Anti-Contractile Mechanism of Resveratrol in Non-Vascular Smooth Muscle Under α1-Adrenoceptor Stimulation involves IP 3 -Receptor, Protein Kinase-C and NADPH Oxidase

con-Abstract Reactive oxygen species (ROS) are products from enzymatic systems that are responsible for several biological disturbances when uncontrolled. Superoxide anion (O 2- ), 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 Ca 2+ mobilization, PKC activation and NADPH oxidase that are all sensitive to RESV.


Introduction
Reactive oxygen species (ROS) are products of most metabolic reactions [1]. 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 [2]. The production of ROS, however, is not always harmful, as can be demonstrated by phagocytic cell actions against invading microorganisms [3], 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 Ca 2+ concentration in the cell cytoplasm [6,7]. Ca 2+ 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 ([Ca 2+ ] c ) increases triggering of the Ca 2+ -calmodulin complexation, which is essential for the muscle contraction due to activation via phosphorylation of myosin light chain (MLC) [8]. Smooth muscle con-How to cite this article: Carolina  Norepinephrine (NE), non-selectively, and Phenylephrine (PE), selectively, stimulate α 1 -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 [9].
Through Gq protein activation, the phospholipase C (PLC) triggers the production of second messengers: inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol (DAG) [10,11]. The IP 3 interacts with receptors (RIP 3 ) in the sarcoplasmic reticulum, the principal, but not the unique reservoir of intracellular calcium [12], releasing Ca 2+ into the cytoplasm. DAG, in turn, activates protein kinase C (PKC), which leads to the opening of Ca 2+ channels on the plasmatic membrane. Furthermore, DAG activates the enzyme NADPH oxidase located in the membrane to produce ROS [13]. The NADPH oxidase system is commonly recognized as the main source of ROS production in the vessel wall. Decreasing mRNA expression of NADPH oxidase [14] 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 α 1 -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 α 1 is the primary receptor [17][18][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 [21]. 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 [22]. Currently, α 1antagonists 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][28][29] and decrease the activity of the contractile machinery of vascular [30,31] and non-vascular smooth muscle [32,33] by reg-ulating 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 α 1 -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 α 1 -adrenergic stimulation.

Drugs
The following drugs were used: phenylephrine (PE), trans-resveratrol (RESV), prazosin, 2-aminoethoxydiphenyl borate (2-APB) [20]  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.

Tissue preparation and isometric force measurement
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 ventral aspect.
As both anococcygeus smooth muscles were isolated [16] 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 [34]. 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% O 2 and 5% CO 2 ) and warmed (37 °C).

Effect of RESV on the contractile response stimulated with pe
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. This set of protocols was intended to investigate the mechanism involved in the cascade under α 1 -adrenoceptor activation, in which RESV could interfere.

Data presentation and statistical analysis
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 (E max , also referred to as efficacy) and potency ( p D 2 = -log EC 50 ). Contraction values are presented as normalized data (percentage, %) of KCl contraction. To determine whether ROS are involved in the activation of α 1 -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 α 1 -adrenoceptors with PE.

RESV reduced the efficacy and the potency of 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.

RESV in combination with ATV further decreased PE-induced contraction
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.

Discussion
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 α 1 -adrenoceptors stimulation on anococcygeus smooth muscle. Different studies demonstrated ROS release promoted vasoconstriction [30,35]. Furthermore, it has been reported that activation of α 1 -adrenoceptor induces ROS releasing in vascular smooth muscles and some non-vascular smooth muscles [30]. 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 [31], this finding confirms the hypothesis that contraction mediated by α 1 -adrenoceptor activation is positively associated with ROS activity in non-vascular smooth muscle.
It is known that superoxide increases the release of Ca 2+ from intracellular stores and promotes an increase in its inflow to the intracellular environment [36]. Complementarily it is also known that ROS mediate α 1 -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 α 1 -adrenoceptors was already characterized [30] for vascular smooth muscle.

Open Access Journal of Toxicology
Based on this observation, we tested the mechanism of RESV after the α 1 -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 Ca 2+ mobilization via IP 3 , DAG, the participation of PKC, and NA-DPH oxidase activation.
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 [39]. Our original contribution is that, in the presence of RESV in a combination of Pz, PE stimulated contraction through α 1adrenoceptor 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 α 1 -adrenoceptor promotes an increase in [Ca 2+ ] c leading to smooth muscle contraction and ROS play an essential role in this process [40]. The contribution of RIP 3 on PE contraction was evaluated using the RIP 3 antagonist, 2-APB [41]. To test the participation of ROS in the contraction via Ca 2+ mobilization from internal stores, 2-APB was used alone and in combination with RESV. Our results identified a dynamic contribution of RIP 3 on PE-induced contraction since the contraction was strongly reduced when the RIP 3 was antagonized. Moreover, the presence of RESV in combination with 2-APB further decreased this contractile response, suggesting PE-induced contraction through ROS-dependent Ca 2+ mobilization from sarcoplasmic reticulum on rat anococcygeus smooth muscle.
Slater et al. [42] demonstrated that RESV causes inhibition of protein kinase C (PKC) in endothelial cells. Considering that, besides Ca 2+ from internal stores, the contraction induced by α 1adrenoceptor is partially dependent of increasing cytosolic Ca 2+ concentration due to its influx in anococcygeus smooth muscle [12]; we have investigated the involvement of PKC on RESV effects. Similarly, to the IP 3 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 PKC.
The NADPH oxidases are the most important enzymes, whose role is to generate superoxide/ROS [43]. 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. [46], 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.

Conclusion
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 α 1 -adrenoceptors, RIP 3 , PKC, and NADPH oxidase activation. Furthermore, the wellknown antioxidant RESV exerts an anti-contractile effect on PE contraction by mechanisms involving ROS-dependent RIP 3 , PKC and NADPH oxidase activation. Open Access Journal of Toxicology 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 H 2 O 2 production by NADPH oxidase.