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J. Biol. Chem., Vol. 279, Issue 26, 27345-27356, June 25, 2004

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Purinergic Receptors Coupled to Intracellular Ca²⁺ Signals and Exocytosis in Rat Prostate Neuroendocrine Cells^*

Jun Hee Kim, Joo Hyun Nam, Mean-Hwan Kim, Duk-Su Koh, So-Jung Choi¶, Soo Jeong Kim, Ji Eun Lee, Kyeong Min Min, Dae-Yong Uhm, and Sung Joon Kim||

From the Department of Physiology and Center for Molecular Medicine and the ^¶Department of Molecular and Cellular Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 and the Department of Physics, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea

Received for publication, December 11, 2003 , and in revised form, April 5, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Rat prostate neuroendocrine cells (RPNECs) display a variety of ion channels and exhibit -adrenergic regulation of cytosolic Ca²⁺ concentration ([Ca²⁺])_c. In this study, purinergic regulation of [Ca²⁺]_c and exocytosis was investigated in freshly isolated single RPNECs showing chromogranin A immunoreactivity. The presence of P2X and P2Y receptors in RPNECs was verified by the transient activation of Ca²⁺-permeable cationic channels and the release of Ca²⁺ from intracellular stores by extracellular ATP, respectively. The transient inward cationic current was effectively activated by ,-methyleneadenosine 5'-triphosphate (,-MeATP) and blocked by 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, suggesting the presence of a P2X₁ or P2X₃ subtype. For the release of stored Ca²⁺, ATP and UTP were equally potent, indicating the functional expression of the P2Y₂ or P2Y₄ subtype. The mRNAs for P2X₁ and P2Y₂ were confirmed from reverse transcription-PCR analysis of RPNECs. The application of ,-MeATP induced large and transient increases in [Ca²⁺]_c, which were not attenuated by the blockers of voltage-activated Ca²⁺ channels or by depleting intracellular Ca²⁺ stores, but were abolished by omitting extracellular Ca²⁺. The application of UTP increased [Ca²⁺]_c to 55% of the peak [Ca²⁺]_c induced by ,-MeATP. The application of ,-MeATP induced exocytotic responses of RPNECs as monitored by carbon fiber amperometry and capacitance measurements. To our interest, the application of UTP did not induce amperometric currents, but reduced the membrane capacitance, indicating a net endocytosis. From these results, we postulate that a sharp rise in [Ca²⁺]_c by the P2X-mediated Ca²⁺ influx is required for exocytosis, whereas the relatively slow release of stored Ca²⁺ induces endocytosis in RPNECs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Prostate neuroendocrine cells, considered to be a group of amine precursor uptake and decarboxylation cells, are intraepithelial regulatory cells with paracrine properties. Similar to other amine precursor uptake and decarboxylation cells in the gastric gland (1), prostate neuroendocrine cells may also influence the growth of the prostate gland or regulate the exocrine secretion of prostatic fluid (2, 3). Previous immunohistochemical studies suggest that prostate neuroendocrine cells both store and produce neurosecretory products such as serotonin, histamine, bombesin, calcitonin, and parathyroid hormone-related peptides that could regulate the growth, invasiveness, metastatic processes, and angiogenesis related to prostatic carcinoma (4–7).

In a previous study, we initially reported that putative rat prostate neuroendocrine cells (RPNECs)¹ display spontaneous action potentials and voltage-activated Ca²⁺ channels (both L and N types) in accordance with the electrically excitable properties of neuroendocrine cells (8). Also, pharmacological studies clearly demonstrated that RPNECs functionally express ₁- and ₂-adrenergic receptors, which are linked to the release of stored Ca²⁺ and the inhibition of N-type Ca²⁺ channels, respectively (9). Despite the aforementioned electrophysiological evidence of excitable cells, the genuine property of neuroendocrine cells, viz. exocytosis, has not yet been directly investigated in RPNECs.

ATP is frequently colocalized with noradrenaline in postganglionic sympathetic nerve fibers and acts as a cotransmitter in several tissues (10). The purinoreceptors (P2), receptors for extracellular ATP, are divided into two distinct receptor families: G-protein-coupled metabotropic receptors (P2Y receptors) and receptor ion channels with a nonselective permeability to cations, including Ca²⁺ (P2X receptors) (10). In some neurons and neuroendocrine cells, extracellular ATP induces exocytosis in a Ca²⁺-dependent manner. In the sympathetic neurons, for example, it has been suggested that the P2X receptors mediate a positive modulation of noradrenaline release, whereas the G-protein-coupled P2Y receptors mediate the opposite response (11). In the case of adrenal chromaffin cells displaying heterogeneous expression of P2X and P2Y receptors, P2X channels are preferentially localized to noradrenaline-secreting cells (12). The pathophysiological significance of purinoreceptors has also been suggested by the Ca²⁺ release and growth regulation of prostate tumor cells by extracellular ATP (13).

