The Prostate 57:99 ^110 (2003)

Adrenergic Regulationof the Intracellular [Ca2+] andVoltage-OperatedCa2+Channel Currents in theRat

ProstateNeuroendocrineCells

Jun Hee Kim,1 Sun Young Shin,1 Joo Hyun Nam,1 Eun-Kyung Hong,3

Young-Shin Chung,3 Jeong Yun Jeong,2 Jeongyoon Kang,2 Dae-Yong Uhm,1

and Sung Joon Kim1*1Departmentof Physiology, SungkyunkwanUniversity SchoolofMedicine, Suwon,Korea

2DepartmentofUrology, Eulji University SchoolofMedicine, Eulji GeneralHospital, Seoul,Korea3Medvill Central Research Laboratory, Pyungchang-Dong, Seoul,Korea

BACKGROUND. The prostate gland contains numerous neuroendocrine cells (PNECs)innervated by adrenergic neurons. PNECs are believed to influence the growth andphysiological function of the prostate gland via paracrine release of hormones.MATERIALS AND METHODS. Using fura-2 fluorescence measurement and patch-clamptechniques, we investigated the effects of adrenergic stimulation on cytosolic concentration ofCa2þ ([Ca2þ]c) and high voltage-activated Ca2þ channel currents (HVA-ICa) of the putative ratprostate neuroendocrine cells (RPNECs) freshly isolated by an enzymic digestion.RESULTS. Noradrenaline (NA, 1 mM) induced a sharp, transient increase of [Ca2þ]c measuredby the fura-2 fluorescence. Pharmacological studies showed that a1-adrenoceptors (a1-ARs)coupled with PLC/IP3 signaling pathway induce the release of stored Ca2þ, which subsequ-ently recruits store-operated Ca2þ entry pathways. In thewhole-cell voltage clamp experiment,NA decreased the amplitude of HVA-ICa by 40%, which was mimicked by an a2-AR agonist(UK14304) but not by an a1-AR agonist (phenyleprine). After selective blockade ofN-type Ca2þ

channels by o-conotoxin GVIA, the addition of NA showed no further inhibition on theremaining L-type Ca2þ channel currents. The adrenergic inhibition of HVA-ICa was partiallyprevented by the pretreatment with pertussis toxin (PTX) (5 mg/ml, 4 hr, 378C).CONCLUSIONS. RPNECs express both a1- and a2-ARs, signaling the release of stored Ca2þ

and the inhibition of N-type Ca2þ channels, respectively. Prostate 57: 99–110, 2003.# 2003 Wiley-Liss, Inc.

KEY WORDS: prostate; neuroendocrine cell; adrenoceptors, voltage-activated Ca2þ

channel; intracellular Ca2þ concentration

INTRODUCTION

The prostate gland consists of a complex ductalsystem lined with exocrine epithelial cells, basal cells,and neuroendocrine cells embedded in a stromalmatrix [1,2]. Compared with its clinical importance,the physiological significance of the normal prostategland and its secretions are largely unknown, andstudies characterizing electrophysiological propertiesof freshly isolated prostate cells have been performedonly recently [3–5].

Sung Joon Kim is an Assistant Professor.

Grant sponsor: Plant Diversity Research Center of 21st CenturyFrontier Research Program funded by Ministry of Science andTechnology of Korean government; Grant number: PF002108-03.

*Correspondence to: Sung Joon Kim, Department of Physiology,Sungkyunkwan University School of Medicine, Suwon 440-746,Korea. E-mail: sjoonkim@med.skku.ac.krReceived 25 November 2002; Accepted 25 February 2003DOI 10.1002/pros.10277

� 2003 Wiley-Liss, Inc.

The prostate neuroendocrine cells (PNECs), consid-ered as a group of amine precusor uptake anddecarboxylation (APUD) cells, are intraepithelial reg-ulatory cells with paracrine properties. Similar toneuroendocrine cells of other APUD system (e.g.,enterochromaffin-like cells of gastric gland) [6], PNECsmay influence the growth of prostate gland or regulatethe exocrine secretion of prostatic fluid [7,8]. Theclinical importance of PNECs has been consistentlyemphasized because the presence of PNECs in theprostatic carcinoma could imply a bad prognosticsignificance [8,9].

