Martin Sarah R, Emanuel Krzysztof, Sears Claire E, Zhang Yin-Hua, Casadei Barbara
Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.
Cardiovasc Res. 2006 Apr 1;70(1):97-106. doi: 10.1016/j.cardiores.2006.02.002. Epub 2006 Mar 20.
The role of constitutive nitric oxide (NO) production in the regulation of beta-adrenergic and muscarinic responses remains controversial. Conflicting data in left ventricular (LV) myocytes from eNOS knockout mice (eNOS-/-) have been ascribed to inconsistent experimental conditions (i.e., differences in the choice of controls, age of the mice, myocytes' stimulation frequency, and in the level of beta-adrenergic stimulation); however, the recent identification of a neuronal-like NO synthase (nNOS) in the LV myocardium has raised the possibility that this isoform may be involved in the modulation of beta-adrenergic and muscarinic responses.
To address these issues we recorded sarcomere shortening at 35 degrees C under basal conditions, in the presence of isoproterenol (ISO, 10-100 nmol/L) and of ISO plus carbamylcholine (CCh, 1 micromol/L) in LV myocytes isolated from eNOS-/- and nNOS-/- mice, their wild type littermates (eNOS+/+ and nNOS+/+) or C57BL/6J mice. eNOS-/- and control myocytes were studied at 1 and 3 Hz, in the presence of 10 and 100 nmol/L ISO, and responses were compared between young (3 months) and old (> or =12 months) mice.
Contraction did not differ between young eNOS-/- and eNOS+/+ mice at all stages of the experimental protocol, either at 1 or 3 Hz or in response to 10 or 100 nmol/L ISO. However, myocytes from old eNOS-/- mice showed a reduced inotropic response to ISO compared with age-matched eNOS+/+ mice (P = 0.02). Similarly, there was a significant difference in the ISO response between eNOS+/+ and C57BL/6J myocytes (P < 0.01), suggesting that experimental variables such as age and the choice of control animals may have contributed to the inconsistency in the results reported in the literature. In contrast, nNOS-/- myocytes showed greater contraction and slower relaxation at all stages of the experimental protocol (P = 0.0003 and P = 0.01 vs. nNOS+/+ myocytes).
Constitutive eNOS expression in murine LV myocytes is not essential for the muscarinic-mediated inhibition of beta-adrenergic signalling and does not appear to play a significant role in the regulation of basal and beta-adrenergic myocardial contraction. Our data suggest that nNOS is the myocardial constitutive isoform responsible for the NO-mediated autocrine regulation of myocardial inotropy and relaxation.
内源性一氧化氮（NO）生成在β-肾上腺素能和毒蕈碱能反应调节中的作用仍存在争议。来自内皮型一氧化氮合酶基因敲除小鼠（eNOS-/-）左心室（LV）心肌细胞的相互矛盾的数据归因于实验条件不一致（即对照选择、小鼠年龄、心肌细胞刺激频率以及β-肾上腺素能刺激水平的差异）；然而，最近在LV心肌中发现了一种神经元样一氧化氮合酶（nNOS），这增加了这种同工型可能参与β-肾上腺素能和毒蕈碱能反应调节的可能性。
为解决这些问题，我们在35℃基础条件下、存在异丙肾上腺素（ISO，10 - 100 nmol/L）以及ISO加氨甲酰胆碱（CCh，1 μmol/L）的情况下，记录从eNOS-/-和nNOS-/-小鼠、它们的野生型同窝小鼠（eNOS+/+和nNOS+/+）或C57BL/6J小鼠分离的LV心肌细胞中的肌节缩短情况。eNOS-/-和对照心肌细胞在1 Hz和3 Hz频率下、存在10和100 nmol/L ISO的情况下进行研究，并比较年轻（3个月）和年老（≥12个月）小鼠之间的反应。
在实验方案的所有阶段，年轻的eNOS-/-和eNOS+/+小鼠之间的收缩情况在1 Hz或3 Hz时以及对10或100 nmol/L ISO的反应中均无差异。然而，与年龄匹配的eNOS+/+小鼠相比，年老的eNOS-/-小鼠的心肌细胞对ISO的变力反应降低（P = 0.02）。同样，eNOS+/+和C57BL/6J心肌细胞之间的ISO反应存在显著差异（P < 0.01），这表明年龄和对照动物选择等实验变量可能导致了文献报道结果的不一致。相比之下，nNOS-/-心肌细胞在实验方案的所有阶段均表现出更大的收缩和更慢的舒张（与nNOS+/+心肌细胞相比，P = 0.0003和P = 0.01）。
鼠LV心肌细胞中内源性eNOS表达对于毒蕈碱介导的β-肾上腺素能信号抑制并非必需，并且在基础和β-肾上腺素能心肌收缩调节中似乎不发挥重要作用。我们的数据表明，nNOS是负责NO介导的心肌收缩力和舒张自分泌调节的心肌组成型同工型。
