Cellular Signalling Vol. 4, No. 5, pp. 543-551, 1992. Printed in Great Britain.

0898-6568/92 $5.00 + 0.00 ~ 1992 Pergamon P ~ s Ltd

ADENOSINE INHIBITS DIVALENT CATION INFLUX ACROSS H U M A N NEUTROPHIL PLASMA MEMBRANE VIA SURFACE ADENOSINE A2 RECEPTORS SATORU TSURUTA,*~f SETSUKOITO~ and HARUKI MIKAWA* *Department of Paediatrics, Kyoto University Hospital, 56 Shogoin Kawaharamachi, Sakyo-ku, Kyoto City 606, Japan and ~Ijinkai Takeda Hospital, 28-1 Ishidamoriminamicho, Fushimi-ku, Kyoto, Japan (Received 23 January 1992; and accepted 9 April 1992) Abstract--Adenosine and its analogues inhibited increases in divalent cation influx stimulated by plateletactivating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (FMLP) in a dose-dependent fashion. This effect was antagonized by theophylline, an adenosine receptor antagonist. When extracellular adenosine was removed by adenosine deaminase, the effect of adenosine was completely abolished. Two adenosine analogues with different affinities for adenosine receptor subtypes, 5'-N-ethylcarboxamideadenosine (NECA) and L-NLphenylisopropyladenosine (PIA), also inhibited divalent cation influx, NECA being more potent than PlA. These results suggest that adenosine and its analogues inhibit divalent cation influx across neutrophil plasma membranes via surface adenosine A2 receptors. Adenosine had little effect on the initial peaks of intracellular free calcium rises induced by chemoattractants, but it inhibited the subsequent rise in free calcium. Since calcium influx through the divalent cation channels or neutrophil plasma membranes is responsible for maintaining free calcium concentration following the initial peaks, we suggest that adenosine modulates neutrophil function by interfering with this calcium influx. Key words: Neutrophil, adenosine, calcium influx, divalent cation channel.

INTRODUCTION

types. Adenosine has been shown to exert its function through modulating intracellular cAMP level and then cAMP-dependent protein kinase [11-13]. In human neutrophils, however, adenosine pretreatment does not affect intracellular cAMP level sufficiently to modulate neutrophil functions, and its mechanism of action has not been elucidated [9]. As adenosine appears to alter only those neutrophil functions which are mediated by intracellular free calcium mobilization [4, 6], there is a possibility that the effects of adenosine are mediated by its interference with intracellular free calcium mobilization. To determine the effects of adenosine on intracellular free calcium homeostasis, we investigated its effects on [Ca2+]i and divalent cation influx across the plasma membrane of human neutrophils stimulated by two chemoattractants, platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (FMLP).

naturally occurring purine nucleoside, has been reported to alter a wide variety of biological functions [I-3]. It has been proposed that these responses are mediated via specific adenosine receptors on the cell membrane. Human neutrophils have surface adenosine receptors, and adenosine modulates neutrophil functions by occupying these receptors. Adenosine inhibits superoxide generation [4--7], neutrophil aggregation [8], membrane depolarization [9], and neutrophil adherence to endothelial cells [10]. In general, adenosine receptors are coupled with adenylate cyclase and the occupancy of adenosine receptors changes intracellular cAMP level in various cell ADENOSINE, a

?Current address: Division of Infectious Diseases, Allergy and Clinical Immunology, Shizuoka Children's Hospital, 860 Urushiyama, Shizuoka-city,420 Japan; and author to whom correspondenceshould be addressed. 543

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S. TSURUTAet al.

MATERIALS AND METHODS

Materials Adenosine, 2-chloroadenosine, L-NS-phenylisopropyladenosine (PIA), theophylline, and FMLP were purchased from Sigma Chemical Co. Adenosine deaminase (ADA) from calf intestines and 5'-Nethylcarboxamideadenosine (NECA) were purchased from Boehringer Mannheim Biochemicais. PAF was obtained from Bachern Fine Chemicals and fura-2/ AM was obtained from Molecular Probes. FMLP and PAF were dissolved in DMSO and phosphatebuffered saline (PBS), respectively, at 10mM, and were stored at -80°C until required for use.

