Acta Pharmacologica Sinica

Full-length article

Acta Pharmacologica Sinica 2006 July; 27 (7): 939-944

Localized Ca²⁺ uncaging induces Ca²⁺release through IP₃R in smooth muscle

Min WANG, Zheng CHEN, Yan XING, Xu ZHANG, Xian-zhi DONG, Guang-ju JI¹

Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

¹Correspondence to Prof Guang-ju JI.
Phn/Fax 86-10-6484-6720.
E-mail gji28@ibp.ac.cn
Received 2006-04-17
Accepted 2006-05-19
doi: 10.1111/j.1745-7254.2006.00389.x

Abstract

Aim: Our previous study indicated that there are two types of Ca²⁺ release events seen in intact mouse bladder tissue. In this study our aim is to investigate the mechanism that underlies the phenomena of Ca²⁺ release in smooth muscle. Methods: Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm×0.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca²⁺ indicator and caged Ca²⁺ by incubation with 10 µmol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm. Results: Local uncaging of caged Ca²⁺was able to trigger Ca²⁺ release events in both single cells and tissue strips from mouse bladder. The Ca²⁺ release events could not be blocked by ryanodine alone, but the property of the Ca²⁺ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca²⁺ release events both in single cell and tissue experiments. Conclusion: (1) Two photon flash photolysis (TPFP) triggers Ca²⁺ induced Ca²⁺ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca²⁺ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.

Key words

two photon flash photolysis; DMNP-EGTA; calcium release; smooth muscle; IP₃R; mice

Extract

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Introduction
As in other muscle types, the major Ca²⁺-store in smooth muscle is the sarcoplasmic reticulum (SR). Evidence indicates that there are two types of Ca²⁺ release receptors, ryanodine receptor (RYR) and inositol 1,4,5-trisphosphate receptor (IP₃R), in SR in the smooth myomytes^[1_4]. Three receptor subtypes, or isoforms, have been identified for each type of receptor^[5_8]. The release of SR-Ca²⁺ results from activation of either RYR or IP₃R. However, the role of IP₃R in regulation of Ca²⁺ release in smooth muscle, especially in mouse bladder smooth muscle, has not been identified, although it has been well established for type 2 ryanodine receptor (RYR2). In urinary bladder myocytes, activation of the voltage-dependent Ca²⁺ current (I_Ca) evokes Ca²⁺ induced Ca²⁺ release (CICR) in the form of Ca²⁺ sparks or global Ca²⁺ waves in a graded fashion^[9]. Genetic evidence indicates that RYR2 channel proteins play a predominate role in SR Ca²⁺ release in bladder myocytes^[10], similar to CICR in heart cells.
We previously reported that there are two types of Ca²⁺ release events in smooth muscle^[11]. One is rapid, whole-cell Ca²⁺ transients, or "Ca²⁺ flashes" and the other is slowly propagating Ca²⁺ waves. The Ca²⁺ flashes occur through RYR-mediated Ca²⁺ release, whereas Ca²⁺ waves arise from IP₃-mediated Ca²⁺ release. However, ours and other studies suggest that IP₃R is not involved in Ca²⁺ release-induced either by stretching or by membrane voltage depolarization. Evidence of this has been found using 2-APB, an IP₃R inhibitor that does not block Ca²⁺ release in mouse bladder single cell studies^[12]. There is no evidence to date that IP₃R is functional in bladder smooth myocytes.
Two photon flash photolysis (TPFP) provides the capability to photorelease molecules in a subcellular volume on the order of 1 femtoliter and this method has been used to release and examine CICR in cardiac myocytes^[13,14]. In the present study, we used TPFP to formally test the hypothesis that localized increases in Ca²⁺ in a small subcellular domain evokes Ca²⁺ release from the SR in smooth muscle independent of the gating of sarcolemmal Ca²⁺ channels. We also aimed to examine the mechanism underlying this process. We reported that TPFP is capable of triggering CICR and results in the release of Ca²⁺ through IP₃R in the absence of PLC in smooth muscle.