The previous immunohistochemical studies suggestthat PNECs produce and secrete a variety of neurose-cretory products like serotonin, histamine, calcitonin,and parathyroid hormone-related peptides [10,11].Concerning the development of prostate gland, theorigin of PNECs is still in debate. Some investigatorssuggest that common stem cells (e.g., basal cells) differ-entiate into both exocrine epithelial cells and neuroen-docrine cells, while others claim the neurogenic originof PNECs, distinct from the urogenital sinus-derivedexocrine secretory and basal cells [8,12].

The physiological and pathophysiological functionof prostate is under the control of autonomic nervoussystem as well as the hormonal state [13–15]. Thesecretion of prostatic fluid, for example, is positivelyregulated by acetylcholine via muscarinic receptors inthe secretory epithelial cells [16,17]. On the other hand,a1-adrenergic stimulation induces the smooth musclecontraction that would help the secreted fluid to beexpelled from the lumen of gland [16,18]. The a1-ARmediated smooth muscle contraction has been therational ground for the use of a1-blockers to relieve thesymptom of benign prostatic hyperplasia [19]. Com-pared with the knowledge of prostate smooth muscle,however, the role of adrenoceptors (ARs) in the PNECshas been largely overlooked.

The regulation of paracrine secretion from PNECsby autonomic nerves could be a crucial step in phy-siological functioning of the prostate gland. In humanprostate tissue, for example, the stimulation of ARswith NA induces the release of histamine that isbelieved to originate from PNECs. In that paper, theeffect of NA was blocked by an a1-AR antagonistwhereas the addition of a2-AR antagonist enhancedthe release of histamine. Moreover, both basal- andNA-induced release of histamine were suppressed byclonidine, an a2-AR agonist [10].

The aforementioned results by Polge et al. [10]suggest that multiple subtypes of ARs are present andoppositely coupled with the process of exocytosis inPNECs. However, the precise knowledge of intracel-lular signal transduction fromARs is largelymissing inPNECs. Like other endocrine cells, it is supposed that

the hormonal release of PNECs is under the control ofcytosolic Ca2þ concentration ([Ca2þ]c). An increase of[Ca2þ]c can be attained by the release from intracellularstores or the influx through plasma membrane Ca2þ

channels, for example, voltage-activatedCa2þ channelsor store-operated Ca2þ entry (SOCE) mechanisms. Theinvestigation of [Ca2þ]c and its regulation by neuro-transmitters, however, have not been attempted inPNECs yet.

In previous studies, we firstly recorded membraneion channel currents of the secretory epithelial cells andputative neuroendocrine cells freshly isolated from therat ventral prostate [3–5].After an enzymic digestion ofthe ventral lobe of rat prostate, the chromogranin-Apositive round-shaped cells (putative PNECs) wereclearly discriminated from the elongated columnarcells (secretory epithelial cells) and could be applied forthe electrophysiological study. The putative rat PNECs(RPNECs) display typical properties of excitable cells;spontaneous action potentials, various kinds of vol-tage-activated ion channel currents including transientoutward Kþ current (Ito), high-voltage activated Ca2þ

currents (HVA-ICa), and a TTX-resistant type ofvoltage-activated Naþ current (TTX-R INa) [5]. In cont-rast, the elongated epithelial cells predominantlyexpress Ca2þ-activated Kþ channels and Ca2þ-acti-vated Cl� channels but no evidence of voltage-activated Ca2þ, or Naþ currents [3,4].

In this study, employing the fura-2 fluorescencemeasurement and the whole-cell patch clamp techni-que, we investigated the effects of adrenergic stimula-tion on [Ca2þ]c andHVA-ICa in freshly isolatedputativeRPNECs. Our results clearly demonstrate the func-tional expression of a1- and a2-ARs that are coupled tothe release of intracellular stores of calcium andinhibition of HVA-ICa, respectively. The physiologicalmeaning of adrenergic regulation in PNECs will bediscussed and compared with other neuroendocrinecells.

MATERIALSANDMETHODS

Cell Isolation

All procedures on experimental animals were per-formed in accordancewith the guidelines of the Institu-tional Animal Care and Use Committee (IACUC) ofSungkyunkwan University. Male Sprage–Dawley rats(350–400 g, n¼ 55) were killed by 100%CO2 inhalationand the ventral lobe of the prostate gland removedrapidly thereafter. The fibrous capsule was removedand the tissue was cut with scissors into small pieces(1–2mm3) in a phosphate-buffered Ca2þ-free Tyrode’ssolution. The tissue was digested for 25 min at 378C inCa2þ-free Tyrode’s solution containing collagenase(2 mg/ml, Wako, Japan), trypsin inhibitor (1 mg/ml,

