Detection of m2 muscarinic acetylcholine receptor mRNA in human corpus cavernosum by in-situ hybridization

Life Sciences, Vol. 55, No. 8, pp. 621-627, 1994

Pergamon

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DETECTION OF m2 MUSCARINIC ACETYLCHOLINE RECEPTOR mRNA IN HUMAN CORPUS CAVERNOSUM BY IN-SITU HYBRIDIZATION Paul Toselli 1, Robert Moreland 2, and Abdulmaged M. Traish 1'2 1Department of Biochemistry and 2Department of Urology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118 U.S.A.

(Received in final form June 10, 1994)

Summary Relaxation and contraction of the smooth muscle of human corpus cavernosum (HCC) of the penis is essential for penile erection and detumescence. Relaxation of the smooth muscle is controlled, locally, by cholinergic, adrenergic and nonadrenergic, noncholinergic neurotransmitters as well as by the vascular endothelium, which lines the lacunar spaces and releases endothelium-derived relaxing factor. Cholinergic neurotransmitters are postulated to act on other neuroeffectors and on the endothelium rather than act directly on the trabecular smooth muscle. We use in-situ hybridization to determine the presence and distribution of muscarinic acetylcholine receptor (mAChR) subtype mRNA in HCC. First, we verified that the riboprobe used for insitu hybridization is specific for m2 mAChR subtype using Chinese Hamster Ovary cells transfected with m2 mAChR subtype. Then, we validated that the tissue obtained from surgical specimens is adequate for in-situ hybridization. The data demonstrate that m2 m A C h R subtype mRNA is expressed in HCC smooth muscle cells, m2 mAChR subtype mRNA expression is not detected in endothelium or nerves. It is possible that this receptor subtype plays a role in smooth muscle contraction. Key Words: muscarinic acetylcholine receptor, in-situ hybridization, corpus cavernosum The tone of the corpus cavernosum smooth muscle controls the state of penile erection. Smooth muscle tone is controlled locally by the release of cholinergic, adrenergic, and noncholinergic, nonadrenergic neurotransmitters, and by substances released from endothelial cells lining the lacunar spaces (blood sinuses) (for review, see 1,2). Acetylcholine (ACh) release from neurons participates in the neurogenic relaxation of corpus cavernous smooth muscle cell (3). Also, exogenous acetylcholine was shown to relax human corpus cavernosum (HCC) and rabbit corpus cavernosum in organ chambers (2,3). Cholinergic mechanisms, therefore, play an important, although non-exclusive, role in penile erection (4). Atropine blocks the physiological action of ACh in HCC, suggesting that ACh acts directly by binding to muscarinic acetylcholine receptor (mAChR) (3). Recent studies have suggested that mAChR on the endothelium may regulate the synthesis of nitric oxide, a substance shown to relax smooth muscle in organ chamber experiments (5). Using binding studies, we have demonstrated that HCC tissue and endothelial cells derived from this tissue express mAChR (6); and, we suggested that mAChR on the endothelial cells are likely to be of the m3 mAChR subtype (6). Recently, we demonstrated that mAChR on bovine aortic endothelial cells in culture exhibited the characteristics of m3 subtype (7). It remains unclear, Corresponding Author: Paul Toselli, Department of Biochemistry, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118.

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however, which subtype of mAChR is expressed in HCC smooth muscle cells and what is the function of this protein is these cells. The cloning of five distinct mAChR subtypes from animal and human genomes has been reported (8). It was shown that different subtypes mediate different responses (9). It is important, therefore, to identify which receptor subtype(s) is/are present in the various cellular elements of HCC. B i n d i n g studies in tissue or c e l l - h o m o g e n a t e s , although i m p o r t a n t for receptor characterization, do not fully identify receptor subtype(s) in various HCC cells. Similarly, in-vitro autoradiography studies which utilize non-specific muscarinic ligands cannot precisely identify or localize specific receptor subtype(s) (10). Identification and localization of a specific mAChR subtype in HCC requires the use of new approaches, such as immunocytochemistry (with receptor subtype-specific antibodies) or in-situ hybridization. The aim of this study is to determine the expression of m2 mAChR subtype mRNA in HCC by in-situ hybridization analysis.

Methods Muscarinic acetylcholine receptors in transfected CHO cells. Chinese Hamster Ovary (CHO) cells transfected with m2 subtype mAChR were provided by Dr. M.R. Brann (University of Vermont). Briefly, the cells were grown at 37 °C in air/CO s (95/5%) in tissue culture medium F l 2 / D u l b e c c o Minimal Essential Medium (50:50 v / v ) , supplemented with 10% fetal bovine serum, 50 units of penicillin and 50 units of streptomycin. CHO cells transfected with m2 mAChR subtype were used as controls. Transfected CHO cells were grown on Lab-Tek chamber glass slides for 3 days, fixed in 4% formaldehyde in PBS, dehydrated and stored in 100% methanol at -20 °C until used for experimentation. Tissue procurement and histological preparation. HCC was obtained from patients undergoing prosthesis implantation. In the operating room, HCC specimens (approximately 8 mm s) were placed in chilled phosphate buffered saline (PBS) and transported to the laboratory. The samples were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) for 30 min in PBS, pH 7.4, at 4 °C. The samples were dehydrated and embedded in Paraplast as described by Sassoon et al. (11 ). Sections, 3 um thick, were sectioned and stored at 4 °C until used for experimentation. l n - s i t u hybridization.

To insure that tissue obtained at surgery contains mRNA suitable for in-situ hybridization, we employed rat alphal(I) procollagen coding sequence (12). A 600 bp cDNA fragment coding the cterminal non-belical region was subcloned into pGEM (kindly provided by B.D. Smith) and used as a positive control. To synthesize anti-sense (ssS)-labeled probe (complimentary riboprobe), DNA template linearized with Pstl was transcribed by T7 RNA polymerase. We also used a 270 bp H i n c I I / E c o R I fragment of rat fibronectin cloned into p-SR270 (13) as a positive control. To synthesize anti-sense (asS)-labeled probe, DNA template linearized with H/ncII was transcribed by SP6 RNA polymerase. Both the rat alphaI procollagen and rat fibronectin coding sequences used for our in-situ hybridization studies cross-react, respectively, with either human alpha 1 procollagen or human fibronectin when examined by Northern blot analysis (data not shown).

Preparation of mAChR riboprobes. The probe for the human m2 mAChR subtype was prepared by cloning a 238 bp S s p I / S a c I restriction cDNA fragment (amino acids $280 through N357) into S m a I / S a c I digested pGEM3zf(-). To synthesize anti-sense (ssS)-labeled RNA probe (complementary riboprobe), DNA linearized with SacI was transcribed by SP6 RNA polymerase. To synthesize sense (ssS)-labeled RNA probe (homologous riboprobe), DNA linearized with PstI was transcribed by T7 RNA polymerase. Three-hundred nanograms of linearized DNA templates were transcribed in 36 mM Tris, pH 7.5, 5 mM MgCI2, 2 mM Spermidine, 9 mM NaCI, 9 mM dithiothreitol, 1 unit human placenta ribonuclease inhibitor, 0.5 mM each of CTP, ATP, GTP, 2.3 mM (ssS)-alpha UTP (250 Ci/mM; NEN) and either "1"3 or T7 RNA polymerase. The transcription reaction mixture was incubated 1-2 hours at 37 °C. This procedure yielded RNA transcripts with a specific activity greater than 5 x l0 s cpm/ug. For removal of DNA template, RNase-free DNase (1 unit/ug DNA) was added and the mixture incubated at 37 °C for 15 minutes. Total yeast RNA was added to obtain a concentration of

