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The pathogenesis of diabetic erectile dysfunction (ED) includes neuropathy, but the molecular basis for neurogenic ED is incompletely understood. The RhoA/ROCK pathway has been implicated in diabetic neuropathy and in ED, but its role in diabetic neurogenic ED is not known.The aim of this study was to determine whether hydroxyl fasudil, a ROCK inhibitor, affects diabetic neuropathy-related ED.Type 1 diabetes mellitus was induced in male rats by streptozotocin (75 mg/kg, intraperitoneally). After 8 weeks, diabetic rats were administered hydroxyl fasudil, a selective ROCK inhibitor (10 mg/kg/day, intraperitoneally) or vehicle, for 4 weeks. Age-matched control, nondiabetic, rats were treated intraperitoneally for 4 weeks with saline. At week 12, after a 2 day washout, neuro-stimulated erectile function was evaluated. Major pelvic ganglia (MPG) were collected for Western blot analysis of RhoA, ROCK-1, ROCK-2, phospho (P)-AKT (Ser(473) ), and P-phosphatase and tensin homolog (P-PTEN) (Ser(380) /Thr(382/383) ).Effect of ROCK inhibitor hydroxyl fasudil on erectile function and ROCK/P-AKT/P-PTEN pathway in the MPG of diabetic rats.Erectile response was significantly (P 0.05) reduced in diabetic rats compared with nondiabetic rats and was preserved (P 0.05) in diabetic rats treated with hydroxyl fasudil. In diabetic rats, RhoA and ROCK-2 protein expressions in MPG were increased (P 0.05) and remained increased in hydroxyl fasudil-treated rats. P-AKT (Ser(473) ) expression was decreased (P 0.05), whereas P-PTEN (Ser(380) /Thr(382/383) ) expression was increased (P 0.05) in MPG of diabetic rats compared with nondiabetic rats, and both were reversed (P 0.05) in diabetic rats treated with hydroxyl fasudil.Improved erectile function and restored P-AKT and P-PTEN in the MPG with hydroxyl fasudil treatment suggest the role of Rho signaling via PTEN/AKT pathway in neurogenic diabetic ED.

We evaluated the therapeutic potential of a sustained nitric oxide (NO)-releasing compound to correct the molecular hallmarks and pathophysiology of priapism, an important but poorly characterized erectile disorder. 1,5-Bis-(dihexyl-N-nitrosoamino)-2,4-dinitrobenzene (C6′) and an inactive form of the compound [1,5-bis-(dihexylamino)-2,4-dinitrobenzene (C6)] were tested in neuronal cell cultures and penile lysates for NO release (Griess assay) and biological activity (cGMP production). The effect of local depot C6′ or C6 was evaluated in mice with a priapic phenotype due to double neuronal and endothelial NO synthase deletion (dNOS−/−) or human sickle hemoglobin transgenic expression (Sickle). Changes in NO signaling molecules and reactive oxygen species (ROS) surrogates were assessed by Western blot. The physiological response after C6′ treatment was assessed using an established model of electrically stimulated penile erection. C6′ generated NO, increased cGMP, and dose dependently increased NO metabolites. C6′ treatment reversed abnormalities in key penile erection signaling molecules, including phosphodiesterase type 5, phosphorylated endothelial nitric oxide synthase, and phosphorylated vasodilator-stimulated phosphoprotein. In Sickle mice, C6′ also attenuated the increased ROS markers gp91phox, 4-hydroxynonenal, and 3-nitrotyrosine. Finally, C6′ corrected the excessive priapic erection response of dNOS−/− mice. Exogenous sustained NO release from C6′ corrects pathological erectile signaling in mouse models of priapism and suggests novel approaches to human therapy.—Lagoda, G., Sezen, S. F., Hurt, K. J., Cabrini, M. R., Mohanty, D. K., Burnett, A. L. Sustained nitric oxide (NO)-releasing compound reverses dysregulated NO signal transduction in priapism.

