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(Circulation Research. 2004;95:e11.)
© 2004 American Heart Association, Inc.

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Subcortical Ca²⁺ Waves Sneaking Under the Plasma Membrane in Endothelial Cells

Masashi Isshiki, Akiko Mutoh, Toshiro Fujita

From the Department of Nephrology and Endocrinology, Faculty of Medicine, Tokyo University, Tokyo, Japan.

Correspondence to M. Isshiki, Department of Nephrology and Endocrinology, Faculty of Medicine, Tokyo University, Tokyo 113-8655, Japan. E-mail isshiki-tky{at}umin.ac.jp

	Abstract

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Subplasmalemmal Ca²⁺, dynamically equilibrated with extracellular Ca²⁺, affects numerous signaling molecules, effectors, and events within this restricted space. We demonstrated the presence of a novel Ca²⁺ wave propagating beneath the plasma membrane in response to acute elevation of extracellular [Ca²⁺], by targeting a Ca²⁺ sensor, cameleon, to the endothelial plasmalemma. These subcortical waves, spatially distinct from classical cytosolic Ca²⁺ waves, originated in localized regions and propagated throughout the subplasmalemma. Translocation of an expressed GFP fused with a PH domain of PLC from the plasma membrane to the cytosol accompanied these subcortical waves, and U73122 attenuated not only the GFP-PH translocation, but also the peak amplitude of the subcortical Ca²⁺ waves; this finding suggests the involvement of local IP₃ production through PLC-mediated PIP₂ hydrolysis in the initiation of these waves. Changes in NO production as well as PKCß-GFP translocation from the cytosol to the plasma membrane, but not of GFP-PLA₂ to perinuclear endomembranes, were associated with the subplasmalemmal Ca²⁺ changes. Thus, extracellular Ca²⁺ maintains the basal PLC activity of the plasma membrane, is involved in the initiation of compartmentalized subcortical Ca²⁺ waves, and regulates Ca²⁺-dependent signaling molecules residing in or translocated to the plasma membrane. The full text of this article is available online at http://circres.ahajournals.org.

Key Words: subplasmalemmal Ca²⁺ • FRET • cameleon • endothelium • phospholipase C

	Introduction

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Intracellular Ca²⁺ is a second messenger that is spatially and temporally organized for controlling numerous cellular processes. Because of its complex organization consisting of amplitude, frequency, timing, and spatiotemporal patterning, cells display functional specificity and versatility.¹ Ca²⁺ dynamics just beneath the plasma membrane seem to be especially important, because Ca²⁺-sensitive molecular effectors and cellular processes are compartmentalized in this restricted environment.

Across the plasma membrane, the average intracellular Ca²⁺ concentration ([Ca²⁺]_i) is generally maintained at a level approximately 10 000 times lower than that of extracellular Ca²⁺ ([Ca²⁺]_o) by Ca²⁺ regulators such as Ca²⁺-ATPase or the Na⁺-Ca²⁺ exchanger, which actively regulate the gradients to achieve equilibrium. Although the cytoplasmic surface of the plasma membrane appears to be spatially continuous with the cytosol, it is actually not part of the cytosol and does not have the same [Ca²⁺] level. It is actually a distinct compartment, differing from other cytosolic regions in terms of the [Ca²⁺]. The subplasmalemmal Ca²⁺ concentration ([Ca²⁺]_spm) in unstimulated cells is 10 times higher than the mean cytosolic Ca²⁺ concentration.² Changes in [Ca²⁺]_spm are expected to be quite different from those of the overall cytosolic [Ca²⁺], based on the proximity of Ca²⁺ channels.³ The heterogeneous structure and biochemical components of the plasma membrane have the potential to create marked differences in Ca²⁺ regulation in the subplasmalemmal space.

Localization of Ca²⁺-sensitive effector molecules downstream from spatially organized local Ca²⁺ signaling is presumed to be critical for proper cellular functioning. One example is endothelial nitric oxide synthase (eNOS), which is normally localized to the cytoplasmic surfaces of caveolae, specialized microdomains in the plasma membrane, via the acylation process.⁴ Mislocalization of eNOS outside the caveolar membrane in nonacylating mutants markedly reduces the sensitivity of the enzyme to Ca²⁺ influx as a trigger for the NO production, a fundamental determinant of cardiovascular homeostasis. In addition to eNOS, some types of adenylyl cyclase localized in cholesterol-rich caveolae, are also sensitive to capacitative Ca²⁺ influx.⁵ Not only molecules such as eNOS, which are semifixed to the plasma membrane, but also diffusible cytoplasmic molecules such as PKCß, which are recruited to the plasma membrane in response to an increase in [Ca²⁺], would be directly affected by Ca²⁺ influx. However, how those molecules are regulated under basal conditions or in response to the much more localized subplasmalemmal [Ca²⁺] change is still unclear.

In the last few decades, chelating Ca²⁺ indicators have contributed significantly to research in this field by uncovering information about the spatiotemporal dynamics of cytosolic Ca²⁺ at the single-cell level. Furthermore, the Ca²⁺-sensitive photoprotein aequorin, with a SNAP-25 targeting motif, has been used for analyzing subplasmalemmal Ca²⁺ in grouped cells.³ However, the rapid dynamics of subplasmalemmal Ca²⁺ have yet to be visualized with sufficient resolution at the single-cell level, because aequorin is not suitable for imaging because of its weak emission. Recently, however, Miyawaki et al⁶ reported on the GFP-based Ca²⁺-indicating protein, cameleon, one of its advantages being targetability to subcellular locations without loss of imaging at the single-cell level.

To investigate the spatial and temporal aspects of endothelial subplasmalemmal Ca²⁺ dynamics, we used yellow cameleon, which shows intramolecular fluorescence resonance energy transfer (FRET) from ECFP to EYFP in response to Ca²⁺. Downstream effectors, including eNOS, PKC, and PLA₂, that might also be regulated in response to localized subplasmalemmal Ca²⁺ changes, were likewise examined.

	Materials and Methods

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Solutions and Chemicals
For experiments that required physiological Ca²⁺, we used Hank balanced salt solution (HBSS, Invitrogen) containing 1.2 mol/L Ca²⁺, 1.05 mmol/L MgCl₂, and 0.83 mmol/L MgSO₄. For experiments that required Ca²⁺-free conditions, the [MgCl₂] of HBSS was adjusted to 1.88 mmol/L. 2-Aminoethoxydiphenyl borate (2APB) and ATP was from Sigma Chemical Co. Ionomycin was from Calbiochem.

