SHORT-TERM CARDIOVASCULAR EFFECTS OF SELECTIVE PHOSPHODIESTERASE 3 INHIBITOR OLPRINONE VERSUS NON-SELECTIVE PHOSPHODIESTERASE INHIBITOR AMINOPHYLLINE IN A MECONIUM-INDUCED ACUTE LUNG INJURY

Meconium aspiration syndrome (MAS) in the newborns is characterized by airway obstruction, surfactant dysfunction, inflammation, pulmonary vasoconstriction, and lung edema. Thus, similarly to acute lung injury of other origin, clinical picture of MAS is characterized by refractory hypoxemia, diffuse pulmonary infiltrates, and high permeability pulmonary edema. Because of complex pathomechanisms of MAS, treatment is usually multi-agent and covers ventilation support and support of vital functions. In severe cases of MAS, therapeutic protocol may be widened of conventional or high-frequency ventilation with higher concentrations of oxygen, administration of exogenous surfactant, inhalation of nitric oxide, liquid ventilation, or use of extracorporeal membrane oxygenation (ECMO) (1). In addition, anti-inflammatory agents, such as glucocorticoids, or non-selective and selective phosphodiesterase (PDE) inhibitors (1), have been increasingly used.

Non-selective PDE inhibitors, e.g., theophylline, are useful for treatment of bronchial asthma and chronic bronchopulmonary disease (2). Theophylline, an active component of aminophylline molecule, elevates levels of cAMP and cGMP and lessens concentrations of intracellular calcium, acetylcholine, and monoamines. It finally results in bronchodilation, vasodilation, enhanced surfactant production and mucociliary transport, and some anti-inflammatory effects (3). As a non-selective PDE inhibitor, theophylline influences several phosphodiesterases at once. Therefore, its administration may be associated with severe adverse effects, e.g., gastrointestinal signs and headache. Other side effects may be caused by antagonism with adenosine receptors (dysrhythmias and increased gastric secretion) (3, 4).

To minimize undesirable effects of the treatment, selective PDE inhibitors have been generated with expectation to keep the therapeutic action in lower occurrence of adverse effects, as they target the specific tissue with lower impact to other tissues or systems. In the respiratory system, PDE3, PDE4, and PDE5 are of particular importance. Therefore, selective inhibitors of these PDEs have been used for treatment of various respiratory diseases (5-7). Representatives of non-selective PDE inhibitors (8, 9), selective PDE3 inhibitors olprinone (10) and milrinone (11), and PDE5 inhibitor sildenafil (12) have been administered also in MAS, where they improved lung functions and reduced oxidative stress.

PDE3 is expressed in myocardium, alveolar macrophages, smooth muscle cells, endothelial cells, fat tissue, and platelets. As inhibition of PDE3 enhances myocardial contraction, produces vasodilation, and reduces platelets aggregation, selective PDE3 inhibitors are used as cardiotonic agents and vasodilators (13, 14). Logically, majority of adverse reactions associated with selective PDE3 inhibitors (e.g., olprinone) involves the cardiovascular system, i.e. tachycardia, ventricular tachyarrhythmias, premature ventricular contraction, or hypotension (13).

Nevertheless, despite increasing use of PDE inhibitors, there is insufficient information on their possible adverse effects in MAS. In our previous study, acute increase in blood pressure, heart rate, and heart rate variability was observed after aminophylline administration in a rabbit model of MAS (15). Aim of the present study was to compare effects of intravenously administered selective PDE3 inhibitor olprinone and non-selective PDE inhibitor aminophylline on cardiovascular functions in the meconium-instilled rabbits. Short-term changes in blood pressure and heart rate were monitored during and immediately after the administration and within 5 hours lasting period after administration of the treatments. In addition, fluctuations of the heart rate around its average value, i.e. heart rate variability (HRV), representing a sensitive marker of cardiac sympathovagal control mechanisms (16), were analyzed.

To extend our understanding of mechanisms involved in the adverse cardiovascular effects of PDE inhibitors, concentrations of several heart-associated substances were determined in the blood plasma. First of them, aldosterone, is referred as a key cardiovascular hormone (17), as it contributes to hypertension and participates in endothelial dysfunction, atherosclerosis, inflammation, and myocardial ischemia in coronary artery disease. Furthermore, enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) were measured, as they may also indicate cardiovascular risk and oxidative stress (18, 19).

