D609-phosphatidylcholine-specific phospholipase C inhibitor attenuates thapsigargin-induced sodium influx in human lymphocytes

Previously, we reported that the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl xanthogenate (D609) potentiates thapsigargin-induced Ca(2+) influx in human lymphocytes. In the present study we examined the effect of D609 on the thapsigargin-induced Na(+) entry. We found that the early phase of the thapsigargin-induced increase in the intracellular Na(+) concentration (approx. 1-2 min after stimulation) was attenuated after preincubation of lymphocytes with D609. By contrast, thapsigargin-induced Na(+) influx was not affected in the presence butan-1-ol, which inhibits phosphatidylcholine-specific phospholipase D (PC-PLD). The thapsigargin-induced Na(+) influx could be mimicked by PC-PLC exogenously added to the lymphocyte suspension, whereas addition of PC-PLD had no effect. In addition, thapsigargin stimulated formation of the physiological PC-PLC products, diacylglycerol. Cell-permeable diacylglycerol analogue, dioctanoyl-glycerol (DOG), produced time- and concentration-dependent increase in the intracellular Na(+) concentration. Both thapsigargin- and DOG-induced Na(+) increases were not affected in the presence of Na(+)/H(+) antiport inhibitor, HOE609, or Na(+)/Ca(2+) antiport inhibitor, dimethylthiourea, as well as in the presence of Co(2+) and Ni(2+), which block store-operated Ca(2+) entry. By contrast, markedly reduced thapsigargin- and DOG-induced Na(+) influx were noted in the presence of flufenamic acid, which blocks the non-selective cation current (I(CRANC)). In conclusion, our results suggest that diacylglycerol released due to the PC-PLC activation contributes to the thapsigargin-induced Na(+) entry.     

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).     

Novel effects of propranolol. Release of internal Ca(2+) followed by activation of capacitative Ca(2+) entry in Madin Darby canine kidney cells

The effect of propranolol on Ca(2+) signalling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Propranolol increased cytosolic free Ca(2+) levels ([Ca(2+)](i)) in a concentration-dependent manner between 0.1 and 1 mM. The response was partly inhibited by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with 0.2 mM propranolol partly inhibited the [Ca(2+)](i) rise induced by 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with thapsigargin abolished propranolol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 0.2 mM propranolol in Ca(2+)-free medium. Propranolol (0.2 mM) inhibited 25% of thapsigargin-induced capacitative Ca(2+) entry. Suppression of 1,4,5-trisphosphate (IP(3)) formation by 2 microM U73122, a phospholipase C inhibitor, did not alter 0.2 mM propranolol-induced internal Ca(2+) release. Propranolol (1 mM) also increased [Ca(2+)](i) in human neutrophils. Collectively, we have found that 0.2 mM propranolol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from thapsigargin-sensitive Ca(2+) stores in an IP(3)-independent manner, followed by Ca(2+) influx from external space. Independently, propranolol was able to inhibit thapsigargin-induced capacitative Ca(2+) entry.     

Thapsigargin decreases the Na(+)- Ca(2+) exchanger mediated Ca(2+) entry in pig coronary artery smooth muscle

Na(+)- Ca(2+) exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca(2+) pool along with the SER Ca(2+) pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca(2+) depletion on NCX-SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na(+)-loaded and then placed in either a Na(+)-containing or in a Na(+)-substituted solution. Subsequently, the difference in Ca(2+) entry between the two groups was examined and defined as the NCX mediated Ca(2+) entry. The NCX mediated Ca(2+) entry in the smooth muscle cells was monitored using two methods: Ca(2+)sensitive fluorescence dye Fluo-4 and radioactive Ca(2+). Ca(2+)-entry was greater in the Na(+)-substituted cells than in the Na(+)-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca(2+) entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na(+)-substituted solution with or without thapsigargin. SER Ca(2+) depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca(2+) entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca(2+) entry may protect the cells against Ca(2+)-overload during ischemia-reperfusion when SERCA2 is known to be damaged.     

Store-operated calcium entry in vascular endothelial cells is inhibited by cGMP via a protein kinase G-dependent mechanism

Store-operated Ca(2+) entry in vascular endothelial cells not only serves to refill the intracellular Ca(2+) stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca(2+) entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca(2+)-ATPase, were used to induce store-operated Ca(2+) entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca(2+) entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 microM) or H-8 (10 microM), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca(2+) entry. Addition of S-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca(2+) influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca(2+)-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca(2+) entry in vascular endothelial cells.     

