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Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP₂ depletion.

ABSTRACT: Brain capillaries play a critical role in sensing neural activity and translating it into dynamic changes in cerebral blood flow to serve the metabolic needs of the brain. The molecular cornerstone of this mechanism is the capillary endothelial cell inward rectifier K⁺ (Kir2.1) channel, which is activated by neuronal activity-dependent increases in external K⁺ concentration, producing a propagating hyperpolarizing electrical signal that dilates upstream arterioles. Here, we identify a key regulator of this process, demonstrating that phosphatidylinositol 4,5-bisphosphate (PIP₂) is an intrinsic modulator of capillary Kir2.1-mediated signaling. We further show that PIP₂ depletion through activation of G_q protein-coupled receptors (G_qPCRs) cripples capillary-to-arteriole signal transduction in vitro and in vivo, highlighting the potential regulatory linkage between G_qPCR-dependent and electrical neurovascular-coupling mechanisms. These results collectively show that PIP₂ sets the gain of capillary-initiated electrical signaling by modulating Kir2.1 channels. Endothelial PIP₂ levels would therefore shape the extent of retrograde signaling and modulate cerebral blood flow.

SUBMITTER: Harraz OF

PROVIDER: S-EPMC5899484 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP2 depletion.

Harraz Osama F OF Longden Thomas A TA Dabertrand Fabrice F Hill-Eubanks David D Nelson Mark T MT

Proceedings of the National Academy of Sciences of the United States of America 20180326 15

Brain capillaries play a critical role in sensing neural activity and translating it into dynamic changes in cerebral blood flow to serve the metabolic needs of the brain. The molecular cornerstone of this mechanism is the capillary endothelial cell inward rectifier K+ (Kir2.1) channel, which is activated by neuronal activity-dependent increases in external K+ concentration, producing a propagating hyperpolarizing electrical signal that dilates upstream arterioles. Here, we ...[more]

PMID: 29581272

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Project description:BackgroundPrimary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity.MethodsPrimary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation.ResultsThe cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer.ConclusionsThe data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

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Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP₂ depletion.

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Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP<sub>2</sub> depletion.

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Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP2 depletion.

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Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP&lt;sub&gt;2&lt;/sub&gt; depletion.

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OmicsDI is part of the ELIXIR infrastructure

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Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP₂ depletion.

Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP<sub>2</sub> depletion.