MATERIALS AND METHODS

General design of experiments

Design of experiments was approved by the local Ethics Committee of Jessenius Faculty of Medicine and National Veterinary Board.

Meconium was collected from healthy term neonates, lyophilized and stored at –20°C. Before use, meconium was suspended in 0.9% NaCl at a concentration of 25 mg/ml.

Adult rabbits (chinchilla) of 2.5±0.3 kg body weight (b.w.) were anesthetized with intramuscular ketamine (20 mg/kg b.w.; Narketan, Vetoquinol, UK) and xylazine (5 mg/kg b.w.; Xylariem, Riemser, Germany), followed by infusion of ketamine (20 mg/kg/h). Tracheotomy was performed and catheters were inserted into a femoral artery and right atrium for sampling the blood, and into a femoral vein to administer anesthetics. Animals were then paralyzed with pipecuronium bromide (0.3 mg/kg b.w./30 min; Arduan, Gedeon Richter, Hungary) and subjected to a pressure-controlled ventilator (Beat-2, Chirana, Slovakia). All animals were ventilated with a frequency of 30/min, fraction of inspired oxygen (FiO2) of 0.21, inspiration time Ti 50%, peak inspiratory pressure (PIP) to keep a tidal volume (VT) between 7–9 mL/kg b.w. and no positive end-expiratory pressure (PEEP) at this stage of experiment.

After 15 min stabilization, cardiopulmonary parameters were recorded and blood gases were analyzed (RapidLab 348, Siemens, Germany). Then, rabbits were intratracheally administered 4 mL/kg b.w. of meconium suspension (25 mg/mL) or saline (served as controls). From this moment on, animals were ventilated with FiO2 1.0 and PEEP 0.3 kPa. In the meconium-instilled animals, respiratory failure developed within 30 min, defined as >30% decrease in dynamic lung-thorax compliance (Cdyn) and PaO2<10 kPa at FiO2 1.0. After recording the parameters, meconium-instilled animals were treated with intravenous aminophylline (each of 2.0 mg/kg b.w.; Syntophyllin, Hoechst-Biotika, Slovakia), due to a short biological half-time given in two doses at 0.5 and 2.5 h after intratracheal meconium instillation (Mec+Amin group, n=6), or received a single dose of olprinone (0.2 mg/kg b.w.; Olprinone hydrochloride, Sigma Aldrich, Germany) 0.5 h after meconium instillation (Mec+Olp group, n=6). Both drugs were diluted in normal saline up to a total volume of 1 mL, or animals received the same volume of saline (1 mL) at corresponding time points to treated animals (referred to as sham-treated animals; Mec+Sal group, n=6, and Sal+Sal group, n=5). Treatment was delivered slowly over a period of 5 min. Immediate cardiovascular changes associated with treatment were evaluated within two intervals (A1 and A2) of 2.5 min of administration (A) of aminophylline or olprinone (5 min in total) and within two intervals (PA1 and PA2) of 2.5 min after finishing the treatment administration (post administration (PA); 5 min in total) (Fig. 1). All animals were oxygen-ventilated for additional 5 hours after the first dose of treatment. Cardiorespiratory parameters were recorded at 0.5, 1, 2, 3, 4, and 5 hours to investigate early effects of the treatment. At the end of experiments, animals were sacrificed by an overdose of anesthetics.

Fig. 1. Scheme of treatment administration.      

Measurement and evaluation of cardiopulmonary parameters

Tracheal airflow and VT were measured by a heated Fleisch head connected to a pneumotachograph. Airway pressure was registered via a pneumatic catheter placed in the tracheal tube and connected to electromanometer. Cdyn was calculated as a ratio between VT (adjusted per kg b.w.) and airway pressure gradient (PIP–PEEP). Mean airway pressure (MAP) was calculated as: MAP = (PIP+PEEP)/2 and oxygenation index (OI) as OI = (MAP×FiO2)/PaO2. Right-to-left pulmonary shunts were calculated by computer program using Fick equation: (CcO2–CaO2)/(CcO2–CvO2)×100, where CcO2, CaO2 and CvO2 are concentrations of oxygen in pulmonary capillaries, arterial and mixed blood. CcO2 was calculated by using PAO2 (alveolar partial pressure of oxygen) from the equation: PAO2 = (PB–PH2O) × (FiO2–PaCO2) × (FiO2+(1–FiO2)/R), where PB is barometric pressure and PH2O the pressure of water vapour. Respiratory exchange ratio (R) was assumed to be 0.8 and the current value of hemoglobin necessary for calculating the oxygen concentration in the blood was measured by combined analyzer (RapidLab 348, Siemens, Germany).