Priapism is a vasculopathy that occurs in approximately 40% of patients with sickle cell disease. Mouse models suggest that dysregulated nitric oxide synthase and RhoA/ROCK signaling as well as increased oxidative stress may contribute to the mechanisms of sickle cell disease associated priapism. We examined changes in the protein expression of nitric oxide synthase and ROCK signaling pathways, and a source of oxidative stress, NADPH oxidase, in penile erectile tissue from patients with a priapism history etiologically related and unrelated to sickle cell disease.Human penile erectile tissue was obtained from 5 patients with sickle cell disease associated priapism and from 6 with priapism of other etiologies during nonemergent penile prosthesis surgery for erectile dysfunction or priapism management and urethroplasty. Tissue was also obtained from 5 control patients without a priapism history during penectomy for penile cancer. Samples were collected, immediately placed in cold buffer and then frozen in liquid nitrogen. The expression of phosphodiesterase 5, endothelial nitric oxide synthase, neuronal nitric oxide synthase, inducible nitric oxide synthase, RhoA, ROCK1, ROCK2, p47(phox), p67(phox), gp91(phox) and β-actin were determined by Western blot analysis. Nitric oxide was measured using the Griess reaction.In the sickle cell disease group phosphodiesterase 5 (p0.05), endothelial nitric oxide synthase (p0.01) and RhoA (p0.01) expression was significantly decreased, while gp91(phox) expression (p0.05) was significantly increased compared to control values. In the nonsickle cell disease group endothelial nitric oxide synthase, ROCK1 and p47(phox) expression (each p0.05) was significantly decreased compared to control values. Total nitric oxide levels were not significantly different between the study groups.Mechanisms of sickle cell disease associated priapism in the human penis may involve dysfunctional nitric oxide synthase and ROCK signaling, and increased oxidative stress associated with NADPH oxidase mediated signaling.

Nitric oxide (NO) generated by neuronal NO synthase (nNOS) initiates penile erection, but has not been thought to participate in the sustained erection required for normal sexual performance. We now show that cAMP-dependent phosphorylation of nNOS mediates erectile physiology, including sustained erection. nNOS is phosphorylated by cAMP-dependent protein kinase (PKA) at serine(S)1412. Electrical stimulation of the penile innervation increases S1412 phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolin also activates nNOS phosphorylation. Sustained penile erection elicited by either intracavernous forskolin injection, or augmented by forskolin during cavernous nerve electrical stimulation, is prevented by the NOS inhibitor l -NAME or in nNOS-deleted mice. Thus, nNOS mediates both initiation and maintenance of penile erection, implying unique approaches for treating erectile dysfunction.

Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown.We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis.SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes.Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis.Relative to hemi mice, SCD increased (P0.05) protein expression of NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) , 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P0.05) the abnormalities in protein expressions of p47(phox) , gp91(phox) (but not p67(phox) ) and 4-HNE, but only slightly (P0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters.NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis.

Nitric oxide is the major neuronal mediator of penile erection but its role in erectile function status after cavernous nerve injury is uncertain. We determined the function of neuronal nitric oxide signaling in the pathobiology of erectile function recovery after partial cavernous nerve injury using genetic and pharmacological mouse experimental paradigms.Erectile function was evaluated in 5 to 7 wild-type and neuronal nitric oxide synthase-α knockout mice per group 1, 3 and 7 days after unilateral crush or sham injury, at day 7 in wild-type mice treated with the nitric oxide synthase inhibitor L-NAME (l-nitro arginine methyl ester) (Sigma-Aldrich®) at baseline and for 6 days after unilateral crush injury. Apoptosis in the penis was evaluated by Western blot analysis of p-Akt-S473, 3-nitrotyrosine and caspase-3 after bilateral crush injury.Intracavernous pressure was significantly decreased at 1, 3 and 7 days in wild-type mice but only at day 1 in knockout mice after unilateral crush injury compared with sham treatment values (p0.05). L-NAME treated wild-type mice had improved erectile function compared with the vehicle treated group at day 7 after unilateral crush injury (p0.05). In penes p-Akt-S473 was significantly decreased in vehicle treated (p0.05) but not in L-NAME treated wild-type mice. In penes 3-nitrotyrosine was significantly decreased in L-NAME treated wild-type and vehicle treated knockout mice (p0.05). Caspase-3 in penes was significantly increased in vehicle treated (p0.05) but not in L-NAME treated wild-type mice and vehicle treated knockout mice.Neuronal nitric oxide signaling regulates erectile function recovery early after partial cavernous nerve injury, exerting an inhibitory role via the induction of apoptotic change in penile tissue. Therapeutic strategies to improve erectile function recovery after radical prostatectomy may consider targeting pathogenic sites of nitric oxide neurobiology.

Introduction Immunophilin ligands such as FK506 (FK) preserve erectile function (EF) following cavernous nerve injury (CNI), although the precise mechanisms are unclear. We examined whether the thioredoxin (Trx) and glutathione (GSH) redox systems mediate this effect after CNI.

