Short-Term Cardiovascular Effects of Selective

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2013, 64, 6, 751-759 www.jpp.krakow.pl

D. MOKRA1, I. TONHAJZEROVA1, H. PISTEKOVA1, Z. VISNOVCOVA1, J. MOKRY2, A. DRGOVA3, M. REPCAKOVA3, A. CALKOVSKA1

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

1Department of Physiology, Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Martin, Slovakia; 2Department of Pharmacology, Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Martin, Slovakia; 3Department of Medical Biochemistry, Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Martin, Slovakia

Various anti-inflammatory drugs have been used for treatment of neonatal meconium aspiration syndrome (MAS). As their adverse effects are poorly described, this study compared effects of selective phosphodiesterase (PDE) 3 inhibitor olprinone and non-selective PDE inhibitor aminophylline on cardiovascular parameters in animal model of MAS. Oxygen-ventilated rabbits were intratracheally instilled 4 mL/kg of meconium (25 mg/mL) or saline. Thirty minutes later, meconium-instilled animals were intravenously given olprinone (0.2 mg/kg) at a single dose at 0.5 h after meconium instillation, or aminophylline (2.0 mg/kg) at two doses at 0.5 and 2.5 h after meconium instillation, or were left without treatment. Cardiovascular changes were evaluated within 5 min of administration and 5 min after finishing the administration. Furthermore, respiratory and cardiovascular parameters were measured within 5 hours following treatment delivery. Oxidation markers (thiobarbituric acid-reactive substances (TBARS), and total antioxidant status) and markers of cardiovascular injury (aldosterone, gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT)) were determined in the plasma. Meconium instillation induced acute lung injury associated with oxidative stress, elevated aldosterone, and slightly increased GGT and AST levels. Both aminophylline and olprinone improved lung functions and reduced oxidation stress. However, the PDE inhibitors acutely increased blood pressure and heart rate, whereas heart rate variability remained higher till the end of experiment and correlated well with markers of cardiovascular injury. Considering that systemic administration of olprinone and aminophylline was accompanied by acute cardiovascular changes in the meconium-instilled animals, use of PDE inhibitors in the newborns with MAS should be carefully monitored.

Key words: meconium aspiration, phosphodiesterase inhibitor, aminophylline, olprinone, cardiovascular, blood pressure, heart rate, heart rate variability

INTRODUCTION Non-selective PDE inhibitors, e.g., theophylline, are useful for treatment of bronchial asthma and chronic Meconium aspiration syndrome (MAS) in the newborns is bronchopulmonary disease (2). Theophylline, an active characterized by airway obstruction, surfactant dysfunction, component of aminophylline molecule, elevates levels of cAMP inflammation, pulmonary vasoconstriction, and lung edema. and cGMP and lessens concentrations of intracellular calcium, Thus, similarly to acute lung injury of other origin, clinical acetylcholine, and monoamines. It finally results in picture of MAS is characterized by refractory hypoxemia, diffuse bronchodilation, vasodilation, enhanced surfactant production pulmonary infiltrates, and high permeability pulmonary edema. and mucociliary transport, and some anti-inflammatory effects Because of complex pathomechanisms of MAS, treatment is (3). As a non-selective PDE inhibitor, theophylline influences usually multi-agent and covers ventilation support and support of several phosphodiesterases at once. Therefore, its administration vital functions. In severe cases of MAS, therapeutic protocol may may be associated with severe adverse effects, e.g., be widened of conventional or high-frequency ventilation with gastrointestinal signs and headache. Other side effects may be higher concentrations of oxygen, administration of exogenous caused by antagonism with adenosine receptors (dysrhythmias surfactant, inhalation of nitric oxide, liquid ventilation, or use of and increased gastric secretion) (3, 4). extracorporeal membrane oxygenation (ECMO) (1). In addition, To minimize undesirable effects of the treatment, selective anti-inflammatory agents, such as glucocorticoids, or non- PDE inhibitors have been generated with expectation to keep the selective and selective phosphodiesterase (PDE) inhibitors (1), therapeutic action in lower occurrence of adverse effects, as they have been increasingly used. target the specific tissue with lower impact to other tissues or 752

