Protein kinase Calpha participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells

Rong Ma, Patrick E. Kudlacek, and Steven C. Sansom

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein kinase C (PKC) plays an important role in activating store-operated Ca2+ channels (SOC) in human mesangial cells (MC). The present study was performed to determine the specific isoform(s) of conventional PKC involved in activating SOC in MC. Fura 2 fluorescence ratiometry showed that the thapsigargin-induced Ca2+ entry (equivalent to SOC) was significantly inhibited by 1 µM Gö-6976 (a specific PKCalpha and beta I inhibitor) and PKCalpha antisense treatment (2.5 nM for 24-48 h). However, LY-379196 (PKCbeta inhibitor) and 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether (HBDDE; PKCalpha and gamma  inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKCalpha antisense significantly depressed thapsigargin-induced activation of SOC. However, LY-379196 and HBDDE did not affect the SOC responses. In inside-out patches, application of purified PKCalpha or beta I, but not beta II or gamma , significantly rescued SOC from postexcision rundown. Western blot analysis revealed that thapsigargin evoked a decrease in cytosolic expression with a corresponding increase in membrane expression of PKCalpha and gamma . However, the translocation from cytosol to membranes was not detected for PKCbeta I or beta II. These results suggest that PKCalpha participates in the intracellular signaling pathway for activating SOC upon release of intracellular stores of Ca2+.

thapsigargin; patch clamp; fura 2 fluorescence


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

STORE-OPERATED CA2+ channels (SOC), identified in a variety of excitable and nonexcitable cells, have multiple physiological functions that include participating in proliferation, immunoreaction, muscle contraction, and secretion (10, 11, 22, 30, 41, 52). In human glomerular mesangial cells (MC), SOC have been described using both electrophysiological and fura 2 techniques (28, 31, 33). MC are specialized renal cells that surround glomerular capillaries and regulate filtration rate by contracting or relaxing in response to agents like angiotensin II or nitric oxide (32, 44).

Protein kinase C (PKC) is composed of a family of related isoenzymes, grouped into three major classes of conventional Ca2+-dependent PKCs (alpha , beta I, beta II, and gamma ), novel Ca2+-independent PKCs (delta , eta , theta , and epsilon ), and atypical Ca2+-and lipid-independent PKCs (lambda , zeta , µ, and iota ) (6, 35, 36). All isoforms express distinct enzymological properties, differential tissue distribution, different substrate specificity, and specific subcellular localization with distinct modes of cellular regulation (4, 6, 9, 18, 23, 36, 38). For example, PKCalpha , delta , epsilon , and zeta , but not PKCbeta , which is strongly expressed in cardiomycytes, were detected in rat MC as determined by Western blotting (18, 19, 42). In renal epithelial cells, PKCdelta , epsilon , and alpha  are all localized in the cytoskeletal compartment; however, only PKCdelta and alpha  are able to translocate from the cytosol to membranes on activation by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; Refs. 4 and 34). Moreover, PKCalpha is a positive mediator of vascular smooth muscle proliferation (37), whereas PKCbeta II is inhibitory (50). Whereas PKCalpha promotes cell growth in vascular smooth muscle, PKCalpha depresses proliferation of a human colonic adenocarcinoma cell line (43). These differences in structure, enzymatic properties, and intracellular localization illustrate that each of the PKC isoforms possess specific cellular functions.

Previous studies from this laboratory have demonstrated that PKC mediates epidermal growth factor and thapsigargin-induced activation of SOC via a phosphorylation mechanism, measured by fura 2 fluorescence and patch clamping (26, 27). The present study was performed to determine which specific isoform of PKC is the intermediary messenger in this signaling pathway. Fura 2 fluorescence and conventional patch clamping were combined with biochemical approaches to examine the involvement of the classic isoforms PKC alpha , beta I, beta II, and gamma . Because obtaining whole cell currents is technically difficult in MC, single-channel current recordings and whole cell Ca2+ measurements with fluorescent dyes were employed in the present study.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Preparation of Cultures of MC

The details regarding the procedures and methods for culturing MC were described in a previous study (13). Briefly, MC were purchased from Biowhittaker (Walkersville, MD) and cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical, St. Louis, MO) supplemented with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.0 mM glutamine, 0.66 U/ml insulin, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20% fetal bovine serum (pH 7.2-7.4). Only subpassages of MC <= 11 generations were used. Upon achieving confluence, cells were passed onto 22 ×22-1 mm cover- slips (Fisher, Pittsburgh, PA) and studied within 56 h. The cover slips served as the floor of a perfusion chamber (Warner RC-2OH, 23°C) used in both fura 2 and patch-clamp experiments.

