Ca²⁺-activated potassium (K_Ca) channels of small and intermediate conductance influence proliferation, apoptosis, and cell metabolism. We analysed whether prolonged activation of K_Ca channels by zoxazolamine (ZOX) induces differentiation of mouse embryonic stem (ES) cells towards cardiomyocytes. ZOX treatment of ES cells dose-dependent increased the number and diameter of cardiac foci, the frequency of contractions as well as mRNA expression of the cardiac transcription factor Nkx-2.5, the cardiac markers cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), ventricular myosin light chain-2 (MLC2v), and the pacemaker hyperpolarization-activated, cyclic nucleotide-gated 4 channel (HCN4). ZOX induced hyperpolarization of membrane potential due to activation of IK_Ca, raised intracellular Ca²⁺ concentration ([Ca²⁺]_i) and nitric oxide (NO) in a Ca²⁺-dependent manner. The Ca²⁺ response to ZOX was inhibited by chelation of Ca²⁺ with BAPTA-AM, release of Ca²⁺ from intracellular stores by thapsigargin and the phospholipase C (PLC) antagonist U73,122. Moreover, the ZOX-induced Ca²⁺ response was blunted by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) as well as the specific P2Y₁ antagonist MRS 2,179, suggesting purinergic receptor-stimulated signal transduction. Consequently, ZOX initiated ATP release from differentiating ES cells, which was inhibited by the chloride channel inhibitor NPPB and the gap junction inhibitor carbenoxolone (CBX). The stimulation of cardiomyogenesis by ZOX was blunted by the nitric oxide synthase (NOS) inhibitor l-NAME, as well as CBX and NPPB. In summary, our data suggest that ZOX enhances cardiomyogenesis of ES cells by ATP release presumably through gap junctional hemichannels, purinergic receptor activation and intracellular Ca²⁺ response, thus promoting NO generation.

中小型电导的Ca ²⁺活化钾（K _Ca）通道影响增殖，凋亡和细胞代谢。我们分析了钾_钙是否长时间激活唑唑胺（ZOX）的通道可诱导小鼠胚胎干（ES）细胞向心肌细胞分化。ZOX处理ES细胞的剂量依赖性增加了心脏灶的数量和直径，收缩的频率以及心脏转录因子Nkx-2.5，心脏标志物心肌肌钙蛋白I（cTnI），α-肌球蛋白重链的mRNA表达（α-MHC），心室肌球蛋白轻链2（MLC2v）和起搏器超极化激活的环状核苷酸门控4通道（HCN4）。ZOX通过激活IK _Ca诱导膜电位超极化，以Ca ²⁺依赖性方式升高细胞内Ca ²⁺浓度（[Ca ²⁺ ] _i）和一氧化氮（NO）。Ca ²⁺通过BAPTA-AM与Ca ²⁺的螯合，毒胡萝卜素和磷脂酶C（PLC）拮抗剂U73,122的毒胡萝卜素从细胞内储库释放Ca ²⁺来抑制对ZOX的应答。此外，嘌呤能受体拮抗剂吡2醛磷酸盐-6-偶氮苯基-2'，4'-二磺酸（PPADS）以及特定的P2Y ₁拮抗剂MRS 2,179使ZOX诱导的Ca ²⁺反应减弱，表明嘌呤能受体被刺激。信号转导。因此，ZOX从分化的ES细胞中引发了ATP释放，这被氯离子通道抑制剂NPPB和间隙连接抑制剂羧苄索隆（CBX）抑制。一氧化氮合酶（NOS）抑制剂1使ZOX对心肌生成的刺激减弱-NAME，以及CBX和NPPB。总而言之，我们的数据表明ZOX可能通过间隙连接半通道，嘌呤能受体激活和细胞内Ca ²⁺响应通过ATP释放来增强ES细胞的心肌发生，从而促进NO的产生。