Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153
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ABSTRACT |
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The subcellular spatial and temporal organization of
agonist-induced Ca2+ signals was
investigated in single cultured vascular endothelial cells.
Extracellular application of ATP initiated a rapid increase of
intracellular Ca2+ concentration
([Ca2+]i)
in peripheral cytoplasmic processes from where activation propagated as
a
[Ca2+]i
wave toward the central regions of the cell. The average propagation velocity of the
[Ca2+]i
wave in the peripheral processes was 20-60 µm/s, whereas in the
central region the wave propagated at <10 µm/s. The time course of
the recovery of
[Ca2+]i
depended on the cell geometry. In the peripheral processes (i.e.,
regions with a high surface-to-volume ratio)
[Ca2+]i
declined monotonically, whereas in the central region
[Ca2+]i
decreased in an oscillatory fashion. Propagating
[Ca2+]i
waves were preceded by small, highly localized
[Ca2+]i
transients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events of
Ca2+ release was 23 nM, and the
underlying flux of Ca2+ amounted
to ~1-2 × 1018
mol/s or ~0.3 pA, consistent with a
Ca2+ flux through a single or
small number of endoplasmic reticulum Ca2+-release channels.
confocal microscopy; fluo 3; endoplasmic reticulum; inositol trisphosphate
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INTRODUCTION |
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INTRACELLULAR CALCIUM is the most common intracellular signaling molecule that controls a wide array of cellular processes such as contraction, cell proliferation, and secretion (1, 2, 11). The regulation of many of these processes occurs through binding of Ca2+ to specific intracellular regulatory proteins. The vascular endothelium, for example, controls vascular tone through the release of nitric oxide, an endothelium-derived relaxing factor that is synthesized by the endothelial Ca2+/calmodulin-dependent nitric oxide synthase (5, 13).
Many of the regulatory actions of Ca2+ are highly localized within cellular subcompartments due to inhomogeneous distribution of Ca2+-containing compartments, Ca2+ binding proteins, and buffers, as well as Ca2+ channels and transport mechanisms. Recent advances in imaging techniques, especially through the use of confocal microscopy, have contributed significantly to our understanding of the subcellular Ca2+ signaling pathways in many cell types and tissues. In particular, the improved spatial resolution provided by these techniques has led to the characterization of elementary events of Ca2+ release in a variety of excitable and nonexcitable cell types (for review see Refs. 2 and 21). Such discrete events of Ca2+ release have been described for both classes of intracellular Ca2+-release channels, the ryanodine receptor (10, 17, 22, 33) and the inositol trisphosphate (IP3) receptor (28, 29, 34). A general concept is evolving that spatially restricted Ca2+ signals as well as complex temporal and spatial macroscopic patterns of intracellular Ca2+ signaling, such as intracellular Ca2+ concentration ([Ca2+]i) oscillations and [Ca2+]i waves, can be accounted for by precisely regulated recruitment of stereotypical elementary events of Ca2+ signaling, caused by the short opening of individual intracellular Ca2+-release channels to liberate a small pulse of Ca2+. The progressive recruitment of elementary events allows a graded, stimulus-dependent magnitude of the intracellular Ca2+ signal and forms the molecular basis for "quantal" Ca2+ release (26).
These elementary events of Ca2+ signaling appear to have a hierarchical organization that depends on the stimulus intensity that triggers them. At low levels of stimulation, localized [Ca2+]i transients result from the opening of a single (or a very small group) of release channels. These smallest [Ca2+]i signals have been characterized as Ca2+ "blips" for release events through the IP3 receptor (29) and "triadic Ca2+ transients" from ryanodine receptors in skeletal muscle (33). [For cardiac cells the concept of "Ca2+ quarks" as the Ca2+ signal resulting from the opening of an individual ryanodine receptor has been proposed (see Ref. 21); however, to this date the existence of such events has not been demonstrated.] At the next level of organization the concerted opening of small clusters of release channels leads to elementary events termed Ca2+ "sparks" for different muscle cell types (10, 17, 19, 22, 27) and Ca2+ "puffs" when occurring through the IP3 receptor (34). Global cellular Ca2+ signals such as [Ca2+]i waves and spatially homogeneous [Ca2+]i transients are due to the coordinated recruitment of a large number of elementary events (for a recent discussion of the hierarchical organization of cellular Ca2+ signaling events see Refs. 2 and 21). Whether the elementary events of Ca2+ signaling that have been visualized to date represent opening and closing of single Ca2+-release channels remains to be determined.
