Cholinergic actions of false neurotransmitters: Acetyldiethylcholine

Neuroscience:Letgers, 28 (1982) 5 i --56

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Elsevier/North,Holland Scientific Pubfisher~s Ltd,

AL; ~~ N S OF FALSE N~UNOTNz-aNSM~. ]'A~S. .

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RO~ERT:$; ARONSTAM-ar~d-JERRY J. BUCCAFU$CO

Department o f P!~rmacology, 'Medical (2'ofh~geo f Gretna,. A ug~.~sW,GA 30907 (U, ~.A.) (N~ceivecl September 7th, t 981; AceeptN October ~th, ~98 !)

Acetyldiethytcholine ,AcDE~h), a. false tra~smi¢~er at eholinergic syna:oses, binds ~o central muscarinic recePtors with 14-fold tower affinit)." ~ha~ ace~,ylchotia~ (AcCb), The p~operti,es of ~!~ binding,, including the ~imi~ed extent of re.o-'~ ..,~en~,., ~ specificity and bi.nd~ng a ctM~atior~ oy N-e~hyb a,.~o.~s,s maMmide, a ~ ihose associated with wea~" ~' ."' ~nd antagonists, imr~cerebrovem ~'icu~.ar injecfie~ of AcDECh or AcCh produces an increase h~ ax~et~! iNood pressure which is b[ocke~i by ~rior ad~niuistration of atropine (i0 nmot), Wh~ie AcDECh is F7-fotd less Fo~e~r ~h:an AcCh in prcAucing ~.he pre.~sor response, the maximum pressm'e changes ar~d time courses are comparable. AcDc.C~ a~so bi,nd~ ?o nicotinic receptors of Torpedo electric organs thereby inducing conformaiiona~ changes in ~he receptor-ion channel complex tha~ .are associated wi~h pos~synap~ic actb, atio~, la ~hesc. nieoti~dc ac~.ions, however, AeDECh is 320-fold less ac~,ive tha~ AcCh. ,.

The goal of manipulating cholinergic ne~..wotransmission through the formation of false trans~nitters has been pursued for a number of year;. A pharmacologically useful false transmitter precursor would be expected to be a favored substrate for the high affinity choline uptake and acetylation systems, while the false transmi~¢er weutd be stored and released in response to appropriate stimuti but possess only limited postsynaptic activity. We have been using acetylcholine analogues a~d derivatives to aiter ct~otinergic function and to probe tt~e molecular bases of cl~cAinergic receptor aetivaticm [6]. (2-HydroaTethyt)methyldiethylammonium (diethylcholi~_e) .,~.,~a~ possesses a i~igb affinity for the eh~bne --" ~ system and is acetytated by choline acetyltransferase t4, 8, 9, 12, 13], whi~e acety!dietiwlchoiine (AcDECh) possesses very limited cholinergic activity [II1]. In the pr~-~,.nt study the interactions ef AcDECh with chNinergic receptors (muscarinic aa~ ~:~eetinic) and its abili~.y ~o eiici~ conformational char~ges in the receptor-ion channel ~empiex ,,~f r~icotinic synapse are compared m tho~e of a~e~:ici~o,me (AcC~0. ~:n :~aamc,a= ~he h'dlueace of intraeerebr,oVentricular iNection of AcDECh and AcCh on a weii-defh~ed centra~ muscaa'i~c f!esponse, ~:.~eeteva~:io~, of arterial bieod a~ressu,~e ~.,~, is described Die~;hy!amine~thaaot was prepared by refl.ax of N,N.diethyIethav~oi..amine and acetic anhydride in diethyl e~he;o AcDECh ~odide was formed by addition of

iodoethane in diethylaminoethanol in ether and was recrystalltzed from methylethyl ~etone. P'roduct purity was assessed by thin layer chromatography. The interactions of AcDECh with muscafinic receptors from rat brain were C" :hteedre,cYp;tandar~rc~dures | | usLng~'H|3"qumuehd~rlYlbCrizflat:e([:H]Q') ~iet tot pro (Fig. 1; ab|e|).AcDECh:~inhibi~O:O5~:~nM~:|3H]QNB : binding to muscarinie~receptors in membranes isolated from:rat brain:cerebral cortex with an iC5o of 22 gM; the corresponding value for AcCh was 1.6 uM. The binding of agonists to neural receptors follows complicated khaetics which are frequently interpreted as indicating mult|p e (2 or 3) receptor populationswhich differ primarily in their affinity for agonists [5]. Alkylation of neural~membranes~ with N-ethylmaleimide (NEM) increases the affinity of agonists for muscarinic receptors in a manner consistent w~th conversion of low affinity receptors to high affinity receptors |1]. NEM treatment shifted the IC50 for AcCh inhibition of specific [3H]QNB binding fi-om 1.6 to 0.1 #M (Fig. 1) whflethe AcDECh inhibition curve was only slightly shifted (the IC~0 decreased from 22 to 10 ~M). There is a regional heterogeneity of neural muscafinic r~eptors with respect to agonist binding affinity [3, 5]; for instance, the overall affinity for A¢Ch of muscarinic receptors from the brainstem is almost 10-fold greater than that of cortex receptors ~.

