CHOLINE ACETYLTRANSFERASE ACTIVITY IN GUINEA-PIG HEART IN VITRO R. RIKKOSKI.JH.. H. E. MAYERand P. G.SCHMID Department, of Biochemistry and Medicine. The University of Iowa. Iowa City. IA 52242 U.S.A. (Kect~iwd22 Mu!. 1974. Accrprcd 2X May 1974) Ahstract Chi>line;icctyltransfcrase (EC' 2.3.1.6) catallw, the following reversible rcaction: acetyl coen- /jmc A + choline e acctylcholine + cocn7yme A. Enzyme activity in the atria and ventricles of guinea- pig hurt wried indcpendcntly of the biochemically rclatcd carnitine acetyltransferasc (EC 2.3.1.7). Cho- line acctyltransfcrasc activity was greatest in right atrium. intermediate in right ventricle and left atrium and lowest iii left ventricle (4.05.2.33. 1.77 and 1.33 nmol min- lg I, respcctively). Carnitine aoetyltransfer- ase activity was greatest in the right and left ventricle and least in the right and left atria (8.86. 827, 3.18 and ?3X mniol min- 'g ~ I, respectively). Carnitine acetyltransferase activity was 800- to 6000-fold greater than that of the choline acetyltransferase. depending on the chamber. Bromoacetylcholine inhibited ace- tylcholine. but not acetylcarnitine hiosynthe>ts in i:im>.Contrariwise. acetylcarnitine inhibited carnitine. but not cholinc acetyltransfcrasc. These results demonstrate the feasibility and specificity of measuring the differences in choline acetyltransferase activity in dialysed homogenates prepared from the four chambers of the heart. ACFTYLCHOLINEis the neurotransmitter of the pre- and removing the heart. samples were taken from the right atrial post-ganglionic components of the parasympathetic appendage (excluding the sinoatrial node). the left atrial nervous system and the preganglionic component of appendage, and the free wall of the right and left ventricle the slmpathetic nervous system (cf. IVERSON.1970). between the apex and bax (excluding papillary muscle). The samples (S@200 mg) were blotted, weighed. and stored in The biosynthesis of acetylcholine is catalysed by the liquid nitrogen before use. To prepare the tissues for assay, ervyme choline acetyltransferase (EC 2.3.1.6)with the the samples were disrupted at liquid nitrogen temperatures stoichiometry given in the following chemical equa- in a tight-fitting stainless steel pulverizer. The samples were tion: acctyl coenzyme A + choline =acetylcholine + subsequentlj maintained at 0 4 C unless otherwise noted coenzyme A. The activity of this enzyme irr tiirru. and transfers were quantitative. The powder was further dis- which is used as a marker for cholinergic neurons, par- rupted with a Potter Elvehjelm (Teflon to glass) motor- allels nerve activity iit ciro in preganglionic sympathe- driven homogeni7cr in a minimum amount (less than 3 ml) tic fihres(Oi.scti & THOt.Nm, 1973: OtSCH. 19741. It is of Buffer A (5 mM potassium phosphate. 0.1 mM EDTA. pH also prohablc that choline acetyltransferase is a 7.4) containing I ",, ti-butanol. The homogenatcs were dia- marker for cholinergic innervation of the heart. Carni- lysed against 200 vol. of Buffer A for 4 h to remove low molecular weight substrate. The buffer was changed once. tine acctyltransferase (EC 2.3.1.7). which uses choline The assaq for ['4C]acetylcholinc end [''Clacetylcarni- as an alternate substrate irt rim(WHITL & Wu. 1973~). tine hiosynthesis was previously described (ROSKOSKI. 1973) may contribute to the apparent activity of choline acc- using 50 pi ['4c'Jacrtyl coen7yme A and 2 mM choline or tyltransferasc in myocardial homogcnatcs. 2 mM L-carnitine in the final incubation mixture. To resolve The present experiments wcrc initiated to investigate labelled acctjlcholine. acctjlcarnitinc and ;I the iictirity or cholinc and carnitinc ;icctyltransrcrase 4. low voltagc (3(1 V cm: 20 minl paper in the heart. Thcsc studies >how that choline acetjl- (Whatman No. I1 in I \I formic acid I \I acetic acid solution tr:tnurcr:tsc ciin hc distinguished from carnitine acct}l- was used. Acetylcholine and ;icut!lcarnitinc standards transferax in guinea-pig heart iir rirro by (I ) dialysing migrated 65 em and 5.5 cin. resprctivrl\.. using a Gclman the hotnogcnates to remove endogenous carnitinc. (2) Elux electrophorssih apparatus. Resolution with the regu- lar Gelman apparatus was not as good under the same con- using low choline concentr:ltions in the assay mixture. ditions. and improving the electrophoretic resolution of (31 Racemic acctklcnrnitine and pigeon breast muscle carni- products. tinc acetyltranskmic (FC 2.3.1.7) wrre purchnrcd from METHODS Sigma Chemical Co. and L-c;irnitine. Mann Rewiirch Malc guinea-pigs (750 850 g) were sacriliccd h) cervical Lahoratorics. Inc The sourws of the othcr rn;iteri:ils Ii;i\c dislocation and then daxpiratcd and exs.inguin;itcd. hlicr hwn previously given (Rosrosul. 1973. 197401. 11Y7 1 I98 R. Ros~toslci.JR.. H. E. MAYERand P. G. SCHMIIJ Protein was determined b) the method of LOWR~Ct a/. TABL~1. EFFECT OF ORGANIC SOLVENT ~XTRACTIONON CHO- (1951)Or WAKHl KG & CHRISTIAN (1941). LINK ACETYLTKANSFt.RASE ACTIVITb Acetylcholine RESULTS Addition (pmol) Gmwl c.hrr~~ic.tt~r.i.