Blocking Nitric Oxide Synthesis Research Articles

Systemic administration of choline acetyltransferase decreases blood pressure in murine hypertension

Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker l-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension.

CEREBROPROTECTIVE ACTIVITY OF OUINAZOLINE-4(3H)-ONE DERIVATIVES WITH A GUANIDINE SUBSTITUENT IN RATS WITH BILATERAL COMMON CAROTID ARTERY OCCLUSION

From derivatives of quinazolin-4 (3H) -one, 2 substances were isolated: VMA-13-15 (N-[2-[4-oxo-3(4H)-quinazolinyl] propionyl]guanidine) and VMA-13-17 (N-[2-[6-bromo-4-oxo-3(4H)-quinazolinyl]acetyl]guanidine). With the therapeutic administration of the compounds, the survival rate and neurological condition of animals with bilateral occlusion of the common carotid arteries were better than in the control group. Also 72 hours after surgery and treatment, the level of cerebral blood flow and the severity of the reactions of cerebral vessels to the introduction of a stimulant and a blocker of nitric oxide synthesis were higher. Compounds VMA-13-15 and VMA-13-17 are promising for further study of their therapeutic potential in cerebrovascular accidents.

Вплив кальцієвого навантаження на утворення мітохондріальних пор in situ і експресію генів мітохондріальних роз’єднувальних білків у серці тренованих щурів

We have studied the effect of calcium load (1.7 to 15 mmol/l in perfusate) on isolated heart function, mitochondrial factor release (as a marker of mitochondrial permeability transition pore, MPTP), and cardiac uncoupling proteins (UCP2/3) mRNA expression in untrained and trained rats (swimming for 4 weeks). It was found that the improvement in the isolated heart function of trained rats was accompanied by an increase in the expression of UCP3, but not UCP2. A gradual increase of the calcium content in the perfusate led to an increase in contractile function, more pronounced in trained rats. However, 10 mmol/l and higher concentration of calcium led to arrhythmia and drastic decrease in contractility of isolated heart more obvious in untrained rats. Swimming course prevented the calcium-induced release of mitochondrial factor exerting a stabilizing effect on mitochondrial membranes which was, however, diminished by a nitric oxide synthesis blocker (L-NAME). We have found that UCPs genes expression is calcium-sensitive: an increase in UCP3 mRNA at 5 mmol of calcium and a sharp decrease in UCP2/3 expression at 12.5 mmol/l of calcium in perfusate in both trained and untrained rats indicating the participation of UCPs in the regulation of calcium homeostasis. Our data suggest that the calcium load may serve as a test for in situ MPTP titration. Activation of UCPs together with up-regulated nitric oxide may play a protective role against increasing extracellular calcium inhibiting MPTP formation during physical trainings.

Nitric oxide as a messenger between neurons and satellite glial cells in dorsal root ganglia

Abnormal neuronal activity in sensory ganglia contributes to chronic pain. There is evidence that signals can spread between cells in these ganglia, which may contribute to this activity. Satellite glial cells (SGCs) in sensory ganglia undergo activation following peripheral injury and participate in cellular communication via gap junctions and chemical signaling. Nitric oxide (NO) is released from neurons in dorsal root ganglia (DRG) and induces cyclic GMP (cGMP) production in SCGs, but its role in SGC activation and neuronal excitability has not been explored. It was previously reported that induction of intestinal inflammation with dinitrobenzoate sulfonate (DNBS) increased gap junctional communications among SGCs, which contributed to neuronal excitability and pain. Here we show that DNBS induced SGC activation in mouse DRG, as assayed by glial fibrillary acidic protein upregulation. DNBS also upregulated cGMP level in SGCs, consistent with NO production. In vitro studies on intact ganglia from DNBS-treated mice showed that blocking NO synthesis inhibited both SGCs activation and cGMP upregulation, indicating an ongoing NO production. Application of NO donor in vitro induced SGC activation, augmented gap junctional communications, and raised neuronal excitability, as assessed by electrical recordings. The cGMP analog 8-Br-cGMP mimicked these actions, confirming the role of the NO-cGMP pathway in intraganglionic communications. NO also augmented Ca2+ waves propagation in DRG cultures. It is proposed that NO synthesis in DRG neurons increases after peripheral inflammation and that NO induces SGC activation, which in turn contributes to neuronal hyperexcitability. Thus, NO plays a major role in neuron-SGC communication.

