Correspondence to: Haruo Kasai, Department of Physiology, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan., hkasai{at}m.u-tokyo.ac.jp (E-mail), +81-3-5841-3460 (phone), +81-3-5841-3325 (fax)
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Abstract |
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The mechanisms of agonist-induced Ca2+ spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP3) and a low-affinity Ca2+ indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP3 was able to reproduce acetylcholine (ACh)-induced three forms of Ca2+ spikes: local Ca2+ spikes and submicromolar (<1 µM) and micromolar (115 µM) global Ca2+ spikes (Ca2+ waves). These observations indicate that subcellular gradients of IP3 sensitivity underlie all forms of ACh-induced Ca2+ spikes, and that the amplitude and extent of Ca2+ spikes are determined by the concentration of IP3. IP3-induced local Ca2+ spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca2+-induced Ca2+ release in local Ca2+ spikes. In contrast, IP3- induced global Ca2+ spikes were consistently faster than those evoked with ACh at all concentrations of IP3 and ACh, suggesting that production of IP3 via phospholipase C was slow and limited the spread of the Ca2+ spikes. Indeed, gradual photolysis of caged IP3 reproduced ACh-induced slow Ca2+ spikes. Thus, local and global Ca2+ spikes involve distinct mechanisms, and the kinetics of global Ca2+ spikes depends on that of IP3 production particularly in those cells such as acinar cells where heterogeneity in IP3 sensitivity plays critical role.
Key Words: Ca2+ waves, caged-IP3, Ca2+ spikes, secretion, inositol trisphosphate
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Introduction |
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AGONIST receptors induce the release of Ca2+ from intracellular stores and thereby generate Ca2+ spikes, waves, or oscillations that play important roles in many cellular functions (
Pancreatic acinar cells represent an ideal system for investigating the mechanisms of agonist-induced generation of Ca2+ spikes. First, agonist-induced increases in the cytosolic concentration of Ca2+ ([Ca2+]i) in these cells are mostly attributable to the generation of IP3 from phosphatidylinositol 4,5-bisphosphate in a reaction catalyzed by phospholipase C (PLC) (
We have now characterized the Ca2+ spikes induced by spatially uniform and rapid increases in [IP3]i, generated by photolysis of caged IP3, and compared them with the Ca2+ spikes induced by a natural stimulus, acetylcholine (ACh). If CICR mechanisms play a dominant role in ACh-induced Ca2+ spikes, then the time course of such spikes should resemble that of those induced by IP3. We found that this was indeed the case for local Ca2+ spikes, but not for global Ca2+ spikes. Ca2+ imaging was performed with a low-affinity Ca2+ indicator, benzothiazole coumarin (BTC), that minimizes the effects of changes in intrinsic Ca2+ buffering in the cells and allowed us to quantify large increases in [Ca2+]i without the problem of dye saturation (
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Materials and Methods |
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Preparation of Acinar Cells
Acinar cells were dissociated from the pancreas of 57-wk-old mice by enzymatic treatment as described (-glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate, P4(5)-1-(2-nitrophenyl)-ethyl ester; Calbiochem-Novabiochem] was also added to the basic internal solution. Osmolarities of the external and internal solutions were estimated to be ~310 mOsM after addition of all chemicals (Semi-Micro Osmometer; Knauer). All experiments were performed under yellow light illumination (FL40S-Y-F; National) at room temperature (2225°C).
Ca2+ Imaging
Confocal Ca2+ imaging was performed as described (
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(1) |
where K and [Ca2+]0 were assumed to be 0.39 and 0.1 µM, respectively. Values of Fmax/Fmin were estimated in vivo by assuming that the maximal [Ca2+]i achieved in the presence of ACh (10 µM) was 10 µM (see Figure 6A and Figure B). The mean value of Fmax/Fmin thus obtained was 6.5 and was used to calibrate local Ca2+ spikes induced with a low concentration of IP3 (see Figure 1 A).
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Ca2+ imaging with a cooled CCD camera was performed as described (R were then calculated by subtracting the distribution of Rmin from that of R. From
R, [Ca2+]i was estimated as KBß·
R/(Rmax m[Rmin]
R).
The [Ca2+]i in Ca2+ images was represented by pseudocolor coding, where 0.1, 0.3, 1, 3, and 10 µM were expressed as blue, sky blue, green, yellow, and red, respectively (Figure 1, Figure 2, Figure 3, and Figure 6).
