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(Journal of Leukocyte Biology. 2001;69:169-176.)
© 2001 by Society for Leukocyte Biology

Modulation of H2 histamine receptor-mediated cAMP generation and granulocytic differentiation by extracellular nucleotides via activation of protein kinase C

Byung-Chang Suh, Hyosang Lee, Ihn-Soon Lee and Kyong-Tai Kim

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular ATP exerts a variety of biological actions through several kinds of P2 receptor in HL-60 promyelocytes. We show that stimulation of P2Y2 receptors with ATP and analogs resulted in the inhibition of a subsequently histamine-induced cAMP production and functional differentiation. Treatment of the cells with phorbol 12-myristate 13-acetate also blocked the histamine-mediated cAMP generation just as ATP did. Incubation of the cells with the protein kinase C inhibitor bisindolylmaleimide (GF109203X) abolished the inhibitory effects of extracellular nucleotides, suggesting that protein kinase C may act as an inter-regulator between two receptors. However, ATP did not affect the binding affinity or total binding of [3H]histamine to membrane receptors; it also did not heterologously desensitize H2 receptors. The ATP treatment synergistically elevated the cAMP levels induced directly by forskolin or indirectly by G protein activation after cholera toxin treatment. This indicates that the site of the protein kinase C action is not the G protein or effector enzyme. Co-stimulation of the cells with nucleotides and histamine inhibited histamine-mediated granulocytic differentiation, which was evaluated by looking at the extent of N-formyl-methionyl-leucyl-phenylalanine responses. Taken together, the results demonstrate that extracellular nucleotides are negatively involved in the modulation of histamine signaling via activation of protein kinase C, probably by inhibiting coupling between receptor and G protein.

Key Words: G protein • cholera toxin • phorbol 12-myristate 13-acetate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular ATP evokes many physiological responses in various cell types. Among them are such different phenomena as platelet aggregation, neurotransmission, and muscle contraction [1 ]. In the inflammatory system, ATP plays important roles in leukocyte functions, which include DNA synthesis, blastogenesis, cell-mediated killing, apoptosis by priming of superoxide release, and the degranulation of mast cells [2 , 3 ]. These various effects of ATP are mediated by plasma membrane P2 receptors. It has been shown that in human HL-60 promyelocytic leukemia cells extracellular ATP increases the intracellular free Ca2+ concentration ([Ca2+]i) via the P2Y2 [3 ] and P2X1 [4 ] receptors. The P2Y2 receptor is functionally coupled to phospholipase C (PLC) through pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. Upon activation, PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol [5 ]. IP3 then increases the [Ca2+]i by mobilizing Ca2+ from the intracellular Ca2+ stores, which is followed by Ca2+ influx through Ca2+-release-activated Ca2+ channels, a process termed capacitative Ca2+ entry [6 ]. The activation of P2X1 receptors results in an inward ion current in immature hemopoietic cells, although the current is also significantly detectable in differentiated cells [4 ]. In addition to P2Y2 and P2X1, we identified a novel type of P2 receptor that elevates cAMP in HL-60 cells [7 ].

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Histamine, prostaglandin E2, ATP, ADPßS, ATP{gamma}S, 2-MeSATP, UTP, BzATP, adenosine, cAMP, ranitidine, thapsigargin, sulfinpyrazone, phorbol 12-myristate 13-acetate (PMA), 4-{alpha}-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 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition by extracellular ATP of histamine-induced cAMP generation
The ability of extracellular ATP and histamine to induce intracellular Ca2+ mobilization from intracellular pools in the absence of extracellular Ca2+ was examined using the fluorescent Ca2+ indicator fura-2 in HL-60 cells. Figure 1A shows that exposure of the cells to ATP resulted in a transient increase in [Ca2+]i in a concentration-dependent manner with a half-maximal effective concentration (EC50) were 8.9 ± 3.4 µM of ATP. However, under these conditions, histamine had no effect in elevating cytosolic Ca2+ levels up to concentrations of 300 µM. Figure 1B shows that the treatment with ATP caused rapid and transient production of IP3. A peak level of IP3 production was achieved 15 s after ATP (300 µM) stimulation, after which the IP3 level decreased slowly to the basal level within 10 min. On the other hand, IP3 generation was barely detected in cells treated with 300 µM histamine. As shown in Figure 1C , histamine and ATP both triggered cAMP production in a concentration-dependent manner with EC50 values of 0.6 ± 0.2 µM and 35.2 ± 6.7 µM, respectively. The hydrolysis-resistant ATP analog ATP{gamma}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 ].



