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The effects of enalapril and losartan on mechanicalventilationeinduced sympathoadrenal activationand oxidative stress in rats

Hale Zerrin Toklu, PhD,a,b,c,* Oh-Sung Kwon, PhD,d Yasemin Sakarya,a,b

Scott K. Powers, PhD,d Katherine Llinas,a Nataliya Kirichenko, MSc,a,b

Kurt J. Sollanek, MSc,d Michael P. Wiggs, PhD,d Ashley J. Smuder, PhD,d

Erin E. Talbert, PhD,d Philip J. Scarpace, PhD,b and Nihal Tumer, PhDa,b,**aMalcom Randall Veterans Affairs Medical Center, Geriatric Research Education and Clinical Center, Gainesville,

FloridabDepartment of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FloridacDepartment of Pharmacology, Marmara University School of Pharmacy, Istanbul, TurkeydDepartment of Applied Physiology and Kinesiology, College of Health & Human Performance, University of Florida,

Gainesville, Florida

a r t i c l e i n f o

Article history:

Received 1 November 2013

Received in revised form

7 January 2014

Accepted 30 January 2014

Available online xxx

Keywords:

Mechanical ventilation

Angiotensin

Losartan

Enalapril

NPY

Thyrosine hydroxylase

Dopamine beta hydroxylase

Sympathetic

Noradrenergic

Norepinephrine

* Corresponding author. Department of PhaGainesville, FL 32610.** Corresponding author. Pharmacology & The

E-mail addresses: haletoklu@yahoo.com0022-4804/$ e see front matter ª 2014 Elsevhttp://dx.doi.org/10.1016/j.jss.2014.01.054

a b s t r a c t

Background: Mechanical ventilation (MV) is a method of maintaining appropriate gas ex-

change in patients who are unable to sustain adequate alveolar ventilation. While life-

saving in the short-term, prolonged MV leads to altered cardiovascular responses and

enhanced lung injury, but the exact mechanism is unknown. Therefore, we investigated

the involvement of the sympathoadrenergic and renineangiotensin system in MV-induced

altered cardiovascular responses.

Methods: SpragueeDawley rats were divided into six groups: (1) spontaneous breathing (SB);

(2) SB þ enalapril (100 mg/kg intravenous infusion); (3) SB þ losartan (100 mg/kg infusion); (4)

12 h of MV; (5) MV þ enalapril; and (6) MV þ losartan. After the animals were sacrificed,

blood and tissue samples were collected. Tyrosine hydroxylase, dopamine beta hydroxy-

lase, and neuropeptide Y were measured in adrenal medulla and hypothalamus, whereas

AT1 was measured in lung tissues by Western blot. Norepinephrine enzyme-linked

immunosorbent assay and total antioxidant capacity were assayed in plasma.

Results: Our findings indicated that MV increases the sympathetic activation markers in ad-

renal medulla and hypothalamus. Moreover, oxidative stress was increased in lung and brain

tissues. Treatment with enalapril or losartan reduced the lipid peroxidation in lung and brain

tissues, while preserving the tissue glutathione content and plasma antioxidant capacity.

Conclusions: These data demonstrate that the inhibition of the renineangiotensin system by

enalapril or losartan may reduce the MV-induced increase in sympathetic activity markers

and oxidative stress, and thus, may have a beneficial effect as adjuvant therapy.

ª 2014 Elsevier Inc. All rights reserved.

rmacology and Therapeutics, College of Medicine, University of Florida, P.O. Box 100267,

rapeutics, College of Medicine, University of Florida, P.O. Box 100267, Gainesville, FL 32610.(H.Z. Toklu), ntumer@ufl.edu (N. Tumer).ier Inc. All rights reserved.

j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1e72

1. Introduction treated with the AT1 antagonist losartan; (4) 12 h of MV; (5)

Mechanical ventilation (MV) is a method to mechanically

assist or replace spontaneous breathing (SB). Although it is

often a lifesaving intervention in critically ill patients, it

carries many potential complications including pneumo-

thorax, acute lung injury, and ventilator-associated pneu-

monia. Other complications include diaphragm atrophy,

decreased cardiac output, and oxygen toxicity [1]. The in-

flammatory response in the lungs due toMVmay lead to distal

organ dysfunction.

