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1.
Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+ 总被引:4,自引:0,他引:4
Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules. 相似文献
2.
The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fc gamma receptor 总被引:9,自引:0,他引:9
H A Wilson D Greenblatt C W Taylor J W Putney R Y Tsien F D Finkelman T M Chused 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(6):1712-1718
We report the cytosolic free calcium, [Ca2+]i, responses of single murine B lymphocytes to whole and F(ab')2 fragments of anti-Ig measured in the flow cytometer with indo-1, a new fluorescent chelator of calcium. The principle advantages of this recording system are these: Indo-1 is highly fluorescent; hence, loading concentrations that introduce artifacts in the reported [Ca2+]i signal may be avoided. The measurement of [Ca2+]i by fluorescence ratio corrects for nonuniform dye uptake, making possible quantitative estimates of [Ca2+]i in single cells and an assessment of the variability of population responses. Baseline recordings of unstimulated lymphocytes indicated a narrow, stable range of [Ca2+]i (75 to 125 nM). The [Ca2+]i rise induced by various anti-Ig preparations exhibited considerable heterogeneity. The initial mean value for F(ab')2 anti-Ig-stimulated cells peaked above 1 microM and was due only to the release of Ca2+ from intracellular stores. A steady state elevation of [Ca2+]i was reached by 5 min and persisted for hours. Cells stimulated with intact anti-Ig reached similar initial peak [Ca2+]i values, but then declined toward baseline. This difference was due to membrane Ig-IgG Fc receptor (mIg-Fc gamma R) cross-linkage, because blocking the Fc gamma R with a monoclonal antibody made the [Ca2+]i responses to F(ab')2 and intact anti-Ig identical. The attenuation of the [Ca2+]i signal by mIg-Fc gamma R cross-linkage is proceeded by a corresponding Fc gamma-mediated reduction in anti-Ig-induced inositol trisphosphate elevation. These findings outline a biochemical basis for mIg- and Fc gamma R-mediated activation and regulation intrinsic to the B cell, and demonstrate the advantages of indo-1 over quin2 for fluorescent measurement of [Ca2+]i in small cells. 相似文献
3.
The affects of volatile anesthetics on mobilization of intracellular Ca2+ was monitored in primary cultures of rat hepatocytes using the fluorescent Ca2+ probe Fura-2. The use of Fura-2 was limited by several factors which complicated the quantitative analysis of the results, such as: (i) a high rate of dye leakage; (ii) changes in the redox state of the hepatocytes which interfered with the fluorescence produced by the dye at various excitation wavelengths; (iii) compartmentalization of the dye producing high local intracellular concentrations; and, of particular importance for this study, (iv) enhanced photobleaching of the dye in the presence of halothane. To aid in the interpretation of the Fura-2 data, the Ca2(+)-sensitive photoprotein aequorin was also used to monitor changes in [Ca2+]i. The aequorin and Fura-2 techniques qualitatively yielded the same result, that the volatile anesthetic agents halothane, enflurane, and isoflurane induce an immediate and transient increase of [Ca2+]i. The durations of these transients were approximately between 5 and 10 min and were not related to any evident acute cell toxicity. The [Ca2+]i increases induced by the volatile anesthetic agents were dose-dependent, with halothane the most potent. The exact mechanism governing these increases in [Ca2+]i induced by these anesthetics in rat hepatocytes is unknown, but is likely to involve effects on both the cell surface membrane and endoplasmic reticulum components of the signal transducing system. 相似文献
4.
The measurement of intracellular calcium ion concentrations [( Ca2+]i) in single living cells using quantitative fluorescence microscopy draws from a diverse set of disciplines, including cellular biology, optical physics, statistics and computer science. Over the last few years, we have devised and built a number of systems for measuring [Ca2+]i with Fura-2, and have applied them in the exploration of a wide range of biological processes controlled by Ca2+. In this report we discuss these systems and their advantages and limitations. We also describe the theoretical and practical problems associated with using Fura-2 to measure [Ca2+]i, and the solutions that we, and others, have developed to overcome them. The approaches described should provide useful guidance for others interested in imaging [Ca2+] distribution in living cells. The factors that limit current methods are discussed, and areas for future development are highlighted. 相似文献
5.
