Download Acetylcholine and Electrolyte Metabolism in the Various Chambers

Acetylcholine and Electrolyte Metabolism in the Various
Chambers of the Frog and Turtle Heart
By
PAUL MAZEL, M.S.,
AND AVILLIAM C. HOLLAND,
M.D.
Evidence has accumulated suggesting- ncetylcholine may be involved in initiation of the
heart bent. Acetyleholine and electrolyte metabolism in the various chambers of the
heart have been studied. The area of highest intrinsic rhythm (sinus) contained greater
amounts of acetylcholine equivalents, true cholinesterase, choline acetylase and sodium;
all of these decreasing in amount in areas of lower intrinsic rhythm. It is concluded
that intrinsic rhythm may be directly correlated with overall acetylcholine metabolism
and sodium content and inversely related to overall energy metabolism and potassium
concentration.
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O
A^ER the past two decades evidence has
accumulated which suggests that acetylcholine plays a role in the initiation of the
heart beat. As early as 1937, Sachs 1 found
that small doses of acetylcholine exerted a
stimulant effect on rabbit atria and AVelsh2
in 1938 reported that small amounts of acetylcholine increased the rate of the crab heart.
Later Abdon and Hammarskjold 3 ' 4 reported
on an acetylcholine precursor which yielded
free acetylcholine upon heating or acidification. The mechanical activity of the heart
appeared to vary directly with the amount of
precursor present. The findings have been
extended and confirmed by Burn, SpadoLini
and coworkers.5' ° I t has been suggested by
the latter group of investigators that acetylcholine induces the automatic rhythm and
regulates the rate and force of beat.
Receutly, it has been postulated by Holland 7 that acetylcholiue might act by virtue
of its ability to increase membrane permeability to sodium aud potassium ions. Thus
the intrinsic metabolism of aeetylcholine iu
pacemaker tissue may be of such a nature
that periodic chauges in permeability occur
which give rise to impulses at regular intervals. If the above hypothesis be true then
one would expect that the region of the heart
with the higher intrinsic rhythm would exhibit a greater overall metabolism of acetylcholine.
There have been scattered observations on
acetylcholine metabolism in the various regions of the heart. In general, these studies
were incomplete in that most of them have
been confined to atria and ventricles. In
order to pursue the matter further we have
made a study of the acetylcholine and electrolyte metabolism in the several chambers
of the frog and turtle heart.
METHODS
The turtle (Psuudemys scripta) and the bull
frog (Rana eastesbiana) were used in all experiments. In these studies pooled sinuses, ntriu and
ventricles were used. Therefore, in a number of
cases, only mean values are presented and no
statistical analysis of the data was undertaken.
The aeetylcholine content of the various chambers
was determined by quickly excising the tissue,
weighing and homogenizing in ice cold 10 per
cent trichloroacetic acid (TCA) 3 to 4 ml./Gni.
tissue. After centrifugation, TCA was removed
by extraction with ether and the solution then
freed of ether by aeration. Aliquots of the extract
were neutralized with a few drops of saturated
sodium bicarbonate solution just prior to assay
on the eserinized (1.0 X 10~5) frog rectus abdominis muscle. The acetylcholine equivalent was
determined using known amounts of acetylcholine
bromide as standard. Precautions of Feldberg
and Hebb8 were observed and the extract again
tested after alkalinization with 1.0 X XaOH and
heating.
Choline acetylase activity was determined by
a modification of the method described by Hebb
and AVaites.8 Acetone dried powders were pre-
From the Department of Pharmacology, Vanderbilt University School of Medicine, Xnshville, Tenn.
Supported by U. S. Public Health Service grant
H-1679(c) and the American Heart Association.
Keeeired for publication April 9, 1958.
684
Circulation. Research, Volume VI. November 1BSS
ACETYLCHOLIXE AND ELECTROLYTE METABOLISM
685
TABLE 1.—Acetylcholine Metabolism of the Sinus, Atria and Ventricle
Average of 5 Observations
Turtle tissue
ACh equlv.
