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Sodium
in smooth
T. S. MA
AND
muscle
relaxation
D. BOSE
Department of Pharmacology and Therapeutics,, University
Manitoba, Canada R3E OW3
Winnipeg,
that this muscle, after undergoing depolarization and
contraction in response to elevated external potassium,
did not relax when the stimulus was removed, although
the membrane potential recovered from depolarization.
The present work was done to elucidate the mechanism
whereby taenia coli fails to relax in the absence of
sodium and also to utilize this phenomenon to elucidate
the nature of movement of calcium in the tissue during
relaxation. A preliminary account of this work has been
presented to the Annual Meeting of the Canadian Federation of Biological Societies (17)
l
MATERIALS
AND
METHODS
Guinea-pig taenia coli, about 20-25 mm in length,
was dissected from adjoining circular muscle and suspended either in organ baths or in test tubes placed
inside aluminum heating blocks. Resting tension was
adjusted to obtain optimum length (L,) for contraction
(usually 0.5 g). In a few experiments, uterine horns
were obtained from albino rats (300 g) injected with
diethylstilboesterol (1 mg/kg ip) 24 and 48 h before the
experiment. The muscles were maintained at pH 7.3
taenia coli; uterus; D600; lanthanum;
Na+-Ca++ exchange
and a temperature of 37°C. One hundred percent oxygen
was used for aerating the various bathing media shown
in Table 1. Both for isometric tension as well as for
CENTRAL
TO THE
UNDERSTANDING
of relaxation in isotope flux studies, the tissues were preincubated in
smooth muscle is the knowledge of how the elevated
normal sodium-containing medium for 30 min followed
cytoplasmic free calcium concentration is reduced. The by switching to the appropriate media indicated in Tamechanisms considered have ranged from active or pas- ble 2. Intracellular calcium was estimated by the lansive transcellular efflux of the ion to intracellular se- thanum method of van Breemen
et al. (26). This estiquestration by sarcoplasmic reticulum and/or mitochonmate may be an approximation of the real value because
dria. Attempts to demonstrate active extracellular
it is not certain whether the disposition of lanthanum is
transport of calcium in taenia coli or uterus have not
wholly extracellular and also because lanthanum may
been successful (11, l&22).
Goodford (11) has proposed a or may not block calcium influx and efflux at the same
two-stage calcium removal process in the membrane
rate (10, 13). However, it is being assumed that such
consisting first of an energy-requiring process followed
error will exist in both the control as well as test cases.
by a passive process, probably involving dissolution of After following the protocols outline in Table 2, lancalcium salts from the membrane. A passive efYlux of thanum (10 mM) was added to 45Ca-containing incubacalcium coupled to influx of sodium has been found in a tion medium for 5 min. This was followed
by three lonumber of excitable tissues, e.g., squid axon and heart
min-long isotope-free washes with the test medium
in
(1, 20). Reuter et al, (19) gave some evidence for the the presence of 10 mM lanthanum. The tissues were
existence of such a mechanism in vascular smooth mus- then removed and lightly
blotted
dry. After weighing,
cle. Lanthanum, which blocks transmembrane influx
they were each dissolved in 300 ~1 of NCS (Amershaml
and efflux of calcium, causes relaxation of a contracted
Searle Corp.) after addition of 20 ~1 of water at 50” C and
muscle. This has been considered, along with other
later neutralized
with 20 ~1 6 N acetic acid to reduce
reasons, to indicate that intracellular sequestration of chemiluminescence and mixed with 10 ml scintillation
calcium can mediate relaxation in the rabbit aorta (25). medium consisting of 6 g 2,5diphenyloxazole (PPO) per
Katase and Tomita (15) studying the effect of sodium- liter of toluene. Radioactivity was measured in a Philfree environment on guinea-pig taenia coli observed lips scintillation counter. Fifty microliters of the radio-
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MA,
T. S., AND D. BOSE. Sodium in smooth muscle reluxation. Am. J. Physiol. 232(l): C59-C66, 1977 or Am. J. Physiol.: Cell Physiol. l(1): C59-C66, 1977. -The
contraction
of
guinea-pig taenia coli due to high K+ could not be reversed by
washing when Na+ was absent from the medium. Reintroduction of Na+ (7 mM or more) caused relaxation.
