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.

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line.

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.

Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages.

This study was designed to compare the effects of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and native PAPC on the inducible nitric oxide synthase (iNOS) in the macrophage cell line RAW 264.7. Macrophages stimulated by bacterial lipopolysaccharide (1 microg/ml) were incubated with increasing amounts of native or oxidized PAPC (oxPAPC, 10-20 microg/ml). Cells incubated with oxPAPC showed a dose-dependent inhibition of inducible nitric oxide synthesis, as well as reduced iNOS protein expression and mRNA levels. Additionally, chromatin immunoprecipitation assay revealed that oxPAPC reduced the interaction of the active NF-kappaB subunit p65 with the iNOS promoter region when compared to native PAPC.

A subset of highly effective propafenone-type multidrug resistance modulators lacks effects on cardiac action potential and mechanical twitch parameters of rat papillary muscles.

In this study, we tested a series of 12 previously identified, highly effective propafenone-type multidrug resistance (MDR) modulators for their possible undesirable effects on cardiac tissue. We used rat papillary muscle preparations and quantitatively determined the potency of these substances to block action potential (AP) upstroke velocity (Vmax) and to prolong APD50. Simultaneously, the effects on isometric twitch parameters were evaluated. Concentration-response curves were obtained for all parameters. Within a subset of the compounds, we found a significant rank correlation (r' = 0.87; p < 0.05) between potencies to block Vmax (kiVmax) and to inhibit daunomycin efflux in MDR cells (IC50). Surprisingly, the most lipophilic compounds with additional aromatic side chains completely lacked effects on AP and mechanical twitch parameters, although they are the most effective MDR modulators. Additional structural modifications such as fluoride substitution of the aromatic ring, introduction of arylpiperazine or piperidine side chains, as well as modifying the hydrogen bond acceptor strength of the carbonyl group did not reestablish cardiac side effects. In contrast, when these substances were truncated at the phenylpropiophenone moiety of the propafenone core structure, cardiac effects reoccurred. We conclude that aromatic substituents in the vicinity of the nitrogen atom prevent interaction with ion channels, likely due to steric hindrance, and are thus a prerequisite for eliminating unwanted cardiac effects.