Endogenous Nitric Oxide Signaling Research Articles

Nitric oxide signal is required for glutathione-induced enhancement of photosynthesis in salt-stressed Solanum lycopersicum L.

Reduced glutathione (γ-glutamyl-cysteinyl-glycine, GSH), the primary non-protein sulfhydryl group in organisms, plays a pivotal role in the plant salt stress response. This study aimed to explore the impact of GSH on the photosynthetic apparatus, and carbon assimilation in tomato plants under salt stress, and then investigate the role of nitric oxide (NO) in this process. The investigation involved foliar application of 5 mM GSH, 0.1% (w/v) hemoglobin (Hb, a nitric oxide scavenger), and GSH+Hb on the endogenous NO levels, rapid chlorophyll fluorescence, enzyme activities, and gene expression related to the Calvin cycle in tomato seedlings (Solanum lycopersicum L. cv. 'Zhongshu No. 4') subjected short-term salt stress (100 mM NaCl) for 24, 48 and 72 hours. GSH treatment notably boosted nitrate reductase (NR) and NO synthase (NOS) activities, elevating endogenous NO signaling in salt-stressed tomato seedling leaves. It also mitigated chlorophyll fluorescence (OJIP) curve distortion and damage to the oxygen-evolving complex (OEC) induced by salt stress. Furthermore, GSH improved photosystem II (PSII) electron transfer efficiency, reduced QA - accumulation, and countered salt stress effects on photosystem I (PSI) redox properties, enhancing the light energy absorption index (PIabs). Additionally, GSH enhanced key enzyme activities in the Calvin cycle and upregulated their genes. Exogenous GSH optimized PSII energy utilization via endogenous NO, safeguarded the photosynthetic reaction center, improved photochemical and energy efficiency, and boosted carbon assimilation, ultimately enhancing net photosynthetic efficiency (Pn) in salt-stressed tomato seedling leaves. Conversely, Hb hindered Pn reduction and NO signaling under salt stress and weakened the positive effects of GSH on NO levels, photosynthetic apparatus, and carbon assimilation in tomato plants. Thus, the positive regulation of photosynthesis in tomato seedlings under salt stress by GSH requires the involvement of NO.

SMA-BmobaSNO: an intelligent photoresponsive nitric oxide releasing polymer for drug nanoencapsulation and targeted delivery

Nitric oxide (NO) is an important biological signalling molecule that acts to vasodilate blood vessels and change the permeability of the blood vessel wall. Due to these cardiovascular actions, co-administering NO with a therapeutic could enhance drug uptake. However current NO donors are not suitable for targeted drug delivery as they systemically release NO. To overcome this limitation we report the development of a smart polymer, SMA-BmobaSNO, designed to release NO in response to a photostimulus. The polymer’s NO releasing functionality is an S-nitrosothiol group that, at 10 mg ml−1, is highly resistant to both thermal (t 1/2 16 d) and metabolic (t 1/2 32 h) decomposition, but rapidly brakes down under photoactivation (2700 W m−2, halogen source) to release NO (t 1/2 25 min). Photoresponsive NO release from SMA-BmobaSNO was confirmed in a cardiovascular preparation, where irradiation resulted in a 12-fold decrease in vasorelaxation EC50 (from 5.2 μM to 420 nM). To demonstrate the polymer’s utility for drug delivery we then used SMA-BmobaSNO to fabricate a nanoparticle containing the probe Nile Red (NR). The resulting SMA-BmobaSNO-NR nanoparticle exhibited spherical morphology (180 nm diameter) and sustained NR release (≈20% over 5 d). Targeted delivery was characterised in an abdominal preparation, where photoactivation (450 W m−2) caused localized increases in vasodilation and blood vessel permeability, resulting in a 3-fold increase in NR uptake into photoactivated tissue. Nanoparticles fabricated from SMA-BmobaSNO therefore display highly photoresponsive NO release and can apply the Trojan Horse paradigm by using endogenous NO signalling pathways to smuggle a therapeutic cargo into target tissue.

