Grape seed proanthocyanidin extract inhibits glutamate-induced cell death through inhibition of calcium signals and nitric oxide formation in cultured rat hippocampal neurons.

BACKGROUND: Proanthocyanidin is a polyphenolic bioflavonoid with known antioxidant activity. Some flavonoids have a modulatory effect on [Ca²âº]i. Although proanthocyanidin extract from blueberries reportedly affects Ca²âº buffering capacity, there are no reports on the effects of proanthocyanidin on glutamate-induced [Ca²âº]i or cell death. In the present study, the effects of grape seed proanthocyanidin extract (GSPE) on glutamate-induced excitotoxicity was investigated through calcium signals and nitric oxide (NO) in cultured rat hippocampal neurons. RESULTS: Pretreatment with GSPE (0.3-10 µg/ml) for 5 min inhibited the [Ca²âº]i increase normally induced by treatment with glutamate (100 µM) for 1 min, in a concentration-dependent manner. Pretreatment with GSPE (6 µg/ml) for 5 min significantly decreased the [Ca²âº]i increase normally induced by two ionotropic glutamate receptor agonists, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). GSPE further decreased AMPA-induced response in the presence of 1 µM nimodipine. However, GSPE did not affect the 50 mM K+-induced increase in [Ca²âº]i. GSPE significantly decreased the metabotropic glutamate receptor agonist (RS)-3,5-Dihydroxyphenylglycine-induced increase in [Ca²âº]i, but it did not affect caffeine-induced response. GSPE (0.3-6 µg/ml) significantly inhibited synaptically induced [Ca²âº]i spikes by 0.1 mM [Mg²âº]o. In addition, pretreatment with GSPE (6 µg/ml) for 5 min inhibited 0.1 mM [Mg²âº]o- and glutamate-induced formation of NO. Treatment with GSPE (6 µg/ml) significantly inhibited 0.1 mM [Mg²âº]o- and oxygen glucose deprivation-induced neuronal cell death. CONCLUSIONS: All these data suggest that GSPE inhibits 0.1 mM [Mg²âº]o- and oxygen glucose deprivation-induced neurotoxicity through inhibition of calcium signals and NO formation in cultured rat hippocampal neurons.

Effects of lobeline, a nicotinic receptor ligand, on the cloned Kv1.5.

The goal of the present study was to examine the effects of lobeline, an agonist at nicotinic receptors, on cloned Kv channels, Kv1.5, Kv3.1, Kv4.3, and human ether-a-gogo-related gene (HERG), which are stably expressed in Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. Whole-cell patch-clamp experiments revealed that lobeline accelerated the decay rate of Kv1.5 inactivation, decreasing the current amplitude at the end of the pulse in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) value of 15.1 µM. Using a time constant for the time course of drug-channel interaction, the apparent association (k(+1)), and dissociation rate (k(-1)) constants were 2.4 µΜ(-1) s(-1) and 40.9 s(-1), respectively. The calculated K(D) was 17.0 µΜ. Lobeline slowed the decay rate of the tail current, resulting in a tail crossover phenomenon. The inhibition of Kv1.5 by lobeline steeply increased at potentials between -10 and +10 mV, which corresponds to the voltage range of channel activation. At more depolarized potentials a weaker voltage dependence was observed (δ=0.26). The voltage dependence of the steady-state activation curve was not affected by lobeline, but lobeline shifted the steady-state inactivation curve of Kv1.5 in the hyperpolarizing direction. Lobeline produced use-dependent inhibition of Kv1.5 at frequencies of 1 and 2 Hz, and slowed the recovery from inactivation. Lobeline also inhibited Kv3.1, Kv4.3, and HERG in a concentration-dependent manner, with IC(50) values of 21.7, 28.2, and 0.34 µM, respectively. These results indicate that lobeline produces a concentration-, time-, voltage-, and use-dependent inhibition of Kv1.5, which can be interpreted as an open-channel block mechanism.

