Regulation of Ryanodine Receptor–Mediated Ca Release in Vas

Regulation of Ryanodine Receptor–Mediated Ca2+ Release in Vas Deferens Smooth Muscle Cells

Akitoshi Ohno1, Susumu Ohya1, Hisao Yamamura1, and Yuji Imaizumi1,* 1Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan

Received February 4, 2009; Accepted March 9, 2009

Abstract. Ca2+ release from intracellular store sites via the ryanodine receptor (RyR) and hormonal regulation by flutamide, an androgen-receptor (AR) antagonist, on it were examined in vas deferens (VD) smooth muscle cells (SMCs). VD and VDSMCs were obtained from two groups of male rats that were treated p.o. with 100 mg/kg flutamide (Flu) or vehicle (Vehicle). Both spontaneous and caffeine-induced Ca2+ releases were markedly smaller in single VDSMCs 2+ from Flu than in those from Vehicle. Interestingly, [Ca ]i rise by 100 μM norepinephrine in VDSMCs from Flu was larger than that in those from Vehicle. The contractions induced by direct electrical stimulation in tissue preparations from Flu showed lower susceptibility to 30 μM ryanodine than those from Vehicle. Real-time PCR analyses revealed that the transcripts of ryanodine receptor (RyR) type 2 and type 3 (RyR2 and RyR3) were expressed in VD and markedly reduced in Flu. The protein expression of total RyR was significantly reduced by flutamide treatment, but that of inositol 1,4,5-trisphosphate receptor (IP3R) was not affected. It can be strongly suggested that long term block of AR by flutamide reduced the expression of RyR and its contribution to the contraction, but not those of IP3R in VDSMCs.

Keywords: ryanodine receptor, Ca2+ release, flutamide, vas deferens, smooth muscle

Introduction signal following the Ca2+ influx through the voltage- dependent Ca2+ channel (VDCC) during the E-C The ryanodine receptor (RyR) is one of the major coupling by the so-called Ca2+-induced Ca2+ release Ca2+-releasing channels on the membrane of endo- (CICR) mechanism due to its Ca2+ sensitivity for open- plasmic/sarcoplasmic reticulum (ER/SR) and plays ing in cardiac myocytes. Impact of RyR2 as a thera- central roles in the regulation of intracellular Ca2+ peutic target is considerably increasing, particularly in 2+ concentration ([Ca ]i) in various types of excitable the treatment of cardiac arrhythmia and heart failure (4). cells (1), particularly in excitation–contraction (E-C) RyR3 has the lower Ca2+ sensitivity and thereby may act coupling as an essential component of “Ca2+-releasing as the buster component of CICR (5). units” in muscles (2, 3). Among the three types of RyRs, The functions of CICR and the contribution of RyR to RyR type 1 and type 2 (RyR1 and RyR2) are abundantly E-C coupling in smooth muscle cells (SMCs) have been expressed in skeletal and cardiac muscles, respectively, also defined in highly excitable SMCs such as vas whereas the expression of type 3 (RyR3) is ubiquitous deferens (VD) and urinary bladder (6, 7). The elevation 2+ but low (1). RyR1 in skeletal muscle couples with of [Ca ]i triggered by a single action potential is highly dihydropyridine receptor on the transverse tubular dependent on CICR through RyR2 in urinary bladder membrane by conformational links to translate the SMCs (8 – 10). In SMCs of some organs, RyR3 may action potential signal to Ca2+ release as the key step of have more significant roles than in cardiac and skeletal E-C coupling. RyR2 in cardiac myocytes amplifies Ca2+ muscles. For example, RyR3 is more abundantly expressed than RyR2 in uterine SMCs (1). Moreover, *Corresponding author. [email protected] a dominant negative splice variant of RyR3 is also Published online in J-STAGE expressed in some smooth muscles and may negatively doi: 10.1254/jphs.09037FP regulate the Ca2+ release functions by forming a hetero-

