Effect of Nitric Oxide on the Expression of Matrix Metalloproteinase and Its Association with Migration of Cultured Trabecular Meshwork Cells

PURPOSE: To determine the effect of exogenous nitric oxide (NO) on the migration of trabecular meshwork (TM) cells and its association with expression of matrix metalloproteinases (MMPs). METHODS: Primary human TM cells treated with 1 or 10 microM S-nitroso-N-acetyl-penicillamine (SNAP) and examined for changes in adherence. TM cells were seeded onto transwell culture inserts, and changes in their migratory activity were quantified. Reverse transcription polymerase chain reaction was performed to determine the relative changes in mRNA expression of MMPs and tissue inhibitor of metalloproteinases (TIMPs). RESULTS: Treatment with SNAP did not significantly suppress TM cell adhesion or migration (p > 0.05). Treatment of TM cells with 10 microM SNAP decreased expression of MMP-2 and increased expression of membrane type MMP-1 and TIMP-2. Treatment with interleukin-1alpha triggered MMP-3 expression but did not exert significant effects on MMP-3 activation in response to SNAP. CONCLUSIONS: These data suggest that NO revealed no significant effect on the migration of TM cells because NO decreased MMP-2 and increased TIMP-2 expression. Although expression of certain MMPs and TIMPs change in response to NO donors, NO may modulate trabecular outflow by changing the cellular production of extracellular matrix without having a significant effect on the migration of TM cells.

Effect of Nitric Oxide on Adhesion and Migration of Trabecular Meshwork Cells

PURPOSE: To investigate the effect of nitric oxide (NO) on the adhesion and migration of cultured human trabecular meshwork cells (HTMC). METHODS: For adhesion assay, primarily cultured HTMC were attached to culture dishes for 1 hr, cells were rinsed, and the remaining adherent cells were assessed with MTT assay. Degree of cellular migration was assessed under normal and stressed conditions using microchemoattraction chambers. Effect of NO on the adhesion and migration was assessed with or without co-exposure of S-nitroso-N-acetylpenicillamine (SNAP). RESULTS: NO did not affect the degree of adhesion or migration of HTMC (p > 0.05). The degree of adhesion increased although the degree of migration decreased with 1% serum (p < 0.05). Degrees of migrations decreased after mechanical stress (p < 0.05). CONCLUSIONS: NO may not affect the adhesion or migration of HTMC.

Nitric Oxide Modulation of GABAergic Synaptic Transmission in Mechanically Isolated Rat Auditory Cortical Neurons

The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3',5'-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic K+ channels by 4-aminopyridine, a K+ channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic Ca2+ channels by Cd2+, a general voltage-dependent Ca2+ channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic Ca2+ channels in the presynaptic nerve terminals of A1 neurons.

Nitric oxide induces heat shock protein 72 production and delayed protection against myocardial ischemia in rabbits via activating protein kinase C / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nitric oxide (NO) is a biologically active molecule which has been reported to protect the heart against ischemia and reperfusion injury in different species. This study aimed to test the hypothesis that nitric oxide may induce the expression of heat shock protein 72 (HSP72) which may protect the heart against ischemia.</p><p><b>METHODS</b>Rabbits were given intravenous saline or S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, or Zaprinast, an inhibitor of cyclic guanosine monophosphate (GMP)-phosphodiesterase, which may increase myocardial cyclic GMP content. Twenty-four hours later, the rabbits were either sampled to measure HSP72, or induced with a 30-minute coronary occlusion followed by a 120-minute reperfusion, and then the infarct size was measured. Meanwhile, chelerythrine (CHE, an inhibitor of protein kinase C) was given intravenously 5 minutes before SNAP injection and the effect on HSP72 expression and infarct size was determined.</p><p><b>RESULTS</b>Twenty-four hours after pretreatment, immunoblotting showed HSP72 expression increased in the SNAP group compared with control groups, and this was blocked by CHE. Myocardial infarct size in the SNAP group was smaller than that of the control group ((32.4 +/- 5.8)% vs (51.1 +/- 4.7)%, P < 0.05). Pretreated with CHE abolished the infarct size-limiting effect of SNAP ((46.0 +/- 5.1)%). Pretreatment with Zaprinast neither induced HSP72 expression nor reduced infarct size ((55.4 +/- 5.4)%).</p><p><b>CONCLUSION</b>NO induced HSP72 expression and a delayed protection to the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.</p>

