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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: amaya@med.ucm.es (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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