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Bovine bronchial epithelial cells metabolize L-arginine to L-citrulline: Possible role of nitric oxide synthase
Life Sciences, Vol. 52, pp. 709-716 Printed in the USA
BOVINE BRONCHIAL L-CITRULLINE:
G.L.
Pergamon Press
EPITHELIAL CELLS METABOLIZE L-ARGININE POSSIBLE ROLE OF NITRIC OXIDE SYNTHASE
R.A. Robbins*, F.G. Hamel, A.A. Gossman, K.J. Nelson, S. Belenky,
TO
Floreani, and I. R u b i n s t e i n
R e s e a r c h Service, Omaha DVA Medical Center and D e p a r t m e n t of Internal Medicine, P u l m o n a r y and critical Care M e d i c i n e Section, U n i v e r s i t y of N e b r a s k a Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-2465. (Received in final form December 4, 1992) Summar 7
The c o n v e r s i o n of L-arginine to L-citrulline is c a t a l y z e d by nitric oxide synthase (NOS), and results in the release of nitric oxide (NO). We h y p o t h e s i z e d that b r o n c h i a l epithelial cells m e t a b o l i z e L-arginine to Lcitrulline. We found that cell lysates obtained from unstimulated, cultured bovine bronchial epithelial cells (BBECs) c o n v e r t e d L-[3H]arginine to L-[3H]citrulline. This c o n v e r s i o n was a t t e n u a t e d by three c o m p e t i t i v e NOS inhibitors and m o d u l a t e d by lipopolysaccharide and c i g a r e t t e smoke extract (p<0.01, all comparisons). These data d e m o n s t r a t e that BBECs m e t a b o l i z e L - a r g i n i n e to Lc i t r u l l i n e and implicate a role for the L - a r g i n i n e : N O S b i o s y n t h e t i c pathway in m o d u l a t i n g airway responses. L - A r g i n i n e is c o n v e r t e d to L-citrulline by the cytosolic enzyme nitric oxide synthase (NOS) releasing nitric oxide (NO) (i). This h i g h l y soluble gas has been shown to elicit a number of b i o l o g i c a l responses in the lung, such as p u l m o n a r y arterial v a s o d i l a t i o n and b r o n c h o d i l a t i o n (2,3). Belvisi et al ~ ) have r e c e n t l y shown that L-arginine and the NOS inhibitor, L-N"-nitroa r g i n i n e - m e t h y l - e s t e r (L-NAME), modulate human airway smooth muscle tone in vitro. In addition, NO has been d e t e c t e d in the expired air of normal humans (5). The c e l l u l a r origin of NO in the airway is u n k n o w n (4,6). We hypothesized that isolated bovine bronchial epithelial cells (BBECs) metabolize L-arginine to L-citrulline via the Larginine:NOS biosynthetic pathway thereby releasing NO. In addition, we investigated w h e t h e r the c o n v e r s i o n of L - a r g i n i n e to L - c i t r u l l i n e by BBECs is m o d u l a t e d by l i p o p o l y s a c c h a r i d e and c i g a r e t t e smoke extract.
*To w h o m reprint requests may be addressed. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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Materials and Methods Culture of bovine bronchial epithelial cells. BBECs were o b t a i n e d a c c o r d i n g to a m o d i f i c a t i o n of the m e t h o d of W u et al (7,8). Briefly, the alveoli and i n t e r s t i t i a l structures were removed f r o m t h e lungs of f r e s h l y slaughtered c o w s to o b t a i n bronchial s e g m e n t s w h i c h w e r e cut into 3-5 cm lengths. These s e g m e n t s w e r e t h e n i n c u b a t e d o v e r n i g h t in 0.1% b a c t e r i a l p r o t e a s e ( S t r e p t o m y c e s q r i s e u s t y p e XIV, S i g m a C h e m i c a l Company, St. Louis, MO). A f t e r 16 h of i n c u b a t i o n at 4°C, the l u m e n a l s u r f a c e of the b r o n c h i a l s e g m e n t s w e r e w a s h e d r e p e a t e d l y to o b t a i n BBECs. Cells o b t a i n e d by t h i s m e t h o d w e r e g r e a t e r t h a n 95% k e r a t i n p o s i t i v e and vimentin negative by immunohistological techniques (8). A p p r o x i m a t e l y 30% of t h e B B E C s w e r e ciliated, a n d m o r e t h a n 95% of t h e m w e r e v i a b l e by t r y p a n b l u e e x c l u s i o n . Cell lysates were obtained by vigorous vortexing and u l t r a s o n i c s o n i f i c a t i o n of 1.67 x 106 B B E C s in 1 ml of 50 m M H E P E S b u f f e r in p h o s p h a t e - b u f f e r e d saline (PBS) at pH=7.4. T h e cell suspension was centrifuged (1500g, I0 min) to r e m o v e l a r g e cell debris. T h e r e s u l t i n g cell s u p e r n a t a n t fluids w e r e a s p i r a t e d and f r o z e n at - 8 0 ° C u n t i l assayed.
Conversion of L - [ ~ ] a r g i n i n e to L - [ ~ ] c i t r u l l i n e . T h e c a p a c i t y of B B E C s to c o n v e r t L - a r g i n i n e to L - c i t r u l l i n e w a s e v a l u a t e d by a m o d i f i c a t i o n of t h e m e t h o d of Ma et al (9). Briefly, 750~i of B B E C l y s a t e w a s a d d e d to 250~i of 8 m M N A D P H (Sigma) in 50 m M H E P E S in PBS (pH=7.4) a n d i0 ~i of L- [3H]arginine (i00 ~ci/ml; s p e c i f i c a c t i v i t y = 290 m C i / m g ; A m e r s h a m , Inc., A r l i n g t o n H e i g h t s , IL). The r e a c t i o n m i x t u r e w a s i n c u b a t e d for 30 m i n at 37°C. Thereafter, the r e a c t i o n w a s s t o p p e d by a d d i n g 1 ml of 20 m M HEPES, 3 m M E D T A in PBS (pH=5.5). T h e r e a c t i o n m i x t u r e w a s t h e n a d j u s t e d to pH 5.5 w i t h 1 N HCI a n d p a s s e d o v e r a 2 ml D o w e x A G - 5 0 c o l u m n (Na÷; BioRad, Inc., R i c h m o n d , CA). A f t e r w a s h i n g the c o l u m n w i t h 3 ml of t h e r e a c t i o n b u f f e r at pH 5.5, L - [ 3 H ] c i t r u l l i n e w a s e l u t e d by w a s h i n g w i t h 3 ml of d i s t i l l e d water. T h e L - [ 3 H ] a r g i n i n e to L[3H]citrulline conversion was expressed as the amount of L[3H]citrulline r e c o v e r e d (dpm). To d e t e r m i n e if the c o n v e r s i o n of L - a r g i n i n e to L - c i t r u l l i n e is i n h i b i t e d b y NOS inhibitors, N G - m o n o m e t h y l - L - a r g i n i n e (L-NMMA, C a l b i o c h e m Inc., S a n Diego, CA), N G - n i t r o - L - a r g i n i n e - m e t h y l - e s t e r (L-NAME, Sigma), or L - c a n a v a n i n e (Sigma) ~ere added in s o m e experiments to t h e B B E C s l y s a t e s (each, i0-M) b e f o r e a d d i n g L[~H]arginine as d e s c r i b e d a b o v e (1,5,6). In s o m e e x p e r i m e n t s , the effects of t h e i n a c t i v e D-isomer of L-NMMA, D-NMMA (10 .4 M; a g e n e r o u s g i f t of Dr. M o n c a d a , T h e W e l l c o m e R e s e a r c h L a b o r a t o r i e s , B e c k e n h a m , UK) w a s a l s o evaluated. To a s c e r t a i n t h a t L - [ 3 H ] c i t r u l l i n e w a s e l u t e d from t h e column, t h i n l a y e r c h r o m a t o g r a p h y w a s p e r f o r m e d a c c o r d i n g to t h e m e t h o d of I y e n g a r et al (i0).
