Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Effect of mitochondria poisoning by FCCP on Ca2+ signaling in mouse skeletal muscle fibers

We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca²⁺ signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca²⁺]_i and MagFluo-4AM to measure Ca²⁺ transients. Exposure to FCCP (2 μM, 2 min) caused a continuous increase in [Ca²⁺]_i at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca²⁺ transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca²⁺]_i = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca²⁺ release. FCCP diminished the rate but not the amount of Ca²⁺ released, indicating that depletion of Ca²⁺ stores did not cause the decrease in Ca²⁺ transient amplitude. Ca²⁺ transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca²⁺]_i induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca²⁺ in the FCCP effect on transient amplitude.     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca²⁺ ([Ca²⁺]_i). The increases of [Ca²⁺]_i evoked by TG was associated with significant changes of plasma membrane Ca²⁺ permeability, with [Ca²⁺]_i values following changes in extracellular [Ca²⁺]. Plasma membrane Ca²⁺ extrusion is activated rapidly as a consequence of the rise in [Ca²⁺]_i evoked by TG and the rate of extrusion is linearly dependent on [Ca²⁺]_i up to 1 μM Ca²⁺. In contrast, the activation of the Ca²⁺ entry pathway is delayed and the apparent rate of Ca²⁺ entry is independent of [Ca²⁺]_i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP₃), the increase of [Ca²⁺]_i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca²⁺]_i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca²⁺]_i increase (from 12.3±1.0 to 6.1±0.6 nM Ca²⁺/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca²⁺]_i. The amplitude of the [Ca²⁺]_i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La³⁺, which is known to inhibit the plasma membrane Ca²⁺-activated adenosine triphosphatase, induced a much higher peak of [Ca²⁺]_i. This increase was associated with an augmentation of the apparent rate of [Ca²⁺]_i increase (from 12.3±1.2 to 16.1±1.9 nM Ca²⁺/s). The inhibitory effect of caffeine, as well as the increase in [Ca²⁺]_i observed on caffeine removal was not affected by the presence of 1 mM La³⁺. These data indicate that an important component of the TG-evoked [Ca²⁺]_i increase is due to InsP₃-sensitive Ca²⁺ release which is probably mediated by the resting levels of InsP₃.     

Fura-2 imaging of spontaneous and electrically induced oscillations of intracellular free Ca2+ in rat myotubes

Rat myotubes have a resting [Ca²⁺]_i of about 82 nM. Myotubes 3–5 days old (quiescent myotubes) display electrically induced and spontaneous transients in the intracellular concentration of free Ca²⁺ ions ([Ca²⁺]_i) uncoupled to any detectable contraction. By contrast, 1-to 2-day-old myotubes are insensitive to electrical stimuli and, after 6 days in culture, stimulated myotubes always show [Ca²⁺]_i transients and twitch contractions. The spatial distribution of [Ca²⁺]_i variations in quiescent myotubes is heterogeneous, local increases in [Ca²⁺]_i being mainly observed near the periphery of the cell. The small effect of different external Ca²⁺ concentrations and of Cd²⁺ on the amplitude of the [Ca²⁺]_i oscillation indicates that the main source of Ca²⁺ may be the sarcoplasmic reticulum. This conclusion is supported by the close similarity between electrically induced and caffeine-induced [Ca²⁺]_i maps. These findings suggest that, at an early stage of myotube ontogenesis, a part of the excitation/contraction coupling, as membrane ionic channels, voltage sensors and Ca²⁺ release and reuptake mechanisms, is functional but, apparently, still uncoupled to the contractile machinery.This work was supported by the Centre National de la Recherche Scientifique, the Ministère de la Recherche et de la Technologie, the Conseil Régional de l'Aquitaine and the Association Française contre les Myopathies     

