Life Sciences 94 (2014) 2429

Contents lists available at ScienceDirect

Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Resistance exercise acutely enhances mesenteric artery insulin-induced

relaxation in healthy rats
M.T. Fontes a, T.L.B.T. Silva a, M.M. Mota a, A.S. Barreto a, L.V. Rossoni b, M.R.V. Santos a,
a
b

Department of Physiology, Federal University of Sergipe, 49100-000, So Cristvo, SE, Brazil

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, 05508-900, So Paulo, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 3 June 2013
Accepted 21 November 2013
Keywords:
Exercise
Mesenteric
Insulin
Vascular endothelium

a b s t r a c t
Aims: We evaluated the mechanisms involved in insulin-induced vasodilatation after acute resistance exercise in
healthy rats.
Main methods: Wistar rats were divided into 3 groups: control (CT), electrically stimulated (ES) and resistance
exercise (RE). Immediately after acute RE (15 sets with 10 repetitions at 70% of maximal intensity), the animals
were sacriced and rings of mesenteric artery were mounted in an isometric system. After this, concentration
response curves to insulin were performed in control condition and in the presence of LY294002 (PI3K inhibitor),
L-NAME (NOS inhibitor), L-NAME + TEA (K+ channels inhibitor), LY294002 + BQ123 (ET-A antagonist) or
ouabain (Na+/K+ ATPase inhibitor).
Key ndings: Acute RE increased insulin-induced vasorelaxation as compared to control (CT: Rmax = 7.3 0.4%
and RE: Rmax = 15.8 0.8%; p b 0.001). NOS inhibition reduced (p b 0.001) this vasorelaxation from both
groups (CT: Rmax = 2.0 0.3%, and RE: Rmax = 1.2 0.1%), while PI3K inhibition abolished the vasorelaxation in CT (Rmax = 0.1 0.3%, p b 0.001), and caused vasoconstriction in RE (Rmax = 6.5 0.6%). That
insulin-induced vasoconstriction on PI3K inhibition was abolished (p b 0.001) by the ET-A antagonist
(Rmax = 2.9 0.4%). Additionally, acute RE enhanced (p b 0.001) the functional activity of the ouabainsensitive Na+/K+ ATPase activity (Rmax = 10.7 0.4%) and of the K+ channels (Rmax = 6.1 0.5%;
p b 0.001) in the insulin-induced vasorelaxation as compared to CT.
Signicance: Such results suggest that acute RE promotes enhanced insulin-induced vasodilatation, which could
act as a ne tuning to vascular tone.
2014 Elsevier Inc. All rights reserved.

Introduction
Several authors have demonstrated the ability of exercise to prevent
cardiovascular risk factors, among them endothelial dysfunction (Di
Francescomarino et al., 2009; Golbidi and Laher, in press; Green et al.,
2004; Zanesco and Antunes, 2007). The literature has demonstrated
the ability of both chronic and acute aerobic exercise to improve the
insulin signaling pathway involved not only in the glucose metabolism
but also in the vascular modulation (Caponi et al., 2013; Pauli et al.,
2010; Yang et al., 2006, 2010). In particular, resistance exercise has
been also used for improvement of diabetes, hypertension and obesity
(Westcott, 2012). Nevertheless, the signaling pathways are not clear.
Hemodynamic effects of insulin occur for two different endotheliumdependent signaling pathways: IR/PI3K/eNOS, responsible for the relaxant
effect, and IR/MAPK/ET-1, responsible for the contractile effect (Chaudhuri
et al., 2012; Montagnani et al., 2001; Muniyappa and Quon, 2007; Salt,
2013). Thus, the balance between the release of NO and ET-1 plays an
Corresponding author at: Federal University of Sergipe, Department of Physiology,
Cidade Universitria Prof. Jos Alosio de Campos, Rosa Elze, Cep: 49100-000, So
Cristvo, Sergipe, Brazil. Tel.: +55 79 21056842.
E-mail address: marcio@infonet.com.br (M.R.V. Santos).
0024-3205/$ see front matter 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.lfs.2013.11.017

important role for the control of vascular tone and blood ow adjustments
in response to the exercise (Mather et al., 2001; Muniyappa and Sowers.,
2013).
Previous studies have shown that insulin-induced vasorelaxation is
enhanced in animals after aerobic exercise. This enhancement is caused
by the increase of NO release, associated to K+ channels-induced hyperpolarization (Ghafouri et al., 2011; Rossi et al., 2005; Yang et al., 2006,
2010). Additionally, Aughey et al. (2007) showed that aerobic exercise can change the activity and expression of skeletal muscle Na+/
K+-ATPase in humans. In vascular smooth muscle, Garland et al.
(2011), Marn and Redondo (1999), and Smith et al. (1997) demonstrated that Na+/K+-ATPase activity may be inuenced by the endothelium and K+ channels. However, there are no data in the literature
showing the effect of resistance exercise on the insulin-induced relaxation nor the pathways involved in this response.
Previous research has shown the ability of resistance exercise to
promote changes in vascular function in rats (Faria Tde et al., 2010;
Harris et al., 2010). Interestingly, these changes can be produced in
blood vessel far from the skeletal muscle used during the exercise,
such as mesenteric or caudal vascular beds (Arajo et al., 2013; Faria
Tde et al., 2010). Moreover, it is related in the literature that results
obtained in mesenteric vascular bed may have physiological relevance