Pentraxin-3 and endothelial dysfunction.

Pentraxin 3 (PTX3) is involved in vascular inflammation and endothelial dysfunction through various mechanisms. Until now, most studies confirmed an important link between PTX3 and endothelial dysfunction and identified several pathogenetic pathways. PTX3 modulates inflammatory cells, thus stimulating vascular inflammation. Within endothelial cells, it decreases nitric oxide (NO) synthesis, inhibits cell proliferation and alters their functions. PTX3 blocks the effect of fibroblast growth factor 2 (FGF2) by making a molecular complex with these molecules inactivating them. However, there are substances like the tumor necrosis factor-inducible gene 6 protein (TSG-6) that block the PTX3-FGF2 interaction. Interacting with P-selectin, it promotes vascular inflammatory response and endothelial dysfunction. PTX3 also increases the matrix metalloproteinases synthesis directly or by blocking NO synthesis. From a clinical point of view, PTX3 positively correlates with arterial hypertension, flow mediated dilation and, with intima media thickness. Therefore, the involvement of PTX3 in the pathogenesis and evaluation of endothelial dysfunction is clear, and it may become a biomarker in this direction, but further studies are needed to determine its reliability in this direction. Last but not least, PTX3 could become an effective therapeutic target for preventing this dysfunction, but further research needs to be conducted.

Serotonin Mediates Depression of Aggression After Acute and Chronic Social Defeat Stress in a Model Insect.

In all animals, losers of a conflict against a conspecific exhibit reduced aggressiveness, often coupled with depression-like symptoms, particularly after multiple defeats. While serotonin (5HT) is involved, discovering its natural role in aggression and depression has proven elusive. We show how 5HT influences aggression in male crickets, before, and after single and multiple defeats using serotonergic drugs, at dosages that had no obvious deleterious effect on general motility: the 5HT synthesis inhibitor alpha-methyltryptophan (AMTP), the 5HT2 receptor blocker ketanserin, methiothepin which blocks 5HT receptor subtypes other than 5HT2, 5HT's precursor 5-hydroxytryptophan (5HTP) and re-uptake inhibitor fluoxetine. Contrasting reports for other invertebrates, none of the drugs influenced aggression at the first encounter. However, the recovery of aggression after single defeat, which normally requires 3 h in crickets, was severely affected. Losers that received ketanserin or AMTP regained their aggressiveness sooner, whereas those that received fluoxetine, 5HTP, or methiothepin failed to recover within 3 h. Furthermore, compared to controls, which show long term aggressive depression 24 h after 6 defeats at 1 h intervals, crickets that received AMTP or ketanserin regained their full aggressiveness and were thus more resilient to chronic defeat stress. In contrast, 5HTP and fluoxetine treated crickets showed long term aggressive depression 24 h after only 2 defeats, and were thus more susceptible to defeat stress. We conclude that 5HT acts after social defeat via a 5HT2 like receptor to maintain depressed aggressiveness after defeat, and to promote the susceptibility to and establishment of long-term depression after chronic social defeat. It is known that the decision to flee and establishment of loser depression in crickets is controlled by nitric oxide (NO), whereas dopamine (DA), but not octopamine (OA) is necessary for recovery after defeat. Here we show that blocking NO synthesis, just like ketanserin, affords resilience to multiple defeat stress, whereas blocking DA receptors, but not OA receptors, increases susceptibility, just like fluoxetine. We discuss the possible interplay between 5HT, NO, DA, and OA in controlling aggression after defeat, as well as similarities and differences to findings in mammals and other invertebrate model systems.

Comparison of endothelin and nitric oxide synthase blockers on hemorheological parameters in endotoxemic rats.