Role of three isoforms of phospholipase A2 in capacitative calcium influx in human T-cells.

The present study was conducted on human Jurkat T-cell lines in order to elucidate the role of phospholipase A2 in capacitative calcium entry. We have employed thapsigargin (TG) that induces increases in [Ca2+]i by emptying the calcium pool of endoplasmic reticulum, followed by capacitative calcium entry. We designed a Ca2+ free/Ca2+ reintroduction (CFCR) protocol for the experiments, conducted in Ca2+-free medium. By employing CFCR protocol, we observed that addition of exogenous arachidonic acid (AA) stimulated TG-induced capacitative calcium influx. The liberation of endogenous AA and its autocrine action seems to be implicated during TG-induced capacitative calcium influx: TG potentiates the induction of constitutively expressed mRNA of four PLA2 isoforms (type 1B, IV, V, VI), the inhibitors of the three PLA2 isotypes (type 1B, V, VI) inhibit TG-induced release of [3H]AA into the extracellular medium, and finally, these PLA2 inhibitors do curtail TG-stimulated capacitative calcium entry in these cells. These results suggest that stimulation of three isoforms of PLA2 by thapsigargin liberates free AA that, in turn, induces capacitative calcium influx in human T-cells.     

Caffeine activates a mechanosensitive Ca(2+) channel in human red cells

Caffeine is known to activate influx of both mono- and divalent cations in various cell types, suggesting that this xanthine opens non-selective cation channels at the plasma membrane. This possibility was investigated in human erythrocytes, studying the caffeine action on net Ca(2+), Na(+) and K(+) movements in ATP-depleted cells. Whole populations and subpopulations of young and old erythrocytes were employed. Caffeine was tested in the presence of known mechanosensitive channel blockers (Gd(3+), neomycin and amiloride) and ruthenium red as a possible inhibitor. Caffeine enhanced net cation fluxes in a concentration-dependent way. In whole populations, the Ca(2+) entry elicited by 20 mM caffeine was fully suppressed by Gd(3+) (5 microM), amiloride (250 microM) and ruthenium red (100 microM) and partially blocked by neomycin (100 microM). The above blockers also inhibited caffeine-dependent Na(+) entry whilst showing antagonistic effects on the corresponding K(+) efflux. These compounds fully suppressed hypotonically-induced (-35 mOsm/kg) Ca(2+) influx at nearly the same concentrations completely blocking caffeine-stimulated Ca(2+) entry. The effect of inhibitors on Ca(2+) influx in young cells exceeded that in old cells at similar concentrations. The results clearly show that caffeine stimulates a stretch-activated Ca(2+) channel in human red cells and that aged cells are less susceptible to mechanosensitive channel blockers.     

Genistein inhibits lysosomal enzyme release by suppressing Ca2+ influx in HL-60 granulocytes

The tyrosine kinase inhibitor genistein (5-200 microM) suppressed Ca(2+)-dependent fMLP (1 microM) and ATP (100 microM)-induced release of the lysosomal enzyme, beta-glucuronidase from neutrophil-like HL-60 granulocytes. Agonist-induced Ca2+ mobilization resulted from the release of intracellular Ca2+ stores and the influx of extracellular Ca2+. Genistein (200 microM) suppressed fMLP (1 microM) and ATP (100 microM)-induced Ca2+ mobilization, by 30-40%. Ca2+ release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+ (Mn2+) influx. Consistent with these findings, genistein (200 microM) or removal of extracellular Ca2+ (EGTA 1 mM), inhibited Ca(2+)-dependent agonist-induced beta-glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced beta-glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+ (Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+ influx induced by thapsigargin (0.5 microM). These data indicate that agonist-induced Ca2+ influx and store-operated Ca2+ influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL-60 granulocytes.     

Capacitative calcium entry mechanism in porcine oocytes

The presence of the capacitative Ca(2+) entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca(2+)-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca(2+) entry. A similar divalent cation influx could also be detected with the Mn(2+)-quench technique after inositol 1,4,5-triphosphate-induced Ca(2+) release. In both cases, lanthanum, the Ca(2+) permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca(2+) influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca(2+) entry mechanism might help in refilling the intracellular stores after the release of Ca(2+) from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca(2+) entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca(2+) entry mechanism and thus contributes to Ca(2+) influx.     

