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1.
Glucose-induced insuline release, glucose-induced rises in intracellular free Ca 2+ concentration ([Ca 2+] i), and voltage-dependent Ca 2+ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca 2+] i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca 2+ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca 2+] 2 rise and, like deprivation of extracellular Ca 2+, prevented the glucose-induced rise in [Ca 2+] i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca 2+ channels was demonstrated directly by measuring Ca 2+ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca 2+] 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 Ca 2+ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells. 相似文献
2.
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
3.
By mediating the Ca 2+ influx that triggers exocytotic fusion, Ca 2+ channels play a central role in a wide range of secretory processes. Ca 2+ channels consist of a complex of protein subunits, including an 1 subunit that constitutes the voltage-dependent Ca 2+-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 Ca 2+ 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 Ca 2+ 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 Ca 2+ channel structure and function in exocytotic secretion. We discuss Ca 2+ channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca 2+ 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 Ca 2+ channel structure and function is central to our understanding of the fundamental process of exocytotic secretion. 相似文献
4.
We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca 2+ ([Ca 2+] i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the β-glucuronidase substrate 4-methylumbelliferyl-β-
-glucuronide. We found that the transient rise in [Ca 2+] i induced by fMLP is a necessary signal to obtain to obtain maximal degranulation. When this Ca 2+ transient is prevented by the Ca 2+ chelator BAPTA, degranulation can still be induced by a stimulated Ca 2+ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of β-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca 2+ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca 2+] i], an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils. 相似文献
5.
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
6.
Ca 2+ 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 Ca 2+ changes across the cell, suggesting the presence of significant spatial Ca 2+ gradients. Treatment with 20 μM ryanodine, an inhibitor of Ca 2+ release from the SR, and 10 μM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca 2+] i elicited by membrane depolarization. The overall spatial distribution of [Ca 2+] i changes appeared unchanged. Ca 2+ 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 Ca 2+ transients increased. Ca 2+ transients elicited by membrane depolarization and largely mediated by reverse operation of Na +-Ca 2+ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These findings suggest that in intact guinea pig cardiac cells, Ca 2+ 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
7.
We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca 2+ ([Ca 2+] i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca 2+] i which was abolished by omission of Ca 2+ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca 2+] i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca 2+ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca 2+] i and DAG had returned to control values suggesting that additional mechanisms may be involved. 相似文献
8.
Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations of cytosolic free Ca 2+ concentration ([Ca 2+] i) produced by 2,5-di(tert-butyl)-l.4-benzohydroquinone (tBuBHQ), a potent inhibitor of hepatic microsomal Ca 2+ sequestration (Moore. G.A., McConkey. D.J., Kass, G.E.N., OBrien, P.J. and Orrenius, S. FEBS Lett., 224, 331-336). (1987). Addition of tBuBHQ to isolated hepatocytes caused a rapid increase in [Ca 2+] i which was similar in magnitude to the [Ca 2+] i elevation induced by the Ca 2+ mobilizing hormone, vasopressin. In contrast with vasopressin which caused a Ca 2+ transient, tBuBHQ elevated [Ca 2+] i to a new steady state that was maintained for up to 15-20min. When vasopressin was administered during the tBuBHQ-induced period of elevated [Ca 2+] i. [Ca 2+] i, rapidly returned to basal levels. Similarly, if vasopressin was administered just prior to tBuBHQ, the resultant tBuBHQ-dependent change in [Ca 2+] i was transient. and not sustained. The hydroquinone mobilized the same intracellular Ca 2+ pool as inositol 1,4,5-trisphosphate. but tBuBHQ did not produce any detectable inositol polyphosphate accumulation. IBuBHQ stimulated glucose release from perifused hepatocytes. mimicking the effect of vasopressin. In the perfused liver, tBuBHQ infusion produced a single, slow and prolonged release of Ca 2+ into the perfusate and inhibition of subsequent vasopressin-induced Ca 2+ effluxes. Inhibition of the response to vasopressin was reversed over time, and closely correlated with the extent of inhibition of both Ca 2+ sequestration and (Ca 2+-Mg 2+)-ATPase activity in microsomes isolated from the isolated perfused liver. The present results are consistent with tBuBHQ inhibiting ATP-dependent Ca 2+ sequestration by a direct effect on the endoplasmic reticular Ca 2+ pump, which results in net Ca 2+ release and elevation of [Ca 2+] i. Furthermore. vasopressin appears to stimulate active removal of increased [Ca 2+] from the hepatocyte cytosol by a mechanism which does not depend on reuptake of Ca 2+ into the endoplasmic reticulum
2,5-Di( tert-butyl) -l,4-benmhydroquinone. calcium. hepatocytes. perfused liver, endoplasmic reticulum 相似文献
9.
A new compound containing a cubane tungsten chalcogenide cluster [W 4(μ 3-Te) 4(CN) 12] 6− and Ca 2+ complex units has been prepared by the reaction of aqueous solution of K 6[W 4(μ 3-Te) 4(CN) 12] · 5H 2O with the solution of a Ca(NO 3) 2 and phen(1,10-phenanthroline) (1:2 molar ratio) in a solvent mixture of H 2O/EtOH. The structure of [{Ca(phen) 2(H 2O)}{Ca(phen)(H 2O) 4}{Ca(phen) 2(H 2O) 3}][W 4Te 4(CN) 12] · 5H 2O 1 has been determined by X-ray crystallography. Compound 1 contains [{Ca(phen)(H 2O) 4}{Ca(phen) 2(H 2O) 3}][W 4(μ 3- Te) 4(CN) 12] units bridged by {Ca(phen) 2(H 2O)} 2+ units to form an one-dimensional zigzag chain structure. Interestingly, compound 1 showed a heterogeneous catalytic activity in the transesterification of a range of esters with methanol under the mild conditions. Moreover, it can be reused without any loss of activity through 10 runs with ester. 相似文献
10.
We examined the profile of two bombesin (BN) antagonists, (CH 3) 2CHCO-His-Trp-Ala-Val-
-Ala-His-Leu-NHCH 3] (ICI 216140) and [
-Phe 6,des-Met 14]BN(6–14)ethylamide (DPDM-BN EA), against neuromedin B-induced Ca 2+ mobilization in the small cell lung cancer (SCLC) line NCI-H345. Neuromedin B (NMB), a BN-like peptide sharing sequence homology with ranatensin, elicited a concentration-dependent Ca 2+ release (in part) from intracellular stores. Sequential addition of NMB attenuated Ca 2+ mobilization. Desensitization occurred between BN and NMB; depletion of intracellular Ca 2+ is a likely mechanism because thapsigargin stimulated Ca 2+ release after a maximally desensitizing dose of NMB. ICI 216140 and DPDM-BN EA competitively inhibited BN-induced Ca 2+ transients. In contrast, these compounds antagonized NMB-stimulated Ca 2+ transients in a noncompetitive manner. The pharmacological profiles obtained support receptor heterogeneity for BN-like peptides on this SCLC line, underscoring the need for thorough examination of dose-response relationships when investigating effects of BN analogues on intact cells. 相似文献
11.
Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca 2+ concentration ([Ca 2+] i) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca 2+] i increase via Ca 2+ release from the sarcoplasmic reticulum through InsP 3 receptors. Several models have been developed to explain the characteristics of InsP 3-induced [Ca 2+] i responses, in particular Ca 2+ oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca 2+ handling, i.e., InsP 3 receptors, SERCAs, mitochondria and Ca 2+-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca 2+ handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP 3 receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca 2+ clearance in the pattern of [Ca 2+] i variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca 2+ influx. Stimulation of this model by simulated increase in [Ca 2+] i predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca 2+ channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents. 相似文献
12.
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
13.
The activation and deactivation of Ca 2+- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved F?rster resonance energy transfer (FRET), we determined the occurrence of Ca 2+-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca 2+ concentrations ([Ca 2+] i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca 2+] i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation. 相似文献
14.
A wasp venom, mastoparan, rapidly increased the cytosolic free Ca 2+ concentration ([Ca 2+] i) and activated phosphorylase in rat hepatocytes in a concentration-dependent manner. Mastoparan could increase [Ca 2+] i even in the absence of extracellular Ca 2+, but a larger increase was observed in the presence of extracellular Ca 2+. Thus, mastoparan mobilized Ca 2+ from intracellular and extracellular Ca 2+ stores. It also activated inositol triphosphate (IP 3) accumulation, but did not stimulate cAMP production. From these results, we conclude that mastoparan activates rat hepatic glycogenolysis mediated by the accumulation of IP 3, which causes an increase of [Ca 2+] i but not that mediated by cAMP. 相似文献
15.
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
16.