100 Kimet al.

Sigma, St. Louis), bovine serum albumin (3 mg/ml,Sigma) and dithiothreitol (1 mg/ml, Sigma). Followingdigestion and subsequent removal of supernatant,tissue segments were transferred to fresh Tyrode’ssolution and agitated gently using a fire-polished widebore (1–2 mm) Pasteur pipette. Cells were isolateddaily then stored in fresh solution at 48C for up to 6 hr.Dispersed cells were moved into the experimentchamber and examined by using an inverted micro-scope (IX-70, Olympus, Japan). After the digestionprocedure, most of isolated single cells had elongatedcolumnar shape, a typical feature of secretory epithelialcells. Besides columnar cells, we could identify roundor oval shape cells with relatively dark cytoplasm thatwere regarded as RPNECs in the present study. Theputative RPNECs were carefully discriminated fromdamaged or swollen epithelial cells that showed alsoround or oval shapes but with pale cytoplasm. Thereliability of above criteria for identifying RPNECswere assured by constant responses of chosen cells tonoradrenaline in [Ca2þ]c or voltage-activated Ca2þ

currents (see Results). Such response to NA was neverobserved in columnar epithelial cells [3,5].

Intracellular Ca2þMeasurement

Dispersed single cells were loaded with acetoxy-methyl ester form of fura-2 (2 mM) in the Ringer’ssolution for 20 min at room temperature and thenwashed out with fresh solution. The putative RPNECswere identified as above criteria, and the region ofinterest for fura-2 experiment was set so that thefluorescence from the single RPNEC could be collectedselectively. The recording of [Ca2þ]c was performedwith amicrofluorimetric system consisting of an invert-ed fluorescence microscope (Olympus IX-70, Japan)with a dry-type fluorescence objective lens (�40, NA0.85), a photomultiplier tube (typeR 1527,Hamamatsu,Japan) and Deltascan illuminator (Photon TechnologyInternational, Inc.). Light was provided by a 75-Wxenon lamp (Ushino, Japan) and a chopper wheelalternated the light path to monochromators (340 and380 nm) with a frequency of 5 Hz, and intensity ofemitted light at 510 nm was measured. As a measureof [Ca2þ]c the fluorescence emission ratio at 340 nm/380 nm excitation (F340/380) is presented.

Patch-ClampMethods

Isolated cellswere transferred into a bath situated onthe stage of an inverted microscope (IX-70, Olympus).The bath (approximately 0.3 ml) was superfused at10 ml/min and voltage clamp experiments were per-formed at room temperature (22–258C). Patch pipettes(with a free-tip resistance 2.5–3MO) were connected tothe head stage of a patch clampamplifier (Axopath 1-D,

Axon Instruments, Foster City). Liquid junction poten-tials were corrected with an offset circuit before eachexperiment. For the perforatedwhole-cell patch clamp,a stock solution of nystatin in dimethylsulfoxide(15 mg/ml) was added to the pipette solution yieldinga final concentration of 0.15 mg/ml. A steady-stateperforation was usually achieved within 10 minutesafter making a giga-seal. pCLAMP software v.7.0 andDigidata-1200A (both from Axon Instruments) wereused for the acquisition of data and the application ofcommand pulses. The voltage and current data werelow-pass filtered (5 kHz) and displayed on a computermonitor. Current traceswere stored in aPentium-gradecomputer and analyzed using Origin v. 6.1 (MicrocalSoftware, Inc., Northampton).

Solution andDrugs

All the experiments were performed in Tyrode’ssolution containing 145 mM NaCl, 1.6 mM K2HPO4,0.4 mM KH2PO4, 1 mM MgCl2, and 5 mM D-glucoseat pH 7.4 titrated with NaOH. CaCl2 was omitted inthe enzymatic isolation of single prostate cells. Thepipette solution for recording Ca2þ current in nystatin-perforated conditions contained 130 mM CsCl; 1 mMMgCl2, 0.5 mM EGTA, 5 mM D-glucose, and 10 mMHEPES at pH 7.2 titrated with CsOH. The pipettesolution for a conventional whole-cell mode experi-ments contained 130 mM CsCl, 1 mM MgCl2, 10 mMEGTA, 5 mM D-glucose, and 10 mM HEPES at pH 7.2titrated with CsOH. U73122 and o-conotoxin GVIAwere purchased from Calbiochem (La Jolla) and Tocris(Bristol, UK), respectively. All other drugs and chemi-cals used were bought from Sigma (St. Louis).

