Inhibition of redox cycling of methoxatin (PQQ), and of superoxide release by phagocytic white cells

Free RadicalBiology& Medicine,Vol. 18, No. 3, pp. 617-620, 1995 Copyright© 1995 ElsevierScienceLtd Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00

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Brief Communication INHIBITION OF REDOX CYCLING OF METHOXATIN (PQQ), A N D OF SUPEROXIDE RELEASE B Y PHAGOCYTIC WHITE CELLS AMY BISHOP, *~ MERCEDES A. PAZ, §~ PAUL M. GALLOP,§~ and MANFRED L. KARNOVSKY* Departments of *Genetics and *Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA; *Laboratory of Human Biochemistry, Children's Hospital, Boston, MA, USA; ~Department of Oral Biology and Pathophysiology, Harvard School of Dental Medicine, Boston, MA, USA (Received 18 March 1994; Revised 18 July 1994; Accepted 25 July 1994)

Abstract--The iodonium compounds diphenyleneiodonium and diphenyliodonium, and the amine compounds, 4,5-dimethyl phenylene diamine, N,N-dimethyl 1,4-phenylene diamine, 1,2-diamino-4,5-methyleneoxybenzene, and aminomalononitrile inhibit methoxatin's (PQQ's) redox activity in vitro, that is, the methoxatin-coupled oxidation of glycine and reduction of nitroblue tetrazolium to formazan. The compounds mentioned above also inhibit phorbol myristate acetate (PMA) stimulated superoxide release by phagocytic white cells--determined mainly as the superoxide dismutase sensitive reduction of ferricytochrome C. Related compounds, 3,4-diaminopyridine and 4-dimethylamino-benzylamine, did not inhibit redox activity of PQQ in vitro, nor did they inhibit PMA stimulated superoxide production in monocytes or neutrophils. Thus, there is a correlation between an agent's ability to inhibit PQQ redox cycling and its ability to inhibit superoxide release by phagocytes. The findings are a further indication that PQQ is involved in the respiratory burst of phagocytic cells. Keywords--Methoxatin, Pyrroloquinolinequinone (PQQ), Neutrophil, Monocyte, Iodonium compounds, Free radicals m o r e recently other proteins, tz'13 It has been d e m o n strated that P Q Q reacts with a r y l - i o d o n i u m c o m p o u n d s such as D P I Y W h e n D P I was incubated with guinea pig neutrophils, a D P I - P Q Q adduct could be isolated. 3 P Q Q has been shown also to react with o - d i a m i n e s such as 4 , 5 - d i m e t h y l - l , 2 - p h e n y l e n e d i a m i n e ( D I M P D A ) . D I M P D A inhibited superoxide production b y guinea pig neutrophils and a D I M P D A - P Q Q adduct was isolated from those cells. 3 Thus, P Q Q was implicated as a m a j o r target o f these agents that inhibit the respiratory burst o f neutrophils. This article augments data on the role o f P Q Q in the respiratory b u r s t ) DPI, BPI, and the a m i n e compounds, DIMPDA, N,N-dimethyl-l,4-phenylened i a m i n e ( N N D M D A ) , 1,2-diamino-4,5-methylenedio x y b e n z e n e ( F D A ) , a m i n o m a l o n o n i t r i l e ( A M N ) , 3,4d i a m i n o p y r i d i n e (DAP), and 4 - d i m e t h y l a m i n o b e n z y l a m i n e ( D M A B A ) were tested with regard to their ability to inhibit P Q Q redox c y c l i n g in vitro. T h e y were also e x a m i n e d to d e t e r m i n e if they inhibit s u p e r o x i d e production in a phagocytic cell t y p e such as the m o n o c y t e - - a s well as in the neutrophil.

