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• W2057989750 abstract "Extracellular ATP increases intracellular Ca2+ ([Ca2+]i) in HL-60 cells. When cells are stimulated with supramaximal concentrations of ATP, although the initial [Ca2+]i increase is similar over a range of 30, 100, and 300 μm ATP, the rate of the return to basal [Ca2+]i level is faster in cells treated with higher concentrations of ATP. This probably results from differences in Ca2+ influx rather than Ca2+release, since the influx of the unidirectional Ca2+surrogates Ba2+ and Mn2+ also exhibit similar responses. Furthermore, while 300 μm ATP had an inhibitory effect on the thapsigargin-induced capacitative Ca2+ entry, 30 μm ATP potentiated the response. However, the inhibitory action of 300 μm ATP was blocked by protein kinase C (PKC) inhibitors, such as GF 109203X and chelerythrine, and the potentiating action of 30 μmATP was blocked by protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. The PKC inhibitors also slowed the decay rate of the Ca2+response induced by 300 μm ATP, and the PKA inhibitors increased it when induced by 30 μm ATP. In the measurements of PKA and PKC activity, 30 μm ATP activates only PKA, while 300 μm ATP activates both kinases. Taken together, these data suggest that the changes in the ATP-induced Ca2+ response result from differential modulation of ATP-induced capacitative Ca2+ entry by PKC and PKA in HL-60 cells. Extracellular ATP increases intracellular Ca2+ ([Ca2+]i) in HL-60 cells. When cells are stimulated with supramaximal concentrations of ATP, although the initial [Ca2+]i increase is similar over a range of 30, 100, and 300 μm ATP, the rate of the return to basal [Ca2+]i level is faster in cells treated with higher concentrations of ATP. This probably results from differences in Ca2+ influx rather than Ca2+release, since the influx of the unidirectional Ca2+surrogates Ba2+ and Mn2+ also exhibit similar responses. Furthermore, while 300 μm ATP had an inhibitory effect on the thapsigargin-induced capacitative Ca2+ entry, 30 μm ATP potentiated the response. However, the inhibitory action of 300 μm ATP was blocked by protein kinase C (PKC) inhibitors, such as GF 109203X and chelerythrine, and the potentiating action of 30 μmATP was blocked by protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. The PKC inhibitors also slowed the decay rate of the Ca2+response induced by 300 μm ATP, and the PKA inhibitors increased it when induced by 30 μm ATP. In the measurements of PKA and PKC activity, 30 μm ATP activates only PKA, while 300 μm ATP activates both kinases. Taken together, these data suggest that the changes in the ATP-induced Ca2+ response result from differential modulation of ATP-induced capacitative Ca2+ entry by PKC and PKA in HL-60 cells. Extracellular ATP evokes many physiological effects such as platelet aggregation, neurotransmission, inflammation, and muscle contraction in numerous cell types (1Gordon J.L. Biochem. J. 1986; 233: 309-319Crossref PubMed Scopus (1408) Google Scholar). These various effects of ATP are mediated by plasma membrane P2 purinergic receptors (2Dubyak G.R. Cowen D.S. Meuller L.M. J. Biol. Chem. 1988; 263: 18108-18117Abstract Full Text PDF PubMed Google Scholar). Six subtypes of P2 purinergic receptors, P2X, P2Y, P2D, P2T, P2Z, and P2U, were identified in pharmacological and functional studies and supported by cloning data (3Fredholm B.B. Abbracchio M.P. Burnstock G. Daly J.W. Harden T.K. Jacobson K.A. Leff P. Williams M. Pharmacol. Rev. 1994; 46: 143-156PubMed Google Scholar). It has been reported that in HL-60 cells extracellular ATP increases the intracellular free Ca2+ concentration ([Ca2+]i) 1The abbreviations used are: [Ca2+]i, intracellular free Ca2+concentration; CRAC, Ca2+ release-activated Ca2+ channel; fura-2/AM, fura-2 pentaacetoxymethyl ester; IP3, inositol 1,4,5-trisphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; MOPS, 3-[N-morpholino]propanesulfonic acid; ATPγS, 5′-O-(3-thiotriphosphate); Rp-cAMPS, 3′,5′-cyclic monophosphothioate.via plasma membrane P2U and P2X1 type receptors (4Dubyak G. El-moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar, 5Buell G. Michel A.D. Lewis C. Collo G. Humphrey P.P. A Surprenant A. Blood. 