Jan 15, 2018 - media, chronic granulomatous disease cells did not dis- ... In several cell types, including lymphoid (7, 8), fibroblastic ..... Under these con-.
OF BIOLOGICAL CHEMISTRY THE JOURNAL Vol. 261 No. 2 Issue of January 15 pp. 512-514 1986 Q 1986 by Thekmerikan Society of Biolokeal Chemists, Inc. Printed in U.S.A.
Communication Cytoplasmic pH Regulation in Normal and Abnormal Neutrophils
but is markedly activated by cytoplasmic acidification (3-5), consistent with a role in pHi homeostasis. This behavior is thought to be determined by a pH-sensitive allosteric site which faces the cytoplasmic compartment (2, 6). The degree of protonation of this site, known as the “modifier,” detercountertransport, with high rates ROLE OF SUPEROXIDE GENERATION AND Na+/H+ mines the rate ofNa’/H’ at acidic pHi and virtual quiescence at pHi = 7.2, the “set EXCHANGE* point” of the modifier (Refs. 3-5; see Ref. 2 for alternative (Received for publication, August 23, 1985) model). In several cell types, including lymphoid (7, 8), fibroblastic Sergio Grinstein$, Wendy Furuya, and W. Douglas Biggar (9), and epithelial(10) cells, addition of phorbol diesters such as 12-0-tetradecanoylphorbol13-acetate(TPA) raises pHi From the Departmentsof Cell Biology and Infectious Diseases, T h e Hospital for Sick Children, above the physiological set point. This is thought to reflect Toronto M5G 1x8 and the Departmentsof Biochemistry stimulation of the Na’/H+ antiport inasmuch as the alkalinand Pediatrics, Uniuersity of Toronto, Ontario, Canada ization is: ( a ) dependentonextracellular Na’; ( b ) accompanied by uptake of Na’; and (c) inhibited by amiloride, a The cytoplasmic pH of human neutrophils was deter- relatively specific inhibitor of the exchanger (11). The actimined fluorometrically using carboxylated fluorescein vation of the previously quiescent antiport has been interderivatives. When normal neutrophils were activated preted as a change in the pHi sensitivity of the modifier site, by the phorbol ester 12-0-tetradecanoylphorbol13- i.e. an alkaline shift in the set point. acetate (TPA) in Na+-containing medium, the cytoIn neutrophils, TPA elicits a biphasic change in pHi; an plasmic pH initially decreased but then returned to initial acidification is superseded by a recovery toward normal near normal values. In Na+-free media or in Na+ medium containing amiloride, TPA induced a marked pHi, occasionally overshooting to more alkaline levels (12). monophasic intracellular acidification. The cyto- The recovery phase is Na+-dependent andamiloride-sensitive plasmic acidification is associated with net H+ equiva- and has therefore been attributed to Na+/H’ countertranslent efflux, suggesting metabolic acid generation. The port. It is not clear if the antiport is directly stimulated by metabolic pathways responsible for the acidification the phorbol ester,as in other cells, or whether the activation were investigated by comparing normal to chronic is secondary to the cytoplasmic acidification. The nature of granulomatous disease neutrophils. These cells are un- the acidification is not well understood, but pharmacological able to oxidize NADPH and generate superoxide. When evidencesuggests that it reflects increased metabolicacid treated with TPA in Na+-freeor amiloride-containing production.‘ The major metabolicpathways stimulated during oxidative metabolism: media, chronic granulomatousdisease cells did notdis- neutrophil activation are dependent on play a cytoplasmic acidification. This suggests that in NADPH isoxidized, the hexose monophosphate shunt (HMS) normal cells NADPH oxidation and/or the accompa- is stimulated, and radicalsof oxygen are generated (13). nying activation of the hexose monophosphate shunt In this report,we compare the pH< response of neutrophils are linked to the acidification. Unlike normal neutro- from normal donors and from patients with chronic granuphils, chronic granulomatousdisease cells treated with lomatous disease (CGD). Neutrophilsfrom CGDpatients lack TPA in Na+-containing medium displayed a significant one of the components of the NADPH oxidase system (Xcytoplasmic alkalinization. The alkalinization was linked CGD) or havedefective stimulus-response coupling Na+-dependentand amiloride-sensitive, indicating ac- (autosomal recessive CGD) andtherefore fail togenerate tivation of Na+/H+exchange. Thus, the Na+/H+anti- superoxide during activation (14).This enabled us to verify port, which can be indirectly stimulated by the meta- whether the TPA-induced acidification is indeed associated bolic cytoplasmic acidification, is also directly actiwith NADPH oxidation and stimulationof the HMS. Morevated by the phorbol ester. over, it became possible to determine if the effect of TPA on Na’/H+ exchange is direct or secondary to metabolic acidification. It was reasonedthat, in the absence of acid generation, a direct activation of the exchangerwould be manifested asa An antiport that exchanges extracellular Na’ for internal consistent and significantcytoplasmic alkalinization. H+ plays an essentialrole in theregulation of cytoplasmic p H (pH,)’ in most mammalian cells (1, 2 ) . In nonepithelial cells, MATERIALS AND METHODS the antiport is largely quiescent near the physiological pHi, * This work was supported by the Medical Research Council of Canada. The costsof publication of this articlewere defrayed in part by the paymentof page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. $ Recipient of a Medical Research Council Scientist Award. ‘The abbreviations used are: pHi, cytoplasmic pH; TPA, 12-0tetradecanoylphorbol 13-acetate; CGD, chronic granulomatous disacid; ease; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic HMS, hexose monophosphateshunt;BCECF, 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein.
