Mechanism of rhinovirus-induced changes in airway smooth muscle responsiveness

An important interplay exists between specific viral respiratory infections and altered airway responsiveness in the development and exacerbations of asthma. However, the mechanistic basis of this interplay remains to be identified. This study addressed the hypothesis that rhinovirus (RV), the most common viral respiratory pathogen associated with acute asthma attacks, directly affects airway smooth muscle (ASM) to produce proasthmatic changes in receptor-coupled ASM responsiveness. Isolated rabbit and human ASM tissue and cultured ASM cells were inoculated with human RV (serotype 16) or adenovirus, each for 6 or 24 h. In contrast to adenovirus, which had no effect, inoculation of ASM tissue with RV induced heightened ASM tissue constrictor responsiveness to acetylcholine and attenuated the dose-dependent relaxation of ASM to beta-adrenoceptor stimulation with isoproterenol. These RV-induced changes in ASM responsiveness were largely prevented by pretreating the tissues with pertussis toxin or with a monoclonal blocking antibody to intercellular adhesion molecule-1 (ICAM-1), the principal endogenous receptor for most RVs. In extended studies, we found that the RV-induced changes in ASM responsiveness were associated with diminished cAMP accumulation in response to dose-dependent administration of isoproterenol, and this effect was accompanied by autologously upregulated expression of the Gi protein subtype, Gialpha3, in the ASM. Finally, in separate experiments, we found that the RV-induced effects on ASM responsiveness were also accompanied by autologously induced upregulated mRNA and cell surface protein expression of ICAM-1. Taken together, these findings provide new evidence that RV directly induces proasthmatic phenotypic changes in ASM responsiveness, that this effect is triggered by binding of RV to its ICAM-1 receptor in ASM, and that this binding is associated with the induced endogenously upregulated expression of ICAM-1 and enhanced expression and activation of Gi protein in the RV-infected ASM.     

Effects of glucocorticoids and beta-adrenoceptor agonists on the proliferation of airway smooth muscle

An increase in airway smooth muscle is a characteristic feature of asthma. Because beta-adrenoceptor agonists and corticosteroids are commonly used in the treatment of asthma we have studied the effects of these medicines on the growth of airway smooth muscle. These agents were incubated with bovine airway smooth muscle cells for 40 h for measurement of thymidine incorporation and 64 h for measurement of cell counts. Salbutamol inhibited thymidine incorporation (IC50 = 60 nM) and led to a reduction in cell number (IC50 = 10 nM). At 10 microM there was a 14.6 +/- 2.6% reduction in cell number. Salmeterol also inhibited the growth of the airway smooth muscle cells but the effect did not plateau at 10 microM. At this concentration there was an 89.5 +/- 3.6% reduction in thymidine incorporation and a 44.1 +/- 5.2% reduction in cell number. Cortisol and beclomethasone dipropionate were more potent than salbutamol in inhibiting thymidine incorporation with IC50 values of 5 nM and 0.2 nM respectively. Cortisol 100 nM led to a 16.6 +/- 6.5% reduction and beclomethasone dipropionate 3 nM led to a 17.8 +/- 5.8% reduction in cell number. If similar effects occur in man and in vivo, these medicines could act directly on airway smooth muscle to inhibit the development of hyperplasia.     

Independent modulation of horse airway smooth muscle by epithelium and prostanoids

The effects of epithelial removal and cyclooxygenase inhibition on contractions induced by exogenous acetylcholine (ACh) and electrical field stimulation (EFS) were evaluated in horse tracheal strips and bronchial rings. Epithelial removal potentiated the response to ACh but had no influence on the response to EFS. The effect of epithelial removal was not altered by pretreating the tissues with meclofenamate, a cyclooxygenase inhibitor. In trachealis strips, meclofenamate augmented contractions induced by EFS but not by ACh. In bronchial rings, meclofenamate augmented EFS-induced contraction to a greater extent than ACh-induced contraction. These effects of meclofenamate were epithelium-independent. We conclude that horse airway epithelium produces a relaxant factor that is not a prostanoid. Endogenous prostanoids originating from non-epithelial sites inhibit only cholinergic nerves in the trachea but both parasympathetic nerves and smooth muscle in the bronchi.     

