| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
Submitted 19 February 2003 ; accepted in final form 11 November 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
0.7 pA/pF at –90 mV was activated when a Ca2+ chelator or inositol (1,4,5)-trisphosphate was included in the pipette or when Ca2+ stores were depleted with a Ca2+-ATPase inhibitor and ionophore. The current was maximal at hyperpolarizing voltages and inwardly rectified. The channel was relatively permeant to Ca2+ and Ba2+ but only poorly to Mg2+ or Mn2+. Taken together, these data support the existence of CCE in adult cardiomyocytes, a finding with likely implications to physiological responses to phospholipase C-generating agonists.
heart; store operated channels
InsP3 receptors are activated when cardiomyocytes are stimulated with agonists having receptors mediated by phospholipase C, such as 
-adrenergic agents thrombin, endothelin-1, and angiotensin II (ANG II) (9). Despite the limitations noted above, these agonists result in increases in [Ca2+]i (25, 28) and contribute to positive inotropic responses, (14, 33) arrhythmias (15, 35), cardiac damage following ischemia-reperfusion (36), and changes in gene expression (18).
The initial increase in [Ca2+]i in response to InsP3-generating agonists is due to the release of Ca2+ from the endoplasmic reticulum (ER) and/or the SR (termed ER-SR). In most cell types prolonged or repetitive stimulation and the subsequent depletion of ER-SR Ca2+ stores results in an influx of extracellular Ca2+ into the cytoplasm that allows for a sustained elevation in [Ca2+]i, a process termed store-operated or capacitative calcium entry (CCE) (21). This process is regulated by the depletion of ER-SR Ca2+ stores and can be activated by store depletion even in the absence of agonist. Such depletion can be achieved by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) responsible for the recovery of Ca2+ by the ER-SR, because in most cell types there is a substantive rate of leakage of sequestered ER Ca2+ (21). Recently, CCE was identified and shown to be dependent on SR Ca2+ stores in both smooth (6, 30) and skeletal (11, 31) muscle.
Hunton et al. (7) recently showed that CCE characterizes neonatal rat cardiomyocytes and linked CCE to myocyte hypertrophy through the calcineurin/nuclear factor of activated T-cells (NFAT) pathway. These authors asked here whether SR Ca2+ store depletion, achieved in adult rat ventricular myocytes (ARVMs) through treatment with SERCA inhibitors and an ionophore, an InsP3-generating agonist, or caffeine, activates CCE. We also utilized whole cell patch-clamp and inhibitors to partially characterize the channels responsible for CCE.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Primary cardiomyocyte cultures. Animal procedures conformed to the Guide for Care and Use of Laboratory Animals, issued by The National Institutes of Health. Primary cultures of ARVMs were obtained from 250- to 300-g male Sprague-Dawley rats. Myocytes were isolated from ventricular tissue by enzymatic dissociation as previously described (38). Briefly, hearts were rapidly excised, and a Langendorff retrograde perfusion was performed at 37°C for 5–7 min with Ca2+-free Tyrode solution equilibrated with 95% O2-5% CO2. The perfusion buffer was then changed to Tyrode solution containing collagenase (0.3 mg/ml collagenase type I, Sigma) and 25 µM Ca2+ for 30–40 min. The dissociated cells were filtered and then centrifuged. Resuspended cells were incubated with 0.1 mg/ml protease at 37°C for 5–10 min. Rod-shaped healthy myocytes were used after 1 h. All experiments were carried out at room temperature.
Patch-clamp recording. The whole cell configuration of the patch-clamp technique was used to record ensemble membrane currents. Patch-clamp electrodes were pulled by using a two-step puller (Narishige PP-830), and after being filled with pipette solution, the electrodes had a tip resistance of 3–5 M
. The junction potential was set at zero before formation of the membrane-pipette seal. After the whole cell configuration was achieved, electrical signals were recorded with a patch amplifier (Axopatch 200B), digitized, and analyzed using PClamp (8.02) and PClampfit software (Axon Instruments).
