Physical activity guidelines and cardiovascular risk in children: a cross sectional analysis to determine whether 60 minutes is enough.

BACKGROUND: Physical activity reduces cardiovascular mortality and morbidity. The World Health Organisation (WHO) recommends children engage in 60 min daily moderate-to-vigorous physical activity (MVPA). The effect of compliance with this recommendation on childhood cardiovascular risk has not been empirically tested. To evaluate whether achieving recommendations results in reduced composite-cardiovascular risk score (CCVR) in children, and to examine if vigorous PA (VPA) has independent risk-reduction effects. METHODS: PA was measured using accelerometry in 182 children (9-11 years). Subjects were grouped according to achievement of 60 min daily MVPA (active) or not (inactive). CCVR was calculated (sum of z-scores: DXA body fat %, blood pressure, VO2peak, flow mediated dilation, left ventricular diastolic function; CVR score ≥ 1SD indicated 'higher risk'). The cohort was further split into quintiles for VPA and odds ratios (OR) calculated for each quintile. RESULTS: Active children (92 (53 boys)) undertook more MVPA (38 ± 11 min, P < 0.001), had greater VO2peak (4.5 ± 0.8 ml/kg/min P < 0.001), and lower fat % (3.9 ± 1.1 %, P < 0.001) than inactive. No difference were observed between active and inactive for CCVR or OR (P > 0.05). CCVR in the lowest VPA quintile was significantly greater than the highest quintile (3.9 ± 0.6, P < 0.05), and the OR was 4.7 times higher. CONCLUSION: Achievement of current guidelines has positive effects on body composition and cardiorespiratory fitness, but not CCVR. Vigorous physical activity appears to have beneficial effects on CVD risk, independent of moderate PA, implying a more prescriptive approach may be needed for future VPA guidelines.

Assessment of biochemical liver markers, physical activity, fitness and body mass index for a cardiometabolic risk model in childhood.

AIM: The aim of this study was to investigate clustered cardiometabolic risk scores in healthy 10- to 12-year-olds using anthropometric characteristics, measurements of cardiorespiratory fitness (CRF) and physical activity and blood markers of metabolic disease. We also evaluated how including markers of liver cell injury would affect the clustered cardiometabolic risk assessment model. METHODS: This cross-sectional study focused on 99 children aged 10-12 years. The main outcome included assessing participants with increased and low cardiometabolic risk factors using a clustered risk score model that incorporated markers implicated in metabolic syndrome pathogenesis. Two clustered risk scores were calculated, one incorporating markers of liver cell injury. RESULTS: Children classified as 'increased risk' exhibited significantly lower CRF and higher body mass index Z-scores than their 'low-risk' peers. No significant differences in physical activity were observed. This trend remained unchanged when markers of liver injury were included in the clustered risk assessment model. CONCLUSION: The clustered risk score model is a scientifically robust method of cardiometabolic risk assessment, which reiterates the importance of weight reduction and CRF promotion in childhood. Our study did not show a significant contribution of liver injury markers, and further research is needed to evaluate their effect on cardiometabolic risk stratification in childhood.

Effects of 6 months glucagon-like peptide-1 receptor agonist treatment on endothelial function in type 2 diabetes mellitus patients.

Glucagon-like peptide-1 receptor agonists (GLP-1 RA) are used for treatment in type 2 diabetes mellitus (T2DM). Little is known about their cardiovascular (CV) impact. We sought to determine the effects of chronic treatment on vascular function in T2DM. Brachial artery endothelial-dependent flow-mediated dilation (FMD) and endothelial-independent glyceryl trinitrate (GTN) function and carotid intima-medial thickness (cIMT) were assessed in 11 severely obese T2DMs (4 females, 7 males: 55 ± 8 years, diabetes duration 8.3 ± 4.7 years mean ± s.d.) before and after 6 months GLP-1 RA. Body weight (5.3 ± 1.2 kg; p < 0.05) and magnetic resonance imaging determined total and subcutaneous fat, but not visceral fat, decreased. Glycaemic control improved. There were no significant changes in FMD, GTN and cIMT (-1.1 ± 0.4%, 0.3 ± 3.0% and 0.00 ± 0.04 mm, respectively). Despite significant improvements in body composition and glycaemic control, 6 months GLP-1 RA treatment did not modulate vascular function. Alternative strategies may therefore be needed to reduce the burden of CV risk in severely obese patients with long-standing T2DM.

Impact of exercise training on endothelial function and body composition in young people: a study of mono- and di-zygotic twins.

Flow mediated dilation (FMD) is a surrogate marker of arterial function which can be improved by exercise training. To date, no study has assessed the magnitude of FMD changes in response to exercise training between groups of mono- (MZ) and di-zygotic (DZ) twins. The purpose of this study was therefore to compare FMD in MZ- and DZ twins before and after identical exercise training interventions. At baseline, FMD was assessed using high resolution Duplex ultrasound in 12 twin pairs (6 MZ pairs 13.5 ± 0.8 years, 6 DZ pairs 13.4 ± 0.8 years). Twins completed 8 weeks of exercise training (65-85% HR(max)), consisting of three 45-min sessions per week. Change (Δ) scores were entered into twin versus twin intraclass correlation analyses by group. Change in %body fat (r = 0.63, P = 0.05) was significantly correlated in the MZ, but not the DZ group (r = 0.31, P = 0.23). Change in FMD was also highly correlated in MZ (r = 0.74, P = 0.02) but not in the DZ group (r = 0.37, P = 0.18). Heritability of ΔFMD was estimated at 0.74. Exercise induced changes in FMD were similar within sets of monozygotic twins but not dizygotic twins. These data suggest that a significant portion of the arterial function response to exercise training may be genetically determined.

Does brachial artery flow-mediated dilation scale to anthropometric characteristics?

Flow-mediated dilation (FMD) assesses the health of the vascular endothelium. Despite widespread adoption of scaling practices in cardiac research, scaling for body size or composition has not been used for FMD. The present study investigated the relationships between brachial FMD and body composition in 129 children aged 9-10 (75 female symbol, 54 male symbol), and 50 men aged 16-49. Body composition variables (total, lean, fat mass in the whole body, arm, forearm) were assessed by dual-energy X-ray absorptiometry, FMD was measured in the brachial artery using high-resolution ultrasound. FMD was scaled using simple ratios (y/x) and allometric approaches (y/x ( b )) after log-log least squares linear regression produced allometric exponents (b). Size independence was confirmed via bivariate correlations (x:y/x; x:y/x ( b )). No relationships were evident between FMD and body composition variables in adults. Small correlations existed between FMD and measures of segmental fat mass in children (r = -0.18 to -0.19, p < 0.05), there were no significant relationships between FMD and measures of lean or total mass in children. For all significant relationships, b-exponents were different from 1 (CIs -0.36 to 0.07), suggesting ratio scaling approaches were flawed. This was confirmed when ratio scaling produced negative residual size correlations, whereas allometric scaling produced size-independent indices. Correlations between FMD and body composition were weak in children and insignificant in adults. As the results of this study are limited to the populations examined, our findings do not support the adoption of scaling procedures to correct FMD.

Does conduit artery diameter vary according to the anthropometric characteristics of children or men?

Arterial measurements are commonly undertaken to assess acute and chronic adaptations to exercise. Despite the widespread adoption of scaling practices in cardiac research, the relevance of scaling for body size and/or composition has not been addressed for arterial measures. We therefore investigated the relationships between brachial artery diameter and body composition in 129 children aged 9 to 10 yr (75 girls and 54 boys), and 50 men aged 16-49 yr. Body composition variables (total, lean, and fat mass in the whole body, arm, and forearm) were assessed by dual-energy X-ray absorptiometry, and brachial artery diameter was measured using high-resolution ultrasound. Bivariate correlations were performed, and arterial diameter was then scaled using simple ratios (y/x) and allometric approaches after log-log least squares linear regression and production of allometric exponents (b) and construction of power function ratios (y/xb). Size independence was checked via bivariate correlations (x:y/x; x:y/xb). As a result, significant correlations existed between brachial artery diameter and measures of body mass and lean mass in both cohorts (r=0.21-0.48, P<0.05). There were no significant relationships between diameter and fat mass. All b exponents were significantly different from 1 (0.08-0.50), suggesting that simple ratio scaling approaches were likely to be flawed. This was confirmed when ratio scaling produced negative residual size correlations, whereas allometric scaling produced size-independent indexes (r=0.00 to 0.03, P>0.05). In conclusion, when between- or within-group comparisons are performed under circumstances where it is important to control for differences in body size or composition, allometric scaling of artery diameter should be adopted rather than ratio scaling. Our data also suggest that scaling for lean or total mass may be more appropriate than scaling for indexes of fat mass.

Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish.

The zebrafish is rapidly becoming a popular model system for the study of vertebrate development because it is ideal for both embryological studies and genetic analysis. To determine if a retroviral vector pseudotyped with the envelope glycoprotein of the vesicular stomatitis virus could infect zebrafish embryos, and in particular, the cells destined to become the germ line, a pseudotyped virus was injected into blastula-stage zebrafish embryos. Fifty-one embryos were allowed to develop and eight transmitted proviral DNA to their progeny. Founders were mosaic, but as expected, transgenic F1's transmitted proviral DNA in a Mendelian fashion to the F2 progeny. Transgenic F1 fish inherited a single integrated provirus, and a single founder could transmit more than one viral integration to its progeny. These results demonstrate that this pantropic pseudotyped vector, originally developed for human gene therapy, will make the use of retroviral vectors in zebrafish possible.

RTT-led IGRT for cervix cancer; training, implementation and validation.

INTRODUCTION: IGRT in cervical cancer treatment delivery is complex due to significant target and organs at risk (OAR) motion. Implementing image assessment of soft-tissue target and OAR position to improve accuracy is recommended. We report the development and refinement of a training and competency programme (TCP), leading to on-line Radiation Therapist (RTT) led soft-tissue assessment, evaluated by a prospective audit. METHODS AND MATERIALS: The TCP comprised didactic lectures and practical sessions, supported by a comprehensive workbook. The content was decided by a team comprised of Clinical Oncologists, RTTs, and Physicists. On completion of training, RTT soft-tissue review proficiency (after bony anatomy registration) was assessed against a clinician gold-standard from a database of 20 cervical cancer CBCT images. Reviews were graded pass or fail based on PTV coverage assessment and decision taken in concordance with the gold-standard. Parity was set at ≥80% agreement.The initial TCP (stage one) focussed on offline verification and decision making. Sixteen RTTs completed this stage, four achieved ≥80%. This was not sufficient to support clinical implementation.The TCP was redesigned, more stringent review guidelines and greater anatomy teaching was added. TCP stage two focussed on online verification and decision making supported by a decision flowchart. Twenty-one RTTs completed this TCP, all achieved ≥80%. This supported clinical implementation of RTT-led soft-tissue review under prospective audit conditions.The prospective audit was conducted between March 2017 and August 2017. Daily online review was performed by two trained RTTs. Online review and decision making proficiency was evaluated by a clinician. RESULTS: Thirteen patients were included in the audit. Daily online RTT-led IGRT was achieved for all 343 fractions. Two-hundred CBCT images were reviewed offline by the clinician; the mean number of reviews per patient was 15. 192/200 (96%) RTT image reviews were in agreement with clinician review, presenting excellent concordance. DISCUSSION AND CONCLUSION: Multidisciplinary involvement in training development, redesign of the TCP and inclusion of summative competency assessment were important factors to support RTT skill development. Consequently, RTT-led cervical cancer soft-tissue IGRT was clinically implemented in the hospital.

Proviral insertions in the zebrafish hagoromo gene, encoding an F-box/WD40-repeat protein, cause stripe pattern anomalies.

The zebrafish, Danio rerio, has three types of pigment cells (melanophores, xanthophores and iridophores) and, in adult fish, these cells are organized into a stripe pattern. The mechanisms underlying formation of the stripe pattern are largely unknown. We report here the identification and characterization of a novel dominant zebrafish mutation, hagoromo (hag), which was generated by insertional mutagenesis using a pseudotyped retrovirus. The hag mutation caused disorganized stripe patterns. Two hag mutant alleles were isolated independently and proviruses were located within the fifth intron of a novel gene, which we named hag, encoding an F-box/WD40-repeat protein. The hag gene was mapped to linkage group (LG)13, close to fgf8 and pax2.1. Amino acid sequence similarity, conserved exon-intron boundaries and conserved synteny indicated that zebrafish hag is an ortholog of mouse Dactylin, the gene mutated in the Dactylaplasia (Dac) mouse [1]. The Dac mutation is dominant and causes defects in digit formation in fore- and hindlimbs. This study revealed that the hag locus is important for pattern formation in fish but is involved in distinct morphogenetic events in different vertebrates.

Regulation of endothelial cell cyclic nucleotide metabolism by prostacyclin.

An analysis of prostaglandin-stimulated adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation in cultured human umbilical vein endothelial cells showed prostacyclin (PGI2) to be the most potent agonist followed by prostaglandin (PG)H2, which was more potent than PGE2, while PGD2 was essentially inactive. The endothelial cells studied apparently have a high rate of cyclic AMP phosphodiesterase activity because significant PGI2-mediated increases in cyclic AMP could not be shown in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX). Endoperoxide PGH2-stimulation of cyclic AMP accumulation was inhibited 75--80% by the prostacyclin synthetase inhibitors 12-hydroperoxyeicosatetraenoic acid or 9,11-azoprosta-5,13-dienoic acid. These data indicate that the PGH2-stimulation is due primarily to conversion to PGI2. The beta-adrenergic agonist L-isoproterenol stimulated cyclic AMP accumulation in the endothelial cells. This accumulation was completely blocked by propranolol. However, stimulation of cyclic AMP accumulation by the beta-adrenergic agent did not equal that induced by PGI2. Furthermore, the PGI2 response could not be blocked by propranolol. Thrombin-stimulated PGI2 biosynthesis was attenuated by PGE1 or isoproterenol in the presence of MIX. MIX alone was less effective than a combination of PGE1 or isoproterenol plus MIX. These data suggest two potential effects of PGI2 biosynthesis by endothelial cells: first, the PGI2 can elevate cyclic AMP in platelets, and second, endothelial cell cyclic AMP can be elevated as well, so that subsequent PGI2 synthesis will be attenuated.

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2).

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3',5'-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4.

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I2, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B4 synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B4 itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B4 is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B4 synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B4 can limit their own activity in neutrophils.

Acetyl glyceryl ether phosphorylcholine (PAF-acether) and leukotriene B4-mediated neutrophil chemotaxis through an intact endothelial cell monolayer.

Human endothelial cell monolayers were grown on nucleopore filters, and used to partition the two halves of a modified Boyden chamber. Human neutrophil chemotaxis through the monolayer was studied in response to leukotriene B4 and acetyl glyceryl ether phosphorylcholine (PAF-acether). Both leukotriene B4 and PAF-acether concentration-dependently stimulated neutrophil chemotaxis through intact monolayer. The biologically inactive lyso-PAF, and leukotriene C4 and D4 were inactive as chemotactic agents. Leukotriene A4 was weakly chemotactic. In the absence of chemotaxin, little penetration of the monolayer by neutrophils was observed. Agents that elevate neutrophil cyclic AMP levels inhibit both leukotriene B4 and PAF-acether-stimulated chemotaxis through the endothelial cell monolayer. The specific 5-lipoxygenase inhibitor, 6,8-de-epoxy-6,9-(phenylimino) delta 6,8-prostaglandin I1 (U-60257), inhibits PAF-acether, but not leukotriene B4-mediated chemotaxis. These data suggest that an intact 5-lipoxygenase may be required for normal PAF-acether-mediated chemotaxis, but leukotriene B4-mediated chemotaxis is independent of 5-lipoxygenase activity. This system may prove to be a useful model for the study of neutrophil-endothelial cell interactions.

