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.
Cell Movement/*drug effects ; Cell Survival/drug effects ; Cells, Cultured ; DNA Primers/chemistry ; Gene Expression Regulation, Enzymologic/*physiology ; Humans ; Matrix Metalloproteinases/*genetics ; Nitric Oxide Donors/*pharmacology ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; S-Nitroso-N-Acetylpenicillamine/*pharmacology ; Tissue Inhibitor of Metalloproteinase-2/*genetics ; Trabecular Meshwork/cytology/*drug effects/enzymology

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

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.
4-Aminopyridine ; Animals ; Auditory Cortex ; Benzoates ; Bicuculline ; gamma-Aminobutyric Acid ; Guanosine ; Humans ; Imidazoles ; Inhibitory Postsynaptic Potentials ; Membranes ; Neurons ; Nitric Oxide ; Patch-Clamp Techniques ; Rats ; S-Nitroso-N-Acetylpenicillamine ; Synaptic Transmission ; Tissue Donors

BACKGROUNDNitric 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.

METHODSRabbits 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.

RESULTSTwenty-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)%).

CONCLUSIONNO induced HSP72 expression and a delayed protection to the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.

Animals ; Benzophenanthridines ; pharmacology ; Cyclic GMP ; metabolism ; HSP72 Heat-Shock Proteins ; biosynthesis ; Hemodynamics ; Male ; Myocardial Infarction ; metabolism ; physiopathology ; prevention & control ; Myocardial Ischemia ; metabolism ; physiopathology ; prevention & control ; Nitric Oxide ; metabolism ; Nitric Oxide Donors ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; Protein Kinase C ; metabolism ; Purinones ; pharmacology ; Rabbits ; S-Nitroso-N-Acetylpenicillamine ; pharmacology

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

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

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*

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

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.
Cell Survival ; Cells, Cultured ; Cornea/*cytology ; DNA Damage ; Dose-Response Relationship, Drug ; Fibroblasts/cytology/drug effects/*metabolism ; Heat ; Heat-Shock Proteins 70/genetics/*metabolism ; Humans ; In Situ Nick-End Labeling ; Nitric Oxide/metabolism ; Nitric Oxide Donors/pharmacology ; *Oxidative Stress ; Reactive Oxygen Species/metabolism ; Research Support, Non-U.S. Gov't ; S-Nitroso-N-Acetylpenicillamine/pharmacology ; Xanthine/pharmacology ; Xanthine Oxidase/pharmacology

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