/iK./Gm.
(Wet weieht)
Sinus venosus
5.0
(3.15 - 8.30)
Cholinesterase activity
p\. COi/Gro./hr.
ACh
154
Ventricle
0.99
(.51 • 2.29)
81
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#
Pabgt Laboratories, Milwaukee, Wisconsin.
280
(263 - 291)
52.S
188
(126 - 316)
36.0
104
(54.0 - 160.0)
that of the atria while the latter is, in turn,
above that of the ventricle.
The quantity of acetylcholiue (ACh), cholinesterase and choline acetylase activities in
the various chambers of the frog and turtle
heart are summarized in tables 1 and 2. It
should be noted that the ACh equivalent in
the sinus is more than twice that of the atria
and the atria, in turn, greater than the
ventricle. The cholinesterase activities show
a similar distribution pattern as did choline
acetylase activity.
The results on the frog shown in table 2
presented a similar pattern. Acetylcholine
equivalent in the sinus M'as greater than that
of the atrium and the atrium above that of
the ventricle. The frog ventricle displayed
a proportionately greater ACh equivalent
than did the turtle ventricle.
Table 3 summarizes the studies on cation
distribution in the turtle heart. It should be
noted that the sodium content of the sinus
is much greater than the atria and the atria,
TABLE 2.—Acetylcholine Content and Cholinenteraxe
Activity of the Sinus, Atria and Ventricle of the Frog
Heart. Average of 5 Observations
Froff tissue
RESULTS
The inherent rhythmicity of various heart
areas has been studied for many years. It
is now generally accepted that the rate of
intrinsic beat of the sinus is greater than
MeCh
193
2.0
(1.53 • 3.0)
Heart.
Choline acetylase
H%. ACh equiv.
formed/Gin, powder/hr.
480
Atria
pared by grinding the tissue in 100 volumes of
ice cold acetone (—10 C.) in a Waring Blendor.
This solution was then filtered through a Buehner
funnel and the powder dried in air several min.
and stored in vaeuo until ready for use. The
powder was then weighed and an extract prepared
by grinding in cysteine-saline (3 ing. Z-cysteine/
ml.). The suspension was centrifuged and the
supernatant diluted to contain approximately 50
to 250 ing. powder/ml. Coenzyme A was obtained
commercially* and prepared so each 0.1 ml. contained 70 units. The incubation mixture was the
same as described by Hebb and Waites" except
for the frozen liver enzyme. The time of incubation WILS 1 hour at 37 C. The amount of acetylcholine formed was then measured on the frog
rectus as previously described.
Cholinesterase activity of pooled sinuses, atria
and ventricular slices was determined manometrically using Ringer-Bicarbonate buffer at 37 C.
in an atmosphere of carbon dioxide and nitrogen.
Acetylcholine bromide (ACh) and aeetyl-B-methy]
choline (MeCh) were used as substrates in final
concentration of 5 X 10~:s M.
Sodium and potassium content was determined
by analyzing TCA extract of the appropriate
tissue by flame speetrophotometry. Magnesium was
determined by the Titan Yellow method of Orange
and Rhein10 using a Beckman Model B spectrophotometer. Calcium was determined by a colorimeter adaptation of the ammonium-purpurate
titration described by Mendelsohn11 using a KlettSummerson colorimeter with blue filter. The
indicator was stabilized by dissolving it in a wateralcohol mixture as suggested by Williams.12
of the Turtle
ACh equiv.
(/ij./Gm,,
wet weight)
Cholinesterase
(/il. COi/Gm./hr.)
