F16357, a novel protease-activated receptor 1 antagonist, improves urodynamic parameters in a rat model of interstitial cystitis.

BACKGROUND AND PURPOSE: The aims of the present study were to characterize the role of PAR1 in rat bladder under inflammatory conditions and determine whether a selective PAR1 antagonist, F16357, can prevent the pathophysiological symptoms of cyclophosphamide-induced interstitial cystitis (IC). EXPERIMENTAL APPROACH: Immunohistochemistry, contractile activity in isolated bladder and urodynamics were determined before and after cyclophosphamide treatment. F16357 was administered intravesically during the acute phase of inflammation, and effects on PAR1 and PAR1-related bladder contraction evaluated 24 h after cyclophosphamide injection. Urodynamics and associated voided volumes were recorded 7 and 24 h after cyclophosphamide. KEY RESULTS: In control conditions, PAR1 was present only in some umbrella cells. Cyclophosphamide disrupted the urothelium and expression of PAR1 by all remaining urothelial cells. After F16357 treatment, urothelial damage was absent and PAR1 immunoreactivity similar to control tissues. Thrombin and TFLLR-NH2 induced bladder contractions. These were increased in inflammatory conditions and antagonized by F16357 in a concentration-dependent manner. In telemetric experiments, furosemide increased urine production and voiding frequency for 60 min, 7 h after cyclophosphamide injection. Intravesical administration of F16357 blocked these changes with a return to a physiological profile; 24 h after cyclophosphamide, the volume of micturition was still lower with no increase in number of micturitions. F16357 30 µM reduced the number of micturitions and improved bladder capacity, but did not affect diuresis. Under similar experimental conditions, lidocaine 2% induced comparable effects. CONCLUSIONS AND IMPLICATIONS: PAR1 is expressed in rat bladder, overactivated in inflammatory conditions and involved in bladder function and sensation. F16357 could represent an interesting candidate for IC treatment.

Pharmacological analysis of wild-type alpha, gamma and delta subtypes of the human peroxisome proliferator-activated receptor.

Three distinct peroxisome proliferator-activated receptor (PPAR) cDNAs were isolated from human brain RNA. Whereas the PPARdelta subtype perfectly matched the amino acid sequences reported in the Genbank database, several differences were found for the PPARalpha (Lys(123)Met, Ala(268)Val, Gly(296)Ala and Val(444)Ala) and PPARgamma2 (Met(8)Ile, Pro(9)Ala, Met(186)Ile, Pro(187)Ala and the deletion of a Gln(213) residue) subtypes. A pharmacological analysis was undertaken by co-expressing each PPAR subtype with a reporter plasmid containing a luciferase gene under the transcriptional control of a synthetic, triplicated PPAR response element in either HepG2 or Cos-7 cells. Whereas fenofibrate unselectively activated the PPARalpha and PPARdelta subtypes, the related BM-17.0744 compound was more potent and selective for PPARalpha. The thiazolidine dione derivatives rosiglitazone and pioglitazone were potent and selective PPARgamma2 agonists. L-165041, reported as a selective and potent PPARdelta ligand, displayed in this specified transactivation system, apart from its highly efficacious PPARdelta agonist activity, partial and full agonism at, respectively, PPARalpha and PPARgamma2 subtypes. In conclusion, transcriptional control of a luciferase gene by wild-type PPAR subtypes provides powerful recombinant assays to evaluate ligand's efficacy at these nuclear receptors.

Lack of toxic effects of F 12511, a novel potent inhibitor of acyl-coenzyme A: cholesterol O-acyltransferase, on human adrenocortical cells in culture.

Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 (S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4-72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition (IC50) values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the IC(50) value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3',5'-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 microM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 microM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.

F 12511, a novel ACAT inhibitor, and atorvastatin regulate endogenous hypercholesterolemia in a synergistic manner in New Zealand rabbits fed a casein-enriched diet.

