Stimulation-induced Relaxation Research Articles

Dual Strategy with Adipose-Derived Stem Cells and l-arginine Recovered Cavernosal Functions in a Rat Model of Radical Prostatectomy.

As standard therapy for prostate cancer, radical prostatectomy causes cavernous nerve (CN) injury and increases fibrosis and hypoxia-induced penile structural alterations. This study aimed to determine the potential beneficial effects of adipose-derived stem cells (ADSCs) and l-arginine alone or in combination on the penile erection in a rat model of erectile dysfunction caused by bilateral cavernous nerve transection (CNT). Male rats (n = 35) were randomized into five groups: Sham-operated; CNT (4-weeks); CNT plus ADSCs (1 × 106 cells by intracavernosal injection); CNT plus l-arginine (4 weeks, 10 mg/kg/day, oral); and ADSCs combined with l-arginine in CNT. In vivo erectile responses and in vitro relaxant responses were measured. Western blot and immunohistochemistry analyses were used to determine the expression and localization of endothelial nitric oxide synthase, neuronal nitric oxide synthase, transforming growth factor-beta 1, hypoxia-inducible factor-1 alpha (HIF-1α), and apoptosis markers (Bax and Bcl-2). The ratio of smooth muscle to collagen and nerve regeneration were calculated using Masson's trichrome and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. The combined treatment restored diminished erectile responses, endothelium-dependent acetylcholine, and electrical field stimulation-induced relaxation of the corpus cavernosum in rats with CNT, whereas either monotherapy produced only partial improvements. All treatment regimens restored increases in the protein expression of HIF-1 and Bax in rats with CNT. The decrease in smooth muscle mass and NADPH-diaphorase-positive nerve fibers was partially ameliorated by monotherapy, whereas combined therapy led to recovery. These findings indicate that combined treatment with ADSCs and l-arginine may restore erectile function in rats with CNT by inhibiting hypoxia-induced neurotoxicity and preserving endothelium function and smooth muscle content.

Macrophage-Specific Toll Like Receptor 9 (TLR9) Causes Corpus Cavernosum Dysfunction in Mice Fed a High Fat Diet

BackgroundErectile dysfunction (ED) has been shown to be related with inflammatory markers in humans. Chronic infusion of TNF-α caused ED in mice while TNF-α knockout mice exhibited improvement in the relaxation of the corpus cavernosum (CC). AimSince obesity triggers an inflammatory process, we aimed to investigate the hypothesis that in obesity, Toll-like receptor 9 (TLR9) activation leads to increased TNF-α levels and impairment in CC reactivity. MethodsFour-week old male C57BL6 (WT) and TLR9 mutant (TLR9MUT) mice were fed a standard chow or high fat diet (HFD) for 12 weeks. Body weight and nonfasting blood glucose were analyzed. Contractile and relaxation responses of the CC were evaluated by electrical field stimulation and concentration response curves to phenylephrine and acetylcholine. Protein expression of nNOS, TNF-α, TNF-R1, TLR9 and MyD88 were measured by western blot. Plasma levels of TNF-α were measured by ELISA. OutcomeIn obesity, impaired cavernosal relaxation is associated with the activation of the innate immune system, by increasing the production of TNF-α through the activation of TLR9 in the macrophages. ResultsAfter 12 weeks of HFD both WT and TLR9MUT mice had increased body weight and nonfasting blood glucose compared to standard chow. In the CC, acetylcholine-induced relaxation was not changed. A trend to increased contraction to phenylephrine and KCl was seen in WT HFD only. electrical field stimulation-induced relaxation of the CC was decreased in WT HFD as well as nNOS expression in the CC of WT HFD, but not in TLR9MUT HFD. In the CC, protein expression of TLR9 and MyD88 was similar in all groups. While circulating levels of TNF-α presented only a trend to increase in mice fed HFD, the CC expression of TNF-α was increased only in WT HFD mice. Clinical TranslationThe innate immune system can be a target for the treatment of erectile complications in obesity. Strengths and LimitationsThis is the first study demonstrating that activation of TLR9 expressed in macrophages leads to impaired cavernosal relaxation. The main limitation of the study is the lack of understanding about the source/expression of the macrophages in the cavernous tissue. Further, herein, the experiments were performed only in isolated cavernous tissue (in vitro), thus the lack of knowledge on how the TLR9 modulates the in vivo response of the erectile tissue is another limitation of this study. ConclusionOur findings indicate that CC dysfunction observed in obesity is at least in part mediated by the production of TNF-α upon activation of TLR9 expressed in the macrophages. Priviero F, Calmasini F, Dela Justina V, et al. Macrophage-Specific Toll Like Receptor 9 (TLR9) Causes Corpus Cavernosum Dysfunction in Mice Fed a High Fat Diet. J Sex Med 2021;18:723-731.

