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Spontaneous Ca2+ Signaling of Interstitial Cells in the Guinea Pig Prostate
Spontaneous Ca2ⴙ Signaling of Interstitial Cells in the Guinea Pig Prostate Michelle Lam, Yusuke Shigemasa, Betty Exintaris, Richard J. Lang and Hikaru Hashitani* From Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences (ML, BE), Parkville and Department of Physiology, Monash University (RJL), Clayton, Victoria, Australia, and Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences (YS, HH), Nagoya, Japan
Abbreviations and Acronyms [Ca2⫹]i ⫽ intracellular concentration of free calcium ion CPA ⫽ cyclopiazonic acid DS ⫽ dispersal solution ER ⫽ endoplasmic reticulum IC ⫽ interstitial cell ICC ⫽ IC of Cajal PIC ⫽ prostate IC PSS ⫽ physiological salt solution STD ⫽ spontaneous transient depolarization Submitted for publication March 11, 2011. Study received Nagoya City University animal experimentation ethics committee approval. Supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) 22390304 (HH) and the National Health and Medical Research Council (RJL). Supplementary material for this article can be obtained at http://www.med.nagoya-cu.ac.jp/ physiol1.dir/Suppl_videos201106/Suppl_video.html. * Correspondence: Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan (telephone: 81-52-8538131; FAX: 81-52-8421538; e-mail: [email protected]).
Purpose: We investigated whether prostate interstitial cells generate spontaneous Ca2⫹ oscillation, a proposed mechanism underlying pacemaker potentials to drive spontaneous activity in stromal smooth muscle cells. Materials and Methods: Intracellular free Ca2⫹ in portions of guinea pig prostate and freshly isolated, single prostate interstitial cells were visualized using fluo-4 Ca2⫹ fluorescence. Spontaneous electrical activity was recorded in situ with intracellular microelectrodes. Results: In whole tissue preparations spontaneous Ca2⫹ flashes firing synchronously across all smooth muscle cells within the field of view resulted in muscle wall contractions. Nonpropagating Ca2⫹ waves were also recorded in individual smooth muscle cells. Nifedipine (Sigma®) (1 M) largely decreased or abolished these Ca2⫹ flashes and suppressed slow wave discharge upon blockade of their superimposed action potentials. Isolated prostate interstitial cells were readily distinguished from smooth muscle cells by their spiky processes and lack of contraction during intracellular Ca2⫹ increases. Prostate interstitial cells generated spontaneous Ca2⫹ transients in the form of whole cell flashes, intracellular Ca2⫹ waves or localized Ca2⫹ sparks. All 3 Ca2⫹ signals were abolished by nicardipine (1 M), cyclopiazonic acid (10 M), caffeine (Sigma) (10 mM) or extracellular Ca2⫹ removal. Conclusions: Prostate interstitial cells generate spontaneous Ca2⫹ transients that occur at a frequency comparable to Ca2⫹ flashes in situ or slow waves relying on functional internal Ca2⫹ stores. However, unlike other interstitial cells in the urinary tract, Ca2⫹ influx through L-type Ca2⫹ channels is fundamental to Ca2⫹ transient firings in prostate interstitial cells. Thus, it is not possible to conclude that prostate interstitial cells are responsible for pacemaker potential generation. Key Words: prostate; calcium channels, L-type; muscle contraction; action potentials; myocytes, smooth muscle
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SMOOTH muscles in the urinary tract and male genital organs develop spontaneous contractions that have often been referred to as myogenic activity in terms of their lack of sensitivity to various blockers of neurotransmitter action or release. In the gastrointestinal
tract spontaneous contractions of the smooth muscle wall are considered to result from slow wave generation, which is electrically driven by ICC networks.1 Identifying ICs in many urogenital organs by their Kit immunoreactivity and/or morphologi-
0022-5347/11/1866-2478/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 2478-2486, December 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.07.082
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RESEARCH, INC.
SPONTANEOUS CA2⫹ SIGNALING OF INTERSTITIAL CELLS IN PROSTATE
cal characteristics similar to those of ICCs has highlighted their putative role in generating spontaneous myogenic activity.2 However, there is considerable variety in the origin and nature of spontaneous activity in the various tissues and organs of the urogenital system. ICs or ICC-like cells in the urethra fire STDs3 upon the opening of Ca2⫹ activated Cl⫺ currents triggered by transient increases in [Ca2⫹].4,5 However, since a low temporal correlation exists between spontaneous Ca2⫹ signaling of ICs and adjacent smooth muscle cells in situ, urethral ICs may act as less secure electrical drivers to increase smooth muscle excitability rather than as directly coupled pacemaker cells.6 In the bladder and corpus cavernosum smooth muscle cells generate their own spontaneous electrical activity and Ca2⫹ transients,7–9 as do ICs distributed in the bladder, although at a slower frequency than neighboring smooth muscle cells.2,10,11 Peristaltic contractions of the renal pelvis are likely driven by atypical smooth muscle cells that generate STDs triggered by intracellular Ca2⫹ transients. The role of neighboring ICs that also generate spontaneous Ca2⫹ transients, again at a lower frequency, remains unknown.12 Smooth muscle cells of the prostate stroma develop spontaneous contractions. Intracellular recording studies showed the generation of 2 distinct patterns of electrical activity, ie pacemaker potentials and slow waves.13–15 Due to the electrical quiescence of isolated smooth muscle cells16 and the relative insensitivity of pacemaker potentials and slow waves to nifedipine compared to their superimposed action potentials13–15 it was suggested that PICs generate pacemaker potentials to drive slow wave firing in stromal smooth muscle cells. However, to our knowledge the electrical and Ca2⫹ signaling of single PICs has not been reported. Thus, their role as pacemaker cells has yet to be established. In the current study spontaneous Ca2⫹ signaling and electrical activity in intact tissue preparations of the guinea pig prostate were recorded separately using fluo-4 fluorescence and intracellular microelectrode techniques. To our knowledge enzymatically isolated single PICs, which have morphological features that readily distinguish them from smooth muscle cells, were examined for the first time in terms of the pharmacological properties of their spontaneous Ca2⫹ signals.
MATERIALS AND METHODS Tissue Preparation Male guinea pigs weighing 250 to 300 gm were sacrificed by exsanguination under sevoflurane anesthesia according to the procedure approved by the Nagoya City University animal experimentation ethics committee. The pros-
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tate was removed and pinned in a dissecting dish. The outer connective tissue layer was dissected from the prostate and the individual lobes were cut open.
Cell Isolation Single PICs were dispersed from 5 mm2 portions of prostate lobes in Ca2⫹-free HEPES buffered DS containing 3 mg collagenase (Worthington type I), 0.2 mg protease (type XXIV), 2 mg bovine serum albumin and 2 mg trypsin inhibitor (Sigma) at 37C. The portions were placed in low Ca2⫹ DS ([Ca2⫹] ⫽ 100 M) at room temperature and subjected to gentle agitation with a pipette. Single PICs were allowed 10 to 20 minutes to settle on a glass bottomed dish.
