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Background Doxorubicin (DOX), is a chemotherapeutic agent, which evokes oxidative stress and cell apoptosis in testicular tissue. It is known that the activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP), leading to apoptosis induced by DOX. The aim of the present study is to investigate whether PARP pathway has a role in DOX-induced testicular damage and infertility utilizing pharmacological PARP-1 inhibitor, PJ34, and PARP-1 knockout mice model. Methods Firstly, we assessed the activation of PARP pathway after DOX-induction at various hours by immunohistochemistry and western blot analysis. Secondly, we evaluated the role of PARP pathway in DOX-induced testicular damage, sperm motility, and fertility with pharmacological inhibition of PARP by using PJ34. Finally, we aimed to correlate a functional relationship between PARP-1 and DOX using PARP-1 knockout mice in DOX-induced testicular damage. Doxorubicin levels in plasma and testis tissue were also assessed. Results In DOX-induced group; PARP-1, PAR and apoptotic pathway protein expressions, increased significantly. In DOX + PJ34 group; PAR, cytochrome c, and AIF levels decreased significantly. Testicular weights, sperm motility, and mean the number of pups per litter decreased in DOX-induced group after 28 days, however they were similar to the control group in DOX-PJ34 group. In PARP-1 KO group, seminiferous tubule morphology was impaired significantly after 28 days of DOX-administration. Conclusions Our study indicates that DOX-induced testicular damage may be related to over-activation of PARP-1. PJ34 application was effective in preventing severe testicular damage caused by DOX-injection and may be considered for fertility protection against DOX-induced testicular damage.

Background: This study aimed to examine the effects of nesfatin-1 on thoracic aorta vasoreactivity and to investigate the inotropic and chronotropic effects of nesfatin-1 on the spontaneous contractions of the isolated rat atria.Methods: Isolated right atria and thoracic aorta were used in organ baths. The reactivity of the thoracic aorta was evaluated by potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh), and sodium nitroprusside (SNP). The effects of nesfatin-1 on the spontaneous contractions of the rat atria were also examined.Results: Nesfatin-1 (0.1–100 ng/ml) produced a concentration-dependent relaxation response in rat thoracic aorta. The relaxant responses to nesfatin-1 were inhibited by the removal of endothelium, NO synthase blocker N-nitro-L-arginine methyl ester (L-NAME, 10−4 M), and soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10–5 M). Nesfatin-1 (10 ng/ml, 30 min) increased the relaxation responses to either ACh or SNP, and the...

The present study was designed to evaluate the cardioprotective effects of nesfatin-1, a novel peptide with anorexigenic properties, in rats with isoproterenol (ISO)-induced myocardial infarction (MI), and to further investigate the role of Akt/GSK-3β signaling pathway in the protective effect of nesfatin-1. To induce MI, ISO was subcutaneously injected into the rats for two consecutive days at a dosage of 85mg/kg/day. ISO-induced myocardial damage was indicated by elevated levels of cardiac specific troponin-T, enhanced myocardial expression of proinflammatory cytokines (interleukin-1β, interleukin-6 and tumor necrosis factor-α), and increased number of cells with apoptotic and necrotic appearance in the myocardial tissue. Levels of p-Akt/Akt and p-GSK-3β/GSK-3β significantly decreased in heart tissue after ISO-induced MI. However, intraperitoneal administration of nesfatin-1 (10μg/kg/day) elicited a significant cardioprotective activity by lowering the levels of cardiac troponin-T and proinflammatory cytokines, indicating the protective effect of nesfatin-1 against ISO-induced MI. The biochemical findings were further confirmed by histopathological examination, which was demonstrated by reduced number of apoptotic and necrotic cells. Moreover, expressions of p-Akt/Akt and p-GSK-3β/GSK-3β in the myocardium of MI group rats were significantly increased by nesfatin-1 administration, suggesting that nesfatin-1, which appears to possess anti-apoptotic and anti-inflammatory properties, may confer protection against ISO-induced MI via an Akt/GSK-3β-dependent mechanism.

The aim of this study was to investigate the effect of resveratrol (RVT) on sildenafil-induced relaxations of isolated corpus cavernosum in non-diabetic and diabetic aged rats. A total of 13 male aged rats (72–80 weeks old) were randomized into two groups including non-diabetic aged rats and diabetic aged rats. Diabetes was induced in aged rats by streptozotocin (single i.p. dose of 45 mg/kg body weight) administration. At the end of the 12th week, corpus cavernosum strips of rats were suspended in an organ bath system. The corpus cavernosum relaxation was evaluated by sildenafil in the presence or absence of RVT (10−4 M) for 45 min. Induction of diabetes resulted in significant inhibition of sildenafil-induced corpus cavernosum relaxation in aged rats. The diminished relaxation in response to sildenafil was significantly improved by acute RVT incubation in both non-diabetic and diabetic aged rats; however, the magnitude of potentiation induced by RVT was more pronounced in diabetic aged rats. The potentiating effect of RVT was significantly inhibited by L-NG-nitroarginine methyl ester (L-NAME, 10−4 M, for 30 min) incubation in both groups. After the L-NAME incubation, the relaxation response of corporal tissues evoked by sildenafil was found to be similar in diabetic and non-diabetic aged rats. RVT improves sildenafil-induced relaxations of corpus cavernosum in both diabetic and non-diabetic aged rats probably by potentiating the activity of NOS, and this effect seems to be more manifest in diabetic aged group.

