Fluorescent Ca Indicator Research Articles

The calcium feedback signal in the phototransduction cascade of vertebrate rods

Intracellular free Ca (Ca i) was measured in functionally intact rod outer segments in darkness and during light responses using the fluorescent Ca indicator Indo-dextran. In darkness, Ca i was 554 ± 25 nM (n = 28) for −85 ± 2 pA of circulating dark current (I d) and declined in saturating light to a minimum value of ∼50 nM with a time course that paralleled the fall in Na:Ca,K exchange current. During a subsaturating flash response that reduced I d by 70%, Ca i fell to a minimum of ∼325 nM and recovered incompletely to a plateau of ∼450 nM that lasted ∼15 s after full recovery of I d. During a 60 s step that caused ∼7-fold reduction in sensitivity of superimposed flash responses, Ca i reached a steady-state level of ∼252 nM.

Visualization of calcium transients controlling orientation of ciliary beat.

To image changes in intraciliary Ca controlling ciliary motility, we microinjected Ca Green dextran, a visible wavelength fluorescent Ca indicator, into eggs or two cell stages of the ctenophore Mnemiopsis leidyi. The embryos developed normally into free-swimming, approximately 0.5 mm cydippid larvae with cells and ciliary comb plates (approximately 100 microns long) loaded with the dye. Comb plates of larvae, like those of adult ctenophores, undergo spontaneous or electrically stimulated reversal of beat direction, triggered by Ca influx through voltage-sensitive Ca channels. Comb plates of larvae loaded with Ca Green dextran emit spontaneous or electrically stimulated fluorescent flashes along the entire length of their cilia, correlated with ciliary reversal. Fluorescence intensity peaks rapidly (34-50 ms), then slowly falls to resting level in approximately 1 s. Electrically stimulated Ca Green emissions often increase in steps to a maximum value near the end of the stimulus pulse train, and slowly decline in 1-2 s. In both spontaneous and electrically stimulated flashes, measurements at multiple sites along a single comb plate show that Ca Green fluorescence rises within 17 ms (1 video field) and to a similar relative extent above resting level from base to tip of the cilia. The decline of fluorescence intensity also begins simultaneously and proceeds at similar rates along the ciliary length. Ca-free sea water reversibly abolishes spontaneous and electrically stimulated Ca Green ciliary emissions as well as reversed beating. Calculations of Ca diffusion from the ciliary base show that Ca must enter the comb plate along the entire length of the ciliary membranes. The voltage-dependent Ca channels mediating changes in beat direction are therefore distributed over the length of the comb plate cilia. The observed rapid and virtually instantaneous Ca signal throughout the intraciliary space may be necessary for reprogramming the pattern of dynein activity responsible for reorientation of the ciliary beat cycle.

Intracellular calcium transients recorded with Fura-2 in spontaneously active myocytes isolated from the atrioventricular node of the rabbit heart

We have used the fluorescent Ca indicator Fura-2 to assess the changes in intracellular calcium (Cai) in single spontaneously active myocytes isolated from the rabbit atrioventricular node (AVN). Simultaneous recordings of membrane potential and the Fura-2 ratio signal (which reflects Cai) showed that a transient rise of Cai occurred with each spontaneous action potential (AP). The AP upstroke preceded the rise in Cai and repolarization of the AP occurred faster than the decline of Cai. The level of Cai remained raised and progressively declined towards a baseline diastolic level during the subsequent pacemaker depolarization. The Fura-2 (Cai) transient in spontaneously active AVN cells had a time-to-peak of 49.2 +/- 5.4 ms (mean +/- s.e.m.; n = 7) and declined with a single exponential time course (time constant = 139.8 +/- 23.9 ms; n = 7). Application of 10 microM ryanodine completely and irreversibly abolished the Cai transient, identifying the sarcoplasmic reticulum (SR) as the major source of releasable Ca. Both removal of external Ca and block of L-type Ca channels (with 2 microM nifedipine) also abolished Cai transients, suggesting that Ca entry via L-type Ca-channels is involved in triggering the SR Ca release underlying the Cai transient. Removal of external Na (in the presence of 20 microM nifedipine to block L-type Ca channels) caused a reversible increase in Cai, showing that Na/Ca exchange is present in AVN cells and that it is involved in Cai regulation. Spontaneous Cai transients were abolished by 1 microM acetylcholine, and this was associated with a hyperpolarization of membrane potential and cessation of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.