Intracellular Imaging Calcium. Whole tissue preparations of prostate lobes were incubated in low Ca2⫹ bicarbonate buffered PSS ([Ca2⫹]o ⫽ 0.5 mM) containing 10 M fluo-4 AM (Dojindo, Kumamotu, Japan) and Cremophor EL® (0.01%) for 60 minutes at 36C. Single PICs were incubated in HEPES buffered low Ca2⫹ PSS ([Ca2⫹] ⫽ 100 M) containing 1 M fluo-4 AM for 5 to 10 minutes at room temperature. After incubation tissue preparations and isolated PICs were superfused with dye-free PSS ([Ca2⫹] ⫽ 2.5 mM) warmed to 36C and buffered with bicarbonate and HEPES, respectively. After illumination at 495 nm fluorescence emissions above 515 nm were detected, as described previously.6,17 Relative changes in intracellular Ca2⫹ are shown as Ft/F0, where Ft represents the fluorescence generated by an event and F0 represents baseline. The amplitude of Ca2⫹ transients (⌬Ft/F0) was calculated by subtracting the baseline fluorescence ratio from the peak values of the events. Mitochondria, ER and nuclei. To assess the intracellular distribution of mitochondria, ER and nuclei PICs were incubated with 10 nM MitoTracker™ Red for 10 minutes, 0.1 M BODIPY® FL thapsigargin for 30 minutes and 100 nM Hoechst 33342 (Invitrogen™) for 5 minutes at room temperature, respectively. PICs were superfused with dye-free HEPES buffered PSS ([Ca2⫹] ⫽ 2.5 mM) warmed to 36C. Images of fluorescent organelles were acquired using the same epifluorescence microscope and objectives as described. MitoTracker Red was excited at 580 nm and emission was collected at 600 nm. BODIPY FL thapsigargin was excited at 490 nm and emission was detected at 520 nm. Hoechst 33342 was excited at 360 nm and emission was detected at 460 nm.
Intracellular Recording Individual stromal cells of whole tissue preparations of prostate lobes were impaled with glass capillary microelectrodes and filled with 0.5 M KCl (tip resistance 150 to 250 M⍀). Membrane potential changes were recorded as described previously.11
Solutions Bicarbonate buffered PSS was composed of 137.5 mM Na⫹, 4.7 mM K⫹, 2.5 mM Ca2⫹, 1.2 mM Mg2⫹, 15.5 mM HCO3⫺, 1.2 mM H2PO4⫺, 134 mM Cl⫺ and 15 mM glucose. The pH of PSS was 7.2 when bubbled with 95% O2 and 5% CO2. The measured pH of the recording bath was
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approximately 7.4. HEPES buffered PSS was composed of 137.5 mM Na⫹, 5.9 mM K⫹, 2.5 mM Ca2⫹, 1.2 mM Mg2⫹, 1.2 mM H2PO4⫺, 134 mM Cl⫺, 10 mM glucose and 10 mM HEPES. DS was composed of 126 mM Na⫹, 6 mM K⫹, 1 mM Mg2⫹, 134 mM Cl⫺, 10 mM glucose and 10 mM HEPES. PSS and DS pH was adjusted to 7.3 to 7.35 with NaOH. The drugs used were caffeine, CPA, nicardipine and nifedipine. CPA and nicardipine were dissolved in dimethyl sulfoxide while nifedipine was dissolved in absolute ethanol. Caffeine was directly dissolved in PSS just before use. The final concentration of these solvents in the PSS did not exceed 1:1,000.
Calculations and Statistics Measured values are shown as the mean ⫾ SD. Statistical significance was tested using the paired t test and considered significant at p ⬍0.05.
RESULTS In Situ Prostate Stromal Cell Spontaneous Ca2ⴙ Transients In whole mount prostate preparations stromal cells showed spontaneous Ca2⫹ flashes that occurred almost synchronously across all cells within the field of view (fig. 1, A and B). Spontaneous Ca2⫹ flashes occurred at a mean of 4.4 ⫾ 1.2 minute⫺1 with a mean amplitude of 1.03 ⫾ 0.35 Ft/F0 in 18 animals (18 preparations). It was clear that Ca2⫹ flashes occurred simultaneously along the full length of individual spindle-shaped smooth muscle cells (fig. 1, C and D), which also showed nonpropagating Ca2⫹ transients (fig. 1, E). Nifedipine (1 M) abolished (4 preparations) or largely suppressed (6 preparations) spontaneous Ca2⫹ flashes (fig. 1, F). In 6 preparations in which nifedipine did not abolish spontaneous Ca2⫹ flashes residual Ca2⫹ transients were still recorded synchronously across the field of view at a mean frequency of 2.1 ⫾ 0.84 minute⫺1 and mean amplitude of 0.18 ⫾ 0.08 Ft/F0 (mean 14.8% ⫾ 3.6% of control values). In some cells nonpropagating Ca2⫹ transients that had no temporal correlation with Ca2⫹ flashes were less affected by Ca2⫹ channel blockers (fig. 1, F). However, we did not further examine their properties. Intracellular Recording We made intracellular microelectrode recordings from the prostate stroma to confirm previous observations of the effects of nifedipine on slow wave discharge. In this series of experiments stromal smooth muscle cells had a mean resting membrane potential of ⫺51.7 ⫾ 4.6 mV in 8 animals (8 preparations) and generated spontaneous slow waves with superimposed action potentials (mean total amplitude 51.5 ⫾ 3.7 mV) and STDs (fig. 2, A and B). Nifedipine (1 M) abolished action potential firing and greatly suppressed slow wave amplitude (fig. 2, B and C), such that the mean
amplitude in nifedipine was 7.1 ⫾ 5.8 mV in 6 animals (6 preparations) but abolished in 2 (2 preparations). Notably suppressed slow waves were still associated with muscle contractions when viewed under microscopy, suggesting incomplete blockade of L-type Ca2⫹ channels in smooth muscle. In 6 experiments electrical couplings between cells were investigated by impaling prostate stromal preparations with 2 independent microelectrodes in 6 animals. Slow waves and superimposed action potentials recorded from the 2 electrodes occurred almost concurrently (fig. 2, D to F). However, STDs failed to propagate from 1 cell to another (fig. 2, G). Isolated PIC Morphological Characteristics Single PICs had spindle-shaped cell bodies and were distinguished from smooth muscle cells by their spiky processes (fig. 3, A and B). Circular epithelial cells with a diameter of less than 10 m were also frequently seen. PICs had a long, thin cell body with a mean length of 159.3 ⫾ 6.0 m and a maximum width at the nucleus of 11.3 ⫾ 0.3 m in 21 animals (49 preparations). In some cases the location of nuclei was confirmed by Hoechst 33342 staining (fig. 3, C and D). Upon staining with MitoTracker Red mitochondria were observed to be clustered around the nucleus while other short filamentous mitochondria were scattered in the cell periphery (fig. 3, D). ER stained with BODIPY FL thapsigargin was sparsely distributed in the cell periphery and preferentially distributed around the nucleus (fig. 3, D). PICs were also distinguished from smooth muscle cells by not contracting when [Ca2⫹]i was raised. Increasing the extracellular concentration of K ions from 6 to 30 mM increased [Ca2⫹]i in PICs and smooth muscle cells (fig. 3, E to J). In contrast to smooth muscle cells, which contracted vigorously in this high K solution, PICs did not contract (fig. 3, E to J). Spontaneous Ca2ⴙ Signaling in PICs Isolated PICs generated spontaneous Ca2⫹ signals in the form of Ca2⫹ waves, whole cell Ca2⫹ flashes or Ca2⫹ sparks. Approximately 45% of PICs generated Ca2⫹ waves that originated from any region and propagated toward the cell periphery (fig. 4, A to C). Spontaneous Ca2⫹ waves were generated at a mean of 3.3 ⫾ 1.0 minute⫺1 with a mean amplitude of 1.6 ⫾ 0.63 Ft/F0 in 17 animals (37 preparations) (fig. 4, G). Another 40% of PICs generated whole cell Ca2⫹ flashes, which occurred almost simultaneously along the cell length (fig. 4, D to F). Spontaneous Ca2⫹ flashes often fired in bursts (fig. 4, F). Whole cell Ca2⫹ flashes occurred at a mean of 6.5 ⫾ 2.1 minute⫺1 with a mean amplitude of 0.75 ⫾ 0.32 Ft/F0 in 18 animals (31 preparations), which was significantly smaller than Ca2⫹ waves (p ⬍0.05, fig. 4, G). Irrespective of the dominant pattern of spontaneous Ca2⫹ signals PICs also generated Ca2⫹
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Figure 1. Spontaneous Ca2⫹ transients in prostate stromal cells in situ. Sequential fluo-4 fluorescent images show spontaneous Ca2⫹ flash in prostate preparation with 100-millisecond frame interval (A). Ca2⫹ transients recorded from 3 regions occurred almost concurrently (B). Sequential images show spontaneous Ca2⫹ flash in another prostate preparation with 100-millisecond frame interval (C). Ca2⫹ transients recorded from 3 smooth muscle cells occurred almost simultaneously (D). s, seconds. In another preparation in which Ca2⫹ flashes and nonpropagating Ca2⫹ transients were generated (E) nifedipine (1 M) abolished Ca2⫹ flashes in 2 cells but not nonpropagating Ca2⫹ transients in another (F).
