Synergistic regulation of NOS II expression by IL-1beta and TNF-alpha in cultured rat colonic smooth muscle cells

John F. Kuemmerle

Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Interleukin-1beta (IL-1beta ), tumor necrosis factor-alpha (TNF-alpha ), and lipopolysaccharide (LPS) were examined for their ability to regulate the activity and protein levels of inducible nitric oxide synthase (NOS II) in cultured rat colonic smooth muscle cells. Treatment with these agents resulted in a time-dependent increase in NOS II activity. After 48 h, NOS II activity, measured as L-[3H]citrulline production, was increased 24.3 ± 6.9 pmol · min-1 · mg protein-1 by 1 nM IL-1beta and 3.2 ± 1.1 pmol · min-1 · mg protein-1 by 1 nM TNF-alpha , and increased synergistically by a combination of the two (51.8 ± 7.3 pmol · min-1 · mg protein-1). Measurement of NOS II activity as nitrite production yielded similar results: IL-1beta , 27.2 ± 1.2; TNF-alpha , 1.6 ± 0.1; and IL-1beta + TNF-alpha , 46.8 ± 3.2 pmol · min-1 · mg protein-1 above basal. LPS (10 µg/ml) had a small but significant effect at 48 h that was only additive with that of IL-1beta . The increase in NOS II activity induced by IL-1beta and TNF-alpha was inhibited 73-86% by transforming growth factor-beta 1 (TGF-beta 1). The NOS isoform induced by IL-1beta and TNF-alpha was identified as NOS II by Western immunoblot analysis and confirmed by its 66-97% inhibition by 100 µM S-methylisothiourea, a selective NOS II inhibitor, and its Ca2+-independent activity. We conclude that the cytokines IL-1beta and TNF-alpha act independently and synergistically to stimulate NOS II expression and enzymatic activity in rat colonic smooth muscle through a mechanism sensitive to inhibition by TGF-beta 1.

nitric oxide; inducible nitric oxide synthase; lipopolysaccharide; transforming growth factor-beta ; interleukin-1beta ; tumor necrosis factor-alpha

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THREE DISTINCT ISOFORMS of nitric oxide synthase (NOS I, II, and III), the enzyme responsible for production of nitric oxide (NO), have been identified (2, 19, 23). The NOS I and NOS III isoforms are constitutively expressed, and the expression of the NOS II isoform is inducible. NOS I is expressed by neurons of the central and peripheral nervous system (2) and by fast muscle fibers of skeletal muscle (12). Within the gastrointestinal tract, NOS I is expressed by nerves of the myenteric plexus (21). NOS III is expressed by endothelial cells (23), smooth muscle cells (26), epithelial cells of the bronchial tree (24), and pyramidal cells of the hippocampus (5). Within the gastrointestinal tract, NOS III is expressed by smooth muscle cells (26) and by the interstitial cells of Cajal (33). The activity of NOS I and NOS III is regulated by the level of cytosolic Ca2+. NOS II may be expressed in macrophages (8) as well as in a number of cell types throughout the body. Within the gastrointestinal tract NOS II expression can be induced in epithelial cells (27) and in cells of the muscularis propria (30). In contrast to NOS I and NOS III, NOS II, once expressed, is present in much greater amounts and is continuously active due to the tight binding of calmodulin even at basal levels of cytosolic Ca2+. These properties result in the production of much greater amounts of NO by NOS II, typically within the micromolar range, compared with NOS I and NOS III (3).

NOS I is found in nerves of the myenteric plexus, where NO acts as an inhibitory neurotransmitter and facilitates vasoactive intestinal polypeptide (VIP) release from nerve terminals (11). NOS III is found within the smooth muscle cells (11, 26), where it is activated by VIP and pituitary adenylate cyclase-activating polypeptide, leading to generation of NO and muscle relaxation (11, 16), and in the interstitial cells of Cajal (33) located at the boundary between muscle and nerve, which are responsible for electrical slow wave activity in the gastrointestinal tract. The role of NOS III in the interstitial cells of Cajal is not clear.

NOS II expression is induced in the intestine in ulcerative colitis and Crohn's disease (1, 15). NOS II expression has been induced in in vivo models of colitis in animals after treatment with agents such as 2,4,6-trinitrobenzenesulfonic acid, lipopolysaccharide (LPS), or peptidoglycan-polysaccharide (10, 14, 27, 31). NOS II expression and activity in these settings is induced in mucosal cells, tissue macrophages, the myenteric plexus, and the muscularis propria. NOS II induction results in muscle hyperplasia and decreased contractility, effects that are inhibited by NOS inhibitors (10, 31). These studies were performed in whole thicknesses of intestine or in muscle strips where, in addition to muscle cells, other cell types such as epithelial and inflammatory cells, neurons, and glia may contribute to the observed events. NOS II expression can be induced directly in cultured vascular, pulmonary, and uterine smooth muscle cells by exposure to the cytokines interleukin-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ) or LPS (17-20). The ability of cytokines to cause induction of NOS II expression in gastrointestinal smooth muscle cells has not been demonstrated directly.

In the present study IL-1beta , TNF-alpha , and LPS from Escherichia coli serotype O55:B5 were examined for their ability to directly induce NOS II expression and NO generation in smooth muscle cells cultured from the rat colon. The results show that IL-1beta , TNF-alpha , and LPS induce NOS II expression and increase NO production by cultured colonic smooth muscle cells in a time-dependent fashion. The effects of IL-1beta and TNF-alpha are synergistic. Cytokine-induced NOS II expression occurs through a TGF-beta 1-sensitive mechanism.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Preparation of isolated muscle cells from rat colon. Muscle cells were isolated separately from the longitudinal and circular muscle layers of the colon of male Sprague-Dawley rats by adaptation of methods described previously (11, 13, 16). Briefly, the colon was cut into 2-cm segments, and the longitudinal layer was removed by tangential stroking. The segments were opened longitudinally, and the mucosa was removed by blunt dissection. The remaining circular muscle segments were incubated at 31°C in 25 ml of medium containing 0.15% collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. The medium consisted of (in mM) 120 NaCl, 4 KCl, 2.6 KH2PO4, 2 CaCl2, 0.6 MgCl2, 25 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 14 glucose, and 2.1% Eagle's essential amino acid mixture. After a 60-min incubation, the partially digested muscle strips were washed free of enzymes and incubated in enzyme-free medium for 30 min to allow the cells to disperse spontaneously.

