role of na+/k+-atpase in the high extracellular calcium-induced impairment of rabbit aorta...
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Vascular Pharmacology 41 (2004) 75–81
Role of Na+/K+-ATPase in the high extracellular calcium-induced
impairment of rabbit aorta contractile activity
Ana Ortega, Amaya Aleixandre*
Departamento de Farmacologı́a, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain
Received 30 July 2002; received in revised form 18 March 2004; accepted 18 March 2004
Abstract
The present study was designed to prove whether the activation of the sarcolemmal Na + /K + -ATPase in the rabbit aorta could explain the
decreased contraction caused in this tissue by high extracellular calcium. To demonstrate this hypothesis, we evaluate the modification in the
contractile responses to KCl and a1-adrenoceptor agonists (methoxamine and phenylephrine) produced by a high extracellular Ca2 +
concentration (10 mM) in isolated rabbit aorta rings when the Na + /K + -ATPase is inhibited with ouabain.
Ouabain 10� 4 M caused an initial rapid increase in tone in the rabbit aorta rings, which could be linked to the release of catecholamines
provoked when the Na + /K + -ATPase in the nerve terminal was blocked. This glycoside also caused a delayed contractile response in the
preparations that could be linked to the inhibition of the Na + /K + -ATPase in the sarcolemma of the smooth muscle. The maximum inhibition
of the sarcolemmal pump was fixed 2 h and 15 min after ouabain 10� 4 M administration. Both responses were smaller with the 10-mM
Ca2 + concentration than with the 2.5-mM Ca2 + concentration.
The contractions elicited by KCl and the a1-adrenoceptor agonists were higher in the aorta ring preparations incubated with the 2.5-mM
Ca2 + solution than in the aorta ring preparations incubated with the 10-mM Ca2 + solution. When the Ca2 + concentration in the organ bath
was 2.5 mM, 10� 4 M ouabain administration caused a decrease in the responses to KCl and a1-adrenoceptor agonists. By contrast, when the
Ca2 + concentration in the organ bath was 10 mM, 10� 4 M ouabain did not modify these responses. As a consequence, the contractions
elicited by KCl were very similar in all the ouabain-treated preparations and those elicited by the a1-adrenoceptor agonists in ouabain-treated
preparations were even higher when the Ca2 + concentration in the organ bath was 10 mM than when the Ca2 + concentration in the organ
bath was 2.5 mM.
The results of this study suggest that the increase in extracellular Ca2 + concentration may facilitate the functioning of the Na + /K + -
ATPase in the vascular smooth muscle (VSM) and produces opposite effects to ouabain. This effect of high extracellular Ca2 + concentration
on the sarcolemmal pump may explain the decrease in the contractile responses elicited by depolarization and a1-adrenoceptor stimulation
observed in rabbit aorta ring preparations.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Vascular smooth muscle; High extracellular calcium; Na+/K+-ATPase; Ouabain
1. Introduction
The ability of elevated levels of external calcium to
reduce the contractile response of vascular smooth muscle
(VSM) has been recognized for a long time. In 1911, Cow
(1911) observed that excess calcium in the muscle bath
depressed the contractile response to noradrenaline in sheep
splenic artery. A few years later, Bohr (1963) demonstrated
the depressant effect of elevated calcium concentration on
the noradrenaline contractile response of rabbit aorta and
1537-1891/$ – see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.vph.2004.03.005
* Corresponding author. Tel.: +34-91-3941475; fax: +34-91-3941463.
E-mail address: [email protected] (A. Aleixandre).
applied the concept of membrane stabilization by calcium to
VSM. A possible mechanism of this membrane stabilization
was proposed to be an alteration in the conductance of major
ions through the cell membrane (Hurwitz, 1965; Jones and
Hart, 1975). In 1971, Blum and Hoffman (1971) described
an increase in K + exchange rate across the cellular mem-
brane when extracellular Ca2 + concentration increased; and
in 1975, Lindenmayer and Schwartz (1975) proposed that
such an increase could stimulate Na + /K + -ATPase activity.