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Figure 2. Nifedipine effects on spontaneous electrical activity. In whole tissue preparation of 1 prostate lobe smooth muscle generated spontaneous slow waves with superimposed action potentials and STDs (A and B). One M nifedipine abolished action potentials and suppressed slow waves but scarcely affected STD generation (A and C). s, seconds. Synchronized slow waves and superimposed action potentials were recorded from 2 cells in prostate lobe preparation (D and E). Each pair of action potentials occurred almost simultaneously (E). In another preparation synchronized slow waves were recorded with 2 electrodes (F). In another preparation STDs were recorded with first but not second electrode (G).
sparks that occurred locally (fig. 4, B and E). The remaining 15% of PICs showed only Ca2⫹ sparks without generating Ca2⫹ waves or whole cell Ca2⫹ flashes (fig. 4, H). Properties. Spontaneous Ca2⫹ signals in PICs, including 5 Ca2⫹ wave cells in 4 animals and 6 Ca2⫹ flash cells in 4, were readily abolished by nicardipine (1 M) (fig. 5, A and B). Nominally Ca2⫹-free solu-
tion also prevented the generation of spontaneous Ca2⫹ signals and decreased baseline Ca2⫹, ie 3 Ca2⫹ wave cells and 3 Ca2⫹ flash cells in 3 animals each (fig. 5, C). These results suggest that PIC spontaneous Ca2⫹ signals fundamentally rely on Ca2⫹ influx through voltage dependent L-type Ca2⫹ channels. Spontaneous Ca2⫹ signals were abolished by CPA (10 M) without changes in baseline Ca2⫹, ie 7 Ca2⫹
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Figure 3. Isolated PIC morphological characteristics. PICs had spindle-shaped cell bodies and were distinguished from smooth muscle cells by spiky processes (A and B). Small round epithelial cells were also seen. Another PIC had spindle-shaped cell body and several processes (C). Overlay shows intracellular distribution of nucleus (blue areas), mitochondria (red areas) and ER (green areas) in same PIC (D). Scale bar (A) represents 30 m (A to D). Bright field (E) and fluo-4 fluorescence (F) images show PIC. Upon [Ca2⫹]i increase in high K solution PIC did not contract (G). Fluorescence images show smooth muscle cell in normal (H) and high K (I and J) solution. Note increased [Ca2⫹]i and vigorous smooth muscle cell contraction.
wave cells in 5 animals and 5 Ca2⫹ flash cells in 4 (fig. 5, D). Also, caffeine (10 mM) abolished the generation of spontaneous Ca2⫹ signals in PICs, ie 5 Ca2⫹ wave cells in 4 animals and 5 Ca2⫹ flash cells in 3 (fig. 5, E), suggesting that the generation of spontaneous Ca2⫹ signals of PICs crucially depends on Ca2⫹ release from ER stores.
DISCUSSION In intact preparations of prostate spontaneous Ca2⫹ flashes generated almost synchronously in all stromal cells in our field of view, and slow waves and their superimposed action potentials were recorded concurrently using 2 independent electrodes. Nifedipine (1 M) abolished action potentials and greatly suppressed the spontaneous firing of Ca2⫹ flashes and slow waves in situ. Thus, it is reasonable to suggest that these Ca2⫹ flashes in individual smooth muscle cells reflect an increase in [Ca2⫹]i during the firing of bursts of action potentials. Previous studies using intracellular recording microelectrodes consistently revealed that nifedipine (1 M) abolished action potential generation but had variable effects on slow wave amplitudes, presumably depending on the distance between the site of pacemaker potential generation and the site of microelectrode penentration.13–15 Small residual Ca2⫹ flashes and suppressed slow waves were detected in the presence of nifedipine (1 M) but these spontaneous signals were associated
with small muscle contractions. Thus, we believe that these residual Ca2⫹ flashes were Ca2⫹ signals recorded from smooth muscle cells, reflecting incomplete blockade of L-type Ca2⫹ channels. Precedence for this incomplete block occurs in the bladder detrusor, in which individual action potentials are readily abolished by nifedipine (1 M) while bursting action potentials require higher nifedipine concentrations (10 M).18 In the mouse renal pelvis nifedipine concentrations greater than 3 M are sometimes required to completely block action potential and Ca2⫹ transient discharge in the typical smooth muscle cells of the muscle wall.12 By analogy with Ca2⫹ signaling in ICC-like cells in the rabbit urethra,6 guinea pig bladder11 and ICC networks in several gut preparations in situ19 one might have expected some form of nifedipine resistant pacemaker Ca2⫹ signal that correlated in its timing and kinetics with the pacemaker potentials or prostate slow waves recorded in the presence of nifedipine. Nonpropagating Ca2⫹ transients in individual cells were occasionally seen in the intact preparation in the absence of nifedipine. However, they showed no temporal correlation with Ca2⫹ flashes in control solutions, indicating that these waves were unlikely to act as genuine pacemaker signals. To our knowledge this report represents the first time that PICs were enzymatically isolated from guinea pig prostate for experimentation. Although we did not examine their Kit immunoreactivity, iso-
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Figure 4. PIC spontaneous Ca2⫹ transients. Sequential fluo-4 fluorescent images show spontaneous Ca2⫹ wave in isolated PIC with 100-millisecond frame interval (A). Spontaneous Ca2⫹ waves and Ca2⫹ sparks were generated (B). When shown on expanded time scale, delays between Ca2⫹ waves were obvious in 3 intracellular regions (C). Sequential images show whole cell Ca2⫹ flash in another isolated PIC with 100-millisecond frame interval (D). Spontaneous Ca2⫹ flashes and Ca2⫹ sparks were generated (E). When shown on expanded time scale, multiple Ca2⫹ flashes in 3 intracellular regions occurred almost simultaneously (F). s, seconds. Note Ca2⫹ wave and Ca2⫹ flash frequency and amplitude (G). Another PIC generated only Ca2⫹ sparks (H). X and Y axes (H) also refer to B and E.
SPONTANEOUS CA2⫹ SIGNALING OF INTERSTITIAL CELLS IN PROSTATE
Figure 5. PIC spontaneous Ca2⫹ transient properties. Nicardipine abolished spontaneous Ca2⫹ signals in PICs generating Ca2⫹ waves (A) and flashes (B). Switching from normal PSS to nominally Ca2⫹-free solution prevented spontaneous Ca2⫹ wave generation in PIC associated with decreased baseline [Ca2⫹]i (C). Upon re-addition of 2.5 mM Ca2⫹ spontaneous Ca2⫹ waves were restored. In another PIC generating spontaneous Ca2⫹ waves 10 M CPA abolished Ca2⫹ waves with only small increase in baseline [Ca2⫹]i (D). In another PIC 10 mM caffeine abolished Ca2⫹ wave spontaneous generation (E).
