Inhibitors of the leukocyte superoxide generating oxidase: Mechanisms of action and methods for their elucidation

Free Radical Biology & Medicine, Vol. 8, pp. 71-93, 1990

0891-5849/90 $3.00+ .00 © 1990PergamonPress plc

Printed in the USA. All rights reserved.

Review Article INHIBITORS

OF

THE

LEUKOCYTE

GENERATING Mechanisms

SUPEROXIDE

OXIDASE:

of Action and Methods

for Their Elucidation

ANDREW R. CROSS Department Biochemistry, University of Bristol, Medical School, University Walk, Bristol BS8 1TD, U.K. (Received 15 May 1989; Revised and accepted 21 August 1989)

Abstract--Oxygen radical production by phagocytic cells is currently receiving a great deal of attention as the role of radical damage is becoming apparent in inflammatory diseases, reperfusion injury, cancer, and aging. A large number of inhibitors of the superoxide generating oxidase are known, including standard and experimental anti-inflammatory and anti-rheumatic drugs, natural products, anaesthetics, tranquillizers and antibiotics, in addition to compounds used as experimental tools. The composition of the oxidase and the possible sites of inhibition of these compounds are discussed together with the possible mechanisms of activation of the oxidase and the effects these agents may have on these pathways. Use of these compounds has provided a great deal of information about the components and the nature of the activation processes involved in the stimulation of radical production by leukocytes, as well as pointing to possible targets for the production of novel anti-inflammatory agents. It is clear however that further understanding of the precise nature of the activation pathways and the extent of the involvement of leukocyte-derived oxygen radicals in disease processes will require more specific inhibitors than most of those currently available. Keywords--Free radical, NADPH oxidase, Inhibition, Anti-inflammatory, Phagocytes

The neutrophil superoxide generating system has evolved to respond to a very wide range of foreign stimuli with one a i m - - t o destroy them. Because of the need to respond to such a wide range of chemically and physically different agonists it appears that there are a number of different stimulus-dependent transduction mechanisms which can be involved in the activation process. As a direct consequence of this complexity there are a large number of more or less specific inhibitors of the activation of the oxidase which interact with these different transduction pathways. Many of these compounds are known to be inhibitors of particular enzyme systems (e.g.,

trifluoperazine, calmodulin antagonist: aspirin, cyclooxygenase inhibitor: N D G A , lipoxygenase inhibitor) and their effects on neutrophils are often attributed to their known inhibitory properties. Unfortunately it is clear that many of these compounds are capable of interacting with the oxidase in ways which are quite independent of their recognised mechanisms and their description as " s p e c i f i c " inhibitors must be treated with caution ~'2 (see also Table 2). In addition, perhaps because of the multi-component nature of the oxidase itself, there are also a number of compounds which are capable of inhibiting the oxidase directly. The inhibitors can broadly be divided into two groups, those which inhibit the activation of the oxidase in whole cells (but do not inhibit the isolated enzyme) and those which exert a direct inhibitory effect. The former group includes compounds which may interfere at all stages of the activation process; inhibition or interference with ligand binding, signal transduction, second messenger formation, activation of protein kinases etc. and compounds which may interfere with the production of the N A D P H required for the oxidase. This can occur due

Born on April 8th 1954 in Aldershot, Hampshire, Sandy Cross obtained his degree in Physiology and Biochemistry at Southampton University where he also obtained his PhD. He moved to his present position as Research Associate in Professor Owen Jones' laboratory in the Biochemistry Department at the University of Bristol in 1979. During the last 10 years he has worked extensively on the characterization of the phagocyte NADPH oxidase particularly with respect to the role of cytochrome b-245. More recently he has concentrated on the flavoprotein component and its interaction with inhibitors. He is married with 3 children. 71

72

A.R. CROSS

to inhibition of one of the enzymes of the hexose monophosphate shunt or by inhibition of glucose uptake. The mechanism of action of those compounds which exert a direct effect has provided insights into the function of the oxidase and can in turn be subdivided into classes. Use of these inhibitors has enabled us to propose the order of the components of the electron transport pathway (Scheme 1). Compounds which inhibit the flavoprotein part of the oxidase prevent electrons passing into cytochrome b_245 and thus prevent its reduction. On the other hand inhibitors of the cytochrome do not effect flavoprotein reduction. It is the purpose of this review to give a brief description of the nature of the oxidase itself, the mechanisms of its activation, types of inhibitors and how they have been used to improve our understanding of the superoxide generating system. Throughout the text those inhibitors which have been more extensively studied are marked with an asterisk. It has become evident in the last few

years that the inappropriate production of oxygen radicals from phagocytes can have severe pathological consequences in disease and the inhibition of radical generation has become a major pharmaceutical target.3

COMPOSITION OF THE OXIDASE

The superoxide generating oxidase of phagocytic cells is a membrane bound enzyme system believed to consist of at least two redox active components, a FAD containing flavoprotein and a unique low potential cytochrome; cytochrome b_>5. The field has been the subject of two excellent recent reviews. 29 This oxidase is present in an inactive or resting state in all the professional phagocytic cells (neutrophils, monocytes, macrophages, and eosinophils) and can be activated by a large number of agents to consume oxygen (the respiratory burst) and produce the superoxide anion radical (formed by the one electron reduction of oxygen), u~ The source of electrons is NADPH, formed by the hexose monophosphate pathway, the activity of which is greatly stimulated during the respiratory burst. The oxidase is thought to function by the flavoprotein accepting two electrons from NADPH and transferring them one at a time to cytochrome b--24s which in turn reduces oxygen to superoxide as shown in Scheme I. The stoichiometry of the system is thus: NADPH + 202

> NADP + + H + + 20~-

Scheme 1. NADPH _ Flavoprotein NADP +

°chr°mb
~

The flavoprotein and cytochrome undergo characteristic changes in light absorbance when going from their oxidised to their reduced forms which has enabled researchers to investigate more closely the effects inhibitors which interact directly with these oxidase components, lnhibitors which affect the oxidase directly are distinguished by inhibiting subcellular preparations of the oxidase prepared from activated cells. These preparations are usually particulate fractions (or detergent solubilized preparations derived from them) obtained by centrifugation of disrupted activated cells. There is apparently no difference in the properties of the activated isolated oxidase regardless of the stimulating agent. If an agent is capable of inhibiting such isolated oxidase preparations its action also must be independent of the activation mechanism. It should be remembered however that in such isolated preparations the inhibitor has no membrane barrier to cross (unless the preparation is in the form of right-side out mem-

brahe vesicles) and inhibitors which have activity against the isolated oxidase may not be effective in whole cells. A case in point is the effect of sulphydryl reagents, all of which will inhibit the isolated oxidase, but not all will inhibit stimulated whole cell oxidase activity, although they may also inhibit other parts of the activation process, possibly through interactions with receptors, t~-~5 The effect of inhibitors against the activation processes induced by various stimuli or their direct effect on the isolated oxidase is given in Table 1. A. Direct Inhibitors 1. Flavoprotein. The compounds thought to directly inhibit the flavoprotein part of the oxidase have provided some useful information about its properties. Antibodies raised to the microsomal enzyme cytochrome P450 reductase inhibited particulate superox-

mm:kl red~e

Alter mmNbrane

.m~y,

Ca2~

S¢svongers

""""'P

~ s )qbem I q l e ~ i

/ I

ofo,

Cvtochrome

FiavoDroteilt

|

NADPH

an~p.es

/

H"

H°

Cheta~s

~

NADPH

"

\

Glucose

Glucose

Fig. 1. Mechanisms of inhibition of the phagocyte superoxide generating system. The figure summarizes the possible sites of action of inhibitors of the superoxide generating system. Fuller descriptions of the mechanisms of action and the compounds involved are given in the text.

Deactivate

FAD analogues

Catcium tag°nists

KInlse . inhibitors

Second~' messenger

i~Mbitors

~il~Se

Sulphydryl

"~

.:pmmk~

>

CI-922 pCMB Chloroquine Colchieine C o m p o u n d 48/8(I Coumarin Cross-linkers Dexamethasonc Diamide Diazepam Diclofenac Disulfiram Doxyclyclinc/tetracyclines Ebselen Eseuletin Ethanol ETYA NEM§ G o l d sodium thiomalate Hy dr o x y c h l o r o q u i n e H7 Ibuprofen lndomethacin lo do a c e ta t e Juglone K a d s u r e n o n e , BN52021 Lapachol L ys o z om o tr o p i c weak bases Mannosc Meclolenac Mefanamic acid

Chlorhexidine

Aspirin Auranofin Benzydamine Bilirubin Biscoclaurinc alkaloids p-Bromophenacyl bromide BW755C C-I Cetiedil CHIP

cAMP

AA861 Acrolein Adenosine Adenyl cyclase toxin + ve alkylamines Amphoteracin B Anaesthetics

Compound

70

400

5

100 10

0.4 70

<300

190 itg/ml 21)00 100

2(10

150

25 /lg*

Oil/latex

( mM

1(I

-

75 ltg/ml

13 u g / m l

100

1 mM 100 m M 30 70

< 100 / 1 (37°C)~ 40 lO0/ 50

1% <'20 5

<50

300

100

100

/1 (37°C)+ 100 2/tg

<5 <'5

/3000 <2

3e/~

1

0.6

<50

fMLP

< 100

-

<5

<2

IgG

< 100

100/tg

<5 l(/

10 < 10

5/tg/ml 1 ltg/ml

OZ

<. 100

<5

C5a

ConA

< 100

<50

.

I e/~ <3(J

< 1()((

1 /tg/ml p o l y h i s t i d i n e

<5

4(1 n g / m l

3 m M cytochalasin E

PAF

.

<30

400

Dig

.

1 mM I(I m M

.

1% <50

100

1 raM/-

< 100

100/tg

<5

-

+

-

A23187

.

1 mM 10 m M

- > 100 l/~0

....1(1 Mac:? < 10

25

100

550

64

- : <. ( ~:

>40 ng/ml

5

1

<20

PMA

<. 1

14

AA

I mM

100

I~

10(I

F

Table 1. Stimulus-Specific Effects of Inhibitors of Superoxide Production. Concentration Required to Inhibit Stimulus (,.M)

"

?

+ 5 +

I <5

300

?

?

5 15 510

+

25(10

< I0(I0

D

('3

>

<50 5/100 itg/ml <250 2

3(10

15 50 <75 20,000 < 160

<5(1

1

3/63 1 mM

3

< 10

25 ,ug/ml

< 10(I 100 2(I

2.2 20

< 10

>.2 100 <5(/

< 16(1 < 10

< 16(J < 10

5(1

< 1(10

500

-

10 <50 95 pM

2 ltg/ml

<10

< 10(1 < 100

100 95 pM

20

.2 ltg ml J

1000

25

1(1(I 10 10

m l - I

95 pM

O. 1 i t g

I

<160

30

<5(I

2 50

Cytochalasin E <4 mM

15 Itg ml -

25

7

<16(I

20

20

1(1

--

<1(10

25

-/.4

1(1

2(1

200

<(0 1(1(I 1 5 3

.15

< 160 <10

<50 13 73/<75

mg/ml

( )

5O

16 2O

75

37

<10 3

25

< 16(I

-(1

10

2500 7 ? ?

9.5

mM)

4(1 +

?

+

4(I

3/25(I

Note. Entries in the table arc those concentrations of compound reported to inhibit 50% of the superoxidc production induced by the stimulus in the absence of inhibitor (ECs0). [ + ] indicates inhibition but the EC50 has not been determined, [ ] signifies that the compound does not inhibit supcroxide production in response to that stimulus, [--] signifies increased superoxide production relative to control. Abbreviations: Oil/latcx--phagocytosable paraffin oil droplets or latex beads; OZ--serum opsonized zymosan; lgG immunoglobulin G; fMLP--N-formyl methionyl-leucyl-phenylalanine; C5 a -complement fragment: PAF--platclet activating factor: ConA--concanavalin A; Dig digitonin; A23187--calcium ionophore; PMA--phorbol myristate acetate; AA--arachidonic acid; F--fluoride; D--direct inhibitor. *nactcria were used as the stimulus. ¢lnhibition was only observed during incubations at 37°C. $tnhibition only observed in macrophages. §Some species differences. IqTrimethoprim/sulfamethoxazote. yeast cells were used as the stimulus.

