[Frequency and nature of drug related problems with corticosteroids in Belgian community pharmacies]. / Fréquence et nature des problems liés aux corticosteroïdes dans les officines publiques de Belgique.

Aim Aims are: 1] Identify causes of Drug Related Problems (DRPs), interventions performed by pharmacists and results of corticosteroid- related problems and 2] distinguish between problems related to inhaled and general corticosteroids. Methods During 5 days of their internship, 534 final year students of pharmaceutical sciences in six Belgian universities collected DRPs encountered in community pharmacies, as well as related interventions performed by pharmacists and the result of the intervention. The DRPs' electronic registration was done through an adapted tool for Belgium based on the classification of Pharmaceutical Care Network Europe [PCNE- v 6.2]. Findings The frequency of DRPs is 24,8%. 766 DRPs (4,8%) related to corticosteroids, of which 351 were inhaled corticosteroids. The most common causes of corticosteroid-related problems (53- 59%) were technical causes. The most represented category of clinical causes was the inappropriate choice of drug [33-41%]. Pharmacists' intervention was similar for inhaled and general corticosteroids. Pharmacists intervened orally with patients in 38-40% of total interventions, and in writing in 16% of interventions. Pharmacists did not react in 16% of corticosteroid-related problems. 81-83% of PLMS were resolved partially or completely. Conclusion In conclusion, DRPs detected in community pharmacies related to corticosteroid are infrequent (4,8% of DRPs) but 82% of detected problems have been resolved. Furthermore, the study shows the importance for the Belgian health system to introduce an official DRPs classification and software facilitating their documentation in community pharmacies.

[Drug related problems in Belgian community pharmacies]. / Les problèmes liés aux médicaments dans les officines belges.

INTRODUCTION: The identification, the management and if possible the prevention of drug related problems (DRP), are the main responsibilities of pharmacists. AIM: The aims of the study were 1/to investigate the frequency and nature of drug related problems detected by community pharmacists, 2/to inventories the frequency and nature of the interventions by community pharmacists on prescribed medicines, and 3/to evaluate whether there is a difference between DRP detection at the moment of dispensing versus in a quiet setting (a posteriori detection). METHOD: All trainees of the participating universities of Belgian were asked to contribute to a observational study. Participating pharmacists quantified DRP's and their interventions on prescribed medicines for 5 days. Registrations were made by using a web tool based on an adapted version of the classification list of PCNE. The registration took place in two phases, at the time of delivery as well as in an a posteriori verification of the prescriptions with the pharmaceutical record file of the patients. RESULTS: The study was conducted from November 2012 to April 2013 in 534 community-pharmacies with internship. During this period 9.869 prescriptions (15%) with at least one DRP were detected on a total of 64.962 prescriptions treated by tutor pharmacists. Since there could be more than one problem on a prescription, 15.952 DRP's were registered. 2.597 of the DRP's were detected by a posteriori verification. 75% of all problems had a technical cause and 37% were clinical in nature. Under the technical causes an incomplete prescription was the most common. The most frequently registered clinical causes were a drug interaction, an inopportune time of intake, a too high or too low dose and an unsuitable drug. Participating pharmacists solved almost 3 of the 4 detected DRP's. In more than half of the DRP's, the patient was verbally and/or written informed. In 44% of the a posteriori discovered problems, the pharmacist intervened. CONCLUSION: Pharmacist detected one or more DRP's with 15% of the prescriptions. Analysis of a prescription prior to dispensing the medicines therefore appears necessary. The active intervention of the pharmacist in 83% of the problems indicates that he contributes to the optimization of drug therapy with a potential increase in the quality of life of the patient and a reduction in the cost of healthcare. The a posteriori discovered DRP's demonstrate the need for pharmacist lead meditation reviews possibly together with the physician and/or patient.

[Opinions and attitudes of Flemish pharmacists and general practitioners towards INN prescribing. A survery in Antwerp and East Flanders]. / Avis et attitudes des pharmaciens et des médecins généralistes vis-à-vis de la prescription en DCI. Enquête réalisée à Anvers et en Flandre Orientate.

INTRODUCTION: Since 2002 in Belgium, physicians are allowed to prescribe by International Non-proprietary Name (INN). In 2005, the conditions for this decree were set. Examples from other countries have shown that INN prescribing can significantly contribute to controlling pharmaceutical expenditures. The share of INN prescriptions remains low in Belgium (7% in 2011). OBJECTIVE: To formulate an answer to the question: what are the opinions and attitudes of pharmacists and general practitioners [GP's] with regards to INN prescribing? METHOD: In the winter of 2011-2012, a questionnaire with closed-ended questions was send to pharmacists and GP's in the provinces of Antwerp and East-Flanders, through training days and personal visits. Pharmacists and GP's scored a list of statements with a 5-point Likert scale. The themes of the statements related to: delivering INN prescriptions, legislation, impact on expenditures, choices regarding patient concerns and interprofessional relations. RESULTS: In total, 353 questionnaires were completed and returned of which 228 165%1 were by pharmacists and 125 (35%1 by GP's. Although both declared to be sufficiently up to date with regulations to prescribe (84%) or to deliver (95%] a INN prescription, only 13% of the pharmacists said all prescription they receive contain the correct information. Less GP's [36%) than pharmacists (82%] feel aided by their software program when prescribing or delivering an INN prescription. GP's rely mostly on NIHDI (National Institute for Health and Disability Insurance) as the main source for information on INN prescribing, pharmacists rely on the [Local) pharmacists association. The pharmacists and GP's in the study who relied on NIHDI as main information source, were less aware of legislation concerning INN [N2, p<0,05] than those who rely on the local professional association [N2, p<0,0001]. All pharmacists in the study said to consider the patients medication history when delivering an INN prescription for chronic treatment. However, 57% of the GP's preferred not to prescribe by INN for the reason that they are not sure whether the pharmacist will always consider the patients medication history in case of an INN prescription. Although the GP's showed certain motivation to prescribe by INN, it was no greater than for generic prescribing. And INN prescribing has no added value compared to generic prescribing, according to the GP's. For the pharmacists, INN prescribing does contain an opportunity. With the increase in numbers of dosages and sorts of packaging of generic products, it becomes more and more difficult for pharmacists to manage their stock. In case of an INN prescription, the pharmacist can choose between the different packages in his stock. This offers opportunities especially for acute conditions. CONCLUSION: INN prescribing is a good example of where the collaboration between pharmacists and GP's still contains a lot of opportunities, as well for the two professions, as the government and the patient in terms of controlling the pharmaceutical expenditures. Also the education for pharmacist or GP can further contribute to the sensitization of INN prescribing. In practice, there remain a number of issues and differences in opinions between pharmacists and general practitioners regarding INN prescribing. GP's feel few motivation to prescribe by INN and the government has put no imperative demands towards prescribers. Further evaluation of the practicaL feasibility of the current conditions for prescribing and delivering INN prescriptions is needed.

[Critical evaluation of interaction screening software available in pharmacies]. / Evaluation critique des logiciels d'interactions médicamenteuses a l'officine.

Due to the increasing complexity of medication regimens it is not always easy for the pharmacist to quickly and effectively screen the drug use of a particular patient for interactions. By means of a survey and a comparison, the advantages and disadvantages of the most common software packages available in Flanders were analysed. Major stumbling blocks of the currently available software are the high number of false positive signals, the absence of a history regarding the management of interactions, the lack of timely updates of the database and the absence of clear guidelines for the management of an interaction. Based on this research, we make the following recommendations: (1) signal fatigue should be reduced by interaction screening based on the duration of therapy in addition to the ability to suppress signals, (2) a log, coupled with the prescription-register, should be implemented, (3) software companies should help pharmacists more in configuring software preferences and provide them with better information bout the available options, (4) the underlying databases must be updated more quickly. (5) OTC medications, especially in the context of polypharmacy, should be registered in the patient record by the pharmacist, (6) note that food supplements are not included in the interaction screening software, unlike registered medication. (7) the knowledge of pharmacists regarding interactions should be maintained and improved.

Reproducible determination of transpulmonary pressures.

