INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IN VASCULAR SMOOTH MUSCLE, the induction of inducible nitric oxide synthase (iNOS) is thought to be responsible, at least in part, for the irreversible hypotension that occurs in the setting of shock induced in humans or other mammals by endotoxin or septicemia (31). In the hypotensive process, tissue-derived (monocyte/macrophage) cytokines such as interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) are also thought to play a vasoregulatory role (3, 5). A number of previous studies have examined the induction of iNOS in vascular tissue, using cultured smooth muscle cell systems (24, 27) or intact vascular tissue obtained from animals that had been either pretreated with endotoxin in vivo (16, 31) or treated with lipopolysaccharide (LPS) or cytokines in vitro (3, 8, 9, 23, 25, 26, 35). The inducible form of the biosynthetic enzyme (iNOS or NOS2) in smooth muscle, responsible for the synthesis of nitric oxide from the precursor L-arginine, has been found to be the same as the enzyme originally found to be induced in macrophages and cultured fibroblasts (11, 27, 29) but distinct from the enzyme present in the endothelium (eNOS or NOS3) or in neurons (nNOS or NOS1) (10, 14).

In previous work, we studied the induction of iNOS in endothelium-denuded rat aorta (RA) rings maintained in an organ bath in vitro (39). We observed a spontaneous induction of iNOS over a 3- to 6-h time period, as documented both functionally (L-arginine-induced relaxation) and biochemically (iNOS mRNA upregulation). It was our conclusion that the trauma of tissue dissection probably represented part of the trigger for iNOS induction. This trauma may reflect the situation in vivo, for instance, when the endothelium is injured during angioplasty. In preliminary experiments aimed at determining an upstream stimulus for the induction of iNOS in the endothelium-denuded RA ring preparation, we observed that the addition of IL-1 to the organ bath (but not TNF-) enhanced the induction of iNOS in the tissue. The induction of "functional" iNOS was indicated by an enhanced relaxation, on the addition of L-arginine to a tissue bath that was preconstricted with phenylephrine. Based on this preliminary observation, we hypothesized that IL-1, originating in the tissue itself, might play a role in the induction of iNOS. In the work we describe in this report we aimed to test this hypothesis 1) with the use of an interleukin-1 receptor antagonist (IL-1ra) (2) and 2) by evaluating the time course of induction of IL-1 mRNA, iNOS mRNA, and functional iNOS, as assessed by the appearance of an aminoguanidine-sensitive L-arginine-induced relaxation. Furthermore, because of our interest in the role of tyrosine kinase and tyrosine phosphatase signal pathways in the induction of iNOS (8, 24, 26, 39), we evaluated the effects of several tyrosine kinase inhibitors: Tyrphostin 47 (TP 47/AG 213, Ref. 20), genistein (1), and the sarcoma virus tyrosine kinase (Src) family-selective tyrosine kinase inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; Ref. 12). In addition, because of our interest in other transmembrane signal pathways, we assessed the effects of several signal transduction pathway probes targeted to the following: phosphatidyl inositol 3'-kinase (PI3-K), mitogen-activated protein kinase kinase (MEK), and nuclear factor B (NF-B).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Bioassay procedures and induction of iNOS. The endothelium-denuded RA ring tissue was prepared as described previously (18, 40). The male Sprague-Dawley rats used for our work, weighing ~250 g, were cared for according to the recommendations of the Canadian Council on Animal Care. After death by cervical dislocation, the animals were exsanguinated from the common carotid arteries, and the aorta tissue was isolated for further dissection. In brief, RA rings (2 mm × 3 mm) rubbed free of endothelium were mounted at 37°C in a gassed (95% O₂-5% CO₂) Krebs-Henseleit buffer (4 ml) of the following composition (in mM): 118.0 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 25.0 NaHCO₃, 1.2 KH₂PO₄, and 10.0 glucose. At hourly intervals, rings were precontracted with 1 µM phenylephrine followed by a tissue wash. The absence of an intact endothelium in the RA preparation was ascertained by monitoring an absence of a relaxation response to 1 µM ACh. For each condition outlined in the following section (i.e., each time point or each test of a putative inhibitor), studies were done using a minimum of 8-10 aorta ring preparations from at least eight different animals. Each bioassay experiment of this kind to monitor contractility was repeated on at least eight separate occasions. Values shown either as histograms or data points represent the average response ± SE, as recorded in the figure legends. Comparisons between groups were assessed using the Student's t-test. With all statistical analyses, an associated probability (P value) <0.01 was considered highly significant.