1996; 87: 2659-2664Crossref PubMed Google Scholar). We have also shown that extracellular ATP elevates cAMP through a novel type of receptor (6Choi S.Y. Kim K.T. Biochem. Pharmacol. 1997; 53: 429-432Crossref PubMed Scopus (25) Google Scholar). The P2U receptor is functionally coupled to phospholipase C (PLC) through pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. The IP3 produced increases the [Ca2+]i by mobilizing Ca2+ from the intracellular Ca2+stores. This Ca2+ mobilization activates the plasma membrane Ca2+ influx pathway through Ca2+release-activated Ca2+ channels (CRAC) and is termed capacitative Ca2+ entry (7Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 8Putney J.W. Bird G. St J. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (394) Google Scholar). The degree of Ca2+ entry is determined by the filling status of the intracellular Ca2+ store. The P2X1 receptor triggers entry of cations; however, it has been shown that the activity is very weak in undifferentiated HL-60 cells. Thus, ATP increases intracellular Ca2+ in HL-60 cells by mobilizing it from the intracellular stores and by influx from the extracellular space. We observed a different rate of decrease in the Ca2+ response, while the peak level remained the same, when HL-60 cells were stimulated with supramaximal concentrations of ATP. There are several mechanisms responsible for Ca2+ removal from the cytosol after the elevation of the [Ca2+]i. These mechanisms include sequestering of Ca2+ into intracellular stores, binding to various Ca2+-binding proteins, and actions by the Ca2+ pump and Na+/Ca2+ exchanger (9Carafoli E. Annu. Rev. Biochem. 1897; 56: 395-433Crossref Scopus (1776) Google Scholar). Among these, the Ca2+ pump, which transports ions across the plasma membrane and into intracellular stores, plays a critical role in reducing the elevated [Ca2+]i. The plasma membrane Na+/Ca2+ exchanger also plays an important role in the control of the intracellular free Ca2+concentration, exchanging three Na+ for one Ca2+. It appears to have a lower affinity for Ca2+ than the plasma membrane Ca2+ pump and a high capacity for removing increased Ca2+. Thus it operates efficiently when [Ca2+]i is increased beyond 10−8m. A number of Ca2+-binding proteins are also involved in buffering the cytosolic Ca2+concentration. We studied the mechanism by which the different patterns of decrease in Ca2+ occur upon stimulation with supramaximal concentrations of ATP in HL-60 cells. Our results suggest that this difference is not due to the cytosolic Ca2+removal system, but that it is instead mainly due to changes in capacitative Ca2+ entry by actions of PKA and PKC, which are differentially activated by ATP itself. ATP, UTP, thapsigargin, Triton X-100, Trizma (Tris base), trichloroacetic acid, EGTA, EDTA, sulfinpyrazone, MOPS, sodium fluoride, sodium orthovanadate, sodium pyrophosphate, β-glycerophosphate, leupeptin, pepstatin A, aprotinin, phenylmethylsulfonyl fluoride, and IP3 were purchased from Sigma. Fura-2 pentaacetoxymethyl ester was from Molecular Probes (Eugene, OR), and [3H]IP3 and [γ-32P]ATP were from NEN Life Science Products. PMA, chelerythrine, Rp-cAMPS, GF 109203X, KN62, and benzamil were obtained from Research Biochemicals Inc. (Natick, MA), and 1.4-dithiothreitol was from Boehringer Mannheim (Mannheim, Germany). H89 was purchased from Seikagaku Co. (Tokyo, Japan), and P81 phosphocellulose paper was purchased from Whatman. Nonidet P-40 was purchased from U. S. Biochemical Corp. Human promyelocytic leukemia HL-60 cells were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 20% (v/v) heat-inactivated bovine calf serum (Hyclone, Logan, UT) plus 1% (v/v) penicillin/streptomycin (Life Technologies, Inc.) under a humidified atmosphere of 5% CO2 at 37 °C. [Ca2+]i level was determined using the fluorescent Ca2+ indicator fura-2 as reported previously (10Suh B.C. Kim K.T. Biochem. Pharmacol. 