AlF 4 − induces Ca2+ oscillations in guinea-pig ileal smooth muscle

The effects of different compounds that inhibit the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase on the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F^–, in the presence of Al³⁺, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca²⁺]_i while the phasic contractions were accompanied by concomitant oscillations in [Ca²⁺]_i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca²⁺]_i and of force elicited by AlF ₄ ^– were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca²⁺ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF ₄ ^– , vanadate and calmidazolium induce cytoplasmic Ca²⁺ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase. The generation of membrane-linked Ca²⁺ oscillations could therefore be related to an inhibition of the plasma-membrane Ca²⁺ pump resulting in an increase of [Ca²⁺]_i. This change in [Ca²⁺]_i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca²⁺]_i to a stable plateau of 10–20 nmol/l above resting [Ca²⁺]_i was observed. Lower osmolalities resulted in a triphasic increase of [Ca²⁺]_i to a peak value. In a first phase after the volume change, lasting 20–40 s, [Ca²⁺]_i increased slowly by about 30 nmol/l. Thereafter [Ca²⁺]_i increased more rapidly within 20–30 s to a peak value. This peak was 189±45 nmol/l (190 mosmol/l, n=9) and 243±41 nmol/l (160 mosmol/l, n=20) above resting [Ca²⁺]_i. The peak was then followed by a decline of [Ca²⁺]_i over the next 2–3 min to a stable plateau value of 28±6 (n=6) and 32±11 nmol/l (n=11) above resting [Ca²⁺]_i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca²⁺-free bath conditions the peak value for the cell-swelling-induced [Ca²⁺]_i transient was reached significantly later (60–100 s, compared to 40–60 s under control conditions). The peak values under Ca²⁺-free conditions were not significantly lower. This indicates that the [Ca²⁺]_i peak was mostly of intracellular origin. No [Ca²⁺]_i plateau phase was observed under Ca²⁺-free bath conditions. With the use of the fura-2-Mn ²⁺ quenching technique an increased Ca²⁺ influx induced by hypotonic cell swelling was shown (160 mosmol/l; n=4). This influx started immediately after or simultaneously with the cell swelling and preceded the [Ca²⁺]_i peak for more than 50 s.This study was supported by DFG grant Gr 480/10.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Endothelin-B receptor-mediated Ca2+ signaling in human melanocytes

 The mechanism of an endothelin-1- (ET-1-) induced intracellular Ca²⁺ ([Ca²⁺]_i) increase and the receptor subtype(s) responsible for this effect in single human melanocytes were studied using fura-2/AM. ET-1 induced a transient increase in [Ca²⁺]_i in a concentration-dependent manner. The transient [Ca²⁺]_i increase was followed by a sustained plateau level of [Ca²⁺]_i which was higher than the initial [Ca²⁺]_i level. IRL-1620, a specific ET-B receptor agonist, increased [Ca²⁺]_i in a dose-dependent manner. BQ-788, a specific ET-B receptor antagonist, abolished the ET-1-induced [Ca²⁺]_i increase, but BQ-123, a specific ET-A receptor antagonist, failed to prevent it. U73122, an inhibitor of phospholipase C (PLC), inhibited the ET-1-induced [Ca²⁺]_i rise in a dose-dependent manner. Prior depletion of intracellular Ca²⁺ stores with thapsigargin, an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum, abolished the ET-1-induced Ca²⁺ transient, whereas removal of extracellular Ca²⁺ with EGTA eliminated the sustained rise. These results suggest that in cultured human melanocytes the binding of ET-1 to ET-B receptors and the subsequent activation of PLC mediate ET-1-induced [Ca²⁺]_i increase. The transient [Ca²⁺]_i increase is attributed to mobilization of Ca²⁺ from inositol 1,4,5-trisphosphate-sensitive intracellular Ca²⁺ stores, and the sustained [Ca²⁺]_i level may be related to the influx of extracellular Ca²⁺. Received: 21 July 1997 / Received after revision and accepted: 16 September 1997     

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca²⁺]_i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca²⁺]_i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca²⁺]_i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca²⁺, whilst at higher concentrations the responses consisted of a rapid rise in [Ca²⁺]_i followed by a smaller more sustained increase. Without external Ca²⁺, 100 M acetylcholine caused only a transient rise in [Ca²⁺]_i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca²⁺]_i oscillations. Acetylcholine-evoked Ca²⁺ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca²⁺]_i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca²⁺]_i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca²⁺ levels, but the Ca²⁺-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca²⁺]_i levels. These data demonstrate for the first time cytosolic Ca²⁺ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca²⁺ influx and Ca²⁺ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.     

Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse

We compared the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), as measured with the fluorescent Ca²⁺ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K²⁺ depolarization and by agonists. Resting [Ca²⁺]_i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca²⁺ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca²⁺]_i increase, the agonist stimulation induced a higher force development than the K⁺ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca²⁺]_o. Our results indicate that the Ca²⁺ threshold for force development was about 150% of the resting [Ca²⁺]_i in both cell types. The resting [Ca²⁺]_i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca²⁺ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca²⁺ sensitivity during contraction. The desensitization to Ca²⁺ was specific for phasic muscle, in which it occurred as an early, timeand Ca²⁺-dependent process that was partially reversible.     

