Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes

Jesús Ródenas, M. Teresa Mitjavila, and Teresa Carbonell

Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nitric oxide (NO ·) has a complex role in the inflammatory response. In this study, we modified the levels of endogenous NO · in vivo in an acute model of inflammation and evaluated the interactions between NO · and superoxide anion (O<SUP>−</SUP><SUB>2</SUB>⋅) produced by polymorphonuclear leukocytes (PMNs) accumulated in the inflamed area. We injected phosphate-buffered saline (control group), 6 µmol of L-N5-(1-iminoethyl)ornithine (L-NIO group), or 6 µmol of L-arginine (L-arginine group) into the granuloma pouch induced by carrageenan in rats. NO<SUP>−</SUP><SUB>2</SUB> plus NO<SUP>−</SUP><SUB>3</SUB> (indicative of NO · generation) was 188 nmol in the exudate of the control group, but it decreased in the L-NIO group (P < 0.05) and increased in the L-arginine group (P < 0.05). When PMNs from treated rats were incubated in vitro, the production of superoxide anion (O<SUP>−</SUP><SUB>2</SUB>⋅) decreased by ~46% in the L-arginine group. Furthermore, O<SUP>−</SUP><SUB>2</SUB>⋅ was inhibited in PMNs when L-arginine was added to the incubation medium before phorbol 12-myristate 13-acetate stimulation but not when added simultaneously. Our results suggest a protective role for NO · in inflammation, through the inactivation of NADPH oxidase and the consequent impairment of O<SUP>−</SUP><SUB>2</SUB>⋅ production for cell-mediated injury.

inflammatory cell-mediated injury; granuloma; peroxynitrite; polymorphonuclear neutrophils; NADPH

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

OXIDATIVE STRESS IN THE extracellular space appears to be an important factor in pathological conditions such as inflammatory cell-mediated injury (7, 22). When polymorphonuclear leukocytes (PMNs) are exposed to inflammatory mediators, respiratory burst takes place and the primary production of O<SUP>−</SUP><SUB>2</SUB>⋅ increases the steady-state concentrations of oxyradicals, resulting in oxidation of key cellular components and cell death (2). During the acute phase of inflammation, large numbers of PMNs migrate from the blood and accumulate in the exudate. When collected and incubated in vitro, these cells release nitric oxide (NO ·) and superoxide anion (O<SUP>−</SUP><SUB>2</SUB>⋅), both in rats (19) and in humans (3), although the amount produced depends on the stimulating agent used (18). There is extensive evidence that production of NO · in the presence of O<SUP>−</SUP><SUB>2</SUB>⋅ leads to cytotoxic events (17, 23) due to peroxynitrite (ONOO-) generation (11). However, anti-inflammatory properties have been reported for NO ·, which limits injury to isolated rat lungs when associated with the production of reactive oxygen species (10) and improves oxygenation in high-altitude pulmonary edema (21). These findings suggest that NO · may have therapeutic properties. We therefore evaluated NO · generation in vivo during an acute inflammatory process in rats and studied whether changes in endogenous NO · production affected the respiratory burst of PMNs that had accumulated in the inflammatory exudate. L-N5-(1-iminoethyl)ornithine (L-NIO), a potent inhibitor of NO · synthesis (15), and L-arginine, a precursor of NO · (16), were injected into the granuloma pouch. Cells were then collected, and the in vitro production of free radicals in response to phorbol 12-myristate 13-acetate (PMA) stimulation was measured.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Induction of inflammation and sampling of exudate. The granuloma pouch was induced in the dorsum of young male Sprague-Dawley rats (200-225 g) by subcutaneous administration of 6 ml of air, followed 24 h later by 4 ml of carrageenan (2% wt/vol, in sterile saline; Marine Colloids, Springfield, NJ) as previously described (8). Animals had free access to tap water and were starved for 12 h before samples were taken. Manipulations and experimental procedures were in accordance with the European Community regulations for the use and handling of experimental animals and were approved by the Ethical Committee of the Faculty of Biology of the University of Barcelona. Rats were divided into three groups of six rats each, and, 23 h after induction of inflammation, 1 ml of phosphate-buffered saline (PBS; control group), 6 µmol of L-NIO (Cayman Chemical, Ann Arbor, MI) in 1 ml PBS (L-NIO group), or 6 µmol of L-arginine (Sigma Chemical, St. Louis, MO) in 1 ml PBS (L-arginine group) were injected into the granuloma pouch. One hour later, rats were killed by ether anesthesia. The exudate was harvested with a heparinized syringe, the pouch was washed with cold saline, and inflammatory cells were obtained by centrifugation at 800 g for 10 min at 4°C.

