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RECEPTORS AND SIGNAL TRANSDUCTION

Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca²⁺ signaling in bovine vascular endothelial cells

Department of Physiology, Loyola University Chicago, Maywood, Illinois

Submitted 23 October 2006 ; accepted in final form 28 February 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The role of glycolytically generated ATP in Ca²⁺/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca²⁺ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca²⁺ concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + -hydroxybutyrate (-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca²⁺]_i. The rise of [Ca²⁺]_i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca²⁺]_i. In the absence of extracellular Ca²⁺ 2-DG caused an increase in [Ca²⁺]_i, suggesting that inhibition of glycolysis directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. The inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca²⁺ response. Ca²⁺ release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca²⁺]_i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca²⁺]_i. Propagating [Ca²⁺]_i waves were preceded by [Ca²⁺]_i oscillations and small, highly localized elevations of [Ca²⁺]_i (Ca²⁺ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca²⁺ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca²⁺ release was attenuated. Similar results were obtained with pyr + -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg²⁺ concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca²⁺ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP₃R.

Ca²⁺/calmodulin-dependent kinase II; glycolysis; calcium regulation

Rather than regulation of IP₃-dependent Ca²⁺ release by direct binding of Ca²⁺/CaM to the channel (61), protein kinase-dependent phosphorylation of the IP₃R provides an additional mechanism for its functional regulation (1, 3, 5, 18, 19, 39–41, 85): the IP₃R can be phosphorylated by protein kinase A (PKA), protein kinase C (PKC), Ca²⁺/CaM-dependent protein kinase II (CaMKII) as well as by nitric oxide/cyclic GMP-dependent protein kinase (PKG), although the functional consequences are unclear.

Recent reports (1, 5) have shown that CaMKII inhibits the IP₃R and the regulation appears to involve a phosphorylation process. However, the precise mechanisms underlying this regulation are largely unknown. In cardiac and vascular smooth muscle cells, the amount of glycolytically generated ATP is significantly lower than the ATP produced by mitochondrial metabolism (32). Despite this relationship glycolysis has been shown to be the preferential ATP source for modulation of membrane functions such as ion transport in cardiac and vascular smooth muscle cells (13, 35, 36, 42, 68, 79). The tight functional coupling between glycolysis and membrane functions is thought to be due to the close association between glycolytic enzymes, ER/sarcoplasmic reticulum (SR) membranes (24, 62, 72), several kinases including CaMKII (72), and ion transport pathways such as the sarcolemmal ATP-sensitive K⁺ (K_ATP) channel (80, 81), the Na⁺-K⁺ pump (22), as well as the ER/SR Ca²⁺ pump (35, 83). Thus, by controlling local ATP production in the microenvironment of surface membrane ion transporters, ryanodine receptor (RyR), IP₃R, and the ER/SR Ca²⁺ pump, glycolytically generated ATP may act as a key modulator of Ca²⁺ homeostasis.

In the present study we have examined the effects of the interplay between glycolysis and CaMKII on [Ca²⁺]_i, using a number of different pharmacological blockers. The data suggest a central role for glycolytically generated ATP in the inhibition of IP₃R-dependent ER Ca²⁺ release by CaMKII in CPAE cells. Previous accounts of this work have been presented in abstract form (2, 4).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell Culture

All experiments were carried out on single cultured CPAE cells. The CPAE cell line was obtained as passage 15 from American Type Culture Collection (ATCC, CCL-209, Manassas, VA). Cells were maintained in Eagle's minimum essential medium, which was supplemented with 20% fetal bovine serum (GIBCO, Grand Island, NY) and 2 mM L-glutamine, at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Once a week cells were dispersed with a Ca²⁺-free (0.1% EDTA) 0.25% trypsin solution and plated onto glass coverslips for later experimentation. Cells were passaged up to six times after they were obtained from ATCC. All experiments were carried out at room temperature (20–22°C).

Measurements of Intracellular Ca²⁺ and Mg²⁺ Concentrations

[Ca²⁺]_i measurements with indo-1. Spatially averaged [Ca²⁺]_i measurements from single CPAE cells were performed with the ratiometric Ca²⁺ indicator indo-1. Cells were loaded by exposure to 1 ml of standard Tyrode solution containing 5 µM indo-1 acetoxymethyl ester (indo-1 AM; Molecular Probes/Invitrogen, Carlsbad, CA) and 5 µl of a Pluronic F-127 stock solution [0.2 g/ml Pluronic F-127 dissolved in dimethyl sulfoxide (DMSO)] for 20 min at room temperature. [Ca²⁺]_i was measured by exciting indo-1 fluorescence with light of 360-nm wavelength and measuring emitted fluorescence signals simultaneously at 405 nm (F₄₀₅) and 485 nm (F₄₈₅). Single-cell fluorescence signals were recorded with photomultiplier tubes (model no. R2693; Hamamatsu, Bridgewater, NJ), and changes in [Ca²⁺]_i are expressed as the ratio R = F₄₀₅/F₄₈₅.

[Ca²⁺]_i measurements with fluo-3. CPAE cells were loaded with the Ca²⁺ indicator by exposure to 5 µM fluo-3 AM (Molecular Probes/Invitrogen) for 20 min at 20°C. For [Ca²⁺]_i measurements, a coverslip with cells was mounted on the stage of an inverted microscope (Eclipse TE 300; Nikon) equipped with a x40 objective (Plan Fluor, oil, numerical aperture = 1.3; Nikon). The microscope was attached to a confocal scanning unit (Radiance 2000/MP; Bio-Rad). With the confocal aperture chosen, the axial resolution was 1 µm at full-width half-maximum (point-spread function determined experimentally). The average pixel size used was 0.25 µm. 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. Changes in [Ca²⁺]_i are reported as the fluo-3 ratio F/F₀, where F is fluorescence intensity and F₀ is the fluo-3 fluorescence under basal conditions recorded under steady-state conditions at the beginning of an experiment.

