254
Electron Probe Analysis of Sodium and Other
Elements in Hypertrophied and Sodium-Loaded
Smooth Muscle
James L. Junker, Arthur J. Wasserman, Peter F. Berner, and Andrew P. Somlyo
From the Pennsylvania Muscle Institute and Departments of Physiology and Pathology, University of Pennsylvania, School of Medicine,
Philadelphia, Pennsylvania
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SUMMARY. The composition of normal, hypertrophied, or sodium-loaded rabbit portal anterior
mesenteric vein and of normal and sodium-loaded guinea pig taenia coli smooth muscle was
measured in cryosections with electron probe analysis, and the effects of wash with cold sodiumfree (Lithium) solutions were determined. There was no significant difference in the cytoplasmic,
nuclear, or mitochondrial concentrations of any of the measured elements (sodium, potassium,
chloride, magnesium, calcium, phosphorus, sulfur) between hypertrophied, sham-operated, or
control veins. The cytoplasmic potassium:sodium:chloride ratio in rabbit portal anterior mesenteric
vein was 1:0.26:0.46, and the average sodium concentration (198 mmol/kg dry cytoplasmic
weight) was nearly twice as high as estimated from ion flux measurements. The cytoplasmic
sodium concentration of normal guinea pig taenia coli was 61 mmol/kg dry weight. The existence
of a rapidly exchanging, relatively low affinity, and temperature-insensitive component of
cytoplasmic sodium efflux was indicated by the reduction in cytoplasmic sodium after washout
in cold, sodium-free (lithium or Tris-substituted) solutions. This reduction, by 62% in normal,
71% in sodium-loaded portal anterior mesenteric vein, and 36% in sodium-loaded guinea pig
taenia coli smooth muscle, suggests that the lithium wash method can underestimate cell sodium.
In sodium-loaded guinea pig taenia coli and portal anterior mesenteric vein smooth muscles, the
cytopiasmic sodium analyzed in individual cells showed a bimodal distribution; in cryosections,
the cells having the highest sodium and lowest potassium and phosphorus content also had a
more electronlucent (light cell) appearance. (Ci'rc Res 54: 254-266, 1984)
THE possibility that the monovalent ion content and
permeability of vascular smooth muscle plays a role
in hypertension has been generally recognized
(Jones, 1981; Hypertension Task Force, 1979), and
there is a particularly large body of literature dealing
with the relationship of sodium to hypertension
(Blaustein, 1977; Jones, 1981, 1982; Hypertension
Task Force, 1979; Genest et al., 1977; Folkow, 1982).
However, the quantification of cellular sodium, and,
to a lesser extent, other ions in vascular smooth
muscle, has been difficult, due to the large extracellular space in blood vessels, the possibility of ion
binding, and the very rapid efflux of radioisotopes
(Jones, 1980; Brading, 1981). Electron probe microanalysis (EPMA) of thin cryosections of rapidly
frozen cells permits the direct quantification of the
contents of cellular organelles and of the cytoplasm
(Somlyo et al., 1979; Somlyo et al., 1981; for review,
see Hutchinson and Somlyo, 1981; Hall and Gupta,
1979; Somlyo and Shuman, 1982), and is a powerful
method for obtaining information about the elemental content of hypertensive vascular smooth muscle.
In order to assess the role and significance, if any,
of changes in vascular smooth muscle composition
in the course of idiopathic, hormonally induced or
renal hypertension, it is necessary to know what
effect an elevation in distending pressure itself may
have on vascular smooth muscle composition. To
answer this question, we used portal anterior-mesenteric veins (PAMV) for this initial study, because
of the extensive functional, compositional, and
structural information already available about this
preparation (Somlyo and Somlyo, 1968; Jones, et
al., 1973; Somlyo et al., 1979), the relative ease in
inducing hypertrophy (Johansson, 1976; Berner et
al., 1981), and the possible additional bearing of the
information obtained on the effect of increased (arterial) pressure on veins used for coronary bypass
grafts.
Our results, like those using radioactive tracer
studies of hypertensive vessels (Jones and Miller,
1978; Jones, 1980, 1981, 1982), showed no significant effect of smooth muscle hypertrophy on cytoplasmic Na or K. However, as in our previous electron probe study of normal vascular smooth muscle
(Somlyo et al., 1979), the cellular Na content or
Na:K ratio measured in both control and hypertrophied vessels was higher than that obtained in radioisotope flux studies designed to separate the extracellular from the cellular Na through washing
tissues with ice cold Na+-free (or nonradioactive)
solutions (Friedman, 1974; Friedman et al., 1974;
Junker et al./Sodium in Smooth Muscle
Jones and Miller, 1978; Jones, 1980). Therefore, in
an attempt to reconcile the different values of cellular Na obtained with different methods, we have
also performed EPMA measurements on normal and
on Na+-loaded rabbit PAMV and guinea pig taenia
coli washed in ice cold Na+-free (Li+) solutions.
These results revealed the presence of a low affinity,
temperature-insensitive Na efflux system in smooth
muscle, and raise serious doubt about the reliability
of Li+ washout as a method for accurately measuring
cell Na.
Methods
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Partial ligation of rabbit PAMV or sham operation was
performed as previously described (Johansson, 1976; Berneretal., 1981). On the 14th postoperative day, the rabbits
were killed by cervical dislocation and the PAMV was
removed. Longitudinal strips were trimmed of advenriria
suspended in a solution of oxygenated Krebs saline of the
following composition (ITIM): NaCl, 125.1; KC1, 4.7;
KH2PO,, 1.2; MgSO4, 1.2; dextrose, 5.6; NaHCO3, 18.7;
CaCl2, 1.2, pH 7.4; stretched to 1.7X slack length (or 1.0X
slack length; see Results), and incubated at 37°C for a
minimum of 90 minutes. Four percent bovine serum albumin (BSA) (pH 7.4) was added to the incubation solution at least 15 minutes before freezing. Strips of guinea
pig taenia coli 1 cm long were removed from the cecum,
cleaned of connective tissue, and cut in half longitudinally.
