Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

Ademuyiwa A. S. Aromolaran and Lothar A. Blatter

Department of Physiology, Loyola University Chicago, Maywood, Illinois

Submitted 3 June 2005 ; accepted in final form 3 August 2005


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 ABSTRACT
 MATERIALS AND METHODS
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The effects of inhibitors of CaMKII on intracellular Ca2+ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca2+]i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca2+ resulted in a dose-dependent increase of [Ca2+]i consisting of a rapid and transient Ca2+ spike followed by a small sustained plateau phase of elevated [Ca2+]i. Exposure to KN-93 in the absence of extracellular Ca2+ caused a transient rise of [Ca2+]i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca2+ from intracellular endoplasmic reticulum (ER) Ca2+ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca2+ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP3) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca2+ response, suggesting that exposure to KN-93 affects Ca2+ release from an IP3-sensitive store. Depletion of Ca2+ stores by exposure to ATP or to the ER Ca2+ pump inhibitor thapsigargin triggered robust capacitative Ca2+ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca2+ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca2+ from intracellular stores and activation of CCE.

Ca2+/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry


THE VASOACTIVE AGONIST ATP evokes a rise in intracellular free Ca2+ concentration ([Ca2+]i) in vascular endothelial cells (VECs). In the presence of physiological levels of extracellular Ca2+ ([Ca2+]o), the ATP-induced Ca2+ response is characterized by two distinct components: an initial rapid and transient rise of [Ca2+]i, which is due predominantly to Ca2+ release from the endoplasmic reticulum (ER) through inositol 1,4,5-trisphosphate receptor (IP3) receptors (IP3Rs), followed by a sustained phase due to Ca2+ influx from the extracellular space. The latter was previously characterized as capacitative Ca2+ entry (CCE) (25) in response to Ca2+ release and depletion of the ER Ca2+ stores, and plays a key role in maintaining cellular Ca2+ homeostasis (19). Both of these Ca2+-regulating processes, ER Ca2+ release and CCE, are complex and highly regulated processes. Ca2+ itself modulates IP3-dependent Ca2+ release, which is characterized by the bell-shaped relationship between [Ca2+]i and IP3-dependent Ca2+ release (6). Moreover, the IP3R has been shown to be regulated by several other proteins, including the Ca2+-binding protein CaM (33), and several kinases, including CaMKII (3, 12, 58).

The sustained phase of the Ca2+ response to agonist stimulation is carried by CCE resulting from ER Ca2+ store depletion. In VECs, CCE leads to replenishing of intracellular Ca2+ stores; activates important second messengers, including nitric oxide (NO) release (10); and plays a role in cell volume regulation and cell proliferation (5). Although CCE has been demonstrated in various cell types, the underlying molecular mechanisms of its activation and regulation are still elusive in many aspects (19, 40). Several signaling pathways have been proposed for the regulation of CCE, including conformational coupling between ER and surface membrane ion channel proteins or an ER resident generation of a diffusible Ca2+ influx factor (CIF). CIF diffuses to the plasma membrane to activate CCE through store-operated Ca2+ channels and appears to require protein phosphorylation to maintain its activity (37). Several protein kinases, including tyrosine kinase (44), protein kinase C (1), and myosin light-chain kinase (MLCK) (51), have been implicated as playing important roles in the regulation of CCE. CaMKII has been shown to regulate ion channels that may be involved in CCE. In chick skeletal muscle cells, inhibitors of CaM (trifluoperazine) and CaMKII {1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62)} completely abolished store-operated Ca2+ influx (52). Similarly, in thyroid FRTL-5 cells (Fischer rat thyroid line 5), KN-62 inhibited Ca2+ entry after ATP- and thapsigargin-dependent Ca2+ store depletion (50).

Having established previously that Ca2+/CaM plays crucial roles in NO production and CCE regulation in calf pulmonary artery endothelial (CPAE) cells (10, 42), in the present study, we have focused specifically on the elucidation of the role of CaMKII in the complex interplay between agonist-induced intracellular Ca2+ release, Ca2+ store depletion, and activation of CCE in this cell type. We found that various inhibitors of CaMKII altered the time course of ATP-induced [Ca2+]i transients, primarily by reducing its sustained component, thus inhibiting CCE. CaMKII inhibitors themselves, however, were also able to cause intracellular Ca2+ release, presumably from an IP3-dependent Ca2+ store. The data suggest that CaMKII activity plays an important regulatory role for both Ca2+ release and CCE. A previous account of this work was presented previously in abstract form (2).


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
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Cell culture. All experiments were performed on single cultured CPAE VECs. The CPAE cell line was obtained at passage 15 from the American Type Culture Collection (catalog no. CCL-209; ATCC, Manassas, VA). Cells were maintained in MEM supplemented with 10% FBS (GIBCO, Grand Island, NY) and 2 mM L-glutamine at 37°C in a humidified atmosphere of 95% air-5% CO2. Once per week, cells were dispersed using 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 the ATCC. All experiments were performed at room temperature (20–22°C).