Vectors and Transfection
The motif used to target YC to the plasma membrane was the 20-amino acid-long, doubly palmitoylated^7,8 amino terminal portion of neuromodulin (also called as GAP43). This motif is known to target GFP mutants to the plasma membrane^2,9 and the chimeric protein of YC with the membrane-targeting motif at its N-terminus was designated NYC. NYC was generated as described previously² from native YC2.1 (kindly provided by Drs R. Tsien, Howard Hughes Medical Institute, UCSD, La Jolla, Calif, and A. Miyawaki, RIKEN Wako Institutes, Saitama, Japan). The mitochondrial targeting sequence (MTS) was amplified by PCR using a pECFP-Mit vector (Clontech) as a template and the PCR product with HindIII sites at both ends was subcloned into the HindIII sites of YC2.1-pcDNA3, which is 18 nucleotides upstream from the initiation codon of YC2.1. The expression vectors for CFPmem, YFPmem, and PKCß-GFP were purchased from Clontech. The vectors for PLC1PH-GFP¹⁰ and GFP-PLA₂ were kindly provided by Drs T. Balla (National Institute of Child Health and Development, Bethesda, Md) and T. Hirabayashi (Chiba University, Chiba, Japan), respectively. Subconfluent endothelial cells were transiently transfected with the indicated cDNA using lipofectamine 2000 (Invitrogen Corp). One day after transfection, the cells were transferred to 35-mm glass-bottom culture dishes (Mat Tex Corp).

DAF-2 Loading
For NO measurement, loading of DAF-2 (Daiichi Pure Chemicals Co, Ltd) into the cell was performed as previously described.²

Imaging
All of the images were obtained with a Leica confocal microscopy system (Leica) equipped with an argon laser, inverted microscope, galvano stage, and oil immersion objectives of x100 and x40 (PL APO, Leica) with NA values of 1.4 and 1.25, respectively.

Images in Figures 1 and 4 were obtained with a x100 objective, and their lateral and axial resolutions are theoretically 0.17 and 0.82 µm, respectively, under optimal optical conditions.^11,12 The pixel size of such images is 0.24 µm/pixel, and the z-distance between image planes in Figure 1B is 1.5 µm. x-z images were obtained using the galvano stage. Other images were obtained with a x40 objective, and their lateral and axial resolutions are 0.20 and 1.03 µm, respectively. Pixel size ranges from 0.37 to 0.73 µm depending on the magnification for each image.

View larger version (38K): [in this window] [in a new window]   Figure 1. Targeting of yellow cameleon to the plasma membrane. A, Constructs consisting of GFP mutants (YC, NYC). Shaded box at the 5' end of NYC represents a cDNA encoding the 20 amino acids of neuromodulin. B, Confocal x-y images of an endothelial cell transiently expressing YC or NYC. Five confocal x-y sliced images from the apex to the base of the cell are shown above the merged image at the bottom (Bar=20 µm). C, Confocal x-z image of a YC- or NYC-expressing cell. NYC is correctly targeted to the plasma membrane including the lamellipodium (red arrow), and an x-y image of the same cell is shown for reference.

View larger version (27K): [in this window] [in a new window]   Figure 4. Yellow cameleon (YC) targeted to mitochondria detects [Ca2+] associated with portion of cytosol not including subplasmalemma. A, Constructs consisting of mitochondria-targetable YC (mit-YC). Shaded box at the 5' end of mit-YC represents a cDNA encoding a mitochondrial targeting sequence (MTS) derived from the precursor of subunit VIII of human cytochrome C oxidase. B, Confocal x-y and x-z images of an endothelial cell transiently expressing mit-YC (excitation wavelength 514 nm, emission wavelength 530 to 550 nm, Bar=20 µm) C, ATP-induced Ca2+ transient detected by mit-YC. D, Acute change in [Ca2+]o does not affect intramitochondrial [Ca2+].

Cells transfected with Ca²⁺ indicating sensors (YC, NYC, and mitYC) were excited at 514 nm and an emission wavelength of 530 to 550 nm was acquired for subcellular localization of the sensors. For the analysis of FRET, cells expressing the sensors were excited at 456 nm and image pairs of donor CFP (480±10 nm) and acceptor YFP (515±10 nm) were acquired simultaneously. We designated the 505 to 525 nm/470 to 490 nm (acceptor/donor) emission ratio, which is a measure of bound Ca²⁺, as the FRET image. To capture the propagation of subplasmalemmal Ca²⁺ waves, images of 128x128 pixels were obtained at the fastest interval of 0.14 seconds by the reciprocal mode of 1000-Hz line scanning.

Cells loaded with DAF-2 or expressing GFP chimeras, including PKCß-GFP and GFP-PLA₂, were excited at 488 nm, and a 500- to 540-nm emission was acquired.

Regions of interest (ROI) were traced on the monitor and the ratio value of YFP/CFP for each ROI was calculated using a built-in accessory program in the confocal system. The processing of ratio images was done off-line using the ratio imaging module of OpenLab software (Improvision Inc). Recordings of subplasmalemmal Ca²⁺ waves were made in more than 30 cells, and experiments other than DAF-2 monitoring were performed in 10 cells. NO recording in DAF-2-loaded cells was performed 20 times in grouped cells (200 cells). All of the data presented are representative of multiple experiments yielding similar results.

	Results

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The chimeric YC protein with the membrane-targeting motif at its N-terminus was generated as described in Materials and Methods (Figure 1A, NYC). Endothelial cells were transiently transfected with YC or NYC. NYC was uniformly distributed on the cell surface, whereas native YC was expressed predominantly in the cytosol as revealed by thin-slice x-y confocal images (Figure 1B and 1C). An x-z image of cells expressing NYC revealed the dome-like distribution of this chimeric protein above the cell body, which was 10 µm in height and often had thin edges or lamellipodia (Figure 1C, red arrow). In intact cells, attachment of the 20 amino acid-long tag to the N-terminus of YC did not affect the Ca²⁺-dependent increase in the emission ratio, which reflects FRET efficiency.²