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Previous intracellular microelectrode studies demonstrated that prostate slow waves intimately depend on Ca2⫹ release from ER Ca2⫹ stores as well as on the uptake and release of Ca2⫹ from mitochondria.20 This intimate relationship is perhaps indicated by the preferential clustering of ER and mitochondria around the nucleus when visualized using specific fluorescent dyes (fig. 3). These results also confirm similar clustering of ER and mitochondria in the perinuclear region of single ICC-like cells isolated from the rabbit urethra,17 which also develop spontaneous activity depending on the Ca2⫹ uptake and release of each organelle.21,22 However, in contrast with Ca2⫹ signaling in rabbit urethral ICC-like cells, Ca2⫹ release in the prostate is driven by 1,4,5-triphosphate dependent release mechanisms and not by the release of stored Ca2⫹ via ryanodine receptors.20 In the current experiments CPA and caffeine abolished spontaneous Ca2⫹ signals in isolated PICs, consistent with this dependence on the release of stored Ca2⫹. Again unlike urethral ICC-like cells, neither CPA nor caffeine evoked large increases in baseline Ca2⫹ in single PICs before blocking their spontaneous Ca2⫹ signals. This suggests that the capacity of intracellular Ca2⫹ stores of PICs may be smaller than that of urethral ICC-like cells. Alternatively PICs may have stronger Ca2⫹ extrusion systems than urethral ICC-like cells. Smaller Ca2⫹ stores in PICs or their relative lack of ryanodine receptor release channels may well explain their greater dependence on Ca2⫹ influx through L-type Ca2⫹ channels to generate their spontaneous Ca2⫹ signals.
CONCLUSIONS lated PICs were readily distinguished from neighboring single smooth muscle cells or epithelial cells by their spiky processes and lack of contraction upon raising [Ca2⫹]i with high [K⫹]o PSS (fig. 3). ICC-like cells dispersed from the rabbit urethra or the guinea pig bladder show spontaneous Ca2⫹ waves and sparks as well as Ca2⫹ activated chloride currents that do not rely on Ca2⫹ influx through L-type Ca2⫹ channels.4,5,10 Freshly isolated PICs also generated whole cell Ca2⫹ flashes, intracellular Ca2⫹ waves and high frequency localized Ca2⫹ sparks. Pacemaker potentials can be recorded in prostate preparations in situ in the presence of nifedipine. Thus, isolated PICs, if they are indeed responsible for pacemaker generation, should be able to develop some form of substantial Ca2⫹ signals that do not fundamentally rely on L-type Ca2⫹ channels. Therefore, it was surprising that almost all spontaneous Ca2⫹ signals recorded in isolated PICs were abolished by nicardipine.
We suggest that in whole mount preparations of prostate in situ the spontaneous Ca2⫹ flashes that occur at frequencies similar to the firing of slow waves reflect Ca2⫹ influx during the synchronous discharge of action potentials in individual smooth muscle cells. Isolated PICs developed spontaneous Ca2⫹ signals that depended on ER Ca2⫹ release. The signals were readily abolished by nicardipine, indicating that Ca2⫹ influx through L-type Ca2⫹ channels has a fundamental role in generating PIC Ca2⫹ signals. However, this dependence of Ca2⫹ signaling on Ca2⫹ influx does not support previous intracellular recording studies in which pacemaker potentials in situ were recorded in the presence of nifedipine. Also, mechanisms underlying the spontaneous activity of single PICs differ significantly from those of other interstitial cells in the urinary tract. Each phenomenon merits further investigation. Thus, we cannot currently conclude that PICs act as pacemaker cells that directly drive spontaneous activity in prostate stromal smooth muscle.
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REFERENCES 1. Sanders KM, Koh SD and Ward SM: Interstitial cells of Cajal as pacemakers in the gastrointestinal tract. Annu Rev Physiol 2006; 68: 307.
9. Karkanis T, DeYoung L, Brock GB et al: Ca2⫹activated Cl⫺ channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection. J Appl Physiol 2003; 94: 301.
2. Brading AF and McCloskey KD: Mechanisms of disease: specialized interstitial cells of the urinary tract—an assessment of current knowledge. Nat Clin Pract Urol 2005; 2: 546.
10. Wu C, Sui GP and Fry CH: Purinergic regulation of guinea pig suburothelial myofibroblasts. J Physiol 2004; 559: 231.
3. Hashitani H, Van Helden DF and Suzuki H: Properties of spontaneous depolarisations in circular smooth muscle cells of rabbit urethra. Br J Pharmacol 1996; 118: 1627.
11. Hashitani H, Yanai Y and Suzuki H: Role of interstitial cells and gap junctions in the transmission of spontaneous Ca2⫹ signals in detrusor smooth muscles of the guinea-pig urinary bladder. J Physiol 2004; 559: 567.
4. Sergeant GP, Hollywood MA, McCloskey KD et al: Specialised pacemaking cells in the rabbit urethra. J Physiol 2000; 526: 359. 5. Johnston L, Sergeant GP, Hollywood MA et al: Calcium oscillations in interstitial cells of the rabbit urethra. J Physiol 2005; 565: 449. 6. Hashitani H and Suzuki H: Properties of spontaneous Ca2⫹ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ. J Physiol 2007; 583: 505. 7. Montgomery BS and Fry CH: The action potential and net membrane currents in isolated human detrusor smooth muscle cells. J Urol 1992; 147: 176. 8. Sui G, Fry CH, Malone-Lee J et al: Aberrant Ca2⫹ oscillations in smooth muscle cells from overactive human bladders. Cell Calcium 2009; 45: 456.
12. Lang RJ, Hashitani H, Tonta MA et al: Spontaneous electrical and Ca2⫹ signals in typical and atypical smooth muscle cells and interstitial cell of Cajal-like cells of mouse renal pelvis. J Physiol 2007; 583: 1049. 13. Exintaris B, Klemm MF and Lang RJ: Spontaneous slow wave and contractile activity of the guinea pig prostate. J Urol 2002; 168: 315. 14. Lang RJ, Nguyen DT, Matsuyama H et al: Characterization of spontaneous depolarizations in smooth muscle cells of the Guinea pig prostate. J Urol 2006; 175: 370. 15. Nguyen DT, Lang RJ and Exintaris B: ␣1-adrenoceptor modulation of spontaneous electrical waveforms in the guinea-pig prostate. Eur J Pharmacol 2009; 608: 62. 16. Lang RJ, Mulholland E and Exintaris B: Characterization of the ion channel currents in single
myocytes of the guinea pig prostate. J Urol 2004; 172: 1179. 17. Hashitani H, Lang RJ and Suzuki H: Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2⫹ waves in interstitial cells of Cajal-like cells of the rabbit urethra. Br J Pharmacol 2010; 161: 680. 18. Hashitani H, Brading AF and Suzuki H: Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea-pig bladder. Br J Pharmacol 2004; 141: 183. 19. Hennig GW, Hirst GD, Park KJ et al: Propagation of pacemaker activity in the guinea-pig antrum. J Physiol 2004; 556: 585. 20. Exintaris B, Nguyen DT, Lam M et al: Inositol trisphosphate-dependent Ca stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland. Br J Pharmacol 2009; 156: 1098. 21. Sergeant GP, Bradley E, Thornbury KD et al: Role of mitochondria in modulation of spontaneous Ca2⫹ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra. J Physiol 2008; 586: 4631. 22. Sergeant GP, Hollywood MA, McCloskey KD et al: Role of IP3 in modulation of spontaneous activity in pacemaker cells of rabbit urethra. Am J Physiol Cell Physiol 2001; 280: C1349.