systems. In the respiratory system, PDE3, PDE4, and PDE5 are of inspired oxygen (FiO2) of 0.21, inspiration time Ti 50%, peak of particular importance. Therefore, selective inhibitors of these inspiratory pressure (PIP) to keep a tidal volume (VT) between PDEs have been used for treatment of various respiratory 7–9 mL/kg b.w. and no positive end-expiratory pressure (PEEP) diseases (5-7). Representatives of non-selective PDE inhibitors at this stage of experiment. (8, 9), selective PDE3 inhibitors olprinone (10) and milrinone After 15 min stabilization, cardiopulmonary parameters (11), and PDE5 inhibitor sildenafil (12) have been administered were recorded and blood gases were analyzed (RapidLab 348, also in MAS, where they improved lung functions and reduced Siemens, Germany). Then, rabbits were intratracheally oxidative stress. administered 4 mL/kg b.w. of meconium suspension (25 PDE3 is expressed in myocardium, alveolar macrophages, mg/mL) or saline (served as controls). From this moment on, smooth muscle cells, endothelial cells, fat tissue, and platelets. animals were ventilated with FiO2 1.0 and PEEP 0.3 kPa. In the As inhibition of PDE3 enhances myocardial contraction, meconium-instilled animals, respiratory failure developed produces vasodilation, and reduces platelets aggregation, within 30 min, defined as >30% decrease in dynamic lung- selective PDE3 inhibitors are used as cardiotonic agents and thorax compliance (Cdyn) and PaO2<10 kPa at FiO2 1.0. After vasodilators (13, 14). Logically, majority of adverse reactions recording the parameters, meconium-instilled animals were associated with selective PDE3 inhibitors (e.g., olprinone) treated with intravenous aminophylline (each of 2.0 mg/kg b.w.; involves the cardiovascular system, i.e. tachycardia, ventricular Syntophyllin, Hoechst-Biotika, Slovakia), due to a short tachyarrhythmias, premature ventricular contraction, or biological half-time given in two doses at 0.5 and 2.5 h after hypotension (13). intratracheal meconium instillation (Mec+Amin group, n=6), or Nevertheless, despite increasing use of PDE inhibitors, there received a single dose of olprinone (0.2 mg/kg b.w.; Olprinone is insufficient information on their possible adverse effects in hydrochloride, Sigma Aldrich, Germany) 0.5 h after meconium MAS. In our previous study, acute increase in blood pressure, instillation (Mec+Olp group, n=6). Both drugs were diluted in heart rate, and heart rate variability was observed after normal saline up to a total volume of 1 mL, or animals received aminophylline administration in a rabbit model of MAS (15). the same volume of saline (1 mL) at corresponding time points Aim of the present study was to compare effects of intravenously to treated animals (referred to as sham-treated animals; administered selective PDE3 inhibitor olprinone and non- Mec+Sal group, n=6, and Sal+Sal group, n=5). Treatment was selective PDE inhibitor aminophylline on cardiovascular delivered slowly over a period of 5 min. Immediate functions in the meconium-instilled rabbits. Short-term changes cardiovascular changes associated with treatment were in blood pressure and heart rate were monitored during and evaluated within two intervals (A1 and A2) of 2.5 min of immediately after the administration and within 5 hours lasting administration (A) of aminophylline or olprinone (5 min in period after administration of the treatments. In addition, total) and within two intervals (PA1 and PA2) of 2.5 min after fluctuations of the heart rate around its average value, i.e. heart finishing the treatment administration (post administration rate variability (HRV), representing a sensitive marker of cardiac (PA); 5 min in total) (Fig. 1). All animals were oxygen- sympathovagal control mechanisms (16), were analyzed. ventilated for additional 5 hours after the first dose of treatment. To extend our understanding of mechanisms involved in the Cardiorespiratory parameters were recorded at 0.5, 1, 2, 3, 4, adverse cardiovascular effects of PDE inhibitors, concentrations and 5 hours to investigate early effects of the treatment. At the of several heart-associated substances were determined in the end of experiments, animals were sacrificed by an overdose of blood plasma. First of them, aldosterone, is referred as a key anesthetics. cardiovascular hormone (17), as it contributes to hypertension and participates in endothelial dysfunction, atherosclerosis, Measurement and evaluation of cardiopulmonary parameters inflammation, and myocardial ischemia in coronary artery disease. Furthermore, enzymes aspartate aminotransferase Tracheal airflow and VT were measured by a heated Fleisch (AST), alanine aminotransferase (ALT), and gamma- head connected to a pneumotachograph. Airway pressure was glutamyltransferase (GGT) were measured, as they may also registered via a pneumatic catheter placed in the tracheal tube indicate cardiovascular risk and oxidative stress (18, 19). 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 MATERIALS AND METHODS calculated as: MAP = (PIP+PEEP)/2 and oxygenation index (OI)

as OI = (MAP×FiO2)/PaO2. Right-to-left pulmonary shunts were General design of experiments calculated by computer program using Fick equation: (CcO2–CaO2)/(CcO2–CvO2)×100, where CcO2, CaO2 and CvO2 Design of experiments was approved by the local Ethics are concentrations of oxygen in pulmonary capillaries, arterial

Committee of Jessenius Faculty of Medicine and National and mixed blood. CcO2 was calculated by using PAO2 (alveolar Veterinary Board. partial pressure of oxygen) from the equation: PAO2 = Meconium was collected from healthy term neonates, (PB–PH2O) × (FiO2–PaCO2) × (FiO2+(1–FiO2)/R), where PB is lyophilized and stored at –20°C. Before use, meconium was barometric pressure and PH2O the pressure of water vapour. suspended in 0.9% NaCl at a concentration of 25 mg/ml. Respiratory exchange ratio (R) was assumed to be 0.8 and the Adult rabbits (chinchilla) of 2.5±0.3 kg body weight (b.w.) current value of hemoglobin necessary for calculating the were anesthetized with intramuscular ketamine (20 mg/kg b.w.; oxygen concentration in the blood was measured by combined Narketan, Vetoquinol, UK) and xylazine (5 mg/kg b.w.; analyzer (RapidLab 348, Siemens, Germany). Xylariem, Riemser, Germany), followed by infusion of ketamine Systolic (SBP) and diastolic (DBP) blood pressures were (20 mg/kg/h). Tracheotomy was performed and catheters were measured via a catheter in the femoral artery connected to inserted into a femoral artery and right atrium for sampling the electromanometer, and the mean arterial blood pressure (MABP) blood, and into a femoral vein to administer anesthetics. Animals was calculated as MABP = DBP+1/3(SBP–DBP). Heart rate were then paralyzed with pipecuronium bromide (0.3 mg/kg (HR) was obtained from ECG recorded by subcutaneous b.w./30 min; Arduan, Gedeon Richter, Hungary) and subjected electrodes. HRV was evaluated using a computer system to a pressure-controlled ventilator (Beat-2, Chirana, Slovakia). (VariaPulse TF3, Sima Media, Czech Republic). Parameters of All animals were ventilated with a frequency of 30/min, fraction time analysis, mean duration of R-R interval (RR) and mean 753 squared successive difference (MSSD), and parameters of RESULTS 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 Body weight and initial values of all cardiopulmonary total spectral power (TP), were analyzed. From the mentioned parameters were comparable between the groups before parameters, MSSD, HF, and TP have been established as intratracheal instillation of meconium or saline (all P>0.05). markers of parasympathetic activity, while LF band represents activity of both branches of autonomic cardiac control and Respiratory parameters expresses activity of baroreceptors, as well (19, 20). Instillation of meconium seriously worsened the lung Biochemical analyses of the blood plasma functions, as demonstrated by increased right-to-left pulmonary shunts and oxygenation index and need for higher ventilatory Quantification of total antioxidant status (TAS) in the plasma pressures in comparison with saline-instilled controls (Table 1). at the end of experiment was carried out using ABTS (2,2’- Administration of both PDE inhibitors significantly reduced azino-di-(3-ethylbenzthiazoline sulphonate) radical formation pulmonary shunting, improved oxygenation and allowed to kinetics (Randox TAS kit, Randox laboratories Ltd., UK) and decrease the ventilatory pressures compared to meconium- expressed in mmol/L. Concentration of thiobarbituric-acid instilled non-treated animals, whereas better effect was observed reactive substances (TBARS) was determined from the in aminophylline (Table 1). absorbance at 532 nm and expressed in nmol/mg protein. Instillation of meconium increased accumulation of liquid in Concentration of aldosterone was measured by aldosterone the lung tissue, as indicated by higher wet-dry lung weight ratio ELISA kit (BioVendor, Czech Republic) and was expressed in in meconium-instilled and non-treated animals (Mec+Sal group; pg/mL. Activities of aspartate aminotranferase (AST), gamma- 7.98±0.15) in comparison with saline-instilled controls (Sal+Sal glutamyltransferase (GGT), and alanine aminotransferase (ALT) group; 5.73±0.17; P<0.001 vs. Mec+Sal). In the treated groups, were measured by biochemical analyzer (Olympus AU640, aminophylline (Mec+Ami group; 6.65±0.28; P<0.001 vs. Beckman Coulter, Switzerland) and were expressed in Mec+Sal) and olprinone (Mec+Olp group; 6.65±0.35; P<0.001 international units (IU)/L. vs. Mec+Sal) significantly reduced the lung edema formation in comparison with non-treated animals. Statistical analysis Cardiovascular parameters Data were tested for normality of distribution by Kolmogorov-Smirnov test. Since distribution of some HRV Before administration of the first dose of treatment, no variables (spectral powers) was extremely skewed, logarithmic significant between-group differences were found. During 5 min of transformation of these data was used to improve normality before treatment administration (two intervals of 2.5 min, A1 and A2), no statistical analysis was performed. Then, between-group significant changes were found in any of recorded cardiovascular differences were analyzed by ANOVA with post-hoc LSD test. parameters. However, within 5 min interval immediately after Within-group differences were evaluated by Wilcoxon test. finishing of the treatment delivery, increase in MABP and tendency Strength of association between biochemical and cardiovascular to elevate HR was observed in both treated groups when compared markers were expressed by Pearson’s correlation coefficient (r) with sham-treated controls. Analysis of heart rate variability and Bonferroni probability (P). A value of P<0.05 was considered showed increase in parameters expressing parasympathetic cardiac statistically significant. Data are expressed as means ± S.E.M. control, such as MSSD, and logHF (Table 2). Similar kind of

