accumulated was found to be unchanged amine under these conditions of incubation, This was shown for NMN in an experiment in which 6 to 8 15 mg cubes of cerebral cortex were incubated for 20 rain with 0.2/aM 3 H-NMN, the tissues were rinsed with saline and homogenized in 70% ethanol and centrifuged for 1 0 m in at 1500g. The supernatant fluid was evaporated to dryness under a stream of N2 and 0.1 ml 70% ethanol was used to dissolve the extract. Fifty/al aliquots were chromatographed in duplicate on thin layer silica gel plates in n-propanoi - l M acehc acid (3: 1, v/v) along with 125/ag authentic dl-NMN. Radiochromatograms of the extracted tissue were found to have a single sharp peak of radioactivity at the Rf of authentic NMN. In another study, accumulated 3H-NE and its metabohtes were measured in cortical slices from normal or 6-HD treated rats after 5 min incubation in 0.1/aM aH-NE, using the ion exchange separahon method o f Taylor and Laverty (1969). Eighty-e~ght per cent and 86% of the radioactivity coming off the columns was associated with unchanged catecholarmne in normal and 6-HD treated tissues respect~vely. Uptake was expressed as the ratio of rnvmoles of 3H taken up by 1 g of tissue per specified time period to mvmoles of 3H per ml o f medium (t~ssue:med~um ratio). The tissue:medmm ratios at O°C were subtracted as diffusional blanks. These were found to be 0.2 for the 30 sec uptake of aH.NMN and 0.8 for the 5 min uptake of 3H-NE, 0.1/aM, in shces o f cerebral cortex and hypothalamus, 2.1.6-Hydroxydopamine pretreatment Male Sprague-Dawley rats ( 1 5 0 - 1 8 0 g) were lightly anesthetized with ether and 20 ~ of a solutton of 6-HD hydrobromide (Regis Chemical Co., Chicago, 111.) was injected into the left lateral ventricle of the

169

brain using a stereotaxic technique. Solutions of 6-HD for intraventricular injection were prepared in sterile acidified Krebs-Henseteit medium (pH 5) with ascorbic acid, 1 mg/ml as described by Uretsky and Iversen (1970). Animals received 250/ag of 6-HD on days 1 and 3 while control animals received an equivalent volume of vehicle only. All animals were killed by decapitation 10 days after the last intraventricular injection. Their brains were removed and the cortex dissected. Tissue samples of cortex or whole brain were assayed for NE using the ton exchange separahon method (Taylor and Laverty, 1969) and the trihydroxyindole fluorometric method (Laverty and Taylor, 1968). Endogenous levels of NE in five control brains were 0.334/ag/g in whole brain

Table 1 Effects of mtraventricular 6-hydroxydopamme on endogenous levels of norepmephrine m whole brain and cerebral cortex m rats. Endogenous NE (tag/g) Rat no. Whole brams

Cerebral cortex

0.35 0.36 0.32 0.32 0.32

0.27 0.27 0.26 0.26 0.21

Control a 1 2 3 4 5

Mean -+S.E.M. 0.334+-0.008 6-Hydroxydopamme b 6 7 8 9 t0

0.11 0.14 0.10 0.11 -

Mean +-S.E.M. 0.115 + 0.008 % Depletion 66% p Value (control vs. 6-HD) (0.001

0.254+0.011 0.06 0.04 0.04 0.06 0.06 0.052 +-0.004 80% <0.001

a Rats were injected intraventncularly on days 1 and 3 with 20 ~I of modified Krebs-Henselelt medmm, pH 2, containmg 1 mg/ml ascorblc acid. Rats were sacrffxeced 10 days after the last injection. b Rats were injected as in (a) except 250 tag 6-hydroxydopamine. HBr were added to the medium.

170

E D.ttendh,y. K M Taylor. S tl Snvder Normetanephrme uptake m brain

and 0.254 in cerebral cortex (table 1). After 6-HD these values were reduced 66% in whole brain and 80% in cerebral cortex. Uptake studies were carried out on samples o f cerebral cortex as described above. The time course for uptake o f 3H-NE in 6-HD treated cortex shoes was similar to that m normal cortex shces except that t~ssue:medium ratios were reduced by 50% to 80% as observed previously by Uretsky and lversen (1969).

3. RESULTS 3.1. Ttme course f o r uptake o f 31LNMN and 3 H-NE Cerebral cortical tissue shoes accumulated 3 H-NMN, 0.1/aM, to levels nearly 3 hmes that of the surroundtng medmm (fig. 1). The m~tial uptake rates were very rapid, and the uptake was linear only up to 1 mm of incubation time. When the mcubahon was carried out at 0°C instead of at 37°C, the accumulahon was much lower, and t,ssue.medlum (T M) ratios never exceeded 0.7. Tissues incubated at 37°C w~th 5/aM 14C-urea, a substance which d,stributes ~tself throughout the b o d y water, or 311-water m tracer quanht,es, gave uptakes similar to those observed with 3H-NMN at 0°C In contrast with the rapid although modest accumulation of 311-NMN the accumulat~on o f 3H-NE, 0.1/aM, was slower ,n onset but reached T M ratios above 8 wlthtn 10 mm (fig. 2). Incubahon o f 3tI-NE at 0°C markedly reduced the uptake of 3H-NE as was the case with 3H-NMN. In studying the effects o f d r u g s or o f v a r y m g incubation conditions, 30 sec accumulation of 3H-NMN and 5 nun accumulation o f 3H-NE were used as mJtml uptake rates.