Measurement of [Ca2+]i. Spatially averaged [Ca2+]i measurements from single CPAE cells were performed using 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, Eugene, OR) and 5 µl of a Pluronic F-127 stock solution (0.2 g/ml Pluronic F-127 dissolved in DMSO) for 20 min at room temperature. [Ca2+]i was measured by exciting indo-1 fluorescence with light emission of 360-nm wavelength and measuring emitted fluorescence signals simultaneously at 405 nm (F405) and 485 nm (F485). Single-cell fluorescence signals were recorded using photomultiplier tubes (model R2693; Hamamatsu, Bridgewater, NJ), and changes in [Ca2+]i are expressed as changes of the ratio R = F405/F485.

Solutions and chemicals. Cells were superfused with standard Tyrode solution that contained (in mM) 135 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, with pH adjusted to 7.3 using NaOH. In Ca2+-free Tyrode solution, CaCl2 was omitted (i.e., nominally Ca2+-free solution). ATP (Na+ salt) and 2-aminoethoxydiphenyl borate (2-APB) were obtained from Sigma (St. Louis, MO); [N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (KN-93), 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), and autocamtide-2-related inhibitory peptide (AIP) were purchased from Calbiochem (San Diego, CA); and thapsigargin was obtained from Alexis (San Diego, CA). ATP, KN-93, and AIP were dissolved in distilled water. KN-62 (10 mM), thapsigargin (10 mM), and 2-APB (20 mM) were dissolved in DMSO and diluted to the final indicated concentrations in normal Tyrode solution. All reagents were prepared on the day of experimentation.

Measurement of Mn2+ influx. Mn2+ was used as a substitute for Ca2+ to characterize unidirectional ion flux through the CCE pathway. Intracellularly, Mn2+ binds to indo-1 and quenches its fluorescence. The rate of Mn2+ entry (1 mM [Mn2+]o) was established previously (43); therefore, CCE activity was inferred from the rate of quenching (–dF424/dt) of indo-1 fluorescence excited at 360 nm and measured at the Ca2+-independent emission wavelength of 424 nm (F424; the Ca2+-isosbestic, Mn2+-sensitive emission wavelength).

Data analysis. Data are reported as means ± SE for the indicated number (n) of cells. Each experiment was performed on a separate coverslip. Statistical differences among the data were determined using Student's t-test for paired and unpaired data, and data were considered significant at P < 0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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ATP-induced [Ca2+]i transients and modulation by CaMKII inhibitors. We tested the effects of pharmacological inhibitors of CaMKII on ATP-induced [Ca2+]i transients in the presence of 2 mM [Ca2+]o. Figure 1A shows a control [Ca2+]i transient evoked by bath application of 5 µM ATP in a CPAE cell. The [Ca2+]i transient was characterized by a rapid increase in [Ca2+]i, which reached a peak with a rise time (10–90%) of 3.1 ± 0.2 s (n = 50) and then declined to a sustained plateau of elevated [Ca2+]i. The ATP-induced [Ca2+]i transient revealed an average ratio amplitude ({Delta}R) of 1.67 ± 0.06 ({Delta}R = Rpeak Rbasal; n = 50). The sustained phase had an average amplitude ratio of {Delta}R = 0.82 ± 0.06 (n = 50).



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Fig. 1. Effects of 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93) on ATP-induced cytosolic Ca2+ concentration ([Ca2+]i) transient in calf pulmonary artery endothelial (CPAE) cells in the presence of 2 mM extracellular Ca2+ ([Ca2+]o). CPAE cells were exposed to 5 µM ATP (open bar) in the absence (A) or presence of 50 µM KN-93 (B; solid bar) in 2 mM Ca2+-containing Tyrode solution. C: average peak levels (open bars) and plateau levels (closed bars representing measurement 2 min after start of exposure to ATP) of [Ca2+]i induced by stimulation with ATP alone and the combined application of ATP with KN-93. In this and subsequent figures, the numbers in parentheses indicate the number of cells tested. *P < 0.05, statistically significant difference of plateau [Ca2+]i compared with stimulation with ATP alone. R = F405/F485; {Delta}R = Rpeak – Rbasal.

 
ATP-induced [Ca2+]i transients were also examined in the presence of increasing KN-93 concentrations ([KN-93]; 5–100 µM). KN-93 inhibits CaM binding to CaMKII and therefore prevents its action. Single CPAE cells were stimulated simultaneously with ATP and KN-93. KN-93 produced a concentration-dependent inhibition of the sustained phase of the ATP-induced [Ca2+]i transient (Fig. 1B), however, it had little effect on the amplitude of the [Ca2+]i peak (summarized in Fig. 1C).

