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1.
The inhibition of testosterone 5α-reductase activity by 3-oxo-4-androstene-17β-carboxylic acid in the male reproductive organs of the rat was demonstrated . The medium for incubation of caput epididymis showed the highest concentration of 5α-dihydrotestosterone (5α-DHT) whereas the highest concentration of testosterone (T) was recorded in medium for incubation of decapsulated testis after two hours of incubation. The 3-oxo-4-androstene-17β-carboxylic acid (1.58 × 10?5M) inhibited the conversion of T to 5α-DHT in all the organs tested (testis, caput and cauda epididymis and ventral prostate) under identical incubation conditions. 相似文献
2.
《Bioorganic & medicinal chemistry letters》2020,30(2):126783
5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17β-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-14C]-3α-diol from [4-14C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC50 = 1.2–14.1 μM). The most potent inhibitor 25 (IC50 = 1.2 μM) possesses a 4-(4-CF3-3-CH3O-benzyl)piperazinyl methyl side chain at C3β and 17β-OH/17α-CCH functionalities at C17 of a 5α-androstane core. 相似文献
3.
45Ca2+ uptake by the human liver fluke Opisthorchis viverrini is enhanced by praziquantel. The drug-induced 45Ca2+ uptake was dependent on the presence of Ca2+ and was attenuated in the presence of 10 mM Mg2+. La3+ and vanadate at concentration of 1mM partially reduced the amount of 45Ca2+ uptake into the liver fluke in response to praziquantel treatment. The stimulating effect of praziquantel was eliminated in the presence of 10 μM verapamil. These findings suggest that praziquantel increases the permeability of the liver fluke tegument to Ca2+ probably by interfering with the mechanism that regulates Ca2+ binding or trnasport across the tegumental membrane. 相似文献
4.
Christopher J. Lynch Richard C. Deth Michael L. Steer 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,757(2):156-163
The relative influences of the in vivo administration of phenoxybenzamine on in vitro binding to α1-adrenergic receptors and α1-receptor-mediated responses were studied. Phenoxybenzamine treatment reduced maximal specific binding of the α1-selective antagonist [3H]prazosin to liver cell membranes. This response was rapid (< 90 min) and half-maximal following a phenoxybenzamine dose of approx. 10 mg/kg. A similar decrease in the ability of phenylephrine to stimulate glucose release and 45Ca2+ efflux from liver slices was also noted after phenoxybenzamine treatment. During the recovery period following administration of 30 mg/kg phenoxybenzamine, [3H]prazosin specific binding and phenylephrine-stimulated glucose release and 45Ca2+ efflux returned to their respective control levels with values of 42, 49 and 38 h, respectively. At all times studied during the recovery period, α1-binding and both of the α1-responses were similar fractions of their respective control values. These observations indicate that a close relationship exists between the density of [3H]prazosin binding sites and the ability of rat liver to respond to α1-stimulation. We suggest that the binding sites identified in studies using the antagonist [3H]prazosin and those through which the agonist phenylephrine stimulates glucose release and 45Ca2+ efflux are either identical or in equilibrium with each other. 相似文献
5.
The effect of regucalcin, a Ca2+-binding protein, on Ca2+ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10-8 to 10-6 M) in the reaction mixture caused a significant increase in Ca2+-ATPase activity and ATP-dependent45 Ca2+ uptake in the microsomes. Regucalcin (10-7 M) increased Ca2+-ATPase activity independently of increasing concentrations of CaCl_2. The microsomal Ca2+-ATPase activity and45 Ca2+ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca2+-ATPase activity and 45Ca2+ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10-7 M). Meanwhile, the microsomal Ca2+-ATPase activity and 45Ca2+ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10-5 and 10-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10-7 and 10-5 M). The effect of regucalcin (10-7 M) on Ca2+ ATPase activity and 45Ca2+ uptake was weakened in the presence of DcAMP or IP3. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca2+ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca2+-ATPase. 相似文献
6.
Ann VanhoorenKristien Vanhee Katrien NoyelleZsuzsa Majer Marcel JoniauIgnace Hanssens 《Biophysical journal》2002,82(1):407-417
Thermodynamic parameters for the unfolding of as well as for the binding of Ca2+ to goat α-lactalbumin (GLA) and bovine α-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25°C. Among the different parameters available, the heat capacity increments (ΔCp) offer the most direct information for the associated conformational changes of the protein variants. The ΔCp values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective ΔCp values for the binding of Ca2+ are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca2+-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca2+-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in ΔCp values for the binding of Ca2+ is presumably in part related to this difference in charge distribution. 相似文献
7.