M.T. Fontes et al. / Life Sciences 94 (2014) 2429

for the entire cardiovascular system (Dohi et al., 1994; Subramanian

and MacLeod, 2003). These studies evaluated the signaling pathway
stimulated by acetylcholine, which is an intracellular calcium-dependent
pathway. On the other hand, the signaling pathway stimulated by insulin
promotes hemodynamic effects without changes intracellular calcium
(Fleming and Busse, 1999).
Therefore, considering it has already been related that resistance
exercise may change the metabolic effects of insulin by the IR/PI3K
signaling pathway (Krisan et al., 2004; Yaspelkis, 2006), the objective
of this study was to evaluate the mechanisms involved in the vasorelaxation induced by insulin after acute resistance exercise in healthy
animals.

Material and methods

Animals
Three-month-old male Wistar rats were obtained from the Central
Animal Facility of the Federal University of Sergipe. Rats were kept
in collective cages (5 animals/cage), temperature-controlled room
(22 2 C) with 12 h light/12 h dark cycle, and received commercial
rodent chow (Labina Purina) and ltered water ad libitum, with free
access to food and water. The rats were randomized into four groups:
control (CT, n = 20), electrically stimulated (ES, n = 5) and resistance
exercise (RE, n = 20). All procedures described in this study are in
agreement with the Brazilian Society of Laboratory Animal Science
and were approved by the Ethics Committee on Animal Research of
the Federal University of Sergipe, Brazil.

Resistance exercise protocol

Animals were exercised following a model described by Tamaki et al.
(1992). Rats in the ER and ES groups were wearing a canvas jacket to be
able to regulate the twisting and exion of their torsos and were xed
by a holder in a standing position on their hinder limbs (Tamaki et al.,
1992; Barauna et al., 2005; Pinter et al., 2008). Electrical stimulation
(20 V, 0.3 s duration, at 3 s intervals) was applied to the tail of the rat
through a surface electrode. The animals underwent three days of
familiarization, where they were placed in the device in the exercise
apparatus starting position and were kept this way for 5 min in order to
reduce the stress caused to the animal for the equipment and handling.
After the familiarization period, the animals performed the test of a maximum repetition (1RM), which consisted in determining the maximum
weight lifted by each rat in the exercise apparatus. The 1RM test is used
to assess maximal muscle strength in humans and animals (ACSM,
2009). After 2 days, the animals were subjected to the exercise protocol.
The RE group was exercised through 15 sets of 10 repetitions with a
180 s resting period between each set, with the intensity of 70% of 1RM.
The animals extended their legs repeatedly, which lifted the weight on
the arm of the exercise apparatus. Animals from ES group underwent
the same conditions of the animals from RE, but without leg extension
movements. The CT group was not subjected to any of these procedures
(Fig. 1).

25

Vascular reactivity studies

Following animal sacrice, the superior mesenteric artery was
removed, stripped from connective and fatty tissues and sectioned
into rings (12 mm). Rings were suspended from ne stainless steel
hooks, connected to a force transducer (Letica, Model TRI210; Barcelona,
Spain) with cotton threads in organ baths containing 10 mL of Tyrode's
solution (composition in mM: NaCl 158.3, KCl 4.0, CaCl2 2.0, NaHCO3
10.0, C6H12O6 5.6, MgCl2 1.05 and NaH2 PO4 0.42). This solution was
continually gassed with carbogen (95% O2 and 5% CO2) and maintained
at 37 C under a resting tension of 0.75 g for 60 min (stabilization
period). During this time, the nutrient solution was changed every
15 min to prevent the interference of metabolites (Altura and Altura,
1970). Isometric tension was recorded through the force transducer
(TRI210, Letica, Barcelona, Spain) coupled to an amplier-recorder
(BD-01, AVS, SP, Brazil).
The functionality of the endothelium was assessed by the ability of acetylcholine (ACh, 1 M) to induce more than 75% relaxation of
phenylephrine (Phe, 1 M)-induced pre-contraction. After that, changes
in vascular reactivity were assessed by obtaining concentrationresponse
curves for insulin (1013106 M).
These same curves were obtained after incubation for 30 min of the following inhibitors: L-NAME was used to evaluate the role of NO (inhibitor of
nitric oxide synthase; 100 M); LY294002, to evaluate the role of the PI3K
pathway (inhibitor of PI3K; 50 M); L-NAME + tetraethylammonium
(TEA), to evaluate the role of NO and K+ channels (potassium channel
inhibitor; 10 M); BQ123, to evaluate the role of endothelin-1 (a selective ETA endothelin receptor antagonist; 1 M); or ouabain, to evaluate
the role of Na+/K+-ATPase activity in the relaxation (Na+/K+-ATPase
inhibitor; 100 M) induced by insulin.
The functional activity of the ouabain-sensitive Na+/K+-ATPase was
indirectly measured in another set of experiments using the method
described by Rossoni et al. (2002). After stabilization in Tyrode's solution with 4.0 mM K+, the arteries were incubated in a K+-free medium
for 30 min. The vessels were then contracted using phenylephrine and
when a plateau was reached, KCl (110 mM) was cumulatively added
to the bath. To evaluate the functional activity of the ouabain-sensitive
Na+/K+-ATPase in these responses, the concentrationresponse curve
to K+ was subsequently determined in vessels pre-incubated for
30 min in a K+-free medium with ouabain (100 M).
It is important to note that each ring received only one inhibitor, and
the cumulative concentrationresponse curve for insulin or K+ was
obtained before and after the inhibitor treatment.
Statistical analysis
Values were expressed as the mean standard error of the mean
(SEM). The maximum response (Rmax values) was calculated by a nonlinear regression analysis of each individual concentrationresponse
curve. AUC were calculated from the individual concentrationresponse
curve plot. Differences of area under the concentrationresponse curves
(dAUC) were expressed between the presence and absence of inhibitors
and were expressed as a percentage of AUC of the corresponding control
situation. Differences between groups were determined using one- or
two-way ANOVA followed by Bonferroni's test or Student's t test, as appropriate (p b 0.05). Statistical analysis was performed using GraphPad
Prism software (San Diego, CA, USA).
Results
Insulin-induced relaxation