Effect of Nitric Oxide on the Migration and Fibroblast-mediated Contraction of Collagen Gels

PURPOSE: To investigate the role of nitric oxide (NO) on the migration of cultured human Tenon's capsule fibroblasts (HTCF) and contraction of collagen gel. METHODS: After artificial wounding, the primarily cultured HTCF were exposed to an NO donor such as sodium nitroprusside (SNP), S-Nitroso-N-acetylpenicillamine (SNAP), or dexamethasone at various concentrations. The cellular migration was measured up to five days. After embedding the cells in the collagen gels, the amount of contraction by the gels was also measured. Cellular survival and NO production were measured with MTT assay and Griess assay, respectively. RESULTS: Cellular survival was decreased by both NO donors but not by dexamethasone. SNP inhibited migration of HTCF in a dose-dependent manner and enhanced contraction of collagen gels. However, SNAP had no effect on the cellular migration or gel contraction. Dexamethasone inhibited cellular migration but did not affect the contraction of collagen gels. CONCLUSIONS: Among the NO donors, only SNP inhibited migration of HTCF and enhanced contraction of collagen gels in vitro. Thus, the effects between the two NO donors on fibroblast induced wound healing differ.

Role of KCa Channels in SNAP-Induced Relaxation of Aorta from Renal Hypertensive Rats / 대한신장학회지

PURPOSE: S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, is thought to relax vascular smooth muscle by stimulation of soluble guanylate cyclase, accumulation of its product cyclic GMP (cGMP) level. Evidence has emerged that NO-induced vasodilatation is also mediated by stimulating Ca2+-activated K+ (KCa) channels directly or indirectly through cGMP. The aim of the present study was to investigate possible involvement or alteration of KCa channels in the mechanism of vasodilation induced by SNAP in two-kidney, one-clip (2K1C) hypertensive rats. METHODS: 2K1C hypertension was made by clipping the left renal artery and age-matched control rats received a sham treatment. Using rings prepared from thoracic aortae, we studied changes in isometric tension of the rings in response to SNAP to evaluate effects of a soluble guanylate cyclase inhibitor methylene blue (MB), and a specific blocker of KCa channel iberiotoxin (ITX). RESULTS: Aortic rings from 2K1C hypertensive and sham-clipped control rats precontracted with phenylephrine showed similar relaxation to SNAP. MB markedly suppressed the SNAP-induced relaxation in both groups, leaving about 30% of MB-resistant relaxation. ITX nearly completely eliminated the MB-resistant relaxation in control rats, but it did not affect 2K1C rats. CONCLUSION: These results suggest that SNAP-induced vasorelaxation is mediated through cGMP- dependent and cGMP-independent KCa channel involving mechanisms, the latter may be altered in 2K1C renal hypertension.

Effect of Nitric Oxide on the Trabecular Meshwork Cell-mediated Contraction of Collagen Gels

PURPOSE: To investigate the effect of nitric oxide (NO) on the contraction of cultured human trabecular meshwork cells (HTMCs). METHODS: After embedding them into collagen gels, primarily cultured HTMCs were exposed to NO donors, such as sodium nitroprusside (SNP) or S-Nitroso-N-acetylpenicillamine (SNAP), for 1 week at various concentrations, and the contraction of the collagen gels was measured. Cellular survival and NO production were measured with MTT assay and Griess assay, respectively. RESULTS: Though SNP and SNAP did not significantly affect cellular survival, they markedly enhanced NO production. Both sodium nitroprusside and SNAP inhibited the contraction of collagen gels by about 10% in dose and time-dependent manners (p<0.05). CONCLUSIONS: NO donors inhibited the contraction of collagen gels in vitro. Thus, NO donors may relax trabecular meshwork and enhance trabecular outflow.

Effects of S-nitroso-N-acetylpenicillamine on the Neuronal Excitability of the Medial Vestibular Nuclear Neuron

