Transforming growth factor-p, inhibits L-arginine-derived relaxing factor(s) from smooth muscle cells D. C. JUNQUERO,
V. B. SCHINI,
T. SCOTT-BURDEN,
Center for Experimental Therapeutics, Department Baylor College of Medicine, Houston, Texas 77030 Junquero, M. Vanhoutte.
D. C., V. B. Schini,
T. Scott-Burden,
and P.
Transforming growth factor-p, inhibits L-arginine-derived relaxing factor(s) from smooth muscle cells. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H1788-H1795, 1992.The effects of human recombinant interleukin-l@ were investigated on the release of nonprostanoid relaxing substances from cultured aortic smooth muscle cells from Wistar rats. Cells cultured on microcarrier beads were packed in columns. The perfusate over these beads was bioassayed by measuring changes in isometric tension of contracted arteries without endothelium. The perfusates from interleukin-I@-treated smooth muscle cells, but not from control cells, evoked relaxations. The relaxations persisted when the transit time between the cultured smooth muscle cells and the detector was increased to 5 min. The effect of relaxing substance(s) was inhibited by cycloheximide, nitro-L-arginine, methylene blue, and transforming growth factor-&. L-Arginine but not D-arginine overcame the blockade by nitro+arginine. Superoxide dismutase potentiated the relaxations. In cells cultured in multiwell plates, interleukin-l@ evoked a time- and concentration-dependent accumulation of nitrite in the extracellular medium that was inhibited dose dependently by transforming growth factor-& These studies demonstrate that cultured smooth muscle cells can be stimulated to produce nitric oxide-related substances and that the inducible pathway is modulated by transforming growth factor-&. interleukin-1; vascular smooth muscle; nitrite; nitric oxide; protein synthesis; bioassay
BOTH ENDOTHELIAL CELLS and circulating blood cells participate in the regulation of the vascular tone by releasing vasoactive substances, such as eicosanoids, nitric oxide (NO), and nonprostanoid relaxing factors other than NO (10, 14, 20). NO is the labile free radical produced by the conversion of L-arginine into L-citrulline by NO synthases. At least two classes of enzymes have been identified: a constitutive calcium- and calmodulin-dependent enzyme that is present in vascular endothelial cells (21, 23) and in the central nervous system (6, 17) and an inducible calcium-insensitive enzyme that is present in macrophages (30, 31). Interleukin-1 and endotoxin impair the responsiveness of isolated blood vessels to contractile agents (4, 19) by activating an L-arginine NO pathway (15); cytokines and endotoxin stimulate the production of cyclic guanosine 3’,5’cyclic monophosphate (cGMP) in vascular smooth muscle (3, 8) and in cultured smooth muscle cells (5, 7, 27). The endothelium-independent relaxations evoked by L-arginine (28) and related compounds (11,33) also suggest the presence of an L-arginine NO pathway(s) in vascular smooth muscle. Transforming growth factor-p is a homodimeric peptide, produced by a variety of cells, including platelets (2) and macrophages (l), that inhibit the induction of NO synthase by interleukin-lp in renal mesangial cells (24) and suppresses the cytotoxic effect of activated macrophages (22). HI788
0363-6135/92
$2.00
Copyright
AND P. M. VANHOUTTE
of Medicine,
The aim of the present study was to characterize the transferable L-arginine-derived relaxing substance(s) generated by vascular smooth muscle cells after treatment with human recombinant interleukin-l@ (27) and to investigate whether transforming growth factor-p may impair its production. MATERIALS AND METHODS Materials. Collagenase, elastase, and human recombinant interleukin- l@ were purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN); transforming growth factor-p, from porcine platelets was purchased from Collaborative Research (Bedford, MA); Cytodex 3 microcarrier beads were purchased from Pharmacia (Piscataway, NJ); fetal bovine serum, Eagle’s minimum essential medium (MEM) , penicillin, phosphate-buffered saline solution (PBS), and streptomycin were obtained from Whittaker Bioproducts (Walkersville, MD). All plasticware was provided by Costar (Pleasanton, CA). Male Wistar rats were purchased from Charles River (Wilmington, MA). Acetylcholine chloride, bovine serum albumin fraction V, cycloheximide, D-arginine, L-arginine, r.,-glutamine, methylene blue, naphthylethylenediamine dihydrochloride, phenylephrine, sodium nitrite, sulfanilamide, superoxide dismutase, and trypsin were purchased from Sigma Chemical (St. Louis, MO); and nitro-L-arginine was purchased from Aldrich Chemical (Milwaukee, WI). Stock solutions of indomethacin were prepared in equimolar (lob3 M) concentrations of Na,CO,; all other drugs were prepared daily and kept at 4°C until used; concentrations were expressed in molar concentrations in the solution perfusing the smooth muscle cells or the bioassay tissues. Cell culture. Procedures used were as described previously (29) with slight modifications. Male rats were killed, and the thoracic aortas were removed and washed in PBS (4°C) containing 200 U/ml of both penicillin and streptomycin and 25 pg/ml fungizone. Once stripped of adventitia, vessels were opened longitudinally and cut into fine pieces. Cell suspensions were obtained after digestion of the tissue pieces with elastase (0.05% wt/vol) for 45 min at 37°C and then by collagenase (0.3% wt/vol) for 3-4 h at 37°C in the presence of heat-inactivated fetal calf serum (10% vol/vol). Digestions were performed in MEM that contained Earle’s balanced salts, 20 mM glutamine, 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-NaOH, 20 mM N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (HEPES)-NaOH (both at pH 7.3), and 100 U/ml of both penicillin and streptomycin as bacteriostatic agents (MEMTH). After centrifugation of cell suspensions, cells were plated into tissue culture flasks (T35, lo6 cells/ flask) or 12-well multiwell plates. Phenotypic characterization (smooth muscle cu-actin determination) was performed in primary cultures as previously described (29), then cells were passaged routinely by using trypsin-EDTA (0.25% wt/vol, 1 mM). Cells from 1st to 16th passage did not change morphologically and were used for experimentation. Cultures were normally maintained in MEMTH containing 10% fetal calf serum, and medium was exchanged every 3 days. The cell numbers were determined by counting aliquots of cell suspensions obtained by enzymatic
0 1992 the
American
Physiological
Society
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TGF-(31, IL-l@,
AND
SMOOTH
dispersion of the cell layers in a Coulter counter. Cells were seeded onto Cytodex 3 microcarrier beads and were maintained in suspension by shaking at 60 rpm. Confluent monolayers on microcarrier beads and in 12-well multiwell plates were obtained after 4-5 days and were used for bioassay experiments and nitrite measurements, respectively, during the next 48 h. Cells were incubated in serum-free medium for 24 h, and this was replaced by medium containing 1% nonmitogenic plasma-derived serum for the duration of the treatment for 2-48 h with interleukin-l@ (l-100 U/ml, equivalent to 0.1-10 rig/ml) or its vehicle (PBS) in the presence of transforming growth factor-p, (0.1-10 rig/ml) or its vehicle (4 mM HCl containing bovine serum albumin 0.1%). Plasma-derived serum was prepared from titrated bovine plasma. Bioassay studies. Mongrel dogs (8-19 kg) and male Wistar rats (365-385 g) were killed. Canine left circumflex coronary arteries and rat thoracic aortas were removed and placed in cold, modified Krebs-Ringer bicarbonate solution of the following composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl,, 1.2 MgSO,, 1.2 KH2P04, 25.0 NaHCO,, 0.026 calcium sodium edetate, 11.1 glucose (control solution). Arteries were cleaned of fat and connective tissue and cut into rings (3 mm long). The endothelium was removed by inserting the tip of a forceps into the lumen and rolling the ring back and forth on paper towel wetted with control solution. Bioassay rings were suspended between two stirrups, one anchored to a steel plate, the other connected to a strain gauge transducer (FT03D, Grass Instruments, Quincy, MA) for recording of isometric force. Bioassay tissues could be moved freely to positions below either one of three vertically mounted polypropylene chromatographic columns (Evergreen Scientific, Los Angeles, CA) that were maintained at 37°C by a glass water jacket. Preparations could be superfused with the perfusate from either columns filled with uncoated microcarrier beads (direct superfusion, direct line) or columns containing microcarrier beads covered with confluent monolayers of smooth muscle cells. Columns of cells were perfused at constant flow (3 ml/min) by means of a roller pump (Gilson Minipuls, Middleton, WI) with control solution at 37°C oxygenated with a 95% 02-5% CO, gas mixture. Bioassay rings (under direct superfusion) were stretched in a stepwise fashion to the optimal point of their length-active tension curve (7-9 and 2.5-3 g for canine coronary arteries and rat aortas, respectively); at each distension level, contractions were evoked by 60 mM KCl. After an equilibration period of at least 45 min, the bioassay tissue was contracted with phenylephrine (lo-” M), and the absence of endothelium was confirmed by the lack of relaxation to acetylcholine (lOA M) given under direct superfusion. Columns of microcarrier beads covered with smooth muscle cells were perfused for 45 min with oxygenated control solution to remove all traces of conditioned culture medium. All experiments were carried out in the presence of indomethacin (10B5 M) to prevent the synthesis of vasoactive prostanoids. Drugs introduced either above columns filled with uncovered beads (direct superfusion) or between columns and detector rings interacted with bioassay tissues only. Drugs injected above the column of microcarrier beads covered with smooth muscle cells interacted with both cells and detector tissues. The transit time between columns of cells on microcarrier beads and bioassay rings could be increased from 1 s to 5 min by changing the length of the polyethylene tube separating the two. The effect of the perfusate of smooth muscle cells was bioassayed either under basal conditions or when cells and/or bioassay tissues were treated with different compounds. The total number of cells present in each column of beads was determined at the end of each experiment using enzymatic dispersion and counting as described above.
