Genistein inhibited hypoxia-inducible factor-1alpha expression induced by hypoxia and cobalt chloride in human retinal pigment epithelium cells.

The time-dependent changes of hypoxia-inducible factor-1alpha (HIF-1alpha) expression induced by hypoxia and CoCl2 treatment and the effects of genistein on the level of HIF-1alpha expression in human retinal pigment epithelium cells were examined. Judging by relative fluorescence using a confocal scanning laser microscope coupled to a computer, HIF-1alpha expression was determined. It was found that hypoxia could markedly increase the expression of HIF-1alpha. The highest expression of HIF-1alpha was detected at 1 h, which was 313.9% +/- 38.2% of the control level. After pretreatment with genistein (50, 100, and 200 micromol/l), the hypoxia-evoked HIF-1alpha expression was concentration-dependently inhibited. CoCl2 treatment could significantly elevate the level of HIF-1alpha expression. At 0.5 h after CoCl2 treatment, the highest level was observed, which was 141.4% +/- 14.1% of the control level. Genistein 50, 100, 200 micromol/L could also suppress HIF-1alpha expression in a concentration-dependent manner. These results suggested that the inhibition of HIF-1alpha protein expression by genistein may partly account for its effect on retinal neovascularization in vivo.

Effects of TMB-8 on constriction of pial arteries in rats.

AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on constriction of pial arteries (PA). METHODS: The change of PA in rats was observed continuously and directly through cranial window using microcircular image-shearing system. RESULTS: Nimodipine (Nim) 1 mumol.L-1 produced dilatation of PA immediately and the maximal response occurred in 2 min. But the diameter was not changed by TMB-8 50 mumol.L-1. PA diameter decreased immediately after the application of CSF containing KCl. TMB-8 25, 50 mumol.L-1 apparently inhibited KCl-induced PA constriction. When persistent constriction was evoked by 5-HT, diameter of PA were increased in a concentration-dependent manner after application of TMB-8, which was inhibited by N omega-nitro-L-arginine methyl ester (L-NAME). CONCLUSION: TMB-8 inhibited the contraction induced by 5-HT or KCl in cerebral artery, probably via its calcium antagonization and nitric oxide release.

Inhibitory effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on intracellular Ca2+ elevated by neurotransmitters in brain cells.

AIM: To study the effects of TMB-8 on [Ca2+]i elevation induced by neurotransmitters in dissociated brain cells. METHODS: The brain cell suspension was made using a gentle trituration for 1 min with a polished pipette. The changes of [Ca2+]i were detected by the fluorescent indicator, Fura 2-AM. RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, sodium glutamate (Glu), histamine (His), and serotonin (5-HT) markedly increased the [Ca2+]i which were reduced by TMB-8 30 mumol.L-1. TMB-8 3 mumol.L-1 produced inhibitory effects on the increase of [Ca2+]i by His and 5-HT in a Ca(2+)-free Hanks' solution. The increase of [Ca2+]i by His and 5-HT was reduced to control level by TMB-8 10 mumol.L-1. CONCLUSION: TMB-8 inhibited the [Ca2+]i elevation induced by Glu, 5-HT, and His in brain cells.

8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate reduced [Ca2+]i elevation induced by histamine, serotonin, and glutamate in cultured calf basilar artery smooth muscle cells.

AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5- trimethoxybenzoate (TMB-8) on intracellular free calcium ([Ca2+]i) in cultured calf basilar artery smooth muscle cells. METHODS: [Ca2+]i was examined by a system of measurement of AR-CM-MIC, using Fura 2-AM as a fluorescent indicator. RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, histamine (His), serotonin (5-HT), and sodium glutamate (Glu) markedly increased the [Ca2+]i which was attenuated by TMB-8. In Ca2+ free Hanks' solution containing egtazic acid 0.1 mmol.L-1, TMB-8 not only reduced the resting [Ca2+]i, but also inhibited the elevation of [Ca2+]i evoked by His and 5-HT. CONCLUSION: TMB-8 reduced the resting [Ca2+]i and attenuated His-, 5-HT-, and Glu-induced increases of [Ca2+]i in basilar artery smooth muscle cells.

[8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate(TMB-8) reduced the elevation of [Ca2+]i induced by BHQ, NE and KCl in cultured single smooth muscle cells of the calf basilar artery].

