All procedures on experimental animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University. Male Sprague-Dawley rats (350-400 g) were killed by 100% CO₂ inhalation and the ventral lobe of the prostate gland removed rapidly thereafter. The fibrous capsule was removed and the tissue was cut with scissors into small pieces (1-2 µL) in a phosphate-buffered Ca²⁺-free Tyrode's solution. The tissue was digested for 25 min at 37℃ in Ca²⁺-free Tyrode's solution containing collagenase (2 mg/mL, Wako, Japan), trypsin inhibitor (1 mg/mL, Sigma, St. Louis, MO, U.S.A.), bovine serum albumin (3 mg/mL, Sigma) and dithiothreitol (DTT, 1 mg/mL, Sigma). Following digestion and subsequent removal of supernatant, tissue segments were transferred to fresh Tyrode's solution and agitated gently using a fire-polished wide bore (1-2 mm) Pasteur pipette. Isolated cells were isolated daily then stored in fresh solution at 4℃ for up to 6 hr. Dispersed cells were moved into the experiment chamber and examined by using an inverted microscope (IX-70, Olympus, Japan). After the digestion procedure, most of isolated single cells had elongated columnar shape, a typical feature of secretory epithelial cells. Besides columnar cells, we could identify round or oval shape cells with relatively dark cytoplasm that were regarded as RPNECs in the present study. The putative RPNECs were carefully discriminated from damaged or swollen epithelial cells that showed also round or oval shapes but with pale cytoplasm. The reliability of above criteria for identifying RPNECs were assured by constant responses of chosen cells to noradrenaline in [Ca²⁺]_c or to α,β-meATP. Such responses to NA and α,β-meATP are not observed in columnar epithelial cells (15, 16).

Dispersed single cells were loaded with acetoxymethyl ester form of Fura-2 (2 µM) in the Ringer's solution for 20 min at room temperature and then washed out with fresh solution. The putative RPNECs were identified as above criteria, and the region of interest for Fura-2 experiment was set so that the fluorescence from the single RPNEC could be collected selectively. The recording of [Ca²⁺]_c was performed with a microfluorimetric system consisting of an inverted fluorescence microscope (Olympus IX-70, Japan) with a dry-type fluorescence objective lens (×40, NA 0.85), a photomultiplier tube (type R 1527, Hamamatsu, Japan) and Deltascan illuminator (Photon Technology International Inc, Monmouth, NJ, U.S.A.). Light was provided by a 75-W xenon lamp (Ushino, Tokyo, Japan) and a chopper wheel alternated the light path to monochromators (340 and 380 nm) with a frequency of 5 Hz, and intensity of emitted light at 510 nm was measured. As a measure of [Ca²⁺]_c the fluorescence emission ratio at 340 nm/380 nm excitation (F_340/380) is presented.

To synthesize first strand cDNA, RPNECs were collected individually by using microelectrodes with inner diameter of about 25-30 µm on the stage of inverted microscope for the patch clamp. After aspiration, the cell was expelled from the pipette into ice cooled 0.2 mL tubes, which contained 2 units of RNase-free DNase (Takara Bio Inc., Shiga, Japan), 40 units of ribonuclease inhibitor (Takara Bio Inc., Otsu, Shiga, Japan), 50 mM Tris-HCl (pH 8.3), 3 mM MgCl₂, 75 mM KCl, and 100 mM DTT. The reaction mixture was incubated at 37℃ for 30 min, followed by 95℃ for 5 min. And then reverse transcription reaction was performed in the presence of 0.8 µg of oligo (dT)₁₅ (Roche Applied Science, Indianapolis, IN, U.S.A.), 10 mM each of dATP, dTTP, dGTP and dCCT (Roche Applied Science), and 200 units of SuperScript™ reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA, U.S.A.). The reaction proceeded for 10 min at 25℃, and for 50 min at 42℃, followed by a 15-min step at 70℃ to inactivate the SuperScript™ reverse transcriptase.

Two rounds of PCR (PTC-0150 MiniCycler™; MJ Research Inc., Waltham, MA, U.S.A.) were performed with outer primers and nested primers of M1, M2, M3, M4, and M5 muscarinic receptors of rat (18, 19). First strand cDNA was used for the first PCR amplification. This mixture was then used for the second nested PCR amplification. PCR reactions were carried out using the 2.5 units of Taq polymerase (Takara Bio Inc.) containing of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 10 mM each of dATP, dTTP, dGTP and dCCT, 100 pM each of sense and antisense primers, and DNA template. Temperature cycling proceeded as follows: 1 cycle at 95℃ for 5 min and 45 cycles at 95℃ for 60 sec, 50℃ (for M4) or 55℃ (for M1, M2, M3, M5) for 60 sec, and 72℃ for 60 sec, followed by 72℃ for 10 min. PCR products were then subjected to gel electrophoresis on a 1.5% agarose gel containing ethidium bromide. Primers for the PCR of M₁, M₂, M₃, M₄, and M₅ are as follows: M₁ sense, 5'-GCA CAG GCA CCC ACC AAG CAG-3'; M₁ antisense, 5'-AGA GCA GCA GCA GGC GGA ACG-3'; M₂ sense, 5'-GGC AAG CAA GAG TAG AAT AAA-3'; M₂ antisense, 5'-GCC AAC AGG ATA GCC AAG ATT-3'; M₃ sense, 5'-CAC AGG CAG TTC TCG AAG CT-3'; M₃ antisense, 5'-AGG ACG GTA GCT TGG TAG AG-3'; M₄ sense, 5'-AGT GCT TCA TCC AGT TCT TGT CCA-3'; M₄ antisense, 5'-CAC ATT CAT TGC CTG TCT GCT TTG-3'; M₅ sense, 5'-CTC ATC ATT GGC ATC TTC TCC A-3'; M₅ antisense, 5'-GGT CCT TGG TTC GCT TCT CTG T-3'; M₁ nested sense, 5'-CCT CCC AAA AGC TCC CCA-3'; M₁ nested antisense, 5'-TGT CCC GGA AGG CTT TGT-3'; M₂ nested sense, 5'-GAA GGA AAA GAA GGA ACC TGT-3'; M₂ nested antisense, 5'-GTC CTG GTC ACT TTC TTT TCC-3'; M₃ nested sense, 5'-CTC GAA GCT GTA GCA GCT ATG-3'; M₃ nested antisense, 5'-GGA GCT ATG GCC TGG ACG CTT GAG G-3'; M₄ nested sense, 5'-ACC CGG CAG TGA CCT TTG-3'; M₄ nested antisense, 5'-TTA CAA TTT GAA TCT TGG ACC AT-3'; M₅ nested sense, 5'-GAA CCT CTA CAC GAC CTA CAT C-3'; M₅ nested antisense, 5'-CCC GGT AGA TCC GGC AGT AG-3'.