2.2. Radioacnve matertals dl-Noreplnephrlne-7-H 3, 6.6 Cl/mmole, dl-normetanephrlne-7-H 3 HCI, 4.2 Ci/mmole, 3H-water, 5 0 / a Q / m l ; and ~4C-urea, 15.4 mCi/mmole were all obtained from New England Nuclear Corporation. I-Normetanephrlne was kindly furnished by Dr. J. Axelrod. The following firms generously donated these compounds: dlbenamme, dibenzyhne and d-amphetamine from Smith Kline and French, Phila. Pa.; MJ1998 and MJ1999 from Mead Johnson Research Center, Evansville, Ind.; phentolamlne from Ciba Pharmaceutical Co.,, Sumrrut, N.J. and 2,5d i m e t h o x y - 4 - m e t h y l a m p h e t a m m e from the Dow Chemical Co. All other drugs were from commercial sources.

Drug doses were expressed as the free base. The t test for statistical significance o f group differences was taken from Snedecor and Cochran (1967).

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E.D.Hendley, K.M. Taylor, S.H.Sn),der, Normetanephrme uptake in bram

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Fig. 2. Time course for the accumulation of 3H-norepinephrme, 0.l pM, m cerebral cortical slices 3H-Normetanephrme data are taken from fig. 1. The accumulanon of 3H-NE at 37 ° in 5 min was 0.53 nmoles per g of tissue. cubes instead o f finely chopped tissue. Single 20 mg cubes o f cortex were tncubated in the usual medium (see METHODS) for 10 or 20 nun, rinsed in fresh nonradioactive medium, weighed, then homogenized in 2 ml absolute ethanol and the supernatant as well as the incubation medium were sampled for 3H concentration. Some of the tissues incubated for 20 min were reserved for washout studies. These were removed from the incubating medium, rinsed thoroughly m fresh chilled nonradioactive medium, then reincubated at 37°C in 2 ml o f fresh nonradioactive medium for 5 or 10 more min. After reincubatlon the shces were rinsed well, weighed and homogenized m 2 ml o f absolute ethanol. The supernatant fluid was analyzed for radioactivity remaining m the slice and the ratios o f the tissue concentrations of radioactivity to that in the original incubating medium were determined (fig. 3). Slower rates o f uptake o f 3H-NMN, aH-NE and t4C-urea occurred in larger slices o f cortex tissue than in f'mely chopped tissue, and confirm previous findings in this laboratory that the in vitro accumulation of 3H-NE is dependent upon the surface area o f the tissue (Snyder et al., 1968a). aH-NE accumulatlon achieved a ratio o f 3 in 20 min, 3H-NMN, 1.85, and 14C-urea, 0.7. After 5 rain washout, 3H-NE fell to a ratio o f 2 and remained at that level at 10 min.

In contrast, 3H-NMN washout was more rapid and prolonged, falling to ratios lower than 0.8 m 5 min and continuing to fall at 10 min. ~4C-Urea washout also continued to fall with time during the 10 min

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r~,E (.~NUTES) Ftg. 3 Washout of radtoactivity was determined m single 20mg cubes of cerebral cortex at 37°C using 0.1uM 3H-NMNand 3H-NE, and 5 pM 14C-urea. Each point represents the average of duphcate determinations. 0°C blanks were not subtracted.

172

E D.Hendley, K M. Taylor, S.H.Snyder. Normetanephrme uptake m bram

washout period. These data are similar to those reported b y Stmmonds and Gdhs (1968) in perfused cocainlzed rat hearts, suggesting that NMN taken up into rat cerebral cortex or into heart is not as firmly b o u n d as NE. 3.3. Regional distribution o f 3H-NMN atu/ 3H-NE m rat brahl There are marked regional differences in the accumulatlon o f 3H-NE by 20 mg cubes o f brain tissue (Snyder et at., 1968a). It was of interest to compare the regional differences m uptake o f 3 H-NMN to those o f NE in finely chopped tissues Seven regions o f the rat brain were dissected according to the procedure of Glowlnskl and lversen (1966). Tissues from each region were finely chopped as described in Methods and incubated with 3H-NMN (0.1 laM) for 30 sec or 3H-NE (0 1 ,uM) for 5 mln (table 2). 3H-NE accumulation showed the same regional differences as reported in larger brain slices (Snyder et al., 1968a) except that the tissue:medium ratios were lugher than in the earlier study with larger tissue shces. The corpus striatum, cerebral cortex and cerebellum had T:M ratios more than double those o f 20 mg slices, T:M ratios in the hypothalamus and medulla-pons were 1.5 times larger and in hippocampus and m~dbram they were 1.1 and 1.3 times greater than in single 20 mg slices, The regaonal distribution of 3H-NMN uptake in Table 2 Regional accumulation of 3H-NMN and 3H-NE m rat bram.

Region

Cerebral cortex tlypothalamus Corpus stnatum lhppocampus M~dbram Cerebellum Medulla-pons

30 sec accumulation 311_NMN tissue medium 1 40 + 0 05 1.10+0.20 1.06-+0.06 0.94 + 0 09 0.88 +- 0.05 0.68 -+ 0.04 0.50 +- 0.04

5 mm accumulation 3ll.N E tissue medmm 5.34 5.54 17.4 3 36 3.47 3.48 2.94

+-0.25 +0.11 +-1.3 -+ 0.21 -+ 0.13 -+ 0.19 +-0.30

Values represent the arithmetic mean -+ S.E.M. of 4 to 8 rephcate determinations. Incubations were carried out In modified Krebs-Henselelt medmm containing 0.1 taM 3H-amines. o°c uptakes were not determined for all regions of the rat brain and have not been subtracted from these values.