BACKGROUND: The medial vestibular nucleus is the largest one among the vestibular nuclei and known to play important roles not only in normal vestibular information processing but also in vestibular compensation. Glutamate is known to have a key role in vestibular compensation via long term potentiation and depression. But the action of nitric oxide related with glutamate is poorly studied. This experiment was designed to explore the effects of nitric oxide on the neuronal activity of a rat medial vestibular nuclear neuron using a nitric oxide enhancing drug, S-nitroso-N-acetylpenicillamine (SNAP). METHODS: Experiments were carried out on Sprague-Dawley rats aged 14 to 17 days. Neurons of MVN were obtained via enzymatic dissociation of a microtomized rat brainstem. Whole-cell membrane potentials were recorded at room temperature by using standard patch-clamp techniques. Action potentials were obtained after administration of SNAP. Changes of potassium currents were recorded using SNAP and ODQ (1H-[1, 2, 4] oxadiazolo [4, 3-a] quinozalin-1-one), an inhibitor of guanylyl cyclase. RESULTS: The mean spike frequency of action potentials was increased by adding SNAP. The mean amplitude of afterhyperpolarization was decreased by adding SNAP. The mean potassium current of medial vestibular nuclear neurons was decreased by SNAP. ODQ inhibited the SNAP-induced potassium currents. CONCLUSIONS: These results suggest that nitric oxide increases the neuronal activity of rat medial vestibular nuclear neurons by inhibiting potassium currents via a cGMP dependent mechanism.

Modulation of glucose uptake in adipose tissue by nitric oxide-generating compounds.

There is increasing evidence that endogenous nitric oxide (NO) influences adipogenesis, lipolysis and insulin-stimulated glucose uptake. We investigated the effect of NO released from S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on basal and insulin-stimulated glucose uptake in adipocytes of normoglycaemic and streptozotocin (STZ)-induced diabetic rats. GSNO and SNAP at 0.2,0.5, and 1 mM brought about a concentration-dependent increase in basal and insulin-stimulated 2-deoxyglucose uptake in adipocytes of normoglycaemic and STZ-induced diabetic rats. SNAP at 1.0 mM significantly elevated basal 2-deoxyglucose uptake (115.8+/-10.4% compared with GSNO at the same concentration (116.1+/-9.4%; P less than 0.05) in STZ-induced diabetic rats. Conversely, SNAP at concentrations of 10 mM and 20 mM significantly decreased basal 2-deoxyglucose uptake by 50.0+/-4.5% and 61.5+/-7.2% respectively in adipocytes of STZ-induced diabetic rats (P less than 0.05). GSNO at concentrations of 10 mM and 20 mM also significantly decreased basal 2-deoxyglucose uptake by 50.8+/-6.4% and 55.2+/-7.8% respectively in adipocytes of STZ-induced diabetic rats (P less than 0.05). These observations indicate that NO released from GSNO and SNAP at 1 mM or less stimulates basal and insulin-stimulated glucose uptake,and at concentrations of 10 mM and 20 mM inhibits basal glucose uptake. The additive effect of GSNO or SNAP, and insulin observed in this study could be due to different mechanisms and warrants further investigation.

Protective Effect of Heat Shock Protein 70 Against Oxidative Stresses in Human Corneal Fibroblasts

We evaluated DNA protection effect of heat shock protein (HSP) against cytotoxic effects of exogenous nitric oxide (NO) and reactive oxygen intermediate (ROI). Cultured human corneal fibroblasts were divided into 4 groups. Control (Group I) was not exposed to a sub-lethal heat treatment. Other 3 groups were exposed to 43 degrees C for 1 hr, then incubated at 37 degrees C during different duration (1, 6, 24 hr, Group II, III, IV, respectively). Expression pattern of HSP 70 was analyzed by Western blot. Cell viability was measured by MTT assay and the relationship between HSP 70 expression and DNA damage was examined by terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick and labeling (TUNEL) stain and single cell gel electrophoresis. Expression pattern of HSP 70 was dependent on recovery times. Cell viability following heat treatment was significantly increased and the TUNEL positive cell number was decreased at 6 hr. In single cell gel electrophoresis, tail moments were increased in a dose-dependent manner by SNAP and X/XO. Following heat treatment, tail moments showed decreased significantly at 6 hr. These results suggest that induction of HSP 70 by sub-lethal heat treatment is closely related with cytoprotective effects against oxidative stresses in human corneal fibroblasts.