The cardiovascular benefits of resveratrol (RSV) have been well established by previous experimental and clinical studies. The aim of this study was to investigate the effectiveness of RSV administration on the impaired endothelial function induced by methylglyoxal (MGO), and to elucidate the role of endothelial nitric oxide synthase (eNOS) on its protective effect. Aged Wistar rats (80 weeks old, n = 15) were used in this study. The thoracic aorta was isolated and cut into rings for organ culture. Aortic segments of rats were incubated with MGO (420 µM) in the presence or absence of RSV (30 µM) for 4 h (short-term) or 24 h (long-term). Isometric tension studies were performed by an isolated organ bath in response to acetylcholine (ACh, an endothelium-dependent vasodilator) and sodium nitroprusside (SNP, an endothelium-independent vasodilator). Beside, expressions of eNOS and phospho-eNOS (p-eNOS) (Ser 1177) in thoracic aorta rings were evaluated by immunohistochemistry. Both short-term and long-term MGO incubation significantly inhibited the relaxation response induced by ACh, while the relaxation to SNP was not significantly altered. In addition, eNOS and p-eNOS expressions decreased significantly in arteries incubated with MGO. The impaired endothelial reactivity as well as decreased expressions of eNOS and p-eNOS in MGO-incubated vessels were significantly improved by RSV treatment. Endothelium-dependent vasodilatation of the thoracic aorta was significantly inhibited by MGO administration, and RSV may improve vascular endothelial function. The protective effect of RSV against MGO-induced endothelial dysfunction seems to be via increased eNOS expression and activity.

Objective The aim of this study was to investigate the effect of chronic exendin-4 (Ex-4) treatment on corpus cavernosum (CC) dysfunction in methylglyoxal (MGO) administered rats. Methods Male rats were divided into four groups as control, MGO (75 mg/kg/day in drinking water for 12 weeks), MGO + low-dose Ex-4 (0.1 μg/kg twice daily subcutaneously for 12 weeks concomitant with MGO), and MGO + high-dose Ex-4 (1 μg/kg twice daily subcutaneously for 12 weeks concomitant with MGO). Nitric oxide (NO)-mediated endothelium-dependent and neurogenic CC relaxations were evaluated by acetylcholine (ACh) and electrical field stimulation (EFS), respectively. Apoptosis was determined by TUNEL. Endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), NADPH oxidase subunit gp91phox (NOX2), and Rho kinase (ROCK2) expressions in CC were investigated by immunohistochemistry. Levels of the malondialdehyde (MDA) and advanced oxidation protein products (AOPP) were also measured. Results In MGO administered rats, both endothelium-dependent and neurogenic CC relaxations were significantly impaired as compared to controls. Apoptotic cell death and levels of MDA and AOPP increased significantly in MGO administered rats. eNOS and p-eNOS expressions decreased significantly in MGO group, while gp91phox expressions increased significantly. The diminished relaxation in response to ACh or EFS as well as the changes in expression of proteins in MGO groups were significantly improved by exendin-4 treatment. TUNEL-positive cells, and levels of MDA and AOPP in MGO group rats were also significantly reduced by exendin-4. Conclusion Exendin-4 treatment improves NO-mediated CC relaxations in MGO administered rats probably by inhibiting NADPH oxidase.

Aim The aim of this study was to investigate whether poly(ADP-ribose) polymerase (PARP) inhibition improves endothelin-1 (ET-1)-induced endothelial dysfunction (ED). Methods Isolated rat thoracic aorta rings were incubated with ET-1 (10 nmol/L) in the presence or absence of either polyethylene glycol–superoxide dismutase (PEG-SOD; a cell-permeable superoxide radical scavenger, 41 U/mL) plus apocynin (a NADPH oxidase inhibitor, 300 µmol/L) or PJ34 (an inhibitor of polyADP-ribose polymerase, 3 µmol/L) for 18 h. Isometric tension studies were performed in response to acetylcholine (ACh; an endothelium-dependent vasodilator), sodium nitroprusside (SNP; an endothelium-independent vasodilator), and phenylephrine (Phe). PARP-1 and PAR (an end-product of PARP activity) expressions were evaluated by both Western blot and immunohistochemistry. Results Incubation of thoracic aorta rings with ET-1 resulted in a significant inhibition of the response to ACh, while SNP-induced relaxation was unaffected. The contractile response to Phe increased in arteries that were incubated with ET-1. PARP-1 and PAR expressions increased after ET-1 incubation. The diminished vasoreactivity as well as changes in expressions of PARP-1 and PAR in ET-1-incubated vessels were improved by both PEG-SOD plus apocynin and PJ34. Conclusion Our studies demonstrate that ED induced by ET-1 seems to be effected via oxidative stress in the thoracic aorta endothelium with subsequent activation of the PARP pathway.

Background: The cardiovascular benefits of Resveratrol (RVT) have been well established by previous experimental and clinical studies. Aims: The goal of this study was to test the effectiveness of RVT administration on the impaired endothelial function induced by lipopolysaccharide (LPS), and to elucidate the role of endothelial nitric oxide synthase (eNOS)/Sirtuin 1 (SIRT1) pathway. Study Design: Animal experiment. Methods: Endotoxemia was induced by intraperitoneal injection of 10 mg/kg LPS, and the thoracic aorta was isolated six hours later. RVT was injected intraperitoneally 15 minutes before LPS administration. Six hours after LPS injection, potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh), and sodium nitroprusside (SNP) were used to examine to vascular reactivity and endothelial function. eNOS, phospho-eNOS (p-eNOS) (Ser 1177), and SIRT1 expressions in thoracic aorta were evaluated by Western blot. Results: LPS administration significantly inhibited the relaxation response induced by ACh, while the relaxation to SNP was not significantly altered. Phe- and KCl-induced contractile responses in the thoracic aorta significantly decreased in LPS-injected group. eNOS and p-eNOS expression decreased significantly in arteries obtained from LPS group rats. The impaired vasoreactivity as well as decreased expressions of eNOS, p-eNOS, and SIRT1 in vessels from LPS-injected rats were improved by RVT treatment. Conclusion: The endothelium-dependent vasodilatation of the thoracic aorta was significantly inhibited by LPS administration, and RVT treatment may improve vascular endothelial function. The protective effect of RVT might be associated with increased eNOS expression and activity.

