Ioannou P, Loh A Y, Osmond D H
Department of Physiology, University of Toronto, Ont., Canada.
Can J Physiol Pharmacol. 1991 Sep;69(9):1331-40. doi: 10.1139/y91-197.
Prorenin determination in rat plasma has been problematic from the outset. Consequently, its existence is questioned by some and its quantity by others, making it difficult for knowledge to advance as to its function relative to the renin system. The present study examines major variables in the determination of rat plasma prorenin and renin, notably different prorenin activation protocols involving blood samples obtained under various conditions from animals under different anesthetics. We found that a trypsin activation step with 5 mg/mL plasma, 60 min at 23 degrees C, followed by a PRA step of 10 min at 37 degrees C, resulted in the highest prorenin estimates, up to approximately 400 ng.mL-1.h-1 in terms of angiotensin I, as compared with published values of 0-190, based on other protocols. These estimates were obtained despite considerable destruction of angiotensinogen (renin substrate) by trypsin. Cryoactivation of prorenin was much less effective than in human plasma but, when followed by trypsin, it facilitated greater activation than with trypsin alone. Comparable fresh and fresh-frozen plasmas had similar prorenin-renin values, but lower values were observed in plasmas that had been repeatedly frozen and thawed. Conscious rats and those anesthetized with Inactin or ether had higher renins and prorenins than those anesthetized with methoxyflurane or halothane. Rats with kidneys in place during blood collection had higher renins (but not prorenins) than those whose kidneys were clamped off, suggesting that last-minute renin release during blood collection had occurred. We conclude that (i) trypsin generates increased renin, or renin-like, activity in plasma, suggesting activation of a precursor; (ii) on this basis, high prorenin levels exist in normal rat plasma; (iii) renin and prorenin levels are variously influenced by different anesthetics and blood handling procedures; (iv) variation in prorenin levels suggests that it is a dynamic (functional?) component of the renin system; (v) prorenin measurements are heavily influenced by methodological variations during the trypsin step or the subsequent PRA step; (vi) using standardized methodology, the rat can serve as a model for investigating the function of prorenin in normotension and hypertension.
从一开始，测定大鼠血浆中的血管紧张素原酶就存在问题。因此，一些人质疑它的存在，另一些人质疑它的数量，这使得关于它相对于肾素系统的功能的知识难以推进。本研究考察了测定大鼠血浆血管紧张素原酶和肾素时的主要变量，特别是涉及在不同麻醉状态下的动物于各种条件下采集的血样的不同血管紧张素原酶激活方案。我们发现，用5 mg/mL血浆在23摄氏度下进行60分钟的胰蛋白酶激活步骤，随后在37摄氏度下进行10分钟的血浆肾素活性（PRA）步骤，可得到最高的血管紧张素原酶估计值，以血管紧张素I计高达约400 ng·mL-1·h-1，相比基于其他方案发表的0 - 190的值。尽管胰蛋白酶对血管紧张素原（肾素底物）有相当程度的破坏，但仍得到了这些估计值。血管紧张素原酶的冷冻激活效果远不如在人血浆中，但在冷冻激活后再用胰蛋白酶处理，其激活效果比单独使用胰蛋白酶时更好。可比的新鲜血浆和新鲜冷冻血浆具有相似的血管紧张素原酶 - 肾素值，但在反复冷冻和解冻的血浆中观察到较低的值。清醒大鼠以及用安泰酮或乙醚麻醉的大鼠比用甲氧氟烷或氟烷麻醉的大鼠具有更高的肾素和血管紧张素原酶。采血时肾脏在位的大鼠比肾脏被夹闭的大鼠具有更高的肾素（但血管紧张素原酶不高），这表明采血过程中出现了最后时刻的肾素释放。我们得出结论：（i）胰蛋白酶在血浆中产生增加的肾素或肾素样活性，提示一种前体被激活；（ii）基于此，正常大鼠血浆中存在高水平的血管紧张素原酶；（iii）肾素和血管紧张素原酶水平受到不同麻醉剂和血液处理程序的不同影响；（iv）血管紧张素原酶水平的变化表明它是肾素系统的一个动态（功能性？）组分；（v）血管紧张素原酶测量受到胰蛋白酶步骤或随后的PRA步骤中方法学变化的严重影响；（vi）使用标准化方法，大鼠可作为研究血管紧张素原酶在正常血压和高血压中的功能的模型。
