Fura-2 Fluorescence Ratio Research Articles

Intracellular Ca 2+ transients in isolated perfused rat heart: measurement using the fluorescent indicator [formula omitted

We have investigated the nature of Fura-2 AM loading into isolated perfused rat heart and the temporal and kinetic relationship between left ventricular [Ca 2+] i dependent fluorescence and isovolumic pressure. The contribution of hydrolysed mitochondrial matrix Fura-2 fluorescence to that measured from the surface of the heart was estimated to be 43.9 ± 5.5% by the addition of 100 μM Mn 2+ to the perfusate. Maximum endothelial Fura-2 fluorescence ratio, estimated by the addition of 3 μM bradykinin to the perfusate, was found to constitute 33.6 ± 2.7% of the maximum myocardial Fura-2 fluorescence ratio. Approximately 11.2% of the 340 nm surface fluorescence was insensitive to 20 mM Mn 2+ in the presence of ionomycin (3 μM) and therefore indicates the degree of partial hydrolysis of Fura-2 AM . Thus, depending on the contribution of endothelial Fura-2 fluorescence at a physiological endothelial calcium concentration, cytosolic fluorescence may comprise between 11–45% of the total cellular fluorescence at 340 nm. Net tissue interference of the Fura-2 fluorescence ratio by NADH emission and myoglobin absorption remained unaltered, providing the oxygenation state of the tissue was unaltered throughout the experiment. The [Ca 2+] i dependent fluorescence decay from peak systole was best fitted to a biexponential decay with fast and slow rate constants of 18.08 ± 1.97 s −1 and 0.23 ± 0.02 s −1, respectively. In addition, a phase shift was observed between temporal and kinetic measurements of the left ventricular isovolumic pressure and calcium dependent fluorescence traces during a contraction-relaxation cycle. We conclude that despite imperfect Fura-2 AM loading, the temporal and kinetic characteristics of intracellular [Ca 2+] transients in normal isolated perfused rat heart are similar to those reported in more controlled preparations such as isolated myocytes and cardiac trabeculae.

Fura‐2 signals evoked by kainate in leech glial cells in the presence of different divalent cations

The glutamate-agonist kainate evokes Ca2+ transients in both neurones and glial cells. Owing to the membrane depolarization elicited by kainate, a Ca2+ influx could occur through voltage-gated Ca2+ channels or through the kainate-gated cation channels directly. We have measured ratio signals of the calcium indicator dye fura-2, injected into giant glial cells of the leech Hirudo medicinalis, as response to kainate (5-20 microM) in the presence of different divalent cations. The responses to kainate increased during the first 2-4 kainate applications, both in unclamped and in voltage-clamped cells. The fura-2 fluorescence ratio (F350/F380) still increased when Ca2+ was replaced by Ba2+ but was suppressed in Ca(2+)-free saline and in the presence of Ni2+ (2 mM). Co2+ and Mn2+ (2 mM) also reduced the kainate-induced fura-2 fluorescence signals, due to entry of these divalent cations into the cells and subsequent quenching the fluorescence of the intracellular dye. It is concluded that Ni2+ blocks the kainate-induced membrane depolarization and Ca2+ transient but apparently does not enter the cells, while Ba2+, Co2+, and Mn2+ appear to permeate the membrane, presumably through the kainate-gated channels.

Glucose-dependent alterations of intracellular free calcium by glucagon-like peptide-1 (7-36amide) in individual [formula omitted] mouse β-cells

Depolarizing concentrations of glucose produce characteristic alterations of intracellular free Ca 2+ ([Ca 2+] i) in pancreatic β-cells. The effects of the proposed incretin, glucagon-like peptide-1 (7-36amide) (GLP-1 a) on [Ca 2+] i were determined from Fura-2 fluorescence ratio imaging of cultured ob ob mouse pancreatic β-cells. In control cells, [Ca 2+] i is low in 3 mM glucose; increasing [glucose] to 8–12 mM results in an initial dip in [Ca 2+] i followed by slow oscillating increases in [Ca 2+] i. GLP-1 a (0.03–10 000 pM) does not alter [Ca 2+] i in 3 mM glucose, but does change the response to elevated glucose (8–12 mM). The time integral of the initial dip is reduced ([GLP-1 a] 10–100 pM), and the integral of the [Ca 2+] i signal is increased ([GLP-1 a] ≥ 1 pM). GLP-1 a increases the frequency of sustained, stable plateau responses to elevated glucose, and the frequency of large, rapid spikes of increased [Ca 2+] i associated with either plateaus, or oscillations. Application of a cAMP analog mimics most of the actions of GLP-1 a. Activation of the GLP-1 a receptor, or application of cAMP alters pancreatic β-cell [Ca 2+] i only when [glucose] is high.

