Calmodulin activates neuronal nitric oxide synthase by enabling transitions between conformational states

Sabadini, Walters and others have developed a categorical, machine based theory of concurrency in which there are four essential aspects: a distributive category of data-types; a bicategory Mach whose objects are data types, and whose arrows are input-output machines built from data types; a semantic category (or categories) Sem, suitable to contain the behaviors of machines, and a functor, “behavior”: Mach→Sem. Suitable operations on machines and semantics are found so that the behavior functor preserves these operations. Then, if each machine is decomposable into primitive machines using these operations, the behavior of a general machine is deducible from the behavior of its parts. The theory of non-deterministic finite state automata provides an example of the paradigm and also throws some light on the classical theory of finite state automata. We describe a bicategory whose objects are natural numbers, in which an arrow M: n→p is a finite state automaton with n input states, p output states, and some additional internal states; we require that no transitions begin at output states or end at input states. A machine is represented by an q+n by q+p matrix. The bicategory supports additional operations: non-deterministic choice, parallel interleaving, and feedback. Enough operations are imposed on machines to show that each machine may be obtained from some atomic ones by means of the operations. The semantic category is the (Bloom-Esik) iteration theory Mat (X✶ whose objects are natural numbers and whose arrows from n to p are n×p matrices with entries in the semiring of languages. The behavior functor associates to a machine M: n→p a matrix |M| of languages, one language to each pair of input and output states. Behavior preserves composition, feedback, takes non-deterministic choice to union, and parallel-interleaving to shuffle. Thus, behavior gives a compositional semantics to a primitive notion of concurrent processes.

While sympathetic stimulation of the heart produces chronotropic, inotropic, and lusitropic effects, increased frequency alone causes a positive force-frequency relationship (FFR) and frequency-dependent acceleration of relaxation (FDAR).1 That is, contraction amplitude and relaxation rate are increased with increasing frequency in most species (including humans). The key mechanism involved in the positive FFR is increased sarcoplasmic reticulum (SR) Ca2+ load, due to increased Ca2+ influx and decreased Ca2+ efflux.1,2 Ca2+ influx increases due to more L-type Ca2+ current ( I Ca) per unit time, while Ca2+ efflux via Na+-Ca2+ exchange (NCX) decreases because the diastolic time is reduced and [Na+]i increases. Enhanced SR Ca2+-pump function causes FDAR and also augments SR Ca2+ loading. Various signaling pathways are involved (eg, CaMKII).3 In human heart failure, the FFR reverses (ie, from positive to negative) due to an inability of the SR to increase Ca2+ content.4 This negative FFR is a main contributor to the loss of contractile reserve in the failing heart. Of many pathways that can modify FFR, nitric oxide (NO) signaling is the topic addressed by Khan et al in this issue of Circulation Research .5 NO synthase (NOS) produces NO from l-arginine, and cardiac myocytes express all three NOS isoforms.6,7 NOS1 (nNOS) and NOS3 (eNOS) are constitutively expressed and produce low amounts of NO (regulated by [Ca2+-calmodulin]i levels). NOS2 (iNOS) …