Septic shock is an important health problem that vastly alters cardiovascular and hemodynamic status. Increased production of nitric oxide (NO) and endothelin is a counterpart of this endotoxemic state. This study was conducted to test the hypothesis that nonselective NO synthesis blocker (L-NAME), inducible NO synthesis blocker (L-canavanine), or endothelin receptor antagonist (bosentan) will reverse the effects of sepsis on hemorheological parameters. Forty-eight male Sprague-Dawley rats were used in 8 groups: saline (control), endotoxin, bosentan, L-NAME, L-canavanine, endotoxin + bosentan, endotoxin + L-NAME, and endotoxin + L-canavanine. Blood was withdrawn at the 4th hour of endotoxemic state. Erythrocyte deformability and erythrocyte aggregation were determined by laser-assisted optical rotational cell analyzer at 37 °C. Plasma viscosity (mPa.s) was measured by a cone-plate viscometer with 0.5 mL of plasma. Endotoxin administration significantly increased aggregation half-time and lowered erythrocyte aggregation amplitude and aggregation index compared to the control, indicating a slower and weaker aggregation pattern. L-NAME and L-canavanine alleviated the effects of endotoxin on erythrocyte aggregation without altering the values in the control animals. However, bosentan did not perform such a restoration. This finding suggests that these restoration effects of the blockers occur via their modulation of nitric oxide synthesis rather than through the endothelin pathway.

P2X Receptor‐Mediated Inhibition Of NaCl Absorption In The Thick Ascending Limb: No Evidence For Nitric Oxide As Second Messenger

BackgroundBasolateral (bl.) P2X receptors acutely and markedly reduce NaCl absorption in mouse medullary thick ascending limb (mTAL). It has been proposed that P2X receptor mediate NO synthesis and subsequent NKCC2 inhibition. P2X receptor stimulation causes an increase in cytosolic Ca2+ and therefore this could be proximal to the generation of NO.ObjectiveHere we tested if blocking NO synthesis, adding an NO donor or hindering Ca2+ influx affects the ATP‐mediated (P2X) transport inhibition.MethodsWe used isolated, perfused mTALs from mice to electrically measure NaCl absorption. By microelectrodes we determined the transepithelial voltage (Vte) and the transepithelial resistance (Rte) and via these the transepithelial Na+ absorption (equivalent short circuit current, I′sc).ResultsWe confirm that bl. ATP (100μM) induced a marked, acute and reversible inhibition of Na+ absorption (28% ± 6%, n=6). In the presence of the NO synthase blocker L‐NAME (100 μM, ~4 min preincubation) the ATP effect remained unaffected (23% ± 9%, n=6). Also higher concentrations and longer incubation with L‐NAME (1 mM, 15min incubation) were without effect. High concentrations of L‐NAME (1 mM) per se reduced transport significantly. Acute addition of the NO donor SNAP (100μM, ~10 min) did not affect tubular transport. Furthermore, the removal of extracellular Ca2+ (100 nM, 2 min preincubation) had no effect on the ATP‐induced transport inhibition ([Ca2+ ]o=100nM: 21% ± 6%, n=5, [Ca2+]o=1μM: 24% ± 4%, n=6).ConclusionWe find no evidence for nitric oxide (NO) as second messenger for P2X receptor‐dependent transport inhibition. Similarly, Ca2+ signaling appears not involved in the ATP effect. It remains undefined how P2X receptors trigger the marked reduction of transport in the TAL.

Linking omentum and ovarian cancer: NO.