Considering the suspected role of ATP as a cotransmitter from the sympathetic nerves that regulate the function of prostate glands, we chose to investigate the operation of multiple Ca²⁺ translocation mechanisms linked to purinoreceptors in RPNECs. In addition, we compared the efficiency of the exocytosis triggered by P2X and P2Y receptors recruiting different sources of calcium ions in RPNECs. The direct measurement of exocytosis in prostate neuroendocrine cells was performed for the first time in this study.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Isolation—Male Sprague-Dawley rats (350–400 g) were killed by 100% CO₂ inhalation, and the ventral lobe of the prostate gland was removed rapidly thereafter. The procedure for single cell isolation was the same as described in our previous reports (8, 9). Briefly, the chopped tissue (1–2 mm³) was digested for 25 min at 37 °C in Ca²⁺-free phosphate-buffered saline (PBS) containing collagenase (2 mg/ml; Wako, Osaka, Japan), trypsin inhibitor (1 mg/ml; Sigma), bovine serum albumin (3 mg/ml; Sigma), and dithiothreitol (1 mg/ml; Sigma). Following digestion, tissue segments were transferred to fresh PBS and agitated gently using a fire-polished wide bore (1–2 mm) Pasteur pipette. Cells were isolated daily and stored in fresh solution at 4 °C for up to 6 h. Dispersed cells were moved into the experiment chamber and examined using an Olympus IX-70 inverted microscope. After the digestion procedure, most of the isolated single cells had an elongated columnar shape, a typical feature of secretory epithelial cells. In addition to columnar cells, we could identify round- or oval-shaped cells with a relatively dark cytoplasm that were regarded as RPNECs in this study.

Immunofluorescence Confocal Microscopy—Chromogranin A was identified using mouse monoclonal antibody LK2H10 (NeoMarkers, Fremont, CA). Cells were maintained at room temperature on a coverslip coated with polylysine (5%), fixed with 2% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and then incubated with PBS containing 3% bovine serum albumin (blocking solution) for 45 min. Cells were incubated with primary antibodies, followed by treatment with bridging Alexa Fluor 546-coupled goat anti-mouse Ig (Molecular Probes, Inc., Eugene, OR) and double staining with fluorescein isothiocyanate (FITC)-conjugated phalloidin. Cells were then mounted onto glass slides and investigated using a Zeiss LSM 510 laser-scanning confocal microscope (FITC, excitation at 488 nm and emission at 520 nm; and Alexa Fluor 546, excitation at 556 nm and emission at 573 nm).

Intracellular Ca²⁺ Measurement—Dispersed single cells were loaded with fura-2 acetoxymethyl ester (2 µM) in PBS for 20 min at room temperature and then washed out with fresh solution. RPNECs were identified following the above criteria, and the region of interest for the fura-2 experiment was set so that the fluorescence from a single RPNEC could be collected selectively. The recording of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) was performed with a microfluorometric system consisting of an Olympus IX-70 inverted fluorescence microscope with a dry-type fluorescence objective lens (x40, numerical aperture of 0.85), a photomultiplier tube (type R1527, Hamamatsu Photonics, Hamamatsu, Japan), and a Deltascan illuminator (Photon Technology International Inc., Lawrenceville, NJ). Light was provided by a 75-watt xenon lamp, and a chopper wheel alternated the light path to monochromators (340 and 380 nm) with a frequency of 10 Hz; the intensity of emitted light at 510 nm was measured. As a measure of [Ca²⁺]_c, the fluorescence emission ratio at 340/380 nm excitation (F_340/380) is presented.

Fluorescence Quenching Experiment—The divalent cation Mn²⁺ (0.1 mM) can be used as a surrogate for Ca²⁺ to trace Ca²⁺ influx (14). Since fura-2 has a high affinity for Mn²⁺ (K_d = 5 nM), essentially all Mn²⁺ ions entering a fura-2-loaded cell are trapped as fura-2·Mn²⁺ complexes. These complexes are virtually non-fluorescent at all wavelengths. Thus, an accelerated attenuation of fura-2 fluorescence provides an estimate of the entry of divalent cations (e.g. Ca²⁺) into a cell.

Single Cell Reverse Transcription (RT)-PCR—To synthesize first strand cDNA, RPNECs were collected individually using microelectrodes with an inner diameter of 25–30 µm on the stage of an inverted microscope for the patch clamp. After aspiration, the cell was expelled from the pipette into ice-cooled 0.2-ml tubes that contained 2 units of RNase-free DNase (Takara Bio Inc., Shiga, Japan), 40 units of ribonuclease inhibitor (Takara Bio Inc.), 50 mM Tris-HCl (pH 8.3), 3 mM MgCl₂, 75 mM KCl, and 100 mM dithiothreitol. The reaction mixture was incubated at 37 °C for 30 min, followed by 95 °C for 5 min. The reverse transcription reaction was then performed in the presence of 0.8 µg of oligo(dT)₁₅ (Roche Applied Science); 10 mM each dATP, dTTP, dGTP, and dCTP (Roche Applied Science); and 200 units of SuperScript^TM reverse transcriptase (Invitrogen). The reaction proceeded for 10 min at 25 °C and for 50 min at 42 °C, followed by a 15-min step at 70 °C to inactivate the SuperScript^TM reverse transcriptase.