A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes. 总被引:11,自引:0,他引:11
We have measured the distribution of cytoplasmic calcium in lily pollen tubes by microinjecting them with indo-1 and performing fluorescence ratio image analysis on them. All of the 16 tubes that were growing at the time of the calcium measurements showed a gradient of [Ca2+]i in the tip region, with Ca2+ being 1.25 to 3.32 times higher at the distal end in 15 cases and more than 5 times higher in one case. The extent of the gradient ranged from 22 to 65 microns. Most of the 15 nongrowing tubes either had no gradient or had lower Ca2+ in the tip region. While we have confirmed a previous report that lily pollen tubes can be loaded with the membrane-permeable acetoxymethyl ester forms of calcium indicators, the dyes loaded in this way are visibly partitioned into organelles and this method of loading is, therefore, not useful for the measurement of [Ca2+]i. Iontophoresis of the dye free acids into tubes produces a more uniform and diffuse fluorescence which does not appear to partition into organelles. Indo-1 remains in the pollen tubes longer than fura-2. The correlation between growth and the [Ca2+]i gradient in the apical portion of the pollen tube is discussed in relation to previous reports that have suggested that such a gradient should exist during polarized growth. 相似文献
6.
The retinal tissue of blowflies was loaded with the fluorescent Ca2+ indicator Fura-2 by incubating cut heads in saline solutions which contained the membrane permeable acetoxymethylester of Fura-2 (Fura-2/AM). The spectral analysis of the tissue fluorescence showed that Fura-2/AM was intracellularly hydrolysed to Fura-2. In order to monitor the intracellular free Ca2+ concentration ([Ca2+]i) the Fura-2 fluorescence was excited by short light flashes. The fluorescence was calibrated by incubating the tissue in Ca2+ buffers of high buffering capacity and subsequent disruption of the cell membranes by freeze/thawing, which gave a dissociation constant for the Ca(2+)-Fura-2 complex of 100 nM. When the extracellular Ca2+ concentration ([Ca2+]o) was altered [Ca2+]i reversibly changed. The changes were most pronounced when [Ca2+]o was varied in the millimolar range, e.g. [Ca2+]i increased from 0.07 microM at [Ca2+]o = 0.1 mM to 1 microM at [Ca2+]o = 10 mM. When extracellular Na+ was replaced by Li+ or other monovalent ions, [Ca2+]i rapidly increased which supports the view that electrogenic Na+/Ca2+ exchange contributes to the control of [Ca2+]i. However, [Ca2+]i decreased again when the tissue was superfused with Na(+)-free media for longer periods, which points to a Ca(2+)-transporting system different from Na+/Ca2+ exchange. Light adaptation had only a small effect on [Ca2+]i. Even after intense stimulation [Ca2+]i increased by a factor of 1.5 only, which is in line with results obtained in the photoreceptors of Balanus and Apis. 相似文献
7.
Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2 总被引:3,自引:0,他引:3
H Komada H Nakabayashi H Nakano M Hara T Yoshida H Takanari K Izutsu 《Cell structure and function》1989,14(2):141-150
The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase. 相似文献
8.
P E Lund E Grapengiesser E Gylfe B Hellman 《Biochemical and biophysical research communications》1991,177(2):777-783
Intracellular free calcium ([Ca2+]i) was measured in individual pancreatic beta-cells from mice using dual emission microfluorometry and the indicator Indo-1 applied by a patch clamp pipette. GTP-gamma-S (100 microM) injected together with 0.3 or 3 mM ATP evoked repetitive [Ca2+]i transients with a frequency of about 1 per min in beta-cells kept at a membrane potential of -70 mV. The oscillatory pattern was unaffected by the Ca2+ channel blocker verapamil (50 microM). When omitting GTP-gamma-S from the pipette medium it became evident that 3 mM ATP alone can induce oscillations. The results provide additional evidence for an important role of ATP in the ionic control of insulin release, indicating that such regulation may also involve activation of G-proteins. 相似文献
9.