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Fig. 1. Interaction of AcDECh with central muscarinic receptors. Binding of 0.05 nM [:~H]QNBto neural receptors was measured in the presence of the indicated concentration of AcDECh or AcCh using a filtration assay described elsewhere [1]. A: inhibition by AcDECh (circles) and AcCh (triangles) of [~H|QNB binding to muscarin;:c AcCh receptors inmembranes isolated from rat cerebral cortex. The membranes were untreated (open symbols) or treated with 1 ram N.ethylmaleimide (closed symbols) for 20 min at B°C. Binding is expressed as fraction of control binding. B: inhibR!on by AcDECh of ~3HIQNB l:inding to muscarinic receptors in membranes isolated from rat brainstem (medulla-ponsmidbrain) which were untreated (o) or treated with~N-ethylmaleimide (e). Each point is the average of 4 determinati ~ns which varied by less than 10%. Fig. 2. Inter:action of AcDECh with nicotinic AcCh receptors from Torpedoelectric organ, Thespecific binding of ~!00~M [3H]AcCh (o)and |3tq|D:,ubocurarine(*) was measurCdbyequ!~!ibriumdiaiYs°isjn the presence of AcDECh at the concentrations indicated On the abscissa. ~Binding is expressedii~::fraetio~ of oral specific (nicofinie-sensitve) binding, and each polar represents the average of,4, determ,lnafions which varied by less than 15:%~ :

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[3, 5]. However, the IC5o values for AcDECh inhibition of [3H]QNB binding in t~:e cortex and brainstem were similar (22 and 13 #M, respectively). Lack of regional variation in muscarinic affinity is generally associated with antagonists and agonists of limited intrinsic activity. The affinity of brainstem receptors for AcDECh was also somewhat increased by NEM treatment: (Fig. 1). -:~ from Torpedo electric organ Were monitored:~o'by measuring the ability of AcDECh to inhibit the binding of 100 nM'PH]AcCh or [3H]u-tubocurarine to cholinergic receptors from Torpedoelectric organs. AcDECh inhibited pH]AcCh and [H]~tubocuranne binding with !C50 valuesof 56and35 #Mrespectwely (F g. 2; Table D. Binding parameters associated withthese mteracfionsare summarized in Table I, The Hill coefficients associated with AcDECh binding WereClose to unity, unlike those associated with the binding of AcCh, nicotine, carbamylcholineand other potent agonists, which are between 1.5 2.0. [3H]Perhydrohistrionicotoxin (pH]HIz-HTX) and [3H]phencyclidine ([3H]PCP) bind tosites associated with the synaptic ion channel of the-nicotinic receptor in Torpedo electric organs [2, i0], This binding is greatly enhanced in the presence of receptor (i.e. AcCh binding site) ligands. For instance, AcCh increases the extent of pH]PCP binding several fold in a manner which closeiy parallels its occupancy of receptor binding sites. Stimulation of 3 nM pH]PCP binding by AcDECh and AcCh

TABLE I INTERACTIONS OF ACETYLDIETHYLCHOLtNE (AcDECh)WITH CHOLINERGIC SYNAPTIC MOLECULES EDS0a

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Muscarinic receptor binding Rat brain: cortex cortex NEM-treated brainstem brainstem NEM.treated

22.4±0.8 10 ±1 12.7 ± 0.9 8 _+ I

0.7 ± 0.1 0.7 _+ O.t 0.61 _+0.08 0.65_+ 0.09

Nicotinic receptor binding PHIAcCh [3H]o.tuboeurafine

56 35

±5 ±7

1.1 ±0.1 1.O ± 0.1

Nicotinic ion channel activation pH]PCP pHIHt2-HTX

48 45

±2 ±2

1.7 ±0.1 1.5 ± 0.I

aConcentrafiora of AcDECh in #M which inhibits by 50% the binding of figands to the Muscafinic ¢,0.05 nM pH]QNB)Ior nicofirfi¢ ( t ( ~ nM [3H]AcCh or pH]D.tub~urafine) receptor~ or the cow:entration of AcDECh which activates half maximally ligand binding to the nicotinic ion channel (3 nM [3HiPCP or 2 bThe Hitl~coeffic~ent for the indicated interaction.