Two rounds of PCR (PTC-0150 MiniCycler^TM, MJ Research, Inc., Waltham, MA) were performed with outer primers of P2Y₂, P2Y₄, P2X₁, and P2X₃ (15, 16) from rat and four sets of nested primers. First strand cDNA was used for the first PCR amplification. This mixture was then used for the second nested PCR amplification. PCRs were carried out using 2.5 units of Taq polymerase (Takara Bio Inc.) containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 10 mM dATP, 10 mM dTTP, 10 mM dGTP, 10 mM dCTP, 100 pM each sense and antisense primer, and DNA template. Temperature cycling proceeded as follows: one cycle at 95 °C for 5 min and 45 cycles at 95 °C for 30 s, 50 °C (for P2X₃) or 55 °C (for P2Y₂, P2Y₄, and P2X₂) for 60 s, and 72 °C for 90 s, followed by 72 °C for 10 min. PCR products were then subjected to gel electrophoresis on a 1.5% agarose gel containing ethidium bromide. The primers used for the PCR of P2Y₂, P2Y₄, P2X₁, and P2X₃ were as follows (15, 16): P2Y₂ sense, 5'-CTG CCA GGC ACC CGT GCT CTA CTT-3'; P2Y₂ antisense, 5'-CTG AGG TCA AGT GAT CGG AAG GAG-3'; P2Y₂ nested sense, 5'-GTC ACC ACC AGC GTG AGA GGG-3'; P2Y₂ nested antisense, 5'-GTA ATA GAG GGT GCG GGT GAC G-3'; P2Y₄ sense, 5'-CAC CGA TAC CTG GGT ATC TGC CAC-3'; P2Y₄ antisense, 5'-CAG ACA GCA AAG ACA GTC AGC ACC-3'; P2Y₄ nested sense, 5'-CCA CTG CGG GCA ATC CGC TGG-3'; P2Y₄ nested antisense, 5'-CCA CAG CAA TGG TAC GGA GGG-3'; P2X₁ sense, 5'-GAA GTG TGA TCT GGA CTG GCA CGT-3'; P2X₁ antisense, 5'-TGC GTC AAG TCC GGA TCT C-3'; P2X₁ nested sense, 5'-CGG ACT GTA TGG GGA GAA GA-3', P2X₁ nested antisense: 5'-CCT CTT AGG CAG GAT GTG GA-3'; P2X₃ sense, 5'-CAA CTT CAG GTT TGC CAA A-3'; P2X₃ antisense, 5'-TGA ACA GTG AGG GCC TAG AT-3'; P2X₃ nested sense, 5'-ATC ATC CCC ACC ATT ATC-3'; and P2X₃ nested antisense, 5'-AAA TAG CAG CCC TTC TTC-3'.

Patch Clamp Methods—Isolated cells were transferred into a bath situated on the stage of an Olympus IX-70 inverted microscope. The bath (0.3 ml) was superfused at 10 ml/min, and voltage clamp experiments were performed at room temperature (22–25 °C). Patch pipettes (with a free tip resistance of 2.5–3 megaohms) were connected to the head stage of an Axopatch 1-D patch clamp amplifier (Axon Instruments, Inc., Foster City, CA). Liquid junction potentials were corrected with an offset circuit prior to each experiment. For the perforated whole cell patch clamp, a stock solution of nystatin in dimethyl sulfoxide (15 mg/ml) was added to the pipette solution, yielding a final concentration of 0.15 mg/ml. A steady-state perforation (series resistance of <20 megaohms) was usually achieved within 10 min after making a giga-seal. pCLAMP Version 7.0 software and Digidata-1200A (both from Axon Instruments, Inc.) were used for data acquisition and the application of command pulses. The voltage and current data were low pass-filtered (5 kHz) and displayed on a computer monitor. Current traces were stored in a Pentium-grade computer and analyzed using Origin Version. 6.1 (Microcal Software Inc., Northampton, MA).

Carbon Fiber Electrode (CFE) Amperometry—CFEs were fabricated from carbon fibers (5–11-µm diameter) and polypropylene micropipettor tips of 10-µl volume as described previously (17). The tip of the electrode was closely apposed to the cell surface to minimize the diffusion distance from the release sites. The amperometric current, generated by the oxidation of released bioamines (e.g. serotonin) at the exposed tip of the CFE, was measured using an EPC-9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) operated in the voltage clamp mode at a holding potential of 600 mV. Amperometric signals were low pass-filtered at 0.1 kHz with a gain of 20 or 50 mV/pA and then sampled at 0.5 kHz. Amperometric recordings were semiautomatically analyzed using software written in Igor (WaveMetrics, Lake Oswego, OR). Some recordings with a small number of amperometric signals were plotted on a fast chart recorder, and the events were counted manually. To evaluate the relative exocytosis, the numbers of exocytotic events in the control and test conditions were averaged for 2 min, respectively.

