Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Korea
Correspondence: Kyong-Tai Kim, Ph.D., Department of Life Science, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang 790-784, South Korea. E-mail: ktk{at}vision.postech.ac.kr
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Key Words: G protein • cholera toxin • phorbol 12-myristate 13-acetate
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Although a physiological role for ATP in bone marrow has not yet been firmly established, the large amount of ATP stored in bone marrow-derived megakaryocytes and its release upon extracellular stimulation suggest functional relevance for extracellular nucleotides in the physiology of hemopoietic cells [8 ]. Recently, it has been shown that P2 purinoceptors are present on various immature bone marrow-derived cells and that they are involved in the regulation of the proliferation of hemopoietic stem cells by causing the release of the histamine from mast cells [9 ]. The released histamine induces the functional differentiation of the stem cells probably through cAMP-dependent gene expression [10 ]. Thus, ATP would be required for the regulation of the start and the rate of differentiation of these cells. Based on the data obtained in this study using the HL-60 promyelocytes, we suggest that the activation of P2 receptors negatively regulates the histamine-induced cAMP generation and thus granulocytic differentiation. Because histamine has been seen to exert power over a host of biological actions, our results provide an example of the importance of the regulation of the histamine signaling pathways and the fine-tuning of the intracellular messenger generation under physiological conditions.
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S,
2-MeSATP, UTP, BzATP, adenosine, cAMP, ranitidine, thapsigargin,
sulfinpyrazone, phorbol 12-myristate 13-acetate (PMA), 4-
-PMA, and
IP3 were purchased from Sigma Chemical (St. Louis, MO).
[3H]IP3, [3H]adenine, and
[3H]histamine were obtained from NEN Life Science
Products (Boston, MA). GF109203X, ionomycin, and isobutylmethylxanthine
(IBMX) were obtained from Research Biochemicals (Natick, MA). Fura-2
pentaacetoxymethylester (fura-2/AM) was purchased from Molecular Probes
(Eugene, OR).
Cell culture
HL-60 promyelocytes were maintained in RPMI 1640 medium (GIBCO,
Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated bovine
calf serum (Hyclone, Logan, UT) plus 1% (v/v) penicillin-streptomycin
(GIBCO) in a humidified atmosphere of 5% CO2 at 37°C.
Fresh medium was added to the culture flasks every 2 days, and cells
were subcultured about once a week.
Measurement of [3H]cAMP
Intracellular cAMP was determined by measuring the formation of
cyclic [3H]AMP from a [3H]adenine
nucleotide pool as we have previously described [11
].
After loading the cells with [3H]adenine (2 µCi/mL) in
complete medium for 24 h, the cells were washed three times with
Locke’s solution (NaCl, 154 mM; KCl, 5.6 mM; MgCl2, 1.2
mM; CaCl2, 2.2 mM; HEPES, 5.0 mM; glucose, 10 mM, pH 7.4)
and stimulated with various drugs for the indicated time. The reaction
was stopped by aspiration of the medium and addition of 1 mL of
ice-cold 5% (v/v) trichloroacetic acid containing 1 µM cold cAMP.
[3H]cAMP and [3H]ATP were separated by
sequential chromatography on Dowex AG50W-X4 (200–400 mesh) cation
exchanger and neutral alumina columns. The [3H]ATP
fraction was obtained from the Dowex column by elution with 2 mL
distilled water. The subsequent elution with 3.5 mL distilled water was
loaded onto the alumina column. The alumina column was washed with 4 mL
imidazole solution (0.1 M, pH 7.2), and the eluate fractions were
collected into scintillation vials containing 15 mL scintillation fluid
for quantitation of the cyclic [3H]AMP. The increase in
intracellular cAMP concentration was calculated as
[3H]cAMP/([3H]ATP +
[3H]cAMP) x 103.
