Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells

It is generally thought that receptor-operated Ca2+ entry is related to store-operated or capacitative Ca2+ entry mechanism. Recent evidence suggests that non-capacitative Ca2+ entry pathways are also involved in receptor activated Ca2+ influx in many different kinds of cells. In this study, we studied whether alpha1-adrenoreceptor (alpha1-AR)-activated Ca2+ entry is coupled to both capacitative and non-capacitative pathways in A10 vascular smooth muscle cells by fura-2 fluorescence probe and conventional whole-cell patch clamp techniques. We found that both thapsigargin (TG) and phenylephrine (Phe) induced transient increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in Ca2+-free medium, and subsequent addition of Ca2+ evoked a sustained [Ca2+]i rise. When the membrane potential was held at -60 mV, both TG and Phe activated inward currents, which were inhibited by GdCl3(Gd3+), 0Na+/0Ca2+ solution and 1-{beta[3-(4-mehtoxyphenyl)propoxy]-4-methoxypheneth-yl}-1H- imidazole hydro-chloride (SK&F96365), but not by nifedipine. When Ca2+ store was depleted by TG in Ca2+-free solution, Phe failed to further evoke [Ca2+]i rise. However, when capacitative Ca2+ entry was activated by TG in the medium containing Ca2+, 10 microM Phe further increased [Ca2+]i. At the same concentration, TG activated an inward cation current, subsequent addition of Phe also further induced an inward cation current. Furthermore, the amplitudes of [Ca2+]i increase and current density induced by Phe in the presence of TG were less than that induced by Phe alone. Our results suggest that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca2+ influx induced by activation of alpha1-AR in A10 vascular smooth muscle cells.     

Effect of the antidepressant sertraline on Ca2+fluxes in Madin-Darby canine renal tubular cells

The effect of the antidepressant sertraline on cytosolic-free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether sertraline changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Sertraline at concentrations between 1and 100 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ implicating Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholiapase A2 but not by store-operated Ca2+ channel blockers and protein kinase C/A modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline partly reduced inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from endoplasmic reticulum. Inhibition of phospholipase C did not much alter sertraline-induced [Ca2+]i rise. Collectively, in MDCK cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels.     

Elevation of cytosolic [Ca2+] due to intracellular Ca2+ release retards carbachol stimulation of divalent cation entry in rat parotid gland acinar cells

This study examines the activation of divalent cation entry into rat parotid gland acinar cells by using Mn2+ as a Ca2+ surrogate cation. Following muscarinic-cholinergic stimulation of dispersed parotid acini with carbachol (10 microM), the onset of internal Ca2+ release (cytosolic [Ca2+], [Ca2+]i, increase) and the stimulation of Mn2+ entry (increase in fura2 quenching) are not simultaneously detected. [Ca2+]i elevation, due to intracellular release, is detected almost immediately following carbachol addition and peak [Ca2+]i increase occurs at 6.0 +/- 0.8 sec. However, there is an interval (apparent lag) between carbachol addition and the detection of stimulated Mn2+ entry. This apparent lag is decreased from 26 +/- 3.1 sec to 9.2 +/- 1.5 sec when external Mn2+ ([Mn2+]0) is increased from 12.5 to 500 microM. It is not decreased further with increase in [Mn2+]0 from 500 microM to 1 mM (9.8 +/- 2.1 sec), although both intracellular free Mn2+ and [Mn2+-fura2]/[fura2] increase. Thus, at [Mn2+]0 < 500 microM, the observed lag time is partially due to a limitation in the magnitude of Mn2+ entry. Furthermore, neither peak [Ca2+]i nor the time required to reach peak [Ca2+]i is significantly altered by [Mn2+]0 (12.5 microM to 1 mM). At every [Mn2+]0 tested (i.e., 12.5 microM-1 mM), the apparent lag is significantly greater than the time required to reach peak [Ca2+]i. However, when carbachol stimulation of the [Ca2+]i increase is attenuated by loading the acini with the Ca2+ chelator, 2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), there is no detectable lag in carbachol stimulation of Mn2+ entry (with 1 mM [Mn2+]0). Importantly, in BAPTA-loaded acini, carbachol stimulates Mn2+ entry via depletion of the internal Ca2+ pool and not via direct activation of other divalent cation entry mechanisms. Based on these results, we suggest that the apparent lag in the detection of carbachol stimulation of Mn2+ entry into parotid acinar cells is due to a retardation of Mn2+ entry by the initial increase in [Ca2+]i, due to internal release, which most likely occurs proximate to the site of divalent cation entry.     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Protease-activated receptor-2-mediated Ca2+ signaling in guinea pig tracheal epithelial cells