Capacitance Measurements—Capacitance measurements were performed using the Lindau-Neher technique implemented as the "sine + dc" mode of the "software lock-in" extension of PULSE software (HEKA Elektronik). A 1 kHz, 35-mV peak-to-peak sinusoid stimulus was applied about a direct current holding potential of -60 mV. For the simultaneous acquisition of membrane current and capacitance, the X-chart program (HEKA Elektronik) was used.

Solutions and Chemicals—All experiments were performed in PBS containing 145 mM NaCl, 1.6 mM K₂HPO₄, 0.4 mM KH₂PO₄, 1 mM MgCl₂, 2 mM CaCl₂, and 5 mM D-glucose (pH 7.4) titrated with NaOH. CaCl₂ was omitted in the enzymatic isolation of single prostate cells. The pipette solution for recording the P2X receptor current and resting membrane potential under nystatin-perforated conditions contained 135 mM KCl, 1 mM MgCl₂, 0.5 mM EGTA, 5 mM D-glucose, and 10 mM HEPES (pH 7.2) titrated with KOH. The conventional whole cell pipette solution for the simultaneous measurement of membrane currents and [Ca²⁺]_c contained 130 mM CsCl, 1 mM MgCl₂, 500 µM K₅-fura-2, 5 mM D-glucose, and 10 mM HEPES (pH 7.2) titrated with CsOH. All drugs and chemicals were purchased from Sigma. The pipette solution for the capacitance measurement contained 110 mM CsCl, 40 mM HEPES, 0.3 mM EGTA, 1 mM MgCl₂, 0.2 mM CaCl₂, 0.35 mM GTP, and 2 mM MgATP with a free [Ca²⁺]_c of 350 nM. For the simultaneous measurement of [Ca²⁺]_c and capacitance, EGTA and CaCl₂ in the pipette solution were replaced with 0.15 mM K₅-fura-2.

Data Analysis and Statistics—The data are presented as original recordings, current-voltage (I-V) curves, histograms, and bar graphs of means ± S.E. (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. p < 0.05 was regarded as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of RPNECs and ATP-induced [Ca²⁺]_c Increase—Among the dispersed prostate cells, we could discriminate between putative neuroendocrine cells with a round shape and relatively dark cytoplasm and the columnar epithelial cells and thin smooth muscle cells (8, 9) The presence of chromogranin A, a representative marker of neuroendocrine cells, was confirmed. Fig. 1A shows a representative confocal image of chromogranin A-positive RPNECs co-stained with FITC-conjugated phalloidin, indicating the distribution of F-actin. It is noteworthy that chromogranin A distributes in a granular fashion at the periphery of the cell, where the signal of F-actin is not strong. The following experiments were performed with putative RPNECs, with which the previous patch clamp study and Ca²⁺ measurement consistently showed characteristics of electrically excitable cells (8, 9).

View larger version (24K): [in this window] [in a new window]   FIG. 1. Identification of RPNECs and ATP-induced increase in [Ca2+]c. A, immunostaining of chromogranin A (panel a, red) and F-actin (panel b, green) in RPNECs. Isolated prostatic cells were stained with anti-chromogranin A mouse IgG and FITC-conjugated phalloidin. Visualization was realized with Alexa Fluor-coupled anti-mouse IgG and FITC. Panel c is a image of chromogranin A and F-actin. Bar = 10 µm. B, representative trace of the fluorescence ratio (F340/380) for ATP (100 µM)(black bars) in the presence and absence of extracellular Ca2+ (Ca2+-free, white bar). Note that the initial transient increase in [Ca2+]c (Ca2+ transient) by ATP (arrowhead) disappeared under the Ca2+-free condition. The trace of the first response overlapped with that of the second response (red dotted line). The time break indicates 5 min. C, fluorescence intensities of fura-2 excited by wavelengths of 340 nm (F340) and 380 nm (F380). The y scale indicates the arbitrary unit (a.u.) of fluorescence. With MnCl2 (0.1 mM) in the Ca2+-free bath solution (hatched bar), the application of ATP (100 µM) immediately attenuated both F340 and F380, followed by a symmetrical transient increase and decrease in F340 and F380, respectively.

P2Y-mediated Release of Ca²⁺ in RPNECs—UTP, a pyrimidine triphosphate, activates some subtypes of P2Y receptors and induces inositol 1,4,5-trisphosphate-mediated Ca²⁺ release (10). Thus, we compared the concentration dependence of stored Ca²⁺ release induced by extracellular ATP and UTP (Fig. 2, A and B). ATP or UTP was applied in the absence of extracellular Ca²⁺ to exclude the Ca²⁺ influx pathways. During the interval of ATP application, CaCl₂ (2 mM) was added back to replenish the intracellular calcium stores. The half-effective concentrations (EC₅₀) of ATP and UTP were 2.8 and 3.8 µM, respectively. The EC₅₀ of UTP in the presence of 2 mM CaCl₂ was 2.2 µM, not significantly different from the EC₅₀ of UTP under the Ca²⁺-free condition (Fig. 2B).

