Diabetic neuropathy: An evaluation of the use of quercetin in the cecum of rats

AIM:To investigate the effect of quercetin supplementation on the myenteric neurons and glia in the cecum of diabetic rats.METHODS:Total preparations of the muscular tunic were prepared from the ceca of twenty-four rats divided into the following groups:control(C),control supplemented with quercetin(200 mg/kg quercetin body weight)(CQ),diabetic(D)and diabetic supplemented with quercetin(DQ).Immunohistochemical double staining technique was performed with HuC/D(general population)/nitric oxide synthase(nNOS),HuC-D/S-100and VIP.Density analysis of the general neuronal population HuC/D-IR,the nNOS-IR(nitrergic subpopulation)and the enteric glial cells(S-100)was performed,and the morphometry and the reduction in varicosity population(VIP-IR)in these populations were analyzed.RESULTS:Diabetes promoted a significant reduction(25%)in the neuronal density of the HuC/D-IR(general population)and the nNOS-IR(nitrergic subpopulation)compared with the C group.Diabetes also significantly increased the areas of neurons,glial cells and VIP-IR varicosities.Supplementation with quercetin in the DQ group prevented neuronal loss in the general population and increased its area(P<0.001)and the area of nitrergic subpopulation(P<0.001),when compared to C group.Quercetin induced a VIP-IR and glial cells areas(P<0.001)in DQ group when compared to C,CQ and D groups.CONCLUSION:In diabetes,quercetin exhibited a neuroprotective effect by maintaining the density of the general neuronal population but did not affect the density of the nNOS subpopulation.     

Neuronal Nitric Oxide Synthase Gene Transfer into the Rat Prostate Using In Vivo Electroporation

Objectives: It has been reported that nitric oxide (NO) mainly contributes to prostate or urethral smooth muscles relaxation, and that nitrergic innervation and neuronal NO synthase (nNOS) levels are decreased in benign prostatic hyperplasia. The purpose of the present study was to evaluate the feasibility to gene therapy for benign prostatic hyperplasia by transferring nNOS gene into the rat prostate with in vivo electroporation (EP) procedure. Methods: Male Sprague–Dawley rats were divided into four groups (sham, only EP, only nNOS injection, and nNOS gene injection with EP groups). Fifty micrograms of luciferase gene and nNOS expression vectors in 50 µL of K‐PBS (potassium‐phosphate buffered saline) were injected into the prostate. Immediately after the injection of these vectors, the vector injection points were electroporated by the two‐square parallel electrodes. Two days after gene transfer, luciferase analysis and an immunohistochemical staining for nNOS were performed, and NO₂^?/NO₃^? (NO_X) release was measured using high‐performance liquid chromatography coupled with the microdialysis procedure. Results: The optimal electric pulse conditions were 50 V, 1 Hz and 10 msec. In vivo EP with these conditions showed the increase in the luciferase gene expression approximately 300‐fold of the control group. In the nNOS gene injection with EP group, the marked nNOS immunoreactivity was observed, and NO_X release was significantly higher, as compared to other groups. Conclusion: The results suggest that EP is a feasible technique for in vivo gene transfer into the rat prostate, and that the transferred nNOS gene functionally expresses and contributes to NO production.     

脉络宁对新生大鼠缺氧缺血性脑损伤后nNOS和c-Fos表达的影响

目的:检测新生大鼠缺血缺氧后神经元型一氧化氮合酶(nNOS)和c-Fos免疫活性表达改变及脉络宁对其的影响。方法:结扎7d龄大鼠右侧颈总动脉1h,然后暴露在8%氧和92%氮的混合气体中2h,建立缺氧缺血脑损伤模型。用免疫组织化学方法检测并比较缺血缺氧后和脉络宁处理后c-Fos和nNOS的免疫活性。结果:缺血缺氧6h后c-Fos表达达高峰;与缺氧缺血组相比,脉络宁处理组c-Fos阳性神经元数量增加,表达c-Fos的时间延长;与假手术组相比,缺氧缺血组nNOS表达水平增高,脉络宁处理组则下降。结论:脉络宁可抑制nNOS表达,增强c-Fos表达,可能对缺血缺氧性脑损伤有保护作用。     

Lower esophageal sphincter is achalasic in nNOS(-/-) and hypotensive in W/W(v) mutant mice.

