Article

Full Access

The activation state of the inositol 1,4,5-trisphosphate receptor regulates the velocity of intracellular Ca²⁺ waves in bovine aortic endothelial cells

Éric Béliveau

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada

Search for more papers by this author

Fanny Lapointe,

Fanny Lapointe

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada

Search for more papers by this author

Gaétan Guillemette,

Corresponding Author

Gaétan Guillemette

[email protected]

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, 3001–12th Avenue North, Sherbrooke, QC, Canada, J1H 5N4Search for more papers by this author

Éric Béliveau,

Éric Béliveau

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada

Search for more papers by this author

Fanny Lapointe,

Fanny Lapointe

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada

Search for more papers by this author

Gaétan Guillemette,

Corresponding Author

Gaétan Guillemette

[email protected]

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada

Faculty of Medicine and Health Sciences, Department of Pharmacology, Université de Sherbrooke, 3001–12th Avenue North, Sherbrooke, QC, Canada, J1H 5N4Search for more papers by this author

First published: 03 August 2011

https://doi.org/10.1002/jcb.23301

Citations: 3

Share a link

Email
Wechat
Bluesky

Abstract

Ca²⁺ is a highly versatile second messenger that plays a key role in the regulation of many cell processes. This versatility resides in the fact that different signals can be encoded spatio-temporally by varying the frequency and amplitude of the Ca²⁺ response. A typical example of an organized Ca²⁺ signal is a Ca²⁺ wave initiated in a given area of a cell that propagates throughout the entire cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP₃R) is responsible for the release of Ca²⁺ from the endoplasmic reticulum. IP₃R activity can be directly modulated in many ways, including by interacting molecules, proteins, and kinases such as PKA, PKC, and mTOR. In the present study, we used a videomicroscopic approach to measure the velocity of Ca²⁺ waves in bovine aortic endothelial cells under various conditions that affect IP₃R function. The velocity of the Ca²⁺ waves increased with the intensity of the stimulus while extracellular Ca²⁺ had no significant impact on wave velocity. Forskolin increased the velocity of IP₃R-dependent Ca²⁺ waves whereas PMA and rapamycin decreased the velocity. We used scatter plots and Pearson's correlation test to visualize and quantify the relationship between the Ca²⁺ peak amplitude and the velocity of Ca²⁺ waves. The velocity of IP₃R-dependent Ca²⁺ waves poorly correlated with the amplitude of the Ca²⁺ response elicited by agonists in all the conditions evaluated, indicating that the velocity depended on the activation state of IP₃R, which can be modulated in many ways. J. Cell. Biochem. 112: 3722–3731, 2011. © 2011 Wiley Periodicals, Inc.

Ca²⁺ is a highly versatile second messenger that plays a key role in the regulation of many cellular processes, including secretion, contraction, proliferation, motility, gene expression, and cell death (Foskett et al., 2007). This versatility resides in the fact that different signals can be encoded spatio-temporally by varying the frequency and amplitude of the Ca²⁺ response (Berridge et al., 2000). A typical example of an organized Ca²⁺ signal is a Ca²⁺ wave initiated in a given area of a cell that propagates throughout the entire cell or within a specific subcellular region (Thomas et al., 1996; Dupont et al., 2007). Ca²⁺ waves have been observed in a wide variety of eukaryotic cell types, and their velocity generally ranges from 1 to 35 µm/s (Jaffe, 2010). Cells use both extracellular and intracellular Ca²⁺ pools to modulate the intracellular Ca²⁺ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP₃R) is responsible for the release of Ca²⁺ from the endoplasmic reticulum, the main intracellular Ca²⁺ store by which the concentration of cytosolic Ca²⁺ is modulated (Clapham, 1995). Three IP₃R subtypes have been identified to date (IP₃R-1, IP₃R-2, and IP₃R-3). They associate into tetramers to form functional Ca²⁺ selective ligand-gated cation channels (Foskett et al., 2007). IP₃R is activated by signaling cascades that generate IP₃. Briefly, an extracellular agonist binds to its specific receptor, which activates phospholipase C (PLC) via a G-protein or tyrosine kinase. PLC then catalyzes the cleavage of phosphatidylinositol-4,5-bisphosphate into diacylglycerol and IP₃, which diffuses into the cytosol and activates IP₃R, its receptor/channel (Berridge et al., 2003). When it is released from the endoplasmic reticulum, Ca²⁺ is rapidly buffered by a number of cytosolic proteins. As such, Ca²⁺ wave propagation cannot be due to simple Ca²⁺ diffusion (Allbritton et al., 1992). IP₃ has a higher diffusion coefficient than Ca²⁺ that has been evaluated at 283 µm²/s in Xenopus oocytes (Allbritton et al., 1992). However, simple IP₃ diffusion is not sufficient to permit the propagation of a Ca²⁺ wave and would require positive feedback to occur (Rooney and Thomas, 1993). Ca²⁺ can participate in this positive feedback by regulating IP₃R activity in a biphasic manner. This regulation confers some Ca²⁺-induced Ca²⁺ release (CICR) properties on IP₃R, allowing the successive activation of IP₃R clusters by Ca²⁺ released from clusters in close proximity in the presence of a minimal concentration of IP₃ (Berridge, 1997; Dupont et al., 2007). IP₃R activity can also be directly modulated in other ways, including via interacting molecules, proteins, and kinases (Choe and Ehrlich, 2006). PKA, PKC, and mTOR have been shown to affect IP₃R activity (Ferris et al., 1991; Hajnóczky et al., 1993; Matter et al., 1993; Cameron et al., 1995; Wojcikiewicz and Luo, 1998; Giovannucci et al., 2000; Poirier et al., 2001; Wagner et al., 2003; Soulsby and Wojcikiewicz, 2005; Arguin et al., 2007; Caron et al., 2007; Chaloux et al., 2007; Regimbald-Dumas et al., 2007, 2011; Betzenhauser et al., 2009; Frégeau et al., 2011).

The endothelium is no longer seen as a passive inner lining of blood vessels, but rather as a multifunctional organ that is actively involved in vital functions of the cardiovascular system, including the modulation of arterial pressure and the maintenance of blood flow (Tran and Watanabe, 2006). As in other tissues, Ca²⁺ plays an important role in many endothelium functions. In endothelial cells, an IP₃R-dependent Ca²⁺ wave is generated in response to ATP, bradykinin (BK), and thrombin (Isshiki et al., 1998; Isshiki et al., 2002; Béliveau and Guillemette, 2009). Endothelial cells express all three IP₃R subtypes and constitute a good model for studying IP₃R-dependent Ca²⁺ wave propagation (Mountian et al., 1999; Laflamme et al., 2002; Grayson et al., 2004; Béliveau and Guillemette, 2009).