Photolysis of Caged IP3
We used a mercury lamp (IX-RFC or IMT-2-RFC; Olympus) as an actinic light source for photolysis of caged IP3. Light from the mercury lamp was filtered through a 360-nm band-pass filter and fed into the second port of the light guide (IX-RFA caged or IMT-2-RFC caged; Olympus). Incorporation of a dichroic mirror (DM400) allowed the light guide to accommodate two light sources, one for photolysis of IP3 and the other for excitation of the Ca2+ indicator. Illumination from the actinic light was gated through an electric shutter (Copal). We estimated that irradiation for 125 ms was necessary and sufficient for full activation of caged IP3. For this calibration experiment, the irradiation was restricted to a recorded cell and not applied to a patch pipette to facilitate recovery of [IP3]i through the pipette, and photolysis was intermittently applied to the same cells. We found that Ca2+ responses depended on the duration of the irradiation, and reached the maximal response at 125 ms. In most experiments, we therefore set the duration of the opening of the shutter at 125 ms to achieve complete photolysis of caged IP3, and the irradiation was applied to whole objective field including the tip of patch pipette to maintain [IP3]i constant as long as possible. In some experiments, a neutral density filter (10, 20, or 50%) was used to reduce the light intensity, in which case the concentration of photolyzed IP3 was obtained by multiplying the concentration of caged IP3 introduced into the cells by the relative light intensity. Only those data obtained from the first photolysis were used to avoid complications of preceding Ca2+ spikes.
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Results |
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IP3-Induced Local Ca2+ Spikes
We first investigated whether homogeneous and constant increases in [IP3]i could produce local Ca2+ spikes in the secretory granule area of pancreatic acinar cells similar to those induced by ACh. Photolysis of caged IP3 was induced 25 min after the establishment of whole-cell perfusion, at which time the concentration of IP3 in the cell should be equilibrated with that in the patch pipette. We monitored [Ca2+]i with a confocal microscope and a high-affinity Ca2+ indicator dye, fluo-3. Local increases in [Ca2+]i confined to small spots within the secretory granule area were detected immediately after photolysis of 5 µM caged IP3 (Figure 1 A). The spatial pattern of the IP3-induced local Ca2+ spikes was similar to those induced by ACh (n = 7, data not shown;
The time course of local Ca2+ spikes induced by photolysis of caged IP3 (Figure 1A and Figure B) also was similar to that of ACh-induced local Ca2+ spikes (
The local Ca2+ spikes also could be detected with the use of the low-affinity Ca2+ indicator BTC and a cooled CCD (charge-coupled device) camera (Figure 1C and Figure D). A focal and transient increase in [Ca2+]i of ~0.5 µM was detected in the trigger zone in response to photolysis of caged IP3 (n = 5). The increases in [Ca2+]i were confirmed by the appearance of Ca2+-dependent Cl- currents (data not shown). The detection of local Ca2+ spikes with BTC allowed us to make a direct comparison with their properties with those of global Ca2+ spikes recorded with BTC.
IP3-Induced Global Ca2+ Spikes
We next examined the effects of rapid photolysis of larger concentrations of IP3 (10100 µM). Ratiometric Ca2+ imaging with BTC was used for reliable estimation of amplitudes and time courses of changes in [Ca2+]i persisting for >20 s. Because of substantial cell-to-cell variability in the responses, these experiments were performed with a large number of cells (n = 41). Photolysis of 100 µM caged IP3 often resulted in large increases in [Ca2+]i throughout the cell that were apparent within 0.24 s (Figure 2A and Figure B), the earliest time at which an image was collected by the CCD camera. The Ca2+ indicator (BTC) was not saturated with Ca2+ at these concentrations (Figure 2 A), and it can therefore be concluded that the increases in [Ca2+]i were relatively homogeneous and exceeded 10 µM throughout the cell. Thus, the capacity for Ca2+ release appeared to be distributed homogeneously throughout the cell. The abundance of IP3 receptors in the basal area was also supported by the previous observation that IP3 injection could directly trigger Ca2+ release in the basal area (Figure 6 C of
Photolysis of caged IP3 at concentrations between 10 and 100 µM induced Ca2+ spikes that were initiated at the trigger zone (Figure 2C, Figure E, and Figure G) as in the case with ACh-induced Ca2+ spikes. In fact, Ca2+ concentrations immediately (0.24 s) after photolysis of caged IP3 were always larger in the trigger zone than in the basal area (Figure 2D, Figure F, and Figure H). Furthermore, the initial Ca2+ concentrations in the trigger zone (initial [Ca2+]t) and the basal area (initial [Ca2+]b) depended on [IP3]i with median effective concentrations of 5 and 50 µM, respectively (Figure 4A and Figure B). These data suggest that IP3 receptors in the basal area were ~10 times less sensitive to IP3 than those in the trigger zone. Gradual increases in [Ca2+]i were detected throughout the cells after photolysis of caged IP3, suggesting positive feedback effect of Ca2+ on Ca2+ release channels.
