Cytoplasmic pH regulation in activated human neutrophils: Effects of adenosine and pertussis toxin on Na+H+ exchange and metabolic acidification

Bio¢~imica et Biophvsi¢~ Acta 889 (1986) 301-309 Elsevier

301

BBA 11841

C y t o p l a s m i c p H r e g u l a t i o n in a c t i v a t e d h u m a n n e u t r o p h i l s : e f f e c t s o f a d e n o s i n e and

pertussis t o x i n o n N a + / H

÷ exchange and metabolic acidification

Sergio Grinstein and Wendy Furuya Departments of Cell Biolog3' and Immunology, Ilospital for Sick Children, 555 Uni~,ersiO"A re, Toronto M5G 1XS, and Department of Biochemist(v, Uni~,ersiO' of Toronto, Toronto (Canada)

(Received 30 June 1986)

Key words: N a + / H ~ exchange; Fluorescent probe: Acidosis: (Leukocyte)

When stimulated, neutrophils undergo a complex change in cytoplasmic pH (pHi): an incipient acidification, followed by an alkalinization which is due to activation of N a + / H ÷ exchange. When the latter is inhibited by amiloride or by removal of extracellular Na ÷, the actual magnitude of the initial acidification can be fully appreciated. The acidification is thought to be of metabolic origin, but the precise origin of the H + (equivalents) remains undefined. We used adenosine, a modulator of neutrophil responsiveness, to identify the source of metabolic acid in cells stimulated by either formylmethionylleucylphenylalanine (fMet-Leu-Phe) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of the cells with adenosine inhibited the fMet-Leu-Phe-induced respiratory burst, but secretion of specific and azurophilic granules, as well as aggregation were unaffected. In fMet-Leu-Phe-treated cells, adenosine reduced the acidification recorded in Na÷-free media, but had no effect on the activation of the N a + / H + antiport. Adenosine had little or no effect on the TPA-induced responses, including the pH i changes. The respiratory burst, as well as the cytoplasmic acidification were also inhibited in parallel by pretreating the cells with 'islet-activating protein' from Bordetella pertussis. It was concluded that activation of the NADPH-oxidase a n d / o r the associated stimulation of the hexose monophosphate shunt play a major role in the metabolic acidification of stimulated neutrophils.

Introduction

Polymorphonuclear leukocytes are attracted to sites of infection, where they undertake a number of bactericidal reactions including phagocytosis, degranulation and the synthesis of reduced oxygen metabolites such as the superoxide anion. In vitro,

Abbreviations: TPA, 12-O-tetradecanoylphorbol 13-acetate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; BCECF, 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein. Correspondence: Sergio Grinstein, Research Institute, Hospital for Sick Children, 555 University Ave, Toronto, Ontario, M5G 1X8, Canada.

these reactions can be triggered by the addition of a variety of soluble or particulate stimuli. The former include the synthetic formylated tripeptide formylmethionylleucylphenylalanine and phorbol esters such as 12-O-tetradecanoylphorbol 13acetate. The purported target of TPA and other /~-phorbol diesters is protein kinase C, a Ca 2+ and phospholipid-dependent enzyme which is thought to be central to the activation process in neutrophils [1]. Stimulation of neutrophils by soluble activators is accompanied by a complex series of cytoplasmic pH (pHi) changes. After treatment with fMetLeu-Phe, rabbit and human neutrophils undergo a transient cytoplasmic acidification, followed by a

0167-4889/86/$03.50 ~'~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)

