Rises in intracellular calcium in primary brain pericytes are caused by ATP
Sofie Hørlyck1 , Hans Christian Helms1 , Sanne Barsballe Jessen2 , Martin Lauritzen2 , Helle Waage-Petersen3 , Birger Brodin1 1
Department of Pharmarcy, København Ø, Denmark 2 Department of Neuroscience and Pharmacology, København Ø, Denmark 3 Department of Drug Design and Pharmacology, København Ø, Denmark
The blood-brain barrier (BBB) separates the brain from the vascular system and issited at the capillaries. The neurovascular unit is composed of endothelial cells, pericytes and astrocytes and cross-talk between these cell types are essential for induction and maintenance of the BBB. Purinergic signalling has earlier been shown to play an important role in maintaining the permeability at the BBB, both in health and disease. The aim of the study is to investigate purinergicsignalling in brain pericytes, and later on, to evaluate the effects of signalling on brain endothelium properties.
Primary pericytes were isolated from bovine brain capillaries and expression ofspecificmarkers were investigated using qRT-PCR and confocal microscopy. Pericytes were loaded with Fura-2 indicator dye in a NovoStar plate reader and intracellular calcium-responses of purinergic agonists and antagonists were measured.
Primary bovine pericytes were cultured for 8 days. They expressedα-SMA and PDGFR-β, and no astrocyte nor endothelial markers. When adding ATP, the pericytes responded in a concentration-dependent manner and this finding contributes to the hypothesis that pericytes might respond to ATP- release from astrocytes; a phenomenon observed in pathological conditions. Adding PPADS, a purinergic antagonist, inhibited the intracellular Ca2+- responses observed when adding ATP and thereby, confirming that the response isinduced by purinergic receptors.
We have established and validated a culture protocol for brain pericytes. ATP signalling via purinergic signalling has been shown. Next steps will be to investigate whether the activated signalling pathways of pericytes are involved in changes in brain endothelium properties.