Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in mouse aortic smooth muscle cells.

BACKGROUND AND PURPOSE: Reduction of intracellular calcium ([Ca(2+)](i)) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca(2+) mobilization during SMC contraction influenced NO efficacy. EXPERIMENTAL APPROACH: Isometric contractions by depolarization (high potassium, K(+)) or alpha-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca(2+)](i) (Fura-2) were measured in aortic segments from C57Bl6 mice. KEY RESULTS: Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K(+) solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca(2+)](i), which was suppressed by sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K(+)-stimulated segments did not parallel a decline of [Ca(2+)](i). However, stimulation (BAY K8644) of L-type Ca(2+) influx diminished, while inhibition (nifedipine, 1-100 nM) augmented the relaxing capacity of NO. CONCLUSIONS AND IMPLICATIONS: In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca(2+) re-uptake into the SR and was prominent when intracellular Ca(2+) was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca(2+)](i), presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca(2+) influx, and L-type Ca(2+) channel blockers increase the vasodilator efficacy of NO.

Chronic inflammation in asthma: a contest of persistence vs resolution.

Recent investigations have highlighted that endogenous anti-inflammatory mediators and immune regulating mechanisms are important for the resolution of inflammatory processes. A disruption of these mechanisms can be causally related not only to the initiation of unnecessary inflammation, but also to the persistence of several chronic inflammatory diseases. In asthma, chronic Th-2 driven eosinophilic inflammation of the airways is one of the central abnormalities. To date, elucidating the role of the different pro-inflammatory mediators involved in orchestrating the inflammatory processes in asthma has been the subject of intense research in both humans and animal models. However, the counter-regulatory mechanisms that co-determine the outcome in the contest of resolution vs persistence of the eosinophilic airway inflammation remain poorly understood. These are currently being investigated in animal models of chronic asthma. Elucidating these mechanisms is of relevance, since it can give rise to a new therapeutic approach in the treatment of chronic airway inflammation in asthmatics. This novel concept of treatment involves the stimulation of endogenous anti-inflammatory pathways, rather than solely antagonising the various pro-inflammatory mediators. Here, we review and discuss the current knowledge about these endogenous anti-inflammatory mediators in clinical and experimental asthma.

The pore of the leaf cavity of Azolla species: teat cell differentiation and cell wall projections.

The differentiation of the specialized secretory teat cells of the leaf cavity pore of Azolla species was investigated at the ultrastructural level with emphasis on their peculiar cell wall projections. The results indicated that the projections are formed as soon as the teat cells complete their differentiation and that their production is principally associated with changes in endoplasmic reticulum profiles. The number of projections increases with the teat cell age and is stimulated under salt and P deficiency stresses. Salt stress also promotes their emergence on Azolla species that under normal conditions do not produce projections. Cytochemical tests on different Azolla species showed that the projection composition is almost identical: proteins, acidic polysaccharides, and pectin are always detected. This study revealed that Azolla teat cell projections differ fundamentally from other types of hitherto described cell wall projections that are considered as remnant structures from cell separation. In contrast, in Azolla teat cells projections are actively produced and compounds are excreted by an exocytotic mechanism. The possible role of the projections in the symbiosis of Azolla spp. with Anabaena azollae is discussed.

Inhibition of all-TRANS-retinoic acid metabolism by R116010 induces antitumour activity.

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

The focal adhesion targeting sequence is the major inhibitory moiety of Fak-related non-kinase.

Focal adhesion kinase (FAK) plays an important role in integrin-mediated signal transduction pathways and its C-terminal noncatalytic domain Fak-related non-kinase (FRNK), which is autonomously expressed, acts as an inhibitor of FAK. A model has been proposed where FAK and FRNK compete for an essential common binding protein. A FRNK variant in which the direct interaction with v-Crk-associated tyrosine kinase substrate (CAS) was disturbed by point mutations still functioned as an inhibitor of FAK, suggesting that FRNK is unlikely to inhibit FAK by sequestering CAS. Deletion variants of FRNK within the region N-terminal to the focal adhesion targeting (FAT) sequence were still able to inhibit FAK function, indicating that this region is dispensable for the inhibitory effect of FRNK. Overexpression of a green fluorescent protein (GFP) fusion protein containing the FAT sequence delayed cell spreading and reduced FAK tyrosine phosphorylation. This indicates that the FAT sequence is the major inhibitory moiety within FRNK.

