L~fe Sciences, Vol. 28, pp. 2257-2263 Pergamon Press

Prmnted in the U.SoA.

NORHARMAN INHIBITION OF [JH]-DIAZEPAN BIN~ING IN MOUSE BRAIN

Anne M. Morln, Irls A. Tanaka and Claude G. Wasterlaln

Neurology and Epllepsy Research Laboratorles Veterans Admlnlstratlon Medlcal Center

Sepulveda, Callfornla and Department of Neurology and Braln Research Instltute,

School of Medlclne, bnlverslty of Californla, Los Angeles, Callfornla

(Recelved In f!nal form February 26, 1981)

Summary

Norharman competltlveiy inhlblts speclflc blndlng of [BH]- dlazepam in mouse braln homogenates. In vlvo thls B-carbollne produces a striking rlgld catatonlc-liKe appearance whlch is abolished by diazepam. It also causes a rapld tremor but has llttle antlconvulsant effect. Measurement of in vivo concentra- tlons and receptor occupancy demonstrate that these biological effects occur at doses whlch occupy a large proportion of benzo- dlazeplne receptors. It may represent a llgand of the benzo- dlazepine receptors WhOSe effects are opposlte those of dlazepam.

Norharman is one of a group of harmala alkaloids, some of whlch are re- sponslble for halluclnatlons, tremors, inhlbltion of monoamlne oxldase, and convulslons (see revlews 1,2). We report here that nornarman inhlblts benzo- dlazeplne bindlng in vitro In mouse bramn mn a competltlve manner and produces tremors and a catatonlc-llke rlgldlty whlch are reversed by dlazepam. Norhar- man contalns an indole rlng structure as do some other llgands of the benzo- dlazeplne receptor such as tryptophan (3) and ethyl B-carbollne-3-carboxyllc acld (4,5). Recent reports have descrlbed the blnding afflnltles of a number of nltrogen contalnlng ringed structures, such as hypoxanthlne and Inosine (6), nlcotlnamlde (8), along with synthetlc compounds such as irazepine (9) dlphenyl- hydan~oln (I0) and trlazolopyrldazines (3) and pentylenetetrazol (8). Our studie indlcate that norharman and dlazepam share a binding site and that diazenam may reverse the In vlvo effects of norharman. Such a dlverslty of actlons by com- pounds which blnd to a con=non slte may be explalned in terms of types of receptor llgand interactlons.

Methods

Preparatlon of Mouse Braln Homo senates:

Adult male C57 black mlce (20-30 gm) were k111ed by decapltat~on, their bralns removed and placed on a chllled surface. Speclflc braln areas were re- moved, welghed and homogenlzed in 20 volumes of 0.05 M Tris HCI buffer pH 7.5. Whole homogenates (50-200 ug proteln) were used in all assays. Protein deter- mlnatlon was accordlng to Lowry et al. (12).

0024-3205181/202257-07502.00/0

2258 Norharman and [~H]-diazepam Binding Vol. 28, No. 20, 1981

Binding Assay"

Bindmng of [3H]-diazepam (87.3 Ci/mmol., New England Nuclear Corp.) to mouse bramn was carrmed out according to the procedure of Squlres and Braestrup with minor modlflcatmons (ii). [3H]-Diazepam concentration was 1.6 nM unless otherwmse stated and specmfic bindlng determlned to be the difference between total blnd~ng and b~ndlng in the presence of 3 uM cold diazepam (non-speciflc blndmng). The assay was carried out in 0.05 M Tris-HCl pH 7.5 mn a final volume of 0.5 ml for 30 minutes at 4°C. The assay mmxture was collected on GF/B What- man glass f~ber f~iter dlscs and washed (3x) w~th 5 ml cold buffer. The radio- act~vlty on each f~iter was then quantitated by liquid scintmllat~on counting • n the presence of PCS (Amersham Corp.): zylene (2:1).

