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.     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Is there a Na+/Ca2+ exchanger in macrophages and in lymphocytes?

In two blood cell types, peritoneal murine macrophages and Jurkat cells (a human T cell line), we have examined whether a Na⁺/Ca²⁺ exchange was present and what could be its functional importance. In nonstimulated macrophages, the intracellular Ca²⁺ concentration, [Ca²⁺]_i, was unchanged when Li⁺ was substituted for external Na⁺. However, after stimulation by platelet-activating factor (PAF), the Ca²⁺ response was larger when the extracellular solution contained Li⁺ rather than Na⁺ ions. In stimulated macrophages, the rate of Ca²⁺ extrusion was smaller in a Li⁺-than in a Na⁺-containing medium. The net electrochemical gradient for ionic movements through the Na⁺/Ca²⁺ exchanger, during the course of the response of macrophages to PAF, was determined by combining the measurements of membrane potential (in patch-clamp), of [Ca²⁺]_i (with fura-2), and of the intracellular Na⁺ concentration (with sodium-binding benzofuran isophthalate). These results show that macrophages possess a Na⁺/Ca²⁺ exchange that only functions as a Ca²⁺ extruder, and this only when [Ca²⁺]_i has been increased, for instance following PAF stimulation. In T lymphocytes, before or after stimulation by an anti-CD3 antibody, no Na⁺/Ca²⁺ activity could be detected by measuring either [Ca²⁺]_i, or the rate of Ca²⁺ extrusion. Even if a Na⁺/ Ca²⁺ exchanger was present in these cells, its equilibrium potential would be such that it would not allow Ca²⁺ influx but only Ca²⁺ extrusion.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Intracellular Ca2+ inactivates an outwardly rectifying K+ current in human adenomatous parathyroid cells

We have used whole-cell patch-clamp techniques to study the conductances in the plasma membranes of human parathyroid cells. With a KCl-rich pipette solution containing Ca²⁺ buffered to a concentration of 0.1 mol/l, the zero current potential was –71.1±0.5 mV (n=19) and the whole-cell current/ voltage (I/V) relation had an inwardly rectifying and an outwardly rectifying component. The inwardly rectifying current activated instantaneously on hyperpolarization of the plasma membrane to potentials more negative than –80 mV, and a semi-logarithmic plot of the reversal potential of the inward current (estimated by extrapolation from the range in which it was linear) as a function of extracellular K⁺ concentration ([K⁺]_o) revealed a linear relation with a slope of 64 mV per decade change in [K⁺]_o, which is not significantly different from the Nernstian slope, demonstrating that the current was carried by K⁺ ions. The conductance exhibited a square root dependence on [K⁺]_o as has been observed for inward rectifiers in other tissues. The current was blocked by the presence of Ba²⁺ (1 mmol/l) or Cs⁺ (1.5 mmol/l) in the bath. The outwardly rectifying current was activated by depolarization of the membrane potential to potentials more positive than –20 mV. It was inhibited by replacement of pipette K⁺ with Cs⁺, indicating that it also was a K⁺ current: it was partially (42%) blocked when tetraethylammonium (TEA⁺, 10 mmol/l) was added to the bath. The outwardly rectifying, but not the inwardly rectifying K⁺ current, was regulated by intracellular free Ca²⁺ concentration ([Ca²⁺]_i) such that increasing [Ca²⁺]_i above 10 nmol/l inhibited the outwardly rectifying current, the half-maximum effect being seen at 1 mol/l. Since it is known that increases in [Ca²⁺]_o produce increases in [Ca²⁺]_i, and that they depolarize parathyroid cells by reducing the membrane K⁺ conductance, we suggest that it is the reduction of the outwardly rectifying K⁺ conductance by increases in [Ca²⁺]_i which is responsible for the reduction in K⁺ conductance seen when [Ca²⁺]_o is increased.     

