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Mechanisms of volume regulation in murine choroid plexus epithelial cells
[Thesis]. Manchester, UK: The University of Manchester; 2010.
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Abstract
The choroid plexuses are largely responsible for cerebrospinal fluid (CSF) secretion and therefore play a fundamental role in brain homeostasis. The membrane proteins involved in CSF secretion are not fully known. Several electroneutral transporters have been identified by molecular methods in choroid plexus epithelial cells but there is a lack of functional data to support their expression making it impossible to elucidate their role in CSF secretion fully. The activity of many of these transporters can be observed in cell volume regulation. Thus, the main aim of the present study was to determine the ability of mammalian choroid plexus epithelial cells to regulate their volume in response to anisosmotic challenge and to investigate the transporters involved.Experiments were performed on cells isolated from the mouse fourth ventricle choroid plexus. Cells were isolated using a combination of manual perturbation, the enzyme dispase and a Ca2+ free incubation to disrupt tight junctions. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71 pL.Cells exhibited a HCO3- dependent regulatory volume increase (RVI) in response to hypertonic challenge. Strong evidence is presented that the Na+/H+ exchanger (NHE1) and the Cl-/HCO3- exchanger (AE2) contribute to the RVI but the Na+K+2Cl- cotransporter (NKCC1) and the epithelial Na+ channel (ENaC) do not. Choroid plexus cells exhibit a HCO3- dependent regulatory volume decrease (RVD) in response to hypotonic challenge. The RVD was unaffected by DIOA (an inhibitor of KCC activity), the K+ channel inhibitors TEA+, Ba2+ or 4AP or the Cl- channel inhibitors DIDS or NPPB. However removal of extracellular Ca2+ completely abolished cell swelling in response to hypotonic challenge. This sensitivity of volume change to Ca2+ was specific to cell swelling as cell shrinkage in hypertonic artificial CSF was unaffected by removal of extracellular Ca2+.Thus functional evidence is presented to further elucidate the role of several proteins in the choroid plexus cell volume regulatory response to anisosmotic challenge.
Layman's Abstract
The brain is surrounded by salty liquid which provides physical and chemical support and protection to the brain. This liquid, called cerebrospinal fluid, also fills ventricles within the brain where it is mainly produced by the choroid plexus tissues. The choroid plexus cells form a tight barrier which prevents cerebrospinal fluid and blood from mixing and thereby disrupting brain function. The components of cerebrospinal fluid are moved across this barrier by several proteins located in the cell membranes of the choroid plexus cells. To investigate the interactions between mouse choroid plexus cells and their surrounding environment, visible changes in cell volume were measured when cells were exposed to different bathing solutions. Choroid plexus cells are able to regulate their volume. When induced to shrink they activate mechanisms to re-swell to their approximate normal volume, this process is known as a regulatory volume increase. Conversely, a regulatory volume decrease occurs when choroid plexus cells are induced to swell and need to correct their volume back towards normal. The identity of several proteins involved in a regulatory volume increase was determined using drugs which inhibit volume regulation. A surprising observation is that calcium must be present for choroid plexus cells to swell but is not necessary for the cells to shrink. This is surprising since previous studies on other cell types have shown that the regulatory volume decrease requires calcium, but have not commented on the calcium dependency of the cell swelling. The present study contributes new knowledge to the growing understanding of the mechanisms which help to keep the brain healthy. Improved knowledge of the proteins involved will enable the development of drugs with fewer side effects and may help in the development of therapies for illnesses as varied as meningitis, Alzheimer’s disease and hydrocephalus.