Modulation of the conversion of L-[3H]arginine to L[~Hlcitrulline. To d e t e r m i n e if g e n e r a t i o n of L - [ J H ] c i t r u l l i n e f r o m L - [ ~ H ] a r g i n i n e by B B E C l y s a t e s c a n be m o d u l a t e d (1,5,6), BBECs w e r e i s o l a t e d as d e s c r i b e d a b o v e and s u s p e n d e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s m e d i u m w i t h 10% h o r s e serum, and c u l t u r e d at 37°C, 5% CO 2 in t h e p r e s e n c e of m e d i a alone, LPS (i00 ~g/ml; E. coli 0127:B8, Difco, Detroit, MI) or a I:i00 d i l u t i o n of a f r e s h l y p r e p a r e d
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c i g a r e t t e smoke extract (8). The cells were incubated for 4 h, and after w a s h i n g r e p e a t e d l y with fresh media, cell lysates were prepared and their c a p a c i t y to convert L-[3H]arginine to L[3H]citrulline was e v a l u a t e d as d e s c r i b e d above.
Data analvsis. All data are e x p r e s s e d as means and one s t a n d a r d error of the mean. Statistical analysis was p e r f o r m e d using a Duncan's analysis of variance. A p < 0.05 was c o n s i d e r e d s t a t i s t i c a l l y significant. Results Conversion of L-[~]arqinine to L-[~]citrulline. BBEC lysates induced a gradual increase in the amount of L-[3H]citrulline g e n e r a t e d from L-[3H]arginine over 30 min (Fig 1). A d d i t i o n of L_NMMA (10-4M) r e s u l t e d in a significant attenuation of L[~H]citrulline g e n e r a t i o n at each time point studied.
A
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FIG. 1 T i m e - d e p e n d e n t g e n e r a t i o n of L-[3H]citrulline from L[3H]arginine by BBEC lysates. L-[3H]citrulline g e n e r a t i o n is e x p r e s s e d as dpm on the vertical axis and time after a d d i t i o n of NADPH is on the horizontal axis. Generation of L-[3H]citrulline by BBEC lysates is r e p r e s e n t e d by the open squares, BBEC lysates with L-NMMA (10-4M) by the diamonds, and the d i f f e r e n c e between the two data points by the square with the inner circle. In control experiments, L-NMMA (10-4M) was added after i n c u b a t i o n of BBECs lysates with L-[~H]arginine and the reaction was stopped after 30 min of incubation. L-NMMA had no s i g n i f i c a n t effect on the amount of L-[3H]citrulline g e n e r a t e d in c o m p a r i s o n to media alone (data not shown). Thin layer c h r o m a t o g r a p h y showed that citrulline, but not arginine, was eluted from the column (data not shown). To ascertain the reproducibility of the results, five d i f f e r e n t cultures of BBECs were obtained from five d i f f e r e n t animals (Fig. 2;p < 0.01). Subtracting the amount of L[3H]citrulline g e n e r a t e d in the p r e s e n c e of L-NMMA c o m p a r e d to media alone r e s u l t e d in specific L-[~H]citrulline ~ e n e r a t i o n of approximately 40-50% of the total amount of L - ~ H ] c i t r u l l i n e g e n e r a t e d from L-[3H]arginine.
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FIG. 2 G e n e r a t i o n of L - [ 3 H ] c i t r u l l i n e from L - [ 3 H ] a r g i n i n e a f t e r 30 m i n i n c u b a t i o n w i t h BBEC l y s a t e s in the p r e s e n c e or a b s e n c e of L - N M M A (104M). L - [ 3 H ] c i t r u l l i n e g e n e r a t i o n is e x p r e s s e d in d p m on the v e r t i c a l axis. G e n e r a t i o n of L[3H]citrulline in the p r e s e n c e of BBEC l y s a t e s o b t a i n e d from c e l l s in m e d i a a l o n e is r e p r e s e n t e d by the b a r on the left, in the p r e s e n c e of L - N M M A by the m i d d l e b a r and the d i f f e r e n c e b e t w e e n the m e d i a and L - N M M A by the bar on the far right. Data are means,± SEM of 5 e x p e r i m e n t s . To a s s e s s the s p e c i f i c i t y of the c o n v e r t i o n of L - [ 3 H ] a r g i n i n e to L - [ 3 H ] c i t r u l l i n e , n o n - r a d i o a c t i v e L - a r g i n i n e (104M) was a d d e d to BBECs lysates. Addition of non-radioactive L-arginine was associated with a decrease in the a m o u n t of L - [ 3 H ] c i t r u l l i n e g e n e r a t e d (22,148 ± 4192 dpm BBEC l y s a t e s a l o n e vs. 13,126 ± 3,528 d p m w i t h L - a r g i n i n e added; n=5; p<0.01). There was a concentration-response relationship between d i l u t i o n s of B B E C s l y s a t e s and g e n e r a t i o n of L-[ 3H ] c i t r u l l i n e from L - [ 3 H ] a r g i n i n e in the p r e s e n c e of L - N M M A (10-4M) : a 1:4 d i l u t i o n of B B E C s l y s a t e r e s u l t e d in 2,780 dpm of L - [ ~ H ] c i t r u l l i n e g e n e r a t e d , a 1:8 d i l u t i o n in 2,157 d p m g e n e r a t e d and