Gender-related differences in the effects of nitric oxide donors on neuroleptic-induced catalepsy in mice

It has been suggested that nigrostriatal dopaminergic transmission is modulated by nitric oxide (NO). Since there is evidence that gonadal hormones can affect extrapyramidal motor behavior in mammals, we investigated the effects of isosorbide dinitrate (ISD), linsidomine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP), three pharmacologically different NO donors, on neuroleptic-induced catalepsy in 60- to 80-day-old male and female albino mice. Catalepsy was induced with haloperidol (1 mg/kg, ip) and measured at 30-min intervals by means of a bar test. Drugs (or appropriate vehicle) were injected ip 30 min before haloperidol, with each animal being used only once. ISD (5, 20 and 50 mg/kg) caused a dose-dependent inhibition of catalepsy in male mice (maximal effect 120 min after haloperidol: 64 percent inhibition). In the females only at the highest dose of ISD was an attenuation of catalepsy observed, which was mild and short lasting. SIN-1 (10 and 50 mg/kg) did not significantly affect catalepsy in female mice, while a significant attenuation was observed in males at the dose of 50 mg/kg (maximal inhibition: 60 percent). SNAP (20 mg/kg) significantly attenuated catalepsy in males 120 min after haloperidol (44 percent inhibition), but had no significant effect on females. These results basically agree with literature data showing that NO facilitates central dopaminergic transmission, although the mechanisms are not fully understood. They also reveal the existence of gender-related differences in this nitrergic modulation in mice, with females being less affected than males

Experimental study on effects of endothelin in the proliferation and collagen synthesis of human scar-derived fibroblasts / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the role of endothelin (ET) in the proliferation and collagen synthesis of human scar-derived fibroblasts and the modulation of its antagonists such as nitric oxide (NO), tetrandrine (Tet).</p><p><b>METHODS</b>With the cultured fibroblasts from the scarring tissue, the cell proliferation was determined by [3H]-TdR incorporation, while the collagen synthesis was evaluated by [3H]-proline incorporation.</p><p><b>RESULTS</b>The ET-1 was significantly increasing the proliferation and collagen synthesis of human scar-derived fibroblasts. The values of [3H]-TdR absorption in the 2.5 ng/ml, 25 ng/ml and 100 ng/ml of ET-1 groups were 1.8 times, 4 times and 4.9 times more than in the control group, respectively (P < 0.01), while the values of the [3H]-proline incorporation were 1.1 times, 3.1 times and 3.8 times respectively (P < 0.01). The fibroblasts, treated with 50 micrograms/ml of S-nitroso-N-acetyl penicillamine(SNAP), were no detectable effect on the basal level of DNA synthesis, but produced decreasing effect on the [3H]-TdR absorption (the rate of inhibition was 22.89%, P < 0.05). It was found that the SNAP inhibited the [3H]-proline incorporation in cultured fibroblasts, but the rate of [3H]-proline incorporation induced by ET-1 was unaltered. The Tet with 3 micrograms/ml, in which does not inhibit the basal level of DNA synthesis, was significantly decreasing the collagen synthesis and decreasing the ET-mediated DNA synthesis (the rate of inhibition was 33.21% (P < 0.01).</p><p><b>CONCLUSION</b>These results indicate that the ET can obviously increase the proliferation and collagen synthesis of human scar-derived fibroblasts, but it can be partially antagonized by NO and Tet.</p>

Effect of Nitric Oxide on the Proliferation of Cultured Porcine Trabecular Meshwork Cells

To investigate the effect of nitric oxide (NO) on the proliferation of trabecular meshwork (TM) cells, primarily cultured porcine TM cells were exposed to NO donor (SNAP, -nitroso-N-acetyl-D, L-penicillamine) with and without its inhibitor (L-NAME, N (w) -Nitro-L-arginine methyl ester). The proliferation of TM cells was quantified by a rapid colorimetric assay. Acridine orange/Hoechest 33342 staining and flow cytometry with annexin-PI were done. As a result, NO inhibited the proliferation of TM cells significantly in a dose-dependent manner and this inhibitory effect was abolished by L-NAME. Fluorescent microscopy and flow cytometric analysis revealed that NO induced apoptotic cell death. The current results suggest that NO inhibit the proliferation of TM cells and apoptosis may be involved in some degree.

Role of nitric oxide and interleukin-1beta on expression of matrix metalloproteinase-2 in TL cell line / 대한산부인과학회잡지