A Phase 2a study evaluating the safety, tolerability, pharmacokinetics, and central nervous system activity of CY6463 when administered to participants with Alzheimer’s disease with vascular pathology

AbstractBackgroundThe soluble guanylate cyclase (sGC) stimulator, CY6463, is being developed as a symptomatic and potentially disease‐modifying therapy for serious central nervous system (CNS) diseases, including Alzheimer’s disease (AD). Nitric oxide (NO)‐sGC‐cyclic guanosine monophosphate (cGMP) is a fundamental neurotransmitter system critical to neuronal function. Impaired signaling of this pathway is known to play a role in the pathogenesis of many neurodegenerative diseases. CY6463, a positive allosteric modulator of sGC, amplifies endogenous NO signaling to increase cGMP production. In preclinical models, CY6463 improved neuronal function, cerebral blood flow, neuroinflammation, and cellular bioenergetics.CY6463 has been evaluated in two Phase 1 studies (110 healthy younger adult volunteers and 24 healthy elderly volunteers >65 years of age) assessing the safety, tolerability, pharmacokinetics (PK), and CNS activity of CY6463. Once‐daily CY6463 administration was well tolerated across dose levels, led to target CNS concentrations, achieved predictable/linear food‐independent PK, and had positive impacts on saccadic eye movements as well as brain neurophysiology as measured by electroencephalography (EEG).MethodThis 12‐week, randomized, placebo‐controlled study will evaluate safety, tolerability, PK, and CNS activity of CY6463 in approximately 40 patients with a combination of AD pathology, sub‐cortical vascular disease, and cardiovascular risk factors. This defined subset of the larger AD population, referred to as AD with vascular pathology (ADv), is hypothesized to be more likely to respond to CY6463 treatment due to CY6463’s expected impact on vascular and CNS biology. This study is funded in part by a Translational Research Award from the Alzheimer’s Association Part the Cloud‐Gates Partnership Grant Program.ResultThe primary objective, safety and tolerability, will be evaluated based on adverse event reporting. Exploratory evaluations assessed variously at baseline and weeks 2, 4, 8, and 12, will include plasma and CSF biomarkers of disease; PK; quantitative EEG and event related potentials as biomarkers of neurophysiology; functional magnetic resonance imaging measures of cerebral perfusion and brain connectivity; and cognitive performance tests of attention, executive function, and memory.ConclusionResults from this study in patients with ADv will inform the design of larger, longer studies evaluating the potential for CY6463 to improve cognition in this patient population.

Molybdenum-induced endogenous nitric oxide (NO) signaling coordinately enhances resilience through chlorophyll metabolism, osmolyte accumulation and antioxidant system in arsenate stressed-wheat (Triticum aestivum L.) seedlings

There is little information available to decipher the interaction between molybdenum (Mo) and nitric oxide (NO) in mitigating arsenic (AsV) stress in plants. The present work highlights the associative role of exogenous Mo and endogenous NO signaling in regulating AsV tolerance in wheat seedlings. Application of Mo (1 μM) on 25-day-old wheat seedlings grown in the presence (5 μM) or absence of AsV stress caused improvement of photosynthetic pigment metabolism, reduction of electrolytic leakage and reactive oxygen species (ROS), and higher accumulation of osmolytes (proline and total soluble sugars). The molybdenum treatment upregulated antioxidative enzymes, such as superoxide dismutase, ascorbate peroxidase and glutathione reductase. In addition, the accumulation of nonenzymatic antioxidants (ascorbate and glutathione) was correlated with an increase in ascorbate peroxidase and glutathione reductase activity. The application of cPTIO (endogenous NO scavenger; 100 μM) reversed the Mo-mediated effects, thus indicating that endogenous NO may accompany Mo-induced mitigation of AsV stress. Mo treatment stimulated the accumulation of endogenous NO in the presence of AsV stress. Thus, it is evident that Mo and NO-mediated AsV stress tolerance in wheat seedlings are primarily operative through chlorophyll restoration, osmolytes accumulation, reduced electrolytic leakage, and ROS homeostasis.

