M. A. Del Monte

Molecular identity and calmodulin-mediated regulation of the taurine transporter in a human retinal pigment epithelial cell line

The molecular identity and calmodulin-mediated regulation of the taurine transporter were investigated in a human retinal pigment epithelial cell line (HRPE). Reverse transcription-polymerase chain reaction amplification of HRPE cell mRNA using primer specific for a taurine transporter cloned from human placenta yielded a product of expected size (approximately 0.9 kb) which hybridized to the placental cDNA probe under high stringency conditions. The nucleotide sequence of the product was identical to the sequence of the portion of the placental taurine transporter cDNA flanked by the specific primers. The taurine transporter expressed in the HRPE cell line thus appears to be identical to the transporter cloned from the placenta. Treatment of the HRPE cells with a selective calmodulin antagonist CGS 9343 B (CGS) led to a marked decrease in taurine transport activity. This effect could be reproduced with W-7, another calmodulin antagonist. The inhibition caused by CGS occurred rapidly (t1/2 approximately 10 min). Treatment of the cells with CGS did not affect the transport of leucine, and amino acid not recognized by the taurine transporter as a substrate. The CGS-induced inhibition of taurine transport was accompanied by a decrease in the maximal velocity of the transporter with no detectable change in the substrate affinity. The steady state levels of the transporter mRNA however remained unaffected by CGS treatment. It is concluded that the HRPE cell line expressed a taurine transporter identical to the transporter describe in the human placenta and that the function of this transporter is regulated by calmodulin-dependent processes.

Properties of taurine transport in a human retinal pigment epithelial cell line

The characteristics of taurine transport were studied in a human retinal pigment epithelial cell line (HRPE). Uptake of taurine into monolayer cultures of the HRPE cells was markedly stimulated by the presence of NaCl in the uptake medium whereas the uptake was negligible in its absence. This NaCl-dependent uptake was an active process as the cells were able to accumulate taurine against a concentration gradient. The uptake rate of taurine was found to be many-fold greater than that of gamma-aminobutyric acid (GABA). Unlabeled taurine and GABA competed with radiolabeled taurine for the uptake process, the former being more effective than the latter. However, uptake of radiolabeled GABA was not affected by unlabeled taurine and GABA. Substrate specificity studies revealed strong interaction of beta-amino acids with the transport system responsible for taurine uptake. alpha-Amino acids failed to inhibit taurine uptake. A specific anion requirement was observed for optimal activity of the taurine transport system and Cl- was the most supportive among several anions tested. Kinetic analyses showed that multiple Na+ and one Cl- were involved in transfer of one taurine molecule. The transport process consisted of a single saturable system with a Michaelis-Menten constant of 2.0 +/- 0.1 microM. These results show that the HRPE cell line expresses a high-affinity taurine transport system. This is the first demonstration of the presence of the taurine transporter in the human retinal pigment epithelium and the HRPE cell line may provide a useful model system for future studies involving taurine transport in the retinal pigment epithelium.

Cyclic AMP-dependent up-regulation of the taurine transporter in a human retinal pigment epithelial cell line

This investigation was undertaken to study the role of cAMP in the regulation of the taurine transporter expressed in a human retinal pigment epithelial (HRPE) cell line. Treatment of the HRPE cells with cholera toxin for 24 h was found to stimulate the taurine transporter activity, as measured by taurine transport into the cells in the presence of NaCl, to a significant extent. The stimulation was 50-60% at 100 ng/ml cholera toxin. This stimulation was specific to the taurine transporter since the transport of two other amino acids (leucine and alanine), which are not substrates for the taurine transporter, was not affected by cholera toxin under similar conditions. Exposure of the cells to cholera toxin for a time period > 4 h was needed to elicit the stimulatory effect. The cholera toxin-induced stimulation of the taurine transporter activity was associated with an increase in the maximal velocity of the transport system. The affinity of the transporter for taurine was not altered by the treatment. The stimulatory effect was markedly blunted when the treatment of the cells with cholera toxin was done in the presence of actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of translation. The increase in the taurine transporter activity induced by cholera toxin was associated with a 2.6-fold increase in the steady state levels of the transporter mRNA. Measurement of cyclic nucleotides in control and cholera toxin-treated cells revealed that the toxin caused a 20-fold increase in the cellular levels of cAMP, the levels of cGMP remaining unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular matrix mediated growth and differentiation in human pigment epithelial cell line 0041