Calcium imaging. ARVMs were isolated as described in Primary cardiomyocyte cultures and maintained in 10 mM HEPES-buffered Ca2+-free Hanks balanced salt solution (HBSS) for 2 h. They were then made Ca2+ tolerant by increasing the HBSS Ca2+ concentration in four graded steps to a final concentration of 1.8 mM over 1 h at room temperature. Cells were then loaded for 20 min with the acetoxymethyl ester of fura-2 (2 µM) [dissociation constant (Kd) for Ca2+ = 0.14 µM; Molecular Probes] in 10 mM HBSS at room temperature. The solution was then exchanged to dye-free HBSS, and the cells were allowed to deesterify the indicator for an additional 30 min. To assess the intraluminal SR Ca2+ concentration simultaneously with [Ca2+]i, cells were incubated with 5 µM mag-fluo-4 AM (Kd = 22 µM; Molecular Probes) at 37°C for 40 min, washed with HBSS, and stored 6–8 h at 4°C to preferentially localize mag-fluo-4 to the SR (26). The cells were then loaded with 2 µM fura-2 AM at room temperature for 20 min and maintained in HBSS for an additional 40 min. Cells were allowed to settle on laminin-coated coverslips in a perfusion chamber and imaged on an Olympus IX70 inverted microscope through a x20 Uplan APO objective. Cells were illuminated at 340 and 380 nm for fura-2 excitation and at 465 nm for mag-fluo-4 excitation. Emissions were monitored at 510 nm. Excitation duration for each wavelength was 50 ms with a 1-s delay between frame sets. For analysis, the perimeter of each cell was traced and defined as the region of interest. For each individual, cell data for fura-2 were normalized to the initial 340-to-380 nm fluorescence ratio (R0) and reported as R (340/380 nm ratio)/R0 as a function of time during the experiment. This facilitated the combinination of data from multiple cells. Differential loading of mag-fluo-4 into the SR was verified by permeabilizing the surface membrane with saponin and affirming an insignificant change in the mag-fluo-4 fluorescence. In experiments utilizing caffeine to deplete SR Ca2+ stores, we used multiple applications in conjunction with the SERCA inhibitor cyclopiazonic acid (CPA) to prevent store refilling and achieve maximal store depletion. Cells were perfused with Ca2+-free HBSS, and caffeine was applied for 30 s at 90-s intervals, with 60 s allowed between stimulations for a total of six applications over a 9-min period.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the absence of extracellular Ca2+, cells responding to 1 µM ANG II displayed a peak increase in fluorescence 30–60 s after treatment (Fig. 1). The average peak response of 21 cells from three experiments was an increase in the fura-2 fluorescence ratio of 13 ± 2%. Without exception, all the cells returned to within 2% of the original baseline within 2 min after the addition of ANG II. In the presence of extracellular Ca2+, the average peak response to 1 µM ANG II of 26 cells was 17 ± 3% and was not significantly different from the response in the absence of extracellular Ca2+. However, there was a surprising degree of variability with respect to the capacity of the cells to maintain sustained elevations in [Ca2+]i. In 6 of 26 cells, a sustained plateau 10% or more greater than the original baseline persisted for minutes. In another 7 cells the plateau was >2% but <10% of the initial fluorescence ratio, whereas in 13 cells no sustained plateau was seen. An overall plateau average of 6% was calculated, which is consistent with the previous report (25) and reinforcing the conclusion that despite substantial cell-to-cell variability, treatment with ANG II in the presence of 1.8 mM extracellular Ca2+ results in an average sustained [Ca2+]i increase in ARVMs not seen in the absence of extracellular Ca2+. This averaged response is thus similar to that seen in other cell types in which InsP3-generating agonists initiate a release of Ca2+ from the ER, which in turn triggers a sustained increase in [Ca2+]i due to CCE (21). Cardiomyocytes are not the only cell type in which experiments with fura-2 that produce smooth results when performed in suspension with hundreds of thousands of cells yield significant variability when individual cells are followed, both with respect to a single cell's temporal response and among different cells (24).
