Cyclic GMP Pathway Research Articles

Ghrelin reduces voltage-gated calcium currents in GH3 cells via cyclic GMP pathways

Ghrelin reduces voltage-gated calcium currents in GH3 cells via cyclic GMP pathways

222 EXPRESSION AND DISTRIBUTION OF PHOSPHODIESTERASE (PDE) ISOENZYMES IN THE HUMAN URETHRA

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology1 Apr 2011222 EXPRESSION AND DISTRIBUTION OF PHOSPHODIESTERASE (PDE) ISOENZYMES IN THE HUMAN URETHRA George Kedia, Markus Kuczyk, Petter Hedlund, and Stefan Ückert George KediaGeorge Kedia Hannover, Germany More articles by this author , Markus KuczykMarkus Kuczyk Hannover, Germany More articles by this author , Petter HedlundPetter Hedlund Lund, Sweden More articles by this author , and Stefan ÜckertStefan Ückert Hannover, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.332AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Up untill today, only a very few studies have addressed the physiology/pharmacology of the human urethra (Hassouna M. et al., J. Urol. 129: 1262–1264, 1983; Abdel-Hakim A. et al., J. Urol. 130: 988–991, 1983; Werkström V. et al., BJU Int. 98: 414–423, 2006). An involvement of the nitric oxide (NO)/cyclic GMP pathway in mediating the relaxation of the bladder outlet and urethra during the micturition phase has been supposed. The present study aimed to evaluate in the human male urethra by means of immunohistochemistry the expression and distribution of cyclic AMP and cyclic GMP phosphodiesterase (PDE) isoenzymes in relation to key mediators/proteins of the cyclic GMP and cyclic AMP pathways. METHODS Using conventional immunohistochemistry (double-labelling technique, laser fluorescence microscopy), thin sections (8 μm – 10 μm) of the human male urethra were exposed to anti-PDE1A, -PDE2A, -PDE3A, -PDE4A, -PDE4B, -PDE5A, anti-nNOS (neuronal nitric oxide synthase), -VIP (vasoactive intestinal polypeptide) and -CGRP (calcitonin gene-related peptide) antibodies followed by the application of fluorochrome-labelled secondary antibodies. RESULTS In the smooth muscle (SM) portion of the urethra, immunosignals specific for PDE1A (cyclic AMP/cyclic GMP PDE, calcium/calmodulin-dependent), PDE4A, PDE4B (cyclic AMP PDEs) and PDE5A (cyclic GMP PDE) were registered. SM bundles were seen innervated by varicose nerve fibers characterized by the expression of nNOS. Some of these nerves also presented staining related to VIP or CGRP. No immunoreactivities specific for PDE2A or PDE3A were detected. CONCLUSIONS PDE isoenzymes type 1A, 4A, 4B and 5A are expressed in human urethral smooth muscle. The findings implicate that the cyclic AMP and cyclic GMP pathways work synergistically together in the control of urethral smooth muscle function. This might be of importance with regard to the identification of new pharmacological avenues to facilitate the contraction and relaxation of the human outflow region. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e91 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information George Kedia Hannover, Germany More articles by this author Markus Kuczyk Hannover, Germany More articles by this author Petter Hedlund Lund, Sweden More articles by this author Stefan Ückert Hannover, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

