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PubMed1999

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.

Animals↗
PubMed2004

Subpopulation of store-operated Ca2+ channels regulate Ca2+-induced Ca2+ release in non-excitable cells.

Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.

Animals↗
PubMed1992

A caffeine- and ryanodine-sensitive Ca2+ store in bullfrog sympathetic neurones modulates effects of Ca2+ entry on [Ca2+]i.

1. We studied how in changes in cytosolic free Ca2+ concentration ([Ca2+]i) produced by voltage-dependent Ca2+ entry are influenced by a caffeine-sensitive Ca2+ store in bullfrog sympathetic neurones. Ca2+ influx was elicited by K+ depolarization and the store was manipulated with either caffeine or ryanodine. 2. For a time after discharging the store with caffeine and switching to a caffeine-free medium: (a) [Ca2+]i was depressed by up to 40-50 nM below the resting level, (b) caffeine responsiveness was diminished, and (c) brief K+ applications elicited [Ca2+]i responses with slower onset and faster recovery than controls. These effects were more pronounced as the conditioning caffeine concentration was increased over the range 1-30 mM. 3. [Ca2+]i, caffeine and K+ responsiveness recovered in parallel with a half-time of approximately 2 min. Recovery required external Ca2+ and was speeded by increasing the availability of cytosolic Ca2+, suggesting that it reflected replenishment of the store at the expense of cytosolic Ca2+. 4. During recovery, Ca2+ entry stimulated by depolarization had the least effect on [Ca2+]i when the store was filling most rapidly. This suggests that the effect of Ca2+ entry on [Ca2+]i is modified, at least in part, because some of the Ca2+ which enters the cytosol during stimulation is taken up by the store as it refills. 5. Further experiments were carried out to investigate whether the store can also release Ca2+ in response to stimulated Ca2+ entry. In the continued presence of caffeine at a low concentration (1 mM), high K+ elicited a faster and larger [Ca2+]i response compared to controls; at higher concentrations of caffeine (10 and 30 mM) responses were depressed. 6. Ryanodine (1 microM) reduced the rate at which [Ca2+]i increased with Ca2+ entry, but not to the degree observed after discharging the store. At this concentration, ryanodine completely blocked responses to caffeine but had no detectable effect on Ca2+ channel current or the steady [Ca2+]i level achieved during depolarization. 7. We propose that, depending on its Ca2+ content, the caffeine-sensitive store can either attenuate or potentiate responses to depolarization. When depleted and in the process of refilling, the store reduces the impact of Ca2+ entry as some of the Ca2+ entering the cytosol during stimulation is captured by the store.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells.

We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40-50% of total Ca2+ uptake was inhibited by TG up to 10 nM (apparent Ki approximately 4.2 nM, Ca2+ pool I). An additional increase of inhibition up to 85-90% of total Ca2+ uptake could be achieved at 15 to 20 nM of TG (apparent Ki approximately 12.1 nM, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki approximately 10 microM). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30-40% of total Ca2+ uptake was inhibited by 100 microM of vanadate (apparent Ki approximately 18 microM, Ca2+ pool II). The remaining 60-70% could be inhibited either by vanadate at concentrations up to 1 mM (apparent Ki approximately 300 microM) or by TG up to 10 nM (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at approximately 25% over a wide range of Ca2+ filling. About 10-20% remained unreleasable by IP3. Reduction of IP3-releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki approximately 3.0 nM for IP3-induced Ca2+ release as compared to approximately 4.2 nM for Ca2+ uptake at TG up to 10 nM) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations > 10 nM which blocked Ca2+ pool II the apparent Ki values were approximately 11.3 and approximately 12.1 nM, respectively. For inhibition by vanadate up to 100 microM the apparent Ki values were approximately 18 microM for Ca2+ uptake and approximately 7 microM for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mM the apparent Ki values were approximately 300 and approximately 200 microM, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 microM. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

The measurement of Ca2+ inflow across the liver cell plasma membrane by using quin2 and studies of the roles of Na+ and extracellular Ca2+ in the mechanism of Ca2+ inflow.