Umbelliferone isolated from Zosima absinthifolia roots partially restored erectile dysfunction in streptozotocin-induced diabetic rats

Erectile dysfunction (ED) is a common problem in diabetic men, which affects the quality of life. Zosima absinthifolia (Vent.) Link (syn. Zosima orientalis Hoffm.) is the only member of Zosima genus growing in Turkey. Its fruits and aerial parts have been used in folk medicine as a sedative and digestive problems. This study aimed to investigate the possible beneficial effect of intracavernosal injection of umbelliferone isolated from Z. absinthifolia Link roots on ED of streptozotocin-induced diabetic rats. Adult Sprague–Dawley (n = 20) rats were divided into control and streptozotocin-induced diabetic groups. In vivo erectile responses were also repeated after intracavernosal injection of umbelliferone (1 µM). Umbelliferone-induced relaxant responses in the presence of inhibitors of nitric oxide synthase (NOS) and cyclic guanosine monophosphate (cGMP) were assessed in corpus cavernosum (CC) strips from control rats. The relaxant responses of CC strips from both groups were evaluated in the presence or absence of umbelliferone (100 μM). In vivo erectile responses in diabetic rats were significantly lower than in controls, which were partially prevented by umbelliferone. Umbelliferone resulted in a relaxation of CC in a concentration-dependent manner. NOS and cGMP inhibitors antagonized the umbelliferone-induced relaxations in CC. Acetylcholine, sodium nitroprusside, a phosphodiesterase type 5 inhibitor, sildenafil and electrical field stimulation-induced relaxation responses in diabetic CC strips were potentialized after pre-incubation with umbelliferone. In conclusion, intracavernosal administration of umbelliferone may prevent ED in diabetic rats by recovering of NO/cGMP pathway. These results may be supported by further studies using combinations of umbelliferone and phosphodiesterase type 5 inhibitors for the treatment of diabetic ED.

P.446 - Slow relaxation kinetics of sarcomeres contribute to muscle slowness in NEM6 patients

Nemaline myopathy (NEM) is among the most common non-dystrophic congenital myopathies and is characterized by muscle weakness. Recently, a novel implicated gene was discovered – KBTBD13. The majority of NM patients with KBTBD13 mutations (NEM6) carry the Dutch founder mutation (c.1222C>T, p.Arg408Cys missense mutation). NEM6 patients display a typical slowness of movement. Here, we investigated the pathophysiology of muscle weakness and slowness of movement in twelve NEM6 patients. Handgrip dynamometry was used to measure contraction and transcranial magnetic stimulation- induced relaxation properties of finger flexors. Muscle biopsies of vastus lateralis were obtained from patients to determine the contractile properties of individual muscle fibers and of individual myofibrils. Transcranial magnetic stimulation-induced relaxation kinetics of finger flexors was significantly slower in patients with NEM6 compared to control subjects. Individual muscle fibers of NEM6 patients had lower maximal active force, increased calcium sensitivity of force, and slower relaxation kinetics. To minimize effects of ultrastructural damage and of impaired calcium diffusion on contractility of fibers, we also studied the contractility of individual myofibrils. Myofbrils, a collection of serially linked sarcomeres, of NEM6 patients had significantly lower maximal force and, importantly, slower relaxation kinetics. Our findings indicate that the typical muscle slowness in NEM6 patients is caused by changes in the functioning of the sarcomeres.

The effect of muscle length on transcranial magnetic stimulation-induced relaxation rate in the plantar flexors.

Transcranial magnetic stimulation (TMS) of the motor cortex during a maximal voluntary contraction (MVC) permits functionally relevant measurements of muscle group relaxation rate (i.e., when muscles are actively contracting under voluntary control). This study's purpose was twofold: (1) to explore the impact of muscle length on TMS‐induced plantar flexor relaxation rate; and (2) to incorporate ultrasonography to measure relaxation‐induced lengthening of medial gastrocnemius (MG) fascicles and displacement of the muscle–tendon junction (MTJ). Eleven males (24.8 ± 7.0 years) performed 21 brief isometric plantar flexor MVCs. Trials were block‐randomized every three MVCs among 20° dorsiflexion (DF), a neutral ankle position, and 30° plantar flexion (PF). During each MVC, TMS was delivered and ultrasound video recordings captured MG fascicles or MTJ length changes. Peak relaxation rate was calculated as the steepest slope of the TMS‐induced drop in plantar flexor torque or the rate of length change for MG fascicles and MTJ. Torque relaxation rate was slower for PF (−804 ± 162 Nm·s−1) than neutral and DF (−1896 ± 298 and −2008 ± 692 Nm·s−1, respectively). Similarly, MG fascicle relaxation rate was slower for PF (−2.80 ± 1.10 cm·s−1) than neutral and DF (−5.35 ± 1.10 and −4.81 ± 1.87 cm·s−1, respectively). MTJ displacement rate showed a similar trend (P = 0.06), with 3.89 ± 1.93 cm·s−1 for PF compared to rates of 6.87 ± 1.55 and 6.36 ± 2.97 cm·s−1 for neutral and DF, respectively. These findings indicate muscle length affects the torque relaxation rate recorded after TMS during an MVC. Comparable results were obtained from muscle fascicles, indicating ultrasound imaging is suitable for measuring evoked contractile properties during voluntary contraction.

