open access

https://doi.org/10.14943/doctoral.k14543

https://hdl.handle.net/2115/81964

Adrenergic regulation of the process formation by astrocytes

日本語

2021-03-25

Hokkaido University

58p

Astrocytes are the most abundant glial cells in the central nervous system (CNS). Astrocytes have numerous fine processes through which they contact blood vessels and neurons to regulate the functions of blood-brain barrier and synapse, respectively. These processes have plasticity depending on neural activity and change dynamically under physiological and pathological conditions, which is closely related to the CNS functions. The mechanisms regulating astrocyte morphology are of great interest because of the relationship between their morphological diversity and the CNS functions. Noradrenaline is a major neurotransmitter in the CNS, and astrocytes express all type of adrenoceptors (ARs), i.e., α1-, α2- and β-ARs. Although all receptors are activated by noradrenaline, each receptor generally activates distinct intracellular signal. For example, β-ARs activate adenylate cyclase/cyclic AMP (cAMP) signal, while α2-ARs inhibit it. It has been reported that the activation of β-ARs induces the process formation of cultured astrocytes via cAMP signal and expansion of astrocyte processes in situ, and contributes to astrocyte hypertrophy under pathological conditions. On the other hand, the role of α-ARs in the morphology of astrocyte processes has not been elucidated. Here, we employed pharmacological techniques to examine the role of α-ARs in the process formation by cultured astrocytes isolated from spinal cord and cerebral cortex of neonatal rats. Process formation of astrocytes was evaluated by phalloidin staining for actin-cytoskeleton with a fluorescence microscopy. Both cultured astrocytes were positive for glial fibrillary acidic protein (GFAP) (expression level: cortical astrocytes > spinal cord astrocytes) and exhibited polygonal shape without any processes. In both cultured astrocytes, application of noradrenaline (NA; 1 µM) and the β-agonist isoproterenol (ISO; 1 µM) for 3 h transformed polygonal astrocytes into process-bearing ones with GFAP-positive major processes and actin-rich fine processes like astrocytes in vivo. The percentages of cells exhibiting process formation induced by NA (1 µM) and ISO (1 µM) peaked at 1–3 h after treatment. NA (1 µM) and ISO (1 µM) also increased the intracellular cAMP concentrations, which peaked within 30 min after treatment. In general, the effects of NA and ISO were more potent in cortical astrocytes than in spinal cord astrocytes. In addition, the effect of ISO was more potent than that of NA, particularly in spinal cord astrocytes. Forskolin, an adenylate cyclase activator, and dibutyryl-cAMP, a cell-permeable cAMP analog, induced process formation in a concentration-dependent manner, which suggests that the increase in intracellular cAMP levels is related to the process formation induced by NA and ISO. Next, the effects of AR-antagonists on NA (1 µM)-induced process formation and cAMP elevation were examined in spinal cord astrocytes. The β-antagonist propranolol (PROP; 10 µM) abolished NA-induced process formation and cAMP elevation. By contrast, the α1-antagonist prazosin (PRAZ; 1 or 10 µM) or the α2-antagonist atipamezole (ATIP; 10 µM) enhanced the NA-induced process formation, and ATIP enhanced NA-induced cAMP elevation. In cortical astrocytes, PROP (10 µM) abolished NA-induced process formation, whereas PRAZ (1 µM) and ATIP (10 µM) had no effect on it. The effects of α-agonists on ISO (1 µM)-induced process formation and cAMP elevation were examined in spinal cord astrocytes. The α2-agonist dexmedetomidine (DEX; 1 µM), but not α1-agonist phenylephrine (1 µM), inhibited ISO-induced process formation and cAMP elevation. DEX (1 µM) also inhibited ISO-induced process formation in cortical astrocytes. These results suggest that activation of β-ARs induces process formation via adenylate cyclase/cAMP signal, which is negatively regulated by α-ARs, especially α2-ARs. A reduction of intracellular cAMP levels contributes to the α2-AR-mediated inhibition of process formation. To ascertain if a mechanism(s) other than intracellular cAMP reduction is involved in α2-AR-mediated inhibition of process formation, the effects of DEX on process formation induced by dibutyryl-cAMP, adenosine and a Rho-associated kinase (ROCK) inhibitor, Y27632, were examined. In both cultured astrocytes, process formation induced by dibutyryl-cAMP (0.1 or 0.3 mM) was inhibited by DEX (1 µM) but not in the presence of ATIP (10 µM). Adenosine (30 µM) and Y27632 (10 µM) induced process formation almost without affecting the intracellular cAMP levels. DEX inhibited these cAMP-independent process formations but not in the presence of ATIP. Activation of α-ARs by NA also inhibited Y27632-induced process formation. In conclusion, in contrast to β-ARs, α-ARs are responsible for inhibiting the formation of processes by cultured astrocytes. Especially, α2-ARs inhibited process formation via inhibition of adenylate cyclase/cAMP signal and cAMP-independent mechanism(s). NA bidirectionally affects astrocyte processes via α- and β-ARs. Noradrenergic regulation of astrocyte morphology likely depends on the balance of functional expression of ARs, which may be related to the CNS functions under physiological and pathological conditions.

2021-03-25

甲第14543号

博士(獣医学)

(主査) 教授 昆 泰寛, 教授 乙黒 兼一, 准教授 岡松 優子, 助教 長谷部 理絵（遺伝子病制御研究所）

獣医学院（獣医学専攻）

博士論文

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