|
ANG II depletes sequestered Ca2+ stores and activates calcium influx in selected ARVMs. To further investigate the mechanism responsible for the persistent elevation in [Ca2+]i in response to ANG II and perhaps to elucidate the unexplained cell-to-cell variability, we employed a differential dye-loading protocol similar to that used by Shmigol et al. (26) in primary uterine smooth muscle cells to determine how [Ca2+]i changes correlated with Ca2+ store depletion. This permitted the simultaneous assessment of [Ca2+]i responses using the high-affinity dye fura-2 and of SR Ca2+ stores with low-affinity mag-fluo-4. A conventional Ca2+ add-back protocol, in which the experiment is initiated in the absence of extracellular Ca2+, was used to identify the extracellular component of the [Ca2+]i increases (21). Exposure to 1 µM ANG II in the absence of Ca2+ resulted in a transient increase in [Ca2+]i in all cells that were analyzed and a decrease in sequestered Ca2+ that was partially reversed (Fig. 2A), likely due to reuptake by SERCA following Ca2+ release (27). This level of Ca2+ store depletion was sufficient upon the readdition of extracellular Ca2+ to activate a Ca2+ influx leading to a change in the fluorescence ratio of >10% in only 7 of 36 cells (Fig. 2A and data not shown), consistent with the experiments reported in Fig. 1 in which Ca2+ was always present. There were no significant differences in the extent of store depletion observed between the cells exhibiting such a plateau and those not. Thus an explanation for the observed cell-to-cell variability was not forthcoming. In control experiments in which extracellular Ca2+ was removed, no agonist was added, and then extracellular Ca2+ was replaced, none of the tested cells showed an increase in ratio of >2% upon the readdition of Ca2+ (data not shown).
|
To achieve more complete SR store depletion, Ca2+ store refilling was prevented by including the SERCA inhibitor thapsigargin. Interestingly, this addition to the protocol greatly reduced the cell-to-cell variability described above. In the presence of 5 µM thapsigargin the average decrease in the mag-fluo-4 signal 5 min after the addition of 1 µM ANG II was 8 ± 1%, and the influx of Ca2+ on add back caused an average [Ca2+]i increase in the fura-2 ratio of 38 ± 6% (n = 13). In the example shown in Fig. 2B, ANG II caused an immediate drop in Ca2+ stores that was followed by a gradual and continuing decrease. A similar pattern of Ca2+ store depletion was seen when treatment with ANG II was coupled with the reversible SERCA inhibitor CPA at 5 µM (Fig. 2C). In these experiments the average decrease in the mag-fluo-4 signal 5 min after ANG II was 9 ± 2%, whereas Ca2+ influx resulted in an average increase in the fura-2 ratio of 44 ± 7% (n = 17). When CPA was washed out before Ca2+ repletion, there was evidence of Ca2+ store refilling, based on an increase in the mag-fluo-4 signal, upon readdition of extracellular Ca2+ (Fig. 2C). This demonstrates that the presence of SERCA inhibitors facilitates ANG II-mediated Ca2+ store depletion and leads to a significantly larger influx of extracellular Ca2+.