The Effect of Muscle Length on Transcranial Magnetic Stimulation-induced Muscle Relaxation Rate

The Effect of Muscle Length on Transcranial Magnetic Stimulation-induced Muscle Relaxation Rate

In Vitro Conditioned Bone Marrow-Derived Mesenchymal Stem Cells Promote De Novo Functional Enteric Nerve Regeneration, but Not Through Direct-Transdifferentiation.

Injury or neurodegenerative disorders of the enteric nervous system (ENS) cause gastrointestinal dysfunctions for which there is no effective therapy. This study, using the benzalkonium chloride-induced rat gastric denervation model, aimed to determine whether transplantation of bone marrow-derived mesenchymal stem cells (BMSC) could promote ENS neuron regeneration and if so, to elucidate the mechanism. Fluorescently labeled BMSC, isolated from either WT (BMSC labeled with bis-benzimide [BBM]) or green fluorescent protein (GFP)-transgenic rats, were preconditioned in vitro using fetal gut culture media containing glial cell-derived neurotrophic factor (GDNF), and transplanted subserosally into the denervated area of rat pylorus. In the nerve-ablated pylorus, grafted BMSC survived and migrated from the subserosa to the submucosa 28 days after transplantation, without apparent dedifferentiation. A massive number of PGP9.5/NSE/HuC/D/Tuj1-positive (but GFP- and BBM-negative) neurons were effectively regenerated in denervated pylorus grafted with preconditioned BMSC, suggesting that they were regenerated de novo, not originating from trans-differentiation of the transplanted BMSC. BMSC transplantation restored both basal pyloric contractility and electric field stimulation-induced relaxation. High levels of GDNF were induced in both in vitro-preconditioned BMSC as well as the previously denervated pylorus after transplantation of preconditioned BMSC. Thus, a BMSC-initiated GDNF-positive feedback mechanism is suggested to promote neuron regeneration and growth. In summary, we have demonstrated that allogeneically transplanted preconditioned BMSC initiate de novo regeneration of gastric neuronal cells/structures that in turn restore gastric contractility in pylorus-denervated rats. These neuronal structures did not originate from the grafted BMSC. Our data suggest that preconditioned allogeneic BMSC may have therapeutic value in treating enteric nerve disorders.

Successful implantation of an engineered tubular neuromuscular tissue composed of human cells and chitosan scaffold

There is an urgent need for gut lengthening secondary to massive resections of the gastrointestinal tract. In this study, we propose to evaluate the remodeling, vascularization, and functionality of a chitosan-based, tubular neuromuscular tissue on subcutaneous implantation in the back of athymic rats. Aligned innervated smooth muscle sheets were bioengineered with the use of human smooth muscle and neural progenitor cells. The innervated sheets were wrapped around tubular chitosan scaffolds. The engineered tubular neuromuscular tissue was implanted subcutaneously in the back of athymic rats. The implant was harvested after 14 days and assessed for morphology, vascularization, and functionality. Gross examination of the implants showed healthy color with no signs of inflammation. The implanted tissue became vascularized as demonstrated by gross and histologic analysis. Chitosan supported the luminal patency of the tissue. The innervated muscle remodeled around the tubular chitosan scaffold. Smooth muscle maintained its circumferential alignment and contractile phenotype. The functionality of the implant was characterized further by the use of real-time force generation. A cholinergic response was demonstrated by robust contraction in response to acetylcholine. Vasoactive intestinal peptide-, and electrical field stimulation-caused relaxation. In the presence of neurotoxin tetrodotoxin, the magnitude of acetylcholine-induced contraction and vasoactive intestinal peptide-induced relaxation was attenuated whereas electrical field stimulation-induced relaxation was completely abolished, indicating neuronal contribution to the response. Our results indicated the successful subcutaneous implantation of engineered tubular neuromuscular tissues. The tissues became vascularized and maintained their myogenic and neurogenic phenotype and function, which provides potential therapeutic prospects for providing implantable replacement GI segments for treating GI motility disorders.

