Isil Ozakca

The effects of chronic trimetazidine treatment on mechanical function and fatty acid oxidation in diabetic rat hearts

Clinical and experimental evidence suggest that increased rates of fatty acid oxidation in the myocardium result in impaired contractile function in both normal and diabetic hearts. Glucose utilization is decreased in type 1 diabetes, and fatty acid oxidation dominates for energy production at the expense of an increase in oxygen requirement. The objective of this study was to examine the effect of chronic treatment with trimetazidine (TMZ) on cardiac mechanical function and fatty acid oxidation in streptozocin (STZ)-diabetic rats. Spontaneously beating hearts from male Sprague-Dawley rats were subjected to a 60-minute aerobic perfusion period with a recirculating Krebs-Henseleit solution containing 11 mmol/L glucose, 100 muU/mL insulin, and 0.8 mmol/L palmitate prebound to 3% bovine serum albumin (BSA). Mechanical function of the hearts, as cardiac output x heart rate (in (mL/min).(beats/min).10-2), was deteriorated in diabetic (73 +/- 4) and TMZ-treated diabetic (61 +/- 7) groups compared with control (119 +/- 3) and TMZ-treated controls (131 +/- 6). TMZ treatment increased coronary flow in TMZ-treated control (23 +/- 1 mL/min) hearts compared with untreated controls (18 +/- 1 mL/min). The mRNA expression of 3-ketoacyl-CoA thiolase (3-KAT) was increased in diabetic hearts. The inhibitory effect of TMZ on fatty acid oxidation was not detected at 0.8 mmol/L palmitate in the perfusate. Addition of 1 mumol/L TMZ 30 min into the perfusion did not affect fatty acid oxidation rates, cardiac work, or coronary flow. Our results suggest that higher expression of 3-KAT in diabetic rats might require increased concentrations of TMZ for the inhibitory effect on fatty acid oxidation. A detailed kinetic analysis of 3-KAT using different concentrations of fatty acid will determine the fatty acid inhibitory concentration of TMZ in diabetic state where plasma fatty acid levels are increased.

Nebivolol prevents desensitization of β-adrenoceptor signaling and induction of cardiac hypertrophy in response to isoprenaline beyond β1-adrenoceptor blockage.

The importance of chronic stimulation of β-adrenoceptors in the development of cardiac dysfunction is the rationale for the use of β-blockers in the treatment of heart failure. Nebivolol is a third-generation β-blocker, which has further properties including stimulation of endothelial nitric oxide synthase and/or β3-adrenoceptors. The aim of this study was to investigate whether nebivolol has additional effects on β-adrenoceptor-mediated functional responses along with morphologic and molecular determinants of cardiac hypertrophy compared with those of metoprolol, a selective β1-adrenoceptor blocker. Rats infused by isoprenaline (100 μg·kg(-1)·day(-1), 14 days) were randomized into three groups according to the treatment with metoprolol (30 mg·kg(-1)·day(-1)), nebivolol (10 mg·kg(-1)·day(-1)), or placebo for 13 days starting on day 1 after implantation of minipump. Both metoprolol and nebivolol caused a similar reduction on heart rate. Nebivolol mediated a significant improvement on cardiac mass, coronary flow, mRNA expression levels of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and atrial natriuretic peptide and phospholamban (PLN)/SERCA2a and phospho-PLN/PLN ratio compared with metoprolol and placebo. Nebivolol prevented the detrimental effects of isoprenaline infusion on isoprenaline (68% of control at 30 μM), BRL37344 (63% of control at 0.1 μM), and forskolin (64% of control at 1 μM) responses compared with metoprolol (isoprenaline, 34% of control; BRL37344, no response; forskolin, 26% of control) and placebo (isoprenaline, 33% of control; BRL37344, 28% of control; forskolin, 12% of control). Both β-blockers improved the changes in mRNA expressions of β1- and β3-adrenoceptors. Our results suggest that nebivolol partially protects the responsiveness of β-adrenoceptor signaling and the development of cardiac hypertrophy independent of its β1-adrenoceptor blocking effect.

