Salmerón Cristina, Harter Till S, Kwan Garfield T, Roa Jinae N, Blair Salvatore D, Rummer Jodie L, Shiels Holly A, Goss Greg G, Wilson Rod W, Tresguerres Martin
Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA.
Department of Pharmacology, University of California San Diego, San Diego, CA, USA.
Interface Focus. 2021 Apr 6;11(2):20200026. doi: 10.1098/rsfs.2020.0026. Epub 2021 Feb 12.
Soluble adenylyl cyclase (sAC) is a -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid-base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by at physiologically relevant levels for fish internal fluids (EC ∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid-base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.
可溶性腺苷酸环化酶（sAC）是一种受β-刺激的酶，可产生普遍存在的信号分子环磷酸腺苷（cAMP），并被认为是一种进化上保守的酸碱传感器。然而，在脊椎动物中种类最多、数量最丰富的硬骨鱼类中，其存在尚未得到证实。在此，我们在各种软骨鱼、辐鳍鱼和肉鳍鱼物种中鉴定出了sAC基因。接下来，我们聚焦于虹鳟鱼的sAC（rtsAC），并鉴定出20种潜在的可变剪接mRNA，它们编码的蛋白质异构体大小从28 kDa到186 kDa不等。对纯化的重组rtsAC蛋白进行的生化和动力学分析确定，在与鱼类内部液体生理相关的水平（EC ∼ 7 mM）下，其受到β-的刺激。rtsAC活性对KH7、LRE1和DIDS（其他生物体中已确定的sAC抑制剂）敏感，而对福斯可林和2,5-二脱氧腺苷（跨膜腺苷酸环化酶的调节剂）不敏感。蛋白质免疫印迹和免疫细胞化学显示，rtsAC在鳃离子转运细胞、肝细胞、红细胞、肌细胞和心肌细胞中高表达。在细胞系RTgill-W1中的分析表明，一些较长的rtsAC异构体可能优先定位于细胞核、高尔基体和足体。这些结果表明，sAC有望介导硬骨鱼类的多种酸碱稳态反应，并为哺乳动物中潜在的新功能提供线索。
