细胞外Ca^2＋内流入胞质的机制

细胞外Ｃａ＾２＋主要是通过塌压依赖性Ｃａ＾２＋通道和钙池耗竭依赖性Ｃａ＾２＋通道而内流的。前者主要见于电兴奋细胞,这一过程比较清楚;后者主要见非兴奋细胞,情况远较复杂：外来信号激活内贮钙池,钙池在释放Ｃａ＾２＋同时通过目前尚不清楚的途径将直接或间接传至质膜Ｃａ＾２＋通道,而诱发Ｃａ＾２＋内流。     

小麦与玉米及鸭茅状摩擦禾远缘杂交的胚发育过程中同工酶和蛋白质的变化

通过比较小麦与玉米及鸭茅状摩擦禾属间杂交获得的胚与小麦正常自交的胚之间在不同发育时期过氧化物酶和酯酶的同工酶谱,发现过氧化物酶同工酶表现出时空顺序的特异性变化。在同一发育时期,远缘杂交的具胚子房和无胚子房之间存在过氧化物酶同工酶谱的差异,这可能涉及到与胚发育相关的同工酶的出现。远缘杂交的具胚子房和正常自交的小麦子房之间也有一定的酶谱差异。同时,同一材料还表现出不同发育时期的过氧化物酶酶谱差别。在远缘杂交后的胚发育期间,酯酶同工酶的时空表达不如过氧化物酶显著。此外,对远缘杂交后的胚中的水溶性蛋白质进行了SD S-PAGE分析,初步的分析结果表明,可能存在与胚发育相关的蛋白质。     

Treatment of Gaucher disease with an enzyme inhibitor

The hypothesis is offered predicting that Caucher patients could be treated with a drug that slows the synthesis of glucosylceramide, the lipid that accumulates in this disorder. The present therapeutic approach involves augmenting the defective enzyme, glucosylceramide -glucosidase, with exogenous -glucosidase isolated from human tissue. This spectacularly expensive mode of treatment should be replaceable with a suitable enzyme inhibitor that simply slows formation of the lipid and matches the rate of synthesis with the rate of the defective, slowly working -glucosidase. Several drugs that possess this ability are available, the best known of which is 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a designer inhibitor that resembles the synthase's substrate and product. PDMP has been found to be effective in mice, rats, fish, and a wide variety of cultured cells. Its use, at suitable dosages, seems to be harmless, although long-term tests have not been made. The lack of suitable animal models of Gaucher disease has made it difficult to test the hypothesis adequately, but PDMP does rapidly lower the levels of glucosylceramide in normal animal tissues and the animals evidently do well with the lowered levels of glucosylceramide and its more complex glycolipid metabolites.Abbreviations PDMP 1-phenyl-2-decanoylamino-3-morpholino-1-propanol - GlcCer glucosylceramide - i.p. intraperitoneal     

Influence of microtubule-associated proteins on the differential effects of paclitaxel and docetaxel

Microtubules are complex structures arising in part from the polymerization of tubulin dimers. Tubulin binds to a wide range of drugs which have been used as probes for tubulin conformation and assembly properties. There is some evidence that taxol and taxotere have differing effects on tubulin conformation. Previous work has shown that MAP2 and Tau, although they both induce microtubule assembly, have qualitatively different effects on tubulin's behavior. Since most microtubulesin vivo are likely to be associated with MAPs, we decided to characterize the differential effects of MAP2, Tau, taxol, and taxotere on tubulin polymerization with the aim of understanding the mechanisms through which these agents stimulate microtubule assembly. Furthermore, the inhibitive effect of calcium has been used to elucidate the ability of the two drugs to force tubulin assembly. These observations suggest that docetaxel, in addition to its greater efficiency in tubulin assembly, may have the capacity to differently alter certain classes of microtubules. Tau and MAP2 accessory proteins may represent important cofactors modulating the effects of taxoids.     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Platelet calcium pump activity in essential hypertensives and their first-degree relatives

Intracellular free Ca²⁺ concentration has been shown to be elevated in platelets of patients with essential hypertension. This study was designed to characterize Ca²⁺-pump activity of the platelet membranes (surface and intracellular) in these patients. A double-blind study was carried out. Untreated and treated (on R-blockers) essential hypertensives were studied in comparison with normotensive control subjects. First degree blood relatives of essential hypertensives were also studied. The Ca²⁺-activation kinetics of the enzyme showed a significant decrease in the V_max. (for the plasma- and intracellular membranes) and Km (for the intracellular membranes) in the essential hypertensive patients. Increased platelet membrane cholesterol content was observed in these patients. Lowered Ca²⁺-efflux by Ca²⁺-ATPase may lead to elevated intracellular free Ca²⁺-levels in platelets of essential hypertensives. A lowered Ca²⁺-ATPase activity may emerge as a marker for essential hypertension.     

Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca²⁺ transporting ATPase which carries out active Ca²⁺ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe²⁺ + H₂O₂ HO· + OH^–+ Fe³⁺) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H₂O₂ plus 50 M Fe²⁺ and 6 mM ascorbate. Ca²⁺ uptake carried out by the Ca²⁺-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca²⁺ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca²⁺ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of ⁴⁵Ca²⁺ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca²⁺-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca²⁺-ATPase. Electrophoretic analysis of oxidized Ca²⁺-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca²⁺-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca²⁺-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP acetylphosphate - BHT butylhydroxytoluene - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - SR sarcoplasmic reticulum - SRV sarcoplasmic reticulum vesicles - TBA thiobarbituric acid - TBARS thiobarbituric acid-reactive substances - TFP trifluoperazine     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

Modulation of maize roots H+-ATPase by sulfated polysaccharides

Vesicles derived from maize roots retain a membrane bound H⁺-ATPase that is able to pump H⁺ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H⁺-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent K_m for ATP from 0.28 to 0.95 mM and a decrease of the V_max from 14.5 to 7.1 mol P_i/mg · 30 min^–1. At pH 7.0 dextran 8000 promotes an increase in V_max from 6.7 to 11.7 mol Pi/mg · 30 min^–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K⁺ and Na⁺. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K⁺>Na⁺Li⁺).Abbreviations Mops 4-morpholinopropanesulfonic acid - EDTA ethylenediaminetetraacetic acid - ACMA 9-amino-6-chloro-2-methoxyacridine - FCCP carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone