MnDPDP: Contrast Agent for Imaging and Protection of Viable Tissue.

The semistable chelate manganese (Mn) dipyridoxyl diphosphate (MnDPDP, mangafodipir), previously used as an intravenous (i.v.) contrast agent (Teslascan™, GE Healthcare) for Mn-ion-enhanced MRI (MEMRI), should be reappraised for clinical use but now as a diagnostic drug with cytoprotective properties. Approved for imaging of the liver and pancreas, MnDPDP enhances contrast also in other targets such as the heart, kidney, glandular tissue, and potentially retina and brain. Transmetallation releases paramagnetic Mn2+ for cellular uptake in competition with calcium (Ca2+), and intracellular (IC) macromolecular Mn2+ adducts lower myocardial T 1 to midway between native values and values obtained with gadolinium (Gd3+). What is essential is that T 1 mapping and, to a lesser degree, T 1 weighted imaging enable quantification of viability at a cellular or even molecular level. IC Mn2+ retention for hours provides delayed imaging as another advantage. Examples in humans include quantitative imaging of cardiomyocyte remodeling and of Ca2+ channel activity, capabilities beyond the scope of Gd3+ based or native MRI. In addition, MnDPDP and the metabolite Mn dipyridoxyl diethyl-diamine (MnPLED) act as catalytic antioxidants enabling prevention and treatment of oxidative stress caused by tissue injury and inflammation. Tested applications in humans include protection of normal cells during chemotherapy of cancer and, potentially, of ischemic tissues during reperfusion. Theragnostic use combining therapy with delayed imaging remains to be explored. This review updates MnDPDP and its clinical potential with emphasis on the working mode of an exquisite chelate in the diagnosis of heart disease and in the treatment of oxidative stress.

Fodipir and its dephosphorylated derivative dipyridoxyl ethyldiamine are involved in mangafodipir-mediated cytoprotection against 7ß-hydroxycholesterol-induced cell death.

OBJECTIVE: Mangafodipir exerts pharmacological effects, including vascular relaxation and protection against oxidative stress and cell death induced by oxysterols. Additionally, mangafodipir has been proposed for cardiovascular imaging. The primary metabolites of mangafodipir, manganese dipyridoxyl ethyldiamine (MnPLED) and its constituent dipyridoxyl diphosphate (Dp-dp) also known as fodipir, are pharmacologically active. However, whether they affect oxysterol-induced cytotoxicity is currently unknown. In this study, we examine whether the mangafodipir metabolite affects 7ß-hydroxycholesterol (7ß-OH)-induced cell death and identify the underlying mechanisms. METHODS: U937 cells were pretreated or not with mangafodipir substrate (Ms; 200 µm), MnPLED (100 µmol/l) or Dp-dp (100 µmol/l) for 8 h and then exposed to 7ß-OH (28 µmol/l) for 18 h. RESULTS: Our results revealed that pretreatment with MnPLED or Dp-dp protected against 7ß-OH-induced cellular reactive oxygen species (ROS) production, apoptosis, and lysosomal membrane permeabilization (LMP). MnPLED and Dp-dp, in par with Ms, confer protection against 7ß-OH-induced cytotoxicity by reducing cellular ROS and stabilization of the lysosomal membrane. CONCLUSION: These results suggest that fodipir is the pharmacologically active part in the structure of mangafodipir, which prevents 7ß-OH-induced cell death by attenuating cellular ROS and by preventing LMP. In addition, MnPLED, which is the dephosphorylated product of fodipir, exerts a similar protective effect against 7ß-OH-induced cytotoxicity. This result indicates that dephosphorylation of fodipir does not affect its pharmacological actions. Altogether our result confirms the cytoprotective effect of mangafodipir and justifies its potential use as a cytoprotective adjuvant.

Effects of cocoa extract and dark chocolate on angiotensin-converting enzyme and nitric oxide in human endothelial cells and healthy volunteers--a nutrigenomics perspective.

