Inhibitor Of Endoplasmic Reticulum Ca2 Research Articles

Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5-triphosphate receptors in the neurotoxic effects induced by the amyloid-beta peptide.

Studies with in-vitro-cultured neurons treated with amyloid-beta (A beta) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca(2+) homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)-specific apoptotic pathway mediated by caspase-12, which is activated upon the perturbation of ER Ca(2+) homeostasis, may contribute to A beta toxicity. To elucidate the involvement of deregulation of ER Ca(2+) homeostasis in neuronal death induced by A beta peptides, we have performed a comparative study using the synthetic peptides A beta(25-35) or A beta(1-40) and thapsigargin, a selective inhibitor of Ca(2+) uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 microM) increased the intracellular Ca(2+) levels and activated caspase-3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the A beta peptides (A beta(25-35), 25 microM; A beta(1-40), 0.5 microM), we observed a significant increase in [Ca(2+)](i), in caspase-3-like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca(2+) release through ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (IP(3)R) in A beta neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca(2+) release through RyR or IP(3)R, were able to prevent the increase in [Ca(2+)](i) and the activation of caspase-3 and to protect partially against apoptosis induced by treatment with A beta(25-35) or A beta(1-40). In conclusion, our results demonstrate that the release of Ca(2+) from the ER, mediated by both RyR and IP(3)R, is involved in A beta toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to A beta-induced neuronal death. This study suggests that A beta accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca(2+) homeostasis.

Antitumor effects of amlodipine, a Ca2+ channel blocker, on human epidermoid carcinoma A431 cells in vitro and in vivo

Amlodipine, a dihydropyridine Ca2+ channel blocker, is reported to inhibit proliferation of human epidermoid carcinoma A431 cells, and specifically attenuates Ca2+ responses evoked by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. In this study, we further examined the possible mechanism of the antiproliferative action of amlodipine and its antitumor effect on A431 xenografts in nude mice. Amlodipine reduced BrdU incorporation into nucleic acids in serum-starved A431 cells, and the reduction was diminished by uridine 5′-triphosphate (UTP), a phospholipase C (PLC)-linked agonist. Fluorometric measurement of intracellular free Ca2+ concentration revealed that amlodipine blunted the UTP-induced Ca2+ release from the internal Ca2+ stores and consequently Ca2+ influx through Ca2+-permeable channels on the plasma membrane. Although amlodipine alone caused Ca2+ release from thapsigargin-sensitive Ca2+ stores, such an effect was not reproduced by other dihydropyridine Ca2+ channel blockers, including nicardipine and nimodipine, despite their antiproliferative effects in the cells. Daily intraperitoneal administration of amlodipine (10 mg/kg) for 20 days into mice bearing A431 xenografts retarded tumor growth and prolonged the survival of mice. Our results suggest a potential antitumor action for amlodipine in vitro and in vivo, which may be in part mediated by inhibiting Ca2+ influx evoked by the passive depletion of internal Ca2+ stores and by PLC-linked agonist stimulation.

Protective Effect of DY-9760e, a Calmodulin Antagonist, against Neuronal Cell Death

An excessive elevation of intracellular Ca(2+) levels is known to play a key role in the pathological events following cerebral ischemia. DY-9760e, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, is a potent calmodulin antagonist that attenuates brain damage in focal ischemia models. In the present study, we investigated the effect of DY-9760e on neuronal cell death induced by a variety of cell-toxic stimuli that increase intracellular Ca(2+). Cell death was induced by the exposure of primary cultured neurons to excitotoxic agents such as glutamate and N-methyl-D-aspartate, membrane-depolarizing agents such as veratridine and high KCl, or thapsigargin an endoplasmic reticulum Ca(2+)-ATPase inhibitor. Treatment with DY-9760e resulted in a dose-dependent prevention of neuronal cell death elicited by excitotoxicity, voltage-gated channel opening, and inhibition of endoplasmic reticulum Ca(2+)-ATPase. These results indicate that DY-9760e can rescue neurons from various types of cell-toxic stimuli, which may contribute to attenuation of brain injury after cerebral ischemia.