While Ca²⁺ waves have been observed in a wide variety of cell types, little attention has been paid to their velocity. There is a lack of information on how the velocity of Ca²⁺ waves can be actively modulated and on which elements of the Ca²⁺ signaling toolkit may modulate it. The velocity of Ca²⁺ waves is certainly an important aspect of the spatiotemporal distribution of Ca²⁺ within cells. Intuitively, the velocity of Ca²⁺ waves should also be an important determinant of the rate of Ca²⁺ oscillations.

In the present study, we showed that the velocity of IP₃R-dependent Ca²⁺ waves induced by ATP or BK increases with the intensity of the stimulus, that extracellular Ca²⁺ does not have a significant impact on Ca²⁺ wave velocity, and more importantly, that endogenous enhancers or inhibitors of IP₃R activity can modulate IP₃R-dependent Ca²⁺ wave velocity. Forskolin increased whereas PMA and rapamycin decreased the velocity of IP₃R-dependent Ca²⁺ waves. Our results also suggested that the velocity of IP₃R-dependent Ca²⁺ waves is not directly correlated with the amplitude of the Ca²⁺ response elicited by an agonist but, rather, that the velocity of IP₃R-dependent Ca²⁺ waves depends on the sensitivity of IP₃R, a state that can be modulated in many ways.

MATERIALS AND METHODS

Materials

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin–glutamine were from Gibco Life Technologies (Gaithersburg, MD). Fura-2/AM and phorbol 12-myristate 13-acetate (PMA) were from Calbiochem (San Diego, CA). Rapamycin was from USBiological (Swampscott, MA). ATP, BK, and forskolin were from Sigma–Aldrich (Oakville, ON, Canada).

Cell Cultures

Bovine aortic endothelial cells (BAECs) were isolated from bovine thoracic aortas and were characterized as previously described (Briand et al., 1999). The cells were maintained in low-glucose DMEM containing 2 mM L-glutamine, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. They were used between the 5th and 20th passages.

Dynamic Video Imaging of Cytosolic Ca²⁺

BAEC grown on glass coverslips were washed twice with HBSS and loaded with 0.4 µM fura 2-AM for 30 min at room temperature in the dark. The cells were then washed and bathed in fresh HBSS for 30 min to ensure complete hydrolysis of the fura-2/AM before placing the coverslips in a circular open-bottom chamber mounted on the stage of a Olympus IX71 microscope fitted with a MetaFluor digital imaging and photometry system (Olympus, Markham, ON, Canada). Fluorescence from isolated fura-2-loaded cells was monitored by videomicroscopy using 334 nm and 380 nm excitatory wavelengths, and emitted fluorescence was recorded at 510 nm. All experiments were performed at room temperature. The data are expressed as the intracellular free Ca²⁺ concentration (nM) calculated from the 334/380 fluorescence ratio according to Grynkiewicz et al. (1985). Under some conditions, cells were pretreated for 3 min with 10 µM forskolin, 5 min with 2 µM PMA, or 5 min with 10 µM rapamycin.

Data Analysis

All experiments were performed at least three times. Results are expressed as means ± standard deviations (SD). When needed, the data were analyzed using an analysis of variance, and pairwise comparisons were performed using Dunnet's test. In all cases, results were considered statistically significant when P < 0.05 (*).

Scatter plot analyses and the Pearson's correlation test were used to analyze the relationship between the amplitudes of the Ca²⁺ signals and the velocities of the Ca²⁺ waves. A Pearson's correlation coefficient (r) higher than 0.8 indicates a strong direct correlation, an r between 0.5 and 0.8 indicates a moderate correlation, an r between 0.3 and 0.5 indicates a weak correlation, an r between 0.1 and 0.3 indicates a poor correlation, and an r below 0.1 indicates no correlation.

RESULTS

Velocities of ATP-Induced Ca²⁺ Waves in Single BAECs

ATP is a Ca²⁺-mobilizing hormone that activates a functional purinergic receptor on BAECs. We used a videomicroscopic system to monitor the fluorescence of fura-2 in real-time in single cells stimulated with ATP. Figure 1 shows typical Ca²⁺ responses of two BAECs (A,A′) stimulated with 200 nM ATP. Pseudocolored images taken at different times showed that the Ca²⁺ responses are organized as Ca²⁺ waves that propagated gradually from a focal point near the edges of the cells through the cytosol to the opposite side of the cells. The experiment was performed in a nominally Ca²⁺-free extracellular medium, indicating that the Ca²⁺ response was exclusively due to intracellular Ca²⁺ release via IP₃R. The intracellular Ca²⁺ concentration was monitored in three different regions in each cell along the axis of propagation of the wave (white squares in Fig. 1B,B′) in order to measure the velocities of the Ca²⁺ waves. The first region was located near the initiation site of the wave, the second in the perinuclear region (midway between regions 1 and 3), and the third in region 3 where the wave ended. In cell A, the distance between regions 1 and 2 (d₁) was 19.8 µm, and the distance between regions 2 and 3 (d₂) was 19.9 µm. Within a single cell, peak amplitude variations of ∼150 nM Ca²⁺ were frequently observed among the different regions. Figure 1C shows that the peak amplitude of the intracellular Ca²⁺ response was different in the three regions in cell A. Figure 1C′ also shows different peak amplitudes in the different regions of cell A′. While not true for all cells, we generally observed the highest Ca²⁺ amplitude in the perinuclear region and the lowest in the region where the wave ended (as seen in cell A′). The average peak amplitude measured in all the BAECs (146 cells) stimulated with 200 nM ATP was 319 ± 62 nM Ca²⁺. Figure 1D shows a time-scale expansion of the results shown in Figure 1C, except that the intracellular Ca²⁺ levels are expressed as percentages of the maximal Ca²⁺ amplitude in each region. This conversion was done to facilitate the comparison of results obtained in the different regions. To determine the exact time at which a Ca²⁺ wave reached a selected region, we chose an arbitrary amplitude set at 50% of the maximal amplitude in this region. For the wave shown in Figure 1A, the time needed to cross-distance d₁ (Δt₁) was 1.00 s and the time needed to cross-distance d₂ (Δt₂) was 1.17 s. The wave velocity between regions 1 and 2 was thus 19.9 µm/s, while it was 16.9 µm/s between regions 2 and 3 (Fig. 1E). While slightly slower in the second portion of the cell, the velocity remained relatively constant from beginning to end. To further verify whether the wave velocity remained constant across the entire cell, we measured it between five other regions (along the propagation axis) in cell A. The average wave velocity was 18.8 ± 2.0 µm/s (data not shown). Figure 1C′–E′ shows that the wave velocity was not identical from cell to cell. The same approach was used to measure Ca²⁺ wave velocity in cell A′ (Fig. 1E′). As with cell A, the wave velocity in cell A′ remained relatively constant. To further verify whether the wave velocity remained constant across the entire cell, we measured it between five other regions (along the propagation axis) in cell A′. The average velocity was 50.9 ± 2.5 µm/s (data not shown). This was approximately 2.5 times higher than in cell A, indicating that, in cells of similar size, the same concentration of ATP can induce intracellular Ca²⁺ waves that propagate at significantly different velocities. Ca²⁺ wave velocities must thus be measured in a large number of cells to obtain results that are representative of a cell population tested under a given experimental condition. In subsequent experiments, Ca²⁺ amplitudes and wave velocities were measured in at least 40 cells. Similar results were obtained when individual cells were stimulated with 10 nM BK, another Ca²⁺-mobilizing hormone that activates a functional receptor on BAECs (data not shown).