View larger version (30K): [in this window] [in a new window]   FIG. 2. Subtypes of P2Y receptors coupled to the release of stored Ca2+. A, representative trace of F340/380 demonstrating the concentration-dependent effects of ATP (black bars) in the Ca2+-free external solution (white bars). B, concentration-F340/380 relationships of ATP (; n = 11) and UTP (; n = 6) in the Ca2+-free external solution and UTP in the control solution (; n = 6). The peak amplitudes of F340/380 (F340/380) were normalized to the maximum effect by 100 µM ATP or UTP, and the mean values were fitted by a function (normalized F340/380 = 1/(1 + (tested concentration/EC50))). C, summary of F340/380 by various purinergic agonists under the Ca2+-free condition. In each cell, F340/380 was normalized to that by 10 µM noradrenaline in the control solution. D, summary of the blocking effects of suramin (50 µM) on UTP-induced F340/380 (n = 7). E, single cell RT-PCR analysis of P2Y2 and P2Y4 receptors in RPNECs. 2-MeS-ATP, 2-methylthio-ATP.

Ca²⁺-permeable P2X Channels in RPNECs—Under the nystatin-perforated or conventional whole cell patch clamp conditions, extracellular ATP (10 µM) induced a fast transient inward current (I_ATP) (Fig. 3A). In some cases, to ensure fast solution exchange, 10 µM ATP was applied by puffing from a thin polyethylene tube (tip diameter of 100 µm) located 50–100 µm from the recorded cell. The "local puff" method, combined with a nystatin-perforated whole cell clamp, was required to prevent the rundown of I_ATP following repetitive stimulation (e.g. Fig. 3D).

View larger version (46K): [in this window] [in a new window]   FIG. 3. P2X receptors and ATP-induced inward current (IATP). A, original trace of IATP induced by a local puff of ATP (10 µM) under nystatin-perforated patch conditions. B, I-V relationship of IATP obtained under conventional whole cell patch conditions. Ramp depolarization from -100 to 60 mV (0.32 V/s, holding potential of -60 mV) was applied every second, and the I-V curve was obtained by the subtraction of the control current from IATP. C, ,-MeATP (10 µM)-induced transient inward current (holding potential if -60 mV) similar to IATP. D, inhibition of IATP by TNP-ATP pretreatment. TNP-ATP (3 nM) was applied for 3 min (white bar) before the addition of ATP (black bars). The time break indicates 5 min. E, RT-PCR analysis of P2X1 and P2X3 subtypes in single RPNECs. P2X1 signals were found in RPNECs. The signal for P2Y2 was confirmed as a positive control. F, I-V relationship of the P2X receptor current obtained by ramp depolarization under bi-ionic conditions (pipette solution, 140 mM Cs+; and bath solution, 110 mM Ca2+). G, concentration dependence of ,-MeATP on [Ca2+]c with () or without () apyrase pretreatment (left panel). Changes in F340/380 (F340/380) induced by various concentrations of ,-MeATP (right panel) are plotted and were fitted to obtain the EC50.

The fast decay of I_ATP in RPNECs is a typical trait of P2X₁ and P2X₃ subtypes (20). Also, the application of ,-MeATP (AMP-CPP; 10 µM), a potent agonist of P2X₁ and P2X₃ receptors (20), induced a similar transient inward current with peak amplitude of 2.1 ± 0.22 nA at -60 mV (n = 22) (Fig. 3C). ,-MeATP (10 µM), another selective agonist of the P2X₁ and P2X₃ subtypes, also induced a similar inward current (n = 4) (data not shown). TNP-ATP (3 nM), a relatively selective antagonist of P2X₁ and P2X₃ (20), abolished 91 ± 2.4% (n = 4) of the I_ATP in a reversible manner (Fig. 3D). Although the above pharmacological profiles do not discriminate between P2X₁ and P2X₃ subtypes, the single RT-PCR analysis indicated the presence of P2X₁ (but not P2X₃) transcripts in RPNECs (Fig. 3E).

P2X channels are usually more permeable to Ca²⁺ than monovalent cations and provide significant Ca²⁺ influx pathways in many kinds of cells (20–22). In RPNECs, after replacing the NaCl bath solution with 110 mM CaCl₂ solution, the bath application of 10 µM ,-MeATP induced a huge inward current with a mean amplitude of 2.04 ± 0.41 nA (n = 7) at -60 mV. The I-V curve showed an inwardly rectifying property with a reversal potential of 18 ± 3.0 mV (n = 7) (Fig. 3F). Under this bi-ionic condition, where Cs⁺ (140 mM) and Ca²⁺ (110 mM) are the only permeable cations at the intracellular and extracellular sides, respectively, the calculated permeability ratio of Ca²⁺ to Cs⁺ (P_Ca/P_Cs) for the P2X channel is 2.0 (23). In line with the high Ca²⁺ permeability of P2X channels, the application of ,-MeATP increased the Ca²⁺ concentration of RPNECs in a concentration-dependent manner (Fig. 3G). In some cases of this experiment, to prevent the spontaneous desensitization of P2X receptors by ATP released from nearby cells, apyrase (20 µg/ml) was included in the extracellular solution throughout the cell isolation and incubation period (22). The EC₅₀ of ,-MeATP was 0.13 and 0.12 µM in the absence and presence of apyrase, respectively, indicating that the P2X channels were not significantly desensitized after single cell isolation (Fig. 3G). In the following experiments, considering the fast desensitization, 10 µM ,-MeATP was applied via the bath perfusate to achieve maximum and simultaneous stimulation of P2X receptors.