INTRODUCTION

P y r r o l o q u i n o l i n e q u i n o n e (PQQ) is a redox cycling, water soluble, anionic orthoquinone ~'2 (for a m o r e c o m plete r e v i e w see ref. 3). It also appears to be a dissociable r e d o x - c o f a c t o r o f the N A D P H o x i d a s e system o f nitric o x i d e synthetase (NOS) 4 as well as the m i t o c h o n drial N A D H - C o Q reductase. 5 P h a g o c y t i c cells, such as m o n o c y t e s and neutrophils, generate s u p e r o x i d e in response to stimuli such as p h o r b o l m y r i s t a t e acetate ( P M A ; e.g., ref. 6). There is an o x i d a s e s y s t e m for r e d u c e d p y r i d i n e nucleotides in white cells that generates superoxide. 6-9 The o x i d a s e s y s t e m catalyzes the one-electron reduction o f d i o x y gen to superoxide. This N A D P H o x i d a s e s y s t e m has been shown to be c o m p r i s e d o f m e m b r a n e - b o u n d h e m e - / a n d flavin-containing proteins, other n e w l y disc o v e r e d c y t o s o l i c proteins, 7'8 and, according to recent findings, pQQ.3 D i p h e n y l e n e i o d o n i u m (DPI) and d i p h e n y l i o d o n i u m (BPI) inhibit the N A D P H o x i d a s e s y s t e m that drives s u p e r o x i d e p r o d u c t i o n in phagocytes.1°-13 The target(s) o f D P I have b e e n suggested to be a flavoprotein and

MATERIALS AND METHODS

Materials

Address correspondence to: Manfred L. Karnovsky, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

P Q Q was p u r c h a s e d f r o m Fluka, Switzerland. Nitroblue t e t r a z o l i u m (NBT), p h o r b o l myristate acetate

617

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(PMA), horse heart ferricytochrome C, superoxide dismutase (SOD), horse radish peroxidase (HPO), alphanaphthyl butyrate ester, vitamin D (1,25(OH)2D3), scopoletin, and most other reagents were purchased from Sigma. DPI, BPI, DIMPDA, NNDMDA, FDA, AMN, DAP, and D M A B A were purchased from Aldrich.

Elicitation of guinea pig neutrophils Thirty milliliters of sterile 12% sodium caseinate was injected into the peritoneum of 500 g Hartley guinea pigs obtained from Charles River. The next day the peritoneum was opened and the elicited neutrophils gathered there were harvested and treated as described earlier. 6'14 The neutrophils, after removal of erythrocytes, 634 were suspended in PBS pH 7.4 at a concentration of 3 × 1 0 7 cells/5 ml. Cell preparations from the peritoneum were more than 90% neutrophils, and viability was more than 90% as determined by trypan blue exclusion. These preparations produced 19 + 4 (range) nmoles 02"- • 1 0 - 7 cells • min -1 (cytochrome method, see later).

Induction of monocytes from HL-60 promyelocytic leukemia cells The human promyelocytic leukemia cell line obtained from American Type Culture Collection was grown in RPMI 1640 medium with 20% heat inactivated fetal calf serum and 2 mM glutamine. The populations were split every 2 - 3 days at a concentration of 2 × 105 cells/ml. This cell line was induced to monocytes by 4 days of treatment with 10 nM or 100 nM vitamin D as described. 15-17

Characterization of monocytes induced from HL-60 cells The conversion of HL-60 cells to monocytes was quantified by visual inspection under the microscope and by assaying for esterase activity with alpha-naphthyl butyrate ester as described. 18After 4 days of treatment with vitamin D, the cells were about 95% monocytes. These preparations produced 6.9 _+ 1.1 (range) nmoles 02"- • 1 0 - 7 cells • min -1 (see formazan method later).

Reaction of PQQ as measured by Nitroblue tetrazolium reduction in vitro (no cells) Samples containing PQQ and PQQ inhibitors were incubated with excess glycine and NBT. PQQ was

Table 1. Agents Inhibiting PQQ Cycling Agent DPI DPI BPI DIMPDA DIMPDA NNDMDA FDA AMN DAP DMABA

Concentration (#M)

% Inhibition

1.5 10.0 10.0 100.0 1000.0 100.0 200.0 200.0 200.0 200.0

50 _+ 4 85 _+ 1 50 +_ 10 50 _+ 5 95 _+ 5 99 _+ 1 99 _+ 1 50 _+ 10 0 _+ 0 0 _+ 0

reduced by the glycine. It then reduced the oxygen present in the solution to superoxide, which in turn reduced nitroblue tetrazolium to a formazan that is purple in color. The formazan's color was read at a wavelength of 530 nm and quantified against a standard curve of known concentrations of PQQ. This assay was performed as originally described. 2'19

Nitroblue tetrazolium assay in cell culture This assay was used to measure superoxide production by PMA stimulated monocytes. The monocytes were spun down gently and the pellet resuspended at a concentration of 1 - 3 × 107 cells/in 400 #1 Hepes Buffered Saline Solution (HBSS). 1.5 ml of NBT (1 mg/5 ml) was added. 20 #1 of SOD (2 #g/ml) was added to every other tube. The cells were stimulated with PMA, and the superoxide produced was measured by the SOD-sensitive reduction of NBT to formazan.

