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Biomedical subjects

R Busse

Publications and source records attributed to R Busse.

At least 91 records · Page 5Linked to original sources
PubMed1998

Sentinel practices in evaluating longer periods of care: quality of life and drug therapy of terminally ill persons in Lower Saxony (Germany).

STUDY OBJECTIVES: (1) To study the feasibility of using sentinel practice networks to evaluate longer periods of care. (2) To assess the quality of life and drug therapy of community dwelling terminally ill persons. DESIGN: Prospective longitudinal design with GPs in an existing sentinel practice network identifying "terminally ill" persons and recording the following data: age, sex, diagnoses, and ongoing drug therapy initially, time, place, duration, services, and drug changes for every contact, quality of life (HRCA-QL Index, Spitzer Index, uniscale), pain intensity and frequency on a weekly basis, and time and circumstances of death. SETTING: 26 GP practices in Lower Saxony, Germany. PATIENTS: 47 patients (age: mean 76 years, range 31 to 98; sex: 21 male, 26 female; diagnosis: 35 with cancer) with 582 contacts. Mean of recorded time before death was 70 days (median 50). MAIN RESULTS: Average number of physician-patient contacts increased from 0.7 a week three months before death to 2.4 in the final week. Quality of life decreased during that period (HRCA-QL Index: 5.1 to 0.8; Spitzer: 4.4 to 0.8; uniscale: 37 to 9). In the last week of life, no person was free of pain; analgetic therapy was "successful" in 57% of cases. CONCLUSIONS: (1) The sentinel practice approach is feasible for evaluating longer periods of care. The generalisibility, however, may be limited to certain subgroups. (2) The observed trends in quality of life, pain, and analgetic treatment should be compared with those in other settings and countries to identify the scope of care improvement.

Adult↗
PubMed1997

Aggregating human platelets stimulate the expression of thrombin receptors in cultured vascular smooth muscle cells via the release of transforming growth factor-beta1 and platelet-derived growth factorAB.

BACKGROUND: Thrombin and the thrombin receptor have been implicated in the proliferation of vascular smooth muscle cells (VSMCs) observed after angioplasty and in atherosclerosis. Because thrombin receptor activation is an irreversible proteolytic event, the marked upregulation of the smooth muscle cell thrombin receptor after vascular injury may account for the maintained mitogenic activity of thrombin. The present study was designed to determine whether aggregating platelets stimulate thrombin receptor expression in cultured VSMCs and, if so, to identify the mediators. METHODS AND RESULTS: Thrombin receptor expression was assessed by Northern and Western blot analyses and functionally by measuring the release of 6-keto prostaglandin F1alpha. Platelet-derived products (PDPs) released by aggregating human platelets enhanced thrombin receptor mRNA levels in a time- and concentration-dependent manner, an effect that was potentiated by transient acidification of PDPs, which release bioactive transforming growth factor (TGF)-beta1, and that was slightly inhibited by ketanserin. Among several factors known to be released by aggregating platelets, only TGF-beta1, platelet-derived growth factorAB (PDGF(AB)), and serotonin mimicked the PDP effect. The level of membrane thrombin receptor protein was increased in TGF-beta1-treated VSMCs. Pretreatment of VSMCs with either acidified PDP, or TGF-beta1 increased the alpha-thrombin-stimulated release of 6-keto prostaglandin F1alpha. This effect was blunted by incubating acidified PDP with either a TGF-beta- or a PDGF-neutralizing antibody. CONCLUSIONS: Aggregating human platelets stimulate the expression of thrombin receptors in VSMCs through the release of TGF-beta1, PDGF(AB), and, to a lesser extent, serotonin. The upregulation of the thrombin receptor by products released by aggregating platelets may sustain the mitogenic activity of thrombin in the vascular wall at sites of injury.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

Platelet-derived growth factor-stimulated superoxide anion production modulates activation of transcription factor NF-kappaB and expression of monocyte chemoattractant protein 1 in human aortic smooth muscle cells.

BACKGROUND: Platelet-derived growth factor (PDGF) and superoxide anion (O2.-) have been implicated in vascular diseases. We investigated whether PDGF stimulates the production of O2.- in human aortic smooth muscle cells (HSMCs) and whether O2.- leads in this way to the activation of nuclear factor-kappaB (NF-kappaB) and induction of monocyte chemoattractant protein 1 (MCP-1) in PDGF-stimulated HSMCs. METHODS AND RESULTS: PDGF-AB concentration- and time-dependently stimulated O2.- generation from HSMCs. The stimulatory effect of PDGF-AB was mimicked by PDGF-BB but not by PDGF-AA. The generation of O2.- by PDGF-AB was attenuated by the NAD(P)H oxidase inhibitor iodonium diphenyl, the specific protein kinase C (PKC) inhibitor Ro 31-8220, and the phosphatidylinositol 3-kinase inhibitor wortmannin. Allopurinol and nifedipine had no effect on PDGF-AB-induced O2.- release, whereas indomethacin potentiated this response. Gel mobility shift assay revealed that PDGF-AB increased the binding activity of NF-kappaB, which contained predominantly the p50/p65 heterodimer in nuclear extracts from HSMCs. Superoxide dismutase as well as iodonium diphenyl, Ro 31-8220, and wortmannin attenuated PDGF-AB-induced activation of NF-kappaB and expression of MCP-1 mRNA. In contrast, superoxide dismutase did not inhibit the interleukin-1beta-induced NF-kappaB activation. CONCLUSIONS: The results demonstrate that PDGF stimulates O2.- generation in HSMCs via PKC-dependent and wortmannin-sensitive pathways involving flavoenzyme(s). This PDGF-induced O2.- production may be involved in vascular lesion formation by mediating, at least in part, NF-kappaB activation and MCP-1 induction.

Allopurinol↗
PubMed1997

Inhibition of glutathione reductase by dinitrosyl-iron-dithiolate complex.

The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-L-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 microM, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 microM, and a k3 of 1.3 x 10(-3) s-1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/or S-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specifically N-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein.

Animals↗
PubMed1997

Tyrosine phosphorylation and bradykinin-induced signaling in endothelial cells.

It is generally accepted that in endothelial cells the occupation of bradykinin B2 receptors, which are linked to the guanine nucleotide-dependent regulatory proteins, Gi and Gq, results in the activation of phospholipase C-beta1 (PLC-beta1), followed by a transient increase in the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. The PLC-beta1 isoform, in contrast to the gamma1 isoform, is present only at a low level in cultured endothelial cells, implying that PLC-gamma1 activation may play an important role in endothelial signaling pathways. In cultured human endothelial cells, bradykinin induced a rapid increase in the tyrosine phosphorylation of several Triton-soluble proteins. Immunoprecipitation of tyrosine-phosphorylated proteins from bradykinin-stimulated cells followed by Western blotting using the respective antibodies facilitated the identification of a 77 kiloDalton (kDa) protein as paxillin, a 130 kDa protein as PLC-gamma1, and a 42/44 kDa doublet as mitogen-activated protein (MAP) kinase. The bradykinin-induced tyrosine phosphorylation of PLC-gamma1 was relatively transient and was associated with an increase in intracellular levels of IP3. Bradykinin also induced the rapid and transient activation of phosphotyrosine phosphatases localized mainly in the Triton X-100-soluble cell fraction; this tyrosine phosphatase activity was apparently initiated after the release of Ca2+ from intracellular stores.

Analysis of Variance↗
PubMed1997

Nitric oxide and endothelium-derived hyperpolarizing factor: formation and interactions.

Nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) represent the two major endothelial autacoids involved in the local control of vascular tone. Here we describe a novel pathway leading to the calcium-independent activation of the endothelial NO synthase by shear stress and enhanced cellular tyrosine phosphorylation. In addition we present evidence that NO inhibits the formation of EDHF, which we have characterized as a transferable, beta-naphthoflavone-inducible P450-dependent metabolite of arachidonic acid.

Animals↗
PubMed1997

Effect of YC-1, an NO-independent, superoxide-sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators.

1. We studied the effects of 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine-3':5' cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or superoxide anions on these parameters. 2. YC-1 elicited a direct concentration-dependent activation of sGC (EC50 18.6 +/- 2.0 microM), which was rapid in onset and quickly reversible upon dilution. YC-1 altered the enzyme kinetics with respect to GTP by decreasing KM and increasing Vmax. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC-1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H-(1,2,4)-oxdiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (NS 2028), abolished activation of the enzyme by either compound. 3. YC-1 induced a concentration-dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increases in response to SNP and YC-1 were additive. 4. YC-1 relaxed preconstricted endothelium-denuded rabbit aortic rings in a concentration-dependent manner (50% at 20 microM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (3 microM), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP- and NTG-induced cyclic GMP formation. 5. Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O-2) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (SOD). YC-1-stimulated sGC was also sensitive to inhibition by endogenously generated (O-2 in rat preconstricted endothelium-denuded aortic rings. Relaxation to YC-1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O-2 at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC-1. 6. In conclusion, these results indicate that YC-1 is an NO-independent, O-2-sensitive, direct activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic responses to NO-donors and YC-1 in cultured VSMC and isolated aortic rings apparently reflect the direct synergistic action of YC-1 and NO on the sGC. The synergism revealed in this in vitro study suggests that low doses of YC-1 may be of therapeutic value by permitting the reduction of nitrovasodilator dosage.