Protein kinase C activates store-operated Ca(2+) channels in human glomerular mesangial cells

Store-operated Ca(2+) channels (SOC) are expressed in cultured human mesangial cells and activated by epidermal growth factor through a pathway involving protein kinase C (PKC). We used fura-2 fluorescence and patch clamp experiments to determine the role of PKC in mediating the activation of SOC after depletion of internal stores by thapsigargin. The measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) revealed that the thapsigargin-induced Ca(2+) entry pathway was abolished by calphostin C, a protein kinase C inhibitor. The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a Ca(2+) influx that was significantly attenuated by calphostin C and La(3+) but not by diltiazem. Neither PMA nor calphostin C altered the thapsigargin-induced initial transient rise in [Ca(2+)](i). In cell-attached patch clamp experiments, the thapsigargin-induced activation of SOC was potentiated by PMA and abolished by both calphostin C and staurosporine. However, SOC was unaffected by thapsigargin when clamping [Ca(2+)](i) with 1,2-bis (o-Aminophenoxy)ethane-N,N,N',N'tetraacetic acid tetra(acetoxymethyl)ester. In the absence of thapsigargin, PMA and phorbol 12, 13-didecanoate evoked a significant increase in NP(O) of SOC, whereas calphostin C did not affect base-line channel activity. In inside-out patches, SOC activity ran down immediately upon excision but was reactivated significantly after adding the catalytic subunit of 0.1 unit/ml of PKC plus 100 microm ATP. Neither ATP alone nor ATP with heat-inactivated PKC rescued a rundown of SOC. Metavanadate, a general protein phosphatase inhibitor, also enhanced SOC activity in inside-out patches. Bath [Ca(2+)] did not significantly affect the channel activity in inside-out patch. These results indicate that the depletion of Ca(2+) stores activates SOC by PKC-mediated phosphorylation of the channel proteins or a membrane-associated complex.     

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

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

Superoxide anion impairs contractility in cultured aortic smooth muscle cells

We examined the effects of superoxide anion (O) generated by xanthine plus xanthine oxidase (X/XO) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and muscle contractility in cultured bovine aortic smooth muscle cells (BASMC). Cells were grown on collagen-coated dish for the measurement of [Ca(2+)](i). Pretreatment with X/XO inhibited ATP-induced Ca(2+) transient and Ca(2+) release-activated Ca(2+) entry (CRAC) after thapsigargin-induced store depletion, both of which were reversed by superoxide dismutase (SOD). In contrast, Ca(2+) transients induced by high-K(+) solution and Ca(2+) ionophore A-23187 were not affected by X/XO. BASMC-embedded collagen gel lattice, which was pretreated with xanthine alone, showed contraction in response to ATP, thapsigargin, high-K(+) solution, and A-23187. Pretreatment of the gel with X/XO impaired gel contraction not only by ATP and thapsigargin, but also by high-K(+) solution and A-23187. The X/XO-treated gel showed normal contraction; however, when SOD was present during the pretreatment period. These results indicate that O(2)(-) attenuates smooth muscle contraction by impairing CRAC, ATP-induced Ca(2+) transient, and Ca(2+) sensitivity in BASMC.     

Versatile regulation of cytosolic Ca2+ by vanilloid receptor I in rat dorsal root ganglion neurons

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca(2+). The VR1 agonist capsaicin induced Ca(2+) mobilization from intracellular stores in the absence of extracellular Ca(2+), and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca(2+) mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca(2+) release receptor. The main part of the capsaicin-releasable Ca(2+) store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca(2+)-ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca(2+) store. In addition, VR1 was observed to behave as a store-operated Ca(2+) influx channel. In DRG neurons, capsazepine attenuated Ca(2+) influx following thapsigargin-induced Ca(2+) store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca(2+) influx and inward currents following Ca(2+) store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca(2+) with the plasma membrane-associated form behaving as a store-operated Ca(2+) influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca(2+) release receptor in sensory neurons.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.     

Induction of a sodium ion influx by progesterone in human spermatozoa

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.     

Effect of thapsigargin on Ca²+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2?](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2?-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 μM increased [Ca2?](i) in a concentration-dependent fashion. The Ca2? signal was reduced partly by removing extracellular Ca2? indicating that Ca2? entry and release both contributed to the [Ca2?](i) rise. This Ca2? influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2? channels or by modulation of protein kinase C activity. In Ca2?-free medium, pretreatment with the endoplasmic reticulum Ca2? pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2? release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2?](i) rise, suggesting that thapsigargin released Ca2? from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca2?](i) rise. At concentrations of 1-10 μM, thapsigargin induced cell death that was partly reversed by chelation of Ca2? with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca2?](i) rises by causing phospholipase C-independent Ca2? release from the endoplasmic reticulum and Ca2? influx via phospholipase A2-sensitive Ca2? channels. Thapsigargin also induced cell death via Ca2?-dependent pathways and Ca2?-independent apoptotic pathways.     