DataAnalysis and Statistics

The data are presented as original recordings,current-voltage (I-V) curves and bar graphs of mean�SEM (for n cells tested). When necessary, Student’st-test for paired samples was applied since control andtest recordings were made from the same cell. P< 0.05was regarded as significant.

RESULTS

Effects ofNAon [Ca2þ]c of RPNECs:a1-ARMediated Response

The fluorescence ratio (F340/380, see Materials andMethods) of RPNECs was measured from individualcells and was regarded as a variable representing[Ca2þ]c. The mean of F340/380 in control conditionswas 1.27� 0.019 (n¼ 85). Membrane depolarizationby the bath perfusion of 60 mM KCl (replaced withthe same concentration of NaCl) induced a tonicincrease of [Ca2þ]c (Fig. 1A, n¼ 12), consistent with

Noradrenaline and ProstateNeuroendocrine Cells 101

the presence of voltage-activated Ca2þ channels asreported previously [5].

In the putative RPNECs examined, the applicationof 1 mM NA induced an increase in [Ca2þ]c comprisedof a sharp initial peak and subsequent plateau aboveresting level (Fig. 1A). ThemeanofmaximumchangeofF340/380 (peak-DF340/380) and themean of plateau aboveresting level (plateau-DF340/380) were 2.4� 0.18 and0.9� 0.36 (n¼ 30), respectively. The concentration-response relation was obtained by plotting meanvalues of peak-DF340/380 against concentrations of NAwhere the half effective concentration (EC50) ofNAwas0.2 mM(n¼ 12, Fig. 1B,C). In contrast to the responses ofRPNECs toNA, the [Ca2þ]c of elongated columnar cells(secretory epithelial cells) was not affected by theapplication of NA (n¼ 10, data not shown).

In the next experiment, subtype-selective agonistsand antagonists for ARswere tested [20,21]. The effectsof NA on [Ca2þ]c was mimicked by 5 mMphenyleprine(a1-selective agonist, n¼ 4) and was completely block-edby thepretreatmentwith 1mMprazosin (a1-selectiveantagonist, n¼ 4) in a reversible manner (Fig. 2A,B). Incontrast to the responses to a1-adrenergic stimulation,neither a2-AR selective agonist (UK14304, 1 mM) [21]nor b-AR selective agonist (isoprenaline, 10 mM)had aneffect on [Ca2þ]c of RPNECs (Fig. 2C,D, n¼ 4, respec-tively). Above results indicate that the a1-ARs mediatethe NA-induced increase of [Ca2þ]c in RPNECs.

In various kinds of cells expressing a1-ARs, thestimulation of receptors are linked to a phospholipaseCb (PLCb)-InsP3 signaling pathway releasing storedCa2þ from endoplasmic reticulum [20]. Therefore, itwas examined whether similar signaling pathways areutilized in RPNECs. In an effort to elucidate the sourceof Ca2þ, NAwas applied in the absence of extracellularCaCl2; CaCl2 was omitted from the bath solution and0.1 mM EGTA was added. In this condition, theaforementioned plateau-like increase in [Ca2þ]c waslargely abolished whereas the initial transient increasewas conserved (Fig. 3A, n¼ 5). Such responses indicatethat the initial sharp increase in [Ca2þ]c was due tothe release of storedCa2þ byNA,while the platuea-like

Fig. 1. Effects ofNAon [Ca2þ]c ofRPNECs.A: Arepresentativetrace of fluorescence ratio (F340/380) induced by high Kþ-induceddepolarization (60mMKCl, open bar) or byNA (1 mM, closedbar).The typicalresponseof [Ca2þ]c toNAwas composedof initialpeakincrease (peak-F340/380) and subsequentplateau (plateau F340/380) asindicated by arrows.B: A representative trace of F340/380 demon-strating the concentration-dependent effects of NA.C: SummaryofCa2þ-response from12RPNECs.Thepeak-DF340/380wasnormal-izedto themaximumeffectby10mMofNA(normalizedDFpeak),andthemeanvalueswereplottedandfittedby thefunction{normalizedDFpeak¼1/[1þ (tested concentration/EC50)

n]} to obtain the 50%effectiveconcentration (EC50)ofNA.