Introduction Immunophilin ligands provide potentially new alternatives for the treatment of erectile dysfunction (ED), which occurs after injury of the cavernous nerves (CNs). Aim To review and update current knowledge of the neurotrophic effects and likely mechanism of action of immunophilin proteins with emphasis on the FK506‐binding protein (FKBP) subfamily and the role of immunophilin ligands for the treatment of CN injury‐induced ED. Methods Review of available reports of studies investigating the effects and neurotrophic mechanisms of immunophilin ligands involved in erectile function recovery in rodent models of CN injury. Main Outcome Measures Erection parameters and molecular correlations associated with CN injury and functional recovery. Results Treatment with prototype immunosuppressive immunophilin ligands FK506 (FK) and rapamycin (Rapa) improve erectile function in animal models of CN injury. Similarly, non‐immunosuppressive analogs such as GPI‐1046 and FK1706 are effective in recovery of erections after CN injury. Neuronal nitric oxide may influence the erection recovery effects of immunophilin ligands after CN injury. FKBPs 38 and 65 expression changes in the penis and its innervation coincide with the neurotrophic effects of immunophilin ligands. Antioxidative actions of immunophilin ligands contribute to their neurotrophic effects. Immunophilins are localized to nerves coursing in human prostate and penile tissue. Conclusions The findings support the hypothesis that immunophilin ligands, working through specific receptor mechanisms that are specific to injured CN, are potentially useful to sustain erectile function in men following radical prostatectomy. Sezen SF, Lagoda G, and Burnett AL. Role of immunophilins in recovery of erectile function after cavernous nerve injury.

OBJECTIVE To determine if FK506-binding proteins (FKBPs) are localized to the autonomic nerve supply of the human penis because FK506 (an immunosuppressant drug) has been linked to enhanced nerve regeneration after nerve injury and neurodegenerative diseases by binding to FKBPs, a select group of immunophilins. MATERIALS AND METHODS Human lower genitourinary tract specimens were obtained and embedded in paraffin wax. The tissue was sectioned (10 µm) and processed for immunohistochemistry using antibodies for FKBPs 12, 38, 52, 65, 135 and neuronal nitric oxide synthase (nNOS). To confirm specificity of the antibody, we processed some tissue in the absence of primary antibody, with mouse or rabbit IgG, and with a blocking peptide for FKBP12. RESULTS In the pelvis, immunoreactivity for all the FKBPs and nNOS was localized to the periprostatic ganglia although FKBP12 was the only FKBP localized to nerve bundles in this location. In penile tissue, immunoreactivity for all five FKBPs and nNOS was localized to nerves, although immunoreactivity for FKBP38 was minimal. The FKBPs were also evident in epithelial, endothelial and smooth muscle cells of the prostate and penis. Negative controls did not produce staining. CONCLUSIONS Identification and localization of immunophilins to nerves coursing in prostate and penile tissue suggest a likely molecular basis to apply immunophilin ligand therapy to protect or regenerate cavernosal nerves. Our findings support the hypothesis that immunosuppressant drugs such as FK506, working via specific receptor mechanisms, are potentially useful to sustain erectile function in men after radical prostatectomy.

Conflicting information exists regarding the long-term efficacy of phosphodiesterase-5 (PDE5) inhibitor therapy for erectile dysfunction, particularly in regard to whether therapeutic resistance occurs. We investigated the erectile response, and cavernous PDE5 expression and activity after continuous long-term administration of sildenafil at verified therapeutic plasma concentrations, applying an in vivo rat model of age related erectile dysfunction.Male Fisher 344 young (4 months old) and aged (19 months old) rats (National Institute of Aging, Bethesda, Maryland) were injected with sildenafil mesylate (20 mg/kg) or saline subcutaneously every 8 hours for 3 weeks. After a 10 to 18-hour washout period electrical stimulation of the cavernous nerve was performed to assess penile erection. Penes were excised to measure PDE5 protein expression and activity, and blood was collected for sildenafil measurement. Responses were compared with those determined 30 minutes after a single sildenafil injection.Chronic sildenafil treatment increased the detumescence phase in young and aged rats (p0.05), although aged rats showed a greater increase than young rats. Baseline cavernous PDE5 expression and activity were greater in aged vs young rats (p0.05). After chronic sildenafil treatment cavernous PDE5 expression was increased in young (p0.05) but not in aged rats. Chronic and acute sildenafil treatment similarly inhibited PDE5 activity in the penis of young and aged rats (p0.05), coincident with its free plasma concentrations equivalent to clinically therapeutic ranges.Pharmacological PDE5 inhibitor therapy with sildenafil chronically does not result in treatment resistance. Rather, therapeutic efficacy is maintained and apparently more pronounced with erectile impairment than with normal erectile ability.