Abbreviations and Acronyms [Ca2⫹]i ⫽ intracellular concentration of free calcium ion CPA ⫽ cyclopiazonic acid DS ⫽ dispersal solution ER ⫽ endoplasmic reticulum IC ⫽ interstitial cell ICC ⫽ IC of Cajal PIC ⫽ prostate IC PSS ⫽ physiological salt solution STD ⫽ spontaneous transient depolarization Submitted for publication March 11, 2011. Study received Nagoya City University animal experimentation ethics committee approval. Supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) 22390304 (HH) and the National Health and Medical Research Council (RJL). Supplementary material for this article can be obtained at http://www.med.nagoya-cu.ac.jp/ physiol1.dir/Suppl_videos201106/Suppl_video.html. * Correspondence: Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan (telephone: 81-52-8538131; FAX: 81-52-8421538; e-mail: [email protected]).
Purpose: We investigated whether prostate interstitial cells generate spontaneous Ca2⫹ oscillation, a proposed mechanism underlying pacemaker potentials to drive spontaneous activity in stromal smooth muscle cells. Materials and Methods: Intracellular free Ca2⫹ in portions of guinea pig prostate and freshly isolated, single prostate interstitial cells were visualized using fluo-4 Ca2⫹ fluorescence. Spontaneous electrical activity was recorded in situ with intracellular microelectrodes. Results: In whole tissue preparations spontaneous Ca2⫹ flashes firing synchronously across all smooth muscle cells within the field of view resulted in muscle wall contractions. Nonpropagating Ca2⫹ waves were also recorded in individual smooth muscle cells. Nifedipine (Sigma®) (1 M) largely decreased or abolished these Ca2⫹ flashes and suppressed slow wave discharge upon blockade of their superimposed action potentials. Isolated prostate interstitial cells were readily distinguished from smooth muscle cells by their spiky processes and lack of contraction during intracellular Ca2⫹ increases. Prostate interstitial cells generated spontaneous Ca2⫹ transients in the form of whole cell flashes, intracellular Ca2⫹ waves or localized Ca2⫹ sparks. All 3 Ca2⫹ signals were abolished by nicardipine (1 M), cyclopiazonic acid (10 M), caffeine (Sigma) (10 mM) or extracellular Ca2⫹ removal. Conclusions: Prostate interstitial cells generate spontaneous Ca2⫹ transients that occur at a frequency comparable to Ca2⫹ flashes in situ or slow waves relying on functional internal Ca2⫹ stores. However, unlike other interstitial cells in the urinary tract, Ca2⫹ influx through L-type Ca2⫹ channels is fundamental to Ca2⫹ transient firings in prostate interstitial cells. Thus, it is not possible to conclude that prostate interstitial cells are responsible for pacemaker potential generation. Key Words: prostate; calcium channels, L-type; muscle contraction; action potentials; myocytes, smooth muscle
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SMOOTH muscles in the urinary tract and male genital organs develop spontaneous contractions that have often been referred to as myogenic activity in terms of their lack of sensitivity to various blockers of neurotransmitter action or release. In the gastrointestinal
tract spontaneous contractions of the smooth muscle wall are considered to result from slow wave generation, which is electrically driven by ICC networks.1 Identifying ICs in many urogenital organs by their Kit immunoreactivity and/or morphologi-
0022-5347/11/1866-2478/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 2478-2486, December 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.07.082
AND
RESEARCH, INC.
SPONTANEOUS CA2⫹ SIGNALING OF INTERSTITIAL CELLS IN PROSTATE
cal characteristics similar to those of ICCs has highlighted their putative role in generating spontaneous myogenic activity.2 However, there is considerable variety in the origin and nature of spontaneous activity in the various tissues and organs of the urogenital system. ICs or ICC-like cells in the urethra fire STDs3 upon the opening of Ca2⫹ activated Cl⫺ currents triggered by transient increases in [Ca2⫹].4,5 However, since a low temporal correlation exists between spontaneous Ca2⫹ signaling of ICs and adjacent smooth muscle cells in situ, urethral ICs may act as less secure electrical drivers to increase smooth muscle excitability rather than as directly coupled pacemaker cells.6 In the bladder and corpus cavernosum smooth muscle cells generate their own spontaneous electrical activity and Ca2⫹ transients,7–9 as do ICs distributed in the bladder, although at a slower frequency than neighboring smooth muscle cells.2,10,11 Peristaltic contractions of the renal pelvis are likely driven by atypical smooth muscle cells that generate STDs triggered by intracellular Ca2⫹ transients. The role of neighboring ICs that also generate spontaneous Ca2⫹ transients, again at a lower frequency, remains unknown.12 Smooth muscle cells of the prostate stroma develop spontaneous contractions. Intracellular recording studies showed the generation of 2 distinct patterns of electrical activity, ie pacemaker potentials and slow waves.13–15 Due to the electrical quiescence of isolated smooth muscle cells16 and the relative insensitivity of pacemaker potentials and slow waves to nifedipine compared to their superimposed action potentials13–15 it was suggested that PICs generate pacemaker potentials to drive slow wave firing in stromal smooth muscle cells. However, to our knowledge the electrical and Ca2⫹ signaling of single PICs has not been reported. Thus, their role as pacemaker cells has yet to be established. In the current study spontaneous Ca2⫹ signaling and electrical activity in intact tissue preparations of the guinea pig prostate were recorded separately using fluo-4 fluorescence and intracellular microelectrode techniques. To our knowledge enzymatically isolated single PICs, which have morphological features that readily distinguish them from smooth muscle cells, were examined for the first time in terms of the pharmacological properties of their spontaneous Ca2⫹ signals.
MATERIALS AND METHODS Tissue Preparation Male guinea pigs weighing 250 to 300 gm were sacrificed by exsanguination under sevoflurane anesthesia according to the procedure approved by the Nagoya City University animal experimentation ethics committee. The pros-
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tate was removed and pinned in a dissecting dish. The outer connective tissue layer was dissected from the prostate and the individual lobes were cut open.
Cell Isolation Single PICs were dispersed from 5 mm2 portions of prostate lobes in Ca2⫹-free HEPES buffered DS containing 3 mg collagenase (Worthington type I), 0.2 mg protease (type XXIV), 2 mg bovine serum albumin and 2 mg trypsin inhibitor (Sigma) at 37C. The portions were placed in low Ca2⫹ DS ([Ca2⫹] ⫽ 100 M) at room temperature and subjected to gentle agitation with a pipette. Single PICs were allowed 10 to 20 minutes to settle on a glass bottomed dish.
