evidence for high affinity [3h] imipramine binding sites in human lung

Vol. 120, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 16, 1984 Pages 926-932

EVIDENCE FOR HIGH AFFINITY [3H] IMIPRAMINE BINDING SITES IN HUMAN LUNG

Didier MORIN 1, Roland ZINI 1, Jacques LANGE 2 and Jean-Paul TILLEMENT

1 Service de Pharmacologie, Facult~ de M~decine de Paris XII, 8 rue du G~ngral Sarrail, 94010 CRETEIL, France

2 Clinique du Sud, 112 Avenue du G~n~ral de Gaulle, 94320 THIAIS, France

Received March 23, 1984

SUMMARY : [3H]imipramine exhib i ts both saturable and high a f f i n i t y binding ~ n human lung with a maximal number of binding sites of 7.50 pmoles/mg protein and a dissociat ion constant of 1.74 nM. Displacement studies indicate that these sites can be considered as specif ic of imipramine, t r i c y c l i c compounds and also monoamine uptake inh ib i to rs : f luoxet ine and nisoxetine. Atypical antidepressants were inact ive as ligands of main known receptors.

INTRODUCTION : High a f f i n i t y binding sites have been already described for

imipramine in rat brain (1,2), human brain (3,4) and human platelets (5,6).

Recently, the presence of high a f f i n i t y imipramine binding sites has been also

reported in rat lung (7). For most authors, these sites can be considered as

specif ic ones, related to 5-HT uptake mechanisms (8,9). Imipramine was shown

to i nh i b i t 5-HT uptake mechanisms in the central nervous system and th is ef fect

was related to i t s antidepressant properties. Analogous 5-HT uptake blockade

was also observed in platelets (both in v i t ro and in vivo) and the in tens i t y

of th is ef fect was correlated with the improvement of treated patients. We

checked the presence of s imi lar binding sites in human lung considering that

in th is t issue, imipramine is highly concentrated and 5-HT uptake mechanism is

present, But as imipramine exhibi ts also chol inergic and ~ adrenergic blocking

ef fects, the poss ib i l i t y of binding to the corresponding sites was also checked.

Simi lar ly as imipramine is extensively metabolized in the body, the poss ib i l i t y

of metabolic sites was also considered. Our results are consistent with the

presence of high a f f i n i t y binding sites in human lung, shared by other t r i c y -

c l i c compounds and two amine uptake inh ib i to rs .

MATERIAL AND METHODS : [3H] imipramine (70.7 Ci/mmole) was purchased from New England Nuclear Chemicals. Various other compounds were selected in order to assess the se lec t i v i t y of imipramine binding. They include other antidepressants, e i ther of t r i c y c l i c structure as clomipramine, desipramine and ami t r ip ty l ine , e i ther not as vi loxazine and mianserin. Other specif ic ligands were also checked, as ~obes of adrenergic (~ and B), muscarinic, histaminergic, dopaminergic, 5-HT, Ca " channels receptors. Specific amine uptake inh ib i to rs were also used, respectively f luoxet ine for 5-HT and nisoxetine for norepinephrine (NA) carr iers. Chlorpromazine, a neuroleptic chemically related to imipramine, was also used. Six human lung samples, obtained during operation, were immediatly dissected

0006-291X/84 $1.50 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. 926

Vol. 120, No. 3, 1 9 8 4 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on ice, and then extensively washed to avoid blood contamination, specially platelets. The sample (about 1 g) was homogenized in 20 ml of ice cold buffer (50 mM Tris-HCl, 120 mM NaCI, and 5 mM KCl, pH 7.4) using an Ultra-Turrax- Ikawerk (4 x 20 sac). The homogenate was centrifuged at i000 g for 10 minutes and the resulting pellet was discarded. The supernatant was centrifuged at 5000 g for I0 minutes and the resulting pellet was discarded again. Final ly, the supernatant was centrifuged at 50.000 g for 15 minutes and the pel let was kept and washed twice by the same procedure. The f inal pel let was resuspended in such a buffer volume that the f inal protein concentratinn was about I mg/ml. P~otein was estimated by the method of Lowry e~al.(lO). The b~nding of [mH] imipramine was carried out at O°c for 60 min in a total volume of 500 ~I containing ~H] imipramine (0,1-12 nM), lung homogenate (0,1 mg/ml) and Tris-buffer. After incubation 400 ~I were rapidly f i l te red through Whatman GF/F glass f iber f i l t e r s . Each f i l t e r was washed with an additional volume of twice I0 ml of ice cold buffer and counted in a l iquid sc in t i l l a t ion counter PACKARD 460 CD. Specific binding was defined as the difference between the binding observed in the presence and absence of 1 ~M clomipramine. This concentration has been determined from previous inh ib i t ion curves of [3HI imipramine binding. E~periments in duplicate were systematically carried out. Inhibi t ion of [~HI imipramine binding by di f ferent compounds was measured under equilibrium condltlons as descrlbed above, in the presence of 2 nM ~H] Imlpramlne. All binding parameters were estimated by means of the non-linear least-squares method using a Gauss-Newton algorithm ( i i ) .