Animals↗
PubMed1997

ACE inhibitor potentiation of bradykinin-induced venoconstriction.

1. Angiotensin-converting enzyme (ACE) inhibitors exert their cardiovascular effects not only by preventing the formation of angiotensin II (AII), but also by promoting the accumulation of bradykinin in or at the vessel wall. In addition, certain ACE inhibitors have been shown to augment the vasodilator response to bradykinin, presumably by an interaction at the level of the B2 receptor. We have investigated whether this is a specific effect of the ACE inhibitor class of compounds in isolated endothelium-denuded segments of the rabbit jugular vein where bradykinin elicits a constrictor response which is exclusively mediated by activation of the B2 receptor. 2. Moexiprilat and ramiprilat (< or = 3 nM) enhanced the constrictor response to bradykinin three to four fold. Captopril and enalaprilat were less active by approximately one and quinaprilat by two orders of magnitude. Moexiprilat and ramiprilat, on the other hand, had no effect on the constrictor response to AII or the dilator response to acetylcholine. 3. The bradykinin-potentiating effect of the ACE inhibitors was not mimicked by inhibitors of amino-, carboxy-, metallo- or serine peptidases or the synthetic ACE substrate, hippuryl-L-histidyl-L-leucine, at a concentration which almost abolished the residual ACE activity in the vessel wall. In contrast, angiotensin-(1-7) (10 microM), an angiotensin I metabolite, significantly enhanced the constrictor response to bradykinin. 4. Ramiprilat did not alter the binding of [3H]-bradykinin to a membrane fraction prepared from endothelium-denuded rabbit jugular veins or to cultured fibroblasts, and there was no ACE inhibitor-sensitive, bradykinin-induced cleavage of the B2 receptor in cultured endothelial cells. 5. These findings demonstrate that ACE inhibitors selectively potentiate the B2 receptor-mediated vascular effects of bradykinin. Their relative efficacy appears to be independent of their ACE-inhibiting properties and might be related to differences in molecule structure. Moreover, the potentiation of the biological activity of bradykinin by this class of compounds does not seem to be mediated by a shift in affinity of the B2 receptor or a prevention of its desensitization, but may involve an increase in the intrinsic activity of unoccupied B2 receptor molecules.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1997

Effects of barbiturates on the expression of the inducible nitric oxide synthase in vascular smooth muscle.

Certain cytokines stimulate the expression of the inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs) and in many other cell types. The large amounts of nitric oxide (NO) generated by iNOS in the vascular wall contribute to the unrelenting hypotension in septic shock. Because septic patients are often treated with barbiturates, we examined the effect of these anesthetic agents on the expression of iNOS in VSMCs. The induction of iNOS was elicited either in cultured rat aortic SMCs [interleukin-1beta (IL-1beta), 60 U/ml for 24 h] or in endothelium-denuded segments of the rabbit carotid artery [IL-1beta (100 U/ml) for 7 h]. The activity of iNOS was assessed by the accumulation of nitrite in the conditioned medium of cultured VSMCs and by the hyporeactivity of carotid arteries to phenylephrine. Moreover, iNOS protein abundance was determined by Western blot analysis, iNOS messenger RNA (mRNA) by reverse transcription followed by the polymerase chain reaction (PCR), and activation of the transcription factor NF-kappaB by gel electrophoretic mobility-shift analysis of nuclear extracts from VSMCs. The IL-1beta-stimulated increase in nitrite formation, iNOS protein, and mRNA abundance in VSMCs was significantly augmented in the presence of thiopental (100 microM), whereas methohexital, hexobarbital, pentobarbital, and phenobarbital were without effect. The potentiating effect of thiopental was observed only when the barbiturate was administered during the first 2 h of the 24-h incubation period of cultured VSMCs with IL-1beta. Thiopental did not affect the IL-1beta-stimulated activation of NF-kappaB in VSMCs. This barbiturate also significantly augmented the hyporeactivity to phenylephrine in carotid arteries exposed to IL-1beta, an effect that was abolished by N(G)-nitro-L-arginine. Exposure of either cultured or native VSMCs to thiopental alone did not stimulate iNOS expression. These findings demonstrate that the thiobarbiturate thiopental, but not oxybarbiturates, augments the IL-1beta-stimulated synthesis of NO in both cultured and native VSMCs. This effect of thiopental is the result of an increased expression of iNOS, involving most likely mechanisms distinct from NF-kappaB activation. The use of thiopental for long-term treatment of septic patients might possibly potentiate the biosynthesis of NO in the vascular wall and thus cause a further deterioration of the hemodynamic state.