78 Ryanodine Receptor in Vas Deferens Smooth Muscle 79 tetramer with RyR2 (11). 10 mM HEPES. The pH was adjusted to 7.4 with NaOH. It has been reported that testosterone substantially High K+ solution was prepared by replacing 77.8 mM modulates Ca2+ handling in both cardiac (12, 13) and NaCl with equimolar KCl (total 80 mM K+) in the skeletal muscles (14) and thereby is, at least, partly HEPES-buffered solution. 2+ responsible for gender-dependent differences in [Ca ]i regulation. RyR1 expression in skeletal muscle is Total RNA extraction, reverse transcription, and real- positively regulated by testosterone (14). In cardiac time PCR myocytes, testosterone up-regulates the expression of Total RNA was extracted from rat tissues and reverse- VDCCs and Na+-Ca2+ exchanger (15), and lines of transcribed as previously reported (19). The PCR ampli- evidence for non-genomic effects of testosterone on fication profile was as follows: 15 s at 95°C and 60 s at ion channels in cardiac myocytes have been also well 60°C, according to the user protocol of AmpliTaq accumulated recently (16). However, direct evidence for Gold® (Applied Biosystems, Foster City, CA, USA). the regulation of RyR2 expression by testosterone has Each amplified product was sequenced with an ABI not been demonstrated. PRIZM 3100 genetic analyzer (Applied Biosystems). Since the activation of androgen receptor (AR) Real-time quantitative PCR analysis was performed with facilitates the development of prostate cancer in some the use of Syber Green chemistry on an ABI 7000 states, the treatment with anti-androgen drugs including sequence detector (Applied Biosystems) (19). Unknown flutamide is common therapy (17). Although the quantities relative to the standard curve for a particular influence of anti-androgen therapy to male genital set of primers were calculated, yielding transcriptional organs is of particular importance from the clinical quantitation of gene products relative to the endogenous aspects, little is known about effects of the sustained standard (β-actin). Qualitative PCR analysis was treatment with these drugs on Ca2+ handling in SMCs. performed to determine the expression of RyR3 spliced The present study was undertaken to elucidate genomic isoforms. The PCR amplification profile was as follows: effects of testosterone deficiency by treatment with 20 s at 95°C, 20 s at 60°C, and 40 s at 72°C for 40 cycles. flutamide, an AR-antagonist, on the Ca2+-release mecha- Gene products were analyzed by 2.0% agarose gel nism in VDSMCs, in which AR is highly expressed (18). electrophoresis.

