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Journal of Physiology (2001), 534.2, pp. 313-326
© Copyright 2001 The Physiological Society
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ABSTRACT |
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-subunit (BK) revealed a spot-like pattern on the plasmalemma, in contrast to the uniform staining of voltage-dependent Ca2+ channel 1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BK and RyRs revealed spot-like co-localization on/beneath the plasmalemma.
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INTRODUCTION |
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The pivotal roles in cellular functions of local Ca2+ release and the subsequent changes in Ca2+ distribution with time have been revealed in a variety of cells based on subcellular Ca2+ imaging on a faster time scale and at higher resolution (Berridge, 1997; Karaki et al. 1997). Ca2+ sparks are local and transient Ca2+ release events from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) (Cheng et al. 1993; Lopez-Lopez et al. 1994). In cardiac myocytes, Ca2+ sparks are recognized as elementary units of Ca2+-induced Ca2+ release (CICR) by the opening of RyRs in junctional SR, which is triggered by Ca2+-influx through L-type Ca2+ channels into the narrow space between the transverse tubule and junctional SR (Cannel et al. 1994; Shacklock et al. 1995). Ca2+ sparks have, therefore, been described as evidence for the existence of micro-architecture for the local control of excitation- contraction coupling (Cannel et al. 1995; Cheng et al. 1996b).
In contrast, Ca2+ sparks in smooth muscle have been reported mainly in relation to large conductance Ca2+-dependent K+ (BK) channel activation and muscle relaxation (Nelson et al. 1995; Imaizumi et al. 1999; Jaggar et al. 2000). A spontaneous Ca2+ spark in the superficial area activates 10-100 BK channels nearby and induces membrane hyperpolarization (Pérez et al. 1999; ZhuGe et al. 1999; Fürstenau et al. 2000), which reduces Ca2+ channel activity. This K+ current is termed the spontaneous transient outward current (STOC) (Benham & Bolton, 1986; Bolton & Imaizumi, 1996) and is considered to be a factor relating to vasodilatation (Nelson et al. 1995). Indeed, in pressurized cerebral arteries, inhibition of Ca2+ sparks gives rise to depolarization and induces vasoconstriction (Knot et al. 1998), and increasing the frequency of Ca2+ sparks induces vasodilatation (Porter et al. 1998). Although the functional coupling between BK channels on the cell membrane and RyRs in subplasmalemmal SR has been suggested (Pérez et al. 1999; Jaggar et al. 2000), the spatial distribution of BK channels and that of RyRs have not been exactly compared with each other.
In single smooth muscle cells of guinea-pig vas deferens and urinary bladder, Ca2+ entry through voltage-dependent Ca2+ channels in the early stages of an action potential may evoke CICR from discrete subplasmalemmal Ca2+ storage sites and generate local Ca2+ transients that spread over the cell to initiate a contraction (Imaizumi et al. 1998; Bolton et al. 1999; Collier et al. 2000). In addition, the subplasmalemmal Ca2+ transients activate BK channels nearby, which results in the activation of Ca2+-dependent K+ current (IK,Ca), a major outward current responsible for action potential repolarization and afterhyperpolarization (Imaizumi et al. 1996, 1998; Heppner et al. 1997). The two local Ca2+ release events, Ca2+ sparks at rest and Ca2+ transients upon depolarization, share physiological roles to activate BK channels and induce membrane hyperpolarization.
The present study was undertaken to examine whether Ca2+ storage sites related to Ca2+ sparks at rest are the origin of Ca2+ transients during depolarization in smooth muscle cells of guinea-pig vas deferens and urinary bladder, as in cardiac muscle (Cheng et al. 1996a). In addition, the distribution of BK channels and that of RyRs in myocytes were compared by the use of immunocytochemical methods and confocal imaging. The results suggest the punctate co-localization of BK channels and RyRs in discrete junctional areas of the plasmalemma and peripheral SR fragments. BK channel clustering was also detected in inside-out patches where BK channel activation was elicited by Ca2+ release from the SR fragment.
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METHODS |
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Cell isolation
Smooth muscle cells were isolated from vas deferens and urinary bladder using a previously described method with slight modification (Imaizumi et al. 1989). All experiments were carried out in accordance with the guiding principles for the care and use of laboratory animals (the Science and International Affairs Bureau of the Japanese Ministry of Education, Science, Sports and Culture) and also with the approval of the ethics committee at Nagoya City University. In brief, guinea-pigs were anaesthetised with ether and killed by decaptitation. Vas deferens and urinary bladder were dissected out from male guinea-pigs and freed from other tissues in Ca2+-free Krebs solution. The tissue was immersed in Ca2+-free Krebs solution for 10-60 min in a test tube at 37 °C. Subsequently, the solution was replaced with Ca2+-free solution containing 0.2 or 0.3 % collagenase, 0.3 % albumin and 0.3 % trypsin inhibitor. After 20-60 min treatment with these agents, the solution was replaced with Ca2+- and collagenase-free Krebs solution. The tissue was gently triturated using a glass pipette to isolate cells. At the start of each experiment, a few drops of the cell suspension were placed in a recording chamber (0.5 ml) mounted on the stage of a phase-contrast microscope equipped for confocal microscopy (Nikon RCM8000). Cells were continuously perfused with Hepes-buffered solution at a constant flow of 5 ml min-1.