ATP-dependent Ca2+ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN3. In contrast, rat kidney mitochondrial Ca2+ uptake is blocked by NaN3. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca2+ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent Km values for Mg2+-ATP differ in the two preparations with a higher affinity for Mg2+-ATP found in the plasma membrane Ca2+ uptake activity of the plasma membrane preparation is readily inhibited by Na+. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca2+ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca2+ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone. 相似文献
8.
Morgan EL Mace OJ Helliwell PA Affleck J Kellett GL 《Biochemical and biophysical research communications》2003,312(2):487-493
Active Ca2+ absorption through epithelial Ca2+ channels TRPV5/6 in duodenum is activated by hyperpolarisation. However, when diet and Ca2+ are plentiful, digestion products cause depolarisation. We therefore used homology-based PCR from a rat jejunal mucosal cDNA preparation to reveal the presence of the neuroendocrine L-type isoform Cav1.3α1. Immunocytochemical labelling and immunoblotting localised Cav1.3α1 protein in apical membrane from proximal jejunum to mid ileum. Perfusion studies in vivo with 1.25 mM luminal Ca2+ revealed L-type channel activity. Inhibition of glucose absorption with phloridzin strongly inhibited 45Ca2+ absorption; absorption was inhibited by nifedipine and Mg2+ and activated by Bay K 8644, none of which affect TRPV5/6. At 10 mM Ca2+, nifedipine inhibited 45Ca2+ absorption with a time course similar to that at 1.25 mM Ca2+: absorption was therefore channel-mediated rather than paracellular. We suggest that in times of dietary sufficiency, Cav1.3 may mediate a significant route of Ca2+ absorption into the body. 相似文献
9.
Sarcoplasmic reticulum (SR) Ca2+-ATPase was phosphorylated by Pi at pH 8.0 in the presence of dimethyl sulfoxide (Me2SO). Under these conditions, it was possible to measure transient 45Ca2+ binding to the phosphoenzyme. Binding reached 1.2 Ca2+ per phosphoenzyme (E-PCax) within 10 min in 30% Me2SO, 20 mM MgCl2 and 0.1 mM Pi and the phosphoenzyme only decreased by 23% during this period. This Ca2+ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca2+-ATPase. At 40% Me2SO, simultaneous addition of Ca2+ and ADP increased Ca2+ binding up to almost four Ca2+ per phosphoenzyme (ADPE-PCay), revealing a species bearing simultaneously four Ca2+ sites. Both E-PCax and ADPE-PCay were further identified as distinct species by (2′,3′-O-2(2,4,6-trinitrophenyl)adenosine 5′-triphosphate) fluorescence, which revealed long-range modifications in the Ca2+-transport sites induced by ADP binding to E-P. In addition, E-PCax was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me2SO was diluted. These findings indicate that more than two functional Ca2+-sites exist on the functional Ca2+-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca2+-ATPase allowing simultaneous binding of Ca2+ at lumenal and cytosolic sites. The stoichiometries for Ca2+ binding found here could either be interpreted as binding of four Ca2+ on a Ca2+-ATPase monomer considered as the functional unit or as binding of two Ca2+ per monomer of a functional dimer. 相似文献
10.
The alteration of the plasma membrane (Ca2+-Mg2+)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl4) solution was investigated. Rats received a single oral administration of CCl4 (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl4 administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca2+-Mg2+)-ATPase activity, indicating that the increased Ca2+ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca2+-Mg2+)-ATPase activity increased by CCl4 administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca2+-Mg2+)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl4-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca2+-Mg2+)-ATPase activity in the liver obtained from CCl4-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca2+ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl4 administration. 相似文献
11.
Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle 总被引:2,自引:0,他引:2
The role of calmodulin in the regulation of microsomal 45Ca2+ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent 45Ca2+ uptake and (Ca2+Mg2+)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent 45Ca2+ uptake and (Ca2+Mg2+)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca2+Mg2+)-ATPase and ATP-dependent 45Ca2+ uptake activities are probably related as they exhibited similar [Ca2+]free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca2+]free in canine tracheal smooth muscle. 相似文献
12.
5α-Dihydrotestosterone (5α-DHT) was rendered antigenic by covalent attachment to bovine serum albumin (BSA) through position 1 of the steroid. Nucleophilic attack by β-mercaptopropionic acid on the 1,2-dehydro derivative of 5α-DHT yielded the corresponding 1α-thioether alkanoic acid which was coupled to bovine serum albumin by use of the carbodiimide reagent. The method should be generally applicable to 3-oxosteroids. Immunization of rabbits with 5α-DHT-1α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for 5α-DHT (Ka= 1.4 × 109 1/mol) that showed little cross reaction with 17β-hydroxy-5β-androstan-3-one (3%), and with a variety of 17-oxoandrostane compounds (≤0.5%). However the serum cross-reacted significantly with testosterone (10%) and with 5α-androstene-3α, 17β-diol (16%). A radioimmunoassay procedure for the determination of 5α-DHT in plasma is described. Chromatographic purification of the plasma extracts proved necessary for obtaining valid results. The plasma level of 5α-DHT(pg/ml; ean ± S.D.) was 364±79 (n = 7) in normal human adult males and 188 ± 62 (n = 5) in normal non-pregnant women. 相似文献
13.