Nimesulide (4-nitro-2-phenoxymethanesulfonanilide) is an atypical NSAID lacking a carboxylic acid moiety. It has a good gastric tolerability due to selective inhibition of COX-2. The study objectives in the present work were to characterize the metabolism of nimesulide in rat plasma at certain time intervals. In vitro studies were also carried out to examine if nitroreduction takes place in vitro using rat hepatic subcellular fractions (microsomal and S9 fraction) besides aromatic hydroxylation. This communication describes detection and characterization of nimesulide metabolites isolated from plasma and hepatic subcellular post-incubates by the use of HPLC-UV/diode array and LC-MS/MS. Hydroxynimesulide was the major metabolite both in vivo and in vitro whereas nitroreduction was observed only in vitro with subcellular fractions under anaerobic conditions.

L’indagine conoscitiva realizzata in una area di 12,5 ettari, sita nel comune di Carpiano, ha permesso di ottenere uno schedario molto ampio di dati chimici e fisici quali metalli pesanti, metalloidi, macroelementi, radionuclidi, sostanza organica (carbonio totale e carbonio organico), diossine, furani, DDT, PCBs, pesticidi orgaclorurati, erbicidi, pH e tessitura. I parametri biologici hanno insistito sugli aspetti connessi alla Biodiversità e alla Bioindicazione come ad esempio i batteri e i funghi, l’utilizzo dei biosensori e degli artropodi, l’analisi dell’attività enzimatica e i lombrichi sino alla coltivazione di piantine di trifoglio per valutare eventuali variazioni del patrimonio genetico (DNA) causate dai contaminanti presenti nel suolo. Le informazioni, nel loro concetto olistico, hanno permesso di osservare uno stato di compromissione del suolo e della stessa vita degli organismi vegetali e animali che sono presenti nel suolo. Tale malessere del suolo è presente a differenti livelli di intensità e copre buona parte dell’area oggetto di indagine.

To evaluate whether FK506 and other immunophilin ligands may have potential therapeutic efficacy for erectile function preservation after penile nerve injury, we demonstrated localizations of the immunophilin FK506 binding protein 12 (FKBP 12) in intact and injured rat penile nerves and correlated these findings with localizations of neuronal nitric oxide synthase (nNOS), which neuronally forms nitric oxide for mediation of penile erection, in response to systemically administered FK506. Adult male Sprague-Dawley rats were subjected to unilateral right cavernous nerve forceps crush injury and administered FK506 (1 mg/kg i.p.) or saline at the same time and daily up to 7 days. At 1, 3 and 7 days after injury, bilateral cavernous nerves and major pelvic ganglia were collected for nNOS immunohistochemistry, FKBP 12 immunohistochemistry, and FKBP 12 in situ hybridisation. Protein expressions of nNOS and FKBP 12 were observed in major pelvic ganglion, cavernous nerve and nerve terminals within the rat penis as well as mRNA expression of FKBP 12 observed in the rat major pelvic ganglion neuronal cell bodies to a minimal extent at baseline conditions. After cavernous nerve injury, nNOS immunoreactivity was observed to be slightly diminished in ipsilateral penile nerve structures at only one day following injury while both FKBP 12 protein and mRNA expressions were observed to be increased at each interval of study. FK506 treatment did not affect staining of intact or injured nerves. Our demonstration that FKBP 12 is localized to penile innervation in the rat and becomes upregulated following cavernous nerve crush injury, independent of FK506 treatment, suggests that this immunophilin mediates a neurotrophic mechanism. Whether FK506 affords neuroprotection that preserves penile erection through FKBP 12 upregulation is unclear.

Objective To characterize transforming growth factor beta 1 (TGFβ1) and related signaling pathway proteins in a large cohort of human penile tissue (HPT) samples. Methods HPT was collected from patients undergoing penile prosthesis implantation for erectile dysfunction (ED) and divided into the following 2 groups: postradical prostatectomy ED (RP-ED; n = 57) and organic ED (O-ED; n = 30). HPT from patients undergoing partial penectomy without ED was used as controls (CON; n = 6). Western blot analysis was performed to investigate the protein expressions of TGFβ1, thrombospondin 1 (TSP1; an activator of TGFβ1), fibronectin (an extracellular matrix glycoprotein induced by TGFβ1), and a family of transcriptional factors activated by TGFβ1 (Smad2, phospho-Smad2-serine-465/467 [pSmad2], Smad3, phospho-Smad3-serine-423/425 [pSmad3]). Results Expressions of TGFβ1 and TSP1 were significantly higher in RP-ED (P

The dual-scaled hydrid scaffold fabrication technology based on combination of 3D printing (fused deposition modeling) and electrospining have been recently introduced. We report here the design, fabrication, mechanical testing, in vitro and in vivo biocompatibility testing of novel auricular implants for treatment microtia fabricated by dual scaled hydbrid scaffold fabrication technology.