Changes in circulating insulin-like growth factor-binding protein-1 (IGFBP-1) during prolonged exercise: effect of carbohydrate feeding.

Plasma levels of glucose, insulin, the insulin-like growth factor (IGF-I and -II) and IGFBP-1 were determined in four young healthy males performing cycle exercise to fatigue while being fed either placebo (trial C) or glucose polymer solution (trial G). There was a significant decline in glucose and insulin from rest to fatigue in C (P < 0.01 and P < 0.05, respectively), but not in G. IGF-I or IGF-II levels did not change significantly in either of the trials. IGFBP-1 levels increased 12-fold in C (11.4 +/- 1.6 ng/ml at rest to 136.5 +/- 19.7 ng/ml at fatigue P < 0.01), and 5.6-fold in G (11.0 +/- 2.3 ng/ml to 62.2 +/- 15 ng/ml, P < 0.05). In C a significant negative correlation was found between IGFBP-1 and glucose (r = 0.69, P < 0.01) and IGFBP-1 and insulin (r = -0.612, P < 0.05) in C, but not in G. These results suggest that during prolonged exercise factors other than insulin or glucose may regulate IGFBP-1 and that IGFBP-1 may serve a role other than to prevent the hypoglycaemic action of the IGFs.

Serial dexamethasone suppression tests in initial suppressors and nonsuppressors treated with electroconvulsive therapy.

Four different methods of quantifying the 1-mg Dexamethasone Suppression Test (DST) were contrasted with serial testing in endogenous depressives receiving electroconvulsive therapy (ECT). Of three continuous measures in 38 patients with pretreatment DSTs, only the log-transformed value for plasma cortisol was normally distributed, indicating that it possessed superior psychometric properties. Pretreatment Hamilton Depression Rating Scores (HAM-D) correlated positively with pretreatment DST status, with a similar association noted between posttreatment DST status and HAM-D scores. There was no uniform effect of ECT on the DST. Although pretreatment nonsuppressors showed a trend toward decreased postdexamethasone cortisol values, initial suppressors (cutoff: 5 micrograms/dl) evidenced a significant increase in these values, and 35.3% of initial suppressors were nonsuppressors at final DST assessment. These trends were noted in the DST assessment done following the third ECT treatment, suggesting an effect of regression to the mean. The findings highlight the importance of following initial DST suppressors in studies of this type.

Urinary excretion of 6-hydroxychlorzoxazone as an index of CYP2E1 activity.

OBJECTIVE: To determine whether the urinary excretion of 6-hydroxychlorzoxazone is an index of CYP2E1 activity in vivo. METHODS: Male volunteers (n = 27; age range, 17 to 36 years) who were abstinent from alcohol were studied. Chlorzoxazone, 500 mg, was given orally and plasma was collected at 31/2, 41/2, 51/2, and 61/2 hours after dosing. Urine was collected for 8 hours. Ten volunteers participated in full kinetic studies to define the absorption phase and plasma area under the concentration-time curve of chlorzoxazone and the urinary kinetics of the 6-hydroxy metabolite. Chlorzoxazone and the 6-hydroxy metabolite were measured by high-performance liquid chromatography. CYP2E1 activity was expressed as a hydroxylation index (HI = mmole oral chlorzoxazone dose/mmole 6-hydroxychlorzoxazone in 8-hour urine). RESULTS: There was a significant positive correlation between plasma elimination rate constant for chlorzoxazone (Ke) and urinary excretion of the metabolite (n = 27, r = 0.42, p < 0.03) and a significant negative correlation between plasma Ke and HI (n = 27, r = -0.41, p < 0.04). The mean absorption rate constant for chlorzoxazone of 3.11 +/- 4.67 hr-1 was fivefold greater than the plasma Ke of 0.57 +/- 0.17 hr-1 for the full kinetic studies. The formation clearance of the 6-hydroxy metabolite was negative between plasma Ke of the parent compound and disposition rate constant for urinary excretion of the 6-hydroxy metabolite (n = 15, r = 0.85, p < 0.0001). CONCLUSIONS: The urinary excretion of 6-hydroxychlorzoxazone is limited by formation rate and may be useful as an in vivo probe of CYP2E1 activity.