Thiomalate b thujaplin TPCK TLCK Triethylphosphine gold cpds. Trifluoperazinc Trim/Sul ycastl] Trithicnyl butane dione Tropolonc U60,257 Verapamil W-7 Zn

Theophyllinc

Olsalazine Opiate antagonists Organotin cpds. Oxyphenbutazone Penicillamine Pertussis toxin Phenidone Phenothiazines Phenyl butazone Piroxicam PDGF Polymixin Primaquine Propyl gallate Pyocyanine Quercetin Quinacrine Retinal Salicyl hydroxamate SBTI Sphingoid Spirogermanium Staurosporine Steroids Sulfasalazine Tetracyclincs

50

NDGA

3(I-5(I

10

200 itg/ml

Methotrexate Morphinc Naloxone Neomycin

gr~

~7 -r

Z

A . R . Cross

76

ide generation suggesting a structural similarity between the two enzymes. ~6 Interestingly, it has been suggested that the phagocyte flavoprotein may be functionally organized such that one flavoprotein molecule may reduce many cytochrome b_245 molecules which is known to be the case for cytochrome P450. J7 There are significant differences in these proteins however. The flavoprotein of the oxidase is known to contain only FAD and not FMN as the prosthetic group whereas P450 reductase contains 1 mol of each. ~8 The oxidase flavoprotein is thought not to exhibit any dye reductase (diaphorase) activity, unlike P450 reductase, which is capable of reducing a number of artificial electron acceptors.~2° There are also marked differences in the sensitivity toward sulphydryl reagents and P450 reductase has a lower Km for NADPH. 2~ The FAD analogue, 5-carba-deaza-FAD, can bind in place of the natural FAD cofactor in detergent solubilized preparations and inhibits electron transfer. 22 5-deaza-FAD is only capable of two-electron transfer and not single electron transfers, confirming the essential role of FAD in mediating the two electron to one electron transfers necessary in going from the obligatory two electron donor NADPH, to the obligatory one electron acceptor, cytochrome b_ 245. Interestingly, work using other substituted FAD analogues has shown that increased rates of electron transfer through the oxidase can occur with flavins which have higher redox potentials than FAD, suggesting that in the fully activated oxidase the rate limiting step is electron transfer from NADPH to cytochrome b 245.23 Diphenylene iodonium is a potent specific inhibitor of the oxidase system in whole cells or isolated preparations of the oxidase. 19,24.25The site of action is known to be at the level of, or prior to, the flavoprotein since it prevents reduction of both the cytochrome b 245 and flavin in steady state experiments. Radiolabelled diphenylene iodonium has been used to covalently label a polypeptide of Mr45,000 in SDS PAGE which may be the flavoprotein polypeptide. The specificity of diphenylene iodonium has been exploited to investigate the relative importance of oxidative damage as opposed to non-oxidative damage produced by activated phagocytes. 26,27 Quinacrine potentially has a number of inhibitory sites of action, but it is known to be a direct inhibitor of the isolated oxidase possibly due to its similarity in structure to FAD.t3

b_245. The terminal component of the oxidase system, cytochrome b 245, reacts with oxygen to form superoxide. It is a heterodymeric protein consisting of a small (23kD) subunit and a heavily glycosylated large subunit (76-92kD). 29 3~ Both subunits are absent in the more common type of Chronic Granulomatous Disease, the X chromosome-linked form, in which the gene coding for the large subunit is faulty. Despite the amino-acid sequences of both subunits being known it is still not clear which subunit is responsible for haem-binding, as the sequences bear no obvious similarities to other known proteins. 32'33 In common with many terminal oxidases the cytochrome can form complexes with ligands other than molecular oxygen, but with rather low affinities. The Km of the oxidase for oxygen is around 5 - 3 0 / / M 34 37 but for the classic cytochrome oxidase inhibitor, carbon monoxide, the Km is much higher (1.4 mM) and it is not inhibitory in the presence of oxygen. 3~ Such resistance to CO is not common among oxygen liganding haemoproteins but is by no means unique. In fact all haemoproteins have a dramatically reduced affinity for CO compared to model haem compounds. This reduction in affinity is due to steric hindrance of the linear F e - - C O complex by amino-acid residues near the binding site which favour the bent Fe--O2 ligand. 39'4°Other haem ligands are effective inhibitors however, and these have been shown to cause characteristic spectroscopic complexes with cytochrome b_245, t3.41 Pyridine and imidazole completely abolish superoxide production when complexed to the haem, whilst butyl isocyanide inhibits in a concentration dependent manner (35% at 20mM). 13 This concentration is sufficient to complex all the haem under anaerobic conditions. The fact that it does not totally inhibit superoxide production aerobically suggests there may be competition for the Fe-ligand between butyl isocyanide and oxygen in the aerobic state. The other type of inhibition of cytochrome b_245 has been exhibited by sulphydryl reagents, notably p-chloromercuribenzoate (pCMB). This reagent has been shown to inhibit the isolated oxidase system at very low concentrations and causes an alteration in the redox potential of the cytochrome indicating there may be a reactive thiol close to the haem. 12 The use of inhibitors which interact with cytochrome b 245 have confirmed its essential role in the oxidase.

2. Cytochrome

Haem ligands: Flavoprotein inhibitors: AB to P450 reductase ~6 5 - D e a z a - F A D ~-2 *Diphenylene iodonium j9'24 *Quinacrine'-" ,4.~

Butyl isocyanide ~3 Imidazole 4~ Pyridine 4~ Reacting close to the haem *pCMB~2 ,~

NADPH oxidase inhibitors

3. Phospholipid. The oxidase has been shown to exhibit an absolute requirement for phospholipid to show activity, 42 as might be expected for a membrane protein. There may also be a crucial role to lipid in the activation process, vide infra. This dependence on the correct hydrophobic environment is another route by which inhibitors may act by disruption of lipid-protein interactions. Detergents such as cetyltrimethylammonium bromide (CTAB) are very powerful inhibitors of the isolated oxidase and it is possible that the real site of action of other hydrophobic inhibitors is due to these effects rather than their ability to inhibit (for instance) phospholipid metabolism. Hydrophobic agents known to act directly CTAB~3,t~ Phenothiazines; Chlorpromazine 43, *Trifluoperazine~4,43-45

4. Magnesium. It is known that some NAD(P)H utilizing enzymes have a requirement for Mg 2+ in order to bind FAD or their substrates. The superoxide generating oxidase has also been shown to exhibit an absolute requirement for Mg2+46~47; activity lost after incubation with EDTA can be restored by the addition of magnesium but not calcium ions 48 (although calcium is required for activation by some stimuli, section B4). Experiments using the cell-free activation system have shown that Mg 2+ is also required during the assembly of the active oxidase as well as for the maintainance of activity. 49.189 A possible role for non-haem iron has been postulated in view of the inhibitory effect of the lipophilic iron chelators (Table 2) but these effects may be due to their disruption of lipid protein interactions rather than their chelation properties as no evidence for the presence of non-haem iron has been found in several ESR studies. 5°.51 Direct acting chelators B athophenanthroline ~4 *EDTA, EGTA, DTPA 46,52,53 *Quercetin, Esculetin and other flavonoids 54 56

5. Thiol. As described above sulphydryl reagents can interact with sensitive thiol groups on the components of the oxidase. Some flavoproteins have thiol groups which are involved in electron transfer reactions and this is one potential site of inhibition. 57 As mentioned above pCMB at low concentrations appears to interact with a thiol group close to the haem of cytochrome b-245.12 The sites of interactions of other thiol reactive compounds is not known. Although the second line anti-arthritic drugs are known to be thiol reagents and their mode of action is thought to depend, at least in

77

part on this activity, it is unlikely that they exert their inhibitory effects on whole phagocytes by direct interaction with components of the oxidase itself. Results obtained with thiol reagents should also be treated with caution as they can act as radical scavengers or chelators and can directly reduce cytochrome c in the superoxide assay. Directly acting thiol reagents Auranofins8 ,pCMBL2 ~5 Disulfiram54 Ebselen 59 *NEM53,60.61 Gold sodium thiomalate 58 Penicillamine 58 *Quercetin, Esculetin and other flavonoids 54 56 Thiomalate 5s Triethyl phosphine gold 5~

6. NADPH analogues. Compounds which resemble NADPH in structure clearly have the capacity to inhibit superoxide production in the isolated enzyme in a competitive manner. ADP and Cibacron Blue (which is recognised by the dinucleotide binding fold of many NADPH binding enzymes) have been shown to compete with NADPH in oxidase preparations. 62,63 Phenothiazines have been shown to he competitive inhibitors of NADPH in other systems and are likely to be competitive inhibitors of the oxidase. 64 Recently dialdehyde derivatives of NADPH have been used in attempts to identify the NADPH-binding polypeptide of the oxidase. The arylazido and 2,3-dialdehyde derivatives of NADPH were shown to inhibit activated membranes of neutrophils and by the use of radiolabelling it was suggested that the NADPH-binding polypeptide had a molecular weight of 66kD. 65-67 More recent work has indicated that the NADPH-binding component of the oxidase is probably not the membrane 66kD protein 68 and may be a cytosolic component of either 6 6 - 7 0 , 55 or 45kD. 69 NADPH analogues ADP ~2 2,3-dialdehyde NADPH 66-69 Cibacron Blue 63 Phenothiazines; chlorpromazine, promethazine, *trifluoperazine ~4,43-45,e4 Pyocyanine?44

7. Charge translocation. Henderson and collegues have recently shown that the generation of superoxide by the oxidase involves the uncompensated charge transfer across the plasma membrane of an electron from internal NADPH to oxygen on the external face and is thus electrogenic. 7°'71 If no mechanism existed

Doxycycline Ebselen ETYA E t han o l

Disulfiram

Coumarin Dexamethasone 5-Deaza-FAD D iam i de Diazepam Diclofe n ac Diphenylene iodonium

C o m p o u n d 48/80

AA861 Acrolein Adenosine A d eny l cyclase toxin ADP + ve A l k y l a m i n e s Amphoteracin B Anaesthetic A b to P450 r ed uct as e cAMP Aspirin Auranofin Bathophenanthroline Benzydamine Bilirubin Biscoclaurine a l k a l o i d s p-Bromo phenacyl b r o m i d e Butyl isocyanide BW755C C-1 Cetiedil CTAB Chelators CHIP* Chlorhexidine Cibacron Blue CI-922 pCMB Chloroquine Chlorpromazine Colchicine

++

++

++

++

+++

Recep. Expr.

++

+++

+++

++

+++

- SH

+ ++

++

++ ++

++ +++

++

-

Phosph. L ip a s e

++ +++

+÷+

++ ++

Ca & Mg

+++

+++

+++

CO

Ca 2+

++ ++

+++

+++ +++

+++ +++

+++ +++ +++

Memb . Effect

++

mast cell d e g r a n u l a t o r

++ ++

+++

++

Protein Kinase

++ +++

++

++

++

+++

+++

+++

Calmodulin

i n h i b i t o r of pl a nt a l t e r n a t i v e o x id as e

++

+++

++

+++

LO

A ) A c t i n g on A c t i v a t i o n P a t h w a y s

Table 2. Sites of I n h i b i t o r y A c t i o n

+++

+++

Deactivator

Metab. I n h ib .

+++

++

+++

+++

+++ ++

+++

+++

+++

+ +++

+++

+++

+++

D ir ect

7~

>

B-Thujaplicin

Thiomalatc

Thenoyl trifluoroacetone

Steroids Sulfasalazine Tetracyclines

Spirogermanium

Sphingoid

Salicylhydroxamate

Pyridine Ouercetin Ouinacrine Retinal

Pyocyanine

Protease inhibitors

Propyl gallate

Esculetin EDTA NEM Gold sodium thiomalate H-7 Hydroxy chloroquine Ibuprofen Imidazole Indomethacin Iodoacetate Juglone Kadsurenone Lepachol Lysozomotropic weak bases Mannose Meclofenac Mefanamic acid Neomycin Nordihydroguaiaretic acid Olsalazine Opiate antagonists Organotin cpds. Oxyphenbutazone Penicillamine Pertussis toxin Phenidone Phenothiazines Phenylbutazone Piroxicam PDGF Polymyxin B Primaquine

+

++ ++

+++

+++

++

+++

++

+++

+

+++

+++

+++ +++

+++

++ +++

++ ++

"~

+++

++ ++ +++ +

++

+++

++

++ +++

+++

+++

+++

+++

+++

+++

+++

+++

1ON

++

+++

+++

radical scavenger

+++

+++

+++ +++

+++

+

++

+

++

++

++

+++

++

redox active

heavy metal chelator; quinone inhibitor?

+

anti-rheumatic

inhibitor of plant alternative oxidase +++ +++

++ ++ +++

redox active; phenothiazine analogue

pKc inhibitors & pLA2 inhibitors

+++

+++

+++

+++

+++

+++

+

+++

+++

++

+++ +++ ++

+++

+++

+++

+++

+++

++

++

+++

z

z

+++

++

++

÷

++

Phosph. Lipase CO

÷++

LO

Ca 2'

Memb. Effect

++

++

antioxidant

+÷+

redox active

n o n - h a e m iron chelator

++

Protein Kinase

÷÷+

+++

Calmodulin

Deactivator

Metab. lnhib.

+ +++

Direct

p - B r o m o phenacyl b r o m i d e Butyl isocyanide CTAB Chelators Cibacron Blue pCMB Chloroquine Chlorpromazine C o m p o u n d 48/80

Bathophenanthroline

Auranofin

ADP Ab to P450 reductase

DIRECT

+

+

Flavin

Cyt

Mg :+

- SH

+

NADPH

?

Fe chelator

- S H ; d e c r e a s e s fMLP binding; inhibits LTB prdn

Lipid

B) C a n d i d a Albicans H y p h a c Inhibitory Protein Redox

+

+

UK

lipoxygenase; protein kinasc--protcin kinase C or M; Ca 2*-calcium release or influx: Mcmb. cffcct--compounds affect membrane fluidity or stability: calmodulin-calmodulin antagonist or inhibitor of calmodulin dependent protein kinasc: deactivator--stimulation of dcactivation pathway(s); m e t a b inhib.--inhibition of metabolic pathways leading to NADPH generation; direct--direct inhibition of the oxidasc. *Candida albicans hyphae inhibitory protein.