746 PHOSPHODIESTERASE TYPE 1, THE CALCITONIN-GENE RELATED PEPTIDE AND VASOACTIVE INTESTINAL POLYPEPTIDE ARE INVOLVED IN THE CONTROL OF HUMAN VAGINAL ARTERIAL VESSELS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 2011746 PHOSPHODIESTERASE TYPE 1, THE CALCITONIN-GENE RELATED PEPTIDE AND VASOACTIVE INTESTINAL POLYPEPTIDE ARE INVOLVED IN THE CONTROL OF HUMAN VAGINAL ARTERIAL VESSELS Stefan Ückert, Matthias Oelke, Markus Kuczyk, and Petter Hedlund Stefan ÜckertStefan Ückert Hannover, Germany More articles by this author , Matthias OelkeMatthias Oelke Hannover, Germany More articles by this author , Markus KuczykMarkus Kuczyk Hannover, Germany More articles by this author , and Petter HedlundPetter Hedlund Milan, Italy More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1771AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The vagina contributes mainly to the normal female sexual response cycle. An increase in vaginal blood flow is considered a key event in the mechanism of sexual arousal. Recent research has focused mainly on the cyclic GMP pathway and the phosphodiesterase type 5 (PDE5, cyclic GMP-specific PDE) in the control of vaginal vascular smooth muscle. In contrast, only little is known on the role of other key proteins and mediators of the cyclic nucleotide mediated signaling in this process. The aim of the present study was to evaluate in the human vagina, by means of immunohistochemistry, the expression and distribution of the phosphodiesterase type 1 (PDE1, known to hydrolize both cyclic AMP and cyclic GMP) in relation to the calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and protein gene product 9.5 (PGP 9.5). METHODS Cryostat sections of the human vagina (full wall specimens) were incubated with antibodies directed against PDE1, CGRP, VIP, PGP 9.5 and alpha-actin, followed by exposure to fluorochrome-labelled secondary antibodies. Visualization was commenced by means of laser fluorescence microscopy. RESULTS Microscopical examination revealed a dense meshwork of PGP 9.5-positive nerve fibers innervating the sections of the vaginal wall. Small vessels interspersing the tissue presented dense staining for PDE1 in their smooth musculature while no signals were observed in the non-vascular smooth muscle. The vessels were found innervated by PGP-positive varicose nerve fibers characterized by the expression of CGRP. Some fibers also presented immunosignals specific for VIP. CONCLUSIONS Key mediators of the cyclic AMP and cyclic GMP pathways are co-localized in nerves seen in close proximity to vascular smooth muscle expressing PDE1. These findings give hint that both signaling cascades are involved in the control of vaginal blood flow. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e300 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stefan Ückert Hannover, Germany More articles by this author Matthias Oelke Hannover, Germany More articles by this author Markus Kuczyk Hannover, Germany More articles by this author Petter Hedlund Milan, Italy More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Modulating the nitric oxide - cyclic GMP pathway in the pressure-overloaded left ventricle and group II pulmonary hypertension

Group II pulmonary hypertension (PH) commonly occurs in the setting of a pressure-overloaded left ventricle (LV) which is also conducive to the development of heart failure with preserved ejection fraction. Population trends and a high prevalence of underlying causative conditions, such as essential hypertension or aortic stenosis, have increased the awareness of the pressure-overloaded LV and associated group II pulmonary hypertension. Patients often exhibit poor exercise tolerance and signs of heart failure indistinguishable from systolic heart failure; but effective medical treatments in this area have been lacking. Recent preclinical work has shed light on how the down-regulated nitric oxide - cyclic GMP pathway (within the myocardium and pulmonary vasculature) contributes to the pathophysiology of these associated conditions. This article will discuss the impact of the nitric oxide - cyclic GMP pathway on the pathogenesis of the pressure-overloaded LV and group II pulmonary hypertension, and will also introduce the potential therapeutic value of modulating this pathway.