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Measurement of intracellular Ca²⁺ wave velocity. BAECs were loaded with fura-2/AM and were imaged using an Olympus IX71 microscope (100× oil immersion objective) coupled to a MetaFluor imaging system. A,A′: Serial pseudocolored images taken at specific times of two selected cells stimulated with 200 nM ATP (at 0 s) in the absence of extracellular Ca²⁺. B,B′: Three discrete regions delimited by the white squares were selected in each cell to monitor fura-2 fluorescence in real time. C,C′: Real-time free Ca²⁺ concentrations in the selected regions identified in B,B′. D,D′: Scale time expansion of the early Ca²⁺ response (delimited by the shaded rectangle) in the selected regions in cells A,A′. The time needed for the Ca²⁺ wave to cross-distances d₁ and d₂ is indicated respectively by Δt₁ and Δt₂. E,E′: Calculation of the intracellular Ca²⁺ wave velocities measured for distances d₁ and d₂ in each cell. These results are representative of experiments performed with 24 BAEC-coated coverslips in three independent experiments.

Modulation of ATP- and BK-Induced Ca²⁺ Responses by Forskolin, Rapamycin, and PMA

The intensity of the intracellular Ca²⁺ signal depends on the concentration of ATP, BK, or other Ca²⁺-mobilizing hormone used to generate IP₃. Kinases such as PKA, PKC, and mTOR can modulate IP₃R-induced Ca²⁺ release activity and, as such, the intensity of the Ca²⁺ signal. However, the relationship between the intensity of the Ca²⁺ signal and the velocity of the Ca²⁺ wave is not clear. Figure 2A shows that, in the absence of extracellular Ca²⁺, 200 nM ATP produced a Ca²⁺ response with a peak amplitude of 280 ± 64 nM. Interestingly, 2 mM Ca²⁺ in the extracellular medium did not significantly affect the magnitude of the Ca²⁺ response elicited by 200 nM ATP (peak amplitude of 319 ± 62 nM). In the absence of extracellular Ca²⁺, a near-maximal concentration of ATP (1 µM) produced a Ca²⁺ response with a peak amplitude of 593 ± 50 nM. Forskolin (10 µM), a PKA activator, significantly increased the peak amplitude of the Ca²⁺ response elicited by 200 nM ATP to 400 ± 51 nM, while 10 µM rapamycin (mTOR inhibitor) and 2 µM PMA (PKC activator) significantly decreased the peak amplitude of the Ca²⁺ response produced by 1 µM ATP to 481 ± 47 nM and 338 ± 22 nM, respectively.

Ca²⁺ wave velocity was measured in parallel with peak amplitude in the same ATP-stimulated BAECs. Figure 2B shows that, in the absence of extracellular Ca²⁺, 200 nM ATP induced Ca²⁺ waves that propagated with an average velocity of 18.8 ± 1.1 µm/s. The presence of extracellular Ca²⁺ did not significantly affect the average wave velocity (21.0 ± 1.8 µm/s). In the absence of extracellular Ca²⁺, a near-maximal concentration of ATP (1 µM) significantly increased the average wave velocity to 37.9 ± 4.9 µm/s. Forskolin (10 µM) significantly increased the wave velocity obtained with 200 nM ATP to 26.3 ± 1.4 µm/s, while 10 µM rapamycin and 2 µM PMA significantly decreased the wave velocity obtained with 1 µM ATP to 26.8 ± 2.5 µm/s and 22.6 ± 1.1 µm/s, respectively. These results showed that wave velocity increases with increasing concentrations of ATP, and that forskolin, rapamycin, and PMA modulate the Ca²⁺ wave velocity in the same direction as they modulate the Ca²⁺ peak amplitude.

Similar experiments were performed with BAECs stimulated with BK. Figure 2A′ shows that, in the absence of extracellular Ca²⁺, 2 nM BK produced a Ca²⁺ response with peak amplitude of 137 ± 16 nM. Interestingly, the presence of 2 mM Ca²⁺ in the extracellular medium significantly increased the magnitude of the Ca²⁺ response elicited by 2 nM BK (peak amplitude of 207 ± 14 nM), suggesting that BK is a good activator of an efficient Ca²⁺ entry pathway in BAECs. In the absence of extracellular Ca²⁺, a near-maximal concentration of BK (10 nM) produced a Ca²⁺ response with a peak amplitude of 250 ± 14 nM. Forskolin significantly increased the peak amplitude of the Ca²⁺ response elicited by 2 nM BK to 184 ± 16 nM, while rapamycin and PMA significantly decreased the peak amplitude of the Ca²⁺ response elicited by 10 nM BK to 204 ± 9 nM and 162 ± 11 nM, respectively.

Ca²⁺ wave velocity was measured in parallel with peak amplitude in the same BK-stimulated BAECs. Figure 2B′ shows that, in the absence of extracellular Ca²⁺, 2 nM BK elicited Ca²⁺ waves that propagated with an average velocity of 15.7 ± 1.5 µm/s. While the presence of 2 mM Ca²⁺ in the extracellular medium increased the amplitude of the Ca²⁺ response, it did not significantly modify the velocity of the Ca²⁺ waves elicited with 2 nM BK (16.9 ± 0.7 µm/s), suggesting that there is no direct correlation between the amplitude of the Ca²⁺ response and the velocity of the Ca²⁺ wave. In the absence of extracellular Ca²⁺, a near-maximal concentration of BK (10 nM) significantly increased the average Ca²⁺ wave velocity to 25.3 ± 1.5 µm/s. Forskolin (10 µM) increased the Ca²⁺ wave velocity obtained with 2 nM BK to 19.5 ± 1.9 µm/s, while 10 µM rapamycin and 2 µM PMA significantly decreased the Ca²⁺ wave velocity obtained with 10 nM BK to 21.7 ± 1.8 µm/s and 21.0 ± 1.9 µm/s, respectively, showing that the Ca²⁺ wave velocity increased with increasing concentrations of BK, as was observed with ATP. These results further confirmed that forskolin, rapamycin, and PMA modulate the Ca²⁺ wave velocity in the same direction as they modulate the Ca²⁺ peak amplitude. However, the significant Ca²⁺ entry into BK-stimulated cells did not significantly modify the Ca²⁺ wave velocity.

