Kittl Michael, Jakab Martin, Steininger Tanja S, Ritter Markus, Kerschbaum Hubert H
Department of Biosciences, University of Salzburg, Salzburg, Austria.
Institute of Physiology and Pathophysiology, Paracelsus Medical University, Salzburg, Austria.
Cell Physiol Biochem. 2019;52(5):951-969. doi: 10.33594/000000066.
BACKGROUND/AIMS: Volume-regulated anion channels (VRACs) are of particular importance in regulating the cell volume (CV) and give rise to the swelling-activated Cl- current (I), a main component driving global regulatory volume decrease (RVD) during cell swelling. Because I affects numerous CV-regulated processes like migration, we assume that its role is also indispensable for phagocytosis which requires local cell swelling. Noradrenaline (NA) modulates phagocytosis in macrophages and microglial cells, macrophage-related cells in the central nervous system. Therefore we evaluated whether NA modulates I and phagocytosis in microglia.
Experiments were performed in murine microglial BV-2 and primary mouse microglial cells. Patch clamp experiments were performed in BV-2 cells using the amphotericin-perforated method to minimize cytosolic disturbances. Phagocytosis was quantified by scanning electron microscopy.
Following activation of I by a hypotonic bath solution, noradrenaline, as well as the β-adrenergic agonist isoproterenol, evoked a transient decrease of I. Repeated application of adrenergic agonists caused a decline of this electrical response. Application of the agonist of exchange protein directly activated by cAMP (Epac), 8-pCPT-2-O-Me-cAMP, or the protein kinase A inhibitor H89 caused a persistent suppression of I. When isoproterenol was added concomitantly with the hypotonic saline, I developed more rapidly compared to control conditions. Uptake of IgG-coated beads was suppressed by NA or H89 when quantified after 15 min of exposure.
The activation of β-adrenergic receptors in microglial cells triggers a cAMP-Epac-dependent and a cAMP-PKA-dependent cascade which affects phagocytosis via modulation of the swelling-activated Cl- current I.
背景/目的：容积调节性阴离子通道（VRACs）在调节细胞容积（CV）方面尤为重要，并产生肿胀激活的Cl-电流（I），这是细胞肿胀期间驱动整体调节性容积减小（RVD）的主要成分。由于I影响众多如迁移等受CV调节的过程，我们推测其作用对于需要局部细胞肿胀的吞噬作用也是不可或缺的。去甲肾上腺素（NA）可调节巨噬细胞和小胶质细胞（中枢神经系统中与巨噬细胞相关的细胞）的吞噬作用。因此，我们评估了NA是否调节小胶质细胞中的I和吞噬作用。
实验在小鼠小胶质细胞BV-2和原代小鼠小胶质细胞中进行。使用两性霉素穿孔法在BV-2细胞中进行膜片钳实验，以尽量减少胞质干扰。通过扫描电子显微镜对吞噬作用进行定量。
用低渗浴液激活I后，去甲肾上腺素以及β-肾上腺素能激动剂异丙肾上腺素引起I的短暂下降。重复应用肾上腺素能激动剂导致这种电反应下降。应用cAMP直接激活的交换蛋白（Epac）激动剂8-pCPT-2-O-Me-cAMP或蛋白激酶A抑制剂H89导致I持续受到抑制。当异丙肾上腺素与低渗盐水同时添加时，与对照条件相比，I发展得更快。暴露15分钟后进行定量时，NA或H89抑制了IgG包被珠的摄取。
小胶质细胞中β-肾上腺素能受体的激活触发了cAMP-Epac依赖性和cAMP-PKA依赖性级联反应，该反应通过调节肿胀激活的Cl-电流I来影响吞噬作用。
