Nitric Oxide-cyclic Guanosine Monophosphate Signaling Research Articles

CGMP Signaling in the Neurovascular Unit-Implications for Retinal Ganglion Cell Survival in Glaucoma.

Glaucoma is a progressive age-related disease of the visual system and the leading cause of irreversible blindness worldwide. Currently, intraocular pressure (IOP) is the only modifiable risk factor for the disease, but even as IOP is lowered, the pathology of the disease often progresses. Hence, effective clinical targets for the treatment of glaucoma remain elusive. Glaucoma shares comorbidities with a multitude of vascular diseases, and evidence in humans and animal models demonstrates an association between vascular dysfunction of the retina and glaucoma pathology. Integral to the survival of retinal ganglion cells (RGCs) is functional neurovascular coupling (NVC), providing RGCs with metabolic support in response to neuronal activity. NVC is mediated by cells of the neurovascular unit (NVU), which include vascular cells, glial cells, and neurons. Nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling is a prime mediator of NVC between endothelial cells and neurons, but emerging evidence suggests that cGMP signaling is also important in the physiology of other cells of the NVU. NO-cGMP signaling has been implicated in glaucomatous neurodegeneration in humans and mice. In this review, we explore the role of cGMP signaling in the different cell types of the NVU and investigate the potential links between cGMP signaling, breakdown of neurovascular function, and glaucoma pathology.

Probable mechanisms involved in the antiepileptic activity of Clerodendrum polycephalum Baker (Labiatae) leaf extract in mice exposed to chemical-induced seizures.

The leaves of Clerodendrum polycephalum Baker (Labiatae) are used as a dietary legume supplement and applied ethnomedicinally for the management of epilepsy, convulsion, and spasms. This study is aimed at evaluating the effects of Clerodendrum polycephalum (CP) leaf extract on chemical-induced seizures in mice and the possible mechanisms of action. Swiss albino mice were pretreated with CP (50, 100, or 500 mg/kg, p.o.) prior to intraperitoneal injection of picrotoxin (PTX) or pentylenetetrazole (PTZ). However, the most effective dose was used to elucidate the role of GABAergic and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling mechanisms in mice brains. Accordingly, we evaluated the preventive and reversal effects of CP on kainic acid (KA)-induced temporal lobe epilepsy (TLE), oxidative stress, and neuroinflammatory in mice. The pretreatment of mice with CP delayed the latencies to PTX and PTZ-induced seizures and decrement in the period of tonic-clonic attacks. Interestingly, CP (100 mg/kg) completely prevented PTZ-induced tonic-clonic seizures. Contrastingly, flumazenil (benzodiazepine receptor antagonist), NG -nitro-L-Arginine (L-NNA) (10 mg/kg., neuronal nitric oxide synthase inhibitor), and methylene blue (MB) (2 mg/kg, a soluble guanylyl cyclase inhibitor) but not L-arginine (150 mg/kg., nitric oxide precursor) reversed CP-induced anticonvulsant-like effect in PTZ model. Furthermore, KA-elicited TLE was prevented by CP treatment. CP also attenuated KA-induced oxidative stress, cyooxygenase-2 (COX-2), and nuclear factor kappa-B (NF-κB) elevated expressions in the hippocampus. The study revealed that the ethanolic leaf extract of CP produced anticonvulsant actions through enhancement of antioxidant defense, GABAergic, and NO-cGMP signaling pathways as well as attenuation of inflammatory processes. PRACTICAL APPLICATIONS: The leaves of Clerodendrum polycephalum Baker (Labiatae) are used as a dietary legume supplement and applied ethnomedicinally for the management of epilepsy, convulsion, and spasms. For this reason, we believe that supplementation of the Clerodendrum polycephalum leaf extract would prevent epileptic-related disorders in mice induced with epileptic conditions using kainic acid and other behavioral phenotypic models. Here, our findings clearly revealed that Clerodendrum polycephalum leaf extract protects against conditions of epileptic-related disorders and thus might be relevant as a dietary supplement in the prevention or delay of the onset of seizures and epileptic behavior.