1994; 47: 1262-1266Crossref PubMed Scopus (18) Google Scholar). HL-60 cells were incubated with 3 μmfura-2/AM in complete medium at 37 °C with stirring for 60 min. The final concentration of dimethyl sulfoxide (Me2SO) in the incubation medium was 0.3%. After the loading, cells were washed twice with Locke's solution (154 mm NaCl, 2.2 mmCaCl2, 5.6 mm KCl, 5.0 mm HEPES, 10 mm glucose, 1.2 mm MgCl2, pH 7.4) to remove extracellular dye. Sulfinpyrazone was added to the washing solution to a final concentration of 250 μm to prevent dye leakage (11Di Virgilio F. Fasolato C. Steinberg T.H. Biochem. J. 1988; 256: 959-963Crossref PubMed Scopus (126) Google Scholar). The fluorescence ratio was recorded at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 500 nm. [Ca2+]i was calculated according to Grynkiewiczet al. (12Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). In Ca2+-free experiments, cells were bathed in Ca2+-free Locke's solution (156.2 mm NaCl, 5.6 mm KCl, 5.0 mm HEPES, 10 mm glucose, 1.2 mm MgCl2, pH 7.4) instead of Ca2+-containing Locke's solution. Cells loaded with fura-2/AM as described above were stimulated with ATP in the presence of 2 mm Mn2+, and fluorescence quenching was measured at an excitation wavelength of 360 nm, which is an isosbestic wavelength, and at an emission wavelength of 500 nm (13Sage S.O. Merritt J.E. Hallam T.J. Rink T.J. Biochem. J. 1989; 258: 923-926Crossref PubMed Scopus (168) Google Scholar). IP3mobilization was determined by competition assay with [3H]IP3 in binding to IP3-binding protein as described previously (14Suh B.C. Kim K.T. J. Neurochem. 1995; 64: 2500-2508Crossref PubMed Scopus (19) Google Scholar). To determine IP3production, 2 × 106 cells per sample were harvested and stimulated with ATP. The reaction was terminated by the addition of ice-cold 15% (w/v) trichloroacetic acid containing 10 mmEGTA. After centrifugation at 2,000 × g for 5 min, the supernatant was obtained. The trichloroacetic acid was removed by three extractions with diethyl ether. The final extract was neutralized with 200 mm Trizma base and its pH adjusted to about 7.4. 20 μl of the cell extract was added to 20 μl of assay buffer (0.1m Tris buffer containing 4 mm EDTA) and 20 μl of [3H]IP3 (0.1 μCi/ml). Finally, 20 μl of binding protein solution was added. The IP3-binding protein was prepared from bovine adrenal cortex according to the method of Challiss et al. (15Challiss R.A. Chilvers E.R. Willcocks A.L. Nahorski S.R. Biochem. J. 1990; 265: 421-427Crossref PubMed Scopus (127) Google Scholar). The mixture was incubated for 15 min on ice and then centrifuged at 2,000 × g for 10 min. 100 μl of water and 1 ml of scintillation mixture were added to the pellet to measure the radioactivity. The IP3concentration of the sample was determined by comparison with a standard curve and expressed as picomoles/mg of protein. The total cellular protein concentration was measured by the Bradford method after sonication of 2 × 106 cells. Intracellular cAMP was determined by measuring the formation of [3H]cAMP from [3H]adenine nucleotide pools as we have described previously (16Suh B.C. Lee C.O. Kim K.T. J. Neurochem. 1995; 64: 1071-1079Crossref PubMed Scopus (36) Google Scholar). The cells were grown in complete medium and loaded with [3H]adenine (2 μCi/ml) for 24 h. After the loading, cells were washed twice with Locke's solution and then stimulated with agonist. The reaction was stopped by adding 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 column. The increase in intracellular cAMP was calculated as [3H]cAMP/([3H]ATP + [3H]cAMP) × 103. PKA activity was determined by measuring the incorporation of 32P from [γ-32P]ATP into the PKA-specific peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), using a procedure described previously (17Roskoski Jr., R. Methods Enzymol. 1983; 99: 3-6Crossref PubMed Scopus (691) Google Scholar, 18Davies W.A. Berghorn K.A. Albrecht E.D. Pepe G.J. Endocrinology. 1993; 132: 2491-2497Crossref PubMed Scopus (16) Google Scholar), with some modifications. Briefly, HL-60 cells (1 × 107 cells/tube) were harvested and treated with inhibitor mixture containing 1 μm GF 109203X and 1 μm KN62, inhibitors of PKC and Ca2+/calmodulin-dependent protein kinase, respectively, for 5 min. They were then stimulated with 30 or 300 μm ATP for 3 min. After the stimulation, the cells were washed twice with Locke's solution within another 2 min, then resuspended in 100 μl of buffer I containing 20 mmTris-HCl, pH 7.5, 0.25 m sucrose, 10 mm EGTA, 2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, 200 μm sodium pyrophosphate, 200 μm sodium fluoride, 1 mm dithiothreitol. The cells were sonicated and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was saved as the PKA fraction and used for in vitro PKA activity measurements. All of the following procedures were performed on ice unless stated otherwise. The reaction was initiated by the addition of 10 μl of cell extract to the 30 μl of a test mixture consisting of 10 μl of Mg2+/ATP mixture containing 75 mm MgCl2, 500 μmATP, 50 μCi of [γ-32P]ATP (3,000 Ci/mmol), 10 μl of 500 μm Kemptide, and 10 μl of inhibitor mixture containing 0.02 μm GF 109203X, 0.9 μm KN62. 10 μm cAMP were added to the reaction mixture with Kemptide for a positive control, and 10 μl of buffer II instead of Kemptide was added to determine the endogenous PKA substrate. All assay components were prepared by using buffer II that contained 20 mm MOPS, pH 7.2, 25 mm β-glycerol phosphate, 5 mm EGTA, 1 mm sodium orthovanadate, and 1 mm dithiothreitol. The reaction mixture was gently vortexed and placed in a 30 °C water bath for 10 min. Then 25 μl of the reaction mixture was transferred to 1 × 3-cm P81 phosphocellulose strips, which were immediately immersed into 0.75% phosphoric acid. The strips were washed three times with 0.75% phosphoric acid and then dehydrated in 95% ethanol, air-dried, and placed into liquid scintillation vials. The radioactivity was quantified in a Beckman LS 8000 liquid scintillation counter. PKC activity was measured by determining the incorporation of 32P from [γ-32P]ATP into histone IIIS as described previously (17Roskoski Jr., R. Methods Enzymol. 1983; 99: 3-6Crossref PubMed Scopus (691) Google Scholar, 19Noland Jr., T.A. Dimino M.J. Biol. Reprod. 1986; 35: 863-872Crossref PubMed Scopus (36) Google Scholar), with some modifications. HL-60 cells were harvested and treated with inhibitor mixture containing 10 μm H89 and 1 μm KN62 and then stimulated with 30 or 300 μm ATP and 100 nm PMA. After the stimulation, the cells were washed three times with Locke's solution and then resuspended in 200 μl of buffer I, which is described in the PKA assay. The cells were sonicated and centrifuged at 100,000 ×g for 1 h at 4 °C, and the pellet was saved as the membrane fraction and then was solubilized with the above buffer I containing 1% Nonidet P-40. The reaction was initiated by the addition of 10 μl of solubilized membrane fraction to the 40 μl of reaction mixture containing 10 μl of 500 μm histone IIIS, 10 μl of inhibitor mixture containing 2 μm PKI, PKA inhibitor peptide and 0.9 μm KN62, 10 μl of 500 nm PMA, and 10 μl of the Mg2+/ATP mixture containing 75 mm MgCl2, 500 μmATP, and 100 μCi of [γ-32P]ATP. All assay components were prepared using buffer II described in the assay of PKA. The reaction mixture was incubated at 30 °C for 10 min, and 25 μl of the reaction mixture was transferred to the P81 phosphocellulose strips. The strips were immersed into the 0.75% phosphoric acid and washed three times for 10 min. After washing, they were rinsed in 95% ethanol, air-dried, and quantified by measuring the radioactivity in a liquid scintillation counter. Data are summarized as the means ± S.E. EC50 was calculated with the AllFit program (20De Lean A. Munson P.J. Rodbard D. Am. J. Physiol. 1978; 235: E97-E102Crossref PubMed Google Scholar). We considered differences significant at p < 0.05. In HL-60 cells, ATP increased the [Ca2+]i in a concentration-dependent manner with maximal and half-maximal effective concentrations (EC50) seen at approximately 10 μm and 85 nm, respectively (Fig. 1 A). Fig. 1 Billustrates the typical changes in [Ca2+]iobserved in fura-2-loaded HL-60 cells stimulated with maximal concentrations of ATP. Initially, the [Ca2+]iincreased rapidly to a peak level and then completely returned to the basal Ca2+ level, even if the stimulant remained present. Notably, the changes in cytosolic Ca2+ exhibited a different desensitization pattern in response to supramaximal concentrations of ATP as compared with the lower concentrations. Although the peak levels were similar, the rate of return to the basal [Ca2+]i level was faster in cells treated with the higher concentration of ATP. This phenomenon is clearly seen in Fig. 1 C. We measured the time from peak response of the Ca2+ signal to the 70% desensitized [Ca2+]i level as indicated in theinset of Fig. 1 C. The data show that the times for return to 30% of the peak level became less in stimulations with increasing concentration of ATP. In other words, the higher the ATP concentration, the faster the return rate. We further analyzed whether changes in the desensitization rate are elicited by Ca2+release from intracellular stores or Ca2+ influx from the extracellular space, since ATP increases [Ca2+]ivia both pathways. To measure Ca2+ release from the intracellular stores, cells were stimulated with ATP in the absence of extracellular Ca2+. Subsequently, 3 mm Ca2+ was introduced to the medium to assess the activity of the Ca2+ influx. As shown in Fig. 2 A, when cells were stimulated with 30, 100, and 300 μm ATP in Ca2+-free medium, there were no significant differences in Ca2+release, but large differences occurred in Ca2+ influx between the different ATP concentrations. Ca2+ influx stimulated by 300 μm ATP was 62.5% less than when stimulated with 30 μm ATP. The data indicate that the differences in the falling state of the Ca2+ responses caused by the supramaximal concentration of ATP resulted from changes in Ca2+ influx. Since the amount of Ca2+release was small, it is possible that undetectable differences could exist between those stimulations. Thus, we measured the IP3production of cells stimulated with ATP. Fig. 2 B shows the time course for IP3 production when cells were stimulated with 30 and 300 μm ATP. At both concentrations, maximal IP3 generation was obtained after 15 s. At that time, 300 μm ATP generated approximately 2.3 times the amount of IP3 than 30 μm ATP. Furthermore, the intracellular IP3 level was more sustained in the stimulation with 300 μm as compared with the stimulation with 30 μm. Thus, from the result of IP3production, it seems likely that 300 μm ATP stimulation can cause more Ca2+ release than 30 μm ATP or, at least, can trigger the release of a similar amount of Ca2+ from the internal stores, while IP3produced by 30 μm ATP is enough to maximally mobilize Ca2+. Therefore, we conclude that the 300 μmATP-induced Ca2+ release is not less than the 30 μm ATP-induced one and that the difference detected in the Ca2+ decay rate is due to changes in the Ca2+ influx from the extracellular space. To test whether differences in the falling state of the Ca2+ response are due to modulation of the Na+/Ca2+ exchanger and Ca2+/ATPase activity, we measured Mn2+ and Ba2+ influx after the addition of ATP. Mn2+ and Ba2+ are good Ca2+ surrogates, since they are not pumped out of the cell, so they can be considered as selective tracers for entry (21Suh B.-C. Song S.-K. Kim Y.-K. Kim K.-T. J. Biol. Chem. 1996; 271: 32753-32759Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 22Schilling W.P. Rajan L. Strobl-Jager E. J. Biol. Chem. 1989; 264: 12838-12848Abstract Full Text PDF PubMed Google Scholar). Mn2+ uptake was estimated by the quenching of the fura-2 fluorescence when excited at the 360-nm wavelength, which is an isosbestic wavelength and insensitive to variations in Ca2+ concentration. Ba2+ uptake was estimated by the increase in the fura-2 fluorescence ratio when excited at the 340- and 380-nm wavelength. Fig. 3 A shows the fluorescence quenching by Mn2+ influx when cells were stimulated with 30, 100, and 300 μm ATP. As 2 mmMn2+ was applied to the medium, it entered the cell slowly. Subsequent stimulation of the cells with 30 μm ATP accelerated the Mn2+ entry as compared with the untreated control (dotted trace). 100 μm ATP also accelerated the entry, however, with a slower rate than 30 μm ATP. In contrast, 300 μm ATP stimulation had little effect on fluorescence quenching in comparison with the untreated control. The data indicate that the lower concentration of ATP activates the divalent cation influx. This result was also supported by the Ba2+ uptake. To measure Ba2+influx, cells were stimulated with ATP in the absence of external Ca2+. When the Ba2+ was added to the medium, it caused an increase in the fluorescence intensity reflecting Ba2+ uptake. The influx of Ba2+ elicited by ATP shows a concentration-dependent pattern with the higher concentrations of ATP triggering less Ba2+ uptake. This is similar to the result of Ca2+ influx as shown in Fig. 