The goal of the present study was to investigate aspects of the spatiotemporal organization of ATP-induced [Ca2+]i transients in single cultured vascular endothelial cells. ATP is a physiological vasoactive agonist that causes endothelium-mediated vasodilation (e.g. see Ref. 13). ATP causes an increase of [Ca2+]i in vascular endothelial cells (15, 23, 25) by releasing Ca2+ from IP3-sensitive intracellular Ca2+ stores (endoplasmic reticulum). Using laser-scanning confocal fluorescence microscopy, we characterized ATP-induced [Ca2+]i waves in terms of initiation sites, rise time, propagation velocities, and spatiotemporal patterns of decline of [Ca2+]i. Furthermore, in this study we describe, for the first time in a nonexcitable cell type of the cardiovascular system (the vascular endothelium), the characteristics of elementary Ca2+-release events. These elementary Ca2+ signals reflect Ca2+ release from a single intracellular endoplasmic Ca2+-release channel or a very small group of channels.
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METHODS |
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Cultured vascular endothelial cells. Experiments were performed on single cultured CPAE vascular endothelial cells. The CPAE cell line was originally derived from bovine pulmonary artery endothelium and was purchased from American Type Culture Collection (Rockville, MD; CCL-209). The cells were cultured in Eagle's minimum essential medium supplemented with 20% fetal bovine serum (GIBCO, Grand Island, NY) and L-glutamine (2 mM) and were kept at 37°C in an atmosphere of 5% CO2-95% air. Once a week the cells were dispersed using a Ca2+-free (0.1% EDTA) 0.25% trypsin solution and were subcultured onto glass coverslips for later experimentation. Cells from passages 3-6 were used. Experiments were carried out within 1 wk after plating the cells onto coverslips. All experiments were performed at room temperature (20-22°C) on single cells in nonconfluent cultures.
Ca2+ measurements. Endothelial cells were loaded with Ca2+ indicator by exposure to 5 µM fluo 3-acetoxymethyl ester (fluo 3-AM; Molecular Probes, Eugene, OR) for 15-20 min at 20°C. The cells were subsequently washed for 20 min in extracellular solution to allow sufficient time for deesterification. For fluorescence measurements, a coverslip with cells was mounted on the stage of an inverted microscope (Axiovert 100; Carl Zeiss) equipped with a ×40 objective (Plan-Neofluar, oil, numerical aperture = 1.3; Carl Zeiss). The microscope was attached to a confocal laser-scanning unit (LSM 410; Carl Zeiss). Fluo 3 fluorescence was excited with the 488-nm line of an argon ion laser. Emitted fluo 3 fluorescence was measured at wavelengths >515 nm.
[Ca2+]i images were calculated according to the formula (10) [Ca2+]i = KDR /(KD /[Ca2+]restCa2+-release flux calculations. Ca2+-release fluxes, as a function of time and space, underlying localized nonpropagating [Ca2+]i transients such as Ca2+ blips and puffs were derived from the amplitude of the Ca2+ signals. Estimations of release flux from the fluo 3 fluorescence signal were based on methods developed previously (4, 33) and were extrapolated from signals measured in skeletal and cardiac muscle, using the same indicator dye, optics, and confocal imaging system.
