Biomedical subjects

H K Mishima

Publications and source records attributed to H K Mishima.

At least 37 records · Page 2Linked to original sources

PubMed1999

Attenuation by PACAP of glutamate-induced neurotoxicity in cultured retinal neurons.

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs: PACAP27, PACAP38) on glutamate-induced neurotoxicity were examined using cultured retinal neurons obtained from 3- to 5-day old Wistar rats. Cell viability was evaluated by double staining with fluorescein diacetate and propidium iodide. Effects of PACAPs on the increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in retinal neurons was investigated using the Ca(2+) image analyzing system with fura-2. The cAMP contents and the mitogen-activated protein (MAP) kinase activity in retinal cultures were measured by radioimmunoassay. Concomitant application of PACAPs (10 nM-1 microM) with glutamate (1 mM) for 10 min inhibited the delayed death of retinal neurons, which was observed 24 h after glutamate (1 mM) treatment in a dose-dependent manner. Protection by PACAPs (100 nM) against glutamate-induced neurotoxicity was antagonized by PACAP6-38 (1 microM), a PACAP antagonist, and H-89 (1 microM), a protein kinase A (PKA) inhibitor. However, PACAPs did not affect the glutamate-induced increase in [Ca(2+)](i), but PACAPs (1-100 nM) increased the cAMP levels in a dose-dependent manner. In addition, activation of MAP kinase by PACAP38 (1 microM) was inhibited by simultaneous application with H-89 (1 microM). These findings suggest that PACAPs attenuate glutamate-induced delayed neurotoxicity in cultured retinal neurons by activating MAP kinase through the activation of cAMP-stimulated PKA.

Animals↗

PubMed1999

Rat retinal ganglion cells culture enriched with the magnetic cell sorter.

The magnetic cell sorter (MACS) technique was applied to isolate retinal ganglion cells (RGCs) for culture. RGCs were labeled retrogradely with 1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). Subsequently retinal cell suspensions were incubated with biotinylated anti-rat Thy-1 antibody and MACS Streptavidin MicroBeads, and then applied onto the column in the magnetic fields. Cells attached on the column were flashed out without magnetism and plated on glass cover slips. RGCs were enriched to 31.0% of all cells with MACS from 0.55% before applying onto the magnetic column. Mean diameters of Dil-labeled cells were significantly larger than those of unlabeled cells. All cells with soma diameter over 11 microm were labeled. The number of viable RGCs were counted in the 10 fields of six cultures at a magnification of x200; the mean numbers on the 2nd, 7th and 14th culture-day were 53+/-3, 24+/-2 and 21+/-3, respectively (mean +/- SEM, n = 6). Thus, the MACS technique was confirmed to be useful for enrichment of RGCs and long-term study of cultured RGCs.

Animals↗

PubMed1999

Role of P-glycoprotein in distribution of rhodamine 123 into aqueous humor in rabbits.

PURPOSE: Examination of the contribution of functional P-glycoprotein (P-gp), an ATP-dependent efflux pump, in blood-aqueous barrier in rabbits. METHODS: Rhodamine-123 (Rho-123), a P-gp substrate, was administered intravenously via the marginal ear vein of rabbits. Rhodamine B (Rho-B), an analogue of Rho-123, was also injected with the same dose, as a reference compound. Quinidine at different concentrations was applied topically to the corneal surface by eye drops prior to the intravenous administration of a Rho compound. The aqueous distribution (a ratio of concentration in aqueous humor to that in plasma) of these Rho compounds was analyzed in relation to the aqueous concentration of quinidine. Transport study across Caco-2 cell monolayers was carried out to examine the involvement of P-gp in Rho-B transport. RESULTS: It was proved that Rho-B is not a P-gp substrate by a transport study across Caco-2 cell monolayers, in contrast to Rho-123 (a P-gp substrate). The aqueous distribution of Rho-123 given intravenously was significantly lower than that of Rho-B. Topical quinidine (a P-gp inhibitor) markedly increased the aqueous distribution of Rho-123, depending on the aqueous concentrations of quinidine, though it did not affect the aqueous distribution of Rho-B. CONCLUSIONS: The contribution of functional P-gp in blood-aqueous barrier was clearly demonstrated by analyzing the aqueous distribution of Rho-123 in the presence or absence of quinidine. These experiments only allow us to address one part of the blood-aqueous barrier, the capillary endothelium, and, to do so by using different substrates for P-gp, a sort of chemical analogy with the presumed blood-aqueous barrier across capillary endothelia. The alteration of P-gp function by pharmacotherapy or in pathological state should be considered in the ophthalmic medical treatment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Protective effects of vasoactive intestinal peptide against delayed glutamate neurotoxicity in cultured retina.

