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Stimulation of acetylcholine synthesis by blockade of presynaptic muscarinic inhibitory autoreceptors: Observations in rat and human brain preparations and comparison with the effect of choline
Life Sciences, Vol. 30, pp. 1517-1524 Printed in the U.S.A.
Pergamon Press
STIMULATION OF ACETYLCHOLINE SYNTHESIS BY BLOCKADE OF PRESYNAPTIC MUSCARINIC INHIBITORY AUTORECEPTORS: OBSERVATIONS IN RAT AND HUMAN BRAIN PREPARATIONS AND COMPARISON WITH THE EFFECT OF CHOLINE. Kenneth L. Marek, David M. Bowen, Nell R. Sims and Alan N. Davison Department of Neurochemistry, Institute of Neurology, Queen Square, London WCIN 3BG (Received in final form February 18, 1982) Summary The central presynaptic muscarinic inhibitory autoreceptor has been monitored by measuring the effects of muscarinic agents on acetylcholine (ACh) synthesis by rat and human neocortical tissue prisms. Quinuclidinyl benzilate (QNB), the antlmuscarinic which of 20 tested caused the most marked stimulation of ACh synthesis in rat, significantly increased ACh synthesis in human prisms over a range of concentrations of 0.i ~M-IO ~M. This data provides the first evidence that human brain contains presynaptic muscarinic receptors. However, the most marked effect of QNB was to increase synthesis to only 112% of control (value without drug) which was much less than in rat (to 140% of control). ACh synthesis is reduced to 50% of control in neocortex from Alzheimer patients so none of the antimuscarinics tested seem to be potentially capable of appreciably reversing this deficit. A high concentration of choline (iO mM) stimulated synthesis in rat prisms to about the same extent as QNB. Moreover, the ACh precursor was at least as effective in stimulating synthesis in human prisms (including those from a patient with Alzheimer's disease). This suggests that an elevated intracellular concentration of choline is likely to be much more effective than an antimuscarinic agent in stimulating synthesis in Alzheimer brain. Rodent brain has been shown to contain presynaptic autoreceptors that modulate the release and synthesis of putative neurotransmitters during nerve stimulation (1,2). Thus presynaptic receptors are now being considered as potential target receptors for the development of new selective drugs (3,4). Whether presynaptic receptors occur in human brain is thus important but has not been determined for most transmitters. We have investigated the cholinergic system because it has been implicated in a number of neurological and psychiatric disorders, especially those associated with ageing. A prominant example is the non-treatable dementing condition of Alzheimer's disease, where there is evidence of a clinically relevant reduction, of about 50%, in the formation of acetylcholine (ACh) in the brain (5-7). The reduction of ACh synthesis appears to be due to loss of presynaptic cholinergic nerve endings. The remaining cholinergic terminals seem to retain normal function and postsynaptic muscarinic receptors are apparently spared (4,5). Thus clinical improvement may be brought about by stimulating ACh synthesis in the few remaining nerve endings. It has been proposed that these objectives could be achieved by either a high choline diet (8,9) or by manipulating the presynaptic muscarinic receptor (4). Muscarinic receptor antagonists
stimulate ACh release and synthesis
0024-3205/82/181517-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
in
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Vol. 30, No. 18, 1982
vitro in rat brain preparations (i0,ii). Acting even in the presence of tetrodotoxin (I0), their effect is reversed by muscarinic agonists (12). Thus there are thought to be muscarinic inhibitory autoreceptors located on the cholinergic nerve terminal (13), in addition to the classical postsynaptic muscarinic receptor (14). The objective of the present work ~as to establish the extent to which antimuscarinic agents stimulate ACh synthesis and to compare this with the effect of choline (15). Using a rat brain preparation a number of antimuscarinic agents and drugs with varying postsynaptic muscarinic potencies and of various structure (16-18), have been screened for their influence on ACh synthesis in vitro. The most effective substance was examined for its effect on synthesis by preparations of fresh normal appearing human brain. Antimuscarinics act optimally in brain tissue preparations that have been depolarized (19, 20). Consequently, the interaction of the substances with the presynaptic receptor has been monitored by using K + stimulated neocortical tissue prisms (6). The prisms enabled the cholinergic nerve endings to be studied free from the influences of the cell body. The incorporation of U-14C -glucose into 14C -ACh and 14CO2 was measured to determine the ACh synthesized and total glucose metabolized, respectively C6). Materials and Methods U-14C -glucose ( 230 Ci/mol) was from The Radiochemical Centre, Amersham, U.K. and was diluted appropriately with glucose (BDH Chemicals, Poole, U.K.). Paraoxon, ACh bromide, choline chloride, acetylcholinesterase (Type VI-S from electric eel), atropine and scopolamine were from the Sigma Chemical Co., Poole, U.K. Oxotremorine was from the Aldrich Chemical Co., Ltd., Gillingham, U.K. Antimuscarinic Agents and Drugs The gifts of clozapine and thioridazine HCI (Sandoz Products, U.K.), amitriptyline (Roche Products, U.K.), trihexylphenidyl HCI (Lederle Labs., U.K.), maprotiline HCI (CIBA Labs., U.K.), benztropine mesylate (Merck, Sharp and Dohme, U.K.), and nomifensine maleate CHoechst, U.K.) are gratefully acknowledged. Apart from scopolamine, Dr. T.O. Inch and Dr. D.M. Green (C.D.E., U.K.) generously provided the other substances listed in Table I and benactyzine