Administration Post-administration Administration Post-administration

Before1 A1 A2 PA1 PA2 Before2 A1 A2 PA1 PA2

1st dose 2nd dose M/S i.t. of treatment i.v. of treatment i.v. inst illat ion (Ami, Olp or Sal) (Ami or S al)

Before After 0.5 h 1 h 2 h 3 h 4 h 5 h M/S M/S

Period of treatment

Fig. 1. Scheme of treatment administration. 754

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.

Before M/S After M/S 30 min 1 hour 2 hours 3 hours 4 hours 5 hours MAP (kPa) Sal+ Sal 0.26±0.02 0.59±0.05 0.55±0.03 0.58±0.03 0.55±0.01 0.51±0.03 0.54±0.04 0.57±0.03 Mec+Sal 0.30±0.01 0.91±0.03c 0.97±0.04 c 1.00±0.03c 1.03±0.03c 1.02±0.04c 1.04±0.01c 1.09±0.02c Mec+ Ami 0.28±0.02 0.82±0.04 0.86±0.04ej 0.82±0.05fj 0.82±0.05fk 0.83±0.05f 0.81±0.05fk 0.80±0.05fl Mec+ Olp 0.27±0.02 0.89±0.03 0.92±0.02 0.91±0.02 0.93±0.03g 0.89±0.04g 0.94±0.03 0.96±0.02e OI Sal+ Sal 0.62±0.08 3.05±0.62 3.26±0.66 2.10±0.45 1.81±0.26 1.68±0.18 2.23±0.35 1.40±0.07 Mec+Sal 0.77±0.06 13.8±1.43 c 14.2±0.94c 14.7±0.94c 16.6±1.12c 17.1±1.62c 16.5±1.25c 18.1±1.33c Mec+ Ami 0.76±0.07 12.4±0.82 10.7±0.93f 9.56±1.06f 9.90±1.50f 9.33±1.41f 8.53±1.24f 8.14±1.16f Mec+ Olp 0.63±0.03 11.8±0.74 9.84±0.76i 9.97±0.84i 10.1±0.83i 9.74±0.71i 10.8±0.79i 10.9±0.78i RLS (%) Sal+ Sal 17.6±4.99 38.2±1.65 36.8±3.52 31.2±3.58 30.1±3.16 27.0±12.81 28.3±1.99 23.2±2.66 Mec+Sal 16.9±3.12 54.8±6.02 b 58.5±6.81b 57.7±4.34c 60.6±2.41c 60.0±1.85c 61.4±1.74c 63.1±3.42c Mec+ Ami 17.3±2.20 55.8±2.85 44.8±4.56d 43.3±4.68d 41.6±5.13e 39.1±2.63f 34.4±2.39fj 31.6±2.99fj Mec+ Olp 14.1±2.97 49.2±4.53 42.9±3.80g 43.0±5.38g 37.7±4.13i 39.3±4.18i 43.3±3.34i 41.8±2.80i

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.

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).