30 sec differed from that o f 3H-NE uptake in 5 mm The T.M ratios o f 3H-NMN accumulation were highest m cerebral cortex and considerably lower (p < 0.01) m the corpus strlatum where 3H-NE accumulatton was exceedingly high. The difference m 3H-NMN accumulation between cerebral cortex and hypothalamus was not significant (p = 0.2). 3 H-NMN accumulation m the cerebellum exceeded that in medulla-pons (p < 0.02) but was lower than in mldbrain (p < 0.05). 3.4. Characteristics o f the uptakes o f 3H-NMN and

3H-NE Experiments were conducted to characterize and contrast the uptakes o f aH-NMN and 3H-NE in rat cerebral cortex. When 95% N: - 5 % CO2 was substltuted for 95% O : - 5 % CO: both in the preparation o f the tissue shces and during the incubation procedure there was no inhibition of the accumulation o f either 3H-NMN or aH-NE (table 3). Hamberger (1967) had previously reported that the energy derived from anaerobic glycolysls was sufficient 1o support the NE uptake system. When glucose was orrutted from the incubation medium aH-NE uptake was greatly reduced whereas the uptake o f aH-NMN was decreased only 22%. When sodium was replaced completely by hthmm in the Krebs-Henseleit medium 3H-NE uptake was reduced 81% whereas 3H-NMN uptake was decreased only 20%. Ouabam ( 10-4 M), an inhibitor o f N a ÷ - K ÷actwated ATPase, reduced 3H-NE uptake as much as d~d omission o f sodium, and whde ouabam produced a lesser reduction m 3H-NMN uptake, its effect was greater than omission o f sodmm. Uptakes o f 3H-NMN and 3H-NE were both temperature-dependent (figs. 1,2; table 3), and reduced about 85% by incubation at 0°C. The temperature coefficient (Q lO) o f both uptakes was deternuned from the ratio of the accumulation o f 3H-amine at 37°C and 27°C. The Q io for 3H-NE accumulation was 3.8, a value that is consistent with an actwe transport mechamsm, while the Q~o for 3[t-NMN accumulation was 1.6. This finding as well as the minimal effects o f removal o f sodium or of glucose on 3H-NMN uptake suggest that the apparent uphill movement o f NMN into brain tissue occurs by means o f a facilitated diffusion rather than an active uptake. Cerebral cortex prepared from rats pretreated with

E.D.Hendley, K.M.Taylor, S.H.Snyder, Normetanephrme uptake in brain

173

Table 3 Characteristics of 3H-NMN and 3H-NE uptakes m cerebral cortical slices.

Standard incubation medmm a

Less oxygen Less glucose Na replaced with L1 Plus ouabam (10 -4 M) 27°C Reserpmized tissue (5 mg/kg l.p. 18 hr prey.)

3H.Normetanephrme, 30 sec accumulation

3H.Norepmephrme 5 mm accumulatton

% Inhibition b

pc

% Inhibition b

pc

11 22 20 44 37 (Q10 = 1.6)

>0.05 <0.02 ~ 0.05 <0.05 ~0.05

0 86 81 81 74 (QI0 = 3.8)

<0.001 ~ 0.01 <0.001 <0.01

0

-

72

d 0 01

a Standard incubation medmm contained modified Krebs-Henseleit bicarbonate buffer, at 37°C, a gas phase of 95% 0 2 - 5 % CO2, nialamlde and 0.1 taM all-amines. Control tissue: medmm ratios were 1.5 -+ 0.10 (12) for the 30 sec uptake of 3H-NMNand 5.1 -+ 0.04 (12) for the 5 min uptake of 3H-NE. These represented accumulation of 0.15 nmoles/g/30 sec for NMN and 0.51 nmoles/g/5 min for NE b % Inhibttlon was determined as % decrease m tissue medmm ratios as a result of altermg the incubation medium, and after the subtraction of 0°C T:M uptake ratios as blanks (0.2 for 3H-NMN and 0.8 for 3H-NE). c p Values were determined by t test and were based on data from at least four determlnattons.

reserpine, 5 mg/kg i.p. 18 hr previously, showed a m a r k e d r e d u c t i o n in 3H-NE a c c u m u l a t i o n but n o t in 3H-NMN a c c u m u l a t i o n w h e n measured at 30 sec. A f t e r 5 min i n c u b a t i o n aH-NMN a c c u m u l a t i o n was reduced 21% in reserpinized tissues. Reserpine interferes with the binding o f N E m intraneuronal granules, suggesting that NMN is n o t taken up into granular sites to any appreciable e x t e n t . When larger slices o f tissue were used ( S n y d e r et al., 1968a) the 5 min a c c u m u l a t i o n o f 3H-NE was reserpine-resistant

whereas at 20 min reserpine reduced a H - N E uptake considerably. In finely c h o p p e d tissues m the present study the time course for uptake is speeded up so that in 5 min N E can be a c c u m u l a t e d into granular sLtes. 3.5. Subcellular distribution of aH-NMN and aH-NE The distribution o f a c c u m u l a t e d a H-NE, 3H-NMN and 14C-urea b e t w e e n the supernatant (S) and particulate (P) fractions o f cerebral c o r t e x t~ssue was

Table 4 Subcellular dlstributton of 3H-NMN and 3H-NE in cerebral cortex tissue. P/S Ratio a Incubation conditions

37°C, 30 mm 0 °, 30 mm 0 °, 30 mm, without 3H.amines b

3H-NMN

3H-NE

14C_Urea c

0.29 + 0.028 0.18 4-_0.017

1.46 -+ 0.09 0.31 +- 0.014

0.08 +- 0.0001 -

0.10 -+ 0.001

0.16 + 0.002

-

a P/S calculated as ratio of 3H concentrahon of sucrose-rinsed pellets to 3H concentratton of supernatant fluid after incubation m 0.4 uM 3H-amines, homogenization m 0.32 M sucrose and centrifugation at 100,000 g for 1 hr. Ratios are expressed as the mean -+ S.E.M. of triplicate determinations. b 3H_Amme s were added during homogenization in sucrose. c From Kuhar and Snyder (1970), in experiments conducted under the same con&tlons as m comparable experiments with 3H-NE and 3[t-NMN.

