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MitoTEMPO Increases the Gastrointestinal Motility in Aged Rats.

INTRODUCTION

Aging is one of the greatest risk factors for many organ dysfunctions and physiological functions in organs and tissues. Thus, the number of nerve cells in the human colon decreases with age (1). The time for food to pass through the colon is prolonged in aged rats, and therefore the colonic motility may be also affected by aging (2). Reports imply that more frequent chronic functional constipation in elderly people has been observed (3). In aging, the gradual loss of the [Ca.sup.+2] balance in cells alters cellular physiological functions (4, 5). Reports showed that during aging, there was a decrease in the [Ca.sup.+2] release according to [Ca.sup.+2]-induction (6) in addition to nitric oxide, the most vital mediator in the longitudinal muscle of young individuals, which losses its significance with aging (7).Mitochondria are exposed to oxidative stress, which is involved in the normal aging process. Under pathological conditions, reactive oxygen species are over produced by the mitochondrial electron-transport chain uncoupling, the activation of the xanthine--oxidoreductase system, or by excessive stimulation of the NAD(P)H, which can overwhelm the endogenous antioxidant defense mechanisms (8). However, the role of mitochondrial dysfunction still remains unclear (9).

The mitoTEMPO has been used as an antioxidant. It is a nitroxide, conjugated to a triphenylphosphonium moiety, which is mitochondrion targeted. As nitroxides are the superoxide-dismutase mimetics, mitoTEMPO could act as a mitochondrial superoxide scavenger and therefore protect mitochondria from the age-induced oxidative damage (10). Previous studies showed that mitoTEMPO lowers mitochondrial superoxide levels and increases the survival rate in septic mice (11).

In this work, we report that aging might have been associated with the reduction of the contractile effects of acetylcholine and relaxation effects of phenylephrine in isolated ileal and duodenal segments.

MATERIALS AND METHODS

This study has received the approval by the local ethics committee of Ankara University (reference number, 20l6-l8-l65).Healthy adult male Wistar rats (150-250 g) were housed under standard conditions. Two experimental sets were designed. The first set of animals was 6 month old (10 male rats weighing 340[+ or -]11g) and was considered the adult set. The second experimental set was 24 months old (10 male rats weighing 350[+ or -]8 g), and it was considered the aged set. The blood glucose level of the adult group compared to the aged group was 80.5[+ or -]1.4 mg/dL and 83.9[+ or -]2.5 mg/dL, respectively

Pentobarbital (30 mg/kg intraperitoneally) was used for anesthetizing. The ileum and duodenum segments (0.3-0.5-mm-long pieces) were removed. Then, the tissue segments were suspended in an isolated tissue bath containing l5 mL of the Krebs-Henseleit solution (mM NaCl 118, KCl 4.7, Ca[Cl.sub.2] 2.5, [KH.sub.2]P[O.sub.4] l.2, NaHC[O.sub.3] 25, MgS[O.sub.4] 1.2 and glucose 11.1), and a 95% [O.sub.2], 5% C[O.sub.2] mixture at 37[degrees]C, pH 7.4, was passed into the solution. The segments were brought into equilibrium for 60 min under an optimal resting tension of lg. After equilibration, the ileum and duodenum segments were contracted with acetylcholine (ACh) ([10.sup.-4]-[10.sup.-7],6 M) and relaxed with phenylephrine ([10.sup.-4]-[10.sup.-7],6 M). These doses were considered as maximal doses, after the cumulative addition of ACh and phenylephrine into the control group. Contraction and relaxation changes were recorded in the isometric tension recorder on the Biopac version 3.7.0. Then, mitoTEMPO (SML 0737, Sigma) was added into the perfusion medium. The isolated ileal and duodenal segments were incubated for 1 h in the medium, and then the contraction and relaxation responses were recorded.

Statistical analyses were performed with an unpaired t-test. Significance values were accepted as *p<0.0 5vs. adult and p<0.05 vs. aged. Values were presented as mean[+ or -]standard error of mean.

RESULTS

The effects of mitoTEMPO on the gastrointestinal contractility of the isolated ileal and duodenal segments of adult and aged rats were examined.

From Figure 1, it can be observed that no significant differences between the responses of the ACh-induced contractions in the adult and aged duodenal segments were found. When the adult and aged duodenal segments were incubated with mitoTEMPO, significant increases in aged-doudenal ACh-induced contractions compared to adult rats were observed.

When comparing the ACh-induced contraction of the ileal segments of adult and aged rats, it was shown that the responses of adult ileal segments were higher when compared to the aged rats. When the adult and aged ileal segments were incubated with mitoTEMPO, the responses of the ACh-induced contractions in aged ileal segments were significantly higher when compared to the adult segments, as shown in Figure 2.

Figure 3 shows that the responses of the phenylephrine-induced relaxations were lower in adult duodenal segments compared to the aged duodenal segments. When the adult and aged duodenal segments were incubated with mitoTEMPO, the responses of phenylephrine-induced relaxation in aged ileal segments were significantly higher when compared to the adult duodenal segments.

As shown in Figure 4, the responses of the phenylephrine-induced relaxations were similar in adult and aged ileal segments. But when the ileal segments were incubated with the mitoTEMPO, the values were significantly higher in the aged ileal segments when compared to the adult ileal phenylephrine-induced relaxations.

DISCUSSION

The effects of mitoTEMPO on the contractility of adult and aged isolated ileal and duodenal segments in rats were investigated in this study. Our results indicate that the contractility in the aged group decreased and that the relaxation was greater than in the adult group. The results also showed that with the incubation for l h with mitoTEMPO, ileal and duodenal contractility increased significantly in aged rats.