Intracellular free Mg²⁺ concentration measurements. Intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured with the fluorescent Mg²⁺ indicator mag-fluo-4 AM (20 µM; Molecular Probes/Invitrogen). Loading procedure and recording conditions were the same as for fluo-3. Cells were pretreated with 10 µM thapsigargin (Tg), and measurements were conducted in the absence of extracellular Ca²⁺ to avoid interference from changes in [Ca²⁺]_i with the mag-fluo-4 signal.

Solutions and Chemicals

Cells were superfused continuously with normal Tyrode (NT) solution that contained (mM) 135 NaCl; 4 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 10 HEPES, and the pH was adjusted to 7.3 with NaOH. In Ca²⁺-free Tyrode solution CaCl₂ was omitted from NT solution (i.e., nominally Ca²⁺-free solution). ATP (Na salt) was obtained from Sigma-Aldrich (St. Louis, MO). KN-93 was obtained from Calbiochem (San Diego, CA). W-7 was purchased from Alexis (San Diego, CA). ATP and KN-93 were dissolved in distilled water. W-7 (10 mM) was dissolved in DMSO and diluted in the superfusion solution to the final concentration of 50 µM. 2-aminoethoxydiphenyl borate (2-APB) was obtained from Sigma-Aldrich.

Glycolytic Inhibitors

Glycolysis was inhibited with NT 1) in which glucose was replaced with 10 mM 2-deoxy-D-glucose (2-DG), 2) in which glucose was replaced by the combination of 10 mM pyruvate (pyr, Na salt) and 10 mM -hydroxybutyrate (-HB, Na salt; in this solution NaCl was reduced to 125 mM to keep osmolarity constant), or 3) containing 1 mM iodoacetic acid (IAA, Na salt). All inhibitors were from Sigma-Aldrich. Focal application of glycolytic inhibitors was performed by pressure ejection from a glass pipette (1.5-µm tip diameter) positioned close to the cell membrane of a cytoplasmic peripheral process. The bulk solution flow in the tissue bath was directed to restrict exposure to the inhibitors to only a peripheral section of an individual cell process. Control experiments using NT as the ejected fluid revealed that the ejection procedure by itself failed to elicit any changes of [Ca²⁺]_i.

Data Analysis

All data are reported as means ± SE obtained from n different cells. Each experiment was performed on a separate coverslip. Statistical differences among the data were determined with the Student's t-test for paired and unpaired data and considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Effects of Extracellular ATP and Inhibitors of Glycolysis on [Ca²⁺]_i

Figure 1 Aa shows an example of a [Ca²⁺]_i transient evoked by bath application of 5 µM ATP_e, a well-established mobilizer of Ca²⁺ in CPAE cells, acting via P_2y and P_2u receptors to generate IP₃ (1, 4, 46) and to release Ca²⁺ from IP₃R-controlled Ca²⁺ stores. In the presence of 2 mM extracellular Ca²⁺ the [Ca²⁺]_i transient was characterized by a rapid initial increase in [Ca²⁺]_i, which is predominantly due to ER Ca²⁺ release through the IP₃R, and a sustained component of elevated Ca²⁺ due to Ca²⁺ entry across the plasma membrane through CCE (1, 29). As reported previously (1) the average [Ca²⁺]_i transient amplitude elicited with extracellular ATP was R = 1.67 ± 0.06 (n = 50; R = R_peak – R_basal).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Effects of extracellular adenosine triphosphate (ATPe) and glycolytic inhibitors on intracellular Ca2+ concentration ([Ca2+]i). A: control response to ATPe (5 µM) and to glycolytic inhibitors in 2 mM Ca2+-containing Tyrode solution. The Ca2+ response to ATPe (a) was characterized by a rapid increase of [Ca2+]i that reached a peak with a rise time (10–90%) of 3 s and then declined to a sustained plateau of elevated [Ca2+]i. Superfusion of cells with the glycolytic inhibitors 2-deoxy-D-glucose (2-DG, b), pyruvate (pyr) + -hydroxybutyrate (-HB) (c), or iodoacetic acid (IAA, d) also induced an increase in [Ca2+]i that reached a maximum within <30 s, but almost completely lacked the sustained phase [extracellular Ca2+ concentration ([Ca2+]o) = 2 mM]. B: [Ca2+]i transient elicited with 2-DG in Ca2+-free external solution. R = ratio of fluorescence at 405 nm to fluorescence at 485 nm.

The second protocol involved selectively interrupting glycolysis without affecting mitochondrial metabolism. This was done by exchanging glucose with the combination of pyr + -HB. With pyr + -HB as metabolic fuels the flux through glycolysis is markedly inhibited. This inhibitory effect is thought to be caused by increased intracellular concentrations of citrate, ATP, and intermediates of the tricarboxylic acid cycle (55). The third glycolytic blocker used was IAA, which inhibits glyceraldehyde-3-phosphate dehydrogenase. Similar to 2-DG, exposure of cells to pyr (10 mM) + -HB (10 mM) evoked two types of responses. In about one-half of the cells (9/16) the response was small in amplitude (R < 0.3). In contrast, in a second population of cells (7/16) the response was much more pronounced (Fig. 1Ac) and amounted on average to R = 1.05 ± 0.24 (63 ± 14% of the ATP response). The average [Ca²⁺]_i transient amplitude evoked with the glycolytic inhibitor IAA (Fig. 1Ad) was R = 2.25 ± 0.22 (135 ± 13% of ATP response; n = 7). In summary, the results presented here indicate that inhibition of glycolysis can induce an increase of [Ca²⁺]_i in CPAE cells.