HCI; aprophen HCI; N-methyl-piperidin-4-yl-2-cyclohexyl-2-hydroxy-2-phenylacetate HCI; N-ethyl-3-piperidyl benzilate HCI; N-methyl-3-piperidyl benzilate HCI; dimethylamino-ethyl-2-cyclohexyl-2-hydroxy-2-phenylacetate HCI; D-trans-4-S-(S-l-cyclohexyl-l-hydroxy-l-phenyl) methyl-l, 3-dioxolan (trans-dioxolan) and D-cis-4-5dimethyl-aminoethyl-2-R-(R-l-cyclohexyl-l-phenyl) methyl-l, 3-dioxolan (cisdioxolan). Control Human Brain Tissue Samples of normal appearing neocortex plus underlying white matter (6) were obtained from patients aged 17-65 at surgery (7 on temporal lobe, 6 on front al lobe, and 1 on occipital lobe) for 5 craniopharyngiomas, 3 meningiomas, 2 astrocytomas, 2 gliomas, 1 basilar aneurysm and 1 pituitary tumor. Alzheimer Brain Tissue The sample of temporal neocortex was taken at diagnostic craniotomy from a demented patient aged 64. Histopathology of adjacent tissue samples revealed the presence of senile plaques and neurofibrillary degeneration, confirming that the patient had Alzheimer's disease. Measurement of ACh Synthesis Male Wistar rats (Porton strain, 6-9 months) were decapitated and the
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brains rapidly removed. Rat and human specimens were placed in ice-cold, modified Krebs Ringer phosphate buffer (141 mM NaCI, 5 mM KCI, 1.3 mM CaCI2, 1.3 mM MgSO 4 and iO mM Na2HP04, pH 7.4) containing 2.5 mM glucose and freshly gassed with 100% oxygen. Neocortex, including all cortical layers, was dissected. Preparation of tissue prisms, preincubation and measurement of incorporation of U-14C -glucose into 14C -ACh and 14C02 in the presence of 31 mM K + and 40 ~M paraoxon was carried out using a slight modification of the method of Sims et al (6). The incubation period was for 30 min instead of I h and prisms were maintained at room temperature after preincubation rather than cooling on ice. The modification to incubation time was made because in incubations terminated after 30 min, i DM atropine enhanced 14C -ACh formation by 39% ± 14% (mean S.D., n = 4), whereas the value was lower (31% ± 8%) for incubations of I h. When preincubated prisms were maintained on ice prior to assay, i uM atropine enhanced 14C -ACh formation by only 18% ± 7% (mean ~ S.D., n = 4) of the control value (prisms without the drug). The value was higher (42% ! 11%), using prisms maintained at room temperature. The control values were independent of the temperature at which the preincubated prisms were maintained. These results were consistent with those of Ei-Fakahany & Richelson (21). Test incubations were performed in quadruplicate and blanks in triplicate. The results are based upon data from at least 3 independent experiments, unless stated otherwise. The method of Sims et al (6) was used to determine the effect of choline on 14C ACh synthesis in human tissue prisms. The incubation medium contained 31 mM K +. The radioactivity isolated as 14C -ACh from incubation of rat prisms with i ~M QNB and without drug was hydrolysed by acetylcholinesterase (6) by 95% ! 2% and 95% ~ 3% (mean ~ S.D., n = 3), respectively. This indicated that essentially all of the radioactivity was attributable to labelled ACh. 14C -ACh synthesis and 14C02 formation are expressed as the mean ! S.D. with I unit being equivalent to i d.p.m./mg protein/min (using 2.5 mM glucose and 2.8 and 11.2 ~ci of U-14C-glucose/flask in incubations with rat and human prisms, respectively). Unless stated otherwise, statistical analysis was by the Student's t-test for unpaired observations; the null hypothesis was rejected for probability values less than O.O1. Due to the variability in 14C -ACh formation by individual control human brains (6) and the small effect of QNB on synthesis in this species, results for human brain (as well as those for rat in Fig. i) were analyzed by the Paired t-test. Results in the text are mean ! S.D. Results Effect of Various Antimuscarinic
Agents and Drugs on
14C -ACh Synthesis
The 20 substances tested (Materials and Methods and Table I) were incubated with rat prisms for 30 mins in medium containing 31 mM K + and 2 mM choline. At a concentration of I ~M 17 compounds caused a significant increase in synthesis to between 120%-140% of the control value. Quinuclidinyl benzilate (QNB) caused the most marked stimulation. The substances without significant effect were maprotoline, trans-dioxolan and cis-dioxolan (detailed elsewhere, 22). At a concentration of O.01 ~M only 4 agents caused a significant increase in the formation of labelled ester (Table I), the most potent agent was again QNB. None of the substances had a significant effect on the production of 14C02 (Table I). Effect of Potent Muscarinic Agents on
crease
14C -ACh Synthesis in Rat and Human Prism~
The effect of QNB, measured over a dose range of 0.OO1-I0 ~M, was to in14C -ACh formation in a dose dependent manner (Fig. i). The most effect-
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Stimulation of Acetylcholine Synthesis
2
4
Vol. 30, No. 18, 1982
CHOLINE (mM} 6 8
•
I
10
I
I
--~140 0
0 o
0
o~ 1 2 0 GO
~
•
o
LU
"r'lO 0
I-Z >-
Ill
$
Z
-
.1
80-
0 -r
o~
0 ._1
>-
w
0 <
60~-
0
I I 0"001 0-01 MUSCARI
I 0.1 NIC
l 1-0
AGENT
I 10
| 6 ,I 100 1000
(juM)
FIG. i. Muscarinic agent and choline dose-response curves for acetylcholine synthesis by tissue prisms prepared from human and rat neocortex. 0 , human prisms incubated with QNB; O ' rat prisms incubated with QNB; w~it' rat prisms incubated with oxotremorine. • , rat prisms incubated h choline. The points represent the data from three (unnumbered) to ten independent determinations. Asterisks identify significant differences (calculated from data expressed in absolute units) from control values (incubations with either no muscarinic agent or 2 mM choline). *p at least < 0.05, **p at least < O.01.
