Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Am J Physiol Renal Physiol. 2013 Mar 1;304(5):F543-52. doi: 10.1152/ajprenal.00530.2012. Epub 2012 Dec 12.
We used the patch-clamp technique to examine the role of carbon monoxide (CO) in regulating Ca(2+)-activated big-conductance K (BK) channels in the principal cell of the cortical collecting duct (CCD). Application of CORM3 or CORM2, a CO donor, activated BK channels in the CCD, whereas adding inactivated CORM2/3 had no effect. Superfusion of the CCD with CO-bubbled bath solution also activated the BK channels in the cell-attached patches. The effect of CO on BK channels was not dependent on nitric oxide synthase (NOS) because the effect of CORM3 was also observed in the CCD treated with l-NAME, an agent that inhibits the NOS. Adding a membrane-permeable cGMP analog, 8-bromo-cGMP, significantly increased the BK channel in the CCD. However, inhibition of soluble guanylate cyclase failed to abolish the stimulatory effect of CORM3 on BK channels. Moreover, inhibition of cGMP-dependent protein kinase G did not block the stimulatory effect of CORM3 on the BK channels, suggesting that the stimulatory effect of CO on the BK channels was, at least partially, induced by a cGMP-independent mechanism. Western blot demonstrated that heme oxygenase type 1 (HO-1) and HO-2 were expressed in the kidney. Moreover, a high-K (HK) intake increased the expression of HO-1 but not HO-2 in the kidney. A HK intake also increased renal HO activity defined by NADPH-dependent CO generation following addition of heme in the cell lysate from renal cortex and outer medulla. The role of HO in regulating BK channel activity in the CCD was also suggested by experiments in which application of hemin increased the BK channels. The stimulatory effect of hemin on the BK channels was blocked by SnMP, a HO inhibitor. But, adding CORM3 was still able to activate the BK channels in the presence of SnMP. We conclude that CO activates the BK channels, at least partially, through a NO-cGMP-independent pathway and that HO plays a role in mediating the effect of HK intake on the BK channels in the CCD.
我们使用膜片钳技术研究了一氧化碳（CO）在调节皮质集合管（CCD）主细胞中钙激活大电导钾（BK）通道中的作用。CO 供体 CORM3 或 CORM2 的应用激活了 CCD 中的 BK 通道，而失活的 CORM2/3 则没有作用。将 CO 饱和浴液灌流到 CCD 中也能激活细胞贴附斑中的 BK 通道。CO 对 BK 通道的作用不依赖于一氧化氮合酶（NOS），因为 CORM3 的作用也在使用 NOS 抑制剂 l-NAME 处理的 CCD 中观察到。添加膜通透性 cGMP 类似物 8-溴-cGMP 可显著增加 CCD 中的 BK 通道。然而，可溶性鸟苷酸环化酶的抑制作用并不能消除 CORM3 对 BK 通道的刺激作用。此外，cGMP 依赖性蛋白激酶 G 的抑制作用并未阻断 CORM3 对 BK 通道的刺激作用，表明 CO 对 BK 通道的刺激作用至少部分是由 cGMP 非依赖性机制诱导的。Western blot 表明肾脏中表达血红素加氧酶 1（HO-1）和 HO-2。此外，高钾（HK）摄入增加了肾脏中 HO-1 的表达，但没有增加 HO-2 的表达。HK 摄入还增加了肾皮质和外髓质细胞裂解物中血红素加合酶依赖性 CO 生成定义的肾 HO 活性。血红素加氧酶在调节 CCD 中 BK 通道活性中的作用也通过应用血红素增加 BK 通道的实验得到了证实。血红素对 BK 通道的刺激作用被 HO 抑制剂 SnMP 阻断，但在 SnMP 存在下，CORM3 仍能激活 BK 通道。我们得出结论，CO 通过一种非 NO-cGMP 途径至少部分激活 BK 通道，HO 在介导 HK 摄入对 CCD 中 BK 通道的作用中发挥作用。