Inhibition of calcium influx and calcium current by gamma-aminobutyric acid in single synaptic terminals.

Inhibition of Ca influx and Ca current by gamma-aminobutyric acid (GABA) was studied in single synaptic terminals of isolated retinal bipolar neurons. Measurements of intracellular Ca concentration [( Ca]i) using the fluorescent Ca indicator fura-2 showed that GABA potently inhibited Ca influx into the terminal elicited by high extracellular K concentration ([K]o). This inhibition was attributed to GABA type A (GABAA) receptor-activated chloride ion conductance that prevented bipolar neurons from depolarizing sufficiently to activate the Ca current, even in response to increased [K]o. Patch-clamp recordings of the Ca current revealed a second effect of GABA: GTP-dependent inhibition of the Ca current. This inhibition was not mediated by GABAA receptors, but baclofen, which binds to the GABA type B (GABAB) receptor and is known to inhibit the Ca current in other systems, was not able to mimic the action of GABA. This suggests the involvement of a different type of GABAB-like receptor in the inhibition of Ca current by GABA. GABA did not cause an overall suppression of the Ca current; rather, the voltage-dependence of Ca-channel activation was shifted to more depolarized potentials. Thus, maximal inhibition of the Ca current by GABA occurred in the physiological range of potential.

Serotonin-stimulated Ca 2+ response is increased in the blood platelets of depressed patients

Abnormalities in central serotonergic function have long been suspected of playing a role in the pathogenesis of depression (Aprison et al 1978). The readily accessible human blood platelet possesses serotonin: (5-HT.,) receptors that mediate the aggregatory res~nses to serotonin (5-HT) (Geaney et al 1984) and has been suggested as a possible model for the central serotonergic neuron because of its p h ~ a c o logical similarities with b ~ prep,a-ations (Stahl 1977). Stimulation of 5-HT2 receptors in human platelets increases phosphoinositide hydrolysis (De Courcelles et al 1985), producing inositol uiphosphate, which mobilizes intracellular Ca 2 + from internal storage sites (Affolter et al 1984). Kagaya et al (1990), using the fluorescent Ca indicator fura-2, have indicated that 5-HT-induced intracellular Ca -+ increase in human platelets was mediated by 5-HT2 receptors. We recently found that this response was not significantly affected by sampling time, sampling season, gender difference, meal, or exercise (vmp,,hli~h,~d ao,~ T~,o..~,r.-..... the 5-HT-stimulated Ca-' + response in human platelets appears to be useful for studying 5-HT2 receptor function in patients. In the present study, we have assessed 5-HTinduced Ca 2+ response in the ~latelets of unmedicated depressive patients and normal con-

Propagation of Transient Ca2+ Increase in Sea Urchin Eggs upon Fertilization and Its Regulation by Microinjecting EGTA Solution.

Upon fertilization, the concentration of intracellular Ca2+ (Cai) in sea urchin eggs increased up to 3 microM when measured with fura-2, a fluorescent Ca indicator and the increase in Cai traversed from the sperm entry point as a wave over the entire egg at the mean propagation velocities of 5.0 microns/sec in C. japonicus egg and 5.3 microns/sec in H. pulcherrimus egg. However, the velocity was not uniform; i.e., it was rapid in the vicinity of the sperm entry point and the opposite point, but slow in the central region of the egg. Microinjecting a Ca-EGTA buffer and an IP3 solution into the C. japonicus egg induced the transient Cai increase more rapidly than that upon fertilization, due perhaps to the diffusion of the injectates. In order to investigate Ca2+ release during Cai increase upon fertilization, EGTA solutions were microinjected into unfertilized or fertilizing eggs. Microinjecting 100 mM EGTA (final concentration of 1 mM) not only suppressed the transient Cai increase, but also reduced the increased Cai rapidly, and never induced egg activation after insemination, whereas 10 mM EGTA (final concentration of 0.1 mM) did not significantly affect the Cai increase or the activation. Ca2+ released upon fertilization was estimated to be 150-170 microM in the egg cytoplasm from the amount of microinjected EGTA and fura-2. It was concluded that although more than 150 microM of Ca2+ was released intracellularly upon fertilization, Cai increased to only a few microM because most of the released Ca2+ was sequestered by intracellular Ca2+ binding substances.

