Adrenoceptors in brain: Cellular gene expression and effects on astrocytic metabolism and [Ca2+]i

Abstract

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789-2795, renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α 1 -, α 2 -and β-adrenergic receptors and their subtypes was determined in freshlyisolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10-12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α 1A -Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α 1B -receptors were not expressed in any cell population. Among α 2 -receptors only α 2A -receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β 1 -Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β 2 -adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in welldifferentiated primary cultures of mouse astrocytes, showing that stimulation of α 2 -adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca 2+ , whereas α 1 -adrenoceptor stimulation enhances glutamate uptake, and β-adrenoceptor activation causes glycogenolysis and increased Na + ,K + -ATPase activity. The Ca 2+ -and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized.

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

“…For primary astrocytes we used a method based on Hertz. 66 Briefly, brain from P0 to P1 pup of C57/Bl6 mouse was removed by decapitation and was immediately put in ice-cold HBSS (without Ca and Mg). Optical bulb, hind brain and meninges were removed under a stereomicroscope (Zeiss stemi 2000, Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) and both the cortical hemispheres minced and digested for 30 min at 37°C in 0.25% Trypsin (GIBCO, Bridgewater, NJ, USA).…”

The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells

“…Glutamate is the main excitatory neurotransmitter in the mammalian CNS and is therefore strictly regulated to prevent neuronal over-excitability and excitotoxicity (Choi 1988;Greene and Greenamyre 1996;Michaelis 1998). a 1 -AR stimulation has previously been shown to result in increased glutamate uptake into cells (Hertz et al 2010). Thus, an increase in intracellular glutamic acid may originate from glucose conversion into glutamate, via transamination of a-ketoglutarate with branchedamino acids such as alanine, thereby reducing the levels of glucose and alanine whilst increasing intracellular levels of glutamic acid.…”

“…In addition, glial a 1 -AR activation by NA promotes intracellular calcium release with downstream effects on membrane depolarisation, neurotransmitter release and cell proliferation (Legendre et al 1988;Stone et al 1999;Alpini et al 2011). It has also been demonstrated that a 1 -AR stimulation enhances glutamate uptake into cultured astrocytes (Hansson and Ronnback 1989;Fahrig 1993;Hertz et al 2010), and Ohashi et al (2007) showed that stimulation of a 1 -AR by phenylephrine suppressed stress-induced death and promoted cell survival in embryonic neural progenitor cells. In experiments using cultured keratinocytes Li et al (2013) showed that exposure to NA led to increased expression of multiple adrenergic receptor subtypes on these cells as shown by real-time PCR.…”

“…Three different isoforms of the a 1 -adrenergic receptor (a 1 -AR) have been identified using molecular cloning techniques (a 1A -AR, a 1B -AR, and a 1D -AR) (Michel et al 1989;Schwinn et al 1990;Pieribone et al 1994;Chang et al 1998;Xiao et al 1998). The distribution of subtypes has been described both in humans and a variety of experimental animals, with subtype-specific differences in various brain regions (Pieribone et al 1994;Day et al 1997;Suzuki et al 1997;Schambra et al 2005;Khan et al 2007;Hertz et al 2010). Neurons and microglia of the CNS have both been shown to express a 1 -AR (Feldstein et al 1986;Day et al 1997;Papay et al 2006;Hertz et al 2010).…”

“…The distribution of subtypes has been described both in humans and a variety of experimental animals, with subtype-specific differences in various brain regions (Pieribone et al 1994;Day et al 1997;Suzuki et al 1997;Schambra et al 2005;Khan et al 2007;Hertz et al 2010). Neurons and microglia of the CNS have both been shown to express a 1 -AR (Feldstein et al 1986;Day et al 1997;Papay et al 2006;Hertz et al 2010).…”