OBJECTIVE: We studied to investigate whether nitric oxide (NO) and IL-1beta modulate MMP-2 and MMP-9 using TL cell line obtained from the normal term placenta. METHODS: After culturing TL cell line for 4 hours, we treated 0.1 mM of SNAP (NO donor) and 50 ng/ml of IL-1beta for 0, 1, 3, 6, and 12 hours, for investigating changes from time. We treated SNAP of 0, 0.01, 0.1, and 0.5 mM for 12 hours and IL-1beta of 0, 1, 10, and 50 ng/ml, for investigating changes from concentration. After extraction of total RNA, we performed reverse transcriptase-polymerase chain reaction (RT-PCR), gelatine zymography and Western blot analysis, for investigating expression of MMP-2 and MMP-9. RESULTS: MMP-9 was not observed in TL cell line. The expressions of MMP-2 mRNA and protein were gradually increased according to the culture time in SNAP treated group. The expressions of MMP-2 mRNA and protein were gradually increased according to the culture time in IL-1beta treated group. The expression of MMP-2 protein was not more increase in SNAP/IL-1beta-treated group than in IL-1beta treated group. The expression of MMP-2 protein was more reduced in SNAP/hemoglobin treated group than in SNAP treated group. MMP-2 protein activity was only increase in SNAP treated group. CONCLUSION: These results indicate that NO, rather than IL-1beta, upregulates the MMP-2 in TL cell line and furthermore may influence in the invasive process of trophoblasts.

Suppressed DNA Repair Mechanisms in Rheumatoid Arthritis

BACKGROUND: Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity. Key members of the MMR system include MutS alpha (comprised of hMSH2 and hMSH6), which can sense and repair single base mismatches and 8-oxoguanine, and MutS beta (comprised of hMSH2 and hMSH3), which repairs longer insertion/deletion loops. METHODS: To provide further evidence of DNA damage, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells (PBC) of RA patients using specific primer sequences for 5 key microsatellites. RESULTS: Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis (OA) tissue. Western blot analysis of the same tissues for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Western blot analysis demonstrated constitutive expression of hMSH2, 3 and 6 in RA and OA FLS. When FLS were cultured with SNAP, the RA synovial pattern of MMR expression was reproduced (high hMSH3, low hMSH6). CONCLUSION: Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

Effect of heme oxygenase induction by NO donor on the aortic contractility

Carbon monoxide (CO) binds to soluble guanylate cyclase to lead its activation and elicits smooth muscle relaxation. The vascular tissues have a high capacity to produce CO, since heme oxygenase-2 (HO-2) is constitutively expressed in endothelial and smooth muscle cells, and HO-1 can be greatly up-regulated by oxidative stress. Moreover, the substrate of HO, heme, is readily available for catalysis in vascular tissue. Although the activation of heme oxygenase pathway under various stress conditions may provide a defence mechanism in compromised tissues, the specific role of HO-1-derived CO in the control of aortic contractility still remains to be elucidated. The present study was done to determine the effect of HO-1 induction on the aortic contractility. Thus, the effects of incubation of aortic tissue with S-nitroso-N-acetylpenicillamine (SNAP) for 1 hr on the aortic contractile response to phenylephrine were studied. The preincubation with SNAP resulted in depression of the vasoconstrictor response to phenylephrine. This effect was restored by HO inhibitor or methylene blue but not by NOS inhibitor. The attenuation of vascular reactivity by preincubation with SNAP was also revealed in endothelium-free rings. AlF4--evoked contraction in control did not differ from that in SNP-treated group. These results suggest that increased production of CO was responsible for the reduction of the contractile response to phenylephrine in aortic ring preincubated with SNAP and this effect of SNAP was independent on endothelium.

The Role of Calcium Affecting Signal Pathway Related to Nitric Oxide-induced Cytotoxicity in H9c2 Cardiac Myoblast / 대한해부학회지

Nitric oxide (NO) elevates intracellular calcium. But the actions of calcium in NO-induced cell death are not well understood. This study was carried out to investigate the signal transduction pathways of calcium and NO-induced cytotoxicity in H9c2 cardiac myoblasts by using NO donor compounds such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP). Pretreatment of intracellular calcium chelating agent (BAPTA/AM) or L-type calcium channel blockers (nicardipine, nifedipine, diltiazem and veraparmil) or T-type calcium channel blocker (flunarizine) blocked SNP-induced cytotoxicity respectively only in a three hours. However, thapsigargin (TG), which inhibits endoplasmic reticulum dependent Ca(2+)-ATPase and thereby increases cytosolic Ca(2+), augmented SNP-induced cytotoxicity. The protective effect of BAPTA/AM was inhibited by treatment of protein synthesis inhibitor, cyclohexamide. In addition, pyrrolidine dithiocarbamate (PDTC), NF-kB inhibitor, attenuates the protective effect of BAPTA/AM against SNP-induced cytotoxicity. It is indicated that the protective effect of BAPTA/AM against NO-induced cytotoxicity might be due to the expression of protein related to activation of NFkB. From these results, it is concluded that SNP-induced cytotoxicity is mediated by calcium in a 3 hours via down regulation of protein expression rleated to activation of NFkB.

