首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca2+ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca2+]i in a concentration-dependent manner. The [Ca2+]i signal was biphasic with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by 41%. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with clomiphene in Ca2+-free medium, confirming that clomiphene induced Ca2+ entry. In Ca2+-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca2+ release. Conversely, pretreatment with 50 μM clomiphene in Ca2+-free medium abolished the [Ca2+]i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca2+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 [Ca2+]i increases in PC3 cells by releasing store Ca2+ from multiple stores in an phospholipase C-independent manner, and by activating Ca2+ influx; and clomiphene was of mild cytotoxicity.  相似文献   

2.
Chao YY  Jan CR  Ko YC  Chen JJ  Jiann BP  Lu YC  Chen WC  Su W  Chen IS 《Life sciences》2002,70(26):4367-3121
The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca2+ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca2+–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca2+ levels ([Ca2+]i) without changing 25 μM clomiphene-induced [Ca2+]i increase. 17β-estradiol and tamoxifen increased [Ca2+]i by causing Ca2+ influx and Ca2+ release because their responses were partly reduced by removing extracellular Ca2+. In contrast, clomiphene solely induced Ca2+ release. The effect of the lignans on these two Ca2+ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca2+]i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca2+ release, and 17β-estradiol-induced Ca2+ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca2+ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca2+ signaling in human neutrophils in a multiple manner.  相似文献   

3.
The mechanisms of intracellular calcium store depletion and store-related Ca2+ 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 Ca2+ by BAPTA/AM (50 μM), or La3+ (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 Ca2+ 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 Ca2+ concentrations ([Ca2+]i).  相似文献   

4.
Glucose-induced insuline release, glucose-induced rises in intracellular free Ca2+ concentration ([Ca2+]i), and voltage-dependent Ca2+ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca2+]i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca2+ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca2+]2 rise and, like deprivation of extracellular Ca2+, prevented the glucose-induced rise in [Ca2+]i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca2+ channels was demonstrated directly by measuring Ca2+ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca2+]1 after membrane depolarization by 45 mMm K+ or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca2+ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.  相似文献   

5.
Extracellular ATP dose dependently stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E2 (PGE2). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca2+ 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 PGE2 synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE2. These results strongly suggest that extracellular ATP stimulates Ca2+ 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 PGE2 synthesis.  相似文献   

6.
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 B2 receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B1 receptor antagonist [des-Arg10]-HOE140 (26.3±15.5%). Intracellular Ca2+ 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-Arg10]-HOE140. The elevation of intracellular Ca2+ by ovokinin(2–7) was dependent on Ca2+ entry from extracellular space as it was reduced in a Ca2+-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca2+ increase by the peptide. PA phosphohydrolase and phospholipase A2 inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B2 bradykinin receptors.  相似文献   

7.
Stably transfected PC12D cell lines overexpressing a catecholamine biosynthesis regulatory protein, V-1, were used to examine the functional role of V-1 in catecholamine secretion. High K+-induced dopamine secretion in V-1 overexpressing clones was shown to be markedly potentiated compared with control clones carried with a vector alone. As assayed intracellular calcium concentration ([Ca2+]i) using fura-PE3, V-1 overexpression was observed to enhance high K+-elicited [Ca2+]i elevation. Electron microscopic analysis revealed an increase in dense-cored vesicle formation by V-1 overexpression. These results suggest that the enhancement of high K+-induced dopamine secretion by V-1 overexpression results from the potentiation of high K+-induced [Ca2+]i elevation and the increase in the number of dense-cored vesicles.  相似文献   

8.
Increase in cytoplasmic cyclic AMP concentration stimulates Ca2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca2+ 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 Ca2+ influx. Cyclic AMP evoked discharge of Ca2+ 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 Ca2+, while Ca2+ lower than 0.1 μM did not affect the Ca2+-release. The Ca2+ flux across plasma membrane was restored from this Ca2+-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca2+ 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 Ca2+ channel.  相似文献   

9.
We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca2+]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 [Ca2+]i. The mechanical stress-induced increase in [Ca2+]i in the presence of LPA was inhibited by removing extracellular Ca2+ or by addition of Gd3+, 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 Ca2+ influx through cation-selective mechanosensitive channels, but does not sensitise Ca2+ 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 Ca2+ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca2+ 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 [Ca2+]i induced by mechanical stress.  相似文献   

10.
Luis Vaca 《FEBS letters》1996,390(3):289-293
Utilizing the whole-cell configuration of the patch-clamp technique the effect of calmodulin (CaM) on thapsigargin-induced Ca2+ current has been studied. Addition of several concentrations of CaM to the patch pipette induced concentration-dependent inhibition of thapsigargin-induced Ca2+ current in bovine aortic endothelial cells. The effect of CaM was Ca2+ dependent and was not observed when the intracellular Ca2+ was buffered to 1 nM with EGTA. CaM produced two major effects on the thapsigargin-induced Ca2+ current. First CaM slow down activation of the current by thapsigargin from a control value of 16 ± 5 to 31 ± 6 s with 1 μM CaM in the pipette solution. The second effect of CaM was to reduce the current amplitude in a concentration-dependent manner. The inhibition of Ca2+ current was observed at the peak of the current and at the sustained current level. The reduction of current at the sustained level was observed 15–20 s after onset of the thapsigargin response. The half inhibitory concentration determined from these experiments was 0.1 μM. These results indicate that CaM can modulate thapsigargin-induced Ca2+ current in this endothelium, suggesting a possible role for CaM in the regulation of store-operated Ca2+ influx.  相似文献   