a 1:16 d i l u t i o n in 24 d p m generated. The enantomeric specificity of L - N M M A was also evaluated. D - N M M A (the b i o l o g i c a l l y i n a c t i v e e n a n t i o m e r of LNMMA) induced no significant decrease in the amount of L[3H]citrulline g e n e r a t e d from L - [ 3 H ] a r g i n i n e c o m p a r e d to c o n t r o l (105±5% of c o n t r o l values; n=5). To d e t e r m i n e if o t h e r NOS i n h i b i t o r s also a t t e n u a t e L[3H]citrulline g e n e r a t i o n from L - [ 3 H ] a r g i n i n e by B B E C s lysates, Lc a n a v a n i n e and L - N A M E w e r e evaluated. I n c u b a t i o n w i t h e i t h e r of t h e s e NOS i n h i b i t o r s r e s u l t e d in a p p r o x i m a t e l y 4 0 - 5 0 % i n h i b i t i o n of L - [ 3 H ] c i t r u l l i n e g e n e r a t i o n c o m p a r e d to BBEC l y s a t e s in m e d i a a l o n e (Fig. 3, n=5; p < 0.01, all c o m p a r i s o n s ) . T h e r e w a s no significant difference b e t w e e n the a m o u n t of L - [ 3 H ] c i t r u l l i n e generated from L - [ 3 H ] a r g i n i n e in the p r e s e n c e of L-NMMA, Lc a n a v a n i n e , or L-NAME. To d e t e r m i n e if the a m o u n t of L - [ 3 H ] c i t r u l l i n e g e n e r a t e d from L - [ 3 H ] a r g i n i n e by BBECs l y s a t e s is s i m i l a r to t h a t of o t h e r c e l l s in the a i r w a y k n o w n to c o n v e r t L - a r g i n i n e to L - c i t r u l l i n e , the c a p a c i t y of h u m a n a l v e o l a r m a c r o p h a g e s o b t a i n e d by b r o n c h o a l v e o l a r l a v a g e to c o n v e r t L - [ 3 H ] a r g i n i n e to L - [ 3 H ] c i t r u l l i n e was e v a l u a t e d at equal cell n u m b e r s c o m p a r e d to B B E C s i s o l a t e d on the s a m e day. We found that both cell lysates (1:4 dilution) converted a p p r o x i m a t e l y the same a m o u n t of L - [ 3 H ] c i t r u l l i n e (2,967 dpm,
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1~^ 1 o
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FIG. 3 Decrease in L-[3H]citrulline generation from L[3H]arginine by addition of the NOS inhibitors, L-NMMA, L-canavanine, and L-NAME (each, IO'4M) to the BBEC lysates. G e n e r a t i o n of L-[3H]citrulline e x p r e s s e d as p e r c e n t of control on the vertical axis. Data are means ± SEM of 5 experiments. a l v e o l a r m a c r o p h a g e s vs. 2,416 dpm, BBECs). The rate of c o n v e r t i o n by both cell lysates was a t t e n u a t e d by L-NMMA (IO'4M) (1,859 dpm, a l v e o l a r m a c r o p h a g e s vs. 1,530 dpm, BBECs).
Modulation of the oonvertion of L-[~]arginine to L[~]oitrulline. Two irritants associated with airway disease, LPS and c i g a r e t t e smoke extract (8,11), were e v a l u a t e d for their c a p a c i t y to m o d u l a t e BBECs g e n e r a t i o n of L - c i t r u l l i n e from Larginine. C u l t u r e d BBECs were suspended in Dulbecco's M o d i f i e d Eagle's M e d i a with 10% horse serum alone, or with LPS (100 ~g/ml) or c i g a r e t t e smoke extract (final dilution, 1:100) added. Both LPS and c i g a r e t t e smoke extract induced a s i g n i f i c a n t d e c r e a s e of L[3H]citrulline g e n e r a t i o n (Fig. 4; n=5; p<0.01, c o m p a r e d to media alone). No s i g n i f i c a n t c y t o t o x i c i t y by both agents could be 8000
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FIG. 4 Decrease in L-[3H]citrulline generation from L[3H]arginine by BBEC lysates obtained from cells cultured in the p r e s e n c e of LPS and cigarette smoke extract. L[3H~citrulline g e n e r a t i o n in dpm is on the vertical axis. L-[~H]citrulline g e n e r a t e d in the p r e s e n c e of BBECs lysates o b t a i n e d after 4 h of culture in media alone (control), LPS (I00 ~g/ml), or cigarette smoke extract (final dilution, I:I00) are r e p r e s e n t e d by the left, middle, and right bars, respectively. Data are means ± SEM of 5 experiments.
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d e t e c t e d by trypan blue exclusion. A d d i t i o n of LPS or cigarette smoke extract to BBECs lysates after adding L-[3H]arginine for 30 min was not a s s o c i a t e d with any significant decrease in the amount of L-[3H]citrulline recovered (data not shown). C o n c e n t r a t i o n s of LPS as low as I00 pg/ml were also a s s o c i a t e d w i t h s i g n i f i c a n t a t t e n u a t i o n of L-[~H]arginine to L-[3H]citrulline by BBECs lysates (data not shown). Likewise, dilutions of c i g a r e t t e smoke extract as low as 1:500 were a s s o c i a t e d with s i g n i f i c a n t a t t e n u a t i o n of L[3H]arginine c o n v e r t i o n to L-[3H]citrulline (data not shown).