F 12511, a novel ACAT inhibitor, lowers plasma cholesterol levels in New Zealand rabbits fed a cholesterol-free casein-rich diet. In rabbits endogenous hypercholesterolemia pre-established for 8 weeks was used to compare treatments with F 12511 and atorvastatin for a further 8-week period, and to determine whether both agents act synergistically. F 12511 appears to be 3-4-fold more potent than atorvastatin in reducing total plasma cholesterol (active doses ranging from 0.16 to 2.5 and from 1.25 to 10 mg/kg per day, respectively) while the hypocholesterolemic efficacy of both compounds at 2.5 mg/kg per day amounted to 70 and 45%, respectively. A reduction by as much as 75% of esterified cholesterol in liver mediated by F 12511 could account for the decrease of plasma VLDL, LDL and apo B-100, whereas a reduction of the LDL production rate has been described as the main mechanism underlying the atorvastatin effect. F 12511 modified adrenal cholesterol balance only at the largest dose studied. In a further experiment the co-administration of threshold doses of F 12511 and atorvastatin (0.63 and 1.25 mg/kg per day, respectively) lowered plasma total cholesterol and apo B-100 containing lipoproteins to a greater extent and more rapidly than either agent alone. In the liver a decrease by atorvastatin in free cholesterol substrate for ACAT may amplify the effect of F 12511 on cholesteryl ester content leading to a diminution, in at least an additive manner, of the assembly and secretion of atherogenic lipoproteins in New Zealand rabbits which have developed an endogenous hypercholesterolemia. Thus, the combination of the ACAT inhibitor F 12511 with atorvastatin can represent a better approach than either agent alone to regulate lipoprotein metabolism in certain pathophysiological situations.

Pharmacological profile of F 12511, (S)-2',3', 5'-trimethyl-4'-hydroxy-alpha-dodecylthioacetanilide a powerful and systemic acylcoenzyme A: cholesterol acyltransferase inhibitor.

The pharmacological profile of F 12511 (S)-2',3', 5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, a new inhibitor of acyl-CoA: cholesterol acyltransferase (EC 2.3.1.26; ACAT), was evaluated by using different in vitro and in vivo models. In vitro, F 12511 was shown to be a highly potent inhibitor of ACAT activity in microsomal preparations from various animal species as well as of cholesterol esterification in relevant human cell lines in culture. The concentrations of F 12511 required to produce 50% inhibition of ACAT activity (IC(50) values) in microsomal preparations ranged from 41nM for hypercholesterolemic rabbit intestine to 223 nM for normocholesterolemic hamster liver. In whole cell assays using hepatic (Hep G2), intestinal (CaCo-2) and macrophagic (THP-1) cell lines, F 12511 inhibited ACAT activity with IC(50) values of 3, 7, and 71 nM, respectively. In vivo, orally administered F 12511 displayed high potency and efficacy as an antihypercholesterolemic compound in different cholesterol-fed animals (rat, guinea-pig, rabbit). For instance, in guinea-pigs the dose required to reduce plasma cholesterol levels by 30% (ED(30) value) was 0.008 mg.kg(-1.) In rabbits, an animal species prone to atherosclerosis, the hypocholesterolemic effect was accompanied by a dose-related reduction in the incidence of aortic fatty streaks that reached asymptote at 2.5 mg.kg(-1) and by an improvement of the impaired endothelial function. When given orally to chow-fed hamsters, F 12511 elicited a dose-related decrease in plasma cholesterol from 9% at 0.63 mg.kg(-1) up to 31% at 40 mg.kg(-1) associated with a preferential reduction in atherogenic lipoproteins, very low density lipoproteins (VLDL) and low density lipoproteins (LDL). Moreover, in the same dose range, F 12511 decreased hepatic cholesteryl ester concentrations and reduced liver ex vivo ACAT activity. By using a bioassay, ACAT inhibitory activity was present in plasma of treated hamsters 1 hr after oral administration of F 12511. Hence, the results in chow-fed hamsters are consistent with systemic and direct hepatic effects of F 12511. In guinea-pigs, an adreno-sensitive species, F 12511 did not impair the adrenal function (adrenocorticotrophic hormone challenge) at doses up to 2.5 mg.kg(-1,) far higher than those eliciting hypocholesterolemic effects in the same species. In conclusion, the results suggest that F 12511, a powerful and systemic ACAT inhibitor, constitutes an appropriate tool to determine whether the inhibition of ACAT constitutes an effective therapy for the treatment of hypercholesterolemia and of atherosclerosis in man.