Note. [ ] Shown not to havc inhibitory activity. [ +++] potcnt inhibition, [ ++ ] moderatc inhibition, [ + ] slight inhibition. Abbreviations: Recep. cxpr.--rcceptor expression: SH--sulphydryl group: Phosph/lipasc--phospholipase A_~ or phospholipase C: CO--cyclo-oxygcnasc; L O - -

W-7 Zinc

Vitamin E

U60,257 Verapamil

Tropolone

Trithienyl butane dione

Triethyl-phosphine gold Trifluoperazine

-SH

Recep. Expr.

Table 2 (Continued).

7~

>.

+

+

+

?

+

antioxidant

n o n - h a e m iron chelator

+

heavy m e t a l chelator; q u i n o n e inhibitor? +

inhibitor of plant alternative oxidase

+

+ +

+

+

inhibitor of plant alternative oxidase +

+

+

+

+

+ + +

+ +

t

+

-i-

nesium;

SH--reaction with essential thiol; NADPH--NADPH analogue; Redox--redox active compounds; UK

unknown mechanism of action.

Note. [ + ] Proposed site of action. Abbreviations: Flavin--flavoprotein or FAD analogue; Cyt--cytochrome b 2~ lipid--removal or replacement of essential lipid; MgZ+--removal of essential mag-

W-7

Vitamin E

Tropolone

Trithienyl b u t a n e dione

Thiomalate B-Thujaplicin Triethyl p h o s p h i n e gold Trifluoperazine

T h e n o y l trifluoroacetone

Salicylhydroxamate

Ebselen Esculetin EDTA NEM Gold s o d i u m t h i o m a l a t e H y d r o x y chloroquine Imidazole Iodoacetate Juglone Lapachol Nordihydro-guaiaretic acid Penicillamine Phenothiazines Primaquine Propyl gallate Pyocyanine Pyridine Quercetin Quinacrine

Disulfiram

Coumarin 5-Deaza-FAD Diamide Diphenylene iodonium

.g

r,

=

"0

Z >

82

A.R.

for the balancing of the movement of negative charge to the outside and the generation of internal protons from the oxidation of NADPH, there would be a massive depolarization of the membrane potential and a fall in the internal pH. The charge compensation is provided by a Z n 2+ and Cd 2+ sensitive proton channel releasing protons to the external face of the membrane. Limiting of the efflux of protons by these metal ions results in an inhibition of the oxidase activity which can be reversed by the addition of an uncoupler such as CCCP. 72 In this respect the superoxide generating oxidase resembles the mitochondrial electron transport chain in that it exhibits respiratory control. The existence of such an ion channel opens up the possibility of a new family of inhibitors of the oxidase which could exert their effects by blocking this channel. It is conceivable that the inhibitory action of weak bases (which are accumulated within the cell) is by sequestering protons within the cytosol which would normally be translocated outwards to compensate for the electrogenic charge movement, and thus resulting in inhibition of the oxidase through a dramatic membrane depolarization. Possible effectors o f c h a r g e t r a n s l o c a t i o n + ve A l k y l a m i n e s 73 B i s c o c l a u r i n e a l k a l o i d s 74 ,ChloroquineS~ 75 7~ H y d r o x y c h l o r o q u i n C 5 7, O t h e r l y s o z o m o t r o p i c w e a k bases 767~

8. Redox active compounds. Compounds can accept or donate electrons can interact with the oxidase through a number of mechanisms. Firstly, they may accept electrons directly from the redox components of the oxidase thereby diverting electrons from the pathway to oxygen. Secondly, they may react with superoxide, either competing with the detection system for the radical or acting as a superoxide dismutase (2,3). Thirdly, they may react with the detecting molecule, competing with superoxide (4), or reforming the detection molecule from its superoxide reaction product (5). In the first case whilst there has been controversy in the literature over many years, it now seems probable that the flavoprotein exhibits little if any intrinsic dye reductase activity 192° unless partially denatured with detergent or salt. 8° There is some evidence that the cytochrome can donate electrons to artificial acceptors but this only occurs at significant rates with high concentrations of acceptors under anaerobic conditions. 81,82 Those compounds reacting with superoxide directly will affect any assay which involves the detection of superoxide (4), rather than the oxidation of NADPH (1) and may also affect assays which depend on mea-

CROSS

surement of oxygen uptake, depending on the nature of the reaction with superoxide, since reoxidation of superoxide back to oxygen would appear to block oxygen uptake (2). Compounds which react with the detecting molecule may act by effectively removing it from the assay (6), or may react with its superoxide product to regenerate the initial detecting agent (5). NADPH +

0 2

Og + X 202

+

Y

~-

Cyt C3+

)'

O2-

(1)

) 02 + X > Y

+ H202

(2) +

02

(3)

Cyt C2+

(4)

Cyt c 2+ -~- Z

> Cyt c 3+ + Z -

(5)

C y t c 3+ + A

~ C y t c 2+ + A +

(6)

0 2

" )' 0 2 ~-

In addition it should be borne in mind that some of these "inhibitors" are used at high concentration where they may have effects on membrane organisation. R e d o x active " i n h i b i t o r s " J u g l o n e ~3 LapachoP ~ * N o r d i h y d r o g u i a r e t i c acid ~4 Propyl gallate ~' P y o c y a n i n e 4~ * Q u e r c e t i n , esculetin a n d o t h e r f l a v o n o i d s 54 5~ T h e n o y l t r i f l u o r o a c e t o n e ~4 B-Thujaplicin ~ Tropolon¢ 3 Vitamin E ~'

B. Activation That the NADPH oxidase is only in the active state following stimulation of the cell surface has been known for many years, but it is only recently that the transduction mechanisms by which it is transformed from a resting to an activated state have become clearer although not fully defined. It is known that a complex series of biochemical changes takes place after exposure to the stimulating agent which involves stimulusspecific rises in intracellular free calcium, changes in membrane potential due to ion movements, phospholipid hydrolysis, alterations in the levels of cyclic nucleotides, protein phosphorylation, intracellular translocation of enzymes and activation of GTP-binding proteins. These changes result in activation of a number of target functions of the phagocyte, dependent

N A D P H oxidase inhibitors

on the transduction pathways activated by the stimulus, such as chemotaxis, phagocytosis, degranulation, adhesin and respiratory burst (recently reviewed2'9).

1. Receptor effects. Many of the physiological stimuli of the phagocyte are mediated through specific cell surface receptors (Table 3). Although the existence of these receptors are known, only the chemotactic peptide (fMLP) receptor has been studied in any detail with regard to inhibitor action. The receptors exist in high and low affinity states which are regulated by the levels of guanine nucleotides. 96'97 The transmission of the signal from the ligand receptor complex also requires the involvement of guanine nucleotide binding proteins (G-proteins) which are sensitive to pertussis and cholera toxins. These processes are fully detailed by Snyderman, Jesaitis, and Casey. 98-j°° Clearly analogues of receptor ligands which prevent their binding will inhibit cellular responses. Such inhibitors have been described for the fMLP receptor, (e.g., t-boc-Phe-LeuPhe j°l) and for the PAF receptor (kaduresone, BN52021, L6521°2). As mentioned above pertussis toxin prevents the transduction process by virtue of its interaction with the G-protein and this has provided a useful means of distinguishing which stimuli involve G-proteins. It appears that of those stimuli studied in this way all those which have "conventional" cell surface receptors are sensitive to pertussis toxin (fMLP, LTB4, PAF, C5a or monomeric IgG) but stimuli such as Con A, PMA, A23187 or IgG aggregates are insensitive, suggesting they can bypass the G-protein step. 1o3-107 Cross-linking agents may also act by preventing correct interaction of receptor with G-protein. A second group of inhibitors may exert their action, at least in part, by preventing the expression of active receptors on the cell surface. Sulphydryl reagents may act by reaction with essential thiols of the receptor but there are also inhibitors which seem to reduce the number of receptors expressed on the cell surface through an unknown mechanism. This phenomenon has not been studied for many inhibitors but it may be signifT a b l e 3. H u m a n P M N R e c e p t o r s Stimulus

Reference

C5a (complement fragment) C3b (stimulates phagocytosis not O2~ production C3b~ fMLP Fc component of IgG Cell-derived chemotatic factor Leukotriene B4 (LTB4) Platelet activating factor (PAF) Platelet derived growth factor

87 88 89 90 91 92 93 94 95

83

icant that many of the cyclooxygenase inhibitors have this property in common. a) C o m p e t i t i v e inhibitors K a d s u r e n o n e ~°2 f M L P analogues ~°~ b) E x p r e s s i o n / b i n d i n g modifiers and potential modifiers (thiol reagents) Acrolein t°~ .Auranofin53.~2,~09 H3 Candida albicans hyphae inhibitory product ( C H I P ) H4 D i a m i d e ~5 Diclofenac HS.H6 Disulfiram 54 Gold sodium thiomalate 53"~ N7 Iodoacetate "3 .lndomethacinH~ ,~ H~ ~-~ M e c l o f e n a c ~z~ * NEM ' 1.5~,6(} 61.118 127 128 Olsalazine ~2~ Penicillamine ~, ,,, Phenylbutazone H~ ~3~, PiroxicamT, ~,H,~ ,3, Protease i n h i b i t o r s ~32'~3~ 2~ .QuercetinS~.56 3~,~,4 ~,~ Sulfasalazine '-''~ T h i o m a l a t e ~3 Triethyl phosphine gold ~ ""~'~7 c) Transduction *Pertussis toxin "'' .,s.,37

2. Phospholipid metabolism. After interaction with stimuli there are a number of modifications of lipid metabolism at the plasma membrane, primarily activation of phospholipase C,t38 ~40 phospholipase A2,121'141'142 and phospholipase D. 143-145 Phospholipase C causes the hydrolysis of phosphatidyl inositides releasing inositol 1,4,5 triphosphate and diacylglycerol which act as second messengers for Ca 2+ release and protein kinase activation. Phospholipase A2 causes the release of lysophospholipids and arachidonic acid which can be metabolized to leukotrienes and prostaglandins by enzymes of the lipoxygenase and cyclooxygenase pathways respectively. Phospholipase D breaks the terminal phosphodiester bond of glycerophospholipids to release phosphatidic acid and the appropriate base. The action of the phospholipases also has effects on the physical properties of the membrane which may in turn have direct effects on membrane associated enzymes. The importance of the activities of the phospholipases can be inferred from the stimulatory effects of exogenous phospholipase C, 146 diacylglycerol,147 fatty acids,148 and detergents; 149and the inhibitory effects of phospholipase inhibitors, particularly of phospholipase A2 (see below). There is increasing evidence that arachidonate (a product of phospholipase A2) and other long chain polyunsaturated fatty acids play a direct role in the activation of

84

A.R.

the oxidase. Much of this evidence comes from work using a cell-free activation system which requires NADPH, a membrane fraction, a cytosolic fraction and fatty acid (or SDS) which suggests that the fatty acid causes a selective conformational change in the oxidase which allows the functional interaction of the components. 15°-~59 The concentration of arachidonate required in these experimental systems is most unlikely to be achieved in vivo, although this does not preclude some kind of localized production of arachidonate. The almost universal inhibitory effect of phospholipase A2 antagonists is strongly suggestive of an essential role for arachidonate. Even stimulating agents such as naphthalene are inhibited by phospholipase A~ inhibitors with restoration of activity by the addition of exogenous arachidonate (Jones, O.T.G., personal communication). Several reports argue against this essential role for phospholipase A2 however, surprisingly, arachidonate stimulation of whole cells is inhibited by the phospholipase A2 inhibitor *p-bromophenacyl bromide, t22 Secondly, fluoride stimulated cells are not inhibited by the phospholipase inhibitor quinacrine (mepacrine) 7~ or show an enhanced response in the presence of a cocktail of inhibitors.~6° Evidence has also been presented to suggest release of arachidonate and production of superoxide are independent events. ~20 What role products of the lipoxygenase and cyclooxygenase pathway play in the activation process is not clear, but compounds which interfere with these pathways certainly have inhibitory effects on activation of superoxide generation. It is difficult to draw a very clear picture with regard to their effects due to their general lack of specificity; typically a CO inhibitor will have at least some lipoxygenase (LO) and phospholipase inhibitory activity, and frequently can be shown to inhibit several other potential intermediates. Inhibitors of phospholipid metabolism A A 8 6 1 ~5 . A s p i r i n ~~,~,8 ,L,,].,9 B e n z y d a m i n e "~ B i s c o c l a u r i n e a l k a l o i d s TM * p - B r o m o p h e n a c y l b r o m i d e m'~2° m L~ BW755Ct2L~22 * C h l o r o q u i n e ~6~ C o l c h i c i n e 58.~2 D i c l o f e n a c "S,'6 ETYA55,. 9,~_~,~ E s c u l e t i n 5s

G o l d s o d i u m t h i o m a l a t e ~'~c~'~ i b u p r o f e n ] . . HSa~7,H9 . i n d o m e t h a c i n m , . ~ , . 8 ,,-~ Meclofenac ~ Mefanamic acid m'm N e o m y c i n ~°~ N o r d i h y d r o g u a i a r e t i c a c i d ~'83's~ ~2° O l s a l a z i n e ~29 O x y p h e n b u t a z o n e "~