Fast, single cell, measurement of the average cytosolic [Ca2+]i with the Fura-2 technique suggests that the depolarization induced [Ca2+]i rise is entirely due to entry through the voltage-activated Ca2+ channels. Involvement of a Ca(2+)-induced Ca(2+)-release process is not evident. Under physiological cytosolic buffering the current-induced [Ca2+]i rise persists for seconds and decays exponentially (tau = 7 s). Analysis of the [Ca2+]i changes during two-pulse protocols indicates that the purely voltage-dependent inactivation of the high voltage-activated (HVA) channels, in the range -80/+70 mV, is a slow process (0.2-1 s) which removes at most 40% of the current. On the contrary, Ca(2+)-dependent inactivation acts in a fast way and it is therefore responsible for the fast inactivating phase of the current; this phase disappears under sustained [Ca2+]i loads, and reappears when redistribution of free Ca2+ takes place. A suitable correction may be devised to compensate for the Ca(2+)-dependent inactivation. 相似文献
10.
F C Praeger L Reinlib M Donowitz B A Gilchrest 《Biochemical and biophysical research communications》1989,159(2):862-870
A transient rise in intracellular free Ca2+ concentration ([Ca2+]i) has been implicated in mitogenic induction of cell division. Individual human foreskin fibroblasts in confluent cultures examined with the Ca2+ indicator Fura-2 and a fluorescence microscope-imaging system had a basal [Ca2+]i which varied markedly from cell-to-cell. A transient serum-induced rise in [Ca2+]i was demonstrated the magnitude of which was directly correlated with the basal [Ca2+]i level. In contrast to serum-induced increase in [Ca2+]i, exposure to an elevated level of extracellular Ca2+, which is at least equally mitogenic for fibroblasts, did not alter the basal [Ca2+]i of single subconfluent cells or confluent cells. Elevated extracellular Ca2+ does not exert its mitogenicity via a transient rise in [Ca2+]i. 相似文献
11.
N C Guérineau R Bouali-Benazzouz J B Corcuff M C Audy M Bonnin P Mollard 《Cell calcium》1992,13(8):521-529
Gonadotropin-releasing hormone (GnRH)-stimulated changes in the cytosolic free Ca2+ concentration ([Ca2+]i) were studied in gonadotrophs cultured from 3-week ovariectomized rat pituitaries. One animal was used per cell preparation. [Ca2+]i was monitored in individual gonadotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe. A short stimulation with GnRH evoked a complex concentration-dependent Ca2+ response in individual gonadotrophs. 0.1-1 nM GnRH triggered a series of sinusoidal-like [Ca2+]i oscillations superimposed upon a modest slow [Ca2+]i rise--the oscillating response mode--while 10-100 nM GnRH caused a biphasic increase in [Ca2+]i consisting of a monophasic transient and oscillations--the transient/oscillating response mode. Despite the consistency of Ca2+ responses, an inter-preparation heterogeneity of [Ca2+]i oscillations frequency was noticed. Moreover, we observed that, within a given cell preparation, the frequency of [Ca2+]i oscillations was independent of GnRH concentration whereas both peak [Ca2+]i and area under the [Ca2+]i versus time curve were concentration-dependent. Thus, in gonadotrophs, the presence of the GnRH signal would lead to [Ca2+]i oscillations, while the amplitude of the [Ca2+]i responses would code for the concentration of agonist. Both transient and oscillating components of GnRH responses depended on releasing activity of Ca(2+)-sequestering pools in as much as GnRH responses were unaffected by brief removal of external Ca2+, but suppressed by chelating intracellular free Ca2+ with BAPTA. However, prolonged exposure to a Ca(2+)-free medium suppressed the transient component while leaving the oscillating component unaffected. We therefore propose that gonadotrophs employ Ca(2+)-sequestering pools, whose maintenance depends on a slow Ca(2+)-entry, to give an amplitude-coded Ca2+ rise in response to a short GnRH stimulation. 相似文献
12.
Intracellular calcium 'signatures' evoked by different agonists in isolated bovine aortic endothelial cells. 总被引:3,自引:0,他引:3
Agonist induced increases in intracellular free calcium, [Ca2+]i, were measured in single Fura-2 loaded bovine aortic endothelial (BAE) cells by dual wavelength microspectrofluorimetry. Low doses of ATP (less than 10 microM) induced complex changes in [Ca2+]i. These changes usually consisted of a large initial transient peak with subsequent fluctuations superimposed upon a maintained rise in [Ca2+]i. Higher doses of ATP (greater than 50 microM) produced much simpler biphasic increases in [Ca2+]i in individual cells. Acetylcholine and bradykinin also elicited increases in [Ca2+]i in single cells in confluent monolayers of endothelial cells. However, only acetylcholine produced complex fluctuations. High doses of acetylcholine evoked simple rises in [Ca2+]i similar to those seen with high doses of ATP. In contrast, bradykinin evoked relatively simple rises in [Ca2+]i at all doses used. These results indicate that the mechanisms responsible for generating agonist induced increases in [Ca2+]i in BAE cells are not homogeneous. ATP and acetylcholine produced more complex Ca2+ changes or 'signatures' in single confluent bovine aortic endothelial cells than bradykinin. All three agonists appeared to release Ca2+ from intracellular stores as well as stimulating Ca2+ influx. The possible mechanisms underlying these phenomena are discussed. 相似文献
13.