713 EXPRESSION AND DISTRIBUTION OF KEY ENZYMES OF THE CYCLIC GMP SIGNALING IN THE HUMAN CLITORIS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research I1 Apr 2010713 EXPRESSION AND DISTRIBUTION OF KEY ENZYMES OF THE CYCLIC GMP SIGNALING IN THE HUMAN CLITORIS Stefan Ückert, Matthias Oelke, Knut Albrecht, Dirk Breitmeier, Markus Kuczyk, and Petter Hedlund Stefan ÜckertStefan Ückert Hannover, Germany More articles by this author , Matthias OelkeMatthias Oelke Hannover, Germany More articles by this author , Knut AlbrechtKnut Albrecht Hannover, Germany More articles by this author , Dirk BreitmeierDirk Breitmeier Hannover, Germany More articles by this author , Markus KuczykMarkus Kuczyk Hannover, Germany More articles by this author , and Petter HedlundPetter Hedlund Milan, Italy More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1154AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The human clitoris contributes mainly to the normal female sexual response cycle. A significance of cyclic GMP has been assumed in the control of clitoral vascular smooth muscle. Recent research presented the expression of the phosphodiesterase type 5 (PDE5, cyclic GMP-specific PDE) in clitoral erectile tissue (UROLOGY 67: 1111-1116, 2006). However, knowledge on the mechanisms controlling this particular female genital organ is still sparse. This prompted us to evaluate in the human clitoris by means of immunohistochemistry the expression and distribution in relation to PDE5 of key enzymes of the cyclic GMP pathway: the endothelial nitric oxide synthase (eNOS), PDE2 (cyclic GMP-binding PDE), PDE5, and protein kinase G (cGK, cyclic GMP-dependent protein kinase). METHODS Human clitoral tissue was obtained from four female cadavers (age at death 18 y - 42 y). Sections of clitoral tissue were incubated (for 24 h) with antibodies directed against eNOS, PDE2, PDE5 and cGK followed by exposure for 2 h to fluorochrome-labeled (fluorescein isothiocyanate, Texas Red) secondary antibodies. Visualization was commenced by means of laser fluorescence microscopy. RESULTS Immunohistochemistry revealed the presence of PDE2, PDE5 and cGK in the smooth muscle wall of blood vessels transversing the clitoral supepithelial space. These vessels were characterized by the expression of NOS in the cells of the endothelial layer. Immunoreactivity specific for PDE2 was also identified in a meshwork of interstitial-like cells located in the basal epithelial layer. No signals specific for PDE2 and cGK were detected in non-vascular clitoral smooth muscle. CONCLUSIONS The results are in favour of a role of the cyclic GMP signaling in the control of clitoral blood flow. It seems likely that the cyclic GMP-binding PDE2 is also involved in the mechanism of local afferent transmission in the clitoris. The data may provide further rationale for the use of drugs interfering with the cyclic GMP pathway in the pharmacotherapy of female sexual arousal disorders. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e278 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stefan Ückert Hannover, Germany More articles by this author Matthias Oelke Hannover, Germany More articles by this author Knut Albrecht Hannover, Germany More articles by this author Dirk Breitmeier Hannover, Germany More articles by this author Markus Kuczyk Hannover, Germany More articles by this author Petter Hedlund Milan, Italy More articles by this author Expand All Advertisement Advertisement Loading ...

An Important Role of the Src Family Kinase Lyn in Stimulating Platelet Granule Secretion

The Src family kinases (SFKs) have been proposed to play stimulatory and inhibitory roles in platelet activation. The mechanisms for these apparently contradictory roles are unclear. Here we show that SFK, mainly Lyn, is important in stimulating a common signaling pathway leading to secretion of platelet granules. Lyn knock-out or an isoform-nonselective SFK inhibitor, PP2, inhibited platelet secretion of both dense and alpha granules and the secretion-dependent platelet aggregation induced by thrombin, collagen, and thromboxane A(2). The inhibitory effect of Lyn knock-out on platelet aggregation was reversed by supplementing granule content ADP, indicating that the primary role of Lyn is to stimulate granule secretion. Inhibitory effect of PP2 on platelet aggregation induced by thrombin and thromboxane A(2) were also reversed by supplementing ADP. Furthermore, PP2 treatment or Lyn knock-out diminished agonist-induced Akt activation and cyclic GMP production. The inhibitory effect of PP2 or Lyn knock-out on platelet response can be corrected by supplementing cyclic GMP. These data indicate that Lyn stimulates platelet secretion by activating the phosphoinositide 3-kinase-Akt-nitric oxide (NO)-cyclic GMP pathway and also provide an explanation why Lyn can both stimulate and inhibit platelet activation.