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  		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.

 
In a sequential stimulation of the two receptors, we found that ATP treatment inhibited the subsequently histamine-induced cAMP generation. Figure 2A shows that histamine stimulation increased the level of cAMP eightfold within 3 min in comparison to the unstimulated control, after which the cAMP was metabolized and slowly decreased to near basal level within 30 min. However, pretreatment of the cells with ATP (300 µM) for 30 min significantly inhibited subsequent histamine induction of cAMP production, which reached a maximal level that was only threefold over that of the unstimulated control. This amounts to ~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.



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  		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.

 
Inhibition of histamine responses by PLC-coupled P2Y2 purinoceptor activation
Because ATP elicits phosphoinositide turnover via the PLC-linked P2Y2 purinoceptor [3 , 18 ] and because it produces cAMP through an adenylyl cyclase-linked receptor in HL-60 cells [7 , 19 ], we treated the cells with various nucleotide analogs in an attempt to determine which signaling pathway was responsible for the inhibitory action described above. Our studies established the following order of potency for nucleotides and ATP analogs in terms of the inhibitory effect on histamine-induced cAMP production: ATP, adenosine 5’-O-(3-thiotriphosphate) (ATP{gamma}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 {alpha},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.


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  		Table 1. Effect of Nucleotides on Histamine-induced cAMP Generation

 
To examine whether the activation of P2Y2 receptors also affects other receptor-mediated cAMP accumulation, we pretreated the cells with the P2Y2 selective agonist UTP and then measured the subsequent BzATP-, prostaglandin E2-, and isoproterenol-stimulated cAMP generation. Figure 3 shows that UTP treatment decreased the prostaglandin E2- and isoproterenol-induced responses by 40–50%, whereas it enhanced the BzATP-stimulated cAMP generation. The results indicate that P2Y2 stimulation differentially regulates the histamine-, prostaglandin-, and ß-adrenergic receptor-mediated signaling and the P2Y11 receptor-mediated signaling in HL-60 cells.



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  		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.

 
Involvement of protein kinase C (PKC) in inhibition of the coupling between histamine receptors and G proteins
Because elevated Ca2+ and diacylglycerol generated by activated PLC activates PKC, we tested the involvement of PKC using PMA, a selective activator of PKC. Figure 4A shows that treatment of the cells with 30 nM PMA significantly inhibited the histamine-induced cAMP production, whereas 4-{alpha}-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.



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  		Figure 4. Inhibition of histamine-induced cAMP generation by PMA. (A) Cells preincubated with vehicle (0.2% DMSO), PMA, or 4-{alpha}-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-{alpha}-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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  		Table 2. Effect of GF109203X on Histamine-induced cAMP Generation

 
To determine whether the ATP-induced inhibition of the histamine-mediated cAMP generation was the result of a decreased number of histamine receptors or of a change in the binding affinity of the receptors, we measured the specific binding of histamine to the cells. Figure 5A shows that ranitidine, a histamine H2 receptor antagonist, almost completely blocked [3H]histamine binding, indicating that the cells mainly used the histamine H2 receptor to bind histamine. However, pretreatment of the cells with 300 µM ATP or UTP did not affect histamine binding. Scatchard plots of the equilibrium saturation binding data showed that the Kd values for the untreated control cells and the ATP-treated cells were 95.4 ± 15.2 and 87.2 ± 10.5 nM, respectively. Thus, there was no real difference between the control cells and the ATP-treated cells. This was confirmed by measuring cAMP generation. Figure 5B shows that pretreatment of the cells with histamine for 24 h prevented subsequent histamine-stimulated cAMP generation, whereas pretreatment with UTP or ATP had little effect on the histamine response. The results, therefore, indicate that the histamine receptors of the HL-60 cells were not heterologously desensitized by treatment with nucleotides.