Materials and Methods PCR primers The following PCR primers were used for real-time Animals PCR analysis: RyR2 (GenBank accession no. Wistar/ST male rats (3-week-old) were treated daily NM_032078, 13011 – 13155), amplicon = 145 bp; with either 100 mg/kg flutamide in sesame oil (Flu), p.o. RyR3 (XM_001080527, 12902 – 13022), amplicon = or with vehicle alone (Vehicle), p.o. After 4 weeks, rats 121 bp; IP3R1 (NM_001007235, 5959 – 6072), of each group were anesthetized with ether and killed amplicon = 114 bp; IP3R3 (NM_013138, 6982 – 7103), by bleeding. All experiments were carried out in accor- amplicon = 122 bp; β-actin (NM_031144, 338 – 438), dance with the guiding principles for the care and use of amplicon = 101 bp. The following PCR primers were laboratory animals (the Science and International Affairs used for the detection of RyR3 spliced isoforms: Bureau of the Japanese Ministry of Education, Culture, XM_342491, 13150 – 13716, 567 bp. Sports, Science, and Technology) and also with the approval of the ethics committee of Nagoya City Western blotting University. Western blotting analyses were performed in a similar manner as described previously (20). Membrane frac- Solutions tions of the rat VD were prepared as according to the Krebs solution contained 112 mM NaCl, 4.7 mM Alomone Labs protocol (Jerusalem, Israel). The protein KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, samples (50 μg/lane) were subjected to SDS-PAGE 2+ 1.2 mM KH2PO4, and 14 mM glucose. Ca -free Krebs on 10% polyacrylamide gel and the proteins were then solution was prepared by omitting Ca2+ from the Krebs transferred to a PVDF membrane (Amersham Bio- solution. Standard and Ca2+-free Krebs solutions were sciences, Piscataway, NJ, USA). Membranes were 2+ equilibrated with a 95% O2 –5% CO2 mixture. For Ca blocked with 0.1% Tween 20 in PBS. The blots were imaging and electrical recording in single cells, HEPES- incubated with the affinity purified polyclonal anti- buffered solution of the following composition was used bodies specific for RyR (Affinity BioRegents, Rockfold, as the external solution: 137 mM NaCl, 5.9 mM KCl, IL, USA) and IP3R (Chemicon International, Temecula, 2.2 mM CaCl2, 1.2 mM MgCl2, 14 mM glucose, and CA, USA) overnight and then incubated with anti- 80 A Ohno et al mouse horseradish peroxidise–conjugated IgG for 1 h. for 10 s using a 10-ms train of 300-mA pulses at 5 Hz An enhanced chemiluminescence detection system in Krebs solution that contained the following neuro- (Amersham Biosciences) was used for the detection of transmitter antagonists: 1 μM atropine, 1 μM phento- the bound antibody. Resulting images were analyzed lamine, 1 μM propranolol, 1 μM tetrodotoxin (TTX), by a LAS-1000 (FUJIFILM, Tokyo) and the digitized and 10 µM suramin and also K+ channel blockers, 0.1 mM signals were quantitated with Image Gauge software 4-aminopyridine (4-AP) and 1 mM TEA. Contractile (Ver. 3.3, FUJIFILM). responses were recorded in a similar manner as reported previously (22) using a strain gauge transducer on an Cell isolation and measurement of Ca2+ signal from ink-writing recorder (Toa Electronics, Kobe). All of the single cells experiments were performed at 36 ± 1°C. Measurement Single SMCs were enzymatically isolated from the of twitch contractions was performed just before the prostatic part of VD of the male rats (7-week-old) (6). addition of ryanodine and at the end of ryanodine Ca2+ images were obtained using a fast laser-scanning treatment. confocal microscope (RCM 8000; Nikon, Tokyo) and ratio3 software (Nikon) in the same manner as reported Statistics previously (6, 7). A myocyte was loaded with 100 μM Pooled data are expressed as means ± S.E.M. The fluo-4-AM in standard HEPES-buffered solution for number of cells and animals used for experiments are 20 min at room temperature (23 ± 1°C). Excitation light indicated as “n” and “N”, respectively. Statistical of 488 nm from an argon ion laser was delivered through significance between two or multiple groups was a water-immersion objective (Nikon Fluo ×40, 1.15 determined by Student’s t-test or Scheffé’s test after NA). Emission light of >515 nm was detected by a one-way ANOVA, respectively. P<0.05 indicates photomultiplier. Fluorescence intensity (F) in a selected statistical significance. area was measured as an average from pixels included in the area. It took 33 ms to scan one frame (512 × 512 Drugs and chemicals pixels). Ca2+ signals were also recorded using fura-2- The sources of pharmacological agents were as AM and an Argus/HiSCA imaging system (Hamamatsu follows: CdCl2, caffeine, TEA chloride, TTX, 4-AP, and Photonics, Hamamatsu) (21). The frequency of image ryanodine (Wako Pure Chemical Industries, Osaka); acquisition was constant at one image every 1 s. The atropine, phentolamine, propranolol, norepinephrine intensity of emission fluorescence >510 nm to the (NE), penitrem A, and suramine (Sigma Chemical Co., alternate excitation (340 and 380 nm) was measured. St. Louis, MO, USA); EGTA and Hepes (Dojin, The experiments were carried out at room temperature. Kumamoto); fluo-4-AM and fura-2-AM (Molecular Probes, Eugene, OR, USA). Electrophysiology Whole-cell voltage clamp was applied to single cells Results using a CEZ-2400 amplifier (Nihon Kohden, Tokyo) (7). Ca2+ currents were recorded using the pipette Effects of flutamide-treatment on spontaneous and 2+ solution of following composition: 120 mM CsCl2, caffeine-induced Ca releases in VDSMCs 20 mM tetraethylammonium (TEA), 4 mM MgCl2, To examine effects of the treatment with flutamide 2+ 2+ 5 mM Na2ATP, 5 mM EGTA, 10 mM HEPES (pH 7.2). on Ca release events in VD, we first examined Ca All experiments were done at room temperature (23 ± release events in single VDSMCs loaded with fluo-4- 1°C). Data acquisition and analyses were performed as AM using a fast scanning confocal fluorescent micro- reported previously (10). scope. Spontaneous local Ca2+ transients, so called Ca2+ sparks, were often observed in VDSMCs from Vehicle, Measurement of contraction from tissue preparation as previously shown in VDSMCs from different species After the VD was dissected and opened longitudinally (7) (Fig. 1A). From two-dimensional images (upper in Ca2+-free Krebs solution, tissue segments of 2–3cm images in Fig. 1Aa) and line scan images (middle in length and proximal to the prostate were prepared images with the time course of fluorescent intensity in by removing the mucosal layer. One end of the tissue lower panel), it is clear that Ca2+ sparks repetitively segment was pinned to a rubber plate at the bottom of occurred in distinct sites. The frequency of Ca2+ sparks the organ bath (approximately 5 ml), and the other end in 13 spark sites of 8 cells was 0.52 ± 0.04 Hz. The was connected to a force transducer. Segments were size and time-course were 2 μm or less in diameter and equilibrated at a resting load of approximately 1 mN. 50 – 150 ms in duration at half-maximal intensity. One To elicit contractions, a tissue segment was stimulated or more Ca2+ spark sites were detected in 8 out of 9 Ryanodine Receptor in Vas Deferens Smooth Muscle 81