Ovarian cancers are one of the most lethal gynecological cancers because they are usually diagnosed in advanced stages when metastasis has already occurred in the peritoneal cavity. Interestingly, ovarian cancers frequently invade the fatty pad of adipose tissue along the stomach called the omentum [1]. Recent studies have shown that omentum contains a population of stem cell-like cells such as adipose stromal cells (ASCs) that engraft in tumors and encourage cancer progression [2]. Omentum-derived ASCs (O-ASCs) have been demonstrated to contribute to the formation of a hospitable environment for the development of ovarian cancer metastasis [2]. Previous studies have shown that O-ASCs enhance proliferation and migration of ovarian cancer cells while reducing their response to chemotherapy and radiation [2]. However, the mechanistic underpinnings of O-ASCs’ role in tumor progression and growth are still unclear. Recently we demonstrated that nitric oxide (NO)-mediated metabolic coupling between O-ASCs and ovarian cancer cells contributes to ovarian cancers’ growth and increased chemosensitivity [3]. Our study revealed that O-ASCs secreted arginine, which was used by cancer cells for NO generation. NO is generated endogenously by an enzymatic conversion of arginine into citrulline through nitric oxide synthase (NOS). NOS has been found to be differentially expressed in obese and non-obese individuals and has been shown to be overexpressed in aggressive ovarian tumors [4]. We revealed that O-ASCs secreted arginine could rescue reduced proliferation rates of ovarian cancers caused by arginine deprivation [3]. Notably, overweight O-ASCs were able to rescue the reduced proliferation of cancer cells more than their lean counterparts. However, more studies are required to shed light on the role of obesity in ovarian cancer development and progression. Several studies indicate that NO is a double-edged molecule. It is a tumor promoter at lower concentrations (less than 500 nM), but damages DNA and induces apoptosis at higher concentrations (millimolar range) [5]. We found that ovarian cancer cells in cocultures with O-ASCs had significantly higher NO levels compared to their non-coculture counterparts. We previously showed that NO synthesis upregulates Warburg effect in ovarian cancers [5]. Interestingly, coculture of cancer cells with O-ASCs reduced oxygen consumption rates (OCR) in cancer cells. We confirmed this shift in ovarian cancers’ metabolism from oxidative phosphorylation (OXPHOS) to glycolysis by comparing the contributions of both pathways towards cellular ATP generation and our data elucidated that O-ASCs increased NO synthesis in cancer cells, resulting in suppression of mitochondrial respiration. Previously, we showed that O-ASCs induced chemo-resistance in cancer cells [2]. Interestingly, in recent studies we found that O-ASCs-mediated chemoresistance can be deregulated by disrupting NO homeostasis [3]. We added L-arginase in direct-contact cocultures of O-ASCs and cancer cells. Both L-arginase (which depletes any secreted arginine by O-ASCs and thereby blocks NO synthesis in cancer cells) and L-NAME (a NO synthase inhibitor) in direct-contact cocultures of O-ASCs and ovarian cancer cells, increased chemosensitivity of paclitaxel in cancer cells. Our results suggest that combined approach of depleting arginine using L-arginase, along with inhibiting NO synthesis in cancer cells using L-NAME, may be a viable therapeutic approach for targeting ovarian cancers, to disrupt the communication between cancer cell and O-ASCs. Arginine is not the only player in the metabolic coupling of O-ASCs and ovarian cancers. Surprisingly, O-ASCs-secreted arginine when is used for NO synthesis in cancer cells, generates citrulline as a byproduct, which is secreted by cancer cells and in turn is ingested by O-ASCs. We hypothesized that this high amount of secreted citrulline concentrations may be beneficial for NO metabolism in O-ASCs. As mentioned before, O-ASCs are multipotent population of mesenchymal stem cells that can differentiate into adipocytes. Surprisingly, we found that secreted citrulline by cancer cells significantly increased adipogenesis capacity of O-ASCs and stimulated the lipid droplet accumulation within O-ASCs. Studies have shown that NO inhibits lipolysis of lipid depots in subcutaneous adipose tissue [6]. This highlights the metabolic symbiosis between omentum-derived ASCs and cancer cells in maintaining NO homeostasis in tumors. Future studies are needed to investigate the mechanism behind the modulation of ovarian tumor's metabolism by NO. The post-translational modification of metabolic enzymes caused by binding of NO with their cysteine residue (s-nitrosylation) may play a crucial role. S-nitrosylation has been known to alter the functions of enzymes in fatty acid metabolism as well as other central energy pathways [7]. The impact of obesity in ovarian cancer initiation and progression has been studied and further studies are needed to explore the role of NO metabolism in this modulation. Furthermore, the link between omentum and other hormonal tumors such prostate cancers needs to be further studied. Even as new mechanisms implicating omentum for ovarian cancer metastasis keep emerging, many aspect of its biology still remains to be unveiled [8]. Figure 1 Crosstalk between ovarian cancers and O-ASCs in tumor microenvironment

Nitric oxide signaling is recruited as a compensatory mechanism for sustaining synaptic plasticity in Alzheimer's disease mice.

