High concentration of injected titanium dioxide in abdominal lymph nodes.

In the rat the greatest accumulation, in any anatomical structure, of titanium dioxide following its intravenous injection was found in two small clusters of lymph nodes in upper abdomen behind the peritoneum. These are the lymph nodes of the liver. This extraordinary quantitative characteristic of the abdominal clusters is attributed to their topography which results in progressive filtration of particulate matter from hepatic lymph.

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis.

BACKGROUND: Despite limiting elastic recoil and late vascular remodeling after angioplasty, coronary stents remain vulnerable to restenosis, caused primarily by neointimal hyperplasia. Paclitaxel, a microtubule-stabilizing drug, has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal hyperplasia. We tested whether paclitaxel-coated coronary stents are effective at preventing neointimal proliferation in a porcine model of restenosis. METHODS AND RESULTS: Palmaz-Schatz stents were dip-coated with paclitaxel (0, 0.2, 15, or 187 microgram/stent) by immersion in ethanolic paclitaxel and evaporation of the solvent. Stents were deployed with mild oversizing in the left anterior descending coronary artery (LAD) of 41 minipigs. The treatment effect was assessed 4 weeks after stent implantation. The angiographic late loss index (mean luminal diameter) decreased with increasing paclitaxel dose (P<0.0028 by ANOVA), declining by 84.3% (from 0.352 to 0.055, P<0.05) at the highest level tested (187 microgram/stent versus control). Accompanying this change, the neointimal area decreased (by 39.5%, high-dose versus control; P<0.05) with increasing dose (P<0.040 by ANOVA), whereas the luminal area increased (by 90.4%, high-dose versus control; P<0.05) with escalating dose (P<0.0004 by ANOVA). Inflammatory cells were seen infrequently, and there were no cases of aneurysm or thrombosis. CONCLUSIONS: Paclitaxel-coated coronary stents produced a significant dose-dependent inhibition of neointimal hyperplasia and luminal encroachment in the pig LAD 28 days after implantation; later effects require further study. These results demonstrate the potential therapeutic benefit of paclitaxel-coated coronary stents in the prevention and treatment of human coronary restenosis.

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ.

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-->E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s-1.

Transient state kinetic effects of calcium ion on sarcoplasmic reticulum adenosine triphosphatase.

A rapid mixing technique was used to investigate the effects of Ca2+ ion on the kinetics of ATP hydrolysis by sarcoplasmic reticulum vesicles. "Basic" ATPase measured in the absence of Ca2+ showed an initial burst of inorganic phosphate production. Similarities in the transient state kinetic properties of basic and "extra" or Ca2+-dependent ATPase suggest that the two activities represent a single enzyme species. At low concentrations of Ca2+ (less than 10(-6) M) the time course of the partial reactions of extra ATPase appeared to fit a simple scheme in which the acid-stable, phosphorylated enzyme (E approximately P) breaks down directly to inorganic phosphate and free enzyme. A similar mechanism seemed to apply to moderate levels of ATP and high external concentrations of Ca2+ known to inhibit transport activity. In the intermediate range of Ca2+ concentrations inorganic phosphate production was resolved into two phases consisting of a fast initial rate (burst) and slow steady state. Acid-stable phosphorylated protein showed a transient decay which coincided with the appearance of the burst. This behavior is consistent with a scheme in which E approximately P breaks down to an acid-labile or noncovalent intermediate state (E-P). A slow secondary increase in phosphorylation followed the transient decay in E approximately P. This late phase of protein labeling was eliminated following pretreatment with Triton X-100, sodium oxalate, or diethyl ether which decrease or prevent the formation of a transport gradient. An analysis of the dependence of the steady state level of phosphorylation and rate of inorganic phosphate production on Ca2+ concentration indicated that the phosphorylation mechanism involves interaction of two Ca2+ ions with the enzymatic carrier. The pathway by which E approximately P breaks down, i.e. whether it goes to E + Pi or E-P, may depend on the extent to which these sites are occupied by Ca2+. The transport of Ca2+ is discussed in terms of a flip-flop mechanism in which E approximately P and E-P represent high and low affinity Ca2+ binding states occurring in separate halves of an enzyme dimer.

Transient-state kinetics of the ADP-insensitive phosphoenzyme in sarcoplasmic reticulum: implications for transient-state calcium translocation.

