Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.     

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.     

Stretch-elicited calcium responses in the intact mouse thoracic aorta

Stretch-elicited intracellular calcium ([Ca(2+)](i)) changes in individual smooth muscle cells in a ring of aorta were measured simultaneously with the force developed by the ring. A phasic increase in [Ca(2+)](i) was observed in 30% of the cells and a sustained one in 10%. Depletion of intracellular calcium store by thapsigargin and caffeine decreased phasic and increased sustained calcium responses. The inhibition of calcium entry either by stretching the aorta in a calcium-free medium or by the inhibition of stretch-activated, non-selective cationic channels by 5 microM GsMtx-4 toxin, decreased the proportion of sustained [Ca(2+)](i) responses but increased transient responses. In this condition, a third of the cells responded to stretch by a bursts of [Ca(2+)](i) spikes. The decrease of calcium influx triggered the generation of burst of calcium spikes after the application of stretch steps to the vascular wall. We conclude that progressive recruitment of smooth muscle cells is the mechanism underlying the force-generating part of the myogenic response. Two types of stretch-elicited calcium responses were observed during the recruitment of the smooth muscle cells. One was a phasic calcium discharge generated by the sarcoplasmic reticulum. The second was a tonic response produced by the activation of the stretch-sensitive cationic channels allowing extracellular Ca(2+) entry.     

Mechanisms of ATP-induced calcium signaling and growth arrest in human prostate cancer cells

This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.     

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine. Loading cells with low concentrations of the cell-permeant esters of the artificial Ca(2+)-buffers EGTA or BAPTA, resulted in fewer cells with a distinct "baseline" oscillatory pattern, and loading with higher concentrations of BAPTA almost completely abolished them. In HeLa-CaBP cells, stimulation with 1 microM histamine resulted in individual Ca(2+) spikes that had a flattened profile when compared to control cells; peak [Ca(2+)](i) was lowered, the rate of increase in [Ca(2+)](i) was slower and transients were prolonged. When compared to HeLa-pCINeo cells, loading with EGTA or BAPTA, or transfection of CaBP, significantly reduced the propagation velocity (by up to 60%) of Ca(2+) waves induced by exposure to 100 microM histamine. We conclude that intracellular Ca(2+) buffering exerts a significant influence on global Ca(2+) responses in HeLa cells and the propagation of Ca(2+) waves that underlie them. The relative effectiveness of different Ca(2+) buffers, including CaBP, appears to be particularly dependent upon the rapidity of their binding kinetics, with BAPTA being the most effective.     

Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages.

A concentration-dependent elevation of intracellular calcium ([Ca2+]i) and oxidation of NAD(P)H occurred in alveolar macrophages during exposure to sublethal tert-butylhydroperoxide concentrations (tBOOH) (< or = 100 microM in 1 ml with 1 x 10(6) cells). Oxidation of NAD(P)H preceded a rise in [Ca2+]i. The elevation of [Ca2+]i was reversible at < 50 microM tBOOH exposure and the return to the steady state [Ca2+]i correlated temporally with repletion of NAD(P)H. At > 50 microM tBOOH, the changes in NAD(P)H and [Ca2+]i were sustained. The relative contributions of NADPH and NADH oxidation were examined by varying the substrates supplying reducing equivalents and by inhibiting glutathione reductase activity. The results suggested that at < 50 microM tBOOH, oxidation of NADPH predominated, while at > 50 microM tBOOH, NADH oxidation predominated. A complex relationship between the relative roles of NADPH and NADH oxidation and the elevation of [Ca2+]i was revealed: (i) reversible oxidation of NADPH is associated with the initial and reversible elevation of [Ca2+]i at < 50 microM tBOOH; (ii) the sustained elevation of [Ca2+]i at > 50 microM tBOOH correlates with the sustained oxidation of NADH; and (iii) the changes in [Ca2+]i did not depend on influx of extracellular Ca2+. We speculate that at low tBOOH, Ca2+ was released from the NADPH/NADP(+)-sensitive mitochondrial Ca2+ pool while higher tBOOH caused additional Ca2+ release from GSH/GSSG-sensitive nonmitochondrial Ca2+ pools with sustained elevation of [Ca2+]i due to decreased mitochondrial Ca2+ reuptake.     

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

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

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Effect of NPC-15199 on Ca2+ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).     

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6-23 cells

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.     

Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase- arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.     

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.     

Reduction of K+-Stimulated 45Ca2+ Influx in Synaptosomes with Age Involves Inactivating and Noninactivating Calcium Channels and Is Correlated with Temporal Modifications in Protein Dephosphorylation

The voltage-dependent calcium uptake in rat brain synaptosomes was measured under conditions in which [Ca2+]o/[Na+]i exchange was minimized to characterize the voltage-sensitive calcium channels from rats of different ages. In solutions of CaCl2 concentrations of less than 500 microM, the initial (5-s) calcium uptake declined by approximately 20-50% in 12- and 24-month-old rats relative to 3-month-old adults. Depolarization of synaptosomes from 3-month-old rats in a calcium-free medium or in the presence of 0.5 mM CaCl2 led to an exponential decline of the calcium uptake rate after 20 s (voltage- or voltage-and-calcium-dependent inactivation) to approximately 66 and 34% of the initial value with a t1/2 of 1.6 or 0.7 s, respectively. The presence of 1 microM nifedipine resulted in a 15-25% reduction of 45Ca2+ uptake rates, which appeared to affect noninactivating calcium channels, but addition of the calcium channel agonist Bay K 8644 was without effect. In 24-month-old rats, inactivation of 45Ca2+ uptake in calcium-free media was nondetectable, and in the presence of 0.5 mM CaCl2, the rate and extent of inactivation were also much lower than in 3-month-old animals (the t1/2 was 0.9 s, and the calcium uptake rate at 20 s was 55% of its initial value). Moreover, the presence of 1 microM nifedipine was without effect on initial calcium uptake or inactivation in synaptosomes from 24-month-old rats. These results indicate that the decrease in calcium channel-mediated 45Ca2+ uptake involves an inhibition or block of both dihydropyridine-resistant and -sensitive calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished     

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.     

Characterization of pressure-induced calcium response in neuronal cell lines

BACKGROUND: Cation channels that respond to mechanical stress have been described in neuronal and nonneuronal cells. These nonselective cation ([C+(SA)]) channels are believed to regulate volume and osmolarity of cells in the central nervous system and are therefore believed to be involved in brain injury, resulting in intracellular calcium accumulation and cell death. METHODS: Activation of pressure-sensitive channels was monitored as an increase in [Ca2+](i) by flow cytometry using indo-1. Several neuronal cell lines including NH15-CA2 neuroblastoma x glioma cells were stimulated by rectangular pressure increase. RESULTS: Neuronal cell lines showed a pressure-sensitive increase in [Ca2+](i) but no pressure sensitivity was found in fibroblasts and embryonic P19 cells. [C+(SA)] channels in NH15-CA2 cells were not blocked by inhibitors of voltage-dependent calcium channels and G-proteins. Depletion of extracellular calcium and of internal Ca2+ stores inhibited pressure-induced [Ca2+](i) increase. Elevated [C+(SA)] channel activity was also observed in confluent NH15-CA2 thus accumulated in the G(0)/G(1)-phase of the cell cycle. P19 cells showed occurrence of [C+(SA)] channel activity only after neuronal differentiation. CONCLUSION: Pressure-sensitive channel activity is present in cells of neuronal origin. This activity depends on neuronal differentiation and might have a pivotal role in neuronal development and differentiation.     

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.     

Intracellular calcium puffs in osteoclasts

We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.