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1.
Ca transients in cardiac myocytes measured with a low affinity fluorescent indicator, furaptra. 总被引:3,自引:1,他引:3
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Intracellular calcium ion ([Ca2+]i) transients were measured in single rat ventricular myocytes with the fluorescent indicator furaptra. Cells were voltage clamped with a single patch electrode containing the K+ salt of furaptra and fluorescence at 500 nm was measured during illumination with 350 and 370 nm light. Depolarizing voltage-clamp pulses elicited [Ca2+]-dependent fluorescent transients in 30 of 33 cells tested. The peak change in [Ca2+]i elicited by 50-ms depolarizations from -70 to +10 mV was 1.52 +/- 0.25 microM (mean +/- SEM, n = 7). The size of the [Ca2+]i transient increased in response to 10 microM isoproterenol, prolongation of the depolarization, and increasing pipette [Na+]. Because furaptra is sensitive to Ca2+ and Mg2+, changes in [Mg2+]i during the [Ca2+]i transient could not be measured. Instead, a single-compartment model was developed to simulate changes in [Mg2+] during [Ca2+] transients. The simulations predicted that a 2 microM [Ca2+] transient was accompanied by a slow increase in [Mg2+] (14-29 microM), which became larger as basal [Mg2+] increased (0.5-2.0 mM). The [Mg2+] transient reached a peak approximately 1 s after the peak of the [Ca2+] transient with the slow changes in [Mg2+] dominated by competition at the Ca2+/Mg2+ sites of Troponin. These changes in [Mg2+], however, were so small and slow that they were unlikely to affect the furaptra fluorescence signal at the peak of the [Ca2+]i transient. The [Ca2+]i transient reported by furaptra appears to be larger than that reported by other Ca2+ indicators; however, we conclude this larger transient is at least as accurate as [Ca2+]i transients reported by the other indicators. 相似文献
2.
The kinetics of the intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMCs) in rat small mesenteric arteries was investigated by confocal laser scanning microscopy using the fluorescent Ca2+ indicator fluo-3 AM. One micromole noradrenaline (NA) induced randomly distributed transient elevations of [Ca2+]i in several single VSMCs which were weakly temporally coupled. Higher NA concentrations of 3 or 10 microM, however, induced strongly synchronised [Ca2+]i oscillations in VSMCs. In preparations with intact endothelium, the synchronisation of [Ca2+]i signals was attenuated by acetylcholine (ACh) but augmented by the NO synthase antagonist L-NAME, pointing to a desynchronising effect of the endothelium even under basal conditions. In preparations with or without intact endothelium sodium nitroprusside (SNP) as well as the gap-junction uncoupler heptanol reversibly desynchronised the [Ca2+]i transients. The effect of ACh but not that of SNP was influenced by L-NAME. Propagated intracellular [Ca2+]i waves had a velocity of 25 microm/s. The phase shift of [Ca2+]i oscillations between single VSMCs were maximally 2s and independent of the distance of up to 90 microm between individual cells. Therefore, we consider intercellular [Ca2+]i waves to be too slow to account for the synchronisation of [Ca2+]i oscillations.We conclude that the coupling of [Ca2+]i signals in vascular smooth muscle cells is not constant but highly regulated by NA and by endothelium derived NO. Oscillations of vessel contraction at high sympathetic tone may be induced by synchronisation of [Ca2+]i transients of distinct VSMCs whereas endothelium derived NO inhibits vasomotion by desynchronising [Ca2+]i transients of single VSMCs. 相似文献
3.