Factors affecting SOCE activation in mammalian skeletal muscle fibers

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca²⁺ entry, SOCE. Sarcoplasmic reticulum (SR) Ca²⁺ stores were depleted by repetitive exposures to high K⁺ or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca²⁺. SR Ca²⁺ store replenishment was substantially reduced using 5 μM cyclopiazonic acid (CPA). Readmission of external Ca²⁺ (5 mM) increased basal [Ca²⁺]_i under two modalities. In mode 1 [Ca²⁺]_i initially increased at a rate of 0.8 ± 0.1 nM/s and later at a rate of 12.3 ± 2.6 nM/s, reaching a final value of 477.8 ± 36.8 nM in 215.7 ± 25.9 s. In mode 2, [Ca²⁺]_i increased at a rate of 0.8 ± 0.1 nM/s to a value of 204.9 ± 20.6 nM in 185.4 ± 21.1 s. FCCP, 2 μM, reduced this Ca²⁺ entry. In nine FCCP-poisoned fibers, the initial rate of Ca²⁺ increase was 0.34 ± 0.1 nM/s (mean ± SEM), reaching a plateau of 149.2 ± 14.1 nM in 217 ± 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca²⁺, we show that during depletion [Ca²⁺]_mito initially increases and later diminishes. Finally, we show that the increase in basal [Ca²⁺]_i, associated with SOCE activation, diminishes upon external Na⁺ withdrawal. Na⁺ entry through the SOCE pathway and activation of the reversal of Na⁺/Ca²⁺ exchanger could explain this SOCE modulation by Na⁺.     

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 Ca²⁺-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, V _bl, 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 mol/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 V _bl 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 V_bl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of V _bl (n=4). Several other putative agonists of pancreatic HCO ₃ ^– secretion were also tested for their effects on [Ca²⁺]_i. Bombesin (10 nmol/l) increased the fura-2 fluorescence ratio by 0.24±0.04 (n=8), neurotensin (10 nmol/l) by 0.25±0.04 (n=6), substance P (0.1 mol/l) by 0.22±0.06 (n=6), and cholecystokinin (10 nmol/l) by 0.14±0.03 (n=7). Taken together, our studies show that Ca²⁺ homeostasis plays a role in pancreatic ducts. The most important finding is that carbachol and ATP markedly increase [Ca²⁺]_i, but their different electrophysiological responses indicate that intracellular signalling pathways may differ.Preliminary reports of the present study have been presented at the 72nd Meeting of the German Physiological Society, March 1993     

Effect of parathyroid hormone on the connecting tubule from the rabbit kidney: biphasic response of transmural voltage

The effect of parathyroid hormone (PTH) on ion transport was examined by observing transmural (V _T) and basolateral membrane voltage (V _B) in the in vitro perfused rabbit connecting tubule. Addition of 10 nmol/l PTH to the bath induced a biphasic response of V _T, with hyperpolarization followed by depolarization. Chlorophenylthioadenosine cyclic 3,5-monophosphate mimicked the effect of PTH, which did not change the V _B in the connecting tubule cell, but mainly caused changes in the apical membrane voltage. The V _T of distal convoluted tubule and the cortical collecting duct were not affected by PTH. Elimination of Na⁺ from the lumen abolished the PTH-induced V _T responses in the connecting tubule. In the presence of 10 mol/l amiloride, PTH caused an initial hyperpolarization but did not induce the late depolarization. The same was seen in the absence of luminal Ca²⁺. Either addition of 0.1 mmol/l ouabain to the bath or elimination of bath Na⁺ completely abolished the PTH-induced V _T changes. The presence of 5 mmol/l Ba²⁺ in the lumen did not affect the response to PTH. These findings indicate that the initial hyperpolarization may be caused by an increase in Na⁺ influx across the luminal membrane through an amiloride-insensitive Na⁺ conductive pathway and that the late depolarization may be caused by the decrease in Na⁺ influx through the amiloride-sensitive Na⁺ conductive pathway. Luminal Ca²⁺ is necessary for the late depolarization caused by PTH. On the basis of these observations, we suggest that PTH initially increase influxes of both Na⁺ and Ca²⁺ across the luminal membrane and that an increase in intracellular Ca²⁺ in turn suppresses Na⁺ entry through the luminal amiloride-sensitive Na⁺ channel.     

