Division of Nephrology, University of Utah Health Sciences Center, 1900 East 30 North, Salt Lake City, UT 84132, USA.
Am J Physiol Renal Physiol. 2010 Apr;298(4):F859-67. doi: 10.1152/ajprenal.00109.2009. Epub 2009 Dec 2.
Little is known about collecting duct adenylyl cyclase (AC) isoforms or regulation in the mouse. We performed RT-PCR for AC isoforms 1-9 in microdissected cortical (CCD) and outer medullary (OMCD) and acutely isolated inner medullary (IMCD) collecting duct. All collecting duct regions contained AC3, AC4, and AC6 mRNA, while CCD and OMCD, but not IMCD, also contained AC5 mRNA. Acutely isolated IMCD expressed AC3, AC4, and AC6 proteins by Western blot analysis. The mIMCD3 cell line expressed AC2, AC3, AC4, AC5, and AC6 mRNA; M-1 CCD cells expressed AC2, 3, 4, and 6, while mpkCCD cell lines contained AC3, AC4, and AC6 mRNA. AVP stimulated cAMP accumulation in acutely isolated mouse IMCD; this was reduced by chelation of extracellular calcium (EGTA) and almost completely abolished by blockade of calmodulin (W-7). Blockade of calmodulin kinase with KN-93 or endoplasmic reticulum calcium ATPase (thapsigargin) also reduced the AVP response. A similar inhibitory effect of W-7, KN-93, and thapsigargin was seen on forskolin-stimulated cAMP content in acutely isolated mouse IMCD. These three agents had the same pattern of blockade of AVP- or forskolin-stimulated AC activity in acutely isolated rat IMCD. AVP responsiveness in primary cultures of mouse IMCD was also reduced by W-7, KN-93, and thapsigargin. Small interfering RNA (siRNA) designed to knock down AC3 or AC6 in primary cultured mouse IMCD significantly reduced AVP-stimulated cAMP accumulation. Together, these data are consistent with a role of AC3 and AC6 in the activation of mouse collecting duct by AVP.
关于收集管腺苷酸环化酶 (AC) 同工型或调节在小鼠中的作用知之甚少。我们对微分离的皮质（CCD）和外髓（OMCD）和急性分离的内髓（IMCD）收集管中 AC 同工型 1-9 进行了 RT-PCR。所有收集管区域均含有 AC3、AC4 和 AC6 mRNA，而 CCD 和 OMCD 但不是 IMCD 也含有 AC5 mRNA。通过 Western blot 分析，急性分离的 IMCD 表达 AC3、AC4 和 AC6 蛋白。mIMCD3 细胞系表达 AC2、AC3、AC4、AC5 和 AC6 mRNA；M-1 CCD 细胞表达 AC2、3、4 和 6，而 mpkCCD 细胞系含有 AC3、AC4 和 AC6 mRNA。AVP 刺激急性分离的小鼠 IMCD 中的 cAMP 积累；这一过程被细胞外钙螯合剂（EGTA）抑制，并用钙调蛋白（W-7）阻断几乎完全消除。用 KN-93 或内质网钙 ATP 酶（他普西龙）阻断钙调蛋白激酶也降低了 AVP 反应。W-7、KN-93 和他普西龙对急性分离的小鼠 IMCD 中 forskolin 刺激的 cAMP 含量也有类似的抑制作用。这三种药物对急性分离的大鼠 IMCD 中 AVP 或 forskolin 刺激的 AC 活性也有相同的抑制作用。W-7、KN-93 和他普西龙也降低了原代培养的小鼠 IMCD 对 AVP 的反应性。针对原代培养的小鼠 IMCD 中的 AC3 或 AC6 设计的小干扰 RNA (siRNA) 显著降低了 AVP 刺激的 cAMP 积累。总的来说，这些数据一致表明 AC3 和 AC6 在 AVP 激活小鼠收集管中的作用。