In brain, several cell types produce tumor necrosis factor-alpha (TNFalpha) after injury or exposure to endotoxin. TNFalpha alone or in combination with endotoxin or other cytokines can cause expression of inducible nitric oxide (NO) synthase. We have previously demonstrated that endotoxin caused NO-dependent dilatation of cerebral arterioles in vivo. In the present study we examined the hypothesis that TNFalpha causes NO-mediated dilatation of cerebral arterioles in vivo. Cranial windows were implanted in anesthetized rats and used to measure the diameter of cerebral arterioles. Windows were flushed every 30 minutes for 4 hours with artificial cerebrospinal fluid (aCSF) (n=6); aCSF with TNFalpha (100 ng/mL; n=10); aCSF with TNFalpha and aminoguanidine (0.3 mmol/L; n=5), an inhibitor of inducible NO synthase; or aCSF with TNFalpha and dexamethasone (1 micromol/L; n=6), which attenuates expression of inducible NO synthase. In some animals, brain from beneath the cranial window was examined by immunocytochemistry for inducible NO synthase expression. Application of TNFalpha caused marked, progressive dilatation of cerebral arterioles, with a maximum increase in diameter of 46+/-9% (mean+/-SEM) at 4 hours. Coapplication of either aminoguanidine or dexamethasone with TNFalpha prevented dilatation of cerebral arterioles compared with TNFalpha alone (4+/-2% and 1+/-1% dilatation at 4 hours, respectively; P<.05). Dexamethasone did not inhibit dilatation of cerebral arterioles in response to adenosine diphosphate. However, 2 hours of aminoguanidine treatment produced moderate inhibition of adenosine diphosphate-induced dilatation of cerebral arterioles. After treatment with TNFalpha, immunocytochemistry for inducible NO synthase demonstrated expression in perivascular and arachnoid cells but not brain cells. There was no detectable expression of inducible NO synthase after treatment with aCSF. The present study indicates that TNFalpha causes cerebral vasodilatation and expression of inducible NO synthase in perivascular and arachnoid cells. Inhibition of TNFalpha-induced dilatation by aminoguanidine and dexamethasone suggests that the vasodilatation was due predominantly to expression of inducible NO synthase. These findings support the concept that cerebral vasodilatation that occurs during pathophysiological conditions associated with increased TNFalpha production in brain is mediated by expression of inducible NO synthase.

1. Experiments were performed to examine the effects of anti-fungal imidazole compounds (clotrimazole, econazole and miconazole) on the induction of nitric oxide (NO) synthase and subsequent production of NO in the cultured murine monocyte/macrophage cell line J774 using a specific cDNA probe for inducible NO synthase mRNA and by monitoring nitrite production. 2. Stimulation of J774 cells with lipopolysaccharide (LPS, 10 micrograms ml-1) resulted in the induction of NO synthase activity as determined by nitrite accumulation in the culture medium (48 +/- 3 nmol per 10(6) cells over 24 h). Production of nitrite was inhibited by co-incubation of cells with LPS (10 micrograms ml-1) and either dexamethasone (10 microM) or NG-monomethyl-L-arginine (L-NMMA; 0.1 mM), however, only L-NMMA was an effective inhibitor of nitrite production when added after induction of NO synthase had occurred. 3. Co-incubation of J774 cells with LPS (10 micrograms ml-1) and either clotrimazole, econazole or miconazole (1-10 microM) resulted in a concentration-dependent inhibition of nitrite production over the subsequent 24 h without any evidence for a cytotoxic effect. However, addition of these imidazoles after induction of NO synthase did not inhibit nitrite production. 4. Messenger RNA for inducible NO synthase was not detected in unstimulated J774 cells. Treatment with LPS (10 micrograms ml-1) for 4 h resulted in significant expression of mRNA for inducible NO synthase which was not altered in the presence of econazole (10 microM) but was reduced significantly by dexamethasone (10 microM). 5. These results demonstrate that anti-fungal imidazoles inhibit the production of nitric oxide by cultured J774 cells by a mechanism which appears to differ from that of dexamethasone and substrate type inhibitors of NO synthase. Furthermore, the presence of mRNA for NO synthase does not indicate the presence of functionally active NO synthase.

Nitrergic neurons of the dorsal raphe nucleus (DRN) may play a role in physiological stress responses. The caudal lateral wings (CLW) are unique compared to other rostral-caudal DRN sub-regions because they contain distinct nitric oxide (NO) synthase (NOS) populations that are independent of tryptophan hydroxylase (TPH). NOS neurons in the CLW are also highly activated during acute restraint stress. However, the effects of acute stress duration on NOS activation in the CLW are unclear. Here NADPH-d, an index of NOS activity, is used to show that sub-regions of the DRN have differential NOS activation in response to 6 hours of restraint stress in rats. We report increased NOS activity through 6 hours of restraint in the caudal lateral wings and ventromedial sub-regions. These data suggest that, NOS neurons may play a dynamic role in the response to stress duration.