Discussion The results of the present study show that u n s t i m u l a t e d c u l t u r e d BBECs m e t a b o l i z e L-arginine to L - c i t r u l l i n e in vitro. This c o n v e r s i o n was s i g n i f i c a n t l y a t t e n u a t e d by c o m p e t i t i v e NOS inhibitors s u g g e s t i n g that a c o n s t i t u t i v e NOS plays a s i g n i f i c a n t role in NO release from unstimulated BBECs. Furthermore, c o n v e r s i o n of L-arginine to L-citrulline was m o d u l a t e d by exposing c u l t u r e d BBECs to LPS and cigarette smoke extract, two irritants that are a s s o c i a t e d with airway injury and inflammation and m o d u l a t e BBECs responses in vitro (8,11). Nitric oxide synthase activity has been d e t e c t e d in several cells that have been suggested to play a role in the p a t h o g e n e s i s of airway injury and inflammation, including v a s c u l a r endothelial cells, p o l y m o r p h o n u c l e a r leukocytes, mast cells, nerve cells and a l v e o l a r m a c r o p h a g e s (1,5,6,12,17,18). For example, Kaplan et al (12) showed that the L-arginine:NOS b i o s y n t h e t i c p a t h w a y m o d u l a t e d p o l y m o r p h o n u c l e a r leukocytes chemotaxis in vitro. Belvisi and her c o l l e a g u e s (4) showed that the L-arginine:NOS b i o s y n t h e t i c p a t h w a y m o d u l a t e d human tracheal smooth muscle r e l a x a t i o n in vitro. Masini et al (17) showed that in mast cells d e c r e a s e d NO g e n e r a t i o n and release is associated with increased stimulated release of histamine. The results of the present study extend these o b s e r v a t i o n s by showing that cultured bovine bronchial epithelial cells have the capacity to generate and release NO. Taken together, these data suggest that NO may play an important role in m o d u l a t i n g airway responses (1-3,5,6,12-18). The other airway cells which are known to contain NOS and could p o t e n t i a l l y c o n t a m i n a t e the BBEC p r e p a r a t i o n and account for the c i t r u l l i n e g e n e r a t i o n include m a c r o p h a g e s and neutrophils. N e i t h e r was observed in the preparation. The short h a l f - l i f e of the neutrophil combined with the m u l t i p l e w a s h i n g s used to obtain the BBECs (which should remove any NOS released) suggest that contamination by neutrophils could not explain the observed results. The o b s e r v a t i o n that >95% of the cells m a r k keratin positive and that m a c r o p h a g e s result in similar arginine to c i t r u l l i n e c o n v e r s i o n on a per cellular basis c o m p a r e d to BBECs, suggest that m a c r o p h a g e activation also could not account for the entire citrulline generation observed. Nevertheless, it is p o s s i b l e that an u n i d e n t i f i e d cell c o n t a i n i n g large amounts of NOS could p o t e n t i a l l y c o n t a m i n a t e the p r e p a r a t i o n a c c o u n t i n g for a s i g n i f i c a n t p o r t i o n of the citrulline generation. The c o n c e n t r a t i o n of the NOS inhibitors used was large to ensure complete inhibition of NOS. Despite these large amounts of inhibitors, some citrulline was still generated. Similar o b s e r v a t i o n s have been made with murine m a c r o p h a g e s s e c o n d a r y to arginine m e t a b o l i s m to ornithine by arginase w h i c h can be converted
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to c i t r u l l i n e by t r a n s c a r b a m o y l a s e (19). R e c e n t l y we have d e t e c t e d arginase in the BBECs (Robbins RA, unpublished observation) suggesting that a similar explanation may account for the c i t r u l l i n e g e n e r a t i o n desite NOS inhibition. Two forms of NOS have been d e s c r i b e d so far in m a m a l i a n tissues (6). The first is a constitutive enzyme found predominantly in endothelial and nerve cells and c a t a l y z e s the c o n t i n u o u s r e l e a s e of n a n o m o l a r c o n c e n t r a t i o n s of NO (6). The results of the p r e s e n t study suggest the p r e s e n c e of a c o n s t i t u t i v e NOS in unstimulated, c u l t u r e d BBECs, b e c a u s e these cells c o n v e r t e d L - a r g i n i n e to L - c i t r u l l i n e (Figs. 2 and 3). The second form is an inducible e n z y m e found in i n f l a m m a t o r y cells (6). W h i c h NOS enzyme(s) is p r e s e n t and m o d u l a t e d in resting and stimulated, c u l t u r e d BBECs is p r e s e n t l y unknown. We found d e c r e a s e d c o n v e r s i o n of L - a r g i n i n e to L - c i t r u l l i n e u p o n e x p o s u r e of c u l t u r e d BBECs to LPS and c i g a r e t t e smoke extract (Fig. 4). The r e s u l t a n t d e c r e a s e in NO g e n e r a t i o n may have important i m p l i c a t i o n s in the p a t h o g e n e s i s of airway d i s e a s e s such as asthma and chronic b r o n c h i t i s (6,8,11,17,18). However, the net effect of a d e c r e a s e in NO is unclear. A l t h o u g h NO r e l e a s e d from BBECs m i g h t be e x p e c t e d to be a bronchodilator, it is also clear that NO can effect the functions of i n f l a m m a t o r y cells and can itself be c y t o t o x i c (6). In the p r e s e n t study, we did not attempt to m e a s u r e the amount of NO r e l e a s e d by c u l t u r e d BBECs or its c a p a c i t y to relax airway smooth muscle tone, the so-called airway epithelium-derived r e l a x i n g factor (2,6,13-16). Previous studies have s u g g e s t e d the p r e s e n c e of airway e p i t h e l i u m - d e r i v e d relaxing factors other than NO, such as p r o s t a n o i d s and m e m b r a n e h y p e r p o l a r i z i n g factors (6,1316). Clearly, investigations to c h a r a c t e r i z e the p a t t e r n s of r e l e a s e of NO and other airway smooth m u s c l e relaxing factors from b r o n c h i a l e p i t h e l i a l cells under resting and s t i m u l a t e d c o n d i t i o n s are indicated. In summary, we found that unstimulated, cultured BBECs c o n v e r t e d L - a r g i n i n e to L - c i t r u l l i n e and that this c o n v e r s i o n was a t t e n u a t e d by c o m p e t i t i v e inhibitors of NOS and m o d u l a t e d by LPS and c i g a r e t t e smoke extract. These data suggest a role for the La r g i n i n e : N O S b i o s y n t h e t i c p a t h w a y in m o d u l a t i n g airway responses. Acknowledqements We t h a n k Debra H a r r e r for typing the manuscript. This study was s u p p o r t e d by Grants from the D e p a r t m e n t of V e t e r a n Affairs, American Heart Association-Nebraska affiliate (NE-91-G-16), A m e r i c a n C a n c e r s o c i e t y (IRG-165D) and by funds from the U n i v e r s i t y of N e b r a s k a M e d i c a l Center. References i. 2. 3.