Fig. 1. Effects of flutamide-treatment on Ca2+ release in VDSMCs. The images of spontaneous 2+ 2+ 2+ Ca release (Ca sparks) and [Ca ]i responses to caffeine and 80 mM K+ were obtained by use of a confocal fluorescent microscope in single VDSMCs freshly isolated from Vehicle (A) and Flu (B). a: Two-dimensional Ca2+ images of sparks (top panels) were obtained at the times indicated (red circles) in the time course of fluo 4–fluorescence changes (F/F0) (bottom panel). The line-scan images (X-Y in upper image) are indicated in the middle panel. * indicates a Ca2+ spark. b: Two-dimensional Ca2+ images (top panels), line scan images (middle panel), and time courses of F/F0 in the responses to 10 mM caffeine. c: The response to 80 mM K+. Note that in Flu, Ca2+ spark and the response to caffeine were not detected, but the response to 80 mM K+ was clearly observed. 82 A Ohno et al cells from 4 rats. In contrast, it is a surprising finding that only one Ca2+ spark site was detected in 11 cells from 3 rats (Fig. 1B). In addition, application of 10 mM caffeine induced a 2+ very small rise of [Ca ]i in Flu, whereas it induced a marked rise in Vehicle (Fig. 1Ab and Bb). The responses to 80 mM K+ solution appeared to be comparable between these two groups (Fig. 1Ac, Bc).

Effects of flutamide-treatment on evoked Ca2+ transients in VDSMCs To examine the influence of flutamide-treatment on 2+ the contribution of RyR to the rise of [Ca ]i, effects of 30 mM caffeine were compared in VDSMCs from 2+ Vehicle and Flu (Fig. 2A). The [Ca ]i rise in VDSMCs from Flu was markedly smaller (Δratio: 0.19 ± 0.02, n = 13, N = 3) than that from Vehicle (Δratio: 0.47 ± 0.04, n = 28, N = 4, P<0.01). Unexpectedly, the rise of 2+ [Ca ]i by 100 μM NE was significantly higher in Flu than Vehicle (P<0.01; Fig. 2B). In contract, the peak of 2+ + [Ca ]i rise induced by 80 mM K was comparable between two groups (Δratio: Vehicle, 0.35 ± 0.03, n = 23, N = 4; Flu, 0.32 ± 0.03, n = 21, N = 4; P>0.05).

Effects of flutamide-treatment on expression of RyR and IP3R in VD The influence of flutamide-treatment on the expression of RyR2 and RyR3 was examined by quantitative real-time PCR. Both transcripts of RyR2 and RyR3 were markedly reduced by flutamid treatment (P<0.01; Fig. 3Aa, Ab). It is notable that the RyR3 transcript was more abundant than RyR2. Since a short splice variant of RyR3 (RyR3S) has been identified in some smooth muscle tissues (11), we also examined the transcripts of the full length RyR3 (RyR3L) and RyR3S by RT-PCR