Mitogen-activated signaling in cultured airway smooth muscle cells

Airway hyperresponsiveness and excess smooth muscle mass coexist in patients with asthma and bronchopulmonary dysplasia. This increase in airway smooth muscle mass, which in part relates to smooth muscle proliferation, may increase bronchoconstrictor-induced airway narrowing, even in the absence of excessive force generation. Thus, there is need for a precise understanding of the events involved in airway smooth muscle mitogenesis. This review examines the inflammatory substances and growth factors that induce airway smooth muscle proliferation, and the signaling pathways that may be involved in the transduction of these extracellular signals to the cell nucleus. Also discussed are various antimitogenic substances and potential mechanisms underlying the inhibition of cell proliferation. Central to the discussion are the extracellular signal regulated kinases (ERKs), serine/threonine kinases of the mitogen-activated protein kinase (MAP kinase) superfamily, which upon activation, translocate from the cytoplasm to the nucleus after mitogenic stimulation. Insight gained from studies of cultured airway smooth muscle growth and mitogen-activated signaling may shed light on parallel mechanisms that may operate in asthma and in bronchopulmonary dysplasia, and may lead to therapeutic interventions against airway remodeling.     

Isometric and isotonic contractions in airway smooth muscle

Canine tracheal smooth muscle was used as an in vitro model of smooth muscle in intrapulmonary airways to determine whether active tension curves derived from isometric and isotonic muscles are similar, and thus resemble striated muscle in this respect. Isometric, isotonic after-loaded, and isotonic free-loaded contractions elicited at different lengths and loads, were analysed. The data demonstrate that length-tension (L-T) diagrams were different in these various types of contractions for electrically and carbachol driven tracheal smooth muscles strips. In general, at any given length active tension is less in isotonic and free-loaded modes of contraction as compared with isometric. We conclude that the ability to actively develop tension at a given length in airway smooth muscle depends on the mode of contraction.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K+ channel have been described in vascular smooth muscle: Voltage-activated K+ channels (Kv) are encoded by the Kv gene family, Ca(2+)-activated K+ channels (BKCa) are encoded by the slo gene, inward rectifiers (KIR) by Kir2.0, and ATP-sensitive K+ channels (KATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4-1.6, Kv2.1, Kv9.3, Kv beta 1-beta 4, slo alpha and beta, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K+ channels are modulated by physiological vasodilators, which increase K+ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate KATP channels. These include adenosine, calcitonin gene-related peptide, and beta-adrenoceptor agonists. beta-adrenoceptors can also activate BKCa and Kv channels. Several vasoconstrictors that activate protein kinase C inhibit KATP channels, and inhibition of BKCa and Kv channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BKCa channels, and possibly KATP channels. Hypoxia leads to activation of KATP channels, and activation of BKCa channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of Kv channels. Vasodilation to increased external K+ involves KIR channels. Endothelium-derived hyperpolarizing factor activates K+ channels that are not yet clearly defined. Such K+ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

Ketamine relaxes airway smooth muscle contracted by endothelin

Endothelins (ETs) are synthesized not only in vascular endothelial cells but also in airway epithelial cells. Increased ET-1 has been demonstrated in bronchial epithelium of asthmatic patients, and, in severe asthma attacks, ET-1 increases in plasma and bronchoalveolar lavage fluid. In this study, we investigated whether ketamine (KET) relaxes ET-induced tracheal contractions. Female guinea pigs were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into two strips that were mounted in an organ bath filled with Krebs-bicarbonate buffer. The response of each strip to 10(-7) M carbachol was taken as 100% contraction to which the response to ET was referred. The contribution of the epithelium to the relaxant effect of KET was studied in denuded tracheae or in the presence of 5 x 10(-5) M indomethacin. ET-1 (3 x 10(-8) M) induced contractions that were 76 +/- 3% of those induced by carbachol. KET reversed the response to ET-1 in a dose-dependent fashion. Similarly, ET-2 (3 x 10(-8) M) induced contractions that were 74 +/- 5% of those induced by carbachol, and KET also reversed this response in a dose-dependent manner. In epithelium-denuded strips, ET-1 induced contractions that were 104 +/- 3% of those induced by carbachol, and KET still reversed this response. The tonic phase of the response to ET-1 was equal (100 +/- 6%) to the response to carbachol, and KET did not affect it significantly. In the presence of ryanodine, KET reduced the ET-1-induced contraction from 67 +/- 2% to 36 +/- 3.%, P < 0.01. In the presence of nicardipine, KET also inhibited the ET-1-induced contraction. We conclude that KET relaxes the tracheal smooth muscle contracted by ETs via a mechanism that is independent of the tracheal epithelium. The relaxant effect of KET on the ET-induced contraction of the trachealis muscle is not dependent upon blockade of 1) sarcolemma influx of Ca2+ through the dihydropyridine Ca2+ channel or 2) the release of intracellular Ca2+ through the ryanodine-sensitive intracellular Ca2+ channel. It is likely that the action of KET relaxing ET-induced tracheal contractions is at some point of the inositol 1,4,5-trisphosphate signaling pathway.     

