Download Regional Distribution of the Molecular Forms of Acetylcholinesterase

55
Regional Distribution of the Molecular Forms
of Acetylcholinesterase in Adult Rat Heart
Cynthia Nyquist-Battie and Karl Trans-Saltzmann
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Acetylcholinesterase (AChE), the enzyme that degrades acetylcholine, exists as multiple
molecular forms that differ in their quaternary structure and mode of attachment to the cell
surface. The distribution of the individual molecular forms of AChE in various cardiac regions
with distinct anatomical characteristics was investigated. The results confirmed those of others
by showing that the total pool of cardiac AChE had a nonuniform distribution in heart that
paralleled the distribution of choline acetyltransferase. The rank order of this distribution was
right a trial appendage >interatrial septum > left atrial appendage=right ventricle=interventricular septum > left ventricle. Velocity sedimentation in sucrose gradients of extracts from selected
cardiac areas showed that four molecular forms were present in all areas but that the
proportions of these forms differed as a function of area. The right and left ventricular walls,
the apical portion of the Interventricular septum, and the left atrial appendage contained G, and
G4 (globular) AChE in near-equal proportions, but in the basal portion of interventricular
septum, the contribution of G4 AChE was greater than that of G, AChE. The right atrial
appendage and the interatrial septum had the largest amount of activity attributable to G4
AChE and the lowest amount attributable to Gt AChE. In all cardiac regions, A u (asymmetric)
AChE comprised 8-10% of the total AChE pool. The results of this research show that the
composition of the globular AChE pool varies in heart as a function of region and that cardiac
regions that have the highest AChE activity and that contain parasympathetic ganglia and nodal
areas have different pools of globular AChE molecules than other areas. In addition, it appears
that there is a uniform proportion of Au AChE in all areas of rat heart. (Circulation Research
1989;65:55-62)
T
he parasympathetic preganglionic nerve supply to the mammalian heart is cholinergic,
arises in the brainstem, and travels in the
vagus nerve to ganglia embedded in the walls of the
atria.1 The postganglionic nerve fibers arising from
these ganglia are also cholinergic and are distributed in a nonuniform manner to the various regions
of the heart.1-4 Vagal stimulation and cholinergic
agonists have well-established negative chronotropic, dromotropic, and inotropic effects on mammalian
heart.2 Acetylcholinesterase (AChE; acetylcholine
acetylhydrolase EC 3.1.1.7) hydrolizes acetylcholine
(ACh) and thereby terminates the action of this
neurotransmitter at the cholinergic neuroeffector junctions of the heart.
From the Division of Structural and Systems Biology, School
of Basic Life Sciences, University of Missouri-Kansas City,
Kansas City, Missouri.
Supported by National Institutes of Health Grant HL-35598
and by the Lettie B. Mclrvain Frederic Fund.
Address for correspondence: Cynthia Nyquist-Battie, PhD,
Structural and Systems Biology, University of Missouri-Kansas
City, 2411 Holmes Ave., Kansas City, MO 64108.
Received August 27, 1988; accepted December 10, 1988.
AChE exhibits an extensive polymorphism with
molecular determinants that allow this enzyme to
be placed at more than one functional cellular
location.5-6 The distribution of AChE forms differs
from tissue to tissue probably reflecting the operational needs of the various types of cholinergic
neuroeffector junctions.3-6 Structural forms of AChE
differ in sedimentation coefficients, reflecting differences in quaternary structure,3 and can be classified
as asymmetric (A) with a covalently linked collagenlike tail or as globular (G) because they lack a tail
structure. Both globular and asymmetric forms of
AChE can be extracted from all chambers of rat
heart. The major globular forms present in rat heart
sediment at 4, 6, and 10S, corresponding to G^ G2,
and G4 AChE, consist of monomers, dimers, and
tetramers of the catalytic subunit, respectively.
Similar to other tissues, a percentage of globular
AChE in rat heart requires detergent for extraction,
an indication that membrane-bound globular forms
as well as soluble forms are present in rat heart.