Solutions
Standard Krebs solution contained (mM): NaCl, 112; KCl, 4.7; CaCl2, 2.2; MgCl2, 1.2; NaHCO3, 25; KH2PO4, 1.2; and glucose, 14. Ca2+-free Krebs solution was prepared by omitting Ca2+ from standard Krebs solution. Standard and modified Krebs solutions were gassed with a mixture of 95 % O2 and 5 % CO2. For electrical recordings, Hepes-buffered solution having the following composition was used as the external solution (mM): NaCl, 137; KCl, 5.9; CaCl2, 2.2; MgCl2, 1.2; glucose 14; and Hepes, 10. The pH was adjusted to 7.4 with NaOH. The pipette solution contained (mM): KCl, 140; MgCl2, 1; Na2ATP, 2; Hepes, 10; and fluo-3, 0.1. The pH was adjusted to 7.2 with KOH.
For the recording of single BK channel current in inside-out patch clamp mode, the pipette solution contained standard Hepes-buffered solution and the bathing solution contained (mM): KCl, 145; MgCl2, 1.2; Na2ATP, 2; glucose, 14; Hepes, 10; and EGTA, 5. Each pCa of the bathing solution was obtained by adding CaCl2 and pH was adjusted to 7.2 with KOH (Imaizumi et al. 1996). In some experiments, the bathing solution contained 50 or 100 µM EGTA and no additional CaCl2 except contamination of < 50 µM.
Electrical recording and data analysis
Whole-cell voltage clamp was applied to single cells with patch pipettes (Hamill et al. 1981) using a CEZ-2400 (Nihon Kohden, Japan) amplifier. The pipette resistance ranged from 3 to 5 M
when filled with the pipette solution. The seal resistance was approximately 30 G. Series resistance was between 5 and 8 M and was partly compensated. Single channel recording of BK channels was performed mainly in the inside-out configuration. The patch pipette resistance ranged between 7 and 15 M for regular recordings. Occasionally, single channel currents were recorded from large excised patches using pipettes with a resistance of 3-5 M. Data were stored and analysed using menu-drive software as previously reported (Imaizumi et al. 1996). Membrane currents were digitized using a pulse code modulator (PCM) recording system (PCM-501ES; Sony, Tokyo, Japan), which was modified to give a frequency response from DC to 20 kHz, and stored on videotape. Data on tape were replayed onto a personal computer using data acquisition software. Data analysis was done on a computer using software (Cell-Soft) developed at the University of Calgary, Canada. Leakage currents at potentials positive to -60 mV were subtracted on the computer, assuming a linear relationship between current and voltage in the range of -90 to -60 mV. All experiments were done at room temperature (23 ± 1 °C).
Measurement of fluo-3 signal from single cells
Ca2+ images were obtained using a fast laser-scanning confocal microscope (RCM 8000; Nikon, Japan) and ratio3 software (Nikon) in the same manner as reported previously (Imaizumi et al. 1998). A myocyte was loaded with 100 µM fluo-3 by diffusion from the recording pipette. Excitation light of 488 nm from an argon ion laser was delivered through a water-immersion objective (Nikon Fluo 
40, 1.15 NA). Emission light of > 515 nm was detected by a photomultiplier. Fluorescence intensity (F) in a selected area was measured as an average from pixels included in the area. The data are shown as F/F0, where F0 is the basal F value obtained as the average from five images of the whole cell area, in which particular Ca2+ events were not observed. It took 33 ms to scan one full frame (512 pixels 512 pixels). Using 1/2, 1/4 and 1/8 band scan modes, frames that correspond to areas of 170 µm 55, 27.5 or 13.8 µm were obtained every 16.5, 8.25 and 4.13 ms, respectively. The resolution of the microscope was approximately 0.4 µm 0.3 µm 1.2 µm (x, y and z). The confocal plane through the cell was usually set where the width of the cell was largest, 2-3 µm from its lowest point. In some experiments, images were obtained by 1/2 band scan mode with a larger confocal aperture to obtain Ca2+ images of the whole cell with a larger measurement depth of about 5 µm, which included the majority of the cell in the z-axis. Recordings were started at least 3 min after rupturing the patch membrane to make fluo-3 diffuse into the cell.
Ca2+ images were stored on optical disk cartridge (LM-A410, Panasonic, Japan) by a rewritable optical disk recorder (LQ-4100A, Panasonic). The images on the optical disks were replayed later and analysed using ratio3 software. Some analyses were performed using GLOBAL LAB image (Data Translation, Marlboro, MA, USA) on an IBM-AT compatible computer. Selected records were printed out by inkjet colour printer (Hewlett Packard Deskjet 720C). During the recording of fluorescence images, the cell shape was also monitored using red/infrared light, having a wavelength range of > 600 nm, and an IR-CCD video camera module (Sony, XC-77BB), which was attached to the microscope, and recorded on a videotape. Video capturing and analysis of cell shape changes were performed later on an IBM-AT compatible computer using an AD translation board (Data Translation, DT-55) and GLOBAL LAB imaging software.