These were stretched from slack length to in vivo length
and mounted and equilibrated in the same manner as the
rabbit PAMV. Freezing in supercooled Freon was performed as previously described (Somlyo et al., 1979; SomIyo et al., 1977). Incubation took place inside an environmental chamber which was mounted above the freezing
solution. The chamber was designed to maintain a constant temperature and high relative humidity to prevent
cooling by evaporation, and ensured that the strip was
exposed to warm, humid air, rather than room air, during
the few seconds between the time the incubation solution
was withdrawn and the freezing solution was shot up to
the strip. Two strips could be incubated and frozen simultaneously. Usually a sham and an experimental strip
were frozen together. The chamber temperature at the
strips was between 35° and 37°C up to the time of
freezing.
"Na+-free Krebs" solutions were prepared with either
Li+ or tris(hydroxymethylamino)methane (Tris): for the
"lithium Krebs" (ITIM): LiCl, 106.4; U2CO3, 18.7. For the
"Tris Krebs': Tris base, 106.4; (Tris)2 CO3/ 18.7 + cone;
HC1 to pH 7.4 at 0°C. K+-free, Na+-loading solution was
prepared with (ITIM): NaCl, 129.8; NaH2PO4, 1.2; and
NaHCO3, 18.7. Since BSA contains some residual sodium,
4% polyvinylpyrrolidone (PVP), mol wt = 40,000, was
added, instead, to the Na+-free solutions, as a cryoprotectant. No cryoprotectant was used for the Na+-loaded
series. The Na+-free solutions were shown to contain less
than 100 /*M Na+ by atomic absorption spectroscopy.
Strips were prepared and incubated in normal Krebs solution as above. The exchange of normal Krebs at 37°C
for ice cold (between 0°C and 5°C) Na+-free Krebs was
performed inside the warm environmental chamber. During the subsequent incubation period (30 minutes, except
where noted), the *Na+-free Krebs' was kept cold in an
ice-water bath, and the temperature of the environmental
255
chamber was lowered to 5°C. The temperature at the
strips was between 3° and 7°C at the time of freezing.
Strips were Na+-loaded in an ice water bath in K^-free
Krebs solution for 3 or 4 hours (see Results) at 2°C, then
frozen in the environmental chamber. Na+-loaded strips
to be Li+ washed were transferred directly to 2°C "Li+Krebs" in the environmental chamber.
Frozen sections (100-200 nm thick) were cut and prepared for EPMA as previously described (Somlyo et al.,
1977) and modified for transverse cryoultramicrotomy
(Karp et al., 1982). Freeze-dried specimens were analyzed
on a Philips electron microscope (EM400) equipped with
a Kevex Si(Li) X-ray detector (Kevex Corp.) and a liquid
N2-cooled specimen holder. The methods used for quantitative EPMA have been published in detail (Shuman et
al., 1976, 1977; Somlyo et al., 1979) and are based on the
linear relationship between the characteristic Xrayxontinuum ratio and elemental concentration (Hall,
1971).
The sources and magnitude of errors induced by instrumental effects and biological variations have also been
discussed in detail (Somlyo et al., 1979; Somlyo et al.,
1981). The measurement of low concentrations of Ca++ in
the presence of high concentrations of K+ can be affected
by instrumental drift in detector calibration. This small
error normally can be corrected (Kitazawa et al., 1983),
but this could not be done in the present study, because
the raw spectra were lost in a computer disk crash. In
addition, for the measurement of low (less than 3 mmol/
kg) dry weight concentration of calcium, the carbon films
have to be analyzed before mounting cryosections, to
ascertain the absence of environmental contamination
with calcium. Since all the carbon films were not checked
in this study and the effects of minor shifts in detector
calibration were not corrected, we cannot exclude the
possibility of an overestimate of cell Ca^+ measurements
by, at most, 2 mmol/kg dry weight.
In the hypertrophy experiment, data on 21 animals
from three time periods were analyzed. Two-way analysis
of variance is a standard statistical method used to separate the variation in the data into distinct components. For
this application, a treatment group component and a
residual, or error, component were analyzed. This method
was used to compute treatment group means adjusted for
differences between time periods, called least squares
means. The F statistic was used to test for statistical
significance of treatment differences (Winer, 1971). Computations were carried out on an IBM 3081 computer,
using the Statistical Analysis System (Statistical Analysis
System Institute, Inc.). For evaluation of paired experiments showing the effects of Na+-free wash, statistical
significance was determined by t-test (Winer, 1971).
Inspection showed that the wall thickness of the ligated
veins was increased, indicative of the hypertrophy consistently produced by this method (Bemer et al., 1981),
and the few rabbits that developed collaterals were discarded. In addition, light microscopic stereological analysis
of \-fim-thick sections obtained from (randomly selected)
three hypertrophied and three sham-operated veins (portions of which were used for cryoultramicrotomy and
electron probe analysis) that had been freeze-subsrituted
(Franzini-Armstrong et al., 1978), embedded in Epon, and
stained with toluidine blue showed that the cross-sectional
area in 155 cells from sham-operated veins was 11.8 ±
6.1 (SD) Mm2, as compared to 16.0 ± 7.2 (imJ in 150 cells
of ligated (hypertrophied) veins. The increase in cell cross
256
sectional area was significant (P < 0.001), confirming the
previous observations.