! 74

L:D.Hendley. K 111.fa)'lor. S tI.Sn.vder, Normetauephrtue uptake tu bratn

determined (table4). Six cubes (15 mg each) of cerebral cortex were incubated in tnphcate at 37°C or at 0 ° as described in METHODS for 30 mm m 0.4/aM all-amines. The tissues were rinsed and blotted lightly, then homogenized gently m 0.32 M sucrose with a teflon pestle, and centrifuged at 100,O00g for 1 hr. The supernatant lluld and sucrose-rinsed pellets were sampled for 311 concentration after extracting the pellet with 0 4 N perchlonc acid. In a separate control experiment, tissues were incubated at 0 ° without all-amine, which was subsequently added during the homogenization procedure in order to determine whether the radioactivity was artffactually redistributed during the homogenization procedure, After incubation at 37 ° the P/S ratio for NE was 5 times that for NMN (p < 0.001). The P/S ratio for NMN was triple that of samples in which 3 H-NMN was added to homogenates in the cold, but less than double the P/S ratios obtained after incubating shoes with 3||-NMN at O°C. Although the low P/S ratio for alt-NMN accumulation at 37°C indicated that the accumulation was mainly cytoplasmic, the P/S ratio for ~4C-urea (Kuhar and Snyder, 1970) was considerably lower than that ofaH-NMN (t9 < 0.01). 3.6. Kinetic constants The K m and V'max values for the uptakes of 3|I-NMN and aH-NE were determined m cerebr,,d cortex slices by means of double-reciprocal plots of initial uptake velocities at varying concentrations of all-amines, after subtracting 0°C uptakes at each concentration as blanks. Straight hne plots were obtained with positive ordinate intercepts for both 3H-NMN and aH-NE, indicating a saturable uptake mechanism for both these substrates. The Mlchaelis constant (Km) for dI-NE was 4.6 X 10-7 M, a value close to that reported for larger slices of rat cerebral cortex (Snyder et al., 1968a) or mouse cerebral cortex (Ross and Renyi, 1964), for synaptosomes in homogenates of rat cerebral cortex (Snyder and Coyle, 1969; Coyle and Snyder, 1969) and for isolated perfused rat hearts (lversen, 1963). The K m for di-NMN uptake was 1.4 X 10-a M, a value 3000 times greater than that for dI-NE uptake. Vma × was 0.48 nmoles]g]min for dI-NE and 3.6/amoles/g/min for dI-NMN,

3 7. t:']]ect c~] anah~gs o] Itormetat~ephrme ~m the uptakes o f 3II-NJIN and 3tt-NI;' Several structural analogs o1" normetanephrme were tested as possible mhibltors of the uptakes of both 3H-NMN and 3H-NE in rat cerebral cortex T,ssues were premcubated for 2 to 5 mm with 3 or 4 concentrations of each test drug and % inhtbition of the 30 sec accumulation of 3H-NMN or of the 5 nnn accumulatK)n of 3 H-NE was determined. IDs0 values were estimated by plotting % inhibition against concentration of mtubltor on log prober paper (Miller and Tamter, 1944). The IDso'S are tabulated m table 5 and the relatwe potency of each inhibitor was calculated m arbitrary units from the ratio of IDso of dI-NMN for each uptake to IDs0 of inhibitor. This ratio was taken as a numerical estimate of the affinity of each an'dog for the NMN and NE uptakes relative to the afl'mitles of those uptake systems for dI-NMN. The 1Ds0 for dI-NMN reduction of 3H-NMN accumulatlon was higher than for 3II-NE uptake, and tts value was close to the Km for "~H-NMN uptake. A lumted amount of I-NMN was avadable to test the stereospeclficlty of the NMN uptake system In contrast with the higher affmlty of the lew)-lsomer of NE for the NE uptake system both m heart (Iversen, 1963) and m cerebral cortex (Coyle and Snyder, 1969), the IDso for I-NMN reduction of 3H-dI-NMN accumulation was no different from that of dl-NMN. dI-NE was a very poor inhibitor of 3H-NMN uptake and its affinity for the NE uptake system was nearly 600 times greater than for 3 H-NMN uptake. With dl-metanephrme, the addition of an N-methyl substituent on NMN enhanced the affinities for both 3H-NMN and 3H-NE uptakes relative to their affinity for dI-NMN. Removal of the /3-hydroxy group ot" NMN (3-methoxy-4-hydroxyphenylethylamme) also enhanced affimtles for both uptakes. Shifting the methoxy group m the latter compound from the meta to the para position (3-hydroxy-4-methoxyphenylethylamme) increased the afflmties for both uptakes, more for 3H-NE than 3H-NMN. From this limited series of analogs of NMN it was generalized that the removal of polar groups or the addition of non-polar substltuents enhanced the affmity for both NMN and NE uptakes relative to their affimtles for dI-NMN. Furthermore, NMN uptake did not exhibit stereospecificity.

