Simultaneous flow cytometric measurements of thrombin-induced cytosolic pH and Ca2+ fluxes in human platelets

Human platelets exhibit an extremely rapid increase in cytoplasmic Ca2+ concentrations ((Ca2+]in) and a dose-dependent cytoplasmic pH change ((pH]in) upon thrombin stimulation. A cytoplasmic alkalinization, maximal by 60 s, is preceded by a very rapid acidification, which is masked by the alkalinization when saturating thrombin doses are used. Using the pH probe 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein we report here the kinetics of simultaneous cytoplasmic pH and Ca2+ changes in thrombin-stimulated platelets, measured in single cells by flow cytometry. This permits analysis of the responding subpopulation. Maximal thrombin stimulation (greater than or equal to 4.5 nM) induces a dose-dependent increase in pHin from approximately 7.0 to 7.30 and a maximal [Ca2+]in transient of up to 800 nM. The Ca2+ transient coincides temporally with the rapid initial acidification, while the alkalinization is maximal considerably later. The Ca2+ transients occur maximally in each responding cell, but occur only in a subpopulation of the platelets at subsaturating (less than 4.5 nM) thrombin doses; in contrast, the dose-dependent cytoplasmic acidification appears to occur uniformly in all platelets. The rapid increase in [Ca2+]in is not dependent on the alkalinization, and the former occurs maximally in amiloride treated, Na+/H+ exchange inhibited human platelets. These results indicate that the acidification and the rise in [Ca2+]in may be interrelated, whereas the cytoplasmic alkalinization (maximal considerably later than either the acidification or the [Ca2+]in rise) may be independent of these earlier, temporally correlated increases in H+ and Ca2+ concentrations.     

Luminal Ca2+ controls the activation of the inositol 1,4,5-trisphosphate receptor by cytosolic Ca2+.

Luminal Ca2+ controls the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3-induced Ca2+ release is also controlled by cytosolic Ca2+; low concentrations of Ca2+ stimulate the release. The aim of this work was to investigate whether luminal Ca2+ would affect the stimulation of the Ins(1,4,5)P3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. We also report that the Ins(1,4,5)P3 receptor in A7r5 cells is activated by low concentrations of cytosolic Ca2+. Cytoplasmic Ca2+ increases the Ins(1,4,5)P3 sensitivity without affecting the cooperativity. The increase in Ins(1,4,5)P3 sensitivity becomes relatively more pronounced when the Ca2+ content of the stores decreases. This modulatory effect of luminal Ca2+ on the responsiveness to cytosolic Ca2+ is an intrinsic property of the Ins(1,4,5)P3 receptor.     

Determination of cytosolic Mg2+ activity and buffering in BC3H-1 cells with mag-fura-2

The magnesium buffer coefficient (B _Mg) was calculated for BC₃H-1 cells from the rise in cytosolic Mg²⁺ activity observed when magnesium was released from ATP after iodoacetate (IAA) and NaCN treatment. The basal cytosolic Mg²⁺ activity (0.54±0.1 mM) measured with mag-fura-2 doubled when 4.54 mM magnesium was liberated from ATP:B _Mg was 12.9 indicating that a 1 mM increase in Mg²⁺ activity requires an addition of about 13 mM magnesium. The accuracy of this value depends on these assumptions: (a) all of the magnesium released from ATP stayed in the cells; (b) the rise in Mg²⁺ was not secondary to pH-induced changes inB _Mg; (c) mag-fura-2 measured Mg²⁺ and not Ca²⁺; and (d) the accuracy of the mag-fura-2 calibration. Total magnesium did not change in response to IAA/CN treatment, thus the change in Mg²⁺ activity reflected a redistribution of cell magnesium. pH changes induced by NH₄Cl pulse and removal had little effect on Mg²⁺ activity and the changes were slower than and opposite to pH-induced changes in Ca²⁺ activity measured by fura-2. Ca²⁺ responses were temporally uncopled from Mg²⁺ responses when the cells were treated with IAA only and in no cases did Ca²⁺ levels rise above 1 M, showing that the mag-fura-2 is responding to Mg²⁺. Additional studies demonstrated that 90% of the mag-fura-2 signal was cytosolic in origin. The remaining non-diffusible mag-fura-2 either was bound to cytosolic membranes or sequestered in organelles with the fluorescence characteristics of the Mg²⁺-complexed form, even when cytosolic free Mg²⁺ activity was approximately 0.5 mM. This bound mag-fura-2 would appear to increase the K_d and thus clearly limits the accuracy of our estimmate forB _Mg. Despite this limitation, we demonstrate that Mg²⁺ is tightly regulated in face of large changes in extracellular Mg²⁺, and that the interplay observed between pH, Ca²⁺ and Mg²⁺ activities strongly supports the hypothesis that these factors interact through a shared buffer capacity of the cell.     