Environmental exposure to pesticides may cause serious health risks including fertility and reproductive function. The aim of this study was to highlight whether there is a relationship between exposure to abamectin and male fertility parameters of farmworkers. Twenty male farmworkers who were using abamectin and 20 men not exposed to pesticides were recruited as experimental and control groups, respectively. Semen analysis, molecular markers of sperm maturity and serum reproductive hormone levels were evaluated. In experimental group, high plasma abamectin levels were detected. These men have decreased sperm motility. Moreover, diminished molecular markers of sperm maturity, such as decreased hyaluronic acid (HA) binding of sperm, increased numbers of aniline blue positive sperm and increased percentage of creatine kinase (CK) positive sperm, were observed in abamectin-exposed men. Their serum testosterone, LH and FSH levels did not change significantly. We conclude that exposure to abamectin may impair male fertility by effecting semen quality.

Despite the known adverse effects of abamectin pesticide, little is known about its action on male fertility. To explore the effects of exposure to abamectin on male fertility and its mechanism, low (1 mg/kg/day) and high dose (4 mg/kg/day) abamectin were applied to male rats by oral gavage for 1 week and for 6 weeks. Weight of testes, serum reproductive hormone levels, sperm dynamics and histopathology of testes were used to evaluate the reproductive efficiency of abamectin-exposed rats. Abamectin level was determined at high concentrations in plasma and testicular tissues of male rats exposed to this pesticide. The testes weights of animals and serum testosterone concentrations did not show any significant changes after abamectin exposure. Abamectin administration was associated with decreased sperm count and motility and increased seminiferous tubule damage. In addition, significant elevations in the 4-hydroxy-2-nonenal (4-HNE)-modified proteins and poly(ADP-ribose) (PAR) expression, as markers for oxidative stress and poly(ADP-ribose) polymerase (PARP) activation, were observed in testes of rats exposed to abamectin. These results showed that abamectin exposure induces testicular damage and affects sperm dynamics. Oxidative stress-mediated PARP activation might be one of the possible mechanism(s) underlying testicular damage induced by abamectin. (C) 2011 Elsevier Inc. All rights reserved.

Recent studies have clearly reported that there is a relationship between endotoxemia and acute renal injury. The aim of this study was to investigate whether treatment with the new potent PARP inhibitor PJ34 could prevent the acute renal injury induced by lipopolysaccharide (LPS). Endotoxemia was induced by LPS injection (10 mg/kg, i.v.). LPS increased blood urea nitrogen (BUN) levels from 22 +/- 0.54 mg/dL to 45.7 +/- 5.79 mg/dL (p < 0.05). The plasma creatinine levels were 0.38 +/- 0.02 mg/dL and 0.47 +/- 0.03 mg/dL for the control and LPS groups, respectively. In addition, urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG, a marker of renal tubular damage) was increased after LPS injection. By light microscopy, structural renal damage was observed in the LPS-treated group. However, PJ34 treatment (10 mg/kg, i.p.) attenuated LPS-induced renal injury, as indicated by plasma BUN and creatinine levels, urinary NAG excretion, and renal histology. These results indicated that the overactivation of the PARP pathway may have a role in LPS-induced renal impairment. Hence, pharmacological inhibition of this pathway might be an effective intervention to prevent endotoxin-induced acute renal injury.

This study was aimed to examine the effects of homocysteine (Hcy) on vascular responsiveness of guinea-pig isolated pulmonary arteries and to investigate possible underlying mechanisms. In order to evaluate vascular reactivity, isometric tension studies were performed in response to potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh), and sodium nitroprusside (SNP). Incubation of pulmonary artery rings with Hcy (10(-3)M, 180min) resulted in significant inhibition of response to ACh (an endothelium-dependent vasodilator)(E(max): 55.3+/-6.7 vs. 13.1+/-2.0(*), P

We prepared diet-induced hyperhomocysteinemia (hHcy) in adult male Wistar rats and investigated the effects of hHcy on the adenosinergic and adrenergic responses in vitro and in vivo. The responsiveness of right atria from hHcy rats to the negative chronotropic effects of adenosine (Ado) was found to be significantly greater in hHcy rats than in controls. The pD2 value and maximum effect of Ado were significantly increased in 12-week hHcy right atria when compared with those from age-matched controls. The vasodilatory effect of Ado on rat thoracic aorta was also increased in hHcy rats. In the presence of dipyridamole, an Ado uptake inhibitor, the negative chronotropic and vasodilatory effects of Ado were significantly potentiated in the hHcy rats much more than in the control rats. In anesthetized rats, Ado and dipyridamole, given as a rapid bolus into the femoral artery, led to reduction in mean blood pressure and heart rate. This effect was significantly pronounced in hHcy rats when compared with control animals. Otherwise, hHcy atria were found to have increased responsiveness to the positive chronotropic response to isoproterenol, an beta-adrenoceptor agonist. However, there were no significant differences between two groups in the vasoconstrictor effects to phenylephrine, an alpha-adrenoceptor agonist. On the basis of these results, we concluded that hHcy rats were significantly more sensitive to the negative chronotropic and vasorelaxant effects of Ado, possibly because of accelerated cellular Ado uptake and/or a change in Ado receptor-G protein system. This change may be related with the increased responsiveness to beta-adrenergic agonists in hHcy rats, and might contribute to the harmful cardiac effects of hHcy.

