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1.
The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca 2+ levels ([Ca 2+] i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca 2+ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca 2+] i in a concentration-dependent manner. The [Ca 2+] i signal was biphasic with an initial rise and a slow decay. Ca 2+ removal inhibited the Ca 2+ signal by 41%. Adding 3 mM Ca 2+ increased [Ca 2+] i in cells pretreated with clomiphene in Ca 2+-free medium, confirming that clomiphene induced Ca 2+ entry. In Ca 2+-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca 2+ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca 2+ release. Conversely, pretreatment with 50 μM clomiphene in Ca 2+-free medium abolished the [Ca 2+] i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca 2+release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca 2+] i increases in PC3 cells by releasing store Ca 2+ from multiple stores in an phospholipase C-independent manner, and by activating Ca 2+ influx; and clomiphene was of mild cytotoxicity. 相似文献
2.
The intracellular free Ca 2+ ion concentration ([Ca 2+]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca 2+]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca 2+ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca 2+]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in [Ca 2+]i were rapid, transient and were blocked by atropine at 10 -6M. A brief exposure to 50 mM K + resulted in a transient increase in [Ca 2+]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10 -4M or 10 -3M) induced [Ca 2+]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca 2+ free medium, the source of the increase in [Ca 2+]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca 2+]i. These data strongly suggest that ACh exerted a direct action on [Ca 2+]i in normal rat pancreatic β-cells and support a role for Ca 2+ as a second messenger in the action of ACh. 相似文献
3.
We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca 2+ concentration ([Ca 2+] i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca 2+] i in the presence of LPA and this increase was concentration-dependent (1–10 μM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca 2+] i. The mechanical stress-induced increase in [Ca 2+] i in the presence of LPA was inhibited by removing extracellular Ca 2+ or by addition of Gd 3+, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca 2+ influx through cation-selective mechanosensitive channels, but does not sensitise Ca 2+ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca 2+ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca 2+ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca 2+] i induced by mechanical stress. 相似文献
4.
The mechanisms of intracellular calcium store depletion and store-related Ca 2+ dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca 2+ by BAPTA/AM (50 μM), or La 3+ (100 μM) enhanced while dantrolene (100 μM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca 2+ stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca 2+ concentrations ([Ca 2+] i). 相似文献
5.
The stiffness of the sarcomeres was studied during the diastolic interval of 18 stimulated (0.5 Hz) cardiac trabeculae of rat (pH 7.4; temperature = 25°C). Sarcomere length (SL) and force ( F) were measured using, respectively, laser diffraction techniques (resolution: 4 nm) and a silicon strain gauge (resolution: 0.63 μN). Sinusoidal perturbations (frequency = 500 Hz) were imposed to the length of the preparation. The stiffness was evaluated from the corresponding F and SL sinusoids by analysis of both signals together either in the time domain or in the frequency domain. A short burst (duration = 30 ms) of sinusoidal perturbations was repeated at 5 predetermined times during diastole providing 5 measurements of stiffness during the time interval separating two twitches. These measurements revealed that stiffness increases by 30% during diastole, while a simultaneous expansion of the sarcomeres (amplitude = 10-60 nm) was detected. Measurements of the fluorescence of fura-2 under the same conditions revealed a continuous exponential decline of [Ca 2+] i from 210 to 90 nM (constant of time 300 ms) during diastole. In order to test the possibility that the increase of sarcomere stiffness and the decline of [Ca 2+] i were coupled during diastole of intact trabeculae, we studied the effect of different free Ca 2+-concentrations ([Ca 2+]) between 1 and 430 nM on sarcomere stiffness in rat cardiac trabeculae skinned by saponin ( n = 17). Stiffness was studied using 500 Hz sinusoidal perturbations of muscle length (ML). We found that, below 70 nM, the stiffness was independent of [Ca 2+]; between 70 and 200 nM, the stiffness declined with increase of [Ca 2+]; above 200 nM, the stiffness increased steeply with [Ca 2+]. The data fitted accurately to the sum of two sigmoids (Hill functions): (1) at [Ca 2+] < 200 nM the stiffness decreased with [Ca 2+] (EC 50 = 160 ± 13 nM; n = −2.6±0.7) and (2) at [Ca 2+] > 200 nM, stiffness increased with [Ca 2+] (EC 50 = 3.4±0.3 μM; n = 2.1±0.2) due to attachment of cross-bridges. From these results, it was possible to reproduce accurately the time course of diastolic stiffness observed in intact trabeculae and to predict the effect on stiffness of a spontaneous elevation of the diastolic [Ca 2+]. Identical stiffness measurements were performed in 4 skinned preparations exposed to a cloned fragment of titin (Ti I-II) which has been shown to exhibit a strong interaction with F-actin in vitro. It was anticipated that Ti I-II would compete with endogenous titin for the same binding site on actin in the I-band. Below 200 nM, Ti I-II (2 μM) eliminated the Ca 2+-dependence of stiffness. These results are consistent with the hypothesis that the Ca 2+-sensitivity of the sarcomeres at [Ca 2+] < 200 nM, i.e. where the myocytes in intact muscle operate during diastole, involves an association between titin molecules and the thin filament. 相似文献
6.