Cyclic GMP pathways in hepatic encephalopathy. Neurological and therapeutic implications

Cyclic GMP (cGMP) modulates important cerebral processes including some forms of learning and memory. cGMP pathways are strongly altered in hyperammonemia and hepatic encephalopathy (HE). Patients with liver cirrhosis show reduced intracellular cGMP in lymphocytes, increased cGMP in plasma and increased activation of soluble guanylate cyclase by nitric oxide (NO) in lymphocytes, which correlates with minimal HE assessed by psychometric tests. Activation of soluble guanylate cyclase by NO is also increased in cerebral cortex, but reduced in cerebellum, from patients who died with HE. This opposite alteration is reproduced in vivo in rats with chronic hyperammonemia or HE. A main pathway modulating cGMP levels in brain is the glutamate-NO-cGMP pathway. The function of this pathway is impaired both in cerebellum and cortex of rats with hyperammonemia or HE. Impairment of this pathway is responsible for reduced ability to learn some types of tasks. Restoring the pathway and cGMP levels in brain restores learning ability. This may be achieved by administering phosphodiesterase inhibitors (zaprinast, sildenafil), cGMP, anti-inflammatories (ibuprofen) or antagonists of GABAA receptors (bicuculline). These data support that increasing cGMP by safe pharmacological means may be a new therapeutic approach to improve cognitive function in patients with minimal or clinical HE.

TRPV1 and TRPA1 Mediate Peripheral Nitric Oxide-Induced Nociception in Mice

Nitric oxide (NO) can induce acute pain in humans and plays an important role in pain sensitization caused by inflammation and injury in animal models. There is evidence that NO acts both in the central nervous system via a cyclic GMP pathway and in the periphery on sensory neurons through unknown mechanisms. It has recently been suggested that TRPV1 and TRPA1, two polymodal ion channels that sense noxious stimuli impinging on peripheral nociceptors, are activated by NO in heterologous systems. Here, we investigate the relevance of this activation. We demonstrate that NO donors directly activate TRPV1 and TRPA1 in isolated inside-out patch recordings. Cultured primary sensory neurons display both TRPV1- and TRPA1-dependent responses to NO donors. BH4, an essential co-factor for NO production, causes activation of a subset of DRG neurons as assayed by calcium imaging, and this activation is at least partly dependent on nitric oxide synthase activity. We show that BH4-induced calcium influx is ablated in DRG neurons from TRPA1/TRPV1 double knockout mice, suggesting that production of endogenous levels of NO can activate these ion channels. In behavioral assays, peripheral NO-induced nociception is compromised when TRPV1 and TRPA1 are both ablated. These results provide genetic evidence that the peripheral nociceptive action of NO is mediated by both TRPV1 and TRPA1.

Regulate Axon Branching by the Cyclic GMP Pathway via Inhibition of Glycogen Synthase Kinase 3 in Dorsal Root Ganglion Sensory Neurons

Cyclic GMP has been proposed to regulate axonal development, but the molecular and cellular mechanisms underlying the formation of axon branches are not well understood. Here, we report the use of rodent embryonic sensory neurons from the dorsal root ganglion (DRG) to demonstrate the role of cGMP signaling in axon branching and to identify the downstream molecular pathway mediating this novel regulation. Pharmacologically, a specific cGMP analog promotes DRG axon branching in culture, and this activity can be achieved by activating the endogenous soluble guanylyl cyclase that produces cGMP. At the molecular level, the cGMP-dependent protein kinase 1 (PrkG1) mediates this activity, as DRG neurons isolated from the kinase-deficient mouse fail to respond to cGMP activation to make branches, whereas overexpression of a PrkG1 mutant with a higher-than-normal basal kinase activity is sufficient to induce branching. In addition, cGMP activation in DRG neurons leads to phosphorylation of glycogen synthase kinase 3 (GSK3), a protein that normally suppresses branching. This interaction is direct, because PrkG1 binds GSK3 in heterologous cells and the purified kinase can phosphorylate GSK3 in vitro. More importantly, overexpression of a dominant active form of GSK3 suppresses cGMP-dependent branching in DRG neurons. Thus, our study establishes an intrinsic signaling cascade that links cGMP activation to GSK3 inhibition in controlling axon branching during sensory axon development.

