共查询到20条相似文献,搜索用时 31 毫秒
1.
Julio Berrios Nelson Osses Carlos Opazo Gloria Arenas Luis Mercado Dale J Benos Juan G Reyes 《Biology of the cell / under the auspices of the European Cell Biology Organization》1998,90(5):391-398
Intracellular calcium, [Ca2+]i, can regulate meiotic progression of mammalian oocytes. However, the role of [Ca2+]i in the regulation of the spermatogenic process and its cellular homeostatic mechanisms in spermatogenic cells has not been elucidated. Using intracellular fluorescent probes for Ca2+ and immunodetection of plasma membrane (PM) Ca2+-ATPases, we report that: a) rat round spermatids maintain [Ca2+]i levels of 60 ± 5 nM (SEM), as estimated with fluo-3 in single cells or fura-2 in cells in suspension; b) these cells regulate [Ca2+]i by actively extruding it using a PM Ca2+-ATPase; c) rat spermatids also actively transport Ca2+ by sarco-endoplasmic reticulum type ATPases (SERCA); d) rat spermatids possess non-mitochondrial intracellular Ca2+i stores insensitive to thapsigargin but releasable by ionomycin; and e) rat spermatids do not activate Ca2+ entry mechanisms by the release of Ca2+ from SERCA-regulated stores. These results demonstrate that rat round spermatids can generate modulated intracellular Ca2+ signals upon activation of Ca2+ channels or Ca2+ release from intracellular stores. 相似文献
2.
Ross WN Nakamura T Watanabe S Larkum M Lasser-Ross N 《Cellular and molecular neurobiology》2005,25(2):283-295
Synaptically activated postsynaptic [Ca2+]i increases occur through three main pathways: Ca2+ entry through voltage-gated Ca2+ channels, Ca2+ entry through ligand-gated channels, and Ca2+ release from internal stores. The first two pathways have been studied intensively; release from stores has been the subject of more recent investigations.Ca2+ release from stores in CNS neurons primarily occurs as a result of IP3 mobilized by activation of metabotropic glutamatergic and/or cholingergic receptors coupled to PLC. Ca2+ release is localized near spines in Purkinje cells and occurs as a wave in the primary apical dendrites of pyramidal cells in the hippocampus and cortex. The amplitude of the [Ca2+]i increase can reach several micromolar, significantly larger than the increase due to backpropagating spikes.The large amplitude, long duration, and unique location of the [Ca2+]i increases due to Ca2+ release from stores suggests that these increases can affect specific downstream signaling mechanisms in neurons. 相似文献
3.
The interplay between Ca2+ efflux mechanisms of the plasma membrane (PM) and transient changes of the cytosolic concentration of ionized calcium ([Ca2+]i) was studied in suspensions of human neutrophils loaded with the [Ca2+]i indicator, Fura-2. To reveal Ca2+ efflux through PM the interference of intracellular Ca stores was prevented by preincubating the cells in the presence of EGTA, thapsigargin, and ionomycin. Addition of econazole prevented varying entry of divalent cations regulated by the filling state of Ca stores. The preincubation seemed to empty and permeabilize virtually all Ca stores, ensuring that the monitored changes of [Ca2+]i were caused exclusively by PM Ca2+ transporters. Following preincubation, the addition of CaCl2 induced, mediated by ionomycin, a transient rise of [Ca2+]i, a spike, eventually decreasing to an intermediary [Ca2+]i level. The ATP-dependent decrease of [Ca2+]i terminating the spike was abolished by the calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), but not by the protein kinase C inhibitor, staurosporine, nor by Na+-free medium, suggesting that neither activity of protein kinase C nor
exchange was necessary for generation of the Ca2+ spike. In conclusion, the PM Ca2+ pump was responsible for the Ca2+ spike by responding to the rapid rise of [Ca2+]i by a delayed activation, possibly involving calmodulin. This characteristic feature of the PM pump may be important for the generation of cellular [Ca2+]i spikes in general. 相似文献
4.
