Measurement of free Ca2+ changes and total Ca2+ release in a single striated muscle fibre using the fluorescent indicator quin 2

The fluorescent Ca2+ indicator, quin 2, has been used in isolated striated muscle fibres. There is a distinct quin 2 fluorescence peak at lambda 500 nm upon excitation at lambda 339 nm after axial injection of the potassium salt of quin 2, pH 7.1. Single voltage-clamp or current clamp electrical stimulation resulted in a distinct transient change in the fluorescence at lambda 500 nm which was not observed at lambda 400 nm, the peak of the fibre autofluorescence. Ca2+ buffering is marked at high quin 2 concentrations (greater than or equal to 400 microM) producing a slow decay of force and fluorescence. At lower concentrations (8-30 microM) of quin, the decay of force is within the range observed in non-injected control fibres. A Kd of 457 nM at 5 mM free Mg2+ suggests an upper resting free Ca2+ concentration of 310 nM at 12 degrees C.     

Intracellular calibration of the fluorescent calcium indicator Fura-2

We present the techniques we have used and the problems we have encountered in our laboratories in the in vivo calibration of the fluorescent Ca2(+)-indicator Fura-2. These techniques include the use of potentiometric methods for the precise control and determination of Ca2+ levels in bathing solutions, in association with methods for the equilibration of internal and external solutions with ionophores (Br-A23187, ionomycin, monensin and nigericin). A by-product of these techniques has been the development of a simple procedure that utilizes Fura-2 as a general indicator of ionized Ca2+ concentrations within the physiological range (pCa 7.5 to 5.5), in other experimental solutions. The major advantages of this relatively simple procedure are that it is (i) rapidly performed, (ii) independent of the total EGTA concentration within each experimental solution, (iii) independent of the absolute EGTA purity, and (iv) unaffected by a large number of potentially interfering cations (i.e. Mg2+, H+, K+, Na+) within the test solutions.     

Assessment of Fura-2 for measurements of cytosolic free calcium

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytoplasmic free calcium in single cells of centric diatoms. The use of fura-2

Summary Cytoplasmic free calcium concentration has been measured in centric diatoms using Fura-2. Cells hydrolysed Fura-2 acetoxymethyl ester and accumulated free Fura-2 in the cytoplasm. The [Ca²⁺] distribution in two species (Coccinodiscus concinnus andGuinardia flaccida) has been mapped using digital image analysis.Abbreviations [Ca _cyt ²⁺ ] Cytoplasmic free calcium concentration - Quin-2/AM Quin-2 acetoxymethyl ester - Fura-2/AM Fura-2 acetoxymethyl ester DMSO dimethyl sulphoxide - CCTV closed circuit television     

Activity-dependent calcium transients in central nervous system myelinated axons revealed by the calcium indicator Fura-2.

Optical measurements from rat optic nerve, loaded with the new Ca2+ indicator Fura-2, provide the first evidence for the presence of activity-dependent fast intracellular [Ca2+] transients in mammalian central nervous system (CNS) myelinated axons. The results suggest that voltage-dependent Ca2+ channels are present in some of the myelinated axons. Optical measurements from axons stained with anterogradely transported voltage-sensitive dye suggest the presence of Ca2+-dependent potassium conductances in these axons. This report also demonstrates that Fura-2 can readily detect changes in [Ca2+] inside cells as a result of electrical activity, and establishes its suitability for measurements of intracellular Ca2+ transients in the millisecond time domain.     

Intracellular free calcium concentration in the blowfly retina studied by Fura-2.

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.     

Metal cations as synaptosomal calcium blockers in studies with Fura-2

Metal ions are often used to block calcium channels in various tissues, including synaptosomes. In the present study, Fura-2 was used to determine the effectiveness of various metal ions as calcium channel blockers in rat brain synaptosomes in vitro. In buffer solutions, La³⁺ and Cd²⁺ increased the Fura-2 fluorescence in a manner similar to Ca²⁺. Ni²⁺ and Mn²⁺ appeared to be fluorescence quenching cations, and Sr²⁺ and Co²⁺ had little effect on the fluorescence of Fura-2. In suspensions of synaptosomes under resting conditions, Cd²⁺, Ni²⁺ and Mn²⁺ were found to be not suitable for use in synaptosome studies. On the other hand, La³⁺ and Co²⁺ had little effect on the Fura-2 fluorescence of resting synaptosomes, and under depolarizing conditions, La³⁺ and Co²⁺ decreased the Fura-2 fluorescence. These resuls, therefore, suggest that La³⁺ and Co²⁺ may be suitable as calcium channel blockers in synaptosome studies.     

Intracellular pH and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy

Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations [( Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.     

