86Rb+摄取法不适用于测定受神经支配组织中的（Na+ + K+）-ATP酶活性。

Unsuitability of the 86Rb+ uptake method for estimation of (Na+ + K+)-ATPase activity in innervated tissues.

作者信息

Powis D A, Madsen G M

出版信息

Biochim Biophys Acta. 1986 Oct 9;861(2):251-8. doi: 10.1016/0005-2736(86)90427-x.

PMID:3019401

Abstract

(Na+ + K+)-ATPase activity was estimated by 86Rb+ uptake in dog saphenous vein to determine the validity of the technique in tissues that have a sympathetic innervation. When saphenous vein rings were incubated at 37 degrees C in Krebs' solution containing 86Rb+, the cardenolide acetylstrophanthidin caused a concentration-dependent inhibition of Rb+ uptake. The threshold for inhibition was approx. 10 nM acetylstrophanthidin and the maximum effect was obtained at 9 microM. In the upper part of this concentration range (greater than 1 microM) acetylstrophanthidin released noradrenaline from the sympathetic nerve terminals associated with the tissue. In this upper part of the acetylstrophanthidin concentration range the alpha-adrenoceptor antagonist phentolamine (8 microM) reduced, by up to 25%, the degree of 86Rb+ uptake inhibition caused by the cardenolide. In other experiments, saphenous vein strips were loaded with 86Rb+ and perifused with Krebs' solution containing acetylstrophanthidin. At concentrations which release noradrenaline, acetylstrophanthidin increased the efflux of 86Rb+. Phentolamine (8 microM) prevented the acetylstrophanthidin-evoked efflux of the isotope as did prior in vitro denervation of 86Rb+ loaded strips with 6-hydroxydopamine. Exogenous noradrenaline (1-100 microM) added to the perifusing fluid also caused an efflux of 86Rb+ that was attenuated by phentolamine. The data indicate for dog saphenous vein that with low concentrations of acetylstrophanthidin the extent of 86Rb+ accumulation might accurately reflect prevailing (Na+ + K+)-ATPase activity. At higher concentrations of acetylstrophanthidin, however, noradrenaline is released from the nerve endings and causes 86Rb+ efflux from the smooth muscle cells consequent upon alpha-adrenoceptor activation. Since this efflux reduces the extent of Rb+ accumulation, measurement of the latter does not adequately reflect uptake mediated by the activity of (Na+ + K+)-ATPase. This is significant because in most applications of the 86Rb+ uptake method it is the estimate of Rb+ accumulation made in the presence of a high concentration of cardenolide that forms the basis of all subsequent calculations with respect to (Na+ + K+)-ATPase activity.

摘要

通过狗隐静脉对⁸⁶Rb⁺的摄取来估计（Na⁺ + K⁺）-ATP酶活性，以确定该技术在具有交感神经支配的组织中的有效性。当隐静脉环在含有⁸⁶Rb⁺的 Krebs 溶液中于 37℃孵育时，强心甾烯毒毛旋花子苷会引起 Rb⁺摄取的浓度依赖性抑制。抑制阈值约为 10 nM 强心甾烯毒毛旋花子苷，在 9 μM 时可获得最大效应。在该浓度范围的上限（大于 1 μM），强心甾烯毒毛旋花子苷会从与该组织相关的交感神经末梢释放去甲肾上腺素。在强心甾烯毒毛旋花子苷浓度范围的这个上限部分，α-肾上腺素能受体拮抗剂酚妥拉明（8 μM）可将强心甾烯引起的⁸⁶Rb⁺摄取抑制程度降低多达 25%。在其他实验中，隐静脉条带加载⁸⁶Rb⁺并用含有强心甾烯毒毛旋花子苷的 Krebs 溶液进行灌流。在释放去甲肾上腺素的浓度下，强心甾烯毒毛旋花子苷会增加⁸⁶Rb⁺的流出。酚妥拉明（8 μM）可阻止强心甾烯毒毛旋花子苷引起的同位素流出，用 6-羟基多巴胺对加载⁸⁶Rb⁺的条带进行体外预先去神经处理也有同样效果。添加到灌流液中的外源性去甲肾上腺素（1 - 100 μM）也会引起⁸⁶Rb⁺的流出，而酚妥拉明可使其减弱。数据表明，对于狗隐静脉，低浓度的强心甾烯毒毛旋花子苷时，⁸⁶Rb⁺积累程度可能准确反映当时的（Na⁺ + K⁺）-ATP酶活性。然而，在强心甾烯毒毛旋花子苷浓度较高时，去甲肾上腺素会从神经末梢释放，并在α-肾上腺素能受体激活后导致⁸⁶Rb⁺从平滑肌细胞流出。由于这种流出会降低 Rb⁺积累程度，因此对后者的测量不能充分反映由（Na⁺ + K⁺）-ATP酶活性介导的摄取。这一点很重要，因为在⁸⁶Rb⁺摄取方法的大多数应用中，正是在高浓度强心甾烯存在下对 Rb⁺积累的估计构成了所有后续关于（Na⁺ + K⁺）-ATP酶活性计算的基础。