Dopamine Receptor Supports the Potentiation of Intrinsic Excitability and Synaptic LTD in Temporoammonic-CA1 Synapse

Dopaminergic projection to the hippocampus from the ventral tegmental area or locus ceruleus has been considered to play an essential role in the acquisition of novel information. Hence, the dopaminergic modulation of synaptic plasticity in the hippocampus has been widely studied. We examined how the D1 and D2 receptors influenced the mGluR5-mediated synaptic plasticity of the temporoammonic-CA1 synapses and showed that the dopaminergic modulation of the temporoammonic-CA1 synapses was expressed in various ways. Our findings suggest that the dopaminergic system in the hippocampal CA1 region regulates the long-term synaptic plasticity and processing of the novel information.

Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic beta-cells

AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic beta-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic beta-cells.

Selective Cytotoxic Mechanism of Homocysteine to Motor Neuronal Cells Expressing the Mutant Cu,Zn-superoxide Dismutase

BACKGROUND: Mutations in Cu, Zn-superoxide dismutase (SOD1) cause about 20% of familial amyotrophic lateral sclerosis (FALS) cases. The mechanism of late-onset disease manifestation despite the innate mutation has no clear explanation. The relationship between homocysteine (HC) and amyotrophic lateral sclerosis (ALS) has not been investigated fully, in spite of the similarity in their pathogenesis. METHODS: We investigated the effect of HC on the motor neuronal cell-line (VSC4.1) transfected with SOD1 of either wild-type or mutant forms (G93A and A4V) using various methods including the MTT assay for the cytotoxic assay, the immunocytochemical staining using anti-SOD1 for the aggregation of SOD1, the western blotting using anti-nitrotyrosine and anti-DNPH for the oxidative protein damage, and the measurement of the intracellular Ca2+ concentration using Fura2-AM. RESULTS: In the MTT assay, the HC induced significant cytotoxicity in the mutants, as compared with wild-type. This HC-induced cytotoxicity was inhibited by the trolox and the bathocuproinedisulfonate (BC). HC increased the carbonylation and nitrosylation of the mutant proteins. HC also increased significant SOD1-aggregation in mutants. This HC-induced SOD1-aggregation in mutants was inhibited by trolox, N-nitro-L-arginine methyl ester, BC, and z-VAD-FMK. HC did not change the intracellular concentration of Ca2+ in the mutants compared with the wild-type. CONCLUSIONS: The authors showed that the vulnerability of the SOD1 mutant motor neuronal cells to HC involves the copper-mediated oxygen radical toxicity, and that HC may be a lifelong precipitating factor in some forms of FALS, suggesting a possible treatment modality with vitamin supplements.

Two Components of Voltage Dependent Outward K+ Current in Isolated Human Atrial Myocytes

BACKGROUND: The cardiac electrophysiological characteristics differ significantly among mammalian species or among various disease processes. However, difficulties in the procedures for harvesting and isolating tissue have precluded studies using human cardiac specimens. METHODS: The outward K+ -currents were recorded in human atrial myocytes isolated from patients undergoing open heart surgery. The electrophysiological characteristics of the voltage-dependent outward currents were investigated using a whole-cell patchclamp technique. RESULTS: Using depolarizing step pulses, the transient outward currents were activated within 10 msec, which slowly inactivated thereafter. After inactivation, the sustained components of the outward currents remained for up to 5.0 seconds of depolarizing step pulses. While the inactivating component was almost completely inactivated at potentials >+30 mV, the non-inactivating component showed only 10-15% inactivation. The non-inactivating component was highly sensitive to 4-AP and was inhibited by >80% at a concentration of 0.2 mM, while the inactivating component was inhibited by only 25%. The delayed rectifier potassium currents were not recorded. The ratios of the amplitudes of the inactivating and non-inactivating components varied. CONCLUSION: Two components of the voltage dependent outward K+ currents in human cardiac tissue were identified, which could be separated according to their kinetic and pharmacologic properties.