R.M.J. PALMER, D. S. ASHTON, and S. MONCADA, N a t u r e (Lond.), 333 664-666 (1988). K. J. MORRISON, Y. GAO, and P. M. VANHOUTTE, Am. J. Physiol. 258 L254-L262 (1990). M. -D. FRATACCI, C. G. FROSTELL, T. -Y. CHEN, J. C. WAIN, JR.,
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M. G. B E L V I S I , C. D. S T R E T T O N , M. YACOUB, a n d P. J. BARNES, Eur. J. P h a r m a c o l . 2 1 0 2 2 1 - 2 2 2 (1992). L. E. G U S T A F S S O N , A. M. LEONE, M. G. P E R S S O N , N. P. WIKLUND, a n d S. M O N C A D A , B i o c h e m . Biophys. Re~. C o m m u n . 181 8 5 2 - 8 5 7 (1991) . S. M O N C A D A , R. M. J. PALMER, a n d E. A. HIGGS, P h a r m a c o l . Rev. 4__33109-142 (1991). R. WU, E. NALON, a n d C. TURNER, J. Cell. P h y s i o l . 1 2 5 1 6 7 181
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S. K O Y A M A , S. I. R E N N A R D , S. SHOJI, D. R O M B E R G E R , J. LINDER, R. ERTL, a n d R. A. R O B B I N S , Am. J. P h y s i o l . 257 L I 3 0 - L I 3 6 (1989). L. MA, Y. I S H I Z A K I , I. MORITA, and S. M U R O T A , N e u r o s c i . Lett. 13223-25 (1991). R. I Y E N G A R , D. J. STUEHR, and M. A. M A R L E T T A , Proc. Natl. Acad. Sci. (USA) 8_446369-6373 (1987). S. SHOJI, R. F. ERTL, a n d S. I. R E N N A R D , Clin. Res. 3 - 5 5 3 9 A (Abstract) (1987). S. S. KAPLAN, T. BILLIAR, R. D. CURRAN, U. E. Z D Z I A R S K I , R. L. S I M M O N S , a n d R. E. BASFORD, B l o o d 7__441885-1887 (1989). R. R. LORENZ, Y. GAO, a n d P. M. V A N O U T T E , P h y s i o l o g i s t 3_!iA 1 2 4 (Abstract) (1988). Y. GAO, a n d P. M. V A N H O U T T E , Am. Rev. R e s p i r . Dis. 139 A 3 5 4 (Abstract) (1989). M. ILHAN, a n d I. SAHIN, Eur. J. P h a r m a c o l . 1 3 1 2 9 3 - 2 9 6 (1986). C. BOULANGER, H. HENDRICKSON, R. R. LORENZ, and P. M. V A N H O U T T E , Circ. Res. 6__441070-1078 (1989). E. M A S I N I , P. F. M A N N A I O N I , A. P I S T E L L I , D. S A L V E R M I N I , a n d J. VANE, Biochem. Biophys. Res. Commun. 1 7 7 1 1 7 8 - 1 1 8 2 (1991). P. G. J O R E N S , F. J. V A N O V E R V E L D , H. BULT, P. A. V E R M E I R E , a n d A. G. H E R M A N , Eur. J. P h a r m a c o l . 2 0 0 2 0 5 - 2 0 9 (1991). D. L. G R A N G E R , J. B. HIBBS, JR., J. R. P E R F E C T , a n d D. T. DURACK, J. Clin. Invest. 8 5 2 6 4 - 2 7 3 (1990).
BOVINE BRONCHIAL L-CITRULLINE:
G.L.
Pergamon Press
EPITHELIAL CELLS METABOLIZE L-ARGININE POSSIBLE ROLE OF NITRIC OXIDE SYNTHASE
R.A. Robbins*, F.G. Hamel, A.A. Gossman, K.J. Nelson, S. Belenky,
TO
Floreani, and I. R u b i n s t e i n
R e s e a r c h Service, Omaha DVA Medical Center and D e p a r t m e n t of Internal Medicine, P u l m o n a r y and critical Care M e d i c i n e Section, U n i v e r s i t y of N e b r a s k a Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-2465. (Received in final form December 4, 1992) Summar 7
The c o n v e r s i o n of L-arginine to L-citrulline is c a t a l y z e d by nitric oxide synthase (NOS), and results in the release of nitric oxide (NO). We h y p o t h e s i z e d that b r o n c h i a l epithelial cells m e t a b o l i z e L-arginine to Lcitrulline. We found that cell lysates obtained from unstimulated, cultured bovine bronchial epithelial cells (BBECs) c o n v e r t e d L-[3H]arginine to L-[3H]citrulline. This c o n v e r s i o n was a t t e n u a t e d by three c o m p e t i t i v e NOS inhibitors and m o d u l a t e d by lipopolysaccharide and c i g a r e t t e smoke extract (p<0.01, all comparisons). These data d e m o n s t r a t e that BBECs m e t a b o l i z e L - a r g i n i n e to Lc i t r u l l i n e and implicate a role for the L - a r g i n i n e : N O S b i o s y n t h e t i c pathway in m o d u l a t i n g airway responses. L - A r g i n i n e is c o n v e r t e d to L-citrulline by the cytosolic enzyme nitric oxide synthase (NOS) releasing nitric oxide (NO) (i). This h i g h l y soluble gas has been shown to elicit a number of b i o l o g i c a l responses in the lung, such as p u l m o n a r y arterial v a s o d i l a t i o n and b r o n c h o d i l a t i o n (2,3). Belvisi et al ~ ) have r e c e n t l y shown that L-arginine and the NOS inhibitor, L-N"-nitroa r g i n i n e - m e t h y l - e s t e r (L-NAME), modulate human airway smooth muscle tone in vitro. In addition, NO has been d e t e c t e d in the expired air of normal humans (5). The c e l l u l a r origin of NO in the airway is u n k n o w n (4,6). We hypothesized that isolated bovine bronchial epithelial cells (BBECs) metabolize L-arginine to L-citrulline via the Larginine:NOS biosynthetic pathway thereby releasing NO. In addition, we investigated w h e t h e r the c o n v e r s i o n of L - a r g i n i n e to L - c i t r u l l i n e by BBECs is m o d u l a t e d by l i p o p o l y s a c c h a r i d e and c i g a r e t t e smoke extract.
*To w h o m reprint requests may be addressed. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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Materials and Methods Culture of bovine bronchial epithelial cells. BBECs were o b t a i n e d a c c o r d i n g to a m o d i f i c a t i o n of the m e t h o d of W u et al (7,8). Briefly, the alveoli and i n t e r s t i t i a l structures were removed f r o m t h e lungs of f r e s h l y slaughtered c o w s to o b t a i n bronchial s e g m e n t s w h i c h w e r e cut into 3-5 cm lengths. These s e g m e n t s w e r e t h e n i n c u b a t e d o v e r n i g h t in 0.1% b a c t e r i a l p r o t e a s e ( S t r e p t o m y c e s q r i s e u s t y p e XIV, S i g m a C h e m i c a l Company, St. Louis, MO). A f t e r 16 h of i n c u b a t i o n at 4°C, the l u m e n a l s u r f a c e of the b r o n c h i a l s e g m e n t s w e r e w a s h e d r e p e a t e d l y to o b t a i n BBECs. Cells o b t a i n e d by t h i s m e t h o d w e r e g r e a t e r t h a n 95% k e r a t i n p o s i t i v e and vimentin negative by immunohistological techniques (8). A p p r o x i m a t e l y 30% of t h e B B E C s w e r e ciliated, a n d m o r e t h a n 95% of t h e m w e r e v i a b l e by t r y p a n b l u e e x c l u s i o n . Cell lysates were obtained by vigorous vortexing and u l t r a s o n i c s o n i f i c a t i o n of 1.67 x 106 B B E C s in 1 ml of 50 m M H E P E S b u f f e r in p h o s p h a t e - b u f f e r e d saline (PBS) at pH=7.4. T h e cell suspension was centrifuged (1500g, I0 min) to r e m o v e l a r g e cell debris. T h e r e s u l t i n g cell s u p e r n a t a n t fluids w e r e a s p i r a t e d and f r o z e n at - 8 0 ° C u n t i l assayed.