Solutions and chemicals. The cells were superfused continuously with a physiological salt solution (standard Tyrode solution) composed of (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid titrated to pH 7.3 with NaOH. In the nominally Ca2+-free Tyrode solution CaCl2 was omitted. Stock concentrations of ATP (Sigma Chemical, St. Louis, MO) were dissolved in distilled water. ![]() |
RESULTS |
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Spatial and temporal organization of global [Ca2+]i transients induced by extracellular ATP. The vasoactive agonist ATP has been shown to release Ca2+ from intracellular Ca2+ stores, i.e., the endoplasmic reticulum, through activation of the inositol phosphate signaling cascade (6, 15, 23-25) linked to P2Y and P2U purinergic surface membrane receptors (23). Figure 1, A-D, shows that activation of a single endothelial cell by superfusion of ATP (250 nM) caused a spatially inhomogeneous increase of [Ca2+]i. In this experiment agonist-induced changes of [Ca2+]i were recorded in the line scan mode of the confocal microscope. A vascular endothelial cell loaded with the fluorescent Ca2+ indicator fluo 3 was scanned repetitively (10 Hz) along the line shown in Fig. 1A. The resulting line scan [Ca2+]i image (Fig. 1B, with time running from left to right) revealed that [Ca2+]i started to rise at two distinct sites (a and d) located in the peripheral fine cytoplasmic processes of the cell. From these sites of initial release of Ca2+, a wave of elevated [Ca2+]i propagated toward more central regions of the cell. Figure 1B, right, shows that the velocity of [Ca2+]i wave propagation was not constant and varied depending on the cell region. In peripheral processes, at the sites of wave initiation, the propagation velocity was highest. In this experiment, at sites 1 and 2 in the fine processes, the velocities (averaged over the distances indicated by the gray bars) were 61 and 24 µm/s, respectively. As the wave of elevated [Ca2+]i advanced toward more central regions of the cell, propagation velocity decreased and was found to be lowest in the nuclear region (site 4, 7 µm/s).
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Elementary events of Ca2+ release from endoplasmic reticulum. High-resolution confocal imaging was used to further investigate the initiation sites of propagating [Ca2+]i waves. In the example shown in Fig. 2Ba, three sites of wave initiation after exposure to ATP (250 nM) could be distinguished. At each initiation site the massive rise of [Ca2+]i was preceded by one or very few localized, nonpropagating [Ca2+]i transients, reminiscent of Ca2+ blips or small Ca2+ puffs (2). These localized transient elevations of [Ca2+]i revealed a spatial spread of 1-3 µm and duration of typically <100 ms (duration at half amplitude). The average amplitude of these local events was 23 nM (see Fig. 3), although the events immediately preceding a propagating [Ca2+]i wave sometimes were larger in amplitude (50-100 nM), possibly due to summation of a small number of elementary events occurring in close proximity. As illustrated by the line profiles in Fig. 2Bb, these events could occur with a clearly resolvable time gap before the onset of the [Ca2+]i wave (e.g., top trace). However, in many instances, these events tended to fuse partially with the rising phase of the [Ca2+]i wave, forming a footlike elevation of [Ca2+]i before the onset of the wave. Figure 2Bb (top of inset) shows the profile of a localized [Ca2+]i transient (red) superimposed on the change of baseline [Ca2+]i (black) that was recorded from a region a few micrometers away. The bottom trace is the result of subtracting baseline [Ca2+]i from the localized [Ca2+]i transient, revealing a [Ca2+]i transient of amplitude and time course resembling a Ca2+ blip or a small Ca2+ puff. Figure 2Bc shows a three-dimensional view of the initiation sites and the localized [Ca2+]i transients preceding the propagating [Ca2+]i wave.
Figure 2C illustrates an example of elementary events of Ca2+ release occurring in the maintained presence of ATP. As shown by the two-dimensional (Fig. 2Ca) as well as the three-dimensional (Fig. 2Cc) representation of the line scan image, localized nonpropagating [Ca2+]i transients occurred in temporal isolation (blips), or several transients appeared in short succession at different sites in close proximity. Together these local release transients gave rise to an elevation of [Ca2+]i of wider spatial spread and longer duration due to spatial and temporal summation, reminiscent of Ca2+ puffs. Nevertheless, the rise of [Ca2+]i remained localized, only spreading over a distance of ~30 µm, and failed to propagate as a [Ca2+]i wave. The [Ca2+]i profiles in Fig. 2Cb show sites where individual Ca2+ blips occurred (e.g., bottom trace). Detailed analysis of the Ca2+ puff revealed distinct steplike increases of [Ca2+]i of ~10 nM amplitude (inset), providing evidence of the quantal nature of Ca2+ release and supporting the hypothesis that Ca2+ puffs represent the temporal and spatial summation of individual elementary Ca2+-release events, i.e., Ca2+ blips.Quantification of elementary
Ca2+-release
events.
