Journal of Nrurochemistry, 1977. Vol. 29, pp 1105-1 109. Pergamon Press. Printed in Great Britain.

IDENTIFICATION OF 5-HYDROXYTRYPTAMINE BINDING SUBSTANCES FROM RAT BRAIN STEM’

DAVID A. JOHNSON, TAE MOOK CHO and HORACE H. LOH Langley Porter Neuropsychiatric Institute and Departments of Pharmacology and Psychiatry,

University of California, San Francisco, CA 94143, U.S.A.

(Received 18 November 1976. Accepted 14 May 1976)

Abstract-GODWIN & SNEDDON (1975) reported the binding of 5-hydro~y-[~H]tryptamine (5-HT) on a Sephadex LH-20 column to ‘proteolipid material’ extracted with n-butanol from rat brain stem. An examination of this ‘proteolipid material’ with TLC showed the main constituents to be cerebroside sulfate (CS), monophosphoinositide (PI), and diphosphoinositide. The elution profiles of C3H]5-HT incubated with purified CS or with a mixture of CS and PI were similar to that of the brain extract on the same column. Because the elution profile of the mixture of CS and PI was more similar to that of the brain extract, it was concluded that what was suggested to be a possible proteolipid ‘5-HT receptor’ was mainly two acidic lipids. The elution profile of C3H]5-HT incubated with purified PI, however, was similar to C3H]5-HT eluted alone. This suggested that either PI did not bind to 5-HT or that the PI-5-HT complex possesses different chromatographic behavior than PI. To test this latter possibility, [“‘C]5-HT and C3H]PI were incubated then eluted on a Sephadex LH-20 column with a continuous gradient of increasing polarity. The gradient first eluted PI, then an apparent PI-5-HT complex, and finally 5-HT. This demonstrated that PI will bind to 5-HT on a Sephadex LH-20 column and that the PI-5-HT complex is probably more polar than PI.

FOLLOWING incubation of C3H]5-HT with butanol- water extracts of rat brain stem and resolution of the components with Sephadex LH-20 chromatography, a peak representing approx 70% of the eluted radioac- tivity has been found in chloroform-methanol (CM) 4 : l (v/v) (FISZER & DE ROBERTIS, 1969; GODWIN & SNEDDON, 1975). Based on the partial retention of [3H]5-HT incubated with butanol brain extracts on a TLC plate and based on the presence of Lowry- positive material in the major peak of radioactivity, FISZER & DE ROBERTIS (1969) concluded that 5-HT was binding to a special proteolipid. This conclusion was further supported by the observation that the 5-HT binding material was precipitated by ice-cold ether, a procedure known to precipitate protein, and by the failure to find this same peak in identically prepared extracts from rat diaphragm (GODWIN & SNEDDON, 1975). However, inconsistent with a proteo- lipid type of 5-HT binding material was the failure to observe any clear relationship between the major peak of C3H]5-HT and a discrete peak of protein and the inability to saturate the C3H]5-HT binding peak even in the presence of 5 x 1 0 - 5 ~ - 5 - H T (GODWIN & SNEDDON, 1975).

Since our laboratory has demonstrated that what was reported to be a proteolipid receptor for opiates based on Sephadex LH-20 chromatography was actually the acidic lipid cerebroside sulfate (LOH et

This is Publication No. 77-10 from the Department of Pharmacology, University of California, San Francisco.

Abbreviations used: CS, cerebroside sulfate, PI, mono- phosphoinositide; 5-HT, 5-hydroxytryptamine.

al., 1974), and has shown that 5-HT binds saturably and with high affinity to acidic lipids (JOHNSON et al., 1977), we examined the major C3H]5-HT binding peak for acidic lipids. Following the procedure out- lined by GODWIN & SNEDDON (1975), we detected cer- ebroside sulfate (CS), phosphoinositol (PI), and a trace amount of diphosphoinositide in the major C3H]5-HT binding peak. Incubation of C3H]5-HT with the same amount of CS and PI in the n-butanol- water brain extract followed by Sephadex LH-20 chromatography produced an elution pattern very similar to that observed with the n-butanol brain extract.