However, the effect of calcium on Na + /K + -pump activa-
tion has always been controversial. High calcium might
induce the inhibition of Na + /K + -ATPase activity in several
cell types (Stemmer and Akera, 1988; Knudsen and Johan-
sen, 1989; Petersen et al., 1991), but this inhibition has not
A. Ortega, A. Aleixandre / Vascular Pharmacology 41 (2004) 75–8176
been described in VSM. Some researchers have indicated
that stimulation of the Na + /K + -ATPase exchanger could
contribute to the vasodilation produced by calcium dietary
supplements (Porsti et al., 1992). More recently, it has also
been suggested that the hyperpolarization caused by the
activation of this pump in the VSM membrane may be
responsible for the relaxation of isolated arteries observed
when calcium concentration is increased in the organ bath in
in vitro studies (Yildirim et al., 1998; Zakharenko and
Reznik, 1998).
It has also been shown that the relaxing effect caused by
high extracellular calcium concentrations may be linked to
the increase in the release of endothelium-derived relaxing
factors (Furchgott, 1981; Singer and Peach, 1982; Luckhoff
and Busse, 1990). Several years ago, however, our research
group carried out a study which demonstrated that the
release of endothelium-derived relaxing factors in rabbit
aorta does not seem to be the main mechanism implicated
in the Ca2 + -induced reduction of its contractile activity
(Ortega et al., 1997). The present study was designed to
test whether the activation of the Na + /K + -ATPase in this
tissue could explain the decreased contraction caused there
by high extracellular calcium.
To demonstrate the abovementioned hypothesis, we
should evaluate the modification in the contractile responses
to KCl and a1-adrenoceptor agonists produced by a high
extracellular Ca2 + concentration in rabbit aorta when the
Na + /K + -ATPase is inhibited with ouabain. Some re-
searchers have already used this glycoside in isolated
arteries and have observed that it produces a biphasic
contractile response. The first phase of tension induced
by ouabain in the VSM appears to be mediated by the
release of catecholamines as this compound also inhibits
the Na + /K + -ATPase located in nerve terminals. According
to these researchers, the second phase of tension induced
by ouabain would be that which may be linked to the
inhibition of Na + /K + -ATPase in the VSM sarcolemma
(Toda, 1980; Motley et al., 1993; Stewart et al., 1993).
Bearing in mind this information, before carrying out the
experiments with KCl and with the a1-adrenoceptor ago-
nists, in this study we have characterized the responses to
ouabain in rabbit aorta and we have determined the
moment when this drug causes the maximum inhibition
of the Na + /K + -ATPase of the smooth muscle of this
artery using physiological and supraphysiological extracel-
lular calcium concentrations.
2. Materials and methods
2.1. Preparation
Adult New Zealand white rabbits were sacrificed with 40
mg/kg iv pentobarbital sodium injected in the ear. The
thorax was opened and the aorta from the aortic arch to
the diaphragm was rapidly excised and transferred to a
beaker containing a low-bicarbonate physiological salt so-
lution of the following composition (mM): 118.2, NaCl; 4.7,
KCl; 2.5, CaCl2; 6.25, NaHCO; and 10.0 glucose. This
portion of the aorta was cleaned of surrounding connective
and fat tissue and cut into rings about 5 mm in width. The
aorta rings were suspended between two stainless steel
hooks immersed in 25-ml organ baths, which also contained
the same medium kept at 37 jC, and constantly bubbled
with 95% O2 and 5% CO2 (pH 7.3). The preparations were
mounted with a resting tension of 4 g and allowed to
equilibrate for a 90-min period. The protocol for the various
experiments appears below. The low bicarbonate concen-
tration in the bathing solution allows the calcium concen-
tration to increase without precipitation, and as we shall see
in some of these experiments after the stabilization period,
the 2.5-mM Ca2 + solution was replaced by another similar
one containing a 10-mM Ca2 + concentration.
