Anti-Contractile Mechanism of Resveratrol in Non-Vascular Smooth Muscle Under α1-Adrenoceptor Stimulation involves IP3-Receptor, Protein Kinase-C and NADPH Oxidase
Carolina Baraldi Araújo Restini1,2*, David Fernando de Almeida Vieira2, Aline Oliveira Domingos2,3, Brain Barnett1 and Juliano M Martins2
1Department of Pharmacology & Toxicology, Michigan State University, USA
2Department of Biotechnology and Medical School, University of Ribeirão Preto, Brazil
3Hospital de Amor, Brazil
Submission: March 14, 2019; Published: April 08, 2019
*Corresponding author: Carolina Baraldi Araujo Restini, PharmD, Ph.D, Department of Pharmacology & Toxicology, College of Osteopathic Medicine, Michigan State University, 44575 Garfield Road, Building UC4, Clinton Township, MI 48038, USA
How to cite this article: Carolina B A R, David F d A V, Aline O D, Brain B, Juliano M M. Anti-Contractile Mechanism of Resveratrol in Non-Vascular
Smooth Muscle Under α1-Adrenoceptor Stimulation involves IP3-Receptor, Protein Kinase-C and NADPH Oxidase. Open Acc J of Toxicol. 2019; 4(1): 555627. DOI: 10.19080/OAJT.2019.04.555627.
Reactive oxygen species (ROS) are products from enzymatic systems that are responsible for several biological disturbances when uncontrolled. Superoxide anion (O2-), increases intracellular calcium-regulated contractile/relaxation responses in smooth muscles. Activation of α1-adrenoceptors promotes these contractions through the Gq pathway involving protein kinase C (PKC) and calcium mobilization. It has been reported that Gq pathway is related to ROS production. It has also been shown that resveratrol (RESV), an antioxidant agent, decreases vascular smooth muscle contraction. The effect of RESV was not yet demonstrated in the anococcygeus smooth muscle contraction. Since ROS are present in rat anococcygeus smooth muscle and RESV has an antioxidant effect, the hypothesis for the current work is that the contractile response, under α1-adrenoceptor stimulation, can be decreased by RESV through decreasing ROS production related to the pathways of PKC and calcium mobilization. Thus, the aims were to investigate if the RESV interferes with the non-vascular smooth muscle contractile reactivity stimulated by the α1-adrenoceptor agonist, phenylephrine (PE) and to analyze its potential mechanisms. Anococcygeus smooth muscles were isolated from male Wistar rats and placed in organ baths to evaluate isometric tension. RESV enhanced the decreased contractions after incubation with α1-adrenoceptor and IP3-receptor (RIP3) antagonists as well as PKC and NADPH oxidase inhibitors. Under α1-adrenoceptor stimulation, the anococcygeus smooth muscle contractions are indeed related to the pathway of ROS production, involving inhibition of Ca2+ mobilization, PKC activation and NADPH oxidase that are all sensitive to RESV.
Reactive oxygen species (ROS) are products of most metabolic reactions . When their production exceeds the physiological antioxidant protection that cells can withstand, harmful and damaging effects occur, such as lipid peroxidation and DNA oxidation. This imbalance is responsible for several diseases such as cancer, cardiovascular diseases like atherosclerosis and hypertension, and several complications of Diabetes Mellitus . The production of ROS, however, is not always harmful, as can be demonstrated by phagocytic cell actions against invading microorganisms , and by the physiological effects of nitric oxide (NO) as an important neurotransmitter and vasodilator [4,5].
Important mechanisms induce an influx of ions from the extracellular environment to the intracellular medium, directly or indirectly, through the production of ROS, which results in increased Ca2+ concentration in the cell cytoplasm [6,7].
Ca2+ mobilization is not only related to diseases and worrisome lesions, but also to muscle contraction, which will be covered in this article. Cytosolic calcium concentration ([Ca2+]c) increases triggering of the Ca2+-calmodulin complexation, which is essential for the muscle contraction due to activation via phosphorylation of myosin light chain (MLC) . Smooth muscle contractions are substantially dependent on adrenergic stimulation,
Norepinephrine (NE), non-selectively, and Phenylephrine
(PE), selectively, stimulate α1-adrenoceptors coupled to Gq protein,
to produce second messengers. Such receptors are located
primarily in vascular and non-vascular smooth muscle but have
also been found in cardiomyocytes .