Neutrophil preparation Ncutrophils were prepared from the heparin-anticoagulated venous blood of healthy adult donors, by dextran sedimentation followed by centrifugation on Ficoll-Hypaque gradients [14], The contaminating erythrocytes were then lysed by the addition of hypotonic saline. Neutrophils were finally suspended in Hanks' balanced salt solution (HBSS), pH 7.4, at a concentration of 107 cells/ml. The ieukocytes obtained in this manner consisted of more than 95% neutrophils without a significant number of platelets.

Measuring intraceilularfree calcium with Fura-2 Cytoplasmic free calcium was measured fluorimetilcally with fura-2, as described by Grynkiewicz et al. [15]. Neutrophils were loaded with fura-2 by incubating a cell suspension (107 cells/ml) with 1 #M fura-2/AM for 30rain at 37°C. Then cells were washed once with HBSS and resuspended at 2.5 × 107/ml in the same buffer and stored at room temperature until use. Neutrophil suspension (5 × 106/ml) in HBSS was placed in a thermostatically controlled (37°C) cuvette holder in a fluorescence speetrofluorometer (Model RF-510, Shimadzu) and the cells were kept in suspension with a magnetic stirrer. Changes in the ratio (R) of emission signals detected at 510 nm after excitation at 340 nm (F340) and 380rim (F380) reflected changes in [Ca2+]j. After the measurtnnems were obtained, calibration was carried out by disrupting the cells with 0.2% Triton X-100 to obtain the ratio in the saturating concentrations of calcium (R,~). EGTA (10mM, pH 10.0) was subsequently added to obtain the ratio in the absence of calcium (R~,). [Ca2+]i was calculated from the formula below, using the dissociation constant (KD) of 224 nmol/I [8]: [Ca2+] ~ = K o ( R - R.~,) F,~,380 (Rmax -- R ) Fro,x380"

Divalent cation influx assessed by Mn influx methods Divalent cation influx across the neutrophil plasma membrane was assessed by the method described by Merritt et al. [16]. Briefly, the fluorescence of neutrophils loaded with fura-2, as described above, was monitored continuously with 510nm emission and 360 nm excitation. Under these conditions, the fluorescence of fura-2 loaded neutrophils was insensitive to changes of [Ca2+]i. When Mn 2+ (100/aM) was added to the medium, it slowly entered the cells and quenched the fluorescence of fura-2. When neutrophils were stimulated with PAF or FMLP, the Mn 2+ influx was rapidly accelerated and the fluorescence decreased quickly. Divalent cation influx was quantified as a percentage of the decreases in fluorescence following chemotactic stimuli. Statistical analysis of data All experiments were performed in triplicate and were done at least three times using neutrophils from different donors. Differences between means were analysed using Student's t-test.

RESULTS

Effects of adenosine on intracellular free calcium mobilization Figure 1 shows the effects of adenosine on intracellular free calcium mobilization in human neutrophils stimulated with P A F and FMLP. Adenosine had little effect on the basal levels and initial peaks of [Ca2+]~, but in adenosine-treated cells, [Ca2+]i declined more rapidly from its peaks. This effect of adenosine was dose-dependent and was more pronounced in the case of P A F stimulation.

Effects of adenosine on divalent cation influx across the plasma membrane of neutrophils stimulated with P A F and F M L P With fura-2, elevations in [Ca~+]~ were shown by an increase in fluorescence at an excitation wavelength of 340 nm and a decrease at 380 nm. At 360 nm, the fluorescence of fura-2 loaded cells remained constant, despite [Ca2÷]~ changes due to chemotactic stimulants. Figure 2 shows typical examples of the fluorescence traces (510 nm emission) o f fura-2 loaded neutrophils with excitation wavelengths

Adenosine inhibits divalent cation influx (a) PAF stimulation

545

(b) FMLP stimulation

(nM) 4O0-

(nM) 30O

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•.-e.- control .-e-- O.Ol pM 0.1 pM .-~.- lpM .-m.- lOpM

1~2

o2 0

K,

50

100 150 200 250 300 TIME(seconds)

0

50

100 150 200 250 300 TIME( ~ )

FIG. 1. Neutrophils were incubated with various concentrations of adenosine for 15 min at 37°C before stimulation with PAF or FMLP (10 -7 M) was carried out. Each point represents the mean value of triplicate determinations.

of 340, 360, and 380 nm. PAF evoked a transient rise in [Ca2+]i, as evidenced by the transient increase in fluorescence at 340nm excitation, with a corresponding decrease at