2.2. Experimental protocol
In this study, we carried out two batches of trials. The
first was designed to characterize the responses to ouabain
in rabbit aorta rings with the physiological extracellular
Ca2 + concentration (2.5 mM) and to evaluate the possible
modification of these responses when extracellular Ca2 +
concentration increased to reach a much higher level (10
mM). Therefore, for these trials, after the equilibration
period, some preparations remained with the 2.5-mM
Ca2 + solution, and in others (as mentioned above), this
solution was changed for a similar one containing 10 mM
Ca2 + . We studied the 20-h course and magnitude of the
dose-dependent response to ouabain in the aorta ring prep-
arations incubated with the 2.5-mM Ca2 + solution. In these
experiments, ouabain was used in concentrations ranging
from 10� 7 to 10� 4 M, and individual rings were exposed
to only a single concentration of the drug. The results
obtained in these experiments using the different doses of
ouabain permitted us to choose the 10� 4-M concentration
of this drug to carry out the remaining experiments in our
study. This concentration of ouabain was also tested on
aorta ring preparations incubated with 2.5 mM Ca2 + , which
had been obtained from rabbits previously treated with
reserpine (1.5 mg kg� 1 day � 1 ip) for 3 days before being
sacrificed, following the method described by Berkowitz et
al. (1971). We also studied the 20-h course and magnitude
of the response to ouabain 10 � 4 M when the Ca2 +
concentration in the bath was 10 mM using preparations
from untreated and reserpine-treated rabbits. We always
measured the magnitude of the contractile response every
5 min for the first hour following ouabain administration,
and then every 15 min for the next 4 h, and every hour after
that.
The second batch of trials was designed to evaluate the
concentration-dependent responses to KCl and to the a1-
adrenoceptor agonists in rabbit aorta ring preparations
incubated with 2.5 mM Ca2 + and with 10 mM Ca2 + , some
A. Ortega, A. Aleixandre / Vascular Pharmacology 41 (2004) 75–81 77
of which had been pretreated with ouabain and some of
which had not. For the experiments with this drug, the
concentration 10� 4 M was administered in the organ bath
following the stabilization period, and KCl (30 and 80 mM)
or the a1-adrenoceptor agonist (10� 7–10� 4 M) was ad-
ministered in increasing concentrations 2 h and 15 min after
the ouabain administration. To make comparable the results
obtained with and without ouabain, in the latter, KCl and the
a1-adrenoceptor agonists were also added to the organ bath
2 h and 15 min after the equilibration period. As a1-
adrenoceptor agonists, methoxamine and phenylephrine
were used. In all these experiments, the responses produced
by the different administrations of KCl and a1-adrenoceptor
agonists were expressed in grams.
2.3. Drugs
The following drugs were used in this study: methox-
amine HCl (Sigma), phenylephrine HCl (Sigma), ouabain
(Sigma), and reserpine (Sigma). The drugs administered in
the organ bath were prepared and diluted in distilled water
daily. The reserpine solutions were also prepared daily but,
in this case, we used dimethylsulfoxide as a vehicle, and the
dose was always injected in 1 ml of solution.
2.4. Statistics
Data are expressed as mean valuesF S.E.M. for 7–9
experiments and the results of the experiments carried out
with KCl and with the a1-adrenoceptor agonists were
analyzed by one-way analysis of variance (ANOVA). Dif-
ferences between groups were assessed by the Bonferroni
test, considering these differences to be significant when
P < .05.