First, we examined how the extracellular Ca²⁺ concentration ([Ca²⁺]_o) might affect the subplasmalemmal Ca²⁺ concentration ([Ca²⁺]_spm), by exposing NYC-expressing cells sequentially to medium containing nominally no Ca²⁺, 0.1 mmol/L EGTA, and then 1.2 mmol/L Ca²⁺. In nominally Ca²⁺-free medium, the ratio images revealed heterogeneous values for the ratio within single cells. For example, the subcellular region at a portion near the edges showed a somewhat higher emission ratio than other regions (Figure 2A, 0.57 seconds; Figure 2B, 0.88 in R1 and 0.84 in R2 and R3). The difference was estimated to be 150 nmol/L based on our previously reported calibrating method.² Images collected at 0.14-second intervals showed the differential sensitivity of the plasma membrane to changes in the [Ca²⁺]_o. Acute depletion of extracellular Ca²⁺ with 0.1 mmol/L EGTA decreased the [Ca²⁺]_spm within a second, but with a slight lag of 1 frame (0.14 seconds) in the R3 region, which appeared to represent the lamellipodium (Figure 2A, 1.29, 1.43 seconds). Most strikingly, repletion with 1.2 mmol/L Ca²⁺ initiated a simultaneous increase in [Ca²⁺]_spm in R1 and R2 (pink arrows at 13.30 seconds in Figure 2A), which then propagated throughout the plasma membrane in the form of Ca²⁺ waves (Figure 2A, 13.30 to 14.87 seconds). The two Ca²⁺ waves originating from such regions fused and finally propagated to R3, representing the lamellipodium. The movie file of this experiment can be found in the online data supplement at http://circres.ahajournals.org. There is usually a single initiation site in a cell, but 3 of the 11 cells examined had multiple sites: two with two hot spots and one with three. Based on our observation of multiple cells, the velocity of Ca²⁺ wave propagation was determined to be (mean±SD) 56±26 µm/s (range, 24 to 105 µm/s). Throughout the recording of traces of the emission intensity of ECFP and EYFP at R1, R2, and R3, mirror images were created to ensure that the ratio changes reflected real changes in FRET from donor ECFP to acceptor EYFP (Figure 2C). Furthermore, because DsRed can be used as a FRET partner with GFP,¹³ we changed the donor and acceptor pairs in the sensor molecule NYC from ECFP/EYFP to EGFP/DsRed. We also confirmed correct membrane targeting of the new sensor, which detected similar [Ca²⁺]_o-dependent FRET changes and subplasmalemmal waves (data not shown). Such FRET changes were, however, not observable in cells doubly transfected with ECFPmem and EYFPmem, control molecules of ECFP and EYFP, respectively. Each has the same membrane-targeting motif at the N-terminus as NYC (data not shown). Thus, we can rule out artifacts possibly caused by pH-dependent changes in YFP fluorescence.¹⁴ Interestingly, [Ca²⁺]_spm are similar at the apical aspect of the cell body and at the lamellipodium, whereas [Ca²⁺]_spm at the basal aspect of the cell body is lower and changes more gradually than in other regions during experimental manipulation of [Ca²⁺]_o (Figure 2D). Thus, the subplasmalemmal Ca²⁺ waves with sharp wavefronts presumably propagate preferentially to the apical side of the subplasmalemma, where [Ca²⁺]_o changes can be directly and rapidly affected. An x-z image of a YC-expressing cell revealed the existence of a subplasmalemmal region beneath the apical side of the plasma membrane with a somewhat higher [Ca²⁺] (Figure 2E). However, [Ca²⁺]_o changes hardly affected [Ca²⁺] in any cytosolic regions in YC-expressing cells (Figure 2E), suggesting the Ca²⁺ wave detected by NYC propagates through a restricted subplasmalemmal space with less vertical thickness than the optical resolution of our experimental system.

View larger version (25K): [in this window] [in a new window]   Figure 2. Spatio-temporal imaging of subplasmalemmal Ca2+. A, Cell expressing NYC was excited at 456 nm and image pairs of donor CFP (480±10 nm) and acceptor YFP (515±10 nm) were collected 0.14 seconds apart. Ratio images of YFP/CFP are shown. Time at the lower right-hand corner of each ratio image corresponds to the time-point of the trace shown in B. Increasing [Ca2+]o causes the appearance of subcortical Ca2+ waves originating from small localized regions (indicated with pink arrows at 13.3 seconds) propagating throughout the cell (Bar=50 µm). B, Time-course of subcellular [Ca2+]spm changes in the NYC-expressing cell. Three distinct subcellular regions are designated R1, R2, and R3, as indicated in the inset. In response to the indicated addition of 0.1 mmol/L EGTA and 1.2 mmol/L CaCl2, the [Ca2+]spm in each of the three regions (R1 in red, R2 in green, and R3 in blue) decreased, and then increased again with different kinetics. C, Time-course of changes in the fluorescence intensity (F, arbitrary unit) of CFP and YFP in each of the three regions in R1-3 in B. D, Difference in kinetics of [Ca2+]spm between the apical and basal aspects of NYC-expressing cells. Time-courses of changes in the emission ratios for 4 different subcellular regions, (1 through 4), as indicated in the false-colored ratio x-z image, are shown (Bar=20 µm). In response to an increase in [Ca2+]o, [Ca2+]spm in the apical aspect of the cell (2, green line) begins to increase first, whereas the increase in the basal aspect (3, blue line) is slower and the peak [Ca2+]spm in the basal aspect is lower than that in the apical membrane (1, 2, and 4). E, Time-courses of changes in the emission ratios for two cytosolic regions near (1, red) and apart from (2, green) the apical plasma membrane in x-z images of a YC-expressing cell. Initial false-colored ratio image with indicated ROIs is shown on the right. Dotted circle in the cell indicates the nucleus (Bar=10 µm).

Cells expressing YC in bulk cytosol can detect only small, if any, increases in the emission ratio in response to an acute increase in [Ca²⁺]_o from 0 to 1.2 mmol/L (Figure 3A). This is in contrast to the dramatic ratio increase detected by NYC (Figure 2B). Although the minimum (R_min) and maximum ratio value (R_max) are required for calibration in each experiment, R_min in YC- or NYC-expressing cells is usually very close to the ratio obtained when the cells are in Ca²⁺-free extracellular condition with EGTA.² Thus, if we assume the minimum and maximum ratio values in Figure 3A to be 1.08 and 1.7, respectively, the percent emission ratio increase (ERI) in YC-expressing cells in response to 1.2 mmol/L CaCl₂ is only 6%. In contrast, NYC-expressing cells usually experience more than 50% ERI, as calculated with R_max obtained by adding 10 µmol/L ionomycin+10 mmol/L CaCl₂ (data not shown). Thus, bulk cytosolic YC compromises the resolution of NYC-detectable subplasmalemmal Ca²⁺ waves because the proportion of the subplasmalemmal volume is small and such a localized signal becomes less clear because of the background fluorescence of a much larger fraction of nonsubplasmalemmal cytosol. Interestingly, NYC cannot detect the YC-detectable global [Ca²⁺] transient induced by ATP under nominally Ca²⁺ free conditions (Figure 3B). This suggests a steep Ca²⁺ gradient of global waves, with lower [Ca²⁺] at the outer rim of the wavefront not exceeding [Ca²⁺]_spm. 2-APB, a putative modulator of the IP₃ receptor,¹⁵ the Ca²⁺-release activated Ca²⁺ channel,¹⁶ or some of the transient receptor potential (TRP) superfamily,¹⁷ dose-dependently decreased the basal level of [Ca²⁺]_spm (Figure 3C) with little effect on bulk cytosolic [Ca²⁺] (data not shown), suggesting the involvement of 2-APB-sensitive mechanisms in the maintenance of the steep [Ca²⁺] gradient between the subplasmalemma and the bulk cytosol.