The effect of 8-(N, N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on the elevation of [Ca2+]i induced by 2, 5-di (tert-butyl)-1, 4-benzohydroquinone (BHQ), norepinephrine (NE), KCl in cultured single smooth muscle cells of the calf basilar artery was studied by a system of measurement of AR-CM-MIC, using Fura-2/AM as a fluoresent indicator. In the presence of extracellular Ca2+ 1.3 mmol.L-1, the resting [Ca2+]i was not changed by TMB-8 (10, 30 and 100 mumol.L-1), but the elevation of [Ca2+]i induced by BHQ, NE and KCl were reduced by TMB-8 (30 mumol.L-1) significantly. In Ca2+ free Hank's solution containing EGTA 0.1 mmol.L-1, the resting [Ca2+]i was markedly reduced by TMB-8 (10, 30 and 100 mumol.L-1), and the increase of [Ca2+]i evoked by BHQ and NE was blocked completely by TMB-8 (30 mumol.L-1). The result suggested that TMB-8 inhibited the Ca2+ release from intracellular stores or increased the up-take of Ca2+ into sarcoplasmic reticulum and the inhibition of Ca(2+)-influx from extracellular site may be an indirect machanism.

[Effect of TMB-8 on the increase of intracellular free Ca2+ induced by NE and BHQ in dissociated single rat brain cell].

The inhibitory effect and mechanism of 8-(N, N'-diethylamino) octyl 3, 4, 5-trimethoxybenzoate hydrochloride (TMB-8) on the elevation of single intracellular free Ca2+ concentration ([Ca2+]i) induced by High K+, Norepinephrine(NE) and 2, 5-Di(tert-butyl)-1, 4-benzohydroquinone (BHQ) in dissociated single rat brain cells were studied. The changes of [Ca2+]i were reflected by the fluorescent indicator, Fura-2/AM, employed. In the absence of extracellular Ca2+, Ca-free Hank's solution, preincubation with TMB-8 (10, 30 mumol.L-1) for 20 min significantly decreased the resting [Ca2+]i from 79 +/- 13 nmol.L-1 to 65 +/- 11 and 61 +/- 6 nmol.L-1, respectively. [Ca2+]i were markedly increased by NE and BHQ and reduced significantly to control level by TMB-8. On the other hand, when the cells were incubated in Hank's solution containing Ca2+ 1.3 mmol.L-1, TMB-8(30, 100 mumol.L-1) suppressed the increase of [Ca2+]i induced by NE (0.0001-0.1 mumol.L-1). TMB-8 showed no significant effect on [Ca2+]i elevation induced by KCl and BHQ in Hank's solution containing Ca2+ 1.3 mmol.L-1. These results indicate that TMB-8 reduced [Ca2+]i via increase of the sarcoplasmic reticulum (SR) sequestration, which blocked the release of intracellular store from the SR. However, the inhibitory effect of TMB-8 on Ca-influx from extracellular medium seems to be an indirect action from the saturation of SR with calcium.

Tetrandrine inhibits breakdown of blood-aqueous barrier induced by endotoxin and interleukin-1 alpha in rats.

Tetrandrine was shown to significantly inhibit uveitis induced by endotoxin and interleukin-1 alpha (IL-1 alpha) in rats. The dose-response curve of IL-1 alpha-induced uveitis was inhibited in a non-competitive manner. The maximum inflammation induced by IL-1 alpha was suppressed to 58.4%, 38.3% and 18.3% of the control peak by 5 mg/kg, 10 mg/kg and 20 mg/kg t.i.d. of tetrandrine, respectively. The maximum inflammation induced by endotoxin was suppressed to 56.5% and 38.0% by 5 mg/kg and 10 mg/kg t.i.d. of tetrandrine, respectively. The mechanism of tetrandrine's anti-inflammation could involve numerous pathways of inflammation processes and multiple inflammatory mediators. The results of this study indicate that tetrandrine appears to be a broad spectrum, non-steroidal, novel ocular anti-inflammatory agent.

Inhibitory effects of tetrandrine on intracellular free Ca2+ increase induced by glutamate, serotonin and histamine in dissociated retina cells.