MUSCLE-DERIVED
NO
H1789
In preliminary experiments, superfusion of the phenylephrine-contracted bioassay rings with perfusates from interleukinI@-treated smooth muscle cells evoked relaxation of the bioassay tissues; the relaxing activity depended on 1) the number of cells (0.2-6.6 X lo6 cells/column), 2) the concentration of interleukin-l@ (l-20 U/ml), and 3) the time of treatment of the cells with interleukin-l@ before bioassay experiments (2-24 h) and 4) was inversely related to the time of continuous perfusion of the column of cells with control solution (l-5 h) (data not shown). Some parameters were standardized for the additional experiments, and they included the number of cells per column (from 2 x lo6 to 3 X 106), the concentration of interleukin-lb (10 U/ml), and the time of treatment of smooth muscle cells with interleukin-lp before bioassay experiments (~3 h). The transit time was 1 s if not otherwise stated. Measurement of nitrite. Nitrite production was estimated by a calorimetric assay (12). Aliquots of cell supernatants (400 ~1) were collected and mixed with an equal volume of Griess reagent [ 1% sulfanilamide and 0.1% N-( 1-naphthyl) -ethylenediamine dihydrochloride in 2% phosphoric acid]. The mixture was incubated at room temperature for 10 min, and the absorbance was measured at 540 nm. Concentrations were determined by comparison with a standard curve obtained with sodium nitrite in water, and the background nitrite values corresponding to serum-free medium were substracted from experimental values. Statistical analysis. Results are expressed as means t SE, and the number of separate experiments (n) includes cell isolates from different animals. Statistical analysis was performed by using Student’s paired t test (2 tailed) or an analysis of variance when more than two groups were compared. P values ~0.05 were considered statistically significant. RESULTS
Basal release of relaxing factor(s). With a l-s transit time, the perfusates from columns of beads covered with cultured smooth muscle cells that had been treated with interleukin-lp (10 U/ml, equivalent to 1 rig/ml) for 24 h induced complete relaxations of canine coronary bioassay rings contracted with phenylephrine (10m6 M) and suppressed endogenous myogenic tone (Fig. 1). Full relaxations were observed even when continuous perfusion of column of cells with control solution was performed for up to 4 h (data not shown). Perfusates from columns containing cultured smooth muscle cells that had been treated with interleukin-lb (10 U/ml) for 12 h, washed twice by culture medium exchange to remove interleukinl& and incubated for 36 h in culture medium before use still induced complete relaxations of contracted bioassay rings (data not shown). The isometric tension of contracted detector rings was not modified by either direct superfusion (perfusion with control solution of columns of uncoated beads; data not shown) or superfusion with perfusates from columns of untreated smooth muscle cells (Fig. 1). Relaxations induced by interleukin- lp-treated cells were similar when the transit time between the donor columns of cells and the detector tissues was increased from 1 s to 5 min, when tested during the first 3 h of perfusion (Fig. 1). Pharmacological characteristics of the relaxing factor(s) and effect of varying transit time. The simultaneous addi-
tion of cycloheximide (5 pg/ml) and interleukin-l@ (10 U/ml) during the 24-h treatment of cells before the bioassay experiments abolished the relaxation (Fig. 1). Treatments of equilibrated columns of interleukin-lp-
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H1790
TGF-fii,
IL-l&
AND SMOOTH
MUSCLE-DERIVED
NO
before contraction with phenylephrine induced contractions in canine coronary arteries but not in rat aortas (Fig. 2). Methylene blue abolished the relaxations of rat aortic detector rings evoked by perfusates from interleukin-l@ (10 U/ml)-stimulated smooth muscle cells and significantly reduced them when canine coronary arteries were used as bioassay tissues (Fig. 2). With a l-s transit time, addition of nitro-L-arginine (10m4 M) to the column of cells reversed the relaxation induced by the perfusates from cells treated with interleukin- l/3 (10 U/ml) for 24 h before bioassay experiments. The subsequent addition of L-arginine at equimolar concentration (10m4 M) to the cells reversed the blockade evoked by nitro-L-arginine. D-Arginine (lop4 to 10d3 M) did not counteract the inhibitory effect of nitro-L-arginine (Fig. 3). When the transit time was increased to 5 min, nitro+arginine (10m4 M) abolished the relaxations evoked by the perfusates from interleukin-la-treated cells (Fig. 4). At transit time >l s, L-arginine did not neutralize the effect of the synthase inhibitor nitro-LFig. 1. Relaxations of canine coronary artery bioassay tissues superfused with perfusates from cultured vascular smooth muscle cells grown on arginine (Fig. 4). The same observations were made when microcarrier beads. Cells were incubated with interleukin-la (IL-l& 10 Wistar rat aortic rings were used as detector tissues (data U/ml) for 24 h in presence or absence of cycloheximide (5 pg/ml). not shown). Arginine-related compounds alone had no Effects of nitro-L-arainine (10m4 M) infused through column of cells for significant direct effect on the isometric tension of canine 1 h and a prolonged?ransit time between donor and detector tissues (5 coronary bioassay rings (data not shown); the additional min) were also determined. Results are presented as means rt SE of 3 separate experiments and are expressed in % contraction induced by contraction observed after the blockade of the relaxation phenylephrine (10-s M); no. of cells per column was 2.6 f 0.2 X 106. by nitro-L-arginine appeared to be related not to the * Statistically significant differences from control (untreated cells). treatments by increasing concentrations of D-arginine but rather to the development of spontaneous myogenic tone in most of the canine bioassay preparations. Relaxstimulated cells for 1 h with nitro-L-arginine (10e4 M), a specific inhibitor of NO production, significantly ation of bioassay rings was not evoked by perfusates from decreased the relaxing activity of the perfusates (Fig. 1). columns of smooth muscle cells treated with L-a&nine Treatments of detector rings with methylene blue (10m5 (10m5 to 10m3 M) unless they had been exposed to interM), an inhibitor of soluble guanylate cyclase, for 30 min leukin-l@ (10 U/ml) for at least 3 h (data not shown).
Fig. 2. Effects of methylene blue (MB, 10-5 M) on isometric tension of a canine coronary artery (A) and a rat aorta used as bioassay tissues (B) and modulation of relaxations elicited by perfusates from columns of cultured vascular smooth muscle cells on microcarrier beads and treated with IL-10 (10 U/ml) for 24 h. Results are expressed as means f SE (n = 4-3 separate expta); no. of cells per column was 1.9 + 0.1 X lo6 and 2.4 + 0.4 X lo6 for experiments with canine and rat arteries, respectively. * Statistically significant differences from contracted level. 6
-2 :
Without
MB
With MB
-2 !
Without
MB
With MB
n Methylene blue 10 pM q Phenylephrine 1 @A 0
Phenylephrine 1 pM + IL-1 B-treated cells
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TGF-81, IL-l@, AND SMOOTH
MUSCLE-DERIVED
B
A
D-erg IL-16
W4Y I)-srg
treated
L-erg
H1791
NO TRANSIT
TIME
1 SECOND
104M
4xW4M
SYC ;
nttro-L-wg
1Q4Y
tmatttmo1aocond PE m-h
Fig. 3. A: experiment with a canine coronary artery as bioassay ring (performed with a transit time of 1 s) showing effect of nitro-L-arginine (nitro+Arg, 10m4 M), r.,-arginine (L-Arg, lo-* M), and D-arginine (D-Arg, 10m4 to 1O-3 M) on relaxing effect of perfusates from IL-l&treated (10 U/ml for 24 h) smooth muscle cells (SMC) grown on microcarrier beads. PE, phenylephrine. B: modulation of relaxations induced by perfusates from IL-l&treated cells in presence or absence of arginine-related compounds. Results are presented as means + SE of 4 separate experiments and are expressed in % contraction induced by PE (10-a M); no. of cells per column was 2.5 f 0.8 x 106. * Statistically significant differences from relaxed level.