Measurement of L-arginine and NO · in the exudate. The supernatant from the exudate was filtered through Millipore filters (Bedford, MA) and used to determine the levels of L-arginine and NO ·. Separation of L-arginine was performed by ion-exchange chromatography and analyzed in an autoanalyzer (Alpha Plus Two, Pharmacia LKB Biotechnology). NO<SUP>−</SUP><SUB>2</SUB> and NO<SUP>−</SUP><SUB>3</SUB> were measured as indicators of NO · generation (14). Aliquots of 0.1 ml were incubated with 0.1 ml Griess reagent [0.1% N-(1-naphthyl)ethylenediamine and sulfanilamide in 2.5% H3PO4] at room temperature for 10 min to measure NO<SUP>−</SUP><SUB>2</SUB> concentration. NO<SUP>−</SUP><SUB>3</SUB> was measured by its conversion to NO<SUP>−</SUP><SUB>2</SUB> using nitrate reductase (Cayman Chemical) (9). Sodium nitrite was used as a standard. Results are expressed as nanomoles of NO<SUP>−</SUP><SUB>2</SUB> plus NO<SUP>−</SUP><SUB>3</SUB> in exudate.

Measurement of NO · and O<SUP>−</SUP><SUB>2</SUB>⋅ produced by PMNs in vitro. After hyposmotic lysis of contaminating erythrocytes, cells were resuspended in PBS without Ca2+ or Mg2+ and washed twice in the same buffer. Viability was assessed by the trypan blue exclusion test, and cells were divided into aliquots for further studies on NO · and O<SUP>−</SUP><SUB>2</SUB>⋅ production.

NO · was determined by the oxyhemoglobin method (6). Inflammatory cells (1 × 106 cells) were incubated for 1 h at 37°C in 1 ml PBS containing 15 µM oxyhemoglobin and 0.6 mM L-arginine together with 100 U/ml catalase and 60 U/ml superoxide dismutase (SOD) (all from Sigma) to prevent peroxynitrite (ONOO-) generation. Cells were stimulated by adding 0.1 mg/ml PMA (Sigma) to the incubation medium. The reaction was stopped by immersion of the tubes in ice, followed by cold centrifugation. The supernatant was collected and the oxyhemoglobin-methemoglobin conversion (indicating NO · formation) was measured by the change in absorbance at 578 nm vs. 592 nm, using the molar absorption coefficient of 11.2 mM-1 · cm-1. The amount of NO · released was calculated from the difference between the absorbance of the samples with and without 0.6 mM L-NIO. The incubation medium without cells was used as a blank. In some cases, NO<SUP>−</SUP><SUB>2</SUB> and NO<SUP>−</SUP><SUB>3</SUB> were also determined by the technique described above (14).

O<SUP>−</SUP><SUB>2</SUB>⋅ was measured by the SOD-inhibitable reduction of ferricytochrome c (13). Cells were incubated in 1 ml PBS containing 0.15 mM ferricytochrome c (Sigma). When indicated, 0.6 mM L-NIO or 0.6 mM L-arginine was added to the medium, which allowed the detection of interferences with NO ·. Cells were stimulated by adding 0.1 mg/ml PMA (Sigma). After incubation for 1 h at 37°C in a water bath, the reaction was stopped by immersion of the tubes in ice, followed by cold centrifugation. Reduction of ferricytochrome c in the incubation medium was measured at 550 nm in a spectrophotometer, using the molar absorption coefficient of 21 mM-1 · cm-1. The amount of O<SUP>−</SUP><SUB>2</SUB>⋅ released was calculated from the difference between the absorbance of the samples with and without 60 U/ml SOD. The incubation medium without cells was used as a blank. Results are expressed as nanomoles of NO · and O<SUP>−</SUP><SUB>2</SUB>⋅ produced per hour by 1 × 106 cells.

Statistical analysis. Data were analyzed by one-way or two-way analysis of variance, using the Student-Newman-Keuls test to identify differences between groups. A P value <0.05 was considered significant. Values are the means ± SE of 6 separate experiments.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Measurements in the exudate. The volume of the exudate (Table 1) did not significantly change between the control, L-NIO, or L-arginine groups. The concentration of cells and their viability (Table 1) were also unaffected by treatment. The concentration of NO<SUP>−</SUP><SUB>2</SUB> plus NO<SUP>−</SUP><SUB>3</SUB> in the exudate of control rats (188 ± 19 nmol; Fig. 1) decreased in the L-NIO group (124 ± 9 nmol) and increased in the L-arginine group (248 ± 22 nmol) (P < 0.05). The amount of L-arginine in the exudate of the L-arginine group (3.62 ± 0.07 µmol, 44% of the total injected) was higher than in the control group (0.83 ± 0.06 µmol; data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Volume and cell concentration in the exudate and cell viability of three groups of rats with different treatments