The effects of divalent cations on voltage-activated Ca2+ channels and depolarization-evoked cytoplasmic [Ca2+] elevations were studied in pyramidal neurones isolated from the dorsal cochlear nucleus of the rat. Ca2+ currents were recorded using the whole-cell configuration of the patch-clamp technique. 10 micromol x l(-1) Cd2+ exerted a greater blocking effect on the high-voltage activated (HVA) currents than on the low-voltage activated (LVA) ones (decrease to 26.6+/-2.5% and to 87.8+/-2.1%, respectively). The blocking effect of 200 micromol x l(-1) Cd2+ was more pronounced and the difference between the effect on the HVA and LVA currents became smaller (decrease to 11.7+/-2.1% and to 32.4+/-2.7%, respectively). 200 micromol x l(-1) Ni2+ reduced the LVA component more effectively (to 77.6+/-5.4%) than the HVA one (to 86.9+/-2.6%). Cytoplasmic [Ca2+] changes were measured applying a fluorimetric technique (Fura-2). 10 micromol x l(-1) Cd2+ decreased the peak values of 50 mmol x l(-1) K+ depolarization-induced [Ca2]+i transients to 30.4+/-1.4% while 200 micromol x l(-1) Cd2+ caused a drop to 2.5+/-0.2%. 200 micromol x l(-1) Ni2+ decreased the peak of the transients to 69.6+/-2.9%. Comparison of the blocking effects of divalent cations on Ca2+ currents and [Ca2+]i transients supports further the conclusion that the depolarization-induced [Ca2+]i changes are produced mainly by the activation of the HVA Ca2+ channels. 相似文献
4.
Bramlage P Joss G Staudt A Jarrin A Podlowski S Baumann G Stangl K Felix SB Stangl V 《Biotechnology progress》2001,17(5):929-934
The contractile cycle of the cardiac myocyte is essentially controlled by the concentration of intracellular calcium ([Ca2+]i). Measurement of [Ca2+]i using Ca2+-dependent fluorescence and simultaneous monitoring of cell dynamics enable characterization of a variety of substances interacting with ion channels and contractile proteins. In this report we describe a novel method featuring up to 480 frames/s for monitoring rapid changes in cellular calcium and cell length, in which every individual cycle allows effective evaluation of major cell parameters. Computers aid in determination of time to peak (in ms), time to 50% decrease (ms), diastolic Ca2+ (relative fluorescence units, rfu), systolic Ca2+ (rfu), Ca2+ transients (rfu), DeltaCa2+/Delta(t) rise (rfu/s), and DeltaCa2+/Delta(t) fall (rfu/s). Contractile parameters are as follows: maximum cell length (microm), minimum cell length (microm), absolute cell shortening (microm), peak DeltaL/Delta(t) shortening (microm/s), and peak DeltaL/Delta(t) relaxation (microm/s). In summary, we succeeded in demonstrating that this system is a unique and valuable tool that allows simultaneous and accurate assessment of contractile parameters and of calcium movements of isolated adult cardiac myocytes. 相似文献
5.
The effect of capacitative Ca2+ entry on cytosolic free Ca2+ concentration ([Ca2+]c) was examined in calf pulmonary artery endothelial cells treated with thapsigargin. Restoration of extracellular Ca2+ evoked an overshoot in [Ca2+]c: the initial rate of Ca2+ influx was 12.4 +/- 0.5 nM/s as [Ca2+]c rose monoexponentially (time constant, tau = 36 +/- 2 s) to a peak (322 +/- 16 nM) before declining to 109 +/- 14 nM after 2000 s. Rates of Ca2+ removal from the cytosol were measured throughout the overshoot by recording the monoexponential decrease in [Ca2+]c after rapid removal of extracellular Ca2+. The time constant for recovery (tau rec decreased from 54 +/- 4 s when Ca2+ was removed after 10 s to its limiting value of 8.8 +/- 1.0 s when it was removed after 2000 s. The time dependence of the changes in tau rec indicate that an increase in [Ca2+]c is followed by a delayed (tau = 408 s) stimulation of Ca2+ removal, which fully reverses (tau approximately 185 s) after Ca2+ entry ceases. Numerical simulation indicated that the changes in Ca2+ removal were largely responsible for the overshooting pattern of [Ca2+]c. Because prolonged (30 min) Ca2+ entry did not increase the total 45Ca2+ content of the cells, an increased rate of Ca2+ extrusion across the plasma membrane most likely mediates the Ca2+ removal, and since it persists in the absence of extracellular Na+, it probably results from stimulation of a plasma membrane Ca2+ pump. We conclude that delayed stimulation of a plasma membrane Ca2+ pump by capacitative Ca2+ entry may protect cells from excessive increases in [Ca2+]c and contribute to oscillatory changes in [Ca2+]c. 相似文献
6.
Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells. 总被引:7,自引:0,他引:7
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Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes. 相似文献
7.
Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.
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The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis. 相似文献
8.
The regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was investigated by microfluorimetry in single cerebellar granule neurons exposed to various treatments (high K+, glutamate, or acetylcholine) and drugs. The responses to the treatments developed asynchronously during cell culture, with high K+ and glutamate reaching their maxima at 6 and 7 days in vitro and acetylcholine at 9 days in vitro. The biphasic [Ca2+]i transients induced by high K+ (an initial peak, followed by a plateau 30-40% of the peak, both sustained by dihydropyridine-sensitive voltage-gated Ca2+ channels) were dissipated by washing with fresh medium or, more rapidly, by addition of excess EGTA (t1/2 = 11 +/- 2 and 3 +/- 0.6 s, respectively). Compared to those induced by high K+, the [Ca2+]i transients induced by glutamate administered in Mg2(+)-free medium were much more variable. An initial peak, sustained by voltage-gated Ca2+ channels, was visible in only approximately 50% of the cells and disappeared when multiple glutamate pulses were administered. In the rest of the population, the transients were monophasic, with persistent plateaus sustained only in part (30-40%) by voltage-gated Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
A more complete understanding of calcium's role in cell division requires knowledge of the timing, magnitude, and duration of changes in cytoplasmic-free calcium, [Ca2+]i, associated with specific mitotic events. To define the temporal relationship of changes in [Ca2+]i to cellular and chromosomal movements, we have measured [Ca2+]i every 6-7 s in single-dividing Pt K2 cells using fura-2 and microspectrophotometry, coupling each calcium measurement with a bright-field observation. In the 12 min before discernable chromosome some separation, 90% of metaphase cells show at least one transient of increased [Ca2+]i, 72% show their last transient within 5 min, and a peak of activity is seen at 3 min before chromosome separation. The mean [Ca2+]i of the metaphase transients is 148 +/- 31 nM (61 transients in 35 cells) with an average duration of 21 +/- 14 s. The timing of these increases makes it unlikely that these transient increases in [Ca2+]i are acting directly to trigger the start of anaphase. However, it is possible that a transient rise in calcium during late metaphase is part of a more complex progression to anaphase. In addition to these transient changes, a gradual increase in [Ca2+]i was observed starting in late anaphase. Within the 2 min surrounding cytokinesis onset, 82% of cells show a transient increase in [Ca2+]i to 171 +/- 48 nM (53 transients in 32 cells). The close temporal correlation of these changes with cleavage is consistent with a more direct role for calcium in this event, possibly by activating the contractile system. To assess the specificity of these changes to the mitotic cycle, we examined calcium changes in interphase cells. Two-thirds of interphase cells show no transient increases in calcium with a mean [Ca2+]i of 100 +/- 18 nM (n = 12). However, one-third demonstrate dramatic and repeated transient increases in [Ca2+]i. The mean peak [Ca2+]i of these transients is 389 +/- 70 nM with an average duration of 77 s. The necessity of any of these transient changes in calcium for the completion of mitotic or interphase activities remains under investigation. 相似文献
10.
Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators. 总被引:1,自引:1,他引:0
Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters. 相似文献
11.
Conwin K Vanterpool William J Pearce John N Buchholz 《Journal of applied physiology》2005,99(3):963-971
Intracellular calcium concentration ([Ca2+]i) release from smooth endoplasmic reticulum (SER) stores plays an important role in cell signaling. These stores are rapidly refilled via influx through voltage-gated calcium channels or spontaneously via store-operated calcium channels and subsequent pumping by SER Ca2+-ATPases. We measured [Ca2+]i transients in isolated fura 2-loaded superior cervical ganglion cells from 6-, 12-, 20-, and 24-mo-old Fischer 344 rats. For rapid refilling, [Ca2+]i transients were elicited by a 1) 5-s exposure to K+, 2) caffeine to release Ca2+ from SER stores, 3) K+ to refill SER Ca2+ stores, and 4) caffeine. The percent difference between the peak and rate of rise of the first and second caffeine-evoked [Ca2+]i transient significantly declined over the age range of 12-24 mo. To estimate spontaneous refilling, cells were depolarized for 5 s with 68 mM K+ (control), followed by a 10-s exposure to 10 mM caffeine "conditioning stimulus" to deplete [Ca2+]i stores. Caffeine was then rapidly applied for 5 s at defined intervals from 60 to 300 s. Integrated caffeine-evoked [Ca2+]i transients were measured and plotted as a percentage of the K+ response vs. time. The derivative of the refilling time curves significantly declined over the age range from 12-24 mo. Overall, these data suggest that the ability of superior cervical ganglion cells to sustain release of [Ca2+]i following rapid or spontaneous refilling declines with advancing age. Compromised ability to sustain calcium signaling may possibly alter the overall function of adrenergic neurons innervating the cerebrovasculature. 相似文献
12.