Cell culture of rat colonic muscle cells. Primary cultures of rat colonic muscle cells were initiated and maintained by modification of previously described methods (13). Cells dispersed from the circular muscle layer were harvested by filtration through 500-µm Nitex mesh and then centrifuged at 150 g for 5 min. Cells were resuspended and washed twice by centrifugation at 150 g for 5 min and resuspension in Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamicin, and 2.5 µg/ml amphotericin B. After the final washing, cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (DMEM-10) and the same antibiotics. The cells were plated at a concentration of 5 × 105 cells/ml as determined by counting in a hemacytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 was replaced every 3 days until the cells reached confluence.

Primary cultures of colonic muscle cells were passaged on reaching confluence by first washing three times with phosphate-buffered saline (PBS). After the PBS was removed, cells were treated for 2 min with 0.05% trypsin and 0.53 mM EDTA. The trypsin activity was neutralized by addition of a fourfold excess of DMEM-10. The resulting cell suspension was centrifuged at 350 g for 10 min at 4°C. The pellet was washed twice by centrifugation at 350 g and resuspension in HBSS. The final cell pellet was resuspended in DMEM-10 at a concentration of 2.5 × 106 cells/ml and plated in either 100-mm dishes or 24- or six-well plates, depending on the experiment to be performed. The medium was changed after 24 h and then every 3 days thereafter. All subsequent studies were performed in cultured muscle cells in first passage on day 7, when cells attained confluence.

Previous studies (13) have shown that muscle cells treated in this manner yield cultures of smooth muscle cells, with 97 ± 2% of cells expressing phenotypic markers characteristic of smooth muscle cells when examined by immunofluorescence. The cultures are essentially devoid of nonmuscle, i.e., neural, epithelial, and inflammatory cells.

Induction of NOS activity. Confluent muscle cell cultures were washed three times with PBS and then incubated in phenol red-free DMEM-10 in the presence of 1 nM IL-1beta , 1 nM TNF-alpha , and 10 µg/ml LPS alone and in combination for various time periods of 0, 1, 2, 4, 6, 12, 24, or 48 h. The effect of inhibitors was examined after a 1-h preincubation with inhibitor followed by treatment with IL-1beta , TNF-alpha , or LPS.

Measurement of NO formation. NOS activity was measured in cultures of muscle cells loaded with L-[3H]arginine and expressed as the amount of L-[3H]citrulline produced, by modification of previously described techniques (11, 16). L-[3H]citrulline and NO are produced in equimolar amounts by the action of NOS on L-[3H]arginine. Muscle cells growing in six-well dishes were treated with cytokines as described above. The medium was aspirated, and the cells were washed three times with 2 ml of HBSS. The cells were then incubated for 30 min at 37°C with L-arginine-free Earle's balanced salt solution (EBSS). This medium was aspirated, and the cells were incubated at 37°C for various time periods for up to 30 min in 0.75 ml EBSS containing 5 µM L-arginine and 2 µCi/ml L-[3H]arginine (sp act 44.2 Ci/mmol). The reaction was terminated, and the cells were extracted by addition of 0.75 ml of 10% tricholoracetic acid at 4°C. The extracts were centrifuged at 10,000 g for 15 min at 4°C, and 1-ml aliquots of the resulting supernatant were removed and extracted three times with 2 ml of water-saturated diethyl ether. Aliquots (0.5 ml) of the resulting aqueous phase were removed and neutralized by addition of 2 ml of 20 mM HEPES (pH 6) solution. L-[3H]citrulline was separated from unconverted L-[3H]arginine by flow-through chromatography in Dowex (AG50W-X8) columns. The amount of L-[3H]citrulline was taken to represent the stoichiometric production of NO. Results were expressed as the increase in NOS activity in picomoles L-[3H]citrulline per minute per milligram protein above basal levels in untreated control cultures.

In some experiments the Ca2+ dependence of the conversion of L-[3H]arginine to L-[3H]citrulline was investigated in cells incubated in Ca2+-free EBSS containing the chelating agent ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0 Ca2+, 4 mM EGTA). The ability of selective inhibitors of NOS activity, NG-nitro-L-arginine methyl ester (L-NAME) and S-methylisothiourea (SMT), to inhibit L-[3H]citrulline conversion was examined in other experiments. L-NAME and SMT inhibit NOS activity by virtue of their actions as competitive substrate inhibitors at the L-arginine binding site (25). SMT exhibits 10- to 30-fold higher potency as an inhibitor of NOS II in vascular smooth muscle cells, with a half-maximal inhibitory concentration of 2 µM, than for NOS I or III (25). In concentrations of up to 1 mM SMT does not inhibit the activity of other cellular enzymes, including xanthine oxidase, diaphorase, lactate dehydrogenase, monoamine oxidase, catalase, cytochrome P-450, or superoxide dismutase (25).

Measurement of total nitrite production. NO was also measured from total nitrite formation with the use of the Greiss reaction, by modification of the techniques of Green et al. (9). Briefly, cultures of muscle cells growing in six-well plates were washed free of phenol red and incubated with cytokines as described above. At various time points the culture medium was aspirated and centrifuged at 10,000 g for 5 min to remove cellular debris. A 1-ml aliquot was removed and added to a 1-ml aliquot of Greiss reagent. Greiss reagent was prepared fresh daily by mixing equal volumes of a 0.1% solution of N-(1-naphthyl)ethylenediamine dihydrochloride and 1% solution of sulfanilamide in 5% phosphoric acid. Absorbance was measured at 545 nm, and nitrite concentration was calculated from a standard curve of sodium nitrite. The response was linear over the range 0.1 to 100 µM nitrite. The amount of nitrite was taken to represent the production of NO (9). Results were expressed as the increase in NOS activity in picomoles nitrite per minute per milligram protein above basal levels in untreated control cultures.

In some experiments, the effect of selective inhibitors of NOS activity, L-NAME (1 mM) and SMT (10 µM), on nitrite production was examined by addition of inhibitor 1 h before the addition of cytokine or LPS. Although inhibitors of NOS interfere with the analysis of nitrite by the Greiss reaction in some systems, no changes in the nitrite standard curve were observed in the presence of 1 mM L-NAME or 100 µM SMT in the present study.

Western blotting of NOS II. The expression of NOS II protein was examined by Western blot analysis in cultured cells and in cells freshly dispersed from the circular layer of rat colon. Cultured but not freshly dispersed cells were treated for 48 h with 1 nM IL-1beta alone and in combination with 1 nM TNF-alpha . The freshly dispersed muscle cells or cultured muscle cells growing in 100-mm plates were washed three times with ice-cold PBS, and whole cell lysates were prepared by adding 1.5 ml of boiling sample buffer to the cells. The resulting cell lysates were boiled for an additional 5 min and then placed on ice. Samples were sonicated to shear DNA and then centrifuged at 12,000 g at 4°C for 15 min. The resulting supernatant was heated to 95°C for 5 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4-12% gradient acrylamide gels in aliquots (20 µl) containing equal amounts of protein (30 µg). The separated proteins were electrotransferred to polyvinylidene difluoride membranes. After transfer the membranes were incubated in blocking buffer consisting of PBS, pH 7.6, and containing 10% milk protein for 1 h at room temperature. The membranes were then incubated for 2 h at room temperature in blocking solution containing a 1:1,000 dilution of a polyclonal immunoglobulin G (IgG) antibody raised in rabbits to a synthetic peptide derived from the COOH-terminal end of the murine macrophage NOS, amino acids 1131-1144. This antibody reacts fully with rat NOS II protein but has no cross-reactivity with either NOS I or NOS III protein. Membranes were washed three times with PBS containing 0.1% Tween 20 and 1% milk protein. Membranes were incubated for 1 h in blocking buffer containing a 1:2,000 dilution of a horseradish peroxidase-conjugated antibody to rabbit IgG raised in goat. The membranes were washed an additional three times in PBS containing 0.3% Tween 20 and 1% milk protein. The protein bands were visualized by enhanced chemiluminescence.

Statistical and densitometric analysis. Values are means ± SE of n experiments, where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student's t-test for either paired or unpaired values as appropriate. Densitometric analysis was performed using computerized densitometry and ImageQuant NT software (Molecular Dynamics). Densitometric values for protein bands were determined in areas of equal size and are reported in arbitrary units above background values.

