Silent information regulator 2-related enzyme 1 (SIRT1) is an aging-related protein activated with aging. Herein, we evaluated the role of SIRT1 in aging-related erectile dysfunction. The expression of SIRT1 was modulated in aged Sprague-Dawley rats following intragastric administration of resveratrol (Res; 5 mg kg^-1), niacinamide (NAM; 500 mg kg^-1) or Res (5 mg kg^-1) + tadalafil (Tad; phosphodiesterase-5 [PDE5] inhibitor; 5 mg kg^-1) for 8 weeks. Then, we determined erectile function by the ratio of intracavernosal pressure (ICP)/mean systemic arterial pressure (MAP). Cavernosal tissues were extracted to evaluate histological changes, cell apoptosis, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP), the superoxide dismutase (SOD)/3,4-methylenedioxyamphetamine (MDA) level, and the expression of SIRT1, p53, and forkhead box O3 (FOXO3a) using immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL), enzyme-linked immunosorbent assays, and western blot analysis. Compared with the control, Res treatment significantly improved erectile function, reflected by an increased content of smooth muscle and endothelium, NO/cGMP and SOD activity, and reduced cell apoptosis and MDA levels. The effect of Res was improved by adding Tad. In addition, the protein expression of SIRT1 was increased in the Res group, accompanied by decreased p53 and FOXO3a levels. In addition, inhibition of SIRT1 by NAM treatment resulted in adverse results compared with Res treatment. SIRT1 activation ameliorated aging-related erectile dysfunction, supporting the potential of SIRT1 as a target for erectile dysfunction treatment.
Animals ; Male ; Rats ; Cyclic GMP/metabolism* ; Erectile Dysfunction/metabolism* ; Nitric Oxide/metabolism* ; Penile Erection ; Penis/pathology* ; Phosphodiesterase 5 Inhibitors/pharmacology* ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Superoxide Dismutase/metabolism* ; Tumor Suppressor Protein p53/metabolism*

OBJECTIVETo investigate the effect and potential mechanisms of rutaecarpine (Rut) in a rat artery balloon-injury model.

METHODSThe intimal hyperplasia model was established by rubbing the endothelia with a balloon catheter in the common carotid artery (CCA) of rats. Fifty rats were randomly divided into five groups, ie. sham, model, Rut (25, 50 and 75 mg/kg) with 10 rats of each group. The rats were treated with or without Rut (25, 50, 75 mg/kg) by intragastric administration for 14 consecutive days following injury. The morphological changes of the intima were evaluated by hematoxylin-eosin staining. The expressions of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM) α-actin in the ateries were assayed by immunohistochemical staining. The mRNA expressions of c-myc, extracellular signal-regulated kinase 2 (ERK2), MAPK phosphatase-1 (MKP-1) and endothelial nitric oxide synthase (eNOS) were determined by real-time reverse transcription-polymerase chain reaction. The protein expressions of MKP-1 and phosphorylated ERK2 (p-ERK2) were examined by Western blotting. The plasma contents of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) were also determined.

RESULTSCompared with the model group, Rut treatment significantly decreased intimal thickening and ameliorated endothelial injury (P<0.05 or P<0.01). The positive expression rate of PCNA was decreased, while the expression rate of SM α-actin obviously increased in the vascular wall after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01). Furthermore, the mRNA expressions of c-myc, ERK2 and PCNA were downregulated while the expressions of eNOS and MKP-1 were upregulated (P<0.05 or P<0.01). The protein expressions of MKP-1 and the phosphorylation of ERK2 were upregulated and downregulated after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01), respectively. In addition, Rut dramatically reversed balloon injury-induced decrease of NO and cGMP in the plasma (P<0.05 or P<0.01).

CONCLUSIONRut could inhibit the balloon injury-induced carotid intimal hyperplasia in rats, possibly mediated by promotion of NO production and inhibiting ERK2 signal transduction pathways.

Actins ; metabolism ; Animals ; Carotid Arteries ; drug effects ; metabolism ; pathology ; Carotid Artery Injuries ; drug therapy ; genetics ; pathology ; Cyclic GMP ; blood ; Disease Models, Animal ; Gene Expression Regulation ; drug effects ; Hyperplasia ; Indole Alkaloids ; pharmacology ; therapeutic use ; Male ; Nitric Oxide ; blood ; Phosphorylation ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Quinazolines ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Tunica Intima ; drug effects ; pathology

OBJECTIVETo investigate whether the methanol extract of Berberis amurensis Rupr. (BAR) augments penile erection using in vitro and in vivo experiments.

