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1.

Background

The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/Principal Findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca2+-handling proteins. Detailed laser confocal Ca2+ imaging demonstrated spontaneous whole-cell [Ca2+]i transients. These transients required Ca2+ influx via L-type Ca2+ channels, as demonstrated by their elimination in the absence of extracellular Ca2+ or by administration of the L-type Ca2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ store, contributing to [Ca2+]i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca2+) and ryanodine (decreasing [Ca2+]i). Similarly, the importance of Ca2+ reuptake into the SR via the SR Ca2+ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca2+]i transients elimination. Finally, the presence of an IP3-releasable Ca2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca2+]i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor U73122.

Conclusions/Significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca2+]i transients in hiPSC-CMs on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release.  相似文献   

2.
This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP3R)-operated Ca2+ stores interact as coupled oscillators to produce near-synchronous Ca2+ release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP3, to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca2+ transients and/or intracellular Ca2+ waves. Larger near-synchronous Ca2+ transients then occurred leading to global synchronous Ca2+ transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca2+ channels with nifedipine prevented ET-1 from inducing near-synchronous Ca2+ transients and resultant action potentials, leaving only asynchronous Ca2+ transients and local Ca2+ waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca2+ release from IP3R-operated Ca2+ stores, which causes depolarization; 2), L-type Ca2+ channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca2+ channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca2+ store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca2+ stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca2+ store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca2+ release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca2+ release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells.  相似文献   

3.
The term excitation-coupled Ca2+ entry (ECCE) designates the entry of extracellular Ca2+ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca2+ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn2+. Here, we have examined the effects of these agents on the L-type Ca2+ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La3+, and Gd3+) and dantrolene all inhibited the L-type Ca2+ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca2+ channel displays permeability to Mn2+ in the absence of external Ca2+ and produces a Ca2+ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca2+ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca2+; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca2+-free solution and were unaffected by Ca2+ removal; and (3) after block of SR Ca2+ release by 200 μM ryanodine, normal myotubes still displayed a large Ca2+ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca2+ entry attributed to ECCE.  相似文献   

4.
The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.  相似文献   

5.
In cardiac muscle, intracellular Ca2+ and Mg2+ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca2+] in the SR ([Ca2+]L) stimulates the Ca2+ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg2+, which has not been regarded as an important regulator of Ca2+ release.  相似文献   

6.

Background

In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, α-RyR and β-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/Principal Findings

In this study, we expressed either or both α-RyR and β-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that α-RyR and β-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca2+ imaging studies, each isoform exhibited caffeine-induced Ca2+ transients, an indicative of Ca2+-induced Ca2+ release (CICR). However, the fashion of Ca2+ release events was fundamentally different: α-RyR mediated graded and sustained Ca2+ release observed uniformly throughout the cytoplasm, whereas β-RyR supported all-or-none type regenerative Ca2+ oscillations and waves. α-RyR but not β-RyR exhibited Ca2+ transients triggered by membrane depolarization with high [K+]o that were nifedipine-sensitive, indicating that only α-RyR mediates depolarization-induced Ca2+ release. Myotubes co-expressing α-RyR and β-RyR demonstrated high [K+]o-induced Ca2+ transients which were indistinguishable from those with myotubes expressing α-RyR alone. Furthermore, procaine did not affect the peak height of high [K+]o-induced Ca2+ transients, suggesting minor amplification of Ca2+ release by β-RyR via CICR in 1B5 myotubes.

Conclusions/Significance

These findings suggest that α-RyR and β-RyR provide distinct intracellular Ca2+ signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.  相似文献   

7.
ICRAC (the best characterized Ca2+ current activated by store depletion) was monitored concurrently for the first time with [Ca2+] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca2+] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca2+ chelator N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca2+] within the ER can be lowered and restored to its original level within 10–15 s without modifications of Ca2+ pumps or release channels. Using these new tools, we demonstrate here that Ca2+ release–activated Ca2+ current (ICRAC) is activated (a) solely by reduction of free [Ca2+] within the ER and (b) by any measurable decrease in [Ca2+]ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.  相似文献   

8.
The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca2+ entry evoked by alkaline depolarization. In the absence of external Ca2+, Na+ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na+]i-reporting probe SBFI in populations of intact sperm. Removal of external Ca2+ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na+]i is greater for sperm alkalinized with NH4Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na+]i rise is slowed by candidate CatSper blocker HC-056456 (IC50 ∼3 µM). HC-056456 similarly slows the rise of [Ca2+]i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO3 but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca2+ entry through the CatSper channel is terminated by removal of external Ca2+. Thus, open CatSper channels and entry of external Ca2+ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.  相似文献   

9.

Background

Ca2+ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca2+ concentration ([Ca2+]c) in the prefusion phase, the occurrence and significance of Ca2+ signals in the postfusion phase have not been described before.

Methodology/Principal Findings

We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca2+- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca2+]c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t1/2 of decay = 3.2 s) rise of localized [Ca2+]c originating at the site of lamellar body fusion. [Ca2+]c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca2+ from, or addition of Ni2+ to the extracellular solution, strongly inhibited these [Ca2+]c transients, whereas Ca2+ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca2+]c. Both effects were reduced by the non-specific Ca2+ channel blocker SKF96365.