CRoss

P h e n i d o n e ' :(~ Phenothiazines; Chlorpromazine, 7~.1~3 ,~6.2~

,Trifluoperazin¢,4.434~4~.6~7~ P h e n y l b u t a z o n e ~~.~, Piroxicam'67 Primaquine m . Q u i n a c r i n e ~ 2 , 4 :~,7~7~,,2~, ,2_, S t e r o i d s ' ~2, ,~ S u l f a s a l a z i n e '2~ U60,257 ~

3. Protein kinase C. There is a good deal of evidence

which implicates protein kinase C in the activation of the respiratory burst which can be summarized as follows: 1. The receptor for the potent stimuli PMA and diacyglycerol is protein kinase C; 168'169 2. Activation is accompanied by the translocation of protein kinase C from the cytosol to the plasma membrane ~7° and the phosphorylation of a number of proteins, 17]-175 one of which is absent in the chronic granulomatous disease, a deficiency of NADPH oxi-

dase;156,159,172376 3. Addition of purified protein kinase C stimulates NADPH oxidase activity in isolated membranes ~77 or the cell-free system; ~78 4. Inhibitors of protein kinase C block the respiratory burst. 1.]2~.164.165,179-183 There are also several pieces of evidence which argue against the obligatory involvement of protein kinase C: 1. The respiratory burst induced by fMLP, OZ or A23187 is not inhibited by H - 7 ; 121'165'183'184 fMLP or C5a by C-I; 18° fMLP by polymyxin B; 179 o r retinal 164 (although a recent report suggests a crucial temperature dependence for fMLP inhibition by H-7 and C-1~85). In fact retinal activates at low doses. 164.186 2. Mezerein inhibits pKC but activates superoxide production; ~87 3. Activation by Con A, LTB4 and PAF occurs in the absence of pKC translocation;~88 4. Activation by arachidonate or SDS in a cell-free system does not require ATP. 189 Specific protein kinase C inhibitors have been important in showing that pathways exist which bypass pKC. Inhibitors such as W-7 which are dual calmodulindependent protein kinase and pKC inhibitors, prevent activation by all stimuli, but the specificity of all these protein kinase inhibitors has been called into question L2 and must be treated with caution. Siefert and Schachtele concluded that protein kinase C inhibitors exhibit cell type specificity, stimulus dependency and lack of

NADPH oxidase inhibitors

correlation with in vivo inhibition of pKC.I The situation is complicated by the presence of several isoforms of protein kinase C which may show different inhibitory sensitivities and cofactor requirements.~9° It is known that protein kinase C can undergo limited proteolysis on the plasma membrane to give a form which is fully active in the absence of Ca 2+ and phospholipid. ~9~ It is also known that a wide variety of proteinase inhibitors are inhibitory to the activation process and it is possible that they exert their effects through this mechanism, although many also have sulphydryl reactivity and membrane perturbing effects. 1 6 7 , 1 9 2 . 2 4 0 Protein kinase C inhibitors a) " S p e c i f i c " StaurosporinC 9s b) Moderate specificity C_1~80 * H_71.,22,t65.~s3,,84

Polymyxin B '5' Retinal ,~,~s6 Sphingoid ~8' :¢W_743,45.165 166A84

c) Nonspecific Ebselen 59 Hydroxychloroquine 7~-77 Phenothiazines; chlorpromazine,

,trifluoperazine~4,43

45.48,64,163 166

Protease inhibitors 132,~33.~67.t92,239,'-4° .Quercetin~4.56.83.134 ~36 ,Quinacrinet ,.-14.28.75.78.120 t22 VerapamiP 6:.'94

85

stimuli, such as protein kinase C, 2°3 but are not sufficient in themselves to activate the oxidase.2°4 Calcium fluxes do appear to have a permissive or priming role for certain stimuli z°5-2°7 and they may play a more important role in the activation of other cellular responses such as chemotaxisfl °7 Ca -'+ inhibitors CetiediF °~ *Chelators 4652.53 Doxycycline, tetracycline t~.2o9.,_~6 Polymyxin B ~~7~t8: ,Quercetin~.~6.~3.,34 ~3~ TMB_8 :~ VerapamiP '~

5. Membrane potential changes. An association between changes in potassium and sodium fluxes through the plasma membrane and the activation of the oxidase has been suspected for several years but the precise relationship is not clear. 9 The most striking support for this relationship is the absence of membrane potential changes in both autosomal and X-linked forms of CGD. 2~°'2t2 As described in section A7 it has recently been found that a major part of the membrane depolarization is more likely to be a result of the activity of the oxidase than to be a mechanism for its activation, but this does not exclude a permissive role for ionic events in the activation process. Possible modifiers of membrane potential

4. Calcium changes. A role for calcium changes was first postulated many years ago following the discovery that calcium ionophores such as A23187 are inducers of the respiratory burst. 195.196 It was later found that most stimuli cause a rise in intracellular free calcium which preceeds the onset of superoxide production. 197 Verapamil and TMB-8 which prevent these calcium changes inhibit the response to Y/VILP, 194'198 a s do depletion of cellular calcium with calcium chelat o r s . 46'52'53 In this respect it is pertinent to note that several pharmacological and antibacterial agents can chelate calcium in addition to their better known mechanisms of action, (for instance the inhibitory effects of tetracyclines can be reversed by the addition of calcium199). The rise in calcium is not an obligatory requirement for activation however, PMA stimulates the respiratory response in calcium depleted neutrophils and the ionophore ionomycin is effective in increasing intracellular calcium but does not elicit a respiratory burst. 2°°'2°~ In electropermeabilized cells a respiratory burst can be induced by fMLP in the absence of an increase in cytosolic calcium. 2°2 Present evidence suggests that rises in free calcium are involved in the generation of other signals for certain

+ ve Alkylamines 73 Biscoclaurine alkaloids TM .ChloroquineS~.7~ 7~ HydroxychloroquineT~ 7~ Other lysozomotropic weak bases 76

6. Calmodulin. The use of calmodulin antagonists has suggested a role for calmodulin regulated proteins in the activation of the superoxide generating system at a point after the generation of secondary messengers such as Ca 2+ and inositol p h o s p h a t e s . 45'164-166'1s°'184 The problem with these data is again one of specificity (Table 2). The most specific calmodulin antagonist, CI-922, does not inhibit PMA stimulation suggesting that protein kinase C either bypasses this calmodulin step or lies after it. 213'214 As it has been shown that rises in intracellular Ca 2÷ are not obligatory for all stimuli it seems that calmodulin cannot lie on the central route for activation. Calmodulin antagonists C1_922213.214 Penothiazines; chlorpromazine, ,trifluoperazine14.43_45.4s.e4.163 166 *H_T2H65 Polymyxin B 1"179'182

86

A.R. CRoss • Quercetin54.5~,s3.J34 ~36

~W_743.45,165,166A84

7. Membrane fusion and mobility. It is widely b e l i e v e d that one of the final processes in the activation of the oxidase is an association o f the c o m p o n e n t s at the p l a s m a m e m b r a n e , whether by association of granule proteins with the m e m b r a n e , or the association of a cytosolic c o m p o n e n t with c y t o c h r o m e b-245 as appears to occur in the cell free activation systems. 150,152,189.215.216 in support of this idea, cross-linking agents prevent activation of the oxidase without inhibiting p r e v i o u s l y activated e n z y m e . 2~v After cleavage of the cross-linker normal activation can be achieved. A g e n t s which have effects on m e m b r a n e stability or fusion could function by inhibition of these fusion processes. They may also inhibit m e m b r a n e associated e n z y m e s involved in the activation process, such as P 1A 2. Compounds with potential membrane effects + ve AIkylamines7~ Amphoteracin B2~ Anaesthetic s 14.64.21~-':l Bilirubin2-'~Biscoclaurine alkaloidsTM CTAB~3.~ ChlorhexidineTM Cross-linkers -'~7 Diazepam-'23 Doxycycline; tetracyclines ~9,-''~°,-'36 Ethanol224 Lysozomotropic weak bases7~ Phenothiazines; chlorpromazine43.s

tivity of the hexose m o n o p h o s p h a t e pathway and this can be inhibited by p h e n y l b u t a z o n e or glucose analogues such as 2 - d e o x y g l u c o s e . Certain c o m p o u n d s have been shown to interfere with the uptake of glucose by p h a g o c y t e s and this in turn restricts the supply of glucose for the HMP. Metabolic inhibitors MannosC 3~ Phenylbutazone' TM ,Quinacrine,2

14.28,7578,120 122

D. Unknown Mechanisms In addition to the inhibitors whose mechanism o f action is known or can be h y p o t h e s i z e d there are a number o f inhibitory substances which do not have obvious targets. Compounds 48/80 ~4 Coumarin~4 Disulfiram~4 Methotrexate"= Opiate agonists/antagonists 2'~ Organotin cpds. TM Propyl gallatC ~ Protease inhibitors ~33~,v,~_,,,,~9_,~o Spirogermanium' '~ Thenoyl trifluoracetone u Trithienyl butane dione~

mast cell degranulator inhibitor of plant alternative oxidasc

radical scavenger inhibitor of plant alternative oxidase non haem iron chelator

Zinc-~,~

,trifluoperazinC4.43-4~,48,64,~6~ ~6 Sphingoid ~8~

8. Deactivation. The process of deactivating the respiratory burst may involve an increase in cAMP, since agents which increase c A M P have inhibitory effects. Recent e v i d e n c e obtained in the cell-free activation system suggests there is a rapid continuous deactivation of the o x i d a s e c o m p l e x occurring in intact neutrophils. 225 The increase in c A M P may activate a protein kinase A which may inactivate the signalling p a t h w a y ( s ) , leading to a net rate o f deactivation. Deactivating agents Adenosine226,227 Adenyl cylase toxinz2s cAMP229 PDGF230 Theophylline z29

TYPES OF ASSAY AND PITFALLS There are three principal types of assay of the superoxide generating system o f leukocytes; measurement of substrate N A D P H or o x y g e n utilization and detection o f product superoxide. Each has advantages and d i s a d v a n t a g e s as described below:

A. Oxygen uptake The o x y g e n electrode is relatively insensitive requiring rather large quantities of material, but can be used for whole cell or subcellular assays. It has the advantage of being unaffected by diaphorase activity, radical scavengers or redox active c o m p o u n d s unless they are o f the type which regenerate o x y g e n from superoxide. Caution should be exercised when using thiol reagents as they can poison the electrode.

C. Metabolism

B. NADPH oxidation (absorbance at 340nm)

Since the s u p e r o x i d e generating system is dependent on N A D P H as a substrate, prevention of its formation will be inhibitory. The N A D P H is supplied by the ac-

Quite sensitive but suitable for subcellular preparations only and therefore not useful for inhibitors of activation processes. It is generally free from artifacts

NADPH oxidase inhibitors unless the test c o m p o u n d u n d e r g o e s c h a n g e s at the m e a s u r i n g w a v e l e n g t h .

absorption

C. Radical production

1. L u m i n o l e n h a n c e d c h e m i l u m i n e s c e n c e : Very s e n s i t i v e but d i f f i c u l t to q u a n t i f y . V e r y s u s c e p t i b l e to s c a v e n g i n g e f f e c t s , q u e n c h i n g and i n t e r f e r e n c e o f c o m p o u n d s w i t h p e r o x i d a s e s and or the d e g r a n u l a t i o n req u i r e d to r e l e a s e t h e m to p a r t i c i p a t e in the l u m i n o l reaction. 2. S O D s e n s i t i v e c y t o c h r o m e c or N B T r e d u c t i o n : S e n s i t i v e . S u s c e p t i b l e to s c a v e n g i n g and s u p e r o x i d e i n d e p e n d e n t c y t o c h r o m e c (or N B T ) r e d u c t i o n by thiol r e a g e n t s and r e d o x a c t i v e c o m p o u n d s . P o s s i b l e i n t e r f e r e n c e in the assay can be c h e c k e d with x a n t h i n e / x a n t h i n e o x i d a s e as s u p e r o x i d e source.

13.

14.

15.

16.

17. 18.

FINAL REMARKS

19. I w o u l d like to take this o p p o r t u n i t y to firstly apolo g i s e to t h o s e authors w h o s e w o r k I h a v e o v e r l o o k e d and s e c o n d l y to ask if t h e y , or any o t h e r i n v e s t i g a t o r s w h o h a v e data on i n h i b i t o r y e f f e c t s o f o t h e r c o m p o u n d s , w o u l d be w i l l i n g to m a k e the i n f o r m a t i o n a v a i l a b l e to me.

20.

21.

Acknowledgement--I am very grateful to the Arthritis and Rhu-

matism Council for financial support.

22.