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Stimulation of Acetylcholine
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ive concentrations in both species were 0.i-i.0 ~M. In incubations with rat prisms, the antimuscarinic agent caused a maximal increase in synthesis to 140% of the control value. There was a maximal stimulation to only 112% in the human samples. Oxotremorine, a muscarinic agonist, caused a dose-dependent decrease in incorporation of U-14C -glucose into 14C -ACh by rat brain prisms, over a dose range of 5-500 ~M (Fig. i). The maximal inhibition was to 39% of the control value and occurred at 500 ~M. In medium containing human prisms and 50 ~M oxotremorine the synthesis value (6.83 ~ 0.93 units) was significantly reduced (p < 0.05, n = 7) to 81% of the value without inhibitor (8.12 J 1.42 units). Thus at the concentration tested the effect of oxotremorine was less marked than that on rat prisms. QNB and oxotremorine has no significant effect on the production of 14C02, except in human prisms at the highest concentration of QNB tested. At this concentration (IO ~M) the formation of 14C02 was significantly (p < O.O1, n = 3) reduced to 1793 ± 102 units from the value without agent of 2083 ± 44 units, suggesting that respiration and glycolysis are impaired (23). TABLE I. Effect of potent antimuscarinic agents on the incorporation of U-14C -glucose into 14C -ACh and 14CO2 by rat neocortical tissue prisms
14C -ACh (d.p°m./mg protein/min)
Antimuscarinic Agent (0.O1 ~M)
14CO2 (d.p.m./mg protein/min)
11.87 j 0.86
(8)*
947 ~ 140 (5)
Dexetimide
11.55 ~ 0.74
(7)*
787 ~ 154 (6)
N-methyl-piperidin-4-yl-(R)2-cyclohexyl-2-hydroxy-2phenylacetate HCI
10.66 + 1.19 (4)*
757 ~ 125 (5)
Scopolamine HBr
10.51 j 1.12 (4)*
907 ! 188 (5)
Quinuclidynl
benzilate
No drug
(QNB)
8.67 ! 0.90
(12)
838 ~ 108 (12)
Results are mean J S.D., with the number of independent determinations in parenthesis. * Significant differences (p < O.01) from incubations without drug. Effect of Choline on
14C -ACh Synthesis
Rat prisms were incubated with various concentrations of choline under the same conditions as were used in the previous experiments. In medium containing 2 mM choline the synthesis value (9.70 ~ 1.59 units, n = 5) was significant ly increased to 208% of the value obtained with no added choline (4.78 ! I.IO units, n = 5). Fig. 1 shows that as the choline concentration was increased further synthesis progressively rises when at IO mM choline the synthesis value was significantly increased to 144% of that with 2 mM choline. A similar result was obtained with prisms from 2 samples of control human temporal neocortex (synthesis values with 10 mM choline were 160-166% of those with 2 mM choline). The effect was apparently larger with prisms from the Alzheimer brain tissue, where in medium containing i0 mM choline the synthesis value (7.3 units) was
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increased to 235% of that obtained with 2 m M choline (3.1 units). The syn thesis values obtained with 2 and i0 m M choline fall within our published values (obtained with 2 mM choline; 6, 24) for Alzheimer (3.4 ! 1.2 range 1.8 - 4.5 units, n = 13) and control (7.3 ± 1.4, range 5.1 - 9.3 units, n = 20) samples, respectively. Discussion Oxotremorine inhibited 14C-ACh synthesis in prisms prepared from rat and human neocortex, as has been shown for the release of ACh from rat neocortical slices (25). As estimates of synthesis of ACh have been made using a radioactive tracer, apparent declines in synthesis may result from dilutions of the label by pools of intermediate compounds in the synthetic pathway. Investigations with this (6) and other related preparations (26-28), indicate that label from glucose is not diluted in synthesis of acetylcholine in control incubations. Thus, while the appearance of such diluting pools may conceivably explain a decrease in the formation of the labelled ester (as with oxotremorine), an increase in the formation of 14C -ACh must be due to a genuine increase in synthesis. QNB, the antimuscarinic which caused the most marked stimulation of ACh synthesis in rat, significantly increased ACh synthesis in human prisms over a range of concentrations of 0.i DM - iO ~M. The data shows that the antimuscarinics have a specific effect on 14C-ACh formation for at all but the highest concentration the production of 14C02 was not affected. This data provides the first evidence that human brain contains presynaptic muscarinic receptors. However, the most marked effect of QNB, was to increase synthesis to only 112% of the control value, which was much less than in rat. Similarly, oxotremorine was less effective as an inhibitor of synthesis in the human specimens. This suggests that if presynaptic regulation is not restricted to a specific pool or compartment of ACh, then the receptor (particularly in human brain) has relatively limited control over ACh synthesis. Thus, none of the specific classes of antimuscarinic compound and drugs tested seem to be potentially capable of appreciably reversing the deficit in ACh synthesis in Alzheimer's disease. High concentrations of choline stimulated ACh synthesis in rat prisms to about the same extent as the most effective antimuscarinic agent. Moreover, the ACh precursor was at least as effective in stimulating synthesis in human prisms (including those from a patient with Alzheimer's disease). The value for ACh synthesis we obtain with rat prisms, incubated in medium containing 2 m M choline, agrees closely with estimates of the turnover rate of ACh in neocortex (6). Stimulation of synthesis above this value in vitro is clearly dependent upon adding millimolar concentrations of choline to the medium. By comparison the extracellular concentration of choline is low (O.I mM, 29). Thus, an exceptionally large increase in the extracellular concentration of choline would seem to be required to appreciably stimulate synthesis. If similar relationships are true for human tissue, then the present data (showing the extent to which i0 mM choline stimulates synthesis), considered with our previous finding (that synthesis in Alzheimer's disease is about half normal), suggests that for synthesis to occur at apparently normal rates in Alzheimer's disease the extracellular concentration of choline would have to be increased by over 50 fold. It is unlikely that a change of this magnitude could be achieved by increasing the dietary intake of choline for the administration of up to 20 g of the substance/ day fails even to double the concentration in CSF (30,31). In conclusion, the present data fail to support the view that high choline-containing diets may be of benefit in Alzheimer's disease. They suggest, however, that an elevated intracellular concentration of choline (which presum-
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Stimulation of Acetylcholine Synthesis
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ably ensued from incubating the prisms in I0 mM choline) is more effective than an antimuscarinic agent in stimulating the synthesis of ACh in human prisms. Two other independent studies also point to the importance of a high intracellular concentration of choline (29,32) so alternative strategies for increasing the size of this pool should now be investigated. Acknowledgements We wish to thank Miss Y. Thompson for expert technical assistance and Dr. D. Neary and various neurosurgeons who provided the Alzheimer and control human material. The work was supported by the Medical Research Council with supplementary grants from the Brain Research Trust, Miriam Marks Charitable Trust and Sandoz Pharmaceuticals. References i. 2. 3. 4.