Role of Beta-3-Adrenoceptor in Catecholamine-Induced Relaxations in Gastric Fundus from Control and Diabetic Rats

The contribution of β-adrenoceptor subtypes to the catecholamine-mediated relaxations in gastric fundus from control and streptozotocin (STZ)-induced diabetic rats were investigated. Isolated organ bath studies and molecular techniques were used to characterize the β-adrenoceptor subtypes mediating relaxation of rat gastric fundus. Isoprenaline-mediated relaxation was not significantly changed by nadolol (β1-/β2-adrenoceptor antagonist; 1 µmol/l) but only shifted to the right by SR59230A (3-(2-ethylphenoxy)-1-[[(1S)-1,2,3,4-tetrahydronaphth-1-yl]amino]-(2S)-2-propanol oxalate salt, 0.1–1 µmol/l), a selective β3-adrenoceptor antagonist, in a competitive manner. Relaxant responses to noradrenaline were antagonized in a concentration-dependent manner by SR59230A (0.1–1 µmol/l), but not by metoprolol (selective β1-adrenoceptor antagonist; 0.1–1 µmol/l) and ICI-118551 (1-[2,3-(dihydro-7-methyl-1Hinden- 4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride, selective β2-adrenoceptor antagonist; 0.1–1 µmol/l). SR59230A (1 µmol/l) also caused a significant rightward shift in fenoterol-induced relaxation while ICI-118551 (1 µmol/l) did not have any effect. Selective β3-adrenoceptor agonist, BRL37344 ([4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid), caused biphasic relaxation which was not affected by nadolol (1 µmol/l). SR59230A (1 µmol/l) abolished only the first phase of BRL37344 response. β1-, β2- and β3-adrenoceptor mRNA expressions have been detected in a similar intensity in gastric fundus from control rats. Experimental diabetes caused a significant decrease in Emax and pD2 values of isoprenaline and noradrenaline. Diabetes also reduced Emax but not pD2 value of the first component of BRL37344-induced relaxation response. The band intensity of mRNA transcript of β3-adrenoceptor was reduced in diabetics while no alteration has been found for β1- and β2-adrenoceptor mRNA transcripts between groups. These results show that functional β-adrenoceptor subtype involved in catecholamine-mediated relaxations is β3-adrenoceptor, and its function and mRNA expression are decreased in diabetes.

β3-Adrenoceptor-mediated responses in diabetic rat heart.

β3-adrenoceptors mediate negative inotropic effect in contrast to classical β1- and β2-adrenoceptors. Cardiac β3-adrenoceptors are upregulated in experimental diabetes. Thus, cardiodepressant effect mediated by β3-adrenoceptors has been proposed to contribute to the impaired cardiac function in this pathology. In our study, we investigated the influence of streptozotocin-diabetes on cardiac contractility to β3-adrenoceptors stimulation by using Langendorff-perfused rat hearts. BRL 37344, a selective β3-adrenoceptor agonist, induced dose-dependent decreases in left ventricular developed pressure (LVDP) in hearts from control rats. BRL 37344 also dose-dependently decreased +dP/dt and -dP/dt values. Effects of BRL 37344 were abolished by SR 59230, but not altered by nadolol pre-treatment. On the other hand, these effects of BRL 37344 were all significantly increased in hearts from diabetic rats. We also observed that diabetes significantly increased the mRNA levels encoding cardiac β3-adrenoceptors. In addition, Giα2 mRNA expressions were found to be increased in the cardiac tissue of diabetic rats as well. The effect of BRL 37344 on cardiac contractility was normalized upon treatment of diabetic rats with insulin. These data demonstrate an increased effect of β3-adrenoceptor stimulation on hemodynamic function of the heart in accordance with an increased mRNA levels encoding cardiac β3-adrenoceptors in 8-week diabetic rats.

Effects of silodosin, a selective alpha‐1A adrenoceptor antagonist, on erectile function in a rat model of partial bladder outlet obstruction

We investigated the effects of silodosin (selective α1A‐adrenoceptor antagonist) on erectile dysfunction (ED) in a rat model of bladder outlet obstruction.

Cardioprotective effects of 44Bu, a newly synthesized compound, in rat heart subjected to ischemia/reperfusion injury.

Excessive intracellular Na+ accumulation followed by Ca2+ overload in cardiac tissue is one of the important mechanisms leading to ischemia/reperfusion injury. In the present study, the cardioprotective effects of 44Bu, 2-hydroxy-3-(butylamino) propyl-4-{(butoxycarbonyl)amino}benzoate hydrochloride, a novel Na+ channel blocker, on ischemia/reperfusion injury were investigated and compared to lidocaine. Isolated rat hearts perfused at the constant flow were exposed to global ischemia for 60 min followed by 30 min of reperfusion. In control hearts, ischemia/reperfusion markedly decreased left ventricular developed pressure and increased left ventricular end-diastolic pressure, and caused lactate dehydrogenase release and infarction. 44Bu (0.1, 0.3 and 1 microM) or lidocaine (1 and 200 microM) was administrated during the last 10 min before ischemia and the first 5 min of the reperfusion period. A significant post-ischemic functional recovery in the same degree was elicited by 0.3 and 1 microM 44Bu or 200 microM lidocaine. These effects of 44Bu and lidocaine closely correlated with the reduction in the infarct size and lactate dehydrogenase release. In contrast, 44Bu (0.1 microM) or lidocaine (1 microM) treatment did not result in a significant recovery in any of the examined parameters. In accordance with functional results, our electrophysiological data demonstrated that 44Bu was a more potent agent than lidocaine in terms of transient Na+ current inhibition. On the other hand, 44Bu did not cause any change in Ca2+ currents and on Na+/H+ exchange activity. These results show that 44Bu, as a novel Na+ channel blocker, has cardioprotective effects against ischemia/reperfusion injury.