Nociceptin (orphanin FQ) is a novel peptide isolated from brain tissue that has an amino acid sequence most similar to that of the endogenous opioid peptide dynorphin A. Aside from this similarity, the association of nociceptin to the endogenous opioid peptide systems and the functional importance of this new peptide in vivo are not completely known. Here we report that nociceptin is physiologically active in vivo and produces marked changes in the renal excretion of water and sodium. In conscious Sprague-Dawley rats, intravenous infusion of nociceptin produced a profound increase in urine flow rate and decrease in urinary sodium excretion. In further studies, intracerebroventricular (i.c.v.) injection of nociceptin into conscious rats produced a concurrent diuresis (dose-dependent) and antinatriuresis. The magnitude and pattern of the central nociceptin-induced water diuresis was similar to that produced by i.c.v. dynorphin A. Whereas i.c.v. pretreatment with the selective kappa-opioid receptor antagonist, nor-binaltorphimine, completely prevented the renal responses produced by dynorphin A, this antagonist did not alter the diuresis or antinatriuresis produced by central nociceptin. Thus, these results indicate that in conscious rats, nociceptin produces a selective water diuresis via a central nervous system mechanism independent of kappa-opioid receptors. Together, these observations suggest that endogenous nociceptin may be a novel peptide involved in the central control of water balance and ultimately in the regulation of arterial blood pressure. In the future, analogues of nociceptin may prove to be the first clinically useful water diuretics for patients with water-retaining diseases.

Microinjections of Leu-enkephalin into the dorsal vagal complex induced hypotension and bradycardia. Both naloxone, given at a dose conferring selectivity for mu receptors, and the delta antagonist ICI 154,129 prevented the cardiovascular effects of Leu-enkephalin. Naloxone was also found to decrease the gain of the baroreflex. These results suggest that Leu-enkephalin is involved in cardiovascular regulation through activation of delta-, and possibly mu-, opioid receptors in the dorsal vagal complex.

INTRODUCTION AND OBJECTIVES: Penile erection is mediated by nitric oxide (NO) that is formed by both neuronal NO synthase (nNOS) and endothelial NOS (eNOS) in the penis. We have previously shown that eNOS is phosphorylated by protein kinase (PK)B/Akt in response to increased blood flow in the penis, leading to activation of eNOS and maintenance of erection. This study investigates the regulation and mechanism of nNOS activation by phosphorylation using rodent models of penile erection. METHODS: Male Sprague-Dawley rats (300–375g) or 8–10 weeks old C57BL6/J (wild type, wt) and nNOS knockout (ko) mice were used. Penile erection was monitored as intracavernous pressure (ICP) via a needle inserted to right corpus cavernosum. For electricallyinduced penile erection studies, cavernous nerve (CN) or major pelvic ganglion (MPG) was stimulated at various stimulation parameters and durations. Inhibition of nNOS phosphorylation was tested after perigangliar injection of Akt inhibitors [LY294002 (LY, 50 uM) and wortmannin (WT, 1uM)] and PKA inhibitors [H-89 (30 uM) and PKA inhibitor peptide (PKA-I, 60uM)]. For pharmacologically-induced erections, papaverine (1.5mg), forskolin (FSK, 0.25-5ug) or deoxy-FSK (dFSK, 0.25-5ug) were injected intracavernosally (ic) or directly beneath the MPG. Some mice were pretreated with L-NAME (100mg/kg, ip) 30 min prior to FSK injection. MPG and penis from various experiments were collected and processed for phospho(P)-protein analysis with western blot or for immunohistochemistry. RESULTS: P-specific antibody to nNOS-S1412 (homologous to S1177 in eNOS) revealed a 5–10 fold increase in P-nNOS-S1412 after CN electrical stimulation (ES) in rat MPG (p 0.05). nNOS-S1412 phosphorylation was voltageand time-dependent and sustained for 3–5 min after ES. Papaverine, a direct vasorelaxant, injected ic stimulated P-eNOS-S1177 in the rat penis, but did not increase P-nNOSS1412. Perigangliar injection of FSK, an activator of PKA, increased P-nNOS-S1412, while Akt inhibitors, WT and LY, had no effect. Furthermore, ic injection of FSK dose-dependently increased ICP in wt mice (p 0.05) while d-FSK, an inactive analog of FSK, had no effect. Pretreatment with L-NAME (100mg/kg, ip) abolished the effect of FSK in wt. In nNOS-ko mice, the ICP increase after FSK injection was 30–50% of the wt response. CONCLUSIONS: Our data suggests a novel mechanism of nNOS activation which involves PKA-dependent phosphorylation and reveals differentially regulated pools of NOS activity mediating penile erection.

