Pierre M. Laduron replies

52

TIPS-

Langer, S. Z. (19811 Brain Res. 21(1, 49:~198 9 Rainbow, T. and Bicgon, A. (Ig['L~)Brain R~:s'. 262, 319-322 10 Fuxc, K., Calza, L., Benfenati. F.. Zini, I. and Agnatti, L. F. (It~83) Prcn: Nat/ Acad. &'i. USA 80, 383(~-384(I 11 Sette, M., Raisman, R , Briley, M, S. and Langer, S. Z. (198111. Neurochem. 37,411--42 12 Gross, H., Golhert, M , Ender, H.-C. and Schumann, H.-J. (1981) Naunyn.Schmiedetwrgs

Arrh. Pha,nakol. 317, 3111-314 13 Bruncllo, M., ('huang, D. and Cosla, E ( 19821 Science 215, I 112-[115 14 Laduron, P. M., Robbyns. M. and Schotte, A (1982) Eur. J. Pharmacol. 78.4~11--493 15 Rchavi, M., Skolnick, P. and Paul, S. M. (19831 Eur. J. Pharmacol, 87, 3Mv-7139 16 Sette, M,, Rubcrg, M., Raisman. R.. Scatton, B., Zivkovic, B., Agid, Y. and Langer, S. Z. ( 1~831/='ur. ,I. Pharmacol t~5, ,1I-51

P i e r r e M. L a d u r o n r e p l i e s [~H]lmipramine and [~H]desipnlmine binding has undoubtedly generated an impressive number of papers; perhaps the reason for this is simply the lack of specificity of the ligands to label amine uptake sites. It is well-known that imipramine is a more potent blocker of NA-uptake tban of 5HT-uptake ~-3. Moreover, the receptor profile of imipramine clearly shows that its affinity for histamine-H~, adrenergic-a~ and serotonln-S, receptors is higher than for the 5HT-uptake sites 3. in these conditions, impramine might not be expected to label 5HT-uptake sites selectively. It is thus not surprising that pyrilamine and phentolamine were found to be potent displacers in the 13H]imipramine binding experiments "s and that zimelidine, a well-known 5HT reuptake blocker, was much less active than pyrilamine and chiorproInazine 5 which are not, as far as 1 kpo~,, antidepressant drug,,s. The lack of specificity of the [3H]desipramine binding is clearly shown in Fig. 1. lmipramine and desipramine are almost as actwe on the NA-uptake as on the [3H]desipramine binding. In cGntrast, chlorpromazine is more active,

even more so than imipramine, on the binding. Obviously, chlorpromazine is chemically related to the tricyclic antidepressants, although it does not belong to the same cla~ of drug. More interesting is the case of imafen and amphetamine which are not chemically related to the tricyclic series. They are much more active on the NA-uptake; for instance the o-enantiomer of imafen is about ! (~) times more potent on the uptake than on the binding; this is quite surprising since this compound was found to be approximately equipotent to desipramine as an antagonist of ptosis in the reserpine test ~'. it should also be noted that the stereospecificity for both imafen enantiomers observed in the NA-uptake experiments was not apparent in the binding experiments. This dissociation between bM,itro tests had already been observed with amphetamine7 but was not considered to be a lack of binding specificity. One may conclude that the ["H]desipramine binding is not specifically related to NA uptake but to various sites including high-affinity recognition sites for tricyclic drugs. The same is also true of the [3H]imipramine IC50 (nM] 3H-DMI BINDING

DE SIPRAMINE

NA UPTAKE

30

2O

360

790

130

1,800

700

30

1,400

2

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70

i CH~CH:~CH~ NH'CH 3

IMIPRAMINE

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CHLORPROMAZINE

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H N ~-,T.N -..