Ferricytochrome assay of

02 °-

release by cells

The amount of superoxide produced by PMA stimulated guinea pig neutrophils was also measured by the SOD-sensitive reduction of ferricytochrome as described. 2°,21

Scopoletin assay The peroxide produced by cells was measured by the scopoletin assay performed as previously describedY RESULTS

Agents that inhibit PQQ redox cycling in vitro A 33-nM solution of PQQ was incubated with the inhibitor to be studied, and the color change resulting from the oxidation of glycine and the reduction of nitroblue tetrazolium to formazan was followed. DPI, BPI, DIMPDA, NNDMDA, FDA, and AMN all inhibited the redox cycling activity of PQQ as shown in Table 1. DAP and D M A B A did not. The results are

Inhibition of redox cycling

619

Table 2. Agents Inhibiting Superoxide Production by Monocytes

Table 3. Agents Inhibiting Superoxide Production by Neutrophils

Agent

Concentration (#M)

Agent

Concentration (#M)

DPI DPI DPI DIMPDA DIMPDA NNDMDA NNDMDA FDA FDA AMN DAP DMABA

2.0 10.0 200.0 200.0 1000.0 40.0 1000.0 20.0 100.0 200.0 200.0 200.0

DPIa BPIa NNDMDA NNDMDA NNDMDA DIMPDA FDA FDA FDA AMN DAP DMABA

1.0 50.0 1.0 10.0 100.0 200.0 10.0 50.0 200.0 40.0 1000.0 1000.0

% Inhibition 0 50 100 50 50 100 100 100 100 100 0 0

± 0 ± 2 _ 0 ___2 ± 2 ± 0 _+ 0 ± 0 ± 0 ± 0 _ 0 ± 0

a

the average o f duplicate experiments, and the ranges are given.

DPI and various diamines inhibit superoxide production by monocytes M o n o c y t e s were i n c u b a t e d with the inhibitors for 25 min at 37°C. T h e cells were stimulated with P M A , and the a m o u n t o f s u p e r o x i d e was m e a s u r e d b y the reduction o f nitroblue t e t r a z o l i u m to f o r m a z a n (see T a b l e 2). D P I inhibited m o n o c y t e s u p e r o x i d e production p r o f o u n d l y , and in a d o s e - d e p e n d e n t manner. N o n e o f the inhibitors directly r e d u c e d N B T e x c e p t for N N D M D A . B e c a u s e there was S O D present, this d i d not matter. T h e average was taken o f duplicate experiments, and the range is given. W h e r e the inhibition was 100% there was no variation p r o b a b l y b e c a u s e the cells were m a x i m a l l y inhibited b y the concentration o f inhibitor used.

DPI and various diamines inhibit superoxide production by neutrophils D P I and D I M P D A were reported to inhibit neutrophil s u p e r o x i d e production, p r e s u m a b l y b y binding pQQ.3 The inhibitory effects o f additional a m i n e c o m p o u n d s were studied (Table 3). N N D M D A is a m o r e potent inhibitor o f neutrophil s u p e r o x i d e production than D I M P D A but is a less potent inhibitor than D P I or BPI. 3 F D A is a m o r e potent inhibitor than D I M P D A and N N D M D A but is still a m u c h less potent inhibitor than BPI or DPI. 3 A m i n o m a l o n o n i t r i l e is a m o r e potent inhibitor than the other amines. Interestingly, the dia m i n e s D A P and D M A B A did not inhibit neutrophil s u p e r o x i d e p r o d u c t i o n or P Q Q r e d o x cycling. The exp e r i m e n t s were done in triplicate and the average was taken. T h e range is given.