Efforts to grow differentiated pigment epithelial cells have led to a characterization of the growth kinetics of spontaneously established, continuously growing, human retinal pigment epithelial (PE) cell line 0041 on several biomatrices. These substrates were prepared from (a) placental and amniotic membrane, (b) commercially available basement membrane matrix (Matrigel), (c) dishes coated with extracellular matrix secreted by endothelial cells (ECM), (d) dishes coated with collagen IV and/or laminin, (e) dishes coated with collagen I and/or fibronectin. Our findings suggest that tissue culture plastic and dishes coated with collagen IV alone promote higher cell densities, while highest plating efficiency (24 hrs) was seen on tissue culture plastic and Matrigel. The highest degree of differentiation (epithelioid appearance, apical villi and junctional complexes) was seen in cells plated on dishes coated with collagen IV and extracellular matrix secreted by endothelial cells. Cells were epithelioid and polarized on those two substrates; they expressed fine finger-shaped villi and the highest degree of cell contact (in the form of junctions). Cells grown on Matrigel looked like fibroblasts and became deeply pigmented; however, the nature of the pigment remains to be determined. Collagen IV and ECM coated dishes, therefore, are most suitable for cultures of human PE cell line 0041 because they provide higher cell densities while retaining the differentiated state. This is the first report where an established pigmented epithelial cell line has been induced to become differentiated by use of extracellular matrices and extracellular matrix components.

Elevated glucose activates the P38 MAP kinase pathway in cultured human retinal pigment epithelial cells

Purpose: Proliferative diabetic retinopathy (PDR) is a serious complication of diabetes that can result in blindness. Elevated glucose, as seen in diabetes mellitus, activates the p38 mitogen-activated protein kinase (P38) pathway in various kinds of cells. Since retinal pigment epithelial cells (hRPE) have been implicated in the pathogenesis of PDR, we examined the production of P38 in cultured hRPE cells under high glucose conditions. Methods: hRPE cells were isolated and cultured from three human eyes. The hRPE were exposed to fetal bovine serum (FBS) (0-10%) and FBS (10%) supplemented with 20mM glucose for 48 hours with or without SB203580, an inhibitor of P38. Some experimental hRPE cells were labeled with 14C-methionine to assess P38 production by immunoprecipitation of 14C-methionine p38 (14C-P38) with anti-P38. hRPE viability and proliferation were evaluated by trypan blue exclusion method (T) and 3H-thymidine (3H-thy) incorporation respectively. Light microscopy was done to assess cell phenotype. Data were analyzed by student t-test. p < 0.05 was considered as significant difference. results: hRPE cells studied in FBS (0-10%) with or without high (20mM) glucose showed similar epitheloid phenotype and remained viable as determined by T. Increasing concentrations of FBS stimulated hRPE proliferation in a dose dependent manner as determined by 3H-thy. Increasing concentration of FBS also stimulated 14C-P38 production in a dose dependent manner. FBS (10%)+ high glucose (20 mM) stimulated significantly greater 14C-P38 production compared to FBS (10%) alone (2106.40±107.12 vs. 1127.00±400.43, CPM±SEM, n=4, p=0.01). FBS (10%)+glucose (20 mM)+SB203580 (20μM) stimulated 14C-P38 even greater production compared to FBS (10%)+glucose (20 mM) alone (2654.69±333.88 vs. 2198.36±226.23, CPM±SEM, n=4, p=0.02). In addition, exposure of hRPE cells to high glucose stimulated increased proliferation, as measured by 3H-thy incorporation, in parallel with the increased 14C-P38 production. conclusion: Exposure of hRPE cells to elevated glucose stimulates increased proliferation as well as increased P38 production. This activation of the P38 MAP Kinase pathway may play a role in the pathogenesis of PDR.