Electrolytes and Arterial Muscle Contractility
By W. ALAN DODD, AND EDWIN E. DANIEL, P H . D .
With the technical assistance of Kathleen Robinson
HAT changes in ionic gradients affect
contractility and excitability has been
shown in skeletal muscle,1'4 cardiac muscle5' °
and nerve.7"10 The reduction of external sodium causes reduction and eventual loss of excitability in nerve and skeletal muscle, supposedly because depolarization is due to the
entry of sodium into the cell. The precise
effect of alterations in external sodium on contractility of muscle is not known. Contraction
of skeletal muscle and cardiac muscle is associated with potassium loss. Increasing the
external potassium or decreasing internal
potassium tends to cause contracture and a decrease in excitability in cardiac and skeletal
muscle.11"12
There are only a few studies of arterial muscle contractility and ionic gradients. Eecent
studies by Leonard13 have shown that potassium-free solutions enhance contractility induced by electrical stimulation and inhibit relaxation resulting eventually in contracture.
This change in contractility was postulated
to be the result of lowered intracelluar potassium. Bohr et al.14 have obtained somewhat
different results finding a marked and progressive reduction in contractility (to epinephrine) in potassium-free solution. These
workers suggested the increase of the Ki/Ko
ratio increased the threshold for response.
The role of the sodium ion has been investigated by Bohr et al.14 A decrease of sodium
(to 85 mEq./L.) produced an increased response to epinephrine; and an increase of
sodium (from normal of 115 mEq./L. to 155
mEq./L), a decrease in response. The purpose
of this series of experiments is first, to study
arterial contractility (not excitability) in al-
tered ionic gradients (varying sodium and
potassium) ; second, by tissue analysis to
demonstrate any relation between tissue electrolytes and manifest response; and third, to
attempt to further elucidate the electrolyte
distribution in arterial muscle.
T
Methods
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Tissue Preparation
Male rabbits, 1.5 to 2 Kg., were used. The animals were killed by a blow at the foramen magnum
and the entire thoracic aorta from the arch to
the posterior attachment of the diaphragm rapidly
removed. Control tissues were taken immediately,
blotted free of blood and weighed. Spiral strips
of thoracic aorta were prepared,15 keeping the
tissues in oxygenated Krebs-Binger medium at
35 C. Strips 2.5 X 0.2 X 0.05 cm. were set up
in a bath to record tension changes.
Tension Recordings
The arterial muscle strip was suspended in a
muscle bath at a constant temperature under a
tension of 1 Gm. An RCA No. 5734 transducer
was used to convert mechanical tension changes
into electrical recording on the Sanborn Recorder
Model No. 60-1300-B.
Tissue Equilibrium
The strips were equilibrated for 2 hours at
35 C, in buffered Krebs-Ringer (pH 7.4) oxygenated with 5 per cent dioxide in oxygen. This
equilibration period allowed for stretching and
increase in sensitivity as noted by Furehgott.18
If the applied tension diminished during this
period, it was restored by stretching the muscle
strip.
Drugs and Stimulation
The drugs used for stimulation were 1-epinephrine bitartrate, histamine acid phosphate, acetylcholine bromide and pitressin. They were diluted
with distilled water in such concentrations that a
constant volume of 0.2 ml. was added to a bath
of 35 ml. The concentrations, expressed in terms
of the salt, were those which would produce maximal contractions (as determined by preliminary
experiments) : epinephrine 10~5 Gm./ml. bath solution, histamine 10"4 Gm./ml. bath solution, acetylcholine 10"3 Gm./ml. bath solution and pitressin
6 X 10~2 units/ml, bath solution.
From the Department of Pharmacology, University
of British Columbia, Vancouver, B. C.
Supported by the Life Insurance Medical Research
Fund.
Beceivod for publication November 30, 1959.
Circulation Research, Volume VIII, March I960
451
DODD, DANIEL
452
Table 1
Relationship of Drugs to Tension and Maximum Bate of Contraction in Krebs-Binger
Max.1
Gm.
2.1
2.6
2.8
6.0
3.3
3.3
Epinephrine
Katet
Gm./sec.
0.05
0.07
0.09
0.11
.08
. 0.06
rate
100
100
100
3 00
100
100
100
100
92
36
100
100
Hiistamine
%
•%
t. max.
rate
81
95
86
57
60
58
100
88
100
100
72
94
Acetylcholine
%
%
t. max.
rate
71
58
57
50
46
52
Pitressin
%
%
t. max.
rate
44
65
38
27
43
52
0
0
0
0
0
0
0
0
0
0
0
0
* T-max. = maximum tension developed in grams,
t rate = maximum rate of tension increase in Gm./sec.
