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Brecher, B. & Bessis, M. (1972) Blood40,333-344 Longster, G . H., Buckley, T., Sikorski, J. & Tovey, L. A. D. (1972) Vox Sung. 22,161-170 Rumsby, M. G., Trotter, J.. Allen, D. & Michell, R. H. (1977) Biochem. SOC.Trans.5 , 126-128 Standefer, J. C. & Vanderjagt, D. (1977) Clin. Chem. 23,749-751 Trotter, J. (1978) D.Phil. Thesis, University of York

Effects of Lithium and Haloperidol on Blood Platelet Function H. WILLIAM READING and ROBERTA ROSlE M R C Brain Metabolism Unit, Thomas Clouston Clinic, 153 Morningside Drive, Edinburgh, EHlO SLC, Scotland, U.K.

The therapeutic value of lithium in treating mania is well established (Schou, 1978). The neuroleptics are also potent anti-manic drugs and their combined use with lithium has enjoyed some success. However, toxic neurological reactions have followed (London & Waring, 1976). Geisler & Klysner (1977) suggested that this adverse reaction may be related to a combined inhibition of striatal adenylate cyclase. Lithium and the neuroleptic a-flupenthixol both inhibit rat striatal dopamine-activated adenylate cyclase, and combination of the drugs produces potentiation of inhibition. Neuroleptics are potent blockers of cerebral dopamine receptors and are thought to act by competitively inhibiting dopamine-sensitive adenylate cyclase (Iversen, 1977). The action of lithium is not so clear-cut, but lithium does antagonize PGE,-induced stimulation of platelet adenylate cyclase (Wang et af., 1974), inhibit fluoride- and noradrenaline-induced adenylate cyclase in rat and rabbit cerebral cortex homogenates (Forn & Valdecasas, 1971), in rat cortex synaptosomes (Reading et al., 1975) and cyclic AMP formation in response to adrenaline in humans (Ebstein et al., 1976). Since both lithium and the neuroleptics possess the property of affecting brain dopamine-sensitive adenylate cyclase, we decided to examine their effects on a peripheral system which might respond in a similar manner to brain. The blood platelet responds by changes in function (aggregation, release and uptake processes) to both lithium and dopamine (Bouillin et al., 1975; Imandt et a f . , 1977; Reading, 1979). Platelet-rich plasma (PRP) was prepared from freshly voided rabbit blood with sodium citrate used as anti-coagulant (0.38 % final concentration). Blood was centrifuged 120g x 20min at room temperature and the PRP withdrawn with a plastic pipette. Platelet-poor plasma (PPP) was prepared by centrifuging an aliquot of PRP at 2000g x 15min. Platelet counts were determined with a Hawksley counting chamber to B.S. 748. Aggregation was measured by a modification of the method of Born (1962) in a Bryston aggregometer coupled to a Vitatron recorder. Each sample of plasma was calibrated separately and drug incubations were carried out at 37°C. Extents of aggregation and disaggregation are expressed as percentage changes in transmission (100 % change = PPP-PRP). Maximum aggregation in response to challenge by 1 ~ M - A D P is given as T,,,. and disaggregation after standard time (5min) as T d i , . ;the higher the latter, the less the disaggregation. Calcium release was determined on washed platelet suspensions in isoosmotic Tris/ HCI buffer, pH7.4 ( lo6 platelets/mm3). Calcium estimations were made on plateletfree solutions, obtained by rapid filtration through polycarbonate membranes (pore size 0.4pm). Calcium was determined fluorimetrically with chlortetracycline as a fluorescent probe (Reading, 1978). When PRP is incubated for 2h at 37°C there is a steady decline in aggregation in response to challenge by ADP. Lithium preserved the sensitivity of the platelet to ADP-induced aggregation over this period (seeTable 1). However, potentiation occurred after a short period of incubation: lOmin incubation of PRP with 10-3~-lithiumand Abbreviations used : PPP, platelet-poor plasma; PRP, platelet-rich plasma.