Evidence suggests that cocoa from the bean of Theobroma cacao L. has beneficial effects on cardiovascular disease. The aim of this study was to investigate if cocoa extract and dark chocolate influence angiotensin-converting enzyme (ACE) and nitric oxide (NO) in human endothelial cells (in vitro) and in healthy volunteers (in vivo). ACE activity was analyzed with a commercial radioenzymatic assay and measured in human endothelial cells from umbilical veins (HUVEC) after 10 minutes of incubation with cocoa extract. NO was measured after 24 hours of incubation. ACE activity and NO were measured at baseline and after 30, 60, and 180 minutes in 16 healthy volunteers after a single intake of 75 g of dark chocolate containing 72% cocoa. Significant inhibition of ACE activity (P < 0.01) and significant increase of NO (P < 0.001) were seen in HUVEC. In the study subjects, a significant inhibition of ACE activity (mean 18%) 3 hours after intake of dark chocolate was seen, but no significant change in NO was seen. According to ACE genotype, significant inhibition of ACE activity was seen after 3 hours in individuals with genotype insertion/insertion and deletion/deletion (mean 21% and 28%, respectively). Data suggest that intake of dark chocolate containing high amount of cocoa inhibits ACE activity in vitro and in vivo.

Prevention of 7ß-hydroxycholesterol-induced cell death by mangafodipir is mediated through lysosomal and mitochondrial pathways.

Mangafodipir, a MRI contrast agent, has been used as a viability marker in patients with myocardial infarction and showed vascular relaxation effect. It confers myocardial protection against oxidative stress. However mechanisms underlying such protection have not yet been investigated. In this investigation we first studied whether mangafodipir inhibits apoptosis induced by 7beta-hydroxycholesterol (7betaOH), a cytotoxic cholesterol oxidation product found in atherosclerotic lesions in humans and in heart of ethanol-fed rats. We then focused on whether mangafodipir influences the production of reactive oxygen species, lysosomal and mitochondrial membrane permeabilities in the cell model. Our results revealed that pre-treatment with mangafodipir (400 microM) protected against cellular reactive oxygen species production, apoptosis, and permeabilization of lysosomal and mitochondrial membranes induced by 7betaOH. In conclusion, a novel effect of mangafodipir on 7betaOH-induced apoptosis is via reduction of cellular reactive oxygen species and stabilization of lysosomal and mitochondrial membranes. This is the first report to show the additional cytoprotective effect of mangafodipir, which may suggest possible use of the drug.

Effects of green tea, black tea and Rooibos tea on angiotensin-converting enzyme and nitric oxide in healthy volunteers.

OBJECTIVE: Tea has been reported to reduce cardiovascular mortality, but the underlying mechanisms are largely unknown. The aim of the current project was to investigate the effect of green tea (Japanese Sencha), black tea (Indian Assam B.O.P.) and Rooibos tea (South Africa) on angiotensin-converting enzyme (ACE) and nitric oxide (NO). DESIGN: Seventeen healthy volunteers received a single oral dose of 400 ml green tea, black tea or Rooibos tea in a randomized, three-phase, crossover study. ACE activity and NO concentration were measured (at 0, 30, 60 and 180 min) in all phases. ACE activity was analysed by means of a commercial radioenzymatic assay. Nitrite was analysed as a marker of NO concentration. In addition, ACE genotype was determined using a PCR method. RESULTS: Oral intake of a single dose of Rooibos tea significantly inhibited ACE activity after 30 min (P < 0.01) and after 60 min (P < 0.05). A significant inhibition of ACE activity was seen with green tea for the ACE II genotype 30 min after intake of the tea (P < 0.05) and for the ACE ID genotype 60 min after intake (P < 0.05). A significant inhibition of ACE activity was also seen with Rooibos tea for the ACE II genotype 60 min after intake (P < 0.05). No significant effect on NO concentration was seen. CONCLUSIONS: These results suggest that green tea and Rooibos tea may have cardiovascular effects through inhibition of ACE activity.