Intracellular Imaging Calcium. Whole tissue preparations of prostate lobes were incubated in low Ca2⫹ bicarbonate buffered PSS ([Ca2⫹]o ⫽ 0.5 mM) containing 10 M fluo-4 AM (Dojindo, Kumamotu, Japan) and Cremophor EL® (0.01%) for 60 minutes at 36C. Single PICs were incubated in HEPES buffered low Ca2⫹ PSS ([Ca2⫹] ⫽ 100 M) containing 1 M fluo-4 AM for 5 to 10 minutes at room temperature. After incubation tissue preparations and isolated PICs were superfused with dye-free PSS ([Ca2⫹] ⫽ 2.5 mM) warmed to 36C and buffered with bicarbonate and HEPES, respectively. After illumination at 495 nm fluorescence emissions above 515 nm were detected, as described previously.6,17 Relative changes in intracellular Ca2⫹ are shown as Ft/F0, where Ft represents the fluorescence generated by an event and F0 represents baseline. The amplitude of Ca2⫹ transients (⌬Ft/F0) was calculated by subtracting the baseline fluorescence ratio from the peak values of the events. Mitochondria, ER and nuclei. To assess the intracellular distribution of mitochondria, ER and nuclei PICs were incubated with 10 nM MitoTracker™ Red for 10 minutes, 0.1 M BODIPY® FL thapsigargin for 30 minutes and 100 nM Hoechst 33342 (Invitrogen™) for 5 minutes at room temperature, respectively. PICs were superfused with dye-free HEPES buffered PSS ([Ca2⫹] ⫽ 2.5 mM) warmed to 36C. Images of fluorescent organelles were acquired using the same epifluorescence microscope and objectives as described. MitoTracker Red was excited at 580 nm and emission was collected at 600 nm. BODIPY FL thapsigargin was excited at 490 nm and emission was detected at 520 nm. Hoechst 33342 was excited at 360 nm and emission was detected at 460 nm.
Intracellular Recording Individual stromal cells of whole tissue preparations of prostate lobes were impaled with glass capillary microelectrodes and filled with 0.5 M KCl (tip resistance 150 to 250 M⍀). Membrane potential changes were recorded as described previously.11
Solutions Bicarbonate buffered PSS was composed of 137.5 mM Na⫹, 4.7 mM K⫹, 2.5 mM Ca2⫹, 1.2 mM Mg2⫹, 15.5 mM HCO3⫺, 1.2 mM H2PO4⫺, 134 mM Cl⫺ and 15 mM glucose. The pH of PSS was 7.2 when bubbled with 95% O2 and 5% CO2. The measured pH of the recording bath was
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approximately 7.4. HEPES buffered PSS was composed of 137.5 mM Na⫹, 5.9 mM K⫹, 2.5 mM Ca2⫹, 1.2 mM Mg2⫹, 1.2 mM H2PO4⫺, 134 mM Cl⫺, 10 mM glucose and 10 mM HEPES. DS was composed of 126 mM Na⫹, 6 mM K⫹, 1 mM Mg2⫹, 134 mM Cl⫺, 10 mM glucose and 10 mM HEPES. PSS and DS pH was adjusted to 7.3 to 7.35 with NaOH. The drugs used were caffeine, CPA, nicardipine and nifedipine. CPA and nicardipine were dissolved in dimethyl sulfoxide while nifedipine was dissolved in absolute ethanol. Caffeine was directly dissolved in PSS just before use. The final concentration of these solvents in the PSS did not exceed 1:1,000.
Calculations and Statistics Measured values are shown as the mean ⫾ SD. Statistical significance was tested using the paired t test and considered significant at p ⬍0.05.
RESULTS In Situ Prostate Stromal Cell Spontaneous Ca2ⴙ Transients In whole mount prostate preparations stromal cells showed spontaneous Ca2⫹ flashes that occurred almost synchronously across all cells within the field of view (fig. 1, A and B). Spontaneous Ca2⫹ flashes occurred at a mean of 4.4 ⫾ 1.2 minute⫺1 with a mean amplitude of 1.03 ⫾ 0.35 Ft/F0 in 18 animals (18 preparations). It was clear that Ca2⫹ flashes occurred simultaneously along the full length of individual spindle-shaped smooth muscle cells (fig. 1, C and D), which also showed nonpropagating Ca2⫹ transients (fig. 1, E). Nifedipine (1 M) abolished (4 preparations) or largely suppressed (6 preparations) spontaneous Ca2⫹ flashes (fig. 1, F). In 6 preparations in which nifedipine did not abolish spontaneous Ca2⫹ flashes residual Ca2⫹ transients were still recorded synchronously across the field of view at a mean frequency of 2.1 ⫾ 0.84 minute⫺1 and mean amplitude of 0.18 ⫾ 0.08 Ft/F0 (mean 14.8% ⫾ 3.6% of control values). In some cells nonpropagating Ca2⫹ transients that had no temporal correlation with Ca2⫹ flashes were less affected by Ca2⫹ channel blockers (fig. 1, F). However, we did not further examine their properties. Intracellular Recording We made intracellular microelectrode recordings from the prostate stroma to confirm previous observations of the effects of nifedipine on slow wave discharge. In this series of experiments stromal smooth muscle cells had a mean resting membrane potential of ⫺51.7 ⫾ 4.6 mV in 8 animals (8 preparations) and generated spontaneous slow waves with superimposed action potentials (mean total amplitude 51.5 ⫾ 3.7 mV) and STDs (fig. 2, A and B). Nifedipine (1 M) abolished action potential firing and greatly suppressed slow wave amplitude (fig. 2, B and C), such that the mean
amplitude in nifedipine was 7.1 ⫾ 5.8 mV in 6 animals (6 preparations) but abolished in 2 (2 preparations). Notably suppressed slow waves were still associated with muscle contractions when viewed under microscopy, suggesting incomplete blockade of L-type Ca2⫹ channels in smooth muscle. In 6 experiments electrical couplings between cells were investigated by impaling prostate stromal preparations with 2 independent microelectrodes in 6 animals. Slow waves and superimposed action potentials recorded from the 2 electrodes occurred almost concurrently (fig. 2, D to F). However, STDs failed to propagate from 1 cell to another (fig. 2, G). Isolated PIC Morphological Characteristics Single PICs had spindle-shaped cell bodies and were distinguished from smooth muscle cells by their spiky processes (fig. 3, A and B). Circular epithelial cells with a diameter of less than 10 m were also frequently seen. PICs had a long, thin cell body with a mean length of 159.3 ⫾ 6.0 m and a maximum width at the nucleus of 11.3 ⫾ 0.3 m in 21 animals (49 preparations). In some cases the location of nuclei was confirmed by Hoechst 33342 staining (fig. 3, C and D). Upon staining with MitoTracker Red mitochondria were observed to be clustered around the nucleus while other short filamentous mitochondria were scattered in the cell periphery (fig. 3, D). ER stained with BODIPY FL thapsigargin was sparsely distributed in the cell periphery and preferentially distributed around the nucleus (fig. 3, D). PICs were also distinguished from smooth muscle cells by not contracting when [Ca2⫹]i was raised. Increasing the extracellular concentration of K ions from 6 to 30 mM increased [Ca2⫹]i in PICs and smooth muscle cells (fig. 3, E to J). In contrast to smooth muscle cells, which contracted vigorously in this high K solution, PICs did not contract (fig. 3, E to J). Spontaneous Ca2ⴙ Signaling in PICs Isolated PICs generated spontaneous Ca2⫹ signals in the form of Ca2⫹ waves, whole cell Ca2⫹ flashes or Ca2⫹ sparks. Approximately 45% of PICs generated Ca2⫹ waves that originated from any region and propagated toward the cell periphery (fig. 4, A to C). Spontaneous Ca2⫹ waves were generated at a mean of 3.3 ⫾ 1.0 minute⫺1 with a mean amplitude of 1.6 ⫾ 0.63 Ft/F0 in 17 animals (37 preparations) (fig. 4, G). Another 40% of PICs generated whole cell Ca2⫹ flashes, which occurred almost simultaneously along the cell length (fig. 4, D to F). Spontaneous Ca2⫹ flashes often fired in bursts (fig. 4, F). Whole cell Ca2⫹ flashes occurred at a mean of 6.5 ⫾ 2.1 minute⫺1 with a mean amplitude of 0.75 ⫾ 0.32 Ft/F0 in 18 animals (31 preparations), which was significantly smaller than Ca2⫹ waves (p ⬍0.05, fig. 4, G). Irrespective of the dominant pattern of spontaneous Ca2⫹ signals PICs also generated Ca2⫹
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Figure 1. Spontaneous Ca2⫹ transients in prostate stromal cells in situ. Sequential fluo-4 fluorescent images show spontaneous Ca2⫹ flash in prostate preparation with 100-millisecond frame interval (A). Ca2⫹ transients recorded from 3 regions occurred almost concurrently (B). Sequential images show spontaneous Ca2⫹ flash in another prostate preparation with 100-millisecond frame interval (C). Ca2⫹ transients recorded from 3 smooth muscle cells occurred almost simultaneously (D). s, seconds. In another preparation in which Ca2⫹ flashes and nonpropagating Ca2⫹ transients were generated (E) nifedipine (1 M) abolished Ca2⫹ flashes in 2 cells but not nonpropagating Ca2⫹ transients in another (F).
