Biomedical subjects

Myung-Suk Kim

Publications and source records attributed to Myung-Suk Kim.

At least 19 recordsLinked to original sources

PubMed2006

ATP6V0C competes with von Hippel-Lindau protein in hypoxia-inducible factor 1alpha (HIF-1alpha) binding and mediates HIF-1alpha expression by bafilomycin A1.

HIF-1alpha not only enables cells to survive under hypoxic conditions but also promotes cell cycle arrest and apoptosis. Therefore, its expression should be controlled at optimal levels in growing tumors. We recently reported that bafilomycin A1 exorbitantly expressed HIF-1alpha and induced the p21(WAF1/Cip1)-mediated growth arrest of tumors (Mol Pharmacol 70:1856-1865, 2006). In the present study, we addressed the mechanism underlying bafilomycin-induced HIF-1alpha expression. Bafilomycin stabilized HIF-1alpha under normoxic conditions without changes in intracellular pH. However, when ATP6V0C, the target protein of bafilomycin, was knocked down, this bafilomycin effect was significantly attenuated. Inversely, ATP6V0C expression increased HIF-1alpha levels in a gene dose-dependent manner. ATP6V0C competed with Von Hippel-Lindau protein in HIF-1alpha binding by directly interacting with HIF-1alpha, which was stimulated by bafilomycin. In confocal images, ATP6V0C was normally present in the cytoplasm but was translocated in company with HIF-1alpha to the nucleus by bafilomycin. The N-terminal end (amino acids 1-16) of HIF-1alpha was identified as the ATP6V0C-interacting motif. These results suggest that ATP6V0C, a novel regulator of HIF-1alpha, mediates HIF-1alpha expression by bafilomycin.

Amino Acid Motifs↗

PubMed2006

Calcineurin-independent inhibition of KV1.3 by FK-506 (tacrolimus): a novel pharmacological property.

The interaction of FK-506 with K(V)1.3, stably expressed in Chinese hamster ovary cells, was investigated with the whole cell patch-clamp technique. FK-506 inhibited K(V)1.3 in a reversible, concentration-dependent manner with an IC(50) of 5.6 microM. Rapamycin, another immunosuppressant, produced effects that were similar to those of FK-506 (IC(50) = 6.7 microM). Other calcineurin inhibitors (cypermethrin or calcineurin autoinhibitory peptide) alone had no effect on the amplitude or kinetics of K(V)1.3. In addition, the inhibitory action of FK-506 continued, even after the inhibition of calcineurin activity. The inhibition produced by FK-506 was voltage dependent, increasing in the voltage range for channel activation. At potentials positive to 0 mV (where maximal conductance is reached), however, no voltage-dependent inhibition was found. FK-506 exhibited a strong use-dependent inhibition of K(V)1.3. FK-506 shifted the steady-state inactivation curves of K(V)1.3 in the hyperpolarizing direction in a concentration-dependent manner. The apparent dissociation constant for FK-506 to inhibit K(V)1.3 in the inactivated state was estimated from the concentration-dependent shift in the steady-state inactivation curve and was calculated to be 0.37 microM. Moreover, the rate of recovery from inactivation of K(V)1.3 was decreased. In inside-out patches, FK-506 not only reduced the current amplitude but also accelerated the rate of inactivation during depolarization. FK-506 also inhibited K(V)1.5 and K(V)4.3 in a concentration-dependent manner with IC(50) of 4.6 and 53.9 microM, respectively. The present results indicate that FK-506 inhibits K(V)1.3 directly and that this effect is not mediated via the inhibition of the phosphatase activity of calcineurin.

Animals↗

PubMed2006

Open channel block of Kv1.3 by rosiglitazone and troglitazone: Kv1.3 as the pharmacological target for rosiglitazone.

The effects of rosiglitazone and troglitazone were examined on cloned Kv1.3 channels stably expressed in Chinese hamster ovary cells using the whole-cell configuration of the patch-clamp technique. Rosiglitazone decreased the Kv1.3 currents and accelerated the decay rate of current inactivation in a concentration-dependent manner with an IC(50) of 18.6 microM. These effects were reversible after washout of the drug. Troglitazone caused the block of Kv1.3 with a similar pattern but was five times more potent than rosiglitazone with an IC(50) of 3.5 microM. The block of Kv1.3 by rosiglitazone and troglitazone was voltage-dependent at a membrane potential coinciding with the activation of the channels. Both drugs decreased the tail current amplitude and slowed the deactivation process of Kv1.3, resulting in a tail crossover phenomenon. These results indicate that rosiglitazone and troglitazone block the open state of Kv1.3 channels, suggesting that it is an important pharmacological target for rosiglitazone as a potent blocker of Kv1.3 channels.