Recent research indicates a cross talk between lungs and

other organs, including brain [2].The hypothalamic sym-

pathoadrenal medullary axis leads tomarked activation of the

adrenal medulla and sympathetic ganglia characterized by

elevated activity of the catecholamine biosynthesizing en-

zymes such as tyrosine hydroxylase (TH) and dopamine beta

hydroxylase (DbH), resulting in a rise in circulating epineph-

rine and norepinephrine (NE). TH is the rate-limiting step

in catecholamine biosynthesis as it catalyzes the hydroxyl-

ation of tyrosine to dopamine, whereas DbH catalyzes the

conversion of dopamine to NE. In addition to catecholamines,

neuropeptide Y (NPY) is synthesized in the adrenal medulla

and is co-released with epinephrine and NE. The previously

mentioned factors, TH, DbH, and NPY are considered as bio-

markers of sympathetic nervous system activity [3].

Sympathoactivation contributes to systemic stress and

cardiovascular complications. The patients who have pro-

longed MV display blood pressure alterations and abnormal

autonomic responses [4], which may be because of MV-

associated activation of the hypothalamic-pituitary-adrenal

axis or hypothalamic sympathoadrenal medullary axis.

Whether MV activates these axis is unknown.

The present study was aimed to test the hypothesis that a

12 h exposure to MV results in increased activation of the

hypothalamic sympathoadrenal medullary axis besides

increasing oxidative stress in plasma and other organs. Thus,

we measured TH, DbH, and NPY protein expression in the

adrenalmedulla and hypothalamus, lung angiotensin II type 1

(AT1) receptor protein levels, oxidative stress in lung and

brain, and plasma NE. In addition, the effects of enalapril-

angiotensin converting enzyme inhibitor (ACE) and losartan-

AT1 receptor blocker on MV-induced changes were evaluated.

2. Materials and methods

2.1. Animals

Adult female SpragueeDawley rats were obtained from

Charles River Labs and were aged 4e6 mo and w300 g at the

time of sacrifice. All animals were housed at the University of

Florida Animal Care Services Center according to the guide-

lines set forth by the Institutional Animal Care and Use

Committee. Animals were maintained on a 12-h lightedark

cycle and provided food (AIN93 diet) and water ad libitum

throughout the experimental protocol. Animals were divided

into six groups (10 animals/group) as follows: (1) 12 h of SB; (2)

SB group treated with the ACE inhibitor enalapril; (3) SB group

12 h of MV with the ACE inhibitor enalapril; and (6) 12 h of MV

with the AT1 antagonist losartan.

Losartan group received an intraperitoneal priming dose

(30 mg/kg) followed by intravenous infusion (100 mg/kg/min,

infusion rate 0.30 mL/h), and enalapril group received

enalapril (40 mg/kg) followed by an intravenous infusion

(100 mg/kg/min, infusion rate 0.30 mL/h) while during the 12 h

experimental period.

2.2. Experimental protocol

Animals were anesthetized with sodium pentobarbital

(60mg/kg, intraperitoneally). After reaching a surgical plane of

anesthesia, tracheostomy was performed on the animals

using aseptic techniques and mechanically ventilated with a

controlled pressure-driven ventilator (Servoventilator 300;

Siemens; Bridgewater, NJ) for 12 h with the following settings:

upper airway pressure limit, 20 cm H2O; pressure control level

above positive end-expiratory pressure 4e6 cm H2O; respira-

tory rate, 80 beats/min; and positive end-expiratory pressure

1.0 cm H2O. In general, we estimate that these ventilator

settings result in a tidal volume of w1 mL/100 g of body

weight. All surgical procedures were performed as previously

described in detail [15]. Briefly, cannulas were inserted into

the carotid artery to permit continuousmeasurement of blood

pressure and the collection of periodic arterial blood samples

during MV. Blood samples were analyzed for pH, pO2, and

pCO2 using an electronic blood gas analyzer (GEM Premier

3000; Instrumentation Laboratory; Lexington, MA). If neces-

sary, adjustments were made to the ventilator to ensure that

the arterial blood gas and pH measures were within the

desired physiological ranges. PaO2 was maintained at 70 mm

Hg throughout the experiment by adjustments in FiO2 (22%e

25% oxygen). Cannulas were inserted into the jugular vein for

the constant infusion of sodium pentobarbital (w10 mg/kg/h).

Body temperature was maintained between 36�C and 37�C by

using a recirculating heating blanket. Continuous care during

the MV protocol included lubricating the eyes, expressing the

bladder, removing airway mucus, rotating the animal, and

passive limb movement. After 12 h of MV, the animals were

immediately killed, and blood and tissue samples were

collected.

2.3. Enzyme-linked immunosorbent assaymeasurements of plasma NE

Blood samples were taken via cardiac puncture at the time of

sacrifice and centrifuged at 5000 rpm for 10 min at 4�C. Thesamples were stored at �80�C until further analysis by an

enzyme-linked immunosorbent assay kit (Rocky Mountain

Diagnostics, Inc Colorado Springs, CO) following the in-

structions of the manufacturer.