(a)

380 nm (Fig. 2a,c). When cells were excited at a wavelength of 360nm, the fluorescence was insensitive to changes in [Ca2+]i and remained constant despite stimulation with PAF. When a

(d)

~Mn 360 nm

340 nm

(b)

360 nm

(e)

~

PAF 360 nm

(c)

380 nm

1 1 min

FIG. 2. Examples of typical fluorescence recordings of fura-2 loaded neutrophils stimulated with 10-7 M PAF. PAF stimulation caused a rapid increase in [Ca2+]i, shown by the transient increase in fluorescence at 340 nm excitation (a) with a corresponding decrease at 380 nm excitation (c). Fluorescence is insensitive to changes in [Ca2+]i at an excitation wavelength of 360 nm (b). Mn 2+ addition caused a slow decline in fluorescence in unstimulated neutrophils, presumably due to the slow basal influx of Mn '+ into the cells (d). When neutrophils were stimulated with PAF 1 min after the addition of Mn 2+, a rapid decrease in fluorescence was observed (e). Arrows indicate times when PAF 0 0 -7 M) or MnCI2 (100 pM) were added. All traces were recorded with an emission wavelength of 510 nm.

s. TSURUTAet al.

546

final concentration of 100/zM MnCI: was added to the medium, the fluorescence decreased slowly due to the slow basal leak of Mn 2÷ into the cell and the quenching of fura-2 fluorescence due to the binding of Mn 2+ and fura-2 (Fig. 2d). When neutrophils were stimulated with the chemoattractants PAF or FMLP, Mn 2÷ influx was rapidly accelerated due to the increased permeability of divalent cations across the plasma membrane; the fluorescence decreased rapidly (Fig. 2e). Since the change in [Ca2+]i did not influence the fluorescence at this excitation wavelength, the speed of fluorescence decrease reflected the permeability of the neutrophil membrane to divalent cations. Essentially the same results were obtained in the case of F M L P stimulation. Figure 3 shows the dose-response relationships of PAF and F M L P in their acceleration of the divalent cation influx. Both PAF and F M L P caused a rapid influx of Mn 2+ across the neutrophil plasma membrane; the effects were almost maximum for both stimulants at a concentration of 10-TM. When using the same

(a)

batch of neutrophils, PAF was always more potent than F M L P with regard to this effect, as shown in Fig. 3. Figure 4 shows the effects of adenosine on the divalent cation influx across the plasma membrane induced by PAF and FMLP. Adenosine, in concentrations as low as 0.1/zM, inhibited the divalent cation influx across the neutrophil plasma membrane induced by both PAF and FMLP, and this inhibition was dose-dependent for both stimulants.

Influence of theophylline on the effects of adenosine Theophylline, which is a potent adenosine receptor antagonist, has been shown to inhibit various receptor-mediated effects of adenosine. Figure 5 shows the influence of theophylline on 0.1/zM adenosine inhibition o f PAF-induced divalent cation influx. The effects of this concentration of adenosine on divalent cation influx were significantly reversed by both 10

(b)

~

Mn

Mn

I

101°M 10 -9 M

10 9 M 10 "8M

.

10 -8 M 10.7 M lO .6 M

10 -7 M 10 -6 M

FIG. 3. Both PAF and FMLP stimulation caused an acceleration of Mn 2+ influx in a dosc-dcpcndeJlt mann©r, as shown by the increased quenching of fluorescvnoc (emission, 510 nm; excitation, 360nm). Arrows indicate times when MnCI 2 (100 #M) and the indicated concentrations of PAF or FMLP were added. Chemoattractants were added 1 rain after the addition of Mn 2+.

Adenosine inhibits divalent cation influx

(a)

547

(b) Mn

Mn

F.,P

I

AR 10 pM

1 ml

AR 1 uM AR 10pM

AR 0.1pM

AR 1 pM

AR ( - )

AR 0.1 pM

AR (-) FIG. 4. Neutrophils were incubated with various concentrations of adenosine for 15 min at 37°C before stimulation with PAF or FMLP. Arrows indicate times when MnCI2 (100/~M) and PAF (10 -7 M) or FMLP (10 -7 M) were added. Mn 2+ was added I min before the addition of FMLP or PAF. AR--adenosine.

and 100/~M theophylline. Since, at these concentrations, theophylline acts as an adenosine receptor antagonist, not as a phosphodiesterase inhibitor, these results suggest that adenosine inhibited the divalent cation influx via adenosine receptors on the neutrophil plasma membrane.