Pharmacological developments in antihypertensive treatment through nitric oxide-cGMP modulation.

Treatment of hypertension until now has been directed at inhibition of vasoconstriction, of cardiac contractility and of blood volume regulation. Despite the arsenal of drugs available for this purpose, the control of target blood pressure is still a difficult goal to reach in outpatients. The nitric oxide-cyclic guanosine monophosphate signaling is one of the most important mediators of vasodilation. It might therefore be a potential and most welcome drug target for optimization of the treatment of hypertension. In this chapter we review the problems that can occur in this signaling system, the attempts that have been made to correct these problems, and those that are still under investigation. Recently developed, clinically safe medicines that are currently approved for other applications, such as myocardial infarction, await to be tested for essential systemic hypertension. We conclude that despite many years of research without translation, stimulation of nitric oxide-cyclic guanosine monophosphate is still a viable strategy in the prevention of the health risk posed by chronic hypertension.

Latent Sex Differences in CaMKII-nNOS Signaling That Underlie Antidepressant-Like Effects of Yueju-Ganmaidazao Decoction in the Hippocampus.

Previous studies have demonstrated that Yueju-Ganmaidazao (YG) decoction induces rapid antidepressant-like effects, and the antidepressant response is mostly dependent on the suppression of nitric oxide-cyclic guanosine monophosphate signaling in male mice. This study aimed to investigate the sex difference mediated by calcium/calmodulin-dependent protein kinase II (CaMKII)-neuronal nitric oxide synthase (nNOS) signaling involved in the antidepressant-like effect of YG in mice. We found that the immobility times in the tail suspension test (TST) were found to be decreased after the single injection of YG in male and female mice with the same dosage. Additionally, chronic administration for 4 days of subthreshold dosage of YG and escitalopram (ES) also significantly decreased the immobility time in mice of both sexes. Chronic subthreshold dosage of YG and ES in LPS-treated mice and in chronic unpredictable stress (CUS) mice both decreased the immobility time, which was increased by stress. Meanwhile, in CUS-treated mice, sucrose preference test, forced swimming test, and open field test were applied to further confirm the antidepressant-like effects of YG and ES. Moreover, CUS significantly decreased the expression of nNOS and CaMKII, and both YG and ES could enhance the expression in the hippocampus of female mice, which was opposite to that in male mice, while endothelial nitric oxide synthase expression was not affected by stress or drug treatment neither in male mice nor in female mice. Finally, subthreshold dosage of YG combined with 7-nitroindazole (nNOS inhibitor) induced the antidepressant-like effects both in female and in male mice, while the single use of YG or 7-NI did not display any effect. However, pretreatment with KN-93 (CaMKII inhibitor) only blocked the antidepressant-like effect of high-dosage YG in female mice. Meanwhile, in CUS mice, chronic stress caused NR1 overexpression and inhibited cAMP response element binding protein action, which were both reversed by YG and ES in male and female mice, implying that YG and ES produced the same antidepressant-like effect in mice of both sexes. The study revealed that chronic treatment with a subthreshold dose of YG also produced antidepressant-like effects in female mice, and these effects depended on the regulation of the CaMKII-nNOS signaling pathway.

Nitric Oxide-cGMP Signaling in Hypertension: Current and Future Options for Pharmacotherapy.

For the treatment of systemic hypertension, pharmacological intervention in nitric oxide-cyclic guanosine monophosphate signaling is a well-explored but unexploited option. In this review, we present the identified drug targets, including oxidases, mitochondria, soluble guanylyl cyclase, phosphodiesterase 1 and 5, and protein kinase G, important compounds that modulate them, and the current status of (pre)clinical development. The mode of action of these compounds is discussed, and based upon this, the clinical opportunities. We conclude that drugs that directly target the enzymes of the nitric oxide-cyclic guanosine monophosphate cascade are currently the most promising compounds, but that none of these compounds is under investigation as a treatment option for systemic hypertension.

Penile Erection Induced by Scoparone from Artemisia capillaris through the Nitric Oxide-Cyclic Guanosine Monophosphate Signaling Pathway.