2.4. Total antioxidant capacity in the plasma

Total antioxidant capacity in the plasma was evaluated by

Oxiselect kit (Biocell Laboratories, San Diego, CA) according to

the manufacturer’s instructions.

Table e Plasma total antioxidant capacity (tAOC) expressed as copper reducing equivalent (CRE) and NE concentrations inSB, enalapril, losartan, and MV groups at 12 h. Each group consists of six to eight rats.*P < 0.05 versus SB and D P < 0.05versus MV. Values are presented as mean ± standard error of mean.

Plasma concentration SB SBE SBL MV MVE MVL

tAOC (CRE mmol/mL) 218.10 � 12.8 225.60 � 19.8 193.00 � 12.4 125.90 � 18.6* 209.10 � 24.2 þ 196.90 � 21.5þ

NE (ng/mL) 2.32 � 0.9 2.34 � 0.6 2.48 � 0.9 1.86 � 0.9 2.95 � 0.9 3.00 � 1.1þ

MVE¼MVwith the ACE inhibitor enalapril; MVL¼MVwith the AT1 antagonist losartan; SBE¼ SB group treatedwith the ACE inhibitor enalapril;

SBL ¼ SB group treated with the AT1 antagonist losartan.

j o u rn a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1e7 3

2.5. Tissue preparation

After completion of the experimental protocol, animals were

overanesthetized with pentobarbital (120 mg/kg intraperito-

neally) and the adrenal medulla, hypothalamus, and lung

rapidly removed, immediately frozen in liquid nitrogen and

stored at �80�C until subsequent analyses. Before homoge-

nization, adrenals were decapsulated and the medullae were

separated from the cortex.

2.6. Western blot analysis

Adrenal medulla, hypothalamus, and lung were homogenized

and assayed for protein levels of the catecholamine biosyn-

thetic enzymes, TH and DbH, along with NPY using antibodies

directed to TH (1/8000; Pel-Freez Biologicals, Rogers, AR), DbH

(1/2000; Novus Biologicals, Littleton, CO), NPY (1/500; Santa

Cruz Biotechnology, Santa Cruz, CA), and AT1(1/2000; Abcam,

Cambridge, MA). All secondary antibodies were used in a

concentration of 1/2000). An equal amount of protein for each

adrenal medulla homogenate (2 mg protein for TH, 10 mg for

DbH, and 35 mg for NPY), hypothalamus homogenate (4 mg

protein for TH, 0.2 mg protein for DbH, and 40 mg for NPY), and

lung (40 mg protein for AT1) was applied to the gels.

2.7. Malondialdehyde (MDA) and glutathione (GSH)assays

Tissue sampleswere homogenizedwith potassiumphosphate

buffer (pH 6.0) and ice-cold trichloroacetic acid (1 g tissue plus

10 mL 10% trichloroacetic acid) in an ultrasonic tissue ho-

mogenizer. The MDA levels were assayed for products of lipid

peroxidation by monitoring thiobarbituric acid reactive sub-

stance formation as described previously [5]. Lipid peroxida-

tion is expressed in terms of MDA equivalents using an

extinction coefficient of 1.56 � 105 /M/cm and the results are

expressed as nmol MDA/g tissue.

GSH measurements were performed using a modification

of the Ellman procedure [6]. Briefly, after centrifugation at

2000g for 10 min, 0.5 mL of supernatant was added to 2 mL of

0.3 mol/L Na2HPO4 2H2O solution. A 0.2 mL solution of dithio-

bisnitrobenzoate (0.4 mg/mL 1% sodium citrate) was added,

and the absorbance at 412 nm was measured immediately

after mixing. The results are expressed in mmol GSH/g tissue.

2.8. Statistics

Data are presented as mean � standard error of mean. Com-

parisons between groups were done by analysis of variance

followed by Tukey multiple comparison test or Student t-test

using a GraphPad Prism 5.0 software (GraphPad, San Diego,

CA). Significance was established at P < 0.05.

3. Results

MV caused a decrease in the total antioxidant capacity of the

plasma in MV group. Enalapril and losartan treatments

significantly restored the plasma antioxidant capacity (Table).

Moreover, the plasmaNE levels had a tendency to decrease

in the MV group; however, this decrease was not significant

because of the large variation between samples. Treatment

with enalapril or losartan prevented this decrease (Table).