The kinetics of formation of the ADP-sensitive (EP) and ADP-insensitive (E*P) phosphoenzyme intermediates of the CaATPase in sarcoplasmic reticulum (SR) were investigated by means of the quenched-flow technique. At 21 degrees C, addition of saturating ADP to SR vesicles phosphorylated for 116 ms with 10 microM ATP gave a triphasic pattern of dephosphorylation in which EP and E*P accounted for 33% and 60% of the total phosphoenzyme, respectively. Inorganic phosphate (Pi) release was less than stoichiometric with respect to E*P decay and was not increased by preincubation with Ca2+ ionophore. The fraction of E*P present after only 6 ms of phosphoenzyme formation was similar to that at 116 ms, indicating that isomerization of EP to E*P occurs very rapidly. Comparison of the time course of E*P formation with intravesicular Ca2+ accumulation measured by quenching with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid + ADP revealed that Ca2+ release on the inside of the vesicle was delayed with respect to E*P formation. Since Ca2+ should dissociate rapidly dissociation from the low-affinity transport sites, these results suggest that Ca2+ remains "occluded" after phosphoenzyme isomerization and that a subsequent slow transition controls the rate of Ca2+ release at the intravesicular membrane surface. Analysis of the forward and reverse rate constants for the EP to E*P transition gave an expected steady-state distribution of phosphoenzymes strongly favoring the ADP-insensitive form. In contrast, the observed ratio of EP to E*P was about 1:2. To account for this discrepancy, a mechanism is proposed in which stabilization of the ADP-sensitive phosphoenzyme is brought about by a conformational interaction between adjacent subunits in a dimer.

The partial reactions of the Na(+)- and Na(+) + K(+)-activated adenosine triphosphatases.

Kinetic investigations carried out in a number of laboratories have accumulated evidence favoring modification of the Albers-Post mechanism. The results of the rapid mixing studies involving the eel enzyme indicate that the complex kinetic behavior is confined to the Na(+)-activated reaction pathway (Na-ATPase). The main conceptual problem in interpreting the dephosphorylation experiments involves the intermediate component, which turns over too slowly to account for the overall velocity of Pi production in the presence of Na+ and K+ and exhibits behavior compatible with an ADP-insensitive phosphoenzyme. Attempts to simulate the dephosphorylation reaction using schemes in which the intermediate component represents a precursor to the K(+)-sensitive phosphoenzyme, E2P, were unsuccessful in reproducing both the pre-steady-state and steady-state time dependence. When Na+ and K+ were both present during phosphorylation, the time course of dephosphorylation showed no evidence of an intermediate decay component, implying that K+ either prevents its formation or accelerates its turnover. Complex kinetic behavior was also observed in the phosphorylation reaction under conditions where the reaction was initiated by the simultaneous addition of ATP, Na+, and Mg2+. Preincubation with Na+ eliminated the biexponential pattern of accumulation so that only the fast phase was seen. The proportion of EP in the slow phase of phosphorylation was approximately equal to the fraction of EP in the intermediate phase of dephosphorylation (roughly one-third of the sites), suggesting that the two may be related to the same catalytic activity. To try to explain these observations using recent modifications to the Albers-Post mechanism is difficult without invoking additional complex effects of the transported ions. We propose that a series model for phosphorylation is inadequate and that further modification of the mechanism is required. The alternative to a consecutive mechanism is a parallel pathway scheme: [sequence: see text] In this model the enzyme exists in two distinct forms which are distributed in the upper and lower pathways in a ratio of 2:1. In the lower pathway the rates of phosphorylation and E2P hydrolysis are controlled by the kinetics of ligand binding because of a structural constraint (ion channel?) imposed by the transport protein. When phosphorylation is carried out in the presence of Na+ alone, E2P and E2P' accumulates rapidly and give rise to the fast and intermediate components of dephosphorylation, respectively. Preincubation with Na+ and K+ eliminates the functional differences between these pathways by removing the kinetic dependence of ligand binding, resulting in behavior that conforms to the Albers-Post mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Time-resolved charge translocation by the Ca-ATPase from sarcoplasmic reticulum after an ATP concentration jump.