Through Gq protein activation, the phospholipase C (PLC)
triggers the production of second messengers: inositol 1,4,5-triphosphate
(IP3) and diacylglycerol (DAG) [10,11]. The IP3 interacts
with receptors (RIP3) in the sarcoplasmic reticulum, the principal,
but not the unique reservoir of intracellular calcium ,
releasing Ca2+ into the cytoplasm. DAG, in turn, activates protein
kinase C (PKC), which leads to the opening of Ca2+ channels on
the plasmatic membrane. Furthermore, DAG activates the enzyme
NADPH oxidase located in the membrane to produce ROS .
The NADPH oxidase system is commonly recognized as the
main source of ROS production in the vessel wall. Decreasing
mRNA expression of NADPH oxidase  has been successfully
studied by using atorvastatin, the synthetic 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase inhibitor. Besides its
well-known lipid-lowering effects, atorvastatin presents pleiotropic
effects including anti-atherogenic anti-inflammatory actions,
inhibition of the in vitro oxidation of LDL, and reduction in various
oxidative stress markers [15,16].
Studies aiming to associate α1-adrenoceptors and ROS production
are greatly benefited by preparations presenting sympathetic
innervation and/or high populations of such receptors.
Anococcygeus smooth muscle is a smooth muscle widely used as
an essential tool for studying the mechanisms involving sympathetic
activation. Its adrenergic innervation corresponds to 60%
compared to other innervations and α1 is the primary receptor
[17-19]. Furthermore, another advantage of the anococcygeus
smooth muscle is its easy isolation and the presence of a long lineal
structure with a thin layer of muscle cells, allowing for prompt
diffusion of drugs and ions [11,20]. As retractor penis muscles,
the anococcygeus is part of the erectile machinery in male rodents
. Concerning the potential clinical importance of anococcygeus
smooth muscle and its relation to male genital apparatus,
thus, it is a useful pharmacological and physiological tool to study
issues such as benign prostate hypertrophy, etc . Currently, α1-
antagonists are commonly used to relax the smooth muscle in the
prostate for treating benign prostate hypertrophy related to lower
urinary tract symptoms [23,24].
Antioxidant substances are being investigated with the intent
to minimize or even solve pathological effects triggered by
ROS. Among these is Resveratrol (RESV), an agent that has been
attracting researchers’ attention since the 80’s after the “French
paradox”, which linked red wine consumption to the reduction of
cardiovascular problems [25,26]. RESV inactivates free radicals
[27-29] and decrease the activity of the contractile machinery of
vascular [30,31] and non-vascular smooth muscle [32,33] by regulating
the phosphorylation of MLC stimulated by PE. Nevertheless,
many of these data were obtained from studies in vascular
smooth muscle. Moreover, there are few studies of non-vascular
smooth muscle in the literature and the correlation between RESV
effects and α1-adrenoceptors stimulation.
It is believed that RESV may be able to prevent the action of
ROS related to its activation by adrenergic stimulation. In this
sense, RESV could lead to a reduction in contractile response in
non-vascular smooth muscle. Thus, this study aimed to investigate
the mechanism of RESV on the contractile reactivity in isolated
rat anococcygeus muscle after α1-adrenergic stimulation.
The following drugs were used: phenylephrine (PE), trans-resveratrol
(RESV), prazosin, 2-aminoethoxydiphenyl borate (2-APB)
 and 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-
3-yl)-maleimide (GF109203X) (Sigma-Aldrich, Inc., St. Louis,
MO, USA), atorvastatin (ATV; Fagron, São Paulo-SP, Brazil), NaCl,
KCl, KH2PO4, CaCl2, MgSO4, NaHCO3 and C6H12O6 (Lab Synth®, Diadema-
SP, Brazil), isoflurane (AstraZeneca®, Cotia-SP, Brazil).
Phenylephrine was dissolved in distilled water; resveratrol was
dissolved in 70% v/v ethanol, and 2-APB was dissolved in methanol
with further dilution in distilled water before use. Working
concentrations of ethanol and methanol in the bath were <0.01%
(v/v). Previous experiments showed that the solvents used had no
effects on preparations at the applied concentrations.