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Fig. !. Effects of various drugs upon IJH/desib mine binding and noradrenaline uptake. © t ~ . ~.w= s = ~ ~

B.v. x=st=,~ ol65 - 6 1 ~ . ®

F e b n m r y 1984

17 I_,mgcr, S. Z , Morel, C , Raisman. R., Dul~covich, M. I.. and Briley. M. Sr (19811) Science 21(I, ! 133-! 135 !8 L,ingt'r, S. Z. and Raisman. R. (10831 Nemo. I)harmacohpgv 22,407-413 Ig Raisman, R., Sct,e, M . Pimoulc, C , Brilc~, M. S and Langcr, S. Z, (Ig82) Eur. J. Pharma¢ol. 78, 34.~351 20 Lee. C. M., Javitch, J. A. and Snyder, S. tl. 1198211. NeuroscL 10, 151~1525

binding. Saturation was achieved in the binding assay at a concentration of about I0 nM of imipramine~: il all the 13Hlimipramine binding sites were related to 5HT-uptake, one would expect

a complete blockade of the amine , ,,take at 10 riM. In fact this was not the case since 240 nM of imipramine was needed to inhibit 511% of the 5HTuptake-'. As a rule. the tricyclic drugs are much more active in the binding assay than in the uptake assay", whereas the opposite is true fi)r non-tricyclic drugs, such as indalpine or citalopram. Numerous reports have revealed a marked difference, sometimes even a complete dis~iation, between the mvitro as~ys, in patients suffering from alcoholic cirrhosis, the uptake of 5HT into platelets was found to be markedh reduced whereas the binding of [~H iimipramine was unchanged compared with control volunteers '~. The Bm,,x of [~H]imipramine was decreased and the Vm,~xof 5HT-uptake was increased after two daily injections of imipramine or desipramine for 3 weeksUL The ontogenetic development of the binding and the uptake was not parallel ~. The 5HTuptake of synaptosomes from Fawnhooded rats was significantly greater than that of Sprague-Dawley rats whereas the Bmax-value of [3H]imipramine binding was lower in the former ~2. Subcellular fractionation studies showed that 5HT-uptake was mainly recovered in the mitochondrial (M and L) fractions whereas [~H]imipramine binding was mainly enriched in the nuclear fraction L~. More recent studies s'~4 did not exactly confirm our results but did not, however, reveal an identical subcellular profile for both entities. A large proportion of ['~H]imipramine sites was found in the microsomal fraction (P), which surprisingly disappeared after chemical lesion with 5,7-dihydroxytryptamine, even more than in the mitochondrial fractions8 where the serotonergic nerve endings were recovered. In the second study z4, although no marker enzymes were used and the myelin and mitochondrial fractions were indicated erroneously, there was no parallel distribution profile

7"1PS - February 1984

for [~H]imipramine binding and 5HTuptake. If all the [-~H]imipramine sites were associated with serotonergic neurones, one would expect an identical disappeaiance of SliT and [~H]imipramine after chemical denervation: in fact [~H]imipramine binding was only reduced b ) 3 6 % whereas more than 90% of 5HT disappeared s. Although a chemical denervation may be extremely useful in assessing a neuronal localization, it does not provide any support for the specificity of the binding, simply because ,sometimes non-specific binding also disappears after lesion. It is very difficult to understand why desipramine (10 gM) was used for measuring non-specific bindin~ in the [3Hlimipramine binding assay" since desipramine is believed to label a different site. Another unexplained point is the occurrence, in rat liver, of [3H]imipramine binding sites (unpublished results) which are of high affinity and displaceabfe by 500 nM chlor-

4 Rat,.man. R . Bnk:~ M b and L,m~cr % / llqrslt) Fur J Pharm~,,l e,l. 3":* L~D r, Paul. S M Rchaxl M Sk~qnkk. P ,~nd t h . a ~ m . F K llte¢tl IJ~'e ~~ 2h. t~L'q~,t~ 6 (~dlXa.-n. F ( . I£n,,~'rz~. F M . %wmc~'c,.:r,

imipramine ~. In order to explain the results showing discrepancy between 5HT-uptake and [~H]imipraminc binding. Langet h~ pr(q~ed a high~ spet'ulative two-site model, according to which the imipramine site should be the, modulator unit of the 5HT-uptake me '~. It will certainly lake a vcry long time to prove this model. Another. perhaps more realistic, approach is to simph, admit that l~Hlimipramine and I'ttidesipramine arc not selective enough ligands to label the amine uptake sites specifically.