2 A. These results suggest that ATP regulates the amount of Ca2+ influx, but does not modulate the activity of Na+/Ca2+ exchanger and Ca2+/ATPase. To investigate the involvement of CRAC, we tested the effect of various metal ions on ATP-induced Ca2+ signaling, because metal ions are known to block CRAC. Cells were treated with 30 μm La3+, Cd2+, Co2+, or Ni2+ for 1 min and then stimulated with ATP in Ca2+-containing medium. The difference between the 30 and 300 μm ATP-induced Ca2+ signals disappeared in the presence of 30 μm La3+, whereas the other metal ions had little or negligible effects (data not shown). The data, thus, suggested that changes in CRAC activity could be the main cause for the rapid desensitization of the Ca2+ response induced by higher concentrations of ATP. In the above experiments, we found that different Ca2+ decay rates were caused by changes in Ca2+ influx. ATP induces capacitative Ca2+entry through CRAC, which is stimulated by a Ca2+ influx factor liberated from the depleted intracellular Ca2+stores by action of IP3. To study the regulation of capacitative Ca2+ entry, we used thapsigargin, which depletes intracellular Ca2+ stores by inhibiting the microsomal Ca2+/ATPase and induces Ca2+ influx (23Takemura H. Hughes A.R. Thastrup O. Putney Jr., J.W. J. Biol. Chem. 1989; 264: 12266-12271Abstract Full Text PDF PubMed Google Scholar). The differences in Ca2+ influx could also be demonstrated when we measured the effect of ATP pretreatment on thapsigargin-induced capacitative Ca2+ entry. As shown in Fig. 4, cells were incubated with 100 nm thapsigargin in a Ca2+-free medium, which resulted in a transient [Ca2+]i elevation. After reaching a peak, the [Ca2+]i decreased slowly to the basal level, which reflects the emptying of the intracellular Ca2+ stores. The subsequent addition of 3 mmCa2+ to the medium induced a marked and sustained Ca2+ rise (dotted trace). Treatment with 300 μm ATP for 1 min prior to the extracellular Ca2+ application significantly diminished the thapsigargin-induced capacitative Ca2+ entry by 25.7% as compared with the untreated control (dotted trace). 100 μm ATP had also a slightly inhibitory effect. However, 30 μm ATP substantially potentiated the thapsigargin-induced capacitative Ca2+ entry to 152.8% over the control cells. These results indicate that ATP has a biphasic effect on the thapsigargin-induced capacitative Ca2+ entry linked to its concentration. Therefore, we speculated that ATP itself might potentiate and inhibit capacitative Ca2+ entry that it evokes, forming both a positive and a negative feedback loop. At 30 μm, ATP potentiates Ca2+ influx, which slows down the desensitization of the [Ca2+]i. Whereas, at 300 μm, ATP inhibits Ca2+ influx, which speeds up the desensitization of the [Ca2+]i. ATP activates PLC and produces IP3 and diacylglycerol, which subsequently activates PKC. We have also shown that extracellular ATP triggers elevation of cAMP in HL-60 cells (6Choi S.Y. Kim K.T. Biochem. Pharmacol. 1997; 53: 429-432Crossref PubMed Scopus (25) Google Scholar). To assess the involvement of PKC and PKA in modulation of capacitative Ca2+ entry, we used inhibitors specific for those kinases. GF 109203X and chelerythrine, selective PKC inhibitors, were used to characterize the inhibitory or stimulatory effect of ATP on the capacitative Ca2+ entry. Fig. 5 A shows what effect pretreatment with protein kinase inhibitors has on the Ca2+transient elicited by thapsigargin. 300 μm ATP has a substantial inhibitory effect on thapsigargin-induced capacitative Ca2+ entry as seen in Fig. 4. This inhibitory action was antagonized by pretreatment with 1 μm GF 109203X, and Ca2+ influx was even potentiated in the presence of GF 109203X. Similar effects were obtained when 1 μmchelerythrine was used in place of GF 109203X. The results suggest that PKC, when activated by 300 μm ATP, inhibits thapsigargin-induced capacitative Ca2+ entry. The involvement of PKA in the 30 μm ATP-induced potentiation of capacitative Ca2+ entry was investigated by testing the effect of the PKA inhibitors H89 and Rp-cAMPS. Fig. 5 B shows the effect that H89 and Rp-cAMPS has on the enhancement of the thapsigargin-induced capacitative Ca2+entry by 30 μm ATP. In cells treated with H89, this potentiation