This study was approved by the Ethics Committee of Animal
Experiments of UNAERP-CEP/UNAERP number 019/2012. Male
Wistar rats (200g) were anesthetized with isoflurane and killed
by decapitation. Anococcygeus is a paired smooth muscle arising
from the vertebral column to the ventral side of the colon. It
comes from tendinous origins on the posterior sacral vertebrae
and runs caudad around both sides of the rectum to unite on its
As both anococcygeus smooth muscles were isolated 
and dissected from each rat, the right muscles were used to test
the drugs while the left ones were used in control experiments in
which the respective drug was dissolved . After isolating the
pair of muscles, they were separated, and each one was carefully
freed of connective tissue, tied at both ends by cotton thread
ligatures and placed in 5mL organ baths, which were oxygenated
(95% O2 and 5% CO2) and warmed (37 °C). The baths of Krebs’
physiological salt solution (PSS) had the following composition
(in mmol/L): 118.0 NaCl, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2
MgSO4, 25.0 NaHCO3 and 2.0 C6H12O6 (pH 7.4). Muscle strips were
connected to a force transducer (Scientific Instruments®, West
Palm Beach-FL, EUA) set to a resting tension of 0.5g and allowed
to equilibrate for 1 hour before the protocols. During the resting
periods, the bath solution was replaced every 15 minutes. After 1
hour, muscle preparations were stimulated twice with 60mmol/L KCl-PSS (equimolar), to generate reproducible contractions and
then washed out back to the resting tension.
This set of experiment was designed to discover the minimal
concentration of RESV would interfere with the potency or efficacy
of the concentration-response curves of PE.
Muscle preparations were stimulated with increasing and cumulative
concentrations of PE (1nmol/L-100μmol/L) before and
after 20 minutes incubation with different concentrations of RESV
(10pmol/L to 1mmol/L). Each concentration of RESV was tested
in different preparations.
Concentration-response curves were performed in anococcygeus
muscles with increasing and cumulative concentrations of
PE (1nmol/L-100μmol/L) before and after 20 minutes incubation
with RESV 100μmol/L in the presence or absence of Prazosin (Pz,
10nmol/L-α1-adrenoceptor antagonist), 2-APB (100μmol/L-IP3
receptor antagonist), GF109203X (5μmol/L - PKC inhibitor) or
Atorvastatin (ATV, 100μmol/L - NADPH oxidase antagonist).
This set of protocols was intended to investigate the mechanism
involved in the cascade under α1-adrenoceptor activation, in
which RESV could interfere.
Data are expressed as mean ± SEM. Significant differences
between two groups (p<0.05 or p<0.001) were determined by
Student two-tailed t-test for paired data or by One-way ANOVA
followed by Newman-Keuls post hoc. The cumulative concentration-
response curves to PE allowed us to analyze the pharmacological
parameters are the maximal effect (Emax, also referred to
as efficacy) and potency (pD2 = -log EC50). Contraction values are
presented as normalized data (percentage, %) of KCl contraction.
To determine whether ROS are involved in the activation of
α1-adrenoceptor upon stimuli with PE, different concentrations of
RESV (10pmol/L to 1mmol/L) were tested. As shown in Figure
1, incubation with RESV 100μmol/L reduced maximal contraction
induced with PE (101.78 ± 1.13% vs. 69.39 ± 5.31%, n=6;
p<0.001), and RESV 1mmol/L reduced efficacy (100.31 ± 0.77%
vs 8.92 ± 4.95%, n=6; p<0,001) and potency (6.47 ± 0.05% vs 5.58
± 0.27%, n=6; p<0.05) of contraction induced with PE. The results
suggest that ROS sensitive to RESV are possibly derived from activation
of α1-adrenoceptors with PE.
The concentrations of RESV that altered the PE-induced contractions
were 100μmol/L and 1mmol/L. At lower concentrations,
neither efficacy nor potency of the concentration-response
curves of PE was changed (data not shown). The next experiments
were done using 100μmol/L of RESV.