The functional gamma-secretase inhibitor prevents production of amyloid beta 1-34 in human and murine cell lines.

The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, beta-secretase and gamma-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid beta (Abeta). Combining immunoprecipitation and mass spectrometry, we characterized soluble Abeta in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Abeta isoforms detected were Abeta11-34, Abeta1-34, Abeta11-40 and Abeta1-40. In this study, we demonstrate that overexpression of human beta-secretase (BACE-1) in HEK293 cells resulted in predominant Abeta cleavage at position Glu(11) rather than Asp(1), as well as increased production of Abeta(x)-34, but not Abeta(x)-40. Incubation of cells with a specific gamma-secretase inhibitor suggests that cleavage of APP at Leu(34) could be mediated by gamma-secretase itself or by a gamma-secretase dependent process.

Heterogeneity in relaxation mechanisms in the carotid and the femoral artery of the mouse.

The participation of prostanoids, nitric oxide and non-prostanoid non-nitric oxide factors in endothelium-dependent relaxations was investigated in phenylephrine (PE)-constricted carotid and femoral arteries of C57BL6 mice. The carotid artery was more sensitive to acetylcholine as compared to the femoral artery, and cyclooxygenase inhibition did not influence the relaxation in either vessel. In the carotid artery, high doses of acetylcholine caused transient constrictions, which were abolished by indomethacin or piroxicam. In the carotid but not the femoral artery, N(omega)-nitro-L-arginine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced PE-induced contractions enormously, suggesting that endogenous nitric oxide production is much higher in the carotid artery. While in the carotid artery all relaxation was abolished by N(omega)-nitro-L-arginine or ODQ, a residual response (34+/-5% and 74+/-4%, respectively) but with a different shape, was maintained in the femoral artery. This N(omega)-nitro-L-arginine-resistant relaxation was abolished by the combination of apamin and charybdotoxin. In both arteries, ODQ abolished relaxation to S-nitroso-N-acetyl-D-penicillamine, while N(omega)-nitro-L-arginine enhanced the sensitivity to this donor of exogenous nitric oxide. In 30 mM KCl, the relaxation to acetylcholine was abolished by N(omega)-nitro-L-arginine or ODQ in either artery. In conclusion, in the carotid artery endothelium-dependent relaxation is mediated predominantly by nitric oxide acting via cyclic GMP-dependent pathways, while in the femoral artery part of the relaxation can be attributed to a non-prostanoid non-nitric oxide factor operating via apamin/charybdotoxin-sensitive potassium channels.

R115866 inhibits all-trans-retinoic acid metabolism and exerts retinoidal effects in rodents.

All-trans-retinoic acid (RA) regulates epithelial differentiation and growth through activation of specific nuclear RA receptors (RARs). Because high-rate metabolism largely impairs the biological efficacy of RA, we have sought for compounds capable of inhibiting the metabolic breakdown of the retinoid. This study identifies R115866 as a novel inhibitor of the cytochrome P450 (CYP)-mediated metabolism of RA. In vitro, nanomolar concentrations of R115866 inhibited the conversion of RA by CYP26, a RA-inducible RA metabolizing enzyme. In vivo, oral administration of R115866 (2.5 mg/kg) to rats induced marked and transient increases of endogenous RA levels in plasma, skin, fat, kidney, and testis. Consistent with its ability to enhance endogenous RA content in tissues, R115866 was found to exert retinoidal activities. Like RA, the title compound: 1) inhibited vaginal keratinization in estrogen-stimulated rats; 2) induced epidermal hyperplasia in mouse ear skin; 3) transformed mouse tail epidermis from a para- to an orthokeratotic skin type; and 4) up-regulated the CYP26 mRNA expression in rat liver. Furthermore, we found that the keratinization-suppressive and CYP26-inducing activities of R115866 could be reversed by concomitant administration of the RAR antagonist, AGN193109. Our data characterize R115866 as a potent, orally active inhibitor of RA metabolism, capable of enhancing RA levels and displaying retinoidal actions. These activities are reversed by RAR antagonism, supporting the idea that the actions of R115866 result from increased availability of endogenous RA and improved RAR triggering.

Accumulation of 3-ketosteroids induced by itraconazole in azole-resistant clinical Candida albicans isolates.