In the majority of the experiments, tension was monitored before and during freezing. During incubation in
Krebs saline, the strips showed the type of spontaneous
activity (Fig. 1) common in this phasic smooth muscle
(Cuthbert and Surter, 1964; Somlyo and Somlyo, 1968;
Somlyo et al., 1969; for review, see Johansson and Somlyo,
1980). This contractile activity persisted, often with
slightly increased amplitude, after the strips were transferred into BSA-Krebs. The environmental chamber was
designed to provide near physiological conditions (i.e.,
warm and humid) for the strips during the seconds prior
to freezing. The persistence of spontaneous contractions,
after removal of BSA solution prior to freezing, indicated
that this requirement was fulfilled.
Results
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Cryosections for electron probe analysis were cut
transversely, because the medial smooth muscle cell
bundles are longitudinally oriented and, therefore,
many more cells are available for probing from a
transverse than from a longitudinal section. These
cells, except at the surface, usually contain some
small ice crystals, but their structural preservation is
sufficient to distinguish cytoplasm from mitochondria and nuclei (Fig. 2). Cytoplasmic (0.5-1.0 pm in
diameter) and mitochondrial (0.15 nm in diameter)
areas were analyzed in each section.
Composition of Normal and Hypertrophied
Portal Vein Smooth Muscle and the Effect of
Cold Na+-Free (Li+) Wash
The results of electron probe analysis of the hypertrophied preparations are shown in Table 1. Each
strip represents a different experimental animal. For
cytoplasmic areas (Table 1), no statistically significant differences were found among experimental,
sham-operated, or control groups. Although there
were no major differences among treatment groups,
there was a large variability from strip to strip, which
is exemplified by the range in sodium concentrations
for the 21 strips in the series (Fig. 3). The F statistic
comparing strip-to-strip with cell-to-cell variations
in the Na content was 2.49, indicating that differences between strips were greater (P < 0.001) than
differences between cells within a single strip. Since
tissues (experimental and control) were obtained in
dralnl
IKrebs
I +bsa
three series, several months apart, it was possible
that strip-to-strip variability due to differences of
unknown origin (e.g., differences between experiments, variations over time in animal populations,
or in instrumental or experimental conditions) could
mask the differences between hypertrophied and
control groups. Therefore, statistical analyses were
performed that take into consideration the possibility of time group differences. The adjusted means
(least squares means) generated by these analyses
also showed no significant (P > 0.05) differences
between hypertrophied and control groups. The
time adjusted mean concentrations of K, Na, and Cl
were (in mmol/kg dry weight), respectively, 803,
198, and 364, and were used in obtaining computed
values.
Mitochondrial data were statistically analyzed in
the same way as cytoplasmic data. The least squares
means adjusted for time group differences showed
no significant differences among experimental
groups, and, specifically, mitochondrial Ca (3.5 ±
0.6 mmol/kg dry weight, n = 110) was not increased
in hypertrophied smooth muscle.
In the Na+-free wash series, normal portal vein
strips were incubated in ice-cold, Li+- or Tris-substituted, Na+-free saline solutions for 30 minutes, 2
hours prior to freezing. The results of these experiments are shown in Table 2. The cytoplasmic Na
concentrations (Table 2) were markedly lower in the
washed preparations, and this was paralleled by a
reduction in the Na content of mitochondria (Fig. 4)
and nuclei (Fig. 4). There were no significant and
systematic differences in cytoplasmic concentrations
between groups after the various wash procedures
(li+ or Tris-Krebs, 0.5- or 2-hour incubation), and
these results were combined (Table 2). The mean
cytoplasmic Na was 74.3 ± 27.4 mmol/kg dry
weight. The over 100 mmol/kg difference between
this value and those found in the preparations not
washed in Na+-free solutions (Table 1) is highly
significant (P < 0.001). There was also a small, but
significant (P < 0.01) reduction in cytoplasmic K in
the strips washed in cold solutions, presumably
reflecting inhibition of the Na pump. The concentrations of the other elements measured, including
Cl~, were (except for a borderline, P < 0.05, decrease
in S) not significantly different after the cold, Na+free wash procedures.
drain!
iA/VuWW/U\AAAMAAA/\A/WwwVwyWW\AAMWW\A/V^
100
1 min
FIGURE 1. Rapid freezing of a portal vein strip. Tension trace of a longitudinal strip of rabbit portal anterior mesenteric vein prior to and at the
time of freezing shoivs normal phasic activity in Krebs' saline, Krebs' salme with 4% added BSA, and in the 37°C humid atmosphere of the
environmental chamber. Strips were less than I mm wide and have stretched lengths of about 1.7 cm. The break in the record after the Kreb's
plus BSA represents 20 minutes.
Junker et al. /Sodium in Smooth Muscle
257
ffit>t*
ii.
- t /
"
•
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FIGURE 2. Transversely cut cryosection of a medial smooth muscle bundle of rabbit portal anterior mesentenc vein. It is possible to probe many
cells within a single section. Circle: typical cytoplasmic area analyzed; N, nucleus; arrows, mitochondria (12.500X).
TABLE 1
Elemental Analysis of Rabbit Portal-Anterior Mescnteric Vein Cytoplasm (mmol/kg dry weight ± SEM)
Experimental
condition
No. of
strips No. of
(n) cells
Na
Cl
Mg
Ca
Hypertrophied
Sham-operated*
Control
10
8
3
136
95
28
811.8 ±40.7
799.5 ±37.0
899.4 ±50.8
218.8 ± 30.1
212 5 ± 37.1
179.9 ± 24.5
369.4 ± 23.1
386.5 ± 27.9
399.6 ± 25.8
50.6 ± 2.6
45.3 ± 3.2
44.1 ± 2.1
3.3 ± 0.8
2.8 ± 0.6
5.7 ±1.2
263.6 ± 15.7
263.0 ± 10.6
294.0 ± 8.3
412.1 ± 17.4
390.6 ± 27.9
400.8 ± 37.2
Total (Mean)
21
259
819.6 ±25.1
210.8 ± 19.9
380.1 ± 15.3
47.6 ± 1.8
3.4 ± 0.5
267.9 ± 8.4
402.3 ± 11.7
* Raw data. Statistical analysis by strip (i.e., n = number of strips).