Dapoxetine induces neuroprotective effects against glutamate-induced neuronal cell death by inhibiting calcium signaling and mitochondrial depolarization in cultured rat hippocampal neurons.

Selective serotonin reuptake inhibitors (SSRIs) have an inhibitory effect on various ion channels including Ca2+ channels. We used fluorescent dye-based digital imaging, whole-cell patch clamping and cytotoxicity assays to examine whether dapoxetine, a novel rapid-acting SSRI, affect glutamate-induced calcium signaling, mitochondrial depolarization and neuronal cell death in cultured rat hippocampal neurons. Pretreatment with dapoxetine for 10min inhibited glutamate-induced intracellular free Ca2+ concentration ([Ca2+]i) increases in a concentration-dependent manner (Half maximal inhibitory concentration=4.79µM). Dapoxetine (5µM) markedly inhibited glutamate-induced [Ca2+]i increases, whereas other SSRIs such as fluoxetine and citalopram only slightly inhibited them. Dapoxetine significantly inhibited the glutamate-induced [Ca2+]i responses following depletion of intracellular Ca2+ stores by treatment with thapsigargin. Dapoxetine markedly inhibited the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine-induced [Ca2+]i increases. Dapoxetine significantly inhibited the glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i responses in either the presence or absence of nimodipine. Dapoxetine also significantly inhibited AMPA-evoked currents. However, dapoxetine slightly inhibited N-methyl-D-aspartate (NMDA)-induced [Ca2+]i increases. Dapoxetine markedly inhibited 50mMK+-induced [Ca2+]i increases. Dapoxetine significantly inhibited glutamate-induced mitochondrial depolarization. In addition, dapoxetine significantly inhibited glutamate-induced neuronal cell death and its neuroprotective effect was significantly greater than fluoxetine. These data suggest that dapoxetine reduces glutamate-induced [Ca2+]i increases by inhibiting multiple pathways mainly through AMPA receptors, voltage-gated L-type Ca2+ channels and metabotropic glutamate receptors, which are involved in neuroprotection against glutamate-induced cell death through mitochondrial depolarization.

Type 2 Diabetes-Associated K+ Channel TALK-1 Modulates ß-Cell Electrical Excitability, Second-Phase Insulin Secretion, and Glucose Homeostasis.

Two-pore domain K+ (K2P) channels play an important role in tuning ß-cell glucose-stimulated insulin secretion (GSIS). The K2P channel TWIK-related alkaline pH-activated K2P (TALK)-1 is linked to type 2 diabetes risk through a coding sequence polymorphism (rs1535500); however, its physiological function has remained elusive. Here, we show that TALK-1 channels are expressed in mouse and human ß-cells, where they serve as key regulators of electrical excitability and GSIS. We find that the rs1535500 polymorphism, which results in an alanine-to-glutamate substitution in the C-terminus of human TALK-1, increases channel activity. Genetic ablation of TALK-1 results in ß-cell membrane potential depolarization, increased islet Ca2+ influx, and enhanced second-phase GSIS. Moreover, mice lacking TALK-1 channels are resistant to high-fat diet-induced elevations in fasting glycemia. These findings reveal TALK-1 channels as important modulators of second-phase insulin secretion and suggest a clinically relevant mechanism for rs1535500, which may increase type 2 diabetes risk by limiting GSIS.

Pergolide block of the cloned Kv1.5 potassium channels.

