CA2+ SIGNALLING IN HUMAN BRAIN PERICYTES: INVESTIGATION OF EFFECTS OF AMYLOID BETA TREATMENT

Abstract

Pericytes are contractile cells that regulate blood flow in the microvasculature. In the central nervous system (CNS), they are an important component of the gliovascular unit, a multicomponent cellular structure that is impacted by amyloid pathology. Here we test the effect of a synthetic human amyloid beta 1:42 peptide (hAβ 1–42) on resting Ca2+ concentrations and G-protein coupled receptor (GPCR)-mediated Ca2+ responses in human CNS pericytes. Human CNS pericytes and cell culture media were purchased from sciencellonline. Cells were grown in flasks according to the supplier's protocols. For Ca2+ imaging, cells were plated onto poly-D-lysine coated glass coverslips and pre-incubated for ∼24 hours with culture medium containing hAβ 1–42 (500 nM) or vehicle (DMSO 0.05%). The pericytes were then loaded with the Ca2+-sensitive ratiometric dye fura-2, before being placed on an inverted microscope equipped for single cell microfluorimetry. Imaging was performed in a standard HEPES-buffered saline solution (HBSS). HBSS variants containing three concentrations of glucose, 0.1, 2.5 and 6 mM, were individually examined. Pericytes exposed to hAβ1–42 displayed a lower basal Ca2+ concentration when experiments were performed in 2.5 and 6 mM glucose, although this was not observed in 0.1 mM glucose. We also tested Ca2+ responses to extracellular applications of a number of GPCR agonists relevant to pericyte function. Robust Ca2+ responses to endothelin1, bradykinin and histamine were noted; the latter producing the biggest overall population responses. Quite similar agonist responses were seen in bath solutions containing the different glucose concentrations. Furthermore, hAβ1–42 treatment produced no notable effect on agonist-induced Ca2+ responses. In summary, our initial data indicate that short term exposure of pericytes to hAβ1–42 can lower resting Ca2+ but has no discernible effect on GPCR-evoked pericyte Ca2+ signalling.