REFERENCES

1. Seifert, R.; Schachtele, C. Studies with protein kinase inhibitors presently available cannot elucidate the role of protein kinase C in the activation of NADPH oxidase. Biochem. Biophys. Res. Comm. 152:585-592; 1988. 2. Rossi, F. The O_,--forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim. Biophys. Acta 853:65-89; 1986. 3. Marx, J. L. Oxygen free radicals linked to many diseases. Science 235:529-531 ; 1987. 4. Weiss, S. J. Oxygen, ischaemia and inflammation. Acta Physiol. Scand. 548:9-37; 1986. 5. Fox, H. B.; De Togni, P.; Babior, B. M. Phagocytes and carcinogenesis, lmmunol. Today 6:327-328; 1986. 6. Halliwell, B. Oxidants and human disease: some new concepts. FASEB J. 1:358-364; 1987. 7. Weiss, S. J. Tissue destruction by neutrophils. N. Engl. J. Med. 320:365-376; 1989. 8. Byczkowski, J. Z.; Gessner, T. Biological role of superoxide ion radical. Int. J. Biochem. 20:569-580; 1988. 9. Bellavite, P. The superoxide-forming enzymatic system of phagocytes. Free Radical Biol. Med. 4:225-261; 1988. 10. Babior, B. M.; Kipnes, R. S.; Curnutte, J. T. Biological defence mechanisms: the production of superoxide, a potential bactericidal agent. J. Clin. Invest. 52:267-271; 1973. 11. Yamashita, T. Effect of maleimide derivatives, sulfhydryl reagents, on stimulation of neutrophil superoxide anion generation with Con A. FEBS Lett. 164:267-271; 1983. 12. Bellavite, P.; Cross, A. R.; Serra, M. C.; Davoli, A.; Jones,

23.

24.

25. 26.

27. 28.

29. 30.

87

O. T. G.; Rossi, F. The cytochrome b and flavin content and properties of the superoxide forming NADPH oxidase solubilized from activated neutrophils. Biochim. Biophys. Acta 746:40-47; 1983. Cross, A. R.; Parkinson, J. E; Jones, O. T. G. The superoxide generating oxidase of leukocytes: NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations. Biochem. J. 223:337-344; 1984. Bellavite, P.; Serra, M. C.; Davoli, A.; Bannister, J. V.; Rossi, F. The NADPH oxidase of guinea-pig polymorphonuclear leucocytes: properties of the deoxycholate extracted enzyme. Mol. Cell. Biochem. 52:17-25; 1983. Wakeyama, H.; Takeshige, K.; Minikami, S. NADPH-dependent reduction of 2, 6, dichlorophenol indophenol by the phagocytic vesicles of pig polymorphonuclear leucocytes. Biochem. J. 210:577-581: 1983. Takayama, H.; Iwaki, S.; Tamoto, K.; Koyama, J. Inhibition of the 02 -generating NADPH oxidase of guinea-pig polymorphonuclear leukocytes by rabbit antibody to homologous liver NADPH-cytochrome c (P-450) reductase. Biochim. Biophys. Acta 799:151-157; 1984. Bellavite, P.; Jones, O. T. G.; Cross, A. R.; Papini, E.; Rossi, F. Composition of partially purified NADPH oxidase from pig neutrophils. Biochem. J. 223:639-648; 1984. lyanagi, T.; Mason, H. S. Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase. Biochemistry 12:2297-2308; 1973. Cross, A. R. The inhibitory effects of some iodonium compounds on the superoxide generating system of neutrophils and their failure to inhibit diaphorase activity. Biochem. Pharmacol. 36:489-493; 1987. Bellavite, P.; Della Bianca, V.; Serra, M. C.; Papini, E.; Rossi, F. NADPH oxidase of neutrophils forms superoxide anion but does not reduce cytochrome c and dichlorophenolindophenol. FEBS Lett. 170:157-161; 1984. Kojima, H.; Takahashi, K.; Sakane. S.; Koyama, J. Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes. J. Biochem. 102:1083-1088; 1987. Light, D. R.; Walsh, C.; O'Callaghan, A. M.; Goetzl, E. J.; Tauber, A. I. Characteristics of the cofactor requirements of the superoxide generating NADPH oxidase of human polymorphonuclear leukocytes. Biochemistry 20:1468-1476: 1981. Parkinson, J. F.; Gabig, T. G. Phagocyte NADPH oxidase. Studies with flavin analogues as active site probes in Triton X-100-solubilized preparations. J. Biol. Chem. 263:88598863; 1988. Cross, A. R.; Jones, O. T. G. The effect of the inhibitor diphenylene iodonium on the superoxide generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase. Biochem. J. 237:111-116; 1986. Hancock, J. T.; Jones, O. T. G. The inhibition by diphenylene iodonium and its analogues of superoxide generation by macrophages. Bioehem. J. 242:103-107; 1987. Ellis, J. A.; Mayer, S. J.; Jones, O. T. G. The effect of NADPH oxidase inhibitor diphenylene iodonium on aerobic and anaerobic microbicidal activities of human neutrophils. Biochem. J. 251:887-891; 1988. Hancock, J. T.; White, J. I.; Jones, O. T. G. Superoxide as the mediator of tumour cell killing. Eur. J. Clin. Invest. 18:A47; 1988. Tauber, A. l.; Simons, E. R. Dissociation of human neutrophil membrane depolarization, respiratory burst stimulation and phospholipid metabolism by quinacrine. FEBS Lett. 156:161164, 1985. Segal, A. W. Absence of both cytochrome b-,.45 subunits from neutrophils in X-linked chronic granulomatous disease. Nature 326:88-91; 1987. Parkos, C. A.; Allen, R. A.; Cochrane, C. G.; Jesaitis, A. J. Purified cytochrome b from human granulocyte plasma membrane is composed of two polypeptides with relative molecular

88

31.

32.

33.

34.

35.

36.

37.

38.

39. 40.

41.

42.

43.

44.

45.

46.

47.

48. 49.

A . R . CRoss weights of 91,000 and 22,000. J. Clin. Invest. 80:732-742; 1987. Parkos, C. A.; Allan, R. A.; Cochrane, C. G.; Jesaitis, A. J. The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes. Biochim. Biophys. Acta. 932:71-83; 1988. Royer-Pokora, B.; Kunkel, L. M.; Monaco, A. P.; Golf, S. C.; Newburger, P. E.; Baehner, R. L.; Cole, F. S.; Curnutte, J. T.; Orkin, S. H. Cloning the gene for an inherited human disorder, chronic granulomatous disease, on the basis of its chromosomal localization. Nature 322:32-38; 1986. Parkos, C. A.; Dinauer, M. C.; Walker, L. E.; Allen, R. A.; Jesaitis, A. J.; Orkin, S. H. Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc. Natl. Acad. Sci. (USA) 85:3319-3323; 1988. Gabig, T. G.; Bearman, S. J.; Babior, B. M. Effects of oxygen tension and pH on the respiratory burst of human neutrophils. Blood 53:1133-1139; 1979. Kakinuma, K.; Kaneda, M. Apparent Kmof leukocyte O2 and H20., forming enzyme for oxygen. In: Rossi, F.; Patriarca, P., eds. Biochemistry and function of phagocytes. New York: Plenum Press; 1982:351-360. Edwards, S. E.; Haliett, M. 13.; Lloyd, D,; Campbell, A. K. Decrease in apparent Km for oxygen after stimulation of respiration of rat polymorphonuclear leukocytes. FEBS Lett. 161:60-64; 1983. Morel, F.; Doussiere, J.; Stasia, M. J.; Vignais, P. V. The respiratory burst of human neutrophils. Role of the b type cytochrome and coenzyme specificity. Eur. J. Biochem. 152:669-679; 1985. Cross, A. R.; Higson, F. K.; Jones, O. T. G.; Harper, A. M.; Segal, A. W. The enzymic reduction and kinetics of oxidation of cytochrome b-24~of neutrophils. Biochem. J. 204:479-485; 1982. Testa, B.; Jenner, P. Inhibitors of cytochrome P-450 and their mechanisms of action. Drug Metab. Rev. 12: I- 117; 1981. Collman, J. P.; Brauman, J. F.; Halbert, T. R.; Suslick, K. S. Nature of 02 and CO binding to metalloporphyrins and haem proteins. Proc. Natl. Acad. Sci. (USA) 73:3333-3337; 1976. lizuka, T.; Kanegasaki, S.; Makino, R.; Tanaka, T.; Ishimura, Y. Pyridine and imidazole reversibly inhibit the respiratory burst in porcine and human neutrophils: evidence for the involvement of cytochrome b55~in the reaction. Biochem. Biophys. Res. Commun. 130:621-626; 1985. Gabig, T. G.; Babior, B. M. The O2--forming oxidase responsible for the respiratory burst in human neutrophils: Properties of the deoxycholate extracted enzyme. J. Biol. Chem. 254:9070-9074; 1979. Jones, H. P.; Ghal, G.; Petrone, W. F.; McCord, J. M. Calmodulin-dependent stimulation of the NADPH oxidase of human neutrophils. Biochim. Biophys. Acta 714:152-156; 1982. Miller, K. M.; Dearborn, D. E.; Sorenson, R. U. In vitro effect of synthetic pyocyanine on neutrophil superoxide production. Infection & Immunity 55:559-563; 1987. Takeshige, K.; Minakami, S. Involvement of calmodulin in the respiratory burst of leukocytes. Biochim. Biophys. Res. Commun. 99:484-490; 1981. Green, T. R.; Wu, D. E.; Wirtz, M. K. The 02 -generating oxidoreductase of human neutrophils: Evidence of an obligatory requirement for Ca 2+ and Mg2+ for catalytic activity. Biochem. Biophys. Res. Commun. 110:973-978; 1983. Suzuki, H.; Pabst, M. J.; Johnston, R. B. Enhancement by Ca `'+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes. J. Biol. Chem. 260:3635-3639; 1985. Parkinson, J. F. NADPH-oxidase; characterization of the solubilized enzyme from pig neutrophils. University of Bristol; 1985. Thesis. Babior, B. M.; Kuver, R. ; Curnutte, J. T. Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils. J. Biol. Chem. 263:1713-1718; 1988.

50. Cross, A. R.; Jones, O. T. G.; Harper, A. M.; Segal, A. W. Oxidation-reduction properties of the cytochrome b found in the plasma membrane fraction of human neutrophils: A possible oxidase in the respiratory burst. Biochem. J. 194:599606; 1981. 51. Kakinuma, K.; Kaneda, M.; Chiba, T.; Ohnishi, T. Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. J. Biol. Chem. 261:9426-9432; 1986. 52. Parkinson, J. F.; Cross, A. R.; unpublished data. 53. Cohen, H. J.; Chovaniec, M. E. Superoxide production by digitonin-stimulated guinea-pig granulocytes. The effect of NEM, divalent cations and glycolytic and mitochondrial inhibitors on the activation of the superoxide generating system. J. Clin. Invest. 61:1088-1096; 1978. 54. Crawford, D. R.; Schneider, D. L. Ubiquinone content and respiratory burst activity of latex filled phagolysozomes isolated from human neutrophils and evidence for the probable involvement of a third granule. J. Biol. Chem. 259:5363 5367; 1983. 55. Ozaki, Y.; Ohashi, T.; Niwa, Y. A comparative study on the effects of the lipoxygenase pathway on neutrophil function. Biochem. Pharmacol. 35:3481-3488; 1986. 56. Tauber, A. 1. Flavonoid inhibition of human neutrophil NADPH-oxidase. Biochem. Pharmacol. 33:1367-1369; 1980. 57. Massey, V.; Palmer, G.; Williams, C. H.; Swoboda, B. E. P.; Sands, R. H. In: Slater, E. C. ed. Flavins andflavoproteins. Amsterdam: Elsevier; 1966:133. 58. Min~a, J. O.; Williams, M. D. Interactions of anti-rheumatic drugs with the superoxide generating system of activated human polymorphonuclear leukocytes. J. Rheumatol. 13:498504; 1986. 59. Cotgreave, 1. A.; Duddy, S. K.; Kass, G. E. N.; Thompson, D.; Moldeus, P. Studies on the anti-inflammatory activity of ebselen. Ebselen interferes with granulocyte oxidative burst by dual inhibition of NADPH oxidase and protein kinase C? Biochem. Pharmacol. 38:649-656; 1989. 60. Higson, F. K. The superoxide generating system of animal blood neutrophils. University of Bristol; 1983. Thesis. 61. Zimmerli, W.; Lew, P. D.; Cohen, H. J.; Waldvogel, F. A. Comparative superoxide-generating system of granulocytes from blood and peritoneal exudates. Infection & lmmunitv 46:625-630; 1984. 62. Cross, A. R.; Jones, O. T. G.; unpublished data. 63. Yamaguchi, T.; Kakinuma, K. Inhibitory effect of Cibacron Blue F3GA on the 02 generating enzyme of guinea-pig polymorphonuclear leukocytes. Biochem. Biophys. Res. Commun. 104:200-205; 1982. 64. Cohen, H. J.; Chovaniec, M. E.; Ellis, S. E. Chlorpromazine inhibition of granulocyte superoxide production. Blood 56 :23-29; 1980. 65. Doussiere, J.; Laporte, F.; Vignais, P. V. Photolabelling of a 02 generating protein in bovine polymorphonuclear neutrophils by an arylazido NADP + analogue. Biochim. Biophys. Acta 139:85-93; 1986. 66. Umei, T.; Takeshige, T.; Minakami, S. NADPH binding component of neutrophil superoxide generating oxidase. J. Biol. Chem. 261:5229-5232; 1986. 67. Umei, T.; Takeshige, T.; Minakami, S. NADPH-binding component of the superoxide-generating oxidase in unstimulated neutrophils and the neutrophils from the patients with chronic granulomatous disease. Biochem. J. 243:467-472; 1987. 68. Smith, R. M.; Curnutte, J. T.; Babior, B. M. Affinity labelling of the cytosolic and membrane components of the respiratory burst oxidase by the 2',3'-dialdehyde derivative of NADPH. Evidence for a cytosolic localization of the nucleotide binding site in the resting cell. J. Biol. Chem. 264:1958-1962; 1989. 69. Takasugi, S.; Ishida, K.; Takeshige, K.; Minakami, S. Effect of 2',Y-dialdehyde NADPH on activation of superoxide-producing NADPH oxidase in a cell-free system. J. Biochem. 105:155-157; 1989. 70. Henderson, L. M.; Chappell, J. B.; Jones, O. T. G. The superoxide-generating NADPH oxidase of human neutrophils is

NADPH oxidase inhibitors

71. 72.