102 Kimet al.

increase was due to the Ca2þ influx from extracellularfluid. In Ca2þ-free conditions, the peak responses torepetitive applications of NA decayed steeply, whichwe interpret that the calcium ions released from intra-cellular stores were pumped out to the extracellularspace and consequently depleted the calcium stores(Fig. 3A, n¼ 4). On replenishing extracellular CaCl2,the [Ca2þ]c was transiently increased and subsequentapplication of NA induced a Ca2þ response similarwith the initial control (Fig. 3A).

The preteatment with U73122 (3 mM), an inhibitorof PLCb [22], completely blocked the NA-inducedincrease in [Ca2þ]c (Fig. 3B, n¼ 3). 2-aminoethoxy-diphenylborate (2-APB, 50 mM), reportedly a mem-brane-permeable InsP3-receptor antagonist [23], also

abolished the response to NA in a reversible manner(Fig. 3C, n¼ 4). All the above results commonlyindicate that the putative RPNECs functionally expressa1-ARs that are coupled with the PLCb-InsP3 signalingpathway to release stored Ca2þ via InsP3 receptors.

Effects ofNAonHVA-ICa

The effects of NA on voltage-operated Ca2þ channelcurrents were then tested using the whole-cell patchclamp technique. All through the experiments,outward Kþ currents were eliminated by using CsCl-pipette solution (see Materials and Methods). In theconventional whole-cell clamp conditions, a sponta-neous decrease of Ca2þ current (run-down) was

Fig. 2. Thesubtypeofadrenoceptors(ARs)mediatingtheincreaseof[Ca2þ]c.A:Theapplicationofphenyleprine(PE,5mM)increased[Ca2þ]csimilarwith theresponse toNA (10mM).B:The effectofNAwas completelyblockedby thepretreatmentwithprazosin (1mM) in a reversiblemanner.NeitherUK14304 (1mM,C)nor isoprenaline (10mM,D)hadaneffecton [Ca2þ]c of the testedRPNECs.

Noradrenaline and ProstateNeuroendocrineCells 103

frequently observed. To minimize the run-down, anystatin-perforatedwhole-cell clampwas also adopted(Fig. 4B,D,F) aswell as the conventionalmodeofwhole-cell clamp (Fig. 4A,C,E). The membrane voltage washeld at �70 mV and various levels of step-like

depolarizing pulses were applied. Under these condi-tions, the voltage-activated Ca2þ currents were record-ed as downward deflections that indicate the influx ofCa2þ from extracellular space. As has been described inour previous paper, the voltage-activated Ca2þ currentstarted to be activated by depolarizing pulses above�40 mV. The Ca2þ channel currents with thresholdabove �40 mV were regarded as HVA-ICa. The peakamplitude of HVA-ICa was maximized at 0 mV, thendecline with further depolarization. The inward cur-rent to voltage relation (I/V curve) showed an invertedbell-shape (Fig. 4E,F). A bath application ofNA (10 mM)decreased the amplitude of HVA-ICa by about 40%(Fig. 4C,D). The inhibitory effects sustained in thepresence ofNAandwerepartly reversedbywashout ofNA (Fig. 4D,F).

The putative RPNECs reportedly express two typesofHVA-Ca2þ channels: the L-type and theN-typeCa2þ

channels that are selectively blocked by nifedipine ando-conotoxin GVIA (o-CTX), respectively [5]. As a nextstep, therefore, we tested whether a specific subtype ofHVA-Ca2þ channel is preferentially regulated by theadrenergic stimulation. Figure 5 depicts the effects ofpretreatment with Ca2þ channel blockers on the NA-induced inhibition of HVA-ICa. In an effort to isolateL-type Ca2þ currents, the cell was pretreated with o-CTX (1 mM, N-type Ca2þ channel blocker), whichdecreased the amplitude of HVA-ICa by some 50%. Inthe presence of o-CTX, the addition of NA did notfurther suppress the remainingHVA-ICa (Fig. 5A,B). InFigure 5A, the inward current resistant to o-CTX andNAwas almost completely abolished by the addition ofnifedipine (1 mM, Fig. 5A). The same experiments weredone in five putative RPNECs and the results summar-ized in the form of I/V curves (Fig. 5B).