Up-Regulation of 3′5′-Cyclic Guanosine Monophosphate-Specific Phosphodiesterase in the Porcine Cumulus-Oocyte Complex Affects Steroidogenesis during in Vitro Maturation

The 3'5'-cyclic GMP (cGMP) pathway is known to influence ovarian functions, including steroidogenesis, ovulation, and granulosa cell proliferation. We show here that cGMP-phosphodiesterase (PDE) activity increased in a gonadotropin-dependent manner more than 3-fold in the cumulus-oocyte complex (COC) after 24 h in vitro maturation (IVM) and up to 5-fold after 48 h. Further characterization of this increase demonstrated that the activity was located primarily in cumulus cells, and was sensitive to sildenafil and zaprinast, two inhibitors specific to both type 5 and 6 PDEs. RT-PCR experiments showed that the mRNAs for cGMP-degrading PDEs 5A and 6C are present in the COC before and after 30 h IVM. Western blotting confirmed the presence of PDE 5A in the COC. Western blotting of PDE 6C revealed a significant up-regulation in the COC during IVM. Isolation and analysis of detergent-resistant membranes suggested that PDE 6C protein, along with half of the total sildenafil-sensitive cGMP-degradation activity, is associated with detergent-resistant membrane in the COC after 30 h IVM. Treatment of porcine COC with sildenafil during IVM caused a significant decrease in gonadotropin-stimulated progesterone secretion. Together, these results constitute the first report exploring the contribution of cGMP-PDE activity in mammalian COC, supporting a functional clustering of the enzyme, and providing the first evidence of its role in steroidogenesis.

Sildenafil augments the beneficial hemodynamic and histopathological effects of amlodipine in nitric oxide-deficient hypertensive rats: Role of nitric oxide–cyclic GMP pathway

The association of erectile dysfunction (ED) with cardiovascular diseases is so common. This study was carried out to investigate possible impact of sildenafil; the prototype phosphodiesterase 5 inhibitor used for treatment of ED, on the beneficial hemodynamic and histopathological effects of the prototype third generation calcium antagonist, amlodipine, in nitric oxide (NO)-deficient hypertensive rats. Hypertension was induced by 4-weeks treatment with N ω-nitro- l-arginine-methyl ester ( l-NAME). Animals were allocated into five groups: normal control, hypertensive control, amlodipine-treated group, sildenafil-treated group and combined treatment group. Drug treatment was started 2 weeks after l-NAME and continued together with l-NAME to the end of the treatment period. Systolic blood pressure (SBP), plasma nitrate/nitrite (NO x ) and plasma cGMP levels were evaluated at the end of the treatment period. Aortic and renal structural alterations were also investigated. l-NAME treatment caused elevation of SBP, reduction in plasma NO x and cGMP levels as well as adverse histological alterations in the tissues studied. Amlodipine normalized SBP, restored plasma NO x and cGMP levels and ameliorated the adverse histological changes seen in NO-deficient rats. When combined with sildenafil, both hemodynamic and histopathological effects of amlodipine were augmented with an underlying enhanced elevation of both plasma NO x and cGMP levels to statistically higher values than amlodipine alone. These results show that sildenafil augments the beneficial hemodynamic and histopathological effects of amlodipine in NO-deficient hypertensive rats with a pivotal role being played by NO–cGMP pathway. Whether this pharmacodynamic interaction could exist in other models of hypertension that do not share such biochemical derangement warrants further investigations.

Developmental exposure to polychlorinated biphenyls or methylmercury, but not to its combination, impairs the glutamate–nitric oxide–cyclic GMP pathway and learning in 3-month-old rats