302 sustained alkalinization of approx. 0.2 units above the resting level [2,3]. Treatment with TPA elicits a similar pattern, but in addition a secondary acidification phase is generally observed [4]. In both cases, the alkalinization has been attributed to activation of the N a + / H + antiport. This conclusion is based on the failure of the cells to become alkaline when suspended in Na+-free media or in media containing amiloride, an inhibitor of the antiport [2 4]. Under these conditions, a large cytoplasmic acidification is unmasked, which is transient in the case of fMet-Leu-Phe but more sustained with TPA. The sustained change evoked by the phorbol ester can account for the secondary acidifying phase, which is absent with fMet-Leu-Phe. The nature of the acidification elicited by the soluble activators is not entirely understood, but it has been suggested to be due to increased metabolic H + (equivalent) production by the activated cells [5]. The evidence supporting this notion can be summarized as follows: (a) the pH i change is accompanied by extracellular acidification, detectable in poorly buffered media; (b) the metabolic burst and the cytoplasmic acidification are inhibited in parallel when the cells are pre-treated with 2-deoxy-D-glucose or with N-ethylmaleimide and (c) the acidification can be mimicked by the addition of permeant redox agents such as phenazine methosulfate, which, like TPA and fMet-LeuPhe, are known to oxidize N A D P H and thereby activate the hexose monophosphate shunt in neutrophils [5]. Because of the complexity of the neutrophil response, it has been difficult to correlate the cytoplasmic acidification with specific reactions or metabolic pathways. However, more definitive information may be obtained by using modulators of the activation process, such as adenosine. Using macrophages, Pike and Snyderman [6] were the first to realize that adenosine can inhibit superoxide production in activated leukocytes. These observations were later extended to neutrophils by Cronstein et al. [7] who found that superoxide production in response to fMet-Leu-Phe, ionophore A23187, concanavalin A or zymosanactivated serum, but not TPA, was greatly inhibited by pre-treating the cells with adenosine. These authors also found that, in contrast to the

marked inhibition of superoxide generation, adenosine had little or no effect on cell aggregation and enzyme secretion. Because of the selective nature of the inhibition, this modulator could in principle be used to correlate the occurrence of cytoplasmic acidification with particular aspects of the neutrophil response. A different type of modulator of the neutrophil response is the 'islet-activating protein' isolated from Bordetella pertussis. This toxin is known to inhibit the receptor-mediated stimulation of phospholipase C by soluble activators, through ADPribosylation of a guanosine nucleotide-binding protein essential for signal transduction [8]. IAP has been reported to block the fMet-Leu-Phe-induced stimulation of the Na +/H + antiport [9,10], but its effects on the metabolic acidification have not been investigated. In this report, we have used fluorimetric techniques to determine the effects of adenosine and islet-activating protein on the pH~ changes elicited in neutrophils by fMet-Leu-Phe, an activator requiring interaction with a surface r e c e p t o r a n d m e d i a t i o n by t r a n s d u c i n g nucleotide-binding proteins, and by TPA, a direct activator of protein kinase C. The data indicate that the stimulation of the NADPH-oxidase a n d / o r the associated burst of the hexose monophosphate shunt are largely responsible for the acidification. Materials and Methods

Reagents. Solution RPMI 1640 (HCO free) was purchased from GIBCO. Ficoll 400 and Dextran T500 were from Pharmacia. TPA, fMet-LeuPhe, cytochalasin B, adenosine, N-t-BocL-alanine-p-nitrophenyl ester, phenolphthalein glucuronic acid and dried Micrococcus lysodeikticus were from Sigma Chemical Co. Islet-activating protein from Bordetella pertussis was obtained from LIST Biochemicals. Nigericin was from Calbiochem-Behring. 2'-7'-biscarboxyethyl-5(6)carboxyfluorescein (BCECF) acetoxymethyl ester was obtained from Molecular Probes, Junction City, OR. Amiloride was the kind gift of MerckFrosst, Montreal, Canada. Solutions. Where indicated, H C O 3 -free medium R P M I 1640 was buffered to pH 7.3 by the addition of 10 mM Hepes-Na. The Na+-solution

