(Received for publication, December 30, 1996, and in revised form, March 24, 1997)
From the Departments of Pathology and
¶ Pharmacology, University of Virginia School of Medicine,
Charlottesville, Virginia 22908
T cell activation leading to cytokine production
and cellular proliferation involves a regulated increase and subsequent
decrease in the intracellular concentration of Ca2+
([Ca2+]i). While much is understood about
agonist-induced increases in [Ca2+]i, less is
known about down-regulation of this pathway. Understanding the
mechanism of this down-regulation is critical to the prevention of cell
death that can be the consequence of a sustained elevation in
[Ca2+]i. Protein kinase C (PKC), activated by the
diacylglycerol produced as a consequence of T cell receptor engagement,
has long been presumed to be involved in this down-regulation, although the precise mechanism is not wholly clear. In this report we
demonstrate that activation of PKC by phorbol esters slightly decreases
the rate of Ca2+ efflux from the cytosol of Jurkat T cells
following stimulation through the T cell receptor or stimulation in a
receptor-independent manner by thapsigargin. On the other hand, phorbol
ester treatment dramatically reduces the rate of Ca2+
influx following stimulation. Phorbol ester treatment is without an
effect on Ca2+ influx in a different T cell line, HSB.
Down-regulation of PKCI expression by 18-h phorbol ester treatment
is associated with a loss of the response to acute phorbol ester
treatment in Jurkat cells, suggesting that PKC
I may be the isozyme
responsible for the effects on Ca2+ influx. Electroporation
of an anti-PKC
I antibody, but not antibodies against PKC
or
PKC
, led to an increase in the rate of Ca2+ influx
following stimulation. Taken together, these data suggest that PKC
I
may be a component of the down-regulation of increases in
[Ca2+]i associated with Jurkat T cell
activation.
Activation of T lymphocytes via stimulation through the T cell receptor for antigen (TCR)1 leading to proliferation, cytokine production, or effector function requires a regulated increase and subsequent decrease in the intracellular concentration of Ca2+ ([Ca2+]i). The biochemical events associated with activation of T lymphocytes and leading to Ca2+ entry have been extensively studied (1). Engagement of the TCR leads to the activation of phospholipase C, which subsequently cleaves phosphatidylinositol bisphosphate into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3, binding to an intracellular receptor, induces the release of Ca2+ from an intracellular storage depot (2). In a receptor-independent manner, the Ca2+/ATPase inhibitor thapsigargin (3) causes an uncompensated leak of Ca2+ from this internal storage pool. In T lymphocytes, as well as in most electrically nonexcitable cells, this depletion of the intracellular storage pool induces the opening of the plasma membrane Ca2+ entry pathway and permits influx of extracellular Ca2+ (1). Previously, we have provided evidence that the pathway between release of intracellular Ca2+ and influx of extracellular Ca2+ is mediated by Ca2+-activated calmodulin (4), which permits Ca2+ entry carried by a current we have called IT (5).
The DAG produced subsequent to TCR stimulation is the naturally
occurring activator of a family of serine-threonine kinases known
collectively as protein kinase C (PKC). It has been proposed that DAG,
via activation of PKC, represents the down-regulatory arm of
Ca2+ signaling initiated by IP3 generation (for
example, see Ref. 6). Four points in Ca2+ signaling have
been implicated as targets for PKC action. First is a PKC-mediated
phosphorylation of the subunit of the TCR·CD3 complex and
subsequent down-regulation of this complex at the surface of the cell
(7, 8). Second is the PKC-dependent phosphorylation of
phospholipase C, with a consequent reduction in phospholipase C
activity and IP3 generation (9). Third is the
phosphorylation of the plasma membrane Ca2+/ATPase, leading
to its activation and a facilitation of efflux of Ca2+
(10). Depending upon the methodology used and the cell type examined,
activation of PKC can (11) or cannot (12) be shown to affect the rate
of Ca2+ efflux following stimulation-induced increases in
[Ca2+]i. The fourth potential target for a PKC
effect is on the rate of Ca2+ influx itself, as suggested
for HPB-ALL cells (13). In this report, we show that in Jurkat T cells
the rate of Ca2+ influx is reduced by approximately 50%
following activation of PKC.
The PKC family consists of at least 11 isozymes that vary in their
requirements for lipid and Ca2+ for activation and are
differentially expressed in various cell types (reviewed in Refs. 14
and 15). The study of the functions of this family of isozymes in
cellular regulation has been facilitated by the use of phorbol esters,
such as phorbol myristate acetate (PMA), which activate PKC, although
with little specificity for any individual isozyme (14). In the past,
the lack of isozyme-specific inhibitors (16) has made assignment of a
PKC effect to a specific isozyme difficult. However, the recent
development of some isozyme-specific inhibitors (17) and the use of
technology such as RNA aptamers to block transcription of individual
isozymes (18) is beginning to allow for the assignment of specific
functions within a given cell type to a specific isozyme. For example,
the development of a specific isozyme inhibitor has implicated PKC
directly as a mediator of the vascular dysfunctions seen in diabetic
rats (17).
In T lymphocytes, expression of the various isozymes of PKC depends upon the cell lineage and the state of differentiation of the cell line (for example, see Ref. 19). The state of differentiation also confers upon T lymphocytes varying degrees of susceptibility to apoptosis induced by increases in [Ca2+]i (20). While apoptosis is used by the immune system to regulate the expression of specific subsets of T lymphocytes during development, disregulation of this process can lead to immune suppression (21). To further define the role of PKC in T cell activation, we have examined the expression of PKC isozymes in Jurkat T lymphocytes and begun the determination of which isozyme(s) may function in the normal down-regulation of antigen- or agonist-induced increases in [Ca2+]i.
Jurkat E6.1 and HSB cells were purchased from ATCC (Rockville, MD). Cells were maintained in RPMI 1640 (BioWhitaker, Walkersville, MD) containing 5% fetal bovine serum (Hyclone, Logan, UT), SerXtend (Irvine Scientific, Santa Ana, CA), and glutamine (BioWhitaker, Walkersville, MD) at 37 °C in a CO2 incubator.
ReagentsIndo-1 and BAPTA were purchased from Molecular Probes (Eugene, OR). Anti-PKC isoform-specific antibodies and the peptides used for immunization were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Unless otherwise indicated, all other reagents were purchased from Sigma.
Intracellular [Ca2+]i MeasurementsCells at a concentration of 4 × 106/ml were incubated with 1 µM indo-1/AM for 1 h at 37 °C in culture medium. Cells were washed three times in buffer A (10 mM HEPES, pH 7.4, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 140 mM NaCl, 0.1% glucose, 1% fetal bovine serum) and suspended at a final concentration of 1 × 106/ml in buffer A. The [Ca2+]i was determined from the fluorescence ratio (398/480 nm) with excitation at 360 nm in an SLM 8100 spectrofluorometer (SLM/Aminco, Urbana, IL) in the T format as described previously (4, 22). Calibration was conducted as described previously (22, 23) using the equation developed for an earlier generation of Ca2+ indicator dyes (24, 25).
Western BlottingCells were lysed in SDS-polyacrylamide gel electrophoresis sample buffer and 50 µl, representing 5 × 105 cells, were applied to a 10% acrylamide gel. Proteins were then transferred to blotting membrane and probed for isoform expression using enhanced chemiluminescence (ECL) detection (Amersham Corp.) according to the manufacturer's directions.