Time-resolved measurements of currents generated by Ca-ATPase from fragmented sarcoplasmic reticulum (SR) are described. SR vesicles spontaneously adsorb to a black lipid membrane acting as a capacitive electrode. Charge translocation by the enzyme is initiated by an ATP concentration jump performed by the light-induced conversion of an inactive precursor (caged ATP) to ATP with a time constant of 2.0 ms at pH 6.2 and 24 degrees C. The shape of the current signal is triphasic, an initial current flow into the vesicle lumen is followed by an outward current and a second slow inward current. The time course of the current signal can be described by five relaxation rate constants, lambda1 to lambda5 plus a fixed delay D approximately 1-3 ms. The electrical signal shows that 1) the reaction cycle of the Ca-ATPase contains two electrogenic steps; 2) positive charge is moved toward the luminal side in the first rapid step and toward the cytoplasmic side in the second slow step; 3) at least one electroneutral reaction precedes the electrogenic steps. Relaxation rate constant lambda3 reflects ATP binding, with lambda(3,max) approximately 100 s(-1). This step is electroneutral. Comparison with the kinetics of the reaction cycle shows that the first electrogenic step (inward current) occurs before the decay of E2P. Candidates are the formation of phosphoenzyme from E1ATP (lambda2 approximately 200 s[-1]) and the E1P --> E2P transition (D approximately 1 ms or lambda1 approximately 300 s[-1]). The second electrogenic transition (outward current) follows the formation of E2P (lambda4 approximately 3 s[-1]) and is tentatively assigned to H+ countertransport after the dissociation of Ca2+. Quenched flow experiments performed under the conditions of the electrical measurements 1) demonstrate competition by caged ATP for ATP-dependent phosphoenzyme formation and 2) yield a rate constant for phosphoenzyme formation of 200 s(-1). These results indicate that ATP and caged ATP compete for the substrate binding site, as suggested by the ATP dependence of lambda3 and favor correlation of lambda2 with phosphoenzyme formation.

Transient state kinetic evidence for an oligomer in the mechanism of Na+-H+ exchange.

Pre-steady-state kinetic measurements of 22Na+ uptake by the amiloride-sensitive Na+-H+ exchanger in renal brush border membrane vesicles (BBMV) were performed at 0 degrees C to characterize the intermediate reactions of the exchange cycle. At 1 mM Na+, the initial time course of Na+ uptake was resolved into three separate components: (i) a lag phase, (ii) an exponential or "burst" phase, and (iii) a constant velocity or steady-state phase. Pulse-chase experiments using partially loaded BBMV showed no evidence for 22Na+ back-flux, suggesting that the decline in the rate of Na+ uptake rate following the burst represents completion of the first turnover of the exchanger. Gramicidin completely abolished Na+ uptake, indicating that the burst phase results from the translocation of Na+ rather than from residual Na+ binding to external sites. Raising the [Na+] from 1 to 10 mM at constant pH (internal pH 5.7; external pH 7.7) produced a sigmoidal increase in the amplitude of the burst phase without affecting the lag duration or the apparent burst rate. In contrast, Na+ uptake in the steady state obeyed Michaelis-Menten kinetics. These results suggest that a minimum of two Na+ transport sites must be occupied to activate Na+ uptake in the pre-steady state. The transition to Michaelis-Menten kinetics in the steady state can be explained by a "flip-flop" or alternating site mechanism in which the functional transport unit is an oligomer and only one promoter per cycle is allowed to form a translocation complex with Na+ after the first turnover.

Initial products of photophosphorylation with AMP and [32P]Pi.

The role of AMP in photophosphorylation was studied using rapid mixing acid quench techniques. Fragmented spinach chloroplast membranes or subchloroplast particles were illuminated and rapidly mixed with [32P]orthophosphate and AMP at pH 7 for 10 ms to 60 s after which time perchloric acid was added to quench the reaction. ATP was found to be the primary and predominant nucleotide labeled. It was found that after illumination, an adenylate kinase-like activity carried out an AMP-dependent conversion of labeled ATP to labeled ADP which was inhibited by the presence of ADP. This reaction was characterized as being similar to chloroplast adenylate kinase in Mg2+ dependency and in sensitivity to phlorizin and tentoxin and distinct from chloroplast coupling factor 1. The small amounts of adenylate kinase activity present in fragmented well washed chloroplast membranes were found to be sufficient to carry out this rapid reaction. These results necessitated a reinterpretation of the earlier findings of Tiefert and Moudrianakis (Tiefert, M.A., and Moudrianakis, E.N. (1979) J. Biol. Chem. 254, 9500-9508) and no longer support the role of ADP as a phosphorylated intermediate in ATP synthesis.

Effects of oligomycin on the partial reactions of the sodium plus potassium-stimulated adenosine triphosphatase.

