STIM and Orai proteins regulate calcium entry into a human sweat gland secretory cell line – NCL-SG3. The production of sweat fluid by eccrine sweat glands in humans, in response to heat or exercise, is crucial in regulating body temperature by evaporative heat loss. A key component in the production and release of sweat is a biphasic increase in the level of intracellular Ca2+ ([Ca2+]i). This increase in [Ca2+]i is brought about by an initial agonist induced release of Ca2+ from internal stores, followed by an influx of Ca2+ through the plasma membrane from the interstitial fluid1. Regulation of Ca2+ entry across the plasma membrane, has been shown, in other exocrine tissues, to be initiated by the emptying of intracellular Ca2+ stores and maintained through a process known as store-operated Ca2+ entry (SOCE) via the interaction of STIM and Orai proteins.2,3 However, to date the mechanisms regulating the Ca2+ entry process into human sweat gland cells is not well understood. The aim of this study was to investigate SOCE in a cell line (NCL-SG3 cells) derived from the secretory portion of human sweat glands. Cells were used for PCR, Western blot analysis and immunofluorescence to identify the presence and localisation of STIM and Orai proteins. The functionality of the SOCE pathway in the cell line was also investigated using calcium-imaging techniques in conjunction with the ER Ca2+-ATPase inhibitor thapsigargin (Tg), and a blocker of SOCE activity 2-APB. The results indicated the presence of STIM 1 and to a lesser extent STIM 2, as well as Orai 1 and 3 mRNA and protein expression. Immunofluorescence studies demonstrated the localisation of STIM1 in the ER and Orai 1 and 3 in the plasma membrane of the cells and that agonist induced stimulation brought about a translocation of the STIM1 molecules to bring them in to close proximity with Orai1 & 3 proteins at the plasma membrane. The FURA-2 calcium imaging experiments demonstrated that an influx of Ca2+ across the plasma membrane, initiated by Tg induced emptying of the ER stores, could be abolished in the presence the 2-APB. The results demonstrate that STIM1 and Orai proteins are present in the sweat gland secretory cell line and that they are key components of the SOCE pathway. This is supported by the finding that 2-APB, a known blocker of SOCE, blocked influx of calcium into these cells. It has been shown that the activation of intracellular enzymes by a sustained increase in [Ca2+]i via SOCE, can exacerbate pathological processes in cells, resulting in further damage4. It is therefore possible that alterations in SOCE activity may underpin hyperhidrosis, a condition whereby sufferers experience excessive fluid secretion from eccrine sweat glands.
Physiology 2015 (Cardiff, UK) (2015) Proc Physiol Soc 34, PC050
Poster Communications: STIM and Orai proteins regulate calcium entry into a human sweat gland cell line – NCL-SG3
D. Bovell1, J. Robertson2, F. Mohamed2
1. Medical Education, Weill Cornell Medical College Qatar, Doha, Qatar. 2. Life Sciences, Glasgow Caledonian University, Glasgow, United Kingdom.
View other abstracts by:
Where applicable, experiments conform with Society ethical requirements.