RESULTS AND DISCUSSION : Association and dissociation kinetics. Association

kinetics of [3HI imipramine to lung membranes reached equilibrium at 30 min.

at O°c (Fig. 1). These studies were carried out with a [3H] imipramine concen-

tration[LT] of 4.35 nM and a receptor concentration [RT] of 1064 nM corres-

ponding to the maximal concentration of speci f ical ly bound [3HI imipramine

[LRe] in kinetic experiments. So, the association rate constant was calculated

from the equation (12) :

In ([LRe]([LT] [LR])/[LT] ([LRe] - [LR])) =k+l t ( [ L R ] - [LRe] )

where[L~represents the ligand-receptor complex at time t . k+l value was 5.3 x 105 M -1 sac - I .

The revers ib i l i t y of [3H] imipramine binding was demonstrated (Fig. 1) after

a previous incubation of 30 min., by adding I ~M unlabelled clomipramine.

The dissociation process was monophasic with a dissociation rate constant

k_l = 1.11 x lO-3sec - I and t l / 2 = 10 min. These values were estimated from

the equation (12) : I , ([L~ / [LRe] ) = - k_l t .

The value of the equilibrium constant calculated from kinetic data is

K D = 2.11 nM.

Equilibrium studies of [3H] imipramine binding to lung membranes. Fig. 2A shows a typical binding experiment of [3H] imipramine to human lung membranes.

This binding was saturable with a l inear Scatchard plot (Fig. 2B) showing one

class of binding sites. The parameters calculated by the non-linear method

revealed an equilibrium dissodationconstant (KD) of 1.74 ± 0.35 nM with

927

Vol. 120, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

B (%) lOO

5o

0 t ! , |

0 10 20 30 t ime (mln)

Fig. 1 : Kinetic studies of [3H]imipramine binding to human lung membranes. ~ s ~ t i o n kinet ic (e-O) was run in incubating membranes (0.1 mg/ml) and [JH] imipramine (4.35 nM) for 30 min at O°c. Non-specific binding was measured in the presence of 1%M clomipramine and substracted from total binding. After equilibrium has been reached, dissociation kinet ic (0 -0 ) was run in adding clomipramine to get a f inal concentration of 1 uM.

densi ty of binding s i tes (Bmax) of 7.50 ] 4.00 pmoles/mg p ro te in . H i l l analy-

s is showed no coopera t i v i t y wi th a H i l l number (nH) of 1.04 (Fig. 2C).

The value of th i s equ i l i b r ium d issoc ia t ion constant is in good agreement wi th

6

o

=z 4 E

o E

2

B

J o f

3 6 9 12 F (nM)

• • • ~ J

4~ e ~ e ~ 2 a 4

J . . . . 0 1.5 :3 4.5 6

B

Fig. 2 : Equilibrium bindin 9 of [3HI imipramine to human lung membranes. ~ b r i u m binding study was performed at O°c for 60 minutes using pH] imipramine at various concentrations for 0,1 to 12 nM (proteins = 0. i mg/ml). A, specif ic (O--0) and non-specific (©-0) bindinq3were calculated as indicated-in the text ; B and F represent bound and free [ H] imipramine respectively. B, Scatchard plot for specif ic FJH] imipramine binding to human lung membranesT C, Hil l plot of specif ic [3H]Limipramine binding : the l ine was f i t ted by l inear regression with the method of least-squares. The Hi l l constant is n H = 1.04.

928


the d issoc ia t ion constant calculated from k ine t i c data (2.11 nM).

Close values of K D have been already found for ~ imipramine binding to

ra t and human brain (1, 3) to human p la te le ts (5) and ra t lung (7). But

the capacity of these binding s i tes is higher. This resu l t can be due to the

membrane's preparation which is l i gh t l y d i f f e ren t and more pur i f i ed than those

used in previous works.

In re turn, th is high Bma x could explain the large imipramine concentrations

found in human lung (13).