ACh
MeCh
Sinus venosus
5.0
(4.78 - 5.34)
794
322.5
Atria
3.1
(2.16-4.01)
280
80.0
Ventricle
2.9
(2.33-3.30)
132
50.0
686
TABLE 3.—Cation Distribution in the Various Chambers of the Turtle Heart, Determinations Made on
Trichloroacetic
Acid Extract of 6 Pooled
Sinuses,
Atria and Ventricles
Turtle tissue
Na+
Sinus venosus 129.5
Atria
64.00
Ventricle
49.8
56.8
57.8
66.0
2.29
1.11
0.75
3.5
3.5
1.4
2.3
3.2
5.0
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TABLE 4.—Cation Distribution in the Various Chambers of the Frog Heart, Determinations Made on Trichloroacetic Acid Extract of 6 Pooled Sinmes, Atria
and
Ventricles
Sinus venosus
Atria
Ventricle
Na+
3 24
87
49.7
"I
Cations, (mM/Kj., tissue)
K+
Na+/K+ Ca++ Mg+
in turn, greater than the ventricles. The
reverse is true with potassium; potassium
being greatest in the ventricle but approximately the same in the atria and sinus. The
sodium-potassium ratio shows a progressive
decline from sinus to atria to ventricle. Similar sodium aud potassium distributions were
observed in the frog heart (table 4).
The high concentration of Na in the sinus
could be the result of a large extracellular
space in this tissue. This seems unlikely in
view of the data presented in table 5. In
these experiments, sinuses and atria were incubated for one hour in an Na deficient medium (28 mMolar). Isotonicity was maintained by the addition of sucrose. Even
though incubating the tissues in a low Na
medium reduces the Na content in both
chambers, the ratio of Na concentration in
the sinus to that in the atria remains essentially the same. (Compare data, in table 5
with those in table 3.)
Calcium was found to be identical in
amount in the sinus and atria with far less
in the ventricle. With respect to magnesium,
the content was similar in the sinus and atria
but greater in the ventricle.
Figure 1 is a summary of some of the data.
Here acetylcholine equivalents, eholinesterase
Frog tissue
HOLLAND
MAZFJJ,
Cations
K+
44.5
42.2
68.2
(mM/Kg.)
Na+/K*
2.79
1.97
0.73
O
IC
20
1.8
1.8
2.0
RHYT™
d'O
50
SO
BEATS/VIUMJTE
FIG. 1. Summary of data. Intrinsic rhythm of
turtle sinus = 46; atria = 29; ventricle = 13.
These are mean values obtained from observations
on 3 different preparations.
and choline acetylase activities and Na/K
ratios are plotted against intrinsic rhythm
of the various chambers. It should be noted
that the tissue with a higher inherent rhythm
(sinus) contains a greater amount of ACh
equivalents, eholinesterase and choline acetylase activity and higher sodium-potassium
ratio; all of these factors decreasing in
amount in those areas of lowered inherent
rhythm (atria and ventricle).
DISCUSSION
On more than one occasion, attempts have
been made to correlate intrinsic rhythm of
the various chambers of the heart with metabolic patterns in these separate regions. For
example, glycogen is said to be highest in
pacemaker tissue.18 On the other hand, an
inverse correlation has been found for succinic dehydrogenase,14 oxygen consumption,15
and content of adenosine and its derivatives.18
TABLE 5.—The Effect of Na Deficient Medium on
the Na and K Content of Sinuses and Atria of the
Turtle Heart. Temperature 27 C. Na Concentration
in Medium: 28m Molar. Duration of Incubation 1
Hour. Medium Changed Every 10 Minutes
Cations
Ca«
30
INTTHN9C
Tissue experiments
Sin us
Atria
8
8
(Mm/Kg. ,
Na
68.6 ± 12
36.4 ± 8
wet tissue)
Ni
K
56
63.6
•+2
7
9
1..22
0..57
AC'ETYU'HOLIXE AXD ELECTROLYTE METABOLISM
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In fact, the work load of the several chambers
bears a direct correlation with overall energy
metabolism. In the present study evidence
is presented which shows a direct relation
between the rate of intrinsic rhythm and
aeetyleholine metabolism. On the other hand,
these investigations may well represent a
comparison of the distribution of nervous
elements in these regions of the heart. This
is reasonable in light of the known role of
acetyleholine in nervous activity. However,
it has been shown histochemically and enzymatically18 that pacemaker and Purkinje
tissue contain large amounts of acetylcholinesterase. In addition, acetyleholine is not
always associated with nervous tissue as
evident by the large amounts found in the
placenta17 and spleen. Burn" has recently
reported the presence of the acetylcholinecholinesterase system in the gill plates of
the mussel Mytilus edulis, a nerveless structure. Girvin and Stevenson18 have described
a choline acetylating system in Lactobacillus
plant arum.