1. Rates of Ca2+ inflow across the hepatocyte plasma membrane in the presence of vasopressin were estimated by using quin2. 2. Plots of the rate of Ca2+ inflow as a function of the intracellular quin2 concentration reached a plateau at about 1.7 mM intracellular quin2. Ca2+ inflow was inhibited by 60% in the presence of 400 microM-verapamil. 3. A plot of the rate of Ca2+ inflow as a function of the concentration of extracellular Ca2+ ([Ca2+]o) was biphasic. The second (slower) phase showed no sign of saturation at values of [Ca2+]o up to 5 mM. It is concluded that, in the presence of vasopressin, Ca2+ flows into the liver cell by two different processes, one of which is not readily saturated by Ca2+o. 4. The effect of the replacement of extracellular NaCl by choline or tetramethylammonium chloride on cellular Ca2+ movement was found to depend on the presence or absence of intracellular quin2. 5. In cells loaded with quin2 and incubated in the presence of choline or tetramethylammonium chloride, a small decrease in the basal intracellular free Ca2+ concentration ([Ca2+]i) was observed, and the increase in [Ca2+]i caused by the addition of vasopressin was considerably diminished when compared with cells incubated in the presence of NaCl. In cells loaded with quin2, replacement of NaCl by choline chloride caused a decrease in Ca2+ inflow in the presence of vasopressin, as measured by using quin2 or 45Ca2+ exchange, whereas no change in Ca2+ inflow was observed in the absence of vasopressin. 6. In cells not loaded with quin2, replacement of NaCl by choline chloride did not alter Ca2+ inflow either in the presence or in the absence of vasopressin. 7. It is concluded that (i) Ca2+ inflow through the basal and receptor-activated Ca2+ inflow systems does not involve the inward movement of Ca2+ in exchange for Na+ or the induction of Ca2+ inflow by intracellular Na+, and (ii) the presence of both intracellular quin2 and extracellular choline or tetramethylammonium chloride (in place of NaCl) inhibits Ca2+ inflow through the receptor-activated Ca2+ inflow system but not through the basal Ca2+ inflow system, and inhibits the release of Ca2+ from intracellular stores.

Aminoquinolines↗
PubMed1999

Overshooting cytosolic Ca2+ signals evoked by capacitative Ca2+ entry result from delayed stimulation of a plasma membrane Ca2+ pump.

The effect of capacitative Ca2+ entry on cytosolic free Ca2+ concentration ([Ca2+]c) was examined in calf pulmonary artery endothelial cells treated with thapsigargin. Restoration of extracellular Ca2+ evoked an overshoot in [Ca2+]c: the initial rate of Ca2+ influx was 12.4 +/- 0.5 nM/s as [Ca2+]c rose monoexponentially (time constant, tau = 36 +/- 2 s) to a peak (322 +/- 16 nM) before declining to 109 +/- 14 nM after 2000 s. Rates of Ca2+ removal from the cytosol were measured throughout the overshoot by recording the monoexponential decrease in [Ca2+]c after rapid removal of extracellular Ca2+. The time constant for recovery (tau rec decreased from 54 +/- 4 s when Ca2+ was removed after 10 s to its limiting value of 8.8 +/- 1.0 s when it was removed after 2000 s. The time dependence of the changes in tau rec indicate that an increase in [Ca2+]c is followed by a delayed (tau = 408 s) stimulation of Ca2+ removal, which fully reverses (tau approximately 185 s) after Ca2+ entry ceases. Numerical simulation indicated that the changes in Ca2+ removal were largely responsible for the overshooting pattern of [Ca2+]c. Because prolonged (30 min) Ca2+ entry did not increase the total 45Ca2+ content of the cells, an increased rate of Ca2+ extrusion across the plasma membrane most likely mediates the Ca2+ removal, and since it persists in the absence of extracellular Na+, it probably results from stimulation of a plasma membrane Ca2+ pump. We conclude that delayed stimulation of a plasma membrane Ca2+ pump by capacitative Ca2+ entry may protect cells from excessive increases in [Ca2+]c and contribute to oscillatory changes in [Ca2+]c.

Animals↗
PubMed2000

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons.

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.

Amphotericin B↗
PubMed1993

Ca2+ and Sr2+ entry induced Ca2+ release from the intracellular Ca2+ store in smooth muscle cells of rat portal vein.

1. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) induced by membrane depolarizations were investigated using indo-1 microspectrofluorimetry in single patch-clamped smooth muscle cells of rat portal vein at room temperature (20-21 degrees C) and in the presence of 2 mM Ca2+. 2. During a 1 s depolarization from -50 to -30 mV [Ca2+]i rose, but, although the Ca2+ current was terminated by repolarization to -50 mV, [Ca2+]i continued to increase in a regenerative manner. The delay between the end of the voltage step and the peak of the [Ca2+]i rise was reduced by increasing the depolarization. 3. When a second identical depolarization was rapidly applied (8-13s) after the first one, it induced an identical Ca2+ current but a smaller increase in [Ca2+]i which started to decay upon repolarization. 4. A low concentration of caffeine (0.05 mM), applied to cells showing a small depolarization-induced [Ca2+]i transient which reached a peak at the end of the voltage step, produced an increase in amplitude and in duration of the [Ca2+]i rise without changing the amplitude of the depolarization-induced Ca2+ current. 5. The depolarization-induced [Ca2+]i rise was shortened and reduced in amplitude after noradrenaline- (NA 10 microM) or caffeine- (5 mM) induced release of Ca2+ store and when the patch pipette solution contained ryanodine (100 microM). Under these conditions, the depolarization-induced [Ca2+]i transient was maximal at the end of the voltage step and declined immediately when the membrane was repolarized at -50 mV. 6. Experiments were done by replacing extracellular Ca2+ by Sr2+. Depolarization-induced Sr2+ entry through voltage-dependent Ca2+ channels could evoke an increase in indo-1 fluorescence which occurred after the termination of the voltage step. This delayed component of fluorescence increase displayed properties similar to those of the regenerative [Ca2+]i rise recorded in the Ca(2+)-containing solution. 7. The inefficiency of the second of two successive depolarizations to produce the delayed component of [Ca2+]i rise was not due to the emptiness of the intracellular Ca2+ store since, under these conditions, caffeine was still able to induce a Ca2+ release. 8. It is concluded that depolarization-evoked Ca2+ or Sr2+ entry through voltage-dependent Ca2+ channels induced the release of Ca2+ from an intracellular store, which could occur in a regenerative manner, independent of the termination of the triggering current.

Animals↗
PubMed1996

Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat ventricular myocytes.

1. We sought to evaluate the contribution of the sarcoplasmic reticulum (SR) Ca2+ pump (vs. diffusion) to the kinetics of [Ca24]i decline during Ca2+ sparks, which are due to spontaneous local SR Ca2+ release, in isolated rat ventricular myocytes measured using fluo-3 and laser scanning confocal microscopy. 2. Resting Ca2+ sparks were compared before (control) and after the SR Ca2(+)-ATPase was either completely blocked by 5 microM thapsigargin (TG) or stimulated by isoprenaline. Na(+)-Ca2+ exchange was blocked using Na(+)-free, Ca(2+)-free solution (0 Na+, O Ca2+) and conditions were arranged so that the SR Ca2+ content was the same under all conditions when Ca2+ sparks were measured. 3. The control Ca2+ spark amplitude (281 +/- 13 nM) was not changed by TG (270 +/- 21 nM) or isoprenaline (302 +/- 10 nM). However, the time constant of [Ca2+]i decline was significantly slower in the presence of TG (29.3 +/- 4.3 ms) compared with control (21.6 +/- 1.5 ms) and faster with isoprenaline (14.5 +/- 0.9 ms), but in all cases was much faster than the global [Ca2+]i decline during a control twitch (177 +/- 10 ms). 4. The spatial spread of Ca2+ during the Ca2+ spark was also influenced by the SR Ca2+ pump. The apparent 'space constant' of the Ca2+ sparks was longest when the SR Ca2+ pump was blocked, intermediate in control and shortest with isoprenaline. 5. We conclude that while Ca2+ diffusion from the source of Ca2+ release is the dominant process in local [Ca2+]i decline during the Ca2+ spark, Ca2+ transport by the SR contributes significantly to both the kinetics and spatial distribution of [Ca2+]i during the Ca2+ spark.

Adrenergic beta-Agonists↗
PubMed2000

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons.

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.