Similar results
were obtained with rat uterus. The effect of sodium replacement was not due to change in ionic strength because equal or
higher osmoles of choline, Ca++, K+, Mn++, or Mg++ had little
or no effect. Persistence of contraction in the Na+-free medium
was not due to a “catch state” of the contractile
apparatus.
Impairment
of Ca+ removal from the cytoplasm rather than
persistent increase in Ca+ influx seemed to sustain the mechanical response. This was because D600 (a calcium influx
blocker) failed to completely relax K+-induced
contraction
in
the absence of Na+ and also because the ability of EGTA to
produce relaxation
was reduced in the absence of Na+. Measurement
of tissue calcium
content using the lanthanum
method revealed coincident, decrease in tissue calcium and
tension to control level during Na+-mediated
relaxation.
The
results suggest a role for transmembrane
Na+-Ca++ exchange
in causing the Na+-mediated
relaxation
of taenia undergoing
Na+-free contracture.
of Manitoba,
C60
TABLE
T. S. MA
1. Composition
Medium
NaCl
of bathing
Sum
crose
“pF*
repeated 4 times or more unless specifically
in RESULTS.
media
KC1
MgSO,
CaC12
Glu-
cow
TrisHCl
4.5
4.5
5.9
5.9
1.2
1.2
1.2
1.2
2.5
2.5
2.5
2.5
11.5
11.5
11.5
11.5
10
10
10
10
4
TABLE
120
0
120
0
0
240
0
240
2. Time in various
0-Na
i- K” +
%a
1.4
1.4
0
0
media
mentioned
Normal Na +
Wa
0-Na +
45Ca
0-Na
+ Lat
45Ca
+
Normal Na +
4sCa
1
58
0
0
0
0
2
3
4
5
30
30
30
30
10
10
10
IO
18
16
14
0
0
0
4
0
2
4
4
min
10
t 10 mM.
active incubation
medium were treated in the same
manner as above before counting.
22Na+ uptake by the blotted whole tissue was measured by y-spectrometry.
Uptake per gram of tissue was
expressed as a percent of the counts per milliliter
of the
medium.
Quick-release
experiments
were performed to assess
the intensity of the active state by the method of Ritchie
(21). The lower end of the muscle was fixed to a Grass
FT-03C force displacement
transducer
while the upper
end was connected by a jeweler’s
chain to a rotary
solenoid described more fully by Bose and Bose (4). This
device was capable of making rapid length reductions of
adjustable magnitude within 10 ms when activated by a
gated DC power supply.
Electron
microscopic
localization
of lanthanum
in
taenia was done by exposing two normal as well as two
taenia strips, depleted of sodium for 30 min, to LaCl, (20
mM) for 30 min. At the end of this period, glutaraldehyde was added to the bathing medium in a concentration of 3%. After 10 min the bathing temperature
was
reduced to 4”C, and after 50 min the muscles were
washed with cacodylate buffer. The muscles were postfixed in osmic acid for 30 min and stained with uranyl
acetate for 60 min. After dehydration
in alcohol, muscle
pieces were embedded in Epon, and ultrathin
sections
were examined in a Hitachi HS-8 electron microscope.
At least 200 cells were examined in each muscle, Only
cells with reasonably
intact membrane boundary were
studied. Drugs and chemicals used in this study were:
ethylene glycol-bis-( P-aminoethylether)-N,
N’-tetraacetic acid (EGTA; Eastman);
D600 (Knoll); diethylstilbesterol (Hanovol;
Homer);
45Ca and 22Na (New England Nuclear Corp); inorganic chemicals were Analar
grade from Fisher Scientific Co., and sucrose was obtained from Sigma Chemical Co+
Statistical
evaluation
for multiple grouped data was
done by analysis of variance in conjunction
with Duncan’s new multiple range test. Experiments
involving
comparisons
between two sets of data were done using a
t test designed for unpaired observation
with unequal
variances or a paired-t test (19)* Every experiment
was
Guinea-pig taenia coli exhibited spontaneous activity
in the normal sodium-containing
medium buffered with
Tris-HCl,
The rate was more rapid than that observed
in Krebs-Henseleit
solution containing bicarbonate as a
buffer. Addition of KC1 (70 mM) caused a rapid phasic
contraction
followed by a sustained tonic contraction,
When the excess potassium was washed out, the muscle
promptly
relaxed to the base line (Fig+ 1). In muscle
exposed to the sodium-free
medium containing an additional 70 mM potassium,
there was an initial contraction which persisted
over an observation
period of 60
min when the extra potassium was washed out after ZO30 min. Changing
over to normal sodium-containing
medium rapidly relaxed the muscle. Relaxation
at a
slower rate was also seen by raising sodium concentration to as little as 7 mM.
