Regulation of Capacitative Ca2+ Influx in Human Neutrophil Granulocytes
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26471
ISSN1083-351X
AutoresMiklós Geiszt, András Kapùs, Katalin Német, Lóránt Farkas, Erzsébet Ligeti,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoCa2+ entry through the capacitative (store-regulated) pathway was shown to be inhibited in neutrophil granulocytes by the protein kinase C activator phorbol 12-myristate 13-acetate and the chemoattractantN-formyl-methionyl-leucyl-phenylalanine (fMLP) by a hitherto unknown mechanism. Measuring both Ca2+ and Mn2+ entry into store-depleted cells we show in the present study that inhibition of the capacitative pathway is absent in various forms of chronic granulomatous disease. To establish the possible relationship between inhibition of the capacitative pathway and ability of O·̄2 production and consequent membrane depolarization, gradual changes of the membrane potential were evoked in neutrophils of healthy individuals. This was accomplished by pharmacological manipulation of the membrane potential and by variations of the concentration and type of the stimulant. Close relationship was observed between membrane depolarization and inhibition of Mn2+ entry through the capacitative transport route. Our results provide an explanation for the inhibitory action of fMLP and phorbol 12-myristate 13-acetate on capacitative cation influx and reveal that upon physiological stimulation, Ca2+ entry into neutrophils is restricted by the depolarization accompanying O·̄2 production. Ca2+ entry through the capacitative (store-regulated) pathway was shown to be inhibited in neutrophil granulocytes by the protein kinase C activator phorbol 12-myristate 13-acetate and the chemoattractantN-formyl-methionyl-leucyl-phenylalanine (fMLP) by a hitherto unknown mechanism. Measuring both Ca2+ and Mn2+ entry into store-depleted cells we show in the present study that inhibition of the capacitative pathway is absent in various forms of chronic granulomatous disease. To establish the possible relationship between inhibition of the capacitative pathway and ability of O·̄2 production and consequent membrane depolarization, gradual changes of the membrane potential were evoked in neutrophils of healthy individuals. This was accomplished by pharmacological manipulation of the membrane potential and by variations of the concentration and type of the stimulant. Close relationship was observed between membrane depolarization and inhibition of Mn2+ entry through the capacitative transport route. Our results provide an explanation for the inhibitory action of fMLP and phorbol 12-myristate 13-acetate on capacitative cation influx and reveal that upon physiological stimulation, Ca2+ entry into neutrophils is restricted by the depolarization accompanying O·̄2 production. Stimulation of neutrophil granulocytes by various receptor agonists is accompanied by an increase of intracellular Ca2+ concentration. This Ca2+ transient is a crucial element in the transduction of extracellular signals, leading to the activation of different effector responses (e.g.degranulation, superoxide production) of the cells (1Scharff O. Foder B. Physiol. Rev. 1993; 73: 547-582Crossref PubMed Scopus (63) Google Scholar, 2Pozzan T. Lew D.P. Wollheim C.B. Tsien R.Y. Science. 1983; 221: 1413-1415Crossref PubMed Scopus (285) Google Scholar, 3Sengelov H. Kjeldsen L. Borregaard N. J. Immunol. 1993; 150: 1535-1543PubMed Google Scholar). The Ca2+ signal of neutrophils consists of two components: (i) a rapid, transient increase of [Ca2+] i 1The abbreviations used are: [Ca2+] i, intracellular Ca2+; CGD, chronic granulomatous disease; di-O-C5(3), 3–3′-dipentyloxacarbocyanine; DPI, diphenylene iodonium; fMLP,N-formyl-methionyl-leucyl-phenylalanine; phox, phagocyte oxidase; PMA, phorbol 12-myristate 13-acetate; TG, thapsigargin. due to Ca2+ release from the internal stores induced by inositol 1,4,5-trisphosphate and (ii) a sustained elevation of [Ca2+] i due to Ca2+ influx from the extracellular space (1Scharff O. Foder B. Physiol. Rev. 1993; 73: 547-582Crossref PubMed Scopus (63) Google Scholar). Neutrophil granulocytes do not possess voltage-dependent Ca2+ channels, but there is growing evidence that emptying of the internal Ca2+ stores initiates Ca2+ influx from the extracellular milieu,i.e. the Ca2+ content of the stores regulates Ca2+-conducting channels in the plasma membrane (4Montero M. Alvarez J. Garcia Sancho J. Biochem. J. 1991; 277: 73-79Crossref PubMed Scopus (114) Google Scholar, 5Demaurex N. Monod A. Lew D.P. Krause K.H. Biochem. J. 1994; 297: 595-601Crossref PubMed Scopus (81) Google Scholar, 6Demaurex N. Schlegel W. Várnai P. Mayr G. Lew D.P. Krause K.H. J. Clin. Invest. 