Teruyo Ohuchi

Heterogeneous response in calcium signaling by adrenergic and cholinergic stimulation in cultured bovine trabecular cells

Adrenergic and cholinergic drug-dependent Ca(2+)-signaling in cultured bovine trabecular cells was studied using a fluorescent calcium dye, fura-2, and digital video-imaging system. The resting calcium concentration in the cultured cell was 178.7 +/- 1.8 nM. Upon stimulation with norepinephrine, isoproterenol and carbachol trabecular cells responded heterogeneously. Many types of reaction to the stimuli were found; about 25% of the cells responded to the stimuli and about 30% showed no response to all of the stimuli. Carbachol induced response was blocked with pretreatment of atropine, and timolol inhibited the isoproterenol-induced response. Ca2+ signaling in trabecular cells may mediate the action of adrenergic and cholinergic drugs and possibly play significant roles in the regulation of intraocular pressure.

Neuropeptide-induced cytosolic Ca2+ transients and phosphatidylinositol turnover in cultured human retinal pigment ephithelial cells

Neuropeptide-induced mobilization of cytosolic free Ca2+ concentration ([Ca2+]i) and phosphatidylinositol (PI) turnover in cultured human retinal pigment epithelial (RPE) cells were studied and their temporal relationship was compared. After RPE cells were loaded with fura-2/AM, [Ca2+]i was analyzed using a digital imaging microscopy system. Bombesin-related peptides which include bombesin, neuromedin B, and neuromedin C induced significant [Ca2+]i transients in RPE cells, whereas other neuropeptides, neuropeptide Y, vasoactive intestinal polypeptide (VIP), and substance P were not effective to produce [Ca2+]i transients. The percentage of reactive cells which showed positive [Ca2+]i transients induced by bombesin-related peptides was around 50%. Bombesin (1 microM) showed a peak concentration of 663 +/- 27.0 nM (mean +/- S.E.M., n = 61), neuromedin B (1 microM), 327 +/- 28.7 nM (mean +/- S.E.M., n = 38), and neuromedin C (1 microM), 357 +/- 22.7 nM (mean +/- S.E.M., n = 32). Ca2+ transients occurred within 30 s and lasted less than 5 min after the application of the neuropeptides. Chelation of the extracellular Ca2+ by EGTA significantly shortened the total time of [Ca2+]i transients induced by the above. The measurements of phosphoinositides in RPE cells revealed that neuropeptide-induced PI turnover was as quick as [Ca2+]i transients. Inositol biphosphate (IP2) and inositol triphosphate (IP3) in RPE cells showed transient increases at 15 s after the stimulation by bombesin-related peptides. These data show that changes in [Ca2+]i and PI turnover are directly linked and both are important in the signal transduction system of bombesin-related peptides in RPE cells. The data also suggest that bombesin-related peptides may play some possible roles in RPE cells.

Mass Cultivation of Human Retinal Pigment Epithelial Cells with Microcarrier

Microcarrier cell culture permits mass cultivation of anchorage-dependent cells. In this study, mass cultivation of human retinal pigment epithelial (RPE) cells was studied using Cytodex 3 (Pharmacia) as a microcarrier. Human RPE cells were established from aborted fetuses and cultured in Dulbeccos modified Eagles Medium (DMEM). After the 3rd-5th passages, RPE cells were suspended in 50 ml of DMEM in a spinner flask at a density of 2 x 10(5)/ml, and Cytodex 3 was added to the spinner flask at a bead density of 10 mg/ml. Cultures were maintained at 20-50 rpm (final speed) on a magnetic stirrer, and DMEM was added up to 100 ml. Fifty milliliters of DMEM were decanted and replaced with fresh DMEM every 2 days. After 1 week, a cell density of 10(6)/ml DMEM was obtained. Phase contrast microscopy showed bridging formation between microcarriers, which suggests tight cell adhesion. Microcarrier cell culture has a variety of advantages which include greater cell production, use of less medium and less risk of contamination compared to the conventional monolayer culture technique, and it also allows passaging without using proteases. Using this culture system, greater possibilities for wider application of new cell cultures can be expected.

Evaluation of radiation therapy for experimental proliferative vitreoretinopathy in rabbits

We evaluated effects of radiation therapy on experimental proliferative vitreoretinopathy (PVR) induced in rabbits by double gas compression of the vitreous followed by homologous dermal-skin fibroblast injection. Electrons were irradiated in two rabbit groups. Group A animals (20 eyes) received 1000 cGy of irradiation immediately after cell injection; group B rabbits (9 eyes), which showed pucker formation 7 days after cell injection, were irradiated on that day at the same dose as was given to group A rabbits. Control animals (14 eyes) were not irradiated. The incidences of traction retinal detachment on day 28 were: control, 86%; group A, 10%; and group B, 22%. There were statistically significant differences between control and group A values and between control and group B values. No significant difference was found between group A and group B. Irradiation of 1000 cGy did not alter the histological picture of experimental PVR. The results showed that radiation suppressed the development of PVR when applied not only immediately after cell injection but also during pucker stages.

The effect of X-ray irradiation on human retinal pigment epithelial cell proliferation.

To obtain a rationale for the use of radiation in the treatment of proliferative vitreoretinopathy (PVR) and to determine the appropriate dose of irradiation, we studied the in vitro growth of cultured human retinal pigment epithelial (RPE) cells after various doses of irradiation. At 48 h after plating, doses of 100–3000 cGy were given to the cells by Linac X-ray. On days 1, 3, and 7 after irradiation, cell growth was evaluated by cell number and DNA synthesis. At 7 days after irradiation, the cell number declined to 42% and 3.7% of control values after doses of 300 and 1000 cGy, respectively. Almost the same inhibitory effects were observed following doses of 1000 cGy and 3000 cGy. DNA synthesis was suppressed to 67.6% and 2.8% of control values on the 7th day after radiation doses of 500 and 1000 cGy, respectively. Low-dose irradiation can be used as adjunctive therapy for PVR, and doses of 500–1000 cGy are most likely needed.