Relationship Between the Amplitude of the Ca²⁺ Signal and the Velocity of the Ca²⁺ Wave

Increasing concentrations of Ca²⁺-mobilizing agonists increased the intensity (amplitude) of the IP₃R-dependent Ca²⁺ signal and the velocity of the Ca²⁺ wave, suggesting that these two parameters are directly related. On the other hand, in the presence of extracellular Ca²⁺, which increases the intensity of the Ca²⁺ signal elicited by BK, the velocity of the Ca²⁺ wave was not significantly modified, indicating that there is no direct relation between the amplitude of the Ca²⁺ signal and the velocity of the Ca²⁺ wave. To better address this question, we re-evaluated our results using scatter plots and Pearson's correlation test in order to visualize and quantify the relation between the peak amplitude of the Ca²⁺ signal and the velocity of Ca²⁺ wave.

Figure 3A shows a scatter plot that graphically locates each cell that generated a Ca²⁺ wave in response to 2 nM BK (cells that did not respond or that responded so weakly that a Ca²⁺ wave could not be detected were not included) based on their peak amplitude and wave velocity. The graph is divided into quadrants delimited by the median value of the wave velocity and the median value of the peak amplitude. The upper left quadrant (UL) groups together cells that responded with a high peak amplitude and a slow wave velocity, the upper right quadrant (UR) groups together cells that responded with a high peak amplitude and a fast wave velocity, the lower right quadrant (LR) groups together cells that responded with a low peak amplitude and a fast wave velocity, and the lower left quadrant (LR) groups together cells that responded with a low peak amplitude and a slow wave velocity. In the absence of extracellular Ca²⁺, 28% of the cells stimulated with 2 nM BK were in the UL quadrant, 22% in the UR quadrant, 28% in the LR quadrant, and 22% in the LL quadrant. In addition, Pearson's correlation test indicated that there is a poor correlation between peak amplitude and wave velocity (r = 0.157, n = 182, P < 0.05).

Using the same quadrants as set in Figure 3A, cells stimulated with 2 nM BK in the presence of extracellular Ca²⁺, were graphically located. Figure 3B shows that 30% of these cells were in the UL quadrant, 31% in the UR quadrant, 23% in the LR quadrant, and 16% in the LL quadrant. In the presence of extracellular Ca²⁺, 11% of the cells relocated to the high amplitude quadrants whereas 3% of the cells relocated to the slow wave velocity quadrants. This further suggested that the Ca²⁺ entry pathway does not directly influence the velocity of the Ca²⁺ wave. In addition, Pearson's correlation test indicated that there is a poor correlation between peak amplitude and wave velocity (r = 0.247, n = 233, P < 0.05).

Using the same quadrants as set in Figure 3A, cells stimulated with 2 nM BK after a pretreatment with forskolin, were graphically located. Figure 3C shows that 19.5% of these cells were in the UL quadrant, 37.4% in the UR quadrant, 17.9% in the LR quadrant, and 25.2% in the LL quadrant. Following the treatment with forskolin, 6.9% of the cells relocated to the high peak amplitude quadrants whereas 12.6% of the cells relocated to the fast wave velocity quadrants, indicating that forskolin had a stronger potentiating effect on wave velocity than on peak amplitude. In addition, Pearson's correlation test indicated that there is a poor correlation between peak amplitude and wave velocity (r = 0.166, n = 246, P < 0.05).

Using the same quadrants as set in Figure 3A, cells stimulated with a high concentration of BK (10 nM) were graphically located. Figure 3D shows that 10.1% of these cells were in the UL quadrant, 63.5% in the UR quadrant, 14.4% in the LR quadrant, and 12.0% in the LL quadrant. This higher concentration of BK relocated 23.6% of the cells to the high amplitude quadrants and 27.9% to the fast wave velocity quadrants, indicating that both peak amplitude and wave velocity increased when the cells were stimulated with a higher concentration of BK. Once again, Pearson's correlation test indicated that there is a poor correlation between peak amplitude and velocity (r = 0.139, n = 208, P < 0.05).

Figure 3E shows the same data as Figure 3D except that the graph is divided into quadrants delimited by the median values of cells stimulated with 10 nM BK in the absence of extracellular Ca²⁺. This scatter plot is the reference for the inhibitory effects of rapamycin and PMA. Figure 3E shows that 20.2% of the cells were in the UL quadrant, 29.8% in the UR quadrant, 29.8% in the LR quadrant, and 20.2% in the LL quadrant. Figure 3F shows that, following a pretreatment with rapamycin and a stimulation with 10 nM BK, 12.7% of the cells were in the UL quadrant, 16.2% in the UR quadrant, 27.7% in the LR quadrant and 43.5% in the LL quadrant. Rapamycin caused a relocation of 21.2% of the cells to the low peak amplitude quadrants whereas it relocated only 6.2% of the cells to the slow wave velocity quadrants. While it affected both parameters, mTOR inhibition had a stronger inhibitory effect on Ca²⁺ peak amplitude than on Ca²⁺ wave velocity. Once again, Pearson's correlation test indicated that there is a poor correlation between the Ca²⁺ peak amplitude and the Ca²⁺ wave velocity (r = 0.241, n = 260, P < 0.05).

Figure 3G shows that 7.5% of the cells stimulated with 10 nM BK following a pretreatment with 10 µM forskolin were in the UL quadrant, 13.9% in the UR quadrant, 29.4% in the LR quadrant, and 49.3% in the LL quadrant. PMA (2 µM) relocated 28.7% of the cells to the low peak amplitude quadrants and 6.8% to the slow wave velocity quadrants. While it affected both parameters, PKC had a stronger inhibitory effect on peak amplitude than on wave velocity. In addition, Pearson's correlation indicated that there is a poor correlation between peak amplitude and wave velocity (r = 0.382, n = 201, P < 0.05).

These findings showed that, under all the conditions tested, the correlation between peak amplitude and wave velocity is relatively poor. The magnitude of the Ca²⁺ release was thus not directly responsible for the velocity of Ca²⁺ waves.

