The Effects of Protriptyline and Clomipramine in Vitro on the Uptake of 5-Hydroxytryptamine and Dopamine in Human Platelet-Rich Plasma

Psychopharmacologia (Berl.) 43, 89-93 (1975) by Springer-Verlag 1975

The Effects of Protriptyline and Clomipramine in vitro on the Uptake of 5-Hydroxytryptamine and Dopamine in Human Platelet-Rich Plasma

ANN TRENCHARD* and P. TURNER Department of Clinical Pharmacology

C. M. B. PARE Department of Psychological Medicine, St. Bartholomew's Hospital, London, England

M. HILLS British Museum (Natural History), London, England

Received September 24, 1974

Abstract. The effects of protriptyline and clomipramine, at The activity of both compounds was competitive but it was concentrations of 10 -7 M to 10 .4 M, were studied in vitro on thought unlikely that they acted through tryptamine receptor the uptake of 5-hydroxytryptamine and dopamine uptake in sites as methysergide 2.5 x 10 .8 M had very little effect on human platelet-rich plasma. It was found that the tertiary 5-hydroxytryptamine Uptake. Neither tricyclic antidepressant amine, clomipramine, was a more potent inhibitor of 5-hy- had any marked effect on dopamine uptake. droxytryptamine uptake than the secondary amine, protriptyline.

Key words: Uptake - Platelets - 5-Hydroxytryptamine - Dopamine - Inhibition - Protriptyline - Clomipramine.

Introduction amine, protriptyline, and the tertiary amine, clomipramine, on the uptake of 5-HT and dopamine in PRP Many workers have compared the ability of certain from normal, healthy volunteers who had not taken neurones to take up and store biogenic amines against any medication for at least three weeks. a concentration gradient with similar properties in blood platelets. Sneddon (1973), in his review of the literature, concluded that the platelets may provide Method a suitable model for the serotoninergic neurone, but Plastic syringes and tubes were used throughout to prevent there is not enough evidence to compare them with platelet aggregation. 30 ml blood was taken by venepuncture catecholamine-storing neurones. from each volunteer. It was put into a centrifuge tube contain- In preliminary experiments with human platelet- ing 4 ml anticoagulant, (25 g trisodium citrate, 13.7 g citric rich plasma (PRP) (Trenchard and Turner, unpub- acid and 20 g glucose in I 1 distilled water; Aster and Jandl, 1964). The blood was left at room temperature for 2- 3 hrs to lished) we confirmed the results of Stacey (1961) and separate out. The PRP was removed and the remaining blood Boullin and O'Brien (1970) who have shown that centrifuged at 156 g for 5-10 min. The plasma layer was 5-hydroxytryptamine (5-HT) and dopamine are taken removed and added to the PRP already collected. In order to up in human platelets by energy-dependent mecha- obtain comparable results for both amines at each concentration of inhibitor, PRP from several subjects was pooled. nisms, but were unable to confirm that similar mecha- The tube was swirled gently to ensure even dispersal of the nisms were involved in the uptake of L-noradrenaline, platelets. A small volume, about 0.2 ml, was taken for counting contrary to the findings of Abrams and Solomon in a Coulter Platelet Counter, model B. This method yielded (1969). In this study we have examined the activities about 4 to 6 x 108 platelets per ml plasma. PRP was dispensed of two tricyclic antidepressant drugs, the secondary in 1 ml aliquots and the tubes incubated at 37~ in a water bath with a shaker. They were allowed to equilibrate for 10 min * A.T. was supported by a grant from the Mental Health before adding the inhibitor. The tritiated amine was added Research Fund. 15 min later. (Control tubes were incubated for 25 min before 90 Psychopharmacologia (Berl.), Vol. 43, Fasc. i (1975)

adding the tritiated amine.) Uptake of the amine was stopped _•-,- 5-HT ,, dopamine after 15 min by adding 2 ml ice-cold saline to each tube. The platelets were centrifuged out at 8900 g for 3 rain. The super- natant was decanted and the tube wiped dry with tissue. 1 ml N/I potassium hydroxide was added to the remaining pellet and left overnight. The platelets were resuspended with i ~,,' a s a rota mixer and 100 gl sample put into 10 ml scintillation fluid, (8 g butyl BPD, 160 g naphthalene, 800 ml 2-methoxy- ethanol and 1200 ml toluene). Each sample was counted twice in a Beckman LS 200 liquid scintillation counter. The readings were converted to d.p.m, per 108 platelets.