View larger version (14K): [in this window] [in a new window]   Figure 3. YC and NYC detect distinctly compartmentalized Ca2+. A, Cell expressing YC in nominally Ca2+-free medium was sequentially exposed to 0.1 mmol/L EGTA, 1.2 mmol/L CaCl2, and then 10 µmol/L ionomycin+10 mmol/L CaCl2 (thick bar). B, Cell expressing either NYC or YC was stimulated with 10 µmol/L ATP in medium nominally free of extracellular Ca2+, and then sequentially exposed to 0.1 mmol/L EGTA, and 10 µmol/L ionomycin+10 mmol/L CaCl2, as indicated. C, 2-APB decreased the basal level of [Ca2+]spm detected by NYC.

To confirm that the Ca²⁺ waves detected only with NYC are confined to the subplasmalemmal space, we examined whether treatment with 1.2 mmol/L CaCl₂ also affected the intramitochondrial Ca²⁺ concentration using mit-YC, a mitochondria-targeting version of YC (Figure 4A). Expression of mit-YC was found by confocal microscopy to be localized to the thin tubes, typical architectural features of mitochondria. Close examination of serial x-z images of a mit-YC-expressing cell revealed a number of dots, which are cross sections of such tubes (Figure 4B). Some of the dots, distributed within the cell, were very close to but apparently not touching the plasma membrane. In nominally Ca²⁺-free medium, 10 µmol/L ATP evoked an intramitochondrial Ca²⁺ transient (Figure 4C), similar in shape to that detected by YC (Figure 3B, bottom), suggesting that subdomains with high cytoplasmic [Ca²⁺] are associated with a [Ca²⁺] rise in the neighboring mitochondria and that the signal detected by mit-YC is another reflection of [Ca²⁺] in a portion of the cytosol not including the subplasmalemma. Rapid exchange of Ca²⁺ free extracellular medium to 1.2 mmol/L-containing medium did not affect [Ca²⁺] detected by mit-YC (Figure 4D), suggesting that the subplasmalemmal Ca²⁺ wave detected by NYC propagates through a very confined space beneath the plasma membrane not reaching the mit-YC-detectable subdomains.

Next, to examine whether phosphatidylinositol 1,4,5-trisphosphate (IP₃) plays a role in the origin of subcortical Ca²⁺ waves, we monitored spatiotemporal changes in the plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP₂) pool and its hydrolyzed product, IP₃. The PLC pleckstrin homology (PH) domain-GFP fusion protein expressed in a cell was exposed to treatments inducing the propagation of subcortical Ca²⁺ waves. PLCPH is known to specifically bind with PIP₂ or IP₃, and to be rapidly redistributed from the plasma membrane to the cytosol in response to PLC activation by receptor stimulation, or by an increase in Ca²⁺ itself.¹⁰ Translocation of PLCPH-GFP also reflects an increase in the cytoplasmic IP₃ concentration ([IP₃]_i), and Ca²⁺-mediated enhancement and suppression of [IP₃]_i have been proposed to be involved in organizing complex Ca²⁺ signaling patterns.¹⁸ As in other cell types, most of the PLCPH-GFP fluorescence expressed in the endothelium was confined to the plasma membrane in the Ca²⁺-free medium (Figure 5A). An acute increase in [Ca²⁺]_o from 0 to 1.2 mmol/L caused translocation of PLCPH-GFP from the plasma membrane to the cytosol, indicative of PIP₂ hydrolysis and IP₃ production through PLC activation, which was 20% of the maximum translocation produced by 10 µmol/L ATP (Figure 5B and 5C). An increase in [Ca²⁺]_spm can mediate PIP₂ hydrolysis through PLC activation because PLC activity is dependent on Ca²⁺.¹⁹ The opposite could also occur if locally produced IP₃ causes local Ca²⁺ influx, as discussed later. In fact, U73122, an inhibitor of PLC, attenuated the rate of increase and the peak value of the [Ca²⁺]_o-dependent [Ca²⁺]_spm increase (Figure 5D), as well as translocation of PLC-GFP from the plasma membrane to the cytosol (Figure 5C). Accordingly, PLC hydrolysis may be at least partially associated with the positive feedback system of subcortical Ca²⁺ wave formation.

View larger version (26K): [in this window] [in a new window]   Figure 5. PIP2 hydrolysis contributing to the organization of subcortical Ca2+ waves in response to extracellular Ca2+ levels. A, Confocal image of an endothelial cell in nominally Ca2+-free medium transiently expressing PLCPH-GFP (Bar=20 µm). B, False-color ratio images were generated by dividing each image by the initial image at t=0 seconds. Cell was sequentially exposed to 0.1 mmol/L EGTA, 1.2 mmol/L CaCl2, and 10 µmol/L ATP. Time shown at the lower right-hand corner of each image corresponds to the time-point of the trace shown on the left of C. Translocation of PLCPH-GFP was monitored by the cytosol (cyt) to plasma membrane (mem) fluorescence ratio shown in the rectangular areas in the right-hand image in B. C, EGTA (0.1 mmol/L), 1.2 mmol/L Ca2+, and then 10 µmol/L ATP were added to the nominally Ca2+-free medium as indicated, with or without 10 µmol/L U73122. D, Effect of 10 µmol/L U73122 (10 µmol/L) on the subplasmalemmal Ca2+ wave.