E D.Hendley, K.M. Taylor, S.H.Snyder, Normetanephrine uptake in brain

175

Table 5 RelaUve potencies of mhtbitors of 3H-NMN uptake in rat cerebral cortical slices. 3H-NMN, 30 sec uptake ID50 a (M)

Relative potency b

3H-lqE, 5 min uptake ID50 a (M)

Relative potency b

Normetanephrine analogs dl-Metanephrine 3-Hydroxy-4-methoxyphenylethylamme 3-Methoxy-4-hydroxyphenylethylamine dI-Normetanephrine I-Normetanephnne dl-Norepinephrme

8.1 X 8.9 X 1.2 X 2.1 X 2.6 X >5.0 X

l0 -4 10 -4 l0 -3 10 -3 10 -3 10 -3

2.6 2.4 1.8 1.0 0.8 ~0.4

4.4 X 10-5 5.5 X 10 ~s 4.6 X 10 -s 2.6 X 10-4 4.4 X 10 --/

5.9 47.3 5.6 1.0 591.0

5.0 X 2.5 X 3.6 X 6.0 X 1.8 X 2.4 X

10-s 10-4 10-4 10 -4 10 -3 10 -3

42.0 8.4 5.9 3.5 1.2 0.9

1.3 X 10 -s -

20.0 -

4.4 X 10-4 4.8 X 10-4 5.0 X 10-4

4.8 4.4 4.2

2.0 X 10 -7 2.0 X 10 ''3 6.0 X I0 -a

Adrenergzc blocking agents Propranolol DIchlorolsoproterenol Phentolamine Yohimbine MJ 1999 MJ 1998

Others d-Amphetamine 2,5-Dimethoxy-4-methylamphetamme (DOM) Desmethyltmipramine

1300 13 4300

a iDso values were derived from log probit plots of % inhibition at 3 or 4 concentraUons of inlu'bitor with 0.1/~M '3H-lqMN or 3H-NE, in quadruplicate, after subtracUon of 0°C uptakes as blank. Data presented are the mean of 2 - 4 such independent determinations of 16 to 20 mcubaUons each in which IDs0 values did not differ by more than 20%. Control T:M ratios in the absence of drugs ranged from 1.2 to 1.7 for the 30 sec uptake of 3H-NMN and from 5.0 to 5.6 for the 5 rain uptake of 3H-NE. These represented accumulation of 0.12 to 0.17 mvmoles NMN/g/30 sec and 0.50 to 0.56 mtamolesNE/g/5 rain. b ID5o dI_NMN/ID50 inhibitor. 3.8. Adrenergic blocking agents The best inhibitors of aH-NMN uptake m the present study were found to be the beta-blockers propranolol and dichloroisoproterenol (DCI) (table5). The alpha-blockers phentolamine and yohimbine were intermediate in relative potency and MJ 1999 and MJ 1998, two powerful beta-blockers, were very poor mhibttors. Thus there was no correlation between effectiveness against alpha or beta adrenergic receptors and inhibition of NMN uptake in rat cerebral cortex. The alpha-blockers phenoxybenzamine, dibenamine and ergotamine tartrate were also tested; however it was not possible to determine IDso values for these compounds, due to their poor solubility in Krebs-Henseleit medium. At 10.-4 M concentratton these 3 alpha-blockers inhibited 3H-NMN uptake by less than 20% whereas propranolol at this concen-

tratlon inhibited uptake b y nearly 60%. In a limited series of experiments the adrenergie blocking agents were found to be good inhibitors of 3 H-NE uptake. At a fixed concentration of 10-4 M, DCI, propranolol, phentolamine and phenoxybenzamine were equipotent ( 6 3 - 6 8 % inlu'bition) and dibenamine and yohimbine i n h ~ i t e d a H-NE uptake b y about 50%. In the 6-HD treated rat cortex the IDso for propranolol was 2.4 X IO s M, a value half that of its IDs0 for 3H-NMN uptake and twice that for 3H-NE uptake in normal cortex. 3.9. Other drugs d-Amphetamine and desmethylimipramine are very powerful inkibitors of intraneuronal NE uptake (Burgen and Iversen, 1965; l w r s e n , 1965b). Much higher doses were required to inlu'bit aH-NMN than 3 H-NE uptake.

176

E.D.Hendley. K M. Taylor. S tt Sn.vder. Normetanephrme uptake m brain

2,5-Dimethoxy-4-methylamphetamine (DOM, "STP"), a powerful psychotomimettc agent in man (Snyder et ai., 1967; 1968b) was found to have a high affinity for both 3H-NMN and 3H-NE uptakes relative to NMN. However, when compared to amphetamine itself, the methoxy-methyl substltuents markedly diminished affmtty for the NE uptake system, but not for the NMN uptake system. A report of the study of a homologous series of psychotropic trtmethoxyamphetamines as mhibttors of both these uptakes is currently m preparatton,

proterenol intermediate (about half as active as eplnephrlne), while dI-NE, dopamine and metarammol were least active, less than half as potent as isoproterenol. To assess the possible slnulartttes of these uptake systems to NMN and NE uptake m brain shces, a series of NE analogs was tested for their effect on the uptake of 3H.NMN and 3H.NE m brain slices of controls or of rats pretreated with 6-HD. The rank order for reduction of 3H-NE accumulation into cortex slices of normal rats was the same as for 3H-NE in the isolated perfused heart (table 6 ) ( B u r -

3.10. A n a l o g s o ] norepmephrme Burgen and lversen (1965) reported marked differences among NE analogs tn their affinities for mtraneuronal NE uptake and for Uptake2 of NE. In a series of analogs, affmtty for intraneuronal NE uptake decreased m the order" dl-metaranunol > dopamine > I-NE > l-epinephrine > d-NE > dl-tsoproterenol. The rank order of affinity for Uptake2 was markedly different, with dl-epinephrme most potent, iso-

gen and Iversen, 1965). At 10-7 M concentrations l-metaraminol was the most active, followed by dopamine, 1-NE and l-epmephrme. I-NE was more active than the d-isomer, and dl-lsoproterenol was the weakest, producing no inhibition of NE uptake at 10-7 M. in rats treated with 6-HD, the NE analogs were about 100 times less efficient in reducmg N'E accumulation, requirmg 10-s M concentrations to produce effects comparable to those obtained m control rats. More-