was blocked. The potentiation also disappeared in 20 μm Rp-cAMP-treated cells. These results suggest the involvement of PKA in the 30 μm ATP-induced enhancement of the capacitative Ca2+ entry. The effects of protein kinase inhibitors on ATP activity in the desensitization pattern of [Ca2+]i were also investigated. Inhibition of PKA by pretreatment with 2 μmH89 significantly accelerated the decay rate of the 30 μmATP-induced [Ca2+]i level with little effect on the peak Ca2+ level (Fig. 6 A). In contrast, pretreatment with 1 μm GF 109203X slowed the decay rate induced by 300 μm ATP, resulting in a Ca2+ response similar to the 30 μm ATP-evoked response (Fig. 6 B). The inhibitory action of 300 μm ATP was slightly enhanced in the presence of PKA inhibitors, while the potentiating effect of 30 μm ATP became even more activated in the presence of PKC inhibitors (data not shown). Thus, the slower decay rate of the 30 μm ATP-induced Ca2+ signal may be the result of the potentiating action of PKA as it increases the capacitative Ca2+ entry induced by ATP, whereas the rapid decay rate in the 300 μm ATP-induced Ca2+ signal might be the result of an inhibitory action by PKC as it blocks the ATP-induced capacitative Ca2+ entry. Taken together, the different desensitization rates of the ATP-induced Ca2+ signals after peak level could be the result of an interplay between inhibition by PKC and activation by PKA of the capacitative Ca2+entry. Since it has been shown that P2U receptors were present and coupled to PLC in HL-60 cells, we treated the cells with UTP and measured the return rate of the [Ca2+]i level. Fig. 7 A illustrates the times for return to 30% of the peak level in response to supramaximal concentrations of UTP: the higher the UTP concentration, the faster the return rate. However, although the phenomenon was similar to that of ATP, the rate of the return to the basal [Ca2+]ilevel was not as remarkable compared with that induced by ATP in Fig. 1 C. We further analyzed whether changes in the desensitization rate involve PKC and PKA in the modulation of the UTP-induced capacitative Ca2+ entry using kinase inhibitors. Fig. 7 B shows that pretreatment with GF 109203X slowed the decay rate induced by 300 μm UTP. However, inhibition of PKA by pretreatment with H89 had no effect on the return rate of the 30 μm UTP-induced [Ca2+]i level (data not shown). The difference in the desensitization pattern between ATP and UTP might result from different activations of effector enzymes, such as PLC and adenylyl cyclase. In HL-60 cells, UTP has no effect on cAMP production (6Choi S.Y. Kim K.T. Biochem. Pharmacol. 1997; 53: 429-432Crossref PubMed Scopus (25) Google Scholar). Therefore, the results suggest that the effect of PKA increasing the capacitative Ca2+ entry is not involved in UTP-treated cells and that PKC alone acts in the desensitization of the UTP-induced Ca2+ response. To assess agonist concentration-dependent differential activation of PKC and PKA, we measured the production of cAMP and IP3. Fig. 8 shows the production of cAMP and IP3 induced by various concentrations of ATP. The maximal increase of cAMP was obtained with 300 μm ATP. The EC50 value was 19.2 μm. Particularly, for 30 and 300 μm ATP, respectively, the cAMP levels reached 137.2 ± 9.3 and 190.2 ± 7.7 over the basal cAMP level of 25.3 ± 4.2. The amounts of IP3 caused by 30 and 300 μm ATP were 27.7 ± 5.7 and 67.0 ± 5.5 pmol/mg of protein, respectively, while the basal IP3 level was 18.0 ± 3.1. There was only a slight increase in the IP3 level in the response to 30 μm ATP, whereas the cAMP level was already significantly increased at that concentration. On the other hand, during stimulation with 300 μm ATP, cAMP was produced maximally, and IP3was also" @default.
• W2057989750 created "2016-06-24" @default.
• W2057989750 creator A5003536922 @default.
• W2057989750 creator A5022553207 @default.
• W2057989750 creator A5087880937 @default.
• W2057989750 date "1997-08-01" @default.
• W2057989750 modified "2023-10-17" @default.
• W2057989750 title "Feedback Regulation of ATP-induced Ca2+ Signaling in HL-60 Cells Is Mediated by Protein Kinase A- and C-mediated Changes in Capacitative Ca2+ Entry" @default.
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