Nok l u l

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s 3o Divalent Cationinflux(%)

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Fro. 5. Neutrophils were preincubated with 0.1/JM adenosine alone or with 0.1/~M adenosine plus 10 or 100/aM theophylline for 15min at 37°C prior to stimulation with PAF. Mn 2+ was added I min before stimulation; divalent cation influx is shown as the percentage fluorescence decrease for the first 2 min after stimulation. Theophylline significantly reversed the inhibition produced by 0. l/zM adenosine. The graph shows the mean values; the error bars shows standard deviations from three determinations. *P < 0.05 (adenosine 0.1/aM vs adenosine 0.1/zM+theophylline 10/~M). **P < 0.01 (adenosine 0.1/aM vs adenosine 0.1/tM + theophylline 100 #M).

Effects of adenosine, 2-chloroadenosine, and ADA 2-Chloroadenosine, an adenosine derivative which is a poor substrate for ADA, was as potent as adenosine in inhibiting the divalent cation influx induced by chemotactic stimuli. When A D A was added to the medium with adenosine, the effect of adenosine was totally reversed. However, A D A treatment had relatively little effect on the inhibition by 2-chloroadenosine (Fig. 6). These results showed that intact molecules of adenosine or its analogues were required in the extracellular medium to exert the effects and that adenosine did not

548

S. Tsul~trrA et al.

(a)

(b) gn

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1 min ~

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.

CI-AR 1 Ci-AFI+A 0

5

10

15

20

25

30

35

40

45

50

Diwiem Cmion Influx (%) FIG. 6. Neutrophils were incubated with adenosine (AR) or 2-chloroadcaosine (CI-AR) with or without 0.25 U/ml ADA for 15min at 37°C. ADA treatmcat completely abolished the inhibition produced by 1 ttM adenosine (*P < 0.01) (a), but had only a slight effect on the inhibition produced by !/~M 2-chloroadenosine (b). The graph (c) shows the effects of various treatments on divalent cation influx, which is shown as the percentage decrease in fluorescence for 2 rain after the addition of PAF or FMLP. The graph shows the mean values; the error bars show standard deviations from three determinations. need to be metabolized by ADA to exert its inhibitory activity.

manner. NECA was more potent than PIA (Table 1); this order of potency was consistent with that of adenosine A2 receptors.

Effects of NECA and PIA NECA and PIA, adenosine analogues which have different affinities for adenosine receptor subtypes, also inhibited the divalent cation influx stimulated by PAF in a dose-dependent

DISCUSSION Although adenosine and its analogues have been shown to modulate human neutrophil functions via surface adenosine receptors, the

Adenosine inhibits divalent cation influx

549

TABLE 1. EFFECTSOF NECA AND PIA ON DIVALENTCATIONINFLUXACROSS NEUTROPHIL PLASMA MEMBRANE

Concentration (/~M)

PIA

0

0.1 1 10

61.66___

55.53-1-0.82 45.985-1.50 40.09-t-0.57

NECA

P value

1.05 51.63_+2.21 39.71 _+1.68 36.79+2.09

< 0.05 < 0.01 < 0.10

Neutrophils were incubated with various concentrations of PIA or NECA at 37°C for 15 rain before stimulation with 10-7 M PAF. The percentage decreases of fluorescence for the first 2 min after stimulation were compared. NECA was a more potent inhibitor of divalent cation influx than PIA. The values represent the means and standard deviations of three determinations. This table is representative of four separate experiments with different donors, which, essentially, had the same results.

mechanisms by which adenosine receptor occupancy modulates these functions are not well understood. Stimulation of neutrophils with chemoattractants such as F M L P or PAF results in an increase in [Ca2÷]i that is due both to release from internal stores and to entry through plasma membrane Ca 2+ channels [16, 17]. Previous studies have shown that adenosine had little effect on initial [Ca2÷]i peaks following stimulation [8, 9, 18]. Our results with fura-2 loaded human neutrophils following PAF and F M L P stimulation also showed that the initial peaks of [Ca2+]i following such stimulation were not significantly different with or without adenosine treatment. However, [Ca2+]i declined faster in adenosine-treated than in control neutrophils, and this effect of adenosine was dose-dependent. Since the influx of extracellular calcium across the neutrophil plasma membrane is necessary to maintain [Ca2+]i following the initial peaks, this effect of adenosine may be explained by its interference with calcium flux across the plasma membrane. In this study, we investigated the effects of adenosine and its analogues on divalent cation influx across plasma membranes stimulated with chemoattractants, using Mn 2+, according to the method described by Merritt et al. [16]. This method has been used previously in several types of cells, including neutrophils, to analyse the permeability of plasma membranes to

