Connolly E, Nånberg E, Nedergaard J
J Biol Chem. 1986 Nov 5;261(31):14377-85.
To examine the involvement of Na+ ions in adrenergic responses in brown adipose tissue, a method is described for measuring Na+ influx into isolated brown adipocytes, using short (30 s) incubations with 22Na+, followed by a two-step centrifugation recovery procedure. Using this method, a clear norepinephrine-stimulated accumulation of intracellular 22Na+ was observed, which was enhanced by the addition of ouabain, was insensitive to amiloride (a Na+/H+ exchange blocker), and could not be mimicked by the total removal of oxygen from the incubation medium. The norepinephrine-stimulated Na+ influx was dose-dependent for the hormone with an EC50 of 250 nM, was blocked by the beta-antagonist propranolol but not by the alpha 1-antagonist prazosin, and could be induced by adrenergic agonists with the order of potency: isoproterenol greater than norepinephrine greater than phenylephrine, indicating a beta-receptor-mediated process. The Na+ influx was found to be cAMP-dependent since it could be induced by both theophylline (a phosphodiesterase inhibitor) and forskolin (an adenylate cyclase activator), but it was independent of other known cellular cAMP-dependent responses since neither addition of fatty acid substrates (octanoate or palmitate), nor of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone could induce the phenomenon, despite having significant stimulatory effects on cellular respiration. Furthermore, total respiratory inhibition with rotenone, or total oxygen depletion of the medium with dithionite, did not prevent the normal norepinephrine-induced Na+ influx. The possibility that this beta-mediated norepinephrine-stimulated Na+ influx plays an important physiological role in brown adipose tissue activity is discussed, perhaps as one of the, as yet undefined, signals initiating tissue growth in the chronically beta-stimulated tissue of animals facing long-term increases in thermogenic demands.
为研究钠离子在棕色脂肪组织肾上腺素能反应中的作用，本文描述了一种测量钠离子流入分离棕色脂肪细胞的方法，即使用²²Na⁺进行短时间（30秒）孵育，随后通过两步离心回收程序进行测量。使用该方法，观察到去甲肾上腺素明显刺激细胞内²²Na⁺的积累，添加哇巴因可增强该积累，对阿米洛利（一种Na⁺/H⁺交换阻滞剂）不敏感，且不能通过从孵育培养基中完全去除氧气来模拟。去甲肾上腺素刺激的Na⁺流入对该激素呈剂量依赖性，EC50为250 nM，被β拮抗剂普萘洛尔阻断，但不被α1拮抗剂哌唑嗪阻断，并且可由肾上腺素能激动剂诱导，效力顺序为：异丙肾上腺素＞去甲肾上腺素＞苯肾上腺素，表明这是一个β受体介导的过程。发现Na⁺流入依赖于cAMP，因为它可由茶碱（一种磷酸二酯酶抑制剂）和福斯高林（一种腺苷酸环化酶激活剂）诱导，但它独立于其他已知的细胞cAMP依赖性反应，因为添加脂肪酸底物（辛酸或棕榈酸）或线粒体解偶联剂羰基氰化物对三氟甲氧基苯基腙均不能诱导该现象，尽管它们对细胞呼吸有显著刺激作用。此外，用鱼藤酮完全抑制呼吸或用连二亚硫酸钠使培养基完全耗尽氧气，均不能阻止去甲肾上腺素正常诱导的Na⁺流入。本文讨论了这种β介导的去甲肾上腺素刺激的Na⁺流入在棕色脂肪组织活性中发挥重要生理作用的可能性，也许它是在面临长期产热需求增加的动物的慢性β刺激组织中启动组织生长的尚未明确的信号之一。