BACKGROUND AND AIMS: It has been proposed that nitrergic nerves mediate lower esophageal sphincter (LES) relaxation with intramuscular interstitial cells of Cajal (ICC-IM) as an intermediary. Dysfunction of the nitrergic pathway has been shown to cause LES hypertension and impaired relaxation in achalasia. We determined whether mice with neuronal nitric oxide synthase gene disruption (nNOS(-/-)) and W/W(v) mice lacking ICC-IM have achalasia-like LES dysfunction. METHODS: Intraluminal manometry using a customized micro-sized catheter assembly was performed in anesthetized mice. Basal LES pressure and swallow- and vagal-evoked LES relaxations were quantified in wild-type, Nomega-nitro-L-arginine methyl ester HCl salt (L-NAME)-treated, nNOS(-/-), and W/W(v) mice. RESULTS: Wild-type mouse LES maintained a basal pressure (24 +/- 3 mm Hg; N = 8) and relaxed normally to swallow (87% +/- 3%; N = 8) and vagal stimulation (91% +/- 4% mm Hg; N = 6). Pretreatment with L-NAME (100 mg/kg, intravenously) attenuated LES relaxation to both stimuli (P < 0.05). The LES in nNOS(-/-) was significantly hypertensive (36 +/- 5 mm Hg; N = 10; P < 0.05) with a markedly impaired relaxation (P < 0.05). In contrast, W/W(v) mouse LES was significantly hypotensive (11 +/- 2 mm Hg; N = 6; P < 0.05) with normal relaxation that was blocked by L-NAME. CONCLUSIONS: nNOS(-/-) mice have LES hypertension with impaired relaxation resembling achalasia. In contrast, W/W(v) mice have hypotensive LES with unimpaired relaxation, suggesting that ICC-IM do not play a role in nitrergic neurotransmission.     

大鼠脑缺血再灌注后神经细胞NOS表达与细胞凋亡及米帕明的保护作用

目的探讨大鼠局灶性脑缺向再灌注后神经细胞一氧化氮合酶（NOS）的表达与神经细胞凋亡的关系及米帕明的保护作用。方法采用大脑中动脉内栓线阻断法（MCAO）造成局灶性脑缺血再灌注模型。用原位细胞凋亡检测方法观察神经细胞凋亡;用免疫组织化学方法检测大鼠神经细胞（nNOS、iNOS）的阳性表达的细胞数目。结果与假手术对照组比较,脑缺血再灌注2h后缺血侧神经细胞nNOS、iNOS表达升高,并出现神经细胞凋亡,随着再灌注时间的延长,神经细胞iNOS的表达明显增强,凋亡神经细胞数逐渐增多,至24h达高峰,但神经细胞nNOS的表达并未见明显增强。米帕明保护组神经细胞nNOS、iNOS的表达和凋亡神经细胞数明显低于缺血再灌组（p〈0．01）。结论脑缺血再灌注后缺血侧神经细胞nNOS的表达增强,iNOS的表达显著升高,使NO的形成增加,这可能是介导脑缺血再灌注后神经细胞凋亡的机制之一。米帕明具有下调神经细胞nNOS、iNOS的表达,减少NO的生成、抑制细胞凋亡．减轻缺血再灌注对大鼠神经细胞损伤的作用。     

Evidence for Forebrain Cholinergic Neuronal Loss in Congenital Ornithine Transcarbamylase Deficiency

