Long-acting phosphodiesterase 5 inhibitor,tadalafil,and superoxide dismutase mimetic,tempol,protect against acute hypoxia-induced pulmonary hypertension in rats

Abstract

Long-acting phosphodiesterase 5 (PDE5) inhibitor, tadalafil, was recently approved for the treatment of pulmonary hypertension. Apart from being a PDE5 inhibitor, tadalafil also possesses antioxidant activity. The aim of this study was to probe whether tadalafil has any beneficial effect over tempol owing to its antioxidant action in addition to PDE5 inhibitory activity. Albino Wistar rats were pretreated with tadalafil (10 mg/kg) or vehicle 2 h before hypoxic exposure, whereas tempol (20 mg/kg) was given 5 min before induction of hypoxia. Right ventricular systolic pressure (RVSP), mean arterial pressure (MAP), heart rate (HR), right ventricular contractility (RVdP/dtmax) and cardiac output (CO) were recorded while subjecting rats to acute hypoxia for 30 min. Lipid peroxidation and reduced glutathione were estimated in serum before and after hypoxia exposure. Tadalafil as well as tempol significantly prevented hypoxia-induced rise in RVSP (p < 0.001) and RVdP/dtmax (p < 0.05). Both tadalafil and tempol pretreatment partially prevented (p < 0.01) the rise in CO due to hypoxia. Tadalafil did not produce any significant change in MAP, whereas tempol led to a significant fall (p < 0.01) in MAP. Acute hypoxia increased the oxidative stress levels. Tadalafil pretreatment partially prevented hypoxia-induced oxidative stress, while tempol pretreatment completely prevented hypoxia-induced oxidative stress. Results suggest that tadalafil because of its antioxidant action in addition to PDE5 inhibitory activity is more appropriate for the prevention of hypoxic pulmonary hypertension than tempol. Tempol also produced undesirable systemic hypotension as side effect, which was not seen with tadalafil because of its pulmonary selective action.

Keywords

pulmonary hypertension phosphodiesterase 5 inhibitor tadalafil tempol acute hypoxia

Introduction

Pulmonary hypertension (PH), a progressive disease of various causative factors, is characterized by increased pulmonary vascular resistance (PVR), vascular remodelling of pulmonary arterioles and results in premature death from right ventricular failure.¹ The estimated incidence of primary PH is 1–2 cases per 1 million persons in the general population.² However, the overall prevalence of secondary PH in the general population is unknown, owing to the heterogeneity of the disease.³ It has been established that increased circulatory levels of endothelin 1, upregulation of phosphodiesterase 5 (PDE5) receptors, dysregulation of potassium channels, increased oxidative stress, reduced expression of nitric oxide synthase and reduction in the enzyme prostacyclin synthase are involved in the pathogenesis of PH.⁴ Current therapy for PH includes prostacyclin receptor agonists, calcium channel blockers, endothelin receptor antagonists and nitric oxide (NO). But unfortunately, existing therapeutic alternatives still face effectiveness limitations and are associated with complications and high cost.⁵ Inhaled NO therapy has found broad clinical utility, yet its use is limited by potential toxicities.⁶ Main disadvantage in the use of prostacyclin analogues for treatment of PH is that their usage requires complicated delivery devices.⁷ Bosentan, an oral, nonselective endothelin receptor antagonist has been approved for the treatment of PH, but long-term use of this vasodilator is limited by liver toxicity. Calcium channel blockers lack pulmonary specificity. Therefore, there is a need of an ‘ideal’ pulmonary vasodilator that combines high treatment efficacy, pulmonary selectivity, simplicity of administration and reduced side effects.⁸

PDE5 is an enzyme that inactivates cGMP and is found in abundance in pulmonary circuit. In PH, there is increased expression of PDE5 receptors, thus limiting the duration of action of cGMP (because of increased degradation by PDE5). As a result, agents that block the activity of PDE5 enzyme have received significant interest for their potential therapeutic benefits in PH. PDE5 inhibitor, sildenafil, has shown good results in the treatment of PH.^9–11 However, short half-life of sildenafil requires frequent daily administration, thus reducing compliance and increasing cost of therapy.^5,12,13 Tadalafil (a long-acting PDE5 inhibitor) has been introduced for treatment of erectile dysfunction. Its main advantage over sildenafil is that because of its longer duration of action it requires single daily administration and is more selective than sildenafil for PDE5 relative to PDE6.¹⁴ Several case reports provide anecdotal evidence of acute and chronic reductions in PVR, improved functional capacity and increased exercise capacity after administration of tadalafil 10–20 mg every 36–48 h.¹⁵

A growing body of evidence indicates that superoxide and other reactive oxygen species (ROS) contribute both to acute hypoxic vasoconstriction and to PH associated with chronic hypoxia.^16,17 An excess of ROS through the increment of cytosolic calcium in the pulmonary arterial myocyte has been suggested to trigger pulmonary arterial vasoconstriction, which operates as an autoregulatory mechanism to limit blood flow to hypoxic alveoli and to preserve ventilation–perfusion matching. Superoxide dismutase (SOD) mimetic, tempol, being an efficient scavenger of free radicals has been shown to inhibit hypoxic pulmonary vasoconstriction independent of its effect on NO production.¹⁸ A preliminary study established that tadalafil exerts a beneficial acute effect on the cardiovascular system by reducing serum levels of oxidative stress and increasing serum levels of paraoxonase 1 in men with erectile dysfunction.¹⁹ However, the effect of tadalafil on oxidative stress associated with acute hypoxic pulmonary vasoconstriction has not been studied.

Therefore, this study was carried out to investigate the effect of PDE5 inhibitor, tadalafil, and SOD mimetic and an efficient free radical scavenging agent, tempol,²⁰ on pulmonary hypertensive response induced by acute hypoxia in rats and to probe whether tadalafil because of its antioxidant effect in addition to its PDE5 inhibitory activity has any beneficial effect over tempol on acute hypoxia-induced PH. Comparison was made between the effects of tadalafil and tempol on hemodynamic and oxidative stress parameters.

Materials and methods

Animals

Healthy Wistar albino rats weighing between 200 and 250 g were obtained from the animal house of Vallabhbhai Patel Chest Institute. Animals were housed in polyethylene cages in groups of 4 rats per cage and were kept in room temperature maintained at 25 ± 2°C with a 12-h light/dark cycle.

Experiments were performed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India, after approval by Institutional Animal Ethical Committee.

Hemodynamic parameters studied

Acute hypoxia-induced pulmonary vasoconstriction was assessed by measuring right ventricular systolic pressure (RVSP), which was taken as an index for pulmonary arterial pressure. To study the effect of hypoxia and drug treatments on right ventricular contractility, maximal rate of rise in right ventricular pressure (RVdP/dtmax) was recorded online by differentiating RVP. Hemodynamic parameters recorded to study the systemic circulation including systolic blood pressure, diastolic blood pressure, mean arterial pressure (MAP), heart rate (HR) and cardiac output (CO).

Assessment of arterial blood pressure and heart rate

Rats were anesthetized with urethane dissolved in distilled water and were injected intraperitoneally at a dose of 1 g/kg body weight. ²¹ A middle line incision was given in the neck region; retracting the pretracheal muscle exposing trachea and a transverse incision was given in between the two rings. A tracheal cannula (PE-240 tubing) was introduced into the opening to allow free breathing without obstruction. Body temperature of the rat was maintained at 37–38°C. Femoral artery of one side was exposed and a polyethylene catheter filled with heparin solution (500 IU/ml, v/v) was inserted in the artery through a small incision for recording arterial blood pressure. The catheter was attached to a 23-gauge needle connected via 3-way stopcock to a pressure Statham-P23D transducer (Schlumberger, Norcross, Georgia). Femoral vein of the other limb was cannulated for injecting drugs.

The pressure recording system was calibrated with the help of a mercury manometer before each experiment. Arterial blood pressure was measured after 30 min of stabilization period. Systolic, diastolic, MAPs and HR were displayed and recorded on a Power Lab Data-Acquisition System (4SP, AD Instruments, Australia) with a computerized analysis program (Chart 5.4.2, AD Instruments, Australia).

Assessment of right ventricular functions

For recording RVSP (which is taken as index for pulmonary arterial pressure), right jugular vein was cannulated and a catheter connected to a pressure tranducer was pushed inside until placed in right ventricle. The maximal rate of rise in right ventricular pressure was recorded online by differentiating right ventricular pressure as a marker of myocardial contractility.

Assessment of cardiac output

For measurement of CO, an incision at the level of the third intercostal space was made, ribs were retracted and a 3-mm Transonic flow-meter probe (model T101, Transonic Systems, Ithaca, NY, USA) was placed around the ascending aorta, perpendicular to the curvature of the arch. CO (ml/kg/min) was estimated from continuous aortic blood flow measurements.

Assessment of oxidative stress

To assess oxidative stress, biochemical evaluations such as lipid peroxidation (LPO) and reduced glutathione (GSH) were carried out in serum from animals exposed to acute hypoxia.

Malondialdehyde (MDA) is a stable product of LPO, which is used as an index of free radical production. LPO was estimated in serum according to the method of Wright et al.²² and expressed in µmol/L. The concentration of GSH in serum was measured by the standard method of Jollow et al.²³

Exposure to acute hypoxia for inducing pulmonary vasoconstriction

Hypoxia-induced PH in rats is a reliable and well-accepted model of PH. This model mimics PH experienced by patients suffering from obstructive sleep apnea disease, hence used to carry out this study. Rats were anesthetized with urethane (1 g/kg), trachea was cannulated and rats were ventilated with room air (mean tidal volume = 5 ± 1 ml/kg), using a small animal respirator (Columbus instruments, USA). For exposure to acute hypoxia, rats were ventilated with a hypoxic mixture of oxygen (10% O₂) through the ventilator for 30 min.^24–26

Experimental protocol

Study was performed on the animals divided into 4 groups having 10 animals in each.

Control group. Hemodynamic parameters were recorded as baseline measurements when rats were breathing normal room air and then animals were subjected to hypoxia using a small animal respirator for a 30-min period. The effect of hypoxia (10% O₂) on cardiopulmonary responses was then assessed during the period of 30 min of hypoxia. Serum biochemical estimations were performed by collecting blood before and after hypoxia exposure of 30 min.

Vehicle (carboxymethyl cellulose) control group

In this group, animals were pretreated with carboxymethyl cellulose (vehicle for tadalafil) 2 h prior to hypoxic exposure for 30 min. The effect of vehicle (if any) on all hemodynamic parameters was recorded at basal level during normoxia and during 30 min of hypoxia. For determination of oxidative stress, biochemical estimations were performed in serum before and after hypoxia exposure.

Tadalafil-treated group

Animals were pretreated with tadalafil (10 mg/kg orally) 2 h before starting hypoxic exposure. Dose of tadalafil was standardized by doing pilot experiments. The effect of tadalafil on various hemodynamic parameters was assessed both before and during 30 min of hypoxic (10% O₂) exposure. Serum biochemical estimations were performed by collecting blood before and after hypoxia exposure.

Tempol-treated group

In this group, the animals were pretreated with tempol (20 mg/kg intravenously) 5 min prior to the start of hypoxic exposure for 30 min. The effect of tempol on hemodynamic and biochemical parameters was assessed before and during hypoxic exposure.

Drugs

Tadalafil

Tadalafil (gift by Ranbaxy, Gurgaon, India) was dispersed in 0.5% Carboxymethyl cellulose (CMC) suspension.

Tempol

Tempol (Sigma Aldrich, USA) was dissolved in saline. Dose of tadalafil and tempol was standardized by doing pilot study. On the basis of these experiments, an effective dose of both tadalafil (10 mg/kg) and tempol (20 mg/kg), which provided an adequate protection against acute hypoxia induced rise in RVSP, was used for further studies.

Statistical analysis

The results were expressed as mean ± SEM. Means were compared using one-way analysis of variance for multiple comparisons, followed by Tukey–Krammer multiple comparison test. Paired t test was used for comparison within the group, normoxia and during hypoxic exposure. p < 0.05 was considered statistically significant.

Results

Pulmonary vasoconstriction induced by acute hypoxia

In rats subjected to acute hypoxic exposure, a significant increase (p < 0.001) in RVSP was observed (Figure 1A). A significant rise (p < 0.05) in right ventricular contractility was also observed following acute hypoxia for 30 min (Figure 1B). Hypoxia did not produce any significant change in HR (Table 1). However, a fall in MAP was observed during hypoxic exposure, but it was not statistically significant (p > 0.05) when compared to basal values (Table 1).

Figure 1.

Changes in the hemodynamic parameters induced by acute hypoxia of 30 min in control group. (A) Right ventricular systolic pressure (mmHg), (B) right ventricular contractility (mmHg/s) and (C) cardiac output (ml/kg/min). Data represent mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 versus normoxia. Comparison was made between normoxia and during hypoxia readings.

Table 1.

Effect of hypoxia (10% O2) on mean arterial pressure (mmHg) and heart rate (BPM) in control, vehicle, tempol-pretreated and tadalafil-pretreated groups^a

Parameters	Control		Vehicle		Tempol pretreatment				Tadalafil pretreatment
Parameters	Con	Hyp	Con	Hyp	Con	Hyp	Tem + Con	Tem + Hyp	Con	Hyp
Mean arterial pressure (mmHg)	62 ± 9	46 ± 4	68 ± 4	53 ± 3	70 ± 4	59 ± 6	40 ± 3^b	45 ± 4^b	78 ± 9	69 ± 3
Heart rate (BPM)	321 ± 28	343 ± 21	325 ± 7	345 ± 15	352 ± 17	367 ± 17	328 ± 18	364 ± 21	339 ± 11	342 ± 16

Con: control, Hyp: hypoxia, Tem + Con: tempol + control, Tem + Hyp: tempol + hypoxia.

^aThe values are the mean ± SEM.

^b p < 0.01 versus control (tempol pretreated group).

A significant fall (p < 0.01) in CO was also observed in rats during hypoxic exposure (Figure 1C). Acute hypoxia of 30 min led to a significant (p < 0.001) rise in MDA levels in comparison to the controls. There was a decrease in serum GSH content observed in rats subjected to acute hypoxia. However, this decrease in GSH content was not found to be statistically significant compared to control rats (Table 2).

Table 2.

Effect of acute hypoxia on concentrations of malondialdehyde (MDA) and reduced glutathione (GSH) in serum of rats pretreated with tadalafil and tempol^a

Parameters	Control	Vehicle	Tada	Temp	Hypoxia	Hyp + V	Hyp + Tad	Hyp + Tem
MDA (µmol/L)	0.36 ± 0.02	0.41 ± 0.03	0.39 ± 0.03	0.4 ± 0.017	0.63 ± 0.02b	0.6 ± 0.02c	0.52 ± 0.01d,e	0.47 ± 0.01f
GSH (µmol/L)	2.4 ± 0.12	2.4 ± 0.13	2.3 ± 0.13	2.4 ± 0.14	2 ± 0.09	2 ± 0.14	2.1 ± 0.2	2 ± 0.1

^aThe values are mean ± SEM. p < 0.001 versus hypoxia.

^b p < 0.001 versus control.

^c p < 0.001 versus vehicle.

^d p < 0.05 versus Hyp + veh (hypoxia + vehicle).

^e p < 0.05.

^f p < 0.001 versus Hyp + Tad (hypoxia + tadalafil).

Effect of vehicle on various hemodynamic parameters

Carboxymethyl cellulose vehicle per se did not show any significant change in RVSP, RVdP/dtmax, MAP, HR, CO, MDA and GSH when compared to the values of control group.

Effect of tadalafil on hypoxia-induced pulmonary vasoconstriction

Tadalafil per se at the dose of 10 mg/kg did not produce any change in any of the hemodynamic or biochemical parameters studied. Tadalafil-pretreated rats significantly prevented (p < 0.001) the rise in RVSP (Figure 2A) and RVdP/dtmax (p < 0.05; Figure 2B) following hypoxia compared to the vehicle control group.

Figure 2.

Changes in the hemodynamic parameters induced by acute hypoxia for 30 min in control, vehicle and tadalafil-pretreated groups. (A) Right ventricular systolic pressure (mmHg), (B) right ventricular contractility (mmHg/s) and (C) cardiac output (ml/kg/min). Data represent mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 control versus hypoxia. ^$$$ p < 0.001, ^$$ p < 0.01, ^$ p < 0.05 tadalafil pretreated + hypoxic exposure group versus hypoxia.

A significant reduction (p < 0.001) in CO was observed in both control and vehicle control group following hypoxia. Tadalafil pretreatment significantly attenuated (p < 0.01) this fall in CO compared to hypoxia exposure values of vehicle control group, but the baseline value of before hypoxia was not achieved (p < 0.01) by tadalafil pretreatment (Figure 2C).

There were no significant differences in MAP and HR observed between tadalafil and vehicle control groups either at baseline or during acute hypoxic exposure (Table 1).

Tadalafil pretreatment significantly prevented the hypoxia-induced rise (p < 0.05) in serum MDA levels in comparison to vehicle control rats. Acute hypoxia did not produce any significant effect on serum GSH levels in tadalafil-pretreated rats (Table 2).

Effect of tempol on hypoxia-induced pulmonary vasoconstriction

In rats, administration of tempol 20 mg/kg per se significantly decreased (p < 0.01) MAP from baseline values. This fall in MAP persisted even after subjecting tempol-pretreated rats to hypoxia (Table 1).

Tempol pretreatment markedly prevented (p < 0.001) the rise in RVSP after hypoxic exposure, but the baseline value of RVSP was not achieved by tempol pretreatment (Figure 3A).

Figure 3.

Changes in the hemodynamic parameters induced by acute hypoxia for 30 min before and after tempol pretreatment. (A) Right ventricular systolic pressure (mmHg), (B) right ventricular contractility (mmHg/s) and (C) cardiac output (ml/kg/min). Data represent mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 versus control. ^$$$ p < 0.001, ^$$ p < 0.01 tempol pretreatment + hypoxia versus hypoxia. ^$ p < 0.05 for tempol pretreatment + hypoxia vs hypoxia p < 0.01 tempol pretreatment + hypoxia versus tempol pretreatment + control.

Right ventricular contractility was significantly increased (p < 0.01) following hypoxia. Tempol pretreatment prevented this rise in RV contractility following hypoxia (Figure 3B).

Tempol pretreatment partially prevented (p < 0.01) the fall in CO which was associated with hypoxia exposure (Figure 3C).

Tempol pretreatment significantly prevented the hypoxia-induced rise (p < 0.001) in serum MDA levels in comparison to control rats. Acute hypoxia did not produce any significant effect on serum GSH levels in tempol-pretreated rats (Table 2).

Discussion

The PDE5 is a major PDE subtype and is predominantly expressed in pulmonary vasculature,^27,28 thus generating interest in the use of PDE5 inhibitors for PH and other vascular pathology.²⁹ Injury mediated by free radicals to pulmonary vasculature is also an important mechanism in hypoxia-induced lung damage.³⁰ Tempol, being a superoxide scavenger has been shown to inhibit hypoxic pulmonary vasoconstriction.¹⁸

Present study was designed to find out whether tadalafil, because of its antioxidant activity in addition to its PDE5 inhibitory action, may prove as a better therapeutic option for hypoxic PH in comparison to that of tempol.

Acute exposure to hypoxia produced a rapid rise in RVSP in anesthetized rats. Tadalafil completely inhibited the hypoxia-induced rise in RVSP, while tempol partially inhibited the hypoxia-induced rise in RVSP. This effect of the orally active PDE5 inhibitor tadalafil on RVSP in the present study is comparable to previous in vitro studies (where tadalafil inhibited hypoxia-induced pulmonary vasoconstriction), showing the beneficial effect of tadalafil in pulmonary vasoconstriction.³¹ The hypoxic challenge was timed to coincide with peak plasma levels of tadalafil after oral dosing.³² Tadalafil, because of its pulmonary selectivity, did not produce any fall in systemic blood pressure, while tempol markedly decreased MAP.