MV caused an increase in sympathetic activation markers,

that is, TH, DbH, and NPY in adrenal medulla, therapeutic

intervention with either enalapril or losartan reversed these

MV-induced changes (Fig. 1). In contrast, TH remained un-

changed in hypothalamus, whereas DBH and NPY were

elevated with MV. In the hypothalamus, enalapril failed to

inhibit the increases in DbH and NPY while losartan had effi-

cacy (Fig. 1).

AT1 protein was assessed in the lungs. AT1 receptor pro-

tein was elevated with MV, and although there was no change

with drug treatment in the absence of MV, enalapril and los-

artan reversed the MV-induced increase (Fig. 2).

MV caused an increase in both lung and brain tissues lipid

peroxidation as evidenced by the increase in the malondial-

dehyde content, that is, thiobarbituric acid reactive substance.

Also there was a concomitant decrease in GSH content in

these tissues. Treatment with either enalapril or losartan

prevented the decrease in GSH and reduced the lipid peroxi-

dation (Fig. 3).

4. Discussion

MV not only causes an injury in the lung, but also may lead to

oxidative stress and injury in distant organs [7]. It is known

that there are a multiple pathways enabling cross talk be-

tween lung and other organs including the brain. An insult to

the lung may alter physiological factors and may lead to an

imbalance in the brain. Because the integrity of the brain

function mainly depends on O2 and glucose, a peripheral ho-

meostatic imbalance likely triggers an inflammatory response

through cytokines and other mediators [2,8].

Previous reports have shown that MV increases the

oxidative stress and causes ventilation-induced acute lung

injury and secondary injuries in other organs such as

Fig. 1 eWestern blot analysis of the protein levels of TH, DßH, and NPY in the adrenal medulla and hypothalamus tissues of

the saline or enalapril (E) or losartan (L)-treated SB or MV groups. Each group consists of n [ 6e8 rats. *: P < 0.05 versus SB

and D: P < 0.05 versus MV.

j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1e74

diaphragm, heart, or kidney [7,9,10]. It has been also shown

that MV leads to a decrease in tissue GSH levels [11]. In the

present study, we have consistently observed that MV caused

a decrease in GSH levels in lung and brain, and a concomitant

increase in lipid peroxidation as evidenced by the increase in

the malondialdehyde content, a thiobarbituric acid reactive

substance. Moreover, plasma antioxidant capacity was

decreased probably because of the extensive oxidative stress

in the body. GSH is one of the principle endogenous antioxi-

dants in all tissues, and the oxidative injury is a consequence

of GSH depletion [12]. Therefore, antioxidants and GSH ana-

logs have been widely studied to prevent or reduce oxidative

injury in various models.

In the present study, both losartan and enalapril were

effective in reducing lipid peroxidation and partially in

preserving the GSH content in lung. On the other hand, in

the brain, losartan demonstrated similar results, but not

enalapril. Although both losartan and enalapril do not have a

sulfhydryl moiety have previously been shown to exert anti-

oxidant effects in various studies [13,14]. Therefore, our

finding that losartan and enalapril reduce oxidative stress and

increase antioxidant capacity was consistent with these pre-

vious reports [15]. However, the failure of enalapril to protect

brain tissue could be a result of its inability to cross the blood

brain barrier. For instance, it has been claimed that losartan

effects are limited to the periphery except for the cases with

brain injury [16]. On the other hand, there are several studies

that support the central effects of losartan. Kucuk et al. [17]

showed that losartan treatment attenuated hypertension-

induced bloodebrain barrier permeability. Moreover, func-

tional evidence has been shown that systemic administration

of losartan crosses bloodebrain barrier in the rat and

attenuates the pressor response to electrical stimulation of

subfornical organ [18].

Fig. 2 e Western blot analysis of the protein levels of AT1

receptor in the lung tissues of the saline or enalapril (E)-or

losartan (L)-treated SB or MV groups. Each group consists

of n [ 6e8 rats. **: P < 0.01 versus SB and D: P < 0.05

versus MV.

j o u rn a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1e7 5

MV caused an increase in sympathetic activation markers,

that is, TH, DbH, and NPY in adrenal medulla. Although TH

remained unchanged in hypothalamus, DbH and NPY were

elevated. Both drugs (enalapril and losartan) reversed these

MV-induced changes in the adrenal medulla. However, ena-

lapril failed to inhibit the increase in DbH and NPY while

Fig. 3 eMalonedialdehyde and GSH levels in lung (A and C) and b

(L)-treated groups. Each group consists of n [ 6e8 rats. *: P < 0

losartan had some efficacy. This increase in the protein levels

of the biosynthetic enzymes (TH and DbH) was probably

because of a compensatory mechanism to restore NE plasma

levels; we observed that plasma NE levels tended to decrease

at 12 h after MV.