The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca 2+ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca 2+–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca 2+ levels ([Ca 2+] i) without changing 25 μM clomiphene-induced [Ca 2+] i increase. 17β-estradiol and tamoxifen increased [Ca 2+] i by causing Ca 2+ influx and Ca 2+ release because their responses were partly reduced by removing extracellular Ca 2+. In contrast, clomiphene solely induced Ca 2+ release. The effect of the lignans on these two Ca 2+ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca 2+] i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca 2+ release, and 17β-estradiol-induced Ca 2+ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca 2+ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca 2+ signaling in human neutrophils in a multiple manner. 相似文献
7.
In this study we investigated the release of Ca 2+ in brain microsomes after Ca 2+ loading by the Ca 2+-ATPase or by the Na +/Ca 2+ exchanger. The results show that in microsomes loaded with Ca 2+ by the Ca 2+-ATPase, Ins(1,4,5)P 3 (5 μM) release 21±2% of the total Ca 2+ accumulated, and that in the microsomes loaded with Ca 2+ by the Na 2+/Ca 2+ exchanger, Ins(1,4,5)P 3 released 28±3% of the total Ca 2+ accumulated. These results suggest that receptors of Ins(1,4,5)P 3 may be co-localized with the Na 2+/Ca 2+ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P 3 receptors in the plasma membrane where the Na 2+/Ca 2+ exchanger is normally present, or both. We also found that Ins(1,4,5)P 3 inhibited the Ca 2+-ATPase by 33.7%, but that it had no significant effect on the Na 2+/Ca 2+ exchanger. 相似文献
8.
Regulation of the increase in inositol phosphate (IP) production and intracellular Ca 2+ concentration ([Ca 2+] i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca 2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC 50) and maximal inhibition of BK induced an increase in [Ca 2+] i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca 2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the KD and Bmax of the BK receptor for binding (control: KD = 1.7 ± 0.2 nM; Bmax = 47.3 ± 4.4 fmol/ mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca 2+ mobilisation in VSMCs. 相似文献
9.
Increase in cytoplasmic cyclic AMP concentration stimulates Ca 2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca 2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca 2+ influx. Cyclic AMP evoked discharge of Ca 2+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 μM of free Ca 2+, while Ca 2+ lower than 0.1 μM did not affect the Ca 2+-release. The Ca 2+ flux across plasma membrane was restored from this Ca 2+-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca 2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca 2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca 2+ channel. 相似文献
10.
1. 1. (Mg2+ + Ca2+) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg2+ + Ca2+) ATPase seen during development was greatest in the synaptic membrane preparations. 2. 2. At 37°C both Na+ or K+ at concentrations higher than 30 mM inhibited the microsomal Mg2+ ATPase, but the (Mg2+ + Ca2+) ATPase was stimulated by both Na+ and K+. Synaptic membrane Mg2+ ATPase was inhibited by concentrations higher than 100 mM K+; Na+ however stimulated this enzyme at all concentrations. Much of this Na+ stimulated activity was ouabain sensitive. Synaptic membrane (Mg2+ + Ca2+) ATPase was stimulated by Na+ or K+, this stimulation follows approximate saturation kinetics with an apparent Km of 18.8 mM Na+ or K+. 3. 3. Arrhenius plots of microsomal (Mg2+ + Ca2+) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole−1 above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg2+ + Ca2+) ATPase from both immature and adult brain.