A method for measuring sarcoplasmic reticulum calcium uptake in skeletal muscle using Fura-2

We have presented an assay for measuring the rate of sarcoplasmic reticulum (SR) Ca²⁺ uptake and Ca²⁺ release in skeletal muscle homogenates using the fluorescent Ca²⁺ probe Fura-2. Using this assay, we investigated the effects of an elevated temperature (40°C) and lowered pH (6.8), two factors proposed to be involved in skeletal muscle fatigue, on SR Ca²⁺ uptake. The EDL muscle was found to have a higher rate of Ca²⁺ uptake than the soleus (34%). Exposure of the muscles to a raised temperature, but not a reduced pH, resulted in a reduction in the rate of Ca²⁺ uptake in both the EDL and soleus homogenates. This uptake process was blocked by cyclopiazonic acid (CPA) a specific inhibitor of the major transport protein of the sarcoplasmic reticulum, the Ca²⁺-ATPase. Calcium release was induced using AgNO₃ after loading of the vesicles during the uptake process. It was found that AgNO₃ was only effective in producing Ca²⁺ release in the EDL muscles. The soleus muscles did not release Ca²⁺ under varying [Mg²⁺] or with Hg²⁺ substitution for Ag⁺, suggesting that fast- and slow-twitch muscle fibres require different conditions for maximum Ca²⁺-release, or that different isoforms of the Ca²⁺ release channels are present in the different fibres.     

Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers.

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (approximately 3.8 microns) and bathed in a 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slow diffusion coefficient in myoplasm, 0.16 (+/- 0.01, SEM) x 10(-6) cm2 s-1 (N = 5; 16 degrees C), it is estimated that approximately 85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3- to 4-fold, the indicator's apparent dissociation constant for Ca2+ (KD), which is estimated to be 1.1-1.6 microM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate fr, the fraction of fura red molecules in the Ca2+-bound form at rest. In 3 fibers thought to be minimally damaged by the micro-injection, fr was estimated to be 0.15 (+/- 0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 microM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length (approximately 2.7 microns), and containing a nonperturbing concentration of indicator (< or = 0.2 mM), [Ca2+]r is estimated to be 0.18-0.27 microM. This range is higher than estimated previously in frog fibers with other techniques. In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission intensity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimulation and compared with absorbance measurements made from the same fiber region. The analysis implies that RMIN and RMAX (the values of R that would be measured if all indicator molecules were in the Ca(2+)-free and Ca(2+)-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+]r from R are analyzed and discussed.     

The use of Fura-2 to estimate myoplasmic [Ca2+] in human skeletal muscle

Fura-2 was used to estimate myoplasmic [Ca2+] in intact fibers and fiber segments from normal and diseased human muscles. Small muscle bundles (20-50 fibers) were loaded with the membrane-permeant form of the dye (Fura-2 AM). High-performance liquid chromatography was utilized to study the ability of these cells to hydrolyze Fura-2 AM. Immediately after the 30 min loading period, Fura-2 (the Ca2+ indicator) was the predominant form of the dye in all preparations and the concentration within these fibers remained stable for over 4 1/2 hours. In addition, the resting myoplasmic [Ca2+] in fiber segments from normal subjects and those susceptible to malignant hyperthermia were the same. However, halothane administration (1.5%) induced correlated increases in myoplasmic [Ca2+] and force only in fibers from the susceptible patients. In contrast, caffeine administration causes correlated increases in myoplasmic [Ca2+] and force in both types of muscle, but lower concentrations were needed to do so in the fibers from the susceptible patients. The effects of halothane and caffeine were reversible. We conclude that Fura-2 can be used successfully to estimate resting levels and changes in myoplasmic [Ca2+] in human skeletal muscle.     

Muscle calcium transient. Effect of post-stimulus length changes in single fibers

We examined the effects of post-stimulus length changes on voltage-clamped, aequorin-injected single muscle fibers from the barnacle Balanus nubilus. Extra light (extra calcium) is seen when the fiber is allowed to shorten (a small percentage) during the declining phase of the calcium transient. The opposite is observed when the fiber is stretched. Increasing the extent of shortening increases the amount of extra calcium, as does decreasing the temperature. The extra calcium probably comes from the myofilaments and not from the sarcoplasmic reticulum because (a) there is a strong correlation between the extra calcium and the level of activation; (b) there is a strong correlation between the extra calcium and the amount of force redeveloped after a length change; and (c) the time course of the appearance of the extra calcium is intermediate between that of the free calcium concentration and that of force. We suggest (a) that the calcium binding to the activating myofibrillar proteins is sensitive to muscle length or muscle force, and (b) that there is a pool of bound calcium (activating calcium) that waxes and wanes with a time course intermediate between the free calcium concentration and force.     

ATP-induced calcium transient in cultured rat aortic smooth muscle cells

To characterize the excitatory purinoceptors in vascular smooth muscle cells and the biochemical mechanisms of their actions, the effects of ATP and other nucleotides on Ca2+ mobilization in cultured smooth muscle cells mainly from rat aorta were investigated. ATP induced a transient and dose-dependent increase in the cytosolic Ca2+ concentration. ATP also induced a rapid production of inositol trisphosphate (IP3). The agonist form of ATP was metal-free ATP and its half-maximal effect was obtained at about 0.1 microM. 4-beta-Phorbol 12-myristate 13-acetate (PMA) or 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both Ca2+ response and IP3 production. In addition, TMB-8 but not PMA, significantly decreased the amount of releasable Ca2+ presumably in the sarcoplasmic reticulum. Pertussis toxin also inhibited the Ca2+ response. Based on the dose-dependent effects of various nucleotides and adenosine on the Ca2+ response, it was concluded that the P2 subclass of purinoceptor is involved in the observed ATP effects. In addition, the observed absence or very weak effect of alpha, beta-methylene ATP relative to the effect of ATP suggests that the excitatory P2-purinoceptors in vascular smooth muscle cells do not form a homogeneous group, because the opposite order of potency for these two nucleotides was reported previously for the P2 purinoceptors involved in contraction of some isolated blood vessels.     