RPMI 1640 (HCOB-free) was purchased from GIBCO. Ficoll-Hypaque and dextran T-500 were from Pharmacia, Uppsala, Sweden. Phenazine methosulfate and TPA were from Sigma. N-Methyl+ glucamine was from Aldrich. Nigericin was from Calbiochem-Behring. 2’,7’-Bis(carboxyethyl)-5(6)-carboxyfluorescein(BCECF) acetoxymethyl ester was obtained from the Hospital for Sick Children, Research Development Corp., Toronto, Canada. Amiloride was the kind gift of Merck Frosst, Montreal, Canada. Na+solution contained 140 mM NaCl, 5 mM KCI, 10 mM glucose, and 10 mM HEPES, pH
* S. Grinstein and W. Furuya, manuscript submitted tion.
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for publica-
p H Regulation and Exchange Na+/H+
513
7.3. N-Methyl-D-glucamine+ solution andK+ solution were prepared in Fig. 1A was not always observed. Only the acidifying phase by isoosmotic replacement of NaCl by the chloride saltsof N-methyl- was detectable when the phorbol ester was added to cells in o-glutamine+ and K+, respectively, hut were otherwiseidentical. Na' solution containing amiloride (Fig. 1B) or in Na+-free These media were nominally Caz+- and M F - f r e e t o minimize light media (Fig. IC). Similar resultswere obtained whether K+or scattering due tocell aggregation during the spectroscopic assays. The patients with CGDwere all males. They had a typical clinical N-methyl-D-glucamine was used as substitutes. The acidifihistory of recurringbacterialinfectionsassociatedwithcatalasecation was somewhat larger in Na+-free solution, suggesting positive bacteria. The diagnosis in all patients was confirmed by that either the concentration of amilorideused (300 ~ L M ) defective bacterial killing of Staphylococcus aureus 502A in vitro (15) produced incomplete inhibition of Na+/H+ exchange or the and abnormal reduction of nitro blue tetrazolium dye (16, 17). None diuretic partially inhibited the acid-generating system. Taken had evidence of bacterial infection when studied. One male patient together, these results indicate that in normal neutrophils hadanaffectedbrotherwithCGDand a mother identified as a TPA activatesa process that tends toacidify the cytoplasmic carrier. Carrier status was established by demonstrating neutrophil mosaicism by nitro blue tetrazolium dye reduction (17). This patient compartment and that under normal conditions is pH; mainlikely acquired CGD as an X-linked inheritance. The other three tained near the resting level by acid extrusion through Na+/ males had no family history of CGD, and their mothers had normal H' exchange. nitro blue tetrazolium reduction by blood neutrophils. The resting pHi of neutrophils from four CGDpatients was Neutrophils from healthyadultsandpatientswith CGD were isolated from fresh heparinized blood by dextran sedimentation fol- 7.07 f 0.07, which is not significantly different from that of lowed by Ficoll-Hypaque gradient centrifugation (18). Contaminating normal cells (7.15 & 0.03). Addition of TPA to CGD cells in red cells were then removed by ammonium chloride lysis. The cells Na+ solution induced a monophasic cytoplasmic alkalinizawere washed andresuspendedinnominally HC0;-free, HEPES- tion (Fig. l A , top trace),resembling the effects of the phorbol buffered RPMI 1640 a t 10' cells/ml and maintained in this medium esteronnormal lymphocytes andfibroblasts (8, 9). The a t room temperature for up to 5 h. For fluorometric pH; determinaelevated pHi was consistently observed and statistically sigBCECF by incubation with the tions, the cells were loaded with parent acetoxymethyl ester(3 pg/ml, final concentration) for 30 min nificant (Table I). This alkalinization is likely due to activaa t 37 "C. After washing, 0.5-1 X lo6 cells were used for fluorescence tion of the Na'/H' antiport, since it was eliminated by determination as described (8, 12). The nigericin/potassium method amiloride (Fig. 1B) and by the replacement of extracellular of Thomas et al. (19) was used for calibration of fluorescence versus Na' with N-methyl-D-glucamine+(Fig. 1C)or K' (not pH,. The cytoplasmic buffering power was measured in intact cells shown). The marked acidification observed when normal cells by titration with NH: (1, 5). All the experiments were performed a t are stimulated in the presence of amiloride was not present 37 "C. RESULTSANDDISCUSSION
The pHiof neutrophils from healthy adults inNa' solution averaged 7.15 f 0.03 (mean f S.E.) in nine determinations. As reported (la), addition of TPA (IO-' M ) t o these cells produced a moderate biphasic pHi change (Fig. 1A): a n incipient acidification followed by a recovery toward the resting level. The slight overshoot to a more alkaline p H illustrated a.