From the above results, it was clear that RPNECs express both P2X (P2X₁) and P2Y (P2Y₂) receptors that link to fast Ca²⁺ influx and relatively slow release of stored Ca²⁺, respectively. As a next step, we asked whether the P2X and P2Y receptors play a subtype-specific role in the physiological responses of RPNECs, viz. exocytosis. To address this question, we tested whether ,-MeATP is a selective activator of P2X receptors without activating stored Ca²⁺ release. In the absence of extracellular Ca²⁺, ,-MeATP had no effect on the [Ca²⁺]_c of RPNECs at 10 µM (n = 7) (Fig. 4A)orat100 µM (n = 3) (data not shown). In another experiment, RPNECs were pretreated with noradrenaline (10 µM) in the presence of thapsigargin (1 µM), a potent inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum. After confirming that noradrenaline did not release any additional Ca²⁺, ,-MeATP (10 µM) was applied, which still induced a robust Ca²⁺ response (Fig. 4B). These results indicate that ,-MeATP could be used as a selective agonist of P2X receptors mediating Ca²⁺ influx in RPNECs.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Comparison of [Ca2+]c responses induced by P2Y and P2X stimulations. A, no effect of ,-MeATP (10 µM; black bars) on [Ca2+]c in the absence of extracellular CaCl2 (white bar). B, effects of noradrenaline (NA; 10 µM) and ,-MeATP (10 µM; black bar) on [Ca2+]c upon pretreatment with thapsigargin (TG; 1 µM; white bars) (left panel). Note that the second noradrenaline application had no effect, and the removal of extracellular Ca2+ (white bars) reversed the tonic increase in [Ca2+]c by thapsigargin and noradrenaline. Summarized results are shown (right panel). C, representative response of [Ca2+]c to, ,-MeATP (100 µM; black bars) and UTP (100 µM; black bar) (left panel). The second application of ,-MeATP in the presence of nifedipine (Nif; 1 µM) and -conotoxin GVIA (-CTX; 1 µM; white bar) induced a large increase in[Ca2+]c similar to the first response to ,-MeATP. A summary of [Ca2+]c responses to ,-MeATP, UTP (n = 25), or ,-MeATP combined with -conotoxin GVIA and nifedipine (n = 7) is shown (right panel). D, [Ca2+]c responses to ,-MeATP (black bar) under voltage clamp at -60 mV and to the depolarizing step pulses to 0 mV with various durations (from 50 to 500 ms) (left panel). The simultaneously recorded membrane currents are also shown (inset). A summary of [Ca2+]c responses to ,-MeATP (at -60 mV) and step depolarization (0 mV, 300 ms) obtained in five RPNECs is shown (right panel).

To compare the peak amplitudes of [Ca²⁺]_c responses to P2X- and P2Y-selective stimulations, we applied ,-MeATP (10 µM) and UTP (100 µM), alternating the order of drug application in each cell. Although both agents induced substantial increases in [Ca²⁺]_c in all the putative RPNECs tested, the peak F_340/380 induced by ,-MeATP was larger than that induced by UTP (F_340/380 = 3.8 ± 0.25 for ,-MeATP and 2.1 ± 0.19 for UTP, n = 25; p < 0.05), as summarized in Fig. 5C. Apart from the larger peak amplitude, the recovery of the Ca²⁺ response after the washout of ,-MeATP generally took longer time than that after the washout of UTP, the reason for which is not clear yet. In >90% of RPNECs tested for UTP and ,-MeATP, both agonists induced significant increases in [Ca²⁺]_c, indicating that P2X and P2Y receptors are coexpressed in the same cell.

View larger version (24K): [in this window] [in a new window]   FIG. 5. Amperometric measurement of exocytosis induced by ATP. A, shown is an original trace of the amperometric record. B, shown is a histogram of the number of current spikes above the base line (10 s of bin width). The CFE was held at 600 mV, and the upward current spikes indicate exocytotic events releasing oxidizable compounds from the nearby RPNEC. Inset, a typical current spike with an expanded time scale illustrating the parameters of peak amplitude and total charge for the analysis below. ATP (100 µM) was applied as indicated (black bar). C, quantal events are plotted at a higher time resolution in the control (left panel) and after the application of ATP (100 µM) (right panel) in same cell. D, in the same cell shown in C, the peak amplitude (left panel) and total charge (right panel) of individual current spikes (inset) were analyzed, and the number of events is plotted against the size of each parameter in histograms. The total recording times were 500 and 200 s for the control (gray bars) and ATP application (white bars), respectively. pC, picocoulomb.