Before1 A1 A2 PA1 PA2 MABP (kPa) Sal+ Sal 8.3±0.8 7.8±0.8 8.4±0.9 8.8±0.9 8.9±1.0 Mec+ Sal 8.5±0.7 8.3±0.7 8.3±0.7 8.3±0.7 8.3±0.7 Mec+ Ami 8.1±0.3 8.3±1.0 9.1±0.8 10.8±0.4c 11.4±0.8dl Mec+ Olp 9.6±0.5 8.1±0.9 9.3±1.0 10.4±0.8f 10.9±0.7f HR (bpm) Sal+ Sal 210±12 212±12 214±11 213±9 211±9 Mec+ Sal 214±14 216±14 216±14 208±10 208±10 Mec+ Ami 212±8 218±7 218±7 222±12 223±9 Mec+ Olp 209±9 226±10 233±5 240±8 f 223±12 MSSD (ms 2) Sal+ Sal 1.9±0.4 1.7±0.5 1.6±0.6 1.7±0.5 1.6±0.4 Mec+ Sal 2.4±0.5 3.3±0.7 3.8±0.8 3.7±0.7 2.9±0.7 Mec+ Ami 3.9±1.1 3.3±0.7 4.3±2.7 6.9±2.4 8.2±1.2 Mec+ Olp 2.8±0.8 2.2±0.9 4.3±2.8 8.4±4.6 14.3±4.7gp log LF Sal+Sal -1.1±0.6 -1.1±0.6 -0.2±0.3 -0.4±0.6 -0.8±0.6 Mec+Sal -1.9±0.6 -1.9±0.6 -1.1±0.6 -0.3±0.6 -0.2±0.4 Mec+Ami -0.8±0.6 -0.8±0.6 -1.1±0.5 -0.1±0.2 1.3±0.4 cm Mec+Olp -1.5±1.1 -1.5±1.1 -1.5±1.0 -0.7±0.7 0.8±0.5 o logHF Sal+ Sal 0.5±0.1 0.4±0.2 0.5±0.2 0.6±0.1 0.6±0.2 Mec+ Sal 0.7±0.1 0.3±0.1 0.3±0.2 0.9±0.4 0.4±0.3 Mec+ Ami 0.6±0.5 0.8±0.4 -0.1±0.3 0.9±0.3 2.1±0.8 dl Mec+ Olp -0.2±0.6 0.2±0.5 -0.5±0.6 0.0±0.6 1.3±0.6

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. 755

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).

Before2 A1 A2 PA1 PA2 MABP (kPa) Sal+ Sal 8.6±0.9 8.3±0.9 8.7±1.0 8.7±0.9 8.7±0.9 Mec+ Sal 7.5±0.5 7.3±0.6 7.6±0.5 7.9±0.4 7.8±0.4 Mec+ Ami 8.3±0.8 8.8±1.2 9.5±1.2 10.3±1.2 10.1±1.0c HR (bpm) Sal+ Sal 231±18 231±19 231±18 233±18 230±18 Mec+ Sal 232±17 229±18 234±16 232±16 234±16 Mec+ Ami 216±10 220±10 227±12 222±15 222±19 MSSD (ms 2) Sal+ Sal 1.8±0.2 1.9±0.2 1.7±0.1 1.7±0.1 1.8±0.1 Mec+ Sal 1.5±0.2 1.8±0.4 2.3±0.4 2.4±0.5 2.5±0.7 Mec+ Ami 6.4±0.8 en 4.4±0.9dm 7.3±1.2en 20.5±3.2en 16.2±6.5cl log LF Sal+Sal 50.4±0.4 -0.1±0.3 0.1±0.3 -0.3±0.6 -0.6±0.5 Mec+Sal -1.8±0.4 a -1.7±0.7 -1.7±0.8 -1.5±0.8 -0.6±0.6 Mec+Ami 0.1±0.2 d 0.4±0.5c -0.5±0.5 0.4±0.9 0.3±0.4 logHF Sal+ Sal 0.2±0.2 0.2±0.2 0.3±0.2 0.4±0.1 0.5±0.1 Mec+ Sal 0.5±0.1 0.4±0.2 0.4±0.1 0.5±0.2 0.8±0.1 Mec+ Ami 1.0±0.2 l 0.8±0.3 0.7±0.4 2.3±0.5dm 2.3±0.5dm

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.

response was demonstrated in administration of the second dose of Correlations between biochemical markers and cardiovascular aminophylline 2 hours after the first dose. Again, there was a parameters tendency to increase MABP, HR, and HRV parameters. However, significant differences between aminophylline-treated group and Pearson’s evaluation of association between the biochemical sham-treated animals were already found before administration of markers showed negative correlations between TAS vs. TBARS the second dose of treatment (Table 3). (r= –0.786, P<0.001), vs. aldosterone (r= –0.773, P<0.001), vs. In the further course of experiment, most of cardiovascular GGT (r= –0.764, P<0.001), vs. AST (r= –0.694, P<0.001), and changes in the treated groups gradually adjusted to the values vs. ALT (r= –0.504, P<0.05). Positive correlations were found comparable with sham-treated groups. However, parameters of between TBARS vs. aldosterone (r=0.682, P<0.001), vs. GGT HRV, particularly MSSD, remained higher till the end of (r=0.543, P<0.05), and vs. AST (r=0.714, P<0.001); between experiment (Table 4). 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. Biochemical markers of meconium-induced injury ALT (r=0.489, P<0.05). Correlations between ALT vs. TBARS, aldosterone, and GGT were not significant (P>0.05). In the meconium-instilled and sham-treated animals (Mec+Sal To evaluate relation of biochemical markers to group), increased TBARS (P<0.001 vs. Sal+Sal) and slightly lower cardiovascular changes, MSSD as the most sensitive TAS (P>0.05 vs. Sal+Sal) in the plasma demonstrated influence of cardiovascular marker was chosen. MSSD at 5 hours of the oxidation processes and deterioration of antioxidant capacity also treatment administration correlated borderly with GGT on the systemic level. Both PDE inhibitors showed a trend to (r=0.503, P=0.040) and a slight tendency to correlate was reduce oxidation and to prevent decline in TAS, with more potent observed in relation to aldosterone (r=0.394, P=0.085), TAS (r= effect observed in olprinone (Table 5). –0.400, P= 0.100), and TBARS (r=0.313, P=0.168). 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 DISCUSSION olprinone reduced aldosterone level (Table 5). To evaluate extent of cardiovascular injury in this model of MAS, plasma levels of PDE inhibitors may be beneficial for treatment of MAS, as enzymes having their origin in the heart were measured. In they improve lung functions and alleviate inflammation and Mec+Sal group, non-significantly higher values of AST and oxidative injury (8-12). Nevertheless, there is little information GGT were found compared with other groups (all P>0.05; Table on their adverse effects in the conditions of meconium-induced 5). To assess liver injury potentially associated with MAS, acute lung injury. Many newborns with MAS suffer from plasma levels of ALT were determined. However, no differences hemodynamic instability and thus, sudden cardiovascular were found between the groups (all P>0.05; Table 5). changes may be life-threatening for them. Therefore, adverse 756

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.