MATERIALS AND METHODS

Compounds. Different batches of 5-hydroxylside ~hain-l,2-~Hltryptamine were purchased from New Eng- land Nuclear (Boston, MA), ranging in specific activity from 5 to 26.7 Ci/mmol. [U- ’4C]M~n~pho~pho in~~ i t ide with a specific activity of 5mCi/mg was purchased from Applied Sciences Lab (Inglewood, CA). Cerebroside sulfate (bovine brain) was purchased from Analabs (North Haven, CT), and 5-HT creatinine sulfate and PI (plant) were obtained from Sigma (St. Louis, MO). All solvents were analytical grade and were used without further purifica- tion.

Preparation of brain extracts. Rat brain stems were pre- pared as reported by GODWIN & SNEDDON (1975). Briefly, male Sprague-Dawley rats (2W300 g) were decapitated, and their brains quickly removed and placed in ice-cold distilled water. The cerebral cortex and cerebellum were discarded. All procedures were carried out at 4°C unless otherwise stated. The brain stems were homogenized with

1106 DAVID A. JOHNSON, TAE MOOK CHO and HORACE H. LOH

a Polytron for 15 s in 20 vol of distilled water (w/v), then centrifuged at 30,000 g for 15 min. The supernatant was discarded, The pellet was rehomogenized in distilled water and centrifuged as in the previous step. The supernatant was again discarded, and the pellet was resuspended in a 50% sucrose solution (w/v) (2 ml/g wet brain stem) and extracted with 10 vol of water saturated n-butanol for 2 h at room temperature by continuous and vigorous stirring. The mixture was centrifuged at 1000 g for 5 min and the butanol phase isolated. This was concentrated under reduced pressure at 38°C to approximately one-third of its original volume and stored at 4°C until use.

Zncubation procedures. For the discontinuous chromato- graphic procedures samples of the brain extract (6.0 ml) were combined with 0.5 ml water and 3 nmol of C3H]5-HT and were incubated for 12 min before a 6.0 ml sample was placed on the column. Purified lipids were dissolved in 6.5 ml of butanol saturated with water and incubated with 3 nmol or [3H]5-HT for 12 min. A 6.0ml sample was then loaded on the column.

For the continuous gradient procedure, 0.5ml of PI (2 mg/ml) and 0.1 ml [14C]PI (0.15 pCi/ml) both in chloro- form-methanol (CM) 2:l (v/v) were combined and evapor- ated with nitrogen in a test tube. One ml of a 2 m ~ solu- tion of C3H]5-HT (65 pCi/ml) in n-butanol saturated with water was added to this test tube, and this was incubated for 12 min before being placed on the column.

Preparation of column and chromatography. For the dis- continuous chromatographic procedure, Sephadex LH-20 (42.5g) resin was equilibrated in chloroform and was loaded into a 2.5 cm diameter glass column. After incuba- tion with C3H]5-HT, the sample was loaded onto the column and was eluted with the following series of sol- vents: 100ml chloroform, 50ml each of CM 15:1, lO:l, 6:1, then 300ml CM 4:1 (v/v), and finally 200ml methanol. Fractions of 5.0 ml were collected. A 0.2 ml aliquot was taken from each fraction to determine radioactivity by liquid scintillation spectroscopy, a 3 ml aliquot to deter- mine lipid phosphorus by the method of BARTLETT (1959) and a 1 ml aliquot to determine CS by the method of KEAN (1968).

Continuous column chromatography was performed by equilibrating 20 g Sephadex LH-20 in chloroform and loading the resin into a 2.0 cm diameter column. After in- cubation with [3H]5-HT, a 0.8 ml sample was loaded on the column. After 20 ml of chloroform had been collected and an additional 10ml of chloroform was placed on top of the column, the column was connected to a closed sys- tem gradient mixer. The mixing chamber contained 100 ml chloroform into which methanol was fed. Fractions of 3.0 ml were collected directly into scintillation vials, and radioactivity was determined following evaporation of the solvent.