73.

74.

75.

76. 77.

78. 79.

80.

81. 82.

83. 84. 85. 86.

87. 88. 89. 90.

electrogenic and associated with a H ÷ channel. Biochem. J. 246:325-329; 1987. Henderson, L. M.; Chappell, J. B.; Jones, O. T. G. Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Biochem. J. 251:563-567; 1988. Henderson, L. M.; Chappell, J. B.; Jones, O. T. G. Superoxide generation by the electrogenic NADPH oxidase of human neutrophils is limited by the movement of a compensating charge. Biochem. J. 225:285-290; 1988. Miyahara, M.; Watanabe, S.; Okimasu, E.; Utsumi, K. Charge-dependent regulation of NADPH oxidase activity in guinea-pig polymorphonuciear leukocytes. Biochem. Biophys. Res. Commun. 929:253-262; 1987. Matsuno, T.; Orita, K.; Sato, E.; Nobori, K.; Inoue, B.; Utsumi, K. Inhibition of metabolic response of polymorphonuclear leukocyte by biscoclaurine alkaloids. Biochem. Pharmacol. 36:1613-1616; 1987. Hurst, N. P.; French, J. K.; Bell, A. L.; Nuki, G.; O'Donnell, M. L.; Betts, W. H.; Cleland, L. G. Differential effects of mepacrine, chloroquine and hydroxychloroquine on superoxide anion generation, phospholipid methylation and arachidonic acid release by human blood monocytes. Biochem. Pharmacol. 35:3083-3089; 1986. Styrt, B.; Klempner, M. S. Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases. Blood 67:334342; 1986. Hurst, N. P.; French, J. K.; Gorjatschko, L.; Betts, W. H. Chloroquine and hydroxychloroquine inhibit multiple sites in metabolic pathways leading to neutrophil superoxide release. J. Rheumatol. 15:23-27; 1988. Miyachi, Y.; Yoshioka, A.; Imamura, S.; Niwa, Y. Antioxidant action of antimalarials. Annals Rheum. Dis 45:244-248; 1986. Goultschin, J.; Levy, H. Inhibition of superoxide generation by human polymorphonuclear leukocytes with chlorhexidine: its possible relationship to peridontal disease. J. Periodontol. 57:422-425; 1986. Gabig, T. G. ; Lefker, B. A. Catalytic properties of the resolved flavoprotein and cytochrome b components of the NADPH dependent 02- generating oxidase from human neutrophils. Biochem. Biophys. Res. Commun. 118:430-436; 1984. Murakami, M.; Nakamura, M.; Minakami, S. 2,6-Dichlorophenolindophenol-reducing activity of phagocytosis-associated NADPH oxidase. J. Biochem. 100:1493-1497; 1986. Murakami, M.; Nakamura, M.; Minakami, S. NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone- 1 under anaerobic conditions. Biochem. J. 237:541 - 545; 1986. Crawford, D. R.; Schneider, D. L. Identification of UQs0 in human neutrophils and its role in microbicidal events. J. Biol. Chem. 257:6662-6668; 1982. Rossi, F~; Della Bianca, V.; Bellavite P. Inhibition of the respiratory burst and of phagocytosis by nordihydroguaiaretic acid in neutrophils. FEBS Lett. 127:183-187; 1981. Beswick, P. H.; Slater, T. F. The "metabolic burst" in bovine blood polymorphonuclear leucocytes when stimulated by phagocytosis. Biochem. Soc. Trans. 5:1299-1301; 1977. Butterick, C. J.; Baehner, R. L.; Boxer, L. A.; Jersild, R. A. Vitamin E - - a selective inhibitor of the NADPH oxidoreductase enzyme system in human granulocytes. Am. J. Pathol. 112:287-293; 1983. Chenoweth, D. E.; Hugli, T. E. Demonstration of a specific C5a receptor on intact human polymorphonuclear phagocytes. Proc. Natl. Acad. Sci. (USA) 75:3943-3947; 1978. Wright, S. D.; Silverstein, S. C. Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes. J. Exp. Med. 158:2016-2023; 1983. Riches, D. W. H.; Channon, J. Y.; Leslie, C. C.; Henson, P. M. Receptor mediated signal transduction in mononuclear phagocytes. Prog. Allergy 42:65-122; 1988. Williams, L. T.; Snyderman, R.; Pike, M. C.; Lefkowitz, R. J. Specific receptor sites for chemotactic peptides on human

91. 92. 93. 94.

95.

96.

97. 98. 99. 100.

101. 102.

103. 104.

105.

106.

107. 108.

109. 110.

89

polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. (USA) 74:1204-1208; 1977. Lobuglio, A. F.; Cotran, R. S.; Jandl, J. H. Red cells coated with immunoglobulin G: Binding and sphering by mononuclear cells in man. Science 158:1582-1585; 1967. Spilberg, I.; Mehta, J. Specific neutrophil receptor for cellderived growth factor. J. Clin. Invest 63:85-88; 1979. Goldman, D. W.; Goetzl, E. J. Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes. J. lmmunol. 129:1600-1604; 1982. Goetzl, E. J.; Derian, C. K.; Tauber, A. I.; Valone, F. H. Novel effects of l-o-hexadecyl-2-acyl-sn-glycero-3-phosphorylcholine mediators on human leukocyte function: delineation of the specific roles of the acyl substituents. Biochem. Biophys. Res. Commun. 94:881-888; 1980. Tzeng, D. Y.; Deuel, T. F.; Huang, J. S.; Senior, R. M.; Boxer, L. A.; Baehner, R. L. Platelet-derived growth factor promotes polymorphonuclear leukocyte activation. Blood 64:11231128; 1984. Koo, C.; Lefkowitz, R. J.; Snyderman, R. The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states. Fed. Proc. 41:27; 1982. Sklar, L. A.; Mueller, H.; Omann, G.; Oades, Z. Three states for the formyl peptide receptor on intact cells. J. Biol. Chem. 264:8483-8486; 1989. Snyderman, R.; Pike, M. C. Tranductional mechanisms of chemoattractant receptors on leukocytes. Curr. Topics lmmunobiol. 14:1-28; 1984. Casey, P. J.; Gilman, A. G. G protein involvement in receptoreffector coupling. J. Biol. Chem. 263:2577-2580; 1988. Jesaitis, A. J.; Allen, R. A. Activation of the neutrophil respiratory burst by chemoattractants: regulation of the N-formyl peptide receptor in the plasma membrane. J. Bioenerg. Biomemb. 20:679-707; 1988. Becket, E. L. A multifunctional receptor on the neutrophil for synthetic chemotactic oligopeptides. J. Reticuloendothel. Soc. 26:701-709; 1979. Vercellotti, G. M.; Yin, H. Q.; Gustafson, K. S.; Nelson, R. D.; Jacobs, H. S. Platelet-activating factor primes neutrophil responses to agonists: Role in promoting neutrophil-mediated endothelial damage. Blood 71:1100-1107; 1988. Blackburn, W. D.; Heck, L. W. Neutrophil activation by surface bound IgG: Pertussis toxin insensitive activation. Biochem. Biophys. Res. Commun. 152:136-142; 1988. Molski, R. F. P.; Naccache, P. H.; Marsh, M. L.; Kermode, J.; Becker, E. L.; Sha'afi, R. I. Pertussis toxin inhibits the rise in the intraceliular concentration of free calcium that is induced by chemotactic factors in rabbit neutrophils: possible role of the "G-proteins" in calcium mobilization. Biochem. Biophys. Res. Commun. 124:644-650; 1984. Fulop, T.; Varga, Z.; Nagy, J. T.; Foris, G. Studies on opsonized zymozan, fMLP, carbachol, PMA and A23187 stimulated respiratory burst in humans PMNLs. Biochem. International 17:419-426; 1988. Shirato, M.; Takahashi, K.; Nagawasawa, S.; Koyama, J. Different sensitivities of the responses of human neutrophils stimulated with immune complex and C5a anaphylatoxin to pertussis toxin. FEBS Lett. 234:231-234; 1988. Holian, A. Leukotriene B4 stimulation of phosphatidylinositol turnover in macrophages and inhibition by pertussis toxin. FEBS Lett. 201:15-19; 1986. Witz, G.; Lawrie, N. J.; Amuroso, M. A.; Goldstein, B. D. Inhibition by reactive aldehydes of superoxide production from stimulated polmorphonuclear leukocytes and pulmonary alveolar macrophages. Biochem. Pharmacol. 36:721-726; 1987. Sung, C-P.; Mirabelli, C. K.; Badger, A. M. Effect of gold compounds on PMA activated superoxide production by mouse peritoneal macrophages. J. Rheumatol. 11:153-157; 1984. Hafstrom, I.; Seligmann, B. E.; Friedman, M. M.; Gallin, J. I. Auranofin affects early events in human polymorphonuclear

90

A.R. CROSS neutrophil activation by receptor mediated stimuli. J. lmmu1984. Neal, T. M.; Vissers, M. C. M.; Winterbourne, C. C. Inhibition by nonsteroidal anti-inflammatory drugs of superoxide production and granule release by polymorphonuclear leukocytes stimulated with immune complexes or fMLP. Biochem. Pharmacol. 36:2511-2517; 1987. Coates, T. D.; Wolach, B.; Tzeng, D. Y.; Higgins, C.: Baehner, R. L.; Boxer, L. A. The mechanism of action of the antiinflammatory agents dexametbasone and auranofin in human polymorphonuclear leukocytes. Blood 62:1070-1077; 1987. Mirabelli, C. K.; Sung, C-P.; Picker, D. H.; Barnard, C.: Hydes, P.; Badger, A. M. Effect of metal containing compounds on superoxide release from phorbol myristate acetate stimulated murine peritoneal macrophages: Inhibition by auranofin and spirogermanium. J. Rheumatol. 15:1064-1069: 1988. Smail, E. H.; Melnick, D. A.; Ruggeri, R.; Diamond, R. D. A novel natural inhibitor from Candida albicans hyphae causing dissociation of the neutrophil respiratory burst response to chemotactic peptides from other post-activation events. J. lmmunol. 140:3893-3899; 1988. Oyanagui, Y. Inhibition of superoxide anion production in nonstimulated guinea-pig peritoneal exudate cells by anti-inflammatory drugs. Biochem. Pharmacol. 27:777 782: 1978. Perianin, A.: Gougerot-Pocidalo, M-A.; Giroud, J-P.: Hakim, J. Diclofenac binding to human polymorpbonuclear neutrophils: Effect on respiratory burst and N-formylated pepetide binding. Biochem. Pharmacol. 36:2609-2615; 1987. Davis, P.; Johnston, C.; Miller, C. L.; Wong, K. Effects of gold compounds on the function of phagocytic cells. II. Inhibition of superoxide radical generation by tripeptide-activated polymorphonuclear leukocytes. Arthritis Rheumatism. 26:82-86: 1983. Simcbovitz, L.; Mehta, J.; Spilberg, 1. Chemotactic factor induced generation of superoxide radicals by human neutrophils. Effect of metabolic inhibitors and anti-inflammatory drugs. Arthritis Rheumatism 22:755 763; 1979. Kaplan, H. B.; Edelson, H. S.; Korchak, H. M.; Given, W. P. ; Abramson, S.; Weissmann, G. Effects of non-steroidal antiinflammatory agents on human neutrophil functions in vitro and in vivo. Biochem. Pharmacol 33:371 378: 1984. Tsunawaki, S.; Nathan, C. F. Release of aracbidonate and reduction of oxygen: independent metabolic bursts of the mouse peritoneal macrophage. J. Biol. Chem. 261:1156311570; 1986. Maridonneau-Parini, I.; Tringale, S. M.; Tauber. A. I. Identification of distinct activation pathways of the human neutrophil NADPH-oxidase. J. lmmunol. 137:2925 2929; 1986. Maridonneau-Parini, I; Tauber, A. I. Activation of NADPHoxidase by arachidonate acid involves phospholipase A_, in human neutrophils but not in the cell free system. Biochem. Biophys. Res. Commun. 138:1099- I 105; 1986. Perianin, A.; Gaudry, M.; Marquetty, C.; Giroud, J-P.; Hakim, J. Protective effect of indomethacin against chemotactic deactivation of human neutrophils induced by formylated peptide. Biochem. Pharmacol. 37:1693-1698; 1988. Dale, M. M.; Penfield, A. Superoxide generation by either Ioleoyl-2-acetylglycerol or A23187 in human neutrophils is enhanced by indomethacin. FEBS Lett. 185:213-217; 1985. Smolen, J. E.; Weissmann, G. Effects of indomethacin, 5:8:11:14 eicosatetrayenoic acid and p-bromophenacyl bromide on lysozomal enzyme release and superoxide generation by human polymorphonuclear leukocytes. Biochem. Pharmacol. 29:533-538; 1980. Perez, H. D.; Elfman, E; Marder, S. Meclofenac sodium monohydrate inhibits chemotactic factor-induced human polymorphonuclear phagocyte function. A possible explanation for its anti-inflammatory effect. Arthritis Rheum. 30:1023 1031; 1987. Yamashita, T.; Someya, A.; Tsuzawa-Kido, Y. Response of superoxide anion production by guinea-pig eosinophils to varnol. 132:2007-2014;

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124. 125.