Before M/S After M/S 30 min 1 hour 2 hours 3 hours 4 hours 5 hours MABP (kPa) Sal+ Sal 9.5±0.6 9.6±0.6 9.2±0.7 9.8±0.8 9.0±0.9 9.5±0.0.7 9.6±0.3 9.9±0.7 Mec+ Sal 9.0±0.1 9.0±0.1 8.4±0.8 8.6±0.4 9.3±0.6 8.6±0.4 8.9±0.4 9.1±0.3 Mec+ Ami 9.0±0.4 9.0±0.4 8.8±0.5 9.8±0.6 9.8±0.5 8.8±0.4 9.3±0.7 9.5±0.4 Mec+ Olp 9.6±0.2 9.7±0.2 10.0±0.4 9.2±0.8 9.7±0.5 7.9±0.7 8.5±0.7 8.7±0.6 HR (bpm) Sal+ Sal 211±13 207±9 212±7 219±5 237±11 227±8 230±9 235±9 Mec+Sal 200±10 201±6 206±3 205±6 213±10 220±8 231±16 234±14 Mec+ Ami 197±14 217±10 245±13 lc 228±13 230±11 232±11 238±12 235±14 Mec+ Olp 203±9 207±9 231±17 216±18 230±14 229±12 230±17 235±10 MSSD (ms 2) Sal+ Sal 2.9±0.8 1.4±0.3 0.8±0.2 1.4±0.5 1.3±0.3 1.5±0.3 0.9±0.2 1.5±0.3 Mec+ Sal 2.8±0.4 1.9±0.4 1.8±0.4 1.8±0.4 1.1±0.3 1.1±0.2 1.4±0.3 1.9±0.6 Mec+ Ami 3.0±0.7 2.9±0.8 5.6±1.6 lc 5.9±1.9cl 5.9±1.8dm 5.5±1.5enj 6.0±1.9dm 3.8±2.2 Mec+ Olp 3.7±0.5 2.7±0.6 6.1±1.5 gp 5.0±1.3fo 3.8±1.3 2.4±0.6 4.7±1.3fo 5.1±1.2fo log LF Sal+Sal -0.5±0.6 -0.7±0.5 -0.6±0.6 -0.3±0.3 0.2±0.5 0.1±0.4 -0.3±0.4 -0.5±0.4 Mec+Sal -0.7±0.4 -1.1±0.7 -0.9±0.5 - 0.8±0.5 -0.3±0.4 -0.2±0.4 -0.5±0.5 -0.3±0.3 Mec+ Ami -1.5±0.8 0.1±0.6 -0.2±0.4 0.6±0.4 -0.3±0.8 -0.3±0.5 1.2±0.2 c 0.8±0.2 Mec+ Olp -1.7±0.8 -0.2±0.9 0.0±0.6 0.0±0.6 -0.9±0.7 -0.4±0.6 -0.1±0.8 0.3±0.7 logHF Sal+Sal 0.5±0.2 -0.2±0.4 -0.5±0.3 -0.4±0.7 0.2±0.6 0.1±0.1 -0.3±0.3 -0.5±0.4 Mec+Sal 0.1±0.1 -0.3±0.3 0.0±0.3 0.0±0.3 0.1±0.3 0.0±0.3 -0.2±0.4 0.2±0.4 Mec+ Ami 0.2±0.2 0.6±0.3 0.7±0.2 1.1±0.2cl 0.7±0.5 1.4±0.2dmi 0.9±0.2 0.7±0.4 Mec+ Olp 0.6±0.3 -0.1±0.4 0.7±0.6 0.6±0.4 -0.4±0.4 0.2±0.4 0.3±0.6 0.9±0.5 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.

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. effects of perspective medicaments should be thoroughly various animal models (22, 23). These findings may seem to be studied before they might be recommended for the use. In this controversial due to relatively unrelated tachycardic reaction study, both selective PDE3 inhibitor olprinone and non- and simultaneous parasympathetic excitation. However, cardiac selective PDE inhibitor aminophylline increased blood activity is an integrated signal that is influenced not only by two pressure, heart rate, and heart rate variability immediately after branches of the autonomic nervous system, but also by other finishing the treatment delivery. Similarly, in aminophylline- underlying physiological mechanisms and various extrinsic treated adults, tachycardia, hypertension, and generation of factors (16). Thus, the heart rate variability cannot be explained extrasystoles were observed (4). In newborns with severe by peculiarities in sympathovagal balance, but it is determined respiratory distress syndrome (20), aminophylline elevated by more universal mechanisms (24) leading to arrhythmias. heart rate and some parameters of HRV, particularly spectral Importantly, the blood pressure and mean heart rate have power in HF band. Aminophylline increased heart rate and stabilized within several minutes, but changes in heart rate cardiac output, and influenced vascular resistance also in variability were observed till the end of experiment. 757