Peter W. Marks Benjamin A. Kruskal Frederick R. Maxfield 《Journal of cellular physiology》1988,136(3):519-525
The calcium-sensitive fluorescent indicator fura-2 and a microscope equipped for rapidly changing excitation wavelengths were used to look at the effects of growth factors on cytosolic free calcium ([Ca2+]i,) in NRK-49F cells. In these cells bradykinin induced a rapid increase in [Ca2+]i, which generally decayed to near basal [Ca2+]i within 3 minutes. The initial rise in [Ca2+]i in response to bradykinin was relatively independent of extracellular calcium; however, the decay to basal [Ca2+]i was more rapid in the absence of extracellular calcium. Measurements made on individual cells showed a heterogeneity in the response to bradykinin. Epidermal growth factor (EGF) had no effect on [Ca2+]i in NRK-49F cells when added alone in the presence of extracellular calcium. Simultaneous addition of bradykinin and EGF produced a more prolonged increase in [Ca2+]i than bradykinin alone. The prolongation was dependent on the presence of extracellular calcium and did not occur in its absence. Transient increases in [Ca2+]i occurring after the initial peak were occasionally seen in these cells. Our results indicate that there is rapid interaction between the signaling mechanisms for bradykinin and EGF. When this occurs, one effect is the transport of calcium into the cell from the extracellular environment, causing a more prolonged rise in [Ca2+]i. This effect occurs within 1 minute after combined addition of bradykinin and EGF. 相似文献
5.
Changes in [Ca2+]i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca2+]i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca2+]i response was due to the release of Ca2+ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca2+]i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca2+]i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca2+]i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca2+ release may be due to nanopore formation in the endoplasmic reticulum. 相似文献
6.
Stéphane Allouche Jocelyne Polastron Philippe Jauzac 《Journal of neurochemistry》1996,67(6):2461-2470
Abstract: Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca2+-channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing δ-opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid-induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gi, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca2+-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, δ-opioid receptors mobilize Ca2+ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of Gi/Go and Gs proteins and protein kinase A activation. 相似文献
7.
This study examined [Ca2+]i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced
oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca2+]i and K
Ca currents in both cell types. Low IP3, but not thapsigargin (Tg), induced [Ca2+]i oscillations, whereas Tg blocked CCh-stimulated [Ca2+]i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca2+ from HSY cells (i) did not affect CCh-stimulated [Ca2+]i oscillations or internal Ca2+ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca2+]i into oscillations. CCh- or thapsigargin-induced Ca2+ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic
reticulum Ca2+ pump (SERCA) and inositoltrisphosphate receptors (IP3Rs) than HSG cells.
These data demonstrate that [Ca2+]i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca2+ from, and release of Ca2+ into, the cytosol by the SERCA and IP3R activities, respectively. In HSY cells, Ca2+ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca2+]i. In HSG cells, [Ca2+]i oscillations directly depend on Ca2+ influx; Ca2+ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences
in the mechanism of [Ca2+]i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca2+ stores.
Received: 30 October 2000/Revised: 26 February 2001 相似文献
8.
The mechanism underlying the generation of cytosolic free Ca2+ ([Ca2+i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca2+]i oscillations with variable patterns. The bombesin-induced [Ca2+]i oscillations could last more than 1 h and glucose was required for maintaining these [Ca2+ fluctuations. Bombesin-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+ entry and were attenuated by membrane hype rpolarization or by L-type Ca2+ channel blockers. These [Ca2+]i oscillations were apparently not associated with fluctuations in plasma membrane Ca2+ permeability as monitored by the Mn2+ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca2+ stores, respectively, by inhibiting Ca2+-ATPase of endoplasmic reticulum and by affecting Ca2+-induced Ca2+ release, disrupted bombesin-induced [Ca2+]i oscillations. 4-chloro-m-resol raised [Ca2+]i by mobilizing an intracellular Ca2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca2+]i It raised [Ca2+]i by promoting Ca2+ entry while inhibiting bombesin-elicited [Ca2+]i oscillations. Our results suggest that in bombesin-elicited [Ca2+]i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca2+ stores; and (ii) the Ca2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca2+]i oscillations. The interplay between intracellular Ca2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca2+ entry determines the [Ca2+]i oscillations evoked by bombesin. 相似文献
9.
Changes in cytosolic Ca2+ concentration ([Ca2+]i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca2+, the increase in [Ca2+]i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca2+ wave toward the basolateral region. The [Ca2+]i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca2+ was eliminated from the extracellular medium, the Ca2+ wave was a result of Ca2+ release from intracellular stores. The magnitude and velocity of the Ca2+ wave decreased with decreasing concentration of CCh, and the increase in [Ca2+]i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca2+ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca2+ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca2+ pump, caused a slow and homogeneous increase in [Ca2+]i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca2+]i suggesting that the inositol-1,4,5-trisphosphate (InsP3) and ThG-sensitive Ca2+ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP3 is essential to trigger the Ca2+ wave and that the development of the Ca2+ wave may be attributed to regional differences in InsP3 sensitivity of Ca2+ stores. The agonist-induced Ca2+ wave is probably a general phenomenon in exocrine acinar cells. 相似文献
10.