Congenital ornithine transcarbamylase (OTC) deficiency in humans results in failure to thrive, hypotonia, seizures and mental retardation. Neuropathologic evaluation reveals significant cerebral cortical atrophy, delayed myelination and Alzheimer type II astrocytosis. Using an animal model of congenital OTC deficiency, the sparse fur (spf) mouse, studies reveal convincing evidence of a loss of forebrain cholinergic neurons in this condition. Evidence includes (i) reduced activities of the cholinergic nerve terminal enzyme choline acetyltransferase (ChAT), (ii) a 25% loss of ChAT immunostaining, (iii) reduced high affinity transport of [³H]choline by cortical synaptosomes and (iv) a selective reduction in densities of presynaptic muscarinic M₂ binding sites, inspf mouse brain compared to controls. A partial correction of the cholinergic deficit was observed following treatment with acetyl-L-carnitine. Possible mechanisms responsible for cholinergic neuronal loss in congenital OTC deficiency include decreased synthesis of the ChAT substrate acetyl CoA, impaired cerebral energy metabolism and NMDA receptor-mediated excitotoxicity. Loss of forebrain cholinergic neurons is consistent with the severe cognitive impairment characteristic of congenital OTC deficiency.     

Gender Bias in Gastroparesis: Is Nitric Oxide the Answer?

Accumulating evidence suggests that gender-related differences are prominent in gastric motility functions in both health and disease. Women are more susceptible to gastroparesis than men. Though the mechanism(s) involved are not fully understood, impairment of the nitrergic system is one of the main factors responsible for the disease. Uncoupling of neuronal nitric oxide synthase (nNOS) causes a decreased synthesis of NO leading to a reduction in smooth muscle relaxation. Tetrahydrobiopterin (BH₄) (an essential cofactor for nNOS) is a key regulator of nNOS activity for stomach dysfunction and gastroparesis. In addition, BH₄ has been shown to be a potent antioxidant and anti-inflammatory agent. Well established by results from our laboratory, a diminished intracellular (BH₄:total biopterin) ratio in diabetic female rats significantly impairs nNOS activity and function. Recent research has been focused on BH₄ biosynthesis and gastroparesis because reduced BH₄ cofactor levels can alter the production of NO by nNOS. Researchers are now paying more attention to the possibility of using BH₄ as a therapeutic strategy in gastroparesis. The purpose of this review is to provide an overview of the regulation and function of nNOS by sex hormones and BH₄ and its potential role in the treatment of gastroparesis.     

Hypothyroidism induces selective oxidative stress in amygdala and hippocampus of rat

The effects of hypothyroidism on lipid peroxidation (LP), reactive oxygen species (ROS), and nitric oxide synthase (NOS), levels and expression, in rat brain were examined. Hypothyroidism was induced by administering methimazole in drinking water (60 mg/kg/day). In striatum, motor cortex and cerebellum of hypothyroid rats LP was not modified, whereas LP and ROS increased in amygdala and hippocampus of hypothyroid rats at the third week of treatment with methimazole as compared to euthyroid group values. Regarding NOS participation, only hippocampal constitutive-NOS activity was increased, accompanied by an augmentation in nNOS expression. Results show that hypothyroidism induces selective oxidative stress in both the hippocampus and amygdala, where the nitrergic system is involved.     

应激对大鼠结肠神经系统nNOS表达的影响

目的:探讨应激对大鼠结肠神经系统nNOS表达的影响. 方法:SD大鼠30只随机分为对照组,应激组和L-NAME 组,采用水浸-束缚应激(WRS)动物模型,用免疫组织化学ABC法检测nNOS在大鼠结肠黏膜下神经丛和肌间神经丛的表达,应用计算机图像分析系统对其表达进行定量分析．结果:与对照组比较,应激组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的灰度值明显减少(P=0．02或P =0．005),阳性神经元细胞数的平均密度增加(P=0．04 或P=0．01),表达增强,且在黏膜上皮细胞、固有层淋巴细胞也有nNOS表达．L-NAME组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的灰度值较应激组增加 (P=0．04),平均密度下降(P=0．04或P=0．03),表达减弱,而与对照组比较均无明显差异(P>0．05)．结论:应激可引起大鼠结肠神经系统nNOS表达增强, 提示一氧化氮(NO)在应激所致的结肠功能失调中可能起重要作用．     

Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: A new insight into selective nitrergic neuropathy in diabetes

