the subcellular distribution of some monoamines following their intraventricular injection into the...

The Subcellular Distribution of Some Monoamines Following their Intraventricular Injection into the Rat

ALAN A. BOULTBN AND GLEN B. BAKER Pvchiatric Research Unit, University Hospital, Saskntoon, Saskntcllewan S7N 0 W 8

Received October I , I973

Boulton, A. A. & Baker, G. B. C 1974) The Subcellular Distribution of Some Msnoamines Following their 1ntraventricular Injection into the Rat. Can. 1. Biocherat. 52, 288-293

The subcellular distribution of radioactively labelled p-tyramine, octopamine, p-phenylethylamine, tryptamine, arid N-methyltryptamine, 38 min after their intraventricuIar injection, was investigated and compared with the distributions of equimolar amounts of the putative transmitters noradrenaline, dopamine, and 5-hydroxytryptamine injected under identical conditions. The amount of each labelled amine, and any formed labelled amine metabolites, in each subcellular fraction was assessed using chromatographic techniques. The effect of the presence of Na+ and various buffer solutions on the distribution of radioactivity between particulate and supernatant fractions, particularly with respect to tryptamine, was investigated.

Boulton, A. A. & Baker, 6. B. ( 1974) The Subcellular Distribution of Some Monoamines Following their Intraventricular Injection into the Rat. Can. J . Biochem. 52,288-293

Trente minutes aprks leur injection intravemtriculaire, nous avons reckerchC Ia distribution subcellulaire des amines marquCes suivantes : p-tyramine, octopamine, p-phCnyH- Cthylamine, tryptamine et N-mCthyltryptarnine et nous avons compark cette distribution B e l l e de quantitCs Cquimolaires de composCs dits midiateurs : noradrCnaHine, dopamine et 5-kydroxytryptamine, inject& dam les mernes conditions. A I'aide de techniques chromatographiques, nous avons CvaluC dans chacune des fractions subcellulaires, le taux de toutes les amines marquees et de tous les mCtabolites aminis marquis formCs. Nous avons CtudiC l'effet de la presence du Na+ et de divers tampons sur la distribution de la radio- activit6 dans les fractions particulaire et surnageante, particuli~rement pour ce qui est de la tryptarnine. [Traduit par le journal]

Introduction in vikro and in viva (21 ), and a preliminary Recently, attention has become focused on investigation of the subcellular distribution of

the aryl alkyl amines and the phenolic enes tryptamine isolated from whole rat brain homog-

because some of them are thought to be hpl i - enates indicated its association with the myelin

cated in some neurological and psychiatric and synaptosomal fractions. Because of this we

disease states (1-5) ; they have been identified decided to investigate more thoroughly9 and on

in brain (6-10) and some of the enzymes a comparative basis, the subcellular distribution

utilized in the of the catecholamines of a number of amines following their intra-

also methylate, hydroxylate, and these ventricular introduction into the rat brain.

other monoamines. It has dso been known for some time that tryptamine and phenylethylamine possess excitatory and mgketamine-like properties (5, 1 1-1 3 ) . Methylated derivatives of many of these amines are potent hallucinogens and methylated indoleamines are synthesized in vkero and in vivo ( 14-1 9). It has been postulated that octopamine and pkenylethanolamine might act as co-neurotransmitters (4,20).

Previous work from our laboratory has indicated that tryptamine, and to a lesser extent phenylethylamine and p-tyramine, are capable of forming relatively stable complexes with particulate materials isolated from rat brain both

Materials and Methods Chemicals and Solvents

A11 chemicals and solvents were of the purest grade commercially available. Aqueous solutions were prepared from water distilled in a glass apparatus. Buffer solutions were prepared as described by Gomori (22). Pargyline was kindly donated by Abbstt Laboratories, North Chicago, 111.