The aim of this study was to investigate whether the low-molecular-weight heparins (LMWHs) (eg, nadroparin, enoxaparin, and dalteparin) cause a vasodilatory effect in human internal mammary artery (IMA) and to further compare its effect with unfractioned heparin (UFH). Samples of redundant IMA obtained from 20 patients undergoing a coronary artery bypass graft surgery were cut into 3-mm-wide rings and suspended in 20-mL organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. LMWHs (0.5-6 U/mL) caused a concentration-dependent relaxation in the endothelium-intact human IMA rings, which were precontracted with Phe (10(-6) M) (P < 0.05). The vasodilator potency of LMWHs seems to be nearly similar while the maximal effect produced by LMWHs was less pronounced compared with that produced by UFH. Removal of endothelium totally abolished the responses of human IMA to LMWHs as well as UFH (P < 0.05). LMWHs-induced vasodilator effect was significantly attenuated by Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) but not indomethacin (10(-5) M). Our results have shown that LMWHs cause a dose-dependent relaxation in human IMA but are less effective than that produced by UFH. The vasorelaxant effects induced by each of LMWH are nearly similar and seem to be via endothelium-dependent mechanisms, including generation of nitric oxide.

The aim of this study was to investigate the acute direct effects of homocysteine (Hcy) on the vascular responsiveness of human internal mammary artery (IMA) and to define the possible underlying mechanisms. The contractile response to both phenylephrine (Phe) (-36%) and KCl (-18%) was significantly reduced in arteries that were incubated with Hcy (10 (-4)M, 30 minutes), compared with controls (P0.05). Removal of endothelium did not significantly alter the responses of human IMA to Phe. Hcy (10 (-6)M) also caused a relaxation response in human IMA rings precontracted with Phe (10 (-4) M) and this effect was not inhibited by N-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), by l-NAME (10 (-4)M) + indomethacin (10 (-4)M), or by intimal rubbing. In addition, contractions induced by stepwise addition to calcium (Ca2+) to high KCl solution with no Ca(2+) were significantly inhibited by Hcy incubation as well as contractions induced by Phe in the absence of extracellular Ca (2+) (P0.05). On the other hand, Hcy (10 M, 30 minutes) did not significantly inhibit the relaxation responses to either acetylcholine (ACh) or sodium nitroprusside (SNP) (P0.05). These results demonstrated that short-term exposure to Hcy significantly decreased vascular responsiveness in human IMA without affecting endothelium-dependent and -independent vasorelaxation. This effect is not NO-, prostaglandin- or endothelium-dependent. The mechanism is uncertain but seems to depend on the interactions of Hcy with Ca(2+) influxes and/or other undefined direct effects in this tissue.

UNLABELLED To test our hypothesis that the abnormally small efficacy of mu-opioid agonists in diabetic rats may be due to functional changes in the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway, we evaluated the effects of N-iminoethyl-L-ornithine, methylene blue, and 3-morpholino-sydnonimine on [D-Ala(2), NMePhe(4), Gly-ol(5)]enkephalin (DAMGO)-induced antinociception in both streptozotocin (STZ)-diabetic and nondiabetic rats. Animals were rendered diabetic by an injection of STZ (60 mg/kg intraperitoneally). Antinociception was evaluated by the formalin test. The mu-opioid receptor agonist DAMGO (1 microg per paw) suppressed the agitation response in the second phase. The antinociceptive effect of DAMGO in STZ-diabetic rats was significantly less than in nondiabetic rats. N-Iminoethyl-L-ornithine (100 microg per paw), an NO synthase inhibitor, or methylene blue (500 microg per paw), a guanylyl cyclase inhibitor, significantly decreased DAMGO-induced antinociception in both diabetic and nondiabetic rats. Furthermore, 3-morpholino-sydnonimine (200 microg per paw), an NO donor, enhanced the antinociceptive effect of DAMGO in nondiabetic rats but did not change in diabetic rats. These results suggest that the peripheral antinociceptive effect of DAMGO may result from activation of the L-arginine/NO/cGMP pathway and dysfunction of this pathway; also, events that are followed by cGMP activation may have contributed to the demonstrated poor antinociceptive response of diabetic rats to mu-opioid agonists. IMPLICATIONS This is the first study on the role of the nitric oxide (NO)/cyclic guanosine monophosphate pathway on [D-Ala(2), NMePhe(4), Gly-ol(5)]enkephalin (DAMGO)-induced peripheral antinociception and the effect of diabetes on this pathway. The study suggests a possible role of DAMGO as a peripherally-acting analgesic drug.

1. The aim of the present study was to investigate the role of several possible neurotransmitters in mediating non-adrenergic, non-cholinergic (NANC) relaxation, and the effects of phosphodiesterase (PDE) III and V inhibitors on adrenergic and NANC relaxation in branch pulmonary artery (PA) of guinea-pig. 2. Under the NANC conditions, electrical field stimulation (EFS, 60 V, 0.2 ms, 20 Hz) induced a tetrodotoxin-sensitive relaxation of the histamine-precontracted PA rings. The nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 10(-4) m) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10(-5) m) partially inhibited the EFS-induced relaxation. The inhibitory effect of l-NAME was reversed completely by l-arginine (10(-3) m), but not d-arginine (10(-3) m). 3. This NANC relaxation was attenuated by 8-phenyltheophylline (10(-5) m), a P1-purinoceptor antagonist. 4. The NANC response was potentiated by 10-6 m zaprinast, a type V PDE inhibitor, but was unaffected by 3 x 10-6 m milrinone, a type III PDE inhibitor. 5. Sodium nitroprusside (SNP) caused a concentration-dependent vasodilator effect which was potentiated by zaprinast, but unaffected by milrinone. Moreover, the effect of combination of zaprinast with milrinone was not significantly different from that observed with zaprinast alone. 6. Isoprenaline produced a concentration-dependent vasodilatation in branch PA of guinea-pig which was potentiated by both zaprinast and milrinone, the efficacy of milrinone being greater than zaprinast. 7. These results suggest that both nitrergic and purinergic pathways are involved in mediating the NANC relaxation in branch PA of guinea-pig. The combination of PDE III or V inhibitors with vasorelaxant drugs may be a hopeful approach for the treatment of pulmonary hypertension.