Alteration of vascular reactivity in heart failure: role of phosphodiesterases 3 and 4.

This study examined the role of the main vascular cAMP-hydrolysing phosphodiesterases (cAMP-PDE) in the regulation of basal vascular tone and relaxation of rat aorta mediated by β-adrenoceptors, following heart failure (HF). Twenty-two weeks after proximal aortic stenosis, to induce HF, or SHAM surgery in rats, we evaluated the expression, activity and function of cAMP-PDE in the descending thoracic aorta. HF rat aortas exhibited signs of endothelial dysfunction, with alterations of the NO pathway, and alteration of PDE3 and PDE4 subtype expression, without changing total aortic cAMP-hydrolytic activity and PDE1, PDE3 and PDE4 activities. Vascular reactivity experiments using PDE inhibitors showed that PDE3 and PDE4 controlled the level of PGF2α -stimulated contraction in SHAM aorta. PDE3 function was partially inhibited by endothelial NO, whereas PDE4 function required a functional endothelium and was under the negative control of PDE3. In HF, PDE3 function was preserved, but its regulation by endothelial NO was altered. PDE4 function was abolished and restored by PDE3 inhibition. In PGF2α -precontracted arteries, β-adrenoceptor stimulation-induced relaxation in SHAM aorta, which was abolished in the absence of functional endothelium, as well as in HF aortas, but restored after PDE3 inhibition in all unresponsive arteries. Our study underlines the key role of the endothelium in controlling the contribution of smooth muscle PDE to contractile function. In HF, endothelial dysfunction had a major effect on PDE3 function and PDE3 inhibition restored a functional relaxation to β-adrenoceptor stimulation.

The effect of pentoxifylline on penile cavernosal tissues in ischemic priapism-induced rat model.

Priapism is defined as persisting (>4 h), painful and abnormal tumescence that can occur without sexual stimulation. Three subtypes priapisms are seen-the non-ischemic priapism, intermittent and the ischemic priapism. In ischemic priapism, there is an abnormality in the veno-occlusive mechanism, resulting in venous stasis and accumulation of deoxygenated blood within the penile cavernosal tissue. Cavernosal tissue necrosis develops after extended period of ischemia and is eventually replaced by fibrotic tissue. It may results in erectile dysfunction if not treated promptly. Although, standard treatment of the ischemic priapism is penile aspiration and intracavernosal alpha-adrenergic agents, new oral agents have been investigated to reduce the cavernosal damage. In this study, the effect of different doses of pentoxifylline on cavernosal tissues was evaluated. Thirty-six male Wistar albino rats, age 5.5-6 months and weighing 250-300 g, were used in this study. The rats were randomly divided into five groups. In Group 1 (n = 7), the control group, only penectomy was performed. In Group 2 (n = 8), after 1 h of ischemic priapism, penectomy was performed. Group 3 (n = 7) received daily a 10 mg oral pentoxifylline for 4 weeks after 1 h of ischemic priapism, group 4 (n = 7) received a daily 30 mg oral pentoxifylline for 4 weeks after 1 h of ischemic priapism, and group 5 (n = 7) received a daily 100 mg oral pentoxifylline for 4 weeks after 1 h of ischemic priapism. At the completion of a 4-week period, penile tissues were obtained. Before penile tissues were obtained, intracavernosal pressures measured with electrical field stimulation and smooth muscle collagen ratio were evaluated pathologically. Electrical field stimulation-induced intracavernosal relaxation decreased in group 2 compared with group 1 (p < 0.05). Electrical field stimulation-induced relaxation enhanced in the group 3, 4 and 5 compared to group 2 (p < 0.05). In group 2, the collagen density was significantly higher than group 1. Administration of pentoxifylline reduced the collagen density caused by ischemic priapism in groups 3, 4 and 5 compared with group 2. The results of the present study showed that ischemic priapism caused damage in the penile tissues of rats, and treatment with pentoxifylline reduced the harmful effects of ischemic priapism.