Conversion of L - [ ~ ] a r g i n i n e to L - [ ~ ] c i t r u l l i n e . T h e c a p a c i t y of B B E C s to c o n v e r t L - a r g i n i n e to L - c i t r u l l i n e w a s e v a l u a t e d by a m o d i f i c a t i o n of t h e m e t h o d of Ma et al (9). Briefly, 750~i of B B E C l y s a t e w a s a d d e d to 250~i of 8 m M N A D P H (Sigma) in 50 m M H E P E S in PBS (pH=7.4) a n d i0 ~i of L- [3H]arginine (i00 ~ci/ml; s p e c i f i c a c t i v i t y = 290 m C i / m g ; A m e r s h a m , Inc., A r l i n g t o n H e i g h t s , IL). The r e a c t i o n m i x t u r e w a s i n c u b a t e d for 30 m i n at 37°C. Thereafter, the r e a c t i o n w a s s t o p p e d by a d d i n g 1 ml of 20 m M HEPES, 3 m M E D T A in PBS (pH=5.5). T h e r e a c t i o n m i x t u r e w a s t h e n a d j u s t e d to pH 5.5 w i t h 1 N HCI a n d p a s s e d o v e r a 2 ml D o w e x A G - 5 0 c o l u m n (Na÷; BioRad, Inc., R i c h m o n d , CA). A f t e r w a s h i n g the c o l u m n w i t h 3 ml of t h e r e a c t i o n b u f f e r at pH 5.5, L - [ 3 H ] c i t r u l l i n e w a s e l u t e d by w a s h i n g w i t h 3 ml of d i s t i l l e d water. T h e L - [ 3 H ] a r g i n i n e to L[3H]citrulline conversion was expressed as the amount of L[3H]citrulline r e c o v e r e d (dpm). To d e t e r m i n e if the c o n v e r s i o n of L - a r g i n i n e to L - c i t r u l l i n e is i n h i b i t e d b y NOS inhibitors, N G - m o n o m e t h y l - L - a r g i n i n e (L-NMMA, C a l b i o c h e m Inc., S a n Diego, CA), N G - n i t r o - L - a r g i n i n e - m e t h y l - e s t e r (L-NAME, Sigma), or L - c a n a v a n i n e (Sigma) ~ere added in s o m e experiments to t h e B B E C s l y s a t e s (each, i0-M) b e f o r e a d d i n g L[~H]arginine as d e s c r i b e d a b o v e (1,5,6). In s o m e e x p e r i m e n t s , the effects of t h e i n a c t i v e D-isomer of L-NMMA, D-NMMA (10 .4 M; a g e n e r o u s g i f t of Dr. M o n c a d a , T h e W e l l c o m e R e s e a r c h L a b o r a t o r i e s , B e c k e n h a m , UK) w a s a l s o evaluated. To a s c e r t a i n t h a t L - [ 3 H ] c i t r u l l i n e w a s e l u t e d from t h e column, t h i n l a y e r c h r o m a t o g r a p h y w a s p e r f o r m e d a c c o r d i n g to t h e m e t h o d of I y e n g a r et al (i0).
Modulation of the conversion of L-[3H]arginine to L[~Hlcitrulline. To d e t e r m i n e if g e n e r a t i o n of L - [ J H ] c i t r u l l i n e f r o m L - [ ~ H ] a r g i n i n e by B B E C l y s a t e s c a n be m o d u l a t e d (1,5,6), BBECs w e r e i s o l a t e d as d e s c r i b e d a b o v e and s u s p e n d e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s m e d i u m w i t h 10% h o r s e serum, and c u l t u r e d at 37°C, 5% CO 2 in t h e p r e s e n c e of m e d i a alone, LPS (i00 ~g/ml; E. coli 0127:B8, Difco, Detroit, MI) or a I:i00 d i l u t i o n of a f r e s h l y p r e p a r e d
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c i g a r e t t e smoke extract (8). The cells were incubated for 4 h, and after w a s h i n g r e p e a t e d l y with fresh media, cell lysates were prepared and their c a p a c i t y to convert L-[3H]arginine to L[3H]citrulline was e v a l u a t e d as d e s c r i b e d above.
Data analvsis. All data are e x p r e s s e d as means and one s t a n d a r d error of the mean. Statistical analysis was p e r f o r m e d using a Duncan's analysis of variance. A p < 0.05 was c o n s i d e r e d s t a t i s t i c a l l y significant. Results Conversion of L-[~]arqinine to L-[~]citrulline. BBEC lysates induced a gradual increase in the amount of L-[3H]citrulline g e n e r a t e d from L-[3H]arginine over 30 min (Fig 1). A d d i t i o n of L_NMMA (10-4M) r e s u l t e d in a significant attenuation of L[~H]citrulline g e n e r a t i o n at each time point studied.
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FIG. 1 T i m e - d e p e n d e n t g e n e r a t i o n of L-[3H]citrulline from L[3H]arginine by BBEC lysates. L-[3H]citrulline g e n e r a t i o n is e x p r e s s e d as dpm on the vertical axis and time after a d d i t i o n of NADPH is on the horizontal axis. Generation of L-[3H]citrulline by BBEC lysates is r e p r e s e n t e d by the open squares, BBEC lysates with L-NMMA (10-4M) by the diamonds, and the d i f f e r e n c e between the two data points by the square with the inner circle. In control experiments, L-NMMA (10-4M) was added after i n c u b a t i o n of BBECs lysates with L-[~H]arginine and the reaction was stopped after 30 min of incubation. L-NMMA had no s i g n i f i c a n t effect on the amount of L-[3H]citrulline g e n e r a t e d in c o m p a r i s o n to media alone (data not shown). Thin layer c h r o m a t o g r a p h y showed that citrulline, but not arginine, was eluted from the column (data not shown). To ascertain the reproducibility of the results, five d i f f e r e n t cultures of BBECs were obtained from five d i f f e r e n t animals (Fig. 2;p < 0.01). Subtracting the amount of L[3H]citrulline g e n e r a t e d in the p r e s e n c e of L-NMMA c o m p a r e d to media alone r e s u l t e d in specific L-[~H]citrulline ~ e n e r a t i o n of approximately 40-50% of the total amount of L - ~ H ] c i t r u l l i n e g e n e r a t e d from L-[3H]arginine.