Effect of external sodium and calcium on calcium efflux in frog striated muscle

Summary The effect of media with different ionic composition on calcium efflux from the dorsal head of semitendinosus muscles ofRana pipiens was studied. The reduction in the fractional loss of⁴⁵Ca, when going from normal Ringer's solution to an ONa–OCa medium, was 60%. Withdrawal of only Na or Ca from the external medium also caused a significant drop in the fractional loss (33% and 34%, respectively). The effect of different concentrations of Ca (studied in the absence of the external Na) was also studied. It was found that a linear function could describe the relationship between the calcium-dependent calcium efflux and the external calcium concentration. These results indicate that calcium efflux from frog muscle fibers consists of three major components: one that is dependent on the presence of calcium in the external medium, one that is dependent on the presence of sodium in the external medium, and one that persists in the absence of these two cations.     

胃动素对胃窦平滑肌细胞内Ca2+浓度的影响及机制

目的：观察胃动素对新生大鼠胃窦平滑肌细胞内Ca^2＋浓度的影响及机制。方法：采用激光共聚焦显微镜结合细胞内荧光染色观察不同条件下相同浓度胃动素对培养大鼠胃窦平滑肌细胞内Ca^2＋浓度的影响。结果：细胞膜Ca^2＋通道阻断剂维拉帕米、含Ca^2＋螯合剂EGTA的D-Hank’s液及细胞内Ca^2＋释放阻断剂TMB-8均可不同程度抑制MTL对胃窦平滑肌细胞内Ca^2＋的升高作用。结论：MTL具有升高胃窦平滑肌细胞内Ca^2＋的作用,细胞外Ca^2＋内流和内Ca^2＋释放参与了这种作用。     

Fura-2 used as a probe to show elevated intracellular free calcium in platelets of Dahl-sensitive rats fed a high salt diet

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.     

Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2

Cut muscle fibers from Rana temporaria (sarcomere length, 3.4-4.2 microns) were mounted in a double Vaseline-gap chamber (14-15 degrees C) and equilibrated with end-pool solutions that contained 20 mM EGTA and 1.76 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from changes in pH (Pape, P. C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:000-000). Although the amplitude and duration of the [Ca] transient, as well as its spatial spread from the release sites, are reduced by EGTA, SR Ca release elicited by either depolarizing voltage-clamp pulses or action potentials behaved in a manner consistent with Ca inactivation of Ca release. After a step depolarization to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca depletion, reached a peak value within 5-15 ms and then rapidly decreased to a quasi-steady level that was about half the peak value; the time constant of the last half of the decrease was usually 2- 4 ms. Immediately after an action potential or a 10-15 ms prepulse to - 20 mV, the peak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The rising phase of the rate of release was also reduced, suggesting that at least 0.9 of the ability of the SR to release Ca had been inactivated by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were not caused by changes in its voltage activation. These effects of a first stimulation on the rate of SR Ca release elicited by a second stimulation recovered during repolarization to -90 mV; the time constant of recovery was approximately 25 ms in the action- potential experiments and approximately 50 ms in the voltage-clamp experiments. Fura-2, which is able to bind Ca more rapidly than EGTA and hence reduce the amplitude of the [Ca] transient and its spatial spread from release sites by a greater amount, did not prevent Ca inactivation of Ca release, even at concentrations as large as 6-8 mM. These effects of Ca inactivation of Ca release can be simulated by the three-state, two-step model proposed by Schneider, M. F., and B. J. Simon (1988, Journal of Physiology. 405:727-745), in which SR Ca channels function as a single uniform population of channels. (ABSTRACT TRUNCATED AT 400 WORDS)     

The elevation of the cytoplasmic calcium ions in vascular smooth muscle cells in SHR--measurement of the free calcium ions in single living cells by lasermicrofluorospectrometry

We have developed an accurate and sensitive system for the measurement of cytoplasmic free calcium concentrations ([Ca++]i) of a single cell by using UV-laser and Indo-1. By this method, we made the first successful measurement of [Ca++]i of single living vascular smooth muscle cells. [Ca++]i in spontaneously hypertensive rats was elevated and maintained after the 6th passage culture. However, [Ca++]i in Goldblatt hypertensive rats was not elevated. Thus, these results suggest that the maintenance of high [Ca++]i levels of vascular smooth muscle cells in spontaneously hypertensive rats is genetically regulated and that it is one of the mechanisms for hypertension.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.