In contrast to the aforementioned effects of o-CTX,a selective blockade of L-type Ca2þ channels failed toprevent the inhibitory effects of NA on HVA-ICa. Thepretreatment with nifedipine decreased the amplitudeof HVA-ICa to 40% of the control, and the addition ofNA further reduced the HVA-ICa to 20% of the initialcontrol (Fig. 5C). In another series of experiments, wechanged the sequence of application of nifedipine andNA. After confirming the inhibitory effects of NA, theaddition of nifedipine further decreased the ampli-tudes of HVA-ICa to less than 10% of the initial control(Fig. 5D). Above results strongly suggest that N-typeCa2þ channels are preferentially inhibited by theadrenergic stimulation in RPNECs.

a2-ARsMediate the Inhibitory Effects onHVA-ICa

Next, we investigated the effects of a1- or a2-ARspecific agonists and antagonists on HVA-ICa. Theresults are summarized as changes of the peak

Fig. 3. The signaling mechanism of the NA-induced increase of[Ca2þ]c.A:NA (10 mM)wasrepetitively applied (filledbar), and theCa2þ was removed from the bath solution as indicated by an openbar. Note the transient increase of [Ca2þ]c on re-adding CaCl2 tothebath solution.ThepretreatmentwithU73122 (3 mM,B) or with2-APB (50 mM, C) completely blocked the Ca2þ-response ofRPNECs toNA.

104 Kimet al.

amplitudes of inward current (Fig. 6). Under thenystatin-perforated conditions, the membrane voltageof RPNECwas held at�70mV and a depolarizing steppulse (0 mV, 40 msec) was applied repetitively (15 sec

interval). After confirming the inhibitory effects onHVA-ICa, 10 mM prazosin (a1-AR antagonist) wasadded, which failed to recover the amplitude ofHVA-ICa (Fig. 6A, n¼ 4). In contrast, the addition of

Fig. 4. EffectsofNAonHVA-ICainRPNECs.A,B:WithCsClpipette solution,a step-likedepolarizationfrom�70to0mV(see theprotocolaboveA)inducedcurrent,theamplitudeofwhichwasreducedbythebathapplicationofNA(10mM).C,D:Timecoursesof thepeakamplitudeofHVA-ICa. Steppulse to0mVwasappliedatevery15sec andthemeansof amplitudesnormalizedto thecontrolcurrentrecordedat30sec(I/I30s)wereplotted.Filledbarsindicate theapplicationofNA.E,F:I/V curvesofpeakinwardcurrents on step-depolarizations and theeffectsofNA.A,C,E,conventionalwhole-cellclamp;B,D,F,nystatin-perforatedwhole-cellclamp.Numbersofcells testedforeachexperimentweredirectlyindicatedin the figure.

Noradrenaline and ProstateNeuroendocrineCells 105

yohimbine (1 mM, a2-AR antagonist) could partlyreverse the inhibitory effect of NA (Fig. 6B, n¼ 4). Inagreement with the effect of yohimbine, UK14304(1 mM, a2-AR agonist) decreased the amplitude ofHVA-ICa, which was reversed by the addition ofyohimbine (1 mM, Fig. 6C, n¼ 3). It thus appears thata2-ARs are coupled with the inhibitory effects onHVA-ICa in RPNECs.

In some kinds of neuronal cell, the a2-adrenergicinhibition of HVA-ICa has been well described and, issupposed to be responsible for the presynaptic inhibi-tion of neurotransmitter release. In those cases, a directinteraction of G-protein subunits with Ca2þ channelproteins has been suggested as the underlyingmechan-ism [21,24]. The class of G-proteins mediating theinhibitory effects of AR is generally thought to be apertussis toxin (PTX)-sensitive one. However, there

also are reports supporting the involvement of PTX-insensitive G proteins in the a2-adrenergic regulationof Ca2þ channels [21,25]. As a next step, therefore, wetested the effects of PTX. Isolated cells were pretreatedwith 5 mg/ml PTX at 378C for 4–6 hr. In seven putativeRPNECs displaying HVA-ICa (222� 53.3 pA at 0 mV),the pretreatment with PTX appeared to attenuatethe inhibitory effects of NA but failed to prevent itcompletely. In average, under the pretreatment withPTX, the peak amplitude of HVA-ICa was reduced to79� 4.5% of the initial control (n¼ 7).