303 contained (in mM): 140 NaC1, 5 KC1, 1 CaC12, 10 glucose and 10 Hepes (pH 7.3) at 37°C. Nmethyl-D-glucamine + solution was prepared by isoosmotic replacement of NaC1 by N-methyl-Dglucamine-chloride, but was otherwise identical. These media were nominally Mg2+-free to minimize light scattering due to cell aggregation during the spectroscopic assays. The osmolarity of the media was adjusted to 290 + 5 mosM with the major salt. Cell isolation and characterization. Neutrophils were isolated from fresh heparinized blood from human donors by Dextran sedimentation followed by Ficoll-Hypaque gradient centrifugation [11]. Contaminating red cells were then removed by ammonium chloride lysis. The cells were washed and resuspended in nominally HCO 3-free, Hepes-buffered medium RPMI-1640 at 107 cells/ ml and maintained with gentle rotation at room temperature. The cells were sized and counted using a Coulter model ZM counter and the C1000 Channelyzer (Hialeah, FL). Where indicated, the cells (no more than 107/ml in Hepes-buffered RPMI 1640) were pre-treated with 500 n g / m l of islet-activating protein for 2 h at 37°C or for 5-10 min with 10 /xM adenosine. Similar effects were obtained at various times within this range. Cytoplasmic pH measurements. Intracellular pH (pHi) was determined fluorimetrically using BCECF. The cells (107/ml), were loaded with the probe by incubation with the precursor BCECFacetoxymethyl ester (1 t~g/ml) for 30 rain at 37 ° C. After washing, approx. 1 • 1 0 6 cells were used for fluorescence measurement in Perkin Elmer 650-40 or LS-5 spectrofluorimeters with excitation at 485 nm and emission at 540 nm, using 5 nm and 10 nm slits, respectively. These wavelengths and slits were chosen to minimize artifacts due to alterations in light scattering produced by fMet-LeuPhe or TPA-induced shape changes a n d / o r aggregation. The nigericin/K + method of Thomas et al. [12] was used to calibrate pH i. Secretion and aggregation measurements. For the experiments described in this section, the cells were pre-incubated with cytochalasin B (5/~g/ml) for 10 min at 37°C, to maximize the response. The secretion of elastase, an azurophil granule marker, was monitored continuously by spectro-

photometrically determining the release of pnitrophenol from the substrate N-t-boc-L-alaninep-nitrophenyl ester by the method of Korchak et al. [13]. The release of fi-glucuronidase was determined using phenolphthalein glucuronidate as a substrate. Briefly, 3-10 6 cells in the indicated medium were pre-treated with or without 10 jaM adenosine, followed by the addition of either TPA (2- 10 s M) or fMet-Leu-Phe (2- 10- 7 M). After 10 min at 37°C, the cells were sedimented and the supernatant saved for determination of enzymatic activity by the method of Brittinger et al. [14]. For the determination of lysozyme secretion, aliquots of 1 0 7 cells were pre-treated with or without adenosine and then activated with TPA or fMetLeu-Phe as above. Following sedimentation, the supernatants were used for measurement of lysozyme activity, determined as the rate of lysis of M. lysodeikticus, measured by the decrease of absorbance at 450 nm [15]. In all cases, the background activity of supernatants from untreated cells was subtracted. Aggregation was measured using a Model 300B Payton dual channel aggregometer module (Scarborough, Ontario). The cells were stirred continuously and the temperature maintained at 37°C. After the baseline transmittance was established, the cells were activated with TPA or fMet-Leu-Phe while recording continued. Oxygen consumption and other methods. Oxygen consumption was measured with a Model 53 Biological Oxygen Monitor (Yellow Springs Instruments), which utilizes a Clark type polarographic electrode. The cells ( 4 . 1 0 6) w e r e suspended in 2 ml of the indicated medium at 37°C and stirred magnetically. Oxygen uptake was monitored continuously using a Y vs. time chart recorder. 02 consumption was calculated using a solubility coefficient of 0.024 ml O2/ml medium at 37°C. Unless otherwise specified, all measurements were made at least three times using different donors. The data are presented as means + S.E. of the number of determinations indicated, or as representative traces of pH i , aggregation or 02 consumption. Results

Effect of adenosine on the respiratory burst In order to determine the effects of adenosine