We examined the effect of two other highly potent and specific CaMKII blockers with different structures or modes of action. Both blockers were applied together with ATP. The structurally unrelated KN-62 (50 µM, Fig. 2A), which acts by a mechanism similar to that of KN-93, reduced the average {Delta}R of the sustained phase from 0.82 ± 0.06 (n = 50) to 0.36 ± 0.14 (n = 6). AIP (20 µM), a nonphosphorylatable, competitive substrate for autophosphorylation of CaMKII resulted in a similar effect. The average {Delta}R of the sustained phase of the [Ca2+]i transient was only 0.32 ± 0.09 (n = 10) in the presence of AIP. Similarly to KN-93, KN-62 and AIP had little effect on the peak of the [Ca2+]i transient.



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Fig. 2. Effects of CaMKII inhibitors 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and autocamtide-2-related inhibitory peptide (AIP) on ATP-induced [Ca2+]i transients. [Ca2+]i transients elicited by exposure to ATP (open bars) in the presence of 50 µM KN-62 (Aa) or 20 µM AIP (Ba) in standard Tyrode solution (2 mM [Ca2+]o). Average peak (open bar) and sustained plateau levels (closed bar) of ATP-induced [Ca2+]i transients in the presence of KN-62 (Ab) and AIP (Bb). *P < 0.05, statistically significant difference of plateau [Ca2+]i compared with stimulation with ATP alone.

 
The effects of CaMKII blockers alone on [Ca2+]i were also investigated. Interestingly, 50 µM KN-93 produced a robust Ca2+ response (Fig. 3 Aa), which was qualitatively similar to the response when applied together with ATP, although smaller in amplitude. [KN-93] between 5 and 50 µM caused a rapid rise of [Ca2+]i, which then declined to a sustained plateau. For example, with 50 µM external [KN-93], a [Ca2+]i transient with an average peak {Delta}R = 1.24 ± 0.12, a sustained plateau of {Delta}R = 0.32 ± 0.06, and a rise time (10–90%) of 11 ± 2 s (n = 20) was recorded. Across the same concentration range, the average peak amplitudes were overall smaller than when KN-93 was applied together with ATP; however, the peak {Delta}R increased with increasing [KN-93]. The average peak and plateau levels are summarized in Fig. 3Ab. AIP (20 µM) also caused a [Ca2+]i transient (Fig. 3B) with a pronounced peak (average {Delta}R = 1.32 ± 0.12; n = 6) but almost completely lacked the sustained phase ({Delta}R = 0.14 ± 0.06).



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Fig. 3. Effects of KN-93 and AIP on [Ca2+]i. A: [Ca2+]i transient (Aa) evoked by bath application of 50 µM KN-93 in standard Tyrode solution (2 mM [Ca2+]o). The Ca2+ response was characterized by a rapid increase in [Ca2+]i, which reached a maximum within <30 s, followed by a relatively rapid decay to a sustained plateau of elevated [Ca2+]i. The average peak level (open bar) and plateau level (closed bar) were plotted as a function of KN-93 concentration ([KN-93]) in Ab. B: [Ca2+]i transient (Ba) evoked by exposure to 20 µM AIP. In the presence of AIP, the sustained component was absent. The average peak level (open bar) and the sustained level (closed bar) are summarized in Bb. CPAE cells were exposed to KN-93 or AIP for the durations denoted by horizontal bars.

 
As shown in Figs. 13, the effects of CaMKII blockers appeared to affect predominantly the sustained plateau phase of the [Ca2+]i transients. To quantify the relative effect of the inhibitors on plateau [Ca2+]i levels, we calculated plateau [Ca2+]i (b) as a fraction of peak [Ca2+]i (a). The ratio b/a, a measure of the inhibitory effect on plateau [Ca2+]i, was calculated for the different experimental conditions and inhibitor concentrations. Figure 4A shows that under control conditions (i.e., exposure to ATP alone) average plateau [Ca2+]i amounted to ~50% of peak [Ca2+]i. When applied together with ATP all three CaMKII inhibitors reduced b/a. A dose-dependent effect was observed with KN-93. The effects of KN-62 (50 µM) and AIP (20 µM) were similar to those of 50 µM KN-93 and decreased plateau [Ca2+]i to ~20% of peak [Ca2+]i.



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Fig. 4. Quantification of the inhibitory action of CaMKII inhibitors on ATP-induced [Ca2+]i transients. The inhibitory action of CaMKII inhibitors on ATP-induced [Ca2+]i transients was quantified by the ratio b/a, in which a represents the maximum peak amplitude (see A, inset) and b is the amplitude of the sustained component. The ratio b/a was calculated for KN-93, KN-62, and AIP and then plotted for the various concentrations of CaMKII inhibitors shown in A. B: b/a ratio as a function of several concentrations of KN-93 and AIP (20 µM) in the absence of ATP. Numbers in parentheses indicate number of cells examined.

 
A similar picture emerged for the effect of CaMKII inhibitors alone (i.e., in the absence of ATP) (Fig. 4B). KN-93 decreased b/a in a dose-dependent fashion. In the presence of 20 µM AIP, b/a averaged only 0.09 ± 0.04 (n = 6), consistent with the results shown in Fig. 3B.

