Acetylcholinesterase molecular forms in muscle and non-muscle cells of rat heart

J Mel

Cell

Cardiol

21, 987-994

( 1989)

Acetylcholinesterase Non-muscle Cynthia

Nyquist-Battid,

Molecular Cells Russel

Forms in Muscle of Rat Heart

T. DoweW

and Hugo

and

Fernandeti

‘School of Basic Lif Sciences, University of Missouri, Kansas City, MO, USA; ‘Department oj Physiolqgy, University of North Carolina, Chapel Hill, flC, USA; 3.Neuroscience Research Laboratory, L’Pterans Administration Medical Center, Kansas City MO, USA and Department of P&siolosl), lJniversi[v of Kansas Medical Center, Kansas Cit_y, KS, USA i Received 14 September 1988, accepted in revised form 22 May 1989) C. NYQCIST-BATTIE, R. T. DOWELL AND H. FERNANDEZ. Acetylcholinesterase Molecular Forms in Muscle and Non-muscle Cells of Rat Heart. Journal of Molecular and Cellular Gzrdiologv (1989) 21, 9877994. Experiments were performed to determine the cellular associations of the molecular forms of acetylcholinesterase (AChE) in adult rat heart. For this purpose, a cardiac muscle and a non-muscle fraction were isolated from rat heart ventricles after perfusion with collagenase and hyaluronidase, extracts of these fractions were subjected to ultracentrifugation on linear density gradients ofsucrose (5-2Oq,,), and fractions ofthese gradients were analyzed for AChE activity. The results show that only globular AChE molecular forms were present in isolated cardiac muscle cells. Clohular AChE forms were also present in the non-muscle cells fraction but in different proportions. The proportions of globular AChE forms plus the high specific activity of choline acetyltransferase in the non-muscle cell fraction suggest that this fraction contains cholinergic nerve fragments. The results of this study also show that asymmetric AChE is released during the perfusion of heart with the digestive enzymes, which suggests that asymmetric AChE is bound to the extracellular matrix of heart.

KEY LVORDS: Acetylcholinrsterase; Parasympathetic system; Heart.

Rat;

Adult

cardiac

Introduction

Acetylcholinesterase (acetylcholine acetylhydrolase; EC 3.1.1.7; AChE), an important component of the parasympathetic nervous system, is responsible for hydrolizing acetylcholine (,ACh) at the cardiac cholinergic neuro-effector junctions between the preganglionic and postganglionic parasympathetic neurons, and between postganglionic fibers and cardiac muscle cells [5, 19, 28, 34, 351. AChE has been shown by histochemical techniques to be associated in heart with nerve fibers, the neuronal cell bodiesof the parasympathetic ganglia, and with the specialized nodal cardiomyocytes; but, normal cardiac musclecells stain only faintly for AChE [I, 7, II, 12, 17, 18, 211. Since the histochemical methods of determining the cellular associations of AChE may not be ideal becauseof Please address all correspondence to: Cynthia Nyquist-Battie, 2411 Holmes, Kansas City, MO 64108-2792, U.S.A. 0022-2828/89/100987

+ 08 $03.00/O

myocytrs;

Isolated

heart

cells;

Cholinergic

nerves;

diffusion artifacts and because of the possibility of preferential staining of certain poolsof AChE molecules, it is not known with certainty to what extent AChE is associatedwith and synthesized by the muscle cells or the cholinergic nerve fibers of the mammalian heart. Yet, knowledge of the cellular placement ofAChE at neuro-effector junctions may help explain ACh turnover rates and the responsetime of cardiac cells to this neurotransmitter [21, 281. Acetylcholinesterase is a secreted glycoprotein that exists as multiple molecular forms, which can be distinguished by sucrose gradient centrifugation. The presenceof more than one structural form of AChE is thought to be important for variations in the extracellular placement of this enzyme [4, 1.5,24, 361.Asymmetric forms, consisting of catalytic School of Basic Life Sciences,