To examine whether the increase of [Ca²⁺]_i observed during glycolytic inhibition was due to Ca²⁺ influx or to release of Ca²⁺ from an intracellular Ca²⁺ store, 2-DG was applied to CPAE cells in the absence of extracellular Ca²⁺. Figure 1B shows that bath application of 2-DG (10 mM) in Ca²⁺-free external solution was capable of evoking a [Ca²⁺]_i transient. The 2-DG-induced [Ca²⁺]_i transient was characterized by a rapid and pronounced initial rise in [Ca²⁺]_i that then decayed completely to the prestimulatory level, even in the maintained presence of 2-DG. The average amplitude induced by 2-DG in the absence of extracellular Ca²⁺ was R = 1.18 ± 0.01 (n = 18) and amounts to 84% of the response evoked with ATP_e in the absence of extracellular Ca²⁺, as reported previously (1). This finding indicates that the observed increase in [Ca²⁺]_i is not directly dependent on Ca²⁺ entry and suggests that glycolytic inhibitors are capable of releasing Ca²⁺ from an intracellular Ca²⁺ store. The following experiments were designed 1) to explore further the spatiotemporal organization of ER Ca²⁺ release induced by inhibition of glycolysis and 2) to test the hypothesis that glycolysis is an important contributor to the regulation of the IP₃-sensitive Ca²⁺ release mechanism.

Spatiotemporal Properties of 2-DG-Induced [Ca²⁺]_i Transients

The data presented in Fig. 1B clearly indicated that the glycolytic inhibitor 2-DG can cause substantial release of Ca²⁺ from intracellular stores. Using confocal microscopy, we characterized the spatiotemporal properties of the 2-DG-induced Ca²⁺ response and compared it with the response to stimulation with the vasoactive agonist ATP. Figure 2A illustrates the spatial and temporal organization of the Ca²⁺ response to stimulation with ATP_e. In this experiment a single CPAE cell loaded with fluo-3 was stimulated with ATP_e in the absence of extracellular Ca²⁺. Figure 2A shows a series of two-dimensional confocal [Ca²⁺]_i images, recorded at the indicated times (in s) after switching to a superfusion solution containing ATP (1 µM). The Ca²⁺ response to ATP_e stimulation was spatially inhomogeneous. ATP_e consistently caused an increase of [Ca²⁺]_i first in the cell periphery (preferentially in the peripheral cellular processes) from where the [Ca²⁺]_i elevation propagated in a wavelike fashion toward the cell center. In the example shown, the first localized increase of [Ca²⁺]_i was detected in the narrow cytoplasmic process (at time t = 3 s; location a), followed by a localized increase of [Ca²⁺]_i at a second distant site (t = 3.4 s; site b). These two initiation sites (4.7 s) together eventually gave rise to a [Ca²⁺]_i wave that propagated from both ends toward the central region of the cell (t = 6.8 s). This ATP_e-induced Ca²⁺ response is known to be mediated by IP₃ generation (1, 30) and subsequent release of Ca²⁺ from intracellular Ca²⁺ stores. Thus the data confirmed prior observations that ATP_e causes spatially inhomogeneous increase of [Ca²⁺]_i in CPAE cells (30).

View larger version (41K): [in this window] [in a new window]   Fig. 2. Effects of ATPe and global inhibition of glycolysis on the spatiotemporal pattern of [Ca2+]i in Ca2+-free external solution. A: series of 2-dimensional confocal [Ca2+]i images recorded at the indicated times (in s) after switching to a superfusion solution containing 1 µM ATPe ([Ca2+]o = 0). A rise of [Ca2+]i was initiated in the peripheral cytoplasmic process of the cell and subsequently propagated as a [Ca2+]i wave toward central regions of the cell. Superfusion of cells with Ca2+-free extracellular solution in which glucose was replaced with either 2-DG (B) or pyr + -HB (C) also induced an increase in [Ca2+]i with the same general spatiotemporal properties as seen with ATPe. The pseudocolor representations at bottom of images indicate changes in [Ca2+]i expressed in arbitrary fluorescence units (a.u.). Arrows indicate initiation sites of [Ca2+]i transients.