To investigate whether the physiological degree
of stretch applied in this study caused cell Na/K to
increase, as compared to flux studies done at slack
length, we prepared four strips from one vein. Two
were stretched to 1.7X slack length and two were
attached to holders a 1.0X slack length. In each of
two freezes, one 1.7x-stretched strip was paired to
a 1.0X strip. The results of large cytoplasmic analyses of these four strips are shown in Table 3 and
show that 1.7x stretch did not cause an increase in
cell Na.
Table 4 compares K:Na:Cl ratios measured by
EPMA (Table 1) and by isotope flux (Jones, 1980).
The K/Na ratio obtained with EPMA is lower in
normal (and hypertrophied) smooth muscle than the
value derived from isotope flux studies, but the
highest ratio is that obtained with EPMA after Na+free wash.
The Effect of Na + -Free (Li+) Wash on
Na+-Loaded Rabbit Portal Anterior Mesentenc
Vein and Guinea Pig Taenia Coli Smooth
Circulation Research/Vol. 54, No. 3, March 1984
258
content by 246 mmol/kg, and the difference was
highly significant (P < 0.0005).
Compositional differences (see below) between
individual Na+-loaded guinea pig taenia coli cells
were accompanied by visible ultrastructural differences in the unstained cryosections: the cells containing the highest Na (and lowest K and P) concentrations were light (relatively electronlucent), while
the more 'normal* cells had a dark (more electron
opaque) appearance (Fig. 5). For example, the Na
Mg, K, P, and Ca concentrations in five light cells in
one strip were, respectively (mmol/kg dry weight ±
SEM): 1643 ± 50.7, 15 ± 5.5, 11 ± 2.4, 64 ± 13.5
and 23.43 ± 3; compared with the values in five
dark cells in the same preparation 422 ± 16.7, 51 ±
6.2, 312 ± 44.2, 203 ± 22.5, and 5.93 ± 1.65.
Muscle and the Composition of Normal Guinea
Pig Taenia Coli
We wished to determine the effect of cold Li
wash on a smooth muscle (guinea pig taenia coli) in
which preliminary electron probe analysis showed
cytoplasmic Na to be relatively low, and, also, in
smooth muscles that were Na+-loaded prior to freezing. The results of electron probe analysis of taenia
coli (Table 5) show in this smooth muscle a normal
cytoplasmic Na (61 mmol/kg dry weight) that is
significantly lower than in rabbit portal veins, and
is not significantly reduced by cold Li+ wash (second
row, Table 5). In contrast, in Na+-loaded guinea pig
taenia coli smooth muscle, the wash in Na+-free
(Li+) solution caused a reduction in cytoplasmic Na
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Cn
o2-
75 100
150
200
250
300
350
400
Na (mmol/Kg dry weight)
FIGURE 3. Distribution of cytoplasmic Na* concentrations in rabbit portal anterior mesentenc vein of control, sham-operated and liypertropliied
animals. Mean sodium concentrations from 21 strips (data from Table 1) representing 21 animals (259 cells) are presented Mean [Na+] = 798
mmol/kg dry weight = [Na+]i = 46 niM calculated for 81% cell H2O. Median sodium concentration is 175 mmol/kg dry weight = [Na*]l = 41
mu.
TABLE 2
Effects of Cold Na-Free Wash on the Elemental Composition of Normal Rabbit Portal Anterior Mesenteric Vein Cytoplasm
(mmol/kg dry weight ± SD)
Experimental
condition
No. of
strips No. of
(n)
cells
K
Na
Cl
Mg
Ca
P
S
Li wash
2hrs
1
6
663.5 ±84.4
68.7 ± 18.9
386.7 ± 75.9
48.8 ± 12.6
4.1 ± 2.8
312.2 ± 56.6
350.3 ± 67.9
li + wash
1 hr
1
12
705.7 ±71.1
67.5 ± 14.9
327.9 ± 57.1
46.7 ± 9.4
3.0 ± 4.0
259.0 ± 32.8
371.1 ±38.3
Li+ wash
30 min
2
27
646.3 ±71.8
73.0 ±21.3
412.0 ± 52.3
47.8 ± 9.6
3.7 ± 2.7
358.3 ± 42.4
372.9 ± 33.9
Tris wash
30 min
3
41
657.6 ±125 9
78.0 ± 34.0
340.3 ± 64.3
43.9 ± 13.3
4.1 ± 4.7
239.3 ± 59.4
338.0 ± 42.8
7
86
661.2 ±102.3
74.3 ± 27.4
364.3 ± 68.5
45.9 ±11.7
3.8 ± 3.9
284.5 ± 73.5
354.4 ± 44.2
Total (Mean)
Junker et al. /Sodium in Smooth Muscle
259
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The large cell-to-cell variations in cytoplasmic Na
content (cell values ranging from 324 to 1813 mmol/
kg dry weight) in Na+-loaded guinea pig taenia coli
smooth muscle, showed a bimodal frequency distribution (Fig. 6). Furthermore, the cells having the
highest Na content had the lowest K, Mg, and
abnormally low P levels, but higher Cl and Ca
contents, as indicated by the correlation coefficients
(Table 6) of cytoplasmic Na with these other elements. After Na+-free (Li+) wash, the frequency
distribution of the cytoplasmic Na concentration
(Fig. 6) was no longer bimodal. Comparison of this
frequency distribution with that of the nonwashed,
Na-loaded cells suggests that most of the Na + has
been removed from the light cells that had originally
contained the highest concentrations of Na. The
median Na concentration also shifted from 600-800
to 400-600 mmol/kg dry weight (Fig. 6), indicating
that cold Li+ wash reduced the Na content even in
the (dark) cells that were not extremely loaded with
Na.