Oesophageal pressures, as measured in an oesophageal balloon catheter, are a validated substitute for pleural pressures. Transpulmonary pressures, indispensable to improve our understanding of ventilatory physiology, are therefore typically calculated as the difference between airway and oesophageal pressures. The oesophageal pressure signal, however, features a superimposed oscillation due to cardiac motion, not representative for pleural pressure. Additionally, oesophageal contractions or surgical manipulation can alter the signal. In practice, transpulmonary pressures are therefore manually determined from the pressure-time graphic by visual inspection of the waves and averaging a limited number of samples. We suggest an approach to extract the end-expiratory transpulmonary pressure from the raw monitoring data.•Our approach reproducibly determines end-expiratory transpulmonary pressures at a given level of set positive end-expiratory pressure at the ventilator.•Our approach ignores surgical disturbance and cardiac oscillations in the oesophageal pressure signal.

[18F]ZCDD083: A PFKFB3-Targeted PET Tracer for Atherosclerotic Plaque Imaging.

PFKFB3, a glycolysis-related enzyme upregulated in inflammatory conditions and angiogenesis, is an emerging target for diagnosis and therapy of atherosclerosis. The fluorinated phenoxindazole [18F]ZCDD083 was synthesized, radiolabeled in 17 ± 5% radiochemical yield and >99% radiochemical purity, and formulated for preclinical PET/CT imaging in mice. In vivo stability analysis showed no significant metabolite formation. Biodistribution studies showed high blood pool activity and slow hepatobiliary clearance. Significant activity was detected in the lung 2 h postinjection (pi) (11.0 ± 1.5%ID/g), while at 6 h pi no pulmonary background was observed. Ex vivo autoradiography at 6 h pi showed significant high uptake of [18F]ZCDD083 in the arch region and brachiocephalic artery of atherosclerotic mice, and no uptake in control mice, matching plaques distribution seen by lipid staining along with PFKFB3 expression seen by immunofluorescent staining. In vivo PET scans showed higher aortic region uptake of [18F]ZCDD083 in atherosclerotic ApoE-/-Fbn1C1039G+/- than in control mice (0.78 ± 0.05 vs 0.44 ± 0.09%ID/g). [18F]ZCDD083 was detected in aortic arch and brachiocephalic artery of ApoE-/- (with moderate atherosclerosis) and ApoE-/-Fbn1C1039G+/- (with severe, advanced atherosclerosis) mice, suggesting this tracer may be useful for the noninvasive detection of atherosclerotic plaques in vivo.

First report of a Mikrocytos-like parasite in European oysters Ostrea edulis from Canada after transport and quarantine in France.

As part of a disease resistance experiment, 112 apparently healthy European flat oysters Ostrea edulis L. were exported from Canada (Nova Scotia) into France to test their susceptibility to Bonamia ostreae infection. Twelve oysters died in transit and 17 others died within 2 wk of laboratory quarantine acclimation. All oysters were examined histologically, and the 17 that died during quarantine were assayed for microcells (Bonamia sp. and Mikrocytos mackini) using molecular techniques. A microcell parasite was detected in the connective tissue of 5 of the 112 oysters. Morphological appearance, tissue affinity and molecular characterization through PCR, in situ hybridization (ISH), fluorescence in situ hybridization (FISH) and sequencing revealed a protist related to M. mackini. This is the first report of a parasite of the genus Mikrocytos in a species belonging to the genus Ostrea from the Atlantic Ocean.

Nitric oxide selectively depletes macrophages in atherosclerotic plaques via induction of endoplasmic reticulum stress.

BACKGROUND AND PURPOSE: Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism. EXPERIMENTAL APPROACH: Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis. KEY RESULTS: Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin. CONCLUSIONS AND IMPLICATIONS: Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.

Oxidative DNA damage and repair in experimental atherosclerosis are reversed by dietary lipid lowering.

Increased oxidative stress is a major characteristic of hypercholesterolemia-induced atherosclerosis. The oxidative environment is mainly created by the production of reactive oxygen species, which are assumed to mediate vascular tissue injury. Oxidative DNA damage resulting from free radical attack remains, however, a poorly examined field in atherosclerosis. Male New Zealand White rabbits were fed a cholesterol-rich diet (0.3%) for 24 weeks. The induced atherosclerotic plaques showed elevated levels of the DNA damage marker 7,8-dihydro-8-oxoguanine (8-oxoG) as demonstrated by immunohistochemistry. 8-oxoG immunoreactivity was found predominantly in the superficial layer of the plaque containing numerous macrophage-derived foam cells but not in the media or in arteries of age-matched control animals. Alkaline single-cell gel electrophoresis revealed that the number of DNA strand breaks was significantly higher in the plaque as compared with control samples of normolipemic animals. These changes were associated with the upregulation of DNA repair enzymes (poly[ADP-ribose] polymerase-1, p53, phospho-p53 [phosphorylated at Ser392], and XRCC1 [x-ray repair cross-complementing 1]). DNA strand breaks normalized after 4 weeks of dietary lipid lowering. However, a significant reduction of 8-oxoG immunoreactivity was only observed after a prolonged period of lipid lowering (12 to 24 weeks). Repair pathways started to decline progressively when cholesterol-fed animals were placed on a normal diet. In conclusion, oxidative DNA damage and increased levels of DNA repair, both associated with diet-induced hypercholesterolemia, are strongly reduced during dietary lipid lowering. These findings may provide a better insight into the benefits of lipid-lowering therapy on plaque stabilization.

Inducible nitric oxide synthase colocalizes with signs of lipid oxidation/peroxidation in human atherosclerotic plaques.

OBJECTIVE: Advanced human atherosclerotic plaques are characterized by the abundant presence of the autofluorescent non-soluble lipid pigment ceroid, consisting of oxidized lipoproteins. The aim of the present study was to examine the topographical and cellular distribution of inducible nitric oxide synthase (iNOS or NOS II) within different stages of atherosclerosis and its colocalization with ceroid deposits and nitrotyrosine. METHODS AND RESULTS: Different stages of atherosclerosis were studied by immunohistochemistry on whole-mount longitudinal sections of carotid endarterectomy specimens. In the adaptive intimal thickening the predominant cell type were smooth muscle cells. The fatty streaks contained both smooth muscle cells and macrophages with an extremely low NOS II immunoreactivity. The advanced atherosclerotic plaques however, showed a very dense infiltration by macrophages, of which a subpopulation expressed NOS II as a vesicular immunoreactivity in their cytoplasm. These were mainly present around the necrotic core, in association with ceroid accumulation and nitrotyrosine. Fluorescence quenching microscopy showed the presence of NOS II on autofluorescent ceroid vesicles in the macrophages. Large extracellular ceroid granules were not NOS II immunoreactive. NOS II mRNA was detected by RT-PCR and the protein by Western blot in the plaque tissue but not in mammary arteries used as controls. CONCLUSION: Ceroid, nitrotyrosine and NOS II colocalized in late stages of atherosclerosis and were found around the necrotic core in the plaque. This could suggest that NOS II expression in macrophages is involved in oxidation and peroxidation of lipids, leading to ceroid formation.

Fibrillin-1 impairment enhances blood-brain barrier permeability and xanthoma formation in brains of apolipoprotein E-deficient mice.

We recently reported that apolipoprotein E (ApoE)-deficient mice with a mutation in the fibrillin-1 gene (ApoE(-/-)Fbn1(C1039G+/-)) develop accelerated atherosclerosis with enhanced inflammation, atherosclerotic plaque rupture, myocardial infarction and sudden death. In the brain, fibrillin-1 functions as an attachment protein in the basement membrane, providing structural support to the blood-brain barrier (BBB). Here, we investigated whether fibrillin-1 impairment affects the permeability of the BBB proper and the blood-cerebrospinal fluid barrier (BCSFB), and whether this leads to the accelerated accumulation of lipids (xanthomas) in the brain. ApoE(-/-) (n=61) and ApoE(-/-)Fbn1(C1039G+/-) (n=73) mice were fed a Western-type diet (WD). After 14 weeks WD, a significantly higher permeability of the BBB was observed in ApoE(-/-)Fbn1(C1039G+/-) mice compared to age-matched ApoE(-/-) mice. This was accompanied by leukocyte infiltration, enhanced expression of pro-inflammatory cytokines, matrix metalloproteinases and transforming growth factor-ß, and by decreased expression of tight junction proteins claudin-5 and occludin. After 20 weeks WD, 83% of ApoE(-/-)Fbn1(C1039G+/-) mice showed xanthomas in the brain, compared to 23% of their ApoE(-/-) littermates. Xanthomas were mainly located in fibrillin-1-rich regions, such as the choroid plexus and the neocortex. Our findings demonstrate that dysfunctional fibrillin-1 impairs BBB/BCSFB integrity, facilitating peripheral leukocyte infiltration, which further degrades the BBB/BCSFB. As a consequence, lipoproteins can enter the brain, resulting in accelerated formation of xanthomas.

