The noncanonical nucleotide binding site 1 of the bile salt export pump is optimized for proper function of the transporter.

The bile salt export pump (ABCB11/BSEP) is a hepatocyte plasma membrane-resident protein translocating bile salts into bile canaliculi. The sequence alignment of the four full-length transporters of the ABCB subfamily (ABCB1, ABCB4, ABCB5 and ABCB11) indicates that the NBD-NBD contact interface of ABCB11 differs from that of other members in only four residues. Notably, these are all located in the noncanonical nucleotide binding site 1 (NBS1). Substitution of all four deviant residues with canonical ones (quadruple mutant) significantly decreased the transport activity of the protein. In this study, we mutated two deviant residues in the signature sequence to generate a double mutant (R1221G/E1223Q). Furthermore, a triple mutant (E502S/R1221G/E1223Q) was generated, in which the deviant residues of the signature sequence and Q-loop were mutated concurrently to canonical residues. The double and triple mutants showed 80% and 60%, respectively, of the activity of wild-type BSEP. As expected, an increasing number of mutations gradually impair transport as an intricate network of interactions within the ABC proteins ensures proper functioning.

Mephedrone induces partial release at human dopamine transporters but full release at human serotonin transporters.

Mephedrone (4-methylmethcathinone) is a cathinone derivative that is recreationally consumed for its energizing and empathogenic effects. The stimulating properties are believed to arise from the ability of mephedrone to interact with the high-affinity transporters for dopamine (DA) (DAT) and norepinephrine (NET), whereas the entactogenic effect presumably relies on its activity at the serotonin (5-HT) transporter (SERT). Early studies found that mephedrone acts as a releaser at NET, DAT and SERT, and thus promotes efflux of the respective monoamines. Evidence linked drug-induced reverse transport of 5-HT via SERT to prosocial effects, whereas activity at DAT is strongly correlated with abuse liability. Consequently, we sought to evaluate the pharmacology of mephedrone at human (h) DAT and SERT, heterologously expressed in human embryonic kidney 293 cells, in further detail. In line with previous studies, we report that mephedrone evokes carrier-mediated release via hDAT and hSERT. We found this effect to be sensitive to the protein kinase C inhibitor GF109203X. Electrophysiological recordings revealed that mephedrone is actively transported by hDAT and hSERT. However, mephedrone acts as a full substrate of hSERT but as a partial substrate of hDAT. Furthermore, when compared to fully efficacious releasing agents at hDAT and hSERT (i.e. S(+)-amphetamine and para-chloroamphetamine, respectively) mephedrone displays greater efficacy as a releaser at hSERT than at hDAT. In summary, this study provides additional insights into the molecular mechanism of action of mephedrone at hDAT and hSERT.

α-PPP and its derivatives are selective partial releasers at the human norepinephrine transporter: A pharmacological characterization of interactions between pyrrolidinopropiophenones and high and low affinity monoamine transporters.

While classical cathinones, such as methcathinone, have been shown to be monoamine releasing agents at human monoamine transporters, the subgroup of α-pyrrolidinophenones has thus far solely been characterized as monoamine transporter reuptake inhibitors. Herein, we report data from previously undescribed α-pyrrolidinopropiophenone (α-PPP) derivatives and compare them with the pharmacologically well-researched α-PVP (α-pyrrolidinovalerophenone). Radiotracer-based in vitro uptake inhibition assays in HEK293 cells show that the investigated α-PPP derivatives inhibit the human high-affinity transporters of dopamine (hDAT) and norepinephrine (hNET) in the low micromolar range, with α-PVP being ten times more potent. Similar to α-PVP, no relevant pharmacological activity was found at the human serotonin transporter (hSERT). Unexpectedly, radiotracer-based in vitro release assays reveal α-PPP, MDPPP and 3Br-PPP, but not α-PVP, to be partial releasing agents at hNET (EC50 values in the low micromolar range). Furthermore, uptake inhibition assays at low-affinity monoamine transporters, i.e., the human organic cation transporters (hOCT) 1-3 and human plasma membrane monoamine transporter (hPMAT), bring to light that all compounds inhibit hOCT1 and 2 (IC50 values in the low micromolar range) while less potently interacting with hPMAT and hOCT3. In conclusion, this study describes (i) three new hybrid compounds that efficaciously block hDAT while being partial releasers at hNET, and (ii) highlights the interactions of α-PPP-derivatives with low-affinity monoamine transporters, giving impetus to further studies investigating the interaction of drugs of abuse with OCT1-3 and PMAT.