The major asymmetric form extracted from rat
heart is identical to that which is found in skeletal
muscle, namely A12 AChE. This form sediments at
56
Circulation Research Vol 65, No 1, July 1989
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16S and contains three tetrameric catalytic subunits
bound to a collagen-like tail.5
Since it appears that cardiac cholinergic neuroeffector junctions exhibit different properties than
those of skeletal muscle,2 it seems of interest to
study the composition of the AChE pool in cardiac
muscle. Although the AChE molecular forms of
skeletal muscle have been well studied, little is
known regarding cardiac AChE molecular forms.
For example, how each of the various AChE forms
are related anatomically to the cholinergic neuroeffector junctions of the heart is unknown, as is the
cellular site of synthesis of each form. In addition,
little is known in heart about what regulates the
synthesis of AChE or what constitutes the external
or functional pool of this enzyme. The aim of this
study was to determine the distribution of the
individual AChE molecular forms in regions of rat
heart with different anatomical components. This
study should help to determine if the cardiac areas
that contain the cholinergic neuroeffector junctions
of the parasympathetic ganglia, nodal, and conducting areas have different pools of AChE molecular
forms than those of contractile areas. To this end, a
study of the distribution of the molecular forms of
AChE in distinct anatomical regions of rat heart
was undertaken, and this distribution was correlated with the quantity of cholinergic nerve fibers in
each area, which was monitored by choline acetyltransferase activity.
Materials and Methods
Biochemicals were obtained as follows: [3H]acetylcholine iodide and [14C]-acetyl coenzyme A
from New England Nuclear, Inc, Boston, Massachusetts; ultra-pure sucrose from Schwartz-Mann
Company, Cleveland, Ohio, and all other biochemicals from Sigma Chemical Company, St. Louis,
Missouri. Other chemicals were obtained from Fisher
Scientific Company, St. Louis, Missouri, or Aldrich
Chemical Company, Milwaukee, Wisconsin.
Animals and Tissue Preparation
Sprague-Dawley male rats (150-200 g) were
obtained from Sasco, Inc, Omaha, Nebraska, and
were maintained with free access to Purina laboratory chow and water. Each animal was anesthetized
with sodium pentobarbital (40 mg/kg) administered
by intraperitoneal injection. When each animal was
deeply comatose, the thoracic cavity was opened,
and the heart was perfused through its apex with 60100 ml ice-cold heparinized saline (10 units/sodium
heparin/ml). The use of heparin in the perfusion
buffer did not alter the amount of asymmetric AChE
extracted from heart (authors' unpublished observation). After perfusion, the right and left vagus nerves
were removed in one piece from the cervical and
thoracic regions. Secondly, the right atrial appendage (RAA), including the sinoatrial node (S-A node),
was removed followed by the left atrial appendage
(LAA). Subsequently, the rest of the heart was
removed from the thoracic cavity, and the interatrial
septum-posterior atrial wall (IAS), interventricular
septum (IVS), left ventricular free wall (LV), and
right ventricular free wall (RV) were obtained. The
dissection was performed so that the IVS contained
the atrioventricular node (A-V node).9 In separate
experiments, the A-V and S-A nodal areas and the
apical half of the IVS were used as samples. All tissue
samples were rinsed, blotted, and weighed. Tissue
samples were either used for AChE molecular form
analysis or for assay of acetylcoenzyme A choline0-acetyltransferase (CAT), EC 2.3.1.6, as a biochemical marker for cholinergic nerve fibers.8
AChE Extraction and Assay
Tissue samples were homogenized with a glassglass motor driven homogenizer at 1/15 (wt/vol) in
50 mM Tris-HCl pH 7.3,10 ml/1 Triton X-100,1 mM
NaCl, and 5 mM EDTA. Homogenates were centrifuged at 24,00Qg for 30 minutes (4° C), and aliquots of the supernatants were either assayed for
AChE activity or subjected to velocity sedimentation analysis. The assay of supernatant AChE activity was performed as described in Nyquist-Battie et
al7 by incubating triplicate samples in a 400 /A final
reaction volume containing 50 mM potassium phosphate, pH 6.8, with 1.0 ml/1 Triton X-100 and 0.1
mM ISO-OMPA (tetraisopropylpyrophosphoramide) to inhibit butyrylcholinesterase.10 After a
20-minute preincubation in the presence of ISOOMPA, reactions were started by the addition of
[3H]-acetylcholine iodide (100 mCi/mmol) at a final
concentration of 0.1 mM. Assays, conducted at
37° C, were terminated after 20 minutes by the
addition of 400 fA of 50 mM glycine with 1M NaCl
at pH 1.25. The [3H]-acetate product was quantified
by liquid scintillation counting. Protein was determined by the method of Markwell et al. In one
series of early experiments, a combination of protease inhibitors (0.2 mg/ml aprotinin, 2.0 mg/ml
benzamidine-HCl, and 0.1 mg/ml bacitracin) was
used in all buffers, but the addition of this mixture
did not alter the sedimentation profiles or AChE
activity. Protease inhibitors have also been shown
not to be important in the extraction and isolation of
AChE forms from skeletal muscle.