Muscarinic hyperresponsiveness of antigen-sensitized feline airway smooth muscle in vitro

OBJECTIVE: To determine the effect of in vivo antigen sensitization (Ascaris suum) of cats on tracheal smooth muscle (TSM) and bronchial smooth muscle (BSM) muscarinic reactivity in vitro. ANIMALS: Healthy domestic shorthair cats of either sex. PROCEDURE: Cats were sensitized and were long-term antigen (or sham) challenge exposed for 6 weeks by aerosolization with soluble Ascaris suum. Tracheal and BSM preparations were obtained and stimulated in vitro by electrical field stimulation (EFS), acetylcholine (ACh, a muscarinic agonist), and physostigmine (an AChase inhibitor). Responses were compared with responses of comparable tissues from sham antigen challenge-exposed cats. RESULTS: Tracheal and BSM from sensitized, compared with sham-sensitized (control), cats had greater isometric contraction (expressed as percentage of the response observed for isotonic, 63 mM KCl-elicited contraction [% KCl]) in response to endogenous (EFS) and exogenous muscarinic receptor activation (ACh). Contractions in response to EFS by TSM from control cats were 74% KCl vs 97% KCl for antigen-sensitized TSM (P < 0.04). Muscarinic responses were augmented comparably by in vivo sensitization; TSM from control cats contracted to 190% KCl vs 230% KCl (P < 0.03) for TSM from immune-sensitized cats. Physostigmine augmented responses of all tissues to ACh so that TSM from control (290% KCl) and antigen-sensitized (257% KCl) cats were similar. Responses of BSM from antigen-sensitized cats had similar augmentation of contractile response to EFS and ACh. CONCLUSIONS: Long-term in vivo antigen sensitization increases numbers of muscarinic receptors on airway smooth muscle or decreases the availability or activity of AChase in cats. CLINICAL RELEVANCE: Modulation of muscarinic receptors may be useful for treatment of asthmatic cats with in vivo airway hyperreactivity.     

Arachidonic acid-induced calcium signalling in human airway smooth muscle cells

Until now computer-assisted parasite identification was based on database applications requiring data specification on an individual basis, thus limiting the ability of the system to handle rule-based knowledge as humans are used to do. A new Expert PArasite IdentificatiON (EPAION: Greek term for expert) system was developed to serve as an interface between the database and the user, where the database is a repository for bionomic and morphological facts about the parasites for the expert system. The system was developed by using a logic-based computer language which allows the definition of rules and facts to assist the creation of queries to the database. The components of the system are the knowledge base, the multimedia data base, the inference mechanism, and the graphical user interface. The operational modules of the system are the Parasite Identifier and the system Utilities. This expert system facilitates knowledge incorporation in a manner simulating the natural mental process, thus allowing the checking of the accuracy of the information that the user feeds to the computer and the creation of intelligent queries to the database. These characteristics accelerate focusing and optimize the parasite identification scheme regardless of the user's profile of competency.     

Purinergic modulation of rat urinary bladder detrusor smooth muscle

This paper reviews the use of economic evaluations in the Swedish health care system. The most important actors are defined and examples are given how economic evaluations have played a role in the decision making process. The introduction of extracorporal shock-wave lithotripsy (ESWL) is used as an example on how economic evaluation was used for recommendations to the county councils to adopt this technology. Mammography is used as an example of how the evaluation is used in the political process following an initiative in the parliament. A study of the cost-effectiveness of hypertension treatment illustrates how economic evaluations are included as part of a medical technology assessment by SBU, the Swedish Council on Technology Assessment in Health Care. The role of economic evaluations for drug reimbursement and pricing is also reviewed. The main conclusions are that economic evaluations are one of several factors influencing a decision making process that have a strong strive for consensus. It is thus difficult to make a definitive statement of the contribution of such study to the outcome of the decision making process, and there is no evidence that the evaluation was the decisive factor. However, a number of changes in the way resources are allocated in the Swedish health care system speaks for an increasing role for such studies in the future. The county councils are identified as the main target for economic evaluations, and SBU has a key role in supplying the county councils with high quality assessment of new and old technologies.     