We then examined how such a locally organized Ca²⁺ increase exerts specificity of downstream signaling. PKC and PLA₂, both of which are Ca²⁺-sensitive signaling molecules, are known to be localized in the cytosol in the unstimulated state. Through the common C2 domain, PKC and PLA₂ are translocated to different membrane domains, the plasma membrane, and the perinuclear endomembranes, respectively. Translocation of these molecules in response to treatment that can evoke subcortical Ca²⁺ waves was monitored by fusing GFP to each molecule. When transiently expressed in endothelial cells, PKCß-GFP was found in the cytosol (Figure 6A). Stimulation-induced translocation of PKCß, one of several Ca²⁺-sensitive PKC subtypes, was evaluated by the plasma membrane to cytosol fluorescence ratio. In contrast to the rapid and dramatic translocation of PKCß-GFP from the cytosol to the plasma membrane induced by ATP or ionomycin, both of which can elevate cytosolic [Ca²⁺]_i, an acute increase in [Ca²⁺]_o from 0 to 1.2 mmol/L transiently induced 5% to 10% of the maximum translocation induced by ATP in the presence of a physiological level of [Ca²⁺]_o (Figure 6B and 6C). Similar to PKCß-GFP, GFP-PLA₂ also appeared to be homogeneously expressed in the cytosol in unstimulated endothelial cells (Figure 7A). Stimulation-induced translocation of PLA₂ was evaluated by the perinuclear endomembrane to whole cell fluorescence ratio. When stimulated with ATP and thapsigargin under conditions nominally free of extracellular Ca²⁺, intended to mobilize intracellular Ca²⁺ from intracellular ER Ca²⁺ stores, an acute but transient accumulation of fluorescence in the perinuclear endomembranes was observed (Figure 7B, t=64 seconds, arrow, Figure 7C). After restoration to the basal level, the addition of 1.2 mmol/L extracellular Ca²⁺, which can induce capacitative Ca²⁺ entry, fluorescence again accumulated in the same perinuclear endomembranes, but with slower kinetics and a lower peak intensity (Figure 7B, t=354 seconds, Figure 7C). A final addition of ionomycin induced rapid and more intense fluorescence accumulation (Figure 7B, t=435 seconds, Figure 7C). In contrast to this translocation that is sensitive to bulk cytosolic Ca²⁺, however, acute elevation of [Ca²⁺]_o, which can increase [Ca²⁺]_spm, did not induce GFP-PLA₂ accumulation in perinuclear endomembranes (Figure 7D). This is in marked contrast to the partial translocation of PKCß-GFP in response to an increase in [Ca²⁺]_spm.

View larger version (38K): [in this window] [in a new window]   Figure 6. PKCß-GFP translocation from cytosol to the plasma membrane occurs in association with an acute increase in [Ca2+]spm. A, Confocal cell image in nominally Ca2+-free medium transiently expressing PKCß-GFP (Bar=20 µm). B, False-color ratio images were generated by dividing by the initial image at t=0 seconds shown in A. Cell was sequentially exposed to 0.1 mmol/L EGTA, 1.2 mmol/L CaCl2, and 50 µmol/L ATP. Time shown at the lower left-hand corner of each image corresponds to the time-point of the trace shown in C. Increase in [Ca2+]o from 0 to 1.2 mmol/L induced an increase in fluorescence at the cell edges (1.2 mmol/L CaCl2, arrows), with a slight decrease in the perinuclear cytosolic area. C, Translocation of PKCß-GFP from the cytosol to the plasma membrane was monitored by the plasma membrane (Fmem, solid line) to cytosol (Fcyt, dotted line) fluorescence ratio. EGTA (0.1 mmol/L), 1.2 mmol/L Ca2+, 50 µmol/L ATP, and then 10 µmol/L ionomycin were added to the nominally Ca2+-free medium, as indicated.

View larger version (25K): [in this window] [in a new window]   Figure 7. GFP-PLA2 accumulation in perinuclear endomembranes is not sensitive to an acute increase in [Ca2+]spm. A, Image of an endothelial cell in nominally Ca2+-free medium transiently expressing GFP-PLA2 (Bar=20 µm). B, Cell was sequentially exposed to 50 µmol/L ATP and 4 µmol/L thapsigargin (ATP+TG), 1.2 mmol/L CaCl2, and 10 µmol/L ionomycin. Time shown at the lower right-hand corner of each image corresponds to the time-point of the trace shown in C. Note that the translocation of GFP-PLA2 to perinuclear endomembranes appears to be sensitive to the global [Ca2+] increase, especially Ca2+ release from intracellular stores. C, Endothelial cell in nominally Ca2+-free medium transiently expressing GFP-PLA2 was sequentially exposed to 50 µmol/L ATP and 4 µmol/L thapsigargin (TG), 1.2 mmol/L CaCl2, and 10 µmol/L ionomycin. Trace indicates the time-course of fluorescence accumulation in perinuclear endomembranes, expressed as the perinuclear region to whole cell fluorescence ratio. D, GFP-PLA2 accumulation is not sensitive to [Ca2+]o manipulation, which can induce subcortical Ca2+ waves.

Lastly, we examined how the activity of eNOS, a Ca²⁺-sensitive molecule localized to the inner surfaces of endothelial membrane microdomains, may be affected by [Ca²⁺]_o. Endothelial cells loaded with the NO indicator DAF-2 were sequentially exposed to nominally Ca²⁺-free medium, 0.1 mmol/L EGTA, 1.2 mmol/L Ca²⁺, and then 10 mmol/L Ca²⁺+10 µmol/L ionomycin (Figure 8). In response to the acute increase in [Ca²⁺]_o, DAF-2 fluorescence gradually increased, which was abolished by L-NAME, indicating that [Ca²⁺]_o-dependent increases in [Ca²⁺]_spm can affect eNOS activity. Although the relative DAF-2 fluorescence increase induced by this [Ca²⁺]_o manipulation was less than 30% of the maximum activation by 10 µmol/L ionomycin+10 mmol/L Ca²⁺, NO production is clearly sensitive to [Ca²⁺]_o.

View larger version (16K): [in this window] [in a new window]   Figure 8. NO production follows the appearance of subcortical Ca2+ waves. Endothelial cells loaded with DAF-2 were monitored by their fluorescence in the absence (solid line) or presence (dotted line) of 10 mmol/L L-NAME. The y-axis denotes the ratio of fluorescence increase divided by the initial fluorescence (dF/F0) normalized by the rate of photobleaching.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The subplasmalemmal spatiotemporal aspects of Ca²⁺ dynamics sensitive to [Ca²⁺]_o were examined using cameleon and a FRET-based technique. Our observations appear to be consistent with those of a previous study using aequorin, in which a dramatic increase in [Ca²⁺]_spm in A7r5 cells was detectable only when the sensor was expressed on the cytoplasmic face of the plasma membrane.³ Cameleon is superior to aequorin in terms of its imaging feasibility based on the intense fluorescence emitted from GFP mutants; consequently, subplasmalemmal Ca²⁺ waves were observed and characterized for the first time in this study. These subcortical Ca²⁺ waves cannot be detected by conventional imaging methods using indicators such as Indo-1, Fluo-4, or native YC that are nonspecifically loaded or expressed in the cytosol. Bulk cytosolic Ca²⁺ waves, which have been well described, basically consist of Ca²⁺ released from internal stores. Unlike the bulk cytosolic Ca²⁺ waves, the Ca²⁺ source for subplasmalemmal waves is extracellular Ca²⁺, and these waves propagate within a very shallow subcortical area between plasma membranes and the cytosol. ATP, which is known to initiate Ca²⁺ release in the form of cytosolic Ca²⁺ waves even in the absence of extracellular Ca²⁺, does not affect subplasmalemmal [Ca²⁺]. The narrow subplasmalemmal space with small subcortical Ca²⁺ waves and the cytosol, where huge Ca²⁺ waves form, represent two distinct Ca²⁺ compartments even though they are spatially continuous.