Effect of various factors on serotonin-induced Ca 2+ response in human platelets

Serotonin (5-HT)-stimulated intracellular Ca 2+ concentration change was studied in the platelets of healthy subjects, using fluorescent Ca indicator fura- 2. 5-HT increased the Ca 2+ response in a concentration- dependent manner. 10 μM of 5-HT induced the maximal response and its EC 5 0 value was 0.4 μM. This response was potently inhibited by selective 5-HT 2 antagonists, suggesting that 5-HT-induced Ca 2+ mobilization in human platelets is mediated by 5-HT 2 receptors. This 5-HT 2- mediated Ca 2+ response was not significantly affected by the time of blood sampling, gender, meal or exercise. However, this response declined with time after blood drawing, suggesting that it must be measured as soon as possible after sampling. These results indicate that 5-HT-stimulated Ca 2+ response in human platelets is a stable parameter and that it will be suitable for assessing 5-HT 2 receptor function in depressed patients.

Intracellular Ca transients in rat cardiac myocytes: role of Na-Ca exchange in excitation-contraction coupling

Membrane current and intracellular Ca concentration ([Ca]i) transients were recorded from isolated rat ventricular myocytes under voltage-clamp control. The cells were dialyzed by the patch pipette solution, which contained the fluorescent Ca indicator indo-1 and 0.5 mM Na. Under these experimental conditions, Ca entry via Na-Ca exchange did not appear to be appreciable even in the absence of extracellular Na. Increasing the duration of voltage-clamp pulses from 5 to 80 ms produced [Ca]i transients of increasing amplitude, while the peak Ca current was not changed. This duration dependence of the [Ca]i transient was most demonstrable at more negative test potentials (e.g., -20 to -30 mV) and was not qualitatively modified by Na-free solutions. This latter result indicates that Ca extrusion by Na-Ca exchange is not responsible for the smaller [Ca]i transients observed when the membrane is repolarized after very brief depolarizations. Although the peak Ca current was not changed by increasing pulse duration, the integrated Ca current was increased. These observations are consistent with a Ca-release mechanism in cardiac excitation-contraction coupling in which 1) the Ca-release process can be modulated by membrane potential or 2) the Ca entering the cell via Ca channels has a preferential access [compared with Ca from the sarcoplasmic reticulum (SR)] to the site(s) that control SR Ca release. The role of Na-Ca exchange in the decline of [Ca]i during relaxation was also explored. Removal of extracellular Na (Nao) resulted in 20% slowing of the decline in [Ca]i during relaxation. From this, we conclude that the Na-Ca exchange competes with SR to remove Ca from the cytoplasm and that under our control conditions the exchanger may account for 20% of this decline. The Nao dependence of relaxation was reduced at more positive membrane potentials and increased by SR Ca loading.

Imaging spatial distribution of release in single muscle fibres from Balanus nubilus using image intensification