Hypercholesterolemia induces erectile dysfunction (ED) mostly by increasing oxidative stress and impairing endothelial function in the penis, but the mechanisms regulating reactive oxygen species (ROS) production in the penis are not understood.We evaluated whether hypercholesterolemia activates nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the penis, providing an initial source of ROS to induce endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction resulting in ED.Low-density-lipoprotein receptor (LDLR)-null mice were fed Western diet for 4 weeks to induce early-stage hyperlipidemia. Wild type (WT) mice fed regular chow served as controls. Mice received NAD(P)H oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Erectile function was assessed in response to cavernous nerve electrical stimulation. Markers of endothelial function (phospho [P]-vasodilator-stimulated-protein [VASP]-Ser-239), oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NAD[P]H oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), P-eNOS-Ser-1177, and eNOS were measured by Western blot in penes.The main outcome measures are the molecular mechanisms of ROS generation and endothelial dysfunction in hypercholesterolemia-induced ED.Erectile response was significantly (P0.05) reduced in hypercholesterolemic LDLR-null mice compared with WT mice. Relative to WT mice, hypercholesterolemia increased (P0.05) protein expressions of NAD(P)H oxidase subunits p67(phox) , p47(phox) and gp91(phox) , eNOS uncoupling, and 4-HNE-modified proteins, and reduced (P0.05) P-VASP-Ser-239 expression in the penis. Apocynin treatment of LDLR-null mice preserved (P0.05) maximal intracavernosal pressure, and reversed (P0.05) the abnormalities in protein expressions of gp67(phox) and gp47(phox) , 4-HNE, P-VASP-Ser-239, and eNOS uncoupling in the penis. Apocynin treatment of WT mice did not affect any of these parameters. Protein expressions of P-eNOS-Ser-1177 and total eNOS were unaffected by hypercholesterolemia.Activated NAD(P)H oxidase in the penis is an initial source of oxidative stress resulting in eNOS uncoupling, thus providing a mechanism of eNOS uncoupling and endothelial dysfunction in hypercholesterolemia-induced ED.

Hyperglycemia is a common defining feature in the development of endothelial dysfunction which plays a key role in the pathogenesis of both type 1 and type 2 diabetes. Caveolin-1 is the main structural component of caveolae which might be involved in the pathophysiology of macrovascular complications of diabetes. In this study we aimed to observe the effect of caveolin-1 on functional responses of aorta and corpus cavernosum in the streptozotocin and fructose-induced diabetes groups. Type 1 diabetes was induced by intraperitoneal administration of streptozotocin (60 mg/kg),. Type 2 diabetes by adding fructose in the rat's drinking water (10% (w/v)) for 8 weeks. For insulin treatment; rats were treated with insulin (6 U/kg) for 8 weeks. In Type I and Type II diabetic groups the contractile responses of corpus cavernosum strips to phenylephrine (EC(50):1.82 x 10(-5)M;1.47 x 10(-5)M, respectively)and relaxation responses to acetylcholine (EC(50):7.5 x 10(-5)M;4.48 x 10(-5)M, respectively)were significantly impaired. Contractile responses of aorticstrips to phenylephrine in diabetic groups were markedly decreased (EC(50):3.7.10(-7)M;2.61.10(-7)M respectively) and dose-dependent relaxation responses to acetylcholine were also attenuated (EC(50):3.23.10(-6)M; 2.0.10(-6)M respectively). Treatment with insulin improved the functional responses in the aorta and corpus cavernosum. Protein expression of caveolin-1 was increased in the aorta and corpus cavernosum of the diabetic groups, but this increase seen in the streptozotocin group was more significant than the fructose group. Our findings indicate that an attenuation of the functional responses in both diabetes groups were probably associated with an enhanced expression of caveolin-1, and therefore a decrease in the eNOS activity with a concomitant decrease in NO synthesis.