Measurement of Norharman Concentration (in vivo):

Norharman CSigma Co.) was dlssolved in 1.5 N HCI, brought to pH 6.0 wlth sodium bicarbonate and immedlately injected l.p. in doses ranging from 50-200 mg/kg. After 2 mlnutes, the anlmals were k111ed, by decapitation, their bralns placed on a chilled surface. The cortex was removed, weighed and homogenized. ~ole cortex homogenates (i0 ul) from anlmals receivlng different doses of norharman were assayed for [3H]-dlazepam blndlng. Allquots of homogenate were spotted onto a slllca gel TLC plate (Brlnkman) and developed in n-butanol: acetlc acid: H20 (4:1:1). Similar plates were prepared with varylng concentratlons (0-20 nMoles) of norharman as standard. Norharman was detected wlth U~ llght and migrated %Ith an Rf of 0.6. Each spot was eluted overnlght with 1 ml of spectrofluorometrlc grade methanol: water (i:i) The fluorescent emlssion and excitation profiles were obtained for the standard norharman using the Farrand spectrofluorometer. Under our conditions there were three excitation peaks (emlsslon 450 rim); 310 nm > 260 nm> 380 nm, and two emission peaks (excitation 310 nm)" 450 nm > 375 nm. The profile of a representative TLC sample from cortex of an anlmal receiving 200 mg/kg norharman was then obtalned and found to be essentlally identlcal to the standard (Insert Fig. 3) The relatlve fluorescence of each experlmental tube was then determined (excltation 310 nm and emlsslon 450 nm). The fluorometrlc readlngs were converted to norharman concentrations

using the curve obtalned from TLC standards.

Results

Norharman dlsplaces speclflcally bound [3H]-dlazemam in homogenates of mouse forebraln (Fig. i) when assayed In vltro accordlng to the procedure descrlbed by Squires and Braestrup (Ii) and Mohler and Okada (13) The EC50 value (Table I) ks hlgher than that of S-carbol~ne-3-carboxv!ate (EC50=7~IO-9M) but similar to other substltuted ~-carbollnes and tetrahvdro-E-carbollnes (4) and lower than some endogenous compounds (7,8) Norharman appears to displace all [3H]-dlaze- pam blndlng but wlth a much lo~er potency than benzodlazeplnes.

Examinatlon of saturatlon studles ~n mouse forebra~n homogenate (Fig. 2) w~th varying concentrations of [3H]-dlazepam and norharman (5 ~M) ~nd~cate that the presence of norharman result in an apparent decrease ~n receptor affinity w~th an ~ncrease ~n Kd values from 3.4 ~ 0.8 nM to 6.8 i 2.0 rum (n=3, P <.025). Bmax values are 752.2 ± 31.8 femtomoles/mg for control and 693.9 ± 56.5 femto-

moles/mB mn the presence of norharman.

Saturation studies in cortex, cerebellum and hi~pocampus (data not shown) demon- strate an increase in Kd ~alues but not in Bmax values In these areas ~lle £-carbo!Ine-carboxyllc acic may 81ve evidence of negat!ve cooperatlvelv in hlppocamDu~ (as demonstrated by curv111near Scatchard plots> (57 nornarman blnds

Vol. 28, No 20, 1981 Nerharman and [3H]-dlaze~am Blndlng 2259

IO0-

. .J o 80- D: F- z 0 6 0 - 0

I,l.. 0 40-

2 0 -

on

I I I I I I I I

- I0 -9 -8 -7 -6 -5 -4 -3

CONCENTRATION (log)

FIG. i

Displacement of specmfically bound [3H]-dzazepam mn mouse forebrazn homogenate (200 uB protein) by norharman and benzodlazepznes. Homogena~e was incubated in the presence of 1.6 ~i [3H]-dlazepam and varylng concentratlons of displaclng drugs accordlng to the procedures described in Methods. Polnts In each curve represent the average of three separate determlnatlons each performed in duplicate. EC50 values were determlned from the slopes of logit-log plots.

TABLE I

Compound EC50 Hzll Coefflc~ent Corr. Coeff.

(M)

Dzazepam 3.6 ± 0,09xi0-9(3) 0.89 Z .01 0.99

Flurazepam 7.5 Z 0.84xi0-9(3) 0.82 ± .12 0.98

Norharman 1.6 ± 0.26xi0-6(4) 0.67 ± .05 0.98

Displacement of [3H]-dlazepam o!ndmng by dlazepam, flurazepam and norharman are expressed as mean i SE and were determined from the slopes of logit-log plots of data from separate experlments (number zn parenthesls) each performed in duplmcate.