MP43-07 PROTECTIVE EFFECT OF TADALAFIL IN A NOVEL RAT MODEL OF CHRONIC PENILE ISCHEMIA

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research I1 Apr 2014MP43-07 PROTECTIVE EFFECT OF TADALAFIL IN A NOVEL RAT MODEL OF CHRONIC PENILE ISCHEMIA Norifumi Sawada, Masanori Nomiya, Mona Zarifpour, Masayuki Takeda, and Karl-Erik Andersson Norifumi SawadaNorifumi Sawada More articles by this author , Masanori NomiyaMasanori Nomiya More articles by this author , Mona ZarifpourMona Zarifpour More articles by this author , Masayuki TakedaMasayuki Takeda More articles by this author , and Karl-Erik AnderssonKarl-Erik Andersson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1164AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Oxidative stress induced by atherosclerosis accelerates ischemia and structural damage of penile corporal tissue and may produce erectile dysfunction. We tested the protective effects of tadalafil in a novel rat model of chronic penile ischemia. Methods Adult male Sprague-Dawley rats were divided into three groups (control, arterial injury: AI; AI with tadalafil treatment: AI-tada). The AI and AI-tada groups underwent endothelial bilateral injury of the iliac arteries and received a 2% cholesterol diet. Tadalafil (2 mg/kg/day) was administered for 8 weeks. The control group received a regular diet. After 8 weeks, apomorphine test was performed in each group. Corporal tissues were then processed for histological, pharmacological, and immunohistochemical studies. Results The iliac arteries from the AI showed neo-intimal proliferation and vascular occlusion. This was not prevented by tadalafil treatment. Erectile responses in AI group were significantly reduced, but were normalized in the in AI-tadalafil group (Figure 1AB). The percentage of collagen was significantly increased in AI the group but was almost normalized in the AI-tada group. (Figure 2AB) Electrical field stimulation-induced relaxation was decreased in AI group, but improved significantly in the AI-tada group (P < 0.05). (Figure 3) Conclusions Arterial occlusive disease caused chronic penile ischemia and resulted in reduced erectile function and structural changes in corporal tissue. These changes could be prevented by tadalafil treatment. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e479 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Norifumi Sawada More articles by this author Masanori Nomiya More articles by this author Mona Zarifpour More articles by this author Masayuki Takeda More articles by this author Karl-Erik Andersson More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Overexpression of Bcl-2 promotes survival and differentiation of neuroepithelial stem cells after transplantation into rat aganglionic colon

IntroductionNeural stem cell transplantation is a promising tool for the restoration of the enteric nervous system in a variety of motility disorders. However, limited cell viability after transplantation has restricted its regenerative capacity. The aim of this study was to evaluate the effect of transplantation of neuroepithelial stem cell (NESC) overexpressing anti-apoptotic gene Bcl-2 on the survival, differentiation and function of grafted cells in rat aganglionic colon.MethodsNESCs were isolated from neural tube of embryonic rat (embryonic day 11.5) and manipulated to overexpress the Bcl-2 gene. After transplantation into the benzalkonium chloride-induced rat aganglionic colon, grafted cells were visualized in colonic sections. Apoptosis and differentiation of the implanted cells were assessed 1, 4 and 8 weeks post transplantation, respectively. Eight weeks post transplantation, neuronal function of the colon was assessed by measuring the response of muscle strips to electrical field stimulation.ResultsTransplantation with Bcl-2-NESCs reduced apoptosis within the transplant at 1 week compared with the vector-NESC grafted group. Our findings also indicated that overexpression of Bcl-2 in the transplanted NESCs enhanced differentiation into PGP9.5-positive and neuronal nitric oxide synthase-positive neurons at 8-week assessment. Moreover, electrical field stimulation-induced relaxation of colonic strips was also significantly increased in the Bcl-2-NESC grafted group.ConclusionTransplantation of NESCs genetically modified to overexpress Bcl-2 may have value for enhancing survival and neurogenesis of grafted cells in the adult gut environment and for improving the efficacy of stem cell therapy following a broad range of gastrointestinal motility disorders.

Vasoactive Intestinal Polypeptide as Possible Mediator of Relaxation in the Rat Gastric Fundus

Relaxations were induced in longitudinal muscle strips of the rat gastric fundus by stimulation of non-adrenergic non-cholinergic (NANC) neurons and by administration of vasoactive intestinal polypeptide (VIP) or isoprenaline. The effect of antiserum against vasoactive intestinal polypeptide (VIP antiserum) and of control serum on these relaxations was investigated. Incubation with VIP antiserum (dilution 1/50) for 1 h almost completely prevented the relaxation by VIP. It partially prevented the relaxation evoked by electrical stimulation while the relaxation induced by isoprenaline was not influenced. Control serum decreased the VIP- and stimulation-induced relaxations much less than did VIP antiserum. In addition, the effect of the putative VIP antagonist (4Cl-D-Phe6, Leu17) VIP was studied on the relaxations induced by NANC neuron stimulation and by VIP. The VIP antagonist (3 x 10(-5) M, incubation time 10 min) had a relaxatory effect itself but had no influence on either VIP- or stimulation-induced relaxations. The results with VIP antiserum confirm the involvement of VIP in the inhibitory NANC neurotransmission of the rat gastric fundus.