The amplitude of the relaxations induced by perfusates from interleukin-l&treated cells was inversely proportional to the duration of perfusion with control solution when transit times of 5 min were used in bioassay experiments (Fig. 5). However, a maximal relaxation could be restored by infusion of superoxide dismutase (7.5-15 U/ml) between the donor columns of cells and the bioassay tissues (Fig. 5B). Also, reduction of the transit time from 5 min to 1 s mimicked the effect of the scavenger of oxygen-derived free radicals (Fig. 5C). When transforming growth factor-p1 (10 rig/ml) was added to the cells simultaneously with interleukin-l/3 (10 U/ml, 24 h before bioassay experiments), it inhibited the relaxing effect evoked by the perfusates from cells treated with the cytokine (Fig. 6A); perfusates from smooth muscle cells treated with transforming growth factor-p1 (10 rig/ml) or its vehicle (4 mM HCl containing bovine serum albumin 0.1%) for 24 h before bioassay experiments did not elicit changes in tension of contracted bioassay tissues. Perfusates from 31’3 fibroblasts cultured on Cytodex microcarrier beads and treated in an analogous manner to vascular smooth muscle cells did not elicit relaxations of either the canine coronary artery or the rat aorta bioassay tissues, even after addition of r.,-arginine ( 10m5 to 10m3 M; data not shown). Production of nitrite. Treatment of cultured vascular smooth muscle cells with interleukin-l@ (10 U/ml) induced a time-dependent accumulation of nitrite in the culture medium (Fig. 7A). After a 48-h exposure of the cells to interleukin-l/3 or its vehicle, the production of
nitrite was 3.28 + 0.79 and 10.73 + 0.75 nmol/106 cells in supernatants from control and treated cells, respectively. The accumulation of nitrite, measured after 24 h, was concentration dependent, and the threshold concentration of interleukin-la was 1 U/ml (Fig. 723). Treatment of cultured cells with transforming growth factor-& (0.1-10 rig/ml) or its vehicle did not evoke significant accumulation of nitrite in the extracellular medium after 24 h incubation; when transforming growth factor-& was added to the cells simultaneously with interleukin- l/3 (10 U/ml), it abolished the production of nitrite induced by the cytokine in a concentration-dependent manner with a maximal effect at 4 rig/ml (Fig. 6B). DISCUSSION
The present data demonstrate that interleukin-l/3 stimulates the production of potent nonprostanoid relaxing substance(s) in cultured smooth muscle cells from Wistar rat aortas. The induction of the relaxing activity by the cytokine probably involves protein synthesis, since the production of the relaxing agent(s) is abolished by cycloheximide and the relaxation of contracted bioassay rings is detected after 13 h of treatment of the smooth muscle cells with interleukin-l@. The blockade of the relaxing activity evoked by cycloheximide was correlated with a 97% inhibition of protein synthesis in treated cells (data not shown), and at the low levels of the compound used there were no apparent cytotoxic effect on the cultured cells. Relaxations evoked by the perfusates are attenuated by treatment of the cells but not the bioassay rings with
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H1792
TGF-j3i, IL-l&
AND SMOOTH
MUSCLE-DERIVED
A
NO
B Parg
IL-18
treated
xl-41 D-•rg
L-W 4XlO”Y
lo”64 L-srg
translt
time
TRANSIT
TIME
5 MINUTES
2 min 30 se5 g ‘;
4XlO’M
106
Fig. 4. A: experiment with a canine coronary artery as bioassay ring (performed with a transit time first of 5 min, then of 1 s) showing effect of nitro+Arg (1O-4 M), L-Arg (10e4 to 4 X 10e4 M), and D-Arg (10m4to 10e3 M) on relaxing effect of perfusates from IL-l&treated (10 U/ml for 24 h) smooth muscle cells grown on microcarrier beads. B: modulation of relaxations induced by perfusates from IL-l&treated cells in presence or absence of arginine-related compounds. Results are expressed as means f SE of 3 separate experiments, and are expressed as % contraction induced by PE (10v6 M); no. of cells per column was 2.5 sf: 0.8 X 10s. * Statistically significant differences from relaxed level.
nitro+arginine, an inhibitor of the production of nitric Infusion of nitro-L-arginine oxide from L-arginine. through the columns of cells also reversed the relaxations evoked by the perfusates, an effect that was overcome stereospecifically by the addition of an equimolar concentration of L-arginine. The relaxing activity produced by interleukin-l&treated cells was inhibited by methylene blue, an inhibitor of soluble guanylate cyclase, suggesting that the relaxation is associated with an increase of cGMP in the tissues. These results confirm previous studies showing that interleukin-la induces an L-arginine NO-like pathway in cultured smooth muscle cells (5, 7) that evokes a sustained production of relaxing factor(s) A
and stimulates the formation of cGMP (27). These observations may account for previous data suggesting that the impairment of contractions in isolated blood vessels exposed to endotoxin and cytokines involve protein synthesis (4, 19) and are mediated by an L-arginine NO pathway (15). In canine coronary bioassay tissues, methylene blue induces significant increases in tension. Only a partial reduction of the relaxation elicited by perfusates of interleukin-lb-treated cells could be detected in canine bioassay tissues treated with the vital dye. These data emphasize the heterogeneity in vascular responses, and the two bioassay tissues used mav exnress different isoforms of the soluble guanylate cyclase, which may C
B
IL-14 IL-1 6 lrbatbd
SMC I SOD 7.5 U/ml I
I
treated sm.
SOD 15 U/ml I
1 second I
QL 2 mln
I
‘1. 3
-PE
Fig. 5. Relaxation of a canine coronary artery ring without endothelium (bioassay tissue) contracted with PE (lOvs M), evoked by perfusats from cultured vascular SMC grown on microcarrier beads, and treated with IL-lb (10 U/ml) for 24 h. Influence of transit time (1 s, 5 min), and of infusion of superoxide dismutase (SOD, 7.5-15 U/ml) between donor cells and detector tissue were determined according to time of perfusion (l-4 h) with control solution. Traces were recorded after 1 h (A), 2.5 h (I?), and 4 h (C) of continuous perfusion of donor cells and superfusion of detector tissue.
mlnutu.
lo-%-
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TGF-/3,, IL-l&
AND SMOOTH
MUSCLE-DERIVED
H1793
NO
B
A
0 conbd n IL-1 D tr*amd SLIC
IL-lO+TGF-RI sue
treated
IL-lR(lOLvfr4
IL.IR treated SMC
PE 10”M TGF-81
(rig/ml)
Fig. 6. A: relaxation of a canine coronary artery ring without endothelium (bioassay tissue) contracted with PE (10e6 M), evoked by perfusate from cultured vascular SMC grown on microcarrier beads, and treated with IL-10 [(lo U/ml for 24 h) or its vehicle (control)] in presence of transforming growth factor-& [TGF-PI (10 rig/ml for 24 h), or its vehicle (control)]. Period of time that is defined by horizontal strait “direct line” corresponds to washing procedure of bioassay preparation superfused with physiological solution passing through a column of cell-free microcarrier beads. B: effect of increasing concentrations of TGF-0, on accumulation of nitrite in extracellular medium evoked by a 24-h exposure of cultured cells to IL-l@. Results are expressed as means +- SE of 3 separate experiments performed in triplicate. * Statistically significant differences from control.
account for the observed differences in the action of methylene blue. However, it can also be speculated that the(se) vasoactive agent(s) may induce smooth muscle relaxation by other mechanism(s) than those involving soluble guanylate cyclase. Complete relaxations were evoked by perfusates from interleukin-lp-treated smooth muscle cells when the transit time between the donor and the detector tissues was increased from 1 s to 5 min. These results suggest either that the released substance(s) is (are) relatively stable and thus differs from NO per se, whose biological half-life ranges between 6 and 50 s (9, 13), or that a large
amount of NO is produced by the interleukin-lp-treated cells and even after 5 min of transit time the concentration of the free radical is still sufficient to elicit maximal relaxations. Because nitro-r,-a&nine rapidly reversed the relaxation observed after 5 min, the active entity present in the perfusates most likely is derived from the metabolism of L-arginine. This interpretation is supported by 1) the stereoselective restoration of the relaxation evoked by exogenous L-arginine observed with a l-s transit time, 2) the effect of superoxide dismutase, which potentiates the relaxing activity of perfusates when the transit time was prolonged to 5 min probably by preventing the deg-
A 12 1
-O-
B
Control
Fig. 7. A: time course of accumulation of nitrite in supernatants from control and IL-la-treated cultured SMC from rat aortas. B: accumulation of nitrite in extracellular medium evoked by a 24-h exposure of cultured cells to increasing concentrations of IL-l@ Results are expressed as means + SE of 3 separate experiments performed in triplicate.