The alteration of plasma epinephrine and NE levels

because ofMVwas previously reported by several researchers.

Aneman et al. [19] showed NE was inconsistent or unstable in

patients. Monteverde [20] showed that the NE requirement

was increased in pediatric patients in prolonged MV and

Barbieri et al. [21] showed that the plasma epinephrine and NE

increased at the first hour of MV in patients, and this increase

was restored to baseline levels by the termination of MV.

Moreover, Wieske et al. [22] have proposed that the autonomic

dysfunction caused abnormal heart rates in intensive care

unit patients.

Activation of angiotensin system contributes to several

pathophysiological effects in the cardiovascular system via the

activation of sympathetic nervous system [23].Previous studies

have reported an increase in lung or bronchoalveolar fluid

angiotensin II levels at 2 or 4 h of MV [24e27]. However, lung

ACE levels were found to be unchanged [25] or increased in

bronchoalveolar fluid [26]. Moreover, captopril was effective in

reducing theMV-induced lung injury and elevatedACE activity,

and blockade of bradykinin receptors did not attenuate the

effect of captopril. Moreover, the serum ACE levels were un-

changed, although angiotensin II levels were increased in the

bronchoalveolar fluid. Thus, indicating the role of a non-ACE

pathway for the production of angiotensin II [26].

A recent study has also shown that angiotensin 1e7 ana-

logs protected the lung against acute lung injury, thereby

increasing the ratio of ACE2/ACE activity and reducing the

rain (B and D) tissues of the salineorenalapril (E) or losartan

.05 versus SB and D: P < 0.05 versus MV.

Fig. 4 e Mechanical ventilation increases the sympathetic nervous system (SNS) tonus as a result of increased

norepinephrine (NE) turnover. The levels of biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta

hydroxylase (DßH) are increased, along with an increase in NPY, which is co-released with NE. Sympathoactivation also

activates the renin-angiotensin system.

j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1e76

bioavailability of angiotensin II and AT1 receptor actions [28].

Increased angiotensin or AT1 levels have been correlated with

the severity of the injury. Both receptor types AT1 and AT2 in

the lungswere also shown to increase after MV [25,27]. Similar

to treatment with ACE inhibitors, treatment with an angio-

tensin receptor blocker, losartan, has been shown to reduce

the lung edema, inflammation, and injury score and AT1 re-

ceptor messenger RNA because of MV [25,27].

In the present study, we did not measure the ACE activity

but we found that lung AT1 receptor protein was increased

in the lung at 12 h of MV, and both enalapril and losartan

treatments were effective in preventing this increase. As we

previously discussed, both treatments also reduced the

oxidative stress in the lungs and sympathetic nervous sys-

tem activity markers in the adrenal medulla. On the other

hand, it would have been valuable to evaluate the AT1 re-

ceptor protein in the aorta and to evaluate the sympathetic

nervous system activity in different brain regions other than

hypothalamus.

5. Conclusions

In summary, these data, for the first time, demonstrate that

inhibition of renineangiotensin system by enalapril or los-

artan may reduce the MV-induced increase in sympathetic

activity markers and oxidative stress, and thus, may have a

beneficial effect as adjuvant therapy.

On the other hand, in humans and a number of species,

including the hamster, quantitatively important chymase-

independent Ang II formation from Ang I occurs in the

heart, arteries, and kidney. However, chymase differs in rats

and rabbits and is not active in the conversion of Ang I to Ang

II, but is involved in Ang II degradation. Consequently, one

would anticipate that blockade of the system at the ACE step

in rats would be equivalent to inhibition of the Ang II receptor

in humans. On the other hand, AT1 receptor blockers may be

more effective than ACE inhibitors in humans [29].

Although the preliminary data of this study are important

to in terms of showing the involvement of the interaction of

j o u rn a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1e7 7

renineangiotensin system with the MV-induced sympathetic

activation (Fig. 4), further studies are needed to elucidate the

detailed mechanisms and implement it into the clinical

setting.

Acknowledgment

This work was supported by the Medical Research Service of

the Department of Veterans Affairs. The authors declare that

they have no conflict of interest.

Author contributions: H.Z.T. collected the data, performed

statistical analysis, and prepared the manuscript. O-S.K.

adapted the animal model, performed the surgery, and

collected the data. Y.S., K.L., and N.K. collected the data and

performed analysis. K.J.S., M.P.W., A.J.S., and E.E.T performed

the surgery, followup the animals, and collected the data. S.P.,

P.J.S., and N.T. were responsible for experimental design and

prepared the manuscript.

Disclosure

The authors reported no proprietary or commercial interest in

any product mentioned or concept discussed in this article.

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