Sulfonamide derivatives of benzylamine block cholesterol biosynthesis in HepG2 cells: a new type of potent squalene epoxidase inhibitors.

Sulfonamide derivatives of ene-yne benzylamine 1 have been prepared and identified as a new class of potent SE inhibitors having demonstrated activity in HepG2 cells as cholesterol biosynthesis inhibitors.

Mildly oxidized LDL evokes a sustained Ca(2+)-dependent retraction of vascular smooth muscle cells.

Oxidized low density lipoprotein (LDL) is thought to play a major role in atherogenesis. Atherosclerotic arteries exhibit structural changes associated with profound alterations in vascular tone that are potentially involved in arterial spasm and ischemic heart disease. We report here the role of oxidized LDL in the retraction of vascular smooth muscle cells. Mildly oxidized LDL elicited a broad and sustained peak in cytosolic calcium concentration ([Ca2+]i) in cultured arterial smooth muscle cells. Concomitant with the [Ca2+]i rise, oxidized LDL evoked a sustained and intense retraction of smooth muscle cells, as shown by the changes in cross-sectional area of single cells. Cell retraction was dependent on time, the concentration of oxidized LDL, and the level of LDL oxidation (native LDL induced neither a significant [Ca2+]i rise nor cell retraction). Oxidized LDL but not native LDL also elicited a delayed (12 +/- 2 hours) and sustained (14 +/- 2 hours) increase in isometric tension in deendothelialized arterial rings only, thus suggesting a protective role of intact endothelium. When triggered by nontoxic doses of oxidized LDL, retraction of cultured cells and the contractile response of aortic rings was reversible, whereas with higher (toxic) doses (> or = 200 micrograms apoB/mL), cell retraction was irreversible and led progressively to detachment and cell death. Cell retraction can be prevented in three ways: (1) by inhibiting LDL oxidation with supplements of antioxidants (indirect inhibition); (2) by blocking the pathogenic intracellular signaling elicited by oxidized LDL (direct inhibition), eg, by inhibiting calcium influx with EGTA or the calcium channel blocker nisoldipine or by blocking intracellular signaling (at a still-unknown step) by the lipophilic antioxidant alpha-tocopherol; and (3) by directly inhibiting myosin light chain kinase by 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1, 4-diazepine. In conclusion, oxidized LDL evoked a sustained and intense calcium-dependent retraction of cultured smooth muscle cell, which can be prevented by inhibiting LDL oxidation or by blocking the intracellular signaling induced by oxidized LDL.

(Aryloxy)methylsilane derivatives as new cholesterol biosynthesis inhibitors: synthesis and hypocholesterolemic activity of a new class of squalene epoxidase inhibitors.

A series of 3-substituted (aryloxy)silane derivatives of benzylamine (4, 4', or 4") was synthesized and evaluated for hypocholesterolemic activity. Most of the new silane derivatives were identified as potent inhibitors of pig liver squalene epoxidase with IC50 values in the submicromolar range. In vitro inhibition of cholesterol biosynthesis in Hep-G2 cells was observed with a very good potency for the ene-yne derivatives 4a, 4i, 4n, 4q, and 4u as well as for the yne-yne compound 4". In vivo, 4i, 4u, 4', and 4" were found to decrease cholesterol biosynthesis in rats upon oral administration with ED50 values in the range of 2-7 mg/kg. Therefore, these new (aryloxy)methylsilane derivatives of benzylamine represent a new class of potent squalene epoxidase inhibitors with promising hypocholesterolemic properties.

Endothelium-dependent contractions are associated with both augmented expression of prostaglandin H synthase-1 and hypersensitivity to prostaglandin H2 in the SHR aorta.