The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.     

Nordihydroguaiaretic acid-induced Ca2+ handling and cytotoxicity in human prostate cancer cells

The effect of nordihydroguaiaretic acid (NDGA), a compound commonly used as a lipoxygenases inhibitor, on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells was investigated. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. NDGA increased [Ca2+]i in a concentration-dependent manner with an EC50 of 30 microM. The Ca2+ signal comprised a gradual and sustained increase. Removal of extracellular Ca2+ partly decreased the NDGA-induced [Ca2+]i increase, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and intracellular Ca2+ release. NDGA-induced Ca2+ influx was independently confirmed by measuring NDGA-induced Mn2+ -coupled quench of fura-2 fluorescence. The NDGA-induced Ca2+ influx was not affected by L-type Ca2+ channel blockers. In Ca2+ -free medium, the NDGA-induced [Ca2+]i increase was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with NDGA abolished thapsigargin-induced [Ca2+]i increase. NDGA-induced intracellular Ca2+ release was not altered by inhibition of phospholipase C. Overnight treatment with 20-50 microM NDGA inhibited cell proliferation rate in a concentration-dependent manner. Several other lipoxygenases inhibitors did not alter [Ca2+]i. Collectively, this study shows that in prostate cells, NDGA induced a [Ca2+]i increase via releasing stored Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. NDGA also caused cytotoxicity at higher concentrations.     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals

Ca(2+)-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activation by conditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRs are primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyR3 in muscle fibers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in intracellular Ca2+ ([Ca2+]i) induced by a short tetanus (50 Hz, 1-2s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 15s every 20s) produced repetitive rises in [Ca2+]i, whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning components. Ryanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15s) in a low Ca2+, high Mg2+ solution. KN-62, a blocker of Ca(2+)/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [Ca2+]i, but markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likewise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency during long tetanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminals and greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis and its plasticity.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

Spatial microenvironment defines Ca2+ entry and Ca2+ release in salivary gland cells

The difference of Ca(2+) mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic-cholinergic agonist, induced intracellular Ca(2+) release and extracellular Ca(2+) entry through store-operated Ca(2+) entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca(2+) entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca(2+) stores, the machinery for intracellular Ca(2+) release was intact in the duct cells. By immunocytochemical experiments, IP(3)R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP(3)R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP(3)R2. These results suggest that the expression of the IP(3)Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca(2+) stores. Therefore, the microenvironment probably affects intracellular Ca(2+) release and Ca(2+) entry.     

Effect of olvanil (N-vanillyl-cis-9-octadecenoamide) on cytosolic Ca2+ increase in renal tubular cells

In canine renal tubular cells, effect of olvanil, a presumed cannabinoid and vanilloid receptor modulator, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Olvanil (5-100 microM) caused a rapid and sustained [Ca2+]i rise in a concentration-dependent manner. Olvanil-induced [Ca2+]i rise was prevented by 70 and 90% by removal of extracellular Ca2+ and La3+, respectively, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of olvanil on [Ca2+]i was abolished; also, pretreatment with olvanil partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phoispholipase C, abrogated ATP-, but partly inhibited olvanil-, induced [Ca2+]i rise. Two cannabinoid receptor antagonists (AM251 and AM281; 5 microM) and a vanilloid receptor antagonist (capsazepine; 100 microM) did not alter olvanil (50 microM)-induced [Ca2+]i rise. These results suggest that olvanil rapidly increases [Ca2+]i in renal tubular cells, by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via mechanism(s) independent of stimulation of cannabinoid and vanilloid receptors.     