0
10
20
Time
30
(hours)
40
50
Interleukin-10
(U/ml)
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H1794
TGF-PI, IL-l&
AND SMOOTH
radation of NO by superoxide anions (26), and 3) the restoration of activity (when it has decayed with a transit time of 5 min) by return to a l-s transit time. The fact that L-arginine overcomes the blockade by nitro+arginine only when the transit time is 1 s suggests that the enzymatic conversion of the amino acid generates a labile relaxing agent that may be NO. Another consideration that suggests NO is the most likely candidate as a relaxing agent is the extracellular accumulation of nitrite in a time- and concentration-dependent manner after treatment of the cells with interleukin-lfi. NO may be stabilized by thiols and iron to form a transferable complex whose biological (except stability) and chemical properties appear similar to those of endothelium-derived NO; this complex will release the free entity, NO per se, to elicit the biological activities (18, 32). This may well occur with the L-arginine-derived relaxing factor(s) produced by smooth muscle cells since the relaxations of the superfused bioassay preparations are present despite a transit time of 5 min. The fact that the production of relaxing factor(s) is cycloheximide sensitive and requires lo-12 h to reach maximum effect is in accordance with the delayed onset of the production of cGMP evoked by interleukin-l@ in cultured smooth muscle cells from rat aorta [maximum levels after 9-10 h (27)], the potential second messenger of the relaxing factor(s). These observations raise the possibility that interleukin-lp may not stimulate the expression of an L-arginine NO synthase-like enzyme directly. The possible intermediates in such an activating process have not yet been identified, but interleukin-l@ induces the production of potent second-message stimulators, one of which, platelet-derived growth factor, plays a major role in the regulation of the metabolism of smooth muscle (25). The production of the relaxing factor(s) by the inducible synthase does not depend on the continuous presence of the cytokine, since the production of the active agent(s) continues for at least 36 h after a 12-h exposure of the cells to interleukin-I& Furthermore, the production and release of the vasoactive substance(s) are sustained, since perfusates from interleukin- I@treated cells relax bioassay tissues after 4 h of continuous perfusion without repletion by L-arginine. Although the intracellular concentration of L-arginine in cultured cells is -1 mM, continuous perfusion of the cells on Cytodex microcarrier beads could eventually lead to depletion of L-arginine stores. A reduction of the intracellular stores of L-arginine by metabolism and/or cellular efflux could account for the decreased potency of the relaxing substance(s) after perfusion of the column of cells for several hours. Replenishment of the stores then would restore the production of a NO-like factor. These results suggest that the inducible NO synthase depends on the availability of the substrate L-arginine to produce vasodilator agent(s) under basal conditions, as is true for the noninduced NO synthase (11, 28, 33). Transforming growth factor-& inhibits interleukin-lpinduced production of nitrite in cultured smooth muscle cells in a concentration-dependent manner and suppresses the relaxing activity of the perfusates from cytokine-treated cells. These results are in accordance with
MUSCLE-DERIVED
NO
previous data reporting an inhibitory effect of the peptide of cGMP in rat renal mesangial cells elicited by interleukin-16 and tumor necrosis factor-a! (24) and 2) on the cytotoxic properties of activated macrophages related to the production of nitrogen oxides (22). Whether transforming growth factor-& interferes with the initial steps after interactions of interleukin-l@ with its receptor or modulates the L-arginine NO pathway once it is induced is unknown. It may affect synthesis and/or activation of proteins involved in the generation of NO. The release of transforming growth factor-p during platelet degranulation, a consequence of adhesion of these cells to the vascular wall during inflammatory or immunological reactions, may counteract endotoxin and cytokine stimulation of NO. These present data suggest that the L-arginine-derived relaxing factor(s) is most likely a close precursor or a complexed form of NO, leading to the elevation of nitrite levels in conditioned medium of interleukin-1P-treated cultured smooth muscle cells. Interleukin-lfl appears to stimulate the inducible NO synthase isoform in the cultured smooth muscle cells from the rat aorta as cytokines and microbial products do in rabbit aortic smooth muscle cells (7) and macrophages (30). By contrast, under the present experimental conditions, such a pathway was not detectable in 3T3 fibroblasts either in the presence or absence of L-arginine (10 -3 M). Furthermore, since interleukin-1P impairs the contractility of rat aortas to the same extent in the presence or absence of endothelium (4) and is a poor direct stimulator of the production of nitrite in cultured endothelial cells from murine brain microvessels (16), it is likely that interleukin-10 exerts a selective effect on the smooth muscle cells of the vascular wall. Although the exact nature of the smooth muscle-derived relaxing factor(s) has not been determined, the L-arginine derived relaxing substance(s) might not act exclusively at a local area and could exert long-term modulation of vascular homeostasis once its synthesis has been induced by a cytokine-mediated pathway. 1) on the production
The authors thank B. Desta and G. Green for technical assistance. This work was supported by National Heart, Lung, and Blood Institute Grants HL-31183, HL-35614, and HL-46356, by an unrestricted Research Award from the Bristol-Myers-Squibb Research Institute, and by the Laboratoire Chauvin (France). Received 15 July 1991; accepted in final form 28 January 1992. REFERENCES 1. Assoian, R. K., B. E. Fleurdelys, H. C. Stevenson, P. J. Miller, D. K. Madtes, E. W. Raines, R. Ross, and M. B. Sporn. Expression and secretion of type fl transforming growth factor p by activated human macrophages. Proc. N&l. Ad. Sci. USA 84: 6020-6024, 1987. 2. Assoian, R. K., A. Komoriya, C. A. Meyers, D. M. Miller, and M. B. Sporn. Transforming growth factor-p in human platelets: identification of a major site, purification and characterization. J. Biol. Chem. 258: 7155-7160, 1983. 3. Beasley, D. Interleukin 1 and endotoxin activate soluble guanylate cyclase in vascular smooth muscle. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R38-R44, 1990. 4. Beasley, D., R. A. Cohen, and N. G. Levinski. Interleukin 1 inhibits contraction of vascular smooth muscle. J. Clin. Invest. 83: 331-335, 1989. 5. Beasley, D., J. H. Schwartz, and B. M. Brenner. Interleukin-l induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth
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TGF-&, 6.
IL-l&
AND SMOOTH
muscle cells. J. Clin. Inuest. 87: 602-608, 1991. Bredt, D. S., and S. H. Snyder. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc. Nutl. Acad. Sci. USA 87: 682-685,
MUSCLE-DERIVED 20.
Moncada, S., R. M. J. Palmer, and E. A. Higgs. Biosynthesis of nitric oxide from L-arginine: a pathway for the regulation of cell function and communication. Biochem. Phurmucol. 38: 1709-
21.
Mulsch, A., E. Bassenge, and R. Busse. Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism. Nuunyn-Schmiedeberg’s Arch.
1990.
1715,
7.
Busse, R., and A. Mulsch. Induction of nitric oxide synthase by cytokines in vascular smooth muscle. FE&S Lett. 275: 87-90,
8.
Fleming, I., G. A. Gray, G. Julou-Schaeffer, J. R. Parratt, and J. C. Stoclet. Incubation with endotoxin activates the L-arginine pathway in vascular tissue. Biochem. Biophys. Res.
1990.
Commun.
171: 562-568,
1990.
Forstermann, U., G. Trogisch, and R. Busse. Species-dependent differences in the nature of endothelium-derived relaxing factor. Eur. J. Pharmacol. 106: 639-643, 1985. 10. Furchgott, R. F., and P. M. Vanhoutte. Endothelium-derived relaxing and contracting factors. FASEB J. 3: 2007-2018, 1989. 11. Gold, M. E., K. S. Wood, R. E. Byrns, G. M. Buga, and L. J. Ignarro. L-Arginine-dependent vascular smooth muscle relaxation and cGMP formation. Am. J. Physiol. 259 (Heart Circ. 9.
Physiol. 12.
28):
H1813-H1821,
1990.
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.
Griffith, T. M., D. H. Edwards, M. J. Lewis, A. C. Newby, and A. H. Henderson. The nature of endothelium-derived vascular relaxant factor. Nature Lond. 308: 645-647, 1984. 14. Ignarro, L. J. Biological actions and properties of endotheliumderived nitric oxide formed and released from artery and vein. 13.
Circ.
Res. 65: l-20,
1989.
15.
Julou-Schaeffer, G., G. A. Gray, I. Fleming, C. Schott, J. R. Parratt, and J. C. Stoclet. Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway. Am. J.
16.
Kilbourn, R. G., and P. Belloni. Endothelial cell production of nitrogen oxides in response to interferon y in combination with tumor necrosis factor, interleukin-1, or endotoxin. J. Nutl. Cancer
Physiol.
Inst. 17.
259
(Heart
82: 772-776,
Circ.
Physiol.
28):
H1038-H1043,
1990.
1990.
Knowles, R. G., M. Palacios, R. M. J. Palmer, and S. Moncada. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Nutl. Acud. Sci. USA 86: 51595162,
1989.
Lancaster, J. R., and J. B. Hibbs. EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proc. Nutl. Acud. Sci. USA 87: 1223-1227, 1990. 19. McKenna, T. M. Prolonged exposure of rat aorta to low levels of endotoxin in vitro results in impaired contractility. Association with vascular cytokine release. J. Clin. Inuest. 86: 160-168, 1990. 18.
H1795
NO
1989.
Phurmucol.
340:
767-770,
1989.
Nelson, B. J., P. Ralph, S. J. Green, and C. A. Nacy. Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-&. J. Immunol. 146: 1849-1857, 1991. R. M. J., D. S. Ashton, and S. Moncada. Vascular 23. Palmer, endothelial cells synthesize nitric oxide from L-arginine. Nature 22.
Land. 24.
333:664-666,
1988.
Pfeilschifter, J., and K. Vosbeck. Transforming growth factor p2 inhibits interleukin lp- and tumor necrosis factor a-induction of nitric oxide in rat renal mesengial cells. Biochem. Biophys. Res. Commun.
175: 372-379,
1991.
Raines, E. W., S. K. Dower, and R. Ross. Interleukin-1 mitogenie activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science Wash. DC 243: 393-396, 1989. 26. Rubanyi, G. M., and P. M. Vanhoutte. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am. J. 25.
Physiol.
250
(Heart
Circ.
Physiol.
19): H822-H827,
1986.
Schini, V. B., D. C. Junquero, T. Scott-Burden, and P. M. Vanhoutte. Interleukin-lp induces the production of an L-arginine-derived relaxing factor from cultured smooth cells from rat aorta. Biochem. Biophys. Res. Commun. 176: 114-121, 1991. L-Arginine evokes both 28. Schini, V. B., and P. M. Vanhoutte. endothelium-dependent and independent relaxations in L-arginine-depleted aortas of the rat. Circ. Res. 68: 209-216, 1991. 29. Scott-Burden, T., T. J. Resink, U. Baur, M. Burgin, and F. Buhler. Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats. Hypertension 27.
13: 295-304,
1989.
Stuehr, D. J., and M. A. Marletta. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon gamma. J. Immunol. 139: 518-527, 1987. 31. Tayeh, M. A., and M. A. Marletta. Macrophage oxidation of L-arginine to nitric oxide, nitrite and nitrate. Tetrahydrobiopterin is required as cofactor. J. Biol. Chem. 264: 19654-19658, 1989. 32. Vanin, A. F. Endothelium-derived relaxing factor is a nitrosyl iron complex with thiol ligands. FEBS Lett. 289: l-3, 1991. 33. Wood, K. S., G. M. Buga, R. E. Byrns, and L. J. Ignarro. Vascular smooth muscle-derived relaxing factor (MDRF) and its close similarity to nitric oxide. Biochem. Biophys. Res. Commun. 30.