Portal anterior mesenteric vein smooth muscle
gained Na and lost K after Na+-loading (Table 7),
and cold, Na+-free (Li+) wash caused an even
greater reduction (405 mmol/kg dry weight, P <
0.005) in its cytoplasmic Na content. Because the
I
Na concentrations of Na-loaded PAMV cells were
also bimodally distributed (Fig. 7), comparison of
the frequency distributions provides a more correct
estimate of the effect of Na+-free (Li+) wash than
comparison of the mean values, and shows that
Na+-free (Li+) wash removed nearly all the Na+
from cells that had been massively (nearly 10-fold)
Na+-loaded in cold, K+-free solution. The Na+ content of the Na+-loaded rabbit portal anterior mesenteric vein after cold Li+ wash was comparable to that
of the control values without Na+-free (Li+) wash
(cf. Table 1), but higher than the Na concentrations
in normal smooth muscles after cold Na+-free (Li+)
wash (cf. Table 2). Na+-free (Li+) wash at 37°C
reduced the cytoplasmic Na content to 16 mmol/kg
dry weight (Table 7).
The Na concentrations of the various (normal and
Na-loaded) smooth muscles and the effects of Na+free (li + ) wash are graphically summarized in Figure
Discussion
The major conclusions drawn from this study are
(1) that the ionic composition of vascular smooth
muscle is unchanged by increased distending pressure at a time when marked structural effects have
taken place (Berner et al., 1981), (2) that the cytoplasmic sodium concentration of rabbit portal anterior mesenteric vein is relatively high and includes
a rapidly exchanging component that is not readily
quantifiable by ion flux studies, and (3) that Na +
loading of (taenia coli and portal anterior mesenteric
vein) smooth muscle for 3-4 hours results in a
heterogeneous population of cells and (4) procedures designed to estimate cell Na by attempting to
•° 150TABLE 4
2 100-
Comparison of Cytoplasmic K:Na:Cl Ratios in Rabbit Portal
Anterior Mesenteric Vein
MITOCHONDRIA
K:Na:Cl
NUCLEI
Rabbit Fbrtal Anterior Mesenteric Vein
^CONTROL
H
L'
WA
SH
FIGURE 4. The effect of cold Na*-free Li* wash on mitochondrial and
nuclear Na+ content of rabbit PAMV smooth muscle. Mitochondria
and nuclei were analyzed in the same preparations as summarized in
Tables 1 and 2. Note that both the mitochondrial and the nuclear
Na* content is lower after cold Na*-free (U+) wash.
[f
Hypertrophy series
(Table 1) Total
1:0.26:0.46
46
Na+-free wash
(LJ+ and Tris):
(Table 2)
1:0.11:0.64
16
Isotope flux*
1.0.15:0.38
26
•Jones, A.W. (1980).
TABLE 3
Elemental Analysis of Rabbit Portal Anterior Mesenteric Vein Cytoplasm (Stretch series—mmol/kg dry weight ± SD)
Experimental
condition
1 Ox stretch
1.7x stretch
No. of strips No of cells
22
20
716.8 ± 139 4
886 8 ± 120.0
Na
Cl
Ratio of K:Na:Cl
269.9 ± 87.7
185.0 ± 78.0
334.8 ± 102.6
364.3 ± 97.3
1:0.38:0.47
1:0.21:0.41
TABLE 5
Elemental Analysis of Cytoplasm: Normal and Na*-Loaded Guinea Pig Taenia Coli (mmol/kg dry weight ± SEM)
No. of No. of
strips cells
Na
Cl
Mg
Ca
Control
3
88
843.3 ±8.7
61.4 ± 2.3
323.1+4.2
47.5 ± 0.9
5.7 ± 0.8
253.6 ± 3.4
398.5
± 3.3
Control*; Li+
wash 2°C
2
66
793.2 ±8.8
56.8 ± 2.4
306.5 ± 7.0
50.5 ± 1.2
5.5 ± 0.3
272.4 ± 5.0
414.1
± 5.0
Na + -loadedt
3
80
84.6 ± 8.9
693.5 ± 33.0
250.4 ± 20.1
44.7 ± 8.9
6.5 ± 0.8
206.3 ± 12.1
370.0
± 6.7
Na+-loadedJ; U +
wash 2°C
3
71
114.1 ±8.2
447.1 ± 13.9
237.1 ± 15.1
56.4 ± 1.1
3.9 ± 0.7
280.6 ± 9.5
388.7
± 5.8
Na + -loaded§; U +
wash 37°C
1
25
196.6 ±5.6
12.1 ± 5.6
396.6 ± 21.0
61.9 ± 2.0
2.5 ± 1.0
291.0 ±5.8
322.4
± 7.0
* Washed 30 minutes at 2°C in Na+-free Ii+-Krebs; no previdous Na+-loading.
f Na+-loaded in K+-free Krebs for 3 hours at 2°C.
j Na+-loaded 3 hours at 2°C; washed 30 minutes at 2°C in Na+-free Li+-Krebs.
§ Na+-loaded 3 hours at 2°C; washed 30 minutes at 37°C in Na+-free Li+-Krebs.