% Inhibition 74 64 0 33 94 84 48 64 100 76 0 0

+__ 5 ± 2 ± 0 ___ 3 _ 3 ± 1 ± 3 ± 2 ± 0 ± 10 _+ 0 ± 0

Previously reported in ref. 3.

Controls for effects of inhibitory agents: Extracellular dismutation of superoxide W e w a n t e d to d e t e r m i n e if the inhibitors resulted in the c o n v e r s i o n o f superoxide p r o d u c e d to p e r o x i d e in a d i s m u t a s e reaction and exerted their inhibitory effects in that noncellular manner. F o r m a t i o n o f h y d r o gen p e r o x i d e was a s s a y e d b y the scopoletin assay. 23 N o n e o f the inhibitors studied resulted in conversion o f s u p e r o x i d e to peroxide.

DISCUSSION

O u r data correlated inhibition o f P Q Q redox cycling in an in vitro assay system with inhibition o f the superoxide production o f m o n o c y t e s and neutrophils. W i t h i n the list o f inhibitors e x a m i n e d , agents that inhibited P Q Q r e d o x c y c l i n g in an in vitro assay s y s t e m at a level o f 200 # M or less also inhibited m o n o c y t e and neutrophil s u p e r o x i d e production at that concentration. A g e n t s found not to inhibit P Q Q r e d o x c y c l i n g in vitro also failed to inhibit m o n o c y t e and neutrophil superoxide production. W e chose inhibitory agents for the study o f P Q Q in p h a g o c y t i c cells b a s e d on their ability to bind P Q Q in vitro. 2'3 D P I is a planar cation that should bind PQQ. In fact, it did sequester P Q Q and has been shown to form an adduct with P Q Q with sufficient stability to be obtained from w h o l e cells. 3 D I M P D A and F D A are p l a n a r aromatic orthodiamines, whereas P Q Q is a planar ortho quinone. The d i a m i n e s should fit P Q Q and sequester it; D I M P D A was studied and shown to do so. L i k e DPI, D I M P D A has been shown to form an adduct with P Q Q that is extractable from w h o l e c e l l s ) F D A , though a p o w e r f u l inhibitor o f s u p e r o x i d e release b y neutrophils, and o f

620

A. B~SHOPet al.

PQQ redox cycling, has not been examined with regard to a d d u c t f o r m a t i o n w i t h P Q Q . We have shown that DPI inhibits monocyte superoxide production in a dose-dependent manner. Possibly t h e t a r g e t o f D P I a n d D I M P D A , i n m o n o c y t e s as w e l l as n e u t r o p h i l s , is P Q Q . A t t h e v e r y l e a s t w e h a v e d e m o n s t r a t e d t h a t D P I is a n i n h i b i t o r o f a n o n n e u t r o p h i l p h a g o c y t i c c e l l t y p e , w h i c h a d d s to p a s t w o r k o n D P I t h a t w a s s h o w n to i n h i b i t s u p e r o x i d e p r o d u c t i o n o f p o r c i n e n e u t r o p h i l s a n d r a t m a c r o p h a g e s jl 13.24,25 as w e l l as g u i n e a - p i g n e u t r o p h i l s . 3 F u r t h e r , s e v e r a l i n h i b i t o r s o f r e d o x c y c l i n g o f P Q Q w e r e d e m o n s t r a t e d to b e blocking agents for superoxide release by both stimulated neutrophils and monocytes. This suggests that P Q Q is i n v o l v e d i n t h e r e s p i r a t o r y b u r s t o f b o t h m a c r o phages and neutrophils.

12. 13.

14.

15. 16.

17.

Acknowledgements--This research was supported by grants from

the National Institutes of Health, partially funded by the National Dairy Promotion and Research Board, and administered in cooperation with the National Dairy Council.

18. 19.

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AMN--aminomalononitrile p toluenesulfate BPI-- diphenyliodonium DAP-- 3,4-diaminopyridine DIMPDA--4,5-dimethylphenylenediamine DMABA-- 4-dimethylaminobenzylamine DPI-- diphenyleneiodonium FDA-- 1,2-diamino-4,5-methyleneoxybenzene NNDMPDA-N,N-dimethyl- 1,4-phenylenediamine