Owicki J C, Parce J W, Kercso K M, Sigal G B, Muir V C, Venter J C, Fraser C M, McConnell H M
Molecular Devices Corp., Menlo Park, CA 94025.
Proc Natl Acad Sci U S A. 1990 May;87(10):4007-11. doi: 10.1073/pnas.87.10.4007.
Activation of beta-adrenergic or muscarinic acetylcholine receptors expressed in transfected cells or epidermal growth factor receptors in human keratinocytes produces 15% to 200% changes in cellular metabolic rates. Changes in cell metabolism were monitored continuously with a previously described silicon-based microphysiometer that detects small changes in extracellular pH. The amplitude and kinetics of the metabolic changes depend upon several factors including pretreatment of the cells prior to receptor stimulation, the dose of hormone/neurotransmitter used, and the receptor complement of the cells. Responses are receptor specific; cells transfected with receptor genes respond only to the appropriate hormone/transmitter, whereas control (nontransfected) cells or cells transfected with different receptors exhibit no response. The specificity of the responses was further documented by using pharmacological antagonists. In Chinese hamster ovary (CHO) cells transfected with human beta 2-adrenergic receptors, isoproterenol produces a 20-60% increase in the rate of extracellular acidification with an EC50 of 4 nM, a response that is competitively antagonized by (-)-propranolol. The EC50 for the isoproterenol response is shifted from 4 nM to 100 nM in the presence of 3 nM (-)-propranolol. The kinetics of the metabolic response induced by beta-adrenergic receptor stimulation are markedly slower than those elicited by muscarinic receptor agonists. The maximal metabolic response in cells transfected with beta-adrenergic receptors peaks at approximately 12 min as compared with less than 30 sec in cells transfected with muscarinic receptors, perhaps reflecting activation of different second-messenger pathways. These findings illustrate an alternative means of studying cellular responses to hormones and neurotransmitters and suggest that metabolic changes will be generally useful for detecting the consequences of receptor-ligand interactions.
在转染细胞中表达的β-肾上腺素能或毒蕈碱型乙酰胆碱受体，或人角质形成细胞中的表皮生长因子受体被激活后，细胞代谢率会发生15%至200%的变化。利用先前描述的基于硅的微生理计连续监测细胞代谢的变化，该微生理计可检测细胞外pH值的微小变化。代谢变化的幅度和动力学取决于几个因素，包括受体刺激前细胞的预处理、所用激素/神经递质的剂量以及细胞的受体组成。反应具有受体特异性；转染了受体基因的细胞仅对相应的激素/递质产生反应，而对照（未转染）细胞或转染了不同受体的细胞则无反应。通过使用药理学拮抗剂进一步证明了反应的特异性。在转染了人β2-肾上腺素能受体的中国仓鼠卵巢（CHO）细胞中，异丙肾上腺素使细胞外酸化速率增加20%至60%，EC50为4 nM，该反应可被（-）-普萘洛尔竞争性拮抗。在存在3 nM（-）-普萘洛尔的情况下，异丙肾上腺素反应的EC50从4 nM变为100 nM。β-肾上腺素能受体刺激诱导的代谢反应动力学明显慢于毒蕈碱受体激动剂引发的反应动力学。转染了β-肾上腺素能受体的细胞中的最大代谢反应在约12分钟时达到峰值，而转染了毒蕈碱受体的细胞中该峰值不到30秒，这可能反映了不同第二信使途径的激活。这些发现说明了研究细胞对激素和神经递质反应的另一种方法，并表明代谢变化通常可用于检测受体-配体相互作用的结果。
