Spatiotemporal Alterations of the NO/NOS Neuronal Pools Following Transient Abdominal Aorta Occlusion: Morphological and Biochemical Studies in the Rabbit

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments.2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [³H] l-arginine to [³H] l-citrulline.3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult.4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min).5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.     

Release of nitric oxide and expression of constitutive nitric oxide synthase of human endothelial cells: Enhancement by a 14-membered ring macrolide

A 14-membered ring macrolide, erythromycin, acts not only as an antibacterial but also as an anti-inflammatory agent. We have previously reported that erythromycin modulates neutrophil functions and ameliorates neutrophil-induced endothelial cell damage through the action of cyclic AMP-dependent protein kinase (PKA) and nitric oxide (NO). We investigated the effect of erythromycin on human endothelial cell functions. Erythromycin enhanced intracellular calcium ion concentration ([Ca²⁺]_i) of endothelial cells and NO release from endothelial cells. The enhancement of NO release from endothelial cells by erythromycin was abolished by addition of EGTA in the medium and was partially reduced by addition of H-89, an inhibitor of PKA. These results suggest that erythromycin enhances NO release from endothelial cells through the action of PKA and [Ca²⁺]_i. In addition, constitutive NO synthase (cNOS) protein expression of endothelial cells was dose-dependently enhanced by treatment with erythromycin, which might also contribute to the enhancement of NO release from endothelial cells by erythromycin. The effect of erythromycin as an anti-inflammatory agent might be partially mediated through the enhancement of NO release from endothelial cells and the drug might be a useful tool for the investigation of cNOS of endothelial cells.     

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.     

Restoration of nitric oxide production by aldose reductase inhibitor in human endothelial cells cultured in high-glucose medium

The effects of elevated glucose and aldose reductase inhibitor (ARI:ONO-2235) on nitric oxide (NO) production in cultured human umbilical endothelial cells (HUVEC) were evaluated. Aldose reductase and nitric oxide synthase(NOS) share NADPH as an obligate cofactor, therefore it is suggested that the enhanced of glucose flux (27.5 mM) by aldose reductase inhibited NO production by blunting NOS activity. However, the addition of ONO-2235 (100 μM) prevented the inhibition of [NO_2⁻] production. Since ARI decreases glucose-mediated inhibition of NO production in HUVEC, this agent might ameliorate endothelial function associated with diabetes.     

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca²⁺ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca²⁺ mobilization study shows that l-NAME inhibited the reduction of Ca²⁺-concentration in VSM cells and reduced the increase in Ca²⁺-concentration in endothelial cells induced by SNP. This enhancement in Ca²⁺-concentration in the endothelial cells is due to a voltage-dependent Ca²⁺ channels activation. The present findings indicate that the relaxation and [Ca²⁺]_i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.     

Aged garlic extract enhances production of nitric oxide

Nitric oxide (NO) controls several physiological functions of the cardiovascular system. Three kinds of NO synthases (NOSs), neuronal constitutive NOS (ncNOS), inducible NOS (iNOS) and endothelial constitutive NOS (ecNOS), were responsible for NO biosynthesis. This study investigated the effect of aged garlic extract (AGE) on NO production by measuring the NO metabolites nitrite and nitrate in the plasma of mice. AGE (2.86 g/kg, p.o.) temporarily increased NO production by 30-40% from 15 to 60 min after administration. The time course of the fluctuation in NO levels in the AGE-treated group was clearly different to that in a group of mice treated with lipopolysaccharides, a typical iNOS inducer. Arginine (63 mg/kg, p.o.) at the equivalent dose of AGE did not increase NO production. However diphenyleneiodonium chloride (1 mg/kg, i.p.), a selective cNOS inhibitor, administered prior to AGE, overcame the effect of AGE. These results indicate that AGE increased NO production by activating cNOS, but not iNOS. The arginine contained in AGE was not responsible for the effect. AGE may be a useful tool for the prevention of cardiovascular disease.     

Opposite caudal versus rostral brain nitric oxide synthase response to generalized seizures in a novel rodent model of reflex epilepsy