Author Keywords: Brain; ATPase; temperature; development; synaptic membranes 相似文献
11.
Intravenous administration of ovokinin(2–7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1–5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2–7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B 2 receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B 1 receptor antagonist [des-Arg 10]-HOE140 (26.3±15.5%). Intracellular Ca 2+ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2–7). The increases were abolished by HOE140 and unaffected by [des-Arg 10]-HOE140. The elevation of intracellular Ca 2+ by ovokinin(2–7) was dependent on Ca 2+ entry from extracellular space as it was reduced in a Ca 2+-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca 2+ increase by the peptide. PA phosphohydrolase and phospholipase A 2 inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B 2 bradykinin receptors. 相似文献
12.
The relationship between the agonist-sensitive Ca 2+ pool and those discharged by the Ca 2+-ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca 2+ ([Ca 2+] i), followed by a sustained plateau phase that was dependent on extracellular Ca 2+. In such cells, TG produced a concentration-dependent increase in [Ca 2+] i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca 2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca 2+] i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca 2+-ATPase inhibitors, cyclopiazonic acid and 2,5-di- t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca 2+-influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca 2+ stores is sufficient for activation of Ca 2+ influx. Some characteristics of the Ca 2+-influx activated by depletion of internal Ca 2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca 2+ influx was inhibited by La 3+, membrane depolarisation, and the novel Ca 2+-influx blocker 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca 2+ influx by TG also was blocked by La 3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca 2+ stores in TSMCs is sufficient for activation of Ca 2+ influx, and (2) the agonist-activated Ca 2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca 2+ pool are indistinguishable. 相似文献
13.
Release of [ 3H]dopamine ([ 3H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [ 3H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca 2+] 0-independent release of [ 3H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 ± 4.15 to 6.98 ± 1.83 mol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C. under glucose-free conditions was significantly reduced from 0.73 ± 0.04 to 0.44 ± 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 ± 0.11 mol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 ± 0.37 nmol/mg protein. The resting release was enhanced in a [Ca 2+] 0-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 μM ouabain, inhibitor of Na +/K +-activated ATPase, enhanced the release of [ 3H]DA at rest in a [Ca 2+] 0-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na +/K +-ATPase that results in reversal of the uptake processes and in [Ca 2+] 0-independent [ 3H]DA release from the axon terminals. 相似文献
14.
A human skeletal actin · tropomyosin · troponin complex was phosphorylated in the presence of [γ- 32P]ATP, Mg 2+, adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 μM cyclic AMP. In the presence of 10 −7 M Ca 2+ and protein kinase 0.1 mole of [ 32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [ 32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca 2+, 5 · 10 −5 M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [ 32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [ 32P]phosphate. Increasing the Ca 2+ concentration did not significantly decrease the [ 32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstituted human skeletal actomyosin made with the [ 32P]phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca 2+. 相似文献
15.
This study was undertaken to assess the role of calcium channels in the contractile response induced by substance P in the isolated rat iris. Substance P produced graded and sustained contraction in the rat iris. Pre-incubation of preparations with thapsigargin (1 μM), verapamil (1 μM), isradipine (1 μM) or with ω-conotoxin MCIIA (0.1 μM) did not significantly inhibit substance P-mediated contraction in the isolated rat iris. However, pre-incubation of the preparations with nicardipine (1 μM) or ruthenium red (1 mM) caused parallel displacement to the right of the substance P concentration–response curve without affecting its maximal response. In contrast, amiloride (1 μM), markedly inhibited substance P-mediated contraction (73±5%), while econazole (1 mM) also significantly inhibited (44±11%) substance P-mediated contraction in the isolated rat iris. Collectively, these results suggest that substance P-mediated contractile response in the isolated rat iris depends largely on the influx of external Ca 2+, by a mechanism which might involve the T-type calcium channels. 相似文献
16.