We investigated the effect of nitric oxide (NO) on the responses of isolated tracheas to acetylcholine and to electrical field stimulation in streptozotocin-diabetic and controls rats. The contractile responses to acetylcholine were neither different nor affected by the NO synthase blocker, N ω -nitro- l -arginine methyl ester ( l -NAME), in the two groups. Diabetic rat tracheas were supersensitive to field stimulation. l -NAME enhanced field stimulation-induced contractions at low frequencies in control rat tracheas, but had no effect in diabetic rat tracheas. After l -NAME treatment, there was no difference in sensitivity to field stimulation between the groups. The relaxation responses to sodium nitroprusside in acetylcholine-precontracted tracheas were not different between the groups. However, diabetic rat trachea was supersensitive to the relaxant effect of sodium nitroprusside on contractile responses to field stimulation. These results suggested that the increase in sensitivity to field stimulation in tracheas from diabetic rats might be due to impairment in the production and/or release of an endogenous NO-like factor.

Aim The aim of this study was to investigate the effect of pravastatin treatment on diminished corpus cavernosum (CC) function associated with aging. Methods Male rats were divided into three groups as adult rats (12-14 weeks old), aged rats (72-80 weeks old) and aged rats given 10 mg/kg/d pravastatin in drinking water for six weeks. Blood pressure was measured by tail-cuff method. Total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, triglycerides and testosterone levels were estimated in blood. Changes in expression levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS) (Ser-1177), neuronal nitric oxide synthase (nNOS), NADPH oxidase subunit gp91(phox), Rho A and Rho kinase (ROCK2) in CC were assessed by immunohistochemistry. Nitric oxide (NO)-mediated endothelium-dependent and neurogenic CC relaxation were evaluated by acetylcholine (ACh, 0.1 nM-100 µM) and electrical field stimulation (EFS; 30 V, 5 ms, 2-32 Hz), respectively. Results In aged rats, NO-mediated, both endothelium-dependent and neurogenic CC relaxation, were significantly impaired as compared to adult rats. Besides, eNOS, p-eNOS and nNOS expressions decreased significantly in CC from aged rats, while gp91(phox), RhoA and ROCK2 expressions increased significantly. The diminished relaxation in response to ACh or EFS as well as the changes in expression of these proteins in aged rats were significantly improved by pravastatin treatment. Conclusion Pravastatin improves NO-mediated CC relaxations of aged rats probably by inhibiting NADPH oxidase/Rho kinase pathways, and this effect does not seem to be associated with lipid lowering effect of this drug.

e90 Volume 35 Number 8S to study the availability of information about the studied population, clinical experience, pharmacokinetic properties, and drug–drug interactions. Using the Systematic Information for Monitoring score, available information was considered clinically applicable if it provided information that could be applied in clinical practice. Missing information was either a statement that information was absent, unjustifiably nonavailable information, or hollow statements (eg, “caution in older patients”). Descriptive statistics (frequencies) were applied using SPSS 20.0. Results: The availability of relevant and clinically applicable information ranged between 10% (Belgian Repertorium and BNF) and 20% (FK), except for the PL (mean, 66%). The PL, which is comparable to the European SmPC, appeared a more extensive document (7–32 pages) than the other handbooks (2 pages). In the handbooks, most information was present about drug–drug interactions (range, 30%–75%). Information about patient characteristics and about experience in older people was present in < 7% of the handbook texts, except for the PL (48% and 81%, respectively). Clinically applicable information (what to monitor, critical value, how to respond) concerning renal impairment ranged from 4% to 29%. Conclusion: This study found that the availability and clinical applicability of information about older people for rational prescribing of medicines is incomplete in the investigated European and American handbooks. Because these handbooks are the primary documents that guide prescribing in actual medical practice, the availability and clinical applicability of the information on older individuals should be improved. Disclosure of Interest: J. Boer: None declared. E. Beers: Grant/ research support from: Erna Beers has received a grant of the Dutch Society of Clinical Pharmacology & Biopharmacy (NVKF&B) for her training in clinical pharmacology and therapeutics. This manuscript was written in the context of her training. The Expertise Centre Pharmacotherapy in Old Persons (EPHOR) is financially supported by The Netherlands Organisation for Health Research and Development (ZonMW). The authors’ work was independent of the NVKF&B and ZonMW. T. Egberts: None declared. H. Leufkens: None declared. P. Jansen: Grant/research support from: The Expertise Centre Pharmacotherapy in Old Persons (EPHOR) is financially supported by The Netherlands Organisation for Health Research and Development (ZonMW).

Although the oxidative stress and inflammation are closely related with breast cancer, there is no study directly examining the possible changes in vascular functions in the presence of breast carcinoma. The goal of the present study was to evaluate changes in vascular reactivity in tumor-bearing mice. In this study, highly metastatic breast carcinoma cells which were derived from liver or brain metastasis of 4T1 murine breast carcinoma (4TLM and 4TBM, respectively), and 67NR cells which were tumorigenic but non-metastatic cells were used. Female Balb-c mice 8-10weeks old were divided into following groups: (1) control, (2) injected with 67NR, (3) injected with 4TLM, and (4) injected with 4TBM orthotopically. Thoracic aorta was removed 23-25days after injection of tumor cells. Isometric tension studies were performed in response to potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh, an endothelium-dependent vasodilator), and sodium nitroprusside (SNP, an endothelium-independent vasodilator). Endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser 1177) (p-eNOS), gp91(phox), and tumor necrosis factor-α (TNF-α) expressions in aortic tissues were demonstrated by immunohistochemistry. The level of TNF-α in vascular tissue was measured by ELISA. The presence of tumor was resulted in significant inhibition of response to ACh in both 4TLM and 4TBM injected mice, but not 67NR injected mice. Furthermore, both KCl and Phe-induced contraction of thoracic aorta was not changed significantly in tumor-bearing animals. eNOS and p-eNOS expressions decreased while gp91(phox) and TNF-α expressions increased in endothelium of 4TLM and 4TBM mice compared to 67NR injected and control mice. Moreover, TNF-α levels of thoracic aorta in mice with metastatic breast carcinoma were significantly higher than that of 67NR mice. Tumor-induced endothelial dysfunction determined by ACh-induced relaxation improved by superoxide dismutase (SOD), apocynin (a NADPH oxidase inhibitor), and infliximab (a TNF-α monoclonal antibody). The findings of this study suggest that the presence of metastatic breast carcinoma may cause a significant reduction in endothelium-dependent relaxation of thoracic aorta via NADPH oxidase-mediated oxidative stress and TNF-α production.