Active transport of rhodamine 123 by the human multidrug transporter P-glycoprotein involves two independent outer gates.

Human P-glycoprotein (P-gp) is a multispecific drug-efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo-electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry-related outer gates. Experiments were aided by availability of the X-ray structure of a homodimeric eukaryotic homolog of P-gp from red alga (CmABCB1), which defined the role of an apical tyrosine residue (Y358) in outer gate formation. We mutated analogous tyrosine residues in each half of the human full-length transporter (Y310, Y953) to alanine. These mutants were introduced in engineered transporters which bind rhodamine 123 in one of two symmetry-related binding modes only. Outer gate dysfunction was detected by a loss of active transport characteristics, while these mutants retained the ability for outward downhill transport. Our data demonstrate that symmetric tyrosine residues Y310 and Y953 are involved in formation of two distinct symmetry-related outer gates, which operate contingent on the rhodamine 123 binding mode. Hence, the rotationally symmetric architecture of P-gp, which determines duality in ATP binding and rhodamine 123 interaction, also forms the basis for the existence of two independently operating outer gates.

"Polytox" synthetic cathinone abuse: A potential role for organic cation transporter 3 in combined cathinone-induced efflux.

Synthetic cathinone derivatives are a new class of psychoactive substances (NPS), also known as "bath salts", designed to exert psychostimulant effects resembling those of well-known psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). As major constituents of bath salts, the cathinone derivatives 3,4-methylenedioxypyrovalerone (MDPV) and 4-methylmethcathinone (mephedrone), have received considerable media attention. MDPV and mephedrone interfere with the function of the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT), resulting in increased extracellular levels of these monoamines, though their mechanism of action differs. MDPV acts as a non-transported inhibitor of DAT, NET and SERT, whereas mephedrone promotes transporter-mediated release in an amphetamine-like fashion. MDPV and mephedrone are often taken together, creating a conundrum in as much as non-transported inhibitors, like MDPV, prevent mephedrone-induced reverse transport via DAT, NET and SERT. Here we provide evidence supporting a role for organic cation transporter 3 (OCT3) in the actions of mephedrone, which may account for its ability to enhance effects of MDPV. We show that mephedrone can induce substrate efflux via OCT3 in the presence of MDPV. Real-time recordings of the fluorescent OCT3 substrate (4-(4-dimethylamino)styryl)-N-methylpyridinium (ASP+) and radiotracer-flux studies using [3H]1-methyl-4-phenyl-pyridinium (MPP+), demonstrated that OCT3 is MDPV-insensitive when expressed in human embryonic kidney (HEK293) cells. Ex vivo experiments performed in cultured superior cervical ganglia (SCG) cells, rich in NET and OCT3, revealed that mephedrone induces [3H]MPP+ release in an OCT3-dependent manner when NET is fully occupied with MDPV. These results extend our recent findings that OCT3 is key in the mechanism of action of amphetamine-induced substrate release. OCT3 likewise appears to be a mechanism through which mephedrone can induce release of monoamines, thereby accounting for the paradoxically more potent psychostimulant effects of MDPV taken together with mephedrone, and greater risk for deleterious side effects.

An unsuspected role for organic cation transporter 3 in the actions of amphetamine.