On the basis of the knowledge that the peak phase of the ATP-induced [Ca2+]i transient results predominately from Ca2+ release from internal stores, whereas the sustained plateau phase results from Ca2+ entry via CCE, the data presented thus far suggest that CaMKII inhibitors are capable of releasing Ca2+ but also had a pronounced inhibitory effect on CCE. The following experiments were designed to test the hypothesis that 1) inhibition of CaMKII activity can indeed release Ca2+ from ER Ca2+ stores and 2) CaMKII is an important contributor to the activation of CCE.

ATP- and KN-93-induced [Ca2+]i transients in the absence of extracellular Ca2+. To examine the effects of CaMKII inhibitors exclusively on Ca2+ release from internal stores, ATP and KN-93 were applied to CPAE cells in the absence of extracellular Ca2+. Figure 5A shows an example of a control [Ca2+]i transient evoked by bath application of ATP (5 µM) in Ca2+-free external solution. The ATP-induced [Ca2+]i transient was characterized by a rapid and pronounced initial rise in [Ca2+]i, which then declined completely to the prestimulatory level even in the maintained presence of extracellular ATP. When ATP was applied simultaneously with KN-93, a [Ca2+]i transient of similar amplitude was observed (Fig. 5B). On average (Fig. 5D), the amplitude of the [Ca2+]i transient evoked by ATP was {Delta}R = 1.40 ± 0.08 (n = 53). The amplitude evoked with ATP plus KN-93 was {Delta}R = 1.50 ± 0.14 (n = 13). The average amplitude was {Delta}R = 1.24 ± 0.09 (n = 31) when KN-93 was applied alone (Fig. 5C). Even though these average amplitudes were not significantly different (P > 0.05), they suggest that the action of ATP and KN-93 was slightly additive. However, the rate of decay was different. In the presence of KN-93, the decline of the [Ca2+]i transient was accelerated. This difference in the rates of decline suggests that KN-93 accelerated Ca2+ sequestration, possibly by enhancing the refilling of ER Ca2+ stores. The CaMKII inhibitor AIP (20 µM; data not shown) was also capable of triggering a [Ca2+]i transient similar to that triggered by KN-93, suggesting that the [Ca2+]i transients were the result of inhibition of CaMKII and not due to an unspecific effect of the inhibitors.



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Fig. 5. Effects of KN-93 on [Ca2+]i in Ca2+-free Tyrode solution. CPAE cells were exposed to ATP in the absence (A) and presence of KN-93 (B) in Ca2+-free Tyrode solution. C: Ca2+ response to bath-applied KN-93 (50 µM) in the absence of ATP. D: average peak levels of [Ca2+]i transients induced by stimulation with ATP alone (open bar), ATP + KN-93 (gray bar), and KN-93 alone (solid bar). Numbers in parentheses indicate number of cells tested.

 
In summary, the results presented herein indicate that inhibition of endogenous CaMKII activity causes Ca2+ release from an intracellular Ca2+ store and facilitates ATP-induced Ca2+ release from the ER in CPAE cells.

Intracellular Ca2+ source for KN-93-induced Ca2+ response. The data presented in Fig. 5C clearly indicated that the CaMKII inhibitor KN-93 caused substantial release of Ca2+. We tested whether KN-93 and ATP increased [Ca2+]i by mobilizing Ca2+ from the same IP3-sensitive intracellular Ca2+ store (10, 28). As shown in Fig. 6A, repetitive, brief KN-93 applications (100 µM, 10 s; n = 3 cells) in Ca2+-free conditions evoked [Ca2+]i transients with progressively decreasing amplitudes due to store depletion. Subsequent application of ATP induced only a small increase in [Ca2+]i. In three separate experiments, the subsequent application of ATP to KN-93-treated cells induced a Ca2+ response that had a mean peak amplitude of {Delta}R = 0.12 ± 0.04; i.e., <10% of typical [Ca2+]i transient amplitude. This suggests that repetitive stimulation with KN-93 had depleted the same Ca2+ store from which stimulation with ATP releases Ca2+. Alternatively, after repetitive ATP application (5 µM, 10-s applications), subsequent exposure to KN-93 failed to increase [Ca2+]i (n = 5) (Fig. 6B). The data indicate that ATP and KN-93 induced Ca2+ release from a common intracellular Ca2+ store.



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Fig. 6. Effects of KN-93 and ATP on intracellular Ca2+ stores. A: repetitive 100 µM KN-93 stimulation (10-s application; {blacksquare}) in Ca2+-free solution elicited [Ca2+]i transients with successively decreasing amplitudes resulting from progressive store depletion. Subsequent application of 5 µM ATP ({square}) induced a small increase in [Ca2+]i. B: when intracellular Ca2+ stores were depleted by repetitive 5 µM ATP stimulation (10-s applications), subsequent application of KN-93 failed to induce a Ca2+ response. C: average peak amplitudes (from experiments shown in A and B) of subsequent [Ca2+]i transients elicited with repetitive exposure to KN-93 (n = 3) and ATP (n = 5). Amplitudes were normalized to the first [Ca2+]i transient elicited with a brief pulse of KN-93 or ATP. *P < 0.05, statistically significant difference.