Store depletion with thapsigargin and ionomycin also leads to calcium influx. We next asked whether Ca2+ store depletion in the absence of InsP3-generating agonists could activate a calcium influx. Initial experiments indicated that, similar to skeletal muscle (11), treatment with 5 µM thapsigargin or 5 µM CPA alone was insufficient to cause rapid Ca2+ store depletion in ARVMs. To facilitate more rapid store depletion in conjunction with 5 µM thapsigargin, 100 nM ionomyocin was used, a concentration that selectively permeabilizes ER-SR Ca2+ stores (21). In the absence of extracellular Ca2+, sequestered Ca2+ levels rapidly decreased by an average of 16 ± 2% at 5 min, whereas the concurrent increase in [Ca2+]i was transient (Fig. 3A). The modest and short-lived response in [Ca2+]i relative to the magnitude of the release of Ca2+ from stores likely reflects both the high level of Ca2+ buffering that characterizes the cytoplasm as well as the removal of cytoplasmic Ca2+ by Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPases (2). The subsequent addition of extracellular Ca2+ resulted in a sustained increase in [Ca2+]i averaging 32% ± 5% in 61 cells, consistent with activation of CCE. Because of the irreversible effects of thapsigargin, there was no significant increase in sequestered Ca2+ after Ca2+ repletion (Fig. 3A).
|
Depletion of caffeine-sensitive SR Ca2+ stores activates calcium influx. We next asked whether triggering Ca2+ store depletion through RyRs would activate a calcium influx. ARVMs were treated with both 5 µM CPA and 20 mM caffeine in Ca2+-free HBSS. Simultaneous assessments of sequestered Ca2+ and [Ca2+]i showed a drop in mag-fluo-4 fluorescence that averaged 21 ± 2%, significantly larger than that seen with ANG II in the presence of SERCA inhibition. The transient increase in the fura-2 ratio was also larger. When extracellular Ca2+ was restored, an increase in the fura-2 ratio of 21 ± 2% (n = 27) was observed (Fig. 3B). These data indicate that depletion of caffeine-sensitive Ca2+ stores activates CCE, although this level of Ca2+ depletion is clearly not physiological.
Previous studies in ARVMs (13) and other cell types (3) suggest that there may be spatially and functionally distinct stores of sequestered Ca2+. To address whether caffeine and ANG II were accessing the same or different SR Ca2+ pools, we used 5 µM thapsigargin to prevent refilling and measured the extent of SR Ca2+ store depletion by each agonist. Treatment with 1 µM ANG II in the absence of extracellular Ca2+ and in the presence of thapsigargin caused a rapid drop in stored Ca2+ that was followed by a continuing gradual decline (Fig. 3C). At 5 min after the addition of ANG II, this amounted to a 7% decrease in the mag-fluo-4 signal (n = 19). Concomitantly, there was a transient increase in [Ca2+]i. Subsequent addition of 20 mM caffeine generated a much larger drop in stored Ca2+ (14%), indicating that ongoing (Fig. 3C) or repetitive (data not shown) treatment with ANG II does not efficiently empty all caffeine-sensitive stores. Interestingly, the partial Ca2+ store depletion with ANG II was sufficient to activate a calcium influx, although an equivalent degree of Ca2+ store depletion achieved with caffeine in the absence of CPA was ineffective (data not shown).
Next, the order of treatments was reversed to determine whether depletion of caffeine-sensitive Ca2+ stores eliminated ANG II-sensitive stores. In none of the 52 cells examined was more than a slight additional drop in stored Ca2+ apparent when treatment with 1 µM ANG II followed 20 mM caffeine in the presence of 5 µM thapsigargin (data not shown). These data are consistent with the findings of Lipp et al. (13) using immunofluorescent antibodies demonstrating that InsP3 receptors are present only on a portion of the SR, but that RyRs are present on all stores, both positive and negative for InsP3 receptors.
L-type channels or the Na+/Ca2+ exchanger do not mediate store depletion-induced Ca2+ influx. ARVMs loaded with fura-2 were treated with 20 mM caffeine in the presence of the reversible SERCA inhibitor CPA (10 µM) in the absence of extracellular Ca2+. As noted above, this protocol was determined to maximally deplete Ca2+ stores and to activate CCE. At the same time that 1.8 mM Ca2+ was restored to the medium, CPA was removed so as to allow the Ca2+ stores to partially refill following Ca2+ entry. After 90 s, cells were subjected to an additional 20 mM caffeine treatment to assess the extent to which SR stores had been refilled and then to 30 mM KCl to assess influx due to L-type voltage-gated channels. Control cells showed a robust increase in [Ca2+]i after Ca2+ was restored to the media and an additional increase in [Ca2+]i in response to caffeine, indicating that the SR Ca2+ stores had been at least partly refilled. Additionally, a robust [Ca2+]i increase following depolarization with KCl showed that L-type channels were functional (Fig. 4A).