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Fig. 3. 'nfleence of AcDECh and AcCh on the binding to Torpedoeleetroplax membranes of a probe for the ion channel associated with the nicotinic AcCh rc~:epzor. Binding is expressed as the fraction of the maximum specific {amantadine-sensitive) 3 nM pH]phencyclidine (PHIPCP) binding, measured by a f~!tration pr~xedure as described elsewhere [10l. In this tissue sample, PHIPCP binding was increased by up to 2.5~fold in the presence of A,zDECh (o) and AcCh (®); the concentrations which produce half of maxima! stmmla.on " ° "; were 48 and O. 15 .M for AcDECh and AcCh, respectively. Higher concentrations of both drugs decreased ~he blr~,|ing stimulation. The time-dependent 'desensitization' of ion channel binding produced by incubating the membranes with AcDECh for various times before the addition of PH]PCP is illustrated in the inset to Fig. 2L in which binding is expressed a pmol of pH]PCP bound/rag protein. Each point is the average of 5 determinations which varied by 5-20%.

are compared in ;?ig. 3. Channel binding was stimulated in the same concentration ranges at which AcDECh and AcCh occupy the AcCh receptor (i.e. inhibit [:~H]AcCh binding). The concentration of AcDECh which stimulates [3H]PCP binding to ha|~ of the maximum extent (the ED~0 was 48 gM (Table I)). [3H]PCP binding was i~lhibited by AcDECh at concentrations above 320 #M; at 10 mM AcDECI~, ~on channel binding was less than in the absence of AcDECh. Exposure of Torpedo membranes to nicotinic agonists for prolonged times or in excessive concentrations leads to a 'desensitization' process which is reflected in a time- and concentration.-dependent attentuation of ion channel binding [2]. While desensitization may be responsible for some of the decline in channel binding at high AcDECh concentrations (Fig. 3), direct effects on the channel cannot be ruled out. in the present experiments, preincubation with 100 ~M AcDECh reduced [3H]PCP binding in a time-dependent fashion (Fig. 3, inset). The exten~ of this decrease, howe,,er, was substantially less than that seen with equivalent concentrations of AcCh (i.e. concentrations which stimulate [3H]PCP to the same extent upon simuhaneous addition of agonist and [3H]PCP). Male Sprague-Dawley rats weighing 3t10-400 g were anesthetized with methohexital (65 mg/kg, ii.p.), placed in a stereotaxic instrument and a stainless steel canaula guide implanted in the skull over the left lateral cerebral ventricle. After 5-7 days the animals, again were anesthetizedand polyethylene catheters filled with heparinized saline (2(1(~units/ml) inserted into the common carotid artery and exteriorized at ~he nape of the neck. On the following day unrestrained rats were

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Fig. 4. Pressor response to intracerebroventricular injection of AcDECh and AcCh. The change in meanarterial blood pressure was measured in conscious, unrestrained rms after a I0 ~l i.c.v, injection of AcDECh (o) or AcCh (A) in the undirected amount. The duration of the pressor response to AcDECh (o) and AcCh ( A ) is i,dicated by the scale on the right, and was similar with either compound. Each poim and bar represents the mean ± standard error of fl~e responses of 4 rats.

placed in individual plastic boxes and the arterial line connected to a pressure transducer coupled to a recorder. Each rat received no more than 2-3 mndomty assigned injections of AcCh (5-150 nmol) or AcDECh (60-1000 nmot) spaced over a 30 h period. Drugs were dissolved in 10 #1 of saline and injected into the cerebral ventricles through a 28-gauge injection ¢annula inserted through ~he cannuta guide. injections were made over a 30 sec period. The basal mean arterial pressure in 12 naive rats was 127 :t 3 mm Hg. Both compounds evoked dose-related pressor responses up to 30 mm fgg ~about the maximal response which can be produced by any cholinergic agorfists). However, the dose-response curve for AcDECh was shifted to the ~ight in a parallel fashion, AcDECh being approximately 17ofold less potent than AcCh (Fig. 4). Also, at doses producir~g equivalent increases in arterial pressure, the duration of this effect was similar fc.r the two drugs. Central injection of atropine (10 nmol) did not alter blood pressure but completely prevented the pressor responses to aubsequent injections of AcDECh. demonstrating the muscarinic nature of these effects. Two factors which contribute to the effectiveness of the false transmitters as modulators of cholinergic activity are receptor affinity and intrinsic activity. The present biochemical and pharmacological evidence indicates that the weak cholinomime:tic activity of AcDECh compared to AcCh in central muscarinic ~nd nicotinic dectropla× organ functions priroarily re~ects a tow ~ffinity for chofinergic receptors. The potency of A~zDECh in elevath~g blood pressure after i.c.vo injection and in inducing conformationa~ changes in postsynapdc nicotir-,ic comple×es parallels dosely its occupancy of the AcCh receptor, h is inte~esting that whi~e