When a different
experimental
protocol was employed, taenia coli was transferred
from normal sodium
medium to Na+-free medium. A rapid contraction which
lasted for 15-20 min ensued. When tension returned to
base line, addition of extra KC1 (70 mM) resulted in a
sustained
contraction
which, as in the previous case,
persisted even though the high external potassium was
washed out with the Na+-free medium, These persistent
contractions
in the Na+-free
medium after KC1 was
washed out will be referred to as Na+-free contractures
These experiments
confirm the work of Katase and
Tomita (15).
Experiments
similar to the above were also done on
six rat uterine horns, Contractions
were elicited in response to KC1 (70 mM) in the presence or after 30-40
min absence of external Na+. The KCl-induced
contractile response was reversed on washing when Na+ was
present but not when Na+ was absent (Fig. 2).
Further
experiments
were done in taenia strips to
investigate
whether
the persistent
contraction
in the
absence of sodium was due to: a) effect of low ionic
strength;
b) “catch
state”
analogous
to molluscan
smooth muscle; c) increased membrane permeability
to
calcium; d) decreased calcium removal from the cytoplasm either by intracellular
sequestration
or transcellular extrusion.
Effect of low ionic strength.
To examine the mechanism of sodium-mediated
relaxation of Na+-free contrac-
t -Ad t
NK/K
NK
SK/K
+
SK
-L
NK
FIG. 1. Effect
of KC1 (70 mM) on guinea-pig
taenia coli in presence of normal
Na (NK/K)
or Na-free
medium
(SK/K).
KC1 washed
out with sodium-containing
(NK) or Na-free
medium
(SK) as indicated. Maintained
contracture
in presence
of SK was rapidly
reversed by introduction
of normal
sodium at NK.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017
Group
* 70 mM.
D. BOSE
RESULTS
mM
Normal Na
Na free
PO, free, normal Na
PO, free, Na free
AND
SODIUM
AND
SMOOTH
MUSCLE
C61
RELAXATION
A
t
NK/K
--- __t-----1 min
FIG. 2. Effect of KC1 (70 mM) on rat uterus: A: in
presence
of normal
Na (NK/K)
or (B) in absence of
(SK/K).
KC1 washed
out with NK or SK as indicated.
NK
I3
SK/K
T-r-SK
tures, it was considered necessary to examine whether
it was an ionic strength effect or due to Na+ per se. Na+free contractures were obtained in eight preparations as
described earlier. Various ions were added to the medium. NaCl, KCl, choline chloride, MgSOq, MnCl,, or
CaCl, were added in concentrations of 10 mM which
resulted in ionic strength increases of 0.02 for the first
three salts, 0.04 for MgS04, and 0.03 for the last two
salts. Relaxation was measured at the end of 5 min
NaCl was the most potent relaxation of all agents
tested. Choline chloride, KCl, and CaC12 had minimal
relaxant effect. MgS04 and MnCl, were intermediate in
effect (Fig. 3). It should be pointed out that these
smaller effects were obtained with amounts of these
salts that caused much larger increases in ionic
strength than NaCl. These results suggest that the
relaxant action of NaCl is a distinct effect of Na+ ions,
although there may be a small effect due to a contribution to ionic strength.