Measurement of [Ca2+]i

The intracellular Ca2+ concentration [Ca2+]i was monitored in MC using fura 2 and dual excitation wavelength fluorescence microscopy, as previously described (3, 12). In brief, MC were incubated with physiological saline solution containing 7 µM fura 2-AM, 0.09 g/dl DMSO, and 0.018 g/dl Pluronic F-127 (Molecular Probes, Eugene, OR) for 60 min at 23°C. A selected individual cell was illuminated alternately at excitation wavelengths of 340 and 380 nm (bandwidth = 3 nm) provided by a DeltaScan dual monochromator system (Photon Technology International, Monmouth Junction, NJ). The emission wavelength was 510 nm. Background-corrected data were collected at a rate of 5 points/s, stored, and analyzed using the FeliX software package (Photon Technologies). Calibration of the fura 2 signal was performed according to established methods previously described (3, 12).

Patch-Clamp Procedures

Conventional cell-attached and inside-out patch configurations were used in the present study. Glass pipettes (plain; Fisher Scientific, Pittsburgh, PA) were prepared with a pipette puller and polisher (PP-830 and MF-830, respectively; Narishige, Tokyo, Japan). The internal diameter of the pipette tip was ~0.5 µm.

Single-channel currents were recorded and analyzed using standard patch-clamp techniques (13, 14). The patch pipette, partially filled with 90 mM BaCl2 solution, was in contact with a Ag-AgCl wire on a polycarbonate holder connected to the head stage of the patch clamp (PC-501A; Warner Instrument, Hamden, CT). The pipettes were lowered onto the cell membrane and suction was applied to obtain a high resistance (>10 GOmega ) seal. All experiments were conducted at room temperature (22-23°C). Data were digitized for single-channel analysis using an analog-to-digital interface (Axon Instruments, Foster City, CA) and recorded by a computer system. Low-pass filter was set at 500 Hz.

Single-Channel Analysis

The unitary current (i), defined as zero for the closed state (C), was determined as the mean of the best-fit Gaussian distribution of the amplitude histograms. Channels were considered open (O) when the total current (I) was >(n - 1/2)I and <(n + 1/2)I, where n is the maximal number of current levels observed. The open probability (Po) was defined as the time spent in an open state divided by the total time of the analyzed record. The channel activity was calculated as NPo = Sigma nPn, where Pn is the probability of finding n channels open. The Axoscope acquisition program and pCLAMP program set 6.02 (Axon Instruments, Foster City, CA) were used to record and analyze currents.

Western Blot Analysis

When cell monolayers, grown in 150-ml flasks, were 80% confluent, the medium was replaced by serum-free DMEM. After 24 h, the medium was replaced by fresh serum-free DMEM with or without thapsigargin (1 µM for 3-5 min). Cells were scraped in PBS with the appropriate amount of protein kinase inhibitor. After centrifuging the cell suspension at 500 g for 10 min at 4°C, the cell pellets were sonicated five times for 10 s each in 180 µl of PBS plus 20 µl of protein kinase inhibitor. The membrane and cytosolic fraction were isolated by centrifugation at 100,000 g for 30 min at 4°C. The membrane pellet was solubilized in a lysis buffer. Equal amounts of proteins, quantified using the Bio-Rad protein assay, were loaded to the 12% SDS-PAGE gel. The proteins were then transferred to the nitrocellulose membrane. The membranes were probed with primary rabbit or mouse monoclonal or polyclonal antibodies (depending on the isoform of PKC) specific for a classic PKC isoform at an appropriate dilution (1:50). A horseradish peroxidase-labeled goat anti-rabbit or anti-mouse IgG secondary antibody was then used to react with PKC antibodies at 1:50,000 dilution. The immunoblots were labeled by enhanced chemiluminescent (ECL) reagents and then placed against reflection autoradiography film and developed in a Kodak M35A X-OMAT processor. The isoforms of PKC in cytosolic and membrane fractions were quantified by measuring densitometry of specific bands using Quantity One 4.1 software. In each group, the total optical densities of a PKC isoform in the cytosolic and membrane fractions were counted as 100%. The amount of individual isoform in either fraction was expressed as a percentage of the total optical density.