Adverse effects of aminophylline occur particularly at high exerts better anti-inflammatory and antioxidative effects at lower doses, i.e. in plasma concentrations of theophylline exceeding plasma concentrations than used in this study (3, 9, 35). 15–20 mg/l (3). Theophylline is metabolized by first-order Furthermore, several heart-associated biochemical markers kinetics in liver. Its half-life in the blood is several hours, e.g. were measured to estimate extent of cardiovascular injury in about 3 hours for intravenously administered dose of 2.5 mg/kg relation to MAS and given therapy. For instance, aldosterone, in rabbits (25). In humans, maximum plasma concentration is which chronic overproduction leads to hypertension. In addition, reached 20 min following intravenous administration (26). via rapid, non-genomically mediated mechanisms aldosterone Nevertheless, in the newborns and in some concurrent situations participates in many processes in vascular smooth muscle cells, (e.g., acute lung edema) theophylline clearance may be reduced. cardiac myocytes, and endothelial cells. As a result, increased As mentioned above, some cardiovascular effects of intracellular Ca2+ and cAMP in smooth muscle cells (17), leads aminophylline are caused by PDE inhibition, as increased cAMP to elevation of vascular resistance, modulation of sympathovagal in myocardium has positive chronotropic and inotropic effects. balance towards potentiating the sympathetic activity, changes in In addition, accumulation of cAMP potentiates action of heart rate and baroreflex sensitivity within several minutes and neurotransmitters and hormones, e.g. catecholamines (13, 27). potentiation of effects of catecholamines (36, 37). Secretion of Some changes in HRV and generation of arrhythmias are caused aldosterone may be stimulated by oxidized fatty acids (38). by antagonism with adenosine receptors, as well. Theophylline Thus, increased plasma aldosterone in the meconium-instilled presumably acts on adenosine A1 and A2A receptors, however, animals could be partially explained by lipoperoxidation, as participation of adenosine receptors antagonism in the indicated by correlations with increased TBARS and decreased cardiovascular action of theophylline (or aminophylline) is not TAS. However, reason for finding of high plasma aldosterone in fully elucidated (3, 28). the aminophylline-treated group and low aldosterone in the On the other hand, increased concentration of cAMP due to olprinone-treated group is unknown. We may presume PDE3 inhibition activates protein kinase A, which stimulates participation of the cross-talk between renin-angiotensin- potential-dependent Ca2+ channels on the membranes of cardiac aldosterone and adrenergic systems (36), as aminophylline muscle cells, leading to facilitated inward of Ca2+ into the cells elevates circulating catecholamines (3). In addition, synthesis of and cardiac contraction. Contrary, PDE3 inhibitors decrease aldosterone is mediated through activation of cAMP-dependent cytosolic free Ca2+in vascular smooth muscle, thus causing signaling pathways leading to induction of genes encoding vasodilation. In addition to other mechanisms, sensitivity of enzymes involved in the conversion of cholesterol to steroids vascular smooth muscle to cGMP or cAMP-dependent (39). Several phosphodiesterases, e.g., PDE2, PDE8B, and vasodilators (including PDE inhibitors) may be modulated by PDE11A are expressed in adrenal cortex (40). Therefore, exposure to NO (29). Olprinone lowers mean aortic and different PDE inhibitors may have different capacity to influence pulmonary artery pressures, but exerts differential vasodilatory aldosterone production through the above mentioned effects on peripheral vessels in each organ, based on different mechanisms. Additionally, by antagonism of adenosine distribution of PDE3 among the organs (30). In asthmatic receptors theophylline (or aminophylline) stimulates secretion of patients, olprinone decreased diastolic blood pressure and renin and later also aldosterone (41), whereas olprinone has no increased heart rate (31). In these experiments, olprinone effect on adenosine receptors. increased blood pressure, heart rate, and heart rate variability to Besides aldosterone, plasma levels of alanine a comparable extent to aminophylline within several minutes aminotransferase (ALT), aspartate aminotransferase (AST), and after the administration. At the moment, we may only speculate gamma-glutamyltransferase (GGT) were investigated, as they on mechanisms behind the short-term cardiovascular changes. elevate in cardiovascular and metabolic diseases (42, 43). In the As mentioned above, cardiovascular effects of olprinone are heart or liver injury, AST increases proportionally to extent of attributable to PDE3 inhibition, while aminophylline action is the injury, whereas the rise is detectable about 6 hours after the mediated by both PDE inhibition and antagonism with insult (43). Elevated levels of GGT and ALT are sensitive adenosine receptors. In addition, olprinone and aminophylline markers of systemic inflammation and oxidative stress (18), and exert different effect on catecholamines. Aminophylline predictors of cardiovascular risk (19). GGT is a key enzyme in increases epinephrine levels, but olprinone has no effect on the catabolism of glutathione modulating the redox status. epinephrine or norepinephrine (27). Based on comparable Production of ROS leads to depletion of glutathione, induces changes in blood pressure, heart rate, and HRV parameters we expression of GGT, and subsequently elevates plasma activity of may presume that the short-term cardiovascular side effects are GGT (44). ALT may be associated with development of predominantly mediated by PDE3 inhibition. Nevertheless, metabolic syndrome, type 2 diabetes, and hypertension (18, 44). further research is needed to elucidate participation of the However, despite slightly increased values of GGT and AST, no mentioned mechanisms in the cardiovascular side effects of PDE significant between-group differences were observed, probably inhibitors more in detail. due to short-term observation and/or absence of morphological On the other hand, aminophylline and olprinone improved changes on the heart structures. In the study by Yamada et al. lung functions, i.e., reduced right-to-left pulmonary shunting, (18), higher ALT and GGT correlated with C-reactive protein improved oxygenation, decreased requirements for ventilation, and markers of lipoperoxidation, indicating cardiovascular risk. and diminished lung edema formation. In addition, both In this study, oxidation markers correlated well with GGT and treatments reduced oxidative stress in meconium-injured lungs. AST, but less with ALT, probably due to a fact that metabolic Biochemical markers of oxidation were measured also in the liver-associated processes are of lower importance in MAS than plasma, as they may influence indirectly also the cardiac activity in the above mentioned diseases. However, significant (21). Systemic consequences of MAS were demonstrated by correlations of GGT and AST with oxidation markers and higher TBARS, a marker of lipid peroxidation, and reduced total aldosterone indicate that these commonly used