Catch state. In molluscan smooth muscle, the catch
state tension can be maintained at very little energy
cost and is associated with a markedly reduced “active
state” (14). To test whether such a phenomenon maintained Na-free contracture in the taenia, active state
was estimated by the “quick-release method.” Notwithstanding the criticism (5) that the method of testing can
itself alter the active state, it was assumed that any
error would have been present in both the control and
test conditions. Four preparations were stimulated by
the addition of KC1 (70 mM) to the normal Na-containing solution. After a steady contraction was obtained,
the muscle length was rapidly reduced by 6%. The tension rapidly decreased followed by a secondary increase
which was measured over the next 10 s, In the presence
of Na, the muscle redeveloped 0.9 t 0.1 g tension, and
the total active tension attained was 62% of the level
before release. The strips were then transferred to Nafree medium for 30 min, and Na-free contractures were
obtained. Quick release of these muscles caused similar
initial decrease in tension followed by an increase of 0.7
2 0.2 g tension (Fig. 4). Active state as measured by this
method was not significantly reduced by Na-free medium (P > 0.05; n = 4). Na-free contracture, therefore,
does not fulfill one of the most important criteria for the
catch state.
50
I
NaCl
CholineCI
KCI
Cdl,
MnCI,
MgSO,
FIG.
3. Effect of various
ions (10 mM) on Na-free
contracture
in
taenia.
Each bar represents
mean * SE of 3-4 experiments.
Asterisks (*P < 0.05; ** P < 0.01) denote significant
difference
(t test for
unequal
variance)
from relaxation
due to NaCl.
normal
Na
Na-f
ree
15
gr
FIG. 4. Effect
of quick
induced
tension
in taenia
deprivation
of Na.
6%
SEC
qr
release
(qr) (6% of L,) on KC1 (70 mM)
in presence
of normal
Na or after 30 min
Effect on Ca++ permeability. Katase and Tomita (15)
suggested that the Na-free contracture was maintained
by a sustained elevation of Ca permeability. Experiments were done with D600, a methoxy derivative of
verapamil known to decrease activated Ca influx (9).
Eight strips of taenia were divided into two groups. One
group was exposed to normal Na-containing medium
while the other was exposed to Na-free medium for 20
min. Both groups of muscles were then exposed to KC1
(70 mM). After a steady contraction was obtained, D600
(0.4 PM) was added to the medium and the rate of
relaxation was measured. Complete relaxation took
place in the presence of Na in 19.3 -+ 1.5 min (Fig. 5).
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<-t
C62
T. S, MA
0600
0.4uM
_
1
191
5min
D600
SK
1
SK/K
_-_----I-_
Incomplete relaxation was obtained with D600 (51.0 t
8.8%; P > 0,OS) in the absence of Na, TI,2 for relaxation
in the presence or absence of external Na were 4.7 t 0*3
and 25.5 -+ 0.2 min, respectively (P < O.Ol)*
These results indicate that part of the Na-free contracture was dependent on continued Ca influx. An
observation that dictated against increase in Ca permeability being the major factor was that EGTA (15 mM)
relaxed a KC1 contraction in the presence of Na much
more rapidly than a Na-free contracture (Tllz of 0.66 t
0.07 min vs. 4.81 -+ 0,96 min; P < 0.01, n = 4, Fig. 6).
The incomplete relaxation by D600 in the Na-free medium is highly suggestive of impaired Ca removal from
the cytoplasm being an important factor in Na-free
contracture. Whether this effect of Na was on the
plasma membrane or some intracellular
site (s) was
examined in the following experiments.
Differentiation of transsarcolemmal Ca extrusion and
intracellular Ca sequestration. Taenia strips, divided
into five groups, were exposed to various media, some of
which contained tracer amounts of 45Ca, as indicated in
Table 2. 45Caexchange was terminated by adding La (10
mM) to the incubation medium as described in METHODS.As shown in Table 3, tissues in group 2 exhibited
Na-free contracture of 3.0 t 0.3 g when the extra KC1
was washed out. This was associated with a significant
increase in tissue 45Cacontent. Addition of normal Na to
:4iiL
t
NK/K
___-------t
NK
1
NK
muscle in groups 3 and 4 for 2 or 4 min caused relaxation of 86.8 t 1.5 and 96 t 1.7%, respectively. Tissue
calcium elevation was completely reversed under these
conditions. Lanthanum (10 mM) was administered for 4
min to muscles of group 5 prior to reintroduction of
normal sodium. Not only was the relaxation caused by a
4-min exposure to sodium reduced (44.4 t 2.7% compared to 96 t 1.7% with Na+), but also the 45Cacontent
of the tissue did not decrease as much as in the absence
of lanthanum pretreatment (Table 3).