1992; 90: 830-839Crossref PubMed Scopus (22) Google Scholar, 7Geiszt M. Káldi K. Szeberényi J.B. Ligeti E. Biochem. J. 1995; 305: 525-528Crossref PubMed Scopus (37) Google Scholar). This Ca2+ entry is often referred to as capacitative or store-operated Ca2+ influx (8Putney J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar). In human neutrophil granulocytes the regulation of capacitative Ca2+ influx is more complex: agonists of several chemotactic receptors,N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor, initiate Ca2+ influx by emptying the internal stores in an inositol 1,4,5-trisphosphate-dependent manner while fMLP, but not platelet activating factor, was also shown to inhibit capacitative Ca2+ influx (9Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 13055-13061Abstract Full Text PDF PubMed Google Scholar). A similar action of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) has been reported, too (9Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 13055-13061Abstract Full Text PDF PubMed Google Scholar, 10McCarthy S.A. Hallam T.J. Merritt J.E. Biochem. J. 1989; 264: 357-364Crossref PubMed Scopus (60) Google Scholar, 11Foder B. Scharff O. Thastrup O. Cell Calcium. 1989; 10: 477-490Crossref PubMed Scopus (64) Google Scholar, 12Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1994; 269: 3963-3967Abstract Full Text PDF PubMed Google Scholar, 13Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 26911-26919Abstract Full Text PDF PubMed Google Scholar). The inhibitory effect of both fMLP and PMA is absent in undifferentiated HL-60 cells and develops gradually during the differentiation induced by dimethyl sulfoxide (13Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 26911-26919Abstract Full Text PDF PubMed Google Scholar). Despite the similarity in the basic feature, i.e. blocking the capacitative Ca2+ influx, several differences were also described in the characteristics of the inhibition brought about by the two compounds. (i) The fMLP-induced inhibition is transient while the effect of PMA is sustained (9Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 13055-13061Abstract Full Text PDF PubMed Google Scholar). (ii) Staurosporine, an inhibitor of protein kinase C prevents the inhibitory effect of fMLP only partially but blocks the effect of PMA completely (9Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 13055-13061Abstract Full Text PDF PubMed Google Scholar). (iii) The inhibitory action of fMLP is sensitive to pertussis toxin whereas the effect of PMA is not influenced by the toxin (14Alonso M.T. Mollinedo F. Barasoain I. Alvarez J. Garcia Sancho J. J. Leuk. Biol. 1996; 60: 323-327Crossref Scopus (2) Google Scholar). (iv) Okadaic acid, an inhibitor of protein phosphatases prolongs the inhibition elicited by fMLP but it does not affect the PMA-induced response (12Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1994; 269: 3963-3967Abstract Full Text PDF PubMed Google Scholar). On the basis of these observations a role of protein phosphorylation was suggested but the intervening kinase(s) remained unidentified (9Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 13055-13061Abstract Full Text PDF PubMed Google Scholar, 12Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1994; 269: 3963-3967Abstract Full Text PDF PubMed Google Scholar, 13Montero M. Garcia Sancho J. Alvarez J. J. Biol. Chem. 1993; 268: 26911-26919Abstract Full Text PDF PubMed Google Scholar, 14Alonso M.T. Mollinedo F. Barasoain I. Alvarez J. Garcia Sancho J. J. Leuk. Biol. 1996; 60: 323-327Crossref Scopus (2) Google Scholar). The characteristics of Ca2+ influx inhibition by fMLP and PMA are remarkably similar to the features of superoxide production, evoked by the same agents (15Thelen M. Dewald B. Baggiolini M. Physiol. Rev. 1993; 73: 797-821Crossref PubMed Scopus (260) Google Scholar, 16Morel F. Doussiere J. Vignais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (527) Google Scholar). Generation of superoxide by the NADPH oxidase induces rapid depolarization of the cells mainly due to electron transfer from the intracellular milieu to the extracellular environment (17Seligmann B.E. Gallin J.I. J. Clin. Invest. 