biochemical antioxidant status (TAS) in the plasma. Despite both PDE markers may be useful in estimation of MAS severity. inhibitors decreased formation of TBARS and prevented a Nevertheless, we are aware of several limitations of our decline in TAS compared with non-treated animals, stronger study. Firstly, due to inter-species differences in autonomic effect was observed in olprinone. Potent anti-inflammatory and cardiac regulation between rabbits and humans the antioxidant action of olprinone has been proven at different doses cardiovascular response to administration of PDE inhibitors may in various models of injury (10, 32-34), while aminophylline differ. Secondly, despite the used model of MAS is acceptable, it 758 cannot fully resemble the clinical MAS on the background of 11. Bassler D, Choong K, McNamara P, Kirpalani H. Neonatal postnatal changes in hemodynamics. Therefore, eventual persistent pulmonary hypertension treated with milrinone: cardiovascular adverse effects of this treatment should be four case report. Biol Neonate 2006; 89: 1-5. studied also in the neonates with MAS. Finally, period of 12. Shekerdemian LS, Ravn HB, Penny DJ. Intravenous observation of cardiorespiratory changes, which was limited in sildenafil lowers pulmonary vascular resistance in a model this study to several hours after the treatment administration, of neonatal pulmonary hypertension. Am J Respir Crit Care should be prolonged in both experimental and clinical studies. Med 2002; 165: 1098-1102. Thereafter, positive and negative effects of the treatment should 13. Mizushige K, Ueda T, Yukiiri K, Suzuki H. Olprinone: a be critically evaluated. phosphodiesterase III inhibitor with positive inotropic and In conclusion, intravenous administration of both selective vasodilator effects. Cardiovasc Drug Rev 2002; 20: 163-174. PDE3 inhibitor olprinone and non-selective PDE inhibitor 14. Rudzinski P, Wegrzyn P, Lis GJ, et al. Vasodilatory effect aminophylline significantly improved the lung functions in and endothelial integrity in papaverine- and milrinone- meconium-instilled rabbits. However, olprinone to, showed no treated human radial arteries. J Physiol Pharmacol 2013; 64: clear benefit to aminophylline in extent of cardiovascular 41-45. adverse effects as both treatments exerted comparable short-term 15. Mokra D, Tonhajzerova I, Mokry J, Petraskova M, Hutko M, increase in blood pressure, heart rate, and heart rate variability. Calkovska A. Cardiovascular side effects of aminophylline Therefore, if PDE inhibitors are used for treatment of MAS, in meconium-induced acute lung injury. Adv Exp Med Biol cardiovascular parameters should be monitored carefully, 2013; 756: 341-347. particularly in patients with cardiovascular instability. 16. von Borell E, Langbein J, Despres G et al. Heart rate variability as a measure of autonomic regulation of cardiac Acknowledgements: Authors thank M. Petraskova, M. activity for assessing stress and welfare in farm animals - a Hutko, D. Kuliskova and Z. Remisova for technical assistance. review. Physiol Behav 2007; 92: 293-316. Study was supported by Project „Center of Excellence in 17. Connell JM, Davies E. The new biology of aldosterone. Perinatology Research (CEPV II) No. 26220120036, co- J Endocrinol 2005; 186: 1-20. financed from EU sources, by Project APVV-0435-11, and Grant 18. Yamada J, Tomiyama H, Yambe M, et al. Elevated serum VEGA No. 1/0057/11. levels of alanine aminotransferase and gamma glutamyltransferase are markers of inflammation and Conflict of interests: None declared. oxidative stress independent of the metabolic syndrome. Atherosclerosis 2006; 189: 198-205. 19. Saely CH, Vonbank A, Rein P, et al. Alanine REFERENCES aminotransferase and gamma-glutamyl transferase are associated with the metabolic syndrome but not with 1. Mokra D, Mokry J. Meconium Aspiration Syndrome: From angiographically determined coronary atherosclerosis. Clin Pathomechanisms to Treatment. New York, Nova Chim Acta 2008; 397: 82-86. Biomedical Books, 2010. 20. Kibblewhite DP, Sleigh JW. Heart rate variability in 2. Mroz RM, Holownia A, Chyczewska E, Braszko JJ. Chronic premature neonates pre- and postmethylxanthine obstructive pulmonary disease: an update on nuclear administration. Paediatr Anaesth 1996; 6: 399-403. signaling related to inflammation and anti-inflammatory 21. Thayer JF, Loerbroks A, Sternberg EM. Inflammation and treatment. J Physiol Pharmacol 2008; 59(Suppl. 6): 35-42. cardiorespiratory control: the role of the vagus nerve. Respir 3. Barnes PJ. Theophylline in chronic obstructive pulmonary Physiol Neurobiol 2011; 178: 387-394. disease: new horizons. Proc Am Thorac Soc 2005; 2: 22. Nadareishvili KS, Meskhishvili II, Kakhiani DD, et al. Heart 334-339. rate variability in Chinchilla rabbits. Bull Exp Biol Med 4. Minton NA, Henry JA. Acute and chronic human toxicity of 2002; 134: 568-570. theophylline. Hum Exp Toxicol 1996; 15: 471-481. 23. Spalding MB, Ala-Kokko TI, Kiviluoma K, Ruskoaho H, 5. Bender AT, Beavo JA. Cyclic nucleotide Alahuhta S. The effect of aminophylline on right heart phosphodiesterases: molecular regulation to clinical use. function in young pigs after ligation of the right coronary Pharmacol Rev 2006; 58: 488-520. artery. Pharmacol Toxicol 2000; 86: 192-194. 6. Mokry J, Mokra D, Nosalova G, Beharkova M, Feherova Z. 24. Takahashi S, Fujii Y, Hoshi T, Inomata S, Miyabe M, Toyooka Influence of selective inhibitors of phosphodiesterase 3 and H. Modifications of the hemodynamic consequences of 4 on cough and airway reactivity. J Physiol Pharmacol 2008; theophylline intoxication with landiolol in halothane- 59(Suppl. 6): 473-482. anesthetized dogs. Can J Anaesth 2000; 47: 265-272. 7. Paplinska M, Chazan R, Grubek-Jaworska H. Effect of 25. Barakat M, El-Kadi AO, Du Souich P. In vivo, ex vivo, and phosphodiesterase 4 (PDE4) inhibitors on eotaxin in vitro effects of L-NAME and L-arginine on the expression in human bronchial epithelial cells. JPhysiol metabolism of theophylline in the rabbit. Drug Metab Pharmacol 2011; 62: 303-311. Dispos 1997; 25: 191-195. 8. Korhonen K, Kiuru A, Svedstrom E, Kaapa P. 26. Muller CE, Jacobson KA. Recent developments in Pentoxyfylline reduces regional inflammatory and adenosine receptor ligands and their potential as novel drugs. ventilatory disturbances in meconium-exposed piglet lungs. Biochim Biophys Acta 2011; 1808: 1290-1308. Pediatr Res 2004; 56: 901-906. 27. Hirota K, Yoshioka H, Kabara S, Kudo T, Ishihara H, Matsuki 9. Mokra D, Drgova A, Mokry J, et al. Comparison of low-dose A. A comparison of the relaxant effects of olprinone and vs high-dose aminophylline on lung function in aminophylline on methacholine-induced bronchoconstriction experimental meconium aspiration syndrome. J Physiol in dogs. Anesth Analg 2001; 93: 230-233. Pharmacol 2008; 59(Suppl. 6): 449-459. 28. Riksen NP, Smits P, Rongen GA. The cardiovascular effects 10. Mokra D, Drgova A, Pullmann R Sr, Calkovska A. Selective of methylxanthines. Handb Exp Pharmacol 2011; 200: phosphodiesterase 3 inhibitor olprinone attenuates 413-437. meconium-induced oxidative lung injury. Pulm Pharmacol 29. Busch CJ, Graveline AR, Jiramongkolchai K, Liu H, Ther 2012; 25: 216-222. Sanchez LS, Bloch KD. Phosphodiesterase 3A expression is 759

