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Organic-anion transport inhibitors to facilitate measurement of cytosolic free Ca2+ with fura-2.
No full textThe fluorescent reporter dyes quin2 and fura-2 are used by many investigators to quantitate the cytosolic free-calcium concentration [Ca2+]i in different types of cells. For the dyes to measure accurately [Ca2+]i, they must be selectively and uniformly distributed within the cytoplasmic matrix of the cells. However, in a number of cell types fura-2 does not remain within the cytoplasmic matrix; it accumulates within intracellular compartments, is secreted from the cells entirely, or both. This chapter reviews the evidence for fura-2 sequestration and secretion by cells and describes the use of organic-anion transport inhibitors to ameliorate problems caused by these processes. Mouse macrophages possess organic-anion transporters that remove fluorescent dyes, including fura-2, from the cytoplasmic matrix of these cells. The dyes are sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. The transporters that promote dye sequestration and secretion are inhibi..
Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells
Full text linkAbstract
The neuroblastoma-like cell line N2A and the pheochromocytoma-like cell line PC12 excrete about 20-25% of the intracellular fluorescent Ca2+ indicator fura-2 during 10 min of incubation at 37 degrees C. The drug probenecid, known to inhibit membrane systems for the transport of organic anions [Cunningham, Israili & Dayton (1981) Clin. Pharmacol. 6, 135-151], inhibited fura-2 excretion in both cell types. However, probenecid also had untoward effects on intracellular Ca2+ homeostasis in N2A and PC12 cells. We therefore tested the drug sulphinpyrazone, another known inhibitor of organic-anion transport systems. Sulphinpyrazone fully inhibited excretion of fura-2 at 250 microM, a concentration one order of magnitude lower than that of probenecid. At this concentration and for incubation times up to 20 min, sulphinpyrazone had no untoward effects on cell viability and metabolic functions. Fura-2 was also loaded into the cytoplasm of N2A cells by permeabilization of the plasma membrane with extracellular ATP. In this case as well, the dye was rapidly released from the cells and the efflux was blocked by sulphinpyrazone. These findings suggest that N2A and PC12 cells possess a membrane system for the transport of the free-acid form of fura-2. This transport system is probably responsible for the excretion of fura-2 from these cells. Incubation of N2A and PC12 cells with sulphinpyrazone may help overcome problems arising in the investigation of [Ca2+]i homeostasis in these cell types
Cell-mediated cytotoxicity: ATP as an effector and the role of target cells
No full textCell-mediated cytotoxicity involves a number of distinct mechanisms as well as the active participation of the target cell. Recently, several investigators have demonstrated that extracellular ATP can act as a cytotoxic effector
H2O2 modulates purinergic-dependent calcium signalling in osteoblast-like cells
No full textReactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis
of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand,
increasingly recognized. Here we investigated the effects of H2O2 and extracellular nucleotides on calcium signalling in four osteoblastic cell
lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H2O2 induced oscillatory
increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity
was induced by micromolar H2O2 doses. The H2O2-induced calcium signals, due to both release from intracellular stores and influx from the
extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing
enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H2O2 failed to evoke calcium signals and millimolar H2O2 induced a slowly
developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and
ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with
the evidence that these cells lack functional purinergic receptors. In these cells, H2O2 up to 1mM did not increase the cytosolic calcium
concentration. In ROS/P2Y2 cells, stably expressing the P2Y2 receptor, spontaneous calcium oscillations were observed in 38% of the population
and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were
inhibited by suramin and apyrase. In these cells H2O2 induced oscillatory calcium activity that was blocked by suramin and apyrase. The
sensitivity of ROS/P2Y2 cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous
calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited
by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in
osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y2 receptor
Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions
No full textAbstract
Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types
Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms
No full textWe have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only
Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers
No full textFura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facilitate the use of Fura-2 in a variety of cell types
Role of extracellular ATP in cell-mediated cytotoxicity: A study with atp- sensitive and ATP-resistant macrophages
No full textAbstract
It has been proposed that extracellular ATP (ATPo) may function as a cytotoxic molecule in Ca(2+)-independent cell-mediated lysis by activating plasma membrane P2 purinergic receptors. In the present study the involvement of the P2z purinergic receptor in ATPo as well as cell-mediated lysis was investigated by using the J774 mouse macrophage cell line, which expresses this receptor, and a panel of J774-derived mutant cell clones selected for the lack of P2z receptor activity. We confirmed that the P2z receptor in J774 is associated with ATPo-induced colloido-osmotic lysis but not with apoptosis. Furthermore, we observed that the lack or the inhibition of the P2z purinergic receptor does not affect lytic activity mediated by different types of cytotoxic cell populations. These results on the whole indicate that the P2z receptor is involved in cell membrane damage induced by ATPo but not in cell-mediated cytotoxicity
CHARACTERIZATION OF THE CYTOTOXIC EFFECT OF EXTRACELLULAR ATP IN J774-MOUSE MACROPHAGES
No full textExtracellular ATP (ATPo) is known to be cytotoxic to many cell types through a mechanism which is largely unknown. Very recently this nucleotide has been shown to cause cell death by apoptosis, probably by interacting with specific cell-surface receptors. In the present study we have investigated the mechanism of ATPo-dependent cytotoxicity in the macrophage-like mouse cell line J774. It has been previously reported that in this cell type ATPo activates trans-membrane Ca2+ and Na+ fluxes and a drastic increase in the plasma-membrane permeability to hydrophilic solutes smaller than 900 Da. These changes are followed by cell swelling and lysis. We show in the present study that, although this nucleotide triggers a rise in the cytoplasmic Ca2+ concentration, neither cell swelling nor lysis is Ca(2+)-dependent. Furthermore, cell lysis is not dependent on Na+ influx, as it is not prevented by iso-osmotic replacement of extracellular Na+ with choline or N-methylglucamine. On the contrary, ATPo-dependent cytotoxicity, but not the ATPo-dependent increase in plasma-membrane permeability, is completely abrogated in sucrose medium. Under our experimental conditions ATPo does not cause DNA fragmentation in J774 cells. We conclude from these findings that ATPo does not cause apoptosis of J774 macrophages and promotes a Ca(2+)- and Na(+)-independent colloido-osmotic lysis
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