PurposeThe objective of this study was to evaluate the relaxant effect of scoparone from Artemisia capillaris on rabbit penile corpus cavernosum smooth muscle (PCCSM) and to elucidate the mechanism of action of scoparone for the treatment of erectile dysfunction (ED).Materials and MethodsPCCSM that had been precontracted with phenylephrine was treated with 3 Artemisia herbs (A. princeps, A. capillaris, and A. iwayomogi) and 3 fractions (n-hexane, ethyl acetate, and n-butanol) with different concentrations (0.1, 0.5, 1.0, and 2.0 mg/mL). Four components (esculetin, scopoletin, capillarisin, and scoparone) isolated from A. capillaris were also evaluated. The PCCSM was preincubated with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). Cyclic nucleotides in the perfusate were measured by a radioimmunoassay. The interactions of scoparone with udenafil and rolipram were also evaluated.ResultsA. capillaris extract relaxed PCCSM in a concentration-dependent manner. Scoparone had the highest relaxant effect on PCCSM among the 4 components (esculetin, scopoletin, capillarisin, and scoparone) isolated from the ethyl acetate fraction. The application of scoparone on PCCSM pretreated with L-NAME and ODQ led to significantly less relaxation. Scoparone also increased the cyclic guanosine monophosphate (cGMP) levels in the perfusate in a concentration-dependent manner. Furthermore, scoparone enhanced udenafil- and rolipram-induced relaxation of the PCCSM.ConclusionsScoparone relaxed the PCCSM mainly by activating the nitric oxide-cGMP signaling pathway, and it may be a new promising treatment for ED patients who do not completely respond to udenafil.

Sympathetic Hyperactivity, Increased Tyrosine Hydroxylase and Exaggerated Corpus Cavernosum Relaxations Associated with Oxidative Stress Plays a Major Role in the Penis Dysfunction in Townes Sickle Cell Mouse.

BackgroundSickle cell disease patients display priapism that may progress to erectile dysfunction. However, little is known about the pathophysiological alterations of corpus cavernosum in sickle cell disease.ObjectiveThus, this study aimed to evaluate the functional and molecular alterations of sympathetic machinery and nitric oxide—cyclic guanosine monophosphate signaling pathway in Townes transgenic sickle cell disease mice.MethodsConcentration–response curves to contractile (phenylephrine) and relaxant agents (acetylcholine and sodium nitroprusside) were obtained in corpus cavernosum strips from sickle and C57BL/6 (control) mice. Neurogenic contractions and nitrergic relaxations were obtained using electrical-field stimulation. Measurements of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), phosphodiesterase-5 (PDE5) and α1A-, α1B- and α1D-adrenoceptor mRNA expressions and reactive-oxygen species were performed. Tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expressions in cavernosal tissues were also measured.ResultsThe neurogenic contractions were higher in the sickle cell disease group, in association with elevated tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expression, as well as increased tyrosine hydroxylase mRNA expression. Likewise, phenylephrine-induced contractions were greater in the sickle mice, whereas α1A-, α1B- and α1D-adrenoceptor mRNA expression remained unchanged. Cavernosal relaxations to acetylcholine, sodium nitroprusside and EFS were higher in sickle mice, accompanied by decreased eNOS and nNOS, along with lower PDE5 mRNA expression. An increase of about 40% in reactive-oxygen species generation in corpus cavernosum from sickle mice was also detected.ConclusionOur study shows that decreased nitric oxide bioavailability in erectile tissue due to increased oxidative stress leads to both sympathetic hyperactivity and dysregulation of nitric oxide signaling in corpus cavernosum from Townes sickle mice.

The role of NMDA receptor and nitric oxide/cyclic guanosine monophosphate pathway in the antidepressant-like effect of dextromethorphan in mice forced swimming test and tail suspension test.