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   NO<SUP>−</SUP><SUB>2</SUB> + NO<SUP>−</SUP><SUB>3</SUB> in exudate of inflamed rats. Rats received 1 ml of phosphate-buffered saline (PBS; control group), 1 ml of PBS containing 6 µmol of L-N5-(1-iminoethyl)ornithine (L-NIO), or 1 ml of PBS containing 6 µmol of L-Arg 1 h before being killed. Values are means ± SE of 6 separate experiments performed in duplicate. Data were analyzed by 1-way analysis of variance (ANOVA), using Student-Newman-Keuls test. Different letters above bars indicate significant differences (P < 0.05).

Production of NO · and O<SUP>−</SUP><SUB>2</SUB>⋅ by PMNs in vitro. Cells from the control group produced 2.93 ± 0.52 nmol NO · · h-1 · 106 cells-1, as measured by the oxyhemoglobin method (Fig. 2). No differences were found if NO · was measured as NO<SUP>−</SUP><SUB>2</SUB> plus NO<SUP>−</SUP><SUB>3</SUB> (3.28 ± 0.58 nmol · h-1 · 106 cells-1), when NO<SUP>−</SUP><SUB>2</SUB> accounted for ~60% of the total NO · (data not shown). Low levels of NO · were generated by cells from the L-NIO-treated group (0.73 ± 0.27 nmol · h-1 · 106 cells-1). Cells from the L-arginine group produced the same amount of NO · as their control (Fig. 2).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   NO · generated by inflammatory cells in vitro. Cells from exudates of 3 different treatments (control, L-NIO, and L-Arg groups) were incubated in PBS with 0.6 mM L-Arg for 1 h. Values (nmol NO · · h-1 · 106 cells-1) are means ± SE of 6 separate experiments performed in duplicate. Data were analyzed by 1-way ANOVA, using Student-Newman-Keuls test to identify significant differences between groups. Different letters above bars indicate significant differences (P < 0.05).

The production of O<SUP>−</SUP><SUB>2</SUB>⋅ in L-NIO-treated rats showed no difference with respect to the control group (Table 2). When L-arginine was injected into the granuloma pouch, cells produced 46% less O<SUP>−</SUP><SUB>2</SUB>⋅ than the control group (Table 2). The presence of L-NIO in the incubation medium did not increase O<SUP>−</SUP><SUB>2</SUB>⋅ generation even in the L-arginine-treated group. The addition of L-arginine (0.6 mM) to the incubation medium of cells from the control group decreased the release of O<SUP>−</SUP><SUB>2</SUB>⋅ (Fig. 3) only when cells were preincubated with L-arginine for 1 h before PMA stimulation (0.24 nmol O<SUP>−</SUP><SUB>2</SUB>⋅ · h-1 · 106 cells-1) and not when L-arginine was added simultaneously with PMA (4.22 nmol O<SUP>−</SUP><SUB>2</SUB>⋅ · h-1 · 106 cells-1).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Superoxide generated by inflammatory cells in vitro


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of L-Arg on O<SUP>−</SUP><SUB>2</SUB>⋅ generation by inflammatory cells in vitro. O<SUP>−</SUP><SUB>2</SUB>⋅ was measured in cells from control group in presence or in absence of 0.6 mM L-Arg. L-Arg was added to medium 1 h before phorbol 12-myristate 13-acetate (preincubated) or added simultaneously (incubated with L-Arg). Values (nmol O<SUP>−</SUP><SUB>2</SUB>⋅ · h-1 · 106 cells-1) are means ± SE of 6 separate experiments performed in duplicate. Data were analyzed by 1-way ANOVA, using Student-Newman-Keuls test to identify significant differences. Different letters indicate significant differences (P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

During the first 24 h of the acute phase of inflammation, the granuloma pouch contains large numbers of PMNs (1). When cells are exposed to inflammatory mediators generated in response to carrageenan, respiratory burst takes place. The levels of NO<SUP>−</SUP><SUB>2</SUB> plus NO<SUP>−</SUP><SUB>3</SUB> found in the exudate of inflamed rats give clear evidence that NO · is produced in vivo.

