The role of nitric oxide in hepatic metabolism

Nitric oxide (NO) may regulate hepatic metabolism directly by causing alterations in hepatocellular (hepatocyte and Kupffer cell) metabolism and function or indirectly as a result of its vasodilator properties. Its release from the endothelium can be elicited by numerous autacoids such as histamine, vasoactive intestinal peptide, adenosine, ATP, 5-HT, substance P, bradykinin, and calcitonin gene-related peptide. In addition, NO may be released from the hepatic vascular endothelium, platelets, nerve endings, mast cells, and Kupffer cells as a response to various stimuli such as endotoxemia, ischemia-reperfusion injury, and circulatory shock. It is synthesized by nitric oxide synthase (NOS), which has three distinguishable isoforms: NOS-1 (ncNOS), a constitutive isoform originally isolated from neuronal sources; NOS-2 (iNOS), an inducible isoform that may generate large quantities of NO and may be induced in a variety of cell types throughout the body by the action of inflammatory stimuli such as tumor necrosis factor and interleukin (IL)-1 and -6; and NOS-3 (ecNOS), a constitutive isoform originally located in endothelial cells. Another basis for differentiation between the constitutive and inducible enzymes is the requirement for calcium binding to calmodulin in the former. NO is vulnerable to a plethora of biologic reactions, the most important being those involving higher nitrogen oxides (NO2-), nitrosothiol, and nitrosyl iron-cysteine complexes, the products of which (for example, peroxynitrite), are believed to be highly cytotoxic. The ability of NO to react with iron complexes renders the cytochrome P450 series of microsomal enzymes natural targets for inhibition by NO. It is believed that this mechanism provides negative feedback control of NO synthesis. In addition, NO may regulate prostaglandin synthesis because the cyclooxygenases are other hem-containing enzymes. It may also be possible that NO-induced release of IL-1 inhibits cytochrome P450 production, which ultimately renders the liver less resistant to trauma. It is believed that Kupffer cells are the main source of NO during endotoxemic shock and that selective inhibition of this stimulation may have future beneficial therapeutic implications. NO release in small quantities may be beneficial because it has been shown to decrease tumor cell growth and levels of prostaglandin E2 and F2 alpha (proinflammatory products) and to increase protein synthesis and DNA-repair enzymes in isolated hepatocytes. NO may possess both cytoprotective and cytotoxic properties depending on the amount and the isoform of NOS by which it is produced. The mechanisms by which these properties are regulated are important in the maintenance of whole body homeostasis and remain to be elucidated.     

The role of nitric oxide in ischemia-reperfused heart

The objective of this study was to estimate the dominance variance for postweaning gain in Limousin cattle. Data included 215,326 records of postweaning gain from 205 to 365 d, provided by the North American Limousin Foundation. Parental dominance subclasses were formed and related using the method of Hoeschele and VanRaden. Variance components were estimated using Method R based on six samples of 50%. Fixed effects in the model included contemporary group and covariates for inbreeding and breed composition (percentage Limousin). Heterozygosity was negatively correlated with breed composition (< -.99) and was therefore not included in the model. Two types of contemporary groups used as original groups from the National Cattle Evaluation were partially based on breed composition. Original contemporary groups that were too homogeneous for breed composition were replaced by herd-year-sex classes. Two models were used with the two data sets. Model 1 contained the fixed effects described above and an additive genetic effect. Model 2 included a dominance effect in addition to the effects contained in Model 1. In total, four combinations of contemporary group x model were used. Dominance variance was computed as being four times the estimated parental subclass variance. Estimates for inbreeding depression and breed composition (percentage Limousin) were all small and not greatly affected by inclusion of dominance effects or changes in contemporary groups. Estimates of the additive variance (expressed as percentage of the phenotypic variance) were only slightly affected, with values between 20 and 21%. Dominance estimates were highly affected when passing from original (10%) and to alternative contemporary groups (18%). Such large values may indicate that dominance is important for postweaning gain. Results showed the advantage of an individual dominance approach based on sire-dam combinations; therefore, expected gains through the use of specific combination ability as a part of the mating selection criteria for growth might be high.     

The role of nitric oxide in vascular biology and pathobiology

Brain natriuretic peptide (BNP) is a novel cardiac hormone secreted predominantly from the ventricle. We examined the plasma levels of BNP and atrial natriuretic peptide (ANP) in 13 patients with aortic stenosis undergoing corrective surgery. Preoperative plasma BNP and ANP levels correlated highly with preoperative left ventricular end-systolic wall stress (ESS) (r = 0.96, p < 0.0001 and r = 0.95, p < 0.0001, respectively). Moreover, between preoperative and late postoperative states, the difference of the plasma levels of BNP and ANP correlated with the difference of ESS. In two patients with elevated ESS and quite high preoperative plasma BNP (> 1000 pg/ml), rapid decrease of the plasma level after operation was observed. These results suggest that synthesis and secretion of BNP and ANP are stimulated by the increase of left ventricular end-systolic wall stress in patients with aortic stenosis.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