|
In other cell types (32) as well as in neonatal cardiomyocytes (7), glucosamine (GlcNH2) has been shown to inhibit CCE. To determine its effects on calcium influx in ARVMs, we repeated this protocol with 5 mM GlcNH2 being added 2 min before caffeine. The response to caffeine was normal, showing that GlcNH2 had little effect on SR Ca2+ stores. However, [Ca2+]i did not increase when Ca2+ was returned to the media, and caffeine failed to induce a response. This suggests that GlcNH2 was able to efficiently block Ca2+ entry and SR store refilling. A subsequent exposure to 30 mM KCl produced a robust [Ca2+]i increase, indicating, as seen previously in neonatal cells (7), that L-type channels were not impaired by the presence of GlcNH2 (Fig. 4B).
Treatment with another inhibitor of CCE, 20 µM gadolinium (21), also effectively blocked Ca2+ influx and SR Ca2+ store refilling without affecting L-type channels (Fig. 4C). SKF-96365, still another CCE inhibitor (21), did the same (Fig. 4D). Lanthanum at 100 µM (21) was also effective, although a gradual increase in fluorescence ratio, probably due to the entry of lanthanum via the Na+/Ca2+ exchanger and its interaction with fura-2 (22), confounded the analysis (data not shown). In contrast, neither nifedipine (10 µM) nor verapamil (10 µM) prevented the influx of Ca2+ or refilling of SR stores, although L-type channels were clearly blocked as depolarization failed to elicit a response (Fig. 4, E and F).
Previous evidence has suggested a role for the Na+/Ca2+ exchanger working in reverse mode in controlling [Ca2+]i responses to InsP3-generating agonists (12). However, treatment with unspecific inhibitors of the Na+/Ca2+ exchanger amiloride (10 µM) and benzamil (10 µM) caused a delay in the removal of Ca2+ following treatment with caffeine but failed to prevent [Ca2+]i increases on Ca2+ repletion, suggesting that the reverse mode of the Na/Ca2+ exchanger is unlikely to be contributing substantially to the calcium influx observed with store depletion (Fig. 4, G and H).
A compound that has received much attention in studies of calcium overload recently is KB-R7943 (8), which preferentially inhibits the reverse mode of the Na+/Ca2+ exchanger responsible for Ca2+ influx. As with the unspecific inhibitors of the exchanger, KB-R7943 at 5 µM was ineffective in blunting the increase in [Ca2+]i resulting from the Ca2+ influx that followed caffeine-induced store depletion (Fig. 5A).
|
To complement this point, we asked whether the agents we used to inhibit the putative CCE influx pathway had effects on Ca2+ influx attributable to the reverse mode of the Na+/Ca2+ exchanger in ARVMs. We utilized the protocol of Eigel and Hadley (4), in which the concentration of intracellular Na+ is elevated by removing K+ from the bath. K+ (5 mM) is then returned to the medium, and Na+ is removed. The resulting Ca2+ influx (Fig. 5B) is attributable to the reverse mode of the Na+/Ca2+ exchanger (4). This increase in [Ca2+]i was sensitive to 5 µM KB-R7943. However, neither 5 mM GlcNH2 nor 1 µM SKF-96365 resulted in a significant decrease in this mode of entry. These results provide further evidence that the Ca2+ influx observed with store depletion is independent of the Na+/Ca2+ exchanger.