Materials. Recombinant human IL-1beta and TNF-alpha and human TGF-beta 1 were obtained from Collaborative Biomedical Products (Bedford, MA); horseradish peroxidase-conjugated goat anti-rabbit IgG was obtained from Amersham (Arlington Heights, IL); rabbit polyclonal antibody to inducible NOS was obtained from Affinity Bioreagents (Neshanic Station, NJ); L-[3H]arginine (sp act 44.2 Ci/mmol) was obtained from DuPont-NEN (Boston, MA); Bio-Rad protein reagent was obtained from Bio-Rad Laboratories (Hercules, CA); DMEM, HBSS, and EBSS were obtained from Mediatech (Herndon, VA); fetal bovine serum was obtained from BioWhittaker (Walkersville, MD); collagenase (CLS type II) and soybean trypsin inhibitor were obtained from Worthington Biochemicals (Freehold, NJ); HEPES was obtained from Research Organics (Cleveland, OH); and culture plasticware was obtained from Corning (Corning, NY). LPS from E. coli serotype O55:B5, cyclohexamide, and all other chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Stimulation of NOS activity by TNF-alpha , IL-1beta , and LPS. Confluent cultures of rat colonic muscle cells were treated for 0, 1, 2, 4, 6, 12, 24, and 48 h with TNF-alpha (1 nM), IL-1beta (1 nM), or a combination of both cytokines. Basal nitrite production in cultured muscle cells remained constant in the absence of cytokines (range: 1.6 ± 0.6 to 4.1 ± 2.5 pmol nitrite · min-1 · mg protein-1). IL-1beta caused a time-dependent increase in NOS activity (Fig. 1A) that became significant after 6 h (2.7 ± 1.1 pmol · min-1 · mg protein-1 above basal; P < 0.05) and was sustained for 48 h (27.2 ± 1.6 pmol · min-1 · mg protein-1 above basal; P < 0.001). TNF-alpha caused a small increase in NOS activity (Fig. 1A) that became significant after 48-h incubation (1.6 ± 0.1 pmol · min-1 · mg protein-1 above basal; P < 0.01). Addition of a combination of 1 nM IL-1beta and 1 nM TNF-alpha caused a significant increase in NOS activity over that observed with IL-1beta alone (24 h: 31.6 ± 3.5 vs. 23.9 ± 2.5 pmol · min-1 · mg protein-1 above basal; P < 0.05; 48 h: 46.8 ± 3.2 vs. 27.2 ± 1.0 pmol · min-1 · mg protein-1 above basal; P < 0.005) (Figs. 1A and 2A). The increase after 48 h was synergistic and was significantly greater than the sum of the responses to IL-1beta and TNF-alpha (sum: 28.4 ± 1.1 pmol · min-1 · mg protein-1 above basal; combination: 46.8 ± 3.2 pmol · min-1 · mg protein-1 above basal; P < 0.001). LPS (10 µg/ml) caused a small increase in NOS activity after 48 h (2.6 ± 0.5 pmol · min-1 · mg protein-1 above basal; P < 0.01) (Fig. 2A). Unlike TNF-alpha , the combination of LPS and IL-1beta was additive rather than synergistic after 48-h incubation (sum: 29.8 ± 1.2 pmol · min-1 · mg protein-1 above basal; combination: 31.3 ± 1.6 pmol · min-1 · mg protein-1 above basal) (Fig. 2A).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Nitric oxide synthase (NOS) activity in confluent cultures of rat colonic muscle cells treated with 1 nM tumor necrosis factor-alpha (TNF-alpha ; black-triangle), 1 nM interleukin-1beta (IL-1beta ; black-square), and a combination of IL-1beta and TNF-alpha (bullet ) for various periods up to 48 h. A: increase in NOS activity was measured from increase in total nitrite production by the Greiss reaction. Results are expressed as increase in nitrite production above basal levels (range: 1.6 ± 0.6 to 4.1 ± 2.5 pmol nitrite · min-1 · mg protein-1), which remained constant over 48 h in untreated control cultures. TNF-alpha caused an increase in nitrite production that became significant after 48-h incubation. IL-1beta caused a time-dependent increase in nitrite production that was significant by 6 h. TNF-alpha  + IL-1beta combination was synergistic after 24- and 48-h incubation. B: increase in NOS activity was measured by conversion of L-[3H]arginine to L-[3H]citrulline. Results are expressed as increase above basal L-[3H]citrulline production, which remained constant over 48 h in untreated control cultures. TNF-alpha caused a small increase in L-[3H]citrulline production that was significant after 48-h incubation. IL-1beta caused a time-dependent increase in L-[3H]citrulline production that was significant after 6 h. Combination of TNF-alpha and IL-1beta was synergistic after 24- and 48-h incubation. Values are means ± SE of 4-6 experiments done in duplicate. * P < 0.05, ** P < 0.01, and +P < 0.001 above basal.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Synergistic stimulation of NOS activity by TNF-alpha and IL-1beta . Confluent muscle cells were incubated with 1 nM IL-1beta , 1 nM TNF-alpha , or 10 µg/ml lipopolysaccharide (LPS) alone and in combination for 48 h. A: increase in NOS activity was measured as total nitrite production. Results are expressed as increase in total nitrite production above basal levels (range: 1.6 ± 0.6 to 4.1 ± 2.5 pmol nitrite · min-1 · mg protein-1). B: NOS activity was also measured from L-[3H]citrulline production. Results are expressed as increase in L-[3H]citrulline production above basal values (range: 23.9 ± 0.9 to 25.2 ± 1.1 pmol citrulline · min-1 · mg protein-1). Whether measured as nitrite or L-[3H]citrulline production, the effect of LPS alone on NOS activity, like that of TNF-alpha , was small but significant. The effect of IL-1beta was additive with that of LPS but synergistic with that of TNF-alpha . Values are means ± SE of 4-7 experiments done in duplicate. * P < 0.05, ** P < 0.01, +P < 0.001 above basal.

A similar pattern of NOS stimulation was observed when NOS activity was measured by the conversion of L-[3H]arginine to L-[3H]citrulline. Basal NOS activity remained constant in the absence of cytokines (range: 23.9 ± 0.8 to 25.2 ± 1.1 pmol L-[3H]citrulline · min-1 · mg protein-1; not significant). IL-1beta caused a time-dependent increase in NOS activity (Fig. 1B), which became significant after 6 h (2.4 ± 1.1 pmol · min-1 · mg protein-1 above basal, P = 0.05) and was sustained for 48 h (24.3 ± 6.9 pmol · min-1 · mg protein-1 above basal; P < 0.01). TNF-alpha caused a small increase in NOS activity (Fig. 1B) that became significant only after 48-h incubation (3.2 ± 1.1 pmol · min-1 · mg protein-1 above basal; P < 0.05). Addition of a combination of 1 nM IL-1beta and 1 nM TNF-alpha for 48 h caused a significant increase in NOS activity over that observed with IL-1beta alone (51.8 ± 7.3 vs. 24.3 ± 6.9 pmol · min-1 · mg protein-1 above basal; P < 0.02) (Figs. 1B and 2B). The increase after 48 h was synergistic and was significantly greater than the sum of the responses to IL-1beta and TNF-alpha (sum: 27.3 ± 3.8 pmol · min-1 · mg protein-1 above basal; combination: 51.8 ± 7.3 pmol · min-1 · mg protein-1 above basal; P < 0.01). LPS (10 µg/ml) caused a small increase in NOS activity after 48 h (4.0 ± 1.6 pmol · min-1 · mg protein-1 above basal; P < 0.05) (Fig. 2B). Unlike TNF-alpha , the combination of LPS and IL-1beta was additive rather than synergistic after 48-h incubation (sum: 25.8 ± 3.3 pmol · min-1 · mg protein-1 above basal; combination: 31.3 ± 1.6 pmol · min-1 · mg protein-1 above basal) (Fig. 2B).

The time-dependent increase in NOS activity stimulated by 1 nM IL-1beta was inhibited in the presence of 20 µg/ml cyclohexamide at all time periods. The effect of cyclohexamide on IL-1beta -stimulated NOS activity was most pronounced after 48-h incubation, by which time cytokine-stimulated nitrite production was inhibited 96 ± 1% (P < 0.001) and L-[3H]citrulline production was inhibited 64 ± 3% (P < 0.001). A similar pattern of effect of cyclohexamide was observed on the synergistic effects of a combination of 1 nM IL-1beta and 1 nM TNF-alpha . After 48-h incubation in the presence of cyclohexamide, nitrite production stimulated by the combination of IL-1beta and TNF-alpha was inhibited 95 ± 1% (P < 0.001), and L-[3H]citrulline production was inhibited 86 ± 7% (P < 0.001). The results imply that the increase in NOS activity stimulated by IL-1beta and TNF-alpha involved new NOS protein synthesis.