1980; 66: 493-503Crossref PubMed Scopus (119) Google Scholar, 18Whitin J.C. Chapman C.E. Simons E.R. Chovaniec M.E. Cohen H.J. J. Biol. Chem. 1980; 255: 1874-1878Abstract Full Text PDF PubMed Google Scholar, 19Lew P.D. Wollheim C. Seger R.A. Pozzan T. Blood. 1984; 63: 231-233Crossref PubMed Google Scholar, 20Nanda A. Grinstein S. Cell. Physiol. Biochem. 1991; 1: 65-75Crossref Scopus (11) Google Scholar, 21Di Virgilio F. Lew P.D. Andersson T. Pozzan T. J. Biol. Chem. 1987; 262: 4574-4579Abstract Full Text PDF PubMed Google Scholar). Depolarization itself was shown to inhibit the agonist-induced Ca2+ influx in several cell types (22Kovács T. Tordai A. Szász I. Sarkadi B. Gárdos G. FEBS Lett. 1990; 266: 171-174Crossref PubMed Scopus (9) Google Scholar, 23Sarkadi B. Tordai A. Gárdos G. Biochim. Biophys. Acta. 1990; 1027: 130-140Crossref PubMed Scopus (33) Google Scholar, 24Merritt J.E. Rink T.J. J. Biol. Chem. 1987; 262: 17362-17369Abstract Full Text PDF PubMed Google Scholar). However, the possibility of a causal relationship between inhibition of capacitative Ca2+ influx by fMLP and PMA and depolarization coupled to superoxide generation elicited by the same agents has never been raised. In the present study two approaches were used to test this hypothesis. First the effect of fMLP and PMA on capacitative Ca2+influx was investigated in neutrophil granulocytes of patients suffering from chronic granulomatous disease (CGD), i.e. in cells which are unable to produce superoxide, but possess the capacitative Ca2+ entry mechanism. The second approach consisted of testing the inhibitory action of fMLP and PMA on Ca2+ influx in healthy neutrophils, where O·̄2production or membrane potential have been modulated by pharmacological means. The obtained results reveal a close relationship between plasma membrane depolarization and inhibition of Ca2+ influx into neutrophil granulocytes. The suggested mechanism may play a significant autoregulatory role in healthy granulocytes, whereas in CGD cells the lack of inhibition of capacitative Ca2+ entry may represent an alteration of cellular Ca2+ metabolism. To our knowledge this is the first report showing disturbed regulation of capacitative Ca2+ entry in a human disease. Fura-2/AM was obtained from Calbiochem; Percoll from Pharmacia; dimethyl sulfoxide, PMA, fMLP, cytochrome c, and thapsigargin were from Sigma and di-O-C5(3) from Molecular Probes. Diphenylene iodonium (DPI) was the kind gift of Dr. Arvind Nanda (Hospital for Sick Children, Toronto, Canada). All the other reagents were of research grade. The routinely used medium (referred to as H medium) contained in mm: NaCl 145, KCl 5, MgCl2 1, CaCl20.8, HEPES 10, glucose 5, pH 7.4. The Ca2+-free medium consisted of the same constituents except for CaCl2 and was supplemented with 0.5 mm EGTA. In the KCl based H medium NaCl was replaced by KCl. Human neutrophils were prepared from blood of healthy volunteers by dextran sedimentation followed by Percoll gradient centrifugation according to the procedure described in Ref.25Hjorth R. Jonsson A.K. Vretblad P. J. Immunol. Methods. 1981; 43: 95-101Crossref PubMed Scopus (239) Google Scholar. Contaminating red cells were removed by hypotonic lysis. Cells were finally suspended in H medium and kept at room temperature. Preparations contained more than 95% neutrophils, viability as determined by erythrosin B dye exclusion exceeded 97%. The diagnosis of CGD was established on the basis of serious reduction of O·̄2 production and by detection of the molecular defect using Western blot analysis. Qualitative investigation of O·̄2 generation was carried out by the nitro blue tetrazolium slide test as described in Ref. 26Johansen K.S. Acta Pathol. Microbiol. Immunol. Scand. C. 1983; 91: 349-354Google Scholar. Quantitative measurements consisted of determination of chemiluminescence (27Allen R.C. Methods Enzymol. 