Effect of ATP, carbachol and other agonists on intracellular calcium activity and membrane voltage of pancreatic ducts.

The pancreatic duct has been regarded as a typical cAMP-regulated epithelium, and our knowledge about its Ca2+ homeostasis is limited. Hence, we studied the regulation of intracellular calcium, [Ca2+]i, in perfused rat pancreatic ducts using the Ca(2+)-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, Vbl, of individual cells. The resting basal [Ca2+]i was relatively high, corresponding to 263 +/- 28 nmol/l, and it decreased rapidly to 106 +/- 28 nmol/l after removal of Ca2+ from the bathing medium (n = 31). Carbachol increased [Ca2+]i in a concentration-dependent manner. At 10 mumol/l the fura-2 fluorescence ratio increased by 0.49 +/- 0.06 (n = 24), corresponding to an increase in [Ca2+]i by 111 +/- 15 nmol/l (n = 17). ATP, added to the basolateral side at 0.1 mmol/l and 1 mmol/l, increased the fluorescence ratio by 0.67 +/- 0.06 and 1.01 +/- 14 (n = 46; 12), corresponding to a [Ca2+]i increase of 136 +/- 22 nmol/l and 294 +/- 73 nmol/l respectively (n = 15; 10). Microelectrode measurements showed that ATP (0.1 mmol/l) hyperpolarized Vbl from -62 +/- 3 mV to -70 +/- 3 mV, an effect which was in some cases only transient (n = 7). This effect of ATP was different from that of carbachol, which depolarized Vbl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of Vbl (n = 4). Several other putative agonists of pancreatic HCO3- secretion were also tested for their effects on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Accommodation of mouse DRG growth cones to electrically induced collapse: Kinetic analysis of calcium transients and set‐point theory

Electrical stimulation causes growth cones of mouse dorsal root ganglion neurons to collapse. During chronic stimulation, however, growth cones resume motility. In addition, these growth cones are now resistant to the collapsing effects of subsequent stimulation, a process we term accommodation. We compared the kinetics of electrically induced Ca2+ transients in naive and accommodated growth cones in order to determine whether the accommodation process results from a change in the Ca2+ transient, or a change in the Ca2+ sensitivity of the growth cones. Three kinetics were determined: (1) the initial increase to peak Ca2+ levels produced by 10 Hz stimulation; (2) recovery from peak Ca2+ levels during stimulus trains lasting 15 min; and (3) clearing of Ca2+ from growth cones after terminating the stimulus. These kinetics were analyzed using single exponential fits to changes in fura-2 fluorescence ratios. The electrically evoked increase in Ca2+ was significantly slower in accommodated growth cones (tau = 6.0 s) compared to naive growth cones (tau = 1.4 s). Despite the slower increase of [Ca2+]i in accommodated growth cones, peak [Ca2+]i was similar to that reached in naive growth cones, and the steady-state Ca2+ level was significantly elevated after chronic stimulation. Thus, accommodated growth cones maintained outgrowth at [Ca2+]i that caused collapse initially. Time course experiments show that accommodation is a slow process (t 1/2 = about 3 h). Accommodation did not induce measurable changes in the rates of Ca2+ homeostasis during or after stimulus trains. The kinetics of Ca2+ recovery during (tau = 90 s) and after 15 min of stimulation (tau = 8.5 s) was not significantly different in accommodated versus naive growth cones. Rates of 45Ca2+ efflux were also similar in both types of growth cones. These results suggest two regulatory processes contributing to growth cone motility during chronic stimulation: (1) recovery of [Ca2+]i to levels permissive to neurite outgrowth, and (2) an increase in the range of optimal [Ca2+]i for growth cone motility. These adaptive responses of mammalian growth cones to chronic stimulation could be involved in the modulation of CNS development by electrical activity of neurons.

Characteristics of Ca2+ release induced by Ca2+ influx in cultured bullfrog sympathetic neurones.

1. A rise in intracellular Ca2+ ([Ca2+]i) and a Ca2+ current (ICa) induced by a depolarizing pulse were simultaneously recorded by fura-2 or indo-1 fluorescence and whole-cell patch clamp techniques in cultured bullfrog sympathetic ganglion cells. 2. [Ca2+]i (calculated from the ratio of fura-2 fluorescences excited at 380 and 340 nm and recorded with a photomultiplier at > 492 nm) rose regeneratively (in most cells) during a command pulse (from -60 to 0 mV, 100 ms), continued to rise thereafter, peaked at 666 ms (on average) and decayed slowly with a half-decay time of 22.8 s. 3. Scanning a single horizontal line across the cytoplasm with an ultraviolet argon ion laser (351 nm) and recording indo-1 fluorescences at two wavelengths (peaked at 410 and 475 nm) with a confocal microscope demonstrated that [Ca2+]i beneath the cell membrane rose much faster than that in the deeper cytoplasm. The time course of the spatial integral of [Ca2+]i, however, corresponded well with that recorded with fura-2 fluorescence using a photomultiplier. 4. [Ca2+]i measured by fura-2 fluorescence ratio using a photomultiplier did not increase during a strong depolarizing pulse (-60 to +80 mV), but sometimes rose after the pulse. A depolarization-induced rise in [Ca2+]i ([Ca2+]i transient) was blocked in a Ca(2+)-free, EGTA solution, reduced by lowering the extracellular Ca2+ concentration ([Ca2+]o) to 0.45 or 0.9 mM and enhanced by raising [Ca2+]o to 7.2 or 14.4 nM. 5. The extracellular Ca2+ dependence was non-linear when long depolarizing pulses (up to 500 ms) were applied; the amplitude of [Ca2+]i transient/Ca2+ entry (unit [Ca2+]i transient) increased with an increase in Ca2+ entry. 6. Increasing the duration of depolarization (-50 or -60 to 0 mV) from 20 to 500 ms enhanced asymptotically the integral of ICa (due to inactivation), and progressively the magnitude of [Ca2+]i transients, leading to the apparent non-linear dependence of unit [Ca2+]i transient on Ca2+ entry as well as on the duration of membrane depolarization. The peak time of [Ca2+]i transient was unchanged for pulse durations up to 300 ms, but prolonged with an increase in pulse duration to 500 ms. 7. Inhibitors of Ca2+ release from intracellular Ca2+ reservoirs, dantrolene (10 microM) and ryanodine (50 microM), blocked the [Ca2+]i transient to 56 and 30%, respectively, of the control. 8. The higher the basal [Ca2+]i level, the greater was the magnitude of the [Ca2+]i transients.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca 2+-dependent inhibition of Ca 2+-independent contraction in uterine smooth muscle

Uterine smooth muscle of the rat shows Ca 2+-independent contraction in response to oxytocin in Ca 2+-free medium. Micromolar Ca 2+ inhibits this contraction. We now tested whether Ca 2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca 2+, was increased in parallel with the degree of inhibition by Ca 2+. When inhibition was elicited by Ca 2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca 2+-free solution and that in the medium with 1 μM Ca 2+ showed that the inhibition by Ca + is non-competitive. EGTA chelation of the intracellular Ca 2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca 2+. EGTA suppressed the Ca 2+-induced contraction but did not affect Ca 2+-independent contraction. It is concluded that the inhibition is induced by intracellular Ca 2+ itself.