METHODSThe ex vivo study used corpus cavernosum strips prepared from adult male New Zealand White rabbits. In in vivo studies for intracavernous pressure (ICP), blood pressure, mean arterial pressure (MAP), and increase of peak ICP were continuously monitored during electrical stimulation of Sprague-Dawley rats.

RESULTSPreconstricted with phenylephrine (PE) in isolated endotheliumintact rabbit corus cavernosum, BAR relaxed penile smooth muscle in a dose-dependent manner, which was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one, a soluble guanylyl cclase inhibitor. BAR significantly relaxed penile smooth muscles dose-dependently in ex vivo, and this was inhibited by pretreatment with L-NAME H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one. BAR-induced relaxation was significantly attenuated by pretreatment with tetraethylammonium (TEA, P<0.01), a nonselective K channel blocker, 4-aminopyridine (4-AP, P<0.01), a voltage-dependent K channel blocker, and charybdotoxin (P<0.01), a large and intermediate conductance Ca sensitive-K channel blocker, respectively. BAR induced an increase in peak ICP, ICP/MAP ratio and area under the curve dose dependently.

CONCLUSIONBAR augments penile erection via the nitric oxide/cyclic guanosine monophosphate system and Ca sensitive-K (BK and IK) channels in the corpus cavernosum.

Animals ; Area Under Curve ; Berberis ; chemistry ; Blood Pressure ; drug effects ; Cyclic GMP ; metabolism ; Epoprostenol ; pharmacology ; In Vitro Techniques ; Indomethacin ; pharmacology ; Male ; Models, Biological ; Muscle Relaxation ; drug effects ; Muscle, Smooth ; drug effects ; physiology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Penile Erection ; drug effects ; Phenylephrine ; pharmacology ; Plant Extracts ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Pressure ; Rabbits

The aim of this study was to explore the mechanism of the nervous system lesions induced by formaldehyde (FA). Male Balb/c mice were exposed to gaseous formaldehyde for 7 days (8 h/d) with three different concentrations (0, 0.5 and 3.0 mg/m(3)). A group of animals injected with the nitric oxide synthase inhibitor L-NMMA (0.01 mL/g) was also set and exposed to 3.0 mg/m(3) FA. The concentrations of cAMP, cGMP, NO and the activity of NOS in cerebral cortex, hippocampus and brain stem were determined by corresponding assay kits. The results showed that, compared with the control (0 mg/m(3) FA) group, the cAMP contents in cerebral cortex and brain stem were significantly increased in 0.5 mg/m(3) FA group (P < 0.05), but decreased in 3.0 mg/m(3) FA group (P < 0.05); The concentration of cAMP in hippocampus was significantly decreased in 3.0 mg/m(3) FA group (P < 0.05). In comparison with the control group, L-NMMA group showed unchanged cAMP contents and NOS activities in different brain regions, but showed increased cGMP contents in hippocampus and NO contents in cerebral cortex (P < 0.05). In addition, compared with 3.0 mg/m(3) FA group, L-NMMA group showed increased contents of cAMP and reduced NOS activities in different brain regions, as well as significantly decreased cGMP contents in cerebral cortex and brain stem and NO content in brain stem. These results suggest that the toxicity of FA on mouse nervous system is related to NO/cGMP and cAMP signaling pathways.
Animals ; Brain Stem ; chemistry ; drug effects ; Cerebral Cortex ; chemistry ; drug effects ; Cyclic AMP ; chemistry ; Cyclic GMP ; chemistry ; Formaldehyde ; toxicity ; Hippocampus ; chemistry ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; chemistry ; Nitric Oxide Synthase ; antagonists & inhibitors ; omega-N-Methylarginine ; pharmacology