Table 6 Inhibition of 3H-normetanephrme and 311-norepmephrme uptake into cerebral cortical slices by catecholamme analogs. 3tt-Normetanephrme, 30 sec uptake

3H-Norepmephrme, 5 mm uptake

Normal rats

Control rats g

6-11D treated rats g

iO 3 M

% Inhibition h

10 -7 M

% Inhibition h

10 -5 M

% Inhibition h

l-Epinephrine

61 -+4 (16)

dl-Metararnmol

45 + 3 (10)

d-Norepmephrme dl-lsoproterenol

41 + 4 ( I 0) 40 +- 5 (10)

dl-lsoproterenol a

46 +-4 (16)

Dopamme c

35 +- 3 (10)

l-Epinephrine

38 +-4 (9)

Doparnme

41 +-5 (18)

l-Norepmephrme

34 +- 3 (10)

l-Norepinephrme

35 - 5 (10)

dl-Metarammol

39 -+4 (18)

l-Epinephrine

31 +- 3 ( l 0)

Dopamme

35 + 4 (10)

d-Norepmephrme b

25 -+5 (15)

d-Norepmephrme d

25 +- 3 (10)

dI-Metarammol f

23 +- 3 (10)

l-Norepmephrme

23 -+5 (16)

dl-lsoproterenol e

0 +-5 (10)

a l-Epinephrine versus dlqsoproterenol: p < 0 02. b dl-Metarammol versus d-norepmephrme p < 0.05. dl-Metarammol versus dopamme p <0.05. d l-Norepmephrine versus d-norepmephrme p '~0 05 e d-Norepmephrme versu~ dl-lsoproterenol p <0.001 f Dopamme versus dl-metarammol p '~0.05. g Two 6-HD treated and two control rats were rejected lntraventrlcularly w,th 6-HD or with vehicle as described m footnotes to table 1 h Control T M ratios in the absence of mhlbitors were 1.5 +- 0.05 (18) for 311-NMN, 4.95 +-0.36 (10) for 3H-NE m control rats and 1.74 -+0.21 (10) for 3H-NE m 6-1tD rats. Figures m parentheses are the numbers of samples m each group.

E.D.Hendley, K.M. Taylor, S.H.Snyder, Normetanephrine uptake in brain

over, there was a marked difference in the relative activity of the various drugs. Metaraminol, the most potent inh~itor of NE uptake in control cortex slices was the least actwe in shces from 6-HD treated rats. There was no sigmficant difference in potency among the other agents. Thus, unlike control slices, d-and 1-NE were equally active m reducing 3H-NE accumulation m 6-HD treated rats. The pattern of inhibition of 3H-NMN uptake by the NE analogs resembled more closely the effects of these agents on 3H-NE uptake by 6-HD treated rats than by control animals, l-Epinephrine was the most active agent; dopanune and dl-metarammol were intermediate; and d- and l-NE were least active,

4. DISCUSSION A mechanism has been described for the accumulatlon of NMN in rat cerebral cortical slices to levels higher in the tissues than in the surrounding medium, It was not possible to determine whether this represented net uptake or exchange with endogenous stores. The characteristics of NMN uptake contrasted sharply with those of NE uptake: (1) initial uptake rates of NMN were more rapid than those of NE and steady-state levels were lower; (2)NMN accumulated in the tissues was more easdy washed out of tissues than was NE and it was distributed more in the cytoplasm than in particulate fractions; (3) NMN accumulatton was unaffected by reserpine; (4) the uptake of NMN was inhibited by desmethylirrupramine only at fairly high concentrations unlike the marked sensitivity to this drug of NE uptake;(5) the regional distribution of NMN uptake in the brain differed markedly from that of NE. The striatum had the highest T:M ratios for 3H-NE whereas the cerebral cortex showed the highest uptake of 3H-NMN; (6) although the uptake of NMN was saturable, as was NE uptake, the NMN transport had much higher K m and Vma x values; (7) unhke NE uptake, NMN accumulation was not greatly reduced by omission of glucose or sodium; (8) NMN uptake was temperaturedependent (Qlo was 1.6), but less so than NE uptake suggestmg that it may involve a facilitated diffusion rather than active uptake; (9) while cerebral cortex in vitro accumulates I-NE in preference to d-NE (Coyle and Snyder, 1969) (table 6) there was no stereo-

177

specificity for NMN uptake. In addition to the well known mtraneuronal uptake system for NE (Iversen, 1967) three other uptakes have been described that have a number of common characteristics: Uptake2 of NE in isolated rat hearts perfused with high concentrations of 3H-NE (lversen, 1965a); the extraneuronal uptake of 3H.NE in isolated rat hearts perfused with cocaine (Eisenfeld et al., 1967a, b, c) and the uptake of 3H-NMN in cocamlzed perfused rat hearts (Simmonds and Gillis, 1968) and in rat cerebral cortex shces m the present study. The findings reported here suggest that there ms a common mechanism for the three latter systems. The uptake of NMN in brain slices shares the following characteristics with the extraneuronal uptake of NE in heart and with Iversen's Uptake2 in heart: (1)initial uptake rates are very rapid; (2) the accumulated amines are readily washed out of the tissues. This was also observed in denervated rat salivary glands (Fischer et al., 1965) and in certain tissues of lmmunosympathectomized rats (lversen et al., 1966); (3) Uptake2 and NMN uptake are saturable with high values for K m and Vma x . However, the kinetic constants for NMN uptake in cerebral cortex were considerably higher than the constants for Uptake2 in perfused rat hearts (lversen, 1965a); (4) extraneuronal NE uptake in heart and NMN uptake in brain were poorly distributed into the particulate fraction of the tissues; (5)the rank order of analogs of NE as mhibitors of aH.NMN uptake was i-epinephrine > dl-isoproterenol = dopamine = dlmetararrunol> d-norepinephrine = 1-norepinephrme. Their rank order as inhibltors of Uptake2 was dlepinephrine > dl-isoproterenol > dl-norepinephrme > dopamine > l-metaraminol. With the exception that d- and I-NE were less potent than doparmne and dl-metarammol in the present study as compared wnh Uptakes, these analogs had similar relative afflmties for Uptakes m heart and for 3H.NMN uptake in brain. Although some adrenerglc blocking agents were the best mhibitors of 3H-NMN uptake reported m the present study in brain slices, phenoxybenzamine which was a potent inhibitor of Uptake2 m the heart (Lightman and Iversen, 1969) was a poor inhibitor of NMN uptake m cerebral cortex. Another discrepancy between Uptakes m the heart and NMN uptake in brain is the higher affinity ofdl-NMN for Uptake2 in