Effect of maprotiline on Ca(2+) movement in human neuroblastoma cells

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2+ concentrations ([Ca2+]i) was explored by using the Ca2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50 = 200 microM). Maprotiline-induced [Ca2+]i rise was reduced by 50% by removal of extracellular Ca2+. Maprotiline-induced [Ca2+]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of maprotiline on [Ca2+]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2+]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.     

Characteristics of lysophosphatidylcholine-induced Ca2+ response in human neuroblastoma SH-SY5Y cells

Lysophosphatidylcholine (LPC) is an important bioactive lipid. In the nervous system, elevated levels of LPC have been shown to produce demyelination. In the present study, we examined the effect of exogenous LPC on intracellular Ca2+ mobilization in human neuroblastoma SH-SY5Y cells. In Ca2+-containing medium, introduction of LPC induced a steady rise in cytosolic Ca2+ levels ([Ca2+]i) in a dose-dependent manner, and this rise was provoked by LPC itself, not by its hydrolysis product produced by lysophospholipase. The increase in [Ca2+]i was reduced by 36% by removal of extracellular Ca2+, while preincubation of the cells with verapamil, an L-type Ca2+ channel blocker, inhibited the response by 23%, part of the Ca2+ influx. Conversely, Ni2+, which inhibits the Na+-Ca2+ exchanger, or Na+-deprivation did not affect LPC-induced Ca2+ influx. In Ca2+-free medium, depletion of Ca2+ stores in the endoplasmic reticulum (ER) by thapsigargin, an ER Ca2+-ATPase inhibitor, abolished the Ca2+ increase. Moreover, LPC-induced [Ca2+]i increase was fully blocked by ruthenium red and procaine, inhibitors of ryanodine receptor (RyR), but was not affected by 2-aminoethoxydiphenyl borate, an inhibitor of inositol triphosphate receptor, or by pertussis toxin, a G(i/o) protein inhibitor. Combined treatment with verapamil plus thapsigargin markedly inhibited but did not abolish the LPC-induced Ca2+ response. These findings indicate that LPC-induced [Ca2+]i increase depends on both external Ca2+ influx and Ca2+ release from ER Ca2+ stores, in which L-type Ca2+ channels and RyRs may be involved. However, in digitonin-permeabilized SH-SY5Y cells, LPC could not induce any [Ca2+]i increase in Ca2+-free medium, suggesting that LPC may act indirectly on RyRs of ER.     

Heregulinbeta activates store-operated Ca2+ channels through c-erbB2 receptor level-dependent pathway in human breast cancer cells

The heregulinbeta (HRGbeta) is a ligand to activate c-erbB2/c-erbB3 interaction and can subsequently increases cytosolic [Ca(2+)](i). In the two human breast cancer cell lines, MCF-7 shows a low c-erbB2 expression level, whereas SK-BR-3 overexpress c-erbB2 receptor. In this article, we have found that in MCF-7, HRGbeta induced Ca(2+) release from the endoplasmic reticulums (ER) and subsequently activated Ca(2+) entry via store-operated Ca(2+) channel (SOC). However, in SK-BR-3, HRGbeta failed to induce Ca(2+) release and Ca(2+)entry. RNA interference to decrease c-erbB2 level in SK-BR-3 resulted in reactivation of HRGbeta-evoked Ca(2+) release and Ca(2+) entry via SOC, which was similar to that of MCF-7. In addition, in the absence of HRGbeta, a constitutive activation of SOC was observed in SK-BR-3 rather than in MCF-7 and c-erbB2-siRNA treated SK-BR-3. Compared to the cells with low c-erbB2 level, c-erbB2 might tend to interact with c-erbB3 in the resting state in the cells with high c-erbB2 level, which resulted in different [Ca(2+)](i) responses to HRGbeta. In SK-BR-3, the Ca(2+) mobilization in the presence or in the absence of HRGbeta was completely blocked by PLC inhibitor U73122. In summary, our results indicate that HRGbeta-induced SOC was regulated by c-erbB2 level and dependent on activation of PLC in human breast cancer cells.     