Velocity Sedimentation
Aliquots (300-500 jxl) of the supernatants were
layered on linear 5-20% (wt/vol) sucrose gradients
containing the AChE homogenization buffer. Sedimentation was performed in a SW 40 rotor at
230,00O#m« to a art value of 1.09xl0 u rad7sec or
for approximately 22 hours at 4° C. Forty 300 -jil
fractions were collected from the top of each gradient. AChE sedimentation coefficients were estimated by comparison with those of bovine serum
albumin (4.41S) and catalase (11.3S), which were
added to each gradient before centrifugation. The
relative proportion of each AChE molecular form
was determined by comparing the enzymatic activ-
Nyquist-Battie and Trans-Saltzmann Acetylcholinesterase in Heart
TABLE 1. Chollne Acetyltransferaie (CAT) and AcetylchoUnesterase (AChE) Actfvitie* In Varioui Regions of Adult Rat Heart
CAT
(pmol/min/mg wet wt)
AChE
(pmol/min/mg wet wt)
RAA
15.2±0.95
331±13
IAS
LAA
RV
TVS
LV
9.18±1.20
5.10±0.45
4.76±0.30
4.35±0.32
2.90±0.21
270±10
95.2±5.0
89.5±5.4
80.1+6.8
53.4±5
Region
RAA, right atrial appendage; IAS, interatrial septum; LAA,
left atrial appendage; RV, right ventricular free wall; IVS,
interventricular septum; LV, left ventricular free wall.
Each value represents the mean±SEM of four experiments.
Enzyme activities were assayed in tissue homogenates for CAT
and in supernatants for AChE.
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ities under each peak14 with that under the entire
sedimentation profile. The activity of each molecular form was derived from supernatant AChE
activity based on the relative proportional data.
CAT Extraction and Assay
Tissue samples, obtained from separate animals
than those used for AChE assays, were homogenized
at a 1/20 (wt/vol) dilution with a glass-glass motor
driven homogenizer in 5 mM potassium phosphate,
pH 7.4, with 0.1 mM EDTA. Levels of acetylcholine
synthesis were quantified by a method based on that
of Roskoski et al,8-13-16 using 0.4 mM [14C]-acetyl
coenzyme A and 2 mM choline. Incubations were
performed in duplicate at 37° C for 15 minutes. To
separate the [14C]-acetylcholine product from other
radioactive metabolites and the substrate, samples
were subjected to low voltage (35 V/cm) paper electrophoresis. In this method, a 1/20 tissue dilution, 2
mM choline, and paper electrophoresis are used to
minimize the contribution of carnitine acetyltransferase to the estimation of CAT activity.8.15-16
Results
Regional Differences in the Activities of Cardiac
AChE and CAT
The activities of CAT and AChE per unit wet
weight in various regions of the rat heart are given
in Table 1. It can be seen that the distribution of
these two enzymes in the selected heart regions was
similar. The regional activities of the two enzymes
had the following relations: RAA>IAS>LAA=
RV=IVS>LV (p<0.01 by analysis of variance).