Monitoring of iNOS induction using the relaxation response to L-arginine. After the preparations were mounted in the organ bath, tissues were precontracted with 1 µM phenylephrine. At the plateau of the contractile response, L-arginine (1 mM) was added to the organ bath; a relaxant response on adding L-arginine served as a pharmacological index of functional iNOS induction. This L-arginine-based pharmacological test, as a sensitive "on-line" functional index of iNOS induction, has been validated previously by us (39, 40) and by others (26). Tissues were then washed three times to remove agonist and excess L-arginine from the organ bath. This L-arginine-induced relaxation response, which is sensitive to 1 mM aminoguanidine, has been found to provide an accurate alternative to biochemical measurements (nitrite production, Western blot analysis) for monitoring the induction of iNOS (26, 39, 40). The time course of the induction of iNOS was determined pharmacologically by measuring L-arginine-induced relaxation every hour during the incubation of tissue in the organ bath; tissues were washed at 45-min intervals. All inhibitors described below, when present, were added to the organ bath at the beginning of an experiment. Actinomycin D and cycloheximide were both obtained from Sigma Chemical (St. Louis, MO). The tyrosine kinase inhibitors TP 47/AG 213, PP1, and genistein and the MEK inhibitor PD-98059 were from Calbiochem (La Jolla, CA). The PI3-K inhibitor wortmannin and the tyrosine phosphatase inhibitor sodium orthovanadate were from Sigma Chemical. Tosylphenylalanylchloromethyl ketone (TPCK; Sigma Chemical) and pyrrolidinedithiocarbamate (PDTC; Sigma Chemical), known to block the activation of NF-B, were also assessed by adding these reagents at the start of a prolonged incubation period (5 h). After either timed intervals (0.5-1 h) or at 5 h, the tissue was washed free from these reagents immediately before the assessment of iNOS induction by pharmacological (i.e., L-arginine-induced relaxation) or biochemical (RT-PCR evaluation of mRNA presence) procedures. As a routine monitor of iNOS induction, either the iNOS inhibitor aminoguanidine (Sigma Chemical) or the guanylyl cyclase inhibitor LY-83583 (Sigma Chemical) was added to the organ bath after the induction period to establish the blockade of L-arginine (1 mM)-induced relaxation by the inhibition of either iNOS or guanylyl cyclase. The recombinant human IL-1 receptor antagonist, IL-1ra, was generously provided by AMGEN (Boulder, CO); Dr. Nilofer Qureshi (University of Wisconsin, Madison, WI) kindly provided the diphosphoryl lipid A from Rhodobacter sphaeroides (13, 19).

Preparation of tissue RNA and RT-PCR analysis. Tissues were either processed immediately on isolation or harvested from the organ bath at the times indicated for processing. For each condition, at least four aorta rings from separate animals were pooled and processed for RNA preparation. Each RT-PCR experiment, incorporating four different tissues for each condition, was repeated a minimum of four times (i.e., representing a minimum of 16 independent tissue preparations, processed for RT-PCR as four pools). Total tissue RNA from the pooled aorta ring preparations was extracted with the TRI-reagent protocol (Molecular Research Center, Cincinnati, OH). The RNA (1 µg of each sample) was reverse transcribed with a first-strand cDNA synthesis kit using pd(N)6 primer (Pharmacia LKB Biotechnology, Uppsala, Sweden) according to manufacturer's recommendations at 37°C for 60 min; 2 µl of the resulting RT reaction product from each sample, containing cDNA, were used to amplify the iNOS, IL-1, and TNF- cDNA fragments. The sequences of the forward (5'-CCAGGGGCAAGCCATGTC-3') and reverse (5'-CTCCAGGCCATCTTGGTGGC-3') iNOS primers (578-bp product) were based on the published RA smooth muscle iNOS cDNA sequence (29). Sequencing of cDNA subcloned into the pBluescript SK phagemid was done previously (39), using the dideoxynucleotide sequencing method (33) and employing a T₇DNA polymerase sequencing kit (Pharmacia) to establish the identity of the RT-PCR fragment as rat iNOS. The PCR primer pairs for IL-1 (expected product, 519 bp) were 5'-CCAGGATGAGGACCCAAGCA-3' (forward) and 5'-TCCCGACCATTGCTGTTTCC-3' (reverse). The RT-PCR signals obtained from the aorta tissue were standardized according to the PCR signal generated concurrently by an actin primer pair (37) that spans an intron: forward primer, 5'-CGTGGGCCGCCCTAGGCACCA-3'; reverse primer, 5'-TTGGCCTTAGGGTTCAGGGGG-3'. The detection of a 243-bp PCR actin product using this primer pair can establish the absence of intron sequences in the RT product obtained from tissue RNA so as to confirm an absence of genomic DNA in the RNA preparations. The number of PCR cycles used (35 cycles of a primer extension period of 1 min at 72°C) was within the linear range for the PCR signals yielded for actin, IL-1, and iNOS. The PCR products were separated by 1% agarose gel electrophoresis and visualized with the use of ethidium bromide staining followed by photographic documentation. A minimum of four independent RT-PCR experiments were done with separate tissue preparations for each mRNA studied. Scanning densitometry of separately run samples was done (Pharmacia Image Master-1D desktop scanning system) to estimate the abundance of the visualized (film negatives used for densitometry) PCR products (%, relative to the signal for actin in each gel). Densitometric measurements were performed for each of four or more independently conducted experiments (comprising tissues from a minimum of 16 different animals, as outlined above). Averaged densitometric values (%, relative to actin) ± SE are shown either as histograms or data points, as elaborated on in the figure legends.