~rjCSof ucc,tylcho/irir mid acetyl- ccirrririiil, hios~~rirli~~sis.Several workers have reported None that the apparent activity of choline acetyltransferase I"<, ti-Butanol 0.5",, Triton X-I00 in undial>sed heart extracts is greater than that of ner- I", Ether vous tissue (EKST~OM,1970; MAHONEYet d..1971; WHITI.&Wu. 1973~).WHITE& Wu(l973u)showed that Right atrial myocardium was prepared for cholinc acetyl- the incomplete resolution of labelled acetylcarnitine transferase activity as described in Methods with buffer con- and acetylcholine synthesized in uirro accounted for taining the specified concentration of organic solvent in each of the steps. Then 10 PI aliquots containing 60 pg pro- this adventitious activity. The apparent choline acetyl- tein were assayed in a mixture containing the specified sol- transferase activity was reduced by removing endo- vent for I5 min. genous carnitine by dialysis. In contrast to these results. acetylcholine biosynthesis was nil in the undia- To release occluded or otherwise inaccessible cho- lysed extracts in the present experiments because the line acetyltransferase. the heart was pulverized into a labelled acetyl coenzyme A was depleted by conversion fine powder and homogenized in hypotonic buffer con- to adtylcarnitine which.was resolved from carrier ace- taining lo;, 17-butanol (v/v). The ti-butanol increases tylcholine. Choline acetyltransferase activity. however. apparent enzyme activity by 30 per cent: Triton X-100. was detectable in the dialysed extracts. The rate of ace- 15 per cent and ether treatment decreases the activity tylcholine biosynthesis was linear for 45 min and that by 30 per cent (Table I). HERB(1972) reviewed the effi- for acetylcarnitine biosynthesis for 60 min (Fig. I). In- cacy of these procedures in other tissues. cubation times of 30 min or less were used in the pres- Commercial crystalline pigeon breast muscle carni- ent studies. tine acetyltransferase was assayed for its abilit) to use choline as substrate. Polyacrylamide gel clectrophor- esis of the carnitine acetyltransferase revealed a single 60 P TABLE3. CHoLlNL AND CARNlTlNE ACtTYLTKANSFtKASE ACTIVITY IN GI!INtA-PIG HEART CHAMBERS Acetyltransrerase activity Choline Carnitine (nmol min-lg-') (mmol min-lg-') Right atrium 445 k 043* 3.18 f 0.19 Left atrium 1.77 + 020 2.38 f 0.24 Right ventricle 233 + w14t 8x6 o.4~ TIME, min Left ventricle 1.33 014 8-27 * 0.34 FIG.1. Time course of acetylcholine and acetylcarnitine Samples from the specified chambers were homogeniced, biosynthesis in dialysed homogenates of guinea-pig right dialysed. and assayed as described in Methods. The rcsults represent the mean s.I:.M. of 5 animals. auricle. The tissue was prepared and assayed as described * P < 005 for differences between values in right atrium in Methods. The extracts for choline (-0) and carnitine and other chambers. (t-0)acetyltransferase contained 60 and 006 pg of pro- t P < 005 for differences between values in right and Id1 tein, respectively. ventricle. TABLE2. BIOSYNTHESISOF ACETYLCHOLINE BY PIGEON BREAST MUSCLE CARNITlNt ACLTYLTRANSFLRASI Carnitine ACh Acetylcarnitine Acceptor substrate acetyltransferase (prnol min-l) (pmol min- I) L-Carnitine 025 ng <O.I Ill Choline 300 ng <5 <O.I None 300 ng <O.I 10.1 Crystalline pigeon breast muscle carnitine acetyltransferase (100 units per mg protem) was assayed by the procedure out- lined in Methods. The acetylcholine and acetylcarnitine were resolved by paper electrophoresis. Choline acetyltransferaseactivity I199 TABLE4. BROMOACETYLCHOLINE INHIBITION OF CHOLIM ACETYLTRANSFERASE Acetyltransferase Choline (pmol min-') Carnitine (nmol min-I) Control 17.1 14.2 0.1 mM Bromoacetylcholine 6.8 13.9 ~~ ~~ Dialysed right atrial extracts (50 pg protein) in 50 mM potassium phosphate. 100 mM KCI. 0.1 mM EDTA 0.1 mM eserine were incubated with specified concentration of bro- . moacetylcholine for 3 min at 37". The choline acetyltransferase activity was measured during a IS min incubation and carnitine enzyme activity during a 30 s incubation. band which comigrated with enzyme activity and the choline and carnitinc acetyltransferase activities in enrymc wasjudged to be greater than 99 per cent pure. heart, differential effects of inhibitors were studied. WtIrTt & Wu (IY73a) reported that the apparent K, Bromoacetylcholine is a potent inhibitor of placcntal of this enzyme for I.-carnitine is 0.4 mM and that its (MORRIS& GREWAAL.1971) and bovine brain (Ros- apparent K, for the alternate substrate, choline, is 8 KOSKI, 1974b) choline acetyltransferase. CHASE & mb4. To minimize adventitious acetylation, a low cho- TURBS(1969. 1970) reported that bromoacetyl-~-carni- line conccntration (2 mM) was used throughout these tine (but not bromoacetyl-u-carnitine) inhibits the experiments. Under these conditions, carnitine acetyl- pigeon breast muscle carnitine acetyltransferase. Un- transferase had 0.004 per cent the activity with the der conditions where carnitine acetyltransferase acti- alternate substrate choline (Table 2). vity is not altered. bromoacetylcholine inhibits acetyl- Choline and carnirine acetylaansferase activity in the choline biosynthesis by 66 per cent (Table 4).