Animals↗

PubMed2006

Bafilomycin induces the p21-mediated growth inhibition of cancer cells under hypoxic conditions by expressing hypoxia-inducible factor-1alpha.

Bafilomycin A1, a macrolide antibiotic isolated from Streptomyces species, has been used as an inhibitor of vacuolar H(+) ATPase (V-ATPase). Bafilomycin has been also evaluated as a potential anticancer agent because it inhibits cell proliferation and tumor growth. Although these anticancer effects of bafilomycin are considered to be attributable to the intracellular acidosis by V-ATPase inhibition, the exact mechanism remains unclear. In the present study, we tested the possibility that bafilomycin targets a tumor-promoting factor, hypoxia-inducible factor-1alpha (HIF-1alpha). Bafilomycin A1 and its analog, concanamycin A, were found to up-regulate HIF-1alpha in eight human cancer cell-lines, and this effect is attributed to inhibited degradation of HIF-1alpha protein. Furthermore, the HIF-1alpha induction by bafilomycin was augmented by hypoxia, which caused a robust induction of p21 and cell cycle arrest in cancer cells. The cell cycle inhibition was shown only in cancer cells expressing both HIF-1alpha and p21. In HIF-1alpha(+/+) or HIF-1alpha(-/-) fibrosarcomas grafted in nude mice, bafilomycin showed the HIF-1alpha-dependent anticancer effect. Based on these results, the exorbitant expression of HIF-1alpha is likely to contribute to the anticancer action of bafilomycin.

Animals↗

PubMed2006

Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene.

Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic beta-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between -73 and -46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5'-GTACGTCA-3') located at -69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at -139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser(133) to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser(133) or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser(133) is necessary for induction of the EGR-1 transcription by exendin-4.

Animals↗

PubMed2006

Desensitization of somatostatin-induced inhibition of low extracellular magnesium concentration-induced calcium spikes in cultured rat hippocampal neurons.

Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg(2+) concentration ([Mg(2+)](o))-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg(2+)](o) to 0.1 mM elicited repetitive [Ca(2+)](i) spikes. These [Ca(2+)](i) spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18-24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca(2+)](i) spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 microM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCepsilon translocation inhibitor peptide (1 microM). In addition, suramin (3 microM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 microM) completely blocked the low [Mg(2+)](o)-induced [Ca(2+)](i) spikes, glutamate-induced [Ca(2+)](i) transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg(2+)](o)-induced [Ca(2+)](i) spikes through the activation of GRK2 and partly a novel PKCepsilon in cultured rat hippocampal neurons.

Action Potentials↗

PubMed2006

Curcumin inhibits hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear translocator: a mechanism of tumor growth inhibition.

Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT), plays a key role in cell survival and angiogenesis in hypoxic tumors, and many efforts have been made to develop anticancer agents that target HIF-1alpha. However, although ARNT is also required for HIF-1 activity, ARNT has been disregarded as a therapeutic target. Curcumin is a commonly used spice and coloring agent with a variety of beneficial biological effects, which include tumor inhibition. In the present study, we tested the possibility that curcumin inhibits tumor growth by targeting HIF-1. The effects of curcumin on HIF-1 activity and expression were examined in cancer cell lines and in xenografted tumors. We found that curcumin inhibits HIF-1 activity and that this in turn down-regulates genes targeted by HIF-1. Moreover, of the two HIF-1 subunits, only ARNT was found to be destabilized by curcumin in several cancer cell types, and furthermore, ARNT expression rescued HIF-1 repression by curcumin. We also found that curcumin stimulated the proteasomal degradation of ARNT via oxidation and ubiquitination processes. In mice bearing Hep3B hepatoma, curcumin retarded tumor growth and suppressed ARNT, erythropoietin, and vascular endothelial growth factor in tumors. These results suggest that the anticancer activity of curcumin is attributable to HIF-1 inactivation by ARNT degradation.

Animals↗

PubMed2006

Interaction of riluzole with the closed inactivated state of Kv4.3 channels.