The physical location of the sites for Ca2+ release, triggering the contraction of a muscle fibre, is a key issue in understanding tbe physiological control mechanisms of excitation-contraction (EC) coupling [l]. The isolation of the ryanodine receptor from rabbit SR fractions [2] shows the terminal cistemal SR to contain ryanodine-sensitive Ca channels with a 1OOpS conductance. A second type of Ca channel, which has a 1OpS conductance and is IP3 sensitive at polarised potentials, has also been identified in the T system [3]. Electron microscopy has shown the ryanodine receptor to be located at tire triad, accounting for at least part of the foot structures [4]. Studies of the 1ocaliWion of Ca2+ release sites have been carried out in a number of ways. Somlyo et al. [5, 61 used an electron microprobe to compare the localisation of calcium at rest and during a tetanus. More recently, Sommer and Nassar [7] using a quick-freezing technique, which reportedly showed the appearance of ‘pits’ over the entire area of the SR examined, within the first millisecond after stimulation. Both these techniques have the drawback that they require fmation of the fibre. The aim of this study was to determine the spatial distribution of the calcium release sites in a single, intact_ electrical1 I+ excited muscle fibre, using the fluorescent Ca indicators Quin-2 and Fura[8] by employing an image intensifier. Single barnacle muscle cell sarcomere length is some g-10 pm [9] and this long sarcomere length provides the advantage that, using conventional light microscope optics, intrasarcomere detail could be better-resolved than for the shorter sarcomeres present in amphibian fibres. Prehminary accounts of the methods and results have already appeared [lo, 111.

Cytosolic Ca2+in Melanotrophs: Pharmacological Insights into Regulatory Influences of Electrical Activity and Ion Channels*

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in melanotrophs, the characteristic endocrine cells of the pars intermedia of the rat pituitary gland, using the fluorescent Ca indicator fura-2. The resting [Ca2+]i was 211 +/- 8 nM and was little affected by tetrodotoxin (TTX; 5 or 10 microM), which inhibits the spontaneous action potentials that occur in these cells. Removal of extracellular Ca2+ (by chelation with EGTA) or addition of the Ca channel blocker nimodipine (1 microM) produced a rapid fall in [Ca2+]i, which occurred whether TTX was present or not. Excess K+ (60 mM), veratridine (10 or 100 microM) and BAY K 8644 (1 microM) each caused a rapid rise in [Ca2+]i, which was blocked or truncated by EGTA or nimodipine. TTX blocked or truncated the increases in [Ca2+]i induced by veratridine, but not those induced by either excess K+ or BAY K 8644. The results show that manipulations that increase or decrease hormone output increase or decrease [Ca2+]i. Furthermore, the resting [Ca2+]i appears to depend importantly on Ca influx, since it is rapidly and markedly reduced by removal of extracellular Ca2+ or addition of a Ca channel blocker.

Effects of E-1020, a new cyclic AMP-specific phosphodiesterase inhibitor, on cyclic amp and cytosolic free calcium of cultured vascular smooth muscle cells.

The purpose of this study is to clarify the pharmacological effects of E-1020 a new cAMP-specific phosphodiesterase (PDE) inhibitor, on cAMP or cytosolic free calcium (Ca) of cultured vascular smooth muscle cells (VSMC). VSMC were separated from the thoracic aorta of 2- to 3-month-old Wistar Kyoto rats (WKY). cAMP was measured by using the 125I-cAMP radioimmunoassay kit. The cytosolic free Ca of VSMC was measured by using the fluorescence Ca indicator, Fura-2/acetoxymethyl ester (Fura-2/AM). As a result, E-1020 increased the cAMP of VSMC in a dose-dependent manner, and about two fold increase in cAMP was observed by 10(-4) M E-1020. E-1020 decreased the cytosolic free concentration of Ca in a dose-dependent manner, and a significant decrease in cytosolic free Ca was observed by greater than 10(-6) M E-1020. These effects of E-1020 on cAMP and cytosolic free Ca were enhanced in the presence of low concentration (10(-8) M) of DL-noradrenaline. That is, a more prominent decrease in cytosolic free Ca than that of E-1020 alone was observed. Our results show that E-1020 decreases cytosolic free Ca of VSMC in parallel with increase in cAMP, and thereby suggest a potential vasodilating activity in vivo.

