1.Effect of Nitric Oxide on Adhesion and Migration of Trabecular Meshwork Cells.
Journal of the Korean Ophthalmological Society 2013;54(4):639-644
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.
Humans
;
Nitric Oxide
;
S-Nitroso-N-Acetylpenicillamine
;
Stress, Mechanical
;
Trabecular Meshwork
2.Correlation of Cavernous Relaxation with Nitric Oxide Concentration in the Rabbit.
Hee Chang JUNG ; Dong Heon LEE ; Hyoung Chul CHOI ; Woo Sung JEON ; Ki Hak MOON ; Tong Choon PARK ; Kwang Youn LEE ; JUn Kyu SUH
Korean Journal of Urology 1997;38(6):585-594
Nitric oxide (NO) is known to act as an important neural mediator of penile erection. However, the degree of penile erection related to the concentration of NO released from corpus cavernosum has not been known yet. The present study was undertaken to correlate the degree of relaxation of the corpus cavernosum with concentration of NO by treatment of various NO releasing agents. Isometric tension of the rabbit cavernous strips was measured by polygraph system. The concentration of NO released from the same strips was simultaneously measured using an electrochemical method (Iso NO meter), which allows to detect change of NO gas level in perfusate of the organ bath. The cumulative additions of endothelial dependent agents, both acetylcholine and bradykinin at concentration from 0.00000001 to 0.0001M relaxed the cavernous strips precontracted by 0.000001M phenylephrine in concentration-dependent manner, which were highly correlated with the concentration of NO (38.9 +/- 15.2 nM at 0.00000001M - 74.5 +/- 18.4 nM at 0.0001M of acetylcholine; 30.2 +/- 5.8 nM at 0.00000001M - 90.5 +/- 10.2 at 0.0001M of bradykinin) released from the same strips, Furthermore, 3-[(3-cholamido propyl)]-l-propane sulfonate (CHAPS), a deendothelial agent, markedly suppressed both acetylcholine or bradykinin-induced cavernous relaxation and abolished NO release. In contrast, endothelial independent agent such as sodium nitroprusside (SNP), N-ethoxycarbonyl-3-morpholino-sydonimine (SIN-l) and s-nitroso-N-acetylpenicillamine (SNAP) at concentration from 0.00000001 to 0.001M relaxed the cavernous strips in` concentration dependent manner, without altering the basal concentration of NO in perfusate. From these results, it appears that the degree of cavernous relaxation induced by acetylcholine or bradykinin is highly correlated with the concentration of NO released from the cavernous endothelium. Furthermore, the direct electrochemical measurement of NO concentration in perfusate may be useful for further NO research in association with penile erection.
Acetylcholine
;
Baths
;
Bradykinin
;
Endothelium
;
Male
;
Nitric Oxide*
;
Nitroprusside
;
Penile Erection
;
Phenylephrine
;
Relaxation*
;
S-Nitroso-N-Acetylpenicillamine
3.Effect of Nitric Oxide on the Trabecular Meshwork Cell-mediated Contraction of Collagen Gels.
Jae Woo KIM ; Woo Seok CHANG ; Su Yoon LEE
Journal of the Korean Ophthalmological Society 2007;48(11):1548-1553
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.
Collagen*
;
Gels*
;
Humans
;
Nitric Oxide*
;
Nitroprusside
;
S-Nitroso-N-Acetylpenicillamine
;
Tissue Donors
;
Trabecular Meshwork*
4.Effect of Nitric Oxide on the Migration and Fibroblast-mediated Contraction of Collagen Gels.
Jeong Il LEE ; Soo Yoon LEE ; Jae Woo KIM
Journal of the Korean Ophthalmological Society 2008;49(4):661-668
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.
Collagen
;
Contracts
;
Dexamethasone
;
Fibroblasts
;
Gels
;
Humans
;
Nitric Oxide
;
Nitroprusside
;
S-Nitroso-N-Acetylpenicillamine
;
Tenon Capsule
;
Tissue Donors
;
Wound Healing
5.Role of nitric oxide and interleukin-1beta on expression of matrix metalloproteinase-2 in TL cell line.
Jae Dong LEE ; Jong Chul SHIN ; Dong Eun YANG ; Hee Bong MOON ; Jee Hyun LEE ; Hyun Young AHN ; Gui SeRa LEE ; Sa Jin KIM ; Su Pyung KIM
Korean Journal of Obstetrics and Gynecology 2002;45(12):2225-2230
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.
Blotting, Western
;
Cell Line*
;
Gelatin
;
Interleukin-1beta*
;
Matrix Metalloproteinase 2*
;
Nitric Oxide*
;
Placenta
;
RNA
;
RNA, Messenger
;
S-Nitroso-N-Acetylpenicillamine
;
Trophoblasts
6.Suppressed DNA Repair Mechanisms in Rheumatoid Arthritis.
Sang Heon LEE ; Gary S FIRESTEIN
Immune Network 2002;2(4):208-216
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.
Arthritis
;
Arthritis, Rheumatoid*
;
Blood Cells
;
Blotting, Western
;
DNA Adducts
;
DNA Damage
;
DNA Mismatch Repair
;
DNA Repair*
;
DNA*
;
Humans
;
Microsatellite Instability
;
Microsatellite Repeats
;
Nitric Oxide
;
Nitrogen
;
Osteoarthritis
;
Oxidative Stress
;
Oxygen
;
Polymerase Chain Reaction
;
S-Nitroso-N-Acetylpenicillamine
;
Synovial Membrane
;
Tissue Donors
7.Role of KCa Channels in SNAP-Induced Relaxation of Aorta from Renal Hypertensive Rats.
Seok CHOI ; Hyoung KIM ; Jae Yeoul JUN ; Pyung Jin YOON ; Hyun Lee KIM ; Jong Hoon CHUNG ; Cheol Ho YEUM
Korean Journal of Nephrology 2007;26(4):398-403
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.
Animals
;
Aorta*
;
Aorta, Thoracic
;
Cyclic GMP
;
Guanylate Cyclase
;
Humans
;
Hypertension
;
Hypertension, Renal
;
Methylene Blue
;
Muscle, Smooth, Vascular
;
Nitric Oxide
;
Phenylephrine
;
Placebos
;
Potassium Channels, Calcium-Activated
;
Rats*
;
Relaxation*
;
Renal Artery
;
S-Nitroso-N-Acetylpenicillamine
;
Tissue Donors
;
Vasodilation
8.Effect of Nitric Oxide on the Proliferation of Cultured Porcine Trabecular Meshwork Cells.
Jae Woo KIM ; Hyun HEO ; Hyo Won LEE
Korean Journal of Ophthalmology 2003;17(1):1-6
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.
Acridine Orange
;
Animals
;
Benzimidazoles
;
Cell Division/drug effects
;
Cell Survival/drug effects
;
Cells, Cultured
;
Flow Cytometry
;
Fluorescent Dyes
;
Nitric Oxide/*pharmacology
;
Nitric Oxide Donors/pharmacology
;
S-Nitroso-N-Acetylpenicillamine/pharmacology
;
Swine
;
Trabecular Meshwork/*cytology/physiology
9.Effects of S-nitroso-N-acetylpenicillamine on the Neuronal Excitability of the Medial Vestibular Nuclear Neuron.
Hoo Won KIM ; Ki Hyun CHO ; Sujeong JANG ; Han Seong JEONG ; Jong Seong PARK
Journal of the Korean Neurological Association 2007;25(2):199-205
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.
Action Potentials
;
Animals
;
Automatic Data Processing
;
Brain Stem
;
Compensation and Redress
;
Depression
;
Glutamic Acid
;
Guanylate Cyclase
;
Long-Term Potentiation
;
Membrane Potentials
;
Neurons*
;
Nitric Oxide
;
Patch-Clamp Techniques
;
Potassium
;
Rats
;
Rats, Sprague-Dawley
;
S-Nitroso-N-Acetylpenicillamine*
;
Vestibular Nuclei
10.Experimental study on effects of endothelin in the proliferation and collagen synthesis of human scar-derived fibroblasts.
Chinese Journal of Plastic Surgery 2003;19(1):51-53
OBJECTIVETo 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).
METHODSWith 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.
RESULTSThe 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).
CONCLUSIONThese 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.
Benzylisoquinolines ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; pathology ; Collagen ; biosynthesis ; DNA ; biosynthesis ; Endothelins ; antagonists & inhibitors ; pharmacology ; Fibroblasts ; cytology ; radiation effects ; Humans ; Nitric Oxide ; metabolism ; pharmacology ; Proline ; metabolism ; S-Nitroso-N-Acetylpenicillamine ; pharmacology