All the experiments were performed in phosphate-buffered saline (PBS) containing 145 mM NaCl, 1.6 mM K₂HPO₄, 0.4 mM KH₂PO₄, 1 mM MgCl₂, 2 mM CaCl₂ and 5 mM D-glucose at pH 7.4 titrated with NaOH. CaCl₂ was omitted in the enzymatic isolation of single prostate cells.

The following drugs were used: acetylcholine chloride, para-fluoro-hexahydrosiladifenidol (pF-HHSiD) hydrochloride, 4-hydroxy-2-butynyl-1-trimethylammonium-m-chlorocarbamilate chloride (McN-A-343), telenzepine dihydrochloride, himbacine hydrochloride, and 4-diphenyl acetoxy-N-methyl piperidine methobromide (4-DAMP) (Sigma-Aldrich, Seoul, Korea). All other drugs except pF-HHSiD were dissolved in distilled water. Stock concentration (1 mM) of pF-HHSiD was prepared in dimethylsulfoxide. Dilutions to working concentrations were made in PBS solution.

The data are presented as original recordings, current-voltage (I-V) curves, histograms and bar graphs of mean±S.E.M. (for n cells tested). When necessary, Student's t-test for paired samples was applied, since the control and test recordings were made from the same cell. Probability value below 0.05 (p<0.05) was regarded as significant.

Among the dispersed prostate cells, we could discriminate the putative neuroendocrine cells with a round shape and relatively dark cytoplasm from the columnar epithelial cells and the thin smooth muscle cells as has been described previously (12, 14-16). The Fura-2 fluorescence ratio (F_340/380, see Materials and methods) was measured in RPNECs. An application of ACh (5 µM) induced a transient increase in [Ca²⁺]_c which is followed by a plateau-like level above the control (Fig. 1). In the same RPNECs, the effects of high K⁺ (60 mM), phenylephrine (PhE, 5 µM), UTP (20 µM), and α,β-meATP (0.5 µM were confirmed since the adrenergic and purinergic (both P2X and P2Y) stimulations as well as the membrane depolarization increase the [Ca²⁺]_c of RPNECs (15, 16). Summary of Ca²⁺ responses to various stimuli demonstrate that the effect of ACh is stronger than the KCl-induced depolarization (p< 0.05) and similar with the responses to PhE, UTP and α,β-meATP (Fig. 1B).

The ACh-induced increase in [Ca²⁺]_c (ACh-Δ[Ca²⁺]_c) was not affected by the removal of extracellular Ca²⁺ (Fig. 2A), indicating a mechanism of Ca²⁺ release from the intracellular stores (e.g. endoplasmic reticulum). In the adrenal chromaffin cells, ACh activates nicotinic ionotropic receptors that induce membrane-depolarization as well as direct Ca²⁺ influx (20). In RPNECs, however, the pretreatment with atropine (1 µM), a representative antagonist of muscarinic receptors, completely blocked ACh-Δ[Ca²⁺]_c (Fig. 2B). This result excludes the involvement of nicotinic ACh receptors in RPNECs (Fig. 2B).

To identify the subtype(s) of muscarinic receptors in RPNECs, effects of subtype-specific pharmacological agents were examined (21, 22). A pretreatment with telenzepine (1 µM), an M₁-sepecific antagonist, did not affect the amplitude of the ACh-Δ[Ca²⁺]_c (93±9.0% of control, n=4, Fig. 3A). Also, the M₁-specific agonist (McN-A-343 1 µM) had no effect on the [Ca²⁺]_c of RPNECs (Fig. 3B, n=3). In the same RPNEC where telezepine had negligible effect, a pretreatment with pF-HHSiD (0.3 µM), a specific M₃ antagonist, completely blocked the ACh-Δ[Ca²⁺]_c in a reversible manner (Fig. 3A, n=5). A lower concentration of pF-HHSiD (0.1 µM) showed partial inhibitory effects (38±20.5% of control, n=4). Also, 4-DAMP (1 µM), another subtype-selective antagonist for M₃ receptors, blocked the ACh-Δ[Ca²⁺]_c (n=2, data not shown). Finally, himbacine (1 µM), an antagonist for M₂ and M₄ receptors, showed a partial inhibitory effect on the ACh-Δ[Ca²⁺]_c (Fig. 3C, 64±9.1% of control, n=5).

Next, to further identify the muscrainic receptor subtype in RPNECs, a nested RT-PCR was performed with isolated RPNECs (see Materials and methods). Among the tested primers, only the M₃-specific pairs showed a positive result (Fig. 4). The same results were confirmed in three separate experiments.