Calcium Release Mechanisms in Smooth Muscle

Properties of the intracellular Ca store were studied using saponinskinned fiber bundles of guinea pig smooth muscles using a fluorescent Ca indicator method. There exist two Ca release mechanisms in the Ca store: Ca-induced Ca release (CICR) and inositol 1,4,5-trisphosphate (IP3)-induced Ca release (NCR) mechanisms. The smooth muscle Ca store consists of two compartments: one (Sa) has both CICR and NCR, and the other (Sβ) has only NCR. The smooth muscle CICR is activated by >1μM Ca2+, has essentially the same properties with that in striated muscles, and is open-locked by ryanodine. After ryanodine treatment, therefore, the Ca uptake capacity of Sa is selectively lost ('functional removal') with no effect on Sβ. The IICR is Ca2+-dependent: Ca2+ enhances the NCR below 300 nM, but has also an inhibitory effect above this concentration. Therefore, Ca2+ acts on the IICR in a positive feedback manner when muscle tension is about to rise, making IP3 more effective, but this feedback is cut off as the tension approaches the maximum. ATP enhances the IICR as is the case in the CICR. 'Functional removal' of Sa in intact bundles by ryanodine was used to estimate the role of Ca release in agonist-induced contractions. Ca release from the Sa is important at least in the initial phase of contractions; and in the pulmonary artery, most of the activator Ca2+ originates from Sα.

Stimulation-Induced Regional Alteration of Ca2+ Levels in Single Arterial Smooth Muscle Cells

Contraction in vascular smooth muscle is normally triggered by an increase in the cytosolic free Ca2+ concentration. We employed the fluorescent Ca indicator, fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in arterial myocytes and the changes elicited by norepinephrine (NE) and by alterations in the Na− electrochemical gradient. In modified Kreb's solution containing 1.8 mM Ca2+, these bovine tail artery myocytes were relaxed and able to maintain normal membrane potentials. The cells contracted rapidly when exposed to NE or high K+ solution. They then relaxed slowly when the activator was washed away. A rise in Ca2+ concentration, [Ca2+], was elicited by NE as well as by Ca2+ entry via Na+/Ca2+ exchange. Digital analysis of images of cellular fura-2 fluorescence revealed that the intracellular [Ca2+] was relatively uniformly distributed in resting cells prior to activation. During NE-evoked contractions, intracellular [Ca2+] in-creased an average of two- to threefold, and the distribution of [Ca2+] became much more heterogeneous. Upon recovery from activation, the cells relaxed, usually attaining >90% of their original resting length. In contrast to the relatively uniform Ca2+ distribution observed prior to NE activation, discrete regions of elevated [Ca2+] were observed throughout the recovered cells. Similarly, reducing the Na+ electrochemical gradient and, presumably, activating Na+/Ca2+ exchange elicited a rise in [Ca2+]. In cells exposed to increased [Na+]i and reduced [Na+]o, as in cells following recovery from NE stimulation, discrete areas of high [Ca2+] were apparent. The large spatial variation induced by cell activation implies that Ca2+ was sequestered at localized sites throughout the cell.

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches.

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.

Calcium dependent inositol trisphosphate-induced calcium release in the guinea-pig taenia caeci

The properties of the Ca stores in the skinned fiber bundles of the guinea-pig taenia caeci were studied by a novel method to measure Ca released from the stores using a fluorescent Ca indicator, fura-2. It is found that the capacity of the Ca stores amounts some 170μ moles/liter cell water. There are at least two Ca release mechanisms, Ca-induced Ca release (CICR) and Ca release induced by inositol-1,4,5-trisphosphate (IP 3). Only about a half of the Ca is released by the Ca-induced Ca release, while IP 3 releases most of the Ca. The rate of the IP 3-induced Ca release is found to be strongly enhanced by submicromolar concentrations of Ca 2+.

The regulation of cytosolic calcium in rat brain synaptosomes by sodium‐dependent calcium efflux.