 
Figure 6C presents the average peak amplitudes of subsequent [Ca2+]i transients elicited with the first four repetitive exposures to ATP or KN-93 in these experiments. The amplitudes were normalized to the first [Ca2+]i transient elicited with a brief pulse of ATP or KN-93. The figure shows that the amplitude declined more slowly as a function of the number of consecutive exposures to KN-93 than it did in response to ATP, suggesting that repetitive exposure to ATP led to a faster depletion of the Ca2+ stores. This observation is in line with the faster decline of the KN-93-evoked [Ca2+]i transient (Fig. 5) and is consistent with the idea that in the presence of KN-93, intracellular Ca2+ sequestration might be enhanced.

Effect of inhibition of IP3 receptor on ATP- and KN-93-induced Ca2+ response. It was shown previously (20, 28) that in CPAE cells, the pathway that links the surface membrane purinoceptors to intracellular Ca2+ release involves the IP3 signaling cascade. Therefore, we investigated whether the KN-93-induced increase in [Ca2+]i involved IP3-dependent Ca2+ release. For these experiments, we used the membrane-permeant IP3R blocker 2-aminoethoxydiphenyl borate (2-APB) (27, 29, 59). Figure 7A shows repetitive stimulation with ATP (5 µM) in Ca2+-free conditions. Between exposures to ATP, cells were placed in 2 mM [Ca2+]o for 20 min to allow the Ca2+ stores to fully reload (10). Repetitive exposure to ATP caused reproducible [Ca2+]i transients of virtually identical magnitude and kinetics. Exposure to 2-APB (20 µM) 10 min before the third stimulation with ATP led to a significant reduction of the amplitude of the ATP-induced [Ca2+]i transient (Fig. 7C). On average, the [Ca2+]i transient was reduced by 64 ± 6% (P < 0.05). These results confirm prior observations that the ATP-induced [Ca2+]i transient is due to Ca2+ release through IP3Rs (24, 32, 35, 36, 39, 46). Using the same protocol, we investigated the potential role of IP3Rs for mediating the effect of KN-93 on [Ca2+]i. Figure 7B shows control [Ca2+]i transients with similar amplitudes induced by two subsequent exposures to KN-93 (50 µM). Incubation with 20 µM [2-APB] for 10 min before the third stimulation with KN-93 also resulted in a significant inhibition of the KN-93-induced [Ca2+]i transient (Fig. 7C). On average, the [Ca2+]i transient amplitude was reduced by 91 ± 6% (P < 0.05). Thus the significant inhibition of the KN-93 induced [Ca2+]i transient by 2-APB indicated that KN-93 caused IP3-dependent Ca2+ release.



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Fig. 7. Effect of 2-aminoethoxydiphenyl borate (2-APB) on ATP- and KN-93-induced intracellular Ca2+ release. A: CPAE cell was stimulated 3 times with 5 µM ATP in the absence of extracellular Ca2+. Between ATP stimulations, intracellular Ca2+ stores were allowed to refill in the presence of 2 mM extracellular Ca2+ for 20 min. 2-APB (20 µM), which inhibited the ATP-induced Ca2+ response, was applied 10 min before the third ATP stimulation. B: repetitive 50 µM KN-93 stimulation using the same protocol used in A. As with ATP, application of 2-APB before the third KN-93 stimulation caused a significant inhibition of the KN-93-induced intracellular Ca2+ release. C: summary of effects of 2-APB (gray bars) on ATP- and KN-93-induced Ca2+ release. Data are presented as average percentages ± SE of control (ATP, open bar; KN-93, solid bar), i.e., the amplitude of the Ca2+ response measured in the absence of 2-APB. Numbers in parentheses indicate number of individual cells examined. *P < 0.05, statistically significant difference.

 
Effects of inhibitors of CaMKII on ATP-induced Ca2+ entry. As shown in Figs. 1, 3, and 4, KN-93 had a pronounced inhibitory effect on the sustained plateau phase of [Ca2+]i transients evoked in the presence of 2 mM [Ca2+]o. This observation suggested an inhibitory effect of KN-93 on CCE, because the plateau phase is mainly carried by store depletion-dependent Ca2+ influx. However, an undefined contribution from Ca2+ release was still present during the plateau phase, thus precluding an unequivocal conclusion that KN-93 solely affected CCE. To test the effect of KN-93 on CCE directly, we applied several protocols that allowed us to separate Ca2+ release from Ca2+ influx (i.e., CCE). These protocols enabled us to separate in time the depletion of intracellular Ca2+ stores from activation of CCE.