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Figure 2. Nifedipine effects on spontaneous electrical activity. In whole tissue preparation of 1 prostate lobe smooth muscle generated spontaneous slow waves with superimposed action potentials and STDs (A and B). One M nifedipine abolished action potentials and suppressed slow waves but scarcely affected STD generation (A and C). s, seconds. Synchronized slow waves and superimposed action potentials were recorded from 2 cells in prostate lobe preparation (D and E). Each pair of action potentials occurred almost simultaneously (E). In another preparation synchronized slow waves were recorded with 2 electrodes (F). In another preparation STDs were recorded with first but not second electrode (G).
sparks that occurred locally (fig. 4, B and E). The remaining 15% of PICs showed only Ca2⫹ sparks without generating Ca2⫹ waves or whole cell Ca2⫹ flashes (fig. 4, H). Properties. Spontaneous Ca2⫹ signals in PICs, including 5 Ca2⫹ wave cells in 4 animals and 6 Ca2⫹ flash cells in 4, were readily abolished by nicardipine (1 M) (fig. 5, A and B). Nominally Ca2⫹-free solu-
tion also prevented the generation of spontaneous Ca2⫹ signals and decreased baseline Ca2⫹, ie 3 Ca2⫹ wave cells and 3 Ca2⫹ flash cells in 3 animals each (fig. 5, C). These results suggest that PIC spontaneous Ca2⫹ signals fundamentally rely on Ca2⫹ influx through voltage dependent L-type Ca2⫹ channels. Spontaneous Ca2⫹ signals were abolished by CPA (10 M) without changes in baseline Ca2⫹, ie 7 Ca2⫹
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Figure 3. Isolated PIC morphological characteristics. PICs had spindle-shaped cell bodies and were distinguished from smooth muscle cells by spiky processes (A and B). Small round epithelial cells were also seen. Another PIC had spindle-shaped cell body and several processes (C). Overlay shows intracellular distribution of nucleus (blue areas), mitochondria (red areas) and ER (green areas) in same PIC (D). Scale bar (A) represents 30 m (A to D). Bright field (E) and fluo-4 fluorescence (F) images show PIC. Upon [Ca2⫹]i increase in high K solution PIC did not contract (G). Fluorescence images show smooth muscle cell in normal (H) and high K (I and J) solution. Note increased [Ca2⫹]i and vigorous smooth muscle cell contraction.
wave cells in 5 animals and 5 Ca2⫹ flash cells in 4 (fig. 5, D). Also, caffeine (10 mM) abolished the generation of spontaneous Ca2⫹ signals in PICs, ie 5 Ca2⫹ wave cells in 4 animals and 5 Ca2⫹ flash cells in 3 (fig. 5, E), suggesting that the generation of spontaneous Ca2⫹ signals of PICs crucially depends on Ca2⫹ release from ER stores.
DISCUSSION In intact preparations of prostate spontaneous Ca2⫹ flashes generated almost synchronously in all stromal cells in our field of view, and slow waves and their superimposed action potentials were recorded concurrently using 2 independent electrodes. Nifedipine (1 M) abolished action potentials and greatly suppressed the spontaneous firing of Ca2⫹ flashes and slow waves in situ. Thus, it is reasonable to suggest that these Ca2⫹ flashes in individual smooth muscle cells reflect an increase in [Ca2⫹]i during the firing of bursts of action potentials. Previous studies using intracellular recording microelectrodes consistently revealed that nifedipine (1 M) abolished action potential generation but had variable effects on slow wave amplitudes, presumably depending on the distance between the site of pacemaker potential generation and the site of microelectrode penentration.13–15 Small residual Ca2⫹ flashes and suppressed slow waves were detected in the presence of nifedipine (1 M) but these spontaneous signals were associated
with small muscle contractions. Thus, we believe that these residual Ca2⫹ flashes were Ca2⫹ signals recorded from smooth muscle cells, reflecting incomplete blockade of L-type Ca2⫹ channels. Precedence for this incomplete block occurs in the bladder detrusor, in which individual action potentials are readily abolished by nifedipine (1 M) while bursting action potentials require higher nifedipine concentrations (10 M).18 In the mouse renal pelvis nifedipine concentrations greater than 3 M are sometimes required to completely block action potential and Ca2⫹ transient discharge in the typical smooth muscle cells of the muscle wall.12 By analogy with Ca2⫹ signaling in ICC-like cells in the rabbit urethra,6 guinea pig bladder11 and ICC networks in several gut preparations in situ19 one might have expected some form of nifedipine resistant pacemaker Ca2⫹ signal that correlated in its timing and kinetics with the pacemaker potentials or prostate slow waves recorded in the presence of nifedipine. Nonpropagating Ca2⫹ transients in individual cells were occasionally seen in the intact preparation in the absence of nifedipine. However, they showed no temporal correlation with Ca2⫹ flashes in control solutions, indicating that these waves were unlikely to act as genuine pacemaker signals. To our knowledge this report represents the first time that PICs were enzymatically isolated from guinea pig prostate for experimentation. Although we did not examine their Kit immunoreactivity, iso-
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Figure 4. PIC spontaneous Ca2⫹ transients. Sequential fluo-4 fluorescent images show spontaneous Ca2⫹ wave in isolated PIC with 100-millisecond frame interval (A). Spontaneous Ca2⫹ waves and Ca2⫹ sparks were generated (B). When shown on expanded time scale, delays between Ca2⫹ waves were obvious in 3 intracellular regions (C). Sequential images show whole cell Ca2⫹ flash in another isolated PIC with 100-millisecond frame interval (D). Spontaneous Ca2⫹ flashes and Ca2⫹ sparks were generated (E). When shown on expanded time scale, multiple Ca2⫹ flashes in 3 intracellular regions occurred almost simultaneously (F). s, seconds. Note Ca2⫹ wave and Ca2⫹ flash frequency and amplitude (G). Another PIC generated only Ca2⫹ sparks (H). X and Y axes (H) also refer to B and E.
SPONTANEOUS CA2⫹ SIGNALING OF INTERSTITIAL CELLS IN PROSTATE
Figure 5. PIC spontaneous Ca2⫹ transient properties. Nicardipine abolished spontaneous Ca2⫹ signals in PICs generating Ca2⫹ waves (A) and flashes (B). Switching from normal PSS to nominally Ca2⫹-free solution prevented spontaneous Ca2⫹ wave generation in PIC associated with decreased baseline [Ca2⫹]i (C). Upon re-addition of 2.5 mM Ca2⫹ spontaneous Ca2⫹ waves were restored. In another PIC generating spontaneous Ca2⫹ waves 10 M CPA abolished Ca2⫹ waves with only small increase in baseline [Ca2⫹]i (D). In another PIC 10 mM caffeine abolished Ca2⫹ wave spontaneous generation (E).