AimsNitric oxide (NO) is synthesized from L-arginine (L-Arg) by three different isoforms of NO synthase (NOS), i.e. the constitutive neuronal and endothelial NOS (nNOS and eNOS) and the inducible NOS (iNOS). NO has been involved in the pathophysiology of epilepsy, but available data are conflicting and the actual role of NO in epilepsy still remains to be clarified. In this study we investigated the basal and post-seizure levels of constitutive NOS (cNOS) activity as well as the expression of the cNOS isoforms across brain regions in a novel model of epilepsy.Main methodscNOS activity was assessed in various brain areas along the rostro-caudal axis in control wild type hamsters, unstimulated generalized audiogenic seizure prone hamsters, Salamanca strain, GASH:Sal and GASH:Sal after 10 sound-induced epileptic seizures. Additionally, Western blot experiments for nNOS and eNOS were performed in those areas where relevant changes in cNOS activity were found.Key findingsIn the GASH:Sal, cNOS activity increased in the mesencephalic areas studied while cNOS activity decreased in both the striatum and cerebral cortex after 10 sound-induced epileptic seizures. nNOS (but not eNOS) expression paralleled the variations in cNOS activity. The same sound stimulation had no effect on control hamsters.SignificanceThese results suggest a different NOS response in the regions close to the original epileptic focus (caudal, in our auditory model) versus the remote areas (rostral) possibly recruited at later stages or after repeated crises. These findings may account for some of the discrepancies found regarding the role of NO in epilepsy.     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

In vitro induction of nitric oxide by fructose-1,6-diphosphate in the cardiovascular system of rats

Nitric oxide (NO) functions as a cellular messenger in a number of organs and cell systems in the cardiovascular system (CVS); it is a significant determinant of basal vascular tone and regulates myocardial contractility and platelet aggregation. The present study focused upon understanding the in vitro effects of fructose-1,6-diphosphate (FDP) on the rat cellular NO pathway. The iNOS activity was measured by monitoring the formation of (³H)-citrulline in 50,000 g soluble fractions of crude homogenates of endothelial (ET) and smooth muscle cells (SMC) from the arteries of rats, and macrophages (MAC) and lymphocytes (LYM) from rat blood. FDP in concentrations of 10-1000 M stimulated rat cellular iNOS activity in a concentration-dependent manner. FDP-stimulated rat cellular iNOS was found to be completely reversed by 5 M concentration of NG-monomethyl-L-arginine (L- NMMA), the potent mammalian NOS inhibitor. These studies demonstrated that FDP may induce the formation of NO by stimulating rat cardiovascular iNOS activity.     

Regulation of M1 Muscarinic Receptor-Mediated Signaling in Intact Cells by Exogenous,but Not Endogenously Produced,Nitric Oxide

Regulation of nitric oxide (NO) formation is critical to ensure maintenance of appropriate cellular concentrations of this labile, signaling molecule. This study investigated the role exogenous and endogenously produced NO have in feeding back to regulate NO synthesis in intact cells. Two NO donors inhibited activation of neuronal NO synthase (nNOS) in response to the muscarinic receptor agonist carbachol in Chinese hamster ovary (CHO) cells stably transfected with the M₁ muscarinic receptor and nNOS. The presence of the NO scavenger [2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide · potassium salt] (C-PTIO) potentiated carbachol-induced activation of nNOS in transfected CHO cells. C-PTIO also potentiated nNOS activity in response to the Ca²⁺ ionophore ionomycin. In contrast, the NO scavenger oxyhemoglobin depressed carbachol- and ionomycin-induced NO formation. These discrepant results suggest that it is unlikely that endogenously produced NO induces feed back inhibition at the level of nNOS activation itself. Exogenous sources of NO inhibited carbachol-induced inositol phosphates formation. However, endogenously produced NO did not appear to feed back to regulate phosphoinositide hydrolysis as there was no difference in [³H]inositol phosphates formation between cells that do or do not express nNOS. There was also no change in carbachol-induced [³H]inositol phosphates formation in the presence or absence of a NOS inhibitor or the NO scavenger C-PTIO. A decrease in the carbachol-mediated transient Ca²⁺ peak was observed in cells that express nNOS as compared to cells lacking the enzyme, suggesting that endogenous NO might inhibit receptor mediated Ca²⁺ signaling. This conclusion, however, was not supported by the lack of ability of a NOS inhibitor to modulate carbachol-induced Ca²⁺ elevations. Taken together, these results highlight differences in the regulation of the nNOS activation cascade by endogenous vs. exogenous sources of NO.     

Anomalous Increase in Nitric Oxide Synthase Activity by Certain Nitric Oxide-Generating Compounds in Intact Neuronal Cells

Abstract: It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E-115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of l -[³H]arginine into l -[³H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN-1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN-1 and SNP markedly stimulated l -[³H]citrulline formation in a time- and concentration-dependent manner when cells were incubated with the compounds for >1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E-115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN-1 or SNP in intact cells was dependent on the presence of extracellular Ca²⁺, suggesting that it might be due to increased Ca²⁺ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN-1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken-cell preparations and in intact cells. Thus, modulation of NOS activity by NO-releasing compounds is not dependent on cyclic GMP formation and might not be related in a simple fashion to NO generation. Alternatively, activation of guanylate cyclase and stimulation of NOS activity might require different redox species of NO. Our present findings might be of clinical relevance in relation to long-term use of NO-generating compounds as therapeutic agents.     

Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

A calcium channel blocker activates both ecto-5(')-nucleotidase and NO synthase in HUVEC

Since amlodipine, a long-acting Ca channel blocker, increases both NO and adenosine production in canine hearts, we investigated that amlodipine activates both ecto-5(')-nucleotidase responsible for adenosine production and NO synthase (NOS) for NO production in human umbilical venous endothelial cells (HUVECs), and its cellular signaling. We measured activities of ecto-5(')-nucleotidase and NOS in HUVECs in the condition with additions of xanthine (100 microM)+xanthine oxidase (1.6 x 10(-3)U/ml) in the presence or absence of amlodipine (1 x 10(-9)-1 x 10(-6)M). Amlodipine increased both ecto-5(')-nucleotidase and NOS activities. Xanthine+xanthine oxidase deactivated both NOS and ecto-5(')-nucleotidase, and amlodipine increased both activities of NOS and ecto-5(')-nucleotidase by 117+/-33% and 48+/-6%, respectively. Amlodipine phosphorylated p38MAP kinase and that an inhibitor of p38MAP kinase inhibited the amlodipine-induced activation of both NOS and ecto-5(')-nucleotidase. Furthermore, amlodipine increased both adenosine and NO production in the canine ischemic hearts. We concluded that amlodipine activates both NOS and ecto-5(')-nucleotidase via p38MAP kinase in vitro and enhances both NO and adenosine production in vivo.     

Acute hypoxia increases intracellular L-arginine content in cultured porcine pulmonary artery endothelial cells

Exposure to hypoxia (0% O₂) for 4–24 h resulted in increased intracellular L-arginine content and increased activity of calpain, a calcium-dependent neutral cysteine protease, in pulmonary artery endothelial cells. Calpain-inhibitor I abolished the increased L-arginine content in hypoxic cells. When endothelial cell proteins were labeled with [³H]-L-arginine and the cells exposed to hypoxia, we observed an increase in free [³H]-L-arginine and a decrease in [³H]-L-arginine-labeled proteins. Once again, calpain-inhibitor I prevented the increases in free [³H]-L-arginine and the decreases in [³H]-L-arginine-labeled proteins in hypoxic cells. Hypoxia also inhibited the synthesis of L-arginine-containing proteins. Thus, the increase in intracellular L-arginine content in hypoxic pulmonary artery endothelial cells is caused by an increase in proteolysis secondary to calpain and a decrease in protein synthesis. These results indicate that hypoxia can modulate the availability of free intracellular L-arginine, the exclusive precursor of nitric oxide (NO) and the primary substrate of NO synthase, by affecting the synthesis and degradation of cellular proteins. © 1996 Wiley-Liss, Inc.     

PDZ-dependent activation of nitric-oxide synthases by the serotonin 2B receptor

Taking advantage of three cellular systems, we established that 5-HT(2B) receptors are coupled with NO signaling pathways. In the 1C11 serotonergic cell line and Mastomys natalensis carcinoid cells, which naturally express the 5-HT(2B) receptor, as well as in transfected LMTK(-) fibroblasts, stimulation of the 5-HT(2B) receptor triggers intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the C terminus of the 5-HT(2B) receptor is required for recruitment of the cNOS and iNOS transduction pathways. Indeed, the 5-HT(2B) receptor-mediated NO coupling is abolished not only upon introduction of a competitor C-terminal 5-HT(2B) peptide in the three cell types but also in LMTK(-) fibroblasts expressing a receptor C-terminally truncated or harboring a point mutation within the PDZ domain. The occurrence of a direct functional coupling between the receptor and cNOS activity is supported by highly significant correlations between the binding constants of drugs on the receptor and their effects on cNOS activity. The 5-HT(2B)/iNOS coupling mechanisms appear more complex because neutralization of endogenous Galpha(13) by specific antibodies cancels the cellular iNOS response while not interfering with cNOS activities. These findings may shed light on physiological links between the 5-HT(2B) receptor and NO and constitute the first demonstration that PDZ interactions participate in downstream transductional pathways of a G protein-coupled receptor.     

Urotensin-II activates L-arginine/nitric oxide pathway in isolated rat aortic adventitia

Urotensin-II (U-II), a cyclic peptide widely expressed in blood vessels, has diverse vascular actions that range from potent vasoconstriction to vasodilation. Although, U-II-induced vasodilation has been shown to be partially dependent on nitric oxide (NO), the involvement of vascular adventitia-derived NO, remains unknown. The present study aimed to elucidate the activation of U-II on L-arginine/NO pathway in isolated rat aortic adventitia. In adventitia of thoracic and abdominal aortas, the l-arginine/NO pathway was similarly characterized: the uptake of l-[(3)H]arginine was Na(+)-independent, with the peak occurring over around 40 min incubation; the total NO synthase (NOS) activity was mostly calcium-independent (>90%), and significantly inhibited by a specific iNOS inhibitor AMT; the production of NO metabolites nitrate and nitrite (NO(x)) was stimulated by L-arginine but not by D-arginine. In aortic adventitia exposed to rat U-II (10(-9) and 10(-8)M) for 6 h, the V(max) of l-[(3)H]arginine uptake over 40 min incubation was significantly increased, while the K(m) of l-[(3)H]arginine uptake showed no significant change. Besides, the iNOS mRNA level was up-regulated, the total NOS activity, largely calcium-independent, was significantly induced, and the NO(x) production was significantly stimulated by U-II. According to the same protocol as U-II, the positive control lipopolysaccharide (LPS, 10 microg/ml), which had been established to activate adventitial L-arginine/NO pathway, increased l-[(3)H]arginine uptake, iNOS activity and NO(x) production to a greater extent than U-II. In addition, the total NOS activities induced by 3 and 6h incubation of U-II and LPS were significantly inhibited by a specific inhibitor of protein synthesis, actinomycin D. In conclusion, the results showed that rat U-II activated L-arginine/NOS/NO pathway in rat aortic adventitia, suggesting a potential contributive role of adventitia-derived NO in the vasodilator response of U-II.     

Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis

The human neuroblastoma cell line SK-N-BE, after incubation with 10 μM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10–14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [³H]arginine into [³H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor N^G-nitro-L -arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells. J. Cell. Physiol. 174:99–106, 1998. © 1998 Wiley-Liss, Inc.     

Endothelial and Neuronal Nitric Oxide Activate Distinct Pathways on Sympathetic Neurotransmission in Rat Tail and Mesenteric Arteries

Nitric oxide (NO) seems to contribute to vascular homeostasis regulating neurotransmission. This work aimed at assessing the influence of NO from different sources and respective intracellular pathways on sympathetic neurotransmission, in two vascular beds. Electrically-evoked [³H]-noradrenaline release was assessed in rat mesenteric and tail arteries in the presence of NO donors or endothelial/neuronal nitric oxide synthase (NOS) inhibitors. The influence of NO on adenosine-mediated effects was also studied using selective antagonists for adenosine receptors subtypes. Location of neuronal NOS (nNOS) was investigated by immunohistochemistry (with specific antibodies for nNOS and for Schwann cells) and Confocal Microscopy. Results indicated that: 1) in mesenteric arteries, noradrenaline release was reduced by NO donors and it was increased by nNOS inhibitors; the effect of NO donors was only abolished by the adenosine A₁ receptors antagonist; 2) in tail arteries, noradrenaline release was increased by NO donors and it was reduced by eNOS inhibitors; adenosine receptors antagonists were devoid of effect; 3) confocal microscopy showed nNOS staining in adventitial cells, some co-localized with Schwann cells. nNOS staining and its co-localization with Schwann cells were significantly lower in tail compared to mesenteric arteries. In conclusion, in mesenteric arteries, nNOS, mainly located in Schwann cells, seems to be the main source of NO influencing perivascular sympathetic neurotransmission with an inhibitory effect, mediated by adenosine A₁ receptors activation. Instead, in tail arteries endothelial NO seems to play a more relevant role and has a facilitatory effect, independent of adenosine receptors activation.     

Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature

Ischemic stroke from a reduction in blood flow to the brain microvasculature results in a subsequent decreased delivery of oxygen (i.e., hypoxia) and vital nutrients to endothelial, neuronal, and glial cells. Hypoxia associated with stroke has been shown to increase paracellular permeability of the blood-brain barrier, leading to the release of cellular mediators and brain tissue injury. Whereas reperfusion does not occur in all ischemic strokes, increased permeability has been seen in posthypoxic reoxygenation. Currently, it is unknown whether these deleterious effects result from cellular mechanisms stimulated by decreased oxygen during stroke or posthypoxic reoxygenation stress. This study used primary bovine brain microvessel endothelial cells (BBMECs) to examine the involvement of nitric oxide (NO) as a mediator in hypoxia-induced permeability changes. Hypoxia-induced increased transport of [14C]sucrose across BBMEC monolayers compared with normoxia was attenuated by either posthypoxic reoxygenation or inhibition of NO synthase (NOS). The hypoxia-induced permeability effect was further reduced when NOS inhibition was combined with posthypoxic reoxygenation. Additionally, a significant increase in total NO was seen in BBMECs after hypoxic exposure. This correlation was supported by the increased [14C]sucrose permeability observed when BBMECs were exposed to the NO donor diethylenetriaamine NONOate. Western blot analyses of NOS isoforms showed a significant increase in the inducible isoform after hypoxic exposure with a subsequent reduction in expression on reoxygenation. Results from this study suggest that hypoxia-induced blood-brain barrier breakdown can be diminished by inhibition of NO synthesis, decreased concentration of NO metabolites, and/or reoxygenation.