PKCalpha Antisense Oligonucleotide Treatment

Translation of PKCalpha RNA was inhibited by using a phosphorothioated PKCalpha antisense oligonucleotide (made in the molecular core lab of the Eppley Institute of the University of Nebraska Medical Center, Omaha, NE) complementary to a region from the initiation codon of PKCalpha (nucleotide 49 5'-TAC CGA CTG CAA AAG GGC CCG-3' nucleotide 28). The control was the scrambled nonsense oligonucleotide (5'-GCA TAG TCA TGG CCT TTA AAT). A stock oligonucleotide solution (2.5 µm) was diluted 1,000 times with DMEM supplemented with 20% FBS to a final concentration of 2.5 nM. MC were incubated with the oligonucleotide containing medium for 24-48 h at 37°C before experimentation.

Solutions and Chemicals

For all fura 2 and cell-attached patch experiments, the initial extracellular physiological saline solution (PSS) contained (in mM): 135 NaCl, 5 KCl, 10 HEPES, 2 MgCl2, and 1 CaCl2. For inside-out patches, the bathing solution contained (in mM): 140 KCl, 2 MgCl2, 0.001 CaCl2, and 10 HEPES. The pipette solution for all patch experiments contained 90 mM BaCl2 plus 10 mM HEPES. In fura 2 experiments, the free Ca2+ concentration of the bath was adjusted to <10 nM by buffering PSS with 1.08 mM EGTA, according to the calcium concentration program by MTK Software. The pH in all solutions was adjusted to 7.4. Thapsigargin, Gö-6976, 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE), purified PKCalpha , beta I, beta II, gamma , ATP, and specific primary antibodies to PKCalpha , beta I, beta II, and gamma  were purchased from CalBiochem (La Jolla, CA). LY-379196 was obtained from Eli Lilly (Indianapolis, IN). The secondary antibodies were purchased from Jackson ImmunoResearch Lab (West Groba, PA).

Statistical Analysis

In patch-clamp experiments, all NPo values were calculated from at least 10 s of single-channel recording. Comparisons between two individual groups were performed by using a Student t-test. One-way ANOVA followed by Student-Newman-Keuls tests were used for comparisons among multiple groups. Data are reported as means ± SE; n is the number of cells. Significance was P < 0.05. Statistical analysis was performed using SigmaStat (Jandel Scientific, San Rafael, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fura 2 Experiments

Effects of specific PKC isoform inhibitors on thapsigargin-induced capacitative Ca2+ entry. Thapsigargin, a specific inhibitor of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) (45), has been used as an efficient tool to specifically activate SOC in a variety of cell types (40). Using fura 2 fluorescence ratiometry, the [Ca2+]i response to thapsigargin was monitored in the absence or presence of specific inhibitors to classic PKC isoforms. Figure 1A shows a typical profile of the change in [Ca2+]i induced by thapsigargin and subsequent manipulation of bath calcium concentration ([Ca2+]o). Application of 1 µM thapsigargin in the presence of 1 mM [Ca2+]o evoked a rapid increase in [Ca2+]i to 180 nM, followed by a plateau phase of ~80 nM. On reduction of bath Ca2+ to <10 nM, the [Ca2+]i was lowered from the sustained stage to ~10 nM. Subsequent readmission of 1 mM Ca2+ to the bath induced an immediate increase in [Ca2+]i to 185 nM. This incremental change in [Ca2+]i in response to readmission of Ca2+, defined as Delta [Ca2+]i in the present study, is an indicator of Ca2+ entering the cell through SOC (28) and is equivalent to capacitative Ca2+ entry as depicted by Putney and McKay (41). Thus, in the following experiments using fura 2 ratiometry, we focused on the alteration in Delta [Ca2+]i induced by treatment of specific inhibitors of PKC isoforms.