Anesthetics, Intravenous↗
PubMed1997

Financing reforms in the German hospital sector: from full cost cover principle to prospective case fees.

OBJECTIVES: The authors provide an overview of the hospital sector in Germany with a focus on the impact of recent reform legislation on this sector. METHODS: Data from the Federal Statistics Office, the Ministry of Health, and the Federal Association of Physicians are synthesized with information obtained from a general review of the literature. RESULTS: Before the implementation of recent health-care reforms, the German health-care system has been sharply divided into inpatient and ambulatory care sectors, resulting in a fragmented system of care delivery. All hospital operating costs were fully covered through per diem charges. The 1992 Health Care Structure Act and subsequent pieces of legislation have introduced new mechanisms to improve cost efficiency in the hospital sector and increase coordination between the inpatient and outpatient care. These measures notably include implementing an inpatient prospective payment system and permitting ambulatory surgery and care services to be offered in inpatient settings. CONCLUSIONS: Whereas prospective payments have greatly reduced the length of stay, hospitals were reluctant to offer ambulatory surgery due to budgetary constraints and the high level of ambulatory surgery by office-based physicians. The reforms passed have not yielded substantial cost savings. These reforms offer a natural experiment that could benefit from national and international studies on the impact of hospital sector redesign on management, financing, and patient outcomes.

Financial Management, Hospital↗
PubMed1997

Hospital outcomes research in Germany: results from a retrospective survey among sickness fund beneficiaries.

OBJECTIVES: The authors assess the feasibility of using retrospective, indication-specific patient surveys to conduct hospital outcomes research in Germany. Surgical outcome and patient satisfaction were examined in patients who underwent common elective surgical procedures. METHODS: Using the International Classification of Diseases Ninth Revision coding available in the Schwäbisch Gmünd health insurance data base, all patients for a defined period of time with one of the three following diagnoses were selected and questioned retrospectively using an indication-specific survey instrument: (1) varicose veins of the lower extremity; (2) nasal septum deviation; and (3) inner knee joint damage limited to patients undergoing arthroscopic meniscus repair. Survey content focused on preoperative conditions, pre- and postoperative symptoms, postoperative complications, the nature and duration of postoperative follow-up, and satisfaction with surgical outcome. RESULTS: Significant postoperative improvement of preoperative symptoms was found for all three groups. Complete freedom from symptoms was found in 29.7% of patients treated for varicose veins, 24.1% of patients with meniscus repair, and in only 10.6% of patients with nasal septum deviation. Multivariate analyses indicated that postoperative impairment was the decisive variable governing patient satisfaction for all three groups. CONCLUSIONS: The use of retrospective, indication-specific patient surveys constitutes a time-efficient, cost-effective, and patient-focused option for the systematic acquisition and evaluation of health outcomes in Germany. This methodology holds promise for international and domestic efforts to demonstrate the consequences of restructuring activities in the inpatient sector.

Adult↗
PubMed1997

Regional measurements of NO formed in vivo during brain ischemia.

Nitric oxide formed in vivo in the rat brain regions of hippocampus, striatum, neocortex and cerebellum was spin trapped and measured ex vivo by cryogenic electron paramagnetic resonance spectroscopy. In non-ischemic control animals the rate of nitric oxide (NO) formation in the individual brain regions ranged from 15 to 42 pmol.g-1.min-1. During exposure to global ischemia for 7 min the generation of NO increased in all parts of the brain. In the hippocampus the rate of NO formation during ischemia increased by 6-fold from a control rate of 19 pmol.g-1.min-1. This increase was attenuated 47% by pretreatment with the NO synthase antagonist 7-nitroindazole, whereas pretreatment with the non-NMDA receptor anatogonist NBQX and the Ca2+ channel blocker NS638 did not influence the NO formation. The data show that short-duration ischemia elicits a significant, NO-synthase-dependent formation of NO in all brain regions.