Systolic (SBP) and diastolic (DBP) blood pressures were measured via a catheter in the femoral artery connected to electromanometer, and the mean arterial blood pressure (MABP) was calculated as MABP = DBP+1/3(SBP–DBP). Heart rate (HR) was obtained from ECG recorded by subcutaneous electrodes. HRV was evaluated using a computer system (VariaPulse TF3, Sima Media, Czech Republic). Parameters of time analysis, mean duration of R-R interval (RR) and mean squared successive difference (MSSD), and parameters of spectral analysis, i.e. spectral powers in low frequency (LF: 0.05–0.15 Hz) and high frequency (HF: 0.15–2.0 Hz) bands and total spectral power (TP), were analyzed. From the mentioned parameters, MSSD, HF, and TP have been established as markers of parasympathetic activity, while LF band represents activity of both branches of autonomic cardiac control and expresses activity of baroreceptors, as well (19, 20).

Biochemical analyses of the blood plasma

Quantification of total antioxidant status (TAS) in the plasma at the end of experiment was carried out using ABTS (2,2’-azino-di-(3-ethylbenzthiazoline sulphonate) radical formation kinetics (Randox TAS kit, Randox laboratories Ltd., UK) and expressed in mmol/L. Concentration of thiobarbituric-acid reactive substances (TBARS) was determined from the absorbance at 532 nm and expressed in nmol/mg protein. Concentration of aldosterone was measured by aldosterone ELISA kit (BioVendor, Czech Republic) and was expressed in pg/mL. Activities of aspartate aminotranferase (AST), gamma-glutamyltransferase (GGT), and alanine aminotransferase (ALT) were measured by biochemical analyzer (Olympus AU640, Beckman Coulter, Switzerland) and were expressed in international units (IU)/L.

Statistical analysis

Data were tested for normality of distribution by Kolmogorov-Smirnov test. Since distribution of some HRV variables (spectral powers) was extremely skewed, logarithmic transformation of these data was used to improve normality before statistical analysis was performed. Then, between-group differences were analyzed by ANOVA with post-hoc LSD test. Within-group differences were evaluated by Wilcoxon test. Strength of association between biochemical and cardiovascular markers were expressed by Pearson’s correlation coefficient (r) and Bonferroni probability (P). A value of P<0.05 was considered statistically significant. Data are expressed as means ± S.E.M.

RESULTS

Body weight and initial values of all cardiopulmonary parameters were comparable between the groups before intratracheal instillation of meconium or saline (all P>0.05).

Respiratory parameters

Instillation of meconium seriously worsened the lung functions, as demonstrated by increased right-to-left pulmonary shunts and oxygenation index and need for higher ventilatory pressures in comparison with saline-instilled controls (Table 1). Administration of both PDE inhibitors significantly reduced pulmonary shunting, improved oxygenation and allowed to decrease the ventilatory pressures compared to meconium-instilled non-treated animals, whereas better effect was observed in aminophylline (Table 1).

Table 1. Lung function parameters, i.e. mean airway pressure (MAP), oxygenation index (OI) and right-to-left pulmonary shunts (RLS), in saline-instilled sham-treated animals (Sal+Sal group), and in meconium-instilled sham-treated animals (Mec+Sal group), meconium-instilled aminophylline-treated animals (Mec+Ami group) and meconium-instilled olprinone-treated animals (Mec+Olp group) before and after intratracheal meconium/saline (Before/After M/S) instillation and during 5 hours after the treatment.

For between-group comparisons: Mec+Sal vs. Sal+Sal: aP<0.05, bP<0.01, cP<0.001; Mec+Ami vs. Mec+Sal: dP<0.05, eP<0.01, fP<0.001; Mec+Olp vs. Mec+Sal: gP<0.05, hP<0.01, iP<0.001; Mec+Ami vs. Mec+Olp: jP<0.05, kP<0.01, lP<0.001.