The effect of riluzole on Kv4.3 was examined using the whole-cell patch-clamp technique. Riluzole inhibited the peak amplitude of Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 115.6 microM. Under control conditions, a good fit for the inactivation of Kv4.3 currents to a double exponential function, with the time constants of the fast component (tau(f)) and the slow component (tau(s)), was obtained. tau(f) was not altered by riluzole at concentrations up to 100 microM, but tau(s) became slower with increasing riluzole concentration, resulting in the crossover of the currents. The inhibition increased steeply with increasing channel activation at more positive potentials. In the full activation voltage range positive to (+)30 mV, however, no voltage-dependent inhibition was found. Riluzole shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. However, the slope factor was not affected by riluzole. The K(i) for riluzole for interacting with the inactivated state of Kv4.3 was estimated from the concentration-dependent shift in the steady-state inactivation curve and was determined to be 1.2 muM. Under control conditions, closed state inactivation was fitted to a single exponential function. Riluzole caused a substantial acceleration in the closed state inactivation. In the presence of riluzole, the recovery from inactivation was slower than under control conditions. Riluzole induced a significant use-dependent inhibition of Kv4.3. These results suggest that riluzole inhibits Kv4.3 by binding to the closed inactivated state of the channels and that the unbinding of riluzole occurs from the closed state during depolarization.

Animals↗

PubMed2006

Serotonin inhibits the induction of NMDA receptor-dependent long-term potentiation in the rat primary visual cortex.

An increase in serotonin [5-hydroxytryptamine (5-HT)] levels in the rat visual cortex is correlated with the developmental decrease in long-term potentiation (LTP), and 5-HT may play an important role in the closure of the critical period by regulating LTP. The effect of 5-HT on the induction of N-methyl-D-aspartate receptor (NMDAR)-dependent and metabotropic glutamate receptor (mGluR)-dependent LTP in visual cortex slices from young rats was investigated. The field potential in layer II/III was recorded by stimulating the underlying layer IV. NMDAR-dependent LTP was induced in slices from 3-week-old rats by theta-burst stimulation (TBS) but not in slices from 5-week-old rats. However, LTP was induced in 5-HT-depleted slices from 5-week-old rats by incubation with para-chloroamphetamine (10 microM, 2 h), a 5-HT-depleting agent. The reinstated LTP in 5-HT-depleted slices was inhibited by the application of D-aminopentanoate, an NMDAR antagonist (50 microM) and 5-HT (10 and 30 microM). In contrast, the induction of mGluR-dependent LTP by weak TBS in disinhibited slices with picrotoxin (1 microM) in the bath was not affected by 5-HT application. The coapplication of 5-HT1A and 5-HT2 receptor agonists inhibited the induction of NMDAR-dependent LTP in 5-HT-depleted slices. 5-HT levels in the visual cortex increased with age. Based on these findings, we conclude that NMDAR-dependent LTP is specifically inhibited by coactivation of 5-HT1A and 5-HT2 receptors with the increase in 5-HT levels in the rat visual cortex at the end of the critical period.

Animals↗

PubMed2006

YC-1 induces S cell cycle arrest and apoptosis by activating checkpoint kinases.

Hypoxia-inducible factor-1alpha (HIF-1alpha) seems central to tumor growth and progression because it up-regulates genes essential for angiogenesis and the hypoxic adaptation of cancer cells, which is why HIF-1alpha inhibition is viewed as a cancer therapy strategy. Paradoxically, HIF-1alpha also leads to cell cycle arrest or the apoptosis of cancer cells. Thus, the possibility cannot be ruled out that HIF-1alpha inhibitors unlock cell cycle arrest under hypoxic conditions and prevent cell death, which would limit the anticancer effect of HIF-1alpha inhibitors. Previously, we reported on the development of YC-1 as an anticancer agent that inhibits HIF-1alpha. In the present study, we evaluated the effects of YC-1 on hypoxia-induced cell cycle arrest and cell death. It was found that YC-1 does not reverse the antiproliferative effect of hypoxia, but rather that it induces S-phase arrest and apoptosis at therapeutic concentrations that inhibit HIF-1alpha and tumor growth; however, YC-1 did not stimulate cyclic guanosine 3',5'-monophosphate production in this concentration range. It was also found that YC-1 activates the checkpoint kinase-mediated intra-S-phase checkpoint, independently of ataxia-telangiectasia mutated kinase or ataxia-telangiectasia mutated and Rad3-related kinase. These results imply that YC-1 does not promote the regrowth of hypoxic tumors because of its cell cycle arrest effect. Furthermore, YC-1 may induce the combined anticancer effects of HIF-1alpha inhibition and cell growth inhibition.