5. 6. 7. 8. 9.
I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25.
S.Z. LANGER, Brit.J.Pharmac., 60, 481-497, (1977). J.T. WALTERS and R.H. ROTH, Naunyn-Schmiedebergs Arch.Pharmacol., 296, 5-18 18, (1976). A. DELINI-STULA, P. BAUMANN and O. BUCH, Naunyn-Schmiedebergs Arch. Pharmacol., 307, 115-122, (1979). D.M. BOWEN, Alzheimer's disease. In Molecular Basis of Neuropathology, R.H.S. THOMPSON and A.N. DAVISON (eds.), Pp 649-665, Edward Arnold, London (1981). N.R. SIMS, D.M. BOWEN, C.C.T. SMITH, R.H.A. FLACK, A.N. DAVISON, J.S. SNOWDEN and D. NEARY, Lancet, i, 333-336, (1980). N.R. SIMS, D.M. BOWEN and A.N. DAVISON, Biochem. J., 196, 867-876, (1981). J.A. RICHTER, E.K. PERRY and B.E. TOMLINSON, Life Sci., 26, 1683-1689, (1980). ANONYMOUS AUTHOR, Lancet, i, 293, (1980). G.W. ARBUTHNOTT, W.D. BOYD, A.I.M. GLEN, R.A. ROBINSON, L.J. WHALLEY and J. WILLIAMSON, Alzheimer's disease: Early Recognition of Potentially Reversible Deficits, A.I.M. GLEN and L.J. WHALLEY (eds.), Pp 197-199, Churchill-Livingstone, Edinburgh. (1979). P.C. MOLENAAR and R.L. POLAK, Brit.J.Pharmacol., 40, 406-417, (1970). P. LEFRESNE, J.P. ROSPARS, J.C. BEAUJOUAN, T.C. WESTFALL and J. GLOWINSKI, Naunyn-Schmiedebergs Arch. Pharmacol., 303, 279-285, (1978). J.C. SZERB, P. HADHAZY and J.D. DUDAR, Brain Res., 128, 285-291, (1977). L.L. IVERSEN, In The Neurosciences Third Study Program, F.O. SCHMITT and F.G. WORDEN (eds.), Pp 905-915, Cambridge, Mass., The MIT Press (1974). N.J.M. BIRDSALL and E.C. HULME, J.Neurochem., 27, 7-16, (1976). D.S. GREWAAL and J.H. QUASTEL, Biochem. J., 132, 1-14, (1973). S.H. SNYDER and H.I. YAMAMURA, Arch. Gen. Psych~atry, 34, 236-239, (1977). R.W. BRIMBLECOMBE, T.O. INCH, J. WETHERELL and N. WILLIAMS, J.Pharm. Pharmac., 2_23, 649-661, (1971). R.W. BRIMBLECOMBE, D.M. GREEN, T.O. INCH and P.B.J. THOMPSON, J.Pharm.Pharmac., 23, 745-757, (1971). J.C. SZERB and G.I. SOMOGYI, Nature New Biol., 241, 121-122, (1973). J.P. ROSPARS, P. LEFRESNE, J.C. BEAUJOUAN and J. GLOWINSKI, Naunyn-Schmiedebergs Arch. Pharmacol., 300, 153-161, (1977). E. EL-FAKAHANY and E. RICHELSON, J.Neurochem., 34, 1288-1295, (1980). D.M. BOWEN and K.L. MAREK, Brit.J.Pharmacol., (in press). J.J. O'NEILL, S.H. SIMON and J.T. CUMMINS, Biochem. Pharmacol., 12, 809-820, (1963). D.M. BOWEN, N.R. SIMS, D. NEARY, D.J. THOMAS and A.N. DAVISON, Metabolic Disorders of the Nervous System, C. ROSE,(ed.), Pp 376-381, Pitman, Tunbridge Wells, (1981). P. HADHAZY and J.C. SZERB, Brain Res., 123, 311-322, (1977).
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26. 27. 28. 29. 30. 31.
32.
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E°T. BROWNING and M.P. SCHULMAN, J.Neurochem., 15, 1391-1405, (1968). P. LEFRESNE, P. GUYENET and J. GLOWINSKI, J.Neurochem., 20, 1083-1097, (1973). G.E. GIBSON and J°P. BLASS, J.Neurochem., 26, 1073-1078, (1976). R.M. MARCHBANKS and S. WONNACOTT, In The Cholinergic Synapse. Progress in Brain Research, 49, S. TUCEK (ed.), Pp 77-88, Elsevier, New York, (1979). J.H. GROWDEN, E.L-~. COHEN and R.J. WURTMAN, J.Neurochem., 28, 229, (1977). K.L. DAVIS, P.A. BERGER, L.E. HOLLISTER, J.T. DOAMARAL and J.D. BARCHAS, In Cholinergic Mechanisms and Psychopharmacology, D.J. JENDEN (ed.), Pp 755-779, Plenum Press, New York, (1977). F.C. FRIEDMAN, K.A. SHERMAN, S°H. FERRIS, B. REISBERG, R.T. BARTUS and M.K. SCHNECK, N.Eng.J.Med., 304, 1490-1491, (1981).