The effects of vasoactive intestinal peptide (VIP) on glutamate-induced delayed death were examined using the primary cultures of rat retinal neurons. Effects of VIP on glutamate-induced neurotoxicity were evaluated by double staining with fluorescein diacetate and propidium iodide. Glutamate (1 mM) was applied to the culture for 10 min in the presence and absence of VIP, and visible cells enumerated 24 h after culture in normal medium. Effects of VIP on increase in the intracellular Ca2+ concentration and currents induced by glutamate in retinal neurons were investigated using the Ca2+ image analyzing system with fura-2 and whole-cell patch-clamp recording, respectively. The cAMP contents in retinal cultures were measured by radioimmunoassay. VIP (10 nM-1 microM) dose-dependently protected against glutamate-induced neurotoxicity in cultured retinal neurons. Protection by VIP (100 nM) against glutamate (1 mM)-induced neurotoxicity was antagonized by VIP6-28 (1 microM), a VIP antagonist, and H-89 (100 nM and 1 microM), a protein kinase A inhibitor. However, VIP had no effect on glutamate-induced inward currents nor glutamate-induced increase in the intracellular Ca2+ concentration. A 10-min exposure of VIP (100 nM) with glutamate (1 mM) resulted in an increase in the cAMP level to 446+/-58 from 22+/-1 pmol/mg protein. These findings suggest that VIP protects against the glutamate-induced neurotoxicity in retinal cultures by elevating the cAMP level via VIP receptors and thereby activating protein kinase A.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Effects of retinoic acid and TGF-beta 1 on the proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro.

PURPOSE: The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) and transforming growth factor-beta 1 (TGF-beta 1) on proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. METHODS: Chick RPE cells were cultured in the presence or absence of RA (10(-5) to 10(-7) M) or TGF-beta 1 (1 to 100 U/ml) for a 10-day period. The effects of RA and TGF-beta 1 on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content in the cells. RESULTS: RA significantly inhibited RPE cell proliferation in a time-dependent manner. The most marked inhibition was observed on day 10 of culture, and the most effective concentration of RA was 10(-6) M. Melanin synthesis was increased on day 10, and the most effective concentration of RA was also 10(-6) M. TGF-beta 1 also inhibited RPE cell proliferation and increased melanin synthesis. CONCLUSIONS: These findings indicate that RA and TGF-beta 1 have regulatory effects on the proliferation and differentiation of chick RPE cells in vitro.

Animals↗

PubMed1998

Evaluation of 9-cis retinoic acid for a new remedy of human retinoblastoma.

We investigated the effect of two isomers of retinoic acid (RA), all-trans RA and 9-cis RA, on the proliferation of Y79 human retinoblastoma cells. The two isomers inhibited the cell proliferation in a concentration-dependent manner. The IC50 for this inhibition by all-trans RA and 9-cis RA was 1.50 and 0.15 microM, respectively. The inhibitory effect of 9-cis RA on Y79 cell growth was observed within 24 hr, thereafter the cell number was gradually decreased. In contrast, no inhibition by all-trans RA of Y79 cell growth was observed within 24 hr, thereafter the cell number was slightly increased. In these cases, the cell viability at 4 days after the addition of 9-cis RA and all-trans RA was more than 90% and 95%, respectively. These results indicate that the two RA inhibit the proliferation of Y79 human retinoblastoma cells without inducing the cell death and that the effect of 9-cis RA on the inhibition of Y79 cell growth is much greater than that of all-trans RA.

