Characterization of cardiac beta-adrenergic receptors in the guinea pig heart: application to study of beta-adrenergic receptors in shock models.
Characterization of cardiac beta-adrenergic receptors in the guinea pig heart: application to study of beta-adrenergic receptors in shock models.
复制标题
豚鼠心脏中心脏β-肾上腺素能受体的表征：应用于休克模型中β-肾上腺素能受体的研究。
DOI：
10.1006/jsre.1993.1177
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Horton,JW
中科院分区：
文献类型：
--
作者：
Kaufman,TM;Horton,JW
在许多类型的休克或心力衰竭中，心肌对儿茶酚胺的反应显著减弱。豚鼠心脏是研究休克的理想模型，因为它相对便宜，并且豚鼠的心血管系统与人类的心血管系统最相似。利用这个模型，我们开发了技术来表征和定量的变化，β-肾上腺素能受体（βAR）在豚鼠心脏烧伤后。进行初步实验以确定最佳结合条件，例如，孵育时间和条件、蛋白浓度、冲洗等。使用激动剂和拮抗剂进行额外的实验，以表征βAR的效力和立体特异性的等级顺序。在37°C下，将取自假烧伤和烧伤心脏的膜粗品（50 μg/250μl）与8-10种浓度的125 I-氰基吲哚酚（10-450 pM）孵育1小时。使用阿普洛尔（10 μM）测定非特异性结合。本研究中使用的膜制剂结合激动剂和拮抗剂，其效价和立体特异性具有β-肾上腺素能受体的特征。最后，在存在和不存在Gpp（NH）p的情况下用异丙肾上腺素进行激动剂竞争曲线，以确定Gs蛋白的受体调节。计算机辅助分析显示，烧伤后高亲和力β受体的比例（41.2 ± 4.7%）较假烧伤对照组（54 ± 2%，P <0.023）明显降低。这里描述的方法可以很容易地适用于在许多类型的休克或创伤期间将心脏功能障碍与β-肾上腺素能受体的变化相关联。
The myocardial response to catecholamines is significantly diminished in many types of shock or heart failure. The guinea pig heart is an ideal model for the study of shock, as it is relatively inexpensive, and the cardiovascular system of the guinea pig most closely resembles that of the human. Using this model, we have developed techniques to characterize and quantitate changes in β-adrenergic receptors (βAR) in the guinea pig heart after burn injury. Preliminary experiments were performed to determine the optimum binding conditions, e.g., incubation time and conditions, protein concentrations, rinsing, etc. Additional experintents were conducted using agonists and antagonists to characterize the rank order of potency and stereospecificity of the βAR. Crude membrane preparations (50 μg/250μl) from sham-burned and burned hearts were incubated with 8-10 concentrations of125I-cyanopindolol (10-450 pM) at 37°C for 1 hr. Under these conditions, binding assays were linear with respect to protein concentration and time. Alprenolol (10 μM) was used to determine nonspecific binding. The membrane preparations used in this study bound both agonists and antagonists with a rank order of potency and stereospecificity characteristic of a β-adrenergic receptor. Finally, agonist competition curves were performed with isoproterenol in the presence and absence of Gpp(NH)p to determine receptor regulation by the Gs protein. Analysis using computer-assisted techniques suggests that the fraction of high-affinity β-receptors is significantly reduced after burn injury (41.2 ± 4.7%) compared to sham-burned controls (54 ± 2%,P⩽ 0.023). The methods described here can be easily adapted to correlate cardiac dysfunction with changes in β-adrenergic receptors during many types of shock or trauma.