Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.     

Genistein inhibits lysosomal enzyme release by suppressing Ca2+ influx in HL-60 granulocytes

The tyrosine kinase inhibitor genistein (5-200 microM) suppressed Ca(2+)-dependent fMLP (1 microM) and ATP (100 microM)-induced release of the lysosomal enzyme, beta-glucuronidase from neutrophil-like HL-60 granulocytes. Agonist-induced Ca2+ mobilization resulted from the release of intracellular Ca2+ stores and the influx of extracellular Ca2+. Genistein (200 microM) suppressed fMLP (1 microM) and ATP (100 microM)-induced Ca2+ mobilization, by 30-40%. Ca2+ release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+ (Mn2+) influx. Consistent with these findings, genistein (200 microM) or removal of extracellular Ca2+ (EGTA 1 mM), inhibited Ca(2+)-dependent agonist-induced beta-glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced beta-glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+ (Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+ influx induced by thapsigargin (0.5 microM). These data indicate that agonist-induced Ca2+ influx and store-operated Ca2+ influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL-60 granulocytes.     

Characterization of the inositol 1,4,5-trisphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate.

The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2+ pump and the non-specific Ca2+ leak from the non-mitochondrial Ca2+ stores in permeabilized A7r5 cells

2-Aminoethoxydiphenyl borate (2APB) is a membrane-permeable blocker of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release in bi-directional Ca2+ -flux conditions. We have now studied the effects of 2APB on the 45Ca2+ uptake into, and on the basal and IP(3)-stimulated unidirectional 45Ca2+ efflux from the non-mitochondrial Ca2+ stores in permeabilized A7r5 smooth-muscle cells. 2APB inhibited the IP3 -induced Ca2+ release, with a half maximal inhibition at 36 microM 2APB, without affecting [3H]IP3 binding to the receptor. This inhibition did not depend on the IP3, ATP or free Ca2+ concentration. The Ca2+ pumps of the non-mitochondrial Ca2+ stores were half-maximally inhibited at 91microM 2APB. Higher concentrations of 2APB increased the non-specific leak of Ca2+ from the stores. We conclude that 2APB can not be considered as a selective blocker of the IP3 -induced Ca2+ release. Our results can explain the various effects of 2APB observed in intact cells.     

bcl-2 protects HL-60 cells from apoptosis by stabilizing their intracellular calcium pools.

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis induced under different circumstances. In this study we investigated the effects of bcl-2 overexpression on the homeostasis of subcellular organelles such as ER and mitochondria. In our study, HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Apoptosis was evaluated by both DNA fragmentation and flow cytometry qualitatively and quantitatively, and the intracellular calcium by Fura-2/AM. Thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca2+ pump, and Br-A23187, a calcium ionophore, were used in this study. Our results showed that overexpression of bcl-2 significantly blocked TG- and Br-A23187-induced apoptosis in calcium containing buffer. Measurement of intracellular calcium showed that bcl-2 overexpression could reduce sustained elevation of cytosolic Ca2+ induced by these agents. However, in calcium-free medium, bcl-2 overexpression maintained Ca2+ uptake in ER of both TG- and Br-A23187-treated cells. Moreover, the depletion of Ca2+ by EGTA enhanced TG- and Br-A23187-induced apoptosis, and reduced the anti-apoptotic action of bcl-2, suggesting that cytosolic Ca2+ elevation may be required for optimal ER pool refilling. These findings suggest that bcl-2 facilitates and maintains the replenishment of Ca2+ in intracellular stores and, as a result, influences the intracellular calcium, thus protecting the cells from death. In addition, there were no cytochrome c release from mitochondria into the cytosol in TG- and Br-A23187- induced apoptosis, suggesting that cytochrome c release is not a universal phenomenon in the apoptotic process.     

Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.     

Effects of silver ion on the calcium-induced calcium release channel in isolated sarcoplasmic reticulum

Ag+-induced Ca2+ release in isolated sarcoplasmic reticulum (SR) was studied by the stopped flow method monitoring chlortetracycline fluorescence change. After improving the experimental procedure, the initial rate of Ca2+ release could be determined more precisely than before. Micromolar concentrations of Ag+ specifically enhanced Ca2+ efflux from heavy fraction of SR vesicles (HSR). This specific effect was referred to as Ag+-induced calcium release. The Ag+-induced Ca2+ efflux was activated by caffeine and ATP, but was inhibited by Mg2+ and procaine. Further, Ag+ enhanced the Ca2+-induced Ca2+ release over the whole range of Ca2+ concentrations, similarly to ATP. Parallel to Ca2+ efflux, Mg2+ efflux, measured by the same method, was also activated by Ag+. Choline permeability determined by the light scattering method was also activated by Ag+. The results suggest that Ag+ binds to the activation site of the Ca2+-induced Ca2+ release channel and opens the channel. The Ag+ binding site is different from the Ca2+ binding site but similar to the ATP binding site.     

Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-trisphosphate receptor and the endoplasmic-reticulum Ca(2+) pumps

Xestospongins, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are potent blockers of the inositol 1,4,5-trisphosphate (IP(3))-induced Ca2+ release in bi-directional Ca2+-flux conditions. We have now studied the effects of xestospongin C on the (45)Ca2+ uptake and the uni-directional (45)Ca2+ efflux in permeabilized A7r5 smooth-muscle cells. Xestospongin C not only inhibits the IP(3)-induced Ca2+ release, but is also an equally potent blocker of the endoplasmic-reticulum Ca2+ pump, while it has no effect on the passive Ca2+ leak. The inhibition of the IP(3) receptor did not depend on the IP(3), Ca2+ or ATP concentration. Xestospongin C can, therefore, not be considered as a selective blocker of IP(3) receptors.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: a comparison with skinned muscle fiber studies

Uptake and release of Ca2+ in heavy and light fractions of fragmented sarcoplasmic reticulum (FSR) isolated from frog and rabbit skeletal muscle was studied under conditions similar to those employed in skinned muscle fiber experiments, where ATP and Mg2+ concentrations were considered to be physiological and free Ca2+ concentration was kept constant during the Ca2+ uptake and release. Ca2+ level in FSR monotonously approached a steady state level which depended only on the final experimental conditions. Heavy fractions, but not light fractions, exhibited characteristics similar to those of Ca2+-induced Ca2+ release reported in skinned fiber studies: i) the rate and steady state level of Ca2+ uptake increased with increase in free Ca2+ concentration in the reaction medium up to 10(-6) M. With further increase in free Ca2+ concentration, the steady state level of Ca2+ taken up decreased while the Ca2+ uptake rate increased. ii) The steady state Ca2+ level was decreased by caffeine but increased by procaine or ruthenium red. Parallel measurement of Ca2+-ATPase activity clearly showed that these drugs modify the Ca2+ efflux but hardly affect the Ca2+-pump activity. It was concluded that the Ca2+-induced Ca2+ release mechanism was in operation at as low as 10(-6) M free Ca2+ concentration. Treatment of FSR with 0.6 M KCl did not have any significant effect.     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

Increase in Ca2+ permeability of intracellular Ca2+ store membrane of saponin-treated guinea pig peritoneal macrophages by inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) releases Ca2+ from the non-mitochondrial Ca2+ store site of various types of cells. To study the mechanisms of the Ca2+ release from the store site, the effect of InsP3 on the passive Ca2+ release and influx, and the active Ca2+ uptake in the presence of oxalate, was examined using saponin-treated guinea pig peritoneal macrophages. InsP3 stimulated the passive Ca2+ release and influx. Although InsP3 slightly inhibited the active Ca2+ uptake in the presence of oxalate, it seems unlikely that the Ca2+ release by this agent is caused by the inhibition of the Ca2+ uptake, because the addition of apyrase or hexokinase (which removes ATP within 30 s, so that no more Ca2+ can be accumulated) or vanadate (which inhibits the Ca2+ uptake) resulted in very slow release of Ca2+. These results suggest that the Ca2+ permeability of the Ca2+ store membrane is increased by InsP3. InsP3 did not cause an increase in the Ca2+ permeability of phospholipid vesicles (liposomes), indicating that this agent may bring about Ca2+ release by a specific effect on the physiologically relevant Ca2+ channels or carriers in the non-mitochondrial Ca2+ store site. The passive Ca2+ release by InsP3 was enhanced by ATP and an unhydrolyzable ATP analogue, 5'-adenylyimidodiphosphate, but not by ADP or AMP. The passive Ca2+ release by InsP3 was observed even at 0 degree C.     

Expression level of inositol trisphosphate and inositol tetrakisphosphate receptors and their influence on Ca2+ release in permeabilized HL-60 and T15 cells

To try to further define the mechanism of action of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (InsP4), we have studied its effects in permeabilized cells expressing different levels of inositol trisphosphate receptor (InsP3R) types I and III and of the GTPase-activating protein GAP1IP4BP. During the growth curve of human HL-60 cells and mouse T15 cells there was an increase in these proteins, which was further increased by differentiation (HL-60) and, marginally, by transformation (T15). T15 cells entering the stationary phase showed much lower concentrations of these proteins and expression was below detection in apoptotic HL-60 cells. Rasp21 showed a different pattern of expression. The ratios of InsP3R subtypes seem to affect the dose-response curve for inositol 2,4,5-trisphosphate Ins(2,4,5)P3. In permeabilized T15 cells the curve was approximately 5-fold to the right of that obtained using HL-60 cells. However, permeabilized untreated and differentiated HL-60 cells and T15 cells all showed a comparable synergistic effect of InsP4 on Ca2+ release stimulated by a concentration of Ins(2,4,5)P3, releasing approximately 20% of the Ins(1,4,5)P3 sensitive Ca2+ pool. The data indicate that under these conditions InsP4 is acting independently of cell type, of the ratio of inositol trisphosphate receptor subtypes, and of the concentration of GAP1IP4BP.     