Effect of transforming growth factor-β on calcium homeostasis in prostate carcinoma cells

Ca 2+ plays a fundamental role in the control of a variety of cellular functions, in particular, in energy metabolism and apoptosis. In this study, we show that TGF-β at concentrations of 0.1–1.0 ng/ml transiently increases the level of intracellular Ca 2+ ([Ca 2+] in) in human prostate carcinoma, PC-3U, cells. Experiments with mitochondrial inhibitors (oligomycin and antimycin A) and an inhibitor of endoplasmic reticulum Ca 2+ uptake (BHQ) implied that the effect of TGF-β1 was due to an effect on the mitochondria. TGF-β1 treatment resulted in a decrease in ATP synthesis and to a depolarisation, leading to a release of Ca 2+ from mitochondria and decreased activity of the Ca 2+ pumps. Analysis of the mitochondria within the PC-3U cells by polarography and membrane potential-sensitive dye (Rhodamine 123) confirmed that under these experimental conditions, TGF-β1 inhibited ATP synthesis and depolarised the mitochondria. The results implicate that TGF-β1 affects the function of the mitochondria and may be of significance for the understanding of the proapoptotic effect of TGF-β1 in these cells.

Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death.

We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Ca(2+) chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca(2+) influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger, and KB-R7943, an inhibitor of the Na(+)/Ca(2+) exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Ca(2+ )stores by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca(2+ )release from intracellular Ca(2+ )stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.

Modulation of secretion by the endoplasmic reticulum in mouse chromaffin cells.

The endoplasmic reticulum (ER) has been suggested to modulate secretion either behaving as a Ca2+ sink or as a Ca2+ source in neuronal cells. Working as a Ca2+ sink, through ER-Ca2+ pumping, it may reduce secretion induced by different stimuli. Instead, working as a Ca2+ source through the Ca2+ induced Ca2+ release (CICR) phenomenon, it may potentiate secretion triggered by activation of plasma membrane Ca2+ channels. We have previously demonstrated the presence of CICR in bovine chromaffin cells, but we now find that mouse chromaffin cells almost lack functional caffeine-sensitive ryanodine receptors in the ER and, consistently, no CICR from the ER could be observed. In addition, inhibition of ER Ca2+ pumping with ciclopiazonic acid or thapsigargin strongly stimulated high-K+-evoked catecholamine secretion and cytosolic [Ca2+] ([Ca2+]c) transients. Surprisingly, 5 mm caffeine reduced high-K+-induced [Ca2+]c peaks but considerably potentiated secretion induced by high-K+ stimulation. However, this potentiation was insensitive to ryanodine and additive to that induced by emptying the ER of Ca2+ with thapsigargin, suggesting that it is unrelated to the activation of ryanodine receptors. We conclude that, in mouse chromaffin cells, CICR is not functional and the ER strongly inhibits secretion by acting as a damper of the [Ca2+]c signal.

Acute and chronic alterations in calcium homeostasis in 3-nitropropionic acid-treated human NT2-N neurons

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, induced ATP depletion and both necrosis and apoptosis in human NT2-N neurons. Necrosis occurred predominantly within the first two days, and increased in a dose-dependent fashion with the concentration of 3-NP, whereas apoptosis was observed after 24 h or later at a similar rate in 0.1 mM and 5 mM 3-NP. We focused our efforts on intracellular calcium homeostasis during the first 48 h in 1 mM 3-NP, a period during which 10% of the neurons died by necrosis and 3% by apoptosis. All NT2-N neurons showed a stereotyped [Ca 2+] i rise, from 48±2 to 140±12 nM (mean ±S.E.M.), during the first 2 h in 3-NP. Despite severe ATP depletion, however, [Ca 2+] i remained above 100 nM in only 17% and 25% of the NT2-N neurons after 24 and 48 h in 3-NP, respectively, indicating that most neurons were able to recover from acute [Ca 2+] i rise, and suggesting that chronic [Ca 2+] i dysregulation is a better indicator of subsequent necrosis. Blockade of N-methyl- D-aspartate–glutamate receptor by MK-801 substantially ameliorated 3-NP-induced ATP depletion, subsequent chronic [Ca 2+] i elevation, and survival. Moreover, xestospongin C, an inhibitor of endoplasmic reticulum Ca 2+ release, enhanced the capacity of NT2-N neurons to maintain [Ca 2+] i homeostasis and resist necrosis while subjected to sustained energy deprivation. As far as we know, this report is the first to employ human neurons to study the pathophysiology of 3-NP neurotoxicity.