The effects of tetrandrine (Tet) on the elevation of intracellular free Ca2+ concentration ([Ca2+]i) induced by glutamate, serotonin and histamine in dissociated rabbit retina cells were studied. The changes of [Ca2+]i were reflected by the fluorescent indicator, Fura-2/AM, employed. In the presence of extracellular Ca2+ (1.3 mM), glutamate, serotonin and histamine significantly increased the [Ca2+]i in a dose-dependent manner. Glutamate (100 microM), serotonin (100 microM) and histamine (200 microM) markedly increased the [Ca2+]i of retina cells by 165%, 126% and 58%, respectively. Tet 30 microM significantly inhibited the increase of [Ca2+]i induced by glutamate (100 microM), serotonin (100 microM) and histamine (200 microM) by 28.0%, 46.8% and 29.0%, respectively. A lower concentration (10 microM) of Tet also produced an inhibitory effect on the increase of [Ca2+]i but was less effective than the Tet 30 microM. In Ca(2+)-free Hank's solution, Tet did not produce a significant inhibitory effect on the increase of [Ca2+]i caused by serotonin and histamine. These results indicate that Tet exercises blocking Ca2+ influx from the extracellular site via NMDA, 5-HT2 and H1-receptor operated Ca2+ channels and has no obvious effect on the Ca2+ release from intracellular Ca2+ stores.

Inhibition of high K+ and norepinephrine-induced free intracellular calcium by tetrandrine in dissociated rabbit retina cells.

The effect of tetrandrine (Tet) on the changes of intracellular free Ca2+ concentration ([Ca2+]i) induced by high concentration of K+ and norepinephrine is dissociated rabbit retina cells were studied. The changes of [Ca2+]i were reflected by the fluorescent indicator, Fura-2/AM, employed. The resting [Ca2+]i was 223 +/- 27 nM in Hank's solution containing 1.3 mM Ca2+. Tet at 30 microM did not affect the resting [Ca2+]i when the extracellular Ca2+ concentration was in the range of 0-2 mM. Tet at 1-100 microM suppressed the [Ca2+]i elevation induced by high concentration of K+ in a concentration-dependent manner. When the concentration of K+ was 50 mM IC50 for Tet was 10.9 microM. Although Tet at high concentration (30 microM) suppressed the [Ca2+]i increases induced by norepinephrine (0.1-100 nM) in the presence of Ca2+ at 1.3 mM, failed to do the same at lower concentration of Tet (1-10 microM), however, Tet at 30 microM did not effect the release of intracellular Ca2+ stored in the sarcoplasmic reticulum in the Ca(2+)-free Hank's solution. These results indicate that Tet is able to block Ca2+ influx from extracellular site via voltage-sensitive ionic channels and at high concentration via receptor-operated ionic channels in retina cells.

Mechanisms of antagonism of interleukin-1 alpha by synthetic interleukin-1 blockers.

A series of interleukin-1 blockers, CK-compounds, were shown to inhibit the dose-response curve of IL-1 induced uveitis in a non-competitive manner. CK-compounds showed little or weak effects on PGE2 production, leukocyte chemotaxis, and lipoxygenase activity which were markedly inhibited by indomethacin, REV 9501, and NDGA, respectively. These results indicate that CK-compounds do not fall into the conventional class of non-steroidal arachidonate mediated anti-inflammatory agents. CK-compounds should therefore be classified as novel compounds to block IL-1 non-competitively and belong to non-steroidal, non-arachidonate mediated anti-inflammatory agents.

Prevention of ocular inflammation by matrine, prednisolone, and cyclooxygenase and lipoxygenase inhibitors.

Lens protein-induced ocular inflammation in rabbits was used to study the action mechanism of some anti-inflammatory agents. Indomethacin, a cyclooxygenase inhibitor, markedly reduced PGE2 and PGF2 alpha in the iris and ciliary body at 2 h but PGE2 only at 4 h. REV 5901, a lipoxygenase inhibitor, only significantly reduced PGE2 levels in the ciliary body at 4 h. PGF2 alpha levels were not affected by REV 5901. When indomethacin and REV 5901 were combined, both PGE2 and PGF2 alpha were suppressed at 2 and 4 h in both iris and ciliary body. Neither matrine nor prednisolone produced significant effects on the levels of PGE2 and PGF2 alpha. However, prednisolone exhibited the greatest reduction in chemotaxis of leukocytes followed by REV 5901. Indomethacin, on the contrary, produced a significant increase in chemotaxis of leukocytes. Matrine produced a decrease in leukocyte counts but was not statistically significant. These results indicate that indomethacin is effective in the early phase of inflammation to reduce PG's production whereas prednisolone and REV 5901 were more effective in the late phase of inflammation. Combined use of REV 5901 and indomethacin could become a drug of choice for the treatment of ocular inflammation without inducing corticosteroidal side effects.