The Ca 2+-mobilizing metabolite cyclic ADP-ribose (cADPR) has been shown to release Ca 2+ from ryanodine-sensitive stores in many cells. We show that this metabolite at a concentration of 17μM, but not its precursor β-NAD + nor non-cyclic ADPR at the same concentration, is active in releasing Ca 2+ from rabbit skeletal muscle sarcoplasmic reticulum. The release was not sensitive to Ruthenium red (1μM) nor to the ryanodine receptor-specific scorpion toxin Buthotus1-1 (10 μM). In planar bilayer single channel recordings, concentrations up to 50μM cADPR did not increase the open probability of Ruthenium red and toxin-sensitive Ca 2+ release channels. Thus Ca 2+ release induced by cADPR in skeletal muscle sarcoplasmic reticulum may not involve opening of ryanodine receptors. 相似文献
17.
Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP-dependent, IP3-sensitive, Ca 2+ store in the glucose- and phosphatidylinositol(PI) agonist-sensitive hamster insulinoma cell line HIT-T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1-14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface-derived vesicle fractions were prepared containing 80% of the total cellular Ca 2+-storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well-known distribution of the Ca 2+-storing activity which is then predominantly recovered within the microsomal fraction. The surface-derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na +/K +-ATPase and an immunoreactive fragment of the GluT-1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca 2+ uptake properties of the cell surface-derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca 2+ stores of various cell types. Inositol 1,4,5-trisphosphate (IP3) at 1 μM induced a maximal release of 35–40% of the stored Ca 2+ from these vesicles. 相似文献
18.
The effects of trifluoperazine on hepatic cell metabolism were investigated using isolated perfused rat liver. The following effects of trifluoperazine were found: (1) trifluoperazine inhibited oxygen uptake, the site of action being the mitochondria. Half-maximal inhibition occurred at concentrations around 50 μM; with 100 μM trifluoperazine the effect was already maximal. When Ca 2+ was withdrawn from the perfusion medium and the intracellular Ca 2+ pools were exhausted, the inhibitory action on respiration was no longer observable. The rein-troduction of Ca 2+ restored inhibition. (2) Glycogenolysis and glycolysis were not significantly affected during the infusion of trifluoperazine. After stopping trifluoperazine infusion, however, glycogenolysis (glucose release) experienced a transitory stimulation. (3) Gluconeogenesis from lactate as the carbon source was inhibited by trifluoperazine. This inhibition was approximately proportional to the inhibition of oxygen uptake. Withdrawal of Ca 2+ diminished, but it did not eliminate, inhibition of gluconeogenesis. (4) Ketogenesis was also inhibited in parallel with the inhibition of oxygen uptake. Withdrawal of Ca 2+ from the perfusion fluid also abolished this action. (5) The effects of trifluoperazine were reverted very slowly when its infusion was stopped. The recovery of oxygen uptake at 50 min after cessation of the infusion was only 30%. Uptake of the substance was very fast. Absence of Ca 2+ did not affect uptake. It was concluded that inhibition of mitochondrial energy metabolism is one of the most prominent effects of trifluoperazine in the liver. The fact that this inhibition depends on Ca 2+ is unique. 相似文献
19.
Extracellular ATP dose dependently stimulated 45Ca 2+ influx even in the presence of nifedipine, a Ca 2+ antagonist that inhibits voltage-dependent Ca 2+ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E 2 (PGE 2). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca 2+ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE 2 synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE 2. These results strongly suggest that extracellular ATP stimulates Ca 2+ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE 2 synthesis. 相似文献
20.
The store-mediated Ca 2+ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca 2+ activated ionic membrane currents. In the cells where intracellular Ca 2+ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca 2+-free extracellular condition, an employment of external Ca 2+ in the absence of ACh caused a sustained increase of the K + current without affecting the Cl − current. A renewed ACh challenge without external Ca 2+ caused repetitive spikes of both K + and Cl − currents due to the Ca 2+ release. SK & F 96365 inhibited the generation of the sustained K + current and refilling of the Ca 2+ store following the Ca 2+ readmission. It is suggested that the Ca 2+ enters the cell through the store-mediated pathway near the K + channels and is taken up by the store. Thus, only Ca 2+ released from the store can activate both the K + and Cl − currents. 相似文献
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