Effect of nitric oxide and plant antioxidants on microsomal content of lipid radicals

The antioxidant ability of nitric oxide (NO) generated by a chemical donor and of commercially available antioxidant preparations was assayed. SNAP (S-Nitroso-N-acetylpenicilamine) was used as the NO donor, and Ginkgo biloba, wheat and alfalfa preparations were tested. Lipid peroxidation was assayed by EPR employing a reaction system consisting of rat liver microsomes, ADP, FeCl3, NADPH and POBN in phosphate buffer, pH=7.4. In vitro NO exposure decreased microsomal lipid peroxidation in a dose-dependent manner. The dose responsible for inhibiting the microsomal content of lipid radical adducts by 50% (LD50) for SNAP was 550 microM (NO generation rate 0.1 microM/min). The addition of 50 microM hemoglobin to the incubation media prevented NO effect on lipid peroxidation. The addition of an amount of the antioxidant preparations equivalent to the LD50 doses inhibited lipid peroxidation by 21, 15, and 33% for wheat, alfalfa, ginkgo biloba preparations respectively in the presence of 550 microM SNAP. We detected a decrease in the content of lipid radical adducts after simultaneous supplementation, although it was less than 50%, even when LD50 doses of the products were added. This suggests that NO and the natural antioxidants inhibit lipid peroxidation by a mechanism that has both common and non-shared features.

Modulation of outward potassium currents by nitric oxide in longitudinal smooth muscle cells of guinea-pig ileum

To investigate the possible involvement of outward potassium (K+) currents in nitric oxide-induced relaxation in intestinal smooth muscle, we used whole-cell patch clamp technique in freshly dispersed guinea-pig ileum longitudinal smooth muscle cells. When cells were held at -60 mV and depolarized from - 40 mV to + 50 mV in 10 mV increments, sustained outward K+ currents were evoked. The outward K+ currents were markedly increased by the addition of 10 muM sodium nitroprusside (SNP). 10 muM S-nitroso-N-acetylpenicillamine (SNAP) and 1 mM 8-Bromo-cyclic GMP (8-Br-cGMP) also showed a similar effect to that of SNP. 1 mM tetraethylammonium (TEA) significantly reduced depolarization-activated outward K+ currents. SNP-enhanced outward K+ currents were blocked by the application of TEA. High EGTA containing pipette solution (10 mM) reduced the control currents and also inhibited the SNP-enhanced outward K+ currents. 5 mM 4-aminopyridine (4-AP) significantly reduced the control currents but showed no effect on SNP-enhanced outward K+ currents. 0.3 muM apamin and 10 muM glibenclamide showed no effect on SNP-enhanced outward K+ currents. 1 muM 1H-(1,2,4)oxadiazolo (4,3-a)quinoxaline-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase, significantly blocked SNP-enhanced K+ currents. We conclude that NO donors activate the Ca2+-activated K+ channels in guinea-pig ileal smooth muscle via activation of guanylate cyclase.

Role of nitric oxide in ischemia-evoked release of norepinephrine from rat cortex slices

It has been generally accepted that glutamate mediates the ischemic brain damage, excitotoxicity, and induces release of neurotransmitters, including norepinephrine (NE), in ischemic milieu. In the present study, the role of nitric oxide (NO) in the ischemia-induced (3H)norepinephrine((3H)NE) release from cortex slices of the rat was examined. Ischemia, deprivation of oxygen and glucose from Mg2+/-free artificial cerebrospinal fluid, induced significant release of (3H)NE from cortex slices. This ischemia-induced (3H)NE release was significantly attenuated by glutamatergic neurotransmission modifiers. NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) or 7-nitroindazole, nitric oxide synthase inhibitors attenuated the ischemia-evoked (3H)NE release. Hemoglobin, a NO chelator, and 5, 5-dimethyl-L-pyrroline-N-oxide (DMPO), an electron spin trap, inhibited (3H)NE release dose-dependently. Ischemia-evoked (3H)NE release was inhibited by methylene blue, a soluble guanylate cyclase inhibitor, and potentiated by 8-bromo-cGMP, a cell permeable cGMP analog, zaprinast, a cGMP phosphodiesterase inhibitor, and S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide generator. These results suggest that the ischemia-evoked (3H)NE release is mediated by NMDA receptors, and activation of NO system is involved.