Carvedilol Induces Endogenous Hydrogen Sulfide Tissue Concentration Changes in Various Mouse Organs *

WILIÑSKI B., WILIÑSKI J., SOMOGYI E., PIOTROWSKA J., GÓRALSKAM., MACURAB. 2011. Carvedilol induces endogenous hydrogen sulfide tissue concentration changes in various mouse organs. Folia biologica (Kraków) 59: 151-155. Carvedilol, a third generation non-selective adrenoreceptor blocker, is widely used in cardiology. Its action has been proven to reach beyond adrenergic antagonism and involves multiple biological mechanisms. The interaction between carvedilol and endogenous gasotransmitter hydrogen sulfide (H2S) is unknown. The aim of the study is to assess the influence of carvedilol on the H2S tissue level in mouse brain, liver, heart and kidney. Twenty eight SJL strain female mice were administered intraperitoneal injections of 2.5 mg/kg b.w./d (group D1, n = 7), 5 mg/kg b.w./d (group D2, n = 7) or 10 mg/kg b.w./d of carvedilol (group D3, n = 7). The control group (n = 7) received physiological saline in portions of the same volume (0.2 ml). Measurements of the free tissue H2S concentrations were performed according to the modified method of Siegel. A progressive decline in H2S tissue concentration along with an increase in carvedilol dose was observed in the brain (12.5%, 13.7% and 19.6%, respectively). Only the highest carvedilol dose induced a change in H2S tissue level in the heart  an increase by 75.5%. In the liver medium and high doses of carvedilol increased the H2S level by 48.1% and 11.8%, respectively. In the kidney, group D2 showed a significant decrease of H2S tissue level (22.5%), while in the D3 group the H2S concentration increased by 12.9%. Our study has proven that carvedilol affects H2S tissue concentration in different mouse organs.

Carvedilol, a third generation non-selective adrenoreceptor blocker, is widely used in cardiology and general practice in the treatment of chronic diseases like congestive heart failure and arterial hypertension (CHAKRABORTY et al. 2010).The action of carvedilol has been proven to reach beyond adrenergic antagonism and comprises i.a.antioxidant activity, calcium channel blockade and nitric oxide (NO) production enhancement (KOSTKA--JEZIERNY & TYKARSKI 2009).On the other hand, endogenously formed 'gasotransmitter' hydrogen sulfide (H S) has been identified as a crucial regulator of circulatory, nervous, gastrointestinal and excretory systems.Altered production of H S was observed in arterial hypertension, myocardial ische-mia and atherosclerosis (£OWICKA & BE£TOWSKI 2007).The interaction between carvedilol and endogenous H S is unknown.
The aim of the study is to assess the influence of carvedilol on the endogenous tissue H S concentrations in mouse brain, heart, liver and kidney.

Animals
Twenty eight SJL strain female mice (11-12 week old individuals) of approximately 20 g weight were involved in the study.The animals were housed under standard laboratory conditions and had free access to water and food.They were kept at a temperature of 22-24°C with a light/dark cycle of 12 h.

Study protocol
A non-selective lipophilic â-blocker / á-1 blocker carvedilol (Avedol, Polpharma, Poland) was dissolved in physiological saline.The study design comprised intraperitoneal injections of 2.5 mg per kg of body weight of carvedilol daily (group D1, n = 7), 5 mg per kg of body weight of carvedilol daily (group D2, n = 7) or 10 mg per kg of body weight of carvedilol daily (group D3, n = 7) for 5 consecutive days at the same time of day (10:30 am), each administration consisted of 0.2 ml of the solution.The control population (n = 7) received intraperitoneally physiological saline in portions of the same volume.The individuals were randomly assigned to each group.The animals tolerated the applied doses of carvedilol well and remained in good condition till the end of the experiment.Measurements of the free tissue H S concentration were performed by the use of the modified method of Siegel (SIEGEL 1965;SOMOGYI et al. 2008).The study has been performed in accordance with the guidelines for the care and use of laboratory animals accepted by Bioethical Committee of the Jagiellonian University Medical College (Kraków, Poland).

Tissue sample preparation
Two hours after the last drug or physiological saline injection the animals were killed by cervical dislocation, their brains, hearts, livers and kidneys were quickly removed, homogenized with 0.01 mol/l sodium hydroxide (NaOH): brain tissue in proportion of 1 to 4, liver and kidney of 1 to 5 and heart of 1 to 10 and frozen.Then 50% trichloroacetic acid (TCA) was added (0.5 ml to 2 g of brain or liver samples in tight capsules of 3 ml and 0.25 ml to 1 g of heart or kidney sample in tight capsules of 2 ml), the suspension was shaken and centrifuged.Subsequently, 1.5 ml brain or liver and 0.75 ml heart or kidney supernatant samples were moved to 2 ml tight capsules with 0.15 ml or 0.075 ml of 0.02 mol/l N,N-dimethyl-p-phenyl-diamine sulfate in 7.2 mol/l hydrochloric acid (HCl), then 0.15 ml or 0.075 ml of 0.03 mol/l iron (III) chloride (FeCl ! ) in 1.2 mol/l HCl portions were added, respectively.After 20 minutes in darkness the content was shaken for 1 minute with 1 ml of chloroform.

H S tissue concentration measurements
Absorbance was measured at 650 nm with the Varian Cary 100 spectrophotometer.A standard curve was prepared with an iodometrically determined 0.0001 mol/l sodium sulfide (Na S) solution.For each group of animals four concurrent analyses of each analyzed tissue type were performed.

Statistical analysis
Statistical analysis was performed within the R Environment by the Student's t-test and univariate analysis of variance (ANOVA).Statistical significance was considered when P<0.05.