MERCURIC CHLORIDE ALTERS THE MEMBRANE POTENTIAL AND INTRACELLULAR CALCIUM LEVEL IN MOUSE PANCREATIC ISLET CELLS

In this study, mercuric chloride was applied to the primary cultures of mouse pancreatic islet cells for studying its effects on resting membrane potential and the intracellular free calcium ion concentration ([Ca 2+ ] i ), using the techniques of electrophysiology and fluoi rometry. It was observed that mercuric chloride (1-100 µ M ) caused a rapid and sustained depolarization, and induced a rapid first phase and a large sustained second phase of elevation in fura-2 fluorescence ratio in islet cells. The depolarization and increased [Ca 2+ ] i induced by mercuric chloride could be inhibited by dithiothreitol (a sulf-hydryl-containing reducing agent). Removing Ca 2+ from the external medium inhibited the mercuric chloride-induced elevation of [Ca 2+ ] i . The increased [Ca 2+ ] i may also originate from the endoplasmic reticulum of pancreatic islet cells, since caffeine (an activator of Ca 2+ release from endoplasmic reticulum) and thapsigargin (an inhibitor of endoplasmic reticulum Ca 2+ -ATPase) could antagonize the effect of mercuric chloride. Moreover, in the absence of glucose in the medium, the response of islet cells to mercuric chloride was a rapid first phase of increased [Ca 2+ ] i followed by a small sustained second phase. Readministration of 5 m M glucose was sufficient but transient to restore sustained phase of increased [Ca 2+ ] i . The increase of [Ca 2+ ] i in islet cells induced by a lower concentration of mercuric chloride (5 µ M ) was potentiated in higher glucose (7.5 m M ) medium. Tolbutamide, an inhibitor of the ATP-sensitive K + -channel, could also inhibit the effect of mercuric chloride. These findings suggest that mercuric chloride initially interacts with the sulfhydryl groups of membrane-bound proteins, which may be an ATP-sensitive K + channel, to cause depolarization of the islet cells. This depolarization triggers Ca 2+ influx and then the release of Ca 2+ from the endoplasmic reticulum.

Effect of BCL-2 on Harringtonine-induced apoptosis and intracellular Ca2+ mobilization in human leukemia HL-60 cells

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb Cephalotaxus hainanensis Li, has been used in the clinical treatment of human glanulocytic leukemia and chromic myelocytic leukemia. In this study, we investigated the effect of Bcl-2 on HT-induced apoptosis and Ca2+ mobilization in human leukemia HL-60 cells. 1 μg/ml HT induced the apoptosis of HL-60/Neo cells in a time-dependent manner; while 1 μg/ml HT failed to induce the apoptosis of HL-60/ Bcl-2 cells. HT-, A23187- (a Ca2+ ionophore), Carbonyl cyanide m-chlorophenylhydrazone (CCCP,a specific releaser of Ca2+ from mitochondria) and thapsigargin- (an inhibitor of endoplasmic reticulum Ca2+> -ATPase) induced changes in [Ca2+]i were monitored by using Fluo 3-AM with confocal laser scanning microscopy. The results demonstrated that HL-60 cells with enforced expression of Bcl-2 (HL-60/Bcl-2 cells) had increased Ca2+ permeability and increased intracellular Ca2+ store in comparison with HL-60 cells with negative control vectors (HL-60/Neo cells), suggesting that Bcl-2 might prevent HT-induced apoptosis by increased Ca2+ permeability and increased intracellular Ca2+ buffering capacity.