Depression is a devastating disorder which has a high impact on the wellbeing of overall society. As such, need for innovative therapeutic agents are always there. Most of the researchers focused on N-methyl-d-aspartate receptor to explore the antidepressant like activity of new therapeutic agents. Dextromethorphan is a cough suppressant agent with potential antidepressant activity reported in mouse force swimming test. Considering N-methyl-d-aspartate as a forefront in exploring antidepressant agents, here we focused to unpin the antidepressant mechanism of dextromethorphan targeting N-methyl-d-aspartate receptor induced nitric oxide-cyclic guanosine monophosphate signaling. Dextromethorphan administered at a dose of 10 and 30mg/kg i.p significantly reduced the immobility time. Interestingly, this effect of drug (30mg/kg) was inhibited when the animals were pretreated either with N-methyl-d-aspartate (75mg/kg), or l-arginine (750mg/kg) as a nitric oxide precursor and/or sildenafil (5mg/kg) as a phosphodiesterase 5 inhibitor. However, the antidepressant effect of Dextromethorphan subeffective dose (3mg/kg) was augmented when the animals were administered with either L-NG-Nitroarginine methyl ester (10mg/kg) non-specific nitric oxide synthase inhibitor, 7-Nitroindazole (30mg/kg) specific neural nitric oxide synthase inhibitor, MK-801 (0.05mg/kg) an N-methyl-d-aspartate receptor antagonist but not aminoguanidine (50mg/kg) which is specific inducible nitric oxide synthase inhibitor as compared to the drugs when administered alone. No remarkable effect on locomotor activity was observed during open field test when the drugs were administered at the above mentioned doses. Therefore, it is evident that the antidepressant like effect of Dextromethorphan is owed due to its inhibitory effect on N-methyl-d-aspartate receptor and NO- Cyclic guanosine monophosphate pathway.

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms.

Skeletal muscle nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSμ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSμ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1-/- muscle. Functional analyses of GC1-/- muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA-IIX fiber balance. Force deficits in GC1-/- muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency. Antioxid. Redox Signal. 26, 966-985.

ShRNA Constructs Targeting IGFBP-3 Alleviate Age Related Erectile Dysfunction in the Rat

We investigated whether injecting shRNA constructs targeting IGFBP-3 in the penis of old rats would improve erectile function. The most validated IGFBP-3 shRNA plasmid vector (pGPU6/GFP/Neo-shIGFBP-3) was prepared and injected in penile corpus cavernosum tissue. A total of 30 old (age 24 months) male Sprague Dawley® rats were randomly divided into 3 groups, including 10 each that received phosphate buffered saline only (100 μl), pGPU6/GFP/Neo-shNC (100 μg) and the most validated plasmid constructs pGPU6/GFP/Neo-shIGFBP-3 (100 μg). At 4 weeks the erectile response was measured as intracavernous pressure. The percent ofsmooth muscle in corpus cavernosum tissue was evaluated. Nitric oxide synthase activity and the cGMP concentration in penile tissue were also analyzed. IGFBP-3 was estimated in penile tissue by Western blot, real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. pGPU6/GFP/Neo-shIGFBP-3 corrected the impaired erectile response in aged rats compared with the response in those injected with phosphate buffered saline and pGPU6/GFP/Neo-shNC (each p <0.01). The percent of cavernous smooth muscle was increased in the pGPU6/GFP/Neo-shIGFBP-3 group. Nitric oxide synthase activity and the cGMP concentration were also significantly increased in rats treated with pGPU6/GFP/Neo-shIGFBP-3. IGFBP-3 shRNA effectively reduced IGFBP-3 mRNA and protein expression in penile corpus cavernosum tissue. Decreasing IGFBP-3 expression by plasmid expressed shRNA improved erectile function in aged rats. The therapy may modulate smooth muscle integrity and increase the cGMP concentration. This may be a new direction for treating erectile dysfunction in clinical practice.