Szappanos H Cseri J Deli T Kovács L Csernoch L 《Journal of biochemical and biophysical methods》2004,59(1):89-101
Changes in intracellular calcium concentration ([Ca2+]i) evoked by prolonged depolarisation (120 mM KCl) or by the application of 15 mM caffeine were measured on skeletal muscle cells in primary culture. The extrusion rate (PVmax) of calcium from the myoplasm was determined, which in turn enabled the calculation of the calcium flux (Fl) underlying the measured calcium transients. PVmax was found to increase during differentiation, from 107 +/- 10 microM/s at the early myotube stage to 596 +/- 36 microM/s in secondary myotubes. This was paralleled by a decrease in resting [Ca2+]i from 99 +/- 4 to 51 +/- 2 nM. The depolarisation-evoked Fl rose to peak and then ceased despite the continuous presence of KCl. In contrast, the caffeine-induced Fl showed a peak and a clear steady-level with a peak-to-steady ratio of 5.6 +/- 1.2. Removal of external calcium suppressed the depolarisation--induced flux by 88 +/- 5% indicating that both an influx and a release from the SR underlie the K(+)-evoked calcium transients. Subsequent applications of caffeine resulted in essentially identical fluxes indicating an efficient refilling of the internal stores. Moreover, if a depolarisation-induced calcium transient preceded the second caffeine-evoked release, the latter was significantly larger than the first suggesting that much of the calcium that entered was stored in the SR rather than extruded. 相似文献
13.
Effect of thyroid state on cytosolic free calcium in resting and electrically stimulated cardiac myocytes 总被引:2,自引:0,他引:2
The effects of the thyroid state on the cytosolic free Ca2+ concentration, [Ca2+]i, of resting and K+-depolarized cardiomyocytes were studied using the fluorescent Ca2+ indicator fura2. The mean resting [Ca2+]i in euthyroid myocytes (89 +/- 8 nM) was not significantly different from that in hyperthyroid myocytes (100 +/- 14 nM). The resting O2-consumption rate was identical for both groups when expressed per mg protein, but a 35% higher value was observed in the hyperthyroid group when expressed per cell on account of the cellular hypertrophy induced by thyroid hormone. Potassium induced depolarization (50 mM [K+]0) raised the level of [Ca2+]i by 50% in both groups. When ATP-coupled respiration was blocked with oligomycin, the 50 mM K+-induced rise in [Ca2+]i was accompanied in both groups by a 40% rise in glycolytic activity as inferred from measurement of lactate production. Ca2+-fluorescence transients were recorded from electrically stimulated myocytes of euthyroid, hyperthyroid and hypothyroid rats. The time taken to reach peak fluorescence (TPL) and that to 50% decay of peak fluorescence (RL0.5) decreased in the direction hypothyroid----hyperthyroid, indicating an increase in Ca2+ fluxes in the same direction. Isoproterenol (1 microM) enhanced the peak Ca2+ fluorescence in electrically stimulated hypothyroid and euthyroid myocytes but not in hyperthyroid myocytes. Both the TPL and RL0.5 were decreased by isoproterenol in euthyroid, but more so in hypothyroid myocytes. None of these parameters were influenced by isoproterenol in the hyperthyroid group. We conclude that (1) thyroid hormone increases neither the O2-consumption rate nor the level of [Ca2+]i of resting cardiomyocytes and (2) the effects of the beta-receptor-agonist isoproterenol on Ca2+ transients of electrically stimulated myocytes, are inversely related to the documented changes in beta-receptor density in heart tissue occurring with alterations in the thyroid state. 相似文献
14.