There has been much interest in the role of NO · in inflammation. However, it is not clear whether NO · acts as a pro- or anti-inflammatory agent, since both activities have been reported. A proinflammatory role for NO · has often been inferred, since NO · inhibitors reduce plasma extravasation in carrageenan-induced paw edema inflammation (12). In contrast, anti-inflammatory roles of NO · are suggested by the reduction of atherogenesis in the hypercholesterolemic rabbit following the supplementation of dietary L-arginine, the substrate of NO · (5), and by the beneficial effects of inhaled NO · in pulmonary edema (21). The reaction between O<SUP>−</SUP><SUB>2</SUB>⋅ and NO · to give ONOO- can explain the paradoxical behavior of NO ·. NO · is cytotoxic only if there is sufficient O<SUP>−</SUP><SUB>2</SUB>⋅ to yield ONOO-; if there is not, it produces vasodilation. The production rates and the steady-state concentrations of NO · and O<SUP>−</SUP><SUB>2</SUB>⋅ at sites of injury may resolve whether the net effect is oxidation and destruction of cellular components and tissues or cell repair. It has recently been suggested that an excess of NO · may act as an antioxidant (20). To test this hypothesis, we modified the levels of endogenously produced NO · by injecting L-NIO and L-arginine into the site of an acute inflammation.

When L-NIO was injected into the granuloma pouch, the expected decrease in the concentration of NO<SUP>−</SUP><SUB>2</SUB> plus NO<SUP>−</SUP><SUB>3</SUB> in the exudate (as an index of NO ·) was observed. When PMNs from these exudates were collected and their ability to produce NO · was investigated, an irreversible inhibition of the inducible isoform of nitric oxide synthase activity was detected, such that little NO · was produced even when L-arginine was added to the incubation medium. These results indicate that the L-NIO administered into the pouch was effectively internalized by the cells and confirm that L-NIO may be a useful tool for studying reactions that involve NO · derived from PMNs. Because NO · was very low, an increase in O<SUP>−</SUP><SUB>2</SUB>⋅ production would be expected after incubation of cells from the L-NIO-treated group (18, 19). However, we observed that cells incubated in an L-arginine-free medium produced low and similar levels of NO · in control and L-NIO groups (19). It has been described that nitric oxide synthase can produce O<SUP>−</SUP><SUB>2</SUB>⋅ in an L-arginine-depleted medium (24). If this occurred here, O<SUP>−</SUP><SUB>2</SUB>⋅ detected in the control group may come from NADPH oxidase and from nitric oxide synthase, whereas in the L-NIO group it would come only from NADPH oxidase, which may explain the lack of differences detected in O<SUP>−</SUP><SUB>2</SUB>⋅.

One hour after injection of 6 µmol of L-arginine into the granuloma pouch, we only detected 3.62 µmol in the exudate. The 32% increase in NO · levels in the exudate means that some of the L-arginine injected was very quickly taken up by inflammatory cells. Two of our findings in the present study demonstrate that NO · inactivates a component required for the activation of the NADPH oxidase: first, the 46% reduction in O<SUP>−</SUP><SUB>2</SUB>⋅ after the treatment with L-arginine in vivo, which may be partially reversed by the addition of L-NIO to the incubation medium, in which case only a 32% decrease in O<SUP>−</SUP><SUB>2</SUB>⋅ is found, and, second, the inhibition of O<SUP>−</SUP><SUB>2</SUB>⋅ after preincubation of the cells for 1 h with L-arginine before PMA stimulation. This inhibition effect, according to Clancy et al. (4), takes place before the assembly of the multiprotein NADPH oxidase system. The NADPH oxidase is composed of membrane-bound proteins, including cytochrome b558, that could be iron nitrosylated through the effect of NO ·. This reaction requires oxygen, and we have demonstrated that such aerobic conditions are found in the granuloma pouch (1).

We show in this paper that NO · is produced in vivo in the granuloma pouch during acute inflammation. The excess of endogenously produced NO · will act as an anti-inflammatory mechanism, inhibiting the NADPH oxidase in the membrane of PMNs and limiting the availability of O<SUP>−</SUP><SUB>2</SUB>⋅, and represents a defense against free radical-mediated injury. Our results confirm that the relative rates of NO · and O<SUP>−</SUP><SUB>2</SUB>⋅ may be critical in determining the outcome of biological oxidations.

    ACKNOWLEDGEMENTS

We thank Robin Rycroft of the Language Advisory Service of the University of Barcelona for help in editing the manuscript.

    FOOTNOTES

This work was supported by Fondo de Investigaciones Sanitarias Grant 96-0766.

Address for reprint requests: M. T. Mitjavila, Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain.

Received 16 July 1997; accepted in final form 2 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Alfaro, V., J. Ródenas, J. Pesquero, M. T. Mitjavila, L. Palacios, and T. Carbonell. Factors influencing the acid-base changes in the air-pouch exudate following carrageenan induced inflammation in rats. Inflamm. Res. 45: 405-411, 1996[Medline].