Prostaglandin H2 (PGH2 [endoperoxide]) is an immediate product of prostaglandin H (PGH) synthase activity (cyclooxygenase) and a likely candidate to mediate endothelium-dependent contractions evoked by acetylcholine in the aorta of the spontaneously hypertensive rat (SHR). Experiments were designed to investigate whether or not endothelium-dependent contractions were associated with an increased expression of PGH synthase, an augmented acetylcholine-induced release of PGH2, and/or a hypersensitivity of the smooth muscle to endoperoxides in SHR aorta compared with normotensive Wistar-Kyoto (WKY) aorta. In SHR aorta, endothelium-dependent contractions to acetylcholine were abolished by tenidap (10(-8) mol/L), a preferential PGH synthase-1 inhibitor, but slightly impaired by NS-398 (10(-6) mol/L), a preferential PGH synthase-2 inhibitor. PGH synthase-1 expression, which was evaluated by both reverse transcriptase-polymerase chain reaction and Western blotting, was about twofold greater in preparations with endothelium from SHR than from WKY rats. There was no difference in PGH synthase-1 expression between preparations with and those without endothelium in both strains. In SHR but not WKY aortas, acetylcholine (10(-5) mol/L, 5 minutes) caused a significant endothelium-dependent release of PGH2 as measured by gas chromatography/mass spectrometry. PGH2 evoked more potent contractions in rings without endothelium from SHR than from WKY rats, whereas the thromboxane analogue U46619 and prostaglandin F2 alpha caused a comparable response in both preparations. These results show that endothelium-dependent contractions to acetylcholine in SHR aorta are associated with a greater expression of PGH synthase-1, a significant release of PGH2, and a hypersensitivity of the smooth muscle to the endoperoxide.

Enhanced production of nitric oxide in aortae from spontaneously hypertensive rats by interleukin-1 beta.

Cultured aortic smooth muscle cells from spontaneously hypertensive rats produce more nitrite than cells from Wistar-Kyoto rats in response to interleukin-1 beta. Therefore, the effect of interleukin-1 beta-induced nitric oxide production was compared on the contractility of aortic smooth muscle from spontaneously hypertensive and Wistar-Kyoto rats. Under control conditions, there was no difference in the response of aortic rings (without endothelium) to phenylephrine between both strains. Contractions to 5-hydroxytryptamine were larger in preparations from hypertensive than normotensive animals. Treatment with interleukin-1 beta for 6 h reduced the responsiveness to both vasoconstrictors in a concentration-dependent manner. The depression was more pronounced in rings from spontaneously hypertensive rats: the threshold concentration of the cytokine was lower, and its maximal effect greater. Nitro-L-arginine prevented the inhibitory effect of interleukin-1 beta. The cytokine evoked a time-dependent loss of tone in phenylephrine-contracted rings with the same time of onset in both strains. However, the decay of tension was more pronounced in aortae from hypertensive than normotensive rats. In aortae from both strains, the decay was potentiated by L-arginine, but not D-arginine. Interleukin-1 beta elicited greater concentration-dependent productions of cyclic GMP and nitrite in rings from spontaneously hypertensive than from Wistar-Kyoto rats, and these were inhibited by methylene blue and nitro-L-arginine, respectively. The concentration-relaxation curves to 3-morpholino-sydnonimine were moderately, but significantly, shifted to the left in aortae from spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The sydnonimine C87-3754 evokes endothelium-independent relaxations and prevents endothelium-dependent contractions in blood vessels of the dog.

Experiments were designed to compare the relaxing activities of the new sydnonimine C87-3754 with SIN-1 in arteries and veins of the dog, and to determine whether C87-3754 can prevent endothelium-dependent contractions. Rings of coronary and femoral arteries, and saphenous veins were suspended in organ chambers for the measurement of changes in isometric tension. SIN-1 and C87-3754 evoked concentration-dependent relaxations in all rings of blood vessels contracted with a submaximal concentration of either prostaglandin F2 alpha, endothelin-1, phenylephrine, or norepinephrine. In both arteries and veins, the concentration-relaxation curves to C87-3754 were shifted significantly to the right (by two to three logarithmic units) of that to SIN-1. The presence of endothelium significantly inhibited the relaxations to SIN-1 but did not affect those to C87-3754. The treatment of coronary arteries with methylene blue or oxyhemoglobin significantly impaired the relaxation to SIN-1 and C87-3754. Neither C87-3754 nor its prodrug pirsidomine (CAS 936) affected the membrane potential in coronary arteries. The endothelium-dependent contractions evoked by nitro L-arginine, arachidonic acid, and the calcium ionophore A23187 in basilar arteries of the dog were inhibited by C87-3754. These results indicate that the sydnonimine C87-3754 is a dilator of both arterial and venous smooth muscle, and can prevent endothelium-mediated contractions in cerebral arteries of the dog. The inhibition of vascular tone is likely to involve the activation of soluble guanylate cyclase, causing enhanced production of cyclic guanosine monophosphate in the smooth muscle without a change in membrane potential.