11.
The Ca2+-mobilizing metabolite cyclic ADP-ribose (cADPR) has been shown to release Ca2+ 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 Ca2+ 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 Ca2+ release channels. Thus Ca2+ release induced by cADPR in skeletal muscle sarcoplasmic reticulum may not involve opening of ryanodine receptors.  相似文献   

12.
By mediating the Ca2+ influx that triggers exocytotic fusion, Ca2+ channels play a central role in a wide range of secretory processes. Ca2+ channels consist of a complex of protein subunits, including an 1 subunit that constitutes the voltage-dependent Ca2+-selective membrane pore, and a group of auxiliary subunits, including β, γ, and 2–δ subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca2+ channels are constituted by different combinations of 1 subunits (of which 10 have been identified) and auxiliary subunits, particularly β (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca2+ channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca2+ channel structure and function in exocytotic secretion. We discuss Ca2+ channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca2+ channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca2+ channel structure and function is central to our understanding of the fundamental process of exocytotic secretion.  相似文献   

13.
Ca2+ mobilization elicited by simulation with brief pulses of high K + were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca2+ changes across the cell, suggesting the presence of significant spatial Ca2+ gradients. Treatment with 20 μM ryanodine, an inhibitor of Ca2+ release from the SR, and 10 μM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca2+]i elicited by membrane depolarization. The overall spatial distribution of [Ca2+]i changes appeared unchanged. Ca2+ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 μM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the exchange mechanism. Conversely, when the reversal potential of the exchange was shifted to negative potentials by lowering [Na+]0 or by increasing [Na+]i by treatment with 20 μM monensin, the amplitude of these Ca2+ transients increased. Ca2+ transients elicited by membrane depolarization and largely mediated by reverse operation of Na+-Ca2+ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These findings suggest that in intact guinea pig cardiac cells, Ca2+ influx through the exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.  相似文献   

14.
W K Pollock  S O Sage  T J Rink 《FEBS letters》1987,210(2):132-136
We investigated the restoration of [Ca2+]i in fura-2-loaded human platelets following discharge of internal Ca2+ stores in the absence of external Ca2+. After stimulation by thrombin [Ca2+]i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca2+]i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca2+]i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca2+ efflux, perhaps via protein kinase C actions on a plasma membrane Ca2+ pump.  相似文献   

15.
The effects of PACAPs on [Ca2+]i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca2+]i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca2+]i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca2+. Carbachol also increased [Ca2+]i in a biphasic manner, but it mobilized intracellular Ca2+ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca2+ entry, this being accompanied by a more marked and prolonged elevation of IP3 and IP4 than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca2+ handling through PACAP receptors than with muscarinic M1 receptors.  相似文献   

16.
In this study we seek to elucidate the interaction of capsaicin with the calmodulin mediated signal pathways in macrophages, by comparing its action on macrophage functions with a known calmodulin antagonist, fluphenazine. Kinetics of capsaicin uptake by macrophages (103 cells) revealed that a maximum of 200 μM capsaicin was taken up within 10 min. Ca2+ ionophore triggered generation of superoxide anion and hydrogen peroxide by macrophages was inhibited in a dose-dependent manner by fluphenazine (IC50, 20 μM and 12 μM, respectively) and also by capsaicin (IC50, 30 μM and 9 μM, respectively), suggesting an involvement of calmodulin in the regulation of NADPH oxidase. In vitro both fluphenazine and capsaicin inhibited Ca2+-Mg2+ ATPase and cAMP-phosphodiesterase from macrophages and this inhibition was reversed by exogenous addition of calmodulin. Fluorescence studies revealed a direct Ca2+ dependent interaction of capsaicin with calmodulin. From these results we suggest that capsaicin acts via calmodulin to inhibit stimulus-induced macrophage oxidative burst and also that calmodulin regulates the oxidative burst in macrophages.  相似文献   

17.
There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca2+]i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca2+]i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y2 receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca2+-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y2 receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.  相似文献   

18.
Ravier MA  Henquin JC 《FEBS letters》2002,530(1-3):215-219
Glucose-induced insulin secretion is pulsatile. We investigated how the triggering pathway (rise in β-cell [Ca2+]i) and amplifying pathway (greater Ca2+ efficacy on exocytosis) influence this pulsatility. Repetitive [Ca2+]i pulses were imposed by high K++ diazoxide in single mouse islets. Insulin secretion (measured simultaneously) tightly followed [Ca2+]i changes. Lengthening [Ca2+]i pulses increased the duration but not the amplitude of insulin pulses. Increasing glucose (5–20 mmol/l) augmented the amplitude of insulin pulses without changing that of [Ca2+]i pulses. Larger [Ca2+]i pulses augmented the amplitude of insulin pulses at high, but not low glucose. In conclusion, the amplification pathway ensures amplitude modulation of insulin pulses whose time modulation is achieved by the triggering pathway.  相似文献   

19.
The intracellular free Ca2+ ion concentration ([Ca2+]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 [Ca2+]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca2+ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca2+]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 [Ca2+]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 [Ca2+]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10-4M or 10-3M) induced [Ca2+]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca2+ free medium, the source of the increase in [Ca2+]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca2+]i. These data strongly suggest that ACh exerted a direct action on [Ca2+]i in normal rat pancreatic β-cells and support a role for Ca2+ as a second messenger in the action of ACh.  相似文献   

20.
Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [125I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K+-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca2+-channel activator BAYK-8644, and blocked K+- and BAYK-8644-evoked rise in the intracellular Ca2+ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca2+ channel-mediated Ca2+ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号