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.

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.

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.

Short-term cognitive learning outcomes in team-based learning: is the permanent team important?

Assigning students to work in permanent teams is a design principle in Team-based learning (TBL). It has been assumed that a stable team composition supports the emergence of collaborative problem-solving and learning: when students became more familiar with each other, they shared more information and resolved discrepancies together, which in turn stimulated knowledge acquisition and comprehension. However, this assumption had not been probed by a randomized controlled trial with performance assessment as an outcome. In an online course for second term medical students, 50% of the students were reassigned to new teams for each of the 24 problems to be solved during four classes, thus precluding familiarity. The learning outcome was assessed shortly after the third of four classes by a domain knowledge test. Whether TBL teams were permanent or temporary did not affect the score of a domain knowledge test. As expected, participation in online TBL improved the domain knowledge test results. Overall, the permanent team seems to be less important for cognitive learning outcomes than previously assumed, but this may depend on the specific educational setting. However, team familiarity may still be important for team decision-making. As clinical reasoning in the medical workplace often involves collaborating in changing teams, future research on TBL should focus on how to utilize this format to prepare medical students for decision-making and optimal learning outcomes under these conditions.

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.

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.

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.

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.

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.

"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.

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.

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.

Intestinal transport and metabolism of acrylamide.

There has been an intensive debate whether dietary exposure to acrylamide could increase the risk of human cancer since the first description of the presence of acrylamide in food in 2002. As the intestinal mechanisms of acrylamide absorption are poorly investigated we studied the transport of acrylamide in differentiated Caco-2 cells and its effects on biotransformation enzymes (CYP2E1 and glutathione S-transferase) and glutathione levels. We found that the apparent permeability of [1-(14)C] acrylamide from the basal to the apical compartment was approximately 20% higher compared to that in the opposite direction. No differences were detected for apical-basal transport against a basal gradient. Transport rates from the apical to the basal chamber at 4 degrees C were about 50% lower than at 37 degrees C. Concentration dependent transport from apical to basal was linear. Predominantly, basal to apical transport was decreased when energy metabolism of the cells was inhibited by application of sodium azide and 2-deoxy-d-glucose. Finally, more acrylamide was transported at luminal pH 6 compared to pH 7.4 from basal to the apical direction. Increasing levels of acrylamide showed no effects on the activity of glutathione S-transferase but resulted in a depletion of total glutathione concentrations. In conclusion transport of acrylamide in the intestine is mediated primarily by passive processes possibly combined with a modest energy- and pH-dependent active secretory component. Depletion of cellular glutathione levels may be one potential mechanism for acrylamide (geno)toxicity.

ATP-sensitive potassium channels expressed by human monocytes play a role in stasis-induced thrombogenesis via tissue factor pathway.

Blood stasis is one of the key risk factors in deep vein thrombosis. Localized blood oxygen and glucose depletion are main characteristics observed during stasis. However, the causal chain leading to clot formation is still obscure. According to our hypothesis, energy depletion causes opening of K(ATP) channels present on monocytes, facilitating influx of calcium and triggering tissue factor-(TF)-dependent procoagulatory activity and eventually clot formation. Using Reverse-Transcript-PCR (RT-PCR) in magnetically enriched human monocytes, mRNA transcription of the K(ATP)-channel subunits Kir6.1 and Kir6.2 could be confirmed. Membrane potential and cytosolic calcium were recorded by time-resolved flow cytometry. The specific K(ATP)-channel opener pinacidil caused a glibenclamide-sensitive hyperpolarization of monocytes and a prolongation of cytosolic calcium transients triggered by purinergic stimulation. TF-initiated whole blood clotting time (TiFaCT) was accelerated comparing 2 and 8 h of simulated in vitro blood stasis using blood of male healthy volunteers. Both with and without activation of the monocytes with 100 ng/ml LPS, the K(ATP)-channel blocker glibenclamide resulted in a significantly (p<0.001) prolonged clotting time after 8 h of stasis compared to vehicle control and LPS, respectively. In the course of stasis, flow cytometry showed an increase in monocytes expressing TF (0.1% and 1.3% after 2 and 8 h, respectively). LPS (100 ng/ml) increased the amount of TF expression significantly to 36%, whereas 30 microM glibenclamide partly reversed this increase down to 24%. Phosphatidylserine-exposure (PSE) on monocytes increased strongly during stasis by 11.2 times, a process which glibenclamide attenuated by 23%. LPS increased PSE further by 65%, which glibenclamide reduced by 50%. In conclusion, presence of integral subunits of K(ATP)-channels is demonstrated in human monocytes. These channels are able to enhance Ca(2+)-dependent intracellular signalling and can increase TF-activity and phosphatidylserine exposure thereby accelerating clot formation during stasis by monocytes.

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.

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.

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 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.