304 on p H i , we had to ascertain that under our experimental conditions, the r e p o r t e d effects of the nucleoside on activation would be present. The respiratory burst was measu'red as changes in the rate of oxygen c o n s u m p t i o n , to preclude the possibility of scavenging of reactive oxygen species by the drugs e m p l o y e d , f M e t - L e u - P h e ( 2 . 1 0 7 M) was found to induce a b i p h a s i c change in the rate of oxygen c o n s u m p t i o n : an initial rapid phase, that was c o m p l e t e within 1 min, followed by a slower phase that varied in m a g n i t u d e a m o n g different donors. Consistent with m e a s u r e m e n t s of superoxide p r o d u c t i o n [7] and of l u m i n o l - d e p e n dent chemiluminescence [16], p r e t r e a t m e n t of the neutrophils with 10 ~ M a d e n o s i n e m a r k e d l y reduced the f M e t - L e u - P h e - i n d u c e d increase in the rate of oxygen c o n s u m p t i o n . In 7 experiments the inhibition, m e a s u r e d at 1 rain, averaged 90.3 _+

1.5%. In contrast to fMet-Leu-Phe, stimulation by T P A resulted in a large, sustained increase in the rate of oxygen c o n s u m p t i o n . P r e t r e a t m e n t with a d e n o s i n e p r o d u c e d a smaller, though significant inhibition. In five experiments the rate of T P A stimulated oxygen u p t a k e was inhibited by 37.8 ± 3.9%.

Effect of adenosine on secretion The effects of f M e t - L e u - P h e and T P A on the secretion of elastase, an azurophil granule marker, are illustrated in Fig. 1. The c h e m o t a c t i c trip e p t i d e was consistently m o r e effective in stimulating elastase release than the p h o r b o l ester: both the m a x i m a l rate a n d the total secretion after 8 min were higher in the case of fMet-Leu-Phe. M o r e i m p o r t a n t l y , p r e t r e a t m e n t of the cells with 10/*M adenosine had only small and insignificant effects on the rate or extent of elastase release. In five experiments, the rate of secretion in fMetLeu-Phe-activated cells treated with a d e n o s i n e was 112.6 ± 10.1% of the control (adenosine-free) rate. Similarly, a d e n o s i n e had no effect on elastase secretion from T P A - t r e a t e d cells: the rate in nucleoside-treated cells was 91.8 ± 11% (n = 3) of the control rate. These results were c o n f i r m e d m e a s u r i n g fl-glucuronidase release, a second azurophil granule marker. The rate of f M e t - L e u Phe-induced secretion in a d e n o s i n e treated cells was 102.9 ± 5% (n = 6) of the control, whereas the

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Fig. 1. Effect of adenosine on the secretion of elastase. Paired samples of neutrophils (5-106/ml) suspended in Na '-medium containing 5 k~g/ml of cytochalasin B were placed in the sample and reference cuvettes of a double beam spectrophotometer. After preincubation for 10 min in the presence (open symbols) or absence (solid symbols) of 10 p.M adenosine, the substrate N-t-boc-L-alanine-p-nitrophenylester (150/*M, final) was added to both cuvettes and, to ensure the symmetry of the system, the baseline rate of differential hydrolysis was recorded as the change in absorbance at 400 nm. Where indicated by the arrow, fMet-Leu-Phe (2.10 7 M: e , © ) or TPA (2.10 ~ M: • , ~) were added to the experimental samples, and absorbance recording was continued. Representative of three similar determinations. Temperature: 37°C.

T P A - s t i m u l a t e d secretion was 95.4 ± 7.8% (n = 6) of the control. These d a t a are in good agreement with those of Cronstein et al. [7], who found only a marginal inhibition in the rate of fMet-LeuP h e - s t i m u l a t e d secretion of this enzyme. W e also d e t e r m i n e d the effect of adenosine on the release of lysozyme, an enzyme present in both specific and azurophilic granules. N o significant inhibition was found in either f M e t - L e u - P h e or T P A - s t i m u l a t e d cells. L y s o z y m e secretion in cells p r e t r e a t e d with 10/.tM a d e n o s i n e was 100.5 _+ 3.5% (n = 6) of the control (adenosine-free) in fMetL e u - P h e - a c t i v a t e d cells and 116.3 _+ 4.8% (n = 6) in T P A - a c t i v a t e d cells.