To investigate Ca2+ uptake by Ca2+-depleted bovine chromaffin cells we depleted these cells of Ca2+ by incubating them in Ca2+-free buffer, then measured changes in cytoplasmic Ca2+ concentration ([Ca2+
1)45Ca2+ uptake, and Mn2+ uptake in response to added Ca2+ or MN2+. In depleted cells, the increase in [Ca2+]i after Ca2+ addition, and the Mn2+ and45Ca2+ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca2+ pools in depleted cells increased after Ca2+ addition. The times for [Ca2+]i rise and Mn2+ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca2+ stores. In Ca2+-depleted cells and cells which had been loaded with BAPTA,45Ca2+ uptake was much higher than in control cells. These results suggest that extracellular Ca2+ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn2+ influx depended on the level of filling of the Ca2+ stores, suggesting that some signalling takes place between the intracellular stores and Ca2+ entry pathways through the plasma membrane.Abbreviations used BAPTA
1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid
- BAPTA/AM
acetoxymethyl ester of BAPTA
- [Ca2+]i
cytosolic Ca2+ concentration
- IP3
inositol 1,4,5-trisphosphate
- tBHQ
2,5-di-(t-butyl)-1,4-benzohydroquinone
This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 相似文献
11.
Roberto Sánchez-Olea Herminia Pasantes-Morales Prof. Arne Schousboe 《Neurochemical research》1993,18(2):147-152
The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca2+]i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca2+ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca2+]i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca2+]i after expoxure to 50 mM K+ which was of the same magnitude as the increase in [Ca2+]i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca2+ channels verapamil had no effect on the hyposmolarity induced increase in [Ca2+]i. On the contrary, this increase in [Ca2+]i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca2+ channels Mg2+ and La3+. Dantrolene which prevents release of Ca2+ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca2+ which represents an influx of Ca2+ rather than a release of Ca2+ from internal, dantrolene sensitive stores. 相似文献
12.
Camillo Peracchia 《The Journal of membrane biology》1990,117(1):79-89
Summary Electrical uncoupling of crayfish septate axons with acidification has been shown to cause a substantial increase in [Ca2+]i which closely matches in percent the increase in junctional resistance. To determine the origin of [Ca2+]i increase, septate axons have been exposed either to drugs that influence Ca2+ release from internal stores, caffeine and ryanodine, or to treatments that affect Ca2+ entry. A large increase in junctional resistance and [Ca2+]i maxima above controls resulted from addition of caffeine (10–30mm) to acetate solutions, while a substantial decrease in both parameters was observed when exposure to acetate-caffeine was preceded by caffeine pretreatment. In contrast, ryanodine (1–10 m) always caused a significant decrease in junctional resistance and [Ca2+]i maxima when applied either together with acetate or both before and with acetate. Calcium channel blockers such as La3+, Cd2+ and nisoldipine had no effect, while an increase in the [Ca2+] of acetate solutions either decreased junctional resistance and [Ca2+]i maxima or had no effect. The data suggest that cytoplasmic acidification causes an increase in [Ca2+]i by releasing Ca2+ from caffeine and ryanodine-sensitive Ca2+ stores. The increase in [Ca2+]i results in a decrease in gap junction conductance. 相似文献
13.
The ryanodine-sensitive intracellular Ca2+ stores are known to play a major role in excitation-contraction coupling in muscles. Although these stores are also abundantly
present in central neurons, their functional role in these cells remains unclear. Using fluorometric digital imaging of the
intracellular Ca2+ concentration ([Ca2+]
i
) in rat hippocampal slices, we investigated the dynamic properties of the ryanodine-sensitive Ca2+ stores inCA1 hippocampal pyramidal cells. We found that at rest the ryanodine-sensitive Ca2+ stores are functioning predominantly as a “sink” for Ca ions responding to an increase in [Ca2+]
i
with an increase in the amount of Ca ions accumulated inside the stores. If, however, [Ca2+]
i
increases significantly, as happens during strong neuronal discharges, the ryanodine-sensitive Ca2+ stores respond with Ca2+ release, thus acting as an amplifier of the intracellular Ca2+ signal. 相似文献
14.
M. V. Kustov V. V. Tsvilovskii A. V. Zholos M. F. Shuba T. B. Bolton 《Neurophysiology》2006,38(1):1-8
Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca2+]i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications
of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and
100.0 μM ATP) induced a rise in the [Ca2+]i. Carbachol evoked an increase in the [Ca2+]i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise
in the [Ca2+]i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly
differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally
Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca2+]i in myocytes. The absence of the slow component in the [Ca2+]i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is
not related to activation of the entry of Ca2+ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP
induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca2+]i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted
only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises
in the [Ca2+]i induced by both CCh and ATP; therefore, the release of Ca2+ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the
difference in the release of Ca2+ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and
complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional
effects of the above-mentioned neurotransmitters on smooth muscles.
Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006. 相似文献
15.
《Cell calcium》1996,20(3):303-314
In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca2+]i) followed by a sustained rise. In Ca2+-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca2+]i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca2+]i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca2+]i and inhibited the following [Ca2+]i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca2+]i. Muscarine-induced sustained [Ca2+]i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca2+]i rise and Mn2+ influx in response to muscarine were suppressed by a voltage-dependent Ca2+ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca2+ entry, but also Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca2+ channels may function as one of the Ca2+ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells. 相似文献
16.
Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons 总被引:8,自引:2,他引:6
Abstract: A large body of evidence suggests that disturbances of Ca2+ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca2+]i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage-sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca2+]i but via different mechanisms. By using the drug dantrolene, it has been shown that 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) apparently exclusively stimulates Ca2+ influx through agonist-operated calcium channels and voltage-operated calcium channels. Increased [Ca2+]i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca2+]i may be attributed to a release of Ca2+ from intracellular stores. Quisqualate (QA) stimulates Ca2+ release from an intracellular store that is independent of Ca2+ influx; presumably this store is activated by inositol phosphates. The increase in [Ca2+]i due to exposure to glutamate or N-methyl-d -aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca2+ release from internal stores. Only one of the latter stores is dependent on Ca2+ influx with regard to release of Ca2+, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca2+ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca2+ pool(s). By using this drug it has been possible to show to what extent these Ca2+ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca2+ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca2+ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex. 相似文献
17.
Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells 总被引:2,自引:0,他引:2
Xi Guo Dennis A. Przywara Taruna D. Wakade Arun R. Wakade 《Journal of neurochemistry》1996,67(1):155-162
Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca2+ sources: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) Intracellular stores of Ca2+ were depleted by soaking cells in Ca2+-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca2+]i and secretion in cells with or without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca2+ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca2+]i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ~20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca2+]i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland. 相似文献
18.
Measurements of Ca2+ influx and [Ca2+]i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca2+ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca2+]i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca2+]i levels were dependent on extracellular Ca2+. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca2+ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca2+ channels. The trivalent cation La3+, a non-specific blocker of plasma membrane Ca2+ channels, eliminated the rPTTH-stimulated increase of [Ca2+]i levels in PG cells and so did amiloride, an inhibitor of T-type Ca2+ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca2+]i levels, which was also dependent on extracellular Ca2+ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca2+ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca2+]i levels and one agent’s mediated increase of [Ca2+]i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca2+ release from inositol 1,4,5 trisphosphate (IP3)-sensitive Ca2+ stores, blocked the rPTTH-stimulated increases of [Ca2+]i levels, suggesting an involvement of IP3 in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca2+]i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca2+]i levels, filling state of IP3-sensitive intracellular Ca2+ stores and the PTTH-receptor’s-mediated Ca2+ influx. 相似文献
19.
The P2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca2+]i due to Ca2+ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca2+ release was accompanied by a Ca2+- dependent regulatory activation of Na+/Ca2+ exchange activity: Addition of extracellular Ca2+ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca2+]i in Na+-free media (Li+ substitution) compared to Na+-containing media; no differences between Na+- and Li+-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na+ and Ca2+, the ATP-evoked rise in [Ca2+]i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca2+]i which eventually came to exceed the declining [Ca2+]i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba2+ influx, consistent with regulatory activation of the Na+/Ca2+ exchanger. The acceleration of Ba2+ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na+ or when the Ca2+ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca2+]i under Na+-free conditions, but did not inhibit the ATP-evoked stimulation of Ba2+ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca2+ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca2+ release from intracellular stores through regulatory activation by increased [Ca2+]i. In the absence of extracellular Ca2+, the stimulation of exchange activity is short-lived and follows the time course of the [Ca2+]i transient; in the presence of extracellular Ca2+, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca2+ entry. 相似文献
20.
An increased cytoplasmic Ca2+ concentration ([Ca2+]i) has been implicated in the pathogenesis of cystic fibrosis. We compared the [Ca2+]i levels of normal and cystic fibrosis peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using quin 2, an internally trapped indicator. The [Ca2+]i levels of normal and cystic fibrosis cells were not significantly different. The ionophore-releasable intracellular Ca2+ stores were also comparable in both types of individual. 相似文献