The same regional distribution of enzymatic activities was seen when the data were expressed per
milligram protein (data not shown). Our present
extraction method for AChE has been shown to
solubilize greater than 95% of this enzyme from rat
heart (authors' unpublished observation).
Regional Distribution of AChE Molecular Forms
in Rat Heart
The five anatomical regions of rat heart were also
examined for their content of AChE molecular
57
forms. Extraction of asymmetric AChE forms from
tissue was ensured by the inclusion of 1M NaCl in
the homogenization buffer. The nonionic detergent
Triton X-100 was included to solubilize the
membrane-bound globular forms of AChE. For this
analysis, tissue samples from six animals were
examined. Figure 1 illustrates the activity profiles
typically obtained after sedimentation analysis of
tissue extracts of the selected heart regions. Gradients from all areas contained three major peaks of
AChE activity but in different proportions. In all
cases, the left peak contained AChE that sedimented at 4S and which can be classified as Gj
AChE.5 Additionally, a small shoulder that contained up to approximately 35% of the activity of
the left peak was seen in all cases and was determined by sedimentation coefficient to be G2 AChE.
The middle peak, sedimenting at 10S, represented
G4 AChE. In some instances, a negligible shoulder
representing Ag AChE was observed. The right
peak, seen in all regions, corresponded to Aa
AChE. Three different pools of AChE forms were
seen in our examination of specific heart regions.
The regional differences in the profiles are the result
of differences in the relative peak height of G] and
G4 AChE activities. The RAA and the IAS exhibited a much higher G4 than G! AChE peak, while the
LAA and ventricular free walls had near equal Gj
and G4 peaks. The IVS had a G4 peak slightly higher
than that due to Gj AChE. Since the RV had an
identical profile to that of the LV, this profile is not
shown.
The percent contributions of Gi-G2, G4, and An
AChE to the total AChE activity were calculated
for each region as described in "Materials and
Methods," and these values are given in Table 2.
Since G2 AChE is not well resolved from the Gj
form, the percent contribution of the two forms is
presented together. However, in all cases the contribution of Gi AChE was much greater than that of
G2 AChE. These two globular forms contributed
41-45% of the total activity in the LAA, the RV,
and LV. In the IVS, 37% of the activity was
attributable to the sum of Gj and G2, while the RAA
and the IAS had 28-33% activity resulting from
G r G 2 AChE. On the other hand, G4 AChE was
responsible for 60-64% of the total AChE activity
of the RAA and the IAS, while the G4 AChE
percent activity in the RV, LV, and LAA was 48%.
The G4 form contributed 54% of the AChE activity
of the IVS. In all six heart regions, Au AChE
represented 8-10% of the total activity. The above
analysis confirms that the major difference in AChE
molecular forms between cardiac regions was the
result of differences in the contributions of G! and
G4 AChE.
The areas of the heart with the high G^Gi ratios
were regions that either contain specialized nodal
cells or conducting fibers. With the possible exception of the IVS, these regions also contain the
parasympathetic cardiac ganglia and their associ-
58
Circulation Research Vol 65, No 1, July 1989
20
IVS
LT.VENT.
1
^A
10-
.
0
10
20
A
10-
o,
J
y
ll
30
40
0
50
10
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r
20-
5
10-
Q
N
V
10
20
30
40
SO
FRACTION NO.
FRACTION NO.
2
u
\
IAS
20
30
FRACTION NO.
40
10
50
20
30
FRACTION NO.
40
50
20
30
FRACTION NO.
FIGURE 1. Representative velocity sedimentation profiles of acetylchoUnesterase extracted from the five cardiac
regions. Individual molecular forms of acetylchoUnesterase were separated on 5-20% sucrose gradients as described in
"Materials and Methods." Svedberg constants (S) were determined by the inclusion of bovine serum albumin (4.4S) and
catalase (11.35) in each gradient. Migration was from left to right. Lt. Vent., left ventricular free wall; IVS,
interventricular septum; IAS, interatrial septum; LAA, left atrial appendage; RAA, right atrial appendage.
ated preganglionic cholinergic nerve fibers.1 It is
logical, therefore, to suggest that these components
could be responsible in some manner for the variation in the cardiac pools of AChE molecular forms.