Effect of adenosine on aggregation T h e light t r a n s m i t t a n c e of suspensions of neutrophils increases u p o n a d d i t i o n of f M e t - L e u - P h e (Fig. 2). This change correlates with, a n d is thought to reflect, increased aggregation of the cells as d e t e r m i n e d microscopically [17]. N e i t h e r the rate n o r the extent of the f M e t - L e u - P h e - i n d u c e d aggre-

305

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gation was affected by pretreatment with 10 ~M adenosine (e.g., Fig. 2). In four experiments, the extent of aggregation in adenosine-treated samples was 98.6 + 6.1% of the control, A slower but sustained aggregation was also induced by TPA. Adenosine also failed to influence the response to the phorbol ester: aggregation in the presence of the nucleoside was 97.3 +_ 3.8% of the control (n = 4).

The response to TPA of normal cells suspended in Na+-medium is illustrated in Fig. 4A. The phorbol ester induced an initial acidification, followed by a small alkalinization, which was in turn superseded by a secondary acidifying phase. As described earlier [5,18], the extent and time of onset of the secondary acidification are variable among donors (compare Figs. 4A and 6A). In the absence of external Na +, the cells respond to TPA

Effect of adenosine on cytoplasmic pH A typical response of neutrophil pH i to fMetLeu-Phe is illustrated in Fig. 3. Cells suspended in Na +-containing medium undergo a transient cytoplasmic acidification, followed by a sustained alkalinization (Fig. 3A). The acidification phase becomes more pronounced and sustained when the N a + / H + antiport is impaired by removal of extracellular Na + (N-methyl-D-glucamine + substitution; Fig. 3C) or by addition of amiloride (not illustrated). The response observed in the Na +solution, which is dominated by the activation of the N a + / H + antiport, was largely unaffected by pretreating the cells with 10 /~M adenosine (Fig. 3B). Occasionally, the alkalinization was somewhat faster a n d / o r larger in the treated cells. In contrast, the acidification recorded in Na+-free N-methyl-D-glucamine + medium was markedly inhibited by adenosine (Fig. 3D), resembling the inhibition of the respiratory burst. The acidification after 1 min, which averaged 0.22 + 0.03 pH units in six control experiments, was reduced to 0.07 _+ 0.02 units in paired adenosine-treated samples.

Fig. 2. Effect of adenosine pretreatment on aggregation. A concentrated neutrophil suspension (10 v cells/ml) was preincubated with or without 10 p.M adenosine and stirred in an aggregometer cuvette. After recording the baseline transmittance, fMet-Leu-Phe (2. 10 v M, final) or TPA (2-10 _8 M, final) were added to the cuvette where indicated by the arrows. Upward deflections indicate increased transmittance. Note that thc amplification setting used for fMet-Leu-Phe (full range 75-100% transmittance) and TPA (20-100%) were not the same, so that the traces are not directly comparable. The traces are representative of three experiments. Temperature: 37 ° C.

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6.'/ Fig. 3. Effect of adenosine on fMLP-induced pH i changes. Neutrophils were loaded with BCECF and suspended in either Na+-medium (A and B) or N M G +-medium (C and D). The cells were preincubated at 37°C for 5 min with (B and D) or without (A and C) 10 ~M adenosine. The final minutes of this preincubation were used to establish the baseline pHi, which was determined fluorimetrically. Where indicated by the arrows, the cells were activated by addition of 2 . 1 0 - v M fMet-Leu-Phe. Discontinuities in the trace indicate opening of the sample compartment for additions. Calibration of pH i was performed using nigericin/K ÷ by the method of Thomas et al. I12]. The traces are representative of at least three experiments. Temperature: 37°C: N M G 4 , N-methyl-D-glucamine +.

306

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6.8. 6.6-Fig. 4. Effect of adenosine on TPA-induced pH i changes. BCECF-loaded neutrophils (5-105/ml) were suspended in either Na +-medium (A and B) or in N-methyl-D-glucamine ~medium (C and D), and preincubated with (B and D) or without (A and C) 10 ~M adenosine for 5 min at 37°C. The final minutes of this preincubation were used to establish the baseline pH i. Where indicated by the arrows, 2.10 s M TPA was added and recording was continued. Other details are as in Fig. 3. The traces are representative of at least three experiments of each type. Temperature: 37°C.

with a very pronounced and sustained acidificat i o n (Fig. 4C). P r e t r e a t m e n t w i t h a d e n o s i n e h a d virtually no effect on the TPA-induced response r e c o r d e d in N a ÷ m e d i u m a n d o n l y m o d e r a t e l y i n h i b i t e d t h e a c i d i f i c a t i o n in t h e N - m e t h y l - D glucamine ~ solution. In four experiments the a c i d i f i c a t i o n , m e a s u r e d a t 1 m i n a v e r a g e d 0.22 + 0.03 p H u n i t s in u n t r e a t e d cells a n d 0.19_+ .03 u n i t s in a d e n o s i n e p r e t r e a t e d cells.

Effect of islet-activating protein on the respiratory burst T h e e f f e c t s o f p r e t r e a t m e n t o f t h e cells f o r 2 h w i t h 500 n g / m l i s l e t - a c t i v a t i n g p r o t e i n o n t h e r a t e of fMet-Leu-Phe and TPA-induced oxygen consumption were studied next. Islet-activating protein virtually eliminated the respiratory burst i n d u c e d b y f M e t - L e u - P h e . I n five e x p e r i m e n t s t h e r e s i d u a l a c t i v i t y w a s o n l y 6.0 _+ 3.4% o f t h e c o n trol. H o w e v e r , i n t h e s a m e cells, a n d e v e n a f t e r t h e a d d i t i o n o f f M e t - L e u - P h e , T P A p r o d u c e d a res p o n s e t h a t w a s c o m p a r a b l e to t h a t o f u n t r e a t e d cells. T h e s e d a t a a r e i n g o o d a g r e e m e n t w i t h m e a surements of superoxide production under comp a r a b l e c o n d i t i o n s [8].

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2 min

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°"' / ' - ' , , 7.0 6.8 Fig. 5. Effect of pretreatment with islet-activating protein on fMet-kcu-Phe-induced changes in pHi. Neutrophils were preincubated in the presence (B and D) or absence (A and C) of 500 ng islet-activating protein for 2 h at 37°C. BCECF-AM (1 ~Lg/ml) was included during the last 30 min of the 2 h incubation with or without islet-activating protein. The cells were then sedimented and resuspended at 5-10S/ml in either Na'-medium (A and B) or N-methyl-D-glucamine ÷ medium (C and D) for the fluorimetric determination of pH i, as described for Fig. 3. Where indicated by the arrows, 2.10 7 M fMet-Leu-Phe was added. The traces are representative of three similar experiments. Temperature: 37°C.

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6.6-Fig. 6. Effect of pretreatment with islet-activating protein on TPA-induced changes in pH i. Neutrophils were preincubated in the presence (B and D) or absence (A and C) of islet-activating protein for 2 h as described for Fig. 5. BCECF-AM (1 #g/ml) was included in the medium during the last 30 rain of this incubation. The cells were then sedimented and resus~ pended at 5.105/ml in either Na + medium (A and B) or N-methyl-D-glucamine ~ medium (C and D) for the fluorimetric determination of p h i , as described for Fig. 3. Where indicated by the arrows, the cells were activated by addition ot 2.10 s M TPA. The traces are representative of at least three experiments of each type. Temperature: 37 ° C.

307

Effect of islet-activating protein on cytoplasmic pH As reported by Volpi et al. [9] and by Satoh et al. [10] using weak electrolyte partition methods, pretreatment with islet-activating protein was found to largely abrogate the alkalinization induced by fMet-Leu-Phe in neutrophils suspended in Na +containing medium (Fig. 5A vs. B). In four experiments the response was inhibited 76.6 + 10.3%. Similarly, the acidification observed in N-methylD-glucamine + medium was largely inhibited by pretreatment with the toxin (Fig. 5C vs. D). Isletactivating protein inhibited the acidification by 75.1 + 7% in four experiments. Fig. 6 illustrates the effect of islet-activating protein on the cytoplasmic pH changes elicited by TPA. Neither the complex pattern observed in Na + medium, nor the monotonic acidification recorded in N-methyl-D-glucamine + solution were discernibly affected by pretreatment with the toxin (104.4 +_ 2.4% of control). Discussion