Contribution of Rho-kinase and Adenosine Monophosphate-Activated Protein Kinase Signaling Pathways to Endothelium-Derived Contracting Factors Responses

Vascular tonus is controlled by endothelium-derived relaxing factor (EDRF), endothelium-derived hyperpolarizing factor (EDHF) and endothelium-derived contracting factor (EDCF) under physiological circumstances. In pathological conditions, impairment of endothelium-derived relaxation can be caused by both decrease in EDRF release and increase in EDCF release. The increase in EDCF is observed with diseases such as hypertension and diabetes. The contribution of Rho-kinase and activated protein kinase (AMPK), which have opposite effects, to the increased EDCF responses was investigated. Rho-kinases are the effectors of Rho which is one of the small guanosine triphosphate-binding proteins. They increase cytosolic Ca+2 concentration and cause vascular smooth muscle to contract, keeping myosin light chain (MLC) in phosphorylated state by affecting myosin phosphatase target subunit which dephosphorylates the MLC. The activities of Rho-kinases increase with the increase of EDCF function. AMPK is the energy sensor of the cell. It provides a vasculoprotective effect by causing endothelium-dependent and endothelium-independent relaxation in smooth muscle. In contrast to Rho-kinase pathway activity, AMPK pathway activity decreases with diseases in which the EDCF function increases. In cases such as diabetes and hypertension that endothelial function impairs toward vasocontraction, it is considered that evaluating Rho-kinase and AMPK pathways which mediate contraction and relaxation in vascular smooth muscle respectively, would provide clues on choosing therapeutic target for pathologies in which endothelial dysfunction is observed.

β3-Adrenoceptor Control of Lower Genitourinary Tract Organs and Function in Male: An Overview

The sympathetic nervous system is one component of the nervous regulatory system of the physiological function of the lower genitourinary tract. Our knowledge on the role of this sympathetic system has advanced during the last decade due to the characterization of β3-adrenoceptors (β3-ARs) in the urogenital system. This review focuses on the pharmacological and molecular evidence supporting the functional roles of β3-AR in male genitourinary tissues of various species. An electronic search in two different databases was performed including MEDLINE (PubMed) and EMBASE from 2010 to 2016. β3-agonists may be a promising alternative to antimuscarinics in the treatment of overactive bladder (OAB) based on available evidence. Although more recent studies have evaluated the involvement of β3-ARs in the physiological control and regulation of various tissues of the lower genitourinary tract mainly urinary bladder, penis, urethra, ureter, there are few innovations in the pipe-line. Among the β3-agonists, mirabegron is a unique drug licensed for the treatment of patients with OAB. Many drugs classified as β3-agonists are still under investigations for the treatment of OAB, lower urinary tract symptoms, ureteral stones, benign prostate hyperplasia, prostate cancer and erectile dysfunction. This review discusses the potential roles of β3-AR as new therapeutic targets by evaluating the results of preclinical and clinical studies related to male lower genitourinary tract function. Looking into the future, the potential benefits of β3- AR agonists from experimental and clinical investigations may provide an attractive therapeutic option.

Roles of PKC Isoforms in Development of Diabetes-Induced Cardiovascular Complications

Diabetes-induced cardiovascular abnormalities are the major causes of mortality and morbidity in diabetic populations. Vascular complications of diabetes can be evaluated as microvascular anomalies leading to retinopathy, nephropathy, and neuropathy, and macrovascular anomalies causing atherosclerosis, coronary artery disease, and peripheral vascular disease. Independent of coronary artery disease and hypertension, cardiomyopathy is also an important abnormality that can occur in the diabetic heart. Hyperglycemia, hyperinsulinemia related to insulin resistance, and increased levels of free fatty acids and lipids seem to have prominent roles in the development of microvascular and macrovascular complications and diabetic cardiomyopathy. Several mechanisms can be implicated in these complications, including increased polyol pathway flux, enhanced nonenzymatic glycation, and intracellular formation of advanced glycation end products (AGEs), activation of protein kinase C (PKC) isoforms, and increased hexosamine pathway activity. The focus of this chapter is recent concepts regarding PKC isoform-specific activation mechanisms and actions that have implications for the development of PKC-targeted therapeutics in diabetic complications. The PKC family of serine/threonine kinases have been associated with a diverse array of biological responses in health and disease. In diabetes, activation of different isoforms of PKC is associated with many pathologies seen in the retina, kidneys, vasculature, and heart. Therefore, inhibition of PKC isoforms can be evaluated as a therapeutic target for preventing of diabetic complications. In this regard, clinical trials using ruboxistaurin, a PKC-β isoform inhibitor, have promising results for treatment of diabetic retinopathy, nephropathy, and endothelial dysfunction.