Fig. 1. Contractile responses to different concentrations of ouabain (OUA) (M
preparations incubated with 2.5 mM Ca2 + . The data represent the meanF S.E.M
3. Results
3.1. Characteristics of ouabain responses
Ouabain caused contractile responses of differing inten-
sity in preparations incubated with the 2.5-mM Ca2 +
solution. These responses also appeared at different times
depending on the concentration administered. Ouabain
10� 4 M induced a rapid early response in these prepara-
tions. This response was evident in minutes and in fact
reached the maximum within 30 min. It was characterized
as the first contractile response to ouabain 10� 4 M. After
this peak, there was a variable fall in vascular tone, but the
contractile plateau always remained above baseline, and
thereafter ouabain induced a secondary gradual rise in tone
followed by a well-sustained contraction that reached the
maximum 2 h and 15 min after administration. This
response was characterized as the second response to
ouabain 10� 4 M. After this second peak, there was a fall
in vascular tone and the preparations relaxed until the
baseline was reached. Ouabain concentrations of less than
10� 4 M did not induce the first early contractile response
in the 2.5-mM Ca2 + incubated preparations, and the
magnitude of the delayed contractile response in these
preparations decreased in a concentration-dependent man-
ner (see Fig. 1). The early response to ouabain 10� 4 M
did not appear either in the preparations from reserpinized
animals incubated with 2.5 mM Ca2 + , and the delayed
contractile response was also smaller in these preparations
than in those from non-reserpinized animals incubated with
2.5 mM Ca2 + (see Fig. 2).
When the Ca2 + concentration in the organ bath was
10 mM, the administration of ouabain 10 � 4 M also
caused an initial rapid contractile response and a second
): 10� 7 (x), 10� 6 (z), 10� 5 (E), and 10� 4 (.), in rabbit aorta ring
. for 8–10 experiments.
Fig. 3. Histograms of the contractions produced by 30 mM and 80 mM KCl
in different rabbit aorta ring preparations: nontreated incubated with 2.5
mM Ca2 + ( ), nontreated incubated with 10 mM Ca2 + ( ), 10� 4 M
ouabain (OUA) treated incubated with 2.5 mM Ca2 + ( ), and 10� 4 M
OUA treated incubated with 10 mM Ca2 + ( ). The data represent the
meanF S.E.M. for 8–10 experiments. aPV.05, bPV.001 versus nontreated
preparations incubated with 2.5 mM Ca2 + .
Fig. 2. Contractile responses to 10� 4 M ouabain (OUA) in aorta ring preparations from nontreated (.) and reserpinized rabbits (o) when the Ca2 +
concentration in the organ bath was 2.5 mM (left panel) and when the Ca2 + concentration in the organ bath was 10 mM (right panel). The data represent the
meanF S.E.M. for 8–10 experiments.
A. Ortega, A. Aleixandre / Vascular Pharmacology 41 (2004) 75–8178
delayed one. Nevertheless, both responses were signifi-
cantly smaller than the corresponding ones caused by this
dose of ouabain in preparations incubated with 2.5 mM
Ca2 + solution. As was the case when the concentration
of Ca2 + in the organ bath was 2.5 mM, the early rapid
response to ouabain 10 � 4 M did not appear in the
preparations from reserpinized animals incubated with
the 10-mM Ca2 + solution. However, in these prepara-
tions, the second responses to ouabain 10� 4 M was very
similar to the second response induced by this concentra-
tion of the drug in aorta rings from untreated rabbits (see
Fig. 2).
3.2. Results of the experiments using KCl and a1-adrenoceptor agonists
The contractions elicited by 30 and 80 mM KCl were
higher in aorta ring preparations incubated with the 2.5-
mM Ca2 + solution than in aorta ring preparations incu-
bated with the 10-mM Ca2 + solution. When the Ca2 +
concentration in the organ bath was 2.5 mM, 10� 4 M
ouabain administration caused a very clear decrease in the
responses to KCl; but when the Ca2 + concentration in the
organ bath was 10 mM, this same concentration of the
glycoside did not significantly modify the response to
KCl. As a consequence, KCl contractions were very
similar in all the ouabain-treated preparations, both those
incubated with the 2.5-mM Ca2 + solution and those
incubated with the 10-mM Ca2 + solution. Fig. 3 shows
the results with KCl.