Thapsigargin reveals evidence for fMLP-insensitive calcium pools in human leukocytes

To investigate the relationship between different intracellular Ca 2+ pools, cytosolic free calcium ([Ca 2+] i) was surveyed by means of a Fura-2 fluorescence ratio method on single isolated human leukocytes. Both monocytes and neutrophilic granulocytes (PMN) displayed long lasting spontaneous [Ca 2+] i transient changes (1–2 min). In PMN stimulated with the bacterial peptide fMLP we observed transients with shorter duration (10–30 s) and smaller amplitude often superimposed on the long lasting transients. The time course of changes in [Ca 2+] i was recorded in a large number (149) of single leukocytes prestimulated for 5 min with fMLP and then challenged with thapsigargin (a blocker of Ca 2+ uptake in intracellular pools). Statistical analysis of [Ca 2+] i responses revealed that fMLP-sensitive pools contributed to the long lasting [Ca 2+] i transients seen in both leukocyte types. However, the existence of fMLP-insensitive calcium pools may explain the superimposed transients seen in PMN. Thapsigargin was also added together with EGTA (to impede contribution from extracellular Ca 2+) to 198 fMLP prestimulated and 153 unstimulated PMN. Based on Ca 2+ reglstrations in these cells and a mathematical model (supposing two separate first order responses) the amount of Ca 2+ stored in the various pools and their release kinetics were estimated. The results indicate that fMLP-insensitive calcium pools exist in PMN but not in monocytes. Since the digital imaging technique also depicts cellular motility, an additlonal finding was that the leukocyte's ability to sequestrate the Ca 2+ from the cytosol seemed important to locomotion.

Mastoparan increases membrane permeability in rat parotid cells independently of action on G-proteins

Mastoparan, a peptide toxin from wasp venom, stimulated the accumulation of inositol phosphates in rat parotid acinar cells. Addition of this peptide to fura-2-loaded cells resulted in a rapid increase in the fura-2 fluorescence ratio (340 nm/380 nm), suggesting that mastoparan stimulates an increase in cytosolic Ca2+ concentration. However, this change in the ratio appears to be due, in part, to fura-2 leakage from the cells, because addition of Mn2+, which quenches extracellular fura-2 fluorescence, reduced the increased fluorescence ratio. In addition to the fura-2 leakage, mastoparan caused considerable leakage of lactate dehydrogenase, a cytosolic marker enzyme. Furthermore, mastoparan decreased the number of trypan blue-excluding cells, indicating a decrease in cell viability. These results suggest that mastoparan enhances the membrane permeability by a mechanism independent of the activation of G-proteins.

Determination of Fura-2 dissociation constants following adjustment of the apparent Ca-EGTA association constant for temperature and ionic strength

The accurate calibration of Fura-2 fluorescence in living cells is dependent upon the apparent dissociation constant (K d) of Fura-2 for Ca 2+. If Ca-EGTA calibration buffers are used to construct an in vitro calibration curve, then the calculated value of the apparent Ca-EGTA association constant (K′ CaEGTA) will have an important influence on the K d of Fura-2 and thus the calculated free [Ca 2+] in cells. In order to simulate experimental conditions, the individual proton and Ca 2+ association constants for EGTA in these experiments were adjusted for both ionic strength and temperature using a semi-empirical form of the Debye-Huckel limiting law and the Van't Hoff isochore, respectively, as described by Harrison and Bers [7]. The modified individual binding constants were then employed in the calculation of K′ CaEGTA using the SPECS computer program of Fabatio. At pH = 7.05, ionic strength = 0.15 M, temp = 20°C, K′ CaEGTA = 3.232 × 10 6 M −1; at pH = 6.84, temp = 36°C, K′ CaEGTA = 1.652 × 10 6 M −1. These values differed substantially from those obtained with unadjusted individual association constants. Calibration buffers of varying [Ca 2+] were prepared using the corrected values of K′ CaEGTA, and Fura-2 fluorescence ratios were measured during superfusion of these buffers in the experimental chamber at both 20°C and 37°C. The K d of Fura-2 for Ca 2+ was determined to be 236 nM at 20°C and 285 nM at 37°C, utilizing the value of K′ CaEGTA adjusted by the method of Harrison and Bers. These new K d constants can be applied in conjunction with in vivo measurements of R′ max and R′ min to achieve accurate quantitation of cytoplasmic free Ca 2+ in a variety of cell types under physiological conditions.

Intracellular Calcium Signals Measured with Fura-2 and Aequorin in Frog Skeletal Muscle Fibers.

Intracellular Ca(2+)-related optical signals during and after contraction (twitch and tetanus) were measured in single frog skeletal muscle fibers with fura-2 and aequorin. In twitch response, the peaks of [Ca2+]i estimated from the in vitro calibrations of fura-2 and aequorin were significantly different (0.5 microM for fura-2 and 5 microM for aequorin). Even 30s after twitch response, the fura-2 fluorescence ratio (F340/F380) signal did not recover to the resting level before stimulation. When the stimulation frequency was increased, an increase in the resting fura-2 ratio signal became obvious and after the cessation of stimulation this increase gradually recovered to the level before stimulation. After tetanus (50 Hz for 1 s), a higher fura-2 ratio signal than that before stimulation was sustained longer than 90 s. These results indicate that the dissociation of the released Ca2+ from the intracellular Ca2+ binding sites probably takes longer time than that previously reported. The present results, therefore, demonstrate that fura-2 is advantageous for qualitative monitoring of a slight change in the resting level of [Ca2+]i, which is not easily detectable with aequorin. In addition, the problems encountered in quantitative estimation of [Ca2+]i with fura-2 are also discussed.

Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes.

Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.

Calcium oscillations and morphological transformations in single cultured gastric parietal cells

Calcium is an important regulator of cellular activities including HCl secretion by parietal cells. With cholinergic agonists, a role for calcium is established; however, with histamine, at least two signaling pathways may be involved including calcium and adenosine 3',5'-cyclic monophosphate (cAMP). Because chelation of medium and/or cellular calcium has pronounced inhibitory effects on cholinergic but lesser effects on histamine-stimulated acid secretory responses in cell populations, the calcium pathway may not be of central importance for HCl secretion regulated by histamine. We have used digitized video imaging of fura-2 fluorescence ratios and cellular morphology to determine more precisely the relationship between cellular calcium signaling mechanisms and acid secretion in single cultured rabbit parietal cells. Calcium signaling patterns were found to exhibit striking differences with histamine as compared with the cholinergic agonist carbachol. Maximal doses of histamine initiated repetitive oscillations in intracellular calcium ([Ca2+]i) in approximately 50% of cells, whereas the maximal carbachol response was characterized by a typical initial spike followed by a sustained elevation in [Ca2+]i. Oscillations in response to carbachol were detected only at doses below the half-maximal concentration for initiation of acid secretion. Correlation of gradual expansion of acidic vacuoles with increases in [Ca2+]i in the same cells indicated that approximately 20% of cells increased acid secretory-related activities in response to histamine with no detectable rise in [Ca2+]i. These data suggest two possibilities: 1) a rise in [Ca2+]i is not necessary for histamine-stimulated HCl secretion, or 2) heterogeneous receptor-coupling mechanisms exist in parietal cell populations with either calcium or cAMP mechanisms predominating in different subpopulations. The ability to assess simultaneously acid secretory-related responses and calcium signaling patterns allows, for the first time, correlation of "physiological" and biochemical responses in single parietal cells. This methodology is expected to provide new insight into second messenger control mechanisms that are not possible either in cell populations or acutely isolated parietal cells that do not exhibit morphological transformations detectable at the light microscope level.

Myocyte structure, function, and calcium kinetics in the cardiomyopathic hamster heart

The hereditary cardiomyopathic strain of the Syrian hamster has been studied extensively as a model of cardiomyopathy and heart failure. However, the primary defect underlying the cascade of events leading to heart failure has not been identified. In the present study, isolated cardiac cells were obtained for study from 8-mo-old cardiomyopathic (Bio 14.6) and age-matched normal hamsters (F1B). We examined the relationships among structure, contractile function, and calcium kinetics in these isolated cardiomyocytes. The cardiomyopathic myocytes were wider and longer than controls, and myopathic cells were less calcium tolerant. The sarcoplasmic reticulum and T-tubule systems from myopathic hearts were more abundant as determined by electron microscopy. The amplitude and velocity of contraction was decreased in cardiomyopathic cells, whereas diastolic relaxation velocity was not different between the two groups. The size of the rapidly exchangeable calcium pool determined by 45Ca2+ uptake was significantly increased in cardiomyopathic myocytes. Time-averaged cytosolic Ca2+ in these cells (472 +/- 60 nM) was significantly higher than control (260 +/- 15 nM) as measured by the fura-2 fluorescence ratio. When extracellular Ca2+ ([Ca2+]o) was increased to greater than 1.2 mM, the resulting increase in intracellular calcium concentration was less than that in normal control cells. The corresponding [Ca2+]o-induced increase in amplitude of cell motion was significantly attenuated in the cardiomyopathic cells compared with normal control myocytes. We conclude that distinct abnormalities of contractile function and calcium homeostasis can be identified in single cells isolated from cardiomyopathic hamster hearts.