Fig. 1. Diagram showing the resistance exercise protocol.

Insulin (1013106 M) induced smaller, yet signicant, relaxation in

a concentration-dependent manner in all groups (Fig. 2). The relaxation
induced by insulin was unaltered in the ES group (Rmax = 7.7 0.5%)
as compared to CT group (Rmax = 7.3 0.4%); however, it was

26

M.T. Fontes et al. / Life Sciences 94 (2014) 2429

curves to insulin were obtained in the presence of LY294002 plus

BQ123. The vascular effect of insulin was inhibited in both groups
(CT: Rmax = 7.3 0.4% to 2.0 0.2%, p b 0.001 and RE: Rmax =
15.8 0.8% to 2.9 0.4%, p b 0.001) (Fig. 3B). Moreover, a comparison
of dAUC values between groups revealed that the effect of incubation of
rings with LY294002 plus BQ123 was increased in the RE group
(91.2 5.5%) relative to the CT group (27.6 9.2%) (p b 0.001; inset
in Fig. 3B).
Roles of NOS and K+ channels in insulin-induced relaxation

signicantly enhanced in superior mesenteric arteries from RE group

(Rmax = 15.8 0.8%; p b 0.001) as compared to ES and CT group.
After this set of experiments, as the relaxation induced by insulin
was similar in mesenteric superior arteries from ES and CT groups
(Fig. 2), we performed the experiments using arteries from CT and RE
groups.

NOS inhibition with L-NAME reduced insulin-induced relaxation in

superior mesenteric arteries from both groups (CT: Rmax = 7.3 0.4%
to 2.0 0.3%, p b 0.001 and RE: Rmax = 15.8 0.8% to 1.2 0.1%,
p b 0.001) (Fig. 4A). A comparison of dAUC values indicated that the
involvement of NOS is higher in the RE group (79.6 4.8%), relative
to the CT group (33.9 5.9%) (inset in Fig. 4A; p b 0.001). As described
in the present introduction section, previous studies have shown that
K+ channels are involved in insulin-induced relaxation. We therefore
pre-incubated the rings with L-NAME plus TEA (a nonselective inhibitor
of K+ channels) to assess the role of these channels in the relaxation
induced by insulin. As observed using the PI3K inhibitor, relaxation
induced by insulin was blocked in the CT group upon treatment with
L-NAME plus TEA (Fig. 4B); while in the RE group this treatment resulted
in a smaller but sustained contraction (CT: Rmax = 7.3 0.4% to
1.3 0.6%, p b 0.001 and RE: Rmax = 15.8 0.8% to 6.1 0.5%,
p b 0.001) (Fig. 4B). A comparison of dAUC values revealed that the
involvement of NOS and K+ channels is higher in RE group (115.4
7.5%) as compared to CT group (45.5 9.0%) (inset in Fig. 4B; p b 0.001).

Roles of PI3K and ET-1 in insulin-induced relaxation

Functional activity of the ouabain-sensitive Na+/K+-ATPase and the role of

Na+/K+-ATPase activity on insulin-induced relaxation

LY294002, the PI3K inhibitor, blocked the relaxation in the CT

group (Fig. 3A). On the other hand, in the presence of LY294002,
insulin caused contraction of superior mesenteric arteries from RE
group (CT: Rmax = 7.3 0.4% to 0.1 0.3%, p b 0.001 and RE:
Rmax = 15.8 0.8% to 6.5 0.6%, p b 0.001) (Fig. 3A). The dAUC
values indicated the enhanced role of PI3K in the vasodilatation induced
by insulin in RE group (69.1 7.1%) as compared to CT group
(36.3 7.9%) (inset in Fig. 3A; p b 0.01). To evaluate the possible role
of ET-1 in the relaxation- and/or contraction-induced by insulin in the
absence or presence of LY294002, respectively; concentrationresponse