Effect of Vaccinium myrtillus and its polyphenols on angiotensin-converting enzyme activity in human endothelial cells.

This study investigates if the connection between Vaccinium myrtillus and angiotensin-converting enzyme (ACE) might be an explanation of the pharmacological effects on circulation. Cultured endothelial cells from human umbilical veins were incubated with bilberry 25E extract. The main anthocyanidins combined in myrtillin chloride and separately in cyanidin, delphinidin, and malvidin, respectively, were examined concerning their effects on ACE. After 10 min of incubation with bilberry 25E, a significant, dose-dependent inhibition of ACE activity was seen, and after incubation with myrtillin chloride a significant inhibition was seen. No effect was seen with the anthocyanidins. The effect seems to be dependent on this specific mixture of anthocyanins in the bilberry. V. myrtillus may thus have the potential to prevent and protect against cardiovascular diseases.

Panax ginseng induces anterograde transport of pigment organelles in Xenopus melanophores.

Melanophores from Xenopus laevis are pigmented cells, capable of quick colour changes through cyclic adenosine 3':5'-monophosphate (cAMP) coordinated transport of their intracellular pigment granules, melanosomes. In this study we use the melanophore cell line to evaluate the effects of Panax ginseng extract G115 on organelle transport. Absorbance readings of melanophore-coated microplates, Correlate-EIA direct cAMP enzyme immunoassay kit, and western blot were used to measure the melanosome movement and changes in intracellular signalling. We show that Panax ginseng induces a fast concentration-dependent anterograde transport of the melanosomes. No significant increase in the cAMP level was seen and pre-incubation of melanophores with the protein kinase C (PKC) inhibitor EGF-R Fragment 651-658 (M-EGF) only partly decreased the ginseng-induced dispersion. We also demonstrate that Panax ginseng, endothelin-3 (ET-3) and alpha-melanocyte stimulating hormone (MSH) stimulate an activation of mitogen activated protein kinase (MAPK). Pre-incubation with M-EGF decreased the MAPK activity induced by ET-3 and MSH, but again only marginally affected the response of Panax ginseng. Thus, in melanophores we suggest that Panax ginseng stimulates an anterograde transport of pigment organelles via a non-cAMP and mainly PKC-independent pathway.

Tea flavanols inhibit angiotensin-converting enzyme activity and increase nitric oxide production in human endothelial cells.

A diversity of pharmacological effects on the cardiovascular system have been reported for Camellia sinensis: antioxidative, antiproliferative and anti-angiogenic activity, and nitric oxide synthase activation. The purpose of this study was to investigate if the connection between tea and angiotensin-converting enzyme (ACE) and nitric oxide (NO) might be an explanation of the pharmacological effects of tea on the cardiovascular system. Cultured endothelial cells from human umbilical veins (HUVEC) were incubated with extracts of Japanese Sencha (green tea), Indian Assam Broken Orange Pekoe (black tea) and Rooibos tea, respectively. The main flavanols and purine alkaloids in green and black tea were examined for their effects on ACE and NO. After incubation with green tea, black tea and Rooibos tea for 10 min, a significant and dose-dependent inhibition of ACE activity in HUVEC was seen with the green tea and the black tea. No significant effect on ACE was seen with the Rooibos tea. After 10-min incubation with (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechingallate and (-)-epigallocatechingallate, a dose-dependent inhibition of ACE activity in HUVEC was seen for all four tea catechins. After 24-h incubation, a significantly increased dose-dependent effect on NO production in HUVEC was seen for the green tea, the black tea and the Rooibos tea. After 24-h incubation with (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechingallate and (-)-epigallocatechingallate, a dose-dependent increased NO production in HUVEC was seen. In conclusion, tea extracts from C. sinensis may have the potential to prevent and protect against cardiovascular disease.