The effects on phosphoenzyme (E-P) formation of ligands which activate Electrophorus (Na,K)-ATPase were investigated in the presence of oligomycin. When the enzyme was allowed to bind oligomycin in the presence of NaCl and MgCl2, subsequent addition of ATP plus KCl produced a monoexponential time course of E-P formation with a rate of 56 s-1, similar to the rate obtained in the uninhibited enzyme phosphorylated by ATP in the absence of KCl. Pi liberation under these conditions was slow and showed no initial burst phase, consistent with the inhibitory effect oligomycin has on the E1-P to E2-P conformational transition. Addition to KCl to a preincubation medium containing oligomycin, NaCl, and MgCl2 had no further effect on E-P formation. However, equilibration with oligomycin, KCl, and MgCl2 prior to the addition of NaCl plus ATP gave a much slower rate of E-P formation (5 s-1) and resulted in an initial rapid release of Pi similar to that found in the uninhibited enzyme. The slow increase in E-P level observed after incubation with oligomycin, KCl, and MgCl2 may be due to secondary formation of an inhibition complex following rapid binding of oligomycin. In contrast to the monophasic behavior which resulted from pre-exposure to NaCl or KCl, preincubation with oligomycin in the presence of MgCl2 plus Tris or Tris alone gave a biphasic pattern of E-P formation in which about 50% of the intermediate accumulated at a rate of 56 s-1 and the remainder at a rate of 5 s-1. In addition, the Pi burst amplitude was reduced, indicating partial inhibition of the enzyme. These results suggest that in the absence of Na+ and K+ only half of the enzyme is inhibited by oligomycin while the remainder undergoes inhibition subsequent to initiation of phosphorylation. Since the oligomycin concentration was saturating, the partial inhibition reflected in the biphasic pattern of E-P formation may be due to half-of-the-sites reactivity in which only half of the subunits bind oligomycin in the absence of monovalent cations.

Inhibition by vanadate of the reactions catalyzed by the (Na+ + K+)-stimulated ATPase. A transient state kinetic characterization.

The interaction of vanadate with the (Na+ + K+)-stimulated ATPase from electric organ was investigated using the acid quench-flow technique. At 21 degrees C, incubation of the enzyme with 1.3 to 1.6 muM vanadate in the presence of 75 mM Na+ and 25 mM K+ strongly inhibits phosphorylation by ATP. Enzyme activity remaining under these conditions shows no change in the apparent rates of phosphorylation or dephosphorylation, although effects were noted which suggest that vanadate increases the reverse rate of dephosphorylation. Ten micromolar vanadate, sufficient to inhibit the (Na+ + K+)-stimulated ATPase by more than 98%, has no effect on phosphorylation in the presence of Na+ alone. Phosphoenzyme formed in the presence of Na+ and K+ consists of rapidly and slowly decaying components which differ in sensitivity to vanadate. Up to 2 muM vanadate suppresses predominantly the rapidly decaying phosphoenzyme, while at higher concentrations vanadate inhibits both the rate and level of formation of the slowly decaying phosphoenzyme. These results indicate that vanadate is a useful reagent for distinguishing between these two phosphorylation reactions.

Template specific inhibitors of E. coli RNA polymerase.

Electroneutral analogs of polynucleotides, poly-9-vinyladenine and poly-1-vinyluracil inhibit E. coli RNA polymerase in all combinations where the single stranded polynucleotides are used as templates and the vinyl analogs are complementary to them. As templates both the ribo- and deoxyribopolynucleotides were tested; variation of the template concentration in the presence of vinyl analogs produced a competitive pattern of inhibition. The electroneutral analogs do not inhibit the enzyme activity when non-complementary single stranded polynucleotides or double stranded polynucleotides are used as templates; the latter fully supports transcription even when one of the strands is complementary to the analog.

Potassium-induced changes in phosphorylation and dephosphorylation of (Na+ + K+)-ATPase observed in the transient state.

Effects of K+ and Na+ on the transient state kinetics of (Na+ + K+)-ATPase from Electrophorus electricus were examined. Exposure of the enzyme to K+ for brief intervals prior to the addition of ATP and Na+ converts the enzyme to a form (E2 . K) which is transiently less reactive than when all three ligands are added simultaneously. Enzyme is reconverted to the rapidly reacting (E1) form if Na+ is added prior to ATP. Exposure of the ATPase to K+ without Na+ for 1 to 2 h partially restores the initial phosphate (Pi) burst but greatly depresses the amount of phosphoprotein intermediate (E-P) observed. Experiments in the presence of valinomycin suggest that much of this depression in E-P is related to the presence of sealed vesicles with K+ sites sequestered in the interior. Although the results are largely consistent with a simple model in which ATP hydrolysis occurs only through the phosphoenzyme intermediate, the partial restoration of the Pi burst following long term exposure to K+ appears to be attributable to a slow change which may allow some ATP hydrolysis to occur within formation of a phosphoenzyme intermediate.