Immunocytochemistry
Isolated smooth muscle cells were fixed in 4 % paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature. After washing, cells were incubated with 0.2 % Triton X-100 in PBS for 15 min, followed by incubation with 0.1 mM lysine in PBS for 15 min. Then, the cells were washed and blocked for 1 h with PBS containing 5 % bovine serum albumin (BSA) and 3 % normal goat serum (NGS). After washing, the cells were incubated with primary antibody (1:200 dilution in PBS containing 1 % NGS for antibodies to the BK channel subunit, RyR or voltage-dependent Ca2+ channel 1C subunit) at 4 °C overnight, followed by further washing. For detection of BK and Ca2+ channels, cells were incubated with biotin-conjugated goat anti-rabbit secondary antibody (1:200 dilution in PBS containing 1 % NGS) for 1h. The cells were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated streptavidin (1:50 dilution in PBS containing 1 % NGS) for 2 h. For double staining of BK channels and RyRs, another incubation with Alexa Fluor 568-conjugated goat anti-mouse IgG (1:200 dilution in PBS containing 1 % NGS) was performed for 2 h. To confirm the specific binding of anti-BK antibody, the binding was blocked by addition of corresponding antigenic peptide (1:40 dilution). Immunofluorescence staining was examined using a confocal fluorescence microscope (Zeiss LSM510) equipped with an oil-immersion objective (Nikon Plan apochromat 63, NA 1.4) and optical sectioning was done every 0.4 µm. The resolution was 0.3 µm 0.3 µm 0.8 µm (x, y and z) and one frame contained 512 pixels 512 pixels corresponding to approximately 150 µm 150 µm. Excitation light wavelengths were 488 and 543 nm from an argon ion laser and a helium neon laser, respectively, and emission light wavelengths of 503-530 and > 560 nm were detected and images were stored on magnetic optical disks. In some experiments, images were obtained with a larger confocal aperture, resulting in a larger measurement depth of ~2 µm.
Materials
The sources of pharmacological agents and immunostaining materials were as follows: collagenase (500 U mg-1; Yakult, Tokyo): trypsin inhibitor (Sigma); albumin (Seikagaku Corporation, Tokyo); CdCl2, caffeine, Triton X-100 (Wako Pure Chemical Industries, Osaka); tetraethylammonium chloride (TEA; Tokyokasei, Tokyo); fluo-3, Hepes (Dojin, Kumamoto); iberiotoxin (Peptide Institute, Osaka); polyclonal anti-BK channel -subunit antibody (rabbit IgG) and monoclonal anti-RyR antibody (clone 34-C, mouse IgG), polyclonal anti-voltage-dependent Ca2+ channel 1C subunit (rabbit IgG) (Alomone lab, Israel); biotin-conjugated goat anti-rabbit IgG, FITC-conjugated streptavidin (Chemicon International, CA, USA); bovine serum albumin; normal goat serum (Sigma); and Alexa Fluor 568-conjugated anti-mouse IgG (Molecular Probes, OR, USA).
Statistics
Pooled data are shown as means ± S.D. in the text. Statistical significance was evaluated using Student's t test or 
2 test (P < 0.05 accepted as significant).
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RESULTS |
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Association and dissociation of Ca2+ sparks with STOCs
Two-dimensional confocal images of Ca2+ sparks were obtained together with measurements of STOCs at a holding potential of -40 mV in single myocytes of urinary bladder or vas deferens of the guinea-pig. Since continuous exposure to laser light for over 3 s resulted in profound photobleaching of the fluorescence dye, measurements for 3 s were repeated 3 times at an interval of 3 min. Ca2+ images were obtained every 8 ms during each measurement for 3 s. Figure 1A shows eight sequentially recorded images (over the time scale of 219 to 276 ms shown in Fig. 1B and C) of Ca2+ sparks occurring separately at two sites ( and 
as indicated by arrows) in a urinary bladder myocyte.
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Figure 1. Simultaneous measurement of confocal Ca2+ images and membrane currents in a urinary bladder myocyte A, Ca2+ images were obtained using confocal microscopy and fluo-3 from a urinary bladder myocyte that was voltage clamped at -40 mV. Images were obtained every 8.25 ms during the continuous recording for 3 s and are shown sequentially. The asterisk indicates the point where the recording pipette was attached. Ca2+ sparks were observed at two distinct sites, | ||
Figure 1B shows the time course of the normalized fluorescence intensity (F/F0) at spark sites (red line) and (blue line), and a STOC measured simultaneously (black line). The fluorescence intensity of a spark was measured as the average intensity at pixels in a circular spot of 1.3 µm in diameter, which was located in the centre of the spark site. It was found that the rising phase of the STOC was almost completely overlapped by the increase in F/F0 at spark site , whereas the F/F0 change at spark site appears to be dissociated from the STOC by about 50 ms (Fig. 1B). The time course of changes in membrane current and F/F0 at the spark sites during the first measurement for 3 s is shown in Fig. 1C. The basal F/F0 at site was higher than that at site and other areas of the cell. The number of STOCs that had a peak amplitude over 50 pA was 15 during the total 9 s recording and, interestingly, 12 out of 15 occurred in synchrony with Ca2+ sparks at site . In turn, the number of Ca2+ sparks occurring at site was 12 and all of them were synchronous with STOCs in a one to one manner. These results indicate that about 80 % of STOCs recorded during this period were synchronized with Ca2+ sparks at site . There was no incidence of the co-occurrence of Ca2+ sparks at site and STOCs with a latency of less than 20 ms.
