Biomedical subjects

Paul A Constable

Publications and source records attributed to Paul A Constable.

4 recordsLinked to original sources

PubMed2006

Light and alcohol evoked electro-oculograms in cystic fibrosis.

Cystic fibrosis (CF) is caused by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) which is a chloride channel. CFTR is expressed in the retinal pigment epithelium (RPE) where it is believed to be important in generating the fast oscillations (FOs) and potentially contributing to the light-electro-oculogram (EOG). The role of CFTR in the alcohol-EOG is unknown. We recruited six individuals with CF (three homozygotes for Delta508 and three heterozygous for Delta508) and recorded the light- and alcohol-EOGs as well as the FOs and compared them to a control group. The results showed that in the CF group the amplitude of the alcohol- and light-EOGs were normal. However, the time to peak of the light- and alcohol-rises were significantly faster than in the control group. We conclude that CFTR is not primarily responsible for the alcohol- or light-rises but is involved in altering the timing of these responses. The FOs showed differences between the homozygotes, heterozygotes and the controls. The amplitudes were significantly higher and the time to the dark troughs were significantly slower in the heterozygote group compared to both controls and the homozygotes. In contrast, the homozygotes did not differ in either amplitude or the timing of the FOs compared to the controls.

Adaptation, Ocular↗

PubMed2006

P-Glycoprotein expression in human retinal pigment epithelium cell lines.

P-Glycoprotein (P-gp), an active efflux transporter encoded by the MDR1 gene, has recently been identified in the human and pig retinal pigment epithelium (RPE) in situ. Efflux pumps such as P-gp are major barriers to drug delivery in several tissues. We wished to establish whether human RPE cell lines express P-gp under the culture conditions recommended for each cell line so as to determine their suitability as in vitro models for predicting drug transport across the outer blood-retinal barrier. Three human RPE cell lines, ARPE19, D407 and h1RPE were investigated. Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to determine the expression of MDR1 mRNA. Immunocytochemistry using the P-gp-specific antibody C219 was undertaken to investigate the presence of P-gp protein in each cell type. Uptake of rhodamine 123, a P-gp substrate, in the presence or absence of pre-treatment with a P-gp inhibitor, verapamil, was measured in each cell line to determine functional expression of P-gp. For all experiments, MDCK cells stably transfected with the human MDR1 gene (MDCK-MDR1) were used as a positive control. ARPE19 cells were consistently negative for P-gp as assessed by RT-PCR and immunocytochemistry. By contrast, RT-PCR of D407 and h1RPE samples yielded weak bands corresponding to MDR1; P-gp protein expression, as demonstrated by C219 immunoreactivity, was also present. Rhodamine uptake after treatment with verapamil was significantly greater in D407 and MDCK-MDR1, indicating functional expression of P-gp in these two cell lines. No evidence of functional P-gp was found in ARPE19 and h1RPE. In conclusion, D407 and h1RPE cells express P-gp, though functional activity was demonstrable only in D407 cells. ARPE19 cells do not express P-gp. Of these human RPE cells lines D407 could be considered as a suitable model for in vitro drug transport studies, particularly those involving P-gp substrates, without modification of their usual culture conditions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

The electro-oculogram.

The retinal pigment epithelium (RPE) lying distal to the retina regulates the extracellular environment and provides metabolic support to the outer retina. RPE abnormalities are closely associated with retinal death and it has been claimed several of the most important diseases causing blindness are degenerations of the RPE. Therefore, the study of the RPE is important in Ophthalmology. Although visualisation of the RPE is part of clinical investigations, there are a limited number of methods which have been used to investigate RPE function. One of the most important is a study of the current generated by the RPE. In this it is similar to other secretory epithelia. The RPE current is large and varies as retinal activity alters. It is also affected by drugs and disease. The RPE currents can be studied in cell culture, in animal experimentation but also in clinical situations. The object of this review is to summarise this work, to relate it to the molecular membrane mechanisms of the RPE and to possible mechanisms of disease states.

Animals↗

PubMed2004

Compartmental analysis of taurine transport to the outer retina in the bovine eye.

PURPOSE: To assess the relative resistance presented individually by Bruch's membrane-choroid (BC) and the retinal pigment epithelium (RPE) to movement of taurine between the choroidal circulation and the outer retina. To quantify the effect of light-evoked changes in subretinal potassium concentration on the transepithelial transport of taurine across bovine RPE. METHODS: Transport studies were performed in Ussing chambers with intact and RPE-denuded specimens of BC. RPE viability was monitored by recording transepithelial potential (TEP) and transepithelial resistance (TER). Taurine transport with substrate concentrations in the micro- and millimolar range, reflecting physiological taurine concentrations in plasma, retina, and subretinal space was quantified by high-performance liquid chromatography (HPLC) and radiotracer techniques. Taurine transport was also assessed after apical potassium concentration was lowered from 6.0 to 2.2 mM to mimic the effects of light. RESULTS: Transport of taurine across RPE-BC at a 10-mM substrate concentration increased from 32.92 before to 111.72 nanomoles/4 mm per hour after removal of the RPE. Similarly, at 50 microM taurine, transport rates increased from 0.158 to 0.439 nanomoles/4 mm per hour after removal of the RPE. At both high (10 mM) and low (50 microM) substrate concentrations, lowering of apical potassium was associated with decreased transport of taurine across the RPE. For taurine concentrations greater than 42 microM, the rate-limiting compartment for transport of taurine to the outer retina was the RPE monolayer. Similar rates were observed across each compartment for concentrations <42 microM. CONCLUSIONS: The magnitude and directionality of taurine transport across the RPE is determined solely by the driving taurine concentration gradient and is modulated by subretinal levels of potassium. Such modulation may provide a mechanism for conserving retinal taurine. Processes that increase the resistance to diffusion across Bruch's membrane such as human ageing and increased thickening and deposition of debris associated with age-related macular degeneration (AMD) are likely to affect transport across the RPE, culminating in a secondary retinal taurine deficiency.

Animals↗