These results indicate that the cellular fraction of
calcium elevated due to the absence of Na, and which to
a certain extent was responsible for the maintained
contracture, was completely eliminated from the tissue
during sodium-mediated relaxation. Therefore, an outward transmembrane calcium movement rather than
intracellular sequestration seems to be more important
for relaxation in the taenia under the conditions of this
study.
The possible effect of lanthanum on sodium entry into
the tissue and thereby on Ca efflux was tested with
22Na. Figure 7 depicts the tissue 22Nacontent (expressed
as a % of medium counts) in the presence or absence of
lanthanum. In all experiments (n = 3 in each group) the
tissues were rinsed in isotope-free medium for 30 s before residual radioactivity
was measured. In tissues
preexposed to lanthanum (10 mM) for 5 min followed by
exposure to lanthanum and 22Nafor 30 min, there was a
small decrease in sodium exchange which was statistically significant (P < 0.05). In other experiments, the
tissues were additionally exposed for 30 or 60 min to
ouabain (30 PM) in order to decrease sodium pumping
and thereby increase sodium content of the tissue. Both
3. Effect on intracellular
isometric tension
Ca and
TABLE
Group
Treatment
1
2
3
4
5
NK Control
0-Na contracture
NK 2’
NK 4’
La pretreated
* Data n = 4-15 in each group.
as 0% and in group II as 100%.
Ca Content,
pmolkg
162.2
329.3
151.5
164.7
249.6
i
+
2
+
f
11.4
16.8
22.2
16.8
16.8
% Ca*
0
100
-6.4 2 11.6
1.5 k 8.8
52.3 2 8.8
% Tension+
0
100
13.2 2 1.5
4.0 2 1.7
44.1 2 2.7
Data normalized
by considering
Ca content
t Relative
to 0-Na contracture
tension.
in group I
FIG. 6. Effect of EGTA
(15 mM) on tension response
of taenia due to KC1 (70 mM) in presence of normal
Na
(A) or in absence of Na (B).
8
SK/K
D. BOSE
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017
FIG.
5. Effect of D600 (0.4 PM) on tension
response
of taenia due
to KC1 (70 mM) in presence
of normal
Na (NK/K)
or in absence of Na
(SK/W.
AND
SODIUM
AND
SMOOTH
MUSCLE
RELAXATION
C63
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017
well as removal of the inhibitory effect of troponin on
actin-mediated increase in nucleotide-bound myosin
ATPase activity. This explanation is not applicable to
our results because the contracture can partly be reversed by EGTA or by a calcium antagonist D600. Since
D600 does not affect smooth muscle actomyosin ATPase
(personal communication), the Na-free contracture is
mediated by Ca. Our findings with small concentrations
of these cations differ from those of Gordon et al. (12)
who found K and Na to be equally effective as relaxants
of skeletal muscle and Katase and Tomita (15) who,
using very high concentrations of various ions, found
them all to relax taenia undergoing Na-free contracture. We feel that the ionic strength effects are best
analyzed with submaximal amounts of ions such as we
Na-22
60
90
have done.
Uwabain 30um
30
60
Molluscan smooth muscle exhibits a catch state under
Time
(min)
certain circumstances (24). This condition is characterFIG . 7 . ““Na content
of taenia expressed
as tissue countslmin
g-l
ized by sustained contraction during which active state
as a percent of medium
countslmin
ml-*. Open bar denotes content in
is markedly reduced. This possibility was ruled out in
absence and hatched
bar denotes
content
in presence
of LaC&(lO
the taenia during Na-free contracture because intensity
mM). A is after 30 min exposure
to 22Na; B and C, additional
30 and
60 min after exposure
to 9Ja and ouabain
(3 x IO-” M). Results
of active state was well maintained.
shown are mean + SE for 3 experiments.
In the absence of the above two possible mechanisms,
attention was focused on the possibility of Na-free contracture being due to either an increase in CA permeacontrol and lanthanum&eated
muscles showed significant increase in tissue 22Nacontent over the correspond- bility and/or inhibition of Ca removal from the contractile system. It is not possible to completely rule out the
ing nonouabain-treated ones. However, a small inhibition was seen in the lanthanum-treated
muscle com- first alternative. The inability of D600 to fully relax Nafree contracture contrasts sharply with the effect of this
pared to control muscles. This inhibition was significantly different (P < 0.01) only in the tissues that had drug on a potassium-contracted muscle (in presence of
normal sodium) and dictates against increased permeabeen exposed to ouabain for 60 min.
bility being the sole mechanism for the contracture.