Conclusions/Significance

Fusion-activated Ca2+ entry (FACE) is a new mechanism that leads to [Ca2+]c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca2+ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions.  相似文献   

10.
Modulation of Ca2+ within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca2+ indicators to define changes in perikaryal Ca2+ concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca2+]i levels within the cell body. Arguing against Ca2+ overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca2+ fluxes revealed that degeneration of pde6cw59 mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca2+]i. Analysis of [Ca2+]i in dissociated Pde6βrd1mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca2+ elevations (‘flashes'') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca2+ fluxes diminished. This study thus provides insight into Ca2+ dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca2+]i that occurs in normal cones.  相似文献   

11.
Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca2+ release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca2+ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode''s solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (Em), and intracellular Ca2+ ([Ca2+]i) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl]o. All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14–18 mV; n = 40) and SR Ca2+ release. However, sugar solutions caused a global increase in [Ca2+]i, whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in Em (by 15.04 ± 1.85 mV; n = 8), whereas [Ca2+]i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and Em was associated with LCR. Both global and localized SR Ca2+ release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca2+ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca2+ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in Em. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.  相似文献   

12.
We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+]i) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed “islet imprinting.” We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+]i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a KATP-channel opener that blocks [Ca2+]i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+]i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+]i oscillations. Lastly, to test whether the imprinted [Ca2+]i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+]i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.  相似文献   

13.
《Journal of biomechanics》2014,47(16):3903-3908
Intracellular calcium transient ([Ca2+]i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca2+]i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca2+]i transient in osteoblasts. Fluorescent dyes DiBAC4(3) and fura-2 AM were respectively used to detect membrane potential and [Ca2+]i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca2+]i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca2+]i transients. Replacing extracellular Na+ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca2+]i transients. However, voltage-activated K+ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca2+ or blocking of L-type voltage-sensitive Ca2+ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca2+]i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca2+ mobilization wherein FSS activated SACs to promote Na+ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca2+ influx though L-VSCCs switched on [Ca2+]i response in osteoblasts.  相似文献   

14.
In cardiac muscle, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca2+ transient. The global elevation of the intracellular Ca2+ concentration arises from the spatial and temporal summation of elementary Ca2+ release events, Ca2+ sparks. Ca2+ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca2+, Mg2+, and ATP). Here, we examined by which mechanism the free intracellular Mg2+ ([Mg2+]free) affects various Ca2+ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg2+ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg2+ from the possible activation by ATP and Mg2+-ATP via the adenine binding site of the channel. Changes in [Mg2+]free generally led to biphasic alterations of the Ca2+ spark frequency. For example, lowering [Mg2+]free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca2+ depletion. Fitting the Ca2+ spark inhibition by [Mg2+]free with a Hill equation revealed a Ki of 0.1 mM. In conclusion, our results support the notion that local Ca2+ release and Ca2+ sparks are modulated by Mg2+ in the intracellular environment. This seems to occur predominantly by hindering Ca2+-dependent activation of the RYRs through competitive Mg2+ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg2+ and ATP can change.  相似文献   

15.
16.

Background

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca2+ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca2+ signals in ipRGCs independent of gap junction blockade.

Methodology/Principal Findings

To test the possibility that carbenoxolone directly inhibits light-evoked Ca2+ responses in ipRGCs, the light-evoked rise in intracellular Ca2+ ([Ca2+]i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca2+]i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.

Conclusions/Significance

We demonstrate that the light-evoked rise in [Ca2+]i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca2+]i in isolated ipRGCs is almost entirely due to Ca2+ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca2+]i in ipRGCs by blocking L-type voltage-gated Ca2+ channels. The ability of carbenoxolone to block evoked Ca2+ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca2+]i is the output being measured.  相似文献   

17.
In this study, the functional consequences of the pharmacological modulation of the M‐current (IKM) on cytoplasmic Ca2+ intracellular Ca2+concentration ([Ca2+]i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. Kv7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K+]e, the IKM activator retigabine (RT, 10 μM) inhibited [3H]d ‐aspartate ([3H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the IKM blocker XE‐991 (20 μM). The IKM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K+]e‐induced synaptosomal [Ca2+]i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca2+]i transients. The P/Q‐type voltage‐sensitive Ca2+channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca2+]i increase and [3H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca2+]i and the resulting enhancement of [3H]d ‐Asp release induced by [Ca2+]i mobilization from intracellular stores, or by store‐operated Ca2+channel activation. Collectively, the present data reveal that the pharmacological activation of IKM regulates depolarization‐induced [3H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca2+]i occurring through P/Q‐type VSCCs.  相似文献   

18.
Two recombinant aequorin isoforms with different Ca2+ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca2+ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca2+]er or [Sr2+]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca2+] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca2+ concentration, the [Ca2+]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca2+]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2+ was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation2+]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca2+], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca2+ or Sr2+, is presented. Moreover, a few other key problems concerning the ER Ca2+ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca2+]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca2+] the cation is simultaneously accumulated; (b) stimulation of Ca2+ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation2+]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca2+], observed when the cells are incubated in Ca2+-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).  相似文献   

19.
In the present study we describe the analysis of optically recorded whole cell Ca2+ transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca2+ mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca2+-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca2+ to the myoplasmic space (Ca2+ input flux). The validity of the procedure was confirmed by model simulations using artificial Ca2+ input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca2+ input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca2+ flux carried by the L-type Ca2+ channels, indicating that it consists mainly of voltage-controlled Ca2+ release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca2+ inward current, indicating that Ca2+ release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca2+ release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.  相似文献   

20.
In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.  相似文献   

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