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation

The relationship between the initial increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca²⁺]_i values (400–900 nM) which appears optimal for T cell proliferation; lower [Ca²⁺]_i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca²⁺]_i requirements were similar following anti-CD3 stimulation. [Ca²⁺]_i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400–900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca²⁺]_i responses still cause the cells to proliferate. The possibility that the lower [Ca²⁺]_i requirements observed following anti-CD3 stimulation was due to [Ca²⁺]_i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca²⁺signal is not more mitogenic than a sustained augmentation of [Ca²⁺]_i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca²⁺]_i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca²⁺]_i augmentations are not cytotoxic. A role for the plasmalemmal Ca²⁺ pump in the prevention of cytotoxicity can be demonstrated. In conclusion, the correspondence between the [Ca²⁺]_i response and cell proliferation is entirely different following stimulation by ionomycin and by anti-CD3. In addition, cell proliferation evoked by very low UCHT1 concentration might reveal the existence of a yet unidentified activation pathway.     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

Intracellular Ca2+ concentrations are not elevated in resting cultured muscle from Duchenne (DMD) patients and in MDX mouse muscle fibres

The free intracellular calcium concentration, [Ca²⁺]_i, was studied in single myotubes using the fluorescent Ca²⁺ indicator fura-2. Myotubes cultured from satellite cells of small muscle specimens from Duchenne muscular dystrophy (DMD) patients were compared with human control myotubes and with myotubes cultured from MDX and control mouse muscle satellite cells. The resting [Ca²⁺]_i levels in DMD and control myotubes were not significantly different, i. e. 104 ±26 nM (mean ± SD, n=190 cells from eight DMD patients) compared with 97±25 nM (175/seven controls) and were not significantly lower than the corresponding murine values (154±33 nM, n=135 MDX myotubes; 159±34 nM, n=135 controls). All myotubes reacted to 10 M acetylcholine or 40 mM KCl with fast transient increases of [Ca²⁺]_i. After application of a hyposmotic (130 mOsm) solution, [Ca²⁺]_i was increased 1.5- to 3-fold within 2–3 min, the DMD myotubes tending to stronger reactions (significantly higher [Ca²⁺]_i in 2 out of 6 cases). The response was usually transient, [Ca²⁺]_i decreasing to the initial level within 10 min. Gadolinium (50 M) reduced the response by 50%–70%, indicating that the osmotic shock increased Ca²⁺ influx. During exposure to high (15 mM) [Ca²⁺]e, [Ca²⁺]_i of DMD and control cells was 1.5- to 2-fold higher. Adult muscle fibres from MDX mice and controls showed identical Ca²⁺ resting levels (n=45 fibres from three mice in each case), but did not respond to decreased external osmolarity with a change in [Ca²⁺]_i. The results indicate that lack of dystrophin in muscle fibres does not necessarily lead to increased [Ca²⁺]_i. It is suggested that increased [Ca²⁺]_i is probably a secondary consequence of fibre damage.     

Activity-dependent intracellular Ca2+ transients in unmyelinated nerve fibres of the isolated adult rat vagus nerve

 Confocal laser scanning microscopy was used to follow changes in the free intracellular calcium concentration ([Ca²⁺]_i) in nerve fibres and adjacent Schwann cells in isolated rat vagus nerves. [Ca²⁺]_i was monitored by the Ca²⁺-sensitive fluorescent dyes Calcium Green-1 and Fura Red. Intracellular Ca²⁺ transients were observed during repetitive (1–50 Hz) supramaximal electrical stimulation or by bath application of ATP. Trains of action potentials were more effective at elongated, fibre-like structures of the vagus nerves, whereas ATP-induced Ca²⁺ transients were found predominantly in regions of Schwann cell bodies. Activity-induced Ca²⁺ signals were unaffected by pharmacological manipulation of intracellular Ca²⁺ stores, during long-lasting application of purinergic receptor agonists, or by substitution of extracellular Na⁺ with Li⁺. However, they were abolished in the presence of Ca²⁺-free bathing solution or after the blocking of Ca²⁺ channels with Cd²⁺. Ca²⁺ transients were also observed during Ca²⁺ action potentials. Such ”Ca²⁺ spikes” were elicited by electrical stimulation in the presence of a combination of tetrodotoxin and K⁺ channel blockers. These data suggest that voltage-dependent Ca²⁺ channels, activated during short trains of Na⁺ action potentials, produce an increase in intra-axonal [Ca²⁺] of rat vagus nerves. We did not find evidence for activity-dependent Ca²⁺ transients in the Schwann cells surrounding the unmyelinated axons. Received: 16 September 1997 / Received after revision: 25 November 1997 / Accepted: 27 November 1997