A Metabolic Checkpoint for the Yeast-to-Hyphae Developmental Switch Regulated by Endogenous Nitric Oxide Signaling

The yeast Candida albicans colonizes several sites in the human body and responds to metabolic signals in commensal and pathogenic states. The yeast-to-hyphae transition correlates with virulence, but how metabolic status is integrated with this transition is incompletely understood. We used the putative mitochondrial fission inhibitor mdivi-1 to probe the crosstalk between hyphal signaling and metabolism. Mdivi-1 repressed C.albicans hyphal morphogenesis, but the mechanism was independent of its presumed target, the mitochondrial fission GTPase Dnm1. Instead, mdivi-1 triggered extensive metabolic reprogramming, consistent with metabolic stress, and reduced endogenous nitric oxide (NO) levels. Limiting endogenous NO stabilized the transcriptional repressor Nrg1 and inhibited the yeast-to-hyphae transition. We establish a role for endogenous NO signaling in C.albicans hyphal morphogenesis and suggest that NO regulates a metabolic checkpoint for hyphal growth. Furthermore, identifying NO signaling as an mdivi-1 target could inform its therapeutic applications in human diseases.

Understanding and targeting the Rho kinase pathway in erectile dysfunction.

Erectile dysfunction (ED) is a common disorder that affects a quarter of US men, and has many causes, including endothelial impairment, low testosterone levels, prior surgical manipulation, and/or psychogenic components. Penile erection is a complex process requiring neurally mediated relaxation of arteriolar smooth muscle and engorgement of cavernosal tissues, mediated by nitric oxide (NO). Current medical therapies for ED largely seek to maximize endogenous NO signalling. Certain aetiologies, including diabetes, are difficult to treat with current modalities, emphasizing the need for new molecular targets. Research has demonstrated the importance of RhoA-Rho-associated protein kinase (ROCK) signalling in maintaining a flaccid penile state, and inhibition of RhoA-ROCK signalling potentiates smooth-muscle relaxation in an NO-independent manner. The mechanisms and effects of RhoA-ROCK signalling and inhibition suggest that the RhoA-ROCK pathway could prove to be a new therapeutic target for the treatment of ED.

Direct sGC activation bypasses NO scavenging reactions of intravascular free oxy-hemoglobin and limits vasoconstriction.

Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation. Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 μM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770. These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling.

Nitric Oxide Synthesis and cGMP Production Is Important for Neurite Growth and Synapse Remodeling after Axotomy

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L. stagnalis neurons B1 and B2 induces extensive central growth of neurites that is accompanied by changes in existing electrical connections, the transient formation of novel electrical connections, and the formation of a novel excitatory chemical synapse from B2 to B1 neurons. Partial chronic inhibition of endogenous NO synthesis reduces neurite growth in NO-synthase-expressing B2, but has only minor effects on NOS-negative B1 neurons. Chronic application of an NO donor while inhibiting endogenous NO synthesis rescues neurite extension in B2 neurons and boosts growth of B1 neurons. Blocking soluble guanylate cyclase activity completely suppresses neurite extension and synaptic remodeling after nerve crush, demonstrating the importance of cGMP in these processes. Interestingly, inhibition of cGMP-dependent protein kinase only suppresses chemical synapse formation without effects on neuronal growth and electrical synapse remodeling. We conclude that NO signaling via cGMP is an important modulator of both neurite growth and synaptic remodeling after nerve crush. However, differential effects of cGMP-dependent protein kinase inhibition on neurite growth and synaptic remodeling suggest that these effects are mediated by separate signaling pathways.

Lead-Induced Nitric Oxide Generation Plays a Critical Role in Lead Uptake by Pogonatherum crinitum Root Cells

The effects of lead (Pb) on endogenous nitric oxide (NO) generation, the role of NO in Pb uptake and the origin of Pb-induced NO production in Pogonatherum crinitum root cells were evaluated. Pb treatment induced rapid NO generation, showing that Pb exposure triggered endogenous NO signaling of the cells. Pre-treatment of the cells with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline -1-oxyl-3-oxide (cPTIO) not only abolished the Pb-triggered NO burst but also reduced Pb contents of the cells. Moreover, Pb exposure enhanced nitrate reductase (NR) activity of the cells. The NR inhibitors tungstate and glutamine not only suppressed the Pb-enhanced NR activities but also reduced the Pb-triggered NO generation. Pre-treatment of the cells with tungstate and glutamine suppressed Pb accumulation and the suppression could be restored by application of exogenous NO via its donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Together, our results indicated that Pb exposure enhanced NR activity and triggered the NO burst of P. crinitum root cells. Furthermore, the data demonstrated that NR was responsible for the Pb-triggered NO burst and that NR-mediated NO generation played a critical role in Pb uptake by P. crinitum root cells. Thus, our results suggest a potential strategy for controlling Pb uptake by plants by targeting NR as a source of Pb-triggered NO production.