Electroporation of AntibodiesThe protocol for electroporation was based on that published by Lukas et al. (26). Briefly, cells were washed three times in buffer B (10 mM PO4, pH 7.4, 150 mM NaCl) and suspended to a final concentration of 2 × 108/ml. Cells (50 µl) were mixed with 50 µl of antibody at 100 µg/ml in buffer B plus 0.1% NaN3 and 0.2% gelatin (as supplied by Santa Cruz Biotechnology) and placed in the electroporation chamber. For those experiments using antibody plus peptide, 50 µl of buffer B or 50 µl of peptides (200 µg/ml in buffer B plus 0.1% NaN3 and 200 µg/ml bovine serum albumin as supplied by Santa Cruz Biotechnology) was mixed with the anti-PKC antibodies and incubated on ice for 30 min prior to the addition of 50 µl of cells to the electroporation chamber. Electroporation (CellPorator, Life Technologies, Inc., Bethesda, MD) was carried out with the following settings: high resistance, 270 V, and 800 microfarads. These settings were chosen based on testing of multiple settings and balancing percentage of cell survival with antibody incorporation (determined by Western blotting and immunofluorescence; data not shown). Cells were then removed from the electroporation chamber, suspended to 2 × 106/ml in culture medium, and placed in a CO2 incubator for a period of recovery (6-18 h) prior to use. Controls include cells mixed with antibody but not electroporated or cells electroporated in the absence of antibody.
ImmunofluorescenceCells subjected to the electroporation
procedure and prepared for use in experiments to measure changes in
[Ca2+]i were allowed to settle on
poly-L-lysine-coated microscope slides. The cells were
lightly permeabilized by incubation with 9% buffered formalin and
washed three times in buffer B. Anti-PKC antibody was visualized by
incubating the slide with fluorescein isothiocyanate-labeled goat
anti-rabbit antibody. For the images presented in Fig. 8, all
fluorescence fields were photographed using T-Max 400 film (Eastman
Kodak Co., Rochester, NY) using a 16-s exposure time. Phase contrast
images were taken of the same fields immediately thereafter using a 3-s
exposure time.
Statistics
All statistical analyses were performed using GraphPad Prism or Microsoft Excel.
In most electrically
nonexcitable cells, the Ca2+ entry pathway can be opened by
depletion of the intracellular Ca2+ storage depot (1). In T
cells, the Ca2+ entry pathway can be opened in a
receptor-dependent or -independent manner, using,
respectively, an activating monoclonal antibody to the T cell receptor
(OKT3) or thapsigargin (4). The effects of activation of PKC by the
phorbol ester PMA on these two methods of opening the Ca2+
entry pathway are shown in Fig. 1. The addition of PMA
prior to stimulation of Jurkat cells with thapsigargin results in a concentration-dependent decrease in the maximum
[Ca2+]i reached (Fig. 1A). Fig.
1B demonstrates that the inhibitory effect of PMA also
occurs once the Ca2+ entry pathway has been opened. The
addition of PMA at a time when the storage pool has refilled but the
Ca2+ entry pathway is still open (4) results in a
concentration-dependent reduction of
[Ca2+]i. Similar results were observed when the
Ca2+ entry pathway was opened in a
receptor-dependent manner using the anti-CD3-specific
antibody OKT3 (Fig. 1, C and D). Previously, it
has been reported that such inhibitory effects of PMA are not seen in
all T lymphocytes (19). Therefore, a different T cell line, HSB, was
examined as well. This human T cell line has lost expression of the T
cell receptor for antigen; thus, the Ca2+ entry pathway can
only be opened by thapsigargin treatment. As shown in Fig.
2, there was no effect of up to 3 µM PMA
on the changes in [Ca2+]i induced by thapsigargin
in this cell line.
It has been proposed that the decreased magnitude of agonist-stimulated
changes in [Ca2+]i after PMA pretreatment is due
to a PKC-dependent phosphorylation and inactivation of
phospholipase C (9). Such a phosphorylation would result in decreased
production of IP3 with a corresponding reduction in the
amount of Ca2+ released from the intracellular storage
depot. To measure the amount of Ca2+ released from the
intracellular storage pool without interference from Ca2+
influx, extracellular Ca2+ was chelated with EGTA prior to
stimulation. Consistent with inhibition of phospholipase C, prior PMA
treatment results in a reduction in the amount of Ca2+
released from the intracellular storage pool following receptor stimulation in Jurkat cells (Fig. 3A).