Instillation of meconium increased accumulation of liquid in the lung tissue, as indicated by higher wet-dry lung weight ratio in meconium-instilled and non-treated animals (Mec+Sal group; 7.98±0.15) in comparison with saline-instilled controls (Sal+Sal group; 5.73±0.17; P<0.001 vs. Mec+Sal). In the treated groups, aminophylline (Mec+Ami group; 6.65±0.28; P<0.001 vs. Mec+Sal) and olprinone (Mec+Olp group; 6.65±0.35; P<0.001 vs. Mec+Sal) significantly reduced the lung edema formation in comparison with non-treated animals.

Cardiovascular parameters

Before administration of the first dose of treatment, no significant between-group differences were found. During 5 min of treatment administration (two intervals of 2.5 min, A1 and A2), no significant changes were found in any of recorded cardiovascular parameters. However, within 5 min interval immediately after finishing of the treatment delivery, increase in MABP and tendency to elevate HR was observed in both treated groups when compared with sham-treated controls. Analysis of heart rate variability showed increase in parameters expressing parasympathetic cardiac control, such as MSSD, and logHF (Table 2). Similar kind of response was demonstrated in administration of the second dose of aminophylline 2 hours after the first dose. Again, there was a tendency to increase MABP, HR, and HRV parameters. However, significant differences between aminophylline-treated group and sham-treated animals were already found before administration of the second dose of treatment (Table 3).

Table 2. Cardiovascular parameters in saline-instilled sham-treated animals (Sal+Sal group), in meconium-instilled sham-treated animals (Mec+Sal group), in meconium-instilled aminophylline-treated animals (Mec+Ami group) and in meconium-instilled olprinone-treated animals (Mec+Olp group) before administration of the first dose of treatment (Before1), and during 5 min of the treatment administration (intervals A1 and A2, each of 2.5 min) and immediately after the treatment administration (intervals PA1 and PA2, each of 2.5 min).

For between-group comparisons: Mec+Ami vs. Mec+Sal: cP<0.05, dP<0.01; Mec+Olp vs. Mec+Sal: fP<0.05, gP<0.01; Mec+Ami vs. Sal+Sal: lP<0.05, mP<0.01; Mec+Olp vs. Sal+Sal: oP<0.05, pP<0.01.

Table 3. Cardiovascular parameters in saline-instilled sham-treated animals (Sal+Sal group), and in meconium-instilled sham-treated animals (Mec+Sal group) and meconium-instilled aminophylline-treated animals (Mec+Ami group) before administration of the second dose of treatment (Before2), and during 5 min of the treatment administration (intervals A1 and A2, each of 2.5 min) and immediately after the treatment administration (intervals PA1 and PA2, each of 2.5 min).

For between-group comparisons: Mec+Sal vs. Sal+Sal: aP<0.05; Mec+Ami vs. Mec+Sal: cP<0.05, dP<0.01, eP<0.001; Mec+Ami vs. Sal+Sal: lP<0.05, mP<0.01, nP<0.001.

In the further course of experiment, most of cardiovascular changes in the treated groups gradually adjusted to the values comparable with sham-treated groups. However, parameters of HRV, particularly MSSD, remained higher till the end of experiment (Table 4).

Table 4. Cardiovascular parameters in saline-instilled sham-treated animals (Sal+Sal group), and in meconium-instilled sham-treated animals (Mec+Sal group), meconium-instilled aminophylline-treated animals (Mec+Ami group) and meconium-instilled olprinone-treated animals (Mec+Olp group) before and after intratracheal meconium/saline (Before/After M/S) instillation and during 5 hours after the first dose of the treatment.

For between-group comparisons: Mec+Ami vs. Mec+Sal: cP<0.05, dP<0.01, eP<0.001; Mec+Olp vs. Mec+Sal: fP<0.05, gP<0.01; Mec+Ami vs. Mec+Olp: iP<0.05, jP<0.01; Mec+Ami vs. Sal+Sal: lP<0.05, mP<0.01, nP<0.001; Mec+Olp vs. Sal+Sal: oP<0.05, pP<0.01.