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FIGURE 5. Transverse cryosection showing heterogeneous population of smooth muscle cells in Na*-loaded guinea pig taenia coli. Strip was Na+loaded for 3 hours. Note the presence of light and dark cells. Light cells contained significantly more Na+ and less P, K+, and Mg*~*. (See Fig. 5
and text for further description.)
Junker et al. /Sodium in Smooth Muscle
261
50-1
Cytoplasmic Na Concentration
in Na-Loaded Guinea Pig
Taenia Coli
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200
400
600
800
1000
1200
1400
1600
1600
2O00
2200
2400
Na Concentration (mmol /kg dry wt)
After Cold Na-free (Li) Wash
200
400
600
800
1000
1200
1400
_L
J_
JL
1600
1800
2000
2200
2400
Na Concentration (mmol/kg dry wt)
FICURE 6. Frequency distribution of cytoplasmic Na* concentrations in Na*-loaded guinea pig taenia coli and the effects of cold Na*-free (Li*)
wash. Panel A: Na* distribution in Na*-loaded cells illustrating bimodal distribution (from three animals; 80 cells analyzed). Panel B: paired
Na*-loaded strips washed in Na-free (Li*) solution for 30 minutes at 2O°C (from three animals; 71 cells analyzed).
remove extracellular Na+ through washout in cold
Na+-free solutions frequently lead to severe underestimates of cell Na.
The similar Na and K concentrations found in,
respectively, hypertrophied and control veins parallel the unchanged cellular ion content of arterial
smooth muscle of genetically [Dahl strain (Abel et
al., 1981); SHR (Jones, 1982)] or DOCA-induced
hypertensive rats; although in the latter, the Na+
and K+ permeability of the surface membranes is
increased (Jones and Miller, 1978; for review see
Jones, 1981, 1982). Cytoplasmic, mitochondria], and
nuclear K, Na, Cl, Mg, and Ca all were unchanged
in hypertrophied PAMV. We cannot exclude the
possibility that transient ionic changes may have
occurred during the early stages of hypertrophy,
because we wished to determine whether compositional changes occurred as a secondary effect of
Circulation Research/Vo/. 54, No. 3, March 1984
262
TABLE 6
Correlation Coefficients of Cellular [Na] with Cellular [Mg],
[P], [Cl], [K], and [Ca] in Na-Loaded Smooth Muscle
Guinea pig taenia coli
(n = 80)
Rabbit portal vein
(n = 81)
-0.66*
-0.65*
0.93*
-0.64*
0.82*
- 0 35t
-0.30t
0.93*
- 0 80*
0.69*
Mg
P
Cl
K
Ca
All correlations were statistically significant:
* P < 0.001.
|P<0.01.
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sustained hypertension and, therefore, we analyzed
tissues at a time (2 weeks) when the structural
changes induced by ligation (Johansson, 1976; Berner et al., 1981) or arterialization (Brody et al., 1972)
were well-developed.
The low mitochondrial Ca concentration observed
in hypertrophied (present study), as in control,
smooth muscle (present study; Somlyo et al., 1979,
1982) indicates that the degree of hypertrophy induced in these experiments did not cause the type
of mitochondrial Ca++-loading observed in some
damaged smooth muscles (Somlyo et al., 1979,
1982). Similarly, we found no evidence of changes
in nuclear composition in hypertrophied PAMV.
The total cytoplasmic K, Na, and Cl concentrations measured in this study were some 18-23%
higher than those previously obtained by EPMA in
the same vascular smooth muscle, whereas the
K:Na:Cl ratios are identical (Table 1, c.f. Table 2,
Somlyo et al., 1979). A systematic difference in the
concentrations of the soluble ions could be due
either to the effects of radiation damage that removes organic mass, but not K, Na, or Cl (Shuman
et al., 1976; Somlyo et al., 1979; for review, Hall
and Gupta, 1979), or to differences in cellular hydration. A small, physiological change in cell water
gives rise to relatively large differences in the concentrations measured in dried cryosecrions. In view
of the much smaller differences in the concentration
of P and the higher concentration of S (largely
bound and more susceptible to radiation damage at
room temperature),* we believe that a larger amount
of cell water (perhaps related to the use of the
environmental chamber) is the major cause of the
somewhat higher concentrations measured in the
present study. For example, a cytoplasmic K concentration of 820 mmol/kg dry weight would be equivalent to 245 mmol K+/liter cell H2O if cell water is
77%, and to 180 mmol K+/liter cell H2O if cell water
is 82% (cf. Jones, 1980: [K+], = 183 mmol/liter cell
HiO). Therefore, in the following discussion, we
shall consider—in addition to absolute values of
intracellular sodium—the ratios of K:Na:Cl, because
the latter comparison eliminates errors in EPMA
measurements due to variability in cell hydration or
radiation damage.
The total (time-adjusted) cytoplasmic Na concentration (198 mmol/kg dry weight) measured with
electron probe analysis in PAMV (Table 1) is equivalent to about 46 mmol/liter cell H2O, assuming a
cell hydration of 81% ([K+], = 803 mmol/kg dry
weight in present study; 183 mmol/liter cell H2O:
Jones et al., 1973). The distribution of Na concentrations (Fig. 3) suggested some skewing, but not a
bimodal distribution. This, like our previous study
on normal vascular smooth muscle (Somlyo et al.,
1979), showed examples of high cytoplasmic Na
concentration unaccompanied by evidence of cell
damage such as lowered cytoplasmic K or mitochondrial calcification. These results suggest that [Na+],
in normal cells can vary over a wide range. The
hypothesis that cytoplasmic Ca++ varies as [Na+],3
(Blaustein, 1977) predicts that such variability of
[Na+], can cause very large variations in resting
tension, but, in the course of relatively extensive
physiological studies, we found no evidence of this
(Somlyo and Somlyo, 1968; Somlyo et al., 1969;
Jones et al., 1973; present study).