University

@) 1989 Academic

of Missouri. Press Limited

C. Nyqnist-Battie

988

subunits covalently bound to a collagenhelical peptide [24, 301 and globular forms, without the collagen tail, are present in all chambers of rat heart, although all areas of the atria exhibit higher AChE molecular form specific activities than the ventricles [271.Both amphiphilic and soluble globular speciescan be extracted from rat heart [271. It has also been observed that the percent activities of Gi and G4 AChE, which consistof monomersand tetramers of the catalytic subunits respectively, are not constant in all cardiac regions. In this regard, regions with the largest concentration of cholinergic nerves, as indicated by choline acetyltransferase activity, have the highest proportions of G4 AChE. This finding along with the observation that the vagus nerve contains predominately G4 AChE would suggest that cholinergic nerves may contain a significant proportion of the cardiac globular AChE. The objective of the present study was to determine if AChE is associatedwith rat cardiac myocytes isolated from the ventricles, and secondly, to determine if both asymmetric and globular AChE speciesexist in these isoLated cells. We report here that globular .\ChE speciesare present in the isolated cardiac myocytes, but a large portion of ::symmetric AChE was lost during the isolation procedure, which suggestsan extra,:ellular position for this cardiac AChE molecular form. Our results also show that a non-musclecell fraction, isolated concurrently with the musclecells, may contain cholinergic nerve fragments with a high content of G4 ZChE. This finding suggests a significant contribution of neural AChE to the cardiac ;,‘tol of this enzyme.

et al

0.5 mM KCl, 0.5 mM glucose, 1.5 mM NazHP04, and 0.4 mM NaHzP04, all adjusted to pH 7.4 with 0.2 M NazHPOd [9]. After flushing the hearts with buffer to remove blood, retrograde perfusion with the digestive enzymes was performed for 45-60 min during which the solution was gassedwith O&O2 (95/5%), and maintained at 37°C. Aliquots of the perfusate were saved for enzymatic analysis. After perfusion with digestive enzymes, hearts were flushed with ice cold buffer. All subsequent procedures were performed at 0-4°C. The left ventricle excluding the atrioventricular junction of each heart was homogenized in 10ml of the buffer without digestive enzymes in a Dounce loose fitting homogenizer using lessthan 10 gentle strokes. The homogenate was centrifuged for 3 min at 50 g. The pellet was washed twice with buffer. The resulting pellet was resuspendedin 5 ml of buffer, and this mixture was layered over 5 ml of 3% w/v Ficoll, made up in buffer. The pellet, obtained after centrifugation at 67 g for 3 min, was washed twice with 5 ml of buffer. The final pellet containing cardiac muscle cells was subjected to enzymatic analysis. To obtain the non-muscle cell fraction, the supernatants from the initial three centrifugations were combined and centrifuged at 800 g for 10 min. The resulting pellet was washed twice with 5 ml of buffer and the final pellet was used for assay of either AChE or choline acetyltransferase (ChAT). Aliquots of both musclecell and non-muscle cell fractions were monitored periodically by light microscopic examination to determine the quality of the cellular fractions. Acetylcholinesterase assay and velocity sedimentation analysis

For AChE determinations, the two types of cellular fractions and aliquots of the perfusate Preparation of isolated cellular fractions were homogenized in a conical glass-glass <,nrdiac myocyte and non-myocyte fractions homogenizer at a dilution of 1:lO v/v in a pH ksere prepared essentially as described in 7.3 buffer consisting of 50 mM Tris-HCl, loi i:utilletta et al. [a], using a retrograde perfu(v/v) Triton X-100, 5 mM EDTA and 1 M bion of hearts excised from adult male rats NaCl. The ventricles were homogenized in a 3% 150-200 g). The resulting fractions have been similar manner at a dilution of 1:15 w/v. extensively characterized [9, 101. The diges- Homogenateswere centrifuged at 20,000 g for rive enzyme solution consisted of 0.1% w/v 30 min (4°C). Aliquots of the supernatants collagenase (Type I-Clostridia), 0.1 :I0 w/v were used to determine AChE activity, or hyaluronidase, and 1% w/v bovine serum were subjected to sucrosegradient centrifugalbumin in a buffer consistingof 0.12 M NaCl, ation to separate AChE molecular forms. Materials

