Muscholl E, Racké K, Spira F J
Neuroscience. 1985 Jan;14(1):79-93. doi: 10.1016/0306-4522(85)90165-4.
Isolated rabbit hearts were perfused according to a modified Langendorff method for 1 h (unstimulated hearts). In different hearts, release of dopamine beta-hydroxylase activity into the transmyocardial fluid draining the interstitium was evoked by electrical field stimulation for six periods of 1 min at 30 min intervals (stimulated hearts). The hearts were then homogenized and fractionated into 100,000 g supernatant and sedimented at 4 degrees C. In homogenates from unstimulated hearts, the soluble dopamine beta-hydroxylase (determined in the supernatant) accounted for 17% of the total dopamine beta-hydroxylase (determined in the homogenate). In stimulated hearts the soluble fraction of dopamine beta-hydroxylase was reduced by 65%. The dopamine beta-hydroxylase released into the transmyocardial fluid by electrical stimulation, expressed as fraction of the total activity, corresponded well to the loss of enzyme from the supernatant demonstrating that the soluble dopamine beta-hydroxylase determined from the supernatant represents the releasable pool. Gadolinium ions (Gd3+) added to the homogenization medium of unstimulated hearts reduced the soluble fraction of dopamine beta-hydroxylase up to 63%, with the maximum effect at 200 microM. Similarly, when neurohypophyses were homogenized and spun at 0-4 degrees C, the fraction of vasopressin in the soluble phase was about 50% of the total. Gd3+ reduced this fraction by maximally 60%, an effect which was accompanied by an increase of vasopressin in the sedimentable fraction. When cytochalasin B (10 microM) was present during the homogenization of the hearts the soluble fraction of dopamine beta-hydroxylase was reduced to the same extent as in the presence of Gd3+. However, cytochalasin B had no effect on the distribution of vasopressin in the soluble and sedimentable fractions of homogenates of neurohypophyses. Gallopamil, when present during the homogenization of the hearts at a maximum effective concentration of 1 microM, reduced the soluble fraction of dopamine beta-hydroxylase by only 40%. However, the electrically evoked noradrenaline release from perfused hearts was completely blocked at 100-300 microM gallopamil. When neurohypophyses were homogenized and fractionated at room temperature only 13% of the total vasopressin was found in the soluble fraction and Gd3+ did not further reduce this fraction. When unstimulated hearts were homogenized and fractionated at room temperature the fraction of soluble dopamine beta-hydroxylase was reduced by 40% compared to the experiments at 0-4 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)
按照改良的Langendorff方法对离体兔心灌注1小时（未刺激的心脏）。在不同的心脏中，通过电场刺激，每隔30分钟进行6个1分钟的刺激时段，诱发多巴胺β-羟化酶活性释放到引流间质的跨心肌液中（刺激的心脏）。然后将心脏匀浆，并在4℃下离心分离成100,000g上清液和沉淀物。在未刺激心脏的匀浆中，可溶性多巴胺β-羟化酶（在上清液中测定）占总多巴胺β-羟化酶（在匀浆中测定）的17%。在刺激的心脏中，多巴胺β-羟化酶的可溶性部分减少了65%。通过电刺激释放到跨心肌液中的多巴胺β-羟化酶，以总活性的比例表示，与上清液中酶的损失很好地对应，表明从上清液中测定的可溶性多巴胺β-羟化酶代表可释放池。添加到未刺激心脏匀浆介质中的钆离子（Gd3+）将多巴胺β-羟化酶的可溶性部分降低至63%，在200μM时效果最佳。同样，当神经垂体在0-4℃下匀浆并离心时，可溶性相中加压素的比例约为总量的50%。Gd3+最大可将该比例降低60%，同时可沉降部分中的加压素增加。当心脏匀浆过程中存在细胞松弛素B（10μM）时，多巴胺β-羟化酶的可溶性部分降低程度与存在Gd3+时相同。然而，细胞松弛素B对神经垂体匀浆中加压素在可溶性和可沉降部分的分布没有影响。当心脏匀浆过程中存在最大有效浓度为1μM的加洛帕米时，多巴胺β-羟化酶的可溶性部分仅降低40%。然而，在100-300μM加洛帕米时，灌注心脏的电诱发去甲肾上腺素释放被完全阻断。当神经垂体在室温下匀浆并分离时，可溶性部分中仅发现13%的总加压素，Gd3+不会进一步降低该比例。当未刺激心脏在室温下匀浆并分离时，可溶性多巴胺β-羟化酶的比例与在0-4℃下的实验相比降低了40%。（摘要截短至400字）
