Ortiz-Capisano M Cecilia, Ortiz Pablo A, Harding Pamela, Garvin Jeffrey L, Beierwaltes William H
Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit,MI 48202, USA.
Hypertension. 2007 Jan;49(1):162-9. doi: 10.1161/01.HYP.0000250708.04205.d4. Epub 2006 Nov 6.
Intracellular calcium and cAMP are the 2 second messengers that regulate renin release; cAMP stimulates renin release from juxtaglomerular (JG) cells, whereas increased intracellular calcium inhibits it. We hypothesized that decreased intracellular calcium acts by activating calcium-inhibitable isoforms of adenylyl cyclase, increasing cAMP, and stimulating renin secretion. We used a primary culture of JG cells isolated from C-57/B6 mice. Cells were plated to a density of 70% in serum-free medium and incubated for 2 hours with or without 100 micromol/L of the cytosolic calcium chelator 5'5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Intracellular cAMP content was 4.04+/-0.92 pM/mL per milligram of protein, and it increased by125+/-33% (P<0.01) with BAPTA-AM. Basal renin was 1.28+/-0.40 microg of angiotensin I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 182+/-62% (P<0.025). Western blots using an antibody that recognizes adenylyl cyclase types V and VI yielded a characteristic band of approximately 135 kDa. When primary cultures of isolated JG cells were tested for the calcium-inhibitable isoforms of adenylyl cyclase, they showed intense focal cytoplasmic staining. Cells stained for both renin and adenylyl cyclase V/VI showed colocalization in the cytoplasm, primarily on the granules. An adenylyl cyclase inhibitor (SQ 22,536) completely blocked BAPTA-AM-stimulated renin release and JG cell cAMP content. We conclude that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Thus, decreased intracellular calcium stimulates adenylyl cyclase, resulting in cAMP synthesis and, consequently, renin release.
细胞内钙和环磷酸腺苷（cAMP）是调节肾素释放的两种第二信使；cAMP刺激球旁（JG）细胞释放肾素，而细胞内钙增加则抑制肾素释放。我们推测细胞内钙减少通过激活腺苷酸环化酶的钙抑制亚型、增加cAMP并刺激肾素分泌来发挥作用。我们使用了从C-57/B6小鼠分离的JG细胞原代培养物。将细胞以70%的密度接种于无血清培养基中，在有或无100微摩尔/升胞质钙螯合剂5,5-二甲基-1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA-AM）情况下孵育2小时以降低细胞内钙。通过放射免疫测定法测定JG细胞cAMP含量和肾素释放。细胞内cAMP含量为每毫克蛋白质4.04±0.92皮摩尔/毫升，用BAPTA-AM处理后增加了125±33%（P<0.01）。基础肾素水平为每毫克蛋白质每小时每毫升1.28±0.40微克血管紧张素I，BAPTA-AM使其增加了182±62%（P<0.025）。使用识别V型和VI型腺苷酸环化酶的抗体进行的蛋白质印迹产生了一条约135 kDa的特征条带。当对分离的JG细胞原代培养物检测腺苷酸环化酶的钙抑制亚型时，它们显示出强烈的局灶性细胞质染色。同时对肾素和腺苷酸环化酶V/VI进行染色的细胞在细胞质中显示共定位，主要在颗粒上。一种腺苷酸环化酶抑制剂（SQ 22,536）完全阻断了BAPTA-AM刺激的肾素释放和JG细胞cAMP含量。我们得出结论，JG细胞内存在腺苷酸环化酶的钙抑制亚型（V型和/或VI型）。因此，细胞内钙减少刺激腺苷酸环化酶，导致cAMP合成，进而引起肾素释放。