Pergolide mesylate, an ergot-derivative dopamine receptor agonist, is prescribed for the management of patients with Parkinson's disease. Pergolide caused vasoconstriction in a pulmonary artery. Kv1.5 channel is highly expressed in pulmonary arterial smooth muscle cells, where it plays an important role as a determinant of vascular tone. In the present study, we investigated the effects of pergolide on Kv1.5 stably expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. The Kv1.5 block by pergolide was concentration-, time-, voltage-, and use-dependent. Pergolide blocked Kv1.5 currents in a concentration-dependent manner, with an IC(50) value of 15.4 µM and a Hill coefficient of 1.7. The activation and inactivation of Kv1.5 were significantly accelerated by pergolide in a concentration-dependent manner. The apparent association and dissociation rate constants were 0.43 µM(-1) s(-1) and 8.34 s(-1), respectively, with a K (D) value of 19.1 µM. Pergolide slowed deactivation kinetics of Kv1.5, resulting in a tail crossover phenomenon. The block of Kv1.5 by pergolide was voltage-dependent, increasing significantly at test potentials from -10 to +10 mV, whereas the current was reduced slightly with a shallower voltage dependence in the range between +20 and +50 mV (δ = 0.34). There was a significant hyperpolarizing shift in the voltage dependence of steady-state inactivation of Kv1.5. Pergolide produced a use-dependent Kv1.5 block at 1 and 2 Hz, and also slowed the time course for recovery from inactivation. These results suggest that pergolide has an affinity for the open and inactivated states of Kv1.5 channels.

Effects of fluoxetine on cloned Kv4.3 potassium channels.

Fluoxetine is widely used for the treatment of depression. We examined the action of fluoxetine on cloned Kv4.3 stably expressed in CHO cells using the whole-cell patch-clamp technique. Fluoxetine did not significantly produce a reduction in the peak amplitude of Kv4.3, but increased the rate of current inactivation in a concentration-dependent manner. Thus, the effect of fluoxetine on Kv4.3 was measured from the integral of the current during the depolarizing pulse. The integral of Kv4.3 was reduced by fluoxetine in a concentration-dependent manner with an IC50 of 11.8µM. Using first-order kinetics analysis, the apparent association and dissociation rate constants were 1.5µM(-1)s(-1) and 22.2s(-1), respectively, with a K(D) of 14.2µM, similar to the IC50 value calculated from the concentration-response curve. Under control conditions, the inactivation of Kv4.3 was best fit by a biexponential function. The fast and slow time constants were significantly decreased in the presence of fluoxetine. Time-to-peak and activation kinetics were significantly accelerated by fluoxetine. The block of Kv4.3 by fluoxetine became more prominent as the membrane potential became more depolarized, displaying a shallow voltage dependence (δ=0.29) in the full activation voltage range. Fluoxetine did not affect the steady-state inactivation curves, but significantly accelerated the closed-state inactivation of Kv4.3. The block of Kv4.3 by fluoxetine was use-dependent during repetitive stimulation, which explained the slowing of the recovery from inactivation of Kv4.3. Our results indicate that fluoxetine blocks Kv4.3 by preferentially interacting with the open and accelerating closed-state inactivation of the channel.

ATM and GLUT1-S490 phosphorylation regulate GLUT1 mediated transport in skeletal muscle.

OBJECTIVE: The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1. RESEARCH DESIGN AND METHODS: L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity. RESULTS: In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL. CONCLUSIONS: These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction.

Effects of dapoxetine on cloned Kv1.5 channels expressed in CHO cells.

The effects of dapoxetine were examined on cloned Kv1.5 channels stably expressed in Chinese hamster ovary cells using the whole-cell patch clamp technique. Dapoxetine decreased the peak amplitude of Kv1.5 currents and accelerated the decay rate of current inactivation in a concentration-dependent manner with an IC ( 50 ) of 11.6 µM. Kinetic analysis of the time-dependent effects of dapoxetine on Kv1.5 current decay yielded the apparent association (k (+1 )) and dissociation (k (-1 )) rate constants of 2.8 µM(-1) s(-1) and 34.2 s(-1), respectively. The theoretical K ( D ) value, derived by k (-1 )/k (+1 ), yielded 12.3 µM, which was reasonably similar to the IC ( 50 ) value obtained from the concentration-response curve. Dapoxetine decreased the tail current amplitude and slowed the deactivation process of Kv1.5, which resulted in a tail crossover phenomenon. The block by dapoxetine is voltage-dependent and steeply increased at potentials between -10 and +10 mV, which correspond to the voltage range of channel activation. At more depolarized potentials, a weaker voltage dependence was observed (δ=0.31). Dapoxetine had no effect on the steady-state activation of Kv1.5 but shifted the steady-state inactivation curves in a hyperpolarizing direction. Dapoxetine produced a use-dependent block of Kv1.5 at frequencies of 1 and 2 Hz and slowed the time course for recovery of inactivation. These effects were reversible after washout of the drug. Our results indicate that dapoxetine blocks Kv1.5 currents by interacting with the channel in both the open and inactivated states of the channel.