In the resting state, a gradient between [Ca²⁺]_spm and extracellular [Ca²⁺] is actively maintained by continuous Ca²⁺ influx into and extrusion from the cell, seemingly resulting in no net flux of Ca²⁺ across the plasma membrane. Because the resting [Ca²⁺]_spm is sensitive to 2-APB (Figure 3C), the maintenance of [Ca²⁺] in the subplasmalemmal compartment may be mediated via Ca²⁺ through channels such as the IP₃ receptor, CRAC, or some of the TRP superfamily. In response to [Ca²⁺]_o elevation from 0 to 1.2 mmol/L, calibrated [Ca²⁺] increases detected by YC and NYC are 45 and 350 nmol/L, respectively, based on the method previously described.² Thus, vertical thickness of the subplasmalemmal compartment is estimated to be 0.2 µm, assuming the cell volume is 4500 µm³ and the [Ca²⁺]_o-sensitive compartment is absolutely confined to the subplasmalemma.

As for the bulk cytosolic Ca²⁺ waves, there are presumably mechanisms underlying the regenerative process of subcortical Ca²⁺ wave propagation because Ca²⁺ is a localized second messenger and cannot diffuse freely by itself because of the abundance of binding proteins within the cell. One candidate mediator for the formation of subcortical Ca²⁺ waves is PLC. It has been demonstrated in several cell types that a [Ca²⁺]_i increase stimulates PLC activity.^20,21 For example, voltage-gated Ca²⁺ influx through the AMPA receptor in Purkinje cells directly stimulates PLC-mediated IP₃ production, as demonstrated by the translocation of PLC-GFP from the plasma membrane to the cytosol.²¹ PLCs are ubiquitous enzymes, and all three isozymes (ß, , and ) are expressed in endothelial cells.²² Although the activities of PLCß and PLC are usually regulated by G-proteins and tyrosine phosphorylation, respectively,^19,23 their catalytic properties are dependent on an optimal Ca²⁺ concentration.^22,24 Although the relative level of endothelial PLC activity is 7- to 8-fold lower than that of the ß or isoenzymes,²² its activity is more sensitive to [Ca²⁺].²⁵ In response to [Ca²⁺] ranging from 10^–6 to 10^–4 mol/L, activities of the ß, , and isoenzymes of PLC, measured using PIP₂ as the substrate, were reportedly increased by approximately 50%, 200%, and 300%, respectively, with the maximal increase in activity seen at around 10^–3 mol/L.²² Thus, it is possible that plasmalemmal PLC in endothelial cells is also activated by Ca²⁺ influx. Activated PLC then leads to local production of IP₃, which might be associated with formation of the subcortical Ca²⁺ wave by IP₃-mediated Ca²⁺ influx across the plasma membrane and the sensitization of neighboring Ca²⁺ channels on the inner surface of the plasma membrane. This hypothesis is reminiscent of the widely accepted mechanisms underlying the well-characterized cytosolic Ca²⁺ waves of the IP₃-induced Ca²⁺ release from ER Ca²⁺ stores and sensitization of neighboring Ca²⁺ channels by the released Ca²⁺ itself. Ca²⁺ itself might modify the positive feedback system of regenerative Ca²⁺ propagation. For example, IP₃-induced Ca²⁺ release exhibits biphasic Ca²⁺ dependence, ²⁶ and increased [Ca²⁺]_i directly enhances TRPC3 channels independently from the emptying of Ca²⁺ stores.²⁷ Our proposal is indirectly supported by the observation that subplasmalemmal IP₃ production parallels [Ca²⁺]_o changes, and that the PLC inhibitor attenuates the peak amplitude of subcortical Ca²⁺ waves. Stretch-activated Ca²⁺ channels (SAC) do not appear to play a major role in the formation of subplasmalemmal Ca²⁺ waves because Gd³⁺, the most commonly used SAC blocker, had no effect (data not shown).

G protein-coupled PLC activation in nonexcitable cells results in IP₃ generation, which mobilizes ER Ca²⁺ stores through IP₃ receptors. This Ca²⁺ mobilization from stores is generally associated with Ca²⁺ entry, so-called store-operated or capacitative Ca²⁺ entry (CCE),²⁸ a process for replenishing these stores by as yet unknown mechanisms. It has been proposed that IP₃ and its receptors play essential roles not only in the release of Ca²⁺ from intracellular Ca²⁺ stores, but also in store-release-dependent Ca²⁺ entry.¹ Involvement of IP₃ in non-CCE mechanisms has also been proposed in endothelial cells²⁹ as well as other cell types.³⁰ Thus, IP₃ may directly activate Ca²⁺ entry. In fact, biochemical studies have suggested the presence of IP₃R or an IP₃ receptor-like protein in the plasma membrane of many cell types,^31–34 including endothelial cells,³⁵ although there is little functional evidence for Ca²⁺ signaling. One exception is the documented functions of IP₃-gated Ca²⁺ channels in the olfactory neuron plasma membrane.³⁶ Furthermore, the propagation velocity of subcortical Ca²⁺ waves is in a range similar to that of ATP-induced Ca²⁺ waves (several tens of micrometers per second) consisting of IP₃-mediated Ca²⁺ release from intracellular Ca²⁺ stores,^37,38 suggesting a mechanistic similarity between wave propagation through the bulk cytosol and the subplasmalemma.