Inhibition of NOS activity by TGF-beta 1. In other cell types, including vascular and pulmonary smooth muscle, TGF-beta 1 inhibits cytokine-stimulated NOS II activity (7, 8, 22). Previous studies have shown that cultured intestinal smooth muscle cells secrete TGF-beta 1 in a time-dependent fashion (13). Concentrations of TGF-beta 1 are low early in culture and at confluence (after 7 days in culture), increasing 10-fold in postconfluent cells (after 14 days in culture). To determine whether TGF-beta 1 might inhibit NOS activity stimulated by cytokines in these cells, confluent cultures were treated for 48 h with 1 nM IL-1beta , 1 nM TNF-alpha , or a combination of both cytokines in the presence or absence of 1 nM TGF-beta 1. In the presence of TGF-beta 1 the ability of IL-1beta , TNF-alpha , or LPS to increase NOS activity measured by nitrite production was significantly inhibited (range: 75 ± 13% to 86 ± 6% inhibition) (Fig. 3).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Inhibition by transforming growth factor-beta 1 (TGF-beta 1) of NOS II activity stimulated by TNF-alpha , LPS, and IL-1beta . Cultures of confluent muscle cells were incubated for 48 h with 1 nM TNF-alpha , 1 nM IL-1beta , and 10 µg/ml LPS, alone and in combination in presence (open bars) and absence (solid bars) of 1 nM TGF-beta 1. Results are expressed as increase in total nitrite production over basal untreated levels (range: 1.6 ± 0.6 to 4.1 ± 2.5 pmol nitrite · min-1 · mg protein-1). TGF-beta 1 significantly inhibited stimulation of NOS II activity by TNF-alpha , LPS, and IL-1beta alone or in combination (range 75 ± 13% to 86 ± 6% inhibition). Values are means ± SE of 4-6 experiments done in duplicate. ** P < 0.01 and +P < 0.001 for inhibition of response.

Characterization of NOS II protein by Western blot analysis. The expression of NOS II in both freshly isolated and cultured muscle cells was investigated further by Western blot analysis. The NOS II specific antibody identified a protein band migrating with an apparent molecular mass of ~130 kDa, the known molecular size of authentic rat NOS II protein. In control experiments a band of ~130-kDa molecular mass was identified by the NOS II specific antibody in cell lysates derived from interferon-gamma - and LPS-stimulated RAW264.7 macrophages, which are known to express NOS II protein (data not shown). In freshly isolated colonic smooth muscle cells, NOS II protein was essentially undetectable (Fig. 4A). NOS II protein was detectable in untreated confluent muscle cells in culture. Treatment with either 1 nM IL-1beta or a combination of 1 nM TNF-alpha and IL-1beta caused an increase in the levels of NOS II protein detected (Fig. 4A).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   NOS II protein levels increase in response to IL-1beta and TNF-alpha . A: representative Western blot of NOS II protein in 30-µg aliquots of whole cell lysate prepared from untreated freshly dispersed muscle cells (lane 1) and cultured muscle cells (lane 2) and in cultured cells after 48-h treatment with 1 nM IL-1beta (lane 3) or a combination of 1 nM IL-1beta and 1 nM TNF-alpha (lane 4). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NOS II protein was visualized using an NOS II specific antibody as described in METHODS. NOS II protein was essentially undetectable in freshly dispersed muscle cells; after 7 days in culture an ~130-kDa protein of similar molecular size to that of authentic rat NOS II protein was detected in muscle cells. Levels of NOS II protein were increased by treatment with IL-1beta and TNF-alpha . B: computerized densitometric analysis was used to quantitate relative increases in NOS II protein levels. Results are expressed as the increase in arbitrary units above background as described in METHODS. NOS II protein was essentially undetectable in freshly dispersed cells. Levels of NOS II protein present in cultured muscle cells increased after treatment with 1 nM IL-1beta and were increased further by combination of IL-1beta and 1 nM TNF-alpha . Values are means ± SE of 4 separate experiments.

Densitometric analysis of protein bands revealed a significant increase in NOS II protein levels from 9 ± 1 units above background in freshly isolated cells to 260 ± 56 units in untreated cultured muscle cells (Fig. 4B). After treatment of the cultured cells for 48 h with 1 nM IL-1beta , NOS II protein levels increased to 560 ± 180 units above background. Addition of 1 nM TNF-alpha to IL-1beta caused an additional increase to 814 ± 174 units above background (Fig. 4B).

Biochemical identification of the cytokine-stimulated NOS isoform. Two biochemical approaches were used to identify the NOS isoform stimulated by treatment of muscle cells with IL-1beta and TNF-alpha . The first entailed use of the NOS antagonists L-NAME and SMT (25) and the second used the characteristics of Ca2+ dependence of NOS activity (3).

In the first approach, muscle cells were incubated for 48 h with either IL-1beta , TNF-alpha , or the combination of the two cytokines in the presence of either the NOS inhibitor L-NAME or SMT, a preferential inhibitor of NOS II (25). NOS activity stimulated by IL-1beta (29.3 ± 2.1 pmol nitrite · min-1 · mg protein-1 above basal) was strongly inhibited in the presence of either 100 µM SMT (94.6 ± 0.2% inhibition; P < 0.001) or 1 mM L-NAME (89.7 ± 4.5% inhibition; P < 0.01) (Fig. 5). NOS activity stimulated by 1 nM TNF-alpha (1.5 ± 0.4 pmol nitrite · min-1 · mg protein-1 above basal) was also inhibited in the presence of either SMT (89.2 ± 5.9% inhibition; P < 0.01) or L-NAME (68.2 ± 7.7% inhibition; P < 0.05). NOS activity stimulated by the combination of IL-1beta and TNF-alpha (41 ± 2 pmol nitrite · min-1 · mg-1 above basal) was also inhibited in the presence of either SMT (96.5 ± 1.1% inhibition; P < 0.001) or L-NAME (94.3 ± 4.0% inhibition; P < 0.001).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   Inhibition of cytokine-stimulated NOS activity by the NOS inhibitors S-methylisothiourea (SMT) and NG-nitro-L-arginine methyl ester (L-NAME). Cultures of confluent muscle cells were incubated for 48 h with 1 nM TNF-alpha , 1 nM IL-1beta , or combination of the 2 cytokines in absence (solid bars) or presence of 100 µM SMT, a preferential NOS II antagonist (open bars), or 1 mM L-NAME (hatched bars). NOS activity was measured as total nitrite production. Results are expressed as increase in nitrite production above basal values (3.3 ± 0.6 pmol nitrite · min-1 · mg protein-1). Increase in NOS activity observed in response to TNF-alpha , IL-1beta , or TNF-alpha  + IL-1beta was virtually abolished in presence of either SMT or L-NAME. Values are means ± SE of 4-6 experiments done in duplicate.