Amphetamine abuse is a major public health concern for which there is currently no effective treatment. To develop effective treatments, the mechanisms by which amphetamine produces its abuse-related effects need to be fully understood. It is well known that amphetamine exerts its actions by targeting high-affinity transporters for monoamines, in particular the cocaine-sensitive dopamine transporter. Organic cation transporter 3 (OCT3) has recently been found to play an important role in regulating monoamine signaling. However, whether OCT3 contributes to the actions of amphetamine is unclear. We found that OCT3 is expressed in dopamine neurons. Then, applying a combination of in vivo, ex vivo, and in vitro approaches, we revealed that a substantial component of amphetamine's actions is OCT3-dependent and cocaine insensitive. Our findings support OCT3 as a new player in the actions of amphetamine and encourage investigation of this transporter as a potential new target for the treatment of psychostimulant abuse.

Molecular Mechanism of Taurocholate Transport by the Bile Salt Export Pump, an ABC Transporter Associated with Intrahepatic Cholestasis.

The bile salt export pump (BSEP/ABCB11) transports bile salts from hepatocytes into bile canaliculi. Its malfunction is associated with severe liver disease. One reason for functional impairment of BSEP is systemic administration of drugs, which as a side effect inhibit the transporter. Therefore, drug candidates are routinely screened for potential interaction with this transporter. Hence, understanding the functional biology of BSEP is of key importance. In this study, we engineered the transporter to dissect interdomain communication paths. We introduced mutations in noncanonical and in conserved residues of either of the two nucleotide binding domains and determined the effect on BSEP basal and substrate-stimulated ATPase activity as well as on taurocholate transport. Replacement of the noncanonical methionine residue M584 (Walker B sequence of nucleotide binding site 1) by glutamate imparted hydrolysis competency to this site. Importantly, this mutation was able to sustain 15% of wild-type transport activity, when the catalytic glutamate of the canonical nucleotide binding site 2 was mutated to glutamine. Kinetic modeling of experimental results for the ensuing M584E/E1244Q mutant suggests that a transfer of hydrolytic capacity from the canonical to the noncanonical nucleotide binding site results in loss of active and adoption of facilitative characteristics. This facilitative transport is ATP-gated. To the best of our knowledge, this result is unprecedented in ATP-binding cassette proteins with one noncanonical nucleotide binding site. Our study promotes an understanding of the domain interplay in BSEP as a basis for exploration of drug interactions with this transporter.

Folding correction of ABC-transporter ABCB1 by pharmacological chaperones: a mechanistic concept.

Point mutations of ATP-binding cassette (ABC) proteins are a common cause of human diseases. Available crystal structures indicate a similarity in the architecture of several members of this protein family. Their molecular architecture makes these proteins vulnerable to mutation, when critical structural elements are affected. The latter preferentially involve the two transmembrane domain (TMD)/nucleotide-binding domain (NBD) interfaces (transmission interfaces), formation of which requires engagement of coupling helices of intracellular loops with NBDs. Both, formation of the active sites and engagement of the coupling helices, are contingent on correct positioning of ICLs 2 and 4 and thus an important prerequisite for proper folding. Here, we show that active site compounds are capable of rescuing P-glycoprotein (P-gp) mutants ∆Y490 and ∆Y1133 in a concentration-dependent manner. These trafficking deficient mutations are located at the transmission interface in pseudosymmetric position to each other. In addition, the ability of propafenone analogs to correct folding correlates with their ability to inhibit transport of model substrates. This finding indicates that folding correction and transport inhibition by propafenone analogs are brought about by binding to the active sites. Furthermore, this study demonstrates an asymmetry in folding correction with cis-flupentixol, which reflects the asymmetric binding properties of this modulator to P-gp. Our results suggest a mechanistic model for corrector action in a model ABC transporter based on insights into the molecular architecture of these transporters.

Proliferation of macrophages due to the inhibition of inducible nitric oxide synthesis by oxidized low-density lipoproteins.