Lysophosphatidic acid presynaptically blocks NO uptake during electric field stimulation-induced relaxation via LPA1 receptor in cat lower esophageal sphincter

Lysophosphatidic acid (LPA) is a lipid mediator that is involved in many biological responses, such as, in the stimulation/inhibition of proliferation, in cell migration, antiapoptosis, tumor cell invasion, platelet aggregation, vascular remodeling, and neurotransmitter release. In addition, LPA indirectly enhances the contractility of smooth muscle. Furthermore, electric field stimulation (EFS) causes contractions of isolated cat esophageal smooth muscle and relaxations of isolated cat lower esophageal sphincter (LES). To test whether or not LPA enhances postsynaptically-mediated contraction in cat esophageal smooth muscle and LES, both types of muscle strips were stimulated with muscarinic agonists. However, no significant effects were observed, and therefore, to investigate whether LPA is involved in presynaptic signal transduction, cat esophageal smooth muscle and LES were pretreated with LPA and stimulated using EFS. LPA had no effect on EFS-induced contraction in esophageal smooth muscle but the EFS-induced LES relaxation was dose-dependently inhibited by LPA. To identify the LPA receptor subtype that inhibits EFS-induced LES relaxation, we used the specific LPA(1)/LPA(3) antagonist Ki16425 and the LPA(3) agonist OMPT. Ki16425 significantly blocked the inhibitory effect of LPA on EFS-induced relaxation, but OMPT did not enhance the effect of LPA. These results suggest that LPA inhibits EFS-induced relaxation in LES via LPA(1) receptor-mediated signaling. It is well known that EFS-induced LES relaxation is related to the release of neurotransmitters, such as, nitric oxide, vasoactive intestinal polypeptide, and calcitonin gene-related peptide. We then investigated whether LPA selectively blocks NO-mediated signaling. Sodium nitroprusside-induced LES relaxation was found to be inhibited in the same manner as EFS-induced LES relaxation by LPA. This result suggests that LPA partially blocks NO uptake by presynaptic pathways, and thus, inhibits LES relaxation.

Comparative Relaxing Effects of Sildenafil, Vardenafil, and Tadalafil in Human Corpus Cavernosum: Contribution of Endogenous Nitric Oxide Release

To compare the direct relaxant activity of sildenafil, vardenafil, and tadalafil in the human corpus cavernosum (HCC) and to investigate their modulatory effects on nitric oxide (NO)-mediated responses. Phosphodiesterase (PDE)-5 inhibitors cause cavernosal smooth muscle relaxation and penile erection. HCC strips were mounted in 10-mL organ baths containing Krebs solution and connected to force-displacement transducers. The changes in isometric force were recorded using the Powerlab 400 data acquisition system. Corporeal smooth muscle was precontracted submaximally with phenylephrine (10 micromol/L). All PDE-5 inhibitors tested (0.001-10 micromol/L) relaxed phenylephrine-precontracted HCC with similar values of potency in a concentration-dependent manner. However, the maximal relaxations induced by tadalafil (83% +/- 4%) were significantly lower compared with sildenafil (107% +/- 5%) and vardenafil (111% +/- 3%). The NO synthesis inhibitor N-nitro-l-arginine methyl ester (100 micromol/L) caused significant rightward shifts in the concentration-response curves for sildenafil (4.0-fold), vardenafil (4.6-fold), and tadalafil (3.2-fold) in HCC tissue. The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L) also produced similar rightward shifts for these PDE-5 inhibitors. The cavernosal relaxations evoked by either acetylcholine or the NO donor glyceryl trinitrate were markedly potentiated by sildenafil, vardenafil, and tadalafil (0.1 micromol/L each). All PDE-5 inhibitors significantly increased the duration of electrical field stimulation-induced relaxations (8 Hz). Our findings have shown that sildenafil, vardenafil, and tadalafil relax HCC tissues in a concentration-dependent manner, but the maximal relaxation obtained with tadalafil was significantly lower than that obtained with sildenafil and vardenafil. Moreover, the PDE-5 inhibitors interacted with endogenous and exogenous NO, amplifying its HCC relaxation.