As showed in Fig. 5A, increased extracellular K+ concentration

(110 mM) induced relaxation to a similar extent in superior mesenteric arteries from CT and RE groups (CT: Rmax = 107.5 11.3% and
RE: Rmax = 106.5 4.5%) (Fig. 5A). In addition, ouabain (an inhibitor
of Na+/K+-ATPase activity) signicantly reduced K+-induced relaxation
in arteries from both groups (CT: Rmax = 107.5 11.3% to 79.6 7.2%,
p b 0.001 and RE: Rmax = 106.5 4.5% to 68.6 7.6%, p b 0.001)
(Fig. 5A). A comparison of dAUC values indicated that functional activity
of the ouabain-sensitive Na+/K+-ATPase was enhanced in superior
mesenteric arteries from RE group (82.6 16.3%) as compared to CT

Fig. 2. Concentrationresponse curve to insulin in intact segments obtained from the superior
mesenteric artery of Wistar rats and pre-contracted with phenylephrine (1 M) in the
control (CT), electrically stimulated (ES) and resistance exercise (RE) groups. The results
are expressed as the mean SEM for 810 experiments in each group. ++p b 0.01,
+++
p b 0.001; CT vs. ER.

Fig. 3. Concentrationresponse curve for the response to insulin in intact segments obtained from the superior mesenteric artery of Wistar rats and pre-contracted with phenylephrine
(1 M) for the control (CT) and resistance exercise (RE) groups in the absence or presence of LY294002 (50 M) (A) and in the absence or presence of LY294002 plus BQ123 (50 M
and 10 M, respectively) (B). The results are expressed as the mean SEM for 810 experiments in each group. The difference in the area under the concentrationresponse curve
(dAUC) for the response to insulin in the CT and RE groups is shown; dAUC is expressed as a percentage of the corresponding AUC in the inset. A: **p b 0.01, ***p b 0.001, CT vs. CT
LY294002; or +++p b 0.001, RE vs. RE LY294002. B: **p b 0.01, ***p b 0.001, CT vs. CT LY294002 + BQ123; +p b 0.05, +++p b 0.001, RE vs. RE LY294002 + BQ123. dAUC: ***p b 0.001.

M.T. Fontes et al. / Life Sciences 94 (2014) 2429

27

Fig. 4. Concentrationresponse curve for the response to insulin in intact segments obtained from the superior mesenteric artery of Wistar rats and pre-contracted with phenylephrine
(1 M) in the control (CT) and resistance exercise (RE) groups in the absence or presence of L-NAME (100 M; A) and in the absence or presence of L-NAME + TEA (100 M and
10 M, respectively; B). The results are expressed as the mean SEM for 810 experiments in each group. The difference in the area under the concentrationresponse curve (dAUC)
for the response to insulin is shown for the CT and RE groups; dAUC is expressed as a percentage of the corresponding AUC in the inset. A: **p b 0.01, ***p b 0.001, CT vs. CT L-NAME;
+
p b 0.05, +++p b 0.001, RE vs. RE L-NAME. B: *p b 0.05, ***p b 0.001, CT vs. CT L-NAME + TEA; +p b 0.05, +++p b 0.001, RE vs. RE L-NAME + TEA. dAUC: ***p b 0.001, **p b 0.01.

group (53.0 16.2%) (inset in Fig. 5A; p b 0.05). In addition, ouabain

signicantly reduced insulin-induced relaxation only in the RE group
(CT: Rmax = 7.3 0.4% to 5.7 0.2%, and RE: Rmax = 15.8 0.8% to
10.7 0.4%, p b 0.001) (Fig. 5B). A comparison of dAUC showed no
signicant involvement of Na+/K+-ATPase on the relaxation induced
by insulin between CT group (6.2 5.4%) and RE group (10.8 4.9%)
(inset in Fig. 5B).
Discussion
The results suggest that resistance exercise increased insulin-induced
relaxation via PI3K/eNOS. Moreover, an increase in K+ channel participation and functional activity of the ouabain-sensitive Na+/K+-ATPase
were observed in the relaxation of the superior mesenteric arteries from
animals subjected to exercise. Our study was the rst to demonstrate
that acute resistance exercise increases insulin-induced relaxation in
superior mesenteric arteries from healthy rats.
The exercise model used in the present study has been reported to
promote cardiovascular adjustments that are benecial to health
(Arajo et al., 2013; Barauna et al., 2005; Faria Tde et al., 2010). It is
important to note that the enhanced insulin-induced relaxation observed
in arteries from resistance exercise is independent on electric stimulation,

since the results obtained in the superior mesenteric artery from ES

group were similar to those observed in the control group, conrming
that the acute effects observed in this study are directly related to resistance exercise.
Insulin directly contributes to the maintenance of homeostasis and
vascular tone (Mather et al., 2001; Muniyappa et al., 2007; Ren et al.,
1999). The physiological effect of insulin on the different vascular beds
consists of vasodilation combined with an increase in NO production
through the activation of the PI3K/eNOS signaling pathway (Muniyappa
et al., 2007; Muniyappa and Quon, 2007; Yu et al., 2011). In addition to
the direct effect of this hormone, it has been broadly established that
aerobic exercise can promote molecular adjustments that result in
an increase in the degree of phosphorylation and activity of the Akt
protein, which consequently promotes an increase in the phosphorylation of serine residues in position 1177 of the eNOS. The events are
concurrent with the increase in bioavailability of NO and relaxation of
insulin-responding vascular beds by the activation of the PI3K/eNOS
pathway (Barbosa et al., 2013; Hambrecht et al., 2003; Wang et al.,
2010; Yang et al., 2006, 2008).
NO plays a key role in the control of vascular tone by acting as the
main inducer of relaxation in conductance vascular beds (Sudhir et al.,
1994). We observed a reversion on insulin-induced relaxation in the