Halothane attenuates calcium sensitization in airway smooth muscle by inhibiting G-proteins

BACKGROUND: Halothane directly relaxes airway smooth muscle partly by decreasing the Ca2+ sensitivity. In smooth muscle, receptor stimulation is thought to increase Ca2+ sensitivity via a cascade of heterotrimeric and small monomeric guanine nucleotide-binding proteins (G-proteins). Whether this model is applicable in the airway and where halothane acts in this pathway were investigated. METHODS: A beta-escin-permeabilized canine tracheal smooth muscle preparation was used. Exoenzyme C3 of Clostridium botulinum, which inactivates Rho monomeric G-proteins, was used to evaluate the involvement of this protein in the Ca2+ sensitization pathway. The effects of halothane on different stimulants acting at different levels of signal transduction were compared: acetylcholine on the muscarinic receptor, aluminum fluoride (AIF4-) on heterotrimeric G-proteins, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) on all G-proteins. RESULTS: Exoenzyme C3 equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization, suggesting that these pathways are both mediated by Rho. Halothane applied before stimulation equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization. However, when added after Ca2+ sensitization was established, the effect of halothane was greater during Ca2+ sensitization induced by acetylcholine compared with AIF4-, which, along with the previous result, suggests that halothane may interfere with dissociation of heterotrimeric G-proteins. Halothane applied during GTPgammaS-induced Ca2+ sensitization had no significant effect on force, suggesting that halothane has no effect downstream from monomeric G-proteins. CONCLUSION: Halothane inhibits increases in Ca2+ sensitivity of canine tracheal smooth muscle primarily by interfering with the activation of heterotrimeric G-proteins, probably by inhibiting their dissociation.     

Mechanical strain increases protein tyrosine phosphorylation in airway smooth muscle cells

The 'comet' assay is being increasingly employed for evaluating DNA damage in biological systems. Using this technique, we examined DNA damage in whole in density-separated trout erythrocytes. Results clearly show that all the three considered parameters (tail length, tail intensity and tail moment) increased with the density of the fractions, possibly reflecting different degrees of DNA damage. Probably, this behaviour is due to different periods of exposure of the density fractions to the hazard of active oxygen radicals; older cells have been exposed to oxidative stress for a longer time.     

Molecular mechanisms of the hyperresponsiveness of airway smooth muscle in bronchial asthma

Chronic neurobehavioral effects of acute sarin poisoning were evaluated in 9 male and 9 female patients who were exposed to sarin poisoning in the Tokyo subway incident in Japan. The investigators used nine neurobehavioral tests, as well as a posttraumatic stress disorder checklist, 6-8 mo after the poisoning occurred. Serum cholinesterase activity in patients on the day of poisoning (i.e., March 20, 1995) ranged from 13 to 131 IU/l (mean=72.1 IU/l). The results of analysis covariance, in which age, education level, alcohol consumption, and smoking status (covariates) were controlled in 18 sarin cases and in 18 controls, showed that the score on the digit symbol (psychomotor performance) test was significantly lower in the sarin cases than in controls. Nonetheless, the scores for the General Health Questionnaires, fatigue of Profile of Mood States, and posttraumatic stress disorder checklist were significantly higher in the sarin cases than controls. The investigators added posttraumatic stress disorder to the covariates, and only the score on the digit symbol test was significantly lower in sarin cases. In addition, the results of stepwise multiple regression analysis in 18 sarin cases revealed that scores for the General Health Questionnaires, fatigue of Profile of Mood States (i.e., fatigue, tension-anxiety, depression, and anger-hostility)-together with the paired-associate learning test-were associated significantly with posttraumatic stress disorder. The association did not remain significant for the digit symbol test score. Perhaps a chronic effect on psychomotor performance was caused directly by acute sarin poisoning; on the other hand, the effects on psychiatric symptoms (General Health Questionnaire) and fatigue (Profile of Mood States) appeared to result from posttraumatic stress disorder induced by exposure to sarin.     

Cytokines induce airway smooth muscle cell hyperresponsiveness to contractile agonists

Inflammatory cytokines have been shown to play an important role in the pathogenesis of various inflammatory processes. In throat infections, intracellular inflammatory cytokines have been detected from the sites of inflammation. The present study aimed to evaluate serum cytokine levels of patients with throat infections and correlate them to the inflammatory parameters and type of inflammation. Significantly higher inflammatory cytokine levels (interleukin [IL]-6 > 7 pg/mL, IL-1 > 1 beta pg/mL, tumor necrosis factor alpha > 1 pg/mL) were detected in most of the patients as opposed to healthy controls. Clinical parameters of infection (fever > 38 degrees C, leukocytosis > 11,000 white blood cells per cubic millimeter, polymorphonuclear neutrophils > 75%) were significantly correlated with high levels of inflammatory cytokines: mainly IL-6 and tumor necrosis factor alpha, and to a lesser degree with IL-1 beta. No correlation, however, was found between the type of inflammation and cytokine levels. The present study indicates a role of inflammatory cytokines in the pathogenesis of throat infections and the need for an anti-inflammatory and anticytokine therapeutic approach.     