Tryptamine-2-"C bisuceinate (specific activity 60 mCi/rnmol) , p-phenylethylamine- I -14C hydrochloride (spec. act. 7 mCi/mmol), and ~,~-octoparnine-2-'H (spec. act. 2.89 Ci/mmol) were purchased from the New England Nuclear Corporation, Boston, Mass.; and dopamine-(ethylamine-d-14C) hydrochloride (spec. act. 55 mCi/mmol) , I -noradrenaline- (methylene-"C)

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BOULTON AND BAKER: SUBCELLULAR DISTRIBUTION OF MONOAMHNES

'TABLE 1. Monoamine oxidase, acetylcholinesterase and protein contents of myelin, mitochondria, and synaptosome fractions isolated from whole rat brain

Monoamine oxidase Acetylcholinesterasa: Protein

Myelin 0.27k0.01 0.61 k0.04 23.6kl .2 Synaptosomes 0.93 k0.03 1.44L-0.08 52.7k2.7 Mitochondria 1.90k0.06 0.38+_0.02 23.7kl .8

NOTE: The data prcsentcd in this table represent the fractions P2A (nayelin), P2B (synaptosomes), and P2C (mitochondria) isolated by density gradient separation of the fraction labelled Pn by Whittaker. The proteins are expressed as percentage of the total recovered from the three subfractions (mean + standard deviation, rt = 6) and the enLynies as the percentage of P2 enzyme activity in each subfractio~l divided by the percentage of Pa protein in that subfraction (mean + standard deviation, ra = 3). The percentage protein distribution in the initial fractions, identified as PI, P?, P3, and Sl by Whittaker werc P I , 24.9 + 2.0; P2, 35.1 +_ 1.6; P.;, 12.7 9_ 1 . 3 ; and Sr, 25.4 k 1.2 (mean + standard deviation, ?a = 61, respectively.

bitartrate (spec. act. 57 mCi/mmol), 5-hydroxytryptamine-3-'C creatinine sulphate (spec. act. 57 mCi/ mmol ) and p-t yramine- 1 -14C hydrochloride (spec. act. 44 mCi/mmol) were purchased from the Amersham/ Searle Corporation, Don Mills, Ont. N-methyltryptamine-l'@ (spec. act. 7.5 mCi/mmol) was kindly donated by the Merck Institute for Therapeutic Re- search, Rahway, N.J.

Purificatis~~ of Radioactive Saabstrures All the labelled amines were checked by radio-

chromatography for purity; those exhibiting peaks other than the amine under consideration were purified. Tryptamine, identified by a synthetic sample run along- side and visualized with p-dimethylaminocinnamalde- hyde reagent (23 ) after overnight development in isopropanol - ammonia (10% ) - water, 200: 10:2Q (v/v), was eluted from a Whatman No. 2 strip in 95% methanol containing six drops of acetic acid per 180 ml, dried under a stream of N2, dissolved in saline solution, frozen, and used within 24 h. 5-Hydroxytrypt- amine (5-HT) was isolated similarly except that the solvent used was butanol - acetic acid - water, 12: 3 : 5 (v/v) . The purification of g-tyramine. octopamine, and dopamine was as previously described (24). p-Phenyl- ethylarnine was isolated after a 14 h separation on Whatman No. 2 paper strips in the solvent system isopropyl ether - methyl ethyl ketone - acetic acid - water, 9: 1:5:5 (v/v) by elution in the methanol - acetic acid solution followed by drying under a stream of N,.

Bnj~ction and SzlbceNular Frnctionatio~a Procedures Male Wistar rats (body weight approximately 175-

200 g) were injected intraperitoneally with pargyline (75 mg/kg). Forty-five minutes later the appropriate labelled amine (0.02 pmol) dissolved in 40 pl ghysio- logical saline was injected into the lateral ventricle during light ether anesthesia. After 30 min the rats were stunned and decapitated and the brains removed, chilled, and homogenized in 0,32 A4 sucrose in a variety of buffer solutions (see Tables 3-5). The isolation of the subcellular fractions was according to Whittaker (25), except that the PI pellet was washed twice with an appropriately buffered 0.32 M sucrose solution and the PB fraction isolated by centrifi~gation of the supernatant and washings at 10 000 x g for 30

min. The P2 pellet was then washed once by resuspension in the appropriate buffered 0.32 M sucrose solution followed by centrifugation at 10 000 x g for 30 min.