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  		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.

 
To further analyze the site of the inhibitory action of nucleotides on the histamine-induced adenylyl cyclase activation, we treated the cells with forskolin, which directly activates adenylyl cyclases, and cholera toxin, which activates Gs proteins by ADP-ribosylation of the {alpha}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-{alpha}-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.



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  		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-{alpha}-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.

 
Negative modulation of histamine-induced granulocytic differentiation of HL-60 cells via P2Y2 receptors
To assess the functional importance of cross-talk between the P2Y2 purinoceptor and the H2 receptor, we looked at effects of agonists on cellular differentiation. Granulocytic differentiation of HL-60 promyelocytes results in increased expression of formyl peptide receptors, which can be readily monitored by observing the increased effectiveness of N-formyl-methionyl-leucyl-phenylalanine (fMLP) to induce rises in [Ca2+]i [20 ]. The responsiveness of fMLP of HL-60 cells treated with histamine (100 µM) for 96 h was substantially increased, whereas UTP treatment had little effect on inducing the fMLP response in the form of a rise in [Ca2+]i (Fig. 7A ). However, in cells simultaneously treated with histamine and UTP, the fMLP response as [Ca2+]i increase was decreased. In contrast to UTP, treatment with ATP resulted in a slight increase in the fMLP response, although the response was not further increased in cells simultaneously treated with ATP and histamine. Inclusion of adenosine, which has no effect on PLC or adenylyl cyclase activation, did not effect the histamine-induced differentiation. Consistent with the Ca2+ mobilization, addition of nucleotides together with histamine also resulted in inhibition of the fMLP-stimulated IP3 generation (Fig. 7B) . These results, therefore, indicated that the H2 histamine receptor-mediated granulocytic differentiation could be functionally regulated by stimulation of the P2Y2 purinoceptor.



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  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Multiple types of P2-nucleotide receptor are expressed in blood cells and the expression level of certain P2 receptors can be rapidly modulated during cell activation processes or during differentiation of hematopoietic progenitor cells [21 , 22 ]. This involvement of purinoceptors suggests that released nucleotides might participate in multiple types of paracrine and autocrine signalings between blood cells and other cell types. In this study, we have demonstrated that extracellular nucleotides can specifically regulate histamine-induced cytosolic cAMP generation and granulocytic differentiation of HL-60 promyelocytes. Several lines of evidence suggest that the activation of PLC-coupled P2Y2 purinoceptors is responsible for the inhibitory effect of extracellular nucleotides. First, the ATP concentration-dependence of the inhibition of the histamine-induced cAMP accumulation correlates to the ATP-mediated cytosolic Ca2+ mobilization. In addition, the order of potency of various nucleotides in terms of inhibition of the histamine-induced cAMP accumulation is consistent with that in the IP3 generation but not the cAMP generation and matches the known P2Y2 purinoceptor. Second, addition of the phorbol ester PMA inhibited the histamine-induced cAMP production, whereas inclusion of the PKC inhibitor GF109203X reversed the inhibitory effect of extracellular ATP. However, activation of cAMP-dependent protein kinase A, which also has been known to decrease G-protein-coupled receptor signaling in various cell types [23 ], is not involved in the ATP effect because prostaglandin E2 or BzATP, which elevate internal cAMP levels more than what can be elicited by ATP, did not affect the histamine-induced responses. Third, our studies show that the ATP-induced [Ca2+]i rise is unlike to play a role in the ATP effect, since the Ca2+ ionophores ionomycin or thapsigargin had little effect on the histamine-induced cAMP production. Therefore, we must conclude that PKC activation upon P2Y2 receptor stimulation is linked to the blockade of histamine-mediated signal transduction through the plasma membrane.

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-{gamma} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by the KOSEF and the Korea Research Foundation for the program year of 1999 and Brain Korea 21 Program of the Ministry of Education. This work was also supported by National Research and Development Program and Brain Research Program sponsored by the Ministry of Science and Engineering. We thank Ms. G. Hoschek for editing the manuscript.

Received March 23, 2000; revised August 7, 2000; accepted August 10, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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