Alitretinoin↗

PubMed1998

Protection against glutamate neurotoxicity in retinal cultures by acidic conditions.

We evaluated the effects of extracellular acidic conditions on glutamate-induced death in cultured retinal neurons. Primary retinal cultures, obtained from 3- to 5-day-old Wistar rats, were estimated to be consisted of mainly amacrine cells (90%) together with a small population of horizontal (8%) and ganglion cells (2%). We examined the effects of acidic pH (pH 6.0 to 7.0) on glutamate neurotoxicity by monitoring the delayed death of retinal neurons induced by brief (10 min) exposure to 1 mM glutamate followed by a 24-hr incubation. The glutamate-induced delayed death of cultured retinal neurons was attenuated with an acidic pH between 6.0 and 7.0. Furthermore, whole-cell patch-clamp recordings were taken from retinal neurons to examine the effects of acidic pH on N-methyl-D-aspartate (NMDA) or kainate receptor-mediated currents. NMDA- and kainate-induced currents were suppressed at pH 6.0 to 7.0 and pH 6.0 to 6.5, respectively. The acidity of the medium protected the retinal neurons from glutamate-induced delayed death, probably by inhibiting NMDA and/or kainate receptor activation.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1997

Cytokine- and neuropeptide-mediated differentiation in retinal pigment epithelial cells in vitro.

To determine the mechanism of growth and differentiation of retinal pigment epithelial (RPE) cells it is important to understand the pathogenesis of several retinal diseases. Recently it has been reported that several cytokines and neuropeptides regulate the growth of RPE cells. In this study, the role of cytokines and neuropeptides in melanin synthesis, which is one indication of the RPE cell differentiation, was examined using chick RPE cells in vitro IL-1beta, TNF-alpha, substance P, beta-endorphin and methionine-enkephalin stimulated the melanin synthesis of RPE cells in a dose-dependent manner. The most effective concentrations of these agents on RPE cell melanin synthesis were not the same as that for RPE cell proliferation. These results indicate that cytokines and neuropeptides play an important role not only for the growth but also for the differentiation of RPE cells.

Animals↗

PubMed1997

Circadian intraocular pressure management with latanoprost: diurnal and nocturnal intraocular pressure reduction and increased uveoscleral outflow.

Based on their mechanism of action, the most frequently used ocular hypertensive agents, the beta-blockers, cannot be assumed to reduce IOP during sleep. The need for drugs that reduce IOP around-the-clock is underscored, however, by the fact that inadequate nocturnal ocular perfusion pressure is considered to be one of the likely causes of glaucomatous optic neuropathy especially in some cases of normal tension glaucoma. The studies reviewed here demonstrate that latanoprost, a new ocular hypotensive prostaglandin F2 alpha analogue, applied once a day at a concentration of 0.005%, maintains a statistically highly significant IOP reduction around-the-clock. The magnitude of this IOP reduction was found to be essentially identical during the day and at night, both in patients maintained on timolol and in those not receiving other glaucoma medication. Latanoprost-induced IOP reduction was also found to be associated with increased uveoscleral outflow in normotensive volunteers, both during the day and at night. These circadian studies suggest that this new ocular hypotensive agent can be expected to be particularly useful for the medical management of some forms of glaucoma, such as normal tension glaucoma, when the cause of the glaucomatous damage cannot be linked specifically to diurnal IOP abnormalities.

Administration, Topical↗

PubMed1997

Purification and characterization of alpha,beta-ketoalkene double bond reductases from bovine eyes.