Focal Inhibition of Glycolysis Triggers Subcellularly Restricted Ca²⁺ Release

The results presented in Fig. 2 revealed substantial regional differences in the spatiotemporal characteristics of the Ca²⁺ response to glycolytic inhibition, suggesting that the processes linking glycolysis to ER Ca²⁺ release might be spatially restricted. To test this hypothesis further we focally applied 2-DG, pyr + -HB, or IAA through a pipette to the peripheral end of a narrow cytoplasmic process while the remaining part was superfused with glucose-containing Ca²⁺-free Tyrode solution (arrows in Fig. 3 indicate bulk flow), thus preventing the rest of the cell from being exposed to the inhibitors (see also Refs. 31, 36). Figure 3, left, depicts the outline of the cells redrawn from confocal images of single CPAE cells and the position of the pipette tip. Under these conditions, pressure ejection of 2-DG-containing pipette solution (Fig. 3A) resulted in spatially restricted application to only the peripheral cytoplasmic process. Figure 3A further shows a time series of two-dimensional [Ca²⁺]_i images recorded at the indicated intervals after focal application of 2-DG. 2-DG rapidly initiated Ca²⁺ release at the site of stimulation (first detectable at t = 2.2 s). Ca²⁺ release subsequently extended over a distance of 40 µm (cf. t = 3 s) along the nonstimulated segment of the peripheral process but tapered off in amplitude and failed to extend further than 80 µm (t = 5.2 s) and to propagate into the cell body. In <20 s [Ca²⁺]_i had returned to basal levels. The effects of focal application of pyr (10 mM) + -HB (10 mM) (Fig. 3B) or IAA (1 mM) (Fig. 3C) were similar to 2-DG in terms of kinetics and spatial progression of the response. Thus the data indicate that focal application of glycolytic inhibitors evoked a local, spatially restricted subcellular elevation of [Ca²⁺]_i.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Effect of focal inhibition of glycolysis on the spatiotemporal pattern of [Ca2+]i. Cells were continuously superfused with glucose-containing Ca2+-free Tyrode solution (bulk flow, arrows on left). Focal application of 2-DG (10 mM, A), pyr (10 mM) + -HB (10 mM) (B), or IAA (1 mM, C) through a pipette positioned close to the cell membrane of the cytoplasmic process of the cell induced local elevations of [Ca2+]i near the pipette tip that then propagated as a [Ca2+]i wave along nonstimulated regions of the cytoplasmic process. In contrast to global glycolytic inhibition, propagating [Ca2+]i waves failed to propagate beyond the point where the cell process fused into the larger cell body. Insets show cytoplasmic peripheral processes at higher magnification.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Induction of oscillatory [Ca2+]i waves by 2-DG in Ca2+-free Tyrode solution. A: series of 2-dimensional confocal [Ca2+]i images recorded from a single calf pulmonary artery endothelial (CPAE) cell. a: [Ca2+]i at rest. In Ca2+-free external solution, 2-DG (10 mM) induced an oscillatory Ca2+ response that was initiated and originally restricted to the cytoplasmic peripheral process of the cell [region of interest (ROI) 2, red trace in B], until a [Ca2+]i transient (d) triggered a [Ca2+]i wave that propagated to the central regions of the cell (e–j). B: local [Ca2+]i transients recorded from ROIs representing the cell body (ROI 1, black square in a; black trace) and cytoplasmic process (ROI 2; red square in a; red trace). F/F0, change in ratio of fluorescence intensity to fluorescence under basal steady-state conditions, where F = F – F0.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Extracellular ATP and the glycolytic inhibitors induced local elevations of [Ca2+]i in form of inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca2+ puffs. Confocal line scan images recorded along a peripheral cytoplasmic process from individual CPAE cells are shown. A: [Ca2+]i (F/F0) line scan image (top) and local [Ca2+]i profiles (bottom) recorded from 1.5-µm-wide regions marked by the black boxes (a, b) on left of the line scan image. The image was recorded during stimulation with 25 nM ATPe in the absence of extracellular Ca2+. The whole cell [Ca2+]i wave was initiated in the cytoplasmic process and was preceded by local elevations of [Ca2+]i in form of Ca2+ puffs (*). Line scan [Ca2+]i images and local [Ca2+]i profiles recorded in Ca2+-free external solution containing pyr + -HB (10 mM each, B), 2-DG (10 mM, C), or IAA (1 mM, D) also revealed that the [Ca2+]i rise along the cytoplasmic processes was preceded by IP3R-mediated Ca2+ puffs (*). For these experiments gain setting was increased to record the small-amplitude [Ca2+]i transients at a sufficient signal-to-noise ratio. Consequently, saturation occurred during the global [Ca2+]i wave. B, top, and inset in D are 3-dimensional representations of local Ca2+ release events during exposure to glycolytic inhibitors.

In CPAE cells the ER Ca²⁺-ATPase pump inhibitor thapsigargin (Tg) causes an increase in [Ca²⁺]_i and depletion of ER Ca²⁺ stores (17, 69, 70). The kinetics of Tg-induced [Ca²⁺]_i elevations are typically slow (28) because they result from passive leakage of Ca²⁺ from the ER while reuptake is blocked. Since glycolysis is the preferential ATP source for ER Ca²⁺-ATPase activity in different cell types (13, 26, 42, 68, 79), we tested whether block of glycolysis caused an increase of [Ca²⁺]_i by the same mechanism as Tg, i.e., through passive Ca²⁺ leakage after complete inhibition of ER Ca²⁺-ATPase. For these experiments cells were first exposed to 2-DG [extracellular [Ca²⁺] ([Ca²⁺]_o) = 2 mM] and then to ATP_e. As shown in Fig. 6A, exposure of a cell to 2-DG (10 mM) induced a rapid and transient rise of [Ca²⁺]_i. In the continued presence of 2-DG, however, a brief stimulation with ATP_e (1 µM, for 20 s) evoked a robust Ca²⁺ response, indicating that Ca²⁺ stores were not significantly depleted after the 2-DG-evoked [Ca²⁺]_i transient. This finding is inconsistent with the idea that 2-DG increased [Ca²⁺]_i through a Tg-like mechanism. More likely, the data suggest that glycolytic inhibitors indeed caused an increase of [Ca²⁺]_i by facilitating Ca²⁺ release from an IP₃-sensitive intracellular Ca²⁺ store, a notion that is also supported by the fast kinetics of the response.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Effects of 2-DG and IP3R blocker 2-aminoethoxydiphenyl borate (2-APB) on ATPe- and KN-93-induced Ca2+ responses. A: a CPAE cell was stimulated with 2-DG (10 mM, solid bar) in 2 mM Ca2+-containing external solution. The subsequent repetitive applications of ATPe (20 s, 1 µM) evoked [Ca2+]i transients with successively decreasing amplitudes resulting from progressive depletion of intracellular Ca2+ stores. B: a CPAE cell was stimulated 3 times with KN-93 (50 µM) in the absence of extracellular Ca2+. Between stimulations, intracellular Ca2+ stores were allowed to refill in the presence of 2 mM extracellular Ca2+ for 20 min. 2-APB (20 µM), which abolished the KN-93-induced Ca2+ response, was applied 10 min before the third KN-93 stimulation. C: repetitive 2-DG (10 mM) stimulations with the same protocol as in B. As with KN-93, 2-DG induced reproducible [Ca2+]i transients with virtually identical amplitudes and kinetics. Application of 2-APB (20 µM) before the third 2-DG exposure caused a significant inhibition of the 2-DG-induced intracellular Ca2+ release.