Sildenafil Increases Muscle Protein Synthesis and Reduces Muscle Fatigue

Reductions in skeletal muscle function occur during the course of healthy aging as well as with bed rest or diverse diseases such as cancer, muscular dystrophy, and heart failure. However, there are no accepted pharmacologic therapies to improve impaired skeletal muscle function. Nitric oxide may influence skeletal muscle function through effects on excitation-contraction coupling, myofibrillar function, perfusion, and metabolism. Here we show that augmentation of nitric oxide-cyclic guanosine monophosphate signaling by short-term daily administration of the phosphodiesterase 5 inhibitor sildenafil increases protein synthesis, alters protein expression and nitrosylation, and reduces fatigue in human skeletal muscle. These findings suggest that phosphodiesterase 5 inhibitors represent viable pharmacologic interventions to improve muscle function.

Defective Nitric Oxide–Cyclic Guanosine Monophosphate Signaling in Patients With Bipolar Disorder

Bipolar disorder (BD) is associated with elevated cardiovascular mortality rates. We investigated the modulation of l-arginine-nitric oxide (NO) signaling in platelets from patients with BD at different phases. Platelets obtained from 28 patients with BD and 10 healthy volunteers were analyzed for l-arginine transport, NO synthase (NOS) activity, cyclic guanosine monophosphate content, and biomarkers of oxidative stress. Expressions of NOS isoforms, soluble guanylyl cyclase, and arginase were also measured in platelets. Amino acid and C-reactive protein levels in plasma were assessed. Plasma concentrations of l-arginine (mean [M] ± standard error of the mean [SEM] = 97 ± 10 versus 121 ± 10 µM) and its transport into platelets (median [interquartile range] = 26.0 [28.6] versus 26.5 [43.9] pmol/10(9) cells per minute) did not differ between patients with BD and controls (p > .05). Patients with BD showed reduced NOS activity (M ± SEM = 0.037 ± 0.003 versus 0.135 ± 0.022 pmol/10(8) cells, p < .001), but not endothelial NOS, inducible NOS, and arginase expression, compared with controls (p > .05). Cyclic guanosine monophosphate content was reduced (M ± SEM = 0.022 ± 0.003 versus 0.086 ± 0.020 pmol/10(8) cells, p < .05) despite the absence of changes in soluble guanylyl cyclase expression (median [interquartile range] = 21.6 [15.5] versus 9.5 [9.4] arbitrary units, p > .05) in patients with BD. Superoxide dismutase activity, but not catalase activity, was increased in patients with BD in the manic phase (M ± SEM = 2094 ± 335 versus 172 ± 17 U/mg protein, p < .001). C-reactive protein was elevated only in manic episodes (M ± SEM = 0.8 ± 0.2 versus 0.1 ± 0.02 mg/L, p < .001). Impaired NO generation from platelets, inflammation, and oxidative stress may play pivotal roles in the multifaceted process of cardiovascular events in BD.

Effects of Icariside II on Improving Erectile Function in Rats With Streptozotocin‐Induced Diabetes

Icariin and icariside II (ICA II), 2 active components isolated from herba epimedii, have a closely structural relationship. There is evidence that icariin may be useful in the treatment of erectile dysfunction (ED); however, the study on the therapeutic efficacy of ICA II on ED is currently scant. We investigated the effects of ICA II on improving erectile function of rats with streptozocin-induced diabetes. Fifty 8-week-old Sprague-Dawley rats were randomly distributed into normal control and diabetic groups. Diabetes was induced by a one-time intraperitoneal injection of streptozocin (60 mg/kg). Three days later, the diabetic rats were randomly divided into 4 groups including a saline-treated placebo arm and 3 ICA II-treated models (1, 5, and 10 mg/kg/d). After 3 months, penile hemodynamics was measured by cavernous nerve electrostimulation (CNE) with real time intracorporal pressure assessment. Penises were harvested with subsequent histological examination (picrosirius red stain, Hart elastin stain, and immunohistochemical stain) and Western blots to explore the expression of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and transforming growth factor β1 (TGFβ1)/Smad2 signaling pathways. Diabetes significantly attenuated erectile responses to CNE. Diabetic rats had decreased corpus cavernosum smooth muscle/collagen ratio and endothelial cell content relative to the control group. The ratio of collagen I to III was significantly lower in the corpora of diabetic rats; furthermore, cavernous elastic fibers were fragmented in the diabetic animals. Neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase, and vascular endothelial growth factor were expressed at lower levels in the diabetic group; ICA II-treated diabetic rats had higher expression in the penis relative to placebo-treated diabetic animals. Both the TGFβ1/Smad2/connective tissue growth factor (CTGF) signaling pathway and apoptosis were down-regulated in the penis from ICA II-treated rats. ICA II treatment attenuates diabetes-related impairment of penile hemodynamics, likely by increasing smooth muscle, endothelial function, and nNOS expression. ICA II could alter corpus cavernosum fibrous-muscular pathological structure in diabetic rats, which could be regulated by the TGFβ1/Smad2/CTGF and NO-cGMP signaling pathways.