The isolated myelin, synaptosomal, and mitochondrial fractions were characterized by electron microscopy and the determination, in the absence of pargyline, of monoamine oxidase (26) and acetylcholinesterase (27). Data on the enzyme and protein distribution are listed in Table I. Since the morphology of the fractions as revealed by electron microscopy was closely similar to that reported by Whittaker (281, the electron micrographs have not been included here. Protein was determined according to the procedure described by Lowry et al. (29).

Isolation and As.~essnzent o f Amhe and Atnine Metabolites

To compare the subcellular distribution, equimolar quantities of amines were injected. To characterize likely metabolites, additional experiments were performed, and in the case of p-phenylethylamine and N- methyltryptamine larger quantities (0.2 pmol ) were injected on account of their low specific activity. In all other cases metabolites were isolated from each subcellular fraction after pooling samples obtained from several different rats. Because of the presence of pargyline, acidic metabolites were not considered. Metabolites arising from noradrenaline and doparnine were isolated and identified according to the procedures described by Kopin et al. (30). Subcellular fractions isolated following injection of all the other amines were homogenized in 0.4 N HCIOl containing Triton X-100 (0.2% v/v). supplemented with EDTA and ascorbic acid in the case of 5-hydroxytryptnrnine (3 1 1. The homogenates were then centrifuged at 10 008 X R for 20 min (I.E.C. B-20 centrifuge), the supernatant was adjusted to pH 7.0 and percolated through a Biorad AGSOWX2 column as described by Kakimoto and Armstrong (32). In the case of p-phenylethylamine the amine eluate was obtained using methanol- #el, 73 :27 (v/v) (6) and for tryptamine, 5-hydroxytryptamine, and N-methylt~grptarnine the column was washed with 0.1 M Tris buffer (pH 8.0) rather than 0.1 M sodium acetate and the amines were eluted in 4 N ammonium hydroxide in 65% ethanol.

Metabolites of p-tyramine and octopamine (octop-

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280 CAW. 1. BIOCAEM. VBL. 52, 1974

amine and synephrine) were assessed, after transfer- ring the dried column eluates in methanol-HC1 solution (2 x 100 ~ 1 ) to the origin of a Whatman No. 4 paper strip, by overnight separation in lert-amyl alcohol - ammonia, 4: 1 (v/v) followed, after drying, by visuali- zation with p-nitroanaline reagent (33). The distribution of radioactivity on the chromatogram was then assessed in a radiochromatogram scanner (Packard model 6061 ). Zones possessing significant amounts of radioactivity were then assessed by eluting the zones (obtained from chromatograms not treated with p- nitrsaniline) in 90% methanol which, after drying and trituration in 1.5 ml 0.4 N HC104, were mixed with 15 ml Aquasol (New England Nuclear Corporation) and counted in a liquid scintillation counter (Nuclear Chicago Isocap 300).

The metabolites of tryptamine and N-methyltryptamine (N-methyl- and N,N-dimethyltryptamine) were transferred to chromatograms in 70% ethanol and then separated and assessed as described by Wu and Boulton (15). In the case of 5-hydroxytryptamine the solvent system used to separate it from N-acetylJ-hydroxytryptamine on Whatman No. 2 paper strips was butanol - acetic acid -water, 12:3:5 (v/v).

Phenylethylamine and the phenylethanolamine that might arise from it were isolated from the dried column eluate by dissolving the eluate in 3 ml ethanol, centri- fuging at 1OOO X g for 1 min to remove salts, trans- ferring the supernatant (after drying and resuspension in ethanol) to a Whatman No. 2 paper strip, and separating overnight in isopropyl ether - methyl ethyl ketone - acetic acid - water, 9: 1 :5:5 (v/v). The amine zones were visualized with ninhydrin and then scanned, and the amount of radioactivity assessed as previously described.