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All drugs were left in contact with the tissues
for 5 minutes, then washed out thoroughly. One
ml. of a 1 per cent sodium nitrate solution was
used sometimes to facilitate relaxation of aorta
strips following a contraction. A period of 15
minutes was allowed from the time the strip had
reached basal tension to the next stimulation.
Control responses were run on each tissue, using
epinephrine, histamine, acetylcholine and. pitressin
before altering the external medium.
Extracellular Electrolyte Changes
Changes in bathing media were accomplished
by a complete washing of the chamber and a 15minute period of equilibration to ensure complete
extracellular exchange before stimulation was begun. The increase in potassium content was
achieved by adding potassium chloride as a solid
resulting in final concentrations of 10 mEq./L.
and 20 mEq./L.
Tissue Analysis
Tissues for analysis, taken at various phases
of altered response, were blotted dry and weighed.
Since the test strip was an insufficient amount for
analysis, a. similar strip was subjected to the
same experimental procedure, with the exception
of stretching.
The analytical methods used are described by
Daniel.18 Fat extraction by the method of Lowry
and Hastings was used.17 Microelectrometric titration for chloride was employed.18
Results
Drug Stimulation in Controls
Results showed that epinephrine produced
the largest increase in tension (expressed as
per cent of initial tension,
—ti_:X1100,
where t m is the m a x i m a l tension developed
and ti the initial tension). Histamine produced the next highest increase in tension, and
acetylcholine the least increase in tension. All
drugs produced a sigmoid shaped contraction
and relaxation curve. Pitressin produced no
response. The maximum rates of contraction
and relaxation were 0.11 Gm./sec. and 0.04
Gm./sec, respectively. Occasionally, it was
noted that the muscle would begin to relax in
the presence of acetylcholine after 2 to 3
minutes of exposure. Relaxation after stimulation by epinephrine often did not begin for
5 to 10 minutes after removal of the drug, and
took up to 60 minutes for completion. The
addition of 1 per cent sodium nitrite increased
the rate of relaxation, but relaxation still followed a sigmoid curve (table 1).
Variation in External Sodium
Sodium Chloride Replaced by Sucrose
There was often an initial increase in basal
tension following immersion* of the strip in y*
sucrose-Krebs or in full sucrose-Krebs media,
up to 1.5 Gm. at a rate of 0.01 to 0.02 Gm./sec.
This increment of tension disappeared in 5 to
10 minutes. Responses to all drugs were decreased in y sucrose-Krebs and still further
in full sucrose-Krebs media, but this decrease
in response occurred gradually and contractions still could be produced up to 2 hours
after complete removal of the external sodium
(fig. 1). No potentiation of response was
noted. The time taken to develop maximal
tension and relaxation increased in the sodium-poor and sodium-free solutions. The rate
of tension increase, as well as the maximum
tension developed, were less than in the controls (table 2). Diminution of the response
to drugs in media with reduced sodium was
Circulation Research, Volume VIII, March 19GO
ARTERIAL MUSCLE CONTRACTILITY
453
100 _
80 _
•
:
0/
0/
RESPONSE 6 0 _
X
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40 .
X
A
20 _
'
X
A
o
X
'
1
1
^
^
|
J
2
3
TIME ( Hou.cs)
Figure 1
Response of aortic strips in sodium-free sucrose-Krebs medium.
% response — tm — ti X 100 in Na-free medium,
tm - ti X 100
Control
ti
ivhere tm = maximum tension developed
ti = initial tension
Symbols
X = acetylcholine
o = epinephrine
induced responses.
/ \ = histamine
proportionately the same, irrespective of
whether epinephrine, histamine or acetylcholine was used. Re-immersion in Krebs-Rmger
restored responses to control levels in about
60 minutes.
Sodium Replaced by Choline Chloride
Equilibration (15 minutes) in sodium-free
choline chloride Krebs medium produced a
Circulation Research, Volume VIII, March 1960
variable change in basal tension; frequently
a decrease in tension was noted. As with the
sucrose medium, a decrease in the druginduced tension increment was the only change
noted. The rates of contraction and relaxation
were prolonged (table 2). Responses were obtained for 2 to 2y2 hours, slightly longer
in the choline-Krebs medium than in the
'
DODD, DANIEL
454
Table 2
Alterations in Epinephrine-Induced Contractions
of Babbit Aorta by Variations in the External
Medium
Medium
Basal
tension
Gm.
Max.
rate of
tension
increase
Gm./sec.
Tension
increase
Gm.