Correct targeting of proteins to specific subcellular sites is critical in the regulation of cellular functions. Cytoplasmic signaling proteins such as PKCß or cytosolic PLA₂ rely on C2, one of the modular domains, for correct targeting of the plasma membrane and perinuclear membranes, respectively. PKCß modulates thrombin-induced increases in endothelial permeability³⁹ or insulin-induced eNOS gene expression,⁴⁰ whereas PLA₂ catalyzes the release of arachidonic acid from phospholipids.⁴¹ In response to an increase in the Ca²⁺ concentration, cytosolic PKCß and PLA₂ are translocated to the membrane. This translocation is a hallmark of the activation of such molecules. Although the C2 domain of both PKCß and PLA₂ determines their destination because of the electrostatically distinct lipid composition of phosphatidylserine-rich plasma and phosphatidylcholine-rich perinuclear membranes, respectively,⁴² the two molecules could not be distinguished by differential modes of relatively large [Ca²⁺] increases: release from intracellular Ca²⁺ stores, capacitative Ca²⁺ influx, or a global nonspecific Ca²⁺ increase by ionomycin. However, only PKCß can be recruited to the plasma membrane, with no significant PLA₂ translocation to the endomembranes, by localizing the area that is exposed to [Ca²⁺] elevation in the subplasmalemmal space. This preferential translocation of PKCß to the plasma membrane may be attributable to the region of [Ca²⁺] elevation being the same as the targeting destination of the molecule. In response to acute changes in [Ca²⁺]_o and the resulting increase in subplasmalemmal [Ca²⁺], a small proportion of cytosolic PKCß, which happens to be just beneath the plasma membrane binds with Ca²⁺ and is then translocated to the plasma membrane only a short distance away. Unlike PKCß, even if a small proportion of PLA₂ binds with Ca²⁺, it needs to travel a much longer distance to reach target perinuclear membranes. The duration of Ca²⁺ elevation is also a critical parameter for PLA₂ stabilization at the membranes.⁴¹ In fact, depletion of extracellular Ca²⁺ does not affect the resting level of 6-keto PGF₁, a metabolite of prostacyclin produced through PLA₂ activation.⁴³ Thus, differential regulation of Ca²⁺-sensitive C2-mediated translocation of cytosolic molecules can be accomplished by localizing the Ca²⁺ increase, under the plasma membrane, initiated by the formation of subcortical Ca²⁺ waves.

Effector molecules on the inner surface of the plasma membrane can also be directly affected by localized subplasmalemmal Ca²⁺ changes. Endothelial NO synthase, which is localized to the cytosolic surface of the plasmalemmal caveolae by an acylation-mediated process, is a Ca²⁺-dependent enzyme for producing NO.⁴ Endothelial cells are known to be equipped with a highly specialized and efficient Ca²⁺ delivery system for eNOS regulation. Caveolae in endothelial cells are preferential sites for capacitative Ca²⁺ entry, a mode of Ca²⁺ influx tightly linked to NO production.² The potent stimulating effect of Ca²⁺ influx for NO production is in contrast to that of Ca²⁺ release from internal stores, which evokes very little NO production, suggesting that the spatial factor of Ca²⁺ delivery from outside the plasma membrane is critical for efficient NO production.^2,4 As expected, eNOS activity is also sensitive to [Ca²⁺]_o-dependent acute changes in [Ca²⁺]_spm, although the amplitude of DAF-2 fluorescence is much smaller than that associated with capacitative Ca²⁺ influx or ionomycin treatment. This is consistent with previous findings that the resting level of cGMP, an indirect parameter of NO production, falls after the removal of extracellular Ca²⁺⁴³. Thus, basal eNOS activity is maintained and regulated by subplasmalemmal Ca²⁺ equilibrated with extracellular Ca²⁺.

Finally, we have presented evidence of a novel spatially and temporally organized Ca²⁺ wave propagating through a functionally compartmentalized space beneath the plasma membrane in response to an acute increase in [Ca²⁺]_o. Such acute changes in [Ca²⁺]_o, although not entirely simulating actual physiological conditions, reveal functions maintained by the extracellular Ca²⁺ supply, including basal PLC activity, phospholipid metabolism, the spatiotemporal organization of subplasmalemmal Ca²⁺, Ca²⁺-dependent molecular translocation, and eNOS regulation. The precise mechanisms for the initiation and formation of subcortical Ca²⁺ waves require further investigation.

	Acknowledgments

 
This work was supported by grants from the Japan Heart Foundation/Pfizer Grant for Research on Hypertension and Vascular Metabolism, Takeda Science Foundation, the Research Grant of the Tokyo Hypertension Conference, and the Ministry of Education, Science and Culture Grant-in-Aid for Scientific Research (15590724, 1993).

	Footnotes

 
Original received February 26, 2004; revision received June 24, 2004; accepted June 29, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol. 2000; 1: 11–21.[CrossRef][Medline] [Order article via Infotrieve]

2. Isshiki M, Yin Y, Fujita T, Anderson RGW. A molecular sensor detects signal transduction from caveolae in living cells. J Biol Chem. 2002; 277: 43389–43398.[Abstract/Free Full Text]

3. Marsault R, Murgia M, Pozzan T, Rizzuto R. Domains of high Ca²⁺ beneath the plasma membrane of living A7r5 cells. EMBO J. 1997; 16: 1575–1581.[CrossRef][Medline] [Order article via Infotrieve]

4. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RG, Michel T. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem. 1996; 271: 6518–6522.[Abstract/Free Full Text]

5. Fagan KA, Smith KE, Cooper DM. Regulation of the Ca²⁺-inhibitable adenylyl cyclase type VI by capacitative Ca²⁺ entry requires localization in cholesterol-rich domains. J Biol Chem. 2000; 275: 26530–26537.[Abstract/Free Full Text]

6. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY. Fluorescent indicators for Ca²⁺ based on green fluorescent proteins and calmodulin. Nature. 1997; 388: 882–887.[CrossRef][Medline] [Order article via Infotrieve]

7. Moriyoshi K, Richards LJ, Akazawa C, O’Leary DD, Nakanishi S. Labeling neural cells using adenoviral gene transfer of membrane- targeted GFP. Neuron. 1996; 16: 255–260.[CrossRef][Medline] [Order article via Infotrieve]

8. Skene JH, Virag I. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J Cell Biol. 1989; 108: 613–624.[Abstract/Free Full Text]

9. Zacharias D, Violin J, Newton A, Tsien RY. Partitioning of Lipid-Modified Monomeric GFPs into membrane Microdomains of Living Cells. Science. 2002; 296: 913–916.[Abstract/Free Full Text]

10. Varnai P, Balla T. Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J Cell Biol. 1998; 143: 501–510.[Abstract/Free Full Text]

11. Majlof L, Forsgren PO. Confocal microscopy: important considerations for accurate imaging. In Matsumoto B, ed. Cell Biological Applications of Confocal Microscopy. San Diego, Calif: Academic Press; 1993.

12. Pawley JB. Handbook of Biological Confocal Microscopy (The Language of Science). New York: Kluwer Academic Publishers; 1995: 3.