Increase in right ventricular contractility by acute hypoxia observed in this study is in agreement with an earlier study.³³ Possibly this is a reflex mechanism of homeometric autoregulation and may be due to the release of positive inotropic substances from the endocardial endothelium, stimulation of stretch-activated calcium channels and elevation of sympathetic tone.³⁴ This reflex mechanism may help the right ventricle to preserve pump performance without changing preload as a consequence of high right ventricle afterload conditions. It was observed that both tadalafil and tempol pretreatment inhibited the rise in RV contractility induced by acute hypoxia. This reduction in contractility may be an indirect mechanism caused by the immediate adaptation of RV contractility to match a drug-induced reduction in RV afterload. As far our knowledge, this is the first study to report the effect of tadalafil and tempol on right ventricular contractility in rats.

The effect of hypoxia on CO in the present study is comparable to previous studies showing the detrimental effect of hypoxia on CO.^35,36 Hypoxia leads to pulmonary vasoconstriction by increasing PVR, which results in increased right ventricular afterload and right ventricular end systolic volume. This rise in right ventricular afterload because of pulmonary vasoconstriction hinders the right ventricle to eject blood in pulmonary circuit, thus decreasing stroke volume, which ultimately results in the lowering of CO (CO = stroke volume × HR). Since there was no change observed in HR during 30 min of hypoxia, stroke volume was the determining factor of CO. One important thing to note over here is that in spite of the increased right ventricular contractility induced by hypoxia, a fall in CO was observed. Explanation to this effect may lie in the fact that acute hypoxia leads to such an intense rise in pulmonary arterial pressure that even a rise in right ventricular contractility (reflex mechanism) could not reduce the right ventricular end systolic volume so as to bring back the CO to basal value. Both tadalafil and tempol partially prevented this decrease in CO in rats subjected to acute hypoxia. These hemodynamic effects are probably mediated by decrease in PVR leading to the lowering of right ventricular afterload and right ventricular end systolic volume, which is the most likely mechanism for the observed tadalafil- and tempol-induced benefits. Present finding agree with other studies which have shown that treatment with tadalafil improves CO,^37,38 but these studies have not studied the effect of tadalafil on cardiac contractility. As mentioned above, both tadalafil and tempol lowered the right ventricular contractility in rats exposed to hypoxia, thus supporting our hypothesis that this effect may not be due to the negative inotropic effect of tadalafil or tempol but may be attributed to decreased right ventricular afterload caused by tadalafil and tempol which in turn might lead to decrease in right ventricular contractility (which as explained above can be a reflex action to the increase in pulmonary vasoconstriction).

In our study, animals subjected to acute hypoxia demonstrated an increase in the serum MDA. This finding is in agreement with the previous finding showing involvement of oxidative stress in hypoxia-induced PH.³⁰ Tadalafil was shown to partially prevent oxidative stress, thus suggesting that tadalafil may have some antioxidant property in addition to its PDE5 inhibitory activity due to which it showed such a marked effect on PH. Effect of tadalafil on oxidative stress confirms the findings of Verit et al. which showed the beneficial effect of tadalafil on cardiovascular system owing to its antioxidant activity.¹⁹ Tempol as expected prevented hypoxia-induced increase in oxidative stress due to which it proved to be beneficial in hypoxia-induced PH.

vIn conclusion, the present study demonstrated that oral pretreatment of tadalafil prevented the rise in RVSP during hypoxic exposure without producing any fall in MAP. This effect of selective pulmonary vasodilation by tadalafil may be attributed to increased expression of PDE5 in pulmonary vasculature. Tempol on the other hand partially prevented the rise in RVSP induced by hypoxia and this effect of tempol was associated with the lowering of MAP. Both these drugs allowed the right ventricle to preserve its pump performance at lower energy cost by matching the contractility to the prevailing afterload. As tadalafil’s RVSP-lowering effect was not associated with any fall in MAP, this finding suggests that tadalafil could be a better therapeutic option in comparison to tempol for treatment of hypoxia-induced PH. Tadalafil also showed a partial antioxidant action which was evident from the decrease in MDA levels and increase in GSH levels. Thus, the beneficial effect of tadalafil on PH may be attributed to its antioxidant activity in addition to its PDE5 inhibitory action. More work is required to establish safety and efficacy of tadalafil especially in the settings of chronic hypoxia-induced PH. Tadalafil may prove to be more effective in chronic hypoxia-induced PH because it can inhibit pulmonary vascular remodeling, which is a major cause of chronic PH.

Footnotes

This work was supported by Life Science Research Board, Defense Research and Development Organization.

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