Aims/hypothesis We have previously shown that in diabetes nitrergic neurones innervating the urogenital and gastrointestinal organs undergo a selective degenerative process. This comprises an initial insulin-reversible decrease in neuronal nitric oxide synthase (nNOS) in the axons, followed by apoptosis of the nitrergic neurones, a process that is not reversible by insulin. Since apoptosis was independent of serum glucose concentrations, and advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of diabetic complications, we have now measured AGEs in the serum and penis, pyloric sphincter and pelvic ganglia of diabetic animals at different times after streptozotocin treatment. Furthermore, we have studied their effect in vitro on human neuroblastoma (SH-SY5Y) cells in the presence or absence of nNOS expression.Methods Serum AGEs were measured using fluorometry and ELISA. Accumulation of AGEs in the tissues was evaluated with immunohistochemistry. The viability, apoptosis and oxidative stress in SH-SY5Y cells were measured upon exposure to AGEs or high concentrations of glucose.Results AGEs increased gradually in the serum and tissues of streptozotocin-induced diabetic rats; this process was not affected by delayed insulin treatment. In SH-SY5Y cells, AGEs, but not high glucose concentrations, increased the reactive oxygen species and caspase-3-dependent apoptosis in a synergistic fashion with endogenous nitric oxide (NO). Apoptosis was prevented by treatment with a NOS inhibitor, a pan-caspase inhibitor, a soluble receptor of AGEs or an anti-oxidant, but not an inhibitor of soluble guanylate cyclase.Conclusions/interpretation The synergistic actions of NO and AGEs account for the irreversible nitrergic degeneration in diabetes.Abbreviations AGEs advanced glycation endproducts - eNOS endothelial nitric oxide synthase - HSA-AGEs advanced glycated human serum albumin - iNOS inducible nitric oxide synthase - L-NAME N^G-nitro-L-arginine methyl ester (NOS inhibitor) - MPG major pelvic ganglion - NAC N-acetyl-L-cysteine - NO nitric oxide - NOS nitric oxide synthase - nNOS neuronal nitric oxide synthase - ODQ 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (sGC inhibitor) - RA retinoic acid - ROS reactive oxygen species - sRAGE soluble receptor of advanced glycation endproduct - STZ streptozotocin - Z-VAD-FMK Z-Val-Ala-Asp(OMe)-CH₂F (pan-caspase inhibitor)     

Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase.

Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. We recently identified a brain-specific protein that associates with huntingtin and is designated huntingtin-associated protein (HAP1). We now describe selective neuronal localizations of HAP1. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy.     

藻蓝蛋白在脑缺血再灌注损伤过程中的抗氧化作用

目的研究脑缺血再灌注神经细胞一氧化氮合酶(NOS)的表达以及藻蓝蛋白的抗氧化作用机制。方法随机将52只Wistar大鼠分为假手术组(4只)、对照组(24只)和治疗组(24只);后两组再分为脑缺血1 h再灌注6、12 h,13、、7及14天组,每组4只。应用线栓法制作大鼠大脑中动脉阻塞再灌注(MCAO/R)模型[1],藻蓝蛋白进行干预,采用免疫组织化学方法分别观察神经元型NOS(nNOS)、内皮型NOS(eNOS)、诱导型NOS(iNOS)的表达以及藻蓝蛋白的干预作用。结果假手术组脑组织中nNOSi、NOS和eNOS有微弱表达,脑缺血再灌注后6 h,皮质区和纹状体区nNOSi、NOS和eNOS逐渐表达增强,于24 h达高峰,之后逐渐减弱,至7~14 d仍高于假手术组。治疗组NOS变化趋势与对照组相似,与对照组同一时间点比较,nNOS和iNOS表达明显低,eNOS表达明显增强。结论脑缺血再灌注损伤后,藻蓝蛋白可能通过降低脑组织中nNOS和iNOS表达,上调eNOS的表达,而发挥其抗氧化作用。     

Is l-Glutathione More Effective Than l-Glutamine in Preventing Enteric Diabetic Neuropathy?

Background

Diabetes and its complications appear to be multifactorial. Substances with antioxidant potential have been used to protect enteric neurons in experimental diabetes.

