ATP induces contraction of cultured brain capillary pericytes via activation of P2Y-type purinergic receptors

Abstract

Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. To investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium chelation with BAPTA, indicating that the contraction was mediated via purinergic P2-type receptor-mediated [Ca²⁺]_i signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated the presence of P2Y₁ and P2Y₂ receptors, using ATP, UTP, ADP, and the subtype specific agonists MRS2365 (P2Y₁) and 2-thio-UTP (P2Y₂). Addition of specific P2X agonists only caused an [Ca²⁺]_i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y-type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding of the role of pericytes in vessel constriction and points toward P2Y receptors as potential therapeutic targets.NEW & NOTEWORTHY The study concerns brain capillary pericytes, which have been suggested to play a role in the regulation of cerebral blood flow. We show that extracellular ATP causes contraction of primary brain pericytes by stimulation of purinergic receptors and subsequent release of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The contraction is mainly mediated through activation of P2Y-receptor subtypes, including P2Y₁ and P2Y₂. These findings add more mechanistic understanding of the role of pericytes in regulation of capillary blood flow. ATP was earlier suggested to be involved in capillary constriction in brain pathologies, and our study gives a detailed account of a part of this important mechanism.

Keywords: BAPTA-AM; P2Y receptor; brain pericyte; capillary blood flow; ischemia; neurovascular unit.