Aldosterone influences free intracellular calcium in human mononuclear leukocytes in vitro 总被引:1,自引:0,他引:1
Mineralocorticoid receptors have been detected in human mononuclear leukocytes (HML) and a physiological effector mechanism was demonstrated subsequently by which aldosterone is able to prevent the loss of intracellular sodium, potassium and cell water during incubation in an aldosterone-free medium. In the present paper, free intracellular calcium, [Ca2+]i, was measured in HML from normal subjects by Quin-2 and Fura-2 fluorescence after incubation for 1 h at 37 degrees C in RPMI-1640 medium. In fresh HML, [Ca2+]i was 54 +/- 15 nM (Fura-2, mean +/- SD, n = 26). After incubation without aldosterone, [Ca2+]i in HML was 118 +/- 27 nM (Quin-2, n = 11) and 50 +/- 13 nM (Fura-2). After incubation with 1.4 (Fura-2) or 2.8 nM (Quin-2) aldosterone, [Ca2+]i was 139 +/- 38 nM (Quin-2, P less than 0.05 compared with value after incubation without aldosterone) and 57 +/- 11 nM (Fura-2, P less than 0.00001). The Kd-value for dose-response curve was 0.4 nM. The effect of aldosterone was antagonized by N-ethyl-isopropylamiloride, but not by canrenoate, canrenone, cycloheximide and actinomycin D. It was absent in a sodium-free buffer. Corticosterone and hydrocortisone were active as agonists. These results show that aldosterone exerts an effect on the [Ca2+]i in HML in vitro which could be involved in hemodynamic responses to mineralocorticoids if also present in cardiovascular tissues. 相似文献
14.
Fura-2 used as a probe to show elevated intracellular free calcium in platelets of Dahl-sensitive rats fed a high salt diet 总被引:1,自引:0,他引:1
S Vasdev P Thompson C Triggle P Fernandez P Bolli V S Ananthanarayanan 《Biochemical and biophysical research communications》1988,154(1):380-386
Elevated intracellular free calcium concentration [Ca2+]i in vascular smooth muscle cells has been implicated in the pathophysiology of hypertension. Platelet [Ca2+]i was measured using the fluorescent indicator, Fura-2, in Dahl sensitive (DS) and resistant (DR) rats given high (8% NaCl) and low (0.4% NaCl) salt diets, as well as in the spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. The aim of this study was to show whether [Ca2+]i is elevated in salt induced hypertension. Platelet [Ca2+]i and systolic blood pressure (SBP) were higher (p less than 0.001) in DS rats given a high than low salt diets. In contrast, no changes in platelet [Ca2+]i and SBP were observed in DR rats. In SHR, platelet [Ca2+]i and SBP were higher (p less than 0.001) than in the WKY rats. Platelet [Ca2+]i correlated with SBP in all groups of rats (r = 0.929; p less than 0.001, n = 38). The parallel increase in SBP and [Ca2+]i in the DS high salt rats and the SHR suggests that an increased [Ca2+]i is involved in the pathophysiology of hypertension in the two models which differ with respect to the pathogenesis of their hypertension. This increase in [Ca2+]i therefore seems to reflect an abnormality in [Ca2+]i handling in hypertension regardless of its cause. 相似文献
15.
Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts. 总被引:2,自引:0,他引:2
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The goal of these studies was to examine the effects of several factors that may artifactually influence quantitation of cytosolic [Ca2+], [Ca2+]c, while using the fluorescent calcium indicator Indo-1. The following factors were investigated: 1) a possible fluorescence contribution from unhydrolized Indo-1/AM (by Mn2+ quenching), 2) Ca2+ buffering by Indo-1 (by varying [Indo-1]), 3) endothelial and mitochondrial Indo-1 loading (by bradykinin stimulation and calculations), and 4) effects of changing tissue fluorescence (predominantly NAD(P)H) on calculated [Ca2+]c during hypoxia (by a new method which allowed simultaneous determination of [Ca2+]c and changes in [NAD(P)H]). No significant contribution of Indo-1/AM was found. With increasing [Indo-1], calculated systolic [Ca2+]c fell significantly. Indo-1 incorporation (< 18%) into endothelial cells, caused a slight underestimation of systolic [Ca2+]c, while mitochondrial Indo-1 loading may cause overestimation of [Ca2+]c. With increased tissue fluorescence, during hypoxia, systolic [Ca2+]c may be underestimated by approximately 27% (for Indo-1 loading factors three to five times original tissue fluorescence). These studies suggest conditions in which experimental artifacts could be minimized to allow reliable quantitation of [Ca2+]c in intact perfused hearts using Indo-1 fluorometry. The major problem of obtaining reliable results depended on the ability to correct for changing NAD(P)H fluorescence while keeping [Indo-1] low. 相似文献
16.
Glucose-stimulated efflux of indo-1 from pancreatic beta-cells is reduced by probenecid 总被引:4,自引:0,他引:4
Indo-1 loaded pancreatic beta-cells, isolated from obese hyperglycaemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), efflux of indicator and insulin release. In the absence of glucose there was a continuous efflux of indo-1 which increased upon stimulation with 20 mM of the sugar. The anion exchange inhibitor probenecid reduced both basal efflux of indo-1 and prevented that promoted by glucose. Measurements of [Ca2+]i and insulin release revealed similar results as previously reported with quin-2 and fura-2. Furthermore, probenecid did not influence the [Ca2+]i responses. It is thus possible to reduce efflux of indo-1 probenecid and thereby improve the measurements of [Ca2+]i in pancreatic beta-cells. 相似文献
17.
Indo-1 is widely used to measure intracellular free calcium, [Ca2+]i, by comparing the fluorescence emission at 2 or more wavelengths with the emissions, which are assumed to be known, of Indo-1 when it is fully calcium-bound and when it is fully calcium-free. Accurate quantitation requires that these "reference" values be obtained on intracellular dye, and the full spectra of this study show that the reason is a significant spectral shift of the calcium-free peak, but not the calcium-bound. A mathematical analysis shows that the new peak must be a new state of the Indo-1 molecule, since it cannot be simply due to residual calcium in the cell. When intracellular "reference" spectra were used in the data analysis, [Ca2+]i could be calculated from whole spectra or from the ratio of observations at two wavelengths with good agreement. When extracellular "reference" spectra were used, the value calculated by the ratio method depended on the choice of wavelengths. 相似文献
18.
Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle. 总被引:5,自引:0,他引:5
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Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase. 相似文献
19.
Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells.
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A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion. 相似文献
20.
Heterogeneous changes in [Ca2+]i induced by glucose, tolbutamide and K+ in single rat pancreatic B cells 总被引:1,自引:0,他引:1
The effects of glucose, tolbutamide and K+ on cytosolic free Ca2+ ([Ca2+]i) in single rat pancreatic B cells were examined using Fura-2 and dual wavelength microfluorimetry. At basal glucose concentration (2.8 mM), about half of the cells were found to display spontaneous Ca2+ oscillations. Glucose (greater than or equal to 11.1 mM), tolbutamide (greater than or equal to 50 microM) and K+ (50 mM) induced rises in [Ca2+]i that could be inhibited by the Ca2+ channel blocker D600. The pattern of response and the sensitivity to the secretagogues were characterized by a marked heterogeneity. The majority of the cells responded to glucose and tolbutamide by a progressive increase in [Ca2+]i onto which sinusoidal oscillations were superimposed. The periodicity of these oscillations was about 2.5/min. Occasionally, some cells displayed slow and major waves in Ca2+ levels (about 0.2/min). None of the cells responded to glucose by displaying an initial decrease in [Ca2+]i. Likewise, the sugar failed to decrease [Ca2+]i in the absence of extracellular Ca2+. The present study shows that, despite a large heterogeneity of the response, the majority of the pancreatic B cells respond to different secretagogues by displaying fast [Ca2+]i oscillations that are reminiscent of the bursts of electrical activity recorded in B cells. 相似文献