Northern blot and ribonuclease protection study of alpha 2-adrenoceptor subtypes in cultured cell lines.
Northern blot and ribonuclease protection assay were used to identify alpha 2-adrenoceptor subtypes in human colonic adenocarcinoma (HT29), neuroblastoma x glioma rat-mouse hybrid NG108-15 (NG108) and opossum kidney (OK) cell lines. Radioligand binding studies showed that the alpha 2-adrenoceptor expressed in HT29, NG108 and OK cells represent the pharmacological alpha 2A, alpha 2B and alpha 2C subtypes respectively. In our Northern blot analysis, hybridization of poly(A)+ RNA from HT29, NG108 and OK cells with human kidney alpha 2-adrenoceptor cDNA probe (alpha 2-C4) identified a single band of 4.4, 4.2 and 4.4 kb respectively in each cell line. Hybridization with a human platelet alpha 2-adrenoceptor genomic probe (alpha 2-C10) resulted in two bands for HT29 cells with the size of 4.4 kb and 3.9 kb. No bands were seen for HT29, NG108 and OK cells when hybridized with a third alpha 2-adrenoceptor human genomic DNA probe which is localized in chromosome 2 (alpha 2-C2). For the HT29 cells, the 3.9 kb band was seen only when using the alpha 2-C10 probe. Thus, this band probably represents alpha 2-C10 mRNA. To further characterize the alpha 2-adrenoceptor mRNA expressed in HT29, NG108 and OK cells, the sensitive ribonuclease protection assay was performed. A single band about 900 bp was protected when the poly(A)+ RNA from NG108 and OK cells was hybridized with an alpha 2-C4 RNA probe and digested with RNAases. Hybridization of mRNA from HT29 cells with alpha 2-C10 RNA probe and digestion with RNAases protected a 500 bp fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
Sun
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Department of Medicine, Creighton University, School of Medicine, Omaha, NE 68131.
Umemura
S
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Chun
M
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Pettinger
W A
WA
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000145:classification / Q000235:genetics*; D012260:Ribonucleases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015347:RNA Probes; D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; D012260:Ribonucleases