1986; 133: 449-493Crossref PubMed Scopus (444) Google Scholar) and cytochrome c reduction (28Markert M. Andrews P.C. Babior B.M. Methods Enzymol. 1984; 105: 358-365Crossref PubMed Scopus (376) Google Scholar) following stimulation of the cells by 300 nm PMA. The amount of cytochrome b 558 in the separated membrane fraction was determined on the basis of the heme spectrum as detailed in Ref. 29Roos D. de Boer M. Borregaard N. Bjerrum O.W. Valerius N.H. Seger R.A. Muhlebach T. Belohradsky B.H. Weening R.S. J. Leukocyte Biol. 1992; 51: 164-171Crossref PubMed Scopus (50) Google Scholar. Expression of the different subunits of NADPH oxidase was assessed by Western blot analysis using monoclonal antibodies against gp91 phox and p22 phox and polyclonal antibodies against p47 phox . All the applied antibodies were developed and kindly provided by Professor Dirk Roos (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, Netherlands). Genetic analysis of the mutation underlying CGD was carried out by Professor Dirk Roos (CLB, Amsterdam, Netherlands) and the results were reported in Ref. 30Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Német K. Hossle J.P. Bernatowska Matuszkiewicz E. Middleton Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar. Four patients (N. M., L. F., A. R., and G. B.) investigated in the present study had a mutation on the cytochromebβ (CYBB) gene and expressed neither gp91 phox nor p22 phox in their neutrophils. One patient (I. B.) with p47 phox deficiency had a GT deletion on the gene neutrophil cytosol factor 1 and the cytosolic protein p47 phox was absent in the granulocytes of this patient. Further data on the patients investigated in the present study are detailed in Ref. 30Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Német K. Hossle J.P. Bernatowska Matuszkiewicz E. Middleton Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar. For loading with the fluorescent dye, cells (5 × 107/ml) were incubated in the presence of 4 μm Fura-2/AM for 30 min at 37 °C. Thereafter cells were washed to remove the extracellular dye and resuspended in H medium at a density of 5 × 107/ml. Fura-2-loaded cells were stored at room temperature. For measurement of [Ca2+] i, Fura-2-loaded cells (106) were suspended in 3 ml of H medium and preincubated at 37 °C for 5 min prior to addition of any agent. Changes of the fluorescence were recorded in a Deltascan dual-wavelength spectrofluorimeter (Photon Technology International, South Brunswick NJ) using wavelengths 340 and 380 nm for excitation and 505 nm for emission. Measurements were performed at 37 °C with continuous stirring. [Ca2+] i was calculated from the ratio of fluorescence excited at 340 and 380 nm, following the method detailed in Ref. 31Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar. Data were analyzed with the Felix software (PTI). Mn2+influx measurements were performed under the same conditions as described for [Ca2+] i except that the excitation wavelength was 360 nm. At this wavelength, fluorescence of Fura-2 is quenched by Mn2+ but it is not influenced by changes of [Ca2+] i (31Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Unless otherwise stated, Mn2+ influx was initiated by adding 300 μm MnCl2. Changes of the membrane potential were monitored by the potential sensitive fluorescent dye 3–3′-dipentyloxacarbocyanine (di-O-C5(3)) as desribed in Refs. 17Seligmann B.E. Gallin J.I. J. Clin. Invest. 1980; 66: 493-503Crossref PubMed Scopus (119) Google Scholar and 32Seligmann B.E. Gallin E.K. Martin D.L. Shain W. Gallin J.I. J. Membr. Biol. 1980; 52: 257-272Crossref PubMed Scopus (73) Google Scholar. Cells (106) were suspended in 3 ml of H medium in the presence of 100 nmdi-O-C5(3) and stimuli were added after binding of the probe reached equilibrium and the fluorescence had stabilized. Changes of the fluorescence were recorded in the spectrofluorimeter used for [Ca2+] i measurements or in a Perkin-Elmer 3000 spectrofluorimeter with wavelengths 484 and 510 nm for excitation and emission, respectively. Calibration of the fluorescent signal was carried out in the presence of 2 μg/ml valinomycin and varying K+ concentration in the external medium, as described previously (32Seligmann B.E. Gallin E.K. Martin D.L. Shain W. Gallin J.I. J. Membr. Biol. 1980; 52: 257-272Crossref PubMed Scopus (73) Google Scholar, 33Nanda A. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10816-10820Crossref PubMed Scopus (77) Google Scholar). In accordance with earlier reports (17Seligmann B.E. Gallin J.I. J. Clin. Invest. 1980; 66: 493-503Crossref PubMed Scopus (119) Google Scholar, 32Seligmann B.E. Gallin E.K. Martin D.L. Shain W. Gallin J.I. J. Membr. Biol. 1980; 52: 257-272Crossref PubMed Scopus (73) Google Scholar) generation of O·̄2 by the xanthine/xanthine oxidase system or removal of O·̄2 issued by neutrophils by superoxide dismutase had no measurable effect on the fluorescence of di-O-C5(3) under our experimental conditions. The amount of O·̄2 generated either by neutrophils or the xanthine/xanthine oxidase reaction was determined photometrically as the superoxide dismutase inhibitable reduction of ferricytochrome cdetected at 550 nm (28Markert M. Andrews P.C. Babior B.M. Methods Enzymol. 1984; 105: 358-365Crossref PubMed Scopus (376) Google Scholar). Neutrophils (106 cells) were suspended and preincubated for 5 min at 37 °C in 1.2 ml of H medium supplemented with 100 μm cytochrome c. The absorbance of the sample was followed continuously at 550 nm by a Perkin-Elmer Lambda 2 spectrophotometer. For generation of O·̄2by the xanthine/xanthine oxidase system, 3 ml of H medium supplemented with 100 μm cytochrome c and 500 μm xanthine was incubated with 20 milliunits/ml xanthine oxidase for 5 min at 37 °C. The absorbance of the sample was read after 5 min. O·̄2 production was calculated using an absorption coefficient of 21 mm−1 cm−1 for cytochrome c. Data are presented either as representative traces of the indicated number of experiments performed on cells of different donors or as the mean ± S.E. of the number of determinations indicated (n). For the selective study of the capacitative Ca2+ influx, neutrophil granulocytes were treated with the microsomal Ca2+-ATPase inhibitor thapsigargin (TG). In this way the agonist-responsive internal Ca2+ stores could be depleted even in the absence of increased inositol 1,4,5-trisphosphate generation and full activation of the capacitative pathway could be achieved both in the absence and presence of extracellular Ca2+ (34Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3016) Google Scholar). Comparative experiments were carried out on neutrophil granulocytes of healthy individuals which produced high amounts of O·̄2 upon stimulation by fMLP and PMA, and on cells of CGD patients lacking a functional oxidase. The active O·̄2producing enzyme is a multicomponent structure and genetic defects have been described in both subunits of the membrane-bound cytochromeb 558 (gp91 phox and p21 phox ) and in the two phagocyte-specific cytosolic proteins (p47 phox and p67 phox ) (30Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Német K. Hossle J.P. Bernatowska Matuszkiewicz E. Middleton Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar, 35Segal A.W. Mol. Med. Today. 1996; 2: 129-135Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 36Smith R.M. Curnutte J.T. Blood. 1991; 77: 673-686Crossref PubMed Google Scholar). We carried out measurements on granulocytes of patients with verified defects in the gene of gp91 phox or p47 phox . Fig. 1 shows the effect of fMLP and PMA on the TG-induced capacitative influx of normal (A) and two different types of CGD cells (B and C). In the absence of extracellular Ca2+ TG elicited a transient increase of [Ca2+] i which declined slowly, due to the extrusion of Ca2+ by the plasma membrane pump. The resting level of [Ca2+] i as well as the amplitude and kinetics of TG-induced increase of [Ca2+] i were basically similar in healthy and CGD cells. Addition of 1 mm CaCl2 (raising the free [Ca2+] to approximately 0.5 mm) after 10 min incubation in the presence of TG, induced a rapid increase of [Ca2+] i both in normal (Fig. 1 A, trace 1) and in CGD (Fig. 1, B and C trace 1) cells, indicating capacitative entry of Ca2+ into the neutrophils. In cells not treated with