178

E.D.ltendle~'. K.M. Taylor. S H Snyder, Normetanephrine uptake m bram

heart (IDso = 4 . 2 × 10-6M, Burgen and lversen, 1965) than for aH-NMN uptake in cerebral cortex (K m = 2.1 X 10 -3 M). In the cerebral cortex o f rats treated with 6-HD, endogenous NE was reduced 8(Y~ and a H-NE uptake was decreased 5 0 - 8 0 % . Thus this treatment appeared to have depleted a large proportion o f noradrenergic nerve terminals, although presumably about 20% remaaned intact. Several characteristics o f 3H-NE uptake into cerebral cortex o f rats pretreated with 6-HD closely resembled features of NMN uptake: (1) high concentrations o f NE analogs were required to inhibit uptake o f 3H-NE; (2) I-NE and d-NE were equally potent in reducing a H-NE uptake; (3) l-Epinephrlne and dl-isoproterenol were more potent inhibitors o f aH-NE accumulation than dl-metaraminol, the analog with highest affinity for the NE uptake system in normal tissue. Since the number o f intraneuronal catecholamine uptake sites is markedly reduced by 6-HD and uptake mto such tissues can therefore be considered to be mainly extraneuronal, the resemblances between NMN uptake in normal cortex and NE uptake in 6-HD treated cortex were taken as additional evidence supporting the hypothesis that NMN uptake involves the sites o f extraneuronal NE uptake, The implications of an extraneuronal uptake of NE have already been suggested by Eisenfeld and coworkers, namely that agents which interfere with entry o f NE into extraneuronal sites would regulate the accessibihty o f NE for attachment to extraneuronal receptor sites and also the rate of its extraneuronal metabolism. Kalsner and Nickerson (1969) suggested that extraneuronal uptake o f NE is a major site o f inactivation o f the transmitter at sympathetic nerve endings. Moreover, Lightman and lversen (1969) found that Uptake2 occurs at low concentrations o f NE that we have employed and that at these concentrations Uptake2 accounts for a substantial proportion o f NE accumulated b y perfused rat heart, The failure to detect Uptake2 at low concentrations in earlier studies (Iversen, 1965a)was due to metabolism o f tlae accumulated amine at low concentrations. Thus it is possible that extraneuronal uptake o f NE in brain as elucidated by the NMN uptake system described herein may also play a significant role in inactivating transmitter NE in brain.

ACKNOWLEDGEMENTS The authors acknowledge the excellent techmcal assistance of Miss Molly J. Ruble and the helpful suggestions of Dr. Leshe L. lversen. REFERENCES Bloom, F.E., S. Algeri, A. Groppettl, A. Revuelta and E. Costa, 1969, Lesions of central norepmephnne termmals with 6-Otl-dopamine: Biochemistry and fme structure, Science 166, 1284-1286. Burgen, A.S.V. and L.L. lversen, 1965, The irthibltion of noradrenalme uptake by sympathomtmetic amines In the rat isolated heart, Brit. J. Pharmacol. 25, 34 -49. Clarke, D.E. and C.J. Jones, 1969, Are adrenergic nerves required for Uptake-2 in the Isolated perfused rat heart ~, European J. Pharmacol. 7, 121-124. Coyle, J.T. and S.II. Snyder, 1969, Catecholamlne uptake by synaptosomes m homogenates of rat brain Stereospecff~city in different areas, J. Pharmacoi. Exptl. Therap. 170, 221-231. Dengler, J.J., i.A. Michaelson, H.E. Spiegel and E. Titus, 1962, The uptake of labeled norepmephrme by isolated brain and other tissues of the cat, Intern. J. Neuropharmacol. 1,23-38. Eisenfeid, A.J., L. Krakoff, L.L. lversen and J. Axelrod, 1967a, Inhibition of the extraneuronal metabolism of noradrenalme m the isolated heart by adrenerg~c blocking agents, Nature 213,297-298. Eisenfeid, A.J., J. Axelrod and L. Krakoff, 1967b, lnhibmon of the extraneuronal accumulation and metabolism of norepinephrme by adrenergic blocking agents, J. Pharmacol. Exptl. Thelap. 156, 107-113. E~senfeld,A.J., L. Landsberg and J. Axelrod, 1967c, Effect of drugs on the accumulatton and metabolism of extraneuronal norepinephrme in the rat heart, J. Pharmacol Expfl. Therap. 158,378--385. Farnebo, L.-O. and T. Malmfors, 1969, lhstochemlcai studies on the uptake of noradrenaline and o~-methyl-noradrenaline in the perfused rat heart, European J. Pharmacol 5, 313-320. Fischer, J.E., l.J. Kopin and J. Axelrod, 1965, Ewdence for extraneuronal binding of norepinephrine, J. Pharmacol Exptl. Therap. 147, 181-185. Glowinski, J., l.J. Kopin and J. Axelrod, 1965, Metabohsm of H3-norepinephrine m the rat brain, J. Neurochem. 12, 25-30. Glowinski, J. and L.L. lversen, 1966, Regional studies of catecholammes m the rat brain. I. The disposinon of [3HJ-norepinephrme, [3H]-doparame and [3H]-DOPA m various regions of the brain, J. Neurochem. 13,655-669. Hamberger, B., 1967, Reserpine-resistant uptake ofcatechoiamines m isolated tissues of the rat, Acta Physiol. Scand. Suppl. 295, 7-- 56.