Monitoring NF-B activation using an immunoprecipitation-Western blot approach and Western blot detection of IL-1. RA tissues (a minimum of four aorta rings from four different animals) were isolated and were either processed for immunoprecipitation immediately (Fresh) or mounted in the organ bath for a 3-h incubation period (Induced) in the absence or presence of added reagents (e.g., PP1, IL-1). Fresh tissues and those taken from the organ bath after incubation were quickly frozen on solid CO₂ and stored at 70°C for further analysis. Quick-frozen tissues were rapidly thawed on ice and homogenized (Polytron, Brinkman Instruments, Rexdale, ON, Canada) in 2 ml of ice-cold immunoprecipitation buffer comprising 50 mM Tris · HCl, pH 7.4, supplemented with 1% vol/vol detergent Nonidet P40, 0.25% wt/vol sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml each of aprotinin and leupeptin, and 150 mM NaCl. For immunoprecipitation, equal protein aliquots of tissue extract (100 µg) were added to 1 ml of a saline (300 mM NaCl)-fortified 50 mM sodium phosphate buffer, pH 8.0, and were incubated for 1 h at 4°C with 0.5 µg/ml of a monoclonal antibody targeted to the nuclear localization sequence of the p65 subunit of NF-B (catalog no. 1697838; Boehringer Mannheim, Laval, QC, Canada). This concentration of antibody does not displace NF-B from the NF-B inhibitory subunit, I-B, and recognizes only the active NF-B subunits free from I-B (15). The immune complex containing activated NF-B was then harvested by the addition of protein G-Sepharose beads (100 µl of a 50% suspension in isotonic phosphate buffer, pH 7.4; Sigma Chemical) and incubated overnight at 4°C. Bead-adsorbed protein was collected by centrifugation (10,000 rpm/6,700 × g_av for 3 min at 4 °C, Sorvall microcentrifuge) with resuspension in 100 µl of the NaCl-fortified buffer described above, pH 8.0. This washing procedure was repeated three more times. Protein bound to the washed beads was eluted at 100°C into 30 µl of SDS/mercaptoethanol-containing electrophoresis sample buffer (17), and 25 µl of the eluted sample was subjected to electrophoresis in SDS-containing 11% polyacrylamide gels (1 mm × 6 cm × 8 cm) for 1.5 h at 100 V at room temperature. After electrophoresis, protein was transferred (30 V overnight at 4°C) to nitrocellulose membranes (0.45 µm; Bio-Rad) before immunoblot detection. Membranes were blocked with 10% (wt/vol) bovine serum albumin for 1 h at room temperature and then exposed for 1 h at room temperature to a 1:500 dilution (final concentration 2 µg/ml) of the murine monoclonal antibody described above, targeted to the nuclear localization sequence of the NF-B p65 subunit. After being washed for 45 min at room temperature in 10 mM Tris-saline, pH 7.4, the blot was exposed to the second antibody (1:5,000 dilution of supplied reagent of goat anti-mouse IgG, coupled to horseradish peroxidase; Amersham, Oakville, ON, Canada), and the location of NF-B was visualized by chemiluminescence detection (ECL reagent, Amersham). Western blot analysis for IL-1 immunoreactivity was performed as for NF-B, with prior immunoprecipitation from tissue extracts containing equal amounts of cellular protein (100 µg). Anti-IL-1, used at a concentration of 0.25 µg/ml for immunoprecipitation and 0.5 µg/ml for Western blot analysis, was from R & D Systems (Minneapolis, MN). Three to five independently conducted experiments were done, each of which represents a pool of four aorta ring preparations from different animals. Scanning densitometry of the Western blot fluorograms was done (Pharmacia Image Master-1D desktop scanning system) to estimate the abundance of activated NF-B and IL-1 relative to the Western blot signal for these proteins observed in freshly processed tissue samples. Values shown in the histograms and elaborated on in the figure legends represent averaged densitometric data ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Augmentation of functional iNOS induction by IL-1. In preliminary experiments, we evaluated the effects of adding LPS (100 ng/ml), TNF- (100 ng/ml), and IL-1 (10 ng/ml) to the organ bath on the rate and magnitude of appearance of an L-arginine-induced relaxation (induction of functional iNOS). The time course of induction of functional iNOS in the endothelium-denuded RA tissue incubated in the organ bath for up to 6 h was the same as we had observed previously (39), with a plateau of induction at ~4.5-5 h after the tissue was mounted in the cuvette (Fig. 1). As in our previous work (39), the relaxation caused by the addition of L-arginine was inhibited by both the iNOS inhibitor aminoguanidine (1 mM) and the guanylyl cyclase inhibitor LY-83583 (20 µM) (data not shown). This time course observed for the spontaneous appearance of L-arginine-induced relaxation was not altered by adding either LPS (100 ng/ml) or TNF- (100 ng/ml) to the organ bath. Nor was the spontaneous time course of induction altered when the LPS antagonist diphosphoryl lipid A (1 µg/ml) (13, 19) was added to the organ bath (not shown). Nonetheless, when IL-1 (10 ng/ml; ~0.6 nM) was added to the organ bath, although the time frame of induction was unaltered, there was, at each time point, an enhancement of the L-arginine-induced relaxation response (see Fig. 1). It was on the basis of these preliminary observations that we suspected that endogenous IL-1, acting in an autocrine/paracrine manner in the tissue preparation, might be playing a role in iNOS induction.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Time course of appearance of L-arginine (LR)-induced relaxation: effect of added interleukin-1 (IL-1). At hourly intervals after the equilibration of fresh rat aorta (RA) tissue in the organ bath in the absence or presence of 10 ng/ml IL-1, preparations were tested for the presence of inducible nitric oxide synthase (iNOS) by washing, constricting by the addition of 1 µM phenylephrine (PE), and then looking for a relaxation response on adding 1 mM LR to the organ bath. The tissue was then washed again and reequilibrated in buffer with or without IL-1. The degree of relaxation was expressed as a percentage relative to the magnitude of the contractile response of each tissue to PE [%relaxation = 100 × (tension in the presence of PE alone  tension in the presence of PE with 1 mM LR added)  tension in the presence of PE alone]. Each data point with error bars represents the mean ± SE relaxant response of 10-12 independently incubated RA rings from different animals. * Relaxation response in the presence of added IL-1 was significantly greater (P < 0.01) than the relaxation induced spontaneously.