(Fig. 3Ac). In VD from Vehicle, the RyR3S transcript 2+ Fig. 2. Effects of flutamide-treatment on evoked Ca transients in 2+ was more dominant than that of RyR3L, while only the VDSMCs. A: Effects of caffeine on [Ca ]i responses in rat RyR3L transcript was detected in brain. The transcrip- VDSMCs. a: Typical recordings of fluorescent ratio (F340/F380) of tional expression of RyR2 was markedly lower in Flu fura-2 as Ca2+ response to 30 mM caffeine in VDSMCs of Vehicle 2+ Δ in comparison with that in Vehicle (Fig. 3Aa). The and Flu. b: Summarized data about the peak Ca rise ( ratio) in response to caffeine in Vehicle (n = 28) and Flu (n = 13). **P<0.01. + 2+ transcriptional expression of both RyR3S and RyR3L B: Effects of 100 μM NE and 80 mM K on [Ca ]i responses. a: were also markedly reduced by the flutamide treatment. Typical recordings of Ca2+ response to 100 μM NE in the presence of 50 μM Cd2+ and that to 80 mM K+. b: Summarized data about the The Western-blotting analysis of RyR protein expres- 2+ relative peak [Ca ]i rise in response to NE in Vehicle (n = 23) and sion with anti-RyR antibody, which did not distinguish + Flu (n = 21). The value was normalized by the response to 80 mM K RyR subtypes, revealed the much lower expression in in each cell. **P<0.01. c: Summarized data of Δratio in response to Flu than Vehicle (P<0.01; Fig. 3Ad). 80 mM K+ in Vehicle (n = 23) and Flu (n = 21). P>0.05. The influence of flutamide-treatment on the expression of inositol 1,4,5-trisphosphate receptors (IP3R) was expression was not changed in Flu in comparison with examined. Transcripts of IP3R type 1 and type 3 (IP3R1 that in Vehicle (P>0.05; Fig. 3Bc). and IP3R3) were detected in VDSMCs and the expression was not affected significantly by the treatment Effects of flutamide-treatment on the contraction with flutamide (P>0.05; Fig. 3Ba, Bb). The results of induced by direct electrical stimulation Western blotting also indicate that the IP3R protein The contribution of RyR to the contraction induced Ryanodine Receptor in Vas Deferens Smooth Muscle 83

Fig. 3. Effects of flutamide-treatment on RyR and IP3R expression in VD. A: Effects of flutamide- treatment on transcriptional expression of RyR2 (a) and RyR3 (b) in VD of Vehicle and Flu groups were examined by real-time PCR analysis (n = 6 for each). **P<0.01. c: The transcriptional expressions of full length RyR3 (RyR3L) and its short splice variant (RyR3S) were examined. In brain, only the RyR3L transcript was identified. In VD, the RyR3S transcript was more abundant than RyR3L. d: Protein expression of RyR was examined by Western blotting (n = 4). The optical band density in VD of Vehicle was taken as 1.0. **P<0.01. B: Effect of flutamide on IP3R in VD. Transcriptional expression of IP3R1 (a) and IP3R3 (b) were examined by real-time PCR (n = 4 for each). c: Protein expression of IP3R was examined by Western blotting (n = 5). by direct electrical stimulation was determined in VD was comparable between Vehicle and Flu (Fig. 4A, Ba). tissue preparations. To avoid effects of transmitters The application of 30 μM ryanodine markedly reduced released from nerve endings, 1 μM TTX, 1 μM atropine, the amplitude of contractions in Vehicle (P<0.01), but 1 μM phentolamine, 1 μM propranolol, and 10 μM did not significantly change that in Flu (P>0.05). Of suramin were added to the bathing solution. Moreover, interest is that the remaining contraction after the to minimize the modulation of E-C coupling by changes addition of ryanodine in Flu was significantly larger than in action potential shape due to K+-channel activation, that in Vehicle (P<0.01; Fig. 4Bb). 0.1 mM 4-AP and 1 mM TEA were also added. The L-type Ca2+ channel currents activated by depolariza- possibility that the altered expression of K+ channels in tion from −60 to 0 mV were also measured under whole Flu affects the action potential shape and, thereby, cell voltage clamp. The current component blocked by contractions was avoided. Under these conditions, the 50 μM Cd2+ was measured as Ca2+ currents. The current evoked contractions were almost completely blocked by density was 3.34 ± 0.70 and 3.05 ± 0.23 pA/pF in 100 μM Cd2+ or 1 μM nifedipine (not shown), indicating Vehicle (n = 3, N = 2) and Flu (n = 4, N = 2) (P>0.05), that Ca2+ influx through L-type VDCC was essential for respectively. The cell capacitance was neither affected the contraction. The amplitude of evoked contraction significantly (56.8 ± 7.7 pF and 43.3 ± 4.7 pF in Vehicle 84 A Ohno et al