DISCUSSION

Intracellular Ca²⁺ waves have been observed in various cell types in humans and other species. Depending on the cell type and experimental conditions, different propagation velocities have been reported for intracellular Ca²⁺ waves (Jaffe, 2010). In the present study, we showed that the velocity of a Ca²⁺ wave is relatively constant in a single BAEC despite significant variations in the Ca²⁺ peak amplitude in isolated areas of the cell. These results are consistent with those obtained with confluent human endothelial cells, where intracellular Ca²⁺ waves induced by a wound in the monolayer propagated at a relatively constant velocity from cell to cell while Ca²⁺ peak amplitudes tended to diminish as the waves got farther away from the initiation site (Sammak et al., 1997). Isshiki et al. (1998) also observed that repetitive stimulations of a single BAEC with ATP induced intracellular Ca²⁺ waves with the same initiation site and propagation axis, while the velocities of the waves remained relatively constant during their propagation through the cell. The propagation of Ca²⁺ waves does not rely exclusively on the cytosolic diffusion of Ca²⁺ since Ca²⁺ is rapidly buffered by cytosolic proteins after being released from the endoplasmic reticulum (Allbritton et al., 1992). Since IP₃ has a diffusion coefficient of 283 µm²/s in Xenopus oocytes, IP₃ diffusion is a more plausible mechanism underlying the propagation of Ca²⁺ waves (Allbritton et al., 1992). However, in and of itself, IP₃ diffusion is not sufficient to explain the constant velocity and high Ca²⁺ levels during the propagation of Ca²⁺ waves (Rooney and Thomas, 1993), suggesting that the propagation of intracellular Ca²⁺ waves does not rely exclusively on the diffusion of IP₃ and Ca²⁺ but requires a feed-forward mechanism in order to propagate with a constant velocity. This feed-forward mechanism is likely related to the CICR property of IP₃R, which allows the successive activation of IP₃R clusters by Ca²⁺ released from clusters in close proximity (Berridge, 1997; Dupont et al., 2007). A recent study using caged IP₃ and Ca²⁺ buffers clearly demonstrated that IP₃ and Ca²⁺ are both required for IP₃R-dependent Ca²⁺ wave propagation in smooth muscle cells, which provides support for the CICR mechanism (McCarron et al., 2010). In the present study, we investigated how some elements known to modulate the intracellular Ca²⁺ response can affect the velocity of Ca²⁺ waves.

We showed that, in the absence of extracellular Ca²⁺, increasing the concentration of ATP and BK increases the amplitude of the Ca²⁺ response and the velocity of the Ca²⁺ wave. A stronger stimulation should increase the production of IP₃, which should increase the open probability of IP₃R, which in turn should cause the release of more Ca²⁺ (Foskett et al., 2007). In terms of the CICR mechanism, IP₃ and Ca²⁺ can contribute to generating higher velocity intracellular Ca²⁺ waves, by increasing the open probability of IP₃R.

Typically, in non-excitable cells, Ca²⁺ signals have two distinct phases: Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from the extracellular environment (Putney, 2009). Ca²⁺ entry might thus contribute to shaping the propagation of intracellular Ca²⁺ waves. Under our experimental conditions, the presence of extracellular Ca²⁺ did not significantly modify the amplitude of the Ca²⁺ response to ATP, indicating that ATP does not efficiently activate the Ca²⁺ entry pathway in BAECs. However, the presence of extracellular Ca²⁺ significantly increased the amplitude of the Ca²⁺ response induced by BK, suggesting that BK is an efficient activator of a Ca²⁺ entry pathway in BAECs. Sustained Ca²⁺ entry is important for BK-induced NO production by endothelial cells (Leung et al., 2006). Interestingly, while the presence of extracellular Ca²⁺ increased the amplitude of the intracellular Ca²⁺ response to BK, it did not significantly modify the velocity of the Ca²⁺ wave induced by BK, suggesting that extracellular Ca²⁺ and the mechanism of Ca²⁺ entry do not influence the velocity of the Ca²⁺ wave. They further suggest that the velocity of the Ca²⁺ wave is not directly related to the amplitude of the Ca²⁺ response.

We also verified whether the direct modulation of IP₃R activity can regulate the velocity of the intracellular Ca²⁺ wave. PKA phosphorylates the three IP₃R isoforms and increases their apparent affinity for IP₃ (Ferris et al., 1991; Hajnóczky et al., 1993; Wojcikiewicz and Luo, 1998; Giovannucci et al., 2000; Wagner et al., 2003; Soulsby and Wojcikiewicz, 2005; Chaloux et al., 2007; Regimbald-Dumas et al., 2007; Betzenhauser et al., 2009). Forskolin, an indirect activator of PKA, significantly increased the amplitude of the ATP- and BK-induced Ca²⁺ responses. Forskolin also significantly increased the velocity of the ATP- and BK-induced Ca²⁺ waves. While it is well documented that forskolin is an efficient activator of PKA, it remains that the increased cytosolic level of cAMP induced by forskolin may activate other signaling pathways that could contribute to the effect observed. For example, in excitable cells such as cardiomyocytes and pancreatic beta cells, cAMP enhances the Ca²⁺ response via the activation the guanine nucleotide exchange factor Epac, leading to an increased ryanodine receptor activity in these cells (for review see Gloerich and Bos, 2010). If a similar mechanism also exists in non-excitable cells, forskolin would increase the velocity of Ca²⁺ waves by increasing the activity state of both the IP₃R and the ryanodine receptor, two Ca²⁺ release channels located on the endoplasmic reticulum.

Conventional PKCs are activated by Ca²⁺ and diacylglycerol and thus are activated concomitantly with IP₃R (Gallegos and Newton, 2008), which they phosphorylate (Matter et al., 1993; Cameron et al., 1995; Poirier et al., 2001; Vermassen et al., 2004; Arguin et al., 2007; Caron et al., 2007). Unlike PKA, the effects of PKC depend on the IP₃R isoform phosphorylated. In cells predominantly expressing IP₃R-1, PKC increases IP₃R activity (Cameron et al., 1995; Poirier et al., 2001), whereas in cells predominantly expressing IP₃R-2 or IP₃R-3, PKC decreases IP₃R activity (Arguin et al., 2007; Caron et al., 2007). PMA, a direct activator of PKC, decreased the amplitude of ATP- and BK-induced Ca²⁺ responses in BAECs, which express all three IP₃R isoforms. PMA also significantly decreased the velocity of ATP- and BK-induced Ca²⁺ waves. Although PKC may have substrates other than IP₃R, and these substrates may directly or indirectly influence intracellular Ca²⁺ responses, our results nonetheless show that the velocity of Ca²⁺ waves is modulated under conditions where it is known that PKC modulates the activity state of the IP₃R.

mTOR is a kinase that is activated by diverse signaling pathways under the control of growth factors, cellular stresses, and nutrients (Wullschleger et al., 2006). IP₃R-dependent Ca²⁺ mobilization decreases under conditions where mTOR is inhibited (Dargan et al., 2002; MacMillan et al., 2005; MacMillan and McCarron, 2009). We recently showed that mTOR phosphorylates IP₃R and increases its apparent affinity for IP₃ (Frégeau et al., 2011; Regimbald-Dumas et al., 2011). In the present study, we showed that rapamycin, a selective inhibitor of mTOR, decreased the amplitudes of ATP- and BK-induced Ca²⁺ responses in BAECs. Rapamycin also significantly decreased the velocity of ATP- and BK-induced Ca²⁺ waves.