The peak amplitudes of the IP3-induced Ca2+ spikes also depended on [IP3]i (see Figure 4 C), as those of ACh-induced Ca2+ spikes did on the concentration of ACh (see Figure 4 D). The amplitudes of Ca2+ spikes ranged from micromolar, with concentrations of >10 µM in the trigger zone (Figure 2 C and 3 A), to intermediate (~5 µM; Figure 2 E and 3 C), to submicromolar (<1 µM) (Figure 2 G and 3 E). The amplitudes of the smallest global Ca2+ spikes generated by IP3 or ACh were <1 µM in most regions of the cell (Figure 2 G and 3 E). The peak amplitudes of ACh-induced increases in [Ca2+]i in the trigger zone were always larger than those in the basal area (Figure 3 and Figure 4 F). This Ca2+ gradient was not due to the gradient of [IP3]i, because similar Ca2+ gradients were induced by homogeneous increases in [IP3]i induced by caged IP3 (Figure 2 and Figure 4 E). Thus, IP3 receptors in the basal area was less sensitive to IP3 than those in trigger zone even at the peak of Ca2+ spikes in the respective areas.
Time Courses of Global Ca2+ Spikes
Marked differences were evident in the time courses of the global Ca2+ spikes induced by caged IP3 and of those induced by ACh (Figure 5). First, the time-to-peak for Ca2+ spikes at the trigger zone induced by caged IP3 was <1 s in most experiments, and was independent of [IP3]i (Figure 5 A). In contrast, the time-to-peak for ACh-induced global Ca2+ spikes was >1 s in most experiments, and decreased as the concentration of ACh increased (Figure 5 B). These data indicate that [IP3]i increases gradually during ACh stimulation, and that the rate of this increase is dependent on ACh concentration.
Second, the spread of Ca2+ spikes induced by caged IP3 was faster than that of those induced by ACh. To quantify the rate of spread of Ca2+ spikes (Ca2+ waves), we defined the spike spread time as the difference between the times at which the half-maximal [Ca2+]i was achieved in the trigger zone and in the basal area. The spread time for spikes induced by caged IP3 was <0.7 s in most experiments, and was independent of [IP3]i (P > 0.1; Figure 5 C). In contrast, the spread time for ACh-induced Ca2+ spikes was >0.7 s in most experiments, and it decreased as the concentration of ACh increased (Figure 5 D).
Finally, the onset of Ca2+ spikes in the basal area was always delayed relative to that of Ca2+ spikes in the trigger zone for cells stimulated with ACh (Figure 3), whereas little delay was observed for Ca2+ spikes induced by caged IP3 (Figure 2). We quantified the delay in the onset of Ca2+ spikes in the basal area by measuring the difference between the times at which [Ca2+]i reached 0.5 µM in the trigger zone and in the basal area. The spike delay ranged between 0 and 0.24 s for IP3-induced Ca2+ spikes (Figure 5 E) and between 0.48 and 4 s for ACh-induced Ca2+ spikes (Figure 5 F). Precise measurements of delay and spike spread times were not possible at high IP3 concentrations with our cooled CCD camera operating at an acquisition interval of 0.24 s.
Line-Scan Analysis of Global Ca2+ Spikes
Therefore, we applied the line-scan mode of confocal laser scanning microscopy to analyze, in more detail, the speed of Ca2+ spikes (Ca2+ waves) induced by large concentrations of ACh (10 µM) or IP3 (100 µM). We chose fluo-3 as the Ca2+ indicator for these experiments, because, unlike BTC, it was not excited by the ultraviolet light used for the activation of caged IP3 and therefore permitted visualization of Ca2+ spikes during photolysis. The Ca2+ spikes induced by 10 µM ACh traversed the acinar cells with the spike spread time of 0.9 ± 1 s (mean ± SD, n = 7) and the spike delay of 0.9 ± 0.9 s (Figure 6A and Figure B;
We postulated that the slow spread of ACh-induced Ca2+ spikes is due to slow generation of IP3 and to sequential activation of Ca2+-release channels with heterogeneous sensitivities for IP3. To test this hypothesis, we reduced the rate of photolysis of caged IP3 by decreasing the intensity of the actinic light source to 10% of its original value, so that the increase in [IP3]i occurred over a period of 1 s. As predicted from our hypothesis, the spike spread time of the resulting Ca2+ spikes was increased to 0.7 ± 0.3 s (n = 5; Figure 6E and Figure F). More importantly, the spike delay was also prolonged to 0.8 ± 0.3 s, similar to the spike delay for ACh-induced Ca2+ spikes (Figure 6A and Figure B). Thus, an artificial slow increase in [IP3]i was required to reproduce the time course of ACh-induced global Ca2+ spikes.