PURPOSE: To explore the alpha,beta-ketoalkene double bond reductases responsible for xenobiotic metabolism in bovine ocular tissues using trans-phenyl-l-propenyl ketone as a model substrate. METHODS: A mixture of trans-phenyl-l-propenyl ketone, NADPH or NADH, and an enzyme source in 0.1 M K,Na-phosphate buffer (pH 7.4) was incubated for 15 min at 37 degrees C. Phenyl propyl ketone formed was quantified by high performance liquid chromatography (HPLC). RESULTS: The lens, ciliary body, iris, retinal pigment epithelium (RPE)-choroid, retina, and cornea exhibited alpha,beta-ketoalkene double bond reductase activities in the presence of NADPH or NADH. An alpha,beta-ketoalkene double bond reductase was purified to homogeneity from the iris-ciliary body cytosol. The molecular weight was estimated to be 58,000 by gel filtration, and 40,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was inhibited by dicumarol, quercitrin, indomethacin, disulfiram, and p-chloromercuribenzoic acid. The enzyme exhibited double bond reductase activity toward 2-alkenals as well as alpha,beta-ketoalkenes. Another alpha,beta-ketoalkene double bond reductase was also purified to homogeneity from the lens cytosol. The molecular weight was estimated to be 105,000 by gel filtration and 40,000 by SDS-PAGE. The enzyme activity was inhibited by dicumarol and quercitrin. The enzyme exhibited double bond reductase activity toward some alpha,beta-ketoalkenes. CONCLUSIONS: Two kinds of enzymes responsible for reduction of the carbon-carbon double bond of xenobiotics were purified for the first time from ocular tissues. The molecular weights, substrate specificities, and sensitivities to inhibitors of the enzymes were different from each other.

Alkenes↗

PubMed1997

Incidence of retinal vein occlusion at the Glaucoma Clinic of Hiroshima University.

We prospectively surveyed the incidence of retinal vein occlusion (RVO) at the glaucoma clinic of Hiroshima University between 1986 and 1991. Among 433 glaucoma patients, 18 (4.2%) subsequently presented with RVO, 9 had central retinal vein occlusion (CRVO) and 9 exhibited branch retinal vein occlusion (BRVO). Seven of 87 (8.1%) patients with primary angle closure glaucoma exhibited RVO, showing the highest incidence among glaucoma types. The incidence of RVO detected by the general outpatient clinic was 0.59% during the same period. The BRVO/CRVO ratio in the glaucoma clinic was 1.0, while it was 4.3 in the general outpatient clinic. Glaucoma is an important risk factor for the development of RVO, especially CRVO.

Adult↗

PubMed1996

A comparison of latanoprost and timolol in primary open-angle glaucoma and ocular hypertension. A 12-week study.

OBJECTIVE: To evaluate the intraocular pressure (IOP)-reducing effect and the side effects of latanoprost (PhXA41), a new phenyl-substituted prostaglandin F2 alpha-isopropyl ester analogue, in patients with elevated IOP, using timolol maleate as the reference drug. METHODS: A total of 184 patients with primary open-angle glaucoma or ocular hypertension at 35 medical centers participated in this randomized double-masked study. The patients were randomized to receive either 0.005% latanoprost once daily or 0.5% timolol maleate twice daily, for a period of 12 weeks. Intraocular pressure was measured 24 hours after the administration of timolol, at 2, 4, 8, and 12 weeks of treatment. RESULTS: Latanoprost reduced IOP at the end of 12 weeks by 6.2 +/- 2.7 mm Hg (mean +/- SD) (26.8%), while timolol reduced IOP by 4.4 +/- 2.3 mm Hg (19.9%). At all visits latanoprost reduced IOP significantly more than timolol did. The main ocular side effects observed in both groups were conjunctival hyperemia and smarting. The main systemic side effect was a reduced pulse rate, which occurred in patients treated with timolol. CONCLUSIONS: The results of this study demonstrated that 0.005% latanoprost taken once daily is well tolerated and more effective in reducing IOP than 0.5% timolol taken twice daily. Thus, latanoprost may become an important choice for the medical treatment of glaucoma.