Distribution of cell replication and apoptosis in atherosclerotic plaques of cholesterol-fed rabbits.

In human atherosclerosis the development of a cell-poor lipid-rich core is an important feature of atheromatous plaque formation. The core is characterized by extracellular lipid deposition, cholesterol crystals and cell death and is situated in the deep layer of the plaque. The aim of the present study was to localize apoptotic cell death and cell replication in atherosclerotic plaques of cholesterol-fed rabbits in order to examine the hypothesis that core formation is a consequence of an imbalance between cell replication and apoptosis. New Zealand White male rabbits were fed a diet supplemented with 0.3% cholesterol for 16 (n = 5) and 27 weeks (n = 9). Cell replication and cell types were demonstrated by immunohistochemistry and apoptotic cell death was demonstrated by DNA in situ end-labeling (ISEL) and transmission electron microscopy. Quantification was done using a colour image analysis system. The plaques showed a clear distinction between a luminal layer composed of numerous lipid-rich foam cells of macrophage origin and a deep layer which was fibrous, containing extracellular lipid deposits and few smooth muscle cells. Cell replication (expressed as percentage of total number of nuclei) in the superficial layer was higher then in the deep layer at both 16 (5.1 +/- 1.8% vs. 1.2 +/- 0.8%) and 27 weeks (11.3 +/- 2.1% vs. 4.4 +/- 1.0%). This was also the case for the total number of nuclei per 50000 microns2 cross-sectional intimal area (numerical density): 235 +/- 13 vs. 147 +/- 7 at 16 weeks and 130 +/- 10 vs. 89 +/- 11 at 27 weeks. Apoptotic cell death (expressed as percentage of total number of nuclei) was low and there was no difference between the superficial and the deep layers of the plaques (0.8% +/- 0.2% vs. 0.4% +/- 0.2% at 16 weeks and 0.6 +/- 0.2% vs. 1.7% +/- 0.6% at 27 weeks). Our results indicate that the control of cell number in superficial vs. deep regions of the plaque is mainly a consequence of differences in cell replication. This may be due to a gradient of endothelial and plasma-derived growth factors. Cells can disappear by apoptosis, albeit at a relatively low level, throughout the lesion. This process may contribute to the pronounced cell loss in more advanced human atherosclerotic plaques, setting the base for plaque rupture.

Platelet adhesion to subendothelial structures under flow conditions: no effect of the lipoxygenase product 13-HODE.

It has been shown that endothelial cells can convert linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) and it has been suggested that 13-HODE has non-thrombogenic properties. However, no direct evidence has been presented that indicates that 13-HODE indeed modulates platelet-vessel wall interaction. In this study we have bound a purified 13-HODE to a thrombogenic surface and its effect on platelet adhesion was studied and compared to the effects of an analogous hydroxy fatty acid, 15-hydroxyeicosatetraenoic acid (15-HETE). The effect of 13-HODE on platelet adhesion was studied both under static and flow conditions. In this report we show that binding of up to 40 times the physiological concentration to a thrombogenic surface has no inhibitory effect on platelet adhesion under static or flow conditions. We conclude that 13-HODE is not an important regulatory substance in platelet-subendothelium interaction, although this does not exclude it has a putative anti-adhesive role on intact endothelium.

Collar-induced elevation of mRNA and functional activity of 5-HT(1B) receptor in the rabbit carotid artery.