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Fig. 1.   Effects of specific inhibitors of conventional protein kinase C (PKC) isoforms on thapsigargin-induced Ca2+ entry measured by fura 2 fluorescence. A: typical two phase-response of intracellular Ca2+ concentration ([Ca2+]i) to 1 µM thapsigargin and response of [Ca2+]i to readdition of extracellular Ca2+ in the continued presence of thapsigargin. The thapsigargin response included an initial transient rise of [Ca2+]i, followed by a plateau phase in the presence of thapsigargin. [Ca2+]i declined rapidly on removal of Ca2+ from the bathing solution and increased immediately upon readdition of Ca2+. B: original tracings showing the influence of various inhibitors of PKC isoforms on the thapsigargin-induced Ca2+ influx pathway. Among them, 1 µM Gö-6976 greatly attenuated the thapsigargin-induced response. However, the response was only slightly inhibited in the presence of 500 nM LY-379196 and 100 µM 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether (HBDDE). The time the bath [Ca2+] was increased from <10 nM to 1 mM was designated as 0. C: summary data, showing that the thapsigargin-induced Ca2+ influx was significantly depressed by Gö-6976 but not by LY-379196 or HBDDE. *Significant changes in Delta [Ca2+]i. +Significant difference between the Gö-6976 and thapsigargin groups of data. Thap, thapsigargin.

Figure 1, B and C, shows the effects of inhibiting various PKC isoforms on the capacitative Ca2+ entry triggered by thapsigargin. Gö-6976, a selective inhibitor of both PKCalpha and beta I in the range of 1 µM, significantly attenuated the thapsigargin-induced Ca2+ influx in response to readdition of Ca2+ to the bath (Delta [Ca2+]i: 113.9 ± 23.1 nM vs. 38.3 ± 14.9 nM, thapsigargin vs. thapsigargin plus 1 µM Gö-6976). However, such inhibition of SOC was not observed when treating with 500 nM LY-379196 (Delta [Ca2+]i = 117.0 ± 11.6 nM), an inhibitor of PKCbeta I and beta II, or with 100 µM HBDDE (Delta [Ca2+]i = 90.5 ± 19.7 nM), an inhibitor of PKCalpha and gamma .

Effects of PKCalpha antisense on thapsigargin-induced capacitative Ca2+ entry. The near complete abolishment of Delta [Ca2+]i by Gö-6976 indicated that PKCalpha might be a specific mediator of capacitative Ca2+ entry. To further explore this notion, the thapsigargin-evoked rise in [Ca2+]i was examined in cells treated with PKCalpha antisense and scrambled oligonucleotides. As shown in Fig. 2, pretreatment with PKCalpha antisense (2.5 nM) for 1-2 days greatly depressed Delta [Ca2+]i. However, when MC were treated for 1-2 days with the same dose of scrambled nonsense oligonucleotides, Delta [Ca2+]i was not different from control (82.7 ± 16.8 nM vs. 113.9 ± 23.1 nM, scrambled sequence vs. control, P > 0.05; Fig. 2B).


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Fig. 2.   Thapsigargin-induced Ca2+ entry in PKCalpha antisense or scrambled oligonucleotide-treated human mesanglial cells (HMC; 2.5 nM for 24-48 h). A: representative tracings show [Ca2+]i responses to readdition of Ca2+ to the bath in a cell treated with PKCalpha antisense or scrambled nonsense. Thapsigargin was present in the bath throughout the experiment. Arrow indicates the time of readmission of Ca2+. B: averaged data showing significant inhibition of Delta [Ca2+]i with PKCalpha antisense treatment. *Significant difference between antisense group and thapsigargin group.