One biochemical property that differentiates NOS II from the constitutive NOS I and NOS III isoforms is the lack of dependence of NOS II activity on added Ca2+. Therefore, in the second approach, the characteristic Ca2+-independent nature of the NOS II enzyme was utilized, and measurements of the conversion of L-[3H]arginine to L-[3H]citrulline over time in the presence and absence of Ca2+ were made. Muscle cells were first treated for 48 h with 1 nM IL-1beta and then incubated for 30 min in control Ca2+-containing medium or Ca2+-free medium with 4 mM EGTA. L-[3H]arginine was then added at time 0, and the reaction was terminated at various intervals up to 30 min. Significant time-dependent conversion of L-[3H]arginine to L-[3H]citrulline was observed after 1 min (1.6 ± 0.4 pmol citrulline · min-1 · mg protein-1 above basal; P < 0.05). Elimination of Ca2+ from the medium had no effect on L-[3H]citrulline production for periods up to 30 min (2 mM Ca2+: 17.5 ± 4.6 pmol citrulline · min-1 · mg protein-1 above basal vs. 0 Ca2+-4 mM EGTA: 16.3 ± 4.0 pmol citrulline · min-1 · mg protein-1 above basal) (Fig. 6). L-[3H]citrulline production, however, was virtually abolished in the presence of the preferential NOS II inhibitor SMT (10 µM) for periods up to 30 min (95.2 ± 1.3% inhibition; P < 0.02). Taken together, the results implied that the conversion of arginine to citrulline reflected activation of the Ca2+-independent isoform NOS II.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   IL-1beta -induced NOS II activity is inhibited by SMT and is Ca2+ independent. Cultures of confluent muscle cells were incubated with 1 nM IL-1beta for 48 h. NOS activity was measured by conversion of L-[3H]arginine to L-[3H]citrulline at intervals up to 30 min in presence and absence of the selective NOS II inhibitor SMT (100 µM) and in presence and absence of Ca2+ as described in METHODS. Results are expressed as increase in L-[3H]citrulline production above basal values. In cells treated with IL-1beta , time-dependent L-[3H]citrulline production was observed (bullet ). In presence of SMT (black-square), L-[3H]citrulline production was virtually abolished (95 ± 1% inhibition at 30 min). Conversion of L-[3H]arginine to L-[3H]citrulline by cells occurred independently of Ca2+ (0 Ca2+ + 4 mM EDTA) (open circle ). Values are means ± SE of 4 experiments done in duplicate.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

This study shows that IL-1beta , TNF-alpha , and LPS increase NOS II protein levels and stimulate NOS II activity in cultured smooth muscle cells of the rat colon. The effects of IL-1beta and TNF-alpha on NOS II activity were synergistic, whereas the effects of IL-1beta and LPS were only additive. The increase in NOS II activity stimulated by IL-1beta and TNF-alpha involved new protein synthesis and could be inhibited by cyclohexamide. TGF-beta 1 inhibited the increase in NOS II activity stimulated by IL-1beta , TNF-alpha , and LPS.

The identity of the NOS II protein induced by IL-1beta , TNF-alpha , and LPS was determined by Western blot analysis, and the increase in expression was evaluated by densitometry. The identification of NOS II was supported by the fact that NOS activity measured either as total nitrite production or from the formation of L-[3H]citrulline was virtually abolished by the preferential NOS II inhibitor SMT and was strongly inhibited by L-NAME. NOS activity measured from the formation of L-[3H]citrulline was also Ca2+ independent, consistent with the known characteristics of NOS II.

The mechanism by which TGF-beta 1 inhibited cytokine- and LPS-induced NOS II activity was not examined in the present study. Similar inhibition by TGF-beta 1 was also noted in other cell types, for example, smooth muscle cell cultures of rat aortic (20) and pulmonary artery (7) and cultures of RAW267.4 macrophages (8). In TGF-beta 1 null [TGF-beta 1(-/-)] mice, basal unstimulated NOS II activity and NO production were significantly higher in heart, kidney, and peritoneal macrophages than in control [TGF-beta 1(+/+) and TGF-beta 1(+/-)] littermates (29). Inhibition of cytokine-stimulated NOS II activity by TGF-beta 1 was attributed to three general mechanisms: decreased NOS II mRNA stability, reduced NOS II translation, and increased degradation of NOS II protein (7, 20, 29, 32). The effects of TGF-beta 1 on colonic smooth muscle likely represent similar processes.

Although NOS II protein expression could not be detected in freshly isolated muscle cells from the rat colon, NOS II protein was detectable in cells after growth in culture. This may be attributed to the presence of stimulatory cytokines in the serum used in the culture medium. The expression of NOS II in the cultured muscle cells could also be affected by the ability of intestinal smooth muscle cells in culture to produce TGF-beta 1. Our previous studies have shown that TGF-beta 1 production by rapidly proliferating smooth muscle cells is low, increases twofold in confluent cultures (1 wk in culture), and increases 10-fold in postconfluent cultures (2 wk in culture) (13). The spontaneous increase in NOS II expression observed in the current study therefore occurred during a period of low but increasing endogenous TGF-beta 1 production. This suggests that the level of NOS II expression represents the sum of stimulatory (cytokines such as IL-1beta and TNF-alpha ) and inhibitory (TGF-beta 1) stimuli whereby endogenous TGF-beta 1 may have acted to attenuate the expression of NOS II.