Synaptic plasticity deficits are increasingly recognized as causing the memory impairments which define Alzheimer's disease (AD). In AD mouse models, evidence of abnormal synaptic function is present before the onset of cognitive deficits, and presents as increased synaptic depression revealed only when synaptic homeostasis is challenged, such as with suppression of ryanodine receptor (RyR)-evoked calcium signaling. Otherwise, at early disease stages, the synaptic physiology phenotype appears normal. This suggests compensatory mechanisms are recruited to maintain a functionally normal net output of the hippocampal circuit. A candidate calcium-regulated synaptic modulator is nitric oxide (NO), which acts presynaptically to boost vesicle release and glutamatergic transmission. Here we tested whether there is a feedforward cycle between the increased RyR calcium release seen in presymptomatic AD mice and aberrant NO signaling which augments synaptic plasticity. Using a combination of electrophysiological approaches, two-photon calcium imaging, and protein biochemistry in hippocampal tissue from presymptomatic 3xTg-AD and NonTg mice, we show that blocking NO synthesis results in markedly augmented synaptic depression mediated through presynaptic mechanisms in 3xTg-AD mice. Additionally, blocking NO reduces the augmented synaptically evoked dendritic calcium release mediated by enhanced RyR calcium release. This is accompanied by increased nNOS levels in the AD mice and is reversed upon normalization of RyR-evoked calcium release with chronic dantrolene treatment. Thus, recruitment of NO is serving a compensatory role to boost synaptic transmission and plasticity during early AD stages. However, NO's dual role in neuroprotection and neurodegeneration may convert to maladaptive functions as the disease progresses.

Tumor Cells Upregulate Normoxic HIF-1α in Response to Doxorubicin

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that controls cellular homeostasis. Although its activation benefits normal tissue, HIF-1 activation in tumors is a major risk factor for angiogenesis, therapeutic resistance, and poor prognosis. HIF-1 activity is usually suppressed under normoxic conditions because of rapid oxygen-dependent degradation of HIF-1α. Here, we show that, under normoxic conditions, HIF-1α is upregulated in tumor cells in response to doxorubicin, a chemotherapeutic agent used to treat many cancers. In addition, doxorubicin enhanced VEGF secretion by normoxic tumor cells and stimulated tumor angiogenesis. Doxorubicin-induced accumulation of HIF-1α in normoxic cells was caused by increased expression and activation of STAT1, the activation of which stimulated expression of iNOS and its synthesis of nitric oxide (NO) in tumor cells. Mechanistic investigations established that blocking NO synthesis or STAT1 activation was sufficient to attenuate the HIF-1α accumulation induced by doxorubicin in normoxic cancer cells. To our knowledge, this is the first report that a chemotherapeutic drug can induce HIF-1α accumulation in normoxic cells, an efficacy-limiting activity. Our results argue that HIF-1α-targeting strategies may enhance doxorubicin efficacy. More generally, they suggest a broader perspective on the design of combination chemotherapy approaches with immediate clinical impact.

Characterization of C-alkyl amidines as bioavailable covalent reversible inhibitors of human DDAH-1.

C-Alkyl amidine analogues of asymmetric N(ω),N(ω)-dimethyl-L-arginine are dual-targeted inhibitors of both human DDAH-1 and nitric oxide (NO) synthase, and provide a promising scaffold for the development of therapeutics to control NO overproduction in a variety of pathologies including septic shock and some cancers. Using a two-part click-chemistry-mediated activity probe, a homologated series of C-alkyl amidines were ranked for their ability to inhibit DDAH-1 within cultured HEK 293T cells. N⁵-(1-Iminopentyl)-L-ornithine was determined to be the most potent compound in vitro (K(d)=7 μM) as well as in cultured cells, and the binding conformation and covalent reversible mode of inhibition was investigated by comparison of interactions made with DDAH-1 and a catalytically inactive C274S variant, as gauged by X-ray crystallography and isothermal titration calorimetry. By interrupting the ability of the inhibitor to form a covalent bond, the contribution of this interaction could be estimated. These results suggest that further stabilization of the covalent adduct is a promising strategy for lead optimization in the design of effective reagents to block NO synthesis.

Pathogenesis of Hypertension: Interactions Among Sodium, Potassium, and Aldosterone

Arterial hypertension is a major cause of disease-related morbidity and mortality worldwide. It is nearly absent in populations that consume natural foods low in sodium. However, in industrial countries, where the individual intake of sodium is at least 10 times higher, the prevalence of hypertension is approximately 40%. Major population-based studies link a high-sodium and low-potassium diet to an increase in blood pressure. A hallmark of arterial hypertension is endothelial dysfunction characterized by decreased synthesis of nitric oxide (NO). Plasma sodium and potassium are major determinants for the mechanical stiffness of endothelial cells. High plasma sodium levels stiffen endothelial cells and block NO synthesis. Aldosterone is a prerequisite for this action. However, high plasma potassium levels soften endothelial cells and activate NO release. There is increasing evidence that sodium can be stored transiently in considerable amounts and osmotically inactive in the interstitium. Taken together, it is recommended to maintain plasma sodium levels in the low physiologic range and potassium levels in the high physiologic range while suppressing plasma aldosterone as much as possible. A restriction in sodium intake that is accompanied by increased intake of potassium can profoundly improve the prevalence of hypertension and cardiovascular disease.

