Ischemia deteriorates the spike encoding of rat cerebellar Purkinje cells by raising intracellular Ca2+

Ischemia-induced excitotoxicity at cerebellar Purkinje cells is presumably due to a persistent glutamate action. To the fact that they are more vulnerable to ischemia than other glutamate-innervated neurons, we studied whether additional mechanisms are present and whether cytoplasm Ca²⁺ plays a key role in their ischemic excitotoxicity. Ischemic changes in the excitability of Purkinje cells were measured by whole-cell recording in cerebellar slices of rats with less glutamate action. The role of cytoplasm Ca²⁺ was examined by two-photon cellular imaging and BAPTA infusion in Purkinje cells. Lowering perfusion rate to cerebellar slices deteriorated spike timing and raised spike capacity of Purkinje cells. These changes were associated with the reduction of spike refractory periods and threshold potentials, as well as the loss of their control to spike encoding. Ischemia-induced functional deterioration at Purkinje neurons was accompanied by cytoplasm Ca²⁺ rise and prevented by BAPTA infusion. Therefore, the ischemia destabilizes the spike encoding of Purkinje cells via raising cytoplasm Ca²⁺ without a need for glutamate, which subsequently causes their excitotoxic death.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Regulation of Ca2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca2+-ATPase type 2

Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk.     

Neuroprotective effects of ginseng saponins against L-type Ca2+ channel-mediated cell death in rat cortical neurons

In the present study, we have examined any possible involvement of L-type Ca²⁺ channels in ginseng-mediated neuroprotective actions. Exposure to a 50 mM KCl (high-K) produced neuronal cell death, which was blocked by a selective L-type Ca²⁺ channel blocker in cultured cortical neurons. When cultured cells were co-treated with ginseng total saponin (GTS) and high-K, GTS reduced high-K-induced neuronal death. Using Ca²⁺ imaging techniques, we found that GTS inhibited high-K-mediated acute and long-term [Ca²⁺]_i changes. These GTS-mediated [Ca²⁺]_i changes were diminished by nifedipine. Furthermore, GTS-mediated effects were also diminished by a saturating concentration of Bay K (10 μM). After confirming the protective effect of GTS using a TUNEL assay, we found that ginsenosides Rf and Rg₃ are active components in ginseng-mediated neuroprotection. These results suggest that inhibition of L-type Ca²⁺ channels by ginseng could be one of the mechanisms for ginseng-mediated neuroprotection in cultured rat cortical neurons.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

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.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Investigation of carvedilol-evoked Ca2+ movement and death in human oral cancer cells

The effect of carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was decreased by 50% by removing extracellular Ca²⁺. Carvedilol-induced Ca²⁺ entry was not affected by the store-operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin did not change carvedilol-induced [Ca²⁺]_i rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²⁺]_i release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²⁺]_i rise. Carvedilol at 5–50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²⁺ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²⁺]_i rise by causing phospholipase C-independent Ca²⁺ release from mitochondria and non-endoplasmic reticulum stores, and Ca²⁺ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca²⁺-independent manner that involved apoptosis.     

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.     

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.     

Mitochondrial free Ca2+ concentration in living cells

Evidence has accrued during the past two decades that mitochondrial Ca²⁺ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca²⁺ transport systems might be able to relay the changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) become essential. In this article, we review the results from recent studies attempting to measure [Ca²⁺]_m in living cells. In addition, the significance of each study is discussed.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Ca2+ in quality control

Calcium (Ca²⁺) has long been known as a ubiquitous intracellular second messenger, exploited by cells to control processes as diverse as development, proliferation, learning, muscle contraction and secretion. The spatial and temporal patterns of these Ca²⁺-associated signals, as well as their amplitude, is precisely controlled to create gradients of the ion, varying considerably depending on cell type and function. Tuning of intracellular Ca²⁺ is achieved in part by the buffering role of mitochondria, whose unperturbed function is essential for maintaining cellular energy balance. Quality of mitochondria is ensured by the process of targeted autophagy or mitophagy, which depends on a molecular cascade driving the catabolic process of autophagy toward damaged or deficient organelles for elimination via the lysosomal pathway. Nonspecific and targeted autophagy are highly regulated processes fundamental to cell growth and tissue homeostasis, allowing resources to be reallocated in nutrient-deprived cells as well as being instrumental in the repair of damaged organelles or the elimination of those in excess. Given the role of Ca²⁺ signaling in many fundamental cellular processes requiring precise regulation, the involvement of Ca²⁺ in autophagy is still somewhat ill-defined, and only in the past few years has evidence emerged linking the two. This mini-review aims to summarize recent work implicating Ca²⁺ as an important regulator of autophagy, outlining a role for Ca²⁺ that may be even more critical in the regulation of targeted mitochondrial autophagy.     

The mechanism of protriptyline-induced Ca2+ movement and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Abstract

Protriptyline, a tricyclic anti-depressant, is used primarily to treat the combination of symptoms of anxiety and depression. However, the effect of protriptyline on prostate caner is unknown. This study examined whether the anti-depressant protriptyline altered Ca²⁺ movement and cell viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Protriptyline evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Protriptyline-evoked Ca²⁺ entry was inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365), protein kinase C activator (phorbol 12-myristate 13 acetate, PMA) and protein kinase C inhibitor (GF109203X). Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydr-oquinone (BHQ) in Ca²⁺-free medium inhibited 60% of protriptyline-evoked [Ca²⁺]_i rises. Conversely, treatment with protriptyline abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 suppressed 50% of protriptyline-evoked [Ca²⁺]_i rises. At concentrations of 50–70?µM, protriptyline decreased cell viability in a concentration-dependent manner; which were not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in PC3 cells, protriptyline evoked [Ca²⁺]_i rises by inducing phospholipase C-associated Ca²⁺ release from the endoplasmic reticulum and other stores, and Ca²⁺ influx via protein kinase C-sensitive store-operated Ca²⁺ channels. Protriptyline caused cell death that was independent of [Ca²⁺]_i rises.