Marisa Brini Francesca Di Leva Claudia K. Ortega Teuta Domi Denis Ottolini Emanuela Leonardi Silvio C. E. Tosatto Ernesto Carafoli 《The Journal of biological chemistry》2010,285(40):30779-30791
Acidic phospholipids increase the affinity of the plasma membrane Ca2+-ATPase pump for Ca2+. They interact with the C-terminal region of the pump and with a domain in the loop connecting transmembrane domains 2 and 3 (AL region) next to site A of alternative splicing. The contribution of the two phospholipid-binding sites and the possible interference of splicing inserts at site A with the regulation of the ATPase activity of isoform 2 of the pump by phospholipids have been analyzed. The activity of the full-length z/b variant (no insert at site A), the w/b (with insert at site A), and the w/a variant, containing both the 45-amino acid A-site insert and a C-site insert that truncates the pump in the calmodulin binding domain, has been analyzed in microsomal membranes of overexpressing CHO cells. The A-site insertion did not modify the phospholipid sensitivity of the pump, but the doubly inserted w/a variant became insensitive to acidic phospholipids, even if containing the intact AL phospholipid binding domain. Pump mutants in which 12 amino acids had been deleted, or single lysine mutations introduced, in the AL region were studied by monitoring agonist-induced Ca2+ transients in overexpressing CHO cells. The 12-residue deletion completely abolished the ATPase activity of the w/a variant but only reduced that of the z/b variant, which was also affected by the single lysine substitutions in the same domain. A structural interpretation of the interplay of the pump with phospholipids, and of the mechanism of their activation, is proposed on the basis of molecular modeling studies. 相似文献
14.
Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca2+ sequestration pathways were characterized. The rate of Ca2+ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca2+/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca2+/mg protein sequestered in 60 min. Ca2+ sequestration was 93% inhibited by 180 mM NaCl [I50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl2. Ca 2+ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca 2+ sequestration was dependent on extravesicular Ca 2+ with half-maximal sequestration at pCa2+ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca2+ could be exchanged with external 45Ca2+, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca2+/mg (mean ± S.D., n = 3). The Ca2+/Ca2+ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl2. About 75% of sequestered 45Ca2+ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered 45Ca2+ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na+-induced release of sequestered 45Ca2+ was rapid, t0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with 45Ca2+ and either annexin proteins V or VI resulted in attenuated uptake of 45Ca2+. These results suggest that Ca2+ uptake in adrenal chromaffin granules is regulated by Na+ and Ca2+ gradients and also possibly by annexins V and VI.Abbreviations EGTA
ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid
- SDS
Sodium dodecyl sulphate
- PAGE
Polyacrylamide gel electrophoresis
- BSA
bovine serum albumin
- AI
Annexin I
- AIIt
Annexin II tetramer
- AIII
Annexin III
- AIV
Annexin IV
- AV
Annexin V
- AVI
Annexin VI
- k
first order rate constant
- AT
total extent of Ca2+ uptake (nmol)
- BufferA
300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA
- Buffer B
300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA
- Buffer C
300 mM sucrose, 10 mM potassium phosphate (pH 7.0)
- Buffer D
300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl2
- Buffer E
300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl2 相似文献
15.
Masaru Wada 《Hormones and behavior》1982,16(2):147-157
Castrated male Japanese quail were implanted with Silastic capsules containing testosterone (T), estradiol-17β (E2), 5β-dihydrotestosterone (5β-DHT), Δ4-androstenedione (Δ4) 5α-androstanedione (A), 5α-dihydrotestosterone (5α-DHT) or with empty capsules. Calling, monitored continuously and automatically, was induced significantly by T and Δ4. Locomotor activity, also monitored continuously by floor deflection, was enhanced by T, Δ4, and E2. Additional data concerning heterosexual and homosexual behavior were obtained from castrated quails after implantation of T, Δ4, E2, or 5α-DHT. T and Δ4 restored hetero- and homosexual behavior as did E2 but to a lesser extent. 5α-DHT did not induce either sexual behavior. Growth of the cloacal protrusion was induced in birds implanted with T, Δ4, A, and 5α-DHT but not with 5β-DHT and E2. These results indicate that calling and locomotor activity enhancement (including sexual behavior) are two different components of reproductive behavior which require different androgens or their metabolites to be activated. 相似文献
16.