Adrenergic beta-Antagonists↗

PubMed1996

Ultrasound biomicroscopic study of ciliary body thickness after topical application of pharmacologic agents.

PURPOSE: To examine the changes in ciliary body thickness after topical application of pilocarpine, cyclopentolate hydrochloride, and PhXA41, a prostaglandin F2alpha analog. METHOD: We used high-frequency Humphrey UBM840 ultrasound biomicroscope to examine 36 healthy young Japanese subjects. RESULTS: The mean ciliary body thickness increased from 0.67 +/- 0.07 mm to 0.073 +/- 0.08 mm (P < .01) after application of 2% pilocarpine; 1% cyclopentolate hydrochloride and 0.005% PhXA41 decreased the mean ciliary body thickness from 0.75 +/- 0.07 mm to 0.69 +/- 0.05 mm (P < .05) and from 0.78 +/- 0.06 mm to 0.75 +/- 0.06 mm (P < .01), respectively. CONCLUSIONS: Our ultrasound study clearly indicates that pilocarpine increased comparative thickness of the ciliary body by 8.3%, whereas PhXA41 decreased comparative thickness by 3.3% in a manner similar to cyclopentolate hydrochloride.

Administration, Topical↗

PubMed1996

Stimulation of retinal pigment epithelial cell growth by neuropeptides in vitro.

PURPOSE: The proliferation of many cell types are regulated by cytokines and neuropeptides by autocrine and paracrine mechanisms. Retinal pigment epithelial (RPE) cells are also regulated by cytokines. But RPE cells are very close to the neural retina which has some neuropeptides. The present study was to investigate the effects of neuropeptides on the growth of RPE cells. METHODS: RPE cells were obtained from the eyes of 11 day old chick embryos and cultured in Dulbecco's modified Eagle's culture medium containing 10% fetal calf serum. The growth of RPE cells was evaluated by [3H]-thymidine uptake. RESULTS: Substance P, beta-endorphin and calcitonin gene-related peptide markedly stimulated the growth of RPE cells. The effects of methionine-enkephalin, somatostatin and vasoactive intestinal peptide were intermediate. The strongest effects of substance P, beta-endorphin and calcitonin gene-related peptide were observed at 10(-6) to 10(-7) M. The stimulation of RPE cells with beta-endorphin was inhibited by naloxone, suggesting that the stimulation with beta-endorphin is mediated by an opioid receptor. beta-endorphin and substance P induced RPE cell growth stimulating activity. Leucine-enkephalin and neuropeptide Y did not affect the growth of RPE cells. CONCLUSIONS: These results suggest that neuropeptides play an important role in the regulation of RPE cell growth.

Animals↗

PubMed1995

Characterization of ciliary muscle relaxation induced by various agents in cats.

PURPOSE: To understand the cellular mechanism underlying the relaxation of ciliary muscle, relaxation induced by prostaglandins (PGs) and some other agents was characterized in the cat. METHODS: Tone of isolated ciliary muscle was measured by means of a force-displacement transducer. Adenylate cyclase activity was determined with membrane fraction of ciliary muscle by measuring the formation of cyclic adenosine monophosphate (cAMP). RESULTS: The addition of various PGs and isoproterenol relaxed the ciliary muscle that had been precontracted with 3 x 10(-6) M carbachol. The relaxation was dose dependent, with an EC50 of 2 x 10(-7) M for PGE2. The rank order of potency by which PGs induced relaxation (PGE2 = E1 > D2 > F2 alpha > I2) was identical with that reported for EP type prostaglandin receptor-mediated responses except for PGD2, which was more potent than expected. Agents that increased cellular cAMP, such as forskolin and IBMX, also relaxed the precontracted muscle. Nitric oxide donors, such as sodium nitroprusside and S-nitroso-N-acetyl-DL-penicillamine (SNAP), also caused dose-dependent relaxation. PGs and isoproterenol, but not nitroprusside, stimulated adenylate cyclase. The rank order of potency by which PGs stimulate adenylate cyclase was similar to that observed for muscle relaxation, suggesting that cAMP is the cellular second messenger for the PG-induced muscle relaxation and thus that PG receptors of EP2 and DP type are involved. CONCLUSIONS: Relaxation of cat ciliary muscle is mediated by two independent mechanisms: a cAMP-dependent one, which includes beta-adrenergic, EP2, and DP receptor-mediated responses, and a cAMP-independent one, which includes the nitric oxide-induced mechanism.