A comparison of Cd 2+ and Ca 2+ effects on in vitro rat liver mitochondria function and a further study of their interaction were conducted. Similarity and distinction in action of rotenone, oligomycin, N-ethylmaleimide, dithiothreitol, catalase, dibucaine, ruthenium red, cyclosporin A (CsA), and ADP on Cd 2+ and/or Ca 2+-induced mitochondrial dysfunction were revealed. We found that rotenone exerted a strong protective action both against Ca 2+ and Cd 2+-produced mitochondrial membrane permeabilization (MMP). In contrast to Ca 2+, catalase and dibucaine did not influence on main Cd 2+ effects. In NH 4NO 3 medium N-ethylmaleimide (NEM) at low concentrations increased markedly Cd 2+-produced swelling of non-energized mitochondria, whereas it exhibited a partial reversal effect following energization. In sucrose medium low [NEM] did not change Cd 2+-produced mitochondrial swelling. High [NEM] promoted synergistic increase of the Cd 2+-produced swelling in NH 4NO 3 medium; all above effects were reversed (and prevented) by dithiothreitol, DTT. We shown also that when exogenous Ca 2+ and P i were simultaneously present in NH 4NO 3 medium, DTT reversed only partially Cd 2+-produced swelling of succinate plus rotenone-energized mitochondria, while DTT recovery action was complete when either Ca 2+ or P i were separately administered to the Cd 2+-treated mitochondria. Besides, DTT added following a low Cd 2+ pulse in KCl medium containing exogenous Ca 2+ induced a substantial enhancing of sustained Cd 2+ stimulation of mitochondrial basal respiration and the stimulation was CsA-sensitive, while the activation promoted by low [Cd 2+] alone was totally eliminated by DTT supplement. We observed the similar respiratory activation earlier when high concentrations of Cd 2+ in the absence of added Ca 2+ were used but it was completely CsA-insensitive. A possible involvement of respiratory chain components, namely complex I (P-site) and complex III (S-site) in Cd 2+and/or Ca 2+-produced MMP was discussed. 相似文献
17.
Measurements of Ca 2+ influx and [Ca 2+] i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca 2+ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca 2+] i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca 2+] i levels were dependent on extracellular Ca 2+. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca 2+ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca 2+ channels. The trivalent cation La 3+, a non-specific blocker of plasma membrane Ca 2+ channels, eliminated the rPTTH-stimulated increase of [Ca 2+] i levels in PG cells and so did amiloride, an inhibitor of T-type Ca 2+ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca 2+] i levels, which was also dependent on extracellular Ca 2+ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca 2+ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca 2+] i levels and one agent’s mediated increase of [Ca 2+] i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca 2+ release from inositol 1,4,5 trisphosphate (IP 3)-sensitive Ca 2+ stores, blocked the rPTTH-stimulated increases of [Ca 2+] i levels, suggesting an involvement of IP 3 in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca 2+] i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca 2+] i levels, filling state of IP 3-sensitive intracellular Ca 2+ stores and the PTTH-receptor’s-mediated Ca 2+ influx. 相似文献
18.
We invetigate the mechanisms underlying the intracellular calcium pulse that occurs in response to extracellular adenosine triphosphate (ATP) in osteoclasts. We find that pre-loading of GDP-β-S abolishes the response in Ca 2+-free medium, demonstrating an internal release of Ca 2+ via a pathway that involves a G protein. GDP-β-S does not block in normal Ca 2+-containing medium, suggesting that ATP also induces a Ca 2+ influx across the cell membrane. We confirmed this using the Mn 2+ quenching technique, which shows significant opening of Ca 2+ channels. We find a smaller response to adenosine diphosphate (ADP) and 2-methylthio-ATP (2-MeSATP), but no response to β, γ-methylene-ATP (AMP-PCP), adenosine monophosphate (AMP) or uridine triphosphate (UTP). Prior application of AMP and UTP, but not AMP-PCP, blocks the response to ATP. Our resutls indicate that the receptor is a P 2 subtype that is not characteristic of any previously reported P 2 receptor or combination of P 2 receptors. 相似文献
19.
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
20.
To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs ( Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO 4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10 −8 M). Such an inhibitory effect of DFB on Ca 2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na +, and Li + did not affect the calcium uptake. Among ionophores, agents disrupting H + gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca 2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and γ-S-GTP stimulated Ca 2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca 2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca 2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells. 相似文献
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