Topography of the respiratory and circulatory responses to acetylcholine and nicotine on the ventral surface of the medulla oblongata

The effects of different [Ca²⁺]₀ in the presence of variable [Na⁺]₀ (100 to 37 mM) on the slow action potentials were studied in isolated rat atria in the presence of 25 mM K⁺ plus 10^–6 M isoproterenol. Two modes of electrical stimulation were used, sustained stimulation at 0.5 Hz (steady-state mode), and stimulation by a single stimulus after rest periods of 2 to 5 min (1st response mode). With the first type of stimulation, and for [Ca²⁺]₀ between 0.5 and 10 mM and [Na⁺]₀ between 100 and 65 mM, the slow action potential overshoot increased linearly with the logarithm of the [Ca²⁺]₀ (28.4 mV per 10-fold change in [Ca²⁺]₀). However, elevation of [Ca²⁺]₀ above 10 mM caused depression of the overshoot. This overshoot depression by high [Ca²⁺]₀ was accentuated if [Na⁺]₀ was decreased to 37 mM. With the 1st response mode of stimulation, the overshoot — log [Ca²⁺]₀ relationship was linear within a wider [Ca²⁺]₀ range (0.5 to 25 mM), and was less sensitive to further decreases in [Na⁺]₀. It is suggested that rat atria slow action potentials are generated by selective influx of Ca²⁺ but not Na⁺, and that the depression of amplitudes observed a high [Ca²⁺]₀ and low [Na⁺]₀ is due to a decrease in the Na²⁺ exchange mechanism which results in a higher [Ca²⁺]_i, and not to a decrease in the inward Na⁺ current. Adenosine produced a parallel downward displacement of the overshoot to log [Ca²⁺]₀ relationship. This adenosine effect was concentration dependent, independent of [Ca²⁺]₀ and the frequency of stimulation. In contrast, the effects of 0.4 mM La³⁺ were dependent on the [Ca²⁺]₀ and on the frequency of stimulation. Adenosine also produced a downward shift of the relationship between maximal rate of rise of the slow action potential and membrane resting potential in such a manner that its effects cannot be attributed to changes in inactivation potential of the slow channels. Hence, adenosine and La³⁺ depress the slow Ca²⁺ action potentials by two different mechanisms. Adenosine may act by 1) decreasing the number of functional slow channels, 2) decreasing the conductance of the individual channels, or 3) altering the kinetic properties of these channels. La³⁺ may act by competing with Ca²⁺ for membrane binding sites. These membrane binding sites appear to be characterized by frequency dependence.Supported by Grants NHLBI 10384 and AHA 74-942.Dr. Luiz Belardinelli is the recipient of a fellowship No. 1112. 1273/75 from Conselho Nacional de Desenvolvimento Cientifico e Technologico (CNPq) and Fundacao Universitaria de Cardiologia do Rio Grande do Sul, Brasil.     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca²⁺ activity ([Ca²⁺]_i) changes in HT₂₉ cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca²⁺]_i was measured with the fluorescent Ca²⁺ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1–40Hz). ATP and CCH induced not only a dose-dependent [Ca²⁺]_i peak response, but also changes of the plateau phase. The [Ca²⁺]_i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca²⁺]_i plateau increased at higher CCH concentrations. NT showed (from 10^–10 to 10^–7 mol/l) in most cases only a [Ca²⁺]_i spike lasting 2–3 min. The [Ca²⁺]_i plateau induced by ATP (10^–6 mol/l) and CCH (10^–5 mol/l) was abolished by reducing the Ca²⁺ activity in the bath from 10^–3 to 10^–4 mol/l (n=7). In Ca²⁺-free bathing solution the [Ca²⁺]_i peak value for all three agonists was not altered. Using fura-2 quenching by Mn²⁺ as an indicator of Ca²⁺ influx the [Ca²⁺]_i peak was always reached before Mn²⁺ influx started. Every agonist showed this delayed stimulation of the Ca²⁺ influx with a lag time of 23±1.5 s (n=15) indicating a similar mechanism in each case. Verapamil (10^–6–10^–4 mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca²⁺]_i increase. Short pre-incubation with verapamil augmented the effect on the [Ca²⁺]_i peak, whereas no further influence on the plateau was observed. Ni²⁺ (10^–3 mol/l) reduced the plateau value by 70%.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.     