Modulation of Intracellular Ca2+ Concentration by Vitamin B12 in Rat Thymocytes

ABSTRACTWe have studied several novel effects of vitamin B12 (cyanocobalamin) on cellular Ca2+ homeostasis in rat thymocytes. We determined the effect of various concentrations of vitamin B12 on intracellular Ca2+ concentration ([Ca2+]i) and parameters of Ca2+in signaling using the fluorescent dye Fura-2. The basal [Ca2+]i in Ca2+-containing media was 115 ± 5 nM but in vitamin B12 (10 nM)-treated thymocytes [Ca2+]i was decreased to 60 ± 15 nM (mean ± SEM) during the first 5 min. The decline in [Ca2+]i was accompanied by an increase in the endoplasmic reticulum Ca2+ store, presumably as a result of Ca-ATPase activation. At the same time 100 nM–10 mM B12 induced the accumulation of Ca2+ in mitochondria. Somewhat higher concentrations of B12 (1–10 μM) had no effect on [Ca2+]i. A further increase in B12 concentration with range from 50 μM to 1 mM caused a dose-dependent elevation of [Ca2+]i from the basal level (115 ± 5 nM) up to 200 ± 50 nM in thymocytes, and this elevation was partially blocked in Ca2+-free media. This high concentration of vitamin B12 caused a gradual decrease of endoplasmic reticulum Ca2+ stores by means of Ca-ATPase inhibition. The B12-induced increase in [Ca2+]i was not observed after depletion of intracellular Ca2+ stores, induced by addition of 2′,5′-di(tert-butyl)-1,4-benzohydroquinone (BHQ), an inhibitor of endoplasmic reticulum Ca 2+-ATPase, concanavalin A, or arachidonic acid. These studies show that vitamin B12 regulates [Ca2+]i via several different mechanisms at different B12 concentrations. Participation of G proteins and calmodulin activity in B12-mediated [Ca2+]i increase is discussed.

Failure of endothelin-1 to activate store-operated Ca2+ channels by lack of mobilization from intracellular Ca2+ stores in cultured bovine adrenal chromaffin cells.

We have recently shown that in addition to L-type voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) stimulation opens two types of Ca2+-permeable nonselective cation channels [designated nonselective cation channel-1 (NSCC-1) and NSCC-2]. However, in this Ca2+ entry, the involvement of store-operated Ca2+ channel (SOCC), which is suggested to exist in chromaffin cells, was unclear. Those NSCCs as well as SOCC can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. To clarify whether SOCC should actually exist and play a role in Ca2+ entry in chromaffin cells stimulated with ET-1, we examined the effects of removal of extracellular Ca2+, thapsigargin (TG, an inhibitor of endoplasmic reticulum Ca2+-ATPase), LOE 908 and SK&F 96365 on cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured bovine adrenal chromaffin cells. After the cells were exposed to Ca2+-free medium followed by exposure to TG to deplete Ca2+ from the intracellular Ca2+ store, restoration of extracellular Ca2+ caused a gradual increase in [Ca2+]i (to about 200% of control). The increase was unaffected by LOE 908, but completely abolished by SK&F 96365. In the Ca2+-free medium, no increase in [Ca2+]i by ET-1 was observed, but the subsequent restoration of extracellular Ca2+ induced a rapid increase in [Ca2+]i (to the same level of [Ca2+]i as that evoked by ET-1 in the normal medium (1.0 mM Ca2+)). Since SK&F 96365 is also a blocker of SOCC, these results indicate that in bovine adrenal chromaffin cells, Ca2+ entry through SOCC (Ca2+ influx through the capacitative Ca2+ entry system) occurs but is comparably weak, and that it virtually does not work on the stimulation of ET-1.

Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca2+-ATPase: relationship between global protein synthesis and expression and translation of individual genes

In the physiological state, there appears to be a regulatory link between endoplasmic reticulum (ER) Ca(2+) homoeostasis and the initiation of neuronal protein synthesis. Exposing neuronal cell cultures to thapsigargin (Tg), an irreversible inhibitor of sarcoplasmic/ER Ca(2+)-ATPase (SERCA), induced an almost complete suppression of protein synthesis, which recovered to approx. 60% of control 24 h after Tg exposure. This is an experimental model where the regulatory link between the initiation of protein synthesis and ER Ca(2+) homoeostasis recovers, despite an irreversible suppression of SERCA activity [Doutheil, Treiman, Oschlies and Paschen (1999) Cell Calcium 25, 419--428]. The model was used to investigate the relationship between transcription and translation of various stress genes that respond to conditions causing graded suppression of protein synthesis. Expression patterns revealed three groups of genes. The mRNA levels of genes responding to conditions of ER stress (grp78, grp94, gadd34 and gadd153) were increased up to 200-fold after Tg exposure, whereas those coding for ER-resident proteins (SERCA 2b and Bcl-2) were increased up to 6-fold in treated cultures, and those coding for cytoplasmic proteins (heat-shock protein 70 and p67) were increased only 2--4-fold. Analysis of translation of these mRNAs suggests an imbalance in the synthesis of apoptosis-inducing (GADD153) and tolerance-activating (GRP78 and Bcl-2) proteins after blocking of the ER Ca(2+) pump. The observation that the relationship between Tg-induced changes in mRNA and protein levels varied considerably for the various genes studied implies that translation of the respective transcripts is differently regulated under conditions causing graded suppression of global protein synthesis. Detailed analysis of the response of neuronal cells to transient disturbance of ER Ca(2+) homoeostasis may help to elucidate the mechanisms underlying neuronal cell injury in those neurological disorders in which an impairment of ER function is thought to contribute to the pathological process of deterioration.

Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca2+-ATPase: relationship between global protein synthesis and expression and translation of individual genes

In the physiological state, there appears to be a regulatory link between endoplasmic reticulum (ER) Ca2+ homoeostasis and the initiation of neuronal protein synthesis. Exposing neuronal cell cultures to thapsigargin (Tg), an irreversible inhibitor of sarcoplasmic/ER Ca2+-ATPase (SERCA), induced an almost complete suppression of protein synthesis, which recovered to approx. 60% of control 24h after Tg exposure. This is an experimental model where the regulatory link between the initiation of protein synthesis and ER Ca2+ homoeostasis recovers, despite an irreversible suppression of SERCA activity [Doutheil, Treiman, Oschlies and Paschen (1999) Cell Calcium 25, 419–428]. The model was used to investigate the relationship between transcription and translation of various stress genes that respond to conditions causing graded suppression of protein synthesis. Expression patterns revealed three groups of genes. The mRNA levels of genes responding to conditions of ER stress (grp78, grp94, gadd34 and gadd153) were increased up to 200-fold after Tg exposure, whereas those coding for ER-resident proteins (SERCA 2b and Bcl-2) were increased up to 6-fold in treated cultures, and those coding for cytoplasmic proteins (heat-shock protein 70 and p67) were increased only 2–4-fold. Analysis of translation of these mRNAs suggests an imbalance in the synthesis of apoptosis-inducing (GADD153) and tolerance-activating (GRP78 and Bcl-2) proteins after blocking of the ER Ca2+ pump. The observation that the relationship between Tg-induced changes in mRNA and protein levels varied considerably for the various genes studied implies that translation of the respective transcripts is differently regulated under conditions causing graded suppression of global protein synthesis. Detailed analysis of the response of neuronal cells to transient disturbance of ER Ca2+ homoeostasis may help to elucidate the mechanisms underlying neuronal cell injury in those neurological disorders in which an impairment of ER function is thought to contribute to the pathological process of deterioration.

Activation of P2Y but not P2X(4) nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts.