0.07
1.0
2.0
Per cent of control value*
0 mEq. K+/L. 100
80
122
10 mEq. K+/L. 100
135
248
139
20 mEq. K+/L. 250
110
0 mEq. Na + /L. 100
67
79
Sucrose
+
91
67
0 mEq. Na /L- 100
Choline
Control
Max.
tension
Gm.
3.0
117
116
160
83
72
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*Figures are an average for all responses up to Vi
hour in the various media.
sucrose-Krebs medium. Epinephrine produced
contraction after the tissue no longer contracted in response to the administration of
acetylcholine and histamine (fig. 2).
Variation in External Potassium
Decreased External Potassium
No significant variation in baseline tension
was observed during equilibration. A slight
decrease in rate of maximal tension increase
in response to various drugs was observed. The
relaxation phase was prolonged, specially during the early phase in which relaxation was
more rapid. Sodium nitrite increased the rate
of relaxation in potassium-free medium, but
not to the same extent as in the controls.
Contractions were initially enhanced and
then progressively decreased; however, even
after i y2 hours in potassium-free medium,
substantial responses were obtained (fig. 3).
When histamine was the test drug, the phase
of enhancement was often absent.
Increased External Potassium
Increase in the potassium concentration to 10
mEq./L. caused no change in basal tension
during equilibration. Contractions induced
by epinephrine and histamine were slightly
greater than the controls, and the maximum
tension developed was about 15 per cent
greater than the control (table 2). Relaxation
was slowed and only 80 to 90 per cent com-
pleted after 1% hours (1 hour was the maximum time for relaxation of control responses).
When the external potassium was increased
to 20 mEq./L., there was no initial increase
in basal tension. Epinephrine and histamine
produced responses in which the rate of maximal tension increase and tension developed
was greater than the control (table 2). Relaxation was progressively less complete (even
using sodium nitrite) until the resting tension
was almost the same as the maximal tension at
the height of contraction.
Tissue Analysis
Krebs-Singer Medium
Strips were immersed in Krebs-Ringer for
4 to 6 hours before being subjected to altered
electrolyte medium. A series was run to establish control values for electrolytes of tissues
undisturbed in Krebs-Ringer medium. There
was a significant gain in sodium (96.9 to
126.4 mEq./Kg.) and chloride from (70.2 to
94.6 mEq./Kg. wet weight). Potassium appeared to decrease (27.1 to 21.1 mEq./Kg. wet
weight) but this was not statistically significant. Tissue water was significantly increased
from 663 to 714 ml./Kg. (table 3). That this
electrolyte gain was not in proportion to their
extracellular concentrations (i.e., the medium)
is shown by the fact that the increase in
sodium space is almost 5 times that of the
chloride space. Calculations indicated that
about 15 mEq./Kg. wet weight of the sodium
taken up in Krebs' solution could not be accounted for by expansion of extracellular
fluid. This probably was chiefly sodium which
entered cells in exchange for potassium. At
least 6 to 10 mEq./Kg. wet weight of potassium may have been exchanged in this way,
but the variability of the potassium data make
precise conclusions impossible.
Sodium-Free Solutions (Sucrose-Krebs Choline-Krebs)
The importance of alterations in tissue electrolytes induced by lowering the external sodium concentration in bringing about the observed changes in contractility was the next
question investigated.
Circulation Research, Volume VIII, March 1960
455
ARTERIAL MUSCLE CONTRACTILITY
IfeO _
ACH - Z EcyuaL Responses
®
140 %
120 .
RESPONSE
X
100 _
80 _
•
60-
A
A
•
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A
40-
"
* "
1
X
A
20-
»(
•
AY
n
X
^>
A
1
1
1
I
2
TIME ( Hours)
3
Figure 2
Response of aortic strips in sodium-free choline-Krebs medium.
% response = tm — ti X 100 in Na-free medium
tm - ti X 100
Control
ti
where tm = maximum tension developed
ti = initial tension
Symbols
X = aeetylcholine
o = epinephrine
induced responses.
/\ = histamine
In sodium-free medium, contractions gradually diminished, as previously described. An
attempt was made to discover any correlation
between alteration in contractility and in
tissue electrolyte concentration. The data from
tissues exposed to media were combined in
table 4, arranged according to the contractility of the tissue at the time of analysis. While
no definite relationship was found between
sodium content and degree of depression of
response, a correlation appeared between contractility and potassium content. Tissues with
decreased contractility had decreased potasCirculation Research, Volume VIII, March 1960
sium concentrations. In tissues with potassium concentrations of about 17.0 mBq./Kg.,
or less, no contractions were produced.