Platelet-derived growth factor suppresses and fibroblast growth factor enhances cytokine-induced production of nitric oxide by cultured smooth muscle cells. Effects on cell proliferation.

Stimulation of thymidine incorporation by basic fibroblast growth factor or epidermal growth factor treatment of cultured quiescent smooth muscle cells (rat and human) was attenuated by the concomitant treatment with interleukin-1 beta in the presence of indomethacin. Platelet-derived growth factor-AB and -BB-induced thymidine incorporation was not inhibited by the presence of the cytokine under similar experimental conditions. Elevation of nitrite levels in the conditioned medium of cultures exposed to interleukin-1 beta correlated with the inhibition of thymidine incorporation. Platelet-derived growth factor-AB and -BB inhibited the production of nitric oxide (measured as nitrite levels in conditioned medium) by cells treated simultaneously with interleukin-1 beta and growth factor. However, platelet-derived growth factor-AA neither affected nitrite production nor thymidine incorporation by smooth muscle cells. Levels of cytokine-stimulated nitrite production by smooth muscle cells were increased synergistically by the presence of fibroblast growth factors or epidermal growth factor. The inhibition of thymidine incorporation and concomitant elevation of nitrite production was abolished in the presence of nitro-L-arginine. Cultures maintained in the presence of low levels of the cytokine for 9 days were growth-inhibited, and this was reversed when culture medium was supplemented with nitro-L-arginine. The treatment of smooth muscle cells, which were grown in coculture inserts with the cytokine to induce nitric oxide production, before their combination with other quiescent layers of cells resulted in the inhibition of thymidine incorporation by this second layer of cells regardless of the growth factor used for stimulation. Nitric oxide may act as an endogenous inhibitor of smooth muscle cell proliferation in the vessel wall, and impairment of its production may be one action of potent vascular mitogens such as platelet-derived growth factor.

Inhibition of cytokine-induced nitric oxide production by transforming growth factor-beta 1 in human smooth muscle cells.

1. Experiments were performed to investigate the effects of human recombinant interleukin-1 beta on the production of vasoactive substances by human aortic smooth muscle cells in culture. Smooth muscle cells were cultured either on microcarrier beads for bioassay experiments, or in multiwell plates for the determination of nitrite levels. 2. Cells were grown on microcarrier beads, treated with interleukin-1 beta or vehicle (control) for 24 h, and packed in a column which was perfused with oxygenated Krebs-Ringer solution in the presence of indomethacin. The activity of the perfusates was bioassayed by measuring the changes in tension of a contracted ring of Wistar rat aorta without endothelium, and by evaluating the modulation of thrombin-induced platelet aggregation. 3. Perfusates from interleukin-1 beta treated cells evoked relaxations of the contracted detector tissues, and microcarrier beads covered with treated cells inhibited thrombin-induced platelet aggregation. Superoxide dismutase enhanced these effects whereas Methylene Blue abolished them. Control cells evoke neither relaxation nor inhibition of platelet aggregation. Interleukin-1 beta induced a time- and concentration-dependent production of nitrite. Cycloheximide and nitro-L-arginine inhibited the relaxations and the production of nitrite evoked by interleukin-1 beta-treated cells. L-Arginine but not D-arginine overcame the blockade elicited by nitro-L-arginine. Transforming growth factor-beta 1 reduced the interleukin-1 beta-dependent generation of nitrite by cultured smooth muscle cells and relaxation of contracted bioassay tissues. 4. Interleukin-1 beta, transforming growth factor-beta 1, Methylene Blue and L-arginine-related compounds did not induce significant variations of tension of the detector rings. 5. These data demonstrate that the inflammatory and immunological mediator interleukin-1 can stimulate the production of a nitric oxide-like substance(s) in cultured human smooth muscle cells leading to the activation of soluble guanylate cyclase. Liberation of transforming growth factor-beta by activated platelets may inhibit these reactions.