It was examined whether ROS are directly involved in the activation
of α1-adrenoceptor by using Pz, an α1-receptor antagonist.
As shown in Figure 2, Pz 10nmol/L did not reduce efficacy but
reduced the potency (6.27±0.09 vs. 5.35±0.09, n=6-7; p<0.001) of
On the other hand, pre-treatment with Pz 10 nmol/L and
RESV 100 μmol/L significantly reduced efficacy (100.06±0.68%
vs. 55.78±4.37%, n=6-7; p<0.001) and the potency (6.13±0.06
vs. 5.44±0.05, n=6-7; p<0.001) of cumulative concentration-response
curves to PE.
To verify if ROS, sensitive to RESV, play a role in the intracellular
contractile mechanisms after activation of α1-adrenoceptor
we tested the contribution of IP3 receptors (RIP3) in this response
using 2-APB, a RIP3 antagonist on the cumulative concentration-
response curves to PE. Figure 3 shows that 2-APB 100μmol/L
reduced both the efficacy (101.85±0.73% vs. 16.57±4.83%, n=6-7; p<0.001) and the potency (6.83 ± 0.27 vs. 5.76 ± 0.11, n=6-7;
p<0.001) of PE. Furthermore, RESV 100μmol/L promoted an
additional reduction to the inhibitory effect caused by 2-APB
100μmol/L. Therefore, the efficacy was practically abolished
(99.3 ± 0.57% vs. 5.37±1.42%, n=6-7; p<0.001).
The participation of ROS sensitive to RESV, under α1-
adrenoceptor stimulation, and PKC activation was tested via
GF109203X, a non-selective PKC inhibitor on the cumulative
concentration-response curves to PE. As depicted in Figure 4,
GF109203X 5μmol/L reduced the efficacy (100.21 ± 0.84% vs.
85.64 ± 4.02%, n=6-7; p<0.001) and the potency (6.46 ± 0.10 vs.
5.69 ± 0.08, n=6-7; p<0.001) of PE. The combination of RESV with
GF109203X promoted a further reduction of the efficacy (100.21
± 0.84% vs. 40.55 ± 7.36%, n=6-7; p<0.001). However, PE potency
was not changed.
The contribution of NADPH oxidase as a source of ROS, sensitive
to RESV, was also investigated using ATV as a non-selective
NADPH oxidase inhibitor. As seen in Figure 5, ATV 100 μmol/L did
not reduce the efficacy but decreased the potency (6.37 ± 0.09 vs.
5.78 ± 0.05, n=6-7; p<0.001) of the cumulative concentration-response
curves to PE. However, the addition of RESV 100μmol/L in
a combination of ATV promoted a further decrease of the efficacy
(99.96 ± 0.93% vs. 90.54 ± 4.29%, n=6-7; p<0.001) but did not
change the potency of the contractions stimulated by PE, which
were previously reduced by incubation with ATV alone.
The present study is the first to demonstrate the anti-contractile
effect of RESV, a compound with antioxidant activity, on the
contractile machinery triggered by α1-adrenoceptors stimulation
on anococcygeus smooth muscle. Different studies demonstrated
ROS release promoted vasoconstriction [30,35]. Furthermore,
it has been reported that activation of α1-adrenoceptor induces
ROS releasing in vascular smooth muscles and some non-vascular
smooth muscles . Our data shows that RESV reduces both
the potency and the efficacy of the cumulative concentration-response
curves to PE in smooth muscle. Considering the already
accepted antioxidant property of RESV , this finding confirms
the hypothesis that contraction mediated by α1-adrenoceptor activation
is positively associated with ROS activity in non-vascular
It is known that superoxide increases the release of Ca2+ from
intracellular stores and promotes an increase in its inflow to the
intracellular environment . Complementarily it is also known
that ROS mediate α1-adrenoceptor-stimulated hypertrophy of
vascular smooth muscle and cardiomyocytes, a long-term effect
of catecholamines [9,37,38]. The contribution of ROS to the acute
vasoconstrictor effect of α1-adrenoceptors was already characterized
 for vascular smooth muscle.