The purpose of the present study is to investigate the effect of isoliquiritigenin (ISL) on the cerebral basilar artery in spontaneously hypertensive rats (SHR). The change of SHR systolic pressure was measured by tail artery pressure measurement instrument before and after ISL intervention. After perfusion with 1 × 10(-5) mol/L phenylephrine (PE), 1 × 10(-5) mol/L PE + 1 × 10(-4) mol/L ISL and 1 × 10(-5) mol/L PE, the diameter of the cerebral basilar artery separated from SHR was measured by pressure myograph. The current of large-conductance calcium-activated potassium (BKCa) channel of SHR single vascular smooth muscle cell (VSMC) was recorded by whole-cell patch-clamp technique and the cGMP levels of basilar artery was evaluated by ELISA. The results showed that 1) after intervention with ISL for 14 days, the systolic pressure of SHR was decreased from (218.3 ± 1.6) mmHg to (119.2 ± 1.9) mmHg (P < 0.01), but there was no difference in systolic pressure between ISL-treated SHR and Wistar-Kyoto (WKY) rat; 2) 1 × 10(-4) mol/L ISL relaxed the SHR cerebral basilar artery (P < 0.01); 3) ISL significantly increased the outward current density of VSMC from SHR cerebral basilar artery (P < 0.01, n = 6), and the effect could be reversed by 1 × 10(-3) mol/L TEA (a BKCa channel inhibitor), but 3 × 10(-4) mol/L 4-AP (a Kv channel inhibitor) had no effect on the enhanced current density induced by ISL in VSMC; 4) 1 × 10(-5) mol/L Methylene blue (a sGC inhibitor) significantly inhibited the ISL-enhanced current density in VSMC (P < 0.05, n = 6); 5) ISL significantly increased the cGMP level of SHR basilar artery (P < 0.05, n = 6). The results suggest that the role of the ISL in relaxing the SHR cerebral basilar artery may be related to its effect in enhancing BKCa current by increasing the levels of cGMP in the VSMC.
Animals ; Basilar Artery ; drug effects ; Blood Pressure ; Cerebral Arteries ; drug effects ; Chalcones ; pharmacology ; Cyclic GMP ; physiology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; physiology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Systole

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 μmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 μmol·L(-1)), significantly blocked SCU (10-1 000 μmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 μmol·L(-1)), did not significantly change the effects of SCU (10-1 000 μmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 μmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 μmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.
Animals ; Apigenin ; pharmacology ; Cell Adhesion Molecules ; drug effects ; Cell Hypoxia ; Coronary Vessels ; drug effects ; Cyclic GMP ; analogs & derivatives ; metabolism ; pharmacology ; Cyclic GMP-Dependent Protein Kinases ; Glucuronates ; pharmacology ; Microfilament Proteins ; drug effects ; NG-Nitroarginine Methyl Ester ; metabolism ; pharmacology ; Phosphoproteins ; drug effects ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; physiopathology ; Signal Transduction ; drug effects ; Thionucleotides ; metabolism ; pharmacology ; Vasodilation ; drug effects ; physiology

Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to vascular injury such as angioplasty. Protein kinase G (PKG) has an important role in the process of VSMC phenotype switching. In this study, we examined whether rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, could modulate VSMC phenotype through the PKG pathway to reduce neointimal hyperplasia after angioplasty. In vitro experiments showed that rosiglitazone inhibited the phenotype change of VSMCs from a contractile to a synthetic form. The platelet-derived growth factor (PDGF)-induced reduction of PKG level was reversed by rosiglitazone treatment, resulting in increased PKG activity. This increased activity of PKG resulted in phosphorylation of vasodilator-stimulated phosphoprotein at serine 239, leading to inhibited proliferation of VSMCs. Interestingly, rosiglitazone did not change the level of nitric oxide (NO) or cyclic guanosine monophosphate (cGMP), which are upstream of PKG, suggesting that rosiglitazone influences PKG itself. Chromatin immunoprecipitation assays for the PKG promoter showed that the activation of PKG by rosiglitazone was mediated by the increased binding of Sp1 on the promoter region of PKG. In vivo experiments showed that rosiglitazone significantly inhibited neointimal formation after balloon injury. Immunohistochemistry staining for calponin and thrombospondin showed that this effect of rosiglitazone was mediated by modulating VSMC phenotype. Our findings demonstrate that rosiglitazone is a potent modulator of VSMC phenotype, which is regulated by PKG. This activation of PKG by rosiglitazone results in reduced neointimal hyperplasia after angioplasty. These results provide important mechanistic insight into the cardiovascular-protective effect of PPARgamma.
Animals ; Aorta/injuries/metabolism/*pathology ; Calcium-Binding Proteins/genetics/metabolism ; Cell Proliferation ; Cyclic GMP/metabolism ; Cyclic GMP-Dependent Protein Kinases/genetics/*metabolism ; Hyperplasia/metabolism ; Microfilament Proteins/genetics/metabolism ; Muscle, Smooth, Vascular/metabolism/pathology ; Myocytes, Smooth Muscle/drug effects/*metabolism ; Nitric Oxide/metabolism ; PPAR gamma/agonists/*metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Sp1 Transcription Factor/metabolism ; Thiazolidinediones/pharmacology ; Thrombospondins/genetics/metabolism ; Tunica Intima/metabolism/*pathology ; Vascular System Injuries/*metabolism/pathology