Results and Discussion Rats were killed and subcellular fractions

isolated 30 min after intraventricular injection because this seems to correspond to a period of slower loss of amines (as assessed from turnover rates ( I 5, 34) ), significant amounts of radioactivity still remained in most cases and presum- ably any formed metabolites would exist in a state of equilibrium. Table 2 lists the amount of total radioactivityopresent in the brain after this time interval. It can be seen that approximately 40% of the activity from the hydroxylated amines remained. As expected, the retention of P-ghenylethylamine was much lower presum- ably because of the great permeability of this amine to the blood-brain barrier (B.B.B. ) (3 5). Since tryptamine can also cross the B.B.B. with ease it was somewhat surprising to find that it was retained at about the 40% level. This may be because of the ability of tryptamine to bind to particulate fractions (21 ). The differential loss of some of the amines makes a direct

TABLE 2. Radioactivity (percentage of total injected) recovered from whole rat brain 38 rnin after

intraventricular injection

Amount recovered (5%:ro)* - - - -

5-Hydroxytryptamine Tryptamine N-methyltryptamine Noradrenaline Bctopamine Doparnine Tyramine 8-Phenylethylamine

'Mean f standard deviation (n = 6).

comparison of the amounts remaining in the subcellular fractions difficult even though equimolar amounts were injected. Consequently the amount present in each subcellular fraction has been calculated as a percentage of the total radioactivity recovered from the whole brain homogenate prepared 30 min after the injection.

Earlier studies have shown that some mines possess the ability to form complexes with particulate material (21 ) and that the amount so complexed varies with the conditions of isolation. Accordingly, the retention of tryptamine in the particulate and supernatant fractions isolated from whole rat brains homogenized in a variety of buffered sucrose solutions was investigated. The composition, molarity, and presence or absence of Na+ ions of the pH '7.4 buffered solutions used in the homogenizations are listed in Table 3. It is apparent that sucrose solutions in glass distilled water and the 2.5 mM Tris yielded the highest radioactive content in the synaptosomal, mitochondrial, myelin, and microsomal fractions. High molarities and the presence of Na+ ions on the other hand resulted in the bulk of the label being recovered from the supernatant and the nuclear debris fractions. Concomitant with the reduction in recovered particulate radioactivity was a decrease in the physical size of the myelin, synaptosomal, mitochondrial, and microsomal pellets, particularly the mitochondria. Clumping of synaptosomes in the presence of metal ions has been noted by others (36, 37) and in those cases where Na+ was present it possibly caused some coacervation of the subcellular particles with resultant altera- tion in their sedimentation properties.

In the case of the 5 m M sodium phosphate buffer, the use of double-distilled water (distilled

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BQULTON AND BAKER: SUBCELLULAR DISTRIBUTION OF MBNBAMINES 291

TABLE 3. The effect of the composition of various buffered sucrose solutions on the subcellular distribution of tryptamine-I4C following intraventricular injection

Buffer Nuclear debris Myelin Synaptosomes Mitochondria Microsomes Supernatant

2.5 mM Tris 10 mM Tris Distilled H20 5 mM Sodium phosphate 10 mM Tris+-5 mM Na+ 100 m M Tris* 108 mM Tris+5 mM Na+* 1 0 0 mM Tris-+-100 mM Na+* 10 mMTris-f-100 mM Na+*

*The high osmolarity of these buffer solutions undoubtedly has an effect o n the composition o f the particulate fractions listed in the table. NOTE: Values are mean f standard deviation (n = 3).