Oxidized low-density lipoprotein (ox-LDL) is assumed to be a major causal agent in hypercholesteraemia-induced atherosclerosis. Because the proliferation of lipid-loaden macrophages within atherosclerotic lesions has been described, we investigated the dependence of macrophage proliferation on the inhibition of inducible nitric oxide synthase (iNOS) by hypochlorite oxidized LDL. Ox-LDL induces a dose dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-interferon stimulated mouse macrophages (J774.A1) with concomitant macrophage proliferation as assayed by cell counting, tritiated-thymidine incorporation and measurement of cell protein. Native LDL did not influence macrophage proliferation and inducible nitric oxide synthesis. iNOS protein and mRNA was reduced by HOCl-oxidized LDL (0-40 µg/ml) as revealed by immunoblotting and competitive semiquantitative PCR. Macrophage proliferation was increased by the addition of the iNOS inhibitor L-NAME. The addition of ox-LDL to L-NAME containing incubations induced no further statistically significant increase in cell number. Nitric oxide donors decreased ox-LDL induced macrophage proliferation and nitric oxide scavengers restored macrophage proliferation to the initial values achieved by ox-LDL. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with ox-LDL demonstrates that the proliferative actions of ox-LDL are associated with a decrease of NO-induced apoptosis. Our data show that inhibition of iNOS dependent nitric oxide production caused by hypochlorite oxidized LDL enhances macrophage proliferation. This might be a key event in the pathogenesis of atherosclerotic lesions.

A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.

Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar(+)-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.

Pore-exposed tyrosine residues of P-glycoprotein are important hydrogen-bonding partners for drugs.

The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.

A quantitative model of amphetamine action on the 5-HT transporter.

BACKGROUND AND PURPOSE: Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), noradrenaline (NET) and 5-HT (SERT); influx of amphetamine leads to efflux of substrates. Various models have been proposed to account for this amphetamine-induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine-induced conformation that is not likely in alternative access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis. EXPERIMENTAL APPROACH: Xenopus laevis oocytes and HEK293 cells expressing human (h) SERT were voltage-clamped and exposed to 5-HT, p-chloroamphetamine (pCA) or methylenedioxyamphetamine (MDMA). KEY RESULTS: In contrast to the currents induced by 5-HT, pCA-triggered currents through SERT decayed slowly in Xenopus laevis oocytes once the agonist was removed (consistent with the molecular stent hypothesis). However, when SERT was expressed in HEK293 cells, currents induced by 3 or 100 µM pCA decayed 10 or 100 times faster, respectively, after pCA removal. CONCLUSIONS AND IMPLICATIONS: This discrepancy in decay rates is inconsistent with the molecular stent hypothesis. In contrast, a multistate version of the alternative access model accounts for all the observations and reproduces the kinetic parameters extracted from the electrophysiological recordings. A crucial feature that explains the action of amphetamines is their lipophilic nature, which allows for rapid diffusion through the membrane.

Inhibition of NF-κB-dependent cytokine and inducible nitric oxide synthesis by the macrocyclic ellagitannin oenothein B in TLR-stimulated RAW 264.7 macrophages.

Immunomodulatory effects of oenothein B (1), a macrocyclic ellagitannin from various Onagraceae species, have been described previously. However, the mechanisms underlying the anti-inflammatory activity of 1 have not been fully clarified. The effects of 1 were investigated on inducible nitric oxide synthase, TLR-dependent and TLR-independent signal transduction cascades, and cytokine expression using murine macrophages (RAW 264.7). Compound 1 (10-60 µg/mL) reduced NO production, iNOS mRNA, and iNOS protein levels in a dose-dependent manner, without inhibition of iNOS enzymatic activity. It reduced the binding of the NF-κB p50 subunit to the biotinylated-consensus sequence and decreased nuclear p65 translocation. Gallic acid as a subunit of the macrocyclic ellagitannin 1 showed a far lower inhibitory activity. Nitric oxide production was reduced by 1 after stimulation using TLR2 (Pam2CSK4) and TLR4 (Kdo2) agonists, but this compound did not inhibit inducible nitric oxide synthesis after stimulation using interferon-gamma. IL-1beta, IL-6, and TNF-alpha mRNA synthesis was clearly reduced by the addition of 1. Oenothein B (1) inhibits iNOS after stimulation with LPS, TLR2, and TLR4 agonists via inhibition of TLR/NF-κB-dependent inducible nitric oxide and cytokine synthesis independent from IFN-gamma/JAK/STAT pathways. The full molecular structure of this macrocyclic ellagitannin seems to be required for its immunomodulatory actions.