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250 ug/ml of final mixture concentration. Probe length was reduced to an approximate mean size of 60 nucleotides by alkali hydrolysis, and unincorporated nucleotides were removed by gel filtration on G-50 Sephadex. The radiolabeled riboprobe transcript fragments were concentrated by cold ethanol precipitation and suspended in hybridization buffer (see below). I n - s i t u hybridization procedure. The procedures used for probe preparation (see above), section treatment, hybridization and washing were those described by Sassoon et al. (11) and Toselli et al (14). The Paraplast embedded sections were deparaffinized in xylene, rehydrated through a graded series of ethanols (100% to 30%), rinsed in 0.85% saline solution (5 min), PBS (5 min), and post-fixed in a prepared and filtered solution of 4% formaldehyde (freshly prepared from paraformaldehyde) in PBS buffer (30 rain). Slides carrying sections were then rinsed in PBS (2 x 10 rain) and treated with a fresh solution of proteinase K (20 ug/ml, 7.5 min) in Tris-HCl, EDTA (50 mM, 5 mM, pH 7.2). They were then rinsed in PBS (5 min), refixed in 4% formaldehyde solution in PBS, dipped in distilled water and acetylated in a 0.09 M-solution of triethanolamine with acetic anhydride (1:400 v/v) for 10 minutes. The slides were subsequently rinsed in PBS and 0.85% saline (5 rain each), quickly dehydrated through a series of ethanols (30% to 100%), and allowed to dry at least 2 hours prior to hybridization. Probe was applied directly to tissue sections (20 ul) at a final adjusted concentration of 1 X l0 s cpm in hybridization buffer (50% deionized formamide, 0.3 M-NaCI, 20 mM-Tris-HCi (pH 7.4), 5 mM-EDTA, 10raM NaPO4 (pH 8.0), 10% dextran sulfate, Denhardt's, 50 ug/ml total yeast RNA) and tissue and probe were covered with a siliconized cover-glass (22 x 22 mm). Hybridization was carried out at 52 °C for approximately 16 hours in a humid chamber. Coverslips were gently floated o f f i n a 5X SSC ( I X SSC, 0.15 M-NaCI, 0.015 M-Sodium Citrate), 10 mM-dithiothreitol (D'IT) at 50 °C and subsequently the sections were subjected to a stringent washing at 60 °C in 50% formamide, 2X SSC, 0.1 M-DTT. Slides were then rinsed in STE (0.1 M-NaCI, 0.01-M Tris-HCl, 0.001-M EDTA) washing buffer and treated with RNase A (20ug/ml) in washing buffer for 45 min at 37 °C. Following washes at room temperature in 2X SSC (15 min) and 0. IX SSC (15 rain), respectively, the slides were rapidly placed in a series of ethanols containing 0.3 M-ammonium acetate. The samples were processed for standard light microscopic autoradiography using Kodak NTB-2 nuclear track emulsion. Analysis was carried out using both light- and dark-field optics on a Leitz Research Light Microscope.

Fig. 1 Control h y b r i d i z a t i o n for the d e t e r m i n a t i o n of m2 mAChR subtype riboprobe specificity. Transfected m2 mAChR subtype CHO cells were cultured on Lab-Tek chamber slides and examined using bright-field microscopy. The whole-mount CHO cells were hybridized with either anti-sense (Figure A) or sense (Figure B) m2 AChR riboprobe. Figure A shows signal variation in the individual cultured CHO cells, m2 riboprobe does not cross-react with CHO cells transfected with either ml or m3 subtype mAChR (data not shown). Arrows - hybridization signal.

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Results Determination of m2 subtype mAChR riboprobe specificity. In order to assess m2 mAChR subtype riboprobe specificity, CHO cells, transfected with the m2 mAChR subtype and cultured for 3 days on Lab-Tek chamber glass slides, were used. All insitu hybridization and signal detection procedures were conducted on un-sectioned, "whole mounted" cells. Figure IA shows CHO cells hybridized with complimentary (anti-sense) m2 mAChR subtype riboprobe (positive control). Note the strong hybridization signal present in the three cells with larger nuclei (arrows); also, note some cells with smaller nuclei have a weaker signal. Hybridization signal was not detected in CHO cells transfected with m2 mAChR subtype hybridized with homogeneous (sense) m2 mAChR subtype riboprobe (negative control) (Figure I B). Since we were concerned with mRNA degradation occurring in HCC tissue obtained at surgery and fixed after arriving at the laboratory, we chose to assess our in-situ methodology by examining pathological tissue known to exhibit highly expressed mRNA. For this reason, we obtained HCC from a patient with a clinical diagnosis of systemic sclerosis (scleroderma). Sections were hybridized with either: (i) anti-sense alphal(I) procollagen riboprobe (positive control), or (ii) antisense fibronectin riboprobe (positive control). Figure 2 illustrates the hybridization of anti-sense alphal(I ) procollagen riboprobe to the sclerodermatous HCC. Note the strong hybridization signal in the fibroblast-like cells (straight arrows) seen lying among large collagen bundles (see C, Figure 2).

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Fig. 2 Control hybridization for the determination of cell-specific hybridization in HCC tissue obtained at surgery. Paraffin-embedded 3-micron section through sclerodermatous corpus cavernosum hybridized with anti-sense alphal(I) procollagen riboprobe. Note the hybridization signal present in the fibroblast-like cells (straight arrows). C collagen bundle; curved arrow - endothelial cells with no detectable hybridization signal. Left panel - bright-field microscopy; right panel - dark-field microscopy.

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Figure 3 shows sclerodermatous HCC hybridized with anti-sense fibronectin riboprobe. The fibronectin hybridization signal is present in a pair of easily identifiable smooth muscle cells (SMC) (straight arrows). Also note fibronectin hybridization signal is not detected in neighboring SMC (Figure 3, larger curved arrows).

