Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca2+ concentration of single blood platelets.

Cytoplasmic free Ca2+ concentration, [Ca2+]i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca(2+)-indicator dye Fura-2. Uneven distribution and low level of [Ca2+]i was found in the resting platelet even in the presence of extracellular 1 mM Ca2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca2+]i, which was followed by a secondary and sustained increase in [Ca2+]i. The distribution of increased levels of [Ca2+]i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca2+]i, but completely inhibited the latter phase, a sustained rise in [Ca2+]i. This result shows that the initial rise of [Ca2+]i might not be caused by Ca2+ influx, but might be induced by mobilization of Ca2+ from intracellular Ca2+ storage sites. This speculation is further supported by the fact that the elevated [Ca2+]i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca2+]i is suggested to consist of two different processes, namely the mobilization of Ca2+ from the intracellular storage sites and the successive Ca2+ influx through the receptor activated Ca2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca2+]i, but this effect of ADP was different form that of thrombin. Thus, the ADP induced rise in [Ca2+]i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca2+]i of platelets.     

Fluorescence digital image analysis of the inositol trisphosphate-mediated calcium transient in single permeabilized parietal cells

The myo-inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization in single saponin-permeabilized and fura-2-loaded parietal cells was analysed by a fluorescence digital image processor. When the cells were incubated with ATP, free cytoplasmic Ca2+ concentration [( Ca2+]i) increased in some restricted cytoplasmic regions showing discontinuous plateau and in the peripheral cytoplasm showing continuous [Ca2+]i gradient towards the plasma membranes. When treated with IP3, the high plateau enlarged to the entire cytoplasm and (a) new higher plateau(s) appeared and enlarged again in a transient manner. The IP3-induced Ca2+ transient was also observed by fluorescence microphotometry of the single cells or by fluorescence spectrophotometry and 45Ca2+ uptake experiment of the cell population.     

Measurement of ionized calcium in normal human blood platelets

To measure the concentrations of cytosolic ionized calcium in platelets we used a calcium-sensitive fluorophor (Fura-2) with two different spectrofluorometers (Perkin-Elmer LS-5 and Fluorolog 222). Values obtained by these two instruments for the basal cytosolic ionized calcium concentration of resting platelets and those of agonist-activated platelets did not differ significantly. Both instruments were capable of monitoring the shifts in wavelengths induced by the dye-calcium complex, the ratio between absorbances at the two wavelengths (340/380 nm), and calcium concentrations continuously during agonist-induced platelet activation. We conclude that a relatively inexpensive instrument may be adequate for measuring ionized calcium in cells by this method, although sophisticated kinetic studies may require analytical or research-grade instruments.     

Agonist-induced oscillations in cytoplasmic free calcium concentration in single rat hepatocytes

The effects of the alpha 1-adrenergic agonist phenylephrine and the peptide hormones angiotensin II and arg8-vasopressin on cytoplasmic free calcium concentration were investigated in single rat hepatocytes microinjected with the photoprotein aequorin. Hepatocytes responded to physiological concentrations of the glycogenolytic agonists with a series of repetitive Ca transients. In each transient free Ca rose in 2-3s to above 600 nM from a resting level of 200 nM. Transient duration depended on the agonist and ranged from approximately 7s for phenylephrine to approximately 15s for angiotensin. Transient frequency, but not shape or size, depended on agonist concentration. The period ranged from less than 20s to several minutes. We suggest that the frequency of the Ca transients is the principal determinant of the amplitude of the cellular response to calcium-mobilizing agonists.     

Measurement of ionized calcium in blood platelets with a new generation calcium indicator

Fura 2, a new generation calcium indicator, has a 30 fold brighter fluorescence than Quin 2, shows wavelength shifts upon calcium binding and has a relatively low buffering capacity for free calcium. Quin 2, the most widely used fluorophore, on the other hand, shows no wavelength shifts and has a very high affinity for free calcium. Therefore, we have compared the relative merits of these two fluorophores for monitoring agonist induced alterations in platelet cytosolic calcium. Platelets loaded with Fura 2 showed a significant rise in cytosolic calcium when stirred with agonists such as epinephrine, arachidonate and thrombin, whereas Quin 2 loaded platelets demonstrated a rise in cytosolic calcium only with thrombin stimulation. A rise in agonist induced calcium in Fura 2 loaded platelets was prevented when the cells were exposed first to antagonists such as aspirin or prostaglandin E1. Arachidonate refractory platelets, upon stirring with a single agonist, did not show a significant elevation in cytosolic calcium. However, when refractory platelets were first exposed to epinephrine and then challenged with arachidonate, they revealed a significant elevation in cytosolic calcium. Unlike Quin 2, Fura 2 at the highest concentration tested did not inhibit platelet function. Improved properties of Fura 2 suggest that it may be a useful agent to study agonist induced alterations in cytosolic calcium levels in blood platelets.     