Figure 3 shows an amplitude histogram of
elementary Ca2+-release events
observed in endothelial cells following stimulation with ATP. On
average the localized nonpropagating elementary
[Ca2+]i
transients had an amplitude of 0.29, expressed as the ratio of fluo 3 fluorescence increase over baseline fluorescence
(F/F0). With the use of the calibration
parameters outlined in METHODS, a
F/F0 amplitude of 0.29 corresponds to an average rise of
[Ca2+]i
of 23 nM. The smallest events recorded showed
F/F0 amplitudes of
0.15,
corresponding to
[Ca2+]i
of ~10 nM. The amplitudes of the smallest events encountered are
consistent with the size of the steplike increases of
[Ca2+]i
observed during a Ca2+ puff, as
shown in Fig. 2Cb,
inset. Based on methods of
Ca2+-release flux calculations
developed for Ca2+ release in
muscle (4, 33), we extrapolated a peak
Ca2+-release flux, in intensive
units, of ~0.5-1 mM/s. Integrating the intensive flux over the
associated volume yields a flux of Ca2+ of ~1-2 × 10
18 mol/s or 0.2-0.4
pA. The same calculation would result in a flux of ~0.1 pA for the
smallest events observed. On the basis of estimates of single-channel
conductances for the IP3 receptor
(3, 28, 32), these numbers are consistent with the release of
Ca2+ from a single or a very small
number of release channels.
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DISCUSSION |
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In the present study we investigated the spatial and temporal organization of ATP-induced [Ca2+]i transients in single vascular endothelial cells. The vasoactive agonist ATP activates the inositol phosphate signaling cascade and triggers release of Ca2+ from the endoplasmic reticulum by activation of the IP3-sensitive release channel. We characterized, for the first time in nonexcitable cells of the cardiovascular system, the elementary events of agonist-induced Ca2+ release from the endoplasmic reticulum.
The elementary release events that depend on the activation of the
inositol phosphate pathway have been termed
Ca2+ blips (29). From the limited
spatial spread of Ca2+ blips (a
few µm), their amplitude (several tens of nM), and duration (100 ms
or less), as well as from quantitative estimates of the amount of
Ca2+ that is released during a
Ca2+ blip, it has been suggested
that Ca2+ blips may be the result
of a short opening of a single IP3
receptor (29), although the ultimate confirmation of the single-channel nature of Ca2+ blips is still
missing. We have addressed this fundamental question by estimating
quantitatively the flux of Ca2+
underlying the elementary events of
Ca2+ release observed in vascular
endothelial cells and by comparing our results with available data on
conductance properties of the IP3
receptor. Based on methods we developed for
Ca2+ flux measurements underlying
elementary events of Ca2+ release
in skeletal (33) and cardiac muscle cells (4), we estimated that the
peak release flux during a Ca2+
blip of average amplitude (F/F0 = 0.3 or 23 nM, respectively) was on the order of 0.5-1 mM/s.
Integration of the flux over the associated volume yielded an average
flux of Ca2+ of ~1-2 × 10
18 mol/s or
0.2-0.4 pA. The smallest events that could reliably be resolved in
our experiments had an amplitude of ~10 nM. In this case a
Ca2+ flux of ~0.1 pA was
estimated. This number is somewhat smaller than that estimated for
Ca2+ blips observed in HeLa cells
(9) but is similar to the estimates reached for oocytes (29).
Furthermore, the Ca2+ flux we
calculated for the Ca2+ blips in
endothelial cells are consistent with estimates for the unitary
Ca2+ current through the
IP3 receptor channel (3, 32).
Additional observations support the possibility that the smallest
Ca2+ signals might reflect a
single-channel event. The smallest
Ca2+ blips observed in endothelial
cells are two to three times smaller in amplitude than the average
elementary Ca2+-release events
occurring through a single ryanodine receptor at the triadic junction
in skeletal muscle (33). The notion that the single-channel conductance
of the IP3 receptor is similar to
(albeit smaller than) the conductance of the ryanodine receptor (12) is
consistent with the single-channel origin of
Ca2+ blips. Further support for
the hypothesis that Ca2+ blips
reflect single-channel events stems from the analysis of the frequency
distribution of the amplitudes of elementary
Ca2+-release events. Because the
amplitude of elementary release events is directly related to the open
time of the release channel, one would expect an exponential
distribution of open times, and therefore blip amplitudes, due to the
stochastic nature of channel gating (14), with a larger number of
low-amplitude events and progressively fewer blips of larger magnitude.