ELectron
microscopy. Electron microscopic examinaAdditionally,
EGTA is less effective in relaxing a Nation revealed that most of the lanthanum was localized
free
contracture
than a normal contraction due to eleeither in the extracellular
or micropinocytic vesicles
vated
potassium
which argues against’an increased Ca
(Fig. 8). In several instances, mitochondria were seen
permeability.
approaching these surface vesicles, but no electron
One has now to consider the effect of Na on Ca reopaque lanthanum aggregates were found to associate
moval
mechanism in the taenia. Smooth muscle cells
with these. Occasionally what looked like lanthanum
are
characterized
by three distinct features: large extraaggregates were seen within cytoplasmic regions. However, the clearly rounded outline of these deposits so cellular space, small cell size, and numerous pinocytic
closely resembled those in the pinocytic vesicles that it vesicles in the plasma membrane (21). All these characwas felt that perhaps the former represented deposits of teristics will tend to favor easy exchange between the
cells and the external medium. Considering these, it is
surface vesicles that were overlying the cytoplasmic
attractive to consider the cell membrane as an imporregion. Relative to the pinocytic vesicles, the extracellutant
site for calcium extrusion during relaxation of the
lar region had much less lanthanum. This suggests that
smooth
muscle cells. However, controversy still exists
the lanthanum trapped in these vesicles is not easily
with regard to the presence of Na-Ca exchange mechawashed out by the fixatives and buffer.
nism across the smooth muscle sarcolemmal membrane,
although there is greater acceptance of this mechanism
DISCUSSION
in the squid axon and cardiac muscle (1, 20). Reuter et
Persistence of smooth muscle contracture due to high
al. (19) found that in rabbit aortic smooth muscle 45Ca
K+ in Na-free medium even after the stimulant was efflux decreased in a Na-free medium by approximately
removed has been systematically
analyzed in the 35% and promptly increased when the Na-containing
taenia, and the observation has also been extended to solution was reintroduced, and they suggested a Na+the rat uterus.
sensitive efYlux mechanism for Ca. Van Breemen et al.
Replacement of Na by sucrose caused a decrease in
(26), on the other hand, showed that in rabbit aorta the
external ionic strength. Gordon et al. (12) showed that
Ca gradient does not depend on the Na gradient, espeskinned frog sartorius muscle developed tension in a low cially under conditions of elevated intracellular
calionic strength medium. Weber and Murray (27) have cium. Furthermore, they showed that the relaxation
referred to the fact that tropomyosin can dissociate from
brought about by reintroduction of normal sodium in a
the contractile apparatus when the ionic strength is high-K contracted aortic strip was not accompanied by
reduced. This can lead to loss of calcium sensitivity as Ca extrusion, indicating that transmembrane calcium
C64
T. S. MA
AND
D. BOSE
movement
was not an important
mechanism
for relaxa-
tioil.
Intracellular
Ca concentration
as measured by the La
method can be used reliably as an index for the testing
of transmembrane
Na-Ca coupled exchange only if
other intracellular
Ca sequestering
sites, e.g., mitochondria or sarcoplasmic
reticulum,
do not complicate
the picture. This error may be different in various tissues. It is possible that Na-CA exchange exists in aortic
tissue as has been shown by Reuter et al. (19). Nevertheless, it may not be a mandatory
process for reducing
cellular Ca during relaxation.
The fact that large elastic
arteries contain a particularly
large volume of sarcoplasmic reticulum,
comprising
approximately
5% of the
cell volume (8), suggests a large intracellular
Ca-binding capacity. It is likely that relaxation
in aorta involves two processes: the faster one involving intracellular Ca binding and a slower one involving
transmembrane Na-Ca exchange.
The more extreme argument by van Breemen et al.
(26) and Deth and van Breeman (7) that Ca++ extrusion
is not a necessary process for aortic relaxation
based on
the finding that La, a Ca efflux blocker, at a concentration of 2 mM relaxed tissues contracted
by K+, Li+,
norepinephrine,
high pH, or ouabain, requires further
evaluation
because the time course of the Ca-efflux
blocking effect of La is not known (i.e., it may require a
longer time to block calcium efflux to the same degree as
compared to influx).