Erythrocyte cytosolic free Ca2+ and plasma membrane Ca2+-ATPase activity in cystic fibrosis

The properties of the Ca2+, Mg2+-ATPase of erythrocyte membranes from patients with cystic fibrosis (CF) were extensively compared to that of healthy controls. Following removal of an endogenous membrane inhibitor of the ATPase, activation of the enzyme by Ca2+, calmodulin, limited tryptic digestion or oleic acid, as well as inhibition by trifluoperazine, were studied. The only properties found to be significantly different (CF cells vs controls) were calmodulin-stimulated peak activity (90 vs 101, P less than 0.02) and trypsin-activated peak activity (92 vs 102, P less than 0.02). No significant difference could be measured in the steady-state Ca2+-dependent phosphorylation of CF and control erythrocyte membranes indicating similar numbers of enzyme molecules per cell. The functional state of Ca2+ homeostasis in intact erythrocytes was investigated by measuring the resting cytosolic free Ca2+ levels using quin-2. Both CF and control erythrocytes maintained cytosolic free Ca2+ between 20 to 30 nM. Addition of 50 uM trifluoperazine resulted in an increase in erythrocyte cytosolic free Ca2+ to about 50 nM in both CF and control cells. Estimates of erythrocyte membrane permeability using the steady-state uptake of 45Ca into intact erythrocytes revealed no differences between CF and control cells. These results confirm that there is a small decrease in the calmodulin-stimulated activity of the erythrocyte Ca2+, Mg2+-ATPase in CF. However, this deficit is apparently not large enough to impair the ability of the CF erythrocyte to maintain normal resting levels of cytosolic free Ca2+.     

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.     

Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca2+ concentration

Synergistic interaction between ADP, adrenaline, 5-hydroxytryptamine (5HT) and [8-arginine]vasopressin is not observed for the aggregatory response of aspirin-treated human platelets when this response is estimated directly from the decrease in the number of single platelets in the suspension. This finding is in marked contrast with prior reports of synergistic interaction between these agonists when the rate and extent of the aggregometer response is estimated from the increase in the light transmittance of the suspension, using a platelet aggregometer. We propose that the apparent synergistic response detected using the aggregometer results from the inability of this instrument to respond during the initial phase of aggregation. Significant synergistic interaction is observed for the increase in cytosolic [Ca2+] induced by addition of the ADP/5HT and, to a lesser extent, of the ADP/vasopressin agonist pairs as compared with that caused by addition of the individual agonists. This effect is not, however, typical of the system since increases in cytosolic [Ca2+] induced by addition of the ADP/thrombin or 5HT/vasopressin agonist pairs are no greater than the sum of the responses to these agonists added separately. Addition of collagen prior to ADP or 11,9-epoxymethanoprostaglandin H2 (U46619) fails to enhance the increase in cytosolic [Ca2+] induced by these latter agonists. Adrenaline, when added prior to non-saturating concentrations of U46619, thrombin, vasopressin or ADP, significantly enhances the increase in cytosolic [Ca2+] induced by these agonists in platelets suspended in media containing less than 0.1 microM or 1 mM Ca2+. However, adrenaline fails to enhance the increase in cytosolic [Ca2+] induced by the divalent cation ionophore, ionomycin. Enhancement by adrenaline of Ca2+ influx induced by U46619, thrombin and ADP has been shown by using Mn2+ as probe. Adrenaline also enhances the extent of [3H]5HT secretion induced by U46619, thrombin and vasopressin but fails to increase that induced by ADP in this aspirin-treated preparation. These results are in part consistent with the postulate that adrenaline, acting via an alpha 2-adrenoceptor, modulates receptor--phospholipase-C coupling. However, such modulation does not appear to involve inhibition of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.

A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.     

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.     

Cyclic AMP inhibition of fMet-Leu-Phe-dependent metabolic responses in human neutrophils is not due to its effects on cytosolic Ca2+.

Cyclic AMP powerfully inhibits the fMet-Leu-Phe-dependent respiratory burst and exocytosis of azurophilic and specific granules without affecting Ca2+ release from intracellular stores. The elevation of [Ca2+]i induced by fMet-Leu-Phe is short-lived in cyclic AMP-treated cells and similar to that of untreated cells stimulated in the absence of external Ca2+. Nevertheless, in these latter cells fMet-Leu-Phe induces metabolic activation. We therefore suggest that the inhibitory action of cyclic AMP on neutrophil responses is not due to its effects on [Ca2+]i homoeostasis.     

Activation and inactivation of the osteoclast Ca2+ receptor by the trivalent cation, La3+.