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FIG. 2 G e n e r a t i o n of L - [ 3 H ] c i t r u l l i n e from L - [ 3 H ] a r g i n i n e a f t e r 30 m i n i n c u b a t i o n w i t h BBEC l y s a t e s in the p r e s e n c e or a b s e n c e of L - N M M A (104M). L - [ 3 H ] c i t r u l l i n e g e n e r a t i o n is e x p r e s s e d in d p m on the v e r t i c a l axis. G e n e r a t i o n of L[3H]citrulline in the p r e s e n c e of BBEC l y s a t e s o b t a i n e d from c e l l s in m e d i a a l o n e is r e p r e s e n t e d by the b a r on the left, in the p r e s e n c e of L - N M M A by the m i d d l e b a r and the d i f f e r e n c e b e t w e e n the m e d i a and L - N M M A by the bar on the far right. Data are means,± SEM of 5 e x p e r i m e n t s . To a s s e s s the s p e c i f i c i t y of the c o n v e r t i o n of L - [ 3 H ] a r g i n i n e to L - [ 3 H ] c i t r u l l i n e , n o n - r a d i o a c t i v e L - a r g i n i n e (104M) was a d d e d to BBECs lysates. Addition of non-radioactive L-arginine was associated with a decrease in the a m o u n t of L - [ 3 H ] c i t r u l l i n e g e n e r a t e d (22,148 ± 4192 dpm BBEC l y s a t e s a l o n e vs. 13,126 ± 3,528 d p m w i t h L - a r g i n i n e added; n=5; p<0.01). There was a concentration-response relationship between d i l u t i o n s of B B E C s l y s a t e s and g e n e r a t i o n of L-[ 3H ] c i t r u l l i n e from L - [ 3 H ] a r g i n i n e in the p r e s e n c e of L - N M M A (10-4M) : a 1:4 d i l u t i o n of B B E C s l y s a t e r e s u l t e d in 2,780 dpm of L - [ ~ H ] c i t r u l l i n e g e n e r a t e d , a 1:8 d i l u t i o n in 2,157 d p m g e n e r a t e d and a 1:16 d i l u t i o n in 24 d p m generated. The enantomeric specificity of L - N M M A was also evaluated. D - N M M A (the b i o l o g i c a l l y i n a c t i v e e n a n t i o m e r of LNMMA) induced no significant decrease in the amount of L[3H]citrulline g e n e r a t e d from L - [ 3 H ] a r g i n i n e c o m p a r e d to c o n t r o l (105±5% of c o n t r o l values; n=5). To d e t e r m i n e if o t h e r NOS i n h i b i t o r s also a t t e n u a t e L[3H]citrulline g e n e r a t i o n from L - [ 3 H ] a r g i n i n e by B B E C s lysates, Lc a n a v a n i n e and L - N A M E w e r e evaluated. I n c u b a t i o n w i t h e i t h e r of t h e s e NOS i n h i b i t o r s r e s u l t e d in a p p r o x i m a t e l y 4 0 - 5 0 % i n h i b i t i o n of L - [ 3 H ] c i t r u l l i n e g e n e r a t i o n c o m p a r e d to BBEC l y s a t e s in m e d i a a l o n e (Fig. 3, n=5; p < 0.01, all c o m p a r i s o n s ) . T h e r e w a s no significant difference b e t w e e n the a m o u n t of L - [ 3 H ] c i t r u l l i n e generated from L - [ 3 H ] a r g i n i n e in the p r e s e n c e of L-NMMA, Lc a n a v a n i n e , or L-NAME. To d e t e r m i n e if the a m o u n t of L - [ 3 H ] c i t r u l l i n e g e n e r a t e d from L - [ 3 H ] a r g i n i n e by BBECs l y s a t e s is s i m i l a r to t h a t of o t h e r c e l l s in the a i r w a y k n o w n to c o n v e r t L - a r g i n i n e to L - c i t r u l l i n e , the c a p a c i t y of h u m a n a l v e o l a r m a c r o p h a g e s o b t a i n e d by b r o n c h o a l v e o l a r l a v a g e to c o n v e r t L - [ 3 H ] a r g i n i n e to L - [ 3 H ] c i t r u l l i n e was e v a l u a t e d at equal cell n u m b e r s c o m p a r e d to B B E C s i s o l a t e d on the s a m e day. We found that both cell lysates (1:4 dilution) converted a p p r o x i m a t e l y the same a m o u n t of L - [ 3 H ] c i t r u l l i n e (2,967 dpm,
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1~^ 1 o
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L-CAN
L-NAME
FIG. 3 Decrease in L-[3H]citrulline generation from L[3H]arginine by addition of the NOS inhibitors, L-NMMA, L-canavanine, and L-NAME (each, IO'4M) to the BBEC lysates. G e n e r a t i o n of L-[3H]citrulline e x p r e s s e d as p e r c e n t of control on the vertical axis. Data are means ± SEM of 5 experiments. a l v e o l a r m a c r o p h a g e s vs. 2,416 dpm, BBECs). The rate of c o n v e r t i o n by both cell lysates was a t t e n u a t e d by L-NMMA (IO'4M) (1,859 dpm, a l v e o l a r m a c r o p h a g e s vs. 1,530 dpm, BBECs).
Modulation of the oonvertion of L-[~]arginine to L[~]oitrulline. Two irritants associated with airway disease, LPS and c i g a r e t t e smoke extract (8,11), were e v a l u a t e d for their c a p a c i t y to m o d u l a t e BBECs g e n e r a t i o n of L - c i t r u l l i n e from Larginine. C u l t u r e d BBECs were suspended in Dulbecco's M o d i f i e d Eagle's M e d i a with 10% horse serum alone, or with LPS (100 ~g/ml) or c i g a r e t t e smoke extract (final dilution, 1:100) added. Both LPS and c i g a r e t t e smoke extract induced a s i g n i f i c a n t d e c r e a s e of L[3H]citrulline g e n e r a t i o n (Fig. 4; n=5; p<0.01, c o m p a r e d to media alone). No s i g n i f i c a n t c y t o t o x i c i t y by both agents could be 8000
600( w z
400(
-~
2ooc
c
MEDIA
LPS
SMOKE EXTRACT
FIG. 4 Decrease in L-[3H]citrulline generation from L[3H]arginine by BBEC lysates obtained from cells cultured in the p r e s e n c e of LPS and cigarette smoke extract. L[3H~citrulline g e n e r a t i o n in dpm is on the vertical axis. L-[~H]citrulline g e n e r a t e d in the p r e s e n c e of BBECs lysates o b t a i n e d after 4 h of culture in media alone (control), LPS (I00 ~g/ml), or cigarette smoke extract (final dilution, I:I00) are r e p r e s e n t e d by the left, middle, and right bars, respectively. Data are means ± SEM of 5 experiments.
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d e t e c t e d by trypan blue exclusion. A d d i t i o n of LPS or cigarette smoke extract to BBECs lysates after adding L-[3H]arginine for 30 min was not a s s o c i a t e d with any significant decrease in the amount of L-[3H]citrulline recovered (data not shown). C o n c e n t r a t i o n s of LPS as low as I00 pg/ml were also a s s o c i a t e d w i t h s i g n i f i c a n t a t t e n u a t i o n of L-[~H]arginine to L-[3H]citrulline by BBECs lysates (data not shown). Likewise, dilutions of c i g a r e t t e smoke extract as low as 1:500 were a s s o c i a t e d with s i g n i f i c a n t a t t e n u a t i o n of L[3H]arginine c o n v e r t i o n to L-[3H]citrulline (data not shown).
