Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata-CONICET, Calle 60 y 120, 1900, La Plata, Argentina.
Pflugers Arch. 2020 Jan;472(1):103-115. doi: 10.1007/s00424-019-02331-x. Epub 2019 Nov 22.
The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na/HCO cotransporter (NBC) promotes the cellular co-influx of HCO and Na. Since sAC activity is regulated by HCO, our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca transient (Ca) by epifluorescence, Ca sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pH) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 μM), the selective inhibitor of sAC KH7 (1 μM), and the PKA inhibitor H89 (0.1 μM) induced a negative inotropic effect which was associated with a decrease in Ca. Since PKA increases Ca release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca handling and basal cardiac contractility.
可溶性腺苷酸环化酶（sAC）在心脏中被鉴定为环磷酸腺苷（cAMP）的另一个来源。然而，其心脏生理功能尚不清楚。另一方面，心脏钠/碳酸氢根协同转运蛋白（NBC）促进 HCO 和 Na 的细胞共内流。由于 sAC 活性受 HCO 调节，我们的目的是研究心肌细胞中 NBC 和 sAC 之间的潜在功能关系。用 Fura-2、Fluo-3 或 BCECF 负载大鼠心室肌细胞，分别通过荧光显微镜测量钙瞬变（Ca）、共聚焦显微镜测量钙火花频率（CaSF）或荧光显微镜测量细胞内 pH（pH）。用摄像机测量肌节或细胞缩短作为收缩性的指标。NBC 阻断剂 S0859（10 μM）、sAC 选择性抑制剂 KH7（1 μM）和 PKA 抑制剂 H89（0.1 μM）诱导负性变力作用，伴有 Ca 减少。由于 PKA 通过肌浆网 RyR 通道增加 Ca 释放，因此测量 CaSF 作为 RyR 开放概率的指标。KH7 可阻止 CaSF 的产生。最后，我们研究了 sAC 激活对 NBC 活性的潜在作用。在 KH7 或 H89 存在的情况下，NBC 介导的从酸中毒中的恢复更快，这表明 sAC-PKA 途径负调节 NBC 功能，与激活 sAC 的 HCO 内流的负反馈调节一致。总之，这些结果表明，复杂的 NBC-sAC-PKA 在 Ca 处理和基础心脏收缩性中发挥着重要作用。