We report changes in the cytosolic Ca2+ concentration ([Ca2+]i) of single rat osteoclasts in response to Ca2+ receptor activation by micromolar concentrations of the lanthanide metal cation, La3+. The extracellular application of La3+ induced a concentration-dependent elevation of cytosolic [Ca2+]. Prior conditioning of osteoclasts with La3+ resulted in a concentration-dependent reduction of the response to a subsequent application of a maximally effective concentration of Ni2+, a known agonist of the osteoclast Ca2+ receptor. The results establish that the osteoclast Ca2+ receptor is highly sensitive to activation and inactivation by the trivalent cation, La3+.     

Ability of the Ca2+-selective microelectrodes to measure fast and local Ca2+ transients in nerve cells

The ability of the Ca2+-selective microelectrode to measure fast Ca2+ transients intracellularly is reviewed. In vitro, Ca microelectrodes can respond to Ca2+ injections with time to peaks as small as 40 ms. We present methods to improve the dynamic response of Ca microelectrodes and to make Ca-buffered solutions in high ionic strength. Examples of measurements of intracellular free Ca2+ [( Ca2+]i) transients in Aplysia neurons and in Limulus photoreceptors are shown. To show the validity of those measurements, simultaneous recordings of the Arsenazo III (AIII) absorbance and of the Ca-selective electrode potential were made in voltage-clamped neurons of the abdominal ganglion of Aplysia californica. Pressure injection of AIII to a concentration of 300-500 microM induced a rise in resting [Ca2+]i; injection of higher [AIII] led to buffering of [Ca2+]i transients. Both techniques responded to changes in resting [Ca2+]i in the same direction except that AIII showed an increase in absorbance in 0 [Ca2+]o. Voltage-clamp pulses transiently increased both the AIII absorbance and the Ca2+ electrode potential. Reducing or increasing the driving force for Ca2+ entry changed the magnitude of both signals in the right direction. Examples of spatial localization of [Ca2+]i increases and Ca2+ gradients within the cytoplasm were demonstrated using the Ca electrode. The use of optical techniques to measure local [Ca2+]i changes is briefly reviewed.     

Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration: studies on Ca2+ channels in rat parotid cells

1. Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration [( Ca2+]i)were studied using quin2. 2. Nicardipine (NIC), diltiazem (DIL) and verapamil (VER) had no effect on the rise in [Ca2+]i evoked by carbachol. Methoxamine-elevated [Ca2+]i was inhibited by VER but not by NIC and DIL. 3. All Ca2+ antagonists tested produced a decline of [Ca2+]i elevated by isoproterenol to the resting level. 4. The addition of 30 mM K+ gradually elevated [Ca2+]i in normal and Ca2+-free media, but it did not increase 45Ca2+ uptake into cells. BAY K 8644 did not increase [Ca2+]i. 5. We suggest that voltage-sensitive Ca2+ channels are lacking and that at least 2 distinct receptor-operated Ca2+ channels exist in rat parotid cells.     

Regulation of hormone secretion and cytosolic Ca2+ by extracellular Ca2+ in parathyroid cells and C-cells: role of voltage-sensitive Ca2+ channels

The two dihydropyridine enantiomers, (+)202-791 and (-)202-791, that act as voltage-sensitive Ca2+ channel agonist and antagonist, respectively, were examined for effects on cytosolic Ca2+ concentrations ([Ca2+]i) and on hormones secretion in dispersed bovine parathyroid cells and a rat medullary thyroid carcinoma (rMTC) cell line. In both cell types, small increases in the concentration of extracellular Ca2+ evoked transient followed by sustained increases in [Ca2+]i, as measured with fura-2. Increases in [Ca2+]i obtained by raised extracellular Ca2+ were associated with a stimulation of secretion of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in rMTC cells, but an inhibition of secretion of parathyroid hormone (PTH) in parathyroid cells. The Ca2+ channel agonist (+)202-791 stimulated whereas the antagonist (-)202-791 inhibited both transient and sustained increases in [Ca2+]i induced by extracellular Ca2+ in rMTC cells. Secretion of CT and CGRP was correspondingly enhanced and depressed by (+)202-791 and (-)202-791, respectively. In contrast, neither the agonist nor the antagonist affected [Ca2+]i and PTH secretion in parathyroid cells. Depolarizing concentrations of extracellular K+ increased [Ca2+]i and hormone secretion in rMTC cells and both these responses were potentiated or inhibited by the Ca2+ channel agonist or antagonist, respectively. The results suggest a major role of voltage-sensitive Ca2+ influx in the regulation of cytosolic Ca2+ and hormones secretion in rMTC cells. Parathyroid cells, on the other hand, appear to lack voltage-sensitive Ca2+ influx pathways and regulate PTH secretion by some alternative mechanism.     

Ca2+ activation of membrane-bound (Ca2++Mg2+)-dependent ATPase from human erythrocytes prepared in the presence or absence of Ca2+.