OBJECTIVETo examine whether calcineurin/NFAT signaling pathway mediates endothelin-1 (ET-1)-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) by regulating phosphodiesterase-5 (PDE5) and the effect of the selective calcineurin inhibitor cyclosporine A and PDE5 inhibitor sildenafil on ET-1-induced PASMC proliferation.

METHODSPASMCs were treated with ET-1 to stimulate their proliferation with or without prior treatment of the cells with CsA or sildenafil. Calcineurin activity in the cells was measured using a calcineurin activity assay kit, PDE5 expression examined using immunoblotting, and cGMP level detected using a cGMP direct immunoassay kit. PASMC proliferation following the treatments was determined using [(3)H]thymidine incorporation assay.

RESULTSET-1 caused a 2.05-fold increase in the cellular calcineurin activity, a 1.80-fold increase in PDE5 expression, and a 3.20-fold increase in the DNA synthesis rate, and reduced the cGMP level by 67%. Pretreatment of the cells with Cyclosporine blocked the effects of ET-1, and PDE5 inhibition by sildenafil pretreatment also abolished ET-1-induced reduction of cGMP level in the cells. Both Cyclosporine and sildenafil suppressed ET-1-stimulated PASMC proliferation.

CONCLUSIONActivation of calcineurin/NFAT signaling pathway mediates ET-1-induced PASMC proliferation by stimulating PDE5 expression, which further degrades cGMP. Both Cyclosporine and sildenafil can suppress ET-1-stimulated PASMC proliferation in vitro.

Animals ; Calcineurin ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; metabolism ; Cyclosporine ; DNA ; biosynthesis ; Endothelin-1 ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; enzymology ; NFATC Transcription Factors ; metabolism ; Piperazines ; Pulmonary Artery ; cytology ; Purines ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Sildenafil Citrate ; Sulfones

PURPOSE: The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. METHODS: Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment. RESULTS: After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015). CONCLUSIONS: L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.
Amiloride/*pharmacology ; Analysis of Variance ; Animals ; Arginine/toxicity ; Cell Death/drug effects ; Cells, Cultured ; Cyclic GMP/pharmacology ; Enzyme-Linked Immunosorbent Assay ; Mice ; Retina/cytology/*drug effects ; Tissue Plasminogen Activator/*toxicity

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca2+ channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca2+ current (ICa,L) in a concentration dependent manner with a IC50 of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 microM). DNP did not affect the voltage dependence of activation and inactivation of ICa,L. The alpha1c subunit of cardiac L-type Ca2+ channel proteins was phosphorylated by the treatment of DNP (1 microM), which was completely blocked by KT5823 (1 microM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 +/- 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 microM). These results clearly indicate that DNP inhibits the L-type Ca2+ channel activity by phosphorylating the Ca2+ channel protein via PKG activation.
Action Potentials/drug effects ; Animals ; Biological Transport/drug effects ; Calcium/metabolism ; Calcium Channels, L-Type/*metabolism ; Carbazoles/pharmacology ; Cells, Cultured ; Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors ; Elapid Venoms/*metabolism/pharmacology ; Enzyme Activation ; Heart ; Heart Ventricles/drug effects ; Myocytes, Cardiac/drug effects ; Patch-Clamp Techniques ; Peptides/*metabolism/pharmacology ; Phosphorylation/drug effects ; Rabbits