Animals↗
PubMed1997

Ca2+ release-induced inactivation of Ca2+ current in rat ventricular myocytes: evidence for local Ca2+ signalling.

1. Inactivation of Ca2+ current (ICa) induced by Ca2+ release from sarcoplasmic reticulum (SR) was studied in single rat ventricular myocytes using whole-cell patch-clamp and indo-1 fluorescence measurement techniques. 2. Depolarizing pulses to 0 mV elicited large Ca2+ transients and ICa with biexponential inactivation kinetics. Varying SR Ca2+ loading by a 20 s pulse of caffeine showed that the fast component of ICa inactivation was dependent on the magnitude of Ca2+ release. 3. Inactivation of ICa induced by Ca2+ release was quantified, independently of voltage and Ca2+ entry, using a function termed fractional inhibition of ICa (FICa). The voltage relation of FICa had a negative slope, resembling that of single-channel Ca2+ current (iCa) rather than the bell-shaped current-voltage (I-V) relation of macroscopic ICa and Ca2+ transients. 4. Intracellular dialysis of myocytes with 10 mM EGTA (150 nM free [Ca2+]) had no effect on ICa inactivation induced by Ca2+ release, despite abolition of Ca2+ transients and cell contraction. Dialysis with 3 or 10 mM BAPTA (180 nM free [Ca2+]) attenuated FICa in a concentration-dependent manner, with greater inhibition at positive than at negative potentials, consistent with more effective buffering of Ca2+ microdomains of smaller iCa. 5. Spatial profiles of [Ca2+] near an opened Ca2+ channel were simulated. [Ca2+] reached submillimolar levels at the mouth of the channel, and dropped steeply as radial distance increased. At any given distance from the channel, [Ca2+] was higher at negative than at positive potentials. The radii of Ca2+ microdomains were significantly reduced by 3 or 10 mM BAPTA, but not by 10 mM EGTA. 6. In conclusion, the distinctive voltage dependence and susceptibility of Ca2+ release-induced ICa inactivation to fast and slow Ca2+ buffers suggests that the process is mediated through local changes of [Ca2+] in the vicinity of closely associated Ca2+ channels and ryanodine receptors.

Animals↗
PubMed1985

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport.

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.

Adenine Nucleotides↗
PubMed1989

Pharmacologic differentiation between inositol-1,4,5-trisphosphate-induced Ca2+ release and Ca2+- or caffeine-induced Ca2+ release from intracellular membrane systems.

Various known Ca2+ channel blockers and intracellular Ca2+ antagonists have been tested for effects of inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from isolated canine brain microsomes. In agreement with previous reports, heparin, p-chloromercuribenzoic acid, W-7, cinnarizine, flunarizine, certain local anesthetics, La3+, and Ca2+ inhibit the release of Ca2+ induced by addition of IP3. In addition, we report here pronounced inhibition of IP3-induced Ca2+ release by low levels of Cd2+, by relatively high concentrations of TMB-8, and by phytic acid. In contrast, a number of blockers of other Ca2+ channels (nifedipine, verapamil, dantrolene, dithiothreitol, and ruthenium red) have relatively little or no effect on IP3-induced Ca2+ release from brain microsomes. The relative ineffectiveness of substances that inhibit Ca2+- or caffeine-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum suggests that release of Ca2+ from caffeine- and IP3-sensitive neuronal Ca2+ stores is likely to be mediated by different channels. Further evidence that different channels are involved is presented by way of demonstration of the lack of Ca2+-induced Ca2+ release from these brain microsomes and the lack of effect on sarcoplasmic reticulum caffeine-induced Ca2+ release of certain inhibitors of IP3-induced Ca2+ release used here. Among IP3-induced Ca2+ release blockers, La3+ appeared to be exceptional in its ability to stimulate microsomal Ca2+ uptake sufficiently to attenuate release of Ca2+ induced by IP3. Most blockers of IP3-induced Ca2+ release appear not to function by way of inhibiting K+ counter-ion movements (valinomycin does not reverse the inhibition) but rather by way of direct interaction with the IP3 receptor or the Ca2+ channel that mediates the IP3-induced Ca2+ release. Inhibition of [3H]IP3 binding to the microsomes by phytic acid, heparin, pyrophosphate, p-chloromercuribenzoic acid, and Ca2+ could be demonstrated but not by the other substances tested.