Analysis of Variance↗
PubMed1997

Increased serum NG-hydroxy-L-arginine in patients with rheumatoid arthritis and systemic lupus erythematosus as an index of an increased nitric oxide synthase activity.

OBJECTIVES: To determine the feasibility of monitoring the serum concentration of NG-hydroxy-L-arginine (L-NHA) as an index of an increased nitric oxide (NO) synthase activity in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) compared with nitrate (NO3-), the major circulating metabolite of NO whose concentration is influenced by dietary intake. METHODS: The serum concentrations of L-NHA, L-arginine (L-Arg), and NO3- were determined in 33 patients with RA, 25 patients with SLE and, 29 healthy subjects. RESULTS: Serum L-NHA was significantly increased in RA patients with high disease activity (287% of control, p < 0.01), but not with low disease activity (115%), as well as in patients with SLE (173%, p < 0.01). In contrast, serum NO3- did not differ significantly between either group of patients and the respective control group. CONCLUSION: NO synthase activity or expression, or both, is upregulated in RA patients with high disease activity and in patients with SLE. Serum L-NHA seems to be a more specific and reliable index of an increased activity of this enzyme in patients with acute or chronic inflammatory diseases than NO3-.

Acute Disease↗
PubMed1997

N-alpha-tosyl-L-lysine chloromethylketone prevents expression of iNOS in vascular smooth muscle by blocking activation of NF-kappa B.

Certain cytokines and lipopolysaccharide stimulate expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle, an event that is regulated at the transcriptional level and appears to involve several transcription factors, including nuclear factor kappa B (NF-kappa B). Since proteases play an essential role in NF-kappa B activation, experiments were designed to clarify, in both cultured rat aortic smooth muscle cells (SMCs) and isolated rat aortas, whether protease inhibitors affect the interleukin-1 beta (IL-1 beta)-elicited expression of iNOS. The formation of NO was assessed by nitrite release in cultured SMCs and the attenuation of phenylephrine-induced contraction in aortic rings, the expression of iNOS by Western blot analysis and reverse transcription-polymerase chain reaction, and NF-kappa B activity in nuclear extracts by gel electrophoretic mobility shift assya. Exposure of cultured SMCs to IL-1 beta increased NF-kappa B binding activity within 30 minutes and was associated with nitrite accumulation and the appearance of iNOS protein 24 hours later. These responses were abolished in cells that had been exposed to the cytokine in the presence of the protease inhibitor N-alpha-tosyl-L-lysine chloromethylketone. Aprotinin and p-toluenesulfonyl-L-arginine methyl ester, two other protease inhibitors, also reduced the cytokine-stimulated release of nitrite and the level of iNOS protein. Exposure of rat aortic segments without endothelium to IL-1 beta activated NF-kappa B within 30 minutes and was associated with the appearance of iNOS mRNA and an attenuation of phenylephrine-induced contraction 6 hours later. These responses were blunted when the segments were incubated with the cytokine and N-alpha-tosyl-L-lysine chloromethyl ketone. The present observations indicate that protease inhibitors prevent iNOS expression in both cultured and native vascular SMCs by blocking the activation of NF-kappa B.

Animals↗
PubMed1997

Inhibition of inducible nitric oxide synthase restores endothelium-dependent relaxations in proinflammatory mediator-induced blood vessels.

Endothelium-dependent relaxations mediated by nitric oxide (NO) are attenuated in arteries exposed to proinflammatory mediators. Because proinflammatory mediators stimulate the expression of the inducible NO synthase (iNOS) in vascular cells, the role of iNOS-derived NO in the impaired endothelium-dependent relaxation was examined in arterial ring preparations. Exposure of rabbit carotid arteries to interleukin-1 beta (IL-1 beta; 100 U/mL for 7 hours) and porcine coronary arteries to a combination of tumor necrosis factor-alpha (1000 U/mL), interferon-gamma (500 U/mL), and lipopolysaccharide (10 micrograms/mL) for 15 hours (conditions that are associated with iNOS expression) markedly attenuated relaxations to receptor-dependent agonists, whereas those to the calcium ionophore A23187 and sodium nitroprusside were virtually unchanged. The impaired relaxation was not associated with a reduced level of the constitutive endothelial NOS (cNOS) but was accompanied by a reduced formation of biologically active NO as assessed in a bioassay system. The attenuated relaxation of carotid arteries to acetylcholine was not affected by superoxide dismutase and was neither found in arteries exposed to IL-1 beta for only 15 minutes nor in IL-1 beta-treated arteries for 7 hours followed by a 17-hour incubation period without the cytokine. Furthermore, no impaired relaxation was found in rings exposed to IL-1 beta in combination with either cycloheximide or N-alpha-tosyl-L-lysine chloromethyl ketone or pyrrolidine dithiocarbamate, treatments that prevent iNOS expression. In addition, selective inhibition of iNOS with S-methylisothiourea (10 mumol/L) completely restored acetylcholine-induced relaxations. These findings indicate that the continuous generation of NO induced by proinflammatory mediators plays a major role in the inhibition of endothelium-dependent relaxation, most likely by impairing a step in the signal transduction cascade that links activation of endothelial receptors to the calcium-calmodulin-dependent activation of NOS.