NO Way to Relax

The roles of nitric oxide (NO) in the heart have been studied intensely ever since the first report nearly 2 decades ago showing that endogenous NO modulates cardiac myocyte function.1 All 3 NO synthase (NOS) isoforms have been found in mammalian heart tissues (see reviews in Massion et al2 and Belge et al3). A complex array of myocyte and nonmyocyte cells in the heart express NOS isoforms, and the local generation of NO2,3 and reactive oxygen species (ROS)4 may exert both autocrine and paracrine effects on cellular function. Not only is the endothelial isoform of NOS (eNOS, or NOS3) robustly expressed in the endothelial cells of the cardiac vasculature, but eNOS is also present within cardiac myocytes, where the enzyme associates with the scaffolding/regulatory protein caveolin-3 in T tubules in plasmalemmal caveolae.5 The neuronal NOS (nNOS, or NOS1) is also expressed in cardiac myocytes, where the enzyme appears to be localized in the sarcoplasmic reticulum and modulates phospholamban phosphorylation.6 Although both eNOS and nNOS appear to be physiologically expressed in cardiac tissues, the inflammation-related NOS isoform (iNOS, or NOS2) only appears in the heart after immunoactivation; iNOS may modulate the decline in myocardial function seen in sepsis.7 There have been innumerable studies of the cardiac effects of various NOS inhibitors, NO-donating drugs, and NO itself.2 The cardiac phenotypes of NOS “knockout” mouse models have been characterized extensively, and mice lacking 1, 2, or all 3 NOS isoforms have been generated and characterized.8 In addition, the roles of NOS substrates, cofactors, and allosteric modulators have been explored exhaustively in diverse cardiac model systems. Emerging from this broad range of experimental evidence is an increasingly clear view that NO and NOS are key determinants of cardiac function. Yet the molecular mechanisms and …

Autoantibodies from schizophrenia patients induce cerebral cox-1/iNOS mRNA expression with NO/PGE2/MMP-3 production

We demonstrated that circulating antibodies from schizophrenia patients, which interact with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), trigger production of nitric oxide (NO), prostaglandin E2 (PGE2) and matrix metalloproteinase-3 (MMP-3), and act as inducers of cyclooxygenase-1 (cox-1) and inducible nitric oxide synthase (iNOS) mRNA expression in the rat frontal cortex. The corresponding affinity-purified anti-M1 peptide IgG from schizophrenia patients, while stimulating cerebral M1 mAChRs, increases NOS activity, PGE2 and MMP-3 production associated with iNOS over-activity and mRNA expression. Moreover, PGE2 and MMP-3 production is the result of cox-1 expression and activity. All these effects were inhibited by pirenzepine or haloperidol and mimicked the action of the authentic mAChR agonist. Concurrent analysis of the effects of iNOS, phospholipase C, protein kinase C and calcium/calmodulin inhibition showed that antibody up-regulation of NOS activity, PGE2 and MMP-3 production is under the control of the endogenous NO signalling system. These results provide evidence of the role that cholinergic receptor antibodies play in the development of cerebral inflammation, which shows that an antibody that interacts with cerebral mAChRs can induce expression of pro-inflammatory mediators, and support the participation of an autoimmune process in a particular group of chronic schizophrenia patients.

