Abstract
In humans, thermoregulatory sweating is produced by eccrine sweat glands in the dermis of the skin. In the mechanism of sweat glands secretion, calcium has been identified as a key ‘second messenger’ in intracellular signalling pathways. The binding of agonists to G-Protein-coupled receptors on cells increases the concentration of intracellular calcium, by activating an IP3 pathway, which causes biphasic increases in intracellular Ca2+ involving the release of Ca2+ ions from intracellular stores, followed by Ca2+ entry across the plasma membrane. This influx of calcium from extracellular sources in other cells has been described as store-operated Ca2+ entry (SOCE). The mechanisms regulating the Ca2+ entry process are not well understood, however recent studies identified two main components of SOCE: the endoplasmic reticulum (ER)-localized Ca2+ sensor proteins, STIMs, and the plasma membrane (PM)-localized Ca2+ channel, ORAIs in addition to TRPC1. The aim of the study was to determine the molecular identity, and characterise functionally proteins and understand their roles associated with calcium entry that initiate fluid secretion in human sweat gland secretory cells.A secretory epithelial cell line (NCL-SG3) derived from the secretory portion of human eccrine sweat glands was used for experimentation. Cells were cultured using standard tissue culture techniques. Cells were examined using immunofluorescence and cell lysates were used for Western blotting using antibodies against STIMs ORAIs and TRPC1; mRNA was extracted and used for PCR experiments. ATP lOOuM, Thapsigargin (TG) 3uM and blockers of the calcium entry channels were superfused onto cells then washed out. The cells were transfected with pre-designed short interfering RNA (siRNA) sequence then collected after 48 hrs for further experiments. Any changes in intracellular calcium [Ca2+]j were recorded using calcium imaging techniques. Statistical analyses were carried out using t, test and one-way ANOVA.
PCR showed the presence of STIMs, ORAIs and TRPC1 mRNA in human NCLSG3 cells. Both immunofluorescence and western blotting demonstrated the presence of these proteins in NCL-SG3 cells, and their interaction following treatment with TG. The percentage change in [Ca2+]i was measured following stimulation with TG, and was significantly reduced in the presence of the blockers. The results of siRNA experiments showed that knockdown of STIM1, ORAI1, ORAI3 and TRPC1, reduced [Ca2+]i in response to 3pM TG. These results suggest that these proteins could be mediated store-operated Ca2+ entry (SOCE) in human sweat gland cells.
| Date of Award | 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Douglas Bovell (Supervisor) |