Conformational transitions of the sarcoplasmic reticulum Ca-ATPase studied by time-resolved EPR and quenched-flow kinetics.

We have used time-resolved electron paramagnetic resonance (EPR) and quenched-flow kinetics in order to investigate the dynamics of Ca-ATPase conformational changes involved in Ca2+ pumping in sarcoplasmic reticulum (SR) membranes at 2 degrees C. The Ca-ATPase was selectively labeled with an iodoacetamide spin label (IASL), which yields EPR spectra sensitive to enzyme conformational changes during ATP induced enzymatic cycling. The addition of ATP, AMPPCP, CrATP, or ADP decreased the rotational mobility of a fraction of the probes, indicating a distinct protein conformational state corresponding to this probe population, while Pi under conditions producing "backdoor" phosphorylation produced no spectral change. Transient changes in the amplitude of the restricted component associated with the pre-steady state of Ca2+ pumping were detected with 10 ms time resolution after an [ATP] jump produced by laser flash photolysis of caged ATP in the EPR sample. The laser energy was adjusted to generate 100 microM ATP from 1 mM caged ATP. At 0.1 M KCl, the EPR transient consisted of a brief initial lag phase, a monoexponential phase with a rate of 20 s-1, and a decay back to the initial intensity after the ATP had been consumed. Raising [KCl] from 0.1 to 0.4 M slowed the rate of the exponential phase from 20 to 6 s-1. Lowering the pH from 7 to 6, which increased the rate of caged ATP photolysis, eliminated the lag but did not change the apparent rate of the EPR signal rise. Parallel acid quenched-flow experiments conducted at 0.1 M KCl and 100 microM ATP produced fast (50-58 s-1) and slow (20 s-1) phases of phosphoenzyme formation. Increasing [KCl] from 0.1 to 0.4 M decreased the rate of the slow phase of phosphorylation from 20 to 5 s-1, without affecting the fast phase. The close correlation between the slow phase of phosphorylation and the exponential phase of the EPR signal suggests that the spin probe monitors a conformational event associated with phosphoenzyme formation in a population of catalytic sites with delayed kinetics. We propose that this constraint is imposed by conformational coupling between the catalytic subunits in a Ca-ATPase oligomer and that, consequently, the EPR signal reflects changes in quaternary protein structure as well as changes in secondary and tertiary structure associated with ATP-dependent phosphorylation.

Na+/H+ exchanger: proton modifier site regulation of activity.

The Na+/H+ exchangers (NHE1-6) are integral plasma membrane proteins that catalyze the exchange of extracellular Na+ for intracellular H+. In addition to Na+ and H+ transport sites, NHE has an intracellular allosteric H+ modifier site that increases exchange activity when occupied by H+. NHE activity is also subject to control by a variety of extrinsic factors including hormones, growth factors, cytokines, and pharmacological agents. Many of these factors, working through second messenger pathways acting directly or indirectly on NHE, regulate NHE activity by shifting the apparent affinity of the H+ modifier site to more alkaline or more acid pH. The underlying molecular mechanisms involved in the activation of NHE by the H+ modifier site are poorly understood at this time, but likely involve slow protein conformational changes within a NHE oligomer. In this paper, we present initial experiments measuring intracellular pH-dependent transition rates between active and inactive oligomeric conformations and describe how these transition rates may be important for overall regulation of NHE activity.

Evidence for a new intermediate state in the mechanism of (Na+ + K+)-adenosine triphosphatase.

A rapid mixing technique was used to follow the intermediate formation of phosphorylated enzyme and liberation of inorganic phosphate by a microsomal preparation of (Na+ + K+)-ATPase. In the presence of 100 mM Na+,but without added K+, phosphorylation reaches a constant level at a rate which is dependent on ATP concentration. Inorganic phosphate production lags during the inital phase of phosphorylation and then accumulates at a constant rate. These observations favor a scheme in which Pi is liberated as the result of turnover of the phosphorylated enzyme. In the presence of 100 mM Na+ and 2.5 mM K+ phosphate production was resolved into two phases consisting of an initial 'burst' and late steady state phase...