Biochemical markers of meconium-induced injury

In the meconium-instilled and sham-treated animals (Mec+Sal group), increased TBARS (P<0.001 vs. Sal+Sal) and slightly lower TAS (P>0.05 vs. Sal+Sal) in the plasma demonstrated influence of oxidation processes and deterioration of antioxidant capacity also on the systemic level. Both PDE inhibitors showed a trend to reduce oxidation and to prevent decline in TAS, with more potent effect observed in olprinone (Table 5).

Table 5. Biochemical markers in the plasma in saline-instilled sham-treated animals (Sal+Sal group), and in meconium-instilled sham-treated animals (Mec+Sal group), meconium-instilled aminophylline-treated animals (Mec+Ami group) and meconium-instilled olprinone-treated animals (Mec+Olp group) at the end of experiment.

For between-group comparisons: Mec+Sal vs. Sal+Sal: aP<0.05, bP<0.001; Mec+Ami vs. Sal+Sal: lP<0.05, mP<0.01; Mec+Olp vs. Mec+Sal: gP<0.01; Mec+Ami vs. Mec+Olp: iP<0.05.

Concentrations of aldosterone, a non-specific marker of stress and injury, were higher in both Mec+Sal (P<0.05 vs. Sal+Sal) and Mec+Ami (P<0.01 vs. Sal+Sal) groups, while olprinone reduced aldosterone level (Table 5). To evaluate extent of cardiovascular injury in this model of MAS, plasma levels of enzymes having their origin in the heart were measured. In Mec+Sal group, non-significantly higher values of AST and GGT were found compared with other groups (all P>0.05; Table 5). To assess liver injury potentially associated with MAS, plasma levels of ALT were determined. However, no differences were found between the groups (all P>0.05; Table 5).

Correlations between biochemical markers and cardiovascular parameters

Pearson’s evaluation of association between the biochemical markers showed negative correlations between TAS vs. TBARS (r= –0.786, P<0.001), vs. aldosterone (r= –0.773, P<0.001), vs. GGT (r= –0.764, P<0.001), vs. AST (r= –0.694, P<0.001), and vs. ALT (r= –0.504, P<0.05). Positive correlations were found between TBARS vs. aldosterone (r=0.682, P<0.001), vs. GGT (r=0.543, P<0.05), and vs. AST (r=0.714, P<0.001); between aldosterone vs. GGT (r=0.622, P<0.01), and vs. AST (r=0.577, P<0.01); and between AST vs. GGT (r=0.641, P<0.01), and vs. ALT (r=0.489, P<0.05). Correlations between ALT vs. TBARS, aldosterone, and GGT were not significant (P>0.05).

To evaluate relation of biochemical markers to cardiovascular changes, MSSD as the most sensitive cardiovascular marker was chosen. MSSD at 5 hours of the treatment administration correlated borderly with GGT (r=0.503, P=0.040) and a slight tendency to correlate was observed in relation to aldosterone (r=0.394, P=0.085), TAS (r= –0.400, P= 0.100), and TBARS (r=0.313, P=0.168).

DISCUSSION

PDE inhibitors may be beneficial for treatment of MAS, as they improve lung functions and alleviate inflammation and oxidative injury (8-12). Nevertheless, there is little information on their adverse effects in the conditions of meconium-induced acute lung injury. Many newborns with MAS suffer from hemodynamic instability and thus, sudden cardiovascular changes may be life-threatening for them. Therefore, adverse effects of perspective medicaments should be thoroughly studied before they might be recommended for the use. In this study, both selective PDE3 inhibitor olprinone and non-selective PDE inhibitor aminophylline increased blood pressure, heart rate, and heart rate variability immediately after finishing the treatment delivery. Similarly, in aminophylline-treated adults, tachycardia, hypertension, and generation of extrasystoles were observed (4). In newborns with severe respiratory distress syndrome (20), aminophylline elevated heart rate and some parameters of HRV, particularly spectral power in HF band. Aminophylline increased heart rate and cardiac output, and influenced vascular resistance also in various animal models (22, 23). These findings may seem to be controversial due to relatively unrelated tachycardic reaction and simultaneous parasympathetic excitation. However, cardiac activity is an integrated signal that is influenced not only by two branches of the autonomic nervous system, but also by other underlying physiological mechanisms and various extrinsic factors (16). Thus, the heart rate variability cannot be explained by peculiarities in sympathovagal balance, but it is determined by more universal mechanisms (24) leading to arrhythmias. Importantly, the blood pressure and mean heart rate have stabilized within several minutes, but changes in heart rate variability were observed till the end of experiment.