Fig. 5. Concentrationresponse curve for the response to KCl in intact segments obtained from the superior mesenteric artery of Wistar rats and previously incubated in a K+-free medium
(A) and insulin (B) and pre-contracted with phenylephrine (1 M) for the control (CT) and resistance exercise (RE) groups in the absence or presence of ouabain (100 M). The variation
in the area under the concentrationresponse curve (dAUC) for the response to insulin in the CT and RE groups is shown; dAUC is expressed as a percentage of the corresponding AUC in
the insert. The results are expressed as mean SEM for 810 experiments in each group. A: **p b 0.01, ***p b 0.001, CT vs. CT ouabain; +++p b 0.001, RE vs. RE ouabain. 5B: ++p b 0.01,
RE vs. RE ouabain. dAUC: *p b 0.05.

28

M.T. Fontes et al. / Life Sciences 94 (2014) 2429

presence of L-NAME (NO synthesis inhibitor) in arteries from both

groups. However, in the RE group, this reversion was more intense.
Such a result can demonstrate that RE promoted an increase in NO bioavailability probably involving the activation of the PI3K/eNOS pathway.
Concordant results were found by Yang et al (2006) and Yang et al
(2008), who demonstrated that the exercise-enhanced vasorelaxation
was abolished for L-NAME. However, the increase in NO bioavailability
may also be the result of shear stress triggered by physical exercise;
this event may contribute to a synergistic effect with the PI3K/eNOS
pathway in maintaining relaxation (Boo et al., 2002; Dimmeler et al.,
1999; Padilla et al., 2011).
We evaluated insulin-induced vascular effect in the presence of
LY294002 (PI3K inhibitor) and found that rings from RE animals presented
vasoconstriction. This response can be explained by blood ow redistribution (reactive hyperemia) that occurs during and immediately after the
execution of RE to the required tissues during exercise, increasing shear
stress and consequently the activation of the signaling pathway PI3K/eNOS.
The literature states that insulin may induce vasoconstriction by an
endothelium-dependent mechanism through the activation of the
MAPK/ET-1 pathway (Cardillo et al., 2000; Jiang et al., 2003). This vasoconstriction may be due to a predominance of the MAPK/ET-1 on PI3K/
eNOS pathway. To corroborate this hypothesis, we used BQ123, an ET-A
antagonist, in the presence of LY294002. In this condition, we found that
insulin-induced vasoconstriction was completely inhibited, suggesting
that this effect appears to involve the activation of ET-A receptor
(Mather et al., 2001, 2013; Muniyappa et al., 2007).
Although the activation of ET-A receptor can be involved in the vasoconstriction induced by insulin in arterial rings from exercised animals
in the presence LY294002, the vasorelaxation promoted by this hormone is predominant. This relaxation response can be explained by
blood ow redistribution (reactive hyperemia) that occurs during and
immediately after the execution of RE to the required tissues during
exercise, increasing shear stress and consequently the activation of
the signaling pathway PI3K/eNOS. That suggests that the activation of
ET-A receptor appears to have no negative effect on vascular reactivity
for insulin. Some authors have shown that under normal conditions,
the effect of ET-A activation is compensated by an increase in NO
bioavailability promoted by the activation of the PI3K/eNOS pathway
(Cardillo et al., 2000; Ferri et al., 1995; Muniyappa et al., 2008;
Wolpert et al., 1993). Therefore, it is possible that after RE, the maintenance of vascular tone is ensured by the adjustments promoted by the
PI3K/eNOS and MAPK/ET-1 pathways, and the ow redistribution for
the exercised muscle is dependent on the balance between them
(Mikus et al., 2012).
The literature suggests that NO also acts in ion channel regulation,
such as in ATP-sensitive (KATP) or calcium-sensitive (K2+
Ca ) potassium
channels. When activated, these channels promote hyperpolarization
that ultimately results in relaxation (Bolotina et al., 1994; Fltou and
Vanhoutte, 1999; McKay and Hester, 1996). Some authors suggest that
K+ channels activated during aerobic exercise play a cardioprotective role
(Merkus et al., 2006; Shi et al., 2013). To assess the involvement of these
channels in insulin-induced relaxation, we carried out experiments in
which the K+ channels were blocked with TEA, a non-selective K+ channel
blocker, in the presence of L-NAME. Similar to the results obtained with
LY294002, the concentrationresponse curve for insulin in the presence
of L-NAME plus TEA also induced contraction in exercised animals. That
indicates that resistance exercise increases K+-channel participation in
insulin-induced response and also that this effect was similar to what
was found in experimental models using aerobic exercise (Shi et al.,
2013; Yasui et al., 2008). Such ndings also suggest that in exercised
animals, improvement in vascular function via IR/PI3K/eNOS is caused
by NO and partially mediated by K+ channels.
The Na+/K+-ATPase also plays a key role in controlling vascular tone
by ensuring the Na+ and K+ electrochemical gradient; consequently,
it acts in the maintenance of smooth muscle cell hyperpolarization
and contributes to relaxation of the beds (Golub et al., 1998; Marn