Transforming growth factor-beta 1 inhibits L-arginine-derived relaxing factor(s) from smooth muscle cells.

The effects of human recombinant interleukin-1 beta 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-1 beta-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-beta 1. L-Arginine but not D-arginine overcame the blockade by nitro-L-arginine. Superoxide dismutase potentiated the relaxations. In cells cultured in multiwell plates, interleukin-1 beta evoked a time- and concentration-dependent accumulation of nitrite in the extracellular medium that was inhibited dose dependently by transforming growth factor-beta 1. 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-beta 1.

The induction of nitric oxide synthase activity is inhibited by TGF-beta 1, PDGFAB and PDGFBB in vascular smooth muscle cells.

The effect of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor (PDGF) was investigated on the induction of nitric oxide synthase activity caused by interleukin-1 beta in cultured smooth muscle cells from rat aorta. TGF-beta 1, PDGFAB and PDGFBB but not PDGFAA inhibited in a concentration-dependent manner the production of nitrite, an oxidation product of nitric oxide, evoked by interleukin-1 beta. The growth factors alone did not stimulate the release of nitrite. The addition of interleukin-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated human washed platelets inhibited the aggregation evoked by thrombin whereas no effect was observed with untreated cells. Platelet aggregation was not inhibited by smooth muscle cells that had been pretreated with interleukin-1 beta in combination with either TGF-beta 1, PDGFAB or PDGFBB but not with PDGFAA. These observations demonstrate that platelet-derived products such as TGF-beta and PDGFs inhibit the induction of nitric oxide synthase activity in vascular smooth muscle cells.

Platelet inhibition by an L-arginine-derived substance released by IL-1 beta-treated vascular smooth muscle cells.

Experiments were performed to examine whether stimulation of cultured vascular smooth muscle cells by interleukin (IL)-1 beta would induce platelet inhibitory properties of these cells. Incubation of platelets with untreated rat aortic smooth muscle cells had no effect on thrombin-induced platelet aggregation. In contrast, incubation of platelets with IL-1 beta-pretreated smooth muscle cells or the perfusate from such cells resulted in the inhibition of thrombin-induced platelet aggregation. This effect was potentiated by superoxide dismutase and reversed by incubating the IL-1 beta-treated smooth muscle cells with NG-nitro-L-arginine (L-NNA) or by treating the platelets with methylene blue. Cytokine-treated smooth muscle cells inhibited thrombin-stimulated changes in platelet cytosolic ionized calcium, whereas untreated cells were without effect. Incubating platelets with IL-1 beta-treated smooth muscle cells resulted in a 10-fold increase in platelet guanosine 3',5'-cyclic monophosphate (cGMP) levels, whereas untreated smooth muscle cells had no effect. The elevation of platelet cGMP induced by the IL-1 beta-treated smooth muscle cells was prevented by exposing the cytokine-treated cells to L-NNA or by treating platelets with methylene blue. Treatment of smooth muscle cells with IL-1 beta also resulted in an eightfold increase in nitrite production, which was blocked when the cells were incubated with L-NNA. The addition of cycloheximide to smooth muscle cells during their incubation with IL-1 beta completely inhibited smooth muscle cell nitrite production, the effects of the smooth muscle cells on platelet cGMP levels, and platelet responses to thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Indapamide potentiates the endothelium-dependent production of cyclic guanosine monophosphate by bradykinin in the canine femoral artery.