An abundant, truncated human sulfonylurea receptor 1 splice variant has prodiabetic properties and impairs sulfonylurea action.

An alternatively spliced form of human sulfonylurea receptor (SUR) 1 mRNA lacking exon 2 (SUR1Δ2) has been identified. The omission of exon 2 caused a frame shift and an immediate stop codon in exon 3 leading to translation of a 5.6-kDa peptide that comprises the N-terminal extracellular domain and the first transmembrane helix of SUR1. Based on a weak first splice acceptor site in the human SUR1 gene (ABCC8), RT-PCR revealed a concurrent expression of SUR1Δ2 and SUR1. The SUR1Δ2/(SUR1 + SUR1Δ2) mRNA ratio differed between tissues, and was lowest in pancreas (46%), highest in heart (88%) and negatively correlated with alternative splice factor/splicing factor 2 (ASF/SF2) expression. In COS-7 cells triple transfected with SUR1Δ2/SUR1/Kir6.2, the SUR1Δ2 peptide co-immunoprecipitated with Kir6.2, thereby displacing two of four SUR1 subunits on the cell surface. The ATP sensitivity of these hybrid ATP-sensitive potassium channels (K(ATP)) channels was reduced by about sixfold, as shown with single-channel recordings. RINm5f rat insulinoma cells, which genuinely express SUR1 but not SUR1Δ2, exhibited a strongly increased K(ATP) channel current upon transfection with SUR1Δ2. This led to inhibition of glucose-induced depolarization, calcium flux, insulin release and glibenclamide action. A non-mutagenic SNP on nucleotide position 333 (Pro69Pro) added another exonic splicing enhancer sequence detected by ASF/SF2, reduced relative abundance of SUR1Δ2 and slightly protected from non-insulin dependent diabetes in homozygotic individuals. Thus, SUR1Δ2 represents an endogenous K(ATP)-channel modulator with prodiabetic properties in islet cells. Its predominance in heart may explain why high-affinity sulfonylurea receptors are not found in human cardiac tissue.

Glibenclamide reduces proinflammatory cytokines in an ex vivo model of human endotoxinaemia under hypoxaemic conditions.

AIMS: In vivo application of the K(ATP)-channel blocker glibenclamide can reverse endotoxin-induced hypotension, vascular hyporeactivity and shock in experimental animals. The hypothesis of the present study is, that the drug effects might not only be based on direct inhibition of K(ATP)-channels of vascular smooth muscle cells, but might also reflect reduction of shock-induced excess proinflammatory cytokines and procoagulatory molecules produced in the blood monocytes. MAIN METHODS: Human whole blood (normoxaemic or hypoxaemic) supplemented ex vivo with 100 ng/ml LPS was used to assess glibenclamide (3-100 µM) effects on IL-1 beta, IL-6, TNF-alpha, tissue factor, and plasminogen-activator-inhibitor-2 (PAI-2). Co-incubations with monocytes and erythrocytes and cytosolic calcium measurements were performed to reveal their purinergic intercellular interaction. KEY FINDINGS: In heparinized blood, glibenclamide reduced LPS-induced release of IL-1 beta and TNF-alpha, tissue factor and PAI-2 mRNA in a concentration-dependent manner. When samples were subjected to strong hypoxemia using 95% N(2)/5% CO(2), these parameters became even more sensitive to the drug. No drug effect was observable in citrated blood or in isolated monocytes. IL-1 beta mRNA inhibition by glibenclamide appeared to be dependent on P2X7-receptor activation of monocytes by ATP-releasing erythrocytes during hypoxia. Cytosolic calcium values as well as the duration of calcium transients elicited by P2X7-receptor stimulation in isolated monocytes were strongly increased during hypoxia, both of which could be abolished by glibenclamide. SIGNIFICANCE: We conclude that the anti-inflammatory effect of glibenclamide is mainly based on the reduction of calcium entry by drug-induced depolarization of hypoxic monocytes. Thus, glibenclamide possesses a potentially beneficial shock-specific anti-inflammatory action.

Pinacidil-primed ATP-sensitive potassium channels mediate feedback control of mechanical power output in isolated myocardium of rats and guinea pigs.

We tested the hypothesis, that ATP-sensitive potassium (K(ATP)) channels limit cardiac energy demand by a feedback control of mean power output at increased cardiac rates. We analysed the interrelationships between rising energy demand of adult rat and guinea pig left ventricular papillary muscle and down-regulatory electromechanical effects mediated by K(ATP) channels. Using the K(ATP)-opener pinacidil the stimulation frequency was increased stepwise and the mechanical parameters and action potentials were recorded. Power output was derived from force-length area or force-time integral calculations, respectively. Simultaneously oxygen availability in the preparations was estimated by flavoprotein fluorescence measurements. ADP/ATP ratios were determined by HPLC. We found highly linear relationships between isotonic power output and the effects of pinacidil on isotonic shortening in both rat (r(2)=0.993) and guinea pig muscles (r(2)=0.997). These effects were solely observed for the descending limb of shortening-frequency relationships. In addition, a highly linear correlation between total force-time integral-derived power and pinacidil effects on action potential duration (APD(50), r(2)=0.92) was revealed. Power output became constant and frequency-independent in the presence of pinacidil at higher frequencies. In contrast, the K(ATP)-blocker glibenclamide produced a lengthening of APD(50) and increased force transiently at higher power levels. Pinacidil prevented core hypoxia and a change in ADP/ATP ratio during high frequency stimulation. We conclude, that pinacidil-primed cardiac K(ATP) channels homeostatically control power output during periods of high energy demand. This effect is associated with a reduced development of hypoxic areas inside the heart muscle by adapting cardiac function to a limited energy supply.

Aqueous extracts of Cimicifuga racemosa and phenolcarboxylic constituents inhibit production of proinflammatory cytokines in LPS-stimulated human whole blood.

Cimicifuga racemosa (black cohosh) is commonly used in traditional medicines as treatment for menopausal symptoms and as an antiinflammatory remedy. To clarify the mechanism of action and active principle for the antiinflammatory action, the effects of aqueous C. racemosa root extracts (CRE) and its major constituents on the release of the proinflammatory cytokines IL-6, TNF-alpha, IFN-gamma, and the chemokine IL-8 were investigated in lipopolysaccharide (LPS)-stimulated whole blood of healthy volunteers. CRE (3 microg/microL and 6 microg/microL) reduced LPS-induced release of IL-6 and TNF-alpha in a concentration- and time-dependent manner and almost completely blocked release of IFN-gamma into the plasma supernatant. Except for IFN-gamma, these effects were attenuated at longer incubation periods. IL-8 secretion was stimulated by CRE. As shown by quantitative real-time RT-PCR, effects on cytokines were based on preceding changes in mRNA levels except for IL-8. According to their content in CRE, the phenolcarboxylic compounds caffeic acid, ferulic acid, and isoferulic acid, as well as the triterpene glycosides 23-epi-26-deoxyactein and cimigenol-3-O-xyloside, were tested at representative concentrations. Among these, isoferulic acid was the prominent active principle in CRE, responsible for the observed inhibition of IL-6, TNF-alpha, and IFN-gamma, but not for IL-8 stimulation. The effect of this compound may explain the antiinflammatory activities of CRE and its beneficial actions in rheumatism and other inflammatory diseases.

Inhibition of inducible nitric oxide synthesis by Cimicifuga racemosa (Actaea racemosa, black cohosh) extracts in LPS-stimulated RAW 264.7 macrophages.