Johan Fredrik Brekke William F Jackson Steven S Segal 《Journal of applied physiology》2006,101(1):307-315
Intracellular calcium concentration ([Ca2+]i) governs the contractile status of arteriolar smooth muscle cells (SMC). Although studied in vitro, little is known of SMC [Ca2+]i dynamics during the local control of blood flow. We tested the hypothesis that the rise and fall of SMC [Ca2+]i underlies arteriolar constriction and dilation in vivo. Aparenchymal segments of second-order arterioles (diameter 35 +/- 2 microm) were prepared in the superfused cheek pouch of anesthetized hamsters (n = 18) and perifused with the ratiometric dye fura PE-3 (AM) to load SMC (1 microM, 20 min). Resting SMC [Ca2+]i was 406 +/- 37 nM. Elevating superfusate O2 from 0 to 21% produced constriction (11 +/- 2 microm) that was unaffected by dye loading; [Ca2+]i increased by 108 +/- 53 nM (n = 6, P < 0.05). Cycling of [Ca2+]i during vasomotion (amplitude, 150 +/- 53 nM; n = 4) preceded corresponding diameter changes (7 +/- 1 microm) by approximately 2 s. Microiontophoresis (1 microm pipette tip; 1 microA, 1 s) of phenylephrine (PE) transiently increased [Ca2+]i by 479 +/- 64 nM (n = 8, P < 0.05) with constriction (26 +/- 3 microm). Flushing blood from the lumen with saline increased fluorescence at 510 nm by approximately 45% during excitation at both 340 and 380 nm with no difference in resting [Ca2+]i, diameter or respective responses to PE (n = 7). Acetylcholine microiontophoresis (1 microA, 1 s) transiently reduced resting SMC [Ca2+]i by 131 +/- 21 nM (n = 6, P < 0.05) with vasodilation (17 +/- 1 microm). Superfusion of sodium nitroprusside (10 microM) transiently reduced SMC [Ca2+]i by 124 +/- 18 nM (n = 6, P < 0.05), whereas dilation (23 +/- 5 microm) was sustained. Resolution of arteriolar SMC [Ca2+]i in vivo discriminates key signaling events that govern the local control of tissue blood flow. 相似文献
15.
Measurement of intracellular Ca2+ in cultured arterial smooth muscle cells using Fura-2 and digital imaging microscopy 总被引:3,自引:0,他引:3
A rise in cytosolic free Ca2+ is the immediate trigger for contraction in vascular smooth muscle (VSM). We employed the fluorescent Ca2(+)-indicator, Fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in cultured A7r5 cells and the changes evoked by activation with 5-HT. Several methodological considerations that affect the temporal and spatial resolution of Ca2+ images have been addressed. These include: cytoplasmic distribution of Fura-2, wavelength selection for ratio imaging, signal:noise ratio measurement and the effect of [Ca2+] on the limits of detectability under conditions in which [Ca2+] is changing. The distribution of apparent free Ca2+, [Ca2+]App, in A7r5 cells was heterogeneous. This reflects, in part, different pools of intracellular Ca2+. [Ca2+]App was lowest in the nucleus (113 +/- 14 nM; n = 20 cells) and highest in the organelle-rich perinuclear region (228 +/- 12; n = 20), while the surrounding cytoplasmic area (containing relatively few organelles) had intermediate [Ca2+]app levels (150 +/- 13; n = 20). 5-HT (1 microM) evoked transient increases in [Ca2+]App that began within 11 s as relatively modest elevations of [Ca2+]App in the periphery, near the sarcolemma, and subsequently spread to the entire cell, reaching a peak within 18-24 s. At the peak of the Ca2+ transients, [Ca2+]App was highest in the perinuclear region where it sometimes exceeded the maximal detectable levels of the system (1.9 microM). The average peak Ca2+ transient amplitude in the non-nuclear cytoplasm was 1083 +/- 208 nM (1 microM 5-HT; n = 20 cells). Despite the continued presence of 5-HT following the Ca2+ transients, [Ca2+]App then returned to pre-stimulation levels within 5 min. These observations indicate that digital imaging microscopy enables the study of subcellular regulation of intracellular Ca2+ in VSM. The results provide new insights into the role of localized changes in Ca2+ in the regulation of VSM contractility. 相似文献
16.