Patch-Clamp Experiments

Effects of various PKC isoform inhibitors on thapsigargin-induced activation of SOC in cell-attached patches. The cell-attached configuration was employed to detect single-channel currents of SOC responding to thapsigargin in the presence and absence of specific inhibitors of PKC isoforms. Representative tracings of single channel currents are shown in Fig. 3A. Consistent with previous reports (26, 28), SOC have minimal spontaneous activity in basal conditions (NPo: 0.17). Depletion of internal Ca2+ stores by thapsigargin increased the NPo to 0.26. The thapsigargin-induced response was ablated in the presence of Gö-6976 (Fig. 3, A and B). However, neither LY-379196 nor HBDDE attenuated the currents activated by thapsigargin (Fig. 3, A and B). None of the three inhibitors significantly affected the basal activity of SOC (Fig. 3A).


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Fig. 3.   Effects of specific inhibitors of PKC isoforms on thapsigargin-induced activation of store-operated Ca2+ channels (SOC) in cell-attached patches at -Vp = -80 mV. A: original tracings show the single-channel currents at basal condition, after application of inhibitors and inhibitors plus thapsigargin. Arrows indicate closed state of SOC. Downward deflects represent inward currents. B: summary data show effects of different inhibitors of PKC isoforms on open probability of SOC in the presence of thapsigargin. *Significant increases in NPo of SOC. +Significant difference between Gö-6976 and thapsigargin.

Effects of PKCalpha antisense on SOC in cell-attached patches. The role of PKCalpha in the SOC signaling pathway was examined by pretreating MC with PKCalpha antisense or scrambled nonsense oligonucleotides before detecting the thapsigargin-evoked SOC responses. As shown in Fig. 4, in the presence of the scrambled nonsense sequence, application of thapsigargin still evoked a significant increase in open probability of SOC (by 98.3 ± 33.5%). However, in the group treated with PKCalpha antisense, thapsigargin evoked only a slight increase in NPo (by 9.5 ± 3.3%). No significant difference in basal activity of SOC was detected when comparing the scrambled sequence and antisense-treated groups (NPo: 0.26 ± 0.09 vs. 0.25 ± 0.09).


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Fig. 4.   Responses of SOC currents (cell-attached, -VP = -80 mV) to thapsigargin in HMC treated with PKCalpha antisense or scrambled nonsense oligonucleotides (2.5 nM for 24 to 48 h). A: representative tracings showing thapsigargin-evoked activation of SOC in the PKCalpha scrambled but not antisense treated cells. B: summary data. *Significant increase in open probability of SOC. +Significant difference compared with scrambled group.

Effects of purified PKC isoforms on SOC in inside-out patches. The inside-out configuration was employed to determine the effects of four classic purified isoforms of PKC on the single-channel SOC currents. In these experiments, as reported previously (26), a spontaneous decrease in SOC activity (rundown) was routinely observed after excision. When the channel activity obtained stability after excision, the specific PKC isoform was added to the bath. Because the classic PKCs require phospholipid and Ca2+ to be activated, 1 µM PMA, 100 µM Mg-ATP, and 1 mM Ca2+ were added to the solution with each PKC isoform. A previous study demonstrated that 1 mM Ca2+ or 100 µM Mg-ATP in the bath did not affect SOC activity (26). The data from this series of experiments are summarized in Fig. 5. Among the four classic isoforms, PKCalpha and beta I reactivated SOC from postexcision rundown, whereas PKCbeta II and gamma  failed to restore channel activity. The restoration of SOC activity by PKCalpha and beta I cannot be attributed to PMA because this stimulatory effect was not observed for PKCbeta II and gamma  under the same conditions.


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Fig. 5.   Effects of purified PKC isoforms on open probability of SOC in inside-out patches at -Vp = -80 mV. Extracellular solution contained 1 mM Ca2+, 1 µM PMA, and 100 µM ATP. *Significant increases in NPo of SOC from baseline activity.

