caa a22 4500 475742591 CHVBK 20180406123504.0 cr unu---uuuuu 170329e20000601xx s 000 0 eng 10.1007/s002400000113 doi (NATIONALLICENCE)springer-10.1007/s002400000113 Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells [Elektronische Daten] [K. Obara, K. Arai, N. Miyajima, A. Hatano, Y. Tomita, K. Takahashi] The aim of this study was to investigate the expression of the muscarinic acetylcholine receptor (mAchR) subtypes mRNA in primary culture of human prostate stromal cells using the reverse transcription polymerase chain reaction (RT-PCR), RNA blotting and in situ hybridization (ISH). Using an explant method, we obtained a primary culture of prostate stromal cells from three patients with benign prostatic hypertrophy. Total RNA was extracted using the acid guanidinium method for cDNA synthesis. First-strand cDNA was then used for PCR with primers designed to amplify the fragments of each mAchR subtypes (m1-m5) cDNA sequence. The m2, m3 and m4 subtype expected bands were detected; in particular m2 transcripts was strongly detected in the stromal cell culture. Each of the PCR products were subcloned into the pGEM-T plasmid vector, sequenced and random primer labeled using 32P. Digoxigenin-labeled cRNA probes were synthesized by in vitro transcription. RNA blotting using a m2 muscarinic receptor cDNA probe revealed a 4.5 kb single transcript. However, m3 and m4 probes did not hybridize. Using in situ hybridization (ISH), m2 receptor mRNA signals were detected in several smooth muscle cells. The staining was predominantly localized to the perinuclear cytoplasm. The m3 and m4 probes did not hybridize. These results suggested that m2 receptor subtype plays a role in smooth muscle activity of the human prostate. Springer-Verlag Berlin Heidelberg, 2000 Key words Muscarinic acethylcholine receptor subtypes mRNA expression nationallicence Primary culture of human prostate stromal cells nationallicence Reverse transcription polymerase chain reaction nationallicence RNA blotting In situ hybridization nationallicence Obara K. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut Arai K. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut Miyajima N. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut Hatano A. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut Tomita Y. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut Takahashi K. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut https://doi.org/10.1007/s002400000113 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s002400000113 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Obara K. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut NATIONALLICENCE 700 1- Arai K. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut NATIONALLICENCE 700 1- Miyajima N. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut NATIONALLICENCE 700 1- Hatano A. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut NATIONALLICENCE 700 1- Tomita Y. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut NATIONALLICENCE 700 1- Takahashi K. Department of Urology, Niigata University, School of Medicine, Asahimachi 1, Niigata 951-8510, Japan e-mail: obarak@med.niigata-u.ac.jp Tel.: +81-25-2272289; Fax: +81-25-2270784, JP aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer