Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx

Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating firm adhesion with beta2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent firm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inflammatory action, its effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreatment with taxifolin (1-100 microM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent firm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and firm adhesion by neutrophils. Taxifolin impeded the calcium influx induced by fMLP (a receptor-mediated activator) or AlF(4)(-) (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC(50)) less than 10microM, possibly through impairing the activation of NADPH oxidase, a major ROS-generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium influx may account for the inhibition of Mac-1-dependent neutrophil firm adhesion that confers taxifolin the anti-inflammatory activity.     

Suppression of receptor-mediated Ca2+ mobilization and functional leukocyte responses by hyperforin

We have recently identified hyperforin, a lipophilic constituent of the herb Hypericum perforatum (St. John's wort), as a dual inhibitor of the proinflammatory enzymes cyclooxygenase-1 and 5-lipoxygenase. The aim of the present study was to further elucidate antiinflammatory properties and respective targets of hyperforin. We found that hyperforin inhibited the generation of reactive oxygen species (ROS) as well as the release of leukocyte elastase (degranulation) in human isolated polymorphonuclear leukocytes (PMNL), challenged by the G protein-coupled receptor (GPCR) ligand N-formyl-methionyl-leucyl-phenylalanine (fMLP) with an IC 50 approximately equal 0.3 microM. When PMNL were stimulated with phorbol-12-myristate-13-acetate (PMA) or ionomycin, hyperforin (up to 10 microM) failed to inhibit ROS production and elastase release, respectively. Moreover, hyperforin blocked receptor-mediated Ca(2+) mobilization ( IC 50 approximately equal 0.4 and 4 microM, respectively) in PMNL and monocytic cells, and caused a rapid decline of the intracellular Ca(2+) concentration in resting cells. In contrast, the Ca(2+) influx induced by ionomycin or thapsigargin was not suppressed. Comparative studies with the specific phospholipase C inhibitor U-73122 and hyperforin revealed similarities between both compounds. Thus, U-73122 and hyperforin blocked fMLP- and PAF-induced Ca(2+) mobilization, ROS formation, and elastase release, but failed to suppress these responses when cells were stimulated by PMA or ionomycin. Also, both compounds rapidly decreased basal Ca(2+) levels in resting cells and led to a rapid decline of the Ca(2+) elevations evoked by fMLP or PAF. Our data suggest that hyperforin targets component(s) within G protein signaling cascades that regulate Ca(2+) homeostasis, coupled to proinflammatory leukocyte functions.     

Specificity of action of bisindolylmaleimide protein kinase C inhibitors: do they inhibit the 70kDa ribosomal S6 kinase in cardiac myocytes?

Bisindolylmaleimide protein kinase C (PKC) inhibitors, such as GF109203X and Ro31-8220, are used as pharmacological tools in many cellular systems. However, in vitro, GF109203X and Ro31-8220 also inhibit the 70 kDa ribosomal S6 kinase (p70^S6K) with similar potency. We determined whether GF109203X and Ro31-8220 inhibit p70^S6K activity in intact adult rat ventricular myocytes (ARVM). First, we confirmed that increased phosphorylation of the 40S ribosomal S6 protein (a cellular substrate for both p70^S6K and the 90 kDa ribosomal S6 kinase) in response to stimulation of ARVM by insulin-like growth factor-1 (300 ng/mL; 10 min) occurs specifically through rapamycin-sensitive activation of p70^S6K. Then, using this response as the index of cellular p70^S6K activity, we determined the effects of GF109203X and Ro31-8220 (1, 3 or 10 μM) on such activity. At these concentrations, neither GF109203X nor Ro31-8220 inhibited cellular p70^S6K activity. In contrast, even at 1 μM, cellular PKC activity (stimulated by a 3 min exposure to 30 nM phorbol 12-myristate 13-acetate) was significantly inhibited by each agent. We conclude that; (1) data obtained in vitro may not necessarily be extrapolated to intact cells and (2) inhibition of p70^S6K is unlikely to contribute to the actions of GF109203X and Ro31-8220 in ARVM.     

Activation of latent transforming growth factor beta 1 and inhibition of matrix metalloprotease activity by a thrombospondin-like tripeptide linked to elaidic acid

Impaired wound healing and skin aging are characterized by neutral protease-mediated destruction of matrix macromolecules associated with disturbance in tissue repair. We synthesized a fatty acyl-peptide derivative at aims to simultaneously activate latent TGF-beta through its peptide domain, KFK, and inhibit MMPs through its lipophilic moiety, elaidic acid. Elaidyl-KFK as well as KFK were shown to activate LAP-TGF-beta both in vitro, using a solid phase assay with immobilized LAP-TGF-beta, and ex vivo using human dermal fibroblasts cultures. In both assays, as much as up to 10% of LAP-TGF-beta added could be recovered as active form. KQK, KQFK as well as their lipopeptide counterparts were inactive. Elaidyl-KFK-mediated LAP-TGF-beta activation led to up-regulation of collagen and TIMP-1 production and down regulation of PMA-induced MMP-1 expression in fibroblasts cultures. Those effects could be suppressed by supplementing cell culture medium with blocking TGF-beta antibody. Elaidyl-KFK inhibited MMP-2, MMP-9, MMP-3, MMP-1, in vitro with IC(50) equal to 1.2, 1.0, 0.24 and 8.9 microM, respectively. Its ex vivo inhibitory capacity, as assessed using skin tissue sections, towards the elastin-degrading capacity of MMP-9 was even more pronounced. At a 1 microM concentration, the lipopeptide decreased by up to 80% enzyme activity. Thus, "Lipospondin," i.e. elaidyl-KFK might be considered as a promising model compound to prevent age-associated dermal alterations.     

Paramethoxyamphetamine (PMA) poisoning; a 'party drug' with lethal effects

Among young people in Norway the recreational use of amphetamine derivatives seems to be increasing. Methylenedioxymethamphetamine (MDMA), known as ecstasy, is the dominant substance, having both stimulant and psychedelic properties. Depending on the illegal source of these so-called 'party drugs' the content and purity can vary. This case report describes the first lethal case of paramethoxyamphetamine (PMA) and paramethoxymethamphetamine (PMMA) intoxication reported in Norway. A 16-year-old male was admitted to a local hospital in a coma with seizures and hyperthermia after he had been found undressed and barefooted in a local forest (temperature 2 degrees C). He was intubated and given supportive care. Blood chemistry revealed hypoglycaemia, hypocalcaemia and hyperkalaemia. Shortly after transfer to the central hospital he developed bradycardia with continuous seizures and asystole. Adverse effects of MDMA are well described and include serotonergic and sympathomimetic symptoms with hyperthermia, coagulopathy, rhabdomyolysis and acute kidney and liver failure. Case reports of PMA deaths collectively suggest PMA to be more toxic than MDMA. A delayed effect after intake of PMA compared with MDMA can lead to increased intake. Hypoglycaemia and hyperkalaemia may be specific to PMA poisoning. Increased thermo genesis will result in a search for cooling, which explains the attempt to undress and a desire to submerge in water. In a cool climate this behaviour itself can be lethal. Measures to treat seizures, hypoglycaemia, electrolyte anomalies and hyperthermia are the therapeutic goals. No specific treatment is available.     

Induction by staurosporine of hepatocyte growth factor production in human skin fibroblasts independent of protein kinase inhibition

Staurosporine is one of the most potent and well known inhibitors of protein kinases, and it is often used to study the involvement of protein kinases in signal transduction pathways. We now report that staurosporine can induce the production of hepatocyte growth factor (HGF) independently of protein kinase inhibition. Staurosporine markedly stimulated the production of HGF in various cell types, including human skin fibroblasts. Its effect was accompanied by up-regulation of HGF gene expression. The inhibition of protein kinases appears not to be involved in staurosporine-induced HGF production, because other protein kinase inhibitors, K-252a, H-7, GF 109203X and genistein, had no HGF-inducing activity. UCN-01, 7-hydroxystaurosporine, which differs from staurosporine only in its aglycone moiety, also showed HGF-inducing activity, and inactive K-252a differs from staurosporine only in its sugar moiety. These results indicate that the sugar moiety, a six-atom ring structure, is important in the HGF-inducing activity of staurosporine. Experiments were then carried out to determine whether the characteristics of staurosporine-induced HGF production have similarities to those of HGF production stimulated by other HGF inducers. The effect of staurosporine like that of 8-bromo-cAMP and that of cholera toxin was marked in human skin fibroblasts from all four different sources, whereas the effects of epidermal growth factor and phorbol 12-myristate 13-acetate were variable depending on cells. The net increase in HGF production induced by staurosporine was not reduced in protein kinase C-depleted human skin fibroblasts. Moreover, synergistic induction of HGF was detected between staurosporine and interferon-gamma as well as between 8-bromo-cAMP and interferon-gamma. Staurosporine, however, did not increase intracellular cAMP levels in human skin fibroblasts. These results indicate that staurosporine induced HGF in different cell types via a signaling pathway similar to the cAMP-mediated pathway without increasing cAMP levels.     

Enhancement of proteolytic processing of the beta-amyloid precursor protein by hyperforin

We studied the effect of hyperforin, a component of St. John's wort (Hypericum perforatum) extracts, on the processing of the amyloid precursor protein (APP) in rat pheochromocytoma PC12 cells, stably transfected with human wildtype APP. We observed transiently increased release of secretory APP fragments upon hyperforin treatment. Unique features, like a strong reduction of intracellular APP and the time course of soluble APP release, distinguished the effects of hyperforin from those of alkalizing agents and phorbol esters, well known activators of secretory processing of APP. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a protonophore, induced an almost identical decrease in intracellular pH in PC12 cells as does hyperforin. Despite this, FCCP induced a less pronounced release of soluble APP fragments and only slightly reduced intracellular APP levels. These results suggest that hyperforin is an activator of secretory processing of APP with a novel mechanism of action not solely dependent on its effects on intracellular pH.