Apoptosis↗

PubMed2006

Spatial profile of back-propagating action potential-evoked Ca2+ transients in basal dendrites.

Calcium influx in basal dendrites evoked by back-propagating action potentials is important for understanding the input-specific modulation of synaptic transmission. Calcium transients evoked by back-propagating action potentials were measured for the basal dendrites of layer II/III pyramidal neurons in visual cortex slices. In contrast to apical dendrites, the peak calcium transients evoked by a single somatic action potential increased with distance from the soma. The peak calcium transients evoked by bursts of back-propagating action potentials (three or five pulses, 20 Hz) were greater than those evoked by a single back-propagating action potential. These different spatial profiles for calcium transients triggered by back-propagating action potentials between apical and basal dendrites suggest that the segregated inputs from brain areas might be modulated in different ways, according to their synaptic location.

Action Potentials↗

PubMed2006

In vivo hepatocyte growth factor gene transfer to bladder smooth muscle after bladder outlet obstruction in the rat: a morphometric analysis.

PURPOSE: We determined whether hepatocyte growth factor gene transfer after partial bladder outlet obstruction would prove effective for decreasing transforming growth factor-beta expression and consequently decreasing collagen deposition in partially obstructed rat bladders. MATERIALS AND METHODS: Ten-week-old male Sprague-Dawley rats were divided into 3 groups of 10 each, including group 1--sham operation, group 2--bladder outlet obstruction for 4 weeks and group 3--hepatocyte growth factor gene transfer after bladder outlet obstruction. Two weeks after the onset of bladder outlet obstruction in group 3 hepatocyte growth factor-liposome complex (50 microg human hepatocyte growth factor cDNA) was injected into the smooth muscle of the rats. RESULTS: We noted no difference between groups 2 and 3 with regard to the ratio of bladder weight to body weight. The ratio in groups 2 and 3 was significantly higher than in group 1 (p = 0.043). The mean percent of collagen area +/- SE was 36.32% +/- 1.83%, 27.90% +/- 2.66% and 8.97% +/- 3.35% in groups 1 to 3, respectively (p <0.05). Relative hepatocyte growth factor and c-met mRNA and protein expression were higher in group 3 than in groups 1 and 2. However, the expression of transforming growth factor-beta1 mRNA and protein was higher in group 2 than in groups 1 and 3. CONCLUSIONS: These findings may imply a possible novel therapeutic strategy against bladder dysfunction arising in patients with bladder outlet obstruction.

Animals↗

PubMed2006

In vivo hyperoxic preconditioning prevents myocardial infarction by expressing bcl-2.

Preconditioning with oxidative stress has been demonstrated in vitro to stimulate the cellular adaptation to subsequent severe oxidative stress. However, it is uncertain whether this preconditioning works in vivo. In the present study, we examined in vivo the beneficial effect of oxidative preconditioning. After rats were pretreated with whole-body hyperoxygenation (100% O(2) at 3 atmosphere for 20 mins, four cycles with 20-min intermission), isolated hearts were subjected to 45-min ischemia followed by 90-min reperfusion. This hyperoxic preconditioning significantly reduced infarct size, cytochrome-c release, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTD nick-end labeling-positive cell frequency in the left ventricle, biphasically with an early (30-min) and a delayed (48-hr) effect after the hyperoxygenation. Mechanistically, the NF-kappaB activity and Bcl-2 expression were enhanced in the hearts, and a NF-kappaB inhibitor, pyrrolidine dithiocarbamate, abolished the Bcl-2 induction as well as the infarct-limiting effect. An antioxidant, N-acetylcysteine, and protein kinase C (PKC) inhibitors chelerythrine and Gö 6983 also blocked the preconditioning effects. These results indicate that hyperoxia induces myocardial tolerance against ischemia-reperfusion injury in association with Bcl-2 induction by NF-kappaB activation through reactive oxygen species and PKC-dependent signaling pathway.

Animals↗

PubMed2005

Amphotericin B blunts erythropoietin response to hypoxia by reinforcing FIH-mediated repression of HIF-1.