Table 5 correlates tissue electrolytes and
time of exposure to sodium-free media. Several things of interest were noted here. The
chloride concentration decreased to about 17
mEq./Kg. wet weight within the equilibration period of 20 minutes and remained at
about this value for 2% hours. This suggests
that the diffusible chloride (presumably extracellular in location) equilibrates rapidly
and that a substantial quantity (11 to 13
456
300
-
X
EPl. HIST. ACh.
250
X
X
X
200 -
%
RESPONSE
150 X
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100 _
x>
*
X
H
X A X
*
X
50-
A
•
•
X
n
1
1
2
1
1
3
4
5
( flour's)
TIME
Figure 3
Response of aortic strips in potassium-free Krebs
% response = tm — X 100 in Na-free medium,
1
1
i
6
7
8
medium.
ti
tm - ti X 100
Control
ti
where tm = maximum tension developed
ti = initial tension
Symbols
X = acetylcholine "1
o = epinephrine
A = histamine
mEq./Kg. wet weight) is more firmly fixed in
some manner, either intracellularly or in an
extracellular solid phase. Such a large portion
of noiidiffusible chloride will produce a significant error in calculation of extracellular
space, using the chloride space, and also secondarily in the cellular electrotyte concentration and cell water.
There appears to be a rapid loss of sodium
within the first 20 minutes. Subsequently, the
^
induced responses.
I
loss of sodium continues slowly into both
media. After a period of 20 minutes in
sucrose-Krebs medium, about 94 mEq./L. of
sodium and 78 mEq./L. of chloride had been
lost per Kg. wet weight, i.e., in a ratio of 0.83,
which is not too different from the ratio of
sodium to chloride (0.91) in Krebs-Ringer,
but there may have been some loss of sodium
unaccompanied by chloride during this 20
minutes. Similar calculations could not be
Circulation Research, Volume VIII, March I960
ARTERIAL MUSCLE CONTRACTILITY
457
Table 3
7
7
96.9
±11.7
126.4
± 4.5
aa
+ W .
«gS
27.1*
±8.2
21.1
±4.0
3 Si
70.2
3.
bo
94.6
714
327.4
±44
434.7
±3.3
±11.0
±19.3
±4.8
663
±14
59.5
±15.9
68.7
±12.0
Nasp
*s*
3
Clsp
la
s
Na+
mEq.
dry w
s
'°£
/Kg.
Electrolyte Contents of Control Tissue
633
650
±42.3
±48
760
931
±22
±32
" i n vivo"t
control
"Krebs"
control
*This value is slightly lower than that of other control groups (+35 mEq./Kg.).
t Tissues taken directly from the animal.
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made for loss of sodium into eholine-Krebs
solution, but the fact that more sodium was
lost in this medium suggests that choline ion
but not sucrose may exchange for bound or
cellular sodium. Interpretations, however,
must be made with caution owing to lack of
large numbers of tissues and to the degree of
individual variation. The mechanism of the
apparently greater loss of sodium into
choline-Krebs medium than sucrose-Krebs
medium cannot be decided from these data,
though the possibility has been suggested19
that the choliue ion exchanges for bound or
cellular sodium. At the end of 2y2 hours in
sodium-free solution, about 19.8 mEq./Kg.
wet weight sodium remained in the tissue
(average sucrose- and choline-Krebs). Obviously a substantial portion of aorta sodium
diffuses only very slowly out of the tissue.
This slow loss of sodium suggests that there
is a form of sodium (cellular) which is not
freely diffusible with the extracellular spaces.
Whether or not there is a more firmly fixed
fraction, as with chloride, could not be determined from these data.
Potassium concentration diminished progressively (reaching 17.7 mEq./Kg. wet
weight average sucrose and choline) after 140
minutes. No difference in the rate of potassium loss in sucrose and choline solution is
evident. Tissue water did not change significantly.
'otaxxium-Free Solutions
Correlation potassium content and contractility. Analyses were made of electrolyte concentrations in aortal strips which had been
Circulation Research, Volume VIII, March 1S60
exposed to potassium-free solutions for varying periods.
Evidence from table 6 shows that changes
in tissue electrolytes cannot be definitely correlated with the alterations in contractility
found in this medium. It is interesting to note
that substantial losses in potassium to concentrations of 3.5 mEq./Kg. wet weight are still
compatible with contractility. Such findings
raise doubts as to whether or not the loss of
contractility in Na-free solutions can be attributed solely to loss of potassium, since the
concentrations of potassium averaged about 17
mEq./Kg. wet weight in those instances.
Electrolytes and Exposure. There was no
apparent potassium loss in the 20-minute
equilibration period (21.1 to 20.4 mEq./Kg.
wet weight). A progressive loss continued, so
that after 7% to 8% hours in K-free solutions,
about 3.5 mEq./Kg. wet weight remained.