As was the case with the contractions elicited by KCl,
the contractions elicited by the a1-adrenoceptor agonists,
methoxamine and phenylephrine, were higher in the aorta
ring preparations incubated with the 2.5-mM Ca2 + solu-
tion than in the aorta ring preparations incubated with the
10-mM Ca2 + solution. The contractions elicited by these
agonists when the Ca2 + concentration in the organ bath
was 2.5 mM were also smaller in the ouabain-treated
preparations than in those preparations untreated with this
drug. Moreover, when the Ca2 + concentration in the organ
bath was 10 mM, the contractions elicited by these
agonists were similar in the ouabain-treated preparations
and in those preparations not treated with this drug. In
addition, it should be emphasized that when the prepara-
tions were treated with ouabain, the contractions elicited
by the a1-adrenoceptor agonists were even higher when
the Ca2 + concentration in the organ bath was 10 mM than
when the Ca2 + concentration in the organ bath was
Fig. 4. Cumulative dose– response curves of methoxamine (upper panel)
and phenylephrine (lower panel) in different rabbit aorta ring preparations:
nontreated incubated with 2.5 mM Ca2 + (o), nontreated incubated with 10
mM Ca2 + ( ), 10 � 4 M ouabain (OUA) treated incubated with 2.5 mM
Ca2 + (.), and 10� 4 M OUA-treated incubated with 10 mM Ca2 + ( ).
The data represent the meanF S.E.M. for 8–10 experiments. The letters
show significant differences when comparing the maximal effects. aPV.05,bPV.001 versus nontreated preparations incubated with 2.5 mM Ca2 + ;cPV.001 versus nontreated preparations incubated with 10 mM Ca2 + ;dPV.001 versus 10� 4 M OUA-treated preparations incubated with 2.5 mM
Ca2 + .
A. Ortega, A. Aleixandre / Vascular Pharmacology 41 (2004) 75–81 79
2.5 mM. Fig. 4 shows the results with the a1-adrenoceptor
agonists.
4. Discussion
In this study we, have begun to characterize the
responses to different concentrations of ouabain in rabbit
aorta rings when the calcium concentration in the organ
bath was 2.5 mM. These experiments were carried out in
order to select a dose of this drug that, under physiolog-
ical conditions, would produce a definite blockage of the
Na + /K + -ATPase in the sarcolemma of the smooth muscle
of this artery and to fix the moment when the selected
dose produced maximum inhibition of this pump in the
preparations.
In the preparations incubated with the 2.5-mM Ca2 +
concentration, we tested four different doses of ouabain, and
the highest one 10� 4 M caused two contractile responses
that could be clearly distinguished by the moment at which
the increase in tone was produced following administration
of the drug (see Fig. 1). The first response, which appeared
shortly after ouabain 10� 4 M was added to the bath, did not
appear in preparations from reserpinized rabbits. In these
animals, the vesicles that contain noradrenaline had been
depleted. We can therefore conclude that this initial increase
in tone caused by ouabain in these conditions was linked to
the mass release of catecholamines when the Na + /K + -
ATPase in the nerve terminal was blocked. The concentra-
tion 10� 4 M of ouabain produced a second contractile
response in the preparations incubated with 2.5 mM Ca2 + ,
and the maximum increase in tone in this case was observed
sometime after the drug was administered (2 h and 15 min
later). Ouabain 10� 4 M also caused a late increase in tone
in the preparations from reserpinized rabbits, and this
displayed a coherent relation in time to the second contrac-
tile response caused by this concentration of the drug in
preparations from untreated animals. We may conclude that
this late contractile response, which is also observed when
the vesicles of catecholamines have been depleted with
reserpine, is fundamentally linked to the inhibition of
Na + /K + -ATPase in the sarcolemma of smooth muscle in
the rabbit aorta. However, it should be borne in mind that
the maximum increase in tone corresponding to the late
response to ouabain 10� 4 M in preparations from non-
reserpinized animals was greater than the maximum in-
crease in tone obtained when this dose of the glycoside
was administered in preparations from reserpinized animals.