STOCs over 25 pA were observed at a holding potential of -40 mV in 50-60 % of the cells, and Ca2+ sparks were detected in 41 out of 112 cells (31 out of 87 vas deferens myocytes and 10 out of 25 urinary bladder myocytes; 36 and 40 %, respectively) under the same conditions. There was significant coincidence between Ca2+ sparks and STOCs in 95.5 % of the cells where simultaneous measurements of Ca2+ sparks and STOCs succeeded (21 out of 22 cells; P < 0.001 by 2 test; 16 out of 16 vas deferens myocytes and 5 out of 6 urinary bladder myocytes). Coincidence was determined by the occurrence of more than two Ca2+ sparks at the same site, followed by STOCs in a one to one manner without a time lag longer than 4-8 ms during the continuous recording for 3 s. The averaged peak F/F0 in sparks presumably was 2.25 ± 0.74 (n = 20) and 2.36 ± 0.15 (n = 8, not significant) in myocytes from vas deferens and urinary bladder, respectively. The averaged diameter of Ca2+ sparks at the peak F/F0 was 2.5 ± 0.7 µm (n = 16) and 2.3 ± 0.4 µm (n = 5, not significant), respectively. The peak amplitude of corresponding STOCs was 87 ± 7 pA (n = 16) and 105 ± 30 pA (n = 5, not significant), respectively. STOC amplitude was reduced to approximately 5 % of the control by application of 3 mM TEA or 100 nM iberiotoxin, while Ca2+ sparks were not affected (n = 3 for each; data not shown). Neither Ca2+ sparks nor STOCs were affected by application of 100 µM Cd2+ for at least 3 min (n = 3; not shown).
To determine the number of Ca2+ spark sites in a cell more exactly, 1/2 band scan mode, which covered an area of 170 µm 55 µm every 16.5 ms, was occasionally used with a wider confocal aperture (approximately 5 µm in the z-axis resolution). Figure 2 shows two Ca2+ sparks in the whole cell area, which were both responsible for the activation of STOCs in a vas deferens myocyte. F/F0 in sites and was measured in the same manner as in Fig. 1. Among 13 STOCs having a peak amplitude of over 10 pA during the 3 s recording shown in Fig. 2Bb, four STOCs were attributable to sparks at sites and , as indicated by asterisks, in a one to one manner. Based on the results from six vas deferens myocytes, the approximate number of frequently discharging Ca2+ spark sites was calculated to be five per cell and over 88 % of STOCs with amplitudes over ~25 pA were elicited by Ca2+ sparks at frequently discharging sites at -40 mV during the total recording period of 9 s. Similar results were obtained from five urinary bladder myocytes.
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Figure 2. STOCs were elicited by Ca2+ sparks in small number of discrete sites in the subplasmalemmal area A, a schematic cell image (left) and two confocal Ca2+ images (middle and right) from a vas deferens myocyte in which two Ca2+ spark sites are indicated as | ||
Localization and kinetics of Ca2+ sparks
Figure 3A shows a Ca2+ spark occurring synchronously with a STOC in a vas deferens mycyte (a), the two-dimensional image of the spark (b) and its surface plot (c). The Ca2+ spark occurred at the edge of the cell. Figure 3B illustrates the distribution histogram of the location of Ca2+ sparks (a, vas deferens; b, urinary bladder). The number of spark sites was plotted against the distance from the plasmalemma. At each Ca2+ spark site, events occurred 1-6 times during 3 s. Over 80 % of spark sites were frequently discharging sites (0.3-2 Hz) during the total recording period (9 s). The averaged number of Ca2+ spark sites in one image (170 µm 27.5 µm) was 2.1. More than 70 % of the Ca2+ sparks occurred in the superficial area within 1.0 µm from the plasmalemma. It is notable that all of the superficial sparks were synchronized with STOCs. On the other hand, Ca2+ sparks occurring further away from the plasmalemma (> 1.0 µm) were rather rare and often not associated with STOCs.
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Figure 3. Location of Ca2+ sparks in myocytes A, a Ca2+ spark and concomitant STOC were recorded in a vas deferens myocyte at a holding potential of -40 mV. The time course of F/F0 (red line) at the Ca2+ spark site ( | ||
Figure 4A shows the relationship between F/F0 of Ca2+ sparks and the amplitude of STOCs, which occurred synchronously in 14 pairs of recordings from six separate cells of vas deferens. The largest F/F0 value was taken as 1.0 in each cell. The relative amplitude of the STOCs was also determined by taking the largest as 1.0 in each cell. There was a significant correlation between these two parameters (r = 0.84, P < 0.05 by 2 test). Figure 4B shows the half-rise and half-decay times of changes in fluorescence intensity at the Ca2+ spark site and those of synchronous STOCs at a holding potential of -40 mV. In each myocyte, two or three pairs of Ca2+ sparks and STOCs were selected and the averaged values of the half-rise and -decay times were obtained. The results indicate that the Ca2+ spark and corresponding STOC have similar kinetic properties in these two types of smooth muscle cell.