Effects of substrate-free anoxia and veratridine on intracellular calcium concentration in isolated rat ventricular cardiomyocytes

Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured in freshly isolated rat ventricular cardiomyocytes during substrate-free anoxia. Cardiomyocytes were loaded with fura-2 and incubated in an anoxic chamber in which a pO₂ equal to 0 mmHg was realized by inclusion of Oxyrase. [Ca²⁺]_i was measured in individual cells using digital imaging fluorescence microscopy. During anoxia, the shape of cardiomyocytes changed from a relaxed-elongated form into a rigor configuration within 15 min after the onset of anoxia. After the cells had developed the rigor state, a delayed rise in [Ca²⁺]_i reached a stable maximal level within 45 min. The mean values for the pre-anoxic and maximal anoxic [Ca²⁺ _i were 52±3 nM (N=42) and 2115±59 nM (N=45), respectively. The purported Na⁺ overload blocker R 56865, significantly reduced maximal anoxic [Ca²⁺]_i to 553±56 nM (P<0.05), implicating a role of elevated intracellular Na⁺ in the anoxia-induced increase in [Ca²⁺]_i. Veratridine (30 M), which induces Na⁺ overload, increased [Ca²⁺]_i to 787±39 nM. The compound R 56865 reduced veratridine-induced increases in [Ca²⁺]_i to 152±38 nM. Upon reperfusion, after 45 min of anoxia, two distinct responses were observed. Most often, [Ca²⁺]_i decreased upon reperfusion without a change in morphology or viability, while in the minority of cases, [Ca²⁺]_i increased further followed by hypercontraction and loss of cell viability. The mean value for [Ca²⁺]_i 10 min after reperfusion of the former group, was 752±46 nM (N=38). The cardiomyocyte cell shape could be followed by monitoring changes in the total fura-2 fluorescence (340+380 nm signal). Within 15 min after the onset of anoxia, the total fluorescence signal increased suddenly, before [Ca²⁺]_i started to rise, coinciding with the onset of rigor contraction induced by ATP depletion.     

A delayed all-or-none hyperpolarisation induced by a single Ca action potential in hamster eggs

A transient change of membrane potential and resistance could be evoked after a long latency (ca. 9 s) by a single calcium action potential in some unfertilised hamster eggs. The estimated reversal potential for the delayed response was close to E_K supporting the conclusion that K channels were opened indirectly by the Ca²⁺ influx through voltage-gated channels. A second action potential elicited after the first did not induce a similar response. A number of treatments (insertion of a Ca² pipette, application of Na⁺-free solution, La³⁺ or high external pH) likely to raise [Ca²⁺]_i also induced similar large changes of potential and resistance after which a single action potential failed to evoke a large delayed response. The evidence indicates that a small rise in [Ca²⁺]_i activates a slow process leading to a further large increase in [Ca²⁺]_i.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.     

[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.     

Sodium-dependent recovery of ionised magnesium concentration following magnesium load in rat heart myocytes

Our objectives were to investigate regulation of intracellular ionised Mg²⁺ concentration ([fMg²⁺]_i) in cardiac muscle and cardiac Na⁺/Mg²⁺ antiport stoichiometry. [fMg²⁺]_i was measured at 37°C in isolated rat ventricular myocytes with mag-fura-2. Superfusion of myocytes with Na⁺ and Ca²⁺ free solutions containing 30 mM Mg²⁺ for 15 min more than doubled [fMg²⁺]_i from its basal level (0.75 mM). Re-addition of Na⁺ caused [fMg²⁺]_i to fall exponentially with time to basal level, the rate increasing linearly with [Na⁺]. Log(recovery rate) increased linearly with log([Na⁺]), the slope of 1.06 (95% confidence limits, 0.94–1.17) suggesting one Na⁺ ion is exchanged for each Mg²⁺. [fMg²⁺]_i recovery was complete even if the membrane potential was depolarised to 0 mV or if superfusate [Mg²⁺] was increased to 3 mM. Recovery was rapid in normal Tyrode (0.3 min^–1) with a Q₁₀ of 2.2. It was completely inhibited by 200 M imipramine but was unaffected by 20 M KB-R7943 or 1 M SEA0400, suggesting the Na⁺ /Ca²⁺ antiporter is not involved. Membrane depolarisation by increasing superfusate [K⁺] to 70 mM, or voltage clamp to 0 mV, increased recovery rate in Na⁺ containing solutions more than threefold. We conclude [fMg²⁺]_i recovery is by Mg²⁺ efflux on a 1 Na⁺:1 Mg²⁺ antiport.     