[The ultrastructure of leptospires after freeze-etching].

The ultrastructure of three strains of leptospires, i.e. L. interrogans serovar Lai strain 017, L. biflexa serovar patoc strain Patoc I and L. illini strain 3055, were studied with the technique of freeze-etching replica. The results showed that (1) the ultrastructure of leptospires after freeze-etching are similar to that observed by means of negative staining and ultrasectioning. There are no dramatic differences among strains of leptospires studied. (2) Globular particles exposed on the resulting two inner membrane faces are asymmetrically distributed. Large areas on EF face studied with numerous globular particles, whereas the PF face had few of them. The density of globular particles varies in different parts of leptospires, from 776/microns2 to 2303/microns2. The globular particles, 14.25 +/- 2.25 nm in diameter, represent membrane proteins in outer envelope of leptospires. (3) Axial filament, 20 nm in diameter, were seen closely surrounded by a 7 nm sheath-like structure. (4) There are subcellular structures in cytoplasm of leptospires. They appear to be ribosomes, chromatins and inclusions.

[Purification and SDS-page analysis of axial filaments from leptospires].

A modified method, differential centrifugation followed by sucrose density centrifugation, was used to purify axial filaments from three strains of Leptospires. Ultrastructure of the axial filaments was studied and profiles of the axial filaments were characterized and compared. The results have shown that all the three strains of Leptospires, i.e., L. interrogsans serovar Lai strain 017, L. biflexa serovar patoc strain Patoc I and L. illini strain 3055, have two axial filaments in one cell. The axial filament is 20 nm in diameter. It is the first observation that the end which inserts the cytoplasms cylinder is wider in diameter than the free one. An insertion pore structure is observed. The new method yields 1.5mg axial filaments from 12 g leptospires cells. SDS-PAGE was first employed in the analysis of axial filaments of leptospires. The results have also shown that there are 6 proteins in the axial filaments of strain 017, MW 26,000-50,000 while 7 proteins in the axial filaments of strain Patoc I and strain 3055. MW 29,000-80,000 and 28,500-80,000 respectively. Interestingly, all the axial filaments of the three strains have a common protein band of MW 31,500. The possibility of using axial filament proteins as a new criterion for typing and a serodiagnosis antigen is discussed.

The potential role of adenosine in regulating blood flow in the eye.

Blood flow in the eye has shown a remarkable ability to autoregulate regardless of intraocular pressure, perfusion pressure or alterations in arterial pressure. This study investigates the possibility that adenosine may play a role in regulating ocular blood flow. Ocular blood flow was measured using radio-labelled 85Sr microsphere and laser Doppler techniques. When two adenosine uptake inhibitors, dipyridamole and papaverine were injected intravitreally, the ocular blood flow increased in all ocular tissues tested: iris, iris root-ciliary body, retina and choroid. This increase in blood flow was blocked by the addition of the adenosine antagonist, 8-phenyltheophylline. The increase in flow produced by dipyridamole continued for up to an hour after administration. The increase in blood flow in individual ocular tissues does not appear to be due to shunting (ie. redistribution) of flow because total blood flow to the eye increased but the percent total flow to each individual tissue remained near control values.

Ocular anti-inflammatory actions of matrine.

Matrine is an alkaloid isolated from the root of Sophora subprostrata (Leguminosae) which has been used as a Chinese medicine for the treatment of inflammation. Instillation of 50 microliter of 1% matrine showed significant inhibition of ocular inflammation induced by lens proteins. Unlike corticosteroids, matrine did not facilitate the IOP recovery in rabbit eyes nor did it change the electrical potential difference across rabbit iris-ciliary body. These results indicate that matrine could become a safer ocular antiinflammatory agent than corticosteroids. Further, matrine was found to markedly increase the reaction time of a mouse placed on a hot plate. These results indicate that matrine could be used as an analgesic as well.