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.     

Cyclosporin A-insensitive permeability transition in brain mitochondria: inhibition by 2-aminoethoxydiphenyl borate

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.     

Improved resolution of the initial fast phase of heavy sarcoplasmic reticulum Ca2+ uptake by Ca2+:antipyrylazo III dual-wavelength spectroscopy

The effect of ATP upon difference absorbance due to Ca2+ and Mg2+ complexation with the metallochromic dye, Antipyrylazo III (AP III), was investigated. At divalent cation concentrations appropriate for Sarcoplasmic Reticulum Ca2+ transport, wavelengths (greater than 670 nm) were found whereupon the addition of up to 1mM nucleotide did not alter divalent cation:AP III difference absorbance. At these sample wavelengths an initial rapid uptake of Ca2+ by Heavy SR (HSR) was clearly resolved by dual wavelength spectroscopy of Ca2+:dye difference absorbance. Elimination of ATP interference of Ca2+:AP III absorbance by Mg2+ elevation (3-10mM) was shown to be an inappropriate general strategy for AP III spectroscopic studies of HSR Ca2+ transport due to Mg2+ inhibition of ryanodine receptor mediated Ca2+ release.     

Curcumin: a new cell-permeant inhibitor of the inositol 1,4,5-trisphosphate receptor

Curcumin (diferuoylmethane or 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6-hepatadiene-3,5-dione) is the active ingredient of the spice turmeric. Curcumin has been shown to have a number of pharmacological and therapeutic uses. This study shows that curcumin is a potent inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel (InsP3 receptor). In porcine cerebellar microsomes, the extent of InsP3-induced Ca2+ release (IICR) is almost completely inhibited by 50 microM curcumin (IC50 = 10 microM). As the extent of IICR cannot be restored back to control levels by the addition of excess InsP3 and since it has little effect on [3H]InsP3 binding to cerebellar microsomes, this inhibition is likely to be non-competitive in nature. IICR in cerebellar microsomes is biphasic consisting of a fast and slow component. The rate constants for the two components are both reduced by curcumin to similar extents (by about 70% of control values at 40 microM curcumin). In addition, curcumin also reduces agonist (ATP)-stimulated Ca2+ mobilization from intact HL-60 cells, indicating that curcumin is cell permeant. However, since it also affects intracellular Ca2+ pumps and possibly ryanodine receptors, it may lead to complex Ca2+ transient responses within cells, which may well explain some of its putative therapeutic properties.     

1,25-(OH)_2D_3诱导HL-60细胞分化后胞液Ca~(2+)浓度、磷酸化酶a和微粒体Ca~(2+)-ATP酶活性的改变

1,25-(OH)_2D_3对HL-60细胞具有促分化作用。本文报道了1,25-(OH)_2D_3在促进HL-60细胞分化前后胞液Ca~(2+)浓度、磷酸化酶a和微粒体Ca~(2+)-ATP酶活性的改变。结果表明,1,25-(OH)_2D_3加入HL-60细胞培养液后72小时,细胞NBT染色阳性率高于70％,形态向正常分化的细胞转化。同对,胞液Ca~(2+)浓度和微粒体Ca~(2+)-ATP酶活性明显降低,而磷酸化酶a活性显著升高。结果提示,在1,25-(OH)2_D_3作用下,HL-60细胞不仅杀菌功能增强,细胞内胞液Ca~(2+)浓度趋向正常,与钙恒稳有关的酶活性也同样发生改变。即1,25-(OH)_2D_3对HL-60细胞的诱导作用伴有钙恒稳的改变。这些变化与DMSO的作用相同。     

Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules

Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate.     

Pathways of calcium release from heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle

The active uptake and efflux of Ca2+ from suspensions of vesicles from heavy rabbit muscle sarcoplasmic reticulum have been examined using the antipyrylazo III dye method in the presence of various nucleotide triphosphate substrates to support active Ca2+ accumulation. On addition of ATP, Ca2+ is rapidly accumulated and maintained at high internal concentrations until the substrate for pump protein is exhausted. Ca2+-induced Ca2+ release which is inhibited by ruthenium red can be demonstrated. The kinetics of Ca2+ release via these channels is different from the Ca2+ efflux observed after substrate exhaustion. This rate was found to be dependent on the type of nucleotide triphosphate, decreasing in the order ATP greater than GTP greater than CTP greater than ITP UTP. It is suggested that different conformations of the Ca2+ pump protein induced by the different substrates may result in the creation of pathways for the facilitated diffusion of Ca2+.