and Redondo, 1999; Pagn et al., 2010). Recent studies have shown
that lower activity and/or lower insulin stimulation over the Na+/
K+-ATPase is associated with hypertension and obesity (dos Santos
et al., 2003; Rossoni et al., 2003; Sweeney and Klip, 1998). In the present study, the functionality and participation of the Na+/K+-ATPase
activity were indirectly measured. These experiments were conducted
in the presence and absence of ouabain, a Na+/K+-ATPase inhibitor.
We observed that exercised animals showed an increase in the functional
activity and participation of the ouabain-sensitive Na+/K+-ATPase in
relaxation-induced by insulin. Such evidence is due, in part, to the
resistance exercise protocol used in this study (high-intensity, highvolume sessions). Other authors have reported that increased exercise
intensity is fundamental in promoting adjustments in Na+/K+-ATPase
functionality (Aughey et al., 2007; Rasmussen et al., 2011). Therefore,
an increase in Na+/K+-ATPase activity seems to contribute to the maintenance of vascular tone control after resistance exercise.
Conclusion
Acute resistance exercise promotes adjustments to insulin-induced vascular reactivity through both the PI3K/eNOS and MAPK/ET-1 pathways. In
addition to these effects, there was a predominance of insulin-induced
relaxation effects, and that was due, in part, to the increase in NO bioavailability mediated by both the opening of K+ channels as well as
Na+/K+-ATPase activation. Such adjustments are necessary for maintaining vascular tone control after an acute resistance exercise.
Conict of interest statement
The authors have no conicts of interest to declare.

Acknowledgments
This work was supported by the Fundao de Apoio Pesquisa e
Inovao Tecnolgica do Estado de Sergipe (FAPITEC/SE), the Conselho
Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq), and the
Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES).
We also would like to thank teacher Abilio Borghi for the grammar review
on the English manuscript.
References
Altura BM, Altura BT. Calcium content and force of drug-induced contractions of arterial
muscle during recovery in vitro. Proc Soc Exp Biol Med 1970;135:73944.
American College of Sports Medicine (ACSM). American College of Sports Medicine position stand. Progression models in resistance training for healthy adults. Med Sci
Sports Exerc 2009;41:687708.
Arajo AJS, Santos ACV, Souza KS, Aires MB, Santana-Filho VJ, Fioretto ET, et al. Resistance
training controls arterial blood pressure in rats with L-NAME-induced hypertension.
Arq Bras Cardiol 2013;100:33946.
Aughey RJ, Murphy KT, Clark SA, Garnham AP, Snow RJ, Cameron-Smith D, et al. Muscle
Na+K+-ATPase activity and isoform adaptations to intense interval exercise and
training in well-trained athletes. J Appl Physiol 2007;103:3947.
Barauna VG, Batista Jr ML, Costa Rosa LF, Casarini DE, Krieger JE, Oliveira EM. Cardiovascular adaptations in rats submitted to a resistance-training model. Clin Exp
Pharmacol Physiol 2005;32:24954.
Barbosa VA, Luciano TF, Marques SO, Vitto MF, Souza DR, Silva LA, et al. Acute exercise induce endothelial nitric oxide synthase phosphorylation via Akt and AMP-activated
protein kinase in aorta of rats: role of reactive oxygen species. Int J Cardiol 2013;
167:29838.
Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates
calcium-dependent potassium channels in vascular smooth muscle. Nature 1994;368:
8503.
Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, et al. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent
mechanisms: role of protein kinase A. J Biol Chem 2002;277:338896.
Caponi PW, Lehnen AM, Pinto GH, Borges J, Markoski M, Machado UF, et al. Aerobic exercise training induces metabolic benets in rats with metabolic syndrome independent of dietary changes. Clinics 2013;68:10107.
Cardillo C, Kilcoyne CM, Cannon RO, Panza JA. Interactions between nitric oxide and
endothelin in the regulation of vascular tone of human resistance vessels in vivo.
Hypertension 2000;35:123741.
Chaudhuri A, Dandona P, Fonseca V. Cardiovascular benets of exogenous insulin. J Clin
Endocrinol Metab 2012;97:307991.