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Previous intracellular microelectrode studies demonstrated that prostate slow waves intimately depend on Ca2⫹ release from ER Ca2⫹ stores as well as on the uptake and release of Ca2⫹ from mitochondria.20 This intimate relationship is perhaps indicated by the preferential clustering of ER and mitochondria around the nucleus when visualized using specific fluorescent dyes (fig. 3). These results also confirm similar clustering of ER and mitochondria in the perinuclear region of single ICC-like cells isolated from the rabbit urethra,17 which also develop spontaneous activity depending on the Ca2⫹ uptake and release of each organelle.21,22 However, in contrast with Ca2⫹ signaling in rabbit urethral ICC-like cells, Ca2⫹ release in the prostate is driven by 1,4,5-triphosphate dependent release mechanisms and not by the release of stored Ca2⫹ via ryanodine receptors.20 In the current experiments CPA and caffeine abolished spontaneous Ca2⫹ signals in isolated PICs, consistent with this dependence on the release of stored Ca2⫹. Again unlike urethral ICC-like cells, neither CPA nor caffeine evoked large increases in baseline Ca2⫹ in single PICs before blocking their spontaneous Ca2⫹ signals. This suggests that the capacity of intracellular Ca2⫹ stores of PICs may be smaller than that of urethral ICC-like cells. Alternatively PICs may have stronger Ca2⫹ extrusion systems than urethral ICC-like cells. Smaller Ca2⫹ stores in PICs or their relative lack of ryanodine receptor release channels may well explain their greater dependence on Ca2⫹ influx through L-type Ca2⫹ channels to generate their spontaneous Ca2⫹ signals.
CONCLUSIONS lated PICs were readily distinguished from neighboring single smooth muscle cells or epithelial cells by their spiky processes and lack of contraction upon raising [Ca2⫹]i with high [K⫹]o PSS (fig. 3). ICC-like cells dispersed from the rabbit urethra or the guinea pig bladder show spontaneous Ca2⫹ waves and sparks as well as Ca2⫹ activated chloride currents that do not rely on Ca2⫹ influx through L-type Ca2⫹ channels.4,5,10 Freshly isolated PICs also generated whole cell Ca2⫹ flashes, intracellular Ca2⫹ waves and high frequency localized Ca2⫹ sparks. Pacemaker potentials can be recorded in prostate preparations in situ in the presence of nifedipine. Thus, isolated PICs, if they are indeed responsible for pacemaker generation, should be able to develop some form of substantial Ca2⫹ signals that do not fundamentally rely on L-type Ca2⫹ channels. Therefore, it was surprising that almost all spontaneous Ca2⫹ signals recorded in isolated PICs were abolished by nicardipine.
We suggest that in whole mount preparations of prostate in situ the spontaneous Ca2⫹ flashes that occur at frequencies similar to the firing of slow waves reflect Ca2⫹ influx during the synchronous discharge of action potentials in individual smooth muscle cells. Isolated PICs developed spontaneous Ca2⫹ signals that depended on ER Ca2⫹ release. The signals were readily abolished by nicardipine, indicating that Ca2⫹ influx through L-type Ca2⫹ channels has a fundamental role in generating PIC Ca2⫹ signals. However, this dependence of Ca2⫹ signaling on Ca2⫹ influx does not support previous intracellular recording studies in which pacemaker potentials in situ were recorded in the presence of nifedipine. Also, mechanisms underlying the spontaneous activity of single PICs differ significantly from those of other interstitial cells in the urinary tract. Each phenomenon merits further investigation. Thus, we cannot currently conclude that PICs act as pacemaker cells that directly drive spontaneous activity in prostate stromal smooth muscle.
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REFERENCES 1. Sanders KM, Koh SD and Ward SM: Interstitial cells of Cajal as pacemakers in the gastrointestinal tract. Annu Rev Physiol 2006; 68: 307.
9. Karkanis T, DeYoung L, Brock GB et al: Ca2⫹activated Cl⫺ channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection. J Appl Physiol 2003; 94: 301.
2. Brading AF and McCloskey KD: Mechanisms of disease: specialized interstitial cells of the urinary tract—an assessment of current knowledge. Nat Clin Pract Urol 2005; 2: 546.
10. Wu C, Sui GP and Fry CH: Purinergic regulation of guinea pig suburothelial myofibroblasts. J Physiol 2004; 559: 231.
3. Hashitani H, Van Helden DF and Suzuki H: Properties of spontaneous depolarisations in circular smooth muscle cells of rabbit urethra. Br J Pharmacol 1996; 118: 1627.
11. Hashitani H, Yanai Y and Suzuki H: Role of interstitial cells and gap junctions in the transmission of spontaneous Ca2⫹ signals in detrusor smooth muscles of the guinea-pig urinary bladder. J Physiol 2004; 559: 567.
4. Sergeant GP, Hollywood MA, McCloskey KD et al: Specialised pacemaking cells in the rabbit urethra. J Physiol 2000; 526: 359. 5. Johnston L, Sergeant GP, Hollywood MA et al: Calcium oscillations in interstitial cells of the rabbit urethra. J Physiol 2005; 565: 449. 6. Hashitani H and Suzuki H: Properties of spontaneous Ca2⫹ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ. J Physiol 2007; 583: 505. 7. Montgomery BS and Fry CH: The action potential and net membrane currents in isolated human detrusor smooth muscle cells. J Urol 1992; 147: 176. 8. Sui G, Fry CH, Malone-Lee J et al: Aberrant Ca2⫹ oscillations in smooth muscle cells from overactive human bladders. Cell Calcium 2009; 45: 456.
12. Lang RJ, Hashitani H, Tonta MA et al: Spontaneous electrical and Ca2⫹ signals in typical and atypical smooth muscle cells and interstitial cell of Cajal-like cells of mouse renal pelvis. J Physiol 2007; 583: 1049. 13. Exintaris B, Klemm MF and Lang RJ: Spontaneous slow wave and contractile activity of the guinea pig prostate. J Urol 2002; 168: 315. 14. Lang RJ, Nguyen DT, Matsuyama H et al: Characterization of spontaneous depolarizations in smooth muscle cells of the Guinea pig prostate. J Urol 2006; 175: 370. 15. Nguyen DT, Lang RJ and Exintaris B: ␣1-adrenoceptor modulation of spontaneous electrical waveforms in the guinea-pig prostate. Eur J Pharmacol 2009; 608: 62. 16. Lang RJ, Mulholland E and Exintaris B: Characterization of the ion channel currents in single
myocytes of the guinea pig prostate. J Urol 2004; 172: 1179. 17. Hashitani H, Lang RJ and Suzuki H: Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2⫹ waves in interstitial cells of Cajal-like cells of the rabbit urethra. Br J Pharmacol 2010; 161: 680. 18. Hashitani H, Brading AF and Suzuki H: Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea-pig bladder. Br J Pharmacol 2004; 141: 183. 19. Hennig GW, Hirst GD, Park KJ et al: Propagation of pacemaker activity in the guinea-pig antrum. J Physiol 2004; 556: 585. 20. Exintaris B, Nguyen DT, Lam M et al: Inositol trisphosphate-dependent Ca stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland. Br J Pharmacol 2009; 156: 1098. 21. Sergeant GP, Bradley E, Thornbury KD et al: Role of mitochondria in modulation of spontaneous Ca2⫹ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra. J Physiol 2008; 586: 4631. 22. Sergeant GP, Hollywood MA, McCloskey KD et al: Role of IP3 in modulation of spontaneous activity in pacemaker cells of rabbit urethra. Am J Physiol Cell Physiol 2001; 280: C1349.