Investigation of the relaxant effects of pancuronium, rocuronium, vecuronium and mivacurium on rat thoracic aorta

Pancuronium, vecuronium, mivacurium and rocuronium are nondepolarizing neuromuscular blocking agents, which are competitive antagonists against acetylcholine at nicotinic receptors, and considered to have no direct actions on vascular smooth muscle. We aimed to investigate the relaxant effects and possible underlying mechanisms of these agents on isolated rat thoracic aorta. The preparations were precontracted with prostaglandin F2alpha (10(-7) mol l(-1)) and pancuronium (10(-7)-10(-4) mol l(-1)), rocuronium (10(-7)-10(-4) mol l(-1)), vecuronium (10(-7)-10(-4) mol l(-1)) and mivacurium (10(-7)-10(-4) mol l(-1)) added at cumulative concentrations in the presence or absence of a prostaglandin synthesis inhibitor, indomethacin (10(-6) M), and a nitric oxide synthesis inhibitor, N(omega)-nitro-L-arginine methylester (3 x 10(-5)). The same protocol was applied to both endothelia (+) and endothelia (-) aortic rings. The preparations precontracted with prostaglandin F2alpha (10(-7) mol l(-1)) were stimulated with electrical field stimulation at a frequency of 10 Hz as square-wave pulses of 50 V (0.2 ms) in the presence of a noradrenaline reuptake inhibitor desipramine (10(-7) mol l(-1)) and a nonselective beta-blocker propranolol (10(-6) mol l(-1)). Drugs were added at ineffective concentration of 10(-7) mol l(-1). Tetrodotoxin (10(-7) mol l(-1)) was added to test whether the changes were dependent on the neuronal response. Pancuronium and rocuronium relaxed aortic rings precontracted by prostaglandin F2alpha in a dose-dependent manner, but vecuronium and mivacurium did not. The relaxation effect of pancuronium and rocuronium was endothelium independent because there was not a significant response difference from the endothelium-denuded group. In conclusion, their relaxation effect may be due to an increase in prostaglandin synthesis. The increased relaxation effect of these agents at electrical field stimulation may be by the decreasing effect of noradrenaline reuptake from nerve endings because a noradrenaline reuptake inhibitor desipramine did not change this effect. Also, these neuromuscular agents may affect beta-receptors, because a nonselective beta-blocker agent, propranolol, decreased their electrical field stimulation-induced relaxations.

Presynaptic Prostaglandin E 2 EP 1 -Receptor Facilitation of Cerebral Nitrergic Neurogenic Vasodilation

Prostaglandin E(2) (PGE(2)) modulates autonomic transmission in the peripheral circulation. We investigated the role of endogenous PGE(2) and its presynaptic EP(1) receptor subtype in modulating the autonomic neurotransmission in cerebral vasculature. The standard in vitro tissue-bath technique was used for measuring changes in isolated porcine basilar arterial tone. Calcium imaging and nitric oxide estimation along with immunohistochemical analysis for cyclo-oxygenase-1, cyclo-oxygenase-2, EP(1) receptor, PGE synthase, and neuronal nitric oxide synthase were done in cultured sphenopalatine ganglia and basilar artery. Selective EP(1) receptor antagonists (SC-19220 and SC-51322) inhibited relaxation of endothelium-denuded basilar arterial rings elicited by transmural nerve stimulation (2 and 8 Hz) without affecting that induced by nicotine or sodium nitroprusside (a nitric oxide donor). The SC-19220 inhibition of transmural nerve stimulation-elicited relaxation was blocked by cyclo-oxygenase inhibitors (salicylic acid and naproxen) but was not affected by guanethidine (a sympathetic neuronal blocker) or atropine. Perivascular cyclo-oxygenase-1- and cyclo-oxygenase-2-immunoreactive fibers were observed in basilar arteries. PGE synthase and EP(1) receptor immunoreactivities were coincident with neuronal nitric oxide synthase immunoreactivities in perivascular nerves of the basilar arteries and the sphenopalatine ganglia. omega-conotoxin (an N-type calcium channel blocker) significantly blocked transmural nerve stimulation-induced relaxation, which was further attenuated by SC-19220. In cultured sphenopalatine ganglia neurons, exogenous PGE(2) significantly increased calcium influx and diaminofluorescein fluorescence indicative of nitric oxide synthesis. Both responses were blocked by SC-19220. These results suggest that neuronal PGE(2) facilitates nitric oxide release from the cerebral perivascular parasympathetic nitrergic nerve terminals by increasing neuronal calcium influx through activation of presynaptic EP(1) receptors. PGE(2) may play an important role in regulating the nitrergic neurovascular transmission in the cerebral circulation.