Effect of various compounds on ~H] imipramine binding to lung membranes.

Increasing concentrations of t r i c y c l i c antidepressants gradual ly i nh ib i ted

[3H] imipramine binding to human lung membranes. Half maximum i n h i b i t i o n

(IC50~ for unlabel led imipramine is 4nM giv ing an i n h i b i t i o n constant

K I = 1.86 nM obtained from the Cheng and Prusoff equation (14) :

This K I value is in good agreement wi th K D values found from k ine t i c data

(K D = 2.11 nM) or from equi l ib r ium binding (K D = 1.74 nM). [3~ imipramine

s p e c i f i c a l l y bound, was displaced by low concentrations of clomipramine,

am i t r i p t y l i ne and desipramine (Fig. 3 and Table I ) . The monoamine uptake

i n h i b i t o r s , f luoxet ine and n isoxet ine, also inh ib i ted ~H] imipramine

binding with a high a f f i n i t y . At the reverse, the atypical antidepressants,

lOO

°! .~ 50

' \

O ,

- 1 0 -g - 8 - 7 -6 -5 - 4 log [antldepressantsl

3 Fig. 3 : inn_~DjInh'~tion of [ ~ imipramine bindln 9 by antidepressants. l~Tna~-ng of--l_~]-imipramine (2 nM) to human lung membranes (0,1 mg/ml) was measured under equilibrium conditions in the presence of various concentrations of imipramine (0 -0 ) , clomipramine (©-©), amitr iptyl ine ( s - s ) , desipramine ([ ]-D), mianserin (A--A) and viloxazine (~-~) .

929


TABLE I : Inhibit ion of ~H] imipramine bindin 9 to human lung membranes. .501~values are the concentrations3of the compound required to inh ib i t 50 % of ~he specific binding of 2 nM [H] imioramine to human lung membranes.

COMPOUNDS IC50 (nM) COMPOUNDS IC50 (nM)

IMIPRAMINE 4 MIANSERIN 7900 AMITRIPTYLINE 7.5 VlLOXAZINE 25000 CLOMIPRAMINE 8 ATROPINE 88000 FLUOXETINE i i DOPAMINE >I00000 CHLORPROMAZINE 67 HISTAMINE >I00000 DESIPRAMINE 76 METOPROLOL >I00000 NISOXETINE 145 NICARDIPINE >I00000 PHENTOLAMINE 800 (-) NOREPINEPHRINE >I00000 IPS 339 1800 PRAZOSIN >I00000 (±) PROPRANOLOL 2100 YOHIMBINE >I00000 SEROTONIN 3300

mianserin and v i loxaz ine , were inact ive (Fig. 3). Furthermore competit ion

studies using various neurotransmit ter agonists or antagonists indicated that

[3HI imipramine did not bind to muscarinic, dopaminergic or histaminergic

receptors (Table I ) . However, phentolamine, (~) propranolol and, to a less

extent , 5-HT showed a weak a f f i n i t y for imipramine binding s i tes (Fig. 4).

The B2-adrenergic antagonist , IPS 339, was a more potent i n h i b i t o r than

Bl-adrenergic antagonist , metoprolol , for imipramine s i tes . Propranolol, non

se lect ive B-adrenergic agent, displayed an intermediate a f f i n i t y . On the other hand, the spec i f ic ~-adrenergic antagonists, prazosin (~I) and

yohimbine (m2) did not possess any a f f i n i t y for imipramine receptor (Table I ) .

Chlorpromazine, a neurolept ic wi thout ant idepressive a c t i v i t y , showed a re la-

t i v e l y high a f f i n i t y fo r imipramine s i tes (Fig. 4). This behaviourcould be

explained by i t s chemical s t ructure close to that of imipramine. These resu l ts

show these s i tes can be considered as recogni t ion s i tes of imipramine and

t r i c y c l i c compounds. They seem d i s t i n c t from other tested receptors.

Recently, Sette et al (15) have proposed that imipramine binding s i te and

serotonin uptake ca r r ie r might be interdependent but coupled to each other.

These conclusions were run from many resul ts inc luding the complex i n h i b i t i o n

curves of imipramine by 5-HT with nH<1. As our i n h i b i t i o n curves show a

monophasic process with 0 .95<nH<1.04, our f indings cannot support the same

conclusions. However i t must be kept in mind that t issues and species are

d i f fe ren t between the two studies. Moreover, as imipramine acts as an an t i -

depressant, i t is l i k e l y to postulate that i t s mechanism of action on central

nervous system is d i f fe ren t from that in lung.

Thus,our resul ts would show that imipramine and the high a f f i n i t y i n h i b i t o r

930


100

,.0

i i F= 50

U

o - 1 0 -g - 8 - 7 - 6 - 5 - 4

tog [Inhlbltors]

3 Fig. 4 : Inhibition of [ H] imipramine binding by different compounds. ~ g of--[J~ imipramine (2 nM) to human lung membranes (0,i mg/m]) was measured under equilibrium conditions in the presence of various concentrations of fluoxetine (e-e) , chlorpromazine (©-©), nisoxetine (m- l ) , phentolamine (D-D), IPS 339 (A-A), (t) propranolol (~-~) , 5-H~ (~-~) and atropine (0-0) .

agents, f luoxet ine, nisoxetine and chlorpromazine are solely bound to

speci f ic imipramine binding sites in human lung as proposed i m p l i c i t l y by

Laduron (16). They do not allow to display a re lat ion between these imipramine

sites and amine uptake carr iers in human lung.

Another explanation of th is high imipramine binding could be the presence of

biotransformation si tes of th is drug in lung. Indeed, i t is highly metabolized

in humans and the relevant transformations can be induced (17). They have been

described in l i ve r , but can also take place in lung (18), where drug metabo-

lism also occurs.

ACKNOWLEDGEMENTS ; We are grateful to Dr, G. Leclerc from Department of Pharmacology, Faculty of Medicine, F-67085 ~trasbourg, for his g i f t of IPS 339 and we thank pharmaceutical companies for providing us the i r respective compounds. The study was made possible vdth special grants from Universi ty of Paris XII and from the "Direction de la Recherche au Minist~re de l 'Education Nationale".

REFERENCES :

1. Raisman, R., Br i ley, M.S., and Langer, S.Z. (1979) Nature 281, 148-150. 2. Raisman, R., Bri ley, M.S., and Langer, S.Z. (1980) Eur. J.--P~Tarmacol.

61, 373-380. 3. ~ h a v i , M., Paul, S.M., Skolnick, P., and Goodwin, F.K. (1980) Li fe Sci.

26, 2273-2279. 4. Langer, S.Z., Javoy-Agid, F., Raisman, R., Br i ley, M.So, and Agid, Y.

(1981) J. Neurochem. 37, 267-271.

931


5. Bri ley, M.S., Langer, S.Z., Raisman, R., Sechter, D. and Zari f ian, E. (1980) Science 209, 303-305.

6. Paul, S.M., Reha--vT, M., Skolnick, P., and Goodwin, F.K. (1980) Life Sci. 26, 953-959.

7. Raism~, R., and Langer, S.Z. (1983) Eur. J. Pharmacol. 94, 345-348. 8. Langer, S.Z., Moret, C., Raisman, R., Dubocovich, M.L., ~ d Bri ley, M.

(1980) Science 210, 1133-1135. 9. Barbaccia, M .L .~ando l f i , 0., Chuang, D.M., and Costa, E. (1983)

Proc. Natl. Acad. Sci. USA 80, 5134-5138. I0. Lowry, O.H., Rosenbrough, N~. , Farr, A.L., and Randall, R.J.(1951)

J. Biol. Chem. 193, 265-275. 11. Zini , R., Gault,---T., Ledewyn, S., d'Athis, Ph., and Tillement, J.P.

(1983), Biochem. Pharmacol. 32, 3375-3380. 12. Engel, G., Hoyer, D., BerthoT-c[, R., and Wagner, H. (1981) Naunyn-

Schmiedeberg's Arch. Pharmacol. 317, 277-285. 13. Simon, P., and Colonna, L., in G~ud , J.P., Math~, G., and Meyniel, G.

(1979) Pharmacologie Clinique, Vol. 2, pp. 1633-1649, Expansion Scienti- f ique, Paris.

14. Cheng, Y., and Prusoff, W.H. (1973) Biochem. Pharmacol 22, 3099-3108. 15. Sette, M., Bri ley, M.S., and Langer, S.Z. (1983) J. Neuro-c-hem. 4_0_0,

622-628. 16. Laduron, M., (1984) Trends Pharmacol. Sci 5, 52-53. 17. Boissier, J.R., Tillement, J.P., Marzin, D.~ and Pichot, P. (1975),

J. Pharmacol. Clin. 2, 11-15. 18. Fishman, A.P., and PTetra, G.G. (1974) N. Engl. J. Med. 291, 953-959.

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evidence for high affinity [3h] imipramine binding sites in human lung

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