The aim of this study was to evaluate the relaxant effect of taurine, one of the most commonly employed dietary supplements, in rat corpus cavernosum (CC), and to further investigate the contribution of possible underlying mechanisms. Strips of CC were suspended in an organ bath system for isometric tension studies. Taurine (10-80 mmol/l) produced a concentration-dependent relaxation response in rat CC. Endothelial removal did not cause a significant inhibition of the relaxation response to taurine. Incubation of endothelium-denuded CC strips with the nonselective potassium channel blocker tetraethylammonium (10 mmol/l), the ATP-sensitive potassium (KATP) channel blocker glibenclamide (10 μmol/l), the inward rectifier potassium (Kir) channel inhibitor barium chloride (30 μmol/l), and the large conductance calcium-activated potassium channel inhibitor iberiotoxin (0.1 μmol/l) significantly inhibited the relaxant responses to taurine. However, taurine-induced relaxation was not inhibited by the voltage-dependent potassium channel inhibitor 4-aminopyridine (1 mmol/l). On the other hand, taurine (20 mmol/l, 30 min) inhibited both intracellular and extracellular calcium-dependent contraction in CC strips. These findings suggest that taurine induced relaxation of CC via an endothelium-independent pathway. The activation of KATP channels, Kir channels and calcium channels is thought to play an important role in endothelium-independent relaxation of CC, but other direct effects on calcium dynamics may also contribute to its relaxant effect.

Nephrotoxicity is one of the serious dose-limiting complications of methotrexate (MTX) when used in the treatment of various malignancies and nononcological diseases. The aim of this study was to investigate the role of poly(adenosine diphosphate ribose) polymerase (PARP) activity in MTX-induced nephrotoxicity. Rats were divided into 4 groups as control, MTX treated (MTX, 7 mg/kg per d, intraperitoneally [ip], once daily for 3 consecutive days), MTX plus 1,5-isoquinelinediol (ISO, a PARP inhibitor, 3 mg/kg per d, i.p.) treated, or ISO treated. Histopathology of kidneys was evaluated by light microscopy. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay was used to analyze apoptosis in kidney sections. Blood urea nitrogen (BUN), serum creatinine, and urinary N-acetyl-β-d-glucosaminidase (NAG) were used as biochemical markers of MTX-induced renal injury. Our results showed that MTX administration significantly increased BUN, serum creatinine, and urinary NAG levels. The PARP-1 and PAR (a product of PARP activity) expression and apoptotic cell death were also markedly increased in renal tubules after MTX administration. The ISO treatment attenuated MTX-induced renal injury, as indicated by BUN and serum creatinine levels, urinary NAG excretion, and renal histology. The PARP inhibitor treatment reduced PARP-1 and PAR expression to levels similar to that of controls. These results revealed that ISO may have a protective effect against the nephrotoxic effects of MTX by inhibiting PARP activation. This is the first study that demonstrates the role of PARP activation in MTX-induced nephrotoxicity and tubular apoptosis.

The aim of this study was to evaluate whether the poly(adenosine diphosphate[ADP]-ribose) polymerase (PARP) pathway is activated by experimental left varicocele. Rats underwent partial ligation of the left renal vein to induce experimental varicocele, and left testes were analyzed 13 weeks after surgery. Tubule degeneration was evaluated by Johnsen score. Expression of 4-hydroxy-2-nonenal (4-HNE)-modified proteins, PARP-1, and poly(-ADP-ribose) (PAR) was detected by immunohistochemistry and Western blot. The degree of apoptosis within testes was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling. Light microscopy revealed testicular damage comprising various degrees of seminiferous tubule degeneration. Germ cell apoptotic index and 4-HNE, PAR, and PARP-1 expression in germ cells increased after varicocele induction. Increased oxidative stress and PARP overactivation in testes might be important with regard to impaired testicular function associated with varicocele.

Introduction. The aim of this study was to investigate whether activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) contributes to the development of aldosterone-induced endothelial dysfunction and treatment with the potent PARP inhibitor 1,5-isoquinolinediol (3 mg/kg/day, i.p.) could prevent endothelial dysfunction caused by aldosterone. Methods. Infusion of subpressor doses of aldosterone with subcutaneously implanted mini-osmotic pumps (0.05 mg/kg/day) to rats for 21 days induced the development of endothelial dysfunction. In order to evaluate endothelial function, isometric tension studies were performed in response to acetylcholine and sodium nitroprusside.Additionally, PAR (the end product of activated PARP) and PARP-1 expressions in the endothelium of thoracic aortas were evaluated by immunohistochemistry. Results. There was a significant loss of endothelium-dependent vasodilatation in response to acetylcholine in aldosterone-infused rats. In animals treated with 1,5-isoquinolinediol, the effect of aldosterone on vascular responsiveness was less than the untreated groups. Immunohistochemical studies demonstrated that aldosterone administration increased PAR and PARP-1 expressions in the endothelium of thoracic aortas, whereas PARP inhibition decreased their expressions to control levels. Conclusion. Our results indicate that PARP activation in the vascular system may be a contributory factor to the impaired endothelial function associated with aldosterone administration.