Measurement of IP3
IP3 concentration in the cells was determined by
[3H]IP3 competition assay in binding to
IP3 binding protein [12
]. The HL-60 cells
were stimulated with agonists, and the reaction was terminated by
aspirating the medium off the cells, followed by addition of 0.3 mL
ice-cold 15% (w/v) trichloroacetic acid containing 10 mM EGTA. The
samples were centrifuged at 5,000 g for 10 min at 4°C. The
trichloroacetic acid in the extract was removed by four extractions
with diethyl ether. Finally, the extract was neutralized with 200 mM
Trizma base and its pH adjusted to about 7.4. Twenty microliters of the
cell extract was added to 20 µL of assay buffer [0.1 M
tris(hydroxymethyl)aminomethane buffer containing 4 mM EDTA and 4 mg/mL
bovine serum albumin] and 20 µL of [3H]IP3
(0.1 µCi/mL). Then 20 µL of solution containing the binding protein
was added, and the mixture incubated for 15 min on ice and centrifuged
at 2,000 g for 5 min. The pellet was resuspended in 100 µL
of water, and 1 mL of scintillation cocktail was added to measure the
radioactivity. IP3 concentration in the sample was
determined based on a standard curve and expressed as picomoles per
milligram protein. The IP3 binding protein was prepared
from bovine adrenal cortex according to the method of Challiss et al.
[13
].
Measurement of intracellular Ca2+ level
The level of intracellular Ca2+ was measured
using fura-2/AM as previously described [14
]. Briefly,
cell suspensions were incubated in fresh serum-free RPMI 1640 medium
with 3 µM fura-2/AM at 37°C for 40 min. Sulfinpyrazone (250 µM)
was added to all solutions to prevent dye leakage. Changes in
fluorescence ratios were measured at the dual-excitation wavelengths of
340 and 380 nm and the emission wavelength of 500 nm.
[Ca2+]i was calculated using the equation
[Ca2+]i =
Kd[(R -
Rmin)/(Rmax -
R)](Sf2/Sb2), where
Rmax and Rmin are the
ratio obtained when fura-2 is saturated with Ca2+ and when
EGTA is used to remove Ca2+, respectively. Sf2
and Sb2 are the proportionality coefficients of
Ca2+-free fura-2 and saturated fura-2, respectively.
Calibration of the fluorescence signal in term of
[Ca2+]i was performed according to
Grynkiewicz et al. [15
]. In extracellular
Ca2+-free experiments, the Locke’s solution did not
contain Ca2+ ion but included 200 µM EGTA.
[3H]Histamine binding
The binding of [3H]histamine to intact HL-60
cells was quantified by the method described previously by Mitsuhashi
and Payan [27
] with some modification. Cells were
collected by centrifugation at 1,000 g for 1 min. The
binding assay was carried out at 25°C in a final volume of 100 µL
of Ca2+-free Locke’s solution supplemented with 1 mM EDTA,
0.2% bovine serum albumin, 5 mM histidine, 50 nM
[3H]histamine, and various drugs. Assays were initiated
by the addition of the cells (106 cells/tube) and
terminated after 20 min by vacuum filtration through nitrocellulose
filters (0.45 µm) using the Millipore multiscreen assay system. The
filters were rinsed two times with 150 µL of ice-cold 50 mM
Tris · HCl containing 1 mM EDTA (pH 7.6). The amount of bound
radioactivity was measured in a liquid scintillation cocktail. Specific
binding was defined as the difference in the amount of radioactivity
bound in the absence and presence of 1 mM unlabeled histamine.
Analysis of data
All quantitative data are expressed as mean ±
SEM. Comparison between two groups was analyzed using
Student’s unpaired t test, and comparison among more than
two groups was carried out using one-way analysis of variance (ANOVA).
Differences were considered to be significant when the degree of
confidence in the significance was 95% or better (P <
0.05). Calculation of EC50 was performed with the Allfit
program [16
].
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S also evoked the cAMP production
with an EC50 of 23.7 ± 5.1 µM (data not shown). The
data suggest that in HL-60 cells the P2 receptors are linked to both
PLC and adenylyl cyclase, whereas histamine receptors are prominently
linked to adenylyl cyclase, as has been previously reported
[17
].
 View larger version (22K): [in a new window] |
Figure 1. Intracellular Ca2+ mobilization, IP3
generation, and cAMP production after ATP and histamine treatment of
HL-60 promyelocytes. (A) Concentration-dependent stimulation of
cytosolic Ca2+ mobilization after ATP and histamine
treatment was measured in the absence of extracellular
Ca2+. Typical patterns of Ca2+ mobilization
after treatment with ATP (300 µM) and histamine (300 µM) are
presented. (B) Time-course of IP3 generation evoked by ATP
and histamine. The cells were treated with 300 µM ATP or histamine
for the indicated times (0, 15, 30, 60, 180, 300, and 600 s), and
the reaction was stopped by addition of 15% (wt/vol) TCA containing 10
mM EGTA. (C) Concentration-dependent increase of cAMP levels after ATP
and histamine treatment. [3H]adenine-loaded cells
pretreated with 1 mM IBMX were stimulated with various concentrations
of ATP or histamine for 3 min, and the cAMP generation was measured as
described in Materials and Methods. Each concentration of ATP and
histamine was tested in three independent experiments, and the
means ± SEM are presented.
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40% of the
rise obtained by the histamine stimulation of cells not pretreated with
ATP. Figure 2B
shows that as the concentration of ATP was raised, the
subsequent histamine-induced cAMP response decreased and that
pretreatment of the cells with a supra-maximal concentration of ATP (1
mM) inhibited the subsequent histamine-induced cAMP increase by
75%. In contrast, a stimulation with histamine first and followed
by ATP treatment did not affect the cAMP generation induced by ATP 30
min after the histamine stimulation (Fig. 2C)
. The ATP-stimulated cAMP
production in histamine-pretreated cells showed no change compared with
the just ATP-stimulated control. These data, therefore, suggest that
extracellular ATP specifically inhibited the signaling of the histamine
receptor.
 View larger version (13K): [in a new window] |
Figure 2. Inhibition of histamine-induced cAMP generation by extracellular ATP.
(A) [3H]adenine-loaded HL-60 cells were stimulated with
100 µM histamine for the designated times after a 30-min pretreatment
with (filled circles) or without (open circles) 300 µM ATP. (B)
Concentration dependence of the ATP effect on the inhibition of the
subsequently 100 µM histamine-induced cAMP generation. (C) HL-60
cells were stimulated with 300 µM ATP for the designated times after
a 30-min pretreatment with (filled circles) and without (open circles)
100 µM histamine. Each data point is the mean ± SEM
of three independent experiments. *P <
0.01, compared with the histamine-induced cAMP generation without ATP
treatment in one-way ANOVA.
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S) 
UTP >
2-methylthioATP (2-MeSATP) adenosine
5’-O-(2-thiodiphosphate) (ADPßS) >
3’-O-(4-benzoyl)benzoyl ATP (BzATP) (Table 1
). The order of the analogs’ effects on the histamine response
matches the order of IP3 generation induced by
P2Y2 purinoceptors in HL-60 promyelocytes
[18
]. However, the pattern of the cAMP production
induced by the ATP analogs differed from the pattern of their negative
effect on the histamine-induced cAMP generation. Moreover, treatment of
the cells with prostaglandin E2, which elevates cAMP even
more than ATP, had little effect on the histamine-induced response
(data not shown), indicating that adenylyl cyclase activation is not
involved in the inhibitory effect of the ATP analogs. Adenosine and
,ß-methylene ATP, which did not induce the IP3
generation and cAMP production, had no inhibitory effect on the
subsequent histamine-induced responses. The data, therefore, suggest
that it is PLC activation by the P2Y2 receptor that causes
the inhibition of the subsequent histamine-induced response. To then
examine whether [Ca2+]i rise is responsible
for the nucleotide’s effect on the histamine response, the cells were
treated with ionomycin or thapsigargin before histamine stimulation.
Treatment with the Ca2+ ionophore ionomycin (100 nM)
induced a cytosolic Ca2+ increase similar to that obtained
with ATP (300 µM), but it did not affect the subsequent
histamine-induced cAMP generation (data not shown). Treatment with
thapsigargin, which elevates [Ca2+]i via the
inhibition of microsomal Ca2+/ATPase, had little effect on
the histamine-induced responses (data not shown), indicating that the
ATP-induced [Ca2+]i elevation was not the
cause of the inhibition. |
View this table: [in a new window] |
Table 1. Effect of Nucleotides on Histamine-induced cAMP Generation
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 View larger version (18K): [in a new window] |
Figure 3. Effect of P2Y2 receptor activation on BzATP-, prostaglandin
E2-, and isoproterenol-stimulated cAMP generation in HL-60
cells. Cells preincubated with vehicle (control) or 300 µM UTP for 30
min were stimulated with BzATP (100 µM), prostaglandin E2
(PGE2, 10 µM), and isoproterenol (10 µM) for 20 min,
and the net increase in cAMP generation is expressed as percent of the
level obtained by treatment with BzATP, prostaglandin E2,
or isoproterenol alone. The experiments were carried out three times in
triplicate and data are the means ± SEM.
*P < 0.01, compared with the
agonist-induced cAMP generation without UTP treatment.
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-PMA, an
inactive PMA analog, had no inhibitory effect. It is interesting that,
however, the ATP-induced cAMP production was synergistically enhanced
by pretreatment of the cells with PMA. The concentration-dependent
effect of PMA on the histamine-induced cAMP production shows that
half-maximal inhibition occurred at approximately 15 nM (Fig. 4B)
. The
data suggested that PKC was selectively involved in the negative
regulation of the histamine signaling. To investigate the possibility
that PKC played a role in the ATP-induced inhibitory action, we used a
PKC inhibitor that was administered before the addition of ATP and the
subsequent histamine stimulation. Table 2
shows that pretreatment with ATP (300 µM) for 30 min
significantly inhibited the histamine-stimulated cAMP production and
that addition of 3 µM GF109203X 10 min before the ATP stimulation
almost completely blocked the ATP effect on the subsequent histamine
response. The PKC inhibitor itself had little effect on the basal and
the histamine-stimulated cAMP accumulation. These results, therefore,
suggest that ATP’s inhibitory effect on the histamine-induced cAMP
production might be mediated by PKC.
 View larger version (23K): [in a new window] |
Figure 4. Inhibition of histamine-induced cAMP generation by PMA. (A) Cells
preincubated with vehicle (0.2% DMSO), PMA, or 4--PMA for 5 min
were stimulated with histamine (100 µM) and ATP (300 µM) for 20
min. *P < 0.01, compared with the
histamine-induced cAMP generation without PMA treatment.
**P < 0.01, compared with the
ATP-induced cAMP generation without PMA treatment. (B) Concentration
dependence of the PMA effect in inhibiting the subsequently
histamine-induced cAMP generation. [3H]adenine-loaded
cells preincubated with various concentrations of PMA (filled circles)
for 5 min were stimulated with 100 µM histamine, and the net increase
in cAMP generation is expressed as percent of the level obtained by
treatment with histamine alone. 4--PMA (open circles) had no
inhibitory effect. The experiments were carried out three times in
triplicate and data are the means ± SEM.
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View this table: [in a new window] |
Table 2. Effect of GF109203X on Histamine-induced cAMP Generation
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 View larger version (45K): [in a new window] |
Figure 5. Binding of [3H]histamine to HL-60 cells. (A) Cells
preincubated with 300 µM ATP, UTP, or 100 nM PMA for 30 min were
treated with [3H]histamine or [3H]histamine
plus 10 µM ranitidine at room temperature for 20 min. The data
represent specific binding obtained after subtracting nonspecific
binding from the total binding. Nonspecific binding was determined
after addition of 5 µM unlabeled histamine and was 45.2 ± 5.0
fmol/106 cells. (B) Cells were treated with 100 µM
histamine, 300 µM ATP, UTP, and 100 nM PMA for 12 h, then washed
with Locke’s solution three times followed by incubation in the buffer
for 1 h. Then the cells were stimulated with 100 µM histamine
and the cAMP generation measured as described in Materials and Methods.
Each point is the mean ± SEM of triple experiments.
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s subunit. Figure 6
shows that the addition of 10 µM forskolin and 2 µg/mL cholera
toxin caused a two- to threefold increase in the cAMP production.
However, this cAMP generation was significantly enhanced in ATP-, UTP-,
and PMA-stimulated cells, whereas 4--PMA had little effect. This
result suggests that the site of the inhibition by PKC is not the G
protein or G protein-coupled adenylyl cyclase but rather the coupling
between histamine receptor and G protein.
 View larger version (33K): [in a new window] |
Figure 6. Effect of nucleotides on adenylyl cyclase activity.
[3H]adenine-loaded HL-60 cells were stimulated with
vehicle, 300 µM ATP, UTP, 100 nM PMA, or 4--PMA in the absence or
presence of 10 µM forskolin and 2 µg/mL cholera toxin for 20 min.
The reaction was stopped by addition of 5% (w/v) trichloroacetic acid
containing 1 µM cAMP, and the [3H]cAMP generation was
measured as described in Materials and Methods. The experiment was done
three times in triplicate and the means ± SEM are
presented.
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 View larger version (24K): [in a new window] |
Figure 7. Effect of nucleotide inclusion on histamine-mediated granulocytic
differentiation of HL-60 cells. (A) fMLP-mediated intracellular
Ca2+ mobilization in cells treated with histamine for
96 h in the presence or absence of 300 µM ATP, UTP, adenosine.
Cells were also treated with 1.25% (vol/vol) DMSO to provide controls
for the differentiation of HL-60 cells. Fura-2/AM-loaded cells were
stimulated with 1 µM fMLP and the peak Ca2+ level was
measured as described in Materials and Methods. (B) fMLP-mediated
IP3 generation in HL-60 cells treated with histamine and
with or without various nucleotides. The HL-60 myelocytes were
stimulated with nucleotides (300 µM) for 15 s and the
IP3 generation was measured as described in Materials and
Methods. The experiments were carried out twice and the values
presented are means ± SEM.
*P < 0.01, compared with the
histamine-treated cells.
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Many studies have shown that the signaling pathway of adenylyl cyclase can be enhanced or depressed by a direct PKC activator, phorbol ester [24 ]. However, little is known about the cross-communication between the phosphoinositide turnover and the cAMP production signaling pathways in intact cells. Our experiments with HL-60 cells show that stimulation of the PLC-coupled P2 receptor itself induces a physiological activation of PKC, which then inhibits the histamine receptor-mediated cAMP production and granulocytic differentiation. Clinical findings have shown that long-term treatment with ATP also leads to the functional differentiation of human myeloid cells [25 , 26 ]. Thus, the physiological effect of nucleotides on differentiation might be more complex. At present, it is unknown which signal pathway of ATP receptors leads on cellular differentiation. We found that P2Y2 activation by UTP did not induce expression of fMLP receptors in HL-60 cells, whereas ATP treatment slightly increased the fMLP response. This indicates that the effect of ATP on differentiation was mediated via cAMP generation, whereas the [Ca2+]i rise did not provide a sufficient signal for the induction of differentiation of HL-60 cells. Nevertheless, P2Y2 receptors play an important role in setting the direction of cellular differentiation of myeloid progenitor cells, which are activated by histamine.