Peripheral estradiol induces temporomandibular joint antinociception in rats by activating the nitric oxide/cyclic guanosine monophosphate signaling pathway

Recently, we have reported that high physiological estradiol level during the proestrus phase of the estrous cycle or systemic estradiol administration in ovariectomized rats decreases formalin-induced temporomandibular joint nociception. However, the mechanisms underlying the antinociceptive effect of estradiol are presently unknown. In this study, we used the temporomandibular joint formalin model in rats to investigate whether estradiol decreases nociception by a peripheral non-genomic mechanism, and if so, whether this mechanism is mediated by the activation of the nitric oxide–cyclic guanosine monophosphate signaling pathway and of opioid receptors. The administration of estradiol into the ipsilateral, but not into the contralateral temporomandibular joint significantly reduced formalin-induced temporomandibular joint nociception in ovariectomized and diestrus but not in proestrus females. However, the administration of the estrogen receptor antagonist ICI 182780 into the ipsilateral, but not into the contralateral temporomandibular joint blocked the antinociceptive effect of serum estradiol in proestrus females, suggesting that the physiological effect of estradiol in nociception is mediated, at least in part, by a peripheral mechanism. The administration of estradiol into the ipisilateral temporomandibular joint did not affect formalin-induced nociception in male rats. The antinociceptive effect of temporomandibular joint estradiol administration in ovariectomized and diestrus females was mimicked by estradiol conjugated with bovine serum albumin, which does not diffuse through the plasma membrane, and was blocked by the estrogen receptor antagonist ICI 182780. The administration of the nitric oxide synthase inhibitor (nitro- l-arginine) or of a guanylate cyclase inhibitor (1H-(1,2,4)-oxadiasolo (4,2-a) quinoxalin-1-one) into the ipsilateral, but not into the contralateral temporomandibular joint blocked the antinociceptive effect of estradiol and of estradiol conjugated with bovine serum albumin, while the opioid receptor antagonist naloxone had no effect. These findings suggest that estradiol decreases temporomandibular joint nociception in female rats through a peripheral non-genomic activation of the nitric oxide–cyclic guanosine monophosphate signaling pathway.

Nitric oxide–cyclic guanosine monophosphate signaling in the local circuit of the cricket abdominal nervous system

The distribution of potential nitric oxide (NO) donor neurons and NO-responsive target neurons was revealed in the terminal abdominal ganglion (TAG) of the cricket. The expression of nitric oxide synthase (NOS) in the nervous system was examined by Western blotting using universal nitric oxide synthase (uNOS) antibody that gave about a 130 kDa protein band. Immunohistochemistry using the uNOS antibody detected neurons whose cell bodies are located at the lateral region of the TAG. These neurons expanded their neuronal branches into the dorsal–median region or the dorsal–lateral region of the TAG. NADPH-diaphorase histochemistry was performed to confirm the distribution of NOS-containing neurons. The distributions of cell bodies and stained neuronal branches were similar to those revealed by uNOS immunohistochemistry. NO-induced cGMP immunohistochemistry was performed to reveal NO-responsive target neurons. Most of the cell bodies stained by immunohistochemistry appeared at the dorsal side of the TAG. At the dorsal–median region, some unpaired neuronal cell bodies were strongly stained. Some efferent neurons whose axon innervate into each nerve root were strongly stained. The generation of NO in the TAG was detected by NO electrode. We found that NO is generally produced to maintain a basal concentration of 70 nM. Hemoglobin scavenged released NO from the ganglion. The concentration of NO was partly recovered when hemoglobin was replaced by normal saline. Application of 10 μM l-arginine that is a substrate of NOS increased NO release by approximately 10 nM. Furthermore, an excitatory neurotransmitter acetylcholine (ACh) also increased NO generation by approximately 40–50 nM in concentration in addition to the basal level of 70 nM. Optical imaging with fluorescent NO-indicator demonstrated that ACh-induced enhancement of NO release was transiently observed in the outer-edge region of TAG, where cell bodies of NOS-immunoreactive neurons were located. These results suggest that ACh accelerates NO production via neuronal events activated by ACh in the TAG.