Western blot analysis of expression of PKCalpha , beta I, beta II, and gamma  in cytosol and membrane fractions. The Ca2+ imaging and patch-lamp experiments suggested that PKCalpha is a contributor to thapsigargin-induced activation of SOC. Results from inside-out patches suggested that SOC are activated by PKCalpha as well as beta I. Western blotting was used to determine which PKC isoforms are present endogenously in MC and involved in activating SOC. Because PKC translocates from cytosol to its substrate when activated, it is presumed that the candidate isoform of PKC would respond to thapsigargin with increased expression in the membranes where SOC are located. The representative immunoblotting bands for each isoform and averaged data for control and various treatments are shown in Fig. 6. All four classic PKC isoforms were detected in the cultured MC. In the absence of thapsigargin, PKCalpha and gamma  were approximately evenly distributed within the cytosolic and membrane fractions. PKCbeta I was predominately present in the cytosol, whereas PKCbeta II was primarily in the membrane fraction. In the samples pretreated with 1 µM thapsigargin for 3-5 min, the immunoblotting bands specific for PKCalpha and gamma  were reduced in the cytosol and more intense in the membrane fractions, indicating migration of PKC alpha  and gamma  from the cytosol to the membranes. As a positive control, the samples were pretreated with 1 µM PMA, a strong activator of PKC. As shown in Fig. 6, a similar alteration in distribution of PKC alpha  and gamma  isoforms was observed. However, PKCbeta I was not translocated with thapsigargin treatment, even though its translocation was obtained with PMA treatment. Because PKCbeta II was already nearly 100% in the membrane fraction, thapsigargin-induced translocation could not be observed for this isoform.


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Fig. 6.   Western blotting, showing expression of various PKC isoforms in cytosolic (Cy) and membrane (M) fractions in control, thapsigargin-treated, and PMA-treated HMC. A: immunoblotting bands of PKCalpha , beta I, beta II, and gamma . B: summary data, showing distribution of each PKC isoform in Cy and M in control (C) and thapsigargin (T)-treated samples. *Significant difference in density of PKC bands comparing fractions in thapsigargin-treated and control groups for each PKC isoform.

Expression of PKCalpha under treatment with PKCalpha -blocking peptide or PKCalpha antisense. Analysis with fura 2 fluorescence ratiometry, patch clamping, and Western blotting consistently implicated PKCalpha as a mediator in the activation of SOC by thapsigargin. To further investigate the notion that thapsigargin treatment triggers PKCalpha translocation, PKCalpha antibody was preincubated with specific PKCalpha blocking peptide for 1 h before its addition to the nitrocellulose membrane, which had been transferred with PKCalpha proteins. The immunoblotting bands, present in the cytosolic and membrane compartments of MC, were not detected after preabsorption of PKCalpha antibody, indicating the specificity of the PKCalpha protein detected in the present study.

In the fura 2 fluorescence and patch-clamp experiments, it was demonstrated that the PKCalpha antisense treatment significantly attenuated the thapsigargin-induced activation of SOC. To further illustrate that this depressed response was attributed to deficient PKCalpha , Western blotting was used to detect the expression of PKCalpha in samples pretreated with PKCalpha antisense and scrambled nonsense. As shown in Fig. 7, specific immunoblotting bands for PKCalpha were detected in both cytosolic and membrane fractions from cells treated with scrambled oligonucleotides. However, the bands in both fractions were reduced in antisense treated samples. As shown, no difference in the expressions of PKCbeta I, PKCbeta II, or PKCgamma was observed between scrambled and PKCalpha antisense-treated cells. Therefore, the antisense-induced depression is selective for PKCalpha .


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Fig. 7.   Expression of PKCalpha , beta I, beta II, and gamma  in cytosolic and membrane fractions in HMC treated with PKCalpha antisense and scrambled nonsense oligonucleotides.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Depending on the specifically tested cells and the experimental conditions, variable results have been reported on the modulation of SOC by PKC (1, 2, 7, 39, 46, 51). The differential tissue distribution, intracellular localization, and cellular functions of different isoforms of PKC might also contribute to these discrepancies. Using fura 2 fluorescence measurements combined with patch clamping, we previously demonstrated that PKC activates SOC through a phosphorylation mechanism (26). The previous findings are extended by the current study, which detects specific isoforms of PKC involved in this signaling pathway. The data of the present study showed the following: 1) Gö-6976, a PKCalpha and beta I inhibitor, significantly attenuated thapsigargin-induced capacitative Ca2+ entry measured by fura 2 fluorescence and single-channel analysis; 2) purified PKCalpha and beta I, but not PKCbeta II and gamma , reactivated SOC from postexcision rundown; 3) specific PKCalpha antisense depressed Ca2+ influx stimulated by thapsigargin; and 4) thapsigargin-induced depletion of internal Ca2+ stores triggered translocation of PKCalpha and gamma , but not beta I and beta II, from the cytosolic to membrane cellular fractions. These results indicate that PKCalpha plays an important role in regulating activity of SOC.

