OBJECTIVES: The present study was designed to investigate the effect of ginseng saponin and its major active metabolite on the HPA axis under acute stress-i.c.v. injection stress, and immobilization stress, and to examine whether nitric oxide is involved in the mechanism of ginseng saponin on the HPA axis under acute stress. METHODS: In the experiment to study the effect of ginseng on HPA axis during stress, various dose of GTS were injected intracerebroventricularly(i.c.v.) or intraperitoneally(i.p.). Plasma corticosterone levels were measured 30 min after the i.c.v. injection stress. Immobilization stress was applied for 30 min and then blood was cellected for the assays of plasma corticosterone levels immediately after the completion of immobilization stress. To determine the active ginsenosides that can affect the stressinduced plasma corticosterone levels, various dose of each gisendosides(Rb1, Rb2, Rc, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3) were injected i.c.v. or i.p.. In the experiment to determine the involvement of the nitric oxide in the inhibitory effect of ginseng on the HPA, NG-Nitro-L-arginine methyl ester(L-NAME) and ginsenosides were coadministered i.c.v. or i.p., and plasma corticosterone levels were measured 30 min after stress was applied. RESULTS: First, the present study showed that ginseng total saponin, ginsenoside Rg3(S form), and ginsenoside Rc administered i.c.v. attenuated the intracerebroventricular injection stress-induced increase in plasma corticosterone levels, and these effects were removed by nitric oxide co-injection. Second, ginseng total saponin and ginsenoside Rc administered i.p. attenuated the immobilization stress-induced increase in plasma corticosterone levels, but ginsenoside Rg3(S form) did not attenuate the immobilization stress-induced increase in plasma corticosterone levels. The attenuative effects of ginseng total saponin and ginsenoside Rc in the immobilization stress-induced increase in plasma corticosterone levels were not affected by L-NAME co-injection. CONCLUSION: This study suggests that ginseng saponin attenuated stress-induced increase in plasma corticosterone levels and these effects were mediated by different mechanisms according to the components of ginseng saponin, and routes of administration.
Animals ; Axis, Cervical Vertebra ; Corticosterone* ; Ginsenosides ; Immobilization ; Mice* ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Nitroarginine ; Panax* ; Plasma* ; Saponins*

The aim of this study was to evaluate neurotoxicity of Nitric oxide(NO) on cornea after excimer laser photorefractive keratectomy(PRK). PRK was performed on rabbit eyes. According to the time table, tear samples were collected with microcapillary tubes and corneal sensitivity was measured with a Cochet-Bonnet esthesiometer. No generation in the tear fluid was analyzed. To demonstrate NO Synthase(NOS), immunohistochemical localization was performed on frozen sections from rat eyeball tissue. Western blot analysis was used for detection of peroxynitrite, powerful oxidant of NO. NO generation was increased and reached to a maximum value(0.69+/-0.22micrometer/microgram) after 96 hours of PRK, as compared with in normal subjects(Mean: 0.30+/-0.08micrometer/microgram) and was not increased in the treated group with topical application of Ng-nitro-L-arginine methyl ester(L-NAME), a competitive inhibitor of constitutive NOS(cNOS) and inducible NOS(iNOS). Corneal sensitivity decreased below pretreatment levers after three postoperative days, but it was not observed in the L-NAME applied group. We have confirmed that a very strong iNOS and BNOS immunoreactivity was present in corneal keratocytes. Western blot analysis identifed the bands of nitrotyro-sine-proteins suggesting in vivo peroxynitrite toxicity. Our results suggested that NO generated from the enzyme after PRK decreased corneal sensitivity by damaging corneal sensory nerve through the NO and iths oxidant peroxitrite. Therefore topical application of a NOS inhibitor may be effective in maintaining corneal sensitivity.
Animals ; Blotting, Western ; Cornea ; Corneal Keratocytes ; Frozen Sections ; Lasers, Excimer* ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Nitroarginine ; Peroxynitrous Acid ; Photorefractive Keratectomy* ; Rats