Several mechanisms may contribute to the synergy between IL-1beta and TNF-alpha on NOS II expression: activation of cell surface receptors and generation of second messengers and/or endocytosis of the cytokine and activation of nuclear receptors. Both mechanisms could affect transcriptional regulation of the NOS II gene in smooth muscle cells. IL-1beta receptors on the cell surface are coupled to generation of adenosine 3',5'-cyclic monophosphate (cAMP), and two copies of the cAMP response element have been delineated in the rat NOS II promotor region (6). TNF-alpha also interacts with cell surface receptors, which can generate a variety of intracellular second messengers, including cyclic nucleotides and prostaglandins. Activation of nuclear receptors after receptor-mediated endocytosis of cytokines has been identified in a variety of cells (4). The rat NOS II gene has sites within its promotor region that are putative IL-1beta -responsive sites [two nuclear factor-kappa B (NF-kappa B) binding sites and two TNF-alpha response elements] (6). The potential for activation of multiple regions of the rat NOS II promoter by IL-1beta and TNF-alpha may explain the synergistic effects of these cytokines in the present study. Similarly, the LPS-mediated effects on transcriptional regulation of the rat NOS II gene promoter have been shown to rely on the presence of NF-kappa B binding motifs in the promoter region, the same two NF-kappa B sites in the promoter region with which IL-1beta interacts (6, 32). Translational and posttranslational mechanisms likely also play a role in the regulation of NOS II expression, as seen with TGF-beta 1 (7, 20, 29, 32).

Inflammatory bowel disease in humans is associated with increased NOS II expression in the muscular layers (1, 15). Enterocolitis experimentally induced in rats by agents such as 2,4,6-trinitrobenzenesulfonic acid, acetic acid, and LPS or by intestinal parasites is associated with the release of cytokines and is accompanied by an increase in NOS II expression in various tissue layers, including muscle (10, 14, 27, 30, 31). The present study corroborates these findings and demonstrates the ability of two cytokines, IL-1beta and TNF-alpha , to act synergistically in inducing NOS II expression directly in smooth muscle cells. Conceivably in colitis the various cytokines that are released could also act synergistically to increase NOS II expression. Inhibitory factors such as TGF-beta 1 could act to attenuate such expression.

In summary, the cytokines IL-1beta and TNF-alpha act independently and synergistically to stimulate NOS II expression and enzymatic activity in rat colonic smooth muscle through a mechanism sensitive to inhibition by TGF-beta 1. Similar mechanisms may be involved in inflammatory bowel disease, where the level of NOS II expression might be the net effect of both stimulation and inhibition by various cytokines produced during inflammation.

    ACKNOWLEDGEMENTS

I thank Toni L. Bushman for excellent technical assistance.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49691.

Address reprint requests to PO Box 980711, Division of Gastroenterology, Virginia Commonwealth Univ./MCV, Richmond, VA 23298-0711.

Received 5 March 1997; accepted in final form 13 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Boughton-Smith, N. K., S. M. Evans, C. J. Hawkley, A. T. Cole, M. Balsitis, B. J. R. Whittle, and S. Moncada. Nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Lancet 342: 338-340, 1993[Medline].

2.   Bredt, D. S., P. M. Hwang, C. E. Glatt, C. Lowenstein, R. R. Reed, and S. H. Snyder. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351: 714-718, 1991[Medline].

3.   Cho, H. J., Q. W. Xie, J. Calaycay, R. A. Munford, K. M. Swiderek, T. D. Lee, and C. Nathan. Calmodulin is a subunit of nitric oxide synthase from macrophages. J. Exp. Med. 176: 599-604, 1992[Abstract].

4.   Curtis, B. M., M. B. Widmer, P. DeRoos, and E. E. Quarnstrom. IL-1beta and its receptor are translocated to the nucleus. J. Immunol. 144: 1295-1303, 1990[Abstract/Free Full Text].

5.   Dinerman, J. L., T. D. Dawson, M. J. Schell, A. Snowman, and S. H. Synder. Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc. Natl. Acad. Sci. USA 91: 4214-4218, 1994[Abstract].

6.   Eberhardt, W., D. Kunz, R. Hummel, and J. Pfeilschifter. Molecular cloning of the rat inducible nitric oxide synthase gene promoter. Biochem. Biophys. Res. Commun. 223: 752-756, 1996[Medline].

7.   Finder, J., W. W. Stark, D. K. Nakayama, D. Geller, K. Wasserloos, B. R. Pitt, and P. Davies. TGF-beta regulates production of NO in pulmonary artery by inhibiting expression of NOS. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L862-L867, 1995[Abstract/Free Full Text].

8.   Förstermann, U., H. H. H. W. Schmidt, K. L. Kohlhaas, and F. Murad. Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-beta . Eur. J. Pharmacol. Mol. Pharmacol. Sect. 225: 161-165, 1992[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.   Hogaboam, C. M., K. Jacobson, S. M. Collins, and M. G. Blennerhassett. The selective beneficial effects of nitric oxide inhibition in experimental colitis. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G673-G684, 1995[Abstract/Free Full Text].

11.   Jin, J.-G., K. S. Murthy, J. R. Grider, and G. M. Makhlouf. Stoichiometry of neurally induced VIP release, NO formation and relaxation in rabbit and rat gastric muscle. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G357-G369, 1996[Abstract/Free Full Text].

12.   Kobzik, L., M. B. Reid, D. S. Bredt, and J. S. Stamler. Nitric oxide in skeletal muscle. Nature 372: 546-548, 1994[Medline].