126.

127.

ious soluble stimuli: Comparison to neutrophils. Eur. Y. Biochem. 145:71-76; 1985.

128. Newberger, P. E.: Chovaniec, M. E.; Cohen, H. J. Activity and activation of the granulocyte superoxide-generating system. Blood 55:85-92; 1980. 129. Neal, T. M.; Winterbourne, C. C.: Vissers, M. C. M. Inhibition of neutrophil degranulation and superoxide production by sulfasalazine. Biochem. Pharmacol. 36:2765-2768; 1987. 130. Strauss, R. R. : Paul, B. ; Sbarra, A. J. Effect of phenylbutazone on phagocytosis and intracellutar killing by guinea-pig polymorphonuclear phagocytes. J. Bact. 96:1982-1990; 1968. 131. Biemond, P.; Swaak. A. J. G.;Penders. J. M. A.; Beindorf, C. M.: Koster, J. F. Superoxide production by polymorphonuclear leukocytes in rheumatoid arthritis and ostcoarthritis: in vivo inhibition by the antirheumatic drug piroxicam due to interference with the activation of the NADPH-oxidasc. Annals Rheum. Dis. 45:249 255: 1986. 132. Goldstein, B. D.; Witz, G.: Amuruso, M.; Troll, W. Protease inhibitors antagonize the activation of polymorphonuclear leukocyte oxygen consumption. Biochem. Biophys. Res. Commun. 88:854 860; 1979. 133. Suter. S.: Lew, P. D.: Waldwogel, W. A. Effect of the synthetic inhibitor TPCK on chemotactic peptide receptor activation and superoxide production in human neutrophils. Pediatric Res. 20:848-852: 1986. 134. Long, G. D.: De Chatelet, L.; O'Flaherty, J. T.; McCall, C. E. : Bass. D. A.; Shirley, P. S.; Parce, J. W. Effects of quercetin on magnesium-dependent adenosine triphosphate and the metabolism of human polymorphonuclear leukocytes. Blood 57:561 566: 1981. 135. Pagonis, C.: Tauber, A. I.; Pavlotsky, N.; Simons, E. R. Flavonoid impairment of neutrophil response. Biochem. Pharmacol. 35:237-246; 1986. 136. Blackburn, W. D.; Heck, L. W.; Wallace, R. W. The bioftavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins. Biochem. Biophys. Res. Commun. 144:1229-1236: 1987. 137. Lad, P. M.;Olson, C.V.;Grewat, l.S. Role of pertussis toxin substrate in the control of lectin-induced cap formation in human neutrophils. Biochem. J. 238:29 36; 1986. 138. Volpi, M.; Yassin, R.; Naccache, P. H.; Sha'afi, R. I. Chemotactic factor causes rapid decreases in phosphotidylinositol 4,5.-biphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils. Biochem. Biophys. Res. Commun. 112:957 964: 1983. 139. Besterman, J. F.; Duronio, V.; Cuatrecasas, P. Rapid formation of diacylglycerol from phosphatidylcholine: A pathway for generation of a second messenger. Proc. Natl. Acad. Sei. (USA) 83:6785-6789: 1986. 140. Agwu, D. E.; McPhail, L. C.; Daniel, L. W.; Bass, D. A.; McCall, C. E. Activation of phospholipase C in human neutrophils (PMN) by phorbol myristate acetate. Fed. Proe. 45:4049 abstr. (1986). 141. Walsh, C. E.; Waite, M. M.; Thomas, M. J,; DeChatelet, L. R. Release and metabolism of aracbidonic acid in human neutrophils. J. Biol. Chem. 256:7228-7234; 1981. 142. Alonso, F.; Henson, P. M.; Leslie, C. C. A cytosolic phospholipase in human neutrophils that hydrolyses arachidonoylcontaining phosphatidylcholine. Biochim. Biophys. Acta 878:273-280; 1986. 143. Cockroft, S.; Barrowman, M. M.; Gomperts, B. D. Breakdown and synthesis of polyphosphoinositides in fMetLeuPhestimulated neutrophils. FEBS Lett. 181:259-263; 1985. 144. Balsinde, J.; Diez, E.; Mollinedo, F. Phosphatidylinositolspecific phospholipase D: a pathway for generation of a second messenger. Biochem. Biophys. Res. Commun. 154:502-508; 1988. 145. Pal, J-K.; Siegel, M. I.; Egan, R. W.; Billah, M. M. Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. J. Biol. Chem. 263:12472-12477; 1988.

NADPH oxidase inhibitors 146. Patriaca, P.; Zatti, M.; Cramer, R.; Rossi, F. Stimulation of the respiration of polymorphonuclear leukocytes by phospholipase C. Life Sci. 9:841-849; 1970. 147. Fujita, I.; Irita, K.; Takeshige, K.; Minikami, S. Diacylglycerol, 1-oleoyl-2-acetyl-glycerol, stimulates superoxide generation from human neutrophils. Bioehem. Biophys. Res. Commun. 120:318-324; 1984. 148. Badwey, J. A.; Curnutte, J. T.; Robinson, J. M.; Berde, C. B.; Karnovsky, M. J.; Karnovsky, M. L. Effects of free fatty acids on release of superoxide and on shape change by human neutrophils,. J. Biol. Chem. 259:7870-7877; 1984. 149. Graham, R. C.; Karnovsky, M. J.; Shafer, A. W.; Glass, E. A.; Karnovsky, M. L. Metabolic and morphological observations on the effect of surface active agents on leukocytes. J. Cell Biol. 32:629-647; 1967. 150. Bromberg, Y.; Pick, E. Unsaturated fatty acids stimulate NADPH-dependent superoxide production by a cell-free system derived from macrophages. Cell lmmunol. 88:213-221 ; 1984. 151. Heyneman, R. A.; Vercauteren, R. E. Activation of an NADPH oxidase from horse polymorphonucelar leukocytes in a cell-free system. J. Leuk. Biol. 36:751-759; 1984. 152. McPhail, L. C.; Clayton, C. C.; Snyderman, R. Evidence that activation of human neutrophil NADPH oxidase involves association of a cytosolic factor with membrane components. Clin. Res. 32:315A; 1984. 153. Curnutte, J. T.; Badwey, J. A.; Robinson, J. M.; Karnovsky, M. J.; Karnovsky, M. L. Studies of the mechanism of superoxide release from human neutrophils stimulated with arachidonate. J. Biol. Chem. 259:11851-11857; 1984. 154. Bromberg, Y.; Pick, E. Activation of NADPH superoxide production in a cell-free system by SDS. J. Biol. Chem. 260:13539-13545; 1985. 155. Curnutte, J. T.; Scott, P. J.; Mayo, L. A. Cytosolic components of the respiratory burst oxidase: resolution of four cytosolic components, two of which are missing in complimenting types of chronic granulomatous disease. Proc. Natl. Acad. Sci. (USA) 86:825-829; 1989. 156. Curnutte, J. T.; Berkow, R. L.; Roberts, R. L.; Shurin, S. B.; Scott, P. J. Chronic granulomatous disease due to a defect in the cytosolic factor required for nicotinamide adenine dinucleotide phosphate oxidase activation. J. Clin. Invest. 81:606610; 1988. 157. Nunoi, H.; Rotrosen, D.; Gallin, J. I.; Malech, H. L. Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosolic factors. Science 242:1298-1301 ; 1988. 158. Volpp, B. D.; Nauseef, W. M.; Clark, R. A. Two cytosolic components absent in chronic granulomatous disease. Science 242:1295-1297; 1988. 159. Caldwell, S. E.; McCall, C. E.; Hendiricks, C. L.; Leone, P+ A.; Bass, D. A.; McPhail, L. C. Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease. J. Clin. Invest. 81:1485-1496; 1988, 160. Muid, R. E.; Twomey, B.; Dale, M. M. The effect of inhibition of both diacylglycerol metabolism and phospholipase A_~ activity on superoxide generation by human neutrophils. FEBS Lett. 234:235-240; 1988. 161. Chang, J.; Musser, J. H.; McGregor, H. Phospholipase A~: function and pharmacological regulation. Biochem. Pharmacol. 36:2429-2436; 1987. 162. Malawista, S. E.; Bodel, P. T. Dissociation by colchicine of phagocytosis from increased oxygen consumption in human leukocytes. J. Clin. Invest. 46:786-796; 1967. 163. Ochs, P. L.; Reed, P. W. Inhibition of the neutrophil oxidative burst and degranulation by phenothiazines. Biochem. Biophys. Res. Commun. 102:958-962; 1981. 164+ Cooke, E.; Hallett, M. B. The role of C-kinase in the physiological activation of the neutrophil oxidase: Evidence from using pharmacological manipulation of C-kinase activity in whole cells. Biochem. J. 232:323-327; 1985. 165. Wright, C. D.; Hoffman, M. D. Comparison of the roles of

166.

167.

168.

169.

170.

171.

172.

173.

174.

175.

176.

177.

178.

179.

180.

181.

91

calmodulin and protein kinase C in activation of the human neutrophil respiratory burst. Biochem. Biophys. Res. Commun. 142:53-62; 1987. AIobiadi, T.; Naccache, P. H.; Sha'afi, R. 1. Calmodulin antagonists modulate rabbit neutrophil degranulation, aggregation and stimulated oxygen consumption. Biochim. Biophys. Acta 675:316-321; 1981. Rao, K. M.; Castronova, V. Phenylmethylsulphonyl fluoride inhibits chemotactic peptide-induced actin polymerization and oxidative burst activity in human neutrophils by an effect unrelated to its anti-proteinase activity. Biochim. Biophys. Acta 969:131-138; 1988. Castagna, M.; Takai, Y.; Kaibuchi, K.; Sano, K.; Kikkawa, U.; Nishizuka, Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumour promoting phorbot esters. J. Biol. Chem. 257:7847-7851; 1982. Neidel, J. E.; Kuhn, L. J.; Vanderbark, G. R. Phorbol diester receptor copurifies with protein kinase C. Proc. Natl. Acad. Sci. (USA)80:36-40; 1983. Wolfson+ M.; McPhail, L. C.; Nasrallah, V. N.; Snyderman, R. Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: Potential role in the activation of the respiratory burst enzyme. J. lmmunol. 135:2057-2062; 1985. Helfman, D. M+; Appelbaum, B. D.; Vogler, W. R.; Kuo, J. F. Phospholipid-sensitive Ca-'+-dependent protein kinase and its substrates in human neutrophils. Biochem. Biophys. Res. Comm. 111:847-853; 1983. Heyworth, P. G.; Segal, A. W. Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils. Biochem. J. 239:723-731; 1986. Kramer, I. M.; Verhoeven, A. J.; van der Bend, R+ L.; Weening, R. S.; Roos, D. Purified protein kinase C phosphorylates a 47kD protein in control cytoplasts but not in neutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease. J. Biol. Chem. 263:2352-2357; 1988. Okamura, N.; Malawista, S. E.; Roberts, R, L.; Rosen, H.; Ochs, H. D.; Babior, B. M.; Curnutte, J. T. Phosphorylation of the oxidase-related 48kD phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease. Blood 72:811-816; 1988. Okamura, N.; Curnutte, J. T.; Roberts, R. L.; Babior, B. M. Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48kD proteins in two forms of chronic granulomatous disease. J. Biol. Chem. 263:6777-6782; 1988. Segal, A. W.; Heyworth, P. G.; Cockcroft, S.; Barrowman, M. M. Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate to M, 44,000 protein. Nature 316:547-549; 1985. Cox, J. A.; Jeng, A. Y.; Sharkey, N. A.; Blumberg, P. M.; Tauber, A. I. Activation of the human neutrophil nicotinomide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C. J. Clin. Invest. 76:1932-1938; 1985. Tauber, A. I.; Cox, J. A.; Curnutte, J. T.; Carrol, P. M.; Nakakuma, H.; Warren, B.; Gilbert, H.; Blumberg, P. M. Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase C. Biochem. Biophys. Res. Commun. 158:884-890; 1989. Naccache, P. H.; Molski, M. M.: Sha'afi, R. I. Polymyxin B inhibits phorbol 12-myristate 13-acetate, but not chemotactic factor, induced effects in rabbit neutrophils. FEBS Lett. 193:227-230; 1985. Gerard, C+; McPhail, L. C.; Marfat, A.; Stimler-Gerard, N. D.; Bass, D. A.; McCall, C. E. Role of protein kinetics in stimulation of human polymorphonuclear leukocyte oxidative metabolism by various agonists: Differential effects of a novel protein kinase inhibitor. J. Clin. Invest. 77:61-65; 1986. Wilson, E.; Olcott, M. C.; Bell, R. M.; Merrill, A. J. Jr.; Lambeth, J. D. Inhibition of the oxidative burst in human

92

182.

183.

184.

185.

186.

187.

188.

189.

190.

191.

192.

193.

194.

195. 196.

197.

198.

199. 200.