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca²⁺ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10^–5 mol/l)-induced intracellular Ca²⁺ transient, (2) a reduced fura-2 Mn²⁺-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca²⁺ as a function of membrane voltage, we measured simultaneously the intracellular Ca²⁺ activity ([Ca²⁺]_i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT₂₉ cells. Ca²⁺ influx was either stimulated by ATP (10^–5 mol/l) or thapsigargin (TG, 10^–8 mol/l). After [Ca²⁺]_i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca²⁺]_i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca²⁺]_i (ATP: 27.5±10 nmol/l per 10 mV, n=6; TG: 19 ±7.9 nmol/l per 10 mV, n=12). The summarised data show a linear relationship between the fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca²⁺]_i (n=9). Under extracellular Ca²⁺-free conditions [Ca²⁺]_i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca²⁺ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca²⁺]_i. This may be due either to a change of driving force for Ca²⁺ into the cell, or may reflect voltage-dependent regulation of the respective Ca²⁺ entry mechanism.     

Antidiuretic hormone acts via V1 receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb

The effect of antidiuretic hormone ([Arg]vasopressin, ADH) on intracellular calcium activity [Ca²⁺]_i of isolated perfused rabbit cortical thick ascending limb (cTAL) segments was investigated with the calcium fluorescent dye fura-2. The fluorescence emission ratio at 500–530 nm (R) was monitored as a measure of [Ca²⁺]_i after excitation at 335 nm and 380 nm. In addition the transepithelial potential difference (PD _te) and transepithelial resistance (R _te) of the tubule were measured simultaneously. After addition of ADH (1–4 nmol/l) to the basolateral side of the cTAL R increased rapidly, but transiently, from 0.84±0.05 to 1.36±0.08 (n = 46). Subsequently, within 7–12 min R fell to control values even in the continued presence of ADH. The increase in R evoked by the ADH application corresponded to a rise of [Ca²⁺]_i from a basal level of 155±23 nmol/l [Ca²⁺]_i up to 429±53 nmol/l [Ca²⁺]_i at the peak of the transient, as estimated by intra- or extracellular calibration procedures. The electrical parameters (PD _te and R _te) of the tubules were not changed by ADH. The ADH-induced Ca²⁺ transient was dependent on the presence of Ca²⁺ on the basolateral side, whereas luminal Ca²⁺ had no effect. d(CH₂)₅[Tyr(Me)²]²,Arg⁸vasopressin, a V₁ antagonist (Manning compound, 10 nmol/l), blocked the ADH effect on [Ca²⁺]_i completely (n = 5). The V₂ agonist 1-desamino-[d-Arg⁸]vasopressin (10 nmol/l, n=4), and the cAMP analogues, dibutyryl-cAMP (400 mol/l, n = 4), 8-(4-chlorophenylthio)-cAMP (100 mol/l, n = 1) or 8-bromo-cAMP (200 mol/1, n = 4) had no influence on [Ca²⁺]_i. The ADH-induced [Ca²⁺]_i increase was not sensitive to the calcium-channel blockers nifedipine and verapamil (100 mol/l, n = 4). We conclude that ADH acts via V₁ receptors to increase cytosolic calcium activity transiently in rabbit cortical thick ascending limb segments, possibly by an initial Ca²⁺ release from intracellular stores and by further Ca²⁺ influx through Ca²⁺ channels in the basolateral membrane. These channels are insensitive to L-type Ca²⁺ channel blockers, e.g. nifedipine and verapamil.Supported by DFG GR 480/10