2260 Norharman and [3H]-dlazepam Bzndlng Vol. 28, No. 20, 1981

E g N

9

i t ~

80-

60-

4 0 -

20 -

A • -o

"H-dlazepam concentrahor ~ (nM)

FIG. 2

o24] io ] \

"°°al

f0 20 30 40 50 60 70

fmoles bound

(A) Binding of [°H]-dlazepam to mouse forebraln homogenate (67.7 ug protein) in the presence of varylng amounts of [3H]-dlazepam (o---o) and additionally, 5 uM norharman io---o). (B) Scatchard plot of saturation data from (A). Points represent the mean of duplicate de~ermlnatlons. Thls experiment was repeated 4 times. (See text for Kd and Bmax values.)

in a llnear manner in all the above mentioned areas. Thus, norharman blndlng resembles the llnearlty of [3H]-dlazepam (5) or 3H-flunltrazepam (14) blndlng suggestlng it competes for blndlng to all forms of receptor recognized by these benzodiazeplnes.

The effects of In vlvo admlnlstratlon of norharman are qulte remarkable Wlthln mlnutes of an 1.p. dose norharman (50 mg/kg neutralized to pH 6.0) the animal becomes qulet, tremulods and loses its rlghtlng reflex when placed on its slde. The anlmal has a catatonlc appearance wlth its front and hlnd llmbs stlff and extended. D1azepam is effective In stopping the tremors in a dose depend- ent manner (Table Ii).

TABLE II Norharman Dlazepam* (mg/kg) (m~/k$)

0 0 5 0.75 1.0 2.0 3.5

5o !O/!O 2/2 - 1/8

75 7/7 -,°/°~ 1/2 0/3

1/3 o/3

Dlazepam inhlbltlon of nornar~n induced tremor Tremors appeared wlthln 8-9 m!nutes after norharman dose (i p.) After !0 mlnutes,

varylng doses of dlazepam were glven (~.p) *Results are expressed ~s number of anlmals havln~ tremors as a fraction of total number of anlmals recelvln~ varv~n~ doses of dlazepam.

Vol. 28, No. 20, 1981 Norharman and [3H]-dzazepam Bzndzng 2261

We znvestzgated the relatlonshlp between these behavloral effects, drug concentratlon and receptor occupancy. A_nlmals were gz~en doses of norharman rangmng from 50 mg/kg to 200 mg/kg (z.p.). Wzthln 2 mlnutes all anmmals had responded in the forementloned characterzstlc manner Each animal was then kzlled and the cerebral cortex removed, homogenmzed (20 volumes) and examzned for zn vitro [3H]-diazepam blndzng as descrlbed in Methods.

In vivo concentratlon of norharman present in cortex (20-fold dllutzon wzth homogenization) was determlned as described zn methods. As shown zn Fzg. 3, the Inhlbition of [3H]-dmazepam binding zs proportzonal to the concentratzon of nor- harman in the brazn homogenate (r=.92). It zs unlikely that the znhzbztlon zs due to a degradatmve product formed In vlvo from norharman since the spectro- fluorometrzc excmtation profzle of norharman in cortex (seen zn the inset, Fig, 3) snows the same profile as the norharman standard.

28J 24

55

r= 0.92 o

--I

~ J / i excltat!.on wQvet~ngth(nm)

ro

I i I I

02 O4 O6 O8

norharman concentrahon(~M)

FIG. 3

[3H]-Dmazepam bmnding (1.6 mM) was measured zn cortex homogenates from animals recelving varying doses of norharman in vlvo. The concentratmon of norharman present mn the cortex homogenates (I0 pl) was determined as descrlbed zn Methods and represents a 20-fold dilution of in vivo concentratmons. Norharman concentratzon refers to the amount present in the in vitro [3H]-dlazepam blndzng assay of cortex homogenates. The mnset shows the spec~rofluorometrzc profile of norharman TLC standard ( - ) and the profzle obtamned from norharman obtazned from cortex ( .... ) after mn vlvo Injectzon (see Methods)

2262 Ncrharman and [3H]-d~azepam B~ndlng Vo!. 28, No. 20, !981

Discussion

The inhlbltlon of in vitro [3H]-dlazepam blndlng by norharman suggests that the In vlvo effects of thls ~-carbollne may be mediated through the benzo-

dlazeplne receptors. Norharman binds competitively to the benzodlazeplne reco~- nltlon site. Displacement studies indicate that all speclflc [3H]-d~azepam

binding In forebraln, cortex, h~ppocampus and cerebellum is dlsplaceable by

norharman. Scatchard plots of saturation data obtained wlth the same areas are

linear as opposed to the curv~llnear plots obtained in some brain areas in the presence of S-carboline-3-carboxyl~c acid (A,5) CurvIlinearlty may indicate co-

operatlvlty between receptors, a eharacterlstlc not detectable in the presence of various benzodlazeplnes nor with norharman.

