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participates in activation of store-operated Ca2+ channels in human glomerular mesangial
cells
Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 PKC
and
I inhibitor) and PKC antisense treatment (2.5 nM for 24-48
h). However, LY-379196 (PKC inhibitor) and
2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether
(HBDDE; PKC and 
inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKC 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 PKC or I, but not
II or , 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 PKC and . However, the translocation from cytosol
to membranes was not detected for PKCI or II. These results
suggest that PKC participates in the intracellular signaling pathway
for activating SOC upon release of intracellular stores of
Ca2+.
thapsigargin; patch clamp; fura 2 fluorescence
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 (, I, II, and ), novel
Ca2+-independent PKCs (
, 
, 
, and 
), and
atypical Ca2+-and lipid-independent PKCs (
, 
, µ,
and 
) (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, PKC, , , and , but not PKC,
which is strongly expressed in cardiomycytes, were detected in rat MC
as determined by Western blotting (18, 19, 42). In renal
epithelial cells, PKC, , and are all localized in the
cytoskeletal compartment; however, only PKC and 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, PKC is a positive mediator of
vascular smooth muscle proliferation (37), whereas
PKCII is inhibitory (50). Whereas PKC promotes cell
growth in vascular smooth muscle, PKC 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 ,
I, II, and . 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.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 G
) 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 = 
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.PKC Antisense Oligonucleotide Treatment
RNA was inhibited by using a
phosphorothioated PKC 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 PKC (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 PKC, I, II, , ATP, and specific primary
antibodies to PKC, I, II, and 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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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 
[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
[Ca2+]i induced by treatment of specific
inhibitors of PKC isoforms.
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and I in the range of 1 µM, significantly
attenuated the thapsigargin-induced Ca2+ influx in response
to readdition of Ca2+ to the bath
([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
([Ca2+]i = 117.0 ± 11.6 nM), an
inhibitor of PKCI and II, or with 100 µM HBDDE
([Ca2+]i = 90.5 ± 19.7 nM), an inhibitor of
PKC and .
Effects of PKC antisense on thapsigargin-induced capacitative
Ca2+ entry.
The near complete abolishment of [Ca2+]i
by Gö-6976 indicated that PKC 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 PKC antisense and scrambled oligonucleotides. As shown in Fig. 2,
pretreatment with PKC antisense (2.5 nM) for 1-2 days greatly
depressed [Ca2+]i. However, when MC were
treated for 1-2 days with the same dose of scrambled nonsense
oligonucleotides, [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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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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Effects of PKC antisense on SOC in cell-attached patches.
The role of PKC in the SOC signaling pathway was examined by
pretreating MC with PKC 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 PKC
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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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,
PKC and I reactivated SOC from postexcision rundown, whereas
PKCII and failed to restore channel activity. The restoration of
SOC activity by PKC and I cannot be attributed to PMA because
this stimulatory effect was not observed for PKCII and under the
same conditions.
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Western blot analysis of expression of PKC, I, II, and in cytosol and membrane fractions.
The Ca2+ imaging and patch-lamp experiments suggested that
PKC is a contributor to thapsigargin-induced activation of SOC. Results from inside-out patches suggested that SOC are activated by
PKC as well as 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, PKC and were approximately evenly distributed within the
cytosolic and membrane fractions. PKCI was predominately present in
the cytosol, whereas PKCII was primarily in the membrane fraction. In the samples pretreated with 1 µM thapsigargin for 3-5 min, the immunoblotting bands specific for PKC and were reduced in
the cytosol and more intense in the membrane fractions, indicating migration of PKC and 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 and isoforms was observed. However, PKCI
was not translocated with thapsigargin treatment, even though its
translocation was obtained with PMA treatment. Because PKCII was
already nearly 100% in the membrane fraction, thapsigargin-induced translocation could not be observed for this isoform.
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Expression of PKC under treatment with PKC-blocking peptide
or PKC antisense.