Fig. 4. Effects of flutamide-treatment on contractions evoked by direct electrical stimulation in VD tissues. Effects of 30 μM ryanodine on contractions evoked by direct electrical stimulation were examined in VD tissue segments of Vehicle and Flu. A: Typical traces showing twitch contractions in VD segments of Vehicle (upper) and Flu (bottom). The bathing solution contained the following neurotransmitter antagonists: 1 μM atropine, 1 μM phentolamine, 1 μM propranolol, 1 μM TTX, and 10 μM suramin and voltage dependent K+ channel inhibitors, 0.1 mM 4-AP and 1 mM TEA. Tissue segments were stimulated by 10- ms pulses of 300-mA magnitude at 5 Hz in a train lasting 10 s. The intervals between stimulus trains was 30 s. B: a: Summarized data about the amplitude of contraction in the absence (control) and presence of 30 μM ryanodine (Ry) (n = 6 for each). b: Summarized data about the relative amplitude of remaining components after application of 30 μM ryanodine (n = 6 for each). Data were recalculated from those shown in “a” by taking the amplitude in the control as 100%. **P<0.01

2+ and Flu, respectively) (P>0.05). These results suggest caffeine-induced [Ca ]i rise is comparable between that the functional expression of L-type Ca2+ channel them (12), suggesting no significant gender difference in was not significantly affected by flutamide treatment. RyR2 expression. The expression of IP3R protein was not changed by Discussion flutamide treatment, indicating that only the down- regulation of RyR expression occurred among two types 2+ 2+ 2+ It is a striking finding in this study that Ca sparks of Ca -releasing channels. The reason why [Ca ]i rise 2+ and the [Ca ]i responses to caffeine almost disappeared by NE was larger in VDSMCs from Flu than those from in VDSMCs from Flu. These results were not the Vehicle was not clear. It might be due to the larger Ca2+ consequence of general dysfunction of SR, since the content in SR because of the decreased Ca2+ leak through Ca2+ release by NE in the presence of Cd2+ was rather RyR in SR, which has been considered to be large under increased. Therefore, it is strongly suggested that the pathological conditions but occurs even under normal functional expression of RyR in VDSMCs was markedly conditions (29, 30). Since the up-regulation of L-type reduced by the treatment with flutamide. Ca2+ sparks in VDCC (α1C) expression by testosterone has been SMCs are due to local Ca2+ release from SR through reported in coronary arterial SMCs (31), the decrease in RyR (23, 24) as well as in cardiac (25) and skeletal functional expression of Ca2+ channels in Flu was 2+ muscles (26). Correspondingly, RyR protein expression expected. However, the observation that the [Ca ]i rise in VD was significantly reduced by the treatment with by 80 mM K+ was comparable in VDSMCs from Flu flutamide as shown by Western-blotting analyses. and Vehicle may suggest the unchanged functional Although up-regulation of RyR1 expression by testoster- expression of VDCCs. Moreover, the density of L-type one has been reported in skeletal muscle (14), the Ca2+-channel current in VDSMCs was not affected regulation of RyR2 and/or RyR3 expression by AR significantly in Flu. stimulation has not been reported so far within our The significant contribution of CICR triggered by searches of the literature. Sex-based differences in Ca2+ influx through VDCC to E-C coupling in SMCs myocardial functions are a current topic of great interest have been defined in highly excitable SMCs such as VD (12, 27); and the up-regulated expression of L-type Ca2+ and urinary bladder (6, 7, 9). In contrast, it has been + 2+ 2+ 2+ channel, Na -Ca exchanger and adrenergic β1 receptor suggested that the [Ca ]i rise due to Ca influx through by testosterone has been reported (28). Although the L-type VDCC during an action potential may possibly 2+ peak [Ca ]i rise during E-C coupling is larger in cardiac be large enough to elicit a twitch contraction in some myocytes from male rats than that from females, types of SMCs (32). In this study, the substantial Ryanodine Receptor in Vas Deferens Smooth Muscle 85 contribution of CICR to E-C coupling in rat VDSMCs SR in VDSMCs from Flu was less leaky and keeps the was confirmed by the finding that the contraction high Ca2+ content, the Ca2+ uptake by the pump in the SR evoked by direct electrical stimulation was markedly may also be reduced. This may result in larger contri- 2+ 2+ suppressed by 30 μM ryanodine. Even 100 μM bution of Ca influx through VDCC to [Ca ]i rise ryanodine