170: 80-88,
1990.
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V. B. SCHINI,
T. SCOTT-BURDEN,
Center for Experimental Therapeutics, Department Baylor College of Medicine, Houston, Texas 77030 Junquero, M. Vanhoutte.
D. C., V. B. Schini,
T. Scott-Burden,
and P.
Transforming growth factor-p, inhibits L-arginine-derived relaxing factor(s) from smooth muscle cells. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H1788-H1795, 1992.The effects of human recombinant interleukin-l@ were investigated on the release of nonprostanoid relaxing substances from cultured aortic smooth muscle cells from Wistar rats. Cells cultured on microcarrier beads were packed in columns. The perfusate over these beads was bioassayed by measuring changes in isometric tension of contracted arteries without endothelium. The perfusates from interleukin-I@-treated smooth muscle cells, but not from control cells, evoked relaxations. The relaxations persisted when the transit time between the cultured smooth muscle cells and the detector was increased to 5 min. The effect of relaxing substance(s) was inhibited by cycloheximide, nitro-L-arginine, methylene blue, and transforming growth factor-&. L-Arginine but not D-arginine overcame the blockade by nitro+arginine. Superoxide dismutase potentiated the relaxations. In cells cultured in multiwell plates, interleukin-l@ evoked a time- and concentration-dependent accumulation of nitrite in the extracellular medium that was inhibited dose dependently by transforming growth factor-& These studies demonstrate that cultured smooth muscle cells can be stimulated to produce nitric oxide-related substances and that the inducible pathway is modulated by transforming growth factor-&. interleukin-1; vascular smooth muscle; nitrite; nitric oxide; protein synthesis; bioassay
BOTH ENDOTHELIAL CELLS and circulating blood cells participate in the regulation of the vascular tone by releasing vasoactive substances, such as eicosanoids, nitric oxide (NO), and nonprostanoid relaxing factors other than NO (10, 14, 20). NO is the labile free radical produced by the conversion of L-arginine into L-citrulline by NO synthases. At least two classes of enzymes have been identified: a constitutive calcium- and calmodulin-dependent enzyme that is present in vascular endothelial cells (21, 23) and in the central nervous system (6, 17) and an inducible calcium-insensitive enzyme that is present in macrophages (30, 31). Interleukin-1 and endotoxin impair the responsiveness of isolated blood vessels to contractile agents (4, 19) by activating an L-arginine NO pathway (15); cytokines and endotoxin stimulate the production of cyclic guanosine 3’,5’cyclic monophosphate (cGMP) in vascular smooth muscle (3, 8) and in cultured smooth muscle cells (5, 7, 27). The endothelium-independent relaxations evoked by L-arginine (28) and related compounds (11,33) also suggest the presence of an L-arginine NO pathway(s) in vascular smooth muscle. Transforming growth factor-p is a homodimeric peptide, produced by a variety of cells, including platelets (2) and macrophages (l), that inhibit the induction of NO synthase by interleukin-lp in renal mesangial cells (24) and suppresses the cytotoxic effect of activated macrophages (22). HI788
0363-6135/92
$2.00
Copyright
AND P. M. VANHOUTTE
of Medicine,
The aim of the present study was to characterize the transferable L-arginine-derived relaxing substance(s) generated by vascular smooth muscle cells after treatment with human recombinant interleukin-l@ (27) and to investigate whether transforming growth factor-p may impair its production. MATERIALS AND METHODS Materials. Collagenase, elastase, and human recombinant interleukin- l@ were purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN); transforming growth factor-p, from porcine platelets was purchased from Collaborative Research (Bedford, MA); Cytodex 3 microcarrier beads were purchased from Pharmacia (Piscataway, NJ); fetal bovine serum, Eagle’s minimum essential medium (MEM) , penicillin, phosphate-buffered saline solution (PBS), and streptomycin were obtained from Whittaker Bioproducts (Walkersville, MD). All plasticware was provided by Costar (Pleasanton, CA). Male Wistar rats were purchased from Charles River (Wilmington, MA). Acetylcholine chloride, bovine serum albumin fraction V, cycloheximide, D-arginine, L-arginine, r.,-glutamine, methylene blue, naphthylethylenediamine dihydrochloride, phenylephrine, sodium nitrite, sulfanilamide, superoxide dismutase, and trypsin were purchased from Sigma Chemical (St. Louis, MO); and nitro-L-arginine was purchased from Aldrich Chemical (Milwaukee, WI). Stock solutions of indomethacin were prepared in equimolar (lob3 M) concentrations of Na,CO,; all other drugs were prepared daily and kept at 4°C until used; concentrations were expressed in molar concentrations in the solution perfusing the smooth muscle cells or the bioassay tissues. Cell culture. Procedures used were as described previously (29) with slight modifications. Male rats were killed, and the thoracic aortas were removed and washed in PBS (4°C) containing 200 U/ml of both penicillin and streptomycin and 25 pg/ml fungizone. Once stripped of adventitia, vessels were opened longitudinally and cut into fine pieces. Cell suspensions were obtained after digestion of the tissue pieces with elastase (0.05% wt/vol) for 45 min at 37°C and then by collagenase (0.3% wt/vol) for 3-4 h at 37°C in the presence of heat-inactivated fetal calf serum (10% vol/vol). Digestions were performed in MEM that contained Earle’s balanced salts, 20 mM glutamine, 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-NaOH, 20 mM N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (HEPES)-NaOH (both at pH 7.3), and 100 U/ml of both penicillin and streptomycin as bacteriostatic agents (MEMTH). After centrifugation of cell suspensions, cells were plated into tissue culture flasks (T35, lo6 cells/ flask) or 12-well multiwell plates. Phenotypic characterization (smooth muscle cu-actin determination) was performed in primary cultures as previously described (29), then cells were passaged routinely by using trypsin-EDTA (0.25% wt/vol, 1 mM). Cells from 1st to 16th passage did not change morphologically and were used for experimentation. Cultures were normally maintained in MEMTH containing 10% fetal calf serum, and medium was exchanged every 3 days. The cell numbers were determined by counting aliquots of cell suspensions obtained by enzymatic
0 1992 the
American
Physiological
Society
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TGF-(31, IL-l@,
AND
SMOOTH
dispersion of the cell layers in a Coulter counter. Cells were seeded onto Cytodex 3 microcarrier beads and were maintained in suspension by shaking at 60 rpm. Confluent monolayers on microcarrier beads and in 12-well multiwell plates were obtained after 4-5 days and were used for bioassay experiments and nitrite measurements, respectively, during the next 48 h. Cells were incubated in serum-free medium for 24 h, and this was replaced by medium containing 1% nonmitogenic plasma-derived serum for the duration of the treatment for 2-48 h with interleukin-l@ (l-100 U/ml, equivalent to 0.1-10 rig/ml) or its vehicle (PBS) in the presence of transforming growth factor-p, (0.1-10 rig/ml) or its vehicle (4 mM HCl containing bovine serum albumin 0.1%). Plasma-derived serum was prepared from titrated bovine plasma. Bioassay studies. Mongrel dogs (8-19 kg) and male Wistar rats (365-385 g) were killed. Canine left circumflex coronary arteries and rat thoracic aortas were removed and placed in cold, modified Krebs-Ringer bicarbonate solution of the following composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl,, 1.2 MgSO,, 1.2 KH2P04, 25.0 NaHCO,, 0.026 calcium sodium edetate, 11.1 glucose (control solution). Arteries were cleaned of fat and connective tissue and cut into rings (3 mm long). The endothelium was removed by inserting the tip of a forceps into the lumen and rolling the ring back and forth on paper towel wetted with control solution. Bioassay rings were suspended between two stirrups, one anchored to a steel plate, the other connected to a strain gauge transducer (FT03D, Grass Instruments, Quincy, MA) for recording of isometric force. Bioassay tissues could be moved freely to positions below either one of three vertically mounted polypropylene chromatographic columns (Evergreen Scientific, Los Angeles, CA) that were maintained at 37°C by a glass water jacket. Preparations could be superfused with the perfusate from either columns filled with uncoated microcarrier beads (direct superfusion, direct line) or columns containing microcarrier beads covered with confluent monolayers of smooth muscle cells. Columns of cells were perfused at constant flow (3 ml/min) by means of a roller pump (Gilson Minipuls, Middleton, WI) with control solution at 37°C oxygenated with a 95% 02-5% CO, gas mixture. Bioassay rings (under direct superfusion) were stretched in a stepwise fashion to the optimal point of their length-active tension curve (7-9 and 2.5-3 g for canine coronary arteries and rat aortas, respectively); at each distension level, contractions were evoked by 60 mM KCl. After an equilibration period of at least 45 min, the bioassay tissue was contracted with phenylephrine (lo-” M), and the absence of endothelium was confirmed by the lack of relaxation to acetylcholine (lOA M) given under direct superfusion. Columns of microcarrier beads covered with smooth muscle cells were perfused for 45 min with oxygenated control solution to remove all traces of conditioned culture medium. All experiments were carried out in the presence of indomethacin (10B5 M) to prevent the synthesis of vasoactive prostanoids. Drugs introduced either above columns filled with uncovered beads (direct superfusion) or between columns and detector rings interacted with bioassay tissues only. Drugs injected above the column of microcarrier beads covered with smooth muscle cells interacted with both cells and detector tissues. The transit time between columns of cells on microcarrier beads and bioassay rings could be increased from 1 s to 5 min by changing the length of the polyethylene tube separating the two. The effect of the perfusate of smooth muscle cells was bioassayed either under basal conditions or when cells and/or bioassay tissues were treated with different compounds. The total number of cells present in each column of beads was determined at the end of each experiment using enzymatic dispersion and counting as described above.
