Expression of m2 muscarinic acetylcholine receptor mRNA in primary cultureof human prostate stromal cells

The aim of this study was to investigate the expression of the muscarinic a cetylcholine receptor (mAchR) subtypes mRNA in primary culture of human pro state stromal cells using the reverse transcription polymerase chain reacti on (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 s ubtypes (m1-m5) cDNA sequence. The m2, m3 and m4 subtype expected bands wer e detected: in particular m2 transcripts was strongly detected in the strom al cell culture. Each of the PCR products were subcloned into the pGEM-T pl asmid vector, sequenced and random primer labeled using P-32. Digoxigenin-l abeled cRNA probes were synthesized by in vitro transcription. RNA blotting using a m2 muscarinic receptor cDNA probe revealed a 4.5 kb single transcr ipt. However, m3 and m4 probes did not hybridize. Using in situ hybridizati on (ISH), m2 receptor mRNA signals were detected in several smooth muscle c ells. The staining was predominantly localized to the perinuclear cytoplasm . The m3 and m4 probes did not hybridize. These results suggested that mi r eceptor subtype plays a role in smooth muscle activity of the human prostat e.