OBJECTIVES: Cimicifuga racemosa (Actaea racemosa, black cohosh) is used as an anti-inflammatory, antipyretic and analgesic remedy in traditional medicines. The present study focuses on the effects of C. racemosa root extracts on inducible nitric oxide synthase (iNOS) in lipopolysaccharide-stimulated murine macrophages (RAW 264.7). METHODS: C. racemosa rhizome and phosphate-buffered saline extracts were analysed for phenolcarboxylic acids and triterpene glycosides using an HPLC photodiode array/evaporative light-scattering detector system. iNOS was characterised by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), nitric oxide production (nitrite levels) and nuclear translocation of nuclear factor-kappaB (p65 subunit) protein. KEY FINDINGS: Incubation of lipopolysaccharide-stimulated macrophages with aqueous C. racemosa extracts (0-6 mg/ml) inhibited nitrite accumulation in a concentration-dependent manner. C. racemosa extracts also reduced iNOS protein expression and iNOS mRNA levels in a dose-dependent manner. C. racemosa extracts did not significantly inhibit iNOS activity and did not affect nuclear translocation of nuclear factor-kappaB (p65 subunit) protein. Incubation with the extract was associated with a concentration-dependent reduction of interferon beta and interferon regulatory factor 1 mRNA. Among the triterpene glycosides, 23-epi-26-deoxyactein was identified as an active principle in C. racemosa extracts. CONCLUSIONS: Extracts from the roots of C. racemosa inhibit nitric oxide production by reducing iNOS expression without affecting activity of the enzyme. This might contribute to the anti-inflammatory activities of C. racemosa.

Clock genes display rhythmic expression in human hearts.

Thus far, clock genes in the heart have been described only in rodents, and alterations of these genes have been associated with various myocardial malfunctions. In this study, we analyzed the expression of clock genes in human hearts. Left papillary muscles of 16 patients with coronary heart disease, 39 subjects with cardiomyopathy, and 9 healthy donors (52 males and 12 females, mean age 55.7+/-11.2; 16-70 yrs) were obtained during orthotopic heart transplantation. We assessed the mRNA levels of PER1, PER2, BMAL1, and CRY1 by real time PCR and analyzed their rhythmic expression by sliding means and Cosinor functions. Furthermore, we sought for differences between the three groups (by ANOVAs) for both the total 24 h period and separate time bins. All four clock genes were expressed in human hearts. The acrophases (circadian rhythm peak time) of the PER mRNAs occurred in the morning (PER1: 07:44 h [peak level 187% higher than trough, p = .008]; PER2: 09:42 h [peak 254% higher than trough, p < .0001], and BMAL1 mRNA in the evening at 21:44 h [peak 438% higher than trough; p < .0001]. No differences were found in the rhythmic patterns between the three groups. No circadian rhythm was detected in CRY1 mRNA in any group. PER1, PER2, and BMAL1 mRNAs revealed clear circadian rhythms in the human heart, with their staging being in antiphase to those in rodents. The circadian amplitudes of the mRNA clock gene levels in heart tissue are more distinct than in any other human tissue so far investigated. The acrophase of the myocardial PER mRNAs and the trough of the myocardial BMAL1 coincide to the time of day of most frequent myocardial incidents.

Oxidation by hypochlorite converts protective HDL into a potent platelet agonist.

High density lipoproteins (HDL) represent a protective factor of central importance that counteracts the development of cardiovascular disease, in part by normalizing platelet (hyper)reactivity. As HDL represent an efficient scavenger of the naturally occurring oxidant hypochlorite, this work was intended to investigate the influence of hypochlorite-oxidized HDL on platelet function. Addition of hypochlorite-oxidized HDL to human platelets results in an immediate and transient raise in intracellular calcium, surface expression of P-selectin and platelet aggregation. The observed effects are dose dependent and can be blocked by an antibody directed against the lipoprotein-binding domain of platelet thrombospondin- and scavenger receptor CD36.