Influences of selective inhibitors of various PKC isoforms. Within a restricted concentration range, a selective inhibitor of a specific PKC isoform might still affect another isoform to some extent. This problem must be considered when interpreting results utilizing pharmacological tools. In the present study, 1 µM Gö-6976, an inhibitor of both PKC alpha  and beta I, significantly depressed the thapsigargin-induced capacitative Ca2+ entry assessed by Ca2+ imaging (Fig. 1). This inhibition was corroborated with electrophysiological methods (Fig. 3), implying that either PKCalpha or beta I (or both) mediate the thapsigargin-evoked activation of SOC. Interestingly, inhibiting PKCalpha and gamma  by HBDDE or PKCbeta I and beta II by LY-379196 failed to suppress the thapsigargin-induced responses. These results could be explained by opposing effects of PKCbeta II or gamma  with PKCbeta I and alpha  on SOC, respectively. Thus the stimulatory effects from PKCalpha or beta I were compromised by the inhibitory effects from PKCgamma or beta II. Indeed, opposite effects of different isoforms of PKC on the same cellular events have been reported by many groups of investigators (5, 37, 50). The data from inside-out patches further suggested that PKCalpha and beta I are able to activate SOC directly (Fig. 5). However, the possible inhibitory effects of PKCbeta II and gamma  could not be detected with the inside out configuration because the channel activity had already been minimized after excision.

Identification PKCalpha as a mediator for thapsigargin-induced activation of SOC. When activated, PKC normally translocates to its target site, which, in the case of SOC, is located in the plasma membrane. The results of Western blotting revealed that only PKCalpha and gamma  translocated from cytosol to membranes in response to thapsigargin (Fig. 6). However, this trafficking could not be observed for PKCbeta I and beta II. These experiments suggest that PKCalpha and gamma  are part of the signaling pathway involving the activation of SOC after depleting internal Ca2+ stores.

Two apparent paradoxes remain when comparing the results from Western blot analysis with those from the fura 2 fluorescence and patch-clamp experiments. The first paradox is that PKCbeta I significantly reversed SOC run down after excision but was not translocated from cytosol to membrane in thapsigargin-treated MC. Thus, although PKCbeta I has the capacity to activate SOC, it may not contribute to the activation of SOC when stores are depleted. The second paradox is that PKCgamma translocated from cytosol to membrane in response to thapsigargin in the Western blot experiments but did not reactivate SOC when applied directly to inside-out patches. One explanation is that membrane components other than SOC are substrates for PKCgamma . Alternatively, PKCgamma could inhibit SOC after translocating to the plasma membrane. An inhibitory effect would not be apparent in inside-out patches because SOC runs down nearly completely after excision. It is also possible that a thapsigargin-evoked increase in cytosolic Ca2+ caused PKCgamma to move to the plasma membrane. However, the translocation of PKCgamma was not examined in the presence of BAPTA-AM, the intracellular Ca2+ buffer, in the current study.

The experiments utilizing PKCalpha antisense provided additional support for the notion that PKCalpha is a key component mediating thapsigargin-evoked activation of SOC in MC. Treating MC with antisense oligonucleotides specific for PKCalpha attenuated thapsigargin-induced capacitative Ca2+ influx as measured by fura 2 ratiometry and completely inhibited thapsigargin-evoked activation of SOC determined by the cell attached patch-clamp method.

The PKC superfamily is composed of twelve members, which are further subdivided into three groups: conventional, novel, and atypical (6, 35, 36). Because specific inhibitors are presently available only to conventional PKCs, the possibility that one or more conventional PKCs are involved in the intracellular pathway for activating SOC has been examined in the current study. Even though these data suggest that PKCalpha participates in thapsigargin-induced activation of SOC, the results do not eliminate the possible involvement of other isoforms of PKC or other mechanisms of regulating the channel activity.