Pergamon Press
STIMULATION OF ACETYLCHOLINE SYNTHESIS BY BLOCKADE OF PRESYNAPTIC MUSCARINIC INHIBITORY AUTORECEPTORS: OBSERVATIONS IN RAT AND HUMAN BRAIN PREPARATIONS AND COMPARISON WITH THE EFFECT OF CHOLINE. Kenneth L. Marek, David M. Bowen, Nell R. Sims and Alan N. Davison Department of Neurochemistry, Institute of Neurology, Queen Square, London WCIN 3BG (Received in final form February 18, 1982) Summary The central presynaptic muscarinic inhibitory autoreceptor has been monitored by measuring the effects of muscarinic agents on acetylcholine (ACh) synthesis by rat and human neocortical tissue prisms. Quinuclidinyl benzilate (QNB), the antlmuscarinic which of 20 tested caused the most marked stimulation of ACh synthesis in rat, significantly increased ACh synthesis in human prisms over a range of concentrations of 0.i ~M-IO ~M. This data provides the first evidence that human brain contains presynaptic muscarinic receptors. However, the most marked effect of QNB was to increase synthesis to only 112% of control (value without drug) which was much less than in rat (to 140% of control). ACh synthesis is reduced to 50% of control in neocortex from Alzheimer patients so none of the antimuscarinics tested seem to be potentially capable of appreciably reversing this deficit. A high concentration of choline (iO mM) stimulated synthesis in rat prisms to about the same extent as QNB. Moreover, the ACh precursor was at least as effective in stimulating synthesis in human prisms (including those from a patient with Alzheimer's disease). This suggests that an elevated intracellular concentration of choline is likely to be much more effective than an antimuscarinic agent in stimulating synthesis in Alzheimer brain. Rodent brain has been shown to contain presynaptic autoreceptors that modulate the release and synthesis of putative neurotransmitters during nerve stimulation (1,2). Thus presynaptic receptors are now being considered as potential target receptors for the development of new selective drugs (3,4). Whether presynaptic receptors occur in human brain is thus important but has not been determined for most transmitters. We have investigated the cholinergic system because it has been implicated in a number of neurological and psychiatric disorders, especially those associated with ageing. A prominant example is the non-treatable dementing condition of Alzheimer's disease, where there is evidence of a clinically relevant reduction, of about 50%, in the formation of acetylcholine (ACh) in the brain (5-7). The reduction of ACh synthesis appears to be due to loss of presynaptic cholinergic nerve endings. The remaining cholinergic terminals seem to retain normal function and postsynaptic muscarinic receptors are apparently spared (4,5). Thus clinical improvement may be brought about by stimulating ACh synthesis in the few remaining nerve endings. It has been proposed that these objectives could be achieved by either a high choline diet (8,9) or by manipulating the presynaptic muscarinic receptor (4). Muscarinic receptor antagonists
stimulate ACh release and synthesis
0024-3205/82/181517-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
in
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Stimulation of Acetylcholine Synthesis
Vol. 30, No. 18, 1982
vitro in rat brain preparations (i0,ii). Acting even in the presence of tetrodotoxin (I0), their effect is reversed by muscarinic agonists (12). Thus there are thought to be muscarinic inhibitory autoreceptors located on the cholinergic nerve terminal (13), in addition to the classical postsynaptic muscarinic receptor (14). The objective of the present work ~as to establish the extent to which antimuscarinic agents stimulate ACh synthesis and to compare this with the effect of choline (15). Using a rat brain preparation a number of antimuscarinic agents and drugs with varying postsynaptic muscarinic potencies and of various structure (16-18), have been screened for their influence on ACh synthesis in vitro. The most effective substance was examined for its effect on synthesis by preparations of fresh normal appearing human brain. Antimuscarinics act optimally in brain tissue preparations that have been depolarized (19, 20). Consequently, the interaction of the substances with the presynaptic receptor has been monitored by using K + stimulated neocortical tissue prisms (6). The prisms enabled the cholinergic nerve endings to be studied free from the influences of the cell body. The incorporation of U-14C -glucose into 14C -ACh and 14CO2 was measured to determine the ACh synthesized and total glucose metabolized, respectively C6). Materials and Methods U-14C -glucose ( 230 Ci/mol) was from The Radiochemical Centre, Amersham, U.K. and was diluted appropriately with glucose (BDH Chemicals, Poole, U.K.). Paraoxon, ACh bromide, choline chloride, acetylcholinesterase (Type VI-S from electric eel), atropine and scopolamine were from the Sigma Chemical Co., Poole, U.K. Oxotremorine was from the Aldrich Chemical Co., Ltd., Gillingham, U.K. Antimuscarinic Agents and Drugs The gifts of clozapine and thioridazine HCI (Sandoz Products, U.K.), amitriptyline (Roche Products, U.K.), trihexylphenidyl HCI (Lederle Labs., U.K.), maprotiline HCI (CIBA Labs., U.K.), benztropine mesylate (Merck, Sharp and Dohme, U.K.), and nomifensine maleate CHoechst, U.K.) are gratefully acknowledged. Apart from scopolamine, Dr. T.O. Inch and Dr. D.M. Green (C.D.E., U.K.) generously provided the other substances listed in Table I and benactyzine HCI; aprophen HCI; N-methyl-piperidin-4-yl-2-cyclohexyl-2-hydroxy-2-phenylacetate HCI; N-ethyl-3-piperidyl benzilate