M.T. Fontes et al. / Life Sciences 94 (2014) 2429

Di Francescomarino S, Sciartilli A, Di Valerio V, Di Baldassarre A, Gallina S. The effect of
physical exercise on endothelial function. Sports Med 2009;39:797812.
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric
oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature
1999;399:6015.
Dohi Y, Criscione L, Pfeiffer K, Luscher TF. Angiotensin blockade or calcium antagonists
improve endothelial dysfunction in hypertension: studies in perfused mesenteric resistance arteries. J Cardiovasc Pharmacol 1994;24:3729.
Dos Santos L, Xavier FE, Vassallo DV, Rossoni LV. Cyclooxygenase pathway is involved in
the vascular reactivity and inhibition of the Na+, K+-ATPase activity in the tail artery
from L-NAME-treated rats. Life Sci 2003;74:61327.
Faria Tde O, Targueta GP, Angeli JK, Almeida EA, Stefanon I, Vassallo DV, et al. Acute resistance exercise reduces blood pressure and vascular reactivity, and increases
endothelium-dependent relaxation in spontaneously hypertensive rats. Eur J Appl
Physiol 2010;110:35966.
Fltou M, Vanhoutte PM. The third pathway: endothelium-dependent hyperpolarization. J Physiol Pharmacol 1999;50:52534.
Ferri C, Pittoni V, Piccoli A, Laurenti O, Cassone MR, Bellini C, et al. Insulin stimulates
endothelin-1 secretion from human endothelial cells and modulates its circulating
levels in vivo. J Clin Endocrinol Metab 1995;80:82935.
Fleming I, Busse R. Signal transduction of eNOS activation. Cardiovasc Res 1999;43:53241.
Garland CJ, Hiley CR, Dora KA. EDHF: spreading the inuence of the endothelium. Br J
Pharmacol 2011;164:83952.
Ghafouri S, Hajizadeh S, Mani AR. Enhancement of insulin-induced cutaneous vasorelaxation by exercise in rats: a role for nitric oxide and K(Ca2 +) channels. Eur J
Pharmacol 2011;652:8995.
Golbidi S, Laher I. Exercise and the cardiovascular system. Cardiol Res Pract 2013. [in press].
Golub MS, Chang CT, Tuck ML, Berger ME. Evidence for increased functional vascular
Na+/K+ pump activity in the obese Zucker rat. Hypertens Res 1998;21:2838.
Green DJ, Maiorana A, O'Driscoll G, Taylor R. Effect of exercise training on
endothelium-derived nitric oxide function in humans. J Physiol 2004;561:125.
Hambrecht R, Adams V, Erbs S, Linke A, Krnkel N, Shu Y, et al. Regular physical activity
improves endothelial function in patients with coronary artery disease by increasing
phosphorylation of endothelial nitric oxide synthase. Circulation 2003;107:31528.
Harris MB, Slack KN, Prestosa DT, Hryvniak DJ. Resistance training improves femoral
artery endothelial dysfunction in aged rats. Eur J Appl Physiol 2010;108:53340.
Jiang ZY, He Z, King BL, Kuroki T, Opland DM, Suzuma K, et al. Characterization of multiple
signaling pathways of insulin in the regulation of vascular endothelial growth factor
expression in vascular cells and angiogenesis. J Biol Chem 2003;278:3196471.
Krisan AD, Collins DE, Crain AM, Kwong CC, Singh MK, Bernard JR, et al. Resistance training enhances components of the insulin signaling cascade in normal and high-fat-fed
rodent skeletal muscle. J Appl Physiol 2004;96:1691700.
Marn J, Redondo J. Vascular sodium pump: endothelial modulation and alterations in
some pathological processes and aging. Pharmacol Ther 1999;84:24971.
Mather KJ, Anderson T, Vermac S. Insulin action in the vasculature: physiology and pathophysiology. J Vasc Res 2001;38:41522.
Mather KJ, Steinberg HO, Baron AD. Insulin resistance in the vasculature. J Clin Invest
2013;123(3):10034.
McKay MK, Hester RL. Role of nitric oxide, adenosine, and ATP-sensitive potassium channels in insulin-induced vasodilation. Hypertension 1996;28:2028.
Merkus D, Sorop O, Houweling B, Hoogteijling BA, Duncker DJ. K+
Ca channels contribute to
exercise-induced coronary vasodilation in swine. Am J Physiol Heart Circ Physiol
2006;291:H20907.
Mikus CR, Roseguini BT, Uptergrove GM, Morris EM, Rector RS, Libla JL, et al. Voluntary
wheel running selectively augments insulin-stimulated vasodilation in arterioles
from white skeletal muscle of insulin-resistant rats. Microcirculation 2012;19:72938.
Montagnani M, Chen H, Barr VA, Quon MJ. Insulin-stimulated activation of eNOS is independent of Ca2 + but requires phosphorylation by Akt at Ser(1179). J Biol Chem
2001;276:303928.
Muniyappa R, Quon MJ. Insulin action and insulin resistance in vascular endothelium.
Curr Opin Clin Nutr Metab Care 2007;10:52330.
Muniyappa R, Sowers JR. Role of insulin resistance in endothelial dysfunction. Rev Endocr
Metab Disord 2013;14:512.
Muniyappa R, Montagnani M, Koh KK, Quon MJ. Cardiovascular actions of insulin. Endocr
Rev 2007;28:46391.
Muniyappa R, Iantorno M, Quon MJ. An integrated view of insulin resistance and endothelial dysfunction. Endocrinol Metab Clin North Am 2008;37:685711.