In Fig. 8A, store depletion was achieved by prolonged exposure to 5 µM ATP (see Ref. 43) in Ca2+-free external solution, which elicited a [Ca2+]i transient due to mobilization of Ca2+ from intracellular stores. After recovery of the ATP-induced [Ca2+]i transient readmission of 2 mM [Ca2+]o generated a second elevation of [Ca2+]i, which was identified previously as CCE transient (10, 18, 25, 42). The combined application of ATP and KN-93 (50 µM) in the absence of extracellular Ca2+ (Fig. 8B) caused a robust [Ca2+]i transient of similar magnitude. Subsequent exposure to 2 mM [Ca2+]o (in the maintained presence of KN-93), however, failed to elicit a pronounced CCE transient. As shown in Fig. 8C, KN-93 reduced the amplitude of the CCE transient from {Delta}R = 0.44 ± 0.05 (control; n = 22) to 0.17 ± 0.06 (n = 7; P < 0.05), or by 61%. These results confirmed that KN-93 exerted a profound inhibitory effect on CCE, presumably through inhibition of CaMKII.



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Fig. 8. Effect of KN-93 on ATP-induced capacitative Ca2+ entry (CCE). Cells were exposed to 5 µM ATP in the absence (A) and presence of 50 µM KN-93 (B) to deplete intracellular Ca2+ stores. After recovery of the ATP-induced [Ca2+]i transient, the marked rise of [Ca2+]i after readdition of extracellular Ca2+ indicated the activation of CCE. Pretreatment of cells with KN-93 produced significant inhibition of the amplitude of the CCE transient. C: average CCE transient amplitudes in control (Ctrl) and in the presence of KN-93. *P < 0.05, statistically significant difference.

 
Effect of KN-93 on thapsigargin-induced CCE. To examine the activation of CCE independently of agonist-induced Ca2+ release, ER Ca2+ stores were depleted using thapsigargin, a potent inhibitor of ER Ca2+-ATPase. As shown previously, exposure to 5 µM thapsigargin for >20 min caused complete depletion of intracellular Ca2+ stores in CPAE cells (10). After store depletion, cells were exposed to 2 mM [Ca2+]o, which elicited a typical biphasic CCE transient (Fig. 9A) consisting of a rapid rise of [Ca2+]i followed by a slower decline that, as shown previously (25, 42), results from the delayed Ca2+-dependent activation of the plasmalemmal Ca2+-ATPase (PMCA). Upon removal of extracellular Ca2+, [Ca2+]i decreased quickly to resting level. The same cell was then treated with KN-93 and subsequently exposed for a second time to 2 mM [Ca2+]o. In the presence of 50 µM KN-93, the CCE transient rose more slowly and peaked at a much lower level. After removal of extracellular Ca2+, [Ca2+]i declined more slowly than it did in the absence of KN-93. The slower rate of recovery is likely to be a consequence of the lower amplitude of the CCE transient and therefore of reduced PMCA activity. We have shown previously that in CPAE cells, Ca2+ is extruded predominantly by PMCA and that PMCA activity is strongly Ca2+ dependent (25, 42). In addition, inhibition ofCaMKII is compatible with decreased PMCA activity due to reduced PMCA phosphorylation (38, 55). In three experiments, repeating the exposure to 2 mM [Ca2+]o revealed that the effect of KN-93 on CCE was largely reversible (Fig. 9A).



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Fig. 9. Effect of KN-93 on thapsigargin-induced CCE. A: CCE was triggered by exposure of CPAE cells to 2 mM external Ca2+. Intracellular Ca2+ stores previously depleted by exposure to 5 µM thapsigargin for 20 min in Ca2+-free solution (data not shown). Activation of CCE in the presence of 50 µM KN-93 inhibited CCE, resulting in lower amplitude and slower rate of rise of the CCE transient. After removal of extracellular Ca2+, [Ca2+]i declined at a slower rate in the presence of KN-93. A third exposure to 2 mM Ca2+ in the absence of KN-93 showed that the effects of KN-93 on CCE were largely reversible. B: CCE transients elicited by brief (20 s) exposure to 2 mM extracellular Ca2+ in the presence and absence of KN-93. KN-93 produced a significant inhibition of peak amplitude and rate of rise of thapsigargin-induced CCE transient. In A and B, the protocol for [Ca2+]o changes is shown underneath [Ca2+]i traces. C: normalized peak CCE transient amplitude (Ca) and rate of rise of the CCE transient (Cb) under control conditions (open bars) and in the presence of KN-93 (solid bars). Average data are from experiments shown in B.

 
Figure 9B shows the effects of KN-93 on CCE when cells were exposed to 2 mM [Ca2+]o for only 20 s after store depletion with thapsigargin. On average, the amplitude of CCE transient measured after 20-s exposure to 2 mM [Ca2+]o was {Delta}R = 0.91 ± 0.08 (n = 21). Upon removal of extracellular Ca2+ during the rising phase of the CCE transient, [Ca2+]i immediately started to decline toward basal levels as a result of Ca2+ extrusion by PMCA. Exposure to 2 mM [Ca2+]o was repeated in the presence of KN-93. Under these conditions, [Ca2+]i also increased but the rate of Ca2+ entry was significantly slower (the rate of rise was reduced by 52 ± 5%, n = 21; P < 0.05) (Fig. 9B) and the amplitude of the CCE transient was significantly reduced by 69 ± 5% (n = 21; P < 0.05). In addition, in 3 of 21 experiments, the CCE transient was completely abolished in the presence of KN-93. The degree of inhibition of CCE by KN-93 was similar when Ca2+ stores were depleted with thapsigargin (reduction of CCE amplitude by 69%) (Fig. 9C) or with ATP stimulation (amplitude reduced by 61%) (Fig. 8C). Furthermore, the declining phase of the CCE transient in the presence of KN-93 was slowed, consistent with the results shown in Fig. 9A.