A. Na+-rnedlurn
C. Na+-free medun Nigerian TPA I
J
NMG+
Na + medium
L6.8
-\
6.5
FIG. 1. Effect of TPA on pH3 in neutrophils from healthy adults and CGD patients. Neutrophils were isolated and loaded with BCECF. pH,(vertical axes)was estimated from thefluorescence emission by calibration with K+/nigericin by the method of Thomas et al. (19). A , cells suspended in Na+ solution (140 mM NaCI, 5 mM KCI, 10 mM glucose, and 10 mM HEPES, pH 7.3). After defining the base-linepH,, M TPA was added where indicated by the arrow. The figure is a composite of representativetracesobtainedwith normal and CGDcells. B , cells suspended in Na+ solution containing 300 p~ amiloride. C, cells suspended in either K+ solution or N methyl-D-glucamine' (NMG") solution. These media were prepared by isoosmotic replacement of NaCl by the chloride salts of K' and N-methyl-D-glucamine+, respectively, but were otherwise identical. Where indicated, TPA ( 1 0 - ' ~ ) was added to all thesamples. Nigericin (1 pg/ml) was added to the CGD sample (top trace) where marked. The time scale is applicable to A-C. The temperature was 37 "C. The traces are representativeof at least four experiments. Discontinuities of the trace indicate opening of the sample compartment for additions.
in CGD neutrophils (Fig. 1B andTable I). Similarly, no acidification was recorded when CGD cells were activated in Na+-free media (Fig. 1C and Table I). Failure to detect an acidification cannotbeattributedtoanabnormally high buffering capacity, since direct measurements showed no significant difference between normal and CGDcells. Moreover, a significant fall in pHiwas recorded upon addition of nigericin,aK'/H+-exchangingionophore, to CGDcells in N methyl-D-glucamine' solution (Fig. lC), verifying the integrity of the cells as well as the sensitivity of the recording system. In normal neutrophils, the HMS activation is limited by the availabilityof NADP+. Thus, stimulation of this pathway results when NADPH isoxidized to NADP' during the synthesis of superoxide in activated cells (13). In unstimulated cells, NADPH can also be converted to NADP+ by addition of exogenous permeant oxidizing agents, such as methylene blue or phenazine methosulfate. This manipulation has also been shown to activate the HMS(20). Even though superoxide synthesis is impaired inCGD cells, the HMS is normally functional and can be activated by oxidizing reagents. This enabled us to test whether stimulation of the HMS is sufficient to affect pHi.Addition of phenazine methosulfate (100 TABLE I Effect of ?'PA O R cytoplasmic pH in normal andCGD neutrophils pHi was determined fluorometrically using BCECF in the indicated media (see legend to Fig. 1).ApHi was measured 3 min after addition of 1 0 " ~TPA. Theamiloride concentration was 300 p ~Temperature . was 37 "C. Data are means rt S.E. of the number of experiments in parentheses. Positive values indicate cytoplasmic alkalinization and vice versa. p was calculated using Student's t test for unpaired samples. Medium Donor
Na+ Na+
+ amiloride
N-MethylD-glutamine+
@HI3 min
Normal CGD
0.04 f 0.012 (4) -0.22 f 0.015 (5) -0.27 f 0.024 (5) 0.12 f 0.006 (5) 0.00 f 0.003 (4) -0.02 f 0.012 (4) p < 0.002 p < 0.001 p < 0.001
514
p H Regulation andNa+/H+Exchange
PM) to normal neutrophils suspended in Na+-free medium produced a gradual cytoplasmic acidification.' The decreased fluorescence is not a spectroscopic artifact. If the cells were first lysed with Triton X-100, releasing the BCECF into the medium, addition of the oxidizing agent produced only a marginal, instantaneous decrease in fluorescence emission. Moreover, phenazine methosulfate was also added to cells suspended in K' medium containing nigericin. Under these conditions, which are expected to clamp pHi at or near the external pH, the acidification induced by phenazine methosulfate was drastically reduced. Addition of the redox reagent to CGD cells in Na+-free medium