Even at such concentrations, contractility remained. No significant changes were noted in
the sodium or chloride content or in the total
tissue water (table 7).
Increased External Potassium
Owing to the small numbers of tissues
analyzed after exposure to increases in potassium concentrations of 10 mEq./L. and 20
mEq./L., no statistical data are included at
this time. Preliminary results indicated however, that the electrolyte composition of
aorta exposed to increased external potassium
up to 2 hours is not essentially different from
the composition of strips exposed to KrebsRinger for 4 to 6 hours. No differences were
noted between strips exposed to 10 and 20
mEq./L.
DODD, DANIEL
458
Table 4
Correlation of Tissue Electrolytes and Response in
Sodium-Free Medium''
Per cent
of control
response
No. of
animals
Na+
K>
mEq./Kg. mEq./Kg.
w. wt.
w. wt.
HsO
ml./Kg.
0
(No response)
8
23.3
±4.9
5-40
7
15.6
±4.1
47-.100
30
27.4
±7.9
17.0
±4.1
23.0
±4.2
27.5
±8.2
659
±10
652
±17
658
±13
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*Combination of data from tissues exposed to sucrose-Krebs and choline-Krebs.
Discussion
Distribution of Tissue Electrolytes
The sodium content of aorta (96.9 to 109.2
mEq./Kg. wet weight) is considerably higher
than that of skeletal muscle (15 to 35 mEq./
Kg.).20 The potassium content of aorta (30
to 45 mEq./Kg. wet weight) is less than skeletal muscle potassium content (110 to 114
mEq./Kg.) Aorta chloride (75 mEq./Kg.)
is greater than that of skeletal muscle (10 to
20 mEq./Kg.). The total water of the aorta
(640 to 670 mEq./Kg.) is less than that of
skeletal muscle (750 to 775 mEq./Kg.). The
sum of cations (Na + K) about 140 mEq./Kg.
was about the same in vascular muscle as in
skeletal muscle and less than that of cardiac
muscle, taenia coli, and uterus.20
Chloride Distribution and ECFV
It is generally considered that so much of
the tissue chloride is located extracellularly,
and in a form which is free to equilibrate with
plasma water, that the chloride space is not
significantly different from the ECFV. Evidence from these experiments indicates that
there is a fraction of freely diffusible chloride
which is largely lost within 20 minutes and a
fraction of more tightly bound chloride.
Inclusion of this bound fraction of chloride
in calculations of the chloride space would
yield an erroneously high value for the ECFV,
assuming that the ECFV has equilibrated in
20 minutes, as suggested by Daniel.19 Calcu-
lations, using corrected values for diffusible
chloride, yield an ECFV of 483 ml./Kg. This
is a more reasonable value than 633 ml./Kg.,
since the total tissue water was found to be
about 640 to 660 ml./Kg.
Intracellular potassium calculated, using
the corrected ECFV of 483 ml./Kg., is about
192 mEq./Kg. cell water, which is not an absurd value. Intracellular sodium calculated
in the same manner is 158 mEq./ml. cell water.
The total cation (Na + K) is about 350 mEq./
ml. cell water, which is similar to values of
other smooth muscle.20 It is likely that a substantial portion of tissue univalent cation is
in bound form in view of such values and of
the evidence that animal cells are in osmotic
equilibrium.20
Sodium Distribution
It is not yet possible to describe the distribution of tissue sodium with certainty. However, some indication as to the location of
aorta sodium has been obtained. One fraction,
lost rapidly with chloride, is probably extracellular (and freely diffusible). The remaining sodium is lost more slowly, or not at all.
Some sample calculations will help to elucidate its possible location. The control tissue
sodium was 96.9 mEq./Kg., of which .483 ml./
Kg. X 148 mEq./L. (concentration of Na in
rabbit plasma) or 71.5 mEq./Kg. was in the
ECFV. If the amount of tissue sodium, (17.1
mEq./Kg.) which remains following 2*4> hours
exposure to choline-Krebs medium is assumed
to be bound, then the 8.8 mEq./Kg. which is
not bound, or accounted for in the ECFV, may
be intracellular. Tissues exposed to KrebsRinger medium for 4 to 6 hours had an average total sodium content of 126.4 mEq./Kg.,
of which 620 ml./Kg. X 138.6 mEq./L., or
85.9 mEq., was in the ECFV. Assuming no
change in the amount of bound sodium, approximately 17.4 mEq./Kg. of sodium could
not be accounted for in the ECFV, in exchange for potassium or in a bound form.
This represents an increase of 9.1 mEq./Kg.
of intracellular sodium which is taken up
either in exchange for another cation (choline)
or with an anion.