Agonist-induced calcium response in single human platelets assayed in a microfluidic device

To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.     

Agonist-induced cytosolic calcium oscillations originate from a specific locus in single hepatocytes

Digital imaging fluorescence microscopy of fura-2-loaded hepatocytes in primary culture has been used to examine the changes of cytosolic free Ca2+ ([Ca2+]i) in response to receptor activation by alpha 1-adrenergic agonists and vasopressin at the subcellular level. Agonist-induced Ca2+ oscillations did not occur synchronously within the cell but originated from a specific region adjacent to the cell membrane and then propagated throughout the rest of the cell, with each oscillation within a series originating from the same locus. Furthermore, hormones acting through different receptors produced Ca2+ waves with similar rates of progress (20-25 microns.s-1) which originated from the same subcellular locus. For a given cell, the rate of progress and amplitude of the Ca2+ waves were independent of applied agonist concentration and were unaffected by depletion of extracellular Ca2+. The kinetics of Ca2+ increase at different points within the cell indicated that the Ca2+ waves were not driven by diffusion but were characteristic of a self-propagating mechanism. Significantly, when cells were treated with A1F-4 to directly activate the G-protein which couples receptor occupancy to [Ca2+]i mobilization, the origin and kinetics of the Ca2+ waves were identical to those observed with hormonal stimulation. It is proposed that the spatial organization of the intracellular Ca2+ release mechanisms may have significance in the regulation of the asymmetric metabolic functions of hepatocytes and other functionally polarized cells.     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.     

Effect of various factors on serotonin-induced Ca2+ response in human platelets

Serotonin (5-HT)-stimulated intracellular Ca2+ concentration change was studied in the platelets of healthy subjects, using fluorescent Ca indicator fura-2. 5-HT increased the Ca2+ response in a concentration-dependent manner. 10 microM of 5-HT induced the maximal response and its EC50 value was 0.4 microM. This response was potently inhibited by selective 5-HT2 antagonists, suggesting that 5-HT-induced Ca2+ mobilization in human platelets is mediated by 5-HT2 receptors. This 5-HT2-mediated Ca2+ 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 Ca2+ response in human platelets is a stable parameter and that it will be suitable for assessing 5-HT2 receptor function in depressed patients.     

Fluorescence and single molecule analysis in cell biology

An overview is presented which describes the development of fluorescence spectroscopy at the cellular level from its beginning as a quantitative tool to determine the content of cellular components to its present use. Analysis of individual biomolecules, their transport and kinetics within a single cell is now possible.     

Functional distinction between serotonin uptake and serotonin-induced shape change receptors in rat platelets

Tyramine and dopamine are taken up by rat platelets through the serotonin uptake mechanism while phenethylamine is not taken up. This indicates that an aromatic hydroxyl group is a structural requirement for the uptake of phenethylamine derivatives by rat platelets. Although none of these phenethylamine derivatives induce platelet shape change, they inhibit serotonin-induced shape change and serotonin uptake with the same relative potency ($\text{tyramine}\text{>}\text{phenethylamine}\text{?}\text{dopamine}$). This suggests that the receptors controlling serotonin uptake and serotonin-induced shape change have a common structural component that binds phenethylamine derivatives. However, the fact that phenethylamine derivatives activate the serotonin uptake mechanism but do not induce platelet shape change suggests that serotonin uptake and serotonin-induced shape change are mediated by two distinct activation sites of serotonin receptors.     

Effects of nitroglycerin on intracellular concentration of calcium in human blood platelets]

It is shown that nitroglycerin has a calcium-blocking property in human platelets both at basal and ADP-induced calcium level, determined by fluorescence techniques (Fura-2). The study demonstrated a dose-dependent inhibitory effect of nitroglycerin on calcium rise, induced by ADP. 30-min preincubation with 60 microM nitroglycerin increased IC50 from 44 (without preincubation) to 320 microM, indicating the development of nitrate tolerance. It was suggested, that human platelet may be used as an experimental model of nitrate tolerance.     

Short-term alteration of MAO activity after translocation of calcium in human blood platelets

Incubation of washed human platelets with 5 μM of Ionophore A 23187 leads within 10 min. up to a 100% activation, whereas 50 μM of A 23187 leads to about a 50% inhibition of MAO activity using p-tyramine, tryptamine and benzylamine as substrates. Increase of MAO activity is followed by the calcium dependent activation of cellular phospholipases, the release of arachidonic acid and their subsequent peroxidation into lipoperoxides, prostaglandins and thromboxanes. Inhibition of phospholipase A₂ or lipoxidase and cyclo-oxygenase prevented activation but not inhibition induced by 50 μM of A 23187. The findings indicate that interactions of Ca²⁺ regulated cellular events can lead to short-term activation or inhibition of mitochondrial MAO activity.     

Spectrofluorimetric and image recordings of spontaneous and lectin-induced cytosolic calcium oscillations in Jurkat T cells

Intracellular variations in Ca2+ concentrations have been measured in single Jurkat T lymphocyte variants (77 6.8 and E6.1) using Fura-2 as a probe. Under basal conditions, the cytosolic Ca2+ level is stable but some cells show spontaneous Ca2+ oscillations (frequency, 0.30 +/- 0.06 Hz). These oscillations are sensitive to the external concentration of Ca2+ since they can no longer be observed when the bathing solution is replaced (superfusion) with a Ca(2+)-free medium or when a Ca2+ chelator (EGTA) is added. Various changes in the cytosolic concentration of Ca2+ ([Ca2+]i) can be observed when the cells are exposed to the mitogenic lectin phytohemagglutinin (PHA, 80 nM). For instance, in the case of non-oscillating cells, the lectin induces either a rapid increase in [Ca2+]i that is followed by a sustained response (plateau) or it triggers Ca2+ spikes. In the case of experiments done in Ca(2+)-free medium, only the initial spike was observed. In the case of spontaneously oscillating cells, PHA induces a rapid increase in [Ca2+]i that is followed by a plateau where oscillations are absent. In every case, the PHA-dependent Ca2+ response is abrogated in a Ca(2+)-free medium. Computer simulations based on the model of Goldbeter et al. [27] show that the various Ca2+ responses of Jurkat cells are related to the cytosolic level of free Ca2+. Video imaging analyses show that the cellular Ca2+ responses are not homogeneous whether the observations are made in spontaneously oscillating Jurkat cells or when they are exposed to PHA.     

Noise analysis of cytosolic calcium image data

Cellular Ca²⁺ signals are often constrained to cytosolic micro- or nano-domains where stochastic openings of Ca²⁺ channels cause large fluctuations in local Ca²⁺ concentration (Ca²⁺ ‘noise’). With the advent of TIRF microscopy to image the fluorescence of Ca²⁺-sensitive probes from attoliter volumes it has become possible to directly monitor these signals, which closely track the gating of plasmalemmal and ER Ca²⁺-permeable channels. Nevertheless, it is likely that many physiologically important Ca²⁺ signals are too small to resolve as discrete events in fluorescence recordings. By analogy with noise analysis of electrophysiological data, we explore here the use of statistical approaches to detect and analyze such Ca²⁺ noise in images obtained using Ca²⁺-sensitive indicator dyes. We describe two techniques - power spectrum analysis and spatio-temporal correlation - and demonstrate that both effectively identify discrete, spatially localized calcium release events (Ca²⁺ puffs). Moreover, we show they are able to detect localized noise fluctuations in a case where discrete events cannot directly be resolved.     

A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis

Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.     

Substrate effects on oscillations in metabolism, calcium and secretion in single mouse islets of Langerhans

Glucose induces complex patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i), metabolism and secretion in islets of Langerhans including "slow" and "fast" pulses with period of 2-5 min and 10-20 s respectively. In an effort to elucidate the origin of slow oscillations, individual mouse islets were exposed to different fuels including glyceraldehyde, pyruvate, methyl pyruvate and alpha-ketoisocaproate (KIC), all of which bypass key steps of glycolytic metabolism, while monitoring [Ca2+]i, oxygen consumption and secretion. Glyceraldehyde gave rise to slow oscillations only when substimulatory glucose was also added to the media. Glucosamine, an inhibitor of glucokinase, blocked these slow oscillations. KIC, pyruvate, and methyl pyruvate did not give rise to slow oscillations alone or with glucose present. The addition of glucose to islets bathed in nutrient-rich cell culture media accelerated metabolism and initiated slow oscillations while glyceraldehyde did not. It is concluded that glucose has a special role in accelerating metabolism and generating slow oscillations in isolated islets of Langerhans from mice. Combined with previous observations of Ca2+ dependency for all oscillations in islets, we propose that interactions between Ca2+ influx and glycolysis are responsible for the slow oscillations. In contrast, fast oscillations can occur independent of glycolytic flux.