The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378-2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14alpha-methyl-ergosta-8,24(28)-diene-3beta,6alpha-dio l (3, 6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14alpha-methyl-ergosta-5,7,22,24(28)-tetraene-3betaol, and 14alpha-methyl-fecosterol. In strains C48 and C56, no further increase of 3,6-diol was observed after exposure to itraconazole. Ergosterol synthesis was less sensitive to itraconazole inhibition, as was expected for these azole-resistant isolates which overexpress ATP-binding cassette transporter genes CDR1 and CDR2. In addition to 3,6-diol, substantial amounts of obtusifolione were found after exposure to itraconazole. This toxic 3-ketosteroid was demonstrated previously to accumulate after itraconazole treatment in Cryptococcus neoformans and Histoplasma capsulatum but has not been reported in Candida isolates. Accumulation of obtusifolione correlated with nearly complete growth inhibition in these azole-resistant strains compared to that found in the susceptible parent strain, although the onset of growth inhibition only occurred at higher concentrations of itraconazole. ERG25 and ERG26 are the only genes assigned to the 4-demethylation process, of which the 3-ketoreductase is part. To verify whether mutations in these ERG25 genes contributed to obtusifolione accumulation, their nucleotide sequences were determined in all three related isolates. No mutations in ERG25 alleles of isolates C48 and C56 were found, suggesting that this gene is not involved in obtusifolione accumulation. The molecular basis for the accumulation of this sterol in these two strains remains to be established.

Oxidized low-density lipoprotein enhances intimal thickening and alters vascular reactivity.

Oxidized low density lipoprotein (oxLDL) is present in atherosclerotic lesions and has been implicated in the etiopathogenesis of atherosclerosis mainly based on in vitro studies. In view of the lack of data on the activity of oxLDL in vivo, we decided to study its effects in the rabbit by local application at the level of the vascular wall. Intimal thickening was evoked by the placement of a silicone collar around the carotid arteries during 2 weeks. The collar was connected to an osmotic minipump containing human oxLDL (7 micrograms h-1), LDL (7 micrograms h-1) or phosphate-buffered saline. Collar placement resulted in a thickening of the intima thereby increasing the thickness from 5 +/- 1 to 26 +/- 5 microns with the appearance of alpha-actine positive smooth muscle cells. Perivascular infusion of LDL or oxLDL significantly enhanced the intima, containing large amounts of T-lymphocytes, collagen and smooth muscle cells. The placement of the collar and the infusion of oxLDL during 14 days resulted in an increased sensitivity to serotonin and a decreased sensitivity to acetylcholine. The maximal relaxation to acetylcholine was reduced by 50% whereas the endothelium-independent relaxation to nitroglycerin were not affected. These results show for the first time that the local application of oxLDL in vivo promotes intimal thickening and impairs the endothelium-dependent relaxations thereby supporting the suggestion that oxLDL plays an important role in the morphological and functional changes present in atherosclerotic blood vessels.

Exposure to oxidized low-density lipoprotein in vivo enhances intimal thickening and selectively impairs endothelium-dependent dilation in the rabbit.

OBJECTIVES: Based on in vitro studies, oxidized low-density lipoprotein (oxLDL) has been implicated in atherogenesis and the associated deficiency in endothelium-dependent relaxation. The aim of this study was to investigate the effects of in vivo exposure to oxLDL on intimal thickening and relaxing behaviour. METHODS: Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of the rabbit for 3 or 14 days. OxLDL (Cu(2+)-oxidized, 7 micron/h) or the vehicle phosphate-buffered saline (PBS) was infused in the collars via subdermally implanted osmotic minipumps. RESULTS: The collared vessels receiving PBS developed discrete intimal thickening after 14 days (intima/media (I/M) ratio 11 +/- 2%). OxLDL infusion resulted in intimal thickening after 3 days and significantly enhanced the intimal thickness by 14 days (I/M ratio 98 +/- 16%). Collaring alone for 3 or 14 days and 3 days exposure to oxLDL did not impair the endothelium-dependent relaxations to acetylcholine or calcium ionophore, nor to the NO donors glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP). However, the sensitivity to acetylcholine was decreased after exposure to oxLDL for 14 days (-logEC50 oxLDL 6.95 +/- 0.11 vs. 7.52 +/- 0.11 collar alone) and the maximal relaxation to the endothelium-dependent agonist was reduced by 50%, this in the presence of a virtually intact endothelium. Complete relaxation was still obtained with the nitric oxide donors. CONCLUSION: Our results show for the first time that local vascular exposure to oxLDL in vivo promotes intimal thickening and inhibits endothelium-dependent dilation, thereby supporting an active role for oxLDL in the morphological and functional changes observed in atherosclerotic blood vessels.