Squires et al. (3) in descrlblng the binding proDertles and ~n vlvo actlvity

of the trlazolopyrldazlnes suggest that antlconfllct and antlmetrazol effects may be medlated by one receotor type while sedation and ataxla are mediated Dy other receptor types. We have observed (manuscrlpt in preparation) that norbar- man, unlike dlazepam and the trlazoloDyr~dazlnes, is not ~n effective anticon-

vulsant when animals are treate~ wlth convulsant doses of pentylenetetrazol or

blcuculline. Perhaps norharman as well as other B-carbolInes elicit tremorlgenlc

effects through selective ~nteractlon wlth one type of benzo~lazeplne recognltlon slte, wnlie havlng llttle action at sites ~;hlch mediate the antlconvulsant

propert!es of benzodlazepines.

Norharman is structurally related to harmallne, a compound whose tremorlgenlc actlvlty, mediated through the Inferlor ollve nucleus is well known (16). Dlaze- pam has been shown to block the tremors caused by harmallne (19) and a similar block of tremors caused by norhar~an was also observed. The inhlbltion of diaze- pam binding in cortex by harmaline was measured it was found that at 10-4M

concentration, there Is 41 7% inhlbltlon and at 2.5xlO-~M, 13 6% Inni~itlon. It is possible that the tremorlgenlc actlon of both drugs may be mediated throuzh a

common mechanlsm, which may invo!ve olndlng to the benzodlazeplne receptor.

Acknowledgment

The authors wlsh to thank Dr. Joseph T Cummins for hls he!oful suggestions

and generous gift of nornarman and related f-carbolines. The authors also thank

Inga L Anderson for thougqtful assistance in preparation of thls manuscrlpt. We also acknowledge sumport from the Resesrch Service of the Veterans Admlnlstra-

tlon and by Research Grant N.S. 13227 from NINCDS.

References

i. N. BUCKHOLTZ, Life Scl. 27 893-903 (1980) 2. B.T. HO, Current Developme---nts in Psycholpharmacolosy, vol. 4, p 152-177.

Spectrum Pub. Inc., New York (1977). 3. R. SQUIRES, D. BENSON, C BRAESTRb~P, J. COUPET, C. KLEPNER, V. M~fERS and

B. BEER. Pharmacoi. Blochem, ~ Behav. i0 825-830 (1979~. 4. C. BRAESTRUP, M. NIELSEN and C. OLSEN, Proc. Natl, Acad. Sci. (USA) 77(4)

2288-2292 (1980). 5 M. NIELSEN and C. BRAESTRUP, Nat. 286 606-607 (1980). 6. P. SKOLNICK, P.J. M~RANGOS, F.K. GOODWIN, M. EDWARDS and S PALqu, Life Scl.

23 1473-1480 (1978). 7 H-~ MOHLER, P POLC, R. CUMIN, L. PIERI and R. KETTIES, Nat. 278 563-565 8 P J. MARANGAS, S.M. PAL~L, A M. PAP~MA, F K. GOODWiN, P SYAPIN and

P SKOLNICK, Life Scl. 24 851-858 (1979). 9. K. RICE, A. BROSSI, J. TALL~N, S.M. PAUL and P. SKOLNICK~ Nat 278 854-855

(1979~

Vol. 28, No. 20, 1981 Nornarman and [3H]-dlazepam Blndlng 2263

[0. G TUNNICLIFF, J.A. SMITH and T.T. NGO, B1ochem. Biophys. Res. Comm. 91(3) 1018-1024 (1979).

ii. R.F. SQUIRES and C. BRAESTRUP Nat. 266 732-734 (1977). 12. O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. blol. Chem. 193

265-275 (1951). 13 H. MOHLER AND T OKADA, Scl. 198 849-851 (1977). 14 R.C. SPETH, G.J. WASLEK, ~ C. JOHNSON and H.I. YAMAMURA, Lile Sclo 22

859-866 (1978). 15. R SQUIRES, R. NAQUET, D. RICHE anG C. BRAESTRUP, Epllepsla 20 215-221

(1979]. 16. R LLINAS and R.A VOLKIND, Exp. Brain Res. 18 69-87 (1979). 17. C deMONTIGNY and Y LAMARRE, Braln Res. 53 81-95 (1973). 18. G. BIGGIO and A. GUIDOTTI, Braln Res. 107 365-373 (1976). 19. C.C. liAO, A GUIDOTTI and E. COSTA, Bra~n Res. 83 516-519 (1975).