In this report, we have used adenosine as a modulator of neutrophil function to study the source of the metabolic acidification detected in activated cells. Adenosine was initially thought to interfere with leukocyte function by partaking in transmethylation reactions [6], but subsequent evidence demonstrated that entry of the compound into the cell was not required for inhibition [7,16]. Instead, adenosine is presently thought to reduce superoxide generation through its interaction with A 2 receptors [19]. Two lines of evidence support this claim: (a) theophylline and 8-phenyltheophylline, competitive antagonists of the receptor, reverse the effects of adenosine and (b) 5'-(Nethyl)carboxamidoadenosine and 2-chloroadenosine are more potent inhibitors than either N 6phenylisopropyladenosine or adenosine, a sequence characteristic of the A 2 receptor [16,19]. It is thought that engagement of the A 2 receptors results in activation of adenylate cyclase [19], and inhibition of neutrophil function has been reported when the concentration of cAMP is elevated [20]. Pretreatment of the cells with adenosine produced a marked inhibition of the fMet-Leu-Pheinduced cytoplasmic acidification recorded in

Na+-free solutions (Fig. 3). This indicates that the reactions involved in secretion of either specific or azurophilic granules are unlikely to underlie the acidification, inasmuch as adenosine had little or no effect on elastase, /3-glucuronidase and lysozyme release. This conclusion is consistent with two other observations: first, the TPA-induced secretion of elastase is notably less than that induced by fMet-Leu-Phe (Fig. 1), yet the acidification is more pronounced in cells treated with the phorbol ester (cf. Figs. 5 and 6 and Refs. 3 and 5). Second, the TPA-induced acidification is preserved in both porcine [4] and human [5] cytoplasts, which are neutrophil fragments largely devoid of secretory granules. The aggregation that follows activation of the cells is also unlikely to account for the acidification. This was concluded because, unlike the inhibition of the metabolic acidification, the fMetLeu-Phe-induced aggregation was not influenced by adenosine (Fig. 2). Similarly, the shape changes undergone by activated neutrophils seem to be unrelated to the pH i change, since the latter is not affected by the addition of cytochalasin B (unpublished observations). The data available at present are consistent with a direct role of the oxidative burst a n d / o r the associated stimulation of the hexose monophosphate shunt in the generation of the cytoplasmic acidification detectable in Na+-free media: (a) the time course and magnitude of the oxygen consumption bursts elicited by fMet-Leu-Phe and TPA correlate with the respective changes in p h i ; (b) both phenomena are similarly sensitive to 2deoxyglucose or N-ethylmaleimide pretreatment [5] and (c) the acidification is virtually absent in neutrophils from patients with chronic granulomatous disease [18]. These cells lack the ability to generate superoxide, but have otherwise normal responses to stimulation. Consistent with these findings, oxygen consumption and the acidification were markedly inhibited by adenosine when the cells were stimulated by fMet-Leu-Phe but less so when the stimulus was TPA. It must be pointed out that, under our experimental conditions, the fractional inhibition of oxygen consumption was somewhat larger than that of the pH i change (90.3% vs. 75.1% for fMet-Leu-Phe and 37.8% vs. 14% for TPA). This suggests that other reactions,

309 15 Worthington Enzyme Manual (1972) Freehold N.J.: Worthington Biochemicals, p. 100 16 Roberts, P.A., Newby, A.C., Hallett, M.B. and Campbell, A.K. (1985) Biochem. J. 227,669 17 Hoffstein, S.T., Friedman, R.S. and Weissmann, G. (1982) L Cell Biol. 95, 234 18 Grinstein, S., Furuya, W. and Biggar, W.D. (1986) J. Biol. Chem. 251, 512 19 Cronstein, B.N., Rosenstein, E.D., Kramer, S.B., Weissmann, G. and Hirschhorn, R. (1985) J. Immunol. 135, 1366

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