Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota 58108-6050, USA.
J Pharmacol Exp Ther. 2011 Feb;336(2):372-80. doi: 10.1124/jpet.110.174102. Epub 2010 Nov 3.
Microinjection of apelin-13 into the rostral ventrolateral medulla (RVLM) in the brainstem increases blood pressure in rats. In the present study, we tested the hypotheses that apelin-13 directly stimulates neuronal activity in neurons cultured from the brainstem and that NAD(P)H oxidase-derived reactive oxygen species are involved in this action of apelin-13. Microinjection of apelin-13 into the RVLM resulted in increases in arterial pressure and in renal sympathetic nerve activity in Sprague-Dawley rats. The pressor effect of apelin-13 was attenuated by the specific NAD(P)H-oxidase inhibitor gp91ds-tat. In neurons cultured from the ventral brainstem, spontaneous action potentials were recorded using current-clamp recording. Superfusion of neurons with apelin-13 (100 nM) increased the neuronal firing rate from 0.79 ± 0.14 to 1.45 ± 0.26 Hz (n = 7, P < 0.01) in angiotensin II receptor-like 1-positive neurons, identified with single-cell reverse transcriptase-polymerase chain reaction. Neither the angiotensin II type 1 receptor antagonist losartan nor the angiotensin II type 2 receptor antagonist 1-[[4-(dimethylamino)-3-methylphenyl[methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate (PD123319) altered the positive chronotropic effect of apelin-13. Pretreatment of cells with either the reactive oxygen species scavenger superoxide dismutase [polyethylene glycol-superoxide dismutase (PEG-SOD), 25 U/ml] or with gp91ds-tat significantly attenuated the chronotropic action of apelin-13. PEG-SOD and gp91ds-tat alone had no effect on basal neuronal firing. In addition, apelin-13 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in neuronal cultures. The superoxide generator xanthine-xanthine oxidase also increased neuronal activity in neurons, mimicking the neuronal response to apelin-13. These observations provide the first evidence that apelin-13 directly increases neuronal activity via stimulation of NAD(P)H oxidase-derived superoxide, a cellular signaling mechanism that may be involved in the pressor effect of apelin-13 in the RVLM.
将阿皮林-13 微注射到脑干中的延髓头侧腹外侧区（RVLM）会增加大鼠的血压。在本研究中，我们测试了以下假设：阿皮林-13 直接刺激源自脑干的神经元中的神经元活性，并且 NAD（P）H 氧化酶衍生的活性氧参与了阿皮林-13 的这种作用。将阿皮林-13 微注射到 RVLM 中会导致 Sprague-Dawley 大鼠的动脉压和肾交感神经活性升高。特异性 NAD（P）H 氧化酶抑制剂 gp91ds-tat 可减轻阿皮林-13 的升压作用。在源自腹侧脑干的神经元中，使用电流钳记录记录自发动作电位。用阿皮林-13（100 nM）灌流神经元会使血管紧张素 II 受体样 1 阳性神经元的神经元放电率从 0.79 ± 0.14 增加到 1.45 ± 0.26 Hz（n = 7，P < 0.01），用单细胞逆转录-聚合酶链反应鉴定。血管紧张素 II 受体 1 型拮抗剂洛沙坦（losartan）或血管紧张素 II 受体 2 型拮抗剂 1-[[4-（二甲基氨基）-3-甲基苯基[甲基]-5-（二苯乙酰基）-4,5,6,7-四氢-1H-咪唑并[4,5-c]吡啶-6-羧酸二三氟乙酸酯（PD123319）均未改变阿皮林-13 的阳性变时作用。用活性氧清除剂超氧化物歧化酶[聚乙二醇-超氧化物歧化酶（PEG-SOD），25 U/ml]或 gp91ds-tat 预处理细胞可显著减弱阿皮林-13 的变时作用。PEG-SOD 和 gp91ds-tat 本身对基础神经元放电没有影响。此外，阿皮林-13 可显著增加神经元培养物中的 NAD（P）H 氧化酶活性和细胞内超氧阴离子水平。超氧化物产生剂黄嘌呤-黄嘌呤氧化酶也可增加神经元中的神经元活性，模拟阿皮林-13 对神经元的反应。这些观察结果首次提供了证据，证明阿皮林-13 通过刺激 NAD（P）H 氧化酶衍生的超氧阴离子直接增加神经元活性，这是一种细胞信号机制，可能参与了 RVLM 中阿皮林-13 的升压作用。
