Department of Physiology and Biophysics, University of California at Irvine, Irvine, California 92697
Extracellular ATP (ATPo) elicits a robust
change in the concentration of intracellular Ca2+
([Ca2+]i) in fura-2-loaded mouse thymocytes. Most thymocytes (60%) exposed to ATPo exhibited a biphasic
rise in [Ca2+]i; [Ca2+]i rose slowly at first to a mean
value of 260 nM after 163 s and then increased rapidly
to a peak level of 735 nM. In many cells, a declining plateau, which lasted for more than 10 min, followed the
crest in [Ca2+]i. Experiments performed in the absence
of extracellular [Ca2+]o abolished the rise in thymocyte
[Ca2+]i, indicating that Ca2+ influx, rather than the release of stored Ca2+, is stimulated by ATPo. ATPo-
mediated Ca2+ influx was potentiated as the [Mg2+]o was
reduced, confirming that ATP4 is the active agonist
form. In the absence of Mg2+o, 3
-O-(4-benzoyl)benzoyl-ATP (BzATP) proved to be the most effective agonist of
those tested. The rank order of potency for adenine nucleotides was BzATP4
>ATP4
>MgATP2
>ADP3
,
suggesting purinoreceptors of the P2X7/P2Z class mediate the ATPo response. Phenotyping experiments illustrate that both immature (CD4
CD8
, CD4+CD8+)
and mature (CD4+CD8
, CD4
CD8+) thymocyte populations respond to ATP. Further separation of the
double-positive population by size revealed that the
ATPo-mediated [Ca2+]i response was much more pronounced in large (actively dividing) than in small (terminally differentiated) CD4+CD8+ thymocytes. We
conclude that thymocytes vary in sensitivity to ATPo
depending upon the degree of maturation and suggest
that ATPo may be involved in processes that control
cellular differentiation within the thymus.
EXTRACELLULAR ATP (ATPo)1 and its metabolic
products evoke physiological responses in virtually
all tissues and cell types from central nervous to peripheral organ systems (for review see Dubyak and El-Moatassim, 1993 ATPo elicits a broad spectrum of physiological changes in
cells of the immune system. In mast cells, ATP release has
been shown to mediate cell-to-cell signaling (Osipchuk
and Cahalan, 1992 Based upon a sensitivity profile for purine agonists and
pharmacological agents, lymphocytes are not believed to
possess G protein-linked purinoceptors (El-Moatassim et
al., 1989b In this study, we examined the dynamics of [Ca2+]i
changes elicited by ATPo at the single-cell level in fura-2-
loaded thymocytes. To our surprise, we found that the
ATPo-mediated [Ca2+]i increase varies significantly between individual cells. Moreover, the kinetics of the rise in
[Ca2+]i at the single-cell level is characterized by a biphasic
time course that is not detectable in average profiles. To
correlate stages of thymocyte development with the degree of sensitivity to ATPo, we measured the surface expression of specific T-lymphocyte markers, CD4 and CD8,
before performing Ca2+-imaging experiments. Our data illustrate that thymocytes vary in sensitivity to ATPo depending upon level of maturation and degree of blastogenesis. Small, terminally differentiated, CD4+CD8+ thymocytes
were least sensitive to ATPo, while 90% of the single-positive (CD4+CD8 Preparation of Cells and Fura-2 Loading
Intact thymus glands were extracted from 4-8-wk-old female BALB/c
mice (The Jackson Laboratory, Bar Harbor, Maine). After gentle dissociation of the thymus between two frosted microscope slides, thymocytes
were washed free in RPMI-1640 media (GIBCO BRL, Gaithersburg,
MD) supplemented with 10% fetal calf serum (JR Scientific, Woodland,
CA), 25 mM Hepes, and 2 mM glutamine. Thymocytes in suspension were
centrifuged at 350 g for 10 min, resuspended in complete RPMI to 5 × 106
cells/ml, and then stored for <15 min before dye loading. Cells were maintained at 22°C during preparation, storage, dye loading, phenotyping, and
experiments.
Thymocytes were loaded with 3 µM fura-2/AM (Molecular Probes,
Inc., Eugene, OR) for 20 min. The cells were then washed three times with
RPMI/10% FCS and stored in the dark. The loss of dye and the sequestration of fura-2 into intracellular compartments was minimal during storage
(typically <6 h), as revealed by subsequent experiments. Thymocyte isolation
and Ca2+-imaging experiments were always performed on the same day.
Chemicals and Solutions
Thymocytes were bathed in mammalian Ringer containing (in mM) 160 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 Hepes, and 10 glucose, titrated to pH
7.4 with NaOH (310 mOsmol kg Fura-2 Imaging and Calibration
Fura-2 loaded thymocytes were allowed to settle on poly-D-lysine-(1 mg/
ml) coated #0 coverslips (#0 red Label; Thomas Scientific, Swedesboro,
NJ) for 10 min and then washed with Ringer on the stage of a fluorescent
microscope (Axiovert 35; Zeiss, Inc., Oberkochen, Germany). Illumination was provided by a xenon arc-lamp (Zeiss, Inc.) and transmitted
through a filter wheel unit ( Lymphocyte Phenotyping
The presence or absence of CD4 and CD8 molecules provides a measure
of T-lymphocyte maturation within the thymus (Scollay et al., 1988 We identified thymocyte surface phenotype before performing Ca2+
imaging for several experiments. Thymocytes were labeled with CD4
(L3T4) and CD8 (Lyt-2) monoclonal antibodies (Pharmingen, San Diego,
CA) conjugated with phycoerythrin (PE) or FITC, respectively. 30-µl aliquots of thymocytes (1.5 × 105 cells) were incubated at 22°C with 5 µg/ml
anti-CD4-PE, 5 µg/ml anti-CD8-FITC, and 3 µM fura-2/AM for 20 min.