Local application of LDL promotes intimal thickening in the collared carotid artery of the rabbit.

Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 micrograms h-1, n = 16 arteries), oxLDL (Cu2+ oxidized, 7 micrograms h-1, n = 16), or phosphate-buffered saline (5 microL h-1, n = 16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of alpha-SMC actin-immunoreactive cells in the intima, thereby increasing the intimal thickness from 5 +/- 1 to 26 +/- 5 microns. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5 +/- 2% in the discrete collar-induced intima to approximately 20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.

Induction of the oxidative catabolism of retinoid acid in MCF-7 cells.

Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.

Analysis of the oxidative catabolism of retinoic acid in rat Dunning R3327G prostate tumors.

We studied the enzymatic characteristics of the oxidative catabolism of retinoic acid (RA) and its inhibition by liarozole-fumarate in homogenates of rat Dunning R3327G prostate tumors. Homogenates of rat liver were used as reference material. Both tumor and liver homogenates were able to catabolize retinoic acid. HPLC analysis revealed only very polar metabolites in tumors, while in the liver both metabolites with intermediate polarity and more polar metabolites were found. Kinetic analysis of retinoic acid catabolism showed a K(m) of 1.7 +/- 0.7 microM and a Vmax of 4.2 +/- 4.4 pmol polar RA metabolites/mg protein/hr for Dunning G tumor homogenates. In liver homogenates a K(m) value of 4.3 +/- 0.5 microM and a Vmax value of 290 +/- 120 pmol polar RA metabolites/mg protein/hr were obtained. Liarozole-fumarate inhibited retinoic acid catabolism in Dunning tumors and liver with IC50 values of 0.26 +/- 0.16 microM and 0.14 +/- 0.05, respectively. The results suggest that rat Dunning R3327G tumors are able to metabolize retinoic acid in a manner similar to that found in rat liver but with a lower metabolizing capacity.

Dual effects of oxidized low-density lipoprotein on immune-stimulated nitric oxide and prostaglandin release in macrophages.

Oxidized low-density lipoprotein (LDL) is currently regarded as a tentative key player in atherosclerosis by virtue of its ability to induce intracellular lipid accumulation and to modulate cell functions in the vessel wall. We previously demonstrated that inducible nitric oxide (NO) synthase activity is attenuated in lipid-laden J774 macrophages obtained by incubation with oxidized LDL 200 micrograms ml-1 for 24 h. In the present study we investigated the effect of oxidized LDL in a lower concentration (20 micrograms ml-1) or for a shorter time (6 h) and the possible mediator role of prostaglandin E2 and prostacyclin. Prostaglandins and the NO synthase metabolites citrulline and nitrite were elevated in the 24 h supernatant after immune stimulation with interferon-gamma 100 U ml-1 with or without lipopolysaccharide 10 micrograms ml-1. Pretreatment with oxidized LDL 20 micrograms ml-1 for 18 h decreased nitrite release by 31 +/- 2%, whereas prostaglandin production was not affected. A 6 h pre-exposure to 200 micrograms ml-1 had an opposite effect: it significantly potentiated interferon-gamma-stimulated prostaglandin E2 (10-fold), prostacyclin (7-fold), nitrite (1.5-fold), and citrulline (2.4-fold) release. Indomethacin 10 microM abolished the prostaglandin production and largely prevented the oxidized LDL-dependent increase in NO synthase activity. Acetylated LDL was without effect. The data show that the immune-induced release of NO is potentiated or suppressed, depending on the conditions of exposure to oxidized LDL. The potentiation due to short, high-dose exposure is partly mediated by prostaglandins since indomethacin inhibited both processes.

Effects of storage temperature and fetal calf serum on the endothelium of porcine aortic valves.