The amplitude frequency histogram of
Ca2+ blips observed in our study
(Fig. 3) is clearly asymmetrical and skewed to the right, with a larger
number of events with small amplitudes. The histogram resembles more
closely an exponential distribution of blip amplitudes. Taken together,
we suggest that the smallest events of
Ca2+ release observed in this
study (i.e., Ca2+ blips) reflect
the opening of a single endoplasmic reticulum Ca2+-release channel.
The next level higher in the hierarchy of IP3-dependent Ca2+-signaling events has been termed Ca2+ puff (34). Individual Ca2+ puffs remain local in nature but represent Ca2+-release events of larger spatial spread, duration, and amplitude. More importantly, puffs are the result of temporal summation and spatial recruitment of elementary Ca2+ blips. Our study provides direct experimental evidence for this concept. Figure 2C, for example, shows a localized increase of [Ca2+]i with several local Ca2+ peaks that occur in short temporal succession at different yet closely spaced sites. These individual Ca2+ peaks are wider and reach higher amplitudes (50-100 nM) than individual Ca2+ blips and are reminiscent of Ca2+ puffs observed in oocytes (34) and HeLa cells (9). Furthermore, the [Ca2+]i profiles recorded from different regions of Ca2+ puffs (e.g., Fig. 2Cb, inset) revealed discrete elementary steps of increasing [Ca2+]i of ~10 nM (indicated by the dashed lines). A step increase of [Ca2+]i by 10 nM is consistent with the recruitment of a single Ca2+ blip (or opening of a single release channel). The observation of superposition and local summation of discrete events of identical amplitude is consistent with an incremental release of Ca2+ by small but rather stereotypical amounts and lends support to the model of a graded, thus quantal nature of IP3-mediated release of Ca2+ (26).
Many times the occurrence of Ca2+ puffs correlated with the initiation site of propagating [Ca2+]i waves (Fig. 2B). Ca2+ puffs could precede propagating [Ca2+]i waves by several hundred milliseconds (Fig. 2Bb, top trace). In other instances Ca2+ puffs were observed much closer in time to the onset of the wave. In these cases [Ca2+]i did not completely decrease to baseline levels and tended to fuse in time with the propagating wave (e.g., Fig. 2Bb, inset).
Initiation sites for [Ca2+]i waves were not randomly distributed throughout the cell. Nonconfluent cultured CPAE cells typically developed long cytoplasmic processes of small diameter (see also Ref. 25). ATP-induced propagating [Ca2+]i waves were typically initiated in these thin peripheral processes. The propagation velocity of ATP-induced [Ca2+]i waves was not constant and was influenced by the cell morphology. The highest propagation velocity was found at the wave initiation site, where the wave front propagated toward central regions at velocities of 30-60 µm/s. In more central regions the propagation velocity decreased. The slowest rates of increase of [Ca2+] were found in the nuclear regions (<10 µm/s), consistent with largely diffusional Ca2+ movements and the lack of Ca2+-release units in the nucleoplasm. The propagation velocities found in our study are within the range found in many different cell preparations and tissues (18) and are consistent with a regenerative process of Ca2+ release involving a positive-feedback mechanism. The peripheral processes represent cell regions that are characterized by a high ratio of cell surface area to cell volume (S/V ratio). The most likely explanation for the preferential location of wave initiation sites as well as the high propagation velocity encountered in these regions is that, on stimulation by agonist, the cytoplasmic IP3 concentration rapidly reaches a level that triggers Ca2+ release. The rapid rise of IP3 levels in the fine cytoplasmic processes together with the positive feedback of Ca2+ on the IP3-induced release of Ca2+ (3, 12) leads to an amplification of the release process from neighboring release sites and subsequently to a rapid propagation of release throughout the restricted volume of the cytoplasmic processes. The process of wave propagation is slowed at sites of volume expansion, i.e., at the transition from the narrow peripheral process to the larger central region. This observation reflects a basic feature of propagated reaction-diffusion waves and their modulation by the geometry of the "excitable medium." Generally, in compartments of larger volumes, it takes longer for a given flux of the propagator (Ca2+) to reach the threshold for initiation of the amplifying reaction (Ca2+ release). Although the cell geometry with marked regional differences in the S/V ratio represents the most likely explanation for the observation that propagating [Ca2+]i waves are initiated preferentially in the peripheral processes, other possibilities have to be considered. Missiaen et al. (25), who made similar observations about the spatial organization of ATP-induced [Ca2+]i waves in CPAE cells, suggested that, in addition to regional differences in S/V ratio, the IP3-sensitive Ca2+-release units in the peripheral processes might exhibit a higher agonist sensitivity.