NoEffect ofNAonTTX-ResistantNaþCurrent

In our previous study, it was shown that about 20%of putative RPNECs display TTX-resistant voltage-gated Naþ current (TTX-R INa) [5]. In the prostate cells

Fig. 5. Inhibition of the N-type Ca2þ channel current by NA.A: With CsCl pipette solution, a step pulse from�70 to 0 mV was applied.o-conotoxinGVIA(o-CTX,1mM),NA(1mM),andnifedipine (1mM)were sequentiallyaddedto thebath solution.Note thatNAhadno furthereffectunder thepretreatmentwitho-CTX.B^D: Summaryof theeffectsof sequentialapplicationofCa2þ channelblockers andNAonHVA-ICa of RPNECs. Averaged I/V curves depict the responses too-CTX followedbyNA andnifedipine (B, n¼ 5), nifedipine followedbyNA (C,n¼ 7), andNAfollowedbynifedipine (D,n¼ 5), respectively.

106 Kimet al.

possessing TTX-R INa, a transient inward current couldbe activated by a step depolarization from �90 to�50 mV and above, a threshold which is definitelylower than that of HVA-ICa. The aforementionedinward current shows Naþ-selective permeability butis resistant to TTX (1 mM), a well-known blocker of

voltage-gated Naþ channels [5]. In those RPNECsexpressing both TTX-R INa andHVA-ICa, the I/V curveofpeak inwardcurrentsdisplayed twopeaksat�30mVand at 0 mV (Fig. 8B). A bath application of NA did notchange the amplitude of TTX-R INa (Fig. 8A). Therefore,the amplitude of I/V curve below �30 mV wasunaffected by NA while the inward currents recordedatmore depolarized voltageswere suppressed (Fig. 8B,n¼ 4).

DISCUSSION

In thepresent study,we investigated the effect ofNAon [Ca2þ]c and HVA-ICa of the putative RPNECs aftersingle cell isolation. Our main findings are (1) Thestimulation of a1-AR markedly increases [Ca2þ]c viaInsP3-mediated release of the stored Ca2þ, (2) NAinhibits the activity of N-type Ca2þ channels via a2-ARs. Although not tested simultaneously, above tworesponses were consistently observed in almost everyRPNEC examined (>95%), which suggested that botha1- and a2-ARs are functionally expressed in the samecell.

As mentioned in the Introduction, the NA-inducedrelease of histamine from human prostate gland isblocked by an antagonist of a1-AR while a2-ARantagonist augments the effect [10]. Considering theimportance of Ca2þ in the regulation of hormonalexocytosis, the aforementioned dual effects of NAcouldbepartly explainedbyourpresent results, i.e., theincrease in [Ca2þ]c by a1-ARs and the inhibition ofHVA-ICa by a2-ARs. In spite of the difference in speciesbetween human and rat, it is very intriguing thatcommon mechanisms may operate for the adrenergicregulation of PNECs.

Ca2þRegulationMediatedbya1-ARs

There are three closely related a1-AR subtypes (a1A,a1B, and a1D), all of which couple to Gq/11 family of Gprotein, thereby activating PLCb and generating thesecond messengers, InsP3 and diacylglycerol thatrelease stored intracellular Ca2þ and activate proteinkinase C [20]. Our present results indicate that themajor effect of a1-adrenergic stimulation in RPNECs isreleasing stored Ca2þ via PLCb/IP3 pathway. An a1-AR-induced increase in [Ca2þ]c has been also reportedin some neuroendocrine cells including rat anteriorpituitary cells and pinealocytes [26–28].

In addition to the initial release of stored Ca2þ, therewas a sustained influx of Ca2þwhich keeps the plateauof [Ca2þ]c as long asNA is present (Figs. 1–3). A knownblocker of L-type voltage-activated Ca2þ channels(nifedipine, 1 mM) failed to suppress the sustainedincrease of [Ca2þ]c (n¼ 3, data not shown). Moreover,from our present result, concomitant stimulation ofa2-ARs would have suppressed the HVA-ICa in the

Fig. 6. PharmacologyofARsmediating theinhibitionofHVA-ICa.With CsCl pipette solution, a step pulse from �70 to 0 mV wasapplied repetitively at every10 sec.The peak amplitudes of inwardcurrents were normalized to the initial control at15 sec (I/I15s), andthemeansofnormalizedamplitudeswereplotted.Afterconfirmingthe inhibitoryeffectofNA, ana1-antagonist, prazosin (A, n¼ 4) ora2-antagonist, yohimbine (B, n¼ 4) was added. In (C), the a2-ago-nist,UK14304 showed an inhibitoryeffect thatwasreversedby theadditionof yohimbine (n¼ 4).