Our results suggested that endogenous kinases known to modulate the activity state of IP₃R can modulate the velocity of intracellular Ca²⁺ waves in BAECs. All the conditions that affected the velocity of the Ca²⁺ waves also affected the peak amplitudes of the Ca²⁺ responses. However, it remains to be determined whether the peak amplitude of the Ca²⁺ response is directly responsible for the velocity of the Ca²⁺ wave. Some studies using Ca²⁺ chelators and caged IP₃ have shown that Ca²⁺ released via IP₃R acts as a positive feedback for further Ca²⁺ release and thus plays a critical role in the initiation and propagation of Ca²⁺ waves (Wang and Thompson, 1995; Dargan and Parker, 2003; Dargan et al., 2004; McCarron et al., 2010). Since the release of some Ca²⁺ is essential for initiating a Ca²⁺ wave, intuitively, the release of more Ca²⁺ should increase the velocity of the Ca²⁺ wave. However, our results indicated that the correlation between the peak amplitude of the Ca²⁺ response and the velocity of the Ca²⁺ wave is weak at best. Under all the conditions tested, some cells responded with a high Ca²⁺ peak amplitude and generated a slow wave while others responded with a low Ca²⁺ peak amplitude and generated a fast wave. This indicates that amplitude and velocity are poorly correlated since a more sensitive IP₃R population should necessarily release more Ca²⁺. However, the sensitivity of IP₃R is not the only mechanism that can modulate the peak amplitude of Ca²⁺ responses. Apart from the Ca²⁺ entry pathway and the activity of IP₃Rs, the amplitude of the Ca²⁺ signal can be modulated by several other components, including cytosolic Ca²⁺-binding proteins that modulate the spatial and temporal aspects of increases in cytosolic Ca²⁺ levels (Schwaller, 2010). The activity of Ca²⁺ pumps and Ca²⁺ exchangers that displace Ca²⁺ from the cytosol to the endoplasmic reticulum or extracellular milieu may also contribute to modulating the amplitude of the Ca²⁺ response (Carafoli et al., 2001; Bobe et al., 2005; Dong et al., 2006). These mechanisms may explain why, in the absence of extracellular Ca²⁺, the Ca²⁺ peak amplitude does not depend exclusively on the activity of IP₃R.

Our results suggested that the state of activity of IP₃R is more important than the magnitude of the Ca²⁺ response in modulating the velocity of Ca²⁺ waves in BAECs stimulated with a Ca²⁺-mobilizing agonist. Our results also showed that the velocity of an intracellular Ca²⁺ wave in a given cell remains relatively constant during its propagation, despite variations in Ca²⁺ peak amplitude. Different mechanisms have been proposed to explain the transition of a local Ca²⁺ increase into a Ca²⁺ wave. Some investigators have suggested that the Ca²⁺ level in a particular region of a cell needs to reach a threshold to initiate a Ca²⁺ wave. Others have suggested that a sufficient frequency of elementary Ca²⁺ events in a particular region of the cell is required to initiate a Ca²⁺ wave [for a review, see Dupont et al. (2007)]. Our results with BAECs provide support for these suggestions, in that once the conditions needed to initiate a Ca²⁺ wave are reached, the resulting wave propagates in the cytosol with a sustained velocity. Moreover, the velocity of the Ca²⁺ wave can be modulated by setting conditions that increase or decrease the open probability of IP₃R. Since the velocity of the Ca²⁺ wave and the amplitude of the Ca²⁺ response appear to be independent Ca²⁺ signals, it remains to be determined which of these Ca²⁺ signals is associated with which cellular activity.

Acknowledgements

This work was supported by funding from the Canadian Institutes of Health Research and by the Heart and Stroke Foundation of Quebec and is part of the Ph.D. thesis of E.B.