HCI; N-methyl-3-piperidyl benzilate HCI; dimethylamino-ethyl-2-cyclohexyl-2-hydroxy-2-phenylacetate HCI; D-trans-4-S-(S-l-cyclohexyl-l-hydroxy-l-phenyl) methyl-l, 3-dioxolan (trans-dioxolan) and D-cis-4-5dimethyl-aminoethyl-2-R-(R-l-cyclohexyl-l-phenyl) methyl-l, 3-dioxolan (cisdioxolan). Control Human Brain Tissue Samples of normal appearing neocortex plus underlying white matter (6) were obtained from patients aged 17-65 at surgery (7 on temporal lobe, 6 on front al lobe, and 1 on occipital lobe) for 5 craniopharyngiomas, 3 meningiomas, 2 astrocytomas, 2 gliomas, 1 basilar aneurysm and 1 pituitary tumor. Alzheimer Brain Tissue The sample of temporal neocortex was taken at diagnostic craniotomy from a demented patient aged 64. Histopathology of adjacent tissue samples revealed the presence of senile plaques and neurofibrillary degeneration, confirming that the patient had Alzheimer's disease. Measurement of ACh Synthesis Male Wistar rats (Porton strain, 6-9 months) were decapitated and the
Vol. 30, No. 18, 1982
Stimulation of Acetylcholine
Synthesis
1519
brains rapidly removed. Rat and human specimens were placed in ice-cold, modified Krebs Ringer phosphate buffer (141 mM NaCI, 5 mM KCI, 1.3 mM CaCI2, 1.3 mM MgSO 4 and iO mM Na2HP04, pH 7.4) containing 2.5 mM glucose and freshly gassed with 100% oxygen. Neocortex, including all cortical layers, was dissected. Preparation of tissue prisms, preincubation and measurement of incorporation of U-14C -glucose into 14C -ACh and 14C02 in the presence of 31 mM K + and 40 ~M paraoxon was carried out using a slight modification of the method of Sims et al (6). The incubation period was for 30 min instead of I h and prisms were maintained at room temperature after preincubation rather than cooling on ice. The modification to incubation time was made because in incubations terminated after 30 min, i DM atropine enhanced 14C -ACh formation by 39% ± 14% (mean S.D., n = 4), whereas the value was lower (31% ± 8%) for incubations of I h. When preincubated prisms were maintained on ice prior to assay, i uM atropine enhanced 14C -ACh formation by only 18% ± 7% (mean ~ S.D., n = 4) of the control value (prisms without the drug). The value was higher (42% ! 11%), using prisms maintained at room temperature. The control values were independent of the temperature at which the preincubated prisms were maintained. These results were consistent with those of Ei-Fakahany & Richelson (21). Test incubations were performed in quadruplicate and blanks in triplicate. The results are based upon data from at least 3 independent experiments, unless stated otherwise. The method of Sims et al (6) was used to determine the effect of choline on 14C ACh synthesis in human tissue prisms. The incubation medium contained 31 mM K +. The radioactivity isolated as 14C -ACh from incubation of rat prisms with i ~M QNB and without drug was hydrolysed by acetylcholinesterase (6) by 95% ! 2% and 95% ~ 3% (mean ~ S.D., n = 3), respectively. This indicated that essentially all of the radioactivity was attributable to labelled ACh. 14C -ACh synthesis and 14C02 formation are expressed as the mean ! S.D. with I unit being equivalent to i d.p.m./mg protein/min (using 2.5 mM glucose and 2.8 and 11.2 ~ci of U-14C-glucose/flask in incubations with rat and human prisms, respectively). Unless stated otherwise, statistical analysis was by the Student's t-test for unpaired observations; the null hypothesis was rejected for probability values less than O.O1. Due to the variability in 14C -ACh formation by individual control human brains (6) and the small effect of QNB on synthesis in this species, results for human brain (as well as those for rat in Fig. i) were analyzed by the Paired t-test. Results in the text are mean ! S.D. Results Effect of Various Antimuscarinic
Agents and Drugs on
14C -ACh Synthesis
The 20 substances tested (Materials and Methods and Table I) were incubated with rat prisms for 30 mins in medium containing 31 mM K + and 2 mM choline. At a concentration of I ~M 17 compounds caused a significant increase in synthesis to between 120%-140% of the control value. Quinuclidinyl benzilate (QNB) caused the most marked stimulation. The substances without significant effect were maprotoline, trans-dioxolan and cis-dioxolan (detailed elsewhere, 22). At a concentration of O.01 ~M only 4 agents caused a significant increase in the formation of labelled ester (Table I), the most potent agent was again QNB. None of the substances had a significant effect on the production of 14C02 (Table I). Effect of Potent Muscarinic Agents on
crease
14C -ACh Synthesis in Rat and Human Prism~
The effect of QNB, measured over a dose range of 0.OO1-I0 ~M, was to in14C -ACh formation in a dose dependent manner (Fig. i). The most effect-
1520
Stimulation of Acetylcholine Synthesis
2
4
Vol. 30, No. 18, 1982
CHOLINE (mM} 6 8
•
I
10
I
I
--~140 0
0 o
0
o~ 1 2 0 GO
~
•
o
LU
"r'lO 0
I-Z >-
Ill
$
Z
-
.1
80-
0 -r
o~
0 ._1
>-
w
0 <
60~-
0
I I 0"001 0-01 MUSCARI
I 0.1 NIC
l 1-0
AGENT
I 10
| 6 ,I 100 1000
(juM)
FIG. i. Muscarinic agent and choline dose-response curves for acetylcholine synthesis by tissue prisms prepared from human and rat neocortex. 0 , human prisms incubated with QNB; O ' rat prisms incubated with QNB; w~it' rat prisms incubated with oxotremorine. • , rat prisms incubated h choline. The points represent the data from three (unnumbered) to ten independent determinations. Asterisks identify significant differences (calculated from data expressed in absolute units) from control values (incubations with either no muscarinic agent or 2 mM choline). *p at least < 0.05, **p at least < O.01.