TG, the addition of CaCl2 caused no detectable change of [Ca2+] i confirming the necessity of store depletion for the activation of the influx (not shown). Pretreatment of the normal cells with 10 nm PMA (Fig. 1 A, trace 2) or 1 μm fMLP (Fig. 1 A, trace 3) 1 min prior to addition of CaCl2 reduced the amplitude of the increase in [Ca2+] i by 93.7 ± 5 and 82.5 ± 8.4% (n = 4), respectively. Application of the same protocol in CGD cells resulted in a strikingly different pattern of [Ca2+] i response (Fig. 1, Band C): neither PMA (traces 2) nor fMLP (traces 3) had any inhibitory effect on the increase of [Ca2+] i . Identical results were obtained in all experiments carried out on the neutrophil granulocytes of two patients lacking gp91 phox (G. B. and A. R.) and one patient (I. B.) with p47 phox deficiency. Apparently, the observed alteration in the operation of the capacitative Ca2+pathway of CGD cells did not depend on which subunit of the NADPH oxidase was genetically defective. The rapid rise in [Ca2+] i after addition of CaCl2 to store-depleted cells reflects the sum of two opposing processes: Ca2+ influx through the capacitative pathway and Ca2+ extrusion via the plasma membrane Ca2+pump. The capacitative pathway of many different cell types including human neutrophils is also permeable for Mn2+, whereas Mn2+ is not a substrate for the Ca2+ pump. Thus, for isolated study of the capacitative influx Mn2+ is a better indicator than Ca2+ itself. As the reaction of Fura-2 with Mn2+ results in quenching of the fluorescence of the dye, Mn2+ entry into the cells is represented by and proportional to the detected decrease of Fura-2 fluorescence (5Demaurex N. Monod A. Lew D.P. Krause K.H. Biochem. J. 1994; 297: 595-601Crossref PubMed Scopus (81) Google Scholar). In the following experiments we examined the effect of fMLP and PMA on the TG-evoked Mn2+ entry. Cells were exposed to 100 nm TG in the presence of extracellular Ca2+. After a 10-min incubation, 300 μm MnCl2 was added. As shown in Fig. 2 (traces 1) the addition of MnCl2 induced a rapid quenching of fluorescence representing Mn2+ entry via the capacitative pathway opened by the store depletion. A rapid fall in fluorescence occurred in the first 30 s (about 80% of total decrease of fluorescence), followed by a slower phase of quenching. In control experiments, where the cells were not treated with TG, MnCl2 caused only negligible decrease in fluorescence (data not shown), proving that the plasma membrane of resting neutrophils is relatively impermeable for Mn2+ and the dye is localized almost exclusively in the cytosol. When normal cells were stimulated by 10 nm PMA (Fig. 2 A, trace 2) or 1 μm fMLP (Fig. 2 A, trace 3) 1 min before the addition of MnCl2, the quenching by Mn2+ was strongly reduced (69.2 ± 8.9% inhibition by PMA,n = 4 and 66 ± 3.2% by fMLP n = 4) indicating that both fMLP and PMA inhibited Mn2+ influx into the cells. Fig. 2, B and C (traces 2 and 3), summarizes the effect of the same agonists on the TG-induced Mn2+ entry in gp91 phox (B) and p47 phox -deficient (C) CGD cells. As the nearly parallel experimental recordings show, PMA and fMLP were both ineffective in reducing the TG-induced Mn2+ entry into the cells. In four experiments carried out on gp91 phox -deficient CGD patients the rate of Mn2+entry in the presence of fMLP or PMA was even slightly higher (by 6.7 ± 7 and 6.2 ± 4.5% respectively) than in the absence of any stimulator. In the experiment carried out on p47 phox -deficient neutrophils, fMLP induced 8% and PMA effected 14% inhibition of Mn2+ entry. Both methods, widely used for investigation of Ca2+ entry through the capacitative pathway provided similar results: neither fMLP nor PMA inhibits this transport route in CGD cells. We wished to establish whether the altered regulation of capacitative Ca2+ entry into CGD cells might be related to the characteristic functional defects observed in this disease. To this end O·̄2 production and the consequent membrane potential changes had to be checked in the cells of the CGD patients investigated in this study. When stimulated with PMA, superoxide production was not detectable in the granulocytes of three gp91 phox -deficient patients (N. M., A. R., and G. B.). The neutrophils of one gp91 phox patient (L. F.) produced less than 10%, whereas the cells of the p47 phox -deficient patient (I. B.) produced less than 5% of the control value. Stimulation of intact neutrophils by fMLP or PMA was shown to induce rapid depolarization of the cells, the main component of which seems to be the electrogenic operation of the oxidase. In accordance with this suggestion, CGD cells were reported to undergo only marginal depolarization upon fMLP or PMA treatment (17Seligmann B.E. Gallin J.I. J. Clin. Invest. 