P 23: The possible contribution of thyroid hormones to insulin effect on beta adrenoceptor mediated cardiac responses in diabetic rats

Aims As is well known, diabetes-induced cardiovascular complications are leading factors of morbidity and mortality. Among them atherosclerosis, hypertension and heart failure are most important ones. The very first problem seen in diabetic heart is the decrease of beta adrenoceptor mediated cardiac responses. The decrease of catecholamine stimulated beta adrenoceptor responsiveness is mostly associated with diminished receptor sensitivity and density. Insulin treatment of diabetic rats has been demonstrated to correct these parameters. However, the improving effect of insulin is shown to be abolished in tyhroidectomized diabetic rats. Although the effect of the relationship between these two hormones on some pathways has already been studied, their exact role on beta adrenoceptor mediated cardiac responses is uncertain. Thus in the present study, we aimed to determine the possible relationship between insulin and thyroid hormones to normalization of diabetes induced cardiac abnormalities. Methods Male Sprague-Dawley rats were used for this study. Ten days after surgical thyroidectomy, diabetes was induced with 38mg/kg streptozotocin by a single intravenous tail-vein injection. After 6-weeks of-diabetes induction, some groups (D-I, TxD-I) were treated with insulin (5–20U/kg/day, subcutan), some other groups (TxD-I-T2.5, TxD-I-T5) were administered insulin + thyroid hormone combination (2,5 or 5ug/kg/day T3 via osmotic minipump) for 2 weeks. Cardiac function was determined by the assessment of in vivo pressure-volume (PV) analysis and in vitro left ventricular papillary muscle experiments (isoprenaline, 0,0001uM-3uM; forskoline, 3uM). mRNA expressions of β1-AR and SERCA were evaluated by QPCR. Results Heart rate and end systolic pressure were markedly reduced in diabetic (D) and thyroidectomized diabetic (TxD) groups; however, they were improved with insulin treatment in D group. On the other hand, in TxD group, only insulin and 5ug/kg T3 combination (TxD-I-T5) ameliorated these parameters. End diastolic pressure and ejection fraction were found to be unchanged between the groups. Rate of contraction and relaxation, time constant of left ventricle pressure decay were reduced in D and TxD groups. These parameters were corrected significantly in D-I and TxD-I-T5 groups. Cardiac index was reduced in D and TxD groups. This parameter was not increased markedly in any of the treated groups. End systolic volume index was raised in D and TxD groups, and reduced only in D-I group. End diastolic pressure index was also increased in D and TxD groups. It was significantly decreased in D-I and TxD-I-T5 group. Isoprenaline, a nonselective β-AR agonist, induced concentrationdependent positive inotropic effects on papillary muscles of control rats (C). Maximum response (Emax) was markedly diminished in D and TxD groups. These parameters were ameliorated in D-I group significantly. Furthermore, in TxD-I-T5 group, the contractile response was enhanced compared to TxD group. Forskolin induced positive inotropic effect in all groups. The response was decreased in D and TxD groups. Insulin treatment improved this decrease in D-I and TxD-I-T5 groups. β1-AR mRNA levels were decreased both in D and TxD groups. Insulin treatment increased β1-AR mRNA levels. In TxD-I group insulin did not increase β1-AR mRNA levels. On the other hand, in TxD-I-T5 group β1-AR mRNA levels were significantly increased compared to TxD, TXD-I and TxD-I-T2.5 groups. SERCA mRNA levels were also found to be reduced in D and TxD groups. Insulin treatment did not correct the decrease in mRNA levels of SERCA of D and TxD groups. In TxD-I-T5 group, SERCA mRNA levels were significantly increased compared to TxD, TXD-I and TxD-I-T2.5 groups. Conclusion Both in vivo and in vitro cardiac experiments showed that heart function was impaired in D and TxD groups. Insulin exerted beneficial effects only in D-I and TxD-I-T5 groups. As no improvement was seen in TxD-I group, it could be thought that insulin could not reverse beta adrenoceptor mediated cardiac responses in thyroid deficiency. The slight but still insignificant recovery that we observed in TxD-I-T2.5 group could be interpreted with inadequacy of T3 dose.