Vol. 30, No. 18, 1982
Stimulation of Acetylcholine
Synthesis
1521
ive concentrations in both species were 0.i-i.0 ~M. In incubations with rat prisms, the antimuscarinic agent caused a maximal increase in synthesis to 140% of the control value. There was a maximal stimulation to only 112% in the human samples. Oxotremorine, a muscarinic agonist, caused a dose-dependent decrease in incorporation of U-14C -glucose into 14C -ACh by rat brain prisms, over a dose range of 5-500 ~M (Fig. i). The maximal inhibition was to 39% of the control value and occurred at 500 ~M. In medium containing human prisms and 50 ~M oxotremorine the synthesis value (6.83 ~ 0.93 units) was significantly reduced (p < 0.05, n = 7) to 81% of the value without inhibitor (8.12 J 1.42 units). Thus at the concentration tested the effect of oxotremorine was less marked than that on rat prisms. QNB and oxotremorine has no significant effect on the production of 14C02, except in human prisms at the highest concentration of QNB tested. At this concentration (IO ~M) the formation of 14C02 was significantly (p < O.O1, n = 3) reduced to 1793 ± 102 units from the value without agent of 2083 ± 44 units, suggesting that respiration and glycolysis are impaired (23). TABLE I. Effect of potent antimuscarinic agents on the incorporation of U-14C -glucose into 14C -ACh and 14CO2 by rat neocortical tissue prisms
14C -ACh (d.p°m./mg protein/min)
Antimuscarinic Agent (0.O1 ~M)
14CO2 (d.p.m./mg protein/min)
11.87 j 0.86
(8)*
947 ~ 140 (5)
Dexetimide
11.55 ~ 0.74
(7)*
787 ~ 154 (6)
N-methyl-piperidin-4-yl-(R)2-cyclohexyl-2-hydroxy-2phenylacetate HCI
10.66 + 1.19 (4)*
757 ~ 125 (5)
Scopolamine HBr
10.51 j 1.12 (4)*
907 ! 188 (5)
Quinuclidynl
benzilate
No drug
(QNB)
8.67 ! 0.90
(12)
838 ~ 108 (12)
Results are mean J S.D., with the number of independent determinations in parenthesis. * Significant differences (p < O.01) from incubations without drug. Effect of Choline on
14C -ACh Synthesis
Rat prisms were incubated with various concentrations of choline under the same conditions as were used in the previous experiments. In medium containing 2 mM choline the synthesis value (9.70 ~ 1.59 units, n = 5) was significant ly increased to 208% of the value obtained with no added choline (4.78 ! I.IO units, n = 5). Fig. 1 shows that as the choline concentration was increased further synthesis progressively rises when at IO mM choline the synthesis value was significantly increased to 144% of that with 2 mM choline. A similar result was obtained with prisms from 2 samples of control human temporal neocortex (synthesis values with 10 mM choline were 160-166% of those with 2 mM choline). The effect was apparently larger with prisms from the Alzheimer brain tissue, where in medium containing i0 mM choline the synthesis value (7.3 units) was
1522
Stimulation of Acetylcholine Synthesis
Vol. 30, No. 18, 1982
increased to 235% of that obtained with 2 m M choline (3.1 units). The syn thesis values obtained with 2 and i0 m M choline fall within our published values (obtained with 2 mM choline; 6, 24) for Alzheimer (3.4 ! 1.2 range 1.8 - 4.5 units, n = 13) and control (7.3 ± 1.4, range 5.1 - 9.3 units, n = 20) samples, respectively. Discussion Oxotremorine inhibited 14C-ACh synthesis in prisms prepared from rat and human neocortex, as has been shown for the release of ACh from rat neocortical slices (25). As estimates of synthesis of ACh have been made using a radioactive tracer, apparent declines in synthesis may result from dilutions of the label by pools of intermediate compounds in the synthetic pathway. Investigations with this (6) and other related preparations (26-28), indicate that label from glucose is not diluted in synthesis of acetylcholine in control incubations. Thus, while the appearance of such diluting pools may conceivably explain a decrease in the formation of the labelled ester (as with oxotremorine), an increase in the formation of 14C -ACh must be due to a genuine increase in synthesis. QNB, the antimuscarinic which caused the most marked stimulation of ACh synthesis in rat, significantly increased ACh synthesis in human prisms over a range of concentrations of 0.i DM - iO ~M. The data shows that the antimuscarinics have a specific effect on 14C-ACh formation for at all but the highest concentration the production of 14C02 was not affected. This data provides the first evidence that human brain contains presynaptic muscarinic receptors. However, the most marked effect of QNB, was to increase synthesis to only 112% of the control value, which was much less than in rat. Similarly, oxotremorine was less effective as an inhibitor of synthesis in the human specimens. This suggests that if presynaptic regulation is not restricted to a specific pool or compartment of ACh, then the receptor (particularly in human brain) has relatively limited control over ACh synthesis. Thus, none of the specific classes of antimuscarinic compound and drugs tested seem to be potentially capable of appreciably reversing the deficit in ACh synthesis in Alzheimer's disease. High concentrations of choline stimulated ACh synthesis in rat prisms to about the same extent as the most effective antimuscarinic agent. Moreover, the ACh precursor was at least as effective in stimulating synthesis in human prisms (including those from a patient with Alzheimer's disease). The value for ACh synthesis we obtain with rat prisms, incubated in medium containing 2 m M choline, agrees closely with estimates of the turnover rate of ACh in neocortex (6). Stimulation of synthesis above this value in vitro is clearly dependent upon adding millimolar concentrations of choline to the medium. By comparison the extracellular concentration of choline is low (O.I mM, 29). Thus, an exceptionally large increase in the extracellular concentration of choline would seem to be required to appreciably stimulate synthesis. If similar relationships are true for human tissue, then the present data (showing the extent to which i0 mM choline stimulates synthesis), considered with our previous finding (that synthesis in Alzheimer's disease is about half normal), suggests that for synthesis to occur at apparently normal rates in Alzheimer's disease the extracellular concentration of choline would have to be increased by over 50 fold. It is unlikely that a change of this magnitude could be achieved by increasing the dietary intake of choline for the administration of up to 20 g of the substance/ day fails even to double the concentration in CSF (30,31). In conclusion, the present data fail to support the view that high choline-containing diets may be of benefit in Alzheimer's disease. They suggest, however, that an elevated intracellular concentration of choline (which presum-
Vol. 30, No. 18, 1982
Stimulation of Acetylcholine Synthesis
1523
ably ensued from incubating the prisms in I0 mM choline) is more effective than an antimuscarinic agent in stimulating the synthesis of ACh in human prisms. Two other independent studies also point to the importance of a high intracellular concentration of choline (29,32) so alternative strategies for increasing the size of this pool should now be investigated. Acknowledgements We wish to thank Miss Y. Thompson for expert technical assistance and Dr. D. Neary and various neurosurgeons who provided the Alzheimer and control human material. The work was supported by the Medical Research Council with supplementary grants from the Brain Research Trust, Miriam Marks Charitable Trust and Sandoz Pharmaceuticals. References i. 2. 3. 4.