TABLE 4. Subcellular distribution of several monoarnines following intraventricular injection

Amine Nuclear debris Myelin Synaptosomes Mitochondria Microsomes Supernatant

5-Hydroxytryptamine 9.8 5 0.8 Tryp tamine 10.3f 0 .5 N-methyltryptamine 9.1 f 0 .5 Nor adrenaline 6 .7 f 0 .4 Betopamine 6 . 8 f 0 . 6 Bopamine 17.5f 0 . 6 p-Tyramine 5 . 9 1 0 . 3 ~Phenylethylamine 11.9f 0 .4

NOTE: All fractions were isolated in sucrose buffered with 2.5 mM Tris at pH 7.4. Values are mean f standard deviation (n = 3).

in presence of potassium permanganate to oxidize traces of mines) rather than glass distilled water resulted in larger particulate fractions. This increase was most noticeable with the mitochondria1 pellet, but this tan-colored pellet was highly contaminated with a buff- colored layer, possible representing clumped synaptosomes. Glass-distilled or double-distilled water was equally suitable for the preparation of the 2.5 mM Tris buffer solution.

Because of the difficulty of maintaining sucrose dissolved in distilled water at a constant pH, osmotically stabilized 2.5 m M Tris was selected as the best disruption medium and was used in most subsequent studies. This choice was based on the comparative studies on tryptamine binding indicated in Table 3, but the general effects of high molarity and high concentrations of Na+ in causing a shift from the particulate to solution was demonstrated for each of the amines used, as indicated by a comparison of the data in Tables 4 and 5. In the buffer containing high Na+ concentration, no attempt was made to maintain the osmotic pressure equiva- lent to that of 0.32 M sucrose, and so some of the observed effects are probably a consequence

of hypertonic conditions. The effect of these harsher disruptive conditions on the nuclear debris is more complicated since, in some cases (5-HT, tryptamine, IV-methyltryptamine, dopamine, p-tyramine, and phenylethylamine ) , the amount of label associated with this fraction decreased, whereas in the other cases (noradrenaline and octopamine) the amount in- creased. The reason for this is not clear, although noradrenaline and octopamine are the only amines hydroxylated in the alkyl side chain. In any case, it is clear that considerable care is required in the selection of disrupting media and the maintenance of stable conditions when preparing subcellular fractions. Consequently, the experimental details must be specified when reporting on binding and/or subcellular experiments.

In the comparative study shown in Table 4 it is apparent that all the amines are retained at significant levels in all the particulate fractions. In particular the amount of m i n e retained in the synaptosomes varied in the range 3.&- 12.8 % with 5-HT, tryptmine, noradrenaline, and octopamine being the highest (9.0-1 2.8 % ) and p-tyramine, dopamine, phenylethylamine,

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292 CAN. J. BYBGHEM. VBk. 52, 1954

TABLE 5. Subcellular distribution of several rnonoarnines following irntraventricular injection and isolation in sucrose containing 10 mM Tris and 180 rnM sodium acetate*

Amine Nuclear debris Myelin Synaptosornes Mitochondria Microsornes Supernatant

0.27L0.02 0.30k0.04 0.45f 8.07 0.25k0.03 8.2410.02 8.23 k0.03 0.23 10.03

N.D.

0.0410.00 0.6610.05 0.03f 0.00 O.88k 0.02 0.04fO.W 1.08+0.18 0.03&0.00 0.88kO.lO 0.29k0.02 0.78k0.20

N.D. 8.78 10.08 N.B. 8.23 f 0.02 N.D. N.D.

*Despite the high NaC concentration and hyperosmolarity, the particulate rractions in the table have k e n denoted, for sonve~lience, as the fractions sedimenting in the same position under isoosmcstic conditions.

NOTE: N.B., not detected. Values arc mean f standard deviation ( r t - 3).

and N-methyltryptamine (3.8-5.9 96 ) the lowest. The fact that the P2 pellet was isolated at 18 000 x g in our experiments rather than at a higher value and that many of the mossy- fiber endings arising from the cerebellar cortex probably sedimented with the nuclear debris (37) may have resulted in a reduction in the amount of radioactivity associated with the syrmaptossmal fraction. As can be seen from Table 4 a significant amount of label was associated with the myelin and mitoehondrid pellets. Association of catecholamines and 5- hydroxytryptamine with these fractions has been demonstrated both in vivo and in vitr-0 by others ( 3 8 4 0 ) . Such an association should be considered when the crude P2 pellet is used to represent the synaptosomal fraction in uptake and binding studies.