Fig. 3 Control hybridization for the determination of cell-specific hybridization in HCC tissue obtained at surgery. Paraffin-embedded 3-micron section through sclerodermatous corpus cavernosum hybridized with anti-sense fibronectin riboprobe. Note the hybridization signal present in a pair of smooth muscle cells (straight arrows). C collagen bundle. Larger curved arrows - smooth muscle cells with no detectable hybridization signal; smaller curved arrow - endothelial cells with no detectable hybridization signal. Left panel - bright-field microscopy; right panel - dark-field microscopy. Expression of m2 mAChR subtype mRNA in human corpus c a v e r n o s u m . To identify cells expressing mRNA coding for m2 mAChR subtype in HCC, i n - s i t u hybridization histochemistry was carried out using anti-sense m2 mAChR riboprobe on n o n sclerodermatous corpus cavernosum tissue samples. The microscopic distribution of m2 mAChR mRNA is shown in Figure 4. Note the hybridization signal present in the SMC (large arrows); m2 mAChR mRNA hybridization signal was not detected in the endothelium (curved arrow) or nerves (data not shown). Sclerodermatous HCC hybridized with anti-sense m2 mAChR subtype riboprobe was also examined (data not shown). In this case, the hybridization signal appeared evenly distributed among most neighboring SMC. We did not detect hybridization signal in sclerodermatous tissue sections hybridized with sense m2 subtype mAChR riboprobe (data not shown).

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Fig. 4 In-situ hybridization of non-sclerodermatous corpus cavernosum hybridized sense m2 mAChR subtype riboprobe. Note the hybridization signal present muscle cell bundles (large arrows). Also note endothelial cells have no signal (curved arrow). Left panel - bright-field microscopy; right panel microscopy.

with antiin smooth detectable dark-field

Discussion

Relaxation of the smooth muscle of the corpus cavernosum leads to penile erection (1,2). This mechanism is controlled locally by cholinergic, adrenergic and non-adrenergic, non-cholinergic neurotransmitters. Relaxation of the corpus cavernosum with exogenous acetylcholine is thought to be mediated by the endothelium, through binding to a specific receptor subtype, which causes release of nitric oxide (5). Several studies have reported on the presence of mAChR in HCC (5,6). The specific mAChR subtypes or their cellular location, however, was not identified. It has been suggested that the effects of ACh on HCC is through prejunctional modulation of the adrenergic and non-adrenergic/non-cholinergic neurotransmitters, through specific binding on ACh to mAChR. Currently, five mAChR subtypes have been described to be present in various tissues (8). This study was aimed to determine the expression of m2mAChR subtype mRNA in HCC cellular elements. HCC smooth muscle cells expressed m2 mAChR subtype, as shown by the hybridization signal. The endothelium, however, did not show any hybridization signal, suggesting lack of m2 mAChR mRNA expression. This observation is consistent with our studies in cultured HCC endothelial cells (6) and cultured bovine aortic endothelial endothelium cells (7), in which mAChR exhibited binding characteristics of m3 subtype. Further, the presence of m2 mAChR mRNA in smooth muscle cells is consistent with immunocytochemical localization using antibody raised to a synthetic peptide derived from the sequence of m2 subtype (data not shown).

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The demonstration that HCC smooth muscle expresses the m2 mAChR subtype suggests a role for this receptor in the function of smooth muscle cells. We speculate that m2 mAChR in HCC smooth muscle may function to induce smooth muscle contractions. Thus, ACh may have a dual role in regulating penile erection. Relaxation through prejunctional modulation of the adrenergic and the nonadrenergic/noncholinergic neurotransmitters as well as the endothelium and contraction through direct binding to the smooth muscle cells. The physiological responses of HCC smooth muscle cells to ACh through m2 mAChR, however, remains to be determined. As pointed out by Thomas et al. (15), numerous pharmacological studies have demonstrated that the m2 mAChR subtype is the most abundant muscarinic subtype in a variety of smooth muscle containing tissues, including the gastrointestinal tract, urinary bladder, respiratory tract and vasculature. In summary, we demonstrate for the first time that HCC smooth muscle cells express m2 m A C h R subtype and the e n d o t h e l i u m may express a d i f f e r e n t subtype of mAChR. The physiological role of m2 mAChR in HCC smooth muscle cells and in the control of penile erection, however, remains unknown, and requires further investigations.

Acknowledgments This work was supported by the U.S. Public Health Service NIH DDK Grant DK40025.

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