Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca²⁺ overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca²⁺-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules <800 Da to enter the cell. The cells then lyse not by rupture of the plasmalemma but through the activation of a "death" channel that lets large proteins (e.g., 140–160 kDa) leave the cell. These changes in permeability are accompanied by the formation of membrane blebs. In this study, we took advantage of the well-known differences in affinity of various Ca²⁺-binding proteins for Ca²⁺ and Sr²⁺ vs. Ba²⁺ to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single-cell level (time-lapse videomicroscopy), we found that the replacement of Ca²⁺ with either Sr²⁺ or Ba²⁺ delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated green fluorescent protein. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba²⁺ not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca²⁺-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba²⁺. Taken together, these results suggest that intracellular high-affinity Ca²⁺-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise. necrosis; vital dyes; membrane blebs; time-lapse videomicroscopy; fura 2     

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca²⁺ (Ca_i²⁺) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Ca_i²⁺ (to micromolar concentrations) observed after the addition of ionophore plus Ca²⁺ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Ca_i²⁺, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Ca_i²⁺ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Ca_i²⁺ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Ca_i²⁺. calcium; connexin43; lens gap junctions     

Ciglitizone inhibits cell proliferation in human uterine leiomyoma via activation of store-operated Ca2+ channels

This study investigated the acute effects of a peroxisome proliferator-activated receptor (PPAR)- ligand, ciglitizone, on cell proliferation and intracellular Ca²⁺ signaling in human normal myometrium and uterine leiomyoma. Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured with fura-2 AM, and cellular viabilities were determined by viable cell count and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay. Ciglitizone (100 µM) induced greater inhibition of cell proliferation in uterine leiomyoma than in myometrium. Ciglitizone also dose-dependently increased [Ca²⁺]_i in both myometrium and uterine leiomyoma; these [Ca²⁺]_i increases were inhibited by PPAR- antagonists and raloxifene. Ciglitizone-induced [Ca²⁺]_i increase showed only an initial peak in normal myometrial cells, whereas in uterine leiomyoma there was a second sustained [Ca²⁺]_i increase as well. The initial [Ca²⁺]_i increase in both myometrium and uterine leiomyoma resulted from the release of Ca²⁺ by the sarcoplasmic reticulum via activation of ryanodine receptors. The second [Ca²⁺]_i increase was observed only in uterine leiomyoma because of a Ca²⁺ influx via an activation of store-operated Ca²⁺ channels (SOCCs). Cell proliferation was inhibited and secondary [Ca²⁺]_i increase in uterine leiomyoma was attenuated by cotreatment of ciglitizone with a SOCC blocker, lanthanum. The results suggest that ciglitizone inhibits cell proliferation and increases [Ca²⁺]_i through the activation of SOCCs, especially in human uterine leiomyoma. peroxisome proliferator-activated receptor-; intracellular calcium; uterine cells     

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse

The myoplasmic free Ca²⁺concentration([Ca²⁺]_i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca²⁺ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na⁺ was reducedfrom 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-modeNa⁺/Ca²⁺exchange (Ca²⁺ entry in exchangefor Na⁺ leaving the cell). Undernormal resting conditions, application oflow-Na⁺ solution only increased[Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting[Ca²⁺]_iof 42 nM. In other fibers,[Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca²⁺ release with caffeine (10 mM)and by inhibiting SR Ca²⁺ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa⁺/Ca²⁺exchange (Ca²⁺ removal from thecell in exchange for Na⁺ influx).These two agents caused a large increase in[Ca²⁺]_i,which then declined to a plateau level approximately twice the baseline[Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa²⁺ had been removed, theincrease in[Ca²⁺]_iduring the second exposure was very low, suggesting thatCa²⁺ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na⁺ solution produced a large,sustained increase in[Ca²⁺]_iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na⁺ solution in whichCa²⁺ had been removed, a sustainedincrease in[Ca²⁺]_iwas not observed, although[Ca²⁺]_iremained higher and declined slower than in normalNa⁺ solution. This suggests thatforward-modeNa⁺/Ca²⁺exchange contributed to the fall of[Ca²⁺]_iin normal Na⁺ solution, but whenextracellular Na⁺ was low, aprolonged elevation of[Ca²⁺]_icould activate reverse-modeNa⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess aNa⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.

     

Sphingosine-1-phosphate activates BKCa channels independently of G protein-coupled receptor in human endothelial cells

The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca²⁺]_i. [Ca²⁺]_i and membrane potentials were strongly correlated. In whole cell clamped cells, BK_Ca currents were activated by increasing [Ca²⁺]_i via cell dialysis with pipette solution, and the activated BK_Ca currents were further enhanced by S1P. When [Ca²⁺]_i was buffered at 1 µM, the S1P concentration required to evoke half-maximal activation was 403 ± 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BK_Ca channel activity in a reversible manner and shifted the relationship between Ca²⁺ concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5'-O-(2-thiodiphosphate) (GDPS; 1 mM) using a patch pipette, GDPS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BK_Ca current and channel activation. These results suggest that S1P enhances BK_Ca channel activity by increasing Ca²⁺ sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca²⁺ influx through Ca²⁺ entry channels. Inasmuch as S1P activates BK_Ca channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca²⁺ mobilization in human endothelial cells. sphingolipid metabolites; intracellular second messenger; Ca²⁺ mobilization     

Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca²⁺ ([Ca²⁺]_i) and intracellular pH (pH_i) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) is a major regulator of [Ca²⁺]_i. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca²⁺]_i and pH_i in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca²⁺]_i was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca²⁺]_i increase than in normal astrocytes. This increase was prevented in nominally Ca²⁺-free buffer and by 2-APB, an inhibitor of store-operated Ca²⁺ channels. In addition, TG-activated Ca²⁺ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pH_i was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na⁺/H⁺ exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca²⁺ homeostasis and pH regulation in tumor cells compared with normal human astrocytes. fura-2; BCECF; store-operated calcium channels     

Bidirectional Ca2+ coupling of mitochondria with the endoplasmic reticulum and regulation of multimodal Ca2+ entries in rat brown adipocytes

How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca²⁺ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca²⁺ or Mg²⁺ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) produced by thapsigargin was abolished by removing extracellular Ca²⁺, depressed by depleting extracellular Na⁺, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca²⁺]_i, in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca²⁺-free EGTA solution, and an Na⁺-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca²⁺ and fully blocked by oligomycin but not by thapsigargin or an Na⁺-deficient solution, was accompanied by a rise in cytoplasmic Mg²⁺ concentration, and was summated with a high pH-induced rise in [Ca²⁺]_i, whereas the extracellular Ca²⁺-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca²⁺ but not by thapsigargin, depressed by decreasing Na⁺, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca²⁺]_i in a Ca²⁺-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca²⁺ release, activating Ca²⁺ release from the ER and store-operated Ca²⁺ entry, and directly elicits a novel plasmalemmal Ca²⁺ entry, whereas Ca²⁺ release from the ER activates Ca²⁺ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP     

Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca²⁺]_i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca²⁺]_i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca²⁺]_i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca²⁺]_i, which was sensitive to antioxidants and removal of external Ca²⁺, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca²⁺-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca²⁺ uptake. This suggests that hydrogen peroxide promotes Ca²⁺ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca²⁺ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca²⁺]_i and altered cholecystokinin-evoked [Ca²⁺]_i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca²⁺]_i also upregulated the activity of the plasma membrane Ca²⁺-ATPase in a Ca²⁺-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca²⁺-ATPase. This may be important in facilitating "Ca²⁺ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF-4-induced [Ca2+]i oscillations in rat pancreatic

AlF₄^-is known to generate oscillations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF₄^--evoked [Ca²⁺]_i oscillations were reported to be dependent on extracellular Ca²⁺, which contrasts with the [Ca²⁺]_i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF₄^- generates extracellular Ca²⁺-dependent [Ca²⁺]_i oscillations in rat pancreatic acinar cells. AlF₄^--induced [Ca²⁺]_i oscillations were stopped rapidly by the removal of extracellular Ca²⁺ and were abolished on the addition of 20 mM caffeine and 2 µM thapsigargin, indicating that Ca²⁺ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store is also required. The amount of Ca²⁺ in the intracellular Ca²⁺ store was decreased as the AlF₄^--induced [Ca²⁺]_i oscillations continued. Measurement of ⁴⁵Ca²⁺ influx into isolated microsomes revealed that AlF₄^-directly inhibited sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). The activity of plasma membrane Ca²⁺-ATPase during AlF₄^- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺. This study shows that AlF₄^- induces [Ca²⁺]_i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca²⁺ as a result of the partial inhibition of SERCA. cholecystokinin; plasma membrane adenosine 5'-triphosphatase; G proteins; caffeine     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Elementary events of agonist-induced Ca2+ release in vascular endothelial cells