Circulation Research, Volume VIII, March 1960
ARTERIAL MUSCLE CONTRACTILITY
450
Table 5
Correlation of Electrolytes and Exposure Time in Sodium-Free Media
Time in
sodium-free
20 minutes*
45- 80 minutes
120-150 minutes
Na* mEq ./Kg. w. wt.
Sucrose
Choline
7t
32.9
±18.0
5
36.4
±11.9
5
22.5
± 6.6
5
29.1
±18.0
5
19.4
± 5.8
5
17.1
± 6.2
K+ mEq./Kg. w. wt.
Sucrose
Choline
Cl" mEq./Kg. w. wt.
Sucrose
Choline
±U
686
±12
± 2.S
641
±15
± 6
108.1
± 3.8
±3
31.7
± 6.6
25.3
± 3.8
± 0.1
101.0
± 3.8
30.5
± 0.5
20.5
±10.8
12.5
± 10.8
18.8
± 2.9
16.5
± 5.4
11.5
± 3.8
17.1
H=O ml./Kg.
Choline
Sucrose
644
88.3
636
657
681
±10
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'Tissues were equilibrated for 15 minutes, then stimulated; hence 20 minutes elapsed before
tissues were removed from the medium,
t Number of animals.
The increase in ECFV (calculated by subtracting the bound chloride) may not be
strictly accurate in view of the possibility that
chloride enters intact or damaged cells. Even
so, it seems probable that there is a considerable portion of tissue sodium existing intracelhilarly and in an exchangeable or an unbound
form.
Sodium Gradient and Contractility
It has been postulated that the sodium
gradient (Nao/Nai) of the vascular muscle
cell is a basic determinant of vascular muscle
tone.-1 Evidence in support of this view is offered from the observations with intestinal
muscle that acute reduction of sodium in the
medium results immediately in an increase in
tension followed by a relaxation to basal tension as the tissue equilibrates; and that reponse to drugs was increased following a 3minute equilibration period in sodium-poor
media, and decreased following exposure to
high external sodium concentrations.
The immediate increase in basal tension
upon exposure to Na-free media was often observed in these experiments. To explain this
phenomenon, using the sodium gradient, it
has to be assumed that the extracellular phase
equilibrates with the bathing medium within
seconds—something difficult to accept in view
of the compact histologic structure of the
aorta and data from other smooth muscle.20
If the increase in response to drug stimulaCirculation Research, Volume VIII. March J9S0
tion observed in rat colon,21 and if a similar
increase in response seen in rabbit aorta following a 15-minute equilibration in sodiumpoor medium,14 are due to an altered Nao/Nat
gradient, the same effect was to be expected
when tissues are exposed to sodium-free media
for 15 minutes. This was not the case in these
experiments; instead, a progressive decrease
in response was obtained on exposure to Na• free media.
Previous reference has been made to the
fact that a portion of the chloride is bound
and a re-calculation of the chloride space
yielded a value of 483 ml ./Kg. The amount
of sodium in the ECFV after 4 to 5 hours exposure to Krebs-Ringer medium (allowing for
the increase in chloride space) Mras approximately 85.9 mEq./Kg. The total measured
tissue sodium after exposure to Krebs-Ringer
medium for the same period of time was 126.4
mEq./Kg. Therefore, when all the extracellular diffusible sodium is lost—i.e., when the
ECFV has equilibrated, the tissue sodium
should be about 40.5 mEq./Kg., which is only
slightly higher than the values seen after 20
minutes of exposure to both of the sodum-free
media. This would indicate that little intracellular sodium has been lost in 20 minutes.
If contractility depended upon the presence
of extracellular sodium to maintain the proper
sodium gradient, then removal of external
sodium might be expected to produce loss of
DODD, DANIEL
460
Table 6
Correlation of Tissue Electrolytes and Response in Potassium-Free Medium
Per oent
response
No. of
animals
0
4
7-50
51-100
4
6
Na+
mEq./Ke.
w. wt.
141.5
±17
108.7
±24
111.9
± 5.1
103-150
286-334
6
3
K+
mEq./Ke.
w. wt.
ci-
mEq./Ke.
w. wt.
95.6
HO
ml./Ke.
Cl
space
Na
space
11.8
± 3.0
17.5
± 4.9
107.0
675
776
741
±15.8
± 6.6
±15
±58
±35
7.2
91.4
± 5.5
,± 4.8
108.8
18.6
86.8
± 5.6
±12.6
±16.4
704
742
988
±12.2
±17
±20
721
740
776
± 8.0
±24.0
±49
703
±10
698
±39
±32
741
131.8
14.0
92.5
710
732
900
±13.8
± 13.8
± 9.0
± 9
±73
±88
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response or paralysis. This was not observed
for a considerable period of time. A decrease
of the sodium gradient in the light of the
present study results in depression, but not
immediate abolition, of contractility.