divalent cations [16, 19-25]. Using fura-2 as a calcium indicator, we were able to analyse the divalent cation influx independently of [Ca2+]i changes. Adenosine and its analogues inhibited the increase of divalent cation influx induced by F M L P and PAF in a dose-dependent fashion. This effect was antagonized by theophylline, and adenosine receptor antagonist [5, 11]. When extracellular adenosine was removed by adenosine deaminase, the effect of adenosine was completely abolished, but ADA had little effect on the action o f 2-chloroadenosine, an adenosine derivative which is resistant to ADA degradation. Thus, we concluded that the effects of adenosine were mediated via surface adenosine receptors. NECA was more potent than PIA, suggesting that the receptor was the A2 subtype. Since calcium is considered to enter activated neutrophils through this type of divalent cation channel [16], we concluded that adenosine inhibited the calcium influx via surface adenosine A2 receptors. Adenosine has been shown to modulate calcium flux across the plasma membrane in several cell types [26-29]. However, the effects of adenosine on calcium homeostasis in human neutrophils are not well understood. Adenosine does not inhibit the initial rise in [Ca2+]i or the formation of inositol, 1,4,5-triphosphate following F M L P stimulation [8, 9, 18],

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suggesting that it does not interfere with the initial steps of signal transduction in neutrophils. It has recently been suggested by Laghi Pasini et al. that adenosine interferes with calcium influx across the neutrophil plasma membrane, since they found that the inhibitory effects of adenosine were partially reversed by increasing extracellular calcium concentration, and, further, it appeared that adenosine and a calcium channel blocker, flunarizine, shared a common binding site on the neutrophil cell membrane [30-33]. Although we employed a different methodology (using Mn2÷), our results also suggest that adenosine interferes with calcium influx through divalent cation channels. Since extracellular calcium is required for optimum neutrophil function, interference with calcium influx by adenosine could result in the inhibition of such neutrophil functions as superoxide generation. However, this interference with calcium influx may not be the sole mechanism underlying adenosine inhibition of neutrophil function, since it has been shown, by Cronstein et al. and Ward et al., that adenosine inhibits superoxide generation in the absence of extracellular calcium [9, 34]. Adenosine may inhibit several steps of the signal transduction pathways distal to the release of calcium from internal stores, and inhibition o f divalent cation influx may be only one of these inhibitory mechanisms of adenosine. Further studies are needed to elucidate the mechanisms which link adenosine receptor occupancy, divalent cation influx, and neutrophil functions, such as superoxide generation.

I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

CONCLUSION

21.

Our findings support the hypothesis that adenosine inhibits calcium influx across the neutrophil plasma membrane by interfering with divalent cation influx in activated neutrophils; this effect is mediated via adenosine A2 receptors.

22. 23. 24.

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Adenosine inhibits divalent cation influx 25. Merritt J. E. and Moores K. E. (1991) Cellular Signalling 3, 243-249. 26. Marangos P. J., Finkel M. S., Verma A., Maturi M. F., Patel J. and Patterson R. E. (1984) Life Sci. 35, 1109-1116. 27. Schubert P., Heinemann U. and Kolb R. (1986) Brain Res. 376, 382-386. 28. Ramagopal M. V. and Mustafa S. J. (1988) Am. J. Physiol. 255, HI492-H1498. 29. Ramagopal M. V. and Mustafa S. J. (1990) Can. J. Physiol. Pharmac. 68, 608-613. 30. Laghi Pasini F., Capecchi P. L., Orrico A., Cec,catelli L. and Di Peril T. (1985) J. lmmunopharmac. 7, 203-215.

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Adenosine inhibits divalent cation influx across human neutrophil plasma membrane via surface adenosine A2 receptors.

Adenosine and its analogues inhibited increases in divalent cation influx stimulated by platelet-activating factor (PAF) and formyl-methionyl-leucyl-p...
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