Effect of 2-DG on KN-93-Induced Ca²⁺ Response

Previously we demonstrated (1) that in CPAE cells CaMKII inhibitors cause IP₃-dependent Ca²⁺ release through reducing the tonic inhibition of the IP₃R by CaMKII. Here we investigated whether 2-DG and the CaMKII inhibitor KN-93 share a similar mechanism of action, i.e., reduced ATP levels resulting from inhibition of glycolysis render CaMKII less effective and decrease CaMKII-dependent phosphorylation (and thus inhibition) of the IP₃R. Figure 6B shows [Ca²⁺]_i transients elicited with repetitive stimulation with KN-93 (50 µM) in Ca²⁺-free conditions. Between exposures to KN-93 cells were placed in 2 mM Ca²⁺ for 20 min to allow the Ca²⁺ stores to fully reload (1, 17). Repetitive exposure to KN-93 caused reproducible [Ca²⁺]_i transients of virtually identical magnitude and kinetics. 2-APB (20 µM, 10 min) applied before the third stimulation prevented KN-93-induced [Ca²⁺]_i increase. As shown previously (1), on average the [Ca²⁺]_i transient was reduced by 91 ± 6% in the presence of 2-APB. These results confirmed prior observations that KN-93-induced [Ca²⁺]_i transient was due to Ca²⁺ release through IP₃Rs. Using the same protocol, we also investigated the effect of IP₃R inhibition with 2-APB on the 2-DG-induced Ca²⁺ response. As seen with KN-93, two subsequent exposures of cells to 2-DG (10 mM) in Ca²⁺-free conditions evoked reproducible control [Ca²⁺]_i transients (Fig. 6C) with similar amplitudes and kinetics (Fig. 6B). Incubation with 20 µM 2-APB for 10 min before stimulation with 2-DG also resulted in a significant inhibition of the 2-DG-induced [Ca²⁺]_i transient (Fig. 6C). On average, the [Ca²⁺]_i transient amplitude was reduced by 70 ± 12% (P < 0.05; n = 6). Thus the significant inhibition of the 2-DG-induced [Ca²⁺]_i transient by 2-APB indicated that 2-DG caused intracellular Ca²⁺ release from IP₃R-dependent Ca²⁺ stores.

In a subsequent series of experiments we tested the hypothesis that 2-DG and KN-93 indeed caused IP₃R-dependent Ca²⁺ release by the same mechanism of decreasing the tonic inhibition of the IP₃R by CaMKII. For this purpose, cells were exposed to 2-DG (10 mM) for 40 min in 2 mM Ca²⁺-containing external solution and then challenged with KN-93 (100 µM; Fig. 7 Aa). Exposure of cells to 2-DG induced a pronounced increase in [Ca²⁺]_i that reached a maximum within 30 s, followed by a relatively rapid decline to prestimulatory levels. Subsequent application of KN-93 (100 µM) in the continued presence of 2-DG induced only a relatively small increase of [Ca²⁺]_i. In three separate experiments, the subsequent application of KN-93 to 2-DG-treated cells induced a Ca²⁺ response that had a mean peak amplitude of R = 0.64 ± 0.17, i.e., 50% of the average KN-93-induced [Ca²⁺]_i transient amplitude (R = 1.25 ± 0.27; n = 5; cf. Fig. 7Bb). This suggests that pretreatment of cells with 2-DG for 40 min rendered KN-93 significantly less effective in releasing Ca²⁺, possibly because reduced ATP levels already inhibited CaMKII action and/or depleted the Ca²⁺ stores. Reversing the order of application of 2-DG and KN-93 elicited a similar response (Fig. 7B). Exposure to KN-93 first triggered a typical [Ca²⁺]_i transient (compare to Fig. 6B). Subsequent exposure to 2-DG (after an interval of 40 min, which is normally sufficient for the stores to refill; cf. Fig. 6) in the continued presence of KN-93 caused a smaller [Ca²⁺]_i transient. On average, the amplitude of the [Ca²⁺]_i transient evoked with 2-DG after preincubation with KN-93 was R = 0.93 ± 0.31 (n = 5, Fig. 7Bb). The amplitude evoked when 2-DG was applied alone was R = 1.71 ± 0.26 (n = 3). The data suggest that during inhibition of CaMKII the 2-DG-induced Ca²⁺ response was diminished, presumably because reducing intracellular ATP reserves by inhibiting glycolysis has only a limited effect on the IP₃R when CaMKII is already blocked beforehand.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Combined effects of inhibition of glycolysis and Ca2+/calmodulin-dependent kinase II (CaMKII) on [Ca2+]i. A: a CPAE cell was stimulated with 2-DG (10 mM) in 2 mM Ca2+-containing Tyrode solution for 40 min. The subsequent application of KN-93 (100 µM) induced a relatively small increase in [Ca2+]i. B: alternatively, when cells were exposed to KN-93 for 40 min the subsequent application of 2-DG induced a Ca2+ response that was smaller and significantly different from the 2-DG-induced Ca2+ response in the absence of KN-93 (A). C: pretreatment of cells with the combination of pyr (10 mM) + -HB (10 mM) almost completely abolished the Ca2+ response to W-7 (50 µM) exposure. D: stimulation with W-7 followed by exposure to pyr + -HB. Summary data (b) are presented as average ± SE peak amplitude (R). Numbers in parentheses indicate the number of individual cells examined. *Statistically significant difference at P < 0.05.