Glyceryl trinitrate-induced angiotensin-converting enzyme (ACE) inhibition in healthy volunteers is dependent on ACE genotype.

Evidence concerning the importance of angiotensin-converting enzyme (ACE) genotype in cardiovascular diseases is accumulating. The aim of this study was to investigate if nitric oxide (NO), generated from glyceryl trinitrate (GTN), affects human serum ACE activity in vivo, and if so, whether this effect was dependent on ACE genotype and (or) reflected in blood pressure reduction. A tablet containing 5 mg GTN was bucally administered for 5 minutes to 17 healthy volunteers. Blood pressure (BP) was recorded, and serum ACE activity, ACE genotype, and plasma cGMP was analyzed. GTN administration significantly reduced BP only in individuals with the deletion/deletion (DD) genotype. Sixty minutes after GTN administration, serum ACE activity was reduced in individuals with the insertion/insertion (II) and insertion/deletion (ID) genotypes, but not the DD genotype. Comparing the change in ACE activity over time between the genotypes resulted in the following: II vs. DD, p < 0.01; II vs. ID, p < 0.05; and ID vs. DD, p < 0.05. There was no significant difference in plasma cGMP content neither between the ACE genotypes nor before and after GTN administration. In conclusion, GTN inhibits serum ACE in vivo in individuals with the II and ID, but not the DD genotype.

An indomethacin-sensitive contraction induced by beta-antagonists in guinea pig airways.

Beta-adrenergic receptor (beta-AR) antagonists have been associated with increased airway reactivity in asthmatics and potentiation of contractile stimuli in animal models. In the present study, using an in vitro model of tracheal preparations from guinea pigs, we show that the beta-AR antagonists propranolol and pindolol induce a smooth muscle contraction. A prerequisite for this contraction is that the airway preparations have been pre-treated with an beta-AR agonist. Our data show that the contractile effect of beta-AR antagonists is not a simple consequence of reversing the agonist-induced relaxation. Furthermore, the effect seems to be mediated through interaction with beta2-ARs since the response is stereo-selective, and the selective beta1-AR receptor antagonist atenolol did not induce any contractile response. SQ 29,546, a thromboxane A2 antagonist; MK 886, a lipoxygenase inhibitor; and indomethacin, a cyclooxygenase inhibitor significantly inhibited the contractions of the tracheal preparations induced with propranolol or pindolol. We put forward the hypothesis that the contractile effect of the beta-AR antagonist is a consequence of their inverse agonist activity, which is only evident when the receptor population have a higher basal activity. Our results indicate a novel additional explanation for the known side effect, bronchoconstriction, of beta-AR antagonist.

Lack of stereospecificity in lysophosphatidic acid enantiomer-induced calcium mobilization in human erythroleukemia cells.

Lysophosphatidic acid (LPA) is a lipid mediator that, among several other cellular responses, can stimulate cells to mobilize calcium (Ca2+). LPA is known to activate at least three different subtypes of G protein-coupled receptors. These receptors can then stimulate different kinds of G proteins. In the present study, LPA and LPA analogs were synthesized from (R)- and (S)-glycidol and used to characterize the ability to stimulate Ca2+ mobilization. The cytosolic Ca2+ concentration ([Ca2+]i) was measured in fura-2-acetoxymethylester-loaded human erythroleukemia (HEL) cells. Furthermore, a reverse transcriptase polymerase chain reaction was used to characterize LPA receptor subtypes expressed in HEL cells. The results show that HEL cells mainly express LPA1 and LPA2, although LPA3 might possibly be expressed as well. Moreover, LPA and its analogs concentration-dependently increased [Ca2+]i in HEL cells. The response involved both influx of extracellular Ca2+ and release of Ca2+ from intracellular stores. This is the first time the unnatural (S)-enantiomer of LPA, (S)-3-O-oleoyl-1-O-phosphoryl-glycerol, has been synthesized and studied according to its ability to activate cells. The results indicate that this group of receptors does not discriminate between (R)- and (S)-enantiomers of LPA and its analogs. When comparing ether analogs having different hydrocarbon chain lengths, the tetradecyl analog (14 carbons) was found to be the most effective in increasing [Ca2+]i. Pertussis toxin treatment of the HEL cells resulted in an even more efficient Ca2+ mobilization stimulated by LPA and its analogs. Furthermore, at repeated incubation with the same ligand no further increase in [Ca2+]i was obtained. When combining LPA with the ether analogs no suppression of the new Ca2+ signal occurred. All these findings may be of significance in the process of searching for specific agonists and antagonists of the LPA receptor subtypes.