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BIOCHEMICAL SOCIETY TRANSACTIONS

Table 1. Effect of lithium and haloperidol on A DP-induced aggregation in rabbit platelets Values are expressed as percentage changes in transmission when the difference between PPP and PRP was adjusted to 100 %. Drugs were dissolved in isoosmotic Tris buffer, pH7.4. Additions of buffer solutions, buffer control and saline control to PRP were in the PRP: buffer 10: 1. Incubation was at 37°C. N.D., not determined. Each value is the mean of two determinations.

--Control (Tris/Cl buffer)

Incubation time

10 30 90 120 120 (control)

Lithium (

M)

Lithium M)/haloperidol (2.5 x 1 0 - 4 ~ ) I

74 43 N.D.

29 30

7 11 N.D. 7 5

101 69 N.D. 73

-

37 17 N.D. 10

-

56 68 73 68

-

-2 8 6 9

-

2.5 x 10-4~-haloperidolantagonized the lithiumeffect at 10min. After longer incubation, haloperidol had no action on the lithium effect. Neither lithium nor haloperidol (without ADP) produced any aggregation at any time. Lithium (1O-j~)did not release any calcium from platelet suspensions when incubated for 0-lOmin, but after 2h incubation it released 0.8 x lo7molecules per platelet. Calcium release by haloperidol was dependent on concentration : 1 0 - 6 ~produced virtually the same release as 10-3~-lithium,0.8 x lo7 molecules per platelet, but higher concentration, ( l O P 5 ~gave ) 0.5 x molecules of calcium per platelet irrespective of incubation time. The two agents together, at the concentrations lo-’ M-lithium and 10-6M-haloperidol, gave an additive release of calcium of 1 . 2 lo7 ~ molecules per platelet (2h incubation). Total calcium content per platelet determined by the same fluorescent method was 1.4+ 1.1 x lo7 molecules per platelet. Results must be interpreted taking into account the findings of Imandt e t al. (1977) that lithium takes 90min-2 h in vitro to reach equilibrium intracellular concentration in platelets. Our results, showing potentiation of ADP-induced aggregation after IOmin, suggest an effect of lithium on the outside of the platelet membrane. Haloperidol antagonized this rapid effect of lithium, suggesting the blocking of a common receptor, the nature of which is unclear. Bouillin e t al. (1978) concluded that there are no specific dopamine receptors on the platelet membrane, but that haloperidol probably interacts with 5-hydroxytryptamine receptors on the platelet. It is therefore of interest to examine further the nature of the platelet receptor concerned in the action of lithium and the lithium-haloperidol interact ion. Bouillin, D. J., Green, A. R. & Grimes, R. P. J. (1975) J . Physiol. (London) 2 5 2 , 4 6 ~ 4 7 ~ Bouillin, D. J., Molyneux, D. & Roach, D. (1978) Er. J . Pharmacol. 63, 561-566 Ebstein, R., Belmaker, R., Grunhaus, L. & Rimon, R. (1976) Nature (London) 259,411-413 Forn, J. & Valdecasas, F. G. (1971) Eiochetn. Phurnracol. 20,2773-2779 Geisler, A. & Klysner, R. (1977) Lancet i , 4 3 M 3 1 Imandt, L., Genders, T., Wessels, H. & Haanen, C. (1977) Thrombosis Res. 11,297-308 Iversen, L. L. (1977) J. Neurochetn. 29,5-12 Loudon, J. B. & Waring, H. (1976) Lancet ii, 1088 Reading, H. W. (1978) Proc. Meet. SOC. Neurochem. 2nd, Gottingen, p. 439, Verlag, Chernie, Weinheim, New York Reading, H. W. (1979) Cell. Mol. Biol. (in the press) Reading, H. W., Kinloch, N. & Loose, A. R. (1975) Proc. ISN Meet. 5th, Barcelona 417 Schou, M. (1978) in Lithium in Medical Practice (Johnson, F. N. & Johnson, S . , eds.), pp. 21-39, MTP Press, Lancaster 1979

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Wang, Y.-C., Ghanshayan, N. P., Mendels, J. & Frazer, A. (1974) Biochem. Pharmacol. 23, 845-855