Rachel A. Skowronsky Mechthild Schroeter Tamatha Baxley Yumin Li Joseph M. Chalovich Anne M. Spuches 《Journal of biological inorganic chemistry》2013,18(1):49-58
Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca2+ binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin–tropomyosin–actin complex. Herein, we report a calorimetric study of Ca2+ binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca2+ binding to both the high-affinity and the low-affinity domain of HcTnC. Ca2+ binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca2+ binding to the regulatory domain. This pattern stems from changes that occur at the Ca2+ site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC1–89 support this claim. The values of the Gibbs free energy for Ca2+ binding to the N-domain in the full-length protein and to the isolated domain (HcTnC1–89) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (?C p ) from a plot of ?H verses temperature and may provide evidence for positive cooperativity of Ca2+ binding to the C-domain. 相似文献
17.
The mechanism of increasing effect of CuCl2 on specific [3H]cimetidine binding was examined in brain membranes of rats. CuCl2-Induced elevation of [3H]cimetidine binding was high in Krebs-Ringer solution (pH 7.4) compared to those in 50 mM Na, K-phosphate buffer (pH 7.4) and in 50 mM Tris-HCl buffer (pH 7.4). CaCl2 (5–50 mM) inhibited effect of CuCl2, but NaCl (25–200 mM), KCl (5–100 mM) or MgCl2 (5–50 mM) did not. CuCl2 (50 μM) elevated 9.3- and 2.5-fold the binding in phosphate- and Tris—HCl buffer, respectively. EDTA-2Na decreased the binding elevated by 50 μM CuCl2 in phosphate buffer to the similar level in Tris-HCl buffer, whereas it did not affect those in Tris-HCl buffer. The absorption spectra of cimetidine and CuCl2 mixture showed a peak at 317 nm in phosphate buffer that was not observed in Tris-HCl buffer. It is suggested that cimetidine-Cu2+ chelate complex could be formed in phosphate buffer, resulting in higher amount of binding in phosphate buffer than in Tris-HCl buffer. PdCl2 also caused a marked elevation in [3H]cimetidine binding, seeming to be due to formation of cimetidine-Pd2+ chelate complex. There were two types of [3H]cimetidine binding in the presence of 20 nM PdCl2: high affinity binding with Kd = 0.7 ± 0.1 nM and low affinity binding with Kd = 44.3 ± 3.0 nM. It is suggested that cimetidine-Cu2+ complex binds to cimetidine binding sites in brain with higher affinity than cimetidine alone. 相似文献
18.
Tryptophan 5-monooxygenase in rat brainstem cytosol was activated about twofold by incubation with 0.5 mm ATP and 5 mm MgCl2. The activation required micromolar concentrations of Ca2+ but was not dependent on either cyclic AMP or cyclic GMP. Rat brain cytosol was shown to possess an endogenous protein kinase which was markedly stimulated by the addition of Ca2+ using endogenous protein substrates. Following activation by ATP and Mg2+ in the presence of Ca2+, tryptophan 5-monooxygenase was reversibly deactivated to the original level by incubation at 30 °C after removal of Ca2+ by adding ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid and was then reactivated by incubation at 30 °C after subsequent addition of Ca2+ and ATP. The deactivation was markedly inhibited by the omission of Mg2+ or by the addition of NaF. 相似文献
19.
《Life sciences》1996,58(11):PL217-PL222
The effect of extracellular adenosine 5′-triphosphate (ATP) on Ca2+ efflux from freshly isolated adult rat cardiomyocytes was examined. ATP stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner (0.01–1 mM). The 45Ca2+ efflux from the cells was also stimulated by adenosine-5′-O-(3-thiotriphosphate) (ATP-γs) and α,β-methylene-ATP and adenosine 5′-diphosphate, but not by adenosine 5′-monophosphate and adenosine. The ATP-stimulated 45Ca2+ efflux was not affected by deprivation of the extracellular Ca2+, but was dependent on the presence of extracellular Na+. These results indicate that ATP stimulates extracellular Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane P2 purinoceptors which may couple to Na+/Ca2+ exchange. 相似文献
20.
The thermodynamic efficiency of the calmodulin-activated form of the Ca2+-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca2+ concentration ([Ca2+]i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][Pi]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg2+, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca2+]o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM Pi, variable [ADP]. Under these conditions, with the pump working near itsK
m
of 64nM, the [Ca2+]i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca2+]i/[Ca2+]o ratio against [ATP]/([ADP][Pi]) gave a straight line with a slope of 1.5×107M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×107M). These results demonstrate (1) tight coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2 Ca2+ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC50 of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed. 相似文献