Indapamide is a sulfonamide diuretic agent that has antihypertensive properties. In the canine femoral artery, indomethacin reduces the endothelium-dependent relaxation induced by bradykinin, and indapamide restores the response. The aim of this study was to determine whether indapamide affects the release or the effects of endothelium-derived nitric oxide and prostanoids. The effect of indapamide on the production of endothelium-derived nitric oxide and prostacyclin was assessed indirectly by the measurement of the tissue content of cyclic guanosine monophosphate (GMP) and the accumulation of 6-keto-prostaglandin F1 alpha in the incubation medium, respectively. Indapamide did not affect the basal production of either cyclic GMP, cyclic adenosine monophosphate (AMP), or 6-keto-prostaglandin F1 alpha in the presence or absence of indomethacin. Indomethacin decreased the production of cyclic AMP and the release of 6-keto-prostaglandin F1 alpha induced by bradykinin, and this was unaffected by indapamide. Indapamide enhanced the bradykinin-stimulated production of cyclic GMP in the presence of indomethacin and did not affect that evoked by 3 morpholino-sydnonimine, an exogenous donor of nitric oxide. Indomethacin had no significant effect on the production of cyclic GMP stimulated by either bradykinin or 3 morpholino-sydnonimine. These studies demonstrate that the potentiation by indapamide of the relaxation evoked by bradykinin is associated with an enhanced production of cyclic GMP in the presence of indomethacin, which suggests that the production of endothelium-derived nitric oxide is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta induces the production of an L-arginine-derived relaxing factor from cultured smooth muscle cells from rat aorta.

The effect of interleukin-1 beta on the production of non-prostanoid vasoactive factors by cultured rat aortic smooth muscle cells was investigated. Under bioassay conditions, the perfusate from a column of confluent cells grown on beads and treated with interleukin-1 beta (1 ng/ml for 18 to 24 hr) abolished the contraction of a canine coronary ring without endothelium contracted by phenylephrine (1 microM), while the perfusate from control cells had no effect. The relaxing activity of the perfusate was observed when transit times were increased from 1 sec to 5 min. Nitro L-arginine (100 microM) reversed the relaxations and L-arginine stereoselectively restored the relaxations. Interleukin-1 beta (1 ng/ml) evoked a time-dependent accumulation of cyclic GMP but not cyclic AMP in cultured smooth muscle cells. The transfer of fresh or stored (-70 degrees C) conditioned culture medium from interleukin-1 beta-treated cells but not from control cells, to cultured smooth muscle cells stimulated the production of cyclic GMP. These observations demonstrate that interleukin-1 beta induces the production of transferable factor which relaxes vascular smooth muscle and stimulates the production of cyclic GMP.

Retinoids enhance the number of EGF receptors in corneal endothelial cells.

Corneal endothelium appears to be an important target tissue for retinoids and epidermal growth factor (EGF). We report here that retinoic acid, or its synthetic analogue CBS-211 A (10(-8)-10(-7) M) dose-dependently enhances the mitogenic effect of EGF on cultured bovine corneal endothelial cells (BCEC). Furthermore, retinoid treatment, especially with CBS-211 A, increases the EGF-binding capacity of BCEC without any modification of EGF receptor affinity. The addition of cycloheximide (0.5 microgram ml-1) to cultures simultaneously with retinoids suppressed the retinoid effect on EGF-binding, whereas this protein synthesis inhibitor was ineffective when added 24 hr later. These results suggest that retinoids could induce the expression of EGF receptors on BCEC as an early event. This study, which demonstrates a cooperation between retinoids and EGF in corneal endothelium, could explain the beneficial effect of retinoic acid previously reported on this tissue repair in vivo. Our data could offer clues for new pharmaceutical strategies in ophthalmology therapy.

LPS-stimulated bovine aortic endothelial cells produce IL-1 and IL-6 like activities.

Vascular endothelium is known to closely interact with leukocytes and immunocompetent cells. We report here that cultured bovine aortic endothelial cells (BAEC) synthesize both interleukin 1 (IL-1) and interleukin 6 (IL-6) like activities in response to bacterial lipopolysaccharide stimulation. Our results agree with previous data obtained from human venous endothelia and support the concept that IL-1 and IL-6 synthesis are properties common to endothelial cells from different vascular beds. The IL-1 activity was measured by murine thymocyte proliferation assay and by an indirect bioassay using NOB1 cells, which evidenced higher IL-1 amounts than the former. This discrepancy appeared to be partly due to the simultaneous production of one or more inhibitor(s) of the thymocyte proliferation by BAEC. The IL-6 assay was performed with the murine hybridoma cell line B9. In other respects, the cyclooxygenase inhibitor indomethacin enhanced the IL-1 like production, but was ineffective on IL-6 like production. The present study provides additional evidence that endothelial cells from large arteries may also participate in inflammatory and immunological processes.