Inhibition of iNOS induction by IL-1ra. When IL-1ra (100 ng/ml) was present in the organ bath during the 5-h incubation period, there was no relaxation induced by the addition of 1 mM L-arginine, as was present in the tissues incubated in the absence of IL-1ra (Fig. 2, A and B). The lack of appearance of L-arginine-induced relaxation correlated with a marked reduction in the abundance of iNOS mRNA in the tissues incubated for 5 h in the concurrent presence of IL-1ra compared with the control induced tissue (Fig. 2C). Quantification of the PCR bands by densitometry, relative to the signal yielded for actin in each tissue, indicated that the presence of IL-1ra reduced the appearance of iNOS mRNA by >90% (Fig. 2D).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Inhibitory effect of the IL-1 receptor antagonist (IL-1ra) on iNOS induction. Representative time/tension tracings (A) and average response histograms (B) for appearance of LR-induced relaxation. Freshly dissected RA tissues (A, left) incubated either without (A, top) or with (A, bottom) IL-1ra (100 ng/ml) were first evaluated for a relaxation response to 1 mM LR () after the tissue had been preconstricted with 1 µM PE (). Tissues were then washed and reequilibrated in fresh buffer, without or with IL-1ra, for 5 h at 37°C, at which time the untreated or IL-1ra-treated tissues were again assessed for the presence of an LR-induced relaxation. The scale for time and tension is to the right of the lower tracing. Histograms (B) show mean ± SE relaxation responses for RA rings obtained from 8 different animals, studied in 8 different experiments observed in the presence of 1 mM LR for freshly isolated tissue (Fresh) that had been maintained in the organ bath for 5 h (Induced) and tissue that had been maintained in the organ bath for 5 h in the concurrent presence of 100 ng/ml of IL-1ra (+IL-ra). C: tissue samples incubated under the identical conditions used for bioassay were equilibrated at 37°C for 4 h without or with 100 ng/ml IL-1ra and were harvested and processed for RNA isolation and RT-PCR analysis as outlined in METHODS by using primer pairs for iNOS and actin. Four RA rings from separate animals were used for each condition. Positions in the separating gel (C) of the PCR products for iNOS and actin are shown to the right of the gel. Data shown for the gel (C) are representative of 4 independently conducted experiments, each with 4 tissues (quadruplicate for each condition; 16 tissues represented by each histogram) from different animals. D: estimates by scanning densitometry of the abundance of the iNOS mRNA PCR signal relative to the actin signal (% of actin, ±SE) for freshly isolated tissue (Fresh), for tissue maintained in the organ bath for 5 h (Induced), and for tissue that was maintained for 5 h in the organ bath in the concurrent presence of 100 ng/ml IL-1ra (+IL-1ra). * iNOS mRNA signal that was significantly diminished (P < 0.01), compared with the signal observed in the absence of IL-1ra and that was not significantly different than the iNOS mRNA signal observed in fresh tissue (nondetectable).

Relative time courses of appearance of IL-1 mRNA and iNOS mRNA. Because the IL-1ra was able to block the spontaneous appearance of iNOS mRNA, we hypothesized that the induction of IL-1 mRNA might precede that of iNOS mRNA. To test this hypothesis, we measured, by RT-PCR, the time courses of appearance of both IL-1 and iNOS mRNAs during a 4-h time period in the organ bath (Fig. 3, top). The PCR signals for IL-1 mRNA and iNOS mRNA were normalized in each tissue relative to the PCR signal obtained concurrently for actin. In freshly isolated tissues, we were able to detect only very low amounts, if any, of IL-1 mRNA. A marked increase in IL-1 mRNA was detected as early as 0.5 h after the incubation of the tissue in the organ bath started; the IL-1 mRNA level continued to increase considerably over a 4-h period (Fig. 3, top). This increase in IL-1 mRNA reflected an increase in the amount of IL-1 precursor protein, detected by immunoprecipitation-Western blot analysis for tissue harvested at 3 h (Fig. 3, bottom left). The averaged densitometric data showed an ~2.8-fold increase in protein detected at 3 h by the Western blot (middle histogram, Fig. 3, bottom right). With an immunohistochemical approach like the one we used previously for iNOS (39), we were unfortunately not able to localize with confidence the site(s) of increase of IL-1 in the aorta ring. In contrast with the early increase in IL-1 mRNA, a definitive increase in the amount of iNOS mRNA was not apparent until 2-4 h of incubation (Fig. 3, top).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Time course of the spontaneous induction of IL-1 and iNOS mRNA (top) and Western blot analysis of IL-1 after induction in the absence or presence of the Src family kinase inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) (bottom). Tissues were incubated in the organ bath for the indicated times and were harvested for RNA isolation and RT-PCR analysis, as outlined in the legend for Fig. 2. The RT-PCR signals for IL-1 and iNOS, determined by scanning densitometry of stained photographed gels, were normalized (IL-1 or iNOS/actin: %Actin) to the signal for actin obtained from the same tissues. Data points, showing mean ± SE PCR signal ratios, represent averages obtained from 5 independently conducted experiments, wherein quadruplicate RA tissue samples, each from a different animal, were pooled for each condition. In Western blot analysis, tissues were either processed immediately (Fresh) or incubated in quadruplicate (each of 4 tissues derived from a different animal) in the organ bath at 37°C for 3 h, as for a bioassay, in the absence (Induced) or presence (+PP1) of PP1 (1 µM). Pooled tissue samples were then harvested for immunoprecipitation and Western blot analysis for IL-1 immunoreactivity, as outlined in METHODS. Only the 32-kDa precursor (Pro-IL-1) was detected. Positions of molecular mass markers (kD) and IL-1 immunoreactivity are shown on right and left, respectively, of the representative Western blot (bottom left). Scanning densitometry (bottom right) was used to estimate, relative to the densitometric signal for Pro-IL-1 in freshly isolated tissue (Fresh), the relative abundance (%Fresh Control) of Pro-IL-1 present in tissue that had been maintained in the organ bath for 3 h (Induced) and in tissue that had been maintained in the organ bath for 3 h in the concurrent presence of 1 µM PP1 (+PP1). Histograms show mean ± SE values for data obtained from 5 independently conducted experiments in each of which 4 individual RA rings from different animals were used for each condition (each histogram therefore representing data obtained from 20 individual tissue samples from different animals). * Reduction in the signal for Pro-IL-1 in the presence of PP1 that was significantly different (P < 0.01) from that observed in the absence of PP1 and that did not differ significantly from the Western blot signal observed for freshly isolated tissue.

Effects of tyrosine kinase/tyrosine phosphatase inhibitors on iNOS and IL-1 induction. Given the previous data obtained by us (39) and by others (8, 26) indicating that tyrosine kinase inhibitors could block the appearance of functional iNOS in vascular tissue, we wished to evaluate separately the effects of two distinct classes of tyrosine kinase inhibitors (PP1, which is selective for Src family kinases, and TP 47/AG 213 and genistein, which are nonselective) on 1) the induction of both functional iNOS and iNOS mRNA and 2) the induction of IL-1 mRNA. Furthermore, we wanted to assess the effects of the tyrosine kinase inhibitors on the ability of exogenously added IL-1 (10 ng/ml) to cause the appearance of functional iNOS and iNOS mRNA in tissues maintained in the organ bath. In these experiments, the tissues were treated with the tyrosine kinase inhibitors both during the dissection process and subsequently during the incubation period in the organ bath.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of tyrphostin 47 (TP 47/AG 213) and PP1 on the appearance of functional iNOS in the absence (spontaneous) or presence (IL-1-induced) of added IL-1. Top: representative time-tension tracings. Fresh RA tissues were mounted in the organ bath and tested first for the absence of LR (1 mM, )-induced relaxation in PE (1 µM, )-preconstricted samples (tracings A-F). Incubation in the organ bath was then allowed to proceed for 5 h at 37°C, without (A and D) or with tyrosine kinase inhibitors (B and E: PP1, 1 µM; C and F: TP 47/AG 213, 80 µM) either in the absence (spontaneous induction, A-C) or presence (IL-1-induced, D-F) of 10 ng/ml IL-1. At the end of the 5-h incubation period, all tissues were constricted with PE and again tested for the presence or absence of a relaxation in response to the addition of 1 mM LR, indicative of the induction of functional iNOS. Bottom: histograms showing average relaxation responses, corresponding to the representative tracings (A-F) shown above. For each condition, average relaxation responses to 1 mM LR (% ± SE) were calculated for data from 10 or more separately conducted experiments. Each histogram represents data averaged for 10-12 different RA ring preparations from 10 or more different animals. * Absent relaxation responses that were significantly lower (P < 0.001) than the relaxations observed in control, induced tissues; ** relaxation responses to 1 mM LR, observed in the presence of added IL-1, that were significantly (P < 0.01) greater than the relaxation responses observed in control induced tissues.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Effects of PP1 and TP 47/AG 213 on the induction of iNOS. RA tissues in quadruplicate, each from a different animal, were either harvested immediately on dissection (Fresh) or incubated for 3 h at 37°C in the organ bath, as outlined in the legend to Fig. 4, without or with either PP1 (+PP1, 1 µM; A) or TP 47/AG 213 (+TP 47, 80 µM; C) in the absence (Induced) or presence of 10 ng/ml IL-1. At 3 h, tissues were harvested and processed for RNA preparation and RT-PCR analysis, as outlined in METHODS. Positions of the oligonucleotide size markers (in bp) and of the iNOS and actin PCR products are indicated to the left and right, respectively, of the gels. Histograms (B and D) show mean ± SE iNOS PCR signals (relative to that of actin: %Actin) obtained by scanning densitometry from 4 independently conducted RT-PCR experiments, wherein, in each experiment, 4 different RA rings from different animals were pooled for each condition (each histogram therefore representing data obtained from 16 different tissue samples, pooled in groups of 4). * Complete suppression (significantly lower than the Induced tissues, P < 0.001) of the RT-PCR signal for iNOS mRNA caused by the tyrosine kinase inhibitors; ** increase in iNOS mRNA in the presence of IL-1 that was significantly (P < 0.01) greater than the increase observed in spontaneously induced tissues.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effects of TP 47/AG 213 and PP1 on spontaneous induction of IL-1 mRNA. A: representative gel showing separation of PCR products for IL-1 and actin. Freshly prepared RA tissues were either harvested immediately (Fresh) or incubated for 4 h in the organ bath at 37°C, as for a bioassay, in the absence (Induced) or presence of either TP 47/AG 213 (+TP47) or PP1 (+PP1, 1 µM). Tissues were then harvested and processed for RT-PCR analysis, using primer pairs for actin and IL-1, as outlined in METHODS. B: densitometric scans showing the abundance of IL-1 mRNA relative to that of actin (%Actin), estimated by scanning densitometry for each condition shown in the representative gel in A. Values are means ± SE for 4 independently conducted experiments in each of which 4 RA ring samples from different animals were pooled for each condition (i.e., each histogram was derived from 16 different tissue samples from different animals, grouped in pools of 4). PP1 (but not TP 47/AG 213) caused a complete suppression, as denoted by *, to the level of fresh tissues, of the induced increase in IL-1 mRNA that was observed in the absence of PP1.

View this table: [in this window] [in a new window]   Table 1.   Effects of inhibitors on the induction of IL-1 mRNA, iNOS mRNA, and functional iNOS (LR-induced relaxation)

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effects of signal pathway inhibitors on the induction of iNOS and IL-1 mRNA. Freshly prepared RA tissues were either harvested immediately or incubated in the organ bath for 3 h at 37°C, as for a bioassay, in the absence (Induced) or presence of the following signal pathway inhibitors: Vanadate (Van, 0.1 mM), actinomycin D (ActD, 1 µM), cycloheximide (CHX, 10 µM), and tosylphenylalanylchloromethyl ketone (TPCK, 20 µM). Both fresh and incubated tissues were processed for RNA preparation and RT-PCR analysis, as outlined in METHODS. Representative gels show the PCR products for IL-1 mRNA (A) and iNOS mRNA (B). The positions in the separating gels of the PCR products for IL-1 and actin and for iNOS and actin in the same separating gel are shown on the far left and far right, respectively. C: densitometric analyses of the abundance of IL-1 and iNOS mRNA relative to actin mRNA (%Actin), estimated by scanning densitometry, as outlined in METHODS, for each condition shown in A and B. Values are means ± SE for 4 independently conducted RT-PCR experiments wherein, in each experiment, 4 different RA ring preparations from different animals were pooled for each condition (each histogram thus representing data obtained from 16 tissue samples from different animals). * Complete suppression to control levels (not detectable) of the induction of iNOS mRNA by Van, ActD, and TPCK.

Effects of other inhibitors on the upregulation of iNOS and IL-1 mRNA. As expected for a protein induced via transcriptional activation, actinomycin D (1 µM) blocked the induction of iNOS that occurred either spontaneously in the organ bath or as a result of activating the tissue with added exogenous IL-1; both the appearance of L-arginine-induced relaxation and the appearance of iNOS mRNA were abrogated (not shown; see Ref. 39). In contrast, treatment of the tissue with actinomycin D, if anything, enhanced the abundance of IL-1 mRNA in the tissue (Fig. 7, A and C). Cycloheximide (10 µM), like actinomycin D, blocked both the spontaneous and IL-1-stimulated appearance of functional iNOS in the tissue (not shown). However, cycloheximide did not block the appearance of iNOS mRNA (either spontaneous or IL-1-stimulated; not shown and Fig. 7, B and C). Like actinomycin D, cycloheximide did not block and if anything potentiated the upregulation of IL-1 mRNA that appeared in the tissue (Fig. 7, A and C).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effect of PP1 on nuclear factor -B (NF-B) activation. Freshly dissected RA tissues were either quick-frozen immediately (Fresh) or incubated in quadruplicate (4 tissues from different animals for each condition) in the organ bath for 3 h at 37°C as for a bioassay either without additions (Induced) or with PP1 (+PP1, 1 µM), in the absence or presence of IL-1 (+IL-1, 10 ng/ml). Quick-frozen tissues (fresh or incubated) were extracted, and equal amounts of protein extracts were processed for immunoprecipitation and Western blot analysis, using the NF-B antibody targeted to the NF-B inhibitory subunit binding site, so as to monitor active NF-B. Inset: representative Western blot for the detection of activated NF-B. Scanning densitometry, as outlined in METHODS, was used to estimate the abundance of activated NF-B detected for each condition relative to the signal observed in freshly isolated tissue (%Fresh Control). Values are means ± SE from 3 independently conducted experiments wherein, in each experiment, 4 different RA ring preparations from different animals were pooled for each condition (each histogram therefore derived from data representing 12 RA tissues from different animals). * Significant (P < 0.01) reduction, compared with Induced, in the Western blot signal for activated NF-B caused by PP1; **significant (P < 0.01) increase, above spontaneously induced tissue, in activated NF-B observed on adding IL-1 to the organ bath, with or without the concurrent presence of PP1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The main findings of our study were 1) that the upregulation of IL-1 appears to play an autocrine/paracrine role in the induction of iNOS in rat vascular tissue and 2) that a Src family tyrosine kinase seems to be involved in the induction of IL-1 in the tissue. Furthermore, our results also imply that the distinct tyrosine kinase or phosphatase pathways, inhibited by PP1 on the one hand and by TP 47/AG 213, genistein, and vanadate on the other hand, are responsible for increased IL-1 mRNA and for IL-1-triggered iNOS induction.

In addition to the smooth muscle elements, the production of IL-1 in tissues could also arise from other cell sources (e.g., monocytes or macrophages). It has previously been shown that IL-1 can cause either an acute indomethacin-sensitive relaxation (23) or a reduction in phenylephrine-stimulated tissue contractility that occurs 2 to 3 h after treatment of the tissue with IL-1, presumably because of iNOS induction (3). Thus IL-1 produced in the vasculature could have either a direct or an indirect (via iNOS induction) effect on tissue tension.

Support for the conclusion that IL-1 plays an autocrine/paracrine role in the induction of iNOS in the RA tissue was provided by three lines of evidence: 1) IL-1ra blocked the spontaneous induction of iNOS in the tissue maintained in the organ bath, 2) the time course of induction of IL-1 mRNA preceded that of iNOS mRNA, and 3) the Src family-selective tyrosine kinase inhibitor PP1 blocked the spontaneous upregulation of IL-1 mRNA and the subsequent induction of iNOS but did not block the induction of iNOS stimulated by exogenously added IL-1. Taken together, our data suggest that a principal effect of the initial stimulus leading to iNOS induction in vascular tissue was to upregulate and release IL-1, which in a sequential manner then activated iNOS gene transcription.

Regrettably, we have not yet been able to determine the nature of the stimulus responsible for the spontaneous induction of IL-1 (and subsequently iNOS) in the organ bath. Neither the addition of LPS nor the inclusion of the LPS antagonist diphosphoryl lipid A (13, 19) in the organ bath buffer altered the time course of induction of iNOS. It is possible that 1) residual endotoxin in the organ bath, resistant to antagonism by diphosphoryl lipid A, 2) the presence in vascular tissue of a distinct stimulus for IL-1 induction (e.g., CD40-ligand; see Ref. 34), or 3) the trauma of tissue dissection and endothelium removal may have served as an initial stimulus for the induction of IL-1, which subsequently induces iNOS in the aorta tissue. Any or all such stimuli might potentially play a role in the setting of surgical angioplasty procedures in humans or in inflammatory or endotoxin-induced processes in vivo.

We were unable to ascertain in the tissue, as we had done previously for iNOS (39, 40), the precise location of induced IL-1, despite being able to document an increase in the 31-kDa IL-1 protein precursor in the tissue extract by immunoprecipitation and Western blot analysis (Fig. 3, bottom). Nevertheless, the induction of IL-1 has been observed in the cytosol and on the surface of LPS-treated cultured human vascular smooth muscle cells and blood vessel organ cultures (7, 21, 22). Furthermore, IL-1 mRNA has been detected in aorta tissue harvested from rabbits that had been treated in vivo with LPS (6). It can be noted, in addition, that cultured human vascular smooth muscle cells can produce not only IL-1 but also a form of the receptor antagonist IL-1ra (4). Thus our data, in keeping with the previous results of others, suggest that locally produced IL-1 can play an autocrine/paracrine role, which we now suggest may relate directly to the induction of iNOS in the tissue.

The data we obtained using the various signal transduction pathway inhibitors (summarized in Table 1) not only extend our previous study of the signal pathways involved in iNOS induction in vascular tissue (39) but also permit a comparison between the signals required for iNOS induction and those involved in the upregulation of IL-1 in the same tissue. Neither IL-1 nor iNOS induction appeared to require activation of the mitogen-activated protein kinase pathway (no effect of PD-98059). Moreover, of all the signal pathway probes evaluated, only the Src family-selective inhibitor PP1 blocked the upregulation of IL-1 mRNA; this inhibitor failed to block the upregulation of iNOS mRNA stimulated by exogenous IL-1. It may be of significance in this regard that, of the Src family members, c-Src represents the major component of the tyrosine kinase activity that can be detected in the cytosolic fraction of aorta tissue (30). Furthermore, the steps leading to the induction of iNOS mRNA and the subsequent posttranscriptional modifications required for the production of functional iNOS involve the activation of NF-B, PI3-K, and one or more distinct tyrosine kinase or tyrosine phosphatase enzymes. The signal pathways for iNOS induction appear to be more complex and quite distinct from those leading to IL-1 induction. Because it has already been possible with the use of nonselective tyrosine kinase inhibitors (tyrphostins and genistein) to prevent some of the untoward effects caused by the administration of LPS in vivo (28, 32, 36), one can suggest that the addition of a Src family-targeted inhibitor to one of the tyrphostins (AG 126 or AG 556) may yield a tyrosine kinase inhibitor cocktail that may be even more successful than tyrphostins alone for the treatment of endotoxin-induced shock in vivo.