Bovine retinal pigmented epithelial cells (RPE cells), after activation with interferon gamma (IFN gamma) and lipopolysaccharide (LPS), express an inducible nitric oxide (NO) synthase. This activity can be inhibited by the fibroblast growth factors (FGF), FGF-1 (acidic FGF) and FGF-2 (basic FGF). We have attempted to elucidate the molecular mechanisms involved in the FGF inhibition of NO synthase activity. Analysis by immunocytochemistry and Western blot using a polyclonal antibody against the inducible NO synthase of rat liver reveals that RPE cells co-stimulated with FGF-2 and LPS/IFN gamma accumulate lower levels of NO synthase than in the absence of FGF-2. Northern blot analysis by cross-species hybridization with a mouse macrophage NO synthase cDNA probe shows that a 4.4-kb mRNA accumulates when RPE cells are activated with LPS/IFN gamma. The level of inducible NO synthase mRNA in LPS/IFN gamma-activated RPE cells is markedly reduced by FGF-1 or FGF-2 treatment. Message stability studies revealed that the presence of FGF did not accelerate mRNA degradation, implying that FGF did not act on inducible NO synthase mRNA stability, but more probably on its expression. Furthermore an effect of FGF on IFN gamma receptors was excluded, since IFN gamma binding was not altered by FGF. Since NO acts as a cytostatic compound, FGF, by preventing NO synthase expression in RPE cells, may protect the retina from endotoxin and cytokine-mediated tissue damage.

Performance of quantum teleportation is typically measured by the average fidelity, an overlap between the input and output states. Along with the first moment, we introduce the second moment of fidelity in continuous-variable (CV) teleportation, i.e., the fidelity deviation as the figure of merit to assess the protocol's efficiency. We show that CV states, both Gaussian and non-Gaussian, can be better characterized by considering both average fidelity and fidelity deviation, which is not possible with only average fidelity. Moreover, we shed light on the performance of the teleportation protocol in two different input scenarios: One is when input states are sampled from constrained uniform distribution while the other one is Gaussian suppression of the input states which again leads to a different classification of CV states according to their performance. The entire analysis is carried out in noiseless and noisy scenarios with noise being incorporated in the measurement and the shared channels. We also report that one type of noise can make the protocol robust against the other one, which leads to a ``constructive effect,'' and identify the noise models which are responsible for decreasing average fidelity and the increment in fidelity deviation.

The modulation of myogenic function and cerebral blood flow (CBF) by nitric oxide (NO) synthases (NOS) was assessed in the middle cerebral arteries (MCAs) of Kyoto Wistar stroke prone hypertensive rats (SHRsp) in relation to haemorrhagic stroke development. MCAs were studied with a pressure myograph. CBF in MCA perfusion domain was measured using laser Doppler techniques. NOS isozymes were identified using immunohistochemistry. MCAs expressed endothelial, neuronal, and inducible NOS (eNOS, nNOS, and iNOS, respectively) in the endothelium, nNOS and traces of iNOS in smooth muscle and adventitial cells. Before stroke, MCA pressure-dependent constriction (PDC) was superimposed over basal non-pressure-dependent tone (BNPDT). Endothelial NO generation and non-endothelial nNOS but not iNOS reduced BNPDT and increased the lumen diameter at which PDC initiated without altering the amplitude of PDC. NOS inhibition decreased CBF and increased the upper blood pressure limit of autoregulation. PDC, CBF autoregulation, and NOS dilatory influence were lost, and BNPDT was increased in MCAs from SHRsp with stroke. The expression of NOS isozymes and MCA reactivity to NO donors was not altered. NOS activity was not recovered by in vitro l-arginine or tetrahydrobiopterin supplementation, l-arginase inhibition or superoxide scavengers. The loss of PDC and CBF autoregulation during hypertension may facilitate over-perfusion and cerebral haemorrhage formation in SHRsp. NOS dysfunction in MCAs preceded stroke and involved the inactivation of eNOS and nNOS in areas not subjected to hyper-distension. The elevation in BNPDT due to NOS inactivation may oppose over-perfusion in the absence of CBF autoregulation.

Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.