Requirements for green fluorescent protein detection in transgenic zebrafish embryos.

We have generated transgenic (Tg) lines of zebrafish in which the green fluorescent protein (GFP)-encoding gfp cDNA is driven by the Xenopus laevis ef1 alpha enhancer/promoter; Tg embryos from most of these lines show detectable fluorescence throughout their body. We have investigated the copy number of the Tg genes in fluorescent and non-fluorescent lines, in order to determine how this affects the production of detectable levels of GFP in the zebrafish embryo. Additionally, we have injected purified recombinant GFP into embryos to determine the intracellular GFP concentration required for detection, both when all of the cells in the embryo contain GFP and when only a few do.

Postictal excitement following bilateral and right-unilateral ECT.

Postictal excitement was observed in two patients following bilateral and/or right-unilateral ECT but not following left-unilateral ECT. The findings agree with reports associating a similar syndrome with right-side brain insult.

Endothelium-dependent relaxation and hyperpolarization in guinea-pig coronary artery: role of epoxyeicosatrienoic acid.

1. Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 microM N(omega)-nitro-L-arginine and 10 microM indomethacin. 2. ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H1-receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was significantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 microM). 3. The cytochrome P450 inhibitors proadifen (10 microM) and clotrimazole (10 microM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 microM) and 7-ethoxyresorufin (10 microM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 microM) and LEM (1 microM)-induced hyperpolarization. 4. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 microM), glibenclamide (10 microM) and BaCl2 (50 microM) had no significant effect on responses to ACh, AA and 11,12-EET. 5. IBTX (100 nM) significantly reduced both 11,12-EET (33 microM) and AA (30 microM) hyperpolarization without affecting the ACh (1 microM)-induced hyperpolarization. In contrast, 4-AP significantly reduced the ACh-induced hyperpolarization without affecting either AA or 11,12-EET-induced hyperpolarizations. 6. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO- and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

Suicide inhibitors of cytochrome P450 1A1 and P450 2B1.

The inhibition of the P450 1A1 dependent de-ethylation of 7-ethoxyphenoxazone (7EPO) and the P450 2B1 dependent de-pentylation of 7-pentoxyphenoxazone (7PPO) by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 1-ethynylanthracene (1EA), 2-ethynylanthracene (2EA), 9-ethynylanthracene (9EA), 2-ethynylphenathrene (2EPh), 3-ethynylphenanthrene (3EPh), 9-ethynylphenanthrene (9EPh), 1-ethynylpyrene (1EP) and 2-ethynylpyrene (2EP) was studied in hepatic microsomal preparations from rats. Although all of the polycyclic aromatic acetylenes studied inhibited the dealkylation of 7EPO or 7PPO, only some of the acetylenes produced a mechanism-based irreversible inactivation (suicide inhibition) of the P450 dependent dealkylation of 7EPO or 7PPO. Of the molecules tested, only 1EP, 1EN, 2EN, 2EPh and 3EPh were effective suicide inhibitors of the P450 1A1 dependent de-ethylation of 7EPO and only 1EN, 2EN, 1EA and 9EPh were effective suicide inhibitors of the P450 2B1 dependent de-pentylation of 7PPO. In addition to the size and shape of the polycyclic aromatic ring system, placement of the carbon--carbon triple bond on the ring system was critical for suicide inhibition. In contrast to 1EP, 2EP was not a mechanism-based inhibitor of P450 1A1; 9EPh, but not 2EPh or 3EPh, was a suicide inhibitor of P450 2B1. None of the aryl acetylenes tested produced heme destruction under assay conditions that produced the suicide inhibition of the P450 dependent 7EPO or 7PPO dealkylation activities. Because a precise orientation of the terminal acetylene is required to produce suicide inhibition without heme destruction, acetylenic suicide inhibitors can potentially be used to differentiate between P450 isozymes and to establish some distinguishing geometric features of the active site of these isozymes.