This difference could be explained by the residual contrac-
tile effect of the catecholamines previously released by the
drug in preparations from non-reserpinized rabbits.
It should also be pointed out that concentrations of
ouabain less than 10� 4 M (10 � 5, 10� 6, and 10� 7 M)
only produced a late contractile response. This late increase
in tone decreases in a concentration-dependent manner
when different doses of ouabain were administered and
was hardly noticeable with the concentration 10� 7 M of
the drug. Experiments not shown in this paper permitted us
to show that ouabain (10 � 5–10� 7 M) also produced this
late increase in tone in rabbit aorta rings preparations from
reserpinized and non-reserpinized rabbits, and therefore this
response is also linked to the blockage of the Na + /K + -
ATPase in the sarcolemma of the VSM. In 1993, Motley et
al. (1993) and Stewart et al. (1993) also studied the effect of
A. Ortega, A. Aleixandre / Vascular Pharmacology 41 (2004) 75–8180
ouabain in rabbit aorta strips and in rabbit aorta rings,
respectively. These researchers also established that the
effect of ouabain on this tissue was concentration depen-
dent, and they also concluded that only the second response
to ouabain 10� 4 M in the rabbit aorta could be linked to the
inhibition of the Na + /K + -ATPase in the sarcolemma of the
VSM. Moreover, Motley et al. (1993) also indicated that this
concentration of ouabain was only twofold higher than the
concentration required to inhibit the Na + /K + -ATPase ac-
tivity in isolated sarcolemmal membranes of rabbit aorta.
When Ca2 + concentration in the organ bath was 10 mM,
ouabain also produced two contractile responses, an early
one which did not appear in reserpinized animals and a later
one which was not sensitive to reserpine treatment and
which was therefore due to the Na + /K + -ATPase inhibition
in the sarcolemma of the VSM. When the Ca2 + concentra-
tion in the organ bath was 10 mM, the late increase in tone
caused by ouabain 10� 4 M began approximately 1 h after
the drug administration and remain constant for about four
more hours (see Fig. 2). We can therefore assume that 2
h and 15 min after the addition of the glycoside to the organ
bath, there was also a definitive blockage of the sarcolem-
mal pump with such high extracellular Ca2 + concentrations.
All the results commented on above led us to select the
concentration 10� 4 M of ouabain to carry out the remaining
experiments in our study, in which we attempted to defin-
itively block the Na + /K + -ATPase in the sarcolemma of the
rabbit aorta. At the same time, all the abovementioned
experiments indicated to us that we should wait 2 h and
15 min after adding the drug to the organ bath in order to be
sure that this pump was inhibited in our experimental
conditions.
It is also true that these experiments in preparations
incubated with 10 mM Ca2 + allowed us to established that
ouabain 10� 4 M caused less intense contractile responses
when the extracellular Ca2 + concentration was high (see
Fig. 2). This would suggest that the supraphysiological
Ca2 + concentrations might produce opposite effects to
those of ouabain, thereby stimulating the functioning of
the Na + /K + -ATPase both in nerve terminals and in VSM.
The contractile responses to KCl and the a1-adrenoceptor
agonists were also smaller when the Ca2 + concentration in
the organ bath was 10 mM than when it was 2.5 mM. These
results confirmed that high extracellular Ca2 + concentration
lessens VSM contraction. We also observed that increasing
the extracellular Ca2 + concentration modified the KCl
contractions to a greater degree than the contractions caused
by a1-adrenoceptor agonists. This fact may be linked to the
sources of Ca2 + that these compounds use to produce VSM
contraction. As we are well aware, KCl contractions in the
VSM are due to extracellular Ca2 + entry into the cell, but
stimulation of the a1-adrenoceptors in this tissue also causes
the release of Ca2 + from the sarcoplasmic reticulum (Deth
and Casteels, 1977; Bolton, 1979; Van Breemen et al.,
1982). In 1998, Yildirim et al. (1991) in fact suggested that
high extracellular Ca2 + concentration provoked an increase
in Na + /K + -ATPase activity and thereby hyperpolarization
of the VSM membrane, which made Ca2 + entry through
voltage-gated channels more difficult. The decrease in
contraction when an a1-adrenoceptor agonist is adminis-
tered with high extracellular Ca2 + concentrations would be
consequence of a more indirect effect, but in the long term
these extracellular Ca2 + concentrations would also lead to a
drop in the amount of free cytoplasmic Ca2 + to be used in
the contraction.