Hypoxia Increases Tumor Cell Shedding of MHC Class I Chain-Related Molecule: Role of Nitric Oxide

The MHC class I chain-related (MIC) molecules play important roles in tumor immune surveillance through their interaction with the NKG2D receptor on natural killer and cytotoxic T cells. Thus, shedding of the MIC molecules from the tumor cell membrane represents a potential mechanism of escape from NKG2D-mediated immune surveillance. Tumor hypoxia is associated with a poor clinical outcome for cancer patients. We show that hypoxia contributes to tumor cell shedding of MIC through a mechanism involving impaired nitric oxide (NO) signaling. Whereas hypoxia increased MIC shedding in human prostate cancer cells, activation of NO signaling inhibited hypoxia-mediated MIC shedding. Similar to incubation in hypoxia, pharmacologic inhibition of endogenous NO signaling increased MIC shedding. Parallel studies showed hypoxia-mediated tumor cell resistance to lysis by interleukin 2-activated peripheral blood lymphocytes (PBL) and NO-mediated attenuation of this resistance to lysis. Inhibition of NO production also led to resistance to PBL-mediated lysis. Interference of MIC-NKG2D interaction with a blocking anti-MIC antibody abrogated the effect of hypoxia and NO signaling on tumor cell sensitivity to PBL-mediated lysis. Finally, continuous transdermal delivery of the NO mimetic glyceryl trinitrate (7.3 mug/h) attenuated the growth of xenografted MIC-expressing human prostate tumors. These findings suggest that the hypoxic tumor microenvironment contributes to resistance to immune surveillance and that activation of NO signaling is of potential use in cancer immunotherapy.

Chemosensitization of Cancer In vitro and In vivo by Nitric Oxide Signaling

Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo. Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence. Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography. Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.

Inhaled nitric oxide induced NOS inhibition and rebound pulmonary hypertension: a role for superoxide and peroxynitrite in the intact lamb

Previous in vivo studies indicate that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity and that this decrease is associated with significant increases in pulmonary vascular resistance (PVR) upon the acute withdrawal of inhaled NO (rebound pulmonary hypertension). In vitro studies suggest that superoxide and peroxynitrite production during inhaled NO therapy may mediate these effects, but in vivo data are lacking. The objective of this study was to determine the role of superoxide in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy in vivo. In control lambs, 24 h of inhaled NO (40 ppm) decreased NOS activity by 40% (P<0.05) and increased endothelin-1 levels by 64% (P<0.05). Withdrawal of NO resulted in an acute increase in PVR (60.7%, P<0.05). Associated with these changes, superoxide and peroxynitrite levels increased more than twofold (P<0.05) following 24 h of inhaled NO therapy. However, in lambs treated with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) during inhaled NO therapy, there was no change in NOS activity, no increase in superoxide or peroxynitrite levels, and no increase in PVR upon the withdrawal of inhaled NO. In addition, endothelial NOS nitration was 18-fold higher (P<0.05) in control lambs than in PEG-SOD-treated lambs following 24 h of inhaled NO. These data suggest that superoxide and peroxynitrite participate in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy. Reactive oxygen species scavenging may be a useful therapeutic strategy to ameliorate alterations in endogenous NO signaling during inhaled NO therapy.

H(1)-Receptor activation triggers the endogenous nitric oxide signalling system in the rat submandibular gland.

BACKGROUND: Histamine is released from mast cells by immunologic and non-immunologic stimuli during salivary gland inflammation, regulating salivary secretion. The receptor-secretory mechanism has not been studied in detail. AIMS: The studies reported were directed toward elucidating signal transduction/second messenger pathways within the rat submandibular gland associated with 2-thiazolylethylamine (ThEA)-induced H(1)-receptor responses. MATERIALS AND METHODS: To assess the H(1) receptor subtype expression in the rat submandibular gland, a radioligand binding assay was performed. The study also included inositolphosphates and cyclic GMP accumulation, protein kinase C and nitric oxide synthase activities, and amylase release. RESULTS: The histamine H(1) receptor subtype is expressed on the rat submandibular gland with high-affinity binding sites. The ThEA effect was associated with activation of phosphoinositide-specific phospholipase C, translocation of protein kinase C, stimulation of nitric oxide synthase activity and increased production of cyclic GMP. ThEA stimulation of nitric oxide synthase and cyclic GMP was blunted by agents able to interfere with calcium movilization, while a protein kinase C inhibitor was able to stimulate ThEA action. On the other hand, ThEA stimulation evoked amylase release via the H1 receptor but was not followed by the L-arginine/nitric oxide pathway activation. CONCLUSIONS: These results suggest that, apart from the effect of ThEA on amylase release, it also appears to be a vasoactive chemical mediator that triggers vasodilatation, modulating the course of inflammation.