Amphotericin B (AmB) is widely used for treating severe systemic fungal infections. However, long-term AmB treatment is invariably associated with adverse effects such as anemia. The erythropoietin (EPO) suppression by AmB has been proposed to contribute to the development of anemia. However, the mechanism whereby EPO is suppressed remains obscure. In this study, we investigated the possibility that AmB inhibits the transcription of the EPO gene by inactivating HIF-1, which is a known key transcription factor and regulator of EPO expression. EPO mRNA levels were markedly attenuated by AmB treatment both in rat kidneys and in Hep3B cells. AmB inactivated the transcriptional activity of HIF-1alpha, but did not affect the expression or localization of HIF-1 subunits. Moreover, AmB was found to specifically repress the C-terminal transactivation domain (CAD) of HIF-1alpha, and this repression by AmB required Asn803, a target site of the factor-inhibiting HIF-1 (FIH); moreover, this repressive effect was reversed by FIH inhibitors. Furthermore, AmB stimulated CAD-FIH interaction and inhibited the p300 recruitment by CAD. We propose that this mechanism underlies the unexplained anemia associated with AmB therapy.

Amphotericin B↗

PubMed2005

Differential responses of two degradation domains of HIF-1alpha to hypoxia and iron deficiency.

HIF-1alpha is a transcription factor involved in the cellular adaptation to either hypoxia or iron deficiency. In the presence of oxygen and iron, proline residues in two degradation domains are modified by HIF-1-prolyl hydroxylases (PHDs), resulting in ubiquitination and degradation of HIF-1alpha. Since both molecular oxygen and iron are elements required for this hydroxylation process, HIF-1alpha might be unmodified and stable in conditions lacking oxygen or iron. If so, two degradation domains may respond to hypoxia and iron-depletion in the same way. In this study, however, we found two degradation domains to differentially regulate the stability of HIF-1alpha. The C-terminal domain responded to both hypoxia and iron-depletion, but the N-terminal domain to only iron-depletion. The deletion or point-mutation of the C-terminal domain blunted the hypoxic induction of HIF-1alpha. However, PHD-silencing siRNAs revealed that two degradation domains were not regulated by different types of PHDs. Both domains were regulated mainly by PHD2. The further mutational analysis demonstrated that the ARD1-acetylated motif near the C-terminal degradation domain (CDD) modulates the oxygen-dependent regulation of HIF-1alpha. The oxygen-dependent HIF-1alpha regulation requiring both proline hydroxylation and lysine acetylation may be more complicated than the iron-dependent regulation requiring only proline hydroxylation.

Cell Line↗

PubMed2005

NOX4 as an oxygen sensor to regulate TASK-1 activity.

When oxygen sensing cells are excited by hypoxia, background K+ currents are inhibited. TASK-1, which is commonly expressed in oxygen sensing cells and makes a background K+ current, is inactivated by hypoxia. Thus TASK-1 is a candidate molecule responsible for hypoxic excitation. However, TASK-1 per se cannot sense oxygen and may require a regulatory protein that can. In the present study, we propose that the NADPH oxidase NOX4 functions as an oxygen-sensing partner and that it modulates the oxygen sensitivity of TASK-1. Confocal imaging revealed the co-localization of TASK-1 and NOX4 in the plasma membrane. In HEK293 cells expressing NOX4 endogenously, the activity of expressed TASK-1 was moderately inhibited by hypoxia, and this oxygen response was significantly augmented by NOX4. Moreover, the oxygen sensitivity of TASK-1 was abolished by NOX4 siRNA and NADPH oxidase inhibitors. These results suggest a novel function for NOX4 in the oxygen-dependent regulation of TASK-1 activity.

Biosensing Techniques↗

PubMed2005

Induction of a SSAT isoform in response to hypoxia or iron deficiency and its protective effects on cell death.

Spermidine/spermine N(1)-acetyltransferase (SSAT) is the key enzyme with regard to the maintenance of intracellular polyamine levels. It is an inducible enzyme, which may participate in adaptive responses to environmental stress. However, little is known regarding its responses to oxygen or nutrient deficiencies. Using microarray assays, we discovered that SSAT was enhanced under both oxygen- and iron-deficient conditions. However, RT-PCR revealed that the SSAT mRNA was not induced; rather, an mRNA variant was newly expressed. In this variant, the splicing-in of 110 bases induces early termination, generating a truncated isoform which lacks catalytic motifs. The variant expression occurs in other cancer cells and was irrelevant to both hypoxia-inducible factor 1 and to the redox state. We attempted to determine its role, using stable cell-lines. The expressed isoform was found to promote cell survival under iron-deficient conditions and blocked the cleavage of poly(ADP-ribose) polymerase. This isoform may contribute to the progression of tumors of a more malignant phenotype under poor conditions and may constitute a potential target for anticancer therapy.

Acetyltransferases↗

PubMed2005

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores.

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.

Adenosine Triphosphate↗