However, the same inhibition of Ca2+ release is seen with
thapsigargin treatment (Fig. 3C). Since thapsigargin
increases [Ca2+]i without the involvement of the
T cell receptor, it seems unlikely that this latter effect is due to
inhibition of phospholipase C. Whatever the site of PKC action is, no
significant effect on [Ca2+]i was observed when
PMA was added after stimulation of Ca2+ release in the
absence of Ca2+ influx (Fig. 3, B and
D).
Phorbol Ester Treatment Specifically Affects Ca2+ Influx in Jurkat Cells
It has been proposed that activation of
PKC results in activation of the plasma membrane Ca2+ pump,
such that there is a more rapid efflux of intracellular Ca2+ following PMA treatment (10). Additionally, it is
possible that activation of PKC inhibits Ca2+ influx. To
examine which of these mechanisms might be active in Jurkat cells, it
was necessary to measure influx and efflux separately. To measure
Ca2+ pump activity, cells were stimulated to open the
Ca2+ entry pathway, and then extracellular Ca2+
was rapidly chelated by the addition of 3 mM BAPTA to the
extracellular medium (Fig. 4A). At this
point, the rate at which Ca2+ returns to base line is a
reflection of the activity of the Ca2+ pump, since there is
no Ca2+ entry. Because the rate of pump activity is
dependent primarily upon the [Ca2+]i (27), a
calculation of the exponential rate of decay was used to provide an
estimate of pump activity; i.e. calculation of the
t1/2 for the return of Ca2+ to basal
levels can be used to compare efflux under various experimental conditions (Fig. 4B).
Unlike a calcium channel blocker such as Ni2+ (4), chelation of extracellular Ca2+ with BAPTA does not close the Ca2+ entry pathway. Therefore, readdition of extracellular Ca2+ to the cell suspension (Fig. 4A) allows for the measurement of entry specifically during the first 10-15 s after readdition of Ca2+ (27). Because the activity of the plasma membrane pump is governed by [Ca2+]i, during this time when [Ca2+]i is low, pump activity will be greatly reduced. Therefore, a straight line function can be applied, and the rate of Ca2+ influx in nmol/s can be determined and compared under the various experimental conditions (Fig. 4C).
Influx and efflux rates in the presence and absence of PMA were
calculated from several independent experiments (Fig.
5). PMA caused a slight, but reproducible, decrease in
the rate (increased t1/2) of Ca2+
efflux, measured during the 30 s following the addition of BAPTA in Jurkat cells (Fig. 5B). In paired experiments, this
difference ranged from a 13 to 23% change but was not, overall,
statistically significant. However, PMA caused a pronounced,
statistically significant decrease in the rate of Ca2+
influx measured in Jurkat cells during the 15 s following
readdition of extracellular Ca2+. This reduction ranged
from 37 to 56% when thapsigargin was used and up to 71% when cells
were stimulated through the T cell receptor with OKT3 (Fig.
5A).
Because the release of Ca2+ from the internal stores is reduced in the presence of PMA (Fig. 3), it is possible that this reduction in the rate of influx is merely a consequence of reduced release from internal stores. However, when the rate of influx was measured in cells stimulated with a concentration of OKT3 that resulted in a 50% reduction in the amount of Ca2+ released from internal stores (0.1 µg/ml of OKT3, data not shown), the rate of influx was not different from the rate of influx in the presence of the maximally effective concentration of 1 µg/ml of OKT3 (83-102 nmol/s with 1 µg/ml OKT3 versus 98-114 nmol/s using 0.1 µg/ml OKT3, n = 3).
As a control for the calculations of influx and efflux, the same experiment was performed using HSB cells, which show no response to PMA on thapsigargin-induced changes in [Ca2+]i. No differences in Ca2+ influx or efflux were observed between cells that were treated with 100 nM PMA and those that were not (Fig. 5, right bars). For HSB cells, influx rates in the presence of PMA varied from 82 to 105% of control, and efflux rates varied from 93 to 110% of control.
In many cell types, treatment with PMA for 12-18 h ablates the effects
of acute PMA addition; under most circumstances, this is due to
extensive proteolytic degradation of PKC (28). To determine whether
this same phenomenon occurred in Jurkat cells, cells were treated for
18 h with 250 nM PMA, and the effects of acute PMA
addition on stimulated changes in [Ca2+]i were
examined. Fig. 6 demonstrates that the rapid return to
basal levels that occurs following acute PMA addition (Fig.
6A) was lost in cells that had been treated overnight with PMA (Fig. 6B). Similarly, when influx and efflux were
measured as in Fig. 4, the effect of PMA on the rate of
Ca2+ influx was completely lost in cells that had been
treated for 18 h with PMA prior to stimulation (Fig.
6C).
Involvement of PKC
As outlined above, PKC is a family of serine-threonine
kinases, and although the isozymes appear to be differentially
sensitive to PMA within any single cell type, there is little
consistency among various cell types (29, 30). To determine whether the lack of phorbol ester-induced inhibition of Ca2+ influx in
HSB cells was due to the absence of a certain PKC isozyme, isozyme
expression in Jurkat and HSB cells was compared. As shown in Table
I, although there were some differences in the level of
expression of a specific isozyme (for example, PKC), there were no
clear cut examples of an isozyme present in Jurkat cells but absent in
HSB.
|
Because the effect of acute PMA addition was ablated by 18 h of
PMA exposure, the Western blot analyses were repeated in Jurkat and HSB
cells that had been incubated for 18 h with 250 nM
PMA. Expression of PKC, -
II, and -
were unchanged following
this treatment (data not shown). However, PKC
I expression was
markedly reduced following PMA treatment in Jurkat cells but only
minimally affected in HSB cells (Fig. 7). This lack of
an effect of PMA in HSB cells is similar to that observed by others
(31, 32).
Since isozyme-specific inhibitors (16) are not commercially available,
the effect of electroporation of isozyme-specific antibodies on
Ca2+ entry was examined to garner additional support for a
role for PKCI in inhibition of Ca2+ entry. Jurkat cells
were electroporated in the presence of isozyme-specific antibodies as
outlined under "Materials and Methods." As shown in Fig.
8, cells mixed with antibody but not electroporated
showed no incorporation of antibody (Fig. 8A). However,
electroporation resulted in a reasonably uniform incorporation of
antibody, as visualized by immunofluorescence (Fig. 8B).
Prior incubation of antibody with the peptide used for immunization did
not affect the incorporation of antibody (Fig. 8C).
The effects of the incorporation of antibody on agonist-induced changes
in [Ca2+]i were examined next. Jurkat cells that
had been electroporated in the presence of anti-PKCI showed a
reduced rate of return of [Ca2+]i to base line
compared with cells that had been electroporated in the absence of
antibody or those electroporated in the presence of anti-PKC
antibody (Fig. 9A). In five such experiments,
electroporation in the presence of anti-PKC
I reduced this rate of
decrease in [Ca2+]i by 40-45%, while
electroporation in the presence of anti-PKC
or anti-PKC
had no
significant effect on this rate (Fig. 9B). Furthermore, the
effects of anti-PKC
I antibody could be blocked by a 2-fold mass
excess of the peptide used to generate this antibody, but not the
peptide used to generate the anti-PKC
antibody (Fig.
9B).
To determine if this reduction in rate was due to reduced efflux or
enhanced influx, both were measured in cells electroporated with the
isozyme-specific antibodies using the protocol described for the
experiment of Fig. 4. The rate of efflux of Ca2+ from cells
electroporated in the presence of anti-PKCI was very similar to the
rate observed in cells electroporated in the absence of antibody (Fig.
10B) or in cells electroporated in the
presence of anti-PKC
(data not shown). Inclusion of peptides for
PKC
or -
caused a small but statistically insignificant increase in the efflux time (decrease in rate). There was a slight reduction in
the magnitude of the PMA effect in cells electroporated with either
anti-PKC antibody, compared with cells electroporated without antibody.
However, this effect could not be assigned to a specific isozyme of
PKC.
The effects of electroporation were more striking when the influx of
Ca2+ was examined. In individual experiments, a 25-40%
increase in the rate of Ca2+ influx was observed in cells
electroporated in the presence of anti-PKCI compared with a less
than 5% decrease in Ca2+ influx in cells electroporated in
the presence of anti-PKC
or anti-PKC
(Fig. 10A).
Additionally, the effect of PMA treatment on Ca2+ influx
was lost in cells electroporated in the presence of anti-PKC
I, while
the PMA effect was still seen in cells electroporated with anti-PKC
or anti-PKC
. As with the effects of anti-PKC
I antibody shown in
Fig. 9, the effects of anti-PKC
I on Ca2+ influx could be
blocked by a 2-fold mass excess of the peptide used for immunization
but were not blocked by the peptide used for the production of
anti-PKC
antibody (Fig. 10A).
Control of intracellular free Ca2+ concentration is a critical component of cellular homeostasis for all cell types. Increases in [Ca2+]i subsequent to receptor stimulation are necessary for the regulated progression of most cells through the cell cycle (33), for appropriate expression of gene products such as cytokines in T lymphocytes (1), and for some T cell effector functions, such as perforin-dependent cell killing by cytolytic T lymphocytes (34). Equally necessary is the regulated decrease with a return to prestimulation calcium levels subsequent to any increase. The consequence of a sustained increase in [Ca2+]i is generally cell death, often due to induction of apoptosis (21).
Changes in [Ca2+]i subsequent to engagement of the T cell receptor for antigen are initiated by release of Ca2+ from an internal storage depot mediated by the IP3 generated by receptor-induced activation of phospholipase C. This release of stored Ca2+ is followed by an influx of extracellular Ca2+. This process has been called by a number of names in electrically nonexcitable cells, such as capacitative calcium entry and the storage-operated calcium entry pathway (1). Regardless of the nomenclature, most current models for this component of cellular signaling indicate a feed forward mechanism.
In general, PKC activation, mediated by the other product of phospholipase C, DAG, has been associated with down-regulation of this pathway, although the data are not wholly consistent. The discrepancies may be due to differences in methodologies employed as much as to differences in cell lines examined. For example, in mixed populations of peripheral blood lymphocytes, phorbol ester treatment has been shown to have either an inhibitory (12) or augmenting (8) effect on receptor-mediated changes in [Ca2+]i. These opposing results may be due to the relative state of differentiation of the cells as at least one study has implied (19). The data are more consistent when cell lines are examined. When changes in [Ca2+]i are examined in Jurkat cells using fluorescent dyes, stimulation through the TCR with prior activation of PKC is associated with a reduction in the magnitude of increases in [Ca2+]i (this report and Refs. 8, 11, and 35). These results are in contrast to stimulation of Jurkat cells with a mitogenic lectin, such as concanavalin A, where there appear to be no effects of PKC activation on the subsequent changes in [Ca2+]i (35).
In addition to controversy over the effects of PKC activation, multiple targets of PKC in the Ca2+ entry pathway have been proposed. While in both a murine T lymphoma line (9) and in neutrophils (7, 36) phosphorylation and down-regulation of receptors by PKC has been implicated, this observation cannot explain the effect of PMA on receptor-independent changes in [Ca2+]i. Similarly, a reduction in IP3 generated due to a PKC-dependent phosphorylation of phospholipase C (9) fails to explain the observed reduction in release of Ca2+ from internal stores following thapsigargin treatment (Fig. 3).
The effects of PKC activation on Ca2+ influx versus efflux also have been examined. In peripheral blood lymphocytes, no effect on efflux following PMA treatment was seen when a straight line function was applied to the decrease in [Ca2+]i following chelation of extracellular Ca2+ (12). However, in Jurkat cells, activation of PKC was associated with enhanced efflux of Ca2+ when a nonlinear, biexponential calculation was used (11). Efflux of Ca2+ has been associated with a pump mechanism, most likely the plasma membrane Ca2+/ATPase (27). The activity of the pump has been shown to be dependent upon the intracellular concentration of Ca2+ (27), which we believe makes this application of a nonlinear function more appropriate. The observation of a PMA effect on Ca2+ efflux is confirmed by the data in this report, although the magnitude of the change was slightly less in the current study than observed in the previous one (11), perhaps due to the different curve fitting method used in this study.
There is more consistency when the effects of PKC activation on Ca2+ influx are examined. In nonlymphoid cells, PKC activation was associated with inhibition of Ca2+ influx when the current induced by Ca2+ influx was measured (37). In another study, a current others (38), but not ourselves (5), have implicated as the mediator of storage pool depletion-induced Ca2+ entry was reduced in the presence of activated PKC (38). Consistent with these observations, we observed a 30-50% inhibition of the rate of Ca2+ influx in Jurkat cells following PKC activation with PMA (Fig. 5).
Some of the discrepancies reported among various cell types may be due
to differences in the expression of individual isozymes of PKC.
Differences in the Ca2+ and lipid requirements for
activation of the various isozymes suggest that specific functions are
associated with each. Isozyme-specific functions in T cells were
recently elegantly demonstrated in a study examining a subclone of
Jurkat cells transfected with constitutively active expression vectors
for different isozymes (39). Regulation of some transcription factors
was enhanced in cells that overexpressed PKC but not PKC
(39).
Several observations in the present study support a role for PKCI in
inhibition of Ca2+ influx. First, although PKC isozyme
expression did not significantly differ between Jurkat cells that
respond to PMA and HSB cells that did not, PKC
I was down-regulated
by long term PMA treatment only in Jurkat cells (Fig. 8). Second, this
long term PMA treatment abolished the PMA-induced inhibition of
Ca2+ influx (Fig. 7). Third, electroporation of an antibody
to PKC
I, but not an antibody to PKC
or PKC
, reduced the rate
of Ca2+ influx and abolished the PMA effect (Fig. 10).
The presence of PKCI in HSB cells seems difficult to reconcile with
the failure of these cells to exhibit PMA-induced down-regulation of
[Ca2+]i if this isozyme is responsible for the
effect. However, the failure of PMA to down-regulate this isozyme in
HSB cells (Fig. 7) suggests that PMA may fail also to activate the
isozyme in these cells. This is consistent with previous reports
suggesting that HSB cells fail to respond to PMA (31, 32). Other
possible explanations for the failure of these cell to down-regulate
[Ca2+]i despite the presence of PKC
I include a
defective enzyme and altered or missing downstream substrates.
Collectively, the results of these and other studies provide support
for a closed loop pathway initiated by engagement of the TCR in human
lymphocytes. Following TCR ligation and activation of phospholipase C,
IP3 induces the release of Ca2+ from the
intracellular stores. This released Ca2+ activates
calmodulin, which initiates the influx of extracellular Ca2+ (23), carried by a current we have named
IT (5). This feed forward portion of the pathway
is down-regulated by DAG-activated PKCI, which inhibits
Ca2+ influx. Whether or not this inhibition is by an action
on IT is currently under investigation.
We thank Paul Jung for excellent technical work examining Ca2+ influx and efflux and Chien Ying Lee for assistance with Western blots.