It was interesting that PKCalpha antisense and Gö-6976 completely abolished the thapsigargin-evoked activation of SOC determined by the patch-clamp technique but failed to completely abolish the thapsigargin-induced Ca2+ entry determined by fura 2 measurements. There are two possible explanations for these apparent contradictory results. First, fura 2 measures global intracellular Ca2+ concentration. It is possible that PKCalpha also stimulates the extrusion of Ca2+ via Na+/Ca2+ exchange after it enters the cell via SOC channels. In this case, an inhibitor of PKCalpha would completely prevent the thapsigargin-evoked increase in NPo of SOC, but it would not completely block a rise in [Ca2+]i. Second, in the fura 2 experiments, the residual Gö-6976-insensitive Ca2+ entry could have been through other Ca2+ permeable ion channels, such as a nonselective cation or the voltage-gated Ca2+ channel, previously described in these cells (13).

Recently, one group of investigators reported that activation of phospholipase C (PLC) activated expressed TRP3 channels in DT40 chicken B lymphocytes in which all three inositol 1,4,5-trisphosphate receptors (IP3R) were deleted (49). Activation of TRP3, a reportedly strong candidate for the store-operated channel (48, 49), was blocked by the PLC inhibitor, U-73122. Importantly, the diacylglycerol (DAG) analog 1-oleoyl-2-acetyl-sn-glycerol also activated TRP3 channels independently of IP3R. Because DAG is a crucial cofactor for conventional and novel isoenzymes of PKC, the results from that study support the hypothesis that one or more isoforms of PKC participate in activating SOC after store depletion. However, it should be noted that an earlier study (15) found that DAG directly activated human TRP3 and TRP6 through a PKC-independent mechanism.

It is not understood how PKCalpha is activated after depletion of internal Ca2+ stores. Although cytosolic Ca2+ concentration is elevated on depleting Ca2+ stores, it is probably not a primary mechanism for activating SOC. Previous studies from this laboratory and others have shown that SOC is activated upon store depletion despite the clamping of Ca2+ with intracellular buffers (16, 26). Supporting this notion are the results of one group that recently investigated the regulation of PKCalpha by temporal and spatial changes in [Ca2+]i. Maasch et al. (29) demonstrated that the thapsigargin-induced elevation of cytosolic Ca2+ targeted PKCalpha to distinct intracellular compartments but not the plasma membrane. It is possible, however, that an unknown PKC-stimulating phospholipid is generated on depleting internal Ca2+ stores. It is also possible that, on depletion of ER Ca2+, the cytoskeleton rearranges and activates PKC. It has been shown previously that disruption of the actin cytoskeleton activates PKCalpha in mesenchymal cells (25). Moreover, it was reported that calponin, a cytoskeletal protein, may serve to regulate PKC by facilitating its phosphorylation (24). A recent study revealed that a functional and integral actin microfilament network is essential for translocation of PKCalpha from the cytosol to the plasma membrane (47).

Another question relates to how phosphorylation by PKCalpha activates SOC. PKCalpha might phosphorylate SOC via a direct enzyme-substrate reaction. However, it is more likely that the effect of PKCalpha on SOC is mediated by a specific scaffolding protein. It has been proposed that many PKC-evoked cellular responses require particular receptor proteins that anchor PKC to its specific targets (8, 20, 21). Interestingly, in Drosophila, an eye-specific protein kinase C (InaC) forms a supramolecular complex with TRP and two other proteins, norpA-encoded phospholipase C and InaD protein (17). InaD is a putative substrate of InaC and might serve as an anchoring protein for InaC.

In conclusion, the present study strongly suggests that PKCalpha participates in the intracellular pathway mediating activation of SOC by depletion of internal Ca2+ stores in MC.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49561 (to S. C. Sansom), a fellowship grant from American Heart Association (Heartland Affiliate) (to R. Ma), and National Heart, Lung, and Blood Institute Research Training Grant 1T32-HL-07888 (to P. Kudlacek).


    FOOTNOTES

Address for reprint requests and other correspondence: S. C. Sansom, Dept. of Physiology and Biophysics, Univ. of Nebraska Medical Center, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: ssansom{at}unmc.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.

July 24, 2002;10.1152/ajpcell.00141.2002

Received 14 June 2002; accepted in final form 24 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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