The cytoplasmic Na measured by electron probe
analysis is nearly twice as high as the 26 mmol/kg
H2O estimated through extrapolation of the slow
component of 24Na efflux measured after "extracellular washout' in the cold (Jones and Miller, 1978;
• The lower sulfur contents found in our previous study (obtained
ivith an earlier electron microscope that had an inferior vacuum) may
have reflected radiation damage.
TABLE 7
Elemental Analysis of Cytoplasm: Na+-Loaded Rabbit Portal Anterior Mesenteric Vein (mmol/kg dry weight ± SEM)
No. of No. of
strips cells
+
Na -loaded'
Na+-loadedt; U*
wash 2°C
Na+-loadedt; Li+
wash 37°C
Na
Cl
Mg
Ca
3
2
81
53
356 0 ±27.7
441.3 ±29.2
574.4 ± 38.7
169.3 ± 6.2
359.0 ± 20.0
459.1 ± 1.4
48.9 ± 1.3
48.6 ±1.4
5.0 ±1.1
5.7 ± 0.6
241.8 ± 9 2
256.9 ± 10.1
310.6 ± 4.2
329.4 ± 4.6
1
25
162.5 ±3.1
16.1 ±2.2
284.7 ± 13.5
58.5 ± 2.2
2.3 ± 0.9
299.8 ± 6.4
340.4 ± 3.9
• Na+-loaded in K+-free Krebs 4 hours at 2°C.
•f Na+-loaded 4 hours at 2°C; washed 30 minutes at 2°C in Na*-free Li+-Krebs.
X Na+-loaded 4 hours at 2°C; washed 30 minutes at 37°C in Na+-free LT-Krebs.
263
Junker et al. /Sodium in Smooth Muscle
2018-
Cytoplasmic Na Concentration
in Na-Loaded Rabbit
Fbrtal Anterior Mesenteric Vein
16-
l/IKO12-
o
to-
ot
M
IT)
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100
200
300
400
500
500
700
800
900
1000
1100
1200
1300
Na Concentration (mmol/kg dry wt)
After Cold Na-free (Li) Wash
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
Na Concentration (mmol/kg dry wt)
FIGURE 7. Frequency distribution of cytoplasmic Na* concentrations in Na*-loaded rabbit portal anterior mesenteric vein and the effects of cold
Na*-free (Li*) wash. Panel A: Na* distribution in Na*-loaded cells illustrating bimodal distribution (from one animal; 34 cells analyzed); Panel
B: paired Na*-loaded strip washed in Na*-free (Li*) solution (same animal; 31 cells analyzed).
Jones, 1980, 1981) or the 23 mmol/liter estimated
by chemical dissection (Jones et al., 1973). In the
case of chemical estimates of cellular Na, uncertainties in the measurement of extracellular space can
give rise to relatively large systematic errors. Because
the absolute value of the [K+], estimated on the basis
of our assumption of cell hydration is in very good
agreement with reliable (not subject to large errors
of extracellular space measurement) chemical (Jones,
1980; Brading, 1981), and electrophysiological
(Hermsmeyer, 1976) measurements, and since cytoplasmic [CP], measurements with EPMA (Somlyo
et al., 1979) have also been independently verified
with ion-selective electrodes (Aickin and Brading,
1982), we conclude that the [Na+], measured with
electron probe analysis is also close to the true in
vivo value. The low Na values measured with electron probe analysis in skeletal muscle (Somlyo et al.,
1981) and in normal guinea pig taenia coli (present
study, cf. Casteels, 1969) indicate that extracellular
Circulation Research/Vo/. 54, No. 3, March 1984
264
800-
Na-LOADED
Na-LOADED
750
70065OUJ
1/1600-
; * ISO'S
EAOO-
£.
^350o
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a
g-200>.
° 15010050-
!
0
Guinea Pig TC
Rabbit PAMV
|{§SHAM
H
HYPERTROPHIED
H
30M1N COLD Li WASH
Q
^ |
COLD (2*C) Li WASH. Na-LOADED
•
CONTROL
Na-LOADED
58
WARM (37'C) Li WASH . Na-LOADED
FIGURE 8. Mean Na* concentration in rabbit portal anterior mesenteric vein and guinea pig taenia coli. Shown are the similar Na* contents in
control and in partially ligated (hypertrophied) or sham-operated portal veins, and the effects of Na*-loading on normal portal vein and guinea
pig taenia coll. Also shown are the effects of washing normal and Na*-loaded smooth muscles with Na*-free (Li*) solution at 2°C and at 37°C
(Data from tables 1, 2, 5, and 7; for further explanation, see text.)
Na is not translocated during preparatory procedures and that such translocation is not the cause of
the high cytoplasmic Na found in rabbit PAMV.
Turning to the ratios of the ionic concentrations
(Table 4), we find that the K:Na ratio measured with
electron probe analysis (1:0.26) is lower than the
1:0.15 ratio obtained with the isotope flux method
(Jones, 1981), whereas, after the Na -free wash, the
K:Na ratio (1:0.11), is higher than the ratio obtained
with isotope flux studies, even though cells lose
[K+], at low temperatures.
The sodium concentration of normal PAMV cells
washed (Table 2) in cold, Na+-free solutions (74
mmol/kg dry weight) is about one-third of the
normal values, corresponding to a [Na+]i of 16
mmol/liter cell H2O, snowing that there is a significant efflux of Na+ in 30 minutes, even at low
temperatures.