Animals↗
PubMed1993

Activation of the Ca2+ "receptor" on the osteoclast by Ni2+ elicits cytosolic Ca2+ signals: evidence for receptor activation and inactivation, intracellular Ca2+ redistribution, and divalent cation modulation.

Earlier studies have demonstrated that a high (mM) extracellular Ca2+ concentration triggers intracellular [Ca2+] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni2+, elicited rapid and concentration-dependent elevations of cytosolic [Ca2+]. The peak change in cytosolic [Ca2+] increased monotonically with the application of [Ni2+] in the 50-5,000 microM range in solutions containing 1.25 mM-[Ca2+] and 0.8 mM-[Mg2+]. The resulting concentration-response function suggested Ni(2+)-induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration-dependent inactivation in which conditioning Ni2+ applications in the range 5-1,500 microM-[Ni2+] inhibited subsequent responses to a maximally effective [Ni2+] of 5,000 microM. Ni(2+)-induced cytosolic [Ca2+] responses were not dependent on extracellular [Ca2+]. Thus, when 5,000 microM-[Ni2+] was applied to osteoclasts in Ca(2+)-free, ethylene glycol bis-(aminoethyl ether) tetraacetic acid (EGTA)-containing medium (< or = 5 nM-[Ca2+] and 0.8 mM-[Mg2+]), cytosolic [Ca2+] responses resembled those obtained in the presence of 1.25 mM-[Ca2+]. Prior depletion of intracellular Ca2+ stores by ionomycin prevented Ni(2+)-induced cytosolic [Ca2+] responses, suggesting a major role for intracellular Ca2+ redistribution in the response to Ni2+. The effects of Ni2+ were also modulated by the extracellular concentration of the divalent cations, Ca2+ and Mg2+. When these cations were not added to the culture medium (0 microM-[Ca2+] and [Mg2+]), even low [Ni2+] ranging between 5 pM and 50 microM elicited progressively larger cytosolic [Ca2+] transients. However, the response magnitude decreased at higher, 250-5,000 microM-[Ni2+], resulting in a "hooked" concentration-response curve. Furthermore, increasing extracellular [Mg2+] or [Ca2+] (0-1 mM) diminished the response to 50 microM-[Ni2+], a concentration on the rising phase of the "hook." Similar increases (0-10 mM) in extracellular [Mg2+] or [Ca2+] increased the response to 5,000 microM-[Ni2+], a concentration on the falling phase of the "hook". These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni2+ as well as the divalent cations, Ca2+ and Mg2+. Receptor occupancy apparently activates intracellular Ca2+ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca2+ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca2+ release.

Animals↗
PubMed2005

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.

Adenosine Triphosphate↗
PubMed2000

Fendiline increases [Ca2+]i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca2+ followed by capacitative Ca2+ entry.

The effect of fendiline, a documented inhibitor of L-type Ca2+ channels and calmodulin, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca2+ probe. Fendiline at 5-100 microM significantly increased [Ca2+]i concentration-dependently. The [Ca2+]i rise consisted of an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca2+ influx and internal Ca2+ release. In Ca(2+)-free medium, pretreatment with 50 microM fendiline nearly abolished the [Ca2+]i rise induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca2+. This indicates that the internal Ca2+ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 microM, fendiline caused Mn2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La3+ by 50%, implying that fendiline-induced Ca2+ influx has two components separable by La3+. Consistently, 0.1 mM La3+ pretreatment suppressed fendiline-induced [Ca2+]i rise, and adding La3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 50-100 microM fendiline in Ca(2+)-free medium. However, 50-100 microM fendiline inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 inhibited 50 microM fendiline-induced internal Ca2+ release by 48%, but inhibition of phospholipase C with 2 microM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca2+]i in MDCK cells by releasing internal Ca2+ in a manner independent of inositol-1,4,5-trisphosphate (IP3), followed by external Ca2+ influx.

Animals↗
PubMed2003

Ca2+ signaling mediated by IP3-dependent Ca2+ releasing and store-operated Ca2+ channels in rat odontoblasts.