Analysis with fura 2 fluorescence ratiometry, patch clamping, and
Western blotting consistently implicated PKC as a mediator in the
activation of SOC by thapsigargin. To further investigate the notion
that thapsigargin treatment triggers PKC translocation, PKC
antibody was preincubated with specific PKC blocking peptide for
1 h before its addition to the nitrocellulose membrane, which had
been transferred with PKC proteins. The immunoblotting bands, present in the cytosolic and membrane compartments of MC, were not
detected after preabsorption of PKC antibody, indicating the
specificity of the PKC protein detected in the present study.
antisense treatment significantly attenuated the thapsigargin-induced activation of SOC. To further illustrate that this depressed response was attributed to deficient PKC, Western blotting was used to detect the expression of PKC in
samples pretreated with PKC antisense and scrambled nonsense. As
shown in Fig. 7, specific immunoblotting
bands for PKC 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 PKCI, PKCII, or PKC was
observed between scrambled and PKC antisense-treated cells.
Therefore, the antisense-induced depression is selective for PKC.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 PKC and I
inhibitor, significantly attenuated thapsigargin-induced capacitative
Ca2+ entry measured by fura 2 fluorescence and
single-channel analysis; 2) purified PKC and I, but
not PKCII and , reactivated SOC from postexcision rundown;
3) specific PKC antisense depressed Ca2+
influx stimulated by thapsigargin; and 4)
thapsigargin-induced depletion of internal Ca2+ stores
triggered translocation of PKC and , but not I and II, from
the cytosolic to membrane cellular fractions. These results indicate
that PKC 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 and 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 PKC
or I (or both) mediate the thapsigargin-evoked activation of SOC.
Interestingly, inhibiting PKC and by HBDDE or PKCI and II
by LY-379196 failed to suppress the thapsigargin-induced responses.
These results could be explained by opposing effects of PKCII or with PKCI and on SOC, respectively. Thus the stimulatory effects
from PKC or I were compromised by the inhibitory effects from
PKC or 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 PKC and I are able to activate SOC
directly (Fig. 5). However, the possible inhibitory effects of PKCII
and could not be detected with the inside out configuration because
the channel activity had already been minimized after excision.
Identification PKC 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 PKC and translocated from
cytosol to membranes in response to thapsigargin (Fig. 6). However,
this trafficking could not be observed for PKCI and II. These
experiments suggest that PKC and are part of the signaling
pathway involving the activation of SOC after depleting internal
Ca2+ stores.
I significantly reversed
SOC run down after excision but was not translocated from cytosol to
membrane in thapsigargin-treated MC. Thus, although PKCI has the
capacity to activate SOC, it may not contribute to the activation of
SOC when stores are depleted. The second paradox is that PKC
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 PKC. Alternatively,
PKC 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 PKC to move to the plasma membrane. However, the
translocation of PKC was not examined in the presence of BAPTA-AM,
the intracellular Ca2+ buffer, in the current study.
The experiments utilizing PKC antisense provided additional support
for the notion that PKC is a key component mediating thapsigargin-evoked activation of SOC in MC. Treating MC with antisense
oligonucleotides specific for PKC 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 PKC 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 PKC 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 PKC also
stimulates the extrusion of Ca2+ via
Na+/Ca2+ exchange after it enters the cell via
SOC channels. In this case, an inhibitor of PKC 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 PKC 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
PKC by temporal and spatial changes in
[Ca2+]i. Maasch et al.
(29) demonstrated that the thapsigargin-induced elevation
of cytosolic Ca2+ targeted PKC 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 PKC 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 PKC from the cytosol to the plasma membrane
(47).
Another question relates to how phosphorylation by PKC activates
SOC. PKC might phosphorylate SOC via a direct enzyme-substrate reaction. However, it is more likely that the effect of PKC 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 PKC
participates in the intracellular pathway mediating activation of SOC
by depletion of internal Ca2+ stores in MC.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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1.
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