Endothelial integrity and function may be an important determinant for long-term success of allograft heart valves. To determine the optimal storage temperatures for preservation of long-term endothelial function in porcine aortic valves, different storage temperatures and times were investigated. Fresh valves were either (1) stored at 4 degrees C, with or without 10% fetal calf serum supplement, for 1, 2, 4, 7, 14, 21, or 28 days; (2) cryopreserved for 2, 4, or 8 weeks at -80 degrees C or -170 degrees C; (3) cryopreserved in long-term storage (as long as 1 year), with or without fetal calf serum, at -170 degrees C. Viability of endothelial cells was assessed through measurement of the production of prostacyclin in basal and bradykinin-stimulated conditions, during in vitro incubation of the valve cusps at 37 degrees C. Endothelial morphologic variations in valves stored at 4 degrees C were evaluated by scanning electron microscopy. With storage at 4 degrees C, after 4 days the valves already produced significantly less (p < 0.05) prostacyclin than fresh preparations in both basal (0.21 +/- 0.04 versus 3.56 +/- 0.03 ng.ml-1.cm-2) and stimulated conditions (4.17 +/- 0.36 vs 24.23 +/- 1.83). Morphologic changes could not yet be distinguished with scanning electron microscopy at that time. When the storage period was extended, the levels of prostacyclin further diminished; after 14 days, prostacyclin release could no longer be detected. In cryopreserved valves, prostacyclin production was similar for as long as 2 weeks of storage either at -80 degrees C or at -170 degrees C in basal (2.69 +/- 0.63 vs 2.93 +/- 0.51) and stimulated (16.43 +/- 3.19 vs 16.50 +/- 2.57, = 6) conditions. After 8 weeks, no prostacyclin release could be detected in valves stored at -80 degrees C. After 6 months storage at -170 degrees C, the prostacyclin production was significantly (p < 0.05) reduced compared with fresh valves; it then remained constant for as long as 1 year. The valves stored with fetal calf serum produced significantly (p < 0.05) less prostacyclin than did those without fetal calf serum. For longer cryopreserved banking, we recommend storing heart valves at -170 degrees C instead of at -80 degrees C to maintain viability of endothelial cells. Fetal calf serum would harm endothelial viability during long-term cryopreservation.

Dexamethasone influences intimal thickening and vascular reactivity in the rabbit collared carotid artery.

Intimal thickening predisposes to atherosclerosis and is often associated with alterations of the vascular reactivity of the artery. We investigated whether dexamethasone inhibited the intimal thickening and reactivity changes induced by a silicone collar placed around the left rabbit carotid artery for 2 weeks. The sham-operated, right artery served as control. Dexamethasone (1 mg/kg/day), given in the drinking water (n = 10) or by a subcutaneous minipump (n = 10), abolished intimal thickening compared to that of both placebo groups (n = 10). Both dexamethasone and the collar suppressed the isometric force development of isolated segments elicited by KCl in organ chamber experiments. The collar raised the sensitivity to serotonin (5-hydroxytryptamine, 5-HT) and the maximum force development (Emax) after normalization for the KCl responses. Dexamethasone exerted complex effects on 5-HT contractions in sham arteries: the curves often became biphasic, and sensitivity and Emax of the first phase were depressed by dexamethasone. In contrast, dexamethasone raised the hypersensitivity of collared arteries to 5-HT even further. Collar and dexamethasone did not influence endothelium-dependent relaxations elicited by acetylcholine or the calcium ionophore A-23187. It is concluded that dexamethasone interfered with neo-intima formation in the collar model, presumably by inhibition of smooth muscle cell migration and/or proliferation, without restoring contractile behaviour. Therefore, the collar-induced alterations in the reactivity of the smooth muscle cells in the media appear to be unrelated to the process of intimal thickening.

Thrombin triggers the de novo expression of an inducible NO synthase in porcine aortic valve endothelial cells.

The nitric oxide (NO) production by porcine aortic valve endothelial cells was estimated in cusps incubated at 37 degrees C by measuring their cyclic GMP content and the nitrite levels of the incubation medium. After a stabilization period, incubation for 5 min with acetylcholine, bradykinin, ADP and bovine thrombin resulted in a receptor-mediated increase in cyclic GMP which could be blocked by EGTA, N-omega-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA). Incubation with lipopolysaccharide (endotoxin) from E. coli O111:B4 or bovine for 5 h, dose-dependently increased nitrite production as well as cyclic GMP content. The elevated nitrite production was completely abolished in the presence of the protein synthesis inhibitor cycloheximide, was reduced by more than 50% by dexamethasone but was not affected by EGTA. L-NMMA dose-dependently reduced the increased nitrite production and cyclic GMP content. These results suggest that besides the presence of a constitutive NO synthase in porcine aortic valve endothelial cells thrombin, like lipopolysaccharide, triggers the de novo expression of an inducible Ca(2+)-independent NO synthase.

Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes.

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.