13.   Kuemmerle, J. F. Autocrine regulation of growth in cultured human intestinal muscle by growth factors. Gastroenterology 113: 817-824, 1997[Medline].

14.   Miller, M. J., H. Sadowska-Krowicka, S. Chotinaruemol, J. L. Dakkis, and D. A. Clark. Amelioration of chronic ileitis by nitric oxide synthase inhibition. J. Pharmacol. Exp. Ther. 264: 11-16, 1993[Abstract].

15.   Mourelle, M., F. Casellas, F. Guarner, A. Salas, V. Riveros-Morena, S. Moncada, and J.-R. Malagelada. Induction of nitric oxide synthase in colonic smooth muscle from patients with toxic megacolon. Gastroenterology 109: 1497-1502, 1995[Medline].

16.   Murthy, K. S., and G. M. Makhlouf. VIP/PACAP-mediated activation of membrane-bound NO synthase in smooth muscle is mediated by pertussis toxin-sensitive Gi1-2. J. Biol. Chem. 269: 15977-15988, 1994[Abstract/Free Full Text].

17.   Nakaya, Y., S. Yamamoto, Y. Hamada, M. Kamada, T. Aono, and M. Niwa. Inducible nitric oxide synthase in uterine smooth muscle. Life Sciences 58: PL249-PL255, 1996[Medline].

18.   Nakayama, D. K., D. A. Geller, C. J. Lowenstein, P. Davies, B. R. Pitt, R. L. Simmons, and T. R. Billiar. Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle. Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992[Medline].

19.   Nunakawa, Y., N. Ishida, and S. Tanaka. Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 191: 89-94, 1993[Medline].

20.   Perrela, M. A., M. Yoshizumi, Z. Fen, J.-C. Tsai, C.-M. Hseih, S. Kourembanas, and M.-E. Lee. Transforming growth factor-beta 1, but not dexamethasone, down-regulate nitric oxide synthase mRNA after its induction by interleukin-1beta in rat smooth muscle cells. J. Biol. Chem. 269: 14595-14600, 1994[Abstract/Free Full Text].

21.   Saffrey, M. J., C. J. Hassall, C. H. Hoyle, A. Belai, J. Moss, H. H. Schmidt, U. Förstermann, F. Murad, and G. Burnstock. Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurons. Neuroreport 3: 333-336, 1992[Medline].

22.   Schini, V. B., W. Durante, E. Elizondo, B. T. Scott, D. C. Juniquero, A. I. Schafer, and P. M. Vanhoutte. The induction of nitric oxide synthase activity is inhibited by TGF-beta 1, PDGFAB and PDGFBB in vascular smooth muscle cells. Eur. J. Pharmacol. 216: 379-383, 1992[Medline].

23.   Sessa, W. C., J. K. Harrison, C. M. Barber, D. Zeng, M. E. Durieux, D. D. D'Angelo, K. R. Lynch, and M. J. Peach. Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase. J. Biol. Chem. 267: 15274-15276, 1992[Abstract/Free Full Text].

24.   Shaul, P. W., A. J. North, L. C. Wu, L. B. Wells, T. S. Brannon, K. S. Lau, T. Michel, L. R. Margraf, and R. A. Star. Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium. J. Clin. Invest. 94: 2231-2236, 1994[Medline].

25.   Southan, G. J., C. Szabó, and C. Thiemermann. Isothioureas: potent inhibitors of nitric oxide synthase with variable isoform selectivity. Br. J. Pharmacol. 114: 510-516, 1995[Abstract].

26.   Teng, B., K. S. Murthy, J. F. Kuemmerle, J. R. Grider, and G. M. Makhlouf. Expression of constitutive endothelial nitric oxide synthase (eNOS) in rabbit and human gastrointestinal smooth muscle (Abstract). Gastroenterology 110: A1125, 1996.

27.   Tepperman, B. L., J. F. Brown, and B. J. R. Whittle. Nitric oxide synthase induction and intestinal epithelial cell viability in rats. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G214-G218, 1993[Abstract/Free Full Text].

28.   Tracey, W. R., J. S. Pollock, F. Murad, M. Nakane, and U. Förstermann. Identification of a type III (endothelial-like) particulate nitric oxide synthase in LLC-PK1 kidney tubular epithelial cells. Am. J. Physiol. 267 (Cell Physiol. 36): C22-C26, 1994.

29.   Vodovotz, Y., J. J. Letterio, A. G. Geiser, L. Chesler, A. B. Roberts, and J. Sparrow. Control of nitric oxide production by endogenous TGF-beta 1 and systemic nitric oxide in retinal pigment epithelial cells and peritoneal macrophages. J. Leukoc. Biol. 60: 261-270, 1996[Abstract].

30.   Watson, E. G., P. Kostka, and E. E. Daniel. Effect of endotoxin and time on the induction of nitric oxide synthase in the canine ileum (Abstract). Gastroenterology 104: A1066, 1993.

31.   Weisbrodt, N. W., T. A. Pressley, Y.-F. Li, M. J. Zembowicz, S. C. Higham, A. Zembowicz, R. F. Lodata, and F. G. Moody. Decreased ileal muscle contractility and increased NOS II expression induced by lipopolysaccharide. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G454-G460, 1996[Abstract/Free Full Text].

32.   Xie, Q.-W., Y. Kashiwabara, and C. Nathan. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269: 4705-4708, 1994[Abstract/Free Full Text].

33.   Xue, C., J. Pollock, H. H. H. W. Schmidt, S. M. Ward, and K. M. Sanders. Expression of nitric oxide synthase immunoreactivity by interstitial cells of the canine proximal colon. J. Auton. Nerv. Syst. 49: 1-14, 1994[Medline].


AJP Gastroint Liver Physiol 274(1):G178-G185
0193-1857/98 $5.00 Copyright © 1998 the American Physiological Society