It is not unexpected that significantly more of the indolic amines were found in the myelin fraction. This may reflect either lipid solubility (cf. Fischer ek a/. (41 ) who have reported that the diamines putrescine and spermidine are found in the myelin sheath) or the formation of amine-lipoprotein complexes f 2 1 ) . This latter phenomenon may also explain the relatively high number of counts found to be associated with the microsomes for all of the amines.

It has been assumed that the presence of a monoamine oxidase inhibitor effectively pre- vents significant amounts of oxidation. Other metabolic interconversions, however, occur. The level of unchanged amine and the amount of other formed amines was assessed as described in Materials and Methods. The ratio of p- tyramine to octopamine 30 min after injection of p-tyramine was 9 : 1 in the nuclear debris, 3 : 1 in the synaptosomes, 1 5 : 1 in the high speed super-

natant, and 1 8 : 1 in the microssmes. No oetop- amine was detected in the myelin or mito- ehondrial fractions. After injection of octopamine itself all of the label in all of the fractions was octopamine-3H.

Following injection of tryptamine, N-acetyl- tryptamine was not identified in any fraction, thus confirming earlier observations in whole brain ( 1 5 ) . Smdl amounts of N-methyltryptamine (about 1 % ) were detected in the supernatant, nuclear debris, and synaptosomes and tiny amounts (0.1-0.2 % > of N,N-dimethyltryptamine in the supernatant. In the case of injection of N-methyltryptamine, about 1.5 % of the counts in the high speed supernatant and 0.5% of the counts in the nuclear debris and synaptosomes were located in the chromato- graphically separated N,N-dimethyltryptamine zone.

The ratio of dopamine to noradrenaline to 3-methoxytyramine 30 min after injection of dopamine was 118~2: 1 in the supernatant, 53 : '1 3 : 1 in the synaptosomes, 92 : 5 : 3 in the microsomes, and 85: 15:s in the nuclear debris? whereas after injection of noradrenaline the noradrenaline to normetanephrine ratios were respectively 3 : 1 in the supernatant, 18: 1 in the synaptosomes, 8 : 1 in microsomes, and 10: 1 in the nuclear debris.

5-Hydroxytryptamine appeared unchanged in all fractions. P-Phenylethylamine also appeared unchanged except that about 0.4% of the counts in the supernatant &action migrated on chromatography along with phenylethanolarnine. The above data indicate that in most cases the radioactivity listed in Tables 3-5 was essentially that of the amine intraventricularly injected.

The ability of several didferent amines to

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BBULTQN AND BAKER: SUBCELLULAR DISTRIBUTION OF MONBAMINES 293

distribute themselves and locate in the same subcellular fraction raises the question as to whether they all become stored or bound in the same or different neurones, and whether or not they might perform similar functions. I t is already known that some amines possess the ability to release other amines but the mechan- ism of this action is not clear. Studies designed to investigate the ability of one amine to inhibit or potentiate the subcellular binding of another would be interesting both from an in vitro as well as in vivo point of view. Such experiments could also be performed using subcellular fractions isolated from different cerebral regions. It is also important to realize that all of the data presented in this paper were obtained in the presence of the monoamine oxidase inhibitor pargyline. The effect that this substance may have on the storage, release, and metabolism of the various amines mentioned above is inadequately under- stood. It is very likely, however, that pargyline will affect different amines differently in their various subcellular and regional locations.

We thank the Psychiatric Services Branch, Depart- ment of Health, Province of Saskatchewan, and the Medical Research Coumcii of Canada for continuing financial support.

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the subcellular distribution of some monoamines following their intraventricular injection into the...

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