E.D.Hendley, K.M. Taylor, S.H.Snyder, Normetanephrine uptake in brain Hendley, E.D. and S.H. Snyder, 1968, Relationship between the action of monoamine oxadase inhibitors on the noradrenaline uptake system and their antidepressant efficacy, Nature 220, 1330-1331. Iversen, L.L., 1963, The uptake of noradrenaline by the isolated perfused rat heart, Brit. J. Pharmacol. 2 1 , 5 2 3 - 5 3 7 . lversen, L.L., 1965a, The uptake of catecholammes at high perfusion concentrations in the rat isolated heart: a novel catecholamme uptake process, Brit. J. Pharmacoi. 25, 18-33. Iversen, L.L., 1965b, Inhibition of noradrenaline uptake by drugs, J. Pharm. Pharmacol. 17, 6 2 - 6 4 . lversen, L.L., 1967, The Uptake and Storage of Noradrenahne m Sympathetic Nerves (Cambridge University Press, New York). lversen, L.L., J. Glowmskl and J. Axelrod, 1966, The physiological disposition and metabolism of norepinephrme m immunosympathectomized animals, J. Pharmacol Exptl. Therap. 1 5 1 , 2 7 3 - 2 8 4 . lversen, L.L. and M.J. Neal, 1968, The uptake of [3HIGABA by slices of rat cerebral cortex, J. Neurochem. 15, 1141-1149. Kalsner, S. and M. Nickerson, 1969, Effects of a haloalkyiaimne on responses to and disposition of sympathomtmetic amines, Brit. J. Pharmacoi. 3 5 , 4 4 0 - 4 4 5 . Krebs, H.A. and K. Henseleit, 1932, Untersuchungen uber die ttarnstoffbddung im Tlerkorper, Z. Physiol. Chem. 210, 33-66. Kuhax, M.J. and S.tl. Snyder, 1970, The subcellular distributlon of free 3 H-glutamtc acid m rat cerebral cortical shces, J. Pharmacol. Exptl. Therap. 171,141 - 152. Laverty, R. and K.M. Taylor, 1968, The fluorometric assay of catecholammes and related compounds: Improvements and extensions to the hydroxyindole technique, Anal. Blochem. 32, 2 6 9 - 2 7 9 . Lightman, S. and L.L. lversen, 1969, The role of Uptakea m the extraneuronal metabolism of catecholamines in the isolated rat heart, Brit. J. Pharmacol. 3 7 , 6 3 8 - 6 4 9 . Mcllwain, H. and R. Rodnight, 1962, Practical Neurochemistry (Little, Brown, Boston).

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Miller, L.C. and M.L. Tainter, 1944, EsttmaUon of the EDso and its error by means of logarithmic problt graph paper, Proc. Soc. Exptl. Biol. Med. 5 7 , 2 6 1 - 2 6 4 . Ross, S.B. and A.L. Renyl, 1964, Blocking actton ofsympathomimetlc amines on the uptake of tntiated noradrenaline by mouse cerebral cortex tissues tn vttro, Acta Pharmacoi. Toxicol. 2 1 , 2 2 6 - 2 3 9 . Simmonds, M.A. and C.N. Gilhs, 1968, Uptake of normetanephrine and norepmephrme by cocaine-treated rat heart, J. Pharmacol. Exptl. Therap. 1 5 9 , 2 8 3 - 2 8 9 . Snedecor, G.W. and W.G. Cochran, 1967, Statistical Methods (Iowa State University Press, Ames). Snyder, S.H. and J T. Coyle, 1969, Regional differences in H3-norepmephrme and H3-dopamme uptake into rat brain homogenates, J. Pharmacol. Exptl. Therap. 165, 78-86. Snyder, S.H., L. Fadlace and L. Holhster, 1967, 2,5-Dimethoxy-4-methyl-amphetamme (STP): a new haliucmogenic drug, Science 1 5 8 , 6 6 9 - 6 7 0 . Snyder, S.H., A.I. Green and E.D. Hendley, 1968a, Kinetics of H3-norepmephrme accumulation into slices from dlfferent regions of the rat brain, J. Pharmacol Exptl Therap. 164, 9 0 - 102. Snyder, S.H., L.A. Faillace and H. Wemgartner, 1968b, DOM (STP), a new hallucmogemc drug, and DOET: Effects m normal sublects, Am. J. Psychiat. 1 2 5 , 3 5 7 - 3 6 4 . Taylor, K.M. and R. Laverty, 1969, The metabohsm of trit,ated dopamine m regions of the rat brain tn vivo I. The separation of catecholamines and their metabohtes, J. Neurochem. 16, 1361 - 1366. Tranzer, J.P. and H. Thoenen, 1968, An electron microscopic study of selective, acute degenerataon of s3rmpathetic nerve terminals after admimstration of 6-hydroxydopamme, Expenentm24, 155-156. Uretsky, N.J. and L.L. lversen, 1969, Effects of 6-hydroxyJopamme on noradrenalme-contammg neurones m the rat brain, Nature 2 2 1 , 5 5 7 - 5 5 9 . Uretsky, N.J. and L.L. lversen, 1970, Effects of 6-hydroxydopamme on catecholamme containing neurones in the rat brain, J. Neurochem., m press.