The decrease in H2 receptor-mediated cAMP synthesis after PKC activation does not appear to be due to an alteration in the activity of the histamine receptors. Our experiments with HL-60 cells show that ATP exposure for 30 min did not affect the [3H]histamine binding site number and Kd value. Moreover, long-term treatment of the cells with ATP or UTP did not induce the heterologous desensitization of histamine receptors. The results imply that the uncoupling between receptor and the Gs protein is induced by acute activation of PKC in a manner that does not affect agonist binding to the receptor. This conclusion is supported by previous observations of phorbol esters inhibiting hormone-stimulated adenylyl cyclase by acting on the level of the receptor, although the exact mechanism is not clear. For example, Mitsuhashi and Payan [27 ] reported that the binding affinity and, up to 1 h after PMA treatment, the binding sites for histamine were not affected on cultured DDT1MF-2 smooth muscle cells. In cultured collecting tubular cells, PMA inhibited arginine vasopressin-stimulated adenylyl cyclase activity, presumably by inhibiting the receptor or the coupling of the receptor to Gs protein [28 ]. Other studies have also contributed evidence that homologous desensitization of hormone receptors results from receptor uncoupling rather than from a decrease in receptor numbers or from receptor sequestration at least within a few minutes after the exposure of the cells to the agonist [29 , 30 ].
It has been recognized in a number of G-protein-coupled receptors that activation of the multiple subtypes of PKC by PMA or diacylglycerol, the endogenous product of PLC activation, resulted in phosphorylation of substrate proteins at the terminal consensus sequences [31 ]. The ß2 adrenergic receptor contains, in the third intracellular loop, the consensus phosphorylation sites for PKC, and the phosphorylation of these sites has been reported to be involved in reduction of receptor potency during acute PMA treatment [32 ]. The third intracellular domain is also known to contain the consensus sequence for a protein kinase A phosphorylation site. The histamine H2 receptors, which belong to the family of G-protein-coupled receptors, also contain in the corresponding region a consensus phosphorylation site for PKC, but not for protein kinase A [33 ]. In addition, they have another phosphorylation site for PKC in the carboxy-terminal region. Likewise, although the site of PKC action has not yet been identified in prostaglandin receptors, a rapid uncoupling of these receptors from G proteins may also occur after addition of PMA [34 ]. However, recently we showed that P2Y11 receptor-mediated cAMP generation was potentiated by PKC activation, probably through direct enhancement of the adenylyl cyclase activity [35 ]. In those studies PKC activation did not affect the coupling between P2Y11 receptors and G proteins, suggesting that the signaling pathway to adenylyl cyclase from P2Y11 receptors are differentially regulated by PKC compared to the histamine or prostaglandin receptor signaling in the cells.
HL-60 cells are pluripotent and can differentiate into monocytes or
neutrophils depending on the inducers of differentiation. It has been
shown that treatment with phorbol esters, ganglioside GM(3), or
interferon- causes the cells to differentiate toward monocytes
[36
]. On the other hand, dimethyl sulfoxide, retinoic
acid, and cAMP-generating agonists including histamine and epinephrine
can induce the cells to differentiate toward neutrophils that display
distinctly different biological and morphological characteristics
compared with monocytes [37
]. During this
differentiation, the expression of surface receptors for chemotactic
factors primes the cell for the activation of granulocytic functions
and the triggering of the respiratory burst pathway. It has been known
previously, that histamine induces neutrophilic differentiation of
HL-60 promyelocytes via the activation of H2 receptors and
that it evokes the expression of fMLP receptors [10
]. We
show here that P2Y2 receptor-mediated PKC activation causes
inhibition of the histamine-mediated cellular differentiation. This
means that cross-talk between the adenylyl cyclase system and PKC would
throw a switch in the cellular program that regulates the rate of
differentiation. Many previous studies have shown that
Ca2+-mobilizing ATP receptors are expressed by both normal
bone marrow-derived cells and by leukemic myeloid progenitor cells,
including myeloblasts, promyelocytes, and promonocytes
[38
], suggesting that purinergic regulation of histamine
responses may occur in a variety of different microenvironmental
situations to which the receptors are exposed. In conclusion, our
results provide an important insight into the relevance of the
cross-communication between a PLC-coupled receptor and the cAMP
production signaling pathway during hemopoietic cell differentiation.
Received March 23, 2000; revised August 7, 2000; accepted August 10, 2000.
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