Stimulation of P2X (but Not P2Y) Induces Exocytosis in RPNECs—We then compared the effects of P2X and P2Y stimulations on the amperometrically measured exocytosis in RPNECs. ,-MeATP (10 µM) significantly increased the amplitude and frequency of spikes in 14 of 15 cells tested. In contrast, UTP (100 µM) induced a slight secretory response in only 2 of 15 cells. Fig. 6 depicts the representative results showing that only the stimulation with ,-MeATP induced exocytotic responses; the frequency of spikes increased from 19 to 85 events/min (Fig. 6B). The difference between the two agonists is also obvious upon comparison of the histograms of the amplitudes and total charge of the current spikes (Fig. 6, C and D).

View larger version (23K): [in this window] [in a new window]   FIG. 6. Effects of UTP and ,-MeATP on exocytosis measured by amperometry. A and B, shown are representative traces of amperometric currents during the application of UTP (100 µM) and ,-MeATP (10 µM) recorded in the same RPNEC. Time breaks indicate 5 min. C and D, during the application of UTP and ,-MeATP (120 s each), the peak amplitude and charge of each current spike were analyzed. Black and white bars represent the responses to UTP and ,-MeATP, respectively. fC, femtocoulomb.

View larger version (20K): [in this window] [in a new window]   FIG. 7. Effects of ,-MeATP and UTP on the membrane capacitance (Cm) of RPNECs. A, shown are the original traces of Cm, membrane current (Im), and [Ca2+]c (F340/380) simultaneously recorded in RPNECs. Part of the initial Cm change upon application of ,-MeATP (10 µM) was deleted due to the large transient change in membrane conductance. The time break indicates 100 s. B, shown is a summary of the Cm changes induced by UTP or ,-MeATP. The time-dependent changes in Cm were plotted by subtracting the resting Cm from the Cm obtained at 16, 60, 120, and 180 s after UTP (; n = 15) or ,-MeATP (•; n = 23) application. C, shown is a comparison of Cm and Im responses to ,-MeATP in the presence and absence of extracellular Ca2+. D, shown is a summary of the Cm (upper panel) and inward currents (lower panel) induced by ,-MeATP in the experiments shown in C. E, the decrease in Cm induced by UTP (100 µM) was blocked by treatment with suramin (50 µM; n = 3) (bar a), but not with TNP-ATP (0.1 µM; n = 2) (bar b). Upon pretreatment with TNP-ATP (0.1 µM), the Cm was reduced by ATP (10 µM; n = 6) (bar d). Note that the application of ATP alone induced a net increase in Cm (n = 5) (bar c).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
This study demonstrates that RPNECs express both P2X (P2X₁) and P2Y (P2Y₂) purinoreceptors. The fast Ca²⁺ influx via P2X channels seems to be more efficiently linked to exocytosis in RPNECs, whereas the relatively slow and smaller increase in [Ca²⁺] signaled from P2Y receptors is consistently linked to a net decrease in C_m, viz. endocytosis. Previous studies of the effects of extracellular ATP in the prostate have been confined to epithelial cells (28) and smooth muscle (29). This study is, to our knowledge, the first investigation on the functional role of purinoreceptors in prostate neuroendocrine cells. Thus, like other organs, purinoreceptors seem to be widely distributed in the prostate gland, including neuroendocrine cells.

Both the fast desensitization of I_ATP and the potent activation by ,-MeATP are consistent with the known properties of P2X₁/P2X₃ channels, and single cell RT-PCR confirmed the expression of the P2X₁ subtype in RPNECs. It is noteworthy that the large ,-MeATP-induced increase in [Ca²⁺]_c ([Ca²⁺]_c) was not significantly affected by blocking VOCCs or by depleting intracellular Ca²⁺ stores (Fig. 4). Because 10 or 100 µM ,-MeATP had no effect in the absence of extracellular Ca²⁺, the large Ca²⁺ response activated by ,-MeATP must have been due to the transmembrane influx of Ca²⁺. Considering the fast inactivation kinetics of P2X₁ channels, however, it was surprising that the transient Ca²⁺ influx through P2X₁ channels overwhelmed the contribution of VOCCs that would be concomitantly activated in RPNECs. In contrast to our present results, in GT1 cells heterologously expressing P2X₁ or P2X₃ receptors, the transient [Ca²⁺]_c by extracellular ATP is largely abolished by nifedipine, suggesting that direct influx of Ca²⁺ via P2X₁ and P2X₃ plays a minor role (22). One plausible explanation of the unexpectedly strong influx of Ca²⁺ upon stimulation with ,-MeATP is the huge amplitude of the P2X receptor current in RPNECs. If we simply assume that 10% of the 2 nA square current with a duration of 20 ms is carried by Ca²⁺ (20), the instantaneous entry of Ca²⁺ would be 4.1451 x 10^-16 mol. Such flux of Ca²⁺ into a RPNEC with a diameter of 10 µm would produce an instantaneous increase in [Ca²⁺]_c by 1.056 mM. If only 1% of the entered Ca²⁺ is present as a free ionized form, it will still produce 10 µM [Ca²⁺]_c. Such a large [Ca²⁺]_c might have not only overwhelmed the secondary influx of Ca²⁺ via VOCCs, but also induced Ca²⁺-dependent inactivation of VOCCs (23), which could explain the insignificant effects of Ca²⁺ channel blockers on [Ca²⁺]_c induced by ,-MeATP (Fig. 4C).