To investigate whether experimental hyperhomocysteinemia (HHCY) can induce adverse changes in bone metabolism.Blood and urine samples were collected from rats fed with a methionine-enriched diet (HHCY, n = 18) or an isocaloric control diet (control, n = 10) for 12 weeks. Biochemical bone turnover markers (osteocalcin, hydroxyproline, N-terminal collagen I telopeptides and homocysteine (HCY), folate and vitamin B12) were measured. Whole body bone mineral density (BMD) was assessed by dual energy X-ray absorptiometry.HCY was significantly higher in HHCY than in control rats (16.2 versus 3.2 micromol/L; p = 0.0006). Bone resorption parameters hydroxyproline (1.60 +/- 0.6 versus 0.85 +/- 0.4; p0.05) and N-terminal collagen I telopeptides (150.8 +/- 78 versus 48.1 +/- 26 nmol/L BCE; p0.05) increased, whereas bone formation marker osteocalcin (9.01 +/- 3.8 versus 15.07 +/- 4.2 ng/mL; p0.05) decreased in HHCY compared to control rats. The relation N-terminal collagen I telopeptides/osteocalcin significantly increased in HHCY compared to control rats (13.14 +/- 3.1 versus 4.14 +/- 1.9). BMD measurement did not reveal any differences between groups.These findings demonstrate a significant modification of bone turnover in HHCY rats. The relation between bone resorption and formation indicates a shift toward bone resorption, which might be a plausible explanation for the relation between HHCY and fracture risk.

The effect of hyperhomocysteinemia induced by pretreatment with methionine 12 weeks prior to the study on the responses induced by gamma-aminobutyric acid (GABA), electrical field stimulation (EFS), and ATP have been evaluated in isolated rat duodenum. In the presence of adrenergic and cholinergic blockade, EFS (60 V, 1 ms, 1-3 Hz) induced frequency-dependent relaxations of the preparation. GABA and ATP also caused submaximal relaxation of the rat duodenum. The relaxations induced by GABA, EFS, and ATP were not significantly changed in duodenal tissues from hyperhomocysteinemic rats compared with control rats. GABA- and EFS-induced relaxations were inhibited by N-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M) in both hyperhomocysteinemic and control rats. On the other hand, L-NAME incubation did not affect ATP-induced relaxation. These results suggest that hyperhomocysteinemia does not cause an important impairment on non-adrenergic non-cholinergic innervation of the rat duodenum.

The main function of vitamin K1 is to act a co-factor for gamma-glutamyl carboxylase. However, it has also been shown to lessen oxidative stress. This study was aimed to evaluate the effect of vitamin K1 supplementation on vascular responsiveness and oxidative status in rats that underwent femoral osteotomy. Twenty-four male rats were divided into three groups to serve as sham, osteotomy and vitamin K1 groups. Indices of oxidative stress (catalase), and oxidative damage (malondialdehyde) were analysed in erythrocytes. In order to evaluate vascular reactivity, concentration-response curves to phenylephrine, angiotensin II, 5-hydroxytryptamine, bradykinin and histamine were constructed. The findings of this study clearly show that oxidative stress clearly increases after femoral osteotomy in rats. Also, this operation causes a significant depression in vascular responsiveness to contracting agents and endothelium-dependent vasodilators. However, vitamin K1 supplementation prevents vascular hyporeactivity by reducing oxidative stress and may represent a novel approach during osteotomy healing. Copyright (c) 2006 John Wiley & Sons, Ltd.

Recent studies clearly show that there is a relationship between endotoxemia and impaired vascular responsiveness. The aim of this study was to investigate whether treatment with the new potent PARP inhibitor PJ34 could prevent the vascular hyporesponsiveness induced by lipopolysaccharide (LPS). Endotoxemia was induced in rats by LPS injection (20 mg kg −1 , i.p.). Administration of LPS caused a decrease in mean blood pressure and an increase in heart rate. In endothelium-denuded rings of thoracic aorta from untreated rats, contractile responses to KCl and phenylephrine decreased after LPS injection. Furthermore, there was a significant loss of endothelium-dependent vasodilatation in response to acetylcholine in LPS-treated rats. The animals pretreated with PJ34 (10 mg kg −1 , i.p., 30 min before LPS injection), the effect of LPS on vascular responsiveness was lower than the untreated ones. Pretreating the animals with PJ34 before the LPS challenge prevented the decline in mean blood pressure. However, this did not result in significant changes to the heart rate. The inhibitory effect of LPS treatment on both KCl- and phenylephrine-induced contraction responses was significantly antagonized by PJ34. Additionally, pretreatment of the rats with PJ34 attenuated the LPS-induced endothelial dysfunction in endothelium-intact aorta rings. This study demonstrates that PARP activation in the vascular system is an important contributory factor to the impaired vascular responsiveness associated with endotoxic shock. Hence, the pharmacological inhibition of PARP pathway might be an effective intervention to prevent endotoxin-induced vascular hyporesponsiveness.