The total amount of Na+ removed during the 30minute cold Na+-free (Li+) wash was even greater
in the Na+-loaded smooth muscles; the amounts of
Na lost (average: 246 mmol/kg dry GPTC weight
and 405 mmol/kg dry PAMV weight) greatly ex-
ceeded the normal Na concentrations in these
smooth muscles. Even more massive losses of cytoplasmic Na+ occurred during the cold Na+-free (li + )
wash from the individual, most severely Na-loaded
(light) cells (see below). In contrast, in the normal
GPTC in which cytoplasmic Na was low to begin
with, the reduction in cytoplasmic Na during cold
Na+-free (Li+) wash was insignificant, and the
EPMA measurements were similar to the results of
chemical analysis (Casteels, 1969).
The major conclusion to be drawn from our observations on the effects of cold Na+-free wash is
that there is, in smooth muscle, a relatively temperature-insensitive, fast Na efflux system that operates
at low temperatures that are known to block the NaK pump (Jones and Miller, 1978). The fact that,
during a 30-minute washout, more Na+ is lost from
Na-loaded than from normal tissues, and the incomplete loss of Na+ from normal PAMV smooth
muscle even after 2 hr wash, suggest that this temperature-insensitive, rapid efflux system has a relatively low affinity for Na+. Nearly all the cell Na
can be removed from Na+-loaded smooth muscle
Junker et al. /Sodium in Smooth Muscle
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by the higher affinity Na pump (at 37°C) during 30
minutes in Na+-free solution. The ouabain-insensitive Na-efflux system of the carotid artery has a
lower affinity for Na+ (half maximal activity at
[Na+], = 60 ITIM) than the ouabain-sensitive Na
pump ([Na+],05 = 49 HIM), but this system is temperature-sensitive (Heidlage and Jones, 1981), as are
the strophanthidin-insensitive Na efflux in striated
muscle (Horowicz et al., 1970) and the ouabaininsensitive Na-Na exchange in taenia coli in
smooth muscle (Casteels et al., 1973; Brading, 1975).
The existence of a very rapid Na" efflux system has
been detected in cardiac Purkinje fibers with Na+selective electrodes (Ellis, 1977). Preliminary studies
with Na+-selective electrodes reported after this
manuscript had been submitted also revealed the
existence of rapid Na efflux in response to lowering
the external Na in guinea pig ureter smooth muscle
(C Aickin and A Brading, personal communication).
The presence of a rapid, relatively temperatureinsensitive Na" efflux system can obviously complicate methods that are based on the assumption that
only extracellular Na is removed by wash in cold,
Na+-free (Friedman et al., 1974) or nonradioactive
Na+-containing solutions (Jones, 1974; Jones and
Miller, 1978), and ascribe an "excess fast-exchanging
Na' to the extracellular space (Heidlage and Jones,
1981; Jones and Swain, 1972; for review see: Jones,
1980; Brading, 1981). The use of cold Na+-free (li + )
wash method is likely to lead to unpredictable errors,
because the loss of cytoplasmic Na during the exposure to the cold Na+-free (Li+) solution appears
to be proportional to the cytoplasmic Na concentration (Fig. 8), leading to severe underestimates of
cellular Na in Na-loaded muscles.
The appearance of a heterogeneous (bimodally
distributed with respect to cellular Na content) population of cells in Na-loaded smooth muscle was
an interesting finding, and would further complicate
chemical and ion flux measurements. Light (electronlucent) and dark (relatively electron opaque)
cells have previously been described in fixed material (Somlyo et al., 1971; Garfield and Daniel, 1976),
and had been related to the degree of cellular hydration (see also: Somlyo et al., 1979). In the present
study, the massively Na-loaded light cells contained abnormally low concentrations of P and high
Ca, reflecting the loss of organic phosphates and
gain in calcium by massively swollen and hyperpermeable cells. We considered the possibility that
an abnormal population of hyperpermeable cells
could account for the rapid and temperature-insensitive Na efflux observed, but consider this unlikely
for the following reasons. There were no structurally
distinguishable light or dark cells in normal smooth
muscles that had not been Na-loaded, nor could
two cell populations be distinguished by the distribution of cytoplasmic concentrations of Na (Fig. 3)
or of other elements (unpublished observation) that
were not bimodal. In addition, during washout of
Na+-loaded smooth muscles in Na+-free solution,
265
the Na was lost not only from light cells, but also
from the more moderately Na+-loaded dark cells, as
indicated by a shift in the median values of the Na
concentrations of the lower end of the frequency
distribution curves (Figs. 6 and 7). Therefore, by
currently available criteria, the rapid Na movements
observed appear to reflect a general property of the
membranes of these smooth muscles.
We would like to thank Keith Soperfor advice about and performance of some of the statistical analyses, and A. Brua and V. Betz for
preparing the illustrationsSupported by HL25348; Training Grants HL07499 and
IT32HD07152 to jf and HL15835 to the Pennsylvania Muscle Institute.
Dr. junker's present address is: NCI-FCRF, Department of Public
Health, National Institutes of Health, Frederick, Maryland 21701.
Address for reprints: Dr. Andrew P. Somlyo, Pennsylvania Muscle
Institute, University of Pennsylvania, School of Medicine, B42 Anatomy-Chemistry Building/C3, Philadelphia, Pennsylvania 19104.
Received January 24, 1983; accepted for publication January 4,
1984
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Electron probe analysis of sodium and other elements in hypertrophied and sodium-loaded
smooth muscle.
J L Junker, A J Wasserman, P F Berner and A P Somlyo
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Circ Res. 1984;54:254-266
doi: 10.1161/01.RES.54.3.254
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