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Figure 4. Kinetic parameters of Ca2+ sparks and corresponding STOCs A, the relationship between the relative fluorescence intensity (F/F0) of Ca2+ sparks and the relative amplitude of STOCs occurring synchronously as 14 pairs in six separate myocytes of vas deferens. Different symbols indicate the data obtained from six different cells. F/F0 was measured and the largest F/F0 value was taken as 1.0 in each myocyte. The relative amplitude of STOCs was obtained by taking the largest as 1.0 in each cell. There was a significant correlation between these two parameters (r = 0.84, P < 0.05 by | ||
Relationship between Ca2+ sparks at rest and Ca2+ transients during depolarization
The location of Ca2+ spark sites at -40 mV in a cell was compared with that of early Ca2+ transients during depolarization in the same cell. Figure 5A shows the time course of STOCs and F/F0 at a Ca2+ spark site at a holding potential of -40 mV in a vas deferens myocyte. The fluorescence intensity of the spark was measured as the average intensity at pixels in a circular spot of 1.3 µm in diameter. Figure 5Ab shows a Ca2+ spark image (arrow) obtained at the corresponding time shown in Fig. 5Aa. The large STOC indicated by an asterisk in Fig. 5Aa had a peak amplitude of approximately 150 pA and is attributable to the Ca2+ spark shown in Fig. 5Ab, since three sequential Ca2+ sparks in this site were followed by STOCs in a one to one manner without a delay of more than 8 ms.
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Figure 5. The spatial relationship between Ca2+ sparks at rest and early Ca2+ transients upon depolarization in a vas deferens myocyte (I) A, the time course of membrane current (black line) and that of F/F0 (red line) at a Ca2+ spark site, at a holding potential (Vh) of -40 mV, are shown in a. F/F0 was measured at the Ca2+ spark site indicated by an arrow in b. Ca2+ images, which were obtained at rest (1.28 s) and at the peak of the STOC (1.30 s) in the time course in a are shown in b. The images were adjusted by subtraction of the averaged basal image. B, the membrane current (IK,Ca, black dots), F/F0 at the same site as the Ca2+ spark in A (red circles) and F/F0 in the whole cell area (blue circles) are plotted against time in a. The same myocyte as shown in A was depolarized from -60 to 0 mV for 50 ms. The Ca2+ images shown in b were obtained at the corresponding time in a and adjusted by subtracting the averaged basal image. The arrow in b indicates the spark site in A. | ||
Figure 5B shows images of Ca2+ transients during depolarization and membrane currents in the same myocyte. The depolarization from -60 to 0 mV for 50 ms elicited an initial Ca2+ inward current and a subsequent large outward current (black dots in Fig. 5Ba). Approximately 80 % of the outward current was due to the activation of BK channels, which were blocked by addition of 2 mM TEA or 100 nM iberiotoxin (Imaizumi et al. 1998). Four or more spot-like Ca2+ transients appeared in the peripheral areas within 20 ms from the start of depolarization. The arrow in the image of Fig. 5Bb at 16.1 ms indicates the Ca2+ transient occurring in the same area as that of the Ca2+ spark at rest. F/F0 in this spot was measured in exactly the same place as the spark site and is plotted in Fig. 5Ba (red circles). It is clear that F/F0 in this transient increased with the activation of IK,Ca (black dots), while the increase in F/F0 over the whole cell area (blue circles) occurred more slowly. The longitudinal shortening of the myocyte started approximately 0.5 s after the depolarization and slowly reached the maximum shortening by about 5 % of the cell length within 2 s. Cell relaxation was detected after 15 s. It is noteworthy that spot-like Ca2+ transients appeared in the same areas when myocytes were repetitively depolarized once every 30 s (Imaizumi et al. 1998). The averaged peak F/F0 in the early Ca2+ transients was 3.59 ± 0.58 (n = 10) in vas deferens myocytes. Not only Ca2+ current but also Ca2+ transients and IK,Ca were blocked by the addition of 50 µM Cd2+ to the bathing solution. Pre-treatment with 5 mM caffeine transiently increased F/F0 over the whole cell area and the subsequent depolarization did not elicit a Ca2+ transient and induced only a small IK,Ca (Imaizumi et al. 1998).
Figure 6A and B shows the same images as in Fig. 5A and B, respectively, at a greater magnification. It is clear that the Ca2+ spark site observed at a holding potential of -40 mV was located within the area of the spot-like Ca2+ transient during depolarization to 0 mV. The contour map of 
F/F0 in Fig. 6C clearly shows that the Ca2+ spark site, which is outlined in red (F/F0 > 0.8 at -40 mV), was completely included in the area of the early Ca2+ transient (blue and green lines: F/F0 > 1.5 and 2.5, respectively, at 0 mV). In all vas deferens myocytes examined (n = 10), the sites of Ca2+ sparks occurring in synchrony with STOCs were completely included in the areas of early Ca2+ transients elicited by depolarization to 0 mV. Essentially identical observations were also obtained in urinary bladder myocytes. Figure 7 shows that the site of the Ca2+ spark (A), which was synchronized with a STOC at -40 mV, was exactly included in the area of the early Ca2+ transients during depolarization to 0 mV (B), as also demonstrated in the contour map of F/F0 (C). F/F0 in the early Ca2+ transients during depolarization to 0 mV was 3.33 ± 0.64 in urinary bladder myocytes (n = 6, not significant vs. vas deferens). The overlapping of Ca2+ spark sites with the areas of the early Ca2+ transients was observed in all urinary bladder myocytes examined (n = 6).