BACKGROUND: Etomidate produces minimal cardiovascular effects in clinical uses but their effects on the porcine coronary arteries are not well known yet. We studied the direct effects of etomidate on porcine coronary arterial tone and the underlying mechanism of its vascular relaxation. METHODS: Porcine coronary arterial ring segments (3-4 mm) with or without endothelium were suspended in modified Krebs solution (37oC) and preconstricted with K+ 40 mM. Changes in tension were measured following cumulative administrations of etomidate (10(-5), 5 x 10(-5) and 10(-4) M). Relaxation caused by etomidate (10(-4)M) were measured in the presence of either NG-nitro-L-arginine methyl ether (L-NAME 10(-5)M, n = 13), indomethacin (2.8 x 10(-5)M, n = 12), methylene blue (2 x 10(-5)M, n = 12), tetraethylammonium (TEA, KCa2+ blocker, 20 mM, n = 11), glibenclamide (n = 13) 2.5 x 10(-5)M or 4-aminopyridine (4-AP, K+ DR blocker, 10 4 M, n = 12). Effects of etomidate on Ca2+ influx through the voltage operated channel (VOC) of the vascular cells were also evaluated. RESULTS: Arterial reings were relaxed by etomidate in a concentration-dependent manner and these effects were not affected by endothelium. In the etomidate pretreated group, arterial ring in a calcium-free solution showed no contraction with KCl 40 mM, but the contraction after the administration of calcium 2.5 mM less than without etomidate group (47.2 +/- 8.7% vs 97.7 +/- 10.6%). The other group, not pretreated with etomidate, showed the same vascular tone of the control group in a slow upstroke manner with calcium administration. Pretreatment with either L-NAME, indomethacin and methylene blue did not affect etomidate-induced vasorelaxation. The TEA, glibenclamide and 4-AP pretreated groups also did not affect the vascular relaxation. CONCLUSIONS: Etomidate relaxes the porcine coronary artery in a concentration-dependent manner withor without endothelium, via inhibition of Ca2+ influx through the voltage-operated Ca2+ channel.
4-Aminopyridine ; Calcium ; Coronary Vessels* ; Endothelium ; Ether ; Etomidate* ; Glyburide ; Indomethacin ; Methylene Blue ; NG-Nitroarginine Methyl Ester ; Nitroarginine ; Relaxation ; Tea ; Tetraethylammonium ; Vasodilation

AIMS OF STUDY: During reflex micturition, the urethral outlet remains open (relaxed) to promote urinary emptying. The mechanisms involved in the relaxation of urethral outlet is thought to be complex including nitric oxide (NO) pathway and beta-adrenergic activity. The aims of the study focused on these several issues related to the neural control of urethral outlet in vivo. MATERIALS & METHODS: Female rats weighing 200~300 gm were anesthetized wish urethane. Catheters were inserted into femoral artery for drug administration.4 two-way catheter (16 G angiocath) was inserted into the bladder for saline infusion and pressure monitoring. A separate cannula (PE 50) was placed into the urethra via external urethral meatus or proximal urethrat opening to record urethral pressure. The bladder was filled with saline at a rate of 0.1 ml/min to induce reflex micturition. Urethral pressure was recorded via cannula through which saline was infused at a rate of 0.05 ml/min. Isovolumetric bladder contraction and urethral pressure were recorded simultaneously. After an equilibration period of 30 minutes, baseline intravesical and urethral pressure were recorded for 10 minutes prior to drug administration. NG-nitro-L-arginine methylester (L-NAME, 10 to 15 mg/kilrogram, i.v.), L-arginine (150 mg/kilrogram, i.v.), propranolol (1 microM., 0.1 ml/250 mg, i.a.), and phenylephrine (1 0~100 microM, i.a.) were administrated. RESULTS: During isovolumetric bladder contraction, urethral pressure was decreased simultaneously, and then returned to the resting states in conjunction with end of the bladder contraction. After the administration of L-NAME, the magnitude of reflex urethral relaxation was decreased significantly (42.6 +/- 15.1% of the control, p<0.01), and this effect was reversed by addition of L-arginine. Administration of propranolol also inhibited urethral relaxation (66.4% of the control). Administration of L-NAME followed by propranolol almost completely abolished the urethral relaxation. Administration of phenylephrine increased the resting urethral tone (mean; 4 cmH2O) significantly, and the magnitude of urethral relaxation was decreased substantially. CONCLUSION: These RESULTS suggest that urethral relaxation is mediated by several neural factors. NO seems like to a potent mediator in a reflex relaxation of the urethral smooth muscle during micturition. Also, beta-adrenergic stimulation play an important role for urethral relaxation. alpha-adrenergic nerve discharge, contributed to contraction of urethral smooth muscle, shows inhibitory effect against the reflex urethral relaxation.
Animals ; Arginine ; Catheters ; Female ; Femoral Artery ; Humans ; Muscle, Smooth ; NG-Nitroarginine Methyl Ester ; Nitric Oxide* ; Nitroarginine ; Phenylephrine ; Propranolol ; Rats ; Reflex ; Relaxation* ; Urethane ; Urethra ; Urinary Bladder ; Urination