Innervation of tracheal epithelium and smooth muscle by neurons in airway ganglia

Despite subjective and objective success rates approaching 90%, significant morbidity is well documented using a wire loop for standard transurethral resection of the prostate (TURP). In an effort to minimize patient morbidity as well as limit future healthcare costs, several alternative instrumental techniques have been examined. Recent studies of transurethral electrovaporization of the prostate, a modification of existing transurethral technology, appear encouraging. The modifications which enable larger volumes of tissue to be vaporized with concurrent desiccation and coagulation are an increase in the surface area of the electrode and effective delivery of high electrical energy by electrical generators. The most extensively studied instrument is the VaporTrode. Promising early published reports of TURP-like efficacy without significant morbidity, as well as low cost, have fueled the popularity and wide application of this technique.     

Effect of isoflurane on endothelin-1 mediated airway smooth muscle contraction

Endothelin-1 (ET-1) is an endogenously produced 21 amino acid peptide that can cause significant airway smooth muscle (ASM) constriction. Volatile anesthetics e.g. isoflurane, can attenuate ASM constriction. This investigation was undertaken to study the direct effects of isoflurane, at a clinically relevant dose, on endothelin-induced constriction in rat trachea. Five Sprague-Dawley female rats (weight 325-350 g) were euthanized. Four 2-3 mm rings were excised from the mid portion of the trachea, attached to a force transducer for isometric measurement, and were then suspended in jacketed baths containing 37 degreesC KH solution, bubbled with 95% O2 and 5% CO2. Carbachol dose-response curves (10(-8)-10(-4)M) were obtained to establish the maximal contractility (Cmax) for each tracheal ring. Carbachol was then washed out of the bath solution until the tissue tension returned to the baseline. Subsequently, ET-1 dose response curves (3-200 nM) were generated in the absence (ET-1, control) and in the presence of 2% isoflurane. Our data suggests that isoflurane at 2% concentration, attenuated ET-1 induced tracheal contraction at 100 and 200 nM concentrations by 20%. This is the first demonstration indicating that, at a clinically relevant dose, isoflurane depresses ET-1 induced ASM constriction.     

Variability in the proliferative responsiveness of cultured human vascular smooth muscle cells to alpha-thrombin

alpha-Thrombin, a key enzyme of the coagulation cascade, is also a potent mitogen for many cell types. In the present study, the responsiveness to alpha-thrombin of cultured human vascular smooth muscle cells (HVSMC) derived from either vein or normal and atherosclerotic arteries was investigated. All HVSMC populations examined responded mitogenically to alpha-thrombin. However, the extent of this response varied between different cell populations. No significant differences were observed between HVSMC derived from vein versus artery or atherosclerotic versus normal tissues. The responsiveness of a specific HVSMC culture to alpha-thrombin was not affected by cell density and remained constant over several passages. Unlike platelet-derived growth factor BB (PDGF-BB), alpha-thrombin did not exhibit any significant chemotactic effects on HVSMC or induce their anchorage independent growth in semi-solid medium. The hypothesis that the observed variability in HVSMC responsiveness to alpha-thrombin is due to the heterogeneity of cultured HVSMC is raised and discussed.     

Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells

BACKGROUND: Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. METHODS AND RESULTS: We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. CONCLUSIONS: The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.     

Dietary beta-adrenoceptor agonists have a persistent effect on nitric oxide synthesis in rat cultured smooth muscle cells

Several compounds including lipopolysaccharide and sympathomimetics stimulate the expression of the inducible nitric oxide synthase in vascular smooth muscle cells. We evaluated the effect of clenbuterol on nitric oxide (NO) production by vascular smooth muscle cells of the rat aorta in culture. Wistar rats were divided into three diet groups (control, clenbuterol and washout). Aortic vascular smooth muscle cells from rats from these 3 diet groups were cultured in the presence and absence of lipopolysaccharide and/or beta-adrenoceptor agonists. NO release was measured by Griess reagent. Clenbuterol or salbutamol added to cells from control rats potentiated lipopolysaccharide-induced NO release. Cells from rats fed on clenbuterol, in a medium without beta-adrenoceptor agonists, showed a similar potentiation, even after a 10-day washout period. The addition of beta-adrenoceptor agonists to the latter cells did not increase NO production. NG-Nitro-L-arginine decreased nitrite production in lipopolysaccharide-stimulated cells. Our results demonstrate that dietary clenbuterol has a persistent 'ex vivo' effect on lipopolysaccharide-induced NO production by cultured vascular smooth muscle cells.