Adverse effects of aminophylline occur particularly at high doses, i.e. in plasma concentrations of theophylline exceeding 15–20 mg/l (3). Theophylline is metabolized by first-order kinetics in liver. Its half-life in the blood is several hours, e.g. about 3 hours for intravenously administered dose of 2.5 mg/kg in rabbits (25). In humans, maximum plasma concentration is reached 20 min following intravenous administration (26). Nevertheless, in the newborns and in some concurrent situations (e.g., acute lung edema) theophylline clearance may be reduced. As mentioned above, some cardiovascular effects of aminophylline are caused by PDE inhibition, as increased cAMP in myocardium has positive chronotropic and inotropic effects. In addition, accumulation of cAMP potentiates action of neurotransmitters and hormones, e.g. catecholamines (13, 27). Some changes in HRV and generation of arrhythmias are caused by antagonism with adenosine receptors, as well. Theophylline presumably acts on adenosine A1 and A2A receptors, however, participation of adenosine receptors antagonism in the cardiovascular action of theophylline (or aminophylline) is not fully elucidated (3, 28).

On the other hand, increased concentration of cAMP due to PDE3 inhibition activates protein kinase A, which stimulates potential-dependent Ca2+ channels on the membranes of cardiac muscle cells, leading to facilitated inward of Ca2+ into the cells and cardiac contraction. Contrary, PDE3 inhibitors decrease cytosolic free Ca2+ in vascular smooth muscle, thus causing vasodilation. In addition to other mechanisms, sensitivity of vascular smooth muscle to cGMP or cAMP-dependent vasodilators (including PDE inhibitors) may be modulated by exposure to NO (29). Olprinone lowers mean aortic and pulmonary artery pressures, but exerts differential vasodilatory effects on peripheral vessels in each organ, based on different distribution of PDE3 among the organs (30). In asthmatic patients, olprinone decreased diastolic blood pressure and increased heart rate (31). In these experiments, olprinone increased blood pressure, heart rate, and heart rate variability to a comparable extent to aminophylline within several minutes after the administration. At the moment, we may only speculate on mechanisms behind the short-term cardiovascular changes. As mentioned above, cardiovascular effects of olprinone are attributable to PDE3 inhibition, while aminophylline action is mediated by both PDE inhibition and antagonism with adenosine receptors. In addition, olprinone and aminophylline exert different effect on catecholamines. Aminophylline increases epinephrine levels, but olprinone has no effect on epinephrine or norepinephrine (27). Based on comparable changes in blood pressure, heart rate, and HRV parameters we may presume that the short-term cardiovascular side effects are predominantly mediated by PDE3 inhibition. Nevertheless, further research is needed to elucidate participation of the mentioned mechanisms in the cardiovascular side effects of PDE inhibitors more in detail.

On the other hand, aminophylline and olprinone improved lung functions, i.e., reduced right-to-left pulmonary shunting, improved oxygenation, decreased requirements for ventilation, and diminished lung edema formation. In addition, both treatments reduced oxidative stress in meconium-injured lungs. Biochemical markers of oxidation were measured also in the plasma, as they may influence indirectly also the cardiac activity (21). Systemic consequences of MAS were demonstrated by higher TBARS, a marker of lipid peroxidation, and reduced total antioxidant status (TAS) in the plasma. Despite both PDE inhibitors decreased formation of TBARS and prevented a decline in TAS compared with non-treated animals, stronger effect was observed in olprinone. Potent anti-inflammatory and antioxidant action of olprinone has been proven at different doses in various models of injury (10, 32-34), while aminophylline exerts better anti-inflammatory and antioxidative effects at lower plasma concentrations than used in this study (3, 9, 35).