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+](i)) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of ([Ca2+](i)) persisted on removal of extracellular Ca2+ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of ([Ca2+](i)). After store depletion in the absence of Ca, addition of Ca led to a rise of ([Ca2+](i)) consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La(3+). In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X(4) nucleotide receptors, but with no rise of ([Ca2+](i)). We conclude that nucleotide-induced elevation of [Ca(2+)](i) in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca2+] control that operates between IP3receptor Ca2+release channels (IP3R) and mitochondrial Ca2+uptake sites, and displays functional similarities to synaptic transmission. Activation of IP3R by IP3is known to evoke quantal Ca2+mobilization that is associated with incremental elevations of mitochondrial matrix [Ca2+] ([Ca2+]m). Here we report that activation of IP3R by adenophostin-A (AP) yields non-quantal Ca2+mobilization in mast cells. We also show that the AP-induced continuous Ca2+release causes relatively small [Ca2+]mresponses, in particular, the sustained phase of Ca2+release is not sensed by the mitochondria. Inhibition of ER Ca2+pumps by thapsigargin slightly increases IP3-induced [Ca2+]mresponses, but augments AP-induced [Ca2+]mresponses in a large extent. In adherent permeabilized cells exposed to elevated [Ca2+], ER Ca2+uptake fails to affect global cytosolic [Ca2+], but attenuates [Ca2+]mresponses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca2+pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca2+uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca2+release through the IP3R or during gradual Ca2+influx to the junction between ER and mitochondria.

Mutual Antagonism of Calcium Entry by Capacitative and Arachidonic Acid-mediated Calcium Entry Pathways

In nonexcitable cells, the predominant mechanism for regulated entry of Ca(2+) is capacitative calcium entry, whereby depletion of intracellular Ca(2+) stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca(2+) entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca(2+) pumps, depletion of Ca(2+) stores, or entry of Ca(2+) through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca(2+) overload.

Thapsigargin induces apoptosis in cultured human aortic smooth muscle cells.

Vascular remodeling is a key feature of many pathologic states, including atherosclerosis, or hypertension. Vascular smooth muscle cells participate in determining the vessel structure by several mechanisms such as cell migration, cell growth, or cell death (necrosis or apoptosis). Here we report that thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ -adenosine triphosphatase (ATPase), is able to induce apoptosis in human vascular smooth muscle cells (HVSMCs). Apoptosis was assessed by three different methods: differential chromatin binding dye staining. cytoplasmic histone-associated DNA fragments detection by enzyme-linked immunosorbent assay (ELISA) and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). When HVSMCs were treated for 1 h with thapsigargin (100 nM-10 microM), there was a concentration-dependent increase in both parameters 24 h after the thapsigargin pulse. When a time-course experiment was performed, both parameters were significantly enhanced from 3 to 6 h after the exposure to thapsigargin. We conclude that thapsigargin promotes apoptosis in HVSMCs, providing a useful tool for the study of programmed cell death in human vascular smooth muscle.

Timing of Ca(2+) release from intracellular stores and the electrical response of Limulus ventral photoreceptors to dim flashes.

Light-induced release of Ca(2+) from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca(2+) indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 microm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Ca(i)) after flashes delivering between 10 and 5 x 10(5) effective photons. Peak Ca(i) increased with flash intensity to 138 +/- 76 microM after flashes delivering approximately 10(4) effective photons, while the latent period of the elevation of Ca(i) fell from approximately 140 to 21 ms. The onset of the light-induced elevation of Ca(i) was always highly correlated with that of the receptor potential. The time for Ca(i) to exceed 2 microM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 +/- 6.4 ms). Ca(i) was also measured during steps of light delivering approximately 10(5) effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Ca(i) were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Ca(i) and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Ca(i) resulted from Ca(2+) release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca(2+) pumps, but not by removal of extracellular Ca(2+) ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca(2+) ions that appears necessary for a highly amplified electrical response to dim flashes.

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria

Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage-independent, non-selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea-pig. Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3-dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

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.