MUSCLE-DERIVED
NO
H1789
In preliminary experiments, superfusion of the phenylephrine-contracted bioassay rings with perfusates from interleukinI@-treated smooth muscle cells evoked relaxation of the bioassay tissues; the relaxing activity depended on 1) the number of cells (0.2-6.6 X lo6 cells/column), 2) the concentration of interleukin-l@ (l-20 U/ml), and 3) the time of treatment of the cells with interleukin-l@ before bioassay experiments (2-24 h) and 4) was inversely related to the time of continuous perfusion of the column of cells with control solution (l-5 h) (data not shown). Some parameters were standardized for the additional experiments, and they included the number of cells per column (from 2 x lo6 to 3 X 106), the concentration of interleukin-lb (10 U/ml), and the time of treatment of smooth muscle cells with interleukin-lp before bioassay experiments (~3 h). The transit time was 1 s if not otherwise stated. Measurement of nitrite. Nitrite production was estimated by a calorimetric assay (12). Aliquots of cell supernatants (400 ~1) were collected and mixed with an equal volume of Griess reagent [ 1% sulfanilamide and 0.1% N-( 1-naphthyl) -ethylenediamine dihydrochloride in 2% phosphoric acid]. The mixture was incubated at room temperature for 10 min, and the absorbance was measured at 540 nm. Concentrations were determined by comparison with a standard curve obtained with sodium nitrite in water, and the background nitrite values corresponding to serum-free medium were substracted from experimental values. Statistical analysis. Results are expressed as means t SE, and the number of separate experiments (n) includes cell isolates from different animals. Statistical analysis was performed by using Student’s paired t test (2 tailed) or an analysis of variance when more than two groups were compared. P values ~0.05 were considered statistically significant. RESULTS
Basal release of relaxing factor(s). With a l-s transit time, the perfusates from columns of beads covered with cultured smooth muscle cells that had been treated with interleukin-lp (10 U/ml, equivalent to 1 rig/ml) for 24 h induced complete relaxations of canine coronary bioassay rings contracted with phenylephrine (10m6 M) and suppressed endogenous myogenic tone (Fig. 1). Full relaxations were observed even when continuous perfusion of column of cells with control solution was performed for up to 4 h (data not shown). Perfusates from columns containing cultured smooth muscle cells that had been treated with interleukin-lb (10 U/ml) for 12 h, washed twice by culture medium exchange to remove interleukinl& and incubated for 36 h in culture medium before use still induced complete relaxations of contracted bioassay rings (data not shown). The isometric tension of contracted detector rings was not modified by either direct superfusion (perfusion with control solution of columns of uncoated beads; data not shown) or superfusion with perfusates from columns of untreated smooth muscle cells (Fig. 1). Relaxations induced by interleukin- lp-treated cells were similar when the transit time between the donor columns of cells and the detector tissues was increased from 1 s to 5 min, when tested during the first 3 h of perfusion (Fig. 1). Pharmacological characteristics of the relaxing factor(s) and effect of varying transit time. The simultaneous addi-
tion of cycloheximide (5 pg/ml) and interleukin-l@ (10 U/ml) during the 24-h treatment of cells before the bioassay experiments abolished the relaxation (Fig. 1). Treatments of equilibrated columns of interleukin-lp-
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H1790
TGF-fii,
IL-l&
AND SMOOTH
MUSCLE-DERIVED
NO
before contraction with phenylephrine induced contractions in canine coronary arteries but not in rat aortas (Fig. 2). Methylene blue abolished the relaxations of rat aortic detector rings evoked by perfusates from interleukin-l@ (10 U/ml)-stimulated smooth muscle cells and significantly reduced them when canine coronary arteries were used as bioassay tissues (Fig. 2). With a l-s transit time, addition of nitro-L-arginine (10m4 M) to the column of cells reversed the relaxation induced by the perfusates from cells treated with interleukin- l/3 (10 U/ml) for 24 h before bioassay experiments. The subsequent addition of L-arginine at equimolar concentration (10m4 M) to the cells reversed the blockade evoked by nitro-L-arginine. D-Arginine (lop4 to 10d3 M) did not counteract the inhibitory effect of nitro-L-arginine (Fig. 3). When the transit time was increased to 5 min, nitro+arginine (10m4 M) abolished the relaxations evoked by the perfusates from interleukin-la-treated cells (Fig. 4). At transit time >l s, L-arginine did not neutralize the effect of the synthase inhibitor nitro-LFig. 1. Relaxations of canine coronary artery bioassay tissues superfused with perfusates from cultured vascular smooth muscle cells grown on arginine (Fig. 4). The same observations were made when microcarrier beads. Cells were incubated with interleukin-la (IL-l& 10 Wistar rat aortic rings were used as detector tissues (data U/ml) for 24 h in presence or absence of cycloheximide (5 pg/ml). not shown). Arginine-related compounds alone had no Effects of nitro-L-arainine (10m4 M) infused through column of cells for significant direct effect on the isometric tension of canine 1 h and a prolonged?ransit time between donor and detector tissues (5 coronary bioassay rings (data not shown); the additional min) were also determined. Results are presented as means rt SE of 3 separate experiments and are expressed in % contraction induced by contraction observed after the blockade of the relaxation phenylephrine (10-s M); no. of cells per column was 2.6 f 0.2 X 106. by nitro-L-arginine appeared to be related not to the * Statistically significant differences from control (untreated cells). treatments by increasing concentrations of D-arginine but rather to the development of spontaneous myogenic tone in most of the canine bioassay preparations. Relaxstimulated cells for 1 h with nitro-L-arginine (10e4 M), a specific inhibitor of NO production, significantly ation of bioassay rings was not evoked by perfusates from decreased the relaxing activity of the perfusates (Fig. 1). columns of smooth muscle cells treated with L-a&nine Treatments of detector rings with methylene blue (10m5 (10m5 to 10m3 M) unless they had been exposed to interM), an inhibitor of soluble guanylate cyclase, for 30 min leukin-l@ (10 U/ml) for at least 3 h (data not shown).
Fig. 2. Effects of methylene blue (MB, 10-5 M) on isometric tension of a canine coronary artery (A) and a rat aorta used as bioassay tissues (B) and modulation of relaxations elicited by perfusates from columns of cultured vascular smooth muscle cells on microcarrier beads and treated with IL-10 (10 U/ml) for 24 h. Results are expressed as means f SE (n = 4-3 separate expta); no. of cells per column was 1.9 + 0.1 X lo6 and 2.4 + 0.4 X lo6 for experiments with canine and rat arteries, respectively. * Statistically significant differences from contracted level. 6
-2 :
Without
MB
With MB
-2 !
Without
MB
With MB
n Methylene blue 10 pM q Phenylephrine 1 @A 0
Phenylephrine 1 pM + IL-1 B-treated cells
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TGF-81, IL-l@, AND SMOOTH
MUSCLE-DERIVED
B
A
D-erg IL-16
W4Y I)-srg
treated
L-erg
H1791
NO TRANSIT
TIME
1 SECOND
104M
4xW4M
SYC ;
nttro-L-wg
1Q4Y
tmatttmo1aocond PE m-h
Fig. 3. A: experiment with a canine coronary artery as bioassay ring (performed with a transit time of 1 s) showing effect of nitro-L-arginine (nitro+Arg, 10m4 M), r.,-arginine (L-Arg, lo-* M), and D-arginine (D-Arg, 10m4 to 1O-3 M) on relaxing effect of perfusates from IL-l&treated (10 U/ml for 24 h) smooth muscle cells (SMC) grown on microcarrier beads. PE, phenylephrine. B: modulation of relaxations induced by perfusates from IL-l&treated cells in presence or absence of arginine-related compounds. Results are presented as means + SE of 4 separate experiments and are expressed in % contraction induced by PE (10-a M); no. of cells per column was 2.5 f 0.8 x 106. * Statistically significant differences from relaxed level.