Prenatal exposure to polychlorinated biphenyls (PCBs) or methylmercury (MeHg) contaminated food may affect brain development, leading to long-term alterations in cognitive function. Both types of contaminants, PCBs and MeHg, are often found together contaminating food, especially fish in some polluted areas. Exposure to combinations of neurotoxicants may exert different effects on the developing nervous system than exposure to individual contaminants. Developmental exposure (during pregnancy and lactation) to PCB126 or PCB153 impairs learning ability when the rats are 3 months old. Impairment of learning seems to be a consequence of impairment of the function of the glutamate–nitric oxide (NO)–cGMP pathway in brain in vivo. The aims of the present work were 1) to assess whether perinatal exposure to MeHg also affects the function of the glutamate–NO–cGMP pathway in brain in vivo analyzed by in vivo brain microdialysis and/or the ability to learn the Y maze task when the rats are 3 months old, and 2) to assess whether perinatal exposure to combinations of MeHg with PCB153 or PCB126 potentiates, decreases or does not modify the effects of the individual neurotoxicants. Perinatal exposure to PCB126, PCB153 or MeHg impaired the function of the glutamate–NO–cGMP pathway in cerebellum and learning ability. However, co-exposure to PCB126+MeHg or PCB153+MeHg inhibits the impairment of the pathway or learning ability. These results support that the function of this pathway modulates learning of the Y maze task. Moreover, they show that co-exposure to these PCBs and MeHg does not exacerbate, but reduces the effects on the ability to learn this task.

Effects of epoxyeicosatrienoic acids on volume‐activated chloride channels via cyclic GMP pathway in rat mesenteric artery

Epoxyeicosatrienoic acids (EETs) are one of the proposed candidates of EDHF. It is known that volume‐activated chloride channels (VACCs) play important roles in regulating cell membrane potential, so we hypothesize that VACCs may be involved in EETs‐induced vasorelaxation. Rat mesenteric arteries were dissected out and the arteries rings were relaxed by acetylcholine (ACh, 1 nM to 1 mM) in the presence of L‐NAME and indomethacin. It was of interest to note that the ACh (1 μM)‐induced relaxation was reduced by about 60% when the arteries were precontracted by 60 mM KCl instead of phenylephrine (1 μM). In addition, the ACh (1 μM)‐induced relaxations were almost abolished by EETs antagonist (14, 15 epoxyeicosa‐5(Z)‐enoic acid, 1 μM). Whole cell patch clamping demonstrated that VACC currents in primary cultured rat mesenteric arterial smooth muscle cells were partly inhibited by EETs. However, the inhibition effects were abolished by cyclic GMP (cGMP) antagonists (Rp‐cGMP, 100 μM), and cGMP‐dependent protein kinase inhibitors (KT‐5823, 0.3 μM). Our results suggest that EETs‐induced relaxations of mesenteric arteries were partly mediated by inhibition of VACCs through cGMP pathway. (Supported by the Research Grant Council of Hong Kong)

Role of nitric oxide/cyclic GMP pathway in regulating spontaneous excitations in detrusor smooth muscle of the guinea‐pig bladder

The role of nitric oxide (NO)/cyclic GMP (cGMP) pathway in regulating detrusor smooth muscle (DSM) function is still to be elucidated. We have investigated the effects of NO donors and phosphodiesterase-5 (PDE5) inhibition on spontaneous excitations in DSM. Multibundle DSM of the guinea-pig bladder generated spontaneous phasic contractions. The effects of sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), NO donors, 8-bromo-cyclicGMP (8-Br-cGMP), a cell-permeable cGMP analog and sildenafil, a PDE5 inhibitor on these contractions were examined. The effects of these agents on spontaneous action potentials were also studied using intracellular recording technique in single-bundle DSM. SNP and SIN-1 enhanced spontaneous contractions in multibundle DSM and increased the frequency of spontaneous action potentials in single-bundle DSM. These excitatory effects were not antagonized by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor for guanylate cyclase, but were attenuated by cyclopiazonic acid (CPA), an inhibitor of sarco- and endoplasmic Ca ATPase (SERCA). 8-Br-cGMP invariably suppressed spontaneous contractility. Sildenafil inhibited spontaneous contractions in about 65% of multibundle DSM but had no effects on the remainder. In single-bundle DSM, sildenafil had no effect on spontaneous action potentials. These results suggested that NO caused an enhancement of spontaneous contractions in DSM by accelerating spontaneous action potentials through cGMP-independent mechanisms, which may involve the Ca release from intracellular stores, whilst cGMP itself has inhibitory effects on DSM contractility. Sildenafil may indirectly suppress DSM contractility by diminishing synchronicity between functional units of DSM bundles without inhibiting excitability of DSM themselves.