Epidermal growth factor-induced depletion of the intracellular Ca2+ store fails to activate capacitative Ca2+ entry in a human salivary cell line

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca(2+) ([Ca(2+)](i)). Generally, agonist-mediated Ca(2+) mobilization involves both Ca(2+) release from internal stores and Ca(2+) influx activated by store depletion (i.e. capacitative or store-operated Ca(2+) influx). However, the role of capacitative Ca(2+) entry in EGF-mediated Ca(2+) mobilization is still largely unknown. In this study, we compared [Ca(2+)](i) signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca(2+) entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca(2+)](i) signal without a plateau phase in the presence of extracellular Ca(2+) and also failed to accelerate Mn(2+) entry. Repletion of extracellular Ca(2+) to cells stimulated with EGF in the absence of Ca(2+) elicited an increase in [Ca(2+)](i), indicating that EGF indeed stimulates Ca(2+) influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca(2+) release. Interestingly, the phospholipase C inhibitor completely inhibited Ca(2+) release induced by both EGF and carbachol and also reduced Ca(2+) influx responsive to carbachol but had no effect on Ca(2+) influx induced by EGF. EGF-induced Ca(2+) influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca(2+) influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca(2+) influx responsive to carbachol but did not increase EGF-activated Ca(2+) influx. Both EGF and carbachol depleted internal Ca(2+) stores. Our results demonstrate that EGF-induced Ca(2+) release from internal stores does not activate capacitative Ca(2+) influx. Rather, EGF stimulates Ca(2+) influx via a mechanism distinct from capacitative Ca(2+) influx induced by carbachol and Tg.     

Nitric oxide inhibition of cAMP synthesis in parotid acini: regulation of type 5/6 adenylyl cyclase

The nitric oxide (NO) donor, GEA 3162, inhibited isoproterenol-induced cyclic AMP (cAMP) accumulation in a concentration- and time-dependent manner in mouse parotid acini; SIN-1 mimicked these effects. Inhibition of stimulated cAMP accumulation was independent of phosphodiesterase activity. GEA 3162 also inhibited forskolin-induced cAMP accumulation. Removal of extracellular Ca(2+), addition of La(3+), or the calmodulin (CaM) inhibitor, calmidazolium, did not prevent the NO-mediated response, and addition of the soluble guanylyl inhibitor, ODQ, did not reverse GEA 3162-induced inhibition of cAMP accumulation. GEA 3162 also inhibited adenylyl cyclase in vitro independently of Ca(2+)/CaM. Further studies revealed that the NO synthase (NOS) inhibitor, 7-nitroindazole (7-NI), reduced significantly thapsigargin-induced Ca(2+) release and capacitative Ca(2+) entry and reversed thapsigargin inhibition of the AC Type 5/6 isoform (AC5/6). Data suggest that NO produced endogenously has dual effects on cAMP accumulation in mouse parotid acini, an inhibitory effect on AC activity and a modulatory effect on capacitative Ca(2+) entry resulting in AC5/6 inhibition.     

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely, E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium.     

Mechanisms of the lysophosphatidic acid-induced increase in [Ca(2+)](i) in skeletal muscle cells.

Although lysophosphatidic acid (LPA) is known to increase intracellularfree calcium concentration ([Ca(2+)](i)) in different cell types, the effect of LPA on the skeletal muscle cells is not known. The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells. LPA induced a concentration and time dependent increase in [Ca(2+)](i), which was inhibited by VPC12249, VPC 32183 and dioctanoyl glycerol pyrophosphate, LPA1/3 receptor antagonists. Pertussis toxin, a G(i) protein inhibitor, also inhibited the LPA-induced increase in [Ca(2+)](i). Inhibition of tyrosine kinase activities with tyrphostin A9 and genistein also prevented the increase in [Ca(2+)](i) due to LPA. Likewise, wortmannin and LY 294002, phosphatidylinositol 3-kinase (PI3-K) inhibitors, inhibited [Ca(2+)](i) response to LPA. The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365. However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA. Our data suggest that the LPA-induced increase in [Ca(2+)](i) might occur through G(i)-protein coupled LPA(1/3) receptors that may be linked to tyrosine kinase and PI3-K, and may also involve the Na(+)-Ca(2+) exchanger as well as the ROC. In addition, LPA stimulated C2C12 cell proliferation via PI3-K. Thus, LPA may be an important phospholipid in the regulation of [Ca(2+)](i) and growth of skeletal muscle cells.