Whole cell patch-clamp experiments detect an inward Ca2+ current activated by SR store depletion. The whole cell patch-clamp configuration was achieved by utilizing a pipette solution buffered to between 0 and 80 nM Ca2+ and that also contained 2 mM MgCl2 and 1 mM Mg2+ ATP. A holding potential of +20 mV that had previously been shown to be optimal for store-operated calcium channels (SOCs) (1) and that leads to the rapid inactivation of L-type channels (23) was utilized. Extracellular Ca2+ was 2 mM. Voltage ramps from –100 to +60 mV were applied every 2 s. When the pipette solution was buffered to 80 nM Ca2+ (Fig. 6A), a current of 0.2 pA/pF at –90 mV developed spontaneously over 4 min and remained stable for at least 30 min thereafter (32 of 34 cells) (Fig. 6A). When SR Ca2+ stores from this population of cells were depleted with 5 µM thapsigargin and 100 nM ionomycin, the inward current at –90 mV rapidly increased (Fig. 6A). Little change was seen at +60 mV. The magnitude of the current at –90 mV, usually 0.5–1.5 pA/pF, is small compared with the per beat influx through L-type channels (14) but not dissimilar to currents due to capacitative entry in other cell types in the presence of 2 mM extracellular Ca2+ (21). An average of the additional current induced by store depletion for five cardiomyocytes is shown as a function of time after the addition of thapsigargin and ionomycin in Fig. 6B. Comparable levels of current were seen when store depletion was achieved with 5 µM thapsigargin and 20 mM caffeine (data not shown). Figure 6 also shows that the additional current induced by store depletion is blocked by 100 µM lanthanum, as are many SOCs (21).
|
We further examined the changes in current observed within the first few minutes of achieving the whole cell configuration. When the cytoplasmic solution was buffered to near-zero [Ca2+]i with no Ca2+ and the chelator EGTA at 10 mM, conditions shown in other cell types to lead to so-called passive store depletion (21), activation of a current comparable to that seen with thapsigargin and ionomycin or caffeine was observed (6 of 6 cells) (Fig. 6C). We also facilitated store depletion at an intracellular Ca2+ of 80 nM by including 20 µM InsP3 in the pipette (Fig. 6C). Within 5 min, a current averaging 0.7 ± 0.1 pA/pF at –90 mV was observed (5 cells) when compared with the 0.2 ± 0.04 pA/pF with the 80 nM Ca2+ buffer alone. The initial decrease to 0.2 pA/pF could be lessened by increasing the intracellular Ca2+ above 80 nM (data not shown), but further elucidation of the mechanism underlying the activation of this current was not achieved.
The current-voltage relationship of the store depletion-induced current was further defined. Utilizing voltage ramps, the results were confounded by the presence of both voltage-gated Ca2+ channels and voltage-gated Na+ channels. The former rapidly inactivated at the +20 mV holding potential and could be effectively inhibited by 50 µM nifedipine and/or 50 µM verapamil. The Na+ channel was also inhibited by nifedipineverapamil or by 50 µM tetrodotoxin. The current that developed in the presence of these inhibitors in response to thapsigargin and caffeine or ionomycin minus the current remaining after inhibition by 100 µM lanthanum was computed (Fig. 6D) and averaged 0.6 pA/pF at –90 mV (n = 5). The computed current displayed an inward rectification characteristic of many SOCs (21). In the absence of inhibitors, the current at –90 mV averaged 0.7 pA/pF.
A series of experiments were then performed in which the SOCs were activated with 5 µM thapsigargin and 100 nM ionomycin in the presence of 2 mM Ca2+. The extracellular buffer was then exchanged for one with a higher concentration of Ca2+, an alternate divalent cation, or the complete absence of divalent cations (Fig. 6E). When Ca2+ was increased to 5 mM, the current increased to 162% of that seen with 2 mM Ca2+. When Ba2+ was exchanged for Ca2+, there was an increase of 80%. However, when either Mg2+ or Mn2+ was utilized instead of Ca2+, the current was decreased to 40 and 10% of the original value, respectively. When all divalents were removed, the current increased to 300% of the original value. This increase in the absence of divalent cations is seen in a variety of SOCs (21).