REFERENCES

Allbritton NL, Meyer T, Stryer L. 1992. Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 258: 1812–1815.
10.1126/science.1465619
CAS PubMed Web of Science® Google Scholar
Arguin G, Regimbald-Dumas Y, Fregeau MO, Caron AZ, Guillemette G. 2007. Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca²⁺ in pancreatoma AR4-2J cells. J Endocrinol 192: 659–668.
10.1677/JOE-06-0179
CAS PubMed Web of Science® Google Scholar
Béliveau E, Guillemette G. 2009. Microfilament and microtubule assembly is required for the propagation of inositol trisphosphate receptor-induced Ca²⁺ waves in bovine aortic endothelial cells. J Cell Biochem 106: 344–352.
10.1002/jcb.22011
CAS PubMed Web of Science® Google Scholar
Berridge MJ. 1997. Elementary and global aspects of calcium signalling. J Physiol 499(Pt 2): 291–306.
10.1113/jphysiol.1997.sp021927
CAS PubMed Web of Science® Google Scholar
Berridge MJ, Lipp P, Bootman MD. 2000. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21.
10.1038/35036035
CAS PubMed Web of Science® Google Scholar
Berridge MJ, Bootman MD, Roderick HL. 2003. Calcium signalling: Dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529.
10.1038/nrm1155
CAS PubMed Web of Science® Google Scholar
Betzenhauser MJ, Fike JL, Wagner LE, Yule DI. 2009. Protein kinase A increases type-2 inositol 1,4,5-trisphosphate receptor activity by phosphorylation of serine 937. J Biol Chem 284: 25116–25125.
10.1074/jbc.M109.010132
CAS PubMed Web of Science® Google Scholar
Bobe R, Bredoux R, Corvazier E, Lacabaratz-Porret C, Martin V, Kovács T, Enouf J. 2005. How many Ca²⁺ATPase isoforms are expressed in a cell type? A growing family of membrane proteins illustrated by studies in platelets. Platelets 16: 133–150.
10.1080/09537100400016847
CAS PubMed Web of Science® Google Scholar
Briand SI, Neugebauer W, Guillemette G. 1999. Agonist-dependent AT₄ receptor internalization in bovine aortic endothelial cells. J Cell Biochem 75: 587–597.
10.1002/(SICI)1097-4644(19991215)75:4<587::AID-JCB5>3.0.CO;2-Q
CAS PubMed Web of Science® Google Scholar
Cameron AM, Steiner JP, Roskams AJ, Ali SM, Ronnett GV, Snyder SH. 1995. Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca²⁺ flux. Cell 83: 463–472.
10.1016/0092-8674(95)90124-8
CAS PubMed Web of Science® Google Scholar
Carafoli E, Santella L, Branca D, Brini M. 2001. Generation, control, and processing of cellular calcium signals. Crit Rev Biochem Mol Biol 36: 107–260.
10.1080/20014091074183
CAS PubMed Web of Science® Google Scholar
Caron AZ, Chaloux B, Arguin G, Guillemette G. 2007. Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells. Cell Calcium 42: 323–331.
10.1016/j.ceca.2007.01.002
CAS PubMed Web of Science® Google Scholar
Chaloux B, Caron AZ, Guillemette G. 2007. Protein kinase A increases the binding affinity and the Ca²⁺ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells. Biol Cell 99: 379–388.
10.1042/BC20060121
CAS PubMed Web of Science® Google Scholar
Choe CU, Ehrlich BE. 2006. The inositol 1,4,5-trisphosphate receptor (IP₃R) and its regulators: Sometimes good and sometimes bad teamwork. Sci STKE 2006: re15.
10.1126/stke.3632006re15
PubMed Google Scholar
Clapham DE. 1995. Calcium signaling. Cell 80: 259–268.
10.1016/0092-8674(95)90408-5
CAS PubMed Web of Science® Google Scholar
Dargan SL, Parker I. 2003. Buffer kinetics shape the spatiotemporal patterns of IP₃-evoked Ca²⁺ signals. J Physiol 553: 775–788.
10.1113/jphysiol.2003.054247
CAS PubMed Web of Science® Google Scholar
Dargan SL, Lea EJA, Dawson AP. 2002. Modulation of type-1 Ins(1,4,5)P₃ receptor channels by the FK506-binding protein, FKBP12. Biochem J 361: 401–407.
CAS PubMed Web of Science® Google Scholar
Dargan SL, Schwaller B, Parker I. 2004. Spatiotemporal patterning of IP₃-mediated Ca²⁺ signals in Xenopus oocytes by Ca²⁺-binding proteins. J Physiol 556: 447–461.
10.1113/jphysiol.2003.059204
CAS PubMed Web of Science® Google Scholar
Dong Z, Saikumar P, Weinberg JM, Venkatachalam MA. 2006. Calcium in cell injury and death. Annu Rev Pathol 1: 405–434.
10.1146/annurev.pathol.1.110304.100218
CAS PubMed Web of Science® Google Scholar
Dupont G, Combettes L, Leybaert L. 2007. Calcium dynamics: Spatio-temporal organization from the subcellular to the organ level. Int Rev Cytol 261: 193–245.
10.1016/S0074-7696(07)61005-5
CAS PubMed Web of Science® Google Scholar
Ferris CD, Huganir RL, Bredt DS, Cameron AM, Snyder SH. 1991. Inositol trisphosphate receptor: Phosphorylation by protein kinase C and calcium calmodulin-dependent protein kinases in reconstituted lipid vesicles. Proc Natl Acad Sci USA 88: 2232–2235.
10.1073/pnas.88.6.2232
CAS PubMed Web of Science® Google Scholar
Foskett JK, White C, Cheung KH, Mak DOD. 2007. Inositol trisphosphate receptor Ca²⁺ release channels. Physiol Rev 87: 593–658.
10.1152/physrev.00035.2006
CAS PubMed Web of Science® Google Scholar
Frégeau MO, Régimbald-Dumas Y, Guillemette G. 2011. Positive regulation of inositol 1,4,5-trisphosphate-induced Ca²⁺ release by mammalian target of rapamycin (mTOR) in RINm5F cells. J Cell Biochem 112: 723–733.
10.1002/jcb.23006
CAS PubMed Web of Science® Google Scholar
Gallegos LL, Newton AC. 2008. Spatiotemporal dynamics of lipid signaling: Protein kinase C as a paradigm. IUBMB Life 60: 782–789.
10.1002/iub.122
CAS PubMed Web of Science® Google Scholar
Giovannucci DR, Groblewski GE, Sneyd J, Yule DI. 2000. Targeted phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits localized Ca²⁺ release and shapes oscillatory Ca²⁺ signals. J Biol Chem 275: 33704–33711.
10.1074/jbc.M004278200
CAS PubMed Web of Science® Google Scholar
Gloerich M, Bos JL. 2010. Epac: Defining a new mechanism for cAMP action. Annu Rev Pharmacol Toxicol 50: 355–375.
10.1146/annurev.pharmtox.010909.105714
CAS PubMed Web of Science® Google Scholar
Grayson TH, Haddock RE, Murray TP, Wojcikiewicz RJH, Hill CE. 2004. Inositol 1,4,5-trisphosphate receptor subtypes are differentially distributed between smooth muscle and endothelial layers of rat arteries. Cell Calcium 36: 447–458.
10.1016/j.ceca.2004.04.005
CAS PubMed Web of Science® Google Scholar
Grynkiewicz G, Poenie M, Tsien RY. 1985. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450.
10.1016/S0021-9258(19)83641-4
CAS PubMed Web of Science® Google Scholar
Hajnóczky G, Gao E, Nomura T, Hoek JB, Thomas AP. 1993. Multiple mechanisms by which protein kinase A potentiates inositol 1,4,5-trisphosphate-induced Ca²⁺ mobilization in permeabilized hepatocytes. Biochem J 293(Pt 2): 413–422.