Role of Stretch-Activated Channels in Stretch-Induced Changes of Electrical Activity in Rat Atrial Myocytes

We developed a cardiac cell model to explain the phenomenon of mechano-electric feedback (MEF), based on the experimental data with rat atrial myocytes. It incorporated the activity of ion channels, pumps, exchangers, and changes of intracellular ion concentration. Changes in membrane excitability and Ca2+ transients could then be calculated. In the model, the major ion channels responsible for the stretch-induced changes in electrical activity were the stretch-activated channels (SACs). The relationship between the extent of stretch and activation of SACs was formulated based on the experimental findings. Then, the effects of mechanical stretch on the electrical activity were reproduced. The shape of the action potential (AP) was significantly changed by stretch in the model simulation. The duration was decreased at initial fast phase of repolarization (AP duration at 20% repolarization level from 3.7 to 2.5 ms) and increased at late slow phase of repolarization (AP duration at 90% repolarization level from 62 to 178 ms). The resting potential was depolarized from -75 to -61 mV. This mathematical model of SACs may quantitatively predict changes in cardiomyocytes by mechanical stretch.

Modulation of L-type Ca2+ channel currents by various protein kinase activators and inhibitors in rat clonal pituitary GH3 cell line

L-type Ca2+ channels play an important role in regulating cytosolic Ca2+ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type Ca2+ channel currents in rat pituitary GH3 cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the Ba2+ current through the L-type Ca2+ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the Ba2+ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced Ba2+ currents. The above results show that the L-type Ca2+ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in GH3 cells may be attributable, at least in part, to their effect on L-type Ca2+ channels.

Effect of propofol, an intravenous anesthetic agent, on KATP channels of pancreatic beta-cells in rats

ATP-sensitive potassium channels (KATP channels) play an important role in insulin secretion from pancreatic beta cells. We have investigated the effect of propofol on KATP channels in cultured single pancreatic beta cells of rats. Channel activity was recorded from membrane patches using the patch-clamp technique. In the inside-out configuration bath-applied propofol inhibited the KATP channel activities in a dose-dependent manner. The half-maximal inhibition dose (ED50) was 48.6+/-8.4 micrometer and the Hill coefficient was 0.73 0.11. Single channel conductance calculated from the slope of the relationship between single channel current and pipette potential (+20~+100 mV) was not significantly altered by propofol (control: 60.0+/-2.7 pS, 0.1 mM propofol: 58.7+/-3.5 pS). However, mean closed time was surely increased. Above results indicate that propofol blocks the KATP channels in the pancreatic beta cells in the range of its blood concentrations during anesthesia, suggesting a possible effect on insulin secretion and blood glucose level.

The role of K+ channels on spontaneous action potential in rat clonal pituitary GH3 cell line

The types of K+ channel which determine the pattern of spontaneous action potential (SAP) were investigated using whole-cell variation of patch clamp techniques under current- and voltage-clamp recording conditions in rat clonal pituitary GH3 cells. Heterogeneous pattern of SAP activities was changed into more regular mode with elongation of activity duration and afterhyperpolarization by treatment of TEA (10 mM). Under this condition, exposure of the class III antiarrhythmic agent E-4031 (5 micrometer) to GH3 cells hardly affected SAP activities. On the other hand, the main GH3 stimulator thyrotropin-releasing hormone (TRH) still produced its dual effects (transient hyperpolarization and later increase in SAP frequency) in the presence of TEA. However, addition of BaCl2 (2 mM) in the presence of TEA completely blocked SAP repolarization process and produced membrane depolarization in all tested cells. This effect was observed even in TEA-untreated cells and was not mimicked by higher concentration of TEA (30 mM). Also this barium-induced membrane depolarization effect was still observed after L-type Ca2+ channel was blocked by nicardipine (10 micrometer). These results suggest that barium-sensitive current is important in SAP repolarization process and barium itself may have some depolarizing effect in GH3 cells.

