Biomedical subjects

Yang-Hyeok Jo

Publications and source records attributed to Yang-Hyeok Jo.

At least 19 recordsLinked to original sources

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

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

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

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↗

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↗

PubMed2004

Inhibitory effects of epicatechin on interleukin-1beta-induced inducible nitric oxide synthase expression in RINm5F cells and rat pancreatic islets by down-regulation of NF-kappaB activation.

Cytokines that are released by infiltrating inflammatory cells around the pancreatic islets are involved in the pathogenesis of type 1 diabetes mellitus. Specifically, interleukin-1beta (IL-1beta) stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction, leading to the beta-cell damage. In activating this pathway, nuclear factor-kappaB (NF-kappaB) plays a crucial role, and many of the IL-1beta-sensitive genes contain NF-kappaB binding sites in their promoter regions. We have recently shown that epicatechin, which is a flavonoid, had a protective effect on pancreatic beta-cells in both streptozotocin-treated rats and islets. In the present study, the effects of epicatechin on IL-1beta-induced beta-cell damage were examined. RINm5F cells and islets were pretreated with epicatechin and next incubated with IL-1beta. The released nitrite, iNOS protein and mRNA expression levels were then measured. IkappaBalpha protein, nuclear translocation of NF-kappaB, and NF-kappaB DNA binding activity were also determined. Following the transient transfection of an iNOS promoter into the cells, the iNOS promoter activity was measured. ATP- or D-glucose-induced insulin release was measured in RINm5F cells and islets, respectively. Epicatechin significantly reduced IL-1beta-induced nitrite production, iNOS protein and mRNA expressions, and it also inhibited IL-1beta-induced IkappaBalpha protein degradation, NF-kappaB activation, and iNOS promoter activity. Epicatechin partly restored the IL-1beta-induced inhibition of insulin release. These results suggest that epicatechin inhibits the IL-1beta-induced iNOS expression by down-regulating NF-kappaB activation, and protecting beta-cells from IL-1beta.

Animals↗

PubMed2004

Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons.

The effects of fluoxetine (Prozac) on the transient A-currents (IA) in primary cultured hippocampal neurons were examined using the whole-cell patch clamp technique. Fluoxetine did not significantly decrease the peak amplitude of whole-cell K+ currents, but it accelerated the decay rate of inactivation, and thus decreased the current amplitude at the end of the pulse. For further analysis, IA and delayed rectifier K+ currents (IDR) were isolated from total K+ currents. Fluoxetine decreased IA (the integral of the outward current) in a concentration-dependent manner with an IC50 of 5.54 microM. Norfluoxetine, the major active metabolite of fluoxetine, was a more potent inhibitor of IA than was fluoxetine, with an IC50 of 0.90 microM. Fluoxetine (3 microM) inhibited IA in a voltage-dependent manner over the whole range of membrane potentials tested. Analysis of the time dependence of inhibition gave estimates of 34.72 microM(-1) s(-1) and 116.39 s(-1) for the rate constants of association and dissociation, respectively. The resulting apparent Kd was 3.35 microM, similar to the IC50 value obtained from the concentration-response curve. In current clamp configuration, fluoxetine (3 microM) induced depolarization of resting membrane potential and reduced the rate of action potential. Our results indicate that fluoxetine produces a concentration- and voltage-dependent inhibition of IA, and that this effect could affect the excitability of hippocampal neurons.

Animals↗

PubMed2003

Fluoxetine blocks cloned neuronal A-type K+ channels Kv1.4.

The effects of fluoxetine were studied on cloned K+ channel Kv1.4 stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell configuration of the patch-clamp technique. Extracellular application of various concentrations of fluoxetine inhibited the amplitude of the peak current of Kv1.4 and accelerated its inactivation time course in a concentration-dependent manner. Thus, fluoxetine decreased Kv1.4 (the integral of the outward current) in a concentration-dependent manner; the IC50 was 33.1 +/- 2.5 microM. The inhibitory effect of fluoxetine was time-dependent. The apparent association (k) and dissociation (l) rate constants measured at +40 mV were 3.5 +/- 0.7 microM-1s-1 and 132.5 +/- 13.3 s-1, respectively. The Kd (= l/k) was 37.9 microM, which was close to the value obtained from the concentration-response curve. The block produced by fluoxetine increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. The fluoxetine block was constant at more depolarized potentials, suggesting that the block by fluoxetine was not voltage dependent. Our data indicate that fluoxetine blocks Kv1.4 channels by preferentially binding to open state.