Finally, we studied the effect of KN-93 on CCE with a third experimental protocol that allowed us to monitor the rate of CCE in CPAE cells directly (see Ref. 25). Mn2+ influx after depletion of intracellular stores with thapsigargin was measured. Mn2+ ions enter cells through the CCE pathway. Mn2+ entry can be used to estimate CCE activity from the rate of quenching of indo-1 fluorescence measured at a Ca2+-insensitive emission wavelength (424 nm). As shown in Fig. 10A, the rate of quenching of the indo-1 signal increased after exposure of thapsigargin-treated cells to Mn2+. However, the addition of KN-93 (50 µM) during Mn2+ influx slowed the indo-1 quenching rate, suggesting inhibition of CCE. In a total of 10 cells, the rate (expressed as the change in indo-1 fluorescence with time, –dF/dt) of Mn2+ entry was significantly reduced by 82 ± 6% (P < 0.05) (Fig. 10B) compared with the rate of quenching in the absence of KN-93. Taken together, our results show that a Ca2+/Mn2+-permeable plasma membrane channel activated after store depletion (and therefore CCE) is a target for an inhibitory effect of KN-93 in CPAE cells, suggesting that CaMKII plays a role in the activation of CCE.



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Fig. 10. Effect of KN-93 on thapsigargin-induced Mn2+ entry. A: after [Ca2+]i store depletion with thapsigargin, a single CPAE cell was exposed to 1 mM Mn2+. Indo-1 fluorescence was excited at 360 nm, and emitted fluorescence was measured at 424 nm (Ca2+-isosbestic, Mn2+-sensitive emission wavelength; F424). Mn2+ influx leads to a decrease of indo-1 signal due to Mn2+ entry and cytoplasmic quenching of indo-1 fluorescence (a.u., arbitrary units of F424). When 50 µM KN-93 was added during the Mn2+ influx the rate (–dF/dt) of quenching of indo-1 fluorescence was significantly reduced because of inhibition of the CCE pathway. B: normalized average rates (rate –dF/dt, expressed as %control) of Mn2+ entry in control and after addition of KN-93. The rate of Mn2+ entry (–dF/dt) was determined as the slope of the linear portion of the signal presented in A. *P < 0.05, statistically significant difference.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The present work provides evidence for a role for CaMKII in the regulation of intracellular Ca2+ release and the activation of CCE in CPAE cells. Our data have shown that exposure to specific inhibitors of CaMKII caused release of Ca2+ from the ER, suggesting that under basal conditions, CaMKII has a tonic inhibitory effect on the IP3R or on IP3-dependent Ca2+ release. Furthermore, CaMKII inhibitors had a profound inhibitory effect on CCE, suggesting that CaMKII is involved in the activation of CCE.

CaMKII and intracellular Ca2+ release.

There are several potential mechanisms through which CaMKII can affect Ca2+ release from IP3-sensitive Ca2+ stores. The prime candidate of modulation of IP3-dependent Ca2+ release is protein phosphorylation by CaMKII. Earlier studies have suggested that CaMKII can phosphorylate the IP3R (12, 58); however, these reports on the effect of IP3R phosphorylation for Ca2+ release and IP3R activity are contradictory, because both inhibition (30, 45, 58) and enhancement (9, 11, 48, 53) of Ca2+ release and/or IP3R channel activity have been observed. A recent study (3), however, has shed new light on the effect of IP3R phosphorylation by CaMKII. Bare et al. (3) showed that CaMKII phosphorylates IP3R type-2 (IP3R type 2 is also the predominant isoform in CPAE cells; unpublished results by J. R. Holda, G. A. Mignery, and L. A. Blatter) and incorporation of CaMKII-treated IP3Rs into planar lipid bilayers revealed that the IP3R-mediated channel open probability was reduced >10-fold by phosphorylation via CaMKII. The study presents strong evidence that the IP3R and CaMKII are two central components of a multiprotein signaling complex. The strong inhibition of IP3R channel activity by CaMKII phosphorylation is compatible with the suggestion that in CPAE cells, tonic activity of CaMKII inhibits IP3Rs and inhibition of CaMKII allows basal IP3 levels to cause Ca2+ release. Our observations are similar to the results obtained in rat pulmonary artery endothelial cells (34), in which inhibition of MLCK has been demonstrated to release Ca2+ from an IP3R-controlled Ca2+ store.