Aim

This study evaluated the effects of supplementation with l-glutamine and l-glutathione on enteric neurons in the jejunum in diabetic rats.

Methods

Rats at 90 days of age were distributed into six groups: normoglycemic, normoglycemic supplemented with 2 % l-glutamine, normoglycemic supplemented with 1 % l-glutathione, diabetic (D), diabetic supplemented with 2 % l-glutamine (DG), and diabetic supplemented with 1 % l-glutathione (DGT). After 120 days, the jejunums were immunohistochemically stained for HuC/D+ neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP). Western blot was performed to evaluate nNOS and VIP. Submucosal and myenteric neurons were quantitatively and morphometrically analyzed.

Results

Diabetic neuropathy was observed in myenteric HuC/D, nNOS, and VIP neurons (p < 0.05). In the submucosal plexus, diabetes did not change nitrergic innervation but increased VIPergic neuronal density and body size (p < 0.05). Supplementation with l-glutathione prevented changes in HuC/D neurons in the enteric plexus (p < 0.05), showing that supplementation with l-glutathione was more effective than with l-glutamine. Myenteric nNOS neurons in the DGT group exhibited a reduced density (34.5 %) and reduced area (p < 0.05). Submucosal neurons did not exhibit changes. The increase in VIP-expressing neurons was prevented in the submucosal plexus in the DG and DGT groups (p < 0.05).

Conclusion

Supplementation with l-glutathione exerted a better neuroprotective effect than l-glutamine and may prevent the development of enteric diabetic neuropathy.     

Neuroprotective Effect of Quercetin on the Duodenum Enteric Nervous System of Streptozotocin-Induced Diabetic Rats

Background

In diabetes mellitus (DM), hyperglycemia promotes changes in biochemical mechanisms that induce oxidative stress. Oxidative stress has been closely linked to adverse consequences that affect the function of the gastrointestinal tract caused by injuries to the enteric nervous system (ENS) that in turn cause neurodegeneration and enteric glial loss. Therapeutic approaches have shown that diet supplementation with antioxidants, such as quercetin, reduce oxidative stress.

Aims

This work sought to evaluate neurons and enteric glial cells in the myenteric and submucosal plexuses of the duodenum in diabetic rats supplemented with quercetin.

Methods

The duodenum of 24 rats, including a control group (C), control quercetin supplementation group (CQ), diabetic group (D), and diabetic quercetin supplementation group (DQ), were used to investigate whole mounts of muscular and submucosal layers subjected to immunohistochemistry to detect vasoactive intestinal peptide in the myenteric layer and double-staining for HuC-D/neuronal nitric oxide synthase (nNOS) and HuC-D/S100.

Results

A reduction of the general neuronal population (HuC/D) was found in the myenteric and submucosal plexuses (p?<?0.001) in the D and DQ groups. The nitrergic subpopulation (nNOS) decreased only in the myenteric plexus (p?<?0.001), and glial cells decreased in both plexuses (p?<?0.001) in the D and DQ groups. In diabetic rats, quercetin supplementation reduced neuronal and glial loss. Diabetes promoted an increase in the cell body area of both the general and nitrergic populations. Quercetin supplementation only prevented neuronal hypertrophy in the general population.

Conclusion

Supplementation with quercetin eased the damage caused by diabetes, promoting a neuroprotective effect and reducing enteric glial loss in the duodenum.     

Brain neuronal nitric oxide synthase neuron-mediated sympathoinhibition is enhanced in hypertensive Dahl rats