Discussion The results of the present study show that u n s t i m u l a t e d c u l t u r e d BBECs m e t a b o l i z e L-arginine to L - c i t r u l l i n e in vitro. This c o n v e r s i o n was s i g n i f i c a n t l y a t t e n u a t e d by c o m p e t i t i v e NOS inhibitors s u g g e s t i n g that a c o n s t i t u t i v e NOS plays a s i g n i f i c a n t role in NO release from unstimulated BBECs. Furthermore, c o n v e r s i o n of L-arginine to L-citrulline was m o d u l a t e d by exposing c u l t u r e d BBECs to LPS and cigarette smoke extract, two irritants that are a s s o c i a t e d with airway injury and inflammation and m o d u l a t e BBECs responses in vitro (8,11). Nitric oxide synthase activity has been d e t e c t e d in several cells that have been suggested to play a role in the p a t h o g e n e s i s of airway injury and inflammation, including v a s c u l a r endothelial cells, p o l y m o r p h o n u c l e a r leukocytes, mast cells, nerve cells and a l v e o l a r m a c r o p h a g e s (1,5,6,12,17,18). For example, Kaplan et al (12) showed that the L-arginine:NOS b i o s y n t h e t i c p a t h w a y m o d u l a t e d p o l y m o r p h o n u c l e a r leukocytes chemotaxis in vitro. Belvisi and her c o l l e a g u e s (4) showed that the L-arginine:NOS b i o s y n t h e t i c p a t h w a y m o d u l a t e d human tracheal smooth muscle r e l a x a t i o n in vitro. Masini et al (17) showed that in mast cells d e c r e a s e d NO g e n e r a t i o n and release is associated with increased stimulated release of histamine. The results of the present study extend these o b s e r v a t i o n s by showing that cultured bovine bronchial epithelial cells have the capacity to generate and release NO. Taken together, these data suggest that NO may play an important role in m o d u l a t i n g airway responses (1-3,5,6,12-18). The other airway cells which are known to contain NOS and could p o t e n t i a l l y c o n t a m i n a t e the BBEC p r e p a r a t i o n and account for the c i t r u l l i n e g e n e r a t i o n include m a c r o p h a g e s and neutrophils. N e i t h e r was observed in the preparation. The short h a l f - l i f e of the neutrophil combined with the m u l t i p l e w a s h i n g s used to obtain the BBECs (which should remove any NOS released) suggest that contamination by neutrophils could not explain the observed results. The o b s e r v a t i o n that >95% of the cells m a r k keratin positive and that m a c r o p h a g e s result in similar arginine to c i t r u l l i n e c o n v e r s i o n on a per cellular basis c o m p a r e d to BBECs, suggest that m a c r o p h a g e activation also could not account for the entire citrulline generation observed. Nevertheless, it is p o s s i b l e that an u n i d e n t i f i e d cell c o n t a i n i n g large amounts of NOS could p o t e n t i a l l y c o n t a m i n a t e the p r e p a r a t i o n a c c o u n t i n g for a s i g n i f i c a n t p o r t i o n of the citrulline generation. The c o n c e n t r a t i o n of the NOS inhibitors used was large to ensure complete inhibition of NOS. Despite these large amounts of inhibitors, some citrulline was still generated. Similar o b s e r v a t i o n s have been made with murine m a c r o p h a g e s s e c o n d a r y to arginine m e t a b o l i s m to ornithine by arginase w h i c h can be converted
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to c i t r u l l i n e by t r a n s c a r b a m o y l a s e (19). R e c e n t l y we have d e t e c t e d arginase in the BBECs (Robbins RA, unpublished observation) suggesting that a similar explanation may account for the c i t r u l l i n e g e n e r a t i o n desite NOS inhibition. Two forms of NOS have been d e s c r i b e d so far in m a m a l i a n tissues (6). The first is a constitutive enzyme found predominantly in endothelial and nerve cells and c a t a l y z e s the c o n t i n u o u s r e l e a s e of n a n o m o l a r c o n c e n t r a t i o n s of NO (6). The results of the p r e s e n t study suggest the p r e s e n c e of a c o n s t i t u t i v e NOS in unstimulated, c u l t u r e d BBECs, b e c a u s e these cells c o n v e r t e d L - a r g i n i n e to L - c i t r u l l i n e (Figs. 2 and 3). The second form is an inducible e n z y m e found in i n f l a m m a t o r y cells (6). W h i c h NOS enzyme(s) is p r e s e n t and m o d u l a t e d in resting and stimulated, c u l t u r e d BBECs is p r e s e n t l y unknown. We found d e c r e a s e d c o n v e r s i o n of L - a r g i n i n e to L - c i t r u l l i n e u p o n e x p o s u r e of c u l t u r e d BBECs to LPS and c i g a r e t t e smoke extract (Fig. 4). The r e s u l t a n t d e c r e a s e in NO g e n e r a t i o n may have important i m p l i c a t i o n s in the p a t h o g e n e s i s of airway d i s e a s e s such as asthma and chronic b r o n c h i t i s (6,8,11,17,18). However, the net effect of a d e c r e a s e in NO is unclear. A l t h o u g h NO r e l e a s e d from BBECs m i g h t be e x p e c t e d to be a bronchodilator, it is also clear that NO can effect the functions of i n f l a m m a t o r y cells and can itself be c y t o t o x i c (6). In the p r e s e n t study, we did not attempt to m e a s u r e the amount of NO r e l e a s e d by c u l t u r e d BBECs or its c a p a c i t y to relax airway smooth muscle tone, the so-called airway epithelium-derived r e l a x i n g factor (2,6,13-16). Previous studies have s u g g e s t e d the p r e s e n c e of airway e p i t h e l i u m - d e r i v e d relaxing factors other than NO, such as p r o s t a n o i d s and m e m b r a n e h y p e r p o l a r i z i n g factors (6,1316). Clearly, investigations to c h a r a c t e r i z e the p a t t e r n s of r e l e a s e of NO and other airway smooth m u s c l e relaxing factors from b r o n c h i a l e p i t h e l i a l cells under resting and s t i m u l a t e d c o n d i t i o n s are indicated. In summary, we found that unstimulated, cultured BBECs c o n v e r t e d L - a r g i n i n e to L - c i t r u l l i n e and that this c o n v e r s i o n was a t t e n u a t e d by c o m p e t i t i v e inhibitors of NOS and m o d u l a t e d by LPS and c i g a r e t t e smoke extract. These data suggest a role for the La r g i n i n e : N O S b i o s y n t h e t i c p a t h w a y in m o d u l a t i n g airway responses. Acknowledqements We t h a n k Debra H a r r e r for typing the manuscript. This study was s u p p o r t e d by Grants from the D e p a r t m e n t of V e t e r a n Affairs, American Heart Association-Nebraska affiliate (NE-91-G-16), A m e r i c a n C a n c e r s o c i e t y (IRG-165D) and by funds from the U n i v e r s i t y of N e b r a s k a M e d i c a l Center. References i. 2. 3.
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