Potassium Gradient and Contractility
The relationship between the potassium
gradient and threshold for response has been
studied in nerve,8 skeletal muscle,3'4 uterus22
and taenia coli.23 Basically at equilibrium, log
K|/K o C= resting potential = threshold for
response, where Kj is intracellular potassium,
Ko is extracellular potassium and C is a constant.
Bohr14 suggests that a similar relationship
exists between the potassium gradient and
aortic smooth muscle responsiveness to physiologic stimuli such that as external potassium
increases relative to internal potassium, the
response increases. Strictly speaking, these
experiments were not designed to test the
threshold response. However, an increase in
rate of contraction and in tension developed
was observed in media containing potassium
at concentrations of 10 mEq./L and 20 mEq./
L, which, may indirectly tend to support this
hypothesis.
For the relation between potassium gradient
and contractility to be valid, an opposite effect should be observed when extracellular
potassium is lowered. This is only partly confirmed by the recent study. In fact, instead
of a decrease in contractility, potentiation of
response was usually seen after 15 minutes in
potassium-free media. Since histamine did
not often produce such potentiation, but eqinephrine and acetylcholine invariably did, it is
possible that response to different drugs may
be affected in different ways by altering
potassium concentrations and/or gradients.
That the loss of arterial muscle fiber potassium causes an increased response, a slower
rate of relaxation and eventually contracture
has been suggested.13 Tissue exposed to potassium-free Krebs for 7y2 to 8% hours contained about 3.5 mEq./Kg. potassium (or 37.6
mEq./L cell water) and averaged 85 per cent
of control response. On exposure to sodiumfree medium, when contractility was no longer
present, tissue potassium averaged 17.0 mEq./
Kg. wet weight (121.3 mEq./L cell water).
This latter value may not be entriely accurate
when all possible sources of error are considered (loss of cell water, damaged cells,
etc.), but if the corrections could be applied,
the intracellular potassium would probably be
even greater. Thus the loss of fiber potassium
does not necessarily cause an increase in response ; in fact, the level of intracellular potassium, per se, does not seem greatly to influence arterial contractility.
The actual external potassium in potassiumfree medium bathing the tissue is probably
0.1-0.2 mEq./L., or less, because of constant
washings. The gradient Ki/K o is therefore
greater than 180. In sodium-free solutions,
Circulation Research, Volume VIII, March 1960
ARTERIAL MUSCLE CONTRACTILITY
461
Table 7
Correlation of Electrolytes and Time of Exposure in Potassium-Free Medium
Time in K-free med.
20 minutes
No. of
animals
5
Na+
mEq./Kg.
w. wt.
134.0
± 4.9
20 minutes—2 hours
2% hours—6V& hours
7% hours—8% hours
7
4
4
118.6
it 8.5
132.9
±14.6
112.5
± 7.6
K+
mEd./Kg.
w. wt.
ClmEd./Kg.
w. wt.
HJO
ml./Kg.
Cl
space
Na
space
Average
per cent
of control
response
20.4
± 3.8
16.0
92.7
± 6.6
716
734
±52
920
117
±12
±29
91.1
684
751
839
± 8.5
± 5.2
± 8
±37
±67
9.7
93.3
± 14.6
±12.0
710
±14
738
±29
±80
3.5
86.1
± 7.6
± 7.6
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K,/K o (using 121.3/5.79) is about 21. Even
if the K| in this latter case were actually 100
per cent larger, an unlikely possibility, the
gradient would not be larger than that in
potassium-free media. If so, then the eventual
decrease in contractility in potassium-free
media must be caused by other means. In view
of these figures, it seems possible that the potassium gradient may be correlated with contractility in this case. Because of the lack of
a complete correlation between contractility
and the potassium gradient, the roles of other
cations (Ca + Mg) and/or anions which are
imdoubtedly concerned with arterial contractility, are becoming increasingly more important to investigate.
Many studies have been undertaken seeking
a relation between intracellular and extracellular electrolyte abnormalities and hypertension. In chronic hypertension, 24 " 28 the
chemical composition of the rat aorta is altered in such a way that the sodium, potassium
and water content increases. These increases
are thought to be intracellular.