After staining, cells were washed three times in complete RPMI and resuspended to a final volume of 400 µl; 50 µl aliquots of stained cells were adhered to poly-D-lysine-coated coverslip chambers for 10 min before experiments.
A xenon light source was used to evaluate the CD4/CD8 phenotype of
labeled thymocytes. The filter sets used for phenotyping were optimized
for the separation of PE and FITC signal, while excluding fura-2 fluorescence. The PE set contained a 510- to 560-nm excitation filter, a 580-nm
dichroic beam splitter, and a 590-nm-long pass emission filter. The FITC
set included a 450- to 490-nm excitation filter, a 510-nm dichroic beam
splitter, and a 40-nm-wide, 540-nm emission filter. Red (CD4/PE) and
green (CD8/FITC) fluorescence images of labeled cells were collected before [Ca2+]i measurements with a black and white SIT camera. The use of narrow filters for fura-2 excitation prohibited PE and FITC fluorescence contamination during [Ca2+]i measurements. Cells were classified by visual inspection into four phenotypic categories by toggling between fluorescence images after experiments: (a) lymphocytes exhibiting fluorescence using both filter sets (double positives); (b) cells exhibiting only PE
fluorescence (CD4+CD8
Data Analysis
Numerical values for single-cell [Ca2+]i traces were analyzed with Igor
Pro, an Apple Macintosh computer program (v3.02; WaveMetrics, Inc.,
Lake Oswego, OR). Statistical analysis was performed on data sets using
Excel v5.0 (Microsoft, Redmond, WA), SuperAnova v1.11 (Abacus Concepts, Inc., Berkeley, CA), and Systat v5.1 (Systat Inc., Evanston, IL). The
composite pseudocolor overlay presented in Fig. 4 A was generated with
Photoshop v4.0 (AdobeSystems Inc., San Jose, CA). Data are reported as
mean ±SD. Analysis of variance (ANOVA) was used to compare mean
values. Pairs of means were considered statistically different if P was
<0.05.
ATPo Induces a Rise in [Ca2+]i in Mouse Thymocytes
ATPo is recognized as an effective modulator of [Ca2+]i
levels within lymphocytes (El-Moatassim et al., 1987
A narrow band centered at 260 ± 29 nM marks the position of a distinct inflection in the [Ca2+]i curves of ATPo-sensitive thymocytes (Fig. 2 A). The inflection gave these
graphs a well defined biphasic character and signaled a
faster rate of rise in [Ca2+]i averaging 19 ± 19 nM/s. As illustrated by three single-cell traces in Fig. 2 A, neither the
length of time before reaching the threshold, nor the peak
value after the inflection, correlated with the level of
[Ca2+]i at which the transition occurred. These observations are presented graphically in Fig. 2 B; while peak thymocyte [Ca2+]i levels are scattered randomly, inflection
point [Ca2+]i levels are tightly clustered.
The [Ca2+]i Rise Is Stimulated Most Potently
by BzATPo
To identify the type of purinoceptor expressed in thymocytes, we tested the efficacy of various nucleotides and
ATP analogs in raising [Ca2+]i. Thymocytes were least
sensitive to ADPo; only 20% (n = 4 experiments, 303 cells) of the cells exhibited a rise in [Ca2+]i after the application of 1 mM NaADP (data not shown). [Ca2+]i time
courses were similar for ADPo- and ATPo-sensitive thymocytes, suggesting that both nucleotides stimulate a single purinoceptor subtype. BzATP, in the absence of extracellular Mg2+, proved to be the most effective agonist and
elicited a rapid rise in thymocyte [Ca2+]i when used at 100 µM (Fig. 3 A). Single-cell [Ca2+]i profiles evoked by BzATPo,
ATPo (compare Fig. 3 B with 2 A), and ADPo were comparable; these agonists caused a biphasic [Ca2+]i rise in
thymocytes after crossing a distinct threshold (Fig. 3 C).
On average, 65 ± 2% (n = 2 experiments, 147 cells) of the BzATPo-treated cells exhibited a slow rise in [Ca2+]i to a
threshold level of 263 ± 33 nM. Above the inflection
point, [Ca2+]i increased at a rate of 17 ± 17 nM/s and
reached a peak averaging 702 ± 249 nM. Similarly, in
transformed fibroblasts (Gonzalez et al., 1989
Phenotypic Classes of Thymocytes Differ in Sensitivity
to ATPo
We examined the surface expression of CD4 and CD8
molecules with fluorescently labeled antibodies to address
the hypothesis that stages of thymocyte maturation correlate with sensitivity to ATPo. In Fig. 4 A, fluorescence images of anti-CD4-PE and anti-CD8-FITC labeled thymocytes
were color coded and overlayed to aid phenotyping. The
fluorescence intensity of individual cells varied, indicating unequal surface expression of CD4 and CD8. Most cells in
PE and FITC composite images correspond to small, double-positive thymocytes; these terminally differentiated
thymocytes constitute 50% of the cells in a typical thymus
preparation (Table I). Larger double-positive thymocytes,
representing 13% of the total population, were easily discriminated from smaller cells based upon surface area outlines in PE and FITC images. After recording fluorescence
images, thymocytes were treated with 100 µM BzATPo. A
snapshot pseudocolor image of [Ca2+]i within cells, acquired 100 s after BzATPo application, is displayed in Fig.
4 B. Selected single-cell [Ca2+]i profiles, representing the
five thymocyte populations, are illustrated in Fig. 4 C. As a
group, the small, terminally differentiated, double-positive
thymocytes were least responsive to BzATPo, while CD4 Table I.
Maturational Stages in Thymocyte Development Show Distinct [Ca2+]i Response Profiles
The ATPo-mediated Rise in [Ca2+]i Does Not Occur in
the Absence of Ca2+o
The nucleotide-dependent rise in thymocyte [Ca2+]i may
be generated by Ca2+ influx across the plasma membrane
or Ca2+ release from intracellular reservoirs. We examined the mechanism leading to the [Ca2+]i response by performing experiments under Ca2+o-free conditions. In Fig.
6, thymocytes were treated with 100 µM BzATPo in Ca-free media for 100 s. A slight rise in [Ca2+]i was registered
in a few thymocytes in zero Ca2+o (Fig. 6 A), however the
majority of cells remained at or near pre-agonist [Ca2+]i
levels. Ca2+o readdition to BzATPo-treated thymocytes
caused a vigorous rise in [Ca2+]i, indicating that Ca2+ influx dominates the response. We have shown previously
that stored Ca2+ levels remain high in thymocytes bathed
in Ca-free media over comparable incubation periods
(Ross and Cahalan, 1995
Ca2+ Influx and the Potency of ATPo Increase as Mg2+o
Is Reduced
Extracellular Mg2+ and Ca2+ ions are thought to play an
important role in regulating ATP-mediated responses in
mast cells (Cockcroft and Gomperts, 1979b
We also examined purinoceptor activity after agonist
withdrawal (Fig. 8). A [Ca2+]i rise was evoked by 1 mM
MgATP in the presence of 1 mM [Mg2+]o. Agonist was
then withdrawn, which caused [Ca2+]i to decrease, as expected. Removing Mg2+o resulted in an unanticipated rise
in [Ca2+]i, in the absence of agonist, indicating that purinoceptors remain occupied by ATP. We believe this
ATP to be initially in the form of MgATP2
Given these data, we conclude that MgATP2 We have characterized ATPo-mediated [Ca2+]i responses
in thymocytes at the single-cell level. The [Ca2+]i profile is
distinguished by a biphasic time course with a distinct inflection (Figs. 1, 2, and 3). The rise in [Ca2+]i is generated
primarily by Ca2+ influx, with Ca2+ release from stores
playing little if any role (Fig. 6). The response is potently
stimulated by BzATPo, supporting the conclusion that thymocytes express purinoceptors of the P2X7/P2Z variety (Fig. 3). Interestingly, actively dividing thymocytes are
preferentially targeted by extracellular nucleotides, such
that single-positive cells exhibit the most pronounced
[Ca2+]i increase (Fig. 4 and Table I). More than half of the
small, terminally differentiated, double-positive thymocytes
were insensitive to the effects of ATPo. Of the small double-positive cells that did respond, peak [Ca2+]i levels were
significantly less than those in all other classes of thymocytes (Table I). Consistent with other researchers, we find Mg2+-free ATP4 Extracellular ATP Stimulates a Biphasic Rise in
Thymocyte [Ca2+]i
By examining purinergic responses in thymocytes at the
single-cell level, our work has revealed that [Ca2+]i dynamics follow a more complicated profile than anticipated, based upon previous studies displaying average traces (El-Moatassim et al., 1987 The biphasic nature of the [Ca2+]i rise and the time
course variability among individual cells complicated our
efforts to develop reliable [ATP]o-[Ca2+]i response curves
(data not shown). The generation of precise nucleotide
concentration-response curves is also made problematic by the presence of surface ectonucleotidases, which degrade extracellular nucleotides rapidly, on CD4+CD8 Ca2+ Influx through Thymocyte Purinoceptors Depends
Critically on Mg2+o
One interpretation of the experiments presented in Figs. 7
and 8 is that ATP4 Significance of ATPo-mediated Ca2+ Influx
in Thymocytes
Members of the P2X family of purinoceptors have been
suspected of being associated with programmed death in
cells of the immune system since the work of Brake et al.
(1994) The possible role of ATPo in programmed cell death in
the thymus has been hotly debated. So far, three different
P2 purinoceptors have been detected in thymocytes at either the mRNA or protein level, or by responses to ATPo
stimulation (P2Y2, P2X1, P2X7/P2Z, respectively). The behavior of these multiple receptor subtypes may be responsible for the varied results in the literature. For instance,
estimates of the molecular weight cutoff for the pore function attributable to P2X7/P2Z purinoceptors in thymocytes vary between laboratories, ranging from a low value of 200 D (methylglucamine; Pizzo et al., 1991 Based upon our results we suggest that ATPo acts as a
generalized signal for growth and differentiation in lineages of thymocytes destined to become mature T cells. In
our scheme, double negative cells in the outer cortex of
the thymus are driven to differentiate by ATPo, which
would be expected to be at relatively low levels. It has
been shown that before TCR expression, double-negative thymocytes are resistant to dexamethasone-induced (Cohen et al., 1993 Ca2+-mediated signaling pathways have been implicated
in the positive selection of double positive thymocytes
driven by the interaction of ; Harden et al., 1995
). Tissues and isolated
cells vary in sensitivity to purine agonists. Nucleotides (ATP,
ADP, and AMP) and adenosine, the nucleoside product of
ATP catabolism, elicit distinct responses in target cells by
triggering P2 and P1 purinergic receptors, respectively
(Burnstock, 1978
). P2 purinoceptors can be further separated into two broad categories. The first group, divided
into P2Y and P2U subtypes, couples nucleotide binding
to effector molecules via G proteins. The second P2 category is comprised of nucleotide-sensitive ion channels and
pores. ATP-gated P2 purinoceptors, designated P2X1
through P2X6 (cation channels) and P2X7 (a dual
function cation channel/pore), display extensive sequence
identity (North, 1996
) but disparate tissue distribution, biophysical properties, agonist profiles, and pharmacology
(P2X1, Valera et al., 1994
; P2X2-P2X6, Collo et al., 1996
;
P2X7, Surprenant et al., 1996
). Moreover, P2X receptors functionally resemble acetylcholine- and serotonin-gated channels with respect to gating and ionic permeability
but are structurally unique. Thus, nucleotides, together with acetylcholine, glutamate, GABA, glycine, and serotonin, are included in a small group of compounds that function as agonists for a structurally diverse set of ligand-gated
ion channels and pores, as well as G protein-coupled receptors.
). In lymphocytes, ATPo triggers cellular
depolarization, greater permeability to small organic molecules (<400 D; Wiley et al., 1993
; Chused et al., 1996
),
and a rise in the concentration of intracellular Ca2+ ([Ca2+]i;
El-Moatassim et al., 1987
; Wiley and Dubyak, 1989
). The
ATPo-mediated rise in [Ca2+]i modifies the functional
properties of thymocytes via DNA synthesis (Gregory and
Kern, 1978
, 1981
; Ikehara et al., 1981
) and blastogenesis (El-Moatassim et al., 1987
). Moreover, an increase in
[Ca2+]i has been linked to programmed cell death in thymocyte populations; Ca2+ release from intracellular stores
evoked by thapsigargin, a microsomal Ca2+-ATPase inhibitor, triggers the DNA fragmentation correlated with thymocyte apoptosis (Jiang et al., 1994
; Zhivotovsky et al., 1994
).
). Rather, lymphocytes and related cell lines express purinoceptors of the ion channel/pore subtype (P2X7).
This ATP-gated pathway, originally termed P2Z (Gordon,
1986
), has been characterized in mast cells (Cockcroft and
Gomperts, 1979a
; Tatham and Lindau, 1990
), transformed 3T3 fibroblasts (Heppel et al., 1985
), macrophages (Buisman et al., 1988
), parotid acinar cells (Soltoff et al., 1992
),
and phagocytic cells of the thymic reticulum (Coutinho-Silva et al., 1996
). During whole cell patch-clamp experiments, putative P2Z channels in human B lymphocytes
(Bretschneider et al., 1995
) and rat peritoneal macrophages (Naumov et al., 1995
) exhibit rapid activation kinetics when exposed to ATPo. The ATPo response depends critically upon extracellular divalent cations (Mg2+
and Ca2+), such that cellular depolarization and membrane permeability are greatest in divalent-free media.
The ability of Mg2+- and Ca2+-ATP complexes to reduce
receptor occupancy by lowering the concentration of ATP4
,
the effective form of the nucleotide agonist, is a hallmark of P2X7/P2Z purinoceptor physiology (Cockcroft and Gomperts, 1979b
).
or CD4
CD8+) cells, believed to be the
immediate precursors of mature peripheral T-lymphocytes, exhibited a robust, ATPo-dependent rise in [Ca2+]i.
The in vitro data we have gathered suggest that ATPo may
drive thymocyte differentiation in the intact thymus.
Materials and Methods
1). MgCl2 and CaCl2 were omitted from
divalent free solutions; Mg- and Ca-free solutions were unbuffered with
respect to divalents. Immediately before experiments, nucleotides were
added to bathing solutions. Solution pH was readjusted to 7.4 after nucleotide addition. All chemicals, including the nucleotides 3
-O-(4-benzoyl) benzoyl-ATP (BzATP), Na2ATP, MgATP, and NaADP, were obtained
from Sigma Chemical Co. (St. Louis, MO).
10; Axon Instruments, Inc., Foster City, CA)
containing 360- and 380-nm excitation filters. The filtered light was reflected by a 400-nm dichroic mirror through a 100× oil-immersion objective to illuminate cells. Emitted light >480 nm was received by a SIT camera (C2400; Hamamatsu Photonics, Bridgewater, NJ) and the video
information relayed to an image processing system (Videoprobe; ETM
Systems, Irvine, CA). Full field-of-view 8-bit images, averaged over 16 frames, were collected at 360- and 380-nm wavelengths. Digitally stored
360/380 ratios were constructed from background-corrected 360- and 380-nm images. Single-cell measurements of [Ca2+]i were calculated from the
360/380 ratios using the equation of Grynkiewicz et al. (1985)
and a Kd of
250 nM for fura-2. The minimum fluorescence value at 380 nm and the
minimum 360/380 ratio were measured in single cells after incubation for
10 min in Ca2+-free Ringer containing 2 mM EGTA. Lymphocytes were
then superfused with Ringer containing 1 µM thapsigargin (LC Services,
Woburn, MA), 5 µM ionomycin (Sigma Chemical Co.), and 10 mM Ca2+ to
evaluate the maximum fluorescence at 380 nM and the maximum 360/380
ratio.
). An
early stage of thymocyte development is indicated by the absence of both
CD4 and CD8 surface markers. These cells, designated CD4
CD8
(double
negatives), differentiate into CD4+CD8+ thymocytes (double positives).
During the double-positive stage, thymocytes are positively selected for
proper recognition between T cell receptor (TCR) and self major histocompatibility proteins (MHC). Anomalously high affinity interactions between TCR and MHC target cells for negative selection. The majority of
thymocytes (95%) are not positively selected and die by neglect within the cortex of the thymus by a process termed apoptosis (Osborne, 1996
). Apoptotic thymocytes are small and terminally differentiated. Thymocytes receiving the proper signals continue to develop into medium and large size
double positives, eventually giving rise to single-positive T cell populations. Therefore, five classes of thymocytes can be identified based upon
cell size and the expression of CD4 and CD8: double negatives, small and
large double positives, and single positives.
single positives); (c) thymocytes exhibiting only
FITC fluorescence (CD4
CD8+ single positives); and (d) unlabeled thymocytes showing no fluorescence (double negatives) that only appeared
in fura-2 [Ca2+]i images. Alternatively, PE and FITC were color coded
and overlaid to produce a single composite image (see Fig. 4 A). The volume (V) of each thymocyte was determined from surface area (S) outlines using NIH Image v1.60 (a Macintosh computer program written by Wayne Rasband at the National Institutes of Health and available from
the Internet by anonymous ftp from <ftp;zippy.nimh.nih.gov> or on floppy disc
from NTIS, 5285 Port Royal Rd., Springfield, VA 22161; part no. PB93-504868) and the equation V = (S)3/2. For this relationship to be valid, thymocytes must remain spherical during experiments. Cell outlines in PE
and FITC images were used to determine the size of single- and double-positive thymocytes, while the volume of double negatives was determined from fura-2 [Ca2+]i images. Double-positive cells were further subdivided into large (actively dividing) and small (terminally differentiated) diameter classes.
Fig. 4.
Sensitivity to BzATPo correlates with stages of thymocyte maturation. (A) Thymocytes were labeled with anti-CD4-PE (red)
and anti-CD8-FITC (green) before BzATPo addition. Black and white fluorescence images were recorded, color coded, and then superimposed to aid phenotyping of labeled thymocytes. The surface expression of CD4 and CD8 are variable, leading to unequal fluorescence intensities emanating from CD4+CD8 (red), CD4+CD8+ (yellow), and CD4
CD8+ (green) thymocytes. The bright spot near the
center of the image is caused by an out of focus cell above the layer of thymocytes attached to the coverslip. (B) A pseudocolor display
of [Ca2+]i within fura-2-loaded thymocytes 100 s after BzATPo application. Below the panel is a rainbow bar correlating color with
[Ca2+]i. [Ca2+]i profiles of thymocytes within boxes are displayed in C. These identification boxes, which in some cases overlap with adjacent cells, were not used to evaluate thymocyte [Ca2+]i levels. (C) Representative single-cell [Ca2+]i profiles for five populations of
thymocytes. Small, terminally differentiated, double-positive thymocytes are least responsive, while 90% of the CD4
CD8+ thymocytes
treated with BzATPo exhibit a rapid rise in [Ca2+]i (experiment 9896-2; +/+, Cell 31; +/+ [large], Cell 77;
/
, Cell 64; +/
, Cell 53;
/+,
Cell 26).
[View Larger Versions of these Images (16 + 30K GIF file)]
Results
;
Pizzo et al., 1991
; Wiley et al., 1993
; Chused et al., 1996
). In
addition to activating Ca2+ influx pathways, ATPo is
thought to trigger the opening of pores in the plasma
membrane of thymocytes, which allow molecules of at
least 200 D to permeate (Pizzo et al., 1991
; Nagy et al.,
1995
; Chused et al., 1996
). In our experiments, thymocyte
ATPo-gated pores are narrow enough to restrict the passage of fura-24
(832 D), since we did not observe a decrease in fluorescence intensity at 360 nM during measurements (data not shown). Fig. 1 shows that the majority of
thymocytes (58%) exposed to 1 mM Na2ATP exhibit a robust change in [Ca2+]i at the single-cell level. In Fig. 1 A,
[Ca2+]i profiles for all thymocytes in a typical experiment
illustrate the variability of the ATPo response. From a
resting [Ca2+]i level of 131 ± 13 nM (n = 25 experiments,
2814 cells), ATPo triggered a rise in [Ca2+]i to peak levels
averaging 735 ± 230 nM (n = 3 experiments, 217 cells). A
slowly declining plateau persisted in many cells for more
than 10 min after peak [Ca2+]i (Fig. 1 B). Because the time
of initiation and amplitude of the rise in thymocyte [Ca2+]i
was variable, the average time course over the length of
the experiment is broadened (Fig. 1 C). Average [Ca2+]i
measurements by spectrofluorimetry (El-Moatassim et al.,
1987
; Pizzo et al., 1991
) and flow cytometry (Nagy et al.,
1995
; Chused et al., 1996
) of ATPo-treated thymocytes are
identical in profile with the upper graph in Fig. 1 C.
Fig. 1.
Mouse thymocytes
exhibit an ATPo-induced
rise in [Ca2+]i. (A) [Ca2+]i is
plotted against time for all
cells in a typical experiment to illustrate the range of responses to ATPo at the single-cell level. Superfusion of
ATPo began at 50 s, as indicated by the application bar
above the graph and the vertical dotted line. During the
application of ATPo, the bath
solution was exchanged over
a period of 20 s. The time to
peak [Ca2+]i and the level of
[Ca2+]i at the crest varied significantly between thymocytes. This experiment
was performed with normal
Ringer's solution containing 1 mM Mg2+o. (B) Two populations of thymocytes can be
identified by sensitivity to
ATPo; 58% of the cells show
a rise in [Ca2+]i, while the
balance exhibits little change
in [Ca2+]i over the length of
the experiment. Two representative [Ca2+]i profiles illustrate ATPo-sensitive and
-insensitive thymocytes at
the single-cell level. The time
courses include experimental data (black dots) recorded
at 5-s intervals (experiment
71295-2; Cells 119 and 126).
(C) Average [Ca2+]i is plotted against time for thymocytes exhibiting an ATPo-induced rise in [Ca2+]i and
for nonresponsive cells.
[View Larger Version of this Image (35K GIF file)]
Fig. 2.
ATPo induces a biphasic [Ca2+]i rise in thymocytes. (A) In most ATPo-sensitive thymocytes, [Ca2+]i
rises slowly at first, reaches a
threshold, and then increases rapidly; three representative single-cell traces
are displayed. The threshold
[Ca2+]i level, signified by an
inflection point in these
curves, occurs over a narrow
range. The gray bar across
the graph is centered over
the mean, while the vertical
thickness of the bar indicates ±SD (experiment 71295-2;
Cells 27, 41, and 61). (B)
Peak [Ca2+]i and the [Ca2+]i
level at the inflection point
are plotted against time for 72 cells. The narrow band of
data points denoting threshold [Ca2+]i contrasts sharply
with the scattered distribution of peak [Ca2+]i values.
[View Larger Version of this Image (26K GIF file)]
), parotid
acinar cells (Soltoff et al., 1992
), macrophages (El-Moatassim
and Dubyak, 1992
; Nuttle et al., 1993
), and B-lymphocytes (Wiley et al., 1994
), BzATPo proved to be the most effective purinergic agonist. Since BzATP is believed to be specific for P2X7/P2Z purinoceptors and all three nucleotides
used in our experiments produced an equivalent [Ca2+]i
time course, we conclude that purinoceptors of the ion
channel/pore subtype are expressed in thymocytes.
Fig. 3.
BzATPo potently stimulates Ca2+ influx in thymocytes.
(A) Of the nucleotides tested, BzATPo proved to be the most effective nucleotide agonist stimulating a rise in thymocyte [Ca2+]i.
Individual thymocytes exhibit a nonuniform [Ca2+]i response to
100 µM BzATPo; 137 single-cell traces are displayed. (B) The single-cell [Ca2+]i rise evoked by BzATPo closely resembles the
ATPo-elicited response. A representative single-cell trace during
BzATPo application is shown. (Experiment 71595-7; Cell 30.) (C)
The level of [Ca2+]i at the peak and at the inflection point for 147 BzATPo-sensitive thymocytes are plotted against time. Peak
[Ca2+]i levels show wide dispersion while threshold values are
tightly clustered (experiments 71595-7 and 71595-23).
[View Larger Version of this Image (37K GIF file)]
CD8+ cells exhibited the most robust [Ca2+]i rise. Furthermore, the onset of the BzATPo response correlates with
phenotype; [Ca2+]i in CD4
CD8+ thymocytes reach inflection and peak levels almost twice as fast as small
CD4+CD8+ cells. Fig. 5, A and B display Gaussian curves
fitted to volume histograms for the five thymocyte populations. Small double-positive thymocytes fell within a narrow volume distribution, while larger double-positive cells
were scattered over a wider range of sizes (Fig. 5 A). Similarly, single-cell volumes of double-negative and single-positive classes were dispersed (Fig. 5 B). Table I and Fig.
5 C summarize average [Ca2+]i data for the five thymocyte
populations. These data indicate that actively dividing thymocytes are most sensitive to extracellular nucleotides,
while cells destined for apoptosis are least responsive.
Fig. 5.
Volume distribution of phenotype subclasses, which show distinct
BzATPo-induced [Ca2+]i response profiles. (A) Gaussian
curves fitted to volume-frequency histograms illustrate
the separation of double-positive thymocytes into two distinct populations. Single-cell
volumes, in femtoliters (fl),
were calculated from surface
area outlines of cells appearing in anti-CD4-PE and
anti-CD8-FITC images. (B)
Size distribution for double-negative and single-positive
thymocytes. Single-cell volumes for CD4CD8
cells
were scattered over a wide
range of values, while those for single positives were
more tightly clustered. (C)
Average [Ca2+]i profiles for
the five thymocyte populations. CD4
CD8+ thymocytes exhibited a strong
[Ca2+]i response, while small
double-positive cells were
least sensitive to 100 µM
BzATPo (experiments 9796-2 through -6, 9896-1 through -4, and 9896-10).
[View Larger Version of this Image (23K GIF file)]
). This observation excludes the
possibility that in zero Ca2+o, intracellular Ca2+ stores may
slowly empty below a level necessary to be registered upon subsequent release. Single-cell traces exhibit a biphasic time course followed by a declining plateau; an example is shown in Fig. 6 B. Grouped data illustrate that
the brief period of Ca2+o withdrawal does not significantly
affect the subsequent [Ca2+]i rise. Nevertheless, average
[Ca2+]i reaches the plateau level more slowly, which suggests that during the Ca2+o-free episode, purinergic receptors may become desensitized to nucleotides (Fig. 6 C).
These data are consistent with previous studies showing
that the activation of P2X7/P2Z purinoceptors stimulates
Ca2+ influx and not Ca2+ release in thymocytes (El-Moatassim et al., 1989b
; Pizzo et al., 1991
; Chused et al.,
1996
).
Fig. 6.
Ca2+ influx, rather than Ca2+ release from stores, is
stimulated by BzATPo. (A) A slight rise in [Ca2+]i (<30 nM) was
observed in a few BzATPo-treated thymocytes bathed in Ca-free
media, indicating that Ca2+ release from intracellular stores plays
an insignificant role in the purinergic response. After Ca2+o is reapplied, [Ca2+]i rises quickly in 75% of the cells. Therefore, Ca2+
influx underlies the BzATPo response. (B) Thymocytes respond normally to BzATPo after Ca2+o removal and readdition; a representative single-cell [Ca2+]i time course is displayed (experiment
71595-21; Cell 78). (C) The average [Ca2+]i course for BzATPo-sensitive cells is slower to reach plateau levels, suggesting that purinergic receptors may become desensitized to BzATPo during
the Ca2+o-free episode.
[View Larger Version of this Image (28K GIF file)]
) and T lymphocytes (Steinberg and Di Virgilio, 1991
). We examined
the ability of Mg2+ to affect ATPo-mediated [Ca2+]i responses in thymocytes by reducing [Mg2+]o from 2 to 1 mM (Fig. 7). Approximately 19% of the cells showed a
rise in [Ca2+]i in 2 mM [Mg2+]o; an additional 41% exhibited a rise in [Ca2+]i as [Mg2+]o was lowered to 1 mM. All
of the thymocytes in the original 19% exhibited a further
rise in [Ca2+]i when [Mg2+]o was decreased. These results
agree with the hypothesis that ATP4
is the active agonist
form for P2X7/P2Z purinoceptors on thymocytes.
Fig. 7.
ATPo-mediated Ca2+ influx in thymocytes is dependent
upon [Mg2+]o. The number of thymocytes exhibiting an ATPo-
dependent rise in [Ca2+]i increases as [Mg2+]o is halved from 2 to
1 mM, suggesting that ATP4, rather than MgATP2
, is the active form of the agonist. The top graph presents two representative single-cell traces that illustrate that the threshold of activation for the [Ca2+]i rise varies between thymocytes. Interestingly,
the reduction in [Mg2+]o elicits a further rise in [Ca2+]i in many
cells. The lower graph displays average [Ca2+]i in ATPo-sensitive
and -insensitive thymocytes. Three times as many thymocytes exhibit a rise in [Ca2+]i as [Mg2+]o is reduced. Reducing Mg2+ did
not affect [Ca2+]i levels within unresponsive thymocytes (experiment 71295-3; Cell 15 and 21).
[View Larger Version of this Image (19K GIF file)]
. The removal
of Mg2+o liberates the more potent ATP4
by dissociation,
which then is registered as rising [Ca2+]i as more purinoceptors are activated. Subsequent addition of 100 µM
BzATPo further stimulates purinoceptor activity.
Fig. 8.
Thymocyte purinoceptors remain open after agonist
withdrawal. Purinoceptors were activated by 1 mM MgATP in
the presence of 1 mM [Mg2+]o. A rise in [Ca2+]i was measured in
64% of the cells. Washing thymocytes with Ringer reduced
[Ca2+]i levels. Subsequent removal of all Mg2+o caused [Ca2+]i to
rise, in spite of the absence of agonist. The addition of 100 µM
BzATPo at 400 s increased Ca2+ influx. The upper graph shows a
single-cell trace, while the lower graph presents average [Ca2+]i
profiles (experiment 71595-13; Cell 19).
[View Larger Version of this Image (20K GIF file)]
can bind
to purinoceptors. However, it is unclear whether MgATP2
directly activates the receptors or if low concentrations of ATP4
, in equilibrium with inactive MgATP2
, are actually responsible for purinoceptor operation. Assuming that MgATP2
has a direct effect, the rank order of potency for stimulation of Ca2+ influx in thymocytes by adenine nucleotides is BzATP4
>ATP4
>MgATP2
>ADP3
.
Discussion
to be the active agonist form (Fig.
7). However, our results suggest that the MgATP2
complex binds to the receptor, and may remain bound even after agonist is washed from the bathing solution (Fig. 8).
; Pizzo et al., 1991
; Nagy et al., 1995
;
Chused et al., 1996
). Earlier work conducted on thymocyte
populations gave no indication of a biphasic time course,
instead showing a smoothly rising profile. In contrast, single-cell measurements of thymocyte [Ca2+]i consistently
exhibit a slowly rising phase that leads to a distinct inflection, followed by a more rapid increase (Figs. 1, 2, and 3).
This biphasic profile may be generated by several mechanisms. First, the initial rise in thymocyte [Ca2+]i may appear slower due to a partial masking of influx by Ca2+ sequestration into intracellular organelles, Ca2+ extrusion
through Ca2+ATPases, and buffering by intracellular compounds. Saturation of these processes at a particular
[Ca2+]i level (the inflection point) may lead to the faster
rising phase. Second, early Ca2+ influx through open pathways may induce other ATP-gated purinoceptors to open,
or may act upon already open channels to trigger greater influx through positive feedback. Third, the activation of
Ca2+-activated K+ channels (KCa; Mahaut-Smith and Mason, 1991
) during early Ca2+ influx may hyperpolarize thymocytes and increase the driving force for Ca2+ through
ATP-gated channels to generate the fast-rising phase. Membrane potential hyperpolarization, believed to be associated with the activity of KCa channels, has been observed when thymocytes are exposed to low concentrations of ATPo (0.5 mM in Mg2+-free buffer; Matko et al.,
1993
). However, higher ATPo concentrations (1 mM)
stimulate substantial depolarization (Pizzo et al., 1991
; Matko et al., 1993
; Chused et al., 1996
). Finally, recent reports document the presence of P2Y2 mRNA (mouse; Koshiba et al., 1997
) and P2X1 mRNA (rat; Chvatchko et al.,
1996
; Koshiba et al., 1997
) in dexamethasone-treated thymocytes. In addition, the superantigen staphylococcal enterotoxin B upregulates P2X1 mRNA expression and protein in mouse thymocytes (Chvatchko et al., 1996
). Our data support the theory that mouse thymocytes normally
express P2X7/P2Z purinoceptors, given maximal sensitivity to BzATPo (Fig. 3) and Mg2+o dependence (see below
and Figs. 7 and 8). If two or more types of purinoceptors
are coexpressed, then the biphasic profile we see may reflect the sequential activation of the multiple classes of nucleotide-gated receptors.
,
CD4
CD8+, and CD4
CD8
, but not CD4+CD8+ thymocytes (Dornand et al., 1986
; however, Barankiewicz et
al. [1988] report no ectonucleotidase activity in pooled
thymocyte mixtures). Nevertheless, our data at the single-cell level provide new information concerning the dynamics of thymocyte [Ca2+]i changes induced by ATPo.
, the active form of the agonist, is released from MgATP2
as Mg2+o decreases. The increase in
ATP4
would open more purinoceptors, which would explain the increase in Ca2+ influx in cells that had already
responded, as well as the recruitment of thymocytes not
responding in higher [Mg2+o]. Nevertheless, other interpretations are possible. First, ATP-gated channels in thymocytes may open in a graded fashion as the concentration of agonist increases, allowing more Ca2+ to enter the
cell. Second, since ectoATPases require MgATP2
as substrate, reducing Mg2+o may inhibit nucleotide catabolism
at the surface of thymocytes, allowing a buildup of ATP4
.
Finally, Mg2+ ions may directly block nucleotide-gated
pathways, as suggested by Nuttle and Dubyak (1994)
.
Therefore, a relief of block, measured as a rise in [Ca2+]i,
would be observed as [Mg2+]o is decreased.
, who demonstrated a 40% sequence similarity between RP-2 (Owens et al., 1991
), a gene activated in rat
thymocytes undergoing apoptosis, and a P2X1 clone isolated from a PC12 cDNA expression library. A subsequent study by Chvatchko et al. (1996)
bolstered this hypothesis
by showing that P2X1 mRNA and protein expression were
upregulated in mouse thymocytes induced to die by superantigen staphylococcal enterotoxin B. In addition, dexamethasone-induced upregulation of P2X1 mRNA was
found to occur in rat (Chvatchko et al., 1996
) but not
mouse thymocytes (Koshiba et al., 1997
). The purinoceptors we have characterized, identified as the P2X7/P2Z
subtype (Fig. 3), do not appear to link ATPo with apoptosis in thymocytes, because terminally differentiated double
positive cells are least sensitive to nucleotides. Similarly,
Nagy et al. (1995)
have shown that mature (medullary)
thymocytes exhibit a greater permeability to propidium iodide (668 D) after ATPo treatment than mixed populations, indicating pore function, which in turn suggests the
involvement of P2X7/P2Z purinoceptors. Since we studied
the ATPo response over a limited time course, we cannot
exclude the possibility that ATPo induces the expression
of P2X1 and/or P2Y2 purinoceptors in terminally differentiated thymocytes, which may lead to programmed cell death.
) through 314 D
(ethidium; El-Moatassim et al., 1989a
; Chused et al., 1996
),
and finally to a high limit of 668 D (propidium; Nagy et al.,
1995
). Two of these reports (Nagy et al., 1995
; Chused et
al., 1996
) demonstrate that mature thymocytes are more
permeable to the tested compound than immature precursors. Furthermore, steroid-induced expression patterns of
mRNA for P2Y2 and P2X1 subtypes appear to be species
dependent; two laboratories have shown P2X1 mRNA expression in rat thymocytes (Chvatchko et al., 1996
; Koshiba et al., 1997
), suggesting that P2X1 has RP-2-like activity. In contrast, dexamethasone induces P2Y2, but not
P2X1 mRNA expression in mouse thymocytes (Koshiba et
al., 1997
). In not one report has a causal relationship between ATPo and apoptosis been conclusively demonstrated. In addition, it has been sugggested that ATPo may
antagonize the apoptotic process in thymocytes, by a yet
unknown process (Apasov et al., 1995
). An alternative role for acute ATPo stimulation and the consequent Ca2+
rise in thymocytes may be to drive cellular differentiation
rather than trigger cell death.
) or Ca2+-mediated (Andjelic et al., 1993
)
apoptosis. As cells progress into the inner layers of the
cortex and differentiate into double-positive thymocytes,
they associate in tightly packed cell clusters (Kyewski et
al., 1987
). As many of these cells undergo apoptosis, high levels of ATPo would be released. At this point in thymocyte development, it would be critical for double positive thymocytes, if they are to survive, to lose any response
that would lead to high [Ca2+]i. Terminally differentiated
double-positive thymocytes that are triggered by other
means to begin programmed cell death regain an ATPo response through the expression of P2X1 and/or P2Y2 purinoceptors, perhaps to accelerate the process by elevating
intracellular Ca2+. Cells that survive selection in the inner
cortex and reach the medulla as single positive precursors
to mature T lymphocytes would again be able to respond
to ATPo without danger of undergoing inadvertent cell
death. As ATPo levels are expected to be low in the medulla, these cells would upregulate the expression of P2X7/
P2Z purinoceptors.
-TCR and MHC (for review
see Jameson et al., 1995
; Guidos, 1996
). For example, inhibition of calcineurin, a calcium- and calmodulin-dependent phosphatase, by FK506, specifically blocks positive
but not negative selection of CD4+CD8+ thymocytes in
mice (Wang et al., 1995
). Elevation of [Ca2+]i by ionomycin in conjunction with protein kinase C activation can bypass the requirement for TCR engagement in positive selection leading to mature CD4+CD8
thymocytes from
mutant TCR-
-deficient CD4+CD8+ cells (Takehama
and Nakauchi, 1996). Eichmann (1995)
suggested that the
magnitude of Ca2+ signal required for differentiation is increased during the progression toward more mature phenotypes. In addition, sensitivity to calcium-mediated apoptosis begins as thymocytes express molecules responsible
for thymic selection (Andjelic et al., 1993
). Our data demonstrate differential [Ca2+]i responses to ATPo among thymocyte subsets; both the probability and intensity of ATP-induced Ca2+ signals are reduced in small, double-positive
thymocytes, relative to all other subsets. Thus, we suggest
that ATPo is not a specific trigger for programmed cell
death in the thymus, but rather that ATPo-stimulated
[Ca2+]i responses may act alone or in combination with
TCR/MHC-stimulated signals to drive differentiation and
positive selection of thymocytes.
Received for publication 4 April 1997 and in revised form 1 July 1997.
Please address all correspondence to Dr. Cahalan, Department of Physiology and Biophysics, University of California at Irvine, Irvine, CA 92697. Tel: (714) 824-7776; Fax: (714) 824-8540; E-mail: mcahalan{at}uci.eduWe thank Dr. Miriam Ashley-Ross for extended discussions during manuscript preparation.
This work was supported by National Institutes of Health grants NS14609 and GM41514.
ATPo, extracellular ATP; BzATP, benzoyl-ATP; [Ca2+]i, intracellular calcium concentration; [Ca2+]o, extracellular calcium concentration; MHC, major histocompatibility proteins; TCR, T-cell receptor.
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