and Methods

Acetylcholinesterase

Molecular

For sucrose gradient centrifugation, aliquots (200-400 ~1) of the supernatants were layered on linear 5-20% sucrose gradients containing the same buffer as was used for AChE extraction, but with a lower Triton X100 concentration (0. I o/ov/v). Sedimentation was performed at 4°C and 260,000 g,,, (Beckman L8-70 ultracentrifuge; SW 41 Ti rotor) to a o?t value of 1.06 X 1012rad2/s(approximately 18 h 45 min). Sixty 200 ,uI fractions were collected from the top of each gradient tube using an ISCO-640 fractionator. AChE sedimentation coefficients were estimated by comparison with those of bovine serum albumin (4.41s) and catalase (11.3 S), which were added to each gradient prior to centrifugation [13, II]. The relative proportions of the AChE molecular forms were determined by comparing the enzymatic activities under each peak with that under the entire sedimentation profile. The enzymatic activity of the AChE molecular forms was measured by incubating individual gradient fractions in 50 mM potassium phosphate buffer (pH 6.8) containing 0.1% (v/v) Triton X-100 and ISO-OMPA (tetraisopropylpyrophosphoramide) at a final concentration of 0.1 mM to inhibit butyrylcholinesterase [Z]. After a 20 min preincubation with inhibitor, the reaction (400 ~1 total reaction volume) was initiated by the addition of (SW)-acetylcholine iodide (50 mci/mmol) at a final concentration of 0.1 mM. Assays were performed at 37°C and were stopped after 45-60 min by the addition of 400 ,ul of 50 mM glycine containing 1 M NaCl at pH 1.25 [20]. The activity of supernatant AChE was measuredusing triplicate samplesin a 400 ~1 final reaction volume as described for gradient assays,and which included 200 ~1of the gradient buffer containing 15% (w/v) sucrose. Reaction time was chosen so as not to exceed 15’:; hydrolysis of acetylcholine, thus preventing product inhibition. The (SH)-acetate product of AChE hydrolysis was quantified by liquid scintillation spectrometry. AChE activity is expressedas pmoles or nmoles of (3H)acetate formed per min. The activities of the individual molecular forms of AChE were calculated from the relative proportions (7, activities) of each molecular form obtained from the gradient profiles and from supernarant AChE activities. Protein was determined

Forms

in Cardlomyocytes

989

by a modification of the method of Lowry r231. Choline acetyltransferase assay

Aliquots of the muscle and non-muscle cell fractions, and the per&sate were homogenized in 10 volumes of an ice cold buffer, pH 7.4, consisting of 5 mM potassium phosphate and 0.1 mM EDTA. Enzymatic activity was measuredduring a 15 min incubation at 37°C using 50 mM ( 14C)-acetyl COA and 2 mM choline as described by Roskoski and coworkers [31]. To resolve the ( 14C)acetylcholine product from acetylcarnitine, other products and the substrate, low voltage (30 V/cm : 30min) paper electrophoresis was usedasdeveloped by Roskoski, et al. [31]. The use of a low concentration of choline, the electrophoretic separation of acetylcholine from other products and substrates, and the dilution of sample with adequate amounts of buffer prior to useare thought to be sufficient to negate the contribution of the enzyme carnitine acetyltransferase to acetylcholine synthesis[31,32]. Protein levels were measuredas described above. Chemicals

Chemicals were obtained as follows: (3H)acetylcholine iodide and (14C)-acetyl CoA from the New England Nuclear Division of E. DuPont Co.; other biochemicals were from Sigma Chemical Co. except for ultra-pure sucrose which was obtained from SchwartzMann Co. Results Characteristics of cellular fractions

Microscopic inspection of cell suspensionsindicated that the myocyte fractions routinely contained a visually homogeneous(92-959;) population of birefringent-anisodiametric cells, which were rod-shaped and judged to be cardiac muscle cells. These cells were stained by the Periodic Acid Schiff reagent (PAS), The non-myocyte fraction contained primarily small rounded cells and cellular fragments that were judged to be of non-muscle cell origin, since they were not positive for PAS. The cross-contamination of the two cellular

990

Acetylcholinesterase

Molecular

fractions, seen in this study, was similar to that reported by Cutilletta et al. [8], who used the same isolation procedure. The contamination of the muscle cell fraction by non-muscle cells was less than 5%, and the non-muscle cell fraction had little or no muscle cell contamination. These two fractions of cells have been used by one of the authors (R.T.D.) to show that aerobic metabolic enhancement, which occurs after birth in rat heart, is confined to cardiac muscle cells [9] and that creatine kinase activity is enriched in the cardiac muscle fraction as compared to the nonmuscle fraction [IO]. AChE and ChAT activities in rat ventricles and in muscle and non-muscle cellular fractions The specific activities of AChE in the supernatants from the isolated cellular fractions and ventricles were determined (Table 1). The myocyte fraction contained approximately three-fold lower AChE activity per mg protein than the non-myocyte fraction. The ventricles exhibited a two-fold higher AChE activity than the myocyte fraction, but this was still lower than the non-myocyte fraction. To determine if AChE in the isolated myocyte fraction could be the result of cholinergic neural contamination, choline acetyltransferase (ChAT), a biochemical marker of cholinergic nerves in heart [32], was measured in both cellular fractions. The non-muscle cell fraction contained greater than a hundredfold higher ChAT activity than the muscle cell fraction: 153.67 &- 7.1 nmol/h/mg protein in comparison to 1.19 + 0.1 nmol/h/mg protein,