Analysisfor acidic lipids. The fractions corresponding to the major peak of radioactivity following the elution of the brain extract which had been incubated with C3H]5-HT were combined and evaporated down to 2 ml. Aliquots were spotted on silica gel H (Analabs) TLC plates, which had been dipped in a 1% aqueous solution of potas- sium oxalate and heated for an hour at 130°C. The plates were developed in chloroform-methanol-ammonium hyd- roxide (4 N) (9: 7: 2) (GONZALEZ-SASTRE & FOLCH-PI, 1968). Acidic lipids were identified following spraying separate samples with ninhydrin or periodate-SchitT reagents and by comparison against lipid standards. Quantitative deter- mination of the concentration of CS, PI. and diphos-

phoinositide in the brain extract was made by application and development of the brain extract on the above-cited TLC system, visualization of the spots with iodine, extrac- tion of the spots corresponding to CS, PI, and diphos- phoinositide standards, evaporation of the solvent, and determination of the CS concentration by the method of KEAN (1968) and PI and diphosphoinositide by determina- tion of the total lipid phosphorus as reported by BARTLETT (1959).

RESULTS

The elution profile of radioactivity which was obtained following incubations of [ 3H]5-HT in the presence or absence of the rat brain stem extract is shown in Fig. 1. The brain extract elution pattern is essentially the same as that shown by GODWIN & SNEDDON (1975) with the major binding peak starting approximately at fraction number 67 and continuing over the next 12 fractions. Also, as GODWIN & SNED- DON (1975) had shown, the free 5-HT appeared in the methanol fractions.

An examination of the major peak of radioactivity for acidic lipids revealed the presence of CS, PI, and diphosphoinositide. The elution patterns of CS and total phosphorus from the brain extract is shown in Fig. 2. These elution patterns further confirm the presence of CS and show the probable presence of phospholipids, since at least 40 mg CS and 1 I nmol phosphorus was present in each fraction composing the major peak of radioactivity. The highest levels of CS and of radioactivity. however, do not exactly correspond to each other and are separated by 4 to 7 fractions.

The total amounts of CS and PI in the brain extract loaded on to the column were found to be 4 and 2.2 mg, respectively, in two determinations. The elution pattern of radioactivity and phosphorus when this same amount of PI was incubated with

Fraction No., 5 m l

FIG. 1. Elution profile after discontinuous chromatography of radioactivity from brain extract incubated with C3H]5-HT (solid line) and elution of C3H]5-HT in butanol saturated with water (dashed line). Elution solvents: 100 ml chloroform, 50x11 each of CM 15:1, lO:l, 6:1, 300ml CM 4:1, and 200ml methanol. Fraction number 1 is the void

volume.

Identification of 5-hydroxytryptamine binding substances from rat brain stem I107

Brain e x t r a c t

J

E

F r a c t i o n No., 5 r n L

FIG. 2. Elution of brain extract showing elution profiles of lipid phosphorus (dotted line), cerebroside sulfate (dashed line) and C3H]5-HT (solid line) after discontinuous chromatography. The radioactivity corresponds to that in Fig. 1. The (*) represents those fractions in which a contaminant prevented

phosphorus determination.

[3H]5-HT is shown in Fig. 3. This pattern of radioac- tivity was quite different from the brain extract pat- tern. All detectable phosphorus was eluted in either the chloroform or CM 4:l fractions, while most radioactivity was eluted in the methanol near where the free C3H]5-HT appeared. The elution pattern of the CS (4mg) incubated with C3H]5-HT, shown in

Fig. 4, is, however, similar to that of the brain extract. The percent of radioactivity in CM 4:l fractions, in the methanol fractions, and the total recovery (Table 1) also confirm the similarity of elution profiles. The major difference between the elution patterns of CS and of brain extract was the fraction number where the highest amount of radioactivity appeared. In three determinations the highest radioactivity appeared in

Fraction No., 5 rnL

FIG. 3. Elution of purified monophosphoinositide incu- bated with C3H]5-HT using the discontinuous elution pro- cedure described in Fig. 1. Radioactivity (solid line) and lipid phosphorus (dashed line) were assayed as described

in Methods.

1:

J

E

*' IC 0

€ 4

ro

Y

Fract ion No, 5 m l

FIG. 4. Elution of purified cerebroside sulfate incubated with C3H]5-HT using the discontinuous elution procedure

described in Fig. 1.

TABLE 1. THE MEAN (+ s.E.M.) RECOVERY OF RADIOACTIVITY FOLLOWING DISCONTINUOUS SEPHADEX LH-20 CHROMATOGRAPHY

% total d.p.m. eluted in CM eluted in

% total d.p.m.