Animals↗

PubMed2003

Mechanism of block by fluoxetine of 5-hydroxytryptamine3 (5-HT3)-mediated currents in NCB-20 neuroblastoma cells.

The effect of fluoxetine (Prozac) on 5-hydroxytryptamine(3) (5-HT(3))-mediated currents in NCB-20 neuroblastoma cells was examined using the whole-cell patch-clamp technique. Fluoxetine produced a significant reduction of peak amplitude without altering the activation time course of 5-HT(3)-mediated currents. These effects were concentration-dependent, with an IC(50) value of 4.15 microM. No voltage dependence was evident in fluoxetine's block of 5-HT(3)-mediated currents over the entire voltage range tested. The extent of block by pre-application of fluoxetine was significantly greater than that by co-application. Fluoxetine also increased the apparent rate of current desensitization to 5-HT application. Using a first-order kinetics analysis, the open-channel blocking rate constants were 0.06 microM(-1)s(-1) (k(+1), association rate constant) and 0.05 s(-1) (k(-1), dissociation rate constant), with an apparent K(d) (=k(-1)/k(+1)) of 0.83 microM. This value is close to an IC(50) of 1.11 microM obtained from the reduction in tau, the time constant of desensitization. Intracellular application of fluoxetine for long durations had no effect on the amplitude or kinetics of 5-HT(3)-mediated currents. Similarly, norfluoxetine, the major metabolite of fluoxetine, reduced the peak current, and enhanced the rate of current desensitization in a concentration-dependent manner with an IC(50) of 2.66 microM, indicating that norfluoxetine is more potent than fluoxetine in blocking 5-HT(3)-mediated currents. These results indicate that, at clinically relevant concentrations, fluoxetine and its metabolite, norfluoxetine, block 5-HT(3)-mediated currents in NCB-20 neuroblastoma cells.

Cell Line, Tumor↗

PubMed2003

Epigallocatechin-3-gallate increases intracellular [Ca2+] in U87 cells mainly by influx of extracellular Ca2+ and partly by release of intracellular stores.

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [3H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca2+] ([Ca2+]i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca2+]i. The EGCG-induced [Ca2+]i increases were reduced to 20.9% of control by removal of extracellular Ca2+. The increases were also inhibited markedly by treatment with the non-specific Ca2+ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca2+ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca2+-ATPase thapsigargin (1 micro M) also significantly inhibited the EGCG-induced [Ca2+]i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 micro M) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 micro M) had no effect. EGCG increased [3H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 micro M) and flufenamic acid (100 micro M), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca2+]i increase in non-treated and thapsigargin-treated cells but indomethacin (100 micro M) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca2+]i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca2+ and partly by mobilizing intracellular Ca2+ stores by PLC activation. The EGCG-induced [Ca2+]i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.

Calcium↗

PubMed2003

Endogenous somatostatin receptors mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells.

Somatostatin receptors are members of the G-protein-coupled receptor superfamily and exert their principal effects by coupling to inhibitory G-proteins. We used fura-2-based digital calcium imaging and assayed for [3H]inositol phosphates (IPs) to study the effects of somatostatin on intracellular calcium signaling in neuroblastomaxglioma NG108-15 cells. Both somatostatin-14 and octreotide induced concentration-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Thirty-four percent of the cells responded to treatment with 100 nM somatostatin-14. Somatostatin-induced responses were not blocked by the removal of extracellular calcium; instead, they were abolished by pretreatment with 100 nM thapsigargin, an agent that depletes and prevents refilling of intracellular Ca(2+) stores. Pretreatment with the inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist xestospongin C (10 microM) for 20 min inhibited markedly the somatostatin-induced response. Somatostatin (100 nM) increased [3H]IPs formation. U73122 (1 microM), an inhibitor of phospholipase C (PLC), completely blocked the somatostatin-induced [Ca(2+)](i) increases and the formation of [3H]IPs. Pretreatment with pertussis toxin (PTX, 200 ng/ml) for 24 h blocked the somatostatin-induced responses. Thus, we conclude that activation of endogenous somatostatin receptors in NG108-15 cells induces the release of calcium from IP(3)-sensitive intracellular stores through PTX-sensitive G-protein-coupled PLC.