Acetylcholine↗
PubMed1997

Endothelial dysfunction coincides with an enhanced nitric oxide synthase expression and superoxide anion production.

We investigated the effects of aortic banding-induced hypertension on the endothelium-dependent vasodilator responses in the aorta and coronary circulation of Sprague-Dawley rats. We studied the influence of hypertension on the endothelial nitric oxide synthase (NOS III) expression, assessed by Western blot and reverse transcription-polymerase chain reactions experiments, and on the superoxide anion (O2-) production. Two weeks after aortic banding, the endothelium-dependent relaxations were not altered. At this time, the expression of NOS III in the aorta and in confluent coronary microvascular endothelial cells (RCMECs) exhibited no marked changes, whereas O2- production was enhanced 1.9-fold in aortas from aortic-banded rats. Six weeks after aortic banding, the endothelium-dependent dilations were markedly impaired in the heart (50% decrease) and aorta (35% decrease). Analysis of NOS III protein and mRNA levels revealed marked increases in both aortas and confluent RCMECs (2.6- to 4-fold) from aortic-banded compared with sham-operated rats. There was no further increase in O2production in both the aorta and confluent RCMECs from aortic-banded rats. An enhanced nitrotyrosine protein level was also detected in the aorta from 6-week aortic-banded rats. These findings indicate that in hypertension induced by aortic banding, an enhanced O2- production alone is not sufficient to produce endothelial dysfunction. Endothelial vasodilator hyporesponsiveness was observed only when NOS III expression and O2- production were increased and was associated with the appearance of enhanced nitrotyrosine residues. This would suggest that the development of endothelial dysfunction is linked to an overproduction of not one, but two, endothelium-derived radicals that might lead to the formation of peroxynitrite.

Animals↗
PubMed1997

Endothelium-derived hyperpolarizing factor, but not nitric oxide, is reversibly inhibited by brefeldin A.

The subcellular localization of the enzymes synthesizing endothelium-derived vasodilator autacoids has been proposed to play a role in determining the ability of endothelial cells to enhance autacoid production in response to stimulation. We therefore investigated the effects of brefeldin A-induced disruption of the Golgi apparatus and Golgi-plasma membrane trafficking on the production of nitric oxide (NO), prostacyclin, and the endothelium-derived hyperpolarizing factor (EDHF) by native and cultured endothelial cells. In porcine coronary artery segments, brefeldin A (35 micromol/L, 90 minutes) did not affect relaxations to sodium nitroprusside or the K+ channel opener cromakalim but elicited a rightward shift in the concentration-response curve to bradykinin without altering the maximum vasodilator response (Rmax). Brefeldin A failed to attenuate the bradykinin-induced, NO-mediated relaxation under depolarizing conditions but inhibited the bradykinin response under conditions of combined cyclooxygenase/NO synthase blockade, suggesting that this agent selectively interferes with the production of EDHF. Indeed, incubation of porcine coronary arteries with brefeldin A, which did not affect the bradykinin-induced accumulation of either cyclic GMP or 6-keto-prostaglandin F1alpha, markedly and reversibly attenuated the EDHF-mediated hyperpolarization of detector smooth muscle cells in a patch-clamp bioassay system. The microtubule destabilizer nocodazole also affected both the EC50 and Rmax to bradykinin in porcine coronary arteries. Since EDHF is thought to be a cytochrome P450-derived metabolite of arachidonic acid and both brefeldin A and nocodazole are known to interfere with the targeting of cytochrome P450 from the Golgi apparatus to the plasma membrane, it is conceivable that brefeldin A inhibits EDHF formation by preventing the targeting of the EDHF-synthesizing enzymes to the plasma membrane.

6-Ketoprostaglandin F1 alpha↗