4: 1 methanol Fraction No. % total d.p.m. (Fractions (Fractions Number with highest

eluted 5&110) 111-133) determinations radioactivity

Brain extract 96.3 1.2 81.7 2.6 17.7 k 2.7 3 7 I, 72,74 CS and PI 91 & 2.9 76.3 3.5 6 k 1.5 3 62,71,13 cs 91.5 i 0.5 76.5 5 2.5 11.5 k 1.5 2 78,76

119,119 P1 65 3.5 28 k 13 37 & 12 2 5-HT only 82 26 53 1 123

I108 DAVID A.'JOHNSON, TAE MOOK C H O and HORACE H. LOH

10 "L

Fraction No., 5 m ~

FIG. 5. Elution of purified cerebroside sulfate and mono- phosphoinositide incubated with C3H]5-HT using the dis-

continuous elution procedure described in Fig. 1.

fractions 71, 72, and 74 with the brain extract, while it appeared in fractions number 76 and 78 in two determinations with CS.

To determine whether PI influenced the elution pattern of CS and C3H]5-HT, CS and PI were com- bined and incubated with C3H]5-HT. This elution pattern of radioactivity (Fig. 5 ) is very similar to that of the brain extract. With one exception, the highest level of radioactivity appeared near the same fractions as with the brain extract in three determinations (Fractions 71, 73, and 63, compared to 71, 72, and 74). Though PI by itself did not cause any C3H]5-HT to appear in the CM 4:1 fractions, PI appeared to interact with CS and [3H]5-HT to cause a slightly earlier elution of [3H]5-HT and, thereby, PI with CS produce a tritium elution pattern essentially identical to that seen with the brain extract.

Since we showed elsewhere (JOHNSON et ul., 1977) that 5-HT binds to PI in an organic environment and that PI elutes in either the chloroform or the

f, ' I ! I

F r a c t i o n No, 3 m ~

FIG. 6 . Elution profile of [14C]monophosphoinositide (1 mg) (solid line) incubated with C3H]5-HT (2 pmol) (dashed line) using a continuous gradient formed by con- necting the column to a closed system gradient mixer con- taining 100ml chloroform into which methanol was fed. Fraction No. 1 is the first fraction collected after sample

was loaded on to column.

CM 4 : l fractions (Fig. 3), it was not anticipated that when PI and C3H]5-HT were eluted together, the major peak of radioactivity would appear in the methanol fractions near the free 5-HT. This suggested either that PI was not binding to 5-HT or that PI was binding to 5-HT producing a PI-5-HT complex more polar than the unbound PI. Such a complex would thus be eluted by methanol. To differentiate between these possibilities, [ '"CIPI and C3H]5-HT were eluted with a continuous gradient of increasing polarity. An approximate 1:2 molar ratio of PI to 5-HT was used in order to increase the likelihood of observing free and bound peaks of 5-HT and PI. The failure to observe free and bound peaks of radio- activity in the methanol fractions when PI and C3H]5-HT were eluted in the discontinuous chroma- tography could be explained if all the 5-HT was bound because of the approx 1000-fold excess of PI compared to 5-HT.

The elution pattern from this experiment is shown in Fig. 6. There are two major peaks of C3H]5-HT and two major peaks of ['"CIPI; one of the [14C] peaks corresponds to the first major C3H]5-HT peak. The molar ratio of PI to 5-HT for this later peak is 0.7 (assuming the molecular weight of PI is 860). This suggests that the first major ['"C] peak is free PI, the second ["C] and first major C3H] peak is a PI-5-HT complex, and the second C3H] peak is free 5-HT. Thus, 5-HT can bind to PI on the LH-20 column and, therefore, the observed single peak of radioactivity in the methanol using the discontinuous procedure probably represents binding of PI to 5-HT.

DISCUSSION

FISZER & DE ROBERTIS (1969) and GODWIN & SNEDWN (1975), using different size columns and volumes of solvent, observed that the major peak of radioactivity when C3H]5-HT was incubated with butanol brain extracts occurred in the CM 4:l frac- tions. Based on the inability of 37% [3H]5-HT incu- bated with butanol brain extract to move on silica gel G developed with chloroform :methanol :water and also based on the presence of Lowry positive material in the major radioactive peak on the LH-20 column, FISZER & DE ROBERTIS (1969) concluded that C3H]5-HT was binding to a special proteolipid. GOD- WIN & SNEDWN (1975) came to the same conclusion based on the observations that 5-HT binding material was precipitated by ice-cold ether and based on the presence of Lowry positive material in the residue of the major radioactivity peak.