Atrial natriuretic peptide reduces the basal level of cytosolic free Ca 2+ in guinea pig cardiac myocytes

The cytosolic free Ca 2+ concentration ([Ca 2+] i) was monitored in quiescent atrial and ventricular myocytes isolated from guinea-pig hearts by the fura-2 fluorescence ratio technique. Recombinant human atrial natriuretic peptide (ANP) was found to reduce their basal [Ca 2+] i level in a dose-dependent manner. Dibutyryl-cGMP mimicked the effect of ANP. Neither the prior application of caffeine nor removal of extracellular Na + impaired the ANP effect. ANP had no inhibitory effect on voltage-gated Ca 2+ currents measured by a whole-cell patch clamp technique. The ANP-induced [Ca 2+] i decrease was abolished by orthovanadate. Thus, it is concluded that ANP reduces the basal [Ca 2+] i presumably through the cGMP-mediated activation of the plasma membrane Ca 2+-pump in cardiac myocytes.

GTPγS-induced calcium transients and exocytosis in human neutrophils

Exocytosis and intracellular free calcium ([Ca2+]in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5'-O(3-thiotriphosphate) (GTP gamma S) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of approximately 40 s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca2+]in increases from approximately 150 nM to approximately 600 nM with a sigmoidal time course. The peak concentration is reached within approximately 30 s but the main increase occurs during approximately 3 s. [Ca2+]in subsequently decays within 1-2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTP gamma S-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca2+]in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types.

Cytoplasmic calcium measurements in intact higher plant cells: Results from fluorescence ratio imaging of fura-2

ABSTRACT Roots of seedling tomato (Lycopersicon esculenturn) and oilseed rape (Brassica napus) plants were grown on agarose slopes formed on microscope slides. Root hairs were used for microelectrode insertions, for observations of the rate of cytoplasmic streaming and for measurements of fluorescence of the calcium indicator fura-2 The resting potential across the plasma membrane of root hair cells was between −140 and − 160 mV in both species. Insertion of microelectrodes caused immediate cessation of cytoplasmic streaming; 1–4 min later streaming restarted and quickly recovered its normal velocity of 1–3 μm s−1. Once the electrode had been inserted, current pulses had no obvious effect on streaming. Thus, fura-2 injected by iontophoresis was quickly distributed throughout the length of the hair and the epidermal cell body. Injections were made into the cap of cytoplasm at the tip of the hair. Movement of fura-2 from the cytoplasm into the vacuole was observed; this was marked by a great increase in its fluorescence after excitation at 350 nm and occurred after about 15–30 min in tomato, but more rapidly (5–10 min) in oilseed rape hairs. No fluorescence was associated with the cell walls; this was demonstrated by plasmolysing the cells. The distribution and concentration of Ca2+ was estimated from the ratio of fura-2 fluorescence excited at 350 and 385 nm and from digital image analysis. Resting levels of calcium in the cytoplasm near the tip were in the range 30·90 nM, while much higher levels, which eventually saturated the fura-2 response (>5 μM), were seen in vacuoles once the indicator had crossed the tonoplast. This distribution was rapidly perturbed by the addition of the calcium channel blocker, verapamil, to the outer solution. There was a return to the original distribution as the verapamil was diluted. Separate experiments showed that verapamil inhibited cytoplasmic streaming over a time-course similar to these perturbations in cytoplasmic Ca2+ and caused a small depolarization (by approx. 30 mV) of the membrane potential. The application of 3×10−5 or 3 ×10−4 M-N-ethyl maleimide to the hairs strongly and irreversibly depolarized the plasma membrane electric potential, caused cytoplasmic streaming to stop or slow down and resulted in a very rapid rise in cytoplasmic Ca2+, which became uniformly distributed with saturating 350:385 nm ratios.