Effect of Glutamine Synthesis Inhibition with Methionine Sulfoximine on the Nitric Oxide–Cyclic GMP Pathway in the Rat Striatum Treated Acutely with Ammonia: A Microdialysis Study

Ammonia neurotoxicity is associated with overactivation of N-methyl-D-aspartate (NMDA) receptors leading to enhanced nitric oxide and cyclic GMP synthesis and to accumulation of reactive oxygen and nitrogen species. Ammonia is detoxified in the brain via synthesis of glutamine, which if accumulated in excess contributes to astrocytic swelling, mitochondrial dysfunction and cerebral edema. This study was aimed at testing the hypothesis that the activity of the NMDA/NO/cGMP pathway is controlled by the ammonia-induced production of Gln in the brain. Ammonium chloride (final concentration 5 mM), infused for 40 min to the rat striatum via a microdialysis probe, caused a significant increase in Gln (by 40%), NO oxidation products (nitrite+nitrate=NOx) (by 35%) and cGMP (by 50%) concentration in the microdialysate. A Gln synthetase inhibitor, methionine sulfoximine (MSO, 5 mM), added directly to the microdialysate, completely prevented ammonia-mediated production of Gln, and paradoxically, it increased ammonia-mediated production of NOx and cGMP by 230% and 250%, respectively. Of note, MSO given alone significantly reduced basal Gln concentration in the rat striatum, had no effect on the basal NOx concentration, and attenuated basal concentration of cGMP in the microdialysate by 50%. The results of the present study suggest that Gln, at physiological concentrations, may ameliorate excessive activation of the NO-cGMP pathway by neurotoxic concentrations of ammonia. However, in view of potential direct interference of MSO with the pathway, exogenously added Gln and less toxic modulators of Gln content and/or transport will have to be employed in further studies on the underlying mechanisms.

Cannabinoid receptor-mediated translocation of NO-sensitive guanylyl cyclase and production of cyclic GMP in neuronal cells

Cannabinoid agonists regulate NO and cyclic AMP production in N18TG2 neuroblastoma cells, leading to the hypothesis that neuronal cyclic GMP production could be regulated by CB1 cannabinoid receptors. NO (nitric oxide)-sensitive guanylyl cyclase (GC) is a heterodimeric cytosolic protein that mediates the down-stream effects of NO. Genes of proteins in the cyclic GMP pathway (α1, α2, and β1 subunits of NO-sensitive GC and PKG1, but not PKG2) were expressed in N18TG2 cells, as was the CB1 but not the CB2 cannabinoid receptor. Stimulation of N18TG2 cells by cannabinoid agonists CP55940 and WIN55212-2 increased cyclic GMP levels in an ODQ-sensitive manner. GC-β1 in membrane fractions was increased after 5 or 20min stimulation, and was significantly depleted in the cytosol by 1h. The cytosolic pool of GC-β1 was replenished after 48h of continued cannabinoid drug treatment. Translocation of GC-β1 from the cytosol was blocked by the CB1 antagonist rimonabant (SR141716) and by the Gi/o inactivator pertussis toxin, indicating that the CB1 receptor and Gi/o proteins are required for translocation. Long-term treatment with rimonabant or pertussis toxin reduced the amount of GC-β1 in the cytosolic pool. We conclude that CB1 receptors stimulate cyclic GMP production and that intracellular translocation of GC from cytosol to the membranes is intrinsic to the mechanism and may be a tonically active or endocannabinoid-regulated process.

Interstitial cells of Cajal in the urethra as effectors of the nitric oxide action through the cyclic GMP pathway

Background Specialized cells similar to the interstitial cells of Cajal (ICC) of the gut have been described in the urethra where they seem to act as pacemakers of contractility [1]. Furthermore, nitric oxide (NO) is the main transmitter involved in relaxation of the urethra during micturition [2]. Here we show that urethral ICC are preferential targets of the NO action by the production of cyclic GMP (cGMP) in response to both exogenous addition or endogenous release of NO.

Targets for the Treatment of Erectile Dysfunction: Is NO/cGMP Still the Answer?