Carvedilol blocks the cloned cardiac Kv1.5 channels in a ß-adrenergic receptor-independent manner.

Carvedilol, a non-selective ß-adrenergic blocker, is widely used for the treatment of angina pectoris and hypertension. We examined the action of carvedilol on cloned Kv1.5 expressed in CHO cells, using the whole-cell patch clamp technique. Carvedilol reduced the peak amplitude of Kv1.5 and accelerated the inactivation rate in a concentration-dependent manner with an IC50 of 2.56 µM. Using a first-order kinetics analysis, we calculated k(+1) = 19.68 µM(-1)s(-1) for the association rate constant, and k(-1) = 44.89 s(-1) for the dissociation rate constant. The apparent K(D) (k(-1)/k(+1)) was 2.28 µM, which is similar to the IC50 value. Other ß-adrenergic blockers (alprenolol, oxprenolol and carteolol) had little or no effect on Kv1.5 currents. Carvedilol slowed the deactivation time course, resulting in a tail crossover phenomenon. Carvedilol-induced block was voltage-dependent in the voltage range for channel activation, but voltage-independent in the voltage range for full activation. The voltage dependences for both steady-state activation and inactivation were unchanged by carvedilol. Carvedilol affected Kv1.5 in a use-dependent manner. When stimulation frequencies were increased to quantify a use-dependent block, however, the block by carvedilol was slightly increased with IC50 values of 2.56 µM at 0.1 Hz, 2.38 µM at 1 Hz and 2.03 µM at 2 Hz. Carvedilol also slowed the time course of recovery from inactivation of Kv1.5. These results indicate that carvedilol blocks Kv1.5 in a reversible, concentration-, voltage-, time-, and use-dependent manner, but only at concentrations slightly higher than therapeutic plasma concentrations in humans. These effects are probably relevant to an understanding of the ionic mechanism underlying the antiarrhythmic property of carvedilol.

Block of cloned Kv4.3 potassium channels by dapoxetine.

Dapoxetine, a short-acting selective serotonin reuptake inhibitor, is widely prescribed for the treatment of patients with premature ejaculation. The effects of dapoxetine were examined on cloned Kv4.3 channels stably expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. Dapoxetine not only reduced the peak amplitude of Kv4.3 currents but also accelerated the decay rate of current inactivation in a concentration-dependent manner. Thus, the concentration-dependent reduction in Kv4.3 was measured from the integral of the current during the depolarizing pulse. Dapoxetine decreased the integral of the Kv4.3 currents over the duration of a depolarizing pulse with an IC(50) of 5.3 µM. Analysis of the time dependence of the block gave estimates of an association rate constant (k(+1)) of 3.9 µM(-1)s(-1) and a dissociation rate constant (k(-1)) of 25.6s(-1). The K(D) (k(-1)/k(+1)) was 6.5 µM, similar to the IC(50) value calculated from the concentration-response curve. The block of Kv4.3 by dapoxetine was highly voltage-dependent at a membrane potential coinciding with the activation of the channels. The additional block by dapoxetine displayed a shallow voltage dependence (δ=0.21) in the full activation voltage range. The steady-state inactivation curves were shifted in the hyperpolarizing direction in the presence of dapoxetine. Dapoxetine also caused a substantial acceleration in closed-state inactivation. Dapoxetine produced a significant use-dependent block, which was accompanied by a delayed recovery from inactivation of Kv4.3 currents. These results indicated that dapoxetine potently blocks Kv4.3 currents by both preferentially binding to the open state of the channels and accelerating the closed-state inactivation. These data could provide insight into the mechanism underlying some of the therapeutic actions of this drug.