A . R . CRoss neutrophils by sphingoid long chain bases: role of protein kinase C in activation of the burst. J. Biol. Chem. 261:1261612623; 1986. Weisman, S. J.; Punzo, A.; Ford, C.; Sha'afi, R. I. Intracellular pH changes during neutrophil activation: Na ~/H ÷ antiport. J. Leuk. Biol. 41:25-32; 1987. Molski, T. F. P.; Ford, C.; Weisman, S. J.; Sha'afi, R. I. Cell alkalinization is not necessary and increased sodium influx is not sufficient for stimulated superoxide production. FEBS Lett. 203:267-272; 1986. Wright, C. D. ; Hoffman, M. D. The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation. Biochem. Biophys. Res. Commun, 135:749-755; 1986. Nath, J.; Powledge, A. Temperature-dependent inhibition of fMLP-stimulated superoxide generation by C-I and H-7 in human neutrophils. Biochem. Biophys. Res. Commun. 156:1376-1382; 1988. Badwey, J. A.; Robinson, J. M.; Curnutte, J. T.; Karnovsky, M. J.; Karnovsky, M. L. Retinoids stimulate the release of superoxide by neutrophils and change their morphology. J. Cell. Physiol. 127:223-228; 1986. Balazovitch, K. J.; Smolen, J. E.; Boxer, L. A. Ca -'~ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils. Blood 68:810-817; 1986. Nishihira, J.; McPhail, L. C.; O'Flaherty, J. T. Stimulusdependent mobilization of protein kinase C. Biochem. Biophys. Res, Commun. 1345:587-594; 1986. Curnutte, J. T.; Kuver, R.; Scott, P. J. Activation of neutrophil NADPH oxidase in a cell-free system: Partial purification of components and characterization of the activation process. J. Biol. Chem. 262:5563-5569; 1987. Dianoux, A-C.; Stasia, M-J.; Vignais, P. V. Purification and characterization of an isoform of protein kinase C from bovine neutrophils. Biochemistry 28:424-431 ; 1989. Melloni, E.; Pontremoli, S.; Michetti, M.; Sacco, O.; Sparatore, B.; Salamino, F.; Horecker, B. L. Binding of protein kinase C to neutrophil membranes in the presence of Ca -'+ and its activation by a Ca 2+ requiring proteinase. Proc. Natl. Acad. Sci. (USA)82:6435-6439; 1985. Kitagawa, S.; Takaku, F.; Sakamoto, S. Evidence that proteases are involved in superoxide production by human polymorphonuclear leukocytes and monocytes. J. Clin. Invest. 65:74-81; 1980. Koenderman, L.; Tool, A.; Roos, D.; Verhoeven, A. J. 1,2 Diacylglycerol accumulation in human neutrophils does not correlate with respiratory burst activation. FEBS Lett. 243:399-403; 1989. Della Bianca, V.; Grzeskowiak, M.; DeTogni, P.; Cassatella, M.; Rossi, F. Inhibition by verapamil of neutrophil responses to formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate. Mechanism involving Ca2+ changes, cAMP and protein kinase C. Biochim. Biophys. Acta 845:223--236; 1985. Zabucchi, G.; Soranzo, M. R.; Rossi, F.; Romeo, D. Exocytosis in human polymorphonuclear leukocytes induced by A23187 and calcium. FEBS Lett. 54:44-48; 1975. Schell-Frederick, E. Stimulation of the oxidative metabolism of polymorphonuclear leukocytes by the calcium ionophore A23187. FEBS Lett. 48:37-40; 1974. Smolen, J. E.; Weissmann, G. The effect of various stimuli and calcium antagonists on the fluorescence response of chlorotetracycline-loaded human neutrophils. Biochim. Biophys. Acta 720:172-180; 1982. Otha, H.; Okajima, F.; Ui, M. Inhibition by islet-activating protein of a chemotactic peptide-induced early breakdown of inositol phospholipids and Ca 2. mobilization in guinea pig neutrophils. J. Biol. Chem. 260:15771-15780; 1985. Glette, J.; Sandberg, S.; Hopen, G.; Solberg, C. O. Influence of tetracyclines on human polymorphonuclear leukocyte function. Antimicrob. Ag. Chemother. 25:354-357; 1984. Smolen, J. E.; Korchak, H. M.; Weissmann, G. The roles of

201.

202.

203.

204.

205.

206.

207.

208.

209.

210.

211.

212.

213.

214.

215. 216.

217.

extracellular and intracellular calcium in lysozomal enzyme release and superoxide generation by human neutrophils. Biochim. Biophys. Acta 677:512-520:1981. Pozzan, T.; Lew, D. P.; Wollheim, C. B.; Tsien, R. Y. Is cytosolic ionized calcium regulating neutrophit activation'? Science 221:1413-1415; 1983. Grinstein, S. : Furuya, W. Receptor mediated activation ofelectropermeabilized neutrophils: evidence for a Ca-'~ and protein kinase C-independent signalling pathway. J. Biol Chem. 263:1779-1783; 1988. Wolf, M.; Le Vine, H.; May, S.; Cautrecass, P.; Sahyoun, N. A model for intracellular translocation of protein kinase C involving synergism between Ca -'+ and phorbol esters. Nature 317:546-549; 1985. Korchak, H. M.; Vienne, K.; Rutherford, L. E.; Weissmann, G. Stimulus response coupling in the human neutrophil: Temporal analyses in changes in cystolic calcium and calcium elflux. Fed. Proc. 43:2749-2754; 1984. Hallet, M. B.; Campbell, A. K. Is intracellular Ca 2~ the trigger for oxygen radical production by polymorphonuclear leukocytes? Cell Calcium 5:1-19; 1984. Badwey, J. A.; Robinson, J. M.; Horn, W.; Soberman, R. J.; Karnovsky, M. J.: Karnovsky, M. L. Synergistic stimulation of neutrophils: possible involvement of 5-hydroxy 6, 8, 1 I, 14-eicosatetraenoate in superoxide release. J. Biol. Chem. 263:2779-2786; 1988. Di Vergilio, F.; Treves, S.; Lew, D.; Pozzan, T. Role of calcium in neutrophil activation. In: Hallet, M. B., ed. The neutrophil: cellular biochemistry and physiology. Boca Raton, FL: CRC Press; 1989:199-218. Wolach, J. B.; Coates, T. D.; Tzeng, D. Y.; Baehner, R. L.; Boxer, L. A. Modulation of polymorphonuclear leukocyte function by cetiedil. Blood 62:274-278; 1983. Forsgren, A.: Schmeling, D.; Quie, P. G. Effect of tetracycline on the phagocytic function of human leukocytes. J. Inf. Dis. 130:412-415; 1974. Seligman, B. E.; Gallin, J. I. Use of lipophilic probes of membrane potential to assess human neutrophil activation. Abnormality in chronic granulomatous disease. J. Clin. Invest. 66:493-503; 1980. Whitin, J. C.; Chapman, C. E.; Simons, E. R.; Chovaniec, M. E.; Cohen, H. J. Correlation between membrane potential changes and superoxide production in human granulocytes stimulated with phorbol myristate acetate. Evidence for defective activation in chronic granulomatous disease. J. Biol. Chem. 225:1874-1878; 1980. Erinstein, S.; Furuya, W.; Biggar, W. D. Cytoplasmic pH regulation in normal and abnormal neutrophils: Role of superoxide generation and Na +/H + exchange. J. Biol. Chem. 261:512-516; 1986. Wright, C. D.; Hoffman, M. D.; Thueson, D. O.; Conroy, M. C. Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-922: Differential inhibition of responses to a variety of stimuli. J. Leukocyte Biol. 42:3035; 1987. Wright, C. D.; Hoffman, M. D.; Theusson, D. O.; Conroy, M. C. Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-922; mechanism of inhibitory activity. Biochem. Biophys. Res. Commun. 148:1110,-1117; 1987. Seifert, R.; Rosenthal, W.; Schultz, G. Guanine nucleotides stimulate NADPH oxidase in membranes of human neutrophils. FEBS Lett. 205:161-165; 1986. Clark, R. A.; Leidal, K. G.; Pearson, D. W.; Nauseef, W. M. NADPH oxidase of human neutrophils: Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system. J. Biol. Chem. 262:4065-4074; 1987. Aviram, I.; Simons, E. R.; Babior, B. M. Reversible blockade of the respiratory burst in human neutrophils by a cleavable cross-linking agent. J. Biol. Chem. 259:306-311; 1984.

NADPH oxidase inhibitors 218. Bjorkstein, B.; Rayt, M. C.; Quie, P. G. Inhibition of human neutrophil chemotaxis and chemiluminescence by amphoteracin B. Infect. lmmun. 14:315-317; 1976. 219. Welch, W. D. Halothane reversibly inhibits human neutrophil bacterial killing. Anaethesiology 55:650-654; 1981. 220. White, J. W. C.; Gelb, A. W.; Wexler, H. R.; Stiller, C. R.; Keown, P. A. The effects of intravenous anaesthetic agents on human neutrophil chemiluminescence. Can. Anaesth. Soc. J. 30:506-511; 1983. 221. Nakagawara, M.; Takeshige, K.; Takamatsu, U.; Takahasi, S.; Yoshitake, J.; Minakami, S. Inhibition of superoxide production and Ca 2÷ mobilization in human neutrophils by halothane, enflurane and isoflurane. Anaesthesiol. 64:4-12; 1986. 222. Nakamura, H.; Uetani, Y.; Komura, M.; Takada, S.; Sand, K.; Matsuto, T. Inhibitory action of bilirubin on superoxide production by polymorphonuclear leukocytes. Biol. Neonate 52:273-278; 1987. 223. Goldfarb, G.; Belghiti, J.; Gautero, H.; Boivin, P. In vitro effects of benzodiazepines on polymorphonuclear leukocyte oxidative activity. Anaesthesiol. 60:57-60; 1984. 224. Nilsson, E.; Palmblad, J. Effects of ethanol on mechanisms for secretory and aggregatory responses of human granulocytes. Biochem. Pharmacol. 37:3237-3243; 1988. 225. Akard, U P.; English, D.; Gabig, T. G. Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst. Blood 72:322-327; 1988. 226. Cronstein, B. N.; Kramer, S. B.; Weissmann, G.; Hirschorn, R. Adenosine: a physiological modulator of 02 generation of human neutrophils. J. Exp. Med. 158:1160-1177; 1983. 227. McGarrity, S. T.; McAninch, J. M.; Webster, R. O. Modulation of human neutrophil functions by adenine nucleotides. Fed. Proc. 46:1030; 1987. 228. Galgiani, J. N.; Hewlett, E. L.; Friedman, R. L. Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphlococcus aureus. Infection & Immunity 56:751 755; 1988. 229. Nakagawara, A.; Minikami, S. Generation of superoxide anions by leukocytes treated with cytochalasin E. Biochem. Biophys. Res. Commun. 64:760-767; 1975.

93

230. Wilson, E.; Laster, S. M.; Gooding, L. R.; Lambeth, J. D. Platelet-derived growth factor stimulates phagocytosis and blocks agonist activation of the neutrophil respiratory burst: a possible cellular mechanism to protect against oxygen radical damage. Proc. Natl. Acad. Sci. (USA) 84:2213-2217; 1987. 231. Rest, R. F.; Farrell, C. F.; Naids, F. L. Mannose inhibits the human neutrophil oxidative burst. J. Leuk. Biol. 43:158-164; 1988. 232. Hyams, J. S.; Donaldson, M. H.; Metcalf, J. A.; Root, R. K. Inhibition of human granulocyte function by methotrexate. Cancer Res. 38:650-655; 1978. 233. Simkins, C. O.; Ives, N.; Take, E.; Johnson, M. Naloxone inhibits 02 release from human neutrophils. Life Sci. 37:1381-1386; 1985. 234. Matsui, H.; Wada, O.; Ushijima, Y.; Akazawa, T. Triphenyltin chloride inhibits superoxide production by human neutrophils stimulated with a surface active agent. FEBS Lett. 164:251254; 1983. 235. Beswick, P. H.; Brannen, P. C.; Hurles, S. S. The effects of smoking and zinc on the oxidative reactions of human neutrophils. J. Clin. Lab. lmmunol. 21:71-75; 1986. 236. Seigel, J. P.; Remington, J. S. Effects of antimicrobial agents on chemiluminescence of human polymorphonuclear leukocytes in response to phagocytosis. J. Antimicrob. Chemother. 10:505-515; 1982. 237. Mori, T.; Taki, Y.; Minakuchi, R.; Yu, B.; Nishizuka, Y. Inhibitory action of chlorpromazine, dibucaine, and other phospholipid-interacting drugs on calcium-activated phospholipid-dependent protein kinase. J. Biol. Chem. 255:83788380; 1980. 238. Matsumoto, T.; Takeshige, K.; Minakami, S. Inhibition of phagocytic metabolic changes of leukocytes by an intracellular calcium antagonist 8-(N, N-diethylamine)-octyl-3,4,5,-trimethoxy benzoate. Biochem. Biophys. Res. Commun. 88:974975; 1979. 239. Abramovitz, A. S.; Yavelow, J.; Randolph, V.; Troll, W. Inhibition of superoxide production by human neutrophils by soybean polypeptides. Re-evaluation of the involvement of proteases. J. Biol. Chem. 258:15153-15157; 1983. 240. Solomon, D. H.; O'Brien, C. A.; Weinstein, B. N-a-tosyl-Llysine chlormethyl ketone inhibits protein kinase C. FEBS Lett. 190:342-344; 1985.