The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Calmodulin stimulates PMCA activity and for some isoforms this activation persists following clearance of Ca2+ owing to the slow dissociation of calmodulin. We tested the hypothesis that PMCA-mediated Ca2+ efflux from rat dorsal root ganglion (DRG) neurons in culture would remain stimulated following increases in intracellular Ca2+ concentration ([Ca2+]i). PMCA-mediated Ca2+ extrusion was recorded following brief trains of action potentials using indo-1-based photometry in the presence of cyclopiazonic acid. A priming stimulus that increased [Ca2+]i to 506 +/- 28 nm (>15 min) increased the rate constant for [Ca2+]i recovery by 47 +/- 3%. Ca2+ clearance from subsequent test stimuli remained accelerated for up to an hour despite removal of the priming stimulus and a return to basal [Ca2+]i. The acceleration depended on the magnitude and duration of the priming [Ca2+]i increase, but was independent of the source of Ca2+. Increases in [Ca2+]i evoked by prolonged depolarization, sustained trains of action potentials or activation of vanilloid receptors all accelerated Ca2+ efflux. We conclude that PMCA-mediated Ca2+ efflux in DRG neurons is a dynamic process in which intense stimuli prime the pump for the next Ca2+ challenge. 相似文献
17.
Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers 总被引:20,自引:4,他引:16
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《The Journal of cell biology》1986,103(6):2379-2387
Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1- selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F- actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine. 相似文献
18.
Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties. 总被引:23,自引:6,他引:17
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We have developed a calcium diffusion model for a spherical neuron which incorporates calcium influx and extrusion through the plasma membrane as well as three calcium buffer systems with different capacities, mobilities, and kinetics. The model allows us to calculate the concentration of any of the species involved at all locations in the cell and can be used to account for experimental data obtained with high-speed Ca imaging techniques. The influence of several factors on the Ca2+ transients is studied. The relationship between peak [Ca2+]i and calcium load is shown to be nonlinear and to depend on buffer characteristics. The time course of the Ca2+ signals is also shown to be dependent on buffer properties. In particular, buffer mobility strongly determines the size and time course of Ca2+ signals in the cell interior. The model predicts that the presence of exogenous buffer, such as fura-2, modifies the Ca2+ transients to a variable extent depending on its proportion relative to the natural, intrinsic buffers. The conclusions about natural calcium buffer properties that can be derived from Ca imaging experiments are discussed. 相似文献
19.
Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the stimulating agonist; the differences lie in the rate of fall of [Ca2+]i from its peak. We considered that differential sensitivity of the InsP3 receptor may underlie agonist specificity. The thiol reagent, thimerosal, is known to increase the sensitivity of the Ca2+ stores to InsP3 by increasing the affinity of the InsP3 receptor for InsP3 in rat hepatocytes. We show here that a low dose of thimerosal (1 microM), insufficient alone to elevate [Ca2+]i, potentiates [Ca2+]i oscillations induced by phenylephrine or ATP in single, aequorin-injected, rat hepatocytes. Moreover, thimerosal enhances both the frequency and amplitude of phenylephrine-induced oscillations, whereas, in contrast, ATP-induced oscillations undergo an increase in the duration of the falling phase of individual [Ca2+]i transients. Thimerosal, therefore, enhances, rather than eliminates, agonist-specific differences in the hepatocyte [Ca2+]i oscillator. 相似文献
20.
Su Z Yao A Zubair I Sugishita K Ritter M Li F Hunter JJ Chien KR Barry WH 《American journal of physiology. Heart and circulatory physiology》2001,280(6):H2665-H2673
Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current (I(Ca,L)), Na/Ca exchanger current (I(Na/Ca)), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. I(Ca,L)-voltage relationships, I(Na/Ca) density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 +/- 54 vs. 349 +/- 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure. 相似文献