A series of experiments was conducted to determine if these anatomical components do contain a
different pool of AChE molecular forms. First, to
determine the composition of the pool of AChE
molecular forms associated with preganglionic parasympathetic nerve fibers the right and left vagus
nerves of three animals were studied. The right and
left nerves of each animal were pooled to form one
sample. The specific activity of AChE in these
samples was 698±11 pmol/min/rng wet wt. A representative gradient profile of AChE activity obtained
from the pooled vagus nerves is shown in Figure 2.
This profile differed from those of all heart regions
by exhibiting the largest 10S AChE (G4) component
and having near equal contributions from Gj and G2
AChE. Pooled G, and G2 activities were 20.1 ±1.2%,
G4 was 73±4.1% and A12 represented 6.5±0.3% of
the total gradient AChE activity.
Secondly, AChE activities and molecular forms
were examined in discrete samples of the heart
containing the pacemaker areas. To ensure enough
AChE activity for gradient analysis, tissue from
three animals was pooled. The percent activities of
the AChE molecular forms were calculated as
described above from gradient profiles (gradients
not shown). The pooled supematants of the S-A
Nyquist-Battie and Trans-Saltzmann Acetykholinesterase in Heart
59
TABLE 2. The Activities of Individual Acetylcholinesterase (AChE) Molecalar Forms in Various Areas of
Adult Rat Heart
AChE (pmol/min/mg wet wt)
Region
RAA
107±5
(33±2)
74.3±6
(28±2)
41.8±3
(44±3)
36.7±2
(42±3)
32.7±2
(37±2)
22.1+1
(41 ±1)
97.0±4
(25±1)
50.1±3
(30±2)
IAS
LAA
RV
rvs
LV
S-A node
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A-Vnode
197+6
(60±4)
172±4
(64+3)
45.7±4
(48±2)
42.9+4
(48±2)
48.3±2
(54±1)
25.5±1
(48±3)
260+6
(67±2)
98.5+5
(59±2)
197+6
(8±1)
23.3±3
(9±1)
8.2±1
(8+1)
8.9±1
8.3±1
(9±1)
5.2±1
(10±l)
31.0±2
(8±1)
16.7±1
RAA, right atrial appendage; IAS, interatrial septum; LAA, left atrial appendage; RV, right ventricular free wall;
FVS, interventricular septum; LV, left ventricular free wall; S-A node, sinoatrial node; A-V node, atrioventricular
node.
Values are mean±SEM, n=3-8 experiments. Since the 4S and 6S forms of AChE were not sufficiently resolved
under our sedimentation conditions, the sum of the two AChE activities is presented. The relative proportions of the
major AChE molecular forms in parentheses, were obtained from sedimentation profiles and, subsequently, the
activity of each form was derived from supernatant AChE activity based on the proportional data.
nodal area contained 388±15 pmol/min/mg wet wt
AChE activity. The major AChE molecular form in
this area was G4 AChE, which contributed 67% of
the total activity (Table 2). The contribution of A12
AChE to the total AChE activity of this region was
8%, which was similar to its contribution in other
heart regions. In contrast, the contribution of G!-G2
molecular forms was less than in most other cardiac
regions (Table 2). The A-V nodal area had lower
AChE activity (166±8 pmol/min/mg wet wt) but had
a similar distribution of molecular forms to that of
the S-A node (Table 2). Lastly, a similar analysis of
AChE molecular forms in the apical and basal
halves of the IVS was performed. The apical por-
a.
o
I
FRACTION NO.
2. Representative velocity sedimentation profile
of acetylcholinesterase extracted from the vagus nerve.
Refer to legend of Figure 1 for procedural details.
FIGURE
tion with 59 ±2 pmol/min/mg wet wt AChE activity
contained 42±1% G r G 2 , 47+3% G_, and 10±l%
A12 AChE activity. In contrast, the basal portion
contained 95 ±4 pmol/min/mg wet wt AChE activity
with the following AChE percent activities: 30±3%
G,-G2, 61±3% GA, and 10±0.9% A u .