Freeman
and Daniel (10) found
that 2 mM La do not block efflux completely in rabbit
aorta. Furthermore,
we have observed that the Nainduced relaxation
of taenia undergoing
the Na-free
contracture,
which was blocked by prior incubation with
10 mM La, was not blocked by 2.5 mM La. This suggests
that lower concentrations
of La do not block Ca efflux
fast enough. In the taenia, addition of La (10 mM)
completely
relaxed
a potassium-stimulated
muscle
probably because influx of Ca was blocked more rapidly
than efflux. Relaxation is not seen if Ca efflux mechanism is inhibited
by Na removal.
The data on 22Na
uptake indicate that La probably
inhibits
Ca efflux
directly rather than by inhibition
of Na influx. Katase
and Tomita (15) studied the Na-induced
relaxation
of
Na-free contracture
in guinea-pig taenia coli. They observed that the relaxation
produced by Na at 15°C was
only slightly
slower than at 35°C. An average Q10 of
about 1.4 was obtained between 35 and 15°C. The low
Q1,, suggests that the removal of intracellular
free Ca is
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FIG. 8. Electron
microscopic
wpearance
of taenia exposed to LaCl, (20
mM) in presence of normal
Na. Most of
electron
dense area due to La deposits
( t ) is located
within
micropinocytic
vesicles.
SODIUM
AND
SMOOTH
MUSCLE
C65
RELAXATION
These experiments
themselves
do not argue against the
presence of transmembrane
Na-Ca exchange in living
tissue.
The La method for measuring
intracellular
Ca (25)
has been criticized,
and ca uti on against using the Larest .stant intracellul .ar pool as a measure of Ca invol ved
in EC coupling is probably justified (13). However,
it is
gratifying
to note that the concentration
of lanthanum
employed by us not only attenuated sodium-induced
45Caloss from the taenia, but also reduced relaxation.
The results of electron microscopy in the taenia also
differ from those in the stripped uterine preparation
(13). Although we could not demonstrate deposits of
lanthanum (applied in the ionic form) in intracellular
structures such as mitochondria, we cannot be absolutely certain that some lanthanum did not go inside the
cell, although one would have expected some to be deposited on the inside of the cell membrane or on the
mitochondrial membrane. The discrepancy between our
results and those of Hodgson et al. (13) merely indicates
gross tissue differences in lanthanum permeability. We
intend our results to qualitatively
depict the role of Na
in controlling Ca movement and relaxation and do not
wish to make any quantitative claims about the contractile Ca-pool in this tissue. It is interesting to note
sodium ions are also involved in promoting relaxation in
the rat portal vein (2). Both taenia and portal vein have
a sparse sarcoplasmic reticulum (8). Whether the effect
of sodium relaxation is prominent in muscles where
sarcolemma plays a greater role in relaxation than sarcoplasmic reticulum remains to be established.
Kroeger et al. (16) have proposed that P-adrenergic
relaxation of myometrium by isoprenaline is due to
calcium extrusion, Our own unpublished observation
that isoprenaline fails to relax taenia as well as diethylstilboesterol-treated
uterine muscle in the absence
of Na can be taken to support the role of Na in drugmediated calcium extrusion mechanism(s).
In conclusion, our results are best explained by postulating a role for transmembrane Na+-Ca++ exchange in
causing relaxation of taenia undergoing Na+-free contracture.
This work
was supported
by grants
from the Medical
Research
Council
of Canada
and the University
of Manitoba.
The expert
assistance
of Paul Hazelton
and Maria
Prasher
in doing the electron
microscopy
is gratefully
acknowledged.
T. S. Ma is the recipient
of a University
of Manitoba
graduate
scholarship.
Present
address
of T. S. Ma: Dept. of Pharmacology,
Dalhousie
University,
Halifax,
Nova Scotia, Canada.
Send requests
for reprints
to: D. Bose, Dept. of Pharmacology
and
Therapeutics,
University
of Manitoba,
770 Bannatyne
Ave., Winnipeg, Manitoba,
Canada
R3E OW3.
Received
for publication
24 November
1975.
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