In the phospholipase-C (PLC) signaling system, Ca2+ is mobilized from intracellular Ca2+ stores by an action of inositol 1,4,5-trisphosphate (IP3). The depletion of IP3-sensitive Ca2+ stores activates a store-operated Ca2+ entry (SOCE). However, no direct evidence has been obtained about these signaling pathways in odontoblasts. In this study, we investigate the characteristics of the SOCE and IP3-mediated Ca2+ mobilizations in rat odontoblasts using fura-2 microfluorometry and a nystatin-perforated patch-clamp technique. In the absence of extracellular Ca2+ ([Ca2+]o), thapsigargin (TG) evoked a transient rise in intracellular Ca2+ concentration ([Ca2+]i). After TG treatment to deplete the store, the subsequent application of Ca2+ resulted in a rapid rise in [Ca2+]i caused by SOCE. In the absence of TG treatment, no SOCE was evoked. The Ca2+ influx was dependent on [Ca2+]o (KD = 1.29 mM) and was blocked by an IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate (2-APB), as well as La3+ in a concentration-dependent manner (IC50 = 26 microM). In TG-treated cells, an elevation of [Ca2+]o from 0 to 2.5 mM elicited an inwardly rectifying current at hyperpolarizing potentials with a positive reversal potential. The currents were selective for Ca2+ over the other divalent cations (Ca2+ > Ba2+ > Sr2+ >> Mn2+). In the absence of [Ca2+]o, carbachol, bradykinin, and 2-methylthioadenosine 5'triphosphate activated Ca2+ release from the store; these were inhibited by 2-APB. These results indicate that odontoblasts possessed Ca2+ signaling pathways through the activation of store-operated Ca2+ channels by the depletion of intracellular Ca2+ stores and through the IP3-induced Ca2+ release activated by PLC-coupled receptors.

Animals↗
PubMed1989

Inhibition of the liver cell receptor-activated Ca2+ inflow system by metal ion inhibitors of voltage-operated Ca2+ channels but not by other inhibitors of Ca2+ inflow.

The properties of the receptor-activated Ca2+ inflow system in the liver cell plasma membrane were compared with those of voltage-operated Ca2+ channels and receptor-operated Ca2+ channels present in other cell types by testing the susceptibility of the Ca2+ inflow system to inhibition by other metal ions and known inhibitors of Ca2+ movement across membranes. Co2+ inhibited Ca2+ inflow through the receptor-activated Ca2+ inflow system, as assessed by measurement of (a) the activation by extracellular Ca2+ (Cao2+) of glycogen phosphorylase in the presence of vasopressin and (b) 45Ca2+ exchange in the presence of the hormone. The concentration of Co2+ which gave half-maximal inhibition was 280 microM. The inhibition by Co2+ was reversed by high Cao2+. Co2+ did not inhibit basal Ca2+ inflow as measured by 45Ca2+ exchange in the absence of vasopressin. Zn2+, Cd2+, Ni2+ and Mn2+ each inhibited Ca2+ inflow through the receptor-activated Ca2+ inflow system. The concentrations of these ions which gave half-maximal inhibition were 10, 50, 220 and 400 microM, respectively. Little inhibition of receptor-activated Ca2+ inflow was observed in the presence of Sr2+ or Ba2+. However, substantial amounts of 90Sr2+ were taken up by hepatocytes. Rates of 90Sr2+ uptake increased from 0.5-8 nmol per min per mg wet wt. when the extracellular concentration of Sr2+ was varied from 0.25 to 2.5 mM. Sr2+ uptake was inhibited 50% by Cao2+ with half-maximal inhibition at 100 microM Cao2+, but was not inhibited by verapamil and was not stimulated by vasopressin. The movement of Ca2+ through the receptor-activated Ca2+ inflow system was not inhibited by high concentrations of each of a number of inhibitors of voltage-operated and receptor-operated Ca2+ channels and intracellular Ca2+ movement. It is concluded that while the susceptibility to inhibition by metal ions of the receptor-activated Ca2+ inflow system in the liver cell plasma membrane is similar to that of voltage-operated Ca2+ channels, there are significant differences between the liver cell receptor-activated Ca2+ inflow system and both voltage-operated Ca2+ channels and some other receptor-operated Ca2+ channels with respect to inhibition by organic compounds.

Animals↗