modulated by nitric oxide in rat pulmonary artery smooth 38. Goodfriend TL, Ball DL, Egan BM, Campbell WB, muscle cells. J Physiol Pharmacol 2010; 61: 663-669. Nithipatikom K. Epoxy-keto derivative of linoleic acid 30. Ueda T, Mizushige K. The effects of olprinone, a stimulates aldosterone secretion. Hypertension 2004; 43: phosphodiesterase 3 inhibitor, on systemic and cerebral 358-363. circulation. Curr Vasc Pharmacol 2006; 4: 1-7. 39. Aumo L, Rusten M, Mellgren G, Bakke M, Lewis AE. 31. Myou S, Fujimura M, Kamio Y, et al. Bronchodilator effects Functional roles of protein kinase A (PKA) and exchange of intravenous olprinone, a phosphodiesterase 3 inhibitor, protein directly activated by 3’,5’-cyclic adenosine 5’- with and without aminophylline in asthmatic patients. Br J monophosphate (cAMP) 2 (EPAC2) in cAMP-mediated Clin Pharmacol 2003; 55: 341-346. actions in adrenocortical cells. Endocrinology 2010; 151: 32. Koike T, Nadeen Qutab M, Tsuchida M, Takekubo M, Saito 2151-2161. M, Hayashi J. Pretreatment with olprinone hydrochloride, a 40. Tsai LC, Beavo JA. The roles of cyclic nucleotide phosphodiesterase III inhibitor, attenuates lipopolysaccharide- phosphodiesterases (PDEs) in steroidogenesis. Curr Opin induced lung injury via an anti-inflammatory effect. Pulm Pharmacol 2011; 11: 670-675. Pharmacol Ther 2008; 21: 166-171. 41. Osswald H, Schnermann J. Methylxanthines and the kidney. 33. Miyakawa H, Kira S, Okuda K, Takeshima N, Mori M, Handb Exp Pharmacol 2011; 200: 391-412. Noguchi T. Olprinone decreases elevated concentrations of 42. Lioudaki E, Ganotakis ES, Mikhailidis DP. Liver enzymes: cytokine-induced neutrophil chemoattractant-1 in septic rats. potential cardiovascular risk markers? Curr Pharm Des JAnesth2008; 22: 27-31. 2011; 17: 3632-3643. 34. Oishi H, Takano K, Tomita K, et al. Olprinone and colforsin 43. Cheung BM, Ong KL, Tso AW, et al. Gamma-glutamyl daropate alleviate septic lung inflammation and apoptosis transferase level predicts the development of hypertension in through CREB-independent activation of the Akt pathway. Hong Kong Chinese. Clin Chim Acta 2011; 412: 1326-1331. Am J Physiol Lung Cell Mol Physiol 2012; 303: L130-L140. 44. Turgut O, Yilmaz A, Yalta K, Karadas F, Birhan Yilmaz M. 35. Chorostowska-Wynimko J, Kus J, Skopinska-Rozewska E. Gamma-glutamyltransferase is a promising biomarker for Theophylline inhibits free oxygen radicals production by cardiovascular risk. Med Hypotheses 2006; 67: 1060-1064. human monocytes via phosphodiesterase inhibition. J Physiol Pharmacol 2007; 58(Suppl. 5): 95-103. R e c e i v e d : July 4, 2013 36. Weber MA, Purdy RE. Catecholamine-mediated constrictor Accepted: November 7, 2013 effects of aldosterone on vascular smooth muscle. Life Sci 1982; 30: 2009-2017. Author’s address: Dr. Daniela Mokra, Department of 37. Yee KM, Pringle SD, Struthers AD. Circadian variation in Physiology, Jessenius Faculty of Medicine, Comenius University, the effects of aldosterone blockade on heart rate variability 4 Mala Hora Street, SK-03601 Martin, Slovakia. and QT dispersion in congestive heart failure. JAm Coll E-mail: [email protected] Cardiol 2001; 37: 1800-1807.