8-bromocyclic-gmp and acetovanillone

INTRODUCTION.: Prevalence of erectile dysfunction (ED) increases progressively with aging, but the ED pathophysiology at its early stages is still poorly investigated. AIM.: This study aimed to evaluate the functional and molecular alterations of erectile function at middle age, focusing on the contribution of oxidative stress in erectile tissue for the ED. METHODS.: Young (3.5-month) and middle-aged (10-month) male Wistar rats were used. Rat corpus cavernosum (RCC) was dissected free and mounted in 10-mL organ baths containing Krebs solution. Intracavernosal pressure (ICP) in anesthetized rats was evaluated. MAIN OUTCOME MEASURES.: Concentration-response curves to endothelium-dependent and endothelium-independent agents, as well as to electrical field stimulation (EFS), were obtained in RCC strips. Measurement of cyclic guanosine monophosphate (cGMP) and expressions of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), gp91(phox) and superoxide dismutase-1 (SOD-1) expressions in RCC were evaluated. RESULTS.: ICP was significantly reduced in middle-aged compared with young rats. RCC relaxations to acetylcholine (10(-8) to 10(-2)  M), sodium nitroprusside (10(-8) to 10(-2)  M), sildenafil (10(-9) to 10(-5)  M), BAY 41-2272 (10(-9) to 10(-5)  M), and EFS (4-32 Hz) were decreased in middle-aged group, which were nearly normalized by apocynin (NADPH oxidase inhibitor; 10(-4)  M) or SOD (75 U/mL). Prolonged treatment with apocynin (85 mg/rat/day, 4 weeks) also restored the impaired relaxations in middle-aged rats. Relaxations to 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP; 10(-8) to 3 × 10(-4)  M) remained unchanged between groups. Basal and stimulated cGMP production were lower in middle-aged group, an effect fully restored by apocynin and SOD. Protein expression of nNOS and phosphorylated eNOS (p-eNOS) (Ser-1177) reduced, whereas gp(91phox) mRNA expression increased in RCC from middle-aged rats. CONCLUSIONS.: ED in middle-aged rats is associated with decreased NO bioavailability in erectile tissue due to upregulation of NADPH oxidase subunit gp91(phox) and downregulation of nNOS/p-eNOS. Antioxidant therapies may be a good pharmacological approach to prevent ED at its early stages.

Topics: Acetophenones; Acetylcholine; Aging; Animals; Blood Pressure; Cyclic GMP; Down-Regulation; Electric Stimulation; Endothelium, Vascular; Enzyme Inhibitors; Erectile Dysfunction; Free Radical Scavengers; Male; Membrane Glycoproteins; Muscle Relaxation; NADPH Oxidase 2; NADPH Oxidases; Nitric Oxide Synthase; Nitroprusside; Penile Erection; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Pyrazoles; Pyridines; Rats; RNA, Messenger; Sildenafil Citrate; Sulfones; Superoxide Dismutase; Superoxide Dismutase-1; Up-Regulation; Vasodilator Agents

The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-alpha-activated endothelial cells by inducing mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). The underlying mechanisms are as yet unknown. We aimed to elucidate the signaling pathways leading to an induction of MKP-1 by ANP in primary human endothelial cells. By using antioxidants, generation of reactive oxygen species (ROS) was shown to be crucially involved in MKP-1 upregulation. ANP was found to increase ROS formation in cultured cells as well as in the endothelium of intact rat lung vessels. We applied NAD(P)H oxidase (Nox) inhibitors (apocynin and gp91ds-tat) and revealed this enzyme complex to be crucial for superoxide generation and MKP-1 expression. Moreover, by performing Nox2/4 antisense experiments, we identified Nox2 as the critically involved Nox homologue. Pull-down assays and confocal microscopy showed that ANP activates the small Rho-GTPase Rac1. Transfection of a dominant-negative (RacN17) and constitutively active Rac1 mutant (RacV12) indicated that ANP-induced superoxide generation and MKP-1 expression are mediated via Rac1 activation. ANP-evoked production of superoxide was found to activate c-Jun N-terminal kinase (JNK). Using specific inhibitors, we linked ANP-induced JNK activation to MKP-1 expression and excluded an involvement of protein kinase C, extracellular signal-regulated kinase, and p38 MAPK. MKP-1 induction was shown to depend on activation of the transcription factor activator protein-1 (AP-1) by using electrophoretic mobility shift assay and AP-1 decoys. In summary, our work provides insights into the mechanisms by which ANP induces MKP-1 and shows that ANP is a novel endogenous activator of endothelial Rac1 and Nox/Nox2.

Topics: Acetophenones; Animals; Atrial Natriuretic Factor; Capillaries; Cell Cycle Proteins; Cells, Cultured; Cyclic GMP; Cycloheximide; DNA, Antisense; Dual Specificity Phosphatase 1; Endothelial Cells; Endothelium, Vascular; Enzyme Induction; Glycoproteins; Guanylate Cyclase; Humans; Immediate-Early Proteins; JNK Mitogen-Activated Protein Kinases; Lung; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Glycoproteins; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; NADPH Oxidase 1; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidase 5; NADPH Oxidases; Oligonucleotides, Antisense; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Atrial Natriuretic Factor; Recombinant Fusion Proteins; RNA, Messenger; Transcription Factor AP-1; Transfection; Umbilical Veins