I I I I Illll Radioactive Compounds 10 100 Tritium-labelled 5-hydroxytryptamine creatinine sulphate amine:nmof per ml PRP and dopamine hydrochloride were obtained from the Radio- Fig. 1. The effect of protriptyline on the uptake of 5-HT and chemical Centre, Amersham. Labelled compounds were dilut- dopamine in platelet-rich plasma ed with non-radioactive compounds to give a standard concentration of 50 gCi/lamol amine. i) Effect of Protriptyline and Clomipramine on 5-HT and 5-HT t dopamine Dopamine Uptake. Concentrations of 5-HT used were 1.25, / l P 2.5, 5.0 and 10.0 nmol/ml PRP and for dopamine, 12.5, 25.0, 50.0 and 100.0 nmol/ml PRP. Uptake was estimated in the % presence of either protriptyline hydrochloride (Merck, Sharpe and Dohme Ltd.) or clomipramine hydrochloride (Geigy % g Pharmaceuticals) at concentration ranging from 10 -7 M to // 10 4 M. The effect of each antidepressant on amine uptake was x estimated on four separate samples of PRP. Statistical Analysis. The variation between plasma samples ,' ~,,' tended to increase with increasing uptake. The simplest way to analyse data of this kind is to break each uptake curve into its four components. The first is the overall uptake 05) and is "/ J the straight average of the uptake values for the four concentra- 0 ~ I I I I IIIII tions of 5-HT or dopamine. The second, third and fourth mea- 10 100 sure the linear, quadratic and cubic components, respectively amine : nmol per ml PRP and are the linear contrasts of the uptake values at the four Fig. 2. The ~ffect of clomipramine on the uptake of 5-HT and amine concentrations. For this data the major differences be- dopamine in platelet-rich plasma tween the uptake curves, in presence of the different concentrations of inhibitor, resided in the overall levels and the linear components. These two components showed a high degree of Protriptyline10-6M correlation, - as the overall uptake level rose so did the linear component. It was sufficient, therefore, to analyse the data ~:~-~c2.12!E'~(~Q''~ ~ ~ Cl~ using the overall levels only, ignoring the other components and analysis of variance for this data has been given. Values were scaled by a factor of ] 0- 3 to facilitate calculations. Y ~ ControIMethysergide2"5X10-SM ii) Nature of the Inhibition of 5-HT Uptake. The nature of the inhibition of 5-HT uptake due to tricyclic antidepressant drugs was determined by the method of Lineweaver and Burk (1934). Concentrations of 5-HT used were 12.5, 16.7, 25.0 and 50.0 nmol/ml PRP. Uptake was estimated on two pooled I ~IZ I I I I I -o.o8 -o~, o.o, ~o8 samples of PRP in the absence and presence of protriptyline /['5-HT' PRP] 10 -6 M, clomipramine 10 -8 M and methysergide bimaleate nmol/ml (Sandoz Products Ltd.) 2.5 x 10 -s M. A duplicate series of Fig. 3. Lineweaver-Burk plot of the effect of protriptyline, tubes was incubated at 0~ for each concentration of 5-HT clomipramine and methysergide on the uptake of 5-HT in in order to correct the results for the amount of amine that platelet-rich plasma entered the platelets by physical diffusion.

Results tration of this compound caused a progressive in- i) The Effect of Protriptyline and Clomipramine on the hibition of 5-HT uptake. Almost maximum blockade Uptake of 5-HT and Dopamine. The results for pro- occurred in the presence of protriptyline 10 .4 M. triptyline are shown in Fig. 1. Increasing the concen- Dopamine uptake was not altered by protriptyline. A. Trenchard et al. : Protriptyline and Clomipramine on 5-HT and Dopamine Uptake in Human Platelet-Rich Plasma 91

Table 1. The effect of protriptyline in vitro I. Analysis of variance for 5-HT (Y x 103)

Source of variance Sums of squares df Mean square Variance ratio P

Plasmas 5067.60 3 1689.20 13.08 < 0.001 Protriptyline concentrations 6422.21 4 1605.55 12.43 < 0.001 Residual 1549.97 12 129.16

Total 13 039.78 19

II. Analysis of variance for dopamine @ x 103)

Plasmas 1280.73 3 426.91 40.93 < 0.001 Protriptyline concentrations 48.06 4 12.02 1.15 > 0.2 Residual 125.18 12 10.43