OBJECTIVE: To elucidate the role of central neurons containing neuronal nitric oxide synthase (nNOS neurons) in the sympathetic nervous system in hypertensive Dahl salt-sensitive (DS) rats. DESIGN AND METHODS: Dahl rats were fed either a regular-salt (0.4% NaCl) or high-salt (8% NaCl) diet for 4 weeks. The effect of intracerebroventricular administration of S-methyl-L-thiocitrulline, a selective nNOS inhibitor, on renal sympathetic nerve activity was examined in chronically instrumented conscious DS rats. The activity and protein amount of brain nNOS was evaluated by enzyme assay and western blot analysis. The distribution and number of nNOS neurons in the brainstem were examined immunohistochemically in hypertensive and normotensive DS rats. RESULTS: S-methyl-L-thiocitrulline induced a larger increase in tonic renal sympathetic nerve activity generated before baroreflex-mediated inhibition in hypertensive DS rats than normotensive DS rats. Hypertensive DS rats showed increased nNOS activity in the brainstem, but not in the diencephalon or cerebellum. High nNOS activity was confirmed by an increase in the amount of nNOS protein. nNOS Neurons were localized in several nuclei throughout the brainstem; the dorsolateral periaqueductal gray, pedunculopontine tegmental nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, lateral parabrachial nucleus, rostral ventrolateral medulla, nucleus tractus solitarius and raphe magnus. The number of nNOS neurons in these nuclei, except for the two raphes, was significantly greater in hypertensive than in normotensive DS rats. CONCLUSIONS: These findings suggest that central nNOS-mediated sympathoinhibition may be enhanced in salt-sensitive hypertensive Dahl rats. The upregulated nNOS-mediated inhibition may occur in the central sympathetic control system generated before baroreflex-mediated inhibition.     

Gastric nNOS reduction accompanied by natriuretic peptides signaling pathway upregulation in diabetic mice

AIM:To investigate the relationship between neuronal nitric oxide synthase(nNOS)expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin(STZ)-induced diabetic mice.METHODS:Diabetic mice were induced by injection of STZ solution.Immunofluorescence labeling of HuC/D,nNOS and natriuretic peptide receptor-A,B,C(NPRs)in the gastric fundus(GF)was used to observe nNOS expression and whether NPRs exist on enteric neurons.The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting.An isometric force transducer recorded the electric field stimulation(EFS)-induced relaxation and contraction in the diabetic GF.An intracellular recording method assessed EFSinduced inhibitory junction potentials(IJP)on the GF.GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide(CNP)for 24 h,after which their nNOS expressions were detected by western blotting.RESULTS:Eight weeks after injection,43 diabetic mice were obtained from mouse models injected with STZ.Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF.The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced,while the contraction was enhanced in the diabetic GF.EFSinduced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice.However,both NPR-A and NPR-B were detected on enteric neurons,and their expression levels were upregulated in the diabetic GF.The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP.CONCLUSION:These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.     

Noradrenergic neuron-specific overexpression of nNOS in cardiac sympathetic nerves decreases neurotransmission

Gene transfer of neuronal nitric oxide synthase (nNOS) with nonspecific adenoviral vectors can cause promiscuous transduction. We provide direct evidence that nNOS targeted only to cardiac sympathetic neurons inhibits sympathetic neurotransmission. An adenovirus constructed with a noradrenergic neuron-specific promoter (PRSx8), driving nNOS or enhanced green fluorescence protein (eGFP) gene expression caused exclusive expression in tyrosine hydroxylase (TH) positive rat cardiac sympathetic neurons. There was no detectable leakage of transgene expression in other cell types in the preparation nor did the transgene express in choline acetyltransferase (CHAT)-positive intracardiac cholinergic ganglia. Functionally, Ad.PRS-nNOS gene transfer increased nNOS activity and significantly reduced norephinephrine release evoked by field stimulation of isolated right atria. These effects were reversed by the NOS inhibitor N(omega)-Nitro-L-arginine. Our results demonstrate that noradrenergic cell-specific gene transfer with nNOS can inhibit cardiac sympathetic neurotransmission. This targeted technique may provide a novel method for reducing presynaptic sympathetic hyperactivity.     