In transient hypertension,21*-*1 the electrolyte shifts are not consistent. Pitressin produced an increase in blood pressure Avith an
accompanying increase in aortal sodium. No
changes were found in potassium content. Hypertensive drugs, such as norepinephrine,
failed to produce any significant alterations
in arterial wall sodium, but a produced decrease in potassium content. Thus it seems
that chronic or "fixed" hypertension is accompanied by an increase in tissue sodium,
Circulation Research, Volume VIII, March 1960
723
± 6.0
743
±42
838
720
94
31
85
± 4.3
potassium and water, while acute or "transient" hypertension is not accompanied by any
consistent electrolyte shift. It might not be
unreasonable, then, to suggest that the transition from early hypertension to fixed hypertension is correlated with a more or less permanent increase in tissue cation.
The relation of increased tissue cations (Na
+ K) to arterial responsiveness in "fixed"
hypertension is still being investigated. In
vivo studies have demonstrated vascular
hyper-responsiveness in hypertensive subjects. 32 ' 3S In vitro studies using arterial
strips from hypertensive rats have failed to
confirm this finding.34' 35 The relation of increased arterial cation content to the mechanism of hypertension in terms of altered vascular contractility is still unknown.
Summary
Arterial contractility in response to various drugs was studied in media designed to
alter ionic gradients of sodium and potassium
across the cell membrane. Tissues were
analyzed to determine the effects of these procedures on tissue electrolytes and to demonstrate any correlation between tissue electrolytes and response.
It was found that the contractile responses
progressively decreased in sodium-free media,
disappearing in 2 to 2y2 hours. A decrease in
external potassium, initially caused a potentiation of response. Thereafter, a decreased
response was manifested, yet contractility remained even after 7 to 8 hours in potassium-
DODD, DANIEL
462
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free medium. Increased external potassium
caused an increase in response.
There was no correlation between the content of sodium and potassium and response in
potassium-free solutions, but in sodium-free
solutions a positive correlation between contractility and potassium content existed.
Chloride appeared to exist in 2 fractions, a
diffusible fraction, and a more tightly bound
fraction. Calculation of the ECFV based on
the bound chloride yielded values which otherwise would have been absurd.
Sodium appeared to be distributed in 3
fractions; diffusible and extracellular, not
diffusible over the duration of our experiments and slowly diffusible and presumably
intracellular, the latter fraction being possibly
capable of cation exchange.
Evidence obtained does not suggest that the
sodium gradient, per se, is responsible for
contractility. The concentration of intracellular potassium does not influence contractility
directly. However, the potassium gradient
may in part determine vascular muscle contractility.
Summario in Interlingua
Le coutructilitiitc lie nuisculo arterial in responsa a
viirie drogas esseva studiate in medios preparate con
le objectivo de alterar le gradientes ionic de natrium
e kaliuin ab mi latere al altere del membrana cellular.
Specimens de tissu esseva nnulysate pro determinar le
efEectos del mentionate manovras super le electrolytos
tissutal e pro demonstrar le existentia possibile de
un correlation inter le electrolytos tissutal e le
responsa.
Esseva constatate que le responsas contractile clecresceva progressivemente in medios libcrc de natrium.
Illos dispareva coinpletemente intra 2 a 2M; boras.
Un reduction del kaliuin externe causava initialmente
un potentiation del responsa. Postea un redueite
responsa esseva manifeste, sed tracias de contnictilitate remaneva presente iticsmo post 7 a 8 lioras in
medios libere de kalium. Augmeiito del kaliuin externe causava un augmento del responsa.
Esseva trovate uulle correlation inter le contento
de natrium e le responsa in solutiones libere de
kalium, sed in solutiones libere de natrium le contractilitate esseva positivemente correlationate eon
le contento de kaliuin.
Chloruro pareva exister in 2 frnctionos, un fraction diffusibile e un plus firmemente ligate fraction.
Le calculation del volumine de tluido extracellular
super le base del ligate chloruro producova valores
plausibile. Super le base do ambe fractiones le
valores haberea essite absurde.
Natrium pareva essor dist.ribuite in tres fractiones:
(1) "Diffusibile e extracellular, (2) non diffusibile in
le spatio de tempore de nostre experiinentos, e (3)
lentemente diffusibile e presumitemente intracellular.
Iste ultimo fraction es possibileiuente capace de excanibio cationic.
Le observationes facito in le curso de iste studios
non suggere que le gradiente de natrium es per se
responsabile pro le pbenoineno del contractilitatc. Le
concentration de kalium intracellular non exerce un
influentia directe super le contractilitate. Tamen, le
gradiente de kalium pote detorminar in parte lo contractilitate de musculo vascular.
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Electrolytes and Arterial Muscle Contractility
W. ALAN DODD and EDWIN E. DANIEL
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Circ Res. 1960;8:451-463
doi: 10.1161/01.RES.8.2.451
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