The presence of large amounts of sodium
in pacemaker tissue is very interesting. Similar findings have been described by Davies19
et al. for the ox heart. This could be the
result of the high intrinsic metabolism of
acetyleholine in the sinus venosus as it is
known that acetylcholiue will release potassium from and cause an uptake of sodium
by myoeardial tissue.7 However, the full
significance of these findings requires further
study.
Although the evidence at present is curcumstantial in nature, it is quite possible
that synthesis and destruction of acetyleholine with its known effects on cell permeability may play a fundamental role in the genesis
of the heart beat.
SUMMARY
Acetyleholine metabolism, including cholinesterase and choline acetylase, in the various
chambers of the frog and turtle heart have
been studied. Cation distribution (Xa+, K+,
Ca<+, Mg'*) have been included. In both
species the ACh equivalents (Gm/wet wt.)
6S7
were as follows: turtle, sinus 5.0, atria 2.0,
ventricle 0.99; frog, 5.0, 3.1, 2.9. True cholinesterase values (/il./CO2/Gm./hr.) were:
turtle, 480, 154, 81; frog, 794, 280, 132, respectively. Choline acetylase activities (^.g./
ACh/formed/Gm. powder/hr.) in the turtle
were: 280, 188, and 31.8. Sodium content
for both species was highest in the sinus, less
in the atria and least in the ventricle. The
reverse was true for potassium. Calcium and
magnesium contents of both sinus and atria
were approximately the same. It is concluded that intrinsic rhythmicity of the various chambers may be directly correlated with
over-all ACh metabolism and sodium content,
and inversely related to over-all energy metabolism and potassium concentration.
SUMMARIO IN INTERLINGUA
Esseva studiate le metabolismo de acetylcholina—incluse cholinesterase e cholinacetylase—in le varie cameras del corde de ranas
e tortucas. Le distributiones cationic (Xa+, K*,
CaT', Mg*+) esseva includite in le studio. Le
valores de acetylcholina in sinus, atrios, e
ventriculo—exprimite in /,g per g de peso
humide—esseva 5,0, 2,0, e 0,99, respectivemente. in le caso del tortuca e 5,0, 3,1, e 2,9
in le caso del rana. Le valores correspondente
pro ver cholinesterase—exprimite in fA de
CO2 per g per hora—esseva 480, 154, e 81 in
le caso del tortuca e 794, 280, e 132 in le
caso del rana. Le valores correspondente del
activitate de cholinacetylase—exprimite in /xg
de acetylcholina formate per g de pulvere
per hora—esseva 280, 188, e 31,8 in le caso
del tortuca. In ambe species—tortuco e rana
—le contento de natrium esseva le plus alte
in le sinus e le plus basse in le ventriculo.
Le contrario valeva in le caso de kalium. Le
contento de calcium e de magnesium in sinus
e in le atrios esseva approximativemente le
mesme. Es concludite que le rhythmicitate
intrinsec del varie cameras es forsan directemente correlationate con le metabolismo general de acetylcholina e le contento de natrium
e inversemente con le metabolismo general de
energia e le concentration de kalium.
MAZEL, HOLLAND
688
REFERENCES
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pharmacol. et toxicol. 1: 169, 1945.
4. ABDON, X. O., AND HAMMARSKJOLD, S.
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11. MENDELSOHN, D.: A micromethod for the
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Acetyicholine and Electrolyte Metabolism in the Various Chambers of the Frog and Turtle
Heart
PAUL MAZEL and WILLIAM C. HOLLAND
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Circ Res. 1958;6:684-688
doi: 10.1161/01.RES.6.6.684
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