The kinetics of Ca2+ activation of membrane-bound (Ca2+ + Mg2+)-dependent ATPase (ATP phosphohydrolase EC 3.6.1.3) from human erythrocytes was studied. The ATPase from membrane prepared in the presence of 0.7-500muM Ca2+ showed positively cooperative behaviour and a Km for Ca2+ of between 1 and 4 muM. If the membranes were prepared in the absence of Ca2+ the Km increased, and an enzyme model with at least four calcium-binding sites accounted for the kinetic change assuming that one calcium-binding site decreased its affinity. Mg2+ or Mg-ATP could not replace Ca2+. Continuous-flow centrifugation involving a shear stress on membranes was necessary to obtain the high affinity ATPase activity. Using ordinary centrifugation the Ca2+-prepared membranes behaved as membranes prepared in the absence of Ca2+. The Ca2+-stimulated ATPase from membranes prepared without Ca2+ showed reduced maximum activity, but dialyzed, membrane-free hemolysates, whether prepared with Ca2+ present or not, recovered the activity when the hemolysate was present during the ATPase assay. It is suggested that the different Ca2+-affinities of the Ca2+-stimulated ATPase correspond to two different states of the calcium-pump.     

Effect of anti-Ig on cytosolic Ca2+ in Daudi lymphoblastoid cells

We examined the response in the free intracellular calcium concentration ([Ca2+]i) of Daudi (human lymphoblastoid) cells to antibodies against human immunoglobulins (anti-Ig), and the relationship of [Ca2+]i to anti-Ig-induced capping. At 80 microM intracellular quin-2 (a fluorescent probe for [Ca2+]i), anti-Ig (10 micrograms/ml) caused a rapid increase in [Ca2+]i from 100 to 600 nM; the signal returned to baseline with approximately 1 min. At 450 microM intracellular quin-2, [Ca2+]i rose to only approximately 250 microM, and the signal declined gradually, returning to base line after greater than 7 min. In subsequent experiments, the lower concentrations of quin-2 were employed. Plots of the amplitude of the [Ca2+]i transients and of the binding of 125I-anti-Ig to Daudi cells versus the concentrations of anti-Ig showed similar saturation kinetics, with half-saturation occurring at 2-3 micrograms/ml. Part of the calcium in the transient is derived from the extracellular medium, and part from the nonmitochondrial intracellular stores. Caffeine (4 mM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (0.5 mM) suppressed the release of calcium from internal stores and the entry of calcium from outside the cells, but permitted capping in more than half of the cells. Phorbol esters (1-2 nM) inhibited both capping and the anti-Ig-induced decrease in [Ca2+]i. None of these agents blocked the binding of anti-Ig to the cells. It appears that receptor capping is not dependent on the anti-Ig-induced transient increase in calcium concentration.     

Switch of voltage-gated K+ channel expression in the plasma membrane of chondrogenic cells affects cytosolic Ca2+-oscillations and cartilage formation

Background

Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca²⁺-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation.

Methodology/Principal Findings

Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated Na_V1.4, K_V1.1, K_V1.3 and K_V4.1 channels, although K_V1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of Na_V1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K⁺ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced K_V currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca²⁺-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive K_V1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive K_V4.1 did not affect cartilage formation.

Conclusions/Significance

These data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca²⁺-oscillations, which are modulated by K_V-driven membrane potential changes. K_V1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use.     

Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1

Cells of Saccharomyces cerevisiae were loaded with indo-1, by incubation in a medium of pH 4.5, which contained penta-potassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-1 was achieved. By permeabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-1 was accumulated in the cytosol. It was also shown that intracellular indo-1 did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca2+ was found to be 346 nM. Vacuolar Ca2+ concentration, calculated from indo-1 fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.     

Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded.     

Histamine evoked sustained elevations of cytosolic Ca2+ in bovine adrenal chromaffin cells independently of Ca2+ entry

Whole-cell patch-clamp experiments and optical measurements with the Ca2+ fluorescent dye fura-2 were performed to examine histamine induced cytosolic Ca2+ changes in bovine adrenal chromaffin cells. The purpose of this study was to find out whether the sustained plateau phase, which followed the rapid transient increase, was due to Ca2+ influx. The extracellular Ca2+ dependence appeared to be minor, because substitution of Ca2+ with EGTA or BAPTA did not cause obvious changes in the biphasic Ca2+ response. Application of histamine in a Mn2+ containing external solution did not quench the fura-2 signal. It was neither possible to detect a histamine induced depolarisation, nor a Ca2+ permeable current. Changing the driving force for Ca2+ during the plateau phase did not result in a correlating fura-2 signal. Metal ions like Cd2+, La3+ and Co2+ which are known to block Ca2+ influx were unable to abolish the typical histamine induced Ca2+ response. These results suggest that primarily intracellular Ca2+ was responsible for generating the characteristic biphasic Ca2+ response due to histamine in bovine adrenal chromaffin cells.