The effects of intracellular monocarboxylates on the ATP-sensitive potassium channels in rabbit ventricular myocytes

A regulating mechanism of the ATP-sensitive potassium channels (KATP channels) is yet to fully explained. This study was carried out to investigate the effects of intracellular application of monocarboxylates (acetate, formate, lactate, and pyruvate) on KATP channels in isolated rabbit ventricular myocytes. Single channel currents of KATP channels were recorded using the excised inside-out or permeabilized attached (open-cell) patch-clamp technique at room temperature. Intracellular application of acetate, formate and pyruvate led to an inhibition of channel activity, whereas intracellular application of lactate increased channel activity. These effects were reversible upon washout. Analysis of single channel kinetics showed that monocarboxylates did not affect open-time constant and close-time constant. These results suggest that monocarboxylates participate in modulating KATP channels activity in cardiac cells and that modulation of KATP channels activity may resolve the discrepancy between the low Ki in excised membrane patches and high levels of intracellular ATP concentration during myocardial ischemia or hypoxia.

Effects of cyclic-GMP on hyperpolarization-activated inward current (I|f) in sino-atrial node cells of rabbit

The aim of present study is to investigate the effects of cGMP on hyperpolarization activated inward current (If), pacemaker current of the heart, in rabbit sino-atrial node cells using the whole-cell patch clamp technique. When sodium nitroprusside (SNP, 80 muM), which is known to activate guanylyl cyclase, was added, If amplitude was increased and its activation was accelerated. However, when If was prestimulated by isopreterenol (ISO, 1 muM), SNP reversed the effect of ISO. In the absence of ISO, SNP shifted activation curve rightward. On the contrary in the presence of ISO, SNP shifted activation curve in opposite direction. 8Br-cGMP (100 muM), more potent PKG activator and worse PDE activator than cGMP, also increased basal If but did not reverse stimulatory effect of ISO. It was probable that PKG activation seemed to be involved in SNP-induced basal If increase. The fact that SNP inhibited ISO-stimulated If suggested cGMP antagonize cAMP action via the activation of PDE. This possibility was supported by experiment using 3-isobutyl-1-methylxanthine (IBMX), non-specific PDE inhibitor. SNP did not affect If when If was stimulated by 20 muM IBMX. Therefore, cGMP reversed the stimulatory effect of cAMP via cAMP breakdown by activating cGMP-stimulated PDE. These results suggest that PKG and PDE are involved in the modulation of If by cGMP: PKG may facilitate If and cGMP-stimulated PDE can counteract the stimulatory action of cAMP.

Effect of metabolic inhibition on inward rectifier K current in single rabbit ventricular myocytes

In the present study, we have investigated the effect of metabolic inhibition on the inward rectifer K current (IK1). Using whole cell patch clamp technique we applied voltage ramp from +80 mV to -140 mV at a holding potential of -30 mV and recorded the whole cell current in single ventricular myocytes isolated from the rabbit heart. The current-voltage relationship showed N-shape (a large inward current and little outward current with a negative slope) which is a characteristic of IK1. Application of 0.2 mM dinitrophenol (DNP, an uncoupler of oxidative phosphorylation as a tool for chemical hypoxia) to the bathing solution with the pipette solution containing 5 mM ATP, produced a gradual increase of outward current followed by a gradual decrease of inward current with little change in the reversal potential (-80 mV). The increase of outward current was reversed by glibenclamide (10 muM), suggesting that it is caused by the activation of KATP. When DNP and glibenclamide were applied at the same time or glibenclamide was pretreated, DNP produced same degree of reduction in the magnitude of the inward current. These results show that metabolic inhibition induces not only the increase of KATP channel but also the decrease of IK1. Perfusing the cell with ATP-free pipette solution induced the changes very similar to those observed using DNP. Long exposure of DNP (30 min) or ATP-free pipette solution produced a marked decrease of both inward and outward current with a significant change in the reversal potential. Above results suggest that the decrease of IK1 may contribute to the depolarization of membrane potential during metabolic inhibition.