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Figure 6. The spatial relationship between Ca2+ sparks at rest and early Ca2+ transients upon depolarization in a vas deferens myocyte (II) The location of early Ca2+ transients upon depolarization was exactly compared with that of the Ca2+ spark site. The Ca2+ spark image in Aa and the Ca2+ transient image in Ba are the same as those shown in Fig. 5Ab and Bb, respectively. The boxed area in a is shown at higher magnification in b. C, a contour map of | ||
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Figure 7. The relationship between Ca2+ sparks and Ca2+ transients in urinary bladder myocytes A, a STOC and corresponding Ca2+ spark were measured in urinary bladder myocytes in the same manner as in Fig. 5. F/F0 at the spark site (spot | ||
BK channel cluster with functional Ca2+ storage sites in excised patches
Single channel currents of BK channels were recorded in inside-out patches from vas deferens and urinary bladder myocytes. BK channel characteristics, such as conductance and Ca2+ sensitivity, were identical to those reported previously under the same experimental conditions (Muraki et al. 1992; Suzuki et al. 1992; Hirano et al. 1998). When the Ca2+ concentration of the external medium was pCa 6.0 and the K+ concentrations in the pipette and external solutions were 5 and 140 mM, respectively, the unitary current amplitude and open probability (Po) of BK channels in vas deferens smooth muscle cells at 0 mV were 4.5 ± 0.5 pA and 0.62 ± 0.32 (n = 22), respectively. Those in urinary bladder myocytes were 4.2 ± 0.8 pA and 0.71 ± 0.49 (n = 17), respectively. The Po was increased to 0.91 ± 0.16 (n = 4) and 0.89 ± 0.27 (n = 5) in vas deferens and urinary bladder, respectively, by an increase in [Ca2+]o to pCa 5.0. The slope conductance was 120.5 ± 21.9 pS (n = 9) and 117.6 ± 12.7 pS (n = 7), respectively.
The number of BK channels per patch was 3.8 ± 3.3 (n = 22) and 4.2 ± 3.3 (n = 17) in vas deferens and urinary bladder myocytes, respectively, when the pipette resistance was in the range 7-15 M. By use of pipettes having a relatively low resistance of 3-5 M, inside-out patches where BK channel activities were susceptible to caffeine were obtained as shown in Fig. 8. The application of 5 mM caffeine markedly increased the opening of BK channels at 0 mV in a reproducible manner, while the inward current was elicited only by the first caffeine application. Even spontaneous opening of BK channels was occasionally observed (Fig. 8B). These findings essentially confirmed the results obtained by Xiong et al. (1992). Application of cyclopiazonic acid (CPA), a blocker of the SR Ca2+-ATPase, induced a transient increase and a subsequent decrease in BK channel activity (Suzuki et al. 1992). The caffeine-induced activation was not observed in the presence of CPA. The activity was increased and decreased immediately after the exchange of external solution with those of pCa 5.0 and 8.0, respectively, indicating that the patch configuration was inside-out (not shown). The number of BK channels in this patch could not be detected exactly but was assumed to be over 100, based on the peak current amplitude of ~400 pA in pCa 4.5 solution, Po of ~0.9 and the unitary current amplitude of 4 pA at 0 mV. This regulation of BK channel activities was presumably due to functional Ca2+ storage sites, which were included in the excised patch (Fig. 8C).
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Figure 8. BK channel activity in inside-out patches with functional SR Single channel currents of BK channels were recorded in an inside-out patch isolated from a vas deferens myocyte. The pipette resistance was approximately 4 M | ||
This type of BK channel activation due to Ca2+ release from functional Ca2+ storage sites was observed in five inside-out patches out of ~100 in vas deferens myocytes (~5 %). In these patches, over 100 BK channels appeared to be included. In ~15 % of patches under these experimental conditions, the first application of 5 mM caffeine slightly or markedly increased activity but the second one did not. In the other ~80 % of patches, even the first application of caffeine did not induce changes in BK channel activity. Although the number of channels in these caffeine-insensitive patches could not be determined exactly, the average was presumed to be 10-20 per patch.
Distribution of BK channels and RyRs
The distribution of BK channels in vas deferens myocytes was analysed using anti-BK channel subunit (BK) antibody and confocal microscopy. Figure 9Aa shows the typical staining pattern of BK. The distribution of green fluorescence signals in the myocyte was specific to the periphery and, interestingly, not uniform along the plasmalemma but was often observed to be punctate. The fluorescence disappeared when the cell was incubated with the primary antibody to BK in the presence of the corresponding antigen peptide (Fig. 9Ab). The staining pattern of the voltage-dependent Ca2+ channel 1C subunit was also peripheral but uniform along the plasmalemma (Fig. 9B). Similar results were obtained in 12 and six myocytes of vas deferens and urinary bladder, respectively.