Furthermore, several heart-associated biochemical markers were measured to estimate extent of cardiovascular injury in relation to MAS and given therapy. For instance, aldosterone, which chronic overproduction leads to hypertension. In addition, via rapid, non-genomically mediated mechanisms aldosterone participates in many processes in vascular smooth muscle cells, cardiac myocytes, and endothelial cells. As a result, increased intracellular Ca2+ and cAMP in smooth muscle cells (17), leads to elevation of vascular resistance, modulation of sympathovagal balance towards potentiating the sympathetic activity, changes in heart rate and baroreflex sensitivity within several minutes and potentiation of effects of catecholamines (36, 37). Secretion of aldosterone may be stimulated by oxidized fatty acids (38). Thus, increased plasma aldosterone in the meconium-instilled animals could be partially explained by lipoperoxidation, as indicated by correlations with increased TBARS and decreased TAS. However, reason for finding of high plasma aldosterone in the aminophylline-treated group and low aldosterone in the olprinone-treated group is unknown. We may presume participation of the cross-talk between renin-angiotensin-aldosterone and adrenergic systems (36), as aminophylline elevates circulating catecholamines (3). In addition, synthesis of aldosterone is mediated through activation of cAMP-dependent signaling pathways leading to induction of genes encoding enzymes involved in the conversion of cholesterol to steroids (39). Several phosphodiesterases, e.g., PDE2, PDE8B, and PDE11A are expressed in adrenal cortex (40). Therefore, different PDE inhibitors may have different capacity to influence aldosterone production through the above mentioned mechanisms. Additionally, by antagonism of adenosine receptors theophylline (or aminophylline) stimulates secretion of renin and later also aldosterone (41), whereas olprinone has no effect on adenosine receptors.

Besides aldosterone, plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase (GGT) were investigated, as they elevate in cardiovascular and metabolic diseases (42, 43). In the heart or liver injury, AST increases proportionally to extent of the injury, whereas the rise is detectable about 6 hours after the insult (43). Elevated levels of GGT and ALT are sensitive markers of systemic inflammation and oxidative stress (18), and predictors of cardiovascular risk (19). GGT is a key enzyme in the catabolism of glutathione modulating the redox status. Production of ROS leads to depletion of glutathione, induces expression of GGT, and subsequently elevates plasma activity of GGT (44). ALT may be associated with development of metabolic syndrome, type 2 diabetes, and hypertension (18, 44). However, despite slightly increased values of GGT and AST, no significant between-group differences were observed, probably due to short-term observation and/or absence of morphological changes on the heart structures. In the study by Yamada et al. (18), higher ALT and GGT correlated with C-reactive protein and markers of lipoperoxidation, indicating cardiovascular risk. In this study, oxidation markers correlated well with GGT and AST, but less with ALT, probably due to a fact that metabolic liver-associated processes are of lower importance in MAS than in the above mentioned diseases. However, significant correlations of GGT and AST with oxidation markers and aldosterone indicate that these commonly used biochemical markers may be useful in estimation of MAS severity.

Nevertheless, we are aware of several limitations of our study. Firstly, due to inter-species differences in autonomic cardiac regulation between rabbits and humans the cardiovascular response to administration of PDE inhibitors may differ. Secondly, despite the used model of MAS is acceptable, it cannot fully resemble the clinical MAS on the background of postnatal changes in hemodynamics. Therefore, eventual cardiovascular adverse effects of this treatment should be studied also in the neonates with MAS. Finally, period of observation of cardiorespiratory changes, which was limited in this study to several hours after the treatment administration, should be prolonged in both experimental and clinical studies. Thereafter, positive and negative effects of the treatment should be critically evaluated.

In conclusion, intravenous administration of both selective PDE3 inhibitor olprinone and non-selective PDE inhibitor aminophylline significantly improved the lung functions in meconium-instilled rabbits. However, olprinone to, showed no clear benefit to aminophylline in extent of cardiovascular adverse effects as both treatments exerted comparable short-term increase in blood pressure, heart rate, and heart rate variability. Therefore, if PDE inhibitors are used for treatment of MAS, cardiovascular parameters should be monitored carefully, particularly in patients with cardiovascular instability.

Acknowledgements: Authors thank M. Petraskova, M. Hutko, D. Kuliskova and Z. Remisova for technical assistance. Study was supported by Project „Center of Excellence in Perinatology Research (CEPV II) No. 26220120036, co-financed from EU sources, by Project APVV-0435-11, and Grant VEGA No. 1/0057/11.

Conflict of interests: None declared.

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