Changes in [Mg²⁺]_i and [ATP]_i During Glycolytic Inhibition

It has been demonstrated that phosphorylation of the IP₃R by CaMKII reduces its activity by more than an order of magnitude (5). Furthermore, we have shown (1) that inhibition of CaMKII in CPAE cells causes Ca²⁺ release, possibly through dephosphorylation of the IP₃R and activation of release by basal IP₃ levels. If 2-DG causes Ca²⁺ release by a mechanism that involves inhibition of glycolytic ATP production and consequently reduced phosphorylation of the IP₃R, one would expect that inhibition of glycolysis causes a decline in intracellular [ATP] ([ATP]_i) with a time course similar to, and possibly preceding, the evoked changes in [Ca²⁺]_i. Therefore, we attempted to estimate changes of [ATP]_i experimentally. For this purpose we measured changes of free [Mg²⁺]_i, using the fluorescent Mg²⁺ indicator mag-fluo-4. Intracellularly the vast majority of cellular Mg²⁺ is bound to ATP and only a small fraction is found free (cf. e.g., Refs. 9, 10, 23, 44). Since free [Mg²⁺]_i is kept constant within a rather narrow range, any change in cellular ATP levels leads to a concomitant change in free [Mg²⁺]_i. Thus changes in [Mg²⁺]_i can be interpreted as changes of [ATP]_i. Figure 8A shows that the increase in mag-fluo-4 fluorescence elicited by 2-DG (10 mM) is essentially immediate (<5 s), suggesting that [ATP]_i levels are dropping rapidly. In comparison, a rise of [Ca²⁺]_i on exposure to 2-DG occurred typically with a delay of 20–30 s (see Fig. 2, A and B). Considering the time course of [Mg²⁺]_i, after such a time interval [ATP]_i levels must have already dropped significantly and may have led to changes in the phosphorylation state of the IP₃R. In the presence of 2-DG [Mg²⁺]_i increased on average from F/F₀ = 1 (basal level) to 1.69 ± 0.19 (n = 6), or by 69% (Fig. 8D). Because 2-DG is known also to act as a sink for inorganic phosphate (P_i) it can further inhibit ATP production by limiting available P_i. We therefore repeated the [Mg²⁺]_i measurements with IAA, which should not create a sink for P_i (see, e.g., Refs. 43, 63). As shown in Fig. 8B exposure to IAA (1 mM) increased [Mg²⁺]_i with similar kinetics. The average increase of [Mg²⁺]_i in the presence of IAA was 49 ± 4% (n = 13). In comparison, exposure to FCCP (5 µM; Fig. 8C) to inhibit only mitochondrial ATP production increased [Mg²⁺]_i to F/F₀ = 1.47 ± 0.04 (n = 13; Fig. 8D). The combined application of IAA and FCCP increased the mag-fluo-4 signal by 96 ± 8% (n = 19; data not shown). These data suggest that in CPAE cells ATP production through glycolysis is substantial and may account for up to half of total ATP production.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Effect of 2-DG on intracellular [Mg2+] ([Mg2+]i). [Mg2+]i was measured with the fluorescent Mg2+ indicator mag-fluo-4. A: exposure to 10 mM 2-DG caused a rapid increase of [Mg2+]i and presumably a concomitant decrease of intracellular [ATP]. B: [Mg2+]i increase elicited with 1 mM IAA. C: [Mg2+]i increase elicited with 5 µM FCCP. D: average % increase of [Mg2+]i elicited with 2-DG, IAA, and FCCP. Numbers in parentheses indicate number of experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The present work provides evidence for a cooperative role for CaMKII and glycolysis in the regulation of intracellular Ca²⁺ release. Our data showed that exposure of cells to the inhibitors of CaMKII KN-93 and W-7 caused IP₃R-dependent Ca²⁺ release, suggesting that under control conditions CaMKII has a tonic inhibitory effect on the IP₃R- or IP₃-dependent Ca²⁺ release (1). Inhibition of glycolysis with 2-DG significantly reduced the KN-93-induced Ca²⁺ response, suggesting an involvement of glycolysis in this regulation. Inhibition of glycolysis with 2-DG, pyr + -HB, or IAA caused Ca²⁺ release in the form of localized Ca²⁺ puffs, [Ca²⁺]_i oscillations, and whole cell [Ca²⁺]_i waves. While these inhibitors are known to have additional (60), albeit differing [e.g., 2-DG can act as a sink for P_i and thereby depress ATP synthesis (6); pyruvate has been shown to affect Ca²⁺ release not only via ATP-dependent mechanisms but also through a direct effect on the Ca²⁺ release channel in cardiac cells (87)] effects, the fact that all three inhibitors elicited similar Ca²⁺ responses argues in favor of the hypothesis that the common action of inhibiting glycolysis is responsible for the observed elevation of [Ca²⁺]_i. Our findings are in line with results obtained in rat (86) and porcine (67) aortic and coronary (56) endothelial cells, where inhibition of glycolysis has been demonstrated to release Ca²⁺ from a metabolically sensitive and/or IP₃-controlled Ca²⁺ store. Inhibition of glycolysis through focal application of 2-DG, pyr + -HB, or IAA resulted in elevation of [Ca²⁺]_i that was confined to the region of application of the inhibitors, suggesting that metabolism, in particular glycolysis, controls Ca²⁺ release locally and presumably also the complex interplay between CaMKII and IP₃R-dependent Ca²⁺ release. While both glycolytic and CaMKII inhibitors alone elicited robust Ca²⁺ release responses (Fig. 7), the [Ca²⁺]_i transients elicited through inhibition of glycolysis were significantly reduced in magnitude after prior inhibition of CaMKII and vice versa. This observation suggests that modulation of IP₃R-dependent Ca²⁺ release and glycolysis (or glycolytic ATP production) are linked together. There are several potential mechanisms through which glycolysis could affect Ca²⁺ release from IP₃-sensitive Ca²⁺ stores.