Aim: To investigate the effect of tadalafil in rats administered with daily dutasteride.Methods: Twenty-four Sprague–Dawley male rats were allocated to three groups as control (group C), dutasteride (group D) and dutasteride plus tadalafil (group D + T). After a month of treatment, serum samples were obtained from rats to measure dihydrotestosterone and total testosterone. Nitric oxide (NO) synthase (NOS) immunoreactivity and levels of NOS enzyme isoforms, NO and cyclic guanosine monophosphate (cGMP) were evaluated in the harvested penile tissues. Also, corporal smooth muscle and collagen were examined.Results: Staining intensities of neuronal NOS and endothelial NOS were significantly lower in group D (p < .05). They were similar between group C and group D + T. Immunoreactivity of inducible NOS was observed higher in group D than group C (p = .01) whereas group D + T had the highest iNOS (p<.001). ELISA revealed similar outcomes in terms of NOS enzyme isoform levels. The mean of smooth muscle to collagen ratio was the lowest in group D (p < .001) and it was similar among group C and group D + T (p = .072). Group D had the lowest cGMP and NO levels (p < .05) and they did not differ between group C and group D + T (p>.05). Group D and group D + T had significantly decreased dihydrotestosterone and increased testosterone, compared to group C (p < .001). They were similar between group D and group D + T.Conclusion: Daily treatment with tadalafil improves dutasteride-induced changes in rat penis.

1. The ability of Corynebacterium parvum (C. parvum) to induce nitric oxide (NO) synthase in the macrophage, spleen, liver, aorta, heart and brain, and to elevate plasma NO2-/NO3- in the mouse was investigated. In addition, the relationship between NO synthase activity and blood pressure was studied. 2. C. parvum (100 mg kg-1, i.p.) induced a time-dependent expression of a Ca(2+)-independent NO synthase in the macrophage, spleen, liver, aorta and heart. The time course of induction of the NO synthase varied such that the maximum enzyme activity was at day 8 in the macrophage and liver, day 12 in the spleen and heart and day 16 in the aorta. 3. There was no significant induction of a Ca(2+)-independent NO synthase in the brain, nor was there any change in the Ca(2+)-dependent enzyme in this organ, during the study period. 4. C. parvum produced a gradual decrease in blood pressure, with a maximum fall at day 16 (from 108 +/- 1 mmHg to 79 +/- 3 mmHg), which recovered gradually by day 28. 5. Plasma NO2-/NO3- was significantly elevated between days 8 and 24, with a maximum increase at day 12. 6. These results show that C. parvum induces a Ca(2+)-independent NO synthase in a number of tissues and that this induction occurs initially in macrophages and the liver. This suggests that induction of the NO synthase in the other tissues is secondary and probably the result of activation of macrophages and some cells of the liver. 7. Furthermore, the decrease in blood pressure induced by C. parvum is associated with the induction of NO synthase in the vasculature, whereas the increased concentration of plasma NO2-/NO3- seems to result from the generation of NO by a number of tissues.

The role of constitutive and inducible nitric oxide synthase in senna- and cascara-induced diarrhoea in the rat

1. Partially purified soluble nitric oxide (NO) synthase was isolated from the bovine retractor penis muscle (BRP), a tissue in which the inhibitory response to non-adrenergic non-cholinergic nerve (NANC) stimulation appears to be mediated by NO or NO-like material. 2. NO synthase from BRP used L-arginine as a substrate, required NADPH, tetrahydrobiopterin, and FAD as co-factors and was Ca2+/calmodulin-dependent. The activity of NO synthase was inhibited by NG-methyl-L-arginine and NG-nitro-L-arginine, and haemoglobin blocked the effect of NO formed by the enzyme. 3. On reducing SDS polyacrylamide gel electrophoresis the apparent molecular mass of NO synthase from BRP was 160 +/- 2 kDa, which is similar to that of the cerebellar NO synthase. Protein immunoblot and immunoprecipitation showed that NO synthase from BRP cross-reacted with the selective antiserum to neuronal NO synthase from rat cerebellum. 4. Immunohistochemistry using the same antiserum demonstrated that NO synthase in BRP was located exclusively within nerve fibres. Thus, autonomic nerves synthesizing the NANC neurotransmitter seem to contain an isoform of NO synthase which is similar to that from rat cerebellum.