While this possibility of regulation of IP3R activity through tonic inhibition is intriguing, other actions of CaMKII affect IP3R-mediated Ca2+ release. CaMKII has been suggested to modulate IP3 metabolism by regulating two key enzymes involved in IP3 breakdown: Ins(1,4,5)P3 3-kinase (7) and Ins(1,4,5)P3 5-phosphatase (8). Therefore, changes in CaMKII activity affect IP3 levels directly. Particularly, the phosphorylation of Ins(1,4,5)P3 3-kinase by CaMKII enhances its activity and accelerates the breakdown of IP3. Inhibition of CaMKII in turn is expected to cause an increase in cytosolic IP3 levels that may be sufficient to result in the activation of Ca2+ release. This putative mechanism might be reinforced by altered levels of Ins(1,3,4,5)P4, a product of Ins(1,4,5)P3 3-kinase action and its effect on Ca2+ release (16, 21).

In addition, the possibility must be considered that the CaMKII inhibitor KN-93 may modulate Ca2+ release through a CaMKII-independent mechanism. KN-93 inhibits CaMKII by preventing CaM binding to the enzyme and therefore could interfere with any potential CaM binding site. For example, CaM inhibits PLC (41) and therefore decreases IP3 production. KN-93 binding to PLC could therefore reverse this inhibitory action of CaM and lead to increased IP3 production (31). Furthermore, through a similar mechanism, KN-93 could prevent inhibition of the IP3R by CaM (49). Nonetheless, it seems unlikely that the effects of KN-93 in our present study occurred solely through a CaMKII-independent pathway, because we observed the same effects with the antagonist AIP, which acts through a completely different mechanism. (AIP binds to the substrate-binding site and not to the CaM-binding site of the kinase.) Furthermore, all CaMKII inhibitors were applied in a concentration range that is considered to cause specific and selective inhibition of CaMKII. The effective inhibitory concentration values of [KN-62] and [KN-93] have been determined to be 1 µM (17) and 20 µM (22), respectively, with no significant effect observed at concentrations ≤100 µM on activities of other kinases, such as MLCK, PKC, or cAMP-dependent protein kinase II. At micromolar concentrations, AIP is also a highly selective inhibitor of CaMKII and the concentration used (20 µM) has little effect on the activities of PKA, PKC, and CaMKIV (22). Therefore, the concentrations of inhibitors used in the present study were in the range expected to produce selective inhibition of CaMKII.

CaMKII and CCE.

In addition to Ca2+ release, CaMKII inhibitors caused inhibition of CCE independently of whether CCE was activated after store depletion in response to stimulation with ATP (Fig. 1B) or thapsigargin (Fig. 6A). This finding suggests a point of action by CaMKII in the regulation of the CCE activation signal transduction cascade that is common for receptor-dependent or receptor-independent store depletion. The similar effects on CCE of KN-93, KN-62, and AIP provide additional evidence for a specific role for CaMKII in this process. CaMKII activity has been associated with CCE activation in other cell types, including Chinese hamster ovary cells (14) and Xenopus oocytes (26).

Several mechanisms can be envisioned through which CaMKII can enhance CCE activity. Several protein kinases, including tyrosine kinases (TKs) and MLCK, have been shown to be involved in CCE regulation in endothelial cells (13, 34, 47, 56, 57), and it has become increasingly clear that phosphorylation is involved in the activation of CCE (4, 15, 23, 54). Thus a likely explanation of the mechanism of the effect CaMKII inhibitors on CCE would involve phosphorylation of the CCE channel (or a regulatory protein of the CCE pathway) by CaMKII. The CCE pathway may have even more than one phosphorylation site that would allow for finely tuned regulation by a variety of different kinases, including TKs, MLCK, and CaMKII.

An additional target for CaMKII-dependent regulation may be the signaling cascade linking store depletion to activation of the Ca2+ entry pathway. Although the molecular identity of the underlying mechanism remains elusive, several models have been proposed (for review, see Ref. 19), including conformational coupling of release and entry channel proteins, ER-dependent generation of a soluble cytoplasmic messenger (CIF), and store depletion-activated CCE channel insertion. CIF has been shown to require phosphorylation to maintain activity (37) in which CaMKII could play a role. Although to date there is no evidence for CIF in VECs, this mechanism cannot be ruled out on the basis of the findings of our present study.

Conclusion.

We have demonstrated that CaMKII inhibitors cause the release of Ca2+ from intracellular stores and inhibit CCE. Taken together, we conclude that CaMKII plays an important role in the regulation of intracellular Ca2+ release and the subsequent activation of CCE in VECs, presumably through phosphorylation of two important Ca2+-handling proteins: the IP3 receptor Ca2+ release channel and the CCE membrane channel. Both actions of CaMKII have in common that they favor refilling the ER with Ca2+.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by the National Heart, Lung, and Blood Institute Grant HL-62231 (to L. A. Blatter) and the American Heart Association–Midwest Affiliate Postdoctoral Fellowship 0325564Z (to A. A. S. Aromolaran).


    ACKNOWLEDGMENTS
 
We thank Vezetter Whitaker for building customized equipment and Anne P. Pezalla for expert technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Blatter, Dept. of Physiology, Loyola Univ. Chicago, 2160 South 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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