2.   Babior, B. M. Oxygen-dependent microbial killing of phagocytes. N. Engl. J. Med. 298: 659-668, 1978[Medline].

3.   Carreras, M. C., G. A. Pargament, S. D. Catz, J. J. Poderoso, and A. Boveris. Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils. FEBS Lett. 341: 65-68, 1994[Medline].

4.   Clancy, R. M., J. Leszczynska-Piziak, and S. B. Abramson. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J. Clin. Invest. 90: 1116-1121, 1992[Medline].

5.   Cooke, J. P., A. H. Singer, P. Tsao, P. Zera, R. Rowan, and M. E. Billingham. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J. Clin. Invest. 90: 1168-1172, 1992[Medline].

6.   Feelisch, M., and E. A. Noak. Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur. J. Pharmacol. 139: 19-30, 1987[Medline].

7.   Fridovich, I. Biological effects of the superoxide radical. Arch. Biochem. Biophys. 274: 1-11, 1986.

8.   Fukuhara, M., and S. Tsurufuji. The effect of locally injected antiinflammatory drugs on the carrageenan granuloma in rats. Biochem. Pharmacol. 18: 475-484, 1969[Medline].

9.   Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. Analysis of nitrate, nitrite and 15N nitrate in biological fluids. Anal. Biochem. 126: 131-138, 1982[Medline].

10.   Guidot, D. M., M. J. Repine, B. M. Hybertson, and J. E. Repine. Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L2-L5, 1995[Abstract/Free Full Text].

11.   Huie, R. E., and S. Padmaja. The reaction of NO with superoxide. Free Radic. Res. Commun. 18: 195-199, 1993[Medline].

12.   Ialenti, A., A. Ianaro, S. Moncada, and M. Di Rosa. Modulation of acute inflammation by endogenous nitric oxide. Eur. J. Pharmacol. 211: 177-182, 1992[Medline].

13.   Johnston, R. B., Jr., C. A. Godzik, and Z. A. Cohn. Increased superoxide anion production by immunologically activated and chemically elicited macrophages. J. Exp. Med. 148: 115-119, 1978[Abstract/Free Full Text].

14.   Marletta, M. A., P. S. Yoon, R. Iyengar, C. D. Leaf, and J. S. Wishnok. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 27: 8706-8711, 1988[Medline].

15.   McCall, T. B., M. Feelisch, R. M. J. Palmer, and S. Moncada. Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. Br. J. Pharmacol. 102: 234-238, 1991[Abstract].

16.   Palmer, R. M. J., D. S. Ashton, and S. Moncada. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333: 664-666, 1988[Medline].

17.   Radi, R., J. S. Beckman, K. M. Bush, and B. A. Freeman. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys. 288: 481-487, 1991[Medline].

18.   Ródenas, J., T. Carbonell, and M. T. Mitjavila. Conditions to study nitric oxide generation by polymorphonuclear cells from an inflammatory exudate in rats. Arch. Biochem. Biophys. 327: 292-294, 1996[Medline].

19.   Ródenas, J., M. T. Mitjavila, and T. Carbonell. Simultaneous generation of nitric oxide and superoxide by inflammatory cells in rats. Free Radic. Biol. Med. 18: 869-875, 1995[Medline].

20.   Rubbo, H., V. Darley-Usmar, and B. A. Freeman. Nitric oxide regulation of tissue free radical injury. Chem. Res. Toxicol. 9: 809-820, 1996[Medline].

21.   Scherrer, U., L. Vollenweider, A. Delabays, M. Savcic, U. Eichenberger, G. R. Kleger, A. Firkre, P. E. Ballmer, P. Nicod, and P. Baertsc. Inhaled nitric oxide for high-altitude pulmonary edema. N. Engl. J. Med. 334: 624-629, 1996[Abstract/Free Full Text].

22.   Sies, H. Oxidative stress: introductory remarks. In: Oxidative Stress, edited by H. Sies. San Diego, CA: Academic, 1985, p. 1-7.

23.   Stadler, J., T. R. Billiar, R. D. Curran, D. J. Steuehr, J. B. Ochoa, and R. L. Simmons. Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am. J. Physiol. 260 (Cell Physiol. 29): C910-C916, 1991[Abstract/Free Full Text].

24.   Xia, Y., V. L. Dawson, T. D. Dawson, S. H. Snyder, and J. L. Zweier. Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc. Natl. Acad. Sci. USA 93: 6770-6774, 1996[Abstract/Free Full Text].


AJP Cell Physiol 274(3):C827-C830
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society