Adenylyl Cyclases↗

PubMed1995

Isoforms of glucose transporter in the iris-ciliary body.

Isoforms of the facilitated glucose transporter (GLUT) were identified in the iris-ciliary body. Western blot analysis showed that GLUT1 and GLUT4 proteins were expressed in the rat iris-ciliary body. In addition, the content of GLUT proteins in the rabbit iris-ciliary body was estimated by specific [3H]cytochalasin B binding assay. The content of GLUT proteins was found to be more abundant in the iris-ciliary body than in the cerebral cortex. The utilization of glucose in the iris-ciliary body and the transport of glucose into the aqueous humor are suggested to be mediated by GLUT1 and GLUT4 proteins.

Animals↗

PubMed1995

Ca2+/calmodulin effects on cAMP response in cultured chick ciliary epithelial cells.

The authors investigated the influence of the elevation of intracellular Ca2+ concentration on the production of cyclic adenosine 3',5'-monophosphate (cAMP) using cultured chick embryo ciliary epithelium (CE). We examined the effects of calcium ionophore (A23187) on cAMP production after incubation with vasoactive intestinal peptide (VIP), isoproterenol (ISO), sodium fluoride (NaF) or forskolin (FSK). A23187 had no effect on the basal cAMP level, or the NaF- and FSK-stimulated responses for cAMP level; but A23187 potentiated VIP- and ISO-stimulated cAMP responses. Calmodulin antagonist (W-7) was very effective in inhibiting the potentiating effects of A23187 on VIP and ISO-stimulated production of cAMP. Our present findings suggested that Ca2+/calmodulin may potentiate the receptor-mediated cAMP pathway through Ca2+/calmodulin-dependent protein kinase in the CE.

1-Methyl-3-isobutylxanthine↗

PubMed1995

The effects of prostaglandins on the blood-ocular barrier.

The effects of prostaglandins (PGs) and PG-related compounds on the blood-ocular barriers were examined using pigmented rabbits. Latanoprost (PhXA41), PGF2(alpha)-isopropyl ester (PGF2(alpha)-IE) or PGE2 was topically applied once only or once daily for 8 weeks. Aqueous flare was measured with a laser flare-cell meter, and morphological changes in the ciliary processes after repeated applications of a test drug were investigated by means of light or electron microscopy using horseradish peroxidase (HRP) as a tracer. PGF2(alpha)-IE and PGE2, but not PhXA41, caused an initial rise in the aqueous flare after application. On the other hand, no morphological changes were found in the ciliary processes after 8-week PhXA41 application. After 8-week application of PGF2(alpha)-IE or PGE2 dilation of ciliary channels in the ciliary processes were found. Leakage of intravenously injected fluorescein was measured by a vitreous fluorophotometer after an intravitreal injection of PGE2, PGF2(alpha), or PhXA41. Vitreous fluorescence was significantly higher in treated eyes than in controls after intravitreal injection of PGE2 or PGF2(alpha), while it showed no significant change after intravitreal injection of PhXA41. Only PGE2 caused morphological changes in the retina. These results suggest that PhXA41 does not compromise the integrity of the blood-ocular barriers, and has a potential as a future anti-glaucoma eyedrop.

Administration, Topical↗