Animals↗

PubMed2003

Protective effects of epicatechin against the toxic effects of streptozotocin on rat pancreatic islets: in vivo and in vitro.

INTRODUCTION: Green tea catechins have diverse pharmacological effects such as anticarcinogenic and antioxidant activities. AIM: To study the protective effects of green tea (-)-epicatechin (EC) against the toxic effects of streptozotocin (STZ), a selective beta cell toxin, on pancreatic islets in vivo and in vitro. METHODOLOGY: Rats were randomly divided into four groups: control, EC (30 mg/kg)-treated, STZ (60 mg/kg)-treated, and EC plus STZ (same doses; EC+STZ)-treated rats. EC was administered twice a day for 6 days, and a single injection of STZ was used. In EC+STZ-treated rats, EC was administered 6 hours prior to STZ since posttreatment with EC had no beneficial effects on fully developed diabetes in our unpublished study. Insulin and insulin mRNA were detected by immunohistochemical analysis and in situ hybridization, respectively, and physiologic parameters including blood glucose concentration were measured daily. Following isolation of the islets, insulin release, nitrite levels, and islet morphology were observed in the four groups: control, EC (0.8 mM)-treated, STZ (5 mM)-treated, and EC+STZ (same doses)-treated islets. RESULTS: In EC+STZ-treated rats, hyperglycemia and weight loss were not observed and islet morphology was well preserved compared with STZ-treated rats. Compared with STZ treatment alone, insulin release was increased and nitrite production was decreased in EC+STZ-treated islets. CONCLUSION: EC appears to be helpful in protecting pancreatic islets against exposure to STZ in both in vivo and in vitro systems.

Animals↗

PubMed2003

Transmodulation between phospholipase D and c-Src enhances cell proliferation.

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

Animals↗

PubMed2003

[Altered cholecystokinin-induced calcium signal in streptozotocin-induced diabetic rat pancreatic acini].

BACKGROUND/AIMS: Pancreatic acini of streptozotocin (STZ)-induced diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion has been closely related to the intracellular calcium concentration ([Ca2(+)](i)) of the acinar cell. In the present study, sequential changes of the intracellular calcium signal which probably underlie the altered enzyme secretion in response to CCK-8 were investigated using pancreatic acini from diabetic rats. METHODS: Diabetic rats were prepared by single intravenous injection of STZ (70 mg/kg). Stimulating experiments with CCK-8 were performed 7 days later. Pancreatic acini were isolated by collagenase digestion. Amylase release and [Ca2(+)](i) were measured by colorimethod and calcium imaging, respectively. The geometry of intracellular calcium signal was analyzed. RESULTS: Normal acini exhibited concentration-dependent [Ca2(+)](i) increase and regular oscillatory calcium signal on CCK-8 stimulation. Amylase release was also concentration-dependent. However, diabetic acini showed significantly less [Ca2(+)](i) increase, prolonged time to peak [Ca2(+)](i), decreased calcium spikes number, and decreased amylase release compared with normal acini. The decreased [Ca2(+)](i) in diabetic acini was restored significantly by insulin treatment. CONCLUSIONS: Relatively decreased amylase release in diabetic pancreatic acini in response to CCK, appears to be associated with altered calcium signal due to insulin deficiency.

Amylases↗

PubMed2002

alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells.

alpha-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Although the function of alpha-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of alpha-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type alpha-synuclein or the mutant forms of alpha-synuclein (A53T, A30P) associated with Parkinson's disease. Tyrosine phosphorylation of alpha-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr(125) to Phe slightly increases inhibitory effect of alpha-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing alpha-synuclein than in control cells. Binding of alpha-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Abeta component of alpha-synuclein. Although biologically important, co-transfection studies indicate that the interaction of alpha-synuclein with PLD does not influence the tendency of alpha-synuclein to form pathological inclusions. These results suggest that the association of alpha-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of alpha-synuclein.

Animals↗