Insulin-Like Growth Factor-1 Restores Erectile Function in Aged Rats: Modulation the Integrity of Smooth Muscle and Nitric Oxide-Cyclic Guanosine Monophosphate Signaling Activity

Insulin-like growth factor-1 (IGF-1) is one of the growth factors that have a wide range of biologic effects. We have confirmed that gene transfer of IGF-1 to the penis could improve erectile capacity. However, there are some limitations in gene therapies, such as toxicity or a risk of insertional mutagenesis. Protein treatment may be another choice for decreasing these risks. To investigate whether intracavernosal injection of IGF-1 protein can restore erectile function in the aging rat. Erectile responses, morphological changes, and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathways-related marker were determined. Ten young (4 months) and 30 old (24 months) Sprague-Dawley male rats were enrolled in this study. The old rats were divided into three groups: vehicle-only (N = 10), IGF-1 1 microg/kg (N = 10) and IGF-1 10 microg/kg treatment group (N = 10). After 4 and 8 weeks of single IGF-1 injection treatment, intracavernous pressure (ICP) responses with electrical stimulation to the cavernous nerve were evaluated. The percent of smooth muscle in corpus cavernosum tissue, the expression of mRNA and protein of endothelial nitric oxide synthase (eNOS) were also evaluated. The activity of nitric oxide synthase (NOS) and concentration of guanosine 3',5'-cyclic-monophosphate (cGMP) that act upon the major NO-cGMP signaling pathways in penile tissue were also analyzed. After IGF-1 treatment, the ICP responses was significantly increased as the young control group in both the IGF-1 1 microg/kg and the IGF-1 10 microg/kg group compared with the vehicle-only group at 4 and 8 weeks (P < 0.05). Masson's trichrom staining showed the percentage of cavernosal smooth muscle was increased in IGF-1 treatment group. IGF-1 increased e-NOS expression. NOS activities and cGMP concentrations were also significantly increased in IGF-1 treatment rats. IGF-1 improved erectile function in aged rats via restoration the integrity of smooth muscle of corpus cavernosum and modulation of NO-cGMP pathways.

Bypassing Vasopressin Receptor Signaling Pathways in Nephrogenic Diabetes Insipidus

Water reabsorption in the kidney represents a critical physiological event in the maintenance of body water homeostasis. This highly regulated process relies largely on vasopressin (VP) action and on the VP-sensitive water channel (AQP2) that is expressed in principal cells of the kidney collecting duct. Defects in the VP signaling pathway and/or in AQP2 cell surface expression can lead to an inappropriate reduction in renal water reabsorption and the development of nephrogenic diabetes insipidus, a disease characterized by polyuria and polydipsia. This review focuses on the major regulatory steps that are involved in AQP2 trafficking and function. Specifically, we begin with a discussion on VP-receptor-independent mechanisms of AQP2 trafficking, with special emphasis on the nitric oxide-cyclic guanosine monophosphate signaling pathway, followed by a review of the mechanisms that govern AQP2 endocytosis and exocytosis. We then discuss emerging data illustrating roles played by the actin cytoskeleton on AQP2 trafficking, and lastly we consider elements that affect AQP2 protein expression in cells. Recent advances in each topic are summarized and are presented in the context of their potential to serve as a basis for the development of novel therapies that may ultimately improve life quality of nephrogenic diabetes insipidus patients.