The Subcellular Fractionation of Plant Shoot Tissue by Zonal Rotor Centrifugation KENNETH J. CATTELL,* MARTIN J. CONNOCK,t TREVOR J. HOCKING,? PAUL R. KIRK,? JOHN SHUTTLEWORTH? and ANTHONY P. STURDEES *Department of Science, Bristol Polytechnic, Bristol BS16 1Q Y, U.K.,

i Department oJ’Biologica1Sciences, The Polytechnic, Wolverhampton WV1 1L Y, U.K. and $Department of Biological Sciences, Lanchester Polytechnic, C V1 5FB, U.K. There is considerable interest in the fractionation of plant shoot tissue so that the subcellular distribution of putative plant hormone receptors and enzymes which may be involved in the growth of cells and the synthesis of cell components can be investigated (Ray, 1977; Bowles et a/., 1977; Hartmann-Bouillon & Benveniste, 1978). The aim of the work reported here was to use density-gradient centrifugation in a zonal rotor to prepare large quantities of subcellular fractions from whole homogenates of plant shoot tissue in order to (i) avoid the use of differential centrifugation to concentrate payloads and (ii) prepare sufficient material for a range of assays from a single gradient experiment. Coleoptiles from Zea mays L. or epicotyls from Pisum sativun: were harvested and chopped by hand with a razor blade in a small amount of homogenization medium before gentle grinding in a chilled mortar. The homogenization medium contained Som~-Tris/acetate, pH 8.0, 0.25 M-sucrose, 1mM-EDTA and 0.1 mM-MgC1,. The brei was filtered through muslin and the residue was reground in homogenization medium and filtered. The final ratio of tissue to medium used was 2: 1 (w/w). The combined filtrates were centrifuged at lOOOg for 5 min. This postnuclear supernatant (200cm3) was then loaded into an MSE aluminium BXIV rotor which contained an interface of 17% sucrose resting against 300cm3 of a linear sucrose gradient (20-50%), which in turn rested against a cushion of 60% sucrose. After supplying the overlay the rotor was centrifuged at 30OOOrev./min for 12h at 5°C. The sample and overlay were collected as a single fraction, the interface as 4 x 12cm3fractions and the gradient as 20x 15cm3 fractions. Fractions were assayed for a range of activities, including antimycin-insensitive NADH-cytochrome c reductase (Donaldson et al., 1972), RNA (Ogur & Rosen, 1950), inosine diphosphatase (IDPase; Leonard et al., 1973), succinate dehydrogenase (SDH; Pennington, 1961), UDP-glucose-lipid glucosyltransferase (LGT; Leizica et al., 1976), glucan synthase measured at low (GSI) or high (GSII) UDP-glucose concentrations (Dohrmann et al., 1978) and catalase (Leighton et al., 1968), and for their ability to bind 1-naphthylacetic acid (NAA) and 1-N-naphthylphthalamic acid (NPA; Ray et al., 1977). Samples of each fraction were also taken for electroil microscopy. The results of typical isopycnic separations obtained with maize coleoptile and pea epicotyl post-nuclear supernatants are summarized in Table 1. The distribution of established marker activities together with the appearance of samples from each fraction in the electron microscope show that four subcellular organelles are clearly separated in both preparations. The endoplasmic reticulum is characterized by the peak of NADH-cytochrome c reductase activity, ribosomes by the presence of RNA, mitochondria are indicated by the peak of SDH activity and peroxisomes are indicated by the presence of catalase. Abbreviations used : IDPase, inosine diphosphatase; SDH, succinate dehydrogenase; LGT, UDP-glucose-lipid glucosyltransferase; GS, glucan synthase; NAA, naphthylacetic acid; NPA, naphthylphthalamicacid.

Vol. 7

Effects of lithium and haloperidol on blood platelet function [proceedings].

583rd MEETING, CAMBRIDGE 933 Brecher, B. & Bessis, M. (1972) Blood40,333-344 Longster, G . H., Buckley, T., Sikorski, J. & Tovey, L. A. D. (1972) Vo...
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