29

Padilla J, Simmons GH, Bender SB, Arce-Esquivel AA, Whyte JJ, Laughlin MH. Vascular
effects of exercise: endothelial adaptations beyond active muscle beds. Physiology
(Bethesda) 2011;26:13245.
Pagn RM, Prieto D, Hernndez M, Correa C, Garca-Sacristn A, Benedito S, et al. Regulation
of NO-dependent acetylcholine relaxation by K+ channels and the Na+K+-ATPase
pump in porcine internal mammary artery. Eur J Pharmacol 2010;641:616.
Pauli JR, Ropelle ER, Cintra DE, De Souza CT, da Silva AS, Moraes JC, et al. Acute exercise
reverses aged-induced impairments in insulin signaling in rodent skeletal muscle.
Mech Ageing Dev 2010;131:3239.
Pinter RCCE, Padilha AS, Oliveira EM, Vassallo DM, Lizardo JHF. Cardiovascular adaptive
responses in rats submitted to moderate resistance training. Eur J Appl Physiol
2008;103:60513.
Rasmussen MK, Juel C, Nordsborg NB. Exercise-induced regulation of muscular Na+K+
pump, FXYD1, and NHE1 mRNA and protein expression: importance of training status, intensity, and muscle type. Am J Physiol Regul Integr Comp Physiol 2011;300:
R120920.
Ren J, Samson WK, Sowers JR. Insulin-like growth factor I as a cardiac hormone: physiological and pathophysiological implications in heart disease. J Mol Cell Cardiol
1999;31:204961.
Rossi M, Santoro G, Ricco R, Pentimone F, Carpi A. Effect of chronic aerobic exercise on cutaneous microcirculatory ow response to insulin iontophoresis and to ischemia in
elderly males. Int J Sports Med 2005;26:55862.
Rossoni LV, Salaices M, Marn J, Vassallo DV, Alonso MJ. Alterations in phenylephrineinduced contractions and the vascular expression of Na+, K+-ATPase in ouabaininduced hypertension. Br J Pharmacol 2002;135:77181.
Rossoni LV, dos Santos L, Barker LA, Vassallo DV. Ouabain changes arterial blood pressure
and vascular reactivity to phenylephrine in L-NAME-induced hypertension.
J Cardiovasc Pharmacol 2003;41:10516.
Salt IP. Examining the role of insulin in the regulation of cardiovascular health. Future
Cardiol 2013;9:3952.
Shi L, Liu B, Li N, Xue Z, Liu X. Aerobic exercise increases BK(Ca) channel contribution to
regulation of mesenteric arterial tone by upregulating 1-subunit. Exp Physiol
2013;98:32636.
Smith JM, Paulson DJ, Solar SM. Na+/K+-ATPase activity in vascular smooth muscle from
streptozotocin diabetic rat. Cardiovasc Res 1997;34:13744.
Subramanian R, MacLeod KM. Age-dependent changes in blood pressure and arterial reactivity in obese Zucker rats. Eur J Pharmacol 2003;477:14352.
Sweeney G, Klip A. Regulation of the Na+/K+-ATPase by insulin: why and how? Mol Cell
Biochem 1998;182:12133.
Tamaki T, Uchiyama S, Nakano S. A weight-lifting exercise model for inducing hypertrophy in the hindlimb muscles of rats. Med Sci Sports Exerc 1992;24:8816.
Wang Y, Wang S, Wier WG, Zhang Q, Jiang H, Li Q, et al. Exercise improves the dilatation function of mesenteric arteries in postmyocardial infarction rats via a
PI3K/Akt/eNOS pathway-mediated mechanism. Am J Physiol Heart Circ Physiol
2010;299:H2097106.
Westcott WL. Resistance training is medicine: effects of strength training on health. Curr
Sports Med Rep 2012;11:20916.
Wolpert HA, Steen SN, Istfan NW, Simonson DC. Insulin modulates circulating endothelin1 levels in humans. Metabolism 1993;42:102730.
Yang AL, Su CT, Lin KL, Chao JI, You HP, Lee SD. Exercise training improves insulin-induced
and insulin-like growth factor-1-induced vasorelaxation in rat aortas. Life Sci
2006;79:201721.
Yang AL, Su CT, Lin KL, Lee SD. Enhancement of vascular function mediated by insulin and
insulin-like growth factor-1 following single exercise session. Chin J Physiol 2008;51:
717.
Yang AL, Yeh CK, Su CT, Lo CW, Lin KL, Lee SD. Aerobic exercise acutely improves insulinand insulin-like growth factor-1-mediated vasorelaxation in hypertensive rats. Exp
Physiol 2010;95:6229.
Yaspelkis III BB. Resistance training improves insulin signaling and action in skeletal muscle. Exerc Sport Sci Rev 2006;34:426.
Yasui S, Mawatari K, Kawano T, Morizumi R, Hamamoto A, Furukawa H, et al. Insulin
activates ATP-sensitive potassium channels via phosphatidylinositol 3-kinase in
cultured vascular smooth muscle cells. J Vasc Res 2008;45:23343.
Yu Q, Gao F, Ma XL. Insulin says NO to cardiovascular disease. Cardiovasc Res 2011;89:
51624.
Zanesco A, Antunes E. Effects of exercise training on the cardiovascular system: pharmacological approaches. Pharmacol Ther 2007;114:30717.