Activation of Nitric Oxide—Cyclic Guanosine Monophosphate Signaling in Kidney by Extracorporeal Shock Wave Therapy

We defined whether extracorporeal shock wave therapy (ESWT) to the kidney activates the nitric oxide (NO)-cyclic 3',5'-guanosine monophosphate (cGMP) pathway. A total of 90 male rabbits were randomly divided into group 1--pretreated with normal saline, group 2--pretreated intravenously (i.v.) with Nomega nitro-L-arginine-methyl ester (NAME) (100 mg/kg), group 3--pretreated i.v. with NAME and L-arginine (300 mg/kg) with ESWT to 1 kidney, group 4--pretreated IV with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) (20 microg/kg) and group 5--pretreated with normal saline with ESWT to the bladder. Plasma nitrite, NO metabolite and cGMP were analyzed in peripheral blood samples before, immediately after, and 30 and 60 minutes after ESWT. ESWT to the kidney but not to the bladder caused an increase before, immediately after, and 30 and 60 minutes after ESWT in plasma nitrite in group 1 (186.1 +/- 20.6, 217.5 +/- 21.6, 241.9 +/- 28.4 and 230.5 +/- 25.3 nM) and group 5 (149.0 +/- 14.7, 155.6 +/- 18.4, 131.8 +/- 13.6 and 140.0 +/- 15.7 nM), and in cGMP in group 1 (24.2 +/- 1.9, 33.8 +/- 3.2, 32.9 +/- 2.2 and 29.4 +/- 1.9 pmol/ml) and group 5 (25.5 +/- 2.1, 27.5 +/- 2.5, 28.7 +/- 3.1 and 25.5 +/- 2.6 pmol/ml, respectively). In group 2 NAME significantly inhibited the production of nitrite (113.4 +/- 18.6, 118.2 +/- 19.9, 114.6 +/- 18.3 and 112.5 +/- 17.6 nM) and cGMP (19.4 +/- 2.6, 20.6 +/- 2.8, 19.3 +/- 2.7 and 18.6 +/- 2.6 pmol, respectively). In group 3 inhibited nitrite and cGMP production caused by NAME was recovered with L-arginine. In group 4 ODQ significantly inhibited cGMP production. The results show that ESWT increases the level of NO and cGMP released by the kidney in an animal model.

Regulation of ciliary beat frequency by the nitric oxide-cyclic guanosine monophosphate signaling pathway in rat airway epithelial cells.

Nitric oxide (NO) upregulates ciliary beat frequency (CBF). The present study evaluates mechanisms of the NO-cyclic guanosine monophosphate (cGMP) pathway regulation of CBF. Rat tracheal explants were loaded with 4,5-diaminofluorescein diacetate for the demonstration of NO production by ciliated epithelial cells after L-arginine (L-Arg) stimulation. CBF was measured using phase contrast microscopy and videotape analysis. The roles of NO, soluble guanylate cyclase (sGC), cGMP-dependent protein kinase (PK) G, and phosphodiesterase (PDE) V in regulation of CBF were evaluated. NO synthase (NOS) was activated with L-Arg or inhibited with N(G)-monomethyl-L-Arg. sGC was stimulated with NO donors 1-hydroxy-2-oxo-3- (N-ethyl-2-aminoethyl)-3-ethyl-1-triazene and S-nitroso-L-glutathione or mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP) and inhibited with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one. The effects of the PKG inhibition with KT5823 and PDE V inhibition with Zaprinast were also examined. The studies demonstrate that ciliated epithelial cells produce NO, which is correlated with CBF stimulation. L-Arg dose- and time-dependently increases CBF, and NO donors, 8-Br-cGMP, and Zaprinast also enhance CBF. Inhibitors of NOS, sGC, and PKG can block the stimulant effect of L-Arg on CBF. Thus, NO is a regulator of CBF acting via sGC and PKG. The NO-cGMP signaling pathway regulates CBF in an autocrine manner in cultured rat ciliated airway epithelium.