induced a cytoplasmic acidification similar to that observed in normal cells. These results further validate the sensitivity of the pHi measurements in CGD cells and indicate that the H' equivalents liberated during the stimulation of the HMS can contribute to the cytoplasmic acidification in activated cells. Two main conclusions can be drawn from the present findings. First, the Na+/H+exchanger of TPA-activated neutrophils is stimulated by a dual mechanism: directly by the phorbol ester and indirectly by the acidification of the cytoplasm. The former activation, which accounts for the occasional pHj "overshoots" observed in normal cells, is particularly noticeable in CGD cells, which fail to generate metabolic acid during activation. By analogy with other cells treated with TPA (7-9), it is likely that this stimulation reflects a shift in the pHi sensitivity of the antiport. The activation that results from lowering pHi is another component of the response of normal cells to TPA, inasmuch as a significant initial acidification is always recorded. A similar response can be elicited in resting cells from both normal and CGD donors by artificially acid loading with weak acids or with ionophores (not shown). The second conclusion concerns the source of acid equivalents appearing in thecytoplasm of activated normal neutrophils. Because the acidification was missing in CGD cells, it is most likely that theactivation of NADPH oxidation and/or the attendant stimulation of the HMS are themain source of the H' equivalents. This conclusion is supported by experiments where these pathways were blocked in normal cells using alkylating reagents or deoxyglucose.' Under these conditions, the TPA-induced acidification was also absent.
In summary, a substantial quantity of acid equivalents is generated in the cytoplasm of activated normal neutrophils. In the absence of Na+ in the medium, H+ ions accumulate in the cytoplasm, resulting in a marked acidification. However, under normal conditions, i.e. in Na+-containing media, the Na+/H+ antiport maintains pHi in the physiological range, enabling other cellular responses to proceed normally. This underscores the importance of the Na+/H+exchanger in pH, homeostasis. REFERENCES 1. Roos, A., and Boron, W. F. (1981) Physiol. Reu. 61, 296-434 2. Aronson, P. S. (1985) Annu. Reu. Physiol. 47,545-560 3. Moolenaar, W. H., Tertoolen, L. G. J., and de Laat, S. W. (1984) J. Biol. Chem. 259,7563-7569 4. Paris, S., and Pouyssegur, J . (1983) J . Biol. Chem. 258, 35033508 5. Grinstein, S., Cohen, S., and Rothstein, A. (1984) J. Gen. Physiol. 83,341-369 6. Aronson, P. S., Nee, J., and Suhm, M. A. (1982) Nature 299, 161-163 7. Rosoff, P. M., Stein, L. F., and Cantley, L. C. (1984) J. Biol. Chem. 259,7056-7060 8. Grinstein, S., Cohen, S., Goetz, J. D., Rothstein, A., and Gelfand, E. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1429-1434 9. Moolenaar, W. H., Tertoolen, L. G. J., and de Laat, S. W. (1984) Nature 312, 371-374 LO. Whiteley, B., Casel, D., Zhuang, Y., and Glaser, L. (1984) J. Cell Biol. 99, 1162-1166 11. Benos, D. J. (1982) Am. J . Physiol. 242, C131-Cl45 12. Grinstein, S., Elder, B., and Furuya, W. (1985) Am. J. Physiol. 248, C379-C385 13. Cheson, B. D., Curnutte, J. T., and Babior, B. M. (1977) Prog. Clin. Immunol. 3,l-65 14. Segal, A. W., Cross, A. R., Garcia, R. C., Borregaard, N., Valerius, N. H., Soothill, J. F., and Jones, 0.T. G. (1983) N . Engl. J. Med. 308,245-251 15. Alexander, J. W., Windhorst, D. B., and Good, R. A. (1968) J. Lab. Clin. Med. 72, 236-241 16. Biggar, W. D., Buron, S., and Holmes, B. (1976) J. Pediatr. 88, 63-67 17. Mills, E. L., Rholl, K. S., and Quie, H. (1980) J . Clin. Inuest. 66, 332-340 18. Boyum, A. (1968) J. Clin. Lab. Znuest. 21, Suppl. 97 77-98 19. Thomas, J. A,, Buchsbaum, R. N., Zimniak, A,, and Racker, E. (1979) Biochemistry 18,2210-2218 20. Evans, W. H., and Karnovsky, M. L. (1962) Biochemistry 1,159166