The role of nitric oxide in passive avoidance learning

The role of nitric oxide on passive avoidance learning was studied by administering L-arginine or D-arginine to male rats in a passive avoidance paradigm. L-Arginine administered into the lateral brain ventricle at a dose of 1.25 microg showed a tendency to increase the passive avoidance latency, and 2.5 microg exerted almost maximal action, but the action gradually increased still further up to 20 microg tested. D-Arginine had no action. Peripheral administration (intraperitoneal) of L-arginine facilitated the consolidation of passive avoidance learning in a dose-dependent manner. A significant increase in passive avoidance response was obtained following an injection of 100 mg/kg L-arginine. When L-arginine was given i.c.v. with a selected dose of 5 microg, 30 min prior to a learning trial, the latency of the passive avoidance response was likewise lengthened. However, when L-arginine was given 30 min before the 24-hr testing (retrieval), it was ineffective. It was also ineffective when given 6 hr after the training trial. However, when L-arginine was administered immediately following the training trial, the action in improving the consolidation could be detected 6 hr after the training trial. Nitro-L-arginine, which blocks nitric oxide synthase, can also block the facilitation of consolidation caused by the nitric oxide donor L-arginine. The nitric oxide synthase inhibitor per se in different doses had no action on the learning of a passive avoidance task. The results indicate that nitric oxide is able to facilitate the learning and consolidation of memory in a passive avoidance paradigm, but it is ineffective in retrieval processes. The results also suggest that, under the experimental circumstances used, nitric oxide is involved only in the facilitated learning and memory processes caused by pharmacological effect of L-arginine, and not involved in normal learning processes.     

Argininosuccinate synthetase: localization in astrocytes and role in the production of glial nitric oxide

An antiserum raised against the peptide representing the partial sequence 196-222 of mouse liver argininosuccinate synthetase (ASS) was used to detect and localize the enzyme in cells of neural primary cultures. No ASS immunoreactivity was detected by Western blotting in homogenates of mouse pure astroglial cultures and rat astroglia-rich cultures. However, when the cultures had been treated with bacterial lipopolysaccharide, interferon-gamma, or a combination of both, ASS immunoreactivity was disclosed. Immunocytochemical examination of rat astroglia-rich cultures revealed a colocalization of ASS with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were some GFAP-positive cells showing no specific staining for ASS, and vice versa. Colocalization of ASS with the inducible isoform of nitric oxide synthase in the same cell was shown only occasionally; nitric oxide synthase was predominantly expressed in microglial cells. In rat neuron-rich primary cultures astroglial cells as well as neurons expressed ASS. Cells of mouse pure astroglial cultures were able to synthesize arginine and, consequently, nitric oxide from citrulline, but not from ornithine. The findings demonstrate that ASS is expressed in astroglial cells under conditions that stimulate long-lasting production of nitric oxide; a functional role of this enzyme in the latter process is implicated.     

The role nitric oxide and other neurotransmitter in canine penile erection

The role on nitric oxide and its relative factors (cGMP, cAMP, methylene blue) was studied in canine erection induced by stimulating pelvic nerves, and the effect of cholinergic neuroeffectors and the sinusoidal endothelium was also observed in this experiment. The results indicate that intracavernous injection of nitric oxide can evoke a penile tumescence, which is similar to that of the neurostimulation. The results also suggest that the cholinergic nerves and the sinusoidal endothelium are involved in erection, and the effect of the former must depend on mediation of the latter. The study supports that cholinergic and nonadrenergic noncholinergic (NANC) neuroeffectors take part in penile erection, and nitric oxide may be one of chief NANC neurotransmitters.     

Gastric mucosal blood flow response to stress in streptozotocin diabetic rats: regulatory role of nitric oxide

To investigate cytoprotection against mucosal injuries of the stomach in patients with diabetes, we investigated gastric mucosal blood flow (GMBF), its response to a burn stress, and the involvement of nitric oxide (NO) in streptozotocin (STZ) diabetic rats. GMBF was measured by laser-Doppler velocimetry (LDV) and by the hydrogen gas clearance technique (HGC). The steady-state GMBF of STZ rats decreased according to the duration of diabetes, and insulin treatment blocked this decrease. Burn stress caused a rapid decrease in the GMBF. Reduction of the GMBF and gastric mucosal leakage of Evans blue (EB) after the burn stress were greater in the STZ rats than in the controls, but insulin treatment completely blocked this increase in EB leakage in the STZ rats. There was a significant negative correlation between the percent GMBF 3 h after the burn stress and EB leakage at the same time point. In the controls and the insulin-treated STZ rats, N-nitro-L-arginine (L-NNA), an NO synthase inhibitor, enhanced the decrease in postburn GMBF and EB leakage, but was without effect in the STZ rats. These results suggest that NO may be involved in the regulation of GMBF, and that persistent hyperglycemia may impair this regulation. These findings suggest that patients with diabetes have reduced cytoprotection against a variety of gastric mucosal injuries.     

Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide

There has been confusion as to what role(s) nitric oxide (NO) has in different physiological and pathophysiological mechanisms. Some studies imply that NO has cytotoxic properties and is the genesis of numerous diseases and degenerative states, whereas other reports suggest that NO prevents injurious conditions from developing and promotes events which return tissue to homeostasis. The primary determinant(s) of how NO affects biological systems centers on its chemistry. The chemistry of NO in biological systems is extensive and complex. To simplify this discussion, we have formulated the "chemical biology of NO" to describe the pertinent chemical reactions under specific biological conditions. The chemical biology of NO is divided into two major categories, direct and indirect. Direct effects are defined as those reactions fast enough to occur between NO and specific biological molecules. Indirect effects do not involve NO, but rather are mediated by reactive nitrogen oxide species (RNOS) formed from the reaction of NO either with oxygen or superoxide. RNOS formed from NO can mediate either nitrosative or oxidative stress. This report discusses various aspects of the chemical biology of NO relating to biological molecules such as guanylate cyclase, cytochrome P450, nitric oxide synthase, catalase, and DNA and explores the potential roles of NO in different biological events. Also, the implications of different chemical reactions of NO with cellular processes such as mitochondrial respiration, metal homeostasis, and lipid metabolism are discussed. Finally, a discussion of the chemical biology of NO in different cytotoxic mechanisms is presented.     

The role of neuronal and endothelial nitric oxide synthase in retinal excitotoxicity

PURPOSE: Nitric oxide synthase (NOS) plays an essential role in neuronal function and is critical in the brain for normal and pathologic responses to glutamate. The role of NOS in the retina is less well understood. The retina provides an experimental system in which the intrinsic circuitry is well defined; retinal excitotoxic damage has been well characterized. METHODS: To determine whether neuronal NOS (nNOS) and endothelial NOS (eNOS) are critical in excitotoxic damage in the retina, nNOS- and eNOS-deficient mice were subjected to intravitreal injections of N-methyl-D-aspartate (NMDA) or to arterial occlusions. RESULTS: Retinal ganglion cells in the nNOS-deficient mouse were relatively resistant to NMDA and to arterial occlusion. In contrast, the damage in the eNOS-deficient mouse retina was not distinguishable from that in control animals. Preinjection with an NOS inhibitor was partially protective. CONCLUSIONS: The presence of nNOS is a prerequisite for the full expression of excitotoxicity in the retina; eNOS does not appear to play a significant role.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Protective role of nitric oxide in ocular toxoplasmosis

AIMS: To evaluate the role of nitric oxide (NO) in ocular involvement during systemic toxoplasmosis. METHODS: C57B1/6 mice were infected with Toxoplasma gondii strain ME49. The synthesis of NO was inhibited by an intraperitoneal injection of aminoguanidine every 8 hours, starting on the day of infection. Control infected mice received phosphate buffered saline vehicle alone. After 14 days, the ocular lesions were evaluated by histopathological examination. The expression of NO synthase induced in the spleen by toxoplasma infection was evaluated by immunostaining. The production of NO by the spleen cells of infected mice was measured by the colorimetric assay of Griess in the supernatant of cultures stimulated with toxoplasma antigen or concanavalin A. RESULTS: The inhibition of NO production in T gondii infected mice resulted in a marked increase in the symptoms of ocular inflammation. We observed a strong induction of NO synthase expression in the spleen of infected animals. In culture, the spleen cells from these mice produced high levels of NO in response to T gondii antigens. This elevation of NO synthesis was suppressed in the presence of aminoguanidine. CONCLUSION: This study indicates that NO plays a crucial role in the protection against T gondii infection as reflected by the severity of the ocular involvement.     

The role of nitric oxide in vivo feline erection under hypoxia

Previous in vitro studies have demonstrated that the cavernous relaxation under hypoxia does not involve the endothelium dependent mechanism. However, the mechanism of nitric oxide pathway under hypoxia are not fully evaluated or understood yet in vivo. The changes of intracavernous pressure to various vasoactive substances were monitored in 45 mature male cats in vivo under normoxia and hypoxia (pH: 7.03, PO2: 25.52 mmHg, PCO2: 84.66 mmHg). L-arginine and SNAP (s-nitroso-n-acetyl-penicillamine) produced cavernous relaxation under normoxia, but not under hypoxia (n = 19, P < 0.01). The L-arginine-induced relaxations were inhibited by L-NAME (N omega-nitro-1-arginine-methyl-ester) or methylene blue under normoxia (n = 19, P < 0.01). The cavernous relaxation was 58% suppressed under hypoxia compared to normoxia with 10(-3) M/0.2 ml of acetylcholine (n = 22, P < 0.01). Moreover, L-NAME attenuated the acetylcholine-induced relaxation under normoxia, but not under hypoxia (n = 22, P < 0.05). Epinephrine suppressed the acetylcholine-induced relaxation in both conditions (n = 10, P < 0.01), while indomethacin significantly potentiated the acetylcholine-induced relaxation under normoxia compared to hypoxia (n = 6, P < 0.05). However, none of these substances responded in severe hypoxia (PO2 < 15 mmHg, n = 3). These results suggest that erectile and contractile responses are attenuated under hypoxia. The endothelium derived relaxation via nitric oxide does not play a role in cavernous relaxation under definitive hypoxia with acidosis like in ischemic priapism (PO2 < 30 mmHg, pH < 7.25).