Noradrenaline can restore decreased neurogenic vasoreactivity

Experiments on isolated segments of the tail artery from young (aged 4-6 weeks) rats addressed the effects of noradrenaline (0.01-1.0 microM) on the reactions of these segments to stimulation with an electric field in isometric conditions. These studies showed that noradrenaline increases this reaction after spontaneous or acidosis (pH 6.6)-induced decreases and that the extent of this increase was proportional to the magnitude of the previous reduction in the reaction. The increase in the decrease in the reaction to electrical stimulation was accompanied by widening of the range of potentiating noradrenaline concentrations and an increase in the noradrenaline concentration at which potentiation was maximal. The nitric oxide synthesis blocker NG-nitro-L-arginine did not eliminate the potentiating effect of noradrenaline. These data led to the conclusion that noradrenaline produces a concentration-dependent recovery of decreased neurogenic reactivity of blood vessels and that this effect is not associated with changes in nitric oxide synthesis.

Polarized intrinsic neural reflexes in response to colonic elongation

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca(2+) imaging techniques. Full colons were 158.1 +/- 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s(-1)). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca(2+) waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechanosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Effects and mechanism of colonic electrical stimulation with trains of short-pulses on colonic transit: experiment with rats

To evaluate the effects and mechanism of colonic electrical stimulation (CES) on colonic transit. 76 male SD rats were equipped with colonic electrodes for stimulation and a catheter in the colon and then randomly divided onto 6 groups: normal saline-(NS)/CES group (14 rats), NS-non-CES group (14 rats), N-nitro-L-arginine (L-NNA)/CES group (12 rats), L-NNA/non-CES group (12 rats), atropine/CES group (12 rats), and atropine/non-CES group (12 rats). All rats underwent intraperitoneal injection of NS, L-NNA, nitric oxide synthesis blocker, or atropine before colon transit. Colonic transit was assessed by calculating the output of phenol red from the anus every 10 min for 90 min CES with trains of short-pulses was performed during the first 40 min of the transit test in the 3 CES groups. (1) CES significantly enhanced the colonic transit. The colonic emptying rate 90 min after the injection of phenol red of the NS/CES group was 82% +/- 5%, significantly higher than that of the NS/non-CES group (57% +/- 6%, P = 0.002) with a 43% increase. (2) L-NNA delayed the colonic transit and prevented the accelerative effect of CES on colonic transit. (3) The phenol red emptying rate 90 min after the injection of the atropine/non-CES group was 16% +/- 3%, significantly lower than that of the NS/non-CES group (P < 0.001). The phenol red emptying rate 90 min after the injection of the atropine/CES group was 41% +/- 6%, significantly higher than that of the atropine/non-CES group (16% +/- 3%, P = 0.01). CES has an excitatory effect on colonic transit and this excitatory effect may be mediated via the nitrergic pathway, not the cholinergic pathway.

Lung toxicity of nitrogen mustard may be mediated by nitric oxide and peroxynitrite in rats

Nitric oxide (NO) has previously been shown to be responsible for nitrogen mustard (NM)-induced tissue toxicity. Excessive amounts of NO are known to be able to produce peroxynitrite, an important reactive nitrogen compound, by reacting with superoxide. Previous studies reported that NO synthase inhibitors are able to prevent NM toxicity. The aim of this study was to evaluate whether peroxynitrite is also responsible for NM-induced lung tissue damage in rats. Wistar rats were divided into four groups. NM was injected intratracheally and was treated with the selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG) (intraperitoneal) or the peroxynitrite scavenger ebselen (intragastric). Control animals were exposed to saline only. NM injection caused both oxidative and nitrosative stress, reflected by dramatically increased levels of the lipid peroxidation end product malondialdehyde (MDA), iNOS activation and urine nitrite–nitrate (NOx) values. Histopathological evaluation demonstrated lung damage with NM exposure. AG blocked iNOS activation and decreased urine NOx levels, and resulted in less histopathological changes in the lung. Although the histopathological outcome was found to be similar to AG, ebselen did not change urinary NOx or lung iNOS levels. Furthermore, ebselen was more able than AG to protect against MDA accumulation. In conclusion, the ability of ebselen to prevent against lung damage without blocking NO synthesis suggests that peroxynitrites may have an important role in the pathogenesis of NM toxicity in addition to NO.