Based on this observation, we tested the mechanism of RESV
after the α1-adrenoceptor stimulus. Experimental protocols were
designed to evaluate PE contractions in the presence of RESV, and
the intracellular mechanisms activated after this stimulus, related
to Ca2+ mobilization via IP3, DAG, the participation of PKC, and NADPH
First, to evaluate the direct α1-adrenoceptors involvement,
prazosin (Pz), a selective α1-adrenoceptor antagonist, was used
alone or in combination with RESV so testing the contribution of
ROS to PE-induced contractions. As a result, the concentration-response
curves induced by PE in the presence of Pz presented a
reduced potency, but not efficacy. Pz resulted in rightward shifts
of the concentration-response curves to PE, with no depression
of the maximum response. This finding is supported by literature
. Our original contribution is that, in the presence of RESV
in a combination of Pz, PE stimulated contraction through α1-
adrenoceptor on the anococcygeus smooth muscle isolated from
rats was further reduced, suggesting the contribution of ROS, sensitive
to RESV, on PE-induced contraction.
It is well known the activation of α1-adrenoceptor promotes
an increase in [Ca2+]c leading to smooth muscle contraction and
ROS play an essential role in this process . The contribution
of RIP3 on PE contraction was evaluated using the RIP3 antagonist,
2-APB . To test the participation of ROS in the contraction via
Ca2+ mobilization from internal stores, 2-APB was used alone and
in combination with RESV. Our results identified a dynamic contribution
of RIP3 on PE-induced contraction since the contraction
was strongly reduced when the RIP3 was antagonized. Moreover,
the presence of RESV in combination with 2-APB further decreased
this contractile response, suggesting PE-induced contraction
through ROS-dependent Ca2+ mobilization from sarcoplasmic
reticulum on rat anococcygeus smooth muscle.
Slater et al.  demonstrated that RESV causes inhibition
of protein kinase C (PKC) in endothelial cells. Considering that,
besides Ca2+ from internal stores, the contraction induced by α1-
adrenoceptor is partially dependent of increasing cytosolic Ca2+
concentration due to its influx in anococcygeus smooth muscle
; we have investigated the involvement of PKC on RESV effects.
Similarly, to the IP3 receptor inhibition, PKC is also important to
PE-induced contraction. Interestingly, RESV enhances the reduction
of the PE-induced contraction in the presence of GF109203X,
a non-selective PKC inhibitor, suggesting PE contraction triggers
the production of ROS, and this is important to the activation of
The NADPH oxidases are the most important enzymes, whose
role is to generate superoxide/ROS . Since we observed ROS
participated in the PE contractile response, the possible source of
this ROS was investigated using atorvastatin (ATV) as a non-selective
NADPH oxidase inhibitor [14,44,45]. According to Orallo et
al. , the vasorelaxant activity of RESV enhancing nitric oxide
signaling in the endothelium has been attributed to the inhibition
of the vascular NADH/NADPH oxidase activity, leading to a reduction
in basal superoxide production, and, consequently, decreased
inactivation of nitric oxide. Our data shows that PE-induced contraction
seemed to be partially dependent on NADPH oxidase activity
in rat anococcygeus smooth muscle.
In our hands, in the presence of RESV and ATV, PE contraction
was further reduced, pointing to a ROS-dependent activation of
NADPH oxidase that contributes to PE-induced contraction in a
non-vascular smooth muscle.
The main limitation of the present study is the lack of molecular
and biochemistry studies. However, by pharmacological tools,
it was possible to reach our aims.
The results support the view that ROS play a crucial role in
the PE-induced contraction of rat anococcygeus smooth muscle.
In addition, this contraction was dependent of α1-adrenoceptors,
RIP3, PKC, and NADPH oxidase activation. Furthermore, the wellknown
antioxidant RESV exerts an anti-contractile effect on PE
contraction by mechanisms involving ROS-dependent RIP3, PKC
and NADPH oxidase activation.
Figure 6 depicts, as a graphical conclusion, the proposed
mechanism of RESV acting on the classical intracellular pathway
triggered by alpha1 adrenoreceptor stimulation associated with
H2O2 production by NADPH oxidase.
The authors thank the Brazilian National Research Council
(Conselho Nacional de Pesquisa: CNPq) and University of Ribeirão
Preto (UNAERP). The authors also thank Carla R. K. Antonietto for