13. Erickson MG, Moon DL, Yue DT. DsRed as a potential FRET partner with CFP and GFP. Biophys J. 2003; 85: 599–611.[Medline] [Order article via Infotrieve]

14. Miyawaki A, Griesbeck O, Heim R, Tsien RY. Dynamic and quantitative Ca²⁺ measurements using improved cameleons. Proc Natl Acad Sci U S A. 1999; 96: 2135–2140.[Abstract/Free Full Text]

15. Ma HT, Patterson RL, van Rossum DB, Birnbaumer L, Mikoshiba K, Gill DL. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca²⁺ channels. Science. 2000; 287: 1647–1651.[Abstract/Free Full Text]

16. Prakriya M, Lewis RS. Potentiation and inhibition of Ca²⁺ release-activated Ca²⁺ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP₃ receptors. J Physiol. 2001; 536: 3–19.[Abstract/Free Full Text]

17. Trebak M, Bird GS, McKay RR, Putney JW Jr. Comparison of human TRPC3 channels in receptor-activated and store-operated modes: differential sensitivity to channel blockers suggests fundamental differences in channel composition. J Biol Chem. 2002; 277: 21617–21623.[Abstract/Free Full Text]

18. Hirose K, Kadowaki S, Tanabe M, Takeshima H, Iino M. Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca²⁺ mobilization patterns. Science. 1999; 284: 1527–1530.[Abstract/Free Full Text]

19. Rhee SG, Bae YS. Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem. 1997; 272: 15045–15048.[Free Full Text]

20. Kendall DA, Nahorski SR. Inositol phospholipid hydrolysis in rat cerebral cortical slices, II: calcium requirement. J Neurochem. 1984; 42: 1388–1394.[CrossRef][Medline] [Order article via Infotrieve]

21. Okubo Y, Kakizawa S, Hirose K, Iino M. Visualization of IP₃ dynamics reveals a novel AMPA receptor-triggered IP₃ production pathway mediated by voltage-dependent Ca²⁺ influx in Purkinje cells. Neuron. 2001; 32: 113–122.[CrossRef][Medline] [Order article via Infotrieve]

22. Fu Y, Cheng JX, Hong SL. Characterization of cytosolic phospholipases C from porcine aortic endothelial cells. Thromb Res. 1994; 73: 405–417.[CrossRef][Medline] [Order article via Infotrieve]

23. Rebecchi MJ, Pentyala SN. Structure, function, and control of phosphoinositide-specific phospholipase C. Physiol Rev. 2000; 80: 1291–1335.[Abstract/Free Full Text]

24. Akhtar RA, Abdel-Latif AA. Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates. Biochem J. 1984; 224: 291–300.[Medline] [Order article via Infotrieve]

25. Allen V, Swigart P, Cheung R, Cockcroft S, Katan M. Regulation of inositol lipid-specific phospholipase c by changes in Ca²⁺ ion concentrations. Biochem J. 1997; 327: 545–552.[Medline] [Order article via Infotrieve]

26. Iino M. Biphasic Ca²⁺ dependence of inositol 1,4,5-trisphosphate-induced Ca²⁺ release in smooth muscle cells of the guinea pig taenia caeci. J Gen Physiol. 1990; 95: 1103–1122.[Abstract/Free Full Text]

27. Ohki G, Miyoshi T, Murata M, Ishibashi K, Imai M, Suzuki M. A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart. J Biol Chem. 2000; 275: 39055–39060.[Abstract/Free Full Text]

28. Putney JW Jr. Capacitative calcium entry revisited. Cell Calcium. 1990; 11: 611–624.[CrossRef][Medline] [Order article via Infotrieve]

29. Vaca L, Kunze DL. IP₃-activated Ca²⁺ channels in the plasma membrane of cultured vascular endothelial cells. Am J Physiol. 1995; 269: C733–C738.[Medline] [Order article via Infotrieve]

30. Kuno M, Gardner P. Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes. Nature. 1987; 326: 301–304.[CrossRef][Medline] [Order article via Infotrieve]

31. Khan AA, Steiner JP, Klein MG, Schneider MF, Snyder SH. IP₃ receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor. Science. 1992; 257: 815–818.[Abstract/Free Full Text]

32. Khan AA, Steiner JP, Snyder SH. Plasma membrane inositol 1,4,5-trisphosphate receptor of lymphocytes: selective enrichment in sialic acid and unique binding specificity. Proc Natl Acad Sci U S A. 1992; 89: 2849–2853.[Abstract/Free Full Text]

33. Feng L, Kraus-Friedmann N. Association of the hepatic IP₃ receptor with the plasma membrane: relevance to mode of action. Am J Physiol. 1993; 265: C1588–C1596.[Medline] [Order article via Infotrieve]

34. Mayrleitner M, Schafer R, Fleischer S. IP₃ receptor purified from liver plasma membrane is an (1,4,5)IP₃ activated and (1,3,4,5)IP₄ inhibited calcium permeable ion channel. Cell Calcium. 1995; 17: 141–153.[CrossRef][Medline] [Order article via Infotrieve]

35. Fujimoto T, Nakade S, Miyawaki A, Mikoshiba K, Ogawa K. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol. 1992; 119: 1507–1513.[Abstract/Free Full Text]

36. Okada Y, Teeter JH, Restrepo D. Inositol 1,4,5-trisphosphate-gated conductance in isolated rat olfactory neurons. J Neurophysiol. 1994; 71: 595–602.[Abstract/Free Full Text]

37. Isshiki M, Ando J, Korenaga R, Kogo H, Fujimoto T, Fujita T, Kamiya A. Endothelial Ca²⁺ waves preferentially originate at specific loci in caveolin-rich cell edges. Proc Natl Acad Sci U S A. 1998; 95: 5009–5014.[Abstract/Free Full Text]

38. Jaffe LF, Creton R. On the conservation of calcium wave speeds. Cell Calcium. 1998; 24: 1–8.[CrossRef][Medline] [Order article via Infotrieve]

39. Vuong PT, Malik AB, Nagpala PG, Lum H. Protein kinase C beta modulates thrombin-induced Ca²⁺ signaling and endothelial permeability increase. J Cell Physiol. 1998; 175: 379–387.[CrossRef][Medline] [Order article via Infotrieve]

40. Kuboki K, Jiang ZY, Takahara N, Ha SW, Igarashi M, Yamauchi T, Feener EP, Herbert TP, Rhodes CJ, King GL. Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo: a specific vascular action of insulin. Circulation. 2000; 101: 676–681.[Abstract/Free Full Text]

41. Hirabayashi T, Kume K, Hirose K, Yokomizo T, Iino M, Itoh H, Shimizu T. Critical duration of intracellular Ca²⁺ response required for continuous translocation and activation of cytosolic phospholipase A₂. J Biol Chem. 1999; 274: 5163–5169.[Abstract/Free Full Text]

42. Stahelin RV, Rafter JD, Das S, Cho W. The molecular basis of differential subcellular localization of C2 domains of protein kinase C-alpha and group IVa cytosolic phospholipase A₂. J Biol Chem. 2003; 278: 12452–12460.[Abstract/Free Full Text]

43. White DG, Martin W. Differential control and calcium-dependence of production of endothelium-derived relaxing factor and prostacyclin by pig aortic endothelial cells. Br J Pharmacol. 1989; 97: 683–690.[Medline] [Order article via Infotrieve]

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