Materials and methods
Cell, tissue strip preparation and solutions Animals were euthanized with CO₂and bladders were rapidly removed and dissected in cold water. Single cells were prepared as described previously^[12]. After removing of mucosal and fibrosal layers, bladders were cut into small pieces and digested in enzymatic solution. The enzymatic solution containing (mmol/L) 80 Na-glutamate, 55 NaCl, 6 KCl, 2 MgCl₂, 10HEPES, and 10 glucose. Two-step digestions were used for single cell dissociation. First, detrusor muscle was incubatedfor 20 min at 37 °C in dissociation solution containing 1mg/mL dithioerythreitol, 1 mg/mL papain, and 1 mg/mL bovineserum albumin (Sigma-Aldrich, St Louis, MO, USA ), and the partially digested tissuewas then transferred to a solution containing 1 mg/mL collagenasetype II (Worthington Biochemical, Lakewood, New Jersey,USA), 1 mg/mL bovine serum albumin,and 100 µmol/L Ca²⁺. After incubation for 10 min, the digestedtissue was washed and gently triturated in dissociation solutionto yield single smooth muscle cells. Tissue strip was prepared as reported previously^[11]. Detrusor muscle running along the axis from the neck to the fundus was cut into strips about 0.1 cm×0.5 cm. The strips were transferred into an optical recording chamber and fixed with a Kevlar fiber retaining clip (Warner Instruments, Hamden, CT, USA). The extracellular solution used for single cell and tissue strip perfusion was (mmol/L) 137 NaCl, 5.4 KCl,1.8 CaCl₂, 1.0 MgCl₂, 10 glucose, 10 HEPES, and pH 7.4 adjusted with NaOH.
Imaging and local uncaging of caged Ca²⁺ Single cells and intact tissue strips were co-incubated with Fluo-4 AM 10 µmol/L (Molecular Probes, Eugene, Oregon, USA) and 1-(4,5-dimethoxy-2-nitrophenyl)-1,2-diaminoethane- N’,N,N’,N’,-tetraacetic acid, tetra (acotoxymethyl ester) (DMNP-EDTA, AM) 10 µmol/L (Molecular Probes, Eugene, Oregon, USA ) in a bath solution containing 0.02% pluronic acid for 10 min (single cell) or 60 min (tissue) at room temperature (20_24 ºC). Fluo-4 AM was excited by a 488 nm laser light and emission was collected through a long-pass filter of 500 nm. Confocal images were captured using a Bio-Rad Radiance 2000 confocal head attached to a Nikon TMIII inverted microscope with 60×water immersion objective. Confocal X-y images are 128×20 pixels in size and with the line frequency of 1200 Hz. Line-scan images were acquired at sampling ratesof 0.83 ms/pixel and 0.73 µm/pixel.
Local uncaging of caged Ca²⁺, DMNP-EGTA, was achieved by exposure to an ultraviolet (UV) laser light beam (Mai Tai, Diode-pumped, mode-locked Ti: sapphire laser, Spectra-Physics, Mountain View, CA, USA) at wavelength of 730 nm and a power of 4.5 mW. Image processing, data analysis, and presentation were performed using software of MATLAB 6.5. Data were reported as mean±SEM. Stu-dent’s t-test was applied to determine statistical significance of measured differences. A P-value of less than 0.05 was considered statistically significant.
Results
Ryanodine did not inhibit Ca²⁺ releases induced by two photon flash photolysis in single cells Our previous studies indicated that RYR2, but neither type 3 nor IP₃R, plays an important functional role in smooth muscle^[5]. In the present study, we directly tested the function of RYR and IP₃R Ca²⁺ release channels using localized uncaging of caged Ca²⁺, DMNP-EGTA, in mouse bladder smooth muscles. First we examined the uncaging of caged Ca²⁺effect in single cells. As shown in Figure 1A, cells co-loaded with DMNP-EGTA and Fluo-4 AM repeatedly exposed to UV (30 ms, 4.3 mW) was able to trigger Ca²⁺ release (sparks), which is consistent with our previous study^[12]. Next we tested the effects of Ca²⁺ release receptor inhibitors on the Ca²⁺ release events triggered by exposure to the bleaching laser