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Figure 9. Immunostaining of BK channels, Ca2+ channels and RyRs in vas deferens myocytes A, confocal image of BK channel | ||
Figure 9Ca again shows green fluorescence staining of BK in a vas deferens myocyte. The same cell was also stained with anti-RyR antibody (red signals; Fig. 9Cb). The staining of RyRs was predominantly observed in the periphery but also in the centre of the myocyte. The double-stained image (Fig. 9Cc) indicates that the green and red signals partly overlapped each other and that the distribution of the overlap of these signals (yellow) was detected as spots on/beneath the plasmalemma in the myocyte. Figure 9D demonstrates a spot-like distribution of yellow signals at higher magnification, suggesting the co-localization of BK and RyRs. Among the total pixels stained with fluorescent anti-RyR or anti-BK antibodies, the signals of RyRs (red), BK (green) and areas where they overlap (mixture of red and green, often yellow) were observed in ratios of 16.1 ± 7.4, 8.8 ± 7.3 and 75.1 ± 2.3 %, respectively (n = 6). The spot-like distribution of yellow signals on/beneath the plasmalemma was observed in all myocytes examined in this study (vas deferens, n = 10; urinary bladder, n = 4).
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DISCUSSION |
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The present study clearly shows that, in smooth muscle cells of guinea-pig vas deferens and urinary bladder, Ca2+ sparks frequently occur at resting membrane potentials in discrete sites mainly in subplasmalemmal areas and generate STOCs. Ca2+ spark sites responsible for STOC generation were always located in the same areas of early Ca2+ transients during depolarization. These Ca2+ sparks and early transients may originate from discrete junctional areas between plasmalemma and SR fragments. Results from BK channel recording in inside-out patches including Ca2+ storage sites and immunocytochemical observations suggest that both BK channels and RyRs are densely distributed and co-localize in a spot-like fashion in the junctional areas.
Synchronization of STOCs with Ca2+ sparks in discrete sites
In the present study, STOCs having a peak amplitude over 50 pA at -40 mV were recorded in 50-60 % of cells and Ca2+ sparks were detected in about 40 % of cells. When STOCs (> 50 pA) were recorded in a myocyte, Ca2+ sparks were also detected with about 80 % probability. It was confirmed that Ca2+ sparks occur repetitively at a limited number of discrete sites in a cell (Gordienko et al. 1998; ZhuGe et al. 1998, 1999; Pérez et al. 1999). The finding in this study that approximately 70 % of these discrete sites were located in the subplasmalemmal area (< 1 µm from the cell edge) is similar to that from cerebral artery myocytes (Nelson et al. 1995) but not consistent with those from intestine (Gordienco et al. 1998) and portal vein (Mironneau, 1996).
Of importance is the observation that only Ca2+ sparks that occurred repetitively in discrete sites in the subplasmalemmal area were synchronous with STOCs in a one to one manner without delay (< 4 or 8 ms). The half-rise and -decay times of Ca2+ sparks were almost identical to those of the corresponding STOCs. These results are, however, not consistent with those of other reports (ZhuGe et al. 1998; Pérez et al. 1999). The linear relationship between the relative amplitude of the Ca2+ sparks and corresponding STOCs gave more evidence indicating the direct generation of STOCs by Ca2+ sparks. There is no doubt that, in general, one Ca2+ spark elicits one STOC by the coupling between RyRs and BK channels at the junctions (Jagger et al. 2000). In about 80 % of cells where both STOCs and Ca2+ sparks were measured, most STOCs having an amplitude of over 50 pA could be attributable to Ca2+ sparks occurring in a few (< 5) discrete sites, which were always located in the subplasmalemmal area (< 1 µm). Although the recording period was limited (3 s recording, 3 times with an interval of 3 min in each myocyte), it may be suggested that a relatively small number of discrete SR fragments, which form a kind of junction with the plasmalemma, are totally responsible for the frequent occurrence of Ca2+ sparks activating STOCs under resting conditions in these myocytes.
Spatial relationship between Ca2+ sparks at rest and early transients during depolarization
In cardiac myocytes, Ca2+ sparks occur at the regularly repeated junctions of the transverse tubules and the junctional SR, where L-type Ca2+ channels and RyRs co-localize, as in the local control model of excitation- contraction (E-C) coupling (Shacklock et al. 1995; Cheng et al. 1996b). Since Ca2+ sparks in smooth muscle occur mainly in peripheral areas in contrast to those in cardiac myocytes, the function of Ca2+ sparks in E-C coupling in smooth muscle could be different from that in cardiac myocytes. It has been reported that an action potential in myocytes of vas deferens and urinary bladder elicits several small spots in subplasmalemmal areas, in which [Ca2+]i rises much faster and to a higher level than in other areas of the cell (Imaizumi et al. 1998). This rise in [Ca2+]i in discrete spots was well synchronized with the activation of a large IK,Ca under whole-cell voltage clamp, as confirmed in this study. The large IK,Ca is greatly responsible for action potential repolarization and partly for the afterhyperpolarization (Imaizumi et al. 1996). The early Ca2+ transients during depolarization may be due to the local Ca2+ release from subplasmalemmal SR fragments, which could be triggered by Ca2+ entering through voltage-dependent Ca2+ channels during depolarization via the mechanism of Ca2+-induced Ca2+ release (CICR) (Endo, 1977) through RyRs. The involvement of CICR in E-C coupling in smooth muscle has been also suggested in several other types of preparation (Ganitkevich & Isenberg, 1992; Arnaudeau et al. 1997), including a urinary bladder tissue preparation (Hashitani et al. 2000). The Ca2+ transients activated by depolarization/ an action potential last over several hundred milliseconds and spread into other areas of the cell, resulting in a rise of whole-cell [Ca2+]i and subsequent cell shortening (Bolton et al. 1999).