does not block significantly the Ca2+ current during action potentials in Flu than Vehicle. through L-type VDCC in SMCs (9). Moreover, the Since the activation of AR facilitates the development finding that the evoked contractions in Flu were not of prostate cancer in some stages, the treatment with significantly changed by 30 μM ryanodine may provide anti-androgen drugs including flutamide is common additional evidence supporting that ryanodine does not therapy (17). Although the dose of flutamide used in directly block VDCC in VDSMCs. The possibility that this study was higher than that for the therapeutic dose the Ca2+ release by ryanodine facilitates the Ca2+- (125 mg, 3 times daily), it was in the range of experi- dependent Ca2+-channel inactivation in Vehicle but has mental doses in rats (10 – 100 mg/kg) (38, 39). Of little effect in Flu cannot be ruled out, while it is not the interest is that Ca2+ release from ER via RyR is case in urinary bladder SMCs (9). suggested to be a significant factor to control apoptosis It has been shown that RyR2 is the major transcript of a prostate cancer cell line (LNCaP), which is derived versus RyR3 in mouse urinary bladder SMCs and that from stroma cells and shows testosterone-dependent RyR2 protein expression is significantly decreased in proliferation (40). The relation between the facilitated urinary bladder SMCs from heterozygous RyR2 knock- apoptosis of the prostate cancer cell-line by RyR acti- 2+ out mouse (10). RyR2 mainly contributes to [Ca ]i rise vation and the reduction of RyR expression in vas and subsequent contraction induced by single action deference by flutamide treatment is not clear. However, potential via CICR (10) and RyR3 may contribute little, this study provides important basic information about if any, in mouse urinary bladder SMCs. The functional the modulation of the functional expression of RyR by expression of RyR3 and its contribution to contraction testosterone in male organs and also valuable informa- have been reported in some SMC types (33 – 35). tion for clinical application of anti-androgen therapy. However, the specific expression of a short variant of RyR3 (RyR3S) has been reported in some SMCs (11). Acknowledgments This RyR3S forms a heterotetramer with RyR2 and reduces the Ca2+-releasing–channel activity because of This work was supported by a Grant-in-Aid for Scientific Research its dominant negative features (36). Therefore, the on Priority Areas (20056027) from the Ministry of Education, impact of the co-expression of a dominant negative Culture, Sports, Science, and Technology and by a Grant-in-Aid for Scientific Research (B) (20390027) from Japan Society for the RyR3 variant with RyR2 may possibly be large in Promotion of Science (JSPS) to YI. SO was supported by a Grant-in- VDSMCs like it is in uterine SMCs, in which the Aid for Young Scientists (B) from JSPS (18790058). expression ratio of normal and dominant negative types is changed by pregnancy (37). Surprisingly, the RyR3 transcripts expressed in rat VDSMCs appeared to be References mainly RyR3S, which corresponds to the dominant 1 Fill M, Copello JA. Ryanodine receptor calcium release negative type. Although it is not clear in this study channels. Physiol Rev. 2002;82:893–922. whether RyR3S expressed in rat VDSMCs possesses 2 Dulhunty AF. Excitation-contraction coupling from the 1950s the dominant negative features or not, it was clearly into the new millennium. Clin Exp Pharmacol Physiol. 2006;33: demonstrated that the RyR in Vehicle worked fine to 763–772. evoke Ca2+ sparks and caffeine-induced Ca2+ transient in 3 Endo M. Calcium ion as a second messenger with special single cells and the twitch contraction induced by direct reference to excitation-contraction coupling. J Pharmacol Sci. 2006;100:519–524. stimulation, which was susceptible to ryanodine. 4 Wehrens XH, Marks AR. Altered function and regulation of Therefore, it can be strongly suggested that the cardiac ryanodine receptors in cardiac disease. Trends Biochem decreased susceptibility to caffeine and ryanodine in Sci. 2003;28:671–678. Flu was due to the decrease in functional RyR protein 5 Ogawa Y, Kurebayashi N, Murayama T. Putative roles of type 3 but not the increase in dominant negative variant. The ryanodine receptor isoforms (RyR3). Trends Cardiovasc Med. decrease in RyR protein in Flu was also demonstrated by 2000;10:65–70. Western blotting analysis. 6 Imaizumi Y, Torii Y, Ohi Y, Nagano N, Atsuki K, Yamamura H, et al. 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