Involvement of the endogenous nitric oxide signalling system in bradykinin receptor activation in rat submandibular salivary gland

Biochemical signalling events coupled to the bradykinin B 2-receptor subtype, related to nitric oxide and prostaglandin E 2 generation were studied in rat submandibular gland. Bradykinin stimulation of the B 2-receptor triggered activation of phosphoinositide turnover, translocation of protein kinase C, stimulation of nitric oxide synthase activity, increased production of cGMP and release of prostaglandin E 2. Bradykinin stimulation of nitric oxide synthase and cGMP production was blunted by agents able to interfere with calcium/calmodulin and phospholipase C activities, while a protein kinase C inhibitor was able to stimulate bradykinin action. Moreover, a specific B 2-bradykinin antagonist of the reversible nitric oxide synthase inhibitor abrogated the bradykinin stimulation of nitric oxide synthase activity, cGMP accumulation and prostaglandin E 2 generation. Furthermore, a specific inhibitor of phospholipase A 2 blocked the bradykinin-induced prostaglandin E 2 release. These results suggest that apart, from the direct effect of bradykinin as an inducer of vasopermeability, it also appears to be a vasoactive chemical mediator that triggers, through release of prostaglandin E 2, a feedback mechanism that induces a protective adaptation of the gland, modulating the course of inflammation.

Involvement of endogenous nitric oxide signalling system in brain muscarinic acetylcholine receptor activation.

Biochemical signalling events coupled to muscarinic cholinergic receptors (mAChR), specifically those related to nitric oxide (NO) production, were studied on rat cerebral frontal cortex. The mAChR agonist carbachol was found to exert a specific biphasic action on NO synthase (NOS) activity: low doses ranging between 10(-9) M to 10(-7) M lead to NOS activation while higher doses (>10(-6) M) inhibited enzymatic activity. Carbachol stimulatory action was blunted by agents that interfere with calcium-calmodulin while a protein kinase (PKC) inhibitor, staurosporine was able to abrogate the inhibitory effect. Moreover, PKC activity showed maximum translocation to cerebral frontal cortex membranes with carbachol concentrations that inhibited NO production. Products from phosphoinosite (PI) hydrolysis are involved in these actions as carbachol was found to increase PI turnover in a dose dependent manner. These results would serve as an example of cross-talk between both enzymatic pathways.

Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor‐inotropic response

1. In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)-dependent inhibition of contractility in rat isolated atria. 2. Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose-response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor-dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8-bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4. These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.

Control of cardiac muscle cell function by an endogenous nitric oxide signaling system.

Nitric oxide (NO) synthesized from L-arginine is a ubiquitous intracellular chemical messenger and is involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The role of the NO-signaling pathway in the direct modulation of cardiac function is less well characterized. In this report, the effects of inhibitors of NO synthase (NOS) were examined in isolated neonatal and adult rat ventricular myocytes exposed to either muscarinic or adrenergic agonists. Carbachol (10 microM) caused a 91% inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes. N omega-monomethyl-L-arginine, an L-arginine analog that inhibits NOS, and methylene blue, an inhibitor of NO, blocked the negative chronotropic effect of carbachol but had no effect on the basal beating rate of these cells. The inhibition by N omega-monomethyl-L-arginine of the negative chronotropic effect of carbachol was reversed by adding excess L-arginine. The negative chronotropic effect of carbachol was also mimicked by analogs of cGMP, a second messenger implicated in mediating the action of NO in other cell types. Production of NO could be detected directly in carbachol-stimulated neonatal myocytes by using a reporter cell bioassay. The regulation of adrenergic responsiveness by the NO signaling system was also documented in studies of adult cardiac myocyte contractility. The NOS inhibitor N omega-nitro-L-arginine significantly increased the inotropic effect of the beta-adrenergic agonist isoproterenol on electrically stimulated adult rat ventricular myocytes, whereas this inhibitor had no effect on basal contractility. Inhibition of NO production by N omega-monomethyl-L-arginine in these cells, as measured by reporter cell bioassay, was also reversible with excess L-arginine. Thus, the physiologic response of isolated neonatal and adult ventricular myocytes to both muscarinic cholinergic and beta-adrenergic stimulation is mediated, at least in part, by products of an endogenous NOS.