The aim of this study was to investigate whether unfractioned heparin produces a direct vasodilatory effect on the human internal mammary artery (IMA) and the possible underlying mechanisms. Samples of redundant IMA were obtained from 20 patients undergoing coronary artery bypass graft surgery, and concentration-response curves to unfractioned heparin were constructed. Unfractioned heparin (0.5-6 U/mL) caused a concentration-dependent relaxation in the endothelium-intact human IMA rings precontracted with phenylephrine (10 - 6 M). Removal of endothelium significantly inhibited the responses of human IMA to unfractioned heparin (P < 0.05). N ω -Nitro-L-arginine methyl ester (L-NAME, 10 - 4 M), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 - 5 M) and L-NAME (10 - 4 M) plus ODQ (10 - 5 M) partially reduced unfractioned heparin-induced vasodilatory response in endothelium-intact rings, whereas indomethacin alone had no effect. The vasodilatory effect of unfractioned heparin was completely inhibited by 40 mM KCl in the presence of L-NAME, ODQ, and indomethacin. These results clearly demonstrated that unfractioned heparin causes a concentration-dependent vasodilatation in human internal mammary artery, and this action seems to be via endothelium-dependent mechanisms, including generation of nitric oxide and endothelium-derived hyperpolarizing factor.

Recent studies have clearly shown that there is a relationship between hyperhomocysteinemia and endothelial dysfunction. However, the effect of poly(ADP-ribose) polymerase (PARP) inhibition on homocysteine (Hcy)-induced endothelial damage has not been investigated. In this study, we investigated whether the loss of endothelial function in rat aortic rings preincubated with Hcy is dependent upon the PARP pathway within the vasculature. Preincubation of rat aortic rings with Hcy (1 mmol/l; 180 min) significantly inhibited endothelium-dependent relaxation in this tissue. This inhibitory effect was significantly reduced in the presence of both superoxide dismutase (100 U ml–1) and catalase (100 U ml–1) together with Hcy. Similarly, preincubation for 180 min with either N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride (PJ34; 3 µmol/l) or 3-aminobenzamide (3 mmol/l), structurally different PARP inhibitors, also significantly prevented the development of endothelial dysfunction induced by Hcy. Further incubation of aortic rings with these PARP inhibitors for 60 min after exposure to Hcy for 180 min, at least in part, improved the endothelium-dependent relaxation responses. Thus, our results suggest that intraendothelial PARP activation may be associated with endothelial dysfunction in hyperhomocysteinemic conditions and that inhibition of this pathway may present a novel pharmacological approach to prevent Hcy-induced endothelial damage. Suprisingly, inhibition of the PARP pathway not only prevents the endothelial dysfunction mediated by Hcy, but is also able to rapidly improve it.

SUMMARY 1. In the present study, we investigated the effect of GABA and selective GABA agonists and antagonists on neurally induced tracheal contractions in streptozotocin (STZ) diabetic rats. 2. Contractile responses to electrical field stimulation (EFS) in rat tracheal rings were completely abolished by atropine and tetrodotoxin, but were unaffected by the ganglion blocker hexamethonium, indicating that they were mediated via neuronal release of acetylcholine (ACh). 3. Contractions induced by EFS, but not by exogenous ACh, were inhibited by GABA and the selective GABAB receptor agonist baclofen, but not by the selective GABAA receptor agonist 3-aminopropane sulphonic acid. The inhibitory effects of GABA or baclofen were not affected by the GABAA antagonist bicuculline, but were significantly reversed by the GABAB antagonist phaclofen. 4. The inhibitory effects of both GABA and baclofen were found to be significantly greater in trachea from control rats compared with tissues from diabetic rats. 5. Non-adrenergic, non-cholinergic relaxation responses elicited by EFS in precontracted tracheal rings from diabetic and control rats were similar in magnitude and were unaffected by GABA or GABA analogues. 6. These results suggest that GABA decreases the response to EFS by directly inhibiting the evoked release of ACh through GABAB receptors in rat trachea and that STZ-induced diabetes causes an impairment in the inhibitory effect of GABA on neurally induced contractions in this tissue.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes involved in a wide variety of biological processes, including DNA repair and maintenance of genomic stability following genotoxic stress, and regulates the expression of various proteins at the transcriptional level as well as replication and differentiation. However, excessive activation of PARP has been shown to contribute to the pathogenesis of several diseases associated with oxidative stress (OS), which has been known to play a fundamental role in the etiology of male infertility. Based on the degree and type of the stress stimulus, PARP directs cells to specific fates (such as, DNA repair vs. cell death). A large volume of accumulated evidence indicates the presence of PARP and its homologs in testicular germ line cells and its activity may offer a key mechanism for keeping DNA integrity in spermatogenesis. On the other hand, a possible role of PARP overactivation in OS-induced male reproductive disorders and in human sperm is gaining significance in recent years. In this review, we focus on the findings about the importance of PARP-1 and PARP-2 in male reproduction and possible involvement of PARP overactivation in various clinical conditions associated with male infertility.

Background: Because commercial minimally invasive harvesting equipments significantly increase operation costs, they are not always available in all clinics worldwide. The aim of this study was to investigate whether minimally invasive saphenous vein harvesting using a laryngoscope can be applied efficiently and successfully. Methods:Thirty patients were prospectively randomized into two groups. One group underwent a minimally invasive technique using a laryngoscope; the other, open saphenous vein harvest. A modified bridging technique, in which tissue retraction and illumination is achieved with a sterilized laryngoscope, was used for minimally invasive harvesting. Smooth muscle contractile and endothelial functions were tested in vitro using an organ chamber. Morphology was examined with light microscopy. Results: There was no statistically significant difference in harvest times or length of the vein harvested by either of the abovementioned techniques. Total length of the incision in the minimally invasive group was significantly shorter than that in the open group. In follow-ups, no significant complications occured in either group. Pain and leg edema were significantly less in the minimally invasive group compared to those of the open group. There was no significant difference in response to acetylcholine and 80 mM KCl between veins taken with the laryngoscope compared to veins taken with the traditional open technique. Similarly, histological data was unable to show any significant damage to the vessel wall. Conclusions: Because the laryngoscopic saphenectomy does not harm the harvested graft, it can be applied, instead of other minimally invasive saphenous vein harvesting systems, with a zero cost, efficiently, successfully, and with satisfactory speed and significant reduction of postoperative leg pain and wound complications.