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Discussion |
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We have demonstrated that spatially homogeneous increases in [IP3]i can induce Ca2+ spikes in acinar cells that share most features of those induced by ACh, consistent with the role of IP3 as the Ca2+-mobilizing messenger for this neurotransmitter. Our data have also confirmed that subcellular gradients of IP3 sensitivities are important for the generation of all forms of Ca2+ spikes in these cells, and that IP3 is a long-range messenger and act as a global signal in those cells with diameters less than 20 µM (
Control of Global Ca2+ Spikes by IP3 Production
The time courses of global Ca2+ spikes induced by instantaneous increases in [IP3]i were faster than those of ACh-induced Ca2+ spikes at all concentrations of IP3 and ACh examined. This observation indicates that ACh-induced activation of PLC results in a gradual increase in [IP3]i, and that the kinetics of [IP3]i is a key determinant of the time course of global Ca2+ spikes. Thus, we propose a mechanism for the generation of Ca2+ spikes in which the time course of their spread reflects that of [IP3]i, and in which their extent and amplitude are determined by the maximal [IP3]i (Figure 7). The control of Ca2+ spikes by IP3 production can explain simply the key properties of agonist-induced Ca2+ spikes in exocrine gland cells. First, the spread of Ca2+ spikes is relatively slow (515 µm/s;
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Our data also support role of CICR mechanisms of Ca2+ release channels in global Ca2+ spikes, because gradual increases in [Ca2+]i were induced in response to rapid photolysis of caged IP3 (Figure 2, CH). However, these increases in [Ca2+]i were too fast (Figure 5A, Figure C, and Figure E) to account for ACh-induced global Ca2+ spikes (Figure 5B, Figure D, and Figure F). Thus, it is conceivable that the CICR mechanism locally generates Ca2+ spikes, and that the increases in [IP3]i control the spread of such Ca2+ spikes. Since gradual increases in [IP3]i determine the kinetics of global Ca2+ spikes, it is likely that the positive feedback effect of Ca2+ on PLC plays a role in the generation of global Ca2+ spikes and oscillation in acinar cells as suggested in other preparations (
Given that the production of IP3 by PLC is not instantaneous in any cell type, the resulting time-dependent increase in [IP3]i may be crucial to Ca2+ spikes in general. Moreover, long-range control of Ca2+ spike spread (Figure 7) can be applied to cells in which gradients of IP3 sensitivity exist (
Types of IP3 Receptors
The control of global Ca2+ spikes by [IP3]i in pancreatic acinar cells is consistent with the previous observation that agonists and IP3 each mobilize Ca2+ in a dose-dependent manner (
It has reported that all three types of IP3 receptors were expressed in acinar cells (
Acinar cells may differ from oocytes and smooth muscle cells in that the latter cell types express predominantly one type of IP3 receptor, and Ca2+ spikes in these cells occur in an all-or-nothing manner (
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Acknowledgements |
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We thank T. Kishimoto, A. Tachikawa, H. Maeda, and T. Nemoto for collaboration throughout the experiments, M. Iino and K. Hirose for helpful discussions, and M. Ogawa for technical assistance.
This work was supported by the Research for the Future program of the Japan Society for the Promotion of Science (JSPS), grants-in-aid from the Ministry of Education, Science, and Culture of Japan, a research grant from the Human Frontier Science Program, a grant from the Toyota Foundation, and CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation (JST). K. Ito is a research fellow of JSPS, and is now at School of Life Science, Tokyo University of Pharmacy and Life Science, Hachiooji, Tokyo 192-0392.
Submitted: March 29, 1999; Revised: June 10, 1999; Accepted: June 15, 1999.
1.used in this paper: ACh, acetylcholine; BTC, benzothiazole coumarin; [Ca2+]i, cytosolic Ca2+ concentration; CICR, Ca2+-induced Ca2+ release; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C
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