The subcellular spatial and temporal organization ofagonist-induced Ca²⁺ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca²⁺ concentration([Ca²⁺]_i)in peripheral cytoplasmic processes from where activation propagated asa[Ca²⁺]_iwave toward the central regions of the cell. The average propagation velocity of the[Ca²⁺]_iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca²⁺]_idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca²⁺]_ideclined monotonically, whereas in the central region[Ca²⁺]_idecreased in an oscillatory fashion. Propagating[Ca²⁺]_iwaves were preceded by small, highly localized[Ca²⁺]_itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa²⁺ release was 23 nM, and theunderlying flux of Ca²⁺ amountedto ~1-2 × 10¹⁸mol/s or ~0.3 pA, consistent with aCa²⁺ flux through a single orsmall number of endoplasmic reticulum Ca²⁺-release channels.

     

Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells

The L-type Ca²⁺ channel is the primary voltage-dependent Ca²⁺-influx pathway in many excitable and secretory cells, and direct phosphorylation by different kinases is one of the mechanisms involved in the regulation of its activity. The aim of this study was to evaluate the participation of Ser/Thr kinases and tyrosine kinases (TKs) in depolarization-induced Ca²⁺ influx in the endocrine somatomammotrope cell line GH3. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using a spectrofluorometric method with fura 2-AM, and 12.5 mM KCl (K⁺) was used as a depolarization stimulus. K⁺ induced an abrupt spike (peak) in [Ca²⁺]_i that was abolished in the presence of nifedipine, showing that K⁺ enhances [Ca²⁺]_i, preferably activating L-type Ca²⁺ channels. H89, a selective PKA inhibitor, significantly reduced depolarization-induced Ca²⁺ mobilization in a concentration-related manner when it was applied before or after K⁺, and okadaic acid, an inhibitor of Ser/Thr phosphatases, which has been shown to regulate PKA-stimulated L-type Ca²⁺ channels, increased K⁺-induced Ca²⁺ entry. When PKC was activated by PMA, the K⁺-evoked peak in [Ca²⁺]_i, as well as the plateau phase, was significantly reduced, and chelerythrine (a PKC inhibitor) potentiated the K⁺-induced increase in [Ca²⁺]_i, indicating an inhibitory role of PKC in voltage-dependent Ca²⁺ channel (VDCC) activity. Genistein, a TK inhibitor, reduced the K⁺-evoked increase in [Ca²⁺]_i, but, unexpectedly, the tyrosine phosphatase inhibitor orthovanadate reduced not only basal Ca²⁺ levels but, also, Ca²⁺ influx during the plateau phase. Both results suggest that different TKs may act differentially on VDCC activation. Activation of receptor TKs with epidermal growth factor (EGF) or vascular endothelial growth factor potentiated K⁺-induced Ca²⁺ influx, and AG-1478 (an EGF receptor inhibitor) decreased it. However, inhibition of the non-receptor TK pp60 c-Src enhanced K⁺-induced Ca²⁺ influx. The present study strongly demonstrates that a complex equilibrium among different kinases and phosphatases regulates VDCC activity in the pituitary cell line GH3: PKA and receptor TKs, such as vascular endothelial growth factor receptor and EGF receptor, enhance depolarization-induced Ca²⁺ influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and may therefore participate in the regulation of a plethora of intracellular processes, such as hormone secretion, gene expression, protein synthesis, and cell proliferation, in pituitary cells. phosphatases; protein kinase A; protein kinase C; epidermal growth factor     

Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells

Tunicamycin is anucleoside antibiotic that inhibits protein glycosylation andpalmitoylation. The therapeutic use of tunicamycin is limited inanimals because of its toxic effects, particularly in cerebralvasculature. Tunicamycin decreases palmitoylation of the endothelialisoform of nitric oxide synthase, stimulates nitric oxide synthesis,and increases the concentration of intracellular calcium([Ca²⁺]_i)in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton.FASEB J. 11: A110, 1997). In the present study,we investigated the mechanism by which tunicamycin alters[Ca²⁺]_iusing the Ca²⁺-sensitive dye fura2. We found that tunicamycin increased[Ca²⁺]_iwithout increasing levels of inositol phosphates. When cells wereincubated in the absence of extracellularCa²⁺,[Ca²⁺]_irapidly rose in response to tunicamycin, although a full response wasnot achieved. The pool of intracellularCa²⁺ mobilized by tunicamycinoverlapped with that mobilized by thapsigargin. Extracellular nickelblocked a full response to tunicamycin when cells were incubated in thepresence of extracellular Ca²⁺.The effects of tunicamycin on[Ca²⁺]_iwere partially reversed by washing out the drug, and the remainder ofthe response was inhibited by removing extracellularCa²⁺. These results indicate thattunicamycin mobilizes Ca²⁺ fromintracellular stores in a manner independent of phospholipase Cactivation and increases the influx ofCa²⁺ across the plasma membrane.

     

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

A sustained increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca²⁺ stores by thapsigargin (TG), induced an increase in the [Ca²⁺]_i and cell death. However, the TG-induced death was not related to the [Ca²⁺]_i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca²⁺-dependent anti-apoptotic mechanism. After TG treatment, Ca²⁺-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca²⁺-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca²⁺]_i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca²⁺-dependent MAPK activation counteracts death signaling; removal of Ca²⁺ activated a second caspase-independent death pathway. sarco(endo)plasmic reticulum calcium ion adenosine triphosphatase; cytosolic calcium ion concentration; mitogen-activated protein kinase     

Sperm extract and inositol 1,4,5-triphosphate induce cytosolic calcium rise in the central cell of Torenia fournieri

Microinjection of soluble sperm extract and Calcium Green-1 10 kDa-dextran conjugate (CG-1) into the mature central cell of Torenia fournieri induced a significant rise in cytosolic free calcium concentration ([Ca²⁺]_i). The rise reached a maximum at 20 min after injection and then steadily declined. Nevertheless, a relatively high level of [Ca²⁺]_i was maintained even 40 min after injection. Microinjection of sperm extract of maize into Torenia central cells, however, did not trigger any increase in [Ca²⁺]_i, suggesting the possibility of distinct triggers in different species. We also injected caged inositol 1,4,5-triphosphate (InsP₃) and caged cyclic ADP-ribose (cADPR) into Torenia central cells to compare the pattern of Ca²⁺ rise induced by the sperm extract. The results showed that [Ca²⁺]_i elevation triggered by the release of InsP₃ after photolysis appears much faster than that induced by sperm extract. The increase in [Ca²⁺]_i reached a maximum at 70-80 s and dropped to the resting level within 300 s after photolysis. Microinjection of cADPR, however, did not induce any changes in [Ca²⁺]_i. The results indicate that sperm extract might contain factors triggering the release of Ca²⁺ in the central cell.     

Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+ signaling in bovine vascular endothelial cells

The role of glycolytically generated ATP in Ca²⁺/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca²⁺ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca²⁺ concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + -hydroxybutyrate (-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca²⁺]_i. The rise of [Ca²⁺]_i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca²⁺]_i. In the absence of extracellular Ca²⁺ 2-DG caused an increase in [Ca²⁺]_i, suggesting that inhibition of glycolysis directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. The inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca²⁺ response. Ca²⁺ release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca²⁺]_i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca²⁺]_i. Propagating [Ca²⁺]_i waves were preceded by [Ca²⁺]_i oscillations and small, highly localized elevations of [Ca²⁺]_i (Ca²⁺ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca²⁺ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca²⁺ release was attenuated. Similar results were obtained with pyr + -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg²⁺ concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca²⁺ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP₃R. Ca²⁺/calmodulin-dependent kinase II; glycolysis; calcium regulation     

Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

     

Dual effects of n-alcohols on fluid secretion from guinea pig pancreatic ducts

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [Ca²⁺]_i was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3–10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [Ca²⁺]_i in secretin- or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [Ca²⁺]_i in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3–10 mM concentrations significantly reduced fluid secretion and decreased [Ca²⁺]_i in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [Ca²⁺]_i response to ACh as well as the [Ca²⁺]_i elevation induced by thapsigargin, suggesting that they inhibit Ca²⁺ influx. Removal of extracellular Ca²⁺ reduced [Ca²⁺]_i in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [Ca²⁺]_i in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane Ca²⁺ channel. intracellular calcium; acetylcholine