Estrogen modulates endothelial and neuronal nitric oxide synthase expression via an estrogen receptor beta-dependent mechanism in hypothalamic slice cultures

Although it is evident that estrogen has important physiological effects in the brain, the signaling mechanisms mediating these effects remain unclear. We recently showed that estrogen mediates attenuated blood pressure responses to psychological stress in ovariectomized female rats through brain nitric oxide (NO). An area likely to mediate these effects is the hypothalamic paraventricular nucleus (PVN), because here NO exerts inhibitory effects on autonomic output to the periphery. Because little is known about how estrogen acts on the NO system in the PVN, our aim was to study the effects of estrogen on the NO system in the PVN of hypothalamic slices cultures. We show that 17beta-estradiol (E2; 1 nm) increases endothelial NO synthase (eNOS) protein expression and decreases the numbers of neuronal NOS (nNOS)-positive neurons in the PVN after 8 and 24 h, respectively. Using the nonselective estrogen receptor (ER) antagonist, ICI 182,780 (10 nm), we determined that E2-induced changes in NOS expression in the PVN are ER dependent. Using the ERbeta agonist, genistein (0.1 microm), we determined that activation of ERbeta induces increased eNOS expression and a decreased number of nNOS-positive neurons. We used the selective ERalpha agonist, propyl-pyrazole-triol (10 nm), and antagonist, methyl-piperidino-pyrazole (1 microm), to exclude the possibility that ERalpha is involved in the E2-induced increase in eNOS and nNOS in the PVN. These results demonstrate that E2 induces changes in NOS expression in the PVN and that these effects are ERbeta dependent.     

Carbon monoxide and nitric oxide as coneurotransmitters in the enteric nervous system: evidence from genomic deletion of biosynthetic enzymes

Nitric oxide (NO) and carbon monoxide (CO) seem to be neurotransmitters in the brain. The colocalization of their respective biosynthetic enzymes, neuronal NO synthase (nNOS) and heme oxygenase-2 (HO2), in enteric neurons and altered intestinal function in mice with genomic deletion of the enzymes (nNOS(Delta/Delta) and HO2(Delta/Delta)) suggest neurotransmitter roles for NO and CO in the enteric nervous system. We now establish that NO and CO are both neurotransmitters that interact as cotransmitters. Small intestinal smooth muscle cells from nNOS(Delta/Delta) and HO2(Delta/Delta) mice are depolarized, with apparent additive effects in the double knockouts (HO2(Delta/Delta)/nNOS(Delta/Delta)). Muscle relaxation and inhibitory neurotransmission are reduced in the mutant mice. In HO2(Delta/Delta) preparations, responses to electrical field stimulation are nearly abolished despite persistent nNOS expression, whereas exogenous CO restores normal responses, indicating that the NO system does not function in the absence of CO generation.     

Neuronal nitric oxide synthase gene transfer decreases [Ca2+]i in cardiac sympathetic neurons

Gene transfer of neuronal nitric oxide synthase (nNOS) can decrease cardiac sympathetic outflow and facilitate parasympathetic neurotransmission. The precise pathway responsible for nitric oxide (NO) mediated inhibition of sympathetic neurotransmission is not known, but may be related to NO–cGMP activation of cGMP-stimulated phosphodiesterase (PDE2) that enhances the breakdown of cAMP to deactivate protein kinase A (PKA), resulting in a decrease in Ca²⁺ influx mediated exocytosis of the neurotransmitter. We investigated depolarization evoked Ca²⁺ influx in nNOS gene transduced sympathetic neurons from stellate ganglia with a noradrenergic cell specific vector (Ad.PRS-nNOS or empty vector), and examined how nNOS gene transfer affected cAMP and cGMP levels in these neurons. We found that targeting nNOS into these sympathetic neurons reduced amplitudes of voltage activated Ca²⁺ transients by 44%. nNOS specific inhibition by N-[(4S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]-N′-nitroguanidine (AAAN) reversed this response. nNOS gene transfer also increased intracellular cGMP (47%) and decreased cAMP (29%). A PDE2 specific inhibitor Bay60-7557 reversed the reduction in cAMP caused by Ad.PRS-nNOS. These results suggest that neuronal NO modulates cGMP and PDE2 to regulate voltage gated intracellular Ca²⁺ transients in sympathetic neurons. Therefore, we propose this as a possible key step involved in NO decreasing cardiac sympathetic neurotransmission.