Hypersensitivity to serotonin (5-HT) develops in rabbit collared carotid arteries. Previous data demonstrated the involvement of 5-HT(1)-like receptors which are not active in normal carotid arteries. This study investigated the interaction in the rabbit carotid artery between 5-HT and a moderate tone as this can uncover functional 5-HT(1)-like receptors. Furthermore, the expression of messenger RNA (mRNA) and protein of 5-HT(1B), 5-HT(1D) and 5-HT(2A) receptors was addressed. Silicone collars were placed around the carotid arteries of male New Zealand White rabbits for 1 week. Rings from inside (=collar) and outside (=sham) the collar were either mounted in isolated organ baths for isometric force measurements or frozen in liquid nitrogen to isolate total RNA or proteins which were subsequently analysed by respectively reverse transcriptase-polymerase chain reaction and Western blot analysis. In sham and collared rings concentration-response curves (CRC's) to 5-HT were monophasic. Only in collared segments the presence of a 5-HT(2A) antagonist (spiperone or ketanserin, 0.1 microM) revealed a biphasic CRC which was even more pronounced when a moderate tone was induced by KCl pointing to functional 5-HT(1)-like receptors. The rabbit carotid artery constitutively expressed 5-HT(1B) and 5-HT(2A) mRNA, not 5-HT(1D) mRNA. Manipulation of the carotid artery increased the 5-HT(1B) mRNA level. Collar placement raised it even further. The 5-HT(2A) mRNA level remained unchanged. All the anti-5-HT receptor antibodies tested resulted in variable, non specific patterns with multiple bands. In conclusion, collar placement elevates mRNA expression and activity of the 5-HT(1B) receptor in the rabbit carotid artery.

Influence of chronic treatment with a nitric oxide donor on fatty streak development and reactivity of the rabbit aorta.

1. The influence of chronic treatment with molsidomine, pro-drug of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on fatty streak development and release of NO and prostacyclin (PGI2) was studied in the aorta of normal and cholesterol-fed rabbits. 2. Groups of 10 rabbits received standard diet (150 g day-1), or diets with 0.3% cholesterol, with 0.02% molsidomine or with the combination of cholesterol and molsidomine for 16 weeks. Lesion area and thickness, maximum change in isometric force (Emax) and sensitivity (-log EC50 or pD2) to constricting and relaxing agonists were assessed in segments of arch, thoracic and abdominal aorta. Bioassay was used to assess NO release. 3. Cholesterol-induced fatty streaks tapered off towards the abdominal aorta. Area, thickness, weight and cholesterylester content of the lesions were augmented by the NO donor, whereas the hypercholesterolaemia remained unchanged. The exacerbation was attributed to co-release of superoxide anion from the sydnonimine. 4. As fatty streaks progressed, amplitude and pD2 of acetylcholine (ACh)-induced relaxations decreased, whereas cyclic GMP and cyclic AMP second messenger systems were not influenced, since Emax and sensitivity to SIN-1 and forskolin remained unchanged. However, extensive lesions apparently trapped some NO, as the pD2 of authentic NO decreased. 5. The fatty streaks curtailed the biosynthesis of PGI2 and the overflow of NO from the perfused thoracic aorta. The latter defect was not restored by L-arginine and appears to be consistent with a functional change of the endothelial muscarinic receptors. 6. The NO donor desensitized the aorta to cyclic GMP-mediated relaxations (ACh, SIN-1 and NO), without affecting cyclic AMP-mediated relaxation to forskolin or constrictor responses to phenylephrine and 5-hydroxytryptamine. 7. The drug also suppressed the ACh-induced overflow of NO, without changing PGI2 release. This selective reduction of endothelial NO release and the desensitization of cyclic GMP-mediated relaxations occurred independently of fatty streak formation. 8. The results indicate that chronic exposure to exogenous NO downregulates endothelial NO release and cyclic GMP-mediated relaxations, and provide evidence for the existence of negative feed-back regulations of the L-arginine NO pathway under in vivo conditions.

The role of endothelial cells in the relaxations induced by 13-hydroxy- and 13-hydroperoxylinoleic acid in canine arteries.