1980; 66: 493-503Crossref PubMed Scopus (119) Google Scholar, 18Whitin J.C. Chapman C.E. Simons E.R. Chovaniec M.E. Cohen H.J. J. Biol. Chem. 1980; 255: 1874-1878Abstract Full Text PDF PubMed Google Scholar, 19Lew P.D. Wollheim C. Seger R.A. Pozzan T. Blood. 1984; 63: 231-233Crossref PubMed Google Scholar, 20Nanda A. Grinstein S. Cell. Physiol. Biochem. 1991; 1: 65-75Crossref Scopus (11) Google Scholar, 21Di Virgilio F. Lew P.D. Andersson T. Pozzan T. J. Biol. Chem. 1987; 262: 4574-4579Abstract Full Text PDF PubMed Google Scholar, 37Ahlin A. Gyllenhammar H. Ringertz B. Palmblad J. J. Lab. Clin. Med. 1995; 125: 392-401Google Scholar). In our experiments addition of PMA or fMLP to neutrophils obtained from healthy individuals resulted in a sudden depolarization of similar amplitude but with different kinetics (Fig.3). Whereas PMA induced a stable depolarization lasting over 5 min (Fig. 3 A), the fMLP-evoked depression of the membrane potential showed a partial recovery after 2 min (Fig. 3 B). Both PMA and fMLP had only a marginal effect on the membrane potential of neutrophil granulocytes obtained either from gp91 phox -deficient (Fig. 3) or p47 phox -deficient (not shown) patients. Our measurements carried out on healthy and CGD cells are thus in full agreement with previous reports (17Seligmann B.E. Gallin J.I. J. Clin. Invest. 1980; 66: 493-503Crossref PubMed Scopus (119) Google Scholar,18Whitin J.C. Chapman C.E. Simons E.R. Chovaniec M.E. Cohen H.J. J. Biol. Chem. 1980; 255: 1874-1878Abstract Full Text PDF PubMed Google Scholar, 37Ahlin A. Gyllenhammar H. Ringertz B. Palmblad J. J. Lab. Clin. Med. 1995; 125: 392-401Google Scholar). The experiments carried out on neutrophil granulocytes of CGD patients revealed that both fMLP and PMA failed to inhibit capacitative Ca2+ influx in these defective cells and they both failed to induce any significant depolarization. In our next experiments we wanted to test the correlation of these two parameters in healthy neutrophils under conditions where gradual changes of the membrane potential could be evoked. As a first approach we tried to reduce the agonist induced depolarization by pharmacological means and study the effect of fMLP and PMA on the Ca2+ influx under these conditions. For this purpose we applied a combined pretreatment using two drugs which affect charge movements in two different ways. The NADPH-oxidase inhibitor DPI reduces the rate of electron efflux through the oxidase (38Ellis J.A. Mayer S.J. Jones O.T. Biochem. J. 1988; 251: 887-891Crossref PubMed Scopus (131) Google Scholar) whereas the K+-selective ionophore, valinomycin, allows charge compensation through outward movement of K+. These two agents with different sites of action were previously described to clamp efficiently the PMA-induced depolarization (33Nanda A. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10816-10820Crossref PubMed Scopus (77) Google Scholar). The addition of 10 nm PMA after 10 min incubation with TG induced a rapid depolarization response (Fig. 4 A, trace b) whereas pretreatment of the cells with valinomycin and DPI largely diminished the depolarizing effect of PMA (Fig. 4 A, trace a). Clamping of the membrane potential by these agents was similarly effective when fMLP was used as a stimulus (Fig.4 B). Both DPI and valinomycin, when applied separately, were able to reduce only partially the membrane potential change induced by either stimulating agent (data not shown). The effect of pretreatment with valinomycin and DPI on the PMA-induced inhibition of the capacitative influx is demonstrated in Fig.4 C. Addition of CaCl2 to store-depleted control cells resulted in an increase of [C
Referência(s)