ATP inhibition of a mouse brain large-conductance K+ (mslo) channel variant by a mechanism independent of protein phosphorylation.
(9/6000)
1. We investigated the effect of ATP in the regulation of two closely related cloned mouse brain large conductance calcium- and voltage-activated potassium (BK) channel alpha-subunit variants, expressed in human embryonic kidney (HEK 293) cells, using the excised inside-out configuration of the patch-clamp technique. 2. The mB2 BK channel alpha-subunit variant expressed alone was potently inhibited by application of ATP to the intracellular surface of the patch with an IC50 of 30 microM. The effect of ATP was largely independent of protein phosphorylation events as the effect of ATP was mimicked by the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) and the inhibitory effect of ATPgammaS was reversible. 3. In contrast, under identical conditions, direct nucleotide inhibition was not observed in the closely related mouse brain BK channel alpha-subunit variant mbr5. Furthermore, direct nucleotide regulation was not observed when mB2 was functionally coupled to regulatory beta-subunits. 4. These data suggest that the mB2 alpha-subunit splice variant could provide a dynamic link between cellular metabolism and cell excitability. (+info)
Vitronectin inhibits the thrombotic response to arterial injury in mice.
(10/6000)
Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1) and integrins and may play an important role in the vascular response to injury by regulating fibrinolysis and cell migration. However, the role of VN in the earliest response to vascular injury, thrombosis, is not well characterized. The purpose of this study was to test the hypothesis that variation in vitronectin expression alters the thrombotic response to arterial injury in mice. Ferric chloride (FeCl3) injury was used to induce platelet-rich thrombi in mouse carotid arteries. Wild-type (VN +/+, n = 14) and VN-deficient (VN -/-, n = 15) mice, matched for age and gender, were studied. Time to occlusion after FeCl3 injury was determined by application of a Doppler flowprobe to the carotid artery. Occlusion times of VN -/- mice were significantly shorter than those of VN +/+ mice (6.0 +/- 1.2 minutes v 17.8 +/- 2.3 minutes, respectively, P < .001). Histologic analysis of injured arterial segments showed that thrombi from VN +/+ and VN -/- mice consisted of dense platelet aggregates. In vitro studies of murine VN +/+ and VN -/- platelets showed no significant differences in ADP-induced aggregation, but a trend towards increased thrombin-induced aggregation in VN -/- platelets. Purified, denatured VN inhibited thrombin-induced platelet aggregation, whereas native VN did not. Thrombin times of plasma from VN -/- mice (20.5 +/- 2.1 seconds, n = 4) were significantly shorter than those of VN +/+ mice (34.2 +/- 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN -/- plasma prolonged the thrombin time into the normal range, suggesting that VN inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an explanation for the accelerated thrombosis observed in VN -/- mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity. (+info)
Occurrence of prostasome-like membrane vesicles in equine seminal plasma.
(11/6000)
Equine seminal plasma was shown to contain membrane vesicles that are similar to the well characterized prostasomes in human seminal plasma. Determination of nucleoside and nucleotide concentrations of these particles have shown that ATP, ADP and adenosine are the main components of the nucleotidic pool. 5' nucleotidase, endopeptidase and dipeptidyl peptidase i.v. activities have been found on the surface of the particles. The interaction between these prostasome-like vesicles and spermatozoa was demonstrated by electron micrograph scans which revealed the steps of a fusion-like process leading to mixing of the membranes. In addition, endopeptidase activity, a marker enzyme of these seminal vesicles that is normally absent from equine spermatozoa, was shown to be acquired by these cells after interaction with the vesicles. The addition of these vesicles to equine spermatozoa resulted in the modification of adenylate catabolism. Therefore, a role in stabilizing the energy charge of the spermatozoa thus allowing longer viability is proposed for these organelles. (+info)
Self-regulated polymerization of the actin-related protein Arp1.
(12/6000)
The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic dynein motor machinery [1] [2] [3]. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to conventional actin [4] [5] [6] and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize. In vivo, all cytosolic Arp1 sediments at 20S [7] suggesting that it assembles into oligomers, most likely dynactin - a multiprotein complex known to contain eight or nine Arp1 monomers in a 37 nm filament [8]. The uniform length of Arp1 polymers suggests a novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping protein at one end and a heterotetrameric protein complex containing the p62 subunit (D.M. Eckley, S.R. Gill, J.B.B., J.E. Heuser, T.A.S., unpublished observations) at the other [8]. In the present study, we analyzed the behavior of highly purified, native Arp1. Arp1 was found to polymerize rapidly into short filaments that were similar, but not identical, in length to those in dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional actin filaments. (+info)
Regulation of mitochondrial KATP channel by redox agents.
(13/6000)
The ATP-dependent K+ channel (KATP) was purified from the inner mitochondrial membrane and reconstituted into lipid bilayer membranes. KATP activity was inhibited by high concentrations of ATP and ADP, but activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethylbenzoate (DEB) acted as a KATP opener: at micromolar concentrations, it reversed inhibition by ATP and ADP and it also prevented KATP rundown. Pelargonidine, extracted from flowers of Pelargonium, reduced spontaneous activity of KATP channels and diminished their potentiation by DEB. Their opposite action on KATP corresponded with their opposite redox properties in reactions with free radicals: DEB behaved as an electron donor, whereas pelargonidine acted as an electron acceptor. We hypothesize that thiol groups on mitoKATP are targets for redox-active ligans. (+info)
Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro.
(14/6000)
1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP. (+info)
Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange.
(15/6000)
Growth factor withdrawal is associated with a metabolic arrest that can result in apoptosis. Cell death is preceded by loss of outer mitochondrial membrane integrity and cytochrome c release. These mitochondrial events appear to follow a relative increase in mitochondrial membrane potential. This change in membrane potential results from the failure of the adenine nucleotide translocator (ANT)/voltage-dependent anion channel (VDAC) complex to maintain ATP/ADP exchange. Bcl-xL expression allows growth factor-deprived cells to maintain sufficient mitochondrial ATP/ADP exchange to sustain coupled respiration. These data demonstrate that mitochondrial adenylate transport is under active regulation. Efficient exchange of ADP for ATP is promoted by Bcl-xL expression permitting oxidative phosphorylation to be regulated by cellular ATP/ADP levels and allowing mitochondria to adapt to changes in metabolic demand. (+info)
Inhibition of myosin ATPase by metal fluoride complexes.
(16/6000)
Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics. (+info)