light. Application of Xestospongin C (final concentration 10 µmol/L), a selective IP₃R inhibitor, there was almost no visible effect on Ca²⁺ sparks (data not shown). Surprisingly, application of ryanodine (10 µmol/L) did not completely block Ca²⁺ release induced by uncaging of caged Ca²⁺, but rather markedly altered the characteristics of CICR (Figure 1B). This encouraged us to test what would happen if ryanodine and Xestospongin C were co-applied. The results indicated that in the presence of both ryanodine (10 µmol/L) and Xesto-spongin C (10 µmol/L) Ca²⁺ sparks and/or waves induced by local uncaging of DMNP-EGTA by exposure to UV laser light were completely ablated (Figure 1C). This suggests that in the presence of RYR inhibitor, increase in the cytosolic Ca²⁺ concentration level by local uncaging of caged Ca²⁺ could directly trigger IP₃R Ca²⁺ release channel function and hence, induce Ca²⁺ sparks and/or waves in bladder smooth muscle.
Effect of Ca²⁺ release receptor inhibitors on Ca²⁺ release in intact smooth muscles As mentioned above, local uncaging of caged Ca²⁺ by exposure to UV laser light was capable of triggering Ca²⁺ release events in the presence of RYR or IP₃R alone in mouse bladder single cells. Next we further examined the role of Ca²⁺ release receptor (channel) inhibitors in the regulation of Ca²⁺ release channels in intact tissue strips. Figure 2A shows that exposed DMNP-EGTA and Furo-4 AP co-loaded tissues to UV laser light was also able to cause Ca²⁺ releases (sparks). But the time exposure to UV laser light to trigger Ca²⁺ release was much longer (100 ms) compared to that used in single cell experiments. Similarly, as observed in single cell experiments, Xesto-spongin C (10_30 µmol/L) or ryanodine (30 µmol/L) alone failed to block Ca²⁺ release triggered by uncaging of caged Ca²⁺ in intact tissue segments. Unlike Xestospongin C experiments, more flash times were needed to trigger Ca²⁺ release in the presence of ryanodine, as indicated in Figure 2B, suggesting the property of Ca²⁺ release was altered by ryanodine in intact tissues as well. Our results also indicated that co-application of ryanodine and Xestospongin C Ca²⁺ releases were completely inhibited in intact mouse bladder tissue segments (Figure 2C).
Effect of Ca²⁺release receptor inhibitors on kinetics of Ca²⁺ induced Ca²⁺ release To further understand the role of specific intracellular Ca²⁺ release channels in locally elicited CICR, we analyzed the kinetics (rise time and time to release), Ca²⁺ wave propagation velocity, following TPFP. The rise time of Ca²⁺ release was markedly affected by RYR antagonism, likely associated with a decrease in the underlying rate of SR Ca²⁺ release. Ca²⁺ spark rise time was markedly prolonged in the presence of 10 µmol/L ryanodine (142.23±17.6 ms, n=21) compared to that in control experiments (64.22±6.21 ms, n=17, Figure 1F). Conversely, in the presence of 30 µmol/L xestospongin C the rate of rise of Ca²⁺ release following TPFP was not significantly different from control (60.6±4.33 ms, n=38). This could be observed in both single cell and intact tissue experiments (Figure 1D, 2E).
The hypothesis that CICR is the mechanism underlying Ca²⁺ spark/wave propagation is commonly accepted. In the present study, the speed of Ca²⁺ spark/wave propagation triggered by exposing caged Ca²⁺, DMNP-EGTA, to UV laser light was determined in intact mouse bladder smooth muscle tissue strips by the time taken between the initiating point of Ca²⁺ sparks/waves and their propagating end in the cells. As shown in Figure 4, ryanodine receptor inhibition also markedly slowed the rate of Ca²⁺ wave propagation following TPFP. Wave propagation velocity decreased from 23.2± 3.6 µm/s (n=18) to 4.3±0.8 µm/s (n=16) in the presence of ryanodine, whereas in the presence of xestospongin C, wave propagation was not significantly different from control (P>0.05, n=12).