The amplitude of the relaxations induced by perfusates from interleukin-l&treated cells was inversely proportional to the duration of perfusion with control solution when transit times of 5 min were used in bioassay experiments (Fig. 5). However, a maximal relaxation could be restored by infusion of superoxide dismutase (7.5-15 U/ml) between the donor columns of cells and the bioassay tissues (Fig. 5B). Also, reduction of the transit time from 5 min to 1 s mimicked the effect of the scavenger of oxygen-derived free radicals (Fig. 5C). When transforming growth factor-p1 (10 rig/ml) was added to the cells simultaneously with interleukin-l/3 (10 U/ml, 24 h before bioassay experiments), it inhibited the relaxing effect evoked by the perfusates from cells treated with the cytokine (Fig. 6A); perfusates from smooth muscle cells treated with transforming growth factor-p1 (10 rig/ml) or its vehicle (4 mM HCl containing bovine serum albumin 0.1%) for 24 h before bioassay experiments did not elicit changes in tension of contracted bioassay tissues. Perfusates from 31’3 fibroblasts cultured on Cytodex microcarrier beads and treated in an analogous manner to vascular smooth muscle cells did not elicit relaxations of either the canine coronary artery or the rat aorta bioassay tissues, even after addition of r.,-arginine ( 10m5 to 10m3 M; data not shown). Production of nitrite. Treatment of cultured vascular smooth muscle cells with interleukin-l@ (10 U/ml) induced a time-dependent accumulation of nitrite in the culture medium (Fig. 7A). After a 48-h exposure of the cells to interleukin-l/3 or its vehicle, the production of
nitrite was 3.28 + 0.79 and 10.73 + 0.75 nmol/106 cells in supernatants from control and treated cells, respectively. The accumulation of nitrite, measured after 24 h, was concentration dependent, and the threshold concentration of interleukin-la was 1 U/ml (Fig. 723). Treatment of cultured cells with transforming growth factor-& (0.1-10 rig/ml) or its vehicle did not evoke significant accumulation of nitrite in the extracellular medium after 24 h incubation; when transforming growth factor-& was added to the cells simultaneously with interleukin- l/3 (10 U/ml), it abolished the production of nitrite induced by the cytokine in a concentration-dependent manner with a maximal effect at 4 rig/ml (Fig. 6B). DISCUSSION
The present data demonstrate that interleukin-l/3 stimulates the production of potent nonprostanoid relaxing substance(s) in cultured smooth muscle cells from Wistar rat aortas. The induction of the relaxing activity by the cytokine probably involves protein synthesis, since the production of the relaxing agent(s) is abolished by cycloheximide and the relaxation of contracted bioassay rings is detected after 13 h of treatment of the smooth muscle cells with interleukin-l@. The blockade of the relaxing activity evoked by cycloheximide was correlated with a 97% inhibition of protein synthesis in treated cells (data not shown), and at the low levels of the compound used there were no apparent cytotoxic effect on the cultured cells. Relaxations evoked by the perfusates are attenuated by treatment of the cells but not the bioassay rings with
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H1792
TGF-j3i, IL-l&
AND SMOOTH
MUSCLE-DERIVED
A
NO
B Parg
IL-18
treated
xl-41 D-•rg
L-W 4XlO”Y
lo”64 L-srg
translt
time
TRANSIT
TIME
5 MINUTES
2 min 30 se5 g ‘;
4XlO’M
106
Fig. 4. A: experiment with a canine coronary artery as bioassay ring (performed with a transit time first of 5 min, then of 1 s) showing effect of nitro+Arg (1O-4 M), L-Arg (10e4 to 4 X 10e4 M), and D-Arg (10m4to 10e3 M) on relaxing effect of perfusates from IL-l&treated (10 U/ml for 24 h) smooth muscle cells grown on microcarrier beads. B: modulation of relaxations induced by perfusates from IL-l&treated cells in presence or absence of arginine-related compounds. Results are expressed as means f SE of 3 separate experiments, and are expressed as % contraction induced by PE (10v6 M); no. of cells per column was 2.5 sf: 0.8 X 10s. * Statistically significant differences from relaxed level.
nitro+arginine, an inhibitor of the production of nitric Infusion of nitro-L-arginine oxide from L-arginine. through the columns of cells also reversed the relaxations evoked by the perfusates, an effect that was overcome stereospecifically by the addition of an equimolar concentration of L-arginine. The relaxing activity produced by interleukin-l&treated cells was inhibited by methylene blue, an inhibitor of soluble guanylate cyclase, suggesting that the relaxation is associated with an increase of cGMP in the tissues. These results confirm previous studies showing that interleukin-la induces an L-arginine NO-like pathway in cultured smooth muscle cells (5, 7) that evokes a sustained production of relaxing factor(s) A
and stimulates the formation of cGMP (27). These observations may account for previous data suggesting that the impairment of contractions in isolated blood vessels exposed to endotoxin and cytokines involve protein synthesis (4, 19) and are mediated by an L-arginine NO pathway (15). In canine coronary bioassay tissues, methylene blue induces significant increases in tension. Only a partial reduction of the relaxation elicited by perfusates of interleukin-lb-treated cells could be detected in canine bioassay tissues treated with the vital dye. These data emphasize the heterogeneity in vascular responses, and the two bioassay tissues used mav exnress different isoforms of the soluble guanylate cyclase, which may C
B
IL-14 IL-1 6 lrbatbd
SMC I SOD 7.5 U/ml I
I
treated sm.
SOD 15 U/ml I
1 second I
QL 2 mln
I
‘1. 3
-PE
Fig. 5. Relaxation of a canine coronary artery ring without endothelium (bioassay tissue) contracted with PE (lOvs M), evoked by perfusats from cultured vascular SMC grown on microcarrier beads, and treated with IL-lb (10 U/ml) for 24 h. Influence of transit time (1 s, 5 min), and of infusion of superoxide dismutase (SOD, 7.5-15 U/ml) between donor cells and detector tissue were determined according to time of perfusion (l-4 h) with control solution. Traces were recorded after 1 h (A), 2.5 h (I?), and 4 h (C) of continuous perfusion of donor cells and superfusion of detector tissue.
mlnutu.
lo-%-
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TGF-/3,, IL-l&
AND SMOOTH
MUSCLE-DERIVED
H1793
NO
B
A
0 conbd n IL-1 D tr*amd SLIC
IL-lO+TGF-RI sue
treated
IL-lR(lOLvfr4
IL.IR treated SMC
PE 10”M TGF-81
(rig/ml)
Fig. 6. A: relaxation of a canine coronary artery ring without endothelium (bioassay tissue) contracted with PE (10e6 M), evoked by perfusate from cultured vascular SMC grown on microcarrier beads, and treated with IL-10 [(lo U/ml for 24 h) or its vehicle (control)] in presence of transforming growth factor-& [TGF-PI (10 rig/ml for 24 h), or its vehicle (control)]. Period of time that is defined by horizontal strait “direct line” corresponds to washing procedure of bioassay preparation superfused with physiological solution passing through a column of cell-free microcarrier beads. B: effect of increasing concentrations of TGF-0, on accumulation of nitrite in extracellular medium evoked by a 24-h exposure of cultured cells to IL-l@. Results are expressed as means +- SE of 3 separate experiments performed in triplicate. * Statistically significant differences from control.
account for the observed differences in the action of methylene blue. However, it can also be speculated that the(se) vasoactive agent(s) may induce smooth muscle relaxation by other mechanism(s) than those involving soluble guanylate cyclase. Complete relaxations were evoked by perfusates from interleukin-lp-treated smooth muscle cells when the transit time between the donor and the detector tissues was increased from 1 s to 5 min. These results suggest either that the released substance(s) is (are) relatively stable and thus differs from NO per se, whose biological half-life ranges between 6 and 50 s (9, 13), or that a large
amount of NO is produced by the interleukin-lp-treated cells and even after 5 min of transit time the concentration of the free radical is still sufficient to elicit maximal relaxations. Because nitro-r,-a&nine rapidly reversed the relaxation observed after 5 min, the active entity present in the perfusates most likely is derived from the metabolism of L-arginine. This interpretation is supported by 1) the stereoselective restoration of the relaxation evoked by exogenous L-arginine observed with a l-s transit time, 2) the effect of superoxide dismutase, which potentiates the relaxing activity of perfusates when the transit time was prolonged to 5 min probably by preventing the deg-
A 12 1
-O-
B
Control
Fig. 7. A: time course of accumulation of nitrite in supernatants from control and IL-la-treated cultured SMC from rat aortas. B: accumulation of nitrite in extracellular medium evoked by a 24-h exposure of cultured cells to increasing concentrations of IL-l@ Results are expressed as means + SE of 3 separate experiments performed in triplicate.
0
10
20
Time
30
(hours)
40
50
Interleukin-10
(U/ml)
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H1794
TGF-PI, IL-l&
AND SMOOTH
radation of NO by superoxide anions (26), and 3) the restoration of activity (when it has decayed with a transit time of 5 min) by return to a l-s transit time. The fact that L-arginine overcomes the blockade by nitro+arginine only when the transit time is 1 s suggests that the enzymatic conversion of the amino acid generates a labile relaxing agent that may be NO. Another consideration that suggests NO is the most likely candidate as a relaxing agent is the extracellular accumulation of nitrite in a time- and concentration-dependent manner after treatment of the cells with interleukin-lfi. NO may be stabilized by thiols and iron to form a transferable complex whose biological (except stability) and chemical properties appear similar to those of endothelium-derived NO; this complex will release the free entity, NO per se, to elicit the biological activities (18, 32). This may well occur with the L-arginine-derived relaxing factor(s) produced by smooth muscle cells since the relaxations of the superfused bioassay preparations are present despite a transit time of 5 min. The fact that the production of relaxing factor(s) is cycloheximide sensitive and requires lo-12 h to reach maximum effect is in accordance with the delayed onset of the production of cGMP evoked by interleukin-l@ in cultured smooth muscle cells from rat aorta [maximum levels after 9-10 h (27)], the potential second messenger of the relaxing factor(s). These observations raise the possibility that interleukin-lp may not stimulate the expression of an L-arginine NO synthase-like enzyme directly. The possible intermediates in such an activating process have not yet been identified, but interleukin-l@ induces the production of potent second-message stimulators, one of which, platelet-derived growth factor, plays a major role in the regulation of the metabolism of smooth muscle (25). The production of the relaxing factor(s) by the inducible synthase does not depend on the continuous presence of the cytokine, since the production of the active agent(s) continues for at least 36 h after a 12-h exposure of the cells to interleukin-I& Furthermore, the production and release of the vasoactive substance(s) are sustained, since perfusates from interleukin- I@treated cells relax bioassay tissues after 4 h of continuous perfusion without repletion by L-arginine. Although the intracellular concentration of L-arginine in cultured cells is -1 mM, continuous perfusion of the cells on Cytodex microcarrier beads could eventually lead to depletion of L-arginine stores. A reduction of the intracellular stores of L-arginine by metabolism and/or cellular efflux could account for the decreased potency of the relaxing substance(s) after perfusion of the column of cells for several hours. Replenishment of the stores then would restore the production of a NO-like factor. These results suggest that the inducible NO synthase depends on the availability of the substrate L-arginine to produce vasodilator agent(s) under basal conditions, as is true for the noninduced NO synthase (11, 28, 33). Transforming growth factor-& inhibits interleukin-lpinduced production of nitrite in cultured smooth muscle cells in a concentration-dependent manner and suppresses the relaxing activity of the perfusates from cytokine-treated cells. These results are in accordance with
MUSCLE-DERIVED
NO
previous data reporting an inhibitory effect of the peptide of cGMP in rat renal mesangial cells elicited by interleukin-16 and tumor necrosis factor-a! (24) and 2) on the cytotoxic properties of activated macrophages related to the production of nitrogen oxides (22). Whether transforming growth factor-& interferes with the initial steps after interactions of interleukin-l@ with its receptor or modulates the L-arginine NO pathway once it is induced is unknown. It may affect synthesis and/or activation of proteins involved in the generation of NO. The release of transforming growth factor-p during platelet degranulation, a consequence of adhesion of these cells to the vascular wall during inflammatory or immunological reactions, may counteract endotoxin and cytokine stimulation of NO. These present data suggest that the L-arginine-derived relaxing factor(s) is most likely a close precursor or a complexed form of NO, leading to the elevation of nitrite levels in conditioned medium of interleukin-1P-treated cultured smooth muscle cells. Interleukin-lfl appears to stimulate the inducible NO synthase isoform in the cultured smooth muscle cells from the rat aorta as cytokines and microbial products do in rabbit aortic smooth muscle cells (7) and macrophages (30). By contrast, under the present experimental conditions, such a pathway was not detectable in 3T3 fibroblasts either in the presence or absence of L-arginine (10 -3 M). Furthermore, since interleukin-1P impairs the contractility of rat aortas to the same extent in the presence or absence of endothelium (4) and is a poor direct stimulator of the production of nitrite in cultured endothelial cells from murine brain microvessels (16), it is likely that interleukin-10 exerts a selective effect on the smooth muscle cells of the vascular wall. Although the exact nature of the smooth muscle-derived relaxing factor(s) has not been determined, the L-arginine derived relaxing substance(s) might not act exclusively at a local area and could exert long-term modulation of vascular homeostasis once its synthesis has been induced by a cytokine-mediated pathway. 1) on the production
The authors thank B. Desta and G. Green for technical assistance. This work was supported by National Heart, Lung, and Blood Institute Grants HL-31183, HL-35614, and HL-46356, by an unrestricted Research Award from the Bristol-Myers-Squibb Research Institute, and by the Laboratoire Chauvin (France). Received 15 July 1991; accepted in final form 28 January 1992. REFERENCES 1. Assoian, R. K., B. E. Fleurdelys, H. C. Stevenson, P. J. Miller, D. K. Madtes, E. W. Raines, R. Ross, and M. B. Sporn. Expression and secretion of type fl transforming growth factor p by activated human macrophages. Proc. N&l. Ad. Sci. USA 84: 6020-6024, 1987. 2. Assoian, R. K., A. Komoriya, C. A. Meyers, D. M. Miller, and M. B. Sporn. Transforming growth factor-p in human platelets: identification of a major site, purification and characterization. J. Biol. Chem. 258: 7155-7160, 1983. 3. Beasley, D. Interleukin 1 and endotoxin activate soluble guanylate cyclase in vascular smooth muscle. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R38-R44, 1990. 4. Beasley, D., R. A. Cohen, and N. G. Levinski. Interleukin 1 inhibits contraction of vascular smooth muscle. J. Clin. Invest. 83: 331-335, 1989. 5. Beasley, D., J. H. Schwartz, and B. M. Brenner. Interleukin-l induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
TGF-&, 6.
IL-l&
AND SMOOTH
muscle cells. J. Clin. Inuest. 87: 602-608, 1991. Bredt, D. S., and S. H. Snyder. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc. Nutl. Acad. Sci. USA 87: 682-685,
MUSCLE-DERIVED 20.
Moncada, S., R. M. J. Palmer, and E. A. Higgs. Biosynthesis of nitric oxide from L-arginine: a pathway for the regulation of cell function and communication. Biochem. Phurmucol. 38: 1709-
21.
Mulsch, A., E. Bassenge, and R. Busse. Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism. Nuunyn-Schmiedeberg’s Arch.
1990.
1715,
7.
Busse, R., and A. Mulsch. Induction of nitric oxide synthase by cytokines in vascular smooth muscle. FE&S Lett. 275: 87-90,
8.
Fleming, I., G. A. Gray, G. Julou-Schaeffer, J. R. Parratt, and J. C. Stoclet. Incubation with endotoxin activates the L-arginine pathway in vascular tissue. Biochem. Biophys. Res.
1990.
Commun.
171: 562-568,
1990.
Forstermann, U., G. Trogisch, and R. Busse. Species-dependent differences in the nature of endothelium-derived relaxing factor. Eur. J. Pharmacol. 106: 639-643, 1985. 10. Furchgott, R. F., and P. M. Vanhoutte. Endothelium-derived relaxing and contracting factors. FASEB J. 3: 2007-2018, 1989. 11. Gold, M. E., K. S. Wood, R. E. Byrns, G. M. Buga, and L. J. Ignarro. L-Arginine-dependent vascular smooth muscle relaxation and cGMP formation. Am. J. Physiol. 259 (Heart Circ. 9.
Physiol. 12.
28):
H1813-H1821,
1990.
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.
Griffith, T. M., D. H. Edwards, M. J. Lewis, A. C. Newby, and A. H. Henderson. The nature of endothelium-derived vascular relaxant factor. Nature Lond. 308: 645-647, 1984. 14. Ignarro, L. J. Biological actions and properties of endotheliumderived nitric oxide formed and released from artery and vein. 13.
Circ.
Res. 65: l-20,
1989.
15.
Julou-Schaeffer, G., G. A. Gray, I. Fleming, C. Schott, J. R. Parratt, and J. C. Stoclet. Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway. Am. J.
16.
Kilbourn, R. G., and P. Belloni. Endothelial cell production of nitrogen oxides in response to interferon y in combination with tumor necrosis factor, interleukin-1, or endotoxin. J. Nutl. Cancer
Physiol.
Inst. 17.
259
(Heart
82: 772-776,
Circ.
Physiol.
28):
H1038-H1043,
1990.
1990.
Knowles, R. G., M. Palacios, R. M. J. Palmer, and S. Moncada. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Nutl. Acud. Sci. USA 86: 51595162,
1989.
Lancaster, J. R., and J. B. Hibbs. EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proc. Nutl. Acud. Sci. USA 87: 1223-1227, 1990. 19. McKenna, T. M. Prolonged exposure of rat aorta to low levels of endotoxin in vitro results in impaired contractility. Association with vascular cytokine release. J. Clin. Inuest. 86: 160-168, 1990. 18.
H1795
NO
1989.
Phurmucol.
340:
767-770,
1989.
Nelson, B. J., P. Ralph, S. J. Green, and C. A. Nacy. Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-&. J. Immunol. 146: 1849-1857, 1991. R. M. J., D. S. Ashton, and S. Moncada. Vascular 23. Palmer, endothelial cells synthesize nitric oxide from L-arginine. Nature 22.
Land. 24.
333:664-666,
1988.
Pfeilschifter, J., and K. Vosbeck. Transforming growth factor p2 inhibits interleukin lp- and tumor necrosis factor a-induction of nitric oxide in rat renal mesengial cells. Biochem. Biophys. Res. Commun.
175: 372-379,
1991.
Raines, E. W., S. K. Dower, and R. Ross. Interleukin-1 mitogenie activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science Wash. DC 243: 393-396, 1989. 26. Rubanyi, G. M., and P. M. Vanhoutte. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am. J. 25.
Physiol.
250
(Heart
Circ.
Physiol.
19): H822-H827,
1986.
Schini, V. B., D. C. Junquero, T. Scott-Burden, and P. M. Vanhoutte. Interleukin-lp induces the production of an L-arginine-derived relaxing factor from cultured smooth cells from rat aorta. Biochem. Biophys. Res. Commun. 176: 114-121, 1991. L-Arginine evokes both 28. Schini, V. B., and P. M. Vanhoutte. endothelium-dependent and independent relaxations in L-arginine-depleted aortas of the rat. Circ. Res. 68: 209-216, 1991. 29. Scott-Burden, T., T. J. Resink, U. Baur, M. Burgin, and F. Buhler. Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats. Hypertension 27.
13: 295-304,
1989.
Stuehr, D. J., and M. A. Marletta. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon gamma. J. Immunol. 139: 518-527, 1987. 31. Tayeh, M. A., and M. A. Marletta. Macrophage oxidation of L-arginine to nitric oxide, nitrite and nitrate. Tetrahydrobiopterin is required as cofactor. J. Biol. Chem. 264: 19654-19658, 1989. 32. Vanin, A. F. Endothelium-derived relaxing factor is a nitrosyl iron complex with thiol ligands. FEBS Lett. 289: l-3, 1991. 33. Wood, K. S., G. M. Buga, R. E. Byrns, and L. J. Ignarro. Vascular smooth muscle-derived relaxing factor (MDRF) and its close similarity to nitric oxide. Biochem. Biophys. Res. Commun. 30.
170: 80-88,
1990.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