(

J |

and Jar~,~:n P A J (!~"~,~ Arch

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g ~ l t c . M . Ruhcrg, M . %talhm. B . tT.~k,~.~ B . A p d . ~' ~md l~m~-~. S / I,, q ,Ahtcc I . Bnk~ %1. ~aP,m,|n i~. l c b u , l } and Langcr S Z t i ~ l ~ Ira' %z 2~ 2323-23.~ Ill BaH~coa. Ni l . (ian&,ifi. ( ) . (huung. D M and ( ' ~ i a . |~ l i ~ 3 l Pr~a gaul 4¢,~,

PI~.RRE M. IAI)UR()N I I Mocchcm. I . Brun¢llo, N and R~-agn,. (~ l i r a 2 ) {/ur l Pha.-n~,,I ~3. ic'1-152 12 Anna. r ( . . h , a g . ( ' . Jackman. !1 l St~fl. D and Mclt:cr. i t 3v i l ~ l # e ~, 33.4~-442 13 L,aluron. P M . R c # ~ l m M and S~hotk..X 119821 Ear J Phar,nacu! "~. 4t~1-4~; 14 Rcha~L M . S k o l ~ k . P and Paul. ~ M

Depanmem o/ Bu,chenucai ]anxwn Pham,~'eunca.

PI annac,,I,,g~. B.Z'14O Reer~, Be!gqum

Reading Ust I Maitre, M.. Moser. P . Bauman~. P A and Waldmemr.P. C. (19801 ,4cm Ph~su,I .Wand (Suppl. 1.61-97 2 Raisman. R , Bnlcy. M and l , a n ~ r . S Z (1979) Nature tLondonl 281. 148-1~ 3 Ladumn, P. Bmchem Pharmacol (m pre~sl

15 Lanwr. S Z 1 and Ratsman. R I tq~t3) %e'z,r,* phannaco/oO ~..~CT-4hJ

Vi®w oi Dialogue What follows is another in our occasional series of TIPS 'debates'. Here, John A. Bevan takes issue with T. O. Neild and G. D. S. Hirst on the interpretation of the effects of adrenoceptor agonists on blood vessels.

'The T-connection': are w e r e a d y to t h r o w out the -adrenoceptor in sympathetic m

•

mml

vasoconstrlCglOn.

@

John A. Bevan Oe~ B~,

of Plmnmacology, Co;;egeof Med~.bm Given Medical Building. Un..nm' o/Ve.nom. VT 03405, USA.

In a recem article Angus t has summarized the evidence for a seemingly unique adrenoceptor, called % found in blood vessels and perhaps other adrenergic innervated smooth muscle tissues. The supporting experiments are almost entirely those of Him and Neild

and ~

(see get. 2, for a t o m y ) .

Briefly, they propose a distinctive adrenoceptor, neither ca nor IL exclusively found at the postsynaptic membrane of vascular smooth muscle, that responds to norepinepluine (NE) re-

leased from nerve terminals leading to excitatory junctional potentials (e.j.ps), f l n m ~ which nerve-induced conumiou

may be imitated. By contrast, the contraction of vascular smooth muscle to exogenous NE is mediated through extras.~aaptic, predominantly classical a-adreaoceptors, it is claimed that only the extrasynaptic, a-mediated response is blocked by phentolamine and other aadrenoceptor antagonists and its threshold to agonists, as esidenced by iontophoredc application, is 2-3 orders of magnitude lower than that for the proposed "y. The implication is that s)mpathetic neurogenic vasoconstriction is mediat~ through an entirely different receptor than that to exogenous NE. There are a number of reasons why this interesting proposal should be seriously questioned. (1) Although electroph)~iological studies have been taade on the rat tail artery, and saphenous and ear arteries34 and e.j.ps shown to be resistant to a-adrenoceptor blockade were recorded, the detailed crucial localization expenments have been carried out on only one type of blood vessel in one species, namely the

!~, ~

Somm ~

B.V. Ammmlam 01{6- el,l'~.~t~tO