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AcDECh was a central cholinomirnefic activity 17-fold lower than that of AcCh, the potency ratio in a nicotinic, neuromuscular-type system (the electric organ) is 320. This latter ratio is in good agreement with that shown by Holton and Ing [11 ], who also demonstrated an even lower AcDECh/AcCh potency ratio in peripheral muscarinic systems. These differences in relative cholinomimetic potency in central an~ peripheral cholinergic systems suggest the feasibility of designing chotinergic false transmitters which exhibit a specific profile of cholinergic actions. |3H]HI2-HTX was generously donated by Dr. John W. Daly of th,~ National Institute of Health, The technical assistance of Latha Narayanan and Laura Crouch is gratefully acknowledged. This research was supported by grants from the National Institutes of Health (DA-02834) ~nd the Pharmaceutical Manufacturers Association Foundation. 1 Aronstam, R.S., Abood, L.G. and Hoss, W., Influence of sulfhydryl reagents and heavy metals on ~he fimctiona| state of the muscarinic acetylcholir~e receptor in rat brain, Molec. Pharmacol., 14 (1978) 575-586, 2 Aronstam, R.S., Eldefrawi, A.T., Pessah, I.N., Daly, LW., Albuquerque, E.X. and Eldefrawi, M.E., Regulation of PHlP~rhydrohistrionicotoxin binding to Torpedo ocellato electroplax by effectors of the acelykholine receptor, J. biol. Chem., 256 (1981) 2843-2850. 3 Aronstam, R.S., Kellogg, C. and Abc~crd, L,G.) Development of muscarinic cholinergic receptors in iabrea strains of mice: identification of regional heterogeneity and rela;ion to audiogenic seizure sw~ceptibility, Brain Rc.~., 162 (1979) 231-241. 4 Barker, L.A. and Mittag, T.W., Comparative studies of substrates and inhibitors of choline transport and choline acetyttransferase. J. Pharmac~l. exp. Thor., 192 (1975) 86-94. 5 Birdsalt) N.J.~,~., Hulme, E.C. and Burgea, A.S.V., Character or" the muscarinic receptors in different regions of the rat brain, Proc. roy. Soc. B, 207 (1980) 1-12. 6 Buccaf~sco, J.J. and Aronstam, R.S.~ Cholinergic actions of false aeurotransmitters: acetylpyrrolidine-choline, Neurosci. Left., 23 (1981) 319-324. 7 Buccafusco, J.J. and Brezenoff, H.E,, Pharmacologic study of a cholinergic mechanism within the rat hypothalamic nucleus with mediates a hypertensive response, Brain Res., 165 (1979) 295-310. 8 Chiou, C.Y., Studies on action mechanisms of a possible false cholinergic transmitter, (2-hydroxyethyl)methyldielhy|ammonium, Life Set., 14 (1974) 1721-1733. 9 Chiou, C.Y., Further studies on the pharmacology of a false chotinergic transmitter, (2-hydroxyethy~)methy|diethylammonium, Life Set., 17 (1975) 907-.914. 10 Eldefrawi, M.E., Eldefrawi, A.T., Aronstam, R.S., Maleque, M,A., Warnick, J.E. and Albuquerque, E.X., [3H]Phencyctidine - a probe for the ionic channel of the nicotinic receptor. Proc. nat. Acad. Sci. (Wash.), 77 (|981) 7458-7462. ! 1 Hol~on, P. and Ing, H.R., The specificity of the trimethylammonium group in acetylcholine, Brit. J. Phar~acol., 4 it949) 1¢~-- 196. K2 Ma~thews, R.T. and Chieu, C~Y., Effects of die~hylcholine in two animal model~ of Parkinsonism ~remors, Europ. J, Pharmacol., 56 (1979) 159-162, t3 Sime~t~ J.R., Mi~tag~ T.W. and Kuhar, M.3., Inhibition of synaptosomal uptake of choline by v~ri,oas choline an.a~ogs, Biochem. Pharmacol., 24 (t975) 1139-1142.