TABLE

1. The percent cholinesterase

Forms

in Cardiomyocytes

n = 5 separate experiments. The activity of ChAT in the ventricles was 6.6 f 0.9 nmol/h/mg protein. These data indicate that cholinergic nerve fragments are a significant component of the non-muscle cell fraction, but are probably not a significant contaminant of the myocyte fraction. AChE molecular forms associated with isolated fractions To determine which of the molecular forms of AChE were present in each of the isolated fractions, aliquots of supernatants were applied to sucrose gradients, which in turn were subjected to velocity sedimentation. Typical profiles of AChE activity obtained by velocity sedimentation are shown in Figure 1. The data show that the myocyte fraction contained two peaks of AChE activity. According to sedimentation coefficients, the peak on the left contained both G1 and GZ forms of AChE, while the right peak contained G4 AChE. The first peak with Gr and G2 AChE represented approximately 630,,, of the total AChE activity, while the G4 peak contained approximately 37O4, of the total activity (Table 1). AI2 AChE, which sediments at 16S, was not apparent in extracts of the myocyte fraction. The non-myocyte fraction also contained Gr, G2 and G4 AChE but in different proportions, as is shown in Figure 1 and Table 1. The contribution of G4 to the total AChE activity was 62q; which was higher than that of the myocyte fraction. Additionally, Gz AChE was a larger component of the first peak in the non-myocyte fraction than in the myocyte

activities of the molecular forms of acetylin fractions isolated from rat ventricles Percent activities

Muscle Non-muscle Ventricles

AChE activity pmol/min/mg prot.

G & Gz

G;4

A 12

313 &- 21* 871 + 10 630 + 20

63 + 2* 36 It I 44 + 3

37+ 1* 62 * 3 47 + 3

N.D. 2 + 0.5 9fl

Values are means + SEM, n = 6 separate animals. Activity is expressed as pmol/min/mg protein. Activities were calculated as described in “Methods”. NonSignificant differences (P < 0.05, Student’s t detectable activity is noted by “N.D.". test) between the two cellular fractions are indicated by *.

C. Nyqdst-Battie

Non-muscle

IO

991

cell

30 Fraction

0

et al

20

30 Fraction

FIGURE I. Representative velocity sedimentation profiles fractions under high ionic strength conditions with 5 rnM EDTA Serum Albumin (4.4s) and Catalase (I 1.35): were recovered expressed as percent total CPMs recovered in the gradient.

fraction. The percent activity of the Gr-GZ pool was 36:,&. A small amount of AI2 AChE, sedimenting at 16S, was seen in the nonmuscle cell fraction. At2 AChE represented 20/;, of the total activity in this fraction. AChE molecular forms extracted from the ventricles had different percent activities of the globular forms and a higher percent activity of AI2 AChE, when compared to the isolated fractions. The finding of a greater percent activity of A,2 AChE would seem to indicate that

40 no.

40 no

50

60

70

of AChE extracted from muscle and non-muscle cell and I?/, Triton X-100. Sedimentation markers, Bovinr in Fractions 10 and 25, respectively. AChE activity is

there was a loss of asymmetric AChE during the preparation of the cellular fractions. To determine if this was the case, the perfusate was collected and analyzed for AChE molecular forms, as described in “Methods.” The AChE specific activity in the perfusate was 560 f 21 pmol/min/mg protein, but this fraction did not contain detectable choline acetyltransferase activity. The perfusate contained substantial amounts of G4 AChE (774,,) and minor amounts of Gt-G2 AChE ilS”,,) and