10.1042/bj2930413
CAS PubMed Web of Science® Google Scholar
Isshiki M, Ando J, Korenaga R, Kogo H, Fujimoto T, Fujita T, Kamiya A. 1998. Endothelial Ca²⁺ waves preferentially originate at specific loci in caveolin-rich cell edges. Proc Natl Acad Sci USA 95: 5009–5014.
10.1073/pnas.95.9.5009
CAS PubMed Web of Science® Google Scholar
Isshiki M, Ando J, Yamamoto K, Fujita T, Ying Y, Anderson RGW. 2002. Sites of Ca²⁺ wave initiation move with caveolae to the trailing edge of migrating cells. J Cell Sci 115: 475–484.
CAS PubMed Web of Science® Google Scholar
Jaffe LF. 2010. Fast calcium waves. Cell Calcium 48: 102–113.
10.1016/j.ceca.2010.08.007
CAS PubMed Web of Science® Google Scholar
Laflamme K, Domingue O, Guillemette BI, Guillemette G. 2002. Immunohistochemical localization of type 2 inositol 1,4,5-trisphosphate receptor to the nucleus of different mammalian cells. J Cell Biochem 85: 219–228.
10.1002/jcb.10124
CAS PubMed Web of Science® Google Scholar
Leung PC, Cheng KT, Liu C, Cheung WT, Kwan HY, Lau KL, Huang Y, Yao X. 2006. Mechanism of non-capacitative Ca²⁺ influx in response to bradykinin in vascular endothelial cells. J Vasc Res 43: 367–376.
10.1159/000094096
CAS PubMed Web of Science® Google Scholar
MacMillan D, McCarron JG. 2009. Regulation by FK506 and rapamycin of Ca²⁺ release from the sarcoplasmic reticulum in vascular smooth muscle: The role of FK506 binding proteins and mTOR. Br J Pharmacol 158: 1112–1120.
10.1111/j.1476-5381.2009.00369.x
CAS PubMed Web of Science® Google Scholar
MacMillan D, Currie S, Bradley KN, Muir TC, McCarron JG. 2005. In smooth muscle, FK506-binding protein modulates IP₃ receptor-evoked Ca²⁺ release by mTOR and calcineurin. J Cell Sci 118: 5443–5451.
10.1242/jcs.02657
CAS PubMed Web of Science® Google Scholar
Matter N, Ritz MF, Freyermuth S, Rogue P, Malviya AN. 1993. Stimulation of nuclear protein kinase C leads to phosphorylation of nuclear inositol 1,4,5-trisphosphate receptor and accelerated calcium release by inositol 1,4,5-trisphosphate from isolated rat liver nuclei. J Biol Chem 268: 732–736.
10.1016/S0021-9258(18)54213-7
CAS PubMed Web of Science® Google Scholar
McCarron JG, Chalmers S, MacMillan D, Olson ML. 2010. Agonist-evoked Ca²⁺ wave progression requires Ca²⁺ and IP₃. J Cell Physiol 224: 334–344.
10.1002/jcp.22103
CAS PubMed Web of Science® Google Scholar
Mountian I, Manolopoulos VG, Smedt HD, Parys JB, Missiaen L, Wuytack F. 1999. Expression patterns of sarco/endoplasmic reticulum Ca²⁺-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells. Cell Calcium 25: 371–380.
10.1054/ceca.1999.0034
CAS PubMed Web of Science® Google Scholar
Poirier SN, Poitras M, Chorvatova A, Payet MD, Guillemette G. 2001. FK506 blocks intracellular Ca²⁺ oscillations in bovine adrenal glomerulosa cells. Biochemistry 40: 6486–6492.
10.1021/bi010207k
CAS PubMed Web of Science® Google Scholar
Putney JW. 2009. Capacitative calcium entry: From concept to molecules. Immunol Rev 231: 10–22.
10.1111/j.1600-065X.2009.00810.x
CAS PubMed Web of Science® Google Scholar
Regimbald-Dumas Y, Arguin G, Fregeau MO, Guillemette G. 2007. cAMP-dependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca²⁺ release in AR4-2J cells. J Cell Biochem 101: 609–618.
10.1002/jcb.21221
CAS PubMed Web of Science® Google Scholar
Regimbald-Dumas Y, Frégeau MO, Guillemette G. 2011. Mammalian target of rapamycin mTOR phosphorylates inositol 1,4,5-trisphosphate receptor type 2 and increases its Ca²⁺ release activity. Cell Signal 23: 71–79.
10.1016/j.cellsig.2010.08.005
CAS PubMed Web of Science® Google Scholar
Rooney TA, Thomas AP. 1993. Intracellular calcium waves generated by Ins(1,4,5)P₃-dependent mechanisms. Cell Calcium 14: 674–690.
10.1016/0143-4160(93)90094-M
CAS PubMed Web of Science® Google Scholar
Sammak PJ, Hinman LE, Tran PO, Sjaastad MD, Machen TE. 1997. How do injured cells communicate with the surviving cell monolayer? J Cell Sci 110: 465–475.
CAS PubMed Web of Science® Google Scholar
Schwaller B. 2010. Cytosolic Ca²⁺ buffers. Cold Spring Harb Perspect Biol 2: a004051.
10.1101/cshperspect.a004051
CAS PubMed Web of Science® Google Scholar
Soulsby MD, Wojcikiewicz RJH. 2005. The type III inositol 1,4,5-trisphosphate receptor is phosphorylated by cAMP-dependent protein kinase at three sites. Biochem J 392: 493–497.
10.1042/BJ20051325
CAS PubMed Web of Science® Google Scholar
Thomas AP, Bird GS, Hajnóczky G, Robb-Gaspers LD, Putney JW. 1996. Spatial and temporal aspects of cellular calcium signaling. FASEB J 10: 1505–1517.
10.1096/fasebj.10.13.8940296
CAS PubMed Web of Science® Google Scholar
Tran QK, Watanabe H. 2006. Calcium signalling in the endothelium. In: S Moncada, A Higgs, editors. The vascular endothelium I. Berlin: Springer. pp 145–187.
10.1007/3-540-32967-6_5
Web of Science® Google Scholar
Vermassen E, Fissore RA, Kasri NN, Vanderheyden V, Callewaert G, Missiaen L, Parys JB, Smedt HD. 2004. Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C. Biochem Biophys Res Commun 319: 888–893.
10.1016/j.bbrc.2004.05.071
CAS PubMed Web of Science® Google Scholar
Wagner LE, Li WH, Yule DI. 2003. Phosphorylation of type-1 inositol 1,4,5-trisphosphate receptors by cyclic nucleotide-dependent protein kinases: A mutational analysis of the functionally important sites in the S2+ and S2- splice variants. J Biol Chem 278: 45811–45817.
10.1074/jbc.M306270200
CAS PubMed Web of Science® Google Scholar
Wang SS, Thompson SH. 1995. Local positive feedback by calcium in the propagation of intracellular calcium waves. Biophys J 69: 1683–1697.
10.1016/S0006-3495(95)80086-X
CAS PubMed Web of Science® Google Scholar
Wojcikiewicz RJ, Luo SG. 1998. Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase. Type I, II, and III receptors are differentially susceptible to phosphorylation and are phosphorylated in intact cells. J Biol Chem 273: 5670–5677.
10.1074/jbc.273.10.5670
CAS PubMed Web of Science® Google Scholar
Wullschleger S, Loewith R, Hall MN. 2006. TOR signaling in growth and metabolism. Cell 124: 471–484.
10.1016/j.cell.2006.01.016
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume112, Issue12

December 2011

Pages 3722-3731

The activation state of the inositol 1,4,5-trisphosphate receptor regulates the velocity of intracellular Ca²⁺ waves in bovine aortic endothelial cells

Abstract