Nitric Oxide Signals ThatAplysiaHave Attempted to Eat, a Necessary Component of Memory Formation After Learning That Food Is Inedible

Inhibiting nitric oxide (NO) synthesis during learning that food is inedible in Aplysia blocks subsequent memory formation. To gain insight into the function of NO transmission during learning we tested whether blocking NO synthesis affects aspects of feeding that are expressed both in a nonlearning context and during learning. Inhibiting NO synthesis with L-NAME and blocking guanylyl cyclase with methylene blue decreased the efficacy of ad libitum feeding. D-NAME had no effect. L-NAME also decreased rejection responses frequency, but did not affect rejection amplitude. The effect of L-NAME was explained by a decreased signaling that efforts to swallow are not successful, leading to a decreased rejection rate, and a decreased ability to reposition and subsequently consume food in ad libitum feeding. Signaling that animals have made an effort to swallow is a critical component of learning that food is inedible. Stimulation of the lips with food alone did not produce memory, but stimulation combined with the NO donor SNAP did produce memory. Exogenous NO at a concentration causing memory also excited a key neuron responding to NO, the MCC. Block of the cGMP second-messenger cascade during training by methylene blue also blocked memory formation after learning. Our data indicate that memory arises from the contingency of three events during learning that food is inedible. One of the events is efforts to swallow, which are signaled by NO by cGMP.

Ontogeny of excitatory and inhibitory control of gastrointestinal motility in the African clawed frog,Xenopus laevis

The transparent body wall of Xenopus laevis larvae during the first developmental stages allows in vivo studies of gastrointestinal tract activity. The purpose of this study was to chart the ontogeny of gut motility in Xenopus larvae and to identify the most important control systems during the first developmental stages. Coordinated descending contraction waves first occurred in the gut at Nieuwkoop and Faber stage 43 [0.8 +/- 0.1 contractions/min (cpm)] and increased to 4.9 +/- 0.1 cpm at stage 47. The cholinergic receptor agonist carbachol (5-10 microM) increased contraction frequency already at stage 43, as did neurokinin A (NKA, 0.3-1 microM). The muscarinic antagonist atropine (100 microM) first affected contraction frequency at stage 45, which coincides with the onset of feeding. The tachykinin antagonist MEN-10,376 (6 microM) blocked NKA-induced contractions but not spontaneous motility. Both sodium nitroprusside [nitric oxide (NO) donor, 1-10 microM] and vasoactive intestinal peptide (VIP, 0.1-1 microM) inhibited contractions from the earliest stage onward. Blocking NO synthesis using NG-nitro-L-arginine methyl ester (100 microM) had no effect per se, but antagonized VIP evoked inhibition at stage 47. We conclude that gastrointestinal motility is well developed in the Xenopus laevis larvae before the onset of feeding. Functional muscarinic and tachykinin receptors are present already at the onset of motility, whereas a cholinergic tone develops around the onset of feeding. No endogenous tachykinin tone was found. Functional VIP receptors mediate inhibition at the onset of motility. NO seems to mediate the VIP effect at later stages.

Colonic Electrical Stimulation Regulates Colonic Transit Via the Nitrergic Pathway in Rats

Gastrointestinal electrical stimulation has been proposed for the treatment of gastrointestinal motor disorders. However, little is known about potential roles of colonic electrical stimulation (CES). The aim of this study was to evaluate the effect and mechanism of CES on colonic transit in conscious rats. Male rats (N = 14) were equipped with a pair of colonic serosal electrodes for stimulation and a catheter in the colon. Colonic transit was assessed in four randomized sessions with or without CES and with or without nitric oxide synthesis blocker, L-NNA, by calculating the output of phenol red from the anus every 10 min for 90 min. Results were as follows. (1) CES with trains of short pulses significantly enhanced colonic transit. Colonic emptying was 57.3 +/- 6.1% in the control session and 81.9 +/- 4.6% with CES at 90 min, reflecting a 43% increase. (2) L-NNA delayed colonic transit compared with saline and prevented the accelerative effect of CES on colonic transit. We conclude that CES has an excitatory effect on colonic transit and this excitatory effect may be mediated via the nitrergic pathway.