Regional Activities of the Individual AChE
Molecular Forms
The activities (pmol/min/mg wet wt) of the major
individual AChE molecular forms in the various
heart regions were determined based on the relative
proportional data and the supernatant activities of
AChE (Table 2). Analysis of variance (p<0.0l)
revealed that for the most part the rank order of the
activities of the AChE molecular forms was similar
to that of total AChE activity. In this regard, the
RAA and IAS had much higher globular and asymmetric AChE activities than the other areas.
Although these two atrial areas had similar Au
AChE activities, the globular forms of AChE had
higher activities in the RAA. The LAA, RV, and the
IVS had similar activities for all molecular forms,
while the LV had the lowest activity overall for all
molecular forms. Data from the two nodal areas
were also examined (Table 2). The S-A node contained the highest G4 AChE activity of all heart
regions but had activities similar to the RAA for the
other forms. The activities of AChE molecular
forms in the A-V nodal region were higher than
those of the LAA, but lower than those of the IAS.
60
Circulation Research Vol 65, No 1, July 1989
Discussion
The present work describes regional differences
in the proportions and specific activities of the
molecular forms of AChE in rat heart. In addition,
this work is in agreement with that of Stanley et al17
showing that the activities of CAT and AChE have
a parallel but nonuniform distribution in this organ.
The regional distribution described in the present
work for CAT was similar to that reported by other
investigators.-" Since the level of CAT activity
in heart is a general indication of the amount of
cholinergic nerves in a given area, it can be concluded that rat cardiac regions with the greatest
number of cholinergic nerves also contain the greatest concentration of AChE, for example, the RAA
and the IAS.
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Histochemical studies of AChE in heart tend to
support the biochemical studies regarding AChE
and CAT distribution in heart. The AChE studies
using the histochemical method to map the cholinergic nerves of the heart indicate that the density of
the postganglionic innervation of the heart varies
from region to region with greater levels of AChEpositive nerves in atrial areas.2-3-24-25 Specialized
cardiac muscle cells of the nodal areas found in the
atria and conducting system receive a higher density of cholinergic fibers than contractile cardiac
muscle cells,2-3-24-23 thus helping to account for the
profound effect of vagal stimulation on chronotropic and dromotropic cardiac responses. It is also
thought, based on histochemical studies, that the
ventricular myocardium receives very sparse cholinergic innervation compared with the atrial
myocardium.2'3 However, if one looks at a region of
the atrial myocardium without specialized areas or
ganglia such as the LAA, the levels of CAT activity
are not very different from that of the RV. Additionally, presumptive cholinergic nerve endings are
seen during electron microscopic examination in
the ventricular myocardium of mammals,25-28 and
levels of muscarinic receptors are significant in the
ventricles.2-3-16 Therefore, it may be that AChE
histochemical staining of nerves is not as good in
the ventricular myocardium as in the atrial myocardium, and there is a need for the use of better
methods such as CAT immunohistochemistry to
determine the density of cholinergic nerves in the
heart. In comparing levels of innervation, it may be
important to consider the amount of innervation per
cell rather than density per area. However, in
support of a more dense cholinergic innervation of
the atrial myocardium, vagal stimulation has a
greater effect on atrial contractility than on ventricular contractility.2 Additionally, there are atrioventricular differences in acetylcholine levels and turnover, both before and after vagal stimulation, which
could also be an indication of unequal innervation
of heart chambers.20
The results of the present work show that the
activities of CAT and AChE are highest in atrial
regions which contain parasympathetic preganglionic nerve fibers and their ganglia.1-3-2931 The
ganglia are located in four groups but are primarily
concentrated in the IAS and posterior atrial wall
with some groups located near the specialized nodal
areas.1-2-29-31 The percentage of CAT activity in
these regions attributable to preganglionic fibers has
been estimated to be approximately 30% of the total
activity.22
A nonuniform distribution of G, and G4 AChE
was observed in this study of rat heart. This finding
agrees with that of Skau and Brimijoin,32 who
reported that the left atria and the ventricles had
different proportions of the individual globular forms
than the right atria. In our study, the RAA, nodal
regions, and the IAS had the highest ratios of G4 to
Gi AChE. There are two possible explanations for
the higher G^Gi ratios in certain heart regions.