5. 6. 7. 8. 9.
I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25.
S.Z. LANGER, Brit.J.Pharmac., 60, 481-497, (1977). J.T. WALTERS and R.H. ROTH, Naunyn-Schmiedebergs Arch.Pharmacol., 296, 5-18 18, (1976). A. DELINI-STULA, P. BAUMANN and O. BUCH, Naunyn-Schmiedebergs Arch. Pharmacol., 307, 115-122, (1979). D.M. BOWEN, Alzheimer's disease. In Molecular Basis of Neuropathology, R.H.S. THOMPSON and A.N. DAVISON (eds.), Pp 649-665, Edward Arnold, London (1981). N.R. SIMS, D.M. BOWEN, C.C.T. SMITH, R.H.A. FLACK, A.N. DAVISON, J.S. SNOWDEN and D. NEARY, Lancet, i, 333-336, (1980). N.R. SIMS, D.M. BOWEN and A.N. DAVISON, Biochem. J., 196, 867-876, (1981). J.A. RICHTER, E.K. PERRY and B.E. TOMLINSON, Life Sci., 26, 1683-1689, (1980). ANONYMOUS AUTHOR, Lancet, i, 293, (1980). G.W. ARBUTHNOTT, W.D. BOYD, A.I.M. GLEN, R.A. ROBINSON, L.J. WHALLEY and J. WILLIAMSON, Alzheimer's disease: Early Recognition of Potentially Reversible Deficits, A.I.M. GLEN and L.J. WHALLEY (eds.), Pp 197-199, Churchill-Livingstone, Edinburgh. (1979). P.C. MOLENAAR and R.L. POLAK, Brit.J.Pharmacol., 40, 406-417, (1970). P. LEFRESNE, J.P. ROSPARS, J.C. BEAUJOUAN, T.C. WESTFALL and J. GLOWINSKI, Naunyn-Schmiedebergs Arch. Pharmacol., 303, 279-285, (1978). J.C. SZERB, P. HADHAZY and J.D. DUDAR, Brain Res., 128, 285-291, (1977). L.L. IVERSEN, In The Neurosciences Third Study Program, F.O. SCHMITT and F.G. WORDEN (eds.), Pp 905-915, Cambridge, Mass., The MIT Press (1974). N.J.M. BIRDSALL and E.C. HULME, J.Neurochem., 27, 7-16, (1976). D.S. GREWAAL and J.H. QUASTEL, Biochem. J., 132, 1-14, (1973). S.H. SNYDER and H.I. YAMAMURA, Arch. Gen. Psych~atry, 34, 236-239, (1977). R.W. BRIMBLECOMBE, T.O. INCH, J. WETHERELL and N. WILLIAMS, J.Pharm. Pharmac., 2_23, 649-661, (1971). R.W. BRIMBLECOMBE, D.M. GREEN, T.O. INCH and P.B.J. THOMPSON, J.Pharm.Pharmac., 23, 745-757, (1971). J.C. SZERB and G.I. SOMOGYI, Nature New Biol., 241, 121-122, (1973). J.P. ROSPARS, P. LEFRESNE, J.C. BEAUJOUAN and J. GLOWINSKI, Naunyn-Schmiedebergs Arch. Pharmacol., 300, 153-161, (1977). E. EL-FAKAHANY and E. RICHELSON, J.Neurochem., 34, 1288-1295, (1980). D.M. BOWEN and K.L. MAREK, Brit.J.Pharmacol., (in press). J.J. O'NEILL, S.H. SIMON and J.T. CUMMINS, Biochem. Pharmacol., 12, 809-820, (1963). D.M. BOWEN, N.R. SIMS, D. NEARY, D.J. THOMAS and A.N. DAVISON, Metabolic Disorders of the Nervous System, C. ROSE,(ed.), Pp 376-381, Pitman, Tunbridge Wells, (1981). P. HADHAZY and J.C. SZERB, Brain Res., 123, 311-322, (1977).
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26. 27. 28. 29. 30. 31.
32.
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E°T. BROWNING and M.P. SCHULMAN, J.Neurochem., 15, 1391-1405, (1968). P. LEFRESNE, P. GUYENET and J. GLOWINSKI, J.Neurochem., 20, 1083-1097, (1973). G.E. GIBSON and J°P. BLASS, J.Neurochem., 26, 1073-1078, (1976). R.M. MARCHBANKS and S. WONNACOTT, In The Cholinergic Synapse. Progress in Brain Research, 49, S. TUCEK (ed.), Pp 77-88, Elsevier, New York, (1979). J.H. GROWDEN, E.L-~. COHEN and R.J. WURTMAN, J.Neurochem., 28, 229, (1977). K.L. DAVIS, P.A. BERGER, L.E. HOLLISTER, J.T. DOAMARAL and J.D. BARCHAS, In Cholinergic Mechanisms and Psychopharmacology, D.J. JENDEN (ed.), Pp 755-779, Plenum Press, New York, (1977). F.C. FRIEDMAN, K.A. SHERMAN, S°H. FERRIS, B. REISBERG, R.T. BARTUS and M.K. SCHNECK, N.Eng.J.Med., 304, 1490-1491, (1981).