Introduction Immunophilin ligands function by binding to receptor proteins such as FK506 binding proteins (FKBPs). FKBPs are studied for their roles in neuroprotection. Aim Compare the effect of FK506 (FK) and rapamycin (RAP) on erectile function (EF) recovery and FKBP expressions in penis and major pelvic ganglion (MPG) after cavernous nerve (CN) injury. Methods Adult male rats were divided into four groups: sham surgery (CN exposure only) + vehicle; bilateral CN injury (BCNI; bilateral crush, 3 minutes with hemostat clamp) + vehicle; BCNI + FK (5 mg/kg/day, 5 days, sc); and BCNI + RAP (2 mg/kg/day, 5 days, sc). At both 24 hours (Day 1) or 1 week (Day 7) after BCNI, EF was assessed by intracavernosal pressure measurement and FKBPs 12, 38, 52, and 65 expressions were evaluated by Western blot analysis in collected penises and MPGs. Main Outcome Measures EF and change in protein expressions of FKBPs in the rat penis and MPG after BCNI with and without immunophilin ligand treatment. Results Both FK- and RAP-treated rats had preserved EF compared with vehicle-treated rats after BCNI. FKBPs changed variably following injury and treatment. In particular, in the penis at Day 1, FKBP 38 expression was decreased after BCNI and both FK and RAP attenuated this decrease. In MPG at Day 1, FKBP 38 expression was also decreased after BCNI and FK attenuated the decrease, while at Day 7, FKBP 38 expression was still decreased and RAP attenuated the decrease. Also, in the penis at Day 1, FKBP 65 expression decreased after BCNI and FK attenuated the decrease. In the MPG, FKBP 65 expression increased at both Days 1 and 7 with FK treatment. Conclusions Improved EF after BCNI, as shown with RAP, further suggests a role of immunophilin ligands as a protective therapy of CN injury associated erectile dysfunction. Our findings also suggest that select FKBPs, such as FKBP 38 and FKBP 65, may mediate these effects. Lagoda G, Sezen SF, and Burnett AL. FK506 and rapamycin neuroprotect erection and involve different immunophilins in a rat model of cavernous nerve injury. J Sex Med 2009;6:1914–1923.

Angiotensin II is a known mediator of smooth muscle vasoconstriction and fibrosis. It up-regulates thrombospondin-1, a major activator of latent transforming growth factor-beta. Transforming growth factor-beta induces vascular fibrosis via intracellular SMAD signaling pathways. We evaluated the effect of treatment with the angiotensin II type 1 receptor antagonist losartan on erectile function in the rat following bilateral cavernous nerve injury.A total of 36 adult male rats were divided equally into 6 groups, including group 1-sham surgery with cavernous nerve exposure only plus vehicle, group 2-sham surgery plus oral low dose losartan (10 mg/kg per day), group 3-sham surgery plus high dose losartan (40 mg/kg per day), group 4-bilateral cavernous nerve injury (3-minute crush using a hemostat clamp) plus vehicle, group 5-bilateral cavernous nerve injury plus low dose losartan and group 6-bilateral cavernous nerve injury plus high dose losartan. Seven days following surgery erectile function was measured by electrically stimulating the cavernous nerves and monitoring intracavernous pressure. Penile tissue was collected for Western blot analysis of fibronectin, transforming growth factor-beta, thrombospondin-1, alpha-actin, and phosphorylated and total SMAD2 and SMAD3 expression.Erectile function was significantly decreased after bilateral cavernous nerve injury compared with that after sham surgery (p0.01). Low and high dose losartan preserved erectile function after bilateral cavernous nerve injury compared to that in vehicle controls (p0.01 and0.05, respectively). Fibronectin, pSMAD2, pSMAD3, transforming growth factor-beta-1, thrombospondin-1 and alpha-actin expression was up-regulated, and total SMAD2 and SMAD3 expression was down-regulated in the penis after bilateral cavernous nerve injury. Each dose of losartan after bilateral cavernous nerve injury significantly attenuated the up-regulated expression of fibronectin (p0.01), pSMAD2 (p0.05) and thrombospondin-1 (p0.05), and up-regulated total SMAD2 (p0.05).These data suggest that fibrotic activators in the penis may cause decreased erectile function after bilateral cavernous nerve injury. Angiotensin II type 1 receptor antagonism may counteract this effect and promote erectile function preservation for conditions associated with penile fibrosis.