992

C. Nyquist-Bat-tie

et al

ference in specific activity for AChE between the two fractions was not nearly as dramatic. Another indication that the globular AChE in the muscle cell fraction was associatedwith the muscle cells and not the result of neural contamination is that the proportions of the various globular AChE forms in the myocyte and non-myocyte fractions were quite different. The association of high levels of globular AChE and especially the G4 form with the putative cholinergic nerve endings of rat heart ventricles would suggest a large amount of AChE in the parasympathetic nerves of heart. These neural endings would represent the post-ganglionic parasympathetic nerve fibers because rat heart ventricles do not contain parasympathetic ganglia and associatedpreganglionic fibers [19,21]. Gd AChE is also the predominant molecular form found in the central nervous systemwhere in large measure it is found presynaptically [16, 241. The function of presynaptic AChE is unknown but could be important in regulating the presynaptic pools of acetylcholine or could allow for degradation of this neurotransmitter near the presynaptic re-uptake site for choline [28, 291. Discussion The latter hypothesis is supported by the fact A major result of the present work is the that presynaptic AChE in brain is largely demonstration that cardiac muscle cells, iso- extracellular [29]. It is interesting that the lated from the ventricles of rat heart, contain heart contains significant AChE associated globular AChE. Additionally, the non-muscle with cholinergic nerve endings, while the cell fraction was found to contain high levels of nerve endings of skeletal muscle are not the cholinergic nerve enzymatic maker, thought to contribute significantly to the choline acetyltransferase, along with high AChE pool at the skeletal muscle end-plates levels of globular AChE, suggesting that this [24]. This difference in AChE placement could fraction contained cholinergic nerve frag- help to explain the differences in the response ments with significant globular AChE as well of cardiac and skeletal muscleto acetylcholine ascontaining other non-muscle cells [S]. Last- [Zl, 281. ly, the presence of globular and asymmetric A large pool of AChE bound to the extraAChE molecular forms in the perfusate is cellular matrix in rat heart ventricles is sugevidence for a rather large externalized pool of gested by our study. The composition of this ventricular AChE, which is releasable from extracellular pool of AChE is not entirely clear the extracellular matrix by perfusion with from our results. The fact that so little asymdigestive enzymes. metric AChE is found in both cellular fracThe presenceof globular molecular forms of tions, although found in significant amounts AChE in the myocyte fraction did not appear in the intact ventricles, coupled with the fact to be the result of contamination of this frac- that Ai2 AChE isfound in the perfusate would tion with cholinergic nerve fibers for the fol- suggestthat Ai2 AChE is lost during perfulowing reasons. The neuronal cell marker, sion. At2 AChE could be bound to the extracholine acetyltransferase [32], had a much cellular matrix which is disrupted by enzyme higher specific activity in the non-myocyte treatment. This hypothesis seemsreasonable than in the myocyte fraction, while the difbecausetreatment of other tissueswith collaAiz AChE (7%). The presenceof both globular and asymmetric AChE in the perfusate could indicate the release of AChE from an extracellular location during perfusion. It hasbeen shown in other tissuesthat crude collagenase,containing trypsin, degradesArz AChE to a form that sedimentswith Gb AChE [24]. Whether this was true in heart was examined by homogenizing replicate piecesof the left ventricle with buffer containing the digestive enzymes or with buffer without these enzymes. Tissue homogenateswere incubated for 30 min at 37°C and then mixed 1:l with the AChE extraction buffer, and centrifuged as described in “Methods.” The supernatants were subjected to velocity sedimentation analysis. The results of this experiment show that incubation with the digestive enzymes reduced the percent activity due to Aiz AChE from 9% to 3% and increased the percent activity of G.+AChE from 48% to 65%. This analysis indicates that the perfusion of heart with collagenaseand trypsin could be capable of degrading Ai2 AChE to a form that sediments at 10s.

Acetylcholinesterase

Molecular

genase has been shown to release AChE by disrupting the extracellular matrix, and since digestive enzymes do not easily penetrate cell membranes [15, 251. Globular AChE molecular forms have also been observed to bind to the extracellular matrix in skeletal muscle at least in frog [Zq and therefore globular AChE, bound to extracellular matrix, may be a source of perfusate AChE as well as A12 AChE. The loss of All AChE during cell preparation precludes us from determining the cellular site of synthesisand attachment of this AChE molecular form in heart ventricles. However, since cardiac muscle cells are surrounded by an extracellular matrix [S, 221it is logical to hypothesize that Al2 AChE is synthesized by the cardiac muscle cells and secreted along with other external lamina components.

Forms

in Cardiomyocytes

993

The present work has described significant levels of AChE associatedwith isolated cardisc muscle cells and cholinergic nerves, as well assuggestingthat there exists a significant pool of AChE molecules bound to the extracellular matrix in rat hearts. The importance of these various pools of AChE molecules in the clearance of ACh from rat heart ventricles is not known, and therefore, the importance of these pools to cardiac function needsfurther study. Acknowledgements

This research was supported in part by the Medical Research Service of the Veterans Administration and the University of Kansas Center on Aging (HLF); NIH HL 33677 and NIH HL 28456 (RTD) and by a grant-in-aid from the American Heart Association (CNB).

References I

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Acetylcholinesterase

Molecular

Forms

in Chdiomyocytes

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