Rat peritoneal polymorphonuclear leukocytes (PMN) elicited with oyster glycogen contain a Ca(2+)-independent nitric oxide (NO) synthase which is induced in vivo in a time-dependent manner. When washed PMN containing low levels of enzyme activity were cultured ex vivo further expression of NO synthase was observed. This was inhibited by cycloheximide indicating that de novo synthesis of the enzyme occurred during the ex vivo incubation. Enzyme activity was enhanced by interferon (IFN)-gamma, but not by tumor necrosis factor (TNF)-alpha when added ex vivo. However, IFN-gamma and TNF-alpha synergized to increase further the expression of NO synthase. Treatment of rats with dexamethasone inhibited the induction of NO synthase in elicited PMN. This treatment reduced the accumulation of PMN by approximately 30%, without affecting cell viability. Dexamethasone also inhibited the induction of the NO synthase ex vivo in a concentration-dependent manner. Furthermore, the enhanced enzyme activity following treatment of PMN with cytokines was also inhibited by dexamethasone. Once induced, dexamethasone did not affect enzyme activity. These data indicate that PMN elicited in the rat peritoneum with oyster glycogen express an NO synthase in vivo and ex vivo. The induction of the enzyme can be further stimulated ex vivo with IFN-gamma and TNF-alpha and inhibited by dexamethasone. The inhibition of the induction of NO synthase in the PMN by dexamethasone may contribute to the anti-inflammatory activity of this and other glucocorticoids.

Nitrc oxide is a ubiquitous signaling molecule with both physiological and pathological functions in biological systems. Formed by the enzmatic conversion of arginine to citrlline, NO, has known roles in circulatory, imune and nerous tissues. In the nerous system nitrc oxide has been implicated in long-ter potentiation neurotranmitter release, chanel function, neuronal protection and neuronal degeneration. Much of our work has focused on yet another role for nitrc oxide in cells namely, neuronal differentiation. Dung development, neuronal differentiation is closely coupled with cessation of proliferation. We use nere growth factor (NGF)-induced differentiation ofPC12 pheochromocytoma cells as a model and fid a novel signal tranduction pathway that blocks cell proliferation. Treatment ofPC12 cells with NGF leads to induction of nitric oxide synthase (NOS). The resulting nitrc oxide (NO) acs as a second messenger activating the p21(WAFl) promoter and inducing expression ofp21(WAFl) cyclindependent kiase inibitor. NO acivates the p21 (W AFl) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation ofp53 , activation ofthe p21(WAFl) promoter, expression of neuronal marker, and neurte extenion. To detere whether p2 1 (WAF 1) is required for neurite extension, we prepared a PC 12 line with an inducible p21 (W AFl) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21 (W AFl) with isopropylthio-betaD-galactopyranoside restored this response. Levels of p21 (W AFl) induced by isopropylthio-betaD-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS , p53 and p21 (W AFl) to block cell proliferation; and is required for neuronal differentiation by PC12 cells. In fuher studies of this pathway, we have examed the role of MA kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In NGF-treated PC12 cells, we find that nNOS is induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRA nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ra adenovis. We have also used drgs that block MA kinase pathways and assessed their ability to inibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we fmd that U0126, but not PD98059 blocks nNOS induction and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inibitor, SB 203580, does not block nNOS induction in our clone ofPC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we deterne that the Ras-ERK pathway and not the p38 or JN pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the RaERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the RasERK pathway is required for nNOS induction. The activation of soluble guanylate cyclase and the production of cyclic GMP is one of the best characterized modes of NO action. Havig shown that inhibition of NOS blocks PC12 cell differentiation we tested whether nitric oxide acts through soluble guanylate cyclase to lead to cell cycle arest and neuronal differentiation. Unlike NOS inibition, the inibition of soluble guanylate cy1case does not block the induction of neuronal markers. Moreover, treatent ofNGF-treated, NOS-inhibited PC12 cells with a soluble analog of cyclic GMP was unable to restore differentiation of those cells. Hence cGMP is not a component of this pathway and we had to consider other mechanisms of NO acion. It has become increasingly evident that another manner by which NO may exert its effects is by S-nitrosylation of cysteine residues. We tested, in vitro whether nitric oxide may control p53 by S-nitrosylation and inactivation of the p53 negative regulator Hdm. Treatment of Hdm with a nitric oxide donor inhibits Hdm2-p53 binding, the first step in Hdm regulation of p53. The presence of cysteine or DTT blocks this inhibition of binding. Moreover, nitric oxide inibition ofHdm-p53 binding was found to be reversible. Sulfhydrl-sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a crtical cysteine residue that nitrc oxide modifies in order disrupt Hdm-p53 binding. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity ofHdm to nitric oxide inactivation.