Our examination of this peak for acidic lipids showed the presence of CS, PI, and a little diphos- phoinositide. Incubation of CS and PI with C3H]5-HT gave essentially the same elution pattern as the brain extract. This data, coupled with our pre- vious observation that CS and PI bind saturably and with high affinity to 5-HT, strongly implies that what

Identification of 5-hydroxytryptamine binding substances from rat brain stem I109

has been suggested to be a proteolipid 5-HT receptor is mainly two acidic lipids, CS and PI.

This conclusion is consistent with GODWIN & SNEDDON’S (1975) observations that the major peak of C3H]5-HT was not saturable up to a concentration of 300nmol 5-HT and that there was no clear rela- tionship between the major peak of [3H]5-HT and a discrete peak of either protein or phosphorus.

Other studies (LOH et al., 1974; Wu et a!., 1977) examining what were claimed to be opioid (LOWNEY et a/., 1974) and cholinergic (DE ROBERTIS et GI., 1969) proteolipid receptors, based on Sephadex LH-20 chromatography, have also revealed the presence of acidic lipids. Chromatography of these acidic lipids with opiates or d-methyltubocurarine have shown that virtually all the observed binding could be accounted for by acidic lipids. This, coupled with the data presented here, casts doubt on the proteolipid nature of other putative receptors partially isolated using Sephadex LH-20 chromatography (DE ROBERTIS, 1975).

While we believe all future claims about the proteo- lipid nature of putative receptors based on Sephadex LH-20 chromatography must eliminate possible lipid binding, failure to detect lipids in the apparent bind- ing peak is not, however, sufficient evidence to dis- count lipid binding. Because ligand concentrations are usually very small (a few nanomol) and because lipid-ligand complexes can exhibit chromatographic behavior quite different from unbound lipids, it would be difficult to detect 1 or 2mol of lipid. Phosphorus determinations will not always detect phospholipid- ligand complexes due to the limited sensitivity of the procedure, as was the case with PI and C3H]5-HT (Fig. 3). Ligand concentrations in the binding peak

must be high enough to detect possible lipid com- plexes.

Acknowledgements-The authors wish to acknowledge the editorial and typing assistance of VIRGINIA HAYES and KAYE WELCH. This study was supported in part by USPHS grants DA-00006, DA-00564 and DA-01583. H. H. LOH is the recipient of Career Research Scientist Development Award K2-DA-70554.

REFERENCES

BARTLETT G. R. (1959) J. biol. Chem. 234, 466468. DE ROBERTIS E., FISZER S., PASQUINI J. M. & SOTO E. F.

DE ROBERTIS E. (1975) Synaptic Receptors: Isolated and

FISZER S. & DE ROBERTIS E. (1969) J. Neurochem. 16,

GODWIN S. F. R. & SNEDDON J. M. (1975) J . Neurochem.

GONZALEZ-SASTRE F. & FOLCH-PI J. (1968) J . Lipid Res.

JOHNSON D. A., CHO T. M. & LOH H. H. (1977) J. Neuro-

KEAN E. L. (1968) J. Lipid Rex 9, 319-327. LOH H. H., CHO T. M., Wu Y. C. & WAY E. L. (1974)

LOWNEY L. I., SCHULZ K., LOWERY P. J. & GOLDSTEIN

MARCHBANKS R. M. (1966) J. Neurochem. 13, 1481-1493. MICHELL R. H., HAWTHORNE J. N., COLEMAN R. & KAR-

NOVSKY M. L. (1970) Biochim. biophys. Acta 210, 86-91. WOOLLEY D. W. & GOMMI B. W. (1964) Nature, Land. 202,

10741075. WU Y. C., CHO T. M. & LOH H. H. (1977) J . Neurochem.

(1969) J . Neurobiol. 1, 41-52.

Molecular Biology. Dekker, New York.

1201-1 209.

25, 283-288.

9, 532-533.

chem. 29, 1101-1 103.

Li$e Sci. 14, 2231-2245.

A. (1974) Science 183, 749-753.

29, 589-592.