Noradrenaline and ProstateNeuroendocrine Cells 107

same cell. After excluding the influx of Ca2þ viavoltage-activatedCa2þ channels, a possiblemechanismof the sustained increase in [Ca2þ]c could be a so-calledstore-operated Ca2þ entry (SOCE), a passive Ca2þ

influx mechanism activated by unrevealed signalingfrom the emptied intracellular Ca2þ stores [29,30].Although the precise nature of SOCE is still uncertain,the activation of Ca2þ influx pathways by stored Ca2þ

release has been widely observed in various kinds ofcells. The rebound increase of [Ca2þ]c on re-addingextracellular Ca2þ after depleting intracellular Ca2þ

stores strongly suggests that the SOCE is present inRPNECs (Fig. 3A). However, considering the variety ofCa2þ influx pathways in animal cells [29], furtherinvestigation is necessary to elucidate the Ca2þ influxpathways activated by adrenergic stimulation inRPNECs.

Inhibition ofHVA-ICa bya2-ARs

In addition to the aforementioned increase in[Ca2þ]c, voltage-clamp study revealed a hidden roleof a2-ARs in RPNECs, namely, the inhibition of HVA-ICa (Fig. 6). The competitive inhibition of HVA-ICa byNA and o-CTX indicates that N-type Ca2þ channelswouldbe theprimary targets of adrenergic inhibition inRPNECs (Fig. 5). Consistent with our previous study, aminor portion of RPNECs (ca. 20%) displayed TTX-RINa, whereas theNaþ current was not affected byNA atall. Therefore, it seemed that the inhibitory signalingfrom a2-adrenergic stimulation was selectively linkedto the N-type Ca2þ channels in RPNECs.

In adrenal chromaffin cells, a representative neu-roendocrine type of cells, the inhibition of HVA-ICa bya2-adrenergic stimulation has been also demonstrated[31]. According to the literature, the functional impor-tance of a2-AR is most eminent as an inhibitory auto-receptor in the noradrenergic neurons of sympatheticganglionwhere the presynapticN-typeCa2þ channel isthe target of inhibitory signal from a2-ARs [21].Therefore, although we have not directly measuredthe event of exocytosis yet, it is very likely that thesignaling via a2-ARs may play a regulatory role in thesecretion of paracrine hormones from RPNECs, as hasbeen suggest in human prostate tissue [10]. Since theadrenergic inhibitionofHVA-ICawaspartly blocked bythe pretreatment with PTX in the putative RPNECs(Fig. 7), together with the Gi protein, a PTX-resistant Go

protein might be concomitantly activated by theadrenergic stimulation in RPNECs.

At a first glance, our present study appear to providemutually antagonizing influence of a1 and a2-ARs onthe regulation of [Ca2þ]c. However, the data shown inFigure 2 clearly demonstrate that the a2-AR is of littlesignificance in the global changes in [Ca2þ]c inducedNA. Possibly, without changing global [Ca2þ]c, the

signaling from a2-AR might directly modulate N-typeCa2þ channels and finally regulate the fusion ofexocytotic vesicles in their vicinity. In this respect, itwould be very intriguing if the a2-ARs and N-typeCa2þ channels are spatially localized, and tightlyassociated with the exocytosis triggered by electricalsignaling.

CONCLUSION

Our resultsdemonstrate that there aremultiplepath-ways in adrenergic regulation of the neuroendocrine

Fig. 7. Effects of PTX on the NA-induced inhibition of HVA-ICa.After incubating thecellswith5mg/mlPTXat378Cfor4 ^ 6hr,con-ventionalwhole-cellpatchclampwasapplied.A:Membranevoltagewas held at�70 mVand a step pulse to 0 mVwas applied.HVA-ICawas still reducedbyNA (1 mM) under thepretreatmentwith PTX.B: Summaryof theresponsesofHVA-ICa.Asteppulsefrom�70to0mVwasappliedatevery10sec andthepeakamplitudeofinwardcur-rent was normalized to the control at 40 sec. Mean� SEM wasplottedagainst the time(n¼ 7).

108 Kimet al.

function of rat prostate. The global increase in [Ca2þ]cbya1-adrenergic stimulation originates from the storedCa2þ release and SOCE, could be a primary excitatorymechanism of exocytosis. On the other hand, the a2-adrenergic inhibitory signaling to N-type Ca2þ chan-nels would negatively modulate the voltage-activatedCa2þ influx of neuroendocrine cells, which may atten-uate the hormonal release evoked by firing of actionpotentials.

ACKNOWLEDGMENTS

We greatly appreciate So-Young Lee and Ji Eun Leefor their technical assistance.

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