Metabolism of salbutamol differs between asthmatic patients and healthy volunteers.

Patients with asthma are a target group for medication with beta2-agonists, often in combination with corticosteroids. Salbutamol is commonly marketed as racemate. R-Salbutamol carries beta2-agonistic property whereas S-salbutamol does not. The racemate undergoes stereoselective sulphatisation by sulfotransferases mainly in the gut and liver, so that S-salbutamol rests for a longer time in the body and reaches higher plasma levels than R-salbutamol. Ten patients with mild stable asthma and at present without cortisone medication were given racemic salbutamol as ventoline 4 mg orally. Plasma and urine levels were estimated until 24 hr after ingestion. For comparison healthy volunteers were treated in the same way. The group of asthma patients was then treated with budesonide inhalations 800 microg daily for one week and the initial programme resumed. Non-cortisone-treated asthmatic patients displayed higher levels of both R- and S-salbutamol in plasma than did healthy volunteers after one single ingestion of racemic salbutamol (CMAX both comparisons P<0.05). Plasma levels of salbutamol isomers in cortisone-treated asthmatic patients resembled the levels in volunteers. The most plausible explanation for the discrepancy in values between asthmatic patients and volunteers is a defective metabolic function by asthmatic patients possibly enzymatic in origin.

Biological actions of formoterol isomers.

Racemic beta(2) agonists, composed of equal amounts of (R)- and (S)-isomers, can display anomalous actions that compromise their effectiveness as asthma therapies. Loss of efficacy during regular use is characteristic of isoprenaline, albuterol and terbutaline and has in part been attributed to the biological effects of the (S)-isomer. This hypothesis was applied to the (R,R)- and (S,S)-isomers of formoterol. (R,R)-formoterol had 1000-times greater affinity (2.9 nm) to the human beta(2) adrenoceptor than (S,S)-formoterol (3100 nm), with receptor binding modulating intracellular cAMP levels. The minimum lethal intravenous (IV) dose was determined to be 100 mg/kg for (R,R)- and 50 mg/kg for (S,S)-formoterol, suggesting that the toxicity of (S,S)-formoterol may not be related to the binding of beta(2) adrenoceptors. In tissues pretreated with (S,S)-formoterol but not with (R,R)- or racemic formoterol contractions to high concentrations of carbachol were exaggerated. In vivo experiments with sensitized guinea pigs demonstrated that (R,R)-formoterol inhibited both histamine and antigen-induced bronchoconstriction with greater potency than (R,R/S,S)-formoterol while (S,S)-formoterol was ineffective. Metabolic radiolabeling experiments of (R,R)-, (S,S)- or (R,R/S,S)-formoterol with crude human liver phenolsulfotransferase (PST) determined the V(max)/K(m) values to be (0.151), (0.74) and (0.143), respectively. The reciprocal plot illustrates a 2-fold reduction in sulfation rate when (R,R)-formoterol is present as a single isomer. The data presented here suggest that (R,R)-formoterol binds to the beta(2) adrenoceptor and inhibits the contraction of bronchial tissues by spasmogens. However, (S,S)-formoterol exhibits properties inconsistent as an asthma therapeutic and may antagonize the actions of (R,R)-formoterol.