We can interpret the results obtained with ouabain and
the contractile agents used in this study when the Ca2 +
concentration in the organ bath was 2.5 mM, also by bearing
in mind the modifications that this glycoside causes in
intracellular Ca2 + concentrations in the VSM. It should
be remembered that KCl and a1-adrenoceptor agonists were
administered at the exact moment when inhibition of the
Na + /K + -ATPase in the VSM preparations was at its great-
est. As a result of this inhibition of the pump, intracellular
Ca2 + levels and arterial tone had increased. In these
conditions, under which arterial tone had already increased
and free Ca2 + in the cytoplasm was greater than normal, it
was probably difficult to obtain an additional increase in this
ion by activating its entry from the extracellular space or its
release from the internal pools. Thus, the increase in
extracellular Ca2 + concentration and the administration of
ouabain to the preparations may have opposite effects on the
sarcolemmal Na + /K + -ATPase and may produce both a
decrease and an increase in the free Ca2 + concentration,
respectively, but both measures would bring about a less-
ening of KCl and a1-adrenoceptor agonist contractions. This
fact may seem paradoxical, but it should be borne in mind
that the decrease in contractile responses caused by high
Ca2 + concentrations, and that caused by ouabain, would be
in fact the consequence of their opposing effects on intra-
cellular Ca2 + levels. In the first case, the level of intracel-
lular Ca2 + to be used for contraction when KCl or a1-
adrenoceptor agonists were administered would be lower,
and in the second case the blockage of the sarcolemmal
Na + /K + -ATPase would make it difficult for this ion to
leave the cell and its accumulation inside the smooth muscle
would hinder a new additional contraction.
In order to explain the results obtained with ouabain and
the contractile agents when the Ca2 + concentration in the
organ bath was 10 mM (see Figs. 3 and 4), it should be
remembered that the contractile responses produced by the
administration of ouabain to the preparations that remained
incubated in a supraphysiological Ca2 + concentrations are
much smaller than those caused by this compound in
preparations incubated in the physiological concentration
of this ion. The increase in intracellular Ca2 + caused by
ouabain with such high extracellular Ca2 + concentrations
would be smaller than that caused by the glycoside with 2.5
mM extracellular Ca2 + concentration, and for this reason it
would be easier to bring about an additional increase in tone
in these conditions when KCl and the a1-adrenoceptor
agonists are administered.
A. Ortega, A. Aleixandre / Vascular Pharmacology 41 (2004) 75–81 81
It is therefore clear that in order to interpret our results, it
should always be assumed that the increase in extracellular
Ca2 + concentration may facilitate the functioning of the
Na + /K + -ATPase in the VSM and produces opposite effects
to ouabain. Although it should not be forgotten that these
extracellular Ca2 + concentrations may also inhibit the
release of catecholamines from the nerve terminals, the
results of this study permit us to conclude that the effect
of high extracellular Ca2 + concentration on the VSM
sarcolemmal pump may explain the decrease in the contrac-
tile responses elicited by depolarization and a1-adrenocep-
tor agonists stimulation observed in isolated artery prepara-
tions, and to be precise in rabbit aorta preparations, when
Ca2 + is increased in the organ bath.
Acknowledgements
This work was supported by CAM (08.4/0015.1/99) and
FIS (00/0925) grants.
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