The generation of early Ca2+ transients by depolarization has been suggested to be a key phenomenon in E-C coupling in these smooth muscle cells (Imaizumi et al. 1998). An important question is whether frequently discharging Ca2+ spark sites possess functional roles as the initiation sites of Ca2+ release in the major step of the E-C coupling process in smooth muscle cells, or mainly act as the generator of STOCs to be involved in the relaxation mechanisms. In the present results, all the frequently discharging Ca2+ spark sites were included in the spots of early Ca2+ transients during depolarization. It is strongly suggested that the Ca2+ release during depolarization is initiated in a small number of discrete sites, where the frequent discharge of Ca2+ sparks occurs to elicit STOCs. The Ca2+ release evoked by depolarization presumably via CICR at a spark site may develop into a Ca2+ transient and initiate the spread of subsequent CICR to the whole area of the cell. If it is the case, the frequently discharging sites at rest are suggested to also be the initiation sites of E-C coupling in these smooth muscle cells.
Co-localized BK channels and RyRs at Ca2+ spark sites
One of the most important questions about ion channel regulation by Ca2+ sparks is whether the channels form a cluster on the plasmalemma at the spark site. The peak diameter of Ca2+ sparks and the peak amplitude of the corresponding STOCs were 2.4 µm and 92 pA, respectively, on average. The area of plasmalemma facing a Ca2+ spark can be estimated as 5.7 µm2, taking the contribution of caveolae to the surface area of 50 % into account (Gabella, 1976). BK channel density in the plasmalemma just over the area of the Ca2+ spark is calculated to be 21.3 ± 3.0 channels µm-2 (n = 23), based on a unitary current amplitude of 1.6 pA at -40 mV (Muraki et al. 1992; Hirano et al. 1998) and an open probability of 0.5 at an [Ca2+]i of 10 µM in the junctional area (Carl et al. 1996; Ganitkevich & Isenberg, 1996; Ganitkevich & Hirche, 1996). If the rise in [Ca2+]i in sparks is lower (Jagger et al. 2000), a higher density of BK channels at the site will be estimated. This raises the possibility that BK channels may be clustered in the junctional areas, because the average density of BK channels was 3-4 channels µm-2. BK channel clustering is also strongly suggested from the results that relatively large excised patches occasionally included functional Ca2+ storage sites sensitive to caffeine (Xiong et al. 1992), while the probability was low (5 out of ~100 patches). The density of BK channels may be more than 20 channels µm-2 in such patches with functional Ca2+ storage sites, based on the calculation from the pipette resistance (Sakmann & Neher, 1995).
The immunocytochemical observations using antibodies to the BK channel subunit, Ca2+ channel 1C subunit and RyR suggest that the BK and RyR but not the Ca2+ channel 1C form clusters in myocytes. The immunostaining pattern of RyRs is consistent with the report that RyRs exist predominantly at the periphery in guinea-pig vas deferens myocytes (Lesh et al. 1998). Co-localization of BK and RyRs in a spot-like fashion on/beneath the plasmalemma (Fig. 9Cc) is consistent with the distribution pattern of early Ca2+ transients upon depolarization (Fig. 6Ba). The possibility that the punctate BK fluorescence observed in this study could be an artifact is unlikely since the binding of anti-BK antibody was completely inhibited by the presence of antigen peptide and also since the BK expressed in HEK293 cells uniformly stained the plasmalemma (S. Ohya & Y. Imaizumi, unpublished observation).
In conclusion, we have provided evidence indicating that Ca2+ sparks at rest and spot-like Ca2+ transients upon depolarization in vas deferens and urinary bladder myocytes occur at the same discrete subplasmalemmal areas. Although Ca2+ sparks at rest do not propagate to other regions of the myocytes and not initiate a contraction unlike Ca2+ transients upon depolarization, they share physiological roles in the activation of BK channels and hyperpolarization. Co-localization of BK channels and RyRs in a punctate fashion in the junctional areas is suggested based on both electrophysiological and immunocytochemical analyses. A limited number of discrete junctions between the plasmalemma and SR fragments have obligatory roles in the regulation of membrane potential and E-C coupling in these smooth muscle cells.
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Acknowledgements
This work was supported by Monbushio International Scientific Research Program through Joint Research Grants 10044313 and by Research Grant for Cardiovascular Diseases (11C-1) from the ministry of Health and Welfare. Y.I. was also supported by Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (10672049) and by a research grant from the DAIKO Foundation.
Corresponding author
Y. Imaizumi: Department of Molecular and Cellular Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan.
Email: yimaizum{at}phar.nagoya-cu.ac.jp
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