Acetylcholine is an important neurotransmitter, and it induces the contraction in the gastrointestinal tract. Our results showed that the ACh-induced contraction decreased in aged rats; however, these data conflict with Tezuka et al. (12) who report that the contractile response to acetylcholine has no effect on the jejunum and colon in aged rats.

The processes of aging are not well defined. More than 300 theories have been introduced since Medvedev, but none was accepted (13). In 1954, the free-radical theory of aging was proposed by Harman who stated that "aging results from imperfect protection against tissue damage by free radicals in addition to factors like genetic, activity, nutrition and temperature." Despite many investigations on the relationship between lipid peroxidation and aging, the results were conflicting because aging changes are small early in life but increase quickly with age (14). However, many investigators have accepted that biological aging is the failure of an organism to maintain homeostasis (15). In aging of the submucous plexus of the jejunum--ileum, there is a loss of 38% of the neurons, which is probably due to degenerated mitochondria in old animals (16). However, histological experiments carried out by Lee et al. (17) reported that during aging, a significant loss in the thickness of the intestinal mucosa is accompanied by higher reactive species.

In this work, 24-month-old animals were used and showed that contractility in the ileum decreased. According to reports, aging is characterized by a decrease in glutathione concentrations and an increase in the oxidation of protein with a decrease in the mitochondrial NO content in 28-month-old rats (l8). During the aging process, the oxidative stress takes place, and endogenously produced oxidants in the cells are usually increased (19, 20).

Lopes et al. (4) reported that the activation of muscarinic receptors induces more contractions in aged rats than in adult animals, which conflicts with our data. They showed that the changes in intracellular [Ca.sup.2+] stores affect contraction and may cause dysfunction during the cell-aging damage.

In aging, several aspects of [Ca.sup.2+] homeostasis might be affected, such as the [Ca.sup.2+] influx, release of [Ca.sup.2+] from stores, and [Ca.sup.2+] uptake process by the sarcoplasmic reticulum and mitochondria (21). It is unknown whether similar changes occur in the gastrointestinal system. Although functional and structural changes are known to occur in many functions, the mechanisms responsible for these changes are unclear. Many investigators claimed that the oxidant stress increases, due to an elevated reactive oxygen, species production, or to decrease the function of natural antioxidant pathways (22-24).

mitoTEMPO was used as the antioxidant factor in our experiments. Mitochondria are the most important sources of reactive oxygen species in the body. We assumed that the therapeutic inhibition of mitochondria by a mitochondrial targeted antioxidant mitoTEMPO might be beneficial in the setting of the gastrointestinal dysmotility. mitoTEMPO is a physicochemical compound that has the ability to pass through lipid bilayers easily and accumulate in the mitochondria selectively (10). Both in vitro and in vivo studies have confirmed that mitoTEMPO is a mitochondria-targeted antioxidant with superoxide and alkyl radical scavenger properties (10, 25). It is unknown if there are any off-target effects of mitoTEMPO when it is accumulated in mitochondria, and it is also unclear how much of mitoTEMPO has actually gone to the organs (26).

In this study, we have also demonstrated that therapeutic inhibition of mitochondrial ROS using mitoTEMPO prevented oxidative stress and reduced the gastrointestinal dysmotility in aged animals. Our data strongly indicated that mitochondria-targeted antioxidants have therapeutic effects on aged-related dysmotility; thus, a form of antioxidant therapy such as mitoTEMPO may be useful for treating age-related gastrointestinal dysmotility. To the best of our knowledge, this study is the first to demonstrate that antioxidant treatment, such as mitoTEMPO, can significantly reverse the response to acetylcholine and phenylephrine in isolated gastrointestinal segments in aged rats.

In conclusion, we demonstrated that the treatment with the antioxidant mitoTEMPO can significantly affect the responses to the ACh-induced contractions and phenylephrine-induced relaxations that occur with aging. The results are consistent with the hypothesis over the production of ROS with aging.

Ethics Committee Approval: Ethics committee approval was received for this study from the Ethics Committee of Ankara University School of Medicine (Approval Date: 2016, Approval Number: 18-165).

Informed Consent: N/A.

Peer-review: Externally peer-reviewed.

Author contributions: Concept - E.K., B.T., Y.O.; Design - E.K., B.T., Y.O.; Supervision - B.T., Y.O.; Resource - B.T., Y.O.; Materials - B.T., Y.O.; Data Collection and/or Processing - E.K., Y.O.; Analysis and/or Interpretation - Y.O.; Literature Search - E.K., B.T.; Writing - E.K., B.T., Y.O.; Critical Reviews - E.K., B.T., Y.O.

Conflict of Interest: The authors have no conflicts of interest to declare.

Financial Disclosure: The authors declared that this study has received no financial support.

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Emine Koc (1) [iD], Yusuf Olgar (2) [iD], Belma Turan (2)

(1) Department of Physiology, Near East University School of Medicine, Nicosia, Cyprus

(2) Department of Biophysics, Ankara University School of Medicine, Ankara, Turkey

ORCID IDs of the authors: E.K. 0000-0001-8804-4937; Y.O. 0000-0002-3226-745.

Corresponding Author: Emine Koc

E-mail: ekoc@medicine.ankara.edu.

Received: 06.12.2018

Accepted: 20.12.2018

Cite this article as: Koc E, Olgar Y, Turan B. MitoTEMPO Increases the Gastrointestinal Motility in Aged Rats. Cyprus J Med Sci 2019; 4(1): 24-7.

DOI: 10.5152/cjms.2019.747
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Title Annotation:Original Article
Author:Koc, Emine; Olgar, Yusuf; Turan, Belma
Publication:Cyprus Journal of Medical Sciences
Date:Apr 1, 2019
Words:2627
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