Cell morphology also determined the temporal pattern of recovery of the ATP-induced [Ca2+]i transient (Fig. 1C). After rapidly reaching a peak, [Ca2+]i decayed monotonically to near-baseline levels in the narrow peripheral cytoplasmic processes, whereas in the nuclear and perinuclear regions the overall decline of [Ca2+]i was superimposed by oscillatory changes of [Ca2+]i, presumably due to periodic reactivation of intracellular Ca2+ release. On the basis of a two-dimensional stochastic model of [Ca2+]i oscillations (20), it has been predicted that the cell morphology, specifically the S/V ratio and the presence of the cell nucleus, critically influences temporal and spatial organization of [Ca2+]i oscillations and [Ca2+]i waves. The model predicts that a low S/V ratio and a large nuclear compartment favor the occurrence of oscillations and correlate with an increased oscillation frequency. The nucleoplasm lacks functional Ca2+ stores and the nuclear envelope possibly forms a partial diffusion barrier for cytosolic Ca2+. Furthermore, the diffusional properties of the nuclear envelope appear to be actively regulated and to depend on the filling status of intracellular Ca2+ stores (30). Additional mechanisms for active regulation of nuclear Ca2+ may contribute to the complex inhomogeneities of [Ca2+]i distribution (31). Nevertheless, the above-mentioned model predicts that, in the nucleus, the rate of rise of [Ca2+]i elicited by agonist stimulation is slower and the increase in [Ca2+] lags behind the rise of cytoplasmic [Ca2+] in terms of both time and amplitude. Our experimental results are consistent with these model predictions. We found that 1) the rise of [Ca2+] in the nuclear compartment was slower and lagged behind the [Ca2+]i elevation in the peripheral processes (Fig. 1D); 2) the largest [Ca2+] transient amplitudes were observed in peripheral cytoplasmic regions (Fig. 1B), whereas the nuclear compartment tended to show smaller [Ca2+] elevations (e.g., Fig. 2A); and 3) [Ca2+]i oscillations were found in regions with a low S/V ratio (nuclear and perinuclear region), whereas in the peripheral processes (high S/V ratio) single-spiked [Ca2+]i transients occurred.
In conclusion, with this study we have presented further experimental evidence for the hierarchical organization of intracellular Ca2+ signaling. The characterization of elementary Ca2+-release events in different cell types (see Ref. 2), including Ca2+ blips in vascular endothelial cells, has provided new insights into the way intracellular Ca2+ channels operate in situ and has opened the tantalizing prospect that complex patterns of cellular Ca2+ signaling can be accounted for by the spatially and temporally highly organized recruitment and summation of relatively stereotypical Ca2+-release events. Although the currently available data on Ca2+ blips do not allow unequivocal conclusions on the single-channel nature of these events, they provide strong support for the graded, or quantal nature of IP3-mediated release of Ca2+ from intracellular stores.
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ACKNOWLEDGEMENTS |
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We thank Christine E. Rechenmacher for expert technical help and Drs. Andrey Klishin, Stephen L. Lipsius, Marina Sedova, and Jaclyn R. Holda for critical comments on the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-51941 and by grants from the American Heart Association National Center and the Schweppe Foundation Chicago.
L. A. Blatter is an Established Investigator of the American Heart Association. J. Hüser is a postdoctoral fellow of the Deutsche Forschungsgemeinschaft.
An initial account of this work was presented in abstract form (16).
Address for reprint requests: L. A. Blatter, Dept. of Physiology, Loyola University Chicago, 2160 S. First Ave., Maywood, IL 60153.
Received 24 June 1997; accepted in final form 25 August 1997.
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