Carre Grégoire, Ouedraogo Maurice, Magaud Christophe, Carreyre Hélène, Becq Frédéric, Bois Patrick, Supuran Claudiu T, Thibaudeau Sébastien, Vandebrouck Clarisse, Bescond Jocelyn
Signalisation et Transports Ioniques Membranaires, Université de Poitiers, CNRS ERL 7368; 1 rue Georges Bonnet F-86073 Poitiers Cedex 09, France.
Laboratoire de Physiologie Animale, Université de Ouagadougou, 03 BP 7021, Ouagadougou 01, Burkina Faso.
J Ethnopharmacol. 2015 Jul 1;169:8-17. doi: 10.1016/j.jep.2015.03.037. Epub 2015 Apr 3.
Dodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius (DC.) Polhill and Wiens, a medicinal plant used in traditional medicine for the treatment of hypertension. This dihydropyranone exerts hypotensive and vasorelaxant effects on rats, and two molecular targets have been characterized: the carbonic anhydrase and the L-type calcium channel in cardiomyocytes with biochemical and electrophysiological techniques, respectively. To further evaluate the involvement of these two molecular targets in vasorelaxation, the effect of Ddn on rat vascular smooth muscle was investigated.
The effects of Ddn on L-type calcium current and on resting membrane potential were characterized in A7r5 cell line using the whole-cell patch-clamp configuration. The molecular identities of carbonic anhydrase isozymes in smooth muscle cells were examined with RT-PCR. Vascular response was measured on rat aortic rings in an organ bath apparatus and the effect of Ddn on intracellular pH was determined by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM [2,7-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester].
100µM Ddn reduced calcium current density of about 30%. In addition, carbonic anhydrase II, III, XIII and XIV were shown to be expressed in rat aorta and inhibited in smooth muscle cells by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation, thereby inducing membrane hyperpolarization and vasorelaxation. The results of vascular reactivity experiments obtained with pharmacological tools acting on the L-type calcium current and carbonic anhydrase suggest that Ddn produces its vasorelaxant effect via the inhibition of these two molecular targets.
This study demonstrates that Ddn induced vasorelaxation by targeting two proteins involved in the modulation of excitation-contraction coupling: L-type calcium channels and carbonic anhydrase.
多东宁（Ddn）是从多东叶菟丝子（Agelanthus dodoneifolius (DC.) Polhill and Wiens）中鉴定出的活性化合物之一，该药用植物在传统医学中用于治疗高血压。这种二氢吡喃酮对大鼠具有降压和血管舒张作用，并且已经通过生化和电生理技术分别确定了两个分子靶点：碳酸酐酶和心肌细胞中的L型钙通道。为了进一步评估这两个分子靶点在血管舒张中的作用，研究了Ddn对大鼠血管平滑肌的影响。
使用全细胞膜片钳技术在A7r5细胞系中表征Ddn对L型钙电流和静息膜电位的影响。用逆转录聚合酶链反应（RT-PCR）检测平滑肌细胞中碳酸酐酶同工酶的分子身份。在器官浴装置中测量大鼠主动脉环的血管反应，并使用pH敏感荧光探针BCECF-AM [2,7-双（2-羧乙基）-5-（和-6）-羧基荧光素，乙酰氧基甲基酯]通过流式细胞术测定Ddn对细胞内pH的影响。
100μM Ddn使钙电流密度降低约30%。此外，碳酸酐酶II、III、XIII和XIV在大鼠主动脉中表达，并被Ddn抑制在平滑肌细胞中。这种抑制导致细胞内pH升高约0.31，导致大电导钙激活钾通道（KCa）激活，从而诱导膜超极化和血管舒张。用作用于L型钙电流和碳酸酐酶的药理学工具获得的血管反应性实验结果表明，Ddn通过抑制这两个分子靶点产生其血管舒张作用。
本研究表明，Ddn通过靶向参与兴奋-收缩偶联调节的两种蛋白质诱导血管舒张：L型钙通道和碳酸酐酶。