Ca2+ influx-independent synaptic potentiation mediated by mitochondrial Na(+)-Ca2+ exchanger and protein kinase C

Activity-dependent modulation of synaptic transmission is an essential mechanism underlying many brain functions. Here we report an unusual form of synaptic modulation that depends on Na+ influx and mitochondrial Na(+)-Ca2+ exchanger, but not on Ca2+ influx. In Ca(2+)-free medium, tetanic stimulation of Xenopus motoneurons induced a striking potentiation of transmitter release at neuromuscular synapses. Inhibition of either Na+ influx or the rise of Ca2+ concentrations ([Ca2+]i) at nerve terminals prevented the tetanus-induced synaptic potentiation (TISP). Blockade of Ca2+ release from mitochondrial Na(+)-Ca2+ exchanger, but not from ER Ca2+ stores, also inhibited TISP. Tetanic stimulation in Ca(2+)-free medium elicited an increase in [Ca2+]i, which was prevented by inhibition of Na+ influx or mitochondrial Ca2+ release. Inhibition of PKC blocked the TISP as well as mitochondrial Ca2+ release. These results reveal a novel form of synaptic plasticity and suggest a role of PKC in mitochondrial Ca2+ release during synaptic transmission.     

Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Cell-cycle-dependent expression of the large Ca2+-activated K+ channels in breast cancer cells

In a previous work, we have reported that the ionic nature of the outward current recorded in MCF-7 cells was that of a K+ current. In this study, we have identified a Ca2+-activated K+ channel not yet described in MCF-7 human breast cancer cells. In cells arrested in the early G1 (depolarized cells), increasing [Ca2+]i induced both a shift in the I-V curve toward more negative potentials and an increase in current amplitude at negative and more at positive potential. Currents were inhibited by r-iberiotoxin (r-IbTX, 50 nM) and charybdotoxin (ChTX, 50 nM). These data indicate that human breast cancer cells express large-conductance Ca2+-activated K+ (BK) channels. BK current-density increased in cells synchronized at the end of G1, as compared with those in the early G1 phase. This increased current-density paralleled the enhancement in BK mRNA levels. Blocking BK channels with r-IbTX, ChTX or both induced a slight depolarization in cells arrested in the early G1, late G1, and S phases and accumulated cells in the S phase, but failed to induce cell proliferation. Thus, the expression of the BK channels was cell-cycle-dependent and seems to contribute more to the S phase than to the G1 phase. However, these K+ channels did not regulate the cell proliferation because of their minor role in the membrane potential.     

ATP modulation of mitogen activated protein kinases and intracellular Ca2+ in breast cancer (MCF-7) cells

In the breast tumor cell line MCF-7, extracellular nucleotides induce transient elevations in intracellular calcium concentration ([Ca(2+)](i)). In this study we show that stimulation with ATP or UTP sensitizes MCF-7 cells to mechanical stress leading to an additional transient Ca(2+) influx. ATP> or =ATPgamma-S> or =UTP>ADP=ADPbeta-S elevate [Ca(2+)](i), proving the presence of P2Y(2)/P2Y(4) purinergic receptor subtypes. In addition, cell stimulation with ATP, ATPgamma-S or UTP but not ADPbeta-S induced the phosphorylation of ERK1/2, p38 and JNK1/2 mitogen activated protein kinases (MAPKs). The use of Gd(3+), La(3+) or a Ca(2+)-free medium, inhibited ATP-dependent stress activated Ca(2+) (SAC) influx, but had no effect on MAPK phosphorylation. ATP-induced activation of MAPKs was diminished by two PI-PLC inhibitors and an IP(3) receptor antagonist. These results evidence an ATP-sensitive SAC influx in MCF-7 cells and indicate that phosphorylation of MAPKs by ATP is dependent on PI-PLC/IP(3)/Ca(2+)(i) release but independent of SAC influx in these cells, differently to other cell types.