In recent years male sexual research has increasingly centered on molecular mechanisms operating from the central nervous system to peripheral end-organ levels involved in the penile erectile response. Major progress has been made in the field, and currently a whole host of neurotransmitters, chemical effectors, growth factors, second-messenger molecules, ions, intercellular proteins, and hormones have been characterized as components of the complex physiology of erectile function. Foremost among these mediators is nitric oxide (NO), which was initially characterized as a locally released physiologic mediator of the erectile response. Impaired formation and action of NO is closely associated with erectile dysfunction (ED), which may be caused by a variety of pathogenic factors. The impact of this knowledge has been substantial, leading to the development of several NO-based medical approaches for the treatment of ED. This review will focus on recent patents and current clinical trials involving innovative pharmacological and gene therapies in the field of male ED, particularly targeting the NO/intracellular cyclic GMP pathway, which still represents the most promising therapeutic approach to treat patients with ED.

Activation of soluble guanylate cyclase by nitric oxide is increased in lymphocytes from both rats chronically exposed to 2,5-hexanedione and workers chronically exposed to n-hexane

Although occupational exposure to n-hexane induces neurotoxic effects in the central and peripheral nervous systems, the mechanisms of its neurotoxicity remain unclear. n-Hexane is metabolized to 2,5-hexanedione (2,5-HD), which is the neurotoxic agent and the indicator chosen for the biological monitoring of exposed workers. It has been previously reported that chronic exposure to 2,5-HD impairs the glutamate–nitric oxide–cyclic GMP pathway at the level of activation of soluble guanylate cyclase (sGC) enzyme by nitric oxide (NO), both in cultured neurons and in the cerebellum of rats in vivo. The aim of this study was to assess whether the activation of sGC by NO is also altered in lymphocytes from rats treated with 2,5-HD and/or workers chronically exposed to n-hexane. Lymphocytes were isolated from male Wistar rats treated with 2,5-HD in drinking water, and from blood samples from shoe-factory workers environmentally and chronically exposed to n-hexane. Urine samples were also collected from workers at the end of the shift in order to measure the urinary levels of 2,5-HD. Activation of sGC by NO was significantly higher (p<0.05) in lymphocytes from rats treated with 2,5-HD than in control rats. In isolated lymphocytes from exposed workers the activation of sGC by NO also increases (p<0.05) in contrast to the controls. The results presented here indicate that the activation of lymphocytes could be an indicator of the toxicity produced by being exposed to n-hexane, since the effects observed in workers chronically exposed to n-hexane are similar to those found in rats chronically treated with 2,5-HD in drinking water.

Increased nitric oxide production in eating disorders

Animal studies showed that nitric oxide (NO)/cyclic-GMP (cGMP) pathway is involved in the modulation of eating behavior. To address its role in eating disorders (ED), plasma nitrite and cGMP levels were studied in 50 ED patients (25 with Anorexia Nervosa, AN; 25 with Bulimia Nervosa, BN) and 20 sex- and age-matched controls (C). Nitrites (nmol/mg protein, mean ± S.E.M.: any ED 1.01 ± 0.29; AN 1.15 ± 0.47; BN 0.88 ± 0.36; C 0.25 ± 0.07; p < 0.01) and cGMP (nmol/ml plasma, mean ± S.E.M.: any ED 2.58 ± 0.60; AN 2.81 ± 1.10; BN 2.41 ± 0.70; C 0.11 ± 0.05; p < 0.01) were significantly higher in ED patients than in C. Nitrite and cGMP levels inversely correlated with BMI in AN patients (nitrites: r = −0.62 p < 0.01; cGMP r = −0.45 p < 0.05) but not in BN patients (nitrites: r = −0.15 p = 0.49; cGMP: r = −0.05 p = 0.13) or in control subjects (nitrites: r = 0.11 p = 0.98; cGMP r = 0.37 p = 0.32). Significant correlations were also present in bulimic patients between nitrite levels, frequency of binges and several psychopathological dimensions, as assessed through the EDE. This is the first evidence of an alteration of the NO pathway in ED patients. Further studies are needed to ascertain whether an increase in NO levels plays a possible role in the pathogenesis of ED.