1. One of the major fatty acids in the arterial wall is linoleic acid. It has been shown that its 13-hydroxy metabolite (13-HODE) is generated in significant amounts by cultured endothelial cells. The aim of the present study was to investigate the relaxations to 13-HODE and its hydroperoxyprecursor (13-HPODE) and to examine the role of the endothelial cells. 2. Ring segments of canine circumflex and splenic artery were mounted in organ chambers for isometric tension recording. During contractions induced by prostaglandin F2 alpha or noradrenaline, 13-HODE and 13-HPODE evoked dose-dependent relaxations. Removal of the endothelial cells reduced the relaxations to 13-HODE, but had no effect on those elicited by 13-HPODE. 3. Indomethacin and meclofenamate (0.3 microM to 30 microM) blocked the relaxations evoked by 13-HODE and 13-HPODE in endothelium-denuded rings. In segments with endothelium, both cyclo-oxygenase inhibitors again abolished the relaxations to 13-HODE, but only diminished those to 13-HPODE. 4. Prostacyclin biosynthesis, as measured by radioimmunoassay, increased upon incubation with 13-HODE and 13-HPODE (10 microM). Bioassay of the release of nitric oxide (NO) indicated that NO was not involved in the relaxations elicited by either metabolite. Moreover, L-NG-nitroarginine (100 microM), a specific inhibitor of NO synthesis, did not influence the relaxations to 13-HODE and 13-HPODE. The responses to 13-HPODE were also not altered by superoxide dismutase. 5. In the splenic artery 13-HPODE and 13-HODE induced contractions above 3 microM which were blocked by the thromboxane receptor antagonist, daltroban.In the circumflex artery contractile responses to high concentrations of 13-HODE could be observed only after inhibition of cyclo-oxygenase.6. We conclude that the vasodilatation induced by 13-HODE and 13-HPODE was due to stimulation of prostacyclin biosynthesis both in the endothelium and smooth muscle cells or other subendothelial structures. An additional, unidentified intermediate, which was neither NO nor a cyclo-oxygenase product nor superoxide anion, contributed to the relaxations to 13-HPODE in arteries with endothelium.

Vasoconstrictor responses after neo-intima formation and endothelial removal in the rabbit carotid artery.

1. The present study examined the responses of the rabbit carotid artery to five vasoconstrictors after neo-intima formation induced by perivascular collar treatment and evaluated the role of constitutive and inducible nitric oxide (NO) synthase and endothelial cells (ECs). 2. Ring segments of the rabbit carotid artery were mounted in organ chambers for isometric tension recording. Neo-intima-bearing vessels developed less force (Emax) in response to KCl, the thromboxane-mimetic U-46619 and 5-hydroxytryptamine (5-HT), but not to angiotensin I and II. 3. The collar-treatment increased the sensitivity to 5-HT, and decreased the sensitivity to angiotensin II. The sensitivity to U-46619 and angiotensin I remained unchanged. 4. Mechanical removal of ECs and inhibition of NO biosynthesis by NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NOARG) increased the sensitivity to 5-HT in sham and collar-treated segments to the same extent. The effects of collar-treatment and endothelial removal or treatment with inhibitors of NO biosynthesis were additive. Inhibition of NO biosynthesis failed to augment sensitivity to 5-HT after endothelial denudation. L-NOARG increased the force development to KCl in sham and collar-treated segments to the same extent. However, L-NMMA and L-NOARG failed to augment the contractile responses of neo-intima-bearing vessels to 5-HT and KCl after endothelial removal. 5. The responses to angiotensin I were not altered, either by the neo-intima or by endothelial removal. In arteries with a neo-intima the sensitivity to angiotensin II was decreased. Removal of the endothelium or incubation with L-NOARG counteracted this rightward shift and increased Emax.6. Our results demonstrate that contractions to 5-HT, angiotensin II and KCl are modulated by NO in both sham and neo-intima-bearing vessels. Inhibition of NO biosynthesis and collar treatment resulted in additive effects on the EC50 values, suggesting that the 5-HT and angiotension (AT) receptors on the smooth muscle cells are also modified by the formation of a neo-intima. Furthermore, the reduced contractile responses of segments with a neo-intima are not due to NO formed by an inducible NO synthase in those vessels.

Early atherosclerosis is accompanied by a decreased rather than an increased accumulation of fatty acid hydroxyderivatives.

The content of 13-hydroxylinoleic acid (13-HODE) and 15-hydroxyarachidonic acid (15-HETE) in the rabbit thoracic aorta was measured using high performance liquid chromatography after chronic exposure to cholesterol and a high dose of molsidomine, a donor of nitric oxide (NO). Cholesterol-induced fatty streak formation was accompanied by a decrease in the amounts of esterified 13-HODE and 15-HETE. The reduction of the esterified 13-HODE content correlated significantly with the severity of the lesions. These results do not support the hypothesis that fatty acid hydroperoxides accumulate in the arterial wall during atherosclerosis. On the other hand, the quantity of esterified 13-HODE and 15-HETE was increased markedly after exposure to molsidomine. The high dose of this agent could have initiated radical reactions (via liberation of NO and production of superoxide anions) thereby leading to a raise of the 13-HODE and 15-HETE content of the vessel.