Total 1453.97 19

Table 2. The effect of clomipramine in vitro I. Analysis of variance for 5-HT (Y x 103)

Plasmas 93.78 3 31.26 1.14 > 0.2 Control vs. 4 concentrations clomipramine 5 310.70 1 5 310.70 193.26 < 0.001 Between 4 concentrations clomipramine 43.42 3 14.37 0.53 > 0.2

Between control and 4 concentrations clomipramine 5354.12 4 1338.53 48.71 < 0.001 Residual 329.79 12 27.48

Total 11 131.81 19

II. Analysis of variance for dopamine (Y x 103)

Plasmas 165.75 3 55.25 13.74 < 0.001

Control vs. 4 concentrations clomipramine 215.54 1 215.54 53.62 < 0.001 Between 4 concentrations clomipramine 4.81 3 1.60 0.40 > 0.2 Between control and 4 concentrations clomipramine 220.35 4 55.09 13.70 < 0.001 Residual 48.13 12 4.01

Total 654.48 19

The analysis of variance (Table 1) confirmed that tween the groups containing clomipramine, on 3 df. protriptyline had a significant effect on 5-HT uptake, There was no significant difference in effect on 5-HT P < 0.001, but did not have any significant effect on uptake between the four concentrations of clomi- dopamine uptake, P > 0.2. pramine, P > 0.2. Clomipramine (Fig. 2) was a more potent inhibitor It can be seen from the graph that clomipramine of 5-HT uptake than protriptyline. Almost maximum 10 .7 M caused a slight inhibition of dopamine uptake, "inhibition occurred at all concentrations. This ac- but this effect was not increased by raising the con- tivity was highly significant, P < 0.001. The sums of centration of clomipramine. Statistical analysis has squares for this source of variation, with 4 dr, has shown this effect was significant, P < 0.001 and that been split into two components. The control uptake the source of variation was between the control group was contrasted with uptake in the presence of clomi- and the groups containing clomipramine, P < 0.001. pramine on 1 df This component was highly signifi- There was no significant difference in uptake between cant, P < 0.001. The second compared uptake be- the 4 groups containing clomipramine, P > 0.2. 92 Psychopharmacologia (Bed.), Vol. 43, Fasc. I (1975) ii) Nature of the Inhibition of 5-HT Uptake. Double exert their effect on 5-HT uptake in platelets at reciprocal plots of l/uptake against I/5-HT concen- membrane level. Boullin (1968) has shown that the tration are shown in Fig. 3. Protriptyline 10 -6 M and related compound, imipramine, binds to the outer clomipramine 10-aM both inhibited 5-HT uptake membrane of human platelets and da Prada and without altering the theoretical maximum uptake, but Pletscher (1968) found that it had a much greater they did increase the apparent Kin, indicating that the ability to block membrane uptake than to inhibit nature of the competition was competitive. Methyser- 5-HT entry into the storage vacuoles of rabbit platelets. gide 2.5 x 10 -s M had very little effect against 5-HT These comparisons indicate that there may simi- uptake. larities between platelet and neuronal uptake systems. A comparative study in both systems of compounds Discussion that have been shown to block dopamine-induced The more effective inhibition of 5-HT uptake by cyclic AMP formation, such as fluphenazine and clomipramine than protriptyline confirmed the earlier ftupenthixol (Iversen et al., 1974), may provide further work of Todrick and Tait (1969). They have shown information. that tricyclic compounds with a tertiary amine group Acknowledgements. We would like to thank volunteers from are more effective inhibitors of 5-HT uptake than the G.P.O., City Section, East Central Branch (London) compounds with a secondary amine group in the and from the Blood Bank, St. Bartholomew's Hospital, aliphatic side chain. who gave blood for this research. Drugs were supplied by Baumgartner and Born (1968) have suggested that Geigy Pharmaceuticals, Merck, Sharp and Dohme Ltd. and Sandoz Products Ltd. platelets have specific receptor sites for 5-HT. The competitive nature of the inhibition of 5-HT uptake References by clomipramine and protriptyline at lower molecular concentrations than the levels of 5-HT used, together Abrams, W. B., Solomon, H. M. : The human platelet as a with their selective activity against this amine, provides model for the adrenergic neurone. Clin. Pharmacol. 10, 702- 709 (1969) further evidence to support this theory. The weak Aster, R.H., Jandl, J. H.: Platelet sequestration in man. blocking effect of methysergide, which confirmed the I. Methods. J. din. Invest. 43, 843-855 (1964) work of'Michal and his colleagues (1971), indicated Baumgartner, H., Born, G. V.R.: Effect of 5-hydroxytryp- that 5-HT uptake did not occur through tryptamine tamine on platelet aggregation. Nature (Lond.) 218, 137- receptors. 143 (1968) Boullin, D. J. : The binding of imipramine to the outer mem- Although Boullin and O'Brien (1970) have shown brane of blood platelets. J. Pharm. Pharmacol. 20, that 5-HT and dopamine are mutually antagonistic, 583- 584 (1968) suggesting that they use the same storage sites in the Boullin, D. J., O'Brien, R. A. : Accumulation of dopamine by platelet, the selective inhibition of 5-HT uptake by blood platelets from normal subjects and Parkinsonian patients under treatment with L-Dopa. Brit. J. Pharmacol. the tricyclic compounds, at concentrations that had 39, 779-788 (1970) little or no effect on dopamine uptake in aliquots Carlsson, A., Corrodi, H., Fuxe, K., H6kfelt, T.: Effects from the same samples of PRP, indicates that the of antidepressant drugs on the depletion of intraneuronal uptakes of the two amines are not identical. There 5-hydroxytryptamine stores caused by 4-methyl-c~-ethyl- is a correlation between these results and the findings meta-tyramine. Europ. J. Pharmacol. 5, 357-366 (1969a) Carlsson, A., Corrodi, H., Fuxe, K., H6kfelt, T. : Effects of of other workers studying uptake systems in neurones. some antidepressant drugs on the depletion of intraneuro- Carlsson et al. (1969a), using the selective depletion nal catecholamine stores caused by 4-ct-dimethyl-meta- of biogenic amines by c~-methyl-meta-tyramine deriva- tyramine. Europ. J. Pharmacol. 5, 367-373 (1969b) tives from the central nervous system of conscious Carlsson, A., Fuxe, K., Hamberger, B., Lindqvist, M. : Bio- chemical and histochemical studies on the effects of rats and mice as a model to study uptake systems, imipramine-like drugs and o(+)amphetamine on central found that the ability of the tricyclic antidepressants and peripheral catecholamine neurones. Acta physiol. to block 5-HT depletion was of a similar order to their scand. 67, 481- 479 (1966) inhibition of 5-HT uptake in vitro. They (1969b) have Da Prada, M., Pletscher, A. : Isolated 5-hydroxytryptamine also shown that secondary amines, such as protripty- organelles of rabbit platelets: physiological properties and drug-induced changes. Brit. J. Pharmacol. 34, 591-597 line, were unable to protect against dopamine deple- (1968) tion, but have not reported any results for tertiary Hamberger, B. : Reserpine-resistant uptake of catecholamines amines. in isolated tissues of the rat. A histochemical study. It has been suggested that the tricyclic antidepres- Acta physiol, scand. 68, Suppl. 295, 1 - 56 (1967) sant drugs exert their blocking action on biogenic Iversen, L. J., Miller, R. J., Horn, A. S.: Effects of dopa- minergic agonists and antagonists on cyclic Y,5'-adeno- amine uptake at the level of the neuronal membrane sine monophosphate (cyclic AMP) production in rat brain carrier systems (Carlsson etal., 1966; Hamberger, homogenates. J. Pharmacol. (Paris) 5, Suppl. 1,117-118 1967). Protriptyline and clomipramine also probably (1974) A. Trenchard et al. : Protriptyline and Clomipramine on 5-HT and Dopamine Uptake in Human Platelet-Rich Plasma 93

Lineweaver, A., Burk, D.: The determination of enzyme Stacey, R. S. : The uptake of 5-hydroxytryptamine by platelets. dissociation constants. J. Amer. chem. Soc. 56, 666-685 Brit. J. Pharmacol. 16, 284-295 (1961) (1934) Todrick, A., Tait, A. C. : The inhibition of human platelet Michal, F., Born, G. V. R., Juengjaroen, K. : Platelet recep- 5-hydroxytryptamine uptake by tricyclic antidepressive tors in platelet aggregation. J. Caen, ed. Paris: Masson drugs. The relation between structure and potency. J. and Co. 1971 Pharm. Pharmacol. 21, 751- 762 (1969)

Ann Trenchard, 23, Goldings House, Hatfield, Herts. ALl0 8TZ, U.K.