First, the regions with the highest G^Gi ratios
contain parasympathetic ganglia and associated
preganglionic fibers,1-29 which could account for the
increased proportions of GA AChE since this is the
predominant AChE form observed in nervous
tissue.- Other evidence for this hypothesis is that
we found that the vagus nerve, containing preganglionic cholinergic fibers, contains predominately
G4 AChE. The second possibility is that specialized
pacemaker muscle cells and/or conducting cardiomyocytes have different pools of AChE molecular
forms. It is impossible to rule out either one of these
hypotheses based on the present data because parasympathetic ganglia and preganglionic fibers could
be in all of our samples exhibiting high G^/G] ratios,
and secondly, because our dissection of the interatrial and interventricular regions may not be precise enough to exclude the possibility that the IAS
contains a portion of the A-V node.
It is interesting that a large pool of AChE may be
associated with cholinergic nerve fibers in heart. A
large pool of presynaptic AChE is also present in
the central nervous system34 and may allow for ACh
hydrolysis and choline formation at a location close
to the choline reuptake machinery.35 In autonomic
ganglia, the rate of ACh synthesis may be more
dependent on choline generated by ACh hydrolysis
than is the case in the myocardium,2 and this finding
could be the reason for the high amount of AChE
associated with the preganglionic cardiac nerve
fibers. Nodal areas may also be more dependent on
locally generated choline since ACh turnover may
be quicker in these regions as compared with the
myocardium.The asymmetric form of AChE (A u AChE) was
found in our study to have a wide distribution in rat
heart representing between 7-10% of the total activity in all regions examined. Since the proportional
activity due to A12 AChE was similar in ah1 regions,
the specific activity of this asymmetric form varied
with the amount of total AChE activity in each
region. A12 AChE was also found to contribute 67% of the activity of the AChE pool of the vagus
Nyquist-Battie and Trans-Saltzmann Acetykholinesterase in Heart
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nerve in agreement with the work of Skau and
Brimijoin.32 Since there is a uniform distribution of
A12 AChE, it would appear that this form is associated with both cardiac muscle and nerves.
It is interesting that cardiac muscle, like skeletal
muscle, contains both asymmetric and globular
forms of AChE,5-14 since the cholinergic neuroeffector junctions in the two types of muscle differ in
many aspects. Skeletal muscle junctions are welldefined anatomically while the cardiac cholinergic
junctions of the myocardium are not focal and
consist of varicosities that lie at various distances
from the muscle cells they innervate.2>26>36 In contrast to skeletal muscle, cardiac muscle cells are
uniformly sensitive to ACh.2-36 Additionally, the
time course of ACh clearance is much slower in
cardiac muscle.2-37 However, the proportions of the
various AChE forms do differ between cardiac and
skeletal muscle. In this regard, A12 AChE is a bigger
component of the skeletal muscle pool5-14 than of
the cardiac muscle pool.
The present study demonstrates that besides the
increased G4 AChE content in the areas of the heart
containing preganglionic fibers, parasympathetic ganglia, and nodal tissue, there appears to be a uniform
pool of AChE molecular forms in atrial and ventricular myocardia whose overall activity varies with
the amount of cholinergic innervation. Further work
needs to be done to determine the composition of
the extracellular pool of AChE in heart and to
determine the contributions of muscle and nerve
fibers to this pool.
Acknowledgment
We wish to thank Cynthia L. Pennington for
manuscript preparation.
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KEY WORDS
• heart
acetylcholinesterase • rat
parasympathetic nervous system
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Regional distribution of the molecular forms of acetylcholinesterase in adult rat heart.
C Nyquist-Battie and K Trans-Saltzmann
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 1989;65:55-62
doi: 10.1161/01.RES.65.1.55
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