To more effectively quantify the time to release, intact muscle segments were exposed to UV light for 100 ms (4.3 mW) at the beginning of a prolonged line scan along the cell axis, and the time delay calculated as the time that from the beginning of the 730 nm pulse to the initiation of Ca²⁺ release. The time to release was 71.33± 6.8 ms for control group and 172.66±36.5 ms for ryanodine group, respectively. Whereas, inhibition of IP₃R with xestospongin C had no significant effect (80.32±7.41 ms, n=17, Figure 3B) observed .
Peak Ca²⁺ was not altered by Ca²⁺ release channel inhibitors As shown in Figures 1E, 2D, and 3B (right), the amplitude of the Ca²⁺ spark or Ca²⁺ wave was equivalent in control cells and those incubated with 10 (or 30) µmol/L ryanodine or 30 µmol/L xestospongin C, suggesting that efficient release of SR Ca²⁺ stores is achieved when CICR occurs through the activation of either SR release channel.
Discussion
We report that localized uncaging of caged Ca²⁺ by two photon flash photolysis initiates CICR in urinary bladder myocyte. The most surprising finding in our study was the apparent ability of two photon flash photolysis to activate Ca²⁺ release through IP₃ receptors, as evidenced by the activation of Ca²⁺ that occurred with different kinetics (delay, rise time, and propagation) and required greater amounts of trigger Ca²⁺ in the presence of ryanodine (10_30 µmol/L). The results confirm that the kinetics of Ca²⁺ signals mediated by RyR and IP₃R are very different^[15]. The activation of slowly propagating, asynchronous Ca²⁺ waves in the presence of ryanodine, the marked slowing of wave propagation velocity in the presence of ryanodine, and the complete inhibition of two photon flash photolysis in the presence of Xestospongin C and ryanodine, but not in the presence of ryanodine alone, strongly suggest that IP₃R Ca²⁺ release can be activated by local increases in [Ca²⁺]_i. While substantial data indicate that the gating of IP₃R is augmented by increases in cytosolic Ca^{2+ [16,17]}, this process is thought to be insufficient to activate IP₃R gating in the absence of phospholipase C stimulation and a significant rise in cytosolic IP₃ concentration. The generation of ryanodine-insensitive, slowly propagating waves by two photon flash photolysis may reflect activation of IP₃R Ca²⁺ channels by non-physiologic increases in local [Ca²⁺]_i, as Ca²⁺ release required multiple laser pulses in the presence of ryanodine (Figure 2), particularly as xestospongin C alone had little effect on Ca²⁺ release. While these findings suggest that ryanodine receptors dominate the pattern of Ca²⁺ release following local rises in [Ca²⁺]_i, the ability to trigger release from IP₃R suggests the potential for some involvement of these release channels during CICR, as release through RYR gating would be expected to produce very high local concentrations of Ca²⁺.
IP₃R, like RYR, is a Ca²⁺ release channel that specially responds to the second messenger IP₃. Three structurally and functionally different isoforms of the IP₃R that are expressed in a cell-type specific manner have been identified^[8]. Our data indicate that all isoforms of IP₃R are expressed in mouse bladder smooth myocytes (data not shown). But the role of IP₃R,unlike RYR, in the regulation of Ca²⁺ release is not clear in smooth myocytes. In the present study, we, for the first time, demonstrated that high local cytosolic Ca²⁺ induced by local uncaging of DNMP-EGTA by TPFP triggers Ca²⁺ release through IP₃R in the absence of PLC activation and that this process, while kinetically distinct from RyR release, is capable of supporting robust CICR.

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