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A previous study utilizing thapsigargin to deplete stores in cardiomyocytes did not detect the prolonged increases in [Ca2+]i that characterize CCE (10), perhaps because the concentration of the inhibitor was only 100 nM and/or because the rate of leak from stores in these cells is less than that in others. This may not have resulted in sufficient store depletion to activate CCE. In contrast, several studies have shown a prolonged elevation of [Ca2+]i in response to InsP3-generating agonists such as endothelin-1, phenylephrine, and ANG II (25, 29). These agents only partially deplete Ca2+ stores, but the subset of Ca2+ stores containing InsP3 receptors (13) appears to be more closely linked to CCE activation.
The level of expression of InsP3 receptors in cardiomyocytes is about 50-fold less than that of RyRs (19), and previous reports (13) have described a limited distribution of InsP3 receptors within cardiomyocytes. Blaustein and Golovina (3) have discussed the concept that a subset of the ER-SR might be preferentially linked to the role of Ca2+ in regulating events at the cell surface. In cardiomyocytes it is critical, of course, to allow Ca2+ cycling of troponin C to proceed so as to permit excitation-contraction coupling. It appears, however, that in distinct areas of the cytoplasm, [Ca2+]i remains sufficiently elevated to allow for the sustained activation of calmodulin and calcineurin, allowing, for instance, for the nuclear translocation of NFAT and the hypertrophic response (18). Interestingly, in smooth muscle cells, flux through SOCs has been shown to increase dye-detected [Ca2+]i without causing constriction, even though a similar overall increase of [Ca2+]i induced by depolarization resulted in constriction (6). The authors suggest Ca2+ influx into a "noncontractile" compartment. Such compartmentalization of Ca2+ may allow ARVMs to discriminate between different Ca2+ signals as well. The putative signaling compartment likely communicates with the "contractile" compartment during the signal transduction leading from InsP3-generating agonists to positive inotropic responses. Recent publications have determined that the increases in L-type currents that characterize the response to such agonists are detected in perforated patch recordings but not in the whole cell configuration (14, 33). This suggests that the internal integrity of the cardiomyocyte may be critical. Recent results (Pang et al., unpublished observatons) have determined that CCE inhibitors partially inhibit this response, perhaps by disrupting the link between InsP3 generation and activation of L-type channels.
The previous publication with neonatal cardiomyocytes (7) was the first to suggest that CCE might be active in this cell type, but the notion was not without precedent. Others have previously observed a Ca2+-permeant channel that was activated by InsP3-generating agonists, not voltage-gated, and distinct from L-type channels. Merle et al. (17) found that such channels were activated with basic fibroblast growth factor, whereas Felzen et al. (5) found similar responses to the Fas ligand or a Fas-specific antibody. In the latter report, the channel could be opened by intracellular delivery of InsP3 as was also shown here. Whereas neither of the previous contributions linked channel activation to depletion of SR Ca2+ stores, such a mechanism could easily apply.
We thus suggest that CCE is likely to mediate InsP3-induced and calcineurin-mediated changes in gene expression, such as those responsible for the hypertrophic response (18), and, indirectly, positive inotropic responses to these agonists (14, 33). In addition, transient receptor potential family members are implicated in the influx of Ca2+ that results from Ca2+ store depletion in skeletal muscle (31). They are also responsible for the elevated [Ca2+]i that characterizes a mouse model of muscular dystrophy (31), although the reason for their aberrant activation due to a mutation in dystrophin is not clear (20). Interestingly, [Ca2+]i is also elevated in hearts of this model, and cardiac calcineurin is constantly activated in utrophin-dystrophin knockout mice (20). Finally, Ca2+ stores in cardiomyocytes are depleted by the removal of extracellular Ca2+ (34) and no doubt by the excessive InsP3 accumulation that is integral to ischemia-reperfusion injury (35, 36). CCE might thus be predicted to play a role in Ca2+ overload stemming from the Ca2+ paradox (37) or ischemia-reperfusion (35, 36). Whereas these hypotheses must be tested, there is growing evidence that cardiomyocyte physiology and pathology must take into account this previously unappreciated pathway for Ca2+ entry.
|
ACKNOWLEDGMENTS |
|---|
Present address of D. L. Hunton: Duke University Medical Center, Durham, NC 27710.