1. When pinched-off presynaptic nerve endings (synaptosomes) isolated from rat brain are incubated in a low-Na (24-36 mM) medium, they take up 45Ca in a time-dependent manner. In a medium containing 1 mM-Ca, this Na-dependent 45Ca uptake amounts to approximately 10 nmol/mg protein at 1 min, and to approximately 40 nmol/mg protein at 20 min. The Na-dependent Ca uptake is not reduced when the synaptosomes are loaded with concentrations of quin 2 as high as 2 mM. 2. The increase in 45Ca uptake is paralleled by an increase in the free cytosolic Ca concentration [Ca]i, as monitored with the fluorescent Ca indicators quin 2 or fura 2. [Ca]i increases from the value of approximately 200 to approximately 500 nM within 3-5 min, and thereafter, remains at this elevated level. 3. When synaptosomes that have been loaded with 45Ca (for 1 min, in a low-Na medium) are diluted into an Na-containing medium, there is a rapid efflux of the Ca load. After correcting for Ca that is taken up during the efflux period, calculations show that the total Ca in the synaptosomes returns to the control level within 1 min. Measurements of total chemical Ca parallel the measurements made with radiotracer Ca, and confirm that the Ca loaded into the nerve terminals during a 5 min incubation in a low-Na medium is extruded from the nerve terminals within 1 min in a normal-Na medium. 4. The efflux of Ca from the synaptosomes is paralleled by a drop of [Ca]i to its basal level, also within 1 min. 5. The mitochondrial uncoupler, carbonyl cyanide p-trifluoromethyloxy-phenyl-hydrazone (FCCP, 1 microM), has no effect on either Na-dependent Ca uptake or efflux in synaptosomes. FCCP causes a slight (100-200 nM) increase in [Ca]i in synaptosomes resuspended in either a Na or a low-Na medium. This indicates that little of the Ca that is taken up by the synaptosomes in a low-Na medium is sequestered by the mitochondria. 6. These results suggest that Na-dependent Ca efflux (probably Na-Ca exchange) plays an important role in allowing nerve terminals to recover rapidly from a Ca load.

Effects of calcitonin on cytosolic Ca in a suspension of rabbit medullary thick ascending limb tubules.

A basal level of cytosolic free Ca (Caf) of 112 +/- 7 nM (n = 15) was measured in rabbit medullary thick ascending limb tubules that were loaded with the fluorescent Ca indicator quin 2. Calcitonin addition (10(-6) M) caused a greater than twofold increase in Caf. The KD for calcitonin stimulation of Caf was 8 X 10(-11) M. The rise in Caf had a half time of approximately 1.5 min. Neither parathyroid hormone (10(-6) M), dibutyryladenosine 3',5'-cyclic monophosphate (10(-4) M), nor forskolin (2 X 10(-5) M) had any significant effect on Caf levels, consistent with the rise in Caf being independent of an elevation in adenosine 3',5'-cyclic monophosphate. The effects of ouabain and the mitochondrial uncoupler, 1799, were also tested to obtain further information regarding the regulation of Caf. Ouabain (10(-4) M) caused no significant elevation in Caf; in contrast, 15 min of incubation with 1799 (10(-5) M) produced a twofold increase in Caf.

Sarcoplasmic reticulum interacts with the Ca(2+) indicator precursor fura-2-AM

Fura-2-am, the pentaester precursor of the fluorescent Ca(2+) indicator fura-2, is modified when it is exposed to isolated skeletal muscle sarcoplasmic reticulum vesicles. The modified fura-2-am has enhanced fluorescence, is not sensitive to Ca(2+), and is partially bound to the SR membrane. The isolated product is further converted into fura-2 by esterase. It is suggested that the SR-induced modification is a selective enzymatic hydrolysis of only some of the five ester moieties on fura-2-am. A structure is proposed to account for the results. The potential for this effect of SR on fura-2-am to cause complications in measurements of in vivo intracellular free [Ca(2+)] is noted.

The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading

The Ca concentration ([Ca 2+] i) in single rat peritoneal mast cells was measured by means of the new fluorescent Ca-indicator dye fura-2. Dye-loaded cells were made to degranulate with either antigen or compound 48/80. In cells loaded with extracellularly applied, membrane-permeant fura-2 ester, degranulation was accompanied by a permanent loss of 40–60% of the fluorescence, but comparison of fluorescence at different wavelengths indicated no or only small changes in [Ca 2+] i. When cells were loaded by microinjection of the impermeant potassium salt of the dye, degranulation resulted in no permanent loss of fluorescence, but instead was preceded by transient fluorescence changes that indicate a rapid, large and transient increase in [Ca 2+] i. We suggest that ester-loaded fura-2 accumulates to a significant degree in the secretory granules and is lost from the cell during exocytosis.