PURPOSE: To investigate the role of nitric oxide(NO) on the survival and proliferation of human Tenon capsule fibroblasts(HTCF) in tissue culture. METHODS: Following exposure to NO donor such as sodium nitroprusside(SNP) or to NO synthase inhibitor such as N omega-Nitro-L-arginine methyl ester(L-NAME) at various concentrations for 24 hr in the media with or without serum, the cellular proliferation and nitrite production were assessed by rapid colorimetric assay (MTT assay) and Griess assay, respectively. RESULTS: L-NAME decreased the cellular proliferation of HTCF in a dose-dependent manner. In contrast, SNP increased the proliferation in a dose-dependent manner. The differences of nitrite production were more remarkable in the serum-deprived condition with the L-NAME or SNP administration. CONCLUSIONS: NO donor has a proproliferative, and NOS inhibitor has a antiproliferative activities on the proliferation of HTCF. This suggests that NO may be act as an important modulator in wound healing.
Cell Proliferation ; Fibroblasts* ; Humans* ; NG-Nitroarginine Methyl Ester ; Nitric Oxide Synthase ; Nitric Oxide* ; Nitroarginine ; Nitroprusside ; Sodium ; Tenon Capsule* ; Tissue Donors ; Wound Healing

The present study was aimed to investigate whether and to what extent hypertension affects the relaxation of the corpus cavernosum. The corpus cavernosum was isolated from 12-week 2- kidney, 1-clip hypertensive rats. The corporal strips were isolated and suspended longitudinally in an organ bath. They were precontracted with phenylephrine, and their responses to electrical field stimulation (EFS) were examined. EFS caused a frequency-dependent contraction (60%) or relaxation (40%) of the corpus cavernosum precontracted with phenylephrine. The contraction response was inhibited or abolished and only frequency-dependent relaxation appeared in the presence of atropine (0.00001mol/L) and guanethidine (0.00001mol/L). The relaxation response to EFS of the corporal preparation precontracted with phenylephrine was attenuated or abolished in the presence of L-NAME (0.0001mol/L). The corporal preparation from the hypertensive rats also showed a frequency-dependent relaxation, however, the degree of which was lower at a low frequency of stimulation than that from the normotensive control. These results suggest that endothelium-derived nitric oxide released upon neural stimulation partly mediate the relaxation of the corpus cavernosum. It is also suggested that hypertension is associated with a partly attenuated relaxation response to EFS.
Animals ; Atropine ; Baths ; Guanethidine ; Hypertension ; Kidney ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Phenylephrine ; Rats* ; Relaxation

PURPOSE: To investigate the effects of nitric oxide (NO) on the permeability of cultured human trabecular meshwork cell (HTMC) monolayer. METHODS: HTMCs were cultured until confluency in the Transwell inner chamber and then exposed to 0, 10 or 100 microm S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 0.5 mm L-NG-Nitroarginine methyl ester (L-NAME) for 24 hours. Permeabilities of carboxyfluorescein through the HTMC monolayer were measured using a spectrofluorometer after 2 hours in the outer chamber. Cellular viabilities and production of NO were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Griess assay, respectively. RESULTS: The cellular survival was not affected by 10 or 100 microm SNAP (p > 0.05) but NO production increased in a dose-dependent manner (p < 0.05). SNAP significantly increased the permeability of carboxyfluorescein through the HTMC monolayer in a dose-dependent manner compared with non-exposed control (p < 0.05). The endothelial NO synthase inhibitor L-NAME abolished SNAP-induced increase of the carboxyfluorescein permeability (p > 0.05). CONCLUSIONS: NO increased the permeability of carboxyfluorescein through the HTMC monolayer in a dose-dependent manner. Thus, NO could increase trabecular outflow by increasing the permeability of trabecular cell layer in addition to trabeular messwork (TM) relaxation.
Humans ; NG-Nitroarginine Methyl Ester ; Nitric Oxide Synthase ; Nitric Oxide* ; Permeability* ; Relaxation ; Trabecular Meshwork*