Several studies have shown that CaMKII inhibits Ca²⁺ release through the IP₃R (1, 5, 50, 85), and this inhibitory effect appears to involve a phosphorylation process (5, 74). Phosphorylation depends critically on ATP available to protein kinases (32). In many cell types most of the cellular ATP is derived from mitochondrial oxidative phosphorylation and not from glycolysis, although our data suggest that in CPAE cells ATP generated from glycolysis is substantial compared with ATP generated by mitochondria (Fig. 8). This is in agreement with previous reports that in vascular endothelial cells glycolysis makes a relatively high contribution to overall ATP production (summarized in Ref. 86). For example, under basal conditions up to 99% of glucose can be metabolized to lactate by anaerobic glycolysis (73), and under profound hypoxic conditions with only glucose as substrate the ATP production is not different from that in normoxic conditions with both glycolytic and oxidative substrates present (51). Furthermore, in aortic and coronary endothelial cells glycolytic inhibition (2-DG, IAA) caused ATP levels to drop by 60% (71) or led to complete depletion of the ATP pool (78). Inhibition of oxidative phosphorylation had a relative small effect on ATP levels (<20% decrease), whereas combined inhibition of glycolysis and mitochondrial metabolism led to a rapid depletion of initial ATP reserves (78). This is consistent with our observation presented in Fig. 8. Inhibition of glycolysis with 2-DG or IAA caused a rapid and robust increase of [Mg²⁺]_i, indicating a rapid drop of the cellular ATP reserves. In conclusion, there is strong evidence that in CPAE cells and endothelial cells from other vascular beds (for references see above) glycolysis makes a significant contribution toward cellular energy metabolism and ATP production. Thus the reliance on glycolytic ATP, which can be generated under anoxic conditions, explains the relative tolerance of endothelial cells toward hypoxic conditions (71). This is of particular importance in the intact vasculature (e.g., coronary artery), where it has been shown that endothelium-mediated relaxation of vascular smooth muscle is critically dependent on a complex interplay between oxygen tension and endothelial metabolism (16, 27).

Glycolytically generated ATP has long been recognized as the preferential energy source for membrane functions, for example, in cardiac (79) or vascular smooth muscle (58, 59) cells. The structural and functional prerequisite that would allow the preferential use of glycolytically generated ATP over ATP generated by mitochondrial metabolism for phosphorylation by CaMKII of the IP₃R is still not yet clear; however, subcellular compartmentalization of energy metabolism may be the key (45). An earlier study (72), however, shed new light on the structural interaction between CaMKII and the enzymes of the glycolytic pathway inasmuch as CaMKII associates with enzymes of the glycolytic pathway in functional microcompartments at the SR membrane and specifically assists in the local regulation of ATP involved in the Ca²⁺ regulatory function of the RyR and the IP₃R Ca²⁺-release channels. Additionally, it has been demonstrated (57) that binding of glycolytic enzymes to IP₃R is required for ATP production to alter local IP₃R Ca²⁺ release. These studies present strong evidence that CaMKII, enzymes of the glycolytic pathway, and IP₃R are three central components of a multiprotein signaling complex and strongly support our hypothesis that in CPAE cells glycolysis is the preferred ATP source for the tonic inhibitory effect of CaMKII on the IP₃R. Inhibition of glycolysis is expected to reduce the tonic inhibitory action of CaMKII, which will in turn allow basal IP₃ levels to cause release. In line with this suggestion is our observation that 2-DG caused within seconds an increase of [Mg²⁺]_i that continued for minutes in the presence of 2-DG (Fig. 8). The rise of [Mg²⁺]_i preceded the 2-DG-induced [Ca²⁺]_i transient by 20–30 s. Assuming that the rise of [Mg²⁺]_i is due to a decrease of cellular [ATP]_i (the main cytoplasmic Mg²⁺ buffer), the results would suggest that the Ca²⁺ response is indeed preceded by a substantial drop in [ATP]_i. Our findings are similar to the results obtained in porcine (67) aortic and coronary (56) endothelial cells, where inhibition of glycolysis has been demonstrated to release Ca²⁺ from an IP₃-controlled Ca²⁺ store.

Our conclusions on changes of [ATP]_i levels rely on the validity of the assumption that changes of [Mg²⁺]_i indeed reflect concomitant, but opposite, changes of [ATP]_i levels. While there is strong evidence for this assumption (see, e.g., discussion in Refs. 23, 44) the possibility needs to be considered that the 2-DG-induced rise of [Mg²⁺]_i is the result of inhibition of Mg²⁺ extrusion rather than a decrease of [ATP]_i. Mg²⁺ extrusion is poorly understood, and Na⁺-dependent and Na⁺-independent mechanisms have been proposed (66). The most widely postulated extrusion mechanism is via Na⁺/Mg²⁺ exchange; however, the molecular identity of such a transporter is elusive and its stoichiometry is still a matter of debate, including the question of whether the transport is electrogenic or electroneutral. Na⁺-independent transport mechanisms (in exchange with other extracellular ions) have been suggested; however, solid experimental evidence is lacking. (Three classes of Mg²⁺ transport proteins have been identified and cloned in prokaryotic cells, however, no homologs of these prokaryotic Mg²⁺ transporters have been found in eukaryotic cells.) Furthermore, among the numerous P-type ATPases described in eukaryotic cells, none has been shown to be capable of transporting Mg²⁺ (see Ref. 33). It has been speculated that ATP depletion may inhibit Mg²⁺ extrusion, suggesting a putative transport pathway that requires ATP hydrolysis and/or requires ATP for phosphorylation and activation (for discussion see Ref. 66). However, all studies on Mg²⁺ transport using plasma membrane vesicles from various tissues have failed to show a requirement for intravesicular ATP.

