Barnett J V, Haigh L S, Marsh J D, Galper J B
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
J Biol Chem. 1989 Jun 25;264(18):10779-86.
We have previously demonstrated that cultures of myocytes from embryonic chick atria grown in media supplemented with fetal calf serum from which lipoproteins have been removed demonstrate a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared with cells grown with control medium. This increased response to carbamylcholine was associated with a 1.4-fold increase in total cell cholesterol, a 2-fold increase in the number of muscarinic receptors which bind agonist with high affinity, and a 2-fold increase in the levels of the alpha subunits of Go and Gi (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In the studies reported here, we determined the responsiveness of cells grown in lipoprotein-depleted serum (LPDS) to beta-adrenergic stimulation. Isoproterenol stimulated a contractile response of 58% measured as an increase in amplitude of contraction with a half-maximal effect at 3 x 10(-7) M for cells grown in fetal calf serum, but had no significant effect on amplitude of contraction on cells grown in LPDS. In cells grown in media supplemented with fetal calf serum, isoproterenol (1 x 10(-3) M) stimulated adenylate cyclase activity 100% over basal with an EC50 of 7 x 10(-6) M compared with an increase of 32% in cells grown in media supplemented with LPDS. beta-Adrenergic receptor number as measured by the binding of 125I-pindolol decreased from 24 +/- 3 (+/- S.E., n = 6) fmol/mg protein in cells grown under control conditions to 12 +/- 2 (n = 6) fmol/mg protein in media supplemented with LPDS. The level of alpha s as measured both by ADP-ribosylation with cholera toxin in the presence of 32P-NAD and by immunoblotting with specific antibody to alpha s decreased by 3-fold in cells grown in media supplemented with LPDS compared with control. All of these effects of growth of cells in LPDS were reversed by incubating cells with LPDS plus 30 microM mevinolin, an inhibitor of endogenous cholesterol synthesis. These studies indicate that growth of cells in media supplemented with LPDS results in a coordinate decrease in the levels of beta-adrenergic receptors and alpha s. Taken together with our previous studies these data support the hypothesis that the receptors and guanine nucleotide-binding proteins which mediate sympathetic and parasympathetic responsiveness in the heart are reciprocally regulated.
我们之前已经证明，在添加了去除脂蛋白的胎牛血清的培养基中培养的胚胎鸡心房肌细胞，与在对照培养基中培养的细胞相比，对毒蕈碱型胆碱能激动剂氨甲酰胆碱的搏动敏感性增加了近10倍。对氨甲酰胆碱的这种反应性增加与总细胞胆固醇增加1.4倍、高亲和力结合激动剂的毒蕈碱受体数量增加2倍以及Go和Gi的α亚基水平增加2倍有关（Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608 - 15618）。在本文报道的研究中，我们测定了在脂蛋白缺乏血清（LPDS）中培养的细胞对β-肾上腺素能刺激的反应性。异丙肾上腺素刺激收缩反应，在胎牛血清中培养的细胞，以收缩幅度增加来衡量，收缩反应为58%，在3×10⁻⁷ M时达到半数最大效应，但对在LPDS中培养的细胞的收缩幅度没有显著影响。在添加胎牛血清的培养基中培养的细胞中，异丙肾上腺素（1×10⁻³ M）刺激腺苷酸环化酶活性比基础水平高100%，EC50为7×10⁻⁶ M，而在添加LPDS的培养基中培养的细胞中增加了32%。通过¹²⁵I-吲哚洛尔结合测定的β-肾上腺素能受体数量，在对照条件下培养的细胞中为24±3（±标准误，n = 6）fmol/mg蛋白质，在添加LPDS的培养基中降至12±2（n = 6）fmol/mg蛋白质。通过在³²P-NAD存在下用霍乱毒素进行ADP-核糖基化以及用αs特异性抗体进行免疫印迹测定的αs水平，与对照相比，在添加LPDS的培养基中培养的细胞中降低了3倍。在LPDS中培养细胞的所有这些效应，通过用LPDS加30 μM美伐他汀（一种内源性胆固醇合成抑制剂）孵育细胞而被逆转。这些研究表明，在添加LPDS的培养基中培养细胞会导致β-肾上腺素能受体和αs水平协同降低。与我们之前的研究一起，这些数据支持这样的假设，即介导心脏交感和副交感反应性的受体和鸟嘌呤核苷酸结合蛋白是相互调节的。