GRANTS
This work was supported by National Institutes of Health Grants DK-55647 (to R. B. Marchase) and T32 HL-07918 (to D. L. Hunton) and by the Juvenile Diabetes Research Foundation 1-2000-137 (to R. B. Marchase).
|
FOOTNOTES |
|---|
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.
* D. L. Hunton and L.Y Zou contributed equally to this study. 
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
q, PLC
I, and PKC. Cardiovasc Res 44: 5–9, 1999.1-Adrenergic activation of L-type Ca current in rat ventricular myocytes: perforated patch-clamp recordings. Am J Physiol Heart Circ Physiol 274: H2203–H2207, 1998.This article has been cited by other articles:
|
|
 |
|
  D. Shan, R. B. Marchase, and J. C. Chatham Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes Am J Physiol Cell Physiol, March 1, 2008; 294(3): C833 - C841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cooling, P. Hunter, and E. J. Crampin Modeling Hypertrophic IP3 Transients in the Cardiac Myocyte Biophys. J., November 15, 2007; 93(10): 3421 - 3433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. K. Saini and N. S. Dhalla Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H169 - H181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Fulop, Z. Zhang, R. B. Marchase, and J. C. Chatham Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2227 - H2236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. E. D. J. ter Keurs and P. A. Boyden Calcium and Arrhythmogenesis Physiol Rev, April 1, 2007; 87(2): 457 - 506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Rose, N. Hatano, S. Ohya, Y. Imaizumi, and W. R. Giles C-type natriuretic peptide activates a non-selective cation current in acutely isolated rat cardiac fibroblasts via natriuretic peptide C receptor-mediated signalling J. Physiol., April 1, 2007; 580(1): 255 - 274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-Z. Xu, K. Muraki, F. Zeng, J. Li, P. Sukumar, S. Shah, A. M. Dedman, P. K. Flemming, D. McHugh, J. Naylor, et al. A Sphingosine-1-Phosphate-Activated Calcium Channel Controlling Vascular Smooth Muscle Cell Motility Circ. Res., June 9, 2006; 98(11): 1381 - 1389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Huang, C. van Breemen, K.-H. Kuo, L. Hove-Madsen, and G. F. Tibbits Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes Am J Physiol Cell Physiol, June 1, 2006; 290(6): C1572 - C1582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Gomez-Pinilla, S. Morales, C. Camello-Almaraz, R. Moreno, M. J. Pozo, and P. J. Camello Changes in guinea pig gallbladder smooth muscle Ca2+ homeostasis by acute acalculous cholecystitis Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G14 - G22. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nagy, V. Champattanachai, R. B. Marchase, and J. C. Chatham Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-linked N-acetylglucosamine Am J Physiol Cell Physiol, January 1, 2006; 290(1): C57 - C65. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Cao, K. M. Harnett, L. Cheng, M. T. Kirber, J. Behar, and P. Biancani H2O2: a mediator of esophagitis-induced damage to calcium-release mechanisms in cat lower esophageal sphincter Am J Physiol Gastrointest Liver Physiol, June 1, 2005; 288(6): G1170 - G1178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Bolotina Store-Operated Channels: Diversity and Activation Mechanisms Sci. Signal., July 27, 2004; 2004(243): pe34 - pe34. [Abstract] [Full Text] [PDF]
|
|
| ||||||
8) from 3 independent isolations with error bars representing means ± SE.