We investigated the effects of the orally bioavailable non-immunosuppressive immunophilin ligand GPI 1046 (GPI) on erectile function and cavernous nerve (CN) histology following unilateral or bilateral crush injury (UCI, BCI, respectively) of the CNs.Adult male Sprague-Dawley rats were administered GPI 15 mg/kg intraperitoneally (ip) or 30 mg/kg orally (po), FK506 1 mg/kg, ip, or vehicle controls for each route of administration just prior to UCI or BCI and daily up to 7 d following injury. At day 1 or 7 of treatment, erectile function induced by CN electrical stimulation was measured, and electron microscopic analysis of the injured CN was performed.Intraperitoneal administration of GPI to rats with injured CN protected erectile function, in a fashion similar to the prototypic immunophilin ligand FK506, compared with vehicle-treated animals (93%+/-9% vs. 70%+/-5% vs. 45%+/-1%, p0.01, respectively). Oral administration of GPI elicited the same level of significant protection from CN injury. GPI administered po at 30 mg/kg/d, dosing either once daily or four times daily with 7.5 mg/kg, provided nearly complete protection of erectile function. In a more severe BCI model, po administration of GPI maintained erectile function at 24 h after CN injury. Ultrastructural analysis of injured CNs indicated that GPI administered at the time of CN injury prevents degeneration of about 83% of the unmyelinated axons at 7 d after CN injury.The orally administered immunophilin ligand GPI neuroprotects CNs and maintains erectile function in rats under various conditions of CN crush injury.

A key role for nitric oxide (NO) in penile erection is well established, but the relative roles of the neuronal NO synthase (nNOS) versus endothelial forms of NOS are not clear. nNOS- and endothelial NOS-deficient mice maintain erectile function and reproductive capacity, questioning the importance of NO. Alternatively, residual NO produced by shorter transcripts in the nNOS −/− animals might suffice for normal physiologic function. We show that the β splice variant of nNOS elicits normal erection despite a decrease in stimulus-response characteristics and a 5-fold increased sensitivity to the NOS inhibitor, l -NAME. Residual nNOSβ generates only 10% of the normal NO level in vitro but produces citrulline and diaphorase staining reflecting in vivo NOS activity in pelvic ganglion nerves that is comparable to WT animals. Thus, alternatively spliced forms of nNOS are major mediators of penile erection and so may be targets for therapeutic intervention.

With the current understanding that nitric oxide (NO) mediates penile erection, the endothelial isoform of NO synthase (eNOS) has been implicated in this function. We undertook this study applying transgenic mice with targeted deletion of the eNOS gene (eNOS-/- mice) as an experimental approach to evaluate the importance of eNOS in cholinergically stimulated erectile function in vivo. Combined pharmacostimulation with intracavernosal carbachol (3 ng) administration and submaximal cavernous nerve (CN) electrical stimulation (16 Hz, 5 millisecond, 1 V) simultaneous with intracavernosal pressure (ICP) monitoring, and both biochemical assay of NO synthase activity and Western blot analysis of eNOS protein content in penile tissue, were performed on eNOS-/- mice and wild-type controls. Combined intracavernosal carbachol administration and submaximal CN electrical stimulation raised the recorded ICP, elicited by CN electrical stimulation alone in wild-type mice (from 35.7 +/- 2.7 to 48.1 +/- 5.5 mm Hg, P < .05) but not in eNOS-/ - mice (from 54.9 +/- 6.3 to 51.0 +/- 9.5 mm Hg, not significant [NS]). Pretreatment with the nonselective nitric oxide synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 100 mg intracavernosally) blocked electrically stimulated ICP responses in eNOS-/- mice to baseline levels (37.8 +/- 4.4 vs 12.7 +/- 4.0 mm Hg, P < .05). In penes of eNOS-/- mice, approximately 60% NO synthase activity of wild-type penis levels was retained (NS), and eNOS protein was absent. We concluded that eNOS-/- mice preserve erectile function on the basis of a noncholinergic but NO-dependent mechanism and that eNOS physiologically mediates penile erection under cholinergic stimulation.

The changes in sediment chemical composition of Lake Maggiore (Italy) have been traced by collecting a series of grab samples in 1962-63, 1975, 1986, and 1999. The map of distributions of Si, Al, Ca. K, Fe, Mg, Ti, S, P, Pb, Zn, Cu, Ni, Mn, Cr, Cd, Hg, Sc, Co, As, and V, allows identification of hot spots which can be related either to geological features or point sources of industrial pollution.

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43°C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl2) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl2 expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.