Kurihara Kinji, Nakanishi Nobuo, Amano Osamu, Tonosaki Keiichi
Division of Physiology, Meikai University, School of Dentistry, Sakado, Saitama, Japan.
Arch Oral Biol. 2008 Jul;53(7):593-604. doi: 10.1016/j.archoralbio.2008.01.007. Epub 2008 Mar 4.
We examined the expression of Na(+)/K(+)-ATPase alpha-subunit isoforms in rat salivary glands by RT-PCR. Isoform alpha1 was expressed strongly in all three major salivary glands. The alpha2 isoform was expressed in both submandibular gland (SMG) and sublingual gland (SLG) but faintly in the parotid gland (PG). The alpha3 was detected only in the SLG, and the alpha3 mRNA in the SLG was 1/8 of its level in the brain. Na(+)/K(+)-ATPase alpha3 isoform in the SLG, was localized predominantly on the basolateral plasma membranes in serous cells by immunohistochemical method. We also found the presence of natural antisense RNA of the alpha3 isoform in rat SLG: the 1st-strand cDNA prepared with gene-specific forward primers targeted to the CDS region and to the promoter region of the alpha3 gene in place of oligo(dT) or gene-specific reverse primers produced reasonable PCR products corresponding to the alpha3 cDNA sequence by the subsequent PCR reaction. Synthesis of the 1st-strand cDNA with the gene-specific forward primers was prevented by RNase digestion of the total RNA preparation, indicating that the PCR products in the RT-PCR system were not due to the contaminated genomic DNA, if any. The alpha3 protein level in rat SLG increased with aging, and levels of both alpha3 mRNA (sense RNA) and alpha3 antisense RNA were higher in SLGs of aged rats than in those of young rats, respectively.
我们通过逆转录聚合酶链反应（RT-PCR）检测了大鼠唾液腺中钠钾ATP酶α亚基同工型的表达。同工型α1在所有三大唾液腺中均强烈表达。α2同工型在下颌下腺（SMG）和舌下腺（SLG）中表达，而在腮腺（PG）中表达较弱。α3仅在舌下腺中检测到，舌下腺中的α3 mRNA水平是其在脑中水平的1/8。通过免疫组织化学方法，舌下腺中的钠钾ATP酶α3同工型主要定位于浆液性腺泡细胞的基底外侧质膜上。我们还发现大鼠舌下腺中存在α3同工型的天然反义RNA：用针对α3基因编码区（CDS）和启动子区的基因特异性正向引物代替寡聚（dT）或基因特异性反向引物制备的第一链cDNA，通过后续的PCR反应产生了与α3 cDNA序列相对应的合理PCR产物。用基因特异性正向引物合成第一链cDNA可通过对总RNA制剂进行核糖核酸酶消化来阻止，这表明RT-PCR系统中的PCR产物不是由于污染的基因组DNA（如果有的话）所致。大鼠舌下腺中的α3蛋白水平随年龄增长而增加，老年大鼠舌下腺中α3 mRNA（正义RNA）和α3反义RNA的水平分别高于年轻大鼠。