While it is an intriguing possibility that IP₃R activity is regulated through phosphorylation by CaMKII, other actions of ATP affect IP₃R or IP₃-mediated Ca²⁺ release. At physiological concentrations ATP binds directly to the IP₃R and therefore prevents IP₃ binding to the IP₃R (20, 47, 52, 53). Thus inhibition of glycolysis with subsequent decrease of [ATP]_i levels should allow IP₃ to bind to the IP₃R and to provide an additional avenue through which basal IP₃ levels could cause Ca²⁺ release. The possibility that the IP₃R can respond with measurable Ca²⁺ release to basal levels of [IP₃]_i was further confirmed by the observation that the IP₃R sensitizer thimerosal (12), a thiol-alkylating reagent, induced Ca²⁺ release in CPAE cells (unpublished results by A. S. Aromolaran and L. A. Blatter).

Glycolytic inhibitors caused substantial release of stored ER Ca²⁺, whether they were applied globally (e.g., Figs. 1 and 2) or focally to a restricted area of the cell (Fig. 3). The latter implies that locally restricted decreases of glycolytically generated ATP—possibly even undetectable at the bulk cellular level—might be able to affect ER Ca²⁺ release. This notion is supported by experiments showing that focally applied 2-DG, pyr + -HB, or IAA caused local elevations of [Ca²⁺]_i that were confined to the subcellular region of exposure. Additionally, confocal line scan experiments revealed that propagating pyr + -HB (Fig. 5B)-, 2-DG (Fig. 5C)-, and IAA (Fig. 5D)-induced [Ca²⁺]_i waves are preceded by local elevations of [Ca²⁺]_i reminiscent of elementary IP₃R-dependent Ca²⁺ release events termed Ca²⁺ puffs.

We also entertained the possibility that not the inhibition of glycolysis but rather a sudden drop of available exogenous glucose may activate a hitherto unknown signaling mechanism that leads to Ca²⁺ release and/or cellular energy metabolism switches to glycogenolysis to fuel glycolysis. While both of these mechanisms cannot be excluded in experiments where 2-DG or pyr + -HB replaced glucose in the superfusion solution, this effect is unlikely to occur in experiments where IAA was applied. IAA was applied in the presence of glucose, but the effect on Ca²⁺ release was similar to that of 2-DG or pyr + -HB. Thus the most likely explanation for the similar effects of these agents is the shared property of inhibiting glycolysis. Tapping into glycogen stores to maintain glycolytic flux is unlikely to play a significant role because vascular endothelial cells have insufficient or poorly utilized glycogen reserves (86). Even in the event that glycogenolysis could be significantly turned on, glycolytic inhibitors that act downstream of the hexokinase reaction (such as IAA) would inhibit ATP production by glycolysis fueled by glucose that was mobilized from glycogen.

We also considered the possibility that inhibition of glycolysis with subsequent depletion of intracellular ATP reduced the ER Ca²⁺-ATPase activity (75) thereby inhibiting ER Ca²⁺ uptake and resulting (because of a "leak" of Ca²⁺) in ER Ca²⁺ depletion. While this mechanism undoubtedly makes a contribution, it seems unlikely that glycolytic inhibition would exert the observed effects solely through this pathway (i.e., causing an increase of [Ca²⁺]_i similar to what is observed after treatment with the ER Ca²⁺-ATPase inhibitor Tg). As shown in Fig. 6, ATP_e was capable of evoking a substantial rise of [Ca²⁺]_i even after treatment with 2-DG, suggesting that the 2-DG-induced [Ca²⁺]_i transient did not abruptly deplete the ER. Nonetheless, repetitive stimulation with ATP_e evoked [Ca²⁺]_i transients with progressively decreasing amplitudes, suggesting that depletion and impaired refilling due to reduced intracellular ATP availability for the ER Ca²⁺ ATPase constitutes a contributing factor.

There is yet another effect of the glycolytic inhibitors that requires further comment. In CPAE cells, the ATP_e-induced [Ca²⁺]_i transient is characterized by two components: an initial and rapid rise of [Ca²⁺]_i that is predominantly due to ER Ca²⁺ release through the IP₃R followed by a sustained phase of elevated [Ca²⁺]_i due to Ca²⁺ entry across the plasma membrane through CCE. In contrast, the [Ca²⁺]_i transient induced in response to the glycolytic inhibitors almost completely lacked the sustained phase (Fig. 1). To date the mechanisms involved in the activation of CCE are not yet clear; however, previous reports (21, 49, 82) have demonstrated that a decrease in cellular ATP content leads to a reduction in CCE. Thus it is possible that in CPAE cells glycolytically generated ATP or another metabolite of glucose plays a role in activation of the CCE pathway.

In conclusion, we have demonstrated that inhibition of glycolysis rapidly decreased [ATP]_i and thereby reduced the tonic inhibitory effect of CaMKII on IP₃R- or IP₃-dependent Ca²⁺ release to cause a rise of [Ca²⁺]_i. Thus we conclude that the sensitivity of IP₃R- or IP₃-dependent Ca²⁺ release is regulated by glycolytic flux, and the availability of glycolytically generated ATP may represent the functional link between the IP₃R and phosphorylation by CaMKII.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by the National Heart, Lung, and Blood Institute (HL-62231 to L. A. Blatter) and the American Heart Association (0325564Z to A. S. Aromolaran, 0530309Z to A. V. Zima).

	ACKNOWLEDGMENTS

 
We thank Vezetter Whitaker for his contribution by building customized equipment and Anne Pezalla and William Johnson for expert technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. A. Blatter, Dept. of Physiology, Loyola Univ. Chicago, 2160 S. First Ave., Maywood, IL 60153 (e-mail: lblatte{at}lumc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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