Results and Discussion
Progressive H S tissue concentration decline was observed in the brain along with a rising carvedilol dose (by 12.5%, 13.7% and 19.6%, respectively).In the heart only the highest carvedilol dose induced H S tissue level change, but the increase was spectacular, reaching 75.5%.In the liver, medium and high doses altered the H S concentration -5 mg/kg b.w./d of carvedilol increased the H S level by 48.1% and 10 mg/kg b.w/d by 11.8%.In the kidney, the group D2 showed a significant decrease of H S tissue level (22.5%), while in the D3 group H S concentration increased by 12.9%.In the variance analysis for each tissue type only H S concentration changes within the heart and brain were statistically significant (Table 1).Noteworthy, each organ has different metabolism, paracrine and endocrine regulation, and specific transmitter interactions, thus variable changes of H S concentrations only confirm this complexity and heterogeneity.
H S is endogenously formed from L-cysteine in several enzymatic reactions catalyzed by cystathionine â-synthase (CBS), cystathionine ã-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST), and in non-enzymatic pathways in many tissues.Cytoplasmatic bound sulfur is postulated to absorb and store exogenously applied and endogenously produced H S which is released from the bound sulfur pool in the presence of physiologic concentrations of glutathione and cysteine in slightly alkaline conditions (ISHIGAMI et al. 2009).H S acts as a 'gasotransmitter' and serves as a co-modulator of various physiological and pathophysiological processes such as regulation of vascular tone, myocardial contractility, neurotransmission and perception (FIORUCCI et al. 2006;SHIBUYA et al. 2009).Its biological action comprises numerous intracellular mechanisms including adenosine triphosphate (ATP)-sensitive potassium channels (K )62 ) stimulation, sulfhydration of different proteins and maintaining protein-SH groups in the reduced state, reaction with reactive oxygen and nitrogen species (ROS and RNS) (£OWICKA & BE£TOWSKI 2007;SUN et al. 2008).H S interacts with carbon monoxide (CO) and nitric oxide (NO) in a number of ways including affecting each other's synthesis and biological responses within target tissues.All these three gases bind to haemoglobin and impede mitochondrial oxidative phosphorylation by inhibiting cytochrome c oxidase (LI et al. 2009).Analogically to carvedilol, H S decreases IL-6, TNF-á levels, reduces the activation of NF-êB complex and the activity of MMP-2 and MMP-9 (OH et al. 2006;SEN et al. 2009;SODHA et al. 2009).
Our study has shown that carvedilol's action involves H S, probably via its production rate alteration and release with possible NO share as one of the mechanisms.Some of the biological effects of the beta-blocker and H S are common.It is unknown whether H S mediates any of them and to what extent carvedilol's biology is dependent on the messenger, since research dedicated to the issue has not been done.In the heart, the cardioprotective effects of H S have been demonstrated to involve opening of K )62 channels as well as effects of preserving mitochondrial structure and function (ELROD et al. 2007).It might be an accessory effect to NO beneficial impact on intracellular transcription factors resulting in increased expression of cardioprotective proteins like superoxide dismutase (SOD), inducible NOS (iNOS), cyclooxygenase-2 (COX-2) and heat shock proteins (DAWN & BOLLI 2002).In hepatology, carvedilol appears to be a potentially viable option for treating portal hypertension along with angiotensin-converting enzyme inhibitors (ACEI) (HEMSTREET 2004).H S regulates perfusion pressure in normal and cirrhotic liver in a NO-independent manner; NO and H S are released by different cellular sources and their hemodynamic effects involve different cellular targets.H S generation in cirrhosis is decreased due to a reduced expression/activity of CSE in hepatic stellate cells (HSC) -one of the main sources of H S in the liver (FIORUCCI et al. 2005).In our previous studies a tissue specific ACEI ramipril also enhanced the H S tissue level in the liver, heart and kidney (WILIÑSKI et al. 2010;WILIÑSKI et al. 2008).In kidneys H S has been recognized as an important regulator of renal function affecting both vascular and tubular actions (XIA et al. 2009).Some studies with carvedilol demonstrate attenu- Carvedilol also protected against the renal mitochondrial toxicity induced by cisplatin and daunorubicin-induced cardiotoxicity and nephrotoxicity in rats (AROZAL et al. 2010;RODRIGUES et al. 2010).H S acts as a neuromodulator as well as an intracellular messenger in the central nervous system.Its perturbed metabolism has been investigated in many neurological disorders including Alzheimer's disease (QU et al. 2008).Recently, carvedilol was found to improve neuronal transmission and attenuate brain oligomeric beta-amyloid content and cognitive deterioration in two mouse models of Alzheimer's disease (WANG et al. 2010).Analogically, non-steroidal anti-inflammatory drugs were also demonstrated to exert some anti-amyloidogenic effects and to affect H S biology (BILSKA et al. 2010;SREBRO et al. 2006).
In conclusion, carvedilol affects the H S tissue concentrations in mouse brain, heart, liver and kidney.The involvement of H S makes the biological action of carvedilol more complex and opens new fields for investigation for both of them in neurology, cardiology, hepatology and nephrology.Several biotechnology companies are already developing H S-based therapeutic compounds, and there are ongoing clinical trials investigating the therapeutic potential of H S (PREDMORE & LEFER 2010).

Table 1
Hydrogen sulfide (H S) tissue concentration in mouse brain, heart, liver and kidney following the administration of 2.5 mg/kg b.w. per day, 5 mg/kg b.w. per day or 10 mg/kg b.w. per day of carvedilol (groups D1, D2 and D3 respectively)