BACKGROUND: Endotoxins play important roles in the pathophysiologic alterations associated with sepsis so the authors examined the effects of hydroxocobalamin, NW-nitro-L-arginine-metyl ester (L-NAME) and aminoguanidine on thiopental-induced contractile responses of lipopolysaccharide (LPS)-treated and control rat aortic rings. METHODS: Aortic ring preparation was obtained from LPS-treated (1.5mg/kg, i.p. for 18h) rats. Cumulative doses of thiopental (10-4~3x10- 3M) were added to construct contraction response curves. Hydroxocobalamin (10-5M), L-NAME (10-6M) or aminoguanidine (10-6M) were added as NO scavenger or as NOS inhibitors. Contraction curves by cumulative doses of thiopental (10-4~3x10-3M) were remeasured after treatment of NO scavenger or NOS inhibitors. Statistical significances (p<00.05) were analyzed according to data characteristics by Student's t-test, paired t-test or ANOVA. RESULTS: The vascular responses of cumulative thiopental (10-4~3x10 3M) administration were dose- dependent contraction and LPS-treated rat was less contracted (p<00.05). There was significant increment on vascular contraction induced by thiopental after hydroxocobalamin pretreatment in LPS-treated rat (p<0.05), in spite of L-NAME, aminoguanidine pretreatment was failed to increase contractile forces in control and LPS-treated rats. CONCLUSIONS: From these results, viewed from maintenance of vasomotor tone in septic state, it is suggested that hydroxocobalamin may be candidate for vasopressor during usual induction of general anesthesia.
Anesthesia, General ; Animals ; Aorta, Thoracic* ; Endotoxins ; Hydroxocobalamin* ; NG-Nitroarginine Methyl Ester ; Rats* ; Sepsis ; Thiopental

PURPOSE: We investigated the effects of the superoxide radical on rat whole bladder contractility with duroquinone (superoxide radical generator, Dq) and diethyldithiocarbamate (superoxide dismutase inhibitor, DETCA), and the effects of ginseng saponin (GS) against superoxide radical injury. MATERIALS AND METHODS: Isometric tension changes of isolated rat whole bladders were recorded in an organ bath using a force transducer. The acute effects of Dq and Dq preincubated with DETCA were assessed on resting tension, electrical field stimulation, and bethanechol-, ATP-, and KCl-induced contraction. The effects of Dq and Dq preincubated with DETCA in the presence of sodium nitroprusside and GS were investigated. RESULTS: The resting tension of the muscle was not changed by Dq and Dq preincubated with DETCA. Dq had a harmful effect on only ATP- and KCl-induced detrusor contraction, whereas Dq pretreated with DETCA attenuated the induction of detrusor contraction which was reduced in response to the exogenous NO including GS. In the presence of L-NAME, the effects of GS reduced the Dq-induced inhibition on the detrusor contractility. CONCLUSIONS: It is suggested that the superoxide radical may be the cause of voiding difficulty. GS, as a NO synthesis stimulator, seems to act as a scavenger of the superoxide anion. However further study on the effect of each subfraction of GS is needed for clinical application.
Animals ; Baths ; Ditiocarb ; NG-Nitroarginine Methyl Ester ; Nitroprusside ; Panax* ; Rats* ; Saponins* ; Superoxides* ; Transducers ; Urinary Bladder*

PURPOSE: To evaluate the effect of nitric oxide (NO) on the survival of R28 cells. METHODS: After immunostaining for GFAP, vimentin, S-100, and neurofilament, R28 cells were exposed to S-nitroso-N-acetyl-D, L-penicillamine (SNAP) at various concentrations, with and without the NO inhibitor, Nomega-Nitro-L-arginine methyl ester (L-NAME) for 1 and 3 days. Cellular survival of R28 cells and the production of NO were quantified by rapid colorimetric assays using the MTT and Griess assay, respectively. To evaluate the effect of serum, 10% serum or serum-free media were used separately. RESULTS: R28 cells showed strong immunoreactivity to GFAP and vimentin compared to S-100 or neurofilament. SNAP inhibited the survival of R28 cells in a dose-dependent manner, and this effect of NO on the cellular survival was abolished by L-NAME. These results were similar after exposure for 1 and 3 days, regardless of the presence of serum in the media. CONCLUSIONS: The current results suggest that NO decreased the survival of R28 cells. Further studies are necessary to evaluate the mechanism of cytotoxicity of the R28 cells.
Culture Media, Serum-Free ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Vimentin