Increased Cyclic Guanosine Monophosphate Synthesis and Calcium Entry Blockade Account for the Relaxant Activity of the Nitric Oxide–Independent Soluble Guanylyl Cyclase Stimulator BAY 41-2272 in the Rabbit Penile Urethra

To study the direct relaxant activity of 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) in the rabbit penile urethra and to investigate its modulatory effect on nitric oxide (NO)-mediated responses. Urothelium-intact (U+) and denuded (U-) rings were mounted in 10-mL organ baths for isometric force recording. Intracellular cyclic guanosine monophosphate (cGMP) levels were quantified with specific kits. BAY 41-2272 (0.0001 to 10 micromol/L) caused relaxation of urethral rings contracted with phenylephrine (10 micromol/L), with higher potency (P <0.01) in U+ (pEC(50) 7.77 +/- 0.09) compared with U- (pEC(50) 6.84 +/- 0.19) preparations. The NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (100 micromol/L) or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) (10 micromol/L) had no effect on BAY 41-2272 responses in U+ or U- rings. The phosphodiesterase-5 inhibitor vardenafil (0.1 micromol/L) potentiated the relaxant effects of BAY 41-2272 in both U+ (10-fold) and U- (sevenfold) tissues. Ca(2+)-induced contractions in K(+) depolarized rings were significantly attenuated by BAY 41-2272 (1 micromol/L) in an ODQ-insensitive manner. BAY 41-2272 (0.03-0.3 micromol/L) increased the amplitude and duration of electrical field stimulation-induced relaxations (1 to 32 Hz), as well as those evoked by the NO donor glyceryl trinitrate (0.0001 to 10 micromol/L). BAY 41-2272 induced ODQ-resistant increases in cGMP levels above baseline (approximately twofold) in both U+ and U- rings. BAY 41-2272 relaxes penile urethra in a synergic fashion with NO. Targeting soluble guanylate cyclase with BAY 41-2272 may represent a new therapy in the management of voiding disturbances associated with impaired NO-cGMP signaling.

Role of the l-citrulline/ l-arginine cycle in iNANC nerve-mediated nitric oxide production and airway smooth muscle relaxation in allergic asthma

Nitric oxide synthase (NOS) converts l-arginine into nitric oxide (NO) and l-citrulline. In NO-producing cells, l-citrulline can be recycled to l-arginine in a two-step reaction involving argininosuccinate synthase (ASS) and -lyase (ASL). In guinea pig trachea, l-arginine is a limiting factor in neuronal nNOS-mediated airway smooth muscle relaxation upon inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation. Moreover, in a guinea pig model of asthma iNANC nerve-induced NO production and airway smooth muscle relaxation are impaired after the allergen-induced early asthmatic reaction, due to limitation of l-arginine. Using guinea pig tracheal preparations, we now investigated whether (i) the l-citrulline/ l-arginine cycle is active in airway iNANC nerves and (ii) the NO deficiency after the early asthmatic reaction involves impaired l-citrulline recycling. Electrical field stimulation-induced relaxation was measured in tracheal open-rings precontracted with histamine. l-citrulline as well as the ASL inhibitor succinate did not affect electrical field stimulation-induced relaxation under basal conditions. However, reduced relaxation induced by a submaximal concentration of the NOS inhibitor N ω-nitro- l-arginine was restored by l-citrulline, which was prevented by the additional presence of succinate or the ASS inhibitor α-methyl- d, l-aspartate. Remarkably, the impaired iNANC relaxation after the early asthmatic reaction was restored by l-citrulline. In conclusion, the l-citrulline/ l-arginine cycle is operative in guinea pig iNANC nerves in the airways and may be effective under conditions of low l-arginine utilization by nNOS (caused by NOS inhibitors), and during reduced l-arginine availability after allergen challenge. Enzymatic dysfunction in the l-citrulline/ l-arginine cycle appears not to be involved in the l-arginine limitation and reduced iNANC activity after the early asthmatic reaction.

P2 purinoceptors account for the non-nitrergic NANC relaxation in the rat ileum

The transmitters involved in the non-nitrergic component of the non-adrenergic, non-cholinergic (NANC) inhibitory response of the rat small intestinal longitudinal muscle to electrical field stimulation of its nerves is a matter of controversy. The present study is the first one to utilise a combination of a nitric oxide synthase inhibitor and a P(2) purinoceptor antagonist for studying this response. We found that the P(2) purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 5x10(-5) M) abolished the non-nitrergic NANC relaxation to electrical field stimulation (10 Hz). PPADS alone provided a significant, moderate inhibitory action. PPADS specifically inhibited relaxations due to exogenous adenosine 5'-triphosphate (ATP) or alpha,beta-methylene ATP. The guanylate cyclase blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10(-6) M) did not add to the inhibitory action of N(G)-nitro-L-arginine on field stimulation-induced relaxation. ODQ abolished the relaxant effect of the nitric oxide donors nitroglycerin or sodium nitroprusside. These data indicate that: (1) nitric oxide and ATP fully account for the field stimulation-induced relaxation in the rat ileal strip under the experimental conditions of this study, and (2) no ODQ-sensitive guanylate cyclase-mediated mechanism is involved in the non-nitrergic component of the NANC relaxation.