The results of amperometry indicate that the tested cells are capable of synthesizing and releasing oxidizable bioamines (Fig. 5 and 6), most likely serotonin (4, 24). The single amperometric spikes in RPNECs usually display 5–10 ms of half-amplitude width. Such values are similar to those observed in pancreatic duct epithelial cells (7 ms) and adrenal chromaffin cells (5–15 ms) (17, 27). However, the mean amplitudes of these amperometric current spikes are much smaller than those observed in adrenal chromaffin cells, which often display >10 pA in an individual spike. Presumably, such small spikes may reflect either the less concentrated hormones or the smaller size of secretory vesicles in RPNECs compared with chromaffin cells (30). Another possibility is that RPNECs store oxidizable compounds together with other substances, e.g. neuropeptides (7), that are undetectable by CFE amperometry.

Both in the amperometry and C_m measurements, UTP was found to be far less effective than ,-MeATP in triggering exocytosis from RPNECs. Although the application of UTP also induced a sizable increase in [Ca²⁺]_c, the change in fura-2 fluorescence (F_340/380) by UTP was about half of that induced by ,-MeATP. If we suppose that the level of [Ca²⁺]_c is the key determinant of exocytosis, the above results suggest that the threshold of [Ca²⁺]_c for triggering the exocytosis in RPNECs is relatively high, which might be hard to be attained by P2Y stimulations. In the hair cells of the inner ear, for example, sizable C_m changes occur only after the elevation of [Ca²⁺]_c by >7 µM (31). Another assumptive speculation is that the P2X-mediated Ca²⁺ influx might have induced a huge increase in [Ca²⁺] in the subplasmalemmal space, where the exocytotic machinery and vesicles are likely to be localized. However, we did not observe such a Ca²⁺ gradient when a preliminary Ca²⁺ imaging study was carried out using confocal microcopy (data not shown). Also, a recent study of cellular Ca²⁺ images induced by P2X stimulation in neuroendocrine cells demonstrated a global increase in [Ca²⁺]_c rather than a local subplasmalemmal change (21).

The opposite changes in the C_m of RPNECs in response to ,-MeATP and UTP initially suggest an intriguing possibility that the Ca²⁺ signals due to P2X and P2Y stimulations may be linked to distinctive cellular responses, viz. exocytosis and endocytosis. According to a recent study in the motor nerve endings of Drosophila, exocytosis and endocytosis are separately controlled by different routes of Ca²⁺ influx mechanisms (32). However, in RPNECs, we do not have further evidence to suggest that exocytosis and endocytosis are separately regulated by Ca²⁺ influx and release from stores. Instead of that hypothesis, the slow increase in C_m, sometimes from below the resting level, upon the application of ,-MeATP (Fig. 7B) suggests that a rise in [Ca²⁺]_c might commonly induce endocytotic events irrespective of the Ca²⁺ recruitment pathways. In other words, the concomitant activation of endocytosis might have impeded the initial fast increase in C_m by exocytosis. An induced decrease in C_m below the resting level has also been reported in calf adrenal chromaffin cells (33) and rat melanotrophs (34). The endocytotic events reported in other cells are usually preceded by an initial exocytosis, whereas the UTP-induced endocytosis in the present study displayed a monophasic decrease in C_m. Thus, the UTP-induced endocytosis in RPNECs may not be a retrieval of previously exocytosed membrane, the physiological meaning of which needs further investigations.

In summary, both ionotropic P2X₁ receptors and metabotropic P2Y₂ receptors are present in RPNECs, the stimulation of which increases [Ca²⁺]_c with discriminate kinetics and amplitudes. The strong Ca²⁺ signal from P2X is more effectively linked to exocytosis, whereas the P2Y stimulation results in a net decrease in C_m, i.e. endocytosis. Identification of the components determining the direction of responses and the threshold of [Ca²⁺]_c will be essential to attain a full understanding of exo/endocytosis in RPNECs, a novel type of neuroendocrine cell.

	FOOTNOTES

 
^* This work was supported by Grants 01-PJ1-PG3–21400-0019 and 03-PJ1-PG3–21400-0007 from the Korea Health 21 Research and Development Project, Ministry of Health and Welfare, Republic of Korea, and by Samsung Grant SBRI B-A3-102. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 82-31-299-6104; Fax: 82-31-299-6129; E-mail: sjoonkim{at}med.skku.ac.kr.

¹ The abbreviations used are: RPNECs, rat prostate neuroendocrine cells; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; [Ca²⁺]_c, cytosolic Ca²⁺ concentration; RT, reverse transcription; CFE, carbon fiber electrode; ATPS, adenosine 5'-O-(3-thiotriphosphate); ,-MeATP, ,-methyleneadenosine 5'-triphosphate; AMP-CPP, adenosine 5'-(,-methylenetriphosphate); TNP-ATP, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; VOCCs, voltage-operated Ca²⁺ channels.

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